contact : khairul khairulbiologi75@gmail.com 1 abstract slender walking catfish (clarias leiacanthus) has decreased in its natural habitat. many factors affect the decline of the population, including the conversion of land where natural habitat becomes plantation, the presence of excess arrest, and the quality of water that has been contaminated. the condition of the waters determines fish life, considering that various species of fish can only live on certain conditions. a study related to the correlation of water quality with fish abundance index is needed as a knowledge of determining biological and ecological needs of fish as a biological resource in a water.. the results of this study showed the abundance index of wild-type catfish at january (0.15 ind./m2), february (0.22 ind./m2), and march (0.20 ind./m2). the correlation analysis of water quality showed ph has a correlation (+) with a value of r (0.827). issn : 2580-2410 eissn : 2580-2119 abundance index slender walking catfish with correlation of water quality in natural habitat khairul department of education biology, universitas labuhanbatu, rantauprapat, north sumatra province, indonesia introduction catfish (siluriformes) mostly live in freshwater and marine waters. the siluriformes family is a fish that has a tentacle in its mouth. the group consists of 106 species and is grouped into 35 genera and 12 families, namely bagridae, siluridae, schilbidae, pangasiidae, akysidae, parakysidae, sisoridae, clariidae, chacidae, ariidae, plotosidae and loricariidae (kottelat, et al., 1993). one of the types is the slender walking catfish (c. leiacanthus). the difference with other catfish only lies in its color and complexion, because the slender walking catfish has a brown color and has a yellow leopard pattern. fish limbat also has a delicious flavor and high economical value. nutritional content also has benefits for health including: prevent atherosclerosis, increase muscle mass, prevent autoimmune diseases, brighten and moisturize the skin, nourish the eyes, and banish the disease of diabetes (rianto, 2018). c. leiacanthus is a endemic fish found in the island of sumatra and kalimantan. lately it has begun to be difficult to find in its natural habitat. one factor of the cause is land conversion, where its natural habitat is converted into plantation land. in the riau province due to the opening of peatland into plantations allegedly have an impact on reduced habitat open access international journal of applied biology keyword c.leiacanthus, abundance index, water quality correlation, natural habitat international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. article history received 20 july 2020 accepted 31 december 2020 international journal of applied biology, 4(2), 2020 2 and natural feed sources for fish, increasing turbidity and water temperature (haryono, 2007). research needs to be done regarding the abundance index of c. leiacanthus population with the correlation of environmental conditions in its natural habitat. based on field observations, the natural habitat of c. leiacanthus is found in the white sand village and bandar tinggi village. the data and information of the study is a preliminary step to anticipate the impact of decline in c. leiacanthus population and also as an effort to preserve sustainability in its natural habitat. materials and methods fish sampling was conducted in january-march 2020. this research is an exploratory study, where researchers are looking for a wild type catfish habitat found in its natural habitat. furthermore, the arrest uses the banjur (passive fishing rod) and the bait in the form of earthworm. banjur used as many as 20 pieces. fish samples were taken at 2 locations: station 1 at pasir putih village, silangkitang district, south labuhan batu regency and station 2 at bandar tinggi village, bilah hulu district, labuhanbatu regency. further observation location can be seen in figure 1. figure 1. map of research observasation population estimation using the catch per unit method effort refers to (ricker, 1975); (pauly,1983). data analysis for calculating the abundance index using formulas (tjakrawidjaja & haryono, 2001). ki = ni na where: ki = abundance index; ni = number of individual catches; na = number of fishing rod. some of the water quality parameters in the test are tds, tss, do, bod, cod, (exsitu), ph and temperature (insitu). water samples were analyzed in the testing laboratory the institute for industrial research and standardization of medan. subsequent data processing was analyzed using microsoft excel 2010 and spss version 22 programs. . international journal of applied biology, 4(2), 2020 3 results and discussion abundance index the abundance index of c. leiacanthus in each month shows the difference of january (0.15 individual/m2), february (0.22 individual/m2), and march (0.20 individual/m2). the data can be viewed in figure 1. figure 2 . abudance index of c. leiacanthus the population density of c. leiacanthus based on the results of the study differs in each month, index of abundance c. leiacanthus is still quite high at every research site, where in march is the peak of 0.22 individuals/m2. this research is much higher the index of abundance c. leiacanthus if comparative with fish kaloso with density 0.017 individuals/m2 in the pomo swamp, merauke regency, papua (tjakrawidjaja & haryono, 2001). the abundance and spread of a population in a aquatic ecosystem, determined by several factors among them the level of resource availability, factors or the physical and chemical properties of water that can be tolerated by species in the population (odum, 1953). it is possible that the habitat of these fishes in the form of rivers and swamps is still in good condition as well as catching mostly using a banjur (marhana & kamri, 2017). the difference in the very high rainfall will also change the structure of the fish community because the difference in water surface changes so that it correlates to changing conditions and availability of habitat for fish (jenkins & jupiter, 2011) correlation of water conditions with abundance index results of the analysis of spss application program version 22 used to determine the correlation of water quality condition in the form of: tds, tss, do, bod, cod, ph, and temperature with a abundance index c. leiacanthus at the research observation. more data can be seen in table 1. table 1. correlation water quality with abundance index of c. leiacanthus no. water quality r 1 tds 0.132 2 tss 0.135 0,15 0,22 0,20 0 0,05 0,1 0,15 0,2 0,25 january february march a b u n d a n c e i n d e x ( in d ./ m ²) sampling time international journal of applied biology, 4(2), 2020 4 3 do 0.103 4 bod 0.112 5 cod 0.105 6 ph 0.827 7 temperature 0.161 analysis results indicate the direct correlation (+), meaning the higher the value of the water quality then the value of abundance index c. leiacanthus the higher. the ph value that indicates the highest correlation, it is given the ph as a limiting factor for fish life. the occurrence of change of water quality parameters in nature can also be caused by the forest over the function of plantation land (sari, et al., 2017). the use of fertilizer and anorganic pesticides continuously can adversely affect the environment especially on the quality of groundwater to the occurrence of pollution, especially nitrogen (n) npk fertilizer that is used continuously without any control or control will affect the quality of groundwater periodically, this influence that will trigger an increase in the concentration of nitrate, phosphate, and potassium in the soil (fikri, et al., 2014). this will certainly affect the value of ph in a water. the ph value after the first fertilization ranges from 5.50-6.60 and the ph value after the second fertilization range 6.5-7.2 (jana, et al., 2014). surely it will donate waste or chemical residue to the river and can impact its water quality (alfionita, et al., 2019). optimal ph for catfish life ranges from 6.78.6 (warseno, 2018). conclusions the abundance index of c. leiacanthus is based on march's capture of the highest score, followed by february and january. the abundance index is categorized as high, habitat condition still supportive for its life in nature. it is correlated with a polysistive water quality factor that is ph, catfish that live in the wild need optimal ph for body development and reproduction. international journal of applied biology, 4(2), 2020 5 references alfionita, a.n.a., patang, & kaseng, e. s. (2019). pengaruh eutrofikasi terhadap kualitas air di sungai jeneberang. jurnal pendidikan teknologi pertanian v, 5(1), 9–23. file:///c:/users/user/downloads/8190-19101-1-sm.pdf odum, e.p. (1953). fundamentals of ecology. philadelphia and london, w.b. saunders co. fikri, u., marsudi, & jati, d. r. (2014). pengaruh penggunaan pupuk terhadap kualitas air tanah di lahan pertanian kawasan rawa rasau jaya iii, kabupaten kubu raya. jurnal teknologi lingkungan lahan basah, 2(1), 1–10. https://doi.org/10.26418/jtllb.v2i1.8280 haryono. (2007). komposisi dan kelimpahan jenis ikan air tawar pada lahan gambut diwilayah propinsi riau. berita biologi, 8(4), 231–239. https://doi.org/10.1017/cbo9781107415324.004 jana, i w., sudarmanto, i g., & rusminingsih, n. k. (2014). pengaruh aktivitas pertanian terhadap kualitas air irigasi di subak tegalampit payangan gianyar. jurnal skala husada, 11(1), 34–40. http://poltekkes-denpasar.ac.id/files/jsh/v11n1/i wayan jana1, i gede sudarmanto2, ni ketut rusminingsih3 jsh v11n1.pdf jenkins, a. p., & jupiter, s. d. (2011). spatial and seasonal patterns in freshwater ichthyofaunal communities of a tropical high island in fiji. environmental biology of fishes, 91(3), 261–274. https://doi.org/10.1007/s10641-011-9776-4 kottelat, m., whitten, a. j., kartikasari, s. n., & wirjoatmodjo, s. (1993). freshwater fish of western indonesian & sulawesi. singapura, periplus editions. marhana, w. o., & kamri, s. (2017). kelimpahan dan distribusi ikan gabus ( channa striata) di perairan rawa aopa watumohai desa pewutaa kecamatan angata kabupaten konawe selatan. jurnal manajemen sumber daya perairan, 2(3), 225–234. file:///c:/users/user/downloads/2665-7375-1-pb.pdf pauly, d. (1983). some simple methods for the assessment of tropical fish stocks (issue 234). fao fisheries technical paper. https://s3-us-west2.amazonaws.com/legacy.seaaroundus/doc/researcher+publications/dpauly/pdf/198 3/books+and+chapters/somesimplemethodsassessmenttropicalfishstocks.pdf rianto, a. (2018.). ikan limbat, saudara lele yang miliki banyak manfaat. iws.co.id. retrieved june 20, 2020, from https://www.isw.co.id/post/2018/11/13/ikan-limbatsaudara-lele-yang-miliki-banyak-manfaat international journal of applied biology, 4(2), 2020 6 ricker, w. e. (1975). computation and interpretation of biological statistics of fish populations. in bulletin of the fisheries research board of canada (vol. 191, issue 2706). https://doi.org/10.1038/108070b0 sari, r. d., purnama, a. a., & lestari, r. (2017). jenis-jenis ikan catfish (siluriformes) di sungai kumango rokan hulu riau. jurnal ilmiah mahasiswa fkip prodi biologi, 3(1), 3–6. https://media.neliti.com/media/publications/111679-id-jenis-jenis-ikan-catfishsiluriformes-di.pdf. tjakrawidjaja, a. h. & haryano. (2001). studi populasi ikan kaloso (scleropages jardinii) di rawa pomo kecamatan citak mitak, kabupaten merauke, papua. berita biologi, 5(4), 357–364. https://ejournal.biologi.lipi.go.id/index.php/berita_biologi/article/viewfile/1119/994 warseno, y. (2018). budidaya lele super intensif di lahan sempit. jurnal riset daerah, 17(2), 3065–3088. file:///c:/users/user/downloads/20180818_05budidayalele.pdf contact: diah sudiarti diah.sudiarti23@gmail.com 106 abstract green eggplant (solanum melongena l.) is a food plant grown for its fruit. the fruit of the eggplant depends on the fruit stalk and is a single true fruit, thick, soft, and juicy. this study was a completely randomized design with a three-treatment pattern. the first is a combination of organic fertilizer and arbuscular mycorrhizal fungi, the second is a positive control, namely using npk fertilizer, the third is a negative control, namely without treatment. each treatment was repeated three times and each repetition consisted of 3 plants. plant growth parameters consist of plant height and number of leaves. plant productivity parameters consist of the number of fruit, fruit wet weight, and dry weight. based on the results of the study, it was obtained data that the combination of organic fertilizer and arbuscular mycorrhizal fungi had better growth and productivity results compared to other treatments with an average plant height of 44.46; the average number of leaves 11.12; the average number of fruits was 5.51; mean fruit wet weight 106; and the average dry weight of fruit 84. the results of the anova statistical test showed that the significance value of plant height and a number leaves was 0.000 (α <0.05), and the significance value for the number of fruits was 0.006 (α <0.05), while the significance value of weight wet and dry weight of fruit is 0.000 (α <0.05), this indicates that the combination of organic fertilizer and arbuscular mycorrhizal has a significant effect on the growth and productivity of green eggplant plants. issn : 2580-2410 eissn : 2580-2119 the effectiveness of organic fertilizer and micoriza arbuscula on growth and productivity green eggplant (solanum melongena l.) diah sudiarti., s.pd., m.si1* 1biologi education, jember islam university, jember, jawa timur, indonesia introduction organic fertilizers are fertilizers from organic materials, namely from plant residues, green manure, manure, including microbes such as bacteria and fungi. organic fertilizers have the advantage of increasing crop production and maintaining soil fertility in a sustainable manner (suliasih, 2015). fertilizers of biological organisms as inoculants with active ingredients of living organisms that function to block certain nutrients or facilitate the availability of nutrients in the soil for plants. facilitating the availability of these nutrients can take place through increasing plant access to nutrients (purnomo et al., 2014). arbuscular mycorrhizal fungi are one of the fungi that live in the soil. this fungus is always associated with higher plants and both provide mutual benefits. the benefits of arbuscular mycorrhizal fungi can be grouped into three, namely for plants, ecosystems, and open access international journal of applied biology keyword arbuscular mycorrhizal; green eggplant (solanum melongena); organic fertilizer article history received 29 september 2020 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 107 humans. for plants, arbuscular mycorrhizal fungi are very useful for increasing nutrient uptake, especially elemental phosphate (p). the rate of entry of p nutrients into the arbuscular mycorrhizal fungi hyphae can be up to six times faster in the roots of plants infected with arbuscular mycorrhizal fungi compared to those not infected with arbuscular mycorrhizal fungi. this happens because the external arbuscular mycorrhizal fungi hyphae network can expand the absorption field (musfal, 2010). eggplant or eggplant (solanum melongena l.) is a food plant grown for its fruit. eggplant is a type of vegetable that contains a variety of nutrients. the nutritional composition of eggplant per 100 grams is 92.7 grams of water; iron 0.4 mg; carbohydrates 5.5 grams; 0.2 grams of fat; calories 24.00 cal; phosphorus 37 mg; calcium 15 mg; 1.1 grams of protein; sodium 4.00 mg; vitamin b1 0.04 mg; vitamin a 30 s.i; vitamin c 5.00 mg (haryoto, 2013: 13). the market potential for eggplant can also be seen in terms of prices that are affordable to all levels of society, thus opening up greater opportunities for market and farmer absorption. based on the results of a market survey, it shows that the eggplant plant is one of the most sought after vegetables by the community. the amount of eggplant consumption has increased every year, but this is not accompanied by an increase in production. this is because the majority of farmers use chemical fertilizers for growth. the current availability of chemical fertilizers is very limited and the price is also expensive, so this is what makes farmers reluctant to grow green eggplant because the price is cheap, not comparable to the price of expensive chemical fertilizers. to increase the growth and productivity of green eggplant plants, a combination of organic fertilizers and arbuscular mycorrhizal fungi are used which are cheap and easy to obtain, thereby increasing the amount of green eggplant production and meet market needs. materials and methods the materials used in this study were the seeds of green eggplant (solanum melongena l.), water for watering plants, and organic fertilizer obtained from the laboratory biology of the islamic university of jember, arbuscular mycorrhizal fungi obtained from the botani laboratory of lipi. this type of research is an experimental study using a completely randomized design with a three-treatment pattern. first, using a combination of organic fertilizer and arbuscular mycorrhizal fungi, the second was positive control (k +), namely npk fertilizer, and the third was negative control (k-), which was without treatment. each treatment was repeated three times, where each repetition consisted of three plants. growth observation parameters include plant height and number of leaves. retrieval of plant growth data began when the plants were 7 days after planting (planting cuttings) until the plants were 45 days old. plant growth data were collected every 7 days. productivity observation parameters include the number of fruits per crop, planting wet weight, and dry weight. productivity measurements are carried out during the first harvest, which is 70-80 days after the seeds are planted. furthermore, harvesting is done every 3-7 days. productivity measurements were carried out for 5 harvests. growth and productivity data of eggplant (solanum melongena l) obtained were statistically analyzed using spss 21 through normality and homogeneity tests. if the data is normal and homogeneous, then the data is analyzed using one-way anova (analysis of variance). if the results have a significant effect, it will be continued with the duncan test to compare the results between treatments. international journal of applied biology, 5(1), 2021 108 results and discussion chemical speciation of heavy metals in bioaugmented and nonbioaugmented soils from tb landfill this research was conducted to determine the effect of a combination of organism fertilizer, and arbuscular mycorrhizal fungi on the growth and productivity of green eggplant plants. the results showed that the best average growth and productivity of green eggplant was found in the treatment of the combination of organism fertilizer, and arbuscular mycorrhizal fungi compared to other treatments. this can be observed in the figure below: figure 1. graph of the average growth of green eggplant plants figure 1. graph of the average productivity of green eggplant plants 0 5 10 15 20 25 30 35 40 45 50 organic fertilizer k + k sc o r e treatment average plant height growth plant height number of leaves 0,00 20,00 40,00 60,00 80,00 100,00 120,00 number of fruits dry weight gross weight s c o r e average green eggplant productivity organic fertilizer k+ kinternational journal of applied biology, 5(1), 2021 109 the figure shows a significant difference in the graph between the combination of organic fertilizer, and arbuscular mycorrhizal fungi with chemical fertilizers (k +) and without fertilizers (k-), both in terms of plant height, the number of leaves, the number of fruits, dry weight, and fruit wet weight. then the data is analyzed statistically, the results of the statistical analysis can be observed in the table below: table 1. statistical test results on the growth and productivity of green eggplant perlakuan number of fruit dry weight fruit wet weight plant height number of leaves organic fertilizer 5,51±0,54 b 84±3 b 106±5,03 c 44,66±3,51c 11,12±0,56 c k+ 5,19±0,40 b 78±7 a 91±2,91 b 29,69±0,72 b 9,67±0,04 b k4,60±0,58 a 66±3 a 80±4,3 a 24±1,80 a 7,76±0,08 a note: numbers followed by the same letter are not significantly different, while numbers followed by a different letter mean significantly different. this research consisted of three treatments, namely, application of a combination of organic fertilizer and arbuscular mycorrhizal fungi, positive control (npk fertilizer), and negative control (without fertilizer) on the growth and productivity of eggplant plants. based on the results of the analysis, it is known that the combination of organic fertilizer and arbuscular mycorrhizal fungi has a significant effect on the growth and productivity of green eggplant, then to determine the difference, the duncan test was performed. from the duncan test, it was found that organic fertilizer and arbuscular mycorrhizal fungi were significantly different from other treatments both in terms of plant height and number of leaves. meanwhile, plant productivity data showed that the application of organic fertilizer and arbuscular mycorrhizal fungi was significantly different from other treatments for wet weight and dry weight of fruit, while for the number of fruits it showed that the combination of organic fertilizer and arbuscular mycorrhizal fungi was not significantly different from the positive control (npk fertilizer). in previous trials, arbuscular mycorrhizal fungi were used for the process of rice plant seeding, and organic fertilizer was used for the growth of rice plants. from these trials, it was found that the results of the process of rice seedlings given arbuscular mycorrhizal fungi had better results, namely, the number of roots was more and longer than rice seeds without being given arbuscular mycorrhizal fungi. the growth of rice plants given organic fertilizer had better results compared to chemical fertilizers. besides previous research conducted by sudiarti, diah (2018) entitled "the effect of giving arbuscular mycorrhizal fungi (cma) on the growth of edamame soybean (glycin max)" shows that giving arbuscular mycorrhizal fungi affect the growth of edamame soybean plants. so this research can be said to be in line with previous research, that the application of organic fertilizer and arbuscular mycorrhizal fungi affect the growth and productivity of green eggplant plants. organic fertilizer is a homemade fertilizer consisting of a consortium of several microbes and fermented using natural ingredients that are around. the microbes used in organic fertilizer are known to be able to fix nitrogen free from air and can provide p elements that can be directly used by plants. besides, some microorganisms play an important role because the organic waste that has died is broken down into elements that are returned to the soil (n, p, k, ca, mg, etc.) and the atmosphere (ch4 or co2). ) as nutrients that can be reused by plants, so that the nutrient cycle runs as it should and life international journal of applied biology, 5(1), 2021 110 processes on earth can take place, the activity of organic matter decomposing organisms such as microbes and mesofauna (invertebrate animals) supports the continuity of the nutrient cycle process in the soil, (widowati, et al. 2005). arbuscular mycorrhizal fungi have a positive influence on growth and other physiological processes in plants. the beneficial effect of arbuscular mycorrhizal fungi on plant growth is often associated with increased uptake of nutrients that are not available, especially phosphorus, (rosliani et al. 2009). so that the results of this study are similar to the theory that the use of a combination of organic fertilizers and arbuscular mycorrhizal fungi can increase the growth and productivity of green eggplant plants. so it can be said that the combination of organic fertilizers and arbuscular mycorrhizal fungi is effective on the growth and productivity of green eggplant plants. conclusions based on the results of the study, it was obtained data that the combination of organic fertilizer and arbuscular mycorrhizal fungi had better growth and productivity results compared to other treatments with an average plant height of 44.46; the average number of leaves 11.12; the average number of fruits was 5.51; mean fruit wet weight 106; and the average dry weight of fruit 84. the results of the anova statistical test showed that the significance value of plant height and the number of leaves was 0.000 (α <0.05), and the significance value for the number of fruits was 0.006 (α <0.05), while the significance value of weight wet and dry weight of fruit is 0.000 (α <0.05), this indicates that the combination of organic fertilizer and arbuscular mycorrhizal fungi has a significant effect on the growth and productivity of green eggplant plants. international journal of applied biology, 5(1), 2021 111 references haryoto. 2013. growing eggplant in pot. yogyakarta: kanisius. musfal, 2010. potential of arbuscular mycorrhizal fungi to increase maize yield. journal of agricultural research and development. 29 (4), 154-158. purnomo, rudi, et al. 2013. the effect of various types of organic and inorganic fertilizers on growth and yield of cucumber (cucumis sativus). journal of plant production vol. 1 no. 3. july-2013. issn: 2338-3976 sudiarti, diah. 2018. the effect of arbuscular mycorrhizal fungi (cma) on the growth of edamame soybean (glycin max). journal of sainhealth vol.2 no.2 september 2018 edition. rosliani, r., y. hilman, and n. sumarni. 2009. utilization of mycorrhizae, organic materials, and natural phosphates on yield, cucumber plant nutrient uptake, and chemical properties in ultisol acid soil. journal of horticulture vol. 19 no. 1, pp: 66-74. suliasih, sri widawati. 2015. increasing corn yield using biological organic fertilizer (poh). pros sem nas masy biodiv indon. widowati, l.r., sri widati, u. jaenudin, and w. hartatik. 2005. the effect of organic fertilizer compost enriched with minerals and biological fertilizer on soil properties, nutrient uptake, and organic vegetable production. agribusiness development program research project report, soil research institute, fy 2005 (unpublished). contact : fahruddin fahruddin_science@unhas.ac.id 59 abstract star heavy metal pollution in the industrial area causes problems in the surrounding vegetation. the study aims to determine the absorption of heavy metals lead (pb) which accumulates on green champa leaves (polyalthia longifolia) in the industrial area of makassar city, and its effect on the number of leaf stomata. leaf samples were taken at three stations, the method used was random sampling. measurement of the concentration of heavy metals lead (pb) by the atomic absorption spectrophotometery method (aas), observation of the number of stomata using a microscope. the results showed the concentration of pb that accumulated in green champa leaves, such as 1.21–2.42 µg/g. heavy metals affect stomata on green champa leaves but do not show significant damage (> 0.05). this shows that the green champa plant has benefits in reducing heavy metal pollution in the air, without experiencing damage to the leaf stomata fruit issn : 2580-2410 eissn : 2580-2119 microbiological assessment of roasted dried periwinkle (tympanotonus fuscatus) sold in yenagoa bayelsa state opara christiana ngozi 1, obi theodora 2*, akpoka augustine obhioze 2 1 federal university otuoke, faculty of sciences, department of microbiology, p. m. b.126, yenagoa bayelsa state, nigeria 2igbinedion university, college of natural and applied, department of chemical sciences, p.m.b 0006, okada, benin, edo state, nigeria 3department of chemistry, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesiindonesia. introduction air pollution originates from very large quantitative human activities, which are sourced from transportation, industrial, and burning activities (aaksu, 2015; nurhidayah et al., 2019). air pollution emissions by industry are very dependent on the type of industry and its processing, industrial equipment and their use. various industries use energy and heat from burning gasoline to increase the amount of pollutants in the air (tauqeer et al., 2013; fahruddin, 2020). combustion of coal coming from industries that emerge from the chimney produces various substances such as co, so2, nox, and pollutants in the form of heavy metal compounds such as cadmium (cd), and lead (pb) (zhao et al. 2013; fahruddin et al., 2018). some heavy metals in the environment bioaccumulate in the food chain, so metals will be distributed to human body parts. if this continues for a long period of time, it may endanger human health and the environment (aksu, 2015). heavy metals have toxic effects open access international journal of applied biology keyword lead (pb), green champa, stomata, industrial areas. article history received 18 july 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 60 when biochemical reactions occur in living organisms, namely inhibiting growth, lack of oxygen needed, reproductive disorders and tissue repair. one of them is lead metal (pb) has attracted attention because it is persistent, bioaccumulative, and toxic (javarabad et al., 2013; adawiyah et al., 2017). makassar industrial area or known as pt. kima is one of the areas in the city of makassar in which there are many industries to meet the needs of the people of makassar city and surrounding areas (hapsah et al., 2013). the existence of an industrial area in the middle of community settlements can bring benefits and negative impacts, will harm the surrounding community if not managed properly (shock et al., 2016). if an industrial company does not properly manage its waste disposal, especially waste entering the air containing heavy metals, it will spread to other places in the direction of the wind (javarabad et al., 2013). the main road in the industrial area of makassar city is traversed every day by various vehicles and industrial activities using fossil fuel types, producing heavy metal emissions of cadmium (cd) and lead (pb). then there will be accumulation of heavy metals in the shade plants in the industrial area of makassar city (hapsah et al., 2013; fahruddin et al., 2019). overcome air pollution due to the presence of industry and traffic is to plant trees on the roadside and in the corner of the city. this is certainly beneficial, namely the road environment to be shady and comfortable, air pollution and noise pollution can be overcome (rungruang et al., 2016). some plants are metal hyperaccumulators, namely mahogany (swetenia macrophylla), green champa (polyalthia longifolia), sea putat (baringtonia asiatica), and rain tree (samanea saman) have the ability to accumulate heavy metals in the air (jafarabad et al., 2013). the heavy metal in the form of free particles will partially stick to the plant especially on the leaves. part of road shading plants that are sensitive to pollutants and are most often exposed to heavy metals are leaves (popescu, 2011). therefore leaves can be an indicator of air pollution, marked by physical changes, including changes in leaf tissue, one of which is the amount of leaf stomata (rungruang et al., 2016). most trees used as shade plants in urban areas and in industrial areas are green champa trees. this tree is a type of plant that has roots that can withstand damage, easily grows in hot areas and is resistant to wind, making it suitable for use as a road shade plant that can absorb heavy metal pollution in the air (tauqeer et al., 2013). based on this background, an observational study of green champa leaves was carried out in absorbing heavy metals lead (pb) and its effects on the amount of stomata in the leaves. materials and methods sampling stations the station is determined based on its location in the industrial area of makassar city on green champa leaves (polyalthia longifolia) affected by air pollution. the study was conducted based on a distance of about 1 km between. different locations, namely: station 1 is the entrance of jalan kima 1, station 2 is the intersection of jalan kima 1 and jalan kima 2, and station 3 is jalan kima 4. map of sampling locations in the industrial area of makassar city is seen in figure 1. international journal of applied biology, 4(2), 2020 61 figure1. map of the location of leaf samples of green champa plants (polyalthia longifolia) in the industrial area of makassar leaf sampling sampling of green champa leaves was carried out by random sampling at each station. leaflets are stored in plastic container for further analysis. green champa leaves taken at each station are the leaves in the upper leaf, namely with the code s1a, s2a, s3a as high as 10-25 m and the lower leaf with the codes s1b, s2b, s3b as high as 5-10 m. measurement of heavy metal pb concentration in leaves measurement of heavy metal pb concentrations was carried out on green champa leaves using atomic absorption spectrophotometery (aas). samples of green champa leaves are heated in an oven at 70 °c until they reach a constant dry weight, then weighed as much as 5 g. the dried leaf samples were grayed in a furnace at 600 °c for 24 hours. leaf ash was given demineralized water with 100 ml and 5 ml concentrated h2so4 (65%). the mixture is heated at 20 °c until the sample reaches 10 ml in a beaker, filtered and added aquabides to the 100 ml limit mark. lead content (pb) in the solution was measured with an atomic absorption spectrophotometer with the formula: cy1= 𝐶𝑦 𝑥 where : cy1 = heavy metal content in leaf tissue (μg/g), cy = the concentration of heavy metals measured at aas (μg/ml) v = dilution volume (ml),w= dry weight of the leaf (g). international journal of applied biology, 4(2), 2020 62 determination of stomata amount in the leaves the underside of the green champa leaves is given cutex, then isolated to get a transparent stomata print. then the mold is affixed to the deck glass. this clear layer is then observed under a trinocular microscope with a magnification of 400x, then the number of stomata is calculated. the data obtained are grouped into categories: few (1 50), quite a lot (51 100), many (101 200), very much (201 -> 300), infinite (301 -> 700). results and discussion vegetation and industry conditions of industrial areal based on observations, there are two highway lanes in the industrial area of makassar city. the most dominant shady tree besides road to grow is green champa (polyalthia longifolia) with a tree height between 10-25 m, has the characteristics of a strong trunk, canopy tree diameter of 5-10 m, and has dense leaves and soars upward which grow in groups and form a barrier along the road and partly in the yard of the factory and warehouse. the distance between the tree and the highway is very close, and the distance between the tree and the industrial factory is 5-10 m. according to data from the head office of industrial area in the city of makassar, there are 265 factories in the form of industrial concrete factories, battery factories, paint factories, textile processing plants, electronic factories, accu factories, paper factories, as well as food factories and others. fuels used in managing industries in the combustion process are coal, firewood and petroleum. this can be a source of heavy metal pollution in the air (hapsah et al., 2013; tauqeer et al., 2013). heavy metal pb concentration on green champa leaves from the observations of heavy metal concentrations showed the green champa leaves at each station. in the upper leaf and lower leaf that is relatively the same. at s2a and s3b the pb concentration was 2.42 μg/g, then at s3a the pb concentration was 1.91 μg/g. furthermore in s1b and s2b with pb concentration was 1.81 μg /g, as shown in figure 2. figure 2. heavy metal pb concentration on green champa leaves in each station, i.e. s1 is station 1, s2 is station 2 and s3 is station 3; a is the upper leaf and b is the lower leaf. differences in plant response to types of heavy metals caused by the characteristics of plants to the way of accumulation, as well as the level of tolerance of plants to the toxic effects of heavy metals pb, even at low concentrations can affect plant metabolic processes (fakhry and migahid 2011). the amount of pb in the air is affected by the volume or density of traffic, distance from the highway and industrial areas, engine acceleration and wind international journal of applied biology, 4(2), 2020 63 direction. while the high content of pb in plants is also influenced by sedimentation (alhasnawi et al. 2016). differences in plant response to types of heavy metals caused by the characteristics of plants to the way of accumulation, as well as the level of tolerance of plants to the toxic effects of heavy metals pb, even at low concentrations can affect plant metabolic processes (fakhry and migahid, 2011). the amount of pb in the air is affected by the volume or density of traffic, distance from the highway and industrial areas, engine acceleration and wind direction. while the high content of pb in plants is also influenced by sedimentation (al-hasnawi et al,. 2016). factors that influence the absorption of heavy metals are the high intensity of light and air temperature, as well as low humidity which triggers the number of stomata more, so that more absorption of heavy metals pb accumulates in the leaves. another factor that affects the absorption of heavy metals is the size of the opening of the stomata which has a length of 12 39 µm. having mesophyll thickening in the stomata exposed to pb (attipalli et al., 2010; andriany et al., 2018). heavy metal pollutants can enter through the stomata due to the very small size of heavy metal particulates, which is less than 2μ, while the opening size of the stomata is 10μ x 27μ, so heavy metal particulates can enter easily through the stomata. the thicker mesophyll shows the more metal concentration (kabir et al., 2010). number of stomata the most number of stomata to the concentration of heavy metal pb was at station 3 in the lower header (s3b) of 130 compared to stations 1 and 2 and control. compared to station 1, there were 77 stomata of the upper leaf (s1a) and 73 stomata of the lower leaf (s1b) of 73 stomata, this is quite a number (51 100) which is relatively the same as the control, as shown in figure 3. figure 3. the number of stomata to the concentration of heavy metals pb in green champa leaves at each station: i.e. s1 is station 1, s2 is station 2 and s3 is station 3; a is the upper leaf and b is the lower leaf; ka is the control on the upper leaf and kb is the control on the lower leaf. based on an analysis of the effect of pb on the upper green champa leaves on the number of stomata using statistical analysis, namely a simple linear regression test, the square of r2 is 0.271. the effect of pb heavy metal concentration on the amount of stomata in the leaves is 27%. this means that there is an influence of heavy metal pb concentration on the amount of stomata in the leaves, and the remaining 73% is influenced by other variables, as shown in figure 4a. based on the results of the analysis of variance shows that international journal of applied biology, 4(2), 2020 64 although the concentration of heavy metal pb on the amount of stomata in leaves is 27%, the results do not experience significant damage. based on the analysis of the effect of pb on the bottom green champa leaves on the number of stomata using statistical analysis, namely a simple linear regression test, the results obtained are the square of r2 of 0.629. the effect of heavy metal pb concentration on the amount of stomata in leaves was 62%. this means that there is an influence of heavy metal pb concentrations on the amount of stomata in the leaves. the remaining 38% is influenced by other variables as shown in figure 4b. based on the analysis of the variance which shows that although the concentration of heavy metals pb on the amount of stomata in the leaves is 27%, the results do not experience significant damage. figure 4. analysis of the effect of pb on the number of stomata in green champa leaves in the upper leaf (a) and green champa leaves in the lower leaf (b). more stomata in the lower canopy than the top canopy, because water loss occurs faster through the stomata on the top of a leaf exposed to sunlight, this is in line with previous research (attipalli et al., 2010; tanjung et al., 2019), the number of stomata under the tree canopy is higher than above the canopy in road shade plants, so the higher the number of stomata, the higher the potential to absorb heavy metals or particles in the air. when in the lack of light intensity and low temperature at each station, the amount of stomata is reduced so that absorbs co2 is reduced and the process of photosynthesis is disrupted (aalto et al. 2017; fahruddin and tanjung 2019). conclusions from research on the absorption of heavy metals pb in green champa leaves (polyaltia longifolia), shows that the heavy metals pb concentration is 1,21–2,42 µg/g which accumulates in green champa leaves in the industrial area of makassar city by being categorized as still normal. heavy metals pb affect the number of stomata in green champa leaves, but microscopically the leaf stomata do not show any significant damage (>0.05) that occurs at three stations. international journal of applied biology, 4(2), 2020 65 references aalto, j., riihimäki, h., meineri, e., hylander, k. & luoto, m. 2017.revealing topoclimatic heterogeneity using meteorological station data. international journal of climatology. 37(13):544-566. adawiyah, s. r., fahruddin & kahar, m. 2017. application of bacterial isolate from tamangapa makassar in the household organic waste processing. celebes biodiversitas.1(1): 40-44. aksu, a. 2015. sources of metal pollution in the urban atmosphere (a case study: tuzla, istanbul). journal of environmental health science & engineering. 3(79):1-10. al-hasnawi, s. s., hussain, m. h., al-ansari, n. & knutsson, s. 2016. the effect of the industrial activities on air pollution at baiji and its surrounding areas, iraq. engineering. 8(1):34-44. andriany, fahruddin & as’adi, a. 2018. effect of bioactivators types on the rate of teak tectona grandis leaf ‘seresah’ decomposition, in the campus unhas tamalanrea. bioma: jurnal biologi makassar. 3(2): 31-42. attipalli, r., girish, k.r. & agepati, s. r. 2010. the impact of global elevated co2 concentration on photosynthesis and plant productivity. current science. 99(1):4657. fahruddin, f, abdullah, a. & nafie, n. l. 2018. treatment of acid mine drainage waste using sediment as source of sulfate-reducing bacteria to reduce sulfates. pollotion research. 37(4): 903-907. fahruddin, f. & tanjung, r.e. 2019. the study of bacteria populations in phytoremediation of cadmium using eichhornia crassipes. journal of physics: conference series. 1341(022019):1-8. fahruddin, f. 2020. isolation and characteristics of cellulose degradation bacteria from makassar antang furniture industry waste. serambi engineering. 5(2): 951-956. fahruddin, nur, h., slamet, s. & sri, w. 2019. assay of bacterial isolates growth ability from water and tallo river sediments on lead metal (pb). jurnal ilmu alam dan lingkungan.10(2): 58 – 64. fakhry, m. & migahid, m. 2011.effect of cement-kiln dust pollution on the vegetation in the western mediterranean desert of egypt. international journal of environmental and ecological engineering. 5(9):480-486. hapsah, samang l. & achmad z. 2013. analysis of the level of demand and availability of green open space in the makassar industrial area. jurnal teknik lingkungan. 3:7-12. javarabad, d. m., azadfar, d. & arzanesh, h. m. 2013. the ability to filter heavy metals of lead, copper and zinc in some species of tree and shrub. international journal of advanced biological and biomedical research.1(1):53-60. international journal of applied biology, 4(2), 2020 66 kabir, a. k. l., huda, n.h., jhanker, y. m. & sharmin, k. 2010. vegetation condition around industry area in karaachi. bioresources. 5(3):1618-1625. nurhidayah, lucia, r. w. & fahruddin. 2019. utilization of bacteria isolates from cocoa pulp liquid as bioactivator in composting cocoa fruit waste. celebes biodiversitas. 2(2): 16. popescu, c. g. 2011. relation between vehicle traffic and heavy metal content from the particulate matters. romanian reports in physics. 63(2):471-482. rungruang, j, chantara, s, inta, a. k. & satake, k. 2016. levels of road traffic heavy metals in tree bark layers of cassia fistula tree. international journal of environmental science and development. 7(5):385-388. shock, d. a., leblanc, s. j., leslie, k. e, hand, k., godkin, m. a., coe, j. b. & kelton, d. f. 2016. studying the relationship between on-farm environmental conditions and local meteorological station data during the summer. international journal of dairy science. (3):2169-2179. tanjung, r. e., fahruddin, f. & samawi, m. f. 2019. phytoremediation relationship of lead (pb) by eichhornia crassipes on ph, bod and cod in groundwater. journal of physics: conference series. 1341(022020):1-6. tauqeer, m. h., ali, s., sardar. k., hameed, s., afzal, s., fatima, s., shakoor, b. m. & bharwana, a. s. 2013. heavy metal contamination and what are the impacts on living organisms.greener journal of environmental management and public safety. 2(4):172-179. zhao, p. s., dong, f., he, d., zhao, x. j., zhang, x. l., zhang, w. z., yao, q. & liu, h. y. 2013. characteristics of concentrations and chemical compositions for pm2.5 in the region of beijing, tianjin and hebei, china. atmospheric chemistry and physics. 13(9):4631– 4644. contact : fahruddin fahruddin_science@unhas.ac.id 1 abstract star heavy metal pollution in the industrial area causes problems in the surrounding vegetation. the study aims to determine the absorption of heavy metals lead (pb) which accumulates on green champa leaves (polyalthia longifolia) in the industrial area of makassar city, and its effect on the number of leaf stomata. leaf samples were taken at three stations, the method used was random sampling. measurement of the concentration of heavy metals lead (pb) by the atomic absorption spectrophotometery method (aas), observation of the number of stomata using a microscope. the results showed the concentration of pb that accumulated in green champa leaves, such as 1.21–2.42 µg/g. heavy metals affect stomata on green champa leaves but do not show significant damage (> 0.05). this shows that the green champa plant has benefits in reducing heavy metal pollution in the air, without experiencing damage to the leaf stomata fruit issn : 2580-2410 eissn : 2580-2119 accumulation of heavy metal lead (pb) and effect of stomates number on green champa leaves (polyaltia longifolia) in industrial area of makassar city hardiyanti ym1, fahruddin fahruddin2*,paulina taba3 1department of environmental management, faculty of graduate school, hasanuddin university, makassar, indonesia 2department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia 3department of chemistry, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesiindonesia. introduction air pollution originates from very large quantitative human activities, which are sourced from transportation, industrial, and burning activities (aaksu, 2015; nurhidayah et al., 2019). air pollution emissions by industry are very dependent on the type of industry and its processing, industrial equipment and their use. various industries use energy and heat from burning gasoline to increase the amount of pollutants in the air (tauqeer et al., 2013; fahruddin, 2020). combustion of coal coming from industries that emerge from the chimney produces various substances such as co, so2, nox, and pollutants in the form of heavy metal compounds such as cadmium (cd), and lead (pb) (zhao et al. 2013; fahruddin et al., 2018). open access international journal of applied biology keyword lead (pb) green champa stomata industrial areas article history received 24 june 2020 accepted 10 october 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 2 some heavy metals in the environment bioaccumulate in the food chain, so metals will be distributed to human body parts. if this continues for a long period of time, it may endanger human health and the environment (aksu, 2015). heavy metals have toxic effects when biochemical reactions occur in living organisms, namely inhibiting growth, lack of oxygen needed, reproductive disorders and tissue repair. one of them is lead metal (pb) has attracted attention because it is persistent, bioaccumulative, and toxic (javarabad et al., 2013; adawiyah et al., 2017). makassar industrial area or known as pt. kima is one of the areas in the city of makassar in which there are many industries to meet the needs of the people of makassar city and surrounding areas (hapsah et al., 2013). the existence of an industrial area in the middle of community settlements can bring benefits and negative impacts, will harm the surrounding community if not managed properly (shock et al., 2016). if an industrial company does not properly manage its waste disposal, especially waste entering the air containing heavy metals, it will spread to other places in the direction of the wind (javarabad et al., 2013). the main road in the industrial area of makassar city is traversed every day by various vehicles and industrial activities using fossil fuel types, producing heavy metal emissions of cadmium (cd) and lead (pb). then there will be accumulation of heavy metals in the shade plants in the industrial area of makassar city (hapsah et al., 2013; fahruddin et al., 2019). overcome air pollution due to the presence of industry and traffic is to plant trees on the roadside and in the corner of the city. this is certainly beneficial, namely the road environment to be shady and comfortable, air pollution and noise pollution can be overcome (rungruang et al., 2016). some plants are metal hyperaccumulators, namely mahogany (swetenia macrophylla), green champa (polyalthia longifolia), sea putat (baringtonia asiatica), and rain tree (samanea saman) have the ability to accumulate heavy metals in the air (jafarabad et al., 2013). the heavy metal in the form of free particles will partially stick to the plant especially on the leaves. part of road shading plants that are sensitive to pollutants and are most often exposed to heavy metals are leaves (popescu, 2011). therefore leaves can be an indicator of air pollution, marked by physical changes, including changes in leaf tissue, one of which is the amount of leaf stomata (rungruang et al., 2016). most trees used as shade plants in urban areas and in industrial areas are green champa trees. this tree is a type of plant that has roots that can withstand damage, easily grows in hot areas and is resistant to wind, making it suitable for use as a road shade plant that can absorb heavy metal pollution in the air (tauqeer et al., 2013). based on this background, an observational study of green champa leaves was carried out in absorbing heavy metals lead (pb) and its effects on the amount of stomata in the leaves. materials and methods sampling stations the station is determined based on its location in the industrial area of makassar city on green champa leaves (polyalthia longifolia) affected by air pollution. the study was conducted based on a distance of about 1 km between. different locations, namely: station 1 is the entrance of jalan kima 1, station 2 is the intersection of jalan kima 1 and jalan kima 2, and station 3 is jalan kima 4. map of sampling locations in the industrial area of makassar city is seen in figure 1. international journal of applied biology, 4(2), 2020 3 figure1. map of the location of leaf samples of green champa plants (polyalthia longifolia) in the industrial area of makassar leaf sampling sampling of green champa leaves was carried out by random sampling at each station. leaflets are stored in plastic container for further analysis. green champa leaves taken at each station are the leaves in the upper leaf, namely with the code s1a, s2a, s3a as high as 10-25 m and the lower leaf with the codes s1b, s2b, s3b as high as 5-10 m. measurement of heavy metal pb concentration in leaves measurement of heavy metal pb concentrations was carried out on green champa leaves using atomic absorption spectrophotometery (aas). samples of green champa leaves are heated in an oven at 70 °c until they reach a constant dry weight, then weighed as much as 5 g. the dried leaf samples were grayed in a furnace at 600 °c for 24 hours. leaf ash was given demineralized water with 100 ml and 5 ml concentrated h2so4 (65%). the mixture is heated at 20 °c until the sample reaches 10 ml in a beaker, filtered and added aquabides to the 100 ml limit mark. lead content (pb) in the solution was measured with an atomic absorption spectrophotometer with the formula: cy1= 𝐶𝑦 𝑥 where : cy1 = heavy metal content in leaf tissue (μg/g), cy = the concentration of heavy metals measured at aas (μg/ml) v = dilution volume (ml),w= dry weight of the leaf (g). international journal of applied biology, 4(2), 2020 4 determination of stomata amount in the leaves the underside of the green champa leaves is given cutex, then isolated to get a transparent stomata print. then the mold is affixed to the deck glass. this clear layer is then observed under a trinocular microscope with a magnification of 400x, then the number of stomata is calculated. the data obtained are grouped into categories: few (1 50), quite a lot (51 100), many (101 200), very much (201 -> 300), infinite (301 -> 700). results and discussion vegetation and industry conditions of industrial areal based on observations, there are two highway lanes in the industrial area of makassar city. the most dominant shady tree besides road to grow is green champa (polyalthia longifolia) with a tree height between 10-25 m, has the characteristics of a strong trunk, canopy tree diameter of 5-10 m, and has dense leaves and soars upward which grow in groups and form a barrier along the road and partly in the yard of the factory and warehouse. the distance between the tree and the highway is very close, and the distance between the tree and the industrial factory is 5-10 m. according to data from the head office of industrial area in the city of makassar, there are 265 factories in the form of industrial concrete factories, battery factories, paint factories, textile processing plants, electronic factories, accu factories, paper factories, as well as food factories and others. fuels used in managing industries in the combustion process are coal, firewood and petroleum. this can be a source of heavy metal pollution in the air (hapsah et al., 2013; tauqeer et al., 2013). heavy metal pb concentration on green champa leaves from the observations of heavy metal concentrations showed the green champa leaves at each station. in the upper leaf and lower leaf that is relatively the same. at s2a and s3b the pb concentration was 2.42 μg/g, then at s3a the pb concentration was 1.91 μg/g. furthermore in s1b and s2b with pb concentration was 1.81 μg /g, as shown in figure 2. figure 2. heavy metal pb concentration on green champa leaves in each station, i.e. s1 is station 1, s2 is station 2 and s3 is station 3; a is the upper leaf and b is the lower leaf. differences in plant response to types of heavy metals caused by the characteristics of plants to the way of accumulation, as well as the level of tolerance of plants to the toxic effects of heavy metals pb, even at low concentrations can affect plant metabolic processes (fakhry and migahid 2011). the amount of pb in the air is affected by the volume or density of traffic, distance from the highway and industrial areas, engine acceleration and wind international journal of applied biology, 4(2), 2020 5 direction. while the high content of pb in plants is also influenced by sedimentation (alhasnawi et al. 2016). differences in plant response to types of heavy metals caused by the characteristics of plants to the way of accumulation, as well as the level of tolerance of plants to the toxic effects of heavy metals pb, even at low concentrations can affect plant metabolic processes (fakhry and migahid, 2011). the amount of pb in the air is affected by the volume or density of traffic, distance from the highway and industrial areas, engine acceleration and wind direction. while the high content of pb in plants is also influenced by sedimentation (al-hasnawi et al,. 2016). factors that influence the absorption of heavy metals are the high intensity of light and air temperature, as well as low humidity which triggers the number of stomata more, so that more absorption of heavy metals pb accumulates in the leaves. another factor that affects the absorption of heavy metals is the size of the opening of the stomata which has a length of 12 39 µm. having mesophyll thickening in the stomata exposed to pb (attipalli et al., 2010; andriany et al., 2018). heavy metal pollutants can enter through the stomata due to the very small size of heavy metal particulates, which is less than 2μ, while the opening size of the stomata is 10μ x 27μ, so heavy metal particulates can enter easily through the stomata. the thicker mesophyll shows the more metal concentration (kabir et al., 2010). number of stomata the most number of stomata to the concentration of heavy metal pb was at station 3 in the lower header (s3b) of 130 compared to stations 1 and 2 and control. compared to station 1, there were 77 stomata of the upper leaf (s1a) and 73 stomata of the lower leaf (s1b) of 73 stomata, this is quite a number (51 100) which is relatively the same as the control, as shown in figure 3. figure 3. the number of stomata to the concentration of heavy metals pb in green champa leaves at each station: i.e. s1 is station 1, s2 is station 2 and s3 is station 3; a is the upper leaf and b is the lower leaf; ka is the control on the upper leaf and kb is the control on the lower leaf. based on an analysis of the effect of pb on the upper green champa leaves on the number of stomata using statistical analysis, namely a simple linear regression test, the square of r2 is 0.271. the effect of pb heavy metal concentration on the amount of stomata in the leaves is 27%. this means that there is an influence of heavy metal pb concentration on the amount of stomata in the leaves, and the remaining 73% is influenced by other variables, as shown in figure 4a. based on the results of the analysis of variance shows that international journal of applied biology, 4(2), 2020 6 although the concentration of heavy metal pb on the amount of stomata in leaves is 27%, the results do not experience significant damage. based on the analysis of the effect of pb on the bottom green champa leaves on the number of stomata using statistical analysis, namely a simple linear regression test, the results obtained are the square of r2 of 0.629. the effect of heavy metal pb concentration on the amount of stomata in leaves was 62%. this means that there is an influence of heavy metal pb concentrations on the amount of stomata in the leaves. the remaining 38% is influenced by other variables as shown in figure 4b. based on the analysis of the variance which shows that although the concentration of heavy metals pb on the amount of stomata in the leaves is 27%, the results do not experience significant damage. figure 4. analysis of the effect of pb on the number of stomata in green champa leaves in the upper leaf (a) and green champa leaves in the lower leaf (b). more stomata in the lower canopy than the top canopy, because water loss occurs faster through the stomata on the top of a leaf exposed to sunlight, this is in line with previous research (attipalli et al., 2010; tanjung et al., 2019), the number of stomata under the tree canopy is higher than above the canopy in road shade plants, so the higher the number of stomata, the higher the potential to absorb heavy metals or particles in the air. when in the lack of light intensity and low temperature at each station, the amount of stomata is reduced so that absorbs co2 is reduced and the process of photosynthesis is disrupted (aalto et al. 2017; fahruddin and tanjung 2019). conclusions from research on the absorption of heavy metals pb in green champa leaves (polyaltia longifolia), shows that the heavy metals pb concentration is 1,21–2,42 µg/g which accumulates in green champa leaves in the industrial area of makassar city by being categorized as still normal. heavy metals pb affect the number of stomata in green champa leaves, but microscopically the leaf stomata do not show any significant damage (>0.05) that occurs at three stations. international journal of applied biology, 4(2), 2020 7 references aalto, j., riihimäki, h., meineri, e., hylander, k. & luoto, m. 2017.revealing topoclimatic heterogeneity using meteorological station data. international journal of climatology. 37(13):544-566. adawiyah, s. r., fahruddin & kahar, m. 2017. application of bacterial isolate from tamangapa makassar in the household organic waste processing. celebes biodiversitas.1(1): 40-44. aksu, a. 2015. sources of metal pollution in the urban atmosphere (a case study: tuzla, istanbul). journal of environmental health science & engineering. 3(79):1-10. al-hasnawi, s. s., hussain, m. h., al-ansari, n. & knutsson, s. 2016. the effect of the industrial activities on air pollution at baiji and its surrounding areas, iraq. engineering. 8(1):34-44. andriany, fahruddin & as’adi, a. 2018. effect of bioactivators types on the rate of teak tectona grandis leaf ‘seresah’ decomposition, in the campus unhas tamalanrea. bioma: jurnal biologi makassar. 3(2): 31-42. attipalli, r., girish, k.r. & agepati, s. r. 2010. the impact of global elevated co2 concentration on photosynthesis and plant productivity. current science. 99(1):4657. fahruddin, f, abdullah, a. & nafie, n. l. 2018. treatment of acid mine drainage waste using sediment as source of sulfate-reducing bacteria to reduce sulfates. pollotion research. 37(4): 903-907. fahruddin, f. & tanjung, r.e. 2019. the study of bacteria populations in phytoremediation of cadmium using eichhornia crassipes. journal of physics: conference series. 1341(022019):1-8. fahruddin, f. 2020. isolation and characteristics of cellulose degradation bacteria from makassar antang furniture industry waste. serambi engineering. 5(2): 951-956. fahruddin, nur, h., slamet, s. & sri, w. 2019. assay of bacterial isolates growth ability from water and tallo river sediments on lead metal (pb). jurnal ilmu alam dan lingkungan.10(2): 58 – 64. fakhry, m. & migahid, m. 2011.effect of cement-kiln dust pollution on the vegetation in the western mediterranean desert of egypt. international journal of environmental and ecological engineering. 5(9):480-486. hapsah, samang l. & achmad z. 2013. analysis of the level of demand and availability of green open space in the makassar industrial area. jurnal teknik lingkungan. 3:7-12. javarabad, d. m., azadfar, d. & arzanesh, h. m. 2013. the ability to filter heavy metals of lead, copper and zinc in some species of tree and shrub. international journal of advanced biological and biomedical research.1(1):53-60. international journal of applied biology, 4(2), 2020 8 kabir, a. k. l., huda, n.h., jhanker, y. m. & sharmin, k. 2010. vegetation condition around industry area in 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(3):2169-2179. tanjung, r. e., fahruddin, f. & samawi, m. f. 2019. phytoremediation relationship of lead (pb) by eichhornia crassipes on ph, bod and cod in groundwater. journal of physics: conference series. 1341(022020):1-6. tauqeer, m. h., ali, s., sardar. k., hameed, s., afzal, s., fatima, s., shakoor, b. m. & bharwana, a. s. 2013. heavy metal contamination and what are the impacts on living organisms.greener journal of environmental management and public safety. 2(4):172-179. zhao, p. s., dong, f., he, d., zhao, x. j., zhang, x. l., zhang, w. z., yao, q. & liu, h. y. 2013. characteristics of concentrations and chemical compositions for pm2.5 in the region of beijing, tianjin and hebei, china. atmospheric chemistry and physics. 13(9):4631– 4644. contact : shadrach osalumhense okhuebor osasokhuebor@yahoo.com 1 abstract this study was conducted to analysis of the antibiotic susceptibility pattern of pseudomonas aeroginosa isolated from ready-to-eat food in selected street food vendors in ikpoba-okha lga. the mean total viable plate counts (tvc) for pseudomonas aeroginosa was ascertained with the spread plate methods using nutrient agar with results indicating a mean total viable count (tvc) ranging from 2.20 x 10⁵ to 1.05 x 10³. the highest counts of 2.20 x 10⁵ was obtained in food samples collected from sfl 5 while the lowest count of 1.05 x 10³ was obtained from sfl 1. the organism had a high percentage occurrence of 90% haven been isolated from most food samples (especially soups) from all street food vending location except sfl 3.the data obtained from the gram negative antibiotic susceptibility analysis showed that pseudomonas aeroginosa was more sensitive to ciprofloxacin (10µg) and norfloxacin (10µg), sensitive to gentamycin (10µg), augmentin (30µg), amikacin (30µg) and ceftazidime (30µg) but showed resistance cotrimoxazole (30µg), ceftriazone (30µg) and tetracycline (30µg). the results of this study indicates that most of the ready to eat food samples examined had high contamination of pseudomonas aeroginosa and hence did not meet microbiological quality standards. hence, it is recommended that a more close supervision of ready to eat food from street food vending locations in ikpoba-okha should be carried out by relevant authorities. issn : 2580-2410 eissn : 2580-2119 antibiotic susceptibility pattern of pseudomonas aeroginosa isolated from ready to eat food from selected street vending food locations in ikpoba-okha local government area of edo state shadrach osalumhense okhuebor 1*, izevbuwa, osazee ekundayo 2 1department of microbiology, faculty of life sciences, university of benin, p.m.b. 1154, benin city, edo state, nigeria 2department of microbiology, faculty of natural and applied sciences, igbinedion university okada, p.m.b. 0006, edo state, nigeria introduction ready to eat foods which are sold by different street food vendors and provide a wide range of essential energy needs and nutrients are eaten daily in nigeria and the world over. the food are known to be very affordable for the lower and middle income groups and are appreciated for their unique flavours and convenience (muzaffar et al., 2009). in contrast to these potential benefits, it is also recognized that ready to eat food are often highly contaminated by microbes as a result of food vendors who are often poor, uneducated, and open access international journal of applied biology keyword antibiotic pseudomonas article history received 12 september 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 2 lack knowledge in safe food handling, environment, sanitation and hygiene, mode of food display, food service and hand washing, sources of raw materials, and use of potable water. consequently, ready to eat food are perceived to be a major public health risk (bhowmik, 2010). contamination of ready to eat food is as a result of so many factors such as preparation methods, poor packaging, indiscriminate waste disposal, poor sanitation, poor hygiene of food handlers, exposure of food to open air, contaminated kitchen equipment and utensils, contaminated water used in washing kitchen equipment and utensils and used for preparing food, contaminated food vending surfaces and too many people clustering around the food vending area (omemu and aderoju, 2008). pseudomonas aeroginosa is a known microbial food contaminant which can cause foodborne diseases. it is of high public health significance haven be reported to be the second most common cause of pneumonia, the fourth most cause of urinary tract infection, and the sixth most common blood stream isolate in intensive care units (icus). many potential reservoirs of the organisms have been identified including vegetables (mahmoud et al., 2013). pseudomonas is a gram-negative rod, motile, aerobic gamma-proteobacteria, belonging to the family pseudomonadaceae and containing 191 validly described species and are commonly found in soil and water and also regularly associated with the surfaces of plants and occasionally with surfaces of animals (euzéby, 1997; kenneth, 2008). pseudomonas is an opportunistic pathogen (igbinosa et al., 2011), which can cause urinary tract infections, sepsis (blood stream infection), pneumonia, pharyngitis, pulmonary disease and many other medical problems (moore et al., 2008). there has also been an observed increase in the patronage of ready to eat food vendors within benin-city, edo state, nigeria (wogu et al., 2011) and especially in ikpobaokha local government area (lga) of edo state (okareh et al., 2015) . however, their poorly regulated operations raise serious questions about food safety and hygiene standard, as well as monitoring by relevant authority (barro et al., 2006; abdalla et al., 2008). ikpobaokha is a fast growing urban centre, expanding rapidly in size and population and is characterised by people on the move; this creates a suitable environment for ready to eat food trade which unfortunately operates under unsanitary conditions. this study is aimed at analysing the public health risk of ready to eat food by carrying out an analysis of the antibiotic susceptibility pattern of pseudomonas aeroginosa isolated from ready-to-eat food in selected street food vendors in ikpoba-okha lga. materials and methods study area this study was carried out in ikpoba-okha local government area of edo state, located in the south-south geopolitical zone of nigeria. ikpoba-okha is a densely populated with a total population of 372,080 according to the 2006 population census conducted by the national population commission of nigeria, and with an increase to 487,400 estimated projection for 2016. ikpoba-okha has the second lga with the highest population (npc 2006). the inhabitants of the area are mainly small to medium scale business owners, farmers, artisans, civil servants, bankers, and students. the people are a combination of christians, muslims, and traditionalists. major languages spoken are english, pidgin english and edo. ten (10) street food vending location mostly patronised by consumers in lga were selected for this study. international journal of applied biology, 5(2), 2021 3 sample collection three (3) different food samples were collected from each street food vending location, particularly rice, soup, and beans. making a total of thirty (30) food samples. the food samples were collected with the dishing spoons used by the food vendors, packaged into sterile containers and were immediately transported to the laboratory for microbiological analysis. viable colony count and isolation of microbes 10g of each food sample was weighed and ground in a sterile mortar. 90ml of distilled water was poured into the mortar, the mixture was homogenized and used as stock followed by serial dilutions. serial dilutions of up to 10⁻⁶ were made. 0.1ml of serial dilutions 10⁻³, 10⁻⁴ and 10⁻⁵ were cultured on nutrient agar using the spread plate technique with a sterile glass rod. the petri dishes for the bacterial plate count were incubated at 37℃ for 24h. the number of colonies seen were counted using a colony counter and recorded as colony forming unit per gram (cfu/g). characterization and identification of microbes macroscopic examination and biochemical analysis of different isolates obtained from the different plates were accessed to identify the organism to the species level, using bergey’s manual of determinative bacteriology antibiotic sensitivity pattern of pseudomonas aeroginosa the antibiotic susceptibility test was performed to determine the levels of sensitivity and resistivity of some gram negative drugs on pseudomonas aeroginosa using the mac farland standard results the result of thirty (30) food samples collected from ten (10) street food vending location mostly patronised by consumers in ikpoba-okha lga is shown in the tables below. table 1 shows the different food samples collected and analysed. some of the food samples collected and analysed were beans (without oil) (26.7%), jollof rice (20%), egusi soup (13.3%) table 1. food analysed food samples frequency percentage (%) white rice and stew 3 10 jollof rice 6 20 fried rice 1 3.3 beans (with oil) 2 6.7 beans (without oil) 8 26.7 egusi soup 4 13.3 vegetable soup 3 10 okro soup 3 10 total 30 100 international journal of applied biology, 5(2), 2021 4 there was growth of pseudomonas aeroginosa in many of the food samples analysed. the total viable count of pseudomonas aeroginosa for sfl 5 had the highest count of 2.20 x 10⁵ while the lowest count of 1.05 x 10³ was found in sfl 1. table 2. values for total viable count for pseudomonas aeroginosa sample location mean tvc for bacteria (cfu/g) sfl 1 1.05± 0.14 x 10³ sfl 2 1.90 ± 0.27 x 10⁴ sfl 3 0.00 ± 0.00 sfl 4 0.38 ± 0.01 x 10³ sfl 5 2.20 ± 0.07 x 10⁵ sfl 6 0.33 ± 0.12 x 10⁵ sfl 7 1.57 ± 0.11 x 10⁴ sfl 8 1.50 ± 0.20 x 10⁴ sfl 9 1.99 ± 0.33 x 10³ sfl 10 1.70 ± 0.23 x 10⁴ tvc: total viable counts; mean ± se (standard error); cfu: colony forming units; it was observed that pseudomonas aeroginosa which had occurrence of 90% was isolated in food samples from all street food vending locations except sfl 3. table 3. distribution of bacteria isolates in the different sample location bacteria found sample location sfl 1 sfl 2 sfl 3 sfl 4 sfl 5 sfl 6 sfl 7 sfl 8 sfl 9 sfl 10 pseudomonas aeroginosa + + + + + + + + + table 4 shows the susceptibility of some selected strains of pseudomonas aeroginosa isolated in food samples from the street food locations to different antimicrobial agents (gram negative antibiotics). the result showed that the isolate was sensitive to ciprofloxacin (10µg), gentamycin (10µg), augmentin (30µg), amikacin (30µg), ceftazidime (30µg) and norfloxacin (10µg) whereas, it was resistant to cotrimoxazole (30µg), ceftriazone (30µg), tetracycline (30µg) and erythromycin (10µg). it was observed that the isolate was more susceptible to ciprofloxacin (10µg) and norfloxacin (10µg). table 4. susceptibility of pseudomonas aeroginosa from food samples to antimicrobial agents antibiotics (concentration) susceptibility cotrimoxazole (30µg) r ceftriazone (30µg) r tetracycline (30µg) r ciprofloxacin (10µg) ms erythromycin (10µg) r gentamycin (10µg) s augmentin (30µg) s amikacin (30µg) s ceftazidime (30µg) s norfloxacin (10 µg) ms s= susceptibile, r= resistant, ms= more susceptible international journal of applied biology, 5(2), 2021 5 discussion the results shows that there was pseudomonas aeroginosa growth in many of the food samples analysed (table 1) especially the soup samples. the growth of this organism which may be as a result contaminated vegetables (kemajou et al.,2017) or meat used in cooking the foods (nkanga and uraih, 1981) and also as a result of poor hygiene practises of the food handlers, inadequate heating, secondary contamination via contact with contaminated equipment, utensils or surfaces and inappropriate processing (gopal et al., 2015). it may also be due to microbial contamination of water used to wash equipment and utensils, large number of people crowding serving space or food sale point and long periods between preparation time and consumption time. food samples collected from sfl 5 in this study had the highest mean total viable count of 2.20 x 10⁵ for pseudomonas aeroginosa compared to the count from the other street food vending locations (table 2). the isolate had high occurrence of 90% from all food samples collected from all the street food vending locations (table 3). this is similar to the report kemajou et al., (2017), who reported counts of pseudomonas aeroginosa isolated with other bacteria isolates from vegetables. pseudomonas aeroginosa can be found in nearly everywhere as long as there is enough water. common habitats are moist soils and lakes as well as toilets, sinks, swimming pools, soap dishes and dishwashers (zottola et al., 1994). pseudomonas biofilm formation is been reported to be problematic and a serious public health risk (meliani and bensoltane, 2015; burmolle et al., 2010). pseudomonas biofilm can grow on abiotic surfaces of different equipment and processing surfaces in food industry food. it is also reported that when organisms like pseudomonas form biofilm, they became more resistant to the chemicals and antibiotics. such a biofilm is a potential source of contamination of foods that may lead to spoilage, foodborne diseases and transmission of foodborne pathogens (gunduz and tuncel, 2006; joseph et al., 2001). table 4 showed that pseudomonas aeroginosa was sensitive to ciprofloxacin (10µg), gentamycin (10µg), augmentin (30µg), amikacin (30µg), ceftazidime (30µg) and norfloxacin (10µg). this is in accordance with the previous report of sensitivity of pseudomonas aeroginosa to several antimicrobial agent (kemajou et al., 2017). sensitivity of the isolate to gentamycin, ceftazidime and amikacin were previously been reported by igbinosa et al. (2011) and odjadjare et al., (2012). it was also observed that the isolate was more susceptible to ciprofloxacin (10µg) and norfloxacin (10µg). this agrees with the reports of tamil and murugan (2011) and bekele et al., (2015) who reported that pseudomonas aeroginosa was more susceptible to the antimicrobial agents it also showed that the isolate was resistant to cotrimoxazole (30µg), ceftriazone (30µg), tetracycline (30µg) and erythromycin (10µg). this agrees with the reports of kemajou et al., (2017) and tamil and murugan (2011) who resistance to same drugs. the observations suggests that the food samples from the street food vending locations were a considerable source of multidrug resistant strains of pseudomonas aeroginosa. conclusions the presence of pseudomonas aeroginosa in food sold in street food vending locations which are highly patronised by consumers in ikpoba-okha local government area (lga) of edo state, is a cause for concern as it could lead to serious public health challenges of the consumers. foodborne diseases induced by pseudomonas aeroginosa can become international journal of applied biology, 5(2), 2021 6 problematic especially in the case of multiple drug resistance. it is therefore recommended that edo state government should enforce strict regulations and supervision on good hygiene practices such as the use of good manufacturing practices (gmp) and hazard analysis critical control point (haccp) application in food production and processing, which will mitigate pseudomonas aeroginosa contamination of food and foodborne diseases references abdalla, m.a. suleiman, s.e., alien, y.y. and bakheit, o. 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(2012). prevalence of multiple antibiotics resistant (mar) pseudomonas species in the final effluents of three municipal wastewater treatment facilities in south africa. international journal of environmental research and public health, 9(6):20922107 https://doi.org/10.3390/ijerph9062092 okareh, o.t. and erhahon o.o. (2015). microbiological assessment of food and hand -swabs samples of school food vendors in benin city, nigeria. food and public health 5(1), 23-28 https://doi:10.5923/j.fph.20150501.04 omemu, a.m. and aderoju, s.t. (2008). food safety knowledge and practices of street food vendors in the city of abeokuta, nigeria. food control 19, 396–402 https://doi.org/10.1016/j.foodcont.2007.04.021 https://doi.org/10.1016/s0168-1605(00)00466-9 https://doi.org/10.21767/2573-0320.10004 https://doi.org/10.5171/2013.290047 https://doi.org/10.4172/2375-446x.1000126 https://dx.doi.org/10.1136/jech-2013-202869 https://doi.org/10.5539/ijbm.v4n2p80 https://doi.org/10.4315/0362-028x-44.1.4 https://doi.org/10.4315/0362-028x-44.1.4 https://doi.org/10.3390/ijerph9062092 https://doi:10.5923/j.fph.20150501.04 https://doi.org/10.1016/j.foodcont.2007.04.021 international journal of applied biology, 5(2), 2021 8 tamil, s.s. and murugan, s. (2011). antimicrobial susceptibility patterns of pseudomonas aeruginosa from diabetes patients with foot ulcers. international journal of microbiology, http://doi.org/10.1155/2011/605195 wogu, m.d., omoruyi, m.i., odeh, h.o. and guobadia, j.n. (2011). microbial load in readyto-eat rice sold in benin city. journal of microbiology and antimicrobials 3(2), 29-33 https://www.researchgate.net/publication/280291409 zottola, e.a. (1994). scientific status summary “microbial attachment and biofilm formation: a new problem for the food industry?” food technology, 48, 107-114 http://doi.org/10.1155/2011/605195 https://www.researchgate.net/publication/280291409 contact : sipora petronela telnoni siporatelnoni@yahoo.co.id 57 abstract this study was conducted to evaluate fertility of sk kedu chicken post inseminated by frozen semen using lactated ringer’s-egg yolk with 10% of dmso. frozen semen of lactated ringer’s-egg yolk with 10% of dmso was picked from container of liquid nitrogen (-196 ºc) and thawed in warm water at 37 ºc for 30 second. frozen semen inseminated to 3 arab hens: a1, a2 and a3 with 100 motile sperm cell in 0.25 ml-1 using 2 straws and deposited into reproductive tract of hen as long as 7 cm in intra utein using ai gun. egg was collected in day 2 after ai till day 14, labelled and incubated. sperm fertility was estimated by using % sperm fertility formulation = fertile eggs / incubated eggs x 100, regardless of whether the eggs will hatch or not. egg fertility are checked on 5 day after incubation. this result showed % sperm fertility of sk kedu chicken were a1 (77.78 %), a2 (33.33 %), and a3 (83.33 %). the mean of % sperm fertility was 72.22 %. it can be concluded that lactated ringer’s-egg yolk with 10% of dmso can maintain sperm fertility of sk kedu chicken. issn : 2580-2410 eissn : 2580-2119 sperm fertility of sk kedu chicken in lactated ringer’s-egg yolk extender with 10% of dmso sipora petronela telnoni 1*, r. iis arifiantini 2, sri darwati 3 1 faculty of mathematics and science, san pedro university, kupang, east nusa tenggara. 2 faculty of veterinary medicine, ipb university, bogor, west. 3 faculty of animal science, ipb university, bogor, west java. introduction sperm fertility for chicken is a manifestation of the fertilization process of spermatozoa in the female reproductive tract. best manifestation of fertilization gives contribution for chicken breeding value and to maintain the population. further more, artificial insemination (ai) as a tool of biotechnology which use for breeding in economic purpose and increase population of chicken widely used require ability of sperm fertility. the prediction of sperm fertilizing ability has great economic importance for breeding herds when artificial insemination is used (gadea, 2005). sk kedu chicken from indonesia biodiversity has the good ability for maintaning economic purpose by using ai. sk kedu is a the one of superior cross-breed chickens. sk kedu chicken is a chicken came from the cross breed of 3 indonesia local chickens i.e sentul chicken, kampung chicken, and kedu chicken. 3 local chicken in sk kedu have the characteristics: sentul chicken has advantages, that is as a producer of meat and eggs (dual use type), male sentul chicken body weight 1.3 kg to 3.5 kg and female chickens 0.8 kg to 2.2 kg, egg production 118 eggs per year (diwyanto et al., 2011), kampung chicken can weigh 1 815 g (male) and 1 382 g (female) (mulyono & open access international journal of applied biology keyword lactated ringer’s-egg yolk, dmso, sperm fertility, artificial insemination, chicken. article history received 26 october 2020 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 58 pangestu, 1996). the production of native chicken eggs is 80 eggs per parent per year (sinurat et al., 1992) and kedu chicken can weigh 2.0 kg to 4.0 kg. kedu chicken has the advantage of high egg production is 124 eggs per year (krista & harianto, 2013). sk kedu as cross-breed indonesia native chicken have the potential in breeding. this potential can be developed as breeding using ai. implementation of ai using frozen semen in cryopreservation is an important because it is able to store genetic material of sk kedu chickens which have advantages and a limited number of breeds. the frozen semen is a result of cryopreservation which using to maintance genetic of some rare spesies preserve cell, included by reproductive cell of poultry. poultry semen cryopreservation is considered to be a challenging method to preserve reproductive cells (blesbois, 2011; kowalczyk & łukaszewicz, 2015). cryopreservation of chicken semen requires a number of supporting factors, including the type of diluent, cryoprotectant and concentration of cryoprotectant. these supporting factors will affect the quality of frozen semen. the criteria for the quality of frozen semen that were assessed included of % sperm motility after thawing and % sperm fertility after ai. the ability of sperm fertility is also determined by buffer system, nature of cryoprotectants and additives such as sugars, calcium chelators, antioxidants and milk or egg yolk proteins (holt, 2000). sperm fertility from frozen semen in cryopreservation methods has difference value of sperm motility that indicated % ability of sperm to fertilise ovum. the assessment sperm motility is influenced by the structure and integrity of the sperm function and allows prediction of the success of sperm fertility (froman, 2007; blesbois et al., 2008). the positive result of fertility in chickens depends on several factors including the ability of the spermatozoa to undergo acrosome reactions at fertilization. the acrosome reaction is an exocytotic process that involves membrane fusion and is required to achieve fertilization (lemoine et al., 2011; nguyen et al., 2014). lactated ringer’s and egg yolk have the ability to maintance frozen sperm quality included sperm fertility. the use of egg yolks has shown a good benefit in cryopreservati on of semen, that is as a protector of the plasma membrane and the acrosome of spermatozoa when cold shock occurs (amira et al., 2004). lactated ringer’s contains a water source and electrolytes, produces a metabolic alkalinizing effect, and contains a number of chemical compounds, water, ph value, osmolarity, and energy source. jones (1997) explains that the lactate is one of the energy sources that can be used to replace glucose and fructose for spermatozoa, which fructose is the major fuel for the mitochondrial production of atp. dmso is a very common cpa that is used at various concentrations (lovelock & bishop 1959; mcgann & walterson, 1987) and is exceptionally effective for the cryopreservation of a variety of biological tissues and used universally for stabilizing cell membranes under rapidly changing conditions, preventing intracellular ice crystal formation during freezing and heat release during the period of phase transition (hubalek, 2003; cottler, 2009). ai in chicken is a semen distribution method (plasma semen and sperm) to female reproductive tract. ai in chicken first reported in 1936 (getachew, 2016). ai in chicken is based on 2 steps: 1). semen is collected from rooster and 2). semen inseminated to hen, with the main purpose is collecting the fertile eggs (bakst & dymond, 2013). collecting semen in rooster for ai uses abdominal massage method (bakst & long, 2010). massage do under the back of abdomen to cloaca for phallus exudation, further massage around cloaca’s area for semen excretion. implementation of the ai process, batch of selected international journal of applied biology, 5(1), 2021 59 sperm will be transferred to the main site of sperm at sperm storage tubules (sst). sperm will exit from sst and transported to infidibulum as fertilization site dan it is function as the second site sperm storage. fertilized ovum will be transferred to magnum as albumin secretion site, and continues in to isthmus as the site of shell membrane formation and shell formation of egg till ovoposed. chicken uses 24-26 hours for formation of follicle (first follicle) and will be ovulated (bakst and dymond 2013).. materials and methods sample this study was conducted after approval by animal care and use committee of ipb university, number 028/acuc/10/2016. 5 sk kedu roosters at 48 weeks and 3 arab hens at 30 weeks were used in this study. all roosters and hens fed with 100 g commercial diet (17% crude protein) individual 1 day -1 and water was provided ad libitum. roosters and hens were housed in individual battery cages. artificial insemination (ai) was done at 45 pm using frozen semen in ai procedur with ≥ 40 % motility post thawed and 100 cell concentration of motil sperms in 0.25 ml -1 and positioned in reproduction tract of arab hen using ai gun. ai gun positioned along 7 cm in intra uterin. composisition of frozen semen extender showed in table 1 and table 2. egg was collected in day 2 after ai till day 14, labelled and incubated. sperm fertility was estimated by using % sperm fertility formulation = fertile eggs / incubated eggs x 100 (brillard 2003). results and discussion sperm fertility showed the ability of sperms to ovum fertilization. based on this study, ovum of arab hens was showed it is fertilized by sperm in frozen semen sperm of sk kedu chicken in lactated ringer’s-egg yolk with 10% of dmso post ai. this result indicated that quality of sperm in frozen semen post thawed has the ability to fertilization the ovum (table 3). % sperm fertility between 3 arab hens was showed different, a1 (1 arab hen), a2 (2 arab hen), and a3 (3 arab hen). a1 and a3 resulted % sperm fertility was higher than a2 % sperm fertility. different result in % sperm fertility of ai caused by the best time of ai when positioned in reproduction tract of arab hen using ai gun. holt (2000) explained that the correct insemination time is very important when using frozen semen because the duration of the survival of the frozen spermatozoa is not the same as in fresh semen. % sperm fertility of a2 (2 arab hen) can be caused by inhibition of spermatozoa cells during their abilitity to reach the ovum, example: failure of spermatozoa to reach and enter the sperm storage tubule (sst), ability to reach the fertility site in the infundibulum, to penetrate the perivitelin layer of the ovum and failure of the spermatozoa to form the pronucleus that causes syngamy does not occur. furthermore, (shubash et al., 2005) explained that probable causes of fertility decline perhaps by the reduced capacity of sst to store spermatozoa and the induction of anti-sperm antibodies. mean of % sperm fertility of sk kedu based this study was 72.22%. sperm fertility on this result was not different from the % sperm fertility resulting from frozen semen sandhill crane from america using 10% concentration of dmso with different extenders which is 73.9% (blanco et al., 2012) and another result showed cryopreservation with 8% dmso in indian red jungle fowl was 73% sperm fertility (rakha et al., 2018). the dmso can maximize the replacement of water molecules in the cytoplasma, therefore formation of ice crystal is prevent and the cell membrane structure have a effective protection (hu et al. 2015). international journal of applied biology, 5(1), 2021 60 furthemore, yamashiro et al. 2010 explained that addition of exogenous substrate such as lactate improved sperm fertility in different spesies and may find lactate as suitable substrate for maintaining the energy production. energy in sperm by atp requires to maintain sperm fertility. atp is hydrolyzed by the dynein adenosine triphosphatase, which converts the chemical energy of atp into mechanical energy used for the movement of sperm (storey, 2008). egg yolk have the ability to maintain sperm fertility. egg yolk have the beneficial molecules such as phospholipids and ldl which have a cryoprotective action to protect sperm against cold shock and the lipid-phase transition effect during the freeze– thaw process (moussa et al., 2002). but another, a result by used the lower concentration of dmso was 4.5 % in white leghorn showed 93% of sperm fertility in beltsville poultry semen extender (voorst & leenstra ,1995). lower of % sperm fertility based on result could be due to the use of a higher dmso concentration and the influence of the lactated ringer’s-egg yolk extender composition. higher concentration of dmso perhaps caused toxicity for sperm. donoghue and wishart, 2000) explained that several factors should be taken into consideration of cryoprotective agens toxicity to chicken sperm is cryoprotectant concentration, equilibration temperature, equilibration time, freezing rate, freezing method and post-thaw treatment. further more, chicken sperm also have the difference morphology to mammalian sperm cells and affect in cryopreservation result. the filiform shape of the poultry sperm head is not much wider in diameter than the tail (thurston & hess, 1987). therefore, sperm heads have less cytoplasmic volume, and this means they have less ability to move in the cryoprotectants (cpa). less cytoplasmic volume and therefore have the less ability to move cryoprotectants inside the sperm head perhaps be one of the reasons avian spermatozoa do not survive the freezing process well (donoghue & wishart, 2000). sperm of poultry were not estimated to have the capacitation or motility hyperactivity during fertilization, although sperm of poultry have a long time in the oviduct before the mechanism oocyte penetration, which is more than 3 weeks in hens (lemoine et al., 2008). during the fertilization time, the ovum will be ovulated consecutively at intervals of 24 hours or more in some species and can be fertilized 15 minutes after ovulation (wishart & staines, 1999). table 1: composisition of lactated ringer's-egg yolk (lr-ey) extender constituent ml lactated ringer's* 80.0 egg yolk 20 ph 6.8 total 100.0 *lactated ringer's: commercial solution (pt emjebe pharma) table 2: extender composition of lactated ringer’s-egg yolk with 10% of dmso constituent ml lr-ey (%) 90 dmso (%) 10 penicillin (iu ml -1) 1000 streptomycin (mg ml -1) 1 total 100 international journal of applied biology, 5(1), 2021 61 table 3. sperm fertility using lactated ringer’s-egg yolk with 10% of dmso arab hens total of egg fertile egg sperm fertility (%) a1 9 7 77.78 a2 3 1 33.33 a3 6 5 83.33 mean 18 13 72.22 conclusions % sperm fertility of a1 and a3 was higher than a2 which can be affected by ai time, extender, and concentration of cryoprotectan. the mean of % sperm fertility of sk kedu chicken after inseminated by frozen semen using lactated ringer’s-egg yolk with 10% of dmso was showed 72.22% and can maintain sperm fertility of sk kedu chicken. references amira, l., tainturier, d., jeanneau, l., thorin. c., gerard ,o., courtens, j.l., & anton, m. 2004. bull semen in vitro fertility after cryopreservation using egg yolk ldl: a comparison with optidyl, a commercial egg yolk extender. theriogenology. 61(5):895-907. baskt, m. r., & long, j. a. 2010. techniques for semen evaluation, semen 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r2 = 0.964), 2.30 mg/l (1.844 to 2.518; r2 = 0.866), 2.27 mg/l (2.117 to 2.373; r2 = 0.959) and 1.88 mg/l (0.7704 to 2.211; r2 = 0.74), respectively. after a feeding trial experiment on post-exposed fingerlings, there was no significant difference (p > 0.05) between the feed intake of the control and ddvp exposed groups. except for protein intake, significant differences (p < 0.05) were found in the growth and nutrient utilization parameters. the findings of this study reveal that c. gariepinus fingerlings could recover after acute exposure to dichlorvos at different concentrations. issn : 2580-2410 eissn : 2580-2119 growth response and nutrient utilization of clarias gariepinus fingerlings exposed to dichlorvos ramota titilayo tahir1, prince emeka ndimele 1, abdulwakil olawale saba 2*, musa adamu ibrahim3 1 department of fisheries, lagos state university ojo, lagos state, nigeria. 2 school of agriculture, lagos state university, epe campus, epe, lagos state, nigeria. 3department of biological sciences, university of maiduguri, maiduguri, borno state, nigeria. introduction the globally, the use of chemicals for the control of pests has continued to increase (sharma et al., 2019; al-kawaz, 2019). these chemicals are also utilized in agriculture due to losses of around 30% initiated by insect attacks and other pest organisms (das, 2013; kumar et al., 2016). 2, 2-dichlorovinyl dimethyl phosphate (ddvp) is a commonly used organophosphate pesticide (op) in controlling insect pests in residential and agricultural areas; however, its toxicity has gone beyond the target organism, and this chemical is frequently detected in water bodies (ezike, 2017). compared to other chemicals, organophosphate pesticides (ops) have gained universal popularity especially because they degrade rapidly (das, 2013). regrettably, ops open access international journal of applied biology keyword recovery, postexposure, organophosphate pesticide, acute toxicity article history received 20 january 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 65 can negatively affect both terrestrial and aquatic animals due to their non-target specificity (das, 2013; sturve et al., 2016). dichlorvos, or 2, 2-dichiorovinyl dimethyl phosphate (ddvp) is one of the most commonly applied ops especially in developing countries (das, 2013). it has a wide application including its use in agriculture for crop protection and the protection of animals from parasitic attack. it is also used in aquaculture to eradicate ectoparasites (das, 2013). in nigeria, ddvp is also used for the protection of crops and animals (omoniyi et al., 2013). the aquatic environment receives toxic contaminants that get to the water bodies through agricultural, domestic, and industrial activities (ololade and oginni, 2010, ö fırat et al., 2011). when this chemical gets into the aquatic environment, it may be highly toxic to the aquatic organisms, is capable of inhibiting the health of fish by impairing metabolism, and could lead to death. fishes are practically the most vital aquatic organisms that are frequently exposed and, therefore, are susceptible to the effects of these toxic pesticides (rao et al., 2017). the acute toxicity of dichlorvos to freshwater fish has been studied in many freshwater fish species including mossambique tilapia (tilapia mossambica) (rath and misra, 1981), african air-breathing catfish (heterobranchus longifilis) (oribhabor and ikeogu, 2016), philippine catfish (clarias batrachus) (narra, 2016), and african mud catfish (clarias gariepinus) (omoniyi et al., 2013, ashade et al., 2011; egesi, 2017). the african mud catfish (c. gariepinus) is a freshwater fish species that is of high commercial importance in africa (dadebo et al., 2014). due to their hardy nature, they are known to survive in harsh environmental conditions (ibrahim et al., 2016; basharat et al., 2020). previous studies have sought to estimate the potential hazards of these chemicals as a form of risk assessment through toxicity testing (omoniyi et al., 2013). some of these studies have assessed the toxicity of ddvp on various life stages of c. gariepinus including omoniyi et al. (2013) who studied the lethal toxicity of ddvp on c. gariepinus fingerlings and juveniles. ojesanmi et al. (ojesanmi et al., 2017) studied the mortality rate of c. gariepinus fingerlings exposed to this chemical, while ndimele et al. (2013) studied the effect of dichlorvos-induced stress on the growth of the fingerlings. apart from ndimele et al. (2013), other available studies had only considered the toxicity of the chemical on c. gariepinus and not the recovery of the survived fish postexposure. this study, therefore, adds to the knowledge regarding the post-exposure influence of ddvp on the growth of c. gariepinus fingerlings. in addition to the growth parameters reported by ndimele et al. (2013), this study presents information on the nutrient utilization of the exposed fishes. the objectives of this study are, therefore, to assess the acute toxicity of ddvp on fingerlings of c. gariepinus and the effects of ddvp on the growth and nutrient utilization of surviving individuals, post-exposure. materials and methods brood stocks of c. gariepinus were sourced from lagos state university hatchery. thereafter, artificial reproduction was carried out following de graaf et al. (1995). after the first eight weeks, 1000 fingerlings were transferred to the laboratory of the department of fisheries, faculty of science, lagos state university, ojo, lagos, nigeria. the experimental fish were fed 2.0 mm size commercial pelleted feed (42% crude protein) at 3% body weight (viveen et al., 1985), and this was administered twice daily: morning (09.00 h) and evening (16.00 h). removal of waste food and water exchange was also done daily. international journal of applied biology, 5(1), 2021 66 dicholorvos, 2, 2-dichlorovinyl dimethyl phosphate (ddvp) was obtained from the stocks available at lagos state agricultural inputs supply authority, ojo, lagos, nigeria. concentrations were prepared from stock (10 mg/l). a 96-hour range-finding test was conducted before the definitive test on the selected fingerlings of c. gariepinus (odiete, 1999). for the toxicity test, the experimental set-up consisted of 21 transparent rectangular (61.5 cm × 33.2 cm × 20 cm) plastic tanks with various concentrations of pesticides: 0, 2.4, 2.6, 2.8, 3.0, 3.2, and 3.4 mg/l (control, t1, t2, t3, t4, t5, and t6, respectively) with three replicates each. the control, t7 had no toxicant. each aquarium (containing 10 litres of dechlorinated water) was randomly stocked with 10 c. gariepinus fingerlings (average body weight 3.3g). before the recovery phase, a new set of fingerlings with an average weight of 3.8 g were exposed for 24 hours, which is the time with the least mortality from the previous toxicity experiment. feeding was done twice a day at 3% body weight between 09:00-16:00 h for 56 days. the fishes were fed through an even distribution of the feed on the water surface of each holding tank. this was to give an even feeding opportunity to all the individual fishes. feeding in all tanks was generally completed in about 10-15 min. the mean size of the fish {weight (g) and total length (cm)} for each treatment and its replicates were measured every 2 weeks (ndimele et al., 2012). feed intake (fi): quantity of feed fed per day. this was enumerated as follows; = 3% body weight of fish/day the growth and nutrient utilization parameters were calculated as follows: growth parameters: i. weight gain (wg) = w2 — w1 (g) ii. percentage weight gain % (pwg) = 100 (w2 — w1) iii. specific growth rate (sgr) % per day = (loge w2– loge w1 ) × 100/t iv. average daily gain (adg) = (w2 w1)/ t where: w2= final mean weight (g), w1 = initial mean weight (g), t2 = final time (days) t1 = initial time (days), loge = natural logarithm, t = period of experiment (days) nutrient utilization: i. protein intake (pi): feed intake (g) × % protein in the diet ii. feed conversion ratio (fcr) = weight of dry feed fed (g)/ live weight gained (g) iii. protein efficiency ratio (per) = gain in weight of test fish (g)/ protein consumed (g) statistical analysis the concentrations and mortality were transformed to log10 and percentage, respectively based on the probit model (sprague, 1969). the median lethal concentration (lc50) and median lethal time (lt50), were determined (finney, 1971) using graphpad prism ver. 7.0. analysis of variance (anova) was used to test for significant differences in growth performance and feed utilization. this was after exploring the data to confirm that they satisfied the conditions for a parametric test. the anova was carried out with international journal of applied biology, 5(1), 2021 67 statistical package for social science (spss) for windows (v 15.0). a pairwise comparison was carried out using fisher’s least significant difference (lsd) at p < 0.05. results and discussion this study assessed the toxicity stress of 2, 2-dichlorovinyl dimethyl phosphate (ddvp) on the growth performance and nutrient utilization of c. gariepinus fingerlings. early-life exposure to environmental stressors (either chemical or physical factors) might be reversible or irreversible depending on the physical and chemical properties of the xenobiotic, the duration of exposure, age, and health condition of an organism (rodriguez dominguez et al., 2018). the mortality recorded after every 24 hours was substantially concentrationdependent. however, no mortality was recorded in the control group (figure 1). the median lethal concentration of ddvp on c. gariepinus fingerlings at 24, 48,72, and 96 hours postexposure (hpe) were 3.30 mg/l (3.197 to 3.441; r2 = 0.964), 2.30 mg/l (1.844 to 2.518; r2 = 0.866), 2.27 mg/l (2.117 to 2.373; r2 = 0.959) and 1.88 mg/l (0.7704 to 2.211; r2 = 0.74), respectively. figure 1. time-mortality relationship of dichlorvos to fingerlings of c. gariepinus figure 2 presents the concentration-mortality at different times of exposure. figure 3 shows the median lethal-time (lt50) of the cumulative mortality at 96 hours post-exposure of c. gariepinus fingerlings. the lt50 was 27.23 hours (6.185 to 41.21; r2 = 0.946). fish under the controlled group expectedly did not die, and they survived better than those exposed to ddvp. this is in agreement with the findings of earlier researchers who also reported that the effects of the pesticides could be regarded as the possible cause of death of the test fish (ugwu et al., 2006; khan et al., 2018). ezike (2017) reported that the 96 hour lc50 of ddvp in freshwater and brackish water fish is 0.2 12 mg/l with > 4 mg/l in marine fish, this is similar to the result in this study. this is contrary to the 96-hour lc50 of ddvp c. gariepinus juveniles reported as 0.93 mg/l by ezike (2017). the mortality recorded in this study is in agreement with the 24, 48, 72, and 96 hours mortality observed in tilapia mossambica exposed to ddvt with the lc50 values ranging between 1.42 – 2.53 mg/l. the highest concentration of the pesticide led to the highest mortality and this suggests a international journal of applied biology, 5(1), 2021 68 concentration influenced lethality and dose-dependent survival (khan et al., 2018, ezemonye and tongo, 2009). higher mortality values recorded may be mainly due to the metabolites of dichlorvos that were formed in animals. dichlorvos is a potent enzyme inhibitor capable of killing organisms directly by inhibiting acetyl cholinesterase (kumar et al., 2016). there was no significant difference (p > 0.05) in the feed intake of the control and other dichlorvos concentration groups. except for protein intake, significant differences (p < 0.05) were found in the growth and nutrient utilization parameters. mean + sd values for growth and nutrient utilization indices can be found in table 1. the highest value for weight gain was 2.45 ± 2.30 g. the percentage weight gain (57.84 ± 43.39 %), specific growth rate (0.96 ± 0.84) and average daily growth (0.18 ± 0.17) were recorded in treatment with concentration of 3.2 mg/l while their lowest values (0.07 ± 0.33), (2.32 ± 1.02), sgr (0.03 ± 0.47) and adg (0.00 ± 0.03) were respectively recorded in group exposed to a concentration of 2.8 mg/l. except for the 2.6 mg/l group, the weight gain of the control was significantly different (p < 0.05) from the other groups. the percentage weight gain of the control was only significantly similar to those of 3.0 mg/l and 3.4 mg/l. the specific growth rate of the control was only significantly different from those of 2.4 mg/l and 2.8 mg/l groups, while the average daily gain of the control was only significantly different from the 2.48 mg/l, 2.8 mg/l, and 3.4 mg/l groups. the feed conversion ratio, of control, was only significantly different from that of the 2.8 mg/l group, while the protein efficiency ratio was significantly different from those of 2.4 mg/l, 2.8 mg/l, and 3.2 mg/l. apart from the control group, slow response to feeding by fish exposed to different concentrations of dichlorvos was observed in the first 14 days of the recovery phase, which improved in the second fortnight. this is an indication that feed intake was likely affected by the presence of the toxicant. the feed intake (fi) increased irrespective of dichlorvos concentration (2.8 3.4 mg/l) between days 28 and 56. better or faster response to feeding in the 28-56 days also implied that the effects of dichlorvos fractions in water were reducing and the fish response to feeding has improved. this was also reported by ugwu et al. (2006). table 1. growth and nutrient utilization of clarias gariepinus fingerlings exposed to dichlorvos at 24hp treatm ent (mg/l) wg (g) pwg (%) sgr (%/day) adg (g/day) pi (%/g) fcr per control 1.24±0.41b 22.36±7.19c 0.71±0.27b 0.09±0.29a 6.65±1.09 a 0.02±0.01a 2.52±0.8b 2.4 0.22±0.37a 2.58±9.34a 0.04±0.37a 0.0±0.26a 4.43±0.34 a 0.01±0.02a 0.29±1.01a 2.6 1.73±0.90b 33±14.15b 0.83±0.32b 0.12±0.06b 5.37±0.91 a 0.07±0.06a 3.95±1.6b 2.8 0.07±0.33c 2.32±1.02a 0.03±0.47a 0±0.03a 4.01±0.02 a 0.19±0.12b 0.17±0.10a 3.0 0.84±0.25d 20.34±5.28c 0.6±0.14b 0.06±0.18b 4.98±0.72 a 0.02±0.00a 2.27±0.5b 3.2 2.45±2.03e 57.84±3.39d 0.96±0.84b 0.18±0.17b 6.29±1.12 a 0.01±0.01a 8.73±0.28c 3.4 0.86±0.39d 18.36±9.06c 0.6±0.20b 0.06±0.28a 4.67±0.46 a 0.02±0.02a 2.45±0.8b international journal of applied biology, 5(1), 2021 69 wg;weight gain, pwg; percentage weight gain, sgr; specific growth rate; adg; average daily gain; pi; protein intake fcr; feed conversion ratio, per; protein efficiency ratio. means followed by the same letter in the same column are not significantly different (p < 0.05) using lsd. values present mean ± standard deviation. in this study, the growth and nutrient utilization parameters of c. gariepinus fingerlings were generally not dependent on the concentration group to which they belong. this is different from the finding of ndimele et al. (2012) who reported a concentrationdependent growth for c. gariepinus fingerling exposed to varying concentrations of dichlorvos. this may be due to different conditions to which the fishes were subjected in their study. for example, the time for which the fishes were exposed before the growth trial was not clearly stated in their study as opposed to this study where the time with the lowest record of mortality was selected. as opposed to their report, this study showed that the fishes belonging to the control group did not necessarily record the best growth or nutrient utilization parameters. this finding points to the possible recovery of the fishes that survived exposure to varying levels of the pesticide for 24 hours. figure 2. median lethal concentrations (lc50) of dichlorvos to fingerlings of clarias gariepinus although it is expected that the residue of the pesticide in the tissues of the fish will fade out within 56 days for which the growth trial lasted, this is not to suggest the possible absence of residual effects on the biochemical parameters of this fish. some studies that reported this had ignored the possible impacts of pesticides on the biochemical parameters after a substantial period (post-exposure) as was the case in this study. for example, based on analyses carried out immediately after the exposure period, ndimele et al. (2015) concluded that another organochlorine pesticide known as endosulfan alters the hematology of c. gariepinus fingerlings. likewise, harabawy et al. (2014) only considered the immediate post-exposure hematological, biochemical, and cytogenetic response of c. gariepinus to carbofuran pesticide. although this study adds to what has been known, there is the need for further research into the post-exposure recovery and the assessment of growth, nutrient utilization, and biochemical and hematological parameters for a while during recovery of exposed fishes to either lethal or sublethal concentrations of chemicals to which they are exposed. international journal of applied biology, 5(1), 2021 70 conclusions this study found a dose-dependent survival and concentration graded lethality of dichlorvos based on the 96-hour acute toxicity test. besides, the growth and nutrient utilization parameters of c. gariepinus fingerlings post-exposure were generally not concentration-dependent. this finding reveals that c. gariepinus fingerlings could recover after acute exposure to 2, 2-dichlorovinyl dimethyl phosphate (dichlorvos) at different concentrations. references al-kawaz, j. m. 2019. effect of acute toxicity of glyphosate in gold fish carassius auratus. annals of tropical medicine and health. 22: 137-145. ashade, o. o., ashiru, a. w & obiri, c. m. 2011. the comparative study of the toxic effects of 2-3 diclorovinyl dimethyl phosphate (ddvp) and chlorpyrifos on the behaviour and haematology of african-(clarias gariepinus). international journal of science & society yabatech. 1(1): 38-47. basharat, h., ali, m., shahid, m & ahmed, a. 2020. introduction of african catfish (clarias gariepinus) in aquaculture system of pakistan: its transportation, acclimatization and cannibalism study. pak. j. agri. sci. 56: 1645-1652. dadebo, e., aemro, d., & tekle‐giorgis, y. 2014. food and feeding habits of the a frican catfish c larias gariepinus (b urchell, 1822) (pisces: clariidae) in lake koka, ethiopia. african journal of ecology. 52(4): 471-478. das, s. 2013. a review of dichlorvos toxicity in fish. current world environment. 8(1): 143. de graaf, g., galemoni, f & banzoussi, b. 1995. artificial reproduction and fingerling production of the african catfish, clarias gariepinus (burchell 1822), in protected and unprotected ponds. aquaculture res. 26: 233-242. egesi, oc. 2017. effect of ddvp (2, 3-dichorovinyl dimethyl phosphate) on clarias gariepinus juvenile. int. j. agric. rural dev. 20: 3058-3065. ezemonye, l. i. n & tongo, i. 2009. lethal and sublethal effects of atrazine to amphibian larvae. jordan journal of biological sciences, 2(1): 29-36. ezike, co. 2017. acute toxicity and heamatology of clarias gariepinus (burchell, 1822) exposed to 2, 2-dichlorovinyl dimethyl phosphate (dichlorvos). int. j. fish aquatic stud. 5: 100-105. finney, d. 1971. probit analysis. 3rd ed. cambridge university press, london, 333pp. fırat, ö., cogun, h. y., yüzereroğlu, t. a., gök, g., fırat, ö., kargin, f & kötemen, y. 2011. a comparative study on the effects of a pesticide (cypermethrin) and two metals (copper, lead) to serum biochemistry of nile tilapia, oreochromis niloticus. fish physiology and biochemistry. 37(3): 657-666. international journal of applied biology, 5(1), 2021 71 harabawy, a.s & ibrahim, a.t.a. 2014. sublethal toxicity of carbofuran pesticide on the african catfish clarias gariepinus (burchell, 1822): hematological, biochemical and cytogenetic response. ecotoxicology and environmental safety. 103: 61-67. ibrahim, a., adetola, j. o., emmanuel kolawole, a & adetunji yusuf, a. 2016. natural occurrence of diplostomum spp. in farm-raised african catfish (clarias gariepinus) from oyo state, nigeria. international journal of veterinary science and medicine. 4(2): 41-45. khan, s., qayoom, i & balkhi, m. 2018. dichlorvos (76% ec) induced behavioural toxicity in common carp fingerlings. j. entomol. zool. stud. 6: 1172-1175. kumar, p., swami, d., karade, h. n., singh, m., tiwari, a., & singh, k. p. 2016. protection studies of new bis quaternary 2-(hydroxyimino)-n-(pyridin-3yl) acetamide 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3: 793-821. sturve, j., scarlet, p., halling, m., kreuger, j & macia, a. 2016. environmental monitoring of pesticide exposure and effects on mangrove aquatic organisms of mozambique. marine environmental research. 121: 9-19. ugwu, l. l., mgbenka, b. o., eneje, l. o., ude, e. f & nwenya, j. i. 2006. toxicity, growth and survival of clarias gariepinus juveniles exposed to different concentrations of crude oil fractions-polluted water. animal research international. 3(2): 466-472. viveen, w.j., richter, c.j., van, p.g & ordt, j.a. 1985. manual for the culture of the african catfish, clarias gariepinus, directorate general for international cooperation, the hague, 93pp. contact : erik prasetyo erikprasetyo96@gmail.com 26 abstract baturraden botanical garden is located in kemutuk lor village, baturraden district, banyumas in the southern slopes of mount slamet. this area is mostly composed by the forest cover (natural forests and production forests) and has a high water abundance so in accordance with the habitat of amphibians (order anura). this study aims to determine the species composition of amphibians (order anura) in the area of the baturraden botanical garden. this research was conducted in july 2015 using the visual encounter survey (ves) method by making three line transects. results of the study found 8 species of amphibians (order anura) from 5 families that were families from megrophryidae (leptobrachium haseltii), bufonidae (leptophryne borbonica), dicroglossidae (limnonectes kuhlii), and (limnonectes microdiscus), ranidae (hylarana chalconota and odorrana hosii) and rhacophoridae (polypedates leucomystax and rhacophorus margaritifer). family composition is dominated from family dicroglossidae and ranidae. issn : 2580-2410 eissn : 2580-2119 species composition of amphibians (order anura) in baturraden botanical garden, banyumas erik prasetyo, umniyyatuz zulfa, rizky zulfa roihana, septiana anggraini, rina wahyuningsih, wahyu mustika dewi, zanuar abdul hamid, annisa nurjannah, annisa lintang malinda, erna muktisari, & nurliana biology department, universitas negeri semarang, semarang, central java, indonesia introduction baturraden botanical garden is located on the southern slope of mount slamet. mount slamet is one of the still active volcano in central java. administratively, mount slamet in five areas, that are banyumas, tegal, pemalang, brebes, and purbalingga. the southern slope areas of mount slamet in the district of banyumas has the most extensive forest cover of the five regions. forest coverage in this region is composed of the natural forest cover / forest protection and production of a resin (agathis dammara) and pine (pinus mercusii) (soemarno and girmansyah, 2012). mount slamet is also an area of forest catcher as well as the source of the watershed (gunawan, 2012). such conditions allow regions of baturraden botanical garden have potential high richness of amphibian species (orders anura). amphibian is one of fauna the constituent ecosystems and biodiversity is that can live in marine habitat, land, until arboreal. the existence of amphibians can be used as bioindicators of environmental quality, because amphibians have sensitive skin that is prone to environmental changes. anura is a tailless amphibians which consists of a group of frogs and toads. anura is a group of animals that require the availability of water in its life cycle, for open access international journal of applied biology keyword amphibians, anura, baturraden, species composition article history received 12 october 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 27 growth and development. anura plays an important role in the food chain and maintain the balance of the ecosystem. anura has role as predators of insects and insect larvae and is not directly useful for reducing the natural human being insect pests and agricultural plantations or insect vectors of disease (stebbins and cohen, 1997). documenting the amphibian species (orders anura) in this region are rare. previous data collection has been done is on the southern slope of mount slamet by riyanto (2010), ratna and wijaya (2013) as well as on the eastern slopes by riyanto and trilaksono (2012). based on this, then do research to determine the species composition of amphibians (order anura) in the area of the baturraden botanical garden and is expected to be considered in conducting advanced research. materials and methods study area sample collection time and location the research was held on july 04th – 06th, 2015. location of research in the baturraden botanical garden, kemutuk lor village, baturraden district, banyumas regency, central java, indonesia. research location map can be seen in figure 1 below. figure 1. research location map of baturraden botanical garden collecting data the method used in this research is the visual encounter survey (ves) that record all species of amphibians are found along the aquatic and terrestrial observation (heyer et al., 1994). used 3 line transects that are area around the mess / lodging, around the frogs garden and around liana gardem. for terrestrial habitat made line transect along the 1000 m, while the aquatic habitat made line transect 500 m. observations were carried out at night starting at 07.30 – 11.00 p.m. sampling was done through direct observation of amphibians, the arrest of individuals put in a plastic bag, and documentation with the camera. analysis data the data of the research results analyzed include: the species name, time, number of species, activity, the size of the snout vent length (svl) and environmental factors (temperature, altitude, and humidity). amphibians that were found subsequently identified using identification guide books iskandar (1998). analysis of the data used for the study of international journal of applied biology, 4(2), 2020 28 amphibians (order anura) is a comparative analysis of the species and conservation status. results and disscussion results the found of amphibians (order anura) in this research consists of 5 families, 7 genera and 8 species. the results found 8 species of anura, which is included in 5 families (table 1), that were from family megophryidae (leptobrachium hasseltii), bufonidae (leptophryne borbonica), dicroglossidae (limnonectes kuhlii and limnonectes microdiscus), ranidae (hylarana chalconota and odorrana hosii), and rhacophoridae (polypedates leucomystax and rhacophorus margaritifer). table 1. list of amphibians species (order anura) and conservation status no species family habitat conservation status (iucn) 1 leptobrachium haseltii megophryidae terrestrial least concern (lc) 2 leptophryne borbonica bufonidae semi aquatic least concern (lc) 3 limnonectes microdiscus dicroglossidae semi aquatic least concern (lc) 4 limnonectes kuhlii dicroglossidae semi aquatic least concern (lc) 5 hylarana chalconata ranidae semi aquatic least concern (lc) 6 odorrana hosii ranidae semi aquatic least concern (lc) 7 polypedates leucomystax rhacoporidae arboreal least concern (lc) 8 rhacoporus margaritifer rhacoporidae arboreal least concern (lc) discussion research on the southern slopes of mount slamet previously been done by riyanto (2010) and ratna & wijaya (2013), while on the eastern slopes by riyanto and trilaksono (2012). in the 2010 study in the area katenger found 16 species of anura, whereas in 2013 found 13 species of anura. in a study in 2012 on a climbing lane bambangan, serang river, and tuntunggunung river area found 14 species. the species of anura were found on the eastern slopes of mount slamet (2012), but was not found on the southern slope, that were microhyla palmipes, hylarana ruficeps, and occidozyga sp. the species of anura were found on the southern slopes of mount slamet in 2010 and 2013, but was not found in this study that were duttaphrynus melanostictus, phrynoidis aspera, megrophrys montana, microhyla achatina, huia masonii, fejervarya cancrivora, fejervarya limnocharis, limnonectes macrodon, philautus aurifasciatus, polypedates otiophilus, and rhacophorus reinwardtii. anura species that have not been found in previous studies (2010 and 2013) on the southern slopes of mount slamet was leptophryne borbonica. the different species of anura were found allegedly caused by different types of habitat examined / observed. in a previous study examines the natural forest habitat, production forests, rice fields and albicia garden. while this study examined the type of habitat that focus on production forests, so that the number of species found anura less than previous studies. baturraden botanical garden’s area there are many waterways so as to allow amphibians (order anura) aquatic types can proliferate more optimal than with other types international journal of applied biology, 4(2), 2020 29 anura, causing the family that dominates the composition is family dicroglossidae and ranidae (type of aquatic). for conservation status on the iucn, all amphibians (order anura) status least concern (8 types). conclusions composition species of amphibians (order anura) in baturraden botanical garden found 8 species belonging to 5 families. family is dominated dicroglossidae and ranidae family. amphibians (order anura) were identified in this study is leptophryne borbonica. the conservation status of all kinds is the least concern. references başkale, eyup & uğur k. 2009. richness and distribution of amphibian species in relation to ecological variables in western aegean region of turkey. 17: 25-31. gunawan, d. 2012. komunitas herpetofauna di lereng gunung slamet, jawa tengah. ekologi gunung slamet. jakarta: lipi press. hal 31 – 40. halliday, t. and k adler. 1986. the encyclopedia of reptiles amphibians. andromeda oxford ltd. england. iskandar, d. t. 1998. amfibi jawa dan bali. bogor: puslitbang biologilipi. kurniati, h. 2003. amphibians & reptiles of gunung halimun national park west java, indonesia. reseach center for biology-lipi. nagao natural environment foundationnef. cibinong munir, m. 2011. kekayaan jenis amfibi pada beberapa tipe habitat di gunung ungaran, jawa tengah. semarang: green community ______. 2012. amfibi gunung ungaran jawa tengah. semarang: green community. putra, k., et al. 2012. komunitas anura (amphibia) pada tiga habitat perairan di kawasan hutan harapan jambi. jurnal biologi uniersitas andalas. vol 1 (2). desember: hal 156 – 165. ratna, a. a. g. dan e. a. p. w. wijaya. 2013. survei awal keanekaragaman ordo anura di desa ketenger, batu raden, jawa tengah. indonesian journal of conservation. vol. 2. no. 1. hal 84 – 90. riyanto, a, dan w. trilaksono. 2012. komunitas herpetofauna di lereng timur gunung slamet, jawa tengah. ekologi gunung slamet. jakarta: lipi press. hal 151 – 160. international journal of applied biology, 4(2), 2020 30 riyanto, a. 2010. komunitas herpetofauna dan potensinya bagi sektor ekowisata pada kawasan ketenger–baturraden di selatan kaki gunung slamet, jawa tengah. biosfera. vol. 27. no. 2. hal: 60 – 67. soemarno, s. dan d. girmansyah. 2012. kondisi kawasan hutan alam gunung slamet, jawa tengah. ekologi gunung slamet. jakarta: lipi press. hal 41-61. stebbin, r. c. and n. w. cohen. 1997. a natural history of amphibians. new jersey: princeton university press. page 316. yani, a., et al. 2015. keanekaragaman jenis amfibi ordo anura di kawasan hutan lindung gunung semahung kecamatan sengah temila kabupaten landak kalimantan barat. jurnal hutan lestari. vol 3 (1). hal: 165 – 20. zug, g. r. 1993. herpetology: an introductory biology of ampibians and reptiles. academic press. london, page: 357 – 358. contact : slamet santosa slametsantosa@unhas.ac.id 89 abstract the bats population lives and reproduces on trees in the city center of soppeng regency. the study aims to analyze the potential of community based ecotourism development of bats population. the method of study are the objects and ecotourism attractions, community based ecotourism and ecotourism management analyzes. the :all respondents stated that the bats population can be an ecotourism attraction. it's easy to observe wild bats. ecotourism is an environmentally friendly tourism trip and empowers local communities. accessibility is easy because the location of ecotourism attractions is only distance of 0.5 km from the bus terminal and public transportation. amenities, such as hotel, restaurant, post offices, telecommunication networks, banks and souvenir shop are available at tourist sites. the study concludes that the bats population is potential to be developed as a community based ecotourism object in soppeng regency. issn : 2580-2410 eissn : 2580-2119 potential of community based ecotourism of bats population (megachiroptera) in soppeng regency , indonesia slamet santosa 1*, e.soekendarsi, m.s.hasan 2, fahruddin, d. priosambodo 3 1department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia. 2department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia. 3department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia. introduction the bat population lives and reproduces in city center of soppeng regency. there are thousands and there have been hundreds of years ago. the government of soppeng tries to protect and preserve. as an effort to help the government of soppeng, this bat population can be used as a tourist attraction. ecotourism is a tour with limited participants, environment based and empowering local communities. by becoming an ecotourism object, the life of the bat population will not be disturbed. luchman (2004), ecotourism has its own specificity, namely promoting environmental conservation, environmental education, the welfare of local residents, and respect for local culture. hill and gale (2009), ecotourism can be seen based on its relationship with the 5 of core elements, which are natural, ecologically sustainable, the environment is educative, benefits local communities, and creates tourist satisfaction. philip et. al, (2009), by empowering local communities, good participation will be realized between the local community and the tourism industry. the open access international journal of applied biology keyword ecotourism, bats population, community based, soppeng regency. article history received 08 august 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 90 community involved in decision-making is expected to be a form of better cooperation between the local community and the tourism industry. ecotourism is an environmentally friendly tour and empowers local people. in the development of ecotourism there are several factors to be considered, namely attractions, accessibility and amenities. the bat population is a local resource in soppeng regency that can be used to generate local revenue (foreign exchange), including as an ecotourism destination. satria (2009), an effort to utilize optimal local resources is to develop tourism with the concept of ecotourism. tourism carried out in this context has an inseparable part of conservation efforts, empowerment of the local economy and mutual respect for cultural or cultural differences. shifting the concept of world tourism to ecotourism models, due to the saturation of tourists to visit artificial tourism objects. if this opportunity can be used optimally to attract foreign tourists to visit objects based on nature and culture of the local population. the bat population are two major groups, namely the fruit eating bat (megachiroptera sub order) and the insect eating bat (microchiroptera sub order). there are currently 1001 species of bats in the world. there are 167 species of megachiroptera and 834 species of microcheroptera.. the last number of published species amounted to 1117 including 1 family with 186 species of megachiroptera and 17 families with 931 species of microchiroptera (srinivasulu et al. 2010). bats live in various types of habitats and choose alternative hanging places. certain types of bats such as ‘kalong’ bats; ‘codots’ bats and some species of the sub order megachiroptera choose hanging-places to sleep on large trees. whereas some types of bats from the sub order of microchiroptera prefer shelter in caves, holes in tree trunks, bamboo gaps , dead trees, woven rattan to the ceiling of houses in residential areas (yuliadi et al., 2014). suyanto et.al. (2002 ), bats should live on large trees in the forest or cave. palmer and woinarski (1999), the megachiroptera bat lives in mangrove forests, bamboo forests and rainforest. while the bats population habitat in soppeng regency is not forest but on trees in the city center. its habitat unique in the city center so it is easy to visit the uniqueness of an animal can be a tourist attraction.. materials and methods study area profile the population of soppeng regency in 2016 is 226.305 people consisting of 106.485 men and 119.820 women . lalabata sub district has the largest population 114.845 people. number of working people 15 years old and over by main industry in soppeng regency ie. manufacturing industry 4.372 people, whole sale trade, retail trade, restaurants and hotels 16.209 people, agriculture, forestry , hunting and fisheries 44.716 people and others 8.343 people (anonymous, 2017). international journal of applied biology, 4(2), 2020 91 figure1. soppeng regency of study area source : google map data collection the method of study uses data collection. data collection methods consist of primary and secondary data. primary data is collected by observation, interviews and questionnaires. interviews were carried out with staff from the cultural and tourism offices, lalabata sub district staff, food traders at tourist sites and tourist visitors. the questionnaire respondents using random sampling method, namely 15 of respondents of culture and tourism offices staff, 15 of the lalabata sub district staff, 15 of food traders at the tourist location and 15 of tourist visitors. while secondary data is collected by reviewing documents in the cultural and tourism offices, environmental agencies and the government of soppeng. data analysis analysis of objects and ecotourism attractions this analysis is to find out the potential objects and tourist attraction of the bats population in soppeng regency. the results of the analysis is a description of the potential of bats population as objects and tourist attraction. international journal of applied biology, 4(2), 2020 92 analysis of community based ecotourism development this analysis is to determine of potential of community based ecotourism development in the habitat area of bats population. analysis of the community based ecotourism development using qualitative descriptive statistical methods, based on the results of interviews and questionnaires. the results of the analysis of community based ecotourism development is a descriptions and community participation in the soppeng regency. results and discussion objects and attractiveness of bats population analyzed are attractions, accessibility and amenities. attractions, accessibility and amenities are determinants of visitors to ecotourism destination. results of analyzed of bats population attraction are the unique habitat (figure 2) and beautiful panoramic when the bats population flies in the air space of soppeng regency (figure 3). then the results of observations showed that the bats population lived and reproduces on trees in the city center of soppeng regency. the people of soppeng regency often connect with events that will “occur in the community”. therefore, the existence of the bats population is protected and preserved by the government of soppeng regency. according to suyanto et al. (2002), generally bats live on large trees in forests and caves. the bats in the soppeng regency are fruit eaters, including the megachiroptera sub order. palmer and woinarski (1999), black flying foxes (megachiroptera) have habitat in bambo forest, mangrove forest and rainforest. figure 2. bats population on trees of pterocarpus indicus (a) and gmelina arborea (b) international journal of applied biology, 4(2), 2020 93 figure 3. the beautiful panoramic of bat population flying widagdo, k.g. (2017)., tourist attractions are divided into natural attractions, attractive artificial tourism and culture. tourist attraction is everything that has unique, beauty, and value in the form of diversity of natural, cultural, and man made wealth that are the target or destination of tourist visits. young et al. (2013), there are three main characteristics who build ecotourism and have approved, namely: attraction nature-based tours, and usually focus on ecosystems that are relatively not disturbed or have an endemic still original like charismatic megafauna (for example, giant pandas, koalas, cranes whooping) inhabiting the ecosystem, charismatic megaflora (for example, trees red wood) and megaliths (for example, volcanoes, cliffs) displayed. eugene e. ezebilo (2014), ecotourism is responsible travel to natural areas that conserves the environment and improves the well-being of local people. it involves visits to destinations where animals, plants and cultural heritage are the main attractions. nugroho (2011), tourist attractions can be in the form of endemic species, animal diversity, natural beauty, animal uniqueness, natural landscape and culture. devy, h.a. (2017), tourist attractions is one of the main capital that must be owned in an effort to increase and the development of object and tourism attractions. the existence object and tourist attraction is the most important link in a tourist’s activities. the main factor that makes visitors to visit tourist destinationsis the potential and attractiveness owned by these attractions. poupineau & claire (2013), ecotourists choose their destination in accordance with the types of products offered, generally they are looking for specific experiences they cannot reach in other areas. therefore, providing a high value product and services is very important. hijriati & rina, (2014). ecotourism is a tour that is responsible for environmental sustainability and welfare local community. the active role in managing ecotourism potential is important because of natural knowledge and cultural potential has a selling value as an ecotourism attraction. the development of ecotourism affects society on ecological, social and economic aspects. the results of the interview, the all respondents (100%) stated that bats population tours in soppeng regency were easily seen and visited because they were in the city center.. the number of thousands of bats hanging on several types of trees. the shape and color of his body look clear with his distinctive voice, because these bats hanging on trees that are only 3-5 meters high. the bats population which flew together in the airspace of city center , soppeng regency showed a beautiful panoramic. the uniqueness of the habitat and beautiful panoramic has the potential to become an attraction for the development of international journal of applied biology, 4(2), 2020 94 ecotourism objects. nugroho (2011), attractions in the tourism industry are the main factors for tourist interest. attractions in tourism can take the form of natural beauty, animal diversity, unique fauna and flora and culture. the respondents are representatives of the community in soppeng regency with a different profile (table 1). table 1. profile of respondent number percent (%) gender : male 25 41.67 female 35 58.33 age : 15-19 5 8.33 20-29 23 38.33 30-39 28 46.67 40-49 3 5.00 50-60 1 1.67 education: yunior high school 11 18.33 senior high school 32 53.33 university or college 17 28.34 occupation: house wife/husband 20 33.34 student 5 8.33 employee: tourism office staff 15 25.00 lalabta district staff 15 25.00 unemployed 5 8.33 the location of the ecotourism object is located in the lalabata sub district (city center). distance of ecotourism object location with bus terminal and public transportation is only 0.5 km. the visitors can walk from the bus terminal to the location of the ecotourism object. environmental conditions during the day are cold because soppeng regency is surrounded by mountains. anonymous (2017), soppeng regency is surrounded by mountains, namely nene conang mount with altitude of 1.463 m; laposo mount, altitude of 1000 m; sewo mountain, altitude of 860 m; lapancu mount, altitude of 850 m; bulu dua mount, altitude of 800 m and paowengeng mount, altitude of 760 m. the results of interviews and questionnaires, 90.67% of respondents stated that the road facilities and public transportation availability were good. access from soppeng regency to other regency using public transport is available at the terminal. soppeng regency does not have pioneer seaports and airports. an international airport near soppeng regency is in the city of makassar. traveling from the city of makassar to soppeng regency using buses or other public transportation. the distance from makassar city to soppeng regency is 159.7 km and can be reached 3 hours travels. hotels and restaurants already exist in soppeng regency. anonymous (2017), soppeng regency has 23 of non star hotels with 150 rooms and 406 beds. the number of 34 restaurants spread over 8 sub districts. the location for ecotourism development is in the lalabata sub district which is the city center of the soppeng regency government. the most hotels and restaurants in lalabata sub district. international journal of applied biology, 4(2), 2020 95 hotels within 1-2 km of ecotourism objects are ada hotels, satria hotels, makmur hotels, surya hotels, kahyangan hotels and vera indah hotels. while restaurants in the city center are padang, ojo lali, and bugis makassar. the result of observations show that on the north side of the ecotourism object there are 25 of traders who sell food and drinks. based on the results of surveys and interviews with visitors, 95.33% of respondents stated the menu and taste of food was good and tasty. the results of observation of amenity aspects are the availability of post offices, telecommunication network, exchange of money (bank) and shop of souvenirs. the aspect of amenity in the tourism industry is a factor supporting tourists. anonymous (2017), there are 4 of post offices and 8 of supporting post offices in 4 of sub districts in soppeng regency. there is 1 of post office in lalabata sub district. there are 3 of banks, namely bri, bni and sulselbar bank. in the location of tourism there are 2 of automatic teller machines (atms), owned by bri and bni banks. the communication network has been provided by ‘pt. telkom’ and several cellular providers. the souvenirs in the form of t-shirts, hats and handicrafts illustrated by bats have been sold by traders at the location of ecotourism objects community-based ecotourism needs to be developed to support community welfare and protection, preservation of bat populations. whereas the location of the bat population is in the lalabata sub-district, the most populated in soppeng regency. anonymous (2017), the population of soppeng regency in 2016 is 226.305 people consisting of 106.485 men and 119.820 women. lalabata sub district has the largest population 114.845 people. number of working people 15 years old and over by main industry in soppeng regency ie. manufacturing industry 4.372 people, whole sale trade, retail trade, restaurants and hotels 16.209 people, agriculture, forestry , hunting and fisheries 44.716 people and others 8.343 people. based on these data, the opportunity to empower people to get income from tourism through service providers, souvenirs and tour guides is very large. the local community have been given permission to open a food and beverage business on the north side by the soppeng regency government. the community empowerment aims to increase the economy and at the same time keep the bats population not disturbed by tourists. then making souvenirs with the characteristics of bats has been taught to the community as part of the tourism element .nugroho (2011), ecotourism activities must involve empowering the local economy. food stalls and souvenirs are economic parts that can be developed by the local community. however, the souvenir industry must get supervision because there is a lot of evidence of raw materials for souvenirs exploiting from protected areas tanaya dan iwan (2014), analysis of community aspects is carried out on 4 variables, namely the character of the community, the form of community participation, business opportunities, as well as the quality of tourism and community readiness. the character of the community is assessed by 7 dictators, namely livelihood, education level, behavior, customs, traditions, habits, and awareness of tourism potential. the quality of tourism is assessed by indicators of continuous visits in the region, and the readiness of the community is assessed by analyzing how prepared the community is if the area is made into an ecotourism area. lovelock dan wright (2007), there are eight components in a service management which can have implications in increasing market share. these elements are divided into the following categories: product element, place, process, productivity and quality, people, promotion and education , physical evidence, price and other user outlays. sondakh dan tumbel (2016), tourist managers must pay attention to aspects of service and security to tourists. this is so that tourists can feel comfortable, safe, and care about the international journal of applied biology, 4(2), 2020 96 natural environment. olivia, et al (2015), concluded that the external environment in the form of cultural factors, social class, references, and family will influence tourists.. cristina bittar rodrigues & bruce prideaux (2018), the community based tourism implementation model is able to provide a roadmap of the actions that need to be undertaken and the sequences that need to be followed, if local communities are to firstly become involved in community based tourism projects and secondly be empowered to the extent that they are able to be treated as an equal by other firms operating in the tourism industry. hijriati and rina (2014), community based ecotourism is an ecotourism business that focuses on the active role of the community. this is based on the fact that the community has knowledge about nature and culture which is a potential and selling value as a tourist attraction, so that community involvement is absolute. community-based ecotourism patterns recognize the rights of local communities in managing tourism activities in areas that they have traditionally owned or as managers. the existence of a community-based ecotourism pattern does not mean that the community will run. while the analysis of tourism management aspects is carried out in 3 variables, namely community participation, transparency, and government policies and programs. as many as 70.67% of respondents stated that they had participated in tourism activities, namely in the form of environmental care and culinary providers, and as many as 95.00% had participated more than 4 years. the reasons for participating are divided into 2 dominant reasons, namely as a form of responsibility, and to make a living. however, as many as 85.67% of respondents stated that there was a micro-economic effort as a form of business opportunity to support community activities, most of which were in the form of culinary gifts, and the rest in the form of traditional food and crafts.management transparency is assessed by 3 indicators, namely the benefits gained by the community, distribution of benefits, and community complaints about tourism activities in the region. based on the results of the questionnaire, 75,65% of respondents stated that the benefits obtained were in the form of efforts to promote regional economies. nonetheless, there are still 30.33% who say they have not felt the benefits of tourism activities. in addition, 15.33% of respondents stated that there was no profit distribution system from the tourism activities manager. wood (2002), ecotourism management does require the participation of local people because they know the condition and situation of tourism. local people who must maintain and maintain tourism objects from damage. the policies and goverment programs related to the development of the bat population are already regulated of the master plan for regional tourism development in soppeng regency. but the implementation of these goverment programs have not been fully realized, because they require full involvement of the community. the main obstacles to community based ecotourism development are people who tend to be lazy and do not care about their environment. conclusions the population of bats has number in the thousands, living in large trees in the city center of soppeng. the potential bat population is used as an object of ecotourism which also serves to protect and conserve. international journal of applied biology, 4(2), 2020 97 references anonymous. 2017. soppeng regency in figures 2017.bpsstatistics soppeng regency. . soppeng. cristina bittar rodrigues & bruce prideaux. 2018. a management model to assist local communities developing community-based tourism ventures: a case study from the brazilian amazon, journal of ecotourism, 17(1), 1-19. devy, h.a. 2017. object development and natural tourist attraction as a area tourism objectives in karanganyra regency. dilema, sosioloy journal, 32(1). eugene e. ezebilo. 2014. choosing ecotourism destinations for vacations: a decision-making process.sweden: asian social science, 10 (2). helgen, kristofer m. 2004. on the identity of flying-foxes, genus pteropus (mammalia: chiroptera), from islands in the torres strait, australia." zootaxa 780, 1–14. hijriati, e and rina m. 2014. communitybased ecotourism influence on changes in ecological, social and economicconditions in batusuhunan village, sukabumi. rural sosilogy journal, 2 (3), 146-159. hill, jennifer and gale, tim (eds.). 2009. ecotourism and environmental sustainability: principles and practice. burlington: ashgate. luchman, hakim. 2014. basics of ecotourism.bayumedia publishing. malang. lovelock, c., & l.k. wright. 2007. service marketing management.gramedia publisher. 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(eds.). 2009. an introduction to community development. new york. poupineau sarah danpouzadoux claire.2013. .internal and external factors that influence theecotourist.school of business and engineering:halmstad university. international journal of applied biology, 4(2), 2020 98 satria, d. 2009. strategy for the development of local economy-based ecotourism in the framework of poverty alleviation programs in the malang regency. journal of indonesian applied economics, 3 (1), 37-47. sondakh, p.m.n., & a. tumbel. 2016. service, security and attraction affect tourist interests visiting mount mahawu natural attractions, tomohon. scientific periodic journal of efficiency, 16 (1), 280-288. srinivasulu, paul a. racey&shahroukhmistry. 2010 a key to the bats (mammalia: chiroptera) of south asia, journal of threatened taxa, 2(7), 1001-1076. suyanto, a., yoneda, m., maryanto, i., maharadatunkamsi and sugardjito, j. 2002, checklist of the mammals of indonesia, lipi-jica-phka, bogor. tanaya, d. rukti and iwanrudiarto. 2014. the potential for community-based ecotourism development in the swamp swamp area, semarang regency. pwk engineering journal, 3(1), 71-81. widagdo, k.g. 2017. marketing, attractiveness of ecotourism and visiting tourist interest. business and management journal, 7 (2), 261-276. woods, m.e. 2002. ecotourism: principles, practice, and policies for sustainability. unep. yuliadi, b., t.f. sari and f.d. handayani. 2014. sulawesi bats, types and roles.ministry of health. jakarta. yulianda, f. 2007. marine ecotourism as an alternative for conservation-based coastal resource use. science seminar paper february 21, 2007. department of aquatic resources management, bogor agricultural institute. bogor. young-sook lee, laura j. lawton dan david b.weaver. 2013. evidence for a south korean model of ecotourism. journal of travel research, 52 (4):520-533. 9 contact : m. farid dimjati lusno faridlusno@fkm.unair.ac.id international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. issn : 2580-2410 eissn : 2580-2119 association between smoking and hypertension as a disease burden in sidoarjo: a case-control study m. farid dimjati lusno1*, setya haksama1, anis wulandari1, shyamkumar sriram2, syahrania naura shedysni3, m. rifqo hafidzudin farid3, abdul fattah farid4, syadza zahrah shedyta5 1faculty of public health, universitas airlangga, surabaya, indonesia 2arnold school of public health, university of south carolina, columbia, united states 3faculty of medicine, universitas airlangga, surabaya, indonesia 4faculty of pharmacy, universitas airlangga, surabaya, indonesia 5husada utama hospital, surabaya, indonesia introduction noncommunicable diseases (ncds) continues to be one of the majors caused of premature death worldwide (world health organization, 2014). this burden is rising disproportionately among low-income and lower-middle-income countries (world health organization, 2017). ncds are by far the leading cause of death worldwide. in 2016, they were responsible for 71% (41 million) of the 57 million deaths which occurred globally. the major ncds responsible for these deaths highest reached by cardiovascular diseases (17.9 million deaths, accounting for 44% of all ncd deaths and 31% of all global deaths); followed open access international journal of applied biology abstract noncommunicable diseases (ncds) became major caused of premature death and disease burden which rising disproportionately among low-income and lowermiddle-income countries. there are common modifiable behavioral risk factors include tobacco smoking. in indonesia there were 147.510 cardiovascular (cvd) death caused by tobacco use which is 26% of all cvd deaths each year. the link between smoking and hypertension is still far to be completely identified. this study aims to analyze the relationship of smoking to hypertension. the study conducted with case control approach. the sampling method use probability sampling with the samples 172 respondents consist of 76 cases and 76 control. hypertension patient data is obtained based on secondary data that has been owned by the public health center service (puskesmas). the finding showed that smoking (p=0,01, or=3,1), early age of smoking (p=0,00, or=6,5) with starting 6 – 19 years old higher or then 20-33 years old (p=0,00,or=4), duration of smoking (p=0,00, or=9,9) in a group with 30 – 53 years of smoking higher than 9 – 29 years of smoking (p=0,0, or=8,2), and type of cigarettes (p=0,001, or=3,4). smoking has association to hypertension as the modifiable factor. the decision maker needs to be concerned especially for ncd’s program by preventing hypertension. article history received 8 august 2020 accepted 9 october 2020 keyword disease burden smoking hypertension case-control international journal of applied biology, 4(2), 2020 10 by cancers (9 million deaths, 22% of all ncd deaths and 16% of all global deaths); chronic respiratory diseases (3.8 million deaths, 9% of all ncd deaths and 7% of all global deaths); and diabetes (1.6 million deaths, 4% of all ncd deaths and 3% of all global deaths). there are four major ncds. those are cardiovascular diseases include cancers, diabetes, and chronic respiratory diseases. raised blood pressure, also known as hypertension, is a major risk factor for coronary heart disease, chronic kidney disease, and ischemic, as well as hemorrhagic, stroke. there has been set for further targets relate to the reduction in ncd risk factors including both behavioral risk factors (the harmful use of alcohol, physical inactivity, salt/sodium intake and tobacco use) and metabolic risk factors (raised blood pressure, raised blood glucose and obesity) (damasceno, 2016). the rise of ncds is particularly visible in southeast asia, such as indonesia where caused by limited resources and related with the social determinants of health. there are common modifiable behavioral risk factors (e.g. tobacco smoking, harmful use of alcohol, unhealthy dieting, and physical inactivity) and biological risk factors (e.g. hypertension, overweight/obesity, or a combination of risk factors like the cardio-metabolic syndrome) (schröders et al., 2017). east java province as one of the densest provinces in indonesia has higher prevalence of cardiovascular disease than the national prevalence (riskesdas, 2018). sidoarjo as one of side urban area has high prevalence of hypertension (dinkes, 2018). this also include the importance of public health center service (yunevy & haksama, 2013) hypertension is considered one of the most important preventable causes of morbidity and mortality (ain et al., 2015). hypertension has been found to be associated with an unhealthy lifestyle, including tobacco smoking, a lack of physical activity, and alcohol consumption (world health organization, 2017). the result of previous study showed that among men having quit tobacco use and depressive symptoms were positively associated with hypertension, while current tobacco use was negatively associated with hypertension (peltzer & pengpid, 2018) smoking and hypertension claimed that smoking is a cause of hypertension despite inconsistent results in the literature (sohn, 2018). cigarette smoking is a known risk factor for cardiovascular disease (cvd), but the association between smoking and blood pressure is unclear (li et al., 2017). many observations identify cigarette smoke as a factor causing a functional and initially transient damage primarily of the endothelium and reduced tolerance to exercise stress testing because of the effects of nicotine and carbon monoxide. at the time, the functional damage became an irreversible pathological damage with ischemic lesions of the myocardium and artery vessel atherosclerosis (leone, 2015). in indonesia there were 147.510 cardiovascular (cvd) death caused by tobacco use which is 26% of all cvd deaths each year. mean age at initiation of daily smoking at 17,6 years which imply to increase of earlier risk of heart disease in younger people (world health organization, 2018). the link between smoking and hypertension is still far to be completely identified. usually a smoker begins to smoke as before as the appearance of the blood pressure disorder. therefore, confusion exists to assess whether hypertension will appear spontaneously and independently in the individuals affected or, on the contrary, is a result of smoking habit (leone, 2015). the program of preventing and controlling sickness, death and disability from cardiovascular disease need to put in higher concern. so that, there will be aware of helping current tobacco users to quit tobacco for healthier heart, prevent people from starting tobacco use, and combine tobacco with the following strategies for effective prevention of international journal of applied biology, 4(2), 2020 11 cvd (world health organization, 2018). however, the priority may come easy with real evidence. therefore, this study aims to analyses the association between smoking and hypertension. materials and methods this case–control study included population age ≥ 17 years old. the population are people from community in work area of public health care centre (puskesmas) sedati and waru , sidoarjo district. the majority of respondents are between 36 – 45 years old. sample was calculated using hosmer & lemeshow based on probability sampling which come up total of sample 76 cases and 76 control. all samples total 152 respondents. cases samples were obtained from those who has systolic blood pressure ≥ 140 mmhg and / or diastolic blood pressure ≥ 90 mmhg, on repeated examinations. systolic pressure is the main reference in determining the diagnosis of hypertension. hypertension patient data is obtained based on secondary data that has been owned by the puskesmas. the people who are in cases sample will be evaluated the result based on retrospective study. all research subjects gathered the primary information by answering questionnaire. the research being held on mei 2018. the independent variable consists of smoking status, early age of smoking, duration of smoking, and types of cigarettes. the dependent variable is hypertension. both of primary data from questionnaire and secondary data from puskesmas were analyzed univariate and bivariate. finally, the data was analyzed using relation chi-square and logistic regression tests with spss software. results and discussion the demographic characteristics of the respondents showed that most of the respondents were females 51,30% which is not far differences number than the males 48,30%. based on age group, most of the subjects were in ≥ 35 years old 85,4% specifically between 36-45 years old 35,5% from total subjects. the education background has showed that most subjects 39,5% are graduated from senior high school. the jobs were dominated by factory workers, helpers, pedicab drivers 23% and farmers 20,4%. all modifiable risk factors studied vis‑à‑vis obesity, lack of physical activity, inadequate fruits and vegetable intake, diabetes, smoking, and alcohol use were significantly different in proportion among cases and controls. obesity, lack of physical activity, smoking, and diabetes were found to be significant risk factors for hypertension after adjusting for other risk factors (pilakkadavath & shaffi, 2016). smoking to hypertension based on the chi-square statistical test, the value of p = 0.01 was obtained. this shows a significant relationship between smoking and hypertension with or value of 3.1 obtained in smoking variable. smoker has 3.1 times greater risk of hypertension than nonsmoker. in a fully adjusted logistic regression model, former smokers had increased or (95% ci) of 1.48 (1.01, 2.18) of hypertension and current smokers had not increased or (95% ci) of 0.83 (0.61, 1.12), compared with non-smokers. this result has in line with previous study which mentioned no significant associations were found between smoking and the risk of incident respiratory diseases, hypertension, and myocardial infarction in the group younger than 35 (gao et all, 2017). among study participants aged between 36–55 and 56–80, international journal of applied biology, 4(2), 2020 12 smoking was positively associated with the risk of incident respiratory diseases, hypertension and myocardial infarction from the lifecourse perspective, and the risk increased with age. in contrast, the results from a current view showed inverse associations between smoking and the risk of the diseases mentioned above (gao et al., 2017). bivariate analysis showed that factors significantly related to stroke were smoking status (p-value = 0.011, or = 2.6), past hypertension (p-value = 0.00, or = 6), past diabetes (p-value = 0.015, or = 5.7), and unhealthy diet (p-value = 0.00, or = 5.7). multivariate analysis showed that factors significantly affecting stroke were smoking status, past hypertension, and an unhealthy diet. in conclusion, smoking, past hypertension, and an unhealthy diet are factors affecting the incidence of stroke (imanda et al., 2019). respondents with poor sleep quality had 1.39 higher odds ratio of experiencing hypertension compared to respondents who had good sleep quality after being controlled by variables of gender, age, smoking status, physical activity, and nutritional status. (poor-good sleep quality = 1.39, 95% ci 1.20-1.61 p=0.000) (nugroho et al., 2020). nearly all women were non smokers (96.3%) compared to 32.8% of men (oddo et al., 2019). the study among men only former tobacco use was positively associated with hypertension, while current tobacco use was negatively associated with hypertension. among smokers in this study the number of cigarettes smoked per day (mean=11.7) did not differ between hypertensives and nonhypertensives (mean: 11.9) (p=0.150). it is possible that the impact of current tobacco use on hypertension is delayed, and, thus, current tobacco use may not be closely correlated with hypertension (peltzer & pengpid, 2018). in its turn, hypertension plays harmful effects on the heart, kidney and arterial tree, mainly coronary, carotid and cerebral vascular structures, by its complications, the target organs of which are the same of cigarette smoke. there is evidence that the association of cigarette smoking with hypertension exponentially increases the risk of cardiovascular disease and events when compared to that of each of these factors singly acting (leone, 2015). in men, smoking was the most prevalent risk factor; approximately two-thirds of men were current smokers compared with only 2% of women, which wholly explains the dominance of smoking (hussain et al., 2016). smoking can increase blood pressure by stimulating the sympathetic nervous system. when a person smokes, the active substance interleukin 6, and can lead to oxidative stress. oxidative stress will trigger some reaction in the body, such as insulin resistance. if this happens, bioavailability endothelium which has a function to inhibit atherogenesis and protect blood vessels will decrease which trigger its happening raises blood pressure, atherosclerosis (elisabeth et al., 2017). early smoking to hypertension the age category of early smoking 6-19 years compared with no smoking based on the logistic regression statistical test obtained p value = 0.00. this value indicates that there is a significant influence between the early age of smoking 6-19 years with hypertension. in the early age category of smoking 6-19 years (or = 6,5) showed that the early age of smoking 6-19 years has a risk of hypertension 6.5 times greater than not smoking. the age category of early smoking 20 33 years compared to not smoking based on the logistic regression statistical test obtained p value = 0.008. this value indicates that there is a significant influence between the initial age of smoking 20-33 years with hypertension. in the early age category of smoking 20 33 years (or=4). this shows that the initial age of smoking 20 33 years has a 4 times greater risk of developing hypertension compared to not smoking. international journal of applied biology, 4(2), 2020 13 the short-term adverse health effects caused by cigarette smoking can be observed in smokers immediately or soon after they begin smoking. the health effects of cigarette smoking thus begin at or near the age of initiation of cigarette smoking, which is usually in adolescence. to highlight the immediacy of the adverse impact of smoking on health, this report uses a life-course perspective by considering health effects of smoking according to the various stages of life, which include childhood, adolescence, and young adulthood as well as middle and late adulthood, when most of the chronic disease burden imposed by smoking occurs (public health implications of rising the minimum age of legal access to tobacco products, 2015). the duration of smoking to hypertension the duration of smoking 30 53 years compared to not smoking based on the logistic regression statistical test obtained p value = 0.00. this value shows that there is a significant influence between the duration of smoking 30 53 years with hypertension. based on duration category of smoking 30 53 years (or=9.9) shows that the duration of smoking between 53 and 53 years has a risk of hypertension 9.9 times greater than not smoking. the duration of smoking 9 29 years compared to not smoking based on the logistic regression statistical test obtained the value of p = 0.00. this value indicates that there is a significant influence between the duration of smoking 9-29 years with hypertension. for duration 9 29 years smoking category (or=8.2) shows that 9 29 years smoking has a risk of hypertension 8.2 times greater than not smoking. from the previous study, has been mentioned that it is possible that the impact of current tobacco use on hypertension is delayed, and, thus, current tobacco use may not be closely correlated with hypertension (peltzer & pengpid, 2018). types of cigarettes to hypertension the category of non-filter cigarettes compared to not smoking based on the logistic regression statistical test obtained the value of p = 0.001. this value indicates that there is a significant effect between types of non-filter cigarettes with hypertension. in the category of non-filter cigarettes, an or value of 7.6 was obtained. this shows the type of non-filter cigarettes has a risk of hypertension 7.6 times greater than not smoking. categories of filter cigarette types compared to nonsmoking based on logistic regression statistical tests obtained p value = 0.051. this value indicates that there is no significant effect between filter cigarette types and hypertension. some studies focused on specific groups of smokers such as women, diabetes patients, and physicians. a few studies reported on the use of smokeless tobacco, betel quid chewing, or more recently the use of electronic cigarettes (schröders et al., 2017). in other study, we defined a current smoker if he said “yes” to the question, “have you ever chewed tobacco, smoked a pipe, smoked self-rolled cigarettes, or smoked cigarettes/cigars?” and “still have” to the question, “do you still have the habit or have you totally quit?” otherwise, he was defined as a current non-smoker (sohn, 2018). international journal of applied biology, 4(2), 2020 14 table 1. cross tabulation smoking status, early age of smoking, duration of smoking, and types of cigarettes from cases sample and control samples from puskesmas sedati and waru area , district sidoarjo year 2018. variables variables pvalue or 95% ci category category n % n % smoking status yes 47 61,8 26 34,2 0,01* 3,1 1,607 6,045 no 29 38,2 50 65,8 1 early age of smoking 6-19 years old 30 39,5 8 10,5 0,00* 6,5 2,618 15,969 20 – 33 years old 17 22,4 18 23,7 0,008* 4 1,427 11,050 not smoking 29 38,2 50 65,8 1 duration of smoking 30-53 years 33 45,8 6 8,2 0,00* 9,9 3,696 16,312 9 – 29 years 10 13,9 15 20,5 0,00* 8,2 2,530 26,900 not moking 29 40,3 52 71,2 types of cigarettes non-filter 18 23,7 4 5,3 0,001* 7,6 24,735 filter 28 36,8 21 27,6 0,051 3,4 11,458 not smoking 30 39,5 51 67,1 1 *shows a significant association conclusions the study has showed the association smoking to hypertension. the smoking status has clear association to hypertension with many previous study. the finding of early age of smoking, duration of smoking and types of cigarettes became new detailed result explaining the association mentioned. however, more detailed sample need to put in further research a head. moreover, the categorical need to represent the population. the study would raise concerned from decision maker especially to linkage the program of ncds especially in preventing hypertension. references ain, q., regmi, k., li, g., wang, h., wang, k., wang, w. w., dong, f., qian, y., gong, h., hui, c., gao, k., shi, x., wang, w. w., schröders, j., wall, s., hakimi, m., dewi, f. s. t., weinehall, l., nichter, m., … damasceno, a. 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(2013). analisis kepuasan berdasarkan persepsi dan harapan pasien di puskesmas medokan ayu surabaya quality. jurnal administrasi kesehatan indonesia, 1(1), 9-20 https://doi.org/10.1016/j.gheart.2018.01.00 http://www.searo.who.int/tobacco/data/ino_rtc_reports http://www.searo.who.int/tobacco/data/ino_rtc_reports association between smoking and hypertension as a disease burden in sidoarjo: a case-control study introduction materials and methods results and discussion smoking to hypertension early smoking to hypertension the duration of smoking to hypertension types of cigarettes to hypertension table 1. cross tabulation smoking status, early age of smoking, duration of smoking, and types of cigarettes from cases sample and control samples from puskesmas sedati and waru area , district sidoarjo year 2018. conclusions references contact : muhammad yusril hardiansyah yusrilhardiansyah1@gmail.com 7 abstract plant growth promoting rhizobacteria (pgpr) is a group of beneficial bacteria that actively colonize the rhizosphere and play an important role in enhancing root growth that impacts on plant growth, yields and land fertility. pgpr is very much found in the area around plant roots or rhizosphere especially thorny bamboo rhizosphere (bambusa blumeana). the number of microorganisms contained in pgpr spiked bamboo rhizosphere makes it difficult to know what types of bacteria are the most dominant and most active in influencing plant growth. one method that can be used to identify microorganisms is by biochemical testing processes using the 3% gram koh test method and gram staining test. this study aims to determine the use of the 3% gram koh test method and gram staining test in identifying plant growth promoting rhizobacteria bacteria from thorny bamboo rhizosphere (bambusa blumeana). this test is carried out at the biofertilizer and potential microbial laboratory, ministry of agriculture, faculty of agriculture, hasanuddin university, makassar and takes place from october to december 2019. the method of carrying out the test includes making the media nutrient agar (na), pouring media, planting bacteria, identifying bacteria in 2 gram reaction methods, namely the gram reaction test using 3% koh and using a simple coloring with methylen blue and lugol. the results obtained are the 3% gram koh test method in testing the scratch method of all samples that have slime while the scatter method has 2 samples that have slime. then the gram staining test method on staining with methylene blue and logol produces blue for the genus pseudomonas which means gram positive and pink or purple for the genus bacillus which means gram positive. so it can be concluded that the 3% koh test method and gram staining test have optimum effect in detecting microorganisms. issn : 2580-2410 eissn : 2580-2119 identification of plant growth promoting rhizobacteria from thorny bamboo rhizosphere with 3% koh gram test and gram staining test muhammad yusril hardiansyah, yunus musa & abdul mollah jaya department of agronomy, agrotechnology study program, faculty of agriculture, hasanuddin university, makassar, indonesia *corresponding author (muhammad yusril hardiansyah, department of agronomy, agrotechnology study program, faculty of agriculture, hasanuddin university. open access international journal of applied biology keyword bacteria, koh 3%, gram staining, plant growth promoting rhizobacteria article history received 27 september 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 8 introduction rhizobacteria are more popular plant growth promoters called plant growth promoting rhizobacteria (pgpr) are a group of beneficial bacteria that actively colonize the rhizosphere. pgpr plays an important role in enhancing root development that impacts on plant growth, crop yields and land fertility (wahyudi, 2009). plants with well-developed roots will efficiently absorb nutrients so that plants are not susceptible to pathogens. in addition, increased plant growth by pgpr can occur through one or more mechanisms related to the functional character of pgpr and conditions in the rhizosphere environment (rahni, 2012). pgpr is a biological mirorooganism that can increase plant growth and yield. the bacteria contained in pgpr can be classified based on their effects on plants and the way they interact with roots, pgpr can affect plants directly and indirectly (saharan & nehra, 2011). the role of pgpr in increasing plant growth and production has something to do with the ability to synthesize growth substances, which produces the hormone indol acetic acid (aia) (thakuria et al., 2003). bamboo is a plant that can grow in several regions in indonesia with a variety of functions and species. in indonesia, there are 60 species of bamboo plants from 200 species in southeast asia and can be found in areas that are free from standing water, from the lowlands to the mountains. the high adaptability of bamboo makes this plant grow well in almost every type of soil (widjaja et al. 1995). until now, there have been many reported potential antagonistic microbial origin of bamboo rhizosphere which has antagonistic power against soil borne pathogens through antagonistic mechanisms in the form of life competition, parasitism, antibiosis and induced systemic resistance. in addition to suppressing the development of pathogens, rhizosphere microbes can also increase plant growth through various mechanisms, including through the production of growth stimulant compounds such as phytohormone. in the soil many microbes have the ability to dissolve phosphate and potassium, tether n2 and produce phytohormone. these microbes can enhance plant growth by producing phytohormone compounds indole acetic acid (iaa) as nutrients for plants (aryantha et al. 2004; zahir et al. 2004). research on the existence and diversity of bamboo rhizosphere microbes has been carried out by several researchers before. according to sharma et al. (2010) in the rhizosphere of healthy bamboo plants found antagonistic fungi such as aspergillus, penicillium, trichoderma which are able to suppress the fusarium and phytophthora pathogens. research conducted by asniah et al. (2013) showed that the inoculation of the fungus paecilomyces sp and chaetomium globosum from bamboo rhizosphere into the nursery soil significantly affected the decrease in the index of clubroot disease and increased wet weight of broccoli plants. research conducted by tu et al. (2013) in china against rhizosphere 6 bamboo species showed that the total population of fungi and bacteria and microbial activity in bamboo rhizosphere soils was very high and had a positive effect on plant growth. the number of microorganisms contained in pgpr spiked bamboo rhizosphere makes it difficult to know what types of bacteria are the most dominant and most active in influencing plant growth. this will also have an impact on the difficulty of knowing and selecting good and bad bacteria contained in the pgpr, so that it greatly affects plants. therefore the method that can be used to determine the type of bacteria contained is to identify microorganisms with biochemical test treatments. according to rahayu & gumilar (2017), bacterial biochemistry test is a method or treatment that is carried out to identify and determine a pure bacterial culture that is isolated through its physiological properties. international journal of applied biology, 4(2), 2020 9 biochemical processes are closely related to cell metabolism, namely during chemical reactions carried out by cells that produce energy or that use energy for the synthesis of cell components and for cellular activities, such as movement. one method used in identifying microorganisms is gram test with 3% koh and gram staining test with methylene blue. the type of gram bacteria was tested using a 3% koh test and staining. testing with 3% koh only aims to determine the type of gram, while the staining method is used to determine the type of gram and the shape of bacterial cells. the method of testing with 3% koh is based (suslow et al. 1982) by mixing bacterial isolates with 3% koh on a sterile glass preparation. bacteria can be classified into aerobes and anaerobes. the main difference between the two is the fact that aerobic bacteria need oxygen to stay alive, while anaerobic bacteria do not depend on oxygen for metabolic processes and survival. whereas aerobes can develop in habitats that have abundant oxygen, anaerobes can die in the presence of oxygen. these types of bacteria do have the advantage of growing areas of the body not exposed to oxygen, and they can be virulent pathogens. the difference in capacity to utilize oxygen between aerobes and anaerobes is important in the treatment of bodily infections (lay, 1994) identification and determination of a pure bacterial culture obtained from the results of isolation can be done by observing the morphological characteristics of the colony, the morphology of the bacterial cell, testing its physiological and biochemical properties. in addition, identification can also be done by decomposing the pathogenicity and serology. the growth of bacteria in nature is influenced by several external factors such as susbtrat, growth, ph, temperature, and chemicals. the bacteria that appear can have the same morphology, but the nutritional requirements and ecological requirements are different. for clear observations of bacterial morphology, the body needs to be filled with color paint, this coloring is called arterial painting (hadioetomo, 1990). the gram method with a 3% koh test is a good method of identifying bacteria in determining the dominant type of active bacteria that is characterized by mucus. generally it is quite difficult and confusing to determine whether mucus is present or not even though some cultures react slowly. if the bacteria are mixed too little it will cause errors in the test and allow not react, so in this test the use of a dark color is very good. determination of gram properties with 3% koh has the same results with gram staining test. tests of 3% koh in bacteria indicate that gram (+) bacteria have thick cell walls and thin fat while gram (-) fat is thick and thin-walled cells that are in the periplasma chamber. koh will attack this fat (lipid bilayer) and break down gram (-) cells. the breakdown of cells releases genetic material (dna) which is an abundant substance in bacterial cells. dna molecules are very long sticky strings (like mucus, sap or can mean sticky) which gives results like mucus when removed with an inoculum needle (edwin, 2011). gram staining or gram method is an empirical method to distinguish bacterial species into two large groups namely gram positive and gram negative bacteria. the difference in classification between the two types of bacteria is mainly based on differences in the structure, chemical properties, and physical cell walls of the bacteria (qiqi, 2008). seeing and observing bacteria in a state of life is very difficult, because in addition to bacteria they are colorless also transparent and very small. in addition, living bacteria will contrast with water, where the bacterial cells are suspended. meanwhile, to overcome this problem, a bacterial cell staining technique was developed, so that cells can be seen clearly and easily observed. therefore this bacterial cell staining technique is one of the most important international journal of applied biology, 4(2), 2020 10 ways in microbiological studies. the basic principle of this coloring is the existence of ionic bonds between cellular components of bacteria with active compounds from dyes called chromogens. ion bonds occur because of the electric charge in both cellular components and in dyes. based on the presence of this charge, it can be distinguished from acidic dyes and basic dyes. the gram staining technique can produce red and purple colors, gramnegative bacteria are characterized by red staining while the positive ones are purple (levine, 2000). gram staining is a type of multilevel coloring, so there are stages in the process. gram staining system is carried out based on the stages that have been determined in the use of dyes or bleach. briefly, in this process the spread of bacteria on a fixed fixation sequence is subjected to a purple crystal solution, lugol liquid, alcohol solution (pale material) and safranin or some other suitable coloring agent. in this process aquades are also used in between several colors (suriawiria, 2005). bacteria stained by this method are divided into two groups, namely gram positive bacteria and gram negative bacteria. gram-positive bacteria will retain the violet crystalline dye and will therefore appear dark purple under a microscope. the gram-negative bacteria will lose violet crystalline dyes after washing with alcohol, and when given a coloring agent that is equal to the water coloring agent fuchsin or safranin will appear red. this color difference is caused by differences in the chemical structure of the cell wall (qiqi, 2008). characteristics of gram positive bacteria is a simple cell wall structure, composed of peptidoglycan without a layer of lipopolysaccharide. if given a gram naming, gram-positive bacteria will be purple. members of many gram-positive bacteria that cause disease in humans, for example streptococcus pneumoniae which causes pneumonia (pneumonia). many gram-positive bacteria that produce toxins, such as clostridium botulinum. the toxin produced by the bacteria c. botulinum is very deadly, one gram of toxin can kill more than one million people. besides being able to cause disease and produce toxins, gram-positive bacteria can also produce beneficial ingredients. for example, antibiotics are produced by bacteria from the actinomycetes group. antibiotics kill other gram-positive bacteria by preventing them from forming proteins. proteobacteria are the largest phyla in kingdom eubacteria. all proteobacteria are gram-negative bacteria. but has a variety of shapes (stem, round, and spiral). most move with flagella (strober, 2001). gram-positive bacteria are bacteria that maintain methyl purple dye during the gram staining process. this type of bacteria will be blue or purple under a microscope, while gram-negative bacteria will be red or pink. the difference in classification between the two types of bacteria is mainly based on differences in the structure of bacterial cell walls (qiqi, 2008). gram-negative bacteria are bacteria that do not maintain the methyl purple dye in the gram staining method. gram-positive bacteria will retain a dark purple color after washing with alcohol, while gram-negative bacteria do not. in the gram staining test, a lead dye (counterstain) is added after methyl purple, which makes all gram-negative bacteria turn red or pink. this test is useful for classifying these two types of bacteria based on differences in the structure of their cell walls. many species of gram-negative bacteria are pathogenic, which means they are harmful to host organisms. the nature of this pathogen is generally associated with certain components of the gram negative cell wall, especially the lipopolysaccharide layer (also known as lps or endotoxin) (qiqi, 2008). international journal of applied biology, 4(2), 2020 11 this study aims to determine the use of the 3% gram koh test method and gram staining test in identifying plant growth promoting rhizobacteria bacteria from thorny bamboo rhizosphere (bambusa blumeana). materials and methods this test was conducted at the biofertilizer and potential microbial laboratory, ministry of agriculture, faculty of agriculture, hasanuddin university, makassar. this test takes place from october to december 2019. tools used were analytical scales, mortars, test tubes, test racks, petri dishes, measuring cups, erlenmeyers, ose needles, drop pipettes, micro pipettes, matches, apatulas, autoclaves, ovens, hotplate, vortex, microscope, glass preparation, laminar air flow, storage box, camera and writing stationery. the materials used are plant growth promoting rhizobacteria solution from thorny bamboo rhizosphere (bambusa blumeana), koh 3%, methylen blue, lugol, 70% alcohol, 96% alcohol, water, aquades, nutrient brooth (nb) 8 grams, agar 15 gram, bunsen, tissue, plastic wrap, aluminum foil paper, paper, label paper. the steps for implementing the test are as follows: 1. making nutrient agar (na) media the making of na media was done by weighing 8 grams of nutrient brooth and 20 grams of agar on the analytical balance. furthermore, the weighed material is put into a 1000 ml size erlenmeyer and 500 ml of distilled water is then stirred until the material is dissolved. then the erlenmeyer is heated using a hotplate while stirring and add distilled water little by little until the volume of the solution reaches 1000 ml. when the solution is homogeneous, the erlenmeyer is covered with aluminum foil and then put into an autoclave to be sterilized wet for 15 minutes. 2. media pouring before starting the pouring of the media, first sterilize the laminar air flow by turning on the lights and spraying 70% alcohol to all parts of the laminar air flow and then cleaning it using a tissue. furthermore, laminar air flow is covered with black plastic and the uv lamp is turned on for 30 minutes. after the uv sterilization process is complete, the laminar air flow blower is turned on and put all the tools and materials that will be used in the pouring media. before inserting tools and materials into laminar air flow spraying is done first with 70% alcohol. turn on bunsen with matches and open the na medium which has been sterilized wet and has been at a moderate temperature. next, the na media was poured over a 15-20 ml petri dish, then covered and glued with plastic wrap and labeled. the media is stored in a storage box and allowed to harden. 3. planting bacteria bacteria planting is carried out again in laminar air flow, which is done by two methods, namely the scatter method and the method of scratching. each method is made in 5 petri dishes each. material in the form of pgh rhizosphere bamboo solution that has been made and na media is put into laminar air flow after spraying with 70% alcohol. the spread method is carried out by taking 1 ml of pgpr solution using a micro pipette and then spreading it over na media and flattened using a spatula, while for the scratching method is done by taking an ose needle then sterilizing after incubation in bunsen then dipping it in the pgpr solution and polishing it. zigzags on the surface of the na media in a petri dish. after the bacterial planting is carried out, the cup is stored in a storage box and physical checking and observation of bacterial growth every day. international journal of applied biology, 4(2), 2020 12 4. bacterial identification bacterial identification is carried out in 2 gram reaction methods, namely: a. uji a. gram reaction test using 3% koh testing with 3% koh only aims to find out the type of gram, the method of testing with 3% koh is by mixing bacterial isolates with 3% koh on a sterile glass preparation (suslow et al., 1982). this bacterial identification method is done by placing 1 drop of 3% koh on a glass preparation then the bacteria that have grown from the scatter method and the method of scraping are taken with an ose needle and rubbed in a 3% koh solution and observed. the category of gram negative bacteria is obtained if it produces mucus (positive reaction) and the category of gram positive bacteria if it does not produce mucus (negative reaction). b. bacteria gram reaction test using simple staining gram staining is a physiological characterization of bacteria carried out to distinguish gram positive and gram negative bacteria. the coloring method is carried out using methylene blue. first the glass preparation is washed using 70% alcohol and then sterilized after incubation in bunsen. next take an ose needle then sterilized afterglow then dipped in 96% alcohol. take one of the aquades and put it on the glass preparation. burning the needle back on the bunsen then aired and used to take bacteria then flattened on the glass preparation and let it dry. drop 1-2 drops of methylene blue on the glass preparation and leave to dry. next wash the glass preparations with distilled water and then drop 1-2 drops of lugol solution on the glass preparations and leave for 1 minute then rinse with 70% alcohol for 30 seconds then dry. after drying, put the deglass on the glass preparation then observe it under a microscope with magnification (40 / 0.65) or (160 / 0.17). if there is a change in the color of the bacteria to pink or purple, the bacteria are gram-positive and belong to the pseudomonas group and if there is a blue discoloration, the bacteria are gram-negative and are the bacillus group (meynell & meynell 1970). results and discussion please testing the gram reaction of pgpr bacteria from the thorny bamboo rhizosphere using 3% koh is as follows. table 1. gram reaction test results using 3% koh on plant growth promoting rhizobacteria of thorny bamboo rhizosphere with scatter and scratch methods methods gram reaction sample 1 sample 2 sample 3 sample 4 sample 5 spread + + scratch + + + + + note: plus signs (+) and minus (-) are given if there is or no mucus during testing international journal of applied biology, 4(2), 2020 13 figure 1. observation of bacterial gram test using 3% koh (scratch method) figure 2. observation of bacterial gram test using 3% koh (scatter method) the gram reaction test results (table 1) show that the prickly bamboo rhizosphere pgpr in the scatter method and the scratch method on average produce mucus when testing the gram reaction using 3% koh, but in the scratch method testing all have slime which means that it is entirely gram negative compared with the scatter method that only 2 samples were slimy. the formation of the mucus is due to the breakdown of bacterial cell walls due to being in high alkaline water when given 3% koh. this is in accordance with the statement of soekirno (2008), which states that the gram reaction can be confirmed by the solubility test of potassium hydroxide (cori). by taking one full loop of bacterial culture that is actively growing and mixed with a drop of 3% koh solution on a clean slide glass then stirring until a flat suspension is obtained. if when the nose is removed and mucous threads are seen, then the bacteria are gram-negative, but if a watery suspension is produced and no mucous threads appear after the ose is moved repeatedly, the bacterial culture is grampositive. this is also in line with the statement of suslow et al. (1982), which stated that gram negative bacteria would form mucus when tested using 3% koh due to rupture of the bacterial cell wall due to being in a high alkaline solution (koh 3%). while gram-positive bacteria do not form mucus because gram-positive cell walls have a thick layer of peptidoglycan. based on the results of gram bacteria test using 3% koh, on average pgpr spiked bamboo rhizosphere bacteria have mucus which means it is gram negative, wherein gram negative bacteria consist of enterobactericeae (escherichia coli, salmonella, shigella), pseudomonas, etc. and there are also pgh rhizosphere bamboo bacteria that do not have mucus, which means they are gram-positive, whereas gram-positive bacteria include bacillus, enterococcus, etc. this is in line with the statement of podile and kishore (2006), which states that some genus of rhizobacteria that are pgpr are pseudomonas, enterobacter, bacillus, azospirilum, azotobacter, burkholderia and serratia. so it can be stated that the bacteria in pgpr rhizosphere of bamboo spines are active and play a role in the process of plant growth. testing the gram reaction of pgpr bacteria from the thorny bamboo rhizosphere using a simple coloring is as follows. international journal of applied biology, 4(2), 2020 14 figure 3. microscopic observations of bacteria under a microscope with magnification (40 / 0.65) after a simple staining method bacterial staining generally aims to facilitate the observation of bacterial morphology with the aid of a microscope. bacteria are generally colorless and almost invisible because they lack contrast with the water where they might be located. staining is needed to see bacteria very clearly both for intracellular observations and overall morphology. simple coloring is coloring that uses a single coloring. single dyes that are usually used in simple coloring are methylene blue, basic fuchsin, and crystal violet. all of these dyes can work international journal of applied biology, 4(2), 2020 15 well on bacteria because they are alkaline and alkaline (the chromophore component is positively charged), while the bacterial cytoplasm is basophilic (likes to bases) so there is a tensile force between the chromophore component in the dye and bacterial cells, which causes the bacteria to absorb coloring well. but what is used in this test is coloring with methylene blue. microscopic observations (figure 3) show that the plant growth promoting rhizobacteria of thorny bamboo rhizosphere on staining with methylen blue and lugol produces blue for the genus pseudomonas and pink / purple for the genus bacillus. this is supported by the opinion of meynell and meynell (1970), which states that if there is a change in the color of the bacteria to pink / purple, the bacteria are gram negative and are a group of pseudomonas and if there is a blue color change, the bacteria are gram positive and are the bacillus group. in accordance with the statement of the jambi agricultural training agency (2010), which states that the content of the pgpr is dominated by pseudomonas fluorescens and bacillus polymixa. benefit claims obtained are as bioprotectan, biofertilizer and as biostimulant. in line with the research of susanti et al. (2015), which stated that several genera of bacteria were obtained from the rhizosphere of bamboo plants, including: genera bacillus, pseudomonas, enterobacter. conclusions based on the research results obtained, it can be concluded that the use of the 3% gram koh test method in identifying the rhizobacteria plant growth promoting rhizobacteria bacteria from thorny bamboo rhizosphere (bambusa blumeana) in the scratch method testing all samples that have slime while the scatter method has 2 samples that have slime so that the 3% koh test method has an optimum effect in detecting microorganisms. then the use of gram staining test method in identifying plant growth promoting rhizobacteria bacteria from thorny bamboo rhizosphere (bambusa blumeana) on staining with methylen blue and logol produces a blue color for the genus pseudomonas which means gram positive and pink / purple for the genus bacillus which means gram positive so that the gram staining test method also has an optimum effect 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rhizosphere of rice grown in acidic soils of assam. journal of current sci. 86 (2): 978-985. tu, z., chen, l., yu, x. & zheng, y. 2013. effect of bamboo plantation on rhizosphere soil enzyme and microbial activities in coastal ecosystem. j. food agric environ. 11 (03): 2333-2338. wahyudi, at. 2009. rhizobacteria plant growth boosters: prospects as biostimulator and biocontrol agents. tangerang (id): nano indonesia. widjaja, ea., sastrapradja, s., prawiroatmodjo, s. & soenarko, s. 1995. types of bamboo. jakarta (id): library. zahir, za. khalid, a. & arshad, m. 2004. creening plant growth promoting rhizobacteria for improving growth and yield of wheat. j. appl. microbiol. 96: 473-480. http://repository.unej.ac.id/ http://repository.unej.ac.id/ contact : bishnu hari adhikary adhikary_bishnu@yahoo.com 85 abstract grain yield production of maize is greatly affected by varieties and fertilizer levels. this study was conducted to determine the effects of different rates of fertilizers (nitrogen, phosphorus, potassium fertilizers and farmyard manures) on grain yield and yield attributing traits of different maize varieties during winter seasons of 2009/10 and 2010/011 at the research farm of national maize research program, rampur, chitwan, nepal. six levels of fertilizers [control (zero fertilizer), fym @ 10 t/ha, fym@ 10 t/ha plus 60:30 20 kg npk/ha, fym@ 10 t/ha plus 120: 60: 40 kg npk/ha, fym@ 10 t/ha plus 180: 90: 60 kg npk/ha, and 120: 60: 40 kg npk/ha] and four maize varieties (rampur composite, manakamana-4, across9942 × across 9944 and s99tlyq-b) were evaluated in randomized complete block design with three replications. the results showed that grain yield was non-significant for maize genotypes but the fertilizers levels were highly significant for grain yield. rampur composite produced the highest grain yield (5195 kg/ha), followed by manakamana-4 (5074 kg/ha), across9942 × across9944 (5052 kg/ha) and s99tlyq-b (4789 kg/ha) with the application of npk 180: 90: 60 kg/ha plus fym 10 t/ha. this information is useful in generating suitable fertilization packages for obtaining higher grain yield of maize varieties. issn : 2580-2410 eissn : 2580-2119 productivity of winter maize as affected by varieties and fertilizer levels bishnu hari adhikary, bandhu raj baral, & jiban shrestha national maize research program, rampur, chitwan, nepal. introduction maize (zea mays l.) has the highest productivity per unit area as compared to other cereal crops. it ranked third among the cereal crops in the world after wheat and rice. in nepal, it is the second most important staple food crop in terms of both area and production after rice but it is the first staple crop for hills. in nepal, maize has been grown over an area of 954158 hectares in 2017/18 with a production of 2555847 tons and a productivity of 2.67 t/ha (moald, 2018). the production of maize is low as compared to other countries. the production can be improved or increased through adequate nutrient management practices. maize being the heavy feeder crop, a balanced dose of organic and inorganic application of fertilizer is needed for increased productivity. fertilizer management is crucial for maize cultivation (baral et al., 2015). manures and fertilizers both play an important role in maize cultivation. n is usually applied in 3 equal splits at sowing, knee-high stage and tasseling stage. als/ha of grain yields and likewise, it can be reduced or open access international journal of applied biology keyword fertilizer and manures, maize genotypes, grain yield, yield attributing traits. article history received 30 march 2020 accepted 15 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 86 increased as per its expected yield. phosphorus (p) is the next most important plant nutrient after n which is found difficult in most soils. it has a beneficial effect on root growth and plant health. this nutrient should be applied initially at the early stage because of its low solubility in water. it should be applied in a moist zone to be transformed quickly for early absorption by the plant. the dose of p should be balanced with the dose of n applied. potassium is considered to be the 3rd most essential fertilizer element, it is not found deficient in most of the soils. it is essential for the vigorous growth of the plant and for so many other metabolic activities.k application through fertilizers has been responding satisfactorily (regmi et al., 2002). maize being a high nutrient mining crop it needs a higher amount of npk for its economic production. farmers applying 20-25 t/ha of compost/fym (manures) are not sufficient to replenish the harvested nutrients and hence need a sufficient amount of mineral fertilizer addition with heavy manure application (joshy, 1997). adhikary et al. (2001) reported that the highest maize grain yield (4.65 t/ha) could be obtained when the crop is fertilized by 20 t of compost plus 100: 75: 40 kg/ha of n, p2o5, and k2o in the acidic soils of malepatan, pokhara. adhikary and ranabhat (adhikary and ranabhat, 2004) studied the economics of manure and fertilizer application on maize production and concluded that most economic dose of fertilizer was 100: 75: 40 kg n, p2o5 and k2o/ha from inorganic sources and 20 t/ha of compost that contained 280 kg n, 184 kg p2o5 and 216 kg k2o. adhikary et al. (2007) studied the effect of fertilizer and agricultural lime on grain yield of different maize genotypes in the western hills of nepal and reported that improved maize variety (manakamana-1) did not differ in grain production with the local variety when supplied with fertilizers at 60: 30: 30 kg n, p2o5 and k2o and 4 t/ha of agri-lime. adhikary (2008) also studied the effects of nitrogen on maize inbred (nml-1) and reported that increased seed yield (2.85 t/ha) was obtained with this variety when supplied with 180 kg n and crop planted at the density of 66,666 plants/ha and crop fertilized along with the recommended dose of p and k fertilizers. series of experiments were conducted to evaluate the effects of fertilizers on different maize genotypes during the years 2009 and 2010. the results revealed that the highest grain yield of 6.28 t/ha was produced by the s99tlyq-b when the crop was fertilized with 120: 60: 40 kg n, p2o5 and k2o/ha and 10 t/ha of compost (narc, 2010). hence, balanced dose of fertilizers is needed to increase the crop yield of maize in acid soils. the number of fertilizers to be applied in maize depends largely on the genotypic makeup of plants. the objective of these experiments were to study the response of fertilizer nutrients at different levels on the different maize genotypes in the soil condition of rampur, chitwan, nepal. materials and methods experimental site the site was located in central nepal at 27° 40’ n latitude and 84° 19’ e longitude with an elevation of 228 m above mean sea level and had a subtropical climate (nmrp, 2011). maize was planted on sandy silt loam, acidic soil (ph 5.54). fertilizer was applied in the form of urea, di-ammonium phosphate (dap), and murate of potash (mop). entire dose of dap and mop were applied at the time of sowing while half of the urea was first top-dressed at the knee-high stage and second top-dressed at tasseling stage. the average data derived from both years on maximum temperature ranged from 21.95 (january) to 36.35 0c (april), the minimum temperature varied from 9.4 (january) to 24.65 0c (october). there is no rainfall in international journal of applied biology, 4(1), 2020 87 november and january, minimum rainfall (1.1 mm) occurred in january and maximum rainfall occurred in 99.35 mm (april). similarly, average data on relative humidity showed that minimum humidity (76.8%) occurred in april and maximum relative humidity (99%) occurred in december. the details of weather data of individual year were shown in table 2. table 1. monthly mean weather condition during crop growing season (october-april) in 2009/10 and 2010/11 winter seasons at rampur, chitwan, nepal month maximum temperature (0c) minimum temperature (0c) rainfall (mm) relative humidity (%) 2009/10 2010/11 2009/10 2010/11 2009/10 2010/11 2009/10 2010/11 october 31.4 31.4 26.5 22.8 101 48.6 97.0 97.5 november 27.1 27.1 21.6 17.0 0.0 0.0 99.0 98.8 december 24.0 24.0 16.0 9.1 2.2 0.0 99.0 99.0 january 20.0 23.9 10.3 8.5 0.0 0.0 94.6 100.5 february 25.4 26.1 11.9 15.1 0.0 34.9 89.5 96.3 march 33.1 31.1 19.1 18.9 0.0 34.4 82.2 83.2 april 38.1 34.6 23.3 19.6 165 33.7 75.4 78.2 *source: (nmrp, 2011) experimental design, treatments and crop management this experiment was conducted at the farm of national maize research program, rampur, chitwan, nepal during the winter season of the year 2009/10 and 2010/11.the crop was planted in october and harvested in april. twenty four treatment combinations consisting of six levels of fertilization and four maize genotypes were replicated three times and laid out in a randomized complete block design. the details of the treatment combinations are given in the following table 1. row to row spacing 75 × 25 cm was maintained. the net harvested area was 7.2 m2. the gross plot size was 12 m2. table 2. the details of the treatments used in experiment in 2009/10 and 2010/11 winter seasons at rampur, chitwan, nepal genotypes fertilizer rates v1= rampur composite f1=control (zero fertilizer) v2= manakamana-4 f2= fym @ 10 t/ha v3=across9942 × across9944 f3= fym@ 10 t/ha plus 60:30 20 kg npk/ha v4= s99tlyq-b f4=fym@ 10 t/ha plus 120: 60: 40 kg npk/ha international journal of applied biology, 4(1), 2020 88 f5=fym@ 10 t/ha plus 180: 90: 60 kg npk/ha f6= 120: 60: 40 kg npk/ha data recorded observations were taken on plant height, ear height, cob length, no. of kernel rows per cob, no. of kernels per rows, and grain yield. plant height and ear height were recorded at just near to harvesting and the rest of the data were recorded after harvesting. at maturity, central two rows from each the plot were separately harvested and the fresh ear weight was measured in each plot. grain yield (kg/ha) at 15% moisture content was calculated using fresh ear weight with the help of the below formula given by carangal et al. (1971), shrestha et al. (2019) and upreti et al. (2020). where, fwt : fresh weight of ear in kg per plot at harvest hmp : grain moisture percentage at harvest dmp : desired moisture percentage, i.e. 15% npa : net harvest plot area, m2 scf : shelling coefficient, i.e. 0.8 statistical analysis data were statistically analyzed. analysis of variance for all data was analyzed using the mstat computer program. the significant differences between genotypes were determined using the least significant difference (lsd) test at 1% or 5% level of significance (gomez and gomez 1984; shrestha, 2019). results and discussion the interaction between different fertilizer levels and varieties on grain yield showed that the highest grain yield (5195 kg/ha) was obtained in rampur composite followed by manakamana-4 (5074 kg/ha) and across9942 × across9944 (5052 kg/ha) under treatment of application of npk 180: 90: 60 kg/ha plus fym 10 t/ha. similarly, s99tlyq-b produced the highest grain yield (4789 kg/ha) under the same level of fertilization (figure 1). international journal of applied biology, 4(1), 2020 89 figure 1. schematic diagram for interaction effect between maize genotypes and fertilizer levels. the increasing amount of nitrogen and phosphorus fertilizer application increased yield attributing traits in maize. the highest yield attributing traits namely cob length, no. of kernel rows per cob and no. of kernels per kernel rows were found under the level of fertilization (180:90:60 kg npk plus 10 t fym/ha) in rampur composite, manakamana-4, across9942 × across9944. gokmen et al. (2001) and wajid et al. (2007) reported that 1000 grain weight increased with increasing n levels. gungula et al. (2007) and dawadi (2009) observed a number of kernels/ear and number kernel rows/cob increased with increasing nitrogen levels. the increase in 1000-grain weight with increasing p levels might be due to improvement in the source-sink relationship. these results are supported by the findings of ahmad (1989) and toor (1990). table 3. effect of different level of manures and fertilizers on different maize genotypes in 2009/10 and 2010/11 winter seasons at rampur, chitwan, nepal treatme nts cob length (cm) kernel rows per cob (no.) kernel per kernel row (no.) grain yield (kg/ha) 2009/10 2010/11 2009/10 2010/11 2009/10 2010/11 2009/ 10 2010/ 11 v1f1 11.4 9.66 10.7 10.4 22.6 14.86 2860 1443 v1f2 12.2 11.6 12 11.93 24.2 17.53 3740 2180 v1f3 13.8 14.13 12.8 13.6 31.1 28.33 4960 2927 v1f4 14.1 14.46 13 13.86 30.5 30.4 5840 3863 v1f5 14.2 15.13 13.1 14 31.9 30.8 6260 4130 v1f6 13.8 13.86 12.6 13.73 29.1 29.46 5630 3733 v2f1 10.4 9.06 11.5 10 21.1 15.53 2240 1677 v2f2 11.7 11.6 13.3 11.7 25.3 21.86 3830 2010 v2f3 13.8 14.6 15.2 13.4 28.5 30.2 5340 3333 v2f4 14.3 14.73 15.3 13.86 30.2 31 5960 3837 f6f5f4f3f2f1 5000 4000 3000 2000 v4v3v2v1 5000 4000 3000 2000 variety v3 v4 v1 v2 fertilizers f3 f4 f5 f6 f1 f2 international journal of applied biology, 4(1), 2020 90 v2f5 14.9 14.86 15.5 14.26 31.7 31.73 5990 4157 v2f6 14.2 13 15.1 12.93 30.7 27.2 5280 3240 v3f1 9.7 8.33 11.7 9.2 19.7 13.8 2300 1163 v3f2 11.6 10.8 13.7 11.53 25.5 21.13 3360 1953 v3f3 12.6 13.2 15.7 12.03 29.3 30.4 5120 3033 v3f4 13.3 13.66 15.7 13.33 28.8 29 6190 3630 v3f5 13.4 13.93 16.3 13.43 30.9 29.53 6280 3823 v3f6 13.2 13.2 15.1 13.16 29.1 28.23 5770 3290 v4f1 9.9 7.2 11.6 9.33 20.7 12.66 2750 1460 v4f2 11.7 10.2 13.5 10.13 25 20.06 3920 2083 v4f3 14.3 13.53 14.9 12.26 28.3 25.4 4990 2890 v4f4 14.9 13.73 15.2 12.13 32.5 29.73 5740 3380 v4f5 13.9 13.13 14.9 12.53 30.5 26.06 5760 3817 v4f6 14.1 13.4 14.9 12.03 29.1 28.6 5280 3140 mean 13 12.54 13.9 12.28 27.8 25.14 4810 2925 cv(%) 4.62 9.36 7.52 7.99 6.25 14.63 15.32 17.44 f-test (v) ** ** ** ** ns ns ns ns (f) ** ** ** ** ** ** ** ** (v × f) ns ns ns ns ns ns ns ns lsd (0.05) 0.402 0.788 0.701 0.658 1.428 2.468 605 419 **highly significant at 0.01 level, *significant at 0.05 level and ns: non-significant. in 2009/10 and 2010/11, the effect of genotypes was observed to be non-significant whereas the effect of fertilizers was found to be highly significant. the increasing amount of fertilizer up to 180 n kg/ha, phosphorus 90 kg/ha, and potassium 60 kg/ha increased the grain yield in both years. singh et al. (2000) indicated that grain increased with the increase in nitrogen level from 0-200 kg/ha. khan et al. (2014) found an increasing amount of phosphorus increased the grain yield of maize at a constant level of 150 n kg/ha application; the maximum grain yield (5356 kg/ha) was recorded in jalal variety when it was fertilized with 150:100 n:p kg/ha. in 2009/10 the highest grain yield (6068 kg/ha) was obtained at the highest level of fertilization (180:90:60 npk kg/ha plus fym 10 t/ha). the variety rampur composite produced highest grain yield (4882 kg/ha) followed by across9942 × across 9944 (4837 kg/ha) and manakamana-4 (4773 kg/ha) similarly in 2010/11, grain yield was increased with an increased level of fertilization. the highest grain yield (3873 kg/ha) was obtained at the highest level of fertilization (180:90:60 npk kg/ha plus fym 10 t/ha). the variety rampur composite produced highest grain yield (3046 kg/ha) followed by manakamana-4 (3042 kg/ha) and across9942 × across 9944 (2816 kg/ha) (table 4). international journal of applied biology, 4(1), 2020 91 table 4. grain yield under different fertilizer levels and genotypes in 2009/10 and 2010/11 winter seasons at rampur, chitwan, nepal treatments grain yield (kg/ha) fertilizer levels 2009/10 2010/11 f1 (control) 2538 1436 f2 (fym 10 t /ha) 3713 2057 f3 (60:30:20 npk plus fym 10t/ha) 5103 3046 f4 (120:60:40 npk plus fym 10t/ha) 5938 3787 f5 (180:90:60 npk kg/ha plus fym 10 t/ha) 6068 3873 f6 (120:60:40 npk kg/ha) 5490 3351 cv(%) 15.32 17.4 f-test ** ** lsd (0.05) 221.5 419.1 genotypes v1 (rampur composite) 4882 3046 v2 (manakamana-4) 4773 3042 v3 (across9942 × across9944) 4837 2816 v4 (s99tlyq-b) 4740 2795 cv(%) 15.32 17.4 f-test ns ns lsd (0.05) 605 342.2 **highly significant at 0.01 level, *significant at 0.05 level and ns, non-significant. conclusions the study showed that maize genotypes namely rampur composite, manakamana-4, across9942 × across 9944 and s99tlyq-b produced higher grain yield of 5195, 5074, 5052 and 4789 kg/ha respectively with the application of npk @ 180: 90: 60 kg/ha plus fym 10 t/ha. similarly, produced the highest grain yield (kg/ha). the yield attributing traits namely cob length, no. of kernel rows per cob and no. of kernels per kernel rows were found higher at the fertilization rate of 180: 90: 60 kg npk/ha plus fym 10 t/ha so such fertilization rate is suitable for maize varieties at rampur, chitwan, nepal during winter seasons. acknowledgements the authors would also like to thank national maize research program rampur (nmrp), rampur, chitwan, nepal for the provision of research materials financial supports for conducting this research. references adhikary, b.h. 2008. effect of nitrogen on inbred maize seed production planted at varying densities in the acidic soil at rampur, chitwan. in: proc. of the abstracts. the fifth national conference on science and technology. nepal academy of science and technology (nast), nov. 10-12, 2008. kathmandu, nepal, p. 19. international journal of applied biology, 4(1), 2020 92 adhikary, b.h., gauli, r.c. & bc, b.b. 2001. effects of manures and fertilizers on the grain production of maize in rotation with cowpea in acid soils of malepatan, pokhara. in: proc. of an international maize symposium. sustainable maize production systems for nepal, held december 3-5, 2001, kathmandu, nepal, pp. 160-162. adhikary, b.h., pandey, b.r. & neupane, d.d. 2007. increased productivity of maize genotypes through the use of inorganic fertilizers and agricultural lime in the western hills of nepal. in: proc. of the 25th national summer crops research workshop, held 21-23 june, 2007. nari, narc, khumaltar, lalitpur, nepal, pp. 225-230. adhikary, b.h. & ranabhat, d.b. 2004. an economic perspective of manures and fertilizer application on maize. pp.. in: proc. of the 24th national summer crops research workshop on maize research and production in nepal, organized by nepal agriculture research council (narc) and nmrp, held june 28-30, 2004, kathmandu, nepal, pp. 287-290. ahmad, i. 1989.the effect of phosphorus application in different proportions with nitrogen on the growth and yield of maize. m.sc. 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(hons.) agri. thesis, dept. of agron; university of agriculture, faisalabad– pakistan. upreti, j., acharya, p., upadhyaya, j. & shrestha, j. 2020. heterosis in maize hybrids at farmer’s field in dang district of nepal. fundamental and applied agriculture, 5(2): 188–193. wajid, a., ghaffar, a., maqsood, m., hussain, k. & nasim, w. 2007. yield response of maize hybrids to varying nitrogen rates. pak. j. agri. sci., 44: 217-220. contact : ngangnang ghislain roméo ngaghirom1@yahoo.fr 85 abstract rickettsial bacteria are important tick-transmitted microorganisms causing disease and death in cattle, sheep, goats and dogs in the area where tick vectors are found, becoming a major problem for improvement of animal production in the endemic areas. the study carried out in the western highlands of cameroon was aimed at highlighting anaplasma and ehrlichia species in apparently healthy cattle. a total number of 162 blood samples were collected from cattle and screened via nested-pcr based reverse line blot hybridization (rlb) assay for detection of rickettsial bacteria. four species of these microorganisms were identified with an overall prevalence of 44.44%, anaplasma marginale (41.35%) being the most prevalent species followed by anaplasma sp. ‘omatjenne’ (15.43%), anaplasma centrale (8.64%) and ehrlichia ruminantium (3.08%). single infection (24.69%) was more frequent among the four types of mix infection observed with a significant difference. parasite association was most found between a. marginale + anaplasma sp. ‘omatjenne’ (11.11%). female cattle (44.79%) were more infected than males3.93%) but without significant difference while, yearling cattle (50%) were statistically more infected than adults (44.07%). the high prevalence and diversity of rickettsial organisms identified is evidence that disease and their vectors, the amblyomma and rhipicephalus (formerly boophilus) ticks might be widespread in the western highlands of cameroon. however, these findings with veterinary significance suggest the dire need for further research on the presence of other vectors apart from amblyomma sp. and rhipicephalus sp. in cameroon. issn : 2580-2410 eissn : 2580-2119 molecular characterization of anaplasma and ehrlichia microorganisms in bovine populations of the western highland agro-ecological zone of cameroon ngangnang ghislain roméo 1*, aktas münir 2, ulucesme mehmet can 2, keptcheu tchankwe désiré 1 , fonteh anyangwe florence3, vincent khan payne1 1 department of animal biology, faculty of science, university of dschang, dschang, cameroon. 2 department of parasitology, faculty of veterinary medicine, university of firät, elazig, turkey. 3 department of animal production, faculty of agronomy and agricultural sciences, university of dschang, dschang, cameroon. open access international journal of applied biology keyword anaplasma; ehrlichia; rickettsial bacteria; molecular characterization; rlb; western highlands of cameroon article history received 11 november 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 86 introduction rickettsial diseases of economic importance are ehrlichiosis and anaplasmosis, a tickborne disease caused by obligate intracellular bacteria in the genera ehrlichia and anaplasma respectively. they are emerging tick-borne pathogens in humans and other wild or domesticated animals worldwide. infections caused by these pathogens are deadly if left untreated (iweriebor et al., 2017). these organisms are widespread in nature and are usually maintained in cycles between ticks and reservoir hosts, which can sometimes remain infected for long periods. for many years, ehrlichia and anaplasma species have been known to cause illness in pets and livestock where the consequences of exposure vary from asymptomatic infections to severe, potentially fatal illness. throughout the tropics, an estimated 600 million cattle are exposed to anaplasmosis and babesiosis. in the 1970s, mccallon, (1973) estimated that the disease caused annual losses of over 300 million us dollars to the american cattle industry. in 1989, over a million cattle in eleven countries of eastern, central and southern africa were estimated to have died of tick-borne diseases. the economic cost in livestock losses and funding for control and research programs was estimated at us$168 million that year (ilrad, 1991). furthermore, mukhebi et al., (1999) estimated that the national annual loss due to cowdriosis in zimbabwe could attain 5.6 million usd and more recently, tanzania was estimated to lose 47.3 million usd solely due to the direct costs of bovine anaplasmosis (kivaria, 2006; kenneil, 2015). the distribution of these diseases follows the presence of the vector amblyomma among them amblyomma variegatum is the most important species which is widely distributed in the sub-saharan africa including cameroon. the control of disease involves controlling the tick vector, establishing endemic stability, performing immunization by infection and treatment, and preventing the disease by regular administration of prophylactic antibiotics. most of these methods are subject to failure for various epidemiological reasons, and serious disease outbreaks could occur (dinkisa, 2018). a relative little information is available about these rickettsial bacteria in cameroon and need to be updated. in the current study, a reverse line blot assay (rlb) was performed in order to identify anaplasma and ehrlichia species circulating amongst cattle in the third agroecological zone of cameroon. materials and methods study area the region considered as the western highlands is the third agro-ecological zone (aez) of cameroon (irad, 2008). it comprises the two administrative regions of west and north west, due to their common biotic and abiotic characteristics. it lies between latitudes 5° and 7° north and longitude 9° and 11° east of the equator. with a size of 31,180 km2, they cover 1/16 of the total land area of the country. altitudes range from around 300 to 3 000 m above sea level. the climate of this region is the tropical humid type with two seasons, the dry and rainy seasons. rainfall varies between 1300-3000 mm with peaks occurring between mid-july and mid-september. the rainy season extends from mid-march to mid-november while the dry season runs from end of mid-november to mid-march. the maximum temperatures vary between 20 and 32°c. the dominant vegetation is residual savannah and the region is designated grassland because a greater proportion of the area is covered by grassland than forest. this region is characterized by a rapid population growth (128.5 inhabitants per km2), most of whom live in rural areas (67.8%) and depend on crop international journal of applied biology, 5(2), 2021 87 and livestock activities. it is the third major cattle producing area, with 500,000 zebu cattle, and one of the most important agricultural production zones of the country (irad, 2008; nchinda and mendi, 2008; jiotsa et al., 2016). collection of the samples between march 2019 and january 2021, one hundred and sixty-two (162) zebu cattle (bos indicus), mainly the local breed (aku, gudali and m’bororo) commonly found in the western highlands of cameroon were sampled according to their age and sex for blood sampling. this target population was in extensive management with no or adequate tick control program implemented. five ml of blood samples were collected from jugular or coccygeal vein of cattle into edta tubes, preferably potassium–ethylenediamine tetra-acetic acid (edta/k3) with a concentration of 1.27mg edta/k3 per ml of blood and into dried blood spot (dbs) specimen collection cards prepared for the purpose. simultaneous detection of rickettsial bacteria in the blood samples was done using nested-pcr based rlb hybridization assay in the laboratory of molecular parasitology, department of parasitology, faculty of veterinary medicine, university of firät, elazig, turkey. dna extraction and pcr dna was extracted by a commercial dna isolation kit (invitrogen corporation, carlsbad, ca, usa) following the manufacturer’s instructions. then, for the amplification of anaplasma/ehrlichia spp., a nested pcr was performed using two universal primer pairs. the primers ec9/ec12a were used for the first round pcr amplification of 1462 bp fragments of the 16s rrna gene of anaplasma/ehrlichia spp. the nested amplification, using the primers 16s8fe/bga1b, produced a 492–498 bp fragment in the hypervariable v1 region of the 16s rrna gene of the anaplasma/ehrlichia species. for the second amplification, one μl of first round pcr products was used as a dna template. to reduce non-specific amplification, a touchdown program was performed. touchdown pcr involves the use of an annealing temperature that is higher than the target optimum in early pcr cycles. rlb hybridization probes of catchall, genus and species-specific for anaplasma/ehrlichia were used with a range of 200–800 pmol/150μl concentration and contain n-terminal n(trifluoracetamidohexyl-cyanoethyl,n,n-diisopropyl phosphoramidite [tfa])-c6 amino linker in the study. the oligonucleotide probes were synthesised by the midland certified reagent (midland, texas, usa). preparation, hybridisation and stripping of the rlb membrane were performed as previously described with minor modifications (georges et al., 2001). table 1. oligonucleotide primers and probes used in this study primer sequence (5’-3’) reference ec9 taccttgttacgactt kawahara et al., 2006 ec12a tgatcctggctcagaacgaacg kawahara et al., 2006 16s8fe ggaattcagagttggatcm*tggytcag schouls et al., 1999 b-ga1b biotincgggatcccgagtttgccgggacttcttct schouls et al., 1999 probe modification (5’-3’) reference international journal of applied biology, 5(2), 2021 88 anaplasma/ehrlichia catch-all c6 amino-ggg gga aag att tat cgc ta bekker et al., 2002 a. marginale c6 amino-gac cgt ata cgc agc ttg bekker et al., 2002 a. centrale c6 amino tcg aac gga cca tac gc bekker et al., 2002 a. bovis c6 amino-gta gct tgc tat grg aac a bekker et al., 2002 e. ruminantium c6 amino-agt atc tgt tag tgg cag bekker et al., 2002 ehrlichia sp. ‘omatjenne’ c6 amino-cgg gtt ttt atc ata gct tgc bekker et al., 2002 a. phagocytophilum group c6 aminottg cta trr aga ata rtt agt gg bekker et al., 2002 a. phagocytophilum 1 c6 aminottgctataaagaataattagtgg schouls et al., 1999 a. phagocytophilum 3 c6 aminottgctatgaagaataattagtgg schouls et al., 1999 a. phagocytophilum 5 c6 aminottgctataaagaatagttagtgg schouls et al., 1999 a. phagocytophilum 7 c6 aminottgctatagagaatagttagtgg schouls et al., 1999 a. phagocytophilum a-he c6 amino-gctataaagaatagttagtgg schouls et al., 1999 a. phagocytophilum a-dhe c6 amino-gctatgaagaatagttagtg schouls et al., 1999 statistical analysis statistical calculations were performed using spss v. 23 software and chi-square tests was used to statistically compare different prevalence of infection. result a total number of 162 cattle blood samples were screened for detection of rickettsial organisms. seventy-two (72) were found positive for the presence of 16s rrna gene of anaplasma and ehrlichia species. we then identified after examination these blood samples of two genera of rickettsial bacteria such as anaplasma sp. and ehrlichia sp. (figure 1). international journal of applied biology, 5(2), 2021 89 figure 1. gel electrophoresis of pcr product of anaplasma and ehrlichia species the detection of 16s rrna gene of anaplasma and ehrlichia species is done by nested pcr using genus-specific primers 16s8fe/b-ga1b. m: 500 bp ladder; n1-n2: standard negative controls (n1, dna isolated from uninfected cow blood; n2, sterile deionized water); p1-p2: positive controls (p1, anaplasma marginale; p2, anaplasma phagocytophilum). lanes 1-8: positive field samples signalling anaplasma/ehrlichia catchall probe in the rlb. after confirmation of the presence of 18s rrna gene of anaplasma and ehrlichia species in the blood samples, rlb was performed to identify these parasites at the level of species and so, the following four of them were incriminated: anaplasma marginale, anaplasma centrale, anaplasma sp. ‘omatjenne’ and ehrlichia ruminantium (figure 2). figure 2. detection of anaplasma and ehrlichia species by rlb oligonucleotide probes were applied in columns and pcr products in rows. lanes 14: positive controls (1, a. marginale; 2, a. centrale; 3, anaplasma sp. ‘omatjenne’; 4, a. international journal of applied biology, 5(2), 2021 90 phagocytophilum. lanes 5-6: negative controls (5, dna isolated from uninfected cow blood; 6, sterile deionized water). lanes 7-16: field samples (single and mixed infection) (7, a. marginale; 8, a. centrale; 9, anaplasma sp. ‘omatjenne’; 10, e. ruminantium; 11, a. marginale + a. centrale; 12, a. marginale + anaplasma sp. ‘omatjenne’; 13, a. marginale + a. centrale + anaplasma sp. ‘omatjenne’; 14, a. marginale + anaplasma sp. ‘omatjenne’ + e. ruminantium; 15, a. marginale + a. centrale + anaplasma sp. ‘omatjenne’ + e. ruminantium; 16, a. centrale + e. ruminantium. the overall prevalence of infection in cattle by these rickettsial bacteria was important and assessed to 44.44%. we also noticed that, female cattle (44.79%) were more infected than males (43.93%) with no significant difference while according to age, prevalence of infection was significantly different between yearling (50%) and adult cattle (44.07%) (table 2). table 2. overall prevalence of infection of rickettsial bacteria in the study area number of cattle blood examined infected prevalence (%) age χ2 = 51.681; df = 1; p < 0.0001 yearling 10 5 50 adult 152 67 44.07 sex χ2 = 2.347; df = 1; p = 0.1255 male 66 29 43.93 female 96 43 44.79 total 162 72 44.44 we noted that four species of rickettsial bacteria: anaplasma marginale, anaplasma centrale, anaplasma sp. ‘omatjenne’ and ehrlichia ruminantium were identified. the most prevalent parasite was a. marginale (41.35%), followed by anaplasma sp. ‘omatjenne’ (15.43%), a. centrale (8.64%) and e. ruminantium (3.08%). there was a significant difference between the prevalence of infection between the species identified (table 3). table 3. prevalence of each rickettsial bacteria identified in cattle blood number of cattle blood examin ed infected prevalence (%) rickettsial bacteria χ2 = 81.252; df = 3; p < 0.0001 anaplasma marginale 162 67 41.35 anaplasma sp. ‘omatjenne’ 25 15.43 anaplasma centrale 14 8.64 ehrlichia ruminantium 5 3.08 several types of co-infections were observed following blood examination. we noted four different types of multiple infection and classified as single, double, triple and international journal of applied biology, 5(2), 2021 91 quadruple infections with a prevalence statistically different. we found that, the most prevalent was single infection (24.69%) followed by double infection (16.67%) while triple (1.85%) and quadruple infections (1.23%) were less prevalent. summarily, difference was not significant between the prevalence of single (24.69%) and whole mixed infection (19.75%) (table 4). table 4. prevalence of co-infection in the study area single infection double infection triple infection quadruple infection total no (%) no (%) no (%) no (%) no (%) age yearling 3 30 2 20 5 50 adults 37 24.34 25 16.44 3 1.97 2 1.32 67 44.07 sex male 18 27.27 9 13.63 2 3.03 29 43.93 female 22 22.91 18 18.75 3 3.12 43 44.79 χ2 = 58.111; df = 3; p < 0.0001 total 40 24.69 27 16.67 3 1.85 2 1.23 72 44.44 co-infection single infection mixed infection frequency 40 32 prevalence (%) 24.69 19.75 χ2 = 0.681; df = 1; p = 0.409 considering the single infection (table 5) of these rickettsial infections of cattle blood, we found that the most prevalent parasite was anaplasma marginale (22.22%), while the most prevalent mixed infection was the double infection (16.66%) with the association between a. marginale + a. sp. ‘omatjenne’ (11.11%). international journal of applied biology, 5(2), 2021 92 table 5. prevalence of rickettsial bacteria association in infected cattle age sex total yearling adults male female study area no (%) no (%) no (%) no (%) no (%) rickettsial bacteria anaplasma marginale 3 30 33 21.71 16 24.24 20 20.83 36 22.22 anaplasma centrale 2 1.31 1 1.52 1 1.04 2 1.23 anaplasma sp. ‘omatjenne’ 1 0.66 1 1.04 1 0.62 ehrlichia ruminantium 1 0.66 1 1.52 1 0.62 a. marginale + a. centrale 8 5.27 3 4.54 5 5.21 8 4.94 a. marginale + a. sp. ‘omatjenne’ 2 20 16 10.52 6 9.09 12 12.5 18 11.11 a. centrale + a. sp. ‘omatjenne’ 1 0.66 1 1.04 1 0.62 a. marginale + a. centrale + a. sp. ‘omatjenne’ 1 0.66 1 1.04 1 0.62 a. marginale + a. sp. ‘omatjenne’ + e. ruminantium 2 1.31 2 2.09 2 1.23 a. marginale+ a. centrale + a. sp. ‘omatjenne’ + e. ruminantium 2 1.31 2 3.03 2 1.23 total 5 50 67 44.07 29 43.94 43 44.79 72 44.44 93 discussion the reverse line blot hybridization was performed to specifically identify simultaneously several species of rickettsial bacteria (anaplasma and ehrlichia species). of the 162 blood samples screened, 77 were found positive for at least one rickettsial bacteria. the overall prevalence of infection was 44.44%. this result was similar to 40.76% and 41% reported by hailemariam et al., (2017) in ethiopia and nguyen et al., (2020) in thailand respectively. however, it was highest compared to 9%, 5.3% and 7.1% reported respectively by aktas et al., (2010) in turkey, parvizi et al., (2019) in egypt and zaid et al., (2019) in palestine. furthermore, this prevalence was lower than the 76.1% found in northern cameroon by abanda et al., (2019). the important prevalence of infection of rickettsial bacteria observed might be associated to the presence of its main vectors, the amblyomma and rhipicephalus ticks (ngangnang et al., 2021). however, hyalomma and haemaphysalis tick species are also considered as potential vectors (latif and walker, 2004; lankester et al., 2007) and were identified in the study area (ngangnang et al., 2021). according to this finding, we could conclude that pathogens and vector might be widespread and well established in the western highlands of cameroon and need a great attention for medical and veterinary concern. it had also been noticed that, female cattle (44.79%) were most infected than male (43.93%) but the difference was not statistically significant as found by nguyen et al., (2020) while, the infection was associated to sex as reported nyabongo et al., (2021) in uganda. according to nyabongo et al., (2021), this risk of infection could be explained by the higher number of female cattle sampled compared to male in the study population. moreover, male cattle are provided with better health care due to their higher value, as they are used by farmers for reproduction and sold for meat, whereas females are kept for dairy. the prevalence of infection was high and significantly different between yearling (50%) and adult cattle (44.07%). similarly, nyabongo et al., (2021) report indicated the same observation while it was different from the finding of lorusso et al., (2016) in nigeria. this study showed that yearling cattle had a higher chance of being infected compared to adults. adult cattle that were infected as calves are resistant to re-infection, which could explain the high risk of infection for calves or yearling compared to adult animals. of the 162 cattle blood samples tested using nested pcr-based rlb hybridization assay for detection of rickettsial bacteria, a. marginale, anaplasma sp. ‘omatjenne’, a. centrale and e. ruminantium were identified. the most prevalent rickettsial bacteria identified in this study was a. marginale (41.35%) followed by anaplasma sp. ‘omatjenne’ (15.43%), a. centrale (8.64%) and e. ruminantium (3.08%). this finding was in agreement with the report of lorusso et al., (2016) in nigeria although they found the prevalence in different proportion and might be due to the sample size or the epizootiological situation of disease in each study site. however, the result contrast the previous report of eygelaar et al., (2015) in botswana and teshale et al., (2018) in ethiopia who reported respectively anaplasma centrale and anaplasma sp. ‘omatjenne’ as the most prevalent species. this contrast might be difference among the target population (buffalo and cattle) during each study site and even the epizootiology of vectors. several categories of co-infection were observed and the most prevalent was the single infection (24.69%) followed by double (16.67%), triple (1.85%) and quadruple infection (1.23%). the single infection (24.69%) was most prevalent than the total mixed infection (19.75%) with no significant difference. these results were different from those of international journal of applied biology, 5(2), 2021 94 hailemariam et al., (2017) and nyabongo et al., (2021) and could indicate the severity of rickettsial infection in cattle in the given study area. conclusion there was a high level of prevalence and species composition of rickettsial bacteria in the study area. this prevalence could be associated to the previous identification of amblyomma and rhipicephalus ticks in the study area. likewise, the current description of biological transmission of a. marginale by rhipicephalus microplus ticks such as biological intrastadial and transstadial transmission could affect the persistence of rickettsial bacteria. better prevention and control methods of these microorganisms could be development of new vector control strategies. authorization this study including cattle was authorized by the regional delegate for livestock, fisheries and animal industries of the west region of cameroon (authorization no 02/19/l/drepia-o/srag). author’s contribution ngangnang ghislain roméo conceived the idea of the study, wrote the research proposal, gathered the data, analysed and interpreted the data, prepared the manuscript, searched the literature and finalized the study. vincent khan payne and fonteh anyangwe florence proposed the study, analysed and interpreted the data, revised and approved the final version of the manuscript. aktas munir and ulucesme mehmet can designed the methodology, extracted the dna and performed pcr and rlb assay and revised the final version of the manuscript on molecular biology. keptcheu tchankwe désiré léonard gathered and analysed the data. conflict of interest the authors declare that they have no competing interest on this study. funding not applicable acknowledgement the authors acknowledge farmers for agreeing to participate and consent to collect ectoparasites and blood samples on cattle. we are also grateful to the department of parasitology, faculty of veterinary medicine, university of firät, elazig, turkey for providing laboratory space and reagents necessary for accomplishment of this work. international journal of applied biology, 5(2), 2021 95 reference abanda babette, archile paguem, mamoudou abdoulmoumini, tanyi kingsley, alfons renz and albert eisenbarth 2019. molecular identifcation and prevalence of tick-borne pathogens in zebu and taurine cattle in north cameroon. parasites vectors 12:448. aktas munir, kursat altay, nazir dumanli, 2010. molecular detection and identification of anaplasma and ehrlichia species in cattle from turkey. ticks 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of ehrlichia, borrelia burgdorferi sensu lato, and bartonella species in dutch ixodes ricinus ticks.’’ journal clinical microbiology, 37, 2215-2222. teshale sori, dirk geysen, gobena ameni, pierre dorny and dirk berkvens, 2018. survey of anaplasma phagocytophilum and anaplasma sp. ‘omatjenne’ infection in cattle in africa with special reference to ethiopia. parasites & vectors 11:162. contact : nur ramaliani samsul nurramalianisamsulbahri@gmail.com 26 abstract trigona bees spp has a potential utilization of this bee is not inferior to apis sp. the use of stingless honey bees in south sulawesi has been carried out in several districts such as selayar, maros and gowa. trigona bees spp produce pollen and honey which contain antioxidants, antioxidants are compounds that can neutralize or destroy free radicals, so as to prevent various diseases. this research was conducted to analyze the antioxidant content of pollen and honey produced by the three districts and compare them with each other. antioxidant levels was carried out using the dpph method (2,2-diphenyl-1-picrylhydrazyl) with vitamin c was used as positive control. the results of the determinining the wavelength used using a uv vis spectrophotometer obtained a wavelength of 513 nm used with the test results of antioxidant levels of pollen extract from the selayar location were 1666.33 µg/g, pollen maros 431.70 µg/g and pollen gowa 259.56 µg/g and the antioxidant content of honey from selayar was 43.62 µg/g, maros honey 28.08 µg/g and gowa honey 31.80 µg/g. pollen and honey from selayar had the highest antioxidant levels from the three locations. issn : 2580-2410 eissn : 2580-2119 analysis of antioxidant content in pollen and honey produced by bees trigona spp at several locations in south sulawesi nur ramaliani samsul 1*, eva johanneas 1,syahribulan 1 1 departement of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia introduction indonesia has the most rich species of honey bees from the apis clan in the world and has about 40 types of stingless bees or commonly called kelulut/klanceng/teuweul. sulawesi is one of the islands in indonesia which is famous for its endemicity. more than nine species of stingless honey bees found in indonesia (trigona apicalis, t. melina, t. itama, t. leviceps, t. drescheri, t. terminate and t. fuscibasis), there is one species endemic to sulawesi, namely t. insica. most of them can be developed as honey-producing bees (harjanto et al., 2020). according to rasmussen 2008, indonesia has at least 40 species of stingless bees, divided into several genera, including: geniotrigona, heterotrigona, lepidotrigona, and tetragonula. the use of stingless honey bees in south sulawesi has been carried out in several districts, namely jeneponto (11 farmer groups), palopo (12 farmer groups), luwu (2-5 farmer groups), north luwu (100 people) and east luwu (50 people). and continue to be developed in various potential areas in south sulawesi (hasan et al., 2020). one of the districts producing honey and other products is selayar. honey that contains pollen also has anti-radiation properties, which can protect the body from health damage due to exposure to free radicals. this means that honey and pollen can be expected as free radical scavengers (hartanto, 2017). free radicals tend to have a chain reaction which, if they occur in the body, can cause ongoing and continuous damage. the open access international journal of applied biology keyword trigona spp; honey; pollen; dpph article history received november 30, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 27 number of free radicals can increase due to stress factors, radiation, cigarette smoke and environmental pollution causing the body's existing defense system to be inadequate, so the body requires additional antioxidants from the outside that can protect against free radical attacks (wahdaningsih et al., 2011). observations were made in the khdtk (special purpose forest area) area of kepau jaya that there are plants that have the potential to be a source of food for trigona spp. types of potential nectar-producing species found in khdtk were temon, coconut, israeli grass, and durian and potential sources of pollen found were coconut, oil palm, sugar palm, and putri malu. the types of plants from the palm family are the best sources of pollen because palm plants are able to produce flowers in large quantities and throughout the year so that the pollen produced is abundant (wiratmoko et al., 2018). knowledge of the potential antioxidants contained in pollen and honey is expected to be one of the drivers in increasing the cultivation of trigona spp bees, which can become plant pollinating agents and can increase awareness of the surrounding community to conserve feed plants and increase forest protection as a place to live for feed plants. as an important factor in the development of beekeeping in the region. materials and methods research site the study was carried out in january-july 2021. sampling was carried out in the districts of selayar, gowa and maros, south sulawesi which were tested. the materials used in this study were beehive colonies of trigona spp. which contains pollen and honey, aquadest, 70% alcohol, 95% ethanol, concentrated h2so4 solution, mayer and dragendorff reagents, chloroform, ammonia, concentrated hcl, hcl in, 1% fecl3, mg powder, acetic acid anhydride, 2,2diphenyl-1-picrylhydrazyl (dpph). data collection in manually taking bee pollen by selecting a beehive box with lots of beehives, then pure pollen is taken in the form of colored solids wrapped in propolis. the collected samples were then air-dried in a shady place to avoid direct sunlight to dry for ± 24 hours. after that the sample is ground and mashed, the refining of the sample aims to maximize the interaction of ethanol (solvent) with the bee pollen sample so that it is expected that all secondary metabolites can be extracted, after the sample is mashed then the sample that has been refined is weighed. the sample used in this study was honey taken directly from the beehive of trigona spp. at 3 bee farms in selayar, maros and gowa. by taking a bag of honey. the honey is then removed from the bag using a syringe and a spoon. honey is then filtered using a filter to clean it from dirt. antioxidant test with dpph . radical attenuation method to determine the percentage of dpph radical reduction (% inhibition) of pollen and honey from bees trigona spp. the antioxidant activity test was carried out using the dpph radical reduction method (kumalaningsih, 2006). the dpph radical is soluble in ethanol and has a strong absorbance at a wavelength of 517 nm with a characteristic purple color. after reacting with antioxidant compounds, the dpph will be reduced and the color will turn yellow. international journal of applied biology, 6(1), 2022 28 pollen and honey samples from 3 beekeeping locations in selayar, maros and gowa were weighed as much as 25 grams and then put in a maceration vessel. extraction liquid in the form of ethanol was added as much as 75 ml to 100 ml, put into a vessel stored for 3 days in a closed room, the solution was then concentrated at room temperature (370c) to 50 ml, added 4 ml of 40 g/ml dpph solution in a vial. the solution was incubated for 30 minutes in a dark place. weighed 100 mg standard vitamin c of 1000 g/ml and dissolved with 96% ethanol to 100 ml, added 4 ml of 40 g/ml dpph solution in the vial. the solution was incubated for 30 minutes in the dark. preparation of the standard curve, as much as 4 ml of 40 µg/ml dpph solution was added with 1 ml of 96% ethanol into the vial, then the absorbance was measured in the wavelength range of 400 nm 800 nm. the maximum wavelength and absorbance value of the dpph standard solution will be obtained (sinala and dewi, 2019). determination of antioxidant level pollen and honey extract solutions were measured for absorbance at the maximum wavelength. the same treatment was also carried out on the dpph solution (which did not contain the test material) and vitamin c as a positive control. the data from the analysis of antioxidant levels in the sample per gram is calculated using a formula to calculate the levels of a substance in solution, as follows : 𝐴𝑛𝑡𝑖𝑜𝑥𝑖𝑑𝑎𝑛𝑡 𝑙𝑒𝑣𝑒𝑙𝑠 𝑆𝑎𝑚𝑝𝑙𝑒 𝑐𝑜𝑛𝑠𝑒𝑏𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑥 𝐹𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑆𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 x dilution factor data analysis pollen research data are presented in the form of figures and tables. the value of the determination of antioxidant levels obtained is presented in the form of a bar graph to see the comparison. results and discussion antioxidant content of pollen and honey trigona spp from selayar, maros and gowa. extraction of pollen and honey using maceration method using 96% ethanol liquid. ethanol is an organic solvent that can attract most of the bioactive compounds found in pollen and honey because it has a high polarity, also based on the extraction of phenolic compounds such as flavonoids and tannins from plant tissues using ethanol as a solvent at room temperature by maceration. wavelength measurement ultraviolet-visible (uv-vis) spectrophotometer is an analytical instrument that belongs to absorption spectroscopy. radiation or light is passed through a colored solution, then radiation with a certain wavelength will be absorbed selectively and other radiation will be passed on (syaifuddin, 2015). the results of the wavelength measurement are presented in figure 1 below: international journal of applied biology, 6(1), 2022 29 figure 1. determination of the maximum wavelength of dpph solution using a spectromotometer. spectrophotometer (uv-vis) is based on the absorption of visible light by a colored solution. this method is also known as the colorimetric method, because only the colored solution can be determined by this method. colorless compounds can be made colored by reacting them with reagents that produce colored compounds (syaifuddin, 2015). the light in question is in the form of visible light, uv and infrared while the material is in the form of atoms and molecules but the more important role is the valence electrons (chalid, 2012). determination of the maximum wavelength aims to determine the wavelength that has maximum absorption, that is, when the colored compound formed has been optimum so that maximum sensitivity is obtained. the wavelength of the sample was carried out using a solution of 2,2-diphenyl-1-picrylhydrazyl (dpph) with a solution of 100 l and the data from the analysis of the wavelength used was 513 nm. determination of antioxidant vitamin c the mechanism of ascorbic acid as an antioxidant is by reducing dpph free radicals by donating hydrogen atoms to produce l-ascorbate radicals. the l-ascorbic acid radical will immediately turn into l-ascorbyl radical and dehydro l-ascorbyl acid. the radicals formed are stable. this is due to the ability of radicals to stabilize themselves by resonating (latifah, 2015). charkraborty (2011) said, vitamin c is one of the important non-enzymatic antioxidants, which react with free radicals to form their own radicals which are less reactive than these radicals. they break radical chain reactions by trapping peroxyl and other reactive radicals. vitamin c is essential for the biosynthesis of collagen, carnitine and neurotransmitters. it is possible that this vitamin is consumed in the process of lipid peroxidation caused by oxygen radicals in reperfusion injury by ischemia to prevent tissue damage. vitamin c can be found in most fruits, especially citrus fruits and vegetables. vitamin c is found in tomatoes, eggplant, potatoes, chilies and red peppers. the fennel tribe (apiaceae), the pumpkin tribe (curcubitaceae), brussels sprouts and broccoli are also rich in vitamin c. vitamin c has a free hydroxy group that acts as a free radical scavenger and if it has a polyhydroxy group it will increase antioxidant activity. samples of vitamin c or ascorbic acid international journal of applied biology, 6(1), 2022 30 are positive controls that are used as comparisons and standards for samples tested to determine the potential for antioxidant activity in a sample (julizan et al., 2019) because they function as secondary antioxidants that counteract extracellular free radicals. the results of the analysis of antioxidant levels from the spectrophotometer are shown in figure 2, as follows: figure 2. concentration of vitamin c figure 2 shows the concentration of vitamin c tested using a uv vis spectrophotometer. the concentration value of vitamin c was 86.565 g/g. the use of positive control in testing this antioxidant activity is to determine how strong the antioxidant potential is in the pollen and honey bee extracts of trigona spp from 3 locations selayar, maros and gowa when compared with vitamin c. determination of pollen and honey antioxidant levels the value of the antioxidant content of the sample was calculated by taking pollen and honey samples from three different locations, namely selayar, maros and gowa. the method used is the dpph method because it is simple, easy, fast and sensitive and only requires a small sample. the mechanism of action of the dpph method is that antioxidant compounds will react with dpph radicals through the mechanism of hydrogen atom donation and cause dpph color decay from purple to yellow which is then measured at the maximum wavelength. in this study, a wavelength of 513 nm was used to see the antioxidant levels of pollen and honey from 3 different locations, the results of antioxidant analysis using a uv vis spectrophotometer are shown in table 1, as follows: table 1. scan analysis report results of pollen and honey samples from uv vis spectromotometer sample concentration mg/l f readings pollen gowa 0.8000 0.4886 pollen maros 0.7028 0.4996 pollen selayar 0.7296 0.4965 madu gowa 1.3018 0.4320 madu maros 0.8649 0.4813 madu selayar 0.9662 0.4698 y = -0,1128x + 0,5789 r² = 0,9929 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 0,5 1 1,5 2 2,5 3 a b so rb a n si konsentrasi (mg/l ) konsentrasi vitamin c absorbansi linear (absorbansi) international journal of applied biology, 6(1), 2022 31 the reduced color intensity of the dpph solution can indicate that there is a reaction between the hydrogen atoms released by the test material and the dpph radical molecule to form a yellow compound 2,2-diphenyl-1-picrylhydrazine. according to widyaningsih (2010), the greater the concentration of the test material, the stronger the yellow color produced. the absorbance test of free radical immersion was carried out on honey and pollen extracts as a result of the absorbance obtained from the uv vis spectrophotometer in tabl e 4 and then the antioxidant levels were calculated in grams. the level of antioxidants contained in the sample extract shows its antioxidant ability, the more antioxidants contained in a sample, the higher the sample's ability to capture free radicals. free radicals actively bind electrons so that they can cause a lot of damage to cells so that they can cause many degenerative diseases. the process of making ethanolic extracts of pollen and honey samples was carried out without heating this was done to avoid damage to antioxidant compounds so that they could not function to inhibit dpph free radicals optimally. the level of antioxidant activity is influenced by various factors, including its easily damaged when exposed to oxygen, light, high temperatures, and drying (putri and nurul, 2015). comparison of pollen antioxidant levels after the dpph test using a uv-vis spectrophotometer on honey and pollen samples was obtained, the absorbance value of the sample was obtained, this absorbance value was then used to determine the levels of antioxidants contained in each gram of the sample tested. the results of the antioxidant levels obtained are shown in figure 3 below: figure 3. comparison of pollen antioxidant levels from several locations. comparison of pollen from these three locations can be seen in figure 3. the antioxidant levels in pollen samples from the selayar location were 1666.33 µ/g, maros pollen 431.70 µ/g and gowa pollen 259.56 µ/g. these three samples had higher antioxidant values than vitamin c, which was 86.565 µ/g. this shows that the pollen sample has a higher anti-radiation ability than vitamin c. from figure 3 it can also be seen that the pollen from the selayar location has higher antioxidant levels than the other two locations, namely pollen from the gowa and maros locations. international journal of applied biology, 6(1), 2022 32 comparison of honey's antioxidant levels. honey from the three locations of selayar, maros and gowa was tested for dpph using a uv-vis spectrophotometer and the absorbance value of the sample was obtained which was then used to determine the level of antioxidants contained in each gram of the sample tested. the results of the antioxidant levels obtained are shown in figure 4 below: figure 4 . comparison of antioxidant levels of honey from several locations figure 4 shows the antioxidant content of honey from selayar, gowa and maros locations. antioxidant levels of selayar honey are 43.62 µ/g, gowa honey 28.08 µ/g and maros honey 31.80 µ/g. from the results obtained, it can be seen that the highest antioxidant content is found in honey from the location in selayar. the results of the antioxidant values of the three samples obtained showed that all samples had antioxidant values that were below the antioxidant value of vitamin c of 86.565 µ/g. this shows that the ability to scavenge free radicals in honey samples is lower than vitamin c. determination of locations that produce the highest pollen and honey from selayar, maros and gowa locations. from the test of antioxidant levels of pollen and honey from three locations, namely selayar, gowa and maros, it can be seen that there are differences in the antioxidant content of pollen and honey produced by trigona spp bees. of these three locations. table 2. calculation of pollen and honey antioxidant levels from several locations in south sulawesi. no. sample type selayar maros gowa average 1. polen 1666,33 µg/g 431,70 µg/g 259,56 µg/g 785,86 µg/g 2. madu 43,62 µg/g 28,08 µg/g 31,80 µg/g 51,75 µg/g international journal of applied biology, 6(1), 2022 33 discussion the results of the average antioxidant value obtained when compared to all honey samples from the three locations had a honey antioxidant content value of 51.75 g/g, much lower than the pollen sample from the three sampling locations which was 785.86 g/g as shown in table 5. this shows that the antioxidant content of pollen may vary at each location depending on the type of plant feed consumed by trigona spp bees. the chemical composition of feed plants affects the content of pollen and honey produced. the chemical content of its complex and diverse composition makes bee pollen has various properties, one of which is as an antioxidant (fiergiyanti, 2015). this is in accordance with previous research conducted by idris (2017) where from 5 parts of beehives tested for their antioxidant levels, the highest to lowest antioxidant content were honey bag extract, egg bag extract, propolis extract, pollen bag extract and activity the lowest antioxidant was found in honey extract. although low, honey still contains antioxidants that can be beneficial if consumed. conducted a study and claimed that in bee pollen there are polyphenols (flavonoids and folic acid) with a large capacity that have the potential as antioxidants, while in a study conducted by cahyaningrum (2019) the value of free antiradical activity comes from several antioxidant compounds. such as flavonoids, vitamin e, vitamin c, beta carotene, phenolic acids and so on. the distance of the farm from available feed also affects the frequency of harvest. nunes et al. (2010) also found that the larger the size of the bee, the greater its flight ability. the body size of the bee trigona spp. which reach 5 mm can cover a distance of up to 600 m in search of food sources. so that the quality of the antioxidant value of the surrounding feed which is in the flying radius of the trigona spp bees. greatly affects the content of the collected feed in the form of honey and pollen which can be consumed directly by humans. sources of honey bee feed are plants which include fruit crops, vegetable crops, ornamental plants, food crops, forest plants, and plantation crops. these plants contain nectar and pollen which are very influential in the production of honey that will be produced by honey bees. in this case the forest becomes a home and nesting place and a source of food for honey bees, while bees help in the process of pollinating forest plants, and produce honey for the community (marhiyanto, 2013). the highest yield of antioxidant pollen and bee honey trigona sp. obtained from the selayar location. where the data on potential feed plants collected around the farm from 3 locations shows that the selayar location is dominated by food sources originating from large trees which generally only live in the forest, because the location of the farms is in the forest. in contrast to the other 2 locations, which are located in plantations and rice fields, the location is not too far from residential areas, with plants that have the potential as a source of food from both maros and gowa locations which are almost the same, namely in the form of fruit trees. the difference in altitude where the trigona bees live causes differences in several different types of pollen collected due to differences in plant species that can grow at certain heights (pratama et al. 2013). the location of selayar in kalepadang village is in the bontoharu sub-district with an altitude of 350-600 meters above sea level, which is a plateau in the form of undulating hills. pollen and honey samples from selayar were taken from kalepadang village, where this village is located near a forest that has various types of flowering tree species whose forests are still maintained due to the absence of logging activities. there are many sugar palm and coconut trees which are the preferred food for international journal of applied biology, 6(1), 2022 34 trigona spp bees. which blooms all year round. almost all types of flowering plants can be a source of food for bees, but there are several types of flowering plants that produce toxic compounds, so they are not visited by bees and insects in general (adler, 2000). in addition, plants that are sprayed with disinfectants and other chemicals cannot be visited or can be used as a source of food by bees. honey is taken from plant fluids, namely nectar and pollen collected by bees from pollen or pollen produced by flowering plants. soluble substances in nectar, namely water, ions, carbohydrates, amino acids, and nectar also contain fragrant compounds to attract pollinators' attention and nectar enzymes and antioxidants (carter and thornburg, 2004). the antioxidant content of pollen and bee honey is influenced by the feed consumed. in a study conducted by cahyaningrum (2019), honey with different types of flowers, namely farm honey and longan honey, had different anti-free radical activities. where the anti-free radical activity in longan honey was greater, namely 82.10% compared to animal honey, which was 69.37%. in a study conducted by rahmi (2017), it can be seen that the antioxidant activity of different fruits is due to the presence of different secondary metabolites. antioxidant activity shows that avocado has tannin which is an active compound with antioxidant properties, strawberries and dragon fruit have antioxidant activity because they contain anthocyanin compounds. jamblang fruit contains flavonoids, also contains several other polyphenolic compounds such as tannins. the antioxidant capacity of a material is influenced by the components in the material that are able to act to inhibit oxidation (ariviani and parnanto, 2013). the content of these antioxidants has differences due to geographical differences or the origin of the bees and the time of harvesting. the antioxidant activity is influenced by the value of flavonoids contained in honey and pollen. flavonoids are one of the components of phenolic compounds which are natural antioxidants derived from plants. the content of flavonoids contained in the product produced by bees is influenced by the origin of the plant as raw material. the chemical composition of propolis depends on the specificity of the local flora at the collection site (jaya, 2017). the most abundant plants in the selayar location are coconut (cocos nucifera) types of coconut fruit which are known to be rich in antioxidants in the flesh, coconut water, coir, to the processed product, namely coconut milk. coconut fruit can be used for treatment and beauty because it contains chemical compounds such as polyphenols, flavonoids, tannins, steroids, triterpenoids. these compounds, especially polyphenols and flavonoids, can be used as an alternative source of natural antioxidants (lima, et al., 2015). from the research conducted by jauziyah et al., (2019), it was found that coir extract and coconut pulp have potential as antioxidants with strong antioxidant activity seen from the ic50 value. the ic50 value of coconut coir extract is 63.95 ppm and for coconut pulp extract is 95.44 ppm, it is known that coconut coir extract has a higher antioxidant potential than coconut pulp extract. conclusion the antioxidant content of pollen extract from selayar is 1666.33 µ/g, maros pollen is 431.70 µ/g and gowa pollen is 259.56 µ/g and antioxidant content of honey from selayar is 43.62 µ/g, maros honey is 28,08 µ/g and gowa honey 31.80 µ/g. trigona spp pollen and honey. from selayar had the highest antioxidant content of the three sampling locations. international journal of applied biology, 6(1), 2022 35 acknowledgments the author expresses his deepest gratitude for the financial support from the hasanuddin university campus. international journal of applied biology, 6(1), 2022 36 references adler, l. s. 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(2018). feed source plants for honeybees of trigona spp type in peat swamp forest, kepau jaya district, riau. national seminar on environmental conservation (senpling) 2018| isbn 978-979-792-865-0. contact : osazee ekundayo izevbuwa osazeeizevbuwa@gmail.com 49 abstract this survey was carried out to investigate the occurrence of escherichia coli o157:h7 in meat products from obinze abattoir, imo state, south-eastern nigeria. a total of 10 beef samples, 10 processing water samples, 10 table swabs and 20 entrails samples were collected. pour plate technique was used after a tenfold serial dilution to inoculate on eosin methylene blue (emb) agar and the e. coli isolates were cultured on sorbitol macconkey agar and incubated for 24 hours at 37oc. the isolates were subjected to biochemical tests for identification before antibiotic sensitivity test was carried out using the disc diffusion (kirbybauer) method. from the survey, the entrails had the highest rate of isolation (4.16 ±0.56 log10 cfu/ml) followed by the beef samples with (3.58 ± 0.01 log10 cfu/ml). the processing water and the table swabs yielded no growth of e. coli o157:h7. the percentage occurrence of e. coli o157:h7 was also determined and the entrails samples had the highest with (17, 89.5%) followed by the beef samples with (2, 10.5%). the processing water and table swabs samples did not yield growth of e. coli o157:h7 and their percentages were 0. the data obtained were subjected to statistical analysis using the single sample t test which showed that there was no significant difference (p > 0.05) in the rate of isolation of e. coli o157:h7 from the samples. the e. coli o157:h7 isolated showed 100% sensitivity to all the antibiotics used. the presence of e. coli o157:h7 implies that these food samples from the obinze abattoir, if consumed could be a potential public health hazard to the community. therefore, strict adherence to quality control measures should be implored in order to reduce contamination and food borne illnesses. issn : 2580-2410 eissn : 2580-2119 occurrence of e.coli o157 h7, from meat products sold in obinze abattoir, rivers state, nigeria osazee ekundayo izevbuwa1*, shadrach osalumhense okhuebor 2 1department of microbiology, faculty of natural and applied sciences, igbinedion university okada, p.m.b. 0006, edo state, nigeria 2faculty of life sciences, department of microbiology, university of benin, p.m.b. 1154, benin city, edo state, nigeria introduction escherichia coli are gram-negative rod shaped, facultative anaerobic and nonsporulating bacterium of the family enterobacteriaceae. the cells are about 2 micrometre long and 0.5 in diameters, with cell volume of 0.6-0.7, commonly found in lower part of the intestine of warm blooded animals (uhitil et al., 2001; bavaro, 2012; coia, 1998). based on its virulence, the bacterial organism is classified into five groups, namely, enterotoxigenic e. coli (etec), enteropathogenic e. coli (epec), enterohemorrhagic e. coli (ehec), attaching and effacing e. coli (aeec), and shiga toxin-producing e.coli (stec) (bavaro, 2012; paton and open access international journal of applied biology keyword escherichia coli o157:h7, public health, hazard, contamination, occurrence article history received 29 juny 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 50 paton, 1998)). optimal growth of escherichia coli occurs at 37oc, but some laboratory strains can multiply at temperature of up to 45-49oc. optimal ph requirement range is 5.5 and 7.5 (palumbo et al., 1995). it is considered as one ubiquitous pathogens in humans and animals. it is present in the environment and resides in the gastrointestinal tract (git) of humans and animals. escherichia coli have been associated with a number of diseases syndromes. among these are often severe and sometimes fatal infections such as peheophitis, septicema, meningitis, endocarditis, urinary tract infections, epidemic diarrhoea of adults and children. detection of e. coli in foods intended for human consumption shows poor in hygiene during production, processing or preparation. ultimately, detection of e. coli in food is indicative of faecal contamination and presence of other dangerous pathogenic microorganisms which can compromise the health and wellbeing of consumers. in addition to hygienic indicator, some strains of e. coli are directly pathogenic to humans. one of such strains of e. coli is the escherichia coli o157:h7 (zhao et al., 1995; nataro and kaper, 1998). e. coli o157:h7 is a particular serotype of the group referred to as enterohaemorrhagic e. coli (ehec), a sub group of the serotype referred to as verocytotoxin producing e. coli (vtec) or shiga toxinproducing e.coli (stec) owing to its ability to produce shiga-like toxins that are closely related to the toxin produced by shigella dysenteriae. most ehec isolates are acid tolerant, capable of surviving in acid foods and during passage through the stomach (arnold and kaspar, 1995) and it can cause severe enteric infections with symptoms such as abdominal pain, bloody diarrhea, hemorrhagic colitis, haemolytic uremic syndrome (hus) and thrombotic thrombocytopenic purpura (ttp). (coia, 1998; zhao et al., 1995; nataro and kaper, 1998; farougou et al., 2012). since it was first identified in 1982 as a cause of hemorrhagic colitis during outbreaks of bloody diarrhea in oregon and michigan, usa, following the consumption of undercooked ground beef, several outbreaks of hemorrhagic colitis and hemolytic uremic syndrome caused by this organism have been epidemiologically linked to consumption of undercooked beef (riley et al., 1983; schlundt, 2001) and it has since been a steadily increasing cause of foodborne illness worldwide. cattle appear to be the main reservoir of e. coli o157:h7. contamination of carcasses during slaughter is the primary route that ultimately leads to contamination of ground beef. other foods (e.g., lettuce, sprouts, fruit juices, vegetables, raw milk) and water also have been implicated as vehicles of transmission. person to person is an important mode of transmission, particularly in day care centres. direct contact with animals carrying the organism is also a recognized source of infection (who, 1997). mead et al., (1999) has estimated the incidence of e. coli 0157:h7 to 50% among ehec serotype in relation to public health problems. this survey was carried out to investigate the occurrence of escherichia coli o157:h7 in meat products from obinze abattoir, imo state, south-eastern nigeria. materials and methods collection and transport of samples a total of 50 samples which comprised of 20 entrails samples, 10 beef samples, 10 table swabs and 10 samples of processing water were collected from the obinze abattoir. the beef and entrails samples were received in cellophane papers and kept in coolers containing ice packs. sterile swab sticks were used to swab the slaughter tables and kept in their containers immediately. the processing water samples were collected in sterile international journal of applied biology, 4(2), 2020 51 universal bottles to prevent allochtonous microbial contaminations. the samples were then transported immediately to the microbiology laboratory for the analysis without delay. preparations for dilutions about 9ml of normal saline was dispensed into each of 10 series capped tubes using sterile 10ml pipette. the filled tubes were sterilized alongside with about 500ml normal saline in a glass conical flask at 121°c for 15 minutes in an autoclave. preparation of beef samples for bacterial enumeration a piece of beef or entrails from each sample was removed from the cellophane paper and mashed with a sterile crushable and pestle. 10g of the mashed beef was weighed and aseptically introduced into a sterile 250ml glass beaker and then topped up to 100ml using sterile normal saline which was properly mixed and labelled as 10-1. preparation of processing water samples for bacterial enumeration 10ml of the processing water sample was transferred from the universal bottle into a sterile beaker using a sterile 10ml pipette and 90ml of sterile normal saline was added to the 10ml of sample to form a homogenous mixture of 100ml. preparation of table swab samples for bacterial enumeration eosin methylene blue agar was prepared and sterilized based on the manufacturer’s prescriptions. 18 to 20ml of the agar was poured into sterile petri dishes and allowed to solidify followed by the drying of the plates using the hot air oven. the labelled swab sticks were cultured using the streak plate technique. the plates were labelled and incubated at 37oc for 24 hours. tenfold serial dilution technique 1ml of the sample was transferred from the beaker labelled 10-1 containing 10g of sample in 100ml of normal saline into the first test tube labelled 10-2 containing 9ml of sterile normal saline using a sterile pipette. the tube was properly mixed by shaking the tube with the cap closed after which, 1ml was transferred to the next test tube labelled 10-3. this dilution continued until the last test tube labelled 10-10 where after mixing, 1ml was drawn and discarded, similar to the description of fao, (1979). determination of e. coli counts and total aerobic plate counts (tapc) after the ten-fold serial dilution, 1ml aliquots from 10-2, 10-3 and 10-4 dilutions were aseptically transferred on petri dishes and about 18 to 20ml of eosin methylene blue agar (emb) was poured on the petri dishes and swirled in several directions (clockwise, anticlockwise, up and down) for even distribution and then was allowed to solidify. for the total aerobic plate count (tapc), nutrient agar was used which was prepared and sterilized according to the manufacturers prescription. the plate were inverted and their labels transferred to the other side of the plate containing the agar before the incubation at 37oc for 24 hours then the colonies were counted (karch et al., 1996). purification and preservation of isolates using a sterile wire loop, discrete colonies which grew on eosin methylene blue (emb) agar with a characteristic metallic green sheen were picked and sub cultured on nutrient agar by streaking followed by the incubation at 37oc for 24 hours to obtain pure international journal of applied biology, 4(2), 2020 52 isolates. single colonies were transferred from the sub culture plates into nutrient agar slants aseptically and incubated for 24 hours at 37oc. after incubation, the slants were preserved by wrapping them up within aluminium foil and kept in a refrigerator at 4oc. cultural characteristics of colonies macroscopic characteristics of the microbial growths on the medium were observed and recorded based on their elevations, edges, colours, opaquecity, and shape. the isolates were microscopically investigated by gram staining. biochemical tests some biochemical tests such as motility, indole production and sugar fermentation with gas production were done for the confirmation of e. coli. identification of e. coli o157:h7 some tests were carried out in the identification of e. coli o157:h7 which included the use of sorbitol macconkey agar to detect sorbitol fermentation and the serological identification of e. coli o157:h7 with the use of the o157:h7 latex kit to check for agglutination. identification of e. coli o157:h7 using sorbitol macconkey agar (smac) the sorbitol macconkey agar was prepared and sterilized based on the manufacturers prescription and 18 to 20ml was poured into sterile petri dishes which were allowed to solidify and dried using the hot air oven. single colonies of e. coli were picked from the nutrient agar sub culture plate and was cultured on the smac using the streak plate technique and incubated at 37oc for 24 hours. e. coli o157:h7 does not ferment sorbitol and remained colourless on the sorbitol macconkey agar (wells et al., 2005). serological identification of e. coli o157:h7 using the o157:h7 latex kit (oxoid england, dr0620) e. coli o157:h7 was confirmed by using latex agglutination test kit (antio157:h7 antibody for e. coli 0157:h7) test kit dr0620m (oxoid ltd hamspshire, england) as described by nataro and kaper (1998). antibiotic sensitivity pattern of e. coli o157:h7 the antibiotic susceptibility test was performed to determine the levels of sensitivity and resistivity of some gram negative drugs on e. coli 0157h7 using the mac farland standard. statistical analysis the data obtained from the investigations were edited, coded and subjected to statistical investigation. for the bacteriological analysis, the mean occurrences for the various samples were determined and in the analysis of the variance, single sample t test was used to determine the significance at 95% confidence interval which was not significantly different (p>0.05) using spss package. results and discussion results international journal of applied biology, 4(2), 2020 53 out of a total of 50 samples which comprised of 20 entrails samples, 10 beef samples, 10 table swabs and 10 samples of processing water which were collected from the obinze abattoir, 17 out of the 20 entrails samples yielded growth of e. coli and they were all e. coli o157:h7. 2 out of the 10 beef samples yielded growth of e. coli and they were both o157:h7. none of the processing water and table swabs samples yielded growth of e. coli. in table 1, the mean e. coli counts was determined in which the entrails samples had the highest mean of (4.16 ±0.56 log10 cfu/ml) followed by the beef samples which yielded (3.58 ± 0.01 log10 cfu/ml). the processing water and table swabs samples did not yield growths of e. coli and their means were 0. in table 2, the percentage occurrence of e. coli, e. coli o157:h7 and non e. coli o157:h7 isolated from entrails, beef, table swabs and processing water samples was determined where the entrails samples had the highest percentage of (89.5%) followed by that of beef which gave (10.5%). the processing water and table swabs samples did not yield growths of e. coli and their percentages were 0. in table 3, the antibiotic sensitivity pattern of e. coli o157: h7 isolated from entrails and beef samples was determined where all the gram negative antibiotics which included septrin, chloramphenicol, sparfloxacin, ciprofloxacin, amoxicillin, augumentin, gentamycin, peflacin, tarivid and streptomycin were all 100% sensitive. table 1: the mean occurrence of e. coli counts isolated from entrails, beef, table swabs and processing water samples from the obinze abattoir, imo state. samples log10 cfu/ml e. coli entrails beef processing water table swab 4.16 ±0.56 3.58 ± 0.01 0.0 ± 0 0.0 ±0 table 2: the percentage occurrence of e. coli, e. coli o157:h7 and non e. coli o157:h7 counts isolated from entrails, beef, table swabs and processing water samples from the obinze abattoir, imo state. samples e coli counts (%) o157:h7 (%) non o157:h7 (%) entrails 17 (89.5) 17 (89.5) 0 (0) beef 2 (10.5) 2 (10.5) 0 (0) processing water 0 (0) 0 (0) 0 (0) table swab 0 (0) 0 (0) 0 (0) total table 3: the antibiotic sensitivity pattern of e. coli 1057 h7 isolated from entrails and beef samples from the obinze abattoir, imo state. antibiotics (concentration) susceptible (%) resistant (%) septrin (30µg) 19 (100) 0 (0) international journal of applied biology, 4(2), 2020 54 chloramphenicol (30µg) 19 (100) 0 (0) sparfloxacin (10µg) 19 (100) 0 (0) ciprofloxacin (10µg) 19 (100) 0 (0) amoxicillin (30µg) 19 (100) 0 (0) peflacin (30µg) 19 (100) 0 (0) gentamycin (10µg) 19 (100) 0 (0) augmentin (30µg) 19 (100) 0 (0) tarivid (10µg) 19 (100) 0 (0) streptomycin (30µg) 19 (100) 0 (0) discussion the data obtained revealed that escherichia coli o157: h7 were isolated from meat products sold in obinze abattoir in imo state, nigeria. previous studies also indicated the presence of e. coli o157: h7 in meat and meat products (enabulele and uraih, 2009; itelima and agina, 2011; salome et al., 2014). the occurrence and isolation of escherichia coli o157:h7 from entrails, beef, processing water and table swabs from the obinze abattoir in imo state indicates contamination which is potentially hazardous. the e. coli o157:h7 counts observed from the beef samples may be attributed to poor handling procedure in the abattoir. it is therefore apparent that proper cooking must be done before the consumption of beef purchased from the abattoir. this is in agreement the report of enabulele and uraih (2009), who stated that the high rate e. coli o157:h7 occurrence is indicative of the poor sanitary environment under which the animals are slaughtered and sold. these animals were reported to be slaughtered on the abattoir floor that is not properly disinfected after every kill, with butchers and retailers walking between carcasses as they transact their business, while those in the market are displayed on tables in the open for sale. also, nkanga and uraih (1981) reported that meat is frequently found to be contaminated due to poor sanitary environment during slaughter, transportation and usage and through handling. cattle are the natural reservoir of e. coli o157:h7 with a prevalent rate of more than 35%. various food items contaminated with cattle faeces are implicated as the source of human infections, beef and beef derived products being the most important. also, the abattoir environment is an ideal reservoir harbouring this pathogen. the presence of e. coli o157:h7 in the ground beef obtained from the abattoir indicates that the consumer of beef and beef products from this abattoir if not properly cooked are at risk of acquiring food poisoning. this agrees with elder et al., (2000) report, stating evidence of contamination as the most potential source of e. coli 0157:h7 in beef was hide and or feaces during slaughter process. evidently, e. coli 0157:h7 has been isolated from feaces or gastrointestinal tract of cattle, sheep, horses, pigs, turkeys, dogs and a variety of wild animals (hancock et al., 1998). 17 out of the 20 entrails samples yielded growth of e. coli and they were all e. coli o157:h7 and then 2 out of the 10 beef samples yielded growth of e. coli and they were both o157:h7 and these accounted for 89.5% and 10.5% respectively. the 89.5% prevalence rate in the entrails in this study is a clear indication of heavy contamination and this agrees with a similar report of dahiru et al., (2008) who reported 53%. one possible reason for this high prevalence of e. coli 0157:h7 found in the entrails is contamination from the faeces of the international journal of applied biology, 4(2), 2020 55 animal. the 10.5% prevalence rate in beef samples agrees with a similar report of hancock et al., (1994) who reported a prevalence rate of e. coli 0157:h7 in herds of dairy cattle to be 8.3% and 16% from beef. however some reports highlighted lower prevalence rate (enabulele and uraih, 2009; itelima and agina, 2011; salome et al., 2014). in this study, e. coli o157:h7 from the samples were susceptible to all ten (10) gram negative antibiotics used as seen in table 3 above. total sensitivity of some gram negative antibiotics to e. coli o157:h7 is a great advantage which implies that the early detection and the use of antibiotics will helpful in the treatment of e. coli o157:h7 infections. there are very rare studies that reports susceptibility of e. coli o157:h7 to all same antibiotic agents used in this study. however, some reports have stated the susceptibility of e. coli o157:h7 to gentamycin and ciprofloxacin which were also used in this study (salome et al., 2014; walsh et al., 2006; okolocha et al., 2006). a possible reason for the susceptibility of the isolate to these drugs may be due to the fact that these antibiotics are very expensive compared to others and therefore, not indiscriminately used (okeke et al., 2000). contrary to the findings in this study, salome et al., (2014) reported resistance to chloramphenicol, streptomycin and amoxicillin. also chigor et al., (2010) reported e. coli o157:h7 resistance to ciprofloxacin. this could be attributed to difference in host habitat. conclusions the total occurrence of e. coli o157:h7 from all the e. coli isolates indicates the prevalence of e. coli o157:h7 amongst other strains of e. coli associated with meat. therefore, the need to improve the sanitary condition of the abattoir should be implored in order to prevent the spread of the pathogen. educating the workers in abattoirs on good sanitary practices during the processes of cow slaughtering and the possible dangers of consuming contaminated products should be ensured.the national agency for food and drugs administration and control (nafdac) should see to the compliance of sanitary practices in abattoirs. furthermore preserving the products in safe condition is necessary. federal government should set standard committee to ensure the standard of processed food and the compliance to who specification. also, organizational seminars and conference should be done to enlighten and educate the masses on the dangers of consuming undercooked beef. international journal of applied biology, 4(2), 2020 56 references arnold, k.w. and kaspar, c.w. (1995). starvation and stationary phase induced acid tolerance in escherichia coli o157:h7. applied and environmental microbiology, 61:2037-2039. bavaro, m.f. (2012) e. coli o157:h7 and other toxigenic strains: the curse of global food distribution. current fungal infection reports, 14(4):317-323. chigor, v.n., umoh, v.j., smith, s.i., igbinosa, e.o. and okoh, a.i. (2010). multidrug resistance and plasmid patterns of escherichia coli o157 and other e. coli isolated from diarrhoeal stools and surface waters from some selected sources in zaria, nigeria. intentional journal of environmental public health, 7:3831-3841. coia, j.e. (1998). clinical, microbiological and epidemiological aspects of escherichia coli o157:h7 infection. fems immunology and medical microbiology, 20:1-9. dahiru, m., uraih, n., enabulele, s.a. and shamsudeen, u. (2008). prevalence of escherichia coli 0157:h7 in fresh and roasted beef in kano city, nigeria. bayero journal of pure and applied sciences, 1(1):39-42. enabulele, s.a. and uraih, n. (2009). enterohaemorrhagic escherichia coli 0157:h7 prevalence in meat and vegetables sold in benin city, nigeria. african journal of microbiology, 3(5): 276-279. available at: http://www.academicjournals.org/ajmr elder, r.o., keen, j.e., siragusa, g.r., barkocy-gallagher, g.a., koohmaraie, m. and laegreid, w.w. (2000). correlation of enterohemorrhagic escherichia coli o157 prevalence in feces, hides, and carcasses of beef cattle during processing. proceeding of the national academy sciences, 97:2999-3003. farougou, s., sessou, p., yehouenou, b. and dossa, f. (2012). microbiological quality of raw milk processed from cows raised under extensive system in the republic of benin. research journal of microbiology, 7(7):337-343. food and agricultural organization (fao), (1979). mannuals of food quality control for microbiological analysis. hancock, d.d., besser, t.e., kinsel, m.l., tarr, p.i., rice, d.h., and paros, m.g. (1994). the prevalence of escherichia coli o157.h7 in dairy and beef cattle in washington state. epidemiology and infection, 113:199-207. hancock, d.d., besser, t.e. and rice, d.g. (1998). multiple source of escherichia coli 0157 in feed lots and diary farms in the northrestorn usa. preventive veterinary medicine, 35:11-19. http://www.academicjournals.org/ajmr international journal of applied biology, 4(2), 2020 57 itelima, j.u. and agina, s.e. (2011). the occurrence of escherichia coli o157:h7 in market and abattoir meat in plateau state, nigeria. global journal of environmental sciences, 10(1&2):47-55. karch, h., janetzki-mittmann, c., aleksic, s. and datz, m. (1996). isolation of enterohemorrhagic escherichia coli o157 strains from patients with hemolyticuremic syndrome by using immunomagnetic separation, dna-based methods, and direct culture. journal of clinical microbiology, 34:516-519. mead, p.s., slutsker, l., dietz. v., mccaig, l.f., bresee, j.s., shapiro, c., griffin, p.m. and tauxe, r.v. (1999). food related illness and death in the united states. journal of emerging of infectious disease, 5(5): 607-25. nataro, j.p. and kaper, j.b. (1998). diarrheagenic escherichia coli, clinical microbiology review, 11:142. nkanga, e.j. and uraih, n. 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(1998). pathogenesis and diagnosis of shigatoxin producing escherichia coli infections. clinical microbiology reviews, 11:450-479. riley, l.w., remis, r.s. helgerson, s.d. mcgee, h.b. wells, j.g. davis, b.r. hebert, r.j. olcott, h.m. johnson, l.m. margrett, n.t. blake, p.a. and cohen, m.l. (1983). hemorrhagic colitis associated with a rare escherichia coli serotype. the new england journal of medicine, 308:681-685. available at: http://meridian.allenpress.com/doi/pdf/10.4315/0362-028x-64.6.862 salome, y.t., jacob, k.p.k., mohammed, b., junaidu, k., veronica, j.u., sabo, e.y. and andrew, j.n. (2014). occurrence of escherichia coli o157 in retailed-beef and related meat products in zaria, nigeria. food and nutrition sciences, 5:481-487. schlundt, j. (2001). emerging food borne pathogens. biomedical journal of environmental science, 14(1-2):44-52. http://meridian.allenpress.com/doi/pdf/10.4315/0362-028x-64.6.862 international journal of applied biology, 4(2), 2020 58 uhitil, s., jaksïc s., petrak, t. and botka-petrak, k. (2001). presence of escherichia coli o157:h7 in ground beef and ground baby beef meat. journal of food protection, 64(6):862-864. walsh, c., duffy, g., o`mahony, r., fanming, s., blair, i.s. and mcdowell, d.a. (2006) antibacterial resistance in irish isolates of verotoxigenic escherichia coli– vtec. international journal of microbiology, 109:173-178. wells, j. e., barry, e.d. and varel, v.h. (2005) effects of common forage phenolic acids on escherichia coli 0157:h7 viability in bovine faeces. journal of applied and environmental microbiology, 71(12):7974-7979. who (world health organization). 1997. prevention and control of enterohaemorrhagic escherichia coli (ehec) infections (who/fsf/fos/97.6). food safety unit, programme of food safety and food aid, world health organization, geneva. zhao, t., doyle, m.p., share, j., garter, l., (1995). prevalence of e. coli o157:h7 in a survey of dairy herd. applied and environmental microbiology, 61:1290-1293. contact : khairul khairulbiologi75@gmail.com 94 abstract swamp flooded is a habitat of various types of fish, which as an feeding ground, shelter, and reproduction. diversity of fish types has been utilized by the people to catch fish, as a material of consumption and economic income. one of the flooded swamp that people have utilized in the barumun river area. the purpose of this research is to know the biodiversity of fish in the swamp flooded barumun river. this research is explorative, with the determination of observation stations with purposive sampling methods. fish are captured using hand cast fishing nets, fishing rods, and gill nets. fish are identified using relevant books. total fishing catches during the study amounted to 298 individuals consisting of 13 genera and 17 speisies. the average value of the keanakaragaman index is 1,85 with a low cathedral. the average value of uniformity index (e ') is 0,67 in medium category. the average value of the dominancy index (c) is 0,027 in low category. based on the results of the study concluded the waters of rawa sungai barumun has suffered disruption due to land function and pollution. this is evidenced by the oil palm factory pt. nubika jaya and palm plantations area. issn : 2580-2410 eissn : 2580-2119 fish biodiversity in the swamp ecosystem of barumun river area khairul department of biology education, labuhanbatu university, rantauprapat city, sumatera utara province, indonesia introduction swamp flooded is an area that is a habitat of various types of fish. some types of fish make swamp flooded as areas for eating, breeding, and parenting. according to nurdawati (2008) in his life cycle the swamp habitat flooded needed various types of fish for enlargement and for feeding. swamp water is heavily influenced by the season, when the dry season is drought and when the rainy season floods. utomo (2016) explains there are two groups of fish in the swamp flooded namely the group of swamp fish (black fish) and the river fish group (white fish). the biodiversity of fish in swamped areas is potential to improve the welfare of the people. the communities around the swamp usually utilize swamp flooded to catch fish, where as a source of fulfilling nutritional needs, and some are sold for family income sources. swamp flooded is also one type of common water that has potential freshwater fishery resources that are very important (ditya et al., 2013). swamp flooded in several rivers in indonesia such as kampar river, musi, lempuing, batanghari, rokan, kahayan, barito, mahakam, and kapuas is an ecosystem that plays an important role in the production of open access international journal of applied biology keyword fish biodiversity, swamp ecosystem, barumun river. article history received 15 june 2020 accepted 1 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 95 freshwater fisheries (samita, 2001). fish resources are essential for the balance of ecosystems, food sources, medicinal resources, and livelihood sources. one of the undisclosed swamps of fish diversity is in the barumun river area. the study of biodeiversity of fish is very important, because fish serves as a constituent of ecosystems, germplasm, and can be used as a bioindicator of water pollution (ismail & yusof, 2011); zulkifli et al (2012); (wahyuni & zakaria, 2018). the wealth data of comprehensive fish species in the swamp flooded barumun river has not existed until now, so that there needs to be wealth information for the concrete species of fish to become the cornerstone of further studies, especially those related to efforts to manage fish resources in the barumun river and its flooded swamps. materials and methods time & location this study was held in january to march 2020. location of research in blok 9, sisimut village, kota pinang district, labuhan batu selatan regency, sumatera utara province, indonesia. research location map can be seen in figure 1. figure 1. map of research observasion collecting data this research uses the purposive sampling method. fish sampling using hand cast fishing nets, fishing rods, and gill nets. caught fish are identified using the book saanin (1984), kottelat et al (1993), and iqbal (2011).he tools used in this research were global positioning system (gps), plot size of 50x50 cm, meter roll, sample bags, tweezers, trays, writing instruments, cameras, bivalvia identification books. the materials used are samples of bivalves, formaldehyde, alcohol, and tissue. analysis data the data of the research results analyzed include: biodiversity index (h') using formula (krebs,1999), uniformity index (e'), using the formula (magurran, 1982), and dominance index (c), using the formula (odum,1991). international journal of applied biology, 4(1), 2020 96 results species composition the catch of fish from the research site consists of 13 genera, 17 species, of a total of 298 individuals. the most fish caught are: tricogester trichopterus (75 individuals), here in after followed: brevipora ceeya (68 individuals), helastoma temminckii (31 individuals), oreochromis niloticus (25 individuals), tricogester lieeri (21 individuals), tricogester pectoralis (17 individuals), puntius gemelus (14 individuals), anabas testudineus (11 individuals), aplocheilus panchax (10 individuals), pristolepis grooti (9 individuals), notepterus notepterus (5 individuals), channa striata (4 individuals), clarias betracus (3 individuals), channa lucius (2 individuals), clarias leiachantus (1 individual), monopterus albus (1 individual), and pangasius pangasius (1 individual). more data can be found in table 1. table 1. species composition of fish biodiversity at the swamp of the barumun river. genera spesies local name st. 1 st. 2 st. 3 ∑ anabas anabas testudineus betok/ pirik 6 2 3 11 aplocheilus aplocheilus panchax kepalatimah 1 8 1 10 brevipora brevipora ceeya seluang 2 64 2 68 chana channal ucius bujuk 1 1 0 2 chana channa striata gabus 2 1 1 4 clarias clarias betracus lele 1 1 1 3 clarias clarias leiachantus lembat 0 0 1 1 helastoma helastoma temminckii tambakan 29 0 3 31 oreochromis oreochromi sniloticus nila 1 24 0 25 monopterus monopterus albus belut 1 0 0 1 notepterus notepterus notepterus putak 1 2 2 5 tricogaster tricogester trichopterus sepat rawa 26 15 24 75 tricogaster tricogester lieeri sepat mutiara 11 2 8 21 tricogaster tricogester pectoralis sepat siam 9 0 8 17 pangasius pangasius pangasius patin 0 1 0 1 pristolepis pristolepis grooti sipatung 7 1 1 9 puntius puntius gemelus kemuringan 2 10 2 14 ecological index the observed ecological indices include: diversity index (h') at the station 1 (1,98), station 2 (1,65), and station 3 (1,93). uniformity index (e'), station 1 (0,710, station 2 (0,60), and station 3 (0,69). dominancy index (c), station 1 (0,02), station 2 (0,02), and station 3 (0,04). the results of an ecological index analysis are shown in table 2. international journal of applied biology, 4(1), 2020 97 table 2. average value of the ecological index of fish biodiversity at the swamp of the barumun river. ecological index station h’ category e’ category c category 1 1,98 low 0,71 medium 0,02 low 2 1,65 low 0,60 medium 0,02 low 3 1,93 low 0,70 medium 0,04 low ∑ averange 1,85 low 0,67 medium 0,027 low discussion types of fish that are caught in the swamp flooded river barumun is tricogester trichopterus (75 individuals), belonging to the family osphronemidae. the osphronemidae family is a type of fish that has a maze. the maze of fish is as a form of morphological adaptation, where fish as organisms living in marshes have the ability to adapt to low ph and oxygen. certain types of fish that can use oxygen from the free air, have more durability over dissolved oxygen-deprived waters (wardoyo, 1978). the results of the research of asyari (2006) in rawa muning gained 13 types of fish dominated by black fish such as: trichogaster trichopterus, anabas testudineus, and channa striata. gunawan & jumadi (2016) in his research to obtain the most fish catches is trichogaster trichopterus 77 individuals. next is the brevipora ceeya species (65 individuals) belonging to the family cyprinidae. the family of the cyprinidae are the dominant fish found in the flooded swamp area. according to simanjuntak et al (2007); kottelat et al (1993), family cyprinidae are the world's largest freshwater fish species and are capable of living in a variety of habitats. fish that live in swamp forest are generally types that are resistant to changing water quality (nurdawati & prasetyo, 2007). according to marini & husna (2010), the cyprinidae family is the most common group of fishes found in freshwater and some of these family can survive in degraded environments. the degradation of swamp ecosystem because land clearing and convertion of lands from naturally tobe agriculture as plantation and other usage (sagala, 2015). the diversity index (h') value of the three stations indicates a medium category. based on the criteria of the index value of the diversity of shannon wiener, if h' ≤ 3,32 then the diversity is relatively low. the low diversity index value indicates that the ecosystem is experiencing reduced pressure or condition. according to gunawan & jumadi (2016) low fish biodiversity indicates habitat conditions that have been damaged, for example: habitat degradation, natural disasters, pollution, and others. the uniformity index (e’) values obtained ranged from 0.4 < e ≤ 0.6, so the uniformity index is categorized as moderate, meaning that the community is unstable. dhahiyat et al (2003) states that the condition of community structures that are in an unstable state indicates ecological pressure. the results of the research dominance index indicates a value of 0 < c ≤ 0.5, with low dominancy criteria. this means that there is not a type of fish that dominates in the swamp area flooded with barumun river. suspected capture equipment to catch fish on some less effective stations, so the fish is not much to be caught. according to nugroho et al (2015), the selectivity of the capture tool determines the value of the dominancy. 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https://dictionary.cambridge.org/dictionary/english-indonesian/this https://dictionary.cambridge.org/dictionary/english-indonesian/family https://dictionary.cambridge.org/dictionary/english-indonesian/can https://dictionary.cambridge.org/dictionary/english-indonesian/survive https://dictionary.cambridge.org/dictionary/english-indonesian/in international journal of applied biology, 4(1), 2020 98 conclusions types of fish caught dominated by family osphronemidae and cyprinidae, considering that this fish is a highly adaptable group in the swamp ecosystem. in addition, it is suspected that the swamp ecosystem is a suitable place for feeding, clusters, and shelter for the fishes. sustainability as a habitat of fish needs to be maintained so that the diversity of fish becomes abundant, so it can be utilized by the surrounding community. references asyari. 2006. karakteristik habitat dan jenis ikan pada beberapa suaka perikanan di daerah aliran sungai barito, kalimantan selatan. jurnal 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(1985). ecology the experimental analysis of distribution and abundance. new york: harper & row publisher. magurran, a. 1988. ecological diversity and its measurement. new jersey: princeton university press. marini, m., & husnah. 2010. struktur komunitas ikan dalam hubungannya dengan kualitas air bagian hilir sungai siak, provinsi riau. prosiding seminar nasional limnologi v tahun 2010 (pp. 156 – 173). bogor: lembaga ilmu pengetahuan indonesia. nugroho, h. a., rosyid, a., & fitri, a. d. 2015. analysis of diversity index, domination index and non target catch proportion modified arad in kabupaten kendal waters. journal of fisheries resources utilization management and technology, 4(1), 1-11. nurdawati, s. 2008. fauna lkan di perairan rawa banjiran sungai batanghari, jambi. prosiding seminar nasional lkan v, (pp. 65-76). bogor. nurdawati, s., & prasetyo , d. 2007. fauna ikan ekosistem hutan rawa di sumatera selatan. jurnal iktiologi indonesia, 7(1), 1-8. odum, e. p. 1991. fundamentals of ecology (third eddition ed.). philadelphia: w. b. saunders co. saanin, h. 1984. taksonomi dan kunci identifikasi ikan. bandung: binacipta. international journal of applied biology, 4(1), 2020 99 sagala, e. p. 2015. the biodiversity of nursery ground in swamp areas important to survive the black fishes in the wetland. biovalentia, 1(1), 34-38. samita, a. s. 200. potensi dan tingkat pemanfaatan perikanan perairan umum sekitar lahan persawahan pasang surut sumatera selatan. jurnal penelitian perikanan indonesia, 7(2), 1-11. simanjuntak, c. p., rahardjo, m. f., & sukimin, s. 2007. iktiofauna rawa banjiran sungai kampar kiri. jurnal iktiologi indonesia, 6(2), 99-109. utomo, a. d. (2016). strategi pegelolaan suaka perikanan rawa banjiran di sumatra dan kalimantan. jurnal kebijakan perikanan indonesia, 8(1), 13-20. wahyuni, t. t., & zakaria, a. 2018. keanekaragaman ikan di sungai luk ulo kabupaten kebumen. biosfera, 35(1), 23-28. wardoyo, s. t. h. 1978. kriteria kualitas air untuk keperluan pertanian dan perikanan . prosiding seminar pengendalian pencemaran air (pp. 293-300). jakarta: direktorat jenderal pengairan, departemen pekerjaan umum. zulkifli , s. z., yusuff , f. m., ismail, a., & miyazaki, n. 2012. food preference of the giant mudskipper periophthalmodon schlosseri (teleostei: gobiidae). knowledge and management of aquatic ecosystems, 405(07), 1-10. contact : santa bahadur b.k shanta2015@live.com 67 abstract the genotypic yield potential of maize is influenced by planting dates. a study was conducted in rampur, chitwan, nepal, in 2013/14 to determine the best planting dates for maize genotypes. every week from april, 2013 to march, 2014, four maize genotypes viz hybrid rml-4/rml-17, rml-32/rml-17, across-9944/across-9942, and s99tlyq-b were planted. the maximum grain production (5565 kg/ha) was recorded in august, followed by february (5266 kg/ha), june (4475 kg/ha), and july (4255 kg/ha). the lowest yield was obtained in november planting (2572 kg/ha) followed by december planting (3019 kg/ha). during august planting, the maximum grain yields were obtained in rml-4/rml17 (7392 kg/ha) followed by rml-32/rml17 (6606 kg/ha) and across9944/across-9942 (5004 kg/ha). the qpm genotype s99tlyq-b produced the highest grain yield (4198 kg/ha) in february planting. maize grown in the winter had a larger yield potentiality than maize grown in the rainy season. this study suggests that rml-4/rml-17, rml-32/rml17, and across-9944/across-9942 should be planted in august (winter season) and s99tlyq-b in february (spring season) for maximizing production. issn : 2580-2410 eissn : 2580-2119 grain yield evaluation of maize genotypes at different planting dates santa bahadur b.k 1*, tika bahadur karki 1, jiban shrestha 1, balram bhandari 1, parbati adhikari 1 1nepal agricultural research council, national maize research program, rampur, chitwan, nepal introduction maize is nepal's the second most important cereal crop in terms of both area and productivity. maize cultivation has an area of 8.49 million ha, and a productivity of 2.3 t/ha (moad, 2013). it accounts for around 25% of overall cereal production, 6.54% of agdp, and 3.15% of gdp (moad, 2013). hills account for 70.23% of maize area, followed by terai (19.32%), and mountain (10.45%) (moac, 2009/10). almost all of the maize grown in the mid hills (1.3 million mt) and high hills (0.18 million mt) is consumed by humans, with just a small percentage being fed to animals. however, poultry and animal feeds account for more than 80% of terai production (0.38 million mt), while the remaining 20% is used for industrial (10%) and human use (10%). (nmrp, 2011). it is farmed on 875660 hectares of land, with an average yield of 2.119 t/ha (moac, 2009/10), which is quite low when compared to neighboring nations' yields. because there is a chance that unfavorable climatic circumstances will develop after planting or during the growth season, either early or late planting can result in reduced yield. as a result, determining the best sowing dates for maize types is critical for increased crop production. nepal is a small country with a wide open access international journal of applied biology keyword grain yield, maize, planting dates and varieties. article history received 25 september 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 68 range of natural environments. weather conditions change dramatically throughout the year. maize must be cultivated from ideal sowing dates in order to utilize moisture, nutrients, and sun radiation. the planting date of maize has an impact on plant population, plant development, the time it takes for reproductive organs to develop, pollination, and harvest, hence choosing the right planting time for maize cultivation is critical to its success. it had been claimed that delaying sowing till the end of october reduced maize grain output (mccormick, 1971). tanaka and hara (1974) in india found that when sowing was postponed till the end of october, the 1000 seed weight was reduced, resulting in a variance in maize grain production. in nepal, very little research has been done to establish the best sowing dates for maize genotypes. therefore, this study was carried out to identify the best planting dates for maize genotypes in the terai region of nepal. materials and methods plant materials the maize genotypes used in this study were s99tlyq-b, rml4/rml17, rml32/rml17, and across-9944/across-9942. all these genotypes were received from national maize research program, rampur, chitwan, nepal. experimental site the experiment site has a subtropical climate and is located at 27o40'n latitude, 84o19' e longitude, and 228 masl height. maize was grown on a sandy silt loam with a ph of 5.0, a medium total nitrogen concentration (0.130%), a high soil accessible phosphorous (279 kg/ha), a high soil available potassium (215 kg/ha), and a high organic matter content (2.70%) (nmrp, 2012). climatic observation table 1 shows the meterological data for temperature, rainfall, and relative humidity during the crop growth season. table 1. meterological data at rampur, chitwan, nepal during 2013-2014. month mean temperature (0c) total rainfall (mm) relative humidity (%) maximum minimum average april (2013) 34.6 16.0 25.3 34.2 87.1 may 35.0 23.9 29.45 375.9 89.2 june 34.2 26.3 30.25 667.5 92.2 july 33.3 33.7 33.5 16.1 93.6 august 23.6 28.0 25.8 7.5 88.6 september 30.06 29.30 29.68 13.0 79.90 october 27.33 26.21 26.77 0.4 77.76 november 21.73 20.17 20.95 0.0 72.08 december 17.40 16.66 17.03 0.0 73.85 january (2014) 18.53 16.19 17.36 0.3 70.88 february 18.95 16.97 17.96 5.2 68.78 march 21.93 20.91 21.42 3.4 66.66 (nmrp, 2014) international journal of applied biology, 5(2), 2021 69 experimental design and crop husbandry the trial was planted in 2013/14 at rampur, chitwan, nepal. from the second week of april, 2013 to the last week of march, 2014, four genotypes, s99tlyq-b rml4/rml17, rml32/rml17, and across-9944/across-9942, were planted in every week. the design was randomized complete block design with four replications (weeks as replication). the planting was repeated four times at seven-days intervals each month. two to three seeds were sowed at 75 cm row to row and 25 cm plant to plant spacing, and after two weeks, one plant/hill was thinned. the plot size was 2 rows of 5 meters (1.5 m × 5 m), with the entire plot being used to determine the final harvest. each trial used fertilizer @ fym 10 t/ha and 120:60:40 kg npk/ha. the other half of the nitrogen was divided into two halves and applied at 20-24 and 40-45 days after sowing as a basal dose, with the entire dose of phosphorous and potash applied at the time of final field preparation. weeding and irrigation were carried out according to the guidelines provided by national maize research program, rampur, chitwan, nepal. grain yield (kg/ha) at 15% moisture content was calculated using fresh ear weight with the help of the formula adopted by carangal et al. (1971). data analysis all collected data were entered in microsoft excel 2010 and analyzed by using mstat-c. all the data collected were statistically analyzed using the analysis of variance (anova) procedure described by gomez and gomez (1984) for randomized complete block design (rcbd) experiments. upon significant f-test results, means were compared using least significant difference test (steel and torrie, 1980). results and discussion the effect of genotypes and planting dates on yield was found to be highly significant (table 2). rml-32/rml-17 produced the highest grain yield (4846 kg/ha), followed by rml-4/rml-17 (4837 kg/ha).the grain yield of s99tlyq-b was the lowest (2979 kg/ha). august planting had the highest grain yield production (5565 kg/ha), followed by february (5266 kg/ha), june (4475 kg/ha), and july plantings (4255 kg/ha). the lowest yield (2572 kg/ha) was obtained in november planting, followed by the highest yield (3019 kg/ha) in december planting. the maize grain yield was as a function of genotypes and sowing dates. the genotypes were found highly significant (p< 0.01) for grain yield. differences in ear length, rows per ear, and grains per row, as well as grain size, caused differences in variety for grains (ali et al., 2015). the similar result was reported by akbar et al. (2009) who evaluated, found variation among maize varieties, and identified high yielding maize varieties. different researchers have reported significant amount of variability in different maize populations including top-crosses and open pollinated varieties (sampoux et al., 1989). grzesiak (2001) also observed considerable genotypic variability among various maize genotypes. the coefficient of variation (cv) for grain yield was observed high (28.7%), the higher cv indicated that there was a higher influence of environments in the expression of character i.e., grain yield. the grain yield varied with sowing dates. optimum sowing date resulted in higher grain yield than early and late planting dates (otegui et al. 1995). the highest yield was found when maize varieties were sown in august (lal, 1973; saberi, 2014). maize grown in the winter has a larger yield potential than maize grown in the rainy season. insects, international journal of applied biology, 5(2), 2021 70 diseases, and weeds are not a serious problem throughout the winter, but parrots are a nuisance during the maturity period. during the winter season, the crop receives more sunlight, has a higher rate of photosynthesis, and assimilates more nutrients. in the winter, fertilizer use efficiency is higher. during the winter season, these conditions contribute to increased grain production. the impacts of planting dates and genotypes on each other were found to be highly significant (table 3). the highest grain yields were found in rml4/rml-17 (7392 kg) followed by rml-32/rml17 (6606 kg/ha) and across-9944/across9942 (5004 kg/ha) in february planting. the genotype s99tlyq-b (2084 kg/ha) produced the lowest yield when planted in november. in our study maize genotypes responded differently with planting dates for grain yield. similar results were reported by lauer et al. (1999), graybill et al. (1991) and fairey (1980). the grain yield of maize is the most important and complex quantitative character controlled by numerous genes. the gain yield of maize under different environment conditions may be due to both environmental and genetic effect. different sowing dates might cause different environmental conditions from emergence to seed filling (dahmardeh, 2012). table 2. effect of different genotypes and date of planting on grain yield of maize (kg/ha) at rampur, chitwan, during 2013/14. sn factors levels grain yield (kg/ha) 1 genotypes 1. s99tlyq-b 2979 2. rml-4/rml-17 4837 3. rml32/rml-17 4846 4. across-9944/ across 9942 3029 f-test ** 2 dates of planting 1. april (baisakh) 3913 2. may (jesth) 3370 3. june (aasad) 4475 4. july (shrawan) 4255 5. august (bhadra) 5565 6. september (aswin) 3559 7. october (kartik) 3954 8. november (mangsir) 2572 9. december (paush) 3019 10. january (magh) 3238 11. february (falgun) 5266 12. march (chaitra) 3889 grand mean 3923 international journal of applied biology, 5(2), 2021 71 f test ** lsd (0.05) 2234.1 cv (%) 28.7 ** significant at the 0.01 probability level table 3. interaction effect of different genotypes and date of planting (month) on grain yield (kg/ha) of maize at rampur, chitwan during 2013/14. sowing months genotypes rml-4/rml17 rml32/rml-17 across 9944/ across-9942 s99tlyq-b 1. april (baisakh) 5225 4843 2624 3353 2. may (jesth) 3897 4174 2057 3353 3. june (aasad) 6894 5784 2314 2907 4. july (shrawan) 5541 4934 3375 3169 5. august (bhadra) 7392 6606 5004 3258 6. september (aswin) 3219 3483 3792 3741 7. october (kartik) 3554 5690 4186 2388 8. november (mangsir) 2775 3177 2252 2084 9. december (paush) 3722 3653 2396 2305 10. january (magh) 4217 3897 2252 2584 11. february (falgun) 6649 6520 3698 4198 12. march (chaitra) 4963 5384 2400 2809 mean 4837 4846 3029 2979 grand mean 3923 f test ** lsd (0.05) 2234.1 cv (%) 28.7 ** significant at the 0.01 probability level international journal of applied biology, 5(2), 2021 72 conclusions during august planting, the genotypes rml-4/rml-17, rml-32/rml17 and across9944/across-9942 produced the highest grain yields, while qpm genotype s99tlyq-b produced the highest grain yield in february. based on the result of this experiment it can be concluded that the higher grain yields in hybrids rml-4/rml-17, rml-32/rml-17, and open pollinated variety across-9944/across-9942 can be obtained by planting them in early winter season especially in september and qpm genotype s99tlyq-b in spring season especially in february at chitwan condition of nepal. acknowledgements the authors are thankful to national maize research program, rampur, chitwan, nepal for providing experimental field. nepal agricultural research council was highly acknowledged for research funding. references akbar, m., saleem, m., ashraf, m.y., hussain, a., azhar, f.m. & ahmad, r. 2009. combining ability studies for physiological and grain yield traits in maize at two temperatures. pakistan journal of botany, 41(4): 1817-1829. ali, s., inamullah, a., jan, m. d. & habibullah, m. 2015. yield response of maize (zea mays l.) hybrids sown on various dates during kharif in peshawar-pakistan. journal of environment and earth science, 5: 13-17. carangal, v.r., ali, s.m., koble, a.f., rinke, e.h. & sentz, j.c. 1971. comparison of s1 with testcross evaluation for recurrent selection in maize. crop science, 11: 658-661. dahmardeh, m. 2012. effects of sowing date on the growth and yield of maize cultivars (zea mays l.) and the growth temperature requirements. african journal of biotechnology, 11(61): 12450-12453. fairey, n.a. 1980. the effects of hybrid maturity, date of planting and date of harvesting, on growth and development of forage maize. canadian journal of plant science, 60:1367–1375. gomez, k. a. & gomez, a. a. 1984, statistical procedures for agricultural research. 2nd ed. new york . john wesley and sons, 680p. graybill, j.s., cox, w.j. & otis, d.j. 1991. yield and quality of forage maize as influenced by hybrid, planting date, and plant density. agronomy journal, 83:559–564. grzesiak, s. 2001. genotypic variation between maize (zea mays l.) single-cross hybrids in response to drought stress. acta physiologiae plantarium, 23(4): 443-456. lal, r.1973. effects of seed bed preparation and time of planting on maize (zea mays l.) in western nigeria. experimental agriculture, 9: 303-313. https://doi.org/10.1017/s0014479700010097. international journal of applied biology, 5(2), 2021 73 lauer, j.g., carter, p.r., wood, t.m., diezel, g., wiersma, d.w., rand, p.e. & mlynarek, m.j. 1999. corn hybrid response to planting date in the northern corn belt. agronomy journal, 91:834–839 mccormick, s. j. 1971. the effect of sowing date on maize (zea mays l.) development and yields of silage and grain. proceedings of the agronomy society of new zealand i: 51-65. moac. 2009/10. statistical information on nepalese agriculture. government of nepal. ministry of agriculture and cooperatives (moac), agri-bussiness promotion and statistics division, singh darbar, kathmandu, nepal. moad. 2013. statistical information on nepalese agriculture 2012/2013. agri-business promotion and statistics division. ministry of agriculture and development (moad), singha durbar, kathmandu nepal. nmrp .2011. annual report 2010-11. national maize research program (nmrp), rampur, chitwan, nepal. nmrp. 2012. annual report 2011-12. national maize research program (nmrp), rampur, chitwan, nepal. nmrp. 2014. annual report 2013-14. national maize research program (nmrp), rampur, chitwan, nepal. otegui, m.e., nicolini, m.g., ruiz, r.a. & dodds, p.a. 1995. sowing date effects on grain yield components for different maize genotypes. agronomy journal, 87: 29-33. saberi, a. 2014. the effects of sowing date on yield of silage corn hybrids. journal of advanced botany and zoology, v1i3:1-4. https://doi.org/10.15297/jabz.v1i3.03. sampoux, j.p., gallais, a. & lefort-buson, m. 1989. s1 value combined with topcross value for forage maize selection. agronomie, 9(5): 511-520. steel, r.g.d. & torie j.h. 1980. principals and procedures of statistics. in: a biometrical approach. mcgrew-hill publishing company, ny. tanaka, a. & hara, t.1974. studies on the nutriophysiology of maize plant 10. grain yield as affected by sowing date. field crop abstracts, 27: 565. contact : manish kumar pal ramanpal273@gmail.com 18 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. issn : 2580-2410 eissn : 2580-2119 evaluation of different plant leaf extracts against mustard aphid [lipaphis erysimi (kalt.)] in rapeseed field *1manish kumar pal, 1 kapil kafle, & 2 jiban shrestha 1 tribhuvan university, institute of agriculture and animal science, lamjung campus, lamjung, nepal 2 nepal agricultural research council, kathmandu, nepal article history received 28 september 2020 accepted 31 december 2020 keyword mustard aphid, plant extracts and rapeseed introduction rapeseed (brassica campestris l.var. tori; family: brassicaceae) is the best oilseed crop and has the highest acreage among all the oilseed crops grown in the country, i.e.85% (ghimire et al., 2000). in nepal, the total area under rapeseed cultivation is 173254 ha, its production is 152263 mt and productivity is 879 kg/ha (cbs, 2014). in spite of the importance of oilseed crops, the average productivity (0.87 t/ha) in nepal is low as compared to that of the world of 1.28 t/ha (norp, 2014). among many factors responsible for low yield, open access international journal of applied biology abstract rapeseed is highly vulnerable to attack by various insect pests. the use of botanicals for control of insect pests is an eco-friendly practice, more compatible with the environmental components, and non-hazardous to human beings. this experiment was conducted to evaluate the effects of different plant leaf extracts against mustard aphid (lipaphis erysimi kalt.) from november 2016 to march 2017 at research field of institute of agriculture and animal science, lamjung campus, lamjung, nepal. five different plant leaf extracts derived from neem, bakaino, hattibar, khirro, and bojho, along with cypermethrin 10% ec@ 1.5 ml/l of water; and control (without treatment) were evaluated against mustard aphids. the experiment was conducted in a randomized complete block design with three replications. morang-2 variety of rapeseed was planted in the field and the rapeseed plant was sprayed with prepared plant leaf extracts on 30 days after sowing (das), 45 das and 60 das, and aphid number was counted after 5, 10 and 15 days of each spray from 10 cm apical shoot. the results showed that the application of cypermethrin 10% ec @ @ 1.5 ml/l of water significantly reduced the aphid population followed by the complete mixture and the mixture without khirro. the highest grain yield (1436.75 kg/ha) was found in complete mixture treated plots. it was concluded that all these plant leaf extracts showed insecticidal properties against aphid in rapeseed crops and successfully be integrated as a part of integrated pest management. international journal of applied biology, 4(2), 2020 19 insect pests play a significant role in reducing the yield and this crop is attacked by about 25 species of insect pests resulting in both quantitative and qualitative losses varying from 4550% (pradhan et al., 1960). among them mustard aphid is the most destructive insect pest (biswas et al., 2000). the yield losses of 27-69% due to attack of aphid (bakhetia and brar, 1983) and 15% reduction in its oil content (verma and singh, 1987). chemical insecticides still remain the key tool for the control of this pest. farmers spray insecticides in their fields indiscriminately which causes phytotoxicity, resistance in pest, destruction of beneficial organisms, disruption of agro-ecosystem, human health hazards, and environmental pollution (mcintyre et al., 1989). with several investigations, application traditional organic insecticides recommended as the best alternative to control mustard aphid (khurana et al., 1989). botanicals are comparatively less toxic, less expensive, and also safe for beneficial organisms. among 2400 species of bio-active plants in the world, almost 324 are found in nepal (neupane, 2005). these abundant naturally occurring biologically active plants appear to have a prominent role in the development of future commercial pesticides in nepal, not only for increased productivity but for the safety of the environment and public health. the objective of this experiment was to evaluate the insecticidal properties of different plant leaf extracts against mustard aphid in lamjung, nepal. materials and methods experimental site the experiment was conducted at the horticultural farm of institute of agriculture and animal science (iaas), lamjung campus, lamjung, nepal. this place has a humid tropical climate with an annual rainfall of 280 cm. the geographical position of the farm is at the latitude of 28° 8' 41"n and longitude of 84° 24' 43" e and elevation of 610 masl. experimental design, treatments and crop management the experiment was conducted during the winter season of 2016/17. the field was prepared by plowing, disking and leveling and seeds were sown in each plot with spacing of 30 cm row to row and 10 cm plant to plant spacing. seeds of rapeseed variety morang-2 were sown on 21 st november in 2m×2.1m size plots in a randomized complete block design with 7 treatments and 3 replications. all the crop management practices were followed as recommended by the national oilseed research program, nawalpur, sarlahi. table 1. detail of treatments used in the experiment s.n. detail of treatments 1 t1: complete mixture: mixture of all plants leaf extract derived from neem, bakaino, hattibar and khirro 2 t2: mixture of all plants leaf extract without neem 3 t3: mixture of all plants leaf extract without bakaino 4 t4: mixture of all plants leaf extract without hattibar 5 t5: mixture of all plants leaf extract without khirro 6 t6: chemical i.e. cypermethrin 10% ec 7 t7: control (water spray) international journal of applied biology, 4(2), 2020 20 preparation of leaf extracts the leaves of neem (azadiractin indica), bakaino (melia azedirach), hattibar (agave americana), khirro (sapium insigne), bojho (acorus calamus), and tobacco (nicotiana tabaccum) were chopped separately of 1-2 cm long. these chopped leaves of neem, bakaino, hattibar, khirro @ 150 g while bojho and tobacco leaf @ 75g were used for the preparation of extracts in fresh cow urine (3 litres in each extract). total five extracts were prepared, one by mixing all the leaf while the remaining four extracts were prepared excluding one ingredient in each mixture but keeping tobacco and bojho in all five extracts. the prepared extracts were decomposed for 1 month period by mixing it once in each week. preparation of spray the well-decomposed plant extract was filtered with the muslin cloth and then mixed the filtrate with water at a 1:4 ratio while cypermethrin 10% ec @ 1.5 ml/l of water and then sprayed with the help of hand sprayer (2 lit capacity) at 15 days interval after 1 month (30 days) of sowing three times i.e. 30das, 45das, and 60das. the spray was prepared as below; first spray (30 das):1200 ml water and 300 ml plant leaf extract second spray (45 das):1600 ml water and 400 ml plant leaf extract third spray (60 das):2000 ml water and 500 ml plant leaf extract. similarly, chemical i.e. cypermethrin was prepared as below; first spray (30 das):1.5 ml chemical/ 1 litre of water second spray (45 das): 3 ml chemical / 2 litre water third spray (60 das):4 ml chemical/2.5 litre water data observation the population of mustard aphid was observed and recorded at 5 th , 10 th and 15 th days of each spray from 10 cm apical twig (centre branch) of randomly selected 5 plants of each plot. statistical analysis data were statistically analyzed. analysis of variance for all data was analyzed using the genstat (version 14 th edition; vsn international, hemel hempstead, uk). the significant differences between genotypes were determined using the least significant difference (lsd) test at 1% or 5% level of significance (gomez and gomez, 1984; shrestha, 2019). international journal of applied biology, 4(2), 2020 21 results and discussion effect of plant extracts on reduction of aphid after first spray the effects of different plant leaf extract on aphid population after the first spray was given in table 4. during the first spray, the chemical treated plot was found to be almost free from the attack of mustard aphid while the control plot showed the highest incidence of aphid in all readings. among plant extracts, the complete mixture reduced the aphid number statically the same as chemical. at 15 days after spraying, the application of cypermethrin 10% ec @ @ 1.5ml/l of water significantly reduced the aphid population (0.07) followed by the completemixture (1.20) and mixture without khirro (2.73). biswas (2013) was also found that the chemical treated plot has a minimum number of aphid than locally prepared plant extracts and control treatments. table 1. effects of different plant leaf extracts on aphid population after first spray treatments number of aphid/plant (10 cm apical shoot) 5dasp 10dasp 15dasp complete mixture 1.60 ab 1.73 ab 1.20 a mixture without neem 4.67 ab 7.20 b 10.67 bc mixture without bakaino 4.53 ab 6.00 ab 5.13 ab mixture without hattibar 3.53 ab 4.53 ab 3.87 ab mixture without khirro 2.47 ab 3.53 ab 2.73 ab cypermethrin 10% ec 0.00 a 0.20 a 0.07 a control 9.27 b 14.27 c 14.87 c grand mean 3.72 5.35 5.5 f test * ** ** sem ± 1.58 1.26 1.6 lsd (0.05) 4.87 3.87 4.94 cv (%) 73.6 40.7 50.5 dasp: days after spraying, *significant at 0.05 level, ** significant at 0.01 level effect of plant extracts on reduction of aphid after second spray the effects of different plant leaf extract on aphid population after the second spray was given in table 2. during second spray, altogether of aphid count in each treatment increases as compare to the previous spray reading. the trend of aphid incidence was the same which reflects that the complete mixture can better control mustard aphid than other selected extracts. along with this, it was also analyzed that the effectiveness of plant extracts decreased with passing time of spray which can be justified by an increased in aphid count from 5 dasp to 15 dasp. bhatt and gc (2005) also reported that the effectiveness of botanicals to reduce the aphid population was significantly higher until 5 days of spray and decreases gradually after that. kafle (2015) also found that the effectiveness of insecticides decreased with increasing time of spray. international journal of applied biology, 4(2), 2020 22 table 2. effects of different plant leaf extracts on aphid population after second spray treatments number of aphid /plant(10 cm apical shoot) 5dasp 10dasp 15dasp complete mixture 0.20 a 0.66 a 0.20 a mixture without neem 4.80 b 9.47 b 9.66 b mixture without bakaino 3.60 ab 4.33 ab 5.20 ab mixture without hattibar 1.73 ab 2.53 a 3.73 a mixture without khirro 0.80 a 1.67 a 1.13 a cypermethrin 10% ec 0.07 a 0.67 a 0.00 a control 9.87 c 18.47 c 18.00 c grand mean 3.01 5.31 5.42 f test ** ** ** sem ± 0.80 1.09 1.10 lsd (0.05) 2.48 3.35 3.40 cv (%) 46.5 35.5 35.3 dasp: days after spraying, ** significant at 0.01 level effect of plant extracts on reduction of aphid after third spray the effects of different plant leaf extract on aphid population after the third spray was given in table 4. during the third spray also chemical and complete mixture resulted in maximum aphid reduction than other treatments and had statically similar effect while mixture without neem and control-treated plots showed least effectiveness, remaining other extracts had statically similar effects. the result revealed that excluding neem from the extract mixture (i.e. without neem), the effect was the same as the control which signifies that neem in the mixture must be needed for effective results. table 3. effects of different plant leaf extracts on aphid population after third spray treatments number of aphid /plant (10 cm apical shoot) 5dasp 10dasp 15dasp complete mixture 0.13 ab 0.13 a 0.20 a mixture without neem 4.40 bc 5.93 bc 4.20 b mixture without bakaino 2.60 ab 1.8 ab 2.66 ab mixture without hattibar 0.53 ab 1.13 ab 1.60 ab mixture without khirro 0.00 a 0.46 a 0.06 ab cypermethrin 10% ec 0.00 a 0.00 a 0.00 a control 7.80 c 7.80 c 8.20 c grand mean 2.21 2.47 2.56 f test ** ** ** sem ± 0.86 1.10 0.64 lsd(0.05) 2.67 3.39 1.99 cv (%) 68 77.4 43.7 dasp: days after spraying, *significant at 0.05 level, ** significant at 0.01 level international journal of applied biology, 4(2), 2020 23 saikia et al. (2000) also reported that leaf extract of neem in the plant extracts caused significant mortality of aphid which results in almost the same effect as chemical. the earlier works on the use of plant extracts have been also concluded the same result by pandey et al. (1987). effect of plant extracts on grain yield of rapeseed the effects of different plant leaf extracts on grain yield of rapeseed were given in figure 1. the application of the complete mixture produced the highest grain yield (1436.75 kg/ha) and the control plot produced the lowest grain yield (1126.90 kg/ha). this finding was supported by biswas (2013) and bhatta et al. (2019) who found the use of plant leaf extracts reduced the aphid population and increased the grain yield of rapeseed. the complete mixture produced a higher yield (but not statically different) even though the chemical which was highly effective to reduce aphid population, because of more pollinators and availability of nutrients through the complete mixture. the plots treated with plant extracts and chemical showed that yield was statically at par, it was because of less no of aphid than etl (15 aphids/plant of 10 cm twig) in those plots during all the spray which leads to almost similar yield. figure1. effect of different plant leaf extracts on mean grain yield of rapeseed. (w/o: mixture without) conclusion the incidence of mustard aphids can be managed by spraying the mixture of plant leaf extracts derived from neem, bakaino, hattibar, khirro, and bojho. the complete mixture gave the best results next to the chemical in term of aphid number reduction and higher yield of rapeseed. the use of the complete mixture is an eco-friendly management practice; it is as emerging solution against mustard aphid. this information would be helpful to the mustard growers in nepal. international journal of applied biology, 4(2), 2020 24 acknowledgements authors acknowledge the institute of agriculture and animal science (iaas), lamjung campus, nepal for providing all research facilities to conduct this study. references bakhetia, d.r.c. & brar, k.s.1983. losses of rapeseed/mustard due to lipaphis erysimi (kalt.). international rapeseed conference in paris, 6:1142-1146 bhatta, k., chaulagain, l., kafle, k. & shrestha, j. 2019. bio-efficacy of plant extracts against mustard aphid (lipaphis erysimi kalt.) on rapeseed (brassica campestris linn.) under field and laboratory conditions. syrian journal of agricultural research, 6(4): 557-566 biswas, g.c. 2013.comparative effectiveness of neem and synthetic organic insecticide against mustard aphid. bangladesh journal of agricultural research 38(2):181-187 biswas, g.c., das, g.p., begum s. & islam, s. 2000. resistance of three brassica species to the aphid (lipaphis erysimi).bangladesh journal of zoology, 28(1): 145-151 cbs. 2014. central bureau of statics, 2013/14. statistical year book of nepal ghimire, t. b., chaudhary, r. n. & ray, s.p. 2000. quantification of yield limiting constraints in toria production. annual report, nor, pp.61 gomez, k.a. & gomez, a.a. 1984. statistical procedures for agricultural research. 2nd edn.international rice research institute, college, laguna, pp. 680. kafle, k. 2015.management of mustard aphid (lipaphis erysimi).international journal of applied science and biotechnology, 3(3): 537-540 khurana, a.d. & batra, g.r. 1989. bioefficiency and persistence of insecticides against lipaphis erysimi (kalt.). journal of insect science, 2(2): 139-145 mcintyre, a.n., allison, h. & pebnab, d.r. 1989.pesticides: issues and options for new zealand. ministry of environment,wellington, new zealand, pp.168 neupane, f.p. 2005. jadibuti dwara kira niyantran.sajha prakashan : pulchowk, lalitpur norp. 2014. annual report .national oilseed research program. nawalpur, sarlahi, nepal http://agri-research-journal.net/sjaren/?p=2063 http://agri-research-journal.net/sjaren/?p=2063 http://agri-research-journal.net/sjaren/?p=2063 international journal of applied biology, 4(2), 2020 25 pandey, n. d., singh , y. p. & tripathi , r. a. 1987. effect of certain plant extract against l. erysimi kalt. under lab condition. indian journal of entomology, 49: 238-242. pradhan,s.m., totwani, g. & sarup, f. 1960. control schedule of mustard crop particularly against mustard aphid in india oilseed. india oilseed journal, 4:125-144. saikia,p., das, d. & salikia, l. 2000. evaluation of botanicals and fish oil formulation against bean aphid (aphis craccivora) koch. journal of agricultural science society. north east, india, 13:79-80. shrestha, j. 2019. p-value: a true test of significance in agricultural research. https://www.linkedin.com/pulse/p-valuetest-significance-agricultural-research jibanshrestha/ verma, s.n. & singh, o.p. 1987. estimation of avoidable losses to mustard by the aphid (lipaphis erysimi kalt.) in madhya pradesh. indian journal of plant protection, 15: 8789. http://www.linkedin.com/pulse/p-valuetest-significance-agricultural-researchhttp://www.linkedin.com/pulse/p-valuetest-significance-agricultural-research contact : santosh kumar yadav sy607667@gmail.com 1 abstract soil fertility e valuation is a n important aspect in the context of sustainable agricultural production of an area. this study was carried out to find the soil fertility status of the dhanushadham municipality, dhanusha, nepal located at 26°52’n, 86o02’e using gps and gis. a total of 61 soil samples were c ollected based on land use, slope, and aspects with the use of google earth pr o (gep) a nd arcgis. the soil was analyzed for its texture, soil ph, total nitrogen, available phosphorous, and potassium. the majority of the study area (36.35%) has loam soils followed by (28.17%) sandy loam soil. the soil ph was strongly acidic to nearly neutral with ph value s ranging from 5.2 to 7.5. the soil organic matter (som) varied from 1.14% to 1.83% with a mean value of 1.52% and was medium in most of the soil. t he mean total nitrogen, available phosphorus, and a vailable potassium were 0.08 %, 120.96 kg/ha, and 146.13 kg/ha re spectively. the total nitrogen was found to be medium in content, phosphorus is high in content and potassium is low in content in the study area. to maintain the nutrient status of soil, use of organic manure, reduced use of che mical fertilizers, and different soil management practices should be adopted in this area. the study can c onclude that gps and gis based soil fertility mapping helps farmer s, scientist s, planners, researchers, and stude nts in pr oviding soil test base d fertilizer recommendation for sustainable soil management as well as developing future research strategies in the farm. issn : 2580-2410 eissn : 2580-2119 use of gis for spatial mapping of soil fertility in dhanusha, nepal santosh kumar yadav 1*, karuna kafle 1, abichal poudel 1, rashil gelal 1, bhusan adhikari 1, biplov oli 1, koshraj upadhyay 2 1 g.p kiorala college of agriculture and research centre (gpcar), morang, nepal . 2 department of plant breeding, institute of agriculture and ani mal science, gauradaha, jhapa, nepal. introduction soil fertility is the most important factor for determining soil productivity. fertile and productive soil enhances life whereas, unfertile and unproductive soil decreases soil productivity leading to hunger and fa mine. however different calamities like soil erosion, landslides, flood, and other different soil degradati on factors cause a serious problem in rapid nutrient depleti on and pose a great challenge in soil fertility management. therefore, soil fertility evaluation and its spatial distribution play an important decision-making role in planning a particular land-use system (oli et al., 2020). the evaluation of soil fertility is the measurement of available plant nutrients and estimation of soil capacity to maintain conti nue the supply of plant nutrients for open access international journal of applied biology keyword arcgis; nypa fruticans; soil fertility; soil organic matter; spatial variation article history received november 11, 2021 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 2 agricultural practices. among the various techniques for a soil fertility evaluation, soil testing is the most widely used technique in the world (havlin et al., 2010).the analysis of physical and chemical properties of soil and soil testing is obligatory for the sustainable management of soil (panda, 2010). soil testing provides information regarding the nutrient availability in soils which forms the basis for the fertilizer recommendati ons for maximizing crop yields. the texture, structure, color are important physical parameters of soil while the soil ph, organic matter, macro, and micronutrients are important chemical parameters of soil. these soil parameters are determined only after analyzing them in the laboratory (khadka et al., 2018a).mapping the status and the spatial distribution of soil fertility plays an important role in the sustainable land use planning process (khadka et al., 2018b). the use of new technologies like gis and gps makes it easy in describing the spatial variability of so il fertility for a larger area. the geographic information system (gis) is a powerful software tool for collecting, storing, retrieving, transforming, and displaying data (cone, 1998). collection of soil samples by using gps is very important for prepa ring the soil fertility maps (mishra et al., 2013). the geogra phical information system (gis) is a potential tool to access, retrieve and manipulate voluminous data of natural resources which is difficult to handle manually. the gps and gis technologies have been adopted in agriculture for better management of land and other resources for sustainable crop production (palaniswami et al., 2011). method and methodology study area the study was carried out in dhanushadham municipality dhanusha, nepal (figure 1). the study area is located at 26°52’n, 86o02’e. the climate in the study area is the subtropical type with hot and wet summer and cool, dry winter. average air temperature ranges from a minimum of about 9°c in winter to a maximum of about 40 oc in summer. since rainfall is not uniform throughout the year, more than 85% of rainfall occurs d uring four months (june-september). however, the stability of the landscape for the development of soil is affected by the variability and intensity of rainfall. the major crops of dhanushadham municipality are rice, wheat, mustard, maize, sugarcane, mung, lentils, vegetables, and pulses. figure 1: location of study area international journal of applied biology, 6(2), 2022 3 soil survey methods the total of 61 soil samples (0-20 cm depth) was collected from different location of dhanusha district. the exact locations of the samples were recorded using a handheld gps receiver for the preparation of thematic soil fertility maps and imported to arcgis softwa re. the random method based on the variability of the land was used to c ollect soil samples. a detailed soil survey of the study area was carried out on grid map prepared using arc gis software. the soil sampling locations were decided based on the land system units, morphology, land use condition, and geology. the soil samples were collected for laboratory analysis of soil parameters that include particle size distribution, soil ph, total nitrogen, organic matter, available phosphorus, and available potassium. figure 2. dhanushadham and soil sampling points inside the study area table 1. different rating classes of soil test data adapted soil testing laboratory, nepal sn soil parameters units very low low medium high very high 1 organic matter % <0.75 0.75-1.5 1.5-3.0 3.0-5.0 >5 2 total nitrogen % <0.03 0.03-0.07 0.07-0.15 0.15-0.25 >2.5 3 available p2o5 kg/ ha <11 11-28 28-56 56-112 >112 4 available k2o kg/ ha <55 55-110 110-280 280-500 >500 international journal of applied biology, 6(2), 2022 4 table 2. rating of soil ph adapted by soil testing laboratory, nepal soil reaction (ph) ph range extremely acidic <4.5 very strongly acidic 4.5-5.0 strongly acidic 5.0-5.5 moderately acidic 5.5-6.0 slightly acidic 6.0-6.5 nearly neutral 6.5-7.5 slightly alkaline 7.5-8.0 moderately alkaline 8.0-8.5 strongly alkaline 8.5-9.5 very strongly alkaline 9.5-10 extremely alkaline >10 laboratory soil analysis soil samples collected from the field were air-dried in shade, crushed, and sieved for physic-chemical laboratory analysis. the parameters tested and methods used are given in the table 1 and table 3. table 3. soil parameters and laboratory soil test methods test parameters methods particle size fraction and texture hydrometer (bouyoucos, 1962) and texture classification (usda texture triangle) soil ph 1:2.5 soil water suspension (jackson, 1967) soil organic matter content (%) walkley and black (walkely and black, 1934) total nitrogen content (total n %) micro-kjeldahl (bremner and mulvaney, 1982) available phosphorus (p2o5 kg/ha) olsen (olsen et al., 1954) available potassium (k2o kg/ha) ammonium acetate (jackson, 1967) statistical analysis and soil fertility mapping latitude, longitude, and the data resulting from the soil analysis were entered into the attri buted table in ms-excel professional plus 2016 and processed in arcgis10.8 software. thematic s oil fertility maps and geospatial tools i.e. ordinary kriging (ok) and interpolation (cressie, 1992) was preferred for predicting values for not sampled locations. ordinary kriging is one of the advanced geostatistical tools that create a surface by using spatial correlation from a scattered set of points by incorporating their properties international journal of applied biology, 6(2), 2022 5 (economic and social research institute, 2001). descriptive statistics (minimum value, maximum value, mean and standard deviation) of soil parameters were computed in the ms-excel professional plus 2016 and arcgis10.8 package. rating (very low, low, medium, high, and very high) of determini ng values of different parameters were based on soil testing laboratory, nepal. arc map10.8 with geostatistical analyst extension of arcgis software was used to prepare spatial distribution map of soil parameters, while interpolation method employed was ordinary kriging with stable semi -variogram. results and discussion soils were analyzed for mechanical composition, ph, organic matter, total nitrogen, available phosphorus, and potassium. soil texture soil texture is the proporti on of sand, silt, and clay and is a permanent attribute of soils. crop producti on, land use, and land management are greatly affected by soil texture and also it has a direct role in water infiltration, drainage, and nutrient retention (brady & weil, 2008). the soil texture of the first horizon (0-20cm) was determi ned by the laboratory test using the textural model. seven different classes of soil texture were identified in the study area dominated by loan soil (36.65%), followed by sandy loam (28.17%), silty loam (13.6%), silty clay loam (6.97%), clay(6.83%), sandy clay loam ( 6.34%) and clay loam (1.74%). the highest area was occupied by loam soil (36.35%) and the lowest was occupied by clay loam (1.74%) which is presented in table 4 and figure 4. sandy loam, loa m, sandy clay loam site is good for the cultivation of different kinds of crops however special care shoul d be taken for soil conservation and water management in the sloppy areas. table 4. area occupying different soil textural classes in dhanushadham municipality, dhanusha, nepal texture class area (ha) (percentage) loam 3328.31 36.35% sandy loam 2579.17 28.17% silty loam 1245.21 13.6% silty clay loam 638.28 6.97% clay 625.88 6.83% sandy clay loam 581.25 6.34% clay loam 159.89 1.74% total area 9157.99 100% international journal of applied biology, 6(2), 2022 6 figure 4. spatial distribution of soil texture in dhanushadham municipality, dhanusha, nepal soil ph soil ph refers to the acidity and alkalinity of the soil influenced by the presence of different acid and base-forming cations. soil ph is an important chemical parameter of soil that affects nutrient availability solubility, and plant growth (brady and weil, 2008). higher soil acidity causes loss of nutrients like ca, mg, increases phototoxic elements such as al and mn; reduces the activity of beneficial microbes, destroys the structure of soil leading to unfavorable soil conditions (nduwumuremyi, 2013). hence, soil acidity should be reduced to improve soil fertility for sustainable soil management. therefore, agricultural lime should be applied to make soil ph adequate. the soil ph in the study area varied from 5.2 to 7.5 with a mean value of 6.30 and a standard deviation of 0.69. the soil ph class was distributed f rom strongly acidic to nearly neutral (table 2). the majority of the study area (28.01%) is under moderately acidic soil followed by slightly acidic (28%) and strongly acidic (25.25%). about 18.24% area has nearly neutral soils as shown in figure 5 and table 5. table 5. area occupying different ph classes in dhanushadham municipality, dhanusha, nepal soil reaction (ph) area (ha) (percentage) moderately acidic 2564.8 28.01% slightly acidic 28.01 28% strongly acidic 2312.96 25.25% nearly neutral 1716.15 18.74% total area 9157.99 100% international journal of applied biology, 6(2), 2022 7 figure 5. spatial distribution of soil ph in dhanushadham municipality, dhanusha, nepal soil organic matter soil organic matter (so m) plays a vital role in crop performance and maintaining soil health as it improves different physical, biological and chemical properties (hoyle et al., 2011). som has a direct influence on water holding capacity due to its ability to absorb large amounts of water. the organic matter content varied from 1.14 to 1.83% (table 6) with a mean value of 1.52%. the distribution of organic matter ranged from low to medium, but mostly medium was prevalent (figure 6). about 62.85% of the total area has a medium range of so m content and 37.15% of the total area has a low range of som a s shown in table 6 and figure 6. figure 6. spatial distribution of soil organic matter in dhanushadham municipality, dhanusha, nepal international journal of applied biology, 6(2), 2022 8 total nitrogen nitrogen is one of the major nutrients requi red for the growth and development of plants. nitrogen imparts dark green color in plants and promotes vegetative growth (bloom, 2015). plants get nitrogen from the soil which is added naturally to the soil from n-fixation by soil bacteria and soil legumes. plants absorb nitrogen from the soil as nitrate (no3-) and ammonium (nh4+). the yellowing of plant leaves, retarded growth, reduced apical dominance, and poor vegetative growth of plants are some symptoms of nitrogen deficiency (bloom, 2015). the total nitrogen content in the study area varied from 0.08% to 0.13% with the mean value of 0.08% as shown in figure 7 and table 6. nitrogen content was in the range of low to medium. about 68.91% of the total area has a medium range of nitrogen and 31.09% has a low range of nitrogen in the study area. figure 7. spatial distribution of total nitrogen in dhanushadham municipality, dhanusha, nepal available phosphorous phosphorous, next to nitrogen, is often the most limiting nutrients for the growth and development of plants (sharma et. al., 2017). phosphorous provides a means of using the energy harnessed by photosynthesis to drive the metabolism in plants. phosphorous also helps in the production of legumes, as it increases the activity of nodule bacteria, which fix nitrogen in the soil. the available phosphorous content in the study area ranged from 40.02 to 282.59 kg/ha with a mean value of 120.95 kg/ha as shown in figure 8 and table 6. the available phosphorus content was in the range of medium to very high. international journal of applied biology, 6(2), 2022 9 about 59.83kg/ha of the total area has a high range of phosphorus followed by 36.83 kg/ha has very high phosphorus and 3.34 kg/ha has a medium range of available phosphorus in the study area. figure 8. spatial distribution of available phosphorous in dhanushadham municipality, dhanusha, nepal available potassium potassium (k) is the third most important essential element next to n and p that limits plant productivity (havlin et al., 2010). it plays a vital role in synthesis of amino acids and proteins from ammonium ion which is absorbed from the soil. the available potassium content in the study area ranged from 169.87 to 358.68 kg/ha with a mean value of 146.13 kg/ ha. the available potassium content ranged f rom very low to very high, domina ted by medium range of phosphorous. about 69.82% of the total area has a medium range of phosphorus followed by low (20.49%), high (6.45%), very high (2.5%) and 0.74% has a very low range of available potassium in the study area as shown in figure 9 and table 6 and 7. international journal of applied biology, 6(2), 2022 10 figure 9. spatial distribution of available potassium in dhanushadham municipality, dhanusha, nepal table 6. soil fertility status of dhanushadham municipality, dhanusha, nepal soil parameters units min. value max. value mean value standard deviation soil ph ph scale 5.2 7.5 6.30 0.6889 som % 1.14 1.83 1.52 0.1441 total nitrogen % 0.05 0.13 0.08 0.0147 available p2o5 kg/ ha 40.02 282.50 120.96 60.6420 available k2o kg/ ha 8.44 1026.89 173.79 104.6048 note: som denotes soil organic matter international journal of applied biology, 6(2), 2022 11 table 7. area occupying different classes of soil parameters in dhanushadham municipality, dhanusha, nepal soil parameters som total nitrogen available phosphorus available potassium very low na na na 69.502 (0.74%) low 3402.21 (37.15%) 2848.94 (31.09%) na 1876.21 (20.49%) medium 5755.78 (62.85%) 6309.05 (68.91%) 308.24 (3.37%) 6389.75 (69.82%) high na na 5476.70 (59.80%) 592.19 (6.45%) very high na na 3373.04 (36.83%) 230.34 (2.5%) total area(ha) 9157.99 9157.99 9157.99 9157.99 note: % =percentage, (na) = not applicable conclusion the soil nutrient status of dhanusadham was mapped using gis which can facilitate the management of nutrients. the soil ph of the study area was mostly acidic and ranged from 5.2-7.5. so m, an integral part of soil nutrient va ried from very low to medium throughout the municipality. there was no significant difference in som content in different land types. the total nitrogen content over the municipality ranged from low to medium with a grand mean of 0.08% which is low. there was no significant difference in n-content over land use. similarly, the available phosphorous ranged from medi um to very high over municipality with a mean value of 120.95 kg/ha, and available potassium varied from very low to very high throughout the municipality with a mean of 146.13 kg/ha. acknowledgement we are grateful to pmapmp, piu dhanusha for providing us intern opportunity. also, we would like to extend deepest gratitude towards our advisor mr. biplov oli for his consistent and guidance. furthermore, we are grateful towards our college administration (pu, gpcar). international journal of applied biology, 6(2), 2022 12 references bloom, a. j. 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(2018b). soil fertility assessment and mapping of regional agricultural research station, parwanipur, bara, nepal. journal of nepal agricultural research council, 4, 33–47. https://doi.org/10.3126/jnarc.v4i1.19688 mishra, a., das,d., saren, s., 2013. preparati on of gps and gis based soil fertility maps for khurda district, odisha. indian agriculturist 57(1): 11-20. nduwumuremyi, a. (2013). soil acidification and lime quality: sources of soil acidity, effects on plant nutrients, efficiency of lime and liming requirements. research and reviews: journal of agriculture and allied science, 2(4), 26-34. oli, b., lamichhane, s., & oli, k. (2020). use of gis in soil fertility mapping of rapti municipality, chitwan, nepal. journal of agriculture and applied biology, 1(2), 64–73. https://doi.org/10.11594/jaab.01.02.04 https://doi.org/10.1016/j.pbi.2015.03.002 https://doi.org/10.1111/j.1745%207939.1998.tb02089.x https://doi.org/10.15406/apar.2018.08.00317 https://doi.org/10.3126/jnarc.v4i1.19688 https://doi.org/10.11594/jaab.01.02.04 international journal of applied biology, 6(2), 2022 13 palaniswami, c., gopalasundaram, p., & bhaskaran, a. (2011). application of gps and gis in sugarcane agriculture. sugar tech, 13(4), 360–365. https://doi.org/10.1007/s12355011-0098-9 panda, s. c. (2010). soil management and organic farming. agrobios. bharat printing press, jodhpur, india. sharma, l., bali, s., & zaeen, a. (2017). a case study of potential reasons of increased soil phosphorus levels in the northeast united states. agronomy, 7(4), 85. https://doi.org/10.3390/agronomy7040085 walkley, a., & black, i. a. (1934). an examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. soil science, 37(1), 29-38. https://doi.org/10.1007/s12355-011-0098-9 https://doi.org/10.1007/s12355-011-0098-9 https://doi.org/10.3390/agronomy7040085 contact : abdul mollah abmoljayabiotek@gmail.com 60 abstract microgreens are plants from the group of vegetables or herbal plants that are harvested at the age of 7-21 days. as a horticultural crop, broccoli (brassica oleracea l.) is a type of vegetable that belongs to the cabbage family (brassicaceae). this study aims to determine the effect of natural zpt and differences in growing media on chlorophyll content, number of stomata and stomata openings of broccoli microgreens. this study consists of 2 treatment factors. the first factor of the planting medium consists of husk charcoal (m1). cocopeat (m2) and rockwool (m3). the second factor of natural zpt consists of no zpt (z0), coconut water (z1) and bean sprout extract (z2). the combination treatment of rockwool growing media and natural zpt of bean sprout extract (m3z2) gave the best results on parameters of chlorophyll a, b and total content, each of which was 14 days old. while the combination of treatment with cocopeat growing media and natural zpt of bean sprout extract (m1z2) gave the best results on parameters of chlorophyll a, b and total content, each of which was 21 days after planting. the best parameters for the number of stomata and the area of stomata openings were found in the combination treatment of cocopeta growing media and natural zpt of coconut water (m1z1), 15.00 and 115.13 m2, respectively. issn : 2580-2410 eissn : 2580-2119 effect of natural growth regulatory substance (pgr) and differences of planting media on chlorophil content number of vegetablestomates and area of vegetablestomates microgreens broccoli (brassica oleracea l.) abdul mollah 1*, achmad roihan 1, julianti halik 1, mur fadhil saputra 1 1 department of agricultural cultivation, faculty of agriculture, hasanuddin university. tamalanrea campus jl. pioneer of independence km. 10, makassar 90245 introduction indonesia is a tropical country with a wide area, with high agroclimatic variations, which is a potential area for horticulture development for both lowland and highland crops. this agro-climatic variation is also beneficial for indonesia, because the fruit, vegetable and flower season can last throughout the year. public awareness of health is increasing along with the development of a healthy lifestyle in the millennial era, especially since society is faced with the covid-19 pandemic that has shocked the world. consuming healthy food is increasingly becoming a trend and is the choice of many people as a contemporary healthy open access international journal of applied biology keyword broccoli, microgreens, chlorophyll. article history received 05 october 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 61 lifestyle. the demand for food sources of vitamins and minerals that are important for increasing immunity, such as vegetables and fruits, continues to increase. (rena, 2016). vegetables and fruit are one of the food menus that support health because they have the nutrients and nutritional values that the body needs. microgreens is a plant from a group of vegetables or herbal plants that are harvested at the age of 721 days. this plant can be harvested by cutting the stems using sharp scissors just above the surface of the growth medium. so that what is consumed from microgreens is the stem, cotyledons and the first leaves that have fully opened except for the roots. this group of plants has been very popular in developed countries since the 1980s due to awareness about healthy living including consuming healthy foods, namely microgreens. types of plants such as broccoli, basil, coriander, arugula and wheat are used as microgreens which are harvested after reaching 48 cm in height (lobiucet al., 2017). as a horticultural crop, broccoli (brassica oleracea l.) is a type of vegetable plant belonging to the cabbage family (brassicaceae). broccoli is often referred to as "super vegetable" because it has a high nutritional or nutritional content, namely it contains folate, vitamin c, vitamin c, and vitamins. k, iron and high in potassium (potassium), and contains antioxidant compounds such as sulforaphane. sulforaphane is formed from methionine and glucorafanine which are precursors or precursors to sulforaphane (rajiman, 2018). according to (maspary, 2011) rice husk charcoal is porous, light, not dirty, but has a low ability to absorb water and has large pores so that evaporation in the media is also higher which causes a lot of nutrients to be lost before being absorbed by plants. . the advantage of cocopeat as a planting medium is because of its high water absorption capacity between 6-8 times its dry weight so it saves water and nutrients, supports root growth quickly so it is good for nurseries. in line with the research of prihmantoro (2013), generally cocopeat has micro pores that are able to inhibit the movement of larger water causing higher water availability, cocopeat also has macro pores that are not too dense so that air circulation is very good for plant roots. nugraha et al. (2018), in his research on the effect of the composition of cocopeat and husk charcoal on the growth and yield of kailan (brassica oleracea var. acephala) plants, concluded that the composition of the growing medium cocopeat 75% + 25% husk charcoal gave the best effect on plant height, fresh weight of stover , dry weight of stover and root loss ratio of kailan plants. coconut water as a natural growth regulator is cheaper and easier to obtain. according to lawalata (2011) that coconut water contains auxin and cytokinin hormones. both hormones are used to support the cell division of coconut embryos. coconut water has a high enough potassium content up to 17%. arif's research (2016) reported that the concentration of sprout extract had a significant effect on the growth of the moon orchid. sprout extract concentration of 150 mg/liter gave the best growth results in lunar orchids. the results of arif's research (2016), showed that the use of 150 g/l touge extract gave the highest yield on moon orchids. products from the broccoli plant that we know and are often sold in the market are only the final product in the form of broccoli. people's interest in vegetables increases with the times and lifestyles of people who begin to implement a healthy lifestyle, it encourages people to consume fresh and quality vegetables. the length of time to harvest broccoli encourages farmers to make various innovations, one of which is by planting bro ccoli microgreens. broccoli plants that can be consumed do not have to be the final product, but can also be consumed in the form of microgreens. international journal of applied biology, 5(2), 2021 62 research methods this research was conducted at the experimental garden of the faculty of agriculture, hasanuddin university, the laboratory of biochemistry and plant physiology of the department of agronomy, and the biochemistry laboratory of the faculty of animal husbandry, hasanuddin university. research will last 8 months tool the tools that will be used are tools content chlorophyll meter (ccm 200+), microscope, slide glass, sprayer, harvesting shears. paranet cameras, analytical balances, and writing instruments. ingredients the materials that will be used in this research include broccoli microgreens vegetable seeds, husk charcoal growing media, cocopeat, rockwoll, natural growth regulators made from coconut water and bean sprout extract, ab mix nutrients and others. methods seed selection and germination the selection of good seeds is done by soaking in water then selecting the seeds that sink, after that they are soaked for 24 hours and ripened for 24 hours for germination. preparation of planting media the growing media used consisted of husk charcoal (m1), cocopeat growing media (m2) and rockwool growing media, each of which was put into 3 planting containers which were repeated 3 times so 9 total planting media. natural zpt immersion seeds that have been selected and have gone through the germination process will be divided into 3 parts and then soaked with natural zpt consisting of no zpt/plain water (z0) as a dick, coconut water (z1) and bean sprout extract (z2), each soaked for 20 minutes. planting planting is done by spreading the seeds on the planting medium according to each treatment. for husk charcoal and rokcwool media, after spreading the seeds, add a little planting medium to the top of the seeds until the seeds are not visible and the root growth is strong downwards. for rokwool media before spreading the seeds, rokwool media made small holes in the media using a pointed tool. maintenance maintenance in the form of watering is done every day until harvest in the morning and evening. in addition, at the age of 3 days after planting, ab mix nutrition specifically for vegetables was given with a concentration of 700-800 ppm. international journal of applied biology, 5(2), 2021 63 observation the parameters observed were the content of chlorophyll a, b and total as well as the opening area and the number of stomata. leaf chlorophyll content was observed using content chlorophyll meter (ccm 200+) on young leaves. observations were made on: the content of chlorophyll a (μmol. m-2), chlorophyll b (μmol. m-2) and total leaf chlorophyll ((μmol. m-2), by using the formula: leaf chlorophyll content = a + b (ccl)c, where a, b and c are constants and ccl is leaf chlorophyll index data read on ccm 200+. stomata sampling was performed using clear nail polish and clear tape. sampling was carried out at 14 dap. leaf stomata components include; measurement of stomatal density was carried out using a magnification of 40 times with a field of view diameter of 0.52 mm .2. stomata opening area (μm2), calculated using the formula: stomata opening area = π x length of stomata x width of stomata measurement of the stomatal opening area was carried out using a magnification of 100 times with a field of view diameter of 0.52 mm .2. sampling was carried out on vegetables microgreens broccoli 14 days after each treatment. results and discussion the results of the research data on the effect of giving natural zpt and the difference in media on chlorophyll content, stomata opening area and number of stomata. chlorophyll a gambar 1. average chlorophyll a (μmol. m-2) on p behavior n zpt al ami dan difference media aged 14 hst and 21 hst figure 1 shows that the highest average chlorophyll a of plant age 14 days after planting was found in the treatment of rockwool growing media with natural zpt of bean sprout extract (m3z2) which was 59.21 μmol. m-2 while the lowest mean of chlorophyll a at the age of 14 days after planting was found in the treatment of cocopeat growing media with natural zpt (m2z0), which was 40.7 μmol. m-2 for the highest average chlorophyll a plant age of 21 days after planting, it was found in the treatment of husk charcoal growing media with natural zpt of bean sprout extract (m1z2), which was 127.78 μmol. m-2while the lowest average chlorophyll a at the age of 21 days after planting was found in the treatment of cocopeat growing media with natural zpt (m2z0), which was 87.71 μmol. m-2 150 100 50 96.97 44.5 115.28 127.78 119.02 94.02 102.59 109.86 57.42 53.52 87.71 40.7 91.71 43. 04 45. 22 46.38 52. 44 59. 21 m1z0 m1z1 m1z2 m2z0 m2z1 m2z2 m3z0 m3z1 m3z2 treatment 14 hst 21 hst international journal of applied biology, 5(2), 2021 64 chlorophyll b figure 2. average chlorophyll b (μmol. m-2) on treatment natural zpt and difference media aged 14 hst and 21 hst figure 2 shows that the highest average chlorophyll a at the age of 14 days after planting was found in rockwool growing media with natural zpt of bean sprout extract (m3z2), which was 51.24 μmol. m-2 while the lowest mean of chlorophyll a at the age of 14 days after planting was found in the treatment of cocopeat growing media with natural zpt (m2z0), which was 49.03 μmol. m-2 for the highest average chlorophyll a plant age of 21 days after planting, it was found in the treatment of husk charcoal growing media with natural zpt of bean sprout extract (m1z2), which was 62.89 μmol. m-2while the lowest average chlorophyll a plant age of 21 days after planting was found in the treatment of cocopeat growing media with zpt tanap natural (m2z0) 55.3 μmol. m-2 total chlorophyll figure 3. average total chlorophyll (μmol. m-2) on treatment natural zpt and the difference between the ages of the media 14 hst and 21 hst figure 3 shows that the highest average chlorophyll a at the age of 14 days after planting was found in rockwool growing media with natural zpt of bean sprout extract (m3z2), which was 107.14 μmol. m-2while the lowest average of chlorophyll a at the age of 14 days after planting was found in the treatment of cocopeat growing media with natural zpt 70 60 50 40 30 20 10 56.87 49.46 60.31 51.04 62.89 50.53 55.3 49.03 55.95 49.29 56.35 57.88 49.54 49.67 59.29 50.4 60.87 51.24 m1z0 m1z1 m1z2 m2z0 m2z1 14 hst 21 hst m2z2 m3z0 m3z1 m3z2 250 200 150 176.89 192.94 181.51 153.64 170 142.03 147.02 149.92 160.73 100 89.49 50 105 100.28 107.14 84.98 87.76 90.35 91.72 99 0 m1z0 m1z1 m1z2 m2z0 m2z1 m2z2 m3z0 m3z1 m3z2 14 hst 21 hst international journal of applied biology, 5(2), 2021 65 (m2z0), which was 84.98 μmol. m-2. for the highest average chlorophyll a at the age of 21 days after planting, it was found in the treatment of husk charcoal growing media with natural zpt of bean sprout extract (m1z2), which was 192.94 μmol. m-2. while the lowest average chlorophyll a at 21 days after planting was found in the treatment of cocopeat growing media with natural zpt (m2z0), which was 142.03 μmol. m-2. number of stomata and stomata opening area the results of the analysis of non-stomatal area variance showed that the interaction between the treatment of planting media and natural zpt had a significant effect on the stomatal opening area. table 1. average number of stomata and stomatal opening area (μm2) to natural zpt treatment and the difference in media at the age of 14 days after planting. parameter perlakuan jumlah stomata luas bukaan stomata (μm2) m1z0 7,67 46,05 𝑎 𝑟 m1z1 13,33 73,27 𝑎 𝑞 m1z2 15,00 115,13𝑎 𝑝 m2z0 5,33 52,33𝑎 𝑝 m2z1 6,67 65,94𝑎 𝑝 m2z2 8,33 81,64𝑎 𝑝 m3z0 9,33 60,71𝑎 𝑝 m3z1 17,00 94,20𝑎 𝑝 m3z2 13,00 83,73𝑎 𝑝 description: numbers followed by the same letter in the same column not significantly different based on duncan's test at the bnj test confidence level 0.05. table 1 shows that the highest average number of stomata is found in the treatment of husk charcoal growing media with natural zpt of bean sprout extract (m1z2), which is 15.00, while the lowest average number of stomata is found in the treatment of cocopeat growing media with no zpt (m2z0) which is 5 ,33. for the highest average stomata opening area, it was found in the treatment of husk charcoal planting media with natural zpt of bean sprout extract (m1z2), which was 115, 13 μm2while the lowest average stomata opening area was found in the treatment of cocopeat growing media with no zpt (m2z0), which was 52.33 μm2. conclusion the combination treatment of rockwool growing media and natural zpt of bean sprout extract (m3z2) gave the best results on parameters of chlorophyll a, b and total content, each of which was 14 days old. while the combination of treatment with cocopeat growing media and natural zpt of bean sprout extract (m1z2) gave good results the best on the parameters of chlorophyll a, b and total content, each of which was 21 days after planting. the best parameters for the number of stomata and the area of stomata openings international journal of applied biology, 5(2), 2021 66 were found in the combination of treatment with cocopeta growing media and natural zpt of coconut water (m1z1), 15.00 and 115.13 m, respectively.2. references arif, m, murniati, and ardian. 2016. test some natural growth regulators against the growth of rubber seeds (hevea brasiliensis muell arg) sleeping eye stum. let's faperta 3 (1). lawalata, imelda jeanette. 2011. provision of several zpt combinations to regeneration of gloxinia plants from in vitro stem and leaf explants. j exp. life science. 1(2) :8387. lobiuc, a., vasilache, v., pintilie, o., stoleru, t., burducea, m., oroian, m., & zamfirache, m.m. 2017. blue and red led illumination improves growth and bioactive compounds contents in acyanic and cyanic ocimum basilicum l. microgreens. molecules, 22(12), 2111. microgreens production with low potassium content for patients with impaired kidney function. nutriens, 10(6), e675. prihmantoro and indriani, 2003. effect of media type and intensity of fertilization on the growth of seedlings of love wave anthurium (anthurium plowmanii). faculty of agriculture eleven march. surakarta rajiman. 2018. effect of natural growth regulatory substances (pgr) on yield and onion quality. stpp magelang agricultural extension department, yogyakarta. renna, m., castellino, m., leoni, b., paradiso, vm, & santamaria, p. (2016). wuryaningsih. s. 2008. ornamental plants planting media. contact : hilda. a. emmanuel-akerele akatahhilda@gmail.com 38 abstract physicochemical analysis of ekemazu stream in delta state, nigeria was carried out to assess the water quality between september 2014 and june 2015. physicochemical parameters were analyzed according to standard methods for examination of water and wastewater. the turbidity of all the samples analysed across the seasons showed that the value was highest at the effluent discharge point at the peak of the flood season and least in the upstream at the setting – in of raining season in the following range: upstream; 12 ± 1 ntu to 25 ± 0 ntu, effluent discharge point; 121 ± 0 ntu to 423 ± 39 ntu, domestic activities point; 85 ± 2 ntu to 373 ± 40 ntu and in the downstream; 70 ± 0 ntu to 341 ± 44 ntu. all other parameters analyzed followed similar trend (highest in the effluent discharge point) either during the peak of flood season or peak of raining season and least in the upstream sample at other seasons. the statistical analysis of the difference in the physicochemical parameters of the upstream samples and the values obtained in effluent discharge point, domestic activities point and the downstream were all significant (p<0.05). this research clearly showed that some physicochemical parameters of the stream are higher than the who acceptable limit. this is due to the release of heavily polluted effluent into the stream, seasonal variations as well as some human activities in the water body resulting to high pollution of the stream.metal pollution in the air, without experiencing damage to the leaf stomata fruit issn : 2580-2410 eissn : 2580-2119 physico-chemical analysis of power plant effluent discharge on ekemazu stream in delta state, nigeria hilda. a. emmanuel-akerele1 1 department of biological sciences, anchor university lagos introduction water is one of the most fundamental elements which make man and the entire ecosystem exist on this planet earth (akatah et al., 2018). water is a vital component of the development of an area and as such, human settlement is to a large extent dependent on the availability of reliable sources of water preferably in close proximity to the settled localities (edet et al., 2012). the availability of drinking water is an indispensable feature for preventing epidemic diseases and improving the quality of life (daniel et al., 2012). water also plays very important role in many industries such as power plant, food and cosmetics industries, as well as pharmaceutical industries, hence, its quality is very crucial (ogbonna, 2010). however, these industries and some human activities heighten surface water’s exposure to various kinds of pollution with deleterious chemicals and pathogenic microorganisms (prerana and vijay, 2013). as such, most rural villages in developing countries have poor access to safe clean water supply. chemicals which cause stream open access international journal of applied biology keyword effluent; physico-chemical; pollution; stream article history received november 30, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 39 pollution from industries are ammonia, phosphate, hydrocarbon compounds, herbicides and pesticides (suha, 2013). polluted water bodies pose a very great health risk to people using such water for drinking, bathing, irrigation of crops which are eaten raw, fishing and recreational activities (decker & simmons, 2013). the components and characteristics of polluted water bodies are dependent on the source(s) of its pollutants (osibanjo et al., 2010). the industrial sector is responsible for the release of hazardous substances/wastes resulting in pollution of the receiving water bodies with hazardous components (govindaraju et al., 2011). water bodies polluted by effluent from combined cycle power plant contain compounds such as ammonia, phosphate, dissolved solids, free chlorine, phosphate, and chlorides (mishra, 2010). a study by osibanjo et al., (2010) on the impact of the industries on surface water quality of river ona and river alaro in oluyole industrial estate, ibadan nigeria showed that the concentrations of nitrate in the polluted rivers ranged between 3.00 and 8.55 mg/l, chloride ranged between 7.48 and 11.78 mg/l, total phosphorus ranged between 2.14 and 3.57 mg/l, total solids ranged between 260 and 520 mg/l and oil grease ranged between 381.20 and 430.80 mg/l. another study by andargachew, (2013) on the effects of dashen brewery wastewater treatment effluent on the bacteriological and physicochemical quality of shinta river in gondar, north west ethiopia show that the ph varied between 6.6 and 7.9, total suspended solids fluctuated between 6 and 42 mg/l, the total dissolved solids between 73 and 201 mg/l and the biochemical oxygen demand (bod) ranged between 3.8 and 23.0 mg/l. govindaraju et al., (2011) on their study of waters around kudan kulam nuclear power plant showed that the maximum values of ph was 8.25, temperature was 29oc, salinity was 32.4 ppt, dissolved oxygen (do) was 5.9 ppt, chloride was 1.7 mg/l, phosphate was 59.98 mg/l, calcium was 3300 mg/l, nitrate was 0.79 mg/l and nitrite was 7.03 mg/l. the normal value (acceptable limit) of nitrate is 50 mg/l and free chlorine is 5 mg/l (who, 2011). while conductivity, total dissolved solids and ph is 1000 µs/cm, 500 mg/l and 6.5 to 8.5 respectively (daniel et al., 2012). govindaraju et al., (2011) on their study of waters around kudan kulam nuclear power plant showed that the maximum values of ph was 8.25, temperature was 29oc, salinity was 32.4 ppt, dissolved oxygen (do) was 5.9 ppt, chloride was 1.7 mg/l, phosphate was 59.98 mg/l, calcium was 3300 mg/l, nitrate was 0.79 mg/l and nitrite was 7.03 mg/l.the normal value (acceptable limit) of nitrate is 50 mg/l and free chlorine is 5 mg/l (who, 2011). while conductivity, total dissolved solids and ph is 1000 µs/cm, 500 mg/l and 6.5 to 8.5 respectively (apha, 2005). however, toxicological effects of ammonia are observed at exposures above 200 mg/kg body weight and chloride concentrations in excess of about 250 mg/l can give rise to detectable taste in water. the components of polluted water bodies have significant effects to both the aquatic and human lives. some of the components contain nutrients that support the growth of some harmful microorganisms which are threat and sometimes fatal to human lives (guecker et al., 2000). ekemazu stream located precisely in independent power plant camp in okpai oluchi, ndokwa east local government area of delta state in this research serves as the major source of water for domestic purposes, fishing and recreational activities for the residents in the area is heavily polluted by the independent power plant located along the course of the stream with compounds of ammonia, phosphate, sodium hypochlorite, hydrazine, and dissolved salts from their water and steam treatment processes. the stream is also polluted international journal of applied biology, 6(1), 2022 40 with domestic wastes from the power plant residential lodge and from the local community residents, living near the stream. water for different purposes has its own requirements of composition and purity hence each body of water has to be analysed regularly in order to monitor its quality and ascertain if it is safe to use for domestic and/or industrial purposes (mishra, 2010). it is on these backgrounds that this research is being carried out. the aim of this study was to analyse the physicochemical parameters of the stream during the different seasons of the year. materials and methods period of study this research was conducted between the months of september, 2014 and june, 2015. the choice of the months of the year is to examine the effect of the peak of the flood season, setting in of the dry season, peak of the dry season, and the raining season on the bacteriological and physicochemical properties of the stream study area the study area was a section of ekemazu stream located in independent power plant camp near okpai oluchi, delta state nigeria. the ekemazu stream is the source of water for domestic purposes, for the residents as well as fishing. it receives effluent from independent power plant which contains compounds of ammonia, phosphate, sodium hypochlorite, hydrazine, and dissolved salts from the water and steam treatment process. the polluted water body also contains domestic wastes from the power plant residential lodge and from the local community residents, living near the stream. figure1. map showing the various sampling point international journal of applied biology, 6(1), 2022 41 sample collection, transportation and storage the water samples were collected from four sampling points along the stream in duplicates with sterile containers and designated with numbers 1 to 4. a retort stand clamp mounted on a stick was used to hold the neck of the sampling container tight and the cover of the container aseptically removed with the mouth of the bottle faced upstream. then, the neck was dipped downwards about 30cm below the water surface till the container was completely filled and the cover carefully replaced (decker and simmon, 2013). the samples were collected once in each month of the study period. sampling point 1: upstream (before effluent discharge and human activities point). sampling point 2: midstream a (effluent discharge point). sampling point 3:midstream b (about 1500metres from effluent discharge point): bathing and domestic activities point. sampling point 4: downstream (about 1000metres away from sampling point 3). the samples were transported to the laboratory in ice bag to keep the samples cold and to avoid multiplication of the microorganisms during the period (kasich et al., 2013). analysis of physico-chemical parameters the physico-chemical parameters analysed are conductivity, total dissolved solids (tds), temperature, total suspended solids (tss), turbidity, hydrogen ion concentration (ph), total iron, free chlorine, total hardness, phosphate, nitrate, ammonia and chloride and biochemical oxygen demand (bod). conductivity, total dissolved solids (tds) and temperature the conductivity and total dissolved solids (tds), are closely related and they were determined using conductivity meter, ec71, product of hach lange company, loveland usa. the meter was first calibrated with standard solutions (potassium chloride) of 147 µs/cm and 1413 µs/cm. the conductivity value was determined by dipping the electrode into the sample and waited for a few minutes to stabilize, then the reading taken and recorded in µs/cm. the total dissolved solids (tds) were determined by selecting the tds option from the menu bottom and the value recorded in mg/l. the temperature was measured using the electronically in-built thermistor in the conductivity meter. the value was displayed on the meter and recorded in degree celsius. total suspended solids (tss) the total suspended solids (tss) were analysed using spectrophotometer dr 5000 by hach lange company, loveland, usa. the stored program number for tss concentration analysis 630 was selected and the wavelength 810 nm automatically selected. then the sample was well shaken and about 10ml added into a sample cell. 10ml of demineralised water was added into a second sample cell and used as the blank. the blank was inserted into the cell holder to obtain the zero concentration, then, the prepared sampl e was also inserted into the cell holder to obtain the total suspended solids concentration in mg/l. turbidity the turbidity was determined with turbidimeter, 2100 n, product of hach lange, loveland, usa. the instrument was first calibrated with the manufacturer’s standard solutions, formazin (hexamethylene tetramine). the sample was well shaken and about 50 international journal of applied biology, 6(1), 2022 42 ml added into a sample cell and inserted into the cell holder of the meter, then waited for stability. the turbidity values were recorded in nephelometry turbidity unit (ntu). hydrogen ion concentration (ph) the hydrogen ion concentration (ph) was determined using sension1 ph meter, product of hach lange, loveland, usa. the meter was first calibrated with buffer solutions; 4, 7 and 10. the ph values of the samples were obtained by dipping the electrode into the sample, waited for stability and the value recorded. total iron the total iron concentration was analysed using ammonium thioglycolate and thioglycolic acid reagent in dr 5000 spectrophotometer manufactured by hach lange company, loveland, usa. the stored program number for total iron concentration analysis 260 was selected and the wavelength 562 nm automatically selected. 2 ml of the sample was added into a clean 25-ml sample cell and demineralised water was used to fill the cell to 25-ml mark to obtain 12.5 dilution factors. then, the prepared sample was inserted into the cell holder of the meter and the zero concentration obtained. the reagent was added into the sample, allowed to react for five minutes and the concentration in mg/l obtained was multiplied by the dilution factor in order to obtain the concentration of the original (undiluted) sample. free chlorine the concentration of the free chlorine in the sample was analysed using the n, ndiethyl-p-phenylenediamine (dpd) free chlorine reagent in a dr 5000 spectro-photometer product of hach lange company loveland, usa. the stored program number for free chlorine concentration analysis 80 was selected and the wavelength 530 nm automatically selected. 10ml of the sample was added into a sample cell and the zero concentration obtained from the spectrophotometer. the contents of the dpd free chlorine reagent was then added into the sample and the concentration in mg/l obtained from the spectrophotometer. the contents of the dpd free chlorine reagent was then added into the sample and the concentration in mg/l obtained from the spectrophotometer within 20 seconds. total hardness the total hardness was analysed using the calcium and magnesium indicator (propionic acid), alkali solution for calcium and magnesium test (sodium hydroxide and nitrilotriethanol), ethylenediamine tetra acetate (edta), and ethylene glycol tetra acetic acid (egta) in dr 5000 spectrophotometer product of hach lange company loveland usa. the stored program number for magnesium hardness concentration analysis 225 was selected and the wavelength 522 nm automatically selected. 100 ml of sample was measured into 250 ml clean conical flask and 1 ml each of the indicator and the alkali solution were added into the sample and properly swirled to mix. then 25 ml each of the prepared solution was measure into three 25-ml sample cells. a drop of edta was added into the first sample solution and used as blank for magnesium hardness while a drop of egta was added into the second sample solution and used to obtain the concentration of the magnesium hardness in mg/l. this second sample was as well used as blank for calcium hardness. the stored program number for calcium hardness concentration analysis 220 was international journal of applied biology, 6(1), 2022 43 selected and the wavelength 522 nm automatically selected. then, the concentrate of calcium hardness was obtained from the third sample solution in mg/l. phosphate the phosphate concentration was determined using ascorbic acid, potassium pyrosulfate and sodium molybdate reagents in dr 5000 spectrophotometer product of hach lange, loveland usa. the stored program number for phosphate concentration analysis, 490 was selected and the wavelength 880 nm automatically selected. 10ml of sample was added into a sample cell and the zero concentration obtained from the spectrophotometer. then the content of the phosphate reagent was added into the sample and allowed to react for two minutes and the concentration in mg/l was recorded. nitrate the concentration of the nitrate was analysed using nitrate reagents (magnesium sulphate, potassium sulphate and sulfanilic acid compounds) in dr 5000 spectrophotometer by hach lange company loveland usa. the stored program number for nitrate concentration analysis, 351 was selected and the wavelength 507 nm automatically selected. 10ml of the sample was added into a sample cell and the zero concentration obtained from the spectrophotometer. then, the content of the nitrate reagent was added into the sample and shaken for one minute and allowed to stand for another two minutes for complete reaction. the concentration of the nitrate was then obtained from the spectrophotometer and recorded in mg/l. ammonia the ammonia concentration was determined using mineral stabilizer (sodium tartrate and sodium citrate), polyvinyl alcohol, and nessler reagent in a dr 5000 spectrophotometer product of hach lange company, loveland, usa. the stored program number for ammonia concentration analysis 380 was selected and the wavelength 425 nm automatically selected. 25 ml of the sample was added into a 25-ml mixing graduated cylinder (prepared sample). 25 ml of deionised water was added into another 25-ml mixing graduated cylinder (the blank). three drops of mineral stabilizer (sodium tartrate and sodium citrate) was added into each cylinder, stoppered and inverted several times to mix. also three drops of polyvinyl alcohol was added to each of the cylinder stoppered and inverted several times to mix. then 1 ml of nessler reagent (potassium tetraiodomercurate (ii)) was added into each cylinder, stoppered and inverted several times to mix. they were allowed to react for one minute and each solution poured into sample cell. the blank was placed into the cell holder of the spectrophotometer and the light shield closed to obtain the zero concentration. the prepared sample was also placed into the cell holder, the light shield closed and the concentration in mg/l obtained and recorded. chloride the chloride concentration was determined using mercuric thiocyanate and ferric ion solution in a dr 5000 spectrophotometer, product of hach lange company, loveland, usa. the stored program number for chloride concentration analysis 70 was selected and the wavelength 455 nm automatically selected. a sample cell was filled with 25 ml of the sample and another cell with 25 ml of deionised water (the blank) 2.0 ml of mercuric thiocyanate was added into the each of the sample cells and swirled to mix. also 1.0 ml of international journal of applied biology, 6(1), 2022 44 ferric ion solution was added into each of the cells and swirled to mix. they were allowed to react for two minutes. the blank was placed into the cell holder and the light shield closed. after the two minutes reactions the zero concentration was obtained and the prepared sample was also placed into the cell holder, the light shield closed and the concentrations in mg/l obtained and recorded. biochemical oxygen demand (bod) the biochemical oxygen demand (bod) was determined using bod nutrient buffer solution (calcium chloride, magnesium sulphate, potassium phosphate, sodium phosphate and ferric chloride compounds) in dissolved oxygen meter, hq40d, product of hach lange loveland usa. the meter was calibrated with oxygen saturated water contained in a bod bottle. the sample dilution water was prepared by adding bod nutrient buffer solution into distilled and sterilized water contained in a sterile jug which was previously stored overnight in an incubator at 20oc for it to be saturated with oxygen and well shaken to dissolve the slurry. a serological pipette was used to add 20 ml, 40 ml, 60 ml and 100 ml of each of the samples into 300-ml bod bottles and labelled appropriately. the dilution water was added into each of the samples to the neck of the bod bottles. another 300 ml bod bottle was also filled to the neck with dilution water only to serve as the control. the initial dissolved oxygen reading of the samples and the control were measured using the dissolved oxygen meter and recorded as d1. enough dilution water was added into each of the bottles to the lip, capped and kept in an incubator at 20oc in the dark for five days. when the five days incubation was completed, the dissolved oxygen content in mg/l (dissolved oxygen remaining) in each bottle were determined and recorded as d2. the following formula was used to calculate the biochemical oxygen demand after five days (bod5). 𝐵𝑂𝐷𝑆 = 𝐷1 − 𝐷2 𝜌 where: d1 = dissolved oxygen of the diluted sample immediately after preparation. d2 = dissolved oxygen of the diluted sample after five days incubation at 20oc ρ = decimal volumetric fraction of the sample used which is calculated as 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 300 𝑚𝑙 statistical analysis independent sample t test was used to find the difference between the physicochemical parameters of the upstream samples and the values obtained in the effluent discharge point, domestic activities point and the downstream samples in all the seasons using spss 21.0. international journal of applied biology, 6(1), 2022 45 results vegetation and industry conditions of industrial areal physicochemical parameters the temperature of the stream in all the samples analysed across the seasons varied between 26.7 ± 0.3 oc at the peak of dry season in upstream sample and 38.9 ± 0.3 oc during the peak of raining season in effluent discharge point sample (table1 5) the conductivity of the stream in all the samples analysed across the seasons varied between 65.0 ± 0 µs/cm in upstream sample at the setting in of dry season and 370 ± 2 µs/cm in effluent discharge point sample during the peak of raining season (table1 5). the total dissolved solids of the stream in all the samples analysed across the seasons varied between 32 ± 0 mg/l in upstream sample at the setting in of dry season and 185 ± 1 mg/l in effluent discharge point sample during the peak of raining season (table1 5). the total suspended solids of the stream in all the samples analysed across the seasons varied between 11 ± 1 mg/l in upstream sample during the setting in of the raining season and 392 ± 30 mg/l in effluent discharge point during the peak of raining season (table1 5). the turbidity of the stream in all the samples analysed across the seasons varied between 12 ± 1 ntu in upstream sample at the setting in of the raining season and 423 ± 39 ntu in effluent discharge point during the peak of raining season (table 1 5). the ph of the stream in all the samples analysed across the seasons varied between 6.5 ± 0 in upstream sample at the setting in of the raining season and 8.9 ± 1 in effluent discharge point during the peak of raining season (table 1 5). the total iron concentration of the stream in all the samples analysed across the seasons varied between 7.2 ± 0.2 mg/l in upstream sample at the peak of dry season and 38.7 ± 0.1 mg/l in effluent discharge point at the setting in of raining season (table 1 5). the free chlorine concentration of the stream in all the samples analysed across the seasons varied between 0.00 ± 0 mg/l in the upstream sample throughout the whole seasons and 0.035 ± 0.005 mg/l in effluent discharge point at the peak of the flood season (table 1 5). the total hardness of the stream in all the samples analysed across the seasons varied between 2.35 ± 0.05 mg/l in upstream sample at the peak of the flood season and 5.6 ± 0.2 mg/l in effluent discharge point at the setting-in of the raining season (table 1 5). the phosphate concentration of the stream in all the samples across the seasons varied between 0.005 ± 0.005 mg/l in upstream sample at the setting in of the raining season and 0.57 ± 0.03 mg/l in effluent discharge point also at the setting in of the raining season (table1 5). the nitrate concentration of the stream in all the samples across the seasons varied between 0.89 ± 0.04 mg/l in upstream sample at the peak of dry season and 7.57 ± 0.26 mg/l in effluent discharge point at the peak of the flood season (table 1 5). the ammonia concentration of the stream in all the samples across the seasons varied between 0.00 ± 0 mg/l in upstream in all the seasons and 0.35 ± 0.03 mg/l in effluent discharge point at the setting-in of raining season (table 1 5). the chloride concentration of the stream in all the samples across the seasons varied between 0.14 ± 0 mg/l in upstream at the settingin of dry season (table 4) and 0.42 ± 0.06 mg/l in effluent discharge point at the peak of flood season (table 1 5). the bod5 concentration of the stream in all the samples across the seasons varied between 1.2 ± 0.1 mg/l in upstream at the peak of the dry season (table 3) and 9.5 ± 0.3 mg/l in effluent discharge point at the peak of flood season (table 1 5). the statistical analysis of the difference between the physicochemical parameters of the upstream samples and the values obtained in effluent discharge point, domestic activities point and the downstream using independent sample t – test showed that the results were all significant (p<0.05) at the peak of flood season and the setting – in of dry international journal of applied biology, 6(1), 2022 46 season and very highly significant (p<0.05) at the peak of the dry season and the setting – in of raining season. at the peak of the raining season, the difference with the effluent discharge point was very highly significant and highly significant (p<0.05) with the domestic activities point and the downstream. international journal of applied biology, 6(1), 2022 47 international journal of applied biology, 6(1), 2022 48 discussion the physico-chemical quality of ekemazu stream in delta state, nigeria was investigated in order to determine the impact of effluent discharge and seasonal variations on the stream. the results of the physicochemical parameters of the stream showed that the temperature was in the range of 26.8 to 39.2oc. this is a good temperature range for the growth of mesophilic micro-organisms (ogbonna, 2010). this result is similar to that of prerana and vijay (2013) on their study of physicochemical study of kanhan river water receiving fly ash disposal wastewater of khaperkheda thermal power station of india who recorded 24.2 to 35.3oc in their research. the value was highest in effluent discharge point, followed by domestic activities point, then the downstream point. upstream sample was the lowest. the high temperature results mostly from release of effluent with high temperature into the water body (akatah et al., 2018). international journal of applied biology, 6(1), 2022 49 the conductivity and total dissolved solids of 64 to 372 µs/cm and 32 to 186 mg/l recorded during the period of this research is within who, (2011) limit of 1000 µs/cm and 500 mg/l respectively. this is contrary to the research of prerana and vijay, (2013) who recorded conductivity of 380 to 2330 µs/cm and total dissolved solids of 225 to 1362 mg/l. in this research, although the conductivity and the total dissolved solids are within who acceptable limit the effluent discharge greatly influenced these two parameters because of high content of dissolved solids in the effluent (buecker, 2014). the results of the total suspended solids and turbidity of 8 – 422 mg/l and 10 – 462 ntu respectively are out of who (2011) acceptable limit of 0 mg/l and o ntu. the results of the total suspended solids is similar to that of andargachew and sahile, (2013) on their study of the effects of dashen brewery wastewater treatment effluent on the bacteriological and physicochemical quality of shinta river in gondar, north west ethiopia at the upstream sample of 6 mg/l, but higher than their record after discharge point which they recorded as 42 mg/l. suspended solids of effluent polluted water body can serve as attachment for microbes (daniel et al., 2012). it can also settle and deposit sand and grit into aquatic systems resulting in interruption of natural ecosystem and aquatic lives (kasich et al., 2013). the results of the ph were acidic in the upstream sample but alkaline in other samples (6.4 – 9.0). the result in this research is above who acceptable limit of 6.5 – 8.5. the acidic nature of the upstream sample could be attributed to forest fire, bacteria action on soil, lightning, (mishra, 2010). the alkalinity nature of the effluent discharge point, domestic activities point and the downstream samples could be attributed to the components of the effluent continuously released from the independent power plant such as ammonia, phosphate, sodium hypochlorite etc. which have high ph value (guecker, 2000). the iron content of 7.0 to 39.8 mg/l is high. high iron content serves as good nutrient and ecological nurture for microbes (govindaraju et al., 2011). this research showed that the iron concentration was highest in effluent discharge point. this can be attributed to release of effluent with high iron content resulting from water purification process in the power plant (suha, 2013). the results of other parameters such as the free chlorine, total hardness, phosphate, nitrate, ammonia, chloride and biochemical oxygen demand (bod) are within who acceptable range. however, the effluent release from the independent power plant greatly influenced the effluent discharge points, domestic activities point and the downstream. their concentrations in the stream do not pose any serious effect to human consumption, except the biochemical oxygen demand (kasich et al., 2013; ashraf et al., 2010). presence of ammonia in water is an indicator of possible bacterial, sewage and animal waste pollution. it can compromise disinfection efficiency, result in nitrite formation in distribution systems, cause the failure of filters for the removal of manganese and cause taste and odour problems (who, 2011). there was observed fluctuation in the physicochemical parameters analysed during the research period. apart from human activities in the stream and effluent discharge by the independent power plant, seasonal variation also influenced the water quality. this observation is similar to that of ogbonna, (2010).. conclusions this research clearly showed that some physicochemical quality of ekemazu stream in okpai-oluchi, delta state nigeria is higher than the who acceptable limit. this is due to release of heavily polluted effluent by the independent power plant into the stream as well international journal of applied biology, 6(1), 2022 50 as some human activities in the water body. the value of the total suspended solids, turbidity, iron, and biochemical oxygen demand (bod) can be used to classify ekemazu stream as a polluted water body and as such unfit for any human or domestic use/consumption in accordance to world health organisation water use guidelines standard. this is a gross violation of effluent discharge permit limits employed by the power plant. it is therefore recommended that the independent power plant should unlike their present method of only primary treatment of their wastewater, adequately treat their effluent to tertiary stage of waste treatment before discharging into the stream. it is also required that the environmental monitoring body, federal environmental protection agency, fepa, regularly inspect and certify the company’s effluent before their release into the water body in order to ascertain compliance by the company. this can be employed through enacting of rules and regulations that can guide against improper sanitary and hygiene practice, education of the masses on the implications of polluted water body and the need to live a healthy life. international journal of applied biology, 6(1), 2022 51 references akatah h.a; omonigho s.e; ibrahim a.g; seyinde d.o. (2018). physico-chemical and microbiological analyses of ikpoba river water, benin city, edo state, nigeria. international research journal of biological sciences 7(5): 1-5.issn 2278-3202. andargachew, b. and sahile, s. (2013). effects of dashen brewery wastewater treatment effluent on the bacteriological and physicochemical quality of shinta river in gondar, north west ethiopia: world environment 3(1): 29-36 apha, (2005). standard methods for the examination of water and wastewater. american public health association, washington dc 21pp ashraf, m.a., maah, m.j., yusoff, i. and mehmood, k. (2010). effects of polluted water irrigation on environment and health of people in jamber district kasur, pakistan. international journal of basic and applied science 10(3): 37-57 daniel, m.h., montebolo, a., bernades, m.c., ometto, j.p., decamargo, p.b., krusche, a.v., ballester, m.v., victoria, r.l. and martinelli, l.a. (2012). effects of urban sewage on dissolved oxygen, organic and inorganic carbon and electrical conductivity of small streams along a gradient of urbanization in the piracicaba river basin. water air and soil pollution 136: 189-206 decker, c. and simmons, k. (2013): surface water sampling, 4th ed. u.s. environmental protection agency, science and ecosystem support division, athens, georgia 22pp duncan, d., harvey, f. and walker, m. (2007). environmental protection authority guidelines: regulatory monitoring and testing water and wastewater sampling, environmental protection authority, south australia 58pp edet, e.j., etim, e.e. and titus, o.m. (2012). bacteriological and physicochemical analysis of streams water in nduetong oku community, uyo, akwa ibom state nigeria. international journal of modern chemistry 3(1): 65-73 govindaraju, m., kumar, p., selvaraj, m. and rajina, c. (2011). study on physicochemical parameters along the coastal waters around kudan kulam nuclear power plant: international journal of oceans and oceanography 5(1): 75-83 guecker, b., brauns, m., pusch, m.t. (2000). effects of wastewater treatment plant discharge on ecosystem structure and function of lowland streams. journal of the north american benthological society 25: 313-329 kasich, j.r., taylor, m., and nally, s.j. (2013): surface water field sampling manual for water column chemistry, bacteria and flows. 3rd ed. environmental protection agency ohio usa 41pp international journal of applied biology, 6(1), 2022 52 mishra, a. (2010). assessment of water quality using principal component analysis: a case study of river ganges. soviet journal of water chemistry and technology 32(4): 227234 ogbonna, d.n. (2010). seasonal dynamics of microbial population and physicochemical characteristics of a water body receiving industrial pollutants in port-harcourt, nigeria. agriculture and biology journal of nigeria 1(6): 1333-1339 osibanjo, o., adegbenro, p.d., and adewole, m.g. (2010). impact of industries on surface water quality of river ona and river alaro in oluyole industrial estate, ibadan nigeria: african journal of biotechnology 10(4):696 702 owa, f.n. (2014). water pollution. sources, effects control and management. international letters of natural sciences 8(2014):1-6 prerana, t. and vijay, c.n. (2013). physicochemical study of kanhan river water receiving fly ash disposal wastewater of khaperkheda thermal power station of india: international research journal of environment sciences 2(9): 10-15 suha, a.i. (2013). studying wastewater parameters of thermal power station: australian journal of basic and applied sciences 7(8): 565 – 570 who, (2011): guidelines for drinking water quality, 4th ed. world health organisation, geneva 541pp. contact : gopal saha saha.18@gmail.com 68 abstract the low productivity of aus rice in the tidal ecosystem of bangladesh is mainly due to the difficulties in nitrogen (n) management under high tidal water along with unavailability of irrigation water during the onset of rice cultivation. our present study demonstrated how the productivity of aus rice could be improved using appropriate seedling raising methods and n management practices. the study was laid out in split plot design consisting two factors viz., seedling raising methods (wet seed bed and dry seed bed) and n management practices (six n treatments) in three replications. results indicated that both the seedling raising methods and n management practices had significant effect on all the yield contributing characters of transplanted aus rice under tidal condition. specifically, the combination of seedlings raised in wet seed bed and fertilized with urea super granule (usg) at 10 days after transplanting (dat) i.e., n3w showed the best values for plant height (110.33cm), number of effective tillers hill-1 (14.60), days required 50% flowering (53.00 dat), days to maturity (84.33 dat), panicle length (23.37 cm), number of filled grain panicle-1 (88.13), 1000grain weight (43.17 g), grain yield (4.62 tha-1), straw yield (6.07 tha-1), biological yield (10.67 tha-1), and harvest index (43.17%). however, in considering the productivity/ grain yield, besides n3w, the whole urea application at land preparation along with wet seed bed (n1w) and usg application along with dry seed bed (n3d) also produced statistically similar results (p<0.01) and thus all these three combinations may be practiced for improving productivity and ensuring horizontal expansion of aus rice in the tidal ecosystem of bangladesh. issn : 2580-2410 eissn : 2580-2119 effect of nitrogen management and seedling raising methods on the productivity of aus rice under tidal ecosystem of bangladesh antara sarker, swadesh chandra samanta, & gopal saha department of agronomy, patuakhali science and technology university, dumki, patuakhali, bangladesh-8602 introduction rice (oryza sativa l.) is the most important staple food crops for over half of the world’s population and over 759.6 mt of rice was produced globally in 2017 (fao 2018). near about 90% of annual rice is produced and consumed in asia. the average yield in asia is low compared to global mean yield (haider 2018). more than two billion people in asia are dependent on rice for their livelihood (xiong et al., 2013). the demand for rice is increasing to meet the demand of rising population. and, the possible way to meet this increases demand by improvement of rice yield per hectare (liu et al., 2016). in bangladesh, about 75% of the total cropped area and over 80% of the total irrigated area is occupied by rice (bbs, 2013). thus, rice plays a vital role in the livelihood of the people of bangladesh. there are open access international journal of applied biology keyword aus rice, nitrogen management, wet seed bed, dry seed bed, tidal ecosystem. article history received 25 march 2020 accepted 14 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 69 three rice growing season viz. aus, aman & boro in bangladesh which are cultivated during kharif-1 (april–july), kharif-2 (july–november) and rabi (november–april), respectively. after the 1980s, aus production slowly began to lose priority to the farmers as they shifted to irrigated boro rice cultivation due to its higher yield (brri 2011). the government of bangladesh has given top priority for increasing the area and production of aus rice to reduce the pressure on electricity for irrigation needed for boro rice production during dry season. compare to boro, aus requires less irrigation and the plan is to cut dependency on the underground water for irrigation and arrest the fall in the water level in the aquifer. aus rice in this regard can create scope by scaling up production and shed dependency on the underground water (unb 2017). but the main drawback is the average yield of aus rice (2.16 t ha-1) which is lower than aman and boro rice (bbs 2013). using the latest high yielding varieties of transplanted aus rice along with improved cultivation techniques might be the best possible options to meet this productivity gap of aus rice. bangladesh occupies most lands of the great bengal plane of the ganges delta with affluent alluvial soils. and, the overall natural climate and geographic condition of bangladesh is blissful for growing aus rice. the main problem is insufficient rainfall in april-may with which wet nursery bed preparation and transplanting is very difficult. so, preparation of wet nursery bed and transplant aus rice seedlings using the tidal water may be a good option. since water is not easily available and time of aus planting uncertain, dry nursery bed may be alternative option by the farmers of tidal area. however, till date there is no such comparative study on the yield variability of transplanted rice between wet and dry nursery bed. besides, the management of nitrogenous fertilizer (either as basal or top dressing) is difficult due to high depth of water during cultivation period. nitrogen (n) is the key element in the production of rice. but, the n management in the tidal areas is always a great challenge. to improve rice yield supplementation of n fertilizer under most agro-ecosystem has been suggested (fageria and santos, 2014). nitrogen helps to increase rice productivity through improving leaf n concentration, photosynthetic rate, delaying leaf senescence, and increasing dry matter for grain filling (hasegawa et al., 1994). moreover, n may also play great role in improving panicle size, grain weight and reducing spikelet sterility (fageria, 2009). application of n fertilizer in rice has also been reported to significantly increase the grain and straw n uptake and n use efficiency (hassan et al., 2009). normally, farmers in the developing countries apply n in two ways. one is broadcasting of prilled urea (pu), which is the common practice in bangladesh and another is urea deep placement (udp) in the form of urea super granule (usg). it has been reported that compared to udp, the main problem of pu is the n volatilization loss as ammonia (rochette et al., 2013). basically the use efficiency of urea is very low and the recovery of n, especially in wetland rice field rarely exceeds 40% (de datta, 1989). however, minimizing of n losses in the environment was also possible by applying urea to plants through the foliage (giroux 1984 & millard and robinson 1990). in several researches it has been observed that most of the plants rapidly can absorb liquid urea and hydrolyze the absorbed urea in the cytosol subsequently ensure better assimilation in the plants’ system which help to increase n use efficiency (wittwer et al. 1963, nicoulaud and bloom 1996 and lam et al. 1996) at this point, our present study has been designed to find out the effect of suitable n management practices along with different seedling raising methods viz. wet and dry bed on the yield and yield contributing characters of tidal aus rice and thus, to unlock the potentials of horizontal expansion of aus rice cultivation in the coastal areas of bangladesh. international journal of applied biology, 4(1), 2020 70 materials and methods experimental location and climate the study was carried out at the agronomy field laboratory of patuakhali science and technology university (pstu), patuakhali, bangladesh from april to august, 2017. the experimental field was located under ganges tidal floodplain agro-ecological zone (aez) 13. this region occupies an extensive area of coastal tidal floodplain land in the south–west of the country. the area lies at 0.9 to 2.1 meter above mean sea level. the study area was located under the sub-tropical climate, which is specialized by moderately high temperature and heavy rainfall during the kharif-1 (march to august) and kharif-2 season (aprilseptember) and low rainfall with moderately low temperature during robi season (octobermarch). the experimental field was flooded twice daily by tidal inundation. the magnitude of tidal water depth, flow of water and stagnation period of water depends mainly on the moonmonth, wind speed, wind direction and air pressure that attains peak in may-august (figure 1). the highest water depth of the field were observed at the beginning and mid of the moonmonth. the data were collected every two days interval from transplanting (14th may) to harvesting date (31th july). figure 1. height of water (in cm) in the experimental field of transplant aus rice from transplanting to harvesting experimental procedure the study was a two factor experiment with three replications where two sets of experimental treatments included in the study as factor-a (six nitrogen management practices) and factor-b (two seedling raising methods) have been presented in table 1. the experiment was laid out in a split-plot design where twelve (12) treatment combinations were allocated in 36 plots of 8 m2 size (5m x 1.6m). brridhan55, an aus rice variety developed by bangladesh rice research institute (brri) was used as experimental materials. 3 2 2,67 2 0 2,33 20 15,67 13 8 26,3327,33 32 24 36 35,6734 22 23 24 22 27 38 50 67 48 32 1 4 .0 5 .1 7 1 7 .0 5 .1 7 2 0 .0 5 .1 7 2 3 .0 5 .1 7 2 6 .0 5 .1 7 2 9 .0 5 .1 7 0 1 .0 6 .1 7 0 4 .0 6 .1 7 0 7 .0 6 .1 7 1 0 .0 6 .1 7 1 3 .0 6 .1 7 1 6 .0 6 .1 7 1 9 .0 6 .1 7 2 2 .0 6 1 7 2 5 .0 5 .1 7 2 8 .0 6 .1 7 0 1 .0 7 .1 7 0 4 .0 7 .1 7 0 7 .0 7 .1 7 1 0 .0 7 .1 7 1 3 .0 7 .1 7 1 6 .0 7 .1 7 1 9 .0 7 .1 7 2 2 .0 7 .1 7 2 5 .0 7 .1 7 2 8 .0 7 .1 7 3 1 .0 7 .1 7 water depth (cm) date international journal of applied biology, 4(1), 2020 71 table 1. list of treatments used in the experiment factor-a: six (6) nitrogen management practices n0: no nitrogen n1: whole urea at land preparation @ 150kg ha-1as recommended dose n2: urea super granule (usg) at land preparation @ 112 kg ha-1 n3: usg @ 1.8g (granule) per 4 hill (112kg ha-1) at 10 days after transplanting (dat) n4: 50% urea of recommended dose+ 2% urea spray at 30, 45 and 60 dat (105 kg) n5: 50% urea of recommended dose at land preparation + magic growth spray at 30, 45 and 60 days factor-b: two (2) seedling raising methods w: wet seedbed d: dry seedbed fertilizer management the experimental plots were fertilized as per fertilizer recommendation guide barc (2012). besides, different nitrogenous fertilizers were applied following the procedure as mentioned below urea spray: liquid urea used in the experiment for foliar spray was prepared by mixing 2 kg of prilled urea in 100 l of water as per treatment. the urea was sprayed@ 500l/ha of spray volume at 30, 45 and 60 dat. magic growth spray: magic growth is a solution of different nutrient elements and it contains 10.51% total nitrogen, 5.58% phosphorous, 6.33% potassium, 0.10% sulphur, 0.16% zinc, 0.04%copper, 0.0006% iron, 0.006% manganese, 0.25% boron, 0.07% calcium and 0.007% magnesium. the spray volume of magic growth was prepared by mixing fifty (50) ml magic growth with 1l water. this mixture was sprayed @ 500l /ha at 30, 45 and 60 dat. urea super granule (usg): usg fertilizer is manufactured from a physical modification of prilled urea (pu) fertilizer. the international fertilizer development center (ifdc) has developed it. its nature and properties are similar to that of urea but its granule size is bigger and condensed with some conditions for slow hydrolysis. usg is spherical in shape containing 46% n which is similar to that of pu. preparation of seedbed and sowing wet seed bed (w): wet nursery bed was prepared under irrigated condition. the soil was puddled by three ploughing and cross ploughing with cultivator. the sprouted seeds were sown in the prepared seedbeds on 18th april, 2017. the size of seed bed was 15 m2 (length 5m & width 1.5×2m). dry seed bed (d): the land was first ploughed, cross ploughed and then harrowed until a good tilth condition is attained. the seeds were sown in the prepared dry seedbed on 15th april, 2017. the seed bed size was 15 m2. data collection and statistical analysis different yield and yield contributing data viz. plant height (cm), number of effective tillers hill-1, days to first flowering, days to 50% flowering, number of non-effective tillers hill1, days to maturity, panicle length (cm), number of filled grains panicle-1, number of unfilled grains panicle-1, thousand grain weight (g), grain yield (t ha-1), straw yield ( t ha-1), biological yield (t ha-1) and harvest index (%) were collected and analysis of variance (anova) was calculated with the help of computer software package mstat-c program (russel 1986). the international journal of applied biology, 4(1), 2020 72 mean differences among the treatments were compared by duncan’s multiple range test (dmrt) at 5% level of significant (gomez and gomez 1984). results and discussion in our study different nitrogen management practices and seedling raising methods were found to have significant roles on all the yield and yield contributing characters of aus rice under tidal ecosystem. the results are presented below in respect to different important growth and yield parameters of transplanted aus rice. plant height nitrogen management had significant effect on plant height (p<0.01; figure 2). among all the n management treatments, maximum plant height (109.17 cm) was observed in n3 treatment [usg @ 1.8g (granule) per 4 hill (112kg/ha) at 10 dat] and the shortest plant (97.73 cm) was recorded in case of n0 treatment (without nitrogen) (figure 2). our data is also in accordance with ilaga and daya (1989) regarding plant height in response to nitrogenous fertilizers. plant height was also significantly influenced by seedling raising methods (p<0.01; figure 2) where in case of seedlings raised in wet seed bed were found as longest (104.23cm) compared to the seedlings raised in dry seed bed (99.61 cm). interaction of nitrogen management and seedling raising methods had significant effect on plant height of aus rice (p<0.01; table 2). the maximum plant height (110.33 cm) was found in n3w treatment combination i.e., usg at 10 dat in combination for the seedlings raised in wet bed and the lowest plant height (94.00 cm) was obtained in n0d treatment i.e. without nitrogen in case of the seedlings raised in the dry bed. we also observed highest plant height of 108.00 cm from the treatment n3 the raised seedlings in dry bed (n3d) which was statistically at par with n3w. using of the urea deep placement technology (usg) has already proven increased yield in several studies (bandaogo et al., 2015; alam et al., 2013; gregory et al., 2010; mohanty et al., 1999; savant and stangel, 1990). increased plant height might be the response of enhanced availability of nitrogen from usg. figure 2. effect of different nitrogen management practices and seedling raising methods on the plant height of transplant aus rice. (n0= no nitrogen, n1=whole urea at land preparation @ 150kg/ha, n2=usg at lp @ 112 kg/ha, n3= usg @ 1.8g (granule) per 4 hill (112kg/ha) at 10 dat, n4= 50% urea + 2 % urea spray at 30, 45 and 60 dat, n5=50% urea at land preparation + magic growth spray at 30, 45 and 60 days). same letter on the bars do not differ significantly as per dmrt (p<0.01). international journal of applied biology, 4(1), 2020 73 table 2. interaction effect of nitrogen management and seedling raising methods on plant height and effective tillers of transplant aus rice interaction plant height (cm) no. of effective tillers hill-1 n0w 101.47 bc 13.13 ab n1w 103.67 b 14.47 a n2w 103.33 b 13.33 ab n3w 110.33 a 14.60 a n4w 103.90 b 14.40 n5w 102.67 bc 13.77 ab n0d 94.00 c 10.73 c n1d 100.07 bc 13.07 ab n2d 97.53 bc 12.33 b n3d 108.00 ab 14.20 a n4d 97.70 bc 11.93 bc n5d 100.33 bc 13.33 ab significance level ** ** cv (%) 4.30 8.85 note: in a column, figures with same letter or without letter do not differ significantly as per dmrt; ‘**’ = significant at 1%; n0= no nitrogen, n1=whole urea at land preparation @ 150kg/ha, n2=usg at lp @ 112 kg/ha, n3= usg @ 1.8g (granule) per 4 hill (112kg/ha) at 10 dat, n4= 50% urea + 2 % urea spray at 30, 45 and 60 dat, n5=50% urea at land preparation + magic growth spray at 30, 45 and 60 days. w=wet seed bed and d=dry seed bed; cv (%) = percent coefficient of variation. effective tillers hill-1 in case of effective tillers per hill, the effect of different n management options were highly significant (p<0.01; figure 3). the maximum number of effective tillers hill-1 (14.40) was observed from the application of usg at 10 dat (n3) and the lowest number of effective tillers hill-1 (11.93) was found from no nitrogen treatment (n0). adequacy of nitrogen from usg probably favored the cellular activity during tiller formation and development, which led to increased number of effective tillers hill-1. international journal of applied biology, 4(1), 2020 74 figure 3. effect of different nitrogen management practices and seedling raising methods on effective tillers hill-1 of transplant aus rice. (n0= no nitrogen, n1=whole urea at land preparation @ 150kg/ha, n2=usg at lp @ 112 kg/ha, n3= usg @ 1.8g (granule) per 4 hill (112kg/ha) at 10 dat, n4= 50% urea + 2 % urea spray at 30, 45 and 60 dat, n5=50% urea at land preparation + magic growth spray at 30, 45 and 60 days). same letter on the bars do not differ significantly as per dmrt (p<0.01). ahmed et al. (2005) also reported that number of effective tillers hill-1 increased with the better management of nitrogen. again, between the two seedling raising methods the number of effective tillers hill-1 was also significantly influenced. in case of wet seed bed raised seedling the highest number of effective tillers was recorded (13.95) while this number in case of dry seed bed raised seedlings was 12.60 (figure 3). the interaction effect of nitrogen management and seedling raising method on number of effective tillers hill-1 was significant (table 2). the number of effective tillers hill-1 ranged from 10.73 to 14.60 over the treatments. the highest number of effective tillers hill-1 (14.60) was obtained with n3w which was statistically similar to n4w and n1w and the lowest number of effective tillers hill-1 (10.73) with n0d. increased n absorption had great role in increasing number of tillers per square meter (yoshida et al. 1972). days to 50% flowering and maturity different n management practices had significant effect on flowering and maturity of transplant aus rice var. brridhan55 (p<0.05; table 3). the lowest days required for 50% flowering and maturity (50.50 and 78.33 dat, respectively) were observed in the plants treated with n4 (50% urea + 2 % urea spray at 30, 45 and 60 dat) while highest days were recorded from the treatment n3 (usg at 10 dat) treated plots which were 52.50 and 82.50 dat, respectively. in case of the two seedling raising methods, we observed significant variation for days to 50% flowering (p<0.05; table 4) which was 51.83 dat in wet seed bed seedlings and 51.07 dat in the dry seed bed seedlings. nitrogen management and seedling raising methods had significant effect on time required for 50% flowering (p<0.01; table 5). the treatment combinations of n4d (n4= 50% urea + 2 % urea spray at 30, 45 and 60 dat with dry seedbed) and n5d (50% urea at land preparation + magic growth spray at 30, 45 and 60 dat with dry seedbed) required the lowest days to 50% flowering (50.33 dat). the highest time period for 50% flowering was required in case of treatment combination n3w (53.00 dat). we also found significant variation in attaining maturity due to interaction between nitrogen management and seedling raising method (table 5). n4w required the lowest 11,93 13,77 12,83 14,4 13,17 13,55 0 2 4 6 8 10 12 14 16 n0 n1 n2 n3 n4 n5 n o . o f e ff e ct iv e t ill e rs p e r h ill nitrogen management practices 13,95 12,6 0 2 4 6 8 10 12 14 wet seed bed dry seed bed n o . o f e ff e ct iv e t ill e rs p e r h ill a b a b c d e f international journal of applied biology, 4(1), 2020 75 duration for 80% maturity (78.00 dat) and n3w needed the longest duration (84.33 dat). the level of nitrogen in rice field might influence the flower initiation and maturity of t. aus rice. the flowering and maturity dates were significantly influenced by different n management in several studies (rahman et al. 2016; halder 2013 and rahman et al. 2005). https://scialert.net/fulltextmobile/?doi=ajcs.2016.1.12#1516821_ja contact : gopal saha saha.18@gmail.com 76 table 3. effect of nitrogen management practices on different yield and yield contributing attributes of transplant aus rice treatment days to 50% flowering days to maturity no. of filled grains panicle-1 no. of unfilled grains panicle-1 panicle length (cm) 1000seed weight (g) grain yield (t/ha) straw yield (t/ha) biological yield (t/ha) harvest index (%) n0 51.33 b 79.67 f 55.80 f 27.40 a 19.98 f 19.98 f 2.94 f 5.07 f 8.01 f 35.32 f n1 51.50 b 80.67 b 65.27 b 24.47 d 21.78 b 20.18 b 3.96 b 5.76 b 9.66 b 40.58 b n2 51.83 b 80.83 c 61.70 e 25.63 b 20.62 e 20.05 e 3.38 e 5.41 e 8.78 e 38.33 e n3 52.50 a 82.50 a 78.20 a 20.60 f 22.70 a 20.32 a 4.23 a 5.88 a 10.10 a 41.65 a n4 50.50 d 78.33 e 62.00 d 25.23 c 21.33 d 20.12 d 3.60 d 5.53 d 9.13 d 39.32 d n5 51.00 c 78.67 d 63.60 c 23.37 e 21.45 c 20.15 c 3.74 c 5.66 c 9.38 c 39.83 c significance level * * ** ** ** * ** ** ** ** cv (%) 2.05 2.88 7.85 8.85 5.10 3.49 8.97 7.55 7.13 3.75 note: in a column, figures with same letter or without letter do not differ significantly as per dmrt; ‘ *’ and ‘**’ = significant at 5 and 1%, respectively ; n0= no nitrogen, n1=whole urea at land preparation @ 150kg/ha, n2=usg at lp @ 112 kg/ha, n3= usg @ 1.8g (granule) per 4 hill (112kg/ha) at 10 dat, n4= 50% urea urea + 2 % urea spray at 30, 45 and 60 dat, n5=50% urea at land preparation + magic growth spray at 30, 45 and 60 days. cv (%) = percent coefficient of variation contact : gopal saha saha.18@gmail.com 77 number of filled grains panicle-1 nitrogen management showed significant variation in the number of filled grains panicle-1 (p<0.01; table 3). the highest number of filled grains panicle-1 (78.20) was obtained from the n3 i.e., usg at 10 dat and the lowest number of filled grains panicle-1 (55.80) was obtained from the no urea treated plot. this finding is in contrast with the findings of rajarathinam and balasubramanivan, (1999) where they found application of n increased number of filled grains panicle-1. the average number of filled grains panicle-1 was found significantly different in seedling raising method (p<0.01; table 4). the highest number of filled grains was recorded in the wet seed bed (71.07) and lowest filled grains were in dry seed bed (57.79). interaction effect between nitrogen management and seedling raising method had positive effect on filled grains panicle-1 (table 5). the highest number of filled grains (88.13) was obtained from n3w i.e., when usg applied at 10 dat in combination with the seedlings raised in wet bed and the lowest number of filled grains panicle-1 (50.33) was obtained from n0d i.e. without nitrogen in case of the seedlings raised in the dry bed. our result suggests that highest number of grains panicle-1 might be the response of enhanced availability of nitrogen from usg especially when it applied at 10 dat. subsequently, higher available n might results more photo assimilates and thus produced more dry matter accumulation in the panicle. these findings are also consistent with the findings of other researchers (alam et al., 2013; bandaogo et al., 2014; gregory et al., 2010). number of unfilled grains panicle-1 the number of unfilled grains per panicle was significantly different in the nitrogen management (table 3). the maximum number of unfilled grains per panicle (27.40) was counted in case of no nitrogen and the minimum number of unfilled grains panicle (20.60) was counted in the n3 i.e., usg at 10 dat. nitrogen took part both in grain formation and development and for this reason number of grains per panicle increased with adequate n levels. this result is in conformity with halder (2013) and nori et al., (2008). in case of seedling raising methods, number of unfilled grains per panicle was higher in wet seed bed (24.46) and minimum unfilled grains per panicle in dry seed bed (24.44; table 4). number of unfilled grains per panicle was found to be significantly affected by interaction effect of nitrogen management and seedling raising method (table 5). the highest (28.73) number of unfilled grains per panicle was recorded under the n4w that was statistically at par with n2d (28.67) and n0w (28.40) whereas the lowest number of unfilled grains per panicle (19.87) was counted in the treatment combination n3w. panicle length (cm) the panicle length was significantly affected by nitrogen management practices (p<0.01, table 3). the maximum panicle length (22.70 cm) was observed in usg @ 112 kg/ha at 10 dat and the lowest (19.98 cm) panicle length was obtained from without nitrogen (n0). the variation in panicle length due to the improved management of n was also reported by parvin (2012). in our study, both of the seedling raising methods had significant effect on the panicle length (p<0.01; table 4) where highest length of 22.08 cm was recorded in wet seedbed compared to plants raised in dry seedbed (20.54cm). again panicle length was also influenced significantly in case of the interaction effect of nitrogen management practices and seedling raising methods (p<0.05; table 5). the longest panicle (23.37 cm) was obtained from n3w and the shortest panicle (19.27cm) was obtained from the n2d which was statistically similar to n0d (19.43cm). contact : gopal saha saha.18@gmail.com 78 table 4. effect of seedling raising method on yield and yield contributing attributes of transplant aus rice seedling raising method days to 50% flowering days to maturity no. of filled grains panicle-1 no. of unfilled grains panicle-1 panicle length (cm) 1000seed weight (g) grain yield (t/ha) straw yield (t/ha) biological yield (t/ha) harvest index (%) wet seedbed (w) 51.83 80.39 a 71.07 a 24.46 a 22.08 a 20.42 a 3.93 a 5.70 a 9.61 a 40.32 a dry seedbed (d) 51.07 79.83 b 57.79 b 24.44 b 20.54 b 19.84 b 3.35 b 5.41 b 8.74 b 38.02 b significance level ns * ** ** ** * ** * ** ** cv (%) 2.05 2.88 7.85 8.85 5.10 3.49 8.97 7.55 7.13 3.75 note: in a column, figures with same letter or without letter do not differ significantly as per dmrt; ‘*’ and ‘**’ = signifi cant at 5 and 1%, respectively, ns= non-significant; cv (%) = percent coefficient of variation contact : gopal saha saha.18@gmail.com 79 thousand (1000)-seed weight (gm) the effect of nitrogen management was significant in respect of 1000-grain weight of t. aus rice (p<0.05; table 3). the highest 1000-grain weight (20.32 g) was obtained from the treatment n3 and the lowest 1000-grain weight (19.98g) was found in control (n0). this possibly happened due to the uptake of n by root of the rice plant that influenced the photosynthesis process and subsequently storage of starch in sink cell such as grain. usg provide the highest amount of available n in the field and created a chance to fill the grain properly. hossain and islam (2008) also reported the variation in 1000-grain weight due to the more availability of n in soil. chaturvedi (2005) observed that n play important role in increasing protein percentage, which in turn increased the grain weight. the weight of 1000grains was also significantly influenced (p<0.05; table 4) by the seedling raising method where the seedling raised in wet bed gave higher 1000-grain weight (20.42g) over dry bed method (19.84 g). further, the interaction effect between nitrogen management and seedling raising method gave significant variation regarding the weight of 1000-grains (p<0.05; table 5). the treatment combination n2w gave highest 1000-grain weight (20.63 g) which was statistically similar with n1w, n3w, n4w, n5w, n3d, n4d and n5d. the lowest weight of 1000-grains (19.47 g) was obtained from n2d grain yield grain yield was significantly influenced (p<0.01) by nitrogen management practices (table 3). by usg @ 112kg/ha at 10 dat (n3) produced the highest grain yield (4.23 t ha-1) which was followed by the treatments n1, n5, n4, n2 and n0. the lowest grain yield (2.94 t ha1) was recorded from without nitrogen (n0). the highest grain yield might be due to the resultant effect of highest number of effective tillers per hill and highest number of grains per panicle as obtained in the treatment n3. miah et al. (2006) stated that usg increased an average of 20% rice yield in tidal flooded condition. grain yield variations due to fertilizer management were also reported by shah et al. (2013), das (2011), jun et al. (2011) and tahura (2011). grain yield was also significantly influenced by seedling raising methods (table 4). the highest grain yield (3.93 t ha-1) was obtained from the wet seed bed compared to the yield (3.35 tha-1) obtained by the seedlings raised in dry seed bed. interaction between nitrogen management and seedling raising method also played positive role in promoting grain yield of transplant aus rice. highest grain yield of 4.62 t ha-1 was from the treatment combination n3w which was statistically at par with n1w (the whole urea application at land preparation along with wet seed bed) and n3d (usg application along with dry seed bed) and the lowest grain yield was recorded in n0d (2.56 t ha-1; table 5). straw yield straw yield was significantly (p<0.01) affected by the nitrogen management (table 3). the highest straw yield (5.88 t ha-1) was obtained from the plot applied with usg 112kg/ha at 10 dat (n3) and the least straw yield (5.07 t ha-1) was obtained from the no urea treated plot (n0). availability of nitrogen at vegetative phase during the initiation of primary, secondary and tertiary tillers resulted in increased accumulation of dry matter which probably favored highest straw yield. significant variation in straw yield was also reported by shah et al. (2013) and das (2011). straw yield was significantly influenced (p<0.05) by the seedling raising method (table 4). the highest straw yield (5.70 tha-1) was obtained from the wet seed bed compared to (5.11 t ha-1) dry seed bed. again, the highest straw yield (6.07 tha-1) was recorded from the interaction of seedling raised in wet seed bed and contact : gopal saha saha.18@gmail.com 80 table 5. interaction effect of nitrogen management and seedling raising method on yield and yield contributing attributes of transplant aus rice treatment combination days to 50% flowering days to maturity no. of filled grains panicle-1 no. of unfilled grains panicle-1 panicle length (cm) 1000seed weight (g) grain yield (t/ha) straw yield (t/ha) biological yield (t/ha) harvest index (%) n0w 51.67 ab 79.67 bc 61.27 c 28.40 a 20.53 b 20.17 ab 3.32 bc 5.25 bc 8.57 bc 36.23 bc n1w 51.67 ab 80.33 ab 77.07 b 22.73 bc 22.80 ab 20.37 ab 4.24 ab 5.91 ab 10.13 ab 41.77 ab n2w 52.33 ab 81.67 ab 63.80 bc 22.60 bc 21.97 ab 20.63 a 3.77 bc 5.66 b 9.42 ab 39.93 bc n3w 53.00 a 84.33 a 88.13 a 19.87 c 23.37 a 20.50 ab 4.62 a 6.07 a 10.67 a 43.17 a n4w 50.67 bc 78.00 c 66.13 bc 28.73 a 21.80 ab 20.47 ab 3.78 bc 5.60 b 9.38 ab 40.23 b n5w 51.67 ab 78.33 bc 70.00 24.40 b 22.03 ab 20.40 ab 3.85 bc 5.68 b 9.50 ab 40.60 ab n0d 51.00 b 79.67 bc 50.33 d 26.40 ab 19.43 c 19.80 bc 2.56 c 4.89 c 7.45 c 34.40 c n1d 51.33 ab 81.00 ab 53.47 cd 26.20 ab 20.77 b 20.00 b 3.67 b 5.61 b 9.18 b 39.40 bc n2d 51.33 ab 80.00 b 59.60 cd 28.67 a 19.27 c 19.47 c 2.99 bc 5.15 bc 8.14 bc 36.73 bc n3d 52.00 ab 80.67 bc 68.27 bc 21.33 bc 22.03 ab 20.13 ab 3.83 ab 5.70 ab 9.53 ab 40.13 b n4d 50.33 c 78.67 bc 57.87 cd 21.73 bc 20.87 ab 19.77 bc 3.42 bc 5.46 bc 8.88 bc 38.40 bc n5d 50.33 c 79.00 bc 57.20 cd 22.33 bc 20.87ab 19.90 bc 3.62 b 5.64 b 9.26 b 39.07 bc significance level ** * ** ** * * ** ** ** ** cv (%) 2.05 2.88 7.85 8.03 5.10 3.49 8.97 7.55 7.13 3.75 note: in a column, figures with same letter or without letter do not differ significantly as per dmrt; ‘*’ and ‘**’ = significant at 5 and 1%, respectively and ns= not significant; n0= no nitrogen, n1=whole urea at land preparation @ 150kg/ha, n2=usg at lp @ 112 kg/ha , n3= usg @ 1.8g (granule) per 4 hill (112kg/ha) at 10 dat, n4= 50% urea + 2 % urea spray at 30, 45 and 60 dat, n5= 50% urea at land preparation + magic growth spray at 30, 45 and 60 days. w=wet seed bed and d=dry seed bed; cv (%) = percent coefficient of variation contact : gopal saha saha.18@gmail.com 81 112kg/ha usg at 10 dat i.e., n3w (p<0.01) which was statistically similar with n1w (5.91 tha1) and n3d (5.70 tha-1). the lowest straw yield (4.89 t ha-1) was recorded in n0d (table 5). biological yield significantly highest (p<0.01) biological yield (10.10 tha-1) was found from usg 112kg/ha at 10 dat (n3) and the lowest value (8.01 t ha-1) regarding biological yield was found from the no urea treated plot (n0; table 3). biological yield was significantly influenced by seedling raising method (table 4) where the maximum biological yield (9.61t ha -1) was found in case of wet seed bed and the lowest biological yield (8.74 tha-1) was found from dry seed bed. interaction effect between nitrogen management and seedling raising method was significant in respect of biological yield (table 5). the highest biological yield (10.67 t ha-1) was recorded in n3w treatment combination which was statistically at par with the treatment combinations n1w, n2w, n4w, n5w and n3d and the lowest biological yield (7.45 t ha-1) in the n0d. harvest index nitrogen management produced significant differences (p<0.01) in respect of harvest index (table 3). the highest harvest index (41.65) was found from the n3 treatment and the lowest harvest index (35.32) was found from the control (n0). harvest index was also significantly influenced (p<0.01) by the seedling raising method (table 4). higher harvest index (40.32) was found from the wet seed bed compared to dry seed bed (38.02). in considering the interaction effect, nitrogen management and seedling raising method significantly highest harvest index (43.17) was recorded in the n3w (p<0.01) which was statistically similar with the treatment combinations of n1w and n5w. the lowest harvest index (34.40) was observed in case of n0d (table 5) conclusions the study concludes that seedlings raised in wet seedbed produced highest grain yield with the application of usg at 10 dat and this was statistically similar to the whole urea application at land preparation along with wet seed bed and usg application along with dry seed bed. this suggests that wet seed bed preparation with whole urea application at land preparation or dry seed bed preparation with usg application at 10 dat may be practiced for cost saving in tidal ecosystem of bangladesh to avoid the complexity of usg application and scarcity of water for wet seed bed preparation. acknowledgement the authors would like to express their thanks to the ministry of science and technology, bangladesh for providing financial support for conducting the experiment references ahmed, m., islam, md. m & paul s.k. 2005: effect of nitrogen on yield and other plant characters of local t. aman rice, var. jatai. research journal of agriculture and biological sciences. 1(2): 158-161. bandaogo, a., bidjokazo, f., youl, s., safo, e., abaidoo, r. & andrews, o. 2015. effect of fertilizer deep placement with urea supergranule on nitrogen use efficiency in sourou contact : gopal saha saha.18@gmail.com 82 valley (burkina faso). nutrient cycling in agroecosystems. 102: 79–89. bbs (bangladesh bureau of statistics) 2013: yearbook of agricultural statistics of bangladesh. ministry of planning.govt. of the people’s republic of bangladesh, dhaka. p. 136–140. brri. bangladesh rice research institute, government of the people’s republic of bangladesh, joydebpur, gazipur; 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science society of america, madison, wi, pp: 429-455. xiong, j., ding, c. q., wei, g. b., ding, y.f & wang s. h. 2013. characteristic of dry-matter accumulation and nitrogen-uptake of super-high-yielding early rice in china. agronomy journal. 105(4):1142±50. yoshida, s., cock, j. h & parao f. t. 1972. physiological aspects of high yield. int. rice res. inst. rice breeding, pp. 455-469. contact : pramita setiawan pramitasetiawan_fbs@uwks.ac.id 31 abstract kefir is beverage fermented product, it is usually made from milk. however, there is an arising concern about lactose intolerant. therefore, taking benefit of herbal tea solution as raw material of kefir will give multiple benefits. the used tea solution was made from keji beling leaves (strobilanthes crispus l.) which has many benefits. kefir beverage is probiotic beverage which is beneficial for digestion health and can keep immune system, as well as keji beling tea which is rich in antioxidants. the used design in this research was complete randomized design (rancangan acak lengkap) with 4 levels of treatment namely: concentration of keji beling 0%, 10% and 15% with fermentation duration of 12 hours and 24 hours. based on the research result pointed out the obvious effect between treatment toward ph, total polyphenols, total acetic acid, and total glucose with p score = 0,00. the result of lactic acid bacteria (bal) total is 1,914 x107 cfu/ml and yeast total is 1,532 x 107 cfu/ml on concentration of 15% kefir of keji beling tea with fermentation duration of 24 hours. result for organoleptic test shows the obvious effect (p=0,00) between the treatment toward parameters of taste, aroma, color and power. panelist takes pleasure on the treatment p1 with concentration of horsewhip tea 150% in fermentation duration of 24 hours. issn : 2580-2410 eissn : 2580-2119 kefir of keji beling tea (strobilanthes crispus) as functional beverage for glucose intolerance pramita laksitarahmi isrianto & sunaryo educational program of biology, faculty of language and science, wijaya kusuma university, surabaya, east java,indonesia introduction kefir is fermented milk product which can be made from raw materials of cow milk, goat milk or sheep milk by adding kefir grains which consists of lactic acid bacteria and yeast. according to gulitz et al, (2011), kefir grains formed from cultures of various strains of healthy bacteria and yeast, which existed together on polysaccharide matrix which was made by bacteria. some yeasts which contained in kefir that rich in probiotics namely; kluyveromyces, saccharomyces, acetobacter spp., lactobacillus acidophilus, brevis, casei, fermentum, helveticus, kefiri, kefiranofaciens parakefiri, lactis and leuconostoc mesenteroides (cai, sounderrajan and serventi, 2020). the relationship of microbial symbiosis produced a stable growth culture. microbes change glucose to be lactic acid, alcohol (ethanol) and carbon dioxide which will produce carbonated fermented beverage. the alcohol content of water kefir ranges between 0,5-1% (gulitz et al., 2011). all this time, kefir is usually made from milk. however, there is an arising concern about lactose intolerant and also the growing number open access international journal of applied biology international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. keyword kefir, keji beling tea, strobilanthes crispus l, fermented beverage. article history received 12 october 2020 accepted 31 december 2020 international journal of applied biology, 4(2), 2020 32 of vegetarians which initiates the increasing in availability of non-dairy beverages (mubin, 2016). therefore, the suggested raw materials are from fruits or vegetables as the medium of probiotics growth. water kefir or also known as kefir grains are able to produce alcohol (ethanol) relatively low, carbon dioxide, organic acids (lactic and acetate) and fat content are few in number. the use of water kefir can be used from the compound of herbal tea water, such as keji beling tea. keji beling (strobilanthes crispus l) is one of plants from familia acanthaceae which has spread in indonesia that is used empirically as anti-diabetes and as the ingredients of other traditional medicine that has multiple benefits. from various researches was known that keji beling contains chemical substance such as: potassium, sodium, calcium, silicate acid, alkaloid, saponins, flavonoid and polylenoid. one of plants which is used as traditional medicine is keji beling (setyaningsih, 2008). some contents of keji beling (strobilanthes crispus l.) are phenol compound which has benefit as anti-bacteria, the content of potassium and silica helps to overcome haemorrhoids and dysentery. the content of vitamin c, b1, b2 and catechins makes keji beling potentially as antioxidants (amalia et al, 2015). keji beling leaves have a rich antioxidant activities compared with verbate (herbal tea) and vitamin c. concerning the previous research result of kefir fermentation of black tea with adding carrot juice pointed out the increasing number of lactic acid bacteria (bal) with the low glucose level (subardjo, 2017). the aim of making process of herbal tea kefir on this keji beling is to know effectiveness making process of kefir with raw material of herbal tea which is expected will produce simultaneous benefit for health effect of herbal tea and functional effect of herbal tea kefir. methodology this research was conducted on may until july 2019 at biology laboratory, fbs wijaya kusuma university of surabaya and laboratory of research association and industrial consultation (bpki). materials on this research are solution of keji beling tea brand “herba tazakka” and kefir bacteria from bioteknologi mikroorganisme laboratory, tg2 ubaya. this experimental research used complete randomized design (ral) with 5 times of repetition namely; variation on concentration of horsewhip tea solution 0%, 10% and 15% with fermentation duration of 12 hours and 24 hours. the used chemical ingredients are sucrose buffer ph 4 and buffer ph 7, pp indicator, aquadest, naoh 0,1 n, alcohol 70%. whereas, the used tools are glass bottle, iron sieve, beaker glass, autoclave, measuring cup, ph meter, water thermometer, digital scale, drop pipette, measuring pipe, petri cup, reaction tube, wooden stirrer, plastic spoon, measuring flask 100 ml, ependorf tube, cuvette, big jar, micropipette, spectrophotometer uv-vis, and centrifuge. performing of research was at the first stage as for procedural stage on this research that was as follows: 1. making process of keji beling tea solution (stachytarpheta jamaicensis) with concentration 10% and 15%. 2. adding 150 gram kefir grains bacteria in 1 l. 3. adding 79 raisins as kefir grains’ nutrition on kefir of keji beling tea that has been made. then it stored at room temperature in accordance with the treatment of fermentation duration 12 hours and 24 hours. after that it was continued with measuring acid total by using titration method, polyphenol total by using spectrophotometer uv-vis at the wavelength 760 nm, glucose total by using luff schoorl method, and for measuring ph by using ph meter. the stage of total lactic acid bacteria (bal) analysis that was the sample was diluted into 0,1 % 9 ml peptone international journal of applied biology, 4(2), 2020 33 solution (this solution is 10-1 dilution), then it was continued until 10-8 dilution. on the last three dilution series, each sample was taken1 ml and poured into petri cup which was filled with mrsa media. colony growth was counted by using total plate count (tpc) number into 1 g by multiplying the average number of colonies with dilution factor which was used with colony forming unit, whereas for the stage of yeast total that was kefir sample was diluted into 0,1 % 9 ml peptone solution (this solution is 10-1 dilution), then it was continued until 105 dilution. each of them was taken 1 ml from the last three dilutions and poured into sterile petri cup, then poured with sterile pgya media (warm) until the bottom of cup covered with media. after the media has solidified, it was incubated at 30oc temperature during 48 hours. then, the growth colony was noted (mubin and elok, 2016). parameter data on this research is ph, acetate acid total, glucose total, polyphenol total, lactic acid bacteria (bal) total and yeast total. result and discussion based on the research result of keji beling tea kefir (strobilanthes crispus l.) toward ph, polyphenol total, acetate acid total and glucose total pointed out the obvious effect with p score = 0,00. the result of ph score of keji beling tea on concentration 10% in duration 12 hours is 4,4 while in 24 hours fermentation ph decreased to be 3,78. for concentration 15% in 12 hours ph score is 4,18 and decreased in fermentation duration 24 hours with ph 3,375 (table 1). the thicker of tea concentration and the longer fermentation duration then ph score is more acid. on polyphenol total of kefir of keji beling tea indicated the increasing score by the longer of fermentation process from 0,144% up to 3,47%. whereas, for acetate acid total is around 1,014%-3,26%. however, the longer fermentation proses for glucose total occurred the decreasing from 6,486% until 4,054 on concentration of keji beling tea 10% and for 15% of tea concentration is from 5,08 % until 4 % (table 1). table 1. the average of ph, polyphenol, acetate acid, glucose of kefir keji beling tea treatment ph polyphenol (%) acetate acid (%) glucose (%) k1 4,4d 0,144a 1,014a 6,486c k2 4,18c 2,25b 2,84b 5,08b k3 3,78b 3,30c 3,11c 4,054a k4 3,375a 3,47c 3,26d 4,0a note : k1= concentration of keji beling tea 10% and duration of fermentation 12 hours, k2= concentration of keji beling tea 15% and duration of fermentation 12 hours, k3= concentration of keji beling tea 10% and duration of fermentation 24 hours, k4= concentration of keji beling tea 15% and duration of fermentation 24 hours. from the result of lactic acid bacteria (bal) and yeast total in kefir of keji beling tea on concentration of keji beling tea 10% with duration 12 hours pointed out the bal total is 0,662 x107 cfu/ml and yeast total is 0,4460 x107 cfu/ml,while for duration of fermentation 24 hours international journal of applied biology, 4(2), 2020 34 pointed out bal total is 1,694 x107 cfu/ml and yeast total is 0,798 x107 cfu/ml. the result of concentration of keji beling tea 15% within 12 hours of fermentation is bal total 0,792 x107 cfu/ml and yeast total 0,582 x107 cfu/ml, while within 24 hours of fermentation produces bal total 1,914 x107 cfu/ml and yeast total 1,018 x107 cfu/ml ( table 2). from the result of statistical test, kefir of keji beling tea toward bal and yeast total indicates the obvious effect with p score =0,000. the average of bal total above pointed out that the longer of fermentation duration will increase the growth of lactic acid bacteria. where environmental factor will also affect the growth of bal namely salinity, temperature, ph, availability of carbohydrates as its source of nutrition (pelczar et al.,2005). the average of ph condition approaches 4. from the previous research, the fermentation process produced ph 3,5 4,5 and alcohol content under 1% and was conducted at temperature 25 30 ° c in anaerobic condition. as for yeast strains which included within it are kluyveromyces and saccharomyces and rich in probiotic bacteria, such as lactic acid bacteria acetobacter spp., lactobacillus acidophilus, brevis, casei, fermentum, helveticus, kefiri, kefiranofaciens parakefiri, lactis and leuconostoc mesenteroides. furthermore, yeast strains was from kluyveromyces lactis and saccharomyces cerevisiae (cai, sounderrajan and serventi, 2020). this indicates that in ph 4 on this research, lactic acid bacteria can change sucrose to be lactic acid on kefir of tea media then the growth of bal will be obstructed and this condition is used by yeast for growing to do metabolism (manik, 2005 as cited by nadhiroh, 2018). some of good microorganisms which contained in kefir of tea make it as functional health beverage for glucose intolerant. this kefir of tea is included on water kefir as probiotic beverage which is health and low glucose (laureys, 2014). the extract of keji beling can produce antibacterial because the existence of some chemical compound on this leaves extract, such as; polyphenol, cathechins, caffeine, alkaloid, tannin, β-citosterol and stigmaste (setyawan, a. budi, winarto, 2016). whereas, antibacterial compound which contained in kefir is lactic acid and alcohol. therefore, during the fermentation process between kefir grains bacteria and tea solution also occur the high activities. on picture 1, it is seen the difference of color on concentration 15% which looks slightly brownish while on concentration 10% looks clearer. generally, this taste of kefir is sour and slightly sweet. this is caused by the activities of yeast, bifidobacteria and lactic acid bacteria which involved in kefir fermentation (laureys, 2014). the success of fermentation process in kefir of tea needs to consider the duration of fermentation. if the length of fermentation duration is excessive then it produces the excessive lactic acid bacteria and occurs the reducing lactic acid bacteria is caused by reducing need of nutrition that will cause the failure in fermentation. on fermentation process will occur fission of nutrients that exist in the ingredients (talattof, 2019). table 2. result of bal total of kefir keji beling tea treatment bal total (x7) cfu/ml yeast total (x7) cfu/ml k1 0,662a 0,4460a k2 0,792b 0,582b k3 1,694c 0,798c international journal of applied biology, 4(2), 2020 35 note: k1= concentration of keji beling tea 10% and duration of fermentation 12 hours, k2= concentration of keji beling tea 15% and duration of fermentation 12 hours, k3= concentration of keji beling tea 10% and duration of fermentation 24 hours, k4= concentration of keji beling tea 15% and duration of fermentation 24 hours. picture 1. kefir of keji beling tea (a. concentration 10%, b. concentration 15%) conclusion kefir of keji beling tea (stachytarpheta jamaicensis) with concentration 10% and 15% in duration of fermentation 12 hours and 24 hours gives the obvious effect with p score = 0,00 toward ph total, acetate acid total, glucose total, polyphenol total, bal total and yeast total. the best treatment of keji beling kefir pointed out on concentration 15% within duration 24 hours. for ph of keji beling kefir shows score 3,375, polyphenol total 3,47%, acetate total 3,26% and glucose total 4. whereas, the result of bal (lactic acid bacteria) total is 1,914 x 107 cfu/ml and for yeast total is 1,018 x107 cfu/ml. acknowledgment the researcher acknowledges the great gratitude due to team of lppm wijaya kusuma university, surabaya for financial support that has been given and to dean of languange and science faculty for the assistance of laboratory facilities during the research process. k4 1,914d 1,018d international journal of applied biology, 4(2), 2020 36 references amalia, siti nur, livia s, leni p. 2015. the influence of leaves position toward total of cathechin levels in keji beling leaves (strobilanthes crispa (l).blume). spesia research proceeding. cai, y., sounderrajan, a. and serventi, l. 2020. water kefir: a review of its microbiological profile, antioxidant potential and sensory quality. acta scientifci nutritional health. 4(6), pp. 10–17. doi: 10.31080/asnh.2020.04.0706. gulitz, a., j.stadie,m.wenning,m.a. ehrmann, and r.f. vogel. 2011. the microbial diversity of water kefir. internasional journal of food microbiology.151 (3):284-288. laureys, d. 2014. water kefir as a promising low-sugar probiotic fermented beverage. archives of public health. biomed central ltd, 72(s1), p. p1. doi: 10.1186/2049-325872-s1-p1. musdholifah, elok zubaidah. 2016. study of antioxidants activity of kefir of soursop leaves tea from various brand in the market. journal of food and agro industrial vol.4(1): 29-39. nadhiroh, husnun. 2018. the influence of fermentation time and concentration of moringa leaves (moringa oleifera) toward the characteristic of water kefir of moringa leaves (moringa oleifera). mini thesis. biology department. fst. maulana malik ibrahim state islamic university. setyaningsih, d. (2008) dwi setyaningsih k 100040050 pharmacy faculty setyawan, a. budi, winarto, e. s. l. 2016. verification of kejibeling leaf extract in improving the immune system. kemas,11(2). available at: http://journal.unnes.ac.id/nju/index.php/kemas%5cnhttp://dx.doi.org/10.15294/. subardjo, m. v. k. 2017. black tea water kefir beverage. massey university. talattof, h. a. 2019. quality of water kefir of soursop fruit with algae cristal starter concentration and different duration of fermentation . muhammadiyah surakarta. contact : a.nur ainun andinurainun@gmail.com 73 abstract the synthesis of silver nanoparticles was carried out using silver nitrate solution as a precursor and padina australis seaweed as a bioreductor. this study aims to obtain silver nanoparticles through the green synthesis method using padina australis seaweed extract. the process of forming silver nanoparticles was carried out by adding padina australis seaweed extract to 1mm agno3 solution. the nanoparticles were characterized using a uv-vis spectrophotometer instrument. the results of the uv-vis spectrum analysis showed that the best nanoparticle synthesis was shown in the 0.5% concentration extract with a duration of 30 minutes with a synthesis temperature of 70°c, with an absorbance peak at a wavelength of 445.00 nm. based on visual observation, the extract turned reddish brown issn : 2580-2410 eissn : 2580-2119 synthesis of silver nanoparticles from padina australis seaweed extract as bioreductor a.nur ainun 1*, zaraswati dwyana 1, eva johannes 1 1 departement of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia introduction nanotechnology is an important field in the field of modern research. the development of nanoparticle technology or often called nanotechnology. in general, nanotechnology can be defined as a technology for designing, manufacturing and applying structures/materials with nanometer dimensions. nanotechnology is not only limited to how to produce nanometer-sized materials or particles, but has a broader understanding including how to manufacture and find out the use of new properties that arise from nanomaterials that have been made (ariyanta,2014). nanoparticles are nano-sized particles around 1-100 nm. materials with nanoparticle structures generally have different properties from the original structure. these various properties can be modified through controlling particle size, regulating chemical composition, surface modification, and controlling interactions between particles. currently, various types of nanoparticles have been synthesized, such as gold, silver, iron, zinc, and metal oxide nanoparticles (prasad, 2013). the nanoparticle synthesis method is based on three process approaches, namely chemical, physical, and biological (iravani et al., 2014). however, both these physical and chemical methods use excessive chemicals which can cause environmental pollution. therefore, an alternative method was developed in the synthesis of nanoparticles or nanomaterials based on the concept of green chemistry, namely the green synthesis method. open access international journal of applied biology keyword green synthesis; silver nanoparticles; padina australis; bioreductor article history received december 21, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 74 the green nanoparticle synthesis method is a synthesis method that forms metal nanoparticles with the help of natural materials derived from organisms (plants and microorganisms) both land and sea (asmathunisha & kathiresan, 2013). the synthesis of silver nanoparticles was carried out using silver nitrate (agno3) solution as a precursor and plants as an alternative bioreductant in synthesizing silver nanoparticles. padina australis contains phenolic compounds and high antioxidant activity this antioxidant content is used as an alternative bioreductant in synthesizing silver nanoparticles. the functional groups of these secondary metabolites work by donating electrons to ag+ ions to produce ag nanoparticles (nursid et al., 2017). materials and methods the study was carried out in may-september 2021 in the integrated laboratory, department of biology and science building, faculty of mathematics and natural sciences, hasanuddin university. the materials used in this study were padina australis seaweed, agno3, naoh, aquabidest, aluminum foil and filter paper. seaweed cleaned, dried with aerated. then the dried seaweed is mashed with a blender until it becomes powder, then sieved using a sieve. then, the padina australis seaweed extract was made. first of all, padina australis seaweed simplicia powder was weighed 10 grams. added 80 ml of aquabidest. the addition of aquabidest is done little by little until the powder is completely wetted and submerged. filter the extract using a funnel using filter paper, the pulp obtained is added with 20 ml of aquabidest then filtered into the same erlenmeyer, then added aquabidest to 100 ml. the extract was diluted with an equivalent concentration of 0.125%; 0.25% and 0.5%, the results of these dilutions are then referred to as test extracts. the synthesis of nanoparticles begins with reacting 0.425 grams of agno3 powder was dissolved in aquabides to a volume of 250 ml and homogenized to make 1 mm agno3 solution. 1 mm agno3 solution was pipetted as much as 20 ml for each concentration of seaweed which had been diluted equally (0.125%; 0.25% and 0.5%) and each solution was put into a glass bottle, then 10 ml of grass extract was added sea of padina australis by ratio (1:3). the test extracts of each concentration were added with agno3 with stirring. after obtaining a homogeneous mixture of the test extract and a solution of agno3 metal salt, then heated with stirring at the synthesis temperature (70°c) for 30 minutes. the absorbance of this solution was observed every 15 minutes (t0; t15; t30) with a spectrophotometer, to monitor the formation of silver nanoparticles. results and discussion the synthesis of silver nanoparticles in this study used the green synthesis method. the silver metal precursor used in this study was agno3 salt, a bioreductant from padina australis seaweed extract. the synthesis was started by dissolving agno3 with padina australis seaweed extract. the treatment being tested is to vary the volume of seaweed extract. the formation of silver nanoparticles can be seen from the results of the uv-vis spectrum of the solution. measurements using a uv-vis spectrophotometer on a solution of seaweed extract were carried out in the absorbance range of wavelength 200 nm 800 nm. the results of the uv-vis spectrophotometer indicate that the reduction reaction has taken place and silver nanoparticles have been formed. international journal of applied biology, 6(1), 2022 75 table 1.measurement of absorbance of each concentration of silver nanoparticle solution of padina australis seaweed extract at a temperature of 70°c. no. treatmen (concentration heating duration (minutes 0,15, dan 30) absorbance (nm) 1. 0,5% 0 minutes 449,00 2. 0,5% 15 minutes 445,50 3. 0,5% 30 minutes 445,00 4. 0,25% 0 minutes 451,00 5. 0,25% 15 minutes 449,00 6. 0,25% 30 minutes 445,00 7. 0,125 % 0 minutes 449,00 8. 0,125% 15 minutes 445,00 9. 0,125% 30 minutes 445,00 the results of the uv-vis spectrophotometer listed in table 1 were 0.5%, 0.25% and 0.125% minutes respectively 0.15.30 synthesis of silver nanoparticles from padina australis seaweed extract resulted that the sample showed the formation of silver nanoparticles characterized by the presence of absorption peak absorbance at a wavelength of 445 nm to 451 nm. the results showed that the best absorbance values were at concentrations of 0.5% minute, 0.15 and 30. in table 1 it can be seen that the uv-vis spectra of silver nanoparticles increased the absorption peak value from 0 minutes to 30 minutes. the increased absorbance indicated that the process of formation of silver nanoparticles increased from 0 minutes to 30 minutes. this also shows that the optimum time for the synthesis of silver nanoparticles is at a synthesis time of 30 minutes with a wavelength of 449.00 nm. figure. 1 the absorbance of the solution with increasing time in the synthesis of silver nanoparticles using padina australis seaweed extract at a temperature of 70°c. silver nanoparticles have specific surface plasmon resonance. if a mixture contains silver nanoparticles, absorption will occur at wavelengths between 400-450 nm (notriawan, 2020). this is in accordance with research conducted by oktaviani, et al (2015) where the nm. 200.00 500.00 800.00 a b s . 4.964 2.006 -0.951 1 2 3 12 3 4 12 3 12 34 1 2 3 1 2 1 2 1 2 1 2 international journal of applied biology, 6(1), 2022 76 synthesized samples formed at absorbance wavelengths of 400 nm to 450 nm are silver nanoparticles. the large number of silver nanoparticles can be seen from the absorbance value (saeb et al., 2014) suggesting that the higher the absorbance, the greater the number of silver nanoparticles. according to handaya et al (2010) samples formed at wavelengths of 400 nm to 450 nm are silver nanoparticles (ag0). while the sample formed at a wavelength of 370 nm to 400 nm is a silver ion (ag+). after the synthesis process, the formation of ag+ means that not all ag+ has been reduced to ag0 so that the maximum wavelength of the synthesized colloid is still less than 400 nm. after 30 minutes, 0.2 m naoh was added dropwise and observed for a color change. the formation of silver nanoparticles is not only seen from the results of the uv-vis spectrum. other parameters can also confirm the formation of silver nanoparticles can be seen by changing the color of the solution. figure 2. concentrations of 0.5%, 0.25%, and 0.125% minute 0. figure 3. concentrations of 0.5%, 0.25%, and 0.125% minutes 15. figure 12. concentrations of 0.5%, 0.25%, and 0.125% 30 minutes. based on visual observations of the results of this study in figures 7,8 and 9, the solution of agno3 and padina australis seaweed extract produced has formed silver nanoparticles with a change in the color of the solution with the addition of 0.2 m naoh and over time, heating from light yellow to reddish brown. this is in accordance with research conducted by haryani et., al (2016) which states that one indicator of the formation of silver nanoparticles in this solution is characterized by a change in color from yellowish to reddish brown with increasing time. international journal of applied biology, 6(1), 2022 77 the occurrence of changes in colloid color in the formation of silver nanoparticles is caused by the oxidation-reduction process. the color change of the solution in the synthesis of silver nanoparticles occurs due to surface plasmon resonance and reduction of silver ions (erjaee et al., 2017). conclusion based on the results of this study, it can be concluded that the seaweed extract of padina australis is able to act as a bioreductant in the process of synthesizing silver nanoparticles. the effect of the concentration of silver nanoparticles in padina australis seaweed extract obtained, the greater the concentration of silver nanoparticles in padina australis seaweed extract, the greater the absorbance value. international journal of applied biology, 6(1), 2022 78 references ariyanta, h. a. (2014). i silver nanoparticles preparation by reduction method and its application as antibacterial for cause of wound infection. jurnal mkmi, 1, 36–42. asmathunisha, n., & kathiresan, k. (2013). a review on biosynthesis of nanoparticles by marine organisms. colloids and surfaces b: biointerfaces, 103, 283–287. https://doi.org/10.1016/j.colsurfb.2012.10.030. handaya a, laksmono ja & haryono a. 2011. preparasi koloid nanosilver menggunakan stabilizer polivinil alkohol dan aplikasinya sebagai antibakteri pada bakteri s. aureus dan e. coli. j kim ind. haryani, y., kartika, g. f., yuharmen, y., putri, e. m., alchalish, d. t., & melanie, y. (2016). pemanfaatan ekstrak air rimpang jahe merah (zingiber officinale linn. var. rubrum) pada biosintesis sederhana nanopartikel perak. chimica et natura acta, 4(3), 151155. nursid, m., marasskuranto, e., atmojo, k. b., hartono, m. p., nur meinita, m. d., & r, r. (2017). investigation on antioxidant compounds from marine algae extracts collected from binuangeun coast, banten, indonesia. squalen bulletin of marine and fisheries postharvest and biotechnology, 11(2), 59. https://doi.org/10.15578/squalen.v11i2.243. notriawan, d., ernis, g., wibowo, r. h., pertiwi, r., & malau, t. r. (2020). aktivitas antibakteri nanopartikel perak hasil green synthesis menggunakan ektrak kulit buah kemuning (murraya paniculata (l) jack). bioedusains: jurnal pendidikan biologi dan sains, 3(2), 140-144. oktaviani, d. t., f., d. c., & amrullah, a. (2009). sintesis nano ag dengan metode reduksi kimia, (1), 101–114. prasad, s. b., & aeri, v. (2013). current understanding of synthesis and pharmacological aspects of silver nanoparticles. american journal of phytomedicine and clinical therapeutics, 1(7), 536-547. saeb, a. t. m., alshammari, a. s., al-brahim, h. dan al-rubeaan, k. a. (2014). production of silver nanoparticles with strong and stable antimicrobial activity against highly pathogenic and multidrug resistant bacteria. the scientific journal, 1-9. erjaee, h., rajaian, h., & nazifi, s. (2017). synthesis and characterization of novel silver nanoparticles using chamaemelum nobile extract for antibacterial application. advances in natural sciences: nanoscience and nanotechnology, 8(2), aa690b. https://doi. org/10.1088/2043-6254/aa690b contact : muhamad azmi dwi susanto muhammadazmidwi@gmail.com 43 abstract the sumur bumi punden is a location used for spiritual tourism. the punde n sumur bumi area has a stagnant aquatic ecosystem type and a low level of disturbance and pollution. therefore, the punden sumur bumi area has the potential to be a natural habitat for dragonflies. this study aims to determine the diversity and composition of the dragonfly community in sumur bumi punden. this study uses the visual day-flying observation technique modified by the transect method. the punden sumur bumi area has a moderate diversity index value, with a value of h' = 2.57. in the sumur bumi punden area, 17 species were found with a total of 124 individuals, including the species with the highest relative abundance, namely brachythemis contaminata. meanwhile, the species with the lowest relative abundance was diplacodes trivialis. the composition of dragonflie s showed that the swamp location had the highest species richness and abundance values, namely 16 species and a total of 79 individuals. meanwhile, the grassland location has the lowest species richness, namely only 5 species. issn : 2580-2410 eissn : 2580-2119 diversity and composition of dragonfly (odonata) at the punden sumur bumi area, surabaya, east java muhamad azmi dwi susanto1* 1 department of biology, faculty of mathematics and natural sciences, universitas brawijaya, malang, indonesia introduction dragonflies are flying insects that in their adult phase live terrestrially (choong et al., 2020), while dragonfly eggs and nymphs live aquatically (kietzka et al., 2021). before becoming terrestrial adults, dragonfly nymphs live aquatically for months to three years (akhtar et al., 2021). therefore, dragonflies are highly dependent on freshwater ecosystems (akhtar et al., 2021; thongprem et al., 2021). dragonflies can be found in various freshwater ecosystems such as rivers, ponds, lakes, and swamps (choong et al., 2020). dragonflies are predatory insects in all phases of their life cycle (nymphs and adults) (thongprem et al., 2021). dragonflies play an important role in maintaining the food chain in ecosystems, namely by controlling small insects, such as orthoptera (tephritidae, acrididae, and gryllidae) and diptera (culicidae, muscidae, chironomidae, and tetrigidae) (dalia & leksono, 2014). dragonflies also play a role as predators of disease-vectoring insects, such as mosquitoes (samanmali et al., 2018). in addition, the diversity of species and composition of dragonflies can also be used as an indicator of the quality of an ecosystem's condition (aziz & mohamed, 2018). several dragonfly species are sensitive to changes in environmental quality open access international journal of applied biology keyword freshwater; composition; insect; odonata article history received february 24, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 44 (buczyński et al., 2020). dragonfly diversity can decrease due to anthropogenic impacts such as habitat destruction, agriculture, and urbanization (perez & bautista, 2020). the punden sumur bumi is an area where there are wells or springs used for spiritual tourism activities. the punden sumur bumi area is located near the industrial area and quite far from residential areas. this location is admini stratively located in karangpilang district, surabaya city. at the location of the punden sumur bumi area, there are stagnant aquatic ecosystems, namely swamps and ponds, which can become dragonflies natural habitat. besides that, there are also meadows, s o the presence of grassy vegetation can provide a variety of small insects as dragonfly food. therefore, the sumur bumi punden area has great potential to become a natural dragonfly habitat. but in the sumur bumi punden area, there is no research data on dragonflies. so that this research can become a source of new literature on the diversity and composition of dragonflies in the sumur bumi punden area. materials and methods time and location study the research was conducted in october and november 2021, with two repetitions each month. this research was conducted at 8:00–11:00 a.m., which is the active time of dragonflies. the research was conducted in the punden sumur bumi area, warugunung village, karangpilang district, surabaya, east java, indonesia. the research location was divided into three locations; each location has different habitat characteristics. the three locations are swamp, pond, and grassland. figure 1. the research location, (a) swamp, (b) pond, (c) grassland a b c international journal of applied biology, 6(2), 2022 45 data collection data collection was carried out using the visual encounter survey (ves) method. data collection was carried out by recording the diversity of dragonfly species and the number of individuals at each predetermined location. the determination of the research path was carried out using the transect and belt transect methods. the transect method is an observation method by following a straight line (used at grassland location), and the belt transect method is an observation method by following a circular line (used at swamp and pond research locations). data collection was carried out by capturing dragonflies using sweeping nets, and then each part of the body was documented in detail using a camera. each individual that has been collected is then identified down to the species level. identification of dragonfly species was based on morphological keys, including body color, wing pattern, abdominal color pattern, and tuft shape. the identification activity was carried out using a guidebook (pamungkas, 2016; setiyono et al., 2017). data collection in this study also took into account abiotic factors consisting of air temperature, humidity, and light intensity. the temperature and humid it y factors were measured using a thermohygrometer, and the light intensity factor was measured using a light meter. data analysis dragonflies encountered during the study were analyzed using the relative abundance, diversity index, dominance index, and evenness index. the following is a formula according to (magurran, 1988): relative abundance (ra) 𝑅𝐴 = 𝑛𝑖 𝑁 x 100% information: ra = relative abundance ni = total individuals belonging to the i spesies n = total individuals of population diversity index (h’) h′ = ∑( 𝑝𝑖 𝐼𝑛 𝑝𝑖) information: h’ = diversity index of shannon-wiener pi = ni/n ni = total individuals belonging to the i spesies n = total individuals of population international journal of applied biology, 6(2), 2022 46 dominance index (d) d = σ (pi)2 information: d = dominance index pi = proportion of each species ni = total individuals belonging to the i spesies n = total individuals of population evenness index (e) e = h’/ ln s information: e = evennes index h’ = diversity index s = number of spesies the study also used multivariate principal component analysis (pca) to determine the relationship between the study location, the biotic index, and abiotic factors. next is to analyze the content of dragonfly compounds between research locations using the upgma cluster analysis method using the bray-curtis index. pca and upgma analyses were performed using past 4.11 software. results and discussion based on observations made in the punden sumur bumi area, it was found that the diversity of dragonflies amounted to 17 species with a total of 124 individuals from 3 families, namely gomphidae, libellulidae, and coenagrionidae (table 1). based on the results of relative abundance, it appears that the species with the highest value is brachythemis contaminata (figure 2a), with a value of 25.00% (table 1). meanwhi le, the species with the lowest relative abundance value was diplacodes trivialis (figure 2b), with a value of 0.81% (table 1). based on data from the international union for conservation of nature (iucn), the conservation status of species found in the punden sumur bumi area are 15 species with low risk or learn concern (lc) status and 2 species with a data deficient conservation status (dd) (table 1). species that have dd conservation status are neurothemis feralis and pseudagrion nigrofasciatum. the conservation status of least concern (lc) is a low-risk status, namely the status for species that are not endangered (pradana et al., 2019). least concern (lc) conservation status is the status of an organism when it has been evaluated based on the red list criteria and does not meet the requirements for critically endangered (cr), endangered (en), vulnerable (vu), or near threatened (nt) (iucn, 2021). meanwhile, the conservation status of dd is the status of lack of data, namely the conservation status of species that do not have sufficient information to make an assessment of their extinction risk based on their distribution and population status. species that fall into the dd category need to be studied further to obtain precise data on their abundance and distribution (iucn, 2021). international journal of applied biology, 6(2), 2022 47 table 1. list of dragonfly species and conservation status family species ra (%) swamp pond grassland status anisoptera gomphidae ictinogomphus decoratus 2.42 + lc libellulidae acisoma panorpoides 8.06 + + lc brachythemis contaminata 25.00 + + lc brachydiplax chalybea 5.65 + + lc crocothemis servilia 4.84 + + + lc diplacodes trivialis 0.81 + lc neurothemis feralis 4.03 + dd orthetrum sabina 11.29 + + + lc pantala flavescens 4.03 + + lc potamarcha congener 4.03 + + lc rhyothemis phyllis 2.42 + + lc rhodothemis rufa 5.65 + + lc zygoptera coenagrionidae agriocnemis femina 7.26 + + lc agriocnemis pygmaea 1.61 + lc ischnura senegalensis 8.87 + + + lc pseudagrion nigrofasciatum 2.42 + + dd pseudagrion rubriceps 1.61 + lc informati on: ra = rel ati ve abundance, (+) pres ent, (-) abs ent. cons ervati on status : dd (data defi ci ent) & lc (leas t concern) (iucn, 2022). figure 2. species of brachythemis contaminata (a) and diplacodes trivialis (b) the shannon-wiener diversity index in the punden sumur bumi area has a value of h' = 2.57 (table 3). these results indicate that the diversity of dragonflies at the location of the punden sumur bumi area is classified as medium diversity, so in this study, it still has a fairly good environment and is in accordance with the natural habitat of various types of dragonflies. according to (purnama et al., 2011), the higher the value of diversity in a location, the higher the complexity of the community, this is because in a community there will be more species interactions. international journal of applied biology, 6(2), 2022 48 the evenness index in the punden sumur bumi area shows a value of e = 0.77; this result shows that the evenness of dragonflies in the punden sumur bumi area is quite high. this is in accordance with (kelsubun & warmetan, 2019), which state that if the value of the evenness index is greater than 0.6, the evenness is high. the evenness index is an index used to measure the balance of the individual components of each community and is used to describe the level of variation in the number of species (nurafni et al., 2020). the evenness index value at a location will be higher if all species found have an even number of individuals (setiadi, 2005). the dominance index in the punden sumur bumi area shows a value of d = 0.10. this result indicates that the dominance of dragonflies in the punden sumur bumi area is relatively low. a low dominance index value at a location indicates that the location has a fairly high species richness with an even distribution (purnama et al., 2011). the punden sumur bumi area is a suitable location for the natural habitat of various types of dragonflies. this is due to the existence of stagnant aquatic ecosystems (swamp and pond) where various types of dragonflies are used to continue their life cycle , namely by laying eggs and becoming larvae in the waters. this is in accordance with (laily et al., 2018), who reported that the egg and nymph phases of dragonflies live in water. as a result, adult dragonflies are rarely far from the water (paulson, 2009; pumungkas & ridwan, 2015; samways, 2008). most of the life stages of dragonflies are in the water, so most types of dragonflies have their main habitat type in the aquatic ecosystem. the diversity and abundance of dragonflies found in the swamp at punden sumur bumi are mostly species that are commonly found in various types of habitats and have a fairly high tolerance for changes in environmental quality. this is because the location of the sumur bumi punden swamp is in an urban area, so the environmental quality is not very good but still allows it to be a natural habitat for various species of dragonflies. therefore, the diversity of dragonflies found at the observation site is a type of dragonfly that has adapted to the urban environment and has a fairly low sensitivity to changes in environmental quality and disturbances from human activities. figure 3. abundance percentage at three research stations 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% swamp pond grassland a b u n d a n c e brachythemis contaminata aciso ma pano rpoides agriocnemis femina brachydiplax chalybea rhodothemis rufa ictinogomphus decoratus orthetrum sabina potamarcha congener ischnura senegalensis cro cothemis servilia rhyothemis phyllis agriocnemis pygmaea pseudagrion rub riceps diplacod es trivialis pan tala flavescen s international journal of applied biology, 6(2), 2022 49 figure 4. result of diversity index, evennes index, dominance index, species richness, and abundance figure 5. the results of measurements of light intensity, temperature, and humidity 0,00 0,50 1,00 1,50 2,00 2,50 3,00 d iv e rs it y i n d e x swamp pond grassland 0,00 0,05 0,10 0,15 0,20 0,25 0,00 0,20 0,40 0,60 0,80 1,00 1,20 swamp pond gra ssland d o m in a n c e i n d e x e v e n n e s i n d e x evenness dominance 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 14 16 18 swamp pond grassland a b u n d a n c e s p e c ie s ri ch n e ss species richness abundance 0 5000 10000 15000 20000 25000 30000 35000 l ig h t in te n si ty ( lx ) swamp pond gra ssland 0 10 20 30 40 50 60 27 28 29 30 31 32 33 34 35 36 37 swamp pond grassland h u m id it y ( % ) t e m p e ra tu re ( o c ) temperature humidity international journal of applied biology, 6(2), 2022 50 figure 6. pca ordinations of three locations the results of pca analysis regarding the relationship of abiotic and biotic factors to the community structure of dragonflies showed that the two main component axes contributed a total of 100% (figure 9). the light intensity and temperature variables (abiotic factors) have a negative correlation with the species richness and diversity index variables (figure 9). this indicates that the intensity of sunlight is too high at the study site, causing the value of species richness and the diversity index to be low. this can be because, generally, dragonflies will choose not to do activities such as sunbathing, flying, a nd looking for food if the intensity of sunlight is too high, but there are also certain species that can still carry out their activities. figure 7. level of similarity based on the composition of dragonflies international journal of applied biology, 6(2), 2022 51 figure 8. differences in family composition based on the level of similarity of dragonfly composition, pond and swamp locations have the same composition (figure 7). this could be because the two locations have similar ecosystem types, namely stagnant waters, so the composition of the dragonflies found also has similarities. there are 10 species that can be found in swamp and pond locations, which are acisoma panorpoides, brachythemis contaminata, brachydiplax chalybea, crocothemis servilia, orthetrum sabina, potamarcha congener, rhyothem is phyllis, rhodothemis rufa, ischnura senegalensis, and pseudagrion nigrofasciatum. the composition of the family at each study site was dominated by the libellulidae family (figure 8). the libellulidae family is a very common dragonfly. this is because t he libellulidae family has a high flying ability and is strong in flight (helmiyetti et al., 2019). the species brachythemis contaminata was the most common species found at the study site. brachythemis contaminata species are found in aquatic plant vegetation. this is in accordance with (helmiyetti et al., 2019), who reported that brachythemis contaminata species prefer waters with lots of vegetation around them. in addition, brachythemis contaminata species can also be found in savannas and rice fields (setiawan et al., 2019). brachythemis contaminata species is often found in locations with open canopy types (susanto et al., 2022), and is often found perched on dry tree trunks (wijayanto et al., 2016), aquatic plants (susanto et al., 2022), ponds and streams in clean to polluted water conditions, so it can be said as a dragonfly that is tolerant of all water conditions (kulkarni & subramanian, 2013). the location of the pond and grassland is a location that only consists of 2 families, namely the libellulidae and coenagrionidae families. the swamp location is the research location with the most complete family composition compared to the other two research sites, which consist of the libellulidae, coenagrionidae, and gomphidae families. there is only one species of the gomphidae family found in the swamp, namely ictinogomphus decoratus. the ictinogomphus decoratus species at the study site was found in tree vegetation. this is in accordance with the study of (susanto et al., 2022), who reported that the species ictinogomphus decoratus was found on wooden branches near the edge of the canopied pond. in addition, ictinogomphus decoratus species can also be found in rice fields, such as in bushes and grass (ansari et al., 2016). the ictinogomphus decoratus species is often found in locations with clean-flowing waters (nisita et al., 2020) and is dominated by vegetation. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% swamp pond grassland gomphidae libellulidae coenagrionidae international journal of applied biology, 6(2), 2022 52 conclusions the punden sumur bumi area has a moderate diversity index value with a value of h' = 2.57. in the sumur bumi punden area, 17 species were found with a total of 124 individuals, including the species with the highest relative abundance, namely 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(2016). inventarisasi capung (insecta: odonata) dan variasi habitatnya di resort tegal bunder dan teluk terima taman nasional bali barat (tnbb). 427–434. contact : whisnu febry afrianto whisnuafrianto@apps.ipb.ac.id 74 abstract acacia deccurens wild. has been reported as invasive alien species (ias) in several areas of indonesia. climate change may impact ias to be more invader. the study aimed was to develop a species distribution model of a. deccurens to depict the potential distribution under climate change in indonesia. biodiversity and climate change virtual laboratory (bccvl) was used to examine a species distribution model (sdm) of a. decurrens in indonesia based on climate variables and its naturalized distribution to predict the project distribution under current and future climate conditions. the data was collected from global biodiversity information facility (gbif) to identify the species occurrences. the climate variables used in this study were temperature and precipitation layers based on worldclim, current climate (1950-2000), 2.5 arcmin (~5km). the sdm of the generalized linear model (glm) was utilized to predict the response variable as a function of multiple predictor variables. we selected four ipcc representative concentration pathways (rcp) 2.6, 4.5, 6.0, and 8.5 for 2050. the prediction of the distribution of a. deccurens in 2050 showed that it was likely to decrease in indonesia (mostly found only in sumatra and sulawesi island). almost all climate variables used in this study were responsive to a. decurrens distribution, except b09 mean temperature of the driest quarter. the roc plot showed excellent values (0.99). the information of the potential distribution on ias under current and future climate scenarios can be used for policymakers and stakeholders to manage and handle the invasion. issn : 2580-2410 eissn : 2580-2119 the potential distribution prediction of the invasive alien species acacia decurrens wild., in indonesia whisnu febry afrianto 1* 1 ecosystem and biodiversity (ecosbio), jl. merapi 02/01, datengan, grogol, kediri, 64151 introduction the genus of acacia worldwide includes ± 1300 species and about 960 species come from australia and it spreads in the tropics to the temperate area, namely europe, africa, south asia, and america (wrigley & fagg, 2013). one of the acacia species spread in indonesia is acacia deccurens wild. this species, commonly known as black wattle or green wattle, is a fast-growing tree species of the fabaceae family (bamidele et al., 2017). this species can grow about 6-12 m and easily adapt to acidic soil conditions (endalew et al., 2014). naturally, a. deccurens grows in a lower mountain valley (molla & linger, 2017). a. deccurens is widely planted because it has benefits both for economic and environmental in forestry, agricultural, and ecosystem forestry (nigussie et al., 2016; wondie & mekuria, 2018; nigussie et al., 2020; chanie & abewa, 2021; nigussie et al., open access international journal of applied biology keyword acacia deccurens, climate change, invasive alien species, special distribution model. article history received 12 september 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 75 2021). several studies have reported that a. deccurens cultivated in degraded land can improve soil fertility, increase water quality, and prevent soil erosion (reubens et al., 2011; molla & linger, 2017; bazie et al., 2020). furthermore, according to cabi (2021), a. decurrens can be used as fuels (charcoal and fuelwood), ornamental plants, and materials (tanning and timber) (richardson et al., 2015). on the other side, several publications have reported that a. decurrens is a severe invasive problem (i.e., hawai, new zealand, africa, and indonesia) that this species spreads rapidly through root suckers and seed (richardson and rejma´nek 2011; richardson et al., 2015). the invasion by an alien species of a. decurrens can proliferate as a pioneer plant in an area where the native species can not to adapt to environmental conditions (sunardi et al., 2015; sunardi et al., 2017). it creates negative consequences, especially for native biodiversity (sunardi et al., 2017). in indonesia, the invasiveness of a. decurrens is found several areas such as mount merapi after eruption 2006 (suryanto et al., 2010 a,b) and 2010 (afrianto et al. 2016; sunardi et al., 2017; afrianto et al., 2017, afrianto et al., 2020), mount merbabu (purwaningsih, 2010; untoro et al. 2017), kawah ijen nature tourism park (hapsari et al. 2014), and mount panderman nature tourism (septiadi. 2018). species distribution models (sdms) can predict the effect on potential species distributions under climate change at the single-species and community levels (sung et al., 2018). the study aimed to develop a sdm of a. deccurens to project the potential distribution under climate change in indonesia. therefore, understanding the sdm of the potential distribution of the invasive species under and future climate change can be used as early preventive and management strategies for managing and handling the invasion. materials and methods data collection this study was analyzed by the biodiversity and climate change virtual laboratory (bccvl) (http://www.bccvl.org.au/). bccvl is cloud-based, providing access to modeling tools, large species distribution, climate, the collection of biological and other environmental datasets, and diverse experiment categories to carry out a study into the relationship between biodiversity and climate change (hallgren et al., 2016). the global biodiversity information facility (gbif) (http://www.gbif.org/) dataset of a. decurrens was used to conduct the species occurrence (gbif 2021). worldclim current conditions (19502000) at 2.5 arcmin was used in this simulation. based on the database, a. decurrens has about 17,917 occurrence records and 17,232 geo-referenced. then, this data was imported in bccvl. the climate and environmental data used worldclim, current climate (19502000), 2.5 arcmin (~5km). these bioclimatic variables were generated using 1950 to 2000 from an array of global climate layers (except antarctica). eight climate variables were chosen in the bccvl, such as: 1. b04 (temperature seasonality, standard deviation) 2. b05 (max temperature of warmest month) 3. b06 (min temperature of coldest month) 4. b08 (mean temperature of wettest quarter) 5. b09 (mean temperature of driest quarter) 6. b13 (precipitation of wettest month) 7. b14 (precipitation of driest month) and, 8. b15 (precipitation seasonality, coefficient of variation). the temperature and precipitation data were chosen because they are important international journal of applied biology, 5(2), 2021 76 factors to impact vegetation range and abundance of species (krebs, 1985). afrianto et al., (2017) state that the habitat preferences of a. decurrens were strongly correlated with temperature conditions. because we do not have a true absence dataset for the experiment, we used the pseudo absence configuration (pa models) with the absencepresence ratio of 1, the random pseudo-absence strategy, and the number of background points of 10,000. pseudo-absence points were used to generate for the experiment. data analysis the experiment was conducted by the primary experiment of sdm experiment. for the algorithm of sdm, we used generalized linear model (glm). the glm is a linear regression model to predict the response variable as a function of multiple predictor variables. the glm was used because it has several advantages, namely (1) the response variable be able to all form of the exponential distribution model, (2) can be used in categorical predictors, (3) easy to interpret and understand how each of the indicators is impacting the outcome, (4) less vulnerable to overfitting than for instance cta or mars algorithms. the area under the curve (auc) of the receiver operating characteristics (roc) curve was used to examine model robustness. this curve is a non-parametric thresholdindependent measure of accuracy used to assess sdm (bertelsmeier & courchamp. 2014). the x-axis of the roc plot is a graph of the false positive rate (1specificity), and the y-axis is an actual positive rate (sensitivity). the values above 0.5 means prediction better than random, and the value of 0.5 means a random prediction. the auc score was classified as follow (crego et al., 2014): a. value above 0.9 is excellent b. good 0.9 > auc > 0.8 c. fair 0.8 > auc > 0.7 d. poor 0.7 > auc > 0.6, and e. fail 0.6 > auc > 0.5 further analysis was conducted by the secondary experiment that is the climate experiment, to investigate the distribution of a species under potential future climatic conditions. a climate change experiment predicted a. decurrens distribution with the climate information under climate change scenarios. in this study, we selected four ipcc representative concentration pathways (rcp) 2.6, 4.5, 6.0, and 8.5 for the 2050s. furthermore, we evaluated by (1) worldclim, future projection using ipsl-cm5a-lr rcp 2.6 10 arcmin (2050), (2) worldclim, future projection using ipsl cm5a-lr rcp 4.5, 10 arcmin (2050), (3) worldclim, future projection using ipsl-cm5a-lr rcp 6.0, 10 arcmin (2050), and (4) worldclim, future projection using ipsl-cm5alr rcp 8.5, 10 arcmin (2050). the prediction results based on the current climate condition indicate distribution of suitable habitat. results and disscussion present distribution of a. decurrens in indonesia the introduction of a. decurrens in indonesia is for industrial purposes. this exotic plant species is planted as crops plants or plantations such as mahogany, pine, agathist, coffee, cocoa, palm oil, acacia, african wood, and others. exotic species introduced in indonesia are listed as industrial plants. including a. decurrens since ancient times of dutch colonialism (purwaningsih, 2010). the prediction of the current distribution of a. decurrens showed that mostly this international journal of applied biology, 5(2), 2021 77 species occurred in sumatra and sulawesi island. in sumatra island, a. decurrens distributed to several provinces banda aceh, north sumatera, west sumatera, and bengkulu. in contrast, in sulawesi island, this invasive species spread to west sulawesi, central sulawesi, and some parts of along with southeast sulawesi (figure 1). however, until now, no scientific documents and reports explain the occurrence of a. decurrens in these areas. mostly, the studies of a. decurrens is only found in java island, especially in mount merapi national park. based on the report gbif, in indonesia a. decurrens has less for the invasiveness impact with 42 occurrences reported (gbif, 2021). on the other hand, the highest occurrence is found in colombia of 12,725 occurrences and has the highest invasiveness impact of a. decurrens. figure 1. map of current distribution of ias of a. decurrens under current climate condition in indonesia using glm algorithm in bccvl. darker areas represent a higher potential distribution of a. decurrens. by “invasion pathway” or the stages of invasion, there are five nonexclusive consequences of climate change for invasive species, namely (1) modified transport and initiation mechanisms, (2) establishment stage by new invasive species in the area, (3) modified impact of existing invasive species, (4) the spreading of invasive species, and (5) modified effectiveness of control approach (hellman et al., 2008). the distribution of invasive species under climate change is found more invader in outside protected areas of europe's marine and terrestrial because of the low human accessibility (gallardo et al., 2017). panda et al., (2017) state the phenology and capacity of species to adapt quickly in climate are potentially related to the invasion stage in the future climate. potential future distribution under climate change of a. decurrens in indonesia climate change condition is likely to decrease the potential of the distribution of a. decurrens in indonesia. figure 2 shows that a. deccurens in sumatra island was only found in banda aceh province based on ipcc rcp 2.6, 4.5, 6.0, and 8.5 for 2050, and some part of north sumatera (pemantang siantar) based on ipcc rcp 2.6 and 4.5 for 2050. moreover, figure 3 shows that a. decurrens in sulawesi island were only found in central sulawesi and south sulawesi based on ipcc rcp 2.6, 4.5, and 6.0 for 2050. international journal of applied biology, 5(2), 2021 78 figure 2. comparison of climate change models of a. decurens for current and 2050 in kalimantan island. (a) current distribution, and (b-2) ipcc rcp 2.6, 4.5, 6.0, and 8.5 for the 2050 were evaluated with worldclim data and 10 arcmin resolution. darker areas represent a higher potential distribution of a. decurrens. international journal of applied biology, 5(2), 2021 79 figure 3. comparison of climate change models of a. decurens for current and 2050 in sulawesi island. (a) current distribution, and (b-2) ipcc rcp 2.6, 4.5, 6.0, and 8.5 for the 2050 were evaluated with worldclim data and 10 arcmin resolution. darker areas represent a higher potential distribution of a. decurrens. except for b09 (mean temperature of driest quarter), all climate variables used in this study are responsive to a. decurrens distribution (figure 4). a. decurrens was found in areas that have moderate frost tolerance. it grows in the warm sub-humid to the humid climatic zone. the environmental requirement of a. deccurens needs annual rainfall of 9001150 mm. it can grow with a mean minimum of the coolest month of 1-5°c, or it will even tolerate temperatures as low as -6°c. on the other hand, the mean maximum of the hottest month is 26-30°c. in general, the roc plot showed excellent values (0.99) (figure 5). the climate change predicted will make several species losses in 2050 (gallagher et al. 2012). this phenomenon is because climate change will make a warmer condition where it may impact the flowering and seedling of several plant species (germishuizen and gardner 2015; booth 2017). the short term of climate change is predicted to affect abundance and distribution (blyth et al. 2021). in a different result, sutomo et al. (2017) shows that the distribution prediction of a. nilotica in 2045 will increase, especially in eastern indonesia. kriticos et al., (2003), also state sdm of a. nilotica might rising significantly because of climate change. it is because a. nilotica can grow in temperature around ~35 °c or 5°c warmer rather than a. deccurens. this model prediction does not include other factors that are considered to impact the distribution of a. decurrens. those factors are edaphic, topographic, and dispersal agent. it is because this species has values both economically and ecologically, thence social aspects are also required to be analysed (sutomo et al., 2021 a,b). by adding those variables, it will make the prediction more powerful (booth, 2018). international journal of applied biology, 5(2), 2021 80 figure 4. the response curve for a. decurrens distribution model. figure 5. roc plot of a. decurrens model using glm in bccvl. conclusions a. decurrens will decrease in 2025 based on the current and future climate variables conditions. except for the mean temperature of the driest quarter, all climate variables used in this study were responsive to a. decurrens distribution, and the roc plot showed excellent values (0.99). this study provides great tools to determine the impacts of climate change on the ias of a. decurrens for management purposes. international journal of applied biology, 5(2), 2021 81 references afrianto, w.f., hikmat, a & widyamotoko, d. 2020. plant species diversity and degree of homogeneity after the 2010 eruption of mount merapi, indonesia. biosaintifika: journal of biology & biology education. 12(2) : 274-281. afrianto, w.f., hikmat, a & widyamotoko, d. 2017. growth and habitat preferences of acacia decurrens willd. 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they are good for fungal growth. this media is pretty much needed in fungal breeding both in the laboratory and in agriculture. but the price of this media is quite expensive besides that not all chemical shops provide it, while the needs of pda media are increasing so that another alternative is needed to replace the fungal culture media. this research is instrumental with a cross sectional approach and aims to find out the level of significance of the differences in the growth of candida albicans against alternative media for agar dextrose agar and semi-synthetic potato dextrose agar (pda). the sampling technique was non-random with a purposive sampling design of 46 people. the sample was then isolated on a semi-synthetic pda media and alternative media to see differences in the growth of candida albicans. based on the research that has been carried out, it is concluded that there are 38 samples of respondents detected positively candida albicans on semi-synthetic media (pda) and alternative media, and based on the results of data processing obtained p value 0,000 <0.05 which indicates that there are very significant differences in growth candida albicans between semi-synthetic media (pda) and alternative media. issn : 2580-2410 eissn : 2580-2119 comparison growth of candida albicans in alternative media and semi-synthetic on fisheries cerumen in bastiong karance ternate city erpi nurdin & lailatul ukhdiyah syarifudin medical technology laboratory, poltekkes kemenkes ternate, ternate city, maluku utara introduction indonesia is a country that is geographically located at 6 northern latitudes 11 south latitudes, 95 east longitude to 141 west longitude. with such a geographical position, indonesia is located in the tropics. it affects the climate, weather, humidity and high rainfall and heat throughout the year. this facilitates the emergence of diseases caused by fungi, including otomikosis (windu, 2010). open access international journal of applied biology keyword candida albicans, alternative media, potato dextrose agar. article history received 11 june 2020 accepted 30 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 116 otomycosis is an inflammatory process in the ear canal caused by a fungal infection. otomycosis is a form of disease that is commonly found throughout the world. the frequency varies depending on differences in geographic zones, environmental factors, and also time. otomycosis is often found in ent (ear, nose, throat) clinics and the prevalence reaches 9% of all patients who show symptoms and signs of otitis externa with candida sp and aspergillus sp as the most fungal species obtained from their oscillates (sukardi, 2009). many factors have been identified as predisposing to otomycosis, including humid weather, the presence of cerumen, ear instrumentation or ear equipment such as hearing aids, immunocompromised patient status, hot and humid climate, and increased use of topical steroids and antibiotics. recommended treatment includes local debridoment, stopping use of topical and local or systemic antibiotics (dwi hapsari, 2011). the ear canal is connected to the outside air by a narrow orifice, so that it can function as an excellent breeding ground for bacterial and fungal growth. at a relative humidity above 80%, the epithelial horn layer can absorb water from the air in large quantities. increasing the contents of the keratin liquid in the pilosebaseus causes swelling and obstruction of the orifice which results in the loss or reduction of cerumen formation. the formation of the affected cerumen will cause a fungal infection in the external ear called otomycosis. cerumen contains protein, amino acids, mineral ions, lysozyme, immunoglobulins and fatty acids which can inhibit the growth of bacteria and fungi (sedjawidada, 2009). from previous research data in 2009, it was found that the incidence of fungal colony in ear infection patients in ent clinic (ear, nose, throat) hasanuddin university hospital, from a total sample of 103, a fungal colony was found on culture media with aspergillus niger 26.6 %, aspergillus fumigatus 37.5%, and candida albicans 26.9% (sedjawidada, et al, 2009). the activity of fishermen on the high seas at night or during the day is often at risk of contracting fungal diseases, because their workplaces are in the open air and in wet and humid areas favored by fungi. the work as fishermen also makes them often exposed to direct sunlight so that the body's temperature rises, the body will produce sweat and cause the ear wax to be disturbed. ear canal is one of the places that is easily infected by fungus because the atmosphere is dark and warm makes the fungus easily grow because it gives high humidity which is suitable as a fungal habitat. in addition, most fishermen also pay little attention to personal hygiene and health. bastiong karance is one of the villages located in the coastal area of ternate city, a village located in ternate island subdistrict, ternate city, north maluku province. geographically, the bastiong karance sub-district is located in the south ternate city subdistrict region, and administratively the bastiong karance kelurahan is bordered by north side with bastiong talangame village, west side with ubo-ubo village, east side with sea, and south side with kayu merah village. (profil kota ternate, 2013). demographic information shows that the total population of the bastiong karance urban village is 5,562. the number consisted of 2,785 men and 2,777 women with a sex ratio of 100.29. based on the area and population, it is found that the population density in this village is 4,653 inhabitants / km2. the number of households is 1,187 families and the number of inhabitants in a family is 4.69 inhabitants. motorcycle taxis, civil servants, and housewives. (bappeda kota ternate, 2012). fungal diagnosis can be made based on examination by various methods, namely the trap method, thinner, seedlings, hendrikill’s, directly (using 10% koh) and culture methods. however, in this study using the culture method, because the culture method is the definitive diagnostic method for most bacteria and fungi. the culture method is also included in the international journal of applied biology, 4(1), 2020 117 gold standard method to identify the types of fungi and bacteria, which is then followed by 10% koh staining, because to confirm the presence of fungi growing on culture media (sedjawidada, et al, 2009). some researchers have succeeded in finding alternative media for fungal growth from protein sources, namely cowpea, green beans, and black soybeans (ravimannan et al, 2014). in addition to research with protein sources, various carbohydrate sources have also been used successfully as alternative media such as cassava starch (kwoseh et al, 2012), sago and uwi (tharmila et al, 2011), potatoes and tuber palmirah (martyniuk et al, 2011). previous study from muhammad rizal's in 2018 succeeded in optimizing the optimum concentration of candida albicans growth on alternative potato media at a concentration of 300 grams per liter of media. erpi nurdin's study in 2018 found the most significant growth results of candida albicans found in breadfruit alternative media, compared to alternative media for potatoes, cassava and sago. the results of the breadfruit chemical analysis (artocarpus altilis) showed variations among 18 populations of breadfruit origin (kediri, banyuwangi, bone, shove, ternate, bali, etc.) with an average number of calories (62.65-110.98), carbohydrate ( 7.81-24.32%), protein (1.383.67%), fat (0.31-0.47%), vitamin c (19.56-47.74 mg / 100g), phosphorus (10.14-35-68 mg / 100g), calcium (42.23-251.45 ppm), fiber (1.18-1.84%), and iron (0.51 ppm). the results of the chemical analysis of breadfruit flour showed that the average carbohydrate content (70.44%), protein (6.59%), fat (1.29%) and fiber (6.55%). with high nutritional content, fruit breadfruit is very potential to be developed as an alternative medium in making fungal growth media. (adinugraha and susilawati, 2014). candida albicans examination can be done by three methods, namely the wet mount method, gram staining, and culture. the advantage of a wet mount examination can be done in a simple way, but direct examination should be done immediately after clinical material is obtained because candida albicans develops rapidly at room temperature so that it can give an inappropriate picture. direct examination with gram staining requires less time compared to examination with koh. this examination can see the fungus candida albicans based on its morphology. the culture method can be used with the potato dextrosa agar (pda) medium with fungal growth appearing to be small, round, moist, white, with smooth and flat edges. (vivi keumala mutiawati, 2016). candida albicans is a unicellular fungal species that is often encountered in human infections, so this fungal species is an object that is grown in a variety of alternative and semisynthetic media. in addition, rapid growth for 48 hours can be observed compared to multicellular fungi. medium is a material that consists of a mixture of food substances (nutrients) that function as a place to grow microbes. in addition to growing microbes, the medium can also be used to isolate, multiply, test physiological properties, and calculate the number of microbes (cahyani, 2014). one of the microorganisms that are often cultured in microbiology both in the food industry and in the agricultural industry is fungals. fungal is one of microorganism which is often grown using pda (potato dextrose agar) media. based on its composition, pda is included in the semi-synthetic media because it is composed of natural ingredients (potatoes) and synthesis materials (dextrose and agar). potatoes are a source of carbon (carbohydrates), vitamins and energy, dextrose as a source of sugar and energy, in addition to components that serve to compact the pda medium. each of the three components is very necessary for the growth and propagation of microorganisms, especially fungi (cahyani, 2014). international journal of applied biology, 4(1), 2020 118 fungal growth and development are generally greatly influenced by a number of factors including temperature, light, air, ph and nutrients such as carbon and nitrogen (barnett and hunter, 1998), and simple carbohydrates (kelley, 1977). semi-synthetic media such as pdas have sufficient carbohydrate content so that they are good for fungal growth. this media is pretty much needed in fungal breeding both in the laboratory and in agriculture. however, the price of this media is quite expensive besides that not all chemical shops provide it, while the needs for pda media are increasing so that another alternative is needed to replace the fungal culture media. based on the description above, the researchers intend to examine and find out the difference in the number of growth of the fungus candida albicans isolated from the ear of fishermen in the village of bastiong karance in alternative breadfruit media and pda semisynthetic media. materials and methods tools and materials petri dishes, rounded oceans, methylated lamps, methylates, microscopes, sterile tubes, autoclaves, glass objects and cover glass, 10% koh (merck®), incubators (memmert®), potato dextrosa agar (pda) (oxoid®), breadfruit , sucrose (merck®), agar (oxoid®) methods prepared tools and materials to be used, inoculated samples from culture media, place on glass objects, add 1 drop of 10% koh, cover with glass cover, wait for 10 minutes, and observe under a microscope at 40x magnification (10x ocular lens and 4x objective lens) and 400x (40x ocular lens and 10x objective lens). this microscopic examination was carried out to observe the morphology of the candida sp fungus seen using a microscope. results the research data was obtained from sampling in the form of ear wax taken from the ears of fishermen in bastiong ternate city. while the sample examination was carried out at the integrated laboratory of health polytechnic ministry of health ternate. from taking and examining samples taken from 46 fishermen, the following results were obtained (table 1). from the results of examination of the sample in table 1 shows that from 38 respondents were found positive for candida albicans. based on table 2 there are 38 positive samples of both synthetic and alternative fungi growth media that are overgrown with candida albicans. the research data were processed using paired t-test for comparison of the growth of candida albicans taken from fishermen's research subjects in semi-synthetic media (pda) and alternative media. from the results of data processing, the significance level of the relationship is 0.000 discussion some researchers have succeeded in finding alternative media for fungus growth from protein sources, namely cowpea, green beans, and black soybeans (ravimannan et al, 2014). in addition to research with protein sources, various carbohydrate sources have also been used successfully as alternative media such as cassava starch (kwoseh et al, 2012), sago and uwi (tharmila et al, 2011), potatoes and tuber palmirah (martyniuk et al, 2011). international journal of applied biology, 4(1), 2020 119 one of the microorganisms that are often cultured in microbiology both in the food industry and in the agricultural industry is fungi. fungi is one of microorganism which is often grown using pda (potato dextrose agar) media. based on its composition, pda is included in the semi-synthetic media because it is composed of natural ingredients (potatoes) and synthesis materials (dextrose and agar). potatoes are a source of carbon (carbohydrates), vitamins and energy, dextrose as a source of sugar and energy, in addition to components that serve to compact the pda medium. each of the three components is very necessary for the growth and propagation of microorganisms, especially fungi (cahyani, 2014). fungal diagnosis can be made based on examination by various methods, namely the trap method, thinner, seedlings, hendrikill’s, directly (using 10% koh) and culture methods. however, in this study using the culture method, because the culture method is the definitive diagnostic method for most bacteria and fungi. the culture method is also included in the gold standard method to identify the types of fungi and bacteria, which is then followed by 10% koh staining, because to confirm the presence of fungi growing on culture media (sedjawidada, et al, 2009). muhammad rizal's research (2018) succeeded in optimizing the optimum concentration of candida albicans growth on alternative potato media at a concentration of 300 grams per liter of media. erpi nurdin's research in 2018 found the most significant growth results of candida albicans found in breadfruit alternative media, compared to alternative media for potatoes, cassava and sago. the results of the breadfruit chemical analysis (artocarpus altilis) showed variations among 18 populations of breadfruit origin (kediri, banyuwangi, bone, shove, ternate, bali, etc.) with an average number of calories (62.65-110.98), carbohydrate ( 7.81-24.32%), protein (1.38-3.67%), fat (0.31-0.47%), vitamin c (19.56-47.74 mg / 100g), phosphorus (10.14-35-68 mg / 100g), calcium (42.23-251.45 ppm), fiber (1.18-1.84%), and iron (0.51 ppm). the results of the chemical analysis of breadfruit flour showed that the average carbohydrate content (70.44%), protein (6.59%), fat (1.29%) and fiber (6.55%). with high nutritional content, fruit breadfruit is very potential to be developed as an alternative medium in making fungal growth media. (adinugraha and susilawati, 2014). candida albicans examination can be done by three methods, namely the wet mount method, gram staining, and culture. the advantage of a wet mount examination can be done in a simple way, but direct examination should be done immediately after clinical material is obtained because candida albicans develops rapidly at room temperature so that it can give an inappropriate picture. direct examination with gram staining requires less time compared to examination with koh. this examination can see the fungus candida albicans based on its morphology. the culture method can be used with the potato dextrosa agar (pda) medium with fungal growth appearing to be small, round, moist, white, with smooth and flat edges. (vivi keumala mutiawati. 2016). candida albicans is a unicellular fungal species that is often encountered in human infections, so this fungal species is an object that is grown in a variety of alternative and semisynthetic media. in addition, rapid growth for 48 hours can be observed compared to multicellular fungi. medium is a material that consists of a mixture of food substances (nutrients) that function as a place to grow microbes. in addition to growing microbes, the medium can also be used to multiply isolation, test physiological properties, and calculate the number of microbes (cahyani, 2014). international journal of applied biology, 4(1), 2020 120 the work as fishermen also makes them often exposed to direct sunlight so that the body's temperature rises, the body will produce sweat and cause the ear wax to be disturbed. ear canal is one of the places that is easily infected by fungus because the atmosphere is dark and warm makes the fungus easily grow because it gives high humidity which is suitable as a fungal habitat. in addition, most fishermen also pay little attention to personal hygiene and health. fungal growth and development are generally greatly influenced by a number of factors including temperature, light, air, ph and nutrients such as carbon and nitrogen (barnett and hunter, 1998), and simple carbohydrates (kelley, 1977). semi-synthetic media such as pdas have sufficient carbohydrate content so that they are good for fungal growth. this media is pretty much needed in fungal breeding both in the laboratory and in agriculture. however, the price of this media is quite expensive besides that not all chemical shops provide it, while the needs for pda media are increasing so that another alternative is needed to replace the fungal culture media. based on the research results of 46 samples examined there were 8 negative candida albicans on semi-synthetic and alternative media, while 38 positive samples were overgrown with candida albicans on both fungal growth media. alternative media derived from breadfruit, dextrose, and agar, are very well grown by fungi isolated from ear wax. in addition to candida labicans, the media is also covered with multicellular fungi such as aspergillus sp. the research data were processed using paired t-test for comparison of the growth of candida albicans taken from fishermen's research subjects in semi-synthetic media (pda) and alternative media. from the results of data processing, the significance level of the relationship is 0.000. from the results of statistical tests it was found that the alternative media is very well used as a growth medium for candida albicans fungi. conclusions based on the research that has been carried out, the following conclusions are obtained: there were 38 samples of respondents who were tested positive for candida albicans on semi-synthetic media (pda) and alternative media. based on the data processing results obtained p value 0,000 <0.05 which indicates that 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pada penderita otore dengan berbagai penyebab di poliklinik tht rumah sakit pendidikan unhas, 1 (1): 1-14. sukardi, 2009. ilmu penyakit telinga hidung tenggorok untuk perawat. fakultas kedokteran universitas indonesia : jakarta. supriatin, s, 2010. candida dan kandidiasis pada manusia. balai penerbit jakarta: fakultas kedokteran ui. international journal of applied biology, 4(1), 2020 122 vivi keumala mutiawati. 2016. pemeriksaan mikrobiologi pada candida albicans. jurnal kedokteran syiah kuala. diakses tanggal 28 febriari 2018 windi, 2017. identifikasi jamur aspergillus sp. pada swab liang telinga para petani di kelura han gambesi kecamatan ternate selatan windu, 2010. laporan kasus otomycosis. jurnal kesehatan andalas, 3(2): 101–106. yudhi, 2010. laporan kasus otomycosis primer dan sekunder. jurnal kesehatan international journal of applied biology, 4(1), 2020 123 table and figure table 1. results of the examination of candida albicans in the ear cerumen against semisynthetic media and alternative media no. name (initial) sample code age (year) growth of candida albicans (colony) semi sintetic media (pda) alternative media 1 tn. hr nc1 37 2 1 2 tn. hn nc2 36 3 3 3 tn. rf nc3 37 5 6 4 tn. mc nc4 28 0 0 5 tn. fl nc5 41 17 2 6 tn. ir nc6 35 1 2 7 tn. hm nc7 36 247 155 8 tn. jl nc8 46 54 31 9 tn. dl nc9 48 24 24 10 tn. rl nc10 39 43 31 11 tn. hs nc11 35 5 5 12 tn. mm nc12 35 7 5 13 tn. mr nc13 37 0 0 14 tn. dd nc14 40 5 7 15 tn. bk nc15 35 48 20 16 tn. ld nc16 36 32 31 17 tn. hd nc17 37 21 13 18 tn.kl nc18 36 14 10 19 tn. ad nc19 48 0 0 20 tn. de nc20 49 3 6 21 tn. ot nc21 37 21 11 22 tn. is nc22 39 0 0 23 tn. ae nc23 37 57 20 24 tn. ad nc24 28 122 23 25 tn. ju nc25 25 23 23 26 tn. dn nc26 45 19 2 27 tn. bm nc27 44 299 5 28 tn. rt nc28 37 76 1 29 tn. hl nc29 34 0 0 30 tn. wd nc30 20 0 0 31 tn. el nc31 31 4 2 32 tn. al nc32 41 0 0 33 tn. ds nc33 32 17 17 34 tn. do nc34 35 0 0 35 tn. rf nc35 27 5 11 36 tn. uc nc36 38 199 2 37 tn. zh nc37 46 5 9 38 tn. on nc38 48 3 3 39 tn. jm ns39 45 7 10 40 tn. er ns40 43 6 21 41 tn. yd ns41 45 8 7 42 tn. st ns42 44 3 3 43 tn. al ns43 36 299 299 44 tn. rs ns44 46 2 4 45 tn. am ns45 46 3 3 46 tn. fi ns46 30 1 2 table 2. distribution and frequency of salmonella typhi on children's hygiene candida albicans quantity total negative positive semi-syntetic media (pda) 8 38 46 international journal of applied biology, 4(1), 2020 124 fungal growth fungal growth p ro g re ss iv e alternative media 8 38 36 table 3. t-test measurements for the growth of candida albicans on semi-synthetic media (pdas) and alternative media test value = 0 t df sig. (2-tailed) mean difference 95% confidence interval of the difference lower upper fungus growth 12.728 45 .000 .783 .66 .91 fungus growth 14.620 45 .000 .826 .71 .94 figure 1. candida albicans colonies and microscopes figure 2. growth diagrams of candida albicans on semi-synthetic media (pda) and alternative media contact : lukman daris daris.lukman70@gmail.com 112 abstract in seaweed cultivation, many wives in seaweed farming households also work to help their husbands. therefore, the role of women is quite crucial for seaweed cultivation. this study aims to analyze the role of women in productive, reproductive, and community management activities and the level of household welfare of seaweed cultivators in jeneponto regency, south sulawesi province. the method used in this research is descriptive qualitative, and quantitative. data were analyzed through a correlation test with quantitative data acquisition using a simple random sampling technique. based on the study results, it is known that women are more dominant in the category of minor role (71.25%). only about 3.75% of respondents fall into the category of very role. 25% of the awards fall into the category of playing a role. based on interviews, it is known that women are more involved in productive activities on land. in reproductive activities and management activities, women are included in having a significant role (100% of respondents). based on the results of statistical analysis, it is known that the relationship between income and the level of household welfare of seaweed cultivators has a substantial correlation coefficient value between the two variables with a significance level of less than 0.01. the value of the correlation coefficient between women and the household income of seaweed cultivators shows a positive relationship between the role of women and the household income of seaweed cultivators. however, the correlation value is weak and has no significant effect. issn : 2580-2410 eissn : 2580-2119 the role of women on the level of household welfare of seaweed cultivators in jeneponto regency, indonesia lukman daris1, khairun nisaa2, irwansyah1, wahyuti1, andi masriah2, alpiani1 1fisheries agribusiness program, faculty of fisheries, cokroaminoto makassar university, makassar, indonesia. 2aquaculture program, faculty of fisheries, cokroaminoto makassar university, makassar, indonesia. introduction seaweed is a fishery commodity that has great potential to increase its production, both natural and cultivated. seaweed production needs to be increased due to the increasing global market demand. according to (stévant et al, 2017), global seaweed production is mainly dominated by asian countries, one of which comes from indonesia. seaweed has many benefits as a food ingredient (either in dry or wet form) or as a follow-up process to make other foods mainly picocoloids such as agar, carrageenan, and alginate. in addition, seaweed is also used for fertilizers, medicines, and cosmetics (stévant et al, 2017) and it has potential as raw material for producing sugar (zaenab et al, 2020). coastal communities are very dependent on their geographical conditions, and traditional fishers still dominate many fishery business actors. this condition was caused by open access international journal of applied biology keyword seaweed; household welfare; women; income article history received december 3, 2020 accepted july 3, 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 113 past development politics that were too pro-land, so coastal communities were not given enough attention, and inequality still occurred. the efforts of coastal communities depend on three things, namely environmental conditions, seasons, and markets (msuya and hurtado, 2017). sustainable managemet principle are very important, especially when linked to the impact of unsustainable managemet (hermawan et al, 2021). environmental conditions affect the sustainability of coastal communities' businesses because their geographical conditions facing the sea are very vulnerable to environmental damage such as industrial waste. in addition, the season also has significant implications for the socio-economic needs of coastal communities because it affects the catch of fishers. coastal communities also depend on the market for their lives, which directly sell their business results to meet their daily needs. coastal communities who initially worked as fishers began to work as seaweed cultivators and made it the primary source of livelihood for their families. this condition is because seaweed cultivation does not require special skills, a relatively short planting period, and a reasonable selling price even though there are times when seaweed prices fluctuate (msuya, 2006). the market demand for seaweed production is increasing (shechambo et al, 1996). the development of seaweed cultivation is very much done because the cultivation has advantages. a short maintenance period, not too large a business cost, does not damage the environment and is accessible to market (hugh, 2003). jeneponto regency is located in south sulawesi province. this regency has been producing seaweed since 1987 but has not run optimally due to the 1997 monetary crisis. seaweed cultivation in jeneponto was again encouraged because the seaweed was sold at a high price and can help the community's economy. according to bps data from jeneponto regency (bps, 2021), the production of seaweed in jeneponto regency from 2016 has increased from 15,718.68 tons to 27,236.83 tons in 2020. from the production of seaweed which is relatively abundant, it cannot be separated from the role of women. the purpose of this study was to analyze the role of women in productive, reproductive, and community management activities, as well as the level of household welfare of seaweed cultivators in jeneponto regency, south sulawesi province. materials and methods data collection the research was conducted in bontosunggu village, tamalatea district, jeneponto regency, south sulawesi province. the location selection was carried out purposively (deliberately) by considering several factors because tamalatea district, a sub-district with the most extensive seaweed farmer household (rtp) in jeneponto regency. based on bps 2020 tamalatea district, jeneponto regency, has several fisheries households (rtp) specifically for seaweed cultivation as many as 2059 rtp. the population in this study was women involved in seaweed cultivation activities in bontosunggu village, tamalatea district, jeneponto regency, with about 286 seaweed households. respondents were given a questionnaire because the answers considered representative of the seaweed farmers, and the respondents only provided information related to themselves. international journal of applied biology, 5(1), 2021 114 figure 1. map of the study area (red pin) (source : https://www.google.com/maps/place) the number of respondents is determined using the following slavin formula: 𝑛 = 𝑁 1 + (𝑁𝑥𝑒2) where: n = number of respondents n = total population e = error tolerance (0.05) the slavin formula is used because the target population is homogeneous, namely seaweed farming households whose husbands and wives cultivate seaweed. the research method used is based on (khoirunnisa, 2019) with slight modifications. the types of data collected in this study are primary data and secondary data. primary data is data obtained directly in the field from respondents and informants. preliminary data in the form of structured interviews with respondents through questionnaires have been prepared and in-depth interviews with informants. secondary data is obtained from searching documents or using supporting literature such as the potential and profile of bontosunggu village, data from the central statistics agency (bps), scientific journals, books, and previous/previous research results. the indicators used to measure the role of women are divided into three major groups (khoirunnisa, 2019): 1. the role of women in productive activities includes women's contributions, access and control over resources and benefits in seaweed cultivation. assessment is based on the average value of the percentage obtained. these classifications include: a. less (< 34.30%) b. enough (34.30% 39.26 % ) c. most (> 39.26 % ) 2. the role of women in reproductive activities includes women's contributions, access and control over resources and benefits in domestic cultivation. the assessment is based on the average value of the percentage obtained. these classifications include: a. less ( < 48,84 % ) b. enough (48.84% 52.80% ) c. most (> 52.80%) international journal of applied biology, 5(1), 2021 115 3. the role of women in community management activities includes women's contributions, access and control over resources and benefits in social and political activities. assessment is based on the average value of the percentage obtained. these classifications include: a. less ( < 20,84 % ) b. enough ( 20,84 % 26,12 % ) c. most (> 26.12%) data processing and analysis techniques the data obtained from this study are in the form of quantitative and qualitative data. quantitative data were obtained through questionnaires filled out by respondents, which were then presented in a frequency table. quantitative data processing was carried out using microsoft excel 2013 and ibm spss version 20. variable testing was tested using the pearson correlation test to see the actual relationship between variables in ordinal form. results and discussion the role of women in productive, reproductive, and community management activities in seaweed cultivation activities, reproductive activities are divided into three stages: the preparation stages, maintenance stages, and harvest and post-harvest stages. figure 2. percentage of household division of seaweed cultivators in the preparation stage in bontosunggu village, tamalatea district, jeneponto regency at the preparation stage for seaweed cultivation, seaweed farmers must carry out several steps, including providing seeds, making stretches, justifying stretch ropes, maintaining seeds, and tying seeds on ropes. based on the results in figure 2, it is known that the activities in the preparatory stage are dominantly carried out simultaneously between men (husbands) and women (wives), especially during the provision of seeds (83.75%), making stretches (83.75%), and justifying stretch ropes (68.75%). specifically for installing seedlings on stretched ropes, 50% of respondents explained that women predominantly carried out the activity, the remaining 50% of respondents carried out these activities simultaneously. the division of labour in seaweed farming households is divided into three activities: productive activities, reproductive activities, and community management activities. the division of labour is seen from the contribution made and is classified into five groups, namely international journal of applied biology, 5(1), 2021 116 done by women only, tends to be women, together, tends to men, and men only (khoirunnisa, 2019). productive activities generate salaries or wages from both men and women. figure 3. percentage of household division of seaweed cultivators at the production stage in bontosunggu village, tamalatea district, jeneponto regency at the production stage (figure 3), the dominant activity is carried out by men (husbands). these activities include planting seeds in the sea (58%), caring for seedlings when planted in the ocean (77.5%), and eradicating seaweed pests (100%). specifically for the use of seaweed cultivation locations, it is intended that how much access to the utilization of these resources is. based on the interviews, it is known that the use of seaweed cultivation locations is almost balanced between men and women, namely respondents who use it together as much as 43.75%. at the same time, the rest is dominated by men. based on the results of in-depth interviews with respondents, they generally said that husbands were more likely to allow women to do more activities on land than at sea, only at certain times when women helped with production activities at sea. figure 4. percentage of household work division of seaweed cultivators at the harvest and post-harvest stages in bontosunggu village, tamalatea district, jeneponto regency at the harvest and post-harvest stages, activities are more dominantly carried out jointly between husband and wife. only during the harvesting process did 100% of international journal of applied biology, 5(1), 2021 117 respondents say that the activity was carried out entirely by husbands or men. activities that are all carried out jointly include: a. the drying process b. marketing of the harvest c. setting the money from the sale of seaweed and using the money from seaweed to determine harvest time, 55% of respondents do it jointly between husband and wife, and men dominantly do the remaining 45%. in reproductive activities, respondents said that women more dominantly carry out this activity. specifically for cooking activities, 100% of respondents did it themselves without the help of a man or husband (figure 5). activities accompanying children to learn and raise children, 100% of respondents admitted that these activities are carried out jointly between husband and wife. cleaning the house, washing clothes and dishes, shopping for daily needs and managing family finances are dominantly carried out by women. figure 5. percentage of household work division of seaweed cultivators in reproductive activities in bontosunggu village, tamalatea district, jeneponto regency in figure 6, it is show that 100% of respondents said that pkk, social gathering and recitation activities were all carried out by women. activities to attend invitations, village meetings, mutual assistance, death ceremonies, and the determination of family members to take part in seaweed counselling, 100% of respondents said they were carried out together. the role of women in productive activities is at a moderate level, as evidenced by their contributions to seaweed cultivation, such as tying seeds to ropes, drying seaweed, and marketing the harvest. in reproductive activities, all work is done by women because there is still an assumption that women also have to do housework. in community management activities, the role of women is at a sufficient level. it plays a significant role because there are several activities that women participate in, such as pkk, invitations to celebrations, death to seaweed cultivation counselling. there is a double workload on women in this study because of the two roles they do, namely in productive and reproductive activities. international journal of applied biology, 5(1), 2021 118 figure 6. percentage of household job share of seaweed cultivators in community management activities in bontosunggu village, tamalatea district, jeneponto regency the high role of women is generally carried out by the old age group so that the role of women is not based on age because women with old age have a high part. furthermore, the elevated position of women is also driven by the household size, which is relatively large. similar to the size of the household, this condition is carried out to save expenses so that women as unpaid labour can help their husbands in cultivating seaweed without paying wages for workers. then, the high role of women also comes from households with a high level of welfare. it happens because families with high interest have a large number of stretches. hence, women play a role in seaweed cultivation to monitor the condition of their cultivation. based on the research, it is known that the role of women in seaweed cultivation is based on the nature of women who work hard, want to stay involved in the process to ease the burden on their husbands, and reduce expenses to pay for labour. the greater the number of hours worked, the more women participate in forms of work derived from seaweed to earn higher incomes (eranza et al, 2015). there is a double workload on women in this study because of their contribution to two activities, namely productive and reproductive. figure 7. percentage of women's roles in seaweed cultivating households based on productive, reproductive, and community management activities in bontosunggu village, tamalatea district, jeneponto regency international journal of applied biology, 5(1), 2021 119 based on figure 7, it is known that for productive activities, women are more dominant in the category of minor role (71.25%). only about 3.75% of respondents fall into the category of every part. the rest, as much as 25%, fall into the category of playing a role. based on the results of interviews, it is known that women are more involved in productive activities on land. generally, the work at sea is done by men, and there are only a few female respondents who help their husbands doing activities at sea. in reproductive activities and management activities, women are included in having a significant role (100% of respondents). this is because men are more active in the sea while women are more active on land in helping their husbands in seaweed cultivating households. based on the results in table 1, it is known that the correlation coefficient value of women's roles with the household income of seaweed cultivators is 0.076. the result shows a positive relationship between the role of women and the household income of seaweed cultivators. however, from the correlation results obtained, the correlation value is weak and has no significant effect. it is probably because women's activities are strongly influenced by age and family members (children). children harm family income. it was further explained that the increase in the number of children owned increases family responsibilities. more time is used to take care of children so that the time to trade is reduced due to reduced income (mugniesyah, 2007; msuya, 2011; aming, 2004). table 1. relationship between women's roles and income correlations women’s role income women’s role pearson correlation 1 0.076 sig. (2-tailed) 0.506 n 80 80 income pearson correlation 0.076 1 sig. (2-tailed) 0.506 n 80 80 conclusion the role of women in seaweed cultivating households in jeneponto regency in productive activities is still minimal, only about 3.75%. in contrast, reproductive activities and community management reaches 100% or plays a significant role in these activities. women contribute to seaweed cultivation because the number of household members drives them. by reducing the use of workers, income can focus financing on the daily life of the household. international journal of applied biology, 5(1), 2021 120 references aming, naruddin. 2004. participation of filipino muslim women in seaweed farming in sitangkai, tawi-tawi, philippines (phd dissertation) (laguna: university of the philippines in los banos philippines) 129 pp. bps. 2020. jeneponto regency in figure (jeneponto: bps-statistics of jeneponto regency) 381 pp. bps 2021 jeneponto regency in figure (jeneponto: bps-statistics of jeneponto regency) 385 pp. eranza d r d, alin j, bahron a and mahmud r 2015. determinants of women’s participation in seaweed farming in the regency of jeneponto, south sulawesi, indonesia1. mediterranean journal of social sciences. 6 (5) 43–45. hermawan, david, s. andriawan, m. yusuf, a. d. riana, a. masriah, a. suryahman, & m. sabiq. 2021. sustainability and feasibility analysis of the skipjack (katsuwnus pelamis) fishery in buhung pitoe island, indonesia. aacl bioflux 14 (3) 1513-1523. khoirunnisa, nisrina. 2019 peran perempuan pada rumah tangga petani rumput laut (bogor: departemen ilmu keluarga dan konsumen fakultas ekologi manusia, institut pertanian bogor). mchugh, d j. 2003 a guide to the seaweed industry fao fisheries technical paper 441 (rome: fao) 105 p. mshigeni, keto e. 1976. seaweed farming: a possibility for tanzania’s coastal ujamaa villages tanzania notes and records. leiden university catalogue. 79 (80) 99–105. msuya, flowwer e. 2006 the impact of seaweed farming on the social and economic structure of seaweed farming communities in zanzibar, tanzania world seaweed resources: an authoritative reference system ed a t critchley, m ohno and d b largo (amsterdam: eti bioinformatics). msuya, flower. e. 2011. the impact of seaweed farming on the socioeconomic status of coastal communities in zanzibar, tanzania. world aquaculture, 42(3), 45–48. msuya, flowwer e. & a.q. hurtado. 2017. the role of women in seaweed aquaculture in the western indian ocean and south-east asia. european journal of phycology. 52 (4) 482– 494. mugniesyah, siti s 2007 gender, lingkungan dan pembangunan berkelanjutan dalam ekologi manusia ed s adiwibowo (bogor: fakultas ekologi manusia institut pertanian bogor) 232 pp. shechambo, fannuel, z. ngazy and msuya f. e. 1996. socio economic impacts of seaweed farming in the east coast of zanzibar, tanzania (zanzibar: canadian international development agency (cida), institute of marine sciences, university of dar es salaam) ims 1997/06, 81 pp. stévant, pierrick, c.rebours & a. chapman. 2017. seaweed aquaculture in norway: recent industrial developments and future perspectives. aquacult. int. 25 1373–1390. zaenab, s., tassakka, a.c.m.a.r., sulfahri., kasmiati. 2020. utilization of double fungal treatment by trichoderma harzianum and saccharomycopsis fibuligera to produce biosugar from red seaweed kappaphycus alvarezii. iop conf. series: earth and environmental science 575 (2020) 012015 contact : shkar k.rostam shkar.rostam@univsul.edu.iq 24 abstract aspergillus nidulans recognized as unique cell factory to manufacture several of useful natural products for human beings. also it considered as a rich reservoir for wide range of the unknown secondary metabolites. due to the great capacity of aspergillus nidulans for fabrication of valuable natural products and in order to have deeper insight for biosynthesis of these metabolites, precise identification is required. accurate identification of aspergillus nidulans from local isolates at the species level based on the multi-copies of internal transcribed regions of the rdna unit (its1-5.8s-its2 rdna), was the aim of current study. thus, in this study, morphological features, molecular approach, sequence alignment, phylogenetic analysis and detection of the matrix diversification for amplified its regions, in combination with highest blast bit score, utilized for accurate identification of aspergillus nidulans. therefore, soil samples of erbil province from different agricultural fields subjected for this study. out of 106 samples only 12 of them successfully identified as aspergillus nidulans. similarity and uniformity of isolates determined by aligning sequencing results of amplified its1-5.8s-its2 regions with available sequences of close related species in ncbi genbank database. sequence alignment results demonstrate that, all isolates displayed (%100) similarity with sequence of at least 14 species previously identified as aspergillus nidulans. concurrently, they displaed (%100) similarity with other species in the same genus as well, such as with aspergillus rugulosus, aspergillus variecolor, aspergillus rugulovalvus and emericella dentate. furthermore, maximum likelihood method used for construction of phylogenetic tree for all isolates with referenced strain aspergillus nidulans with accession number (af138289) to ascertain phylogenetic position of isolates. interpretation for outcome of phylogenetic analysis of isolates, determine that, isolates (mw228089, mw228090, mw228094, mw228095, mw228096, mw228097, mw228098, and mw228099) belonged to an its genotype corresponding to the reference strain aspergillus nidulans.while due to the interspecies variation, profound changes, single and multiple substitutions in its1 region of isolates (mw228088, mw228091, mw228092 and mw228093), they displayed divergent phylogenetic position in dendrogram in compare with reference strain. issn : 2580-2410 eissn : 2580-2119 molecular identification of local isolates aspergillus nidulans from erbil province using internal transcribed spacers khattab mustafa 1, shkar k.rostam 2* 1 mycology lab, biology department , faculty of science, school of science at the university of sulaimani, sul, iraq. general director at the ministry of higher education and scientific research-krg 2 mycology lab, biology department ,faculty of science, school of science at the university of sulaimani, of sulaimani, sul, iraq open access international journal of applied biology international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. keyword aspergillus nidulans, molecular identification, its1 and its2, phylogenetic tree. article history received 12 september 2021 accepted 30 december 2021 international journal of applied biology, 5(2), 2021 25 in spite of that, taxonomic analysis results revealed that, all isolates be affiliated with class of aspergillaceae (124 hits). overall in present study we confirmed that, colony morphology, molecular approche, sequence alignment analysis for amplified regions its1 and its2 in combination with highest aligned bit score of species addressed in listed of blast search required for accurate identification of isolates at the species level and distinguishing them from other close related species introduction filamentous fungi such as aspergillus nidulans, considered as well know biological model system to generate variety of complex secondary metabolites. these natural products are appraised as remarkably rich source of medically useful compounds [1, 2]. aspergillus nidulans has substantial capacity of for manufacturing of valuable natural products. essentially, due to the similarity and overlapping the morphological features of aspergillus nidulans with close related species, accurate identification at te species level required. to have deeper insight for biosynthesis of these natural products and exploring for novel metabolites, unambiguous and reliable recognition methods are strongly recommended [3, 4]. several techniques such as conventional microbiological methods and different molecular approaches should apply for authentic identification of aspergillus species. basically, morphological characteristics among close related species of aspergillus species, are almost similar and instable. in the fact of that, long time and optimal standard culture procedure requested for discrimination of colony characteristics and adequate evaluation of morphological features of aspergillus sp [5, 6]. therefore, in compare with culture based methods, molecular approaches based on polymerase chain reaction are required to give sufficient devaluation in time for accurate diagnosis and identification of isolates at the level of species [7, 8]. different molecular approaches have been used for detection of aspergillus species [6, 7]. to delimit the species within the aspergillus sp, most frequently, multiple gene loci such as protein coding regions (β-tubulin, calmodulin gene), and internal transcribed spacer (its) regions of the ribosomal rna gene cluster (rdna) applied for species identification [6,7, 8]. among the dna barcode markers, its regions has been chosen as admissible standard barcode marker for diagnosis of aspergillus species, this is due to that, its region has highest expectation of successful in detection of fungal isolates [8]. also, its regions are separates by 5.8s of rrna genes and located in between the 18s and 28s rrna genes [4, 9, 10]. hereupon, this study was carried out to identify of aspergillus nidulans from our local isolates at the species level by using internal transcribed spacer regions 1 and 2. accordingly, to analyze the genetic diversity and construction of phylogenetic tree for isolates, sequence variation of its regions has been subjected for exploration [4, 7, 8]. materials and methods survey sites soil composite were collected from the surface layer (0.0 -0.3 m) of the dominant cropped soils at different sites within erbil governorate. the sites encompassed for sampling, include soil of different agricultural fields which previously cultivated with different agriculture commodities. to remove impurities and stones, collected soil samples were passed through a sieve, then for further analysis samples putted inside the zip locked international journal of applied biology, 5(2), 2021 26 cover and stored in refrigerator. all samples were collected in between (10th of jan, 2019 10th of feb 2020). fungal isolation isolation fungi from the soil samples soil samples were subjected for fungal isolation within two days after of collection. fungi were isolated by using soil dilution method on potato-dextrose agar (pda) and sabouraud dextrose agar (sda). both potato-dextrose agar and sabouraud dextrose agar, were prepared according to the instructions as indicated by the manufacture. after adding (100 mg\1l) chloramphenicol antibiotic for prevention of bacterial growth, they were sterilized by autoclaving at 121ºc for 15 min under 1.5 bar pressure. tenfold dilution method used for inoculation of plates as described by [13, 18, 19]. the inoculated plates then incubated at 25º c. morphological features examined after (5-7) days of incubation. the desired colonies were sub cultured onto test tubes (18 ×180mm) containing sabouraud dextrose agar for working on fungal purification process [12, 13, 14]. identification of aspergillus species morphological identification morphological features such as growth rate, colony diameter, aerial mycelium, pigmentation of fungi, conidiophore, vesicle, metulae, phialides and conidia are generally basic essential tool for identification of aspergillus species. colony appearance were examined under the dissecting microscope after they have been grown on (pda) at 25o c for seven days [7, 15]. thereafter, for studying of microscopical feature, new subcultures of aspergillus isolates were prepared on (pda). most microscopic characters such as (conidiophore, vesicle, metulae, phialides and conidia) were examined by (slide cultures technique) as described by [17]. slides were prepared and stained with lactophenol cotton blue, then examined under the light microscope. criteria used for identifications of isolated fungi have been reported by [15, 17, 18]. molecular identification culture preparation for the commencement, 50 ml of sabouraud broth has been prepared and inoculated with inoculum from previously cultivated culture, then incubated for 72 h at 28°c. filters with 0.45-mm pore size used for recovering of hyphae from the grown media, subsequently filtrated hyphae washed with sterile saline [4]. dna extraction afterward the genomic dna from the all isolates were extracted and prepared by using dna extraction kit (bio basic) as described by manufacturer's instruction and as reported by [19]. about 100-500 mg (wet weight) of hyphae has been grinded in liquid nitrogen by using a pestle. then the grinded sample was transferred to a clean 1.5 ml microtube. the powder was suspended in 180 μl of universal digestion buffer with 20 μl proteinase k, then mixed thoroughly by vortexing and incubated at 56°c for 30-60 min. following the steps, 100 μl of universal buffer, has been added and mixed by inverting, then incubated at -20°c for 5 minutes. after centrifugation at 12,000 x g for 5 minutes at room temperature, the supernatant was transferred to a new 1.5 ml tube which contained 200 μl international journal of applied biology, 5(2), 2021 27 universal buffer bd. 200 μl of ethanol (96-100%) added to the tube and then mixed thoroughly by vortexing. after that, the mixture from the previous step (including any precipitate) was transferred into ez-10 column and placed in a 2 ml collection tube. subsequently centrifuged at 9,000 x g for 1 min. flow through was discarded, then 500 μl of universal pw solution has been added to the tube, and centrifuged for 1 min at 9,000 x g. flow through was throw out again. 500 μl of universal wash solution was added, and centrifuged for 1 min at 9,000 x g, flow through was throw away. finally 50-100 μl buffer te was added directly onto the center part of ez-10 membrane and incubated at room temperature for 1 min, then centrifuged for 1 min at 9,000 x g. following the extraction, the eluted and purified dna was stored at -20°c for the next step. universal primers its1 and its2 universal fungal primers its1 (forward primer, 5'-tcc gta ggt gaa cct gcg g3') and its4 (reverse primer, 5'-tcc tcc gct tat tga tat g-3') as reported by [4, 20], used for amplification of its 1 and its 2 noncoding regions and intervening region 5.8s gene, of the ribosomal rna (rrna) gene complex. pcr amplification amplification reactions were carried out in volumes of 50 μl containing 5 μl (10 pg– 100 ng) of template dna, 1 μl of each primers (20 mm), 5 μl of 2x pcr reaction buffer (hs prime taq dna polymerase 1 unit/10 μl, 20 mm tris-hcl, 80 mm kcl, 4 mm mgcl2, enzyme stabilizer, sediment, loading dye, ph 9.0), 5 μl of (0.5 mm of each datp, dctp, dgtp, dttp), supplied prime taq premix genetbio (korea). pcr reactions were performed in the eppendorf mastercycler gradient. the pcr amplification protocol was as follow: after initial denaturation of dna 1 cycle for 5 min at 95 °c, 40 cycles performed, each cycle consisted of a denaturation step at 95°c for 30 s, an annealing step at 50°c for 30 s, and an extension step at 72°c for 1 min, with a final extension at 72°c for 5 min following the last cycle. amplified product stored at -20°c until used [4, 20, 21, 23]. pcr products were separated and detected on 2% agarose ethidium bromide gels in tae 1x buffer (tris–acetate 40mm and edta 1.0 mm), and visualized under uv light in gel documentation cabinet. the 100-bp dna ladder genetbio (korea) was used as the molecular size marker. separated amplicons were purified from the gel by using the 96 pcr kit (nucleofast®, macherey-nagel, hoerdt, france), according to the manufacture’s instructions. cloning procedure and insert amplification cloning for purified pcr products were performed by using the pgem® -t easy vector system kit (promega, lyon, france) as clarified by manufacturer’s instructions and explained by [22]. re-suspended 20 μl of bacteria suspension in a tube contain 80 μl of lb, then the suspension was plated onto the lb agar plates supplemented with ampicillin (100 μlg/ml), x-gal (80 μlg/ml), iptg (120 μg/ml) and the plates were incubated overnight at 37°c. finally screening to confirm the presence of the inserts in white colonies fulfilled b y using specified pcr amplification protocol as described above, with the m13 forward primer (5’-gtaaaacgacggccag-3’) and m13 reverse primer (5’-aggaaacagctatgac-3’) as mentioned by [22, 23]. correct sizes of the inserts were purified by using the 96 pcr kit (nucleofast®, macherey-nagel, hoerdt, france), according to the manufacturer’s instructions. international journal of applied biology, 5(2), 2021 28 sequencing for purified pcr products from previous steps purified cloned fragment of both strands of plasmid containing the fungal inserts subjected for sequencing with universal m13 forward and reverse sequence primers as reported by [22, 23]. sequencing performed by macrogen global biotechnology company for research and development of genome analysis. on other hand for direct sequencing of non-cloned amplicons, pcr products were directly sequenced using the its 1 and its 4 pcr primers. the outcome obtained from sequencing were deposited in genbank database [4, 7. 20]. sequence analysis acquired sequences were aligned with partial its sequences available in genbank database by using muscle algorithm [24]. comparison alignment for obtained sequences from sequencing results have been performed with existing sequences in genbank database by using blastn search alignment tool at the website of the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/blast). through this way similarity and uniformity of isolates determined [22, 23]. phylogenetic analysis after alignment for sequencing results for all isolates based on the nucleotide sequence of its1 and its2 regions, and due to the great reliability of its region in distinguishing of close phylogenetic distance, maximum likelihood method has been used for analysis and construction of phylogenetic tree with close related species [6-25-28-29]. data from alignment process imported to the mega x 10.05, for generating maximum likelihood tree. program parameters setup by default [25-29]. nucleotide sequences accession numbers the its 1–5.8s–its 2 gene complex sequences used in this article were submitted in to the genbank database with accession numbers from (mw228088 to mw228099). results and discussion morphological identification of aspergillus nidulans macroscopical identification based on morphological key features as reported by [26, 27, 28], macrospical characterstics of isolates determined. typically colonies of aspergillus nidulans after incubation for 5 days on pda at 25o c, appeared granular in texture, and surface color dark green in center with pale olive green near the margin of colonies figure 1a. revers color of colonies were hyaline or pale green, figure 1b. usually exudate were present with light brown color. international journal of applied biology, 5(2), 2021 29 figure 1.a colony appearance of aspergillus nidulans on pda at 25o c after 5 days of incubation had, granular texture, surface color dark green in center with pale olive green near the margins. b revers color of colony was hyaline or olive pale green. microscopical identification among our isolates aspergillus nidulans characterized microscopically, in such way that they had septate and hyaline hyphae. conidial heads are short and columnar. conidiophores commonly sinuous, near the foot cell and brown smooth-walled. vesicles were hemispherical, with metulae and phialides occurring on the upper portion (biseriate) figure 2 [26, 27, 28]. summary of morphological features illustrated in table 1. figure.2 microscopical features of aspergillus nidulans 1) hemispherical vesicles bearing (metulae and phialides) biseriate conidial head. 2) aspergillum like spore bearing sinuous conidiophore. 3) metulae , 4) phialides. microscopic observation of the fungal isolate under 400x magnification, stained by (lactophenol cotton blue). table 1. morphological features used to identify aspergillus nidulans on pda media. colony color\ texture dark green \ granular colony diameter (mm) 25-30 colony reverse color hyaline, pale olive seriation biseriate vesicle shape hemispherical conidia head shape short 3.metulae 4. phialides 2.sinuous conidiophore 1.hemispherical vesicles international journal of applied biology, 5(2), 2021 30 molecular identification of aspergillus nidulans after morphological detection, based on the specific pcr amplification procedure, isolates in this study identified as aspergillus nidulans with expected dna size (561-565) base pairs as showed in figure 3. out of 106 samples, only 12 of them successfully identified as aspergillus nidulans. m 1 2 3 4 5 6 7 8 9 10 11 12 13 figure 4. pcr-based detection of aspergillus nidulans. lanes (1–12) pcr products with (561-565 bp). pcr products include five regions of rdna complex, ist1-5.8s-its2 and 3' end of the 18s ribosomal dna with 5' end of the 28s rdna gene. lane (13) non dna template. m: (100) bp dna molecular size marker. amplification reactions were carried out in volumes of 50 μl containing 5 μl (10 pg–100 ng) of template dna, 1 μl of each primers (20 mm), 5 μl of 2x pcr reaction buffer (hs prime taq dna polymerase 1 unit/10 μl, 20 mm tris-hcl, 80 mm kcl, 4 mm mgcl2, enzyme stabilizer, sediment, loading dye, ph 9.0), 5 μl of (0.5 mm of each datp, dctp, dgtp, dttp), supplied prime taq premix genetbio (korea). analysis of its regions amplification of its1-5.8s-its2 regions for the 12 isolates which morphologically identified as aspergillus nidulans, generate pcr products ranging in size from (561-565 bp) table 2. sequencing carried out for both cloned amplicons and direct pcr products. table 2. pcr products sizes of aspergillus nidulans isolates isolates accession number pcr product size (bp) mw228088 563 mw228089 564 mw228090 565 mw228091 561 mw228092 562 mw228093 564 mw228094 565 mw228095 565 mw228096 563 mw228097 565 mw228098 565 mw228099 565 500(bp ) 565(bp ) international journal of applied biology, 5(2), 2021 31 potential variations due to the random changes during the sequence amplification process examined for sequencing results of cloned amplicons and direct pcr products, by generating the amplicons with aid of taq polymerase with proofreading capability. copy of sequencing results from cloned amplicons and direct pcr products used for each isolates to determine the variation in their sequences. variation in sequencing results of direct pcr products and cloned amplicons in its1-5.8s-its2 regions illustrated in in table 3. table 3. number of nucleotide variations in sequencing results of its1-5.8s-its2 regions of direct pcr products in compare with sequencing results of cloned amplicons. isolates accession number number of nucleotide variations in sequencing results of its1 region of direct pcr products in compare with sequencing results of cloned amplicons 5 5.8 s number of nucleotide variations in sequencing results of its2 region of direct pcr products in compare with sequencing results of cloned amplicons mw228088 2 0 0 mw228089 2 0 0 mw228090 0 0 0 mw228091 2 0 0 mw228092 3 0 0 mw228093 3 0 1 mw228094 0 0 0 mw228095 0 0 0 mw228096 2 0 0 mw228097 0 0 0 mw228098 0 0 0 mw228099 0 0 0 maximum number of nucleotide sequence variations in between sequencing results of cloned pcr products with direct pcr products, observed in isolates with accession number (mw228092 and mw228093) with (3-4) varied nucleotides respectively. no more than (3) nucleotides seen varied in results of comparison of isolates with accession number (mw228088, mw228089 and mw228096). however, remaining isolates (mw228090, mw228094, mw228095, mw228097, mw228098, and mw228099), had the same sequencing results, with no nucleotide sequence variation. alignments for contiguous sequences of its1-5.8s-its2 regions for all isolates amplified sequence of its1-5.8s-its2 regions of all isolates subjected for blastn alignments with sequences of those closely related species in same taxa in genbank database. our alignments results demonstrated that, sequence of all isolates displayed (%100) similarity with sequence of at least 14 species previously identified as aspergillus nidulans. based on the highest aligned bit score of species addressed in listed of blast search, all isolates in this study identified as aspergillus nidulans. notwithstanding that, similarity with (%100) observed with other related strains as well, such as with (aspergillus rugulosus ku866664.1, aspergillus variecolor hq674656.1, aspergillus rugulovalvus ab248977.1 and emericella dentate ab248999.1) respectively. moreover, two strains of (emericella nidulans) with accession number (hq674655.1, and eu287942.1) and two international journal of applied biology, 5(2), 2021 32 uncultured fungi with accession numbers (jf289097.1 and kf800548.1) present the same similar percentage identity with our isolates. table 4 present the accession numbers, similarity with coverage percentage and size of those 14 strains which had (%100) similarity with our islates and identified as aspergillus nidulans. table 4. present the accession numbers, similarity with coverage percentage and size of those 14 strains which had (%100) similarity with our islates and identified as aspergillus nidulans. accession no. of (%100) similar aligned shot sequences similarity percentage with isolates coverage% length in bp mt316339.1 %100 100 846 mk806488.1 %100 100 571 mh858232.1 %100 100 857 mg991576.1 %100 100 569 ky074657.1 %100 100 1783 ky425597.1 %100 100 566 kp278174.1 %100 100 575 kp165435.1 %100 100 570 kp131596.1 %100 100 577 ay373888.1 %100 100 590 mw493182.1 %100 100 569 fj878641.1 %100 100 590 fj878647.1 %100 100 565 kp131594.1 %100 100 565 determination of sequence diversity in matrix alignments of its1-5.8s-its2 regions for all isolates with referenced strain aspergillus nidulans (atcc 10074) accepted in genbank database with accession number (af138289) matrix diversification of its1-5.8s-its2 regions of isolates exhibited by comparing their alignment results with sequence of referenced strain aspergillus nidulans (atcc 10074) which accepted in genbank database with accession number (af138289). variation in the lengths, and single or multiple nucleotide differences in sequence of rdna complex regions of isolates observed as a results of deletion and insertion in the sequences of isolates. figure 5 illustrate the sequence alignments results of all isolates with referenced strain of aspergillus nidulans in genbank database. https://www.ncbi.nlm.nih.gov/nucleotide/mt316339.1?report=genbank&log$=nucltop&blast_rank=1&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/mk806488.1?report=genbank&log$=nucltop&blast_rank=2&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/mh858232.1?report=genbank&log$=nucltop&blast_rank=3&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/mg991576.1?report=genbank&log$=nucltop&blast_rank=4&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/ky074657.1?report=genbank&log$=nucltop&blast_rank=5&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/ky425597.1?report=genbank&log$=nucltop&blast_rank=6&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/kp278174.1?report=genbank&log$=nucltop&blast_rank=8&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/kp165435.1?report=genbank&log$=nucltop&blast_rank=9&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/kp131596.1?report=genbank&log$=nucltop&blast_rank=10&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/ay373888.1?report=genbank&log$=nucltop&blast_rank=12&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/mw493182.1?report=genbank&log$=nucltop&blast_rank=20&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/fj878641.1?report=genbank&log$=nucltop&blast_rank=24&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/fj878647.1?report=genbank&log$=nucltop&blast_rank=29&rid=a5r55x0m016 https://www.ncbi.nlm.nih.gov/nucleotide/kp131594.1?report=genbank&log$=nucltop&blast_rank=30&rid=a5r55x0m016 international journal of applied biology, 5(2), 2021 33 18 s its1 50 aspergillus nidulans af138289 tccgtaggtgaacctgcggaaaggacatta ccgagtgcgggctgcctccgggcgcc caacctcccacccgtgacta_ctaacactgt aspergillus_nidulans_mw228088 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgagcagcctccgggcgcc _ _acctcccacccgtgactacctaacactgt aspergillus_nidulans_mw228089 tccgtaggtgaacctgcggaaggatcatta cagagtgcgggctgcctccgggcgcc _aacctcccacccgtgactacctaacactgt aspergillus_nidulans_mw228090 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcccaacctcccacccgtgactacc taacactgt aspergillus_nidulans_mw228091 tccgtaggtgaacctgcggaaggattgtaa ccgggtgcg_gctg _ctccgggcgcc _ _ acctcccacccgtgactacctaacactgt aspergillus_nidulans_mw228092 tccgtaggtgaacctgcggaaggatcatta ccgtacgcg_ gctg _ctccgggcgcc_ _ acctcccacccgtgactacctaacactgt aspergillus_nidulans_mw228093 tccgtaggtgaacctgcggaaggatcatta ccgggtgcgggctg _ctccgggcgcc_ _ acctcccacccgtgactacctaacactgt aspergillus_nidulans_mw228094 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcccaacctcccacccgtgactacc taacactgt aspergillus_nidulans_mw228095 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcccaacctcccacccgtgactacc taacactgt aspergillus_nidulans_mw228096 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcc _ _acctcccacccgtgactacctaacactgt aspergillus_nidulans_mw228097 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcccaacctcccacccgtgactacc taacactgt aspergillus_nidulans_mw228098 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcccaacctcccacccgtgactacc taacactgt aspergillus_nidulans_mw228099 tccgtaggtgaacctgcggaaggatcatta ccgagtgcgggctgcctccgggcgcccaacctcccacccgtgactacc taacactgt 100 its1 150 aspergillus nidulans af138289 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228088 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228089 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228090 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228091 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228092 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228093 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228094 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228095 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans mw228096 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans mw228097 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans mw228098 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca aspergillus_nidulans_mw228099 tgcttcggcggggagccccccaggggcgagccgccgggg accactgaacttcatgcctgagagtgatgcagtctgagcctgaatacaaatcagtca its2 350 its2 400 aspergillus nidulans af138289 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228088 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228089 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228090 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228091 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228092 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggctccgtgtccggtcctcgatcgta aspergillus nidulans mw228093 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgagaggcagcggccgctccgcgtccggtcgacgatcgta aspergillus nidulans mw228094 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228095 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228096 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228097 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta c o n se rv e d r e g io n ( 5 .8 s ) e x cl u d e d 1 8 4 -3 4 2 international journal of applied biology, 5(2), 2021 34 aspergillus nidulans mw228098 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta aspergillus nidulans mw228099 gctgccctcaagcccggcttgtgtgttgggtcgtcgtcccccccgggggacgggcccgaaaggcagcggcggcaccgtgtccggtcctcgagcgta 450 500 its2 28s aspergillus nidulans af138289 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228088 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcacgta aspergillus nidulans mw228089 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228090 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228091 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcacgta aspergillus nidulans mw228092 ggt_cgctttgtcacccg_ctcgagtagggctcggccgggcaccagccggcttctccaa_ccttattgt_tctaaggtt gacctcggatcacgtc aspergillus nidulans mw228093 tggtcgctttgtcacccgcctcgattagggc_cgcgccggcaccagccggcgtctccaagccttattttgtctcaggtt gacctcggatcagtcc aspergillus nidulans mw228094 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228095 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228096 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228097 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228098 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta aspergillus nidulans mw228099 tgg_ggctttgtcacccg_ctcgattagggc_cggccgggcgccagccggcgtctccaa_ccttatttt_tctcaggtt gacctcggatcaggta 550 28s 572 aspergillus nidulans af138289 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228088 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228089 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228090 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228091 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228092 gcgatacccgctgaactt_ _gcatatcaataagcggagg aspergillus nidulans mw228093 gggatatccgctgtgctt _ _gcatatcaataagcggagg aspergillus nidulans mw228094 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228095 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228096 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228097 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228098 gggatacccgctgaacttaagcatatcaataagcggagg aspergillus nidulans mw228099 gggatacccgctgaacttaagcatatcaataagcggagg figure 5. sequence alignment of its regions of all isolates with referenced strain of aspergillus nidulans in genbank database with accession number (af138289). complete its1 and its2 regions with 3' end of the 18s ribosomal dna (rdna) gene which incorporate with its 1 primer site, and 5' end of the 28s rdna gene which incorporate with its 4 primer site, present in this figure. conserved region of 5.8s rdna gene sequence has been excluded. numbering of the sequence begins with the first nucleotide in the 3' end of the 18s ribosomal dna and ended with 5' end of the 28s rdna gene. gap in a sequence is marked with hyphen and highlight with yellow ( _ ). however any changes in nucleotide sequences highlighted with red. international journal of applied biology, 4(1), 2020 34 comparison results of its1 region of isolates with referenced strain of aspergillus nidulans with accession number (af138289) reveal that, isolates with accession number (mw228088, mw228089, mw228091, mw228092, mw228093 and mw228096) had the most variations in nucleotide sequence of its1 region in contrast with its2 region. these variations occurred in area of its1 region with (27) nucleotide bases in length, as showed in figure 6. however, all remain isolates displayed (%100) identical similarity with its1 region of referenced strain of aspergillus nidulans. the sequence of highly conserved region 5.8s rdna of all isolates were identical with the referenced strain. except for isolates (mw228092 and mw228093), (%100) identical similarity at its2 region noticed in all isolates in compared with its2 region of referenced strain. in contrast with other, remarkable variations observed in nucleotide sequences of its2 region of isolates (mw228092 and mw228093) in compare with referenced strain of aspergillus nidulans, in such way that (11-16) nucleotides varied in their sequence respectively. nucleotide sequence variations of its2 region of isolate (mw228092 and mw228093) highlighted with red and illustrate in figure 5. figure 6. nucleotide sequence variations of isolates (mw228088, mw228089, mw228091, mw228092, mw228093 and mw228096) in its1 region with referenced aspergillus nidulans with accession number (af138289). variation changes observed only in (27) nucleotide bases of its1 region. construction of phylogenetic tree maximum likelihood method have been used for construction of phylogenetic tree and determination the phylogenetic distance with close related species [6, 25, 28, 29]. its1 and its2 regions subjected for construction and phylogenetic analysis, in view of th e fact that, generally its regions displayed variations between close species. however, its regions present great reliability in distinguishing of close phylogenetic distance. based on phylogenetic result figure 7, phylogenetic position of isolates within the genus and species level determined, regarding to the aligned region of its1 and its2. our results elucidates that, isolates with accession number (mw228089, mw228090, mw228094, mw228095, mw228096, mw228097, mw228098, and mw228099) belonged to an its genotype corresponding to the reference strain aspergillus nidulans (atcc 10074) with accession number (af138289). furthermore, isolates (mw228088, mw228091, mw228092 and international journal of applied biology, 4(1), 2020 35 mw228093), they displayed divergent phylogenetic position in dendrogram in compare with reference strain. figure 7. genetic diversity of local isolates aspergillus nidulans based on the sequence analysis of its1 and its2 region. tree was constructed by maximum likelihood method with 1000 bootstrap replicates. only bootstrap value > %50 are shown. scale bar represents the genetic distance. results of taxonomic analysis for our isolates in genbank database confirm that, our isolates are member of class aspergillaceae (124 hits), and maximum similarity of sequences matched with aspergillus nidulans (37 hits), table 5. international journal of applied biology, 4(1), 2020 36 table 5. taxonomic analysis for isolates based on ncbi genbank database confirmed that, our isolates belonged to class of ascomycetes (124 hits) and maximum similarity of sequences matched with aspergillus nidulans (37 hits). discussion screening and mining for novel secondary metabolites among the filamentous fungi are challenging. unambiguous identification of fungi require for precise exploration and characterization of novel metabolites. aspergillus nidulans recognized as well know biological model for biosynthesis of variety of valuable natural products. because of similarity and overlapping of morphological features of aspergillus nidulans with close relate species and their alike ability for production of secondary metabolites as well, accurate discrimination for aspergillus nidulans among the isolates strongly recommended. current study performed based on premise that authentic identification of aspergillus nidulans at the species level, have great importance and profitable to us to manifest a clear vision for exploration of novel metabolites in aspergillus nidulans. thus, in this study, fungi 130 . . aspergillaceae 124 . . . aspergillus 101 . . . . aspergillus subgen. nidulantes 38 . . . . . aspergillus nidulans 37 aspergillus nidulans hits . . . . aspergillus rugulosus 9 aspergillus rugulosus hits . . . . aspergillus pachycristatus 3 aspergillus pachycristatus hits . . . . aspergillus spinulosporus 1 aspergillus spinulosporus hits . . . . aspergillus stellatus 1 aspergillus stellatus hits . . . . aspergillus rugulovalvus 1 aspergillus rugulovalvus hits . . . . aspergillus quadrilineatus 13 aspergillus quadrilineatus hits . . . . unclassified aspergillus 13 . . . . aspergillus corrugatus 11 aspergillus corrugatus hits . . . . aspergillus niger 2 aspergillus niger hits . . . . aspergillus sublatus 3 aspergillus sublatus hits . . . . aspergillus parvathecius 1 aspergillus parvathecius hits . . . . aspergillus montenegroi 1 aspergillus montenegroi hits . . . . aspergillus miyajii 1 aspergillus miyajii hits . . . . aspergillus fruticulosus 1 aspergillus fruticulosus hits . . . . aspergillus foveolatus 2 aspergillus foveolatus hits . . . emericella 23 . . acrophialophora sp. 4 yhy-2018 1 acrophialophora sp. 4 yhy-2018 hits . uncultured fungus 5 uncultured fungus hits https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=4751 https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1131492 https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=5052 https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=2720870 https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=162425 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=41736 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1810921 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1810908 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1549217 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=369730 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=41735 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=2606521 https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=210208 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=5061 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1810927 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=41740 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1506150 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=359949 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=1250369 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=210207 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=5071 https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=2138179 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi https://www.ncbi.nlm.nih.gov/taxonomy/browser/wwwtax.cgi?id=175245 https://blast.ncbi.nlm.nih.gov/blast.cgi https://blast.ncbi.nlm.nih.gov/blast.cgi international journal of applied biology, 4(1), 2020 37 molecular approach, sequence alignment ,phylogenetic analysis and determination the matrix diversification for amplified regions of its 1 and 2, in combination with highest blast bit score, utilized for accurate identification of aspergillus nidulans at the species level. several methods, such as culture based methods and different molecular based techniques, recommended for identification of aspergillus nidulans. conventional microbiological methods, not quite enough to identify fungi at the species level, therefore molecular based technique is necessary for precise identification of fungi. its regions are most frequently utilized for identification broadest range of fungi, in the fact that, its region present highest probability of correct identification for wide number of fungal lineage [6, 7, 8, 9]. its1 and its2 regions have been chosen as a convenient universal dna marker for detection of fungi at species level. identification of isolates as aspergillus nidulans out of 106 sample, initially only 12 of them identified as aspergillus nidulans based on colony characteristics. concurrently, with aid of the molecular approach for amplification of its1 and its2, and results obtained from sequence alignment analysis for amplified regions, in compare with reference strain aspergillus nidulans (atcc 10074), identity of all isolates determined and confirmed as aspergillus nidulans. isolates in this study morphologically identical, in contrast they were varied in some nucleotide base pairs of its1 and its2 regions. castañeda-ramírez et al., (2016) noticed similar results in such way that, morphological features of their isolates were identical but variations seen in nucleotide sequence of its regions. furthermore, they wrote that, changes in nucleotide sequences took place due to the evolution processes, which influenced by environmental factors. interspecies variations and similarity among the isolates based on its1 and its2 regions in compare with its2 region, its1 area of our isolates with accession number (mw228088, mw228089, mw228091, mw228092, mw228093 and mw228096) displayed greatest interspecies variation. henry, iwen and hinrichs, (2000), disclosed that, depend on alignment results for pathogenic species of aspergillus, interspecies variation were common in its1 region rather than its2 region. conversely, except for isolates (mw228092 and mw228093), (%100) identical similarity at its2 region noticed in all isolates in compared with its2 region of referenced strain. on other hand, frequency of multiple substitutions such as transition, transversion and deletion mutations in its1 region considerably greater than its2. the 5.8s rrna gene sequence region highly conserved region with a little variations in all isolates. however, it had no remarkable impact on the overall the comparison process. rely on our comprehension for comparison and alignments results, we declare that both its1 and its2 regions were necessary for identification of isolates at species level, concurrently this consequence confirmed by [4]. comparing in reading of sequencing results of pcr products with cloned pcr products our results illustrated that, no more than four nucleotides were vary in reading between sequencing results of pcr products with cloned pcr products. accordingly, it was not require to clone the pcr product to acquire the precise reading of the sequences. determination of isolates similarity with close relate species in genbank database species similarity of all isolates determined rely on outcome of blast search in genbank database and highest aligned bit score of species addressed in listed of blast search. international journal of applied biology, 4(1), 2020 38 isolates in this study exhibited (%100) similarity with aspergillus nidulans species in genbank database. concurrently, all isolates displaed (%100) similarity with other species in the same genus as well such as with aspergillus rugulosus, aspergillus variecolor, aspergillus rugulovalvus and emericella dentate. basically isolates of aspergillus nidulans deffirentaites with mentioned closel related species rely on microscopical features such as, smooth walled ascospore with bearing two parallel, equatorial crests with 0.5 to 1.0 μ in width used for distingustion them. determination of taxonomic status and phylogenetic position of isolates with same loci of referenced strain taxonomic status and phylogenetic position of isolates in this study determined by construction of phylogenetic tree with using maximum likelihood method rely on its1 and its2 regions in concordance with same loci of referenced strain. its regions have great reliability in distinguishing of close phylogenetic distance [6, 25, 28, 29]. our results elucidates that, isolates with accession number (mw228089, mw228090, mw228094, mw228095, mw228096, mw228097, mw228098, and mw228099) belonged to an its genotype corresponding to the reference strain aspergillus nidulans (atcc 10074) with accession number (af138289). in spite of that, taxonomic analysis results revealed that, all isolates be affiliated with class of aspergillaceae (124 hits). furthermore, due to the interspecies variation, profound changes, single and multiple substitutions in its1 region of isolates (mw228088, mw228091, mw228092 and mw228093), they displayed divergent phylogenetic position in dendrogram in compare with reference strain. similar to our results, berbee, et al (2000), described that, based on solely morphological features or single gene like rdna, taxonomic status evaluation and identification at species level couldn’t be processed. moreover, alshehri and palanisamy, (2020), suggested that the use of protein coding regions such as b-tubulin and calmodulin genes more accurate for the identification of aspergillus at species level. overall, in present study we confirmed that for accuate identification of isolates as a aspergillus nidulans at the species level, colony morphology, molecular approche, sequence alignment analysis for amplified regions its1 and its2 in combination with highest aligned bit score of species addressed in listed of blast search required for distinguishing it from other close related species. conclusions approaches used in this study provide reliable accurate and efficient methods for identification at the species level. furthermore, except the its regions, multiple gene loci such as protein coding regions (β-tubulin and calmodulin gene) highly recommended for identification of fungi at species level as well. also, for determination of phylogenetic relationships among aspergillus species, igs regions from the nu-rrna gene complex and two less often used mitochondrial gene sequences (rns and cox1) put forwarded. conflict of interest there is no conflict of interest to declare. international journal of applied biology, 4(1), 2020 39 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175 abstract xylitol is a pentose sugar that has many benefits. the production of xylitol in biotechnology is more promising because in its production it only utilizes xylose fermentative yeast. this study aims to obtain yeast isolates and determine their ability to ferment xylose to xylitol. the samples used came from palm sap, coconut sap, soil and sugar press-mud. yeast isolation was carried out by growing on yma media which had added 0.1% chloramphenicol. subsequently, the colony and cell morphology were observed. a total of 12 isolates of palm sap, 12 isolates of coconut sap, 8 isolates from soil and 6 isolates from sugar press-mud were screened on ypx agar and xylose broth media with 3% xylose concentration to see their ability to grow on xylose media. isolates p1, s3, kp3, kp4, t4 and b4 were selected for the fermentation test because they had the best growth in xylose broth as indicated by the highest od value. fermentation was carried out for 72 hours by measuring the ph value, total yeast at intervals of 24 hours. the xylitol levels formed were measured using hplc/uplc. based on the measurement of the xylitol levels formed, the results were obtained in isolates p1 which was 1.28 g/100ml, s3 was 2.05 g/100ml, kp3 was 2.23 g/100ml, kp4 was 2.19 g/100ml, t4 2 was g /100ml and b4 2.47 g/100 ml with xylitol yields p1 which is 0.59 g/g, s3 is 0.67 g/g, kp3 is 0.63 g/g, kp4 is 0.62 g/g, t4 is 0.66 g/g and b4 were 0.64 g/g so that the six isolates isolated from palm sap, coconut sap, soil and sugar press-mud had the ability to produce xylitol. issn : 2580-2410 eissn : 2580-2119 exploration of indigenous yeasts as inoculum in fermentation of sugar factory waste (bagasse) into xylitol low-calorie sugar nur haedar 1*, as’adi abdullah 1, paramita sudirman 1, veni apriliani 1 , ayu mitha lestari 1 1department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia. introduction xylitol is a five-carbon sugar alcohol that has a sweet taste level similar to sucrose, but with lower calories, namely 2.4 cal/g while sucrose is 4 cal/g. xylitol has many benefits such as as an alternative sugar for diabetics because it does not involve insulin in metabolic processes in the body.in addition, xylitol can also prevent otitis, osteoporosis, inflammatory processes and prevent carriers and improve oral health because xylitol can inhibit the growth of streptococcus mutans bacteria that cause open access international journal of applied biology keyword yeast, xylitol , sap, soil, sugar press-mud. article history received 21 november 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 176 plaque on teeth (sasaki et al. 2010; kusumaningsari and handajani 2011; pal et al. 2013). so that currently the production of xylitol has been developed and has a high appeal. xylitol production is carried out by catalytic hydrogenation of pure d-xylose solution under high temperature and pressure. this process is very expensive and energy-intensive. one of the xylitol production strategies that are cheaper and more effective is biotechnology by utilizing microorganisms. among the known xylose-using microorganisms, yeast is the best producer of xylitol and has been studied extensively compared to other microorganisms (guo et al. 2006; dasgupta et al. 2017). yeast can convert xylose into xylitol because it has the enzyme xylose reductase (xr). several types of yeast such as candida boidinii, candida guilliermondii, candida tropicalis, candida magnolia, debaryomyces hansenii, and pichia assignitis are known to have the ability to produce xylitol (kumar et al. 2015). yeast can be found on various substrates such as fruits, seeds, animal skins, air, soil, water, and various substrates containing sugar (suryaningsih et al., 2018). it is recorded in pontoh (2012) that fresh sap contains sucrose 13.9-14.9%, ash content ranges from 0.220.98%, protein ranges from 0.20-0.61% and fat content is 0.02 %. the nutritional content of sap provides suitable conditions for yeast growth. in addition to sap, the presence of yeast in the soil is also very abundant, especially on sugar cane plantations. sugarcane plantation soil is a suitable habitat for yeast growth because it is rich in organic matter derived from the decomposition of sugarcane litter which is also a source of nutrition for microorganisms (botha, 2011). on the other hand, the presence of sugar factory waste around sugar cane plantations and in the community is still common. sugar factory waste can be in the form of solid, liquid, or gaseous waste. solid waste in the form of sugar press mud, furnace ash, fly ash, and bagasse (bagasse). this waste can cause environmental pollution if not managed properly (dharma et al. 2017; fangohoy and wandansari, 2017). sugar press mud waste is one of the wastes that has not been utilized properly but still contains nutritional elements such as na, k, ca, phosphorus and sugar content of about 5-15% and still contains high organic matter, which is around 50%. the organic matter content can also increase the activity of microorganisms (baig et al. 2002; gupta et al. 2011; juradi et al. 2020) the presence of organic matter in sap, soil, and sugar press-mud can be a source of nutrition for yeast. several studies reported the presence of yeast in soil, sap, and sugar press mud, namely those from the genera candida, saccharomyces, yorrowia, brettanomyces, endomycopsis. rhodotula, rotula and debaryomyces. although various studies have shown the presence of yeasts in sap, soil, and sugar press mud, exploration of potential yeasts to produce xylitol is still rare and needs to be developed. therefore, this study was conducted to obtain yeast from sap, sugarcane plantation soil, and sugar press mud waste which has the potential to ferment xylose to xylitol. international journal of applied biology, 5(2), 2021 177 materials and methods materials the sample of this study was yeast isolated from sap, soil, and sugar press mud. the sap used was palm sap and coconut sap obtained from one of the sap farmers in pinrang regency, while for sugarcane plantation land and sugar press mud waste from the bone sugar factory in bone regency. medium yeast malt agar (0.3 g of yeast extract, 0.3 g of meat extract, 0.5 g of peptone, 1 g of glucose, and 2 g of agar for 100 ml). medium yeast peptone xylose agar ( 1 g of yeast extract, 2 g of peptone, 3 g of xylose, 1.5 g of agar for 100 ml). medium xylose broth ( 0.1 g of yeast extract and 3 g of xylose for 100 ml). yeast isolation a total of 1 g of the sample was diluted graded to 10-6. then it was grown on yeast malt agar (yma) medium. after that, it was incubated in an incubator at 37 oc for 1x24 hours. after that, each colony showing a different morphology was regrown on the same medium to obtain pure isolates (kanti and latupapua, 2018). observation of colony morphology and yeast cell morphology the morphological oservations of yeast colonies were carried out using a stereo microscope with 40x magnification, to observe the characteristics of shape, color, margin, and elevation of yeast colonies growing on yeast malt agar (rahmana et al. 2016). while the morphological observations of yeast cell were carried out with methylen blue staining, to observe the characteristics of cell shape, budding, and the presence or absence of pseudohyphae. observations using a binocular microscope with a magnification of 1000x (rahmana et al. 2016; citra, 2019). screening of yeast to grow on media containing xylose preculture isolates were inoculated on yeast peptone xylose agar media with the quadrant scratch method to see the ability of yeast to grow on media containing xylose. the media was incubated at 37 oc for 48 hours. isolate with the best growth on yeast peptone xylose agar media was then carried out a second screening using xylose broth media. selected isolates from preculture were inoculated in sterile distilled water and diluted to obtain an od value of 25% transmittance. a total of 1 ml was inoculated on 5 ml of xylose broth media and then incubated again for 24 hours at 37 oc. cell growth was observed by measuring the optical density (od) of each isolate using a spectrophotometer at a wavelength of 600 nm (guo et al. 2006). xylose fermentation a total of 12 ml of inoculum that had been precultured and had the same od value, was then inoculated on 120 ml of fermentation medium. the fermentation medium used were xylose 5.0 g, yeast extract 0.3 g, kh2po4 0.5 g, mgso4.7h2o 0.1 g, (nh4)2so4 0.3 g for 100 ml. then it was incubated for 72 hours at room temperature with a rotary shaker at a speed of 250 rpm (yulianto et al. 2005). international journal of applied biology, 5(2), 2021 178 analysis of fermentation parameters total yeast calculation calculation of total yeast using the spc (standard plate count) method. a total of 1 ml of the fermented culture was put into 9 ml of sterile distilled water and carried out with graded dilutions. furthermore, the last 3 dilution tubes were taken as much as 1 ml and inoculated with the pour plate method using yeast malt agar (yma) media then incubated at 37 oc for 24 hours. the total yeast calculation was carried out at 0, 24, 48, and 72 hours (wiratno and nofi, 2018) fermentation culture ph measurement the measurement of the ph value was measured with a digital ph-meter. the ph meter was used by dipping the electrode into the sample to be measured. however, before using the tool, it was calibrated with a buffer of ph 7 and ph 4. xylose and xylitol xylose content was measured using hplc (high-performance liquid chromatography) with 80 ± 5% acetonitrile as mobile phase. meanwhile, the measurement of xylitol levels was carried out using the uplc (ultra performance liquid chromatography). data analysis the data from the isolation and observation of the morphology of the colonies and yeast cells were processed in the form of tables and figures and discussed descriptively. while the total yeast calculation data and ph measurements are displayed in graphical form. meanwhile, the data obtained from the analysis of xylose and xylitol levels are presented in tabular form and discussed descriptively. results and discussion yeast isolations the results of yeast isolation from palm sap, coconut sap, sugarcane plantation soil and blotong waste obtained 38 isolates. where 12 isolates were obtained from sugar palm sap, 12 isolates were obtained from coconut sap, 8 isolates were obtained from sugarcane plantation soil, and 6 isolates were obtained from sugar press-mud. the determination of the selection of isolates was based on differences in the morphological characteristics of the colonies which included shape, color, elevation and margins of each of these yeast isolates. observation of yeast colony and cell morphology the morphological observations of yeast colonies were carried out using a stereo microscope with a magnification of 40x. colony morphology was observed directly from the purification results grown on yma media. while the observation of yeast cell morphology using 0.1% methylene blue staining. the addition of methylene blue color to yeast cells was observed to show the difference between live and dead yeasts (wachid & mutia 2019). international journal of applied biology, 5(2), 2021 179 table 1. colony and cell morphology isolate source isolate code colony morphology cell mophology shape margin elevation color shape budding pseudohyphae p1 round flat raised white oval multipolar not found p2 round flat raised white oval monopolar not found p3 round flat raised white oval monopolar not found p4 round flat raised white oval monopolar not found p5 round flat raised white oval monopolar not found palm sap p6 round flat raised white oval monopolar not found s1 round flat raised white oval monopolar not found s2 round flat raised white oval monopolar not found s3 round flat raised white oval monopolar not found s4 round flat raised white oval monopolar not found s5 round flat raised white oval monopolar not found s6 round flat raised white oval monopolar not found kp1 round flat raised white oval monopolar not found kp2 round flat raised white oval monopolar not found kp3 round flat raised white oval monopolar not found kp4 round flat raised white oval monopolar not found kp5 round flat raised white oval monopolar not found coconut sap kp6 round flat raised white oval monopolar not found ks1 round flat raised white oval bipolar not found ks2 round flat raised white oval monopolar not found ks3 round flat raised white oval monopolar not found ks4 round flat raised white oval monopolar not found ks5 round flat raised white oval monopolar not found ks6 round flat raised white oval monopolar not found t1 round filamentous raised creamy oval multipolar found t2 round filamentous convex creamy oval multipolar found t3 round filamentous convex creamy oval multipolar found soil t4 round filamentous raised white oval multipolar found t5 round filamentous convex white oval multipolar found t6 round flat convex creamy oval monopolar not found t7 round flat convex creamy round monopolar not found t8 round flat convex creamy round monopolar not found b1 round flat convex creamy oval monopolar not found b2 round flat convex creamy oval multipolar not found sugar press mud b3 round flat convex creamy oval monopolar not found b4 round filamentous raised creamy oval multipolar not found b5 round flat convex creamy oval monopolar not found b6 round flat flat creamy oval monopolar not found based on the results of observations of colony morphology on 12 yeast isolates obtained from palm sap, in general, they have the same shape, namely round, margins are flat, elevation is raised with the color of the colony, namely white, while for the results of cell morphology observations in general have the same cell shape, namely oval, with budding type that is monopolar except for isolates with code p1 with multipolar budding type, and for the presence of pseudohyphae of the 12 isolates not found. international journal of applied biology, 5(2), 2021 180 the results of observations of colony morphology on 12 yeast isolates obtained from coconut sap in general have the same shape, namely round, margins that are flat, elevation that is raised with the color of the colony that is white, while for the results of cell morphology observations in general have the same cell shape, namely oval. , with budding type that is monopolar except for isolates with code ks1 with budding type that is bipolar, and for the presence of pseudohyphae of the 12 isolates not found. the results of observations of colony morphology on 8 yeast isolates obtained from sugarcane plantation soils generally had the same shape, namely round, with colony margins that were filamentous for isolates t1, t2, t3, t4, and t5 and for isolates t6, t7, and t8, namely flat. the colony elevation was convex for isolates t2, t3, t5, t6, t7, and t8, while for isolates t1 and t4 the elevation was raised with the colony color being cream for isolates t1, t2, t3, t6, t7, and t8 while for isolates t4 and t5 are white. as for the results of cell morphology observations in general, the cell shape is oval, except for isolates with codes t7 and t8 which are round with budding types, namely multipolar for isolates t1, t2, t3, t4, and t5, while for isolates with codes t6, t7 , and monopolar t8. for the presence of pseudohyphae for isolates t1, t2, t3, t4, and t5, pseudohyphae were found while for isolates with codes t6, t7, and t8 were not found. the results of observations of colony morphology on 6 yeast isolates obtained from sugar pressmud generally had the same shape, namely round, with flat colony margins, except for isolate b4 which was filamentous. the colony elevation was convex for isolates b1, b2, b3 and b5, while for isolates b4 raised and b6 flat, the colony color in general was cream. as for the results of cell morphology observations in general, the cell shape is oval, with the type of budding that is monopolar except for isolates b2 and b4 which are multipolar. for the presence of pseudohyphae from the 6 isolates, no pseudohyphae were found. figure1. colony morphology of sap, soil and sugar press-mud international journal of applied biology, 5(2), 2021 181 figure2. cell morphology of sap, soil and sugar press-mud; (a) cell shape; (b) budding; (c) pseudohyphae screening of yeast to grow on media containing xylose the screening process is the stage of selecting yeasts that can consume xylose as a carbon source. this stage was repeated twice by growing on agar and liquid media (broth). the first screening was carried out by growing 38 isolates purified on ypx agar media using the quadrant scratch method and incubating for 2x24 hours. the concentration of xylose used in ypx agar media is 3%. screening on agar media is intended to see the growth of yeast isolates on media containing xylose. based on the results of the first screening, it was found that from 38 isolates grown only 25 isolates were able to grow well on ypxa media, namely all isolates from palm sap, 6 isolates from coconut sap, namely kp1, kp2, kp3, kp4, kp5, kp6, 6 isolates from the soil, namely t1, t2, t3, t4, t5, t6 and one isolate from sugar press mud, namely b4. twentyfive isolates can consume xylose as a carbon source. xylose is a pentose sugar that not all yeasts can consume, it is influenced by the presence of the enzyme xylose reductase (xr) where the enzyme will be activated when there is xylose (muller,2009). after being grown in ypx agar media, the 25 isolates were then continued for a second screening by growing them in xylose broth media with 3% xylose media composition. before being put into the xylose broth medium, the isolate was first diluted to reach a transmittance value of 25% to equalize the cell biomass entering the xylose broth medium. then incubated for 1x24 hours. after incubation, the optical density (od) value of each isolate was measured using a spectrophotometer with a wavelength of 600 nm. the measurement of the od value was carried out to see the level of turbidity of the isolated culture media where the higher the od value, the higher the amount of cell biomass in the culture medium. international journal of applied biology, 5(2), 2021 182 figure 3.graph of od values the results of the measurement of od values are presented in (figure 3) of 25 isolates grown on xylose broth media, 6 isolates were selected from 4 different isolate sources which showed the highest optical density (od) value, namely 2 isolates from palm sap ( p1 and s3) with an od value of 0.95, 2 isolates derived from coconut sap, namely kp3 with an od value of 0.89 and kp4 with an od value of 0.83. b4 from soil with an od value of 0.83, and t4 from blotong with an od value of 0.64. the difference in od values was due to differences in the ability of yeast to use xylose within 24 hours, thus affecting the growth and number of yeast cells in liquid media. mardawati et al. (2018) explained that each yeast has a different metabolic rate in converting xylose to xylitol. fermentation xylose to xylitol the fermentation test is intended to see the ability of yeast to ferment xylose to xylitol within 72 hours. before the fermentation test, the six selected isolates were first grown in preculture media for 1x24 hours to adjust the growth of yeast before entering the fermentation medium. fermentation was carried out by growing isolates p1, s3, kp3, kp4, b4, and t4 which had been precultured into fermentation media with 5% xylose concentration. in addition, a fermentation medium was made without adding yeast isolate as a control (comparison) in the fermentation test. the concentration of cells entering the fermentation medium was equated with an od value of 0.6 or 25% transmittance using a wavelength of 600 nm. during fermentation, total yeast was calculated using the standard plate count (spc) method and measured ph values every 0, 24, 48, and 72 hours or 24-hour intervals for 3 days. the results of fermentation for 72 hours have then measured the levels of xylitol formed and the levels of xylose used using hplc/uplc total yeast from the results of the total yeast calculation, the growth graph is obtained as follows : international journal of applied biology, 5(2), 2021 183 figure4. graph of total yest based on the graph of total yeast growth, it was known that yeast cells increased steadily with increasing fermentation time. the amount of yeast at the beginning of the fermentation continued to increase until the 72nd hour. where isolates p1 and s3 with an initial yeast count of 1.2 x 1010 cfu/ml increased in isolates p1 of 1.8 x 1019 cfu/ml, s3 by 6.8 x 1020 cfu/ml. for isolates, kp3 showed an increase from the initial number of the incubation period of 1.3 x 108 cfu and increased to a total of 3.24 x 1020 cfu/ml and kp4 with an initial amount of yeast of 2.7 x 108 cfu/ml continued to increase up to 1.3 x 1021 cfu/ml. of 9.4 x 1020 cfu/ml. while isolate b4 from the initial yeast count, namely 1.2 x 1010 cfu/ml, continued to increase to 9.4 x 1020 cfu/ml and t4 also showed the same increase in total yeast, where the initial yeast total was 1.8 x 109 cfu/ml to 8.8 x 1020 cfu/ml. as for the control, cell biomass was not added so that it did not show cell growth. based on the graph of the total yeast count (figure 4), it can be seen that the yeast cells of the six isolates were still in the growth phase. the increase in total yeast indicates that during the fermentation process the yeast cells continue to grow. with the increase in the number of cells, metabolic activity increases (usmiati and marwati, 2007). based on the graph of the total yeast count (figure 4), it can be seen that the yeast cells of the six isolates were still in the growth phase. the increase in total yeast indicates that during the fermentation process the yeast cells continue to grow. with the increase in the number of cells, metabolic activity increases (usmiati and marwati, 2007). international journal of applied biology, 5(2), 2021 184 measurement of ph measurement of ph was carried out to determine changes in ph during the fermentation process. the following are the results of measuring the ph value of each fermentation inoculum: figure 5. graph of ph based on the results of ph measurements, it is known that the initial ph of all fermentation media after adding inoculum is ph 5. according to fairus et al. (2013) a good ph value for the growth of yeast candida sp. is a xylose user yeast with a ph range of 4-6. all isolates experienced a decrease in ph to 4 after 72 hours of fermentation. the decrease in ph is caused during the fermentation process which tends to form organic acids which can lower the ph. according to yulianto (2001), during the xylose fermentation process, acetic acid will be formed which can lower the ph. in this study, the optimum ph for the tested yeast is not yet known. however, it is known that the ph of each fermentation medium has met a good ph range for fermenting xylose. analysis of fermentation yield analysis of xylose and xylitol levels in the six isolates and controls was carried out after 72 hours of the fermentation process which was measured using hplc (highperformance liquid chromatography) and uplc (ultra performance liquid chromatography). measurement of final xylose and xylitol levels is intended to see the levels of xylitol produced by yeast by utilizing used xylose within 72 hours. the initial xylose concentration used in the fermentation was 5 g/100ml. the results of xylitol production by yeast isolates are presented in (table 6), which shows that the results of measuring xylose levels obtained final xylose at p1 of 2.85 g/100ml, isolate s3 of 1.94 g/100ml, kp3 of 1.50 g /100ml, kp4 is 1.52 g/100ml, b4 is 1.15 g/100ml and at t4 is 1.97 g/100ml. so it can be seen that the substrate or xylose used in the fermentation has not been consumed by the yeast to produce xylitol during the 72 hour fermentation time. international journal of applied biology, 5(2), 2021 185 table2. analysisof fermentations yield initial xylose concentration (g/100ml) final xylose concentration (g/100ml) xylose used (g/100ml) final xylitol concentration (g/100ml) yield xylitol (y p/s ) (g/g) p1 5 2,85 2,15 1,28 0,59 s3 5 1,94 3,06 2,05 0,67 kp3 5 1,50 3,50 2,23 0,63 kp4 5 1,52 3,48 2,19 0,62 b4 5 1.15 3.85 2.47 0.64 t4 5 1.97 3.03 2 0.66 control 5 5 this is in line with the yeast growth graph (figure 4) which still shows the growth phase so that the consumed xylose has not been used up. the final xylose content in the control was still 5 g/100ml and no xylitol production was found. the results of the measurement of the final xylitol level obtained in s3 are 2.05 g/100ml, p1 is 1.28 g/100ml, kp3 is 2.23 g/100 ml, kp4 is 2.19 g/100 ml, b4 is 2.47 g/100ml and t4 is 2 g/100ml. based on these results, it is known that the isolates that produced the highest xylitol levels were isolated b4 with xylitol levels of 2.47 g/100ml. the results of the calculation of xylitol production in yeast are expressed in xylitol product yield (yp/s) (g/g). according to safitri et al. (2016) product yield (yp/s) is the number of nutrients or substrate (xylose) used by yeast to form products (xylitol). the xylitol yield obtained in isolates p1 was 0.59 g/g, s3 was 0.67 g/g, kp3 was 0.63 g/g, kp4 was 0.62 g/g, b4 was 0.64 g/g while in t4 is 0.66 g/g. the yield obtained in isolate p1 was higher than that in isolate b4, meaning that the efficiency of using xylose in p1 to form xylitol was higher than that in isolate b4 although the final xylitol yield obtained in b4 was higher. according to barbosa et al. (1988) in arifan and nuswantari (2020), stated that the theoretical maximum value of xylitol production yield is 0.917 g/g. based on the xylitol yield value, it was still below the maximum yield of xylose fermentation. this can be influenced by factors that support the production of xylitol. azizah (2019), explained that several factors influence the fermentation process to produce xylitol, namely carbon and nitrogen sources, temperature, substrate concentration, and aeration. in addition, during the fermentation process, it allows the formation of inhibitor compounds such as acetic acid which can reduce xylitol production. silva et al. (2004) explained that acetic acid can affect yeast cell metabolism in producing xylitol where acetic acid will reduce yeast cell growth. thus, this study found that isolates isolated from palm sap, coconut sap, soil, and sugar press mud had the potential to ferment xylose to xylitol for 72 hours of fermentation. conclusions based on this research it can be concluded that: 1. the results of yeast isolation from sap were 24 isolates, consisting of 12 isolates from sugar palm sap samples and 12 isolates from coconut sap samples, 8 isolates from the soil, and 6 isolates from sugar press mud where only 25 isolates were able to ferment international journal of applied biology, 5(2), 2021 186 xylose, namely 12 isolates. palm sap, 6 isolates from coconut sap, 6 isolates from the soil, and 1 isolate from the soil. 2. six yeast isolates from sap, soil and sugar press mud, namely isolates p1, s3, kp3, kp4, t4 and b4 were able to ferment xylose to xylitol with final xylitol levels respectively 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makassar strait with a river length of 78.75 km which allows the presence of many wild animals that live around the river. this study aims to determine the diversity and distribution pattern of birds in the je'neberang watershed as an inventory of bird data. the point count method is used in this study where observations are divided into 5 stations from upstream to downstream, at each station, there are 6 observation spots with a distance of 300 m from each spot, with an observation radius of 50 m. the parameters of species diversity used in this research are shanon biodiversity, mdified hill's ratio index of evenness, dominance index, and morisita index. based on the results of the study found 39 species from 24 families of species diversity (h') of birds in the je'neberang watershed with a value of h' at the five stations from 1.95 to 2.67 (1.0 < h' 3.0) meaning moderate diversity. evenness index (e') bird species in the je'neberang watershed, gowa regency, were classified as unstable with an e value at the five stations, from 0.54 to 0.68. dominance index (d) of bird species in the je'neberang river basin at each observation station, it is known that the five stations have high dominance because the d value at each station is close to 1, which is between 0.80 0.89. the distribution pattern of birds in the je'neberang watershed is mostly clustered, namely 30 species of birds, 5 species with uniform distribution patterns, and 4 species that cannot be analyzed. issn : 2580-2410 eissn : 2580-2119 bird diversity and distribution patterns in the je'neberang river flow region, south sulawesi gowa regency abd rukman 1*, slamet santosa 2, ambeng 2 1postgraduate program, department of biology, faculty of mathematics and natural sciences hasanuddin university makassar 2 department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, science building 3rd floor introduction birds are one of the animals that have had a strong relationship with human life since time immemorial. birds live in almost all habitat types and at altitudes. bird habitat types are highly correlated with their daily life and activities such as resting places, perching, reproduction, eating, sheltering, and nesting (syarifuddin, 2011). birds are scattered from the coast to the top of mountains, in natural or artificial habitats, in urban, suburban, rural areas, mountain forests, lowland forests, beaches, ponds, rivers, lakes, agricultural land, grasslands, and settlements (baskoro, 2018). the je'neberang river area is located in the southern part of south sulawesi province. based on presidential decree no. 12 of 2012 concerning the formation of river open access international journal of applied biology keyword je’neberang river; diversity; distribution pettern; bird article history received november 29, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 13 areas, the je'neberang river is included in the national strategic je'neberang river basin. the area of the je'neberang river is 9,389.47 km2, which includes 58 watersheds. administratively, there are 1 (one) city and 9 (nine) regencies traversed by the je'neberang river, namely makassar city, gowa regency, takalar, maros, jeneponto, bantaeng, bulukumba, sinjai, bone and selayar islands. (bpdas, 2010). based on the data obtained, it is known that until now there is no latest data on bird diversity in the je'neberang watershed. this study aims to determine the diversity and distribution pattern of birds. it is hoped that this research can assist in making an inventory of bird species in the je'neberang watershed materials and methods this research was conducted from october 2020 to june 2021 in the je'neberang river basin from hulu to hilir, gowa regency, south sulawesi with the tools used in the form of observation sheets/tally sheets, monocular binoculars, canon eos d750 camera, canon ultrasonic lens 70 200 mm, stationery, gis application, hygrometer, thermometer, and bird field guide book in wallacea, sulawesi, maluku, and nusa tenggara areas. the materials used in this study were bird species at the observation site. the method of data collection in this study is using the point count method (bibby et al, 2000), by determining 5 stations consisting of 6 observation spots at each station. a map of the location of bird observation points in the je'neberang watershed, gowa regency, south sulawesi is presented in figure 1. figure1. location map placement of bird observation points in the je'neberang watershed, gowa regency, south sulawesi. the workings of research observations in the je'neberang watershed are as follows; preliminary preparation based on the journal rusmendro, 2009. before data collection, preliminary observations were made, namely place observations to know the initial conditions of the research site and determine the observation points wherein each observation point the distance was 300 meters with an observation radius of 50 meters. international journal of applied biology, 6(1), 2022 14 data retrieval observations were made in the morning at 06.00 09.00 wita, continued in the afternoon at 15.00 18.00 wita. bird data was collected using the point count method (mackinnon, et al 2010). bird observation data taken is comprehensive data on bird species and the number of birds found at each observation point. identification of the bird includes morphology, namely beak length, color, and foot color, and recording the number of birds on the tayle sheet that has been provided at each bird encounter. data analysis the data obtained were then analyzed using data analysis as follows: a. diversity index (h') 𝐻′ = ∑ 𝑝𝑖 𝑙𝑛 𝑝𝑖 𝑛 𝑖=1 information : h' : diversity index pi : ni/n ni : number of individuals of type i n ; total number of individuals of all types according to yanto, et al, 2008, the shannon-wienner index has the following indicators: h' < 1.5 ; low level of diversity 1.5 h' 3.5 : moderate level of diversity h' > 3.5 : high level of diversity b. uniformity/evenness index (e') 𝐸′ = 𝐻′ 𝐻𝑚𝑎𝑘𝑠 information : e : uniformity/evenness index hmax : ln s s : number of species indicator uniformity index (magurran, 1982); 𝐸 = 0 − 1 the distribution of individuals between species is uneven/there are certain types of dominant (labile) when approaching 0 the distribution of individuals between species is even if it is close to 1 international journal of applied biology, 6(1), 2022 15 c. dominance index (d) the dominance index is calculated using the simpson dominance formula (odum, 1993): 𝐷 = ∑(𝑛𝑖/𝑁) information : d : simpson's dominance index ni : number of individuals per species n : number of individuals of all species the dominance index ranges from 0 to 1, if the value of the dominance index is smaller, it indicates that there is no dominant species. d. morisita index id = ∑ 𝑥2 − ∑ 𝑥 ∑ 𝑥2 − ∑ 𝑥 𝑀𝑈 = 𝑥 0,975𝑑𝑓 − 𝑛 − ∑ 𝑥𝑖 2 ∑ 𝑥𝑖 − 1 𝑀𝐶 = 𝑥 0,025;𝑑𝑓 − 𝑛 − ∑ 𝑥𝑖 2 ∑ 𝑥𝑖 − 1 information ; id : morisita index mu : uniform distribution pattern index mc : index of aggregative distribution pattern n : sample size xi : number of individuals in the i-th sample unit the decision rules for determining the shape of the distribution pattern of the observed organisms are as follows: 1. if the value of id mc 1, then ip is calculated using the equation; ip = 0.5 + 0.5 [ 𝐼𝑑 − 𝑀𝑐 𝑛 − 𝑀𝑐 ] 2. if the value of mc > id 1, then ip is calculated using the equation: ip = 0.5 [ 𝐼𝑑 − 1 𝑀𝑐 − 1 ] international journal of applied biology, 6(1), 2022 16 3. if 1 > id > mu, then ip is calculated using the equation: ip = −0.5 [ 𝐼𝑑 − 1 𝑀𝑐 − 1 ] 4. if 1 > mu > id, then ip is calculated using the equation: ip = −0.5 + 0.5 [ 𝐼𝑑 − 𝑀𝑢 𝑀𝑢 ] 95% confidence indicator ip value: a.ip < 0, then the distribution pattern is uniform (uniform) b.ip = 0, then the distribution pattern is random c.ip > 0, then the distribution pattern is clumped results and discussion species of birds found in the je'neberang watershed using the point count method at each station (table 1). the number of bird species found in the je'neberang watershed was 39 species of birds with a total of individuals and species at each station, namely station i as many as 15 species with a total of 148 individuals, station ii 20 species with 169 individuals, station iii 20 species with 339 individuals. , station iv has 12 types with 458 individuals, station v has 14 types with 358 individuals table 1. types of bird composition in the je'neberang river basin no lokal name latin name observation stasiun i ii iii iv v 1 anis gunung turdus poliocephalus 2 0 0 0 0 2 ayam hutan merah gallus galus 3 0 0 0 0 3 blekok sawah ardeola speciosa 0 14 41 130 11 4 bondol kepala pucat lonchura pallida 0 3 8 33 15 5 bondol pecking lonchura punctula 0 0 11 6 27 6 bondol rawa lonchura malacca 12 0 54 41 116 7 cabai panggul kuning dicaeum aureolimbatum* 5 10 9 0 0 8 cabai sulawesi dicaeum nehrrkorni* 7 0 0 0 0 9 cekakak sungai halcyon chloris 0 2 9 0 2 10 cerek tilil charadrius alexandrius 0 0 12 0 0 11 cici padi cisticola juncidis 1 6 0 0 4 12 cucak kutilang pycnonotus aurigaster 21 30 36 32 41 13 dara biasa sterna hirundo 0 0 0 0 22 14 decu belang saxicola caprata 2 8 0 0 0 15 decu belang betina saxicola albonata 0 9 0 0 0 16 elang bondol haliastur indus 0 1 0 0 0 17 elang kelabu butastur indicus 0 2 0 0 0 18 elang ular sulawesi spilornis rufipectus* 2 0 0 0 0 19 gagak hutan corvus enca 0 4 1 0 0 international journal of applied biology, 6(1), 2022 17 20 gereja erasia passer montanus 0 7 30 8 15 21 jalak kerbau acriditheres javanicus 2 0 0 0 0 22 kacamata biasa zosterops palpebrosus 14 15 21 32 29 23 kacamata gunung zosterops montanus 16 0 0 0 0 24 kapasan sulawesi lalage leucopygialis* 0 3 8 0 0 25 kapinis rumah apus affinis 0 0 0 10 0 26 kekep babi artamus leucorhynchus 0 0 3 0 0 27 kicuit batu motacilla cinerea 0 0 5 0 0 28 kowak-malam merah nycticorax nycticorax 0 0 0 6 0 29 kuntul kecil egretta garzetta 0 0 1 0 0 30 layang-layang batu hirundo tahitica 0 3 12 18 4 31 madu sriganti nectarinia jugularis 0 4 5 0 0 32 merpati batu columba livia11 0 0 3 5 3 33 raja udang erasia alcedo atthis 0 6 2 0 0 34 sikatan bakau cyornis rufigastra 1 0 0 0 0 35 sikatan pulau eumyias panayensis 4 0 0 0 0 36 terkukur biasa streptopelia chinensis 0 6 0 0 0 37 trinil pantai actitis hypoleucos 0 0 0 0 3 38 trinil semak tringa glareola 0 1 0 0 0 39 walet sapi collocalia esculenta 56 35 68 137 66 . information : * = endemic in the wallacea region based on table 1 above, there are 4 species of birds endemic to sulawesi, namely the sulawesi snake eagle (splornis rufipectus), sulawesi kapasan (lalage leucopygialis), sulawesi chili (dicaeum nerhkoni), and yellow panggul chili (dicaeum aureolimbatum). the types of birds that most often appear at each station, namely 8 bird species can be seen in figure 1 below, as follows. number of individuals 148 169 339 458 358 number of species 15 20 20 12 14 (1) (2) (3) international journal of applied biology, 6(1), 2022 18 figure 2. species that often appear at each observation station (1). blekok sawah (ardeola speciosa), (2). bird bondol kepala pucat (lonchura pallida), (3). bird bondol rawa (lonchura malacca), (4). cucak kutilang (pycnonotus aurigaster), (5). bird layang-layang batu (hirundo tahitica), (6). walet sapi (collocalia esculenta), (7). bird kacamata biasa (zosterops palpebrosus) and bird gereja erasia (passer montanus) . (personal documentation, 2021) heterogeneity (heterogeneity) the heterogeneity of bird communities at the five observation stations in the je'neberang watershed includes the shannon-wienner diversity index, simpson dominance index, and evenness index. the index of bird species diversity at the two five stations ranged from 1.95 to 2.67 which was categorized as moderate diversity (1.0 < h' 3.0). the evenness index (e) obtained has a value close to 0 (0.54 0.68) which is considered to have an uneven distribution. the simpson dominance index (d) obtained has a value close to 1 (0.80 0.89) indicating dominance by one or several species. table 2. values of shannon-wienner diversity index (h'), evenness index (e), simpson's dominance index (d) at the five observation stations in the je'neberang watershed, gowa regency. (4) (5) (6) (7) (8) observation parameter observation station i ii iii iv v species diversity (h') 2,1 2,6 2,5 1,9 2,1 specific evenness index (e') 0,5 0,7 0,6 0,6 0,6 dominance index (d) 0,80 0,89 0,89 0,80 0,82 international journal of applied biology, 6(1), 2022 19 distribution pattern distribution patterns of birds in the je'neberang watershed, gowa regency at each station (table. 3), using the morisita index, namely there are 30 species whose distribution patterns are clustered, there are 5 species with uniform distribution patterns and only 4 species that cannot be analyzed. because the individual abundance of each bird species is below one individual. table 3. bird distribution patterns in the je'neberang watershed, gowa regency, south sulawesi no spesies amount ip information 1 anis gunung (turdus poliocephalus) 2 1 clumped 2 ayam hutan merah (gallus galus) 3 0,12 clumped 3 blekok sawah (ardeola speciosa) 196 0,1 clumped 4 bondol kepala pucat (lonchura pallida) 59 0,12 clumped 5 bondol pecking (lonchura punctula) 44 0,08 clumped 6 bondol rawa (lonchura malacca) 223 0,07 clumped 7 cabai panggul kuning (dicaeum aureolimbatum) 24 0,08 clumped 8 cabai sulawesi (dicaeum nehrrkorni) 7 0,23 clumped 9 cekakak sungai (halcyon chloris) 13 -0,01 uniform 10 cerek tilil (charadrius alexandrius) 12 1 clumped 11 cici padi (cisticola juncidis) 11 0,01 clumped 12 cucak kutilang (pycnonotus aurigaster) 160 0,009 clumped 13 dara biasa (sterna hirundo) 22 1 clumped 14 decu belang (saxicola caprata) 10 0,09 clumped 15 decu belang betina (saxicola albonata) 9 0,19 clumped 16 elang bondol (haliastur indus) 1 17 elang kelabu (butastur indicus) 2 1 clumped 18 elang ular sulawesi (spilornis rufipectus) 2 1 clumped 19 gagak hutan (corvus enca) 5 -0,06 uiniform 20 gereja (passer montanus) 60 0,08 clumped 21 jalak kerbau (acriditheres javanicus) 2 1 clumped 22 kacamata biasa (zosterops palpebrosus) 111 0,042 clumped 23 kacamata gunung (zosterops montanus) 16 0,63 clumped 24 kapasan sulawesi (lalage leucopygialis) 11 0,19 clumped 25 kapinis rumah (apus affinis) 10 1 clumped 26 kekep babi (artamus leucorhynchus) 3 1 clumped 27 kicuit batu (motacilla cinerea) 5 1 clumped 28 kowak-malam merah (nycticorax nycticorax) 6 0,6 clumped 29 kuntul kecil (egretta garzetta) 1 30 layang-layang batu (hirundo tahitica) 37 0,04 clumped 31 madu sriganti (nectarinia jugularis) 9 0,37 clumped 32 merpati batu (columba livia) 11 0,21 clumped 33 raja udang erasia (alcedo atthis) 8 -0,0043 uniform 34 sikatan bakau (cyornis rufigastra) 1 35 sikatan pulau (eumyias panayensis) 4 0,35 clumped 36 terkukur biasa (streptopelia chinensis) 6 -0,007 uniform 37 trinil pantai (actitis hypoleucos) 3 -0,02 uniform international journal of applied biology, 6(1), 2022 20 2,1 2,6 2,5 1,9 2,1 0 0,5 1 1,5 2 2,5 3 i ii iii iv v species diversity index stasiun 38 trinil semak (tringa glareola) 1 39 walet sapi (collocalia esculenta) 362 0,07 clumped discussion composition type birds are members of a group of vertebrate animals (vertebrates) that have feathers and wings (kuswanda, 2010). birds are also wild animals that have survived in all habitats, and have high mobility, and are easy to adapt to a wide variety of environmental types (welty, 1982 in rohiyan et al, 2013). this research was conducted in the je'neberang river basin, gowa regency, from october 2020 to may 2021 by collecting data at five observation stations with six observation points at each station which were divided into six points per station. the data obtained is based on table 1. there are 39 species of birds from 24 families, with a total number of individuals at each station, namely at station 1 there are 148 individuals with a total of 15 species, at station 2 there are 169 individuals with 20 species, at station 3 there are 339 individuals with 20 species, station 4 there are 458 with 12 species, and station 5 there are 356 individuals with 14 species. species found at all observation stations in the je'neberang watershed tend to experience an increase in the number of individuals and a decrease in the number of species, which is fluctuating, it can be seen in table 1. found at station 2 and station 3, this is because at stations 2 and 3 are areas that have many adequate sources of feed because they are close to forest areas, agricultural land, fish ponds, and according to lack, 1971 that the presence of birds in an area depends on the characteristics habitat, the number of species is also influenced by the level of adequate resources. heterogeneity 1. diversity of bird species in the je'neberang river basin based on observations and data analysis, it is known that the diversity of bird species in the je'neberang watershed shows that the five stations have an average value of species diversity that is almost the same, which is between 1.9 to 2.6 (table 2) and overall has a moderate level of diversity, with a moderate level of productivity, fairly balanced ecosystem conditions and moderate ecological pressure (1 < h 3) (figure 3). figure 3. histogram of the bird diversity index (h') at five stations in the je'neberang river basin, gowa regency 2021 international journal of applied biology, 6(1), 2022 21 0,54 0,68 0,61 0,58 0,58 0 0,2 0,4 0,6 0,8 i ii iii iv v species evenity index stasiun based on the analysis of the diversity index at all stations, the data obtained at station one has a diversity of 2.1, station two has a diversity value of 2.6, station three has a diversity of 2.5. station four has a diversity value of 1.9 and station five has a diversity of species that is 2.1, it can be seen in table 2. the results of the study indicate that station two has the highest level of diversity, while station four has a low level of diversity. this is because station four is located with densely populated conditions and human activities are too dense, which causes the availability of food sources for birds to not be fulfilled, and also human interaction in residential areas makes the natural ecosystem for birds to experience competition against the environment and the natural environment of birds is disturbed. habitat conditions greatly affect the high and low diversity of bird species (simanjuntak, et. al, 2013). according to (yoga, 2018), in his journal, the higher the shannon wiener index, the better the carrying capacity of the ecosystem in that location. 2. species evenness index in the je'neberang watershed the species evenness index (e) can be seen in figure 4. where the highest value is 0.68 at station two. then at station three, the evenness value is 0.61, station four, and station five have the same type of evenness, namely 0.58. and the lowest is at station one, which is 0.54, this is because at station one it was caused by extreme weather at the time the research was conducted and the air temperature was too low. in the je'neberang watershed at each observation station, the evenness index obtained has a value close to 0 (0.54 0.68) which means it has an uneven distribution (labile). evenness in a habitat will reach its maximum point and is classified as homogeneous if all species have a balanced number of individuals in all observed habitats (fikriyanti et al., 2018). figure 4. histogram of evenness index (e') of birds at five stations in the je'neberang river basin, gowa regency 2021. 3. simpson's dominance index (d) based on observations made at each station, it was found that at station 1 the dominance value was 0.80, at station 2 and station 3 had the same dominance value of 0.89, at station 4 had a dominance value of 0.80, and station 5 had dominance value of 0.82. the dominance index shows that at each observation station in the je'neberang watershed, it is close to 1 (0.80 0.89) indicating dominance by one or several species. international journal of applied biology, 6(1), 2022 22 0,8 0,89 0,89 0,8 0,82 0,75 0,8 0,85 0,9 i ii iii iv v dominance index stasiun figure 5. histogram of bird dominance index (d') at five stations in the je'neberang watershed, gowa regency 2021. from the observations obtained, it is known that there are several types of birds in the je'neberang watershed that dominate, namely blekok rice fields (ardeola speciosa), pale head bondol (lonchura pallida), swamp bondol (lonchura malacca), cucak finches (pycnonotus aurigaster), ordinary glasses (zosterops palpebrosus), stone kite (hirundo tahitica), church (passer montanus) and cow swallow (collocalia esculenta). from each observation station, it is known that the five stations have high dominance because the d value is close to 1. according to (odum,1971), states that this category can be caused by lack of food and hunting for certain bird species so that it can cause pressure or obstacles to bird species in the five stations. observation. distribution pattern the spread of bird species is strongly influenced by the suitability of birdlife which includes bird adaptation to the environment, ecosystem level, competition, food availability, and natural selection (wisnubudi, 2009). based on the results of research conducted in the je'neberang watershed, it can be seen in table 3. that most of the bird species in the je'neberang watershed are more widely distributed in groups than a uniform distribution pattern. factor. according to (alan, 2012) being influenced by the presence of resources, especially food in a habitat, is a very urgent limiting factor for the distribution and becomes very important in the survival of organisms in that habitat. grouping indicates that organisms gather in several productive habitats, this event is caused by group behavior, diverse environments, reproductive structures that have special characteristics, physical and chemical factors, weather, food availability, social level. and due to the nature to defend themselves from predators and other unfavorable factors (junaidi et al. 2009). the distribution pattern of an organism in nature rarely forms a uniform, but generally forms a clustered pattern (gate, et al, 2007 in the journal putri, et al 2015). this can be seen from the existence of the ecosystem around the je'neberang river which is homogeneous and scattered in each observation, namely the same type of ecosystem at each station. international journal of applied biology, 6(1), 2022 23 conclusions based on the results of research and bird observations in the je'neberang river basin, it can be concluded that; 1. species diversity (h') of birds in the je'neberang watershed with a value of h' at the five stations from 1.95 to 2.67 (1.0 < h' 3.0) means moderate diversity. 2. evenness index (e') bird species in the je'neberang river basin, gowa regency, are in the unstable category with an e value at the five stations, namely 0.54 to 0.68. 3. dominance index (d) bird species in the je'neberang watershed at each observation station, it is known that the five stations have high dominance because the d value at each station is close to 1, which is between 0.80 0.89. 4. the pattern of bird distribution in the je'neberang watershed is 30 species of birds with a group distribution pattern, only 5 species with uniform distribution patterns, and 4 species that cannot be analyzed. international journal of applied biology, 6(1), 2022 24 references bibby, c., d.b. neil and h. david. 2004. bird census techniques. book. the cambridge university press. uk. 255 p. bpdas jeneberang walanae, 2010, review of the characteristics of the jeneberang watershed in 2010, center for management of the jeneberang walanae watershed, makassar. b. alan. 2002. "waders of shores, wetlands and grasslands". american avocet.u.s. fish and wildlife service division of migratory bird management. b. gates, d. ewert, d. granfors, b. russel, b. potter, m. scieldcastle., g. soulliere. 2007. “shorebird habitat conservation strategy”. u.s. fish and wildlife service, fort snelling, mn. 101pp. fikriyanti, m., wulandari, s., fauzai, i., & rahmat, a. 2018. diversity of bird species in various communities on sangiang island, banten province. biodjati, 3(2), 157165. karyadi baskoro. 2008. avifauna semarang raya "atlas of bird biodiversity in the semarang area". haliaster biology nature lover, diponegoro university. uswanda, w. effect of plant composition on bird population in batang gadis national park, north sumatra. journal of forest research and nature conservation, vol.7(2) : 193213. junaidi e, sagala ep, joko. 2009. population abundance and distribution pattern of mussels (corbula sp) in the borang river, banyuasin regency. journal of scientific research. mackinnon, j., k. philips, and b. van balen. 2010. birds in sumatra, java, bali, and kalimantan. lipi-indonesian bird. bogor. odum, e.p. 1971. fundamentals of ecology. third edition. philadelphia. w. b. sounders co. pitcher, t.j., magurran, a.e., & winfield, i.j. 1982. fish in larger shoals finf food faster. bahvioral ecology and sociobiology, 10(2), 149-151. putri ayu jannatul., and aunurohim. 2015. distribution patterns of coastal birds in wonorejo, surabaya as an important bird area (iba). its journal of science and arts vol.4.no.1.,(2015) 2337-3520. rusmendro, h. 2009. comparison of bird diversity in the morning and evening in four habitat types in the pangandaran area, west java. vitalis vis. 1(2) :8-16. rohiyan m, setiawan a, rustiati e.l. 2013. diversity of bird species in pine forest and muarasipongi mixed forest, mandailing natal regency, north sumatra. journal of sylva lestari, 2(2) (89-98). international journal of applied biology, 6(1), 2022 25 simanjuntak, e j., b. nurdjali, and s. siahaan. 2013. diurnal bird species diversity in ptpn xiii palm oil plantation, amboyo inti village, ngabang district, landak regency. sustainable forest journal. 1(13):317-326. syarifuddin, d. 2011. diversity of bird species in several habitat types at tambling wildlife nature conservation (twnc), bukit barisan national park, south lampung. essay. department of forestry and ecotourism, bogor agricultural university. bogor. yanto santoso, eko prasito ramadhan, dede aulia rahman. 2008. study of mammal diversity in several tioe habitats at pondok ambung research station tanjung puting national park, central kalimantan. conservation media vol.13, no.3 1-7. yoga putra aliyani. 2018. “bird community in the sermo kulon progo watershed and its conservation status”. journal of bioma, vol.3 no.2. wisnubudi, g. 2009. use of vegetation strata by birds in the tourism area of mount halimun-salak national park. vis vitalis vol.2, september 2009. issn 1978-9513. 1(1)9-13 !!!!!!!!!!!!!!!!!!contact&:&ayub&wirabuana&&&&&&&awiputra22@gmail.com! ©!international!journal!of!applied!biology! 9! abstract& algae!spyrogyra(hyalina!is!one!species!of!fresh!water!algae!that!is!very!easy!to! find!in!indonesia.!this!species!of!algae!known!to!have!a!lot!of!potential,!which! until!now!unknown.!one!potential!that!tried!to!explore!in!this!research!is!the! ability!of!these!algae!for!decreasing! the! level!of!salt!water!salinity! into!fresh! water.! this! research! is! an! experiment! with! a! completely! randomized! design! (crd),!three!treatments!and!three!replications.!testing!is!done!by!performing! algae!arbsorbtion!using!three!forms!(powder,!granule,!and!paper)!with!different! biomass!(1!g,!2!g,!and!4gr).!preparations!then!coupled!into!a!transparent!pipe.! the! results! showed! the! real! effect! of! the! decreased! levels! of! salinity! brine! through!the!circuit.!filter!with!the!powder!forms!with!4gr!biomass!has!the!highest! salinity!level!reduction!of!15%o,!!then!the!dosage!form!of!granules!with!biomass! 4gr!with!decreased!5.23%o!and!the!third!dosage!form!of!paper!with!1!gram!of! biomass!with!a!decrease!of!4.23%o.forms!with!other!biomass!have!a!decrease!in! the!salinity!level!of!not!more!than!4%o.!preparations!with!powder!form!and!4gr! biomass!can!be!used!to!reduce!the!salinity!level!in!the!salt!water.& issn!:!2580v2410! eissn!:!2580v2119! ! ! biofiltration&efficiency&of&algae&spirogyra)hyalina&to&reduce& salinity&of&sea&water&& ! apriliya&maipa1&,&siti&mushlihah2,&ayu&wulandari1&,&nur&qalbi1&,&ayub&wirabuana1&and&ulfah& ervita3& ! 1!department!of!biology,!faculty!of!mathematics!and!natural!sciences,!hasanuddin! university,!makassar,!indonesia! 2!department!of!environmental!engineering,!institut!teknologi!sepuluh!nopember,! surabaya,!indonesia! 3!department!of!public!health,!faculty!of!public!health,!hasanuddin!university,!makassar,! indonesia! ! & & & & & & & & & & & & & & introduction& waters!are!the!basic!need!of!human!being.!in!fact,!not!all!countries!have!many!water! resources!that!can!be!consumed.!coastal!areas!and!small!islands!in!the!middle!of!the!seas! only!have!a!few!fresh!water!resources.!thus,!this!condition!can!cause!the!problem!of!water! supply.!but!if!we!try!to!see!from!different!point!of!view,!these!area!have!many!sources!of! water!that!come!from!the!sea,!although!it!can!not!be!directly!consumed!because!the!excessive! in!salinity!(asmara!&!hasanuddin,!2012).!the!various!ways!of!increasing!the!quality!of!water! have! been! applied,! such! as! –! ion! exchange,! absorption,! membrane! technology,! reverse! osmosis,! electrochemistry! process,! and! ! solvent! extraction.! those! methods! can! separate! metal!in!excessive!numbers!relatively,!but!they!are!judged!not!yet!effective!and!uneconomical! because!need!the!high!cost!(mawardi!et(al.,!2014).! &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword! biosorpsion,( salinity,( sea(water,( spirogyra(hyalina.! nicotine,! algae.! ! article&history! received!23!may!2017! accepted!10!july!2017!! ! international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international(journal(of(applied(biology,(1(1),(2017( ! 10! based!on!this!problem,!one!of!the!appropriate!method!for!processing!the!salt!water!that! source!from!the!sea!into!fresh!water!is!can!be!conducted!by!using!local!plant!biodiversity!that! widely!available!in!indonesia.!one!of!the!biodiversity!in!indonesia!is!algae!spirogyra(which! can!be!easily!found!in!this!country!and!also!easily!to!be!found!in!fresh!waters!area!on!the! excessive!numbers((oktaviani,!2014).! algae!spirogyra(has!a!shape!of!irregular!surface!texture!and!consists!of!amino(acid,( amide,!carboxylic,!hydroxyl!and!carbonyl(group!that!are!capable!for!binding!a!wide!range!of! heavy! metals! (singh! et( al.,! 2012),! such! as! the! use! of! green! algae! spirogyra! biomass! as! biosorbent!of!heavy!metals!(like!as!v!kation(cu2+,!zn2+,!pb2+!and!cr6+!in!solution!(gupta!&! rastogi,!2008;!mawardi,!2011;!lee!&!chang,!2011;!mawardi!et(al.,!2014).!kumar!&!oommen! (2012),!found!out!that!dried!spirogyra(biomass!have!many!potentials!as!biosorbent!to!absorb! cadmium!(cd),!mercury!(hg),!lead!(pb),!arsenic!(as)!and!cobalt!(co)!from!liquid!solution!which! are! emphasized! on! the! difference! of! heavy! metals! concentrations! and! contact! time! of! biomass.!furthermore,!algae!spirogyra(can!reduce!organic!waste!of!anguilla(sp!cultivation! (apriadi!et(al.,!2014)!and!also!textile!dye!(khalaf,!2008).!research!on!the!utilization!of!algae! spirogyra(hyalina!as!an!agent!in!the!salt!water!filtration!into!fresh!water!has!a!potential.!this! study!aims!to!determanie!the!potential!of!spyrogyra(hyalina(in!different!form!and!different! biomass!for!decreasing!the!salinity!level!of!water!(salt!water!into!fresh!water).!! & materials&and&methods& algae&culturing& algae! spirogyra( hyalina! took! from! the! hasanuddin! university! lake! (05°13'81,58's'! ;119o48'81,17'e).! algae! spirogyra( hyalina! that! obtained! then! identified! by! manual! identification!of!zarina!et(al.!(2007),!to!make!sure!of!the!species.!algae!spirogyra(hyalina! cultured!in!a!medium!using!a!glass!aquarium!with!artificial!lighting!using!3,000!lux!flouresence! light!(duration!of!lighting!12!hours!of!light!and!12!hours!of!dark).!for!algae!culturing!that!used! growth!medium!(kno3!40!mg/l,!p2o5!30!mg/l,!k2o!30!mg/l,!mgso4!2!mg/l,.!ca(no3).4h2o!2! mg/l!and!micronutrient).(algae!were!cultured!for!15!days!with!the!addition!of!growth!medium! every!5!days!(feddbatch(culture).! ! pretreatment& algae&powder& alga!spirogyra(hyalina!which!have!been!harvested!then!dried!using!an!oven!at!80°c! for!24!hours.!algae!spirogyra(hyalina!then!crushed!using!a!hand!mixer!into!powder!form!of! spirogyra(hyalina!with!particle!sizes!up!to!40!mesh.! ! algae&granules& 50!grams!of!spirogyra(hyalina!powder!are!poured!with!100!ml!water!and!mixed!by! using!hand!mixer!for!10!minutes.!after!that,!algae!were!filtered!by!using!silkvscreening.!algae! which!have!been!filtered!then!poured!into!spons!which!have!been!layered!by!fabric.!algae! which!have!been!on!the!fabric!were!covered!by!wet!fabric!and!pressed!by!using!the!board.! after!getting!dried,!those!algae!were!formed!into!granules.!! ! algae&paper& 50!grams!of!spirogyra(hyalina!powder!are!poured!with!100!ml!water!and!mixed!by! using!hand!mixer!for!10!minutes.!after!that,!algae!were!filtered!by!using!silkvscreening.!algae! which!have!been!filtered!then!poured!into!spons!which!have!been!layered!by!fabric.!algae! international(journal(of(applied(biology,(1(1),(2017( ! 11! which!on!the!fabric!were!covered!by!wet!fabric!and!pressed!by!using!the!board!until!getting! dried,!thus!algae!in!form!of!paper!was!gotten.!algae!which!have!been!in!form!of!paper!were! used!as!biosorbent.! ! data&collection! data!collection!of!this!research!was!conducted!by!doing!direct!observation!to!algae! spirogyra(hyaline(which!are!given!the!specific!treatment.!these!observations!were!made!by! measuring!the!salinity!of!sea!water!before!and!after!poured!into!the!pipe!which!has!been! modified!in!such!a!way.! & analysis&of&data! data!processing!!in!this!research!were!obtained!using!analysis!of!variance!(anova).! methods!of!analysis!of!variance!(anova)!was!used!to!determine!the!effect!of!different!forms! of! algae! spirogyra( hyalina( in! absorbing! salt! water! into! fresh! water! and! comparing! the! effectiveness!of!algae!spirogyra(hyalina(based!on!the!quantity!of!absorbent!! & results&and&discussion&& the!use!of!algae!spirogyra(hyalina(as!biosorbent!for!salt!water!filtration!into!fresh! water! has! considerable! potential.! from! the! results! obtained,! the! salt! water! which! early! salinity! is! 25! ‰,! decreased! after! tested! using! algae! spirogyra( hyalina! biorsorbent.( algae! powder!with!4!g!biomass!show!significant!gains!compared!to!stocks!and!other!biomass.!the! analysis!also!showed!that!most!algae!biomass!which!are!4!grams!have!a!real!effect!in!reducing! the!salinity!brines.!this!shows!that!algae!biomass!as!effective!biosorbent!in!decreasing!the! salinity!of!salt!water!if!in!doing!further!testing!allows!to!obtain!fresh!water.!the!results!of! anova!shows!that!there!is!an!influence!(h1!accepted),!thus!the!test!continued!to!duncan! test.! from!the!results!of!anova!test!which!is!continued!with!duncan!test!with!95%!believed! range,!it!was!known!that!from!three!kinds!of!treatment!which!are!given!to!the!algae!extract! did!not!give!any!real!differences!because!all!of!this!treatment!being!in!the!same!subset.! & && & & & & figure&1.&average&of&salinity&in&different&form&& (a.&powder,&b.&paper&and&c.&granule)& & & 0 10 20 30 1!g 2!g 4!g early final 1!g 2!g 4!g1!g 2!g 4!g algae!biomass early final 0 5 10 15 20 25 30 1!g 2!g 4!g sa lin it y a& b& c& international(journal(of(applied(biology,(1(1),(2017( ! 12! discussion& this! research! was! conducted! by! using! three! masses! variation! and! three! forms! variation.!mass!variation!is!given!in!a!dose!of!1!gram,!2!grams!and!4!grams,!while!algae!forms! that!were!given!are!powder,!!paper!and!granules!and!repeated!3!times!using!salt!water!that! has!a!beginning!salinity!25!‰!.!based!on!the!data!obtained!from!this!treatment!as!in!the!table! above,!the!results!of!anova!analysis!showed!algae!biomass!has!significant!results!or!real! influence!on!the!decrease!of!salinity!(f!<0.05),!whereas!no!significant!algae!form!(f>!0.05).! duncan!test!showed!the!effect!of!form!and!the!number!of!algal!biomass!spirogyra( hyalina( to! the! salinity.! the! algae! powder! average! of! final! salinity! is! 20ppm,! algae! paper! average!salinity!is!21ppm!and!algae!granules!average!is!21.44ppm.!it!showed!that!there!is!no! significant!difference!in!the!differences!of!algae!form!to!salinity!decrased!level.!while!in!the! biomass,!algae!with!biomass!1!g,!2!g,!and!4!g!obtained!22.78,!22.78,!and!16.89!average!of!final! salinity.!duncan!analysis!showed!that!the!real!effect!from!4!grams!biomas!of!algae!in!the! powder!forms!is!the!best!form!for!reducing!salinity.! from!the!results!that!showed!in!figure!1,!the!algae!mass!that!used!in!every!form!had! an!influence!to!decrease!salt!water!salinity!level.!that!mean!higher!biomass!that!used!to! decreasing!salinity! level!will!be!very!effected,!and!that!mean!the!effectiveness!of!salinity! biosorption!can!be!increased.!every!form!that!used!in!this!research,!show!us!the!difference!of! effectiveness!in!decreasing!salinity!level.!the!powder!form!is!the!most!effecive!form!that!can! be!used!as!biosorben.!both!of!this!result!supported!by!oscik!&!cooper!(1992),!that!explain! the!active!sites!number!is!as!large!as!biosorbent!area!and!every!active!sites!can!only!adsorb! one!adsorbat!molecule.!in!condition!where!the!adsorbtion!area!was!saturated!the!biosorbent! can!not!maximalized!number!of!compound!that!adsorb.!!! ! study!about!algae!spirogyra( in!biosorption.!algae!spirogyra!can!be!used! in!water! treatment!(gupta!et(al.,!2006;!sulfahri!&(manuhara.,!2013),!waste!water!treatment!(rajfur!et( al.,!2012;!sulfahri!et(al.,!2016),!for!water!monitoring!(koanga!et(al.,!2008;!sulfahri!et(al.,!2017),! and!can!be!used!for!heavy!metal!waste!that!very!commercil!to!published!(singh!et(al.,!2012;! sulfahri!et(al.,!2017).!all!of!this!study!show!us!that!algae!spirogyra!can!be!used!as!water! treatment.!every!study!said!that!this!kind!of!algae!can!adsorb!any!compound!that!dissolved! in!water,!such!as!pb!and!cu.!we!know!that!salt!water!contain!salt!which!made!from!chemical! reaction!of!na+!and!clv.!the!result!of!this!research!show!us!this!algae!can!reduce!the!salinity! level!of!salt!water!which!can!be!meant!this!algae!can!adsorb!na!and!cl!that!dissolved!in!salt! water.!in!the!future,!this!kind!of!algae!can!be!used!to!reduce!salinity!level!of!salt!water.!this! mean!it!can!be!a!solution!for!lack!of!fresh!water!in!certain!area,!especially!in!the!island!that! fresh!water!source!can!not!easily!to!be!found.! !!!!!! conclusions& the!effectiveness!of!algae!spirogyra(hyalina!as!biosorbent!with!some!variations!in! preparations!to!decrease!the!salinity!level!of!seawater!can!be!seen!from!the!results!of!sea! water!salinity!level!changes!before!and!after!treatment.!! & acknowledgment&& the!authors!gratefully!acknowledge!financial!support!from!the!ministry!of!research,! technology! and! higher! education! of! indonesia! with! project! pkm! (program! kreativitas! mahasiswa)!contract!number!547/b3.1/km/2017.! & international(journal(of(applied(biology,(1(1),(2017( ! 13! to&cite&this&article:&& maipa,!a.,!mushlihah,!s.,!wulandari,!a.,!qolby,!n.,!wirabuana,!a.!&!ervita,!u.!2017.!biofiltration! efficiency!of!algae!spirogyra(hyalina!to!reduce!salinity!of!sea!water.!international(journal(of(applied( biology.!1(1):9v13.& & references& apriadi,!t.,!niken,!tm.,!p.!&!sigid,!h.!2014.!phytoremediation!of!wastewater!of!anguilla!sp.! cultivation!by!using!filamentous!algae!(spirogyra!sp.).(depik,!3(1):46v55.! asmara,!b.!&!hasanuddin,!p.!2012.!training!in!making!desalinisation!of!sea!water!in!to!fresh! water!by!using!the!spider’s!nest!method!for!society!of!olele!village,!kabila!district,! regency!of!bone!bolango.!journal(of((sibermas,!6(2):82v86.!! gupta!v.k.,!rastogi!a.,!saini,!v.k.!&!jain!n.!2006.!biosorption!of!cu(ii)!from!aqueous!solutions! by!spirogyra(species.!j.(colloid(interface(sci.!296:59v63.! gupta!v.k.!&!rostogi,!a.!2008.!biosorption!of!lead!from!aqueous!solutions!by!green!algae! spirogyra!species:!kinetics!and!equilibrium!studies.!j.(hazard.(mater.!152:407–414! khalaf,! m.a.! 2008.! reactive! dye! from! textile! wastewater! by! nonvviable! biomass! of! aspergillus(niger!and!spirogyra!sp.!bioresource(technology.!99:6631–6634.! kumar,!n.!&!oommen,!c.!2012.!removal!of!heavy!metals!by!biosorption!using!fresh!water! alga!spirogyra(hyalina.(journal(of(environmental(biology.!33:27v31.! lee,!y.c.!&!chang,!s.p.!2011.!the!biosorption!of!heavy!metals!from!aqueous!solution!by! spirogyra!and!cladophora(filamentous!macroalgae.!bioresource(technology.!102:5297– 5304.! mawardi.! 2011.! biosorpstion! of! kation! cu! 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hyalina( biomass!in!nonvaerated!culture.!journal(of(applied(phytotechnology(in(environmental( sanitation.!2!:!53v58.!! sulfahri,!amin,!m.,!soemitro,!s.b.!&!saptasari,!m.!2017.!comparison!of!biomass!production! from!algae!spirogyra(hyalina!and!spirogyra(peipingensis.!biofuels.!8(3):359v366.! zarina,! a.,! hasana,! m.u.! &! shameel,! m.! 2007.! diversity! of! the! genus! spirogyra( (zygnemophyceae!shameel)!in!the!northveastern!areas!of!pakistan.!proceedings(of(the( pakistan(academy(of(sciences.(44!(4)!:!225!–!248.!! ! ! ! ! ! ! ! contact : hilda. a. emmanuel-akerele akatahhilda@gmail.com 73 abstract in nigeria, the reliance on sanitary landfills is a common phenomenon in the disposal of waste materials. the aim of the study was to ascertain the physicochemical and microbiological effect landfill has on its surrounding soil and water. four water samples and five soil samples were collected each from ile-epo and legacy dumpsites and the adjoining areas. physicochemical parameters determined were temperature, ph, total dissolved solids (tds), total hardness (th), and electrical conductivity. most of these parameters indicated pollution but were below the world health organization (who) limits for consumption in the water. microbiological analysis was carried out using standard microbiological procedures. the mean bacteria count and fungal count for water and soil samples are 26.41 cfu/ml and 10.00 cfu/ml; and 26.30 and 14.50 (cfu/g) respectively. the antibiotic susceptibility pattern of the bacterial isolates against conventional antibiotics displayed varying degree of susceptibility and resistance; the bacterial contaminants were susceptible to augmentin, gentamycin and chloramphenicol and resistant to pefloxacin, amoxacilin, tarivid, streptomycin, sparfloxacin and ciprofloxacin. the results obtained in this study showed that the leachate generated from the landfill site has a minimal impact on the groundwater and soil quality in the locality. issn : 2580-2410 eissn : 2580-2119 microbial and physico-chemical assessment of soil and water around waste dump sites in lagos hilda. a. emmanuel-akerele1*, favour ihotu peter1 1 department of biological sciences, anchor university lagos. introduction increase in the amount of wastes can be attributed to the massive expansion of the population in the towns and cities due to many people moving from the rural areas to the cities (ali et al., 2016). waste is an unwanted or unusable material, substances or byproducts eliminated or discarded as no longer useful or required after the completion of a process. the wastes generated are from residential, commercial, industrial, institutional, construction, demolition, agricultural and municipal services (salam, 2010). some of the wastes in the waste dump sites include; broken glass, raw steel metal, food residue, human waste, wood, plastic, textile, nylon, and so on, that poses threat to human health. the waste dumps serve as breeding ground for rodents, mosquitoes, flies and certain microorganisms which can cause diseases (ayilara et al., 2020). in most developing countries, open dumping has been the only management option for solid waste disposal. ecological impacts such as land degradation, water and air pollution are related to improper management of solid wastes (khajuria et al., 2008). the occurrence of these dump sites deteriorates the soil quality and decreases vegetation abundance. soils open access international journal of applied biology keyword antibiotics, dumpsite, landfill, microbiological, physicochemical. article history received 16 march 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 74 at disposal sites show high ph, total dissolved solids and heavy metal concentrations, i.e., lead (pb), copper (cu), nickel (ni), zinc (zn). the major sources of heavy metals in landfills are the co-disposed industrial wastes, incinerator ashes, mine wastes and household hazardous substances such as batteries, paints, dyes, inks, etc. (alam and ahmade 2013). this causes adverse effects on human health, animals and the soil’s fertility and quality. therefore, solid wastes affect the physicochemical properties of the soil which contributes to poor vegetation (adetutu et al., 2012). absorption of the content of the polluted soil through the root system retards plant growth and hinders the normal metabolism of the plant (salam, 2010). the presence of this waste on the soil also aids in the colonization by fungi and bacteria carrying out the degradation and transformation of biodegradable materials in the waste (ayilara et al., 2020). their metabolic activity of detoxifying materials from complex organic molecules into simpler less toxic molecules is attributed to their high growth rate, metabolism and collective ability to degrade a wide variety of naturally occurring organic materials (adetutu et al., 2012). dumped solid wastes produce leachate; a liquid that drains or leaches from a landfill as a result of water present in the landfill or rainfall, which contains variety of chemicals like detergents, inorganic chemicals, complex organic chemicals and metals (arukwe et al., 2012). during infiltration of water by rainfall, leachate leaves the dumping ground laterally or vertically and finds its way into the ground water or nearby surface water thereby causing contamination. dumped solid wastes release its initial interstitial water gradually and some of its decomposition by-products get into the water moving through the waste deposit (sulam, 2010). leachates percolating into the groundwater is a mixture of highly complex contaminants such as potentially toxic metals (e.g. lead, mercury, cadmium, chromium etc.); persistent organic pollutants (pops) (e.g. dioxins, furans, polycyclic aromatic hydrocarbons (pahs), polybrominated diphenyl ethers (pbdes) etc); inorganic compounds (e.g., ammonium, sulphates, chlorides) as well as bacterial contamination – total coliform and faecal coliform (guerrero et al., 2013; kanmani & gandhimathi, 2013 and oyeku and eludoyin, 2010). many communities in lagos, nigeria depend on groundwater supply for domestic purposes. these dumpsites poses a major threat to groundwater resources receiving a mixture of municipal, commercial and mixed industrial wastes (adewole, 2009). the presence and potential exposures of the community to groundwater contaminants may contribute to the deterioration of human health, from simple poisoning to cancer, heart diseases and tetratogenic abnormalities (su, 2008). some of the wastes in the dump will rot and, in the process, it will smell or generate methane gas which contributes to greenhouse effect and pollute the air. incinerating the wastes also is not advisable because plastics tend to release toxic substances, such as dioxins, when they are burnt. this would pollute the air and contribute to acid rain. the short-term effect of the air pollution due to presence of waste dumps includes; congenital anomalies, asthma and respiratory infection (alam & ahmade, 2013). spore producing microbes around the dump sites can release their spores that will be inhaled by those living around the dumps or those waste picking within the open dump sites and this can pose serious health risk (salam, 2010). in lagos nigeria, there is paucity of information on the types of microorganisms associated with communities situated close to dump sites. there is therefore need to isolate, characterize and identify the types of bacteria and fungi associated with areas situated close to waste dump sites. as wastes disposed in the dumps alter the properties of international journal of applied biology, 5(1), 2021 75 the soil, air and water which affects the plants, animals and humans around it. organic waste may also act as an important breeding site for disease causing vermin including flies, insects and rodents, which are vectors of diseases such as cholera, diarrhea, dysentery and typhoid fever (fobil et al., 2008). this study aims to investigate the microbial and physicochemical characteristics of soil and water around waste dumpsite and how it affects human health. materials and methods sample area samples were collected from two waste dump sites, ile-epo landfill and legacy waste dump. ile-epo landfill, a 3.0 hectares land of 6o36’29”n latitude and 3o17’35”e longitude is located at ile epo busstop along abule egba agbado road, lagos state. it is a large landfill that consists of various kinds of wastes. scavengers are found in this landfill, people live in this landfill and a market where various food items are sold lies side by side with this landfill. this creates a huge concern on the health risks associated with the close proximity of people and food items to this landfill. legacy waste dump is located at legacy road, ipaja, ayobo, lagos state. it is not as large as the ile epo landfill and consists majorly of municipal wastes from homes, shops, etc. it has geographical coordinates of 6° 36' 0" north, 3° 14' 0" east. sample collection water and soil samples were collected from both landfills. five soil samples were collected, the surface debris was removed and subsurface soil dug to a depth of 5 cm into sterile duplicate sampling bottles and labelled. four water samples were collected from taps from houses around the dump site in sterile bottles for analysis. samples were transported to and analysed in the laboratory, soil samples were spread in petri dishes and air dried, the dried soil was grinded and passed through an aluminium sieve with 2mm wire mesh. the sample was stored prior to analysis. physicochemical analysis ph : hanna pocket sized ph meter (hi77700p) (hanna instruments, usa) was used to analyze the ph. the ph electrode of the meter was first calibrated with standard buffer solutions with known ph values. soil was diluted in distilled water and stirred. the electrode was rinsed using distilled water, dried off with clean tissue, placed in the sample solution and the value was recorded. to check ph of water sample, the meter is calibrated using standard buffer, rinsed with distilled water and dried off. the water sample was measured and the electrode inserted for reading and the value was recorded (arukwe et al., 2012). total dissolved solids (tds)/ electrical conductivity (ec)/ temperature: the total dissolved solids, electrical conductivity and temperature of the water sample was measured using the bench top conductivity meter (bante510) (bante instruments, china) and result was recorded. hardness of water: 50 ml of edta (ethylenediaminetetraacetic acid) was prepared into a burette, 10 ml of the test water is pipetted into a conical flask and 5 ml of ammonia based ph 10 buffer is added to it. 2 drops of indicator eriochromschwartz-t was added and the color turned red. edta was transferred through the burette into the flask until it turns sky blue. the end point was recorded and the titration was repeated (anthony, 2012). international journal of applied biology, 5(1), 2021 76 total solids: a clean evaporating dish was used and weighed as weight 1. the sample was poured into the dish and content evaporated using a steam bath. the dish was then placed in the oven at 120oc for 1 hour. the dish was allowed to cool and weighed as weight 2. the two weights were subtracted and result recorded (venkataramaiah & krishnaiah, 2014). microbiological analysis cultivation and enumeration of bacteria and fungi each sample of 1 g freshly air-dried fine soil thoroughly shaken in 9 ml distilled water. an aliquot (1.0 ml) was transferred into the next test tube and diluted serially in onetenth stepwise to 10-10 dilution. from the dilutions of each soil sample, 1 ml was transferred into the petri dish and media; potato dextrose agar (pda) (himedia laboratories, vadhani, india), nutrient agar (neogen heywood, united kingdom), eosin methylene blue (himedia laboratories, vadhani, india), and macconkey agar (axiom medical limited, united kingdom)) using pour plate method. 10 ml of streptomycin was added to 1000 ml of pda media, to prevent the growth of bacteria. the poured plate was gently swirled and the agar left to gel. all inoculated plates were inverted and incubated at 37oc for 24 hours except pda plates that was inoculated at 28oc for 3-7 days. plates were examined for growth and colonies were counted and recorded (cheesbrough, 2006). for the water sample, 1ml of each water sample was diluted in 9 ml distilled water. an aliquot (1.0 ml) was transferred into the next test tube and diluted serially in o ne-tenth stepwise to 10-10 dilution. 1 ml of aliquot is transferred using pour plate method and inoculated using the same media and at the same temperature as was used for soil samples. the preparation of media and cultivation of bacteria and fungi was carried out aseptically. all isolates were characterized and identified according to standard microbiological procedures (cheesbrough, 2006). characterization and identification of fungi isolates pure cultures of fungi were obtained by sub culturing discrete colonies onto freshly prepared potato dextrose agar plates and inoculated at room temperature (28± 2oc). lactophenol cotton blue stain was used in the identification of fungi isolate. a drop of lactophenol solution was placed onto a clean slide. the wire loop was sterilised using bunsen burner with blue fame. using the wire loop a small amount of the fungal culture was removed from the edge (younger colonies). the fungal culture was spread gently on the slide using the wire loop in order to tease out the fungal structures, the coverslip was gently placed on the slide for examination under the microscope. the fungal elemental characteristic was detected, examined and recorded. the identification of fungal isolates was done by comparing the result of their cultural and morphological characteristics with those of known taxa. antibiotics susceptibility testing the antibiotics susceptibility test of the isolates was carried out using the kirbybauer disk diffusion technique according to the methods recommended by clinical laboratory and standards institute (clsi, 2018). discrete colonies of the isolates were inoculated into 5ml of normal saline standardized with 0.5 mcfarland standard suspensions. sterile cotton wool swab was used for the inoculation of the bacterial suspension to freshly prepared mueller-hinton agar plates prepared according to manufacturer's instructions (clsi, 2018). the antibiotic sensitivity discs were aseptically and spaciously placed (20mm international journal of applied biology, 5(1), 2021 77 away from each other) on the inoculated mueller-hinton agar plates. the antibiotic discs used were: sxt; septrin (30μg), r; rocephin (25μg), am; amoxacillin (36μg); cn; gentamycin (10μg), pef; pefloxacin (10μg), apx; ampiclox (30μg), s; streptomycin (30μg), e; erythromycin (10μg) for gram negative isolates. while sxt; septrin (30μg), ch; chloranphenicol (30μg), sp; sparfloxacin (10μg), cpx; ciprofloxacin (30μg), am; amoxacillin (30μg); au; augmentin (10μg), pef; pefloxacin (30μg), ofx; tarivid (10μg) for gram positive isolates. after incubation, the test plates were examined for confluent growth and zone of inhibition. the diameter of each zone of inhibition was measured in millimetre (mm) using a ruler on the underside of the plate. the interpretation of the measurement as sensitive, intermediate and resistant were made according to clsi manual (clsi, 2018). results table 1: the physicochemical results obtained for soil and water samples location samples ph electrical conductivity tempera ture total dissolv ed solids total solids hardness of water ile-epo water sample 1 6.1 456 28.5 228 310 93.6 water sample 2 6.5 457 28.1 230 312 93.9 soil sample 1 8.1 legacy water sample 1 6.1 43.7 25.4 22.0 158 31.5 water sample 2 6.8 12.7 25.8 64.7 123 25.6 soil sample 1 7.9 240 who standard 6.5 8.5 1000µs/cm <32oc 1000 mg/l 1000mg /l ns ns – not specified effect of wastes on the physico-chemical properties of water and soil the effect of the waste on the physic-chemical parameter of water and soil in the locations of study was determined by comparing the values of physicochemical parameters obtained with the standard limit. the result is presented in table 2. table 2: comparison of values of physico-chemical properties obtained with standard limit sample n mean who limit water ph 4 6.38± 0.34 6.58.5 electrical conductivity 4 242.35±247.60 1000 temperature 4 26.95±1.58 < 32 total dissolved 4 136.18±108.60 1000 international journal of applied biology, 5(1), 2021 78 solids hardness 4 61.15±37.72 150(nsdwq) total solids 4 225.750±99.47 1000 dissolved oxygen 4 4.61±1.73 7.5 soil ph 5 8.00±0.22 6.5 8.5 %toc 5 11.25±1.26 table 2 shows that for the water around the waste dump site, mean ph value obtained is 6.38±034 which when compared to the who limit is of between 6.5 to 8.5 shows that the ph is slightly acidic. other physicochemical parameters of the water around the waste dump site are the electrical conductivity (242.35±247.60), temperature (26.95±1.58), total dissolved solid (136.18±108.60), hardness (61.15±37.72), total solids (225.750±99.47) and dissolved oxygen (4.61±1.73) were also less than the recommended values by who/nsdwq (national standards for drinking water quality). for the soil sample, the mean ph is 8.00. the mean percentage of toc (total organic carbon) found in the soil was 11.25%. table 3: total viable count, total coliform, total faecal coliform and total fungal count obtained. location samples dilution factor total viable count total coliform total fecal coliform total fungi count water cfu/ml soil cfu/g ile-epo water sample 1 10-7 290 200 106 25 water sample 2 10-7 300 295 200 11 soil sample 1 10-8 250 300 240 30 soil sample 2 10-8 300 300 239 50 soil sample 3 10-7 290 295 225 46 legacy water sample 1 10-7 290 290 250 12 water sample 2 10-5 300 298 225 20 soil sample 1 10-6 290 300 240 30 soil sample 2 10-8 300 300 250 28 international journal of applied biology, 5(1), 2021 79 table 4: mean bacterial and fungal counts in water and soil samples around waste dump site sample mean bacteria count(108) mean fungi count (108) water 26.41± 5.68 (cfu/ml) 10.00 ± 10.60 (cfu/ml) soil 26.30±7.79 (cfu/g) 14.50±13.61 (cfu/g) total 26.35±6.74 12.57±12.18 table 5: difference in the microbial population of water and soil sample organism sample mean(108) mean diff(108) confidence interval lb(108) ub(108) bacteria water 26.41± 5.68 0.11 -6.87 7.09 soil 26.30±7.79 fungi water 10.00 ± 10.60 -4.50 -19.14 10.14 soil 14.50±13.61 table 5 shows that difference in the bacterial count for water (26.41± 5.68) and soil (26.30±7.79) is 0.11. the confidence interval shows that the lower bound for the bacterial count between the water and soil sample is -6.87, while the upper bound is 7.09. the mean difference values lies between a negative and a positive value, indicating that the difference in bacteria population of the water and soil samples around waste dump site is not significant (negligible). for the fungal population in water and soil sample around the waste dump site, it also shows that the difference in fungal counts between water (10.00 ± 10.60) and soil (14.50±13.61) sample is 4.50. the confidence interval shows that the lower bound for the fungal count between the water and soil sample is -19.14, while the upper bound is 10.14, indicating the mean difference in fungi count between the water and soil sample found around the waste dump site is also not significant. table 6: antibiotics susceptibility test antibiotics organism au cn pef am ofx s sxt ch sp cpx escherichia coli. s s r r r r r s s r klebsiella pneumoniae s s r r r r s s r s salmonella enterica s s r s r r r s s r salmonella paratyphi a s s r s r r r s r r pseudomonas aeruginosa s s r r r r s s r s enterobacter aerogenes s s r r r r s s s r key: r – resistant, s – sensitive, au –augmentin, cn –gentamycin, pef-pefloxacin, am amoxacilin, ofxtarivid, s-streptomycin, sxtseptrin, chchlorophenicol, spsparfloxacin, cpx-ciprofloxacin international journal of applied biology, 5(1), 2021 80 discussion from the analysis carried out on the water samples around the waste dump sites, ph had mean value 6.04 – 6.72, slightly acidic and below who standard (6.5 – 8.5). a similar result was obtained by anthony, (2012), the ph ranged from 4.63 – 7.43. the ph of water is very important because changes in ph values may affect the toxicity of microbial poisons in the water (su, 2008). this slight acidity of the water examined in this study, poses health risk to consumers who use the water for cooking, washing, drinking, bathing and other domestic purposes. the ph value for soil sample was 8.00 which is within the limit set by who. the values for electrical conductivity (ec) and total dissolved solids (tds) were below the permissible limit by who standard, arukwe et al., (2012) observed similar results. ec is a function of magnesium, calcium, sodium and sulphates in water, the conductivity level of the samples reveals that there are moderate dissolved salts in the water, and it is within limits approved for safe drinking water. tds comprises of inorganic salts and small amounts of organic matter that are dissolved in water, it indicates presence of low impurities. results obtained by ali et al., (2016) agrees with the values obtained in this study. the temperature of the water analysed was within the range of 25.37-28.53oc, it corresponds with the temperature of water in the storage tanks 26.0 to 27.6oc, observed in all the sampling points that lie within the range of < 32oc for safe drinking water by venkataramaiah & krishnaiah (2014). the temperature range observed in this work will discourage rate of chemical and biochemical reactions, solubility of gases in the water which could impact negatively on the taste and odour of the water at higher temperatures. based on the who standards, water samples are unacceptable for human consumption when it has high bacterial loads. according to us epa standards, water samples in which coliforms are detected should be considered unacceptable for drinking as they are regarded as the principal indicators of water pollution. the organisms isolated in this study include; e.coli, p. aeruginosa, s. paratyphi a, s. enterica, enterobacter aerogenes, klebsiella pneumonia, rhizopus stolonifera, penicillium sp. and aspergillus niger. the results in table 2 revealed that all the water and soil samples from both areas had very high counts of total and faecal coliforms. oyeku & eludoyin, (2010) isolated enterobacter, pseudomonas, escherichia, salmonella from soil and water samples, this correlates from the results obtained in this work. the presence of faecal coliforms like e. coli and klebsiella sp. indicated pollution by sewage. they are important human pathogens associated with a variety of infectious diseases such as gastroenteritis, typhoid fever, dysentery, urinary tract infection, etc. the high count of these pathogenic bacteria in the water sources could be due to any of the following: improper disposal of sewage and wastewater from domestic activities, discharges from septic tanks and latrines close to some of the bore holes, in-appropriate siting of boreholes very close to dumpsites and extraction of ground water from very shallow aquifers. this is in agreement with the work of kanmani and gandhimathi (2006), which also stated that high bacterial load in borehole water supplies may be due to discharges from septic tanks and waste materials from a nearby dumpsite. adetutu et al., (2012) and su, (2008) in their articles also highlighted the presence of coliforms, faecal coliforms and pathogens (escherichia coli, streptococcus, pseudomonas and salmonella) in large numbers which was attributed to the emanation of these species from some sources such as seepages from septic tanks into household drinking water supply and soil, and unhealthier latrine systems. the presence of these indicator organisms in drinking water sources may provide an indication of water-borne problems which is a direct threat to international journal of applied biology, 5(1), 2021 81 human health and is a matter of serious concern (salam, 2010). results of the water analyses reveals that most of the parameters analysed in the water samples from both areas were not within the acceptable water quality standards and therefore indicate the existence of pollution in these drinking water sources from both study areas. continuous water quality monitoring in and around both dumpsite areas is encouraged. most of the bacteria displayed varying degree of resistance to multiple drugs. escherichia coli were found to be susceptible to chloramphenicol, augmentin, gentamicin and sparfloxacin, but displayed resistance to the other antibiotics tested (pefloxacin, amoxacilin, tarivid, streptomycin, septrin, ciprofloxacin). pseudomonas aeruginosa was susceptible to augmentin, chloramphenicol, gentamicin, septrin and ciprofloxacin but was also resistant to the other antibiotics screened (pefloxacin, amoxacilin, tarivid, streptomycin, sparfloxacin, ciprofloxacin). similarly, salmonella spp. exhibited susceptibility to chloramphenicol, augmentin and gentamycin and amoxicillin and resistance to others. conclusions the results obtained in this study showed that the leachate generated from the landfill site has a minimal impact on the groundwater quality in the locality. the mean ph was observed to be lower than the who standard, which indicates that it to be slightly acidic; hence it needs to be treated for it to be potable. faecal coliforms and fungal population was observed both in the soil and water samples. this indicates that the water and soil are contaminated and the water is unreliable for drinking water supply purposes and therefore puts emphasis on the need to improve on waste management practices and construct properly engineered sanitary landfill sites to curtail the pollution of groundwater and it also encourages the proper treatment of water before usage. landfills should be sited faraway from residential areas. this would help in limiting the impact on health o f humans. sanitary and well-engineered landfills should be constructed, this would help in reducing the impact and influence that it has on soil, groundwater and humans. wastes should be sorted out properly, reused and recycled. if sanitary landfill is properly designed and maintained, it will have less negative impact to man and the environment. references adetutu e.m., ball a.s., weber j., aleer s., dandie c.e. and juhasz a.l. 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(2010), environmental and health impact of solid waste disposal at mangwaneni dumpsite in manzini: swaziland, journal of sustainable development in africa, 12(7): 64-78. su, g.l.s., (2008), “assessing the effect of a dumpsite to groundwater quality in payatas, philippines”. american journal of environmental sciences, 4(2): 276-280. venkataramaiah g. and krishnaiah s. (2014) “characterization of contaminated soil and surface water/ ground water surrounding waste dump sites in bangalore”. international journal of environmental research and development, 4(2): 99-104. https://doi.org/10.3390/su12114456 https://doi.org/10.1007/s13201-012-0072-z 53 abstract the experiments were carried out in 2020 and 2021 rainy seasons to determine the performance of rice as influenced by planting method and weed control treatments. the treatments consists of three planting methods (dibbling broadcasting and transplanting and weed control methods (hoe weeding, pre emergence application of gramazone (200g/l of paraquat/ha), pre and post emergence of gramazone (200g/l of paraquat/ha) and propanil (360g/l of propanil + 200g/l 2-4d) and weedy check). the treatments were laid out in a randomized complete block design (rcbd) and were replicated three times. the results revealed the plots weeded twice and those treated with pre and post emergence application of gramazone and propanil respectively resulted in more number of spikelet per panicle, longer panicle, increase in number of effective tillers per hill, more grains per panicle and grain yield. the effect of planting methods on the rice growth revealed that rice sown under transplanting methods gave more number of effective tiler per hill, longer panicle, more grain per panicle and increases in grain yield compared to the others methods while broadcasting resulted in the least value of the parameters than others methods. therefore, it can be concluded that rice farmers in the sudan savanna zone of nigeria can adopt pre and post emergence application of gramazone and propanil with transplanting method since the combination of these treatments gave better weed control, growth and yield of paddy rice. issn : 2580-2410 eissn : 2580-2119 effect of weed control treatments and planting method on the yield and yield parameters of rice in sudan savannah of nigeria danmaigoro.o 1*, bilyaminu a. s 1, abduljalal.t 2, umar. m.m 2 1 department of crop science, faculty of agriculture, federal university dutse, jigawa 2 department of agricultural extension federal ministry of agriculture abuja. introduction rice belongs to the genus oryza and the tribe oryzeae of the family gramineae also known as poaceae. rice constitutes major staple foods in many parts of the world and it is a source of income to more than 100 million households around the world (abiwon et al., 2016). globally, rice ranks third after wheat and maize in terms of production (ejebe, 2013). the production of the crop has been affected by a number of factors among which include water, nutrient availability and weed management especially in the direct seeded cultivation and this has affected in the yield. weeds also serve as alternate hosts to pests and pathogens which usually affect crops in the field and during storage (akobundu, 2013). previous studies have shown that weed occurrence is a constant component of the ecosystem in comparison to the epidemic nature of other pests which makes farmers open access international journal of applied biology keyword herbicide; length of panicle; number of grain per panicle; grain yield article history received december 5, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 54 unaware of the significant losses they incur from weed infestation (johnson, 1999). however, of all the constraints limiting the production of this crop, weeds, appear to have the most deleterious effect causing between 75 and 100% reduction in potential paddy rice yield (akobundu, 2011; imeakparia, 2011; lavabre, 2011). yield reduction due to weed competition is greater in direct-seeded than in transplanted rice. inadequate land preparation, use of short-stature early maturing cultivars and increased fertilizer use have resulted in severe weed problems in direct – seeded rice. the limited data available indicate that production losses can reach 30-40% for fields that are poorly weeded (anon, 1988). with direct seeding, the germination of rice seeds and the emergence of weeds take place almost at the same time. therefore, weed control at the early stages of the crop growth is important (street and lanham, 2016). hoe removal of weeds still remains the most practical method of weed control in many developing countries. hoe weeding is quite effective if employed at the right time. the number of hoe weeding depends on crop and weed growths and the critical period of crop-weed competition. however, the efficiency of hoe weeding as reported by gianessi, (2013) is often compromised by the continued wet conditions characteristic of the beginning of the rainy season. hoe weeding under wet conditions often causes weed to re‑root and re‑establish, necessitating several rounds of weeding to keep the crop weed‑free and avert yield losses. according to ogwuike et al. (2014) and datta et al. (2017) it is tedious, inefficient, time consuming and associated with high labour demands. in addition, labour for manual weeding is scarce and often too expensive for the average farmer to afford (adigun et al., 2017; daramola et al., 2019). alternatively, herbicides are quite effective and efficient in suppressing weeds in rice if properly used. the underlying strategy behind using herbicides for weed control in rice is to kill or stunt the growth of weedy plants while allowing the rice plants to grow and achieve a competitive height advantage. herbicides reduce drudgery and protect crops from early weed competition according to rodenburg et al. (2011). moreover, herbicides for weed control in rice are often not available to smallholder farmers at the time of need and, when available, farmers lack the requisite knowledge and skill to use herbicides correctly. although herbicide use alleviates the problem of labour for weeding, incorrect use may complicate crop performance (ekeleme, 2009). application characteristics such as herbicide choice, rate and timing, are reported to frequently deviate from the recommendations (rodenburg and johnson, 2009). in a related study by ismaila1 et al. (2012) reported that increasing weed population is caused by wrong use of herbicides, lack of skill in weed identification and correct matching of herbicides with weeds. poor knowledge on how to use chemicals, inability to read and understand instructions and lack of technical skills to operate the chemical spray machines are the major problems associated with the use chemical by farmers. danmaigoro et al., 2018 have reported reduction in weed dry matter production due to application of herbicides in rice in rice crop production, the planting methods have an impact on the growth and yield besides cost of cultivation and labor requirement. awan, et al. (2007) reported that direct seeding has good stand establishment, higher tillering and higher grain yield. other advantages are stable growth, reduced transplanting shock but there is weed problem in direct seeding compared to transplanted rice. in most developing countries, rice transplantation is usually performed by hired expensive labor, which is not specialized to maintain the required plant population to achieve higher productivity (mann et al., 2007). to overcome this problem, direct seeding has been considered as only viable alternatives in international journal of applied biology, 6(1), 2022 55 rescuing farmers (aslam et al., 2008). this technique reduces labour needs by more than 25% in term of working hours. the input requirements and the investment in direct seeded rice are much lower than in transplanted rice (sunil et al., 2002). direct seeded rice, if managed properly, can yield as high as transplanted rice ( ali, et al., 2007). furthermore, a report by rani and jayakiran (2010) transplanting provides crops less competition for growth resources such as sunlight, moisture, and nutrients; and enables easy crop management like weeding, and herbicides as well as pesticide applications, ensures uniform plant stands and gives the rice crop a head start over emerging weeds. ismaila et al. (2012) reported that transplanted rice reduces weed population since the crop has an additional advantage due to its age as the result of covering the ground early. transplanting rice seedlings, gives the crop a 14 to 21 days growth advantage over the weeds, and allows continuous flooding at greater depths. during transplanting, weed seedlings are also trampled and incorporated into the soil. the major planting methods commonly adopted in nigeria are drilling, broadcasting and transplanting. the objectives of this study were to determine to evaluate the effect of different weed control treatments on the yield attributes of rice and to determine the best planting method that promote better yield of rice. materials and methods the experiment was carried out during the 2020 and 2021 raining seasons in the faculty of agriculture research farm, federal university dutse (lat 11°46’39” n, long 9°20’3” e). the experiment consists of three different planting methods (dibbling transplanting and broadcasting ) and four different weed control treatments (hoe weeding at 3 and 6 weeks, pre emergence application of gramazone (200g/l of paraquat/ha), pre and post emergence of gramazone (200g/l of paraquat/ha) and propanil (360g/l of propanil + 200g/l 2-4d and weedy check). the treatments were laid out in a randomize complete block design (rcbd). npk 15:10:10 at the recommended dose in two split was applied. weed control was carried out based on treatments designed for specific plot. harvesting was done when the crop reached physiological maturity. five plant stands were tagged within each plot for growth parameters sampling. the following parameters were taken: number of spikelets per panicle the number of spikelets per panicle was obtained by counting the spikelet on the panicle from the five tagged plants in each net plot at harvest and the average was recorded. number of days to 50% panicle initiation this was taken by making daily observation from the date of sowing/planting to the time of heading and recording the number of days when half the plants in the plot produced panicle from date of sowing. number of effective tillers this refers to those tillers that produced panicle with filled grains. the total number of effective tillers were determined from five tagged plants from each plot at harvest by counting the number of effective tillers per stand and the average was recorded. international journal of applied biology, 6(1), 2022 56 length of panicle per plant (cm) the panicle length was taken from five tagged plants per plot at harvest by measuring the panicle from the base of the panicle to its tip using a ruler and the average was recorded. number of grains per panicle the number of grains per panicle were counted from the tagged plants after harvest and the average was used to give number of grains per panicle. 1000 grains weight (g) after threshing and winnowing in the air, 1000 grains were counted and weighed in a laboratory using electric sensitive weighing balance grain yield harvested paddy from each net plot was threshed after sun drying, winnowed and grain yield obtained from net plot was weighed on a mettle balance and converted to per ha basis expressed in kg/ha. data analysis data collected was subjected to analysis of variance anova (genstat 17th edition). the means obtained were compared and separated using duncan’s multiple range test (dmrt) (duncan, 1995). results number of spikelet per panicle the effect of weed control and planting method on the number of spikelet per panicle is shown in table 1. the result shows that there was significant effect (p<0.001) of weed control methods on the number of spikelet per panicle. hoe weeding at 3 and 6wap gave the highest number of spikelet per panicle and statistically at par with pre and post emergence application of gramazone + propanil, while weedy check gave lowest number of spikelet per panicle. significant effect (p<0.001) of planting methods on the number of spikelet per panicle was observed. transplanting resulted in the highest number of spikelet per panicle though statistically at par with dibbling method while broadcasting gave the lowest. number of effective tillers per stand the effect of weed control and planting method on the number of effective tillers is shown in table 1. the result reveals that there was significant effect (p<0.001) weed control methods on the number of effective tillers. hoe weeding at 3 and 6wap gave the highest number of effective tillers per stand though statistically at par with pre and post emergence application of gramazone + propanil, while weedy check gave lowest number of effective tillers per stand. significant effect (p<0.001) was also observed among the planting methods on the number of effective tillers. transplanting resulted in the highest number of effective tillers per stand while broadcasting gave the lowest. international journal of applied biology, 6(1), 2022 57 number of grains per panicle the effect of weed control and planting method on the number of grains per panicle is shown in table 1. there was significant effect (p<0.001) of weed control methods on number of grains per panicle. pre and post emergence application of gramazole + propanil gave the highest number of grains per panicle though statistically at par with hoe weeding at 3 and 6 wap, while pre emergence application of gramazole gave the lowest and statistically at par with weedy check. significant effect (p<0.001) of planting methods on the number of grains per panicle was also observed. dibbling method gave the highest number of grains per panicle though statistically at par while broadcasting gave the lowest length of panicle per plant the effect of weed control and planting method on the length of panicle per plant is shown in table 1. the result indicates that there was significant effect (p<0.001) of weed control methods on the length of panicle per plant. pre and post emergence application of gramazole + propanil gave the tallest length of panicle per plant but statistically at par with hoe weeding at 3 and 6 wap, while weedy check gave the shortest length of panicle per plant. significant effect (p<0.001) of planting methods on the length of panicle per plant was also observed. transplanting method gave in the tallest length of panicle per plant though statistically at par with dibbling while broadcasting gave the shortest. 1000 grain weight (g) the effect of weed control and planting method on 1000 grain weight is shown in table 3. there was significant effect (p<0.001) of the weed control methods on 1000 grain weight. hoe weeding at 3 and 6wap gave the heavier 1000 grain weight but statistically at par with pre and post emergence application of gramazole + propanil, while weedy check gave the lowest. significant effect (p<0.001) of planting methods on 1000 grain weight was also observed. transplanting method gave the heavier grain weight but statistically at par with dibbling while broadcasting gave the lowest. the interaction between weed control methods and planting methods shown in table 2 was significant (p<0.01). combination of hoe weeding at 3 and 6wap and transplanting gave the heaviest grain weight, while weedy check and broadcasting gave the lowest. days to 50% panicle initiation the effect of weed control and planting methods on days to 50% panicle initiation is shown in table 3. there was significant effect (p<0.001) of weed control methods days to 50% panicle initiation. hoe weeding at 3 and 6 wap resulted in early to reach 50% panicle initiation followed by pre and post emergence application of gramazone + propanil while weedy check took longer days to attain 50% panicle. significant effect (p<0.001) was also observed on planting methods. on days to 50% panicle initiation, transplanting treated plot reached 50% panicle initiation early while broadcasting took longer days. the interaction between weed control methods and planting methods shown in table 1 was significant (p<0.001). hoe weeding at 3 and 6 wap and transplanting resulted in early 50% panicle initiation though statistically at par with pre and post emergence (gramazone + propanil) and transplanting, while weedy check and broadcasting resulted in longer days to 50% panicle.. international journal of applied biology, 6(1), 2022 58 grain yield (kg/ha) the effect of weed control and planting method on grain yield is shown in table 3. the result shows that there was significant effect (p<0.001) of weed control methods on grain yield. pre and post emergence application of gramazole + propanil gave the highest grain yield though statistically at par with hoe weeding at 3 and 6 wap, while weedy check gave the lowest. significant effect (p<0.001) of planting methods on grain yield was also observed. transplanting method gave the highest grain yield which was statistically at par with dibbling while broadcasting gave the lowest yield. the interaction between weed control methods and planting methods shown in table 4 was significant (p<0.001) in 2020. pre and post emergence application of gramazole + propanil and transplanting gave the highest number of grains per panicle while weedy check and broadcasting gave the lowest discussion the present study shown that weeds were adequately and effectively controlled by all the weed methods adopted. the results revealed the plots weeded twice and those treated with pre and post emergence application of gramazone and propanil respectively resulted in taller plant height, large leaf area, higher number of effective tillers and grain yield of rice than the weedy check and also application of gramazone as pre-emergence alone. this is due to the fact that effective weed control was observed by the application of pre and post emergence herbicides which provides large spectrum and long-time effect that suppressed weed infestation in the treated plots and reduced weeds competition for available resources. ishaya et al. 2012 and danmaigoro et al., 2018 have reported reduction in weed dry matter production due to application of herbicides in rice. the failure of gramozone applied as pre emergence to give broad spectrum weed control compared to its followed-up application with others herbicides is due to narrow of its weed control. the performance of rice crop in plots weeded twice can be attributed to effective weed control that minimized competition for growth resource between crops and weed specifically during the critical period of weed interference in rice resulting to greater efficiency in utilizing nutrients. this is in line with earlier report by pamploma et al., (1990) who reported yield increase of maize because the crop was free from weeds during its critical period of weed interference. the result of this trials indicated that weeding of rice field should not be delayed beyond 2wat to avoid yield losses due to weed interference with crop. significantly longer number of days to 50% heading observed in the weedy check compared with weed control treated plots may just be the reason for disparity in the yield because it took longer days for the crop in the weedy check to head. this result is similar to the finding of adeosun (2008) who reported that weed of rice field should not be delayed beyond 3 weeks of crop establishment to avoid weed interference with crop that might prolong days to heading. the yield contributing parameters such as number of tillers per pant, number of spikes were significantly reduced in weedy check probably due to severe weed competition for light, nutrient and moisture during major part of the crop vegetative growth period of the crop life cycle. the effect of planting methods on the rice growth revealed that rice sown under transplanting methods gave higher yield than others methods. this is due to better weed competitive ability of transplanted rice than direct seeding and broadcasting, initial and better crop establishment and ability to use environmental resources better thus resulting in increases growth performance of crop. the similar finding was reported by olorukooba et al., (2012). 59 table 1: effect of weed control treatments and planting methods on days to 50% panicle heading, number of spikelets per pani cle, number of effective tillers per stand and length of panicle per plant of rice at dutse in 2020 and 2021 rainy seasons days to 50% panicle heading number of spikelets per panicle number of effective tillers per stand length of panicle per plant treatments rate/ha 2020 2021 2020 2021 2020 2021 2020 2021 weed control methods hoe weeding at 3 and 6 wap 71.78d 64.78d 20.11a 17.11a 26.33a 30.33a 24.17a 23.89a gramazone 200g/l paraquat 91.44b 84.44b 13.00b 10.00b 8.89b 11.78b 15.46b 15.18b gramazone + propanil 200g/l paraquat + 360g/l propanil + 200g/l 2-4d 74.89c 67.89c 18.56a 15.56a 25.89a 29.89a 23.93a 24.44a weedy check 124.11a 117.11a 7.67c 4.33c 8.33b 3.67c 12.71c 11.89c se± 0.418 0.418 0.634 0.628 0.812 0.665 0.842 0.414 planting methods dibbling 60kg 90.33c 83.33b 16.17a 13.17a 20.25b 21.67b 19.73b 21.03a transplanting 25kg 84.83b 77.83c 16.83a 13.75a 22.42a 24.58a 22.71a 21.52a broadcasting 80kg 96.50a 89.50a 11.50b 8.33b 9.42c 10.50c 14.76c 14.00b se± 0.362 0.362 0.549 0.544 0.703 0.576 0.730 0.358 interaction weed control*planting method ns ns ns ns ns ns ns ns means followed by the same letter(s) are not significantly different at 5% level of probability using dmrt. table 2: interaction effect of weed control treatments and planting methods on 1000 grain weight of rice in dutse in 2020 and 2021 rainy seasons treatments planting methods 2020 2021 dibbling transplanting broadcasting dibbling transplanting broadcasting rate/ha 60kg 25kg 80kg 60kg 25kg 80kg weed control hoe weeding at 3 and 6 wap 31.07a 32.27a 16.10b 35.27a 36.47a 20.30b gramazone 200g/l paraquat 7.90c 8.20c 8.83c 12.00c 12.40c 12.73c gramazone + propanil 200g/l paraquat + 360g/l propanil + 200g/l 2-4d 26.53a 29.47a 15.67b 30.73a 33.67a 19.87b weedy check 5.40c 4.83c 5.83c 7.30c 9.03c 7.20c se± 2.185 2.160 international journal of applied biology, 6(1), 2022 60 means followed by the same letter(s) are not significantly different at 5% level of probability using dmrt. table 3: effect of weed control treatments and planting methods on number of grains per panicle, 1000 grain weight (g) and grain yield (rate/ha) of rice at dutse in 2020 and 2021 rainy seasons number of grains per panicle 1000 grain weight (g) grain yield (rate/ha) treatments rate/ha 2020 2021 2020 2021 2020 2021 weed control methods hoe weeding at 3 and 6 wap 174.6a 186.7a 26.48a 30.68a 3974a 4083a gramazone 200g/l paraquat 87.3b 99.4b 8.31b 12.38b 1006b 1115b gramazone + propanil 200g/l paraquat + 360g/l propanil + 200g/l 2-4d 175.2a 188.1a 23.89a 28.09a 4026a 4135a weedy check 88.6b 100.7b 5.36b 7.84c 430b 478b se± 4.87 4.69 1.262 1.247 224.2 227.4 planting methods dibbling 60kg 152.0a 165.7a 17.73a 21.32a 2812a 2904a transplanting 25kg 152.0a 163.3a 18.69a 22.89a 3070a 3164a broadcasting 80kg 90.2b 102.2b 11.61b 15.02b 1196b 1291b se± 4.22 4.06 1.093 1.080 194.2 197.0 interaction weed control*planting method ns ns ** ** ** ** means followed by the same letter(s) are not significantly different at 5% level of probability using dmrt. international journal of applied biology, 6(1), 2022 61 table 4: interaction effect of weed control treatments and planting methods on grain yield of rice in dutse in 2020 and 2021 rainy seasons treatments planting methods 2020 2021 dibbling transplanting broadcasting dibbling transplanting broadcasting rate/ha 60kg 25kg 80kg 60kg 25kg 80kg weed control hoe weeding at 3 and 6 wap 4842a 5224a 1858b 4951a 5333a 1967b gramazone 200g/l paraquat 996bcd 1039bcd 983bcd 1105bcd 1148bcd 1092bcd gramazone + propanil 200g/l paraquat + 360g/l propanil + 200g/l 2-4d 4922a 5507a 1650bc 5031a 5616a 1759bc weedy check 489cd 511cd 292d 531cd 559cd 345d se± 388.4 393.9 means followed by the same 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(2011): challenges for weed management in african rice systems in a changing climate. journal of agricultural science 149: 427–435. sunil k., s. n. khajanji, g. k. shrivastava, r. s. tripathi and s. kumar (2002). comparative study of different methods of sowing on morphophysiological traits and economics of medium duration rice (oryza sativa) inceptisols. j. interacademicia 6 (2): 244– 249. ismaila1 u, a. c. wada1, e. daniya2 & a. u. gbanguba (2012) meeting the local rice needs in nigeria through effective weed management sustainable agriculture research; vol. 2, no. 2; 2013 contact : bartholomew saanu adeleke microbade@yahoo.com 190 abstract cassava is a tuber crop mainly cultivated in africa countries. the presence of unwanted microorganisms can complicate the control of the fermentation process, which can lead to the production of objectionable odors in steeping water. microbial and physical analyses during fufu production were performed on the steeping water. bacteria count decreased with an increase in the fermentation time. the highest bacterial count of 6.6 × 103 cfu/ml, while the least count of 2.2 × 103 cfu/ml was recorded, respectively. the bacteria isolated include bacillus subtilis, pseudomonas species, lactobacillus fermentum, proteus mirabilis, and klebsiella sp. the ph value of 3.32 was recorded on day four, while the temperature was constant during the fermentation process. a high value of 45.2 mg/l total solids was recorded on day four, while the least value of 15.0 mg/l was obtained on day one. the effect of fermenting microorganisms under a controlled environment reduces the foul odor usually perceived during fufu production. hence, the survival of these microorganisms at low ph can inhibit the growth of unwanted microorganisms, thus contributing to the acceptability of the cassava product, i.e. fufu. issn : 2580-2410 eissn : 2580-2119 characterization of bacteria isolates from fermented cassava steeping water olalekan blessing balogun 1, bartholomew saanu adeleke 2*, ibukun i. owoseni 1 1 department of biological sciences, joseph ayo babalola university, ikeji-arakeji, nigeria 2 department of biological sciences, olusegun agagu university of science and technology, okitipupa, nigeria introduction cassava (manihot esculenta) is a tuber crop serving as a cheap source of carbohydrates and other nutrients in the diet of the teeming population in africa and asia, which provides energy for about 500 million people (achi & akomas, 2006). bamidele et al. (2015) posited cassava as a supplementary staple food to more than 200 million africans aside from its uses as livestock feeds. nigeria is ranked first and largest producer of cassava but with less export compared to thailand (otekunrin & sawicka, 2019). cassava production and processing are usually concentrated in the hands of numerous smallholder farmers located primarily in the south and central regions of nigeria. cassava tuber consists of 64 87% starch depending on the growth stage or at maturity, but with low protein, fats, vitamins, and minerals (aloys & hui ming, 2006). cassava tubers are composed of starch open access international journal of applied biology keyword cassava; fermentation process; food contamination; steeping water; starter culture article history received 05 october 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 191 depending on the growth stage or at maturity and the starch content compared to other starchy carriers (lindeboom et al., 2004). adequate processing of cassava tubers by traditional means has yielded to a variety of edible products, such as fufu, ‘akpu’, lafun, garri, abacha, and tapioca. fermentation is one of the oldest and most important traditional food processing and preservation techniques. employing this technique enhances the nutrient content and reduces the antinutrient contents in cassava (oyetayo, 2006). in nigeria, cassava has been processed into many fermented and unfermented products in many ways. some of the fermented products include cassava flour (lafun), which is produced by drying and milling fermented cassava tubers, cassava flakes (garri), which is produced by grating, soaking, fermenting, and roasting cassava mash. other products include fermented cassava slurry used to produce “fufu”. the product of interest of this research is fufu, a fermented cassava mash, which comes as a wet mash or a dry powder (adegbehingbe et al., 2019) and is most commonly consumed in the eastern and southern regions of nigeria. fermentation of cassava involves the steeping of cassava roots in water for 3 to 4 days, which softened the root to disintegrate the tissue structures in contact with linamarin which is located in the cell walls by the action of linamarase (adeleke & olaniyi, 2018). this enzyme hydrolyses linamarin to glucose and cyanohydrins and subsequently breaks down to ketone and hcn (aloys & hui-ming, 2006). traditionally, african fermented foods and products, for instance, garri and fufu can be obtained from a series of operational procedures, which include grating, dewatering, fermenting, and roasting and these processes generate waste among which is steeping water, wastewater, and solid waste. however, the focus of this study is on steeping water. about one-third of the cassava tubers harvested in nigeria are utilized domestically for fufu production (essers et al., 1995). it is bulky, easily contaminated by microorganisms, and cannot be stored for long due to its high water content, thus susceptible to easy spoilage. the inefficient method of preservation of wet cassava mash can be linked to the microbial growth that causes undesirable odor, and sometimes total spoilage. nigeria has a large, sustainable, and expanding market for cassava fufu, with a population of over 180 million people (ezedinma et al., 2006). in nigeria, the consumption pattern varies according to ecological zones as garri, a roasted granule is widely accepted in both rural and urban areas. interestingly, it can be consumed without any additives or with a variety of additives, such as sugar, groundnut, fish, meat, and stew (graffham et al., 2019). the presence of unwanted microorganisms complicates the control of the fermentation process, thus leading to the production of objectionable odors in steeping water (omar et al., 2000). therefore, to provide basic information on the microorganism present in the fermenting substrates, there is a need to understand the type of the fermenting microbes in the steeping process to ensure improvement in the quality of cassava and cassava products. therefore, this study was designed to isolate, enumerate, and identify microorganisms present in cassava steeping water and also determine the physical properties of the steeping water. materials and methods sample collection and processing this study was carried out in february 2017. cassava tubers were obtained from a cassava processing site in ikeji-arakeji inside sterile plastic bags and then transported to the international journal of applied biology, 5(2), 2021 192 department of microbiology, joseph ayo babalola university, osun state, nigeria for further processing, such as washing, peeling, cutting, and re-washed with sterile distilled water. fermentation and microbial analysis cassava tubers were fermented for four days inside fermenting vessels containing sterile distilled water at room temperature. microbial analyses were performed daily by pipetting 1 ml of the sample stock solution and then serially diluted up to the appropriate dilutions. one milliliter from the dilutions, 10-2 and 10-4 was pipetted, gently dispensed into petri dishes, and then pour plated using nutrient agar (na), salmonella shigella agar (ssa), mannitol salt agar (msa), macconkey agar (ma) and eosin methylene blue agar (emb). the petri plates were incubated at 37oc for 24 hours to determine bacterial growth and morphological details. pure cultures were obtained by repeated streaking of the bacterial inoculum on the fresh bacteriological media and stored on slants inside the refrigerator for further use. furthermore, the pure culture was subjected to various morphological and biochemical characterization tests, such as catalase, oxidase, and indole, hydrogen sulfide, coagulase, and sugar fermentation, to determine the identity of the bacteria isolates (adeleke et al., 2017). physical properties the physical properties of distilled water were used in the soaking of the cassava were carried out in the laboratory. the physical parameters determined were ph, temperature, total dissolved solids, total suspended solids, and total solid according to the modified method of and afuye & mogaji (2015) and adegbehingbe et al. (2019). statistical analysis data were reported as average of triplicate determinations and analysed using analysis of variance (anova) on spss. duncan's multiple tests at 5% level significance were used to determine the significant differences among the samples. results and discussion total bacteria count the population of bacteria isolated from the cassava steeping water indicated a reduction from day one to the last day of the fermentation process. the bacterial count ranged from 2.2 × 103 cfu/ml to 6.6 x 103 cfu/ml. day one had the highest bacterial count of 6.6 × 103 cfu/ml while the least bacterial count of 2.2 × 103 cfu/ml was recorded on day four (table 1). cassava is usually processed into various products through the fermentation process to increase the shelf life, easy packaging, and transportation for economic purposes (aro, 2008). processing of cassava by submerged state fermentation techniques by traditional methods and beyond specified fermentation time usually produce mash, which contains a foul odor resulting from the uncontrolled fermentation and storage techniques (oyewole & odunfa, 1988). during the process of retting of cassava, a decrease in the total viable count of aerobic mesophiles was observed and this can be linked to the increase in acidity of the fermentation medium (adegbehingbe et al., 2019). also, an increase in acidity of the medium can cause a decrease in the growth of pathogenic-like microorganisms (ojo et al., 2019). international journal of applied biology, 5(2), 2021 193 nine bacterial isolates were identified based on colonial and cellular morphological characterization. the identifiable bacterial isolates include, klebsiella pneumonia, lactobacillus species, bacillus subtilis, proteus mirabilis, pseudomonas spp, pseudomonas aeruginosa and staphylococcus aureus (table 2). table 1: mean population of bacteria from the steeping water days mean cfu/ml x 103 1 66 6.6 2 50 5.0 3 30 3.0 4 22 2.2 key: cfu colony-forming unit international journal of applied biology, 5(2), 2021 194 table 2. characterization and identification of isolates is o la te c o d e colonial morphology staining reactions biochemical tests sugar fermentation c it r a te u ti li z a ti o n p r o b a b le i so la te s s h a p e e le v a ti o n c e ll s h a p e c e ll a rr a n g e m e n t c o lo u r g ra m s ta in in g s p o re s ta in in g c a ta la se t e st in d o le p ro d u c ti o n c o a g u la se t e st h 2 s p ro d u c ti o n s u c ro se l a c to se g lu c o se ia cr rd r s cr +ve -ve -ve ve -ve -ve -veg -veg +veg +ve lactobacillus fermentum ib cr rd sr c wh ve -ve + ve ve ve ve +ve +ve + ve -ve klebsiella pneumoniae ic cr rd r s cr ve -ve + ve ve ve ve veg veg -veg +ve pseudomonas sp. id cr f r c wh ve -ve + ve ve ve ve ve ve + ve +ve proteus mirabilis ie ir f r c cr + ve +ve + ve ve ve + ve +veg +veg +veg +ve bacillus subtilis if cr rd sr c wh ve ve + ve ve ve -ve + ve + ve +ve -ve klebsiella pneumoniae ig cr cv cc c ly + ve -ve + ve ve + ve ve + ve + ve + ve -ve staphylococcus aureus ih cr rd sr s cr + ve -ve ve ve ve ve veg veg +veg -ve lactobacillus sp. ii cr rd r s gr -ve -ve + ve -ve ve ve ve ve ve ve pseudomonas aeruginosa international journal of applied biology, 5(2), 2021 195 ezedinma et al. (2006) reported the amylolytic activity of bacillus subtilis, which produces an enzyme necessary for the breakdown of starch to sugar needed for the growth of other fermenting microorganisms, including lactic acid bacteria. in the report of freire et al. (2015), the authors reported some members of enterobacteriaceae during the fermentation of cassava, which is similar to the results obtained in this study. the presence of proteus spp and klebsiella spp could underline microbial activities in the rotting of cassava roots which was consistent with the findings of achi & akomas (2006). bacillus spp and lactobacillus spp were detected at the later stage of the retting process due to their persistence and ability to grow in an acidic medium. obilie et al. (2003) and essers et al. (1995) reported the involvement of bacillus species in the textural modification of cassava roots, which cause a softening of the cassava tissues, which is evident in this study. the presence of staphylococcus aureus, a normal skin microflora in the cassava steeping water may originate from human contact, contamination, poor hygienic conditions, and post-contamination (olopade et al., 2014). identification of s. aureus from diverse fermenting substrates has been reported in many studies (nout, 1994; fowoyo & ogunbanwo, 2017; anyogu et al., 2021). however, the results from this study agree with the findings of oyetayo (2006), who reported some pathogenic bacteria from fermenting cassava steeping water. pseudomonas spp and proteus spp have also been reported in the fermentation of cassava, due to their enzyme activities in the reduction of cyanide (izah et al., 2018). the lactobacillus fermentum isolated from this study agreed to the findings of adeleke et al. (2017) who reported l. fermentum from the fermented cassava peels. the presence of these bacteria can contribute significantly to the fermentation process of cassava wastewater for desirable output. table 3 shows the physicochemical properties of steeping water. a ph value of 3.67 was recorded at day one while at day four a ph value of 3.32 was recorded, respectively. table 3. physicochemical properties of the steeping water parameters days one four ph 3.67 3.32 temperature (oc) 26.2 26.2 international journal of applied biology, 5(2), 2021 196 the temperature was constant. figure 1 shows the chemical properties of the steeping water. the high total solids value of 45.2 mg/l was obtained at day four while the lowest total dissolved solids of 15.0 mg/l were obtained at day one of the fermentation processes. in this study, the ph of the retting liquor (steep water) was acidic after 24 hours of the fermentation process. this explains the high count of lactic acid bacteria in the later stage of the fermentation, probably due to increased acidity of the medium, which favored the growth of the microorganisms (adegbehingbe et al., 2017b). the decrease in ph recorded throughout the fermentation period may be associated with the fermentation by lactobacillus fermentum. an increase in the acidity and decrease in ph of tuber fermenting medium has been documented in several studies (oboh, 2006; olufemi & murtala, 2015; adegbehingbe et al., 2017a). the results of the effect of temperature on cassava retting showed that the optimum temperature was 26˚c. this is an indication that temperature had a very strong effect on the retting time. moreover, the container should be covered immediately after soaking the cassava to prevent contamination and allow the chemical reaction to take place. furthermore, this study showed the chemical parameters of fermented cassava steeping water. the values of total solids were higher in the fermented samples during the fermentation process. a total suspended solids value of 9.28 mg/l in cassava effluents has been reported by (lawal et al., 2018). proper measures should be taken to treat cassava wastewater for irrigation purposes instead to be indiscriminatingly discharged into the environment. figure 1. chemical properties of steeping water. key: tss total suspended solids, tds total dissolved solids, ts total solids 0 5 10 15 20 25 30 35 40 45 50 tss tds ts c o m p o si ti o n ( m g /l ) chemical parameters da… da… international journal of applied biology, 5(2), 2021 197 conclusions in conclusion, this study has revealed microorganisms involved in the fermentation of cassava steeping water. ensuring quality control during fufu production by soaking cassava tubers is essential to reduce contaminants from the fermenting substrates. the presence of lactobacillus species in the fermenting medium can be beneficial in softening the cassava tubers and inhibiting the growth of other pathogens which might cause foul odor during fufu production. the physical properties of the steeping water reveal the survival rate of the isolated microorganism fermentation medium. therefore, the microorganisms identified in this study can further be harnessed as a starter in the authors’ contributions this work was performed in collaboration with all the authors. authors bob and oii conceptualize the study. author oii performed the laboratory work. authors bob, bsa, and aoa managed the literature searches and wrote the first draft of the manuscript. authors bob aoa and oii performed the data analysis. author bob and bsa revised the drafts. all authors read and approved the final manuscript for publication. acknowledgments the authors gratefully acknowledge the academic support of the member of staff in the department of biological sciences of the authors’ affiliations. references achi, o. & akomas, n. 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(1988). microbiological studies on cassava fermentation for ‘lafun’production. food microbiology. 5, 125-133. contact : a k sultan mohideen smdbio@yahoo.co.in 67 abstract the fine structure of trichobothria in the salticid spider marpissa calcuttaensis (tikader 1974) was investigated by scanning electron microscopy (sem). the specimens were collected from the new college campus, chennai, india and kept in the laboratory before processing. the specimens were then fixed in trump’s fixative followed by post fixation in 2% osmium tetraoxide at room temperature for 90 minutes. after that, the specimens were dehydrated in the graded ethanol series and hexamethyldisilazane dried. lastly, the specimens were mounted on aluminum rods with araldite adhesive and coated with a thin layer of gold in a sputter coating unit and viewed under sem. the sem photomicrographs revealed the presence of trichobothria on the dorsal aspect of the first leg segments. the trichobothria were observed to be long and slender, embedded in special sockets. the articulation of the trichobothria in response to air deflections corresponds to that of other spider species. in addition, the slitsensilla and lyriform organ were noticed on the tarsal area of the first leg may react to substrate vibrations which are in accordance to other arachnids. thus, the structural characteristics features of the mechanoreceptors were compared with other arachnids to decipher their possible functional role and physiological significance issn : 2580-2410 eissn : 2580-2119 fine structure of trichobothria in the salticid spider marpissa calcuttaensis a k sultan mohideen 1*, a mohamed habibullah 2, 11department of zoology, faculty of zoology, the new college (autonomous) affiliated to university of madras, chennai-600014 tamil nadu india 2department of zoology, faculty of zoology, the new college (autonomous) affiliated to university of madras, chennai-600014 tamil nadu india introduction the arachnida is the largest and most important class of chelicerates. it includes spiders, scorpions, ticks, mites, harvesters, solifuges and other forms that are less known (cracraft, 2004). the largest group of arachnids is the spiders, which comprise the araneae order and whose bodies are divided into two segments rather than three. there may be more than 45,700 different types of spider species worldwide (world spider catalogue, 2016). spiders are cosmopolitan in distribution and help to manage several crop pests effectively, particularly in rice fields. they serve as potential candidates for the biological control of many farm insect pests and thus considered as friends of farmers. open access international journal of applied biology keyword marpissa calcuttaensis, jumping spider, trichobothria, mechanoreceptors, lyriform organ. article history received 20 august 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 68 spiders are widespread in india and have been recorded in a variety of habitats, such as building walls, grasslands, paddy fields, etc. the specimen chosen for this study belongs to the salticidae family, marpissa calcuttaensis (tikader, 1974) consisting of the spiders. these spiders often live in close competition for resources within the habitat. they normally live in moist microclimates and show a preference for high humidity. however, due to evaporation from the tracheal opening, they lose water quickly in dry air. they are often brown in color with black spots on the back of the opisthosome. the cephalothorax is wide and has large eyes in the anterior row, while the abdomen is long and the legs are stout and strong. the most characteristic feature is the presence of different patterns of spines and filiform hairs or trichobothria throughout the body. the filiform hair or trichobothria vary in size and diversity. on the other hand, all the leg segments are covered with different sensory hairs. most sensilla are movable, articulated setae or bristles that act as mechanoreceptors (touch and vibration). each leg receptor is associated with several sensory cells and, as a result, thousands of separate sensory fibers build up the sensory nerves. the slit sensilla, which occurs in all leg segments individually or in groups, are less conspicuous mechanoreceptors. the slits of the lyriform organs are located near the leg joints measure the stress in surrounding cuticle while the proprioreceptors gather information or the position of a specific leg joint (rathmayer, 1967; rathmayer & koopman, 1970). the trichobothria are extremely fine hairs embedded within the special sockets. they are much less numerous than the common tactile hairs usually arranged in regular rows on certain segments of the legs. the most striking feature is their extreme sensitivity. the long slender hair shaft is embedded in a thin cuticular membrane, so that the slightest air currents will make it quiver. four dendritic nerve endings attached to the specialized hair base and three of the four have specific sensitivity. the air vibrations that an insect produces with its wings suffice to trigger a directed capture response from the spider. they serve the spider to detect and localize prey and predators by sensing the air flow around them with tremendous efficiency (humphrey & barth, 2008). this could be attributed to the presence of numerous trichobothria found on the pedipalps and walking legs of spiders. each leg in agelena labyrinthica bears 25 trichobothria (gorner & andrews, 1969a), while in cupiennius salei each leg consists of 50 trichobothria (barth, 1982). the measurements on spider trichobothria are used in physical and mathematical modeling studies on mechanical hair behavior for fluctuating air flows in biologically relevant frequencies of 10 to 950 hz (barth, 1993). trichobothria was previously thought to represent hearing organs (dahl, 1883), but now it is considered to be a "touch at a distance "receptor because it reacts to air currents and low air vibrations (gorner & andrews, 1969b). materials and methods a total of 10 adult spiders were collected from their natural habitat at the new college campus, chennai, india. the specimens were kept in a screened aquarium and maintained in the laboratory before processing. the specimens were fixed in trump’s fixative (sodium cacodylate buffer, formalin and glutaraldehyde) followed by post fixation in 2% osmium tetraoxide for 90 minutes at room temperature. specimens were then dehydrated with graded ethanol series and chemically dried using hexamethyldisilazane (nation, 1983). international journal of applied biology, 4(2), 2020 69 the specimens were mounted on aluminium stubs with araldite adhesive and coated with a thin layer of gold in a sputter coating unit and viewed under sem. photomicrographs were captured using jeol-jsm: 5300 sem and jfc-1100 e/fine at metallurgy department, indian institute of technology, chennai, india. results and discussion phylum : arthropoda class : arachnida order : araneae family : salticidae genus : marpissa species : marpissa calcuttaensis (tikader, 1974) the spiders collected from the new college campus were identified and authenticated at the post graduate and research department of zoology, the new college, chennai-600014, india. the spider identified as marpissa calcuttaensis sp. belongs to the family salticidae. their body size ranged from 1.0 to 1.2 cm in length and 0.3 to 0.4 cm in width. the cephalothorax is wide and bears large prominent eyes on the anterior row. the abdomen is long and the legs are found to be short, stout and strong. the body is brown in color with black spots on the back of the opisthosoma (posterior part of the body) and covered by spines and filiform hairs or trichobothria. the image of the jumping spider m. calcuttaensis sp. is presented in figure1. figure 1. marpissa calcuttaensis. (araneae: salticidae) the diagnosis of the spider m. calcuttaensis sp. are in accordance to the characteristic features outlined in family salticidae. in fact, salticidae is considered to be the largest spider family in terms of number of species. the vast majority of species are active hunters and only a few make webs (shear, 1986). m. calcuttaensis commonly known as jumping spider is a small, brightly colored, enclothed with numerous filiform hairs on the body. the enlarged eyes possess the best eye sight responsible for acute daytime vision. in addition, jumping spiders react very definitely to visual stimuli like passing insects or the approaching finger of an observer. the short stout legs comprising of front and hind legs used to leap off an edge. in the jumping spider heliophanus, the front legs are raised but only the hind legs provide the thrust for jump (foelix, 1982a). international journal of applied biology, 4(2), 2020 70 the specimen distribution map from the new college campus, chennai, india calculated to be 13° 03′ 10°n, 80°15′ 36°e and altitude 6 m.. the site shows a variety of habitat which includes shrubs, grasses and walls of the building. in addition, the jumping spiders live in close competition for resources within the habitat. they inhabit microclimates and show a preference towards high humidity (figure 2). figure 2. distribution of marpissa calcuttaensis sp. from the new college campus, chennai, india (source: google earth) in the present study, scanning electron micrographs revealed a variety of sensory hairs across the entire leg surface of m. calcuttaensis. the leg segments were mostly composed of fine filiform hairs or trichobothria, tactile hairs, spines, slit sensory organs or sensilla and scopulae (figure 3). figure 3. external morphology of the entire first leg of m. calcuttaensis sp. showing different kinds of hairs and spines. sp-spines. scale bar = 500µm the trichobothria are slender and incorporated in special sockets that are randomly arranged on the dorsal aspect of the leg segments. alternatively, they are more or less regularly arranged on the femur and metatarsus region (figure 4). international journal of applied biology, 4(2), 2020 71 figure 4. trichobothria and cuticular teeth embedded within special sockets of tibia. tr-trichobothria, t-cuticular teeth. scale bar = 10µm. in fact, a large number of filiform hairs or trichobothria was observed in a definite pattern on the dorsal side of the first leg of m. calcuttaensis. in fact, their occurrence and pattern of distribution is unique to each species and therefore of immense taxonomic value. moreover, several investigators have shown a wide range of applications of trichobothria extending from taxonomy to behavioral studies. for instance, in the phylogeny of spiders the trichobothrial count and arrangement has great significance. earlier studies on insect karyotyping showed a correlation between the number of chromosomes and the arrangement of trichobothria (grozeva, 1995). similarly, in adults podopinae, meristic and topographical aspects of trichobothrial pattern showed a reduction of the typical 5+5 ground plan to 2+2 trichobothria per abdominal sternites (foelix, 1970). the filiform hairs or trichobothria also form one dorsal row on each tarsus. moreover, there are three rows of trichobothria present in tibia with different lengths of hair shafts. in addition, cuticular teeth were observed arranged in the form of rows on each side of the tactile hair emerging from underdeveloped sockets. consequently, the length of these hair shafts increases gradually to the end of the leg (figure 5). figure 5. trichobothria with hair shafts of different length, increasing towards the tip of the first leg region. tr-trichobothria. scale bar = 10µm. the trichobothria are mechanosensory in function. they are slender and embedded in special sockets randomly arranged on the dorsal aspect of leg segments. moreover, the long slender hair shaft is suspended in very thin (0.5µm) cuticular membrane (foelix, 1982a). therefore, even the slightest air current will disturb and triggers the movement of these specialized hairs. for this reason, a spider is always alert and capable of exploring the environment very efficiently. international journal of applied biology, 4(2), 2020 72 according to harris and mill (1977), spiders’ trichobothria is ideally suited to detect the presence and direction of prey-stimulated air currents. similarly, c. salei can locate a crawling fly up to 20 cm away by orienting itself towards the fly-triggered air current, by means of its trichobothria barth (1982b). in the same way, sericopelma rubronitens trichobothria present on the dorsal area of tibia and more distal articles in the legs and pedipalps are sensitive enough to detect the movement of the cricket leg which is 2cm away (den otter, 1974). however, in pseudoscorpions enhanced predatory behaviour and feeding rate is directly related to the trichobothrial count and its length (davidova & stys, 1993). the spiders are therefore considered as good hunters and voracious feeders. simultaneously, we also found the tactile hairs emerging from underdeveloped sockets bearing numerous cuticular teeth arranged in rows on each side of the hair shaft. these tactile hairs are strong in nature compared to trichobothria, which is similar to the tactile hairs of the wolf spider lycosa gulosa (foelix, 1970). the slit sensory organs or sensilla are fine hairs seen on the dorsal surface of the leg. the fine hairs were observed in the form of groups on the tarsal area. hence, they are also called as a lyriform organ (figure 6). at the end of the tarsus a pair of claws was seen with a tuft of fine hair called scopulae (figure 7). figure 6. lyriform organ on the tarsal area of m .calcuttaensis sp. ly-lyriform organ. scale bar = 100µm. figure 7 . scopulae at the tip of the tarsus of m calcuttaensis. sp. sc-scopulae. scale bar = 50µm. the slit sense organs or sensilla observed over the tarsal area of the first leg are fine hairs occurring either singly or in groups. when they occur in groups it is called as a lyriform organ. though, all arachnids possess slit sensilla and lyriform organs but only in spiders it international journal of applied biology, 4(2), 2020 73 reaches the fine sophistication. on the contrary, the slit sensilla function varies in different spiders which is usually classified as proprioreceptive mechanoreceptors, connected with the position and movement of the body. but this is not entirely correct because the stimuli do not have to be related to the movement of the spider itself. in addition, responses to airborne sound were shown electrotrophysiologically for a single tarsal slit sensillum (barth, 1967). while some slit sensilla act as gravity receptors, as they probably register the relative movements between prosoma and opisthosoma (barth & libera, 1970). further, in non araneomorph spiders the prey is detected in the pedipalps and legs of these spiders. whereas, slit sensilla such as lyriform organs and single-slit sensilla are important substratum vibration detectors found on the legs of araneoomorph spiders (barth 1982; walcott 1969). likewise, the wandering araneomorph c. salei is able to locate its prey and mates at a range of upto 1 m on banana plants using substrate-vibration detectors (rovner & barth 1981). similarly, brownell (1977) has shown that sand scorpions' basitarsal compound slit organs are sensitive to substrata vibrations that can detect and locate insect’s movement on sand surface up to 50 cm away. in addition to sensilla and trichobothria, spines were also observed throughout the dorsal side of the first leg. in fact these spines become erect when haemolymph pressure increases the nerve impulses of leg spines, unlike those of simple tactile hairs which can be recorded only during the erection phase, but not during the return to the flat resting position. these spines are therefore considered to be haemolymph pressure receptors, rather than touch receptors (schlegel & bauer, 1994). conclusions in conclusion, sem photomicrographs reveal that m. calcutttaensis contains different types of sensory receptors on their first leg. these findings provide clear evidence that salticid spider may perceive air currents sensed by their trichobothria during the movements of a prey or predator. the slit-sensilla may play a role in detecting minute mechanical pressures that facilitates in movement and positioning the body. therefore it is quite evident that mechanoreception, touch, vibration, and the perception of airflow are all different tasks carried out by these sensory hairs. thus the filiform hairs not only facilitate localization of prey but also help to escape from predation. these characteristics features may significantly contribute to their existence and survival of these spider species. acknowledgements i thank the management, principal and head, department of zoology for providing laboratory facilities. i am extremely grateful to my research supervisor prof. a. mohamed habibullah (retd.), professor of zoology for his guidance, support and encouragement. i sincerely thank prof. c.v. gokulrathnam and mrs. shanthi devanathan, department of metallurgy, indian institute of technology for their assistance in electron microscopy. references barth, f.g. 1993. sensory guidance in spider pre-copulatory behaviour. comp. biochem. physiol 104, 717–733. https://doi.org/10.1016/0300-9629(93)90148-w barth, f.g., libera, w. 1970. ein atlas der spaltsinnesorgane von cupiennius salei keys. chelicerata (araneae). z morphol tiere 68, 343–369. google scholar https://doi.org/10.1016/0300-9629(93)90148-w 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press, stanford, california. 1986. p: 428. tikader, b.k. 1974. marpissa calcuttaensis. proc. indian acad. sci, 76, 210. walcott, c. 1969. a spider’s vibration receptor: its anatomy and physiology. am. zool 9, 133-44. doi:10.1093/icb/9.1.133 world spider catalog. 2016. natural history museum bern. accessed on 20 march 2020. https://wsc.nmbe.ch/ https://scholar.google.com/scholar?q=humphrey%20jac%2c%20barth%20fg%20%282008%29%20medium%20flow-sensing%20hairs%3a%20biomechanics%20and%20models.%20in%3a%20casas%20j%2c%20simpson%20sj%20%28eds%29%20advances%20in%20insect%20physiology.%20insect%20mechanics%20and%20control%2c%20vol.%2034%2c%20elsevier%20ltd%2c%20pp%201%e2%80%9380 http://scholar.google.com/scholar_lookup?&title=die%20verteilung%20der%20propriorezeptoren%20im%20spinnenbein.%20untersuchungen%20an%20der%20vogelspinne%20dugesiella%20hentzi%20chamb&journal=z%20morph%20tiere&volume=66&pages=212-223&publication_year=1970&author=rathmayer%2cw&author=koopman%2cj http://scholar.google.com/scholar_lookup?&title=elektrophysiologische%20untersuchungen%20an%20propriorezeptoren%20im%20bein%20einer%20vogelspinne%20%28eurypelma%20hentzi%20chamb.%29&journal=z%20vergl%20physiol&volume=54&pages=438-454&publication_year=1967&author=rathmayer%2cw http://scholar.google.com/scholar_lookup?&title=elektrophysiologische%20untersuchungen%20an%20propriorezeptoren%20im%20bein%20einer%20vogelspinne%20%28eurypelma%20hentzi%20chamb.%29&journal=z%20vergl%20physiol&volume=54&pages=438-454&publication_year=1967&author=rathmayer%2cw https://wsc.nmbe.ch/ https://wsc.nmbe.ch/ contact : c.c. ezemba constancechinyere790@gmail.com 137 abstract this research was carried out to produce wine from banana (musa sapientum) using yeast (saccharomyces cerevisiae) isolated from grape (vitis vinifera). the fermentation of the banana wine lasted for 21days. during fermentation, liquor of the fermenting “must” were removed every 48hours from the fermentor for analysis of ph, titratable acidity, specific gravity and reducing sugar using standard procedures.. the results from the experiment showed that specific gravity of the wine was observed to reduce drastically as the fermentation progresses. the ph of the banana wine during fermentation increased from 4.164.22 while the titrable acidity of the banana wine produced increased from 1.05-1.77. the alcohol content of the wine increased from 0.0 to 9%. the higher the fermentation temperature, the faster the conversion of sugars into alcohol and carbon dioxide by the yeast. the flavor and taste was appreciable. this study showed that acceptable wine can be produced from banana with the yeast saccharomyces cerevisiae isolated from grape. issn : 2580-2410 eissn : 2580-2119 wine production from banana (musa sapientum) using yeast (saccharomyces cerevisiae) isolated from grape (vitis vinifera). c.c. ezemba 1*, ezemba, a.s. 2, ezeokoli, c.m.. e.j. archibong2, okeke, c.a2 1department of microbiology, chukwuemeka odimegwu ojukwu university igbariam (coou), anambra state. nigeria. 2department of applied microbiology and brewing, nnamdi azikiwe university awka, anambra state, nigeria. introduction wine is a product of alcoholic fermentation by yeast of the juice of ripe grapes or any fruit with a good proportion of sugar [1]. wine is one of the most recognizable high value added products from fruits. it can also be used as a substrate for the manufacture of vinegar, a by-product of wine manufacture. fruit juices are fermented to produce wine, an alcoholic beverage. grapes are usually preferred because of the natural chemical balance of the grape juice which aids their fermentation process without addition of sugars, acids, enzymes, or other nutrients. however, fruits such as banana, cucumber, pineapple and other fruits are used in wine production [2]. wine manufacture is challenging in which marketable product can be obtained, but the processes involved in its production are relatively straight forward [3]. highly acceptable wines can be made from practically all fruits. wine can be fermented with yeast that occurs naturally in grape which is the main organism responsible for alcoholic fermentation which belongs to the genus saccharomyces. although, many genera and species of yeast are found in must, saccharomyces cerevisiae is the main yeast strain that is commonly reported to be open access international journal of applied biology keyword banana; fermentation; grape; yeast; fermentor; carbondioxide article history received january 22, 2022 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 138 responsible for alcoholic fermentation [1] and in other countries where grape is not produced, emphasis is usually placed on other fruits for wine making. there are some soft fruits from both temperate and tropical regions whose pigment stability and flavor profiles match those of any wine from grapes, but suffer from the lack of intensive research and development given to grape wine. reports on tropical fruit wines have been mainly on exotic species such as banana, pineapple, citrus, mango, pawpaw, apple, strawberries etc.[4]. wine represents a safe and healthful beverage; it also provides calories and vitamins. during period when life was often strenuous, it offered relaxation and relief from pains. bananas (musa sapientum) are an important staple starchy food in nigeria. it is a seasonal and highly perishable fruit, which can be available all year round. the large quantity of bananas and plantains provides the potential for industrial use [5]. in addition, any application to produce a marketable, value-added product will improve banana farming economies and eliminate the large environmental problem presented by banana waste. banana could then compete in the market, either as banana juice or as mixtures with other juices because of its flavor and aroma [6]. bananas has a lot of nutritional benefits, thus demands in the market are high. they are highly recommended by doctors for patients whose potassium is low, because of its impressive potassium content. potassium is an important component of cell and body fluids that helps control heart beat and blood pressure, countering bad effects of sodium. banana is considered as an important food to boost the health of malnourished children, it contains good amount of soluble dietary fiber that helps normal bowel movements; thereby reducing constipation problems. medicinal uses of banana have positive contribution towards successful treatment of anemia, heartburn, temperature control, ulcer, overweight etc. banana juice can also be applied to wine production; however, banana juice is turbid, gray in color, very viscous, tends to settle during storage and, therefore must be clarified prior to commercialization [6]. the turbidity and viscosity of banana wine are caused mainly by the polysaccharides in banana juice such as pectin and starch and therefore make the clarification process harder. application of pectinase and -amylases that affect the quality of wine is important for improving the process of banana wine production. material and methods sample collection ripe banana fruits used for this experiment were bought from eke market, awka, in anambra state. preparation of must/ fermentation the ripe bananas were washed with distilled water. the bananas were thoroughly disinfected with cotton wool soaked in ethanol. the banana slices were blended with water in a super mark blender. the slurry was filtered through a "muslin cloth” to obtain the juice (must) and autoclaved after which it was allowed to cool and poured into the fermenting vessel and the 250ml inoculum developed was inoculated into the fermenting vessel. 0.14 sodium metabisulphite was added to inhibit the growth of micro-organism and about 100g of sucrose was added to fortify the must and kept for fermentation to commence. the must was analysed for reducing sugar, titratable acidity, specific gravity and alcoholic content before fermentation. international journal of applied biology, 6(1), 2022 139 inoculum development the banana fruits were washed thoroughly with 0.1% sodium metabisulphite in water. the fruits were cut, manually deseeded, blended and filtered to obtain the juice. about 250ml of the juice was introduced into a clean sterile 500 ml conical flask and sterilized by autoclaving. upon cooling, three (3) loopful of the yeast culture isolated from the palm-wine was used to inoculate the juice and incubated in a rotary shaker for 48 hours. all procedures were done under aseptic condition. ph determination the ph was determined using a standard ph meter. the ph meter was standardized with buffer solution, the buffer solution was prepared with ph buffer powder of ph 4.00 at 25oc dissolved in 250ml distilled water. ten ml of the “must” was put into a sterile beaker, the electrode of the ph meter was immersed in the beaker containing the “must”. the ph of the “must” was determined using a digital ph meter (model no: ph s-25) determination of reducing sugar the quantitative estimation of reducing sugar of the wine was determined using the method described by amerine and ough (2000). 1ml of 3,5-dinitro salicyclic acid (dns) is added to 1ml of supernatant of wort (sample) in a test tube and the mixture heated in boiling water for 10mins. the test tube is cooled rapidly in tap water and the volume adjusted to 12ml with distilled water. a blank containing 1ml of distilled water and 1ml of dns is also prepared. the optical density (od) of sample is read against the blank in a spectrophotometer at 540nm absorbance. the concentration of reducing sugar is estimated from a glucose standard curve. determination of specific gravity fifty ml specific gravity bottle was thoroughly cleaned with distilled water, dried in an oven for 50oc and allowed to cool. the weight of the cooled dried bottle (w1) was recorded. the dried bottle was filled with deionized water and surface of the bottle was cleaned with a cotton wool and weighed as (w2). the bottle was empty and cleaned twice with 10ml of the “must” thereafter the bottle was filled to the brim with the “must” and the bottle cleaned with cotton wool and weighed as (w3). the specific gravity (s.g) was calculated. 𝑆.𝐺 = 𝑊3 − 𝑊1 𝑊2 − 𝑊1 = 𝑆 𝑊 where s= weight of volume of must (w3 w1) w= weight of volume of water (w2 w1) estimation of titrable acidity this was determined by the methods described by [7]. 1% of aqueous alcoholic phenolphthalein as indicator was added to 200ml of distilled water. it was titrated with 0.1m of na0h. titration was stopped when a faint but definite pink colour appeared. the titre was taken, this served as the initial titre. 5ml of the must was added to the neutralized solution. the same 0.1m naoh was used to titrate it. the titration was stopped at the appearance of faint, but definite pink colour. the titre was taken. this served as the final titre. the titratable acidity was calculated with reference to tartaric acid. international journal of applied biology, 6(1), 2022 140 alcohol content determination the distillation method according to the association of official analytical chemists methods of analysis [8]. fractional distillation of the wine was carried out and 100ml of the wine was measured into a roundbottom flask with side arms. the flask was connected to the fractioning column and an anti-bumping chip was added to the wine. this ensured adequate dispersal of heat in the wine. it was then heated and the distillate collected. the distillate was poured into a clean dry conical flask and made up to 100ml with distilled water. it was left to stand overnight on the bench. the specific gravity of the distillate was determined and the result was taken to determine the alcoholic strength. results table 1. analysis of the fermenting wine throughout the period of fermentation, ph of the must was within the acidic range. the ph of the fermenting wine as shown in table 1 and fig. 1 indicate a gradual decrease in the ph as the fermentation time increases and increased at the end of fermentation. at day 0, the ph is 4.16. while at day 21 the ph is 4.22. the result of titrable acidity carried out on the sample as shown in table indicates that the titrable acidity ranges from 1.05 at 0 day to 1.77 at day 21 in the fermenter. therefore, the titrable acidity increases as fermentation time increases. the result shows an an increase in the alcohol content of the fermenting sample as shown in the table. number of days ph reducing sugar(mg/ml) titratable acidity specific gravity alcohol content day 0 4.16 0.20 1.05 1.266 0.0 day 2 3.91 0.18 1.09 1.224 0.3 day 4 3.72 0.09 1.12 1.218 2.4 day 6 3.53 0.05 1.23 1.049 5.8 day 8 3.92 0.02 1.32 1.012 7.5 day 14 4.00 0.02 1.44 1.012 7.6 day 21 4.22 0.01 1.77 1.04 9.0 international journal of applied biology, 6(1), 2022 141 figure 1. wine analysis the alcohol content ranges from 0.0 to 9.0% at day 21. in the case of specific gravity of the fruit wine gradual decrease in valves were observed throughout the period of fermentation. these decreases were observed to be irrespective of the yeast strain and the fruits used in the wine production. between 0 day to 21 days of the fermentation, specific gravity values were observed to range from 1.266 to 1.04kgm-3. discussion in this research, it is observed that the fermentation period agrees with the result of [9] who observed that saccharomyces. cerevisiae could strive under low ph. the result of this experiment shows that ph values of banana wine decrease progressively as the fermentation period increases and finally increased at the end of the fermentation. the ph value at day 0 is 4.16 which decreases progressively as fermentation continues and increased at the end of the fermentation. the ph is 4.22 at day 21 (table 1). this shows that the wine became more acidic with the period of fermentation. the drop in ph also records the utilization of the sugar present in the must for growth. this observation is similar to that reported by [10]. result of this study also revealed consistent increase in titratable acidity of the wine throughout the perod of fermentation. the titratable acidity is recorded as 1.05 at day 0 which increased to 1.77 at day 21. it is observed that the titratable acidity increases with the period of fermentation. studies have shown that during fermentation of fruits, low ph is inhibitory to the growth of spoilage organisms but creates conducive environment for the growth of desirable organisms. also, low ph and high acidity are known to give fermenting yeast competitive advantage in natural environment [7]. the alcohol content of the wine increased from 0.00% to 9% on the last day of fermentation (fig 1). this result is found to conform to that of [11] who observed gradual increase in the titratable acidity and the alcohol content in the fermentation of plantain. the specific gravity of the fruit wine produced in this study reduces as the fermentation days of the wine increases. after the 21 days fermentation the specific gravity of the wine reduced drastically to 1.04kgm-3 this was due to the type of yeast used in the wine production. saccharomyces cerevisiae isolated from palm wine has been reported to 0 1 2 3 4 5 6 7 8 9 10 day 0 day 1 day 4 day 6 day 8 day 14day 21 f e r m e n ta ti o n p a r a m e te r s fermentation days ph reducing sugar titratable acidity specific acidity alcohol content international journal of applied biology, 6(1), 2022 142 reduce specific quality of fruit wines during fermentation9. the short shelf-life of beverages are however a major problem faced by their producers and consumers in africa [12]. in texture, the wine produced was completely watery at the end of fermentation. this result is similar to that recorded by [7] that as fermentation rate proceeded, gas was formed and this rose through the liquid then during active fermentation, forth or foam is formed on the surface. the gas carries the cells through the fermenting must cause it to be cloudy and as a result, a strong odor of alcoholic fermentation developed. the result of the banana wine produced by the fermenting with yeast strains isolated from grape (saccharomyces cerevisiae) shows high rate of alcoholic production at the end of day 21. this result is similar to the observation reported by [13, 14] who recorded that pure culture of saccharomyces cerevisiae produces more ethanol and give a faster fermentation than the native yeast. conclusion banana wine is very nutritious and easy to produce and could then compete in the market with other wines because of its flavor, aroma and the successful production of using indigenous fruits as substrates for wine production and the efficiency of locally isolated yeast saccharomyces cerevisiae strains from grape (vitis vinifera) for fruit wine production. the ph of the fermenting must was shown to increase at the last day of fermentation ranging from 4.16 to 4.22. the alcohol content also gradually increased ranging from 0.00% to 9% showing the strength of the fermenting must. banana wine has a lot of nutritional benefits, vitamins including b5, b6, c, a are all present in banana wine and this makes it one of the high ranking beverage over other alcoholic ones. vitamin a helps in restrain of eye sight. banana wine is infused with high taste and health. commercially produced banana wine is a clear, slightly sparkling alcoholic beverage with a long shelf life than banana beer, which is spoiled easily and therefore not stored for long periods. however, there is the need for further research to ascertain the shelf life for the wines. international journal of applied biology, 6(1), 2022 143 references amerine, m. a., berge, h. w., kunkee, c. s., ough, v.l., singleton, b.v and webb, a.c. (1979).the technology of wine making. avi publishing co. west port, usa. amerine, m. a., and ough, c. s. (2000). methods for analysis of must and wines. john wiley and son inc. new york. pp. 334 archibong, e.j., ezemba. c.c.,;chukwujama, i.c. and archibong e.j. (2015). production of wine from mixed fruits: pineapple (ananascomosus) and orange (citrus sinensis) using yeast isolated from palmwine. world journal of pharmacy and pharmaceutical sciences, 4(08):126-136 association of official analytical chemistry {a.o.a.c}.(1990). official methods of analysis (15th edition). washington d.c. 1: 73-74 food and agriculture organization of the united nations (fao) (2003). banana fruits and their uses. retrieved from faostat statistics database accessed on june 22, 2015. lee, w.c., yusuf, s., hamid, n.s. and baharin, b.s. (2006). optimizing conditions for enzymatic clarification of banana juice using response surface methodology. journal of food engineering. 73:53-63. maldonado, o., rolz, c. and schneider de cabnera, s. (1995). wine and vinegar production from tropical fruits. journal of food science. 40: 262-265. mc bryde, c., gardner, j.m., de barros l.m. and jiranek, v. (2006). generation of novel wine yeast strains by adoptive evolution. american journal of enology and viticulture. 4:12-16 obaedo me and ikenebomeh mj (2009). microbiology and production of banana (musa sapientum) wine. nigerian journal of microbiology, 23(1): 18901895. okegbile, w.t. and taiwo, e.a. (1990). fruit wine discussions; food microbiology. aska vino publishing, new york. pp. 175. okoro, p. (2007). the technology of passion fruits and banana wines. american journals of enology and viticultures, 17:27-29. sanni, a.i. and oso, b.a. (2008). production of agadagidi a nigeria fermented beverage. die nahrung. 32:319-326. yuting, l. (2009). discussion about entry: food and fruit rich project. changemaker publishers. usa. pp. 224. zohany, d. and hopf, m.(2000) domestication of plants in the old world, 3rd edition, oxford university press, england. pp.193. contact : nancy taha mohamed nancyt0000@yahoo.com 98 abstract this study aimed to investigate the bioactive compounds in the haemolymph of scarab beetle scarabaeus sacer by using the gas chromatography–mass spectrometry (gs-ms) analysis. the identification of the bioactive compounds is based on peak area, retention time, molecular formula and molecular weight. there are 129 compounds are detected in the haemolymph of scarab beetle and 43 of them were reported to have a bioactivity. the most analyzed bioactive compounds are alcohols, steroids, fatty acids and terpenoids. the current study also test the antimicrobial activity of scarab beetle haemolymph against gram-negative bacteria (escherichia coli, enterobacter cloacae, gram-positive bacteria (staphylococcus aureus, bacillus subtilis), and fungi (aspergillus fumigatus and candida albicans). the haemolymph has highest antibacterial activity against gram negative bacteria enterobacter cloacae, escherichia coli respectively and against gram-positive bacteria bacillus subtilis, staphylococcus aureus respectively. no antifungal activity has been detected. issn : 2580-2410 eissn : 2580-2119 separation of bioactive compounds from haemolymph of scarab beetle scarabaeus sacer (coleoptera: scarabaeidae) by gc-ms and determination of its antimicrobial activity nancy taha mohamed 1*, doaa hassan abdelsalam 1, ahmed salem el-ebiarie 1, mahmoud elaasser 2 1 zoology & entomology department, faculty of science, helwan university. 2 regional centre of mycology and biotechnology, al-azhar university. introduction coleoptera is one of the largest order of insects with about 370,000 insect species described worldwide. the family scarabaeidae encompasses over 30.000 species of beetles worldwide; they are often called scarabs. dung beetles are a major insect group (coleoptera: scarabaeidae) distributed globally except antarctica with a high number of diversity comprising approximately 6,200 species and nearly 267 genera (tarasov and génier, 2015). these species are coprophagous in nature which live freely in soil and mostly feed on both wet and dry dung materials of herbivorous mammals. the undigested excreta of mammals are utilized as food and nesting material throughout their life cycle, hence, they possess many ecologically beneficial functions. the dung beetles play a vital role in nutrient recycling by decaying organic matter and developing soil aeration (manning et al., 2016) thereby, reducing the greenhouse gas fluxes (slade et al., 2016). it also improves plant growth and grain production (koyama et al., 2003). scarabeus sacer is considered a species open access international journal of applied biology keyword haemolymph, gs-ms, gram negative bacteria, gram positive bacteria, antimicrobial activity. article history received 11 november 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 99 of genus scarabaeus, occurs in coastal dunes and marshes around the mediterranean basin. it can be found across north africa, southern europe and parts of asia (afghanistan, corsica, cyprus, france, iran, israel, italy, morocco, sardinia, sicily, sudan and syria). scarabaeus sacer is a species of dung beetle belonging to the family scarabaeidae (long, 1836) insects are known for their ability to resist infection. they protect themselves against bacterial infection by secreting a battery of antimicrobial peptides (amp) into the hemolymph. hemolymph, also known as the insect blood, is a clear fluid, with or without yellow or greenish pigmentation. it constitutes 16-40% of the body weight of certain insects. the volume and component of hemolymph are vary in different types of insects and their developmental stages. it spends much of its time flowing freely within body cavities where it makes direct contact with all internal tissues and organs. therefore the circulation would help to transport the amp to its target site (kurata, 2006). in insects, amps / polypeptides are manufactured mainly in a fat body (similar to mammalian liver) and are released into hemolymph where they play a vital role in innate immune systems and host defense mechanisms, and having a broad spectrum of activity against both gram + ve and gram -ve bacteria and against fungi (januszanis et al., 2013) however, misuse of antibiotics intake has caused many problems, such as the appearance of antibiotic-resistant bacteria, weakening of disease resistance in livestock, and ecosystem pollution (looft et al., 2012). insects exhibit innate immune systems that produce potent amps to protect them from pathogen invasion, and these amps are viewed as strong natural antibiotic applicants (kalsy et al., 2020). the insect innate immune system is categorized into cellular and humoral immunity. cellular immunity involves the phagocytosis of bacteria, fungi, and protozoa, and nodule formation and encapsulation, while humoral immunity involves the secretions of proteins and peptides produced in fat and blood cells to hemolymph in response to infection (wu et al., 2018). amps secreted by the humoral immune response are classified according to their structure and amino acid sequence into cecropins, defensins, proline-rich peptides, glycinerich peptides, and lysozymes and are found in various insect orders including coleoptera, diptera, hymenoptera, and lepidoptera (michael zasloff, 2002). melittin is familiar amp contained in bee venom and its antimicrobial activity was observed greatly in methicillin resistant staphylococcus aureus (mrsa) and gram-positive and gram-negative bacteria (pashaei et al., 2019). amps are small molecules that vary in size, ranging from 10 to 100 amino acid residues and are produced by all living organisms. the rich diversity of insects makes them rich sources of amps. the black soldier fly hermetia illucens l. (diptera: stratiomyidae), particularly, able to live in hostile environments rich in microbial colonies, making it one of the most promising sources of amps (moretta et al., 2020). nowadays, the using of gc-ms technique is important in analyzing and separation the compounds found in plants extracts (eva de rijke et al., 2006)and also the haemolymph of arthropods. this will be a new method for discovering a future drugs to be used in traditional medicine system. in this article, the haemolymph of scarab beetle was analyzed by gc-ms and resulting in many bioactive compounds. materials and methods collection of insects scarab beetle was collected from baltim in the kafr el sheikh governorate, in the north coast of egypt. https://en.wikipedia.org/wiki/scarabaeus https://en.wikipedia.org/wiki/mediterranean_basin https://en.wikipedia.org/wiki/italy https://en.wikipedia.org/wiki/morocco https://en.wikipedia.org/wiki/sardinia https://en.wikipedia.org/wiki/sicily https://en.wikipedia.org/wiki/sudan https://en.wikipedia.org/wiki/syria https://en.wikipedia.org/wiki/species https://en.wikipedia.org/wiki/dung_beetle https://en.wikipedia.org/wiki/scarabaeidae https://en.wikipedia.org/wiki/kafr_el_sheikh_governorate https://en.wikipedia.org/wiki/north_coast_of_egypt https://en.wikipedia.org/wiki/egypt international journal of applied biology, 5(2), 2021 100 withdrawing of hemolymph scarab beetle body surface cleaned with 70% alcohol. then, in order to collect haemolymph, hind pair legs were cut from coxa, and haemolymph fluid was extracted with a capillary tube placed into micro tubes containing edta. haemolymph was centrifuged at 10000 × g for 10 minutes and the supernatant was collected for the antimicrobial testing and stored in 4°c. gas chromatography–mass spectrometry (gc-ms) analysis haemolymph was collected and 30 milligrams were homogenized in 1ml methanol centrifuged at 4500 rpm for 10 minutes, the supernatant was taken to gc-ms. the chemical composition of samples was performed using trace gc1310-isq mass spectrometer (thermo scientific, austin, tx, usa) with a direct capillary column tg–5ms (30 m x 0.25 mm x 0.25 µm film thickness). the column oven temperature was initially held at 50 c and then increased by 5°c /min to 230°c for 2 min. increased to the final temperature 290°c by 30°c /min and hold for 2 min. the injector and ms transfer line temperatures were kept at 250, 260°c respectively; helium was used as a carrier gas at a constant flow rate of 1 ml/min. the solvent delay was 3 min and diluted samples of 1 µl were injected automatically using auto sampler as1300 coupled with gc in the split mode. ei mass spectra were collected at 70 ev ionization voltages over the range of m/z 40–1000 in full scan mode. the ion source temperature was set at 200 °c. the components were identified by comparison of their retention times and mass spectra with those of the wiley 09 and nist 11 mass spectral databases. antimicrobial activity assay: the antimicrobial activity was investigated in the haemolymph against microorganisms. all microbial strains were provided from the culture collection of the regional center for mycology and biotechnology (rcmb), al-azhar university, cairo, egypt. the antimicrobial profile was tested against two gram‐positive bacterial species (bacillus subtilis, staphylococcus aureus), two gram-negative bacterial species (escherichia coli, enterobacter cloacae) and two fungi (aspergillus fumigatus and candida albicans) using a modified well diffusion method. briefly, 100 μl of the test bacteria/or fungi were grown in 10 ml of fresh media until they reached a count of approximately 108 cells/ml for bacteria or 105 cells/ml for fungi (ibrahim et al., 2014). one hundred μl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained and tested for susceptibility by well diffusion method on mueller‐hinton and sabaroud agar (clinical and laboratory standards institute, 2012.) one hundred µl of each sample (at 10 mg/ml) was added to each well (10 mm diameter holes cut in the agar gel). the plates were incubated for 24-48 h at 37 °c (for bacteria and yeast) and for 48 h at 28 °c (for filamentous fungi). after incubation, the microorganism's growth was observed. the resulting inhibition zone diameters were measured in millimeters and used as a criterion for antimicrobial activity. if an organism is placed on the agar, it will not grow in the area around the well if it is susceptible to the chemical. this area of no growth around the disc is known as a "zone of inhibition" or "clear zone". the size of the clear zone is proportional to the inhibitory action of the compound under investigation. solvent controls (dmso) were included in every experiment as negative controls. dmso was used for dissolving the tested compounds and showed no inhibition zones, confirming that it has no influence on growth of the tested international journal of applied biology, 5(2), 2021 101 microorganisms. gentamycin and ketoconazole (sigma aldrich, usa) were used as standard antibacterial and antifungal drugs at 30 and 50ug/ml, respectively. mic determination the tested extract was screened in vitro for their antibacterial and antifungal activities at a different concentration to determine the lowest concentration inhibiting the growth of the organism that recorded as the mic (ibrahim et al., 2014). results and discussion in the present article, the separation of compounds in the haemolymph of scarab beetle by using gc-ms analysis gas separation technique resulting in 43 bioactive compounds as shown in table (1). the identification of the bioactive compounds is based on peak area, retention time, molecular formula and molecular weight. in this study, the antibacterial effectiveness of the haemolymph against gram -v bacteria (escherichia coli, enterobacter cloacae, against gram +v bacteria (staphylococcus aureus, bacillus subtilis) as well as antifungal activity (aspergillus fumigatus, candida albicans) is investigated. antibacterial activity against bacillus subtilis was first, followed by enterobacter cloacae, escherichia coli and staphylococcus aureus respectively. no antifungal activity has been investigated yet (table 2). the minimum inhibitory concentration (mic) is measured for the tested sample and it was 2500 g/ml against e. coli, 1250 g/ml against enterobacter cloacae, bacillus subtilis and 10000 g/ml against staphylococcus aureus respectively (table 3). table 1. bioactive compounds in haemolymph of scarab beetle separating by gc-ms no. compound name molecular formula chemical nature mwt area % rt bioactivity 1 trichloromethane chcl3 trihalomethane 118 3.57 4.06 anti-virus, anti-cancer, anti-mutagenic, antiallergic and anti-ulcer (ali et al., 2015) 2 tert-hexadecanethiol c16h34s thio-alcohol 258 0.11 5.16 enzyme activators (rajendran et al., 2017) 3 ethanol, 2 octadecyloxy c20h42o2 alcohol 314 0.11 5.16 antimicrobial activity (sudhandra karthi, 2016) 4 1-heptatriacotanol c37h76o alcohol 536 0.48 39.87 anticancer, antineoplastic and anti-hiv (kala and ammani, 2017) 5 1,4-benzenediol, 2-(1,1dimethylethyl)-5-(2propenyl) c13h18o2 alcohol 206 2.66 25.14 anticancer, antioxidant activity and pesticidies (swamy et al., 2017) 6 e,e,z-1,3,12nonadecatriene5,14-diol c19h34o2 alcohol 294 0.40 39.20 antimicrobial activity (hadi et al., 2016) 7 3,7,11,15tetramethyl-2hexadecen-1-ol c20h40o terpene alcohol 296 6.43 30.04 antimicrobial (ponmathi sujatha et al., 2017) precursor of synthetic forms of vitamin e and vitamin k1 (devakumar et al., 2017) international journal of applied biology, 5(2), 2021 102 8 dotriacontane c32h66 saturated hydrocarbons 450 0.11 5.16 antimicrobial, antifungal, anti-inflammatory, cytotoxic activity (harris, 1992) 9 isochiapin b c19h22o6 terpenoids 346 0.32 10.83 anti-insect antitumor agent (elsharkawy, 2016) 10 14-á-h-pregna c21h36 steroids 288 0.32 10.83 defense chemical and diabetic retinopathy prevention (durak and kalender, 2007) 11 cycloheptasiloxane, tetradecamethyl c14h42o7si7 organo silicone compound 518 1.69 15.25 antimicrobial, antiseptic, hair conditioning agent, skinconditioning agentemollient and solvent (mary and giri, 2018) 12 4h-1-benzopyran-4 one, 2-(3,4dimethoxyphenyl)-3, 5-dihydroxy-7methoxy c18h16o7 ketonic comound 344 0.38 17.48 antioxidant, antimicrobial, cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries (albergoni et al., 1980) 13 cyclooctasiloxane, hexadecamethyl c16h48o8si8 alkanes 592 2.84 19.68 antimicrobial (al bratty et al., 2020) 14 cyclododecasiloxane, tetracosamethyl c24h72o12si12 alkanes 888 2.84 19.68 hepatoprotective, antispasmotic, antirheumatic (al bratty et al., 2020) 15 9,12 octadecadienoic acid (z,z)-, 2,3bis [(trimethysilyl)oxy]pr opyl ester c27h54o4si2 linoleic acid ester 498 0.74 21.58 anti-inflamation (rani and kapoor, 2019) 16 4h-1-benzopyran-4one, 2-(3,4dimethoxyphenyl)-3, 5-dihydroxy-7meyhoxy c18h16o7 phenolic compound 344 0.28 25.36 antioxidant, antimicrobial, cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries (huang and irwin, 2006) 17 9,12,15octadecatrienoic acid ,2,3 bis[(trimethylsilyl)ox y ]propyl ester, (z,z,z) c27h52o4si2 alpha linoleic acid 496 0.28 25.36 anti-oxidant, anti-diabetic, and anti-inflammatory (rajendran et al., 2017) 18 eicosamethyl,cyclode casiloxane c20h60o10si10 organoheterosilane 740 2.59 27.05 prevent degenerative diseases (budayatin et al., 2021) 19 silcone oil n/a polysiloxane compounds 0 2.59 27.05 toxic for bedbug(insecticide) (zha et al., 2018) 20 1h-purin-6amine,[(2flourophenyl)methyl] c12h10fn5 amino compounds 243 2.59 27.05 anti-oxidant (budayatin et al., 2021) 21 neophytadiene c20h38 alkenes 278 6.43 30.04 analgesic, antipyretic, antiinflammatory, international journal of applied biology, 5(2), 2021 103 antimicrobial, and antioxidant compound (venkata raman et al., 2012) 22 2,6,10-trimethyl,14ethylene-14pentadecene c20h38 alkenes 278 6.43 30.04 antiproliferative activity (devakumar et al., 2017) 23 phytol isomer c20h40o diterpene 296 0.29 30.93 antimicrobial, antioxidant, and anticancer activities (rani and kapoor, 2019) 24 7-methyl-ztetradecen-1-ol acetate c17h32o2 acetate ester 268 0.64 31.36 anticancer, antiinflammatory, hepatoprotective (hameed et al., 2015) 25 2-dodecen-1-yl()succinic anhydride c16h26o3 succinicacid anhydride 266 0.13 33.83 antineoplastic agents, antioxidants, antimicrobial (jatin and sonawani, 2016) 26 hexadecanoic acid, methyl ester c17h34o palmitic acid ester 270 2.09 34.34 anti-oxidant, decrease blood cholesterol, antiinflammatory (hema et al., 2015) 27 pentadecanoic acid, 14-methyl-, methyl ester c17h34o ester 270 2.09 34.34 antimicrobial, antifungal (beschi et al., 2021) 28 dasycarpidan-1methanol, (ester) c20h26n2o2 ester 326 0.74 21.58 antimicrobial (rani and kapoor, 2019) 29 dibutyl phthalate c16h22o4 benzoic acid ester 278 0.98 38.03 use in cosmotics (mary ann liebert, 1985), ectoparasiticide (prabhu et al., 2018) 30 phthalic acid, butyl undecyl ester c23h36o4 ester 376 0.98 38.03 antimicrobial antibacterial & anti-inflammatory (al-gara’awi et al., 2019) 31 phthalic acid, butyl tetradecyl ester c26h42o4 ester 418 0.98 38.03 antimicrobial activity (bekele et al., 2016) 32 11,14-eicosadienoic acid, methyl ester c21h38o2 ester 322 0.40 39.20 anti-inflammatory, antioxidant, anti-arthritic, anticoronary. (chinnasamy p.s et al., 2018) 33 1,2benzenedicarboxylic acid, butyl decyl ester c22h34o4 ester 362 5.04 39.35 antimicrobial activity (shoge et al., 2016) 34 diisooctyl phthalate c24h38o4 phthalic acid derivative (ester) 390 1.80 50.09 antimicrobial activity (ali shafaghat, 2012) 35 9,12,15octadecatrienoic acid, 2,3dihydroxypropyl ester, (z,z,z) c21h36o4 linoleic acid ester 352 0.87 52.61 analgesic, antipyretic, anticonvulsant, antiseptic (srivastava et al., 2015) 36 9,12,15octadecatrienoic acid, 2-phenyl-1,3dioxan-5-yl ester c28h40o4 phenolic ester 440 0.46 58.55 antimicrobial & antiinflammatory (kadhim et al., 2017) 37 cholest-5-en-3-ol, 24propylidene-, (3á) c30h50o fatty acid 426 6.09 63.51 antibacterial activity (hussein et al., 2017) 38 cis-13-eicosenoic acid c20h38o2 fatty acid 310 0.08 33.78 anti-inflammatory activity (sosa et al., 2016) international journal of applied biology, 5(2), 2021 104 39 ppropiolic acid, 3-(1hydroxy-2-isopropyl5 methylcyclohexyl) c13h20o3 fatty acid 224 1.72 37.54 anti-angiogenic activity against solid tumor growth (hussein et al., 2016) 40 2-nonadecanone 2,4dinitrophenylhydrazi ne c25h42n4o4 nitrogen compound 462 0.36 50.66 antimicrobial activity (muthulakshmi et al., 2012) 41 ethyl iso-allocholate c26h44o5 steroid derivative 436 0.48 39.87 antimicrobial,antioxidant, anti-inflammatory & antiarthritic antiasthmatic (sheela and uthayakumari, 2013) 42 1,2-15,16diepoxyhexadecane c16h30o2 epoxide 254 0.40 39.20 antitumor, antiinflammatory (hameed et al., 2016) 43 milbemycin b, 6,28anhydro-15-chloro25-isopropy l-13dehydro-5-odemethyl-4-methyl c33h47clo7 -----590 0.33 58.77 anti mrsa (vilas and amit, 2015) the gc-ms analysis on the haemolymph of scarabeus sacer revealed presence of some bioactive compounds such as alcohols, terpenoids, ketones, phenolic compounds, alkanes, alkenes, amino-compounds, fatty acids and steroids. alcohols were discovered to have antimicrobial activity (gołêbiowski et al., 2012). isochiapin b, 3, 7, 11, 15-tetramethyl2-hexadecen-1-ol and phytol isomer are terpenoids present in haemolymph of s. sacer. terpenoid compounds (phorbol, isochiapin b, stigmasterol acetate, and b-sitosterol) were detected in essential oil of achillea fragmmentissma that well known for their biological activities as anti-insect and anti-tumor agents (elsharkawy, 2016). terpenes are bioactive compounds detected in ulva fasciata, u. lactuca and corallina mediterranea seaweeds extract and steroids were detected in the extracts of u. fasciata, and amphiroa anceps seaweeds (mofeed et al., 2021). most of these compounds exhibit biological activities such as anticancer, antiviral, antioxidant, and anti-inflammatories (jiang et al., 2017). phytol isomer is a diterpenes identified in the haemolymph of adult s. sacer. phytol is a bioactive compound that has a potent anticancer activity (sheeja et al., 2016). it also serve as a chemical attractant for parasitoids, according to research on these species: lucilia sericata (gobiowski et al., 2012c), leptinotarsa decemlineata (nelson et al., 2003). alkanes can help distinguish organisms by acting as a chemical signal (lockey 1988). alkanes were also marked in the surface lipids of liposcelis bostrychophila, cryptolestes ferrugineus (howard and lord 2003) and laelius utilis (howard, 1992). dotriacontane is saturated hydrocarbons present in haemolymph of scarab beetle and reported to has antimicrobial, antifungal, anti inflamatory and cytotoxic activity (harris, 1992). also the hydrocarbons used to distinguish between the male and female of sarcophaga species (moore et al., 2021). alkanes were also marked in the surface lipids of liposcelis bostrychophila, cryptolestes ferrugineus (howard and lord, 2003) and laelius utilis (howard r. w, 1992). larvae of potato beetle contain hydrocarbons of high molecular weight, particularly tetrapentacontane (c54h 110), pentapentacontane (c55h 112) and heptapentacontane (c57h 116) (ardenne et al., 1965). pentadecanoic acid, 9,12,15-octadecatrienoic acid, hexadecanoic acid methyl ester were detected in the haemolymph of adult s. sacer. pentadecanoic acid and 9,12,15octadecatrienoic acid reported to have anti-inflammatory, antimicrobial, antioxidants, and antiproliferative activity (rani and kapoor, 2019). hexadecanoic acid methyl ester is also known as palmitic acid ester and efficiently used as an antioxidant, pesticide, anti international journal of applied biology, 5(2), 2021 105 androgenic, nematicide, flavoring agent, hypocholesterolemic, and lubricant (karthikeyan and sudan, 2017). also hexadeconic acid was the major fatty acid in sargassum granuliferum seaweed which prevents the biofilm forming bacteria (bakar et al., 2017). esters with even longer c22, c24, c42 and c46 carbon chains were determined in aleurotithius timberlakei (nelson et al., 1997). in the haemolymph of scarab beetle, fatty acids such as – cis-13-eicosenoic acid, cholest-5-en-3-ol, 24-propylidene(3á), and ppropiolic acid, 3-(1-hydroxy-2-isopropyl-5 methylcyclohexyl) were detected. in case of leptinotarsa decemlineata (coleoptera: chrysomelidae), the presence of c6, c9, c10, c12, c14, c16 and c18 fatty acids were found. fatty acids c18 and c20 are found also in bombyx mori and blatella germanica (barlow j. s., 1964). fatty acids c18:1, c18:2 and c18:3 were estimated in the larvae of drosophila melanogaster, musca domestica and galleria mellonella (barlow j. s., 1964). fatty acids c16 – c18 have also been determined in the surface lipids of cryptolestes ferrugineus and liposcelis bostrychophila insects (howard and lord, 2003). cholest-5-en-3-ol, 24propylidene have been detected in the methyl extract of sargassum crassifolium (albratty et al., 2021) and (erwan plouguerné et al., 2006) seperated cholest-5-en-3-ol, 24propylidene from the red alga grateloupia turuturu. in the adult scarab beetle haemolymph, there is a ketonic compounds such as 4h-1benzopyran-4 one, 2-(3, 4-dimethoxyphenyl)-3,5-dihydroxy-7-methoxy that act as antioxidant, antimicrobial, cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries (albergoni et al., 1980). also ketones are separated from tessaratoma papillosa (zhang et al., 2009). the relationships of these analyzed compounds in insects play vital roles as they can be transmitters of information and signals (taylor et al., 2012)and also serve as pheromones (noguez et al., 2013). ethyl iso-allocholate is a steroid derivative compounds detected in the haemolymph of adult scarab beetle and in the black fruit of pistacia lentiscus, this steroid compound has antimicrobial, anti-inflammatory, anticancer, antiasthma and diuretic activities (daffodil d almeida et al., 2012) silcone oil, milbemycin b, 6, 28-anhydro-15-chloro-25-isopropyl-13-dehydro-5-odemethyl-4-met-, and dibutyl phthalate were detected in the haemolymph of adult scarab beetle. zha et al., 2018, reported that silicone oil in the bed bug is cytotoxic and has an insecticidal activity that can kill insects by physical mean that affecting on tracheal system causing asphyxiation of insects. anti mrsa activity was reported for milbemycin b, 6, 28anhydro-15-chloro-25-isopropyl-13-dehydro-5-o-demethyl-4-met(vilas and amit , 2015) and dibutyl phthalate act as ectoparasiticide (prabhu et al., 2018). table 2. antimicrobial activity (as a mean zone of inhibition) of the haemolymph of adult scarab beetle sample tested microorganisms heamolyph control fungi ketoconazole aspergillus fumigatus na 17 candida albicans na 20 gram positive bacteria: gentamycin staphylococcus aureus 8 24 http://en.wikipedia.org/wiki/leaf_beetle international journal of applied biology, 5(2), 2021 106 bacillus subtilis rcmb 015 (1) 15 26 gram negatvie bacteria: gentamycin escherichia coli 10 30 enterobacter cloacae 14 27 *na: no activity. table 3. the antimicrobial activity as minimum inhibitory concentration (mic) in g/ml of the tested microorganisms. the test was done using the diffusion agar technique sample tested microorganisms heamolyph control gram positive bacteria: gentamycin staphylococcus aureus 10000 3.9 bacillus subtilis 1250 1.95 gram negatvie bacteria: gentamycin escherichia coli 2500 1.95 enterobacter cloacae 1250 3.9 *na: no activity. the present works approved that the haemolymph of s. sacer possesses antibacterial activity against gram –negative bacteria (escherichia coli, enterobacter cloacae) and against gram-positive bacteria (staphylococcus aureus, bacillus subtilis). no antifungal activity had been investigated against aspergillus fumigatus and candida albicans. there are many works hassling to our work; the methanol extract of oriental hornet vespa orientalis and zophobas mori (coleoptera:tenebrionidae) larva show antibacterial activity against e. coli and no antifungal activity (hassan et al., 2015). contrary to our results, the whole body extract of housefly maggots show no activity against e. coli and exhibit antifungal activity (meylaers et al., 2004). while (hou et al., 2007) documented that the extract of the housefly maggots have higher activity against grampositive bacteria than gram negative bacteria and had not antifungal activity yet. most of insect extracts show antibacterial activity against gram-positive and gramnegative bacteria, the silk worm bombyx mori (seiichi hara and yamakawa, 1995), the european bumble bee, bombus pascuorum (rees ja et al., 1997) and tenebrio molitor larvae (lee et al., 1998). on the other hand, some other insects revealed activity only against gram-positive bacteria as aedes aegypti (lowenberger et al., 1995), chironomus plumosus (lauth et al., 1998) and anopheles gambiae (vizioli et al., 2001). synthetic antibiotics and antimicrobials have contributed to public health and stimulated the growth of livestock. conversely, overuse and abuse of antibiotics and antimicrobial drugs may causes drug-resistant bacteria, which threaten public and livestock health. several studies reported that insects manufacture antimicrobial peptides (amps) international journal of applied biology, 5(2), 2021 107 which act as a natural antibiotic (vetterli et al., 2018). insects not only perform different roles in the environment, but also host a variety of community of microorganisms. the complicated cellular and humoral mechanisms include the innate immune system of an insect (kanost et al., 2004). the cellular mechanism is rely on phagocytosis process which is activated by enzymes and invading microorganisms then encapsulated by the hemolymph. moreover, the humoral response is represented in the production of broad-spectrum antimicrobial peptides (amps), reactive oxygen or nitrogen intermediates, and complex enzymatic cascades that help to regulate hemolymph coagulation or melanization (ahmed mh ali et al., 2020). the presence of microorganisms invading insects causes the fat body to rapidly synthesize amps, which are then released into the hemolymph (hoffmann and reichhart, 2002). previous research shows that each insect species produces a distinct antimicrobial peptide that acts against specific microorganisms (yi et al., 2014). on the other hand, in order to enhance the insect’s defense system against other pathogens, some of the peptides are expressed simultaneously, encouraging synergism (rahnamaeian et al., 2015). as such, amps have a specific modes of action, such as altering the electrochemical gradient at the membrane, producing reactive oxygen/nitrogen species (ros/rns) that cause cell death, inhibiting protein synthesis, and permeabilizing the cell membrane (thevissen et al., 2004). amps have pharmacological properties such as low molecular weight, high water solubility, broad-spectrum antimicrobial activity, and low levels of cytotoxicity (lei et al., 2019). (turillazzi et al., 2004) reported that the antibacterial activity in salivary secretions of polistes dominulus larvae inhibits growth of gram positive bacillus subtilis and gram negative e. coli. there are number of studies that have tested ability of the insect extracts against pathogenic bacteria, especially antimicrobial peptides extracted from various insects maggots (guo et al., 2007), dung beetles (mohtar et al., 2014), red palm weevil (chernysh et al., 2015), pupae of the giant silk moths (sewify et al., 2017). contrary to our study on the haemolymph of adult s. sacer, the non-induced hemolymph of dung beetle, onthophagus taurus did not show inhibitory activity against any of the bacterial strains and fungus. it does not mean that peptides are absent but it may be present in smaller quantity so that no visible action in in-vitro studies is detected (patil and kumar, 2013). but the immune induced hemolymph exhibits activity against all tested bacteria and no activity against fungus. therefore, the peptide is active against prokaryotes and doesn’t affect the fungus which is a eukaryote. many studies on insect species assert that bacteria injected into the haemocoel stimulate the synthesis of number of peptides and proteins which are active singly or in concert against the invaders and are secreted into the hemolymph (gillespie jp et al., 1997). conclusions on conclusion, antimicrobial activity of haemolymph of adult s. sacer may be due to presence of the previous bioactive compounds which separated by the gc-ms technique. future studies are necessary to purify the compounds with antimicrobial activity and investigate their antitumor effect against different cell lines. acknowledgment great thanks and appreciation for the mycology center, al-azhar university for their cooperation. international journal of applied biology, 5(2), 2021 108 references ahmed mh ali, abdur rauf, emad m abdallah, 2020. insects as producers of antimicrobial polypeptides: a short review. gsc biol. pharm. sci. 12, 102–107. https://doi.org/10.30574/gscbps.2020.12.3.0281. al-gara’awi, n.i., abu-serag, n.a., alee shaheed, k.a., bahadly, z.k. al, 2019. analysis of bioactive phytochemical compound of ( cyperus alternifolius l.) by using gas chromatography-mass spectrometry. iop conf. ser. mater. sci. eng. 571. https://doi.org/10.1088/1757-899x/571/1/012047. al bratty, m., makeen, h.a., alhazmi, h.a., alhazmi, h.a., syame, s.m., syame, s.m., abdalla, a.n., abdalla, a.n., homeida, h.e., sultana, s., ahsan, w., khalid, a., khalid, a., 2020. phytochemical, cytotoxic, and antimicrobial evaluation of the fruits of miswak plant, salvadora persica l. j. chem. 2020. https://doi.org/10.1155/2020/4521951. albergoni, v., e, p., o, c., 1980. esponse to heavy metals in organisms-i. excretion and accumulation of physiological and non physiological metals in euglena gracilis. comp biochem physiol c comp pharmacol 67, 121–127. albratty, m., bajawi, a.a.m., marei, t.m.h., shamsher alam, m., alhazmi, h.a., najmi, a., ur rehman, z., sivagurunathan moni, s., 2021. spectral analysis and antibacterial potential of bioactive principles of sargassum crassifolium j. agardh from red sea of jazan origin. saudi j. biol. sci. 28, 5745–5753. https://doi.org/10.1016/j.sjbs.2021.06.017. ali, h.a.m., imad, h.h., salah, a.i., 2015. analysis of bioactive chemical components of two medicinal plants (coriandrum sativum and melia azedarach) leaves using gas chromatography-mass spectrometry (gc-ms). african j. biotechnol. 14, 2812–2830. https://doi.org/10.5897/ajb2015.14956. ali shafaghat, 2012. phytochemical and antimicrobial activities of lavandula officinalis leaves and stems against some pathogenic microorganisms. j. med. plants res. 6, 455–460. https://doi.org/10.5897/jmpr11.1166. ardenne, m. von, osske, g., schreiber, k., steinfelder, k., r tümmler, 1965. no title. j. insect physiol. 11, 1365. bakar, k., mohamad, h., latip, j., tan, h.s., herng, g.m., 2017. fatty acids compositions of sargassum granuliferum and dictyota dichotoma and their anti-fouling activities. j. sustain. sci. manag. 12, 8–16. barlow j. s., 1964. fatty acids in some insect and spider fats. can. j. biochem. 42, 1365– 1374. bekele, d., tekie, h., asfaw, z., petros, b., 2016. bioactive chemical constituents from the leaf of oreosyce africana hook.f (cucurbitaceae) with mosquitocidal activities against adult anopheles arabiensis, the principal malaria vector in ethiopia. j. fertil. pestic. 7, 1–8. https://doi.org/10.4172/2471-2728.1000159. international journal of applied biology, 5(2), 2021 109 beschi, d.a., appavoo, m.r., wilsy, j.i., 2021. gc-ms analysis, collected from kavalkinaru area, tirunelveli district, tamil nadu, india. eur. j. mol. &amp; 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volatile characteristics of tessaratoma papillosa using spme-gc-ms. j. chromatogr. sci. 47, 291–295. contact : chiagoziem ogazirilem emerole chiagoziem.emerole@futo.edu.ng 46 abstract background: long-lasting insecticide-treated nets (llins) is one of the major interventions for the control and elimination of malaria, especially among pregnant women. aim: this study aimed to evaluate the knowledge, occurrence of malaria, utilisation and acquisition of long lasting insecticide treated net (llins) among pregnant women in nwangele l.g.a. imo state. method: this was a cross-sectional study among women attending antenatal care in a primary health facility in nwangele lga, imo state, nigeria. a total of 150 women were interviewed using structured questionnaire to obtain information on their knowledge and the coverage of llins. data analysis was done using spss version 20. results: the mean age of the women was 29years. most (81.3%) of the respondents had at least a secondary education. 92% used the llins at night time. cost of acquiring these nets were free and in good condition (97.3%). distance was listed as a hindrance to acquisition of nets (42.7%). conclusion: malaria is a very serious public health problem; prompt treatment alone cannot guarantee the achievement of the global eradication goal. all strategies must be strengthened and employed in preventing malaria occurrence if the goal is to be achieved. issn : 2580-2410 eissn : 2580-2119 coverage of llin among expectant mothers in nwangele, imo state, nigeria chiagoziem ogazirilem emerole1*, dr. salome ibe1, dr. uchechukwu madukaku chukwuocha1, prof. eunice nwoke1, prof. ikechukwu dozie1, prof. okwuoma abanobi1 1department of public health, school of health technology, federal university of technology, owerri, imo state, nigeria. pmb 1526 introduction malaria is vector – borne disease transmitted by the female anopheles mosquito (greenwood, 1992; iyer et al., 2019; organização mundial da saúde, 2019). in sub – saharan africa, malaria remains a major public health problem that affects every demographic. however, some are at increased risk of severe illness than others. these vulnerable group include under – five children and expecting mothers (feachem, jamison, & bos, 1991; world malaria report 2019, 2019). in expectant mothers, malaria increases the risk of low birth weight (lbw) (beeson, scoullar, & boeuf, 2018; borgella et al., 2013; dombrowski et al., 2018; guyatt & snow, 2004). in nigeria, malaria in pregnancy was responsible for 11% of maternal deaths (agomo, oyibo, anorlu, & agomo, 2009). in 2000, the world health organization recommended a package of interventions to prevent malaria during open access international journal of applied biology keyword llin, malaria, nigeria, maternal health, public health article history received 12 april 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 47 pregnancy. this package includes the promotion of insecticide-treated bed net (llins), intermitted preventive treatment in pregnancy and effective case management of malaria illness (health, 2013; pérez et al., 2017; who/afro, 2004). in nigeria, one of the strategies of the nigerian national strategic plan for malaria control is to create awareness on prevention of malaria in pregnancy through the use of llins, among others. the strategy also promotes the integration of malaria prevention into the safe motherhood package (ankomah et al., 2014). in a study among pregnant women in lagos state, only 11.2% and 37.5% actually used llins in two study hospitals (aina & ayeni, 2011). in some parts of nigeria, the use of bed nets among pregnant women is still low. in a study in northern nigeria, 73% of respondent have never used llin before because of lack of awareness (omotosho, ganiyu, & rabiu, 2009). in another study in ibadan, south west nigeria, only 20.9% pregnant women use llins (aluko & oluwatosin, 2012). a 2015 survey of malaria in nigeria showed that 69% of the population owned llins; of which 37% actually used their nets. imo state had 46% ownership and 6% usage of llins (national malaria elimination program (nigeria) and icf international, 2016). to promote use and uptake of these malaria preventive strategies, mass media campaigns have been initiated to sensitize the general public particularly pregnant women, on the effectiveness and long term benefits of correct and consistent use of the llins during pregnancy. in the same 2015 survey, 36% of women age 15-49 have seen or heard a message about malaria (national malaria elimination program (nigeria) and icf international, 2016). the mass media campaigns message were aired on national radio and television stations in english, pidgin english, and the three main local languages in nigeria. also bill boards with clear messages about the link between mosquitoes and malaria prevention were place at strategic locations in major cities in nigeria to further improve access to correct information. the messages on the bill boards were reproduced into posters and handbill that widely distributed across the country (ankomah et al., 2014). despite these efforts, malaria in nigeria still poses serious threats. the high rate of malaria has been attributed to some factors that affect the distribution, uptake and utilization of in insecticide treated nets (babalola et al., 2019; linn et al., 2019). therefore, the present study aims to evaluate the coverage (access and utilization) of insecticide treated nets among expectant mothers in nwangele local government area of imo state, nigeria. materials and methods study design a descriptive cross sectional study was used to determine the malaria distribution and uptake, and usage of insecticide treated nets among women of child bearing age in the study area. to ensure that the question was consistent in evaluating or testing the objective of the study, a pilot study was conducted in owerri west local government (ihiagwa) and data collected were tested or reliability using the chronbach’s alpha. with the reliability coefficient of 0.5, the questionnaire was found to be reliable. study settings the area of study is nwangele local government area, imo state. it is located at the central part of amaigbo and it is made up of 11 villages comprising of amaigbo community, international journal of applied biology, 5(1), 2021 48 abba community, dimnanume community, isu ancient kingdom community, umuozu community, isiala umuozu community, umunakara community, umudurunna community, abajah community, ogwuaga/ekitiafor community and umunna community. the urban towns of nwangele l.g.a. are abajah, isu, amaigbo, umuozu and abah, the rest are more of rural towns. the women attending antenatal care in a primary health facility in nwangele lga, imo state, nigeria were recruited for the study. sample and sampling technique to be included in the study, participants had to be aged 18+, a resident in the selected village and able to speak either english pidgin or igbo (the local language) fluently. convenience sampling was felt to be the most appropriate strategy for selecting participants, due to the somewhat wide inclusion criteria. additionally, convenience sampling offered more easily available access to data and was the most straightforward to implement. to select participants, the primary health care centre of the study area was located to recruit the participants. this method was used as the study population are expectant mothers and this group visit the health centres for pre – natal care which include intermittent preventive treatment during pregnancy. data collection the researchers administered the questionnaires to respondents found in the healthcare centers. the purpose of the study was explained to them and their consent obtained before administering the questionnaire. data analysis data collected were coded on a spreadsheet and entered into spss version 20 for analysis. quantitative data were represented on tables and charts for clarity. results and discussion the results from the data collected from 150 mothers in the study area are presented in tables and figures below. socio – demographic information is found in table 1; coverage (knowledge, access, affordability, availability, ownership and usage is found in tables 2 – 4. distribution and frequency of malaria in the study population is found in figures 1 and 2. socio-demographic characteristics table 1 shows that 30% of the respondents were aged 19 – 29 years and 66% were aged 30 years and above. 90.7% of them were married and the common occupation among them was business (56%), farming (28.7%) and civil/public servants (15.3%). 81.3% have attained secondary education. about 77.3% of them live in a 3 – 4 person household. table 1: socio-demographic characteristics of the mothers variables frequency percentage age bracket < 18 years 6 4.0 19 29 years 45 30.0 ≥ 30 99 66.0 international journal of applied biology, 5(1), 2021 49 marital status married 136 90.7 widowed 14 9.3 occupation business 84 56.0 farmer 43 28.7 civil/public servant 23 15.3 educational qualification primary 4 2.7 secondary 122 81.3 tertiary 24 16.0 household size 1 – 2 12 8.0 3 – 4 116 77.3 ˃ 5 22 14.7 malaria when asked about having suffered malaria, 57.3% reported that they last had malaria more than two weeks before the study commenced. only 21(14%) had malaria 1 – 7 days prior (figure 1). and although they don’t suffer from this often (97.3%), 2.7% suffer it very often (figure 2). figure 1: malaria distribution in nwangele lga, imo state, nigeria figure 2: frequency of suffering malaria in nwangele lga, imo state, nigeria. 14% 28,70%57,30% malaria distribution 1 7 days ago two weeks ago more than two weeks ago 2,70% 97,30% frequency of suffering malaria very often not often international journal of applied biology, 5(1), 2021 50 knowledge of long-lasting insecticidal net (llin) the common source of information about bed nets in the l.g.a is hospitals and health centers, 134(89.3%) and 80(53.3%) have 1 – 2 bed nets at home while 57(38%) and 13(8.7%) have 3 – 4 and 5 – 6 bed nets at home. most (96.7%) of the mothers believe that everybody in the house needs the net and no one is special. table 2: knowledge of insecticide treated bed nets source of knowledge on mosquito bed nets market 11 7.3 hospital/health center 134 89.3 media 4 2.7 others 1 0.7 right time to use mosquito bed net night 138 92.0 mid-night 4 2.7 other 8 5.3 who need net most mother 5 3.3 everybody 145 96.7 other use of mosquito nets pillow case 2 1.3 fishing net 20 13.3 others 128 85.3 access to long-lasting insecticidal net (llin)s these bed nets are commonly sourced at hospitals and health centres for free but despite the availability of the bed nets, only 43.3% stated that two persons in the house sleep under net while three persons sleeps among 47.3%. distance (42.7%) and bad roads (41.3%) were the major hindrances to access to bed nets. table 2: access to long-lasting insecticidal net (llin)s cost of acquiring nets free of charge 150 100.0 nature of mosquito bed net when received well packaged 146 97.3 partly torn 4 2.7 hindrance to access mosquito bed nets distance 64 42.7 bad road 62 41.3 availability 24 16.0 utilization of long-lasting insecticidal net (llin)s 49.3% of the respondents used llin while 76(50.7%) of the mothers use just treated bet nets and about 138(92%) uses the net every night compared to 8(5.3%) that uses it three time a week. but the common hindrance to sleeping under the net is because at times it’s very hot inside (26.7%) or makes one uncomfortable (36%) and other issues (31.3%) that might influences sleeping under mosquito bed nets. table 3: utilization of mosquito bed net international journal of applied biology, 5(1), 2021 51 variables frequency percentage how many mosquito bed net available in the house 1 – 2 80 53.3 3 – 4 57 38.0 5 – 6 13 8.7 how many person sleeping in a room one 6 4.0 two 65 43.3 three 71 47.3 more than three 8 5.3 frequency of usage of mosquito net every night 138 92.0 once a week 4 2.7 three times a week 8 5.3 how net is hung over the bed 150 100.0 hindrance to sleeping under net very hot inside 40 26.7 bed net disturbs breathing 9 6.0 make one uncomfortable 54 36.0 others 47 31.3 every pregnancy comes with its own risks and pregnant women are 3 times more likely to suffer from severe disease as a result of a malarial infection compared with their non pregnant counterparts (takem & d’alessandro, 2013). malaria in pregnancy compromises the outcome of pregnancy, affecting both the mother and the foetus. in malaria endemic area such as in imo state, nigeria, it is estimated that there is an alarming rate of occurrence of placental malaria (ukaga et al., 2007). the present study sought to provide reference data on the coverage of insecticide treated nets among expectant mothers in nwangele local government area of imo state, nigeria. 66% of the respondents in the present study were aged 30 and above and naturally as one advances in age, childbirth is associated with increased risk of adverse maternal perinatal outcomes, such as postpartum haemorrhage, eclampsia, and cephalopelvic disproportion, as well as adverse infant outcomes including preterm birth, poor foetal growth, low birth weight, and neonatal mortality (cavazos-rehg et al., 2015; cunnington, 2001; fraser, brockert, & ward, 1995). therefore any intervention should focus more of this age group. llin ownership and use in the study reflects on the level of awareness and campaign that goes on in the study area. these awareness campaigns have proven effective in past studies (ankomah et al., 2014). this cannot be said for poor use of llins among pregnant women in enugu (ugwu, ezechukwu, obi, ugwu, & okeke, 2013). two-thirds of all households had at least one llin acquired free of charge (100%) and in good condition (97.3%). when asked about ease of access to llins, 42.7% recorded distance as a hindrance to acquiring more long-lasting insecticidal net (llin). this has been noted in a similar study as a hindrance to accessing timely care for malaria of under – five children in south eastern nigeria (uchechukwu madukaku chukwuocha, okpanma, nwakwuo, & dozie, 2014). the mothers showed enthusiasm to accept and use the llins. this is very promising for the malaria prevention and elimination programmes. factors such as high literacy level and international journal of applied biology, 5(1), 2021 52 awareness about malaria in the area could have contributed to this (uchechukwu m. chukwuocha et al., 2018). llins have been proven to reduce malaria (binka & akweongo, 2006; malaria consortium, 2016). in the present study, reasons for not sleeping under net, there are some beliefs that act as a barrier, such as belief that llins disturbs breathing (60%), very hot inside (26.7%), while (36%) says that mosquito net makes one uncomfortable. these finding were consistent with the result in mukono district, uganda few pregnant women reportedly do not use llins due to the belief that the chemicals used to impregnated nets have dangerous effects on pregnancy and the foetus due to its repellent action against mosquitoes [30], [31]. reported misuse of llins for anything other than protection against malaria was noted. 1.3% of household used mosquito net as pillow case and another 13.3% used it as fishing net. a similar to research from zambia showed only 3% of house holds reported misuse of llins (eisele, thwing, & keating, 2011). these data also supported a review of evidence that shows that widespread llin misuse in africa has been overblown by media report (berthe et al., 2019)(eisele et al., 2011; minakawa, dida, sonye, futami, & kaneko, 2008) malaria is a huge global health burden due to its mortality, morbidity and cost to economies. it is necessary to eliminate the disease in all countries where possible to achieve the world health organization target of > 90% reduction by 2030 (“global technical strategy for malaria 2016–2030,” 2016). with every study there are limitations, one of the limitations of the present study is that these results are likely not to be generalizable to rural populations in imo state or urban populations. in addition, the questions were translated into the local language which creates a risk of loss of meaning through translation. conclusions malaria is a very serious public health problem; prompt treatment alone cannot guarantee the achievement of the global eradication goal. all strategies must be strengthened and employed in preventing malaria occurrence if the goal is to be achieved. therefore llin must be provided for vulnerable groups and awareness improved to counter unhealthy beliefs on use of llins, while environmental management must be given adequate attention so as to reduce the vector population. ethics approval and consent to participate ethical approval was granted by the research ethics committee of the school of health technology, federal university of technology, owerri, nigeria. data handling was in accordance with the data protection act. permission was obtained from the community leaders. informed oral consent was obtained from all the study participants before they were allowed to take part in the study. participants were given an individual identification number, so there was no personal identifiable information attached to the data. consent for publication not applicable. international journal of applied biology, 5(1), 2021 53 availability of data and materials the datasets during and/or analysed during the current study are available from the corresponding author on reasonable request. competing interests no conflicts of interest. funding no funding was received. authors’ contributions dr. umc designed the study and collected the data. prof. en supervised the data collection. prof. id proofread the manuscript. prof. oa interpreted the data. coe drafted the manuscript and analysed the data. all authors read and approved the final manuscript. acknowledgments we want to thank all the patients who participated in this study a primary health facility in nwangele lga, imo state, nigeria, for their expertise and use of their facilities. references agomo, c. o., oyibo, w. a., anorlu, r. i., & agomo, p. u. 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(2019). contact: umm-kulthum abubakar gimba ukgimba@gmail.com 26 abstract heavy metal pollution has led to serious consequences since the dawn of industrialization. the aim of this study is to compare the speciation of heavy metals in bioaugmented and non-bioaugmented soils from taman beringin landfill, malaysia. this is to allow the proper understanding of the specific forms of heavy metals and their mobility in the environment. sequential extraction process based on tessier et al. (1979), tsang et al (2007) and standard iso 11466 (1995) was performed for the determination of eight heavy metals (cr, mn, co, ni, cu, zn, cd and pb) in soils samples. the results revealed that the percentage reduction of the mobile and non mobile forms (f1, f2, f3, f4 and f5) of heavy metals varied for all metals in nonbioaugmented and bioaugmented soils. additionally, the distribution of the specific form of the metals ( for mn, co, ni, cu and pb) changed after bioaugmentation at day 100. issn : 2580-2410 eissn : 2580-2119 chemical speciation of heavy metals in bioaugmented and non-bioaugmented soils from taman beringin landfill umm-kulthum abubakar gimba 1* , sharifah mohammad 3,4, fauziah shahul hamid 2,4, jayanthi barasarathi 2,4, emenike chijioke uche 2,4, siti farhana abdul raof 3 1department federal university of technology minna, p.m.b 65, niger state, nigeria. 2institute of biological sciences, faculty of science, university of malaya, kuala lumpur, malaysia. 3department of chemistry, faculty of science, university of malaya, kuala lumpur 50603, malaysia 4centre for research in waste management, university of malaya, 50603 kuala lumpur, malaysia. introduction global industrial revolution has led to the release of unprecedented amount of toxic substances (pollutants) into the environment. these pollutants not only pose threats to human health but also to the environment (air, soil, flora, fauna and water). major sources of pollutants are attributed to rapid urbanization and increased population growth, invention of new products, methods of waste disposal, agricultural activities such as factory farming, transport and manufacturing industries (ripin et al., 2014; dixit et al., 2015; dhaliwal et al., 2020). an example of a toxic and recalcitrant pollutant of concern is heavy metals. heavy metals are group of metals and metalloids with density greater than 4±1 g/cm³ and typically with atomic number 21 or higher. examples of heavy metals includes cadmium(cd), iron(fe), open access international journal of applied biology keyword speciation, landfill, bioaugmentation, soil, mobility. article history received 03 december 2020 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 27 cobalt(co), chromium(cr), copper(cu), mercury(hg), lead(pb), zinc(zn), arsenic(as), manganese(mn), nickel(ni), selenium(se) and the platinum group metals, which comprises platinum, palladium, rhodium, ruthenium, osmium, and iridium (mosa et al., 2015; wolowiec et al., 2019). there are two major sources through which heavy metals enter into the environment; natural sources and anthropogenic or man-made sources. in nature, trace quantities of some these metal such as fe, ,se, cd, pb, cu,, zn, ,mn, ni, v, cr are some of the essential micronutrients needed by life forms and they are only deleterious in high amounts (masindi & muedi, 2018; singh et al., 2018; rai et al., 2019). according to ripin et al., (2014) and lai et al., (2011) sources and fluxes of anthropogenically generated heavy metals have received significant attention worldwide. this concern has also been raised in malaysia. heavy metal pollution concerns are due to its properties and behavior in the environment. one of the most important properties of these metals which differentiate them from some other toxic pollutants is that they are not biodegradable in the environment. another problem associated with these metals is their potential for bioaccumulation and biomagnification causing heavier exposure for organisms than is present in the environment alone (sameera et al., 2011). natural degradation of pollutants such as heavy metals is becoming increasingly difficult because of the increased quantity, heterogeneity and increased complexity of pollutants released into the environment (masindi & muedi, 2018). various methods have been employed to achieve heavy metal removal from a given environment (dhaliwal et al., 2020). these methods include physicochemical methods such as excavation and landfill thermal treatment, acid leaching, electro reclamation, chemical precipitation, electrochemical treatment, reverse osmosis, freeze crystallization, electrodialysis, cementation, bioremediation, chemical oxidation and reduction, ion exchange, filtration, starch xanthate adsorption, solvent extraction (sinha et al., 2010; mohammed et al., 2011; shah, 2014; li et al., 2019). compared to other methods, bioremediation is considered the safest and best, it is also relatively low-cost and requires low-technology techniques. bioremediation techniques also has high public acceptance (because it is safer for the environment) and can often be carried out on site. (shah, 2014). in this study, speciation analysis of bioaugmented and non bioaugmented leachate contaminated soil from taman beringin landfill site in malaysia was carried out. this is to ascertain whether or not indigenous microbes found in taman beringin landfill site can be used to remove heavy metals such as cr, mn, co, ni, cu, zn, cd and pb that were observed in the leachate contaminated soil. materials and methods description of the sampled landfill taman beringin landfill is located at north jinjang which is about 10 km north west of kuala lumpur city centre. it received wastes from municipal, commercial, agricultural, recreational, domestic and mixed industrial wastes originating from households and industrial premises (ashraf et al., 2019). the landfill covers an area of about 16 hectares. the landfill, which is not a sanitary type, started its disposal activities since 1991 and was closed in early 2005 (thus currently inactive) (ashraf et al., 2019). ex disposal sites such as taman beringin (tb) landfill have been proposed for redevelopment into resid ential buildings, public parks and commercial premises in the national urban policy in malaysia by international journal of applied biology, 5(1), 2021 28 the year 2020 (simis, awang & arifin, 2016). figure 1 shows the three sampled stations (inset figure 1). speciation of heavy metals using sequential extraction technique in this study, a sequential extraction process based on tessier et al. (1979), tsang et al (2007) and standard iso 11466 (1995) was performed for the determination of eight heavy metals (cr, mn, co, ni, cu, zn, cd and pb) in soils samples. equal weight of soil samples taken from the three sampled locations were mixed together. an initial weight of 1.0 g was used in the sequential extraction process and all sample analyses were ran in triplicates. the extraction procedures employed are detailed as follows: fraction 1 (f1) (exchangeable metal fraction) 1g sample was extracted with 10 ml of 1m magnesium chloride (mgcl2) at ph 7 for 2 hours at room temperature with continuous agitation. fraction 2 (f2) (carbonate bound metal fraction) the residue from f1 was continuously agitated with 10 ml of 1m sodium acetate (naoac) adjusted to ph 5 with acetic acid (hoac) at room temperature for 5 hours. fraction 3 (f3) (fe-mn oxides bound metal fraction) residue from f2 was mixed with 20 ml of 0.04m hydroxylammonium hydrochloride (nh2oh-hcl) in 25% (v/v) hoac for 6 hours at 96 °c with occasional agitation. after cooling, the sample was diluted to 20 ml with deionised water. fraction 4 (f4) (organic matter and sulphide bound metal fraction) residue from f3 was extracted with 3 ml of 0.02m nitric acid (hno3) and 5 ml of 30% hydrogen peroxide (h2o2) (adjusted to ph 2 with hno3), heated to 85 °c for 2 hours, then extracted with another 3 ml aliquot of 30% h2o2 (adjusted to ph 2 with hno3) at 85°c for 3 hours with intermittent agitation. after cooling 5 ml of 3.2 m nh4oac in 20% (v/v) hno3 was added, with continuous agitation for 30 minutes. fraction 5 (f5) (residual metal fraction) the residue from f4 was digested with 9 ml of 12 m hydrochloric acid (hcl) followed by 3ml of 15.8 m hno3 added dropwise to reduce foam. after that, 5 ml of 0.5 m hno3 was added and kept for 16 hours at room temperature to oxidize the organic matter in the soil. the mixture was then heated and maintained for 2 hours before cooling. thereafter, samples were centrifuged at 3500 rpm for eight minutes at room temperature for each extraction and the supernatants from each extraction were subjected to icp-ms analysis. prior to the start of the next extraction step, 10 ml deionised water was used to wash samples and then the washing solution was discarded after centrifugation. all the experiments were carried out in triplicates to reduce systematic error. bioaugmentation setup in this study, a blend of a total of fourteen (14) strains of bacteria from tb landfill was used in the bioaugmentation experiment. the following steps were taken to inoculate the blend of microorganisms into the leachate contaminated soil: international journal of applied biology, 5(1), 2021 29 a. leachate contaminated soil from all sampled stations were mixed together and prepared by removing unwanted debris such as stones, plastic, broken glass, grasses etc b. next, equal portions of soil in triplicates were set up under different conditions: ta (leachate contaminated soil amended with all the isolated bacteria strains from tb landfill) and tc (control soil which is leachate contaminated soil without any amendment with micro organisms) c. ta was watered with 200 ml of the inoculum (obtained from equal volumes of pooled discrete species) each containing about 3 x 109 cfu g-1 while tc was not watered with the inoculum d. after the microbial formula was introduced into the designated microcosm’s soils, it marked the start of the bioaugmentation experiment. e. thereafter, portions of the soil microcosms were taken from ta and tc every 20 days during the experimental time-course (until 100 days) for heavy metal analysis using sequential extraction procedure and assessment of the total count of microbial population. f. the soil moisture of the bioaugmentation setup was maintained during the experimental time-course by watering with distilled water. caution was exercised during watering to avoid leaching of metal contents and contamination of the soil. results and discussion chemical speciation of heavy metals in bioaugmented and nonbioaugmented soils from tb landfill the sequential extraction procedure revealed metals in the f1, f2 and f3 which is the mobile fractions or forms of heavy metals in tb landfill. metals observed in these fractions are bioavailable and potentially toxic. the last two fractions (f4 and f5) are the non mobile fractions or forms of metals observed in tb landfill. metals extracted in the f4 and f5 are generally considered neither mobile nor bioavailable because they are bound to silicates and primary minerals in uncontaminated soils and sediments (ashraf et al., 2011) and therefore may not pose immediate threat to the environment. in this study, in order to gain an understanding of the mechanism by which bioremediation of heavy metals in leachate contaminated soil from tb landfill is reduced, sequential extraction procedures was used to identify changes associated with metal speciation in bioaugmented soil. the results of the sequential extraction obtained at day 100 (which marked the end of the bioaugmentation) were compared to day 0 i.e prior to the addition of the microorganism (at the beginning of the bioaugmentation) and with control soils. detailed discussion of each metal and their speciation before and after bioaugmentation is given in subsequent sections. the percentage reduction of the heavy metals at day 100 compared to day 0 was calculated using the following formula: removal % = [(co cf)/ co] × 100…………….equation 1 where co and cf are the initial and final concentration (μg/g) of metal in soil, respectively (salehzadeh, 2013). international journal of applied biology, 5(1), 2021 30 (a) chromium the distribution of cr in tb landfill indicates that its content in various fractions varied in the microcosm at the beginning of the experiment (in non bioaugmented soil at day 0), f4 and f5 had the highest concentration at 1.610 μg/g and 6.640 μg/g, respectively while f1, f2 and f3 were 0.060 μg/g, 0.070 μg/g and 0.700 μg/g of cr, respectively. the f4 and f5 recorded the highest amount of cr and thus the immobile fraction predominates over the mobile fraction in non bioaugmented soil at day 0 (0.360 μg/g in f4 and 0.400 μg/ in f5) and in bioaugmented soil at day100 (0.690 μg/g in f4 and 1.000 μg/g in f5). furthermore, concentration of cr was found to have decreased in bioaugmented soil at day 100 (0.690 μg/g in f4 and 1.000 μg/g in f5) across all fractions. the highest percentage removal was observed in f1 in bioaugmented soil at day 100. similar to this study, jeyasingh and philip (2005) reported that indigenous soil microbial community were able to reduce approximately 97% of hexavalent cr (vi) (mobile and toxic form of cr) in contaminated soil. also bader et al., (1999) reported that reduction in cr concentration in contaminated sites is due to widespread cr (vi) reducing bacteria in such soil and thus decontaminating polluted sites as seen in this study. a decrease in percentage distribution of cr in bioaugmented soil at day 100 (except in f2 and f4) was observed compared to its distribution in non bioaugmented soil at day 0. cr decreased from 73.3% to 63.92% in f5, 7.71 to 7.56% in f3 and 0.66% to 0% in f1. the influence of microorganisms in contaminated soil was also observed to decrease cr distribution in f1, f2, f3 in control soil (insert figure 2). (b) manganese high concentration of mn was observed in tb landfill. sources of manganese in msw can be traced to the disposal of bottle caps, cosmetics, insecticides, paints and pigments along with domestic garbage (kanmani and gandhimathi, 2013). the mobile form of mn was highest with f1, f2, and f3 having 6.560 μg/g, 25.180 μg/g and 22.940 μg/g, respectively in non bioaugmented soil at day 0. additionally, mobile form of mn still dominated over the immobile form even in bioaugmented soil at day 100. according to nadaska (2010) and habibah et al (2014), oxide-mn is readily reduced to available forms and is an important source of mn for plants and so elevated levels observed in non bioaugmented soil at day 0 and in bioaugmented soil at day 100 is easily bioavailable in the environment. the removal efficiency in bioaugmented soil at day 100 was about 49% in f1, 71% in f2 and 64% in f3. reduction in mn immobile forms was at lower rate in f4 (19.802%) as compared to other fractions. in f5, a 76.120% reduction was also observed mn distribution and speciation before and after remediation in tb landfill revealed that mn reduced from 33.74% to 28.85% in f2 and 22.67% to 6.03% in f5. however, an increase in the percentage of f3 and f4 occurred in bioaugmented soil at day 100. addition of the microbial formula could have altered the distribution of mn in soil (insert figure 3). (c) cobalt speciation of co revealed that the most dominant fraction was in the immobile form in non bioaugmented soil at day 0 and in bioaugmented soil at day 100. additionally, complete reduction (100%) of co in f1, f2 and f4 was observed in soil with added microorganisms after day 100. the percentage distribution of co in the various chemical fractions was different from all other metals in tb. co distribution was reduced from 31.3% to 25% in f2 with a international journal of applied biology, 5(1), 2021 31 corresponding increase in the percentage distribution in f5 from 57.39% to 75%.the bioaugmentation of soil reduced the bioavailable form of co in tb landfill. complete reduction of co in control soil can be attributed natural bioattenuation phenomenon (emenike et al., 2016) (insert figure 4). in comparison to other metals in this study, low levels of cobalt recorded maybe due to the fact that typically, msw landfills such as tb may not receive cobalt containing waste such as fossil fuel and waste combustion, aircraft exhausts, cobalt containing alloys, copper and nickel smelting and refining wastes, cobalt chemicals and fertilizers derived from phosphate rocks which have been observed to be the primary source of anthropogenically induced cobalt in soils (sternenberger, 2007). (d) nickel ni was not detected in f1 and f2 in tb landfill and therefore the immobile form was more dominant especially in the residual fraction in non bioaugmented soil at day 0. the only mobile form of this metal observed was associated with the fe-mn oxide bound metal fraction at 0.430 μg/g. in bioaugmented soil at day 100, the immobile form of ni was still observed to be prevalent. further, in bioaugmented soil at day 100, the percentage reduction of ni concentration in the f3 was over 97%, thus eliminating the potential risk of this metal in the environment. similarly, over 70% reduction in the immobile form of ni was observed after day 100 of bioaugmentation (insert figure 5). the chemical fraction of ni changed in bioaugmented soil at day 100 due to the addition of microbial formula to soils. reduced percentage of ni was observed in f3 from 14.04% to 1.47% and an increase in f5 (from 79.53% to 91.18%) was observed in bioaugmented soils at day 100. ni immobilization was observed in bioaugmented and control soils due to microbial action in soil. (e) copper similar to ni, immobile forms of cu were most prevalent in non bioaugmented soil at day 0. the mobile form of cu was still dominant over the immobile form even in bioaugmented soil at day 100 with 3.010 μg/g in f4 and 4.780 μg/g in f5. in the mobile form, concentration of cu was 0.040 μg/g, 0.110 μg/g and 0.570 μg/g in f1, f2 and f3 at day 0, respectively. by day 100, 100% reduction in f1 and f2 of cu was observed while over 85% reduction in f3 was recorded thus making the immobile form still prevalent. because the concentration of cu was majorly associated with the f4 and f5, cu in tb landfill may not pose immediate threat to the environment. the percentage distribution of cu showed there is a difference in the distribution of cu in non bioaugmented soil at day 0 and in bioaugmented soil at day 100. cu was majorly found in the f5 in non bioaugmented soil at day 0. after bioaugmentation at day 100, cu was majorly found in f4 and this could be because cu was completely reduced in f1 and f2 (insert figure 6). (f) zinc similar to ni, the immobile form of zn predominate over all other fractions in non bioaugmented soil at day 0 and in bioaugmented soil at day 100. zn concentration in mobile form was 0.250 μg/g and 2.520 μg/g in f2 and f3, respectively. a complete reduction of zn in f2 was observed after day 100 treatment while almost 70% reduction was observed in f3. international journal of applied biology, 5(1), 2021 32 similar to ni, percentage distribution of zn showed that in non bioaugmented soil at day 0, zn was mostly associated with f5 (65.29%). however, after bioaugmentation at day 100, the pattern of distribution changed and thus zn became mostly associated with f3 (43.56%). complete reduction in percentage distribution of zn in f2 was recorded bioaugmented soil at day 100. this can be attributed to the addition of microbial formula in soil (insert figure 7). (g) lead pb concentration in microcosm at the beginning of the experiment at day 0 was observed to be highest in the immobile form with f4 and f5 having 0.790 μg/g and 1.980 μg/g, respectively. a contrast in the concentration of pb in f1, f4 and f5 was observed in microcosm with microbial formula after day 100 where an increase in pb concentration occurred. although metals confined in the residual fractions (immobile forms) are not usually expected to be released over short period of time under the conditions usually encountered in nature, pb observed in tb may still pose immediate threat to the environment. this is because of its increased concentration in f1 observed after day 100 of bioaugmentation. this result agreed with other studies by kulikwoska and kluimuik (2008) who reported that there was no decrease in pb concentration compared to other metals because pb was observed to form very stable complexes with humic acid. pb distribution was in the order f5>f3>f4>f2>f1 however, a decrease in f2 (from 2.05% to 1.09%) and f3 (26.41% to 6.56%) was observed (insert figure 8). conclusions the chemical speciation of heavy metals in non-bioaugmented and bioaugmented soils taman beringin landfill revealed that the percentage reduction of the mobile and non mobile forms (f1, f2, f3, f4 and f5) varied for all metals and the distribution of the specific form of metals changed after bioaugmentation at day 100. in addition, the percentage of metal distribution before (at day 0) and after bioaugmentation (day 100) highlighted that mobility of mn, co, ni, cu and pb were reduced. however, cr and zn remained highly mobile after bioaugmentation at day 100. these results indicate that microorganisms are able to reduce the mobility of most metals, however it will not completely eliminate the risk and the toxicity that maybe caused by these metals. this study highlights the need for proper assessment of ex disposal sites such taman beringin landfill before they are redeveloped into residential buildings, public parks and commercial premises as proposed in the national urban policy in malaysia by the year 2020. international journal of applied biology, 5(1), 2021 33 references ashraf, m. a., balkhair, k. s., chowdhury, a. j. k., & hanafiah, m. m. 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(2007). modeling cadmium transport in soils using sequential extraction, batch, and miscible displacement experiments. soil science society of america journal, 71(3), 674-681. wołowiec, m., komorowska-kaufman, m., pruss, a., rzepa, g., & bajda, t. (2019). removal of heavy metals and metalloids from water using drinking water treatment residuals as adsorbents: a review. minerals, 9(8), 487. contact : hermawan seftiono hermawan_seftiono@trilogi.ac.id 13 abstract broiler chicken is one of the poultry products that are very susceptible to damage due to exposure to microbes, temperature, and light. in these conditions, indicators are needed to play a role in informing related changes in the product quality. the butterfly pea can be used as an indicator because it contains anthocyanin which can change color due to the influence of ph. the objective of this study was to obtain the best indicator film formulation and to determine the film response in detecting the level of damage of the chicken meat through parameters of color change, thickness, ph, tpc, and tvbn. the methods in this research were the extraction of butterfly pea, making indicator films, and applying the film to chicken meat, and physical and chemical analysis of the product. anthocyanin was applied to the indicator film with levels of pva and chitosan 20:80 with the addition of 5 ml of dye. the change in color of the indicator could be seen at the 24-hour mark, which was a yellowish-green color. the ph value of broiler chicken meat had entered the post-rigor stage at 24 hours with a ph value of 6.595±0.049. the tvbn regression value showed that broiler chicken meat was not safe for consumption after being stored for 4 hours and 23 minutes of 40 mgn/100 g. the tpc test that has been performed in chicken meat showed that the chicken meat was not safe for consumption in storage between 0 to 8 hours at 5.598±0.068 log cfu/g to 8.322± 0.053 log cfu/g. issn : 2580-2410 eissn : 2580-2119 color indicator film from butterfly pea (clitoria ternatea l.) as smart packaging in broiler chicken meat hermawan seftiono1*, della ayu pramesti1, inanpi hidayati sumiasih1 1food technology study program, faculty of bioindustry, trilogi university, jakarta, indonesia introduction the production of broiler chicken in indonesia had been increasing each year. based on statistical data, the production of broiler chicken in 2018 was 2.144.013 tons, an increase compared to 2014 which was 1.544.379 tons (center bureau of statistic 2018). the increase in broiler chicken production indicates that there is a high level of public consumption of chicken meat due to its relatively affordable price, it can be obtained easily to obtain, and its high nutritional content (winda 2016). its high nutritional content causes broiler chicken meat to easily degraded because it is a good medium for microorganisms (barus et al. 2017). furthermore, the decomposition of protein by microorganisms resulted in foulsmelling compounds such as indole, skatole, h2s, and nh3 which accelerate the rotting process of chicken meat (warsiki et al. 2012). open access international journal of applied biology keyword anthocyanin, discoloration, ph indicator, tvbn article history received 31 march 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 14 one of the efforts that can be done to delay the rotting process of food is by using packaging. the development of packaging technology is rapid. nowadays, the packaging is not only required for basic functions, but also required to actively participate in providing protection for the product, and providing information about the condition of the product in it. among technological advances in packaging is smart packaging. smart packaging is designed to provide actual information to consumers regarding the level of freshness and safety of food products, equipped with indicator films that are placed on the internal and external parts of the packaging (riyanto et al. 2014). the development of smart packaging in the form of the film is usually to make it easier to use, film is tinted with additional dye for quality indicator (setiautami 2013). this research used butterfly pea flower as a natural dye for indicator film. the butterfly pea flower was chosen because the researchers deemed it still underutilized and had not been widely studied, even though there is great potential for it to be used as a natural ph indicator. based on research conducted by marpaung (2012), the total anthocyanin content of butterfly pea flower was 40.58 mg/liter, resulted from 10 grams of butterfly pea flower with 40 ml of water and hcl solvent. based on the information above, there was a need for research to study the utilization of butterfly pea flower as indicator film for smart packaging in detecting broiler chicken quality. the existence of this indicator film is expected to be wildly used as the packaging of chicken sold in the market in the future so that it can make it easier for consumers to assess the quality of chicken meat. this research objective is to obtain the best indicator film and to study the response of indicator film in detecting the level of damage in chicken meat through parameters of color, thickness, ph, the total amount of microbial contamination, and total volatile basic nitrogen (tvbn). materials and methods time and place this research was conducted in mei to july 2019, in laboratory of microbiology and biochemistry, food technology study program, faculty of bioindustry, trilogi university jakarta, and in laboratory of testing, bogor center for agricultural research and development. materials the main material for this research was butterfly pea flower obtained from a garden in the kedung halang area and chicken breast meat obtained from lenteng agung market. ingredients for making indicator film were chitosan, polyvinyl alcohol, glycerol, distilled water, and acetate acid. the materials for applying the smart packaging were plastic saran wrap and styrofoam. the materials used for analysis were aluminum foil, whatman filter paper no 1, distilled water, merck plate count agar (pca) media, merck buffer peptone water (bpw) media, merck 95% ethanol, laboratory center rofa potassium carbonate, nitra kimia's trichloroacetic acid 7% (tca), hcl 1.5 n, pudak scientific's 2% boric acid, nitra kimia's bromocresol green (bcg) indicator, pudak scientific's methyl red (mr) indicator, rofa laboratory center vaseline, and merck's 0.02 n hcl. tools for extracting anthocyanin from butterfly pea flower were excalibur dehydrator, kern's analytical balance, stove, pan, filter, stirrer, and thermo ta 288 thermometer. the tools for making color indicator films and smart packaging were kern's international journal of applied biology, 5(1), 2021 15 analytical balance, thermo ta 288 thermometer, beaker, stuart hot plate, magnetic stirrer, measuring cup, and plastic mold 12x12 cm in size. the tools for analysis were agilent technologies ph meter, screw micrometer, tcr 200 chromameter, petri dish, memmert's oven, erlenmeyer, test tube, burner, mortar and pestle, memmert's incubator, micropipette, uv-vis spectrophotometer, conway dish, hirayama autoclave, vortex zx3, burette, stative, and pipette. experimental methods butterfly pea flower extraction (sinha et al. 2012 with modification) butterfly pea flower that had been clean, dried using dehydrator at 60 oc for 1 hour (modification). next, 5 grams of the dried flower was mixed with 250 ml distilled water and heated at 80 oc for 5 to 10 minutes. after heating, the mixture was then cooled and filtered. the filtrate was used for the next step. making color indicator film (warsiki et al. 2013 with modification) polyvinyl alcohol was dissolved in distilled water at 80 oc for 30 minutes using a hot plate. then the chitosan was dissolved using 1% acetic acid, then distilled water was added and heated at 80 oc for 30 minutes. polyvinyl alcohol solution was mixed with the chitosanacetate solution according to the ratio (100: 0), (80:20), (60:40), (40:60), (20:80). the next step was to add 1% glycerol, then homogenization was carried out by stirring. next, butterfly pea flower extract was added as the natural dyes. the technique for staining the film was done by mixing it directly into the film solution before molding. the amount of butterfly pea flower extracts added were 5, 10, 15, and 20 ml. the homogeneous solution was molded with a plastic mold measuring 12x12 cm. modifications were made to the temperature and drying time of the indicator film, the film was dried at room temperature of 25 oc for 48 hours. application of indicator film on chicken meat (octavia 2015 with modification) the application of indicator film was conducted using a method developed by octavia (2015) with modifications. the modifications were the temperature and testing time. indicator film applied to the chicken meat by placing the indicator film sized 3 x 3 cm on the upper side of the packaging. the chicken meat was measured to be 60 grams, put on the styrofoam, and wrapped using plastic saran wrap which has previously been affixed with indicator film. nest, chicken meat was stored at room temperature of 25 ºc, rh of 50% for 48 hours. the observations were conducted at hours 0, 8, 24,32, and 48 to see the changes in indicator film. analytical method the main experiments in this research included the ph test and the anthocyanin content of butterfly pea flower extract (less & francis 1972 in nofrida 2013), indicator film thickness test (nofrida 2013), color analysis of indicator film (hunter 1958 in octavia 2015; nofrida 2013), analysis of quality degradation of meat, including ph test of chicken meat (mega et al. 2009), total plate count test (tpc) (bsn 2008), total volatile basic nitrogen (tvbn) (bsn 2009). international journal of applied biology, 5(1), 2021 16 results and discussion chemical characteristics of butterfly pea flower extract extraction aims to obtain the chemical compound of material using certain solvents (santoso & estiasih 2014). the extraction method used in this research was hot maceration with a temperature of 80 oc for 5 to 10 minutes. according to rahmawati (2017), the best heating process to prevent anthocyanin degradation was by using high temperatures for a short period. the ph value of the butterfly pea flower extract in this study was 5.838 which resulted in a purplish-blue color. the presence of a purplish-blue color in the butterfly pea flower extract indicates the presence of natural pigments. natural pigments that give butterfly pea flowers their purplish-blue color is delphinidin anthocyanins (change 2018). the analysis result of anthocyanin in butterfly pea flower was 218.232 mg/kg. the anthocyanin level obtained was lower than the research result by vankar & srivastava (2010) which was 227.42 mg/kg. the difference was due to different extraction methods, the solvent used, butterfly pea variety, and time of harvest. the extraction process of anthocyanin by vankar & srivastava (2010) used the cold maceration method by keeping the solution at room temperature, in the dark, for 2 to 3 hours, using methanol and 0.1% hcl as the solvent. while in this research, the extraction method used was hot maceration at 80 oc for 5 to 10 minutes with water as the solvent. other than extraction method and solvent, the different anthocyanin levels from each research were could also because of different butterfly pea variety used and harvest time. this was supported by ahmadiani et al. (2014), that the different varieties could result in different concentrations of anthocyanin, and the longer the harvest time, the lower the anthocyanin level. determination of best formulation the experiment was to obtain the best formulation of indicator film based on the ratio of pva, chitosan, and natural dye from butterfly pea flower extract to detect the degradation level of broiler chicken meat in storage. in this research, there were 5 formulations tested, namely f1, f2, f3, f4, and f5 and the addition of butterfly pea flower extract of 5, 10, 15, and 20 ml. the best formulation was determined by the intensity of color change during storage. the intensity of color change from 5 formulations with the addition of 5 ml butterfly pea flower extract was shown in figure 1. the formulation of f5 with the addition of 5 ml butterfly pea flower extract was chosen as the best formulation because the film changed its color from greenish blue to yellowish-green. the change in indicator film was due to the protein degradation process in chicken meat that produced basic volatile compounds (rahardjo & simon 2015). the chosen formulation was then used as an indicator film that would be applied to the smart packaging. figure 1 color changes in indicator film formulations with addition of 5 ml natural dye during storage f1 f2 f3 f4 f5 international journal of applied biology, 5(1), 2021 17 notes: f1= polyvinyl alcohol:chitosan (100:0), f2= polyvinyl alcohol:chitosan (80:20), f3= polyvinyl alcohol:chitosan (60:40), f4= polyvinyl alcohol:chitosan (40:60), f5= polyvinyl alcohol:chitosan (20:80). the color of indicator film during storage the color change in indicator film provides information to the consumers about the degradation of the product or food. the color change occurs due to the interaction of volatile bases produced during the deterioration of chicken meat with the indicator film. the resulting color showed that the range of °hue value of indicator film during 48 hours of storage was in the second quadrant area which shows a yellowish-green color. according to hasnedi (2009), the change in color of the indicator film occurs due to the interaction between the functional groups possessed by anthocyanins and volatile base compounds resulting from rotting chicken meat, so that the interaction involves the oh (hydroxyl) groups of anthocyanins and nh3 (ammonia) from volatile base compounds. this causes volatile base compounds such as amines, diethylamine, and triethylamine to be bound to the hydroxyl group so that the indicator film changes color from green to yellowish-green. the color change is due to the loss of protons (deprotonation) in the butterfly pea flower dye as the environmental conditions around the film become alkaline. table 2 color changes of indicator film during storage storage duration (hour) °hue value smart packaging indicator film 0 173.93 ± 5.76 8 162.56 ± 2.82 24 151.99 ± 1.01 32 149.32 ± 1.75 international journal of applied biology, 5(1), 2021 18 48 147.39 ± 1.21 the value of °hue of indicator film decreased during 48 hours of storage. the decrease of °hue value from 0 to 48 hours was from 173.94±5.76o to 147.39±1.21o which was around green color to yellowish-green color. the result showed that the °hue value of butterfly pea flower indicator film before storage was higher than after storage. it was because the yellow degree of b value increased while the green degree of a value stable, so it affected the °hue value. the indicator film °hue value regression equation was y= -0.5395x+169.13 with a strong correlation value (r2) of 0.8593. this showed that storage duration was correlated with a color change in indicator film. the slope value was negative, meaning that the regression model had a negative correlation. the longer storage duration, the lower °hue value, and this showed in the regression curve from 0 to 48 hours in figure 2. figure 2 the °hue value of indicator film during storage the changes in quality of broiler chicken meat stored for 48 hours at 25 °c with rh of 50%, caused color change in indicator film from before to after. the ∆e value of indicator film increased during 48 hours of storage. the increase of ∆e value was significant at hour 0 to hour 8 which was 0.93 ± 0.48 to 3.59 ± 0.70. this was because the volatile bases produced during the 0 to 8 hours storage were more than in the next hour. the indicator film ∆e value regression equation was y= 0.1361x+1.8394 with a strong correlation value (r2) of 0.8995. it showed that storage duration was correlated with the indicator film total color change. the slope value was positive, meaning that the regression had a positive correlation. the longer the storage duration, the higher the ∆e value, and this showed in the regression curve from 0 to 48 hours in figure 3. 173.94±5.76 162.56±2.82 151.99±1.01 149.32±1.75 147.39±1.21 y = -0,5395x + 169,13 r² = 0,8593 140 145 150 155 160 165 170 175 180 0 8 16 24 32 40 48 56 ºh u e v a lu e storage duration (hour) international journal of applied biology, 5(1), 2021 19 figure 3 indicator film ∆e value during storage indicator film thickness during storage the thickness was one of the test parameters to determine the physical characteristics of the indicator film that has been made. the thickness of the indicator film was influenced by the volume of the solution and the size of the mold used. according to nofrida (2013), the higher the volume of the solution, the thicker the indicator film produced, while the larger the mold size used, the thinner the indicator film would be. the decrease in the indicator film thickness from the 0 to 48 hours storage was 0.17 ± 0.05 to 0.13 ± 0.02, shown in figure 4. the indicator film thickness regression equation was y = -0.0009x + 0.1726 with a strong correlation value (r2) of 0.9809. this showed that the storage time correlates with the decreasing thickness of the indicator film. a negative slope value indicates that the regression model has a negative correlation. the longer the storage duration from 0 to 48 hours, the lower the thickness. according to sitompul & elok (2017), the decrease in indicator film thickness was affected by the type of plasticizer used. the plasticizer used in this research was glycerol. the presence of glycerol in the indicator film could cause the film to be thinner at 25 oc. according to pagliaro & michele (2008), glycerol had melting point at 18.2 oc. meaning that the glycerol in the indicator film caused the film to be moist while in the smart packaging environment. it caused the indicator film to be thinner since the melting point of glycerol is lower than the storage temperature and glycerol in the film became liquid. figure 4 indicator film thickness during storage 0.93±0.48 3.59±0.70 5.14±0.36 7.24±0.70 7.55±1.86 y = 0,1361x + 1,8394 r² = 0,8995 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 0 8 16 24 32 40 48 56 ∆ e storage duration (jam) 0.17±0.05 0.17±0.03 0.15±0.03 0.14±0.03 0.13±0.02 y = -0,0009x + 0,1726 r² = 0,9809 0,00 0,04 0,08 0,12 0,16 0,20 0,24 0,28 0 8 16 24 32 40 48 56 k e te b a la n ( m m ) lama penyimpanan (jam) international journal of applied biology, 5(1), 2021 20 the ph value of chicken meat during storage the ph value is an important parameter in determining meat quality. this is because the ph value could show how much damage happened to broiler chicken meat. the ph value of chicken meat obtained from the 0 to 8 hours observation decreased by 6,039 ± 0.087 to 5,778 ± 0.008. the decrease in the ph value of chicken meat was due to the rigor mortis phase. according to lawrie (2003), a decrease in ph is caused by depletion of oxygen supply as a result of stopped blood circulation to muscle tissue. this condition causes the accumulation of lactic acid due to the breakdown of glycogen through the anaerobic glycolysis process. lactic acid accumulation will stop after muscle glycogen reserves are depleted or reach the final ph value (wowor et al. 2014). there was an increase in ph value during 8 to 48 hours by 5.778±0.008 to 7.440±0.020. the increase in ph value was because the chicken meat had entered the postrigor mortis phase. according to dengen (2015), the increase in ph at the post-rigor mortis phase indicates the chemical compound decomposition process, specifically protein that broken into amino acid through deamination process and resulted in ammonia. this was also supported by riyadi et al. (2014), who stated that the increase of ph value in the post rigor phase is due to bacterial activity that the increase in ph value in the post rigor phase is due to the activity of bacteria that break down the protein with the final result of volatile alkaline compounds such as ammonia. the chicken meat value regression equation was y= 0.034x+5.8063 with a strong correlation value (r2) of 0.9164. it showed that storage duration was positively correlated with the ph value. the slope value was positive, meaning that the regression had a positive correlation. furthermore, this is illustrated in the regression curve with ph value decreased from 0 to 8 hours and increased from 8 to 48 hours in figure 5. figure 5 the ph value of broiler chicken meat during storage tvbn value of broiler chicken meat during storage the total volatile basic nitrogen (tvbn) test is one of the tests to determine the deterioration level of chicken meat quality. the principle of tvbn testing is the evaporation process of volatile alkaline compounds present in the sample during storage (wally et al. 2015). the tvbn value in broiler chicken meat increased during storage for 48 hours at 25 oc with rh 50%. the increase in the tvbn value from the 0 to 48 hours storage was from 30.8 ± 10.7 mgn/100g to 67.2 ± 15.8 mgn/100g, showed in figure 6. the increase of tvbn during storage was due to microbial activity in the chicken 6.039±0.087 5.778±0.008 6.595±0.049 6.989±0.114 7.440±0.020 y = 0,034x + 5,8063 r² = 0,9164 5,500 6,000 6,500 7,000 7,500 8,000 0 8 16 24 32 40 48 56 p h storage duration (hour) international journal of applied biology, 5(1), 2021 21 meat causing the degradation process of protein and produced basic nitrogen compound, including volatile basic nitrogen namely ammonia (nh3), dimethylamine (dma), monomethyl amine (mma), and trimethylamine (tma) (sahliyah 2017). this was supported by waryani et al. (2014), who stated that the increase of tvbn value is because of the protein degradation process due to the enzyme and putrefactive bacterial activity. the chicken meat tvbn value regression equation was y= 0.7034x+36.909 with a strong correlation value (r2) of 0.895. it showed that storage duration was positively correlated with the tvbn value. the slope value was positive, meaning that the regression had a positive correlation. furthermore, this is illustrated in the regression curve with increasing tvbn value from 0 to 48 hours. figure 6 the tvbn value of broiler chicken meat during storage research conducted by balamatsia et al. (2007) stated that the maximum tvbn value of chicken meat was 40 mgn/100g. based on the regression calculation of y= 0.7034x+36.909, broiler chicken meat had been degraded at 4 hour 23 minutes with a tvbn of 40 mgn/100 g, the maximum limit of tvbm value to be consumed by human. the tpc value of broiler chicken meat during storage the total plate count (tpc) test is a parameter to determine how many microbes in a product or food by counting the colony grown in an agar medium. the number of microbes is the main factor to determine the quality of broiler meat during storage at a temperature of 25 oc and rh 50%. the number of microbes increased significantly during storage at 0 to 8 hours, which was from 5.60±0.07 log cfu/g to 8.32±0.05 log cfu/g. that was because of the high availability of nutrients in chicken meat still, causing rapid growth of microbes (sukmawati et al. 2018). the growth of microbes continued to increase from 24 to 48 hours of storage, which was from 9.11±0.06 log cfu/g to 9.55±0.24 log cfu/g. the longer storage duration the more degradation in chicken meat due to the higher number of microbes (jaelani et al. 2016). the growth of microbes in the chicken meat was because chicken meat had high protein content that can be a very good medium for microbial growth. chicken meat tpc value regression correlation was y= 0.0718x+6.8014 with a good correlation value (r2) of 0.6948. it showed that storage duration was positively correlated with the tpc value. the slope value was positive, meaning that the regression had a positive correlation. furthermore, this is illustrated in the regression curve with increasing tpc value from 0 to 48 hours shown in figure 7. 30.8±10.7 47.6±10.7 56.0±9.1 61.6±19.4 67.2±15.8 y = 0,7034x + 36,909 r² = 0,895 10,0 20,0 30,0 40,0 50,0 60,0 70,0 80,0 0 8 16 24 32 40 48 56 t v b n ( m g n /1 0 0 g r) storage duration (hour) international journal of applied biology, 5(1), 2021 22 figure 7 broiler chicken meat tpc value during storage based on indonesian national standard 7388:2009 about the maximum microbial contamination of food, state that the maximum total of microbial contamination on fresh chicken meat for consumption is no more than 1x106 colony/g or equal to 6 log cfu/g. meaning broiler chicken meat in this research storage condition was not safe for consumption at 8 hours with a tpc of 8.23 log cfu/g, because it exceeded the maximum limit of safe microbial contamination. conclusions butterfly pea flower extract could be used as coloring dye in indicator film because it is sensitive to ph changes. the best formulation for indicator film that can detect the damage in broiler chicken meat was polyvinyl alcohol: chitosan ratio of 20:80 with the addition of 5 ml anthocyanin extract from butterfly pea flower. the color change of indicator film from green to yellowish-green on hour 24, showed that the chicken meat was not safe for consumption anymore. the color change in indicator film was correlated with ph, tvbn, and tpc value of broiler chicken meat. based on data obtained, the ph value o f chicken broiler meat had entered the post-rigor mortis phase at hour 24 with a ph of 6.595. if using tvbn regression value, it stated that broiler chicken meat could no longer be consumed after being stored for 4 hours 23 minutes, while in the tpc test, chicken meat is not suitable for consumption at storage between the 0 and 8 hours when tpc value of 5,598 log cfu g became 8,322 log cfu / g. references ahmadiani n, rebecca jr, thomas mc, monica gm. 2014. anthocyanins contents, profiles, and color characteristics of red cabbage extracts from different cultivars and maturity stages. journal of agricultural and food chemistry. 62: 7524─7531. balamatsia cc, apostolos p, michael gk, ioannis ns. 2007. possible role of volatile amines as quality-indicating metabolites in modified atmosphere-packaged chicken fillets:correlation with microbiological and sensory attributes. journal food chemistry.104(4): 1622─1628. 5.60±0.07 8.32±0.05 9.11±0.06 9.46±0.16 9.55±0.24 y = 0,0718x + 6,8014 r² = 0,6948 5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00 9,50 10,00 0 8 16 24 32 40 48 56 t p c (c f u /g ) storage duration (hour) international journal of applied biology, 5(1), 2021 23 [bsn] badan standarisasi nasional. 2008. sni 01-2897-2008. tentang metode pengujian cemaran mikroba dalam daging, telur dan susu, serta hasi olahannya. jakarta (id): badan standarisasi nasional. ______. 2009b. sni 01-2354-2009. tentang cara uji kimia-bagian 8: penentuan kadar tvb pada produk prikanan. jakarta (id): badan standarisasi nasional. badan pusat statistik. 2018. rata-rata konsumsi per kapita seminggu beberapa macam bahan makanan penting tahun 2007-2018. 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wilt. the use of biological agents, such as bacterial groups has been tried in several areas of potato plantations in java. this study aims to obtain bacillus isolates from the potato cropping area, sumber brantas, bumiaji, batu, which had the potential to suppress growth r. solanacearum. bacillus was isolated from the soil rhizosphere of potato plants, while r. solanacearum was isolated from the base of potato stems showing bacterial wilt symptoms on tetrazolium chloride (tzc) selective medium. bacillus spp. and r. solanacearum isolates were tested for hypersensitivity on the leaves of the kr15 tobacco plant. isolates that cause necrosis symptoms in tobacco leaves can be ascertained to be pathogenic. this study succeeded in obtaining 13 bacillus spp. isolates with different colony morphologies. three isolates of bacillus spp. were selected from the 13 isolates obtained, based on their ability to suppress the growth of r. solanacearum and are expected to be potential as biological agents. based on genetic analysis, the 3 bacterial isolates were identified as bacillus mycoides and bacillus weihenstephanensis. issn : 2580-2410 eissn : 2580-2119 exploration indigenous bacillus bumiaji-malang against ralstonia solanacearum causing potato bacterial wilt endang triwahyu prasetyawati1,2, tini surtiningsih3,4, ni’matuzahroh3,4,5*, purkan6, silvia kurnia sari3,4, ana mariatul khiftiyah3,4, 1faculty of agriculture, upn “veteran” jawa timur, surabaya, indonesia 2faculty of science and technology, universitas airlangga, surabaya, indonesia 3department of biology, faculty of science and technology, universitas airlangga, surabaya, indonesia 4university-center of excellenceresearch center for bio-molecule engineering, universitas airlangga, surabaya, indonesia 5faculty of advanced technology and multidiscipline, universitas airlangga, surabaya, indonesia 6department of chemistry, faculty of science and technology, universitas airlangga, surabaya, indonesia introduction one of the factors that can affect plant productivity is pathogen attack. plant pathogenic microbes (phytopathogens) can be bacteria, fungi or viruses. some important phytopathogenic bacteria include ralstonia solanacearum, pseudomonas syringae pv. open access international journal of applied biology keyword bacillus spp; bacterial wilt; pesticide reduction; potato plants; ralstonia solanacearum article history received 20 june 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 96 glycines, pseudomonas fluorescens, erwinia sp., and xanthomonas oryzae. ralstonia solanacearum is a soil borne plant pathogen that is found in many subtropical and tropical areas which naturally infects roots and multiplies in xylem tissue (hayward, 1991; yabuuchi et al. 1995). ralstonia solanacearum is one of the phytopathogens that attack potato plant. r. solanacearum causes bacterial wilt on potatoes. this pathogenic infection is reported to cause huge losses in various potato production centers and a threat to development target areas in indonesia (kuswinanti, et al., 2014). sumber brantas village, bumiaji district, batu, is one of the centers for potato plantations in east java. the use of chemicals by farmers in sumber brantas village to protect crops from pathogens is still being carried out. control of soil borne pathogens such as r. solanacearum using chemicals formulated as pesticides, fungicides, bactericides and others, is still questionable about its safety. excessive use of chemicals is harmful to both the biotic and abiotic environment. one of the efforts to overcome these problems is by using bacteria as biocontrol agents. the mechanisms for controlling pathogens by biocontrol agents include antibiosis, space and nutrition competition, microparasitism, cell wall degrading enzymes, resistance inducers, growth promoters, rhizosphere colonization and anti-quorum sensing (lo, 1998; yin et al, 2010; junaid et al, 2013). bacteria that have the ability as biocontrol agents can be isolated from soil in the plantation land (rahman et al., 2012; saha et al., 2012; ali et al., 2020). several groups of bacteria that are known to have potential as biocontrol agents are agrobacterium, pseudomonas and bacillus (fravel, 2005). the genus bacillus has the ability to synthesize several compounds that are useful in agriculture and industry. several secondary metabolites produced by several species and strains of bacillus show antibacterial and antifungal activity against plant pathogens (yu et al., 2002; ongena & jacques, 2008). bacillus also has different enzymatic capabilities in producing enzymes, including in producing amylase, protease and lipase enzymes. bacillus sp. can produce phytohormones that have the potential to develop sustainable agricultural systems. the phytohormones produced can affect plant growth, either directly or indirectly. indirectly, the phytohormones from bacteria inhibit pathogenic activity in plants, while the direct influence of these phytohormones increases plant growth and can act as a facilitator in the absorption of some nutrients from the environment. the application of bacteria as a biocontrol agent, although it is environmentally friendly, but it’s not widely used. potato farmers in sumber brantas village are also not interested in using it, due to its effects that do not meet their expectations, such as slow response effect. thus, exploration of bacteria as biocontrol agents must be carried out to obtain potential isolates to suppress growth of r. solanacearum which causes bacterial wilt on potato plants in sumber brantas village. the purpose of this study was to obtain bacillus spp. isolates from potato plantation in sumber brantas village, bumiaji district, batu, which were able to suppress the growth of the r. solanacearum which causes bacterial wilt on potato plants, and also to identify bacillus spp. isolates that were the most capable to suppress the growth of r. solanacearum. materials and methods isolation of ralstonia solanacearum diseased plant samples were taken from the potato planting area in the sumber international journal of applied biology, 5(1), 2021 97 brantas area, batu, malang. the base of the potato stems showing wilting symptoms, washed with sterile water then cut into pieces 5 mm x 5 mm in size then crushed with a mortar until smooth and added 1 ml of sterile distilled water. the bacterial suspension in the mortar was taken as much as 0.5 ml and then put in a test tube containing 4.5 ml of mgso4.7h2o. furthermore, it was diluted to reach 10-5 dilution based on the kelman (1953) method. a total of 0.1 ml of the diluted suspension was cultured in a petri dish containing sterile triphenyl tetrazolium cloride (ttc / tzc) medium, and leveled with a sterile lshaped glass rod and incubated at 30°c for 2 x 24 hours. the growing bacterial colonies were separated between virulent and non-virulent colonies on new sterile kelman tzc solid media, and re-incubated according to the previous work step. isolation of bacillus spp. soil samples from the rhizosphere of potato plantation were weighed 10 grams and put in an erlenmeyer, were added 90 ml of sterile distilled water and shaken for 30 minutes at a speed of 150 rpm. furthermore, the sample was heated in a water bath at 80 °c for 15 minutes, then dilutions were carried out in series until 10-5. dilutions of 10-3, 10-4 and 10-5 were taken as much as 100 μl grown on petri dishes containing tryptic soy agar (tsa) medium (singh et al., 2008). petri dishes were incubated at a temperature of approximately 28 oc for 24 hours. hypersensitivity test bacillus spp. isolates were propagated in tsa media, r. solanacearum isolates in tzc media for 48 hours. all colonies were harvested, each isolate was added in a tube of 2 ml of sterile distilled water. the suspension was then infiltrated into tobacco k-15 on the lower surface, incubated for 48 hours and observed for leaf necrosis. bacillus spp. isolates that showed negative reactions (no leaf necrosis were observed) were used for further testing (klement et al, 1990). meanwhile, r. solanacearum isolates used for further research were those that showed a positive reaction (showing symptoms of necrosis). antagonist test the antagonist test was carried out in vitro. r. solanacearum was cultured on tzc medium, made a suspension at λ600nm = 2 or reached a population of about 2.0x108 cfu / ml, added to yeast pepton glucose agar (ypga) medium and poured on a petri dish. ten µl of cells (od600 = 3) bacillus spp. was dropped on sterile filter paper with a diameter of 0.8 cm, and placed in a petri dish that already contained r. solanacearum. furthermore, the petri dishes were incubated for 48 hours at 28 oc and observed for the formation of clear zones and measured the diameter. as a control filter paper was dripped with sterile water and each treatment was repeated three times. in vivo selection of antagonistic bacteria isolates the in vivo test used the kr-15 variety of tobacco as a test plant. prepare 24-hourold bacillus spp. isolates, 48-hour-old r. solanacearum isolates, and 2-month-old tobacco plants in polybags. tobacco plants were placed in a plastic cover in the green house, the plants that were ready are sprayed with bacillus spp. isolate, respectively. according to the treatment, there were three tobacco leaves in each treatment and left for 3 days. furthermore, the tobacco plants, inoculated with r. solanacearum, were placed on the lower surface of the leaf veins. plants remained in plastic cover, observed daily starting from international journal of applied biology, 5(1), 2021 98 two days after treatment until three weeks after inoculation of r. solanacearum. the percentage of inhibition was calculated by the formula: 𝐵 𝐴 ×100% a : leaf area (cm2) b : necrosis area (cm2) characterization and identification of bacteria three bacterial isolates were selected to be identified, of the 13 isolates that showed the worst symptoms on the in planta test. characterization was carried out through colony macroscopic, cell microscopic, physiological, and genetic observations. colony macroscopic characterization was performed by culturing bacteria on nutrient agar for 24 hours. microscopic characterization was conducted by gram staining, while physiological characters were performed using kit a, which include, catalase, oxidase, oxidativefermentation requirements, hydrolysis of gelatin, starch, levan formation, proskauer's voges test, arginine, dehydrolase, motility, tolerance for bacterial growth at several temperatures, ph and hcl concentration, use and overhaul of carbon, citrate and nitrogen compounds (lelliot and stead, 1987; william et al., 1989, chun and vidaver, 2001). identification was continued molecularly, by sequencing the 16s rrna gene. preparation before the sequencing process refers to the protocol kit (genomic dna mini kit, geneaid), 16s rrna was amplified by polymerase chain reaction (pcr) technique using universal primers (27f 5 "-aga gtt tga tcc tgg ctcag-3" and 1429r 5 "-ggt tac ctt gtt acg actt-3"). the pcr mixture was prepared in a 25 µl volume containing 1 µl f primer, 1 µl r primer, 12.5 µl mix (kapa taq ready mix, kapa biosystems, united states), 2.5 µl dna extract, and 8 µl dh2o. then the dna amplification was carried out on a pcr (swift maxi thermal cyber) machine with an initial denaturation for 1 minute at a temperature of 95 oc. then followed by 30 denaturation cycles at 95 oc for 1 minute, annealing at 55 oc for 1 minute and extension at 72oc for 1.5 minutes. followed by an additional extension step at 72oc for 5 minutes. 2 for 5 minutes. pcr products were visualized by electrophoresis on 1% agarose gel which had been added with ethidium bromide and tbe buffer. the sequencing of pcr results was carried out by a sequencing service provider company. dna sequence homology searches were executed using a dna database (genbank) using the blast program from the national center for biotechnology information (https://blast.ncbi.nlm.nih.gov/blast.cgi). results and discussion isolation of bacteria the results of the isolation of r. solanacearum from potato plants, obtained 6 different isolates based on colony morphology. r. solanacearum belongs to a group of gram-negative bacteria, the morphology of short rod-shaped cells, single cells. colony morphology of r. solanacearum was irregularly rounded, milky white, the texture of the colony was slimy / shiny, the edges of the colony were uneven, the elevation was convex. according to ray et al (2013) ralstonia sp. can grow on sucrose peptone agar (spa) media, with irregularly shaped colonies, slightly convex elevation, slimy, creamy milk color, shiny surface, and in terms of physiology ralstonia sp. in the form of a prosthetic with a size of https://blast.ncbi.nlm.nih.gov/blast.cgi international journal of applied biology, 5(1), 2021 99 0.5-0.7 μm. based on these characteristics which were confirmed by the characteristics listed in bergey's manual of determinative bacteriology and as reported by ray et al (2013), the bacteria isolated in this study were identified as the ralstonia genus. meanwhile, bacillus spp., based on differences in colony morphology, there were 13 isolates of bacillus spp. on nutrient agar medium. in general, the colonies have a characteristic whitish cream color and a round and irregular colony. the edges of the isolates were flat and some were irregular. this characterization showed 13 isolates including the genus bacillus sp. hypersensitivity test hypersensitivity test results on tobacco plant leaves showed that three isolates of r. solanacearum were able to cause necrose symptoms, namely isolates rs-3, rs-4 and rs-1, each of which had an incubation period of 4 days after incubation (dai), 8 dai and 11 dai (figure 1). from these results selected rs-3 which has a faster incubation period. according to agrios (2005), environmental conditions that support the growth of pathogens will accelerate the incubation period of the disease, so it will be faster to infect plants. meanwhile, bacillus spp. none of the 13 isolates showed symptoms of necrosis, therefore all of them have the opportunity to be used for further research. antagonist test the in vitro antagonist test results of 13 bacillus spp. isolates against r. solanacearum showed similar results, namely that no isolate was able to produce an inhibition zone. the absence of a zone of inhibition from bacillus spp. isolates does not mean that the bacteria were unable to suppress the development of r. solanacearum. the inability of bacillus spp. isolates to form a zone of inhibition in vitro, presumably due to the condition and nutritional content of the media used, allows bacteria to not produce antibiotic compounds. saputra (2015) stated that bacillus did not show bactericidal against r. solanacearum. these results are also in line with research by zicca et al., (2020) who reported that b. oleronius, b. licheniformis, and b. megaterium did not show a zone of inhibition when grown together with xylella fastidiosa in the same media. the mechanism of inhibition of antagonistic bacteria can also be by competition for space and nutrition, releasing degrading enzymes or through induced resistance mechanisms (lo, 1998). figure 1. hypersensitivity test results for r. solanacearum bacteria isolates in tobacco kr15, symptoms of necrosis caused by isolate (a) rs-6, (b) rs-4, (c) rs-1. international journal of applied biology, 5(1), 2021 100 solanacearum suppression testing in greenhouse the results showed that the ability of bacillus spp. to suppress the growth of r. solanacearum was different. this is indicated by the percentage area of necrosis symptoms that appear varies (figure 2). this difference was due to the different abilities of the thirteen bacillus spp. isolates in suppressing r. solanacearum. according to choudhary and johri (2008) biological control of bacillus spp. through the mechanism of antibiosis, secretion of lysing enzymes and inducers of systemic resistance. figure 2. potential of bacillus isolates in suppressing necrotic symptoms in plants caused by r. solanacearum identification of bacillus spp. three bacillus spp. isolates which had the strongest ability to suppress the r. solanacearum growth, ba-1, ba-2 and ba-11, were selected for further identification. table 1. macroscopic colony and microscopic characters of selected bacteria isolate codes microscopic and macroscopic characteristics of bacillus isolates surface edge shape color gram endospores isolate ba-1 (figures 3a and 4a) flat flat round white + + isolate ba-2 (figures 3b and 4b) flat irregular round white + + isolate ba-11 (figures 3c and 4 d) slimy flat round white + + figure 3. morphological colony of bacillus spp. which had the strongest ability to suppress the growth of r. solanacearum. isolate colonies (a) ba-1, (b) ba-2, (c) ba-11 international journal of applied biology, 5(1), 2021 101 the round colony shape and white colony color generally indicate that the bacteria belong to the genus bacillus sp. according to corbin (2004), the colony of bacillus sp. has the general characteristics of having a whitish cream color and a round and irregular colony shape, flat and uneven colony edges. according to hatmanti (2000), the bacteria bacillus spp. colony has various kinds of flat and uneven edges, the surface is rough and not slimy, there are even tend to be dry and powdery, the colonies are large and not shiny. figure 4. morphology cell of bacillus spp. (a) ba-1, (b) ba-2, (c) ba-11. the three isolates showed a rod-shaped form of bacterial cells, gram positive, forming endospores. according to sofyan et al. (2009), bacillus sp. is a gram-positive bacteria with a short rod to a single rod with a single arrangement. table 2. physiological and biochemical test with kit a. no. characteristics bacillus isolates code 1 2 11 1. oxidase + + + 2. motilitas + + + 3. nitrate + + + 4. lysine 5. ornithine 6. h2s 7. glucose 8. mannitol 9. xylose 10. onpg 11. indole 12. urease 13. vp 14. citrate 15. tda 16. gram test positif positif positif 17. shape rod rod rod 18. endospore present present present international journal of applied biology, 5(1), 2021 102 table 3. molecular identification of bacterial isolates no code isolates results identification (%) 1 ba-1 bacillus weihenstephanensis 99% 2 ba-2 bacillus weihenstephanensis 99% 3 ba-11 bacillus mycoides 99% the homology of ba-1 and ba-2 isolates based on the 16s rrna gene from the blast results showed that these bacteria were related to several bacillus weihenstephanensis. meanwhile, ba-11 isolate is related to bacillus mycoides (table 3). based on the 16 s rrna sequence of bacillus weihenstephanensis, one group with b. cereus, b. mycoides, b. anthracis, b. pseudomycoides and b. cytotoxicus, because they both have the cspa, glpf, gmk, purh, and tpa genes (habazar et al., 2018). in a further development between b. mycoides and b. weihenstephanensis it is stated that they are not different species. b. mycoides grows in the temperature range 10-15 °c to 35-40 °c, while b. weihenstephanensis grows in a temperature range of 7 43 °c (soufiane et al., 2013). according to habazar (2018), research on b. weihenstephanensis as a biological agent is still very limited. b. weihenstephanensis is known to have the potential to inhibit the phytopathogenic growth of verticillium (hollensteiner et al., 2017) and as an insecticidal agent in schistocerca gregaria (mashtoly et al., 2019). genomic analysis conducted by hollensteiner et al. (2017) revealed that b. weihenstephanensis has genes encoding antifungal substances and secondary metabolite gene clusters including non-ribosomal polypeptides. while, b mycoides, able to synthesize protease and cellulase enzymes according to sofiane et al, 2013, it is strongly suspected that the bacillus mycoides and b. weihenstephanensis group's has ability as biological controllers to plant pathogens is associated with induced resistance mechanisms, because they show the high ability to control plant disease. conclusions there were 13 isolates of bacillus spp. with different characteristics which have potential biocontrol agents. among these isolates, there were three best candidates for biocontrol agents, namely ba-1, ba-2, and ba-11. based on the 16s rrna gene sequence analysis, ba-1 and ba-2 isolates have a 99% similarity identification with bacillus weihenstephanensis, while ba-11 shows 99% similarity with bacillus mycoides. references agrios, g.n. 2005. plant pathology. academic press, new york ali, m.a., ren, h., ahmed, t., luo, j., an, q., qi, x & li, b. 2020. antifungal effects of rhizospheric bacillus species against bayberry twig blight pathogen pestalotiopsis versicolor. agronomy, 10, 1811. international journal of applied biology, 5(1), 2021 103 chooudary, d.k & johri b.n. 2009. interaction of bacillus spp. and plants-with special reference to induced systemic resistance (isr). chun, w & vidaver, a.k. 2001. gram positive bacteria: bacillus, in: schaad nw, jones jb, chun w (eds). plant pathogenic bacteria 3nded. aps press, minnesota. corbin, b.d. 2004. identification and characterization bacillus thuringiensis. journal of bacteriology. 186: 7736–7744. fravel, d. 2005. commercialization and implementation of biocontrole. annual review of phytopathology. 43: 337-359. habazar, t., yanti, y & reflinadon. 2018. implementasi tehnik in planta dalam pengembangan strategi pengendalian penyakit layu bakteri (ralstonia solanacearum) pada tanaman cabai dengan rhizobakteri indigenous. laporan penelitian. lppm universitas andalas. hatmanti, a. 2000. pengenalan bacillus spp. balitbang lingkungan laut lipi, jakarta. hayward, a.c. 1991. biology and epidemiology of bacterial wilt causes by pseudomonas solanacearum. annual review of phytopathology 29: 65-87. hollensteiner, j., wemheuer, f., harting, r., kolarzyk, a.m., diaz valerio, s.m., poehlein, a & liesegang, h. 2017. bacillus thuringiensis and bacillus weihenstephanensis inhibit the growth of phytopathogenic verticillium species. frontiers in microbiology, 7, 2171. junaid, j.m, dar, n.a., bhat, t.a., bhat, a.h & bhat, m.a. 2013. commercial biocontrol agents and their mechanism of action in the management of plant pathogens. international journal of modern plant & animal sciences. 1(2): 39-57. kelman, a. 1953. the bacterial wilt caused by pseudomonas solanacearum. a literary review and bibliography. technical bulletin of north carolina agricultural experiment station no. 99. raleigh, north carolina. 194 p. klement, z., rudolp, k & sands, d.c. 1990. methods in phytobacteriology. academical kiado budapest. 547p. kuswinanti, t., baharuddin, b & sukmawati, s. 2014. efektivitas isolat bakteri dari rizosfer dan bahan organik terhadap ralstonia solanacearum dan fusarium oxysporum pada tanaman kentang. jurnal fitopatologi indonesia, 10(2), 68-68. lelliot, r.a & stead, d.e. 1987. methods in plant pathology. vol. 2. methods for the diagnosis of bacterial diseases of plants. british society for plant pathology by blackwell scientific publication, oxford. 216 p. lo, ct. 1998. general mechanism of action of microbial biocontrol agents. plant pathology bull 7: 155-156. mashtoly, t. a., el-zemaity, m. s., abolmaaty, a., abdelatef, g. m., marzouk, a. a & reda, international journal of applied biology, 5(1), 2021 104 m. 2019. phylogenetic characteristics of novel bacillus weihenstephanensis and pseudomonas sp. to desert locust, schistocerca gregaria forskål (orthoptera: acrididae). egyptian journal of biological pest control, 29(1), 1-9. ongena, m & jacques, p. 2008. bacillus lipopeptides: versatile weapons for plant disease biocontrol. trends in microbiology. 16:115-125. rahman, m.m., ali, m.e., khan, a.a., akanda, a.m., uddin, m.k., hashim, u & hamid, s. b.a. 2012. isolation, characterization, and identification of biological control agent for potato soft rot in bangladesh. the scientific world journal. ray, d. k., mueller, n. d., west, p. c & foley, j. a. 2013. yield trends are insufficient to double global crop production by 2050. plos one, 8(6), e66428. saha, d., purkayastha, g.d., ghosh, a., isha, m & saha, a. 2012. isolation and characterization of two new bacillus subtilis strains from the rhizosphere of eggplant as potential biocontrol agents. journal of plant pathology, 94(1): 109-118. saputra, r., arwiyanto, t., wibowo, a. 2015. uji aktifitas antagonistik beberapa isolat bacillus spp terhadap penyakit layu bakteri (ralstonia solanacearum) pada beberapa varietas tomat dan identifikasinya. prosiding seminar nasional masyarakat biodiversitas iv indonesia vol 1, nomor 5, agustus 2015 hal, 11161122. singh, n., pandey, p., dubey, r.c & maheshwari d.k. 2008. biological control of root rot fungus macrophomina phaseolina and growth enhancement of pinus roxburghii (sarg.) by rhizosphere competent bacillus subtilis bn1. world journal of microbiology and biotechnology 24: 1669-1679 sofyan, salmariza, s.y & ardinal. 2009. kombinasi sistem anaerobik filter dan multi soil layering (msl) sebagai alternatif pengolahan limbah cair industri kecil menengah makanan. jurnal riset industri 3(2), 118-127. soufiane, b., baizet, m & côté, j.c. 2013. multilocus sequence analysis of bacillus thuringiensis serovars navarrensis, bolivia and vazensis and bacillus weihenstephanensis reveals a common phylogeny. antonie van leeuwenhoek 103:195–205. williams, t.s., goodfellow, m & alderson, g. 1989. genus streptomyces warksman and henrici, in: williams me, holt jg. bergeys manual of systematic bacteriology. williams and wilkins, london. yabuuchi, e., kosako, y., yano, i., hotta, h & nishiuchi, y. 1995 transfer of two burkholderia and an alcaligenes species to ralstonia genus nov.: proposal of ralstonia pickettii (ralston, palleroni and douderoff 1973) comb.nov., ralstonia solanacearum (smith 1896) comb. nov. and ralstonia eutropha (davis 1969) comb. nov. microbiology immunology 39: 897. international journal of applied biology, 5(1), 2021 105 yin, x.t., xu, l., fan, s.s., xu, l.n., li, d.c & liu, z.y. 2010. isolation and characterization of an ahl lactonase genefrom bacillus amyloliquenfaciueus. world journal of microbiology and biotechnology. 26: 1361-1367 yu, g.y., sinclair, j.b., hartman, g.l & bertagnolli, b.l. 2002. production of iturin a by bacillus amyloliquefaciens suppressing rhizoctonia solani. soil biology and biochemistry. 34: 955. zicca, s., de bellis, p., masiello, m., saponari, m., saldarelli, p., boscia, d & sisto, a. 2020. antagonistic activity of olive endophytic bacteria and of bacillus spp. strains against xylella fastidiosa. microbiological research, 236, 126467. 1(1)1-8 contact&:&syamsul&bahri&&&&&&&sambar2250@gmail.com0 ©0international0journal0of0applied0biology0 10 abstract& algae0 spirogyra) peipingensis0 is0 a0 group0 of0 green0 algae0 from0 the0 order0 zygnematales.0spirogyra0algae0serves0as0a0bioremediation0agent.0in0addition0 spirogyra0 algae0 has0 great0 potential0 in0 reducing0 toxin0 levels0 in0 cigarette0 smoke.0 because0 the0 biomass0 of0 alga0 spirogyra0 has0 an0 irregular0 shaped0 surface0texture0and0consists0of0amino0acid,0amide,0carboxylic,0hydroxyl,0and0 carbonyl0 group0 that0 can0 bind0 various0 heavy0 metals0 and0 other0 harmful0 compounds.0alga0spirogyra)peipingensis0samples0used0are0algae0from0bone,0 south0sulawesi.0prior0to0the0trial,0the0first0pretreatment0and0processed0to0 become0powder.0algae0powder0was0 then0applied0to0cigarette0filters0and0 tested0on0several0respondents.0results0showed0that0there0was0a0significant0 decrease00tar,0nicotine0and0carbon0monoxide0levels0in0cigarette0smoke0by0 using0 spirogyra) peipingensis0 algae0 but0 the0 most0 significant0 decrease0 of0 carbon0monoxide,0while0tar0and0nicotine0decreased0even0though0not0as0high0 as0carbon0monoxide.00 & issn0:02580k24100 eissn0:02580k21190 0 0 0 application&of&cigeratte&filter&from&algae&spirogyra)peipingensis& to&reduce&nicotine,&tar,&and&carbon&monoxide& 0 syamsul&bahri1,&kamsinar1,&sarioja1,&edi&tompo2,&and&nurul&fahmiah1& 0 10department0of0biology,0faculty0of0mathematics0and0natural0sciences,0hasanuddin0 university,0makassar,0indonesia0 20department0of0animal0sciences,0faculty0of0animal0sciences,0hasanuddin0university,0 makassar,0indonesia0 0 0 & & & & & & & & & & & & & introduction& smoking0has0been0known0to0cause0health0problems0(ching,02017;0chen,02010).0this0 health0disorder0can0be0caused0by0nicotine0derived0from0mainstream0smoke0and0secondhand0 smoke0 from0 smokers0 smoked0 cigarettes0 (chen,0 2017).0 thus0 the0 sufferer0 is0 not0 only0 the0 smokers0themselves0(active0smokers)0but0also0people0who0are0in0the0environment0of0tobacco0 smoke0(environmental0tobacco0smoke)0or0called0passive0smokers0(zenzhes,02009;0castilla,0 2005).0 health0 disorders0 can0 include0 chronic0 bronchitis,0 emphysema,0 lung0 cancer,0 larvae,0 mouth,0pharynx,0esophagus,0bladder,0artery0narrowing0and0others0(abedi,02017;0bae,02015).0 however,0there0are0still0many0men0and0women0who0have0not0or0can0not0leave0this0habit.0 various0 efforts0 have0 been0 made0 by0 those0 concerned0 about0 environmental0 health0 from0 cigarette0 smoke,0 such0 as0 smoking0 bans0 in0 public0 places,0 special0 installations,0 and0 others0 (huang,02016).0even0government0warnings0on0tobacco0packaging0stating0that0smoking0can0 harm0health0do0not0get0a0good0response0from0the0community0(huang,02016;0tang,02016).0 &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword0 cigarette)filters,) spirogyra)peipingensis,0 nicotine,0 algae.0 0 article&history0 received080may020170 accepted030july0201700 0 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 0 international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international)journal)of)applied)biology,)1(1),)2017) 0 20 according0 to0 the0 world0 health0 organization0 (who),0 the0 environment0 of0 cigarette0 smoke0 is0 the0 cause0 of0 various0 diseases,0 and0 can0 also0 affect0 healthy0 people0 who0 are0 not0 smokers.0continuous0exposure0to0secondhand0smoke0in0healthy0adults0can0increase0the0risk0 of0lung0disease0and0heart0disease0by020k300percent0(abedi,02017;0atar,02017;0bae,02015).0the0 environment0of0cigarette0smoke0can0worsen0the0condition0of0a0person0with0asthma,0causing0 bronchitis,0and0pneumonia0(su,02016;0stirk,02015).0cigarette0smoke0also0causes0eye0irritation0 and0nasal0passages0for0people0around0them0(wegrezensky,02017;0wang,02016).0cigarettes0 contain0about04,0000types0of0chemicals,0of0which0400are0carcinogenic,0and0at0least02000of0them0 are0hazardous0to0health.0the0main0toxins0in0cigarette0are0tar,0nicotine0and0carbon0monoxide0 (wang,02016;0simsek,02016).0in0addition,0in0a0cigarette0also0contains0other0chemicals0that0are0 not0less0toxic0that0affect0the0health0of0the0body0(damron,02017).0 risks0that0can0be0caused0by0smoking0can0actually0be0reduced0if0known0toxins0levels0in0 cigarette0smoke0such0as0nicotine,0tar,0and0carbon0monoxide.0when0these0levels0are0listed0then0 the0candidate0smokers0can0choose0cigarettes0with0the0content0of0toxins0as0small0as0possible0 or0the0least0content0of0the0types0of0cigarette.0to0overcome0this0problem,0it0is0necessary0to0 make0 a0 new0 innovation0 of0 cigarette0 filter0 made0 from0 algae0 spirogyra) peipingensis0 that0 effectively0reduces0toxins0levels0in0cigarette0smoke0such0as0reducing0the0content0are0nicotine,0 tar,0and0carbon0monoxide0produced0by0cigarette0smoke.00 & materials&and&methods& pretreatment& the0biomass0of0spirogyra)peipingensis0was0dried0using0oven0at080°c0for0480hours.0the0 biomass0of)spirogyra)peipingensis0blended0until0crushed0and0sieved0with0a0mesh0size0of0400 mesh.0spirogyra0algae0biomass0that0passes0400mesh0sieve0is0weighed0as0much0as01000grams0 (sulfahri0et)al.,02017;0zhang0&0feng,02010).0spirogyra0algae0is0then0used0as0raw0material0of0 cigarette0filter0in0accordance0with0the0research0design.0 0 cigarette&filter&creation& cigarette0filters0of00.70in0diameter0and050cm0in0length0are0printed0using0a03d0printer0 with0a0design0such0as0 in0figure01.0spirogyra0powderkshaped0algae0is0then0inserted0into0a0 cigarette0filter0with0an0algae0thickness0of020cm.0the0algae0powder0that0has0been0inside0the0 tube0is0then0pressed0with0an0iron0ballast0to0condense0spirogyra0algae0powder.0at0each0end0of0 the0cigarette0filter0is0covered0with0a0wool0cloth0with0a0diameter0of0wool0cloth0on0each0end0 with0a0length0of030mm.0 0 cigarette&filter&app& various0types0of0cigarettes0were0applied0to0this0cigarette0filter0to0see0the0effect0of0the0 application0 using0 the0 spirogyra) peipigensis0 algae0 filter.0 the0 main0 toxin0 levels0 found0 in0 cigarettes0are0nicotine,0tar0and0carbon0monoxide0which0will0be0filtered0by0using0spirogyra) peipingensis0 algae0 filter0 on0 20 cm0 thickness0 and0 cigarette0 without0 using0 algae0 filter0 then0 measured0nicotine,0tar0and0carbon0monoxide0levels.0 & nicotine&and&tar&levels&analysis& measurement0of0nicotine0and0tar0content0on0clove0and0filter0cigarettes0refers0to0the0 decree0 of0 the0 indonesian0 minister0 of0 industry0 and0 trade0 no.0 62/mpp/kep/2/20040 dated0 february017,020040on0how0to0test0nicotine0and0tar0content0levels0for0kretek0and0filters.0 making0a0solution0extract,0weigh010g0chinaldine0add0100ml0of0ethanol0and0then0apply0it0with0 international)journal)of)applied)biology,)1(1),)2017) 0 30 2kpropanol0in020liter0flask.0making0a0mother0liquor0weighs00.50gr0of0nicotine,02.50gr0of0eugenol0 and0 10 g0 of0 water0 then0 appropriately0 with0 the0 extracting0 solution0 in0 a0 500 ml0 flask.0 make0 standard0solution0for0calibration0curve0pipette0of0mother0liquor0as0much0as010ml,020ml,030ml,040 ml,050ml0and0insert0in0measuring0flask0100mh0then0apply0until0mark0line0with0extract0solution.0 inject02ml0of0the0solution0in0gc0to0obtain0the0calibration0curve.0extraction0filter0pad0from0 smoking0machine0in0500ml0extract0solution,0shake0with0shaker0for0300minutes0with0speed02700 rpm.0enter0the0extract0in0the0vial,0close0the0meeting.0insert020ml0of0the0solution,0then0test0in0 choromotoghrafi0(gc)0gas0to0obtain0nicotine0and0tar0levels0(iso03402k19990&0iso010362k1k 1999).0 & analysis&of&carbon&monoxide&(co)& to0measure0the0concentration0of0co0in0the0air0is0used0drager0multigas0detector0tool0 and0the0results0can0be0read0on0a0scale0of0reading0between01000k030000ppm0for0100times0the0 pressure,0and0between01000k030000ppm0for010time0pressure.0the0tool0is0a0tube0with0a0length0 of0approximately0100cm,0on0the0left0on0the0wall0there0are0scales0of0100,0500,01000,02000,0and0 30000which0states0the0concentration0of0co0in0ppm,0while0on0the0right0on0the0wall0there0is0a0 scale0from0100to0300.0top0is0broken0and0connected0with0a0pressure0pump,0if0this0tool0will0be0 used0 to0 measure0 co0 concentration.0 by0 pressing0 the0 pump0 once0 can0 be0 measured0 air0 concentration0between01000to03000ppm,0while0to0measure0the0concentration0of0100to03000 ppm,0the0pump0is0pressed0100times.0 & tool&saturation&test& the0cigarette0smoke0filter0will0be0tested0from000to0100cigarettes0and0so0on,0then020,0 30,040,0and0500cigarettes,0the0cigarette0filter0will0be0measured0every0100cigarette0intervals0 with0the0measurement0of0nicotine,0tar,0and0carbon0monoxide0(co).0if0the0levels0are0nicotine,0 tar,0and0carbon0monoxide0(co)0content0less0than060%0and0then0the0tool0filter0used0is0already0 saturated0threshold.0if0the0cigarette0filter0tool0 is0saturated0then0cigarette0filter0should0be0 replaced0new.0 & results&and&discussion&& algae&preparation) algae0taken0are0spirogyra)peipingensis0species0in0the0rice0fields0by0using0a0sieve0then0 collected0in0the0bag0and0then0washed0thoroughly.0subsequently0algae0were0identified0using0a0 microscope0and0algae0identification0book.0set0it0algae0dried0in0the0sun0to0dry0and0blend0until0 smooth.0algae0that0have0been0blended0using0400mesh0sieve0to0produce0algae0flour0which0will0 using0as0raw0material0in0making0filter0cigarettes.0 ) cigarette&filter&creation& collecting0waste0wood0from0craftsman0remain0and0other0woods0and0then0cut0into0 pieces0with0a0length0of0100k1320mm,0after0that0drill0the0middle0of0the0wood0with0a0diameter0 7,60k8,000mm0as0a0place0to0discharge0smoke0then0smoothing0the0surface0of0the0wood0with0 sandpaper0and0shaped0like0a0picture0that0has0been0designed.0spirogyra0already0in0powder0 pretreatment0then0put0on0cigarette0filter0with0thickness0of0alga0that0is020cm0by0using0horn0 spoon.0the0algae0powder0that0has0been0inside0the0tube0is0then0inserted0and0compacted.0at0 each0end0of0the0cigarettes0filter0is0covered0with0a0wool0cloth0with0a0diameter0of0wool0cloth0on0 each0end0with0a0length0of030mm.0 0 international)journal)of)applied)biology,)1(1),)2017) 0 40 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 & & & & & & & figure&1.&cigerette&filter&desain& 0 product&trial&results& cigarette0filter0products0that0have0been0made0then0tested0it’s0ability0in0reducing0toxin0 levels0 in0 cigarette0 smoke.0 the0 results0 show0 that,0 there0 is0 a0 significant0 decrease0 between0 cigarette0smoke0before0it0is0filtered0and0after0filter0(figure03).0each0content0of0nicotine030.80 ppm,0tar020.50ppm,0and0carbon0monoxide017.60ppm.0while0the0content0of0cigarette0smoke0 that0has0been0filter0respectively0that0is011.90ppm0nicotine,010.60ppm0tar,0and0carbon0monoxide0 2.40ppm.0this0is0due0to0the0content0contained0in0algae0that0can0absorb0various0compounds0 produced0by0cigarette0smoke.0 this0is0in0accordance0with0opinion0(sulfahri0et)al.,02017)0which0states0that0the0biomass0 of0alga0spirogyra0has0an0irregularkshaped0surface0texture0consisting0of0amino0acid,0amide,0 carboxylic,0hydroxyl,0and0carbonyl0group0capable0of0binding0various0heavy0metals.0it0is0also0in0 line0with0opinion0(ozer0et)al.,02006)0that0spirogyra0algae0can0reduce0blue0textile0dyes0(acid0 blue02900and0acid0blue0324).0the0process0of0remediation0of0acid0blue02900and0acid0blue03240 textile0dyes0is0done0by0using0the0dead0spirogyra0algae.0the0adsorption0process0using0spirogyra0 algae0has0the0same0way0of0working0with0activated0carbon0in0absorbing0various0compounds0in0 a0 solution.0 dry0 spirogyra0 algae0 can0 be0 utilized0 for0 water0 treatment0 (gupta0 et) al.,0 2006).0 international)journal)of)applied)biology,)1(1),)2017) 0 50 wastewater0 treatment,0 for0 surface0 water0 biomonitoring0 and0 as0 heavy0 commercial0 heavy0 metal0waste0adsorbents0for0application.0spirogyra0algae0can0adsorb0cu0and0pb0in0low0or0high0 concentrations.0capacity0of0heavy0metal0absorption0by0alga0spirogyra0are:0cr014.70mg0/0g0d.m.0 (gupta0et0al.,02001),0pb01400mg0/0g0d.m.0(gupta0et)al.,02008),0cu0133.30mg0/0g0d.m.0heavy0metal0 ions0which0are0absorbed0by0the0dried0algae0of0spirogyra0algae0are0mn20+,0cu20+,0zn20+,0and0 cd20+,0using0the0adsorbent0capable0of0absorbing090k95%0metal0ions0(gupta0et)al.,02006).0 0 figure&2.&cigarette&filter&assemble& 0 test&results&of&saturation&tool& cigarette0smoke0filters0will0be0tested0for0saturation0point0from000to050cigarettes0sticks0 and0so0on.0on0the0use0of0filters0on050cigarettes0was0declared0already0saturated0because0the0 algae0inside0the0filter0has0changed0the0color0of0the0green0color0into0a0thick0black0color0and0the0 smell0 of0 algae0 also0 smells0 a0 bit0 stinging.0 then0 the0 filter0 tool0 used0 was0 already0 saturated0 threshold.0if0the0cigarette0filter0tool0is0saturated0then0cigarette0filter0should0be0replaced0new.0 0 0 0 0 0 international)journal)of)applied)biology,)1(1),)2017) 0 60 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 figure&3.&graph&of&laborotorium&analysis&result&of&nicotine&content,&tar,&and&carbon& monoxide&of&cigarette&smoke& 0 organoleptic&test&results& the0organoleptic0test0was0performed0by0hedonic0method0on0100panelists.0the0test0 parameters0included0flavor0and0aroma.0to0the0attachment0presented0cigarettes0and0filter0 samples0 of0 algae0 cigarettes0 then0 the0 panelists0 tried0 to0 suck0 it0 and0 then0 fill0 out0 the0 questionnaire0based0on0the0preferred0levels0according0to0the0rating0scale.0based0on0the0results0 of0organoleptic0tests0that0have0been0done0to0100panelists0obtained0an0average0of04.30/05.00is0 a0comparison0of0numbers0with0a0very0small0difference,0ie00.10means0the0potential0of0this0 cigarettes0filter0to0be0applied0to0the0public.0in0addition0to0reducing0levels0of0toxins0such0as0 nicotine,0tar,0and0carbon0monoxide,0spirogyra)peipingensis0algaekbased0filter0is0also0in0great0 demand0by0the0public.0so0by0applying0this0product0is0expected0to0reduce0the0various0diseases0 of0society0caused0by0cigarettes.0 0 conclusions& spirogyra)peipingensis0algae0cigarette0filter0can0reduce0the0content0of0nicotine,0tar,0 and0carbon0monoxide0in0cigarette0smoke0significantly.0spirogyra)peipingensis0algae0cigarette0 filter0can0reduce0the0content0of0nicotine0by061.360%,0tar0by048.29%0and0co0by086,36%.0the0 decreasing0content0of0toxins0in0cigarette0smoke0caused0by0the0content0contained0in0algae0that0 can0absorb0various0compounds0produced0by0cigarette0smoke.0 & acknowledgment&& the0authors0gratefully0acknowledge0financial0support0from0the0ministry0of0research,0 technology0 and0 higher0 education0 of0 indonesia0 with0 project0 pkm0 (program0 kreativitas0 mahasiswa)0contract0number0547/b3.1/km/2017.0 & references& abedi,0m.02017.&germination)patterns)of)the)scrublands)in)response)to)smoke:)the)role)of) functional)groups)and)the)effect)of)smoke)treatment)method.0south)african)journal)of) botani.0111:01k60 0 5 10 15 20 25 30 35 nicotine tar carbon0monoxide co nc en tr at io ns &(p pm ) toxin&parameters before0filters after0filter international)journal)of)applied)biology,)1(1),)2017) 0 70 amstrong,0b.0k.01984.0smoke)and)health.)bpom.0jakarta0[in0indonesian]0 atar,0y.02017.0effects0of0smoke0generated0by0electrocautery0on0the0larynx.)journal)of)voice.0 31:380k388.0 bae,0 s.0 2015.0 development0 of0 a0 smoke0 effect0 model0 for0 representing0 the0 psychological0 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(zygnemophyceae0shameel)0in0the0northkeastern0areas0of0pakistan.0proceedings)of)the) pakistan)academy)of)sciences.)440(4)0:02250–0248.00 international)journal)of)applied)biology,)1(1),)2017) 0 80 to&cite&this&article:&& bahri,0s.,0kamsinar,0sarioja,0tompo,0e.0&0fahmiah,0n.02017.0application0of0cigeratte0filter0from0algae0 spirogyra)peipingensis0to0reduce0nicotine,0tar,0and0carbon0monoxide.0international)journal)of) applied)biology.01(1):1k8.0 & zenzhes,0 y.0 j.0 2009.& effect0 of0 moxa0 smoke0 produced0 during0 combustion0 of0 aiye0 (folium) artemisiae)argyi)0on0behavioral0changes0in0mice0inhaling0the0smoke.0journal0of0traditional0 chinese0medecine.*36:80k811.& zhang,0k.0&0feng,h.02010.&fermentation0potentials0of0zymomonas)mobilis)and0its0application0 in0ethanol0production0from0lowkcost0raw0sweet0potato.0african)journal)of)biotechnology.) 90(37)0:061220–06128.00 0 0 0 0 0 0 0 0 0 contact : hermawan seftiono hermawan_seftiono@trilogi.ac.id 79 abstract beef is a food source with a high protein content that would be ideal for microbes to flourish. microbes would reduce the quality of a product. therefore, an indicator on smart packaging would be needed to detect the quality of a product. the indicator film in this study used butterfly pea flower extract which contains a natural coloring agent, anthocyanin. the objective of this study was to obtain the indicator film with the best concentration of pva, chitosan, and butterfly pea flower extract as the natural dye, to study the response of indicator film color, ph, and thickness, to the beef ph and tvbn, and to calculate the total microbes as the determinant of the beef quality. the study consisted of three steps, namely, the extraction of butterfly pea flower, the making of indicator film, and the application of indicator film on beef packaging. the best indicator film was obtained with the formulation of pva and chitosan of 20:80 with the addition of 5 ml of butterfly pea extract. the color change was from blue to yellowish-green with °hue of 137.81±19.31o. the thickness of indicator film in 48 hours of storage decreased from 0.171±0.042 to 0.136±0.043. the ph of beef increased after 8 hours of storage from 5.726±0.011 to 7.540±0.351. the tvbn of beef after 8 hours of storage had exceeded the threshold of 30.815±5,602 which indicates that it was not safe for consumption. the tpc of beef from the 8 hours of storage had exceeded the maximum number of 7.338±0.035 log cfu/g. issn : 2580-2410 eissn : 2580-2119 indicator film of natural coloring of butterfly pea (clitoria ternatea l.) as detection of beef damage hermawan seftiono 1*, qorry aina 1, inanpi hidayati sumiasih1 1 trilogi university, jakarta, indonesia introduction beef consisted of water, fat, protein, carbohydrate, vitamin and several minerals (prasetyo et al. 2013; komariah et al. 2009). the chemical compositions of beef are 77.65% water. 14.7% fat and 18.26% protein (prasetyo et al. 2013). the high protein content in beef causes microbes to grow and multiply so that it can reduce product quality. efforts to increase the shelf life of beef to slow down the quality degradation due to contamination can be done by storing at low temperatures, using natural preservatives, and good packaging. the current innovation of packaging is smart packaging. smart packaging is a packaging system that can monitor temperature, freshness, the presence of microbes, and product shelf life (ahmed et al. 2018). smart packaging has a ph indicator that can use natural or synthetic dyes. this study used butterfly pea flower as a natural dye in smart packaging because currently its utilization has not been maximized compared to its big potential to be used as a natural ph indicator. butterfly pea flower has a color pigment open access international journal of applied biology keyword anthocyanin; butterfly pea extract; thickness; volatile bases article history received december 22, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 80 called anthocyanin which can be useful as an indicator of changes in ph. based on research conducted by vankar & jyoti (2010), the anthocyanin levels in the butterfly pea flower are 227.42 mg/kg. based on this information, the use of butterfly pea flower as a natural dye in indicator films needs to be developed. the presence of a natural dye in indicator film on smart packaging plays a role in detecting the quality of the product directly which is proportional to its color change. the objective of this study was to obtain an indicator film with the best concentration of polyvinyl alcohol (pva), chitosan, and butterfly pea flower extract as the natural dye, to study the color response of the indicator film on changes in color, ph, thickness, total volatile base nitrogen (tvbn) of beef, and to calculate the total microbes as a determinant of beef quality.based on this information, the use of butterfly pea flower as a natural dye in indicator films needs to be developed. the presence of a natural dye in indicator film on a smart packaging plays a role in detecting the quality of the product directly which is proportional to its color change. the objective of this study was to obtain an indicator film with the best concentration of polyvinyl alcohol (pva), chitosan, and butterfly pea flower extract as the natural dye, to study the color response of the indicator film on changes in color, ph, thickness, total volatile base nitrogen (tvbn) of beef, and to calculate the total microbes as a determinant of beef quality. materials the study was conducted from may to july 2019 at the laboratory of microbiology and biochemistry, department of food technology, faculty of bioindustry, trilogi university, jakarta, and at testing laboratory of the bogor agricultural postharvest research and development center. the materials used in this study were categorized into four types namely the materials to extract butterfly pea flower dye, the materials to make the film/ packaging, the materials for applying the indicator film, and the materials for analysis. the materials used for extracting the color from butterfly pea flowers were butterfly pea flowers obtained from a garden in the kedung halang region and distilled water. the materials for making the indicator film were chitosan polyvinyl alcohol (pva), acetic acid, distilled water, and glycerol. the materials for applying the indicator film were beef obtained from lenteng agung market, plastic wrap, and styrofoam. the materials for analysis were aluminum foil, whatman's filter paper no. 1, rofa labolatorium centre's vaseline, nitra chemical's 7% tca solution, rofa labolatorium centre k2co3, merck's ethanol 97%, merck's hcl 1.5 n, merck's hcl 0.02 n, pudak scientific's boric acid 3%, nitra chemical's bromocresol green (bcg), pudak scientific's methyl red (mr), merck's peptone water (bpw) buffer media, and merck's plate count agar (pca). tools for extracting the butterfly pea flower were excalibur dehydrator, kern analytical balance, thermo ta288 thermometer, stove, pan, and stirrer. the tools used to make the indicator film are kern analytical balance, beaker glass, stuart hot plate, magnetic stirrer, thermo ta288 thermometer, measuring cup, and plastic mold (size 12x12cm). the tools used for analysis were memmert incubator, hirayama autoclave, agilent technologies ph meter, tricle brand screw micrometer, tcr 200 chromameter, memmert oven, beaker, mortar and pestle, bunsen, test tube, petri dish, burette, stative, vortex mixer. zx3, erlenmeyer, conway dish, micropipette, and uv-vis spectrophotometer. international journal of applied biology, 6(1), 2022 81 methods butterfly pea flower extraction (sinha et al. 2012 modified) the butterfly pea flowers were first dried using a dehydrator at 60 °c for 1 hour (modified from the previous method without drying), and then the flowers were cleaned and weighted to get 5 grams. in a saucepan, 250 ml of distilled water and 5 grams of flowers were added then bring to 80 °c for 5 to 10 minutes. after extraction, the flowers were separated and the extract was used for the next step. the making of indicator film (nofrida 2013 modified) the indicator film was made by using chitosan-acetate, pva, and glycerol. the composition used was the combination of 3% pva (w/v) and 3% chitosan-acetate (w/v) with the addition of glycerol as a plasticizer of 1% (v/v) of the total solution volume. the tested factor was the addition of the dye, using 5, 10, 15, and 20 ml of dye/100 ml of film solution. the dye used was the butterfly pea flower extract. in the first step, pva was dissolved with distilled water at 80 °c for 30 minutes using a magnetic stirrer. next, the chitosan was dissolved in a 1% acetic acid solution. the dissolved pva solution was added with dissolved chitosan with a volume ratio that can be seen in table 2. the next step was to add 1% glycerol and then homogenize it by stirring, then add 5, 10, 15, or 20 ml of natural dyes from butterfly pea flower per 100 ml of film solution. the homogeneous film solution was poured into 12x12 cm plastic molds and dried at room temperature (25±3 °c) with a modified time of 48 hours, while research conducted by nofrida (2013) used 24 hours. the application of indicator film on beef packaging (octavia 2015 modified) beef cutlet of 60 gram was put on styrofoam and covered with cling plastic wrap with 3 x 3 cm of indicator film attached to it on the inside. the beef was then stored at a modified room temperature of (25 ± 2) °c for 48 hours. the storage temperatures in the study by octavia (2015) were in room temperature of (25 ± 2) °c and cold storage of (4 ± 2) °c. the observation at (25 ± 2) °c was conducted at 0, 8, 24, 32, and 48 hours to observe the color changes of the indicator film. analysis methods the main research carried out in this study included testing the ph of the butterfly pea flower extract, measuring the anthocyanin content (less & francis 1972 in nofrida 2013), testing the thickness of the indicator film (nofrida 2013), color analysis of the indicator film (hunter 1958 in octavia 2015; nofrida 2013); the analysis of meat quality degradation includes the ph test of the beef (mega et al. 2009), the total plate count (tpc) test (bsn 2008), and the total volatile basic nitrogen (tvbn) test (bsn 2009). results and discussion chemical characteristic of butterfly pea flower extract the butterfly pea flower is one of the flowers with the potential as a natural dye source. the extract of butterfly pea flower can be used as a natural dye in the making of indicator film due to its anthocyanin content. the chemical characteristic of butterfly pea flower extract had been analyzed by measuring the ph and its anthocyanin content. international journal of applied biology, 6(1), 2022 82 the analyzed butterfly pea flower extract analyzed had a ph value of 5.838 and the produced color was purplish-blue. analysis of the ph aims to see the degree of acidity of the butterfly pea flower extract. the acidity level of the butterfly pea flower extract can affect the stability of the anthocyanin compound. the results of the ph analysis obtained of the butterfly pea flower extract are in the normal ph range because based on the determination of the ph route carried out by nikijuluw (2013) at ph 5 to 7 anthocyanin has stable color as at neutral ph, which was blue so that at that ph it can be used as an indicator film. the result for anthocyanin content of butterfly pea flower was 218.323 mg/kg. the study by vankar & jyoti (2010) obtained higher anthocyanin content which was 227.42 mg/kg. this was due to the difference in the extraction method and the difference in the solvent used. the extraction process of butterfly pea flower used by vankar & jyoti (2010) was maceration methods which kept in room temperature in the dark using methanol solvent and acidified using 0.1% hcl, while in this study the method used was hot maceration by applying heat at 80 °c for 5 to 10 minutes using distilled water as solvent. this study used the hot maceration method because the materials and the technique needed were simple. in addition, the butterfly pea flower is polar so it will be easily dissolved with water in the heating process. the methanol solvent maceration method would get a more concentrated extract color, but the extraction process is quite long because of the evaporation process to evaporate the methanol in the solution. in addition, the evaporation process is feared to leave residual methanol which can affect the further analysis process. determination of the best formulation the objective of this study was to obtain the best indicator film formulation of the pva and chitosan composition with the addition of butterfly pea flower extract as a natural dye. based on the previously determined formulation, the next step was to apply the indicator film. the application was to study the color changes in butterfly pea flower dyed indicator film. the observation results of indicator film in 48 hours showed that there was a color change in the film with 5 ml butterfly pea flower extract while in the film with an additional of 10, 15, and 20 ml of extract did not show any changes. the more concentration of butterfly pea flower extract in the indicator film, the more vibrant the color and resulted in less observable color change. the five formulations (f1-f5) of indicator film showed that the best formulation was f1 with the composition of pva: chitosan of 20:80 (figure 1). the best formulation which was f1 showed color changes from blue to yellowishgreen. the color change in indicator film occurred because of the protein degradation process of beef. the result of the degradation process was the volatile base that would evaporate and react with indicator film (riyanto et al. 2014). figure 1. indicator film with the addition of 5 ml butterfly pea flower extract international journal of applied biology, 6(1), 2022 83 indicator film color change during storage the color change of indicator film indicates the quality changes in the product kept inside the smart packaging. the color change in the film occurred because the meat undergoes a decomposition process. as the beef decayed, it produced an unpleasant aroma from the formation of volatile alkaline compounds such as ammonia, dimethylamine, and trimethylamine. volatile bases were the product of the protein decomposition process into amino acids by bacteria (iskandar 2014). the gas produced during the decomposition process would interact with the indicator film containing anthocyanins. the anthocyanin compounds in the butterfly pea flower are sensitive to changes in the degree of acidity. this can be indicated by the change in color of the anthocyanin in the butterfly pea extract as the ph change from acidic to alkaline. the volatile compounds produced during the decomposition process are collected in the packaging and cause the ph of the indicator film to change. table 1. color change of indicator film with butterfly pea extract during storage duration of storage (hours) °hue value color range* smart packaging indicator film 0 171,03 ± 4,12 green 8 163,84 ± 0,42 green 24 151,48 ± 2,20 yellow to green 32 151,15 ± 0,53 yellow to green international journal of applied biology, 6(1), 2022 84 48 137,81 ± 19,31 yellow to green note: (*) chromatic color range according to hutchings (1999) in nofrida (2013) there was a decline in the °hue value of indicator film after 48 hours of storage. the decline started from hour 0 of 171.03 ±4.12o to hour 8 of 163.84± 0.42o. the °hue value at hour 0 was categorized as green and still green at hour 8. the °hue value continued to decline at hour 24 to 151.48±2.2o, at hour 32 to 151.15±0.53o, and at hour 48 to 137.81±19.3o. the decline in °hue value of indicator film had a regression equation of y= -0.664x + 169.9 with a strong correlation value (r²) of 0.978. this showed that the storage time is correlated with the color change of the indicator film. a negative slope value showed a declining graph model during the storage process from hour 0 to hour 48 which can be seen in figure 2. figure 2 indicator film °hue value during storage the total of indicator film color change during storage can be shown with ∆e value by calculating the changes of l*, a*, and b* value from indicator film during storage. the ∆e value obtained (figure 3) showed that there was a significant increase at hour 0 of 0.7±0.80 to 3.14±1.26 at hour 8, 3.87±0.52 at hour 24, 4.74±0.7 at hour 32, and significantly increase to 7.05±3.55 at hour 48. the regression equation was y= 0.117x + 1.270 with strong correlation value (r2) of 0.937. this showed that duration of storage was correlated with increasing ∆e. a positive slope value showed that the graph model was increasing during the process. the increasing ∆e value caused the color change of indicator film from green to greenish-yellow for 48 hours. the color change in indicator film during storage showed that the beef underwent a decaying process and produced volatile bases that were interacted with the film. international journal of applied biology, 6(1), 2022 85 figure 3. value of indicator film indicator film thickness during storage the indicator film thickness was measured to observe the changes during storage. the volume of solution and the size of the mold affected the film thickness (setiautami 2013). when using molds of the same size could produce different thicknesses depending on the solution volume used. the higher the volume, the thicker the film produced. the indicator film thickness changes during storage were shown in figure 4. the thickness at hour 0 was 0.171 ±0.042 mm and then kept declining up hour 48 to 0.136±0.043 mm. the indicator film thickness had a regression equation of y = -0.0007 + 0.169 with a strong correlation value (r2) of 0.915. this showed that the storage duration correlated with film thickness. a negative slope value showed that the graph model was declining from hour 0 to hour 48. the decline in thickness showed that the film was getting thinner. according to jabbar (2017), the thickness was affected by the film resistance from water vapor, gas, and volatile compound transmission. the thinning of indicator film was caused by the process of water vapor transmission from the product. this process would cause the environmental conditions inside the packaging to become moist so that the indicator film was getting thinner due to interaction with water vapor. in addition, chitosan which was used as the base material for making this indicator film cannot hold water vapor well which causes the film to decompose and causes the indicator film to thin during the storage process (fehragucci 2012). furthermore, ridhawati (2016) stated that the concentration of plasticizers can affect water vapor transmission. the plasticizer used in this indicator film was glycerol. the addition of glycerol as a plasticizer could increase the permeability of indicator film so that evaporated water could get through the film easily and cause the thinning of indicator film. international journal of applied biology, 6(1), 2022 86 figure 4. indicator film thickness during storage beef ph value during storage the ph value is an indicator to determine the level of acidity of the beef meat. analysis of ph became an important factor in determining the quality of beef because the ph value can show the decrease in the quality of stored beef. in addition, analysis of ph in the use of smart packaging was the benchmark for the level of quality changes of beef with changes in the color of the indicator film. the graph of changes in the ph value of beef is shown in figure 5. the ph value of beef at hour 0 was 5.761±0.034 and decline to 5.726±0.011 at hour 8. the decline in beef ph was due to the anaerobic glycolysis process that change glycogen into lactic acid (kurniawan et al. 2014). this process would continue until the glycogen reserves in the meat tissue were depleted. this study was similar to pangestika (2017) which showed that the ph value of meat decreased at hour 8, from ph 7 to 5.6. based on the obtained results, the ph value from hour 8 to hour 48 increased from 5.726±0.011 to 7.540±0.351. the increase in ph value was due to the formation of volatile bases compounds from the decomposition process of protein (azizah 2015). the increase of ph showed the rigor mortis phase had stopped and had entered the post rigor phase. the post rigor phase is characterized by the formation of aroma and the meat becomes soft again (anggraeni 2005). the beef ph value had regression equation of y = 0.039x + 5.597 with strong correlation value (r2) of 0.971. this showed that storage duration is correlated with beef ph value. a positive slope value showed that the graph model was increasing from hour 8 to hour 48. international journal of applied biology, 6(1), 2022 87 figure 5. beef ph value during storage beef tvbn value during storage the freshness of beef can be determined by the total volatile base nitrogen (tvbn) test. the principle of the total volatile base nitrogen (tvbn) test was to evaporate the volatile base nitrogen such as amino-, mono-, di-, and trimethylamine during storage (hasnedi 2009). the presence of those compounds caused the unsavory odor of beef during storage at a temperature of 25 °c with rh 50%. the storage temperature affects the microbial activity which caused the formation of volatile compounds from meat (heising 2014). the value of beef total volatile base nitrogen (tvbn) during 48 hours of storage in temperature of 25 °c and rh 50% showed in figure 6. the tvbn value increased during storage. the first measurement at hour 0 showed a tvbn value of 16.808±6.496 mg n/100 g, which increased to 30.815±5.602 mg n/100 g at hour 8. the increase of total volatile base nitrogen (tvbn) continued to hour 48 which was 58.829±10.728 mg n/100 g. the value of beef tvbn had a regression equation of y = 0.829x + 20.64 with a strong correlation value (r2) of 0.968. this showed that storage duration was correlated with beef tvbn. a positive slope showed that the graph model increased during storage from hour 0 to hour 48. the increase of total volatile base nitrogen (tvbn) was due to the increase in activity of microbes that decompose protein compounds into amino acids which produce volatile base compounds such as ammonia due to the deamination of amino acids during decomposition (cristiana et al 2007). in addition, trimethylamine (tma) compounds are produced by the degradation of decomposition bacteria (jinadasa 2014). the in crease in these compounds correlated with the deterioration of beef quality and the odor produced when the meat entered the rotten phase. based on the study by byun et al. (2003), the limit for tvbm for beef was 20 mg n/100g. the obtained tvbn value at hour 8 had already passed the threshold which was 30.815±5.602 mg n/100 g that indicates that beef had already entered the rotten phase and was not suitable for consumption. international journal of applied biology, 6(1), 2022 88 figure 6 value of beef tvbn during storage beef total plate count (tpc) during storage bacterial activity is responsible for the spoilage of beef during storage. the total plate count (tpc) test was carried out to determine the number of bacteria contained in beef so that the quality of the meat can be determined. the results of the total plate count (tpc) test on beef stored at 25 °c with 50% rh were shown in figure 7. the beef tpc value at hour 0 was 5.483±0.067 log cfu/g. the tpc value then increased significantly at hour 8 to 7.338±0.035 log cfu/g. the increase in total bacteria continued to hour 48 of storage which was 10.474±0.196 log cfu/g. based on that data the regression equation can be obtained, which was y = 0.105x + 6.294 with a strong correlation value (r2) of 0.861 (figure 7). this showed that storage duration was correlated with the number of bacteria on the beef meat. a positive slope value showed that the graph model increased during storage. an increase in the number of bacteria in beef with increasing storage time indicates a decrease in meat quality (anggraeni 2012). parameters that showed the decreasing quality of meat caused by bacteria were changes in color, aroma, texture, formation of a slimy compound, the emergence of gas, and increase in liquid (dengen 2015). according to sni7388-2009, the microbiological requirements contained in beef for consumption should not exceed 1x106 cfu/g or about 6 log cfu/g. the tpc value of beef at hour 8 of storage was 7.338±0.035 log cfu/g, which had exceeded the maximum microbial limit set so that beef was not suitable for consumption anymore because it had been damaged. figure 7. beef tpc value during storage international journal of applied biology, 6(1), 2022 89 conclusions butterfly pea extract can be used as a natural dye in the making of indicator film due to its anthocyanin content of 218.323 mg/kg and ph of 5.838. the best formulation for indicator film was obtained with the composition of polyvinyl alcohol (pva and chitosan 20:80 with the addition of 5 ml of butterfly pea flower extract. based on the data obtained the ph of beef increased after 8 hours of storage from 5.726±.011 to 7.540±0.351 which indicates that the beef had already entered the rotten phase. the total volatile base nitrogen (tvbn) value obtained at hour 8 was 30.815±5.602 mg n/100 g and already exceeded the threshold of 20 mg n/100g. at hour 8 of storage, the beef tpc value was 7.338±0.035 log cfu/g and showed that the number of bacteria had already exceeded the maximum limit of 6 log cfu/g. the tvbn and tpc value at hour 8 of storage showed that the beef was not safe for consumption. the application of indicator film on beef packaging showed that there was a correlation between the decline of beef quality with the color change of indicator film. the color change of indicator film in 48 hours of storage was from green to yellowish-green with film thickness changed from 0.171±0.042 mm to 0.136±0.043 mm. acknowledgment acknowledgments are especially shown for capacity-building research through research and technology and higher education simlitabmas funding in 2018. international journal of applied biology, 6(1), 2022 90 references [bsn] badan standarisasi nasional 2008. metode pengujian cemaran mikroba dalam daging, telur, dan susu serta hasil olahannya. jakarta (id): badan standarisasi nasional. _____. 2009. cara uji kimia-bagian 8: penentuan kadar total volatil base nitrogen (tvb-n) dan trimetil amin nitrogen (tman) pada produk perikanan. jakarta (id): badan standarisasi nasional. ahmed i. lin h. zou l. et al. 2018. an overview of smart packaging technologies for monitoring safety 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[skripsi]. bogor (id): institut pertanian bogor. vankar ps, jyoti s. 2010. evaluation of anthocyanin content in red and blue flowers. journal of food engineering. 6(4):1-11. international journal of applied biology, 6(1), 2022 92 yanti h, hidayati, elfawati. 2008. kualitas daging sapi dengan kemasan plastik pe (polyethylene) dan pp (polyprophylen) di pasar arengka kota pekanbaru. jurnal peternakan. 5(1):22-27. contact : stella magdalena stella.magdalena@atmajaya.ac.id 83 abstract bacteria can interact with each or other microorganisms by releasing, sensing, and reacting to small diffusible chemical signal molecules to alter their community behavior. this process, termed quorum sensing, is influenced by density of other bacteria that present in the environment. one example as a result of this process is the formation of biofilm. biofilm consists of bacterial communities that attach to a surface and envelope themselves in secreted polymers. this formation can be beneficial to pathogenic bacteria because they become highly resistant to antibiotics and human immunity. thus, antibiofilm agents that can inhibit biofilm formation are needed. the objective of this study were to screen and evaluate bacteria from hot spring and crater lakes that have antibiofilm activity against pathogenic bacteria. in this study, 26 isolates were successfully obtained and tested for quorum sensing and quorum quenching activities. based on the result, two isolates, which were km16 and pap26, were found to have quorum quenching activity. further research showed that km16 and pap26 had antibiofilm activity against more than six pathogenic bacteria. from characterization of the bioactive compounds, it is known that different compound from km16 and pap26 have different activity against each pathogen. in molecular identification, isolates km16 and pap26 were identified as bacillus subtilis and pseudomonas sp. through molecular identification. issn : 2580-2410 eissn : 2580-2119 evaluation of the potentials of bacillus subtilis km16 and pseudomonas sp. pap 26 isolated from the hot spring and crater lakes as antibiofilm agents stella magdalena1*, fabiola giovani 2, yogiara3 1department of food technology, faculty of biotechnology, atma jaya catholic university of indonesia 2department of biotechnology, faculty of biotechnology, atma jaya catholic university of indonesia 3department of magister of biotechnology, faculty of biotechnology, atma jaya catholic university of indonesia introduction biofilm is microbial community embedded on inert or living surface and encaged in self-produced extrapolymeric substances (eps) that contains proteins, polysaccharides, and extracellular dna. the eps provided structural strength and defense against environmental condition, host immunity, and antimicrobial agent (davies, 2003; singh et al., 2017). most chronic infection is caused by biofilm-forming microorganisms and it is very difficult to eradicate them by only using conventional antibiotics or other antimicrobial therapy. in open access international journal of applied biology keyword antibiofilm, quorum quenching, quorum sensing, bacillus. article history received 31 march 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 84 biomedical, biofilm can be formed on inanimate surface such as medical device, catheters, and living-tissue associated infection. human immune system cannot correctly kill pathogenic bacteria or fungal and therefore, they may cause damage to encircling tissue (taraszkiewicz et al., 2013). biofilm can also contaminate food processing and attach to water pipe, henceit may cause food-borne and water-borne disease (kokare et al., 2009). several strategies that can be used to inhibit biofilm formation are to interfere with quorum sensing mechanism and using antibiofilm agents. quorum sensing can influence gene expression that is responsible for biofilm formation, virulence, sporulation, and pathogenicity. this process, alters wide-scale behavior of population in response to cell density. therefore, mechanism that can interfere with quorum sensing mechanism, quorum quenching, is needed (brackman & coenye, 2015). thermophilic bacteria are a type of bacteria that can survive at high temperature environment, as in hot spring and crater lake. these bacteria have tremendously gained popularity in pharmaceutical and many industries because they can produce heat-stable bioactive molecule, for example, thermostable protease that does not denature at high temperature, but remains active at such temperature (panda et al., 2013). furthermore, antibiofilm activity of bacteria from hot spring and crater lakes in indonesia has n ot been much explored. the purpose of this study was to screen and evaluate bacteria from hot spring and crater lakes that have antibiofilm activity against pathogenic bacteria. materials and methods water samples and isolation of bacteria water samples were obtained from hot spring and crater lakes at mount pancar, bogor, indonesia (table 1). the media used for isolation were luria broth (lb) (10g tryptone, 5 g yeast extract, and 10 g nacl, and 1000 ml ddh2o) and luria agar (la) (lb with 1.5% (w/v) bacteriological agar). a total 5 ml water sample was transferred into 250 ml conical flask containing 45 ml lb and incubated at 37°c, 70°c, and 80°c for 5 hours. the suspension was plated onto la plates and incubated at 37°c overnight. morphologically different colony that grew on la plate was collected and inoculated by repeated streaking on the same medium. for short term preservation, isolates were streaked on la and stored at 4°c. for long-term preservation, the culture was stored at -80°c in 15% (v/v) glycerol. table 1. water source and condition source location condition (temperature; ph) hot spring mount pancar, bogor, indonesia merah crater lake hitam crater lake natural crater lake mount pancar, bogor, indonesia 67oc; 7 48oc; 7 43oc; 7 quorum sensing assay quorum sensing activity from bacteria sample was determined via cross-feeding assay as sketched by magdalena et al. (2020). n-acyl-homoserine lactone production was examined by streaking the isolate in parallel with a lane of the monitor strains chromobacterium violaceum 026 (cv026) onto brain heart infusion agar (bhia). bhia medium incubated at 28°c for 48 hours. cv026 that showed production of violacein (purple international journal of applied biology, 5(1), 2021 85 color) indicated a positive result of quorum sensing activity. experiments were performed in triplicates. quorum quenching assay the procedure used in this quorum quenching assay was agar well diffusion method which is a modification of the procedure of soundari et al. (2014). each isolate was grown in lb at 37°c until they reached absorbance value of 0.132 at λ=600 nm (mcfarland 0.5). the inoculated lb was centrifuged twice at 12,000×g for 10 minutes and then filtered (0.22 µm) to obtain the cell-free supernatant. c. violaceum was grown in brain heart infusion broth (bhib) at 28°c until it reached absorbance value of 0.5 at λ=600 nm (mcfarland 0.5). one hundred μl of c. violaceum was spotted and streaked on bhia using sterile cotton bud. each well was made using cork borer. a total of 15 μl of cell-free supernatant was loaded into the well. lb was used as negative controls. the plates were incubated 28°c overnight and inhibition of purple pigment production was interpreted as positive results. experiments were performed in triplicates. biofilm inhibition assay the method used in this biofilm inhibition assay was the static biofilm assay which is a modification of the procedure of magdalena et al. (2020). in this method, the biofilm inhibition was observed using 5% (v/v) concentration of cell-free supernatant. pathogenic bacteria that were used in this assay were acinetobacter baumanii atcc 19606, pseudomonas aeruginosa atcc 1637, escherichia coli atcc 4157, salmonella enterica atcc 51741, staphylococcus aureus atcc 25923, bacillus cepacia atcc 25416, and bacillus licheniformis atcc 12759. the test bacteria were grown in lb at 37°c until they reached absorbance value of 0.132 at λ=600 nm (mcfarland 0.5). in 96-well round bottom (u) microplates, 200 μl of tested bacteria were inoculated in each well along with 5% (v/v) cell-free supernatant. in this assay, 200 μl tested bacteria alone and 200 μl of uninoculated lb were used as negative control. the microplate was incubated at 37°c overnight. after incubation, the media and planktonic cells were discarded and the wells were rinsed twice using sterile distilled water and air-dried. then, each well was stained with 200 μl of crystal violet and incubated for 30 minutes. the crystal violet was discarded and the wells were rinsed three times using sterile distilled water and air-dried. afterwards, 200 μl of absolute ethanol was added to each well and incubated for another 30 minutes. finally, the dissolved crystal violet was transferred to a new microplate and measured at λ=595 nm with biorad 680 microplate reader. the percentage of biofilm inhibition was calculated using the following formula from nikolić et al. (2014): % biofilm inhibition = (od growth control − od sample) od growth control × 100 characterization of bioactive compounds this method was adapted from the procedure of jiang et al. (2011). cell-free supernatant of each isolate was treated with proteinase-k (1 mg/ml), nuclease (100 μg/ml dnase and 25 μg/ml rnase), and naio4 (20 mm) separately and incubated in 37°c for 12 hour. after incubation, treated cell-free supernatants were then used in static biofilm inhibition assay with cell-free supernatant concentration of 5% (v/v). international journal of applied biology, 5(1), 2021 86 microscopic observation and biochemistry tests for the microscopic observation the isolates were stained using gram staining and observed under the magnification of 100x10. the biochemistry tests used for this research were catalase test, triple sugar iron agar (tsia) test, carbohydrate fermentation test (glucose, lactose, maltose, and mannitol), and citrate test. molecular identification of km16 and pap26 genomic dna was isolated using wizard® genomic dna purification kit (promega, wisconsin, usa) and used as a dna template in pcr. the identification of the isolates was conducted by amplifying the 16s rrna gene using universal primer sequences 63f (5′caggcctaacacatgcaagtc-3′) and 1387r (5′-gggcggwgtgtacaa ggc-3′) (marchesi et al., 1998). the reaction mixture consisted of 0.5 μl dna template, 2 μl of forward primer 63f, 2 μl reverse primer 1387r, 25 μl of go taq® green (promega) 2x, and 20.5 μl of ddh2o. thermal cycling of 30-cycle pcr, included pre-denaturation at 95°c for 5 minutes, denaturation at 95°c for 30 seconds, annealing at 55°c for 30 seconds, extension at 72°c for 1 minute, and followed by a post-extension at 72°c for 20 minutes. pcr products were then analyzed by gel electrophoresis in 1% agarose gel at 90 volt for 60 minutes using 1x tae buffer, visualized under uv light with the help of florosafe dna stain (1st base), and recorded with gel doc instrument (biorad, usa). the marker used was 100 bp dna ladder (promega). pcr results were sent to 1st base, malaysia for sequence. the sequences were used to identify the isolates using blast (ncbi) and submitted to genbank. results and disscussion isolation of bacteria in this research, water samples were collected from different crater lakes and hot spring at mount pancar, bogor, indonesia, using sterile glass container. the container then kept inside vacuum flask to maintain the water temperature. bacteria were tested using broth media at diverse temperatures, but mostly at high temperature. in high temperature environment, most bacteria are believed to have characteristics of thermophile. this is because thermophilic bacteria have optimum growth temperature in the range of 45 80°c. thermophilic bacteria are known to produce bioactive compound that can worked at high temperature (taylor & vaisman, 2010), such as antimicrobial agent and quorum sensing inhibitor is produced by hot spring cyanobacterial mats (dobretsov, 2010). in this study, 26 bacterial isolates were retrieved. they were 8 isolates from merah crater lake, 12 isolates from hitam crater lake, 3 isolates from natural crater lake, and 3 isolates from hot spring (table 2). all isolates were tested for quorum sensing activity via cross feeding assay using c.violaceum 026 (cv026) as a detector. table 2. isolates from mount pancar, bogor, indonesia source number of isolates code of isolates merah crater lake 8 km 2, 7, 13, 14, 15, 16, 19, 22 hitam crater lake 12 kh 3, 5, 6, 8, 9, 10, 11, 17, 18, 20, 21, 23 natural crater lake 3 kn 1, 4, 12 hot spring 3 pap 24, 25, 26 international journal of applied biology, 5(1), 2021 87 quorum sensing activity ahl-mediated quorum sensing control genes were responsible for colonization in eukaryotes in most bacteria species (anbazhagan et al., 2012). cv026 is ahl negative mutant because the presence of mini-tn5 mutagenesis in cvii (ahl synthase), hence it requires exogenous ahl to produce violacein (purple pigment) (vasavi et al., 2013). based on the result, none of the cv026 produced purple pigment when they were grown together with each isolates. cv026 detected wide range of ahl with n-acyl side chains ranging from c4 to c8 in length, but did not detect ahl with n-acyl side chain raging from c10 to c14 (anbazhagan et al., 2012). this showed that all 26 isolates did not produce short chain ahl molecules. in spite of that, it did not rule out the possibility that these isolates can perform cell-to-cell communication. for example, wild type b. subtilis uses comqxpa and rap-hpr quorum sensing systems to coordinate sporulation and competence. as gram-positive bacteria, b. subtilis uses small peptide as signal molecule (kalamara et al., 2018). thus, it cannot be detected by cv026. another example is pseudomonas species, p. aeruginosa, which uses lasi/lasr and rhii/rhir quorum sensing system to control biofilm and generate extracellular enzymes. although pseudomonas as gram-negative uses ahl as their molecule signal (umesha & shivakumar, 2013), pap26 is likely to produce long chain ahl molecules. bacteria have differences in quorum sensing systems, including signal types, receptors, and signal transduction mechanisms) (waters & bassler, 2005). although they are in the same gram type of bacteria, there is specificity in terms of signal types and receptors structure, hence this can cause inhibition (quorum quenching) because homologous signal molecule interferes with signal binding to receptor and decrease receptor concentration (dong et al., 2007). for example, staphylococcus aureus has been divided into four groups based on the interaction between molecule signal and its receptor. each group produced homologous aip and only activated response in the same group member, but inhibited other group response (umesha & shivakumar, 2013). therefore, quorum sensing activity could be used to inhibit quorum sensing of other bacteria. quorum quenching and biofilm inhibition activity it was found out that bacterial isolates km16 (figure 1) and pap26 had quorum quenching activity. quorum quenching activity can be achieved by inhibiting signal synthesis, degradation of the signal molecule, and preventing signal molecule binding to transcriptional factors (grandclément et al., 2016). figure 1. positive result of the quorum quenching assay of km16 isolate (red arrow) the biofilm inhibition activity was performed using static biofilm inhibition assay to determine whether bacterial isolates km16 and pap26 can prevent biofilm formation of tested bacteria. seven pathogen bacterial species were inoculated with 5% cell-free international journal of applied biology, 5(1), 2021 88 supernatant (v/v) of km16 and pap26. based on the result, bacterial isolates km16 and pap26 had antibiofilm activity towards several pathogenic bacteria (table 3). km16 showed the highest antibiofilm activity against a. baumannii with 82.29% activity. on the other hand, pap26 showed the highest antibiofilm activity against e. coli with 84.09% activity. this activity can be influenced by polysaccharide, protein, or nucleic acid (dna or rna) compound in the isolate supernatant. table 3. biofilm inhibition activity using 5% (v/v) crude extract of bacterial isolates km16 and pap26 pathogenic bacteria % inhibition km16 pap26 a. baumannii atcc 19606 82.29 78.74 p. aeruginosa atcc 1637 48.28 36.72 s. aureus atcc 25923 41.49 44.87 e. coli atcc 4157 78.52 84.09 s. enterica atcc 51741 29.13 34.72 b. cepacia atcc 25416 14.77 b. licheniformis atcc 12759 28.00 42.57 characterization of bioactive compounds afterward, cell-free supernatant from each isolates was pre-treated using naio4, proteinase-k, and nuclease. it is well known that proteinase-k, dnase, and rnase can degrade protein, dna, and rna. naio4 is capable to hydrolyze carbohydrate molecules by oxidizing the carbons bearing hydroxyl groups and cleaving the c-c bonds (jiang et al., 2011). in figure 2(a), reduction of biofilm activity against p. aeruginosa was shown after cellfree supernatant of km16 was pre-treated with proteinase-k and nuclease. on the contrary, cell-free supernatant pre-treated with naio4 presented increasing activity. this can be caused by carbon and energy resources from breakdown of biopolymers used by pathogen to enhanced biofilm formation (rabin et al., 2015). figure 2. naio4, proteinase-k, and nuclease effects on (a) km16 crude extract against p. aeruginosa, (b) pap26 crude extract against s.aureus biofilm. in figure 2(b), pre-treated cell-free supernatant of pap26 with naio4 exhibited decline in antibiofilm activity against s. aureus. this suggested that antibiofilm compound consisted of dna, rna, protein, and polysaccharide (table 4). biofilm matrix components in p. aeruginosa consist of psl and pel proteins that enhance intercellular adhesion and also (a) (b) international journal of applied biology, 5(1), 2021 89 function as a barrier for immune and antibiotic attacks. alginate plays role in structural stability and protection of biofilm. edna is created from random chromosomal dna that serves as a cell-to-cell component united in the matrix biofilm. protein and proteinaceous components serve as adhesion molecules and structural strength in biofilm formation (karygianni et al., 2020; wei & ma, 2013). cell-free supernatant of km16 produced protein and nucleic acid as antibiofilm agent, meanwhile pap26 produce all three biomolecules. s. aureus produce adhesion factor, such as serine-aspartate-repeat (sdr) family, accumulation-associated protein (aap), and autolysin (atl). polysaccharide intercellular adhesion (pia) or pnag together with other polymer such as teichoic acids and proteins form major part of eps in staphylococci. bap is also involved in intercellular adhesion and biofilm formation. controlled cell death in staphylococci released dna that was needed for biofilm formation (otto, 2008). km16 produced polysaccharide and protein as antibiofilm agent. on the other hand, pap26 produced all three biomolecules. some bacteria exopolysaccharides can inhibit and destabilize biofilm from other bacteria without bacteriostatic and bactericidal activities, for example, p. aeruginosa cells degraded biofilm formation by staphylococcus epidermidis and s.aureus. therefore, pel, psl, and alginate do not only facilitate adhesive molecules to form biofilm, but also have antibiofilm properties (rendueles et al., 2013). table 4. characterization of bioactive compounds of km16 and pap26 against seven tested bacteria pathogen bacterial isolates km16 pap26 a. baumannii polysaccharide − − protein √ √ nucleic acid √ √ p. aeruginosa polysaccharide − √ protein √ √ nucleic acid √ √ s. aureus polysaccharide √ √ protein √ √ nucleic acid − √ e. coli polysaccharide √ √ protein − √ nucleic acid √ √ s. enterica polysaccharide √ √ protein √ − nucleic acid √ − b. cepacia polysaccharide √ protein − nucleic acid √ b. licheniformis polysaccharide √ − protein √ √ nucleic acid √ √ *(√) = present, (-) absent, (■) not tested due to not having antibiofilm activity international journal of applied biology, 5(1), 2021 90 there are three modes of action that are involved in polysaccharide antibiofilm activity, which are modifying abiotic and biotic surface properties, acting as signaling molecules that modulate gene expression, and acting as competitive inhibitor in carbohydrate-protein interaction. biosurfactants change surface characteristic (wettability and charge of the surface), thus influencing interaction between bacteria and surface. antibiofilm polysaccharides also alter physical properties of cell surface. for example, b. licheniformis reduced cell surface hydrophobicity, hence reducing p. aeruginosa colonization. bacterial polysaccharides also caused down regulation of several genes that are responsible for biofilm formation, such us adhesion factor. this mechanism brings advantage to bacteria in bacteria competition and biofilm regulation (rendueles et al., 2013). protein acted as antibiofilm in the form of enzyme that degrade eps matrix component and object that was trapped in eps matrix. negative charge of edna can act as repulsive force in initial attachment (rabin et al., 2015). edna also can bind to bacteria adhesive structure and inhibit cell attachment (berne et al., 2010). srnas can interfere translation process by binding to ribosome and promote mrna degradation using rnase. srna can also terminate premature transcription by binding to a nascent mrna (mika & hengge, 2013). identification of bacteria bacterial isolate identification assay was performed by microscopy, biochemistry, and molecular assay (table 5). microscopy observation was done with gram staining. from the result, each isolate had different gram type but similar morphology. based on biochemistry assay, it was known that both isolates had completely different substrate preference. table 5. bacterial isolate identification assay of km16 and pap26 bacterial identification with 16s rrna showed that km16 and pap26 were identified as 99% b. subtilis and pseudomonas sp. b. subtilis is gram positive and catalase positive bacterial isolate identification assay isolate km16 pap26 microscopy gram + shape bacil bacil biochemistry citrat + tsia slant acid alkaline butt acid alkaline gas h2s glucose + lactose maltose + mannitol + catalase + + molecular identification genus b. subtilis pseudomonas sp. accession ku877820.1 ku877821.1 identity 99% 99% international journal of applied biology, 5(1), 2021 91 bacilli. b. subtilis can utilize citrate and produce acid from glucose, sucrose, maltose, and manitol fermentations. however, b. subtilis cannot produce acid from lactose (saleh et al., 2014). on the other hand, km16 fermented lactose, but could not utilize citrate. previous study showed that extracellular α-amylase from b. subtilis induced biofilm inhibition and degradation by disrupting exopolysaccharide in methicillin-resistant staphylococcus aureus (mrsa) and p. aeruginosa (kalpana et al., 2012). cyclic lipopeptide (lp) biosurfactants produced by bacillus, also modified bacterial surface hydrophobicity and affected the development of flagella. thus, it demonstrated significant anti-adhesive and antibiofilm activity (moryl et al., 2015) pseudomonas sp. is a gram negative, rod shaped, catalase positive bacteria, and naturally found in soil and water ecosystem. most species do not require polysaccharide as carbon source. the genus pseudomonas comprises species that are capable of living under diverse environmental conditions. pseudomonas species are capable to biofilm formation and often resistant to antibiotics, disinfectants, detergents, heavy metals, and organic solvents (rocha et al., 2019). the mechanisms of antibiofilm activity on pseudomonas remain unclear. conclusions twenty six isolates were successfully isolated from crater lakes and hot spring at mount pancar, bogor, indonesia. from quorum sensing assay, it was found out that none of the isolates produced short chain ahl molecules. from quorum quenching and biofilm inhibition assay, bacterial isolates km16 and 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https://doi.org/10.1146/annurev.cellbio.21.012704.131001 wei, q., & ma, l. z. 2013. biofilm matrix and its regulation in pseudomonas aeruginosa. international journal of molecular science, 14(10), 1-23. https://doi.org//10.3390/ijms141020983 https://doi.org/10.1146/annurev.cellbio.21.012704.131001 contact : sudirman sudirmancpg@gmail.com 93 abstract japanese taro is an alternative food commodity and has prospects as an export commodity, especially to japan. colchicine is a mutagen that can be used in polyploid mutations but can be toxic in plants. the purpose of this study was to determine the effect of various concentrations of colchicine and immersion time on the growth and morphological characters of japanese taro plantlets in vitro. in this study, there were 2 focus factors of treatment. the first factor is colchicine concentration consisting of 0.0% (k0/control), 0.1% (k1), 0.2% (k2) and 0.3% (k3). the second factor is the immersion time consisting of 48 hours (t1), 72 hours (t2) and 96 hours (t3). the treatment of in vitro shoot immersion with 0.1%, 0.2% and 0.3% concentration of colchicine solution for 48 hours, 72 hours and 96 hours was significantly different from the control. explant growth decreased every week and died at 6 msi on colchicine treatment. the level of concentration and duration of immersion with different colchicine caused different color changes of the explants, the higher the concentration of colchicine and the duration of immersion, the color of the explants would quickly turn brown. issn : 2580-2410 eissn : 2580-2119 the morphology character of japanese taro (colocasia esculenta var. antiquorum) in induction of polyploidization mutations in vitro: a case study of increased concentration and duration of immersion of colchicine mutagens sudirman 1*, nirwansyah amier 1, ihsan syawal rahmat 1 1 study program of agrotechnology, faculty of agriculture, hasanuddin university, jl. perintis kemerdekaan km. 11 tamalanrea, kota makassar, 90245, sulawesi selatan, indonesia introduction japanese taro (colocasia esculenta var. antiquorum) or satoimo taro (japan) is an alternative food commodity that is currently gaining popularity in indonesia. the popularity of japanese taro cultivation is due to its good economic value and prospects, especially as a food ingredient and export commodity to japan. most of the japanese population consume japanese taro as a staple food. based on data from seameo (2013), the demand for japanese taro in japan reaches ±360,000 tons per year, while production capacity in japan continues to decline to 250,000 tons per year. the decrease was due to limited land and climatic factors that made it impossible to plant throughout the year. this opens up opportunities for tropical countries, such as indonesia, to export japanese taro plants to japan. the indonesian government continues to encourage local governments to increase the productivity of japanese taro. several local governments such as kepahiang, cisarua, bantaeng, malang and buleleng (maretta et al., 2016; wahyuni, 2019) encourage their open access international journal of applied biology keyword japanese taro; colchicine; in vitro; polyploidizatio article history received january 9, 2022 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 94 farmers to develop japanese taro as an export commodity. this results in the need for a large and continuous supply of seeds. fulfillment of seeds in large quantities and sustainable is certainly something that must be fulfilled to support productivity. one method that can be used is by in vitro propagation. in vitro propagation can produce seeds in large quantities and in a relatively short time and uniformly so that it can be one solution. on the other hand, the productivity of japanese taro in indonesia is still very low so that innovative solutions are needed to increase the productivity of japanese taro. research to increase the productivity and quality of japanese taro can be done among others through the induction of polyploidization mutations. induction of polyploidization aims to increase crop productivity because polyploid (tetraploid) plants are known to have a larger figure, fruit size, tuber or flower than diploid plants (suryo 2007). polyploidization can occur in plants either naturally or induced by antimitotic chemicals, such as orizalin, trifularin, amiprofos methyl, and colchicine. induction of polyploidization can be done by giving chemical mutagens such as colchicine to plant meristem tissue. colchicine can not only change the number of plant chromosomes but can cause gene mutations on the scale of seeds and vegetatively propagated plants. this induces polyploidy by inhibiting the formation of spindle fibers during cell division, while the number of chromosomes increases but cell division does not occur, resulting in the production of polyploid cells (manzoor et al., 2018). the success of polyploidization induction using colchicine application, depends on the part of the plant used, the species of the plant, the concentration of colchicine used and the duration of exposure to the plant. concentrations that are too high often cause problems in the form of abnormalities in developing seedlings (manzoor et al., 2018). so this needs to be done very carefully by a plant breeder. polyploidy induction of japanese taro has never been done. induction of polyploidy is expected to produce plants that have high productivity and are resistant to various diseases after various stages of selection so that they are superior to diploid taro. this study aimed to study the effect of various levels of colchicine concentration and immersion time on the growth and morphological characters of japanese taro plantlets in vitro. materials and methods this research was conducted at the tissue culture laboratory, department of agrotechnology, faculty of agriculture, hasanuddin university, jl. perintis kemerdekaan km 10 tamalanrea makassar. the study used a 2-factor completely randomized design (ral2f) with 3 replications and 5 explants for each treatment. the first factor is the treatment with colchicine concentration and the second factor is the soaking time treatment. the concentration of colchicine used as the first factor was 0% (control), 0.1%, 0.2% and 0.3%. the immersion time used as the second factor was 48 hours, 72 hours and 96 hours. concentration of 0% (control) was still soaked according to the second factor with sterile distilled water. international journal of applied biology, 6(1), 2022 95 table 1. combination of treatment between colchicine concentration and soaking time concentration immersion time t1 t2 t3 k0 k0t1 k0t2 k0t3 k1 k1t1 k1t2 k1t3 k2 k2t1 k2t2 k2t3 k3 k3t1 k3t2 k3t3 description: k0 (0%), k1 (0.1%), k2 (0.2%), k3 (0.3%), t1 (48 hours), t2 (72 hours), t3 (96 hours) plantlets used as material for polyploidy induction were in vitro shoots of japanese taro from 8 weeks old subculture which were then used as explants. the in vitro shoots were removed from the midrib to 1 cm in size. the shoots were immersed in a culture bottle containing 25 ml of colchicine solution and shaken at 100 rpm on a shaker. after immersion, the explants were washed with sterile distilled water 3 times, removed the outer midrib, and then planted into shoot and root propagation media. the shoot and root propagation medium used was murashige and skoog (ms) media with the addition of 2 mg.l-1 bap and 1 mg.l-1 iba. the medium contains sugar (30 g.l-1), the ph of the media is adjusted to 5.8 and compacted with agar (7 g.l-1). the shoots were kept in an incubation room at a temperature of 25-26°c with 16 hours of light per day. in vitro shoot growth observations of japanese taro after colchicine immersion were carried out during incubation (1-6 msi). observation parameters include the number of compound shoots (saplings), the number of leaves, the number of roots and the number of live/dead shoots. observations were also made on plantlet color after treatment. observational data were analyzed using analysis of variance (anova) followed by a real difference test using 5% bnj. the 5% bnj test was carried out using the statistical tool for agricultural research (star) application. results and disscussion percentage of induction live shoots colchicine is a mutagen that can affect the ability of a plant such as slowing down the ability to sprout, root or leaf formation. in this study, treatment with different concentrations of colchicine with different soaking times affected the percentage of live japanese taro shoots (table 2). the percentage of live shoots in the control treatment and colchicine 0.1% and 0.2% for 2 days of immersion did not show any shoot death up to 2 msi, but at 5-6 msi at a concentration of 0% shoot death occurred. at concentrations of 0.3% (48 hours immersion), 0.2% and 0.3% (72 hours immersion) and 0.1%, 0.2%, 0.3% (96 hours immersion) started to cause death from 1 msi. observations at 1 msi showed that 100% of shoots were alive in the control and treatment with colchicine immersion for 2 days at 0.1% and 0.2% and 3 days immersion in 0.1% colchicine. however, there was shoot death of all explants at a concentration of 0.3% (48 hours immersion) at 3 msi and 0.3% colchicine concentration (72 hours immersion) at 2 msi. at 6 msi all colchicine treatments experienced shoot death except in controls. international journal of applied biology, 6(1), 2022 96 table 2. average life percentage of japanese taro shoots resulting from colchicine treatment and soaking time at age 1 to 6 msi immertion time (hours) colchicine consentration (%) % shoots live at age (week) 1 2 3 4 5 6 kontrol 0 100,0 100,0 100,0 100,0 86,6 86,6 48 0,1 100,0 100,0 53,3 33,3 33,3 00,0 0,2 100,0 100,0 33,3 20,0 13,3 00,0 0,3 86,6 33,3 00,0 00,0 00,0 00,0 72 0,1 100,0 66,6 53,3 33,3 20,0 00,0 0,2 93,3 86,6 46,6 20,0 13,3 00,0 0,3 53,3 00,0 00,0 00,0 00,0 00,0 96 0,1 60,0 60,0 46,6 46,6 20,0 00,0 0,2 86,6 66,6 40,0 13,3 13,3 00,0 0,3 93,3 86,6 33,3 20,0 13,3 00,0 the percentage of live shoots decreased with increasing colchicine concentration and soaking time. the results obtained from this study on japanese taro were an increase in the concentration and duration of immersion of colchicine caused a decrease in the survival percentage of taro shoots to 5 msi and resulted in the death of all plantlets at 6 msi. in some studies using colchicine can produce shoots to form plantlets. however, in this study, they did not grow to become plantlets. this is presumably due to the different conditions of the cells in each plant so that the sensitivity of the cells also differs in responding to the treatment of high concentrations of colchicine and longer immersion time. in addition, the difference in the age of the plantlets used as explants was suspected to be the cause of the difference in tolerance between explants and colchicine so that the explants became weak and reduced their survival percentage. the smaller growth of induced shoots was thought to be due to damage to plant tissue after exposure to colchicine so that it took a longer time for recovery (damayanti and mariska 2003). number of induced roots and shoots root growth in immersion for 48 hours showed an increase in concentration of 0% (control) at the age of 2 msi and constant until the age of 3-4 msi, but decreased at the age of 5-6 msi. the decrease occurred due to wilting which then died in japanese taro plantlets. root growth in the control was greater than the colchicine treatment. at 48 hours of immersion, all concentrations (0.1%, 0.2% and 0.3%) increased at the age of 5-6 msi and 4 msi at concentrations of 0.1% and 0.2% (figure 1a). the highest number of roots in shoots treated with colchicine was found at a concentration of 0.1% with 2 roots and gave the highest average number of roots of 0.8 for colchicine treatment. at 72 hours of immersion, there was also a decrease in the concentration of 0% (control) due to wilting which then died. at 72 hours immersion all concentrations increased and were constant (figure 1b). at the concentration of 0.1%, 0.2% and 0.3% at the age of 1-4 msi did not increase the number of roots. at 0.1% and 0.2% immersion there was an increase at the age of 5-6 msi with 1 root each. at a concentration of 0.3% there was no increase until the age of 5-6 msi. the highest average number of roots for soaking colchicine for 72 hours was at concentrations of 0.1% and 0.2% with an average of 0.2, respectively. at 96 hours of immersion, the concentration of 0% (control) did not decrease until the age of 5-6 msi. in colchicine immersion for 96 hours, it was constant and increased with each age (figure 1c). at international journal of applied biology, 6(1), 2022 97 concentrations of 0.1%, 0.2% and 0.3% there was no increase in the number of roots from 1 msi to 4 msi. however, there was an increase at the age of 5-6 msi, namely 1 root each. figure 1. graph of root number of japanese taro explants resulting from colchicine induction for 6 msi description: a) immersion 48 hours; b) immersion 72 hours; c) immersion 96 hours the results of the annova test analysis showed that the 0% colchicine concentration treatment (control) was significantly different from the 0.1%, 0.2% and 0.3% colchicine treatments in influencing the number of root explants. soaking and concentration of colchicine were significantly different in influencing the number of roots. the interaction between soaking time and colchicine concentration was significantly different in influencing the number of roots. table 3. results of the 5% bnj test analysis of total roots of japanese taro shoots induced by colchicine treatment and immertion time treatment average number of japanese taro shoots at the age of 6 msi np bnj 5% (k) t1 t2 t3 k0 2,33 aq 2,67 aq 8,00 ap 1,56 k1 0,67 bp 0,33 bp 0,33 bp k2 0,33 bp 0,33 bp 0,33 bp k3 0,33 bp 0,00 bp 0,33 bp np bnj 5% (t) 1,56 notes: numbers followed by the same letter in the same column are not significantly different in the 5% bnj test; msi = week after induction international journal of applied biology, 6(1), 2022 98 the results of the 5% bnj follow-up test showed that the addition of the highest number of roots in japanese taro explants obtained k0t3, namely control (0%) with 96 hours of immersion and significantly different from other treatments at the age of 6 msi (table 3). the roots formed in this study were still in the form of root nodules which were identified by paying attention to the shoots. the color of the root nodules is white so that it can be distinguished from compound shoots. root growth was stunted during the study and some were damaged. at 6 msi, the explants died so that the roots did not grow again. according to mugiono (2001), physiological damage caused by colchicine treatment can be caused by chromosomal damage or cell damage outside the chromosome. colchicine is known to be toxic to plants, thereby reducing the vegetative growth of post-induction polyploid plants, such as mung bean (haryanti et al., 2009). number of induced shoots leaf growth in colchicine treatment 0.1% (k1t1, k1t2, k1t3), 0.2% (k2t1, k2t2, k2t3) no leaves. while in k0t1 at 1 msi there were 11 leaves, 2 msi 24 leaves, 3 msi 29 leaves, 4 msi 28 leaves, 5 msi 17 leaves and 6 msi 17 leaves. in k0t2 there were 1 msi 18 leaves, 2 msi 32 leaves, 3 msi 33 leaves, 4 msi 35 leaves, 5 msi 31 leaves and 6 msi 31 leaves. in k0t3 there were 1 msi 7 leaves, 2 msi 16 leaves, 3 msi 30 leaves, 4 msi 32, 5 msi 65 leaves and 6 msi 65 leaves. the decrease in the number of leaves at k0t1 and k0t2 at 5 msi was due to some leaves dying and wilting, but some when entering 6 msi the leaves began to grow again (figure 2). figure 2. graph of leaf number of japanese taro explants result of colchicine induction for 6 msi description: a) immersion 48 hours; b) immersion 72 hours; c) immersion 96 hours international journal of applied biology, 6(1), 2022 99 the results of the annova test analysis showed that the treatment with 0% colchicine concentration (k0t1, k0t2, k0t3) was significantly different from 0.1% colchicine treatment (k1t1, k1t2, k1t3), 0.2% (k2t1, k2t2, k2t3) and 0.3% (k3t1, k3t2 , k3t3) in influencing the number of leaf explants. soaking had no significant effect, but the concentration of colchicine was significantly different in influencing the number of leaves. the interaction between soaking time and colchicine concentration was significantly different in influencing the number of leaves. table 4. analysis results of 5% bnj test number of japanese taro leaf shoots induced by colchicine treatment and immertion time treatment average number of japanese taro shoots at the age of 6 msi np bnj 5% (k) t1 t2 t3 k0 5.67 aq 10.33 aq 21.67 ap 7.16 k1 0.00 ap 0.00 bp 0.00 bp k2 0.00 ap 0.00 bp 0.00 bp k3 0.00 ap 0.00 bp 0.00 bp np bnj 5% (t) 7.16 notes: numbers followed by the same letter in the same column are not significantly different in the 5% bnj test; msi = week after induction the results of the 5% bnj follow-up test showed that the addition of the highest number of leaves in japanese taro explants was obtained by k0t3 and was significantly different from other treatments at the age of 6 msi (table 4). in observing the number of induced shoots, it was found that 0.1% (k1t1, k1t2, k1t3), 0.2% (k2t1, k2t2, k2t3) and 0.3% (k3t1, k3t2, k3t3) treatments had no leaves while at 0% (k0t1 , k0t2, k0t3) there are leaves. the highest number of leaves was found in k0t3 with a total of 21,67. number of induced tillers the growth of tillers (compound shoots) in the colchicine treatment of 0.1% (k1t1, k1t2, k1t3), 0.2% (k2t1, k2t2, k2t3) and 0.3% (k3t1, k3t2, k3t3) showed no tillers. while in control (k0t1) at 1 msi there were 6 tillers, 2 msi 16 tillers, 3 msi 17 tillers, 4 msi 15 tillers, 5 msi 7 tillers and 6 msi 7 tillers. in k0t2 there were 1 msi 10 tillers, 2 msi 32 tillers, 3 msi 32 tillers, 4 msi 32 tillers, 5 msi 25 tillers and 6 msi 25 tillers. in k0t3 there were 1 msi 6 tillers, 2 msi 24 tillers, 3-6 msi 29 tillers. the decrease in the number of tillers at 0% concentration (k0t1 and k0t2) at 5 msi was due to some leaves dying and withering, but some when entering 6 msi the leaves began to grow again. international journal of applied biology, 6(1), 2022 100 figure 3. graph of the number of tillers of japanese taro explants produced by colchicine induction for 6 msi description: a) immersion 48 hours; b) immersion 72 hours; c) immersion 96 hours the results of the annova test analysis showed that the treatment with 0% colchicine concentration (k0t1, k0t2, k0t3) was significantly different from 0.1% colchicine treatment (k1t1, k1t2, k1t3), 0.2% (k2t1, k2t2, k2t3) and 0.3% (k3t1, k3t2 , k3t3) in influencing the number of explant tillers. soaking had no significant effect, but the concentration of colchicine was significantly different in influencing the number of tillers. the interaction between soaking time and colchicine concentration was significantly different in influencing the number of tillers. the results of the 5% bnj follow-up test showed that the addition of the highest number of tillers in japanese taro explants was obtained by k0t3 and was significantly different from other treatments at the age of 6 wap (table 5). table 5. results of the 5% bnj test analysis of the number of japanese taro shoots induced by colchicine treatment and immertion time treatment average number of japanese taro shoots at the age of 6 msi np bnj 5% (k) t1 t2 t3 k0 2.67 aq 8.33 aq 9.67 aq 1.78 k1 0.00 ap 0.00 bp 0.00 bp k2 0.00 ap 0.00 bp 0.00 bp k3 0.00 ap 0.00 bp 0.00 bp np bnj 5% (t) 1.78 notes: numbers followed by the same letter in the same column are not significantly different in the 5% bnj test; msi = week after induction international journal of applied biology, 6(1), 2022 101 explant color during incubation observation of the color of the explants was carried out to see the effect of the concentration of colchicine and the duration of immersion on the color of the explants that occurred. in the study, it was found that the average colchicine treatment had a slightly brownish color in the explants at the end of the immersion and at 2 msi to 6 msi they experienced browning (brown) and when they entered 6 msi, all explants died except for controls (figure 4). figure 4. colors of japanese taro explants result of colchicine induction for 6 msi description: a) colchicine 0% (control); b) colchicine 0.1%; c) colchicine 0.2%; d) colchicine 0.3% based on observations of the color of japanese taro shoots during polyploid induction (figure 4), it was found that there was a change in shoot color in explants treated with various levels of colchicine concentration. the response to changes in explant color every week was different for each concentration. in the control treatment (0% with immersion 24 hours, 72 hours and 96 hours) the color of the explants tended to be stable from the beginning of incubation to the end of incubation. meanwhile, in the treatment of various concentrations of colchicine (0.1%, 0.2% and 0.3% with immersion time of 48 hours, 72 hours and 96 hours) changes were seen every week. the color of the explants at the beginning of incubation was greenish but at 1 msi there was a treatment that showed a color change, namely in some explants at concentrations of 0.2% and 0.3% with a soaking time of 72 hours and 96 hours. at the age of 3 msi to 6 msi, color changes continued to occur at all concentrations of colchicine treatment. at 6 msi, all treatment explants at the colchicine concentration level experienced overall browning and died. some explants that have shown root nodules have also become browning, so that in this study no cytology test (chromosomal analysis) was carried out. colchicine not only helps in the doubling of chromosomes, but also causes mutations in plants. plants that have mutated through colchicine are known as colchi-mutants (ari et al., 2015). the concentration of colchicine for seed treatment is usually in the range of 0.1%-0.8%, but high doses cause malformations and reduce the production of tetraploid plants. so, it is recommended to use colchicine with the lowest possible concentration (pirkoohi et al., 2011). since coccicin is highly toxic to plants, therefore low doses with a long exposure period are considered reliable to reduce its toxic effects and increase the rate of polyploid production (sajjad et al., 2013). a. c. b. d international journal of applied biology, 6(1), 2022 102 conclusions the treatment of in vitro shoot immersion with 0.1%, 0.2% and 0.3% concentration of colchicine solution for 48 hours, 72 hours and 96 hours was significantly different from the control. explant growth decreased every week and died at 6 msi on colchicine treatment. shoot growth after colchicine treatment was slower than control. the level of concentration and duration of immersion with different colchicine causes different color changes of explants, the higher the concentration of colchicine and the duration of immersion, the color of the explants will quickly turn brown. colchicine has toxic properties that can kill plants when applied in high concentrations. each concentration of colchicine and soaking time gave different effects on japanese taro explants. the higher concentration affects the inhibition of the growth of explants and can cause damage to the explants. international journal of applied biology, 6(1), 2022 103 references ari, e., djapo, h., mutlu, n., gurbuz, e., and karaguzel, o. 2015. creation of variation through gamma irradiation and 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wahyuni, y. i. 2019. strategi pengembangan satoimo di desa bonto daeng kecamatan uluere kabupaten bantaeng. [skripsi]. program studi agribisnis, fakultas pertanian, universitas muhammadiyah makassar. contact : nindrahayu nindrahayum@yahoo.com 144 abstract this study aims to measure the biological activity of flavonols in the leaves of sterculia foetida as an antihyperlipidemic drug. the chemical structure of flavonols contained in the leaves of sterculia foetida was taken from the literature. the target protein used was 3-hydroxy 3-methylglutaryl coenzyme a reductase and the control was simvastatin. water molecules have been removed with pymol v2.5.2 software. docking between the target protein and flavonols was performed using pyrx-python prescription 0.8 software. the results showed that flavonol compounds have greater potential for antihyperlipidemia compared to control compounds. the equivalent affinity of 3-hydroxy 3-methylglutaryl coenzyme a reductase for flavonols is -8.3, and the affinity of 3-hydroxy-3methylglutaryl coenzyme a reductase for simvastatin is -7.9. flavonol toxicity studies have shown that flavonols are not potentially carcinogenic and did not cause mutations. the absorption of flavonols in water was higher than that of the control compound. issn : 2580-2410 eissn : 2580-2119 in silico testing on the activity of flavonol in sterculia foetida leaf as natural anti hyperlipidemia compounds nindrahayu 1*, ariyandy a 2, yustisia i 3, sulfahri 4 1 biomedical, graduate school, hasanudin physiology, school of medicine, hasanuddin university, makassar, indonesia 2 department of physiology, faculty of medicine, hasanuddin university, makassar, indonesia 3biomedical, graduate school, hasanudin physiology, school of medicine, hasanuddin university, makassar, indonesia 4 faculty of biology, faculty of science, hasanudin university, makassar, indonesia introduction hyperlipidemia is a condition characterized by an increase in total blood cholesterol level (1). it can cause a variety of chronic diseases such as atherosclerosis, coronary artery disease, diabetes, cancer and stroke. (2) the world health organization (who) showed that in 2008, the incidence of hyperlipidemia in adults increased globally by 37% in men and 40% in women. while in indonesia, the incidence of hyperlipidemia is 32.8% for men and 37.2% for females. (3) by inhibiting the activity of 3-hydroxy 3-methylglutaryl coenzyme a reductase enzyme with simvastatin, the risk of complications of hyperlipidemia can be reduced. however, this drug can cause gastrointestinal disorders and myopathy. therefore, researchers wanted to discover a natural ingredient that could replace the function of simvastatin. one of the plants that may replace the function of simvastatin is sterculia foetida. (4) (5) the leaf extract of sterculia foetida contains various compounds that are beneficial to the body, such as flavonoids, coumarins, organic acids and steroids. (6). the most common open access international journal of applied biology keyword antihyperlipidemia; sterculia foetida; flavonol; 3-hydroxy-3methylglutaryl; coenzyme a reductase article history received february 3, 2022 accepted june, 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 145 types of flavonoids found in the leaves of sterculia foetida are flavonols and quercetin, which can act as antioxidants and antihyperlipidemic. (7) (8). materials and methods ligand preparation the chemical structure of flavonol compounds was collected from published literature. the 3d chemical structure and smiles of flavonol ligands were obtained from the pubchem compound database (https://pubchem.ncbi.nlm.nih.gov/) with id number: cid:11349 and canonical smile : c1=cc=c(c=c1)c2=c(c(=o)c3=cc=cc=c3o2)o. ligand and stereoscopic (3d) of the chemical structure were sketched in avogadro and stored in pdb format. target selection the target protein for docking was generated using published literature and validated using uniport (https://www.uniprot.org). proteins collected and validated using pdb (protein data bank https: ///www.rcsb.org/pdb) are cleaned by removing water molecules from the structure using pymol v2.5.2 software.. in this study, the target protein was 3-hydroxy 3methylglutaryl coenzyme a reductase and the pdb code was 2q1l, an enzyme involved in cholesterol formation in the body. (3) molecular docking molecular docking experiments were performed using pyrx0.8 software. the docking process was performed using the vina wizard feature built into the pyrx 0.8 software. this function responded to the natural flavonol compound, the target protein 3 hydroxy 3 methylglutaryl coenzyme a reductase, and the control compound simvastatin. visualization of interactions between molecules and small molecules a control ligand (flavonol) for the target protein (3 hydroxy 3 methylglutaryl coenzyme a reductase) and a control ligand (sinvastatin) for the target protein (3 hydroxy 3 methyl glutaryl coenzyme a reductase). interactions were visualized and analyzed using pymol software v 2.5. 2. compound properties and admet prediction using admetsar (http://lmmd.ecust.edu.cn/admetsar2/), predictors and importance of compound physicochemical properties, lipophilicity, pharmacokinetics, and drug-like properties predicted descriptors. results and discussion flavonols and quercetin are compounds of the flavonoid group most commonly found in sterculia foetida leaf extracts. flavonol compounds can be used as antihyperlipidemic and antioxidants. as an antihyperlipidemia, flavonols can lower total cholesterol levels and prevent lipid peroxidation through multiple processes. first, it inhibits the formation of mevalonic acid by inhibiting the activity of 3-hydroxy 3-methylglutaryl coenzyme a reductase (hmg-coa reductase), which is an enzyme involved in the formation of cholesterol in the body. the second mechanism is to reduce the need for nadph for the formation of fatty acids and cholesterol. the final mechanism is to increase the ldl receptor and chelate cholesterol https://pubchem.ncbi.nlm.nih.gov/compound/11349 international journal of applied biology, 6(1), 2022 146 acyltransferase (lcat). this also increases ldl uptake and can convert free cholesterol to hdl. (4) (9) the structures of plant compounds, control compounds, and target proteins were taken from pubchem (https://pubchem.ncbi.nlm.nih.gov/) and visualized in 3d by the pymol application. (figure 1). the results of docking with the pyrx application showed that flavonols could interact with the target protein, demonstrating that flavonols could be used as an antihyperlipidemic drug. docking showed that the binding affinity of flavonol compounds was lower than that of simvastatin. this means that flavonols require less energy to bind to the target protein than simvastatin. hmg-coa reductase is responsible for the converting of hmg-coa to mevalonic acid (8). inhibition of hmgcoa reductase causes a decrease in cholesterol synthesis and an increase in the number of ldl receptors present in the cell membranes of the liver and extrahepatic tissues, resulting in a decrease in total and ldl cholesterol levels in plasma. (10) toxicity tests which had been carried out through admet predictions presented that flavonol compounds had no carcinogens or mutagens potential. the absorption rate by the body was higher than simvastatin as a control. however, it is not recommended to extract this compound because it is potentially toxic. (a) (b) figure 1. (a) 3d structure of flavonol compounds, (b) 3d structure of simvastatin control compounds figure 2. results of docking simvastatin (purple) and flavonol (yellow) with protein 3 hydroxy 3 methylglutaryl coenzyme a reductase table.1. results of docking between flavonol compounds and simvastatin with target proteins international journal of applied biology, 6(1), 2022 147 conclusion based on the intermolecular interactions and their affinity levels, it can be concluded that the flavonol compounds in sterculia foetida leaf can be used as antihyperlipidemic drugs. origin of compound ligand binding affinity (kcal/mol) sterculia foetida flavonol -8.3 control simvastatin -7.9 international journal of applied biology, 6(1), 2022 148 references 1. heryani r. effect of red dragon fruit extract on blood lipid profile introduction. 2016;1:8–17. 2. permatasari aaap, widhiantara ig. testosterone therapy increases leydig cell number and spermatogenesis of mice (mus musculus) experiencing hyperlipidemia. junal media science. 2017;1(22):77–83. 3. susilawati e. effect of ethanol extract of rambutan leaves (napaleum lappaceum l.) against ldl and hdl levels in male rats. j pharmacopolium. 2019;1(3):143–8. 4. sutejo ir, rasyada i, yuniar a. antihyperlipidemic and atheroprotective activity of kepuh (sterculia foetida) leaves ethanolic extract on high-fat-diet rat models. j agromedicine med sci. 2017;3(1):44. 5. normaidah, nurmansyah d. in silico study of hylocereus polyrhizus and allium sativum compounds on hmg-coa reductase enzymes. 2021;(october). 6. s'adah diamond nur. effectiveness of kepuh (sterculia foetida l.) leaf ethanol extract on sgot and sgpt levels of male wistar straight rats induced by high fat diet. 2017. 1–55 p. 7. permatasari ta. effectiveness of kepuh leaf (sterculia foetida) ethanolic extract against malondialdehyde levels in rat serum induced by high fat diet. 2017; 8. zulviyati. antioxidant and antihyperlipidemic activity test of kepuh leaf extract (sterculia foetida): dpph method and in vitro lipase inhibition. jember univ. 2015; 9. chen t., liu j., chang m., tsai m., heish pc. the in vitro inhibitory effect of the flavonoid astilbin on 3-hydroxy-3methylglutaryl coenzyme a reductase on vero cells. zhonghua yi xue za zhi. 2000;(march 2014):114. 10. fajrin, fifteen a. aktivitas ekstrak etanol ketan hitam untuk menurunkan kadar kolesterol. over rim. 2017;6:191–9 contact : daniel kamua nganga dankaihura@gmail.com 76 abstract antibiotic resistant bacteria and antibiotic residues are a serious safety problem for animal food products. poultry products have been long recognized as a reservoir for antibiotic resistant commensals and pathogens. antibiotic residues ingested via food animal products expose gut micro-flora to low concentrations of antibiotics, which promote antibiotic resistance. however, there is limited knowledge regarding the potential of chicken products to act as a transmission corridor for the spread of the antibiotic resistant bacteria and antibiotic residues. the present study aimed at profiling antibiotic resistant bacteria and antibiotic residues in raw chicken products sold around kenyatta university, kenya. a total of 32 meat and egg samples were randomly collected from two study sites; km and ku. antibiotic residues in the study samples were detected using two microbiological techniques with bacillus subtilis , staphylococcus aureus, escherichia coli , and salmonella typhi as test organisms. selective and differential media were used to isolate escherichia coli , salmonella and shigella from the samples. antibiotic susceptibility testing of these isolates against commonly used antibiotics was done using kirby-bauer antibiotic discs diffusion method. of the total samples tested, 87.50% meat and 100% egg samples showed presence of antibiotic residues. the mean colony forming units (cfus) of meat samples from km (190.25 x 10² cfu) was higher than that of ku (104.96 x 10² cfu). eggs from km showed contamination (158.88 x 10² cfu) than those sampled (108.29 x 10² cfu) within the university outlets. escherichia coli , salmonella and shigella, were resistant to ampicillin. escherichia coli showed intermediate resistance to tetracycline while escherichia coli and shigella showed intermediate resistance to amikacin. this study reveals the presence of antibiotic residues and antibiotic resistant bacteria in chicken meat and eggs sold in the study area. knowledge generated from this study is helps to develops effective strategies to control antibiotic resistance. issn : 2580-2410 eissn : 2580-2119 profiling antibiotic resistant bacteria and antibiotic residues in raw chicken products sold around kenyatta university, kenya daniel kamua nganga1*, harry asena musonye1, patrick kamau kamande2, lucy muthoni kamau 3 1 department of biochemistry, microbiology and biotechnology, kenyatta university, nairobi, kenya 2 department of food nutrition and dietetics, kenyatta university, nairobi, kenya 3 department of animal health and production, kenyatta university, nairobi, kenya introduction the burden of antibiotic resistance (abr) has a negative impact on the health and socio-economic status of the people (ferri et al., 2017). the emergency and spread of abr is open access international journal of applied biology keyword antibiotics; antibiotic resistance; antibiotic residues article history received 10 august 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 77 associated with the selective pressure exerted by antibiotic use in the community, farm agriculture, veterinary health, aquaculture, hospitals, and the environment. the world health organization has warned that inappropriate use of antibiotics in agriculture and food systems may lead to increased food insecurity and food safety hazard (who, 2017). the most substantial use of antibiotics worldwide is in the production of animals where they are used for therapeutics, prophylaxis or growth promoters in animal feed (silbergeld et al., 2008; cháfer-pericás et al., 2010; abdullahi et al., 2015). the practices risk exposure of human consumers to abr food-borne pathogens and commensals via animal food products and the environment. already, about 95% of the world food-borne infections are associated with ingestion of contaminated animal food products, poultry products and seafood products (akbar and anal, 2011; jans et al. 2018; sugrue et al., 2019). furthermore, incorrect application of antibiotics has led to noticeable deposits of xenobiotics in meat, milk, eggs, cheese and other livestock products. chronic exposure of antibiotics and their derivatives to commensal microorganisms has triggered the development of resistant strains of bacteria because of bioaccumulation of antibiotic residues in animal tissues (pavlov et al., 2008). although humans are not the target organism for these drugs, they consume copious amounts of them as residues in food, which potentially destabilizes and eliminate normal intestinal microflora. other possible pathological effects of these xenobiotics include allergies, carcinogenicity, mutagenicity, teratogenicity, nephropathy, immunological disorders, hepatoxicity, and reproductive disorders (panigrahi et al., 2017). commensal bacteria like escherichia coli and salmonella found in livestock are frequently present in fresh meat products and they may serve as reservoirs for resistant genes that could potentially be transferred to pathogenic organisms in humans. in other cases, the intestinal microflora adapts to these antibiotics and their metabolites by developing resistance and consequently transfer antimicrobial resistance genes to clinically relevant pathogens (manyi-loh et al., 2018). the pathogens can be transferred from animals to humans, indirectly through food or directly during handling and processing, thereby posing a threat to public health (nyamboya et al., 2013). mathur and singh (2005) noted that the food chain is a major route of antibiotic resistance transmission between animals and human populations. subsequently, commensal bacteria are suspected of serving as resistance reservoirs as they can transfer resistance genes to pathogenic bacteria. poultry farming is one of the most important small-scale agricultural businesses in sub-sahara africa. the population in this region depends partially on poultry farming for home consumption and monitory value from the sale of poultry and poultry products. in kenya, small scale poultry farming is practiced in most cities with a per capita consumption of poultry meat and eggs at 1.1 kg and 37.5 kg per annum respectively (mccarron et al., 2015). nonetheless, poultry production in kenya is still constrained by abr. antibiotics are used indiscriminately on poultry treatment and in their feed, raising a lot of concern over their effects on the quality of poultry products. while antibiotic use in food animals may represent a risk to human health, the degree and relative efforts to combat this have not offered optimum solution (darwish et al., 2013). accurate information on the flow of abr strains in food products is still lacks in kenya. there is a need to carry out investigation whose results can help understand the local abr and antibiotic residues in poultry food products to ensure food safety and food security. international journal of applied biology, 4(2), 2020 78 materials and methods study area sample collection was carried out at kenyatta university and adjacent kiwanja market in nairobi, kenya (1°10'59.0"s; 36°55'34.0"e) (geographic positioning system (gps, etrex, usa). these sites constitute the leading vendors of chicken meat and poultry products to the university community of more than 70,000 people. the chicken used for meat and other poultry products in the study area are outsourced from small scale farmers and other national suppliers across the country. sample collection overall, 32 samples of which 16 raw chicken meat and 16 eggs were collected from randomly selected outlets in km open market and ku. the samples were collected in sterile zip-lock bags for two weeks. the samples were labelled and immediately placed in a cool box and transported to kenyatta university microbiology research laboratory for analysis. all the samples collected were within the required date for consumption. detection of antibiotic residues in chicken meat first, 10 g of each meat sample was soaked in 100 ml ethyl acetate and crushed using a pestle in a sterile mortar. the solvent was then centrifuged at 6000 revolutions per minute (rpm) for 10 minutes. the supernatant was then transferred into a fresh sterile bijou bottle and sterile filter paper discs placed inside. the set up was left to stand until the solvent completely evaporated. these paper discs were then placed on spread plates of salmonella typhi, staphylococcus aureus, bacillus subtilis and escherichia coli and incubated for 18 hours at 37°c (kehinde et al., 2012). paper discs dipped in ethyl acetate and subsequently evaporated were used as a negative control. in the second method, 1 mg pieces of fresh meat samples were cut and placed directly on spread plates containing the test organisms and incubated at 37°c overnight. inhibition zones were observed and measured in millimeters (myllyniemi et al., 2001). detection of antibiotic residues in chicken eggs presence of antibiotic residues on eggs was done using two methods. first, pieces of the eggshells were placed on spread plates containing the test organisms and incubated at 37°c for 24 hours. in the second method, wells were punched into spread plates of muller hinton agar with the test organism. a 0.1ml of egg albumen from each egg sample was placed in the wells. the plates were then incubated at 37°c for 24 hours. zones of inhibition around the agar wells on each plate were observed and the absence or presence of antimicrobial residues recorded (idowu et al., 2010). bacterial counts in raw chicken meat and eggs one gram of the meat sample was aseptically homogenized in 99 ml of 0.85% saline; this effected a 10-2 dilution which was further serial diluted to effect a 103 diluent. from the homogenate, 0.1 ml was spread plated on nutrient agar plates in triplicate and incubated at 37°c for 24 hours. the colony forming units (cfus) were then counted from each plate using a colony counter (akusu and wemedo, 2016). for microbial contamination of eggs, each was placed in a separate sterile plastic bag. a 100 ml sterile isotonic saline was added and washed thoroughly. a 0.1 ml of the washing was then spread plated on nutrient agar plates and incubated at 37°c for 24 hours. viable cell counts were determined by international journal of applied biology, 4(2), 2020 79 counting the cfu. the washing was also used for microbiological isolation of other contaminating organisms (englmaierová et al., 2014). isolation and identification of salmonella, shigella and escherichia coli the fresh meat and egg washing from samples were each pre-enriched in buffered peptone water at 370c for 16 hours. the pre-enriched samples were sub-cultured and incubated for 24 hours at 37°c in selenite f broth for selective enrichment of salmonella and shigella. this was then followed by plate streaking on dca and salmonella agar (oxoid, basingstokes, uk) and incubation was done at 37°c for 24 hours. to isolate escherichia coli, the samples were enriched in lactose broth and after incubation sub-cultured on emb agar (oxoid, basingstokes, uk) at 37°c for 24 hours. typical characteristic colonies of escherichia coli, with a green metallic sheen on emb agar (oxoid, basingstokes, uk) and those of salmonella and shigella on dca were purified by sub-culturing on nutrient agar (himedia, mumbai india). the identity of the isolates was further confirmed by subjecting them to biochemical tests; urease test, triple sugar iron and imvic (indole, methyl red, vogesproskauer and citrate). all the media were inoculated with suspected isolates and incubated at 37°c for 18-48 hours, and the result interpreted according to the manufacturer’s instructions (kyung-min et al., 2015). antibiotic sensitivity testing disc diffusion technique was used to determine the susceptibility of isolates to commonly used antibiotics in animal and human medicine as recommended by the clinical and laboratory standards institute guidelines (clsi, 2015). the antibiotic-impregnated discs used include tetracycline (te 30), chloramphenicol (c 50), ciprofloxacin (cip 5) ampicillin (ax 10), amikacin (ak 30) and gentamycin (gen 10). representatives of the isolates were spread plated on muller hinton agar and antibiotic discs placed on the agar and incubated at 370c for 24 hours. after incubation period, the diameter of the zones of inhibition was measured to the nearest millimeter using a digital calliper (0-150 mm).the isolates were classified as resistant, susceptible or intermediate. data analyses antibiotic susceptibility means of inhibition zones, values for the abundance of the isolated pathogen and bacterial load values were subjected to anova with significant difference determination among means using tukey’s honesty significant difference test at p≤ 0.05. the analysis was done using statistical analysis system version 9.1 (sas institute, cary, nc). results antibiotic residues in chicken meat bacillus subtilis and staphyloccocus aureus were inhibited (plate 1). of the total meat samples, 14 (87.50%) showed inhibition zones on bacillus subtilis , 7 from each sampling site. this was observed on both meat pieces tested and the paper discs, although the paper discs showed more distinct and clear inhibition zones. out of the total meat samples, 6 from ku and 5 from km showed inhibition against staphyloccocus aureus (figure 1.1). international journal of applied biology, 4(2), 2020 80 figure 1.1 percentage levels of inhibiting samples of antibiotic residues in chicken meat. antibiotic residues in chicken egg albumen and shells egg albumen of all the sampled eggs showed 100 % inhibition when tested against bacillus subtilis and staphyloccocus aureus (plate 2). of the total egg sampled, only two (6.25%), from different outlets, revealed inhibition against escherichia coli. none of the samples inhibited the growth of salmonella typhi (figure 1.2). the eggshells did not show any inhibition when subjected to the test organisms. international journal of applied biology, 4(2), 2020 81 figure 1.2. percentage levels of inhibiting samples of antibiotic residues in egg albumen/ bacterial counts of meat samples the mean cfus for chicken meat from ku and km were 104.96 x 103/gm and 190.250 x103/gm, respectively (p≤0.05). samples obtained from km were significantly contaminated as compared to those obtained from ku. the highest bacterial load (288.00 x 103) was recorded in samples obtained from km (p < 0.001). the highest bacterial load of the samples obtained from ku was recorded at 157.00 x 103 (p < 0.001). the least contaminated sample had a bacterial load of 57 x 103gm at p <0.001 and was from ku (table 1.1). table 1.1: mean values breakdown of cfus of meat samples for ku and km ku km meat sample mean ± se meat sample mean ± se mu1 111.667 x 103±3.28cdef mm1 127.333 x 103±4.67cde mu2 125.000 x 103±12.34cde mm2 288.000 x 103±5.03a mu3 70.000 x 103±7.10fg mm3 242.00 x 103±26.62ab mu4 100.667 x 103±2.19edfg mm4 286.333 x 103±5.93a mu5 157.000 x 103±6.43c mm5 142.667 x 103±3.76cd mu6 131.667 x 103±4.49cde mm6 210.333 x 103±3.53b mu7 57.000 x 103±2.52g mm7 86.333 x 103±7.62efg mu8 86.667 x 103±7.42efg mm8 138.333 x 103±4.67cd site mean 104.96 x 103±6.77b site mean 190.25 x 103±15.37a p-value< 0.001 p-value < 0.001 key: mu; ku meat, mm; km meat. means values followed by the same letter within the columns are not significantly different according to tukey’s honest significance difference (hsd) at 5% level bacterial counts on eggs there was a significant difference the between bacterial population of egg samples obtained from ku and km at p≤0.05 (table 1.2). the mean bacterial count from egg washing of samples obtained from ku was 108.292 x 103 while that from egg washing of samples international journal of applied biology, 4(2), 2020 82 obtained from km was 158.875 x 103 both at p < 0.001. the highest bacterial load (286.667 x 103) was recorded at km while the lowest bacterial load (52.667 x 103) was recorded at ku (p < 0.001). table 1.2: mean values ± se breakdown of cfus of egg samples for the two study sites ku km egg sample mean ± se egg sample mean ± se eu1 86.667 x 103±2.91hi em1 286.667 x 103±6.36a eu2 145.333 x 103±6.36cde em2 156.667 x 103±1,76cd eu3 117.667x 103±5.90efg em3 93.000 x 103±6.56gh eu4 52.667 x 103±1.76j em4 247.333 x 103±4.33b eu5 170.333x 103±3.84cd em5 115.667 x 103±5.04fg eu6 150.667 x 103±7.69cd em6 136.333 x 103±4.49def eu7 81.000 x 103±8.33hi em7 156.000 x 103±6.43cd eu8 62.000 x 103±3.46ij em8 79.333 x 103±3.53hij site 108.292 x 103±8.74b site 158.875 x 103±14.30a p-value < 0.001 p-value < 0.001 key: eu; ku eggs, em; km eggs, 1-8 sampling points. mean values followed by the same letter within the columns are not significantly different according to tukey’s honest significance difference (hsd) at 5% level. total isolated pathogens of the total samples analyzed, 14 (43.75%) were positive for salmonella. eight of these isolates were from chicken meat samples while six isolates were from the surfaces of eggs samples. about 6 (18.75%) of the samples were positive for shigella. three of the isolates were from chicken meat samples while the other three isolates were from eggs samples. twenty-one samples (62.63%) were contaminated with escherichia coli. out of this, 12 were from chicken meat samples, while 9 were from egg samples (table 1.3). table 1.3: bacterial pathogens isolated from both sites site sample salmonella sp. shigella sp. e. coli ku meat (n = 8) 5 1 6 eggs (n = 8) 3 2 4 km meat (n = 8) 3 2 6 eggs (n = 8) 3 1 5 percentage 43.75% 18.75% 62.63% the abundance of the isolated pathogen the detection level of the isolated bacteria varied across the sampled outlets within ku at p≤0.05. the abundance of pathogens isolated from ku was significantly different (p= 0.0282). escherichia coli were the most abundant at an average mean of 15.6 followed by salmonella and shigella. the abundance trend of isolates from km samples was similar to that of km isolates with e. coli registering the highest abundance (17.19). shigella was the least abundant in km with a mean of 4.69. overall, the abundance of the isolates between ku and km was not significant with both having a mean of 8.203 and 8.984, respectively (table 1.4). international journal of applied biology, 4(2), 2020 83 table 1.4: abundance of pathogenic bacteria isolated from the study sites key: mean values followed by the same letter within a column are not significantly different according to tukey’s honest significance difference (hsd) at 5% level. antibiotic sensitivity testing the results showed that within each isolated bacterial genus, the inhibition was significantly different (p≤0.05) against the tested antibiotics. all the isolates showed significant zones of inhibition against ciprofloxacin, followed by chloramphenicol (table 1.5). the isolates were 100% susceptible to ciprofloxacin and gentamicin. none of the isolates was susceptible to ampicillin. all escherichia coli species and shigella species were resistant to ampicillin, while 83% salmonella were resistant to ampicillin (table 1.5) table 1.5. inhibition zones in mm of the isolates to the tested antibiotics antibiotic test organism salmonella sp. e. coli shigella sp. amikacin (30) 17.5±0.55b 16.833±0.75b 17.333±1.53b ampicillin (30) 7.33±3.27c 6.000±0.00c 8.67±4.62c chloramphenicol (50) 28.667±1.12a 29.667±1.03a 30.667±0.58a ciprofloxacin (30) 30.667±1.21a 29.33±4.23a 30.00±4.36a gentamycin (10) 19.333±1.21b 19.5±1.23b 19.333±0.58b tetracycline (50) 19.333±1.21b 19.17±4.26b 18.333±1.53b p-value < 0.001 < 0.001 < 0.001 key: means followed by the same letter within a column are not significantly different according to tukey’s honest significance difference (hsd) at 5% level. table 1.6. antimicrobial susceptibility profiles of the isolates key: s=sensitive; i=intermediate; r=resistant; n=number of tested isolates isolate ku km e. coli 15.63±3.13a 17.19±1.56a salmonella sp 12.5±3.13ab 9.38±2.56ab shigellasp 4.69±0.56ab 4.69±0.56ab p-value 0.0282 0.0312 site 8.203±2.50 8.984±2.16 p-value 0.0729 percentage inhibition of all the isolates tested antibiotic salmonella sp. (n=6) escherichia coli (n=6) shigella sp. (n=6) s i r s i r s i r ciprofloxacin(30) 100 0 0 100 0 0 100 0 0 tetracycline (30) 100 0 0 84 16 0 100 0 0 gentamicin (10) 100 0 0 100 0 0 100 0 0 chloramphenicol (50) 100 0 0 100 0 0 100 0 0 amikacin (30) 100 0 0 67 33 0 67 33 0 ampicillin (10) 0 17 83 0 0 100 0 0 100 international journal of applied biology, 4(2), 2020 84 discussion the current findings revealed bacillus subtilis as the highly susceptible bacteria to most of the samples of chicken meat and egg albumen. these results correspond with those of a study carried out in khartoum by elnasri et al. (2014) who reported susceptibility of bacillus subtilis to most of the samples of chicken tissue. these results also suggest that the test organisms were subjected to samples which contained unspecific inhibitory substances (myllyniemi et al., 2001). the potential of chicken meat and egg albumen to inhibit the growth of bacillus subtilis, staphyloccocus aureus and escherichia coli depicts poultry products as have unspecified antibiotic residues and therefore, potential reservoir of antibiotic resistance. susceptibility of these bacteria to the chicken meat and egg albumen could be linked to the use of antibiotics in poultry production (mund et al., 2017). samples obtained from km were significantly contaminated as compared to those obtained from ku. the samples evaluated in this study came from two different sites whose outlets serve almost the same clients. the difference in the contamination could be attributed to poor handling of poultry food products. according to jans et al. (2018), transmission of bacteria between food animals and humans occurs during the handling of animal food products at production, distribution, and household levels. there is limited sanitary knowledge for most food handlers at km as opposed to ku food handlers who have some level of knowledge in sanitation as trained caterers. in a related study carried out in kumasi, ghana, it was found that street food was more contaminated with bacteria, especially enteric bacteria caused by improper handling of food (feglo and sakyi, 2012). the most common bacterial contaminant in this study was escherichia coli, followed by salmonella, and shigella, which gives evidence of the presence of enteric bacterial contamination in the tested samples within the study areas. wong et al. (2009) documented that salmonella mostly contaminate meat from faecal material during slaughter and processing. the authors added that salmonella sometimes might be present internally in meat tissue of infected animals. the difference in bacterial contamination of samples in the current study can be associated with the preservation methods used. during sampling, most of the meat samples from km were not refrigerated, while in ku all the samples were kept in a cold room. in a related study, mensah et al. (2002) who investigated the microbial quality of foods sold on streets of accra, ghana and factors predisposing to their contamination reported similar findings. rane, (2011) reported that in developing countries, food sold in open-air markets are mostly contaminated with salmonella sp., escherichia coli, and shigella sp. the researcher associated this with poor handling techniques of food and lack of proper storage facilities. the results revealed a high percentage of salmonella, escherichia coli and shigella resistance to ampicillin and high susceptibility to ciprofloxacin, gentamicin and chloramphenicol. this result corresponds with those of nyamboya et al. (2013) who reported high resistance against ampicillin and susceptibility to chloramphenicol and gentamicin by bacteria isolates from different abattoirs in nairobi. tetracycline and amikacin were moderately resisted by shigella and escherichia coli. the existence of resistant bacteria in the chicken meat and egg albumen samples reflect the potential spread of these bacteria in human consumers as well as the transfer of resistant components among the bacteria in the environment (singer et al., 2016). the incidence of antibiotic resistant bacteria in poultry products as shown in the current study is a threat to public health as the bacteria could disseminate antibiotic resistant genes to other bacteria of human clinical significance (woolhouse et al., 2015). poultry products could, therefore, act international journal of applied biology, 4(2), 2020 85 as conduits for the dissemination of clinically relevant antibiotic resistance to the environment. conclusions there is a high level of antibiotic residues in chicken meat and eggs sold in ku and km. these residues are in contact with commensals and clinically relevant bacteria on the same chicken products. there is, therefore, a high risk of these microbes adapting to the presence of the antibiotics and thus developing resistance to them. the chicken products in the study area are contaminated with bacteria pathogens; escherichia coli, salmonella and shigella, which pose a health risk to the population. the isolates were resistant to ampicillin and relatively resistant to tetracycline. this study findings form the basis upon which intervention tools for monitoring the influence of animal origin food on the development of antibiotic resistant bacteria in the environment can be developed. consent for publication not applicable availability of data and materials the datasets analyzed during the current study are available from the corresponding author on reasonable request competing interests the authors declare that they have no competing interests. funding not applicable author contributions dkn and lmk conceived the idea for the study and designed the experiment. dkn and pkk collected data, performed the experiments and analyzed data. dkn and ham prepared and reviewed the manuscript. all authors read and approved the final manuscript. acknowledgements the authors acknowledge meat and eggs vendors for agreeing to participate and consent for us to collect chicken meat and eggs. we thank the department of biochemistry, microbiology and biotechnology of kenyatta university for allocating laboratory space for sample analysis.. references abdullahi, m., s. olonitola, v. umoh, and i. inabo. 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(2009). salmonella, escherichia coli o157:h7 and e. http://doi.org/10.1016/j.ijfoodmicro.2005.03.008 https://doi.org/10.1039/b306622c http://ir-library.ku.ac.ke/handle/123456789/7336 http://doi.org/10.1007/s12088-011-0154-x international journal of applied biology, 4(2), 2020 88 coli biotype 1 in a pilot survey of imported and new zealand pig meats. food microbiology, 26:177 – 182. woolhouse, m., ward, m., van bunnik, b., and farrar, j. (2015). antimicrobial resistance in humans, livestock and the wider environment. philosophical transactions of the royal society b: biological sciences, 370(1670), 20140083. world health organization. (2017). prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis (no. who/emp/iau/2017.12). world health organization. contact : shkar rzgar k. rostam shkar.rostam@univsul.edu.iq 36 abstract nowadays, infections of the hbv and hcv are one of the major global public health problem. the objective of this study was to determine the prevalence of hbv and hcv virus from the first six months of 2020 in the of erbil province, kurdistan region of iraq. also associations between the prevalence of hbv and hcv in different age groups, has been established. the study was conducted on (101) individuals (63 males and 38 females), of different aged population groups, in erbil city from beginning of january 2020 to the end of june 2020. to detect the positive results of both hbv and hcv (vl), all samples subjected to detect their viral load by genexpert (rt pcr or rtqpcr). out of (101) samples, 24(%23.76) of hbv and 7(%6.93) for hvc in both sex were detect positive respectively. prevalence of both hbv and hcv was highest in males than females. also the highest percentage of hbv found between (19-40) years, 12(%19.04) in males and 5(%13.15) in females. in addition highest number of hcv positive was found between (41-60) years, which 2(%3.17) and 2(%5.26) for males and females respectively, while opposite results were found from other aged groups issn : 2580-2410 eissn : 2580-2119 screening of prevalence hbv and hcv from first six months of 2020 in erbil province kurdistan region /iraq shkar rzgar k. rostam 1*, harem othman smail 2 1department of biology, college of science, university of sulaimani, sulaimani, kurdistan regioniraq. 2department of biology, faculty of science and health, koya university, koya koy45, kurdistan region-f. r. iraq introduction viral hepatitis b and c viruses (hbv and hcv) are responsible for the majority of chronic liver disease worldwide (villar et al., 2015). an infected individual spreads the viruses in contaminated blood and body fluids to an uninfected individual (pardee 2019). worldwide, there are over 350 million people with chronic hbv (hebo et al., 2019). of the approximately 2 billion exposed, more than 600 000 deaths annually; 170 million have chronic hcv, with nearly 500 000 deaths annually (kateera et al., 2015). hbv infection prevalence varies significantly by geographic area, ranging from 0.1% to 20% in different parts of the world and from 2% to 8% in countries with intermediate endemicity for hepatitis b infection (tozun et al., 2015). infections of chronic hepatitis b virus (hbv) and hepatitis c virus (hcv) are typically asymptomatic for decades, and proactive screening can avoid liver disease through prompt open access international journal of applied biology keyword hbv, hcv , genexpert hbv viral load , genexpert® hcv viral load and erbil article history received 04 january 2021 accepted 03 july 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(1), 2021 37 diagnosis and treatment. more reliable estimates of the general population and risk groups for chronic hbv and hcv infections are required (kanda et al. , 2019). the number of deaths caused by the majority of communicable diseases, hepatitis mortality rates worldwide, rose from 0.89 million in 1990 to 1.45 million in 2013 (moradi et al., 2019). few reproducible mortality studies have been carried out in patients with autoimmune hepatitis (aih) and its variants (van et al. ,2019). recently, an epidemiological study of hbv and hcv infection has been performed among individuals between the ages of 10 and 69 living in the five geographic regions of brazil, with average hbsag, anti-hbc and anti-hcv seroprevalence levels of 0.37%, 7.4% and 1.38% respectively (villar et al., 2014). the plan of the global health sector at the who to eradicate viral hepatitis as a major threat to public health by 2030 needs significant efforts from governments, health authorities and communities. to help control the number of people living with hepatitis b virus (hbv) (who 2016), service provision needs to be increased. just 5 million of the estimated 71 million people diagnosed with hcv have currently been treated (popping et al. ,2020). several research showed that in the first years of injection drug use, the incidence of hcv was greatest, but little is known about health seeking actions (enkelmann et al. ,2020). materials and methods sample collection a total of 101 blood samples were collected from suspected patients, who visited the private clinical sectors, from 1 january to 30 june 2020. 10 ml of blood was drawn from each suspected patients. 5 ml of the blood samples collected inside the gel tube, after clotting, centrifuged them for 15 minutes at 5000 rpm. other remained 5 ml of blood collect inside the (edta) tube, centrifuged as mentioned above. a questionnaire was also prepared and used to gather personal data (name (optional), age, and gender) for each case. the samples separated regarding to their gender to (63 males and remains were females), and their ages in to four specific age groups (6-18, 19 -40, 41 -60 and 61 -86). hbv and hcv viral load detection blood samples subjected for detection of hbv and hcv viral load by real-time qpcr (real-time quantitative pcr) technology. regarding to the manufacture instructions catalog number gxhbv-vl-ce-10 targets for hbv dna, and catalog number gxhcv-vl-ce-10 targets for hcv rna ,used for screening and diagnose the viral load of both virus in our samples. for detection of hbv (0.6-1ml plasma edta) for hcv (1ml of plasma of edta) added to their cartridges, by using 1ml micropipette with disposable pipette. cartridges loaded inside the genexpert instrument, which it is full automate system and integrate specimen purification, nucleic acid amplification, and detection of the target sequence in simple or complex specimens using real-time reverse transcriptase pcr (rt-pcr) and (rt-qpcr) which uses fluorescence to detect the rna and dna of interest viruses. the systems require the use of single-use disposable genexpert cartridges that hold the rt-pcr reagents and host the rtpcr processes. xpert hcv and hbv viral load cartridge includes reagents for the detection of hcv rna and hbv dna in specimens as well as two internal controls used for quantitation of hcv rna and hbv dna. the internal controls (high and low) internal quantification standards (iqs-h/iqs-l) monitor recovery and the presence of inhibitor(s) in the rt and pcr international journal of applied biology, 5(1), 2021 38 reactions. to verifies that the correct volume of sample has been added in the sample chamber and ensure the sample was correctly added to the cartridge system has sample volume adequacy (sva) control. also the probe check control (pcc) verifies reagent rehydration, pcr tube filling in the cartridge, probe integrity, and dye stability. because the cartridges are self-contained, cross contamination between samples is minimized. the results are interpreted automatically by the genexpert instrument system from measured fluorescent signals. regarding to who reference material for plasma and serum samples limitation by genexpert system reported as 3.20 iu/ml, 5.99 iu/ml respectively for linear range quantification 10 to 109 iu/ml for hbv and 10 to 108 iu/ml for hcv (who 2016). statistical analysis the chi-square was applied to examine the relationship between the prevalence of hbv and hcv from different sexes and to determine the association the prevalence of hbv and hcv among different aged gropes. p-values < 0.05 were considered to be statistically significant. results table 1. prevalence of hbv and hcv according to gend table 2. prevalence of hbv according to four specific age groups viruses number of screened % positive p value males females total males females total hbv 63(%62.37) 38(%37.62) 101(%100) 19(%30.1) 5(%13.15) 24(%23.76) 0.004 hcv 63(%62.37) 38(%37.62) 101(%100) 3(%4.76) 2(%5.12) 7(%6.93) 0.654 gender different age groups % hbv positive % p value 6-18 19-40 41-60 61-86 total 6-18 19-40 41-60 61-86 total male 5 (%7. 93) 27 (%42. 85) 15 (%28. 8) 16 (%25. 39) 63 (%62. 37) 1 (%1. 58) 12 (%19. 04) 5 (%7.9 3) 1 (%1.5 8) 19 (%30. 1) 0.0007 female 3 (%7. 89) 18 (%47. 36) 15 (%39. 47) 2 (%5.2 6) 38 (%37. 62) 0 (%0) 5 (%13. 15) 0 (%0) 0 (%) 5 (%13. 15) 0.001 international journal of applied biology, 5(1), 2021 39 figure 1. prevalence of hbv and hcv according to genders figure 2. prevalence of hbv according to four specific groups international journal of applied biology, 5(1), 2021 40 figure 3. prevalence of hcv according to four specific groups discussion infection with the hepatitis b virus ( hbv) and hepatitis c virus ( hcv) is a public health problem (police et al. ,2020). many men, 40-80% live with chronic hepatitis b or c, are unaware of their serostatus, and remain infectious to others (ayele et al. ,2020). recent data have shown that about 350 million people are chronically infected with hbv and around 200 million are infected with hcv (hussein et al . ,2017). in recent years, more and more data have been developed in developing countries and high/intermediate endemic areas where the most common route of infection is still vertical transmission from mother to child, and horizontal transmission between children , particularly siblings (franco et al., 2012). previous data from 2006 to 2009 suggest that a low prevalence of hepatitis b and hepatitis c infection exists in iraq's capital city baghdad (ataallah et al., 2011). it also reported similar results in basra (al-rubaye et al . , 2016). hbv and hvc prevalence are investigated in erbil (taher et al. ,2020). the prevalence of hepatitis b surface antigen (hbsag) and hcv antibody (anti-hcv) was identified in the turkish population as 2.3% and 0.37% respectively (yakaryilmaz et al., 2006). at the national level, the age-standardized prevalence of hepatitis b in iran fell from 3.02 % (95 % uncertainty interval; 2.26 to 3.96) in 2000 to 1.09 % (95 %uncertainty intervals; 0.85 to 1.37) in 2016, with a cumulative improvement of -64.84 %. in 2016 the prevalence of hepatitis b in males was more than 1.3 times greater than in females (rezaei et al. ,2020). the total number of screened for hbv and hbc were 101 blood samples, 63(%62.37) of them were males and 38(%37.62) were females. the present study indicated that %23.76 in males and %13.15 in females of the samples were positive for hbv, while the number of hcv positive dramatically decreased to %4.76 in males and %5.12 in females. for both viruses statistically showed significant for hbv the p-value was 0.004, whereas for hcv was non-significant and the p-value 0.654. (table 3.1 and figure 3.1). although the prevalence of hbv and hcv among diabetic patients is higher, it has a low prevalence compared with other patients studied (merza et al. ,2020). at 1992 epidemiology estimated that the prevalence of hbsag in the general chinese population was 75 % (said et al . , 2019). chronic hepatitis b patients reported slightly lower health-related life scores in various areas relative to stable control subjects in the province of al-najaf / iraq (kadhim et international journal of applied biology, 5(1), 2021 41 al. ,2020). there are 3 prequalified rapid diagnostic assays for hepatitis b surface antigen (hbsag) by who that is responsive, accurate, and effective in hbv screening: sd bioline, determines 2, and vikia. pocts are less accurate and sensitive to other biomarkers but have some applicability. the genexpert hbv viral load assay is sensitive and specific with a wide range of dynamics (jackson et al. ,2020). the hbv dna quantification is used to initiate and monitor antiviral treatment (abravanel et al. ,2020). the genexpert hbv-vl test, which has the potential for near-point molecular treatment testing, showed excellent performance and proved to be a reliable tool for quantification of hbv dna (khodare et al. ,2020). the research found a seroprevalence rate of 13.3 % of hbeag among prospective asymptomatic blood donors (rufai et al., 2014). in our results showed that there are association among the different aged group that infected by hbv in male, the statistical of p-value was 0.0007, the highest number was 1940 aged groups 12(%19.04) while in 41-60 ages were 5(%7.93) .on the other hands for two other remains groups have the lowest number of positive hbv 1(%1.58) respectively .in female the prevalence of hbv positive has the largest number was 5(%13.15) in aged groups 19-40 years and three other groups there were no any infected hbv positive and statistically significant ( table 3.2 and figure 3.2). hbv prevalence at 2.2% and carrier rate at 0.3% in men and 0.1% in women (goudeau et al., 1995). at first sex, the number checked and those positive in the age group is as follows: < 10 years (7/25), 11-14 years (24/143), 15-18 years (76/433), 19-22 years (14/103), > 23 years (2/16)(forbi et al., 2008). the prevalence of hepatitis b surface antigen (hbsag) and total anti-hbc (core) was 6.8% and 35.6% in turkish ,kurds, and 2.2% and 12.7% in iraqis respectively (chironna et al., 2003). based on the prevalence in the general population of carriers of hepatitis b surface antigen, countries are classified as high (> 8%), intermediate (2-7%) or low (< 2%) endemic. iraq is among the middle prevalence countries (hussein et al., 2015). also, approximately 5% of the general population in western iran has previous hbv exposure and less than 1% is hbsag carrier (alavian et al., 2012). the prevalence of hepatitis b surface antigen (hbsag) varies according to different reports, but in iranian provinces, a prevalence of 1.2%-5% was registered (ziaee et al . , 2016). at the point of care, molecular testing can turn out to be a game-changer for hcv diagnosis and treatment monitoring through increased sensitivity, reduced turnaround time, and performance ease. one such assay has recently been released on genexpert (gupta et al., 2017). although the rna point-of-care technology for novel hepatitis c virus (hcv) has the potential to improve diagnosis in resource-limited settings (mohamed et al. ,2020). viral load genexpert hcv (within 105 minutes). (latham et al., 1998). for genexpert hcv viral load and venipuncture blood samples, finger-stick whole-blood samples were used (bajis et al . , 2019; calvaruso et al., 2019; saludes et al., 2020). regarding hcv infection 3(%4.76) for different aged groups mostly between 41-60 years, their prevalence was 2(%3.17) and it is higher than from other groups and statistically significant in males the p-value was 0.0008. however for female groups all positive hcv in 41-60 aged groups (2(%5.26) and cannot find any positive hcv from reaming groups, therefor statistically no significant and its p-value was 0.11 (table 3.3 and figure 3.3). in iraq hcv prevalence rises dramatically with maternal age, women around 30 years of age are considered to be a risk group for hcv infection (al-kubaisy 2012).evelopment are people who tend to be lazy and do not care about their environment. international journal of applied biology, 5(1), 2021 42 conclusions we have shown that nearly a quarter of the blood 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cihan university-erbil scientific journal, 4(1), pp.45-51. tozun, n., ozdogan, o., cakaloglu, y., idilman, r., karasu, z., akarca, u.l.u.s., kaymakoglu, s. and ergonul, o., 2015. seroprevalence of hepatitis b and c virus infections and risk factors in turkey: a fieldwork turhep study. clinical microbiology and infection, 21(11), pp.1020-1026. van den brand, f.f., van der veen, k.s., de boer, y.s., van gerven, n.m., lissenberg-witte, b.i., beuers, u., van erpecum, k.j., van buuren, h.r., den ouden, j.w., brouwer, j.t. and vrolijk, j.m., 2019. increased mortality among patients with vs without cirrhosis and autoimmune hepatitis. clinical gastroenterology and hepatology, 17(5), pp.940-947. villar, l.m., amado, l.a., de almeida, a.j., de paula, v.s., lewis-ximenez, l.l. and lampe, e., 2014. low prevalence of hepatitis b and c virus markers among children and adolescents. biomed research international, 2014. villar, l.m., cruz, h.m., barbosa, j.r., bezerra, c.s., portilho, m.m. and de paula scalioni, l., 2015. update on hepatitis b and c virus diagnosis. world journal of virology, 4(4), p.323. world health organization, 2016. global health sector strategy on viral hepatitis 2016-2021. towards ending viral hepatitis (no. who/hiv/2016.06). world health organization. yakaryilmaz, f., alp gurbuz, o., guliter, s., mert, a., songur, y., karakan, t. and keles, h., 2006. prevalence of occult hepatitis b and hepatitis c virus infections in turkish hemodialysis patients. renal failure, 28(8), pp.729-735. ziaee, m., ebrahimzadeh, a., azarkar, z., namaei, m.h., saburi, a., fereidouni, m., bijari, b., karamian, m. and sharifzadeh, g., 2016. seroprevalence and risk factors for hepatitis b in an adult population: the first report from birjand, south khorasan, iran. hepatitis monthly, 16(9). contact : magdalena litaay mlitaay@fmipa.unhas.ac.id 149 abstract tunicates belongs to urochordate that inhibit coral reefs ecosystem. the uniqueness of the tunic can be seen from the body shape like a tube with 2 siphons and the body is covered by a protective layer known as the tunic. the present study aims to observe marine tunicate at waters of badi island, pangkajene regency, south sulawesi. this research using the belt transect method with a length of 20 m and a width of 4 m at a depth of 5 m with 4 station points according to the wind directions. all tunicate inside transect was noted and identified. identification was based on morphological characteristics. the result shows that there were 18 species of tunicates present in the coral reefs ecosystem of badi island. issn : 2580-2410 eissn : 2580-2119 biodiversity of tunicate (ascidiacea) in the waters of badi island, pangkajene regency of south sulawesi rensi piri1, magdalena litaay1*, dody priosambodo1 1 department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar introduction tunicate include animals that are in the primitive chordate phylum with ±3000 species in it (santhanam, 2020). most have habitats in the sea by sessile or free life in the water column and live in colonies. the group of animals that have tunics, hence the name tunicates, is made of cellulose which is rarely found in animals (shin, 2014). urochordata is different from other chordates because there is a larval stage, urochordates have a neural tube and a notochord, but will disappear in the adult phase (collin, 1995). systematically the general classification of tunicates of the phylum chordata, subphylum urochordata with classes divided into 4, namely ascidiacea, thaliacea, appendicularia, sorberacea (santhanam, 2020). these animals usually inhabit littoral waters and in the intertidal to subtidal zones and mostly live sessile or attached to coral reefs (tahir, 2016). according to ali et al (2014), tunicates can also be attached to the hull which will increase the weight of the ship. the spread of tunicates from one place to another is quite fast and good adaptability is carried out by this group. the spread can be through ship transportation and also currents in the waters that carry larvae to other places (saputri, 2019). tunicate that lives on coral reefs are known to contain chemical compounds that are used as antibiotics, anti-tumor, antiinflammatory, immunosuppressant, and anti-cancer (litaay, 2018). one example of a species is didemnum molle which is in symbiosis with bacteria that produce bioactive compounds (used as antifouling and antiviral) (tahir, 2016). tunicate has an ecological role, namely marine life that contributes to maintaining the stability of the marine ecosystem. open access international journal of applied biology keyword ascidian; sangkarang; spermonde archipelago; species distribution article history received march 15, 2022 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 150 this benefit causes the existence of tunicates to be very important for the habitat of marine biota, so scientific information related to this biota is needed. information on biodiversity in sangkarang area is limited. on the other hand information on biodiversity is baseline data to support sustainability of marine resources. therefore, the distribution of species and their habitats, especially on waters of badi island of sangkarang archipelago is required. materials and methods the tools used in this research were stationery, camera, sample bottle, label paper, thermometer, ph meter, drift float, roll meter, bcd, mask, fins, salinity hand-refractometer, do meter, calculator, gps (global positioning system), diving knife, scissors, tunicate identification book. the materials used are 70% alcohol and tunicates. the research was carried out in october 2020-november 2021 on the waters of badi island, pangkajene regency, south sulawesi. the method used is a 20 m long belt transect with a width of 4 m which was carried out at a depth of 5 m at four different stations according to the wind directions. all tunicate that found were noted, and proceeded for identification. species identification was observed morphologically and documented. the tunicates that could not be identified directly at the observation site were taken and preserved using 70% alcohol and further identified in the laboratory. results and discussion the results of observations that have been carried out in the waters of badi island show that there are 18 species of tunicates, especially the ascidiacea class, which were obtained at the 4 stations, namely east, south, west, and north. at station i (east) there were 9 species of tunicates, station ii (south) 6 species, station iii (west) 9 species, at station iv (north) there 11 species of tunicates. number of species in the present study is higher compare to the previous research by hakim (2013), in which only 10 species of tunicates belonging to the ascidiacea class were found in badi island waters. in different study, mawaleda (2014) found 33 species of tunicates from 7 family present in waters of baranglompo island, nearby badi island. litaay et al. (2018) found similar number of tunicate species (18) at waters of samalona island, eventhough some species are different from the present study. the distribution of tunicates in the waters of badi island is quite varied and there are several species that are only found at a some stations. this is related to the presence of several factors such as differences in environmental parameters that affect the distribution of the tunicate. in general, tunicates in the ascidiacea class have habitats on coral reefs, bo th live corals and dead corals, while on sandy, muddy, or broken coral substrates are usually only occupied by certain tunicates. one of the reasons why there are fewer tunicates on badi island compared to barranglompo island because the coral cover which is the habitat of the tunicates is greater on barranglompo island and research by hakim (2013) is thought to be related to the influence of abiotic and biotic factors on badi island. of the 18 types of tunicates found on badi island (figure 1), 3 species that have a wide distribution or can be found at all station location points are polycarpa aurata , didemnum molle , and polycarpa sp., while the other species are only found in several stations and even only found in 1 species. at 1 station, namely aplidium breviventer and ascidia sp. international journal of applied biology, 6(1), 2022 151 figure 1. species of tunicates in the waters of badi island a. polycarpa aurata, is the most common type of tunicate found in water areas with a body shape similar to a heart, having a yellow/white color with blue and purple stripes. the body size is larger than other tunicates, which is about 5-15 cm and lives attached to corals. b. polycarpa morph brown, tunicates that live solitary with a body size of 5-15 cm. p. morph brown is pale brown in color with sediment that almost covers part of the tunic and has a fairly hard tunic. these tunicates usually live attached to corals. c. didemnum molle, a tunicate species that has a high distribution with quite a lot of numbers in the waters. d. molle including solitary tunicates that are in groups and can grow to a range of 1-3 cm. on the part of the tunic is yellowish-white to greenish due to the presence of prochloron sp. which is in symbiosis with d. molle. found in flat areas of coral reefs or areas that are still exposed to sunlight. d. clavelins robusta, one of the tunicates that have a black body which near the siphon is yellowish-white and this tunicate is a solitary tunicate. the size of c. robusta ranges from 1-3 cm. e. herdmania morph red, including tunicates that live solitary and can be found attached to coral substrates. the body color of h. morph red is red and has a fairly long siphon where at the end of the siphon there is a vertical white pattern and the body size is about 5-10 cm. f. herdmania pallida, is a solitary tunicate that lives permanently on corals. the body size is about 5-7 cm with the body covered by the substrate and has a pale red color. g. pyura sp., is a tunicate that lives solitarily in waters. body size ranges from 5-10 cm and there is sediment that covers the body. this tunic has a tunic that is quite hard and looks like a thickening on the siphon. h. rhopalaea crassa, a type of tunicate that has a blue body and a slightly darker siphon, so this species is easy to find. the body size of r. crassa is about 2-5 cm and is usually found attached to corals. international journal of applied biology, 6(1), 2022 152 i. ascidian morph transparent white, this type of tunicate has an almost transparent white color with a size of 2-5 cm and there is a white vertical line on the siphon hole. including solitary tunicates and live in the coral reefs. j. polyclinidae morph black, is a tunicate that lives in colonies and lives attached to corals. has a small body shape and a black tunic. k. diplosoma sp., tunicates that live in groups or in colonies and live attached to coral areas, especially branching corals. diplosoma sp. has a very small body shape and looks blue on each siphon. l. trididemnum solidium, which lives in colonies and is attached to substrates such as corals. t. solidium has a light blue body color and the location of the siphons is not close to each other. m. applidium breviventer, live solitary and attached to coral crevices. the body shape of a. breviventer is that it has small pores on the body. body size is about 2-5 cm with a white body. n. oxycornia fascicularis, tunicates that have a unique body shape, where there are small circles that are gathered on a single stalk resembling a tree. in each circle, there are 2 white siphons and count as 1 individual/species. this type of tunicates can be said to live in colonies with a size of 0.5-2 cm in each individual. o. didemnum albidum, live in colonies and attach to substrates such as coral. d. albidum has a pale white body with small pores. the siphon hole is quite large and has a body size of about 2-5 cm. p. ascidia sp., live solitary and can be found attached to coral crevices. it it has a fairly long siphon and at the end of the siphon it is transparent and has a body size of about 5-8 cm. q. polucarpa contecta, tunicates solitary but form groups or live in colonies. p. contecta is brown with a size of 1-3 cm and lives attached to substrates such as coral. r. polycarpa sp., tunicates that live solitary and can be found on coral reefs, especially massive corals. this type has a body shape/tunic which is quite hard and firmly attached to the substrate. its body size ranges from 5-10 cm and when opened the inside of the chiffon looks purplish pink. environmental parameters environmental parameters is described in the table 1. as can be seen from the table, ph value of 7.8-8.1 which indicates a relatively safe value for tunicates. according to pancawati (2014) waters with a ph that tends to be stable are in the range of 7-8.5 and for marine biota, especially live tunicates, at a ph of 6.7-8.6 (radhalakshmi, 2014). salinity values are in the range of 29.6-30.6 ‰, this is in accordance with kott, et al (2009) which states that tunicates can generally live at 29-32 ‰ salinity. table 1. environmental parameters in study area station ph salinity (‰) temperature ( °c ) do current (m/s) clarity (m) i (east) 7.8 30.6 26 5.8 0.9 8 ii (south) 8.1 29.6 27 5.9 0.16 8.7 iii (west) 7.9 30.3 26 5.8 0.1 10.7 iv (north) 8.1 29.6 27 5.7 0.12 9.7 international journal of applied biology, 6(1), 2022 153 the temperature value ranged from 26-27°c respectively, which according to menklh, 2004 regarding the normal temperature limit in the waters is 28-32°c, so it can be said that the temperature in the waters of badi island is still suitable for tunicate life. do (dissolved oxygen) according to the menklh, 2004 seawater quality standard suitable for marine biota is >5. the results showed in table 1, do ranged 5.7-5.9 indicating a relatively safe value for tunicates. current values at all stations are classified as moderate current speeds. this is supported by padang (2020) that currents that are less than 0.1 m/s are classified as weak, while a value of 0.1-1 m/s are classified as moderate, and a value of 1 m/s are classified as a strong ocean current. the measurement of the clarity level showed in the table is at a value of 8-10.7 m. according to menklh (2004) the clarity of the waters for marine biota is 3-5 m, while the results obtained are above the average value so that it can be said that badi island has waters with a high clarity. conclusions we conclude that 18 tunicates species present at waters of badi island. of these tunicates polycarpa aurata, didemnum molle, and polycarpa sp. show a wide distribution in the study area. international journal of applied biology, 6(1), 2022 154 references ali, h. a. j., m. tamilselvi., and v. sivakumar (2014). non-indigenous ascidians in vo chidambaram port, thoothukudi india. indian journal of geo-marine sciences. 43(11):2147-2157. collin, p. l. and c. anerson. 1995. tropical pacific invertebrates : a fiels guide to the marine invertebrates occuring on tropical pacific coral reef, seagrass and mangrove. california. coral reef press. hakim, m. f. b. a. 2013. distribution and diversity of tunicates (ascidiacea) in different water conditions in badi island, bone batang and lae-lae. bachelor honors skripsi fikp unhas. kott, p. 2009. taxonomic revision of ascidiacea (tunicate) from the upper continental slope off north-western australia. journal of natural history. vol 43:31-32. litaay, m, s. santosa, e. johannes, r. agus, w. moka, j.d.d. tanjung. 2018. biodiversity of marine tunicates in samalona waters sangkarang archipelago, indonesia. spermonde 4(1): 26-31. litaay m. 2018. marine tunicate from sangkarang archipelago indonesia: recent finding and bioprospecting. j. phys.: conf. ser. 979 012003. doi:10.1088/1742-6596/979/1/ 012003 mawaleda, r. 2014. distribution and habitat preferences of ascidiacea class urochordata in coral reef areas of barranglompo island, makassar city. bachelor honors skripsi fikp unhas. menklh. 2004. decree of the minister of the environment number: 51/menlh/2004 of 2004, the determination of sea water quality standards in the set of regulations in the environmental field. jakarta. pancawati, d. n., d. suprapto., and p. w. purnomo., 2014. physical and chemical characteristics of bivalve habitat waters in the wiso river, jepara. diponegoro journal of maquares. vol 3 (4): 141-146. padang, r. w. a., w. nurgayah., n. irawati. biodiversity and vertical distribution of phytoplankton in bokori island waters. sapa laut. vol 5(1): 1-8. radhalakshmi, r., v. sivakumar., and h. a. j. ali., 2014. analysis of selected species of ascidians as bioindicators of metals in marine ecosystem. international journal of current microbiology and applied science. vol 3(8): 755-764 saputri, n. m. p. m.., i. d. n. n. putra., and w. karim., 2019. abundance and diversity of tunicate (ascidiacea) in jemeluk and penuktukan waters, bali. journal of marine and aquatic sciences. vol 5(1): 11-21. santhanam, r. and s. ramesh., 2020. biology and ecology of pharmaceutical marine tunicates. boca raton. crc press. shin, y. k., j. j. park., j. i. myeong., m. a. jeon., and j. s. lee., 2014. digestive gland ultrastructure of the tunicate, halocynthia roretzi (ascidiacea: pyuridae) in relation to function. journal of coastal life medicine. vol 2(12): 947-952. international journal of applied biology, 6(1), 2022 155 tahir, e., m. litaay., r. g. budji., n. haedar., priosambodo, and syahribulan. 2016. potential of tunicate rhopalaea sp. as a source of antimicrobial symbiont fungal inoculum. jurnal spermonde. vol 2(2): 33-37. contact : nadhila idris nadhilaidris14@gmail.com 36 abstract mustard g reens brassica juncea l. one of vegetable that is very easily damaged by microorganisms known as soft rot disease. this causes a decrease in the quality of green mustard so that it cannot last long. a study entitled “the potential of hexadecanoic acid compounds as antimicrobials in bacteria and fungi that cause decay in mustard greens brassica juncea l.". this research aims to specify effect of hexadecanoic acid compounds in inhibiting the growth of bacteria and fungi that cause decay in mustard greens. an inhibition test was carried out on xanthomonas campestris bacteria and fusarium oxysporum fungus using 10%, 20%, and 40% hexadecanoic acid test compounds. the results obtained showed that 10%, 20%, and 40% hexadecanoic acid extracts were able to inhibit the growth of xanthomonas campestris bacteria and the fungus fusarium oxysporum. hexadecanoic acid compounds are bacteriostatic in xanthomonas campestris and fungi fusarium oxysporum are fungistatic. issn : 2580-2410 eissn : 2580-2119 potential of hexadecanoic acid as antimicrobials in bacteria and fungi that cause decay in mustard greens brassica juncea l. nadhila idris1*, eva johannes1, zaraswati dwyana1 1 department of biology, mathematic and natural sciences faculty, hasanuddin university, makassar, indonesia introduction indonesia is an agrarian country, because of that agriculture have an crucial role within the standard the overall wheels of the country's economy. t his can be proven by the large number of people who work in agriculture as a source of livelihood which is also supported by suitable natural conditions that allow residents to plant throughout the yea r (suratman, 2018). green mustard brassica juncea l. is a vegetable commodity that is in great demand by the people of indonesia because it has commercial value and good prospects from various aspects. in addition, mustard has a high demand and is always increasing along with the increasing population in indonesia and increasing public awareness of the importance of nutritional needs (sarif, et al, 2015). open access international journal of applied biology keyword mustard greens brassica juncea l; hexadecanoic acid; xanthomonas campestris; fusarium oxysporum; article history received march 3, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 37 but in its cultivation it often has drawbacks because it is easy for green mustard to be attacked by diseases caused by bacteria and fungi that cause decay to occur in mustard greens so that the quality of mustard greens decreases and the selling price of mustard greens decreases. some of the causes of decay in green mustard a re xanthomonas campestris bacteria, and there is also fusarium oxysporum fungus which can be some of the rots in mustard greens (semangun, 2004). theref ore, natural preservatives are needed to inhibit the bacteria and fungi can cause decay in mustard greens . one of the natural preservatives that can be used to inhibit bacteria and fungi that cause decay in green mustard is hexadecanoic acid which is an isolate from the hydroid aglaophenia cupressina lamoureoux. by johannes, 2009. hexadecanoic acid is a deriva tive of carboxylic acid which has antibacterial and antifungal properties, so it is hoped that this study can extend the shelf life of mustard greens . materials and methods materials the materials used in this study included hexadecanoic acid compounds, xanthomonas campestris bacteria culture (inacc b1449), fusarium oxysporum fungus culture (inacc f641), potato dextrose agar (pda) medium, label paper, nutrient agar (na) medium, sterile distilled water, 70% alcohol, physiological nacl 0.9%, aluminum foil, cotton, cotton swab, ciprofloxacin, and ketoconazole. methods this study used the well diffusion method to test the activity of the hexadecanoic acid compound by looking at the inhibition zone f ormed a round the well. place 5 containers on nutrient agar medium and potato dextrose agar medium in each petri dish. the media containing the suspension of bacteria and fungi pour into each petri dish and are allowed to solidify. the reservoir is removed to form a well for the test solution (pangemanan, et al, 2016). hexadecanoate extract with 3 different concentrations (10%, 20%, 40%), positive control (ciprofloxacin) for antibacterial test, positive control (ketoconazole) for antifungal test, and negative control (aquades) was dripped as much as 50 l on each each different well was carried out in duplicate and then incubated in an incubator at 37 oc for 24 hours for bacteria and 72 hours for fungi. diameter of the inhibition zone formed was observed and measured using a caliper (pangemanan, et al, 2016). results and discussion hexadecanoic acid activity test against xanthomonas campestris bacteria the results of the observation of the inhibition zones formed at extract concentrations of 10%, 20%, and 40% on xanthomonas campestris bacteria after incubation 24 hours and 48 hours can be seen in figure 1 below. international journal of applied biology, 6(2), 2022 38 figure 1. the results of the inhibition test of 10%, 20% and 40% hexadecanoic acid extract and control against xanthomonas campestris after incubation for 24 and 48 hours. table 1. average diameter of inhibition zone of hexadecanoic acid extract with concentrations of 10%, 20%, 40%, and control against xanthomonas campestris bacteria after 24 hours and 48 hours incubation periods. concentrations average diameter of inhibition zone (mm) xanthomonas campestris 24 hours 48 hours 40% extract 48,22 47,87 20% extract 43,35 43,15 10% extract 32,37 32,04 + control 37,4 34,3 control 0 0 baseduonutable 1,uthe measurement resultsuofuthe hexadecanoic acid extract with concentrations of 10%, 20%, and 40% showed a clear zone around the well in the xanthomonas campestris bacterial culture. t he admi nistration of 10%, 20% and 40% hexadecanoic acid extract and positive control (ciprofloxacin) in each well showed a clear zone which was the inhibition zone of each treatment. the measurement results above show a decreaseuin the diameteruof the inhibition zone after observations were made at incubation ti mes of 24 hours and 48 hours. this indicated that the hexadecanoic acid extract was bacteriostatic against xanthomonas campestris bacteria. according to sinurat, et al, (2019), bacteriostatic means a substance can inhibit theugrowth ofubacteria which is characterized decrease in theuarea of the inhibition zone which is directly proportional to the increase in the incubation period. when the administration of antimicrobial compounds is stopped, microbial growth will increase again because the compounds given cannot kill but only inhibit microbial growth. it could be seen that the inhibitionuzone formed will have a larger diameter when given hexadecanoic acid extract with a higher concentration. according to cappucino (1978), the difference in the size of the large or small diameter of the inhibition formed is influenced by the growth rate of the microbe, the sensitivity of the microbe to the active substance, the ability of the active ingredient to diffuse in the medium, and the viscosity of the medium used. the cause of the inhibition by antimicrobial substances is due to interference with cell membranes from microbes, inhibition of enzyme work, disruption of protein and nucleic acid synthesis, or inhibition of cell wall synthesis (pelczar dan chan, 1998). there is a reaction between the hydroxyl group of lipopolysaccharide which is a constituent of the cell wall with hexadecanoic acid, causing changes in the structure of the lipopolysaccharide membrane from the cell wall to being asymmetrical. this causes the cell 24 hours 48 hours international journal of applied biology, 6(2), 2022 39 to become lysed or damaged due to disruption of the balance of the lipid membrane structure so that it will disrupt the integrity of the bacterial cell membrane (sjafaraenan, et al, 2021). based on the observations, it can beuseen that the 40%uconcentration has the largest inhibition area among the other concentrations used, this indicates that the hexadecanoic acid extract has antibacterial activity. of the three concentrations, 10%, 20%, and 40% were categorized as having very strong inhibito ry power because they showed an inhibitory power of more than 20 mm, which means they are classified as very strong (davis dan stout,1971 in palupi dan nugraha, 2014). ,in this study, ciprofloxacin was used as a positive control in the inhibition test of xanthomonas campestris bacteria. ciprofloxacin was used as a comparison of the effects of drugs, antimicrobials, standard with hexadecanoic acid extract test solution. ciprofloxacin is a fluoroquinolone antibiotic that has the ability to inhibit bacterial d na synthesis so that it becomes anti-microbial. ciprofloxacin is also an antibacterial that is can againstugrampositiveuandugramnegativeubacteria, theref ore ciprofloxacin is often used as a treatment for several infections caused by bacteria (castro, et al, 2013). ciprofloxacin was used as a positive control because ciprofloxacin has an antibacterial against xanthomonas campestris. the data obtained above shows a reduction in the area of the inhibition area which indicates that ciprofloxacin is bacteriostatic against xanthomonas campestris bacteria. similar results were reported by rojas, et al. (2019) which stated that ciprofloxacin was bacteriostatic against xanthomonas campestris which was characterized by a reduction in the area of the inhibition area. hexadecanoic acid activity test against fusarium oxysporum fungus figure 2. resultsuof the inhibition test of hexadecanoic aciduextract at concentrationsuof 10%, 20% and 40% and control of the fungus fusarium oxysporum after incubation for 48 and 72 hours. 48 hours 72 hours international journal of applied biology, 6(2), 2022 40 table 2. average diameter of inhibition zone of hexadecanoic acid extract with concentrations of 10%, 20%, 40%, and control against fusarium oxysporum fungus after 48 hours and 72 hours incubation periods. concentrations average diameter of inhibition zone (mm) fusarium oxysporum 48 hours 72 hours 40% extract 22,6 21,45 20% extract 19,25 18,4 10% extract 11,7 10,34 + control 35,4 34,65 control 0 0 baseduonutable 2, it can beuseen that there were clear zones formed in the 10%, 20%, 40% hexadecanoic acid extract and in the positive control (ketoconazole). the hexadecanoic acid extract could inhibit the fungus fusarium oxysporum which was seen from the inhibition zone that appeared around the well. , it can beuseen that the inhibition area formed atua concentra tion of 40% had the largest area of the inhibition zone compa red to other extract treatments. after observing the incubation time of 48 hours and 72 hours, there was a decrease in the area of inhibition because the hexadecanoic acid extract was fungistatic. according to jana, et al (2020) in sjafaraenan (2021), an antimicrobial substance is fungistatic if there is no increase in the a rea of the inhibition zone after incubation and the second observation is because the antimicrobial antimicrobial substances are not able to kill microbial growth. based this results above, the concentrati onuof 40% has the largest inhibition among other concentrations. the area of the inhibition zone at a concentration of 40% is included in the very strong group because it has an inhibitory zone area of more than 20 mm. meanwhile, at concentrati ons of 20% and 10%, it is included in the strong group because it has an area between 10-20 mm according to davis and stout (1971) in palupi and nugraha (2014). hexadecanoic acid has the capability to inhibit fungal accretionby forming complex compounds when it will bind to the active groups of fungal cell walls. fungal cells have chitin compounds in their cell walls. although there is a reaction between hexadecanoic acid and cell wall active groups, this reaction will only reac t with the outer ring structure (co2-oh) and cannot da mage the main structure of chitin in fungal cell walls. this causes the lack of this reaction affects the integrity of the fungal cell wall so that the hexadecanoic acid compound only inhibits or does not kill fungal cells. (johannes, 2013). in wells with positive control, ketoc onazole ha d an antifungal effect. the data obtained above showed a reduction in the inhibiti on area which indicated that ketoconazole was fungistatic against the fungus fusarium oxysporum. ketoconazole was used as a positive control because ketoc onazole is a synthetic broad-spectrum antifungal drug that bel ongs to the imidazole group. imidazoles and triazoles are from the azole group which are synthetic compounds. ketoconazole was the first oral azole to be used clinically from several other drugs (lely, et al, 2017). in addition to the negative control, in this study aquadest was used as negative control which was also used as a solvent for the hexadecanoic acid extract. the purpose of using distilled water as a negative control is to prove that the solvent used does not affect international journal of applied biology, 6(2), 2022 41 the antimicrobial test results of the compounds to be tested. table 1 and table 2 state that distilled water did not show antibacterial and antifungal activityiwhich was indicatedibyithe absence of aniinhibition area formed around the well. thus it can be believed that the use of distilled water as a solvent does not affect the antimicrobial test results of the extract . conclusions extracts of hexadecanoic acid compounds have an effect on inhibiting bacteria and fungi that cause decay in mustard brassica juncea l. based on inhibitory and bacteri ostatic tests on xanthomonas campestris and fungistatic on fusarium oxysporum. international journal of applied biology, 6(2), 2022 42 references cappucino, g. j., dan welsh, c., 2018, microbiology a laboratory manual eleventh edition, pearson education limited, england. castro, w., navarro, m., dan biot, c., 2013, medicinal potential of ciprofloxacin and its derivatives, journal future medicinal chemistry, vol. 5(1). johannes, e., 2013. pemanfaatan senyawa bioaktif hasil isolat hydroid aglaophenia cupressina lamoureoux sebagai bahan sanitizer pada buah dan sayuran segar, disertasi. universitas hasanuddin, makassar. lely, n., pratiwi, r. i., dan imanda, y. l., 2017, efektivitas antijamur kombinasi ketokonazol dengan minyak atsiri sereh wangi (cymbopogon nardus (l.) rendle), indonesian journal of information systems, vol. 7(2). pangalinan, f. r., kojong, n., dan yamlean, p. v. y., uji aktivitas antijamur ekstrak etanol kulit batang rambutan (nephelium lappaceum l.) terhadap jamur candida albicans secara in vitro, pharmacon. pangemanan a., fati mawali., dan budiarso f., 2016, uji daya hambat ekstrak rimpang kunyit (curcuma longa) terhadap pertumbuhan bakteri staphylococcus aureus dan pseudomonas sp., jurnal e-biomedik, vol. 4(1); 81-85. palupi, n. w., dan nugraha, a. s., 2014, pemanfaatan benalu kapas sebagai salah satu sumber bahan antimikroba alami : kajian aktifitas antimikroba, jurnal ilmiah inovasi, vol. 14 (1). pelczar m.j., chan ecs., 1988 . dasar-dasar mikrobiologi 2. diterjemahkan oleh hadioetomo rs, imas t, tjitrosomo ss, angka sl. jakarta: penerbit universitas indonesia. rojas, m., pena, m., vera, m. j. p., sulbaran, m., perez, e., velasquez, c. l., 2019, characterization and determi nation of antimicrobial and metal resistant profiles of xanthomonas strains isolated from natural environments, journal of analytical & pharmaceutical research, vol. 2. sarif, p., hadid, a., dan wahyudi, i., 2015, pertumbuhan dan hasil taanaman sawi (brassica juncea l.) akibat pemberian berbagai dosis pupuk urea, e-journal agrotekbis, vol. 3 (5). semangun, h., 2004, penyakit-penyakit tanaman hortikultura di indonesia, gadjah mada university press,yogyakarta. sinurat, a. a. p., renta, p. p., herliany, n. e., negara, b. f. s. p., purnama, d., 2019, uji aktivitas antibakteri ekstrak metanol rumput laut gracilaria edulis terhadap bakteri aeromonas hydrophila, jurnal enggano, vol. 4(1). sjafaraenan, johannes, e., dan tuwo, m., 2021, efektivitas senyawa asam heksadekanoat dan β-sitosterol isolat dari hydroid aglaophenia cupressina lamoureoux sebagai bahan antimikroba pada bakteri salmonella thypi dan jamur aspergillus flavus, bioma : jurnal biologi makassar, vol. 6(1). suratman, y. y. a., 2018, analisis pendapatan usahatani sawi (brassica juncea l.) di kelurahan landasan ulin utara kecamatan liang anggang kota banjarbaru, jurnal ziraa’ah, vol. 43 (2). contact : evy noviana novianaevy@gmail.com 54 abstract breast cancer is the highest incidence of cancer for among women worldwide. the use of anticancer compounds from natural ingredients as cancer therapeutics is to reduce chemotherapy side effects. indonesia has high biodiversity especially its pharmacologically beneficial such as nipah. nipah (nypa fruticans w.) is a type of palm-shaped mangrove plant which widely found in watershed areas . this study aims to explores the bioactivity of ethanol extract from n. fruticans leaves against mcf-7 breast cancer cells. screening for bioactivecompounds was performed by gas chromatography-mass spectrometry (gc-ms). anticancer pre-screening was assessed by brine shrimp lethality (bsl) test and anticancer activity was assessed by 3-(4,5-dimethylthiazol-2-cyl)-2,5diphenyltetrazolium bromide (mtt) assay. according of gc-ms analysis showed the presence of 54 phytochemical compounds. some of them have bioactivity as anticancer including sitosterol, tocopherol, and phytol. the bslt method showed potent activity with lc50 was 84.25 µg/ml. anticancer activity using mtt assays results the low ic50 value was 88.77 µg/ml that showed high anticancer activity by growth inhibition in mcf-7 breast cancer cells. this study delivers information that ethanol extract from n. fruticans leaves possesses bioactive compounds could be use as a candidate for anticancer compounds for cancer treatment. issn : 2580-2410 eissn : 2580-2119 bioactivity of nypa fruticans leaves as a candidate for anticancer compounds against mcf-7 breast cancer cells evy noviana 1*, eva johannes 1, sjafaraenan 1 1 department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar 90245, indonesia. introduction cancer is an abnormal cell growth and uncontrolled proliferation that will form a tumor mass (greaves m and maley cc, 2012). nearly 90% of cancer-related deaths are caused by metastatic processes that can invade other tissues and cause damage to vital functions (suhail et al., 2019). breast cancer is one of the most common cancers in women worldwide, accounting for approximately 627,000 atau sekitar 11,6 % deaths in 2018 (who, 2018). in indonesia, the highest incidence of cancer for women is breast cancer, which is 42.1 per 100,000 population with an average death rate of 17 per 100,000 population, followed by cervical cancer at 23.4 per 100,000 population (kemenkes ri, 2019). breast tumors usually start from the ductal hyperproliferation, and then develop into benign tumors or even metastatic carcinomas after constantly stimulation by various carcinogenic factors (sun, y. s., et al., 2017). open access international journal of applied biology keyword bioactive compounds; nypa fruticans; breast cancer; mcf-7 cells; gas chromatographymass spectrometry article history received december 8, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 55 cancer treatment have been carried out, namely through surgery, radiation, chemotherapy, and biological therapy. cancer treatment using chemotherapy can cause side effects in the form of damage to healthy cells. some side effects of chemotherapy drugs can induce cardiotoxicity (akazawa, 2017) and heart failure has been documented in women receiving doxorubicin (watkins, 2019). the risk of endometrial cancer, stroke, pulmonary embolism, and deep-vein thrombosis is increased in tamoxifen-treated patients (yang y, 2017). in addition, the use of chemotherapy can also cause problems with gene mutations and drug resistance (fisusi and akala, 2019). better medicines with less adverse effects and afavorable risk-benefit ratio need to be developed in the future. reducing risk factors and taking chemoprevention are two main measures to prevent breast cancer. one of the plants that can be used is nipah (nypa fruticans). research conducted by (edu et al., 2015; ebana et al., 2015; lestari et al., 2016; habibi, 2017; lovly and merlee, 2018; ubulom et al., 2019; gazali et al., 2019) found that nipah leaves extract contains polyphenolic compounds, flavonoids, triterpenoids/ steroids, saponins, alkaloids, tannins, and cardiac glycosides, with various bioactivities such as antioxidant, anticancer, antimicrobial, antifungal, antiviral, and others. in addition to anticancer potential, nipah leaves also have potential as antioxidants. nipah leaves contain high concentrations of phenolic compounds and an antioxidant value with an ic50 value of 0.32 mg/ml (aziz and jack, 2015). the results of putri et al. (2012) showed that the antioxidant activity of nipah leaf extract had an ic50 value of 17.72 ppm where the value was close to the standard value of antioxidant vitamin c. not much different, lovly and merlee (2017) reported that the methanol extract of nipah leaves showed high antioxidant activity with an ic50 value of 6.11 g/ml. supporting the results of previous studies, the results of the antioxidant activity test of the methanol extract of nipah leaves showed an ic50 value of 9.31 g/ml (gazali et al., 2019). anticancer activity can be related to its antioxidant activity. one of the causes of cancer is free radicals that attack the cells of the human body. antioxidants can prevent cells from oxidative damage, which causes cancer and cardiovascular disorders (muniyandi et al., 2019). materials and methods equipment and materials the tools used are ultrasonic homogenizer merek hielscher up200st, filter paper, rotary evaporatory, vacuum pump, chiller, analytical balance, funnel, erlenmeyer, beaker glass, micropipette, dropper pipette, vial bottle, 96-well plate, biosafety cabinet (bsc), centrifuge, co2 incubator, misroscope, multimode reader, uv-vis spectrophotometer, tissue cultureflask, microtube, tube, gc-ms (gas chromatography spectrofotometry massa) qp2010 simadzu. the ingredients used are nypa fruticans leaves powder, ethanol 96 %, artemia salina larvae, artificial sea water, cisplatin, ceftriaxon, dimethil sulfoxide (dmso), phosphate buffered saline (pbs), prestoblue, roswell park memorial institute medium (rpmi), fetal bovine serum (fbs), trypsin edta, tripsin blue. international journal of applied biology, 6(1), 2022 56 research prosedures nipah leaf extraction nipah leaf powder samples as much as 300 grams were extracted using 3 liters of 96% ethanol solvent by ultrasonic method at 100% amplitude and 26 khz frequency for 30 minutes. extraction was carried out for 30 minutes. then the filtrate was filtered and obtained about 800 ml. the filtrate was then concentrated with a rotary evaporator (re) and 70 ml of dark green thick extract was obtained. brine shrimp lethality test (bslt) method preparation of artemia salina larvae was carried out by incubating eggs placed in bottles filled with artificial seawater prepared by dissolving 35 g of sodium chloride in 1 l of distilled water, for 48 hours until hatching was incubated at room temperature (28-30 °c) under aeration conditions. strong and continuous light, larvae (nauplii) hatch within 48 hours. adding 10 artemia salina shrimp larvae to each extract that had been dissolved in dimethyl sulfoxide (dmso) and prepared at concentrations of 0.01, 0.1, 1, 10, 100, 1000 ppm in 3 replicates for control and each extract solution. after incubation for 24 hours, observations were made under light and counted the surviving larvae. mtt method prepared 8 pieces of 1.5 ml microtubes, then each microtube was labeled with the appropriate dilution concentration, then the stock sample was diluted into 8 concentration variants using a media solvent. take out 96 wellplates containing cells from the incubator. labeled on the plate along the left margin for which rows will be standardized and which rows will be sampled. then removed the media from each well. using a micropipette, transfer 100 l of each sample and positive control of cisplatin from the microtube into each of the corresponding wells, 96 well plates containing cells. incubate for 48 hours. discarded media on each well. prepare 9 ml of media in a tube, add 1 ml of prestoblue cell viability reagent then add 100 l of the solution mixture into each well of the microplate and then incubate for 12 hours until changes are seen color. gc-ms analysis the ethanol extract of nipah leaves was analyzed using gcms qp2010 simadzu. identification of phytochemical compounds using the willey database version 7.0 by comparing the mass spectrum pattern and the fragmentation pattern of reference compounds stored in the willey library. results and discussion the results of toxicity test using the brine shrimp lethality test (bslt) method was shown in table i. table i shows that the higher the sample concentration, the higher the mortality percentage of artemia salina larvae. the lowest concentration of nipah leaf extract, 1 g/ml, gave a mortality effect on artemia salina larvae of 13%, while the highest concentration of nipah leaf extract of 100 g/ml gave a mortality effect on artemia salina larvae of 50%. the results showed potent activity with lc50 was 84.25 µg/ml, included in the toxic category and has potential as an anticancer compound. international journal of applied biology, 6(1), 2022 57 table 1. the results of the toxicity test using the brine shrimp lethality test method. sample code [sample concentration] (μg/ml) axis x (log [sample]) % kematian larvae control axis y (probit value) nipah 1 0,00 13 3,87 leaves 10 1,00 33 4,56 extract 100 2,00 50 5,00 the brine shrimp lethality test (bslt) is a preliminary test to determine the toxicity of a compound or extract acutely using shrimp larvae experimental animals (artemia salina nauplii). parameter shown to indicate the presence of biological activity in a compound in artemia salina leach is the number of shrimp larvae mortality due to the influence of administration of the compound at a predetermined dose (kurniawan and ropiqa, 2021). the low value of lc50 of bslt method has been evidenced of the presence of anticancer compounds in plant extracts (geethaa s., et al., 2013). the test results of mcf-7 breast cancer cells using the mtt method was shown in figure i. ethanol extract from nipah leaves has the low value of ic50 about 88.77 µg/ml. it states that viability of mcf-7 cells will decrease in line with increase or decrease in extract concentration from a concentration of 88.77 µg/ml. figure 1. cytotoxicity graph of nipah leaves ethanol extract against mcf-7 cancer cells these results indicate that the higher the concentration of nipah leaves extract, the smaller the percentage of live cells. based on figure 1, showed that as the concentration of the sample increased, the number of living cells decreased. the ic50 value of 88.77 µg/ml was obtained and was included in the very toxic category, which means that it has the potential to inhibit the growth of mcf-7 breast cancer cells and also potential as candidate for anticancer compounds. this is supported by the presence of bioactive compounds which, based on previous research, can act as anticancer compounds. ethanol extract of nipah leaves possessed many bioactive compounds. gc-ms analysis international journal of applied biology, 6(1), 2022 58 revealed the presence of more than fifty four compounds, and some of them found in larger quantity than other compounds which showed in table 2. tabel 2. the majority of phytochemical compounds contained in the ethanol extract of nypa fruticans using the gc-ms method retention time compound name % peak area 11,554 phytol 8.54 18,699 tocopherol 7.01 20,604 sitosterol 12.11 in table 2, it can be seen that the phytochemical compounds that have been identified in the ethanol extract of nypa fruticans leaves by gc-ms method include sitosterol, tocopherol, and phytol. from the results of the chromatogram (figure 2), there is one compound whose composition is larger (indicated by % total the largest, 12.11%) was sitosterol. figure 2. gc-ms chromatogram of nypa fruticans ethanol extract sitosterol is a phytosterol widely distributed throughout the plant and its function to be involved in the stabilization of cell membranes. sitosterol can treating different types of cancer via different pathways (bin sayeed, m.s. and ameen, s.s., 2015). sitosterol function was could inhibits the growth of cultured cancer cell lines that are associated with stimulation of apoptotic cell death (awad, a., et al., 2007), inhibition of cancer cell proliferation at low concentrations with no cytotoxic effect on noncancerous cells (jayaprakasha, g., 2007), arresting of cells at g2/m phase in cancer cells and decreasing free radical generation in vitro (shahdaat m., et al., 2016). dietary sitosterol supplementation could decreased mammary hyperplastic lesions and total tumor burden in female mice fed with a high-fat diet but not in those with a low-fat diet (salehi b., et al., 2020). α-tocopherols (vitamin e) have the role, as a potent antioxidant is to uphold the legitimacy of long-chain fatty acids (unsaturated) in superficial surface (cell membrane) and thus sustaining a normal genetic commotion (batool1 m., et al., 2020). they scavenge lipid peroxyl radicals by donating hydrogen from the phenolic group on the chromanol ring. international journal of applied biology, 6(1), 2022 59 alphatocopherol mainly inhibits the production of new free radicals. oxidation has been linked to numerous possible conditions/diseases including: cancer, ageing, arthritis and cataracts. thus, tocopherols (vitamin e) might help prevent or delay the chronic diseases associated with reactive oxygen species molecules (rizvi s., et al., 2013; jiang, 2014). phytol is terpenoid compounds found in leaf extract. phytol produced the removal of oh∙, exhibiting antioxidant activity which may be capable of inhibiting cell damage caused by this radical (carolina, et al. 2013). many papers indicate also that phytol (1) exhibits cytotoxic potential against certain cancer cell lines: leukemia (mv4-11 and hela), breast (mcf-7), prostate (pc-3) and lungs (a-549)37–39 and is the substance promising for the treatment of cancer (gliszczyńska, et al., 2021). phytol induces apoptosis and reactive oxygen speciesmediated protective autophagy in human gastric adenocarcinoma cells (song and cho, 2015). conclusions based on the results of the study, it can be concluded that nipah leaves extract has potential as an anticancer compounds which is supported by the results of the bslt method and the mtt method with an ic50 value which is 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and fisheries owned by indonesia becomes a massive opportunity in producing various types of commodities provided by natural resources such as the fishing fish, aquaculture, fishery products, and ecotourism. based on this, it is necessary to have synergy between the government, the private sector and the community in managing marine fisheries resources that exist today. of course, with the existing potential, it will become a challenge in its management that can guarantee the sustainability of natural resources and the improvement of the people's economy. this paper examines the opportunities and challenges of the fishing industry in indonesia using the input and output data of the directorate general of maritime and fisheries resources supervision, which are analyzed descriptively using tables and graphs and journal reviews relating to the study. issn : 2580-2410 eissn : 2580-2119 prospects of fisheries industry development in indonesia through online publication media mahmud 1, akriani dewi bau sinrang 2, andi nur apung massiseng 3 1 study program of public relations, faculty of social sciences, economics and humanities, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar sulawesi selatan, indonesia 2 study program of management, faculty of social sciences, economics and humanities, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar sulawesi selatan, indonesia 3 study program of fisheries agribusiness, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar sulawesi selatan, indonesia introduction indonesia has considerable fisheries potential that includes inland fisheries and sea fisheries. one of the advantages of the marine fisheries sector is that indonesia is a maritime country with the most significant sea area and the highest number of islands, around 17 x 103, which are spread across the archipelago (nurkholis et al., 2016). based on this, indonesia has enormous potential from the fisheries sector, namely the cultivation and capture sector, which is a mainstay in supporting the development of the fishing industry in indonesia and is one of the strategic economic activities that deserve to be developed. fisheries resources in indonesia are very abundant because they have a very high level of biodiversity (biodiversity). open access international journal of applied biology keyword fisheries industry, the potential of fisheries, aquaculture, fishing fish, ecotourism industry. article history received 11 november 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 118 indonesia has several main types of commodities that are the mainstay of the fisheries industry in fishing fish, such as shrimp, tuna, tuna and skipjack, squid, cuttlefish, octopus and crab-crab, while in the aquaculture sector has fisheries commodities, namely: seaweed, shrimp, milkfish, snapper, carp, tilapia, carp and catfish. these commodities are the mainstay of exports in indonesia at this time. the second position of the world is occupied by indonesia, being the largest producer of marine fisheries globally; it's just tha t the production of marine fisheries has not significantly contributed to the economy. if seen from the contribution of the marine fisheries sector only gives 2 per cent of the contribution to the national gdp. by looking at this case, it becomes a sign that the management and coordination between marine and fisheries planning and development are still very weak, different if we apply appropriate management and coordination, then indonesia is able to produce the maximum potential of marine wealth that we have even equivalent to developed countries. the tendency of the world community today to consume more fish is cultivated than can consume fish caught. by seeing this phenomenon, fishing fish can be managed sustainably by maximizing the potential of very large aquaculture (ditjen pds-kkp, 2017). innovation is the key to success in increasing competitiveness (barney, 2002; foss et al., 2011, hult, hurley and knight, 2004; molina-castillo and munuera-aleman 2009; shapiro, 2002; rogers, 2003). industrial development in indonesia still has many limitations in designing and creating sustainable innovation (dhanani, 2000). based on indonesian marine and fisheries investment data for 2017, it is known that the value of the production of catches and aquaculture is around rp. 1.23 trillions, while the processing industry rp. 2.29 trillions. the highest value comes from the fisheries processing industry due to high export market demand so that the development of the fisheries processing industry continues to increase. materials potential of aquaculture industry the fisheries sector plays an important role in the indonesian economy through increased income, diversification of livelihoods, supply of animal protein, and foreign exchange earnings. this sector contributes 3.1% of total national gross domestic product (gdp) and 21.0% of total agricultural gdp, creates around 6.4 million direct jobs for the people of indonesia, and obtains the us $ 4.2 billion dollars from food exports sea in 2012 and provided 54.8% of domestic animal protein supply (mmaf, 2013). fish consumption per capita in indonesia has also increased, with annual consumption per capita increasing from 21.0 kg in 2003 to 33.9 kg in 2012 (marine and fisheries statistics book, 2012). fishing fish are currently the dominant source of fish in the domestic market, and the primary source of nutrition is essential for many consumers who are below the poverty line. the decrease in fish consumption because of rising prices will endanger the welfare of the people of indonesia. the study results highlight the importance of policies targeting sustainable fisheries landings and sustainable aquaculture growth. under the "global maritime fulcrum", the indonesian government has taken drastic steps to limit foreign fishing to increase domestic catches, including sinking foreign fishing vessels in its waters and capturing fishermen (connelly, 2015). however, given the limited resources and concerns about the sustainability of fish stocks in indonesia and the consequences that ma y arise from climate change that are projected to have severe impacts on indonesian fishing fish, the government needs to prioritize managing fish in nature (cheung et al. 2010). international journal of applied biology, 5(2), 2021 119 the potential of marine culture, consisting of the possibility of fish farming (snapper, grouper, cobia); shrimp, molluscs (shellfish, pearls, sea cucumbers); and seaweed, with a potential area of cultivation in indonesia of 2 million ha (20% of the total potential of coastal and marine waters within 5 km of the coastline) with a volume of 46.73 million tons per year, and the potential for brackish cultivation (ponds) reaches 913,000 ha. for the possibility of marine biotechnology, there are still great opportunities to be developed, such as the raw material industry for food, the natural feed ingredients industry, and fish and shrimp seeds (lasabuda, 2013). a development zone for aquaculture was created by the government where intensification of production could be supported through investment in private hatcheries, distribution and marketing channels for fisheries, training, information system improvement, and support for product certification and access to capital (nurdiana, 2006). nevertheless, aquaculture in indonesia needs to grow substantially in the future. during the projection period, an increase in aquaculture output will require expansion to occur only in soils that do not result in the loss of important ecosystems, intensification of sustainable agricultural practices and an increase in the efficiency of user inputs for production that is ecologically feasible (bostock et al., 2010). as producer prices increase over time, research investment will also be needed to help farmers reduce production costs and remain competitive. this investment must promote a major transformation of the aquaculture system, policies, and investments that meet the blue economy's principles that align development goals, natural resources, and environmental conservation (adger et al., 2005). based on table 1, it is known that the largest export value comes from the cultivation sector, especially the shrimp commodity. the value is 35.84% with a volume of 197,433,608 kg. this shows that the shrimp aquaculture industry in indonesia is still the prima donna and still has opportunities for continued development because it is supported by research related to disease control which is a major obstacle in the aquaculture industry. table 1. value of main exports of indonesian fisheries commodities no commodity value (usd) % value volume (kg) % volume 1 shrimp 1,742,119,193 35.84% 197,433,608 17.53% 2 tunatongkol cakalang 713,919,147 14.69% 168,433,759 14.96% 3 cumi-stotongoctopus 554,594,192 11.41% 152,108,581 13.51% 4 crab 472,962,123 9.73% 27,791,618 2.47% 5 seaweed 291,837,226 6.00% 212,961,523 18.91% 6 other commodities 1,085,479,049 22.33% 367,349,488 32.62% 7 grand total 4,860,910,930 100.00% 1,126,078,577 100.00% data source: ditjen pds-kkp, 2017 international journal of applied biology, 5(2), 2021 120 figure 1. value and volume export of aquaculture raw materials for 2015-2017 (source: ditjen pds-kkp, 2017) based on figure 1, the largest aquaculture commodity in indonesia in terms of export volume is a seaweed commodity, but the highest export value is shrimp commodity. this indicates that the export value of seaweed is low because what is exported is a raw material or has not been processed, so that even though the export volume is high, the matter remains low compared to shrimp. potential of fishing fish industry indonesia has enormous fisheries potential. the maximum sustainable value (msy) of fishing fish resources is around 12.54 million tons per year. at the same time, the potential that can be exploited (allowable catch) of 80% of msy is about 10,032 million tons per year. based on catch data from 2012 to 2016 (table 2), the value of catches continues to increase each year but is still very low compared to the potential value of fishing fish that 0 100 200 300 400 2015 2016 2017 124 131 138 212 188 192 48 51 52 export volume of aquaculture raw materials (thousand tons) other aquaculture commodity seaweed shrimp international journal of applied biology, 5(2), 2021 121 can be utilized. this shows that the potential for developing the fishing fish industry is still huge. table 2. national fishing fish production value from 2012 to 2017 year production value msy value allowable catch (thousand tons) (million tons/ year) (million tons/ year) 2012 5.829 12.54 10.032 2013 6.105 2014 6.484 2015 6.678 2016 6.58 2017 6.04 data source : ditjen pds-kkp, 2017 although the value of msy is still far above the value of the production of catches, specific fish species are already in the control stage of fishing due to the decreasing amount in nature, for example is grouper. south sulawesi province decreased export activity caused by a decrease in marine fishery yields, which impacted the decline in grouper export results, thereby reducing regional income. along with this, coral reefs experienced a reduction in the area of coral reefs, which affected the catch of coral fish, which decreased by 63 per cent (sutinah, rahmatia, seniwati & kitta, 2017). in addition to production data, the magnitude of opportunities for the development of the fishing industry can be seen from the value of national fish needs. based on the calculation of the national fish demand value (figure 2), it is known that federal fish consumption needs continue to increase every year. this indicates that people are increasingly aware of the importance of fish as a healthier consumption material. this trend is believed to continue to grow so that the development of the fishing fish industry is needed to meet the needs of the national fish. figure 2. data on fish consumption and national fish demand for 2012-2017 (source: ditjen pds-kkp, 2017) 33,89 35,21 38,14 41,11 43,94 46,49 6,92 7,36 7,95 8,58 9,37 10,38 0 10 20 30 40 50 2012 2013 2014 2015 2016 2017 fish consumption (kg/capita) fish demand (million tons) international journal of applied biology, 5(2), 2021 122 figure 3. value export of fishing fish in 2015-2017 (source : ditjen pds-kkp, 2017) the export value of fishing fish commodity tuna-tongkol-cakalang (ttc) ranks second after combining other fishing fish commodities. the fishing fish of the tuna-tongkolcakalang (ttc) commodity is still below the msy limit, so fishing fish can still be made, which must be based on sustainability. potential of fishery product processing industry indonesia is one of the world's largest maritime and island nations, producing hundreds of abundant fisheries commodities. not surprisingly, seaweed and tuna products rank first globally; similarly, crab and shrimp products occupy the second position. according to 2015 food and agriculture organization (fao) data, china ranks first as a shrimp producer globally. various superior fishery products are scattered throughout the archipelago. shrimp production centres are in sumatra and west java. crabs are spread in java, sumatra and kalimantan. the leading producers of seaweed are in the sulawesi and nusa tenggara regions. meanwhile, tuna-producing areas focus on papua, maluku, and north sulawesi. the total indonesian fishery products in 2016 reached more than the us $ 2.9 billion. during the five years from 2011 to 2016, the export value of indonesian fishery products grew 3.5% per year. the five largest importing countries of indonesian fish products in 2016 were the us, japan, china, vietnam and malaysia. among the five, exports to china and vietnam experienced rapid development, while exports to japan declined. 39.4% of indonesia's total exports to the world in 2016 were directed to the us, up from 31.5% five years earlier. the chinese market share rose from 5.3% to 9.4%, vietnam from 4.5% to 6.7%, and malaysia rose from 2.8% to 3.7%. japanese market share fell from 27.0% in 2011 to 16.3% in 2016 (ningsih. 2018). processed fishery products in the excellent category for export are processed shrimp, squid, fish meat, crab. those with the opportunity but not yet maximally fulfilled are: frozen fish, frozen lobster, albacore or frozen long tuna fins, live eel, processed sea cucumbers, and frozen mackerel. processed fish products are determined using three fourdigit hs categories: extracts and juices of meat, fish or crustaceans (hs 1603); processed or preserved fish (hs 1604); and processed or preserved crustaceans and molluscs (hs 1605). figure 3 shows that in these three categories, the export value of hs 1605 reached twice that of hs 1604, while the export value of hs 1603 was not significant (ningsih. 2018). 0 1000 2000 3000 2015 2016 2017 584 566 660 213 337 397 1.594 1.651 1.621 export value of fishing fish (usd million) other fishing fish commudity cumi-sotonggurita ttc international journal of applied biology, 5(2), 2021 123 table 3. world exports of processed fish products by country 2011-2016 no importer total value (us $ million) market share (%) 2011 2012 2013 2014 2015 2016 2011 2016 1 as 302.2 307.3 406.1 534.2 457.6 471.7 40.6 49.0 2 jepang 129.3 147.7 132.3 144 122.8 122.3 17.4 12.7 3 arab saudi 33.2 53.5 59.4 55.3 61.2 57.2 4.5 5.9 4 britania raya 25.9 43.6 58.3 60.6 49.8 48.8 3.5 5.1 5 belanda 36.1 29 49.8 60.1 19.3 27.6 4.8 2.9 6 italia 3.2 3.5 18.0 25.9 29.9 25.5 0.4 2.6 7 thailand 18.7 34.5 34.1 14.9 16.9 24.7 2.5 2.6 8 australia 8.5 15.3 22.2 27.5 29.7 21.4 1.1 2.2 9 jerman 39.3 27.5 36.2 35.5 18.0 15.0 5.3 1.6 10 vietnam 1.9 1.5 2.1 3.2 2.1 11.5 0.3 1.2 11 kanada 9.5 9.7 10.2 7.5 7.8 9 1.3 0.9 other countries / regions in the world 137.1 169.8 160.8 167.0 129.5 127.5 18.4 13.3 world total 744.9 843 989.5 1135.7 944.7 962.3 100.0 100.0 source : international trade centre. 2018 the total export value of indonesian processed fish products in 2016 reached the us $ 962 million. (see table 3). the central destination countries for this product are the us, japan, saudi arabia, the united kingdom and the netherlands. in 2016, total exports to these five countries accounted for 76% of indonesia's exports to the entire world. this represents a small portion (i.e. 0.9%) of indonesia's total exports of processed fish products and is lower than the share recorded in 2011 (ningsih. 2018). potential of ecotourism industry marine and fisheries-based ecotourism in indonesia has been developed into one industry that needs special attention from all stakeholders, considering that tourism in this sector can lead to domestic and foreign tourists. ecotourism, being a solution to care for the natural and cultural environment, can also minimize adverse effects on the environment, besides visitors willingly pay to get a comfortable and quality vacation. in the current conditions, many tourists are not satisfied with the existing ecotourism because of its homogeneous nature. increased human awareness of the importance of preserving nature and culture is a significant factor that also determines the growth of ecotourism, so there must be equality between social and cultural based on the tourist destination created (situmorang, 2007). in developing an ecotourism area, it must be based on local communities empowered through the provision of education, social entrepreneurship and how to preserve the existing culture that can have an impact on improving welfare and improving education. the science of social entrepreneurship can educate local people to be people who are responsible for improving people's lives by how to behave in protecting the environment (situmorang & mirzanti, 2012) international journal of applied biology, 5(2), 2021 124 ecotourism is a place to work with stakeholders in the social world (wood, 2008). gonzalo (n.d.) states that ecotourism is a social endeavour that leads to community organizations. in planning and managing an ecotourism area, local people and natural resources significantly contribute. this shows that the implementation of ecotourism must benefit local communities (asadi and kohan, 2011). people who are empowered through tourism activities become an effective solution in tourism development, where developed tourism can support the preservation of culture and the environment so that sustainable tourism development such as ecotourism is realized. the purpose of organizing communitybased tourism is to see the community build themselves based on ecotourism activities carried out in their area. tourism development will not succeed without local leaders and communities (sutawa, 2012). as one example of a case study in shiraz, iran, this place has good prospects for tourism development; with the participation of the community, the tourism industry in shiraz can increase (aref and ma'rof, 2008). many people are aware of how important tourism is in making social, cultural and environmental treatment and economic change; for this reason, tourism is very dependent on the local community. (beeton, 2006). tourism can be one of the factors of economic development; in this case, leaders who deal directly in the field of action must take an essential role in the organization of environment-based tourism and the community to improve their welfare (godfrey 1996). indonesia, which is known for its wide variety of cultural, ethnic, and marine tourism potentials, has the potential to make this an opportunity that requires good management. based on the current literature review, it is emphasized that the best-applied ecotourism management pattern is community-based ecotourism for ecosystem sustainability and improving the welfare of local communities. methods this study uses input and output data from the directorate general of maritime and fisheries resources supervision, which are analyzed descriptively using tables and graphs and related journal reviews. results and discussion based on the information that has been presented, the government should adopt a development policy based on the perspective of ecological, economic and social development dimensions, or integration of the three dimensions (integrated management) in all sectors, as an example of the case of the results of the study of tran et al. (2017), where researchers developed several approaches to the projection model of indonesian fisheries trends. in this study, the focus is on growth trends in the marine and inland fisheries sector as well as the significant impacts on the social, economic and environmental sectors related to this trend from increased activities in the public and private sectors, which are identified as policies that are guided by the application of the blue economy concept. constraints faced in the development of the fishing industry include the use of ecofriendly fishing gear, which is still very limited, and fishers still prioritize the number of catches compared to the selectivity of pitfalls. in addition, there are not many regulations governing fishing time, so spawning fish are also caught. stocks of fish in the natural world are declining, and this is because the policy regarding determining the right time to see to international journal of applied biology, 5(2), 2021 125 protect the mother fish when spawning does not yet exist. solutions that can be offered related to the development of the fishing industry in the future are; first, the indonesian government needs investment both in the form of research and product development in overcoming the problem of diseases which are the main obstacle to the aquaculture industry, in addition, the government also needs to promote restocking and recovery activities in nature through the concept of sea ranching. increasing human resources (hr) is also very much required by fishermen and fish farmers related to knowledge and skills in their fields in supporting the sustainability of their businesses (tran, 2017). conclusions the current approaches that need to be taken by the government related to the development of the fishing industry are increasing science and technology and human resources, developing the production centre area, improving the quality and food safety for exports, developing sustainable and community-based ecotourism areas, increasing supervision of marine natural resources and fisheries, additional investment in the aquaculture sector, sustainable management of fishing fish, diversifying fishery products and reducing the export of fishery products in the form of raw materials by making partnership patterns to revive domestic industries. this approach is critical to do based on the problems of the existing fishing industry; 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(28.5%), staphylococcus aureus (25%), shigella sp. (21.4%), escherichia coli (17.9%), salmonella sp. (3.6%) and klebsiella sp. (3.6%). four (4) fungal isolates were obtained from the dried periwinkle samples and two belonged to the genus aspergillus, while the other two isolates belong to the genera penicillium and mucor. the percentage (%) occurrence of the fungal isolates were: mucor (41.6%), aspergillus flavus (25%), aspergillus niger (16.7%) and penicillium (16.7%). there was no significant difference in total viable count between one market and another at 5% level of significance (p>0.05). also, there was no significant difference in total viable count between one seller and other sellers. the occurrence of bacillus, shigella, staphylococcus, klebsiella, escherichia coli, salmonella, aspergillus, and mucor species are pinpointing the high pathogenicity and health hazard in consuming the dried periwinkle. due to the soaring demand of this sea food (periwinkle) and the health hazard associated with microorganisms isolated from them as revealed in this study, additional concentration should be paid to safety through proper storage and handling processes and it is important that periwinkle should be properly cooked before consumption. issn : 2580-2410 eissn : 2580-2119 microbiological assessment of roasted dried periwinkle (tympanotonus fuscatus) sold in yenagoa bayelsa state opara christiana ngozi1, obi theodora2*, akpoka augustine obhioze2 1 federal university otuoke, faculty of sciences, department of microbiology, p. m. b.126, yenagoa bayelsa state, nigeria 2igbinedion university, college of natural and applied, department of chemical sciences, p.m.b 0006, okada, benin, edo state, nigeria open access international journal of applied biology keyword microbial assessment, periwinkle, tympanotonus fuscatus article history received 16 july 2020 accepted 31 december 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(2), 2020 38 introduction sea foods are vital source of food in the niger delta. main seafood’s consumed in the region include periwinkle which is an important sources of protein. fish constitutes over 40 % of the animal protein consumed by an average nigerian compared to meat and it is relatively less expensive (adebayo-tayo et al., 2008). this accounts for the mass preference for fish products. they are marine mollusks that are represented in mangrove swamps, lagoons and estuaries by two genera tympanotonus and pachymelania (buchaan et al.,1954). tympanotonus fuscatus is a shellfish dominantly found in brackish waters of the riverine areas of nigeria, where they are highly prolific. this feature had made them a cheap source of protein in many homes when compared to other conventional protein sources (bassey et al., 2007). various methods have been developed to preserve fish. these include refrigeration and drying (ayers et al., 1980). the techniques employed depend on the technological advancement of the people (adebayo-tayo et al., 2008). however, studies on the microbiological quality of shell fishes have shown that they harbor many pathogenic microorganisms, the most prevalent being bacteria and fungi. as a result of pollution of water bodies, pathogenic organisms may be introduced to these aquatic ecosystems from which this sea food is harvested. sources of pollution vary and could include faecal contamination usually from untreated human waste. as a result, water bodies may contain high numbers of coliform and these organisms would also be present in sea foods harvested from such water system (jay et al., 2000). periwinkles are known to contain lot of microorganisms which are usually either due to untreated human wastes which are deposited into the water which the periwinkle inhabits. such microbes as, vibrio sp, bacillus sp, escherichia coli, micrococcus sp, which may be indigenous flora of the water body and are responsible for diseases associated with seafood when their microbial load is high such as cholera, camphylobacterlosis, gastroenteritis, salmonellosis, shigellosis, typhoid fever, brucellosis, amoebiasis and poliomyelitis (ekanem & otti, 1997). in bayelsa state of nigeria the dried periwinkle (tympanotonus fuscatus) are often exposed to sources of contamination like soil, dust and sand due to the fact that the dried periwinkle is not properly covered and handled during sale. the need to inform the public on the health hazard associated with the consumption of dried periwinkle sold in the market which could result in ingestion of pathogenic microorganism’s lead to this study. the aim of this study is to isolate and identify the possible microbial organisms present in dried periwinkle (tympanotonus fuscatus) from the three sources and to evaluate their safety on consumption. materials and methods sampling area the samples were bought randomly from three different markets in yenagoa, bayelsa state, nigeria. sample area 1: opolo market (sample oa, ob and oc), sample area 2: kpansia market (sample ka, kb and kc), and sample area 3: swali market (sample sa, sb and sc). sample collection the sample used for this analysis is dried periwinkle. it is a marine sea food mostly found and eaten in the niger delta region. the samples were bought randomly from three international journal of applied biology, 4(2), 2020 39 different markets (three samples from three different vendors in each market) in yenagoa and were wrapped in sterile aluminum foil, labeled and taken to the laboratory for analysis. preparation of sample microbiological analyses was carried out in triplicates on 1g of samples which were soaked and homogenized with 9 ml sterile normal saline for 3 minutes using a kenwood blender as describe in the bacteriological analytical manual (fda, 1984). tenfold serial dilution was prepared in 9ml normal saline and 1ml of 10-2 was pour plated in nutrient agar and salmonella shigella agar. enumeration of bacterial and fungal colonies aliquot (0.1 ml) of the sample was transferred into sterile plates in duplicates agar was poured on the samples and incubated in inverted position at 37 oc for 24 hours. the isolates from nutrient agar and salmonella shigella agar were sub-cultured in a nutrient agar using streak plate method. the inoculated media were labeled and incubated for 24 hours at 37 oc. for coliform count, 1 ml of the 10-2 dilution for each sample was pour plated in macconkey agar. the plates were labeled and incubated at 37 oc for 24 hours and the colonies were counted. for the enumeration of fungal colonies, 1ml of 10-2 was pour plated in potato dextrose agar and plates were incubated at 28 °c for 5 days and the fungal colonies counted. identification of bacterial and fungal isolates identification of the isolates was based on their cultural morphology, microscopic examination and biochemical tests. references were made to bergey’s manual of determinative bacteriology (1992) for identification of bacteria. morphological studies were carried out on different media plates used for the isolation of the organisms; pure colonies were isolated based on colony size, shape, pigmentation, elevation and texture of the individual organisms after 24 hours of growth at 37 oc. identification of the isolated bacteria was based on cultural characteristics, cell morphology and biochemical tests which include catalase, citrate, oxidase, gram stain, lndole tests (holt et al., 1994). characterization of fungi identification of fungi isolate was based on the morphological and microscopic characterization such as type of mycelium, pigmentation type of sporulating structures and sexual reproduction (if present). they are examined using hand lens to determine those morphological characteristics. fungal isolates were stained with lacto phenol cotton blue and examined microscopically. the isolates were identified based on cultural characteristics, morphology of hyphae, cells and spores and kind of fruiting bodies (barnet and barry, 1972). gram staining and microscopic examination a portion of each discrete colony was thinly smeared in a drop of peptone water on a clean grease-free glass slide with the aid of a sterile wire loop and. the slides were allowed to air dry. the fixed smear was flooded with crystal violet stain for 1 minute and washed off with water. lugol’s iodine was used in flooding it and allowed to stay for 1 minute and rinsed with water. the smear was decolourized with alcohol and rinsed immediately with water. the smear was then counterstained with safranin for 1 minute, and then it was washed off with water and allowed to air dry at room temperature. the stained smear was examined under the microscope using x100 objective with immersion oil. gram international journal of applied biology, 4(2), 2020 40 positive organisms retained the primary stain (blue stain, crystal violet) while the gram negative organisms ones picked up the red or pink stain of the safranin. statistical analysis the data was subjected to test of difference of means using anova with the aid of spss statistical software, to determine the f statistic and probability at 5% significant level (spss, 2010, version 19) results and discussion bacterial and fungal count on the samples from different markets: total heterotrophic bacteria and total fungi using standard plate count of the various samples from yenagoa were shown in table 1. table 1: bacterial and fungal counts for dried periwinkle sold in markets in yenagoa samples market total viable count (10-5cfu/g) total coliform (10-5cfu/g) total salmonella shigella count (10-5cfu/g) total fungi count (10-5cfu/g) oa opolo 22 25 1 1 ob opolo 37 11 11 4 oc opolo 12 15 5 6 sa swali 30 13 3 4 sb swali 29 12 2 5 sc swali 49 30 13 3 ka kpansia 38 17 12 4 kb kpansia 18 28 3 7 kc kpansia 20 10 8 2 key: oa= opolo a, ob = opolo b, oc = opolo c, sa = swali a, sb = swali b, sc = swali c , ka = kpansia a, kb = kpansia b, kc = kpansia c. table 1 shows the levels of microbial load in dried periwinkle samples from three different markets in yanegoa, bayelsa state. total bacterial populations of the samples from opolo market range from 37×10-5 12×10-5 cfu/g, swali range from 49×10-5 29×10-5 cfu/g, while kpansia market range from 38×10-5 18×10-5 cfu/g. for fungi population, opolo market range from 6×10-5 1×10-5 cfu/g, swali market range from 5×10-5 3×10-5 cfu/g, while kpansia market range from 7×10-5 2×10-5cfu/g. the samonella/shigella counts from swali market range from 13×10-5 12×10-5 cfu/g, kpansia market range from 12×10-5 3×10-5 cfu/g, while opolo market range from 11×10-5 1×10-5 cfu/g. the coliform counts from swali market range from 30×10-5 12×10-5 cfu/g, kpansia market range from 28×10-5 10×10-5 cfu/g, while opolo market range from 25×10-5 11×10-5 cfu/g. for fungi population, opolo market range from 6×10-5 1×10-5 cfu/g, swali market range from 9×10-5 3×10-5 cfu/g, while kpansia market range from 7×10-5 2×10-5 cfu/g, this is in accordiance with the fungal count in the work of nrior, iyibo and ngerebara, (2016). bacteriological guideline have the limit for raw molluscan shellfish contamination of not more than 5x105 bacteria/g and less than 230 escherichia coli /100g for sea food harvested from known unpolluted waters, using 5 sample units (seafood network information center, 2008). the microbial load varied from location (market) to another and also from samples of the same location (market), this variation may be due to the processing and handling by different people and the microbial variation in atmosphere of the different localities. international journal of applied biology, 4(2), 2020 41 the morphology, gram reaction and biochemical reactions of the bacterial isolates are shown in table 2. table 2: morphology, gram reaction and biochemical reaction of bacterial isolates cell morphology gram reaction catalase citrate indole oxidase name of isolate rod + + + bacillus species rod + + klebsiella pneumonia rod + + escherichia coli rod + salmonella species rod + + shigella species cocci + + + staphylococcus aureus table 2 shows that all the isolates were rods with the exemption of one. all of the isolates were catalase positive and oxidase negative (table 2). the bacteria belonged to six genera identified as: klebsiella, salmonella, escherichia coli, shigella, staphylococcus and bacillus (table 2). the majority of these bacteria have also been reported by preceding researchers (rhodes and kator, 1988). the microbiological quality of the river, estuaries and seashores from which shellfish are harvested influence the microflora of shellfish samples (adams and moss, 2005). the primary microbial load on ready-to eat foods is important; but factors such as processing, storage and display for sale may influence the microbiological load of readyto-eat foods at the point of sale (beuchat and ryu, 2004). although drying reduces water activity and destroys bacteria in the course of heating, post processing contamination can occur especially during handling and transportation of processed periwinkle to point of sale(obire, nwosu and wemedo, 2017). staphylococcus species was isolated from roasted samples of tympanotonus fuscatus. studies have suggested that the presence of staphylococcus species on ready-toeat food may be as a result of improper handling, cross contamination and poor temperature control (christiansen and king, 1991). market shellfish vendors use their bare hands during measuring and constantly dip their fingers into basins containing fresh and dry seafood, even different types of shellfish during saling which lead to cross contamination of the roasted periwinkle. food handlers with hand infection or with cold or with sore throat may transfer enterotoxigenic strains of staphylococcus to food. when given optimum conditions, it grows, generate toxins and cause staphylococcal intoxication. growth to levels above 106cfu/g is required for toxin formation and since staphylococcus aureus is a mesophilic organism, some degree of temperature abuse precedes intoxication (fao/who, 2003). isolation of bacillus species and shigella species indicated that the seafood was contaminated from wherever they were harvested from. the display of the roasted dried periwinkle meat without any form of packaging could also be attributed to contamination. being frequently displayed and uncovered, the shellfish meat will become prone to contamination from bacterial origin. some strains of bacillus (e.g. bacillus cereus) and staphylococcus aureus are known enterotoxin producers (bryant, 2007). the inherent danger in the association of bacillus cereus and staphylococcus aureus with or without their metabolic products in various foods, without further heat treatment is the possible outbreak of serious food-borne illness. keeping processed seafood for retail free of contamination with staphylococcus species is best ensured by observing proper food international journal of applied biology, 4(2), 2020 42 handling practices involving minimal contact with human skin. the infectious dose of shigella is low, approximately 10 to 100 cells (fao/who, 2003), therefore its presence in food must be avoided. isolation of salmonella species from the shellfish samples can be attributed to possible chronic carriers, from feaces to other persons by the oral-feacal route, which may be water-borne, food borne or by contact with hands and other formites. the findings of the study show that microbial counts were generally lower for samples from opolo market compared to kpansia and swali markets. the morphological characteristics of fungal isolates are shown in table 3. table 3: morpholological characteristics of fungi isolated from samples growth medium colony morphology microscopic appearance identity pda yellowish–green mycelium brush-like conidia, septate branching, conidiophore was, smooth. penicillum species pda effuse black colony simple septate and branched conidia in chain. aspergillus niger pda greenish mycelium conidiophore with vesicles, unbranched conidiophores in chain. aspergillus flavus pda whitish grey mycelium sporangiophore branched with spored sporangium. rhizoids not present. mucor species four (4) fungal isolates were obtained from the dried periwinkle samples, aspergillus flavus, aspergillus niger, penicillium species and mucor species (table 3). they belonged to three genera penicillium, aspergillus and mucor. isolation of aspergillus species indicated that the dried periwinkle was contaminated by air, water or soil because the spores are commonly found in air, water or soil. isolation of penicillium and mucor species indicated that the dried periwinkle was contaminated from air to which the dried periwinkle was exposed to. table 4: mean and standard deviation of total microbial counts sample location total viable count (10-5cfu/g) total colifom count (10-5cfu/g) total salmonella shigella count (10-5cfu/g) total fungi count (10-5cfu/g) opolo market 23.6667 ± 12.58306 17.0000 ± 7.21110 5.6667 ± 5.03322 3.6667± 2.51661 swali market 37.0000 ±10.58301 20.3333 ± 9.07377 8.0000 ± 5.56776 5.6667 ±3.05505 kpansia market 25.3333 ± 11.01514 18.3333 ± 9.07377 7.6667 ± 4.50925 4.3333 ±2.51661 table 4 shows that there were no significant difference in total viable count between one market and another at 5% level of significance (p>0.05). also, there was no significant difference in total viable count between one seller and other sellers. percentage (%) and frequency of occurrence of microbial isolates: the percentage (%) frequencies of the different bacterial and fungal isolates are shown in table 5 6. international journal of applied biology, 4(2), 2020 43 table 5: frequency and percentage (%) occurrence of bacterial isolates from dried periwinkle isolate frequency of occurrence percentage (%) of occurrence bacillus species 8 28.5 staphylococcus aureus 7 25 shigella 6 21.4 echerishia coli 5 17.9 klebsiella pneumonia 1 3.6 salmonella species 1 3.6 total 28 100 the percentage (%) occurence of the bacterial isolates were: bacillus species (28.5%) > staphylococcus aureus (25%), shigella species (21.4%), escherichia coli (17.9%), salmonella species (3.6%) and klebsiella pnuemoniae (3.6%) (table 5 ). table 6: frequency and percentage (%) of occurrence of fungi in roasted dried periwinkle isolate frequency of occurrence percentage (%) of occurrence mucor species 5 41.6 aspergillus niger 2 16.7 aspergillus flavus 3 25 penicillum species 2 16.7 total 12 100 the percentage (%) occurence of the fungal isolates were: mucor (41.6%), aspergillus flavus (25%), aspergillus niger (16.7%), penicillium (16.7%) (table 6). the occurrence of bacillus, shigella, staphylococcus, klebsiella, escherichia coli, salmonella, aspergillus, and mucor species are pinpointing the high pathogenicity and health hazard in consuming the dried periwinkle. due to the soaring demand of this sea food (periwinkle) and the health hazard associated with microorganisms isolated from them as revealed in this study, additional concentration should be paid to safety through proper storage and handling processes and it is important that periwinkle should be properly cooked before consumption. conclusions the result identified fungi and bacteria as the microorganisms associated with dried periwinkle. some of these microorganisms are pathogenic and are able to cause chronic illnesses in human if ingested. contamination is common due to processing, storage and handling which is the major source of cross contamination. this study indicates that this dried periwinkle is not safe for consumption. international journal of applied biology, 4(2), 2020 44 references abolagba, o.j., adekunle, a., dede, a., omoigui, g.o. 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(1999). an outbreak of escherichia coli infection following exposure to contaminated food. jana: 290 2178. contact : harem othman smail harem.othman@koyauniversity.org 100 abstract the experiment was conducted at agronomy field laboratory of patuakhali science and technology university, dumki, patuakhali, bangladesh from july 2018 to november 2018 to find out the effect of sunflower residues and herbicides on weed control and yield of t. aman rice. weed control methods tested were t1= weedy check,t2 = weed free check, t3 = pendimethalin, t4 = pretilachlor, t5 = butachlor, t6 = pyrazosulfuron ethyl, t7 = bensulfuron methyl + acetachlor, t8 = bispyriback sodium, t9 =2,4-d amine, t10 = mcpa, t11 = sunflower residues, t12 = sunflower residues + 100% pyrazosulfuron ethyl, t13 = sunflower residues + 75% pyrazosulfuron ethyl, t14 = sunflower residues + 50% pyrazosulfuron ethyl. the weed spectrum of the experimental field consisted of all the three groups of weeds viz., grasses (15.78%), sedges (59.02%) and broad-leaved weeds (25.2%). the dominants weeds were cynodon dactylon and echinochloa crusgalli among grasses; cyperus difformis, fimbristylis miliacea and scirpus supinus among sedges and jussiaea decurrens among broad-leaved weeds. hand weeding recorded the highest weed control efficiency (99.05%) and weedy check recorded the lowest. among different herbicides applied alone, butachlor had the highest weed control efficiency (87.59%).hand weeding produced the highest grain yield (5.14 t ha-1) which was statistically similar to butachlor, pendimethalin, pretilachlor, bensulfuron methyl + acetachlor and sunflower residues + 100% pyrazosulfuronethyl. application of sunflower residues along with the reduced rate (75 or 50%) of pyrazosulfuron ethyl had effective weed control and satisfactory yield comparable to butachlor. the farmers can use this technology as an eco-friendly approach in transplanted aman rice field. issn : 2580-2410 eissn : 2580-2119 efficacy of sunflower residues and herbicides in controlling weeds in transplanted rice shaon samanta tanu, purnendu biswas, sultan ahmed, & swadesh chandra samanta department of agronomy, patuakhali science and technology university, dumki, patuakhali, bangladesh-8602 introduction weeds are the greatest yield-limiting pest to rice reduction and their control is labor intensive. hand weeding and other traditional control methods are time consuming and involve high labour cost. the rice cropsare severely infested with weeds and can reduce the grain yield by 30-40% for transplanted aman rice in bangladesh (bhuiyan, 2016). weeds not only cause huge reductions in rice yields but also increase the cost of cultivation, reduce input efficiency, interfere with agricultural operations, impair quality, act as alternate hosts for open access international journal of applied biology keyword sunflower residues, herbicides, weed suppression, yield, transplanted aman rice article history received 03 june 2020 accepted 14 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 101 several insect pests, diseases, affect the aesthetic look of the ecosystem as well as native biodiversity, affect human and cattle health (bhuiyan et al., 2017). weeds always compete with crop for resources like light, nutrient, space and moisture causingyield loss. thus, weed is a serious threat for sustaining rice production in bangladesh and necessitates proper weed management for rice production. herbicidal weed control methods offer an advantage to save labour and money and hence regarded as cost effective. chemical weed control is becoming popular in bangladesh mainly due to scarcity of labour during peak growing season and lower cost. due to high labour and huge time requirement for manual weeding, farmers of coastal area, particularly in kalapara upazila are using other alternative measures like chemical weed control. phyrazosulfuron ethyl (a new pre-emergence herbicide) is being used successfully for weed control in t.aus and t. aman rice in this area. however, the indiscriminate use of herbicides poses serious problems, such as environmental pollution, human health hazards, developing weed resistance, depletion of crop diversity and reduction of crop productivity (khanh et al, 2005). singh et al. (2017) reported that continuous and extensive use of herbicides raised concerns for the long-term sustainability of soil fertility.allelopathy is suggested as a potential safer method for weed control compared to the use of synthetic herbicides. many researchers demonstrated that use of sunflower residues alone or in combination with lower rates of herbicides into field soil significantly reduced weed population and dry weight over control (alsaadawi et al., 2011; saif et al., 2016; rawat et al., 2017). however, information available on crop residues and herbicides for weed control in transplanted aman rice is meager under bangladesh conditions. therefore, this study was undertaken to investigate the influence of sunflower residues and herbicides on weed suppression and yield of transplanted aman rice. materials and methods the experiment was carried out at the agronomy field laboratory of patuakhali science and technology university (pstu), dumki, patuakhali, bangladesh in high land during july to november, 2018. the experimental field belongs to the agro-ecological zone13(ganges tidal floodplain). the experimental soil was clay loam in texture having ph 6.0, organic matter 1.38%, total n 0.09%, available p 6.30 ppm, exchangeable k 0.11 me/100g, available s 15.25 ppm and available zn 0.46 ppm. there were fourteen weed control treatments which were as follows: t1= weedy check (unweeded control) t2 = weed free check by hand weeding twice (at 20 and 40 dat) t3 = pendimethalin (pe) t4 = pretilachlor (pe) t5= butachlor (pe) t6 = pyrazosulfuron ethyl (pe) t7 = bensulfuron methyl + acetachlor (pe) t8 = bispyriback sodium ((pe) t9 = 2, 4-d amine (poe) t10 =mcpa (poe) t11 = sunflower residues (pe) t12 = sunflower residues (pe) + 100% pyrazosulfuron ethyl (pe) t13= sunflower residues (pe) + 75% pyrazosulfuron ethyl (pe) international journal of applied biology, 4(1), 2020 102 t14 = sunflower residues (pe) + 50% pyrazosulfuron ethyl (pe) * pe= pre-emergence, poe= post-emergence pyrazosulfuron ethyl was studied in combination with sunflower residues because many of the farmers of this region use this herbicide for weed control in rice. the experiment was laid out in a randomized complete block design with three replications. the layout was completed one day before transplanting. the individual plot size was 4 m × 2.5 m (10 m2). fertilizer was applied @ 90-15-51-9-1 kg n, p, k, s and zn ha-1as urea, triple super phosphate, muriate of potash, gypsum and zinc sulphate, respectively on soil test basis. urea was applied in three equal splits; 1st at 15 days after transplanting (dat), 2nd at 30 dat and 3rd at 45 dat. total amount of triple super phosphate, muriate of potash, gypsum and zinc sulphate was applied during final land preparation. thirty-two day old seedlings of t. aman rice variety brri dhan49 were transplanted on 9 august 2018 using 2-3 seedlings hill-1 at a spacing of 20 × 20 cm. the details of the herbicides are given in table 1. table 1. details of the herbicides used in the study trade name common name chemical family rate of application time of application panida 33ec pendimethalin dinitroaniline 2.50 l ha-1 5 dat super heat 500 ec pretilachlor chloroacetamide 1.0 l ha-1 7 dat amchlor 5g butachlor chloroacetamide 25 kg ha-1 5 dat super power 10wp pyrazosulfuron ethyl sulfonylurea 150 g ha-1 7 dat super mix 18 wp bensulfuran methyl+ acetachlor chloroacetamide + sulfonylurea 0.74 kg ha-1 7 dat extra power 20wp bispyribac sodium pyrimidinylthiobenzoates 148 g ha-1 7 dat fielder 2, 4-d amine phenoxycarboxylic acid 2.25 l ha-1 28 dat mcpa 500 mcpa phenoxycarboxylic acid 988 ml ha-1 28 dat liquid or powdery herbicides were applied uniformly with the spray volume of 500 l ha-1 for pre-emergence spray and 350 l ha-1 for post-emergence spray using a knapsack sprayer. the pre-emergence application was made uniformly on the soil surface with 5 cm water depth. granular herbicide was broadcast uniformly on the soil surface by mixing with dry sand.the weed free treatments were kept weed free up to 40 dat by hand weeding at 20 and 40 days after transplanting. weedy plots were allowed season long weed infestation.at physiological maturity, heads of sunflower plants were removed and the plants were harvested, sun-dried for several days and chopped into 2-3 cm pieces. chopped material was kept under room condition until application. the chopped residues were incorporated into soil 3 days after transplanting of t. aman rice @ 5 t ha-1.data on weed density and dry weight wererecorded at 45 dat randomly from 0.25 m2 (0.5 x 0.5 m) area and then converted to per square metre. dry weight of weeds was taken by drying in an electric oven at 700c until the international journal of applied biology, 4(1), 2020 103 constant weight was recorded.relative weed density (rwd) in control plots was calculated as follows: rwd (%)= density of individual weed species in the community total density of all weed species in the community ×100 weed control efficiency was calculated by using the following formula given by mani et al. (1973). wce (%)= dry weight of weeds in unweeded control − dry weight of weed in treatment plot dry weight of weeds in unweeded control ×100 an area of 5.12 m2 (3.2 m x 1.6 m) was harvested from the central portion of each plot to record grain and straw yields. the fresh weight of grain was recorded just after threshing and cleaning and adjusted to 14 % moisture content.weed index (wi) was calculated by the following formula as suggested by gill and vijay(1969). weed index (%) = x−y x ×100 where, x =total yield from hand weeded, weed free or minimum weed competition plot y = total yield from treated plots since weed population and dry weight have shown high degree of variation, the data were subjected to square-root [(x + 0.5)0.5] transformation to make analysis of variance more valid as suggested by chandel (1984). data were analyzed with star 2.0.1 software for analysis of variance. the mean differencesamong the treatment means were adjudgedby least significant difference (lsd) (gomez and gomez, 1984). correlation was done by the same software. results and discussion weed composition and relative proportions of different weed types in control plots weed flora in transplanted aman rice field was very much diverse. fourteen weed species infested the control plots which belong to 8 families (poaceae, cyperaceae, pontederiaceae, alismataceae, compositae, sphenocleaceae, lythraceae and onagraceae (table 2). among the infesting weed species, three were grasses, five were sedges and the rest six were broad-leaved. the weed species belonging to grass family were cynodon dactylon, echinochloa colonum and paspalum distichum, sedges were fimbristylis miliacea, scirpus mucronatus, scirpus supinus, scirpus acutus and cyperus difformis, broad-leaved weeds were monochoria vaginalis, sagittaria guyanensis, eclipta prostrata, sphenoclea zeylanica and ammannia baccifera and jussiaea decurrens (table 2). similar weed species in transplanted rice during kharif season were also reported by prakash et al. (2013).the relative proportion of different weed species into total weed number is presented in figure 1. the most prominent weed in the control plots was cyperus difformis with 27.82% relative weed density (rwd). the second most important was scirpus supinus with 14.29 % rwd. among the grasses, echinochloa colonum was the highest inrespect of rwd (7.14%) followed by cynodon dactylon (5.26%) and paspalum distichum (3.38%).among the sedges, cyperus difformis had the maximum rwd (27.82%) and scirpus mucronatus had the lowest (1.50%). among the broad-leaved weeds, jussiaea decurrens was the highest with 9.40% rwd and international journal of applied biology, 4(1), 2020 104 table 2. weed composition in control plot of the experimental field of transplanted aman rice local name common name scientific name density (no. m-2) type family durba bermuda grass cynodon dactylon 18.67 grass poaceae khudey shama jungle rice echinochloa colonum 25.33 grass poaceae gitla joint grass paspalum distichum 12.00 grass poaceae joina hoorah grass fimbristylis miliacea 41.33 sedge cyperaceae chechra bog bulrush scirpus mucronatus 5.33 sedge cyperaceae khudey chechra dwarf club rush scirpus supinus 50.67 sedge cyperaceae sakta chechra hard stem bulrush scirpus acutus 13.33 sedge cyperaceae green nakfuli small flower umbrella cyperus difformis 98.67 sedge cyperaceae chota panikachu oval leaf pondweed monochoria vaginalis 8.00 broadleaf pontederiaceae chand mala arrow head sagittaria guyanensis 2.67 broadleaf alismataceae keshuti white eclipta eclipta prostrata 22.67 broadleaf compositae zhil marich goose weed sphenoclea zeylanica 8.00 broadleaf sphenocleacea e datmari tooth cup ammannia baccifera 14.67 broadleaf lythraceae panilong winged water primrose jussiaea decurrens 33.33 broadleaf onagraceae total 354.67 international journal of applied biology, 4(1), 2020 105 figure 1. relative density of different weed species (rwd) in unweeded plot of transplanted aman rice (var. brri dhan49) sagittaria guyanensis was the lowest with 0.75% rwd. based on the relative weed density, the most dominant weed species could be arranged in the order of cyperus difformis > scirpus supinus > fimbristylis miliacea > jussiaea decurrens > echinochloa colonum > eclipta prostrata > cynodon dactylon.the degree of occurrence of different weeds include 15.78% grasses, 59.02% sedges and 2 5.20% broad leaf in weedy check (figure 2). therefore, sedges were the dominant weed species in respect of weed density in weedy check. puniya et al. (2009) also reported sedges as predominant weed flora in transplanted rice. figure 2. relative density of different weed (rwd) types in unweeded plot of transplanted aman rice (var. brri dhan49) 0 5 10 15 20 25 30 5,26 7,14 3,38 11,65 1,5 14,29 3,76 27,82 2,26 0,75 6,39 2,36 4,13 9,31r w d ( % ) different weed species rwd(%) 0 10 20 30 40 50 60 grass sedge broad-leaf 15,78 59,02 25,2 r w d ( % ) weed type rwd(%) international journal of applied biology, 4(1), 2020 106 effect of weed control methods on weed growth species-wise results are presented for the first 5 weeds only as they were dominant in the samples. the data pertaining to the density of individual weed (first five) are presented in table 3. weed density echinochloa colonum (khudey shama) echinochloa colonum was significantly averted by the weed control treatments (table 3). the non-treated weedy check had the highest weed density (25.33 plants m-2). hand weeding and sunflower residues + 100% pyrazosulfuron ethyl showed the best performance in controlling this weed. 100% of this weed plants was controlled by these two treatments. pendimethalin, butachlor and bensulfuron methyl + acetachlor provided satisfactory weed control, next to these treatments. the results are in agreement with raj et al.(2016) who demonstrated that hand weeding was the best in reducing echinochloa colonum while preemergence application of pendimethalin, butachlor and bispyriback sodium significantly reduced the mean density of echinochloa colonum over weedy check . incorporation of sunflower residue into field significantly reduced the weed population by 84.21% over control. however, combination of sunflower residue and pyrazosulfuron ethyl at 75 and 50% of recommended dose significantly reduced weed density by 89.46 and 85.51% over control and both the treatments were statistically similar to recommended dose of pyrazosulfuron ethyl. rawat et al. (2017) observed that sunflower-oat rotation over a 5-year period significantly lowered the density of grassy weeds in fields than in control plots which might be due to allelopathic effect of sunflower. fimbristylis miliacea (joina) the density of this weed was significantly influenced by the treatments (table 3). weedy check treatment had the highest density (41.33 plants m-2) of this weed. pretilachlor, butachlor, bensulfuron methyl + acetachlor, bispyriback sodium, 2,4-d amine, mcpa and sunflower residues + 100% pyrazosulfuron ethyl provided excellent control and reduced fimbristylis miliaceaby 100%. saha and rao (2010) stated that sulfonyl urea herbicide like bensulfuron methyl, pyrazosulfuron ethyl effectively controlled sedges. singh et al. (2016) reported that cyperus spp. could be effectively controlled by butachlor. integration of sunflower residue with 50% pyrazosulfuron ethyl reduced weed density by 93.54% over weedy check and this was statistically similar to recommended dose of pyrazosulfuron ethyl. sunflower residue + 75% pyrazosulfuron ethyl resulted in 96.78% weed control and it was better even than the recommended dose of pyrazosulfuron ethyl. scirpus supinus (khudey chechra) different weed control treatments significantly influenced the density of this weed (table 3). the maximum weed density (50.67 plants m-2) was found in the non-treated weedy plot. no weed of this species was observed in pendimethalin, pretilachlor, butachlor and bensulfuron methyl + acetachlor treated plots which indicate excellent control of scirpus supinus. singh et al. (2016) stated that cyperus spp. was efficiently controlled by butachlor. recommended dose of pyrazosulfuron ethyl, sunflower residue alone, sunflower residue + 100% pyrazosulfuronethyl, sunflower residue + 75% pyrazosulfuron ethyl and sunflower residue + 50% pyrazosulfuron ethyl were statistically similar with respect to weed control. however, incorporation of sunflower residue alone reduced 64.48% of this weed species over international journal of applied biology, 4(1), 2020 107 weedy check. sunflower residue coupled with 100%, 75% and 50% pyrazosulfuron ethyl resulted in 76.32%, 76.32% and 70.40% reduction of weed density over weedy check. cyperus difformis (green nakfuli) all the weed control treatments significantly reduced the density of cyperus difformis compared with the weedy check (table 3). the highest weed density of this weed was observed in weedy check (98.67 plants m-2). butachlor and bensulfuron methyl + acetachlor controlled all plants of this weed which was similar to weed free treatment. singh et al. (2016) also observed that cyperus spp. was efficiently controlled by butachlor. reddy et al. (2000) reported that ethoxysulfuron provided a 100% control of cyperusdifformis in transplanted rice. however, sunflower residue incorporation alone resulted in 74.32 % control of this weed species which was similar to recommended dose of pyrazosulfuron ethyl, sunflower residue + 75% pyrazosulfuron ethyl and sunflower residue + 50% pyrazosulfuron ethyl. sunflower residue along with 100%, 75% and 50% pyrazosulfuron ethyl resulted in 82.43%, 81.08% and 77.02% reduction of weed density over weedy check. jussiaea decurrens (panilong) density of jussiaea decurrens was significantly affected by different weed control treatments (table 3). the highest weed density (33.33 plants m-2) was recorded in weedy check treatment. 100% weed of this species was controlled by pendimethalin, pretilachlor, butachlor, bensulfuron methyl + acetachlor, 2, 4-d amine, mcpa and sunflower residues + 100% pyrazosulfuron ethyl and these were statistically similar to weed free treatment. akbar et al. (2011) reported that butachlor, pendimethalin and pretilachlor effectively controlled broad-leaved weed like eclipta alba. bari (2010) stated that herbicides of phenoxy carboxylic acid group like2,4-d amine and mcpa are broad-leaf killer and they pick broad-leaf weeds. application of sunflower residue into field significantly reduced the weed population by 96% over control. however, combination of sunflower residue and pyrazosulfuron ethyl at 75 and 50% of recommended dose significantly reduced weed density by 84 and 79.99% over control and these were statistically similar to recommended dose of pyrazosulfuron ethyl. rawat et al. (2017) also observed that sunflower-oat rotation over a 5-year period significantly lowered the density of broad-leaved weeds in fields than in control plots which might be due to allelopathic effect of sunflower. others other weeds like cynodon dactylon, paspalum distichum, scirpus mucronatus, monochoria vaginalis etc. were observed in small number in the experiment. total densities of these weeds were significantly influenced by the weed control treatments (table 3). hand weeding and sunflower residues + 100% pyrazosulfuron ethyl, being similar in controlling other weeds significantly reduced the mean density of these weeds as compared to other weed control treatments. these treatments reduced the other weed density to the tune of 2.67-4.00 plants m-2 as compared to 105.34 plants m-2in weedy check. among the herbicides used alone, butachlor, pyrazosulfuron ethyl and bensulfuron methyl + acetachlor had the lowest number of other weeds. however, combination of sunflower residue and 75% pyrazosulfuron ethyl provided other weed population suppression statistically similar to that achieved by the label rate of pyrazosulfuron ethyl. akbar et al. (2011) reported that butachlor, pendimethalin and pretilachlor effectively controlled all types of weeds. international journal of applied biology, 4(1), 2020 108 table 3. effect of weed control methods on weed density (m-2) at 45 days after transplanting of transplanted aman rice treatments echinochloa colonum fimbristylis miliacea scirpus supinus cyperus difformis jussiaea decurrens others total density t1 5.07 a (25.33) 6.46 a (41.33) 7.14 a (50.67) 9.93 a (98.67) 5.80 a (33.33) 10.26 a (105.34) 18.81 a (354.67) t2 0.71 f (0) 1.34 c (1.33) 1.76 f (2.67) 0.71 f (0) 0.71 d (0) 1.77 i (2.67) 2.67 i (6.67) t3 1.29 e (1.33) 1.76 b (2.67) 0.71 g (0) 4.21 c (17.33) 0.71 d (0) 4.37 e-g (18.67) 6.34 fg (40.00) t4 2.90 c (8.00) 0.71 d (0) 0.71 g (0) 2.67 de (6.67) 0.71 d (0) 4.51 ef (20.00) 5.92 g (34.67) t5 1.29 e (1.33) 0.71 d (0) 0.71 g (0) 0.71 f (0) 0.71 d (0) 3.75 h (13.67) 3.90 h (15.00) t6 2.11 d (4.00) 1.76 b (2.67) 3.53 e (12.00) 4.51 bc (20.00) 2.67 b (6.67) 3.93 f-h (15.00) 7.80 de (60.34) t7 1.29 e (1.33) 0.71 d (0) 0.71 g (0) 0.71 f (0) 0.71 d (0) 3.79 gh (14.00) 3.95 h (15.33) t8 2.11 d (4.00) 0.71 d (0) 1.74 f (2.67) 3.13 d (9.33) 2.42 b (5.33) 5.57 c (30.67) 7.24 ef (52.00) t9 4.18 b (17.00) 0.71 d (0) 6.22 b (38.67) 2.90 d (8.00) 0.71 d (0) 4.88 de (23.33) 9.35 bc (87.00) t10 4.49 b (19.67) 0.71 d (0) 4.67 c (21.33) 2.10 e (4.00) 0.71 d (0) 5.10 cd (25.67) 8.40 cd (70.67) t11 2.11 d (4.00) 1.77 b (2.67) 4.30 cd (18.00) 5.06 b (25.33) 1.29 c (1.33) 6.62 b (43.33) 9.75 b (94.66) t12 0.71 f (0) 0.71 d (0) 3.52 e (12.00) 4.18 c (17.33) 0.71 d (0) 2.12 i (4.00) 5.80 g (33.33) t13 1.76 de (2.67) 1.29 c (1.33) 3.52 e (12.00) 4.37 bc (18.67) 2.41 b (5.33) 3.76 h (13.67) 7.33 ef (53.67) t14 2.04 d (3.67) 1.76 b (2.67) 3.92 de (15.00) 4.80 bc (22.67) 2.68 b (6.67) 4.79 de (22. 67) 8.58 cd (73.35) ls ** ** ** ** ** ** ** lsd (0.05) 0.5520 0.3709 0.6518 0.7050 0.3593 0.5927 1.0174 cv (%) 14.36 14.65 12.60 11.76 13.06 7.56 8.02 t1=weedy check, t2 = weed free check by hand weeding twice, t3 = pendimethalin, t4 = pretilachlor, t5= butachlor,t6 = pyrazosulfuron ethyl, t7 = bensulfuron methyl + acetachlor, t8 = bispyriback sodium, t9 =2, 4-d amine, t10 = mcpa, t11 = sunflower residues, t12 = sunflower residues + 100% pyrazosulfuron ethyl, t13 = sunflower residues + 75% pyrazosulfuron ethyl, t14 = sunflower residues + 50% pyrazosulfuron ethyl, ls = level of significance. figures in parentheses are original values. the data were subjected to square root [(x+0.5)0.5] transformation before statistical analysis. figures in a column followed by different letters differ significantly, but with common letter (s) do not differ significantly at 5% level of probability contact : harem othman smail harem.othman@koyauniversity.org 109 total density all the weed control methods reduced the density of total weeds over weedy check (table 3). weedy check registered the highest total mean weed density (354.67 plants m-2) while weed free treatment by hand weeding registered the lowest (6.67 plants m-2). akbar et al. (2011) stated that hand weeding recorded higher weed suppression than chemical weed control. butachlor and bensulfuron methyl + acetachlor were statistically similar in controlling total weed density and significantly reduced the total weed density over weedy check and other herbicides tested alone or in combination with sunflower residues. hasanuzzaman et al. (2008) demonstrated that application of butachlor rendered efficient weed control.both the herbicides reduced the total mean weed density by about 96% over weedy check. the post-emergence herbicide viz. 2, 4-d amine and mcpa were the worst in this regard. 2, 4-d amine and mcpa being broad leaf killer, they only picked broad leaved weeds,while the grasses escaped its control. hence their overall effect was lesser as compared to other herbicides. however, reduced doses of pyrazosulfuron ethyl (75%) in combination with sunflower residue had greater weed suppression than that realized with recommended dose of pyrazosulfuron ethyl used alone. sole application of sunflower residue resulted in 73.31% control of total weed population. sunflower residue along with 100%, 75% and 50% pyrazosulfuron ethyl resulted in 90.60%, 84.87% and 79.32% reduction of total weed density, respectively over weedy check. however, application of 50% of the recommended dose of pyrazosulfuron ethyl coupled with sunflower residue rendered statistically similar suppression of total weed density to that of recommended dose (100%) of the same herbicide used alone. total weed dry weight the data on weed dry matter recorded from different weed control treatments at 45 dat are presented in table 4. all weed control treatments significantly reduced the total weed dry weight compared with non-treated plots. the minimum weed dry weight of weeds was obtained from manual weeding (0.49 g m-2) and the maximum weed dry weight was obtained from weedy check (51.81 g m-2). among the sole herbicide treatments, butachlor recorded the lowest weed dry weight (6.43 g m-2) and 2, 4-d amine the highest (10.98 g m-2). none of the treatments were comparable to manual weeding in reducing total weed dry weight. however, sole application of sunflower residue and sole application of pyrazosulfuron ethyl recorded statistically similar total weed dry weight indicating the allelopathic effect of sunflower. sunflower residues + 50% pyrazosulfuron ethyl, sunflower residues + 75% pyrazosulfuron ethyl, butachlor and bensulfuron methyl + acetachlor were statistically similar and better than all other herbicides. raj et al. (2016) observed that total weed dry biomass at harvesting of rice was significantly reduced by pre-emergence application of pendimethalin, butachlor and bispyriback sodium over weedy check.application of butachlor gave the effective weed control as reported by hasanuzzaman et al. (2008). weed control efficiency (wce) manually weeded plots maintained the highest weed control efficiency (99.05%) followed by sunflower residues + 100% pyrazosulfuron ethyl (93.46%), while the least was maintained by the post-emergence herbicide 2, 4-d amine (table 4).moorthy and saha (2002) also recorded higher weed control efficiency (93.10 %) in hand weeding treatments.herbicide-treated plots rendered higher weed control efficiency than weedy plots.among the sole herbicide treatments, the pre-emergence herbicide butachlor gave the international journal of applied biology, 4(1), 2020 110 best weed control efficiency (87.59%), only next to manual weeding. bari (2010) stated that butachlor provided better weed control efficiency. however, sunflower residues + 75% pyrazosulfuron ethyl and sunflower residues + 50% pyrazosulfuron ethyl applications were better than sole application of pyrazosulfuron ethyl at recommended rate in respect of weed control efficiency. weed control efficiency in sole sunflower residue application was 78.11% which was close to sole application of pyrazosulfuron ethyl at recommended rate. it indicates that a reduced level of pyrazosulfuron ethyl may be feasible for satisfactory weed control. nikneshan et al. (2011) alsodemonstrated that extract of sunflower at 100% concentration suppressed over 80% of narrowand broad-leaved weeds in wheat indicating allelopathic properties of sunflower. table 4. effect of weed control methods on weed dry weight, weed control efficiency, grain yield and weed index of transplanted aman rice treatment weed dry weight (gm-2) weed control efficiency (%) grain yield (t ha-1) % increase yield over control weed index (%) t1 7.22 a (51.81) 0.00 3.38 d 34.24 t2 0.97 h (0.49) 99.05 5.14 a 52.07 0.00 t3 3.05 b-d (8.83) 82.96 4.71 ab 39.35 8.37 t4 2.98 c-e (8.37) 83.84 4.72 ab 39.64 8.17 t5 2.63 ef (6.43) 87.59 4.81 ab 42.31 6.42 t6 3.22 bc (9.91) 79.29 4.41 bc 30.47 14.20 t7 2.68 d-f (6.78) 83.77 4.73 ab 39.94 7.98 t8 3.20 bc (9.80) 81.08 4.51 bc 33.43 12.26 t9 3.39 b (10.98) 77.67 4.01 c 18.64 21.98 t10 3.26 bc (10.15) 80.41 4.11 c 21.60 20.04 t11 3.44 b (11.34) 78.11 4.40 bc 30.18 14.40 t12 1.97 g (3.39) 93.46 4.97 ab 47.04 3.31 t13 2.58 f (6.14) 88.14 4.46 bc 31.95 13.23 t14 2.94 c-f (8.16) 84.25 4.45 bc 31.66 13.42 ls ** ** lsd (0.05) 0.3952 0.58 cv (%) 7.57 7.74 t1=weedy check, t2 = weed free check by hand weeding twice, t3 = pendimethalin, t4 = pretilachlor, t5= butachlor,t6 = pyrazosulfuron ethyl, t7 = bensulfuron methyl + acetachlor, t8 = bispyriback sodium, t9 =2, 4-d amine), t10 = mcpa, t11 = sunflower residues, t12 = sunflower residues + 100% pyrazosulfuron ethyl, t13 = sunflower residues + 75% pyrazosulfuron ethyl, t14 = sunflower residues + 50% pyrazosulfuron ethyl, ls = level of significance.figures in a column followed by different letters differ significantly, but with common letter (s) do not differ significantly at 5% level of probability. effect of weed control methods on grain yield and weed index grain yield weed control treatments had significant effect on grain yield of t. aman rice (table 4). the weed free plots by hand weeding registered the highest grain yield (5.14 t ha-1) and was comparable to pendimethalin, pretilachlor, butachlor, bensulfuron methyl + acetachlor and sunflower residues + 100% pyrazosulfuron ethyl. singh et al. (2008) observed that international journal of applied biology, 4(1), 2020 111 different herbicide treatments gave similar yield to that of weed free plots. the weedy check treatment gave significantly the lowest grain yield (3.38 t ha-1). the minimum yield in unweeded control was due to severe weed competition by the uncontrolled weed growth. among different herbicides, butachlor produced the maximum yield (4.81 t ha-1).bhanu rekha et al. (2004) observed that pre-emergence application of butachlor recorded the highest grain yield (4.73 t ha-1) among different herbicides. 2, 4-d amine and mcpa recorded the lowest grain yields (4.01 and 4.11 t ha-1, respectively).the pre-emergence herbicides (sole) treated plotsincreased the grain yield ranging from 30.47% to 42.31% with an average value of 37.52% over weedy check, while the respective increase in yields under 2, 4-d amine and mcpa were only 18.64% and 21.60%, respectively. the present result confirms the findings of bari (2010) who reported higher grain yield of rice with pre-emergence herbicide like butachlor than with post-emergence herbicide like mcpa. it might be due to the fact that pre-emergence herbicides offered early season weed control up to the period of full canopy cover by rice plants, which might have contributed to higher grain yield. application of post-emergence herbicides at 25 dat could not bring the desired benefits as weeds grew luxuriantly and competed with the crop for nutrients, light, water and space. moreover, they effectively controlled only broad leaved weed as shown in table 3. it is noticeable here that grain yields produced by sunflower residue alone and sunflower residue + pyrazosulfuron ethyl at 75 and 50% of recommended dose were comparable to pyrazosulfuron ethyl used alone. in seems that a reduced rate of pyrazosulfuron ethyl may be feasible for providing satisfactory grain yield when it is applied simultaneously with sunflower residue. these results are in agreement with the findings of alsaadawi and sarbout (2015) who observed that combination of lower rate of trifluralin and sunflower residues at 6 t ha-1 significantly reduced weed density and weed biomass by 79 and 90%,respectively over control. weed index weed index is an ideal parameter to judge the effectiveness of weed control treatments. this is a measure of reduction in the grain yield due to competition stress offered by weeds asagainst weed free treatment. the lower weed index indicates higher effectiveness of a weed control treatments. the crop yield is inversely related to weed index. all the weed control methods showed lower weed index (3.31-21.98%) than the weedy check (34.24%) (table 4). singh et al. (2008) reported that the application of different herbicide treatments recorded lower weed index. the highest weed index in weedy check was due to lowest grain yield associated with uncontrolled weed growth throughout the crop growth period. preemergence herbicides exhibited lower weed index as compared to post-emergence herbicides. however, the lowest weed index was noticed in sunflower residues along with 100% pyrazosulfuron ethyl (3.31%) application followed by butachlor (6.42%) due to satisfactory weed control. the effective control of weeds by these treatments might have enabled the crop to utilize available resources like light, nutrients, moisture and space to a greater extent resulting in higher yield. correlation between pairs of characters the data pertaining to correlation co-efficient values (r) worked out for grain yield versus total weed density, total weed dry weight and weed control efficiency are presented in table 5. grain yield showed significant positive correlation with weed control efficiency (0.843*). grain yield had significant negative correlation with total weed density (-0.929**) and total weed dry weight (-0.902**). mondal et al. (2018) also reported a strong negative international journal of applied biology, 4(1), 2020 112 correlation of weed density and weed dry matter with grain yield. weed control efficiency was negatively correlated with weed density (-0.915**) and weed dry weight (-0.967**). table 5. correlation co-efficient (r) values between pairs of characters correlation grain yield weed density weed dry weight weed control efficiency grain yield 1.000 weed density -0.929** 1.000 weed dry weight -0.902** 0.937** 1.000 weed control efficiency 0.843** -0.915** -0.967** 1.000 ** indicates significant at 0.01 probability level conclusions the results of the study showed that among different herbicides, butachlor produced the highest grain yield of t. aman rice comparable to hand weeding. thus, the use of butachlor may be an alternative in controlling weeds more easily and cheaply when there is a labour crisis. application of sunflower residues in combination with the lower rate (75 or 50%) of pyrazosulfuron ethyl may be considered as an eco-friendly approach in sustainable agriculture.further research may be carried out to investigate possible complementary interaction of allelopathic crop residues with other herbicides against weeds in field crops for further improvement of the efficacy of this technique. references akbar, n., ullah, e. and jabran, k. m.a. 2011. weed management improves yield and quality of direct seeded rice. asian j. crop sc. 5(6): 688-694. alsaadawi, i. s., khaliq, a., al-temimi, a. a. and matloob, a. 2011. integration of sunflower residues with a pre-plant herbicide enhances weed suppression in broad bean. planta daninhavicosa-mg. 29 (4): 849-859. alsaadawi, i.s. and sarbout, a. k. 2015. reducing herbicide rate in combination with allelopathic sunflower residues for weed control in cowpea. j. allelochemical interact. 2 (1): 9-16. bari, m. n. 2010. effects of herbicides on weed suppression and rice yield in transplanted wetland rice. pakistan j. weed 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weed control and enhancement of productivity and profitability of rice. expl. agric. 1 contact : stanley chukwudozie onuoha sconuoha@yahoo.com 1 abstract vibrio species in abattoir and aquaculture environment are of human health significance and may be increasing in pathogenicity and abundance. this study was aimed at determining the multidrug resistance of vibrio species isolated from abattoir and aquaculture environment in ebonyi state. effluent samples were obtained from different abattoir sites within ebonyi state from the butchers’ table, drainage, and wash water, while aquaculture sample were collected from different pond sites within the study area. vibrio isolates were detected using thiosulphate citrate bile salt (tcbs) agar and identified based on the morphology and biochemical characterization. the results revealed a total of 50 isolates from 20 waste water samples. waste water samples from different points of drainage water from abattoir had the highest microbial load of 3.85±0.35x106 cfu/ml. while, samples from butcher’s table and wash water from abattoir had high microbial load of 3.30±0.14x106and 3.25±0.14 x106 cfu/ml. respectively when compared to aquaculture samples which had the least microbial load of 0.6±3.9 x106 cfu/ml. percentage antibiotic susceptibility profile showed vibrio isolates were highly resistant to tobramycin 40(80%), cefoxitin 40(80%), amoxicillin/clavulanic acid 40(80%), meropenem 30(52%), cefepime28(56). abattoir effluents and aquaculture are important reservoirs of multidrug resistant vibrio pathogens. this implies that abattoir effluents could be important contributors to the episodes of epidemic cholera, non-vibrio cholera and vibrio causing illnesses in the study area issn : 2580-2410 eissn : 2580-2119 multidrug resistant vibrio species isolated from abattoir and aquaculture environment in ebonyi state, nigeria stanley chukwudozie onuoha 1*, felicia ngozi oko 2, collins onyebuchi okafor 2, kenneth ndidi ovia 2 1 department of biotechnology, faculty of science, ebonyi state university, abakaliki, ebonyi state, nigeria 2 department of microbiology, college of sciences, evangel university akaeze, ebonyi state, nigeria. introduction vibrio is a genus of gram-negative, curved-rod bacteria, some species may cause food-borne infection which are typically associated with eating undercooked seafood. usually, vibrio species are found in salt water and are facultative anaerobes that test positive for oxidase and do not form spores (thompson et al., 2015).various vibrio species are pathogens. gastroenteritis is associated with some disease-causing strains, but they can also infect open wounds and cause sepsis. many marine animals may carry them, such as open access international journal of applied biology keyword aquaculture; abattoir; vibrio species; ebonyi state article history received november 18, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 2 crabs or prawns, and during exposure they are known to cause fatal infections in humans. the specific pathogenic species of vibrio are v. cholerae, v. parahaemolyticus and v. vulnificus (quilici and robert-pillot, 2011). an abattoir is a special facility designed and licensed for receiving, holding, slaughtering, and inspecting meat animals and meat products before release to the public (witus and vun, 2016). abattoir inspection of live animals (ante-mortem) and carcasses (post-mortem) is critical to surveillance for animal diseases and zoonoses (amini et al., 2011). aquaculture, also known as aquafarming, is the farming of fish, crustaceans, molluscs, aquatic plants, algae, and other organisms (nordstrom et al., 2009). nigeria's aquaculture and abattoir industries are primarily related to their economic gains from supplying domestic and foreign demands and producing steady income for farmers. fish and meat are among the most common protein products that consumers from african countries consume on a daily basis. approximately 75% of global fish and meat production is primarily for human consumption (teh, 2012). fish consumption in nigeria has risen since 1970 and now exceeds 40 kg / capita / year (hajeb et al., 2009). these products have a healthy diet, such as high protein levels, omega-3 fatty acids (n-3), essential vitamins and minerals that an individual requires. the growth and strength of these industries (aquaculture and abattoir) has contributed to immune systems suppression and increases the vulnerability of fish and meat to bacterial infections (finlay and falkow, 2010). antibiotic-resistant infection has become more difficult to treat with existing antibiotics, leading to infections that lead to increased morbidity and mortality, resulting in huge societal costs. some human pathogens, including vibrio species, are involved in this increasing resistance. multidrug-resistant (mdr) bacteria have been a major health issue in medically used antibiotics and a major challenge to drug discovery programs around the world. it is well documented that there are antibiotic resistance traits in both clinical and environmental strains of vibrio (koelle et al., 2016). vibrio species will serve as a reservoir of antibiotic resistance genes for the environment. vibrio species, independent of sero-group, has been found to have a plastic genome and a long history of active plasmid interaction. in other words, vibrio species may be able to acquire and exchange genes through either integrons or integrative and conjugative elements (ice) such as the sxt element, due to its genetic characteristics.(ceccarelli et al.,2013). the problem of antimicrobial resistance among the agent continues to be alarming. there is a growing global occurrence of vibrio spp in aquaculture and abattoir and the strains has been known to be resistant to various antibiotics. despite some studies on diarrhoeal diseases in nigeria, there is lack of adequate information on bacterial enteric pathogens and their antimicrobial resistance trend within the study area. this study will determine the best suitable antibiotics for the treatment of vibrio spp especially within the study area. materials and methods sample collection samples used in this study were obtained from different selected abattoir sites in ebonyi state viz: ezzamgbo, effium, ngbo, new market, kpirikpiri and meat market all in ebonyi state. the samples were obtained from the butchers’ table, drainage sites and international journal of applied biology, 6(1), 2022 3 washwaters, while, the aquaculture samples were collected from selected aquaculture pond sites in different localities in ebonyi state such as josel fish pond nkaliki, matthew pond in izzi, onuebonyi abakaliki, obodo pond in ezzamgbo, and ifeanyi pond in ngbo. samples were collected using sterilized bottles and were preserved in peptone water for 24 hours for enrichment before analysis. sample processing five (5 ml) of freshly prepared peptone water was dispensed into test tubes and were sterilized at 121 ℃ 15 psi for 15 minutes. thereafter, one (1 ml) each of the samples were aseptically weighed into the sterile peptone water and were incubated for 24 hou rs for enrichment of the whole sample. isolation of vibrio species one (1 ml) each of the enriched samples was aseptically measured into test tubes containing 9 ml of sterile distilled water and shaken thoroughly for even distribution of organisms to make a stock. thereafter, ten-fold serial dilution of the samples were carried out and subsequently inoculated on a freshly prepared nutrient agar plates and were incubated at 37 ℃ for 24 hours. after 24 hours of incubation, some colonies were transferred to thiosulphate citrate bile salt (tcbs) agar plates based on their colour and shape under aseptic condition with the aid of a wire loop and incubated at 37 ℃ for 24 hours. after 24 hours, colonies of vibrio in thiosulphate citrate bile salt agar showed yellowish colour. the colonies were picked, sub-cultured and subsequently identified using standard microbiological procedure. antibiotics sensitivity testing susceptibility test was done on muller hinton agar (oxoid, uk) plates by standard disk diffusion method in conformity to the recommended standard of clinical and laboratory standard institute (2005). the antibiotic disks used include; imipenem (10 µg), cefoxitin (30 µg), cefotaxime (30 µg), cefeprime (30 µg), meropenem (10 µg), tobramycin (10 µg) ceftazidime (30 µg) and amoxicillin clavulanic acid (30 µg). all the antibiotics disk were procured from oxoid limited (oxoid, uk). these antibiotics were chosen either because they are used in both medicine and human veterinary practice or as a result of previous studies with reports of microbial resistance to them. colonies of confirmed vibrio isolates were collected using wire loop and were dispensed into test tubes containing 5 ml distilled water. the cell concentration was adjusted to 0.5 macfarland standard. sterile swap stick was used to collect the organisms and these were streaked on freshly prepared mueller-hinton agar plates. the plates were allowed to stand for 15 minutes so that the cells will adapt to the environment of the medium. after this, the standard antibiotic disks were placed 15 mm apart and was incubated at 30oc for 24 hours and the zones of inhibition diameter was measured according to clsi criteria (2005). determination of multiple antibiotic resistance index (mar) multiple antibiotic resistance (mar) index was determined for each isolate by using the formula mar = a/b, where a represents the number of antibiotics to which the test isolate depicted resistance and b represents the total number of antibiotics to which the test isolate has been evaluated for susceptibility. mari of relative ratio >1 is shown to represent potential risk source of resistant strain from the environment. if mar index value international journal of applied biology, 6(1), 2022 4 is between 0.200 and 0.250 it becomes a very risky case where there are equal chances that mar may fall in the high risk and low risk phases (raiz et al, 2011). statistical analysis experimental data was presented as mean±standard deviation, while one way anova procedure will be used to analyze statistical difference in the data generated . results microbial load of the isolated vibrio species from different aquaculture and abattoir samples the result as represented on table 1 revealed that a total of 50 isolates of vibrio were isolated from 20 waste water samples. samples from drainage water from abattoir had the highest microbial load of 3.85±0.35x106, followed by butchers table, 3.30±0.14x106, while wash water from hartchery had the least microbial load of 1.21±0.33 x106. for aquaculture; samples from concrete pond had highest microbial load of 2.2±3.4x106 from ebonyi farm when compared to earthen pond which had the least microbial load of 1.0±3.0 x106 from chiboy farm. hence in comparison of the waste water samples, abattoir samples from drainage water from ezzamgbo had the highest microbial load of 3.85±0.35x106, while aquaculture samples from earthen pond had the least microbial load of1.21±0.33 x106. table1. microbial load of vibrio species obtained from aquaculture and abattoir samples aquaculture fish pond chiboy obodo chuks josel ebonyi chali concrete 2.0±1.4 1.5±4.2 1.6±4.0 0.6±3.9 2.2±3.4 12.0±0.96 earthen 1.0±3.0 1.5±0.6 1.7±04.1 1.5±0.6 1.22±2.9 19.9±0.65 abattoir ww bt dw new market 2.10±1.55 3.30±0.14 2.12±0.03 kpiripkiri 3.25±0.14 1.97±0.41 2.46±0.23 meat market 2.72±0.70 1.85±0.64 2.36±0.90 ezzamgbo 2.30±0.12 2.13±1.23 3.85±0.35 jemenny 2.60± 0.22 2.10±1.30 2.22±0.10 ishieke market 2.85±0.35 1.90±0.14 2.40±0.42 amike-aba 2.87±0.21 2.13±1.65 2.46±0.48 eke-aba 2.87±0.21 1.48±0.64 2.67±0.40 abofia market 2.70±0.42 1.75±0.43 2.32±0.38 azugwu market 2.70±0.22 2.12±1.12 2.56±0.26 international journal of applied biology, 6(1), 2022 5 key: bt = butcher’s table, ww = wash water, dw = drainage water, cfu = colony forming unit, ml = milliliter distribution of vibrio species isolated from samples from abattoir and aquaculture farm the result of occurrence and distribution of vibrio species is represented in (table 2). the results reveal that waste water samples from butchers table showed highest percentage distribution 15(50.00%) from different locations than drainage water 8(26.68%), while wash water had the least percentage distribution 7(23.33%). aquaculture samples showed highest percentage distribution 12(60%) among vibrio species from earthen pond while concrete pond showed least percentage distribution 8(40%). table 2. distribution of the isolates from samples from abattoirs and aquaculture farms abattoir new market kpirikpiri ezzamgbo jemenny meat market total (%) drainage 2 (6.67) 0 (0) 2 (6.67) 2 (6.67) 2 (6.67) 8 (26.68) washwater 2 (6.67) 2 (6.67) 1(3.33) 1(3.33) 2 (6.67) 7(23.33) butchers table 3 (10.0) 4 (13.33) 5 (16.67) 0 (0) 3 (10) 15(50.0) total 6 (20) 3 (10) 6 (20) 7 (23.33) 8 (26.67) 30 (100) fish pond chiboy obodo chucks jossel hartchery ebonyi total concrete 1 (5) 0(0) 1 (5) 2 (10) 1 (5) 1 (5) 8 (40) earthen 2 (10) 2 (10) 1 (5) 3 (15) 2 (10) 1 (5) 12 (60%) total 3 (15) 2 (10) 2 (10) 5 (25) 3 (15) 2 (10) 20 (100) key: ww = wash water, dw = drainage water, bt= butcher’s table, % = percentage, cp = concrete pond, ep = earthen pond, % = percentage. antibiotics susceptibility pattern of the vibrio species to the commonly used antibiotics table 3 shows the result of the antibiotics susceptibility of vibrio species to the respective antibiotics used. the results reveal that the isolates were highly resistant to fox, tob, amc, 40(80%) respectively. some of the isolates showed slight resistance to fep and mem, 28(56%) and 30(52%) respectively, while lesser percentage of the isolates showed least resistance to ipm and caz at 12(24%) respectively. meanwhile, the susceptibility pattern revealed that the isolates had highest susceptibility to ipm and caz,38(76%) respectively while few were slightly susceptible to amc, tob, fox, 10(20%) respectively. umuoghara 2.22±0.10 2.20±1.31 2.40±1.13 ogoja road 2.87±0.21 1.34±0.11 2.34±1.12 hartchery 1.21±0.33 2.22±0.10 2.40±1.13 ngboagbaja market 2.72±0.70 1.47±0.21 2.52±1.03 international journal of applied biology, 6(1), 2022 6 table 3. antibiotics susceptibility pattern of the isolates to the used antibiotics. antibiotics resistance(%) intermediate(%) susceptibility(%) total (%) ipm 12(24) 0(0) 38(76) 50 (100%) fox 40(80) 0(0) 10(20) 50 (100%) ctx 10(20) 5(10) 35(70) 50 (100%) fep 28(56) 10(20) 12(24) 50 (100%) tob 40(80) 0(0) 10(20) 50 (100%) mem 30(52) 5(10) 15(30) 50 (100%) caz 12(24) 0(0) 38(76) 50 (100%) amc 40(80) 0(0) 10(20) 50 (100%) key: ipm = imipenem, fox = cefoxitin, ctx = cefotaxime, fep = cefepime, tob = tobramycin, mem = meropenem, caz = ceftazidime, amc = amoxicillin/clavulanic acid, % = percentage. multiple antibiotics resistance of vibrio species table 4 reveals the results of the respective drugs resistances of the isolated vibrio species with respect to their different samples collection sites. multiple antibiotics resistance (mar) index is a tool that reveals the tendency of an organism to form resistance to more than two antibiotics. from the results, vibrio species obtained from drainage water had the least mar indices of 0.1 and 0.2, while butcher's table and wash water had the highest mar indices of 0.4, 0.5 and 0.6 respectively when compared to aquaculture samples which were highest with multiple antibiotics resistance indices of 0.6 and 0.7. the isolates had high multiple resistance to ipm, fox, tob, mem, caz and fep at a mar index of 0.7, while they showed lower multiple resistance to amc at a mar index of 0.1. table 3. multiple antibiotic resistance index of the isolates from abattoir and aquaculture. sample point mari (n/n) antibiotics chiboy fish farm 0.6 fox ctx tob mem amc ogbodo fish farm 0.6 fox ctx tob caz amc chucks fish farm 0.6 fox ctx tob caz amc jossel fish farm 0.7 ipm fox tob mem caz amc ebonyi fish farm 0.6 fox fep tob mem amc international journal of applied biology, 6(1), 2022 7 jemenny abattoir 0.4 fox tob amc kpirikpiri market 0.5 fox tob mem amc meat market abattoir 0.6 fox fep tob mem amc new market abattoir 0.4 fox fep amc hartchery abattoir 0.2 fox amc amike-aba abattoir 0.2 ipm ctx umuoghara abattoir 0.1 amc eke-aba abattoir 0.2 ipm ctx ogoja road abattoir 0.2 ipm ctx hill-top abattoir 0.4 fox fep amc azu-ugwu abattoir 0.5 fox tob mem amc ezzamgbo abattoir 0.5 fox ctx feo mem ngboagbaja abattoir 0.4 fep tob mem key: ipm = imipenem, fox = cefoxitin, ctx = cefotaxime, fep = cefepime, mem = meropenem, caz = ceftazidime, amc = amoxicillin/clavulanic acid, mari = multiple antibiotics resistance index, n = number of the antibiotics that the isolates were resistant to, n = total number of all the tested antibiotics. discussion vibrio species in abattoir and aquaculture environment are of human health significance and may be increasing in pathogenicity and abundance. vibrio illness originating from dermal contact with vibrio laden waters or through ingestion of seafood originating from such waters and abattoir effluent can cause deleterious health effects, particularly if the strains involved are resistant to clinically important antibiotics. the purpose of this study was to evaluate antimicrobial resistance pattern among these pathogens. the results revealed that a total of 50 different isolates of vibrio species were gotten from aquaculture and abattoir samples within the study area. the result of the microbial load revealed that vibrio species were highest in microbial load among the waste water samples from drainage, while those samples from butchers table and wash water had lower microbial load when compared. although samples from aquaculture showed sligh t difference from those abattoir samples, the result revealed that abattoir and aquaculture is a reservoir for vibrio species which collaborated the work of baumann and schubert (2005) who have advance hypothesis that aquaculture settings serve as foci or reservoir for pathogenic vibrio strains, during certain period of the year. pathogenic vibrio would withstand environmental conditions within aquaculture settings and when favourable environmental conditions established, vibrio would be able to cause disease in wild animal (deepanjali et al., 2005). also, the result revealed that samples from aquaculture had lower percentage distribution while samples from abattoir effluent had higher percentage distribution which is in agreement with the report of (costa et al., 2015), which suggested that the detection international journal of applied biology, 6(1), 2022 8 of virulence and invasive vibrio isolates in abattoir effluent indicates that abattoir effluent is an important repository of pathogenic vibrio species; and could be a considerable contributor to the recurrent episodes of epidemic cholera and other non-vibrio cholerae outbreaks in certain parts of nigeria localities. a total of 50 vibrio species (n=50) were tested against eight commonly used antimicrobials in this study. the isolates exhibited remarkable sensitivity to imipenem, cefepime, meropenem and ceftazidime (table 3), in agreement with the report of chiang and chuang (2003) who observed that imipenem and the cephalosporins, including ceftazidime were effective against vibrio infections. however, contrary to the submission of chiang and chuang (2003), some species in this study exhibited reduced sensitivity and resistance to cefotaxime (20%). it is noteworthy that aquaculture and abattoirs environment within ebonyi state has shown to be a reservoir of antibiotics resistant bacteria. a study conducted by onuoha et al, (2016a, 2016b, 2017) in both abattoir and aquaculture environment in ebonyi state showed high multi-drug resistance which is widespread among majority of the bacteria studied. also, igbinosa et al., 2009 reported considerable resistance of vibrio isolates from municipal wastewater against ampicillin, trimethoprim, and trimethoprim/sulphamethoxazole in south africa; while marin et al., 2013 documented resistance against trimethoprim and trimethoprim/sulphamethoxazole amongst clinical vibrio strains isolated from different parts of nigeria. strains of vibrio tested in this study (except few) were generally resistant to tobramycin, amoxicillin and cefoxitin (80%) in agreement with reports from tanzania and rwanda, but contrary to reports from kenya, south sudan, south africa and somalia, materu et al., (1997). this might be due to the use of this antibiotic for long period of time in the community because it is relatively cheap and easily availability. more so, of the 50 isolates tested for antibiogram against 8 different antibiotics, the highest multiple antibiotics resistance index of the isolated vibrio spp. was shown by the vibrio species from aquaculture samples from earthen pond (0.7) which is in contrast with what was obtained by igbinosa et al., (2019). while butcher's table and wash water had lower mar index when compared to aquaculture samples. consistent with the observation of this study, onuoha et al, 2016b reported that bacteria isolated from their study were completely resistant to tetracycline, cephalothin, penicillin g, and erythromycin with mari values above 0.20 with the highest values in the range of 0.90, which was higher than what was observed in this study. also, igbinosa et al, 2019, reported that the percentage of isolates exhibiting mar was (10 20%) which was relatively higher than that of (0.7) as observed in this study. the isolates had high multiple resistance to imipenem, cefoxitin, tobramycin, meropenem, ceftazidime and cefepime, while they showed lower multiple resistance to amoxicillin. hence, aquaculture and abattoir effluents are considered to be one of such high-risk sources of contamination since they are associated with waste from livestock, which are often feed containing antibiotic additives. residual antibiotics that enter the environment with abattoir waste effluent have been reported (kümmerer, 2003) to exert selective pressure on microbial populations contained therein, thereby enhancing mar, as observed in this study. conclusions although abattoir and aquaculture effluents have been reported to be important environmental reservoirs for vibrio species, there is a dearth of information in the literature on antibiotic susceptibility patterns of vibrio species isolated from aquaculture and abattoir international journal of applied biology, 6(1), 2022 9 effluents in abakaliki, ebonyi state. isolates of the current study exhibited remarkable sensitivity to imipenem, meropenem, cefotaxime and cefepime. the mar index from this work indicated that vibrio species were highly resistant to most of the commonly used antibiotics, which is a clear indication that these microorganisms are rapidly developing resistance to the commonly used antibiotics. funding information the authors received no specific funding for this work. author contribution osc.; conceptualization, methodology, formal analysis, resources, writing (reviewing & editing), supervision, ofn; project administration. oco; formal analysis and investigation, okn; resources and visualization. conflict of interest the authors declare that they have no known competing financial or non-financial, professional, or personal conflicts that could have appeared to influence the work reported in this paper. acknowledgements we wish to acknowledge the owners of the various fish bonds and abattoir workers for granting us accesses to collect samples for the work. 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(2011) antibiotic susceptibility pattern and multiple antibiotic resistances (mar) calculation of extended spectrum β-lactamase (esbl) producing escherichia coli and klebsiella spp. in pakistan. afri j. biotechnol. 10(33): 63256331. witus, w.i and vun, w.l (2016). aquaculture in malaysia: a short review on current policy and legislation, transactions on science and technology, 3(1-2), 150 154 contact : vikash b. joshi vks.joshi1@gmail.com 63 abstract extremely halophilic isolates were obtained from mangrove vegetation of west coast bordi region, maharashtra india. all six isolates were selected for further investigation on the basis of their potential of producing enzymes amylase, protease and lipases respectively. production of enzyme from isolates was performed using basal medium with starch, skimmed milk and tributyrin. ammonium sulphate salt precipitation and dialysis was performed to obtain partially purified crude samples of enzymes. activity of crude enzymes was determined in unit/ml by using standard methods. also the effect of ph and temperature on enzyme activity was also determined. issn : 2580-2410 eissn : 2580-2119 extraction, purification and characterization of amylase, protease, lipase of halophilic bacterial isolates from bordi, india vikas b. joshi1 mrs.dr.anupama pathak1, supriya murkute2, bhoomi das2, zeenat khan2 1school of life science (dst-fist & ugc-sap sponsored) swami ramanand teerth marathwada university nanded, india 2department of microbiology, n.b.mehta science college bordi maharashtra india introduction halophilic prokaryotes has been found in a wide range of saline environment within various geographical locations including salt lakes, marine salterns and saline soils (de la haba, r.r et al.,2011, lizama, c et al.,2001). hypersaline environments host a considerable diversity of extremely halophilicarchea as well as halophilic and halotolerant bacteria (oren, a., 2002). in the past few years, the microbial diversity of such hypersaline environment has been extensively explored using both culture dependent and culture independent techniques. (benlloch, s et al.,1995, borsodi, a.k et al.,2013, burns, d.g et al.,2004, youssef, n.h et al.,2012, boutaiba, s., hacène et al.,2011, oren, a., 1983, oren, a., 2014). halophiles provide a potential source of novel enzyme that function under salt stress condition, such as lipases, proteases, amylases, gelatinase and xylanases with polyextremophilic properties. (dassarma s, dassarma p., 2015). halophilic enzymes are capable of functioning under high salt concentration, wide range of temperature an d ph at which other protein denature. certain enzymes that halophiles synthesize are useful for bioremediation of pollutants in saline habitats ( dastgheib sm et al.,2011). the halophilic enzymes have also been used in many industries including pharmaceutical, textile, detergent, baking, paper and pulp industries (sarwar mk, azam i, iqbal t 2015). a number of halotolerant and halophilic bacteria and archea such as bacillus, halobacillus, halomonas, salinobacillus, haloarcula and haloferax have been explored for open access international journal of applied biology keyword mangrove vegetation; halophile; amylase; protease; lipases article history received december 11, 2021 accepted june 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 64 their ability to hydrolyze enzymes like proteases, amylases and lipases (bozic n, ruiz j, lopezsantin j et al 2011). halophilic enzymes are also considered as important biocatalyst under low water conditions such as hypersaline environment and non-aqueous media (marhuendaege fc, bonete mj 2002). the current investigation focuses on (i) isolating moderate halophiles from saline habitat, such as coastal regions of bordi (maharashtra) (ii) screening for industrially important enzymes (particularly amylases, lipases, and proteases), and (iii) studying few properties of these enzymes. materials and methods the present study was carried out on potential of screening, extraction, purification and characterization of industrially important enzymes from previously isolated and identified halophilic bacteria namely virgibacillusdokdonesis dsw-10(t), kocuriaflava ho9041(t), halomonaspiezotolerans nbt06e8(t),halolactibacillusmiurensis dsm 17074(t), oceanobacilluscaeni s-11(t), planococcusplakortidis dsm 23997(t). these isolates were denoted in this study as isolate bb, bc4, bj, bk, bs213, bs6 respectively. screening of extracellular hydrolytic enzymes the extremozymes production from extremophilic microbes can be completed using the standard method and media. microbes for enzyme production under normal as well as extreme conditions can be screened using diffusion agar plate of basal medium composition: yeast extract 1 gm, kh2po4 1 gm, mgso4.7h2o 0.1 gm, cacl2.h2o 0.05 gm, nacl 10 gm, naco3 1 gm, 18 gm/ lit agar supplemented with different substrates such as starch (0.25%), skimmed milk(10% w/v) and tributyrin (1%) (divjot kour et al.,2019). amylase on basal medium supplemented with 0.25 % starch, isolates were spot inoculated and kept for incubation for 72 hrs. after incubation starch hydrolysis was detected by flooding plates with iodine-potassium iodide solution also known as lugol’s iodine solution containing 1 gm of iodine crystals, 5 gm potassium iodide and 330 ml distilled water. clear zone around the colony indicates hydrolysis of starch (montalvo-rodriguez, r et al.,1998). protease proteolytic or caseinolytic activity of the microbes was tested in skimmed milk plates of basal medium with skimmed milk 10% w/v. clear zone around the growth were considered as an indication of protease activity. for some microbes required more time period for hydrolyzing activity (gonzalez, c et al.,1978). lipase lipase activity detected using basal medium containing 1% tributyrin (v/v). clear orange colored fluorescent halos around colonies were considered as indication of lipase activity. here rhodamine b (0.005%) was used and plates observed under uv light in chamber ( kouker, g. and jaeger, k.-e. 1987, boutaiba, s et al.,2006). production of enzymes basal medium with starch(0.25 %), skimmed milk(10 % w/v) and tributyrin(1 %) was used for the production of enzymes from six isolates. all the isolates were inoculated in the international journal of applied biology, 6(1), 2022 65 erlenmeyer flask containing 50 ml basal medium with substrates. flasks were incub ated on rotary shaker at 370c temperature and speed was maintaining 120 rpm for 8 days. purification of enzymes the cultured broth centrifuged for 10min at 13000 xg in 40c. enzymes were precipitated by 80% saturated ammonium sulphate. the mixtures were left overnight at 40c. the precipitate was centrifuged for 30 min at 12000xg and dissolved in 100 mmtris-hcl buffer (ph 8) and dialyzed to obtain crude partially purified enzymes. these crude and partially purified enzymes were used for determination of enzyme assay and activity at variable ph and temperature (chakraborty s et al.,2009, hmidet n et al.,2008). enzyme assay amylase assay in this assay soluble starch 1% was used as substrate. if amylase digests starch, the maltose was released. di-nitro salicylic acid (dnsa) react with maltose to generate deep orange red colored complex on boiling for 10-15 min, which was measured as an absorbance value on uv-visible spectrophotometer (systronics 117) at 540 nm. a maltose standard calibration curve was performed and constructed to determine amount of maltose released after the amylolytic activity. a series of maltose standard solution at different concentrations ( 100-1000µm) were prepared from the maltose stock solution with deionized water. 1ml of properly diluted (in acetic acid buffer ph 4.9) crude enzyme was incubated for 15min at 400c with 1 ml of soluble starch solution 1% w/v. the produced quantity of reducing sugar released from starch was determined by taking absorbance at 540 nm. as unit of activity (u) of the enzyme amylase is arbitrarily appointed, the quantity of enzyme required for the production of 1µm of maltose in 1 min, when the enzyme was incubated along with the substrate at ph 4.9 and temperature 400c (plummer, d 1978, harvey,r 2005, miller gl 1959). protease assay in this assay, β-casein was used as substrate. if protease digest casein, the aminoacid tyrosine is liberated along with free other peptide fragments. folin’s reagent react with free tyrosine to generate a blue colored product, which was measured as an absorbance value on uv-visible spectrophotometer ( systronics 117) at 660 nm. a tyrosine standard calibration curve was performed and constructed to determine amount of tyrosine released after the proteolytic activity. a series of tyrosine standard solution at different concentrations (40-200 µg/ml) were prepared from the l-tyrosine stock solution with deionized water. 1 ml substrate casein (1%) incubated with 1 ml crude enzyme at 400c for 30min. addition of 5% tca 5ml in reaction mixture allowed it to stand for 10 min at room temperature, then addition of folin’s reagent 0.5ml. measure the absorbance at 660 nm against blank without enzyme. the unit of activity of protease calculated as the amount of enzyme that release one micromole of the product per milliliter of the sample per minute under standard reaction conditions (plummer, d 1978, harvey,r 2005, peterson 1977). lipases assay to test for lipase activity, the crude enzyme preparation was assayed titrimetrically. to 3ml of phosphate buffer (ph 7.5) was added 1ml of substrate (olive oil), 2.5 % gum arabic and 0.5 ml of crude enzyme. the reaction was performed under continuous agitation for 5min. the reaction was terminated by addition of 25 ml acetone:ethanol (1:1) and librated fatty acid was titrated against 50mm naoh. indicator used for titration was thymopthalein international journal of applied biology, 6(1), 2022 66 0.9%. mixture was titrated until light blue colour appears. one unit of lipase activity was equivalent to the amount of enzyme that catalyzes the release of 1µm of fatty acid from the substrate per milliliter in 1 min under the standard assay conditions (gupta, n., mehra, g. and gupta, r., 2004). effect of ph and temperature on enzyme activity the crude enzyme was used for checking activity at variable ph and temperature. ph range used for all three enzymes was 6 to 10 with difference of 0.5. the enzyme stability is depending on the optimum ph. each enzyme has a region of optimum ph for stability. the optimum ph was determined by incubating the enzyme with substrate at ph 6 to 10. the enzyme activity was calculated at each ph on which crude enzyme incubated. curve was plotted of enzyme rate of reaction against the different ph at which the enzyme catalytic reactions are incubated. from the curve optimum ph which had given the maximum activity of the enzyme was noted. enzyme assay with suitable substrate for all three enzymes was performed at a range of temperature 40c, 250c, 370c and 550c according to method describe above. the maximum enzyme activity was found by plotting graph temperature versus enzyme activity. results and discussion all the isolates was used for study related to their extracellular enzyme producing ability were isolated from mangrove rhizosphere and have ability to show growth at high salt concentration up to 15% nacl. these isolates were isolated from bordi region west coast maharashtra india. screening of enzyme was performed with the help of modified basal medium containing 0.25% starch, 10% (w/v) skimmed milk and 1% (v/v) tributyrin. already identified isolates were shown potential of producing all the three enzymes. observation of starch hydrolysis was performed by using lugol’s iodine solution, casein hydrolysis was observed against the brightly illuminated light and lipid hydrolysis by using 0.005% rhodamine b under uv chamber (table 1). enzymes were produced in large quantity by using same media which was used for screening. after incubation of 7 days fermented broth was centrifuged and used for ammonium sulphate precipitation. 80% (w/v) salt was used for obtaining the maximum precipitate. precipitate was dissolved in 100mm tris-hcl buffer (ph 8.0). dissolved precipitate used for dialysis and partially purified enzyme used for assay and for checking activity. activity of enzyme amylase was determined by using standard plot of maltose (1000mm). maximum amylase activity on 1% starch was shown by isolate bj (121.68u/ml) and lowest activity by isolate bb (43.74u/ml) respectively. protease activity was determined by using standard plot of l-tyrosine (200mcg/ml). maximum activity noted for isolate bs213 (43.74 u/ml) and lowest activity for isolate bj (8.33u/ml). lipases activity was determined by using olive oil and arabic gum mixture. maximum activity noted for isolate bs6 (187 u/ml) and lowest activity given by isolate bb (86 u/ml) (table 2) (figure 1). activities of enzymes were also determined on different ph condition. after calculating the activity of each enzyme on different ph condition it was found that ph 8.5 is suitable for production of enzymes as most of them showed maximum activity at ph 8.5. isolate bb and bs213 showed maximum protease activity at ph 8.5 while bs213, bj and bs6 showed maximum lipases activity at ph 8.0. (figure 2,3,4) activity of enzymes was also international journal of applied biology, 6(1), 2022 67 determined at variable temperature; it was found that all enzymes have been given maximum activity at 370c and though even at 500c enzyme is not losing a lot activity (figure5,6,7). table 1. screening of enzyme production by isolates isolate name of isolate amylase protease lipase bb virgibacillusdokdonesis dsw-10(t) + + + bc4 kocuriaflava ho-9041(t) + + + bj halomonaspiezotolerans nbt06e8(t) + + + bk halolactibacillusmiurensis dsm 17074(t) + + + bs213 planococcusplakortidis dsm 23997(t) + + + bs6 oceanobacilluscaeni s-11(t) + + + table 2. enzyme activity of isolates isolate name of isolate amylase u/ml protease u/ml lipase u/ml bb virgibacillusdokdonesis dsw-10(t) 43.74 32.15 86 bc4 kocuriaflava ho-9041(t) 63.02 11.82 129 bj halomonaspiezotolerans nbt06e8(t) 121.68 8.33 165 bk halolactibacillusmiurensis dsm 17074(t) 101.12 12.91 104 bs213 planococcusplakortidis dsm 23997(t) 84.38 43.74 143 bs6 oceanobacilluscaeni s-11(t) 76.39 10.06 187 international journal of applied biology, 6(1), 2022 68 figure 1. enzyme activity figure 2. effect of ph on amylase activity figure 3. effect of ph on protease activity 0 20 40 60 80 100 120 140 160 180 200 bb bc4 bj bk bs213 bs6 e n zy m e a ct iv it y i n u /m l name of isolate enzyme activity amylase protease lipase 0 10 20 30 40 50 60 70 80 90 100 110 120 130 6 6,5 7 7,5 8 8,5 9 9,5 10 e n zy m e a ct iv it y i n u /m l effect of ph on amylase activity bb bc4 bj bk bs213 bs6 ph 0 5 10 15 20 25 30 35 40 45 6 6,5 7 7,5 8 8,5 9 9,5 10 e n zy m e a ct iv it y i n u /m l effect of ph on protease activity bb bc4 bj bk bs213 bs6 ph international journal of applied biology, 6(1), 2022 69 figure 4. effect of ph on lipase activity figure 5. effect of temperature on amylase activity figure 6. effect of temperature on protease activity 0 20 40 60 80 100 120 140 160 180 200 6 6,5 7 7,5 8 8,5 9 9,5 10 e n zy m e a ct iv it y i n u /m l ph effect of ph on lipase activity bb bc4 bj bk bs213 bs6 0 20 40 60 80 100 120 140 4 25 37 50 e n zy m e a ct iv it y i n u /m l temperature in 0c effect of temperature on amylase activity bb bc4 bj bk bs213 bs6 0 5 10 15 20 25 30 35 40 45 4 25 37 50 e n zy m e a ct iv it y i n u /m l temperature in 0c effect of temperature on protease activity bb bc4 bj bk bs213 bs6 international journal of applied biology, 6(1), 2022 70 figure 7. effect of temperature on lipase activity conclusions in recent years, the enzymes from extremophiles have been in very high demand in various sectors like medicine, agriculture, food and paper industries, laundries etc,due to their wide applications in various processes. the enzymes which has studied in this work are also used in various sectors as they show activity at variable temperature and ph. future research should focus on confirming the km value for all three enzyme synthesis, purifying and deeply characterising all three enzymes generated and optimising the growing conditions of halophilic isolates for future applications in various industries. acknowledgement the authors are grateful to vice-chancellor of s.r.t.m. university nanded and principal of gokhale education society’s, n.b.mehta science collegebordi maharashtra india, for providing infrastructural facilities and constant support during work. 0 20 40 60 80 100 120 140 160 180 200 4 25 37 50 e n zy m e a ct iv it y i n u /m l temparature in 0c effect of temperature on lipase activity bb bc4 bj bk bs213 bs6 international journal of applied biology, 6(1), 2022 71 references benlloch, s., martínez-murcia, a.j., rodríguez-valera, f., 1995. sequencing of bacterialandarchaeal 16s rrna genes directly amplified from a hypersalineenvironment.syst. appl. microbiol. 18, 574–581. birbir, m., calli, b., mertoglu, b., bardavid, r.e., oren, a., ogmen, m.n., ogan, a., 2007.extremely halophilicarchaea from tuz lake, turkey, and the adjacent kaldirimandkayaciksalterns. world j. microbiol. biotechnol. 23, 309–316. borsodi, a.k., felfoldi, t., mathe, i., bognar, v., knab, m., krett, g., jurecska, l., toth, e.m., marialigeti, k., 2013. phylogenetic diversity of bacterial and archaealcommunitieinhabiting the saline lake red located in romania sovata. extremophiles 17,87–98. boutaiba, s., bhatnagar, t., hacène, h., mitchell, d.a., baratti, j.c., 2006. preliminarycharacterisation of a lipolytic activity from an extremely halophilicarchaeaon,natronococcus sp. j. mol. catal b. enzym. 41, 21–26. boutaiba, s., hacène, h., bidle, k.a., maupin-furlow, j.a., 2011. microbial diversity ofthehypersalinesidiameur and himalatt salt lakes of the algerian sahara. j. arid.environ. 75, 909–916. bozic n, ruiz j, lopez-santin j et al (2011) production and properties of the highly efficient raw starch digesting α-amylase from a bacillus licheniformis atcc 9945a. biochemeng j 53:203–209. burns, d.g., camakaris, h.m., janssen, p.h., dyall-smith, m.l., 2004. combined use 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extremophiles for hydrolytic enzymes productions: biodiversity and potential biotechnological applications, bioprocessing for biomolecules production 2019 pp.321-372. international journal of applied biology, 6(1), 2022 72 gonzalez, c., gutierrez, c., ramirez, c., 1978. halobacteriumvallismortis sp. nov. an amylolytic and carbohydrate-metabolizing: extremely halophilic bacterium. can. j.microbiol. 24, 710–715. gupta, n., mehra, g. and gupta, r., 2004. a glycerol-inducible thermostable lipase from bacillus sp.: medium optimization by a plackett-burman design and by response surface methodology. can. j. microbiol., 50, 361–368. harvey,r and champe,p. lippincott biochemistry, london.2005. hmidet n, bayoudh a, berrin jg, kanoun s, juge n, nasri m. purification and biochemical characterization of a novel α-amylase from bacillus licheniformisnh1: cloning, nucleotide sequence and expression of amy n gene in escherichia coli. process biochem. 2008;43:499-510. kouker, g. and jaeger, k.-e. (1987). specific and sensitive plate assay for bacterial lipases.appl. environ. microbiol. 53: 211–213. lizama, c., monteoliva-sanchez, m., prado, b., ramos cormenzana, a., weckesser, j.,campos, v., 2001. taxonomic study of extreme halophilicarchaea isolated from thesalar de atacama, chile. syst. appl. microbiol. 24, 464–474. marhuenda-ege fc, bonete mj (2002) extreme halophilic enzymes in organic solvents. curropinbiotechnol 13:385–389. miller gl (1959) use of dinitrosalicylic acid reagent for determination of reducing sugar. anal chem 31:426–428. montalvo-rodriguez, r., vreeland, r.h., oren, a., kessel, m., betancourt, c., lopezgarriga, j., 1998. halogeometricumborinquense gen. nov. sp. nov., a novel halophilicarchaeon from puerto rico. int j. syst. bacteriol. 48, 1305–1312. oren, a., 1983. halobacteriumsodomense sp. nov., a dead sea halobacterium with anextremely high magnesium requirement. int. j. syst. bacteriol. 33, 381–386. oren, a., 2002. diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. j. ind. microbiol. biotechnol. 28, 56–63. oren, a., 2014. taxonomy of halophilicarchaea: current status and future challenges.extremophiles 18, 825–834. peterson, precipitation lowry protein assay, 1977 anal. biochem. 83, 346 plummer, d. an introduction to practical biochemistry. mcgraw-hill, london. 1978 sarwar mk, azam i, iqbal t (2015) biology and applications of halophilic bacteria and archaea: a review. e j bio 11:98–103. youssef, n.h., ashlock-savage, k.n., elshahed, m., 2012. phylogenetic diversities andcommunity structure of members of the extremely halophilicarchaea (orderhalobacteriales) in multiple saline sediment habitats. appl. environ. microbiol. 78,1332–1344. contact : andi ariyandy ariyandyasir@unhas.ac.id 172 abstract this study aims to determine the bioactivity of the compound quercetin in the peel of onion (allium cepa l) as a natural antihypertensive compound. the chemical structure of the quercetin compound found in allium cepa l peel was taken from the literature. the target protein used was angiotensin-converting enzyme (ace), while the control compound was lisinopril. water molecules were removed using pymol v2.5.2 software. docking between the target protein and the compound was carried out using pyrx-python prescription 0.8 software. the results showed that the quercetin compound had more significant potential as an antihypertensive compared to lisinopril as a control compound. the affinity ratio of the angiotensin-converting enzyme with quercetin is -8.1, while the affinity value of the angiotensin-converting enzyme with lisinopril is -7.1. issn : 2580-2410 eissn : 2580-2119 in silico testing on the activity of quercetin in the skin of onion allium cepa l as a natural antihypertensive compound andi ariyandy1, nindrahayu2, ami febriza3, andi irwan muluk4, sulfahri5 1 department of physiology, faculty of medicine, hasanuddin university makassar, indonesia, 2 hasanuddin university hospital, makassar, indonesia, 3 department of physiology, faculty of medicine, university of muhammadiyah makassar, indonesia 4 biomedical sciences, graduate school, hasanuddin university, makassar, indonesia, 5 biological sciences, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia introduction the circulatory system illness known as hypertension raises blood pressure above normal ranges, which makes the heart work harder to circulate blood throughout the body. (haryanto susanto, rehmadanta sitepu fx, 2021). blood pressure is checked twic e, with a five-minute gap, at a peaceful time (putri, 2020). the world health organization (who) defines adults over the age of 18 as having mild hypertension if their systolic pressure is between 140 and 159 mmhg and their diastolic pressure is between 90 and 99 mmhg, moderate hypertension if those numbers are between 160 and 179 mmhg and 100 to 109 mmhg, and severe hypertension if those numbers are above 180 mmhg (harfiantoko & kurnia, 2013). according to who, by 2025, there will be approximately 1.5 billion people affected by hypertension each year (pharmaceutical et al., 2019). patients with hypertension tend open access international journal of applied biology keyword quercetin; angiotensin-converting enzyme (ace); allium cepa l article history received august 13, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 173 to increase year after year, with an estimated 600 million worldwide, and have become a very serious health problem known as "the silent killer." (rahajeng and tuminah, 2019). if not controlled, it can lead to coronary heart disease, stroke, kidney disease, peripheral arterial disease, and blindness (rahajeng & tuminah, 2019;perez-vizcaino et al., 2009). both drug-based and non-drug approaches can be used to treat hypertension. on the other hand, because the treatment is prolonged, it may be risky and result in negative side effects (putri, 2020). lisinopril and other medications in the ace inhibitor class are frequently used for hypertension, but according to braghi, they can occasionally cause taste alterations and cause side effects such a dry cough, which affects roughly 5 -20% of europeans and 40% of asians. (rehmadanta sitepu, fx haryanto susanto 2021). due to its lower cost and lower number of adverse effects, herbal medicine is utilized as an option to treat hypertension (chaudhary et al., 2020). one of the naturally occurring herbal substances that can be utilized as an ace inhibitor is the quercetin found in the peel of onions (allium cepa l), which is beneficial as a natural antioxidant and antihypertensive. (amin et al.2018). according to earlier studies, giving obese prehypertensive patients 54 mg of quercetin three times a day for six weeks decreased their systolic and mean arterial blood pressure. (brühl et al. 2015). smokers' systolic and diastolic blood pressure decreased when given 100 mg of quercetin a day for 10 weeks (lee et al. 2011). onions are widely utilized as herbal medications since they contain elements that are incredibly useful to human health (sari, 2016). the highest quercetin level was found in onions (allium cepa l), particularly when compared to garlic, in a survey of 28 different types of vegetables and fruits by holman et al. (crystal et al. 2003; patil et al.1995). quercetin is the major component of onion skin, which is typically discarded (setiawan et al., 2021). the flavonoid flavonol 25—which is a member of the flavonoid family and has potential as an antihypertensive, antioxidant, antibacterial, anticancer, anticholesterol, and hypoglycemic agent—belongs to quercetin (sari, 2016). materials and methods ligand preparation the pubchem compound database (https://pubchem.ncbi.nlm.nih.gov/) was used to obtain the quercetin chemical structure in the form of a 3d chemical structure and smiles. the molecule's id number is cid:5280343, and its canonical smiles are c1=cc(=c(c=c1c2c3=c(c=c3o2)o)o)o)o)o. three-dimensional (3d) and the chemical structures of the ligands were drawn using avogadro and saved in pdb format. target selection the target protein was prepared for docking using the published literature and the protein data bank, namely www.swisstargetprediction.ch and http://prediction.charite.de, and then confirmed using uniport (https://www.uniprot.org). proteins were gathered and verified with pdb (protein data bank; http://www.rcsb.org/pdb), after which the protein was cleaned up for use by eliminating water molecules from the structure using pymol v2.5.2 software. angiotensin-converting enzyme, with the pdb code 1r42, was the target protein in this investigation. angiotensin ii is produced by the conversion of angiotensin i and induces http://www.swisstargetprediction.ch/ http://prediction.charite.de/ https://www.uniprot.org/ http://www.rcsb.org/pdb) international journal of applied biology, 6(2), 2022 174 the constriction of blood vessels and the release of vasoconstrictors, which raises blood pressure. molecular docking we carried out molecular docking tests with pyrx 0.8 program. the target protein angiotensin-converting enzyme (ace), the natural chemical quercetin, and lisinopril as a control compound are all reacted to during the docking process utilizing the vina wizard tool built into the pyrx 0.8 software (o. trott aj.olson, 2010). visualization of interactions between molecules and small molecules pymol software version 2.5.2 was used to visualize and evaluate the interaction of the control ligand (lisinopril) and the target protein's ligand (quercetin), angiotensinconverting enzyme. compound properties and prediction of admet to predict and significant characteristics of the compounds' physicochemical, lipophilicity, pharmacokinetic, and druglikeness features, admetsar (http://lmmd.ecust.edu.cn/admetsar2/) was utilized. results and discussion length-frequency analysis the pubchem website (https://pubchem.ncbi.nlm.nih.gov/) was used to retrieve the structures of herbal compounds, control compounds, and target proteins, which were then visualized in 3d using the pymol program (fig. 1). quercetin can interact with the target protein angiotensin-converting enzyme (ace), according to the res ults of docking using the pyrx program, suggesting that it can be utilized as an antihypertensive drug. the docking results showed that quercetin had a lower binding affinity than lisinopril, the control molecule, indicating that quercetin needed less energy to bind to the target protein. lisinopril needed more energy to bind to the target protein. (a) (b) figure 2. 3d structure of quercetin compound, (b) 3d structure of control compound lisinopril http://lmmd.ecust.edu.cn/admetsar2/) international journal of applied biology, 6(2), 2022 175 figure 2. results of docking quercetin (blue) and lisinopril (green) with protein angiotensin-converting enzyme (ace) table 1. results of docking between quercetin and lisinopril compounds with the target protein origin of compound ligand binding affinity (kcal/mol) allium cepa l quercetin -8.1 control lisinopril -7.1 it has been demonstrated that quercetin lowers both systolic and diastolic blood pressure in numerous antihypertensive studies. (edwards et al.2007). regarding antihypertensive drugs, quercetin works through a number of mechanisms, including reduced oxidative stress, compromised renin-angiotensin-aldosterone system (raas), and improved vascular function via endothelial cells. (larson et al.2012) the antihypertensive mechanism of quercetin as an ace inhibitor is by inhibiting the activity of angiotensin-converting enzyme (ace), thereby inhibiting the process of angiotensin ii formation. angiotensin ii in circulating blood plasma can cause blood vessel constriction and facilitate the occurrence of natriuretics (garciá-saura et al., 2005). as a result, the absence of angiotensin ii eliminates vasoconstriction and has a negative effect on blood pressure levels (putri, 2020). endothelial cells produce nitric oxide (no) as endothelium-derived relaxing factor (edrf) more frequently when quercetin increases the activity of nitric oxide synthase in those cells. nitric oxide causes blood vessel walls to enlarge and lowers blood pressure by relaxing the smooth muscles in blood vessels (ozarowski et al., 2018; perezvizcaino et al., 2009; putri, 2020). quercetin produces vasorelaxation in blood vessels in addition to the endothelium mechanism by acting directly on vascular smooth muscle and obviating the endothelium.larson et al.2010). according to earlier research on mouse coronary arteries, circulation levels of 10-7 moles/l quercetin will have a vasodilating impact because of elevated amounts of endogenous vasoactive molecules from the coronary artery wall. (lee et al.2011) quercetin also acts as an antioxidant, preventing ldl oxidation reactions by releasing hydrogen ions, which help to stabilize free radicals. this prevents blood coagulation, which international journal of applied biology, 6(2), 2022 176 can lead to fat deposition on blood vessel walls. as a result, atherosclerosis, one of the causes of hypertension, is avoided (putri, 2020). quercetin compounds are not potentially carcinogenic, according to toxicity studies performed using admet projections. it is not advised to extract the chemical directly due to its potential for toxicity. conclusion it is feasible to draw the conclusion that the chemical quercetin present in onion skin (allium cepa l) can serve as a natural antihypertensive based on the results of intermolecular interactions and affinity. international journal of applied biology, 6(2), 2022 177 references amin, m., putra, ks, amin, if, earlia, n., maulina, d., lukiati, b., & lestari, u. (2018). quercetin: the bioactive compound from allium cepa l. as anti -inflammation based on in silico screening. biology, medicine, & natural product chemistry, 7(1), 27–31. https://doi.org/10.14421/biomedich.2018.71.27-31 brüll, v., burak, c., stoffel-wagner, b., wolffram, s., nickenig, g., müller, c., langguth, p., alteheld, b., fimmers, r., naaf, s. ., zimmermann, bf, stehle, p., & egert, s. (2015). effects of a quercetin-rich onion skin extract on 24 h ambulatory blood pressure and endothelial function in overweight-to-obese patients with (pre-)hypertension: a randomized double-blinded placebo-controlled cross-over trial. british journal of nutrition, 114(8), 1263–1277. https://doi.org/10.1017/s0007114515002950 chaudhary, n., sabikhi, l., hussain, sa, & kumar mh, s. (2020). a comparative study of the antioxidant and ace inhibitory activities of selected herbal extracts. journal of herbal medicine, 22(november 2018), 100343. https://doi.org/10.1016/j.hermed.2020.100343 crystal, s., lombard, ka, peffley, eb, & liu, wx (2003). genetic analysis of quercetin in onion (allium cepa l.) 'lady raider. texas journal of agriculture and natural resources, 16(0), 24-28. edwards, rl, lyon, t., litwin, se, rabovsky, a., symons, jd, & jalili, t. (2007). quercetin reduces blood pressure in hypertensive subjects. journal of nutrition, 137(11), 2405– 2411. https://doi.org/10.1093/jn/137.11.2405 pharmacy, ps, nahdlatul, u., sunan, u., bojonegoro, g., & antihypertensive, o. (2019). garciá-saura, mf, galisteo, m., villar, ic, bermejo, a., zarzuelo, a., vargas, f., & duarte, j. (2005). effects of chronic quercetin treatment in experimental renovascular hypertension. molecular and cellular biochemistry, 270(1–2), 147–155. https://doi.org/10.1007/s11010005-4503-0 harfiantoko, mn, & kurnia, e. (2013). degree of hypertension (according to who) affects sleep quality and psychosocial stress. stikes journal, 6(2), 1–12. larson, aj, david symons, j., & jalili, t. (2012). therapeutic potential of quercetin to decrease blood pressure: review of efficacy and mechanisms. advances in nutrition, 3(1), 39–46. https://doi.org/10.3945/an.111.001271 larson, aj, symons, jd, & jalili, t. 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(2018). pharmacological effect of quercetin in hypertension and its potential application in pregnancy-induced hypertension: review of in vitro, in vivo, and clinical studies. evidence-based complementary and alternative medicine, 2018. https://doi.org/10.1155/2018/7421489 patil, bs, pike, lm, & yoo, ks (1995). variation in the quercetin content in different colored onions (allium cepa l.). journal of the american society for horticultural science, 120(6), 909–913. https://doi.org/10.21273/jashs.120.6.909 perez-vizcaino, f., duarte, j., jimenez, r., santos-buelga, c., & osuna, a. (2009). antihypertensive effects of the flavonoid quercetin. pharmacological reports, 61(1), 67– 75. https://doi.org/10.1016/s1734-1140(09)70008-8 putri, sd (2020). quercetin in parasite tea as hypertension therapy. medulla, 10, 307– 311. rahajeng, e., & tuminah, s. (2019). hypertension prevalence and its determinants in indonesia hypertension prevalence and its determinants in indonesia. center for biomedical and pharmacy research institute for health research, ministry of health, jakarta abstract:, 59(12), 581–590. rehmadanta sitepu fx haryanto susanto, es (2021). evaluation of the use of ace inhibitor lisinopril antihypertensive drugs on the occurrence of dry cough in hypertensive patients in the church community in malang regency. wijata journal, 8(2), 51–57. sari, an (2016). various spice plants as a source of natural antioxidants. elkawnie, 2(2), 203. https://doi.org/10.22373/ekw.v2i2.2695 setiawan, ayd, putri, ri, indayani, fd, widasih, nms, anastasia, n., setyaningsih, d., & riswanto, fdo (2021). chemical content and potential of shallots ( allium cepa l.) as an inhibitor of sars-cov-2. indonesian journal of chemometries and pharmaceutical analysis, 1(3), 143–155. contact : st zaenab nhenhabsitti@gmail.com 242 abstract diabetes mellitus (dm) is a chronic disease that occurs when the body experiences insulin resistance. one of the treatment for dm is by inhibiting the performance of the -glucosidase enzyme which is located on the smooth wall. indonesia is famous for having various types of herbal plants that are often used as traditional ingredients, one of which is cinnamon (cinnamomun burman ii) which has compounds such as catechins that are useful as antioxidants. this research method a docking simulation, the ligand was downloaded in the pubmed database and the androgen receptor macromolecule was downloaded in the protein data bank (pdb), the ligand structure preparation, the test ligand docking simulation and the comparison uses the pymol,pyrx program, and lipinski test, toxicity test using the preadmet program. the results of binding affinity for the test compound, namely catechin compounds, obtained a binding affinity of -8.0 kcal/mol, while the comparative test, metformin, obtained a binding affinity of 3.3 kcal/mol and lipinski qualifies as non-carcinogens but can be mutagens. these results indicate that catechin compounds have greater inhibition on alpha glucosidase receptors that cause type 2 diabetes mellitus compared to metformin compounds. issn : 2580-2410 eissn : 2580-2119 antitiabetic activity of catechin from cinnamon (cinnamomun burmanii) by inhibiting alfa glucosidase enzyme zohrah hasyim1, ismayniar2, natalia ratu2, emi eka putri2, st zaenab3* 1 post graduate school, hasanuddin university, makassar, indonesia 2 departemen of biologi, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia 3 study program of aquaculture, faculty of fisheries, cokroaminoto makassar university, makassar, indonesia introduction catechins are natural polyphenolic compounds flavan-3-ols (or flavanols), belonging to the flavonoid family. they are found in abundant concentrations in a variety of fruits, vegetables and plant-based beverages. the name catechin is derived from cutch tree (acacia catechu l.f). catechin chemically consists of two benzene rings (aand b-rings) and a dihydropyran heterocycle (the c-ring) with a hydroxyl group on carbon 3. there are two chiral centers on the molecule on carbons 2 and 3. catechin stereoisomers in cis ((-)-epicatechin) or trans ((+)-catechin) configuration, with respect to carbons 2 and 3, are flavan -3 ol compounds. through esterification with open access international journal of applied biology keyword diabetes mellitus; cinnamomun burmanni; α-glicosidase; catechin; docking article history received november 20, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. mailto:nhenhabsitti@gmail.com international journal of applied biology, 6(2), 2022 243 gallate groups, flavanols can form gallic acid conjugates epica techin gallate (ecg), epigallocatechin (egc), dan epigallocatechin gallate (egcg). condensed catechins are obtained via catechin polymerization. the most common oligomers derived from epicatechin are a-type and b-type procyanidins. in a type dimers, the monomers are linked by both a 4→8 carbon–carbon and a 2→o7 ether bond, and the monomers of the b-type dimers are linked through 4→8 carbon–carbon bonds (bernatoniene and kopustinskiene, 2018). the antioxidant efficacy of catechins is exerted through direct mechanisms scavenging ros, chelating metal ions; and indirect mechanisms inducing antioxidant enzymes, inhibiting pro-oxidant enzymes, and producing phase ii detoxification enzymes and antioxidant enzymes. the incidence of diabetes mellitus (dm) is increa sing every day in the world. based on a report from the international diabetes federation, there are 463 million people (20 -79 years) who currently suffer from diabetes and is expected to increase to 700 million in 2045. the proportion of people with type 2 diabetes is increasing in most countries and 79% of them live in poor and developing countries. as many as 374 million people are currently at risk for type-2 diabetes. dm is a chronic metabolic disorder caused by insulin deficiency or resistance. in type 1 dm there is damage to beta cells in the pancreas resulting in insulin deficiency and in type 2 dm there is resistance or decreased insulin secretion. there are several drug therapies used to treat type 2 dm, namely sulfonylureas, thiazolidinediones, gl p-1 receptor agonists, dpp-4 inhibitors, sglt2 inhibitors, glp-1 receptor agonists or basal insulin, drug selection is based on the specific effects of the drug or the patient's condition. glibenclamide., glibenclamide has an action to increase insulin sec retion, (mrabti et al., 2018). the glucokinase enzyme is known as hexokinase (hk) type iv from a member of the hexokinase family of enzymes. the function of the glucokinase enzyme is to catalyze reactions in glucose metabolism, namely the phosphorylation of glucose to glucose phospha te. anti-inflammatory, cancer and rheumatic analgesic. kaempferol itself is known as an antioxidant, anti-inflammatory, anticancer by inducing apoptosis and is known as a phytoestrogen. apart from gandarusa, kaempferol is also contained in ginkgo biloba, tilia spp., equisetum spp., moringa oleifera, sophora japonica and propolis ( nuraisyah, 2018). other glucose for type 2 diabetes mellitus metformin hydrochloride, a biguanide, is the most popular oral glucose-lowering drug in most countries, widely viewed as the 'baseline therapy' for individuals with newly diagnosed type 2 diabetes mellitus. this reputation results from its effective glucose lowering capabilities, low cost, weight neutrality, safety profile good overall (especially the lack of hypoglycemia as a side effect). the minimum effective dose of metformin is 500 mg daily and maximum efficacy is achievedat a dose of 2000 mg daily. while some pa]\tients may benefit from doses as high as 2500 mg daily, in the present study, overall, there were no major differences in fasting plasma glucose and hba1c when compared to the lower daily dose of 2000 mg. at 500 mg, metformin lowered fasting plasma glucose by an adjusted mean of 1.1 mmol/l and hba1c by 0.9% (9.8 mmol/mol; placebo reduction); at 2000 mg, the corresponding reductions were 4.3 mmol/l and 2.0% (21.9 mmol/mol; p ÿ 0.01) (sumaryada, 2014). international journal of applied biology, 6(2), 2022 244 materials and methods materials and tools the material used in this docking simulation is a two-dimensional structure of the reference ligand in the form of bichalutamide and catechin test ligands and their analogues which can be downloaded at the pubmed database (pmid:18775680) (http://www.ncbi.nlm.nih.gov/pubmed). while the selected macromolecules are androgen receptors with the code pdb 3b67, which can be downloaded at the protein data bank (pdb) (http://www.rscb.org/pdb/). the tools used are hardware and software. ligand structure preparation preparation of the ligand structure of the test compound and the ligand of the comparison compound is the first step that must be carried out. bicalutamide, ketamine compounds, and their analogues (pmid: 18775680) were prepared in a two-dimensional (2d) structure and then transformed into a three-dimensional (3d) structure with the marvinsketch 6.0 program package. test and comparison ligand docking simulations the test ligands were in the form of catechins and their analogues, and the reference ligands in the form of pubchem in pdb format were converted into *.pdbqt format throug h the https://www.rcsb.org/ . the docking method is carried out by tethering each ligand to a catechin compound. each ligand is in a flexible state which will interact with biomacromolecules in a rigid condition. phmol,pyrx used in the docking simulation of the test and comparison ligands for catechin compounds (arwansyah, et al., 2014). lipinski rule analysis and toxicity test lipinski's rule is calculated using the swissadmet web (http://www.swissadme.ch/index.php#). lipiski parameters include mlogp≤4,15, molecular weight ≤500da, hydrogen bond donor ≤5 and hydrogen bond acceptor≤ 10. absorption and distribution tests. and analysis of the toxicity properties of secondary metabolites of cinnamon plants using web preadmet (https://preadmet.webservice.bmdrc.org/toxicity). https://www.rcsb.org/ http://www.swissadme.ch/index.php https://preadmet.webservice.bmdrc.org/toxicity international journal of applied biology, 6(2), 2022 245 results and discussion results table 1. the active compounds of catechins and antidiabetic metformin compounds (drugs) active compound structure comparison ligand macromolecular identity catechin metformin c15h14o6 c4h11n5 table 2. molecular docking test results ligand name binding affinity(kcal/mol) the atoms play a role the role of atoms in ligands catechin -8.0 o=h acceptor h metformin -3,3 h=o donor h table 2 shows the molecular docking results of catechin compounds against α glucosidase (gaa), namely a binding affinity of -8.0kcal/mol, while the results of molecular docking of metforming compounds obtained a binding affinity of -3.3kcal/mol. these results indicate that the catechin contained in cinnamon (cinnamomum burmannii) exhibits interactions with the α-glucosidase (gaa) ligand site. table 3. results of drug-like properties analysis based on lipinski's rule compound lipinski's rule catechin massa molekul≤500da mlogp ≤4,15 akseptor ikatan hydrogen ≤10 donor ikatan hydrogen≤ 5 description 290.27 0,24 6 5 fullfill table 4 result farmakokinetika test (absopstion, distribution) and toxicity compound farmakokinetika toksisitas absorption distribusi bbb ppb catechin high no yes non carsinogenik. mutagen international journal of applied biology, 6(2), 2022 246 figure 1. interaction of catechins with the enzyme α-glucosidase (gaa) figure 1. above shows the active site where the protein enzyme α-glucosidase (gaa) and catechin compounds bind well where it can be seen that the atoms that play a role are o=h figure 2. interaction of metformin (drug) with the enzyme α-glucosidase (gaa) figure 2. above shows the active site where the protein enzyme α-glucosidase (gaa) and the compound metformin bind well where you can see the atom that plays a role, namely h=o. international journal of applied biology, 6(2), 2022 247 discussion one of the plants that can be used as herbal medicine is cinnamon ( cinamomun burmanii) (arrafi and amanatie, 2018). the use of cinnamon plants as a medicine for gout, high blood pressure, ulcers, hermia, asthma, canker sores, constipation, and diabetes mellitus (syarif et al., 2015) molecular docking is a computation to predict a relationship whether the compound has activity before being tested. this experiment with molecular docking was to see if the diabetes mellitus drug in circulation had activity in inhibiting the aldose reductase enzyme. molecular docking can be done with many paid and free software. this study uses pyrx software for docking and uses the pymol programs (karisma et al., 2016). based on the results of docking between the ligand and the receptor, it is obtained from the conformation of the ligand with the lowest energy. binding affinity is a measure of the ability of a drug to bind to a receptor. the lower the binding affinity value, the higher the affinity between the ligand receptors and vice versa, the greater the binding affinity value, the lower the affinity between receptors (karisma et al., 2016). in the results of the pymol and pyrx program (table 2), the results of the binding affinity for the test compound, namely the catechin compound, obtained a binding affinity of -8.0 kcal/mol, while in the comparator test, namely metformin, a binding affinity result of 3.3 kcal/mol. these results indicate that the catechin compound has greater inhibition to the alpha glucosidase receptor causing type 2 diabetes mellitus compared to the metformin compound. table 3. above catechins have a molecular mass of ≤ 290.27, mlogp ≤ 0.24, hydrogen bond acceptors ≤10, and hydrogen bond donors ≤ 5 where these results meet the requirements of the lipinski rule. table 4. catechin absorption exhibits high permeability in penetrating cell membranes. while the results of the distribution of ppb values indicate good absorption in the human body and a good level of absorption in the human intestine. the bbb value indicates the drug's ability not to penetrate the blood-brain barrier. meanwhile, the toxicity profile was examined through mutagenicity and carcinogenicity parameters using the preadmet web. preadmet is widely used to assess the mutagenic potential of a compound using bacteria. the results above indicate that catechin compounds are not carcinogenic but can act as mutagenic and non-carcinogenic. conclusion catechin has the lowest affinity energy, which is -8.0 kcal/mol compared to the metformin test ligand, which is -3.3 kcal/mol, and lipinski qualifies as non-carcinogens but can be mutagens. so that catechin is the most widely tested ligand potential as an antidiabetic drug compared to metformin. these results still need to be tested further using experimental tests. international journal of applied biology, 6(2), 2022 248 references ahmad naufal arrafi and amanatie. uji aktivitas antidiabetes infusa kulit batang kayu manis (cinnamomun burmanii) pada mencit putih jantan secara in vivo. jurnal kimia dasar. 2018;7(2). arwansyah, ambarsari l, sumaryada ti. 2014. simulasi docking senyawa kurkumin dan analognya. current biochemistry. volume 1 (1): 11-19 bernatoniene j, and kopustinskiene dm. the role of catechins in cellular responses to oxidative stress. molecules. 2018;23(4):1-11. doi:10.3390/molecules23040965 karisma enggar s, nurul f, erwinda k, dedy p, dan broto s. 2016. docking molekular potensi antidiabetes melitus tipe 2 turunan zerumbon sebagai inhibitor aldosa reduktase dengan autodock vina. chimica et natura acta. 4(1): 2016.16-20. mrabti hn, jaradat n, fichtali i, et al. separation, identification, and antidiabetic activity of catechin isolated from arbutus unedo l. plant roots. plants. 2018 ;7(2). doi:10.3390/plants7020031 nuraisyah f. faktor risiko diabetes mellitus tipe 2. j kebidanan dan keperawatan aisyiyah. 2018.13(2):120-127. doi:10.31101/jkk.395 sumaryada ti. simulasi docking senyawa kurkumin dan analognya sebagai inhibitor enzim 12-lipoksigenase. curr biochem. 2014. 1(1):11-19. syarif, p., suryotomo, b., & soeprapto, h. 2015. diskripsi dan manfaat tanaman obat di pedesaan sebagai upaya pemberdayaan apotik hidup (studi kasus di kecamatan wonokerto). pena jurnal ilmu, 13. retrieved from http://jurnal.unikal.ac.id/index.php/pena/arti cle/view/49 contact : desalegn amenu wadadesalegn@gmail.com 56 abstract the main objective of the study was to analysis the trends of malaria prevalence and surveillance data from 2015 to 2020 in sibu sire woreda, east wollega zone, and western ethiopia. a retrospective study was conducted on pur pose ly selected hospital and health center s of sibu sire woreda, east w ollega zone zones september 2015 to august 2020. fr om t he total 20,030 cases of malaria reported in sibu sire woreda from 2015 2020, 6,965 confirmed cases were reported, 2,234, 3,203 and 1,528 in chingi health center, sibu sire health center and sibu sire hospital, respectively. the high pre valence of malaria cases in the study are was reported both in autumn a nd spring seasons (april to september), and in relation to year distribution, more confirme d cases were reported during 2011 and 2012. the study demonstrated that malaria is a public health concern, in whic h p. falciparum and p. vivax are the pre dominant species, so, it needs more interventions to interrupt disease transmission and e ventual reduction malaria of malaria cases sibu sire woreda. issn : 2580-2410 eissn : 2580-2119 a retrospective analysis of malaria prevalence from 2015 to 2020 in sibu sire woreda, east wollega zone, and western ethiopia temesgen tafesse1, desalegn amenu2* 1 microbiol ogy and microbial biotechnology, assistant researcher, armauer hansen research institute, addis ababa ethiopian 2 microbiology (food microbiology), college of natural and computational science, biology department, wollega university, ethiopia introduction due to the i mproved coverage of malaria treatments and intervention measures have been expanded throughout the world, in ethiopia prevalence of malaria cases have been decreased in the past two decades (1). in contrast, but still large numbers of the population are suffering and now days large number of death has been reported in ethiopian (2). for instants, in 2016, about 2,927,266 new malaria cases and 4782 deaths were reported, respectively. furthermore, more than 30% of the disabilities cases were reported and still the malaria cases results a huge social and economic development (3). generally, malaria cases is more critical issue for the rural communi ties of the country than urban area since the malaria prevention and control strategy is highly depends on the availability of some infrastructure. t his idea is supported by the research conducted open access international journal of applied biology keyword ethiopia; sibu sire woreda; malaria prevalence; retrospective study; article history received march 17, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 57 temesgen et al.,2019, the authors reported that some economic issues such as having good and well sanitized housing and drainage system and well appropriate expanded personal protection can effective the diagnostic and treatment of malaria cases (17). so, many subsaharan countries including ethiopia are more affected by malaria cases than any other parts of the world since they are characterized by poor housing, lack of sanitation and drainage of surface water that would provide favorable conditions for malaria vector breeding (5). as the research conducted desalegn dabaro (2020), reported that more than 60% of the population has been suffered due to malaria cases. the same authors indicated that the prevalence of malaria rich the highest peak in 2018 with the mouths of transmission from september to december, following the main rainy seasons (june to september). in spite of the widesprea d incidence of malaria in several districts of the region, the general trend of malaria prevalence has not been thoroughly studied in s ibu sire woreda particularly in this respective year. therefore, this study aimed to assess the trends of prevalence and surveillance data of the consecutive six years from 20122017. methods study area and period the study was conducted in sibu sire district, east wollega zone of oromia regional state, based on the clinical data report from 2007 to august 2012. it is one of the districts in east wollega zone and is located 281km in west from addis ababa and 50km east from nekemte, the administration town of east wollega zone. sibu sire has 22 kebeles from these 19 kebeles are rural and 3 municipals. the study area has a total population of 124,304 and from these urban dwellers male 6744 and female 6954, rural dwellers male 54,920 female 55,686 (8). study design and population a retrospective study was conducted to determine the prevalence of malaria over six years (from september 2015 – august 2020) in east wollega zone, sibu sire district. in this study the malaria prevalence and surveillance data from district health services (sibu sire hospital, chingi health center, and sibu sire health center) was employed to determine the six years analysis of malaria prevalence by reviewing blood film malaria record from sibu sire hospital and health center and also chingi health center. t he study participants were all individuals diagnosed for malaria using giemsa stained preparation and the data registered on laboratory registration book during the study peri od (from september 2015 to august 2020). while data collections information such as date of examination, numbers of treated and confirmed cases of malaria in months and years, respectively, malaria species, and patients data (age and sex) were collected. data collection a 6 years (2015–2020) retrospective data on malaria prevalence was collected from september 2015 to august 2020 at sibu sire hospital, chingi health center, and sibu sire health center. in this health centers, peripheral smear examination of blood film has been used as the gold standard in confirming the presence of malaria infection. international journal of applied biology, 6(2), 2022 58 data quality control in this study, the six years data concerning malaria prevalence was taken from malaria registration book in sibu sire health centers. to collect the data in good manner, the data collection format sheet was prepared and has been used for recording the data. all data collectors were adequately trained about the data collection procedures. overall data process and analysis was conducted by principal investigators. always the researchers checked the number of confirmed cases with the number of suspected cases throughout the reviewed data. data analysis data analysis was conducted using microsoft excel data sheets, and analyzed using spss 21 software package. distribution and prevalence of malaria species in terms of sex, age was analysis using figure, tables and charts. furthermore, the trend of malari a case in terms of age, sex, years and months was determined. results and discussion trend of malaria cases in sibu sire woreda from 2015 to 2020 during the study duration conducted from 2015 -2020, a total of 20,030 patients were diagnosed for malaria cases and examined. out of these, 6,965(69.54%) were reported as a positive cases. the prevalence of malaria was fluctuating during the six years of study with minimum (n =240) and maximum (n = 690) number of annual cases were reported (table 1). the highest numbers of malaria-suspected patients were examined in 2019 and, 2020, respectively. table 1. distribution of malaria cases in relation to sex and age groups; sibu sire districts (sibu sire health center, chingi health center, and sibu sire hospital), from 2015 to 2020. sex age groups positive (%) negative (%) positive (%) negative (%) positive (%) negative (%) mal e <5 198(23.00) 694(78.00) 394(27.00) 1086(74.00) 173(34.60) 327(65.40) 5 to 15 242(34.22) 465(66.00) 560(31.00) 1295(70.00) 243(32.00) 517(68.02) >15 704(41.25) 1003(58.80) 800(40.00) 1700(60.00) 420(38.60) 667(62.00) femal e <5 190(24.00) 610(76.20) 301)27.00) 815(73.02) 100(21.00) 373(79.00) 5 to 15 200(28.60) 500(71.42) 447(44.00) 583(56.60) 203(31.40) 444(69.00) >15 700(46.20) 817(54.00) 701(37.00) 1204(63.20) 100(26.00) 289(74.20) total <5 388(23.00) 1304(77.01) 695(27.00) 1901(73.00) 273(28.00) 700(72.00) 5 to 15 442(31.42) 965(68.60) 1007(35.00) 1878(65.00) 446(35.40) 814(64.60) >15 1404(44.00) 1820(56.45) 1501(39.00) 2405(61.60) 809(38.80) 1278(62.00) international journal of applied biology, 6(2), 2022 59 prevalence of malaria cases among sex and age in sibu sire health centers from 2015 to 2020. regarding the prevalence of malaria among sex, there was no significance difference distribution, but in terms of age, the high distribution of malaria was observed in age category greater than 15 years old. age groups ≥15 years were more affected, with a prevalence rate of 3,714 (18.54%), followed by 5-15 years old, and under 5 yea rs’ children with prevalence rates of 1,356 (7%), respectively (table 1). prevalence of malaria in terms of seasonal variation months of two health centers and sibu sire hospital (2015-2020). according to this finding the seasonal distribution of malaria cases is presented in fig. 4. hence, although the prevalence of malaria has been occurred in all seasons, but the prevalence of malaria cases was fluctuating across the four seasons over the l ast 6 years (2015-2020). the highest and the lowest cases of malaria were observed during spring (september, november) (38.19%) and winter (december – february) (10.00%), respectively. higher number of cases of p. falciparum was pragmatic in spring and summer, while more cases of p. vivax were observed in spring, followed by winter. however, the minimum number of p. f alciparum and p. vivax cases were observed during winter (december – february). figure 1. prevalence of malaria in terms of seasonal variation months of two health centers and sibu sire hospital (2015-2020) prevalence of malaria species the prevalence of confirmed malaria cases was higher in sire health center (28%) followed by chingi health center (22%). the highest prevalence of p. falciparum also was reported sire health center. similarly, higher rate of p. vivax infection was recorded si re health center including the mixed infection of both malaria species. 0 500 1000 1500 2000 2500 3000 dec. jan. feb. mar. apr. may jun. jul. aug. sep. oct. w in te r a u n tu m su m m e r sp ri n g confirmed cases total cases international journal of applied biology, 6(2), 2022 60 figure 2. prevalence of malaria species in sibu sire woreda health centers from 2015-2020. table 2. distribution of malaria species among age categorize in sibu sire district health facilities from 2015 to 2020, east wollega zone, ethiopia. according to the this findings, concerning to the trends analysis of malaria species across the age groups revealed that, in all age and sex groups, the most predominate malaria species was p. falciparum followed by p. vivax. a sharp increment of cases in these two species was observed in the age groups 5-15 through >15 yea rs. an increment in mixed infections was also seen in the age groups 5-15 years through >15 (table 2). annual trends analysis of malaria cases in sibu sire districts health facilities, 2015 to 2020 regarding to the trends analysis of malaria in sibu sire woreda from 2015 to 2020, fluctuating trends analysis was occurred and there was not definition of the prevalence as the overall the six years data showed that. it was observed to the highest prevalence rate of malaria were observed in 2017, 2019 and 2020, respectively. the highest peak was observed in 2019 and 2020, with the highest percentage of malaria cases (figure 3). 0 500 1000 1500 2000 2500 p re v a le n ce o f m a lr a ia malaria spcies chingi health center sibu sire health center sibu sire hospital age malaria species positive negative positive negative positive negative <5 p.falciparium 203(12.00) 1489(88.00) 524(20.18) 2072(80.00) 268(27.54) 705(72.46) p.vivax 120(7.09) 1572(92.91) 318(12.25) 2278(88.00) 56(5.76) 917(94.24) mi xed 0.00 1692(100.00) 35(1.35) 2561(98.65) 12(1.23) 961(98.77) 5 to 15 p.falciparium 800(56.86) 607(43.14) 582(20.17) 2303(80.00) 353(28.03) 907(72.00) p.vivax 310(22.03) 1097(77.97) 325(11.27) 2560(88.74) 74(2.87) 1186((64.00) mi xed 64(4.55) 1343(95.45) 37(1.28) 2848(98.72) 16(1.27) 1244(99.00) >15 p.falciparium 540(16.75) 2683(83.25) 776(19.87) 3130(80.13) 585(28.03) 1502(72.00) p.vivax 190(5.90) 3033(94.10) 466(11.93) 3440(88.07) 122(5.85) 1965(94.00) mi xed 17(0.53) 3206(99.47) 52(1.33) 3854(98.67) 26(1.25) 2061(99.00) international journal of applied biology, 6(2), 2022 61 figure 3. annual trend of malaria prevalence at sibu sire district, east wollega zone (20015–2020) discussion the present study was conducted to evaluate the trends of malaria prevalence in sibu sire woreda health centers from 2015 -2020 f or consecutive six years. the overall six years malaria trends analysis of the three health centers of sibu sire woreda indicates that the incidence of the total confirmed cases of the malaria were still increased from 20152020. the overall prevalence of malaria in this study was 69.54% where a total of 6,966 confirmed malaria cases were detected in six years from 2015-2020 years retrospective study, important indicator for existence of malaria burden in sibu sire woreda health center. the result of the present study is similar with the retrospective study conducted in benishangul gumuz regional which reported tha t about 57.5% of the malaria cases were reported (9). in this study in compared to other study as the trends of malaria revealed that, much higher prevalence of the malaria cases was reported and hence, this retrospective study is higher than the study conducted five-year trend study conducted in ataye, north shoa, ethiopia; reported a total prevalence of 8.4%, the seven-year retrospective malaria report from metema hospital, northwest ethiopia; detected a prevalence of 17%, the fifteen-yea r study in ethiopia; and noted a prevalence of 12.5% (10-11). the differences might be due to time variations of the studies, difference in insecticide application in the areas, variations in geographical locations, and differences in population awareness about malaria control methods application and utilizations, its transmission. regarding to the annual prevalence of the malaria trends in this study, the highest prevalence was observed in 2019 followed by 2020. in the current study, the prevalence of malaria was high in august and the least in march over the six years of twelve months. seasonal fluctuations and high prevalence of malaria were observed in spring (september to december) and the least prevalence in autumn ( march to may). this finding is more similar with the study conducted by dabaro et al., the authors revealed that, high prevalence of malaria was reported from september to december and this issue may be related to some environmental conditions such as presence 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2015 2016 2017 2018 2019 2020 p re v a ln ce o f m a la ri a c a se s years ssht sshc chingi international journal of applied biology, 6(2), 2022 62 of stagnant water, favorable temperature, and high vegetation density for mosquito breeding. on the other hand, the least prevalence was observed in spring (march to may); this might be due to drought (18). the study also showed that the prevalence of malaria was high among sex was not shown significance variation, but on other hands the heights prevalence of malaria was observed a mong age category greater than 15 yea rs old over the six years trend. the present finding was much more similar with the study conducted in addi arkay health center and abeshge (12-13). the youngest age the so called productive age groups were most of the ti me more affected than the others, because they are more engaged in filed and as the result they are more exposed. in contrast, less than five years age categories were less exposed, because of their less likely exposure to infected mosquito bite, this may be due their parents care takers on malaria control and prevention activities. in the study area, the number of malaria cases peaked in spring, followed by autumn. these two seasons are the major transmission periods in ethiopia, respectively. in generally speaking as we cited and discussed repeatedly, the main transmission session of malaria cases were heavily seeks for the heavy rain seasons which is from june to september. because, it is this period which creates suitable conditions for the breading of anopheles mosquitoes. in many cases the malaria transmission is usually associated with rainy seasons; in the present study malaria cases were also significant in winter and au tumn, indicating that climatic and environmental factors other than rainfall can also determine the occurrence of malaria (19, 20). concerning to the prevalence of the malaria species of the current study sites, the two species (plasmodium falciparum, p. vivax) were the dominant malaria species identified in the study area. this was in agreement with the nati onal profile of plasmodium species (14). according to the present findings declared that, the prevalence malaria cases (plasmodium species) varied from year to year. there has been an increase in p. falciparum cases in recent years, particularly in 2018 and 2019, and a minor drop in p. vivax cases. due to its clinical difficulties and drug -resistant danger, stakeholders have focused their efforts on combati ng p. falciparum. other plasmodium species, on the other hand, have received less attention. as a result, drug resistance to chloroquine in p. vivax may be developing. conclusion in conclusion, the study found that malaria continues to be a public he alth concern in an area with a high proportion of slide positivity. this would be a strong indication that the area requires additional attention and c oordinated malaria efforts. despite the fact that modern 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"analysis of the trend of malaria prevalence in ataye, north shoa, ethiopia between 2013 and 2017." malaria journal 17.1 (2018): 1-6. contact : mildayati milda.valyt@gmail.com 9 abstract population growth and changes in people's consumption patterns lead to an increase in the volume, types and characteristics of increasingly diverse waste. this increase in the amount of waste that is not followed by the repairment and improvement of waste management facilities and infrastructure has resulted in a complex waste problem. therefore, one of the efforts that can be applied to overcome the waste problem is by taking a source approach. in this approach, waste will be handled (managed) at the upstream (source) before it reaches the landfill (downstream). ways that can be done include sorting the waste and also the 3r (reduce, reuse and recycle) program. this study aims to determine the effectiveness of waste management at the hamlet level in mamasa village, mamasa regency. this study uses a descriptive qualitative approach with data reduction analysis. the results showed that the effectiveness of waste management was 37.5% and the effectiveness of waste reduction was 42%. the study concluded that the effectiveness of waste management at the hamlet level in mamasa village was not very effective. issn : 2580-2410 eissn : 2580-2119 effectiveness of waste management at the hamlet level in mamasa village mamasa regency mildayati 1*, amran achmad 2, m. rijal idrus 3 1environmental management, postgraduate faculty, hasanuddin university 2 faculty of forestry, hasanuddin university 3 faculty of marine and fisheries sciences, hasanuddin university introduction population growth and changes in people's consumption patterns lead to an increase in the volume, types and characteristics of increasingly diverse waste. this increase in the amount of waste that is not followed by the repairment and improvement of waste management facilities and infrastructure has resulted in complex waste problems. the waste problem never ends and is a serious problem, especially in big cities in indonesia. if waste enters the environment (water, air and soil) then the quality of the environment will decrease. this process of waste entry into the environment is known as an environmental pollution phenomenon. the waste problem is a challenge that will determine the environmental sustainability of a city. failure to deal with this waste problem will increase the risk of the city residents dealing with various kinds of diseases that will increase the social costs of health. up to this point, some people still adhere to the old paradigm in waste management, namely waste is collected, transported and then disposed of in the landfill. therefore, one of the efforts that can be applied to overcome the waste problem is by taking a source open access international journal of applied biology keyword effectiveness, management, waste. article history received 12 september 2021 accepted 30 desember 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 10 approach. in this approach, waste will be handled (managed) at the upstream (source) before it reaches the landfill (downstream). ways that can be done include sorting the waste and also the 3r (reduce, reuse and recycle) program. in mamasa regency itself, landfill facilities have been provided by the regency government and have been effectively used, including transport personnel, infrastructure for collecting and transporting waste. however, there are irresponsible parties who still littering in random places such as rivers. this kind of community culture is difficult to change because of a hereditary habit and thus waste ends up in the sea instead of in the landfill. in addition, the waste that is dumped into rivers and sewers has the potential to cause flooding, where the community itself suffers the most. mamasa village is also the capital of mamasa regency which should be arranged in such a way as to be an example for other villages and sub-districts in mamasa regency in terms of waste management. therefore, mamasa village has a high enough urgency to reduce and manage waste so that it can set an example to reduce waste problems in mamasa village in particular and mamasa regency in general. this study aims to determine the effectiveness of waste management at the hamlet level in mamasa village, mamasa regency. materials and methods this study uses a qualitative approach with a descriptive method. the technique of collecting data from respondents/informants used in this research is purposive sampling technique. sources of the data are obtained from primary and secondary sources. primary data primary data is data that is directly collected by researchers from the original source. the source of primary data in this study was obtained directly from the place where the research was carried out, namely in the form of direct interviews with employees of the housing, settlement and land office of mamasa regency, mamasa district environmental and forestry service officer, head of mamasa village, garbage collectors, mamasa village community as many as 4 (four) respondents per hamlet or as many as 24 respondents. secondary data secondary data is data that is not directly from the community, but from reading sources related to the problems in this research as well as other supporting data related to research from related agencies. in this study, the data collection techniques that the author uses consist of 3 (three) methods, namely: observation, interviews and documentation . data analysis technique the data analysis technique used is descriptive qualitative, consisting of three flow of activities that occur simultaneously, namely: data reduction, data presentation, and conclusions drawing or verification. (miles, mb., 2001). in this study, data analysis was carried out using these following techniques: a. data reduction the data obtained through observation and documentation were collected, selected, and grouped and then concluded without eliminating the existing data. reducing data means summarizing, choosing the main things that are important, looking for themes and patterns. collecting data that are obtained from observations, interviews and documentation. international journal of applied biology, 5(2), 2021 11 b. data presentation the presentation of data in qualitative research used by researchers is by making narrative texts. the presentation of this data is carried out in the form of a brief description, compiling all the data obtained in accordance with the planned discussion. c. conclusion and data verification conclusions making and data verification is done by means of data that has been arranged and patterned systematically and then concluded by researchers so that meaningful conclusions are obtained from the data that has been obtained. verify the data that has been collected to be used as the basis for drawing conclusions. effectiveness analysis the analytical tool used to measure the effectiveness of waste management is a formulation that emphasizes the compatibility between the objectives and results of program implementation. subagyo (2000) explains that the level of effectiveness can be calculated using the following effectiveness formula: effectiveness = 𝑅 𝑇 × 100 % explanation: r = realization t = target validity test/data validity test to avoid mistakes or errors in the data that has been collected, the researcher needs to check the validity of the data by means of triangulation techniques, namely the technique of checking the validity of the data based on something outside the data for the purpose of checking or as a comparison against existing data. triangulation in testing the validity of this data is defined as checking data from various sources in various ways, and at various times. with triangulation, researchers will mention sources, documents, and techniques (widiyanto et al, 2017). results and discussion mamasa district profile astronomically, mamasa regency is located between 2°39'216'' south latitude and 3°19'288''south latitude and between 119°0'216'' east longitude and 119°51'17'' east longitude. mamasa regency is 1 of 6 regencies in west sulawesi province, with an altitude of 3000 meters above sea level (masl). the area of mamasa regency is 3,005.88 km2. according to data from mamasa district in figures 2020, in 2019 the population of mamasa district was 27,292 people (sp2010). in 2019, the area of mamasa village is 7.11 km2 which consists of 6 (six) hamletsthat are villages with the largest population, which is 5,272 people. respondent profile based on the results of the questionnaire processing, the profile of the respondents who became the sample in this study was obtained. based on the age, of 24 respondents the age below or equal to 50 years (≤ 50) consist of 15 people or 62.5%. this number is international journal of applied biology, 5(2), 2021 12 relatively larger than the age above 50 years (> 50) which amounted to 9 people or 37.5%. based on the gender of the respondents, there are two gender namely male and female. after the questionnaires were distributed to 24 respondents, the results showed that the respondents were dominated by 14 women or 58.33% and just 10 men or 41.67%. based on the education level of the respondents, most respondents have high school education and college education of 10 people each or 41.67%, followed by respondents with elementary school education as many as 3 people or 12.5% and junior high school as many as 1 person or 4.17%. thus, most of the respondents who were sampled in this study had high school and college education (bachelor). according to the respondent's profile based on occupation, it shows that respondents who work as civil servants/retirees dominate as many as 11 people or 45.83%, entrepreneurs as many as 9 people or 37.3%, followed by respondents who work as housewives as many as 2 people or 8,335% and farmers and general elections commission of indonesia each 1 person or 4.17% each. based on the number of family members, the results showed that respondents who were below or equal to 5 people (≤ 5) were more dominant, namely 14 people or 58.33% and respondents with the number of family members above 5 people (> 5) were 10 people or 41.67%. for more details, see table 1 below. table 1. respondent profile personal background frequency percentage (%) age (year) ≤ 50 15 62,5 > 50 9 37,5 total 24 100 gender male 10 41,67 female 14 58,33 total 24 100 pendidikan elementary 3 12,5 junior high 1 4,17 high 10 41,67 university 10 41,67 total 24 100 occupation entrepreneur 9 37,5 civil servants/retiree 11 45,83 international journal of applied biology, 5(2), 2021 13 farmer 1 4,17 housewives 2 8,33 general elections commission 1 4,17 total 24 100 number of family members (persons) ≤ 5 14 58,33 > 5 10 41,67 total 24 100 source: questionnaire results processed by the author. waste management in mamasa village, mamasa regency waste management aims to improve public health and environmental quality and turn waste into a resource. according to damanhuri and padmi (2015), the success of management does not only depend on technical aspects, but also includes non-technical aspects. a. legal and regulatory aspects the legal basis for waste management that has been issued by the mamasa regency government is 1) mamasa regent regulation no. 12a of 2007 dated april 2, 2007 concerning guidelines for implementing minimum standards in the waste sector. 2) mamasa regent regulation number 06 of 2019 concerning regional policies and strategies in the management of household waste and waste similar to household waste. this regent regulation has not been socialized until the end of 2020, so the implementation of the regulation does not run optimally. according to the head of waste management, b3 waste and capacity building for the environment and forestry agency of mamasa regency, this is due to the diverted funds for handling covid-19. even if it is budgeted, the funds are very minimal. the head of mamasa village hopes that this regulation will soon be socialized so that a waste management mechanism can be formed and can involve the village party. according to the results of interviews conducted by researchers at the hamlet level in mamasa village, there are only a portion of the community, namely around 21% of the total respondents (5 respondents out of a total of 24 respondents) who know that there is a regional regulation on waste management, and even then, they do not specifically know about the waste management regulation that is issued. in this case, one of the respondents understands the waste management regulation, which is about sanctions for irregular waste disposal, in this case the rules/sanctions for throwing garbage anywhere, in accordance with the mandate of law number 18 of 2008 concerning waste management. according to the mamasa village head, the existence of sanctions that are not yet strict on waste management has caused some people to still litter, such as the habit of throwing garbage into rivers. the recommendation to dispose of garbage in its place is only an appeal without clear rules/sanctions for people who violate it. this can be seen from the garbage that is still found piling up and even carried away by river flows which can potentially cause flooding and damage to aquatic ecosystems. rivers that are often polluted international journal of applied biology, 5(2), 2021 14 by garbage from the community are the mamasa river and the tatoa river that pass through mamasa village. laws and regulations are based on the fact that indonesia is a country of law, where the joints of life rely on applicable laws. municipal solid waste management in indonesia requires strength and a legal basis, such as the formation of organizations, collection of retribution, public order, the responsibilities of each agency, individual and so on (anas, 2017). to achieve this, the central government, provincial and regional/district governments must establish policies and strategies for waste management in accordance with the hierarchy of authority, and for now the provincial government of west sulawesi and the regional government of mamasa regency have not issued a local regulation on waste management. b. financing aspect regarding the levy on waste management in mamasa regency, regional regulation number 02 of 2007 that was issued on august 14, 2007 concerning the levy for cleaning services. the collection of user fees for waste services is carried out by the collector of the cleaning section staff in the waste section according to the results of an interview with mr. kombas as the driver of the garbage transporter. the results of this waste retribution are managed directly by the housing office according to the results of an interview with the mamasa village head. the levies collected vary based on the categories contained in the above regulations, namely large, medium and small categories of waste generated by households, restaurants, hotels, markets and offices. in accordance with the statement as the head of the cleanliness section of mamasa regency. from the results of interviews conducted by researchers at the hamlet level in mamasa village, there were 38% of respondents (9 respondents) who admitted that they routinely paid the retribution every month according to the categories stipulated in regional regulation no. 02 of 2007, there were 53% of respondents (13 respondents) who admitted they never paid a levy and 8% of respondents (2 respondents) who claimed to have paid a levy but never again received a bill from a cleaning collector. residents who routinely receive bills are generally residents who are on the main road that is traversed by a transport fleet whose waste is picked up directly from the household. however, some of the respondents who never received a bill came from hamlet 01 to hamlet 04, while the majority came from the hamlet 05 and hamlet 06 areas admitted that they had never received a retribution bill. in these two hamlets, there is 1 laystall which becomes a dumping ground for the people around the hamlet. according to residents, they are ready to pay every month, with the hope that garbage in people's homes and at laystall is routinely collected by garbage officers. they also hope that if the garbage is not picked up at the household, the capacity of the laystall should be enlarged for areas that already have laystall. if waste management is carried out at the source (decentralization) then the financing will be fulfilled from the residents' waste retribution. this means that the polluter fine principle (polluters must pay) has been fulfilled so as to support sustainable management from the economic aspect. however, if waste management is carried out conventionally (centralized) then a special subsidy is needed from the local government. the cost of waste management at the source is cheaper and more effective than the costs that must be incurred for conventional waste management which is purely based on landfill. the cost becomes cheaper because the waste that must be transported to the landfill is international journal of applied biology, 5(2), 2021 15 reduced so that the costs for transportation and disposal are reduced. the cost becomes more effective because one rupiah allocated for waste management at the source will have a greater impact than one rupiah allocated for conventional waste management. even the waste management brings economic added value that can be enjoyed by the actors who are active in it (utami, b., d., et.al, 2008). the general problem that is often encountered in financing is that the collected retribution is very limited and not commensurate with the operational costs, the existing authority and organizational structure are not entitled to manage their own funds and the levy tariff arrangement is not based on the correct method. retribution from the field of cleanliness is one source of original local government revenue in a region. taking into account the number of customers around 1,016 in mamasa regency, this indicates that the number is still relatively small. the relatively small amount of retribution results in fulfilling the need for waste management is still a big burden for the government. the amount of the waste retribution is 1% of the income per household. thus, the amount of retribution for waste varies according to the level of income, the higher the income of a household, the greater the retribution they have to pay because the higher the economic level of a person, the greater the waste produced (anas, 2017). however, for the mamasa regency scale, the regulation on financing, in this case retribution, refers to regional regulation no. 02 of 2007 concerning cleaning service retribution, namely the levy collected varies based on large, medium and small categories of waste generated by customers. however, for the household scale, the tariff is the same, namely rp. 2000, per month, regardless of their income level. basically the government needs to make policy breakthroughs, especially in this financing, such as making regulations at the regional level where local governments can get a special portion of financing or through private and other assistance by empowering and fostering as well as providing operational funding assistance to 3r laystall and waste banks. in accordance with the results of interviews in this study, in general, people admit that waste management is carried out by the government without cooperation with the private sector. c. operational aspect garbage is basically generated by or is a consequence of human activities. every human activity always produces waste or waste whose amount and volume are proportional to our level of consumption of goods and materials that we use daily. likewise with waste, it really depends on the lifestyle and the type of material we consume. based on the sni 19-2454-2002 standard, waste receptacle is an activity to temporarily accommodate waste in an individual or communal container at the source of the waste. this storage is carried out on waste that has been sorted, namely organic, inorganic and hazardous toxic waste. the pattern of accommodation consists of individual patterns and communal patterns. from the interviews, it was found that in general, the community prepared their own containers in the form of buckets, plastic and sacks. garbage is put together in containers that will be transported by officers without distinguishing between organic, inorganic and b3 waste. the requirements for the container material are durable and waterproof, easy to repair, light and easy to lift and economical, easy to obtain or make by the community. communal waste containers are procured by the management agency while individual containers are provided by individuals or management agencies (syafrudin and priyambada, international journal of applied biology, 5(2), 2021 16 2001). the purpose of the container is to make it easier to transport and in addition to using this container, the odor due to decaying garbage which can also attract the attention of flies can be overcome, rainwater which has the potential to increase the water content of the waste can be controlled and mixing of dissimilar waste can be avoided (rahardyan et al. widagdo, 2005). until now, the mamasa regency government has not been able to handle the solid waste problem thoroughly to the villages and even the garbage around the mamasa regency capital has not been fully handled properly. one of the factors that causes this to happen is the limited infrastructure owned by the waste section which has impacted in several routes and areas that are not served. waste handling is only in the capital area of mamasa district with a service area of around 1016 objects. the service areas that have been served by the mamasa regency waste management system currently include residential areas, offices, markets/shops, inner-city roads, religious facilities, tourist facilities, schools and others with large, medium and small categories. this service is carried out mainly in urban areas/district capitals, and even then it is only limited to collection and transportation to laystall and disposal to landfill without any management from upstream, namely segregation from households or management at integrated laystall and 3r laystall. in some areas, especially slum and congested areas, waste collection services are not carried out properly, due to the lack of a garbage collection fleet, the distance from the house which is the source of the waste is far from the collection location, as well as the difficulty of accessing roads outside the city of mamasa and the lack of public awareness of cleanliness. some of the waste generated in the environment is burned and most of it is disposed of in ditches/rivers, which can cause channel blockages and potentially cause flooding and inundation. facilities in the form of a garbage collection fleet consisting of 2 (two) trucks and 2 (two) motorcycles with poor vehicle conditions. previously there were 5 (five) motorbikes and 2 (two) trucks that could reach the city's garbage transportation, but 3 (three) motorbikes were no longer usable (totally damaged), causing a lot of complaints from the community due to the garbage cannot be handled optimally. trash cans that are used to collect garbage from people's homes are also still being prepared privately because there is no procurement from the government/private sector. the community's willingness to prepare trash containers/bins is a form of community participation in waste management (yudianto, et al, 2021). trash cans should conform to standards consisting of 2 or 3 types, namely: organic waste, inorganic waste and b3 waste. but in reality it has not been implemented in mamasa village. the schedule for transporting waste is 1 trip per day. motorcycles carry out door to door transportation, while trucks carry garbage from laystall and also door to door at residents' homes which are carried out according to the distribution area zone starting at 06.00 until finished. in residential areas that can be reached by garbage collectors managed by the department of housing, settlement and land affairs of mamasa regency, the community collects waste in front of their respective homes and will be transported by officers, some of them take the waste to the nearest laystall which will then be transported by the transport fleet to the salubue landfill. garbage in laystall has not been handled, several 3r laystall are already available but have not been able to reduce waste generation before being disposed of to the landfill because they are not operating properly. the location of the landfill in mamasa regency is international journal of applied biology, 5(2), 2021 17 located in salubue, rantepuang padang village, mamasa district, with a landfill area of 2,480 m2, the distance from the landfill to the nearest settlers is 200 m and the distance from the landfill to the nearest river is 700 m. d. institutional aspects (institutions) waste management cannot be separated from the role of institutions. waste management basically involves stakeholders, namely the waste-producing community, ngos, the private sector, local governments and the central government. each of these stakeholders has a role in waste management (puspasari and mussadun, 2016). so far, mamasa village office has not played a significant role in waste management. according to the mamasa village head, the village office is only a source of information from the government to the community or vice versa regarding waste management. furthermore, he hopes that there will be a synergy between the regency government and the village office in terms of waste management. when the party in charge of cleaning will plan or hold activities related to waste management in the mamasa village area, it should involve the village office. the government's role in waste management is very important, because good waste management is a manifestation of the form of urban infrastructure services to the community. the crucial problem of waste management in mamasa village is the less than optimal performance of the institutions that play a role in waste management. the village office still does not have a strategic role in waste management even though this institution has a very close position and is in direct contact with the community. so far, the sub-district only continues the appeal from the office that handles solid waste. the waste and cleanliness section which used to be in the department of housing, settlement and land affairs, mamasa regency, but since january 2021 has joined with environment and forestry agency of mamasa regency is considered the most responsible institution in waste management in mamasa regency. environment and forestry agency is tasked with carrying out the transportation and processing functions at the end, but the number of fleets and personnel owned is very limited. in addition, the volume of waste produced by the community continues to increase, even people have not done any sorting, and the waste is still disposed of in the conventional way (by burning, stockpiling, and throwing it anywhere). so that the burden of environment and forestry agency’s task becomes quite heavy. it is the hope of the community that environment and forestry agency should involve the village office regularly to socialize about waste management so that a change in the mindset of the community in managing waste is formed through socialization (3r) and pioneering the procurement of waste banks. the success of waste management cannot only rely on the role of the government, but community involvement in waste management can be the key to success in waste management. the community, as a source of household waste, must help the government in waste management. the community can play a role by not littering, not throwing garbage in the river, being able to sort organic and inorganic waste when disposing of garbage. the role of the private sector is very much needed in waste management. the presence of the private sector in waste management can help ease the burden on the government. the role of the private sector in waste management can have a positive impact on municipal waste management, because at least the existence of the private sector is able to answer the problems faced by the government so as to create ideal waste management conditions. although in reality the private sector has not played a role in waste management in mamasa village at this time. international journal of applied biology, 5(2), 2021 18 e. aspects of community participation the level of public awareness and concern for waste is still very low. according to the results of interviews with the mamasa village community, the community has not fully reduced waste from the household in terms of implementing 3r and sorting organic and inorganic waste, which in turn burdens the landfill. there are 71% (17 respondents) who know that waste must be sorted but of these 17 respondents only 8 respondents (47%) implemented waste segregation. where wet waste (organic waste and baby diapers) is disposed of in the trash/laystall and dry waste (plastic, paper and cardboard) is burned. there are 8% (2 respondents) who stockpile wet waste and burn dry waste. in the pattern of landfilling, it will cause soil pollution if there is garbage containing hazardous substances classified as b3 waste that enters the soil around residential areas. burning garbage will also cause air pollution, especially if it is carried out in densely populated residential areas/complexes, it can cause uncontrolled fires. according to utami, et. al (2008), the waste incineration system needs to be avoided because it is harmful to human health. burning waste at low temperatures can form toxic gases in the form of carcinogenic dioxins and furans as well as very small dust particles (particulate matter) that can cause acute respiratory infection (ari). some people still throw garbage in any place that causes the environment to become dirty, for example, baby diapers scattered on the road. another phenomenon that also happen is that people still throw garbage into the river. there are 4% (1 respondent) who admitted to throwing garbage into the tatoa river which is 5 meters away from their house. this waste is in the form of fish waste which cannot be stored for long because it will rot if it is not immediately disposed of. by looking at the phenomenon of the large amount of garbage scattered in the river, apart from intentional factors by irresponsible people who throw garbage into the river, there are also unintentional factors, for example on the tatoa river there is one laystall located on the riverbank. this laystall is used by hamlet 05 and hamlet 06 mamasa village, and according to the residents around this laystall it is often also used by people from the 2 closest sub-districts, namely tawalian district and sesenapadang district, who happened to pass the laystall causing the waste in the laystall to exceed it’s capacity. if it exceeds the capacity of the laystall, the garbage will be scattered and fall into the river. mamasa village is traversed by 2 (two) rivers, namely the mamasa river and the tatoa river. mamasa river is a large river with high water discharge. mamasa river passes through the heart of mamasa city and crosses several sub-districts and villages including mamasa village, especially hamlet 1 and 2. in this river you can still see a lot of garbage carried by water and even piled up on the riverbank. the tatoa river is a river that passes through hamlet 4, 5 and 6, and the upstream is located at hamlet 6. this river has a small discharge, so garbage is often found piling up in the river, which during the rainy season often floods due to the accumulated garbage in the river and due to silting of the river. this river passes through ± 2 villages and finally empties into the mamasa river. according to the mamasa village head, there was once a cleaning of the tatoa river by involving school children in the hope that the community would be moved and aware not to throw garbage into the river but this was not enough to bring order to the community. disposal of garbage in any place, especially rivers, will detain the rate of rainwater on the surface so that the flow is only focused on one point. when the rainfall is high, this kind of condition can lead to flooding. the worries not only during the flood but also after the flood. hunger, disease, unemployment, and other social problems become a homework international journal of applied biology, 5(2), 2021 19 that must be solved (silolongan and apriyono, 2019). community involvement in waste management is an important asset given the volume of waste that increase every day, while at the same time the government does not have enough funds to use advanced technology in waste management (prihatin, r.b., 2020). without the participation of the community, all the waste management programs that have been planned will be in vain (anas, 2017). community participation in the waste management system can be divided into several levels based on the involvement or role of the community in it. the level of participation is applying the right habits when handling waste, participation in consultation activities on waste management, participation in administrative and management activities, is the highest level of community participation in the waste management system, participating in decision making during meetings related to waste management programs (maulina, 2012). according to the vision of the environment and forestry agency of mamasa regency in point 3 it reads "improving waste management to create a clean environment by involving community participation". therefore, the collaboration between the government and the community in terms of waste management must continue to be improved. effectiveness of waste management at hamlet level in mamasa village waste management with the new paradigm is carried out by waste management with waste reduction and handling activities. waste reduction includes: restriction activities, reuse, and recycling. waste handling includes: sorting, collecting, transporting, processing, and final processing. a. waste reduction effectiveness as the largest source of waste disposal (household waste), the community needs to receive education about the importance of handling waste that is environmentally friendly and sustainable to help process waste from the source. considering that the largest source of waste in cities in indonesia is household organic waste, various recycling systems such as individual, communal and regional scale composting can be applied. in addition, the reuse of non-organic waste can be done with the waste bank program. for waste reduction, simple techniques such as reducing the use of single-use packaging and using biodegradable packaging. from the results of interviews conducted by researchers, it was found that of the 24 total sample respondents, 16 respondents (67%) claimed to know about the 3r but only 6 respondents (37.5%) had started implementing the 3r. from a total of 24 respondents, only 9 respondents (37.5%) applied 3r in their waste management. the 3r programs that have been started by the community include limiting plastic waste, and using a tumbler when traveling. effectiveness = 𝑅 𝑇 × 100 % explanation: r = realization t = target international journal of applied biology, 5(2), 2021 20 effectiveness = 9 24 × 100 % effectiveness = 37, 5 % in accordance with the effectiveness measurement standard (department of agriculture research and development 1991) the value of the effectiveness of reducing waste below 40% is declared very ineffective, so for waste reduction in mamasa village it is classified as very ineffective with a value of 37.5%. b. waste handling effectiveness waste segregation is a method of reducing waste. from the interviews conducted, it was found that 71% (17 respondents) claimed to know that waste must be sorted, as many as 42% (10 respondents) of the community had started sorting waste. apart from the unavailability of containers/bins to separate the waste, it is also because people think that after separating this garbage, what will they do. ideally, waste sorting is based on 3 (three) types, namely organic waste, inorganic waste and b3 waste. they generally sort paper, cardboard and plastic waste by burning and organic/wet waste and baby diapers to be disposed of in the trash/laystall. through interviews conducted, there were residents who claimed to know that waste must be sorted, but what to do after sorting it out. this indicates that public knowledge about waste management is still very shallow. this becomes an evaluation material for the government, private institutions or environmentalists to continuously disseminate information to the public regarding waste management. how do we treat the waste we produce? garbage that is sorted from the house in the form of organic waste can be converted into compost and inorganic waste can be recycled into useful goods and has economic value because it can be sold. another thing that can be done by the community is that inorganic waste in the form of cardboard, glass, used plastic bottles and papers that have been sorted by the community can be sold to collectors for economic value. in order to reduce the volume of waste in landfills, article 13 of the uups stipulates, "managers of residential areas, commercial areas, industrial areas, special areas, public facilities, social facilities, and other facilities must provide waste sorting facilities". furthermore, in the elucidation of article 13, “settlement areas include residential areas in the form of clusters, apartments, condominiums, dormitories, and the like. the sorting facilities provided are placed somewhere that is easily accessible by the community. the intended sorting facilities are medium-sized facilities such as laystall that can reduce the amount of waste before it is transported to the landfill. the government will conduct socialization and will fix the completeness of the institutions at the 3r laystall and preparations for the development of non-governmental organization (ngo). the calculation of the effectiveness of waste reduction is as follows: effectiveness = 𝑅 𝑇 × 100 % explanation: r = realization t = target international journal of applied biology, 5(2), 2021 21 effectiveness = 10 24 × 100 % effectiveness = 42 % in accordance with the standard effectiveness measure (department of agriculture research and development 1991) the effectiveness value of 40%-59.9% is declared ineffective, so for waste reduction in mamasa village it is classified as ineffective with a value of 42%. waste management effectiveness to determine the effectiveness value of waste management in mamasa village, it can be determined by calculating the average value of the effectiveness of waste handling and effectiveness of waste reduction. the calculations are as follows: average effectiveness = 𝐸1+𝐸2 2 based on the results of the calculation of the waste handling effectiveness of 37.5% and the effectiveness of waste reduction of 42%, the average value of 39.75% is obtained. in accordance with the standard effectiveness measure (department of agriculture research and development 1991) an effectiveness value below 40% is declared very ineffective, so in general the effectiveness of waste management in mamasa village is classified as very ineffective conclusions and suggestion conclussion from this study it can be concluded that the effectiveness of waste management at the hamlet level in mamasa village, mamasa regency is very ineffective. suggestion it is necessary to improve the quality of work of the janitors, to improve waste management infrastructure and to supervise the implementation of regulations on waste management. public awareness must be increased by providing counseling or socialization about waste management and there must be a cooperation between the community and the government in terms of waste management. international journal of applied biology, 5(2), 2021 22 references anas. 2017, akuntabilitas penyelenggaraaan pengelolaan sampah kota di kabupaten mamuju provinsi sulawesi barat. universitas terbuka, jakarta. damanhuri, e., & padmi, t. 2015. pengelolaan sampah terpadu: institut teknologi bandung. mamasa dalam angka 2020. https://mamasakab.bps.go.id, diakses 17 oktober 2020. pukul 23.05 wita maulina, a.s. 2012. identifikasi partisipasi masyarakat dalam pemilahan sampah di kecamatan cimahi utara serta faktor yang mempengaruhinya. jurnal perencanaan wilayah dan kota. 23 (3):177 – 196 miles, mb. 2001. analisis data kualitatif.jakarta:universitas indonesia. peraturan presiden republik indonesia nomor 97 tahun 2017 tentang kebijakan strategi nasional pengelolaan sampah rumah tangga dan sampah sejenis sampah rumah tangga . peraturan pemerintah nomor 81 tahun 2012.pengelolaan sampah rumah tangga dan sampah sejenis rumah tangga. republik indonesia. peraturan bupati mamasa nomor 06 tahun 2019 tentang kebijakan dan strategi daerah dalam pengelolaan sampah rumah tangga dan sampah sejenis rumah tangga. prihatin, r. b. 2010. pengelolaan sampah di kota bertipe sedang: studi kasus di kota cirebon dan kota surakarta, jurnal masalah-masalah sosial. 11 (1) :1-16 puspasari, g.r. dan mussadun. 2016. peran kelembagaan dalam pengelolaan persampahan di kabupaten trenggalek. jurnal pembangunan wilayah dan kota. 12 (4): 385 399 rahardyan b. dan widagdo a.s. (2005). peningkatan pengelolaan persampahan perkotaan melalui pengembangan daur ulang. materi lokakarya 2 pengelolaan persampaham di propinsi dki jakarta. standar nasional indonesia nomor sni-19-2454-2002 tentang tata cara teknik operasional pengelolaan sampah perkotaan, badan standar nasional (bsn) silolongan , r.f. dan apriyono, t. 2019. analisis faktor penghambat efektivitas pengelolaan sampah di kabupaten mimika. jurnal kritis. 3 (2) : 17-39 subagyo, a.w. 2000. efektivitas program penanggulangan kemiskinan dalam pemberdayaan masyarakat pedesaan.yogyakarta : ugm. syafrudin dan priyambada i.b. 2001. pengelolaan limbah padat. diktat kuliah program studi teknik lingkungan. fakultas teknik undip, semarang. international journal of applied biology, 5(2), 2021 23 undang-undang republik indonesia no 18 tahun 2008 tentang pengelolaan sampah. utami, indrasti, n. s., dharmawan, a.h. 2008. pengelolaan sampah rumah tangga berbasis komunitas : teladan dari dua komunitas di sleman dan jakarta selatan. jurnal transdisiplin sosiologi, komunikasi dan ekologi manusia. 2 (1):49-68. widiyanto, a. f., pratiwi o.c., dan yuniarno, s. 2017. model pengelolaan sampah rumah tangga di kabupaten banyumas, prosiding seminar nasional dan call for paper “pengembangan sumber daya perdesaan dan kearifan lokal berkelanjutan vii 17-18 november 2017. universitas jenderal sudirman, purwokwerto. yudianto, t., setyono, p., dan handayani, i.g.a.k.r. 2021. implementasi kebijakan dan strategi dalam pengelolaan sampah di kabupaten blora. jurnal kesehatan lingkungan indonesia. 20 (1) : 21-26 contact : lukman daris daris.lukman70@gmail.com 230 abstract mangrove ecosystems provide various ecological and economic functions, such as environmental services as an ecotourism area. this study aims to determine the factors that influence plastic waste on the socio-economic life of coastal communities in the lantebung area. the data in this study are primary data obtained from interviews with respondents with structured questions and statements. the data collection method with structured interview and data analysis used in this research is an impressional statistical method with an sem approach. the results of the data analysis, the factors that affect plastic waste are the volume of waste with a loading factor value of 0.966. in contrast, the factors that affect the social economy is a loading factor value of 0.732. plastic waste has a significant relationship to the socio-economic community, with a p value of 0.018 less than 0.05. the direction of the relationship between plastic waste and the socio-economic community is negative, with -0.240, meaning that the higher the plastic waste, the socio-economic community in the lantebung mangrove area will decrease. issn : 2580-2410 eissn : 2580-2119 the effect of plastic waste on the coastal socio-economic community of the lantebung in makassar city, west sulawesi province, indonesia lukman daris1*, andi nur apung massiseng1, irwansyah1, wahyuti1, fitria pebriyanti husain1 1 fisheries agrobusiness study program, faculty of fisheries, universitas cokroaminoto makassar, makassar, indonesia introduction coastal areas are rich in potential resources consisting of biological and nonbiological resources. biological resources consist of various fishery products, coral reefs, mangrove forests, and others, while non-biological resources are in the form of petroleum products and mining products and environmental services, which are the primary capital of national development. hence, they are essential for people's livelihoods (kismartini & bugin, 2019). coastal ecosystems generally found are mangroves with much diversity (biodiversity). the types of mangroves that are often seen include mangroves (rhizophora), api-api (avicennia), sonneratia and types of nipah (nypa). mangrove ecosystems have open access international journal of applied biology keyword coastal; lantebung; makassar; plastic waste; socio-economic article history received october 12, 2022 accepted december 8, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 231 ecological and economic functions, including providing environmental services as ecotourism areas. main ecosystems in coastal and marine areas such as mangroves have many functions, including providing nutrients in the waters, breeding and spawning grounds for various types of aquatic biota, protecting the coast from abrasion, protecting the coast from tsunamis and wind, and preventing seawater intrusion. in addition, the function of the mangrove ecosystem economically includes it as a livelihood source and an ecotourism area (utina et al., 2018). according to ihsan et al. (2015), ecotourism is a science that discusses environmental insight by implementing the principles of environmental sustainability and balance. in contrast, according to lappo., et al. (2010), ecotourism is a positive relationship between actors related to alternative tourism concepts by emphasizing social, community, and natural values. in addition to providing benefits from the mangrove ecosystem in the form of an ecotourism area, it also has an impact in the form of garbage or plastic wa ste generated by visitors or tourists. the waste includes mineral water bottles, biscuit packages, cracker packages, glass drinking water packages, bread packaging packages and plastic bags. suppose the disposal of plastic waste is not prevented immediatel y. in that case, it will have a socio-economic impact on the community around the mangrove ecotourism area, such as reduced income, decreased health, and decreased revenue due to fewer visitors. plastic waste contamination can have an impact on marine and mangrove ecology. this happens because plastic waste is the main factor that causes air pollution. after all, the mangrove ecosystem is damaged. besides the ecological impact, marine debris can cause a decrease in tourist visits to carry out tourism activi ties around the mangrove area because the surrounding area is dirty, affecting the decline in people's income (waryono, 2006; putri et al 2020). the above is the basis, so research on "the effect of plastic waste on the socioeconomic conditions of coastal communities in the lantebung mangrove ecotourism area in makassar city" is essential. this study aimed to determine the factors that influence plastic waste and the impact of plastic waste on the socio-economic life of coastal communities in the lantebung area of makassar city. materials and methods time and place this research was carried out within three (3) months, from may to july 2021. data was collected in the mangrove ecotourism area of lantebung, bira village, tamalanrea district, makassar city, south sulawesi province (figure 1). the location was chosen because the lantebung area is one of the mangrove ecotourism development areas in makassar city. international journal of applied biology, 6(2), 2022 232 figure 1. the research location of the lantebung mangrove ecotourism area data types and sources the type of data in this research is primary data. primary data were obtained from direct interviews with predetermined respondents with several structured and structured questions. primary data is obtained directly by researchers or for the first time (yusuf & daris, 2018). meanwhile, according to umar (2013), primary data is obtained from filling out questionnaires (interviews) that come directly from the first source, individuals and groups. this research data comes from the community around ecotourism and visitors who are respondents who provide information about the criteria and indicators studied. methods and data collection the collection method in this study was carried out using a survey method with structured interview techniques. the survey research method uses a questionnaire tool by taking samples from one population (singarimbun & sofian, 1982). according to sugiyono (2018), the meaning of the survey is the method used to obtain data, the behaviour of variable relationships and test the hypotheses obtained quantitatively. information can be obtained through data collection using interviews if the researcher already knows the information for sure. before conducting interviews, a research instrument was first prepared to consist of several questions whose answers had been prepared. the study's respondents comprised 60 people divided into 20 people from the community, 20 visitors and 20 managers. the technique of collecting samples was done by using a random sampling technique (random). sugiyono (2017) states that random sampling is sampling in a population that is carried out randomly without choosing a particular sample. international journal of applied biology, 6(2), 2022 233 data analysis the research data were processed using inferential statistical methods with the sem approach. this method consists of analyzing data and drawing conclusions from a random sample. inferential statistics include: estimating data and testing hypotheses, and making decisions. the discussion of inferential statistics includes analysis of covariance, simple linear regression analysis, analysis of variance, analysis of correlation, and testing the average. structural model equation (sem) is a common cross -sectional statistical modelling technique, incredibly linear, factor analysis, path analysis, and regression. sem is a confirmatory technique and not exploratory. hence, researchers are more inclined to use sem to determine whether a particular model is valid by using sem to find an ideal model (syahrir et al., 2020). the steps that can be carried out in sem analysis are as follows: 1) create designs and models based on theory, determine how to measure constructs, collect data, and then modulate data and output. 2) input data in sempls software 3) interpreting the output of the analysis by measuring the evaluation of the structural model and the equation test. results and discussion characteristics of respondents age respondents (visitors, community, and managers) have diverse characteristics from their teens to their parents' ages; this allows respondents to vary in age. characteristics of respondents based on age are presented in table 1 and figure 2 below: table 1. characteristics of respondents based on age source: primary data, 2021 figure 2. graph of respondent's profile based on age <20 3% 20-40 58% 41-60 37% >60 2% no age respondent percentage 1 <20 2 3% 2 20-40 35 58% 3 41-60 22 37% 4 >60 1 2% total 60 100% international journal of applied biology, 6(2), 2022 234 in figure 2 and table 1 above, it is known that the age of the dominant respondent ranges from 20-40 years, namely 35 people (58%), while the respondent's age is at least over 60 years, one person (2%). this means that the age of 20-40 years is productive. simatupang's statement (2019) states that those aged 35-44 are highly desirable to obtain travel insurance. education each individual has a different education caused by the mindset that influences a person's decisions. the characteristics of respondents based on education are presented in table 2 and figure 3 below: table 2. characteristics of respondents based on education no education respondent percentage 1 no school 9 15% 2 elementary school 12 20% 3 junior high school 4 7% 4 senior high school 26 43% 6 d3 2 3% 7 college students /s1 7 12% total 60 100% source: primary data, 2021 figure 3. graph of respondent's profile based on education table 2 and figure 3 show that the education level of sma/smk occupies the first position with a total of 26 people (43%). at the same time, the education level d3 has several respondents, at least two people (3%). for the elementary education level, there were 12 people (20%), middle school students were 4 people (7%), students/s1 were 7 people (12%), and those who did not go to school were 9 people (15%). arif's research (2017) states that education is related to easy accessibility; tourists with a high level of education will find it easier to access tourist attractions with a high level of accessibility, and tourists with moderate education will more easily reach tourist areas with a moderate level of accessibility. no s chool 15% elementary s c hool 20% junio r high s c hool 7% s enio r high s c hool 43% d3 3% c o llege s tudent/s 1 12% international journal of applied biology, 6(2), 2022 235 work in everyday life, a person's decisions are often influenced by the job one has. the characteristics of respondents based on work are presented in table 3 and figure 4 below: table 3. characteristics of respondents based on work no work respondent percentage 1 does not work 3 4% 2 bookkeeping admin 1 2% 3 midwife assistant 1 2% 4 barber shop 2 3% 5 art 6 10% 6 sales / traders 5 8% 7 employee 5 8% 8 head of lpm bira village 1 2% 9 chairman rw.06 1 2% 10 student 5 8% 11 fisherman 20 33% 12 tour guide 1 2% 13 boat builder 1 2% 14 manager jekomala 2 3% 15 onion peeler 1 2% 16 driver 2 3% 17 parking attendants 1 2% 18 self-employed 1 2% 19 marketing 1 2% total 60 100% source: primary data, 2021 figure 4. graph of respondent profiles by occupation doesnt work 5% b ookkeeping admin 2% midwife assistant 2% b arb er shop 3% irt 10% sales/trader 8% employe 8% head of lpm b ira 2% chairman rw.06 2% student 8% fisherman 33% tour g uide 2% b oot b ulder 2% manag er jekomala 3% onion peeler 2% driver 3% parking attendants 2% self employee 2% marketing 2% international journal of applied biology, 6(2), 2022 236 table 3 and figure 4 show that the most jobs of the respondents are fishermen, as many as 20 people (33%), while the fewest are several types of work such as bookkeeping admin, assistant midwife, head of lpm, head of rw, tour guide, boat maker, onion peeler, parker, entrepreneur and marketing each of 1 person (2%). for the type of job, barbershops, managers and also drivers are 2 people (3%), irt is 6 people (10%), sellers or traders, private employees and also students/students are 5 people each (8%). jayanti's research (2018) and roswiyanti et al (2022) said of states that the community's need factors will influence the type of community work. this encourages people to get more promising jobs in the agricultural and tourism sectors. income one's income certainly has an important role in satisfying one's needs. the characteristics of respondents based on income are presented in table 4 and figure 5 below: table 4. characteristics of respondents based on income source: primary data, 2021 figure 5. graph of respondents' profiles based on income in figure 5 and table 4 above, it can be seen that the income of the majority of respondents ranged from rp. 2,600,000 – rp. 4,600,000, as many as 24 people (40%), while the minor income is erratic, as much as 1 person (2%). it was also found that 7 people (11%) had no income, and people earned less than rp. 500,000 – rp. 2,500,000, as many as 9 people (15%), people who earn between rp. 4,700,000 – rp. 6,700,000 as many as 3 people (5%) no thing 11% unc ertain 2% <500.0002.500.000 15% 2.600.0004.600.000 40% 4.700.0006.700.000 5% 6.800.00010.000.000 15% >10.000.000 12% no income respondent percentage 1 nothing 7 11% 2 uncertain 1 2% 3 <500.000-2.500.000 9 15% 4 2.600.000-4.600.000 24 40% 5 4.700.000-6.700.000 3 5% 6 6.800.000-10.000.000 9 15% 7 >10.000.000 7 12% total 60 100% international journal of applied biology, 6(2), 2022 237 who earn between rp. 6,800,000 – rp. 10,000,000 as many as 9 people (15%) and income of more than rp. 10,000,000, namely 7 people (12%). harvey's research (2013) stated that the level of spending, the amount of working time and the number of family members could affect the income level of people, especially those who use tourism services. income data analysis in the study used smartpls 3.0 software. the model built in this study can be seen in the following figure: figure 6. outer research model factors affecting plastic waste and the socio -economy of the lantebung coastal community the results of the data analysis show that the factors that affect plastic waste and the socio-economic community in the lantebung mangrove ecotourism area can be seen from the factor loading values, where factors that have a value of > 0.700 influence the variable. plastic waste variable this study's influencing factor is the waste volume, with a loading factor value of 0.966. according to fajria et al. (2019), dense activities, especially in the coastal environment, can have a significant influence becaus e they can reduce people's opinion and the ecology of the waters because of the large amount of waste generated by visitors. the ecological impact in the form of mangrove damage and the economic impact of decreasing people's income due to the reduced number of visitors. human behaviour dramatically influences the existence of waste somewhere (adam, 2015). in addition, population growth also affects the volume of waste because it is closely related to lifestyle (hariyanto, 2014). in reducing the volume of waste in the lantebung ecotourism area, there needs to be active and integrated education and outreach, especially to the surrounding community and visitors, so that the awareness of environmental sustainability increases. one that influences the source of waste according to wijaya (2021) is that there is still a lack or low public awareness of environmental cleanliness, so waste is disposed of anywhere. therefore the community must prioritize discipline in disposing of waste in its place. there are two sources of waste in the lantebung ecotourism area: plastic waste generated from community activities or visitors and plastic waste carried from rivers and ocean currents. darwin (2019) states that waste depends on the number of visitors, where to eat, and trash carried from ocean currents, whereas according to subekti (2017), most of the garbage originating from river currents occurs during the rainy season. international journal of applied biology, 6(2), 2022 238 socio-economic variables the influencing factor is health, with a factor loading value of 0.732. improvi ng social welfare in the community must be supported by adequate health because high work productivity and community income are strongly supported by high public health. people's thinking power is greatly influenced by people's daily consumption of food and nutrition (suryandari, 2017). arum & sujiyatini (2009) state that learning capacity and work productivity are influenced by the level of health in both the family and individual environments. being physically fit and healthy affects the level of producti vity at work. at the macro level, health is essential in reducing poverty and increasing economic growth. high economic growth is influenced by the level of good health among the people. health affects economic factors because if health is disrupted, it wi ll also disrupt community performance, whereas if performance is disrupted, the income generated will decrease. for example, it is known that the average coastal community in the lantebung ecotourism area has the main occupation as a fisherman; if the fishermen's health is disturbed, the catch will also decrease. the impact of plastic waste on the socio -economic society of the lantebung mangrove ecotourism area in looking at the relationship between plastic waste and the socio-economic community, a bootstrapping test was carried out on smartpls with a significance of 5% with the following image model: figure 7. the relationship between plastic waste and the socio-economic community table 5. the relationship between plastic waste and the socio -economic community original sample (o) sample mean (m) standard deviation t statistic p values plastic waste --> socioeconomic community -0,240 -0,237 0,101 2,383 0,018 source: path coeffi ci ents boots trappi ng smartpls table 5 can be seen that plastic waste has a significant relationship with the socio economic community with a p value of 0.018, less than 0.05 (h1 is accepted) with the direction of the relationship between plastic waste and the socio-economic community is hostile, meaning that the higher the plastic waste, the social economy of the communi ty will decrease with the original sample (o) value of -0.240. international journal of applied biology, 6(2), 2022 239 plastic waste pollution has an impact on ecologica l aspects and economic aspects. this can cause damage to mangroves and ultimately reduce the number of visitors to tourist sites. according to day (2020), the bad habit of people who have existed for a long time, namely throwing garbage all over the place, will indirectly impact us in the form of losses because marine life experiences digestive system disorders and marine biota growth. according to desy (2018), the ecological impact of the death of the mangrove ecosystem is decreasing the ecosystem's population around the mangroves. furthermore, it was added that mangrove damage would affect the decrease in people's income. conclusion the lantebung mangrove ecotourism area impacts the plastic waste volume with a loading factor value of 0.966. in contrast, health influences socio-economic status with a loading factor value of 0.732. plastic waste has a significant relationship to the socio economic community, with a p value of 0.018 less than 0.05. the relationship between plastic waste and the socio-economic 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alfabeta. suryandari an. 2017. pengaruh pertumbuhan ekonomi, pendidikan, dan kesehatan terhadap tingkat kemiskinan di provinsi daerah istimewa yogyakarta tahun 20042014. skripsi. syahrir., danial., e. yulinda., dan m. yusuf. 2020. aplikasi metode sem-pls dalam pengelolaan sumberdaya pesisir dan lautan. bogor: ipb press. tika pabundu. 1997. metode penelitian geografi. jakarta: pt gramedia pustaka utama umar h. 2013. metode penelitian untuk skripsi dan tesis. jakarta : rajawali. utina r, nusantari e, katili as, dan tamu. 2018. ekosistem dan sumber daya alam pesisir: penerapan pendidikan konservasi. yogyakarta : deepublish. waryono, t. 2006. konsepsi manajemen pemulihan kerusakan mangrove di dki jakarta. yayasan mangrove indonesia. jakarta. wijaya ikwb, prathiwi kdjr & muliadi nm. 2021. pengembangan literasi ekologi siswa sekolah dasar. jurnal adi widya. 6(1) hal. 46-53. contact : alpiani alpiani.pia@gmail.com 104 abstract this study aims to analyze and examine the exchange rate of labour fishers on bagan rambo fishing gear in barru district, barru regency. the fishers' exchange rate is used to measure the basic needs of fishers, which considers the income and expenditure of the households of the bagan rambo labourers. this type of research uses instrumental case studies. analyze the poverty of fishers as an instrument for understanding their lives. the results showed that the exchange rate of fishers (ntn) of bagan rambo workers was lower than one hundred (88,49%), which means the bagan rambo labour fishers cannot meet their basic needs. issn : 2580-2410 eissn : 2580-2119 the exchange rate of bagan rambo fishermen in barru district. barru regency, south sulawesi alpiani alpiani1*, andi nur apung massiseng1, andi ummung1 1faculty of fisheries, university of cokroaminoto makassar, indonesia. jl perintis kemerdekaan km.11, makassar introduction the makassar strait has considerable potential for fishery resources, including the marine area of barru regency, which has a marine area of around 56,000 ha. these coastal resources are utilized by many people (fachry et al., 2021), mainly fishermen. the coastal area of barru regency is inhabited by people who generally work as fishermen. popular fishing gear in barru county is bagan rambo. bagan rambo is used for pelagic fishing (daris et al., 2021). the bagan rambo fishing gear operation requires many workers, namely nahkoda, machine man, and at least 14 labour fishermen (ilhamdi & surahman, 2018). these workers have different social stratification statuses. nahkoda is the arrest operation leader who has the most percentage of income compared to others. however, bagan rambo's efforts are known to be quite successful in barru county. the lifestyle of the business capital owner bagan rambo and his nahkoda is quite luxurious. they have a decent house and some other possessions, and the average vehicle has a vehicle in the form of a private car and a fisherman exchange rate above 100%. however, mo re profoundly, seeing that in the business, there are not only owners of capital and nahkoda but also many labour fishers who deserve to pay attention to their economic conditions. the fishing workers are small-scale fishermen. small-scale fisheries survive as coastal livelihood activities, although there is a decreasing chance due to the long-term intensification of fisheries exploitation (fabinyi et al., 2022). open access international journal of applied biology keyword poperty; labour; bagan-rambo; terms of trade of fisherman article history received june 14, 2022 accepted july 7, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 105 most small-scale fishers belong to the poor category (yonvitner et al., 2021). this is inseparable from the minimal division of responsibilities and wages, and the most affecting is the season for fishers. if the famine season comes, fishers almost do not go to sea, and fishers have no income during the famine season (rohayati et al., 2018; yulianda et al., 2021). labour fishers are very depressed and have difficulty making ends meet, so that borrow from another person or, in this case, the owner of the bagan rambo business (aqmal & prastiyo, 2018). this is quite ironic amid the many potential resources such as aquaculture, which has a vast area of land (zaenab et al., 2021), which can be cultivated by fishers as an alternative livelihood and do not know the famine season. also, other businesses such as processing catches or utilizing waste resources in coastal environments can improve the economy of fishing households (fachry & alpiani, 2021). according to some research results, as stated by aqmal et al., 2020, fishers' income cannot meet their household needs, and the fisherman's income tends to decrease yearly. faried et al. (2018) state that fishers living in coastal areas are below the poverty line and have become marginalized. therefore, the position of the labour fisherman in the bagan rambo business is worthy of attention. the first thing that can be done is to examine how the conditions meet the household's needs. fishers' exchange rates are used to determine and analyze the economy of fishing households, whether the work he is engaged in as a bagan rambo fisherman can meet his basic needs or is it the same as other small-scale fishermen living in poverty. materials and methods this research was conducted in sumpang binangae village, barru district, barru regency, south sulawesi, indonesia. the study was conducted from october to december 2019. site selection is done intentionally (purposive), considering the location is a village centre for bagan rambo labour fishermen who can represent data from barru regency. the type of research used in this study is a type of case study research by analyzing the level of meeting the household needs of fishing communities using the fishermen's exchange rate approach. the case study in this study is an instrumental case study where the case of fishermen's households is an instrument in understanding the living conditions of bagan rambo fishermen, especially their workers. the total population of bagan rambo workers settled down in this study was 155. the sample taken by 30% of the total fishermen of bagan rambo labourers was 47 people in sumpang binangae village, barru district, barru regency. the data obtained from the results of observations and interviews were analyzed using the fishermen's exchange rate approach with the following formula (basuki et al., 2001): ntn = (yt/et) x 100 yt = yft + ynft et = eft + ekt yft = total fishermen's receipts from fisheries business (rp) ynft = total fishermen's receipts from non-fisheries (rp) eft = total fishermen's expenditure for fisheries business (rp) ekt = total fishermen's expenses for fishermen's family consumption (rp) t = time (month, year, etc.) international journal of applied biology, 6(1), 2022 106 if income and expenses are the same, then statistically, the figure that appears in the calculation of ntn is 100. this is because the number 100 describes income and expenses equally. therefore, less than 100 have not been able to meet basic needs, equal to 100 expenses and income equals (there is nothing yet for savings and investments), and more than 100 can meet basic needs. results and discussion description of bagan rambo fishing gear bagan rambo is a fishing gear operated by two vessels. the main ship serves as a chart buffer that does not have a propulsion engine. an introductory ship that serves to pull the bagan ship from the landing base to the catchment area and from the catchment area to the landing base, escorting bagan rambo fishermen from the landing base area to the catchment area and vice versa, taking catches from the bagan rambo as well as delivering operational fishing materials. in this study, the ship measuring 30 m with a net fishing gear measuring 30 x 30 m. the machine used for lighting is a yanmar tf 300 with diesel fuel. delivery ship measuring 22 m, using a car engine with diesel fuel. both ships are made of wood with an economic life of 10 – 15 years. the method of determining the location of the arrest is only based on experience and feelings without adequate technology. the capture location is around the barru area, with a distance of about 2 hours. bagan rambo can be operated on as many as 20 24 trips a month during the fishing season and in good weather. the delivery boat towed bagan rambo to the capture area during the good season and towed back to the landing base during the full moon and western season. the rambo-chart is usually moved twice to different catchment areas before being pulled to the landing base. it is not uncommon for the bagan rambo not to be moved as long as the fish in the catchment area remain numerous. all workers were escorted to the arrest area in the afternoon around 5 pm and picked up in the morning around 5 am. they alternated guarding the bagan ship daily with about three workers per day. the business of the bagan rambo fishermen at the research site is enough to absorb jobs for the natives of sumpang binangae and migrant residents. although, in general, the income of baganrambo workers can only meet the needs of household consumption for workers who do not play a critical role in the operation of this fishing gear. bagan rambo is operated by 12-15 workers. there are several levels of the role of workers in the operation of rambo chart fishing gear, namely punggawa bagan (leader of fishing operations at sea), machine-builder, delivery man and labour fisherman. labourers have the lowest wages of all levels of the worker's role, so the wages are also the least. labour wages for one person are generally only about 1% of the total net profit, with an average monthly wage value ranging from 0-rp. 1,500,000 (alpiani, 2019). exchange rate of bagan rambo labor fishermen in sumpang binangae measurement of the level of fulfilment of basic needs is one of the welfare approaches that must be calculated to determine the level of economic development in a region. fishermen's exchange rate analysis can determine the ability to meet the basic needs of bagan rambo labour fishermen in sumpang binangae village. this approach is not only limited to knowing the ability to meet basic needs but also the factors that affect income and expenses in detail so that it can be used as a benchmark for formulating a policy. it can also be seen the factors that affect income and expenditure in detail so that they can be international journal of applied biology, 6(1), 2022 107 used as a benchmark for formulating a policy. the income and expenditure of labour fishermen on the study can be described as follows: income the source of income seen is the overall source of income, both sources of income from fishery and non-fishery businesses. below is explained regarding the source of income for bagan rambo labour fishermen in sumpang binangae village. income from fisheries the source of income for bagan rambo labour fishermen is from the results of fishing rods at sea and from wages to become labour fishermen. the income from the fishing netting each time you go to sea is rp.0 rp. 150.000,and wages to be a labourer for a month from rp. 0 – rp. 1.500.000some fishing products are directly sold at auctions; usually, some are dried by labour fishermen's wives and for consumption. the income of the bagan-rambo labour fishermen is minimal, and they will not be able to meet their basic needs. however, they still go to the sea as labourers because they do not have enough fishing capital such as engined boats and fishing gear to catch fish. free time is used for fishing because the task of labour fishermen only helps to lift the net with the overall working time of only about 2 hours (alpiani, 2019). for this reason, they use the facilities of bagan rambo activities for fishing. it is through this result that they hope to be able to make ends meet. so, working as a bagan-rambo labour fisherman is not the main livelihood goal of these fishermen, but only so that they can fish on the high seas by utilizing the facilities of the bagan rambo boat. so that minimum wages are not a problem for them. also, bagan rambo workers get security protection from rambo bagan boat owners, who increasingly encourage labourer fishermen to work as labourers in the bagan rambo business. income from non-fisheries all labour fishermen who were respondents did not have side jobs. the wives of the labour fishermen were only housewives. labour fishermen are very dependent on fishing activities. when it does not go to sea, then there is no income. meanwhile, they have to make ends meet. expense all expenditure forms are calculated to determine the exchange rate of bagan-rambo labour fishermen in sumpang binangae village. the expenditures are as follows: fishery business costs the tool needed for fishing is a fishing line along 200 -500 m at around rp.30,000, up to 6 months of use. and one big fishing rod they got at a price of rp.5,000 / fishing rod eye. for one month, the fishing rod eyes are usually only 1. in a month began, rambo labour fishermen needed a fishing capital of rp.10,000. this depends on the strength of the fishing rod eye bond on the fishing line, so it does not come off quickly when pulling fish. fishing baits are small fish caught in the bagan rambo net. fish caught by bagan-rambo fishermen's fishing rods such as tuna, skipjack tuna, mackerel, redfish and other large fish. the price of fish caught by bagan-rambo fishermen in sumpang binangae village ranges from rp.20,000 – rp.50,000/kg. household consumption household consumption of fishermen of bagan rambo workers, namely consumption for daily needs such as rice, instant noodles, sugar, coffee, tea, milk, cooking oil, side dishes, international journal of applied biology, 6(1), 2022 108 fruits, and fuel oil, in this case, all respondents use lpg 3 kg, kitchen spices, soap, toothpaste, toothbrush and shampoo. the average daily consumption expenditure is rice for rp. 218,468, instant noodles for rp. 46,891, sugar for rp. 48,617, coffee, tea and milk for rp. 30,914, cooking oil for rp. 56,808, side dishes for rp. 128,872, fish, egg and chicken for rp.128,723, gas refill for rp. 51,170, kitchen spices for rp. 106,638, bath soap, detergent and shampoo for rp. 147,128, as well as cakes/snacks for rp. 245,766. cigarettes for rp.189,191. so the total average for daily consumption is rp.1,209,000. cigarettes are not a basic need. however, the perspective of the fishermen of bagan rambo considers that cigarettes are a basic need that must be met. school education costs education is a basic human need to be able to knowledgeable and moral. the children of the fishing workers of bagan rambo, who are the respondents, send their children to high school. this is because school fees for elementary school to senior high school have been freed according to government programs. education costs include transport and snacks, stationery and books, uniforms, shoes and school bags. the average expenditure on education costs, namely transportation and snacks, is rp. 183,533, and stationery is worth rp. 3,285 books worth rp. 19,318, as well as uniforms, shoes and school bags worth rp. 35,733. health as with education, medical expenses are also accessible at the village health centre, especially for the poor. fishermen who work in bagan rambo are a group of people whose medical expenses are free of charge. however, the fishermen's families can buy medicines at the stalls around the house. the average cost of medical treatment for fishermen working in bagan rambo in sumpang binangae village is rp. 1,277. house the house is a basic need as a shelter and rest. the condition and size of the fishermen's housing for the bagan rambo workers are elementary. generally, the housing for fishermen and labourers is made of wood and roofed with zinc. there is also housing for fishermen and labourers whose floors are made of cement, walls and roofs of tin. the household electricity consumption of the fishermen of bagan rambo ranges from rp. 20,000 to rp. 50,000. home care is not paid much attention because they focus on meeting food needs. home maintenance costs around rp. 0 20,000 per month. clothes at the fisherman level, bagan rambo workers in sumpang binangae village, clothes are usually bought only at certain times, such as eid al-fitr. their children's clothes are usually gifts from neighbours, family and friends punggawa (owner of bagan rambo business). they buy other types of clothes only when urgent, like torn clothes, and there are no replacements. the average expenditure for clothing for fishing families of bagan rambo fishermen in sumpang binangae village per month is rp. 24,362. the income of labour fishermen is used to meet their basic needs. the percentage of income allocation for fishermen of bagan rambo workers in sumpang binangae village is as follows: international journal of applied biology, 6(1), 2022 109 table 1. average percentage of income allocation of fishermen workers of bagan rambo in sumpang binangae village in october – december 2019 no. output type total (rp.) percentage (%) 1 household consumption 1.398.191 86,55 2 school education costs 150.957 9,34 3 health 1.277 0,08 4 house 40.745 2,52 5 clothes 24.362 1,51 total 1.615.532 100 source: primary data after processing, 2019 all household expenditures for fishing workers of bagan rambo in sumpang binangae village are the highest, which is the fulfilment of consumption with a percentage of 86.55%. engel's theory states that the lower the income level, the more income allocation is focused on consumption (food) needs. the highest fisherman exchange rate is 144.44, with a total income of rp. 1,300,000 and a total expenditure of rp.900,000, while the lowest fisherman exchange rate is 46.07 with a total income of rp.850,000 and a total expenditure of rp.1.835,000. a total of 16 respondents had a fisherman's exchange rate of more than 100, which was around 100.36 – 144.44 and 31 respondents had a fisherman's exchange rate lower than 100 with a value of 94.71 – 46.07. the fisherman's exchange rate is meagre, with less than 60 as many as eight respondents with a value of 59.70 – 46.07. of the 47 fisherman respondents working in bagan rambo, only 16 respondents were able to meet their subsistence needs. this means that many fishermen who work in bagan rambo in sumpang binanagae village have been unable to meet their basic needs. this is also inseparable from the rising prices of necessities and the lower wages of fishermen working in bagan rambo as a result of the increase in fuel oil. from the data processing, it was found that the fisherman's exchange rate for fishing labourers of bagan rambo in the period october december was an average of 88.49. this shows that in that period, the household expenditures of the fishermen of bagan rambo workers were more significant than the income, where the income had not been able to meet the basic needs of the fishermen of the bagan rambo workers. the most influential factor in the size of the exchange rate of fishermen in bagan rambo is the number of family members. the greater the number of dependents in the household, the greater the expenditure for household consumption, especially food. second, the more cigarettes consumed by labour fishermen, the greater the expenditure. third, the more fishing rods caught by labour fishermen, the higher the level of fulfilment of their essential needs. the results of fishing rods between labour fishermen are different because fishing activities are carried out by each labour fisherman and are not group activities. the last factor is the number of loans to punggawa to meet needs. the costs of education, health, housing and clothing also affect the fisherman's exchange rate, but because the total cost is relatively low, it can be said that it does not have much effect. therefore, the fisherman's exchange rate is very well used in making policies to reduce poverty compared to income indicators. the fisherman's exchange rate is used to determine the source of income, the types of expenditure and the factors that influence household income and expenditure by radically examining all related matters. the results of the analysis carried out on fishing labourers of bagan rambo in sumpang binangae village can be seen as the factors that international journal of applied biology, 6(1), 2022 110 affect the exchange rate of fishermen. many factors affect the low exchange rate of fishermen as follows. 1. there is no other source of income apart from fishing. when the season is not fishing, then there is no income. meanwhile, they have to fulfil their substantial needs, especially food needs. 2. limited knowledge, skill and fishing technology. so the catch is less than optimal. in addition, the government has not been able to distribute information related to fishing activities, such as fishing locations, fishing technology, etc. 3. when the season is not fishing, they are unemployed or can be said to be seasonal unemployment. 4. lack of income. so there is no savings and investment because they cannot meet their substantial needs alone. 5. there is no processing of the catch. the price decreases when fish are abundant, and many fish go unsold and rot. there is no thought and skill to process fish when the catch is quite a lot, so it generates income from processed products that can cover needs when not at sea. 6. there is no income contribution from the fishers' wives to increase their income even though they have a lot of free time and sufficient energy to work. 7. there is a fisherman's perception that cigarettes are a necessity for their substance and are far more critical than consuming food. some of these problems are the government's responsibility and those concerned to solve them. the first thing that must be built in the independent awareness of fishermen, especially labour fishermen's families, to increase their income armed with knowledge and skills as well as a strong desire to work in order to produce something that has a selli ng value from their creativity and does not only depend on fishing activities. the government must strive to build awareness of labour fishermen through various ways that can reach changes in the mindset of labour services, both through mass media, environ mental constructs, programs and regulations that are supported by skills training and continuous delivery of information, providing capital in the form of money and goods. and there must be continuous control and sustainable development until finally achieving the goal of independence from fishermen, especially fishermen's families, which are expected to improve their welfare. conclusions the fisherman's exchange rate (ntn) for fishermen working in bagan rambo in sumpang binangae village is 88.49, which means that the household expenses of the fishermen working for bagan rambo are more significant than their income. international journal of applied biology, 6(1), 2022 111 references alpiani, a. 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(2021). pemanfaatan limbah usus ayam untuk pembesaran ikan lele utilization of chicken intestine waste for growth of catfish. 14(2), 193–198. https://doi.org/10.29239/j.agrikan.14.2.185-190 contact : andi masriah andimasriah@gmail.com 249 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. issn : 2580-2410 eissn : 2580-2119 effect of bioslurry concentration in feed on the growth and survival of milkfish (chanos chanos forsskal) st. zaenab1, andi masriah1*, agus suryahman1 1 study program of aquaculture, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar, indonesia. article history received august 14, 2022 accepted december 14, 2022 keyword bioslurry, probiotic, feed, milkfish, liquid, solid introduction one of the primary commodities of pond cultivation in pangkep regency is milkfish. milkfish have a high protein content (fao, 2020). many milkfish farming activities are supported by the area's potential to have ponds reaching 13,000 hectares by 2020 (statistik.kkp.go.id, 2020). currently, the pond cultivation system has developed from a traditional system that utilizes natural feed to an intensive that relies on artificial feed (nasution & yanti, 2015) and the use of a waterwheel. feed is an essential component in the cultivation process because the availability of feed will affect the growth and survival of fish (zaenab & massiseng, 2021). the feed consumed by fish must contain nutrients easily digested and absorbed by fish (masriah, 2020., nasser et al., 2022). commercial feed in the market is costly because of the high price of raw materials. the more complete the feed composition, the higher the feed cost because raw material prices also increase. therefore fish cultivators look for alternative raw materials that can be used as a quality feed that is easily available and of good quality. good feed contains complete open access international journal of applied biology abstract bioslurry is cow excreta waste proce ssed anaerobically as a nutritional by product of biogas production, w hich contains proteins, carbohydrates, fats and minerals. t his study aims to determine the conce ntration of solid bioslurr y in feed and the c oncentration of liquid bioslurry in fee d containing solid bioslurry, which is optimal for increasing the growth and survival of milkfish. the results showed that the be st feed with a concentration of 50% solid bioslurry resulted in a growth of 100.05 grams ± 6.98 and a survival rate of 96.67% ± 1.15. a significant growth increase in fee d with a high bioslurry c ontent indicates that bioslurry c ontains nutrients capable of increasing growth energy in milkfish. the growth of milkfish continue s to increase along with the increasing concentration of bioslurry liquid waste added to the feed as a source of pr obiotic s. adding 50 ml of liquid bioslurry into 100 grams of feed re sulted in the be st growth of 165.33 grams ± 2.08. liquid bioslurry contains probiotic micr obial bioactivators and functions t o increase growth. mailto:andimasriah@gmail.com international journal of applied biology, 6(2), 2022 250 nutrition, especially protein (hussain et al., 2021) and carbohydrates which can support the growth and survival of fish. bioslurry is an alternative that can be used as raw material for maki ng feed. bioslurry is cow excreta waste processed anaerobically as a by-product of biogas production (islam et al., 2020). currently, many biogas reactors produce bioslurry waste as solids and liquids. the cow excreta in the reactor produces nearly the same amount of bioslurry (fa rihah, 2020). t he results of converting cow dung into biogas with a fermentation process in the reactor will produce methane gas (ch4) (seran et al., 2020) and bioslurry. bioslurry contains nutrients in the form of protein, carbohydrates, fatty, calcium (ca) and phosphorus (p) (romadhon et al., 2013; seran et al., 2020). using bioslurry as a raw material for making fish feed can reduce feed costs because it is easy to obtain at a low price and has a complete nutriti onal content. this study aims to determine the concentration of solid bioslurry in feed and the concentration of liquid bioslurry in feed containing solid bioslurry that is optimal for increasing the growth and survival of milkfish. materials and methods research design this research was divided into two research stages, namely (1) the percentage of solid bioslurry waste in feed and (2) the percentage of liquid bioslurry waste in feed containing solid bioslurry waste. the two stages of this study were designed using a completely randomized design (crd). the study's first phase consisted of 4 treatments and 3 replications (12 experimental). the treatment tested was the concentration of solid bioslurry in the feed and used commercial feed as a control (k). the concentration of bioslurry in processed feed is presented in table 1. table 1. the concentration of bioslurry and raw material composition in processed feed raw material feed a (20%) feed b (35%) feed c (50%) solid bioslurry (%) 20 35 50 dregs of tofu (%) 10 10 10 fish flour (%) 5 5 5 fine bran (%) 45 30 15 seaweed waste (%) 10 10 10 fat (%) 10 10 10 total (%) 100 100 100 international journal of applied biology, 6(2), 2022 251 b2 k 1 c3 a2 c2 a3 b1 a1 k3 b3 k2 c1 a2 e1 b1 c3 c1 b3 a3 b2 a1 d3 e3 e2 d2 c2 d1 the layout of the experimental container used in this first stage of research: figure 1. the layout of the experimental set-up during the first phase of the study the second research stage is adding bioslurry liquid as a source of probiotics to the best feed obtained in the first research stage. the mixing process is carried out by spraying the feed as pellets. the research design used in this phase 2 study was randomized entirely, consisting of 5 treatments and 3 replications (15 experimental). the treatments tested in this phase 2 study were as follows, and the layout of the experimental container in figure 2: a : control (without liquid bioslurry probiotics) b : 30 ml of bioslurry probiotic liquid / 100 g of feed c : 40 ml of bioslurry probiotic liquid / 100 g of feed d : 50 ml of bioslurry probiotic liquid / 100 g of feed e : 60 ml of bioslurry probiotic liquid / 100 g of feed figure 2. the layout of the experimental containers in the second phase of the research international journal of applied biology, 6(2), 2022 252 parameters observed in the first and second phase of the research, the parameters observed were as follows: a. absolute weight growth rate absolute weight growth (w) is calculated using the formula arifin and rupawan (1997): w= wt -w0 information: w : growth weight (g) w0 : initial weight (g) wt : final weight (g) b. survival rate survival rate is the percentage of the number of live fish and the number of fish stocked during maintenance, calculated by effendi's formula (2002): 𝑵𝒕 𝑺𝑹 = 𝑵𝟎 x 100 information: sr : seed survival (%) nt : the number of fish stocked at the end of the study (fish) n0 : number of fish stocked at the start of the study (fish) data analysis this study's growth and survival rate data were analyzed for variance (anova) using the spss application. if the data show significant differences between treatments (sig <0.05), proceed with the w-tuckkey test. results and discussions growth and survival of milkfish fed with various concentrations of solid bioslurry the growth and survival of milkfish fed bioslurry solid waste with various concentrations are presented in table 1. table 1. the effect of the concentration of solid bioslurry in feed on the growth and survival of milkfish treatments parameter ± std growth rate (g) survival rate (%) control (commercial feed) 33,42 ± 6.67 a 65,33 ± 6,11 a 20% bioslurry 47,57 ± 7.69 a 68,00 ± 2,00 a 35% bioslurry 64,94 ± 4.01 b 86,67 ± 4,16 b 50% bioslurry 100,05 ± 6.98 c 96,67 ± 1,15 c note: different superscripts in the same column indicate significant differences between treatments (sig. <0.05) at the 95% confidence level. international journal of applied biology, 6(2), 2022 253 based on the results of the analysis of variance (anova), the effect of the concentration of solid bioslurry in the feed had a significant (sig<0.05) effect on the growth and survival of milkfish with a 95% confidence level. the development a nd survival of milkfish increase with the increase in bioslurry solid waste in the feed. the best growth and survival rates were seen in the feed treatment with a 50% solid bioslurry waste concentration compared to the control treatment, which was 20% and 35% solid bioslurry. more specifically, the relationship between the concentration of bioslurry solid in feed and increased growth and survival of milkfish is presented in figure 3. figure 3a. graph of the relationship between the concentration of bioslurry in the feed and the growth of milkfish figure 3b. graph of the relationship between the concentration of bioslurry in feed and the survival of milkfish there was an increase in the growth and survival of milkfish in line with the rise in the concentration of solid bioslurry because it contains nutrients tha t can increase growth energy in milkfish as the organism that consumes it. as (zulaehah and supraptom 2018) mention, several benefits of bioslurry, includi ng complete and quality nutriti on and can be used as an excellent mixed feed ingredient for fish and eel. furthermore (singgih and yusmiati 2018) explained that solid bioslurry derived from biogas digesters has excellent potential to be used as raw material for fish feed because it contains nutrients in the form of amino acids, fatty acids, humic acids, vitamin b12, auxin hormone, cytokinins, antibiotics, and micronutrients such as iron (fe), copper (cu), zinc (zn), manganese (mn), and molybdenum ( mo) which play an essential role in the growth and survival of fish. in addition (alfarizi, repika, and furqan 2022) also reported that fish feed containing 40% bioslurry had a suitable nutritional composition for the growth of tilapia (oreochromis sp.), which contained 29.106% protein and 3.131% fibre. in additi on (rustidja 2003) also explained that fish feed which is formulated with a mixture of sludge (solid bioslurry waste) and other feed raw materials such as fish meal and soy flour does not reduce the quality of feed quality, in fact, the quality is the same as factory feed. international journal of applied biology, 6(2), 2022 254 growth and survival of milkfish fed with 50% solid bioslurry and various concentrations of liquid bioslurry table 2. the effect of concentration of liquid bioslurry as a source of probiotics in feed on the growth and survival of milkfish parameter ± std treatments growth rate (g) survival rate (%) control (without probiotics) 102,06 ± 5,56 a 95,33 ± 1,15 a 30 ml/ 100 g feed 118,86 ± 9,01 b 97,33 ± 1,15 a 40 ml/ 100 g feed 146,67 ± 4,99 c 95,33 ± 1,15 a 50 ml/ 100 g feed 165,33 ± 2,08 d 96,00 ± 2,00 a 60 ml/ 100 g feed 134,65 ± 4,15 c 95,33 ± 1,15 a note: different superscripts in the same column indicate significant differences between treatments (sig. <0.05) at the 95% confidence level. based on the analysis of variance (anova), the effect of the concentration of liquid bioslurry as a source of probiotics on feed containing 50% solid bioslurry waste had a significant (sig<0.05) effect on growth and had no significant impact (sig.> 0.05) on survival milkfish at the 95% confidence level. the growth of milkfish continues to increase along with the increasing concentration of liquid bioslurry waste as a source of probiotics in the feed up to a certain concentration (50 ml/100 gram of dinner). however, if the concentrati on of liquid bioslurry waste is increased, the growth of milkfish will decrease. so the best growth of milkfish was seen in the treatment of 50 ml of liquid bioslurry waste per 100 g of feed compa red to the treatment without liquid bioslurry waste or treatment with a higher concentration (60 ml/100-gram feed). in more detail, a graph of the relationship between the concentration of bioslurry wastewater as a source of probiotics in feed and the growth of milkfish is presented in figure 4. figure 4. graph of the relationship between the concentration of liquid bioslurry in the feed and the growth of milkfish international journal of applied biology, 6(2), 2022 255 the increase in the growth rate of milkfish fed with liquid bioslurry waste indicates that the feed consumed can be digested and absorbed correctly due to microorganisms in the liquid bioslurry waste. it can assist digestion, which increases growth, such as (zulaehah and supraptom 2018) states that liquid bioslurry contains probiotic microbial bioactivators and growth regulators (hormones). in addition, (rustidja 2003) explained that bioslurry contains coenzyme b12, an important substance that can help the growth process in fish. furthermore, (farihah 2020) reported that liquid bi oslurry contains amino acids, micronutrients (fe, mn, cu, zn, co, and mo), b vitamins, various hydrola se enzymes, organic acids, plant hormones (growth), antibiotics and humic acid. conclusion based on the study's results, it can be concluded that the concentrati on of solid bioslurry waste in the feed that can opti mize the growth and survival of milkfish is 50%. in compa rison, the c oncentration of liqui d bioslurry waste in feed containing 50% solid bioslurry can optimize milkfish. the growth rate is 50 ml/100 grams of feed. international journal of applied biology, 6(2), 2022 256 references alfarizi, lalu mariawan, baiq repika, and nurul furqan. 2022. fish feed formulation based on bioslury and its application in fish farmi ng communities. abdinesia: jurnal pengabdian kepada masyarakat 2(2): 4–6. [in indonesian] farihah, turik. 2020. community empowerment through utilization of biogas by-products (bio-slurry) in somoda ran hamlet, purwomarta ni village.. jurnal aplikasi ilmu-ilmu agama 20(1): 47–62. [in indonesian] fao. the state of world fisheries and aquaculture: sustainability in action, fao, rome, 2020, p. 206, https://doi.org/10.4060/ca9229en. hussain, m., hassan, h. u., siddique, m. a. m., mahmood, k., abdel-aziz, m. f. a., laghari, m. y., halima. 2021. effect of varying dietary protein levels on growth performance and survival of milkfish chanos chanos fingerlings reared in brackish water pond ecosystem. the egyptian journal of aquatic research. doi:10.1016/j.ejar.2021.05.001 islam, m. a., biswas, p., sabuj, a.a.m., haque, z. f., saha, c. k., alam, m.m., rahman, m. t., saha, s. 2019. microbial load in bio-slurry from different biogas plants in bangladesh. journal of advanced veterinary and animal research. vol 6(3): 376–383 masriah, a. 2020. addition of cattle rumen fluid waste at various levels of carbohydrates in feed on nutrient retention and chemical composition of milkfish body (c hanos chanos forsskal). octopus : jurnal ilmu perikanan. vol. 9(1):33-38. [in indonesian] nasser s. flefil, asmaa ezzat, ahmad m. aboseif, asmaa negm el-dein, lactobacillus fermented wheat bran, as an economic fish feed ingredient, enhanced dephytinization, micronutrients bioavailability, and tilapia performance in a biofloc system, biocatalysis and agricultural biotechnology, volume 45, 2022, 102521, issn 1878-8181, https://doi.org/10.1016/j.bcab.2022.102521. nasution, z & yanti, b.v.i. 2015. adoption rate of tiger prawn cultured on brackiswater fish pond. j. kebijakan sosek kp vol. 5 (1):1-9 romadhon, i.k., nur koma r., r. yulianingsih. 2013. desain optimal pengolahan sludge padat biogas sebagai bahan baku pelet pakan ikan lele (optimal design of solid sludge from biogas processing as raw catfish feed pellets). jurusan keteknikan pertanian fakultas teknologi pertanian unbra malang. [in indonesian] rustidja. 2003. the effect of giving mud in feed rations on the growth rate of tilapia kado seeds (oreochromis sp.). jurnal perikanan ugm 5(1): 25–32. [in indonesian] seran, p. r.ch., ati, v. m. & kadang, l. 2020. analysis of the nutrient content of solid waste (sludge) in biogas production from bali cattle dung ( bos sondaicus). jurnal biotropikal sains vol. 17(1):1-10. [in indonesian] singgih, bambang, and yusmiati. 2018. utilization of residues/dregs of biogas production from ani mal husbandry waste (bio-slurry) as a source of organic fertilizer. jurnal kelitbangan 6(2): 139–48. [in indonesian] zaenab, s., & massiseng a.n. 2021. utilization of chicken i ntestine waste for growth of catfish. agrikan: jurnal agribisnis perikanan, 14(2): 193-198. doi: https://doi.org/10.29239/ j.agrikan.14.2.193-198. international journal of applied biology, 6(2), 2022 257 zulaehah, ina, and edi supraptom. 2018. “effect of liquid bio-slurry application on cauliflower growth ( brassica oleracea var . botrytis l .) varietas dataran rendah.” pendidikan biologi dan saintek iii: 161–66. [in indonesian] 1(1)14-21 contact&:&magdalena&litaay&&&&&&&mlitaay@fmipa.unhas.ac.id1 ©1international1journal1of1applied1biology1 1 141 abstract& research1 on1 the1 diversity1 of1 invertebrate1 epifaunas1 associating1 with1 mangroves1 in1 the1 balangdatu1 village1 of1 tanakeke1 regency,1 takalar1 district,1 south1 sulawesi,1 has1 been1 conducted1 from1 september1 to1 december12016.1the1objective1of1this1research1was1to1know1the1diversity1 of1 invertebrate1 epifaunas1 associating1 with1 mangrove1 in1 balangdatu1 village1 of1 tanakeke1 islands.1 sample1 collection1 was1 done1 by1 using1 explorative1 quantitatively1 plot1 methods1 with1 size1 21 x1 21 m,1 applied1 at1 three1different1stations.1data1analysis1includes1calculation1of1absolute1 density,1 uniformity1 index1 (shannonowiener),1 dominance1 index,1 and1 distribution1index.1the1results1showed1that1there1were1131species1of1epi1 fauna1of191tribes,1namely1littoraria1sp11,1cerithidea-cingulata,1littoraria1 sp12,1littoraria-pallescens,1episesarma-sp,1saccostrea-cucullata,1pagurus1 sp,1 terebraliasulcata,1 cerithideasp,1 neritaplanospira,1 nistohistrio,1 littoraria-scabra,1and1cassidula-vespertilionis.1the1highest1density1was1 found1in1littoraria-scabra1with1a1density1of10.721ind/m 2 .1diversity1indices1 are1low1which1indicate1depressed1environmental1conditions.1distribution1 index1values1across1stations1<11indicating1that1the1pattern1of1individual1 dispersion1tends1to1be1uniform.1 & issn1:12580o24101 eissn1:12580o21191 1 1 1 diversity&of&invertebrate&epifaunas&associating&with&mangrove&in& balangdatu&village&tanakeke&islands&of&south&sulawesi& 1 crisnawati,&magdalena&litaay,&dody&prisambodo,&and&slamet&santoso& 1 department1of1biology,1faculty1of1mathematics1and1natural1sciences,1hasanuddin1 university,1makassar,1indonesia1 1 1 & & & & & & & & & & & & & & & & introduction& mangrove1ecosystem1is1an1ecosystem1that1is1strongly1influenced1by1water1conditions1 that1 change1 at1 any1 time.1 it1 has1 an1 effect1 on1 the1 living1 water1 biota1 associated1 with1 the1 mangrove1 ecosystem.1 yuniarti1 (2007)1 stated1 that1 coastal1 areas1 are1 productive1 maritime1 environment1 that1 can1 be1 used1 directly1 or1 indirectly.1 potential1 mangroves1 as1 a1 source1 of1 nutrients1for1biota1living1is1as1a1residence,1feeding1ground,1nursery1ground1and1spawning1 ground.1adamy1(2009),1states1that1many1benthic1faunas1are1associated1in1mangrove1forests,1 of1which1has1important1economic1value.1 mangrove1ecosystems1provide1a1good1habitat1for1the1colonization1of1various1fauna1 such1 as1 shade,1 moist1 basic1 substrate,1 tree1 as1 a1 sticking1 point1 and1 most1 importantly1 the1 &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword1 diversity,1marine1 invertebrates,1 mangrove,11 balangdatu,11 tanakeke.1 article&history1 received151may120171 accepted1151july1201711 1 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international-journal-of-applied-biology,-1(1),-20171 151 abundance1of1organic1detritus1as1food1source1(rangan,12010).1thus,1the1mangrove1ecosystem1 has1very1big1influence1for1the1survival1of1every1mangrove1fauna.1 many1living1fauna1attach1themselves1to1mangrove1plants,1but1the1attachment1can1 cause1serious1problems1because1it1can1inhibit1the1survival1of1mangroves.1the1invertebrate1 epifaunas1are1barnacles,1oysters,1crabs1and1other1invertebrate1groups.1barnacle1can1be1a1big1 problem1for1mangroves1because1barnacles1release1the1sticking1liquid1that1can1be1bad1for1 mangrove.1 barnacle1 can1 create1 stress1 of1 mangrove1 plants1 that1 hamper1 the1 process1 of1 photosynthesis1which1should1be1a1source1of1food1for1all1living1creatures1(tapilatu1and1pelasula,1 2012).11 information1on1the1ecology1of1invertebrate1epifaunas1in1mangrove1forests,1especially1 tanakeke1 island1 is1 indispensable1 in1 efforts1 to1 manage1 mangrove1 biological1 resources.1 however,1the1availability1of1this1information1is1still1very1limited.1therefore,1a1research1is1done1 entitled1the1diversity1of1invertebrate1epifaunas1that1is1associated1with1mangrove1in1the1coastal1 area1of1balangdatu1village1of1tanakeke1islands,1takalar1regency,1south1sulawesi.1 & materials&and&methods& tools&and&materials& the1tools1used1in1this1research1are1nylon1rope,1chisel,1scissors,1plastic1sample,1roller1 meter,1 stationery,1 digital1 camera,1 underwater1 camera,1 plot1 size1 21 x1 21 m,1 thermometer,1 bootish,1 permanent1 marker,1 loop1 (magnifying1 glass),1 box,1 identification1 book,1 global1 positioning1system1(gps),1hand1refractometer,1doometer,1phometer.1materials1used1in1this1 study1including170%1alcohol1substrate,1label1paper1and1sample1of1invertebrate1epifaunas.1 1 research&methods& the1research1method1used1is1the1transect1method1combination1plot.1data1collection1 was1done1by1purposive1sampling1method1using1quadratic1transect.1 1 description&of&research&location& the1 research1 area1 was1 located1 in1 balangdatu1 village,1 mappakasunggu1 district,1 tanakeke1 islands,1 takalar1 district,1 south1 sulawesi.1 geomorphologicaly,1 tanakeke1 is1 an1 archipelago1consisting1of1several1islands1linked1to1the1expanse1of1mangrove1forest,1the1total1 land1area1of1tanakeke1islands1reaches18.371km 2 1(nurdin,12014).1the1sampling1point1consists1 of131(three)1stations1(figure11).1station1(1)1one1located1in1the1urban1area1of1cambangloe1 located1near1the1residential1population1with1sandy1mud1substrate.1station121(two)1is1located1 in1balangloe1urban1area1located1near1pond1with1mangrove1condition1which1partially1logged.1 station1 31 (three)1 is1 located1 in1 bungung1 lompoa1 urban1 area1 which1 is1 close1 to1 seaweed1 cultivation1with1mangrove1condition1that1there1is1planting1activity.1 1 1 1 1 1 1 1 1 figure&1.&map&of&tanakeke&island,&takalar&district,&south&sulawesi.& (source:&google&earth,&2016)& international-journal-of-applied-biology,-1(1),-20171 161 work&procedures& the1data1were1collected1by1purposive1sampling1method1(transect1quadrate)1at1three1 stations1by1hand1on1plot1size121x121m1with1transect1length11001m.1the1samples1of1encapsulated1 epifauna1were1inserted1into1plastic1clip1differentiated1between1those1found1in1roots,1stems,1 and1mangrove1leaves.1the1samples1were1then1washed1and1preserved1using170%1alcohol1then1 taken1to1the1laboratory1for1identification1and1analysis.1 1 sample&identification& the1samples,1that1had1been1preserved1using170%1alcohol,1were1then1identified1by1 observing1 morphological1 characteristics1 based1 on1 dharma1 reference1 (2005).1 the1 identification1 was1 conducted1 at1 the1 laboratory1 of1 environmental1 and1 marine1 sciences,1 department1of1biology,1faculty1of1mathematics1and1natural1sciences,1hasanuddin1university.1 1 oceanographic&parameters& environmental1parameters1included1temperature,1salinity,1ph,1dissolved1oxygen1(do)1 and1substrate1were1recorded1during1field1campaign.1 1 data&analysis& the1 collected1 species1 of1 invertebrate1 epifaunas1 were1 identified1 by1 using1 an1 identification1book1according1to1dharma,12005.1data1of1invertebrate1epifaunas1were1then1 analyzed1 by1 using1 absolute1 density1 formula,1 index1 of1 species1 diversity,1 uniformity1 index,1 dominance1index,1and1distribution1index/distribution1type.1 & results&and&discussion&& the1 identification1 of1 invertebrate1 epifaunas1 revealed1 that1 there1 are1 131 species1 of1 91 families1consisting1of1two1phylum1namely1phylum1molluska1and1arthropoda.1the1species1of1 invertebrate1epifaunas1found1in1the1three1research1stations1are1generally1gastropod1species,1 only1a1few1species1of1crustaceans1and1bivalves,1a1total1of1131species1found1among1them1are1in1 station11,1as1many1as1121species,1in1station121are1111species1and1at1station131are171types1(table1 1).1 table&1.&species&composition&of&invertebrate&epifaunas&found&in&balangdatu&waters& note:&o1=1absent11√1=1present1 no& species& st&1& st&2& st&3& total&no&of& ind/species& 11 littoraria-sp11√1 √1 o1 441 21 cerithidea-cingulata√1 √1 o1 501 31 littoraria1sp12√1 √1 o1 191 41 littoraria-pallescens√1 √1 √1 751 51 episesarma-sp√1 √1 √1 481 61 saccostrea-cucullata√1 o1 √1 221 71 pagurus1sp√1 o1 o1 381 81 terebralia-sulcata√1 √1 √1 221 91 cerithidea-sp√1 √1 √1 1191 101 nerita-planospira√1 √1 √1 161 111 nerita-histrio√1 √1 o1 181 121 littoraria-scabra√1 √1 √1 1891 131 cassidula-vespertilioniso1 √1 o1 41 international-journal-of-applied-biology,-1(1),-20171 171 absolute&density& the1observations1showed1absolute1density1with1different1ranges1in1station1i1with1the1 number1of1fauna1species1observed1attached1to1the1 2 nd1root,1721ind/m 2 1on1stem10.961ind/m 2 1 and1 on1 leaves1 0.491 ind/m 2 .1 meanwhile,1 in1 the1 station1 ii,1 the1 number1 of1 observed1 fauna1 attached1at1the1root1is11.791ind/m 2 ,1on1the1stalk1is10.781ind/m 2 1and1on1leaves1is10.291ind/m 2 1 and1at1station1iii1with1the1number1of1individuals1attached1to1the1roots1is11.431ind/m 2 ,1on1stem1 is10.171ind/m 2 ,1and1on1leaves1is10.401ind/m 2 .1based1on1these1data1the1number1of1epifauna1 found1more1in1the1roots1and1stems1of1mangrove1plants1compared1to1the1leaves.1the1absolute1 density1 data1 found1 in1 each1 station1 research1 area1 was1 3.351 ind/m 2 ,1 while1 the1 number1 of1 individual1with1the1lowest1densities1was1found1in1station131with1the1density1of12.131ind/m 2 .1 & index&of&species&diversity&(h')& the1results1showed1that1the1diversity1of1epi1fauna1species1is1low1due1to1the1ecosystem1 condition1has1been1disturbed1by1the1existence1of1human1activities1in1the1area,1such1as1logging1 conducted1in1mangrove1forests1to1be1used1as1firewood,1waste1is1thrown1into1the1waters,1so1 that1the1research1station1obtained1the1number1of1species1diversity1ranged1between11.331to1 1.87.1this1result1corresponds1to1abbas,1a.a.'s1observations.1(2015),1states1that1the1value1of1 low1biodiversity1is1due1to1the1influence1of1human1activities1in1the1study1sites.1furthermore,1 arbi1(2008),1states1that1the1high1value1of1species1diversity1can1be1caused1by1the1number1of1 species1or1individual1fauna1obtained1in1abundant1amounts1than1other1types.1three1important1 ecosystems1in1coastal1areas1are1sea1grass1beds,1coral1reefs1and1mangrove1forests1that1are1the1 main1habitat1of1almost1all1aquatic1fauna.1 according1to1brower1et-al.1(1990),1all1computed1values1have1a1biodiversity1value1of1less1 than121(h1<2).1this1condition1shows1low1species1diversity1which1means1low1community1stability1 and1water1conditions1have1been1disrupted.1 1 uniformity&index&(e)& the1results1showed1stable1category1with1index1values1ranging1from10.571to10.751stable1 community1means1that1the1spread1of1individual1fauna1is1relatively1same1or1uniform1although1 the1type1of1gastropod1is1greater1than1the1other1types.1the1existence1of1relatively1uniform1 environmental1conditions1allows1balance1distribution1of1epi1fauna1species1primarily1in1the1 mangrove1ecosystem1balangdatu1village.1in1the1research1of1yulianti1(2013),1in1marabombang1 waters1of1pinrang1regency1of1south1sulawesi1stated1that1the1result1of1data1analysis1of1epi1fauna1 from1gastropod1type1ranged1from10.37o0.56.1moreover,1pratiwi1(2009),1reported1that1in1the1 mangrove1area1of1delta1mahakam,1east1kalimantan1the1value1of1uniform1index1of1type1of1epi1 fauna1of1crustaceans1species1ranged1from10.53o2.021which1illustrated1that1the1distribution1of1 epi1fauna1in1the1area1was1relatively1the1same1or1evenly1distributed.1 & index&of&dominance&(c)& the1result1of1data1analysis1on1epi1fauna1shows1the1value1of1dominance1index1ranged1 from10,19o0,34.1this1index1value1belongs1to1low1category1and1shows1that1at1research1station1 there1is1no1dominance1of1certain1type1of1epi1fauna1or1community1in1stable1condition1so1that1 there1is1no1certain1species1which1has1a1high1number1of1indiv.1based1on1the1results1of1research1 nurrudin1(2015),1in1the1village1of1tungkal1i1tanjung1jabung1west,1the1dominance1index1(c)1in1 each1study1location1ranged1from10.121to10.221indicating1that1there1is1no1type1which1dominates1 in1the1area1although1the1type1of1gastropoda1much1more1but1not1dominate.1in1contrast1to1the1 results1 of1 research1 gladys1 (2013),1 in1 the1 area1 tongkeina1 manado1 which1 has1 a1 value1 of1 international-journal-of-applied-biology,-1(1),-20171 181 dominance1index1ranged1from10.157o0.3501which1means1that1there1are1almost1no1species1in1 this1community.1 & index&of&distribution/distribution&type&(id)& the1result1of1data1analysis1showed1that1the1pattern1of1dispersion1of1epi1fauna1species1 in1mangrove1plants1in1general1can1be1categorized1uniformly,1with1the1index1value1in1each1 research1 station1 ranged1 from1 0,52o0,81,1 in1 contrast1 to1 research1 of1 fitriana1 (2006),1 which1 stated1index1value1of1spread1in1forest1mangrove1ngurah1rai1bali1forest1park1especially1in1 research1station1has1the1pattern1of1spreading1(>11)1that1is1ranged1between11,584o1,869.1this1 indicates1the1presence1of1resources1to1support1organisms1that1are1clumped1and1unevenly1 distribute1across1all1observation1sites.1 according1to1brower1et-al.1(1998),1if1the1spread1index1is1less1than1one1(id1<1)1then1the1 dispersion1pattern1is1uniform1distribution1pattern,1if1spread1index1is1equal1to1one1(id1=11),1then1 the1dispersion1pattern1is1random,1whereas1if1the1spread1index1is1more1than1one1(id>11),1then1 the1dispersion1pattern1formed1is1clustered.1 thus1it1can1be1concluded1that1the1pattern1of1epifauna1distribution1in1mangrove1plants,1 especially1 in1 the1 research1 station1 located1 in1 balangdatu1 village1 tend1 to1 be1 uniform.1 this1 pattern1of1uniform1distribution1according1to1(odum11993),1occurs1because1of1the1existence1of1 individual1fauna1are1having1competition1so1that1it1encourages1the1division1of1space1evenly.1 1 environmental&parameters& the1result1of1measurement1of1environmental1parameters1in1study1area1is1described1 in1table12.1 1 temperature& the1 results1 of1 the1 measurement1 and1 analysis1 of1 water1 samples1 at1 the1 study1 sites1 showed1 a1 temperature1 range1 between1 30o32 o c,1 this1 range1 is1 still1 feasible1 for1 the1 life1 of1 epifauna1which1can1generally1live1in1a1wide1temperature1range.1according1to1the1ministerial1 decree1no.1511year120041air1quality1/1air1temperature1in1mangrove1area1with1natural1category1 ranged1 between1 28o32 o c.1 while1 based1 on1 research1 conducted1 by1 litaay1 et1 al.1 (2014)1 conducted1in1mangrove1area1of1bontolebang1waters1of1selayar1regency1of1south1sulawesi,1the1 result1of1temperature1measurement1at1each1station1showed1the1same1range1ranging1from131o 38 o c1the1range1is1still1feasible1for1the1life1of1the1sticking1fauna1such1as1molluscs1this1is1due1to1 the1morphological1form1of1the1epi1fauna1that1has1the1shell.1 1 salinity& the1result1of1measurement1of1water1samples1at1each1research1station1obtained1salinity1 range1between125o30º/oo,1salinity1range1in1mangrove1area1in1each1research1station1is1good1for1 mangrove1growth1and1organism1living1in1the1area.1this1is1in1accordance1with1the1decree1of1 the1ministerial1decree1no.1511year12004,1which1stated1salinity1in1mangrove1area1with1natural1 category1ranged1from130o341º/oo.1while1the1results1of1research1conducted1by1muhsin1(2016),1 salinity1value1in1the1mangrove1area1of11kendari1gulf,1especially1at1the1research1station1that1 range1from129.5o301º/oo.1 1 degree&of&acidity&(ph)& the1result1of1data1analysis1of1ph1value1showed1at1all1stations1ranged1from17,9o8,3.1the1 value1is1categorized1appropriate1and1it1still1supports1the1life1of1organism,1especially1epifauna1 international-journal-of-applied-biology,-1(1),-20171 191 at1research1location1in1mangrove1area1of1balang1village1of1tanakeke1archipelago1of1takalar1 regency.1ennawati1et1al.,1(2013)1reported1that1ph1value1in1mangrove1forest1of1tongkeotongke1 village,1sinjai1regency1ranged1between17,40o8,08.1 1 dissolved&oxygen&(do)& the1result1of1dissolved1oxygen1(do)1concentration1in1each1station1is1quite1high1(>151 mg/l)1which1is1between17,7o8,21so1that1it1can1support1the1life1of1the1aquatic1organism.1this1is1 in1accordance1with1the1regulation1of1the1minister1of1environment1no.1511year12004,1dissolved1 oxygen1content1in1mangrove1area1with1natural1category1that1is1more1than1five1(>151mg/l).1 while1 research1 conducted1 by1 adamy1 (2009)1 the1 average1 dissolved1 oxygen1 of1 waters1 in1 panimbang1coastal1mangrove1habitat1ranged1between17,13o7,361mg/l.1 1 substrate&characteristics& substrate1conditions1at1research1stations1in1three1locations1consisting1of1cambangloe,1 balangloe1and1bungunglompoa1have1substrate1quality1of1sandy1mud1in1the1bottom1of1the1sea.1 this1indicates1that1the1basic1substrates1present1in1the1three1research1stations1can1still1support1 the1mangrove1growth1activity1and1biota1life1that1exist1on1the1island,1in1accordance1with1the1 statement1of1bengen1(2004),1mangrove1generally1grown1in1intertidal1areas1of1muddy,1clay1and1 sandy1soils.1 based1on1research1conducted1by1kamalia.m1(2014),1substrate1type1found1at1research1 station1is1mud1and1sandy1mud1found1many1number1of1gastropod1species1so1that1it1can1be1said1 that1type1of1substrate1is1very1good1for1life1of1gastropod1type1living1in1that1area.1 1 table&2.&value&of&environmental&parameter& & station& environmental&parameter&& temperature& (ºc)& salinity& (º/oo)&& ph& do& (mg/l)& substrate& characteristics&& water& air& a11 321 281 251 8,31 8,01 muddy1sand11 a21 311 281 251 8,31 8,01 muddy1sand11 a31 311 261 251 8,31 7,91 muddy1sand11 b11 321 271 291 8,21 7,81 muddy1sand11 b21 321 291 281 8,21 8,01 muddy1sand11 b31 311 281 301 8,21 7,71 muddy1sand11 c11 311 291 281 7,91 8,21 muddy1sand11 c21 321 291 281 7,91 8,21 muddy1sand11 c31 301 281 271 7,91 8,21 muddy1sand11 1 conclusions& the1study1concludes1that1there1are1131species1of1invertebrate1epifaunas1which1belongs1 to191families1are1living1on1mangrove1area1of1balangdatu1village1of1tanakeke1archipelago1of1 takalar1regency1of1south1sulawesi.1the1index1of1species1diversity1in1the1biological1index1of1 each1station1is1low1ranging1from11.331to11.881reveals1that1the1mangrove1area1of1balangdatu1 village1can1be1categorized1as1disturbed.1 & international-journal-of-applied-biology,-1(1),-20171 201 references& abbas.a.a.,12015.1biodiversity1of1gastropoda1epifauna1in1mangrove1area1bontolebang1waters1 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inner1bay1of1 ambon.1department1of1marine1science1and1technology1fpikoipb.1journal1of1tropical1 marine1science1and1technology.14(2):267o279.1 yulianti,12013.1biodiversity1of1gastropoda1in1marabombang1sea1region1of1pinrang1regency,1 south1sulawesi.1journal1of1marine1science1and1fisheries.123(3):167o175.1 international-journal-of-applied-biology,-1(1),-20171 211 to&cite&this&article:&& crisnawati,1 litaay,1 m.,1 prisambodo,1 d.1 &1 santoso,1 s.1 2017.1 diversity1 of1 invertebrate1 epifaunas1 associating1with1mangrove1in1balangdatu1village1tanakeke1islands1of1south1sulawesi.1internationaljournal-of-applied-biology.11(1):14o211 1 yuniarti,12007.1management1coastal1areas1of1indonesia.1(case1study:1community1based1coral1 reef1management1in1riau1islands):1kepri.1381pages.1[in1indonesian]1 1 1 1 1 1 1 1 1 1 contact : olaniyi alaba olopade olaniyi.olopade@uniport.edu.ng 161 abstract some important information on the population biology and stock assessment of african moony fish (monodactylus sebae) was scrutinized based on monthly length frequency data collected from new calabar river, nigeria from february 2020 to march 2021. the estimated von bertalanffy growth parameters were growth performance index 2.91 per year, asymptotic length (l∞) and growth curvature (k) were 36.54 cm and 0.61yr-1 respectively. the estimated theoretical age at birth (to) and longevity for the assessed fish species were 0.55 years and 2.91 years, respectively. the total mortality (z), natural mortality (m), fishing mortality (f), for m. sebae were 1.87 year-1, 1.23 year-1 and 0.64 0.64 year-1 respectively. the length at first capture (lc) was 8.56cm. the exploitation rate (e) and maximum exploitation rate (emax) were calculated as 0.34 and 0.36 respectively. the recruitment pattern occurs throughout the year, with only one recruitment peaks in may with 19.9 % recruits. biological reference points: emsy which depicts exploitation rate producing maximum yield of a cohort indicated that, the exploitation rate which maximizes yield per recruit produced values of emax was 0.36 while e50 was 0.23 for m. sebae indicates that the current exploitation rate was below the maximum sustainable yield, indicating that this stock was underexploited. issn : 2580-2410 eissn : 2580-2119 stock assessment of the african moony fish (monodactylus sebae) (cuvier, 1829) in the new calabar, nigeria olaniyi alaba olopade1, henry eyina dienye1, desire precious dike1 1 university of port harcourt, faculty of agriculture, department of fisheries, choba, eastwest road, pmb 5323, port harcourt, rivers state, nigeria introduction fish population are subject to natural control processes that continually modify and adjust the structure and abundance of population and their life cycle in response to a wide range of factors (milner et al., 2003), apart from those caused by human activi ties such as overfishing and habitat alteration, as well as pollution and lately climate change. stock assessment is the basis for understanding changing fishery patterns and issues such as habitat destruction, predation and optimal harvesting rates (olopa de et al., 2019). regular stock assessment and reference points are required for monitoring and for determining whether the stocks are subject to overfishing or overfished and develop fishery management plans (mohamed et al., 2021). kebtieneh et al., (2016) stated that the basic purpose of stock assessment is to provide decision-makers with the information necessary to make rational choices on the optimum level of exploitation of aquatic living resources such as fish. stock assessment forms the basis for calculations leading to knowledge of the growth, mortality, recruitment and other fundamental parameters of their populations (olopade et al., 2019). open access international journal of applied biology keyword monodactylus sebae; growth; mortality; asymptotic length; maximum sustainable yield; new calabar river article history received august 3, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 162 the fish family monodactylidae contains six extant species in two genera, monodactylus and schuettea. monodactylidae is found in the eastern tropical atlantic ocean along the african coast from senegal to angola and the canary islands (desoutter 1990). monodactylidae is primarily found in estuaries and coastal mangrove habitats, but is able to live in both fres hwater and marine habitats (schneider 1990). reproduction takes place in marshes and lower courses of rivers, sometimes ascending over long distances into freshwater (bauchot 2003). they are laterally compressed with an approximately diamond shape body with a long anal and dorsal fin extended distance that gives this fish a square like look african monny, monodactylus sebae is a member of the family and the only species identified so far in nigerian fresh water (adesulu & sydenham 2007). even though, it is marine species which can survive in fresh water for some times. this species lacks the yellowish coloration in the caudal fin seen in other species of monodactylus (monks 2006). this species is economically important as it can be found in the aquarium trade, and lately, this fish has assumed importance in the niger delta region of nigeria by virtue of its acceptance as a food fish in both fresh and dried conditions. according to the iucn (2021) record, the fish is assessed as at least concern (lc). however, there is massive fishing pressure on this species in the new calabar river and in other water bodies in the niger delta region. this could be attributed to the absence of commonly important fish species. in spite of the importance of the monodactylus sebae in nigeria there is no information on stock assessment in the country or elsewhere. the objective of the present study was to assess the growth parameters, mortality rates, probability of capture, recruitment pattern, yield per recruit, and virtual population analysis of monadactylus sebae in the new calabar river, nigeria. materials and methods time and place the new calabar river, nigeria is a partially mixed estuary system that lies between latitude 4°25′n and longitude 7°16′e (olopade et al. 2019) (figure 1). the entire river course is situated in the coastal area of the niger delta and empties into the atlantic ocean. figure. 1 map of new calabar river, nigeria international journal of applied biology, 6(2), 2022 163 data collection fish samples were collected from the new calabar river at two fishing landing sites, namely choba and ogbogoro from the local fishermen using different gears. these samples were taken twice monthly, starting from the month of february 2020 to march 2021. the species were identified to the species level using the identification keys by monks (2006). fish specimens were immediately iced and transported to the laboratory to measure the weight (to the nearest 0.5 g) and length (to the nearest 1.0 cm) of each specimen. data analysis the length-frequency data for m. sebae were collected monthly from a number of different gears at all sites and then grouped into class intervals for analysis. the data were analyzed using fisat ii (fao-iclarm stock assessment tools) as explained in details by (gayanilo et al. 2005). the von bertalanffy (pauly 1980) growth parameters, asymptotic length l ∞ and annual growth coefficient k were computed by elefan i (electronic length frequency analysis) method (beverton & holt 1966). the total mortality rate (z) was estimated by lengthconverted catch curve (pauly 1984). the natural mortality rate ( m) was also calculated by using pauly’s empirical formula (pauly 1980). the fishing mortality rates ( f) was then calculated by the difference between (z) and (m). the rate of exploitation (e) was calculated by the quotient between fishing and total mortality: e= f/z (pauly 1984). relative yield per recruit (y/r) was estimated using the model of beverton and holt (beverton and h olt 1966) as modified by pauly and soriano (pauly and soriano 1986) and incorporated in the fisat software. lengths at first capture (lc50) and first maturity (lm50): the left ascending part of the length converted catch curve was used to estimate the probabilities of length at 50, 75, and 95 capture which correlates with the cumulative probability at 50, 75 and 95 percent, respectively (pauly 1984). the length at first maturity (lm50) was estimated. the length at first maturity was estimated using the expression: length at first maturity (lm50) = 2 * l ∞ /3 (hoggarth et al. 2006). one year recruitment pattern was obtained by projecting the length frequency data backward on to the time axis as described in the fisat routine. biological reference points: emsy which depicts exploitation rate producing maximum yield of a cohort and e0.5 implying exploitation rate under which the population is reduced to half its virgin biomass were computed together with the corresponding fishing mortality rate (i.e. fmsy and f0. 5).virtual population analysis – vpa the length-based virtual population analysis (vpa) was performed on the pooled annual length frequencies from the fishery to estimate the mean number in the population and the overall fishing mortality by length group. results and discussion results length-frequency analysis a total of m.sebae specimens collected for this study was 390. the size frequency distribution of the m. sebae population (figure 2) shows that it was of the unimodal type. they were grouped into twenty classes of total length frequency with the collected samples falling in the length range of 7.7 to 34 cm and with a mean of 13.53±2.94. the 12.8cm tl size group was numerically dominant, followed by 10.8 cm, and constituted 53.86% of the total population. international journal of applied biology, 6(2), 2022 164 figure 2. graph of respondent's profile based on age estimation of growth parameters and growth performance index figure 3 shows the growth curve generated by elefan i for m. sebae. the asymptotic length (l∞) and growth rate (k) were 36.54 cm tl and 0.61 year-1, respectively (table 1).the growth performance index or phi prime (φ′) for m. sebae recorded from new calabar river was 2.91. the estimated theoretical age at birth (to) and longevity for the assessed fish species were 0.55 and 2.91 respectively (table 1). table 1: population parameters of monodactylus sebae in the new calabar river, nigeria indicators unit value growth rate (k) year-1 0.61 asymptotic length (l∞) cm tl 36.54 age at birth (to) years -0.55 longevity (tmax) years 4.37 growth performance index(phi) 2.91 natural mortality rate (m) year-1 1.23 total mortality rate (z) year-1 1.87 fishing mortality rate (f) year-1 0.64 exploitation rate (e) 0.34 m/k 2.02 e-10 0.26 e-50 0.23 e-max 0.36 l25 cm 7.06 l50 cm 8.56 international journal of applied biology, 6(2), 2022 165 l75 cm 10.20 lm cm 29.75 figure 3 elefan-1growth curve of m. sebae in the new calabar river, nigeria the length-converted catch curve for m. sebae to estimate the annual total mortality rate (z) is shown in figure 4. the natural mortality (m/year) as per pauly’s empirical formula was calculated as 1.23 year-1 and the total mortality (z) as 1.87 year-1 while the fishing mortality (f) was taken by subtraction of m from z and was 0.64 year-1 (table 1). the current exploitation rate (e current) computed as f/z= 0.34 for m.sebae and the m/k ratio found was 2.02. (table1). international journal of applied biology, 6(2), 2022 166 figure 4 growth performance index (kscan routines) of m. sebae in the new calabar river, nigeria the goodness-of-fit was at rn= 0.344 figure 5 length converted catch curves of m. sebae in the new calabar river, nigeria length at first capture l50 for m. sebae in the new calabar river, nigeria the logistic of the probability of capture routine of m. sebae presented in figure 5. the length at which 50% of the stock biomass is vulnerable to capture estimated at l 50 = 8.56cm. the l25 was calculated as 7.06cm while l75 was found to be 10.20cm for m. sebae. the length at first maturity (lm50) was estimated at 24.36 cm. in this study, the reproductive load ratio (l50/ l∞) (8.56/36.54) = 0.23 for m. sebae indicating the length at first capture is quite low for the population (table 1). international journal of applied biology, 6(2), 2022 167 figure 6 length at first capture l50 for m. sebae in the new calabar river, nigeria recruitment pattern of m. sebae in the new calabar river, nigeria as shown in figure 7, the annual recruitment pattern of m. sebae indicated that recruitment occurred throughout the year with only one prominent peak in may with 19.9 % recruits, while the minor peak occurred in september with 16.6 % recruits. figure 7 recruitment pattern of m. sebae in the new calabar river, nigeria relative yield per recruit (y'/r) and biomass per recruit (b'/r) analyses of m.sebae in the new calabar river, nigeria the beverton-holt relative yield per recruit (y’/r) and relative biomass per recruit (b’/r) estimated using selective ogive procedure of fisat for the species is given in the figure below. the analysis indicated that, the exploitation rate which maximizes yield per recruit produced values of emax was 0.36 while e50 was 0.23 for m. sebae (figure 5). international journal of applied biology, 6(2), 2022 168 figure 8 relative yield per recruit (y'/r) and biomass per recruit (b'/r) analyses of m. sebae in the new calabar river, nigeria length-structured virtual population analysis of m. sebae in the new calabar river, nigeria figure 9 showed that natural mortality is the only cause of loss of m. sebae at lengths from 6.8 cm to 9.8 cm. m. sebae was caught by fishing gear in sizes from 6.8 cm, with the highest quantities in lengths from 10.8 cm to 12.8 cm. the fishing mortality were at lowest at 22.8 and 30.8 cm. the smallest length groups have lower catches (harvesting rates) than the largest ones (table 2), indicating that the fishing mortality rate is size specific. natural losses were highest among individuals within the length range of 6.8 to 9.8 cm and then decreased gradually to the length group of 30.8 cm. figure 9: length-structured virtual population analysis of m. sebae in the new calabar river, nigeria discussion l eng thf requenc y di s tri buti ons obs erved i n thi s s tudy provi de s na ps hots of the s i ze s truc ture of the s pec i es. length frequency distribution showed that small size fish were the most abundant in the catches. the 12.8cm tl size group was numerically dominant, followed by 10.8 cm, and constituted 53.86% of the total population. the growth parameters in this study were estimated using the length frequency data in elefan 1 software, revealing the following result: asymptotic length (l∞) as 36.54cm, growth curvature (k) as 0.61 per year, growth performance index as 2.91, and t0 (per year) as -0.55. the growth rate (k) of 0.61 year1 from the current study signifies that m. sebae exhibited a fast growth rate, evinced by the low longevity of 4.37 years. the growth coefficient (k) of m. sebae was high (2.91). the greater of these values indicates that the fish growth rate to achieve the maximum size is faster. the total mortality (z), natural mortality (m/year), fishing mortality (f), and exploitation rate (e) of m. sebae were found to be 1.87, 1.23, 0.64, and 0.34 respectively. natural mortality of fishes becomes much heavier in an unexploited population than in an international journal of applied biology, 6(2), 2022 169 exploited one. however, since value for natural mortality (1.23) exceeded fishing mortality (0.64), the stock is not overexploited. according to (macer 1977) the consistency of the estimated natural mortality rates (𝑀) was ascertained using the 𝑀/𝐾 ratio, which has been reported to be within the range of 1.12 and 2.5 for most fishes. the 𝑀/𝐾 ratio in this study was 2.02 which was within the normal range. exploitation rate allows for determining whether a stock is overfished or not based on the assumption that the optimal value of e is 0.5 (gulland 1971; pauly 1983). based on the exploitation rate (e) of m. sebae in this study (0.34), it clear that the stock is currently underexploited. in this study, the length at first maturity (lm50=29.75 cm) was higher than the length at which this species become vulnerable to the fishing gears (lc50 = 8.56cm) indicating that this species is harvested before they could reach the matured stage, a characteristic feature of growth overfishing (fröese 2004). furthermore, the critical length at capture which is ratio of lc50/ l∞ (8.56/36.54= 0.23), indicated that it was lower than 0.5. this signals the harvesting of more juvenile fish species (pauly & soriano 1986). the presence of many small -sized fish species in the catches could be explained by the unselective use of small mesh sized fishing gears. continuous exploitation of this at this level could result in growth overfishing and eventually lead to a possible collapse. in m.sebae population, a two-peak recruitment pattern was observed and the peaks were during may and september. this result was in line with (pauly 1982) observed a double recruitment pulse per year for tropical fish species and for short lived species. the recruitment pattern has concerned with the spawning time (fiorentino et al . 2008). the present study agrees with spawning seasons reported for tropical fish species. the yield per recruit model is an efficient approach for fish stock assessment, consisting in an important tool to the management of fisheries (sparre & venema 1997). the predicted emax of the selective ogive procedure for m. sebae (0.36) was higher than the current exploitation rate e (0.34) showing that m. sebae was lower than both target reference points. this is a further implication that the stock of the species is underexploited. virtual population analysis (vpa) data were utilised to make management decisions and provide more information about the status of fish stocks in terms of growth, recruitment, and overfishing (chen et al. 2008). according to virtual population analysis (vpa), the 12.8 cm length group was more vulnerable to fishing and more harvested. this implies that more individuals are caught before they reached length at first sexual maturity. this situation is also described by (fröese 2004) as growth overfishing; when fishes are caught before they can realize their full potential. if this condition continues without any efforts to regulate m. sebae stock, the fish species will be threatened in the long term. the protection of juveniles throug h fish size stipulation and mesh size limitation is probably a key factor for the s ustainability of this species. this can be achieved by compliance with or enforcement of the mesh size (7.5 cm) recommended as the standard as the minimum mesh size for all inland water bodies in nigeria by a joint effort between resource users and the governing authority. conclusion the present study is the first effort to evaluate the growth parameters some important information on the population biology and the stock assessment of the species m. sebae from the new calabar river, nigeria. the study revealed that the m. sebae stock was underexploited, and more individuals are caught before they reached length at first sexual maturity. this study suggests that mesh size regulations will be required to protect m.sebae in the new calabar river in nigeria. international journal of applied biology, 6(2), 2022 170 references adesulu, e.a. & sydenham, d.h.j. 2007. the freshwater and fisheries of nigeria, macmillan nigeria publishers, lagos, nigeria, 397. baijot, e. & moreau, j. 1997. biology and demographic status of the main fish species in the reservoirs of burkina-faso. in: baijot, e., moreau, j., bouda, s. 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(2006) stock assessment for fisher\ management-a framework guide to the stock assessment tools of the fisheries management science programme (fmsp). fisheries technical rome. fao pp: 261. iucn 2021. iucn red list of threatened species. downloaded on 05 may, p. 2021. kebtieneh, n.; alemu, y. & tesfa, m. 2016. stock assessment and estimation of maximum sustainable yield for tilapia stock (oreochromis niloticus) in lake hawassa, ethiopia. agric, for fish. 5(4):97-107. international journal of applied biology, 6(2), 2022 171 macer, c.t. 1977. “some aspects of the biology of the horse mackerel [ trachurus trachurus (l.)] in waters around britain,” j of fish biol. 10(1): 51–62. milner, n.j; elliott, j.m.; armstrong, j; gardiner, r.; welton, j.s. & ladle, m. 2003. the natural control of salmon and trout populations in streams. fisheries res. 62:111 125, doi:10.1016/s0165-7836(02)00157-1. monks, n. 2006. brackish-water fishes; an aquarist’s guide to identification, care, and husbandry. t.f.h. publications. neptune city, new jersey. 383p. mohamed, k.s.; sathianandan, t.v.; vivekanandan, e.; kuriakose, s.; ganga, u.; pillai sl, et al. 2021. application of biological and fisheries attributes to assess the vulnerability and resilience of tropical marine fish species. plos one 16(8): e0255879. https://doi.org/10.1371/journal. pone.0255879 olopade, o.a.; dienye, h.e. & amponsah, s.k.k. 2019. a preliminary study of growth pattern, condition factor and population structure of sicklefin mullet, liza falcipinnis (valenciennes, 1836) in the new calabar river, nigeria turkish j of sci. techn. 14(1), 15-22. pauly d. 1980. on the inter-relationships between natural mortality, growth performanc e and mean environmental temperature in 175 fish stock. j of conser. 39 (3): 175-192. [15]. pauly, d. 1982. studying single-species dynamics in a tropical multispecies context. in: pauly, d. and g. l., murphy. eds., theory and management of tropical fisheries: iclarm conference proceedings, iclarm, manila, 1982; 3370. pauly, d. 1983. length-converted catch curves: a powerful tool for fisheries research in the tropics (part 1). iclarm fishbyte, 2, 9-13. pauly, d. & munro, j.l.1984. once more on the comparison of growth in fish and invertebrates. fishbyte, 2 (1), 1–21. pauly, d. & soriano, m.l. 1986. some practical extensions to beverton and holt's relative yieldper-recruit modelin: j.l. maclean, l.b. dizon, l.v. hosillos (eds .), first asianfisheries forum, asian fisheries society, manila, philippines. pp: l49-495. schneider, w. 1990. fao species identification sheets for fishery purposes. field guide to the commercial marine resources of the gulf of guinea. prepared and publis hed with the support of the fao regional office for africa. rome: fao. 268 p. sparre, p. & venema, s.c. 1997. introduction to tropical fish stock assessment part 1: manual. roma: fao, 404 p. documento técnico sobre as pescas, no. 306/1. contact : andi nur faiziah palla andinurfaiziah15@gmail.com 222 issn : 2580-2410 eissn : 2580-2119 protein hydrolyzate of grouper viscera : effects of crude bromelain extract concentration and hydrolysis time on yield and degree of hydrolysis andi nur faiziah palla1*, metusalach2, nursinah amir2 1 fisheries science study program, faculty of marine science and fisheries, hasanuddin university, makassar, indonesia. 2 department of fishery, faculty of marine science and fisheries, hasanuddin university, makassar, indonesia introduction in fish processing industry, demersal fish like grouper is usually processed into fish fillet which produces high amount of by-product such as head, scales, bones, frames, skin, fins, and viscera. this by-product may account up to 45-65% of the total weight of the fish. fish viscera is one of the fish processing by-product that is still unutilized or underutilized despite of its high protein content (mardina et al., 2018). the fish viscera contains high level of protein (atma, 2016), reaching up to 31.20% of protein on a dry basis (aditya, 2018), and making it a highly potential raw material for the production of certain or specified fish produc t such as fish protein hydrolyzate. fish protein is highly digestible and has an excellent essential open access international journal of applied biology keyword by-product; degree of hydrolysis; fish viscera; protein hydrolyzate; proteolytic enzyme article history received november 19, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. abstract grouper is a fishery commodity that has high demand for both domestic and export markets. for export market, the grouper is commonly sold in a fillet form. however, production of the grouper fillet generates substantial amount of by-products, such as skin, scales, bones and viscera. the viscera contains high amount of protein and can be converted into protein hydrolyzate. the enzymatic processing of the protein hydrolyzate requires proteolytic enzymes, such as bromelain. this study aimed to analyze the effect of concentration of the bromelain crude extract and hydrolysis time on yield and degree of hydrolysis of grouper viscera protein hydrolyzate. the experimental design used was a completely randomized factorial design with different enzyme concentrations (4, 6, 8, 10 and 12%) and different length of hydrolysis time (4, 6, 8 and 10 h). the results showed that the concentration of the bromelain enzyme with the length of time in the waterbath had an effect on the yield and degree of hydrolysis of the grouper viscera (p<0.05). the highest yield was 18.17%, while the highest degree of hydrolysis was 89.29%, indicating the potential use of the grouper viscera and the crude extract of bromelain in the production of the fish protein hydrolyzate. international journal of applied biology, 6(2), 2022 223 amino acid (eaa) profile that suits the needs of the human diet as defined by the world health organization (who) (egerton et al., 2017). fish protein hydrolyzate can be produced using chemical or enzymatic hydrolysis. for the enzymatic hydrolysis, a proteolytic enzyme is into the fish or fish parts raw materials to accelerate the hydrolysis process under controlled conditions with the end result being a mixture of protein components (piggot and tucker 1990). proteolytic enzymes that are often used in the food industry include bromelain and papain. of the two enzymes, the papain is more commonly used in the production of the protein hydrolyzates. however, previous research has shown that the papain enzyme leaves a bitter taste (somanjaya, 2013). so the bromelain enzyme is used which is a protease that has considerable potential for application in the food industry. the bromelain is relatively inexpensive (wijayanti et al., 2016), but it causes no bitter taste to the protein hydrolyzate. bromelain can be obtained from the pineapple plant (ananas comosus) including the stalks, skin, leaves, fruit and stems in varying amounts (masniar et al., 2016). research on fish protein hydrolyzate from grouper viscera with bromelain enzymes has not been carried out. this research utilized the grouper viscera for the production of the fish protein hydrolyzate and examined the effects of the concentration of the bromelain crude extract on the yield and degree of hydrolysis of the grouper viscera protein. it is hoped that this research may offer help to produce an added value product for the grouper processing industry. materials and methods this research was carried out from july to november 2022 at the animal microbiology laboratory, faculty of veterinary science, hasanuddin university. materials used in this study were the viscera of grouper, pineapple, k2so4, hgo, h2so4, h3bo3, hcl, ch3cooh, naoh, aquadest. the equipment used included commercial blender, calico cloth, measuring cup, erlenmeyer, hotplate with temperature control, centrifuge and oven, a set of laboratory equipment for chemical analysis of fish hydrolyzate such as kjeldahl flasks, digestion and distillation units, soxhlet, oven, desiccator, porcelain cup, ashing furnace, filter paper, ph meter, stirrer. methods this research used an experimental method, employing the experimental design used was a completely randomized factorial design. the factors used were concentration of the bromelain (crude extract of the pineapple) and hydrolysis time. the bromelain concentration consisted of five levels (4, 6, 8, 10, and 12% of the homogenized viscera weight) while hydrolysis time consisted of three levels (4, 6, 8, and 10 h). all experimental treatments were carried out in three replicates. a certain amount of pre-homogenized grouper viscera was thoroughly mixed with the crude extract of bromelain at a proposed concentration. the mixture was the hydrolyzed in a waterbath at 55°c for a period of time according to the proposed hydrolysis time. at the end of the hydrolysis, the hydrolyzate was recovered by filtration, enzyme-inactivated at 90°c, centrifuged at 3500 rpm, and then the solid hydrolyzate was dried in an oven. after drying, the yield of the protein hydrolyzate was calculated and the degree of hydrolysis was measured. international journal of applied biology, 6(2), 2022 224 preparation of pineapple crude extract the pineapple fruit used was a ripe one, peeled, the peeled fruit was splitted and chopped into small pieces and then homogenized using a commercial blender. to recover the crude extract of the bromelain, the homogenized pineapple was then squeezed and filtered to separate suspended fine particles. the filtrate was then transferred into a tight-capped plastic bottle until used for the experiment. production of fish protein hydrolyzate an approximately 250 g of the pre-homogenized grouper viscera were transferred into a 500 ml glass beaker, added with either 4, 6, 8, 10, or 12% of the bromelain extract and then mixed thoroughly. the beakers containing experimental samples were arranged properly in a waterbath, and then the hydrolysis process was carried out at 55°c for either 4, 6, 8, or 10 hours. the ph of the samples was maintained at 7 during the hydrolysis process, using either ch3cooh or naoh solutions. at the end of each of the proposed hydrolysis time, each sample was filtered through a 160 mesh filter cloth then inactivated the enzyme at 90°c for 20 minutes, and centrifuged at 3500 rpm for 15 minutes. the solid (protein hydrolyzate) was recovered, spresded evenly in a drying pan and dried in an oven at a temperature of 65°c for 24 hours. the dried protein hydrolyzate was then finely grinded to obtain the protein hydrolyzate flour. the parameters tested in this study were the yield and degree of hydrolysis. a. yield the yield of the protein hydrolyzate was calculated by dividing the weight of the hydrolyzate powder with the initial sample weight, and multiplied by 100%. 𝑌𝑖𝑒𝑙𝑑 (%) = ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑧𝑎𝑡𝑒 𝑝𝑜𝑤𝑑𝑒𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) b. hydrolysis degree the degree of hydrolysis was analyzed following amiza et al. (2012) and nurilmala et al. (2018). as much as 20 ml of protein hydrolyzate was thoroughly dissolved in 20 ml of 20% tca (v/v). the mixture was then allowed to stand for 30 minutes for pre cipitation to occur, then centrifuged (7,800 x g) for 15 minutes. the supernatant was analyzed for nitrogen content using the kjeldahl method (aoac 2005). the degree of hydrolysis is calculated using the following formula (hoyle and merritt, 1994). 𝐷𝑒𝑔𝑟𝑒𝑒 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑏𝑙𝑒 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑖𝑛 20% 𝑇𝐶𝐴 𝑇𝑜𝑡𝑎𝑙 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑖𝑛 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋 100 data analysis the experimental design used in this study was a completely randomized factorial design with different concentrations of the bromelain enzyme (4, 6, 8,10 and 12%) and different lengths of hydrolysis (4, 6, 8 and 10 hours). the parameters measured in this study were the yield and degree of hydrolysis. data were analyzed using analysis of variance international journal of applied biology, 6(2), 2022 225 (anova) and if the treatment showed a significant effect, a tukey test was performed. significant difference was determined at 95% level of probability (α = 0.05). results and discussion yield the yield of the protein hydrolyzate of the grouper viscera in this study ranged from 8.00 to 18.17%, where the lowest yield was at 4% bromelain enzyme concentration with 4 hours lengths of hydrolysis time, whereas the highest yield was obtained at 10% bromelain enzyme concentration with 8 hours lengths of hydrolysis time (table 1). table 1. the yield of the grouper viscera protein hydrolyzate produced at various concentration of bromelain and length of hydrolysis time. bromelain enzyme concentration (%) yield (%) 4 hours 6 hours 8 hours 10 hours 4% 8.00 ± 0.24ay 15.22 ± 0.82abw 11.39 ± 0.50ax 9.97 ± 0.50az 6% 11.93 ± 0.54bz 14.86 ± 0.01ay 15.86 ± 012bx 11.40 ± 0.47bz 8% 14.80 ± 1.27cy 16.60 ± 1.33bcy 14.78 ± 0.09cy 13.59 ± 0.22cz 10% 14.92 ± 0.34cy 15.29 ± 0.14by 18.17 ± 0.54dx 10.68 ± 0.23az 12% 14.16 ± 0.78cy 17.97 ± 0.18cx 14.19 ± 0.19ey 10.76 ± 0.40abz val ues fol l owed by the s ame l etter i n the s ame row (x,y,z..) or i n the s ame col umn (a,b…) i ndi cati ng no di fference at 95% confi dence l evel (p>0.05) the results of a two-way anova showed that the concentration of bromelain enzyme and hydrolysis time had a significant (p<0.01) effect on the yield. based on the hydrolysis time, tuckey test showed that at 4 hours hydrolysis time, the yield of the 8, 10, and 12% bromelain concentration was similar (p>0.05) but was significantly higher (p<0.05) as compared those of the 4 and 6% bromelain concentrations. at 6 hours hydrolysis time, the 12% bromelain concentration produced a significantly higher yield than those of other concentration, except with the 8% bromelain concentration. however, significant difference (p<0.05) of the yield existed between all the concentration treatments for the 8 hours hydrolysis time, being highest at 10% and lowest at 4% bromelain concentrations. for the 10 hours hydrolysis time, only the concentration of 8% bromelain produced a significantly higher yield (p<0.05), while the other concentration were relatively similar in their yields (p>0.05). for any concentration tested, the highest yield produced was obta in at either 6 or 8 hours of hydrolysis time. the highest yield of the combination of 10% bromelain enzyme concentration and 8 hours lengths of hydrolysis time may indicate the optimum combination for the production of the protein hydrolyzate from the grouper viscera. wijayanti et al (2016) showed that the increase in the yield protein hydrolyzate of the milkfish meat was primarily due to the increase in the concentration of the bromelain enzyme. similarly, harahap (2022) reported that the increase in the yield was due to the contribution and activity of the bromelain enzyme in the process of protein hydrolysis. the yield of the protein hydrolyzate of using the bromelain enzyme has been reported by several authors, such as wijayanti et al (2016) for international journal of applied biology, 6(2), 2022 226 milkfish meat (11.41%); purbasari (2008) for golden clams (17%); amelia et al (2021) for the viscera of bombay duck fish (12.55%), and widadi (2011) using papain enzyme for african catfish (25%). the yield in this study was much higher compared to the results of anissa et al (2017) for tilapia (5.64%), for milkfish (2.73%) and for sharks (2.83%), and kamini et al. (2016) for siamese catfish viscera (2.77%). however, it may worth to note that these researchers used different concentrations of enzymes and hydrolysis times. degree of hydrolysis the degree of hydrolysis of the grouper viscera in this study ranged from 55.18 to 89.29%, being lowest at 8% bromelain concentration with 10 hours hydrolysis time and highest at 10% bromelain concentration with 8 hours hydrolysis time (table 2). table 2. the degree of hydrolysis of the grouper viscera at different concentration of bromelain and length of hydrolysis time bromelain enzyme concentration (%) hydrolysis degree (%) 4 hours 6 hours 8 hours 10 hours 4% 70.19 ± 4.50ay 80.50 ± 5.75ax 80.72 ± 6.84ax 84.39 ± 1.98acx 6% 63.56 ± 4.03abz 83.80 ± 3.87ay 87.18 ± 2.13abx 79.76 ± 3.57ay 8% 67.73 ± 3.55aby 79.18 ± 3.00ax 83.74 ± 4.19ax 55.18 ± 9.54bz 10% 58.08 ± 9.38bz 74.03 ± 7.48ay 89.29 ± 1.12bx 77.31 ± 5.25ay 12% 67.23 ± 7.65abz 64.39 ± 2.69by 88.02 ± 4.26abx 87.44 ± 2.02cx val ues fol l owed by the s ame l etter i n the s ame row (x,y,z..) or i n the s ame col umn (a,b…) i ndi cating no di fference at 95% confi dence l evel (p>0.05) the results of a two-way anova showed that the concentration of the bromelain and length of hydrolysis time significant affected (p<0.01) the degree of protein hydrolysis of the grouper viscera. the tuckey test showed that some significant differences (p<0.05) existed among the bromelain concentration as well as the hydrolysis time treatments regarding the degree of protein hydrolysis of the grouper viscera. in line with the yield, the highest degree of hydrolysis was also observed for the 10% of the bromelain at 8 hours hydrolysis time . also, the 8 hours hydrolysis time tended to produce higher degree of hydrolysis at any concentration of the bromelain enzyme. the highest degree of protein hydrolysis of the grouper viscera at 10% bromelain with 8 hours hydrolysis time may indicate the optimum conditions for the hydrolysis process. when considering the whole concentration of the bromelain used, the hydrolysis time of 8 hours seems to be the best to obtain a maximum degree of hydrolysis the high degree of hydrolysis in this study may confirm harahap (2022) that the bromelain enzyme is more active in hydrolyzing protein in animals. the high degree of hydrolysis in this study indicated that the protein hydrolysis process of the protein took place efficiently. the degree of hydrolysis obtained in this study was far greater than that of fakhrija (2021) for black sea cucumbers (10.11%), and harahap (2022) for eel (6.46%). international journal of applied biology, 6(2), 2022 227 the availability of the enzyme in sufficient quantity can increase the rate of the hydrolysis. according to hasnaliza et al. (2010), enzyme concentration greatly determines the degree of protein hydrolysis. however, too much of the enzyme does not help in maximizing not only the yield but also the degree of the protein hydrolysis as shown by this study. the concentration of the enzyme required is proportional to the number of peptide bonds that need to be hydrolyzed. the increase in protein content requires an increasing concentration of the added enzyme, but to a certain extent the addition of excess enzyme will produce a constant or even decrease amount of hydrolyzate due to the short of substrate to act on. where the substrate is abundant, the higher the concentration of the enzyme used the greater the amount of amino acid protein hydrolyzate produced as reported by wij aya and yunianta (2015). conclusions the yield and degree of hydrolysis of the grouper viscera protein hydrolyzate were affected by the concentration the bromelain enzyme and the length of the hydrolysis time. the best condition for producing the protein hydrolyzate using viscera of the grouper fish is the bromelain concentrations of 10% with a length of hydrolysis time of 8 hours. international journal of applied biology, 6(2), 2022 228 references aditya, r.p. 2018. aktivitas antioksidan dan antibakteri hidrolisat protein hasil fermentasi telur ikan cakalang [tesis]. bogor (id): institut pertanian bogor. amelia, g. 2021. pembuatan hidrolisat protein ikan lemok harpondo nehereus menggunakan enzim papain dengan waktu hidrolisis berbeda. jurnal universitas riau amiza, m.a., kong, y.l and faazaz, a.l. 2021. effect of hydrolysis on physicochemical properties of cobia (rachycentron canadum) frame hydrolysate. journal international food research, 19(1); 199-206 annisa, s., y.s. darmanto, dan amalia, u. 2017. pengaruh perbedaan spesies ikan terhadap hidrolisat protein ikan dengan penambahan enzim papain. saintek perikanan. vol. 13 no. 1: 24-30. issn: 1858-4748. aoac. 2005. official methods of analysis. the association of official analytical chemists. arlington, virginia usa: aoac inc. atma, y. 2016. pemanfaatan limbah ikan sebagai sumber alternatif produksi gelatin dan peptida bioaktif: review. seminar nasional sains dan teknologi. fakultas teknik universitas muhammadiyah jakarta. egerton, s., sarah, c., jason, w., catherine, s. and paulros, r. 2017. characterization of protein hydrolysates from blue whiting (micromesistius poutassou) and their application in beverage fortification. food chemistry, s0308-8146 (17) 31744-2. fakhrija s. 2021. hidrolisis protein teripang hitam (holothuria edulis) menggunakan bromelin kasar dari batang nanas (ananas comocus l). jurusan kimia fakultas matematika dan ilmu pengetahuan alam universitas tadulako, palu. harahap, m. t., edison dan sukmawati, m,. 2022. pengaruh konsentrasi enzim bromelin terhadap derajat hidrolisis hidrolisat protein belut (monopterus albus). jurnal universitas riau. pekan baru. hasnaliza, h., maskat, m, y., wan,a, m. n dan mamot, s., 2010. mamot s. 2010. the effect of enzyme concentration, temperature and incubation time on nitrogen content and degree of hydrolysis of protein precipate from from cockle (anadara granosa) meat washwater. international food research journal. 17(1): 147-152 hoyle, n. t. and merritt, j. h. 1994. quality of fish-protein hydrolysates from herring (clupea harengus). journal of food science 59(1): 76-79. kamini., p. suptijah., santoso,j dan suseno, s.h. 2016. ekstraksi dry rendering dan karakteristik minyak ikan dari lemak jeroan hasil samping pengolahan salai patin siam pangasius hypophthalmus. jphpi vol. 19 no. 3. mardina, v., fitirani, tisna, h. dan goldha, m. h. 2018. valorisasi pankreas ikan tongkol (eutynnusaffinis) untuk produksi enzim lipase. elkawnie: journal of islamic science and technology, vol. 4 no.2, desember 2018 masniar, m., zainal, a., muchlisin. dan sofyatuddin, k. 2016. pengaruh penambahan ekstrak batang nanas pada pakan terhadap laju pertumbuhan dan daya cerna protein international journal of applied biology, 6(2), 2022 229 pakan ikan betok (anabas testudineus) jurnal ilmiah mahasiswa kelautan dan perikanan unsyiah volume 1, nomor 1: 35-45. nurilmala, m., hizbullah, h.h., karnia, e., kusumaningtyas, e. and ochiai, y. 2020. characterization and antioxidant activity of collagen, gelatin, and the derived peptides from yellowfin tuna (thunnus albacares) skin. marine drugs. 18(2); 98. piggot, g.m. and tucker, b.w. 1990. utility fish flesh effectively while maintaining nutritional qualities. seafood effects of technology on nutrition. newyork: marcel decker, inc. purbasari, d. 2008. produksi dan karakterisasi hidrolisat protein dari kerang mas ngur (atactodeastriata). [skripsi]. fakultas perikanan dan ilmu kelautan ipb. somanjaya, r. 2013. pengaruh enzim papain terhadap keempukan daging. jurnal ilmu pertanian dan peternakan vol.1 no.2 widadi, i.r. 2011. pembuatan dan karakteristik hidrolisat protein dan ikan lele dumbo (clarias gariepinus) menggunakan enzim papain. fakultas perikanan dan ilmu kelautan ipb. wijaya j c, dan yunianta. 2015. pengaruh penambahan enzim bromelin terhadap sifat kimia dan organoleptik tempe gembus (kajian konsentrasi dan lama inkubasi dengan enzim). jurnal pangan dan agroindustri, 3(1), 96–106 wijayanti, i., romadhon, dan rianingsih, l. 2016. karakteristik hidrolisat protein ikan bandeng (chanos chanos forsk) dengan konsentrasi enzim bromelin yang berbeda. jurnal saintek perikanan. vol (11). no.2:1229-133. contact : p.s lama patienncesunday@gmail.com 179 abstract over the years, the use of agrochemicals has been one of the reasons for resistance of mosquitoes to insecticides, a nega tive trend that has bee n on the increase. this study was carried out to determine the effect of azadirachta indica (neem) see d extract on the larvae and adult anopheles mosquitoes in nasarawa local government area of nasarawa state as a possible alternative to already existing insecticide against the parasite. the research was carried out at the insectary laboratory of nasarawa state university keffi. impregnated papers of neem seed oil were used for adult anopheles mosquitoe s and the larviciding effect was also determine d. adult anopheles mosquitoe s expose d to impregnated papers of neem seed extract at differe nt conce ntrations of 20%, 40%, 80% and 100% showed 5%, 29%, 66% and 91% respectively. m ortality rate was recorded at 24h of e xposure w ith lc50 (lethal conce ntration) of 4.45ml. the larvae were also exposed to different conce ntration of neem see d extract of 1%, 4%, 8% and 10% for 72h and the mortality rate was 100% with lc50 value (lethal conce ntration) of 4.16ml. this implie d that adult anopheles mosquitoe s will required more of the extract to achieve 100% mortality than larvae. using azadirachta indica (neem) seed extract as an anti-mosquito agent was more effective on the larvae of anophe les mosquitoe s than the adult anopheles mosquitoes. issn : 2580-2410 eissn : 2580-2119 effects of azadirachta indica (neem) seed extract on larvae and adult anopheles mosquitoes in nasarawa local government area, nasarawa state nigeria. p.s. lama1*, m.d. olayinka1, a. ezekiel1, j.d.c tongjura1, r.j ombugadu1, o akyengo2 1 department of zool ogy, faculty of natural and applied sciences, nasarawa state university keffi, nasarawa state nigeria. 2 department of science laboratory technology, nigerian institute of leather and science technology, samaru, zaria, nigeria. introduction malaria is one of the major causes of mortality and morbidity in nigeria ( morakinyo et al., 2018; who 2018). who has rated nigeria 92% in african region where nigeria accounted for 25% of the global burden (who2018). mosquitoes are not just the vectors of malaria but the vectors of several diseases (an et al., 2020) with anopheles gambiae giles also known as africa mosquito been the most common vector of human malaria in afro tropical region (cdc, 2010). in recent years, research has been going on where botanical metabolites are increasingly realized as potential substitute for chemical insecticides (vivekanandhan et al., 2018). open access international journal of applied biology keyword anopheles mosquitoes; azadirachta indica; lethal concentration; nasarawa. article history received august 24, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. mailto:patienncesunday@gmail.com international journal of applied biology, 6(2), 2022 180 the neem plants belong to the family of meliaceae plants which contain so many varieties of compound tha t shows insecticidal, antifeedant, growth-regulating, and development-modifying properties (nugroho et al., 1999; greger et al., 2001; d'ambrosio et al.,, 2002; nakatani et al., 2004). meliaazedarach l. and azadirachtin indica (sapindales: meliaceae), commonly known as chinaberry or persian lilac tree, are deciduous trees that are native to north western india; and have long been recognized f or their insecticidal properties. these trees grow typically in tropical and subtropical parts of asia, but nowadays they are also cultivated in other warm regions of the world because of their considerable climatic tolerance. fruit extracts of meliaazedarach and azadirachta indica elicit a variety of effects in insects such as anti feedant, growth retardation, reduced fecundity, moulting disorders, morphogenetic defects, and changes of behaviour (schmidt et al., 1998; abou et al., 2001; gajmer et al., 2002; banchio et al., 2003; wandscheer et al., 2004). larval of anopheles mosquitoes were exposed to undiluted extract of seed oil, leaf and bark of aza dirachtin (neem) for 12 hours, with a high mortality rate to seed oil, leaf extract and less mortality to bark of azadirachtin at usman danfodio university sokoto (aleiro,2003). larvae of anopheles mosquitoes were susceptible to the seed and leaf of azadirachtin in kogi state (adobu,et al.,2018). fathelrahman,(2017) also observed in sinnar state -sudan that anopheles arabiensis were susceptible to the extract of leaf and seed of azadirachtin with high susceptibility to the kernel seed. when applied to artificial water bodies in every two weeks within period of three months, emulsified neem oil have the same effect on larval mortality and adult density as the commonly synthetic insecticides used (awad,et al.,2003). a study was also carried out using a neem oil formulation on third and fourth larvae stage of anopheles gambiae and showed 50%inhibition of adult emergence at a concentration of 6ppm (okumu, 2007).in a study using emulsified neem oil, it shows that within a three months, anopheles larvae failed to develop resistance or change their susceptibility to the oil (awad et al., 2003). materials and methods study area the study was carried out in nasarawa local government area of nasarawa state which is located in the western senatorial zone with latitude 08°41ʹ20ʺ to 08°32ʹ10ʺ n and longitude 07°48ʹ43ʺ to 07°50ʹ00ʺ e. it has two seasons rainy seasons starts from april to october while dry season start from october to march. sample collection and procedures the anopheles mosquitoes larva were collected from their natural water habitat in some communities of nasarawa local government area using entomological ladles into a well labelled container, they were transported the insectary laboratory, department of zoology nasarawa state university keffi. the anopheles mosquitoes were reared to adult in a container in the laboratory, the larva were fed with biscuit and yeast while the adult were fed with sugar solution according to who, (1998) procedure for who method for susceptibility of neem oil extract the susceptibility test was carried out using four tubes for each of the concentration (neem seed extract) used with two control tubes each. an aspirator was used to introduce 25 anopheles mosquitoes into the impregnated tubes. a timer was used to record both the international journal of applied biology, 6(2), 2022 181 number of dead a nd alive mosquitoes after 10 minutes for 1hour. af ter which the mosquitoes were transfer into a recovery tubes and were fed for 24 hour and the mortality rate was also recorded. the mortality in the control tubes was recorded as well. (who, 1998). procedure for larviciding distilled water was introduced into four different containers for each of the concentration of the oil used with two controls. a pipette was used to introduce 25 anopheles larvae into the containers and different concentration of neem seed oil was introduce into the containers. mortality was recorded at 60 minutes, 24hour, 48hour, and 72hour. mortality in the control was also recorded. (who, 1998). results effects of azadirachtin indica oil extract on adult anopheles mosquitoes result showed that a total of 400 adult anopheles exposed to a. indica oil extract at different concentration f or 24h. mortali ty was recorded at 10 minutes, 60 minutes and 24h post exposure as shown in table 1. the percentage of adult anopheles mosquitoes to 20%, 40%, 80% and 100% concentration of a. indica oil extract at 60 mins of exposure were 0, 20, 47 and 49 percentage mortality. while the mortality recorded for 24h post exposure were 5, 29, 66 and 91 respectively. table 1. effects of azadirachtin indica oil extract on adult anopheles mosquitoes concentration of oil extract (%) no. of anopheles mosquitoes exposed mortality (%) 60(mins) 24h 20.0 100 0(0.0) 5(5.0) 40.0 100 20(20.0) 29(29.0) 80.0 100 47(47.0) 66(66.0) 100.0 100 49(49.0) 91(91.0) control r1 25 0(0.0) 1(0.25) r2 25 0(0.0) 1(0.25) the larviciding effect of a. indica oil extract the larviciding effect of a. indica oil extract is as shown in table 2 a total of 400 anopheles larvae were exposed to different concentration of neem seed extract ( 1 %, 4%, 8% and 10%) for 24h, 48h and 72h. the percentage mortality of neem seed extract to larvae showed 86.5%, 98.8% and 100% mortality. while 2(1%) mortality in the control was also recorded. international journal of applied biology, 6(2), 2022 182 table 2: larviciding effect of azadirachta indica seed oil extract concentration of oil extract (%) no. of anopheles larval exposed mortality % 24h 48h 72h 1 100 78(88.0) 95(95.0) 100(100.0) 4 100 85(85.0) 100(100.0) 100(100.0) 8 100 88(88.0) 100(100.0) 100(100.0) 10 100 95(95.0) 100(100.0) 100(100.0) control r1 25 0(0.0) 0(0.0) 1(0.25) r2 25 0(0.0) 0(0.0) 1(0.25) discussion adult anopheles mosquitoes exposed to different concentrati on of neem seed oil at 20%, 40%, 80% and 100% f or 60 mi ns and 24h post exposure mosquitoes were highly resistant at different dosage in all the concentra tion for 24h and at 100% they were less resistant. this may be as a result of the effect of agricultural chemicals that were used to control pests in which the neem oil will not be effective enough in the knockdown. this present study shows that adult anopheles mosquitoes were resistant to neem seed oil and in previous study in sudan, fatherahman (2017) observed that a. aranbiasis were susceptible to neem seed oil. larval of anopheles mosquitoes were exposed to different concentration of neem seed oil at 1%, 4%, 8% and 10% for 60 mins and 24h post exposure. in this research, the larval of anopheles mosquitoes were susceptible to neem seed oil, this is because neem seed oil can cause deterrence in insect, repellency, growth disruption, reduced fitness and sterility activities. this confirmed the previous study in sokoto by aleiro (2003) where larval of anopheles mosquitoes were exposed to undilute d extract of neem s eed oil and susceptibility was obtained. it was also observed by adobu et al.,(2018) in kogi state where larval of anopheles mosquitoes were 100% susceptible to neem seed oil. (kela et al., 2019) uses neem oil water dispersible tablet against the third instar larvae of anopheles mosquitoes and its showed 98% mortality. (assalif et al., 2016) also observed that neem powder applied on 1st, 2nd and 3rd instar larvae showed88.9%, 87.9% and 79.4% mortality. the mortality was very high at the first instar larval stage. conclusion from this present study, neem seed extract showed efficacy as an anti -malaria agent by killing the mosquitoes subjected to it. its efficacy was however more pronounced on the anopheles larvae than the adult anopheles mosquitoes. therefore the use of neem seed extract should be encouraged in the control of mosquitoes but for a more appreciable result, the mosquito larvae should be targeted when using the neem seed extract. competing interests the authors wish to declare that there are no competing interests. funding no external funding was received. international journal of applied biology, 6(2), 2022 183 author contributions lps, omd, ea, t.j.d, orj conceived, designed and performed the ex peri ment, ao contributed immensely in the search for the literature. all authors read and approved the final manuscript. acknowledgements our sincere gratitude goes to the staff of who insect laboratory of nasarawa state university keffi international journal of applied biology, 6(2), 2022 184 references aboufakhrhammad, m., zournajian, hand talhouk, s. 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(2004): larvicidal action of ethanolic extracts from fruit e ndocarps of meliaazedarachandazadirachtaindicaagainst the dengue mosquito aedesaegypti. toxicon 44, 829–835. world health organisation (2018). world malaria report. world health organisation geneva. who (1998): test procedures for insecticides resistance in malaria vectors, bio-effecacy and persistence of insecticides on treated surfaces. who/ cds/cpc/ma journal 12: 98 contact : chairunnisah j. lamangantjo chairunnisah@ung.ac.id 138 abstract panua sanctuary is a conservation area, known as a habitat for birds, particularly the diurnal birds. the objectives of this re search are as follow s to figure out the species of diurnal birds, and to know the diversity, diver sity number s, evenne ss, and dominance index of the diur nal birds in panua sanctuary. the research was c onducted fr om february until march 2021. the point c ount method was employed in four observation stations. the research result revealed 10 or dos, 27 familie s, 50 species, and 321 total of individuals. the diversity index in station i was 0.15, station ii 2.10, station iii 2.34, and station iv 2.70. the diversity numbers in station i was 0.15, station ii 0.36, station 0.22, and station iv 0.14. the evenness index in station i was 0.87, station ii 0.79, and station iii 0.8. station i was dominated by yellow-sided flowerpecker with di = 15.44% which was considered a s a dominating type w hile station ii was dominated by yellow-breasted racquet-tail with di = 35.71%. station iii and iv were dominated by cattle egret with di value respectively 21.62% and 14.49%. issn : 2580-2410 eissn : 2580-2119 the diversity of diurnal birds in panua sanctuary conservation area in pohuwato district, gorontalo province, indonesia chairunnisah j. lamangantjo1*, dewi wahyuni k. baderan1, sri yolanda talaa1, sukirman rahim2 1 department biology, faculty mathematics and natural sciences, universitas negeri gorontalo, bone bolango regency, gorontalo province. 2 master of population and e nvironment, universitas negeri gorontalo, gorontalo city gorontalo province. introduction sulawesi is an island with diverse potential in indonesia with an area of 187,882 km2 (whitten et al., 2012). it is also a bio-geography area of wallacea with the highest endemic proportion after irian jaya province in terms of reptiles and endemic birds. located in the northern part of sulawesi, gorontalo province has some wallacean endemic bird species. it consists of six conservation areas, i.e. bogani nani wartabone national park, nantu wildlife reserve, tangale sanctuary, tanjungpanjang sanctua ry, mas popaya raja island sanctuary, and panua sanctuary (natural resources conservation bureau of north sulawesi, 2019). birds, or the so-called aves, are the members of vertebrae which can be used as an indicator of biodiversity, the revolution of a quality environment, and conservation area (safanah et al., 2017) as they are highly sensitive and easily offended (callaghan et al., 2018). birds and animals are classified into two g roups based on their activity patterns: diurnal and nocturnal (active during the days). most bi rds are active during the days yet open access international journal of applied biology keyword birds; panua; point count; indonesia; conservation article history received july 18, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. mailto:chairunnisah@ung.ac.id international journal of applied biology, 6(2), 2022 139 they take a rest at different times. families of strigiformes, podargidae, and caprimulgidae are the only nocturnals (active at night) (saman et al., 2019). the existence of a bird species basically adjusts to a chosen certain environment where they can easily obtain resources to fulfill their life in that area (indra et al., 2020). the presence of birds can also affect an ecosystem. panua sanctuary has several ecosystems, such as beach forest, mangrove forest, lowland, and mountains (natural resources conservation bureau of north sulawesi, 2019). a variety of ecosystems can create various habitats for birds. wibowo et al (2017) claimed that some considerations of a habitat such as the availability of food, breeding, nesting, and self-protecting from preys affect the diversity of bird species. different habitats provide decent spaces for birds (iswandaru et al., 2020). study about birds is very engaging due to their dynamic natures and functions as the predictor of environmental changes of where they live. birds can also be found almost everywhere (hadinoto et al., 2012). based on the background of the study, considering the urgency of the information related to the species of the diurnal birds in the area of pa nua sanctuary, including its diversity, diversity numbers, evenness, and dominance index, this study is needed to be conducted to support the attempts in preserving and protecting the diurnal birds. materials and methods research site the research was conducted in the area of panua sanctuary in pohuwatu regency, an area with a rich species of birds especially the endemic ones as supported by natural resources conservation bureau of north sulawesi (2019). the research took place in four observation stations, i.e. (1) primary area, (2) secondary area, (3) transition area, and (4) distracted area (figure 1). figure 1. location of the research on the structure of diurnal birds community in panua sanctuary, gorontalo province, indonesia international journal of applied biology, 6(2), 2022 140 the observation was determined by birds habitats namely: station 1 i n a pri mary forest, station 2 in a secondary forest, station 3 in a transition area and station 4 in a disturbed area. station i: primary forest it is a natural forest area which has never been expl oited. the area of beach forest located in panua sanctuary was determined as the location for observing the bi rds as part of the primary forest. station ii: secondary forest it is a regeneration forest of the primary forest. it was initially damaged due to natural disaster or the arranged logging to fulfill humans’ needs. this area is located in the northern part of the sanctuary. station iii: transition area it is a transitional area between a sanctuary and produc tion f orest, limited production forest, protection forest, and converted production forest. in this research, several transition areas between the sanctuary and the converted production forest were chosen, in this case plantation. station iv: disturbed area the disturbed is where the plantation lies in the area of panua sanctuary. research method diurnal birds data collection stage the data were collected by using stratified systematic sampling in each observation station. point count method was implemented in the location where the birds were observed and where the researcher stood on a certain place for a certain period of ti me while noting and counting all of the birds seen in each station (colin et al., 2000). observations were conducted from 06:00-09:00 in the morning and f rom 15:00-18:00 in the afternoon.. birds with the most active moves can be seen in the morning and afternoon (john et al., 2020). the duration of the observation lasted for 20 minutes in each observation point. af ter 20 minutes, the observation moved to the next loc ation which included several activities such as noting the date and time of the observation, bird activities, species, as well as the numbers of birds seen in the observation position, and taking the photographs of the invisible birds by using digital came ra. data collection in each station consists of four observational points, the observati on in each observation station lasted for 180 minutes. the observation was repeated for two ti mes. determining the observational station was based on the birds’ habitats such as (1) primary forest, (2) secondary forest, (3) transitional forest, and (4) the distracted area. data analysis identification stage the collected data of the birds were identified based on its morphological characteristics by referring to a book entitled field guide of birds in wallacea area and the list of indonesian birds (sukmantoro, 2007), a book about birds in wallacea area: sulawesi, maluku, and nusa tenggara (coates & bishop, 2000) and a book entitled birds of indonesia international journal of applied biology, 6(2), 2022 141 archipelago to figure out their english names. next, the data of the species and numbers of the species were analyzed by using descriptive statistics. diversity (h’) it measures the diversity index of birds by using shannon-winner formula (1949). description: h’ = diversity index of bird species ni = total individuals of bird n = total individuals of all birds diversity number (pi) diversity number is a total number of the bird individuals found during the observation. to reveal the diversity numbers of each kind of bird, the following formula is applied: evenness (e) index of evenness can be calculated by using a formula as described in the following: description: e = evenness index s = number of species h’ = the diversity of shannon-wienner dominance (di) it determines the value of domina nce which further benefits in determining or setting which species of birds which are dominant, sub-domi nant, or not dominant in an observation track. description: di = dominance index of a bird species ni = number of individuals of a bird species n = number of individuals of all bird species results and discussion results general description of the research site research on diurnal birds in the panua nature reserve area was carried out at 4 observation stations with different habitat types, namely prima ry forest, secondary forest, international journal of applied biology, 6(2), 2022 142 transition areas, and disturbed areas. a description of each station is shown in table 1 and a map of the research sites is presented in figure 1. table 1. general description of diurnal bird research sites in the panua nature reserve area station i station ii station iii station iv located on the coordinate of 121º58’58.1”e and 0º27’31.7”n located on coordinate of 121º58’07.3”e and 0º35’16.4”n. located on coordinate of 121º59’38.9”e and 0º2844,3”n located on coordinate of 121º59’21.5”e and 0º28’48.6”n. primary forest secondary forest transition areas disturbed areas prima ry area (station i) is a beach forest. it is located in the southern part of panua with an altitude of 73.90 amsl and l ocated on the coordinate of 121º58’58.1”e and 0º27’31.7”n. according to the real condition of the area, the prima ry field has a heterogeneous vegetation. the flora consists of beach she-oak and mangrove. the secondary (station ii) area is the secondary forest located in the northern part of panua sanctuary exactly in marisa sub-district. this area is located on the steep slope land (15%-25%) with coordinate of 121º58’07.3”e and 0º35’16.4”n. the tertiary (station iii) area is the transition area between the sanctuary and the farm area located in the northern pa rt of the area with coordinate of 121º59’38.9”e and 0º2844.3”n. according to the condition of the farm field, it is considered as dry farm land which is combined with bushes. the land cover of the forest is also degraded and a little bit cover in the form of young bushes remains on a relatively flat land and old bushes on the rocky steep slope. meanwhile, the sanctuary area is actually a primary dry forest land with diverse vegetations with tight header cover. the distracted area (station iv) is located in the north. it is the area of plantation owned by the people living inside panua sanctuary which is located in the coordinate of 121º59’21.5”e and 0º28’48.6”n. according to the condition of the field, this a rea is dominated with corns and cashew crops, as well as growing grass which are used for the people to feed their farm animal that is cow. this plantation area is more -less 4 hectares. types of diurnal birds in panua sanctuary the existence of a bird species is affected by the habitat. panua sanctuary is a habitat for bi rds, particularly the diurnal birds. based on the field reference book about birds in wallacea area: sulawesi, maluku, and nusa tenggara (coates and bishop, 2000) and birds of the indonesian archipelago: greater sundas and wallacea (eaton et al., 2016), 10 ordos were collected namely charadiiformes, columbiformes, coraciiformes, cuculiformes, galliformes, passeriformes, pelecaniformes, piciformes, psittaciformes, and sulformes. in addition to that, 27 families and 50 species were also collected with the numbers of observed individuals as big as 321 (table 2). international journal of applied biology, 6(2), 2022 143 table 2. diurnal birds in panua sanctuary area in pohuwato district family species total individuals iucn cites scientific names english names st i st ii st iii st iv alcedinidae pelargopsis melanorhyncha great-billed kingfisher 2 1 lc ceyx fallax sulawesi dwarf kingfisher 1 lc anhingidae anhinga melanogaster oriental darter 1 nt ardeidae butorides striatus striated heron 1 lc bubulcus ibis cattle egret 1 16 10 lc artamidae artamus leucorhynchus white-breasted woodswallow 4 lc artamus moachus ivory-backed woodswallow 3 lc bucerotidae rhabdotorrhinus exarhatus sulawesi hornbill 4 vu rhyticeros cassidix knobbed hornbill 1 2 1 vu campephagidae curacina leucopygia white-rumped cuckooshrike 3 1 lc columbidae ptilinopus melanospilus black-naped fruit-dove 3 lc ducula radiata gray imperial-pigeon 1 lc chalcophap sindica asian emerald dove 3 1 1 lc macropygia amboninensis ruddy cuckoo-dove 1 lc treronvernans pink-necked green-pigeon 6 lc geopelia striata zebra dove 3 lc duculaaenea green imperial-pigeon 4 1 2 7 nt turacoena manadensis white-faced cuckoo-dove 1 lc duulaluctuosa silver-tipped imperial-pigeon 5 5 lc streptopelia chinensi spotted dove 1 lc treron griseicauda gray-cheeked green-pigeon 8 lc streptopelia tranquebarica red collared-dove 1 1 lc corvidae corvusenca slender-billed crow 2 2 6 2 lc cuculidae rhamphococcyx calyorhynchus yellow-billed malkoha 1 2 lc centropus bengalensis lesser coucal 3 2 lc dicaeidae dicaeum celebium gray-sided flowerpecker 10 lc dicaeum aureolimatum yellow-sided flowerpecker 21 lc dicruridae dicrurus hottentottus hair-crested drongo 4 9 14 3 lc estrildidae lonchura attricapilla tricolored munia 3 lc lonchura molucca black-faced munia 5 lc hirundinidae hirundo tahitica pacifis swallow 8 lc international journal of applied biology, 6(2), 2022 144 megapodiidae macrocephalon male maleo 2 cr megapodiuseumingii dusky scrubfowl 1 4 lc meropidae meropogonforsteni purple-bearded bee-eater 2 lc monarchidae hypothymispuella pale-blue monarch 7 4 lc motacillidae motacilla cierea gray wagtail 1 1 lc muscicapidae eumyias panayensis turquoise flycatcher 2 lc nectariniidae anthreptes malacensis brown-throated sunbird 17 lc cinnyris jugularis olive-backed sunbird 6 7 lc oriolidae excalfactor chinenesis black-naped oriole 3 lc phanasianidae synoicuschinennesis king quail 2 1 lc picidae mulleripicus fulvus ashy woodpecker 1 lc psittaculidae loriulus stigmatus sulawesi hanging-parrot 3 1 lc prioniturus flavicans yellow-breasted racquettail 15 3 nt pycnonotidae pycnonotus aurigaster sooty-headed bulbul 4 7 8 lc scolopacidae actits hypoleucos common sandpiper 1 lc sturidae aplonis panayensis short-tailed starling 1 lc timaliidae pellorneum celebense sulawesi babbler 1 lc 27 50 13 6 42 74 69 note : st (station) figure 2. bird species in panua sanctuary: a) macrocephalon maleo; b) coracina leucopygia; c) dicaeum aureolimatum; d) ceyx fallax. based on the data (table 1), in station i (the prima ry forest) there were 35 species of birds obtained out of the total individuals of the observed diurnal which was as big as 136 birds. in station ii (the secondary forest), there were 14 species of birds obtained and the total individual of the observed birds as big as 39 birds. in station iii (transition area), there were 15 bird species obtained with the total of the observed individuals as big as 74 birds. international journal of applied biology, 6(2), 2022 145 while in station iv (the distracted area), there were 19 species of diurnal birds collected with total observed individuals as big as 69 birds. figure 3. diversity index of diurnal birds in panua sanctuary area it is evident that there were several differences among each observational station based on the diversity index calculated by shannon wienner (h’) formula. the highest diversity index was on station i with h’= 3.1198 while the lowest diversity index is on station ii with h’= 2.0975. figure 4. index of diversity numbers of diurnal birds in panua sanctuary area the diversity numbers (pi) in station i was as big as 0.15 dominated by yellow-sided flowerpecker (dicaeumaureolimatum). in station ii, the pi value of the yellow-breasted racquet-tailis was as big as 0.36. meanwhile, in station iii and iv, the diversity numbers was dominated by cattle egretwith with pi value respectively 0.22 and 0.14. 3,12 2,10 2,34 2,70 0 0,5 1 1,5 2 2,5 3 3,5 station i station ii station iii station iv d iv e rs it y i n d e x (h ') 0,15 0,36 0,22 0,14 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 station i station ii station iii station iv d iv e r s it y n u m b e r s ( p i) international journal of applied biology, 6(2), 2022 146 figure 5. evenness index of diurnal birds in panua sanctuary area the evenness index of the diurnal birds in panua sanctuary of station i to station iv generated the evenness index which was categorized as almost even. figure 6. dominance index of diurnal birds in panua sanctuary area station i was dominated by yellow-sided flowerpecker with di = 15.44%. station ii was dominated by yellow-breasted racquet-tailwith di = 35.71%. in station iii and iv, there were cattle egret dominated the area with di value respectively 21.62% and 14.49%. discussion panua sanctuary has various types of ecosystems starting from beach forest, mangrove forest, swamp, lowland, to mountains (natural resource conservation, 2019). the types of this various ecosystems create various habitats for birds. this was proven by the discovery of 50 species of diurnal birds and 4 species which were the endemic of sulawesi. according to anugrah, et al (2017), the bigger the animal diversity, the more diverse the structure of their ha bitats (the diversity of plants and its vegetation s tructure). birds use their habitats as the source for food, water, and shelter (kurnia et al., 2021). according to the research result, it was revealed that the value of diversity index from four 0,87 0,79 0,86 0,91 0,72 0,74 0,76 0,78 0,8 0,82 0,84 0,86 0,88 0,9 0,92 stati on i stati on ii stati on iii stati onn iv e v e n n e ss i n d e x ( e ) 15,44% 35,71% 21,62% 14,49% 0,00% 5,00% 10,00% 15,00% 20,00% 25,00% 30,00% 35,00% 40,00% station i station ii station iii station iv d o m in a n ce i n d e x ( d i) international journal of applied biology, 6(2), 2022 147 stations was different. station one has h’ = 3.12 which was categorized high, station two has h’ = 2.10 which was categorized moderate, station three has h’ = 2.34 which was categorized medium, and station four has h’ = 2.70 which was categorized moderate. this is relevant to a criteria by fachrul (2012) which claimed if the value of h’ 1 ≤ h' ≤ 3m, the level of diversity is considered moderate. station 1 provided a natural habitat and sources of food f rom tight trees and heterogeneous vegetation. besides, the absence of humans makes many types of birds live in this area so that the diversity index in this station was considered high. baderan and utina (2021) in their research explained that a community was considered hig h in terms of diversity index if many species exist in that community. according to adelina, et al (2016) the combination of vegetation, appropriate habitats, a nd the existence of various kinds of food source contributed to the existence of variety of bids. putri (2015) and khan and pant (2016) stated that the availability of food significantly affected the diversity of birds. this appeared differently from station ii, iii, and iv with moderate category. this was because the trees are considered rare thus the availability of source of food for birds is limited. in addition to that, huma ns’ activities in this transition area create distraction as well as pressure towards the natural ecosystem of the birds. gardening activities in the distracted area creates changes on vegetation land cover so that it also impacts to the birds’ habitats. humans’ activity would also likely to decrease the diversity of birds and increase distraction towards the existence of birds in a certain area (naithani et al., 2018; issa, 2019). baderan (2016) further claimed that the availability of species components and changes of plants growth in the research location was considered as the consequence of community activities that generated moderate diversity in an ecosys tem. the value of h’ is the calculation result of diversity index which determines a certain species diversity in a certain area. if the value of h’ is bigger or equal to 1, an area is considered moderate in diversity. if the value of h’ is bigger than 3, the diversity of that area is considered high or abundant. if the value of h’ is bigger than 0 yet lesser than 1, the community in that area has a low dive (krebs, 1989; fachrul, 2012). station i with high diversity signifies a tendency of stable condition of an ecosystem in the beach forest. the calculation result of diversity numbers demonstrated differences in each observation station. different vegetation affects the diversity numbers of the birds (khan & pant, 2016). station i, with typical habitat of primary forest, had the highest diversity numbers that was 0.15, particularly yellow-sided flowerpecker (dicaeum aureolimatum). this bird is the type of songbird and rain eater. morning movement of this bird is higher than the afternoon movement. it is because the diurnal birds start their activities in the morning. the availability of the food influences the diversity numbers of yellow-sided flowerpecker. t his is relevant to the condition of beach forest where the trees -beach sheoak (casuarinae quisetifolia), mangrove, and tropical almondmassively grow,. according to the observation result, these birds eat the mangrove grains. yellow-sided flowerpecker is the endemic bird of sulawesi. station ii had the highest diversity numbers of yellow-breasted racquet-tailbirds with pi = 0.36%. this bird is an endemic bird in sulawesi. according to the natural r esources conservation bureau of north sulawesi (2019), primary and secondary beach which is high, bushy, and farm land is the common habitat of this bird. in contrast with station i and ii, station iii and station iv had pi index respectively 0.22 and 0.14. the migrated birds are called cattle egret(bubulcus ibis). the existence of this abundant animals in the distracted and transition areas is due to the availability of food source in this area. cattle egret usually international journal of applied biology, 6(2), 2022 148 hunts for food in dry and open areas such as the distracted and transition areas which have open land-cover. this bird tends to hunt for flies or lice living on cows and other kinds of insects and small animal. the abundant numbers of cattle egretbird were affected by group activity of cattle egretwhen hunting for meals. the evenness index describes the stability of a community which depicts the even species and shapes the community (daget, 1976). in each observation station, the value of evenness index was almost complete. e = 0.87 is for station i, 0.79 for station ii, 0.86 for station iii, and 0.91 for station iv. accordi ng to putu (2017), evenness index is used to measure how stable a species in maintaining their skills to survive. by measuring the evenness index, the spread of diurnal bird species in panua sanctuary was considered almost even. referring to a research by putu (2017), the higher the value of evenness index, the more the stability of the species diversity of that community.the lower the value of evenness, the lower the stability of the diversity of that community. considering the value of evenness index from community perspective, the requi rement is as follows: the oppressed community is 0 < e ≤ 0.5; unstable community is 0,5< e ≤ 0.75; and the stable community is 0.75 < e ≤ 1 (krebs, 1989). panua sanctuary whose evenness is almost even is included as the stable community based on the community research. kinds of birds domi nating each station has different observation result. putu (2017) in his research explained that the domi nance index describes the availability and unavailability of a genus or dominant species in constructing a community in an ecosystem. station i located in the primary forest habitat was dominated by yellow-sided flowerpecker birds with index value di = 15.44%. the dominance of this bird was influenced by the availability of food source that is the grains on mangrove. nugroho (2016) reported that vegetation plays importa nt role as a factor supporti ng the life of birds. the existence of group activity also affects the level of dominance of yellow-sided flowerpecker birds. according to wibowo et al (2017), high diversity numbers of bird is caused by their habits in doing group activity thus the dominance value is high. station ii was dominated by yellowbreasted racquet-tailbird with index value di = 35.71. station iii and iv were dominated by cattle egretbirds with di respectively 21.62 and 14.49 which were categorized dominant. the domina nce of these birds was affected by the source of food in the transition and distracted areas. conclusions the research result revealed 10 ordos, 27 families, 50 species, and the total of individuals were as big as 321. the divercity index of station one was in the high category while stations ii, iii, and iv were in the medium category. the evenne ss index of diurnal birds in panua nature reserve from station i to iv was in almost-even category. the dominance index of station i was dominated by yellow-sided flowerpecker, station ii was dominated by yellow-breasted racquettail, while station iii and iv were dominated by cattle egret dominated. acknowledgments the authors channeled their gratitude to the conservation centre of natural resources of north sulawesi especially the division of area ii conservation of gorontalo who had supported this research. international journal of applied biology, 6(2), 2022 149 references sulfahri, a.m., soemitro, b.s., murni s. 2017. comparison of biomass production from algae spirogyra hyalina and 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soil and maize grains of sulaimani governorate were subjected for our study. primary isolation and identification of isolates performed based on using the morphological features of aspergillus flavus on selective and differential media. pcr-based protocol used for more accurate identification of isolates as aspergillus flavus, which based on the multi-copy internal transcribed region of the rdna unit (its1-5.8s-its2 rdna). different culture and highly specific sensitive methods used for determining aflatoxigenicity of our isolates. as a culture methods, colony fluorescence, ammonia vapor test and characteristics of aspergillus flavus on aspergillus differentiation media have been used. accurate detection of aflatoxigenicity of our isolates confirmed by using conventional pcr for detection of the potential gene markers (afld and aflo),in the aflatoxin biosynthesis. out of eighteen isolates of aspergillus flavus, aflatoxigenicity seven of them have been detected and confirmed by both culture and molecular methods. in conclusion both culture and molecular methods could be used for rapid detection of aflatoxigenicity of aspergillus flavus. issn : 2580-2410 eissn : 2580-2119 culture methods and conventional pcr for detection the aflatoxigenicity of aspergillus flavus in local isolates samples khattab ahmed mustafa shekhany1, shkar rzgar rostam 2* 1 mycology lab, biology department , faculty of science, school of science at the university of sulaimani, sul, iraq. general director at the ministry of higher education and scientific research-krg 2 mycology lab, biology department ,faculty of science, school of science at the university of sulaimani, of sulaimani, sul, iraq introduction air food and feed contamination by mycotoxins is a significant food safety issue in the world and developing countries. mycotoxins such as aflatoxins have pivotal health effect on humans and animals. one of the most common foodborne fungi are the aflatoxins producer [1]. aflatoxins of a.flavus and a.parasiticus are the most widely subjected for investigation, due to that they have carcinogenic, mutagenic and teratogenic properties [23-4-5-34]. several methodologies have been developed for detection of aflatoxigenicity of a. flavus. there are many highly specific and sensitive culture methods can used for open access international journal of applied biology keyword culture based, aspergillus flavus, aflatoxin detection, polymerase chain reaction (pcr). article history received 12 september 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 44 determination of aflatoxins in samples, which allow the determination of mycotoxins, if samples contaminated with aflatoxigenic fungi [6]. culture based methods such as 1) colony fluorescence, appearance of blue fluorescence of aflatoxin b, when it is subjected to ultraviolet (uv) light, produce an intense blue fluorescence [78]. 2) observing of orange color on the reverse side of the aspergillus differentiation agar plates. visualizing of orange color on reverse side of colonies, is due to the reaction of aspergillic acid molecules synthesized by aspergillus species with the ferric ions from ferric citrate of medium. 3) ammonia vapor test, color change of undersides of aflatoxin-producing colonies when exposure to ammonium hydroxide vapor [9-10-11]. in this method, the undersides colonies of aflatoxin-producing fungi, will quickly turn plum-red when the bottom of the petri dish has been inverted over the lid containing the ammonium hydroxide [7]. in many of these methods, more specialty and experts require for detection the presence of aflatoxins. moreover, there are some other advanced and more accurate, but rather expensive and time consuming, techniques used to determine aflatoxigenicity of a. flavus, such as highperformance liquid chromatography (hplc), enzyme-linked immunosorbent assay (elisa), thin-layer chromatography (tlc), and fluorescence polarization assay [8-12]. each of these methods has advantages and limitations in detection of toxigenic strains of fungi. also molecular techniques have been introduced as powerful tools for detecting and identifying aflatoxigenic fungi [8-12]. several research groups [13-14] have tested the possibility of applying pcr-based detection techniques, for the presence of genomic dna by conventional pcr. some of other researchers, used reverse transcription pcr (rt-pcr) or real-time pcr to detect the expressivity of the aflatoxin biosynthetic genes. use of polymerase chain reaction (pcr) to amplify the aflatoxigenic genes, is more easily and more accurately in compare with other methods [12]. hussain et al., [12] developed reliable and quick method for the detection of aflatoxin producing strains in peanuts by using a molecular approach to amplify three target genes (afld, aflm and aflr) involved in the aflatoxin biosynthesis. attempts to decipher and determine the role of potential gene markers in the aflatoxin biosynthetic pathway began shortly after the determination of the structure of this toxin [17]. there are estimated to be 27 enzymatic steps in the aflatoxin biosynthesis. some structural intermediates in biosynthesis of aflatoxins pathway, enzymatically identified [17-18]. at least 30 genes identified that they involve in biosynthesis of aflatoxins. these genes are found on chromosome iii and clustered within a 75-kb region of the a. flavus and a. parasiticus genome. they localized around 80 kb away from telomere [1-17-18-19]. developed multiplex pcr have been used for discrimination between toxigenic and non-toxigenic strains within the a. flavus group [15]. applicability of rt-pcr (reverse transcription-pcr) technique tested to differentiate between aflatoxinproducing and aflatoxin-non-producing strains of a.flavus by scherm et al., [16]. the afld (nor-1) and aflo (omtb) are two potential gene markers play important role in aflatoxins biosynthesis, their determination give the reliable evidence of aflatoxigenicity of isolates [17-18-19-21]. here, we used the most reliable culture methods, and conventional pcr to amplify potential gene markers (afld and aflo) for detection the aflatoxigenicity of a. flavus in local isolates samples, which allowed us to investigate the prevalence the aflatoxigenicity of a. flavus in the local area samples. international journal of applied biology, 5(2), 2021 45 materials and methods survey sites composite soil samples were collected from the surface layer (0.0 -0.3 m) of the dominant cropped soils at different sites within sulaimani governorate that included agricultural soils cultivated with maize and different vegetables. maize grains were collected from corn fields at sulaimani governorate. fungal isolation isolation fungi from the soil samples soil samples were subjected for fungal isolation within two days of collecting. fungi were isolated by using soil dilution plate method on (pda) and (afpa). both potatodextrose agar (pda) and aspergillus differentiation agar media (afpa) were prepared according to the instructions as indicated by the manufacture. after adding (100 mg\1l) chloramphenicol antibiotic for prevention of bacterial growth, they were sterilized by autoclaving at 121ºc for 15 min under 1.5 bar pressure. after inoculation of media by samples, then they were incubated at 28º c. the plates have been observed after (5-7) days [22-23-24-25]. isolation fungi from the maize samples maize grains were collected from three different locations of sulaimani governorate. direct plate method used for isolation of fungi from maize grain samples. th is method previously described by [26]. this step was followed by surface sterilization of the grains in naocl for 120 seconds, then the maize grains were washed off using dh2o. later on, drying was performed by using sterile whatman filter paper, and plated on sterile (pda) at the rate of 10 grains per plate. thirty seeds were plated per plant and incubated at 28º c. identification of aspergillus species morphological identification morphological characteristics such as growth rate, colony diameter, aerial mycelium and pigmentation of fungi or colony reverse color, conidiophore, vesicle, metulae, phialides and conidia are generally basic and essential tool for identification of aspergillus species. macroscopic colony characters were examined under the dissecting microscope after aspergillus isolates have been grown on selective and differential media (afpa), and (pda) at 28ºc and 25ºc respectively for seven days. after aspergillus isolates grown on both media under the mentioned incubation conditions, new subcultures of aspergillus isolates were prepared on (afpa) and (pda) for studying microscopical characters. most microscopic characters such as (conidiophore, vesicle, metulae, phialides and conidia) were examined by (slide cultures technique) as described by shekhany, [27]. slides were prepared from these cultures by using lactophenol cotton blue as a mounting medium then examined under light microscope. macroscopic and microscopical characters have been used for identifications of isolated fungi as reported by [9-28-29-30]. molecular identification as an alternative way to rapid and accurate diagnosis for our isolates, pcr -based methods recommended. due to their high specificity and sensitivity in diagnosis, we decided to use pcr-based protocol for the detection of our isolates of a. flavus [2-9-31]. the international journal of applied biology, 5(2), 2021 46 genomic dna from the all seven isolates were extracted and prepared by using dna extraction kit (bio basic) as described according to manufacturer's instruction. extracted dna from isolates subjected as template for pcr amplification. the pcr protocol designed to differentiate a. flavus from other closely related species in particular from a. parasiticus, and organisms commonly found on grains [2-9-31-32]. multi-copy sequences of (its) (its-1, 5.8 s and its-2) of rdna amplified with specific pcr assays by using the purchased primers fla1 (5’gtagggttcctagcgagcc-3’) and fla2 (5’ggaaaaaga ttgatttgcgttc-3’) for a. flavus. pcr reactions were performed in the eppendorf mastercycler gradient. the pcr amplification protocol for a. flavus was as follows: 1 cycle of 5 min at 95 °c, 26 cycles of 30s at 95 °c (denaturation), 30 s at 58 °c (annealing), 45 s at 72 °c (extension) and, finally, 1 cycle of 5 min at 72 °c [2-32]. amplification reactions were carried out in volumes of 25 μl containing 4 μl (10 pg– 100 ng) of template dna, 1 μl of each primer (20 mm), 2x pcr reaction buffer, 4 mm of mgcl2, enzyme stabilizer, loading dye, and 0. 5 mm of dntps (100mm) and taq dna polymerase (1unit \ 10μl) supplied prime taq premix genetbio (korea). pcr products were detected on 2% agarose ethidium bromide gels in tae 1x buffer (tris–acetate 40mm and edta 1.0 mm). the 100-bp dna ladder genetbio (korea) was used as the molecular size marker. 2.4 determination of aflatoxigenic potential of a.flavus production ability of mycotoxins are vary among the aspergillus species, therefore, there is a need for screening for their toxin production abilities [14]. all identified a.flavus isolates were tested for their aflatoxigenic potential. using colony fluorescence, for detection of aflatoxin producers coconut milk agar media have been used for a primary screening of aflatoxin production according to [33].coconut constituents have effect on the fluorescent emission production in coconut culture [10]. for preparation of coconut milk agar media, 100 ml of coconut’s milk, purchased locally, homogenized for 5 minutes with 1000 ml of hot distilled water. four layers of cheese cloth used for filtration of homogenate, and ph of clear filtrate adjusted to (7) with 2 n naoh. mixture heated till it boiled, then cooled to 50°c, then ph adjusted again to (7). the mixture was then autoclaved for 18 min at 121ºc under 1.5 bar pressure, cooled to about 40 to 45°c, and poured into sterile petridishes [8-10-33]. aflatoxigenicity of isolates, checked by appearance of blue fluorescence of aflatoxins producer colonies when grown on coconut milk agar medium. suitability of solid media such as potato dextrose agar and coconut milk agar have been checked by some of researchers to differentiate aflatoxigenicity of aspergillus species [7-8-10-33]. loop full of a.flavus inoculum transferred to the center of coconut milk agar plate and inoculate with it, then incubated in the dark at 28°c. after seven days of incubations, presence and absence of fluorescence ring in the agar surrounding the colonies under the uv light scored as appositive and negative results [78-10]. using aspergillus differentiation media in detection of aflatoxin producers based on cultural characteristics: aspergillus differentiation media (afpa) is a selective and differential medium, it used for detection of aflatoxin producing aspergillus species from food samples. [8-9-15]. in this method, after seven days of incubation at 28 °c, aflatoxigenic a.flavus species have international journal of applied biology, 5(2), 2021 47 been distinguished from other aspergillus species based on visualizing of orange color on reverse side of colonies, this is due to the reaction of aspergillic acid molecules synthesized by aspergillus species with the ferric ions from ferric citrate of medium [9-10-15]. detection of aflatoxin producers based on the ammonia vapor test for this method, a.flavus was grown on medium such as potato dextrose agar and incubated for seven days. after incubation, the dish was inverted up on the lid contain 1 or 2 drops of concentrated ammonium hydroxide solution. the undersides of aflatoxin producing colonies quickly turn plum-red after the bottom of the petri dish has been inverted over the lid. essentially no color change occurs on the undersides of colonies that are not producing aflatoxins [7]. molecular detection of aflatoxigenic a.flavus isolates recently, molecular methods have been developed to distinguish and identify aflatoxinogenic strains of a.flavus from other foodborne fungi [15]. cluster genes in aflatoxin biosynthesis pathway contain structural marker genes such as (afld nor-1, and aflo omtb) which they are responsible for coding key enzymes in the production of aflatoxins. two primer pairs have been used for specific amplification of (afld and aflo) genes table (1) [35]. as a system control for dna amplification, housekeeping gene tub1 coding for βtubulin has been chosen in the system. amplification reactions were performed by using the prime taq premix genetbio (korea) kit with 25 μl reaction mix containing 4 μl (10 pg–100 ng) of template dna, 1 μl of each primer (20 mm), 2x pcr reaction buffer, 4 mm of mgcl2, enzyme stabilizer, loading dye, and 0. 5 mm of dntps (100mm) and taq dna polymerase (1unit \ 10μl). pcr products were detected on 2% agarose ethidium bromide gels in tae 1x buffer (tris–acetate 40mm and edta 1.0 mm). the 100-bp dna ladder (100-1500) genetbio (korea) was used as the molecular size marker. cycling parameters were: 5 min at 94 °c, for 35 cycles, 30 s at 94 °c, 60 s at 50 °c, 90 s at 72 °c, with a final extension at 72 °c for 7 min in a dna thermal cycler [16]. results morphological detection of a. flavus examination of cultural and morphological features can be used as a key to identify species in the a.flavus group and to differentiate between this group and other fungal groups with similar morphologies [9-28-30-36]. the color of the colonies was used for first identification of a.flavus. after seven days of incubation at 28°c on afpa, cma and at 25°c international journal of applied biology, 5(2), 2021 48 on pda, plates were observed for varieties of macroscopic characteristics such as colony color, colony diameter, colony reverse color figures 1,2 and 3. table 2. morphological features used to identify a. flavus on afpa and pda the size of the colonies are observed after seven days of incubation on both media. figure 1. colony characteristics of a. flavus : name of medium: afpa, colony color: white , colony diameter: (18) mm, colony reverse color: yellowish orange after seven days of incubation at 28°c. figure 2. colony characteristics of a. flavus. name of medium: cma. colony color: green. colony diameter: (40) mm. colony reverse color: hyaline. after seven days of incubation at 28°c. international journal of applied biology, 5(2), 2021 49 figure 3. colony characteristics of a. flavus : name of medium: pda, colony color: yellow to green, colony diameter: (35) mm, colony reverse color: hyaline, after seven days of incubation at 25°c. a. fluvus is morphologically characterized by yellow green woolly or granular colonies on pda. the colonies are flat, with radial grooves. this species is microscopically characterized by conidiophores that are hyaline and globose vesicles bearing chains of conidia. conidia appeared globose to subglobose, pale green in color [30-37]. conidial heads are typically radiate and biseriate figure 4. figure 4. biseriate conidial head of a.flavus: 1) aspergillum like spore bearing conidiophore. 2) globose vesicles. 3) vesicles bearing (metulae and phialides) biseriate conidial head. microscopic observation of the fungal isolate under 400x magnification (lactophenol cotton blue). 3.2 molecular identification of a.flavus: seven out of eighteen a. flavus isolates included in this study were identified by specific amplification of a dna fragment of expected size (500 bp) in pcr by using specific primers fla1 and fla2 figure 5 [2-31].as an alternative way, for rapid, high specific, sensitive diagnosis and detection of aflatoxigenic strains of a. flavus ,pcr based method used [2]. international journal of applied biology, 5(2), 2021 50 figure 5: pcr-based detection of a. flavus. lanes (1–7) pcr amplification product (500 bp) by using primers fla1/fla2 and dna from a. flavus isolates. lane (8) non dna template. m: (100) bp dna molecular size marker. reactions were carried out in volumes of 25 μl containing 4 μl (10 pg–100 ng) of template dna, 1 μl of each primer and pcr master mix.pcr products were detected on 2% agarose ethidium bromide gels in tae 1x buffer. cultural methods for aflatoxin detection various analytical methods have been reported for the detection of aflatoxins. much interest has come in developing and using cultural methods for detecting aflatoxins in fungal cultures. screening was done by using colony fluorescence upon exposure to uv, ammonia vapor test and orange color characteristics in aspergillus differentiation media [810]. detection of aflatoxin producers based on colony fluorescence aflatoxigenicity of isolates, checked by appearance of blue fluorescence of aflatoxins producer colonies when grown on coconut milk agar medium (cma) figure 6. aflatoxins produce an intense blue fluorescence visible at approximately (450 nm), when exposed to long-wavelength (365 nm) ultraviolet (uv) light [7-8]. figure 6 colony fluorescence detection of aflatoxin: coconut milk agar plate inoculated with loop full of a.flavus colonies and then incubated for 7 days at 28°c. a) cma plate with aflatoxin producing fungal colonies showing fluorescence on uv exposure. b) cma plate with non-aflatoxin producing fungal colonies showing no fluorescence can be detected on uv exposure. detection of aflatoxin producers based on reverse color change on afpa aspergillus differentiation media (afpa) is a selective and differential medium which it used for detection of aflatoxin producing aspergillus species. as it can be seen from international journal of applied biology, 5(2), 2021 51 figure 7, in this method, after seven days of incubation at 28 °c, aflatoxigenic a. flavus species distinguished from other aspergillus species based on visualizing of orange color on reverse side of colonies, this is due to the reaction of aspergillic acid molecules synthesized by aspergillus species with the ferric ions from ferric citrate of medium [9-10-15]. figure 7. a. flavus on afpa, after 7 days incubation at 28ºc, with characteristic orange color on reverse side of plate. ammonia vapor test for rapid detection of aflatoxigenic aspergillus: differentiation of aflatoxigenic and non-aflatoxigenic aspergillus by using ammonia vapor test was one of the applicable and rapid technique for this purpose [10]. in this method, the undersides of aflatoxin-producing colonies quickly turn plum-red after the bottom of the petridish has been inverted over the lid containing the ammonium hydroxide. essentially no color change occurs on the undersides of colonies that are not producing aflatoxins figure 8 [7]. figure 8. a.flavus was grown pda incubated for 7 days at 25 ºc. aflatoxinproducing colonies quickly turn plum-red after the bottom of the petri dish has been inverted over the lid containing the ammonium hydroxide. (a)aflatoxigenic fungal isolate before exposed to ammonium vapor. (b)aflatoxigenic fungal isolate producing pink color when exposed to ammonium vapor. detection of aflatoxin producing strains by conventional pcr method: for molecular diagnosis of aflatoxin producing strains of a. flavus, polymerase chain reaction (pcr) was carried out for two structural gene markers (afld, and aflo). as a system control for dna amplification, housekeeping gene tub1 coding for β-tubulin has been chosen in the system. pcr results revealed that all toxigenic isolates of a. flavus included in this study contained both potential gene markers (afld and aflo) figure 9. non dna templates running for each primers have been used to detect there were no any contamination in our pcr results. (b) (a) international journal of applied biology, 5(2), 2021 52 figure 9. pcr-based detection of aflatoxigenic a. flavus. reactions were carried out in volumes of 25 μl containing 4 μl (10 pg–100 ng) of template dna, 1 μl of each primer, 5.5 μl of rna dna free and pcr master mix. pcr products were detected on 2% agarose ethidium bromide gels in tae 1x buffer. gel electrophoresis analysis of pcr products using primers and dna extracted from isolate one of a.flavus. m: (100) bp dna molecular size marker. lanes (1) dna of a. flavus isolate using housekeeping gene tub1primer with 1498bp. lanes (2) dna of a. flavus isolate using afld primer with 990bp. lanes (3) dna of a. flavus isolate using aflo primer with 1333bp. discussion require time for accurate detection of toxicogenic fungi, is a one of the most critical point, it should take in consideration during search for incidence of toxigenic strains among the isolates. take the advantages of classical methods in preliminary determination of toxigenic strain allow us to select the most suitable approaches in diagnosis of aflatoxigenic fungi. culture methods such as colony fluorescence upon exposure to uv light, ammonia vapor test and colony appearance on aspergillus differentiation media, are examples of classical methods, they used for determination of toxigenic strains of our isolates. furthermore to corroborate of our results, two potential gene markers (afld and aflo) which they involved in the aflatoxin biosynthesis pathway have been detected by conventional pcr to convince of aflatoxigenicity of our results. morphological identification of the isolates: the description features reported by [9-28-29-30-37] have been used for morphological identification of isolates. a. fluvus is morphologically characterized by yellow green woolly or granular colonies on pda its colonies are flat, with radial grooves [37]. differences in colony diameter of a. flavus have been observed. colony diameter on afpa was smaller in compared to its diameter on pda, and this is due to the difference in nutrient ingredients in between afpa and pda media. seriation and microscopical features like metulae and phialides used as a primary keys for differentiation among a. flavus and a. parasiticus. rodrigues et al. [9], reported that a. parasiticus is mostly observed having uniserate conidial head. however, a. flavus shows biseriate conidial head. figure 4. molecular identification of the a. flavus: the most widely used dna target regions to identify aspergillus species are the ones in the rdna complex, mainly the internal transcribed spacer regions (its1-5.8s-its2 rdna) [2-5-9-31-38]. seven out of eighteen isolates in this study were identified by specific pcr protocol. extracted dna from isolates subjected as template for pcr amplification. the pcr international journal of applied biology, 5(2), 2021 53 protocol designed to differentiate a. flavus, from other closely related species in particular from a. parasiticus, and organisms commonly found on grains [2-9-31-32]. however, for further and accurate discrimination between those species that they show high degrees of dna relatedness with a. flavus such as a. oryzae, the mentioned protocol was not sufficient for differentiation among a. flavus and a. oryzae [2-9-38]. took the advantage of differences in genes involved in aflatoxins biosynthesis pathway between a. flavus and a. oryzae, allow differentiate them accurately [38].to discriminate between a. flavus and a. oryzae, marker genes in aflatoxins biosynthesis such as (afld, and aflo) have been detected in all seven isolates. molecular methods widely applied in the identification of a large number of aspergillus species with different levels of success. complex regions of rdna and structural aflatoxin genes reported as a markers for identification of a. flavus [9]. al-wathiqi et al., [31] successfully used two different sets of primers for identification of a. flavus, universal primer for (its-1 and its-2) (genus specific primers), and (species specific primers) for variable regions of β-tubulin and calmodulin genes. some genes like topoisomerase 2 calmodulin, as well as β-tubulin are extensively used to identify fungi at species level. however, the use of these genes is limited to distantly relate fungal species [39-40]. el khoury et al., [41] designed gene-specific primers for the aflatoxin biosynthesis genes. in order to distinguish between a. flavus and a. parasiticus. detection of aflatoixgenic a. flavus: three different cultural based methods and conventional pcr, were used in order rapidly differentiate between aflatoxigenic and non aflatoxigenic of aspergillus species. [910-11]. detection of aflatoixgenic a. flavus by using the culture based method colony fluorescence nair et al., [8], davis et al.,[33] describe the method, in which colony of aflatoxigenic strains produced blue fluorescence on the reverse side of the coconut milk agar medium when exposed to uv light. [10]. in our results, we observed that coconut milk agar medium showed an increase in the number of fungal colonies exhibiting blue fluorescence zones. this can be explained by the enhancement of aflatoxin production and detection using uv light in the case of coconut supplemented agar media [33]. aflatoxigenicity of a. flavus on coconut milk agar (cma) media reported by yazdani et al., [10], as a result appearance of fluorescence ring around the colonies. treatment by enhancer such as cyclodextrins agents will substantially improve the fluorescence emission of aflatoxins bl and g1[7].yazdani et al., [10] showed that the cyclodextrins test did not have enough sensitivity for detection of aflatoxins. detection of aflatoixgenic a. flavus by using the culture based method aspergillus differentiation media aflatoxigenic a. flavus can produce yellowish orange color on the reverse side of the colonies, when they have been grown on the (afpa) medium. (afpa) is a selective and differential medium for a. flavus and a. parasiticus. it was used for the rapid detection of aflatoxigenic species. the coloration is due to the reaction of aspergillic acid molecules synthesized by aspergillus species with the ferric ions from ferric citrate of medium [8-9]. both a. flavus and a. parasiticus are capable to produce potent aflatoxins. some of international journal of applied biology, 5(2), 2021 54 researchers stated that, only aflatoxins b1 and b2, synthesized by a. flavus, whereas a. parasiticus has ability to produce aflatoxins b1, b2, g1 and g2 [1-18-42]. our results shows that this medium (afpa) is suitable for primary detection of aflatoxigenic a. flavus. detection of aflatoixgenic a. flavus by using the culture based method (ammonia vapor test) novel method has been introduced by saito and machida, [43], as a rapid and sensitive identification way to differentiate aflatoxin producing and nonproducing strains of a. flavus. plum red color was observed when the (pda) culture of aflatoxigenic a. flavus was inverted over the lid containing the ammonium hydroxide. this change in color is due to anthraquinone intermediates compounds of aflatoxins biosynthesis which act as ph indicator dyes, and are more visible when they have turned red at alkaline ph [1]. essentially no color change occurs on the undersides of the colonies that are not producing aflatoxins [11-44]. yazdani et al., [10] detected the production of toxin by tlc and hplc while negative results of ammonium vapor test for some of the isolates have been observed. based on that, they concluded that, the ammonium hydroxide test was an unreliable test for detection of aflatoxins. in contrast regarding to the our results, we demonstrated that ammonium vapor test can be used as a method for primary screening for detection aflatoxigenic of a. flavus. detection of aflatoixgenic a. flavus by using molecular method (conventional pcr) molecular techniques have been widely applied to distinguish the aflatoxin producing and non-producing strains of a. flavus and related species. this has been through the correlation of presence-absence of one or several genes involved in the aflatoxin biosynthetic pathway and the ability-inability to produce aflatoxins. recently, dna based detection systems have been introduced as powerful tools for detecting and identifying the aflatoxin producing fungi [12-1445]. several pcr based systems have been developed to discriminate between aflatoxin producing and non-producing a. flavus strains. hussain et al., [12], degola et al., [14], and criseo et al., [15], used a multiplex pcr approach based on the amplification of four target dna fragments (afld , aflo, aflp and aflr ) for detection of aflatoxigenic a. flavus. latha et al., [46] reported that the multiplex pcr could be used as a marker to clearly differentiate between the aflatoxin-producing and nonaflatoxigenic a. flavus. mayer et al., [47] have used real-time pcr to monitor the expression of an aflatoxin biosynthetic gene of a. flavus in wheat. houshyarfard et al., [48] reported that the analysis of deletion patterns in aflatoxin gene cluster was a useful marker for the identification of non-aflatoxigenic strains. furthermore, several research groups have adopted reverse transcription pcr technique (rt-pcr) to detect an mrna specific for an aflatoxin biosynthetic gene and differentiate aflatoxin-producing from nonproducing strains of a. flavus [14-16-47]. among the 27 genes involved in aflatoxin biosynthesis only two structural key genes (afld, and aflo) have been used in this study as a target molecular markers to detect the aflatoxigenic a. flavus. scherm et al. [16] studied 13 isolates of a. flavus and found consistency of (afld and aflo) genes in detecting aflatoxin production ability, further indicating them as potential markers. most of the genes in the aflatoxin biosynthetic pathway gene cluster are regulated by (aflr) [18]. (aflo,and aflp) are necessary for final formalities of aflatoxins biosynthesis [19-21]. in the present study, molecular method is proved as a rapid and accurate detection system to detect aflatoxigenic a. flavus. our international journal of applied biology, 5(2), 2021 55 results for all aflatoxigenic isolates shows that bands of the fragments of (afld and aflo) genes visualized at (990, 1333) respectively in figure 9. rodrigues et al., [9], scherm et al. [16], latha et al., [46] reported that the expression profile of the two genes (afld and aflo) were consistently correlated with a strain’s ability to produce aflatoxins. degola et al., [14] shows that one strain that apparently transcribed all the relevant genes (afld and aflo) but did not produce aflatoxin in the medium. according to the houshyarfard et al., [48], biosynthesis of aflatoxins depend on the several factors. firstly, presence of certain genes. secondly, the genes should be intact. it means that any changes for instance deletions or insertions within the gene regions or regions flanking the gene will influence the aflatoxin’s biosynthesis. deletions of several portions of the aflatoxin biosynthesis gene cluster has been reported to be the main cause for the lack of aflatoxin production. degola et al., [14] found that screening tests based on pcr detection of aflatoxin biosynthesis genes are not dependable, this is due to the technique that cannot detect mutations outside the primers’ targeted region of the gene sequence. no sign of aflatoxigenicity can be observed for eleven isolates when we used the one culture method for detection of their toxigenicity. this is the reason why we consider these isolates as non aflatoxigenic isolates. non aflatoxigenicity of these isolates, may be because of a mutation or gene deletion in one or more genes belonging to the biosynthetic gene cluster [14]. houshyarfard et al., [48] said that the analysis of deletions within the aflatoxin biosynthesis gene cluster for the 15 iranian non-aflatoxigenic strains of a. flavus revealed that a. flavus strains had different deletions in the aflatoxin gene cluster. conclusions in this study, it is concluded that seven isolates of a. flavus were able to confirm the aflatoxin production by using culture methods and amplifying the two target genes (afld and aflo) as these genes are considered as indicators of aflatoxin production. conflict of interest the authors declare they have no 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and ehrlich, k. aflatoxin biosynthesis. rev. iberoam micol, (19):.191200, 2002. yu, j., chang, p., ehrlich, k., cary, j., bhatnagar, d., cleveland, t., payne, g., linz, j., woloshuk, c. and bennett, j. clustered pathway genes in aflatoxin biosynthesis. appl. and environm. microbiol 70(3):.1253-1262,2004. 1(1)32-36 contact&:&sabrina&resky&pratiwi&&&&&&&sabrinareskyp7@gmail.com3 ©3international3journal3of3applied3biology3 323 abstract& earthworms3 (lumbricus) rubellus)3 extract3 is3 known3 to3 contain3 bioactive3 protein3 as3 antibacterial3 compounds.one3 of3 the3 disadvantages3 of3 polar3 compounds3is3slow3penetration3into3the3skin3layers3which3can3be3solved3by3 formulating3it3in3the3form3of3ethosomal3drug3delivery3system.3the3aims3of3 this3research3was3to3find3out3information3about3ethanol3concentration3that3 provide3the3highest3entrapment3 efficiency3of3 the3ethosome.3earthworms3 powder3 was3 macerated3 using3 50%3 ethanol3 for3 33 days.3 the3 extract3 was33 formulated3into3ethosome3with3variation3of3ethanol3concentration3that3are3 20%,330%,340%,3and350%,3respectively.3the3measurement3of3entrapment3 efficiency3 was3 conducted3 by3 measuring3 the3 amount3 of3 active3 protein3 of3 earthworms3 extract3 that3 was3 entrapped3 in3 ethosome3 vesicles3 using3 spectrophotometer3 uvnvisible.3 the3 results3 showed3 that3 the3 entrapment3 efficiency3was3directly3proportional3to3ethanol3concentration3in3formula.33 & issn3:32580n24103 eissn3:32580n21193 3 3 3 a&study&on&entrapment&efficiency&of&earthworms&(lumbricus)rubellus)&& extract&in&the&ethosomal&drug&delivery&system) 3 sabrina&resky&pratiwi,&emilia&utomo,&hardyanti,nur&ainiah,&dini&rusdayanti&putri&&&sartini& 3 faculty3of3pharmacy,3hasanuddin3university,3makassar,3indonesia3 3 3 & & & & & & & & & & & & introduction& earthworms3(lumbricus)rubellus)l.)3had3been3used3as3china3traditional3medicine3and3 empirically3used3by3people3to3treat3thypoid3fever3caused3by3salmonella)thyposa3and3acne3vulgaris3 caused3by3propionibacterium)acnes3(immanita,32012;3sun,32015).3this3treatment3was3supported3 by3previous3studies3which3showed3that3earthworms3has3antibacterial3and3antioxidant3activity3 (aldarraji,3 2013;3 mathur,3 2011).3 earthworm3 extract3 in3 concentration3 7.5%3 could3 inhibit3 the3 growth3 of3 propionibacterium) acnes) and3 treat3 inflammation3 due3 to3 acne3 (asmawati,3 2016).3 earthworm3 l.) rubellus3 was3 made3 in3 a3 solid3 dosage3 form3 proved3 to3 have3 broad3 spectrum3 antimicrobial3against3gramnpositive3bacteria3s.)aureus,3gramnnegative3bacteria3e.)coli,3and3fungi3 c.)albicans3(damayanti3et)al.,32008).3besides,3earthworms3also3contained3phenolic3compound3 which3has3antioxidant3activity3(aldarraji,32013).3 the3disadvantages3of3earthworms3extract3are3having3bad3smell3and3slow3absorption3into3 skin3 layer3 caused3 by3 polar3 bioactive3 compounds.3 previous3 research3 has3 conducted3 the3 formulation3of3earthworm3extract3in3the3gel3preparation,3but3it3wasnot3able3to3cover3the3smell3 up3(asmawati,32016).3another3formula3containing3earthworm3extract,3maltodextrin,3distilled3 &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword3 ethosome,3 ethanol3concentration,3 entrapment3efficiency,3 lumbricus)rubellus.3 3 article&history3 received3183june320173 accepted3193august3201733 3 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 3 international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international)journal)of)applied)biology,)vol.)1,)issue)1,)2017) 3 333 water3 (23 :3 53 :3 50)3 was3 made3 by3 encapsulating3 extract3 in3 maltodextrin3 and3 tested3 for3 its3 antibacterial3activity3(istiqomah,32012).33 the3 ethosome3 is3 a3 carrier3 of3 a3 kind3 of3 soft3 vesicle3 contained3 phospholipid,3 high3 concentrations3 of3 alcohol,3 and3 water3 (tiwari,3 20103 ;3 patel,3 2013).3 ethosomal3 drug3 delivery3 system3has3many3advantages3such3as3good3delivery3of3protein3molecules,3containing3non3toxic3 materials,3 increasing3drug3permeation3into3skin3 layers,3and3convenient3to3use.(razavi,32015;3 sujatha,32014).3good3penetration3of3ethosome3is3caused3by3two3mechanisms.3firstly3ethanol3 takes3a3role3as3penetration3enhancer3which3is3penetrate3into3intercelluler3lipid,3increase3lipid3 fluidity3of3cell3membrane,3and3decrease3multilayer3lipid3of3cell3membrane.3secondly,3ethosome3 increases3skin3permeability3so3that3it3is3easily3permeated3into3inner3layer3of3skin3and3release3the3 drug3(parashar,32013).3 entrapment3efficiency3is3the3most3important3thing3in3characterization3study3of3ethosome3 formulation.3it3indicates3the3amount3of3drug3or3bioactive3compounds3which3is3entrapped3in3the3 vesicles.3 it3 will3 absolutely3 affect3 the3 amount3 of3 drug3 that3 reaches3 the3 target3 of3 therapy3 (barupal,2010).3in3order3to3find3out3the3highest3entrapment3efficiency3of3earthworms3extract,3 the3measurement3of3entrapment3efficiency3was3done3to3four3ethosome3formula3with3variation3 of3ethanol3concentration3(20%,330%,340%3and350%).3the3aims3of3this3research3was3to3find3out3 information3about3ethanol3concentration3that3provide3the3highest3entrapment3efficiency3of3the3 ethosome.33 & materials&and&methods& earthworm&preparation& dried3earthworms3(lumbricus)rubellus)l.)3that3were3used3in3this3research3obtained3from3 makassar,3south3sulawesi,3indonesia.33 3 extraction&of&earthworms& earthworms3extraction3was3done3by3using3maceration3method.3a3total3of35003g3dried3 earthworms3was3macerated3using350%3solvent3ethanol3for333days.3the3solvent3was3evaporated3 using3rotary3evaporator3and3continued3with3lyophilization3to3obtain3viscous3extract.3 3 measurement&of&total&protein&content&of&earthworms&extract& & a3total3amount3of3503mg3extract3was3dissolved3in3distilled3water3to3103ml3(530003ppm).3 taken32003µl3of3the3solution3and3then3added3with3reagent3c,3 incubated3for3103minutes3and3 reagen3 d,3 incubate3 for3 203 minutes.3 distilled3 water3 was3 added3 up3 to3 53 ml3 (2003 ppm).3 the3 absorbances3were3measured3using3spectrophotometer3uvnvisible3at37023nm.3 & preparation&of&ethosomal&dispersion& the3composition3of3ethosome3formula3of3earthworms3extract3is3shown3in3table31.3 table&1.&composition&of&ethosome&formula&of&earthworms&extract& compositions& amount& f1& f2& f3& f4& earthworms3extract3 0.53%3 0.53%3 0.53%3 0.53%3 soya3lecithin3 2%3 2%3 2%3 2%3 96%ethanol33 20%3 30%3 40%3 50%3 propyleneglycol3 10%3 10%3 10%3 10%3 distilled3water3up3to3 100%3 100%3 3100%3 3100%3 international)journal)of)applied)biology,)vol.)1,)issue)1,)2017) 3 343 the3ethosome3was3made3using3cold3method.3earthworms3extract3was3dissolved3in3ethanol3and3 homogenized3using3magnetic3stirrer3for353minutes3at37503rpm,340oc.3after3that,3soya3letichin3was3 dispersed3into3the3mixture.3after353minutes,3propyleneglycol3was3added3into3the3mixture3and3 homogenized3for353minutes.3distilled3water3at330oc3was3added3at3last.3the3size3of3ethosome3 vesicles3was3minimized3by3sonicating3for3153minutes.3 3 determination&of&entrapment&efficiency& & an3amount3of313ml3of3ethosome3was3added3to3effendorf3tube,3then3centrifugated3for323 hours3at313.0003rpm,34oc.3taken3250µl3of3the3supernatant,3then3was3added3with3reagen3c,3 incubated3for3103minutes3and3reagen3d,3incubated3for3203minutes.3distilled3water3was3added3up3 to3 53 ml.3 the3 absorbance3 was3 measured3 using3 spectrophotometer3 uvnvisible.3 percentage3 of3 entrapment3efficiency3was3calculated3using3the3formula3below:3 !! = #$%&'(%)*'(+#,,*--+&.#$%&'(%)(%(#/-+&. 0100%& & results&and&discussion&& entrapment3efficiency3is3an3important3thing3in3the3characterization3study3in3ethosomal3 formulation.3it3indicates3the3amount3of3drug3entrapped3in3the3prepared3vesicles3(verma,32011).3 because3of3that,3it3is3expected3that3the3higher3the3entrapment3efficiency,3the3higher3amount3of3 drug3that3reaches3target3of3therapy.3in3this3study,3there3are3four3concentration3of3ethanol3that3 was3used3that3are3f13(20%),3f23(30%),3f33(40%),3and3f43(50%).3 the3results3showed3the3percentage3of3entrapment3efficiency3of3f1,3f2,3f3,3and3f43that3 are335.56%,330.45%,359.16%,3and372.58%,3respectively3as3shown3in3figure31.3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 & & figure&1.&entrapment&efficiency&of&ethosome&of&earthworms&extract& & & the3 results3 indicated3 that3 the3 entrapment3 efficiency3 was3 affected3 by3 ethanol3 concentration3in3formula.3the3highest3entrapment3efficiency3was372.58%3given3by3f43which3 containing350%3of3ethanol.3this3is3likely3due3to3the3solubility3of3extract3in3the3ethosomal3core3was3 higher3in3f43than3other3formulas.3in3this3study,3the3entrapment3efficiency3of3the3formulation3 escalated3along3with3the3increasing3of3alcohol3concentrations.3alcohol3is3a3natural3enhancer,3 which3has3the3property3to3alter3the3skin3permeability.3however,3transdermal3permeability3of3 35.559 30.447 59.163 72.583 0 10 20 30 40 50 60 70 80 90 f1 f2 f3 f4 en tr ap m en t&e ff ic ie nc y& (% ) treatment international)journal)of)applied)biology,)vol.)1,)issue)1,)2017) 3 353 ethosomal3formulations3was3found3to3be3higher3compared3to3hydroalcoholic3drug3solution3which3 indicates3that3alcohol3is3not3the3only3one3contributor3to3increase3the3skin3permeability.33 several3studies3have3investigated3the3possible3mechanism3of3enhancing3skin3permeability3 by3lipid3vesicular3system.3vesicles3can3interact3with3the3stratum3corneum3lipids3and3alter3the3 permeability,3 which3 facilitates3 penetration3 of3 drug3 across3 stratum3 corneum.3 enhanced3 permeation3of3drug3with3ethosomal3formulations3could3be3caused3by3the3combination3effect3of3 alcohol3and3lipid3vesicular3system3(chourasia,2011).3according3to3vijayan3(2015),3entrapment3 efficiency3was3affected3by3molar3ratio3of3lecithin3and3ethanol.3in3addition,3in3the3formulation3of3 serratiopeptidase3enzyme3ethosome,3the3highest3entrapment3efficiency3was375.37%3given3by3 formula3containing3lecithin35%3(w/w)3:3ethanol3203ml,3propyleneglycol3103ml,3active3compound3 0.053 mg3 (vijayan,3 2015).3 another3 previous3 research3 showed3 that3 there3 are3 two3 factors3 influencing3the3entrapment3efficiency3that3are3soya3phosphatidyl3choline3(spc)3and3ethanol3 concentration.3the3amount3of3these3materials3influenced3the3entrapment3of3ketoprofen3inside3 lipid3vesicles3in3a3positive3way.3it3has3been3said3the3entrapment3efficacy3of375n80%3was3provided3 by3 formula3 containing3 30n40%3 of3 alcholo3 and3 2,5n3%3 of3 spc.3 while3 the3 lower3 efficacy3 was3 performed3by3formula3containing3333320n25%3of3alcohol3and31n2%3of3spc3(chourasia,2011).33 in3addition,3research3about3aceclofenac3ethosome3has3shown3that3the3highest3and3the3 lowest3 entrapment3 efficiency3 was3 given3 by3 formula3 containing3 30%3 and3 50%3 of3 ethano3 respectively.3there3was3increase3in3percent3entrapment3efficiency3was3observed3with3an3increase3 of3 ethanol3 concentration,3 but3 when3 ethanol3 concentration3 exceeded3 30%,3 a3 decrease3 in3 entrapment3efficiency3was3observed.3if3ethanol3concentration3increased3above330%3resulting3 into3leakage3of3drug3from3fluidized3bilayer3of3vesicles3(barupal,32010).3the3obtained3results3of3 this3research3was3in3line3with3previous3mentioned3research3which3indicated3that3the3higher3the3 concentration3of3ethanol,3the3higher3its3entrapment3efficiency.3besides,3based3on3mechanism3of3 drug3entrapment3in3ethosome,3hydrophilic3drugs3are3entrapped3in3the3aqueous3core3of3lipid3 carrier3 while3 lipophilic3 drugs3 are3 retained3 in3 the3 nonpolar3 chain3 (chourasia,3 2011).3 so,3 earthworms3extract3which3is3hydrophilic3was3entrapped3in3the3core3of3ehtosomes3vesicle.3 3 conclusion&& 3 the3highest3percentage3of3entrapment3efficiency3of3earthworms3extract3ethosome3was3 72.58%3 presented3 by3 formula3 containing3 earthworms3 extract3 5%,3 soya3 lecithin3 2%,3 propyleneglycol3 10%,3 and3 ethanol3 50%.3 the3 entrapment3 efficiency3 of3 the3 formulations3 was3 observed3to3increase3along3with3the3increasing3concentration3of3alcohol.33 3 acknowledgements& 3 this3 research3 was3 supported3 by3 the3 ministry3 of3 research,3 technology3 and3 higher3 education3of3republic3of3indonesia3for3the3research3grant.3 & references& aldarraji,3 q.m.,3 halimoon,3 n.3 and3 majid,3 n.m.2013.3 antioxidant3 activity3 and3 total3 phenolic3 content3 of3 earthworm3 paste3 of3 lumbricus) rubellus3 (red3 worm)3 and3 eudrilus) eugenia3 (african3night3crawler).3journal3of3entomology3and3nematology.35(3):33n37.3 asmawati3 and3 pratiwi,3 s.r.3 2016.3 development3 of3 earthworm3 (lumbricus) rubellus)3 extract3 formulation3as3antiacne:3its3antiinflammatory3and3antibacterial3activity.3research3grant3 report.3universitas3hasanuddin.3 international)journal)of)applied)biology,)vol.)1,)issue)1,)2017) 3 363 to&cite&this&article:&& pratiwi,3s.r.,3utomo,3e.,3hardyanti,3ainiah,3n.,3putri,3d.r.3&3sartini.32017.3a3study3on3entrapment3 efficiency3 of3 earthworms3 (lumbricus) rubellus)3 extract3 in3 the3 ethosomal3 drug3 delivery3 system.) international)journal)of)applied)biology.31(1)3:332n336.33 & barupal,3a.k.,3gupta.3v.,3and3ramteke,3s.32010.3preparation3and3characterization3of3ethosomes3 for3topical3delivery3of3aceclofenac.3indian3journal3of3pharmaceutical3sciences.372(5):582n 586.3 damayanti,3 e.,3 h.3 julendra,3 &3 a.3 sofyan.3 2008.3 antimicrobial3 activity3 of3 earthworm3 meal3 lumbricus3rubellus3and3its3potency3as3additive3in3poultry3feed.3j.3biosfera325:3123n128)& immanita,3s.,3debora,3k..,3and3rochmanti,3m.32012.3effect3of3earthworms3(lumbricus3sp.)3extract3 antibacterial3activity3against3the3bacteria3salmonella)typhii.)folia3medica3indonesiana.3 48(3):102n108.3 istiqomah,3 l.,3 herdian,3 h.,3 damayanti,3 e.,3 hayati,3 s.n.,3 and3 julendra,3 h.3 2012.3 inhibitory3 of3 encapsulated3 earthworm3 extract3 (lumbricus) rubellus)3 on3 pathogenic3 bacteria3 in) vitro.3 media3peternakan.353(1):31n8.& mathur,3a.,3verma,3s.k.,3singh,3s.k.,3prakash,3a.,3prasad,3g.b.k.s.,3and3dua,3v.k.32011.3antin inflammatory3 activity3 of3 earthworm3 extracts.3 international3 journal3 of3 pharmaceutical3 sciences3and3research.32(2):278n281.3 parashar,3t.,3sachan,3r.,3singh,3v.,3singh,3g.,3tyagi,3s.,3patel,3c.,3and3gupta,3a.32013.3ethosomes:3 a3recent3vesicle3of33transdermal3drug3delivery3system.3international3journal3of3research3 and3development3in3pharmacy3and3life3sciences.2(2):3285n292.& patel,3a.,3sharma,3r.k.,3trivedi,3m.,3shivaprakash,3and3panicker,3a.32013.3ethosomes:3a3novel3 tool3for3transdermal3drug3delivery.3research3journal3of3pharmacy3and3technology.)63(8):3 838n841.3 rajput,3 a.,3 shriwas,3 s.,3 dwivedi,3 s.3 and3 dubey,3 r.3 2016.3 formulation3 and3 evaluation3 of3 ethosomes3of3plumeria3indica3linn.3flowers.3advances3in3pharmacology3and3toxicology.) 17(3):3345n483 razavi,3h.3and3janfaza,3s.32015.3ethosome:3a3nanocarrier3for3transdermal3drug3delivery.3journal3 of3paramedical3sciences.363(2):338n43.3 sujatha,3v.,3vishnuvaravidyadhar,3t.,3parvathi.,3m.,3and3reddy,3s.32014.3a3review3on3transdermal3 drug3delivery3system3by3ethosomes.3pharmatutor3magazine.323(11):350n55.3 sun,3z.32015.3earthworm3as3a3biopharmaceutical:3from3traditional3to3precise.)european)journal) of)biomedical)research.)13(2):328n35.3 tiwari,3 r.k.,3 chauhan,3 n.s.,3 and3 yogesh,3 h.s.3 2010.3 ethosomes:3 a3 potential3 carriers3 for3 transdermal3drug3delivery.3international3journal3of3drug3development3and3research.)23 (2):3448n452.3 verma,p.,deshmukh,k.r.,and3 pandey,a.3 2011.3 entrapment3 efficiency3 study3 of3 hydrophilic3 molecule3in3ethosomal3formulations.3international3journal3of3universal3pharmacy3and3life3 sciences.31(3):351n56.3 vijayan,3v.,3kumar,3k.j.,3muralidharan,3s.,3and3parasuraman,3s.formulation3and3characterization3 of3 novel3 ethanolic3 carrier3 for3 improves3 penetration3 of3 serratiopeptidase.3 research3 &3 reviews:3a3journal3of3drug3formulation,3development3and3production.23(1):31n6.& 3 3 3 3 contact : queen ozioma lawrence queenlawrence8@gmail.com 126 abstract human intestinal parasites are among the most common infectious disease s worldwide. this st udy wa s conducted to determine intestinal parasites prevalence and risk factors in the study area. a total of 350 c onsented inmates aged 18->60 years participated in the study. stool samples were scree ned using wet preparation and formol ether c oncentration method. an overall prevalence of 48.8% was recorded for intestinal parasite, which wa s significantly (p< 0.05) high among females. five species of inte stinal parasite were ide ntifie d. the most common identified was hookworm (95, 33.5%), while the least was trichuris trichiuria (33, 11.6%). age group 51-60 years recorded the highest infection (63.33%) among the male s, while age group 18-20 years had the least (25.0%). age groups 51-60 and >60 years recorded 100% infections among the females. of the 171 infections recorde d, 97(56.7%) had single infection, while 74(43.3%) had multiple infections. questionnaire analysis showe d that inmates who have been in the prison for years were more parasitized. inmates who dew orm monthly, yearly and not at all accounted for 64.2%, 78.7% and 39.2% infections respectively whic h was significant (p< 0.05). the study revealed the prevalence of intestinal parasitic infections in the inmates. improved healthcare a nd awareness is highly recommended inside the facility. issn : 2580-2410 eissn : 2580-2119 intestinal parasites and risk factors among inmates in umuahia abia state, nigeria. queen ozioma lawrence 1*, anthonia nnenna chuks amadi 1, chinenye okosa 1, eberejah juliet john 2, nsikakabasi charles ukpong 3, prisca chimerebere aririguzo 4 1 department of zoology and environmental biology, college of natural sciences, michael okpara university of agriculture umudike, nigeria 2 department of microbiology, faculty of biological sciences, abia state university uturu, nigeria 3 department of ani mal and environmental biology, faculty of sciences, university of uyo, nigeria 4 department of public health, school of health technology, federal university of technology owerri, nigeria introduction the parasitic worms found in the intestine of man or animals are known as intestinal parasites (arora, and arora, 2006) intestinal parasite infections are among the most persistent public health problems and are of public health importance (brookers et al., 1999). either protozoa or helminthes can cause intestinal parasite infection. protozoan parasites are one-celled microscopic organisms belonging to the kingdom protista ( arora, open access international journal of applied biology keyword parasites; hookworm; infection; factors; inmates article history received june 25, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 127 and arora, 2006). they can take over the intestinal tract of thei r host and then migrate to other organs and tissues (arora, and arora, 2006), while helminthes are worms usually with elongated, flat, or round bodies (castro, 1996). among these helminthes are soil transmitted hel minthes; the roundworms, whipworm and hookworm, classified as neglected tropical diseases. neglected tropical diseases (ntds are diverse group of 20 health condi tions that are mainly prevalent in tropical areas, where they mostly affect the impoverished communities and disproportionately affects women and childre n (who, 2002; who, 2022). they are widely distributed in tropical and subtropical areas, with the most significant numbers occurring in sub-saharan africa, the americas, china, and east asia (who, 2002). theses disease causes devastating health, social and economic consequences to more than 1 billion people (al amin and wadhwa, 2022). its study has received less than 1% of the global research budget (al amin and wadhwa, 2022). approxi mately more than 2 billion people are infected by soil-transmithiasis (samuel et al., 2017) the majority of the people in the general population are affected by intestinal parasites to which the main species are entamoeba histolytica, ascaris lumbricoides (roundworm), trichiuris trichiuria (whipworm) and hookworms species (lorainne et al., 2015). intestinal parasite infections can be contracted through faecal-oral rout (eg; eggs or larvae t. trichiuria and a. lumbricoides) transdermal (e.g; larval of hookworm) and vector-borne transmission (eggs of the flukes) (schabussova, 2014; kiani et al., 2016; kumie and ali, 2005). these parasitic intestinal infections are promoted by several factors such as poor personal and community hygiene, poor environmental sanitation, ignorance, and climatic condition (al amin and wadhwa, 2022). other factors tied to the prevalence of the infection are; untreated water supply, poverty, low level of education (kiani et al., 2016). amuta et al. (2010) reported tha t improperly cooked meat exacerbates intestinal parasite infection. his the study further noted that sources of drinking water like the river, well, water bought from vendors and patronizing food vendors were observed as risk factors for the tra nsmission of intestinal parasite infections in the study area. kiani et al. (2016) observed that educational status, contact with domestic animals and soil, age and seasons were significantly related to intestinal parasite infections. signs and symptoms of intestinal parasitic infections can often be unclear and misleading or even asymptomatic. however, some of the symptoms include some common ones like abdominal pain, bl oating, nausea, and vomiting. diarrhoea is also a common infection symptom usually observed in the chronic state. intestinal parasites lead to the development of mild diarrhoea, which ma y last for several days or even months. e. histolytica occasionally may invade the brain, lungs, liver, and other organs forming cysts and giving rise to a disease called amoebic dysentery. other symptoms include abdominal cramps and severe colitis, along with the development of an ulcer. in addition, blood and pus may be seen occasionally in the stool (who, 2022; al amin and wadhwa, 2022). intestinal parasitic infestations do not let the intestines absorb minerals and vitamins, resulting in pale skin and fatigue (arora, and arora, 2006). transmission of parasitic infection can be prevented by employing good hygienic practices, avoiding soil consumption, discriminate defecation, and washing vegetables and fruits with clean water bef ore intake is highly recommended (al amin and wa dhwa, 2022; khan et al., 2022). the constant wearing of shoes or slippers (to prevent hookworm infection) should be instill on the population. campaign progra ms should be employed to educate the population on the need for proper personal and environmental hygiene, i. e. washing hands after going to the toilet, playing outside, and preparing or eating food (al amin and wadhwa, 2022; who, 2022). safe single-dose drugs, such as albendazole, are international journal of applied biology, 6(2), 2022 128 highly effective for treating intestinal parasites, available through healthca re services, school health progra ms, and community interventions directed at vulnerable groups (who,2022; ughava and okon, 2016). the study is aimed at determining the presence and prevalence of intestinal parasites and their risk factors among inmates in a correctional facility (prison) in umuahia, abia state, nigeria. materials and methods a cross sectional study was adopted for the purpose of this study which lasted between februa ry and may 2017. t he study was carried out in umuahia prison located at aba road in afara, in umuahia the capital of abia state, south-eastern nigeria (fig 1). it is located on latitude 5o31’12.0”n and longitude 7o29’16.8”e in nigeria. the average annual rainfall is 133.7mm and temperature 74oc. the study a rea is within the tropical rainforest of eastern nigerian states (within the ecological zones of enugu, akwa ibom, cross river, ebonyi, imo, and rivers states). samples were exa mined in zoology and environmental biology (zeb) postgraduate laboratory, michael okpara university of agriculture umudike (mouau). figure 1. map of umuahia north showing the location of the prison, aba road, umuahia sample collection ethical clearance was sought and obtained from the ethical board of authorities of nigerian correctional services, umuahia, abia state (ref no: abs/ shq/c.37/vol.111/907), ministry of health ethical research committee, abia state (ref no: ab/mh/e&hr/1/17/0 4) and ethical research committee, college of natural sciences ethical research committee, michael okpara university of agriculture umudike (ref no: creec/004/18). consent was sought from the inmates by educating them on the need and relevance of the study. a fresh stool sample was collected using a sterile labeled screwed capped bottle from 350 inmates (280 males and 70 females) aged 18->60 years and was transported to zoology and international journal of applied biology, 6(2), 2022 129 environmental biology postgraduate laboratory, michael okpara university of agriculture umudike, nigeria. samples were assessed macroscopically and reported before laboratory analysis (cheesbrough, 2005). saline wet preparation and formol ether concentration methods were used for laboratory examinations. saline wet preparation technique a drop of normal saline was placed on a clean grease free glass slide, and 2g of faeces was placed and gently mixed with the drop of normal saline. they were gently covered wi th cover slip to avoid air bubbles. the smear was examined under binocular microscopy with x10 and then x40 objectives (arora and arora, 2006). formol ether concentration technique formol ether concentration techniques were used to demonstrate the presence of eggs and cysts of the parasites. about 2g of stool sample was emulsifi ed using a rod in approximately 3ml of 10% formol saline contained in a tube. 3-4ml of 10% formol saline was further added, mixed well, and then sieved. about 7ml was transferred into a test tube, and 3ml of formol ether was added to make it 10ml. covered and shake very well for proper mixing. samples were centrifuged immediately at 1500rpm for five minutes. with a stick, the faecal debris layer was loosed from the side of the tube and then inverted to discard the formol saline and faecal debris. the sediment was transferred to a slide, covered wi th a cover slide and examined microscopically using x10 and x40 objective to examine the small cyst and eggs of the parasites (zongo et al., 2020). statistical analysis the part statistical package (version 3.1) was used for data analysis to test the association between demog raphics, behavioral factors, and intestinal parasite infection. results were analyzed using chi-square. the relationship was considered significant when the p-value was <0.05 (hammer, 2001). results and discussion results a total of 171 inmates were infected with at least one intestinal parasite; giving an overall prevalence rate of 48.8% (table 1). the prevalence rate of intestinal parasites was highest among the females (72.8%) compared to the males (42.8%). there was a significance difference (p < 0.05) in the prevalence of the intestinal parasites among the female and male inmates. table 1. gender related prevalence of intestinal parasites infection among inmates in umuahia correctional facility, abia state gender total no examined total no infected percentage (%) p – value male 280 120 42.8 0.015572 female 70 51 72.8 total 350 171 48.8 df= 1, chi 2=61846, p-val ue= 0.015572 international journal of applied biology, 6(2), 2022 130 age group prevalence among the males showed that age group (51-60) had the highest (63.33%) followed by 31-40 (44.87%), 41-50 (42.18%), 21-30 (36.7%), 60> (33.33%) and 18-20 was the least (25.0%). those in the age groups 51-60 and >60 recorded 100% prevalence among the female inma tes, and those in age groups 31-40 and 41-50 recorded 75.00% prevalence, while those in the 18-20 age group were the least (33.33%) (table 2). however, chi-square analysis showed no association between the prevalence of intestinal parasites and the age of both male and female inmates (p>0.05). table 2. prevalence of intestinal parasites among the inmates by sex and age group the study area male female total no infected (%) age group number examined number infected % number examined number infected % 10-20 4 1 25.00 9 3 33.33 4 (1.4 ) 21-30 98 36 36.7 10 6 60.00 42 (12.0) 31-40 78 35 44.87 20 15 75.00 50 (14.28) 41-50 64 27 42.18 16 12 75.00 39 (11.14) 51-60 30 19 63.33 10 10 100.00 29 (8.22) >60 6 2 33.33 5 5 100.00 7 (2.0) total 280 120 42.85 70 51 72.85 171(48.84) mal e: df = 5, chi 2=2.8128, p-val ue= 0.72882 femal e: df=5 chi 2=2.3604, p-val ue= 0.79735 five species of intestinal parasites was identified in the study which occurred 248 times as a single or multiple infections (polyparasitism) (table 3). hookworm was the most common parasite identified (95, 33.5%), followed by ascaris lumbricoides (57, 20.1%), entamoeba histolytica (56, 19.7%). giardia lamblia (43, 15.1%) and trichuris trichiuria was the least (33, 11.6%). table 3. prevalence of intestinal parasites species identified in umuahia correctional facility, abia state (n=284). parasites species identified no of parasite seen % hookworm 95 33.5 ascaris lumbricoides 57 20.1 trichuris trichiuria 33 11.6 giardia lamblia 43 15.1 entamoeba.histolytica 56 19.7 total 284 100 table four highlighted single and mixed infections recorded in the study area. of the171 inmates that had intestinal parasite infections, 56.7% had a single infection, while 43.27% had mixed infection. hookworm was the most (28.9%) frequent single parasites international journal of applied biology, 6(2), 2022 131 recorded while t. trichiuria (9.3%) was the least (table 4). mixed infections with the combina tions of hookworm (hw) and e. histolytica (eh) was highest (36.5%) while other mixed infection were combinations of more than two parasites (table 4). table 4. prevalence of single and mixed (multiple) infections among inmates in umuahia correctional facility, abia state parasite seen male female total (n=171) no infected % no infected % no infected % single infection protozoal g. lamblia 14 20.3 8 28.6 22 22.6 e.histolytica 14 20.3 6 21.4 20 20.6 helminths hookworm 20 28.9 8 28.6 28 28.9 a.lumbricoides 16 23.2 5 17.9 21 21.6 t.trichiuria 5 7.2 1 3.6 6 9.3 total 69 100 28 100 97 56.7 mixed infection hw+al 17 33.3 3 13.0 20 27.0 hw+tt 5 9.8 6 26.0 11 14.9 hw+gl 8 15.6 0 0 8 10.8 hw+eh 15 29.4 12 52.1 27 36.5 eh+tt 0 0 1 4.3 1 1.4 a.l+tt 1 1.9 0 0 1 1.4 g.l+eh 1 1.9 0 0 1 1.4 hw+al+tt 1 1.9 0 0 1 1.4 hw+eh+gl 1 1.9 0 0 1 1.4 hw+al+eh 1 1.9 0 0 1 1.4 hw+al+tt+eh 1 1.9 1 4.3 2 2.7 total 51 100 23 100 74 43.27 key=hw=hook worm, al = as cari s lumb ricoides, eh=enta moeba histoly tica , gl = giardia la mblia, tt= tri chiuri s trichiuria. out of the 350 questionnaire distributed among the participants, only 220 filled and returned to us. all the respondents agreed that they use borehole water for drinking and other usages. the infected inmates that have been in the correctional facility for years, months, and weeks accounted for varying prevalence rates 74.6%, 40.5%, and 22.2% respectively that was significantly different (p< 0.05) see table 5. those that reported washing their hand after eating only were highly infected (90.0%), followed by those who said 'always' (69.6%), 'sometimes' (57.3%), and those that wash their hands before eating alone were the least infected (33.3%). there was no significant difference (p> 0.05) in this aspect (table 5). inmates who wash their hands sometimes after defecation had the highest international journal of applied biology, 6(2), 2022 132 infection (70.0%) while those who wash thei r hand always recorded the least infection 59.3%. there was also no significant difference (p> 0.05) in this aspect (table 5). inmates that deworm monthly, yearly and not all accounted for 64.2%, 78.7%, and 39.2% of infections respectively. there was a significant difference (p< 0.05) in this aspect. table 5. risk factors of intestinal parasites among the participating inmates in umuahia correctional facility, abia state variables no of respondents (n=220) no infected (%) p-value how long have you been in the prison years 142 106 (74.6) 0.022216 months 69 28 (40.5) weeks 9 2 (22.2) total 220 136 (61.9) how often do you wash hands before eating only 30 10 (33.3) 0.21929 after eating only 10 9 (90.0) sometimes 68 39 (57.3) always 112 78 (69.6) total 220 136 (61.9) how often do you wash hands after defecation sometimes 50 35(70.0) 0.8109 always 150 89 (59.3) not at all 20 12 (60.0) total 220 136 (61.9) how often do you wash your fruits before intake sometimes 52 26 (50.0) 0.32256 always 128 90 (70.3) not at all 40 20 (50.0) total 220 136 (61.9) how often do you deworm yourself monthly 28 18 (64.2) 0.020679 yearly 108 85 (78.7) not at all 84 33 (39.2) total 220 136 (61.9) the inmates reported va rious symptoms associated with intestinal parasite infection: watery stool (50, 29.2%), stool accompanied by blood (11, 6.4%) and abdominal cramps (20, 11.7%), although a greater number 90 (52.6%) of people were asymptomatic. there was no significant difference (p> 0.05) in this aspect (table 6). international journal of applied biology, 6(2), 2022 133 table 6. symptoms associated with intestinal parasite infection among the inmates in umuahia correctional facility, abia state no examined total no infected (%) total no without symptoms (%) characteristic symptoms watery stool no infected (%) stool accompanied with blood no infected (%) abdominal cramps (%) 350 171 (48.9) 90 (52.6) 50 (29.2) 11(6.4) 20 (11.7) df=1, chi 2= 0.32589, p-val ue = 0.57409 discussion intestinal parasites such as soil transmitted hel minthes (sth) remains a public health concern and part of neglected tropical diseases (ntds). among the six 2030 global targets for sth by the world health organization is to achieve and maintain elimination of sth and is part of who global target 2030 (who, 2022). microscopic examination using wet mount and formol ether concentration methods is still the most appropriate techniques widely used for the detection of intestinal parasites despite the advances in intestinal parasites diagnosis (zongo et al., 2020). our study employed the two above menti oned technique to determine the presence and prevalence of intestinal parasites among inmates in umuahia correcti onal facility using stool samples. the study recorded an overall prevalence of 48.8% for intestinal parasitic infections, accounting for almost half of the population harboring at least one intestinal parasite species. the overall prevalence rate of 48.8% was high and agreed with rasha et al. (2011) value that recorded a prevalence rate of 49% in omduruma prison in sudan. the high prevalence of intestinal parasite infection recorded could be linked to poor environmental and personal hygiene (khan et al., 2022), common among prison inmates. these are among the major contributing factors to intestinal parasite transmission (arora, and arora, 2006). however, the prevalence was lower tha n the 72.7% recorded by ma mo (2014) in north central ethiopian prison, 74.85% by amuga (2006) in keffi prison, nasarawa state and 77.0% by okolie (2008) in owerri prison but higher than 33.5% reported by kadio et al. (2021) in central prison of conakry, guinea, 32.84% reported by colman et al. (2013) among inmates of maiduguri prison, 26.6% reported by loraine et al. (2015) in kajang prison in malaysia and 20.2% reported by curval et al. (2017) in brazil. it is expected that prevalence rates should be on the decline in nigeria due to some significant improvements in the level of awareness and personal and environmental hygiene val ues (amuga et al. 2006; okolie, 2008). female inmates recorded a higher prevalence of intestinal parasite than the males. this may be associated with the different exposure rates and unhygienic environment often exposed during household chores (as seen in normal population), general unhygienic nature of the facility and females’ physiology. studies have shown that latrines and unsanitary toilets disposes one to intestinal parasite infection. this is contrary to the findings of colman et al. (2013) in maiduguri, borno state and ughavah et al., (2016) in jos, plateau state, international journal of applied biology, 6(2), 2022 134 which reported higher prevalence among male inmates with a zero infection among female inmates. males in the age group 51-60 years were more infected while age group 31-40 in females recorded 100% infection. on other ha nd, 18-20 years age group recorded the minor infection in both male and females. this finding did not agree with related studies. the highest infection recorded in owerri prisons, imo state was among those under 35 years of age group (okolie, 2008), 15-20 years in keffi prison, nasarawa state (amuga et al., 2006), 21-30 years in omdurman prison, sudan (colman et al., 2013), <20 years in jos prison, plateau state (ughava and okn, 2016) and 15-24 yea rs in north central ethiopia (mamo, 2014). as seen in the present study, the highly infected age group may be because these groups lack proper personal hygiene. the male inmates from this age group are known for lackadaisical attitude of and frustration leading to less care about thei r health beings which may increase parasite transmission. previous studies revealed that high intensity of helminthic parasites, especially hookworm, occurs in adults resulting from farming as an occupation (kadio et al., 2022; amuga et al., 2006; brooker et al., 2006). however, there was no association between the prevalence of intestinal parasites and age group in both male and female inmates (p> 0.05). the most frequent parasite recorded was hookworm followed by a. lumbricoides (20.1%) as observed by okolie (2008) in owerri prisons but differ f rom a. lumbricoides reported as the most common pa rasite by amuga et al. (2016) in keffi prison, nassarawa state. the high occurrence of hookworm infection c ould be due to a simple mode/mechanism of parasite transmission (who, 2022). inmates that have spent years in prison were more parasitized than inmates who have been in the correctional facility for only months or weeks. this finding did not agree with amuga et al. (2006) in keffi prison, nassarawa state that recorded 100% prevalence among inmates that had been in the correcti onal facility for less than one month. this current observati on may be linked to the fact that the immune systems of the newly confined inmates are still strong enough to resist parasitic infections of the intestine than those who have spent years in the correctional facility (kadio et al., 2021). besides, they have not been subjected to the prevailing conditions in the facility. those that wash their hands after eating were more parasitized compared with those that wash their hands sometimes and those that wash their hands before eating. a significant difference was recorded between inmates that wash hands before meals and those that do not in kissi prison, kenya (dickson et al., 2016). maintaining a good routine of hand washing, especially before eating, can reduce intestinal parasite transmission ( al amin and wadhwa, 2022). no significant difference was recorded between the inmates that regularly washed thei r hands and those that do not wash their hands always after defecation (p>0.05). this also differs from the findings of dickson et al. (2016) in kissi prison, kenya. improper washing of hands before eating and after defecation is associated with intestinal parasite infection and can explain the high prevalence recorded a mong those that wash their hands sometimes after defecation (al amin and wadhwa, 2022). the effects of proper washing of fruits before intake on the transmission of intestinal parasite infections were accessed. those that deworm yearly were found more infected than those that deworm monthly. the high prevalence recorded may be linked to an inconsistent deworming at least to a considerable interval; leading to increase parasite multiplication and transmission. deworming practice and intestinal parasite burden were significantly related. international journal of applied biology, 6(2), 2022 135 since all the respondents agreed on using the sa me source of water supply and toilet facility type, we could not investigate or compare the effects of water source and toilet facility on the transmission of intestinal parasites. studies have shown that inmates using boreholes are more prone to intestinal parasite infection (kadio et al., 2021). however, related studies have demonstrated a strong relationship between the prevalence and transmission of intestinal parasitic infection to toilet facility and water source used (amuga et al., 2006; amuta et al., 2010; dickson et al., 2016). a water closet facility assumed to be the best can amplify parasitic infection transmission, especially when the faeces accumulate for a long time or if the toilet is not adequately washed regularly. studies have also opined that the transmission of intestinal parasites is related to the water closet type of toilet used by many household (either at public or private level) a result of splashing of water during defecation (afolabi et al., 2016). related studies revealed various factors responsible for high prevalence of intestinal parasite infection and incidence [brooker et al., 2006; kumie and ali, 2005) these factors include inadequate and untreated water supply (to which borehole belong to, age, poor sanitation, low level of income, low level of education, consumption of unwashed vegetables and fruits, and low personal hygiene practice ( al amin a nd wadhwa, 2022; khan et al., 2021). they are considered significant risk factors for intestinal parasite infections ( al amin and wadhwa, 2022). symptoms of intestinal parasites reported by the inmates were watery stool, stool accompanied with blood and abdominal cramps, though a significant number of the inmates were asymptomatic. the presence of blood in the stool suggests amoebic dysentery, usually caused by e. histolytica. diarrhoea is one of the most reported symptoms of the intestinal parasite to which g. lamblia is responsible (who, 2022) conclusion the presence and prevalence of intestinal parasitic infections has been established among inmates of the correctional facility (prison), umuahia, abia state. females were more infected than males. the most common intestinal parasites were soil transmitted helminthes, especially hookworm. mixed infections of intestinal parasite species were confirmed. variables like inconsistent deworming and duration spent in the prison were considered significant risk factors. the spread of parasitic infection could be controlled, reduced or even elimina ted by proper hygiene, and deworming the rapy latest every three months. we recommend that inmates should be checked and treated adequately (dewormed), especially before being released into the general population. inmates should be educated on the dangers of dangerous behavior that could lead to the spread of this parasite infection. we recommend further research investigating the handling of food pre and post-cooking inside the correctional facility as a risk factor for intestinal parasites. acknowledgement the authors are grateful to all the inmates and staff of umuahia prison, anyawu. e. d., mr. pipi okechukwu and the department of zoology and environmental biology, michael okpara university of agriculture, umudike, nigeria for providing the necessary facilities used in this study. international journal of applied biology, 6(2), 2022 136 references arora, d. r. and arora, b. 2006. medical parasitology textbook. 2 nd edition. cbs publishers and distributors, new delhi, india; 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(accessed 2 june 2022) ughava, j. r. and okon p. e. 2016. prevalence of intestinal helminthiasis among prison inmates in jos, nigeria. african journal of biology. 3(6): 238-242. world health organisation. http:www.who.int/news-room/fact-sheets/detail/solitransmitted-helminth-infections 2022; (accessed 6 may 2022) world health organization. 2002. preventi on and control of schistosomiasis and soil transmitted helminthiasis; report of a who expert committee. geneva. 912: 63. zongo, a. w., ouattara, a.k., yonli, a. t., sorgho, p. a., sombié, h.k, lallogo, t., bado1, p., compaoré, t. r., djigma1, f. w., ouermi, d., millogo, h. and si mporé, j. 2020. realtime multiplex pcr diagnosis of common diarrhea causing parasites in children under five years in ouagadougou, burkina faso. infectious disease and tropical medicine. 6: e641 contact : abdalia liaabda03@gmail.com 213 abstract the objective of this research was to determine the efficiency and productivity of gillnet mesh size in north galesong district, takalar regency. the type of research was survey research with a quantitative descriptive approach to obtain a sample from a population that describes or explains catch production, productivity, and efficiency of gillnet fishing gear. the population of this research was 40 cantrang fishermen who switch to using gillnet fishing gear in north galesong district. the sample was all cantrang fishermen who switched to gillnet fishing gear. the sampling method employed in this study was a census sampling strategy, where all members of the population were used as samples. the methods of data collection were questionnaires, observations, and interviews. the results showed that the most efficient gillnet mesh size of the five mesh sizes 1 inch, 1.5 inch, 2 inch, 2.5 inch and 6 inch is the 1.5 inch mesh size with a value of 3.90. the results of the calculation of the productivity of gillnet fishermen in each mesh size has a different value. the highest gillnet productivity was at 1 inch mesh size with a value of 44.40kg/trip, then the lowest productivity was at 2.5 inch mesh size, which was 24.39 kg/trip. issn : 2580-2410 eissn : 2580-2119 the productivity of gillnet mesh size as a substitute for cantrang in tamasaju village, north galesong district, takalar regency abdalia1, sri suro adhawati1, najamuddin1, sutinah1, aris baso1, hamzah1 1 study program of fisheries science, faculty of marine science and fisheries, hasanuddin university, makassar introduction cantrang is a fishing equipment that operates close to the water's bottom. when using a cantrang, the rope is spread out in a circle, the cantrang net is lowered, and finally the rope's two ends are brought together. the two ends of the rope are pulled towards the ship until all parts of the net bag are lifted (luhur, 2018). the use of cantrang fishing gear can damage the environment by not paying attention to environmental aspects and causing conflicts between traditional fishermen and machine fishermen (rawaeni, 2017). the minister of marine affairs and fisheries on january 9 2015, has promulgated ministerial regulation number: 2/permen/kp/2015. the regulation stated that the use of trawls and seine nets is prohibited in the fisheries management area of the republic of indonesia due to the fact that their usage on indonesian territory has led to a decline in fish resources and jeopardized their environmental sustainability. (permen kp no. 2/2015) open access international journal of applied biology keyword gillnet; cantrang; productivity; efficiency; production article history received november 3, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 214 for fishermen, regulations prohibiting the use of cantrang fishing gear can cause turmoil for fishermen, because cantrang is a tool that fishermen use every day. when cantrang is prohibited, fishermen will be concerned and worried about the work they are experiencing (zulbainarni et al., 2016). therefore, in order to survive, fisherman must alter their equipment or convert to non-prohibited, environmentally acceptable equipment (adhawati, et all, 2017). north galesong district is an area that has had a significant impact from the ban on cantrang fishing gear. the impact of the ban has caused fishermen to switch to fishing gear that is more environmentally friendly and not banned, namely gill nets. gillnet is a fishing gear that has a general rectangular shape with tool parts consisting of main net, top rigging rope, bottom rigging rope, and towing ropes. the gill nets used by fishermen have several types of different mesh sizes. the size of the mesh used by fishermen consists of 5 types of mesh sizes, namely 1 inch, 1.5 inch, 2 inch, 2.5 inch and 6 inch. research on the productivity of gill net mesh sizes in north galesong district, takalar regency is required so that a variety of mesh sizes can be used as an alternative to cantrang fishing gear. materials and methods research location and time the research was conducted in north galesong district, takalar regency which was carried out from september to october 2021. the choice of the research site was made purposively, taking into account that fishermen in the area had previously relied on cantrang fishing gear but have since switched to gillnet fishing gear, which is more environmentall y friendly. data collection the data collection technique used in this research is direct observation by obtaining basic data sourced from respondents in the form of fishermen's activities in takalar regency and interviews to obtain clearer information regarding research problems. the population of this research was all cantrang fishermen who switch to using gillnet fishing gear in north galesong district. in the north galesong district, the most users of fishing gear are located in tamasaju village and aeng batu-batu village. the total population of gillnet fishermen in north galesong district is 40 people. according to arikunto (2014), if the number of a population is less than 100 people, then the number of samples taken is entirely, but if the total population is more than one hundred, then the sample taken is 10-15% or 20-25% of the total population. based on these considerations, the sample taken in this study was 100% of the total population of gillnet fishermen, namely 40 respondents. thus, the sampling technique used in this study was a saturated sampling technique (census), where all members of the population are used as samples (sugiyono, 2014) types of research the research type was survey research using a quantitative descriptive approach to obtain a sample from a population that describes or explains the catch production, income, efficiency, and productivity of gillnet fishermen. international journal of applied biology, 6(2), 2022 215 data types and sources of data there were two data used in this study, namely primary data and secondary data. primary data is data obtained from interviews with respondents based on questionnaires that have been made, including respondents' identities, catches, costs used, length of nets, selling prices, and number of gillnet fishermen's trips. while secondary data is data obtained from institutions or agencies related to research problems. data collection methods the data collection methods used in this study were observation, interviews, and library research. the method of observation or direct observation is a data collection method that obtains basic data sourced from respondents in the form of fishing activitie s in the north galesong district. interview is a method used to obtain clear information in relation to research problems. while the library research is a collection of data obtained by reading literature or research results that are relevant to the research theme. data analysis the data analysis used in this study consisted of several analytical methods in order to achieve the desired results. the data analysis method used is as follow: a. efficiency and economic feasibility of gillnets to answer the first point, descriptive analysis of comparative percentage is used with a sematic differential measurement scale approach by osgood. the assessment criteria use 5 economic indicators (mallawa 2016 and adhawati 2018), which include: annual producti on (x1), annual cost (x2), annual revenue (x3), annual income (x4), net length (x5). in the assessment of all criteria in an integrated manner, standardization of values is carried out. value standardization can be done by using the scoring method. the scoring method in the scoring method starts from the lowest to the highest score (iskandar dahri and ade guntur, 2014). standardization with value functions can be done using the following equation (mangkusubroto & trisandi, 1985; najamuddin et al, 2017): v (x) = 𝑋− 𝑋0 𝑋1− 𝑋0 v (a) = ∑ 𝑉𝑖 (𝑋𝑖) untuk i = 1.2.3. ……., n where: v (x) = the best function of the variable x x = variable x x1 = the best value of criteria x x0 = the worst value of criteria x x v (a) = value function of alternative a vi (xi) = the value function of the alternative on the -i criterion the order of priority starts from the highest value to the lowest value. by using the value function, the priority order is set in order from the alternative with the highest function value to the alternative with the lowest function value. international journal of applied biology, 6(2), 2022 216 b. productivity of gillnet mesh size to determine the productivity of gillnet mesh size, the measurement of gill net productivity includes productivity per unit of fishing gear and per trip, using the following formula (cholik, 1994): productivity = ∑ 𝑁𝑃𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑣𝑎𝑙𝑢𝑒 ∑ 𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑠𝑡 ( 𝑅𝑝 𝑡𝑟𝑖𝑝 ) results and discussion gillnets gill nets are rectangular nets with the same mesh size in all parts of the net. the length of the net is longer than the width or height of the net and the top of the net has a buoy and the bottom has a ballast (mallawa, 2012). fishing equipment operation method the catching process was carried out at 05.30 and at 18.00 central indonesian time to the fishing ground. the boat moved from the fishing base area to the fishing area and took about 30 minutes to catch fish. the gillnet operation method has several stages, namely as follows: a. preparation the first step in using gillnet fishing equipment is preparation, which involves paying attention to water conditions such monitoring depth, currents, etc. b. setting the fishermen start to lower the buoy first, followed by the body of the net, after it is deemed safe to do so. in order to prevent the gillnet from drifting or breaking free from the ship while in use, the ship must move backwards when the body of the net is dropped into the sea. this process ends with a rope connecting the gillnet and the ship. in order to avoid getting trapped and have the gillnet fully extended, the ship goes rearward in a straight line at constant speed. c. immersing immersing is the waiting period for the gillnet to be left in the water until the time specified by the fisherman. the gillnet waiting period is erratic, usually up to 3 to 5 hours. every now and then the fisherman would check the ropes to see if the gillnet was still attached to the boat. d. hauling hauling is the process of lifting gillnets from the waters onto the ship, after a long time the gillnets are left in the waters then the gillnets are lifted onto the ship little by little. until all parts of the gillnet are on board the ship. the operation of gill nets is installed in the fishing area in an upright position facing the biota/cutting currents (matsuganda, 2008). one of the reasons gillnet fishing gear is operated by blocking the current is to block the direction of the fish swimming movement (alwi et al, 2020). the fishing method on gillnet fishing gear is passive, namely by spreading the net in the waters and waiting for the fish to get entangled in the operculum which is carried out during the day and night without the help of light (pramesthy et al, 2020). the operating technique of the fishing gear is simple so as not to endanger the safety of fishermen. international journal of applied biology, 6(2), 2022 217 gillnets are among the environmentally beneficial fishing gear since they are highly selective, don't destroy habitats or fishermen's livelihoods, don't harm consumers, don't impact biodiversity, and don't capture legally protected species. pramesthy et al (2020). catch result based on the results of research that has been carried out, there are several types of fish caught from gill net fishing gear, namely tembang fish (sardinella fimbriata), banyar fish (restrelliger kanagurta), mullet (mugil cephalus), layur fish (trichiurus lepturus), fourthfinger threadfin (eleutheronema tetradactylum), and milkfish (chanos chanos). on average, gillnet boats in north galesong subdistrict, takalar regency, use gypsun boats with a size of 14 gt, with main dimensions ranging from 12 meters long, 2 -2.5 meters wide. the machines used were noncing and yamaha brands. fishermen used several types of mesh sizes when catching fish in order to increase their catch. gillnet catching unit efficiency efficiency is a comparison between output and input used in the production process. the definition of efficiency is very relative, efficiency is defined as the use of the smallest input to get the maximum production (soekartawi, 2003). the variables used to determine the efficiency of the gillnet fishing unit in each mesh size are annual production yield, annual cost, annual revenue, annual income and net length. table 1. data on average production results per year, cost per year, revenue per year, income per year and length of gillnet fishing nets no mesh size v1(x1) range v2(x2) range v3(x3) range v4(x4) range v5(x5) range 1 1 inch 6.993 3.72011.520 19.450.500 1.860.00038.298.090 12.537.627 074.188.800 1.460.549 052.029.162 60 0-450 2 1,5 inch 742 222.688 8.992.873 2.866.63925.570.174 89.700.750 0172.800.000 75.183.750 0155.520.000 356 0-630 3 2 inch 237 181.728 3.123.440 0-989.688 57.822.171 5.268.293144.000.000 52.787.521 4.213.927131.480.000 0 0-450 4 2.5 inch 74 0-817 2.167.897 65.4875.569.972 9.723.400 0100.800.000 6.813.775 093.400.000 165 0-450 5 6 inch 0 0-341 0 0-174.884 0 025.097.561 0 028.006.295 22 0-450 source: proces s ed pri mary data, 2021 table 1 shows the production results of each mesh size. the 1-inch size has the highest production value per trip of 6,993 kg with a catch range of 4,301-11,520 kg/year, followed by a 1.5-inch mesh size of 2,619 kg with a catch range of 1,080-5,760 kg/year. meanwhile, the lowest annual production is at 6-inch mesh size of 891 kg with a catch range of 360-1,800 kg/year. the highest annual cost for each mesh size is 1 inch with a value of rp19.450.500/year while the lowest cost per year is at 6 inch mesh size of rp6.485.625/year. the annual income ranges from rp31.198.125– rp89.700.750. the income per year from gillnet fis hermen is the largest, which is at the size of a 1.5-inch net, which is rp75.183.750 /year while the lowest is at the size of a 6-inch mesh of rp24.712.500/year. this happens because the catch of 1.5 inch mesh size is higher and the selling price is also quite high, while for the 6 inch mesh size the income is lower because the catch is small because this net is used only when the waves are international journal of applied biology, 6(2), 2022 218 strong or in the west season which is around month 11 to month 1. length of net used by gillnet fishermen in north galesong district ranges from 304 meters – 356 meters. table 2. comparison of calculation of economic indicators that determine efficiency in gillnet fishing equipment no mesh size v1(x1) v2(x2) v3(x3) v4(x4) v5(x5) average range average range average range average range average range 1 1 inch 6.993 3.72011.520 19.450.500 1.860.00038.298.090 12.537.627 074.188.800 1.460.549 052.029.162 60 0-450 2 1,5 inch 742 222.688 8.992.873 2.866.63925.570.174 89.700.750 0172.800.000 75.183.750 0155.520.000 356 0-630 3 2 inch 237 181.728 3.123.440 0-989.688 57.822.171 5.268.293144.000.000 52.787.521 4.213.927131.480.000 0 0-450 4 2.5 inch 74 0-817 2.167.897 65.4875.569.972 9.723.400 0100.800.000 6.813.775 093.400.000 165 0-450 5 6 inch 0 0-341 0 0-174.884 0 025.097.561 0 028.006.295 22 0-450 source: proces s ed pri mary data, 202 1 x1 = yield per year (kg) x2 = cost per year (rp) x3 = revenue per year (rp) x4 = income per year (rp) x5 = net length (m) table 2 shows the results of the comparison of economic indicators for each mesh size on gillnet fishing gear. the comparison shows the efficiency level of each mesh size of the gillnet fishing unit against one of the economic indicators used, namely x1 to x5. furthermore, to determine the order of priority of mesh sizes that have the best efficiency, calculations are carried out using the value function of each economic indicator. the calculation results determine the order of efficiency of each mesh size of the g illnet fishing unit, as presented in table 3. table 3. gillnet catching unit efficiency no mesh size x1 x2 x3 x4 x5 average range average range average range average range average range 1 1 inch 1,00 0,801,18 1,00 1,002,07 0,27 0,000,92 0,05 0,000,82 0,19 0,001,00 2 1,5 inch 0,28 0,020,54 0,62 0,281,62 1,00 0,001,00 1,00 0,001,00 1,00 0,001,40 3 2 inch 0,14 0,010,60 0,30 0,000,98 0,76 0,121,00 0,81 0,111,06 0,00 0,001,00 4 2.5 inch 0,06 0,000,28 0,23 0,010,47 0,22 0,001,00 0,20 0,001,14 0,50 0,001,00 5 6 inch 0,00 0,000,19 0,00 0,000,02 0,00 0,000,54 0,00 0,000,73 0,07 0,001,00 source: proces s ed pri mary data, 202 1 table 3 shows the calculation of the efficiency of the gillnet fishing gear unit in the north galesong district. based on table 13 the size of the 1.5-inch mesh has an overall efficiency level of 3.90 and is ranked first. the second rank is the mesh size of 1 inch with a value of 2.51. international journal of applied biology, 6(2), 2022 219 the smallest efficiency level is found in the 6-inch mesh size which has an efficiency value of 0.07. assessment of efficiency is carried out to compare the output and input of each fishing gear unit (suharto 2003). based on the results of efficiency calculations on each mesh size of gillnets, it is known that the most efficient among the five mesh sizes i s 1.5 inch mesh size, this is because at 1.5 inch mesh size the amount of production produced in one year catches more and the selling price is quite high so as to produce a higher level of efficiency compared to other mesh sizes. gillnet catching unit productivity productivity is a universal concept that applies to all economic and social systems. the key factor driving global economic expansion is productivity. increasing productivity is a fundamental challenge for developed countries (mcgowan et al., 2015). the efficiency of the gillnet fishing units in north galesong is measured by comparing it to the average output of the gillnet fishing units per fishing effort, where the effort in this case is the typical annual trip. table 4 shows the annual productivity for gillnets, including average production, trips, and productivity. table 4. average production yield, trips and productivity of gillnet fishermen for each mesh size no mesh size production result (kg) trip/year productivity average range average range average range 1 1 inch 6993 4.320-11.520 158 144-180 44,40 30-40 2 1.5 inch 2619 1080-5760 103 72-144 25,53 10-40 3 2 inch 1728 720-2880 61 36-72 28,24 20-40 4 2.5 inch 1251 180-2880 51 36-72 24,39 7,5-80 5 6 inch 891 120-1800 26 18-36 33,67 15-50 source: proces s ed pri mary data, 202 1 based on table 4, it can be seen that the highest average production of gillnet fishermen in north galesong district is a net with a size of 1 inch which is 6,993 kg/year. while the lowest is the production with a mesh size of 6 inches. this happens because the 6-inch net size is only used at certain times, namely at month 11 to month 1 because at that time it is very suitable for catching large fish. however, the number of trips is low due to very bad weather conditions, so fishermen only go to sea when the weather improves a little. the mesh size of 1 inch has the highest number of trips, which is 158 trips/year due to the fact that nets with a size of 1 inch are often used by fishermen when catching. while the lowest number of trips is at the 6-inch mesh size, which is 26 trips/year. table 4 shows that the highest productivity is at 1 inch mesh size, which is 44.40 kg/trip. this happens because when compared to other mesh sizes, the 1 inch mesh size has the maximum production value and number of trips. while the mesh size of 2.5 inches, which has the lowest productivity at 24.39 kg/trip. based on the research of yohanes et al (2022) on the productivity of gillnet fishing gear in nangamerah waters, bangkoor village, kec. talibura, kab. sikka obtained a catch productivity value of 21.53 kg/trip, which means gillnet fishing gear operating in nangamerah waters, bang koor village, kec. talibuan has the ability to catch fish as much as 21.53 kg/trip.the amount of production/catch that fisherman obtain and the number of trips they make in a year have a significant impact on the productivity differences international journal of applied biology, 6(2), 2022 220 at each mesh size. according to ariandi (2012), productivity is determined by the quantity of fishing trips as well as the amount of production. conclusion the conclusions drawn from the research that has been done are as follows the highest efficiency value for gillnet mesh size 1.5 inches is 3.90, and the lowest efficiency value is for mesh size 2.5 inches, which is 24.39 and he gillnet fishing gear in north galesong district, takalar regency, has a highest productivity value at 1 inch mesh size, which is 44.40 kg/trip, meaning that it has the capacity to catch 44.40 kilogram of fish every trip. international journal of applied biology, 6(2), 2022 221 references adzah rawaeni. implementasi larangan penggunaan alat tangkap cantrang pada jalur penangkapan ikan, skripsi : fakultas hukum universitas hasanuddin, 2017 adhawati s, s. 2018. reconstruction post moratorium of dregged nets at south sulawesi. sekolah pascasarjana. universitas. hasanuddin. makassar adhawati s, s, aris baso, dan achmar mallawa. 2017. social study of cantrang (danish trawl) fisheries post moratorium at makassar straits and bone gulf, south sulawesi province , indonesia. jurnal aacl bioflux vol 10/5. peraturan menteri kelautan dan perikanan no. 2 tahun 2011 jo no. 08 tahun 2011 jo no. 18 tahun 2013 jo no. 42 tahun 2014 tentang perubahan keempat atas peraturan menteri kelautan dan perikanan nomor per. 02/men/2011 tentang jalur penangkapan ikan dan penemuan alat penangkapan ikan di wilayah pengelolaan perikanan negara republik indonesia arikunto, s. 2014. prosedur penelitian suatu penelitian praktik. rineka cipta. jakarta. alwi et al, 2020). spesifikasi dan hasil tangkapan jaring insang di desa prapat tunggal, kabupaten bengkalis, provinsi riau. aurelia journal vol. 2 (1) oktober 2020 : 39-46 cholik abdul, rivai wirasasmita dan ofan sofyan. 1994. evaluasi proyek (suatu pengantar). pioner jaya. bandung. 70 hlm. najamuddin et all.2017. performance of fishing gear on skipjack tuna katsuwonus pelamis in south sulawesi, indonesia. jurnal aacl bioflux vol 10/2. mallawa, a. 2016. kajian pengembangan tehnologi tangkap prioritas. in strategi dan kebijakan pengembangan perikanan tangkap (berkelanjutan). makassar mallawa achmar. 2012. dasar-dasar penangkapan ikan . masagena press. makassar mangkusubroto k., trisnadi c. l., 1985 decision analysis with system approach and project management. ganesa exacta, bandung. martasuganda s. 2008. jaring insang (gill net). institut pertanian bogor minggo yohanes d.b.r, erfin dan febronius florianus m r. 2022. produktivitas alat tangkap gillnet di perairan nangamerah, desa bangkoor, kecamatan talibura, kabupaten sikka. jurnal ilmu kelautan dan perikanan vol.04, no.01. pramesthy et al., 2020. analisis alat tangkap jaring insang (gill net) berdasarkan kode etik tatalaksana perikanan bertanggung jawab di perairan kota dumai. aurelia journal vol. 1 (2) april 2020 : 103-112. soekartawi. 2003. agribisnis teori & aplikasinya. raja grafindo persada. jakarta sugiyono. (2014). metode penelitian pendidikan pendekatan kuantitatif, kualitatif, dan r&d. bandung: alfabeta. zubainarni n, yani,a. faradissa. 2016. kajian valuasi dampak ekonomi dan sosial pelarangan alat tangkap cantrang di jawa tengah. bogor (id) : agro maritim. contact : isnawati isnawati@unesa.ac.id 108 abstract tempeh and tofu are widely consumed by indonesians and easily made using simple tools and method, thus numerous home industries producing are emerging. however, soybean processing in these industries produces high volume of wastewater. this study provides a simple wastewater treatment instrument design that could be used by soybean-based home industries by applying a series of physical, chemical, and biological treatment. physical treatment was performed by filtration, chemical by addition of alum salts, while biological treatment was done by supplementing with lactic acid bacteria (lab) from leftover container of fermented milk. wastewater was recorded by its parameters (temperature, ph, dissolved oxygen/do, biological oxygen demand/bod, turbidity) at every phase of treatment after incubation for two various of periods; 1 hour and 5 days. results showed that wastewater treated using instrument design had changes in all of its parameters observed during the course of treatment. wastewater treated in all phase of instrument incubated for 1 hour showed parameters approaching wastewater quality standards, thus it was safer to be disposed to environment compared to wastewater incubated for 5 days. wastewater treatment as designed in the current study can be applied by soybean-based home industries with short incubation time to remediate wastewater produced during soybean processing before disposal. issn : 2580-2410 eissn : 2580-2119 lactic acid bacteria (lab) from fermented milk leftover for wastewater treatment of soybean-based industries guntur trimulyono, isnawati*, mahanani tri asri, firas khaleyla department of biology, faculty of mathematics and natural science, state university of surabaya, surabaya, jawa timur introduction tempeh and tofu are popular food widely consumed by the indonesian community. both can be made from soybeans using easy method and simple tools. because of that, local home industries focusing on both have products become numerous. however, soybean processing into tempeh and tofu produces high volume of wastewater. wastewater from tempeh and tofu production contains various type of organic materials. wastewater produced by soybean -based home industry was found to contain 34.8% carbohydrate, 34.9% protein, 18.1% fat, and traces of salt, minerals, and other chemicals (utami et al. 2013). various parameters of soybean -based industrial wastewater were found to be above quality standards (purwanti et al. 2018). however, because of their minimum cost and limited tools, soybean-based home industries hardly perform any open access international journal of applied biology keyword wastewater treatment; lactic acid bacteria; soybean-based home industry. article history received january 30, 2023 accepted june 21, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:%20isnawati@unesa.ac.id international journal of applied biology, 7(1), 2023 109 treatment to their wastewater before they dispose it to environment. disposal of untreated wastewater with high organic content can induce eutrophication of water body. continuous eutrophication leads to algal bloom that caused a lot of negative effects towards various species composing aquatic ecosystem (fao & iwmi 2017). simple wastewater treatment can be performed by physical, chemical, and biological treatment. physical treatment can be conducted via filtration using various substrate, such as fabrics, sands, or palm fibers, while chemical treatment by administer alum salts. filtration of wastewater can effectively remove particles and suspended solids (crini & lichtfouse 2019), while alum salts can induce coagulation, causing lower turbidity and total dissolved solid (tds) as performed previously performed in wastewater produced by blueberry and carrot processing (ardley et al. 2019). meanwhile, biological treatment can be conducted by adding microbe culture which is able to degrade organic materials, such as lactic acid bacteria (lab). lactic acid bacteria (lab) is a type of bacteria used widely in fermentation process of various foodstuffs, mainly dairy products, for example yoghurt, cheese, or ya kult. lactic acid bacteria are able to metabolize carbohydrate into lactic acid, acetic acid, and ethanol (ganzle 2015). respiration of lab can occur at minimal level of oxygen. in addition, lab is also able to suppress the growth of other microorganisms b y mean of its rapid metabolism speed and accumulation of lactic acid and acetic acid which their metabolism produces (ganzle 2015). in wastewater of tempeh and tofu industries, various organic materials are dissolved from raw material soybeans into water during their processing, such as protein and carbohydrate during boiling and soaking process, resulting in high level of total suspended solid (tss) (purwanti et al. 2018). organic materials contained in wastewater from soybeanbased home industries can be used by lab as materials for their metabolism. lowering organic materials contained in wastewater can neutralize thus it will be safer to be disposed to environment. one of lab species, lactobacillus plantarum had been studied its ability to remediate wastewater of dairy industry. addition of l. plantarum culture under optimum condition was found to lower sugar level, total protein content, and chemical oxygen demand (cod) significantly (golalikhani & razavi 2015). this study was conducted to design and subsequently test a simple wastewater treatment instrument that could be applied by soybean -based home industries using a series of physical, chemical, and biological treatment using lab culture from leftover container of fermented milk. materials and methods sample wastewater used in this study was wastewater produced by tempeh home industries located in tembarak village, kertosono, east java. wastewater was measured of its parameters (ph, do, temperature, and turbidity) before treatment was performed. simple wastewater treatment instrument was made by combining physical, chemical, and biological treatment into a series of equipment we called ipal-lab (instrumen pengolahan air limbah dengan bakteri asam laktat/wastewater treatment instrument using lactic acid bacteria). first step of treatment was physical treatment which was filtration international journal of applied biology, 7(1), 2023 110 through cotton fabric. second step of treatment was chemical, in which 10% alum salts was added into wastewater. the last step of treatment was biological, in which lab culture from leftover container of fermented milk was added. the series of treatment was designed as schemed in fig. 1. sample wastewater was treated in volume 20 l for each respective replication, for four times replication. after each step of treatment, about 10 ml of wastewater sample was taken and then incubated for 2 various periods; 1 hour and 5 days to see whether incubation time affected parameters of treated wastewater and to measure bod level. water samples from pre-treatment, physical, and chemical step were stored in refrigerator, while samples after biological treatment was incubated in room temperature to let culture active. water parameters (temperature, ph, do, bod, and turbidity) was recorded after samples were incubated and then described q uantitatively. figure 1. series of wastewater instrument using lactic acid bacteria (lab) for wastewater treatment of soybean-based home industries. results and discussion comparation of various water parameters recorded from wastewater of tempeh industries from pre-treatment step up to last step of biological treatment with different incubation time is presented in table 1. international journal of applied biology, 7(1), 2023 111 table 1. parameters of wastewater from tempeh home industries recorded after treatment steps with different incubation time. parameter incubated 1-hour after treatment stepincubated 5-days after treatment step pretreatment physical chemical biological pretreatment physical chemical biological temperature (°c) 28.38±0.25 28.45±0.10 28.18±0.24 28.28±0.22 29.80±0.00 29.80±0.00 29.80±0.00 29.80±0.00 ph 6.73±0.42 7.07±0.16 7.02±0.08 6.31±0.88 5.09±0.00 5.00±0.01 4.22±0.03 4.20±0.01 do (mg/l) 2.80±0.53 3.73±0.10 5.80±1.44 5.18±0.91 1.50±0.08 1.53±0.05 2.20±0.12 2.30±0.08 bod5 (mg/l) n/a n/a n/a n/a 1.30±0.45 2.20±0.14 3.60±1.50 4.88±0.83 turbidity (ntus) 182.00±21.95 133.50±30.65 67.25±15.65 43.35±4.82 157.25±2.22 141.95±6.32 41.32±5.44 57.75±3.40 result of wastewater parameter recording from pre-treatment up to last step of treatment showed changes in some parameters during the course of treatment, both in 1 hour and 5-days incubation time. acidity level did not change significantly in 1 -hour incubation time, but in 5-days, ph was lowered up to acid level (4.20±0.01). dissolved oxygen level was increased from pre-treatment until last step of treatment. higher increase was found in 1-hour incubation time (from 2.80±0.53 to 5.18±0.91) compared to 5-days incubation time (from 1.50±0.08 to 2.30±0.08). along with do raise, bod was also increased along with course of treatment, from 1.30±0.45 mg/ml during pre-treatment up to 4.88±0.83 mg/ml at the last step of treatment. prominent change was observed in turbidity; in 1 -hour incubation time, wastewater turbidity lowered as much as 139.65 ntu, while in 5-days incubation time, turbidity lowered as much as 99.50 ntu. the series of wastewater treatment performed in the current study to remediate tempeh wastewater was based on commonly used process to treat wastewater before being disposed to environment, including physical, chemical, and biological process. water parameters was recorded from pre-treatment and after steps of ipal-lab up to the last step treatment. from various water parameters recorded, all parameter except temperature showed changes. temperature did not alter because treatment steps applied in the instrument did not involve process which could cause temperature change. acidity level of wastewater did not change much in wastewater incubated for 1 hour, however it was decreased up to ph acid level (5.09±0.00 to 4.20±0.01) in wastewater incubated for 5 days. lowest ph was shown by wastewater after lab biological treatment which incubated for 5 days (4.20±0.01). decrease of acidity level was related to lab culture additi on that was able to metabolize carbohydrate and other organic materials contained in tempeh industry wastewater into lactic acid (ganzle 2015). the longer wastewater incubated, lab activity in it was also higher, resulting in higher level of lactic acid produced. increasing level of lactic acid cause wastewater ph to lower up to acidic range. however, this range of ph caused wastewater less than safe to be disposed of, due to unsuitable range as required by water organisms. most water organisms require ph a t normal range at around 7.4 to live. international journal of applied biology, 7(1), 2023 112 wastewater ph level after 5 days incubation also did not meet quality standards as established by local government at range of 6-9 (permen lhk no. 68 year 2016), in opposite to ph of wastewater incubated for 1 hour (6.31±0.88). level of do was significantly raised in treated wastewater incubated for 1 hour. dissolved oxygen level after last treatment and incubated for 1 hour was 5.18±0.91 mg/l. this level of do is still in optimal do range that indicate good water qual ity for most water organisms, at range of 4-6 mg/l (shah & joshi 2017). in the other hand, do level from treated wastewater incubated for 5 days had insignificant change. level of do after last treatment was 2.30±0.08 mg/l. this do level is not suitable to meet oxygen demand of common water organisms and can induce hypoxia in them. only certain organism with specific tolerance for low level of oxygen can survive such level of do, such as bottom-dweller bacteria found in the river floor (connolly et al. 2004). lowering do level of wastewater after biological treatment incubated for 5 days compared to wastewater incubated for 1 hour also indicated microorganism activity in the wastewater. the longer wastewater incubated, both naturally occurred microorganism and lab culture added to it also increased, resulting in lowering do level. level of treated wastewater bod after 5 days incubation was raised from pre treatment step (1.30±0.45 mg/l) up to last step using lab (4.88±0.83 mg/l). although this level was below quality standards (permen lhk no. 68 year 2016), increase of bod level indicated that wastewater treated using instrument in the current study after 5 days incubation become more difficult to be degraded when disposed to environment. the most prominent change in parameters observed was found in turbidity. turbidity had linear relationship with total suspended solid (tss) contained in the water (hannouche et al. 2011), thus can be used as a proxy to measure particles contained in the wastewater (rugner et al. 2013). treated wastewater sample incubated for both 1 hour and 5 days had significant decrease of turbidity (182.00±21.95 to 43.35±4.82 ntus and 157.25±2.22 to 57.75±3.40 ntus, respectively). the most significant decrease occurred during chemical treatment, after alum salts was added into wastewater. alum salts induce coagulation and flocculation of organic materials contained in the waste by forming aluminum hydroxide when it contacts water. aluminum hydroxide is able to remove suspended materials vi a charge neutralization, sweep floc, dan adsorption mechanism (ahmad et al. 2016), resulting in reduced turbidity. treatment using lab culture from leftover container of fermented milk was able to lower turbidity further in wastewater incubated for 1 hour, but on the contrary, it caused turbidity to raise after chemical treatment in sample incubated for 5 days. duration of incubation correlates to lab population in the wastewater. the longer wastewater was incubated, the higher lab population in it, causing turbidity to raise. the utilization of lab culture to remediate waste has been extensively studied, but mostly was in the reuse of solid waste with high content of organic material. lactobacillus amylovorus had been applied to produce lactate from bread wastes (adessi et al. 2018). lactobacillus casei shirota had also been studied for producing lactic acid from food waste international journal of applied biology, 7(1), 2023 113 with high level of glucose, fructose, and amino acid (kwan et al. 2016). in matter of remediation, previous study showed that l. casei was able to reduce cod level of wastewater from dairy industry up to 60% in 25 days (keffala et al. 2017). the series of ipal-lab simple wastewater treatment as designed and described above can be applied to neutralize parameters of wastewater produced by soybean -based home industries because the instrument was easy to be compiled with simple tool and materials. wastewater incubated for 1 hour showed parameters that meet quality standards more than wastewater incubated for 5 days; thus, it was recommended to perform short -period incubation for this treatment. conclusion simple wastewater treatment instrument as designed in the current study can be applied by soybean-based home industries to remediate wastewa ter produced during soybean processing. wastewater should be incubated in short amount of time (1 hour) to obtain parameters closer to government wastewater quality standards. international journal of 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res. eng. technol., 6(4): 108-113. kwan, t. h., hu, y., & lin, c. s. k. 2016. valorisation of food waste via fungal hydrolysis and lactic acid fermentation with lactobacillus casei shirota. bioresour. technol., 217: 129-136. https://doi.org/10.1016/j.biortech.2016.01.134 regulation of indonesian ministry of environment and forestry no. p.68/menlhk/setjen/kum.1/8/2016 on domestic wastewater standards (in indonesian). purwanti, i.f., simamora, d., & kurniawan, s.b. 2018. toxicity test of tempe industrial wastewater on cyperus rotundus and scirpus grossus. int. j. civ. eng. technol., 9(4): 1166-1172. rügner, h., schwientek, m., beckingham, b., kuch, b., & grathwohl, p. 2013. turbidity as a proxy for total suspended solids (tss) and particle facilitated pollutant transport in catchments. environ. earth sci., 69(2): 373-380. shah, k.a. & joshi, g.s. 2017. evaluation of water quality index for river sabarmati, gujarat, india. appl. water sci., 7(2017): 1249-1358. utami, t.s., arbianti, r., herlani, t., & kristin, e. 2013. increased electricity generation in single chamber microbial fuel cell (mfc) using tempe industrial wastewater. proceeding of icce 2013 universitas katolik parahyangan. https://doi.org/10.1016/j.wri.2019.100107 https://doi.org/10.1007/s10311-018-0785-9 https://doi.org/10.1515/ijfe-2015-0109 https://doi.org/10.1016/j.biortech.2016.01.134 1(2)58-66 !!!!!!!!!!!!!!!!!! contact&:&evi&lauw&&&&&&&evi.lauw09@gmail.com! ©!international!journal!of!applied!biology! 58! abstract& lactic!acid!bacteria!are!known!to!have!potential!in!producing!antimicrobial! compounds,! including! antifungal! agents.! this! study! aims! to! determine! whether!the!lactic!acid!bacteria!from!human!oral!mucosa!have!the!potential! to! produce! the! secondary! metabolite! to! inhibit! the! candidiasis,! which! caused! by! pathogen! fungi,! candida& albicans.! lactic! acid! bacteria! were! isolated!from!three!healthy!respondents!based!on!age!difference!that!is!an! infant,! child,! and! adult.! lactic! acid! bacteria! isolation! using! the! demann! rogosa!sharpe!broth!(mrsb)jliquid!medium,!followed!by!purification!stage! with!the!quadrant!scratch!method!using!the!demann!rogosa!sharpe!agar! (mrsa)jsolid! medium.! the! purified! isolates! obtained! were! characterized! and! identified! by! gram! staining,! biochemical! tests,! and! inhibition! zone! diameter! measurement! toward! pathogenic! fungi! candida& albicans.! the! results!were!obtained!from!each!of!the!two!lactic!acid!bacteria!isolates!from! each!respondent!with!characteristics!including!gramjpositive!bacteria!with! short!stem!cell!shape,!possibly!as!lactobacillus&sp.!it!is!conclude!that!the! lactic! acid! bacteria! of! oral! mucosa! from! adult! had! greater! anticandidal! activity!than!infant!and!child.! & issn!:!2580j2410! eissn!:!2580j2119! ! ! ! study&of&lactic&acid&bacteria&activities&from&human&oral& mucosa&for&candida&albicans&inhibition& ! evi&lauw1,&khusnul&hatimah&ilham1,&febliani&taruk&palinggi1,&desy&setiady2&and&sartini1& ! 1!faculty!of!pharmacy,!hasanuddin!university,!makassar,!indonesia! 2!faculty!of!dental!medicine,!hasanuddin!university,!makassar,!indonesia! ! ! & & & & & & & & & & & & & & & introduction& during! the! last! one! decade,! the& prevalence! of! infectious! diseases! caused! by! pathogenic!fungi,!which!is!candidiasis,!has!increased!sharply!(fortún&et&al.,!2012).!candidiasis! is! a! mucosal! fungal! infection! on! humans! of! all! ages! that! affect! the! skin! and! mucous! membranes!of!oropharynx,!oesophagus,!and!vagina!(fortún&et&al.,!2012;!gajer!et&al.,!2012).! there!are!at!least!15!distinct!candida!species!that!cause!human!disease,!but!more!than!90%! of!invasive!disease!is!caused!by!the!five!most!common!pathogens,!c.&albicans,&c.&glabrata,&c.& tropicalis,&c.&parapsilosis,&and&c.&krusei!(silvajdias!et&al.,!2014).!candida&albicans&continues!to! be!the!most!prevalent!and!problematic!of!all!candida!species!(perera!et&al.,!2015;!paul!et&al.,! 2014).!the!pathogenicity!of!candida!species!is!attributed!to!certain!virulence!factors,!such!as! the!ability!to!evade!host!defences,!adhesion!and!biofilm!formation!(on!host!tissues!and!or!on! medical! devices),! and! the! production! of! tissuejdamaging! hydrolytic! enzymes,! such! as! proteases,!phospholipases!and!haemolysins!(paul!et&al.,!2014).!moreover,!the!morbidity!and! &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword! lactic&acid&bacteria&& human&oral&mucosa& antifungal&activity& candida&albicans! article&history! received!23!may!2017! accepted!10!november!2017!! international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international&journal&of&applied&biology,&1(2),&2017& ! 59! mortality!associated!with!candidiasis!are!still!very!high,!even!using!the!actual!antifungal!drugs.! annually,! 50%! of! adults! and! up! to! 30%! of! children! die! of! candidiasis! (paul! et& al.,! 2016;! martinez!et&al.,&2015).! the!major!classes!of!antifungal!drugs!used!for!treatment!of!candida!species!infections! are! azoles,! polyenes,! and! echinocandins! (martinez! et& al.,! 2015).! according! to! previous! experimental!results,!irrational!use!may!lead!to!colonization!of!candida&sp.!that!are!resistant! to!these!drugs.!it!has!been!reported!that!conventional!treatment!for!treating!candidiasis!with! fluconazole!has!a!case!of!resistance!(martinez!et&al.,!2015;!costa!et&al.,!2015).!the!occurrence! of! candida! species! infections! becoming! more! difficult! to! treat! due! to! the! growth! of! immunogenic! diseases,! endocrine! disorders,! malnutrition,! the! disproportionate! use! of! immunosuppressive!drugs,!and!broad!spectrum!antibiotics!(costa!et&al.,!2015).!it!is!believed! that!the!increase!of!c.&albicans&infections!is!due!to!its!intrinsically!low!susceptibility!to!azoles,! including! the! imidazole! and! the! oraljparenteral! triazoles! (e.g.,! fluconazole,! voriconazole)! (gayford! jj! &! harskell! r,! 2010).! additionally,! it! is! known! that! the! acquired! resistance! is! resulted!of!rare!mutations!that!are!selected!by!drug!pressure!(samaranayake,!2012).! the!recent!efforts!of!various!excellent!researches!tremendously!broadened!on!the! complex!mechanisms!of!normal!flora!(scatassa!et&al.,!2015).!they!were!shown!that!living! creatures!without!normal!flora!are!more!susceptible!to!pathogenic!microbes!(kanmani!et&al.,! 2013;! amadou! et& al.,! 2013;! amenu,! 2013).! in! this! sense,! the! authors! highlighted! the! importance!of!normal!flora!and!that!the!addition!of!secondary!metabolite!to!improve!the! efficacy!of!candidasis.!the!result!of!a!study!showed!that!there!was!an!increase!in!oral!and! dental!health,!as!well!as!a!decrease!in!the!number!of!candida&albicans&fungi!in!school!children! who!consumed!milk!or!cheese!containing!lactobacillus&rhamnosus&strain&gg.3!which!belongs! to!the!lactic!acid!bacteria!group!(canabarro!et&al.,!2012).!according!to!the!authors,!some! strains! of! lactic! acid! bacteria! have! a! positive! activity! in! reducing! the! amount! of! candida& albicans!in!the!saliva!of!the!human!oral!cavity!(amenu,!2013;!canabarro!et&al.,!2012).! recent! studies! revealed! the! involvement! lactic! acid! bacteria,! especially! the! genus! lactobacillus,! can! be! isolated! from! animal! and! human! digestive! tracts,! including! upper! gastrointestinal! organs! such! as! the! oral! cavity! (corona! et& al.,! 2016).! it! has! obtained! lactobacillus&gasseri&isolates!identified!using!16sjrrna!sequencing!from!oral!mucosa!of!a!4j monthjold!infant!(settanni!et&al.,!2013).!however,!this!article!showed!antijfungal!activity!of! lactic!acid!bacteria!isolates!from!three!most!potent!sources!as!producers!of!anti!candidiasis! metabolite!compounds!that!are!an!infant,!child,!and!adult.!the!significance!of!this!study!is! due!to!its!contribution!to!knowledge!on!the!determination!of!the!ability!of!lactic!acid!bacteria! isolates!from!the!oral!cavity!of!humans!as!anti!candidiasis!agents.! & materials&and&methods& preparation&of&tools&and&materials! the!tools!used!in!this!research!were!autoclave!(all!american),!incubator!(memmert),! laminary!air!flow,!microscope!(olympus),!oven,!caliper,!analytical!scales!(ohauss),!sterile! swab! (onemed),! petri! disc,! chemical! glass! (pyrex),! bunsen,! cotton,! measuring! cylinder! (pyrex),!handscoon,!electric!stove,!cloth!mask,!round!loop,!straight!loop,!spoon,!test!tube,!vial! tube,!etc.! the!materials!used!in!this!research!were!70%!alcohol,!aqua!distillate,!pure!candida& albicans!culture!(obtained!from!microbiology!laboratory,!faculty!of!pharmacy,!hasanuddin! international&journal&of&applied&biology,&1(2),&2017& ! 60! university),!ketoconazole,!demann!rogosa!sharpe!agar!and!broth!(mrsa!and!mrsb)!media,! and!potato!dextrose!agar!(pda)!medium.! ! experimental&methods& isolation&of&lactic&acid&bacteria& oral!mucous!samples!were!taken!from!each!of!the!three!samples!individually,!infant,! child,!and!adult.!sampling!is!done!by!rubbing!into!the!inside!cheeks!in!samples!using!a!sterile! swab!sticker.!the!swab!result!is!then!inserted!into!a!vial!containing!the!mrsb!medium!as!a! transport!medium,!then!immediately!taken!to!the!laboratory.! samples!of!the!oral!mucosa!were!diluted!by!1!ml!pelleted!and!fed!into!9!ml!of!sterile! distilled!water,!then!diluting!the!dilutions!from!10j1!to!10j5.!a!total!of!1!ml!of!liquid!from!each! dilution!was!introduced!into!petri!dishes!and!10!ml!of!mrsa!and!caco3!1%!were!added!and! homogenized,!then!incubated!at!37!°c!for!1x24!hours.!when!a!clear!zone!is!formed!around! the!colony,!then!the!colony!is!suspected!as!lactic!acid!bacteria.! ! purification&of&lactic&acid&bacteria&isolates! lactate!acid!bacteria!isolates!were!then!inoculated!by!scraping!them!onto!the!medium! in!a!petri!dish!then!incubated!for!1x24!hours!at!37!°c!at!the!aerobic!incubator.!then!purified! by!inoculating!the!mrsa!medium!inclined!in!the!test!tube,!after!which!incubated!for!1!×!24! hours!at!37!°c!in!the!aerobic!incubator.! ! characterizations&and&identifications&of&lactic&acid&bacteria! macroscopic&test! pure!lactic!acid!bacteria!isolates!were!inoculated!on!mrsa!medium!in!petri!dishes,! after!which!incubated!for!1x24!hours!at!37!°c!in!aerobic!incubators.!then,!observed!colonies.! & microscopic&test& gram!staining!method!was!used!by!isolate!growing!on!mrsb!medium!placed!on!the! preparation.!then,!fixed!and!added!2j3!drops!of!gram!a!(crystal!violet),!left!for!1!minute.! wash!with!running!water,!dried!with!filter!paper.!add!gram!b!(mordant).!left!1!minute.!spilled! with!gram!c!(95%!ethanol)!for!10j20!seconds.!wash!with!running!water,!dried!with!filter! paper.!add!gram!d!(safranin)!for!1!minute.!wash!with!running!water,!dried!with!filter!paper.! it!was!observed!under!a!microscope!with!100x!widening!by!using!immersion!oil.!gram!positive! bacteria!will!be!purple!and!gram!negative!will!be!red!(ventimiglia!et&al.,!2015).! & biochemical&test! biochemical!tests!in!this!study!consist!of!the!several!tests!as!following;! & carbohydrate&fermentation&test! cultures!of!lactic!acid!bacteria!inoculated!into!lactose!broth,!sucrose!broth,!glucose! broth,!and!mannitol!broth!media!were!incubated!for!1!x!24!hours!at!37!°c.!thus,!observed! changes!in!medium!color!and!gas!formation.! & motility&test! the!culture!of!lactic!acid!bacteria!was!inoculated!puncture!on!an!erect!sim!medium! and!incubated!for!1!x!24!hours.!if!isolate!growth!spreads!far!from!puncture!indicates!motile! isolate,!whereas!if!growth!only!occurs!puncture!discharge,!it!indicates!no!motile!isolate.! international&journal&of&applied&biology,&1(2),&2017& ! 61! catalase&test! suspension!isolates!drops!as!much!as!two!drops!on!glass!object,!and!then!spilled!3%! h2s2!solution.!the!positive!catalase!reaction!is!shown!by!the!presence!of!foam!or!foam!that! occurs!after!the!addition!of!the!solution!for!1!min.! & test&of&optimum&growth&temperature& a! total! of! 1! ml! of! pure! culture! suspension! were! each! inoculated! into! 4! tubes! containing! 9! ml! mrsb! media! on! which! the! purple! bromjkresol! indicator! was! split.! the! medium!is!incubated!at!a!temperature!of!10!°c,!15!°c,!37!°c!and!45!°c!for!1j5!x!24!hours!under! suitable!conditions.!the!media!color!change!from!purple!to!yellow!signifies!the!growth!of! culture.! & test&of&optimum&growth&ph! a!total!of!1!loop!of!pure!isolates!was!each!inoculated!into!4!tubes!containing!mrsb! media!on!which!the!purple!bromjkresol!indicator!was!split.!the!medium!was!incubated!at!ph! 2,!4,!6!and!8!for!1j!5!x!24!hours!at!37!°c.!the!media!color!change!from!purple!to!yellow!signifies! the!growth!of!culture.! & test&of&isolate&activity&against&candida&albicans! the!isolated!lactic!acid!bacteria!were!grown!in!mrs!broth!media!for!24j48!hours.!the! result! of! the! fermentation! will! be! precipitated! its! cell! mass.! then! the! supernatant! fluid! containing! the! bioactive! component! was! tested! for! its! anticandidal! activity! of! candida& albicans!by!the!paper!discjdiffusion!method.!the!isolates!were!prepared!for!the!test!with!two! replications!of!each!sample.& & results&and&discussion&& lactic&acid&bacteria&isolation&& this! research! began! with! sterilization! as! the! most! important! stage! in! the! implementation!of!research!related!to!microorganisms.!furthermore,!the!manufacture!of!a! special!medium!for!the!growth!of!lactic!acid!bacteria!is!mrsb!(demann!rogosa!sharpe!broth)! as! the! transport! medium.! the! next! stage! is! the! oral! mucosal! sampling! of! three! people! samples;! infant,! child,! and! adult! from! three! volunteers! with! healthy! dental! and! mouth! conditions.!sampling!is!done!by!using!sterile!swab!swabs!and!immediately!inserted!into!the! transport!medium!immediately!after!taking!to!minimize!the!occurrence!of!contamination.! the!purpose!of!this!early!stage!is!to!obtain!lactic!acid!bacteria!from!the!oral!mucosa!and! increase!the!number!of!cells!(cultivation)!before!the!next!stage.& this!process!used!mrsa!(demann!rogosa!sharpe!agar)!medium!in!a!petri!dish!and! calcium!carbonate!(caco3!1%)!added!to!the!medium!in!order!to!identify!differences!of!lactic! acid!bacteria!compared!to!the!other!types!of!bacteria.!the!validation!of!the!lactic!acid!bacteria! was!performed!by!the!muddiness!of!medium!colour!(vestman!et&al.,!2013).!if!it!contained! lactic!acid!bacteria,!the!calcium!from!caco3!in!mrsa!can!react!with!lactic!acid!produced!by! lactic!acid!bacteria!to!form!soluble!calcium!lactate,!which!makes!the!colour!of!the!medium! becomes!transparent!(vestman!et&al.,!2013).! ! & & international&journal&of&applied&biology,&1(2),&2017& ! 62! lactic&acid&bacteria&purification! the!next!stage!was!the!purification!of!isolates.!this!isolation!was!carried!out!each!into! 2!petri!dishes!for!each!sample.!the!observation!of!colony!growth!will!be!done!on!days!1,!3,!5,! and!7.!on!the!5!the!day,!a!transparent!zone!has!been!formed!on!the!mrsa!medium!that!is! thought!to!be!a!colony!of!lactic!acid!bacteria.!the!purification!results!are!repeated!twice!with! the!aim!of!obtaining!more!pure!isolates!and!increasingly!free!from!mixtures!of!other!types!of! bacterial!colonies.! subsequently,! the! isolates! were! moved! into! the! test! tube! containing! the! inclined! mrsa!in!the!test!tube!for!the!further!stages,!i.e.!identification!of!isolates.!isolates!were!named! by!code,!i.e.!infant’s!mouth!isolates!(mb,!mb2),!child’s!mouth!isolates!(ma,!ma),!and!adult’s! mouth!isolates!(md,!md2).! & lactic&acid&bacteria&characterizations&and&identifications& in!this!study,!the!identification!of!lactic!acid!bacteria!microscopically!was!done!by!the! gram!staining!method.!the!staining!results!for!the!three!types!of! isolates!(mb,!ma,!md)! showed!purple!bacterial!cells,!so!the!isolates!in!this!study!included!gramjpositive!bacteria.! this!supports!the!theory!that!lactic!acid!bacteria,!one!of!which!is!from!the!genus!lactobacillus,! is!a!type!of!gramjpositive!bacteria!that!in!gram!staining!will!have!a!purple!appearance!under! an!optical!microscope!(ventimiglia!et&al.,!2015;!norder!et&al.,!2013).! in!addition,!besides!the!microscopic!tests!and!gene!identification!above,!the!isolates! were!tested!for!their!antimicrobial!activity!by!the!diffusion!method!in!order!to!use!paper!discs.! it!was!measured!from!horizontal,!vertical,!and!diagonal,!then!the!calculated!for!the!average! diameter.!based!on!antimicrobial!activity!test!from!oral!mucosa!isolate!to!candida&albicans! fungi!causing!infectious!disease!of!candidiasis,!and!obtained!the!result!of!the!zone!diameter! as!follows.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! figure&1.&inhibition&zone&diameter&of&lactic&acid&bacteria&isolates&from&human&oral&mucosa& & kontrol!1! ma!1! ma!2!kontrol!2!mb!1! mb!2! md!2! md!1!2! international&journal&of&applied&biology,&1(2),&2017& ! 63! table&1.&inhibition&zone&diameter&measurement&of&lactic&acid&bacteria&isolates&from&human& oral&mucosa& types&of&isolate& replication& diameter&±&standard&deviation&(mm)& i& ii& ketoconazole&(control&+)& 19.1! 20.4! & &18.7! 19.3! 19.4! 18.3! average& 19.07& 19.30& 19.19&±&0.16& infant&(mb)& 8.1! 7.3! & &6.5! 7.5! 7.1! 6.8! average& 7.23& 7.20& 7.22&±&0.02& child&(ma)& 9.3! 10.3! & &9.5! 9.9! 9.1! 10.2! average& 9.30& 10.13& 9.72&±&0.58& adult&(md)& 16.4! 15.8! & &16.2! 16.1! 15.9! 16.2! average& 16.17& 16.03& 16.10&±&0.09& ! from!the!table!above,!it!can!be!seen!that!the!average!inhibitory!zone!diameter!of!the! oral!mucosa!of!infants!(mb!and!mb2)!is!7.23!mm!and!7.20!mm!(average!7.22!mm!±!0.02!mm),! lactic!acid!bacteria!isolate!from!the!oral!mucosa!of!children!(ma)!is!9.30!mm!and!10.13!mm! (average!9.72!mm!±!0.58!mm),!and!lactic!acid!bacteria!isolate!from!adult!mouth!mucosa!(md)! were!16.17!mm!and!16.03!mm!(average!16.10!mm!±!0.09!mm).!while!the!results!of!inhibitory! power!using!ketoconazole!antifungal!drugs!as!a!comparison!were!19.07!mm!and!19.30!mm! (average!19.19!mm!±!0.16!mm).!it!is!showed!that!lactic!acid!bacteria!isolates!from!adult!mouth! mucosa!(md)!had!the!best!inhibitory!power!seen!from!their!larger!inhibitory!diameter!zones! compared!to!both!infant!isolate!(mb)!and!child!isolate!(ma).! ! ! ! & & & & & & & & & & & & & & & figure&2.&the&collection&of&potent&lactic&acid&bacteria&isolates&as&anti&candidiasis& international&journal&of&applied&biology,&1(2),&2017& ! 64! for!additional!data,!biochemical!tests!consisting!of!carbohydrate!fermentation!test,! motility!test,!catalase!test,!optimum!ph!test,!and!optimum!temperature!test!were!conducted.! the!aim!of!these!five!biochemical!tests!is!to!confirm!that!the!isolates!obtained!from!the!oral! mucosa!of!humans!in!this!study!can!satisfy!the!characteristics!of!lactic!acid!bacteria!with! specific!physiological!and!metabolic!activities!specifically.! the!results!of!carbohydrate!fermentation!test!on!the!three!isolates!(mb,!ma,!md)!with! medium! lb,! gb,! and! sb! showed! the! three! results! of! each! positive! able! to! ferment! carbohydrate.!the!positive!result!is!marked!by!the!colour!change!of!the!medium!to!yellow! and!the!formation!of!gas!in!the!durham&tube.!this!is!consistent!with!the!reference!that!lactic! acid!bacteria!include!heterofermentative!bacteria,!which!are!capable!of!fermenting!sugars! (monosaccharaides!and!disaccharides)!to!produce!acidic!and!co2!gases.!the!acidic!conditions! in!the!medium!result!in!a!change!of!medium!colour!and!bubble!formation!(minervini!et&al.,! 2012;!pepe!et&al.,!2013).! the!result!of!the!motility!test!of!all! three! isolates!showed!the!growth!of!colonies! perpendicularly!in!the!direction!of!inoculation!using!a!loop,!and!without!spreading!around!the! inoculants.! if!the!bacterial!cell! is!motile,! its!colonies!will!spread!throughout!the!medium.! therefore,!the!three!isolates!(mb,!ma,!md)!are!nonjmotile!bacteria!(minervini!et&al.,!2012;! pepe&et&al.,!2013).! the!result!of!catalase!test!of!lactic!acid!bacteria!isolated!from!oral!mucosa!of!the!three! samples!(mb,!ma,!md)!was!negative!catalase.!this!is!in!accordance!with!the!reference!to!the! fact!that!lactic!acid!bacteria!do!not!produce!catalase!enzymes!so!that!the!test!results!are! negative!as!a!result!of!the!absence!of!h2o2!degradation!by!catalase!enzymes!(minervini!et&al.,! 2012;!pepe!et&al.,!2013).! the!optimum!ph!test!results!in!this!study!were!ph!4.!at!ph!4,!the!most!turbid!visible! medium!was!thought!to!be!the!highest!growth!rate!of!lactic!acid!bacteria!in!this!study.!the! previous! study! stated! that! lactic! acid! compounds! capable! of! inhibiting! the! growth! of! pathogenic! microbes! by! lowering! the! ph! of! the! environment! to! 3! to! 4.5! so! that! other! microbial!growth!will!be!hampered!(minervini!et&al.,!2012;!pepe!et&al.,!2013).! !the!optimum!temperature!test!results!are!at!45!°c,!which!is!the!highest!temperature! of!this!test.!the!highest!growth!of!colonies!in!the!form!of!the!cloudyjlooking!medium!occurred! at!45!°c!compared!to!25!°c!and!37!°c.!based!on!the!reference,!lactic!acid!bacteria!can!grow! at!a!temperature!of!20j50!°c!(minervini!et&al.,!2012;!pepe!et&al.,!2013).! based!on!the!five!biochemical!tests,!the!three!types!of!isolates!are!catalase!negative,! nonmotile,!and!include!gram!positive!(+)!bacteria!with!basil!(stem)!morphology.!in!this!study,! it!can!be!said!that!the!isolates!obtained!from!the!mucosa!of!the!human!mouth!(mb,!ma,!md)! belong!to!the!lactic!acid!bacteria!group,!were!from!the!genus!lactobacillus!based!on!the! biochemical!test!results.! ! conclusions! the!anti!candidiasis!activity!of!lactic!acid!bacteria!isolates!from!the!adult!oral!mucosa! (md)! most! potentially! inhibited! the! growth! of! candida& albicans! that! is! 16.10! mm,! when! compared!to!lactic!acid!bacteria!isolates!from!the!infant!(mb)!and!the!child!(ma)! & acknowledgment&& the!authors!are!grateful!to!ministry!of!research,!technology,!and!higher!education! indonesia!which!has!funded!this!research!through!student!creativity!programjexact!research! international&journal&of&applied&biology,&1(2),&2017& ! 65! with!contract!number!0547/b3.1/km/2017.!we!also!grateful!to!the!lecturer!dr.!sartini,!m.si.,! apt.,!for!her!valuable!help!and!to!hasanuddin!university,!which!has!facilitated!us,!so!that!the! research!could!successfully!conduct.! & references& amadou,!i.,!le,!g.w.!&!shi,!y.h.!2013.!evaluation!of!antimicrobial,!antioxidant!activities,!and! nutritional!values!of!fermented!foxtail!millet!extracts!by!lactobacillus&paracasei&fn032.! int.&j.&food&prop,!16(6):1179j1190.! 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of!oral!lactobacilli!in!breastfed!infants.!bmc&microbiology,!13(1):12.!! samaranayake,!lakshman.!2012.!essential&microbiology&for&dentistry:&fourth&edition.&elsevier,! 3:307.! scatassa,! m.l.,! gaglio,! r.! &! macaluso,! g.! 2015.! transfer,! composition! and! technological! characterization!of!the!lactic!acid!bacterial!populations!of!the!wooden!vats!used!to! produce!traditional!stretched!cheeses.!food&microbiol.!52:31j41.! settanni,! l.,! ventimiglia,! g.,! alfonzo,! a.,! corona,! o.,! miceli,! a.! &! moschetti,! g.! 2013.! an! integrated!technological!approach!to!the!selection!of!lactic!acid!bacteria!of!flour!origin! for!sourdough!production.&food&res.&int,!54:1569–1578.!! silvajdias,! a.,! miranda,! i.m.,! branco,! j.,! cobrado,! l.,! monteirojsoares,! m.,! pinajvaz,! c.! &! rodrigues,!a.g.!2014.!in!vitro!antifungal!activity!and!in!vivo!antibiofilm!activity!of!cerium! nitrate!against!candida!species.!j.&antimicrob.&chemother,!70:1083–1093.! ! ! ! ! ! ! ! ! & ! 1(1)22-31 contact&:&haeriah&&&&&&&haeriah564@gmail.com0 ©0international0journal0of0applied0biology0 220 abstract& chitosan0can0be0obtained0from0chitin0isolated0from0shrimp0waste0through0 the0process0of0deacetylation0of0chitin.0chitosan0can0be0used0as0a0patch0base0 material0 because0 of0 its0 biocompatibility0 and0 biodegradability,0 and0 has0 antibacterial0activity.0the0combination0of0chitosan0and0aloe%vera0extracts0 may0 be0 useful0 in0 patch0 dosage0 forms0 as0 wound0 dressings0 that0 have0 antiinfective0activity.0the0aims0of0this0research0was0to0obtain0patch0from0 combination0of0aloe%vera0l.0leaves0extract0and0chitosan0isolated0from0shrimp0 shell0waste0as0antiinfection0agent.0chitosan0was0obtained0from0shell0waste0 sequentially0 by0 deproteinisation,0 demineralisation,0 and0 deacetylation0 processes,0and0analyzed0its0characteristic,0respectively;0aloe%vera0gel0was0 extracted0 using0 maceration0 methods0 with0 ethanol0 as0 solvent;0 patch0 was0 formulated0using02%0chitosan0in01.5%0glacial0acetic0acid,001.6%00aloe%vera0 extracts0 and0 100 %0 glycerin0 and0 evaluated0 its0 physical0 properties,0 0 skin0 iritation0test,0and0antibacterial0 test0against0 s.aureus.&the0results0showed0 that0patch0made0from0the0combination0of0shrimp0waste0chitosan0and0aloe% vera0had0good0physical0characteristics0and0effectively0inhibit0the0growth0of0 s.aureus.0so,&the&combination0of0aloe%vera%leaves0extract0and0chitosan0from0 shrimp0shell0as0patch0can0be0used0as0antiinfection0wound0healing.0& & issn0:02580q24100 eissn0:02580q21190 0 0 0 the&production&of&chitosan&from&shrimp&shell&waste&and&its& formulation&in&patch&dosageform&combined&with&aloe%vera&extract& as&antiinfection&agent& 0 haeriah1,&muhammad&rahmatullah1,&andi&indardaya1,&emilia&utomo1,&novianti2,&sartini1& 0 10 faculty0of0pharmacy,0hasanuddin0university,0indonesia0 20 faculty0of0mathematics0and0natural0sciences,0hasanuddin0university,0indonesia0 0 0 & & & & & & & & & & & & & & & introduction& the0injury0in0which0skin0is0torn,0cut0or0punctured,0also0called0wound0(katari0et%al.,2014;0 varshney0&0dhyani,02015).0wound0healing0is0the0process0comprising0of0healing0of0dermal0and0 epidermal0tissues0by0their0regeneration0(katari0et%al.,02014;0takeo0et%al.,02015).0it0involves0 consecutive0cascade0of0stages0inflammation,0migration,0proliferation,0and0maturation0(katari0 et%al.,02014;0takeo0et%al.,02015).0skin0itself0repairs0the0wound0but0open0wounds0faces0a0number0 of0 anomalies0 like0 infection0 (sepsis)0 from0 190 air,0 water0 pollutants0 and0 microorganisms,0 its0 spread0to0health0tissues0and0tissue0disruption0(rupture)0and0even0to0others0(katari0et%al.,02014;0 devika0&0koilpillai,02014).0staphylococcus%aureus%is0one0of0the0most0common0bacteria0caused0 infection0(choudury0et%al.,02012;0rajan0et%al.,02016).0previous0research0showed0that0this0type0 &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword0 aloe%vera0l.,0 chitosan,0 wound0dressing,0 patch,0 shrimp0shell0waste.0 0 article&history0 received0230june020170 accepted0190august0201700 0 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 0 international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 230 of0bacteria0caused0mild0skin0infections0and0postoperative0wounds0since01880s0(choudury0et% al.,02012;0vijayalakshmi,02015).00 0 one0of0the0most0popular0local0drug0delivery0system0today0is0transdermal0drug0delivery0 system0in0the0form0of0transdermal0patch0(fathima0et%al.,02017;0sachan0&0bajpai,02013).0it0is0 preferred0because0it0has0many0advantages0compared0to0other0preparations0(e.g.0oral0dosage0 form)0(fathima0et%al.,02017;0sachan0&0bajpai,02013).0one0of0the0polymers0used0in0transdermal0 patch0formulation0is0chitosan0(fathima0et%al.,02017;0daniel0&0hamblin,02016).0chitosan0is0used0 as0 the0 part0 of0 drug0 delivery0 system0 because0 of0 its0 biocompatibility0 and0 biodegradability0 (fathima0et%al.,02017;0daniel0&0hamblin,02016).0 chitosan0can0be0obtained0from0chitin0that0is0isolated0from0shrimp0shell0waste0by0chitin0 deacetylation0(alami0&0permatasari,02016;0ahing0&0wid,02016).0chitin0content0on0shrimp0shell0 waste0is0about015q20%0of0dry0weight0(alami0&0permatasari,02016;0danujatmiko0et%al.,02014).0 shrimp0waste0is0its0head0and0shell0that0cause0environmental0pollution0(alami0&0permatasari,0 2016;0ahing0&0wid,02016).0therefore,0this0waste0can0be0utilized0as0chitosan0source0(alami0&0 permatasari,02016;0ahing0&0wid,02016).0based0on0bellamkonda0et%al.0(2017)0showed0that0the0 obtained0chitosan0has0antibacterial0activity0against0staphylococcus%aureus%(bellamkonda0et% al.,0 2017).0 hence,0 chitosan0 in0 patch0 preparation0 has0 two0 functions,0 as0 polymer0 in0 patch0 formulation0and0as0antibacterial0agent.0 one0of0the0natural0materials0that0has0been0known0to0be0used0in0wound0healing0is0aloe% vera0l.0(silva0et%al.,%2013;0sharma0et%al.,02015).0aloe%vera0l.0has0an0activity0as0antibacterial,0as0 evidenced0by0the0results0of0a0study0conducted0by0jothi0et%al.0(2014)0showing0that0aloe%vera0 can0inhibit0the0growth0of0staphylococcus%aureus0bacteria0with0a0percentage0of0resistance0of0 61q66%0(jothi0et%al.,02014).0aside0from0being0an0antibacterial,0aloe%vera0l.0also0has0increased0 collagen0 content0 within0 the0 wound,0 supporting0 faster0 wound0 healing0 (silva0 et% al.,% 2013;% sharma0et%al.,02015).0aloe%vera0contains0many0active0substances0that0are0essential0for0wound0 healing0(silva0et%al.,%2013;%mahor0et%al.,02016).0based0on0the0results0of0research0conducted0by0 tudose0et0al.0(2009)0on0nctc25440cells0showed0that0cells0given0aloe%vera0extract0experienced0 higher0cell0proliferation0than0control0(tudose0et%al.,02009).0these0results0suggest0that0aloe% vera0can0be0used0as0a0topical0natural0treatment0(tudose0et%al.,02009;0mahor,02016).0both0 activities0possessed0by0aloe%vera0are0very0useful0in0wound0healing0due0to0its0synergistic0effect0 in0this0research.0this0study0aims0to0obtain0patch0from0aloe%vera0extract0combination0and0 chitosan0as0wound0dressing.0 & materials&and&methods& isolation&of&chitosan0 shrimp0shell0was0obtained0from0kima0industry0in0makassar.0the0waste0was0then0washed0 and0cleaned0using0running0water,0and0mashed0using0blender.0next,0the0dried0waste0was0kept0 in0closed0container.0 & isolation&of&chitin& the0process0of0chitin0isolation0involved0two0steps.0firstly,0demineralization0which0was0 done0by0soaking0the0dried0waste0in020liters0of03%0hydrochloric0acid0for0160hours0at0room0 temperature.0after0that,0the0residue0was0washed0and0soaked0in0distilled0water0until0netral.0 secondly,0deproteinization0which0was0done0by0soaking0the0residue0in020liters0of04%0sodium0 hydroxide0solution0for0200hours0at0room0temperature.0 & & international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 240 purification&of&chitosan&(deacetylation)& 0 the0 residue0 was0 washed0 and0 soaked0 in0 distilled0 water0 until0 netral0 then0 dried.0 deacetylation0process0was0done0by0soaking0the0residue0in020litres0of060%0sodium0hydroxide0 solution0for0200hours0at065 o c.0afterwards,0it0was0washed0using0distilled0water0until0netral.0the0 obtained0residue0was0chitosan0which0was0then0dried0for040hours0at0650±05 o c0and0ready0to0 characterize.0 0 analysis&of&chitosan& chitosan0 analysis0 consists0 of0 pharmaceutical0 and0 chemistry0 analysis.0 the0 pharmaceuticals0 analysis0 includes0 ph0 measurement0 of0 chitosan0 solution0 using0 ph0 meter,0 viscosity0measurement0of0chitosan0solution0brookfield ® 0viscometer,0solubility0test0which0was0 done0by0dissolving00.50g0of0chitosan0with0500ml0of01%0acetic0acid,0stirring0for0300min0and0 observed0solubility,0and0morphology0observation0using0scanning0electron0microscopy.0the0 chemistry0 analysis0 includes0 crystallinity0 detection0 using0 xqray0 diffraction0 spectrometer,0 functional0groups0observation0using0infrared0spectroscopy0in040000–04000cm q1 ,0and0degree0of0 deacetylation0calculation0based0on0ftqir0results,0using0the0following0formula:0 0 !!"# % = [(1 − a1655#a3450#0# 1 #1,33 )0#100#]0 0 extraction&of&aloe%vera& aloe%vera0leaves0was0washed0using0distilled0water0to0remove0the0dirts0and0then0peeled.0 the0gel0of0aloe%vera0was0cut0into0small0pieces0and0then0mashed.0after0that,0it0was0dried0in0the0 oven0for0240hours0at080 o c.0the0dried0gel0was0macerated0using0ethanol096%0for0240hours.0the0 solvent0was0evaporated0using0rotary0evaporator0to0obtain0the0viscous0extract.0 & analysis&of&aloe%vera&extract& antibacterial&test& antibacterial0test0was0done0by0using0agar0diffusion0method.0a0total0of0300mg0of0extract0 were0dissolved0in030ml0of0dmso.0ten0microlitres0of0staphylococcus%aureusculture0suspension0 was0added0into0petri0dish0followed0by0150ml0of0mha0media.0a0total0of0400µl0and0800µl0of0extract0 solution0was0added0into0paper0disc0which0was0placed0on0the0agar0surface.0it0was0incubated0 for010x0240hours0at037 o c.0the0formed0inhibit0zone0was0then0observed.0 0 qualitative&test&of&acemannan&in&aloe%vera&extract& a0total0of01000mg0extract0was0added0to010ml0of0distilled0water.0after0that,02.50ml0of00.20 m0 sodium0 hydroxide0 solution0 and0 10 ml0 of0 0.00020 m0 congo0 red0 solution0 were0 added0 consecutively.0the0formed0color0was0observed.0 & formulation&of&patch& two0percents0of0chitosan0was0dissolved0in01.5%0(v/v)0glacial0acetic0acid.0afterwards,0 10%0of0glycerin0and01.6%0of0aloe%vera0extract0were0added0consecutively0while0stirring.0about0 200g0of0the0solution0was0poured0into0petri0dish0and0then0dried0in0the0oven0for0480hours0at040 o c.0 & evaluation&of&patch& the0thickness0of0the0patch0was0measured0using0vernier0calipers0at030different0points.0 the0folding0endurance0of0patch0was0measured0manually0by0cutting0the0patch0by010cm 2 0and0 international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 250 folded0repeatedly0in0the0same0place0till0broken.0the0number0of0folds0without0fracture0was0the0 value0 of0 folding0 ability.0 patch0 was0 weighed0 and0 placed0 in0 container0 containing0 1000 ml0 saturated0 solution0 of0 potassium0 chloride.0 after0 240 hours,0 patch0 then0 weighed0 again.0 the0 percentage0of0moisture0uptake0was0measured0using0the0following0formula:0 0 %456789:;#<=75:>865? =#@6?<a#b;6cℎ8 − e?686<a#b;6cℎ8e?686<a#b;6cℎ8 #0#1000 0 moisture&content&measurement& patch0was0weighed0accurately0and0placed0in0container0containing0silica0gel.0after0240 hours,0patch0then0weighed0again.0the0percentage0of0moisture0loss0was0calculated0using0the0 formula0below:0 0 %456789:;#a577 =#e?686<a#b;6cℎ8 − f6?<a#b;6cℎ8f6?<a#b;6cℎ8 #0#1000 0 skin&irritation&test& the0test0used0three0rats0(rattus%novergicus)0as0test0animals.0the0patch0was0pasted0on0 the0back0of0the0rat0(rattus%novergicus)0for0240hours.0erithema0and0edema0were0observed0after0 240hours0treatment.0 0 antibacterial&test&& antibacterial0 test0 was0 conducted0 using0 staphylococcus% aureus% culture0 in0 mueller0 hilton0agar0media0and0tetracycline0as0a0positive0control.00.10ml0of0bacteria0suspension0was0 added0in0petri0dish0followed0by0mha0media.0circle0shaped0patch0with0diameter05.50mm0was0 placed0on0the0surface0of0the0media.0twenty0microliters0of0300ppm0tetracycline0solution0in0 paper0disk0was0also0placed0on0the0surface0of0medium.0it0was0then0incubated0for010x0240hours0 at037 o c.0the0formed0inhibitory0zone0was0observed.0 & results&and&discussion&& isolation&and&characterization&of&chitosan&from&shrimp&shell&waste& the0percentage0of0yield0of0chitosan0that0obtained0was032.67%.0in0addition,0there0are0 also08%0q010%0of0calcium0phosphate0of0total0anorganic0substances.0deproteinization0purpose0 is0to0break0the0bond0between0protein0and0chitins,0by0adding0sodium0hydroxide.0in0its0extracted0 crude0form,0chitin0has0a0highly0ordered0crystalline0structure,0is0translucent,0resilient0and0quite0 tough.0it0has,0however,0poor0solubility0and0low0reactivity0(abbaas,%2015).0deacetylation0aims0 to0remove0the0acetyl0group0that0attach0in0chitin.0in0deacetylation0process,0high0concentration0 of0sodium0hydroxide0solution0(400–060%)0is0used0to0obtained0chitosan0from0chitin.0chitosan0 characterization0results0is0presented0in0table01.0 0 table&1.&characterization&of&chitosan&isolated&from&shrimp&shell&waste& analysis& result& ph0 40 solubility0in0glacial0acetic0acid0 soluble0in010%0solution0 viscosity0 93.330cps0 0 international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 260 in0table01,0ph0of0chitosan0was04,0while0the0previous0studies0got08.5.0in0the0extraction0 process,0strong0acid0(hydrochloric0acid)0and0strong0base0(sodium0hydroxide)0were0used,0so0 that0different0volume0or0concentration0that0used0can0affect0the0ph0of0chitosan.0the0solubility0 of0chitosan0in0glacial0acetic0acid0was0one0of0the0parameters0that0can0be0used0as0standard0of0 chitosan0quality.0the0highest0the0solubility,0the0better0chitosan0obtained.0in0this0test,0the0 solubility0of0chitosan0sample0and0chitosan0standard0was0the0same0that010gram0of0chitosan0 dissolved0in01000ml0of0glacial0acetic0acid.0the0measurement0of0viscosity0gave0the0result0of0 93.330cps0of0chitosan0sample0and086.670cps0of0chitosan0standard.0 the0result0of0chitosan0characterization0using0ftqir0spectroscopy0was0given0in0figure010 which0was0chitosan0sample0and0chitosan0standard,0respectively.0in0the0analysis0using0ftqir,0it0 can0be0clearly0seen0that0peak0area0of0chitosan0sample0was0in0the0range0of03724.540–0349.120 nm q1 ,0while0the0peak0area0of0chitosan0standard0was0in0the0range0of03722.610–0352.970nm q1 .0for0 the0deacetylation0degree0calculation0is0obtained030%0and037.7%0for0chitosan0sample0and0 chitosan0 standard,0 respectively.0 it0 indicated0 that0 there0 is0 no0 significant0 characteristic0 differences0between0sample0and0standard.00 0 & & & & & & & & & & & & & & & & & & & & & & figure&1.&ftuir&spectra&of&chitosan& (a.!chitosan&sample,&b.0chitosan&standard)& & in0the0analysis0using0xrd,0theoretically0said0that0the0crystal0of0chitin0will0be0decreased0 after0changed0to0chitosan,0which0the0strongest0reflection0index0was0at0the090–0100degrees0 because0 in0that0area0there0was0an0 incorporation0of0water0molecules0 into0crystal0 lattices.0 crystallinity0degree0is0depend0on0deacetylation0degree0of0chitosan;0the0higher0the0degree0of0 deacetylation,0the0higher0the0degree0of0crystallinity0(yuan0et%al.,02011).0this0may0be0caused0by0 the0 fact0 that0 chitosan0 chain0 with0 higher0 deacetylation0 cause0 the0 particles0 become0 more0 a& b& international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 270 flexible0and0have0less0acetyl0groups.0the0results0of0this0analysis0is0presented0in0table020and0 figure02.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 figure&2.&xuray&diffraction&results& &(a.&chitosan&sample,&b.&chitosan&standard)& 0 table&2.&cri%value&of&chitosan&standard&and&chitosan&sample& 0 0 0 0 0 observation0 of0 chitosan0 structure0 using0 scanning0 electron0 microscopy0 (sem),0 theoretically0 said0 that0 chitosan0 has0 smooth0 morphology0 with0 minimum0 residue,0 while0 according0to0research0that0is0conducted0by0islam0et0al.%(2011),0reported0that0chitosan0was0not0 homogen0and0has0a0bit0not0smooth0surface0with0the0presence0of0bond0and0shrinkage0on0the0 surface.0based0on0that0information,0the0result0that0obtained0were0appropriate0(figure03).%% % 0 0 0 0 0 0 0 0 0 0 0 0 0 figure&3.&morphology&observation&results&using&sem& (a.&chitosan&sample,&b.&chitosan&standard)& types&of&chitosan& cri& cri110% cri020% chitosan0standard0 18.51440 q101.250 chitosan0sample0 18.70480 q75.200 a& b& a& b& 0 international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 280 &extraction&of&aloe%vera&leaves& the0results0of0aloe%vera0 leaves0extraction0was0obtained0the0percentage0of0yield0of0 22.73%.0the0qualitative0test0of0acemannan0in0aloe%vera0extract0gave0positive0result0which0was0 showed0by0red0complex0formed0after0the0addition0of0congo0red0in0an0alkaline0situation.0this0 positive0result0indicated0that0aloe%vera0extract0contained0acemannan0as0cell0regeneration0 stimulating0agent.0acemannan0is0a0linear0polysaccharide0composed0by0(1,4)qlinked0mannosyl0 residues,0with0c20or0c30acetylated0and0some0sideqchains0formed0by0galactose0units0attached0 to0c6.0it0is0aβq(1,4)qqlinkedpolydispersed,0highly0acetylated0mannan0with0an0average0molecular0 weight0of0approximately010000kda,0is0obtained0from0the0inner0leaf0of0aloe%vera.%acemannan0 has0 shown0 to0 accelerate0 wound0 healing.0 acemannan0 produces0 immune0 agents0 such0 as0 interferon0and0interleukin0which0help0to0destroy0viruses,0bacteria,0and0tumor0(cancer)0cells 0 (coche0et%al.,02014).0the0result0of0antibacterial0activity0test0is0shown0in0table03.0 0 table&3.&average&diameter&of&inhibitory&zone&of&aloe%vera&extract&(mm)& concentration0of0extract0 diameter0 of0 inhibitory0 zone0 average0diameter0 10 20 800µl0(1.6%)0 7.780 8.840 8.310 400µl0(0.8%)0 00 00 00 0 formulation&and&evaluation&of&transdermal&patch& the0obtained0chitosan0and0aloe%vera0extract0were0then0formulated0in0transdermal0 patch0which0evaluation0results0is0shown0in0table04.0 0 table&4.&evaluation&results&of&transdermal&patch% tests& results& moisture0uptake0test0(%)0 40.030 moisture0content0test0(%)0 23.640 thickness0test0(mm)0 0.450 folding0endurance0test0(times)0 1140 skin0irritation0test00 not0irritating0 % prepared0tansdermal0patch0showed0good0physical0characteristic.0as0in0study0by0jaydatt0 &0sreenivas0(2013)0showed0that0transdermal0patch0have0good0physical0characteristic0with0 thickness0varies0between00.42q0.450mm,0folding0endurance0between0102q1370times0(jaydatt0 &0sreenivas,02013)0while0the0higher0percentage0of0moisture0uptake0and0moisture0content0is0 due0to0its0hydrophobic0nature0of0the0polymer0used0(kumar0et%al.,02012).0 the0antibacterial0activity0test0was0done0using0positive0control0(tetracyline)0in0order0to0 compare0 the0 activity0 of0 patch0 with0 tetracycline.0 tetracycline0 is0 one0 of0 antibiotic0 which0 is0 commonly0used0to0treat0infection0caused0by0s.%aureus.0the0result0of0antibacterial0activity0test0 showed0that0transdermal0patch0of0chitosan0and0aloe%vera0extract0has0antibacterial0effect0 against0 staphylococcus% aureus% with0 the0 average0 diameter0 of0 inhibitory0 zone0 of0 6.39.0 the0 diameter0of0inhibitory0zone0of0positive0control0(tetracycline)0was07.730mm0which0was0not0 significantly0different0from0the0patch.0this0indicated0that0the0ability0of0aloe%vera%extract0and0 chitosan0 to0 inhibit0 the0 growth0 of0 s.% aureus% was0 not0 much0 different0 with0 tetracyline.0 arunkumar0&0mutuselyam0(2009)0showed0that0ethanol0extract0from0aloe%vera%can0inhibit0s.% aureus% (arunkumar0 &0 mutuselyam,0 2009),0 and0 morshed0 et% al.0 (2011)0 showed0 chitosan0 international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 290 isolated0from0shrimp0shells0having0activity0against0s.%aureus0bacteria0with0a0100mm0inhibit0 zone0(morshed0et%al.,%2011).0 the0description0of00results0above0are0supported0by0several0studies,0including:0rahman0 et% al.0 (2017),0 which0 said0 that0 aloe% vera% has0 a0 cell0 regeneration0 effect0 of0 acemannan0 compounds0and0the0antibacterial0effects0of0aloin0compounds 0 (rahman0et%al.,02017).0another0 study,0 hong0 et% al.0 (2017),0 said0 that0 chitosan0 is0 a0 polymer0 that0 is0 often0 used0 in0 topical0 preparations0because0of0its0highly0beneficial0mucoadhesive0properties0against0drug0delivery0 to0mucoidqcovered0organ0targets0(hong0et%al.,02017).0in0addition,0it0has0the0effect0of0wound0 healing0as0well0as0antibacterial.00 research0 has0 shown0 that0 aloe% vera0 increases0 the0 collagen0 content0 and0 promotes0 faster0wound0healing.0in0addition,0chitosan0has0been0widely0used0as0a0base0material0in0matrix0 production0for0wound0management0because0of0its0easy0production,0long0shelf0life0and0the0 intrinsic0nature0of0this0polymer0(silva0et%al.,02013).0therefore,0it0can0be0said0that0the0results0of0 this0research0has0been0in0accordance0with0existing0theories0and0supported0by0some0previous0 research.0 0 conclusion&& 0 combination0 of0 chitosan0 form0 shimp0 shell0 waste0 and0 aloe% vera0 extract0 can0 be0 formulated0into0patch0dosageform0which0had0antibacterial0activity0against0staphylococcus% aureus.% 0 acknowledgements& 0 this0 research0 was0 supported0 by0 ditjen0 belmawa0 kemenristekdikti0 (ministry0 of0 research,0technology,0and0higher0education)0for0research0grant02017.0the0authors0would0 like0to0thank0the0supervisor0as0well0as0the0parties0who0have0helped0the0authors0in0completing0 this0research.0 & references& abbaas.a.a.f.0 2015.0 dynamic0 assessment0 of0 biocidal0 properties0 of0 siliconqchitosanq containing0 hydrogels.0 international0 journal0 of0 innovative0 research0 in0 science,0 engineering0and0technology.04(4):02045q2050.0 ahing,f.a.,0&0n,0wid.02016.0extraction0and0characterization0of0chitosan0from0shrimp0shell0 waste0in0sabah.0transactions0on0science0and0technology.03(1q2):0227q237.0 alami,0 r.0 &0 permatasari,0 l.0 2016.0 industry0 pharmaceuticals0 :0 chitosan0 as0 an0 alternative0 replacement0gelatin0capsules0on0shell.0journal0of0medical0and0bioengineering.5(1):67q 71.0 arunkumar,0 s.0 &0 muthuselvam,0 m.0 2009.0 analysis0 of0 phytochemical0 constituents0 and0 antimicrobial0 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international%journal%of%applied%biology,%vol.%1,%issue%1,%2017% 0 310 to&cite&this&article:&& haeriah,0 rahmatullah,0 m.,0 indardaya,0 a.,0 utomo ,0 e.,0 novianti0 &0 sartini.0 2017.0 the0 production0of0chitosan0from0shrimp0shell0waste0and0its0formulation0in0patch0dosageform0 combined0with0aloe%vera0extract0as0antiinfection0agent.%international%journal%of%applied% biology.01(1)0:022q31.0 tudose,0a.,%christian,0c.,0francesco,0c.,0margherita,0v.,0roberto,0m.,0&0donatella,0p.02009.0 regenerative0 properties0 of0 aloe% vera0 juice0 on0 human0 keratinocyte0 cell0 culture.0 farmacia.057:590–596.0 varshney,0s.,0&0s,0dhyani.02015.0medicinal0herbs0having0incredible0wound0healing0effects.0 international0journal0of0research.06(5):0573q579.0 vijayalakshmi,0p.02015.0incidence0of0staphylococcus%aureus% in0surgical0site0infections0in0a0 teaching0hospital.04(4):032q34.0 yuan,0y.,0betsy,0m.c.,0warren,0o.h.,0&0joel,0d.b.02011.0deacetylation0of0chitosan:material0 characterization0and0in%vitro0evaluation0via0albumin0adsorption0and0preqosteoblastic0 cell0cultures0 0 0 0 0 contact : mark ohioghie aihiokhai markaihiokhai@yahoo.com 89 abstract overtime, the state of maternal health in nigeria is poor and can be attributed to inadequate access to reproductive health services, poverty and in some areas, cultural resistance. medicinal plants used in agbarha-otor, ughelli, nigeria for managing women’s reproductive health during pregnancy, birth and post -partum were investigated in this study. surveys were conducted using semi structured questionnaire as well as guided field-walks where respondents, specifically, traditional birth attendants (tbas), were interviewed. a total of 30 plants belonging to 23 families having properties against different conditions, ailments and peculiarities relating to gynaecological conditions in women were documented. most of the documented plants belong to asteraceae family. also, most of the reported plants were majorly herbs and shrubs which were surveye d plants from the wild. leaves were the most frequently used plants’ part. most of the medications were prepared as decoctions and mostly administered orally. from the survey, it can be deduced that the use medicinal plants for reproductive healthcare and gynaecological conditions in the study area is still in practice. scientific validation of the biological properties of the surveyed plants is highly advocated and cultivation of medicinal plants to minimize the pressure on wild species is also recommended. issn : 2580-2410 eissn : 2580-2119 some plants used for reproductive health by women in agbarha-otor community, delta state mark ohioghie aihiokhai1*, macdonald idu2 and thessy arueyingho3 1,3 department of biological sciences, faculty of natural and applied sciences, michael and cecilia ibru university, agbarha-otor, pmb 100, ughelli, delta state, nigeria. 2 phytomedicine unit, department of plant biology and biotechnology, faculty of l ife sciences, university of benin, pmb 1154, benin city, edo state, nigeria introduction healthcare services in nigeria are often inadequate and unevenly distributed with only a small number of healthcare facilities provide antenatal care, delivery and postnatal services. this deficiency has really contributed to the extremely high maternal mortality ratio, which is among the highest in the world. who (2012), reported that maternal mortality ratio in nigeria is 630 per 100,000 live births, which places the country as the country with the tenth highest mortality ratio. nigeria and other five countries account for almost half (49%) of the overall maternal mortality figure worldwide according to hogan et al. (2010). this unprecedented figure cannot be disassociated from the fact that most nigerian women employ the services of alternative traditional healthcare through the services of traditional birth attendants open access international journal of applied biology keyword reproductive health; traditional birth attendants; traditional maternal healthcare; medicinal plants; agbarha-otor article history received may 19, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 90 (tbas) and traditional healers (ths), who inevitably use medicinal plants for maternal healthcare, particularly in rural areas where orthodox health facilities are either absent or extremely difficult to access. the utilization of plants and their resources for combating various ailments predates written history and they are still in use all over the world (abe and ohtani, 2013). aihiokhai et al. (2019) reported that phytochemical analysis of plant extracts has shown the presence of some secondary metabolites which is responsible for their immense medicinal value. however, medicinal plants are widely used for pre and postnatal care in many parts of the world (zumsteg and weckerle, 2007); thus , various studies have documented many medicinal plants used to treat obstetric and gynecological conditions, such as birth control, complications during pregnancy and child birth and problems associated with infertility (abdillahi and van staden, 2013). indigenous people worldwide have used oral traditions and empirical means to compile detailed knowledge regarding the use of medicinal plants, and this information is disseminated from generation to generation (abel et al., 2005). delta state is one of the oil producing states in nigeria, that is among the niger delta states in the country. culturally, the state houses majorly the anioma, urhobo, ijaw, isoko and itsekiri ethnic people. the study area, agbarha-otor community is made up of the urhobo speaking people and it is under the ughelli north local government area of delta state. despite the effort made by the past and present democratically elected governments in building many health institutions across the state, most people of the state still patronise complementary alternative healthcare services through the use of medicinal plants, which are readily available and considered to be relatively safer than the synthetic drugs that are inevitably used in orthodox medicine. the use of these plants to alleviate medical ailments has resulted in the indiscriminate cutting of plant resources, and this is posing a great danger to plant biodiversity in the state particularly, in rural communities, which is already facing threats from deforestation, desert encroachment, and global warming consequences. although people have been using medicinal plants to cure various diseases in agbarha -otor community since time immemorial, though, their usage is rarely documented, and the information is being passed verbally from generation to generation which is associated with the danger of losing some useful information possibly due to aging and death of the custodians of this knowledge (maregesi et al., 2007), urban migration and lack of interest by the younger generation. these challenges gave credence to this study which was carried out in agbarha -otor community in ughelli north local government area, delta state, nigeria. we aimed to produce an inventory of some of the plants used by the people of agbarha-otor community in treating female reproductive conditions and a documentation of how the identified plants are used by the traditional maternal healthcare providers . we adopted a focused approach and carried out a non-experimental validation of the plants used by examining literature on phytochemical/pharmacological information supporting the medicinal activity of any of the documented plants. however, we have defined reproductive conditions to include those that affect reproductive success through pregnancy, delivery and for the prevention of conception. international journal of applied biology, 6(2), 2022 91 materials and methods study area agbarha-otor community in ughelli north local government area of delta state (latitude: 5.53on; longitude: 6.07oe) with a total population of 75,000 people as at last census in 2006. it is one of the largest communities in urhobo land, both in terms of size and population and widely known for its rich crude oil deposits which is evident in the presence of shell petroleum development company (spdc) in the community. agbarha -otor lies within the tropical rain forest area of the niger-delta regions which experiences high rainfall and humidity most of the year. the climate is equatorial and marked by two distinct seasons: dry and wet seasons. the dry season lasts from about november to april and is significantly marked by the cool "harmattan" dusty and haze from the north-east winds. the rainy season spans from may to october with a brief dry spell in august. the community is mainly inhabited by the urhobo people of nigeria with their major occupation being farming, trading and fishing. figure 1. map of study area adapted from: ohwonohwo (2019) data collection and sampling techniques ethnobotanical data were collected between may and august, 2021 from 5 respondents who were selected purposely based on their rich traditional knowledge of plants and their medicinal applications especially for women’s reproductive health concerns. the selected respondents are traditional birth attendants (tbas) who are well known in the community due to their long practice in providing services related to traditional healthcare. international journal of applied biology, 6(2), 2022 92 the tbas were interviewed separately in urhobo language though often times in english language with the help of a semi-structured questionnaire. basically, the questionnaire contained pertinent questions relating to plants used for gynaecological conditions that occur before and after natal periods. the method of cotton (1996) and martin (1995) was employed. prior to any data collection, informed consent was obtained from respondents. they were informed that collected data on their indigenous knowledge will be stored and disseminated for new scientific research and discoveries geare d towards encouraging conservation efforts to saving their medicinal plants. information provided by the tbas were done without objection, except that they wanted their professional interest to be protected by concealing information on the exact formulations and dosages of their products. in the method followed, guided field walks were carried out by accompanying the practitioners during plant collections. plants were shown with detailed information as to type of management (wild/cultivated), availability (common, sporadic, rare) in the area, whether their occurrence is threatened or not, reproductive health conditions treated, plant part(s) used, whether combinations of plants are used to treat any particular ailment or if any single plant is used to treat multiple ailments, condition of plant part used (fresh/dried), other ingredients or additives (if any), preparation methods, routes of administration, noticeable adverse effects of remedies, taboos/beliefs related to collection and use of plants, source of knowledge, and method of indigenous knowledge transfer. it was observed that the medicinal plants collected by the tbas in agbarha-otor community were collected both from nearby areas to the community, as well as considerable distances (more than 5 km) away from the locality. digital pictures of plant samples were taken in the field and samples were collected, pressed and taken to the department of plant biology and biotechnology, university of benin for identification and authentication. the non experimental validation for the documented plant species was carried out using a modified version of method described in lans (2007). this method mainly involved searching scientific literature for phytochemical/ pharmacological data supporting the medicinal use of a plant species. results and discussion results a total of 30 plants belonging to 23 families having properties against different conditions, ailments and peculiarities related to female reproductive health and gynaecological conditions were documented, and their medicinal activity validated nonexperimentally. the conditions were categorised on the basis of when they occur in the female reproductive cycle, that is, during pregnancy (antenatal), at delivery, and after delivery (post-partum) as presented in table 3. international journal of applied biology, 6(2), 2022 93 table 1. routes of administration of herbal preparations routes of administration frequency percentage orally 26 87 topically 4 13 insertion inhalation n = 30 table 2. plant parts used in the herbal preparations plant parts frequency percentage leaf 20 55.6 seed 1 2.8 fruit 4 11.1 root 1 2.8 flower 1 2.8 whole plant 2 5.6 stem bark 7 19.4 n = 36 table 3: some plants used for female reproductive health in agbarha -otor community of delta state, nigeria s/n condition plant species used family parts used preparation and administration 1 contracepti ve persea americana mi l l . lauraceae barks decocti on of s tem bark taken oral l y 2. pos tpartum hemorrhage citrullus lanatus thumb. cucurbi taceae leaves topi cal appl icati on of l eaves poul ti ce on the s ki n international journal of applied biology, 6(2), 2022 94 3 mi s carriage baphia nitida lodd. fabaceae leaves topi cal appl icati on of dri ed ground l eaves to l ower porti on of the abdomen. 4 labour i nducti on pennisetum purpureum schumach poaceae leaves leaves decocti on mi xed wi th l ocal gi n (ogogoro) taken oral l y 5 impotency/i nferti l i ty citrus aurantifolia chri s tm. rutaceae frui ts and l eaves frui ts and l eaves decocti on mi xed wi th pal m kernel oi l and l ocal gi n taken oral l y 6 uri nary tract i nfecti on zea mays sturtevant. grami neae frui t s i l k decocti on of frui t s i l k taken oral l y 7 res us ci tates uncons ci ous pregnant women alchornea cordifolia (schumach. & thonn.) mül l . arg. euphorbi aceae leaves bl ended l eaves appl i ed topi cal l y 8 uterus pos i ti oni ng ocimum basilicum l. lami aceae leaves leaf i nfus i on taken oral l y 9 umbi l i cal cord i nfecti on bryophyllum pinnatum (lam.) oken cras s ul aceae leaves mi xture of l eaf decocti on and l ocal gi n taken oral l y 10 gonorrhea bambusa vulgaris schrad. ex j.c. wendl . poaceae stem local gi n i nfus ed wi th s tem, tobacco and nati ve s al t taken oral l y 11 fetal macros omi a piper nigrum li nn. pi peraceae leaves fres h l eaf i s chewed 12 iron-defi ci ency anaemi a musa paradisiaca li nn. mus aceae frui t, bark bark i s boi l ed wi th bl ack pepper and fi s h and taken oral l y 13 anaemi a lycopersicon esculentum li nn. sol anaceae frui ts frui t decocti on taken oral l y 14 inducti on of l abour and bl ocked fal l opi an tube brassica oleracea li nn. cruci ferae leaves mi xture of l eaf decocti on and l ocal gi n taken oral l y 15 sedati ve for chi l dren hibiscus rosasinensis li nn. mal vaceae leaves leaf decocti on i s us ed to bath i nfant 16 internal bl eedi ng aspilia africana (pers .) c.d. adams as teraceae leaves leaf decocti on taken oral l y 17 induces l abour vernonia amygdalina del . as teraceae leaves leaves are extracted wi th l ocal gi n and taken oral l y 18 inhi bi ts mens truati on duri ng pregnancy newbouldia laevis (p. beauv.) seem. bi gnoni aceae stem bark gri nded s tem bark i s boi l ed wi th cameroon pepper (yel l ow pepper) and taken oral l y 19 mi s carriage musanga cecropioides r.br.ex tedi l e. urti caceae seeds ,l eaves and root infus i on of gri nded whol e pl ant parts taken oral l y international journal of applied biology, 6(2), 2022 95 20 mi s carriage magnifera indica li nn. anacardi aceae stem bark stem bark are boi l ed and admi ni s tered oral l y 21 anaemi a and reduce naus ea and morni ng s i cknes s duri ng pregnancy musa acuminate col l a. mus aceae stem the s tem i s cut when rotten, ground and i nfus ed i n water 22 mi s carriage laportea aestuans (l.) chew urti caceae leaves decocti on of bark, tobacco l eaf and bl ack pepper taken oral l y 23 headaches and s tabi l ises s ugar l evel duri ng concepti on curcuma longa l. zi ngi beraceae leaves leaves decocti on wi th s ens i ti ve pl ant (mimosa pudica) admi ni s tered oral l y 24 mi s carriages and aborti ons avicennia africana p.beauv. acanthaceae leaves , s tem and fl ower fl owers and l eaves decocti on mi xed wi th pal m kernel oi l and l ocal gi n admi ni s tered oral l y 25 pregnancy nouri s hment talinum triangulare (jacq.) wi l l d. tal i naceae leaves decocti on of l eaves taken oral l y 26 contracepti on jatropha gossypiifolia l. euphorbi aceae leaves leaves decocti on mi xed wi th l ocal gi n taken orally 27 general wel l bei ng ipomoea aquatica fors s k. mal vaceae whol e pl ant water i nfus i on of powdered pl ant taken oral l y 28 mi s carriage, l abour i nducti on and mens trual cramps corchorus olitorius l. logani aceae leaves leaves decocti on taken oral l y 29 sti l l bi rths and anaemi a spigelia anthelmia l. as teraceae leaves local gi n extracti on of l eaves taken oral l y 30 general wel l bei ng ageratum conyzoides l. convol vul aceae whol e pl ant the whol e pl ant i s extracted wi th l ocal gi n and admi ni s tered oral l y international journal of applied biology, 6(2), 2022 96 fig 2. frequencies of families of medicinal plants discussion documentation of ethnomedicinal knowledge is a critical issue owing to its enormous potential to enhance research and development. available literature consulted shows that, the use of ethnomedicinal plant to treat various ailments, particularly, female re productive conditions were not documented in the study area. hence, this study adopted an approach focusing on plants used for female reproductive conditions in agbarha -otor community. during the field survey, respondents, specifically, traditional birth a ttendants (tbas) reported ethnomedicinal data on 30 species of plants (table 3) distributed across 23 families (fig. 2) as having properties against different conditions, ailments and peculiarities related to the women’s reproductive healthcare. among the families reported, asteraceae was represented with the highest number of plant species in conformity with some studies carried out in a locality of cameroon (adjanohoun et al. 1996; focho et al., 2009; noumi, 2010; simbo, 2010). these plant families are among the most commonly seen in south western nigeria used in the treatment of infertility and sexually transmitted diseases (abo et al ., 2000). also, the vast utilisation of species from these families might relate to the presence of effective bioactive secondary metabolites that work against reproductive health–related infections (cowan 1999; gazzaneo et al. 2005; kamatenesi-mugisha and oryem-origa, 2007; kothale et al. 2011; néné bi et al. 2009). most medicinal plants used in the area were majorly herbs, which agrees with results from other studies in nigeria (agize et al. 2013), cameroon (adjanohoun et al . 1996; jiofack et al. 2010; simbo 2010), uganda (kamatenesi–mugisha et al. 2007), ivory coast (djah and danho, 2011), ethiopia (megersa et al., 2013), and democratic republic of congo (kasali et al. 2014). this could imply that herbs are usually more readily available than shrubs and trees that are often prone to regular harvesting from forest patches far away from residential areas. it could also be due to the fact that the respondents live in areas where herbs abound. giday 0 0,5 1 1,5 2 2,5 3 3,5 f re q u e n c ie s families of medicinal plants https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4927590/#cr1 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4927590/#cr28 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4927590/#cr59 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4927590/#cr68 international journal of applied biology, 6(2), 2022 97 et al. (2003) reported that the zay people in ethiopia derive their medicine from herbs partly because of the fact that forests have been degraded, whereas it usually takes much more time and effort to harvest material from medicinal trees. it is true that herbs can grow everywhere (roadside, home garden, farmland, and in wild habitats) and are common in the study area compared with other perennial life forms such as shrubs, trees, and climbers. on the other hand, the present survey is in conflict with the findings of diame (2010), which was conducted on plants used for reproductive health at bia biosphere reserve in the western region of ghana. this survey is also in conflict with the findings of yineger and yewhalaw (2007), who reported that trees and shrubs were the most frequently used growth forms for remedy preparation. it was evident that oral (87%) route was the most used method of administration since the majority of the extracts were alcoholic or aqueous and acceptable through the mouth, while topical (13%) application was the least means of application of the herbal formulation (table 1). leaves were the most frequently used plant part in the preparation of drugs in the study area (table 2). they were administered directly in the form of decoctions, and in few cases applied externally or topically. okoegwale and omefezi (2001) indicated that leaves of plants accumulate inulins, tannins, and other alkaloids more than other parts of plants which may be responsible for their various medicinal properties, hence explaining their wide use. according to our results, the majority of herbal remedies in the research area were prepared from fresh materials. other ethnobotanical inventories (bussmann and glenn, 2010; yineger et al., 2007) have also indicated the wide use of fresh plant material for remedy preparation probably because of the higher efficacy than when using dri ed plant materials. this is because some important chemicals may change in nature or even disappear upon drying. also, the dominant use of medicinal plant decoctions for various ailments associated with the reproductive health system might be related to their proven effectiveness over many years of trial and indigenous knowledge accumulated on the efficacy of such preparations. on the other hand, the frequency of this method of preparation by a majority of respondents may be due to the fact that boiling the ingredients will kill some unwanted microbes that are present on the plant material used (souad et al. 2010; ugulu et al. 2009). however, heat facilitates extraction of active compounds from the plant part that is an ingredient in the remedy (souad et al. 2010). decoction also preserves the prepared medicine longer (muthu et al. 2006). the majority of medicines in the area were administered orally (table 1). similar findings were reported by other researchers (bhattarai et al . 2010; kamatenesi et al. 2011). the choice of oral administration over possible alternatives may be related to the use of some solvents (water, palm oil, or local wine or gin called ogogoro) that are commonly believed to serve as a good vehicle to transport the remedies’ active princ iples. as already indicated, most medicinal plants in the study area are collected for their leaves, such as citrullus lanatus, pennisetum purpureum, alchonea cordifolia, hibiscus rosa-sinensis, laportea aestuans, curcuma longa and so on (table 3). this practice helps to reduce the pressure on these species compared to what would happen if bark or roots were collected. from the overall survey, it could be deduced that the high level of consensus among the tbas about the use of medicinal plants for reproductive healthcare and gynaecological conditions prevalent in the study area suggests that the ethnomedicinal uses of these plants is still in practice. however, scientific validation of the biological properties of the surveyed plants is highly advocated and cultivation of medicinal plants to minimize the pressure on wild species is also recommended. international journal of applied biology, 6(2), 2022 98 references abpsd. 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(1994). factors influencing the amount and distribution of leaf area of pine. copenhagens: ecological bulletins 43. contact : mardiana ethrawaty fachry mardianaethra@gmail.com 79 abstract poverty is still a problem in urban areas. limited skills and employment have caused women from low-income families to also seek work as scavengers or collectors of waste and used goods to help support their family's economy. this study aims to find the characteristics of female scavengers in responding to poor women fulfilling their family's economy. the involvement of children under five in the scavenging process is also a review in finding the distribution of gender roles in scavenger households. this research was conducted in makassar city using a qualitative approach. data were obtained from 25 families of female scavengers. the study found that women in scavenging activities involved children between the ages of 1 and 5 years, which was a domestic role and a strategy to get additional income from the community's compassion. scavenger women make an economic contribution to the family between 40% and 77%. the involvement of children under five in the scavenging process is a form of child exploitation which has yet to become the attention of the makassar city government. issn : 2580-2410 eissn : 2580-2119 strategy for women scavengers in fulfilling the family economy in makassar city (the case of scavengers involving toddlers in urban areas) nur ikhsan1, mardiana ethrawaty fachry2*, mansyur radjab3, herawaty abbas1, fatmawati1, sitti bulkis1 1 graduate school hasanuddin university, south sulawesi indonesia 2 faculty of marine sciences and fisheries, department of socio-economic hasanuddin university, south sulawesi indonesia 3 faculty of social and politic, department of sociology, hasanuddin university, south sulawesi indonesia introduction poverty is a problem in urban areas. makassar city statistical data shows an increase in poor people of around 4.17%, or 74,690 people, over the past three years (bps, 2021). this is caused by various factors such as lack of employment, low skills and education and the presence of covid at the end of 2019 (fikri & gopar, 2021). the impact of the pandemic is tremendous on the economic role of women. around 53.79% of msmes in indonesia are owned by women, with 97% of the workforce being women (azizah, 2022). economically, the contribution of women to the gross domestic product (gdp) is 61%. even so, unemployment in indonesia will still reach 8.42 million in 2021. open access international journal of applied biology keyword the role of women; scavengers; toddlers; economic contribution article history received october 3, 2022 accepted december 28, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(2), 2022 80 the development of people who work as scavengers is one of the results of poverty in big cities, including makassar. the number of scavenger members of the indonesian scavenger association (ipi) in 2019 reached 3.7 million members from 25 provinces. there are still five provinces that still need to be recorded. this number also does not include unrecorded scavengers. the involvement of women in the household economy is a productive role that has become part of life in urban areas (roswiyanti, et al. 2022). increased family needs are the primary basis for women to work apart from carrying out their functions in domestic and social aspects (telaumbanua & nugraheni, 2018: fachry & alwi, 2018). women (wives) mainly work because their husband's income is insufficient (zunaidi & maghfiroh, 2021: kauntu and suraya, 2018). in addition, limited skills, education and capital make them enter the informal sector, such as becoming scavengers. jobs that deal with garbage, dirty, full of flies and maggots they ignore for the sake of getting money. therefore, scavenging is the job rated the lowest, without skills and only done by those who do not have access to other jobs. collecting waste from used goods that can still be used for sale is a job that is starting to develop and is being carried out by many poor women in makassar city. one characteristic of their activities is that they involve children aged under five using carts or some means of conveying goods when collecting or scavenging. involving toddlers in productive activities is a strategy for scavenger families to get money from the community. this can be seen in the increasing number of young children as beggars or vendors of various food products on the streets (yuniarti, 2013: hasanah & amir, 2020). scavenging involving children under five is part of the behaviour of adult beggars who use children to get money, which will create street children as breadwinners. street children become urban communities and become one of the characteristics of poverty in urban life. this research describes the life of scavenger women in their contribution to the family economy by involving children to maintain the sustainability of life in makassar city. materials and methods the research was conducted in panakukang district, makassar city. this study uses a qualitative descriptive approach with a type of phenomenology (moleong, 2010) which aims to understand the behaviour of women's scavenger families involving toddlers in earning income. sampling by purposive sampling and snowball. in the snowball technique, at first, the researcher randomly selected informants and then headed to the next informant with the help of the first informant. the total number of informant families selected was 25 families consisting of female scavengers and scavenger husbands. data analysis used the interactive model milles and huberman (2014). that is analyzing the data through three steps, namely first, condensing data (data condensation), secondly presenting data (data display), and thirdly drawing conclusions or verification (conclusion drawing and confirmation). data condensation refers to selecting, focusing, simplifying, abstracting, and transforming data. international journal of applied biology, 6(2), 2022 81 table 1. primary data collection matrix on female scavenger families no informant/ respondent variable method of collecting data data analysis 1. scavenger woman • characteristics of female scavengers • background as a scavenger bringing a toddler • domestic and public roles • expenditures of scavenger households • family economic contribution deep interview using a guide interactive model of data analysis (miles & huberman, 2014) 2. the husband of a female scavenger • husband's characteristics scavenger • domestic and public roles • income/support for the family economy deep interview interview guide and observation sheet results and discussion the monetary crisis that occurred in 1998 had an impact on all sectors, especially the economy, which caused unemployment everywhere and poverty. the poverty that occurs causes the emergence of social phenomena, one of which is the increase in the number of workers in the informal sector, such as beggars, homeless people and street children. according to dimas (2013), the factors that cause begging activities are entangled in economic problems. meanwhile, according to suhandi & erlita (2021), the factors that cause someone to beg are due to the powerlessness experienced by people who have difficulty meeting their family needs. because they cannot earn income and become a group that cannot get out of the poverty cycle. the poor have no hope of progressing and developing due to their lack of education, skills and an impact on business motivation which can become habitual and entrenched. the inheritance of the culture of poverty will be more lasting because one of the characteristics of culture is that it can be learned (koentjaraningrat, 2002). scavengers are an example of informal sector activities in urban areas. scavengers collect used goods because of demand from industries that recycle used materials (etnayanti et al., 2020), and the amount of waste in urban areas is more significant than in villages. this is directly proportional to the dense urban population (fachry & alpiani, 2021). the presence of scavengers is based on their previous unfavourable work experience due to lack of income and difficulty finding a decent job. thus there is a tendency for the background of women scavengers to be similar, such as the husband's non-permanent job (daily labourer), reasons for taking toddlers along for scavenging, and the size of the contribution to the family's economy. international journal of applied biology, 6(2), 2022 82 characteristics of female scavengers distinguishing factors such as age, education and family responsibilities support someone in choosing a job. according to selvia aprilyanti (2017), age greatly influences the work productivity of the productive period. usually, younger people have a higher level of productivity than older workers because they are physically weak and limited. the results of the study describe that female scavengers and their husbands are at a productive age and are still relatively young, namely 23 to 45 years with an education level of 32% not completing elementary school, 48% completing elementary school and 20% junior high school. this is why it is difficult for scavenger families to access decent jobs. the family dependents, on average, are three people. this amount is quite a burden for low-income families, which can result in children needing help to continue their education and become successors to their parents' work as scavengers and daily labourers. this is what is intended as the inheritance of a culture of poverty. therefore it is essential to have government intervention or nongovernmental organizations to empower the children of scavengers. one of the existing policies is regional regulation no. 2 of 2008 concerning fostering street children, homeless people, beggars and buskers using street children approach models. however, this policy was made in the context of creating order and reducing public anxiety due to acts of violence that have the potential to be committed by homeless children and beggars, so that it does not touch on efforts to empower children and their families. scavenger women and the family economy the involvement of women in economic activities in helping to provide for the family occurs not only in low-income families but also in middle-income families due to increasingly complex needs. in addition, the role of women in the productive sector is an actualization of women's roles besides the domestic part. this role has shown that women have made a significant contribution to their families and the economic development of society (susanti & patonah, 2020). this is in line with sari & anwar (2019), who found that women's involvement in earning a living is not only to meet economic needs but as a desire to work. based on the results of the study, it was found that the main reason for women to become scavengers was economic factors (68%). the husband's income as daily work was insufficient, and the rest was due to the inability to access work due to a lack of skills and low education (12%). apart from that, being a scavenger is also caused by having a job since childhood because of the influence of the environment, so children's character is formed to do what they usually see in their environment (muhtazib et al., 2022). following his parents' work (12%), as shown in the following table. table 2. reasons for women to become scavengers and involve toddlers reasons to be a scavenger total percentage fulfilling family needs 17 68,0 no jobs can be accessed 3 12,0 lacks the skills for a better job 2 8,0 been scavenging since childhood 3 12,0 total 25 100 sources: primary data processed in 2021 international journal of applied biology, 6(2), 2022 83 it was also found that to decide to work as scavengers, respondents made observations and sought information from other scavenger workers. decisions are made through an awareness and assessment process regarding the amount of income to be earned, the time to be used, the location and distance of scavenging activities to the risks that will occur if you become a scavenger. this process is an individual's choice influenced by other individuals or groups to become part of the work as a scavenger. the study's results found that 64% got information from friends, 24% from husbands and the rest from their own experience (12%). this illustrates that external information is very influential in decisionmaking by scavengers. the income earned from scavenger work is still being determined because it depends on the availability of waste or used goods at the search location. plastic and cardboard, as well as bottles that are still fit for sale, are objects of scavenging activities. the price of junk goods varies; for example, plastic bottles per kilogram at the collector level range from rp. 2,000 to rp. 3,000. plastic bags cost rp. 250 to rp. 1,500, and various bottles can reach rp. 3,500 to rp. 6,000. the results of this scavenging are considered sufficient to help the family's economy, in addition to the income from husbands who generally work as non-permanent daily labourers. meanwhile, the income of scavenger families can be averaged in a month between rp. 1,200,000 to rp. 2,050,000. from the expenditure aspect, scavenger households have the largest composition of expenditure on basic needs, namely consumption (28.5%) and the needs of toddlers to purchase milk and snacks (24.4%), housing costs (23.7%) and education costs ( 9.0%). in addition, scavenger families have loans from other scavengers or used goods collectors (13.3%), which illustrates the apprehensive economic conditions. the contribution of scavenger women to the family economy the significant role of women scavengers in the family economy is a form of women's strategy in supporting the sustainability of their household life. this is closely related to the work of scavenger husbands as non-permanent daily workers. so that in a month, there may be no income that the scavenger husband gets. therefore scavenging is a woman's strategy to support her family's economy. women's contribution to the family economy ranges from 31.7% to 77.7%, as shown in the following table. table 3. the contribution of women scavengers to the household economy average income husband (rp.)/month income scavenger (rp.)/week total income of scavenger family (rp./week) woman scavenger contribution (%) total respondents 1.400.000 650.000 2.050.000 31,7 8 1.000.000 720.000 1.720.000 72,0 11 800.000 600.000 1.400.000 75,0 33 650.000 500.000 1.200.000 77,7 2 sources: primary data processed in 2021 scavenger woman strategy the size of the family's dependents and routine expenses that must be incurred every month, such as consumer needs, house rent, electricity and water costs, are demanding to be met. women scavengers use some strategies to get money, namely working from 06.00 to 22.00 at night so that the number of used goods worth selling can be obtained. including international journal of applied biology, 6(2), 2022 84 toddlers in garbage carts to get the community's mercy (100%) and 84% borrow money from fellow scavengers or collectors with the obligation to repay the loan per day with an additional of around 5 to 10%. this is by the concept of a survival strategy as a series of actions that are chosen standardly by individuals and households who are middle to lower socioeconomically. through a strategy carried out by a person, it can increase income by utilizing other sources or reduce expenses by reducing the quantity and quality of goods or services. in addition, the survival strategy applies a double income pattern, which is part of the economic strategy and happens a lot by borrowing money. in low-income families with various limitations and conditions that are very risky to change, a behavioural strategy and a balancing/adaptation strategy are a form of dealing with the pressures of change. the results of the analysis also show that women scavengers, in the face of limited education, skills and economy, can choose scavenger work as a strategy for earning income. the strategy related to domestic activities is carried out by using carts to include children under five, which is also a medium for getting money from the community's compassion. conclusion from an economic point of view, scavengers are informal sector jobs that generate significant income, especially for low-income families who cannot work in other formal or informal sectors due to limited education and skills. the contribution of women scavengers to the family economy is quite significant, reaching 40% to 77%. the strategy of 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[in indonesian] https://doi.org/10.33007/inf.v4i2.1474 https://doi.org/10.15294/komunitas.v4i2.2416 contact : oluwafemi emmanuel ekun oluwafemi.ekun@aaua.edu.ng 101 abstract peptide base d antioxidant s from plant and animal pr oteins are being identifie d as food additives and also as potential alternatives in the reduction of oxidative stress. this study inve stigated the antioxidative potentials of pe ptide digests of albizia lebbeck seed pr otein. the proteins were extracted and isolated from a. lebbeck by de fatting with n-hexane, followed by alkaline solubilization and acid precipitation of the seed meal. the protein isolate was then subjected to enzymatic hydrolysis using four pr oteases – pepsin, trypsin, papain a nd chymotrypsin. the resulting hydrolysates were the n evaluated for the ir abilities to reduce ferric ions, as we ll a s their effects on hydroxyl radicals, 1,1-diphe nyl-2picrylhydrazyl (dpph) and superoxide radicals. hydrolysates obtaine d fr om peptic prote olysis demonstrated the best activitie s against dpph radical a nd ferric ions (57.589 ± 1.286% and 52.000 ± 0.589 mm fe2 + respectively), whereas chymotrypsin hydrolysates scave nged super oxide radicals and hydroxyl radicals better than other protein digests (74.520 ± 0.998% and 36.925 ± 1.880% respectively). the choice of enzyme use d and the pre sence of specific amino acid residue s at certain positions of pe ptides in the hydr olysates influence d their antioxidant capacities. it is conclude d that albizia lebbeck seed proteins enc ode potentially bioactive pe ptides, w hich c ould be harne ssed for numerous therapeutic and nutraceutical benefits. issn : 2580-2410 eissn : 2580-2119 antioxidant properties of albizia lebbeck seed protein hydrolysates oluwafemi emmanuel ekun 1* 1 department of biochemistry, faculty of science, adekunle ajasin university, akungba akoko, ondo state nigeria. introduction free radicals have continuously played roles as both toxic and beneficial compounds in cellular respiration, immune response, drug detoxification, among other physicochemical processes (pham-huy et al., 2008). they are produced f rom the oxidation of biomolecules in the body through metabolism in vivo or from exposure to envi ronmental pollutants, toxicants and other xenobiotics. when there is an apparent shortfall in the ability of the body’s detoxification systems to combat an excess of these oxidants, they accumulate in the body, causing a state referred to as oxidative stress (majhenic et al., 2007). oxidative stress generally describes a state of imbalance between the systemic generation of reactive oxygen and nitrogen species, and the ability of a biological system to quickly neutralize these reactive compounds or to repair the damage caused (chandra et al., 2015). perturbations in the normal cellular redox states can give rise to toxic effects such as dna strand breakage, nucleotide base damage, erythrocyte lysis, among other deleterious open access international journal of applied biology keyword albizia lebbeck; protein; hydrolysate; peptides; antioxidant article history received may 21, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. mailto:oluwafemi.ekun@aaua.edu.ng international journal of applied biology, 6(2), 2022 102 effects (chandra et al., 2015). this is thought to occur through the production of free radicals such as singlet oxygen, peroxides, superoxides, that damage cellular components such as lipids, proteins and dna. furthermore, some oxidative species act as intracellular messengers in redox signaling; and as a result, oxidative stress can cause disruptions in normal mechanisms of cellular signaling. in humans, oxidative s tress is involved directly and/or indirectly in the progression of several diseases, which include diabetes mellitus (olusola et al., 2018), cancer (halliwell, 2007), parkinson’s disease, alzheimers disease (valko et al., 2007, pohanka, 2013) atheroscleros is, myocardial infarction (dean et al., 2011; ramond et al, 2011) among others. it is important that natural antioxidants are utilized as additives and supplements for the reduction of oxidative stress due to disease progression (bains and hall, 2012). as a result, attention has turned to harnessing newer and previously underutilized plant sources for their antioxidant potentials. specifically, protein hydrolysate prepa rations and pepti des from previously underutilized plants are being investigated for their bi ological activities. peptides obtained from hydrolysis of plant and animal proteins have been identified as safer alternatives to synthetic antioxidants. the increased interest of antioxidant peptides obtained from these sources is as a result of their potential roles as dietary supplements (ajibola et al., 2011: olusola et al., 2018). peptide antioxidants have simpler structures than their parent proteins. this enhances greater stability in different situations (heat, protease activities), they elicit no immunoreactions and often ex hibit enhanced nutriti onal and functional properties in addition to their antioxidant activity (xie, et al., 2008). for most peptides, the ability to scavenge free radicals is mostly dependent on the peptide size, with peptides of low molecular weight being more effective than those of larger molecular weight. (alashi et al., 2014). one of the plants whose proteins and peptides have not been previ ously investigated is albizia lebbeck. albizia lebbeck is a large, deciduous, drought – resistant, leguminous tree belonging to the mimosaceae family. it is commonly known as the siris tree or lebbeck tree. it is alternatively known as “women’s tongue” because of the rattling sound its mature seed-containing pods make when the wind blows (verma et al., 2013). it is relatively ubiquitous as it grows well in a wide diversity of regions. it is native to a number of areas especially in the forests of sub – saharan african and asian countries. a. lebbeck is a plant whose leaves, pods, seeds, stem bark and roots are excellent sources of bioactive phytochemicals and other secondary metabolites of pharmacologic significance (zia-ul-haq et al., 2013; verma et al., 2013). its leaves contain flavonoids, saponins, tannins, alkaloids and cyanogenic g lycosides (bobby et al., 2012) just as its roots are reservoirs of lupeol, stigmasterol and echinocystic acid (verma et al., 2013; musa et al., 2020). the seeds of a. lebbeck are rich sources of minerals such as calcium, iron, magnesium and potassium; phyt onutrients which include ascorbic acid and niacin; as well as essential fatty acids (verma et al., 2013). zia-ul-haq and others (2013) found out that that its seeds contained 34.17% c rude protein, 49.07% carbohydrate and 5.62% c rude fibre. its high protein content makes it a good source of potentially bioactive peptides. proteins of a. lebbeck are rich in arginine, lysine, histidine, leucine, isoleucine, phenylalanine, valine, tyrosine, aspartate, glutamate, but limi ting in methionine and cysteine (zia -ul-haq et al., 2013). parts of the plant have been used as feed for rumina nt livestock (hassan et al., 2007) and in traditional medicine (chulet et al., 2010). its leaves have been reported to possess antimicrobial, anticancer, anti-diarrheic activities (bobby et al., 2012) its stem bark extracts have also been reported to demonstrate anti-parasitic potentials, and its use in treating dental infections have been documented (qadri et al., 2005; umar et al., 2009). despite its numerous biological activities, the antioxidative capacities of hydrolysate preparations of its international journal of applied biology, 6(2), 2022 103 proteins have not been evaluated. as a result, this study is geared towards the antioxidant potentials of albizia lebbeck seed protein hydrolysates. materials and methods materials collection of seeds a. lebbeck seeds were obtained from its trees in the botanical garden of adekunle ajasin university akungba akoko, ondo state. nigeria. they were subsequently identified and voucher samples were deposited at the department of plant science and biotechnology, adekunle ajasin university, akungba akoko. chemicals and reagents enzymes: pepsin (from porcine gastric mucosa), trypsin (from bovine panc reas), phymotrypsin (from bovine pa ncreas), papain (from carica papaya) were products of sigmaaldrich laboratories, co-artrim, united kingdom. other reagents: 1-diphenyl-2-picrylhydrazyl (dpph), ascorbic acid, trichloroacetic acid (tca), potassium ferricyanide, ferric chloride, pyrogallol, hydrogen peroxide, ethylene diamine tetraacetic acid (edta) these were a lso products of sigma-aldrich laboratories, coartrim, united kingdom. all chemicals and reagents used were of analytical grade. methods isolation of a. lebbeck seed proteins the seeds were dried, pulverized and kept in a dry container at 4oc. they were defatted using n-hexane as described by olusola et al.,(2018). the meal was extracted twice with n-hexane using twenty grams (20 g) of seed meal suspended in 200 ml of n-hexane. the meal was then dried at 40oc in a vacuum oven and ground again to obtain a fi ne powder, termed defatted seed meal, which was stored at -10oc. the protein component of the defatted meal was extracted using the method reported by ekun et al.(2022). defatted a. lebbeck seed meal was suspended in 0.5 m naoh ph 12.0 at a ratio of 1:10, and stirred for one hour for the purpose of alkaline solubilization, using a magnetic stirrer. this was centrifuged at 18°c and 3000 g for 10 min. one additional extraction of the residue from the centrifugation process was performed with the same volume of 0.5 m naoh and the supernatants were pooled. the ph of the supernatant was adjusted to 4.0 to facilitate acidinduced protein precipitation using 0.5 m hcl solution; the precipitate formed was recovered by centrifugation. the precipitate was washed with distilled water, adjusted to ph 7.0 using 0.1 m naoh, freeze-dried and the protein isolate was then stored at -10°c until required for further analysis. preparation of a. lebbeck seed protein hydrolysates the protein isolate was hydrolysed using the methods described by olusola and ekun (2019) with slight modifications. the conditions for hydrolysis were optimized for each enzyme for maximized activity. hydrolysis was performed using each of pepsin (ph 2.2, 37ºc), trypsin (ph 8.0, 37ºc), chymotrypsin (ph 8.0, 37ºc and papain (ph 6.0, 60ºc). the protein isolate (5% w/v) was dissolved in the appropriate buffer (phosphate buffer, ph 8.0 for trypsin and chymotrypsin, glycine buffer, ph 2.2 for pepsin, phosphate buffer, ph 6.0 for papain). the enzyme was added to the slurry at an enzyme-substrate ra tio (e:s) of 1:50. digestion was performed at the specified conditions for eight (8) hours. the enzyme was international journal of applied biology, 6(2), 2022 104 then inactivated by boiling in water bath (100oc) for fifteen (15) minutes and undigested proteins were precipitated by adjusting the ph to 4.0 with 2 m hcl/2 m naoh followed by centrifugation at 7000 g for 30 min. the supernatant containing the peptides were then collected. the protein content of samples were determined using biuret assay method with bovine serum albumin (bsa) as standard. determination of dpph radical scavenging activity the dpph (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity was measured by using the assay method described by arise et al.(2016a) with slight modifications. 1 ml each protein hydrolysate at different concentrations (0.5 – 2.5 mg/ml) was added to 1 ml 0.1 mm dpph dissolved in 95 % ethanol. the mixture was shaken vigorously and incubated in the dark and at room temperature for 30 min. the absorbance was read at 517 nm. ethanol (95 %) was used as a blank. the control solution consisted of 0.1 ml of 95 % etha nol and 2.9 ml of dpph solution. analyses were carried out in triplicates. percentage inhibition of ddph radical was calculated as follows: % dpph inhibition = (abs control abssample) x 100 / (abscontrol) ec50 values were estimated f rom percentage inhibiti on pl ot, using a non-linear regression plot. determination of ferric reducing antioxidant property the reducing power of the protein hydrolysates was determined according to the method reported by olusola et al.(2018) with slight modification. an aliquot of 1 ml of different concentrati ons (0.5 – 2.5 mg/ml) of the hydrolysates (0.2 m pbs, ph 6.6) was mixed with 1 ml of 1% potassium ferric cyanide solution. the mixture was then incubated at 50°c for 30 minutes followed by the addition of 1 ml 10% (w/v) tca. 1 ml of the incubation mixture was added with 1 ml of distilled water and 0.2 ml of 0.1% (w/v) ferric chloride in test tubes. after a 10 min reaction time, the absorbance of the resulting solution was read at 700 nm. higher absorbance suggested stronger reducing power. ascorbic acid was used as the reference anti oxidant. an aqueous solution of known fe(ii) concentrations (feso4·7h2o; 2.0, 1.0, 0.5, 0.25, 0.125 mm) were used for calibration. results were expressed as mm fe2+/mg hydrolysate. all the tests were performed in triplicate. determination of hydroxyl radical scavenging activity determination of the ability of the hydrolysates to prevent fe2+/h2o2 induced deoxyribose decomposition was carried out using the method described by ogunwa et al.(2016) with some modifications. the reaction mixture contained 120 l of 20 mm deoxyribose, 400 l of 0.1 m phosphate buffer ph 7.4, 40 l of 20 mm hydrogen peroxide and 40 l of 0.5m i ron (ii) tetraoxosulphate (vi). the hydrolysates were added to the mixture. distilled water was then added to make the mixture volume up to 800 l. this was incubated at 37oc for 30 minutes. the reaction was stopped by the addition of 0.5ml of 2.8% thiobarbituric acid, tba. the reaction tubes were inc ubated in boiling water f or 20 mins, after which they were centrifuged at 3000g for 10 minutes. the absorbance was taken at 532 nm using a spectrophotometer. percentage inhibition of the hydroxyl radical was calculated as follows: % oh inhibition = (abs control abssample) x 100 / (abscontrol) international journal of applied biology, 6(2), 2022 105 determination of superoxide radical scavenging activity the method described by xie et al. (2008) was used to determine the abilities of the hydrolysates to scavenge the superoxide radical. samples were each dissolved in 50 mm tris–hcl buffer, ph 8.3 containing 1 mm edta and 80 μl was transferred into a clear bottom microplate well; 80 μl of buffer was added to the blank well. this was followed by addition of 40 μl 1.5 mm pyrogallol (dissolved in 10 mm hcl) into each well in the dark and the change in the rate of reaction was measured immediately at room temperature over a period of 4 minutes using a microplate reader at a wavelength of 420 nm. ascorbic acid was used as control. the superoxide scavenging activity was calculated using the following equation: superoxide scavenging activity (%) = (δabs/minb – δabs/mins)/δabs/minb x 100 where b and s are blank and sample, respectively. statistical analyses results were expressed as mean of triplicate observations ± standard error of mean. the data were statistically analyzed using one way analysis of variance (anova) and duncan’s multiple range tests. differences were considered statistically significant at p<0.05 using graphpad prism version 6.0 (graphpad software, san diego, ca, usa). results and discussion dpph radical scavenging activity the abilities of a. lebbeck seed protein hydrolysates are illustrated in figure 1 and their values of 50% inhibition are also displayed in figure 2. all peptide digests demonstrated significantly (p<0.05) reduced dpph scavenging activities when compared to the control, ascorbic acid. among the hydrol ysates, peptide digests obtained from peptic hydrolysis had the highest (p<0.05) dpph scavenging activities at all the studied concentrations, reaching a maximal effect of 57.589 ± 1.286 % at a final concentration of 2.5 mg ml-1. it had the lowest (p<0.05) ec50 value (1.075 ± 0.021 mg ml-1) among the peptide digests. this was comparable to 56.39 ± 0.45% obtained for peptic hydrolysates of citrullus lanatus seed proteins (arise et al., 2016a), and higher than 48% for peptic digests of yellow fin protein hydrolysates (naqash & nazeer, 2013). the potential bioactivity of a peptide released from enzymatic hydrolysis has been found to be dependent on factors such as nature of proteolytic enzyme utilized, hydrolysis time, as well as identity and position of amino acid residues in the peptide (udenigwe & aluko, 2011; ekun et al., 2022). pepsin being an enzyme which cleaves polypeptides at c -terminals of hydrophobic and to a lesser extent, acidic residues may have cleaved the proteins into several dissimilar peptides with the required ami no acid residues to donate protons to, and consequently scavenge the dpph radical. at higher concentrations, chymotrypsin hydrolysates demonstrated higher (p<0.05) scavenging activities than tryptic and papain digests, displaying an a bility of 37.385±0.887 % at a final concentration of 2.5 mg ml-1. this could be that the aromatic aminoacyl residues (tryptophan, tyrosine, phenylalanine) released during proteolysis took part in scavenging the dpph radical. this is consistent with the report of wu et al. (2011) who reported that, even free aromatic amino acids, tryptophan and tyrosine from egg yolk have antioxidant potentials in foods. international journal of applied biology, 6(2), 2022 106 figure 1. dpph radical scavenging activity of a. lebbeck seed protein hydrolysates bars are expressed as means ± standard error of mean of triplicate determi nations (n=3). compa risons are made among samples of the same concentration only. values with different letters are significantly different from one another. bars carrying the same l etter or symbol are not significantly different (p<0.05). figure 2. fifty percent effective concentration values (ec50) of dpph radical scavenging activity of a. lebbeck seed protein hydrolysates bars are expressed as means ± standard error of mean (sem) of triplicate determinations (n=3). bars with the same letters do not differ significantly while bars with different letters are significantly different (p<0.05) from one another. international journal of applied biology, 6(2), 2022 107 ferric reducing antioxidant property the relative abilities of a. lebbeck seed protein hydrolysates in reducing the ferric ion, in comparison to ascorbate, are depicted in figure 3. all hydrolysates had significantly lower (p<0.05) reducing power when compared to ascorbic acid (control). they also demonstrated a concentrationdependent increase in reducing power. this is consistent with a previous study (razali et al., 2015) which revealed a similar trend of results. the low ferric-reducing property of a. lebbeck seed protein hydrolysates when compared with ascorbate may be attributed to the relatively low a mount of sulfur-containing aminoacyl residues in the hydrolysates, which would have otherwise increased antioxidative activity by donating protons to ferric iron in the reaction medium (lopez-barrios et al., 2014). however, peptic digests exhibited significantly higher (p<0.05) reducing capacities than other hydrolysates, as it converted a maximum of 52.000 ± 0.589% mm of fe3+ to fe2+ at a final concentration of 2.5 mg ml-1. this was comparable to the reducing abilities of peptic digests of moringa oleifera seed proteins (olusola et al., 2018) but higher than those of peptic hydrolysates obtained from cowpea seed proteins(olusola and ekun, 2019). udenigwe and aluko (2011) previously reported that acidic amino acid residues are moderately strong contributors to the reduction of ferric ions. this could be the reason why peptic hydrolysates still exhibited high reducing antioxidant power, even with limiting amounts of sulfur – containg aminoacyl residues such as cysteine and methioni ne. tryptic hydrolysates also reduced 22.483 ±2.265 mm of fe3+ to fe2+ and these were higher (p<0.05) than the reducing properties of chymotryptic and papain hydrolysates, and this is consistent with previous studies on unfractionated hydrolysates of watermelon seed proteins (arise et al., 2016a), and this suggests the presence of some other aminoacyl residues within the peptides, which may have contributed to the reduction of ferric ions. figure 3. ferric reducing antioxidant properties of a. lebbeck seed protein hydrolysates bars are expressed as means ± standard error of mean of triplicate determi nations (n=3). compa risons are made among samples of the same concentration only. values with different letters are significantly different from one another. bars carrying the same letter or symbol are not significantly different (p<0.05). international journal of applied biology, 6(2), 2022 108 hydroxyl radical scavenging activity the hydroxyl radical scavenging capacities of a. lebbeck seed protein hydrolysates are illustrated in figure 4, while their values of 50% inhibition are displayed in figure 5. all hydrolysates demonstrated varying hydroxyl radical scavenging activities, such that chymotrypsin hydrolysates displayed significantly higher scave nging property than other digests, attaining a maximum activity of 36.925 ± 1.880 % at 2.5 mg ml-1. there is relative paucity of information relating to the activities of protein hydrolysates on the hydroxyl radical, when compa red to reports on their effects on other free radical systems. the effects of a. lebbeck seed protein hydrolysates and on the hydroxyl radical indicated that these protein digests had lower oh radical scavenging activities than the standard antioxidant, ascorbic acid. however, chymotrypsin hydrolysates scavenged the oh radical better than the other hydrolysates. chymotrypsin is a specific endoprotease that cleaves proteins at c terminal ends of aromatic amino acid residues (voet et al., 2016). also, li & li (2013) had previously found that the bioc hemical nature of the amino acid(s) at the c -terminal of peptides is more important to its antioxidative ability than that of its n-terminal. in similar fashion, siow and gan (2016) reported that hydrophobic, as well as aromatic aminoacyl residues are strong contributors to the scavenging of free radicals owing to their electron dense branch chain groups. it therefore f ollows that the c -terminal aromatic residues arising from chymotrypsin digestion may have contributed strongly to the quenching of the hydroxyl radical. papain digests and trypsin hydrolysates also recorded maximal scavenging activities of 30.663 ± 0.824 % and 31.544 ± 0.464 % respectively. these activities were significantly higher when compared to the maximum activity of peptic hydrolysates. however, all hydrolysates demonstra ted reduced l ower (p<0.05) hydroxyl radical quenching properties when compared to control. these results translated to ec 50 values of 0.668 ± 0.114 mg ml-1, 6.148 ± 0.3348 mg ml-1, 3.227 ± 0.288 mg ml-1, 2.697 ± 0.098 mg ml-1, and 1.206 ± 0.045 mg ml-1 for ascorbic acid (control), peptic, tryptic, chymotrypsin hydrolysates and papain digests respectively. among the protein digests, papain hydrolysates had the lowest (p<0.05) ec50 value. papain digests also demonstrated hydroxyl ra dical scavenging activities, but these reduced at higher concentrations and this is exemplified by its low ec50 (1.206 ± 0.045 mg ml-1) when compared to other hydrolysates. papain, being a non-specific enzyme capable of releasing quite a lot of short, dissimilar peptides (naik, 2012; voet et al., 2016) could have produced certain peptides which, in turn, might have antagonized the effects of the main biologically active peptides in solution. this phenomenon may have been responsible for the progressive reduction in thei r activities at higher concentrations. going by the cleavage specificities of the enzymes used in the hydrolysis, it could be inferred that these hydrolysates contained peptides with these aminoacyl residues which could scavenge the hydroxyl radical, either by virtue of proton donation and/or by their high hydrophobicities. international journal of applied biology, 6(2), 2022 109 figure 4. hydroxyl radical scavenging activities of a. lebbeck seed protein hydrolysates dots on each line graph are expressed as means ± standard error of mean (sem) of triplicate determinations (n=3). dots belonging to different hydrolysate/fractions with different letters at the same concentration are significantly different (p<0.05) from one another. figure 5. fifty percent effective concentration values (ec50) of hydroxyl radical scavenging activities of a. lebbeck seed protein hydrolysates bars are expressed as means ± standard error of mean (sem) of triplicate determinations (n=3). bars with the same letters do not differ significantly while bars with different letters are significantly different (p<0.05) from one another. superoxide radical scavenging activity the effects of a. lebbeck seed protein digests on the superoxide radical and their values of 50% inhibition a re displayed in figures 6 and 7 respectively. as with other antioxidant activities, all hydrolysates displayed significantly lower (p<0.05) abilities against international journal of applied biology, 6(2), 2022 110 the superoxide radical when compared with the control. peptide digests obtained from chymotrypsin hydrolysis had better (p<0.05) capabilities than other hydrolysates in scavenging the superoxide radical, such that it had a maximum activity of 74.520 ± 0.998% at 2.5 mg ml-1. however, there is a relative paucity of informa tion in the literature about the activities of chymotrypsin – hydrolyzed proteins against the superoxide radical. peptides obtained by chymotrypsin digestion have a number of hydrophobic residues and aromatic residues toward their c-terminals (voet et al., 2016; ekun et al., 2022) and these may have contributed to their increased quenc hing of the superoxide radical as concentrations increased. tryptic digests were also effective in quenching the superoxide radical (58.659 ± 0.334 % at 2.5 mg ml-1) better than peptic hydrolysates and these results were c onsistent with the reports of olusola et al.(2018) in their findings with unfractionated m. oleifera seed protein hydrolysates. at the same time, it was higher than what was obtained for tryptic digests of watermelon seed proteins (< 20% scavenging activity)(arise et al., 2016b). tryptic hydrolysates displayed a concentration – dependent increase in superoxide scavenging activity, attaining a maximum quenching extent of 58.659 ± 0.334 %. udenigwe and aluko (2011) earlier stated that lysine residues are especially important contributors to the quenching of the superoxide radicals. this could explain why tryptic hydrolysates were able to scavenge the superoxide radical, owing to the fact that they contain several peptides having lysine residues on their c-terminals. papain hydrolysates had its maximal scavenging activity of 47.698 ± 1.083 % at a low concentrati on of 0.5 mg ml-1, and the lowest ec50 of 0.970 ± 0.067 mg ml-1. this indicates that the activity of papain digests against the superoxide radical decreased with increasing concentration, and this could be that the continued presence of certain peptides in the solution could have antagonized the activities of peptides that could have contributed positively to the quenching of the superoxide radical. figure 6. superoxide radical scavenging activities of a. lebbeck seed protein hydrolysates dots on each line graph are expressed as means ± standard error of mean (sem) of triplicate determinations (n=3). dots belonging to different hydrolysate/fractions with different letters at the same concentration are significantly different (p<0.05) from one another. international journal of applied biology, 6(2), 2022 111 figure 7. fifty percent effective concentration values (ec50) of superoxide radical scavenging activities of a. lebbeck seed protein hydrolysates bars are expressed as means ± standard error of mean (sem) of triplicate determinations (n=3). bars with the same letters do not differ significantly while bars with different letters are significantly different (p<0.05) from one another. conclusion the results of this 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37: 25-32. 
 contact : omosigho omosighoop@gmail.com 14 abstract the burde n of hepatocellular carcinoma as well a s other health complications associated w ith infections due to hepatis b and c viruses are significant around the w orld. a number of research suggests that assumed healthy living individuals and potential blood donor s are the major reservoirs of the viruse s in the c ommunity. during the cour se of this study, a total of 300 apparently healthy blood donor s w ho pre sented to the blood group serology unit for blood donation across ilorin, kwara state, nigeria, were screened to determine the ser o-prevale nce of transfusion transmissible hepatitis viruse s (hepatitis b and c viruse s). blood samples obtaine d from consented potential blood donor s were collected and tested for hepatitis b surface antigen (hbsag) and anti-hcv using immunochr omatographic methods. the positive sample s after the initial qualitative scree ning were subjected to e nzyme linked immunosorbent assay (elisa) for confirmation of hbv and hcv immunoglobulin g (igg) following standard best practices. the results showed that 10% and 0.7% of the blood sample s tested positive for hbv and hcv respectively. t he male donor s had more hbv infection 24 (8.0%) than the female s 6 (2.0%). donors with o rhesus d positive blood type had more hbv infection 12 (4.0), while blood groups ab negative, b negative and o negative showe d the least prevale nce of 0 (0.0%). the age group of donor s above 30 years of age had more hbv prevalence with 37.3% while the age group of 26 to 30 years showed the least prevalence of 28.7%. data obtained from the piloted structured que stionnaires de signed to study t he socio-demography of the participant s showed that hbv was more prevalent among single blood donor s, civil servant/profe ssional wor kers, and among blood donors that have succe ssfully completed their tertiary e ducation. the data from this research however, showed a negative correlation between the spread of the transfusion transmissible he patitis vir use s and the educational/ occupational standards of the test subjects who were included in this re search. the highest rates of viremia were recorded among blood donor s residing in urban areas however, the prevalence was not statistically different (p≥0.05). . issn : 2580-2410 eissn : 2580-2119 sero-prevalence of transfusion transmissible hepatitis viruses among blood donors in ilorin, kwara state omosigho, o. pius 1,2*, izevbuwa, osazee ekundayo 1, omole, samuel abiodun 2 1 department of medical laboratory science, college of health sciences, igbinedion university, okada 2 department of medical laboratory science, faculty of pure and applied sciences, school of basic medical science. kwara state university, malete open access international journal of applied biology keyword hepatitis c (hcv); hepatitis b (hbv); viruses; blood; prevalence article history received november 7, 2021 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. mailto:omosighoop@gmail.com international journal of applied biology, 6(2), 2022 15 introduction blood donation is an important procedure that saves millions of lives; it involves a person donating blood for the purpose of transfusion. however, unsafe transfusion practices also put millions of people at risk of transfusion-transmissible infections (ttis) (nada and atwa 2013). blood transfusion is the process of transferring blood or blood products into one’s circulation intravenously; transfusion refers to therapeutic infusion of blood, bl ood components and blood products into an individual in order to meet a specific physiologic need (adewoyin and oyewale, 2015). blood transfusion is a cornerstone of modern medical practice essential in almost every field of clinical practice either in emergency situations or as a necessary adjunct to modern and emerging medicare (orkuma et al., 2014). nigeria utilizes about 1.5 million units of blood annually, this requirement cuts across the three levels of healthcare delivery in the country; pri mary, secondary, and tertiary (okocha et al.,2015). transmission of infectious diseases including hepatitis b (hbv) and hepatitis c (hcv) through donated blood is a significant challenge to blood safety. hbv and hcv infections are common serious complications of blood transfusion. prevention of ttvs in developed countries has been achieved by reducing unnecessary transfusions, using only regular voluntary donors, excluding donors with specific risk factors and systematic screening of all donated blood for infection (nada and, atwa, 2013). globally, hepatitis b virus (hbv) and hepatitis c virus (hcv) infection are major causes of acute and chronic liver disease (e.g., cirrhosis and hepatocellular carcinoma [hcc]), resulting in an estimated 1.4 million deaths annually (gbd, 2013). it is esti mated that 248 million people are living with chronic hbv infection (chb) (schweitzer et al., 2015), and that 110 million persons are hcv-antibody positive and 80 million have chronic viraemic hcv infection (gower et al., 2014). to prevent ttvs, mandatory screening tests are performed on the blood bef ore transfusion for hiv and hbv by blood transfusion center and blood banks. qualityguaranteed screening of all donated blood for ttis for both hbv, and hcv, is recommended by the who for the provision of safe and efficacious blood and blood components (biadgo et al.,2017). this includes the selection of eligible blood donors, the collection of blood, the processing and testing of the donated blood, the issuing of compatible blood, and safe administration of the blood to recipients. in response to this strategy, starting with identifying donor cha racteristics such as occupation, test seeking behavior, family history and type of donor. about a third of the world population has been inf ected with hbv and hcv at one point in their life (who, 2009). the world health organization (who) has estimated that more than 2 billion people in the world have been infected with hbv at some time in their lives and about 350 million people suffering from chronic infection worl dwide, also estimated a worldwide prevalence of about 3% with hcv virus affecting 200 million people worldwide and 3 to 4 million new infections each year (augustine et al., 2014) stating clearly that the disease has caused epidemi cs in parts of asia and africa, and it is endemic status in china. in nigeria, the prevalence rate of hbv is 10% and hcv is 2.1% (lavanchy, 2004). estimating the prevalence of ttvs, namely hbv and hcv among blood donors can reveal the problem of unnoticeable infections in healthy-looking members of the general population and also provide data that is important in formulating the strategies for improving the management of a safe blood supply. international journal of applied biology, 6(2), 2022 16 the aim of this study is to estimate the prevalence and associated sociodemographic factors of serological markers of transfusion transmissible hepatitis viruses (hepatitis b (hbv) and hepatitis c (hcv)) among blood donors attending university of ilorin teaching hospital ilorin, kwara state. materials and methods specimen collection about 5mls of fresh whole blood was aseptically collected from the participants and dispensed into ethylene diamine tetra-acetic acid (edta) anticoagulated sample bottles following standard operating procedures for venipuncture. the blood samples were then transported to the hematology laboratory in uith, kwara state for analysis. sample preparation and screening for hbv and hcv hbv and hcv screening was performed using the hbsag and anti -hcv rapid diagnostic test strips respectively. (bio-inteco), uk to qualitatively demonstrate the presence of hbsag and anti-hcv in human whole blood, serum or plasma specimens. the whole blood samples were centrifuged at 3000rpm for 2mins with the plasma extracted and dispensed into appropriately labelled plain screw capped bottles. about 50ul of plasma according to the manufacturer’s prescription was dispensed on the absorbent pad on the immunochromatographic strips using sterile automatic pipettes and disposable tips. one drop of buffer solution (phosphate buffered saline and preservative) provided was also added onto the absorbent pad with the sample. capillary action brought a bout the upward movement of the sample on the test strips and the results were read after 10 mi n according to manufacturer’s instruc tion. double red lines were visible for positive samples (control and test line). negative samples showed single line (control line). the samples that were reactive and give positive result were further c onfirmed using enzyme linked immunosorbent assay (elisa). elisa for hbsag and anti-hcv positive samples obtained after the preliminary screening were further confirmed using elisa (bio-inteco), uk. technique. following the manufacturer’s instructions. result and discussion results table 1. shows the frequency distribution of socio-demographic factors of the selected blood donors is presented below, 34% of the blood donors were between 20 -25 years, 28.7% were between 26-30 years and 37.3% were above 30 years. majority of the blood donor were above 30, however, the mean age was 29.46 years. male were more than female in the study with 57.3% and 42.7% respectively. blood donors with a+, b+ and o+ blood group dominated the study with a joint percentage of 83.3%. 68.7% of the blood donors were living in urban area while 31.3% were living in rural area. majority were single (48%), 42% were married while 10% were divorced/separated. few of the blood donors had prima ry education (5.3%), 26.7% have secondary education while majority, 68% had postsecondary education. 14.7% were unemployed, 20.7% were student/apprentice, 21.3% were artisan/trader and 43.3% were civil servant/ professional workers. international journal of applied biology, 6(2), 2022 17 table 2 shows the prevalence of hepatitis b and hepatitis c among blood donors in ilorin. 270 blood donors were hbv negative and 30 were positive. while for hcv, only 2 blood donors were positive. generally, majority of the blood donors were negative for both hbv and hcv and no co-infection was recorded in this study. table 3 shows gender distribution of hbv among blood donors in ilorin, out of 30 blood donors found to be hbv positive, 24 were male while 6 were female. the distribution tends to be higher among the male with (57.3%) than the female with (42.7%). the 𝑝 − 𝑣𝑎𝑙𝑢𝑒 = 0.061, since the p-value > 0.05, the result indicates that, there is no significant association between blood donors’ gender and hbv positivity. table 4 shows the result of blood donors based on blood group type. out of 30 blood donors found to be hbv positive, 8 were a+, 2 were a-, 2 were ab+, 6 were b+, 12 were o+ while no positive result was recorded for ab-, oand b-. hbv had a highest distribution among blood group o + (4.0%), then followed by blood donor group a+ (2.7%), and blood group b+ (2.0%). the distribution among blood groups aand ab+ both showed (1.0%) each. table 5 shows the result of blood donors based on age group. out of 30 blood donors found to be hbv positive, 10 donors were found to be positive across the three age groups in this study. the blood donors between the age range of 20-25 year had a prevalence of (3.3%), age range between 26-30 had a prevalence of (3.3%) and age range above 30 had a prevalence of (3.3%). table 6 show the results of blood donors based on thei r location of residence. out of 30 blood donors found to be hbv positive, 26 donors from urban areas were found to be positive while 4 donors from rural arears were found to be positive. resulting in a prevalence of 8.7% and 1.3% respectivly. table 7 shows the hbv distribution among blood donors based on marital status. out of 30 blood donors found to be hbv positive, 14(47%), donors were single, 12(40%) were married and 4(13%). were divorced/separated. table 8 shows the hbv distribution among blood donors based on educational status. out of 30 blood donors found to be hbv positive, 2 had primary education, 6 had secondary education and 22 had tertiary education. the distribution is higher among blood donors that attained tertiary education (73%) and lowest among donors with primary education (7%). table 9 shows the result of hbv distribution among blood donors based on occupational status. out of 30 blood donors found to be hbv positive, 6 were unemployed, 2 were students, 4 were artisan/traders and 18 were civil servant/professional worker. the distribution was higher among civil servant/ professional workers (60.5), and unemployed showing the prevalence of (20%), artisan showing a prevalence of (13%) and lowest among student/apprentice (7%). table 1. distribution of hbv and hcv among blood donors in ilorin based on socio demographic factors. factors frequency percentage age 20-25 102 34.0% 26-30 86 28.7% above 30 112 37.3% international journal of applied biology, 6(2), 2022 18 mean age 29.46 gender male 172 57.3% female 128 42.7% blood group type a+ 94 31.3% a10 3.3% ab+ 22 7.3% ab2 0.7% b+ 94 31.3% b6 2.0% o+ 62 20.7% o10 3.3% location urban 206 68.7% rural 94 31.3% marital status single 144 48.0% married 126 42.0% divorced/separated 30 10.0% educational status primary 16 5.3% secondary 80 26.7% tertiary 204 68.0% occupation unemployed 44 14.7% student 62 20.7% artisan/trader 64 21.3% civil servant/professional 130 43.3% international journal of applied biology, 6(2), 2022 19 table 2. prevalence of hbv and hcv among blood donors in ilorin. negative positive total hbv 270(90%) 30 (10%) 300 (100%) hcv 298 (99.3%) 2 (0.7%) 300 (100%) table 3. gender distribution of hbv among blood donors in ilorin. gender negative positive total male 148 (49.3%) 24(8.0%) 172 (57.3%) female 122 (40.7%) 6 (2.0%) 128(42.7%) total 270 (90.0%) 30 (10.0%) 300 (100%) table 4. distribution of hbv among blood donors in ilorin based on blood group. occupation negative positive total a+ 86 (28.7%) 8 (2.7%) 94 (31.3%). a8 (2.7%) 2 (0.7%) 10 (3.3%) ab+ 20 (6.7%) 2 (0.7%) 22 (7.3%) ab2 (0.7%) 0 (0.0%) 2 (0.7%) b+ 88 (29.3%) 6 (2.0%) 94 (31.3%) b6 (2.0%) 0 (0.0%) 6 (2.0%) o+ 50 (16.7%) 12 (4.0%) 62 (20.7%) o10 (3.3%) 0 (0.0%) 10 (3.3%) total 270 (90.0%) 30 (10.0%) 300 (100%) international journal of applied biology, 6(2), 2022 20 table 5. distribution of hbv among blood donors in ilorin based on age group. age group negative positive total 20-25 92 (30.7%) 10 (3.3%) 102 (34.0%) 26-30 76 (25.3%) 10 (3.3%) 86 (28.7%) above 30 102 (34.0%) 10 (3.3%) 112 (37.3%) total 270 (90.0%) 30 (10.0%) 300 (100%) table 6. distribution of hbv among blood donors based on location. location negative positive total urban 180 (60.0%) 26 (8.7%) 206 (68.7%) rural 90 (30.0%) 4 (1.3%) 94 (31.3%) total 270 (90.0%) 30 (10.0%) 300 (100%) key urban: parti ci pants res i ding wi thi n il orin metropol i s rural : parti ci pants res i ding outs i de il orin metropol i s . table 7. distribution of hbv among blood donors in ilorin based on marital status. marital status negative positive total single 130 (43.3%) 14 (47%) 144 (48.0 %) married 114 (38.0%) 12 (40%) 126 (42.0%) divorced/separated 26 (8.7%) 4 (13%) 30 (10.0%) total 270 (90.0%) 30 (10.0%) 300 (100%) table 8. distribution of hbv among blood donors based on educational status. educational attainment negative positive total primary 14 (4.7%) 2 (7%) 16 (5.3%) secondary 74 (24.7%) 6 (20%) 80 (26.7%) tertiary 182 (60.7%) 22 (7.3%) 204 (68.0%) total 270 (90.0%) 30 (10.0%) 300 (100%) international journal of applied biology, 6(2), 2022 21 table 9. distribution of hbv among blood donors based on occupational status. occupation negative positive total unemployed 38 (12.7%) 6 (2.0%) 44 (14.7%). student/apprentice 60 (20.0%) 2 (0.7%) 62 (20.7%) artisan/trader 60 (20.0%) 4 (1.3%) 64 (21.3%) civil servant/ 112 (37.3%) 18 (6.0%) 130 (43.3%) professional worker total 270 (90.0%) 30 (10.0%) 300 (100%) discussion in this present study, the prevalence of hbv among blood donors in ilorin is (10%). the study results were higher than those reported (3%) occult hepatitis b infection among blood donors in lagos, nigeria (akinbami et al., 2019), ( 5.9%) transfusion transmissible viral infections among potential blood donors in ibadan, nigeria (afolabi et al., 2013), (6.16%) hepatitis b virus and hepatitis c virus co-infections among ekiti people in southwestern nigeria (akinbolaji et al., 2015), (9.8%) hepatitis c virus (hcv) antibody and hepatitis b virus surface antigen (hbsag) in blood donors in a southwestern nigerian city (motayo et al., 2015), (8.3%) among commercial blood donors in sokoto, nigeria (augustine et al., 2014), benin, nigeria (umolu et al., 2005), (5.0%) hepatitis b surface antigen and antibodies to hepatitis c in the general population of benue state, central nigeria (malu et al., 2020) and the blood transfusion unit of ahmadu bello university teaching hospital, zaria, kaduna state, nigeria (mukta r et al., 2005), (1.4%) among blood donors in alexandria, egypt (wasfi and sadek, 2011) and (4.2%) a mong blood donors in nairobi, kenya (abdalla et al., 2005).while the results shows a distribution lower than the prevalence of, (10.1%) reported in bida, north central, nigeria (omosigho et al., 2012) found among the commercial blood donors in nigeria, (12.2%) a national survey of hepatitis b infection in nigeria (adebola et al., 2016), and (14.0%) among eligible blood donors in a tertiary healthcare facility in nasarawa state, nigeria (akpu et al., 2017). this study is reporting hbv prevalence among other groups besides blood donors in nigeria include, (7.0%) among attendees of association of reproductive and family health centre in ibadan, nigeria (okonko et al., 2012), (6%) among pregnant women attending the antenatal clinic at nnewi, anambra state, nigeria (oluboyo et al., 2014) and (3.9%) among adolescents in abakalaki, nigeria (ugwuja and ugwu, 2010). the prevalence of hcv reported as 0.7% from this study is l ower than the (1.3%,) hepatitis c virus (hcv) antibody and hepatitis b virus surface antigen (hbsag) in blood donors in a southwestern nigerian city (motayo et al., 2015), (1.4%) transfusion transmissible viral infections among potential blood donors in ibadan, nigeria (afolabi et al., 2013), (1.71%) hepatitis b virus and hepatitis c virus co-infections among ekiti people in south-western nigeria (akinbolaji et al., 2015), (4.0%) among commercial blood donors in sokoto, nigeria (augustine et al., 2014), (2.1%) risk factors and seroprevalence of hepatitis international journal of applied biology, 6(2), 2022 22 c virus antibody among blood donors in lagos, nigeria (balogun et al., 2012), (3.0%) among blood donors from a teaching hospital in the south western nigeria (opaleye et al., 2010), (4.4%) among hiv negative patients at kenyatta national hospital, kenya (karuru et al.2005) and (7.0%) among eligible blood donors in a tertiary healthcare facility in nasarawa state, nigeria ( akpu et al., 2017). however, it is higher compared to (0.4%) among blood donors in kano, nigeria (imoru et al., 2003). reports from different parts of the world vary, with prevalence of hbv and hcv among blood donors being (0.66%-25%) and (1.0%) -(13.3%) respectively (butashirili et al.2001; gupta et al., 2004; matee and mages, 2006). in nigeria prevalence figures of (1.3%-1.49%) and (6%-8.4%) have been reported for hcv and hbv respectively (ejele et al.,2005; chikwem et al.1997; egah et al., 2004; ayolabi et al., 2006; muktar et al., 2005; uneke et al., 2005). in developed countries , hbv and hcv infection risk was estimated at 1 in 3000 transfusions, which is very low. the reasons for this most likely are low prevalence in the general population, awareness among the general public and adequate health care facilities (moradpour et al., 2001). however, in comparison with the developed countries, emerging countries experience higher prevalence rates for hbv and hcv due to inadequate screening of the blood and blood produc ts, improper injection safety practices, high risk sexual behaviors , socio-cultural practices like tattooing, scarification and circumcision practices through the use of unsterilized instruments. differences in prevalence can be most likely associated with variations in the geographic locations and the differences in the diagnostic kits or markers used in detection of hbv and hcv antibodies. also, as there are the differences in the prevalence of hbv and hcv are reported among different population in the same town (karmochki re et al., 2006; fasola et al., 2009; buseri et al., 2009). population selection is also a factor contributing to the variations in prevalence. this study reported that males had a higher prevalence (8.0%) of hbv than their female counterparts (2.0%). our finding is consistent with a previous report among namibian blood donors in which the prevalence of hepatitis b was significantly higher among male compared to female blood donors (mavenyengwa et al., 2014) also with hepatitis c virus (hcv) antibody and hepatitis b virus surface antigen (hbsag) in blood donors in a southwestern nigerian city (motayo et al., 2015), (14.3%) by (akpu et al., 2017) also with that found in sierra leone reported by (tognon et al., 2020) seroprevalence of hepatitis b and hepatitis c among blood donors in sierra leone, but in contrary with the hcv reported (akpu et al., 2017) in nasarawa state which reported high prevalence among the female (11.1%). the reason for this male gender predisposition may be due to the fact tha t polygamy and maintenance of multiple sex partners is preva lent in the area. men are permitted on religious ground to marry up to 4 wives. this increased risk associated with the maintenance of multiple sex partners may play a role in the higher prevalence observed among men compa red to women. therefore, there is no significant association between blood donors’ gender and hbv positivity. this study reported that blood group o+ has the highest prevalence with (4.0%), blood group a+ (2.7%), blood group b+ (2.0%), blood group aand ab+ both with the lowest prevalence (0.7%) while blood group ab-, b-, and ohas no recorded case at all. this is in agreement with that found in sokoto state (57.2%) by (augustine et al., 2014), also in china by jing (jing et al., 2020) abo blood groups and hepatitis b virus infection, in baghdad (aljooani et al., 2012) the infection with hbv and hcv and their relationship to abo blood international journal of applied biology, 6(2), 2022 23 group among blood donors . but in contra ry with that found in nasarawa state reported by (akpu et al., 2017) with high prevalence among blood group o (50%) and a(33.3%), also in turkey reported by genc (genc, 2017) hepatitis b virus infection and abo/rh blood group, where blood group a+ has been reported with the highest prevalence rate. the p-value >0.05, we conclude that there is no significant association between blood donors’ blood group type and hbv positivity. this study also shows that the prevalence of 3.3% hbv occurred across all ages of 18 to 65. it was observed that the prevalence was higher 6.6% between the ages of 18 -30 while the only positive case for anti-hcv antibodies occurred between the ages of 26 to 30 years. this is contrast with the findings of the studies that supported that, in developing countries, the prevalence of hbv was higher and distributed more generally through older populati ons than in the developed settings (abdalla et al., 2005; gupta et al., 2004). however, this is consistent with the findings reported in previous studies in nigeria (busari, 2011), (afolabi et al., 2013) this finding is also in agreement with previous results reported by baba and colleagues (baba et al., 2000) and ejele and coworkers (ejele et al., 2005) in which higher hbv prevalence were observed among youths. the high prevalence of hbv among the youth could be as a result of the high-risk behavior such as maintenance of multi ple sex partners, tattooing, having unprotected sex, intravenous drug abuse and other unhygienic activities involving youths. the economic costs of the failure to control the transmission of infection among young nigerians include increased requirement for medical care, higher level of dependency and the loss of productive labor force, thereby placing heavy burdens on the already overstretched health and social services and on the natural economy (kitchen et al., 2001). there is no significant association between blood donors’ age group and hbv positivity. this study reported that blood donors residing in an urban settlement has the highest prevalence (8.7%) compared with blood donors from rural areas with prevalence of (1.3%), this may be as a result of the test been carried out in an urban location. the discovery of such a high hbv prevalence of (8.7%) in this study, a mong an urban population which supposedly should have a low prevalence rate — unlike rural populations where most practices such as poor adherence to vaccination schedules, relatively low vaccination coverage, sharing of drinking cups, and relatively frequent unprotected sex favour transmission—underscores the endemicity of hepatitis b in nigeria. this is in contrary with that reported in egypt by (nada et al., 2013) with high prevalence (2.6%) and (7.6%) for hbv and hcv respectively among the participant in the rural settlement. the p-value > 0.05. we conclude that, there is no significant association between blood donors’ location and hbv positivity. this study reported that unma rried people tend to have the highest prevalence (4.7%) which is higher than the prevalence among married people (4.0%) and lowest in the divorced/separated couples (1.3%), more than 50% of the blood donors were unmarried and this might be the reason for such occurrence of high prevalence among the singles. this study is in agreement with that reported in ibadan by afolabi (afolabi et al., 2013) transfusion transmissible viral infections among potential blood donors in ibadan, nigeria, and in contrary with (akpu et al., 2017) in nasarawa state, that reported high prevalence (14.3%) among the divorced, also with that reported by (motayo et al., 2015) that reported highest prevalence (7.1%) and (augustine et al., 2014) that reported (78.6%) among the married in sokoto state. the p-value > 0.05. we conclude that, there is no significant association between blood donors’ marital status and hbv positivity. international journal of applied biology, 6(2), 2022 24 this study shows that participant that attained tertiary education has the highest prevalence of (68.0%), while those that attained secondary education came out with (26.7%) and primary education has the lowest prevalence (5.3%). this is in contrary with (motayo et al., 2015) that reported high prevalence among the illiterate (22.2%) in blood donors in abeokuta, nigeria. this study shows that civil servants/professional workers had the highest prevalence with (6.0%), followed by the unempl oyed with (2.0%), artisan/trader (1.3%) and student/apprentice had the lowest prevalence (0.7%). this study in contrary with that conduc ted in nasarawa state reported by (akpu et al., 2017) with higher prevalence among the artisan (17.5%), (augustine et al.2014) that reported high prevalence among farmers (28.6%) in sokoto state, (okocha, et al 2015) that reported high prevalence among traders in nnewi, south-east nigeria. also, with that found in sierra leone where informal workers has the highest prevalence (11.0%) reported by (tognon et al., 2020) seroprevalence of hepatitis b and hepatitis c among blood donors in sierra leone. the p-value > 0.05, we conclude that, there is no significant association between blood donors’ occupation and hbv positivity. blood borne infections like hbv and hcv infections continue to pose a great challenge to transfusion medicine, most particularly in africa, because of a high transfusion demand (fleming, 1997). transfusion-transmitted infections in developed countries have been reduced by preventing unnecessary transfusions, preferring only regular voluntary donors, thereby avoiding donors with specific risk factors and systematic screening of all donated blood for infection. however, in many emerging countries none of these interventions are applied uniformly and the risk of transfusion-transmitted infections remains high (gurol, 2006). hence, strict selection of blood donors, public awareness on hbsag, hcv infections and prevention strategies is a must for the emerging countries like nigeria. based on the findings of the current study, it is recommended that blood donors should always be screened for hbv and hcv. appropriate screening programs based on simple assay formats like agglutination should be encouraged. consequently, international models and standards for organizing blood banks should be adapted to resource poor environments including locally relevant guidelines for counseling and management of hbsag and hcv positive blood donors (allain et al., 2004). overall, there arises a need to c reate an awareness regarding hbv and hcv not only among the high-risk population but also among the general population. emphasis should be placed on these aspects through information education communication campaigns. the high disease burden of such blood borne infections demands formulation of government-supported prevention and control strategies for hbv and hcv, not only in blood banks but also in similar hospital settings. conclusion the result of this study brings light to the high prevalence of hepatitis b (10%) infection among blood donors in ilorin, of the blood donors found to be sero-positive for hbsag, 24(8.0%) were male, 6(3.0%) were female. 26(8.7%) were from urban location, 4(1.3%) were from rural location. there was even distribution between the age group distribution with each age group 20-25, 26-30 and above 30 having 10 (3.3%) respectively. the above study shows decreasing prevalence of hepatitis c infection as antibody to hepatitis c virus was detected in 2 (0.7%) of the blood donors. there was no hbv and hcv co-infection among the participants in this study. socio-demographic characteristics like age international journal of applied biology, 6(2), 2022 25 group, gender, educational status, occupation, blood group type, ma rital status and location were not significant different in this study (p <0.05). the high prevalence of hbv in this study, underscores the importance of emulating global standard practices towards restraining 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of this experiment indicated that salmonella typhii gave a positive result for the biochemical test with tsia media, sim media, mrvp media, citrate, urea, glucose, sucrose, lactose, mannitol at three samples of milkfish out of ten samples taken randomly at the fish market and fish auction in makassar, the contamination level of salmonella typhii bacteria in all observed samples was moderate with a percentage level of 30%. next test antibacterial and antibiotic tests were carried out with mha medium and antibiotics, and the antibiotic ciprofloxacin has a high degree of sensitivity test. issn : 2580-2410 eissn : 2580-2119 the activity of salmonella typhii in milkfish (chanos chanos) with invitro. andi muhammad munawir u1, anastasia a basir2 1department of health analyst, stikes yapika makassar, indonesia 2department of nursing, stikes yapika makassar, indonesia. introduction salmonellosis is a disease caused by salmonella bacterial infection in humans and animals that attacks the gastrointestinal tract, which includes the stomach, small intestine and large intestine or colon (zelpina and noor, 2020). bacteria that are often found in food and drink contaminated one of which is salmonella sp. usually, infection of salmonella sp causes salmonellosis (lestari et al, 2017). salmonellosis is zoonotic, meaning that this disease can be transmitted from animals to humans (dharmojono, 2001). salmonella that plays the most role is s.typhi because it is still a major health problem causing typhoid fever in developing countries. bacteria with a level of danger that can cause death (ariyanti and supar, 2017). typhoid fever is generally found in developing and low income countries, as well as countries with tropical climates, who estimates that there are around 16 million cases annually worldwide and around 600,000 people die (who, 2018). data information from gbd for indonesia itself according to gender in 2019, the value open access international journal of applied biology keyword salmonella typhii, invitro, tsia, mrvp, ciprofloxacin article history received february 22, 2023 accepted june 14, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 7(1), 2023 27 obtained for men is 187.06 dalys per 100,000 while for women it is 122.99 dalys per 100,000 (global burden of disease, 2020). specifically for south sulawesi province, a value of 147.88 dalys per 100,000 was obtained in 2019 (ainil, m. 2021). transmission of bacteria in f oods, there are 39% that are positively contaminated by salmonella typhi. this can be influenced by several factors, namely food processing, selling environmental hygiene, contaminated water, and flies as disease vectors (putri et al, 2019) foodborne disease is a disease caused by microorganisms and is a poison found in food that then enters through the mouth and is digested in the stomach inside the body (muna et al, 2020). fish is foodstuff with a high protein source, containing essential amino acids needed by the body. fish contains high nutrients but is easily decomposed , caused by the presence of microbes found in fish (melawati, et al, 2019). milkfish can also be a source of spreading bacteria if not managed hygienically and safely. the multiplication of bacteria in the body of fish is not only caused by bacteria naturally found in fish (in the stomach contents, gills, and skin) but also by bacteria originating from other sources that contaminate the fish, including pathogenic bacteria, such as salmonella typhii (samiadi, 2021). salmonella species encompasses a group of common rod shape, gram-negative rods that are motile, 1.0 to 3.0 µm long, and 0.8 to 1.0 µm wide (darmawan et al, 2020). salmonella will produce pink bars on gram stain on microscopic examination. salmonella can ferment glucose, produce gas, but do not ferment lactose and sucrose. generally , salmonella sp. produces h2s, is resistant to certain chemicals, and grows at 15-41°c with a ph of 6-8. the formation of gas comes from the reaction of acids which are retained based on media (ulya, 2020). on media containing high protein, salmonella sp. grow quickly and can even grow as many as 90,000 cells in just 6 hours (yunus, et al. 2017). in a culture so that the colonies are large, 2-8 millimeters in diameter, slightly convex round, clear, smooth, on bap media do not cause hemolysis, on mac concey media salmonella sp. does not ferment lactose (nlf), the consistency is smooth salmonella sp. survive in water that is frozen for a long time, these bacteria are resistant to certain chemicals (eg brilliant green, sodium tetrathionate, sodium deoxycholate) that inhibit the growth of other enteric bacteria, but these compounds are useful to be added to the isolation medium salmonella sp. on the stool sample (irianto, 2018). typhoid fever is one of the endemic diseases in indonesia, so it must be given serious attention because it can be a threat to public health. moreover, there has been an increase in relapse cases and there is resistance to the drugs used, which will make it more difficult for various efforts to be made related to this disease (depkes, 2006). salmonella incubation time lasts 7-14 days depending on the number of bacteria, virulence, and immune response (hardianto, 2019) one way to prevent bacterial contamination is to wash your hands (taliya, 2021). contaminated microbes in food are the result of direct or indirect contamination with pollutant sources (amiruddin et al, 2017). cross contamination can also occur if the international journal of applied biology, 7(1), 2023 28 processing and preparation process does not meet the requirements, such as food that is not clean and undercooked (yuniatun, et al. 2017). the objective of this research was to look at the activity of contaminated salmonella typhii in milkfish sold in unclean fish markets, besides that the length of the process from cutting until the milkfish is ready for consumption, is very possible for contamination of milkfish, including contamination at the time of slaughter, namely with the market or unhygienic fish auction places, tools used for cutting, water used for washing, workers' hands, contact with parts that are often contaminated such as scales, internal organs, gill covers, and others. materials and methods experimental location and samples this experiment was conducted in microbiology laboratory at the faculty of medical hasanuddin university, makassar, indonesia, in june 2021. the population is all samples of milkfish scattered in markets and fish auctions in makassar city. samples were taken as ten samples with areas selected by probability sampling. isolation and identification with specific medium isolation and identification with mcconkey agar (mca) or bhib medium, ssa medium, and na medium were incubated at 35oc for 24 hours. biochemical test for salmonella typhii for the identification of s.typhii, specific biochemical tests were conducted according tsia media, sim media, mrvp media, citrate, glucose, sucrose, urea, lactose, mannitol were incubated at 35oc for 24 hours. gram strain gram stain with crystal violet, iodine, safranin, and alcohol 70%. antibacterial and antibiotic tests antibacterial test with mha media (mueller hinton agar) and antibiotics used such as ceftriaxone, sulfamethazole, ampicillin, ciprofloxacin, chloramphenicol (andi munawir, 2021) results and discussion milkfish samples totaled ten samples by taking milkfish (chanos chanos) samples at different locations in the fish market and auction in makassar city by providing sample codes a-j. the sample criteria examined were scales, gill covers, gills, fluids in the fish's mouth, and parts of the fish's intestines figure 1. international journal of applied biology, 7(1), 2023 29 figure 1. samples milkfish (chanos chanos) milkfish samples are put into bhib (brain heart infusion broth) medium , as seen in figure 2, where bhib is a microorganism growth medium which is rich in nutrients which is suitable for various types of microorganisms including bacteria. figure 2. identification test with bhib medium test selective medium ssa (salmonella shigella agar) and na (nutrient agar) is a selective medium for detecting and identifying salmonella and shigella bacteria at figure 3. figure 3. culture on ssa and na selective medium international journal of applied biology, 7(1), 2023 30 salmonella typhii belongs to the gram-negative bacteria in the form of rods as shown in the gram stain with figure 4. figure 4. gram strain at s.typhii biochemical test with tsia media, sim, mr-vp, citrate, glucose, lactose, sucrose, and mannitol in figure 5. specific characteristics of salmonella typhii in that the tsia medium gives a reddish color change with a red slant which includes alkali and a red butt forming h2s gas, then the sim medium changes color, and there is movement of motile bacteria characterized by the spread of bacteria with the spread of growth in the area around the ose puncture, then on the mr-vp medium there is a color change from yellow to reddish, then citrate test, urea test, lactose test, and the sucrose test did not change color, while the glucose and mannitol test gave a color change from red to yellow. figure 5. biochemistry test with tsia, mr-vp, citrate, urea, glucose, lactose, mannitol, sucrose in table 1, samples coded c, g, and j were suspected of salmonella typhii, then sample a was suspected of providencia stuartii bacteria, then samples b, d, e, h , and i were suspected of proteus mirabilis bacteria, with reference to great wohls (1980). international journal of applied biology, 7(1), 2023 31 table 1. table of observation of biochemical test of bacterial samples in figure 6, the measurement of inhibition using the antibiotics ceftriaxone, sulfamethoxazole, ampicillin, ciprofloxacin, chloramphenicol, and tetracycline, can be seen in table 2 the most sensitive antibiotic against salmonella typhii is ciprofloxacin (cip) with a high inhibition value compared to with other antibiotics which refer to the measurement and inhibition of antibiotics with reference to great wohls (1980) or (andre b, and novita, 2022). the level of contamination of salmonella typhii bacteria in milkfish shows a percentage of 30%, which is included in the moderate level, this indicates that the level of contamination with this bacteria is still high. figure 6. antibacterial test with antibiotics cefriaxone, sulfamethazole, ampicillin, ciprofloxacin, chloramphenicol no code bhib media mac conkey agar (mca) media ssa and na media gram stain tsia sim mrvp cit ur ea g l s m interpretation slant butt h2s gas indol motil h2s mr vp 1. a cloudy colourless, concave round, big basil gram (-) alkali acid + + + + + providencia stuartii 2. b cloudy cloud red round, big basil gram (-) acid acid + + + + + + + + + + proteus mirabilis 3. c cloudy colourless, convex round, small basil gram (-) alkali acid + + + + + + salmonella typhii 4. d cloudy cloud red round, big basil gram (-) acid acid + + + + + + + + + + proteus mirabilis 5. e cloudy cloud red round, big basil gram (-) acid acid + + + + + + + + + + proteus mirabilis 6. f cloudy convex red round, small, smooth basil gram (-) acid acid + + + + + + escherechia coli 7. g cloudy colourless, convex round, small basil gram (-) alkali acid + + + + + + salmonella typhii 8. h cloudy cloud red round, big basil gram (-) acid acid + + + + + + + + + + proteus mirabilis 9. i cloudy cloud red round, big basil gram (-) acid acid + + + + + + + + + + proteus mirabilis 10. j cloudy colourless, convex round, small basil gram (-) alkali acid + + + + + + salmonella typhii international journal of applied biology, 7(1), 2023 32 table 2. antibacterial test using certain antibiotics amp te sxt c cip 1. salmonella typhii 17 19 16 18 21 2. proteus mirabilis 8,2 24,4 17,4 16,6 25,5 3. providentia stuartii 6,7 6,3 19,5 22 21 4. escherechia coli 6,9 20,4 22,3 25,5 11,9 description : amp : ampicilin te : tetracyclin sxt : sulfamethazole c : chloromfenikol conclusion samples of salmonella typhii were obtained in 3 samples of the ten samples studied. the level of contamination of salmonella typhii bacteria in all samples observed was moderate, with a percentage level of 30% of samples taken at fish markets and auctions in makassar city. the antibiotic ciprofloxacin has a high sensitiv ity test rate compared to the four antibiotics, namely tetracycline (te), chloramphenicol (c), ampicillin (amp), and sulfamethoxazole (sxt). international journal of applied biology, 7(1), 2023 33 references ainil, m. 2021. angka kejadian demam tifoid berdasarkan kajian serologis di rs unhas makassar, puskesmas tamalanrea tahun 2019-2020. fakultas kedokteran, unhas, makassar. amiruddin, r. r., darniati., dan ismail, 2017. isolasi dan identifikasi salmonella sp. pada ayam bakar di rumah makan kecamatan syiah kuala kota banda aceh. jimvet, 01(3): 265274. andi munawir, anastasia a, 2021. uji aktivitas bakteri salmonella typhii pada sayuran lalapan kemangi (ocimum sanctum l) secara invitro. umi medical journal vo 6, no 2, 31 desember 2021, e-issn: 2685-7581. andre budi, novita l. 2022. pola resistensi bakteri salmonella typhii terhadap antibiotik ceftriaxone dan ciprofloxacin. journal health and science, gorontalo, vol 6 nomer 1, april 2022, e-issn: 2656-9248. ariyanti, t., dan supar. 2017. problematika salmonellosis pada manusia. lokakarya nasional penyakit zoonosis, jakarta. departemen kesehatan republik indonesia, 2006, pedoman pengendalian demam tifoid. menteri kesehatan repubik indonesia, jakarta. darmawan, a., lucia, m., sitti, a., dan hapsari, m. (2020). kontaminasi salmonella sp. pada daging ayam broiler yang dijual di beberapa pasar tradisional di makassar. indonesia medicus veterinus, 9(2) : 168-176. dharmojono, 2001, limabelas penyakit menular dari binatang ke manusia. milenia populer, jakarta. global burned of disesase, 2020. global burden of 369 diseases and injuries in 204 countries and territories. the lancet, 396(10258), pp. 1204–1222. doi : 10.1016/s0140-6736(20) 30925-9. hardianto, d. 2019. telaah metode diagnosis cepat dan pengobatan infeksi salmonella thypi. jurnal bioteknologi dan biosains indonesia, 6(1): 149-158. irianto, k., 2018, epidemiologi penyakit menular dan tidak menular panduan klinis, alfabeta, bandung, jawa barat. lestari, i dewa a.m.d dan hendrayana, made agus, 2017. identifikasi dan diagnos is infeksi bakteri salmonella typhi. skripsi. fakultas kedokteran : universitas udayana denpasar. melawati, b., fakhrurrazi, dan abrar, m., 2019. deteksi bakteri salmonella sp pada ikan asin talang-talang (scomberoides tala) di kecamatan leupung kabupaten aceh besar. jimvet, 3(3): 175-180 muna, fauzul dan khariri. 2020. bakteri patogen penyebab foodborne disease. jurnal uin alaudin. makassar. putri, meiwa r., tri umiana s., syazili mustofa dan ety apriliana. 2019. identifikasi bakteri salmonella typhi pada makanan jajanan gorengan yang dijual di depan sekolah dasar negeri kecamatan kedaton kota bandar lampung. universitas lampung : jurnal agromedicine,vol 6, no. 2. samiadi, l.a. 2021. salmonellosis. artikel kesehatan pencernaan. http://hellosehat.com. salmonellosis.id diakses 15 januari 2022 pukul 11.00 wit taliya putri wardani, 2021. identifikasi salmonella typhii pada swab telapak tangan pedagang makanan di sd kanisus semanggi ii surakarta. stikes nasional, surakarta international journal of applied biology, 7(1), 2023 34 ulya, n. n., fitri, i. dan widyawati, i. d. 2020. gambaran makroskopis dan mikroskopis bakteri salmonella typhi dan salmonella paratyphi pada penderita demam tifoid. jurnal sintesis, 1(2): 40-46. world health organization. 2018. thypoid fever. http://www.who.int. diakses pada tanggal 28 july 2021. yuniatun, t., purwantisari, s., dan yuliawati, s. 2017. hubungan hygiene sanitasi dengan kualitas mikrobiologis pada makanan gado -gado di kecamatan tembalan kota semarang. jurnal kesehatan masyarakat, 5(4) : 491-499. yunus, r., mongan, r., dan rosnani. 2017. cemaran bakteri gram negatif pada jajanan siomay di kota kendari. medical laboratory tecnology journal, 3(1) : 87-92. zelpina, e., dan noor, sm. 2020. non-typhoid salmonella penyebab foodborne disease: pencegahan dan penanggulangannya. wartazoa, 30(4):221-229. contact : charles kwesi koomson charleskoomson@yahoo.co.uk 152 abstract the efficacy of the plant powder of alchornea cordifolia was evaluated under laboratory conditions against the larvae of the leather beetle dermestes maculatus which is a major fish pest that cause serious damage when left uncontrolled. the experime nt was in concentrations of 0g, 1.0g, 2.0g, 2.5g, 3.0g admixed with 15g of smoked fish pow der; while all the treatment containing 0g were without plant powder whic h served as control, all the treatment were in triplicates. newly emerged (0 72 hours old) larvae of d. maculatus were introduce d. evaluation of the potency of the plant pow der was based on larvae mortality and the adult emergence. the weight losse s in fish muscle in the treated and untreated sample s were compared a s index of fish damage during storage. the result showe d that higher plant pow der conce ntration were significantly (p< 0.05) effective in killing larvae stage of t he insect as well as the larvae at 1st and 2nd weeks after infe station (wai). adult emergence was significantly (p<0.05) inhibited in the treated fish and weight loss due to insect infestation was greatly suppressed by the higher dosage rate of the plant powder c ompared to c ontrol. the findings showe d that e fficienc y of a. cordifolia leaf pow der is dosage dependent and is capa ble of c ontrolling the larval stage of d. maculatus in smoked fish (clarias gariepinus) during storage. it is therefore recommende d that plant could be used by poor resource fish farmers, processors and marketers in protecting smoke d-drie d fish against d. maculatus during processing, transportation, marketing and storag e. issn : 2580-2410 eissn : 2580-2119 susceptibility of dermestes maculatus degeer (coleoptera: dermestidea) larvae infesting smoked african catfish (clarias gariepinus) to alchornea cordifolia (schum. & thonn.) leaf powder koomson, c.k. 1*, owusu-fordjour, c. 1, darku, a. 2 1 department of integrated science education, university of education, winneba, p.o. box 25, winneba, central region, ghana 2 department of science education, university of education, winneba, p.o. box 25, winneba, central region, ghana introduction one of the major and cheapest sources of animal protein is fish. this has been used steadily due to its availability and nutritional values; fish is also used to correct protein deficiency in human diets in the tropic area (nta et al., 2019). fish has high protein content, which serves the purpose of natural supplement for meat and cereals in the human diet open access international journal of applied biology keyword alchornea cordifolia; dermestes maculatus; clarias gariepinus; adult emergence; efficacy; larvae mortality; toxicity article history received july 18, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 153 (nta et al., 2019). the consumption of fish provides essential nutrients to a great number of people globally and thus its significant contri bution to nutrition cannot be over-emphasized, a decline in fish availability will have a detrimental effect on the nutritional status in places where fish contributes significantly to the protein intake of the people (nta et al., 2019). stored fish just as any other stored agricultural produc t is infested by several stored product pests (babarinde et al., 2016). notably among them are beetles of the genera, dermestes and necrobia throughout processing, transporta tion, marketing and storage (adesina et al., 2014). d. maculatus infestation causes enormous losses. these include physical loss whereby the a mount of fish available for human consumption is reduced, economic loss whereby the physical loss despite the amount of fish available for sale and or the price commanded for insect damaged fish is below that for unda maged fish, and nutritional loss, which is a direct consequence of the physical and economic loss and causes the retail value of fish to increase beyond the purchasing power of the poor (moses, 1992). attempts at controlling this dreadful insect pest of clarias gariepinus has been overwhelmi ngly relied upon the use of synthetic insecticides. these synthetic products, however, are not without thei r hazards to huma n health and the environment (babarinde et al., 2008). apart from the health and environmental hazards posed by synthetic insecticides, mis-use and over-use by applicators have led to serious problems, including development of insect resistant strains to insecticides, toxic residues on stored products, health hazards to handlers, food poisoning and environmental pollution (ali, 2009). these problems have stimulated research into plants with insecticidal properties grown locally that are readily available, effective, affordable, less poisonous and less detrimental to the environment (tierto, 1994). most plants are rich sources of compounds that have insecticidal properties (obeng ofori et al.; 1997). one of such plants is alchornea cordifolia (schum. & thonn.) which is an important medicinal plant in african traditional medicine and much pha rmacological research has been carried out into its antibacterial, antifungal and antiprotozoal properties, as well as its anti-inflammatory activities, with significant positive results (agbor, 2004). the leaves or leafy stems are also believed to be abortifacient, antispasmodic, blood purifier, diuretic, emetic (in large doses), emmenagogue, oxytocic, purgative, sedative and tonic (agbor, 2004). the crushed fresh leaves or powdered dry leaves are applied externally as a cicatrizant to wounds, to relieve pain, e.g. backache and headache, to fractures to improve healing and to treat eye infections and numerous skin afflictions including venereal diseases, leprosy, sores, abscesses, yaws and filariasis (agbor, 2004). koomson and oppong (2018), koomson, et. al., (2018) as well as koomson (2020) found out the leaves, bark and roots of the plant was effective in controlling the stored products insect pests through suppressing oviposition and progeny development, contact toxic ity and repellency activities. this present study is aimed at determining the larvicidal activity of the leaf powder in order to evaluate its insecticidal potential in suppressing infestation and damage by d. maculatus on stored smoke-dried fish. materials and methods the research was carried out in the biology education department laboratory of the university of education, winneba, central region, ghana. temperature in the laboratory was 30±2°c and relative humidity was 70±5%. the study was carried out fr om may 2021 to august 2021. international journal of applied biology, 6(2), 2022 154 collection and preparation of plant materials leaves of alchornea cordifolia plant were collected from the gomoa otapirow area of the central region of ghana early in the morning. they were rinsed in clean water to remove sand and other i mpurities, air dried a t room temperature in the laboratory f or 15 days, after which, ground into very fine powder using an electric blender. the powders were further sieved to pass through 1mm2 perforations. the powders were packed in plastic containers with tight lids to ensure that the active ingredients are not lost and stored in the laboratory prior to use. insect culture and maintenance the initial source of d. maculatus culture used for this study was obtained from natural infested smoked catfish (c. gariepinus) collected from smoked fish market stall in the mandela market, agona swedru, ghana. it was maintained in a kilner jar covered with muslin cloth under laboratory conditions and kept at a temperature 30+ 2℃ under relative humidity 70 + 5%. all bioassay jars was disinfected in an oven at 80℃ for 2 hours and was allowed to cool at room temperature. new generations was prepared by removing newly emerged (0-72h old) larvae from a stock culture, and placed on fresh uninfected fish, while the parent adult was removed after 2-3weeks oviposition peri od. smoked samples of the fish species (clarias gariepinns) were obtained from smoked fish market stall at the mandela ma rket at agona swedru in the central region of ghana. the fish samples showed no visible presence of neither adult or larvae of d. maculatus infestation. the c ured fish species were sterilized thermally by heating at 10℃ for one hour in a hot air oven (gallenkamp oven) in the laboratory in order to kill any insect pests that may be present (atijegbe, 2004), and allow to cool at room temperature in the laboratory. effect a. cordifolia powder on larvae and adult emergence of d. maculatus the toxic effect of a. cordifolia on larvae d. maculatus was carried out using 250ml plastic containers containing 15g of smoked cat fish with concentration of 1.0g 2.0g, 2.5g and 3.0g a. cordifolia leaf powder. the smoked fish in control dish contain no plant powder. the containers were gently shaken for 2 min to ensure homogenous mixture (adesina et al. 2012) of the smoked fish and treatment powder. ten newly emerged (0-72h old) larvae of d. maculatus was introduced into each treated and control dishes and was covered. each treatment was in triplicate. larvae mortality was counted every 24 hours for 7 days. the insects were confirmed dead when there was no response to probing with blunt forceps at the abdomen (adedire et al., 2011). daily observation was made until adult emergence. the number reaching adult stages was recorded and percentage weight was also recorded. the percentage reduction in adult emergence of f1 progeny was calculated using the formula: percentage adult emergence reducti on = 100 × (no. of adult insect emerged in control dish － no. of adult insect emerged in treated dish) / no. of adult insect emerged in control dish. the % loss in weight was determi ned and recorded using the method described by odeyemi and daramola (2000). % weight loss = 100 × (initial weight of fish sample －final weight of fish sample) / initial weight of fish sample. international journal of applied biology, 6(2), 2022 155 experimental design and data analysis the experiment was laid out in complete randomised design (crd) and each treatment was replicated three (3) times. data were subjected to analysis of variance and where significant differences existed, treatment means were separated using least significant difference (lsd) at 5% probability level. data in percentage were a rcsine transformed, before analysis. results table 1 presents the mortality rate of d. maculatus larva over a period of 120h after infestation. the results showed that the plant powder exert significant (p<0.05) larva mortality with increase in application rate over the time of exposure. fish treated with 2.5g and 3g a. cordifolia had the highest larval mortality, while fish protected with 1g and 2g had the lowest mean values of larval mortality. table 2 shows the larval mortality week after infestation. there was significant difference (p<0.05) at the first and second weeks after infestation (wai) with 3g treated having the highest larval mortality of 86.47% and 94.87% respectively while 1g treatment had the lowest of 25.63% and 48.36% for both the first and second wai. weight loss as a result of the activities of d. maculatus showed a trend that reflected the number of surviving larvae and adult that emerged from the respective treatment dishes. dish treated with a. cordifolia powder had significantly (p<0.05) suppressed adult emergence and lower weight loss as can be found in table 3 and 4 respectively. table 1. percentage larval mortality of d. maculatus treated with different concentration of a. cordifolia powder treatment hours after infestation conc./ 15g fish 24h 48h 72h 96h 120h 0g 0.0 0.0 12.21 13.12 17.94 1.0g 12.68 23.41 41.45 51.34 64.32 2.0g 14.87 25.34 46.32 58.35 68.35 2.5g 15.98 32.01 46.10 62.34 70.32 3.0g 29.45 39.48 56.32 74.98 84.02 lsd 5.21 10.21 14.23 14.98 13.21 the data above (table 1) is summarized in the figure below: figure 1. percentage larval mortality of d. maculatus treated with different concentration of a. cordifolia powder 0 20 40 60 80 100 24h 48h 72h 96h 120h p e rc e n ta g e l a rv a l m o rt a li ty hours after infestation 0g 1.0g 2.0g 2.5g 3.0g international journal of applied biology, 6(2), 2022 156 table 2. percentage larval mortality of d. maculatus treated with different concentration of a. cordifolia powder weeks after infestation treatment weeks after infestation conc./ 15g fish 1st 2nd 0g 8.20 5.3 1.0g 25.63 48.36 2.0g 59.48 75.48 2.5g 78.69 83.94 3.0g 86.47 94.87 lsd 5.02 6.21 the data above (table 2) is summarized in the figure below: figure 2. percentage larval mortality of d. maculatus treated with different concentration of a. cordifolia powder weeks after infestation table 3. percentage adult emergence of d. maculatus treated with different concentration of a. cordifolia powder treatment adult emergence conc. / 15g fish 1 week after infestation 0g 3.20 1.0g 0.87 2.0g 0.71 2.5g 0.58 3.0g 0.31 lsd 0.02 the data above (table 3) is summarized in the figure below: 0 10 20 30 40 50 60 70 80 90 100 0g 1.0g 2.0g 2.5g 3.0gp e rc e n ta g e l a rv a l m o rt a li ty concentration / 15g fish 1st 2nd international journal of applied biology, 6(2), 2022 157 figure 3. percentage adult emergence of d. maculatus treated with different concentration of a. cordifolia powder table 4. percentage weight loss due to fish infestation on samples treated with different concentration of a. cordifolia powder treatment adult emergence conc. / 15g fish 1 week after infestation 0g 13.42 1.0g 6.11 2.0g 5.23 2.5g 5.01 3.0g 4.06 lsd 1.05 the data above (table 4) is summarized in the figure below: figure 4. percentage weight loss due to fish infestation on samples treated with different concentration of a. cordifolia powder 0 0,5 1 1,5 2 2,5 3 3,5 0g 1.0g 2.0g 2.5g 3.0g a d u lt e m e rg e n ce concentration / 15g fish 0 2 4 6 8 10 12 14 16 0g 1.0g 2.0g 2.5g 3.0g a d u lt e m e rg e n ce concentration / 15g fish international journal of applied biology, 6(2), 2022 158 discussion the use of botanicals to control dermestes species is not new especially in developing countries where these botanicals are cheaply available (babarinde, 2016). the powdered plant material was very potent in controlling the various stages of d. maculatus in smoked, c. gariepinus. the plant powder possesses high potency in killing the larvae of d. maculatus in smoked c. gariepinus during storage. the result is in agreement with many other researchers on the use of botanicals against suppression of d. maculatus infestation on smoked-dried fish (muf utau, 2012; ahmed et al., 2013 and adesina 2014). the study clearly indicated that the higher dosage level of the treatment was the most effective in the application rates compared to the untreated control. the emergence of f1 adult from all the treated dishes can also be attributed to the hairs found on the larvae which prevent di rect contact of the powder on the body surface of the larvae as compared to the adult with smooth cuticle (kemabota et al., 2013). furthermore, plants have phytochemicals which act as chemical defense against other organisms (such as insect) in the environment (ogunwenmo et. al., 2007). it is theref ore possible that the strong pungent odour produced by the plant prevented d. maculatus from normal feeding, hence resulting in starvation and subsequent death (nta et al., 2014) and impairing the development of the i mmature stage of the insect pests. fasakin, 2003 reported similar scenario when he used the extract from pipe r guineense, monodora myristica, aframomum melegueta, tithona diversifolia and nicotiana tobaccum as surface protectants against the different stages (adults stage to the eggs) of fish beetle ( dermestes maculatus) (de geer). similar case of weight loss in both the treated and untreated fish muscles (which are used as indexes for calculating the rate of fish damage during storage) was also observed in the present study, and the percentage weight loss in fish during storage was significantly reduced (p<0.05) in fishes treated with the plant powder than the control. the higher percentage of weight loss recorded in untreated fish suggests that the larval stage of the beetle is more destructive than the adult (nwankwo et al., 2011). ala m (2004) reported that if d. maculatus are left undisturbed, they can consume all the flesh and soft tissue of dried fish until only bone and some hard tissue remain. this study revealed that smoked c. gariepinus is susceptible to d. maculatus infestation and thus has to be protected well during storage by techni que that are environmental friendly and safe if nutrient loss is to be avoided. the use of alchornea cordifolia among poor resource fisher farmers, processors and marketers should be advocated since the plant is widely distributed and used among rural folks for its ethno medical importance. international journal of applied biology, 6(2), 2022 159 references adesina, j.m., 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(coleopltera: curculionidae). j. stored prod. res., 30(4): 297-301. contact : saroj thapa thapasaroj1998@gmail.com 115 abstract a study to evaluate the performance of different varieties of potato was conducted in daulichaur, bajhang from february to june, 2021. six commonly grown potato varieties namely cardinal, janakdev, khumal rato-2, khumal ujjwol, ms 42.3 and bajhang local were used for the study. an experiment was conducted in randomized complete block design (rcbd) with 6 treatments replicating 4 times each. the data on plant height, number of main stems per hill, number of leaves, and canopy diameter were taken at different days after planting (dap) and the data on number and weight of tubers were taken after harvesting of potato. data entry and analysis was done in ms-excel and r-studio. significant variation among the varieties in terms of plant height, main stem per hill, canopy diameter and leaves number per plant was observed. number of stems per hill was found maximum (3.88) and minimum (1.63) on bajhang local and janakdev respectively. plant height is found maximum on janakdev (42.75 cm) and minimum on khumal rato-2 (21.45 cm). the maximum (40.90) and minimum (27.55) number of leaves per plant was recorded on khumal ujjwol and cardinal while maximum and minimum canopy diameter was observed on bajhang local (40.90 cm) and khumal rato-2 (36.12 cm). similarly, yield and all the yield attributing parameters was found significant at 5% level of significance. highest number of tuber per plant was observed on bajhang local (17.25) and lowest number of tuber per plant was observed on khumal rato-2 (8.25) in which highest number of marketable size tuber per plant (>25g) was recorded on khumal ujjwol (12.05) and lowest number of marketable size tuber per plant (<25g) was recorded on khumal rato-2 (5.75) but variation among the varieties in case of unmarketable tuber per plant was non-significant. highest tuber weight per plant (485.50g) and lowest tuber weight per plant (306.25g) was recorded from khumal ujjwol and ms 42.3 respectively. the highest tuber yield was recorded from khumal ujjwol (40.45 t/ha) and lowest from ms 42.3 (25.52 t/ha) issn : 2580-2410 eissn : 2580-2119 evaluation of performance of different varieties of potato (solanum tuberosum l.) in bajhang, nepal saroj thapa1*, pradeep raj rokaya2, sandesh parajuli1, binod pokhrel1 and yubraj aryal1 1 agriculture and forestry university, rampur, chitwan, nepal 2 department of horticulture, agriculture and forestry university, rampur, chitwan, nepal introduction nepal is an agricultural country having 65.6 percent population of the country belonging to the farming family (cbs, 2011). nepal's varied climatic circumstances, both within and among the different ecological zones, have created practically limitless open access international journal of applied biology keyword potato; yield; variety; canopy article history received june 6, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 116 opportunities for growing a wide varieties of vegetables including potato throughout the year. this would result in increased exports and import substitution, as well as serve as a raw material base for future agro-industries. furthermore, as the population is increasing geometrically and people become more aware of the nutritional benefits of vegetables, the potential for vegetable production and commercialization in nepal has grown dramatically. potato (solanum tuberosum l.) belongs to the family solanaceae is the world's number one non-cereal crop that feeds more than a billion people daily (fao, 2013). potato ranked as second, third, and fourth position in terms of production, human consumption, and area coverage respectively in the world (faostat, 2016). potato is one of the most important vegetables as well as a staple crop in nepal. it occupies the fifth position in area coverage, second in total production, and first in productivity among the food crops grown in nepal (abpsd, 2019/20). the different varieties of potato mostly cultivated in nepal are cardinal, khumal rato2, janakdev, khumal seto, desiree, kufri jyoti etc. (aicc, 2018). nepal is one of those countries where potato is used to s upplement the normal human diet. the demand for potatoes is increasing as the quality of the potatoes improves, as well as the variety of ways in which they are consumed (shrestha et al., 2020). in the terai region, potatoes are used as a complement to vegetables, whereas in the hill and mountain regions, they are a staple meal too (subedi et al., 2019). therefore, the potato could be a decent option for enhancing the health and nutrition of rural residents. and it is more productive than major cereals and has higher economic value (ghimire & dhakal, 2014). bajhang is one of those districts in the country having highest potential for potato production which is also declared as the potato zone in 2019. the main occupation of the people of the bajhang is agriculture. rice, wheat, finger millet, potato, and barley are the major crops grown here. the total area of potato cultivation in the bajhang district is 1,063 hectares and total production is 17,155 mt with the productivity of 16.14 tons/hectare (abpsd, 2019/20). materials and methods a field experiment was conducted in a farmer’s field at surma rural municipality -1, daulichaur, bajhang, nepal from february 2021 to june 2021. the study site is located at 29.69°n latitude and 81.16°e longitude in the sub-humid sub-tropical zone with an elevation of 1,867 masl. experiment was conducted in a randomized complete block design (rcbd) with six treatments. each treatment was replicated four times. altogether there were 24 individual plots. individual experimental plot area was 3m2, having plot-plot spacing 30 cm, block-block spacing 50cm and 20 cm border, thus the total area for research was 109.81 m2. six commonl y grown varieties of potato viz. cardinal, janakdev, khumal rato-2, khumal ujjwol, ms 42.3 and bajhang local were used. well decomposed fym at the rate of 30 ton/ha was thoroughly mixed during field preparation. recommended dose of fertilizer i.e 40g urea, 65g dap and 30g mop per plot was applied. half dose of urea, full dose of dap and mop was applied as basal dose. split dose of urea was applied on 45 dap. hand weeding at 40 dap and single earthing up was done at 65 dap. by using furrow method of irrigation, field was irrigated thrice at 45, 60 and 75 dap. data on growth parameters were recorded on the field at 45, 60 and 75 dap. the recorded data entry and analysis were done using ms-excel and software package r-studio. international journal of applied biology, 6(2), 2022 117 results and discussion growth parameter number of main stems per hill the effect of different varieties on number of main stems per hill was significant among the treatment (table 5). at 45 dap, maximum number of stems were recorded on bajhang local (2.95) and minimum number of stems were recorded on ms 42.3 (1.30). at 60 dap, maximum number of stems were recorded on bajhang local (3.80) and minimum number of stems were recorded on janakdev (1.50). similarly at 75 dap, maximum number of stems were recorded on bajhang local (3.88) and minimum number of stems were recorded on janakdev (1.63). quality of tuber used and presence of number of buds on the tuber might be the factors that influence the number of main stems per hill. table 1. number of stems per hill of different potato varieties at different days of observations treatments no of stem per hill 45 dap 60 dap 75 dap cardinal 1.85bc 2.05b 2.27b janakdev 1.45c 1.50b 1.63b khumal rato-2 1.70bc 2.00b 2.23b khumal ujjwol 2.30ab 3.15a 3.3a ms 42.3 1.30c 1.70b 1.85b bajhang local 2.95a 3.80a 3.88a sem (±) 0.106 0.116 0.094 f probability <0.01 <0.001 <0.001 lsd (=0.05) 0.786** 0.863*** 0.6977*** cv, % 27.109 24.21 18.27 grand mean 1.92 2.36 2.53 mean fol l owed by common l etter(s ) wi thi n col umns are non -s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel , dap: days after pl anti ng plant height significant variation of plant height was observed among different potato varieties at different days of observations (table 6). at 45 dap maximum avera ge height of plant was recorded in janakdev (6.06 cm) and minimum average height of plant was recorded in khumal rato-2 (3.15 cm) which was at par with ms 42.3 (3.32 cm). at 60 dap, janakdev (19.60 cm) was recorded with maximum height and cardinal (9.55 cm) was recorded with minimum height. at 75 dap, maximum plant height was recorded in janakdev (42.75 cm) and that of minimum was recorded in khumal rato-2 (21.45 cm). lower temperature during earlier months of growing might be the cause of slower earlier growth of potato (banjade, 2019). plant genetics and the quality of plant materials might be the cause for differences in plant height between the varieties (touria, 2017). these results are similar to those reported by s. banjade et.al. janakdev was the variety having highest plant height (banjade, 2019). international journal of applied biology, 6(2), 2022 118 table 2. plant height of different potato varieties at different days of observations treatments plant height (cm) 45 dap 60 dap 75 dap cardinal 4.05bc 9.55d 23.90cd janakdev 6.06a 19.60a 42.75a khumal rato-2 3.15c 11.27cd 21.45d khumal ujjwol 5.22ab 15.20bc 38.15ab ms 42.3 3.32c 13.25bcd 27.50cd bajhang local 4.25bc 16.20ab 32.30bc sem (±) 0.181 0.542 1.125 f probability <0.01 <0.01 <0.001 lsd (=0.05) 1.337** 4.005** 8.31*** cv, % 20.42 18.74 17.78 grand mean 4.34 14.17 31.008 mean fol l owed by common l etter(s ) wi thi n col umns are non -s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel , dap: days after pl anti ng number of leaves per plant the statistical analysis result shows that the number of leaves per plant is significantly different among the varieties at different days of observations (table 7). at 45 dap, maximum number of leaves per plant was recorded on bajhang local (15.05) and minimum number of leaves per plant was recorded on khumal rato-2 (8.95). similar results were observed at 60 dap where, leaves per plant was recorded maximum on bajhang local (30.75) which was at par with khumal ujjwol (29.50) and minimum on khumal rato-2 (16.98) which was at par with janakdev (17.85), ms 42.3 (18.20) and cardinal (18.70). at 75 dap, maximum number of leaves per plant was recorded on khumal ujjwol (40.90) which was at par with bajhang local (39.50) and minimum number of leaves per plant was recorded on cardinal (27.55). temperature and light intensity may interact to influence the number of leaves that grow. plant genetic differences among the varieties may be the cause that contributes to the significant differences in number of leaves that grow per plant. table 3. number of leaves of different potato varieties at different days of observations treatments no. of leaves 45 dap 60 dap 75 dap cardinal 10.90b 18.70b 27.55c janakdev 9.70b 17.85b 35.05abc khumal rato-2 8.95b 16.98b 30.55bc khumal ujjwol 11.85ab 29.50a 40.90a ms 42.3 10.57b 18.20b 30.97bc bajhang local 15.05a 30.75a 39.50ab sem (±) 0.435* 1.0009** 1.213* f probability <0.05 <0.01 <0.05 lsd (=0.05) 3.213 7.390 8.958 cv, % 19.08 22.29 17.43 grand mean 11.17 21.99 34.08 mean fol l owed by common l etter(s ) wi thi n col umns are non -s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel , dap: days after pl anti ng international journal of applied biology, 6(2), 2022 119 canopy diameter significant variation for canopy diameter was observed among potato varieties (table 8). from the combined analysis of variance, at 45 dap maximum canopy diameter was recorded from janakdev (18.65 cm) and minimum canopy diameter was recorded from ms 42.3 (7.15 cm). at 60 dap, widest canopy diameter was recorded on khumal ujjwol (32.40 cm) and narrowest canopy diameter was recorded on ms 42.3 (23.37 cm) which was at par with khumal rato-2 (23.52 cm). similarly, at 75 dap, maximum canopy diameter was observed from bajhang local (47.90 cm) which was at par with khumal ujjwol (47.65 cm) and janakdev (45.20 cm) and minimum canopy diameter was observed from khumal rato-2 (36.12 cm) which was at par with ms 42.3 (37.32 cm). leaf area index (lai) of the plants determines rates of energy and material exchange between plant canopies and the atmosphere (vose et al., 1994). genetic factors as well as environmental factors could be the causes of difference in canopy diameter among the varieties. table 4. canopy diameter (cm) of different potato varieties at different days of observations treatments canopy diameter (cm) 45 dap 60 dap 75 dap cardinal 11.15bc 29.80a 42.25ab janakdev 18.65a 31.30a 45.20ab khumal rato-2 8.77cd 23.52b 36.12b khumal ujjwol 14.52b 32.40a 47.65a ms 42.3 7.15d 23.37b 37.32b bajhang local 13.85b 30.55a 47.90a sem (±) 0.504 0.410 1.203 f probability <0.001 <0.001 <0.05 lsd (=0.05) 3.72*** 3.034*** 8.88* cv, % 20.008 7.066 13.79 grand mean 12.35 28.49 42.74 mean fol l owed by common l etter(s ) wi thi n col umns are non -s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel , dap: days after pl anti ng yield and yield attributing parameter number of tubers per plant by grade basis the analysis of variance for the average number of tubers per plant shows varieties to be significantly different for both marketable tuber and unmarketable tuber (table 9). highest number of marketable size tuber (>25g) was recorded from khumal ujjwol (12.05) which was at par with bajhang local (11.70) and lowest number of marketable size tuber (>25g) was recorded from khumal rato-2 (5.75) which was at par with janakdev (6.05). on the other hand, highest number of unmarketable size tuber (<25g) was recorded from bajhang local (5.55) and lowest number of unmarketable size tuber (<25g) was recorded from both cardinal and khumal rato-2(2.5). these differences in the numbers of tubers of different grades among varieties could be related to the variety's tolerance to the trial site's climatic circumstances, its genetics, or the quality of the potato seed (touria, 2017). international journal of applied biology, 6(2), 2022 120 table 5. number of tubers per plant by grade basis of different potato varieties treatments number of tubers per plant mtn (>25g) umtn (<25g) cardinal 8.40b 2.5b janakdev 6.05c 3.8ab khumal rato-2 5.75c 2.5b khumal ujjwol 12.05a 4.3ab ms 42.3 8.50b 3.7ab bajhang local 11.70a 5.55a sem (±) 0.234 0.305 f probability <0.001 <0.1 lsd (=0.05) 1.73*** 2.25ns cv, % 13.15 40.21 grand mean 8.74 3.72 mean fol l owed by common l etter(s ) wi thi n col umns are non -s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel , mtn: marketabl e tuber number, umtn; unmarketabl e tuber number tuber weight per plant by grade basis the effect of potato varieties on tuber weight by grade basis is presented (table 10). there was significant effect of varieties on marketable and unmarketable tuber weight per plant was observed. highest weight of marketable size tuber (>25g) was recorded from khumal ujjwol (443g) and lowest weight of marketable size tuber (>25g) was recorded from ms 42.3 (281.75g). and, highest weight of unmarketable size tuber (<25g) was recorded from bajhang local (52.25g) and lowest weight of unmarketable size tuber (< 25g) was recorded from khumal rato-2 (21.25g) which was at par with cardinal and ms 42.3 (24.5g) and janakdev (25.75g). table 6. tuber weight per plant (g) by grade basis of different potato varieties treatments tuber weight per plant (g) mtw (>25g) umtw (<25g) cardinal 384.25ab 24.50b janakdev 347.25bc 25.75b khumal rato-2 384.50ab 21.25b khumal ujjwol 443.00a 42.50a ms 42.3 281.75c 24.50b bajhang local 405.00ab 52.25a sem (±) 10.291 1.893 f probability <0.01 <0.01 lsd (=0.05) 75.98** 13.97** cv, % 13.47 29.17 grand mean 374.29 31.79 international journal of applied biology, 6(2), 2022 121 mean fol l owed by common l etter(s ) wi thi n col umns are non -s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel , mtn: marketabl e tuber number, umtn; unmarketabl e tuber number number and weight of tuber per plant the analysis of variance for number and weight of tuber per plant shows varieties to be significantly different (table 11). the highest number of tuber per plant was observed on bajhang local (17.25) and lowest number of tuber per plant was observed on khumal rato-2 (8.25). the highest tuber weight per plant (485.50g) was recorded from khumal ujjwol and lowest tuber weight per plant (306.25g) was recorded from ms 42.3. table 7. number of tuber and weight of tuber per plant of different potato varieties treatments number and weight of tuber number of tubers per plant weight of tubers per plant (g) cardinal 10.9b 408.75ab janakdev 8.35c 373.00bc khumal rato-2 8.25c 405.75ab khumal ujjwol 16.35a 485.50a ms 42.3 12.20b 306.25c bajhang local 17.25a 457.25a sem (±) 0.332 10.17 f probability <0.001 <0.01 lsd (=0.05) 2.45*** 75.09** cv, % 13.34 12.27 grand mean 12.21 406.08 mean fol l owed by common l etter(s ) wi thi n col umns are non-s i gni fi cantly di fferent bas ed on dmrt p=0.05, **si gni fi cant at 0.01 p l evel , ***si gni fi cant at 0.001 p l evel tuber yield (t/ha) the effect of different potato varieties on tuber yield (t/ha) was found to be significant. the highest tuber yield was recorded from khumal ujjwol (40.45 t/ha) which was statistically similar with bajhang local (38.10 t/ha) and lowest tuber yield was recorded from ms 42.3 (25.52 t/ha). the tuber yield is affected by environmental factors such as soil temperature, moisture, light intensity, fertilizer delivery, and disease and pest control (struik & wiersema, 1999). differences in yield among varieties might be genetically determined, the quality of potato seed, or the variety's adaptive responses to the experimental site's climatic conditions were also might be the factors for this result. higher yield in the local cultivar; ba jhang local is consistent with (khatri, 2004) and (shrestha et al., 2020)’s findings. international journal of applied biology, 6(2), 2022 122 table 8. yield (t/ha) of different potato varieties treatments yield (t/ha) cardinal 34.06ab janakdev 31.08bc khumal rato-2 33.81ab khumal ujjwol 40.45a ms 42.3 25.52c bajhang local 38.10a sem (±) 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(1994). factors influencing the amount and distribution of leaf area of pine. copenhagens: ecological bulletins 43. contact : anirban poddar dranirbanpoddar@gmail.com 115 abstract diabetes presents with a constellation of symptoms,hyperglycemia being just one of them.the primary challenge faced by physicians is to achieve a control over this exorbitant blood sugar levels.oral antidiabetic agents & insulin therapy are the primary modalities of treatment available presently. the most reliable diagnostic as well as prognostic marker used for this purpose is hba1c. adenosine deaminase (ada), an enzyme associated with purine metabolism is known to exert potent metabolic effects through its receptors. ada is primarily an established inflammatory marker reflecting t lymphocyte activity .adenosine,the substrate of ada is known to affect cholesterol synthesis in liver,thereby serving as a key hinge in the fine balance between glucose & lipid homeostasis. due to the association of ada with glucose & lipid homeostasis ,the authors believe that studying ada activity in serum of diabetics can reveal many important facts which might open a new dimension in diagnosis & treatment protocol for diabetes mellitus.the purpose of our study was to monitor ada activity in serum of diabetic patients who are on oral antidiabetic drugs & do not have evidence of microvascular or macrovascular complications of diabetes & also determine if any correlation exists between fasting blood glucose,hba1c & serum ada activity. issn : 2580-2410 eissn : 2580-2119 adenosine deaminase activity in serum of diabetics: a reflection of glycemic profile? anirban poddar 1*, and devyani de 2 1demonstrator, dept of biochemistry,deben mahato govt medical college & hospital,purulia,west bengal. 2assistant professor, dept of biochemistry, calcutta national medical college & hospital,kolkata,west bengal. introduction the unholy association of diabetes & obesity & its role in increasing cardiovascular risk is a well known fact(stina johanson,2007). dyslipidemia or an elevated level of triglycerides & free fatty acids in blood is a commonly encountered phenomenon & is du e to a jeopardised lipid metabolism(dennis mcgarry,2002). adenosine’s role as an antilipolytic agent is well established. adenosine exerts multiple effects through its receptors. the a1 receptor of adenosine is expressed exclusively in the adipose tissue & it is through this receptor adenosine exerts its antilipolytic effect (stina johanson,2007). adenosine deaminase (ada) catalyses the irreversible hydrolytic deamination of adenosine to inosine. open access international journal of applied biology keyword adenosine; ada; diabetes; glycemic marker; glycemic profile. article history received december 16, 2022 accepted june 14, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:dranirbanpoddar@gmail.com international journal of applied biology, 7(1), 2023 116 many researchers have in the past have reported elevation of ada activity in serum of patients suffering from diabetes mellitus (madhavi reddy a,1995). the exact reason for the elevation of ada activity in diabetics has not been elucidated clearly till now. insulin administration in diabetic subjects has shown to reduce ada activity (madhavi reddy a,1995). events such as hypoxia stimulate adenosine production & this increased adenosine production inturn acts as a stimulant for increased expression of ada.this phenomenon inturn reveals how ada acts, i.e by regulating the levels of intracellular & extracellular adenosine. diabetes mellitus is characterised by a gross imbalance in energy dynamics resulting in mitochondrial dysfunction,endoplasmic reticulum stress & hypertrophy of adipocytes (sarah de ferranti,2005).these hypertrophied adipocytes are capable of liberating lar ge amount of inflammatory cytokines. the immune cells in close proximity of the adipocytes infiltrate the adipose tissue easily resulting in insulin resistance associated with inflammation. adenosine has been proved to be responsible for glucose uptake in cells(l.vergauwen,1994). therefore in an insulin sensitive tissue,if ada activity is high,it will lead to depletion of adenosine & consequently glucose uptake in these cells will be compromised.ada is a key player involved in lymphocytic proliferation & d ifferentiation.tlymphocytes have been found to posses high ada activity. thus an inference may be drawn that if ada activity in insulin sensitive tissues is suppressed,it may facilitate glucose uptake in the cells resulting in a better utilisation of glucose at the cellular level. materials and methods study design this case control study was carried out at deben mahato govt medical college & hospital,purulia,west bengal over a period of 6 months. the subjects involved in the study were involved into two groups. 1. group athis group comprised of 20 patients with history of diabetes mellitus who presented to the general medicine opd.these patients were being treated with oral antidiabetic drugs & did not have any symptoms of microvascular or macrovascular complications of diabetes. 2. group b this group comprised of 20 age & sex matched healthy controls who presented to the institution for a regular health check up. this group served as the control group. a valid written consent was obtained from all the participants in the study. the study was approved by the institutional ethics committee at deben mahato govt medical college & hospital. the statistical data was analysed using spss v 17.0. inclusion criteria: 1. patients who are known cases of type 2 diabetes mellitus with no obvious symptoms of complications. 2. age>35 years & < 65 years . 3. patient being treated with oral antidiabetic drugs. 4. no h/o tuberculosis,psoriasis,viral hepatitis,rheumatoid arthritis. international journal of applied biology, 7(1), 2023 117 exclusion criteria: 1. diabetic patients who have symptoms of obvious complications of diabetes. 2. h/o rheumatoid arthritis,viral hepatitis,psoriasis,tuberculosis. 3. diabetics on insulin therapy. 4. pregnant women. method 7 ml of venous blood was collected with full aseptic precautions after 12 hours of fasting.2ml of this blood sample was collected in a fluorinated vacutainer for estimation of fasting blood sugar.rest 5 ml was collected in a plain vacutainer which was processed ultimately to obtain serum.this was used for analysing serum ada. estimation of glucose fasting blood glucose estimation was done by god-pod{glucose oxidase peroxidase method}.this is an enzymatic method employed in the clinical laboratory for the es timation of glucose.glucose is oxidized by glucose oxidase to gluconic acid and h2o2 is liberated. the colorimetric indicator, quinonemine is generated from 4 – amino antipyrene and phenol by h2o2 under the catalytic action of peroxidise. intensity of colo ur generated is directly proportional to glucose concentration. normal range in serum or plasma is 70 -100 mg/dl. estimation of ada ada estimation was done by a commercially available kit by tulip diagnostics pvt ltd,goa,which is based on the method describ ed by giusti & galanti (giusti & galanti,1984). adenosine deaminase hydrolyses adenosine to ammonia and inosine. the ammonia formed further reacts with a phenol and hypochlorite in an alkaline medium to form blue indophenols complex with sodium nitroprussi de acting as a catalyst.intensity of the blue coloured indo phenol complex formed is directly proportional to the amount of ada present in the sample. table 1. normal reference range of ada in serum normal <30 u/l strong suspect 30u/l40 u/l suspect positive > 40 u/l60 u/l positive >60 u/l results and discussion adenosine deaminase activity was found to be elevated in the diabetic patients as compared to the healthy controls in our study.increased adenosine deaminase activity would thus lead to depletion of adenosine.adenosine through its receptors exerts potent metabolic effects. a1 receptor agonists have been found to be associated with increased insulin sensitivity. thus,depletion of adenosine due to increased adenosine deaminase activity would mean increase in insulin resistance in the body & subsequent hyperglycemia,which is a hallmark feature of diabetes mellitus.this explains the hyperglycemia encountered in diabetic patients. international journal of applied biology, 7(1), 2023 118 our study also reveals a very strong correlation between fasting blood glucose levels & serum ada activity in patients with uncomplicated type 2 diabetes.a similar correlation also exists between serum ada levels & hba1c. hba1c is considered to be an established glycemic marker used to asses the glycemic status of a diabetic patient & also forms the diagnostic criteria for diabetes mellitus(hba1c>6.5 gm%).since a strong positive correlation exists between fasting blood glucose levels ,hba1c & serum ada,therefore estimation of serum ada might serve as an inexpensive glycemic marker for assessing the glycemic status of a diabetic patient on oral antidiabetic agents who is free from symptoms of microvascular & macrovascular complications of diabetes & also chronic inflammatory conditions in which ada activity tends to flare up.the data for the same is hereby depicted below: table 2. data for group a fasting blood sugar (fbs) serum ada hba1c 167 15 7.2 185 18 6.8 165 17 7.9 220 25 8.2 200 20 7.3 178 19 6.5 189 20 7.0 168 17 7.2 148 15 6.8 210 21 7.9 235 21 8.2 189 19 7.3 176 18 6.5 192 19 7.0 190 17 7.1 165 15 6.8 193 19 7.3 182 17 6.9 176 17 6.7 180 18 7.0 international journal of applied biology, 7(1), 2023 119 mean: 1. fbs185.4000±20.22089 2. serum ada-18.3500±2.39022 3. hba1c-7.1550±0.49362 figure 1. scatter plot showing correlation between fasting blood sugar levels & serum ada levels in patients with uncomplicated type 2 diabetes mellitus (group a). ‘r’ value: -0.832 ‘p’ value: ˂0.01(significant) figure 2. scatter plot showing correlation between serum ada levels & hba1c in patients with uncomplicated type 2 diabetes mellitus (group a). ‘r’ value: -0.755 ‘p’ value: ˂0.01(significant) international journal of applied biology, 7(1), 2023 120 table 3. data for group b fasting blood sugar(fbs) serum ada hba1c 80 5 5.0 76 8 6.1 74 9 5.3 70 7 5.0 69 6 5.3 79 9 5.6 96 9 4.7 90 8 6.0 84 8 5.2 90 9 5.3 69 6 5.0 88 8 6.0 84 7 5.8 90 7 6.1 75 9 4.6 77 8 6.1 72 7 5.8 71 7 5.7 70 9 5.2 73 8 5.1 mean: 1. fbs78.8500±8.42474 2. serum ada-7.9500±1.27630 3. hba1c-5.4450±0.48175 international journal of applied biology, 7(1), 2023 121 conclusion our study revealed a positive correlation between serum ada activity and hba1c levels as well as with fasting blood glucose levels. this raises an important question as to whether ada activity in serum is actually a reflection of the glycemic status of an individual.although our study goes in favour of this hypothesis,but it was done with a very limited sample size over a short span of time .more elaborate studies over longer period of time with a substantially larger sample size at different geographic locations are required to establish the above hypothesis. international journal of applied biology, 7(1), 2023 122 references stina johannson.2007 metabolic roles of adenosine. karolinska institute.,sweden. denis mcgarry j. dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. banting lecture 2001. diabetes 2002; 51 (1): 7-18. tomas dolezal. adenosine deaminase: review of physiological roles. 2001; university of south bohemia, czech republic. john n.fain, paul w. weiser. 1975.effects of adenosine deaminase on cyclic adenosinemonophosphate accumulation, lipolysis, and glucose metabolism of fat cells. the journal of biological chemistry; 250(3): 1027-1034. peter arner, jan ostman.1978. relationship between the tissue level of cyclic amp and the fat cell size of human adipose tissue. journal of lipid research; 19: 613 618. madhavi reddy a, rao y.n., yogendra singh, alpana saxena.1995. adenosine deaminase and protein tyrosine phosphatase activities in liver and peritoneal macrophages of streptozocin induced diabetic mice. indian journal of clinical biochemistry; 10(2):66 71. anjali c. warrier, narasimha y. rao, tarun k. mishra et al.1995. evaluation of adenosine deaminase activity and lipid peroxidation levels in diabetes mellitus. indian journal of clinical biochemistry; 10(1): 9-13. mustafa araz, yuksel ozdemir, mehmet tarakcyoolu et al .2000. elevated adenosine deaminase activity is not implicated in microvascular complications of type 2 diabetes mellitus except hba1c. turkish journal of endocrinology and metabolism; 4(3): 95-99. shiva prakash m, chennaiah s, murthy ysr et al. 2006. altered adenosine deaminase activity in type 2 diabetes mellitus. journal, indian academy of clinical medicin e; 7(2): 114-117. holger k. eltzschig, marion faigle, simone knapp, jorn karhausen, juan ibla, peter rosenberger et al.2006. endothelial catabolism of extracellular adenosine during hypoxia: the role of surface adenosine deaminase and cd26. blood; 108( 5): 16021610. contact : odo joel inya odojoel@gmail.com 79 abstract environment pollution is a bur ning topic of the day. air, water and soil are being polluted alike. soil being a "universal sink" bears the greatest burden of environmental pollution. it is getting polluted in a number of ways. there is urgency in controlling the soil pollution in order to preserve t he soil fertility and increase the productivity. in this re search work, the microbial and physioche mical assessment of soil contaminated with cassava waste water were studied using standard-based method and standard analytical methods. a total of 6 soil sample s were obtained from naka road, north bank and gboko road. three of the soil samples were contaminated with cassava waste water and the remaining three soil samples were used a s control. the isolation a nd enumeration of micr obial population was carried out using standard-ba sed methods. standard analytical methods were used to assay for physicoc hemical properties. t he highest bacterial count of 3.40x103 , 2.85x103 and 2.70x103 cfu/g for naka road, gboko r oad and north bank respectively w hile for uncontaminated soil were 4.70x104 , 2.90x104 and 2.70x104 cfu/g for north bank naka road, and gboko road respectively. there is significant difference in the total viable count between c ontaminated and uncontaminated (p<0.05). the fungal counts for the polluted and c ontrol soil ranged from fungi count 1.16 x 103 ±5.70 x 101 to 1.4×103±2.82×103 cfu/g, re spectively. the fungal counts were significantly lower tha n the bacterial counts (p < 0.05). the bacteria isolates were pseudomonas spp, bacillus spp, micrococcus spp, klebsiella spp, escherichia coli, staphylococcus spp, and proteus spp and for the fungi isolates were aspergillus spp, geotrichum spp, mucor spp and rhizopus spp. the pre sent study shows that the cassava effluent can have an increasing or limiting effect on the microbial diversity of the polluted soil which could also be attributed to the simultaneous impact on the physicochemical parameters of the soil. therefore the release of cassava waste water into the environment should be disc ouraged; processor should be traine d on simple treatment technique on e fflue nts that will make it less harmful to the environment. and there nee d for public awareness on the danger of releasing effluents into the environment. issn : 2580-2410 eissn : 2580-2119 microbial and physicochemical assessment of soil contaminated with cassava waste water in makurdi metropolis ebah esther eneyi 1, odo joel inya 2*, obochi irene ijakuwa 1 1 department of fisheries and aquaculture university of agriculture, makurdi, nigeria. 2 departments of microbiology, university of agriculture, makurdi, nigeria . open access international journal of applied biology keyword cassava; physicochemical; microbial; contamination article history received april 4, 2022 accepted december 14, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 80 introduction environment pollution is a burning topic of the day. air, water and soil are being polluted alike. soil being a "universal sink" bears the greatest burden of environmental pollution. it is getting polluted in a number of ways. there is urgency in controlli ng the soil pollution in order to preserve the soil fertility and increase the produc tivity. polluti on may be defined as an undesirable change in the physical, chemical and biological characteristics of air, water and soil which affect human life, lives of other useful living plants and animals, industrial progress, living conditions and cultural assets. a pollutant is something which adversely interfers with health, comfort, property or environment of the people. generally most pollutants are introduced in the envi ronment by sewage, waste, accidental discharge or else they are by-products or residues from the production of something useful. due to this our precious natural resources like soil, water and air are getting polluted (mohammed et al., 2014) microorganisms are very small forms of life that can sometimes live as single cells, although many also form colonies of cells. a microscope is usually needed to see individual cells of these organisms. many more microorganisms exist in topsoil, where food sources are plentiful, than in subsoil. they are especially abundant in the area immediately next to plant roots called the (rhizosphere), where sloughed-off cells and chemicals released by roots provide ready food sources. these organisms are primary dec omposers of organic matter, but they do other things, such as provide nitrogen through fixation to help growing plants, detoxify ha rmf ul chemicals (toxins), suppress disease organisms, and produce products that might stimulate plant growth. soil microorganisms have had another di rect importance for humans —they are the source of most of the antibiotic medicines we use to fight diseases (fred and harold, 2009). soil microorganisms can be grouped into bacteria, actinomycetes , fungi, algae, protozoa, and nematodes. apart from the dead plant or ani mal residues in soils, soil organic manure is composed of a significant content of living microorganisms and their dead fractions. the humus fraction is resistant to microbial decomposition and persists for thousands of years contri buting to the long -lived carbon pool in soils. soil microorganisms are involved i n the decomposition of soil organic matter, and the rate of decomposition depends both on the nature of microorganisms in soil and the nature of organic matter sources. enhancing the activities of soil fungi has been recognized as one of the potential options for reducing soil organic carbon turnover, thereby increasing carbon sequestration. melanin, chitin, and glomalin are examples of fungal-derived recalcitrant residues that tend to exist for a long time in soils. apart from the humification process, soil microorganisms are involved in mineralization of soil organic manure, thereby resulting in the loss of carbon from soils (thangavel et al., 2019). cassava (manihotesculentacrantz, synonymous with manihotutilissimarhol) belongs to the family euphorbiaceae. the tubers are quite rich in carbohydrates (85 -90%) wi th a very small amount of protein (1.3%) in addition to cyanogenicgluc oside (linamarin and lotaustiallin) which are present in cassava (nwabueze and odunsi, 2007). this high carbohydrate content makes cassava a major food item especially for the lower income earners in most tropical countries especially africa and asia (dess e and taye, 2001). cassava is a starchy food for more than 300 million people in many tropical countries of the world. cassava food products are the most important staples of rural and urban household in southern nigeria. in nigeria, traditional foods processed at home in small scale cottage operation constitute the principal mode of utilization of cassava (inges, 1982). international journal of applied biology, 6(2), 2022 81 it is generally believed to have originated from brazil in south america. cassava has spread to many other tropical countries like west indians, south east asia, and other west africa, especially in nigeria, sierra leone and liberia. in nigeria, cassava is extensively cultured and classified into two kinds: namely sweet cassava ( manihotesculenta) and bitter cassava (manihotutilisssima). bitter cassava contains glucoside which forms hydrocyanic acid during processing. hydrocyanic acid can be removed by cooking or fermenting in water for specific period. there are varieties of cassava which differ significantly in their colour, stem and period of maturity (iita, 2011). cassava processing plant also known as cassava mill was invented in 1919 and planted in 1934 and is extensively used in nigeria, especially in the southern part where cassava is a major agricultural produce. it is used to grind peeled cassava tubers which are drained for 2-4 days and then baked over fire in pans to produce garri a major staple food (fao, 2006). the edible tubers are processed into various forms which include chips, pellets, cakes and flour. the flour could be fried to produce garri or steeped in water to ferment and produce fufu when c ooked (oyewole and afolami, 2001). the produc tion and consequent consumption of cassava have increased extensively in recent times. the increased utilization of processed cassava products has increased the environmental pollution associated with the disposal of effluents. the highly offensive odour emanating from the fermenting effluent calls for regulation in the discharge of waste generated (akanniet al., 2006). in most areas, cassava mills are mainly on small scale basis, owned and managed by individuals who have no basic knowledge of environmental protection. though on small scale basis, there are many of them, which when put together, create enormous impact on the environment. c assava also contains much pollutant such as disease causing pathogens e.g. bacteria and fungi. disposal of agricultural by -products such as cassava waste from processing activities is a concern in nigeria. there is an appreciable high level of contami nation arising from the discharge of effluents on agricultural soil hence the need for proper treatment before discharge and conversion of these cassava wastes into biosorbent that can remove toxic and valuable metals from the effluent. effluent is a liquid or solid waste, especially chemicals produced by factories or from agricultural products or domestic waste. effluents usually contain a wide variety of chemicals, debris and various microorganisms which are mostly emptied on soil or carried away through special underground pipes called sewers. types of effluents include industrial effluent, agricultural effluents, domestic effluent and storm effluent (cheesbrough, 2005). the aim of this study is to determine the microbial and physiochemical assessment of soil contaminated with cassava waste water in makurdi metropolis, benue state. materials and methods study area this study was carried out in makurdi local government area. makurdi local government area has a population of 300,000 persons (npc, 2006), and lies between latitudes 7º40¹n and 7º53¹ n of the equator, and between longitudes 8º22¹e and 8º35¹e of greenwich meridian. it is a163km radius circle, covering 804km² land mass. climatically, makurdi falls within the tropical, sub-humid, wet and dry climate which has two distinct seasons, namely wet and season and dry season. the wet season starts from april and lasts till october, while the dry season starts in november and lasts till march. rainfall ranges from 775 millimeters to1792 millimeters, with a mean a nnual value of 1190 millimeters. international journal of applied biology, 6(2), 2022 82 sampling techniques a total of 6 soil samples were obtained from naka road, north bank and gboko road. three of the soil samples were contaminated with cassava waste water and the remaining three soil samples were used as control. the samples were collected using sterile containers and were transported to the laboratory for analysis. chemicals and reagents nutrient agar, macconkey agar, potato dex trose agar (pda), distilled water, acetone, simon’s citrate agar, urea agar, peptone water, hydrogen peroxide, lacto phenol cotton blue, and picric acid. equipments, apparatus and instruments weighing balance, test tubes and test tube rack, wire loop, heating mantle, conical flask, petri dish, ph meter, sprayer, measuring cylinder, aluminum foil, spectrophotometer, syringe, incubator, pressure pot, sample bottle, spirit la mp, cotton wool, microscope and microscope slide. enumeration of total heterotrophic bacteria and fungi samples of the polluted soil were serially diluted in ten fol ds. total viable heterotrophic aerobic counts were determined by plating in duplicate using pour plate technique. t hen the molten nutrient agar, eosin methylene blue agar at 45 0c and was potato dextrose agar was poured into the petri dishes containing 1ml of the appropriate dilution for isolation of the total heterotrophic bacteria and fungi, coliforms and escherichia coli respectively. they were swirled to mix and colony count was taken after i ncubating the plates at 300c for 48hrs and culture growth was preserved by sub culturing the bacterial isolates into nutrient agar slant which was used for biochemical tests. characterization and identification of bacterial and fungal isolates bacterial isolates were characterized and identify after studying their gram reaction as well as cell micro morphology. other tests like spore formation, motility, and catalase production. citrate utilization, oxidative/fermentative utilization of glucose, indole production, methyl red-voges proskauer reaction, urease and coagulase production, starch hydrolysis, production of h2s from tri ple sugar iron (tsi) agar and sugar fermentation were carried out according to the methods described by ochei and kolhatkar (2008) . fungal isolates were examined macroscopically and microscopically using the needle mounts technique. their identification was performed according to the scheme of apha (2005). determination of the physicochemical parameters a number of physicochemical parameters of the contaminated soil samples were determined. these include ph, conductivity, nitrate, phosphate, sulphate, oil content and exchangeable cations. the ph was measured using ph meter; conductivity was measured using conductivity meter. sulphate, nitrate and phosphate were determined using barium chloride (turbid metric), cadmium reduction and ascorbic acid methods respectively. all analyses were in accordance with apha (2008). international journal of applied biology, 6(2), 2022 83 biochemical tests catalase test: this test was carried out to determine the ability of the test organism to produce enzyme that breaks down hydrogen peroxide to oxygen and water. a drop of hydrogen peroxide was added to the growths isolated on the subculture plate and observation was made after 10-20 seconds. observation of white bubbles confirms positive, while no bubbles with original color gives negative result (cheesbrough, 2005). urease test: this test was done to determine the ability of the test organism to produce enzyme urease, which breaks down urea to ammonia and carbon dioxide. 2ml of urea agar was measured in to a sample bottle, slanted and allow cooling and jelling to occur. the test organism was collected, inoculated on the medium and incubated for 24 hour, after which a pink color was observed for positive and no color change for negative (cheesbrough,2005). indole test: this test was done to differentiate gram negative bacilli. 2ml of peptone water was dispensed in to a sterilized sample bottle and the test organism was inoculated. this was incubated for 24-48 hours at 35-370c after which 0.2ml of kovac’s reagent was added and mixed. a positive test gives pink coloration at the top of the medium, while no color change is an indicative of negative test (s, 2000). citrate test: this test was carried out to determine the ability of the test organism to utilize citrate as its sole source of carbon. si mon’s citrate agar medium was dispensed in a sample bottle and sterilize for 15 minutes at 1210c. the organism was collected and inoculated incubated for 24 hours at 370c. microscopy: after 48 hours of incubation the suspended organisms were seen and were used to prepared smears on clean slides. the slides was cleaned with alcohol, the test organism was placed on each of the clean slides using a sterilized wire l oop and each slide were stained with lactophenol for about 1 minutes. the slide was subjected to the observation of the suspected organism under oil immersion objective lens (x100) of a bright field microscope (baseyet al., 2000). results the mean viable, coliform and fungi count of soil samples contaminated with cassava waste water as presented in table 1, the total heterotrophic bacteria and fungi count range from 2.70 x 103±8.49 x 102 to 3.4×103±8.49×103 cfu/g and fungi count 1.16 x 103 ±5.70 x 101 to 1.4×103±2.82×103 cfu/g. control soil on the other hand ranges from 2.70 x 104±1.56 x 103 for gboko road to 4.0×104 ±2.83×103 cfu/g for naka road samples and 2.5×104 ±7.49×103 cfu/g typically there is significant variation(p<0.05). table 2, presents the prevalence of isolates across locations. table 3, presents the cultural morphology and biochemical characteristics of bacteria isolates. seven genera of bacteria were identified in this study pseudomonas spp, klebsiella spp, bacillus spp, escherichia coli, staphylococcus spp, and proteusspp. table 4, presents the morphology and characteristics of fungi isolates. four (4) genera of fungi were identified in this study: aspe rgillus spp, geotrichum spp, mucor spp and rhizopus spp. table 5, presents the physicochemical parameter of soil samples which were; temperature, soil ph, colour and texture. international journal of applied biology, 6(2), 2022 84 table 1. total viable, coliform and fungi count of samples from the study locations location tvc tcc tfc naka road 3.40 x 103 ±8.49 x 102b 1.60 x 103 ±8.49 x 102b 1.16 x 103 ±5.70 x 101c north bank 2.70 x 103±8.49 x 102b 2.00 x 103 ±1.13 x 103b 3.00 x 103±1.69 x 103c gboko road 2.85 x 103 ±3.54 x 102b 2.15 x 103 ±4.94 x 103b 1.40 x 104±2.82 x 103b control( naka road) 2.90 x 104±9.90 x 103b 4.90 x 104±1. 41 x 103a 1.90 x 103±4.29 x 102c control( naka road) 4.00 x 104±2.83 x 103a 5.95 x 104±1.20 x 104a 2.50 x 104±7.49 x 103a control( gboko road) 2.70 x 104±1.56 x 103a 5.40 x 104±2.82 x 104a 1.22 x 104±1.13 x 103a pvalue 0.008 0.001 0.002 table 2. prevalence of isolates across locations isolates l1 l2 l3 c1 c2 c3 total pseudomonas spp 0(0.00) 0(0.00) 0(0.00) 1(1.56) 2(3.13) 1(1.56) 4(6.35) bacillus spp 1(1.56) 1(1.56) 2(3.13) 3(4.69) 1(1.56) 2(3.13) 10(15.63) micrococcus 0(0.00) 1(1.56) 0(0.00) 1(1.56) 2(3.13) 1(1.56) 5(7.81) klebsiella spp 0(0.00) 1(1.56) 0(0. 00) 2(3.13) 3(4.69) 2(3.13) 8(12.50) escherichia coli 1(1.56) 0(0.00) 1(1.56) 1(1.56) 1(1.56) 0(0.00) 4(6.35) staphylococcus specie 1(2.86) 1(2.86) 0(0.00) 2(5.71) 0(0.00) 1(2.86) 5(7.81) proteus spp 0(0.00) 0(0.00) 1(1.56) 1(1.56) 2(5.71) 1(1.56) 5(7.81) aspergillus spp 1(1.56) 0(0.00) 1(1.56) 1(1.56) 1(1.56) 1(1.56) 5(7.81) geotrichum spp 0(0.00) 1(1.56) 0(0.00) 2(5.71) 1(1.56) 0(0.00) 5(7.81) mucor spp 0(0.00) 0(0.00) 0(0.00) 1(1.56) 0(0.00) 1(1.56) 4(6.35) rhizopus 2(5.71) 1(1.56) 2(5.71) 2(5.71) 2(5.71) 3(4.69) 2 (3.13) total 6(9.38) 6(9.38) 7(10.94) 17(26.56) 15(23.44) 13(20.31) 64(100) key: l1 – naka road l3 – gboko road c2control 2 s pp-s peci es l2 north bank c1control 1 c3control 3 international journal of applied biology, 6(2), 2022 85 table 3. morphology and biochemical characteristics of bacteria isolates. colony colour colony shape morphology gram’s reaction cat cit urease indole oxidase h2s suspected organisms cream ci rcul ar cocci + + + staphylococcus s pp green metal l i c sheen ci rcul ar rod + + escherichia col i yel l ow ci rcul ar rod + + + micrococcus s pp mucoi d ci rcul ar rod + + klebsiella s pp green ci rcul ar rod + + + pseudomonas s pp pal e ci rcul ar rod + + + + proteus s pp whi te irregul ar rod + + + bacillus s pp key: h2shydrogen sul phi de ci tci trate uti l i zati on catcatal as e producti on rxnreacti on table 4. macroscopic and microscopic characteristics of fungi macroscopic microscopic fungi isolates velvety filamentous white growth that sporulates black powdery spores long septate with conidiophores bearing brown spores and phialide at its apex aspergillus spp whitish smooth circular and raised colony or growth presence of arthrospore spores with rounded end geotrichum spp white and wooly aerial growth that darkens as its sporulates non-septate hyphae with straight sporangiophore with many spherical spores. mucor spp long hyphael growth which sporulated within two days to turn to black spore non-septate, branched mycelium with round shaped sporangia rhizopus spp key: s pp – s peci es table 5. physicochemical parameters sample temperature(ºc) soil ph colour texture naka road 28 8.0 brown silt north bank 29 7.0 dark-brown coarse gboko road 25 7.5 dark fine control soil 1 30 8.2 brown silt control soil 2 32 8.5 dark-brown x coarse control soil 3 31 8.4 dark fine international journal of applied biology, 6(2), 2022 86 discussion environment pollution is a burning topic of the day. air, water and soil are being polluted alike. soil being a "universal sink" bears the greatest burden of environmental pollution. (mohammed et al., 2014). the impact of cassava waste water on the physiochemical and microbial quality of the soil around cassava processing zone constitute great concern as it alters the natural environment. this effluent is released indiscriminately into the environment without any form of treatment. this activity of cassava processor has serious impact on the soil as these effluents contain chemical that may affect the biotic components of the soil. the result of this study reveal that the microbial population (total viable count, total coliform count and total fungi count) reduced significantly in soil contaminated with cassava effluent when compa red with control soil from the same area, although thi s contra dict the findings of igbinosa and igiehon (2015) whose findings indicated that there is significant increase observed in the microbial density of the polluted soil.total viable count for contami nated soil where 3.40x103 , 2.70x103 and 2.85x103 cfu/g for naka road, north bank and gboko road respectively while for unconta minated soil were 2.90x10 4 , 4.70x104 and 2.70x104 cfu/g respectively. there is significant difference in the total viable count between contami nated and uncontaminated (p<0.05).altho ugh igbinosa and igiehon (2015) observed that the fungal counts of the polluted soil were significantly lower than the bacterial counts generally(p<0.05). this results shows that cassava waste effluent negatively affect the microbial population. this may be attributed to the negative impact of harsh chemicals like cyanide that is present in the effluent a nd other chemical by-products of cassava fermentation. this finding agrees with (oti, 2002 and goodley,2004). also the study identified pseudomonas specie, bacillus micrococcus, klebsiella, escherichiacoli, staphylococcus and proteus as bacteria genera found in the study area while aspergillusspecie were fungi flora identified in this study. these bacteria and fungi species were isolated by previous authors (knowles, 1988; ehiagbonare et al., 2009). however, not all these soil microbial where found in the contaminated soil. pseudomonas, klebisiellaproteus specie and trichodema were not found at all in the three area studied but were found in the control soil thereby suggesting that these microbial genera could not withstand the negative impact of the effluent. these findings of concern as disruption of the microbial constitute serious threat to the soil. the fungal counts for the polluted and control soil ranged from fungi count 1.16 x 103 ±5.70 x 101 to 1.4×103±2.82×103 cfu/g, respectively. this suggests that the cassava effluent has effects on the fungal diversity of the polluted soil. the fungal counts were significantly lower than the bacterial counts (p < 0.05); and this is in agreement with the report from aiyegoro et al. (2007). conclusion based on the result of this study the following conclusions are reached. i. that release of cassava waste effluents to the soil affects the microbial population as well as the microbial diversities of the soil. ii. this waste contains pollutants that also affect the physicochemical composition of the soil. international journal of applied biology, 6(2), 2022 87 recommendations i. the release of cassava waste water into the environment should be discouraged. ii. cassava processor should be trained on simple treatment technique on effluents that will make it less harmful to the environment. iii. there is the need for public awareness on the danger of releasing effluents into the environment. iv. further research is recommended to find out ways for simple and affordable means of treatment and also ways of converting the effluent into useful substances (waste to wealth) that will benefit mankind. international journal of applied biology, 6(2), 2022 88 references aiyegoro, o. a., akinpelu, d. a., igbinosa, e. o. and ogunmwonyi , h. i. (2007). effect of cassava effluent on the microbial population dynamic and physicochemical characteristic on soil community. sci focus, 12: 98-101. american public health association (apha) standard methods for the examination of water and wastewater. (2005)american public health association, 20th ed. washington usa, pp 5-17. basey, j. m., mendelow, t. n., ramos, c. n. (2000). current trends of community college lab curricula in biology: an analysis of inquiry, technology and content. j bio. educ. 34 (2): 80-86 cheesbrough, m. (2005). district laboratory practice in tropical countries. cambridge university press, united kingdom, pp. 30-41. desse, g. and taye, m. (2001). microbial loads and microflora of cassava (manihotesculentagrantz) and effects of cassava juice on some food borne pathogens. j. food technol. afri.,6(1): 21-24. ehiagbonare, j. e., enabulele., s. a., babatunde, b. b. and adjarhore, r. (2009). effect of cassava effluent on okada denizens. sci res essay, 4: 310313. food and ag ricultural organization, fao. (2006). impact of cassava processing on environment: fao corporate documents repository, 12(4): 56-98. fred, m. and harold, van es. (2009). building soils for better crops, 3rd edition, sustainable agriculture research and education goodley, j. (2004). a compendium dhi-water and environment. 4th edn., fao, canada. international institute of tropical agriculture, iita. (2011). cassava processing in nigeria research for development, 15(7): 54-77. knowles, c. j. (1988). cyanide utilization and degradation by microorganisms. ciba foundation symposium 140: 3-15. nwabueze, t.u. and odunsi, f. o. (2007) optimization of process conditions from cassava (manihotesculenta) lafun production. afri. j. biotechnol., 6(5): 603-611. .ochei, j.o. and kolhatkar, a.a.(2008) medical laboratory science: theory and practice, tata mcgraw-hill publishing company limited, new york, pp. 637-745. oti, e. e. (2002). acute toxicity of cassava mill effluent to the african catfish. oyewole, o.b. and afolami, o.a.(2001) quality and preference of different cassava variety for lafun production. j. food technol. afri., 6(1): 27-29. thangavel, r., nanthis, b., mary, b. k., hasintha, w., manjaiah, k., cherukumalli, s. r., sasidharan, s., jörg, r. y., sik, o.u., choudhur ya, h., wangjk, c., tangl x., wangl, z., song m, o. w., freeman i. i. (2019) soil organic carbon dyna mics: impact of land use changes and management practices: a review, advances in agronomy, volume 156, 2019, pages 1-107 1(2)76-81 !!!!!!!!!!!!!!!!!!contact&:&agus&muji&santoso&&&&&&&agusmujisantoso@unpkediri.ac.id! ©!international!journal!of!applied!biology!! 76! abstract& soil!pathogen!is!one!of!main!problem!in!java!ginseng!production!in!tropical! field.!this!study!was!aimed!to!evaluate!the!impact!of!vam!application!to! java!ginseng!tuber!production.!application!of!vam!was!conducted!in!various! concentrations! (0,! 1,! 2,! and! 3%)! (w/w).! thin! layer! chromatography! was! used!to!detected!saponin!and!spectrophotometry!was!used!to!determine! total!saponin.!biomass,!the!number!of!rotten!tuber,!and!total!saponin!were! determined!after!eleven!months!under!greenhouse!condition.!inoculation! 3%!of!vam!can!enhance!the!number!of!healthy!and!biomass!java!ginseng! tuber!significantly.!but!the!application!of!vam!did!not!increase!total!saponin! content.!! issn!:!2580t2410! eissn!:!2580t2119! ! ! ! talinum(paniculatum((jacq)&gaertn&(java&ginseng)&production& using&vesicularbarbuscular&mycorrhizal& ! sulistiono1,&alfinda&novi&kristanti2&and&agus&muji&santoso1! ! 1!biology!education!department,!faculty!of!training!teacher!and!education,!university!of! nusantara!pgri!kediri,!east!java,!indonesia!! 2!chemistry!department,!faculty!of!science!and!technology,!airlangga!university,!surabaya,! indonesia! ! ! & & & & & & & & & & & introduction& java!ginseng!(portulacaceae)!is!an!herbaceous!medicine!of!tropical!plant.!the!plant!is! widely!used!as!active!compounds!source,!such!as!saponins,!flavonoids,!tannins,!triterpenes! or!sterols,!and!polyphenols.!java!ginseng!has!been!known!for!its!effectiveness!as!inducer!on! spermatogenesis!and!motility!of!sperm,!antitinflammatory,!androgenic!potency,!and!sperm! viability!(saroni!et#al.,!1999;!sumastuti,!1999;!winarni,!2009;!rahmi!et#al.,!2011).!saponins! are!glycosylated!secondary!metabolites!distributed!in!plant!tissues!(hu!et#al.,!2003).!santoso! et#al.#(2016)!was!reported!that!java!ginseng!rich!of!ginsenosides,!an!important!triterpenoid! saponin!as!well!as!the!other!ginsengs.!java!ginseng!is!a!potential!medicinal!plant!with!activity! as!good!as!other!medicinal!plants!such!as!korean!ginseng!or!another!ginseng!from!other! countries.!! there! are! many! previous! studies! revealed! the! efficacy! of! java! ginseng,! but! its! production!in!indonesia!has!not!been!optimal!yet.!there!are!some!factors!which!inhibit!java! ginseng!production!in!indonesia,!such!as!the!lack!information!of!good!cultivation!techniques! in!field.!java!ginseng!plantation!in!field!also!has!its!own!some!challenges,!such!as!tuberteating! nematode!which!attack!the!plant!and!cause!tuber!rotten!as!well!as!soil!pathogens!do.!this! &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword! saponin! vesicular.arbuscular# mycorrhizal! soil!pathogen! talinum#paniculatum algae.! article&history! received!23!november!2017! accepted!30!december!2017!! international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international#journal#of#applied#biology,#1(2),#2017# ! 77! case! has! been! recorded! on! sewon! bantul! yogyakarta! and! plosoklaten! kediri! (east! java! provice)!indonesia!as!the!central!of!java!ginseng!production.!! vesiculartarbuscular! mycorrhizal! (vam)! is! vesicle! formed! by! fungi! which! have! a! benefit!on!plant!growt!(sikes,!2010;!gazey!et#al.,!2004;!altkaraki!&!altraddad,!1997)!because! it!can!absorb!phosphor!in!optimum!amount!plants!needed!to!survive!in!drought!condition! (nurhayati,!2010;!berta!et#al.,!1995).!it!also!protects!plants!from!pathogens!(rianto!et#al.,! 2005;!gazey!et#al.,!2004;!simanungkalit,!1997).!for!example,!a!glomus#sp.!fungus!on!vam!has! capability!in!inhibition!of!several!microbial!pathogens!activity!which!causes!plant!wilting,!such! as!pseudomonas#solanacearum#(rianto!et#al.,!2005).!plant!with!mycorrhizal!roots!can!absorb! zn!and!s!from!the!ground!faster!than!plant!with!nontmycorrhizal!root!(ruiztlozano#et#al.,! 1995;!abbott!et#al.,#1992).!this!means!that!vam!application!is!promising!in!increasing!the! java!ginseng!production!in!indonesia.! one!problem!of!java!ginseng!tuber!production!in!indonesia!is!the!lack!information!of! good!planting!techniques.!this!research!is!important!because!the!results!will!help!to!obtain! information!about!good!planting!techniques!which!can!be!applied!by!farmers!to!provide! healthy!tuber!of!java!ginseng.& & materials&and&methods& application&of&vam! java!ginseng!seeds!were!collected!from!field!in!plosoklaten!kediri!east!java!during!dry! season!from!may!to!august!2013.!java!ginseng!seed!were!obtained!by!cutting!the!dry!flower! panicles.!seeds!were!germinated!in!tray!and!taken!at!21!days!after!germination.!germinated! java!ginseng!then!transferred!into!polytbags!filled!with!soil!and!sand!(1:1)!and!planted!for!2! weeks.!the!seedlings!then!inoculated!with!vam!in!some!concentration!(0,!1,!2,!and!3%)!(w/w).! vam!inoculates!which!used!in!this!research!were!obtained!from!biotechnology!center,!bogor! agricultural!institute.!plants!were!treated!for!8!months!with!liquid!compost!supplementation.! weeding! and! pest! controlling! were! done! during! the! treatment.! java! ginseng! roots! were! harvested!by!cutting!the!stem!base!at!3t4!cm!from!the!surface!of!planting!medium.!planting! medium!with!root!was!given!water!until!saturated!then!the!plastic!bag!was!cut!on!one!side! and!root!can!be!released!slowly.!furthermore,!ginseng!roots!were!cleaned!from!soil!particles! using! tap! water.! roots! were! obtained! biomass! under! 70oc! until! constant! condition! (g).! healthy!tubers!per!plant!was!evaluated!by!counting!of!rotten!bulb!using!this!formula!(rotten! bulb!index!=![nr/nhr];!nr!is!the!number!of!rotten!bulb!of!tuber!per!plant!and!nhr!is!the! number!of!healthy!tuber!per!plant)! & determination&of&saponin&content! saponin!content!of!air!dried!java!ginseng!bulbs!were!measured!through!qualitative,! semi!quantitative!and!quantitative!methods!(santoso,!2012).!one!gram!of!dried!samples!was! grinded!into!fine!powder!in!room!temperature.!extraction!was!done!by!maceration!using!80%! of!ethanol!for!3!times!then!saturated!in!water!bath!on!80oc!until!shrunk!into!0,1!ml.!detection! of!java!ginseng!root!saponin!content!has!been!done!using!thin!layer!chromatography!(tlc)! method!using!propanol!and!water!(14:3).!chromatograms!then!was!removed,!dried!at!room! temperature,!and!sprayed!with!anisaldehidth2o2,!followed!by!heating!at!110°c.!the!saponin! existence!will!be!presented!by!dark!green!to!black!spot.!while!quantitative!test!performed!by! spectrophotometer!(365!nm)!with!80%!of!ethanol!which!reacted!with!vanillin!reagent.!tlc! and! quantitative! test! were! calculated! according! to! the! method! as! described! by! santoso! international#journal#of#applied#biology,#1(2),#2017# ! 78! (2012).!all!chemical!reagents!were!purchase!in!pro!analysis!grade!by!sigma.! & data&analysis& biomass!and!total!saponin!were!performed!by!onetway!analysis!of!variance!(anova)! using!the!statistical!software!spss!for!windows.!duncan’s!multiple!range!tests!were!used!to! evaluate!significant!difference!between!means!at!a!significant!level!(p#<!0.05).!biomass!and! saponin!spot!in!chromatogram!were!evaluated!descriptively. & results&and&discussion&& vam! inoculation! was! conducted! in! java! ginseng! successfully! for! 8! months! under! greenhouse!condition.!application!of!vam!can!enhance!of!biomass!(table!1)!and!the!number! of!healthy!java!ginseng!tuber!(table!2).!there!were!a!significant!increase!of!biomass!and!the! number! of! healthy! java! ginseng! tuber.! in! the! present! research.! higher! biomass! can! be! obtained!by!adding!3%!of!vam!(11,11!±!0,49!g!per!plant)!but!inoculation!of!2%!can!provide! healthy!tuber!of!java!ginseng.!in!control!group,!the!number!of!rotten!bulb!up!to!28%!(0,28)! (figure!1b)!and!tended!to!decrease!in!1%!vam!inoculation.!! !! & & figure&1.&thin&layer&chromatography&of&ethanol&extract&of&java&ginseng&tuber&after&vam& inoculation&on&silica&gel&(standard&saponin&=&s)&(a)&and&spot&of&rotten&bulb&in& control&group&caused&pathogen&(white&arrow)&(b).( & table&1.&biomass&of&java&ginseng&tuber&after&vam&inoculation&(g)& treatment& biomass&(g)& pt0%! 9,16!±!1,42c! pt1%! 10,21!±!0,84a! pt2%! 10,61!±!0,78a! pt3%! 11,11!±!0,49b! *different!letters!in!the!same!column!indicate!significant!differences!at!the!level!of!duncan!test!(p#<!0.05)! ! in!order!to!evaluate!saponin!content,!tlc!was!used!and!be!confirmed!quantitatively! by!using!spectrophotometry!method.!both!of!tlc!and!spectrophotometry!method!were!done! successfully.!all!samples!contain!rich!saponin.!it!can!be!confirmed!by!the!same!color!of!spot! s& pb0%& pb1%& pb2%& pb3%& (a)& (b)& international#journal#of#applied#biology,#1(2),#2017# ! 79! which!appear!on!chromatograms!as!the!color!of!saponin!standard!(figure!1a).!thus,!the!result! indicated! that! there! was! no! significant! total! saponin! of! java! ginseng! tuber! after! vam! inoculation!(table!3).!the!present!study!revealed!that!vam!inoculation!in!java!ginseng!can! enhance!the!biomass!and!improving!the!healthy!tuber!but!did!not!total!saponin.!!!! & table&2.&root&number&of&java&ginseng&on&8&months&after&vam&inoculation.& treatment! rotten&bulb! pt0%! 0,28! pt1%! 0,11! pt2%! t! pt3%! t! *rotten!parts! ! table&3.&saponin&content&on&java&ginseng&tuber&(mg/g&sample)& treatment& saponin&content& pt0%! 13,33!±!0,3055a! pt1%! 13,34!±!0,8083a! pt2%! 13,23!±!0,5133a! pt3%! 13,37!±!0,3055a! *different!letters!in!the!same!column!indicate!significant!differences!at!the!level!of!duncan!test!(p#<!0.05)! ! vam!application!on!plant!is!expected!to!give!benefit!on!plant!growth!(berta!et#al.,! 1995;! michelsen! &! rosendahl,! 1990).! the! benefit! for! plant! is! vam! can! provides! some! essential!nutrients!such!as!p,!mg,!k,!fe,!and!mn!(setiadi,!1989).!vam!has!ability!to!form!colony! as!mycorrhizal!hyphae!on!root!surface!(rianto!et#al.,!2005;!altkaraki!et#al.,#2004;!berta!et#al.,! 1995).! this! hyphae! extension! helps! root! in! nutrients! intake,! especially! in! nutrienttpoor! conditions,!low!ph,!and!lack!of!water!(dearnaley,!2007;!stewart!et#al.,!2005).!mycorrhizal!on! plant!roots!can!increase!zn!and!s!absorption!from!ground!faster!than!nontmycorrhizal!root! plants!(abbott!et#al.,!1992;!michelsen!&!rosendahl,!1990).!application!of!vam!does!not! increase!plant!secondary!metabolites!production,!such!as!saponin!in!java!ginseng!tubers!in! this!study.!the!mechanism!of!saponin!biosynthesis!or!main!function!in!plant!are!unclear!until! now!(santoso!et#al.,!2016).! on! the! other! hand,! vam! application! prevents! rotten! tuber.! rotten! tuber! can! be! detected!by!cut!the!tuber!transversely.!healthy!tubers!do!not!have!any!spots!on!it!when!being! cut!transversely.!brown!or!red!spots!found!in!tuber!when!being!cut!indicate!infection!that!can! spread! and! root! will! become! rotten.! this! result! study! shows! that! vam! application! can! prevents!tuber!damage!caused!by!soil!pathogens!attack!(yadav!et#al.,#2012;!sikes,!2010;!berta! et#al.,!1995),!such!as!fungi!and!nematodes!(michelsen!&!rosendahl,!1990).!previous!study! revealed!that!the!existence!of!several!fungi!such!as!glomus#sp!(rianto,!et#al.,!2005;!altkaraki! et#al.,#2004;!altkaraki,!1998;!altkaraki!&!altraddad,!1997).!futher!research!needed!to!be!done! specially!to!evaluate!the!nutrient!uptake,!physiology,!and!ginsenoside!kinetics!of!java!ginseng! under!vam!inoculation!in!field& && conclusions& this!study!shown!that!vam!application!on!java!ginseng!prevents!tuber!damage!caused! by!pathogen.!however,!there!is!no!significant!difference!on!dry!biomass!data!and!saponin! levels! of! java! ginseng! tuber! between! control! and! treatment! group.! therefore,! further! international#journal#of#applied#biology,#1(2),#2017# ! 80! research!about!the!increasing!of!saponin!level!is!needed,!such!as!drought!stress!techniques.! & acknowledgment&& this!research!has!been!funded!by!the!directorate!of!higher!education,!ministry!of! education!and!culture!of!indonesia!and!thanks!to!febi!kurnia!putri!for!her!excellent!assistant! in!laboratory.!& & references& abbott,!l.k.,!robson,!a.d.,!jasper,!d.a.!&!gazey,!c.!1992.!what!is!the!role!vam!hyphae!in! soil?!in:!read!dj,!lewis!dh,!fitter!ah,!alexander!ij!(eds).!mycorrhizas!in!ecosystems.!cab! international,!uk:!oxford.! altkaraki,!g.n!&!altraddad,!a.!1997.!effects!of!arbuscular!mycorrhizal!fungi!and!drought! stress!on!growth!and!nutrient!uptake!of!two!wheat!genotypes!differing!in!drought! resistance.#mycorrhiza,!7:83–88.!! altkaraki,!g.n.!1998.!benefit,!cost!and!watertuse!efficiency!of!arbuscular!mycorrhizal!durum! wheat!grown!under!drought!stress.!mycorrhiza,!8:41–45.! altkaraki,! g.n,! mcmichael,! b,! &! zak! j.! 2004.! field! response! of! wheat! to! arbuscular! mycorrhizal!fungi!and!drought!stress.!mycorrhiza,!14:263–269.! berta,! g.,! trotta,! a.,! fusconi,! a.,! hooker,! 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(talinum! paniculatum)!to!amount!and!motility!of!spermatozoa!in!mice.!warta#tumbuhan#obat# indonesia,!5:13t14![in!indonesia].! setiadi,! y.! 1989.! utilization! of! microorganisms! in! forestry.! departemen! pendidikan! dan! kebudayaan!direktorat!jenderal!pendidikan!tinggi.!pusat!antar!universitas!bioteknologi! ipb,!bogor,!indonesia![in!indonesia].! sikes,!b.a.!2010.!when!do!arbuscular!mycorrhizal!fungi!protect!plant!roots!from!pathogens.! plant#signaling#&#behavior,!5(6):763t765.!! simanungkalit,! r.d.m.! 1997.! effectiveness! of! 10! species! of! arbuskular! mycorrhizal! fungi! isolated! from! west! java! and! lampung! on! maize! and! soybean.! in:! jenie! ua! (ed)! indonesian!biotechnology!conference!1997:!proceeding!of!the!indonesian!biotechnology! consortium,!iuc!biotechnology!ipb,!bogor,!indonesia.! stewart,!l.i.,!hamel,!c.,!hogue,!r.!&!moutoglis,!p.!2005.!response!of!strawberry!to!inoculation! with! arbuscular! mycorrhizal! fungi! under! very! high! soil! phosphorus! conditions.! mycorrhiza,!15:612–619.! sumastuti,! r.! 1999.! effect! of! inflammation! of! leaf! infusion! and! root! of! somjawa! (t.! paniculatum)! in! white! rats! in! vitro.! warta# tumbuhan# obat# indonesia,# 5:15t17! [[in! indonesia].! winarni,!d.!2009.!androgenic!potential!of!ginseng!javan!root!(talinum#paniculatum#gaertn.)! on!low!testosterone!conditions.!dissertation:!airlangga!university![in!indonesia]! yadav.!k.,!singh,!n.!&!aggarwal,!a.#2012.!arbuscular!mycorrhizal!technology!for!the!growth! enhancement!of!micropropagated!spilanthes#acmella#murr.!plant#protect.#sci.,#48(1):31– 36.! ! ! ! contact : prabin adhikari adkprabin26@gmail.com 65 abstract nepal is a small south asian country with a varied geographical and bioclimatic landscape. invasive plant species have already been introduced in large numbers, causing the extinction of native species and disrupting crop production, as well as degrading endangered animal habitats, changing plant species composition, and interfering with tree seedling regeneration. however, it is critical in the health treatment of rural populations in developing countries. our study will look at and compile knowledge on the therapeutic applications of invasive plants utilized by a range of nepalese communities. we combed through numerous websites, including research gate and google scholar, for historical and contemporary studies on the therapeutic usage of nepal's invasive plants.24 species from 12 families of invasive alien plant species were identified to be utilized for medicinal purposes by nepalese rural people. the 39 diseases for which medicinal plants were reported were diabetes, rheumatism, fever, ulcers, bronchitis, kidney stone, asthma, urinary insufficiency, and others. traditional knowledge of invasive plant species' uses is diminishing due to the negative effects of invasive plant species on the ecosystem; hence, rigorous documentation of ethnomedicinal knowledge on invasive plant species is needed. thus, the medicinal potentialities of unwanted invasive species, which are sometime s neglected by others, will provide a gem for the study world. issn : 2580-2410 eissn : 2580-2119 ethno-medicinal usage of invasive plants in traditional health care practices: a review prabin adhikari1*, swastika acharya1, sabina sigdel1 and ganga kandel1 1 institute of forestry, tribhuvan university, pokhara campus, pokhara, nepal introduction plant-based traditional medical practices are founded on hundreds of years of beliefs and observations that precede the formation and dissemination of modern medicine (aburjai et al., 2007; mukherjee and wahile, 2006), and this knowledge has been passed down orally from generation to generation without the use of any written documents ( samy and ignacimuthu, 2000). over the last few decades, medicinal plants have seen a considerable increase in popularity for use in traditional medicine, as well as contemporary and alternative medicine, since they are inexpensive, effective at curing diseases, and relatively safe with few or no adverse effects (sigdel and acharya, 2021). ethnic diversity and indigenous knowledge in nepal have resulted in extensive ethnobotanical research (pappan and thomas, 2017). because of a global renaissance in traditional and alternative healthcare systems, the market open access international journal of applied biology keyword exotic; invasive plant species; traditional healers; medicinal plants; alien species article history received march 18, 2022 accepted december 14, 2022 i nternational journal of a pplied biology is lic ensed under a c reative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. international journal of applied biology, 6(2), 2022 66 for herbal pharmaceuticals has risen at an amazing rate, and medicinal plants are consequently very important economically (kunwar, 2013). nepal is a tiny south asian country with a diversified geographical and bioclimatic landscape (tiwari et al., 2005). it is home to 118 different habitats and 3.2% of the world's flora (gon/ mofsc, 2014). nepal has already seen the introduction of a huge number of alien plant species due to its location in the heart of the himalayan biodiversity hotspot and between two big economically growing asian countries (china and india) (shrestha et al., 2019a). biological invasions are widespread a nd the fifth most significant cause of global change, with almost one-fifth of the earth's surface vulnerable to plant and animal invaders (ipbes, 2019; sala et al., 2000). richardson and rejmánek (2011) define invasive alien species (ias) as species whose range has been altered by humans, either unintentionally or intentionally, resulting in self-replicating populations capable of spreading across broad regions in their new home. in nepal, 27 species (15%) of the 182 naturalized plant species are invasive. some alien species, which are often farmed, may serve local societies with food, medicine, fuel, or fodder(kull et al., 2007; roder et al., 2007), while some are responsible for the deterioration and extinction of native species, disturbances in crop production, habitat degradation for endangered wildlife (e.g., one-horned rhinoceros; murphy et al., 2013), changes in plant species composition (timsina et al., 2011; shrestha and dangol, 2014; thapa et al., 2016), interference with tree seedling regeneration (thapa et al., 2017), livestock grazing, and human health deterioration (sharma et al.,2005; kohli et al., 2006). iaps are being incorporated into indigenous traditional people's everyday lives (geldenhuys and macdevette, 1989). however, the usage of alien plant species in ethnomedicine has grabbed less attention (shackleton and shackleton, 2018; shackleton et al., 2007; novoa et al., 2016). this study aims to discuss the medical applications of invasive alien plants based on current research and to describe these species' beneficial impact on nepalese rural residents. materials and methods review methodology we conducted a literature study to determine the medicinal uses of invasive species for human health. we looked through both academic and non-academic publications. the phrases "invasive," "alien," "exotic," "medicinal uses," "human well -being," and "benefit" were typed into google scholar and the research portal. only case studies that directly investigated the medical use of invasive species were considered after all of the sources were filtered for relevance. also, an evaluation of further information on the distribution pattern and diversity of invasive alien species was conducted. we also looked for additional sources by going through the reference lists of all of the case study articles that were included. in nepal, there has been very little study on the medicinal uses of invasive species. therefore, the goal is to give a summary of what is currently known and a platform from which to make suggestions for future research. results and discussion our study found a total of 27 invasive alien species, among which 24 plant species were enumerated as having medicinal values belonging to 12 families. the asteraceae family has the most invasive species, with ten, followed by fabaceae with three, and amaranthaceae with two and one species each belonging to papaveracece, pontederiaceae, convolvulaceae, international journal of applied biology, 6(2), 2022 67 verbenaceae, poaceae, lamiaceae, oxalidaceae, araceae, and rubiaceae (shrestha et al., 2018; shrestha 2019a; shrestha and shrestha, 2019) (figure 1). iaps distribution and richness vary with elevation, physiographic (terai, siwalik, middle mountains, and high mountains), and phytogeographic areas (eastern, central, and western nepal)(bhattarai et al., 2014). naturalized plant species richness rises with heig ht, peaks at about 1100m above sea level, and declines as elevation rises (bhattarai et al., 2014) (figure 3). the plant parts used along with their medicinal uses and altitudinal range and phytogeographic regions (eastern, central, and western) are mentioned in table 1 and table 2 respectively. most of the plant species have multiple uses and they are used to treat 39different ailments. diabetes, fever, rheumatism, ulcers, bronchitis, asthma, kidney stone, urinary insufficiency, syphilis, and other disorders were among the 39 ailments for which medicinal plants were documented. almost all the plant parts were used to cure numerous diseases. the most regularly used plant part is a leaf (14 species), followed by a whole plant (7 species), root (4 species), seed, flower (3 species), fruit (1 species), fluid (1 species), and unidentified for 3 species (figure 2). table1. names of plants with their ethnomedicinal uses and parts used s.n sci enti fi c name common name (local name) fami l y parts us ed us es references 1 ageratina adenophora (spreng.) r.m.ki ngandh.r ob. crofton weed (kal o banmara) as teraceae leaf and root treatment of s ki n di s eas es , wound, i tchi ng, meas l es , uteri ne bl eedi ng jami r et al ., 2018 2 ageratum conyzoides l. bi l l ygoat weed (gandhe) as teraceae whol e pl ant treatment of wounds and burns , fever, pneumoni a, rheumati s m, headache, and col i c mi ng, 1999 3 ageratum houstonianum mi l l . bl ue bi l l ygoat weed (ni l ogandhe) as teraceae leaf and whol e pl ant us ed i n cuts and wounds , ski n i nfecti ons andradecetto, 2009 ; dani and ti wari , 2018 4 alternanthera philoxeroides (mart.) gri s eb. al l i gator weed (jal jambu) amaranthace ae whol e pl ant treatment of ni ght bl i ndnes s , mal ari a, hazy vi s i on, fi s tul a, pos t-natal compl ai nts , di arrhea , dys entery, and puerperal fever, the mens trual fl ow, l eucorrhoea, l epros y, eczema, gonorrhea rahman and gul s hana, 2014 5 amaranthus spinosus l. spi ny pi gweed (lunde kanda) amaranthace ae root and leaf to treat burns wounds , boi l s , al egbej o, 2013 international journal of applied biology, 6(2), 2022 68 gonorrhea , eczema, earache, gal actagogue, hemorrhoi ds , s ores , ophthal mi a, menorrhagi a, and anti dote to s nakebi te poi s on 6 argemone mexicana l. mexi can poppy (thakal ) papaveraceae seed and l eaf treatment of s everal di s eas es i ncl udi ng tumors , warts , s ki n di s eas es , i nfl ammati ons , rheumati s m, j aundi ce, l epros y, mi crobi al i nfecti ons , and mal ari a. brahmachari , et al ., 2013 7 bidens pilosa l. hai ry beggar’s s ti ck (kal o kuro) as teraceae leaf and pl ant fl ui d treatment of fever, angi na, di abetes , edema kvi eci ns ki eta l ., 2008 8 chromolaena odorata (spreng.) r.m.ki ngandh.r o si am weed (seto banmara) as teraceae leaf treatment of coughs and col ds , toothache, s ore throat, mal ari a, wounds , di arrhea , s ki n i nfecti on, dys entery, s tomach ache, convul s i ons, and pi l es , omokhua et al ., 2016 9 eichhornia crassipes (mart.) sol ms . water hyaci nth (jal kumbhi ) pontederi ace ae root, leaf, and fl ower treat hai r fragrance, chol era, s ore throat, s nake bi tes , wound, i nhi bi t cel l growth to treat cancer and treatment of l i pid di s order ayanda et al ., 2020 10 erigeron karvinskianus dc. karwi ns ky’s fl eabane (phul e jhar) as teraceae uni denti fi e d treatment of headache, ki dney s tones , bronchi ti s , di arrhea, body pai n, hematuri a, arthri ti s , indi ges ti on, enteri ti s , epi demi c hepati ti s , fauzi ana and sus andari ni, 2019 international journal of applied biology, 6(2), 2022 69 os teoporos i s , and cys ti ti s 11 galinsoga quadriradiata rui z and pav. shaggy sol di er (jhus echi tl ang e) as teraceae fl ower to treat s nake bi te, col d, anemi a, and j aundi ce sal tos et al ., 2016 12 ipomoea carnea s ubs p. fistulosa (mart. ex choi s y) d.f. aus ti n bus h morni nggl ory (bes haram) convol vul ace ae leaf, s eed, and fl ower us ed agai ns t immunodefi ci enc y syndrome (aids), hypertens i on mei ra et al ., 2012 13 lantana camara l. lantana (ban fanda, ki rnekanda) verbenaceae leaf treatment of col d, whoopi ng cough, headache,as thma , chi ckenpox, eye i nj uri es , bronchi ti s , and arteri al hypertens i on kal i ta etal ., 2012 14 leersia hexandra swartz. southern cut gras s (karauteghan s ) poaceae uni denti fi e d treatment of hypertens i on, i mprovement of l i ver and ki dneys functi ons bi l anda et al ., 2019 15 mesosphaerum suaveolens (l.) kuntze bus hmi nt (ban si l am) lami aceae leaf treatment of di ges ti ve tract and res pi ratory di s eas es bezerra et al ., 2020 16 mikania micranthakunth mi l e-a-mi nute weed (lahare banmara) as teraceae leaf us ed to bathe ras hes , and s ki n i tches , to make a poul ti ce for s nake bi tes and s corpi on s ti ng, and heal i ng s ores li et al ., 2013 17 mimosa pudica l. sens i ti ve pl ant (laj j awati ) fabaceae leaf treatment of di abetes , uri nary i nfecti ons , cancer, hepati ti s , and obes i ty muhammad et al ., 2016 18 oxalis latifolia kunth. purpl e wood s orel (thul o chari ami l o) oxal i daceae whol e pl ant treatment ofcuts , fever, col d, cough, di arrhea, traumati c i nj uri es , dys peps i a, pi l es , anemi a, dementi a and convul s i ons,s prai ns , and uri nary tract i nfecti ons sarkar et al ., 2020 19 parthenium hysterophorus l. partheni um (pati jhar) as teraceae uni denti fi e d remedy for uri nary tract i nfecti ons , s ki n patel , 2019 international journal of applied biology, 6(2), 2022 70 i nfl ammati on, rheumati c pai n, mal ari a, di arrhea, dys entery, and neural gi a. 20 pistia stratiotes l. water l ettuce (kumbhi ka, jal akumbhi ) araceae whol e pl ant, l eaves us ed for a s tomach di s order,throat, curi ng ri ngworm, eczema, anodyne for eyewas h, rel i evi ng ear compl ai nts , l epros y, ul cers , pi l es , chroni c dermati ti s , fever i ntes ti nal bacteri al i nfecti ons , and mouth i nfl ammati on tul i ka etal ., 2015 21 senna occidentalis (l.) li nk coffee senna (thul otapre, panwar) fabaceae treatment of paras i tic s kin i nfecti ons es s i en, etal ., 2019 22 senna tora (l.) roxb. si ckl e pod s enna (sano tapre) fabaceae s eed and l eaf treatment of toothache, s ore throat, mal ari a, wounds , di arrhea, s ki n i nfecti on, convul s i ons, dys entery, pi l es , s tomach ache, coughs , and col ds shukl a et al ., 2013 23 spermacoce alataaubl . broadl eaf botton weed (al u pate) rubi aceae whol e pl ant treatment of headache, mal ari a, di arrhea s ki n di s eas es , i nfl ammati on of eye and gums , fever, hemorrhage, uri nary and res pi ratory i nfecti ons cons ervaand jes u, 2012 24 xanthium strumarium l. rough cockl ebur (bhede kuro) as teraceae whol e pl ant,root, and frui t, us ed foreheadache, gas tri c ul cer, urti cari a, rhi niti s, nas al s inus itis, fungal i nfecti ons , rheumati s m bacteri al , and arthri ti s . fan et al ., 2019 international journal of applied biology, 6(2), 2022 71 table 2. distribution of invasive alien plant species (iaps) in nepal s.n scientific name elevation (m asl) phytogeographic regions 1 ageratina adenophora (spreng.) r.m.kingandh.rob. 130-3280 e,c,w 2 ageratum conyzoides l. 75-2140 e,c,w 3 ageratum houstonianum mill. 60-2160 e,c,w 4 alternanthera philoxeroides (mart.) griseb. 65-1505 e,c,w 5 amaranthus spinosus l. 60-2640 e,c,w 6 argemone mexicana l. 65-1400 e,c,w 7 bidens pilosa l. 100-2930 e,c,w 8 chromolaena odorata (spreng.) r.m.king and h.ro 75-1710 e,c,w 9 eichhornia crassipes (mart.) solms. 60-1500 e,c,w 10 erigeron karvinskianus dc. 780-2110 e,c,w 11 galinsoga quadriradiata ruiz and pav. 560-2800 e,c,w 12 ipomoea carnea subsp. fistulosa (mart. ex choisy) d.f. austin 60-1350 e,c,w 13 lantana camara l. 70-1715 e,c,w 14 leersia hexandra swartz. 100-800 e,c 15 mesosphaerum suaveolens (l.) kuntze 75-1065 e,c,w 16 mikania micranthakunth 70-1200 e,c,w 17 mimosa pudica l. 75-1495 e,c,w 18 oxalis latifoliakunth. 600-2570 e,c,w 19 parthenium hysterophorus l. 60-1990 e,c,w 20 pistia stratiotes l. 70-800 e,c,w 21 senna occidentalis (l.) link 70-1405 e,c,w 22 senna tora (l.) roxb. 75-1300 e,c,w 23 spermacoce alataaubl. 110-2000 e,c,w 24 xanthium strumarium l. 60-2500 e,c,w (eas tern=e, wes tern=w and central =c) international journal of applied biology, 6(2), 2022 72 figure 1. division of plant species based on their family figure 2. line graph showing plant parts used 0 2 4 6 8 10 12 asteraceae fabaceae amaranthaceae papaveraceae pontederiaceae convolvulaceae verbenaceae poaceae lamiaceae oxalidac eae araceae rubiaceae 14 7 4 3 3 1 3 0 2 4 6 8 10 12 14 16 leaf whole plant root seed flower fruit unidentified international journal of applied biology, 6(2), 2022 73 figure 3. elevation range of invasive species conclusions for those living in nepal's rural areas, invasive alien plant species are vital sources of medicine. a total of 24 invasive plant species belonging to 12 families have been discovered to play an important part in traditional therapeutic practices.plant components such as roots, leaves, fruits, and even the entire plant have therapeutic properties.invasive plants in new locations change the makeup of indigenous communities, reduce species variety, disrupt ecosystem processes, and cause massive economic and ecological imbalances. on the other side, they can be employed as a medical tool to improve human society's well -being. the advancement of these traditional medical systems with the goals of safety, efficacy, and quality will not only help to maintain this traditional legacy, but will also assist to rationalize the use of natural products in health care. an invasive plant species that poses a threat to native plants and civilization can be exploited to profit in a variety of ways. the usage of invasive plants in this paper may be useful for other people to include these species into their traditional health-care practices, reducing the load on native medicinal plants.in nepal, most people's ethnic knowledge of invasive plant species remains untapped. traditional knowledge of invasive plant species' uses is diminishing due to the negative effects of invasive plant species on the ecosystem; hence, rigorous documentation of ethnomedicinal knowledge on invasive plant species is needed.thus, the medicinal potentialities of unwanted invasive species, which are sometimes neglected by others, will provide a gem for the study world. 0 500 1000 1500 2000 2500 3000 3500 a g e ra ti n a a d e n o p h o ra a g e ra tu m c o n yz o id e s a g e ra tu m h o u st o n ia n u m a lt e rn a n th er a p h ilo xe ro id es a m a ra n th u s sp in o su s a rg e m o n e m ex ic a n a b id en s p ilo sa c h ro m o la e n a o d o ra ta e ic h h o rn ia c ra ss ip es e ri g e ro n k a rv in sk ia n u s g a li n so g a q u a d ri ra d ia ta ip o m o e a c a rn e a s u b sp .… la n ta n a c a m a ra le e rs ia h e xa n d ra m es o sp h a er u m s u a ve o le n s m ik a n ia m ic ra n th a m im o sa p u d ic a o xa lis la ti fo lia p a rt h en iu m h ys te ro p h o ru s p is ti a s tr a ti o te s se n n a o cc id en ta lis se n n a t o ra sp e rm a co ce a la ta x a n th iu m s tr u m a ri u m e le v a ti o n r a n 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https://doi.org/10.1111/wre.12238 timsina, b., shrestha, b. b., rokaya, m. b., & münzbergová, z. 2011. impact of parthenium hysterophorus l. invasion on plant species composition and soil properties of grassland communities in nepal. flora-morphology, distribution, functional ecology of plants, 206(3),233-240. https://doi.org/10.1016/j.flora.2010.09.004 tulika, t., & mala, a. 2015. pharmaceutical potential of aquatic plant pistia stratiotes (l.) and eichhornia crassipes. journal of plant science, special issue: medicinal plants, 3, 10 18. doi: 10.11648/j.jps.s.2015030101.12 http://rdo.psu.ac.th/sjstweb/journal/38-6/38-6-11.pdf https://doi.org/10.1111/wre.12238 https://doi.org/10.1016/j.flora.2010.09.004 contact : sudeep poudel sudip.poudel20@gmail.com 47 abstract excessive synthetic fungicide use reduces soil's antagonistic population, allowing soil-borne plant pathogens to cause significant global losses. despite high fungicide application, plant diseases persist, harming the ecosystem. trichoderma spp, an eco-friendly plant symbiont, can serve as an alternative biocontrol agent. this study evaluated trichoderma harzianum's antagonistic effect against fungal pathogens and its compatibility with fungicides in in-vitro using a completely randomized design with four replications per treatment. trichoderma harzianum showed high antagonistic activity for alternaria brassicicola (70.35%), fusarium solani (70.82%), helminthosporium sorokinana (66.55%), rhizoctonia solani (78.58%), sclerotium rolfsii (92.53%). among the tested fungicides, copper oxychloride and mancoxeb at 400 ppm showed maximum compatibility with growth inhibition per cent (gip) of 2.41% and 7.91%, respectively, after 60 hours of incubation. fungicides viz., carbendazim, hexaconazole, and carbendazim+mancozeb at all concentrations aren’t compatible with 100% gip throughout the experiment, and for metalaxyl+mancozeb, high growth inhibition percent was observed ranging from 42.77% to 78.40% making it incompatible. in integrated disease management, compatible fungicides at recommended doses can be used in combination with t. harzianum. issn : 2580-2410 eissn : 2580-2119 biological control of fungal phytopathogens with trichoderma harzianum and its fungicidal compatibility. sudeep poudel1*, pratit khanal2, bigyan k.c.3, sadikshya pokharel4, sandesh gauli5 1department of plant pathology, pg program, institute of agriculture and animal science, tribhuvan university, chitwan, bagmati province 2institute of agriculture and animal science, lamjung campus, kathmandu, bagmati province 3department of plant breeding, pg program, institute of agriculture and animal science, tribhuvan university, kathmandu, bagmati province 4institute of agriculture and animal science, lamjung campus, pokhara, gandaki province 5institute of agriculture and animal science, gauradaha campus, chitwan, bagma ti province introduction prolonged and continuous application of chemical inputs for enhancing soil fertility and better disease management has resulted in unforeseen hazardous environmental effects. this has led to the reduction of bacterial and fungal antagonistic populations, a s well as an increase in several soil-borne diseases. despite the various noxious effects on the environment, the use of the total amount of fungicides and fertilisers has skyrocketed globally open access international journal of applied biology keyword trichoderma; fungicides; food poisoned technique; integrated disease management; antagonistic effect. article history received april 19, 2023 accepted june 21, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 7(1), 2023 48 to control different plant pathogens and to fulfil the food dem and of the growing world population through intensive agriculture (vitousek et al., 1997; frink et al., 1999). thus, the main challenge in present agriculture is to increase productivity and control plant disease by reducing the use of harmful chemical fun gicides. in this regard, it is imperative to use microbial inoculants such as plant growth -promoting rhizobacteria and trichoderma spp. as an alternative (bagwan, 2010; alori and babalola, 2018). trichoderma spp. is an omnipresent, asexually reproducing fungus found most frequently in the soil which can antagonise a wide range of soil-borne plant pathogens, induce both localised and systemic resistance in various plants with a substantial influence on plant immunity, growth, and development in field and greenhouse conditions (enshasy et al., 2020; tyśkiewicz et al., 2022). since the 1920s, trichoderma spp. has been known for its capability to function as a biocontrol agent (bca) against various plant pathogens, and among its many strains, mainly trichoderma harzianum, trichoderma viride, and trichoderma virens have been identified as promising strains having potential in biological controls because of its multiple modes of action such as mycoparasitism, antibiosis, endophytic survival on host and secretion of enzymes and metabolites (monte & llobell, 2003; benítez et al., 2004; inayati et al., 2020; zaenab et al., 2020). significant growth inhibition of phytophthora capsici and fusarium oxysporum (das et al., 2019), alternaria solani of tomato (imran et al., 2022), plasmopara viticola of vitis vinifera (perazzoli et al. 2012), rhizoctonia solani in tobacco seedlings (gveroska and ziberoski, 2011) by t. harzianum had reported by other authors as well. halifu et al., (2019) reported that applying t. harzianum e15 had increased seedling biomass and height, root structure index, soil nutrients, and soil enzyme activity. various t. harzianum strains, alone or in combination with fungicides, have been playing a pivotal role in the management of various seed and soil-borne diseases in integrated disease management (idm), and these strain’s application not only suppresses diseases but also have comprehensive advantages of improved seed germination, increased plant size, promote crop precocity, augmented leaf area an d weight (samuels, 1996; atomare et al., 1999; banjac et al., 2021; yassin et al., 2021). biocontrol agents, along with a combination of chemical fungicides, would also provide almost similar levels of disease suppression as achieved with higher fungicides (monte, 2001). however, several chemicals harm the growth of trichoderma spp. in nepal, the data suggest that there has been an indiscriminate use of chemicals, especially in vegetable farming (bhandari et al., 2018). this helps to develop fungicide-resistant strains of pathogens, even demanding more toxic chemicals in the coming days. so, it is mandatory to seek compatible fungicides to be used with trichoderma spp. in idm. our long-term goal is to adopt environmental-friendly approaches for disease management, hinder the development of resistance in pathogens to chemicals, build up bacterial and fungal antagonistic populations in the soil, and seek cost -effective methods in disease management. hence, this study aims to evaluate the compatibility of t. harzianum with chemical fungicides for the control of plant pathogens, to develop environment ally friendly and cost-effective disease management strategies. this study has the potential to international journal of applied biology, 7(1), 2023 49 offer valuable insights into the selection of fungicides that do not harm the growth and effectiveness of t. harzianum, enabling the development of integrated strategies that enhance disease suppression while decreasing chemical inputs. materials and methods the experiment was conducted at the central laboratory of the institute of agriculture and animal science, lamjung campus, and paklihawa campus, nepal, in 20 20 and 2021. isolation of pathogens two fungal phytopathogens: helminthosporium sorokiniana and alternaria brassicicola were isolated from the diseased leaf of wheat (agronomy farm of iaas, phaklihawa) and cabbage (horticulture farm of iaas, lamjung). for isolation, diseased samples were cut into pieces of 1 cm and surface sterilised in 1% sodium hypochlorite solution for 1 minute, followed by three consecutive washes in distilled water. samples were then placed on sterilised blotter paper and air-dried inside laminar flow. they were subsequently plated on water agar plates and incubated at 27±1°c. after 48 hours, the growt h of the pathogen was observed, and the pathogen was confirmed based on its microscopic characteristics, as in figure 1. the identified mycelia were later transferred to potato dextrose agar plates to obtain pure cultures of the pathogens. pure cultures of fusarium solani, rhizoctonia solani, and sclerotium rolfsii were obtained from the nepal agriculture research council (narc). figure 1. conidia – (a) helminthosporium sorokiniana, (b) alternaria brassicicola foodpoisoned technique a pure culture of t. harzianum was obtained from narc, and an in-vitro compatibility test was conducted using six commonly used chemical fungicides by nepalese farmers (listed in table 1) in a completely randomised design by food poisoned technique describ ed by nene and thapliyal (1993). international journal of applied biology, 7(1), 2023 50 table 1. chemical fungicides used in an experiment. s.n. trade name active ingredient mode of action 1 uthane m-45 mancozeb 75%wp contact 2 blitox copper oxychloride 50%wp contact 3 saaf carbendazim 12% + mancozeb 63% wp systemic + contact 4 kriloxyl gold metalaxyl 8% + mancozeb 64% wp systemic + contact 5 navistin carbendazim 50% wp systemic 6 hexa hexaconazole 5% ec systemic under dark conditions, pure culture was incubated for four days in a bacteriological incubator at 25±2˚c. further, a subculture was made, and a 5-day-old culture with green mycelia was used for an experiment. for the evaluation of compatibility of chemical fungicides, stock solutions of five fungicides were prepared by dissolving 1 gm of each fungicide in 10 ml of distilled water to make 75,000 mg/l of mancozeb 75% wp and carbendazim 12% + mancozeb 63% wp, 72,000 mg/l of metalaxyl 8% + mancozeb 64% wp and 50,000 mg/l of copper oxychloride 50% wp and carbenazim 50% wp. in the case of hexaconazole, it’s available 5% ec solution was taken as the original stock solution. 80 μl, 160 μl, and 240 μl stock solutions of each mancozeb and carbendazim + mancozeb, 83.3 μl, 166.6 μl, and 249.9 μl stock solutions of metalaxyl + mancozeb, 120 μl, 240 μl, and 360 μl stock solutions of each copper oxychloride, carbendazim and hexaconazole were mixed in lukewarm molten pda to prepare 100 mg/l, 200 mg/l and 300 mg//l of 60 ml a mended media. then in each 90 mm sterilised petri plate, 20 ml of poisoned media with different fungicides was poured and allowed to solidify. the poisoned pda plates amended with fungicides were inoculated aseptically in laminar flow with a 5mm mycelial d isc of t. harzianum grown on subcultured plates. for each treatment, four replications were made, and an unamended pda media served as a control, and all the treated plates along with the control were incubated in a bacteriological incubator at 25±2˚c. measurement of mycelial growth (mm) was measured using a verniercalliperr scale after 24, 36, 48, and 60 hrs of incubation, and growth inhibition per cent (gip) was calculated. gip was calculated using the following formula given by vincent (1947). per cent growth inhibition (%) = a–b/a × 100 where, a = mycelium growth of the t. harzianum on the control plate b= mycelium growth of the t. harzianum on a treated plate after each observation, all the data were entered in ms excel (2013), an analysis of variance was done using rstat software (version 3.5.3), and a mean comparison was made using the fisher-lsd test at a 0.05 level of significance. dual culture five days old cultures of t. harzianum and a week of the culture of each fungal pathogen were taken for the study. a 7 mm disc of pathogens and t. harzianum were placed on solidified pda medium on the opposite ends of the plate at equidistance from the international journal of applied biology, 7(1), 2023 51 periphery. the experiment followed a completely randomised design with three replications for each treatment. plates incubated with the pathogen alone were used as a control. after seven days of incubation, the radial mycelial growth of t. harzianum and the pathogens was measured, and the growth inhibition of fungal pathogens by t. harzianum was calculated using the formula given by vincent (1947). results and discussion in vitro screening of t. harzianum for compatibility with different fungicides all screened fungicides significantly inhibited the radial mycelium growth of t. harzianum over the control, as demonstrated in table 2 and figure 2. table 2. per cent growth inhibition of trichoderma harzianum at various concentrations of different chemical fungicides under in-vitro at different time intervals. s. n. treatments conc (ppm) mycelial diameter(mm) per cent growth inhibition (%) 24 hrs 36 hrs 48 hrs 60 hrs 24 hrs 36 hrs 48 hrs 60 hrs 1 control 52.53 66.76 78.59 83 0.00 0.00 0.00 0.00 2 mancozeb 400 32.66 46.12 67.81 76.44 37.81f 30.92fg 13.71h 7.91h 500 24.02 40.03 57.40 70.63 54.27de 40.03e 26.96f 14.91fg 600 20.4 28.98 51.38 64.54 61.16d 56.59c 34.62e 22.25e 3 carbendazim 400 0.00 0.00 0.00 0.00 100a 100a 100a 100a 500 0.00 0.00 0.00 0.00 100a 100a 100a 100a 600 0.00 0.00 0.00 0.00 100a 100a 100a 100a 4 hexaconazole 400 0.00 0.00 0.00 0.00 100a 100a 100a 100a 500 0.00 0.00 0.00 0.00 100a 100a 100a 100a 600 0.00 0.00 0.00 0.00 100a 100a 100a 100a 5 carbendazi+ mancozeb 400 0.00 0.00 0.00 0.00 100a 100a 100a 100a 500 0.00 0.00 0.00 0.00 100a 100a 100a 100a 600 0.00 0.00 0.00 0.00 100a 100a 100a 100a 6 metalaxyl + mancozeb 400 21.49 34.52 40.65 47.50 59.09d 48.30d 48.28d 42.77d 500 16.37 26.50 32.88 40.78 68.83c 60.30c 58.17c 50.87c 600 11.34 16.98 25.48 34.57 78.40b 74.57b 67.58b 58.35b 7 copper oxychloride 400 39.09 56.10 71.25 81.00 25.57g 15.96h 9.34h 2.41i 500 33.14 49.08 63.75 73.93 36.92f 26.48g 18.89g 10.93gh 600 26.87 43.87 54.50 68.75 48.84e 34.29ef 30.65ef 17.17f grand mean 76.16 71.52 67.12 62.64 cv (%) 4.26 3.64 3.87 3.28 lsd (p≤0.05) 5.37 4.31 4.29 3.40 conc= concentration, cv= coefficient of variation, hrs= hours, lsd= least significance difference, mm= millimetre, ppm= parts per million international journal of applied biology, 7(1), 2023 52 figure 2. mycelial growth diameter of trichoderma harzianum after 60 hours of inoculation in media amended with different concentrations of various chemical fungicides (a) mancozeb 400 ppm, (b) mancozeb 500 ppm, (c) mancozeb 600 ppm, (d) carbendazim 400 ppm, (e) carbendazim 500 ppm, (f) carbendazim 600 ppm, (g) hexaconazole 400 ppm, (h) hexaconazole 500 ppm, (i) hexaconazole 600 ppm, (j) carbendazium+mancozeb 400 ppm, (k) carbendazium+mancozeb 500 ppm, (l) carbendazium+mancozeb 600 ppm, (m) metalaxyl + mancozeb 400 ppm, (n) metalaxyl + mancozeb 500 ppm, (o) metalaxyl + mancozeb 600 ppm, (p) copper oxychloride 400 ppm, (q) copper oxychloride 500 ppm, (r) copper oxychloride 600 ppm, (s) control copper oxychloride at 400 ppm exhibited the highest compatibility with mycelial growth, resulting in diameters of 39.09 mm, 56.10 mm, 71.25 mm, and 81.00 mm, and gip of 25.57%, 15.96%, 9.34%, and 2.41% after 24, 36, 48, and 60 hours of incubation, respectively, among the six chemical fungicides. its 500 ppm concentration also showed promising results and was statistically at par (p≤0.05) with 400 ppm of mancozeb after 24, 36, and 60 hours of incubation. on the other hand, carbendazium, hexaconazole, and carbendazium+mancozeb proved to be extremely lethal, with a gip of 100% throughout the experiment. the fungicide metalaxyl+mancozeb had a varying gip, ranging from 42.77% to 78.40%, and showed a deleterious effect on the mycelial diameter of t. harzianum. overall, the results indicate that copper oxychloride and mancozeb are highly compatible with t. harzianum at all tested concentrations, suggesting that they can be used effectively in integrated disease international journal of applied biology, 7(1), 2023 53 management strategies to control soil-borne plant pathogens while maintaining a healthy soil environment. for fungicides viz., mancozeb, metalaxyl+mancozeb, and copper oxychloride , a significant increase (p≤0.05) was observed in per cent inhibition as the concentration increased, and with time, the gip of these fungicides decreased gradually. this may be, up to our understanding, due to the decreased efficacy of these fungicides an d the increased ability of the t. harzianum to degrade these chemicals with time. for carbendazium, hexaconazole, and carbendazium+mancozeb, there was 100% growth inhibition at all the tested concentrations with no gradual decrease in gip with time. gowdar et al., (2006) and manandhar et al., (2020) also reported that the percentage inhibition of the t. harzainum increased with an increase in concentration and decreased with an increase in the incubation period. antagonistic effect of trichoderma harzianum against pathogen after incubating for seven days, t. harzianum significantly inhibited the mycelial growth of several fungal phytopathogens, such as alternaria brassicicola (by 70.35%), fusarium solani (by 70.82%), helminthosporium sorokinana (by 66.55%), rhizoctonia solani (by 78.58%), and sclerotium rolfsii (by 92.53%), as shown in figure 3 and figure 4. figure 3. growth inhibition of different fungal pathogens by trichoderma harzianum figure 4: growth inhibition of helminthosporium sorokinana by trichoderma harzianum 0 20 40 60 80 100 g ro w th i n h ib it io n p e rc e n ta g e fungal pathogens growth inhibtion percentage of trichoderma harzianum against various fungal pathogens alternaria brassiciola helminthosporium sorokiniana fusarium solani rhizoctonia solani sclerotium rolfsii international journal of applied biology, 7(1), 2023 54 the study found that t. harzianum, a soil antagonistic fungus, is compatible with copper oxychloride and mancozeb but not with carbendazim 50 % wp, hexaconazole 5 %, or carbendazim 12 % + mancozeb 63 % at all the tested concentrations. several earlier reports had also reported good growth of trichoderma spp. at low and moderate levels of different fungicides (srinivasulu et al., 2005; elshahawy et al., 2016; shashikumar et al., 2019). this information holds significance in developing integrated disease management strategies, as soil-borne plant pathogens can be difficult to control with a single method. by using biocontrol agents such as t. harzianum in combination with compatible agrochemicals, farmers can more effectively eradicate plant diseases while also being environmentally conscious. additionally, t. harzianum was found to significantly inhibit the growth of various fungal pathogens, suggesting its potential as a biocontrol agent for crop protection . sonavane and venkataravanappa (2017) found 0% mycelial inhibition of t. harzianum with mancozeb 75wp and copper oxychloride 50 wp even at higher concentrations of 1500ppm. also, they found that carbendazim 50 % wp at 250 ppm, hexaconazole 5 % at 500 ppm, and carbendazim 12 % + mancozeb 63 % wp at 500 ppm completely suppressed the growt h of t. harzianum with 100% mycelial inhibition revealing no compatibility with antagonistic soil fungus, trichoderma. manandhar et al. (2020) also reported mancozeb, copper oxychloride, and metalaxyl + mancozeb as compatible fungicides and hexaconazole, c arbendazium, and carbendazium + mancozeb as incompatible fungicides when tested at 50 and 100 ppm. the high inhibition of benzimidazole compounds, i.e. carbendazim is due to its binding with β tubulin of fungal pathogen and causes inhibition of microtubule assembly which ultimately hinders cell division and may lead to cell death (zhou et al., 2016). moreover, it inhibits dna synthesis and blocks nuclear division (clemons & sisler, 1971; davidse, 1973; hammerschlag & sisler, 1973). similarly, hexaconazole's high inhibition capability is due to the presence of systematic demethylation inhibitors. these inhibitors primarily target the vegetative stage of fungi, disrupting the development of mycelium both internally and externally within the host plant (khalfallaha et al., 1998). saaf, combined fungicide, is a mixture of carbendazim (12%) and mancozeb (63%), so it has a collective effect of systematic and contact fungicides resulting in high mycelium inhibition. t. harzianum significantly suppressed the growth of all the fungal pathogens that were tested. chamoli et al. (2020) also recorded a high gip of 67.22% for alternaria brassicicola by t. harzianum and 73.55% by t. asperellum. singh et al. (2018) reported high growth inhibition per cent of 73.07%, 63.84%, and 69.22% for bipolaris sorokiniana by trichoderma viride, trichoderma harzianum, and trichoderma virens, respectively. bastakoti et al. (2017) reported high growth inhibition of sclerotium rolfsii (100%), rhizoctonia solani (62%), and fusarium solani (68%) by trichoderma spp. similarly, manandhar et al. (2019) also reported more than 80% growth suppression of fusarium solani and rhizoctonia solani by trichoderma isolates. the high growth inhibition of phytopathogens by t. harzianum is due to the rupture of the cell wall of the host fungus by multiple enzymes, mainly glucanolytic and chitinolytic enzymes (monte, 2001; sood et al., 2020). international journal of applied biology, 7(1), 2023 55 the study on the biocontrol potential of trichoderma harzianum against soil-borne phytopathogens has promising implications for sustainable agriculture. however, there are some limitations to be considered, such as the specific strains of t. harzianum used in this study may have different interactions with various agrochemicals and fungal pathogens, and their efficacy may vary across different agroecological zones. additionally, this study tested the efficacy of t. harzianum with a limited number of fungal pathogens, and other soil -borne plant pathogens may respond differently to biocontrol agents. these limitations highlight the importance of conducting further research to investigate the potential of t. harzianum to mitigate different soil-borne phytopathogens and their compatibility with a wider range of chemicals. conclusion the finding reveals that t. harzianum poses high growth inhibition for alternaria brassicicola, bipolaris sorokiniana, sclerotium rolfsii, rhizoctonia solani, and fusarium solani. among the fungicides tested, mancozeb and copper oxychloride are compatible with t. harzianum while hexaconazole, carbendazium, and carbendazi+ mancozeb are extremely lethal with 100 % gip throughout the experiment. metalaxyl + mancozeb also poses high gip throughout the experiment. fungicides with lower gip can be used in combination with t. harzianum in idm to control different fungal phytopathogens. future research should focus on field trials to evaluate the effectiveness of t. harzianum, either independently or in conjunction with compatible chemicals identified in 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https://doi.org/10.3390/ijms23042329 https://doi.org/10.1890/1051-0761(1997)007%5b0737:haotgn%5d2.0.co;2 https://doi.org/10.1890/1051-0761(1997)007%5b0737:haotgn%5d2.0.co;2 https://doi.org/10.1016/j.jksus.2021.101363 https://doi.org/10.1094/phyto-08-15-0186-r 2(1)47-54 international journal of applied biology, 2(1), 2018 47 abstract kappaphycus alvarezii is a red algae that can be used as alternative raw material for bioethanol production. this is because k. alvarezii contains a high carbohydrate that reaches 60%. this study aims to determine the effect of fermentation nutrition and fermentation duration of hydrolysis results of k. alvarezii algae on ph changes, sugar levels, cell biomass and ethanol content. there are two methods was used in this research, namely hydrolysis method and fermentation method. the hydrolysis method was used α-amylases enzyme which is 150 knu/l with 0.5%, 1.0%, 1.5% and 2.0% concentrations. the next method, k. alvarezii algae was fermented by pichia kudriavzevii and used gandasil-d® as an anorganic suplementation and yeast extract which is 0 g/l, 1 g/l, 2 g/l and 3 g/l. the results showed that k. alvarezii can be fermented into bioethanol after enzymatic hydrolysis process. the optimal ethanol content was produced at 48 hours of incubation duration. issn : 2580-2410 eissn : 2580-2119 production of bioethanol from kappaphycus alvarezii algae by using pichia kudriavzevii nurfadillah, winda lestari taufan, muhammad syahdan aska & sulfahri department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia introduction k. alvarezii is a leading commodity produced in various countries, one of that is indonesia whose production reaches 12-18 tons of dry algae/ha/ year (hayashi et al., 2011; lee et al., 2016; ra et al., 2013) . this algae was contains protein, fiber and carbohydrates (ra et al., 2013; fayaz et al., 2005). carbohydrate content of polysaccharide reaches 60.6% which consists of kappa-carrageenan and cellulose (ra et al., 2013; kim et al., 2015; arad & levy-ontman, 2010). these carbohydrates can be broken down into monosugar through a hydrolysis process and will be fermented to produced etanol (kim et al., 2015; gonzalez et al., 2008; ravanal et al., 2016; munoz et al., 2004). bioethanol is an organic compound with the chemical formula c2h5oh that resulting from the fermentation process of starches, sugars and cellulose with the help of certain microorganisms (kumar et al., 2013; sankh et al., 2013). bioethanol production was developed as fuel replacement fuel with fuel grade ethanol ≥ 99.5% to compensate for the scarcity of petroleum resources. it can be an alternative energy because the oxygen content open access international journal of applied biology keyword hydrolysis fermentation α-amylases phicia kudriavzevii article history received 06 june 2018 accepted 12 july 2017 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 48 was high, environmentally friendly, and its energy source was can reneweble (kumar et al., 2013; madigan et al., 2012). bioethanol produced from biomass is usually produced by biochemical processes such as fermentation process. biomass which is used as raw material of bioethanol is corn and sugar cane where the raw material is a food material and it takes a large area to produce it. the existence of microalgae is very potential in the production of bioethanol to replace raw materials that still have high food value. microalgae contains carbohydrates and proteins that can be used as a carbon source in the fermentation process of bioethanol formation (nicolau et al., 2010). enzymatic hydrolysis and fermentation process are effective in breaking cellulose and hemicellulose present in marine algae into sugar (gonzales et al., 2008; ravanal et al., 2016). carbohydrate hydrolysis can be performed by using enzymes such as cellulase to hydrolyze cellulose, while to hydrolysis of hemicellulose can use enzymes such as glucuronidase, acetyl esterase, xylanase, β-xylosidase, galactomannanase, glucomannanase and α-amylase enzymes used to hydrolyze the kappa-carrageenan (ramachandran et al., 2013; wu et al., 2014). kappa-carrageenan is a hydrocolloid composed of d-galactose-4sulphate and 3,6-anhydro-d-galactose-2-sulfate which are potentially fermentable to obtain bioethanol from the fermentation process (loreo et al., 2013; chang et al., 2017; meinita et al., 2012; fayaz et al., 2005). another method that can be used is fermentation. one of the potential microbes in the fermentation process is pichia kudriavzevii. p. kudriavzevii is one of the yeast that is round, elliptical or elongated (pandey et al., 2000). previous studies have shown that p. kudriavzevii contains 29.3% palmitate, 8.89% stearate and oleic acid 41.9%. this yeast can also be used as a mixed starter culture on combinations between ethanol-tolerant (nicolau et al., 2010; ra et al., 2013). therefore, this study aims to determine the effect of fermentation nutrition and fermentation duration of k. alvarezii algae hydrolysis results on ph change, sugar levels, cell biomass and ethanol content. materials and methods kapppaphycus alvarezii this study used the marien algae of k. alvarezii obtained from coastal waters of punaga village, mangarabombang district, takalar regency. algae that have been obtained are then identified that belong to k. alvarezii type. pretreatment kappaphycus alvarezii algae algae biomass washed and soaked in clean water for 2-3 hours. then dried under the sun. algae k. alvarezii that has been dried mashed using a hummer mill and sifted using a 40 mesh sieve. the resulting algae flour was weighed according to a combination of concentrations are 0.5%; 1%; 1.5% and 2% , then dissolved with 50 ml distilled water. hydrolysis kappaphycus alvarezii algae k. alvarezii algae solution with variation concentration of 0.5%, 1.0%, 1.5%, and 2.0%. then, the algae solution was heated using a hot plate at 100 ° c for 90 minutes. thereafter, an enzyme α-amylase of 80 knu/l was added. the added solution of the incubated apparatus for 24 hours, 48 hours and 72 hours. once hydrolyzed, the algae solution is filtered to separate the natan and its supernatant. the supernatant was then international journal of applied biology, 2(1), 2018 49 centrifuged at 9,000 rpm for 10 min. supernatant of centrifugation was taken and measured its sugar content. fermentation process k. alvarezii solution was prepared according to the optimal concentration obtained at the hydrolysis process, which is 0.5% with the addition of 150 knu/l enzyme α-amylase and incubated for 24 hours. then, fermentation method was added nutrients in form of gandasil-d® as an inorganic nutrient and yeast extract as an organic nutrition (0.5 g/l and 1.0 g/l). after that, a starter p. kudriavzevi was activated with 5% concentration into the fermentor bottle. the fermentation process are used various fermentation durations (0 hours, 24 hours, 48 hours and 72 hours). after the incubation period of 0 hours, 24 hours, 48 hours and 72 hours, measurements of sugar levels, ethanol content and cell biomass of p. kudriavzevii. measurement of cell biomass the cellular biomass of p. kudriavzevii was measured using the method of dry cell weight (dcw = dry cell weight). the dry weight of the cell was carried out by first centrifuging the fermentation medium sample by 50 ml at 9,000 rpm for 10 min. after centrifugation, supernatant and pellet cells are obtained. the supernatant is removed by pipette. after that, the resulting pellets are then washed by adding aquadest on the cell pellets and centrifuged at 9,000 rpm for 5 min. the pellet is then suspended with aquadest and vacuumed with a pipette and then transferred into a filter paper with a pore size of 0.47 μm which has previously been diovened to a constant weight (w1). the filter paper containing pellets is then dried in the oven at 80°c for 24 hours and weighed (w2). the dcw result is the difference between the weight of the final filter paper and the weight of the initial filter paper (w2-w1) expressed by the dry weight of the cell with gram/liter (g/l) unit. data analysis this study used ral (complete random design) with three replications. the parameters measured were sugar content and cell biomass. data were analyzed statistically using analysis of variance (anova) with 95% confidence interval (α = 0.05). the analysis was conducted to compare the effect of acid hydrolysis process on sugar content and fermentation process effect in yielding sugar content and cell biomass from algae k. alvarezii. if there is influence then continued with tukey test at 95% confidence level (α = 0.05) to know pair of group the same and different data in each treatment. results and discussion sugar levels in hydrolysis process the results of the measurement of sugar content showed that algae concentration of 0.5% resulted in average sugar content of 0.56 g/g during 24 and 48 hours incubation. while, 72 hour incubation showed the average of different sugar content with other incubation duration, that is 0,47 g/g. then concentration of 1% yields, mean sugar content of 0.56 g/g at 24 hours and 48 hours incubation. while, the duration of incubation 72 hours resulted in the average of sugar content of 0.46 g/g the concentration of 1.5% on 24 hours, 48 hours and 72 hours incubation time resulted in average sugar content of 0.46 g/g. whereas at 2% concentration yield average of sugar content equal to 0,49 g/g during incubation period 24 hours, 48 hours and 72 hours. international journal of applied biology, 2(1), 2018 50 based on anova test at 95% confidence interval it can be seen that the concentration of k. alvarezii algae used has significant effect on sugar content, but the duration of incubation does not affect the sugar content, then continued with tukey test. then, from the tukey test it was concluded that the most optimum sugar content produced was at a concentration of 0.5% and 1% marine algae with an incubation period of 24 hours, 48 hours and 72 hours. therefore, researchers used 0.5% concentration with 24 hours incubation duration. table 2. comparison of sugar result from this study with several references related research in hydrolysis process marine algae treatment sugar (g/g) reff enzyme temp ph time laminaria digitata cellulose 37˚c 5 24 jam 0.09 vanegas et al.,2015 saccarina latissima cellulose 37˚c 5 24 jam 0.12 vanegas et al.,2015 gelidium amansii cellulose 40˚c 5 48 jam 0.13 ra et al., 2012 saccarina japonica α-amylase 45˚c 5 60 menit 0.2 jang et al., 2012 entheromorpha intestinalis viscozyme®l & cellic ctec2 45˚c 5 48 jam 0.2 kim et al.,2015 gracillaria sp. cellulose 50˚c 4.5 6 jam 0.3 wu et al., 2014 kappaphycus alvarezii α-amilase 40˚c 5 24 jam 0.56 penelitian ini research used enzymatic hydrolysis method yields sugar of 0.3 g/g of gracillaria sp. marine algae (wu et al., 2014). while gelidium amansii only produced a sugar content of 0.13 g/g (ra et al., 2013) and a similar study was performed using entheromorpha intestinalis marine algae producing a sugar content of 0.21 g/g (wu et al., 2014). more study by used saccarina latissima just able to produce 0.12 g/g sugar and laminaria digitata produce sugar levels of 0.09 g/g (vanegas et al., 2015). while, in other studies produced sugar content of 0.2 g/g by using the enzyme α-amylase and algae saccarina japonica as raw materials (jang et al., 2012). based on the comparison of some references, it is known that the lowest sugar content is obtained from research using laminaria digitata algae that is 0,09 g/g and reference with highest sugar content from gracillaria sp. of 0.31 g/g of sugar content (vanegas et al., 2015 and wu et al., 2014). so from the comparison, it can be said that research using k. alvarezii algae, proved higher in producing sugar content of 0.56 g/g. k. alvarezii has a greater chance of producing bioethanol from the resulting sugar content. sugar and ethanol levels in fermentation process this fermentation process is done during 24 hour interval, 48 hours and 72 hours. the results showed that the sugar content will decrease with the length of fermentation time and ethanol content will increasingly with the lenght the fermentation time. this means that polysaccharides contained in the algae of k. alvarezii algae produce high sugar international journal of applied biology, 2(1), 2018 51 levels at the beginning of fermentation but because of the use of p. kudriavzevii yeast causes reduced sugar content of yeast to produce ethanol. based on previous research who said that light will be consumed by the microbes at the beginning of the fermentation period (wu et al., 2014). the resulting ethanol content also reaches the optimum level. figure 1. average graph of fermented sugar level with p. kudriavzevii based on anova test at 95% confidence interval it can be estimated that nutritional distance of nutrients and fermentation concentration did not significantly influence to ethanol content. so it can be concluded that the most optimal sugar content produced at hours 0 hours with sugar content of 0.56 ± 0.06 g/g while ethanol content is most generated at 48 hours incubation and ethanol content of 0.28 ± 0.50 g/g. this proves that yeast p. kudriavzevii by using the algae of k. alvarezii algae which has greater potential in producing bioethanol. figure 2. graph of average ethanol levels with inorganic nutrition (gandasil-d®) and organic (yeast ekstract) during fermentation process 0 0.1 0.2 0.3 0.4 0.5 0.6 0 24 48 72 su ga r co nt en t (g /g ) fermentation time (hours) or-0.1 or-0.5 an-0.1 an-0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0 24 48 72 et ha no l ( g/ g) fermentation time (hours) or-0.1 or-0.5 an-0.1 international journal of applied biology, 2(1), 2018 52 biomass levels in fermentation procces the success of fermentation can be seen based on the microbial growth response on the fermentation medium. in this study, the measurement of yeast cell biomass p. kudriavzevii was done by using dry weight method (dry cell weight). cell biomass measurements were performed during fermentation of 0 hours, 24 hours, 48 hours and 72 hours. the results showed that the longer of fermentation duration so the more cell biomass produced. this means that yeast p. kudriavzevii has used carbohydrates from the algae medium to divide. based on the research of widodo et al. (2013) explains that the fermentation time is very influential on the activity of yeast because the longer the fermentation so the more active yeast breeding. based on the anova test at a 95% confidence interval it can provide different values of fermented nutrients or unconverted fermented concentration to the biomass of fermentation time cells affecting the resulting cell biomass. the biomass that produces fermented nutrients does not differ significantly with inorganic supplementation and the use of producing similar biomass. it was concluded that most biomass was produced at 48 hours incubation and yielded cell biomass of 0.27 ± 0.003 g/g. conclusions the enzymatic hydrolysis process yields the most optimum sugar content whic is 0.56 g/g at 0.5% algae concentration. meanwhile, the use of yeast p. kudriavzevii in the fermentation process yields a sugar content which is 0.56 ± 0.06 g/g and ethanol content of 0.28±0.50 g/g at 48 hours incubation duration. acknowledgment the authors gratefully acknowledge financial support from the ministry of research, technology and higher education of indonesia with project pkm (program kreativitas mahasiswa). references arad, s.m., levy-ontman, o. 2010. red microalgal cell-wall polysaccharides: biotechnological aspects. curr.opin.biotechnol. 21 (3): 358–364. chang, v., patrick, n. o., swee-sen, t. 2017. the properties of red seaweed (kappaphycus alvarezii) and its effect on mammary carcinogenesis. biomedicine & pharmacotherapy. 296-301. del monaco, s.m., maria e. rodriguez, chistian a. lopes. 2016. pichia kudriavzevii as a representative yeast of north patagonian winemaking terrior. journal international of food microbiology. 230: 31-39. fayaz, m., k.k. namitha, k.n. chidambara murthy, m. mahadeva swamy, r. sarada, s. khanam, p.v. subbarao, g.a. ravishankar. 2005. chemical composition iron bioavailability, and antioxidant activity of k. alvarezii (doty). journal agriculture food chem. 53: 792–797. gonzalez, c.f., farina, j.i. & figueroa, l.i.c. 2008.optimized amylolytic enzymes production in s. fibuligera dsm-70554.enzyme and microbial technology. 42: 272–277. international journal of applied biology, 2(1), 2018 53 hayashi, l., a.a. santos, g.s.m. faria, b.g. nunes, m.s. souza, a.l.d. fonseca, p.l. m. barreto, e.c. oliveira, z.l. bouzon. 2011. k. alvarezii(rhodophyta, areschougiaceae) cultivated in subtropical waters in southern brazil. j. app. phys. 23: 337–343. jang, j.s., cho, y.k., jeong, g.t., kim, s.k., 2012a. optimization of saccharification andethanol production by simultaneous saccharification and fermentation (ssf)from seaweed, saccharina japonica. bioprocess biosyst. eng. 35, 11–18.jannah, a., m. 2010.proses fermentasi hidrolisat jerami padi untuk menghasilkan bioetanol.jurnal teknik kimia. 17(1): 44. kim, s.w., hong, c., jeon, s. & shin, h. 2015. high-yield production of biosugars from gracilaria verrucosa by acid and enzymatic hydrolysis processes. bioresource technology. 196: 634–641. kumar, s., gupta, r., kumar, g., sahoo, d., kuhad, r.c., 2013.bioethanol productionfrom gracilaria verrucosa, a red alga, in a biorefinery approach.bioresour.technol. 135: 150– 156. lee, sang-bum and gwi, t. j. 2016.production of biosugar from red macro-algae eucheuma cottonii using acid-hydrolysis.microbiolgy and biotechnology letters. 44(1): 48-54. loreo, y. c., ferrari, m. d., guigo, m., fajardo, l., larnaudie, v., ramirez, m. b. & gerreiro, j. m. 2013.evaluation of sweet potato for fuel bioethanol production: hydrolysis and fermentation. springer plus. 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nipah skala 50 liter dengan penambahan tween 80 dan ergosterol pada proses fermentasi menggunakan saccharomyces cerevisiae. 2015. jom teknik. 2:2. 1-8. wu, f.c., wu, j.y., liao, y.j., wang, m.y., shih, i.l., 2014. sequential acid andenzymatic hydrolysis in situ and bioethanol production from gracilariabiomass.bioresour. technol. 156, 123–131. 2(1)55-60 contact : fajrin hasan basri fajrinhasan97@gmail.com � international journal of applied biology 55 abstract utilization of secondary metabolites from actynomycetes mangrove forests can be used as supportive therapy for colon cancer patients that have already through apoptotic testing so it can directly treat the target of illness. this study was prepared in the form of michrosphere bioadhesive using secondary metabolite actynomycetes m10a rhizosphere mangrove. the purpose of this study was to obtain the development of michosphere bioadhesive preparations from secondary metabolites actynomycetes m10a rhizosphere mangrove as supportive therapy for colon cancer patients. method of the research is base on laboratory experiment which started with purification of actynomycetes isolate, secondary metabolite fermentation, michrosphere bioadhesive formulation and evaluation of michrosphere bioadhesive preparation. the concentration of secondary metabolite actynomycetes m10a rhizosphere mangrove that been used for each formula are 400 μl, 500 μl, and 600 μl, where the percentage of submerged in f1, f2, and f3 are 93,94%, 96,96%, and 94, respectively, 12%. particular particle size distribution is f1 (3.1μm), f2 (4.4 μm), f3 (3.9 μm). the rest angles obtained are f1 (0.002o), f2 (0.002o), f3 (0.0016o), respectively. applied efficiency was obtained by f1 (214 nm), f2 (210 nm), f3 (211 nm). dissolution results show the absorbance results at 266 nm wavelength. from the results obtained that meet the specifications seen in formula 2. issn : 2580-2410 eissn : 2580-2119 development michrosphere bioadhesive delivery system from secondary metabolit actynomycetes m10a rhizosphere mangrove as supportive therapy of colon cancer patients fajrin hasan basri, andi nur annisa abbas, annisa fitri, herlina rante faculty of pharmacy, hasanuddin university, makassar, indonesia introduction colon cancer is one of the leading causes of death in both men and women. patients with colon cancer continue to increase as long as the increasing of smoking habits and bad lifestyle (gracia, 2011). in indonesia, colon cancer is included in the third most common cancer attack, after lung cancer and breast cancer. basic health research (riskesdas) in 2013 states that the prevalence of colon cancer in indonesia in the productive age population or under 40 years is high enough to reach 30%. meanwhile, according to data from the american society cancer, in 2017 the total new cases of colorectal cancer patients reached 135,430 with an estimated death of 50,260 cases. the main therapies of colorectal cancer include surgery, radiotherapy, and chemotherapy that require a long therapy time open access international journal of applied biology keyword colon cancer actynomycetes m10a rhizosphere mangrove michosphere bioadhesive. article history received 13 june 2018 accepted 15 july 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 56 and great maintenance costs. although very effective for treating various types of cancer, surgery and radiation are only localized therapies. most patients have a disease that has spread during diagnosis so that local therapy often fails in eliminating overall cancer (dipiro, 2009).through several studies one source of anticancer compounds derived from actynomycetes. actynomycetes is the most antibiotic and anticancer-producing microbial group compared to molds and bacteria especially those from rhizosphere. mangrove forests are a highly productive ecosystem, providing important protection for beaches in tidal areas in the tropics and subtropics. the ecosystem of flora and fauna habitats of various marine, freshwater and terrestrial species. research conducted by (wang et al., 2011; xiao et al., 2009) found that the mangrove environment, especially in rhizosphere sediments containing new actinobacteria and streptomyces avicenniae. the bioactive activity of the rhizosphere increases as a result of exudation of compounds produced by plant roots and through the formation of plant hormones (berg et al,. 2005). (rante et al., 2016) has isolated actynomycetes m10a from rhizosphere mangrove which has activity against colorectal cancer cell widr through apoptotic mechanism. therefore, innovation development of bioadhesive michrosphere formulation of secondary metabolites is expected to increase colorectal anticancer activity without giving side effects that can damage normal cells and the tendency of drug resistance to patients. microsphere is a drug delivery system that has advantages among others because of its size is very small (smaller than the size of blood cells) so that it can be given directly orally or through blood tissue directly to the center of the pain. however, this microsphere system is limited because of the short contact time with the absorption area. it is therefore necessary for a system to be in contact for longer with absorption membrane. this development is done through a microsphere bioadhesive delivery system (parmar, 2010). bioadhesive is a drug delivery system that extends the density of live dosage at the application site or extends the absorption time and facilitates the tight contact between the preparation and the absorption surface so as to improve the performance of drug therapy. this causes a longer stay time of drug molecules to tend to high local concentrations. this strategy can be applied to drug delivery systems targeting the colon. various polymers used for bioadhesive systems are polycarbophils, polyurethanes, polyethylene oxide and polypropylene oxide (parmar, 2010).this study aims to develop a bioengineous michrosphere formula from secondary metabolite actynomycetes m10a rhizosphere mangrove anticancer as supportive for colon cancer patients. materials and methods equipment and materials the tools used are horn spoons, stirring rods, basins, porcelain cups, parchment paper, scissors, watch glasses, erlenmeyer flasks, measuring cups, pumpkin shapes, beaker, ph meters, rotary evaporators, ovens, analytical scales, microsphere size analyzer, uvvisible spectrophotometer, glass preparation, dissolution tool, spoit, and viscometer. the ingredients used are actynomycetes isolate, aquadest, acetic acid, 96% ethanol, polycarbophyls, paraffin liquid, span 80, petroleum ether, cyanoacrylic glue, rat, tissue, gloves, spectrophotometric uv-vis, aluminum foil, whatmann no. 1 and no. 44, silica gel, plastic wrap, and mask. international journal of applied biology, 2(1), 2018 57 research prosedures 1. the process of rejuvenating actynomycetes bacteria actynomycetes isolates were grown into sna medium (starch nitrate agar). as much as 1 ose isolate actynomycetes from new medium sna kemedium until obtained pure isolate, and then incubated for 24 hours. 2. fermentation (production of metabolite compounds) the isolate obtained was transferred to the starter kemedium then transferred to a 500 ml erlenmeyer containing 100 ml of the same medium. fermentation was carried out at 28oc for 11 days under the conditions of the teragity at a rate of 150 rpm. 3. formulation and evaluation of biooadhesive microsphere no. equipment formula f1 f2 f3 1. metabolite sekunder actynomycetes 400 µl 500 µl 600 µl 2. ethyl cellulose 2 g 2 g 2 g 3. aseton 20 ml 20 ml 20 ml 4. span 80 1,5 g 1,5 g 1,5 g 5. paraffin liquid (ad) 100 ml 100 ml 100 ml used actynomycetes bacteria, then mixed with ethyl cellulose that has been dissolved with acetone to form a mixture a. separately mixed liquid paraffin with span 80 then homogenized for 10 minutes and formed a mixture of b. after that, mixed a to a mixture of b while being sensed at 2500 rpm for 30 minutes. the final mixture is filtered using a filter paper, and the formed microsphere is washed with n-hexane until it is nongreasy. 4. evaluation of microsphere bioadhesive (pendyala, yamini. 2012) a. testing the particle size distribution observed microsphere under an optical microscope then calculated the particle size of the microsphere. b. determination of break angle the rest angle is determined by inserting the michrosphere sample into a funnel that has been covered in the bottom, then flattening the surface of the sample. place the semilog paper just below the funnel. then the funnel cap is opened, recorded and calculated the break angle of each formula by the following formula c. determination of percent rendamen the microsphere obtained from each formula is weighed, then calculated percent of rendamen by considering the weight of the active ingredient and the polymer by using the following formula: % rendament = (practice weight) / (theory weight) x 100% d. applied efficiency a sample of 50 mg was dissolved with artificial intestinal fluid with a ph ± 8 then filtered and measured its wavelength according to the standard. e. in-vitro wash-off test testing in-vitro wash off test is done by using organs of animal intestine try the mice (mus musculus) which i then used the intestinal organs to see the bioadhesive michrosphere can be attached to the intestinal organs. international journal of applied biology, 2(1), 2018 58 f. in-vitro dissolution test dissolution test using basket method at 37 ± 0.5oc with rotation speed 100 rpm using ph 8 posfat buffer as dissolution medium. samples of the microsphere 100 mg were used in this test. samples samples are periodically taken over a period of time and analyzed by spectrophotometric method. results and discussion after extracting the actynomicetes isolate, a microsphere bioadhesive formulation was obtained by percent rendamen for each of the formulas, 93.94% (f1), 96.96% (f2), and 94.12% (f3).from the measurement of the particle size distribution, it was found that there was no significant difference of the three tested formulas. the mean particle diameter of f1 (3.1μm), f2 (4.4 μm), and f3 (3.9 μm). these results indicate that the concentration of secondary metabolites actynomicetes does not affect the resulting formula in which of the three formulas and all form in microspheres. the particle size distribution graph of f2 can be seen in figure 1. figure 1: particle size distribution based on the graph, the higher cumulative percentage is 34% in 3 µm particle size. that means most of particle size that found in f2 has 3 µm. the smallest particle shown the good form of microsphere that have been prepared. on the measurement of the velocity of flow and the rest angle, the flow rate is 0.105 g / s (f1), 0.064 g / s (f2), 0.095 g / s (f3) and the fixed angle is 0.002o (f1), 0.002o (f2) , and 0,0016o (f3). a silent angle below 25° represents a good flow rate so it is predicted to produce a good release rate of the drug when formulated. maximum wavelength determination results show that the highest uptake of the formula with in vitro gastric medium without enzim is at 266 nm wavelength. of the three formulas showed insignificants rate of dissolution. further measurements were made on each of the formulas which obtained the highest absorbance of f1 (214 nm), f2 (210 nm), f3 (211 nm). in the 2 3 4 5 6 13.7 34 29.7 18.3 4.3 1 2 3 4 5 total of particle particle size (µm) the particle size dstribution of f2 particle size that shown in f2 cumulative percentage of particle size international journal of applied biology, 2(1), 2018 59 formula 2 the highest absorbance is at 210 nm wavelength indicates the same wavelength as the references so that shown 100% absorption efficiency is found in formula 2. the spectrophotometric graph is shown in figure 2. figure 2. applied efficiency of f2 bioadhesive capability is also tested in microsphere bioadhesive formula. in vitro wash test was performed by orally administered in mice (mus musculus). method that used is to indicate the microshphere attached to the intestine so it was predicted that the bioadhesive properties were obtained in all three formulas. conclusions the results obtained by using secondary metabolite actynomycetes m10a are 400 μl, 500 μl, and 600 μl, respectively, where the percentage of rendamen in f1, f2, and f3 are 93,94%, 96,96%, and 94, respectively, 12%. particular particle size distribution is f1 (3.1μm), f2 (4.4 μm), f3 (3.9 μm). the rest angles obtained are f1 (0.002o), f2 (0.002o), f3 (0.0016o), respectively. applied efficiency was obtained by f1 (214 nm), f2 (210 nm), f3 (211 nm). the dissolution results showed absorbance results at 266 nm wavelengths. while for invitro wash off test result using mucus organs organ (mus musculus) it was found that the microsphere bioadhesive preparation could be attached to the intestinal organ of mouse. based on the evalution results, f2 is the good formula of microspher bioadhesiveactynomycetes m10a rhizosphere mangrove. acknowledgment this research was supported by pharmacy faculty of hasanuddin university, funded by the ministry of research technology and higher education. international journal of applied biology, 2(1), 2018 60 references arora, a., nair., m.g. & strasburg, g.m. 1998. structure–activity relationshipsfor antioxidant 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determination of potency apoptosis induction of secondary metabolite actinomycetes from mangrove rizosphere using flow cytometry method. faculty of pharmacy. hasanuddin university saija, a., scalese, m., lanza, m., marzullo, d, bonina, f & casteli, f. 1995. flavonoids as antioxidant agents: importance of theirinteraction with biomembranes.free radic. biol. & med. 19(4):481–486. wang rong., john a. dearing., peter g. langdon., enlou, zhang., xiangdong, yang., vasilis, dakos., & marten, schefer. 2011. growth propagation of yeast in linear arrays of microfluidic chambers over many generations. biomicrofluidics 5(4):44118-441189 xiao j., chen xw., davies ba., saltiel ar., katzmann dj., & xu z. 2009. structural basis of ist1 function and ist1-did2 interaction in the multivesicular body pathway and cytokinesis. mol biol cell 20(15):3514-3524 contact : wahyuti wahyutiasaf864@gmail.com 1 abstract the mangrove ecosystem in the babana area has the potential for developing ecotourism activities. this study aims to identify the potential of the babana mangrove ecotourism in larompong selatan district, and to analyze the babana mangrove ecotourism development strategy in larompong selatan district. this research was conducted from may to july 2022 and was carried out in the babana mangrove ecotourism area, larompong selatan district, luwu regency, south sulawesi province. data collection was carried out through field surveys and interviews using a questionnaire. the method used in this study was swot analysis. the results of this study indicate that the potential for babana mangrove ecotourism is the uniqueness of the mangrove ecosystem lies in the presence of a rainbow bridge which is the main attraction for babana mangrove ecotourism. the babana mangrove ecotourism development strategy has great opportunities but also has weaknesses. the strategy for developing the babana mangrove ecotourism in larompong selatan district, luwu regency, is to create complex concepts related to the development of mangrove ecotourism by the interests of visitors and involve the community to participate in it, improve facilities and infrastructure and increase human resources for the development of mangrove ecotourism, increase the attractiveness and cleanliness and safety of ecotourism mangroves, and optimizing promotion and interpretation for visitors. issn : 2580-2410 eissn : 2580-2119 development strategy of babana mangrove ecotourism in larompong selatan district, luwu regency wahyuti1*, andi ummung1, andi nur apung massiseng1, lukman daris1, nurwahidah1 1study program of fisheries agrobusiness, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar, indonesia. introduction indonesia is a country that has many kinds of natural potential. these potentials can undoubtedly be developed to impact indonesia's tourism industry positively; indonesia is famous for its various tourism potentials. starting from its beautiful beaches, green mountains, and many historical relics such as temples are also found in indonesia, so it is not strange if indonesia is said to be "zambrut on the equator", which is a tourist attraction that cannot be found in other countries, so many domestic and foreign tourists who want to enjoy this natural beauty (asmaria et al., 2020). ecotourism is a concept created for the development of sustainable tourism, which has the aim of supporting efforts to preserve the environment and increase community open access international journal of applied biology keyword development strategy; mangrove ecotourism; babana; south larompong; luwu regency article history received may 7, 2023 accepted june 13, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 7(1), 2023 2 participation in its management to provide economic benefits to local communities and governments, as well as provide opportunities for present and future generations to utilize and develop them. become a fisheries resource with economic value (permatasari, 2020; massiseng et al., 2022; fachry & alpiani, 2021). one of the ecosystems that has the potential to be developed as an ecotourism area is the mangrove ecosystem. the role of the mangrove ecosystem as a place for biota to interact continuously and as a place for sediment storage makes mangrove forests an ecosystem with a high level of productivity with a variety of economic, social and environmental functions (permatasari, 2020). the mangrove ecosystem is the leading ecosystem that is very productive but vulnerable to changes or external influences. mangrove ecosystems have multiple functions, namely physical, ecological, and socio -economic. physically, mangroves can withstand high waves, storms and tides anytime, reducing coastal abrasion. ecologically, it is a source of germplasm, spawning grounds and nesting sites for marine biota. socio-economically, mangroves can be used as a cultivation area, to maintain fish species with high economic value, or as a natural tourist attraction in ecotourism development, according to sulastini (susi et al., 2018). the development of mangrove ecotourism is an effort to sustainably utilize environmental services from coast al areas. ecotourism in mangrove forests synergizes with concrete forest ecosystem conservation measures (mulyadi & fitriani, 2012). nonetheless, in practice, ecotourism development in mangrove forests must still be managed by avoiding risks and negative e nvironmental impacts, such as by paying attention to suitability and environmental carrying capacity (permatasari, 2020). the development of a tourist village is one of the efforts that can be taken to empower the community and encourage the development of the village's potential, which leads to increasing the community's productivity so that it is more independent. the regen t of luwu is very urgent for tourism development in the luwu regency area, where every village in luwu regency is emphasized to develop tourism by utilizing the existing potential. the development of mangrove ecotourism is one of the environmental uses of coastal areas in a sustainable manner. ecotourism development in mangrove ecosystems can be managed by avoiding risks and negative environmental impacts, such as paying attention to suitability and environmental carrying capacity (asmaria et al., 2020). tembo'e village is one of the villages located in south larompong district, luwu regency, south sulawesi, which has a beach called mangrove beach tourism or commonly called babana tembo'e beach. the development of the mangrove tourism area in tembo'e village uses the budget of the ministry of villages (ri ministry of villages) and village budget sharing and the luwu regency apbd. tembo'e village has good enough mangrove potential to be developed into an ecotourism area in luwu regency, one of which is the man grove ecosystem that needs further development. the objectives of this research are to identify the potential of the babana mangrove ecotourism in larompong selatan district and to analyze the babana mangrove ecotourism development strategy in larompong selatan district. the results of this research can be used as information material and data indicators for interested stakeholders and used for future policy considerations. materials and methods time and location of research this research was conducted for ± 3 months, from may to july 2022. the research location was in the mangrove ecotourism area in tembo'e village, larompong selatan international journal of applied biology, 7(1), 2023 3 district, luwu regency, south sulawesi province. location and field data collection are presented in figure 1. figure 1. research locations tools and materials during this research, the tools used in the field were cameras, recorders, stationery, and several other supporting tools. table 1. tools and materials in research no tools utility 1 2 3 kamera recorder stationery documenting research activities a recording device at the time of the interview record research results materials and equipment used in the study consisted of cameras, recorders, and stationery. cameras are used to document research activities, recorders are used to record the results of interviews with respondents, and stationery is used to record research and interview results with respondents at research locations. research procedure the procedure in the research was to identify the potential and strategies for babana mangrove ecotourism through observation, in -depth and structured interviews using questionnaires with community leaders, traditional leaders, village heads, cultural an d tourism offices of luwu district, south sulawesi province and babana mangrove tourism managers. data analysis this research was conducted using survey methods, observation and documentation. a quantitative and qualitative descriptive approach is used to identify the tourism potential of babana mangroves in the south larompong district. according to sumadi (putri, k.n. et al., international journal of applied biology, 7(1), 2023 4 2020), a descriptive approach is a study based on a systematic, factual and accurate formatted understanding of the objects and facts of a study. the collection of descriptive data includes interviews (interviews) and filling out questionnaires. this method determines the socio economic and cultural conditions related to the babana mangrove management in larompong selatan district. this analysis stage is also an initial observation that describes the condition of the mangroves and can also describe the problems that exist in the research location. according to sugiyono (rizky, 2014), a study using a descriptive statistical approach provides an overview and description of an object to be studied. using swot analysis, analyze the strategy for developing mangrove ecotourism in tembo'e village. swot analysis is an analytical tool used to derive strategies for an activity (putri, k.n. et al., 2020). swot analysis is an advanced analysis stage. based on the analysis results from the descriptive approach, the next step is to identify strategic factors to identify swot (strength, weakness, opportunity, threats). the swot analysis steps are as follows: 1. identify management strategy factors. 2. identify strengths (s), weaknesses (w), opportunities (o), and threats (t) from the results of the observations made. 3. from the identification results, five points were selected which were considered necessary from each of the swot components. 4. next, determine the strategy to be executed by creating a combined matrix of the four swot components. from the results of the combined matrix, we can define strategies in general groups (sw, wo, st, and wt), which will then be described in a more specific form. according to rangkuti (rizky, 2014), swot analysis compares external factors of opportunities and threats with internal factors of strengths and weaknesses. internal factors are entered into the internal strategic factor matrix or ifas (internal strategic factor analysis summary). external factors are entered into the external strategic factor matrix or efas (external strategic factor analysis summary). after the matrix of internal and external strategic factors has been prepared, the results are entered into a quantitative model, namely the swot matrix, to formulate the company's competitive strategy. next is to determine the weight of internal and external factors according to their level of importance. the sum of all weights must be 1.0. after that, give a rating for each factor based on the answer/response effect. these factors affect the mangrove ecosystem's management in the babana ecotourism (score: 4 = very good, 3 = good, 2 = not good, 1 = below average). then multiply the weight with the rating value of each factor to determine the score value and add up all the scores to get the total score. the next stage is data analysis to develop strategic factors, processed as a swot matrix. this matrix can clearly describe how external opportunities and threats may arise, as well as adjustments to the strengths and weaknesses possessed. the matrix can produce four possible alternative strategies in detail in the following table: international journal of applied biology, 7(1), 2023 5 tabel 2. matriks swot ifas efas strength weakness opportunity strategy (so) create strategies that use strengths to take advantage of opportunities— used if the manager is in quadrant i. strategy (wo) create strategies that minimize weaknesses to take advantage of opportunities— used if the manager is in quadrant iii. treat strategy (st) creates a strategy that uses strengths to avoid threats—used if the manager is in quadrant ii. strategy (wt) creates strategies that minimize weaknesses and avoid threats—used if the manager is in quadrant iv. the manager's position in the right quadrant means that the manager can make more appropriate decisions, namely: quadrant i : the strategy must be implemented to support aggressive policies. quadrant ii : using a diversification strategy, the strategy that must be applied is to use power to take advantage of long-term opportunities. quadrant iii : the strategy must be implemented to minimize internal problems. quadrant iii shows that managers have enormous opportunities, but on the other hand, managers have internal weaknesses. quadrant iv : is an unfavourable situation because managers face threats and internal weaknesses. results identification of babana mangrove ecotourism potential in south larompong district a tourist attraction is "something" that exists at a tourism destination/destination location that not only offers/provides something for tourists to see and do but also becomes a magnet that attracts someone to travel (pangastuti, w.m, 2017). the main characteristic of a tourist attraction is that it cannot be moved, and to enjoy it, and tourists must visit the place. the attractiveness of the babana mangrove ecotourism in the south larompong district can be seen from the potential of the plants and the uniqueness of their ecosystem. the mangrove ecosystem is a system consisting of organisms (plants and animals) that interact with environmental factors with each other in a mangrove habitat. bengen (pangastuti, w.m, 2017) states that the mangrove ecosystem is a tropical coastal vegetation community dominated by several species of mangrove trees that can grow and develop in tidal muddy coastal areas. this vegetation community generally grows in areas p rotected from big waves and strong tidal currents. mangrove ecosystems are found on shallow bay beaches, estuaries, deltas and protected coastal areas. from the observations of mangrove plants, several types of mangrove plants were found in the babana ecot ourism area consisting of rhizophora apiculata (figure 2), sonneratia alba, rhizophora spp and rhizophora mucronata. the uniqueness of the babana mangrove ecotourism lies in the presence of a rainbow bridge which is an attraction for tourism. this further beautifies the panorama of the babana mangrove ecotourism. international journal of applied biology, 7(1), 2023 6 figure 2. babana mangrove ecotourism rainbow bridge community potential in developing mangrove ecotourism tembo'e village consists of 5 hamlets, namely ponnori, matali, simoma, lambellang, and tembo'e, of the five hamlets, which border and interacts directly with the babana ecotourism area, is tembo'e hamlet. in 2019 the recorded population was 3800 spread across five hamlets, with 700 households. community characteristics the people interviewed live around the babana mangrove ecotourism in tembo'e hamlet and have activities that can support the development of the babana mangrove ecotourism. the number of respondents is as many as 30, with details of 23 men and seven women with various educational characteristics (table 3). table 3. age characteristics of the tembo'e village community age amount percentage 20-29 30-39 40-49 50-59 60-69 2 7 12 6 3 6,67 23,33 40,00 20,00 10,00 amount 30 100 source: processed from primary data, 2022 most people's age ranges from 40-49 years, with a percentage of 40%. the age range of 20-29 years is 7%, age 30-39 years is 23%, age 40-49 years is 40%, age 50-59 years is 20%, and age >59 years is 10%. a. educational characteristics in general, public education still needs to be improved. this can be seen from the level of community education. elementary and equivalent education was 40%, junior high school was 27%, high school was 17%, undergraduate was 7%, and those who did not attend school were 10% (table 4). international journal of applied biology, 7(1), 2023 7 table 4. the educational characteristics of the tembo'e village community level of education amount percentage elementary school junior high school senior high school bachelor no school 12 8 5 2 3 40,00 26,67 16,66 6,66 10,00 amount 30 100 source: processed from primary data, 2022 the high percentage of people who have graduated from elementary school and its equivalent indicates that the community is aware of the importance of education. however, this needs to be supported by the culture that develops in society. the culture that h as developed in society since long ago was that if there were girls who had graduated from elementary school, they were considered capable of living life. so that if someone proposes to a child, it is taboo to refuse it. this is what causes the decline in the level of education achieved by the people of tembo'e village. some people who are aware of the importance of education prefer to continue their daughters' schooling in the hope that these children will be able to complete higher education so they can rebuild their area again. b. job characteristics based on the interview results, the characteristics of the community's work are fishermen 30%, farmers 23%, entrepreneurs 17%, civil servants 13%, and others (pensioners, drivers) 13% (table 5). table 5. characteristics of the work of the tembo'e village community work amount percentage fisherman farmer self-employed government employees other 9 7 5 4 5 30,00 23,33 16,67 13,33 16,67 amount 30 100 source: processed from primary data, 2022 most people in tembo'e village have their main livelihood as fishermen and farmers who take advantage of the babana mangrove ecotourism. people usually catch fish and crabs in the mangrove ecotourism area. community activities utilizing mangrove ecotourism do not make its use their primary job but as additional work. this is because the community is aware of the importance of the role of mangroves in maintaining the balance of nature. policy analysis (swot analysis) the strategy for developing mangrove ecotourism in babana, temboe village, larompong selatan district, and luwu regency uses swot analysis (strength, weakness, international journal of applied biology, 7(1), 2023 8 opportunity, and threats). the swot analysis stage used in analyzing further data is to collect all information that affects the ecosystem in the study area, both externally and internally. the results of the field study through the analysis of primary and secondary data were carried out based on research methodology, and stakeholder perceptions, nam ely the government, in this case consisting of the village head, luwu regency tourism office, local communities who live around the mangrove ecotourism, and visitors, a swot analysis was carried out. the first thing to do in this analysis is to identify th e internal and external environmental factors that have a real influence on the development of the babana mangrove ecotourism. then formulate alternative strategies to obtain the chosen strategy recommended to the luwu regency government, especially for st akeholders directly related to luwu regency coastal development planning. the following results identify internal and external factors. internal and external factors a. internal factor analysis based on the internal factor analysis of the babana mangrove ecotourism development strategy in larompong selatan district, luwu regency, several identified strengths are as follows: 1) babana mangrove ecotourism has begun to be developed for education -based tourism. 2) the diverse potential of mangrove resources. 3) babana mangrove ecotourism has been designated as a tourist attraction in luwu district. in addition to the potential internal factors that become strengths in the babana mangrove ecotourism development in larompong selatan district, luwu regency, severa l internal factors become weaknesses as follows: 1) babana mangrove ecotourism is a place that is less well -known than other mangrove ecotourism areas in luwu regency, especially tourists from outside the area. 2) south larompong district has many tourist sites, such as ponnori beach. this makes the mangrove ecotourism area less competitive. 3) much garbage left by the visitors. 4) lack of understanding of the community and visitors about ecotourism. 5) lack of maintenance of facilities and infrastructure. b. external factor analysis based on the analysis of the external factors of the babana mangrove ecotourism development strategy in larompong selatan district, luwu regency, there are opportunity factors that can be utilized to implement their functions and r oles. several identified opportunity factors are related to the development of the babana mangrove ecotourism, including: 1) good community participation and support for ecotourism development. 2) the majority of visitors are students in the south larompon g sub-district. 3) the babana mangrove tourism area is near the ponnori beach tour. 4) the number of visitors who come on holiday. 5) natural potential that supports ecotourism activities. in addition to the potential external factors that become opportunities in the babana mangrove ecotourism development strategy in the larompong selatan sub -district, luwu district, there are also external factors that become threats, namely, as follows: 1) other tourist objects are more attractive. 2) negative impacts of mangrove tourism activities (garbage, activities that damage mangroves, etc.). international journal of applied biology, 7(1), 2023 9 3) the community needs to be more responsive to the management and promotion of the babana mangrove ecotourism. matrix of internal factor evaluation strategy (ifas) and external factor evaluation (efas). furthermore, the results of the accumulation of internal and external factors are entered into the ifas and efas matrices to obtain weights, ratings, and scores. t he results of the ifas matrix (table 10) and efas (table 11) are as follows: c. internal factor matrix table 10. matrix of internal strategic factors for babana mangrove ecotourism no internal strategic factors strength (s) weight ratings score 1 babana mangrove ecotourism has begun to be developed for educationbased tourism. 0,15 4,4 0,66 2 the diverse potential of mangrove resources 0,12 3,7 0,444 3 babana mangrove ecotourism has been designated a tourist attraction in the luwu district. 0,13 3,9 0,51 total strength 0,40 1,614 no weakness (w) weight ratings score 1 babana mangrove ecotourism is a place that is less well-known than other mangrove ecotourism in the luwu regency area, especially tourists from outside the area. 0,12 3,5 0,42 2 larompong selatan district has quite a lot of tourist sites, such as ponnori beach etc. this makes the mangrove ecotourism area lose the competition. 0,12 3,4 0,408 3 the amount of garbage visitors leave reduces the beauty of the babana mangrove ecotourism. 0,13 3,9 0,507 4 lack of understanding of the community and visitors about ecotourism 0,11 3,3 0,363 5 lack of maintenance of the infrastructure facilities and infrastructure contained in the babana mangrove tour 0,12 3,4 0,408 total weakness 0,60 2,106 total internal factors (ifas) 1 3,720 table 10 shows the babana mangrove ecotourism development strategy matrix, which has a strength of 1.614 while a weakness indicates a value of 2.106. where this internal factor has an accumulated value of -0.492. from an internal perspective, the development of mangrove ecotourism is fragile, so it requires strength to change existing strategies. the international journal of applied biology, 7(1), 2023 10 results of the accumulation of external factors for the development of the babana mangrove ecotourism can be seen in the following table: table 11. matrix of external strategic factors for babana mangrove ecotourism no external strategic factors opportunity (o) weight ratings score 1 good community participation and support for ecotourism development 0,14 4,1 0,574 2 the majority of visitors are students in the south larompong sub-district 0,11 3,3 0,363 3 the babana mangrove tourist area is adjacent to the ponnori beach tourism 0,12 3,7 0,444 4 the number of visitors who come on holidays 0,12 3,4 0,408 5 the natural potential that supports ecotourism activities 0,14 4,1 0,574 total opportunity 2,363 no threat (t) weight ratings score 1 there are other more exciting tourist objects besides the babana mangrove ecotourism. 0,12 3,6 0,432 2 negative impacts of babana mangrove tourism activities (garbage, activities that damage mangroves, etc.) 0,12 3,7 0,444 3 the community needs to be more responsive to tourism activities in managing and promoting the babana mangrove ecotourism. 0,13 3,7 0,481 total threat 1,357 total external factors 1 3,720 the external strategy matrix in table 11 shows that the value of the opportunity component is 2.363, and the threat component is 1.367. from external factors, an accumulation of 1.006 is obtained. this situation can condition that to take advantage of existing opportunities; one must anticipate threats that might occur so that utilization can run as expected. based on the calculation results, the strategic position is in quadrant iii at point ( 0.492, 1.006). the position of the babana mangrove ecotourism development strategy needs to be analyzed. the analysis in question includes an analysis of the internal and external environment: strengths, weaknesses, opportunities and threats. the four elements must be able to "value" to determine the right strategy to achieve the vision and mission that has been implemented. quadrant iii shows that the situation in the babana mangrove ecotourism development strategy has enormous opportunities, but on the other hand, it also has internal weaknesses. the strategy that must be carried out in this research is to minimize inter nal problems. the swot analysis produces four combinations of strategies, namely: 1. strengths opportunities (so) strategy is a strategy that uses strengths to take advantage of international journal of applied biology, 7(1), 2023 11 opportunities, 2. strengths threats (st) strategy is a strategy that uses str engths to overcome threats, 3. weaknesses opportunities (wo) strategy) is a strategy that minimizes weaknesses to take advantage of opportunities, and 4. the weaknesses threats (wt) strategy is a strategy that minimizes weaknesses and avoids threats. mangrove ecotourism development strategy after knowing the position from the results of the swot matrix analysis, the next step is to determine alternative utilization strategies that will be recommended. the following is a matrix of alternative utilization strategies for mangrove ecotourism areas in table 4.10. based on the swot matrix analysis results with a combination of internal and external factors, the development of the babana mangrove ecotourism is in quadrant iii (figure 4.6). by looking at the considerations between opportunities and weaknesses, what is being done is to support the turn-around – wo (weaknesses and opportunities) strategy, which creates strategies by minimizing weaknesses to take advantage of opportunities. the strategic steps taken to support the babana mangrove ecotourism include those presented in table 4.10 below: table 12. swot matrix support turn-around strategy (-0,492: 1,006) supports aggressive strategy support diversification strategy supports defensive strategy international journal of applied biology, 7(1), 2023 12 strength (s) 1. babana mangrove ecotourism has begun to be developed for education-based tourism 2. the diverse potential of mangrove resources 3. mangrove ecotourism has been designated as a tourist attraction in the luwu district. weakness (w) 1. babana mangrove ecotourism is a place that is not quite as well-known as other mangrove ecotourism in the luwu district, especially tourists from outside the area. 2. larompong selatan district has many tourist sites, such as ponnori beach etc. this makes the babana mangrove ecotoxurism unable to compete 3. lots of trash left by visitors 4. lack of understanding of the community and visitors about ecotourism 5. lack of maintenance of facilities and infrastructure. opportunity (o) 1. good community participation and support for ecotourism development 2. the majority of visitors are students in the sub-district of south larompong 3. the babana mangrove ecotourism area is close to ponnori beach 4. the number of visitors on holidays 5. the natural potential that supports ecotourism activities strategy (so) develop complex concepts related to the development of mangrove ecotourism by the interests of visitors and involve the community to participate in it. strategy (wo) improving facilities and infrastructure, as well as increasing human resources to develop mangrove ecotourism international journal of applied biology, 7(1), 2023 13 threat (t) 1. other tourist objects are more attractive. 2. negative impacts of tourism activities (garbage, activities that damage mangroves, etc.) 3. the community needs to be more responsive to the management and promotion of babana ecotourism. strategy (st) increase the attractiveness, cleanliness, and safety of the babana mangrove ecotourism. strategy (wt) optimizing promotion and interpretation for mangrove ecotourism visitors by the results of the identification of internal and external environmental conditions that affect the babana mangrove ecotourism, four strategies can be formulated, resulting in the swot matrix that can be carried out to develop the babana mangrove ecotou rism, namely: 1. strategic s–o, namely by compiling complex concepts related to the development of mangrove ecotourism by the interests of visitors and involving the community to participate in it. 2. the w–o strategy is to improve facilities and infrastru cture and increase human resources to develop mangrove ecotourism. 3. the s–t strategy is to increase the attractiveness, cleanliness, and safety of the babana mangrove ecotourism. 4. the w-t strategy is to optimize promotion and interpretation for visitor s. conclusion based on the research that has been done, it can be seen from the matrix of internal and external factors in the babana mangrove ecotourism that the results of evaluating internal strategic factors obtained a strength value of 1.614 and a we akness value of 2.106. at the same time, the results of the evaluation of external factors obtained an opportunity value of 2.363 and a threat value of 1.367. so the babana mangrove ecotourism has more vital external factors than internal ones. the babana mangrove ecotourism development strategy obtained through swot analysis, namely develop complex concepts related to the development of mangrove ecotourism according to the interests of visitors and involve the community in it; we are improving facilities and infrastructure and increasing human resources to develop mangrove ecotourism, increasing mangrove ecotourism's attractiveness, cleanliness, and safety; we are optimizing promotion and interpretation for visitors. acknowledgement we want to thank the chancellor of cokroaminoto university makassar for assisting in research funding through the institute for research and community service (lppm) and colleagues and respondents who have helped carry out the research well. international journal of applied biology, 7(1), 2023 14 references asmaria, a., akbar, m. f., & kuswarak, k. 2020. pemanfaatan potensi desa dalam pengembangan desa wisata di desa pulau legundi kecamatan punduh pedada kabupaten pesawaran. in prosiding seminar nasional penelitian dan pengabdian kepada masyarakat (vol. 1, no. 1, pp. 413-420). fachry, m. e., & alpiani, a. (2021). a model of waste management in kapoposang island south sulawesi. jurnal agrikan (agribisnis perikanan),14(2), 427-437. kusumastanto, t. dkk. 2012. pengelolaan wilayah pesisir dan laut. modul materi pokok universitas terbuka cetakan ketiga: tangerang selatan. mandar, k. p., & alfira, r. 2014. identifikasi potensi dan strategi pengembangan ekowisata mangrove pada kawasan suaka margasatwa mampie di kecamatan wonomulyo. massiseng, ana., alpiani a., ummung a., dan zaenab s. 2022. supply chain of tuna fishery resources (thunnus sp) in maumere district, sikka regency, east nusa tenggara province. international journal of applied biology, vol. 6 no. 1, hal. 123-136. nuitja, i. n. s. 2019. manajemen sumber daya perikanan. pt penerbit ipb press. nurhidayah, a. 2017. kajian kesesuaian dan daya dukung kawasan ekowisata pantai dan terumbu karang berbasis masyarakat di pantai bangsring banyuwangi, jawa timur (doctoral dissertation, universitas brawijaya). pangastuti wm, arief h, sunarminto t. 2015. pengembangan ekowisata mangrove di pantai bilik dan sejile resort labuhan merak taman nasional baluran, jawa tim ur. bonorowo wetlands. vol. 6, no. 2, hal. 92–102. permatasari, p. 2020. identifikasi potensi dan strategi pengembangan ekowisata mangrove di desa balang baru kabupaten jeneponto (doctoral dissertation, universitas hasanuddin). putri kn., asgar ma., & massiseng, ana. 2020. study of potential and development strategy of the botolempangan tourist village in maros regency, indonesia. agrikan: jurnal agribisnis perikanan, 13(2), 435-448. ristilia r. 2019. aplikasi pelayanan jasa berbasis android pada bidang perikanan tangkap pada dinas kelautan dan perikanan provinsi kepulauan bangka belitung (doctoral dissertation, stmik atma luhur). rojana arf., & muhsoni ff. 2021. analisis kesesuaian ekowisata pantai di pantai gua manik dan pantai benteng portugis kabupaten jepara. jurnal perikanan dan kelautan . vol 27, no. 1. hal. 6–11. supriadi, b. (2016). pengembangan ekowisata pantai sebagai diversifikasi mata pencaharian. jurnal pariwisata pesona. volume 1 no. 1. hal. 1–20. susi, s., adi, w., & sari, s. p. 2018. potensi kesesuaian mangrove sebagai daerah ekowisata di dusun tanjung tedung sungai selan bangka tengah. akuatik: jurnal sumberdaya perairan. volume 12, no. 1, hal. 65-73. yapanto, l. m. 2021. ekowisata terumbu karang. universitas negeri gorontalo, 15pp. yuniari, s. h. 2017. pengelolaan ekowisata mangrove sebagai penunjang perekonomian masyarakat melalui pendekatan ekologi dan sosial: studi kasus mangrove blok bedul, resort grajakan, taman nasional alas purwo, banyuwangi. agrikan. volume 11, no. 1. hal. 66–78. 1(2)67-75 !!!!!!!!!!!!!!!!!! contact&:&magdalena&litaay&&&&&&&magdalenalitaay@yahoo.com! ©!international!journal!of!applied!biology! 67! abstract& the!research!about!the!biodiversity!of!gastropod!has!been!conducted!in! seagrass!meadow!of!balangdatu!waters,!tanakeke!island,!south!sulawesi.! the!research!aims!to!assess!the!diversity!of!gastropod!species!in!balangdatu! waters.! sampling! was! conducted! using! quadrate! transect! method! systematically.!three!replicates!of!transect!were!applied!for!each!station.! the!result!indicates!there!were!34!species!of!gastropods!from!14!genera!and! 14!families!were!found.!diversity!index!from!every!station!varies!from!1,661! to!2,!899.!these!values!range!from!low!to!moderate.!the!diversity,!evenness,! and! dominance! indices! showed! that! balangdatu! waters! still! in! good! condition!and!sustain!habitat!for!gastropod.! issn!:!2580q2410! eissn!:!2580q2119! ! ! ! biodiversity&and&distribution&of&gastropods&at&seagrass&meadow& of&balangdatu&waters&tanakeke&island&south&sulawesi&indonesia& ! magdalena&litaay,&marwa&deviana&and&dody&priosambodo & ! department!of!biology,!faculty!of!mathematics!and!natural!sciences,!hasanuddin! university,!makassar,!indonesia! ! ! & & & & & & & & & & & & introduction& gastropods!are!known!as!snails!that!has!more!diverse!members!in!the!phylum!of! mollusks.!main!characteristics!of!this!group!are!single!shell,!wellqdeveloped!head!and!its!body! is!completed!by!tentacle,!eyes!and!radula.!some!gastropods!are!economics!important!due!a! valuable!shell!for!ornament!materials!and!souvenirs!or!meat!as!protein!sources!for!human.! therefore,! gastropods! are! recognized! as! potency! group! that! can! contribute! to! coastal! community!commodity!welfare!and!fishery!productivity!at!coastal!areas.!amongst!mollusks! group,!gastropods!is!a!successful!group!in!term!of!widely!distributed.!they!can!be!found!in! almost!all!habitats!including!the!terrestrial,!freshwater,!and!deep!sea.!gastropods!are!also! found!living!in!different!coastal!ecosystem!including!mangrove,!seagrass!meadows!and!coral! reefs.!(tomascik!et#al.,!1997).! coastal!communities!utilized!seagrass!meadows!for!different!purposes!such!as!fishing! ground,!collecting!snail!as!seaweeds!aquaculture.!on!coastal!aquaculture!practice,!this!area! is!often!cleaned!from!unwanted!organisms!including!parasite!of!seaweed.!this!action!will! contribute! to! damage! of! seagrass! meadows.! hence,! seagrass! ecosystems! are! a! threat! worldwide.!many!studies!have!been!conducted!on!mollusca!around!indonesia,!but!still!less! studied!on!seagrass!meadows!of!south!sulawesi!(litaay!2006,!kusnadi!2008,!litaay#et#al.,! &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword! biodiversity## marine#mollusca# seagrass#meadow# spermonde# article&history! received!12!october!2017! accepted!29!december!2017!! international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international#journal#of#applied#biology,#1(2),#2017# ! ! 68! 2010,!arbi!2011,!metungun!et#al.,!2011).!!!on!of!coastal!area!in!south!sulawesi!province!that! has! seagrass! meadows! that! used! for! seaweed! cultivation! is! tanakeke! island! of! takalar! regency.!high!activity!of!human!along!tanakeke!coastal!area!to!some!extent!will!contribute! to!a!change!in!water!quality!that!will!disturb!seagrass!meadow.!information!on!associate!biota! to!seagrass!meadows!from!this!area!is!limited.!information!on!biodiversity!of!associate!biotas! to! different! ecosystems! is! needed! to! support! sustainable! used! of! coastal! and! marine! resources.!the!aim!of!the!present!study!on!gastropods!diversity!that!associates!with!seagrass! meadows! of! balangdatu! water! of! tanakeke! island! south! sulawesi! is! needed! to! support! sustainable!use!these!resources.!!! & materials&and&methods& sampling&site&& sampling!site!was!determined!based!on!water!condition,!topography,!habitat!type,! distribution!of!seagrass!meadows!and!community!activity!along!the!coast!of!balangdatu.!we! divided!sampling!sites!into!three!areas!as!follows:! site!1! :!close!to!lagoon,!substrate!rubble,!seaweed!cultivation!area,! site!ii!! :!seaweed!cultivation!areas,!substrate!sand,! site!iii!!:!close!to!human!settlement,!substrate!muddy!sand,!many!seaweeds!cultivation! ! sampling&methods& sampling! method! of! transect! combination! with! the! plot! was! used! systematically! during! gastropods! collection! (modified! english! et# al.,! 1997,! hemminga! and! duerte! 2000,! priosambodo!2014).!three!transects!and!two!replicates!were!applied!at!each!sampling!site.! each!transect!was!divided!into!5!plot.!a!transect!was!placed!at!seagrass!offshore!until!reach! top!end!that!seagrass!was!found.!!balangdatu!has!4,800!m!coastal!line!in!which!seagrass! meadows!areas,!divided!into!three!sites:!sta.!i:!150!m2,!sta.!2:!300!m2!and!sta.!3:!600!m2.! gastropods!in!all!transect!were!collected,!cleaned!and!preserved!into!70%!alcohol,!brought! to!the!laboratory!for!further!identification.!samples!were!photographed!in!situ!before!being! preserved.!identification!was!done!based!on!main!characters!(dharma,!2005).!! ! environment&parameters& environment!parameters!including!salinity,!temperature,!ph,!dissolved!oxygen!and! sediment!type!were!measured!in!situ,!respectively.!! & data&analysis& ecology!indices!included!absolute!density,!species!diversity,!evenness,!dominance!index,! and!dispersal!pattern!was!applied!in!this!study.!data!was!analysed!according!to!formulas!as! follows!(odum!1993):! a.!density!(di)!!=!ni/a,!where:!! a! =!density!species!i;!! ni! =!total!no!of!ind!species!i;!! l!! =!total!area!of!plot!! ! b.!simpson!dominance!index!c!!=!!!∑!(ni!/!n)2,!!!where!:& ni!! =!!no!of!ind!species!!–!i! n!!! =!!total!no!of!ind!all!species! international#journal#of#applied#biology,#1(2),#2017# ! ! 69! criteria&:& 0! <!!c!!≤0,5!!!!!!!!!=!!low!! 0,5! <!!c!!≤! 0,75!!!!=!!moderate! 0,75!<!!c!!≤! 1,00!!!!=!!high!! ! c.! shanonqwiener!diversity!index!h!=!!q∑!(ni!/!n)!ln!(ni!/!n),!where!:& ni! =!!number!of!spesies!!q!i! n! =!!total!number!of!species!!!! h’! =!!diversity!index! ! criteria&:! ! 0! <!!h’!!≤!!2,0!!!!=!!low!! 2!!!!!<!!h’!!≤!!3,0!!!!=!!moderate! ! 3,0!!<!!h’!!≤!!4,0!!!!=!!high!!! !! d.!morisita!index!id!!!=!!!!!! ( )1 2 − −∑ nn nx n !!!,!!where!:& n! =!no!of!plot/transect!!! ! !!!!=∑f(x)! n! =!total!no!of!indiv!inside!plot/transect!!=!∑[f(x)]!(x)! ∑x2!=!quadrate!no!of!indiv!inside!plot! !!!!=!∑[f(x)]!(x2)!!!!!!!!! & criteria&:& id!!<!!!1,0!!!!=!!randomly!distributed!! id!!=!!!1,0!!!!=!!uniformly!distributed!! id!!>!!!1,0!!!!=!!clumped!distributed!!! ! ! results&and&discussion&& species&composition& we!found!14!families!and!14!genera!of!gastropods!that!consists!of!34!species.!these! species!are!aluco#aluco,#chicoreus#capucinus,#conus#eximus,#conus#ferrugineus,#conus#magus,# conus#varius,#cymbiola#vesvertillo,#cypraea#annulus,#cypraea#talpa,#cypraea#tigris,#cypraea# vitellus,#engina#alveolata,#engina#armilata,#engina#concinna,#lambis#lambis,#lambis#truncata,# littoraria#scabra,#nassarius#arcularius,#nassarius#jacksonianus,#nassarius#olivaceus,#nassarius# reeveanus,#nassarius#stolatus,#nassarius#venustus,#nerita#squamulata,#oliva#tigridella,#oliva# taeniata,# polinices# mammilla,# polinices# melanostomus,# pyrene# decussata,# rhinoclavis# vertagus,#strombus#labiatus,#strombus#urceus,#thais#tuberosa,#trochus#californicus.# amongst!34!species!of!gastropods,!7!species!was!found!at!st.!i,!24!species!was!at!sta.! ii,! and! 13! species! at! sta.! iii.! in! terms! of! species! composition,! gastropoda! from! family! nassaridae!genus!nassarium!are!more!abundance!with!6!species.!on!the!other!hand,!family! ciiridae,!columbellidae,!littorinidae,!neritopsidae,!and!volutidae!only!consist!of!1!species.! this! study! found! strombus# urceus! occurred! frequently,! counted! for! 41.379%! from! total! occurrence.! & & international#journal#of#applied#biology,#1(2),#2017# ! ! 70! density& the!result!shows!that!absolute!density!of!gastropods!at!each!station!are!follows:!sta.! i!0.06q0.8!ind/m2,!sta.!ii!0.06q0.5!ind/m2,!and!sta.!iii!0.06q2.22!ind/m2!(table!1).!strombus# urceus!shows!highest!occurrence!at!sta.!i!(41.379%)!(table!2).!this!in!accordance!with!study! done!by!arbi!(2001).!he!found!that!strombus#urceus!was!one!of!gastropods!that!distributed! widely! at! talise! waters! of! north! sulawesi.! the! lowest! density! was! shown! by! chicoreus# capucinus#and!thais#tuberosa.#seagrass!medows!of!balangdatu!waters!were!situated!nearby! mangrove!and!coral!reefs!may!allow!some!gastropods!that!origin!from!coral!reef!areas!as!hard! substrate!were!found!at!seagrass!areas.!the!last!two!species!chicoreus#capucinus#and!thais# tuberosa#that!present!at!seagrass!meadows!may!due!to!tidal!at!that!location.!supriharyono! (2000)!argued!that!current!have!contributed!significantly!to!a!dispersal!or!movement!from! benthic!fauna!from!one!ecosystem!to!others.!! ! table&1.&occurrences&of&gastropods&at&three&sampling&sites& no& species& sta.&1& sta.&2& sta.&3& 1! aluco#aluco# q! +! +! 2! chicoreus#capucinus# q! +! q! 3! conus#eximus# q! +! q! 4! conus#ferrugineus# +! +! +! 5! conus#magus# q! +! q! 6! conus#varius# q! +! q! 7! cymbiola#vesvertillo# +! q! +! 8! cypreae#annulus# q! +! q! 9! cypreae#talpa# q! +! q! 10! cypreae#tigris# q! q! +! 11! cypreae#vitellus# +! +! +! 12! engina#alveolata# q! +! q! 13! engina#armilata# q! q! +! 14! engina#concinna# q! +! q! 15! lambis#lambis# +! q! +! 16! lambis#struncata# q! +! q! 17! littoraria#scabra# q! +! q! 18! nassarius#arcularius# q! +! q! 19! nassarius#jacksonianus# q! +! q! 20! nassarius#olivaceus# q! +! q! 21! nassarius#reeveanus# q! +! q! 22! nassarius#stolatus# +! q! q! 23! nassarius#venustus# q! +! q! 24! nerita#squamulata# q! +! q! 25! oliva#tigridella# q! +! +! 26! olivia#taeniata# q! +! q! 27! polinices#mammilla# q! +! q! 28! polinices#melanostomus# q! q! +! 29! pyrene#decussata# q! +! q! 30! rhinoclavis#vertagus# q! q! +! 31! strombus#labiatus# +! q! q! 32! strombus#urceus# +! +! +! international#journal#of#applied#biology,#1(2),#2017# ! ! 71! 33! thais#tuberosa# q! q! +! 34! trochus#sp# q! q! +! & table&2.&density&of&gastropods&at&seagrass&meadows&of&balangdatu&waters&tanakeke& no.& gastropods& density&(ind/m 2 )& sta.&i& sta.&ii& sta.&iii& 1! aluco#aluco# 0! 0.200! 0.533! 2! chicoreus#capucinus# 0! 0.066! 0! 3! conus#eximus# 0! 0.266! 0! 4! conus#ferrugineus# 0.200! 0.133! 0.933! 5! conus#magus# 0! 0.200! 0! 6! conus#varius# 0! 0.066! 0! 7! cymbiola#vesvertillo# 0.133! 0! 0.533! 8! cypraea#annulus# 0! 0.666! 0! 9! cypraea#talpa# 0! 0.067! 0! 10! cypraea#tigris# 0! 0! 0.400! 11! cypraea#vitellus# 0.333! 0.133! 0.133! 12! engina#alveolata# 0! 0.466! 0! 13! engina#armilata# 0! 0! 0.200! 14! engina#concinna# 0! 0.333! 0! 15! lambis#lambis# 0.133! 0.000! 0.066! 16! lambis#struncata# 0! 0.066! 0! 17! littoraria#scabra# 0! 0.066! 0! 18! nassarius#arcularius# 0! 0.200! 0! 19! nassarius#jacksonianus# 0! 0.133! 0! 20! nassarius#olivaceus# 0! 0.200! 0! 21! nassarius#reeveanus# 0! 0.066! 0! 22! nassarius#stolatus# 0.267! 0! 0! 23! nassarius#venustus# 0! 0.066! 0! 24! nerita#squamulata# 0! 0.800! 0! 25! oliva#tigridella# 0! 0.533! 0.133! 26! olivia#taeniata# 0! 0.266! 0! 27! polinices#mammilla# 0! 0.400! 0! 28! polinices#melanostomus# 0! 0! 0.466! 29! pyrene#decussata# 0! 0.266! 0! 30! rhinoclavis#vertagus# 0! 0! 0.133! 31! strombus#labiatus# 0.067! 0! 0! 32! strombus#urceus# 0.800! 0.400! 2.200! 33! thais#tuberosa# 0! 0! 0.066! 34! trochus#sp# 0! 0! 0.066! total& 1.933! 6.058! 5.862! & species&diversity&(h’)& diversity!index!ranged!from!1,661!to!2,899!(table!3).!according!to!brower!et#al.,&(1990)! these!values!are!categorized!as!low!to!moderate.!low!diversity!in!sta.!i!may!due!to!habitat! which! dominated! by! rubble.! comparing! to! substrates! at! sta.! ii! and! sta.! iii,! less! nutrient! available!in!this!sta.!i!type!of!substrate!may!contribute!to!species!diversity.!! international#journal#of#applied#biology,#1(2),#2017# ! ! 72! & table&3.&species&diversity&index&(h’)&and&evenness&index&(e)& no.& sta.&& species&diversity&index&(h')& evenness&index&(e)& value& category& value& category& 1! i! 1.661! low! 0.853! stable! 2! ii! 2.899! moderate! 0.912! stable! 3! iii! 2.006! moderate! 0.782! stable! & evenness&index&(e)& this!study!revealed!that!sampling!site!shows!stable!condition!as!shown!by!values!of! evenness!0,782!–!0,912!(table!3).!stable!community!allows!species!to!occupy!equally!or!they! show!distribution!pattern!of!more!or!less!equal.!environmental!condition!that!relatively!same! would!allow!species!to!widely!distributed!at!balangdatu!waters.!different!type!of!habitats! would!have!influence!species!distribution!in!nature.!leaf!density!of!seagrass!as!habitat!is!also! affected! macrozoobenthos! including! gastropods! as! reported! by! hemminga! and! duarte! (2000). & dominance&index&(c)& in!the!present!study,!value!of!dominance!index!ranges!from!0.0001!to!0.140!(table!4).! this! reveals! that! community! in! stable! condition! as! dominance! index! close! to! zero,! as! indication!that!there!is!no!dominant!species.!also,!competition!amongst!species!may!also! result!in!space!distribution!odum!(1993).! ! table&4.!dominance&index&(c)&and&morisita&index&at&different&sampling&sites! no.& gastropods& indices& dominance&(c)& morisita&(id)& sta.&i& sta.&ii& sta.&iii& sta.&i& sta.&ii& sta.&iii& 1! aluco#aluco# 0! 0.001! 0.008! q! 1! 0.33! 2! chicoreus#capucinus# 0! 0.0001! 0! q! ~! q! 3! conus#eximus# 0! 0.001! 0! q! 1! q! 4! conus#ferrugineus# 0.01! 0.00048! 0.025! 1! 1! 0.259! 5! conus#magus# 0! 0.001! 0! q! 1! q! 6! conus#varius# 0! 0.0001! 0! q! ~! q! 7! cymbiola#vesvertillo# 0.004! 0! 0.008! 1! q! 0.485! 8! cypaea#annulus# 0! 0.012! 0! q! 0.684! q! 9! cypraea#talpa# 0! 0.0001! 0! q! ~! q! 10! cypraea#tigris# 0! 0! 0.004! q! q! 0.413! 11! cypraea#vitellus# 0.029! 0.0004! 0.0005! 0.59! 1! q0.06! 12! ensgina#alveolata# 0! 0.005! 0! q! 1! q! 13! engina#armilata# 0! 0! 0.001! q! q! 0.119! 14! engina#concinna# 0! 0.003! 0! q! 0.727! q! 15! lambis#lambis# 0.004! 0! 0.0001! 1! q! ~! 16! lambis#struncata# 0! 0.0001! 0! q! ~! q! 17! littoraria#scabra# 0! 0.0001! 0! q! ~! q! 18! nassarius#arcularius# 0! 0.0001! 0! q! 1! q! 19! nassarius#jacksonianus# 0! 0.0004! 0! q! 1! q! & & international#journal#of#applied#biology,#1(2),#2017# ! ! 73! 20! nassarius#olivaceus# 0! 0.001! 0! q! 1! q! 21! nassarius#reeveanus# 0! 0.0001! 0! q! ~! q! 22! nassarius#stolatus# 0.019! 0! 0! 1! q! q! 23! nassarius#venustus# 0! 0.0001! 0! q! ~! q! 24! nerita#squamulata# 0! 0.017! 0! q! 0.762! q! 25! oliva#tigridella# 0! 0.007! 0.0005! q! 1! q0.06! 26! olivia#taeniata# 0! 0.001! 0! q! 0.624! q! 27! polinices#mammilla# 0! 0.004! 0.006! q! 0.611! q! 28! polinices#melanostomus# 0! 0! 0! q! q! 0.523! 29! pyrene#decussata# 0! 0.001! 0! q! 0.624! q! 30! rhinoclavis#vertagus# 0! 0! 0.0005! q! q! 0,060! 31! strombus#labiatus# 0.001! 0! 0! ~! q! q! 32! strombus#urceus# 0.171! 0.004! 0.14! 0.89! 0.525! 0.517! 33! thais#tuberosa# 0! 0! 0.0001! q! q! ~! 34! trochus#sp# 0! 0! 0.0001! q! q! ~! ! ! distribution&pattern&(id)! ! generally,!gastropods!at!almost!sampling!sites!shows!clumped!distribution,!although! some!species!were!widely!distributed.!clumped!distribution!is!shown!by!value!of!morisita! index!is!close!to!zero,!as!described!in!table!4.!odum!(1993)!explained!that!distribution!pattern! of!species!are!correlated!to!feeding!habit!or!feeding!mode.!species!tends!to!form!a!group! where!more!food!are!available.!beside,!external!reproduction!as!characteristics!of!substrates! may!also!contribute!to!certain!distribution!pattern!of!species!such!as!clumped!distribution.! gastropodas! such! as! cypreae# vitellus,# engina# armilata,# and! olivia# tigridella! show! equal! distribution,!this!pattern!indicating!that!there!is!competition!amongst!species,!hence!space! distribution!equally. & environment&parameters&& environment! parameters! are! shown! in! table! 5.! as! seen! from! table! 5,! water! temperature!ranged!from!31.77!to!34.17°c.!these!temperatures!are! in!favor!to!molluscs! especially!gastropods.!gastropods!are!having!a!thick!shell,!allows!this!group!to!survive!at!quite! high!temperature.!marine!fauna!can!live!normally!in!water!with!temperature!of!26!q!32°c,! although! several! invertebrate! fauna! can! adopt! to! a! high! temperature! odum! (1993).! the! present! study! indicates! that! salinity! of! balangdatu! water! of! about! 33.67! ‰! –! 34.67! ‰,! respectively!(table!5),! in!which!is!suitable!for!macrozoobenthos.!metungun!et#al.,! (2011)! stated!that!salinity!of!25!–!40‰!is!good!for!marine!gastropods.!ph!value!of!sampling!site! varied!from!6.92!to!q7.19!(table!5),!these!ph!ranged!can!be!tolerated!by!marine!organisms,! even!though!ph!marine!water!can!be!varied!between!!7.5!and!8.4.!gross!(1972)!in!metungun# et#al.,!(2011),!most!of!the!aquatic!gastropods!can!live!at!ph!of!5q9.!in!terms!of!dissolved! oxygen!(do),!balangdatu!waters!contain!4.5!to!5.17!mg/l!(table!5).!another!study!at!bone! batang!gusung!at!seagrass!meadows!showed!a!similar!pattern!where!do!ranged!from!4.6!to! 5.5!mg/l.!these!values!support!optimal!growth!for!seagrass.!! furthermore,!the!result!of!sediment!analysis! indicates!that!substrates!at!sampling! sites!were!categorized!as!sandy!clay!(table!5).!sediment!texture!differ!amongst!sampling!sites! as!follows!sand!(65.00!%!q!67.33!%),!dust!(11.04q15!%)!and!clay!(4.40q5.30!%).!the!texture!of! sediment! at! seagrass! meadows! of! balangdatu! differs! from! the! common! structure! at! international#journal#of#applied#biology,#1(2),#2017# ! ! 74! spermonde!archipelago!that!dominated!by!sand,!ruble!and!gravel!(sawall!et#al.,!2011;!2013),! progeroutz!et#al.,!2012).! ! table&5.!environment&parameters&at&seagrass&meadows&of&balangdatu&waters!! parameters& sampling&site& sta.&i& sta.&ii& sta.&iii& temperature!(°c)! 31.77! 33.1! 34.17! ph! 7.19! 6.92! 7.19! salinity!(‰)! 33.67! 34.67! 34.67! do!(mg/l)! 4.5! 5.13! 4.97! substrates! sandy!clay! sandy!clay! sandy!clay! ! conclusions& the! present! study! concludes,! based! on! ecology! indices,! balangdatu! waters! of! tanakeke!takalar!south!sulawesi!is!still!in!good!condition!and!support!marine!gastropods.!!! & acknowledgment&& we!thank!yusran!nurdin!massa,!regista,!suardi,!dan!a.!darmawansyah!from!yayasan! hutan!biru!indonesia,!and!local!people!of!balangdatu!tanakeke!for!assistance!during!field! work.!the!ministry!of!education!and!higher!degree!of!indonesia!for!providing!travel!grant!for! first!author!to!present!this!work!at!wcm!2016.! & references& andy! omar! s.b,! m.! litaay,! n.! anwar.! 2006.! the! distribution! and! abundance! of! tropical! abalone!(haliotis!spp)!at!reef!flat!of!bonetambu!island,!makassar.!torani!spec!publ.! 16(2):142q147.! arbi,!u.y.!!2011.!mollusca!community!structure!at!seagrass!medow!of!talise!waters,!north! sulawesi.!oseanologi#and#limnologi#di#indonesia,!37(1):71q89! brower,!j.s,!j.h.!zar,!n.o.!ende.!1990.!field!and!laboratory!methods!for!general!ecology.! third!edition.!brown.! cappenberg,! a.w.! 2014.! megabenthos! community! structure! in! pangkajene! waters! of! pangkep!district!south!sulawesi.!zoo#indonesia,!23(2):57q67! clark,!j.!1974.!costal!ecosystem.!ecological!consideration!for!management!of!the!coastal! zone.!the!conservation!foundation.!washington,!dc.! dharma,!b.!1988.!indonesia!shell!part!i.!germany.! dharma,!b.!2005.!recent!and!fossil.!indonesian!shells.!conch!book.!germany.! english!s,!c.!wilkinson,!v.!baker.!1997.!survey!manual!for!tropical!marine!resources.!! ! australian!institute!of!marine!science.!townsville.! litaay,!m.!2006.!!macroinvertebrates!associated!with!seagrass!at!awarange!and!labuange! 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un!strait!at!south!east!moluccas.!oseanologi#and#limnologi#in#indonesia,!isbn:!978q602q 98439!:!225q231! odum,!e.p.!1993.!ecology.!gadjah!mada!university!press.!yogyakarta! priosambodo,!d.!2014.!baseline!survey!manual!and!data!analysis!for!seagrass.!yayasan!hutan! biru!indonesia.!makassar.! progeroutz!c,!asmus!h,!ahnelt!h,!kneer!d,!litaay!m.!2012.!the!influence!of!canopy!structure! and! tidal! level! on! fish! assemblages! in! tropical! southeast! asian! seagrass! meadows.! eustarine#coastal#and#shelf#science,!107(2):58q68.! sawall!y,!teichberg!m,!seemann!j,!litaay!m,!jompa!j,!richter!c.!2011.!nutritional!status!and! metabolism! of! the! coral! stylophora# ubseriata! along! a! eutrophication! gradient! in! spermonde!archipelago!(indonesia).!coral#reefs,!30:!841–853.! sawall!y,!c.!richter,!j.!jompa,!m.!litaay.!!2013.!!coral!recruitment!and!potential!recovery!of! eutrophied! and! blast! fishing! impacted! reefs! in! spermonde! archipelago,! indonesia.! marine#pollution#bulletin,!74(1):374q382.! supriharyono.!2000.!conservation!and!management!of!natural!resources!in!tropical!coastal! areas.!pt.!gramedia!pustaka!utama,!jakarta![in!indonesia]! tomascik!t,!a.j.!mah,!a.!nontji,!m.k.!moosa.!1997.!the!ecology!of!the!indonesian!seas.!part! two.!periplus!edition!(hk)!ltd.!singapore! ! ! ! ! ! ! 2(1)1-11 contact : dominggus rumahlatu dominggus_amq@yahoo.co.id ó international journal of applied biology 1 abstract the existence of macrozoobenthos in an ecosystem is closely related to the conditions of the surrounding environment. this research aimed to examine the physical chemical condition of aquatic environment and macrozoobenthos community structure in the coastal waters of marsergu island. this research was conducted in july and december 2016 in 3 stations. the data were collected by using sampling technique to measure the environmental factors (temperature, salinity, ph, and dissolved oxygen of seawater) and enumeration of gastropod species with in-situ. the results of this research showed that macrozoobenthos in the coastal waters of marsegu island consisted of 2 phylum, namely molusca and echinoderms, that is, gastropod with 18 species, bivalvia with 5 species, and echinoderms with 17 species. echinoderms was the dominant macrozoobentos with a percentage of 45%, followed by gastropods 42%, and bivalvia 13%. this suggests that echinoderms and gastropods are the main components that make up the macrozoobenthos community structure in the coastal waters of marsegu island. the results of the calculation of the diversity index of macrozoobenthos in the three research stations showed the highest score was station 1 as much as 3.56, station 2 as much as 3.52 and the lowest was station 3 as much as 2.07. issn : 2580-2410 eissn : 2580-2119 macrozoobenthos community structure in the coastal waters of marsegu island, maluku, indonesia fitry yunita, fredy leiwakabessy & dominggus rumahlatu biology education departement, faculty of teacher training and education science, pattimura university, ambon, maluku, indonesia introduction along with the growing human needs, the economic activity is also growing. various industries emerge with the aim of meeting the human needs. this rapid industrialization has had a negative impact on the environment. the residue of the processing becomes a source of pollution for the surrounding environment, both land, air and water. the environmental quality continues to decline significantly, especially the marine waters. most residues of human activity will be disposed at sea, and this causes environmental problems. sea water pollution is a paradox, because most of the nutrients needed by humans come from the open access international journal of applied biology keyword community structure macrozoobenthos marsegu island article history received 23 march 2018 accepted 18 june 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 2 ocean. therefore, continuous water quality monitoring is required by utilizing bioindicators such as macrozoobenthos. the studies on the role of macrozoobenthos as bioindicator are based on the fact that these organisms have a very important role in nutrient cycling in waters. răescu et al. (2011) explains that macrozoobentos are the main source of nutrients in aquatic ecosystems. macrozoobenthos plays a fundamental role in transfering the energy through the food chain and helps break down various types of material in the sea bed (vyas et al., 2012). dauer (1983) explains that macrozoobenthos has a living behaviour which does not change much, accustomed to make contact with contaminants, and shows different levels of tolerance toward contaminants. the distribution of macrozoobenthos in an aquatic environment is determined by a number of factors, such as the physical properties of the substrate and nutrients in the water column. in addition, trayanova et al. (2007) add that benthos invertebrates are used as environmental indicators because their responses can predict various types of anthropogenic stress. some researches have proved that the presence of macrozoobenthos in an ecosystem is closely related to the conditions of the surrounding environment. the research conducted by cai et al. (2012) found that the total concentrations of nitrogen, phosphate, ammonium and dissolved oxygen is closely related to the macrozoobenthos community structure in caochu china river. taurusman (2010) also found that the structure of macrozoobenthos in eutrophic coastal waters had a significant correlation with the quantity and quality of organic material. the research also proved that macrozoobenthos could respond to eutrophication conditions. one of the coastal areas which have the potential of macrozoobenthos is marsegu island in west seram district, maluku, indonesia, with an area of 240.20 ha and it has marine areas as much as 10.000 hectares. marsegu island is often used as a laboratory for various types of research, namely agriculture research, forestry research, fishery research, conservation research, and as tourism area. therefore, a continuous analysis is required to assess the quality of the waters by utilizing aquatic organisms as bioindicators, and analyzing the physical-chemical factors in marine environment, as well as analyzing the macrozoobenthos community structure in the coastal waters of marsegu island. materials and methods this research was conducted in the coastal waters of marsegu island in july 2016, at coordinate 02°59’03°01’ latitude and 128°02’ 128°03’ longitude (figure 1). the data were collected by using sampling technique to measure the physical chemical factors of the environment (temperature, salinity, ph and dissolved oxygen) and enumeration of gastropods with in-situ at 3 stations of data collection. the data collection was carried out during the lowest tide, beginning with determining the sampling area, and then making the vertical transect line as many as 10 transect lines from the highest tide limit toward the sea with the distance between the transect line with each other 50 m. in each transect were made 10 plots of 1×1 m with the distance between the plots 10 m, to obtain 100 plots for each data collection station. the measurement of the environmental factors (temperature, salinity, ph, and do) was carried out on each plot for 10 transect lines in the morning, afternoon, and late afternoon. the equipments used for the measurement of temperature, salinity, ph, and dissolved oxygen of sea water were thermometer, refragtometer, ph meter, and do meter. international journal of applied biology, 2(1), 2018 3 the data about the types of macrozoobenthos were collected by enumeration, and it was done by taking pictures of each macrozoobenthos in the research sites. figure 1. location of research stations the calculation of the diversity index of macrozoobenthos was done descriptively by using shannon-wiener formula, as follows. η’ = − ∑ pi ln pi (brower et al. 1990) where: p i = n i / n η’ = shannon–wiener diversity, ni = total individuals of species i, n = total number of individuals of all species, ln= logarithm nature, σ = number of individual species, with the criteria of diversity, namely low (h <2) moderate (2 <h <4), and high ( h> 4). in addition, it was also calculated the evenness value, richness value, density, frequency of attendance, and importance values. results and discussion the physical chemical environmental factors the results of the measurement of the physical chemical factors of the aquatic environment in each plot at the three observation stations (table 1) show that there were fluctuations of the physical chemical environmental factors in the coastal waters of marsegu island measured in the morning, afternoon and late afternoon. international journal of applied biology, 2(1), 2018 4 the results of this research (table 1) show that the average temperature of the sea water experienced fluctuation in morning, afternoon and late afternoon. the sea water temperature seemed to be lower in the morning, but it increased in the afternoon, and it decreased again in the late afternoon. the average temperature in the morning ranged from 27.80 8.00oc, in the afternoon ranged from 29.90 30.16oc and in the late afternoon was 26.00oc. the fluctuation of the sea water temperature was influenced by the characteristics of the intertidal zone which had a very extreme temperature fluctuation. this temperature fluctuation was caused by the solar radiation and the co2 content in the water. these fluctuations are among the factors that determine the level of adaptation of organisms in the intertidal zone. temperature is known to play an important role in the various physiological responses of aquatic organisms (szathmary et al., 2009; fly et al., 2012). this is due to climate change which results in increasing the frequency and severity of heat waves (adamo & lovett, 2011). in addition, temperature affects the rate of biochemical and physiological processes and the stability of biomolecules (sanford, 2002). high temperatures in the body of the organism can affect the rate of digestion, respiration, metabolic activity, growth, reproduction and gametogenesis in poikilotherm organisms (christophersen & strand, 2003). table 1. the results of the measurement of the physical chemical environmental factors in the coastal waters of marsergu island variables station morning afternoon late afternoon temperature (oc) 1 27.80 29.90 26.00 2 28.00 30.16 26.00 3 27.80 29. 90 26.00 ph 1 7.00 7.00 7.00 2 7.00 7.00 7.00 3 7.00 7.00 7.00 do (mg/l) 1 4. 16 3.02 2.38 2 4. 06 3.19 2.07 3 3. 79 3.06 2.67 salinity (%o ) 1 31.00 31.00 31.00 2 31.00 31.00 31.00 3 31.00 31.00 31.00 ph factor (table 1) in the coastal waters of marsegu island was relatively stable. there was not any significant ph fluctuation during the measurement in the morning, afternoon and late afternoon. rumahlatu & leiwakabessy (2017) explains that the ph of a waterway affects the distribution of gastropods. based on the results of the measurement, the ph of the sea water was 7. the acidity factor of aquatic environment is strongly influenced by the concentration of co2 in the atmosphere. if the concentration of co2 in the atmosphere is very high, then it will be absorbed into the water, so that the ph becomes reduced, and the saturation of carbonate ions in water will occur. the condition of the seawater that is too acidic will affect the physiological activity of the organisms which live in it (paganini et al., 2014). physiologically, ocean acidification will lead to a change in the structure of organisms in the habitat, food web, and the loss of marine resources that are useful for human being (newell, 2004). international journal of applied biology, 2(1), 2018 5 another environmental factor which did not experience a significant fluctuation was the salinity of sea water. the average salinity measured in the coastal waters of marsegu island was 31%o and did not show any difference among the measurement carried out in the morning, afternoon and late afternoon. although it did not show any significant fluctuation, montory et al. (2014) explain that the organisms that live in the intertidal zone are often faced with the fluctuation of salinity of sea water that causes pressure to physiological conditions. too high salinity and if followed by increased temperature will reduce the availability of oxygen in the tissues and reduce the resistance of organisms (pörtner & farrell, 2008). the fluctuation in the environmental factor appeeared in the dissolved oxygen where fluctuation occured in the morning, afternoon and late afternoon. the dissolved oxygen level was high in the morning and decreased in the afternoon and late afternoon. the average dissolved oxygen in the morning ranged from 3.79-4.16 mg/l, and in the afternoon it decreased to 3.02-3.09 mg/l. the dissolved oxygen continued to decline in the late afternoon to 2.07-2.67 mg/l. oxygen is a factor for the respiration of all living things. if the oxygen concentration in the water is too low, it can cause hypoxia. at the individual level, this condition can cause physiological changes and interfere with the ability of individuals to survive, reduce reproductive system work, and be highly vulnerable to various types of diseases (ekau et al., 2010). hipoksia juga dapat menyebabkan perubahan kepadatan dan distribusi, serta dapat mengganggu komposisi kumunitas dengan cara menghilangkan spesies sensitif serta memberi kesempatan pada spesies toleran untuk berkembang biak (weisberg et al., 2008). types of macrozoobenthos in the coastal waters of marsegu island the individuals of macrozoobenthos were collected and subsequently identified based on the taxon order (table 2). based on the results of identification, macrozoobenthos in the coastal waters of marsegu island consisted of 2 phylum, namely mollusk and echinodermata, with 18 species of gastropods, 5 species of bivalves, and 17 species of echinoderms. table 2. macrozoobenthos species in the coastal waters of marsegu island phylum class order family genus species molluscs gastropods littorinimorpha strombidae canarium canarium urceus (linne, 1758) lambis lambis-lambis (linne, 1758) lentigo lentigo lentiginosus (linne, 1758) cypraeidae cypraea cypraea annulus (linne, 1758) cypraea evrones (linne, 1758) cypraea tigris (linne, 1758) cypraea ovum (gmelin, 1791) cypraea vitellus (linne, 1758) caenogastropoda cerithiidae clypeomorus clypeomorus concisus (hombron & jacquinot, 1848) neogastropoda volutidae cymbiola cymbiola vespertilio (linne, 1758) conidae conus conus eburneus (hwass in bruguière, 1792) conus muriculatus (sowerby, 1833) buccinidae chantarus chantarus undosus (linne, 1758) engina engina medicaria (linne, 1758) nassariidae nassarius nassarius albescens (dunker, 1846) nassarius livescens (philippi, 1849) cycloneritimorpha neritidae nerita nerita sp (linnaeus, 1758) bivalves venerida veneridae gafrarium gafrarium tumidum (röding, 1798) ostreida pinnidae pinna pinna bicolor (gmelin, 1791) pinna muricata (linne, 1758) veneroida tellinidae tellina tellina radiate (linne, 1758) international journal of applied biology, 2(1), 2018 6 phylum class order family genus species mactridae spisula spisula subtruncata (costa, 1778) echinodermata asteroidea valvatida ophidiasteridae linckia linckia laevagata (linne, 1758) linckia multifora (lamarck, 1816) archasteridae arachaster arachaster typicus (müller & troschel, 1840) oriasteridae culcita culcita novaeguinea (müller & troschel, 1842) protoreaster protoreaster lincki (blainville, 1830) protoreaster nodusus (linne, 1758) forcipulatida asteriidae asterias asterias vulgaris (verrill, 1866 ) ophiuroidea ophiurida ophiuridaea ophiura ophiura ophiura (linne, 1758) ophiactidae ophiopholis ophiopholis aculeafa (linne, 1767) echinoidea diadematoida diadematidae diadema diadema setosum (leske, 1778) echinothrix echinothrix calamari (pallas, 1774) astropyga astropyga radiata (leske, 1778) echinoida strongylocentrotidae strongylocentrotus strongylocentrotus purpuratus (stimpson, 1857) holothuriodea apodida chiridotidae chiridota chiridota violacea (müller, 1849) holothuriida holothuriidae holothuria holothuria atra (jaeger, 1833) holothuria nobilis (selenka, 1867) bohadschia bohadschia marmorata (jaeger, 1833) the researc results (table 2) showed that echinoderms were very dominant in the coastal waters of marsegu island with a percentage of 45%, followed by the gastropod class by 42% and bivalves by 13%. this means that echinoderms and gastropods were the main components that made up the macrozoobenthos community structure in the coastal waters of marsegu island. the results of this research were in line with williams and garcía-sais (2010) who explained that echinoderms are very important marine animals, and most of its species are known to become the key species that govern the structure and balance of community that affects the life of other organisms on various types of ecosystems. supono et al. (2014) assert that echinoderms are the major part of marine biodiversity, especially in the context of biomass and macroinvertebrates that play an important role in the functions of ecosystem. echinoderm species are members of the seabed community which are distributed in the polar regions and the tropics regions. echinoderm phylum contains several trophic groups, namely detrivor, filter-feeder, grazer, scavenger and an active predator, and plays an important role in the structure of benthic communities (petovic & cetkovic, 2016). described by bellwood et al. (2004) that echinoderms are an important component in coral reef ecosystems. in aquatic environment, some species of gastropods are basically animals which feed on detritus (detritus feeder) and on the litter of falling leaves and circulate substances that are suspended in water in order to get food. in marine waters, gatropods are more widespread in the littoral region. this is an area affected by tidal activity. this is because the littoral zone area also has a variety of supporting ecosystems and other biodiversity, especially for the typical coastal area in tropical waters and for biota life. in the marine environment, gastropods are also found in various habitats, such as mangrove forests, coral reefs, rocky beaches, sandy beaches, seagrass and deep sea (khade & khade, 2016). diversity index, dominance index, and evenness index of macrozoobenthos species in the coastal waters of marsegu island the results of enumeration of the macrozoobenthos species were then analyzed to determine the diversity index, evenness index and abundance index of macrozoobenthos in the coastal waters of marsegu island (table 3). international journal of applied biology, 2(1), 2018 7 table 3. diversity index, evenness index and abundance index of macrozoobenthos species station diversity index evenness index abundance index 1 3.56 0.97 0.03 2 3.52 0.95 0.03 3 2.07 0.56 0.24 the results of the analysis of community structure (table 3) showed that the diversity index of macrozoobenthos on the three research stations from the highest to the lowest was station 1 as much as 3.56, station 2 as much as 3.52 and the station 3 as much as 2.07. morris et al. (2014) explain that biodiversity represents the diversity and heterogeneity of organisms or characteristics at all levels of the hierarchy of life, from molecules to ecosystems. heterogeneity of organisms also indicates that the ecosystem is more stable. the results of the calculation of evenness index showed that the evenness index at station 1 was as much as 0.965, station 2 as much as 0.954, and station 3 as much as 0.561. heip et al. (1998) explains that the evenness expresses how individuals of different species are evenly distributed within a community. evenness indicates whether or not there is a dominance of particular individuals in the community. the results of the calculation of the abundance index showed that the abundance index at station 1 was as much as 0.03, station 2 as much as 0.03 and station 3 as much as 0.24. aslam (2009) explains that the abundance shows the number of species in a region or at the sampling location. the diversity index, evenness index and abundance index are important ecological indicators to measure the stability of a community. gotelli & chao (2013) explained that information on species diversity is one of efforts to conserve biodiversity along with increasing pressure and climate change environment due to human activities. density, occurrence frequency and importance value index the data from the results of observations at each research station were then used to calculate the density value, abundance value, occurrence frequency and the improtance value index of each species. the summary of the analysis results is presented in table 4 below. table 4. density (d), occurrence frequency (of) and the importance value index (ivi) no species station 1 station 2 station 2 d of ivi d of ivi d of ivi 1 nerita sp 1.01 0.51 15.51 0.97 0.50 14.00 18.38 0.89 81.41 2 tellina radiate 0.92 0.47 14.20 0.92 0.47 13.23 9.67 9.67 78.17 3 strongylecentrotus purpuratus 0.88 0.43 13.76 0.88 0.45 12.99 1.29 0.61 8.71 4 spisula subtruncata 0.88 0.44 13.47 0.89 0.44 12.97 1.18 0.55 7.92 5 gafrarium tumidum 0.82 0.41 12.55 0.88 0.43 12.76 0.49 0.49 5.32 6 conus eburneus 0.76 0.38 12.45 0.82 0.41 12.69 0.66 0.29 4.85 7 deadema setosum 0.82 0.40 12.43 0.83 0.41 12.09 0.68 0.30 4.78 8 echinothrix calamari 0.71 0.36 10.92 0.82 0.40 11.89 0.64 0.28 4.70 9 conus muriculatus 0.62 0.28 10.61 0.72 0.34 10.66 0.62 0.27 4.54 10 canarium urceus 0.69 0.32 10.59 0.71 0.33 10.45 0.61 0.26 4.44 11 nassarius livescens 0.67 0.30 10.15 0.71 0.33 10.45 0.61 0.26 4.44 12 cymbiola vespertilio 0.62 0.29 9.92 0.61 0.27 8.82 0.60 0.26 4.39 13 cypraea evrones 0.55 0.22 8.77 0.56 0.22 8.13 0.35 0.35 4.33 international journal of applied biology, 2(1), 2018 8 14 protoreaster nodusus 0.59 0.24 8.37 0.55 0.21 7.91 0.62 0.23 4.32 15 cypraea tigris 0.54 0.21 8.13 0.55 0.21 7.91 0.59 0.24 4.23 16 cypraea vitellus 0.50 0.19 7.86 0.52 0.20 7.49 0.32 0.32 3.96 17 pinna bicolor 0.55 0.22 7.76 0.50 0.20 7.29 0.28 0.28 3.89 18 cypraea annulus 0.48 0.17 7.39 0.48 0.18 7.26 0.49 0.17 3.57 19 nassarius albescens 0.49 0.18 6.95 0.47 0.17 7.04 0.49 0.17 3.57 20 clypeomorus concisus 0.39 0.13 6.31 0.46 0.15 6.70 0.68 0.31 3.36 21 pinna muricata 0.45 0.14 6.08 0.45 0.14 6.48 0.41 0.15 3.28 22 arachaster typicus 0.46 0.15 6.08 0.45 0.14 6.48 0.42 0.15 3.08 23 lambis-lambis 0.42 0.15 5.71 0.42 0.12 5.92 0.24 0.24 2.97 24 cypraea ovum 0.37 0.12 5.57 0.37 0.11 5.70 0.17 0.17 2.88 25 protoreaster lincki 0.41 0.14 5.49 0.36 0.11 5.58 0.20 0.20 2.78 26 linckia laevagata 0.40 0.14 5.40 0.34 0.10 5.22 0.31 0.10 2.69 27 engina medicaria 0.34 0.11 5.11 0.33 0.10 5.10 0.34 0.11 2.64 28 ophiura ophiura 0.38 0.12 4.98 0.32 0.10 4.98 0.17 0.17 2.36 29 ophiopholis aculeafa 0.37 0.12 4.88 0.31 0.09 4.75 0.17 0.17 2.36 30 asterias vulgaris 0.35 0.12 4.70 0.30 0.09 4.63 0.27 0.09 2.36 31 chantarus undosus 0.30 0.10 4.54 0.28 0.09 4.39 0.26 0.09 2.29 32 holothuria atra 0.34 0.11 4.48 0.28 0.09 4.39 0.22 0.07 2.24 33 holothuria vagabunda 0.27 0.09 4.37 0.27 0.09 4.27 0.16 0.16 2.22 34 chiridota violacea 0.32 0.11 4.30 0.27 0.08 4.15 0.06 0.04 0.91 35 linckia multifora 0.29 0.10 4.04 0.26 0.08 4.03 0.05 0.04 0.79 36 holothuria nobilis 0.28 0.10 3.94 0.24 0.07 3.68 0.04 0.04 0.68 37 lentigo lentiginosus 0.24 0.08 3.88 0.23 0.07 3.56 0.02 0.02 0.52 38 bohadschia marmorata 0.23 0.08 3.76 0.21 0.06 3.20 0.02 0.02 0.52 39 astropyga radiata 0.21 0.07 3.40 0.20 0.06 3.08 0.02 0.02 0.52 40 culcita novaeguinea 0.10 0.05 1.91 0.13 0.05 1.32 0.03 0.03 0.51 the results of this research (table 4) show that the nerita sp species was the main constituent component of the macrozoobenthos community in the coastal waters of marsegu island. this can be seen from the density value, occurrence frequency, and the importance value index of nerita sp which were high on the three research stations. at station 1, the density value was 1.01 and the occurrence frequency was 0.51 and the importance value index was 15.51. furthermore, at station 2, the density value of nerita sp was 0.97, the occurrence frequency was 0.50 and the importance value index was 14.00. at station 3, the density value was 18.38, the occurrence frequency was 0.89 and the importence value index was 81.41. the research results (table 4) also show that the culcita novagaeguinea species had the smallest contribution in the structure of macrozoobenthos community in the coastal waters of marsegu island. it can be seen from the density value, the occurrence frequency, and importance value index of culcita novagaeguinea in the three research stations. at station 1, the density value was 0.10, the occurrence frequency was 0.05 and the importance value index was 1.91. furthermore, at station 2, the density value was 0.13, the occurrence frequency was 0.05, and the importance value index was 1.32. at station 3, the density value was 0.03, the occurrence frequency was 0.03, and the importance value index was 0.05. makrobentos invertebrates are important parts of marine ecosystems (mcdonald et al., international journal of applied biology, 2(1), 2018 9 2012). more than 95% of species in marine ecosystems are benthic organisms. these organisms include epifauna which lives on the surface of the substrate (tagliapietra & sigovini, 2010). these organisms have the capability to attach to the surface of the substrate or can move freely on the surface of the sediment. the organisms are known to have high sensitivity and can provide a fast response to environmental degradation. therefore, these organisms are often used as biological indicators (medrano, 2015). conclusions the based on results and discussion of this research, it can be concluded that the physical chemical environmental factors (temperature, salinity, and dissolved oxygen) in the coastal waters of marsegu island experienced fluctuations, while the ph of seawater did not experience a fluctuation in the measurement which was carried out in the morning, afternoon and evening. the physical chemical environmental factors had an effect on the structure of macrozoobenthos community in waters. the macrozoobenthos found in the coastal waters of marsegu island consisted of 2 phylum, namely mollusk and echinodermata, that is, gastropods with 17 species, bivalves with 5 species, and echinoderms with 18 species. based on the number of the macrozoobenthos found, the results of the diversity index calculation of macrozoobenthos in the three research stations showed that 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ecological status of varna lagoon. transitional waters bulletin, 3:33–36. vyas, v., bharose, s., yousuf, s. & kumar, a. 2012. distribution of macrozoobenthos in river narmada near water intake point. journal of natural sciences research, 2(3):18–24. weisberg, s.b., thompson, b., ranasinghe, j.a., montagne, d.e., cadien, d.b., dauer, d.m., diener, d., oliver, j.s., reish, d.j., velarde, r.g. & word, j.q. 2008. the level of agreement among experts applying best professional judgment to assess the condition of benthic infaunal communities. ecological indicators, 8:389–394. williams, s. & garcía-sais, w. 2010. temporal and spatial distribution patterns of echinoderm larvae in la parguera, puerto rico. rev biol trop, 58:81-88. contact : muh. syahrial msyahrial195@gmail.com abstract the objective of this research was to assess the financial feasibility of tiger prawn polyculture (penaeus monodon) with finfish (milkfish, tilapia, and white snapper) using a leased land system and privately owned land. the sample of respondents was determined by purposive sampling technique in which the number of respondents each was 34 people for privately owned land and leased land. the primary data were collected through direct interviews with respondents. to determine the variations in financial feasibility parameters between the two polyculture cultivation systems, the data were examined using quantitative descriptive analysis, business financial analysis, and a t-test. the results showed that the polyculture business of tiger prawns and finfish using privately owned land with leased land had a significant difference in the income parameters, r/c ratio, and return on investment (roi) and was not significantly different for the payback period (pp) parameter. the average value of the parameters of the financial feasibility analysis are: income of rp. 7.043.771/year, r/c ratio of 1,44, pp of 4 years 1 month, roi of 40,97%, npv of rp. 35.375.216, irr of 77.32%, net b/c 2,98 for privately owned land systems and income of rp. 4.007.466/year, r/c ratio of 1,28, pp of 4 years 10 months, roi of 24,88%, npv of rp. 21.492.203, irr of 50,36%, net b/c 2,12 for leased land system. financially, the cultivation system on private land is more profitable than the polyculture cultivation system on leased land. however, in general, both polyculture cultivation systems are profitable and feasible to be continued and developed. issn : 2580-2410 eissn : 2580-2119 analysis of the feasibility of tiger prawn and finfish polyculture business using land with different ownership status in the coastal area of pinrang regency muh. syahrial1, aris baso1, irmawati1 1 study program of fisheries science, faculty of marine science and fisheries, hasanuddin university, makassar introduction aquaculture is one type of sector that contributed about 50 percent of the world's total fishery production in 2018 with a production growth rate of more than 500% since the 1990s (fao, 2020). one model of cultivation is polyculture. in order to make the most use of the available resources, polyculture is founded on the premise that each species that is stocked has its own feeding niche (anil et al. 2010). based on the concepts of agroecology and aquaculture ecology, the polyculture system is concerned with increasing land productivity, environmental conservation, and animal welfare. efficient polyculture supports synergies between species to obtain optimal productivity (thomas et al., 2020). in line with this, in 2020 the fisheries and marine service of pinrang regency recorded an increasing trend open access international journal of applied biology keyword finfish; financial feasibility; polyculture; tiger prawns article history received march 25, 2022 accepted july 05, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 157 of shrimp and fish from polyculture cultivation in the last 5 years. the data showed an increase in production yields of 6%-15% for tiger prawns and 10%-25% for milkfish. a promising brackish-water aquaculture, shrimp farming offers a high rate of return on investment during a brief development time. for shrimp to grow and produce more, technological advancements in polyculture with other aquaculture biota are required. some types of finfish that are commonly cultivated together with tiger prawns are tilapia and milkfish. in 2018, kkp through the directorate general of aquaculture introduced white snapper (lates calcalifer bloch, 1790) or known locally as bale kanja as a new type of finfish that can be polycultured with tiger prawns. fish with a smooth flesh texture, white flesh without melanin, and few spines make barramundi very popular, and have high market prospects and prices (irmawati et al. 2021). south sulawesi's tiger shrimp production is expected to reach 12 million heads in 2020 (marine and fisheries department of south sulawesi, 2020). pinrang regency is the major tiger shrimp production region in south sulawesi and one of the top regions for tiger shrimp production. the total potential area of ponds in pinrang regency is ±15,853 ha or 16,51% of the total ponds in south sulawesi. this is what makes pinrang regency designated as the center for the development of tiger shrimp production in south sulawesi. the majority of pond cultivators in pinrang regency manage ponds traditionally with a polyculture system. cultivation with a polyculture system is intended to optimize ponds by compaction in order to provide greater income for pond entrepreneurs. in research conducted by islamiyah (2020), polyculture actors of milkfish, tiger shrimp, and seaweed in bonto langkasa village, minasatene sub-district, pangkajene regency earned an income of rp. 24.093.532,-/year and the business was concluded to be feasible with the net present value. (npv) was positive, the net benefit-cost ratio (net b/c ratio) was more than 1, the internal rate of return (irr) was greater than the interest rate, and the payback period (pp) was 3,2 years. in the pinrang regency region, it is unknown with certainty how much money will be made or how feasible polyculture ponds will be. since they neglect the financial component, cultivators are unable to assess the feasibility of their farming efforts. in order to provide pond farmers, relevant agencies, and institutions that support people's efforts to accelerate economic development with information, researchers are encouraged by these issues to learn more clearly. materials and methods research location and time the study was conducted from march to june 2021 in lanrisang and mattirosompe sub-districts, pinrang district, south sulawesi. purposefully, this area was chosen because it is a hub for tiger shrimp development in south sulawesi, particularly for polyculture system farming. research location and time the research used survey research methods (interviews, observations, and literature studies) (singarimbun and effendi, 2006). sampling technique was done by conducting cluster sampling. according to sedgwick (2014), cluster sampling involves taking random cluster samples from the population, with all members of each selected cluster invited to participate and a sampling frame is required that includes all clusters in the population. the sample is then selected at random from the framework that has been made. each cluster has the same international journal of applied biology, 6(1), 2022 158 probability of being selected, regardless of the others. the number of respondents for polyculture businesses that use private and rented land are 34 people so that the total number of respondents in this study is 68 people. respondents are farmers who are actively engaged in the polyculture aquaculture industry, which is one of the factors taken into account. types of data and source of data the data obtained were primary data (respondents) and secondary data (relevant agencies and institutions). secondary data were obtained from the marine affairs and fisheries department (dkp) of pinrang regency and related literature data. primary data were obtained through direct interviews with cultivators who became respondents, namely by using a list of questions (questionnaires). primary data collected from respondents were production results, labor, investment costs, operational costs, and production value. secondary data collected included the production and productivity of shrimp and fish in pinrang regency. data analysis the data analysis used in this research was quantitative descriptive analysis and financial feasibility analysis. descriptive analysis is an analysis used to describe the results of observations in accordance with the reality in the field regarding something being studied. tables and graphs will be used to present all of the analysis's data. financial feasibility analysis includes: business analysis according to febrianto (2008), the components used in conducting business analysis include production costs, business revenues and income derived from fishery businesses. the business analysis consists of revenue and cost balance analysis, payback period (pp) analysis and return of investment (roi) analysis. after analyzing several parameters, the test was continued with a t-test to see the differences that exist in polyculture businesses that use private land and polyculture businesses that use leased land. income analysis income analysis aims to determine the amount of profit obtained from a business activity carried out. business income in the development of aquaculture can be calculated using the following equation (siang and aziz, 2010): 𝜫 = 𝑻𝑹 − 𝑻𝑪 ............................................. (1) note: π = profit tr = total revenue tc = total cost with business criteria: tr > tc : profitable business tr < tc : business suffers a loss tr = tc : work at the break-even point international journal of applied biology, 6(1), 2022 159 analysis of the balance of revenue-cost ratio this analysis aims to determine how far each rupiah value of costs used in business activities can provide a number of revenue values as benefits. the formula used to calculate r/c is the equation (darsono, 2008): 𝑹 𝑪⁄ 𝑹𝒂𝒕𝒊𝒐 = 𝑻𝑹 𝑻𝑪 ............................................. (2) with criteria: r/c > 1, profitable business r/c < 1, business suffers a loss r/c = 1, at the break-even point payback period (pp) analysis according to umar (2003), payback period (pp) is a period required to recoup investment spending using cash flows. payback period (pp) as the ratio between investment expenditures and profits which result in units of time. pp calculation is done by the formula: 𝑷𝑷 = 𝑰𝒏𝒗𝒆𝒔𝒕𝒎𝒆𝒏𝒕 𝒗𝒂𝒍𝒖𝒆 𝑷𝒓𝒐𝒇𝒊𝒕 × 𝟏 𝒚𝒆𝒂𝒓 .................. (3) analysis of return of investment (roi) return on investment (roi) is the ability of the capital invested in all activities to generate a net profit. the formula used to calculate roi is the equation (sutrisno, 2001): 𝑹𝑶𝑰 = 𝑷𝒓𝒐𝒇𝒊𝒕 𝑰𝒏𝒗𝒆𝒔𝒕𝒂𝒕𝒊𝒐𝒏 × 𝟏𝟎𝟎% .......................... (4) analysis of investment in addition to business analysis, the feasibility of a business can be seen using an investment feasibility analysis. the measuring instruments used include net present value (npv), internal rate of return (irr), and net benefit cost ratio (net b/c). this activity is carried out to assess the extent of the benefits that can be obtained in carrying out a business activity. with this feasibility analysis, it is hoped that the risk of failure in polyculture shrimp and fish cultivation at the research site can be avoided. according to thomas (2017), the best practice for investment analysis is to use standard cost categories. standard categories allow manufacturers to more easily identify common costs across their operations. net present value (npv) the net present value is used to assess the benefits of the investment, namely what is the present value of the net benefits of the project expressed in rupiah. the project is declared feasible to continue if the npv>0, while if the npv<0, then the investment is declared unprofitable which means the project is not feasible to be implemented. if the npv value = 0 means that the project only returns capital or no profit and no loss, kadariah et al. (1999). the formula used to calculate npv is the equation according to gittinger (1982): international journal of applied biology, 6(1), 2022 160 𝑵𝑷𝑽 = ∑ 𝑩𝒕−𝑪𝒕 (𝟏+𝒓)𝒕 𝒏 𝒕=𝟏 ..................................... (5) note: bt : benefit of year t ct : cost of year t r : discount rate t : year n : project life indicators: 1. npv > 0 (positive), the business is feasible to carry out, 2. npv < 0 (negative), the business is not feasible to carry out. net benefit-cost ratio (net b/c) according to kadariah et al. (1999), net benefit-cost ratio (net b/c) is the ratio between the present value of net profit in years where net profit is positive and net profit is negative. the formula used is: 𝑵𝒆𝒕 𝑩/𝑪 = ∑ 𝑩𝒕−𝑪𝒕 (𝟏+𝒊)𝒕 (𝑩𝒕−𝑪𝒕)>𝟎 𝒏 𝒕=𝟏 ∑ 𝑩𝒕−𝑪𝒕 (𝟏+𝒊)𝒕 𝒏 𝒕=𝟏 (𝑩𝒕−𝑪𝒕)<𝟎 ..................... (6) with feasiblity criteria: b/c > 1, means the business is worth running b/c < 1, means the business is not worth running b/c = 1, means the implementation decision depends on the investor internal rate of return (irr) the internal rate of return is the value of the interest rate i that makes the npv of the project equal to zero. irr can be defined as the interest rate at which the present value of total costs is equal to the present value of total revenues. irr is also considered as the rate of net return on investment, where a positive net benefit is reinvested in the following year and earns the same rate of return and is given interest for the remaining life of the project. irr can be formulated as follows: 𝑰𝑹𝑹 = 𝒊𝟏 + 𝑵𝑷𝑽𝟏 𝑵𝑷𝑽𝟏−𝑵𝑷𝑽𝟐 (𝒊𝟐 − 𝒊𝟏) ................. (7) note: npv1 = npv that is still positive npv2 = negative npv i1 = discount rate that still gives a positive npv i2 = discount rate that gives a negative npv indicators: 1. irr > the prevailing interest rate, then the business is declared feasible 2. irr < the prevailing interest rate, then the business is declared unfeasible international journal of applied biology, 6(1), 2022 161 results and discussion polyculture cultivation business potential the adoption of polyculture cultivation business is one sort of business in the fisheries industry that aims to improve production outcomes, revenue, and profits. the basic objective of business players is to make money, either directly or indirectly, so that their enterprise can develop and grow. this is in line with the economics of running a business principle, which is to maximize profit at the lowest possible cost. the polyculture activity of tiger prawns with finfish in pinrang regency is carried out throughout the year with one to two growing seasons. in the third month (12th week) after the first stocking, the farmers do partial harvest of tiger prawns one to two times per month. and so on until the sixth month (end of the first cycle). milkfish harvest is carried out at week 14 and at week 16 farmers start harvesting barramundi, with a success rate of 60% to 75%. cultivation land ownership status the area of land owned by milkfish and tiger shrimp cultivators is on average between 1-5 hectares. cultivators who manage their own land are cultivators who have been running their business for generations. meanwhile, cultivators who run polyculture businesses with leased land status, rent land from the local government or from land owners at a rental price of 6-7.5 million/hectare for 1 year. vanderpuye et al. (2020) states that there are several forms of ownership status, namely: 1. joint assets where there are joint assets that are owned, managed and controlled by joint owners, such as groups or associations of individuals. due to this form of joint ownership of property, individuals can resolve and manage conflicts through mutual benefit and enforcement of responsibilities. 2. public property can also be referred to as state property which is generally owned, managed, and controlled by the government of that country. 3. individual or private property describes the rights of access, use and management of individual property. investment cost the investment cost incurred by finfish-shrimp farmers on an area of ±1 ha per year is rp19,357,330,for business on private land and rp18,729,526,for land lease system business. the amount of investment cost used in the two criteria does not show a significant difference. in detail, the differences in the structure of investment costs can be seen in table 1. champo & zuniga-jara (2017) stated that the capital costs in aquaculture generally vary in the range of 10%-15% depending on the project risk (higher in offshore projects). table 1. investment component asset's useful lifw (year) total cost (rp) shrinkage /year (rp) asset's useful lifw (year) total cost (rp) shrinkage /year (rp) 1 guard house/warehouse unit 1 6,2 5.195.238 843.557 4,3 1.814.079 418.750 2 operational motorcycle unit 1 8,0 7.119.048 891.650 10,5 8.041.516 766.781 3 water pump machine unit 1 2,7 2.678.333 992.559 3,6 3.038.087 841.550 4 sluice set 1 4,7 1.025.397 218.983 6,2 1.148.014 185.965 5 net set 1 1,6 441.270 272.549 2,2 533.394 246.250 6 scale set 1 2,5 657.937 264.013 3,3 884.477 270.718 7 fishing nets unit 1 1,9 279.619 150.564 2,4 331.119 135.881 8 fish trap unit 1 1,5 297.540 203.750 1,9 351.444 180.278 9 fish chart unit 1 1,4 365.508 267.756 1,8 362.274 198.713 10 initial working capital ls 1 1.297.441 2.054.122 19.357.330 4.105.380 18.558.526 3.244.887 no description unit total private land total investasi leased land international journal of applied biology, 6(1), 2022 162 fixed costs and variable costs fixed cost components for polyculture cultivation consist of maintenance costs, land rental costs, taxes and levies and annual depreciation of investment costs. the use of fixed costs for polyculture cultivation on leased land is greater than for polyculture cultivation on private land, this is because cultivators have to pay land rent at the beginning of the season before carrying out production activities. details of the fixed costs required for one year are shown in table 2. the use of costs in this business is not only fixed costs but also variable costs. variable costs are incurred when farmers carry out shrimp and fish production activities. the difference in the amount of cost used between businesses on privately owned land and leased land can be seen from the number of stockings and types of biota that are stocked. land owners generally carry out production activities by prioritizing shrimp over fish. meanwhile, land cultivators optimize land by prioritizing fish production. this is done because land cultivators have to create a relatively faster cash flow. the difference in total expenditure used is ±3-4 million per year for a 1 ha pond. the details of the variable cost components for a period of one year are shown in table 2. table 2. components of fixed costs and variable cost table 2 shows that the total value of fixed costs in both land ownership statuses is lower than the total value of variable costs in both land ownership statuses. according to research by miqdad et al. (2020) on fish farming land in muthanna, the total costs incurred for variable costs show a larger percentage compared to fixed costs of 60,78%, while the percentage of fixed costs is only amounting to 39,22% which indicates that variable costs are the largest part of the total costs needed in cultivation, so efforts need to be made to minimize the costs that come through the low cost of one or all variable cost items. quantity annual cos t (rp) quantity annual cos t (rp) 1 ls 1 4.105.380 1 3.244.887 2 ls 0,05 902.994 0,05 825.220 3 ha 1 95.882 1 95.000 4 ha 1 1 6.690.000 5.104.257 10.855.107 1 ha 1 161.765 1 126.250 2 ls 1 3.426.471 1 3.225.000 3 ha 1 1.058.699 1 879.853 4 kg/ha 424 850.642 295 574.675 5 kg/ha 453 731.415 400 678.500 6 ha 1 3.599.438 1 2.443.122 7 liter/ha 17 161.207 15 194.625 8 liter/ha 380 2.660.000 380 2.660.000 9 ls 1 1.000.000 1 1.000.000 13.649.636 11.782.025 18.753.893 22.637.132 fuel cost packing & freight total variable cos t total cos t (expe ns e ) harvest work wages lime procurement cost fertilizer procurement cost seed procurement cost probiotic procurement cost total fixe d cos t b. variable cos t preparatory work wages maintenance work wages a. fixe d cos t investment depreciation cost maintenence cost taxes & levies land lease no de s cription unit private land le as e d land international journal of applied biology, 6(1), 2022 163 table 3. average of production results and annual revenues business analysis the results of the income analysis for polyculture pond cultivation with an average area of ±1 ha per year indicate that this activity provides benefits. this can be seen from the total revenue obtained is greater than the costs incurred. the total revenue of polyculture pond cultivation per year is rp. 30.459.853,for privately owned land and rp. 29.120.882, for leased land. details of the comparison of production results and total revenues can be seen in table 3. according to erwiantono et al. (2020), the practice of polyculture has the potential to provide a lucrative business, leading to increased income and poverty alleviation for small-scale farmers. table 4. analysis of financial feasibility of tiger prawns-finfish polyculture culture in pinrang regency, south sulawesi note : s = significantly different between private land and leased land ns = not significantly different between private land and leased land the level of income calculated through the t-test shows a significant difference between the income level of polyculture cultivation using privately owned land and leased land. the income of polyculture cultivation using privately owned land is greater than the income of farmers using leased land. the amount of income obtained on average is rp. 7.043.771,-/year for private land and rp. 4.007.466,-/year for leased land. the amount of profit earned by cultivators is determined by the amount of shrimp and fish production and the selling price in the market. this amount of production is influenced by the survival rate (sr) of the cultured cultivar which is obtained from the calculation of the number of fish harvested compared to those stocked. the r/c ratio of polyculture businesses that use private land is greater than the r/c ratio of leased land. the average value obtained is 1,44 and 1,18, respectively. that is, every rp. 1,the total costs incurred will result in a total revenue of rp. 1,44,polyculture cultivation price/kg (rp) sr (% ) harvest (kg) total (rp) sr (% ) harvest (kg) total (rp) 1 tiger prawn 75.000 17 182 13.617.353 17 145,2 10.887.353 2 milkfish 18.000 80 448 8.055.000 80 818,5 14.733.529 3 white snapper 50.000 21 99 4.937.500 4 tilapia 28.000 55 138 3.850.000 50 125,0 3.500.000 30.459.853 29.120.882 total sales no commodities private land leased land private land leased land a business analysis income rp 7043771 ± 604927 4007466 ± 165692 0,000 (s) r/c ratio 1,4426 ± 0,0395 1,1856 ± 0,0088 0,000 (s) pp tahun 4,13 ± 0,49 4,85 ± 0,34 0,235 (ns) roi 0,4097 ± 0,0490 0,2488 ± 0,0196 0,004 (s) b investment analysis npv rp 38.375.216 20.778.485 irr % 77,32 50,36 net b/c ratio 2,98 2,12 financial feasibility analysis unit polyculture farming ttest year international journal of applied biology, 6(1), 2022 164 on private land and rp. 1,18,polyculture cultivation business with a rental land system. both systems of polyculture cultivation are feasible because the resulting value is greater than 1 (one). in a study conducted by khairini et al. (2021), the r/c value of the polyculture system between fish, shrimp, and crab is 1.73, which means that each additional input of 1 will produce an output of 1,73. the test results for the payback period do not show a significant difference. the average time needed to return the investment costs for polyculture cultivation businesses using both privately owned land and leased land is ± 4 years. however, this value is included in the good category because the payback period is less than 5 years. this is in line with the opinion of umar (2007), the rate of return on capital is categorized as fast if the pp value is <5 years, while it is slow if the pp value is >5 years. the faster the return on investment costs of a project, the better the project because the smoother the turnover of capital (ibrahim, 2003). the percentage of the ability of the invested capital to generate a net profit (return of investment) based on the test results shows a significant difference. the average roi value for businesses that use private land is greater than polyculture businesses with leased land. the average level of capital ability to generate net profit on each criterion is 40,97% for private land and 24,88% for leased land. this value is the optimal value and shows that this effort is feasible. based on the results of research israel et al. (1985), in polyculture systems between shrimp (penaeus monodon) and milkfish (chanos chanos), the roi values ranged from 8 to 85% and the payback period from 1,1 to 10,5 years. based on the results of the business analysis presented in table 4, it shows that the polyculture shrimp farming business with finfish, both using privately owned land and leased land in lanrisang and mattirosome subdistricts are included in the criteria for development. analysis of investment based on the results of the feasibility analysis of the npv value of polyculture cultivation at the research site, it shows that these business activities will provide a value benefit of rp. 38.375.216,for polyculture cultivation on private land and rp. 20.778.485,for the cultivation business of the rental land system, for 5 (five) years of investment period with a discount rate of 12% per year. based on these results, the npv value is positive because it is more than zero, this indicates that the cultivation business is feasible to be continued or developed. the following graph will show the difference in nvp and irr values between polyculture cultivation on private land and polyculture cultivation on leased land systems. figure 1. private land figure 2. leased land international journal of applied biology, 6(1), 2022 165 in the irr feasibility test, the value of polyculture cultivation at the study site is greater than the discount rate target of 12%. the results obtained were 77,32% for polyculture cultivation on private land and 50,36% for cultivation with leased land systems. these two results indicate that the cultivation business provides good benefits and returns from the value of the investment invested. this is also supported by the net b/c value of each criterion, namely 2,98 for polyculture cultivation on private land and 2,12 for cultivation of leased land systems. this means that the investment business is feasible because the net b/c value is greater than 1 (one). overall, the results of the business analysis and the results of the investment analysis show that polyculture cultivation using privately owned and leased land is still feasible in pinrang regency. if the actual conditions in the field are in accordance with the assumptions, then this is included in the good category for the parameters of the financial feasibility analysis. conclusions the tiger shrimp polyculture with finfish systems business operations on owned land and leased land in pinrang regency demonstrate notable variances in terms of income, r/c ratio, and roi. although there is no difference between private land systems and leased land, the payback period (pp) is still within the ideal range. businesses engaged in polyculture agriculture that employ private land benefit more than those that use leased land based on the metrics of nvp, irr, and net b/c ratio. however, the two polyculture cultivation systems used in pinrang regency offer advantages to business actors and unquestionably show that the enterprise is highly likely to be continued or expanded. international journal of applied biology, 6(1), 2022 166 references anil s. kotiya, b. gunalan, k.l. jetani, g. kuldeep trivedi and p. soundarapandian1. 2010. “determine the economic feasibility of the polyculture system (giant tiger shrimp and mullet).” african journal of basic & applied sciences 124-127. champo, s.r., and zuniga-jara, s. 2017. reviewing capital cost estimations in aquaculture. aquaculture economics & management. 22(1): 72-93. https://doi.org/10.1080/13657305.2017.1300839 darsono. 2008. hubungan perceived service quality dan loyalti (peran trust dan satisfaction sebagai mediator. the national conference ukwms. surabaya erwiantono, o. darmansyah, q. saleha, zulkarnain, e. sulistianto, w. fahrizal, f. maryanto, h. susilo. 2020. impact of shrimp-fish polyculture practices on small-scale farmers’ income in indonesia. aacl bioflux. 13(6). 3407-3419. fao. 2020. the state of world fisheries and aquaculture. sustainability in action. rome febrianto a. 2008. pengembangan usaha perikanan tangkap di kabupaten bangka provinsi kepulauan bangka belitung: suatu pendekatan sistem bisnis perikanan [tesis]. bogor: institut pertanian bogor. 112 hal gittinger, j.p. 1982. economic analysis of agricultur project, baltimore, john hopkins university press. ibrahim, m. y. 2003. studi kelayakan bisnis. rineka cipta. jakarta. irmawati, alimuddin, asmi citra malina a.r tassakka , 2019. budidaya ikan kakap putih ( lates calcarifer bloch, 1790) berbasis ekosistem. islamiyah, n. 2020. analisis pendapatan usaha budidaya polikultur ikan bandeng (chanos chanos), udang windu (penaeus monodon), dan rumput laut (gracilaria sp.) pangkajene dan kepulauan district. skripsi. hasanuddin university israel, d., apud, f., & franco, n. 1985. the economics of different prawn and shrimp pond culture systems: a comparative analysis (abstract only). in taki y., primavera j. h. and llobrera j. a. (eds.), proceedings of the first international conference on the culture of penaeid prawns/shrimps, 4-7 december 1984, iloilo city, philippines (pp. 172-173). iloilo city, philippines: aquaculture department, southeast asian fisheries development center. kadariah, lien k, clive g. 1999. pengantar evaluasi proyek. jakarta: faculty of economic indonesia university. khairini, r., t. supriana, t. sebayang. 2021. analysis of the feasibility of a polyculture pond business (tilapia – tiger shrimp – crab) (case: belawan sicanang village, medan belawan, medan city). iop conf. series: earth and environmental science. 782. 022014. doi:10.1088/1755-1315/782/2/022014 miqdad, j.a., wadhah, r.r., oleiwi abdulridha, m. 2020. costs estimation and economies of scale for fish farming projects in muthanna governorate for 2018. plant archives. 20(1): 926-928. sedgwick p. 2014. cluster sampling. bmj; 348 :g1215 doi:10.1136/bmj.g1215 https://doi.org/10.1080/13657305.2017.1300839 international journal of applied biology, 6(1), 2022 167 siang, r.d. dan n. aziz. 2010. struktur biaya dan profabilitas usaha miniplant rajungan (portunus pelagicus). jurnal bisnis perikanan. fpik uho. 2(1):15-30 singarimbun, m dan effendi, s. 2006. metode penelitian survei lp3es. jakarta. sutrisno. 2001. manajemen keuangan. ekonesia: yogyakarta. thomas, d.s. 2017. investment analysis methods: a practitioner’s guide to understanding the basic principles for investment decisions in manufacturing. national institute of standards and technology. https://doi.org/10.6028/nist.ams.200-5 thomas, m., pasquet, a., aubin, j., nahon, s., & lecocq, t. 2020. when more is more: taking advantage of species diversity to move towards sustainable aquaculture. biological reviews, 96(2), 767–784. doi:10.1111/brv.12677 umar h. 2003. studi kelayakan bisnis. edisi kedua jakarta: pt gramedia pustaka utama. umar h. 2007. studi kelayakan bisnis. edisi 3. pt gramedia pustaka utama. vanderpuye, i.n., darkwah, s.a., živělová, i. 2020. the system of land ownership and its effect on agricultural production: the case of ghana. journal of agricultural science. 12(5): 57. doi: 10.5539/jas.v12n5p57 https://doi.org/10.6028/nist.ams.200-5 1(2)37-45 !!!!!!!!!!!!!!!!!!contact&:&inanpi&hidayati&sumiasih &!!!!!!!inanpihs@trilogi.ac.id! ©!international!journal!of!applied!biology! 37! abstract& citrus!peel!color!is!one!of!the!main!quality!attributes!which!was!caused!by! the!accumulation!of!carotenoids!and!its!derivatives,!especially!β"citraurine.! it!makes!citrus!peel!color!looks!attractive!(orange).!the!orange!color!is!a! mixture! of! β"cryptoxanthin! with! β"citraurin.! the! objectives! of! this! study! were!(1)!to!observe!the!effect!of!precooling!and!duration!of!proper!ethylene! exposure!in!the!formation!of!orange!color!on!citrus!peel,!(2)!to!identify!and! determine!the!β"cryptoxanthin0content!and!total!chlorophyll!on!citrus!peel.! citrus!was!from!tuban,!east!java!while!the!study!was!conducted!at!pkht!ipb! and!lipi.!precooling!and!without!precooling!treatment!prior!to!injection!of! 100!ppm!of!ethylene!exposed!at!15!°c,!duration!of!exposure!control!(0),!24,! and!48!hours.!the!results!show!that!the!best!color!of!the!citrus!color!index! (cci)!is!the!precooling!treatment!and!the!duration!of!ethylene!exposure!for! 24!hours,!which! can!reduce! total!chlorophyll! content!about!8! times!and! proved! to! increase! β"cryptoxanthin! pigment! content! five! times! in! accelerating!the!formation!of!orange!citrus0reticulata0peel!color!to!bright! orange.!degreening!has!no!significant!effect!on!total!dissolved!solids!and!the! firmness!level!of!citrus!fruits.! & issn!:!2580x2410! eissn!:!2580x2119! ! ! ! the&analysis&of&β"cryptoxanthin&and&zeaxanthin0using&hplc&in& the&accumulation&of&orange&color&on&lowland&citrus&& ! inanpi!hidayati!sumiasih1,2,!roedhy!poerwanto1,!darda!efendi1,3,!andria!agusta4!and! sri!yuliani5! ! 1!agronomy!and!horticulture!department,!faculty!of!agriculture,!bogor!agricultural! university,!bogor,!indonesia! 2!agroecotechnology!program,!faculty!of!bioindustry,!trilogi!university,!jakarta,!indonesia! 3!center!for!tropical!horticulture!studies!(pkht)!baranangsiang!campus!of!ipb,!bogor,! indonesia! 4!center!for!biology,!indonesia!institute!of!sciences!(lipi),!cibinong,!indonesia! 5!center!for!agricultural!postharvest!research!and!development,!bogor,!indonesia! ! ! & & & & & & & & & & & & & & & introduction& peel!color!is!one!of!external!quality!attributes!of!citrus!and!plays!important!factor!in! costumer!decision!in!purchasing.!greenxcolored!citrus!is!usually!considered!sourxtasted!and! unripe,!while!orangexcolored!citrus!is!considered!otherwise.!generally,!consumers!choose! orangexcolored!citrus.!therefore,!it!is!essential!to!ensure!that!the!citrus!fulfills!the!criteria! when!it!is!ready!to!enter!the!market.! &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword! β"cryptoxhantin0 citrus0 chlorophyll0 degreening0 ethylene0zeaxanthin! article&history! received!23!november!2017! accepted!21!december!2017!! international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international0journal0of0applied0biology,01(2),020170 ! 38! in!order!to!fulfill!the!consumers’!demand!regarding!the!taste,!the!accumulation!of! orangexcolored!on!citrus!peel!through!postxharvest!treatment!should!be!done.!the!treatment! is!degreening!with!ethylene.!degreening!so!far!is!still!not!succeed!to!form!orangexcolored! citrus.!in!fact,!it!made!the!citrus!to!form!yellowxcolored!peel.!this!result!is!also!unfavorable! because!consumers!consider!yellowxcolored!citrus!is!in!the!verge!of!senescence.!! degreening!in!tropical!region!is!still!not!success!because!βxcitraurin0pigment!as!the!red! color!formation!is!not!formed.!only!yellow!color!pigment!β"cryptoxanthin0is!formed.!(kato!et0 al.,02006;!fanciullino!et0al.,!2008).!yellow!on!citrus!peel!is!caused!by!carotenoids!derivative! cryptoxanthin0 (zhou! et0 al.,! 2010).! orange! color! can! be! formed! due! to! the! mixture! of! β" cryptoxanthin!and!β"citraurine!(stewart!&!wheaton,!1971;!rodrigo!et0al.,02013).!!while!β" cryptoxanthin!and!zeaxanthin0is!the!pigment!contributed!in!the!formation!of!β"citraurin!(ma! et0al.,02013).! the!failure!of!degreening!in!indonesia!is!because!the!citrus!is!not!exposed!to!low! temperature!during!its!growth.!thus,!β"citraurin0pigment!can!not!be!formed.!it!is!only!formed! at!18x24!oc.!to!substitute!the!existence!of!low!temperature,!the!precooling!treatment!was! introduced.!precooling!after!harvest!is!expected!to!substitute!low!temperature!treatment! which!could!not!be!fulfilled!when!the!fruit!is!in!the!field.!ladaniya!(2008)!reported!that!low! temperature!affects!greatly!on!the!color!changes!on!peel.!! according!to!matsumoto!et0al.,!(2009)!research!about!satsuma!fruit!storage!at!30!ºc! was!proven!to!increase!zeaxanthin!and!β"carotene0but!no!β"citraurin0pigment!was!found.!the! main!reason!of!failure!was!that!high!temperature!prevents!the!accumulation!of!β"citraurin! and!reduced!orange!color!(stewart!&!wheaton,!1971).!high!temperature!on!tropical!regions! limits!the!accumulation!of!important!pigments!on!citrus!and!prevents!its!peel!to!achieve!more! favorable!color.!! therefore,! degreening! technology! which! could! induce! citrus! peel! orange,! reduce! chlorophyll! content,! and! identify! β"cryptoxanthin0 as! orange! formation! is! needed.! the! research!aims!to!observe!the!effect!of!precooling!and!obtain!the!proper!ethylene!exposure! duration!and!to!identify!and!determine!β"cryptoxanthin0and!chlorophyll0content!on!citrus! peel!before!and!after!degreening.! & materials&and&methods& the!research!was!done!at!pkht!ipb!and!the!analysis!of!chemical!content!of!peel!was! done!at!lipi!cibinong.!it!was!conducted!from!january!2016!until!july!2017.!materials!used!in! the!research!were!citrus!reticulate!“tejakula”,!ethylene!gas,!and!liquid!nitrogen.!tools!used! were!degreening!box,!cool!storage,!syringe,!refractometer,!color!reader,!spectrophotometer,! and! chromatography,! hplc! (high! performance! liquid! chromatography).! a! 2! factors! randomized! group! design! was! used! as! research! design.! treatments! were! harvest! and! exposure! duration.! harvest! factor! consists! of! 2! levels! that! is! without! precooling! and! precooling.! exposure! duration! factor! consists! of! 3! levels,! without! ethylene! exposure,! exposure!for!24!hours,!and!sxposure!for!48!hours.!there!were!6!combinations!repeated!3! times!and!18!units!of!experiment.& citrus0reticulata!“tejakula”!came!from!low!land!estate!in!singgahan,!tuban,!east!java! (40!m!asl).!after!the!citrus!was!harvested!and!treated,!it!was!brought!to!pkht!lab,!bogor.!it! was!sorted!and!put!into!degreening!box!and!sealed!tightly!(air!tight).!ethylene!gas!100!ppm! was!injected!to!the!degreening!box!using!5!ml!syringe,!then!the!citrus!was!put!in!cool!storage! at!15!°c.!the!fan!was!activated!during!degreening,!which!is!control,!24!hours,!and!48!hours.!! international0journal0of0applied0biology,01(2),020170 ! 39! ! hue&angle&and&citrus&color&indeks0(cci)& changes!in!the!color!quality!of!citrus!peel!was!measured!with!minolta!color!reader! type!310.!this!tool!color!notation!system!is!hunter!notation!system!(color!system!l,!a,!and!b).! the! measurement! results! were! expressed! in! the! citrus! color! index! (cci).! hunter! color! notation!system!is!characterized!by!three!parameters!of!color,!the!brightness!symbolized!by! l,!the!chromatic!colors!symbolized!by!‘a’!notation!and!color!intensity!with!the!notation!‘b’.! notation!‘l’!states!the!parameter!of!brightness!(lightness),!with!value!l:!value!0!means!black! and!100!means!white.!while! ‘a’!notation!stating!chromatic!colors!redxgreen!mix!with!+a! (positive)!from!0!to!+100!indicating!red!color!and!value!(negative)!from!0!to!x80!indicating! green!color,!while!the!notation!‘b’!states!chromatic!color,!the!mixture!of!bluexyellow!value!+b! (positive)!from!0!to!+70!indicating!yellow!color!and!the!xb!(negative)!value!from!0!to!x70! indicating!blue!color!(andarwulan!et0al.,02011).!! the!value!of!citrus!color!index!(cci)!is!a!formula!widely!used!to!see!the!quality!of!citrus! peel!color.!according!to!jimenezxcueata!et0al.,! (1981),!value!citrus!color! index!(cci)! is!a! formula!that!is!widely!used!to!see!the!quality!of!orange!peel!color.!based!on!jimenezxcueata! et0al.,!(1981)!that!the!value!of!cci!is!used!as!followshe!following!equation:!! ! !!"# =#1000.().* 0 0 citrus!color!index!(cci)!using!following!range:!cci≤!x5!(dark!green),!x5!<!cci!≤!0!(green),! 0!<!cci!≤!3!(yellowish!green),!3!<!cci!≤!6!(greenish!yellow),!6!<!cci!≤!8!(yellowish!orange),!8! <cci!≤10!(orange),!dan!cci!>!10!(dark!orange).! & total&solluble&solid& the! fruit! flesh! of! several! samples! from! each! treatment! were! taken! and! the! total! soluble!solids!was!measured!using!a!hand!refractometer.!a!drop!of!juice!was!placed!on!hand! refractometer!prism.!! ! fruit&firmness& fruit! firmness! is! determined! by! fruit! resistance! to! puncturing! the! peel! done! by! penetrometer.! ! total&chlorophyll&& the! total! content! of! chlorophyll! was! measured! using! spectrophotometry! method.! citrus!peel!was!weighed!at!0.5!grams!and!then!crushed!(slurry)!and!extracted!with!2!ml! acetris.! the! extract! then! inserted! into! microtube! and! centrifuged! for! 10! seconds.! the! centrifuged!filtrate!was!inserted!in!a!1xml!reaction!tube,!and!then!3!ml!acetris!was!added! and!placed!in!cuvet!to!be!measured!using!a!spectrophotometer!at!wavelengths!537,!647!and! 663!nm.!according!to!sims!and!gamon!(2002),!after!obtaining!an!absorbance!value,!the!total! content!of!chlorophyll!can!be!calculated!by!the!following!equation:! ! !ℎ,#=#0.01373∗1223# −#0.000897∗1738# −#0.003046∗12;8! !ℎ,< #=#0.02405∗12;8# −#0.004305∗1738#−#0.005507∗12230 ?@a(,#b,@c@de,# =#!ℎ,+ !ℎ,<0 international0journal0of0applied0biology,01(2),020170 ! 40! 0 identification&of&β"cryptoxanthin&and&zeaxanthin0pigments& material&preparation& citrus!peels!are!separated!from!the!edible!portion,!then!immediately!cooled!with! nitrogen!liquid!in!the!dewar!tube!until!next!step.! ! carotenoid&extraction& the!identification!of!carotenoids!was!performed!by!the!method!described!by!kato!et0 al!(2004).!the!pigment!was!extracted!from!the!sample!with!hexane!solution:!aceton:!ethanol! (2:! 1:! 1,! v! /! v)! containing! 0.1%! (w! /! v)! 2.6! ditert! butyl! 4! methylphenol! and! 10%! (w! /! v)! magnesium! carbonate.! after! the! organic! solvent! evaporates! entirely,! the! carotenoidx containing! extract,! which! is! esterified! into! fatty! acid,! is! disacorated! with! 20%! (w! /! v)! methanolic!koh.!waterxsoluble!extracts!are!removed!by!saturated!nacl.!the!pigments!were! partitioned!in!the!dhietyl!ether!phase!and!evaporated.! ! analysis&of&β"cryptoxanthin,0zeaxanthin&and&β"carotene& the!residue!is!redissolved!in!methyl!tert!butyl!ether!solution:!methanol!(4:!6,!v!/!v).!an! aliquot!(30!μl)!is!separated!by!an!hplc!reversed!phase!(shimadzu,!ascentis)!equipped!with! type!cx18,!at!a!1!ml!minx1!flow!rate.!the!standards!of!β"cryptoxanthin,!zeaxanthin!and!β" carotene!were!prepared!based!on!the!method!described!with!fresh!weight!micrograms!per! gram! (preparing! for! calibration! of! the! standard).! quantification! of! β"cryptoxanthin,! zeaxanthin0and!β"carotene!was!performed!in!3!replications.!the!data!were!obtained!from! average!hplc!values!and!calculated!using!the!calibration!equation.!with!standard!pigments! (figure!1),!and!the!calibration!formula!are!as!follows!(table!1):! ! & & & & & & & & figure& 1.& hplc& chromatogram& for& mixture& of& authentic& sample& of& zeaxhantin0 β" cryptoxanthin,&and&β"carotene.& table&1.&the&linier&equation&for&β"carotene,0βqcryptoxanthin,0and0zeaxanthin0pigments& standard& 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0 5 10 15 20 25 30 mv detector+a:450nm+ 6 .8 3 3 /5 9 0 8 0 5 1 0 .7 7 8 /8 9 8 5 7 1 9 .1 5 7 /9 7 0 6 1 zeaxanthin! βxcryptoxhantin! βxcarotene! international0journal0of0applied0biology,01(2),020170 ! 41! ! data&analysis& ! the!results!from!observation!were!tested!by!using!sas!software!(statistical!analysis! system)!the!data!were!analyzed!using!analysis!of!variance!at!5%!significance!level.!when!the! result! showed! significant! effect! of! the! treatment,! it! will! be! tested! further! using! duncan! multiple!range!test!(dmrt)!at!5%!level.! && results&and&discussion&& the!effect!of!precooling!and!exposure!duration!to!citrus0reticulata!of!tuban!origin! showed!the!best!color!change!was!on!the!duration!of!24!hours!exposure!with!precooling!and! degreening! treatment.! citrus! peel! color! with! precooling! treatment! on! 24xhour! ethylene! exposure! resulted! in! orange! peel! color,! whereas! without! precooling! showed! a! yellowish! orange!color!(figure!2,!3).! ! & & & & ! ! ! ! figure&2.&the&change&of&citrus0reticulata&‘tejakula’&peel&color&without&precooling&treatment& on&several&ethylene&exposure&durations&(9&days&after&treatment)& & & & & & & & & figure&3.&the&change&of&citrus0reticulata&‘tejakula’&peel&color&with&precooling&treatment&on& several&ethylene&exposure&durations&(9&days&after&treatment)& & total&soluble&solids&and&and&citrus&firmness& the!measurement!for!measuring!citrus!sweetness!level!is!expressed!in!the!content!of! total!soluble!solids.!based!on!the!precooling!treatment!and!ethylene!exposure!duration!at!the! final!observation,!it!was!shown!that!the!treatments!gave!no!significant!effects!to!total!soluble! solids!and!citrus!firmness.!! & table&2.&the&change&of&citrus0reticulata&“tejakula’&peel&color&with&degreening&treatment&on& several&ethylene&exposure&durations&(0,&12&days&after&treatment)& treatment! total!soluble!solids!(o!brix)! citrus!firmness!(kgf)! !! zeaxantin& criptoxantin& caroten& linier!equation! y!=!86079x!+!44977! y!=!68174x!+!111876! y!=!3915.3x!+!249036! r2! r²!=!0.9938! r²!=!0.9673! r²!=!0.9838! without! degreening! degreening, 24!hours! degreening,! 48!hours! precooling! without! precooling!! without! degreening! ! degreening,! 24!hours! degreening,! 48!hours! international0journal0of0applied0biology,01(2),020170 ! 42! 0! 12! 0! 12! day!after!treatment! precooling! ! ! ! ! x!non!precooling! 10.76! 10.76! 0.58! 0.62! x!precooling! 10.77! 10.72! 0.56! 0.62! exposure!durations! ! ! ! ! x!0!hour! 10.77! 10.73! 0.57! 0.63! x!24!hours! 10.75! 10.76! 0.57! 0.63! x!48!hours! 10.79! 10.74! 0.57! 0.60! 0 citrus&color&index&value0(cci)&& the!use!of!the!citrus!color!index!(cci)!value!described!colors!such!as!red,!orange!or! green.!the!increase!of!cci!value!indicates!that!there!is!a!change!of!color!from!green!to!orange.! precooling!with!degreening!had!an!effect!on!the!cci!value!on!citrus0reticulata!to!form!orange! color!faster!than!treatment!without!precooling.!the!result!of!cci!measurements!of!citrus0 reticulata!with!precooling!treatment!with!degreening!on!24xhour!exposure!duration,!it!could! form!orange!color!on!dayx9!after!degreening!with!value!7.94.!while!the!cci!value!of!citrus0 reticulata!given!treatment!without!precooling!on!24xhour!exposure!duration!could!form!an! orange!color!on!dayx12!of!treatment!with!value!7.99!(data!not!shown).& & total&chlorophyll& the!color!variation!of!citrus!fruits!is!caused!by!the!main!groups!of!chlorophyll!pigments! and! carotenoids.! the! dominant! pigment! on! unripe! or! ripe! green! fruit! peel! is! chlorophyll! (rodrigo!et0al.!2013).!the!total!chlorophyll!of!citrus0reticulata!“tejakula”!was!observed!at!the! time! before! degreening! treatment! (harvest,! 0! days),! without! degreening! (9! days! after! treatment)!and!precooling!treatment!with!degreening!(9!days!after!treatment).!the!total! chlorophyll!in!citrus0reticulata!“tejakula”!with!precooling!and!degreening!treatment!with!24! hours!exposure!decreased!sharply!with!the!lowest!value!compared!to!other!treatments,!with! a!value!of!9.81!ug!g!fwx1!on!dayx9!after!treatment,!initial!chlorophyll!content!value!before! treatment!113.81!ug!g!fwx1!(figure!4).!! ! ! ! ! ! ! ! ! ! ! ! ! ! & figure&4.&the&change&of&total&chlorophyll&on&citrus&peel&& chlorophyll!degradation!causes!color!changes!during!degreening!and!the!appearance! of!carotenoid!pigment!to!produce!orange!color.!these!results!are!also!similar!to!the!study! conducted! by! stewart! and! wheaton! (1972);! rodrigo! and! zacarias! (2007)! that! ethylene! 0 20 40 60 80 100 120 140 before!degreening!(0!day! after!harvest) without!degreening!(9!day! after!harvest) degreening!(9!day!after! harvest) ch lo ro ph yl l!c on te nt (u g! g! fw x1 ) non!precooling precooling international0journal0of0applied0biology,01(2),020170 ! 43! applications! triggered! skin! pigmentation! by! stimulating! chlorophyll! degradation! and! accumulation!of!carotenoids,!including!β"citraurin.0 ! identification&of&βqcarotene,0βqcriptoxanthin0and&zeaxantin0 from! the! result,! we! can! conclude! that! in! order! to! form! orange! color! on! citrus0 reticulata!"tejakula"!in!lowland,!precooling!prior!to!degreening!is!needed.!precooling!and! degreening!treatments!provided!better!and!faster!result!in!orange!color!accumulation.!hplc! curve!on!zeaxanthin!and!βxcryptoxanthin!pigments!(figure!5).!the!content!of!βxcryptoxanthin! on!citrus0reticulata!"tejakula"!with!precooling!and!degreening!treatments!increased!rapidly! around!22.03!ug!g!fwx1!on!day!9!after!treatment!(figure!5).! !! ! ! ! ! ! ! ! & & & & & & & & & & figure&5.&hplc&chromatogram&of&citrus0reticulata&"tejakula"&without&precooling&(a),&and& precooling&(b)&(day&9&after&treatment)&with&degreening&& ! the!content!of!zeaxhantin!on!precooling!and!degreening!treatments!and!exposure! duration! for! 24! hours! resulted! in! 37.65! ug! g! fwx1! on! day! 9! after! treatment.! zeaxhantin! content!without!degreening!shows!lower!value!that!is!12.22!ug!g!fwx1!(data!not!shown).!this! result!is!in!accordance!with!kato!et0al!(2004);!rodrigo!et0al!(2013)!who!stated!that!the!change! of!citrus!peel!color!from!green!to!orange!caused!by!nonphotosyntetic!carotenoid!synthesis,! that! is! βxcitraurin! acts! as! the! color! formation! to! redxorange! on! citrus! nobilis! peel.! the! accumulation!of!this!compound!is!determined!by!precursor!availability!in!carotenoids!which! is! photosynthetic! such! as! zeaxanthin,! βxcryptoxanthin,! karotenoid! nonphotosintetic! (βx citraurin)!which!only!take!place!in!low!temperature.& & conclusions& the!citrus!color!index!(cci)!is!the!precooling!treatment!and!the!duration!of!ethylene! exposure!for!24!hours,!which!can!reduce!total!chlorophyll!content!about!8!times!and!proved! to! increase! β"cryptoxanthin! pigment! content! five! times! in! accelerating! the! formation! of! orange!citrus0reticulata0peel!color!to!bright!orange.!degreening!has!no!significant!effect!on! total!dissolved!solids!and!the!firmness!level!of!citrus!fruits.! & 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0 25 50 75 100 125 150 mv detector+a:450nm+ 9 .0 1 7 /1 0 4 7 4 7 3 1 2 .3 4 3 /7 7 2 2 0 5 1 7 .4 5 7 /8 0 2 0 8 6 (a)! 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 0 25 50 75 100 125 150 175 200 mv detector+a:450nm+ 5 .8 1 6 /2 1 1 1 2 5 8 9 .9 7 4 /1 3 2 3 3 4 1 1 7 .6 1 6 /3 8 4 5 5 9 (b)! zeaxanthin0 β"cryptoxanthin0 zeaxanthin0 β"cryptoxanthin0 international0journal0of0applied0biology,01(2),020170 ! 44! acknowledgment&& the!author!would!like!to!express!gratitude!to!center!for!tropical!horticulture!studies! (pkht)!ipb!and!lipi!cibinong!which!have!facilitated!the!research.!the!author!also!would!like! to! express! gratitude! to! ministry! of! research,! technology,! and! higher! education! for! the! doctoral!dissertation!research!grant!with!research!contract!number!0432/k3/km/2017.!the! author!also!would!like!to!thank!to!ministry!of!agriculture!for!the!partnership!grant!which!help! funding!the!research!with!contract!number!714/lb.620/1.1/2/2013.& & references& andarwulan,!n.,!kusnandar,!f.!&!herawati,!d.!2011.!analysis!of!food.!jakarta.!dian!rakyat![in! indonesian]! gang,! ma,! zhang! l.,! matsuta,! a.,! matsutani,! k.,! yamawaki,! k.,! yahata,! m.,! wahyudi,! a.,! motohashi,!r.!&!kato!m.!2013.!enzymatic!formation!of!βxcitraurin!from!bxcryptoxanthin! and! zeaxanthin! by! carotenoid! cleavage! dioxygenase4! in! the! flavedo! of! citrus! fruit.! american0society0of0plant0biologists.!163(2):682–695.! jiménezxcuesta,!m.j.,!cuquerella,!j.!&!martínezxjávega,!j.m.!1981.!determination!of!a!color! index!for!citrus!fruit!degreening.!in0proc.0of0the0international0society0of0citriculture.!2(5)! :!750x753.! kato,!m.,!ikoma,!y.,!matsumoto,!h.,!sugiura,!m.,!hyodo,!h.!&!yano,!m.!2004.!accumulation!of! carotenoids!and!expression!of!carotenoid!biosynthetic!genes!during!maturation!in!citrus! fruit.!biochemical!processes!and!macromolecular!structures.!134(2)!:!824x837.! kato,! m.,! matsumoto,! h.,! ikoma,! y.,! okuda,! h! &! yano,! m.! 2006.! the! role! of! carotenoid! cleavage! dioxygenase! in! the! regulation! of! carotenoid! profiles! during! maturation! in! citrus!fruit.!journal0of0exprimental0botany,!57(10):2153x2164.! ladaniya,! m.s! 2008.! citrus! fruit.0 biology,! technology! and! evaluation.! principal! scientist! (horticulture).!icar!research!complex!for!goa!:!india.! ministry! of! agriculture.! 2015.! prospect! of! agricultural! commodities! subsector! of! orange! horticulture.!jakarta:!agricultural!data!and!information!system!center!of!the!secretariat! general!–!indonesian!ministry!of!agriculture![in!indonesian]! rodrigo,! m.! j.,! alquezar,! m.,! alos,! e.,! medina,! v.,! carmona,! l.,! bruno! m.! 2013.! a! novel! carotenoid!cleavatage!in!the!biosynthesis!of!citrus!fuit!specific!apocarotenoid!pigments.! experimental0botany,!64:4461x4478.! rodrigo.!m.j!&!zacarias,!l.!2007.!effect!of!postharvest!ethylene!treatment!on!carotenoid! accumulation!and!the!expression!of!carotetenoid!biosynthetic!genes!in!the!flavedo!of! orange!(citrus0cinensis!l.!osbeck)!fruit.!scientia0hortikultura.!43:14x22!! sims,! d.a.,! gamon,! j.a.! 2002.! relationship! between! leaf! pigment! content! and! spectral! reflectance!across!a!wide!range!of!species,!leaf!structures!and!development!stages.!j! remote!sensing!envir.081:337–354.! stewart,! i! &! wheaton,! t.a.! 1971.! effent! of! ethylene! and! temperature! on! carorenoid! pigmentation!of!citrus!pell.!proc.0fla.0state0hort.0soc,!84:264x266.! stewart,! i.! wheaton,! t.a.! 1972.! carotenoids! in! citrus:! their! accumulation! induced! by! ethylene.0j.0agric.0food0chem,!20:448–449.! zhou,!y.j.,!sun,!c.d.,!zhang,!l.l.,!dai,!x.,!xu,!cj!&!chen,!k.s.!2010.!preferential!accumulation! of! orangexcolored! carotenoids! in! ponkan! (citrus0 reticulata)! fruit! peel! following! postharvest!application!of!ethylene!or!ethephon.!j.0scientia0horticulturae,!126:229x235.! ! international0journal0of0applied0biology,01(2),020170 ! 45! to&cite&this&article:&& sumiasih,!i.h.,!poerwanto,!r.,!efendi,!d.!&!agusta,!a.!&!yuliani,!s.!2017.!the!analysis!of!β" cryptoxanthin! and! zeaxanthin0 using! hplc! in! the! accumulation! of! orange! color! on! lowland!citrus.!international0journal0of0applied0biology.!1(2):!37x45.! & ! ! ! 2(2)13-19 contact : jiban shrestha jibshrestha@gmail.com © international journal of applied biology 13 abstract genotypic yield potential of maize varieties is greatly affected by sowing dates. in order to investigate the effects of sowing dates and varieties on the grain yield of maize, the field experiment was carried out at research field of national maize research program (nmrp), rampur, chitwan, nepal from april 2009 to march 2010. three varieties namely rampur composite, arun-2 and gaurav were sown at every week. the results of experiment showed that interaction effect of variety and sowing date on grain yield of maize was significant. rampur composite produced highest grain yield (6.1 t/ha) in august and lowest yield (2.6 t/ha) in may. similarly arun-2 produced highest yield (4.6 t/ha) in august and lowest yield (2.1 t/ha) in may. gaurav produced highest grain yield (5.1 t/ha) in september followed by 4.9, 4.8 and 4.6 t/ha in february, july and august respectively and lowest yield (1.5 t/ha) in november. the sowing date was highly significant on grain production. the highest grain production was 5.1 t/ha in august followed by in february (4.9 t/ha), september (4.6 t/ha) and march (4.4 t/ha) respectively. the lowest grain yield was produced in may (2.4 t/ha). therefore it was concluded that august planting was best for higher grain production of maize varieties (rampur composite, arun-2 and gaurav) in terai region of nepal. issn : 2580-2410 eissn : 2580-2119 productivity of maize (zea mays l.) as affected by varieties and sowing dates dil bahadur gurung1, balram bhandari2, jiban shrestha1,2 & mahendra prasad tripathi2 1nepal agricultural research council, singh darbar plaza, kathmandu, nepal 2nepal agricultural research council, national maize research program, chitwan, nepal introduction maize (zea mays l.) is the second most important staple crops after rice both in terms of area and production. its area, production, and productivity in nepal are 882395 ha, 2145291 t, and 2431 kg (moad 2016) respectively. this productivity is very low compared to that of neighboring countries. for optimum production seed must be sown on proper time. considerable reduction can occur in yield if the crop sown to early or too late (chaudry, 1994). ismail (1996) reported that early sowing of maize lead to an increased maize yield. either early planting or late planting can result in lower yield because the probability exists that unfavourable climatic conditions can occur after planting or during the growing season. in order for crop to best utilize moisture, nutrient and solar radiation, they must be grown from optimum sowing dates. nepal is a small country with large environmental variation. throughout the seasons of a year, weather conditions fluctuate a lot. in this situation, the same varieties may perform differently in different seasons. open access international journal of applied biology keyword grain yield sowing dates varieties article history received 12 july 2018 accepted 10 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 14 for maximization of benefit to farmers, they have to grow best maize varieties in best season of that location. tanaka and hara (1974) in india reported that variation in maize grain yield is due to the reduction in 1000-seed weight when sowing was delayed to the end of october. mascagni and boquet (1996) studied the effect of sowing dates on performance of maize and concluded that delay in sowing reduced yield of maize. in nepal very little work was done on the effect of sowing date and varieties on the performance of maize. the objective of this study was to determine the grain yield responses of maize to sowing dates and varieties. materials and methods description of experimental site the geographical location of the experiment site was located at 27040’n latitude, 84o19’ e and 228 masl and has sub tropical climate. the experimental soil was sandy silt loam, strongly acidic soil (ph 5.0), medium in total nitrogen (0.130%), high in soil available phosphorous (279 kg/ha), high in soil available potassium (215 kg/ha) and high in organic matter content (2.70%) (nmrp, 2011). meterological observation the meterological data in terms of temperature, rainfall and relative humidity during the crop growing period was given in table 1. table 1. meterological data at rampur, chitwan, nepal during 2009/10-2010/11 month mean temperature (0c) total rainfall (mm) relative humidity (%) 2009 2010 2009 2010 2009 2010 maxi min maxi min april 37.4 19.2 38.1 23.3 7.3 165 57.5 75.4 may 35.4 22.5 35.2 25.4 274.2 193 72.3 77.5 june 35.1 24.9 35.4 26.9 179.2 372 81.1 78.7 july 33.5 29.7 33.5 26.5 455 115.0 83 87.4 august 33.5 29.7 33.5 26.7 753 641.2 96 96.0 september 32.8 29 32.8 25.8 126 523.2 90 87.9 october 31.4 26.5 31.4 22.8 101 48.6 97 97.5 november 27.1 21.6 27.1 17.0 0 0.0 99 98.8 december 24 16 24.0 9.1 2.2 0.0 99 99.0 january 20 10.3 23.9 8.5 0 0.0 94.6 100.5 february 25.4 11.9 26.1 15.1 0 34.9 89.5 96.3 march 33.1 19.1 31.1 18.9 0 34.4 82.2 83.2 experimental design and cultural practices three varieties namely rampur composite, arun-2 and gaurav were sown in every week from april to march. the design was randomized complete block design replicated four times. in each month, the planting was done at seven days intervals. spacing 75 cm row to row and 25 cm plant to plant spacing was maintained and two to three seeds are sown and after two weeks thinned one plants/hill. plot size was 6 × 3.6 m out of which 5 × 2.4 was used to assess final harvest. fertilizer @ fym 10 t/ha and 12:60:40 kg npk/ha was applied for each experiment. half dose of nitrogen and full dose of phosphorous and potash was applied as international journal of applied biology, 2(2), 2018 15 basal dose at the time of final land preparation and remaining half of nitrogen was divided into two; one part applied at 20-24 and 40-45 days after sowing. weeding and irrigation was done as per recommendations. field measurements grain yield (kg/ha) at 15% moisture content was calculated using fresh ear weight with the help of the below formula: grain yield ( kg ha ) = f. w. ( kgplot) × (100 − hmp) × s × 10000 (100 − dmp) × npa where, f.w. = fresh weight of ear in kg per plot at harvest hmp = grain moisture percentage at harvest dmp = desired moisture percentage, i.e. 15% npa = net harvest plot area, m2 s = shelling coefficient, i.e. 0.8 this formula was also adopted by carangal et al. (1971) and shrestha et al. (2015) to adjust the grain yield (kg/ha) at 15% moisture content. statistical analysis data regarding the grain yield was statistically analyzed by combined analysis procedure to test differences among and within the different factors. the least significant difference (lsd) was used to separate the means of the main and interaction effects. results and discussion varieties also showed highly significant effect on yield. rampur composite produced the highest 4.1 t/ha yield which was as par with gaurav (3.6 t/ha). arun-2 produced the lowest 3.3 t/ha (figure 1). this results was similar to the results of sadek et al. (1994) and zaki et al. (1999) who reported that maize cultivars differed in their yield in india. figure 1. pooled mean grain yield of different maize varieties in rampur chitwan in 2009 gaurav produced the highest yield (4.3 t/ha) and arun-2 produced the lowest (3.3 t/ha). but in 2010 rampur composite produced the highest amount (4.1 t/ha) and gaurav produced the lowest (3 t/ha).there was highly significant effect of interaction of years and date of sowing on grain yield of maize (table-2). the grain yield differed in yield. table 2. interaction effect of years and variety on grain yield of maize in rampur, chitwan 0.0 5.0 arun-2 rampur composite gaurav 3.3 4.1 3.6 variety g ra in y ie ld (t /h a) lsd (0.05)=0.4295, sem=0.1171 and p value=0.01 international journal of applied biology, 2(2), 2018 16 the highest production was 5.1 t/ha in august followed by february (4.9 t/ha), september (4.6 t/ha) and march (4.4 t/ha) respectively. the lowest yield was of month may (2.4 t/ha) (figure 2). this results was confirmed with the findings of dahmardeh and dahmardeh (2010) who reported that the maximum grain yield (8.84 t/ha) was obtained from the 5 august sowing date that was not statistically on a 20 july and 20 august. winter maize has higher production potential. crop receives longer sunshine duration, higher rate of photosynthesis and assimilates utilization occurs during winter season. fertilizer use efficiency is higher in winter season. these factors contribute higher production during winter season. figure 2. effect of date of sowing (month) on grain yield of maize in rampur chitwan effect of interaction of variety and date of sowing (month) on grain yield of maize was found significant (p ≥ 0.05) (table 3). arun-2 produced highest yield in august and lowest yield in may. similarly, rampur composite produced highest yield in august and lowest yield in may. in the same way gaurav produced highest in september and lowest in november. this result agree with finding by otegui et al. (1995) that optimum planting date resulted in higher grain yield than early and late planting dates because of higher cob numbers and greater kernel numbers per plant). variation in yield of corn varieties at different planting dates was associated with differences in the amount of intercepted radiation. highly significant interaction among year, variety and date of sowing (month) on grain yield of maize was obtained (table 4). arun-2 produced highest yield in august in both of years but lowest yield was in april (1.9 t/ha) in 2009 and in may (1.1 t/ha) in 2010. rampur composite produced highest yield in february in 2009 and in august in 2010 but lowest yield was in november in 2009 and in may of 2010. gaurav produced highest yield in july in 2009 0.0 2.0 4.0 6.0 3.1 2.4 3.1 4.1 5.1 4.6 3.9 2.6 3.1 2.7 4.9 4.4 grain yield (t ha-1) lsd (0.05)=0.8590, sem=0.2343 and p value=0.01 s.n. years varieties arun-2 rampur composite gaurav 1. 2009/10 3.3 4.0 4.3 2. 2010/11 3.3 4.1 3.0 lsd at 0.05 = 0.6074 sem = 0.1656 p value = 0.01 international journal of applied biology, 2(2), 2018 17 and in september in 2010 but lowest yield was in november in 2009 and in may of 2010. table 3. interaction effect of variety and date of sowing (month) on grain yield of maize in rampur, chitwan (2009-2010) s.n. date of sowing (month)(month) variety arun-2 rampur composite gaurav 1. april 3.2 3.7 2.6 2. may 2.1 2.6 2.4 3. june 2.3 2.7 4.2 4. july 3.2 4.3 4.8 5. august 4.6 6.1 4.6 6. september 4.0 4.8 5.1 7. october 3.5 4.4 3.7 8. november 3.2 3.3 1.5 9. december 2.6 3.6 3.1 10. january 2.4 3.2 2.5 11. february 4.5 5.4 4.9 12. march 4.1 4.8 4.3 lsd at 0.05 = 1.129 sem = 0.4057 p value = 0.05 table 4. interaction effect of year, variety and date of sowing (month) on grain yield of maize in rampur, chitwan s.n. date of sowing (month)(m onth) variety and year arun-2 rampur composite gaurav 2009 2010 2009 2010 2009 2010 1. april 1.9 4.4 2.9 4.5 3.3 1.8 2. may 3.1 1.1 3.6 1.6 3.7 1.2 3. june 2.1 2.5 2.8 2.7 5.3 3.1 4. july 4.0 2.4 3.6 5.1 6.6 3.0 5. august 4.4 4.9 4.6 7.5 6.0 3.3 6. september 3.1 4.8 4.8 4.8 5.5 4.8 7. october 3.9 3.1 4.4 4.3 4.6 2.8 8. november 2.4 3.9 2.3 4.2 1.1 1.9 9. december 2.5 2.7 3.3 4.0 1.2 5.0 10. january 3.2 1.5 4.5 1.9 3.6 1.4 11. february 5.1 4.0 6.7 4.1 6.4 3.3 12. march 4.3 4.0 4.8 4.7 4.6 4.0 lsd at 0.05 = 2.104 sem = 0.5738 p value = 0.01 bk et al. (2015) reported that grain yield of maize was affected by genotypes and date of planting at rampur chitwan nepal. lesser grain august was due to less population because a lot of plants were lodged due to the rainy season in month of august. optimum sowing date international journal of applied biology, 2(2), 2018 18 resulted in higher grain yield than early and late planting dates (otegui et al. (1995). the result are in confirmation with jaliya et al. (2008), namakka et al. (2008), aziz et al. (2007), khan et al. (2002) and zaki et al. (1994) who reported that grain yield was reduced by delay in sowing. conclusions based on the result of this experiment it can be concluded that the higher grain yield in rampur composite, gaurav and arun-2 can be obtained by planting them in early winter season especially in the month of august in terai region of nepal. acknowledgments the authors gratefully acknowledge financial support from nepal agricultural research council, nepal. references aziz, a., rahman, h. & khan, n. 2007. maize cultivar response to population density and planting dates for grain and biomass yield. sarhad j. agric. 23(1): 25-30. b.k., s. b., karki, t.b. , shrestha, j. & adhikari, p. 2015. productivity of maize genotypes under different planting dates. our nature. 13(1): 45-49. doi: http://dx.doi.org/10.3126/ on.v13i1.14208 carangal, v.r., ali, s.m., koble, a.f., rinke, e.h. & sentz, j.c. 1971. comparison of s1 with testcross evaluation for recurrent selection in maize. crop science, 11: 658-661. chaudry, f.m. 1994. kharif cereal crops in crop production. national book founda tion, islamabad, pp: 262. chitwan, nepal. dahmardeh, m. & dahmardeh, m. 2010. the effect of sowing date and some growth physiological index on grain yield in three maize hybrids in south eastern iran. asian journal of plant sciences, 9: 432436. ismail, a.a. 1996. gene action and combining ability for flowering and yield in maize under two different sowing dates. assiut j. agric. sci., 27: 91-105. jaliya, m.m., falaki, a.m., mahmud, m. & sani, y.a. 2008. effect of sowing date and npk fertilizer rate on yield and yield components of quality protein maize (zea mays l.). arpn j. agric. bio. sci. 3(2): 23-29. khan, h., arif, m., gul, r., ahmad, n. & khan, i.a. 2002. effect of sowing dates on maize cultivars. sarhad j. agric. 18(1): 11-15. mascagni, jr. h.j. & boquet, d.j. 1996. starter fertilizer and planting date effects on corn rotated with cotton. agron. j., 88: 975-982. moad. 2016. statistical information on nepalese agriculture, 2015/2016 (2072/2073). agribusiness promotion and statistics division, singha durbar, kathmandu, nepal. namakka, a., abubakar, i.u., sadik, a.i., sharifai, a.i. & hassas, a.h. 2008. effect of sowing date and nitrogen level on yield and yield components of two extra early maize varieties (zea mays l.) in sudan savanna of nigeria. arpn j. agric. bio. sci. 3(2):1-5. nmrp. 2011. annual report 2010-11. national maize research program, raampur, otegui, m.e., nicolini, m.g. , ruiz, r.a. & dodds, p.a. 1995. sowing date effects on grain yield components for different maize genotypes. agronomy journal, 87: 29-33. sadek, s.e., el-sherbieny, h.y.s., ahmed, m.a. & younis, m.a. 1994. evaluation of eight yellow maize (zea mays l.) hybrid grown in egypt. growth analysis and grain yield components. journal agriculture science mansoura university, 19: 4154-4160. international journal of applied biology, 2(2), 2018 19 shrestha, j., koirala, k., katuwal, r., dhami, n., pokhrel, b., ghimire, b., prasai, h., paudel, a., pokhrel, k., and kc, g. 2015. performance evaluation of quality protein maize genotypes across various maize production agro ecologies of nepal. journal of maize research and development, 1(1): 21-27. tanaka, a. & hara, t. 1974. studies on the nutrio-physiology of maize plant. 10. grain yield affected by sowing date. fld. crop abstr. 27: p. 565 zaki, m.s., shah, p. & hayat, s. 1994. effect of sowing date on maize and non-flooded land rice. sarhad j. agric. 10(2): 191-199. zaki, n.m., el-gazzar, m.m., gamal, k.m. & ahmed, m.a. 1999. partition and migration of photosynthesis in some maize hybrids. egyption journal application science, 14: 117139. 2(1)22-28 contact : jiban shrestha jibshrestha@gmail.com © international journal of applied biology 22 abstract studies on the effect of different planting dates and varieties of maize on the infestation of maize stem borer (chilo partellus) were carried out at research field of national maize research program, rampur, chitwan, nepal in 2010/11 and 2011/12. three maize varieties namely namely arun-2, rampur composite and gaurav (hybrid) were grown at weekly interval upto 52 weeks from january to december. the results revealed that the damage was higher in arun-2 (38.9%) followed by gaurav (35.4%) and rampur composite (30.8%) respectively. higher damage was observed in april (58.2%) and the damage was gradually decreased toward the month of january/february (13.2 to 25.6%) and november /december (13.3 to 16.5%). lower infestation was observed in winter season (22.5%) followed by spring (47.1%) and summer season (47.0%) respectively. this study suggests that the percent infestation of maize stem borer can be minimized by planting rampur composite variety in winter season. issn : 2580-2410 eissn : 2580-2119 effect of planting dates and varieties on infestation of maize stem borer chilo partellus (swinhoe) pitamber thakur, ghanashyam bhandari, jiban shrestha & buddhi bahadur achhami nepal agricultural research council, national maize research program, rampur, chitwan, nepal introduction the yield of maize crop is adversely affected due to insect pests, diseases, weeds, nematodes and birds etc. the major insect pests that attack maize crop include maize aphids, cutworms, stem/shoot fly, root worm and stem borer. the grain losses range from 10-15% due to insect pests alone (jaipal and dass, 1993). maize stem borer chilo partellus swinhoe is the most serious pest (kumar et al., 1993). the damage due to insect pest complex depends upon their population trends in the field which, in turn, rely upon their dynamically of the physical factors of their immediate environment (isard, 2004). pests of maize are strongly influenced by weather conditions and are very difficult to predict. a thorough understanding of the exact relationship between the change in environmental factors and those in the pest population may not only help anticipate the pest losses to the crop, but also help avoid them open access international journal of applied biology keyword maize infestation maize stem borer planting dates varieties article history received 12 june 2018 accepted 22 july 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 23 through some well-timed pest control measure. insect damage decreases the yield and lower grain quality (rahman et al., 1994). low productivity (2.5 ton/ha) of maize in nepal is attributed to many reasons. among them is the attack of various insect pests. the cumulative effect of pests including some other minor insects on maize yield is reported to be 33-41% at khumaltar (joshi, 1977). among the serious pests, maize stem borer, chilo partellus swinehoe (lepidoptera: pyralidae) is one of the major biotic constraints in successful maize production throughout the country and abroad (pingali 2001, dhaliwal and arora 2001, sekhon and kanta 1997). the larvae (caterpillars) eat through leaves when young and as they grow older, eventually bore into the stem causing it to break or die. various approaches have been made to control maize stem borer including cultural, physical, biological and chemical in an uncoordinated manner throughout the country. not much attempts have been made to look into the level of infestation in the stubble and particularly in the stalks. the use of pesticides in crop protection has certainly contributed for minimizing yield losses however quite often the indiscriminate and unscientific use of pesticides has led to the many problems. keeping in view the importance of maize crop and the severity of damage caused by maize stem borer the present experiment was conducted with the following objectives. 1. to find out a proper sowing date for the maize crop in chitwan, nepal. 2. to find out the link between the sowing date and infestation so as to avoid damage from maize stem borer. materials and methods a field experiment composed of three popular and released maize varieties namely arun-2 (short duration), rampur composite (full season) and gaurav (hybrid) were seeded round the year in weekly interval at national maize research program (nmrp), rampur, chitwan, nepal during two consecutive years of 2010/11 and 2011/12. the experiments were laid out in a randomized complete block design with three replications. individual plot size was 2 rows of 5m length and spacing was 75 × 20 cm between row to row and plant to plant. the field was fertilized with chemical fertilizer @ 120:60:40 kg npk/ha. half dose of nitrogen with full dose of phosphorous and potash was applied at the time of planting and the remaining nitrogen was top dressed at the time of second weeding. all intercultural operation was provided as per recommendation of nmrp for good plant stand. the plots were surveyed at weekly interval for the presence of pests. individual plants in each variety were thoroughly examined and damage done by stem borer. insects damage rating as visual score (0-9 scale), total healthy and damage plants/plots, tunnel length (cm) inside the stalk, exit holes, pin holes, tattering stage and dead heart record were done as ascribed by ampofo and saxena (1987), cimmyt, mexico. the data pertaining to dead heart and infestation was pooled together. general rating was done in 1-9 scale where 1 indicated resistant and 9 as a highly susceptible to the pests. ten randomly selected stalks from two rows of each genotype were taken for tunnel length measurement during harvesting time. each stalk was longitudinally cut by the help of knife and recorded length of tunnel in centimeter scales and numbers of exit holes formed by adult borer inside the stalk. similarly the data on temperature obtained from the meteorological observatory of nmrp, rampur, chitwan, nepal. available observations were collected, summarized in weekly and monthly basis and presented in tabular and graphic forms. international journal of applied biology, 2(1), 2018 24 results and discussion the result of the present study inferred that insects injury was approximately in the same trend in both the years proportional to prevailing temperature. percentage stem borer infestation was gradually increased toward the progress of weeks and more or less remained constant from 10 to 42 weeks and further start declined gradually. the trend of temperature as was also alike to that of insect infestation. thus the temperature was pronounced resulting in higher percentage of infestation (figure 1). thus, it is cleared that the above mentioned temperature is most congenial for pests’ growth and development. these finding was confirmed with the work of tamiru et al. (2012) as most suitable condition for chilo partellus swinehoe development was 26 to 30 oc temperature. regarding the maize growing season, mean of the weeks revealed that borer damage was comparatively less (22.5%) at 21.1oc during winter seeded maize plants than spring (47.08% at 28.78oc) and rainy (47.03% at 29.2oc). the above statement is supported by muhammad et al. (2010) that the infestation of chilo partellus swinehoe was found highest at higher temperature (32.5oc). 2010/11 2011/12 minimum temperature: 13.7oc on january 1st week maximum temperature: 32.05oc on june 1st week average of the year:25.6oc minimum temperature: 12.5oc on december 3rd week maximum temperature: 29.98oc on june 2nd week average of the year: 24.35oc figure 1. effect of temperature fluctuation on insects’ population growth and its result on maize genotypes during two consecutive years of 2010/11 and 2011/12 at nmrp, rampur, chitwan, nepal as shown in table 1, among the varieties tested, mean result of the weeks inferred that arun-2 (short duration) was slightly more sensitive to maize stem borer damage (38.9%) than gaurav (35.4%) and rampur composite (30.8%). the range of damage varied from 8.7 65.2% in arun-2, 3.458.6% in rampur composite and 9.6-72.2% in gaurav. maximum infestation of 72.2% was observed in gaurav variety on 16 weeks at which 29.8oc temperature was recorded followed by 65.2% damage in arun-2 on 15 weeks at 29.2oc and 58.6% damage in rampur composite on 16 weeks at 29.8oc. thus from the above depicted international journal of applied biology, 2(1), 2018 25 figure, it could be clarified that around 29 oc temperature fall on 15-16 weeks (april) was most congenial for the growth and development of borer. rampur composite was found more resistant variety. large number of maize genotypes with resistance to chilo partellus has been reported (kumar, 1993). table 1. response of maize varieties/hybrid to maize stem borer damage (%) in weekly interval seeded experiment at nmrp, rampur, chitwan, nepal (average data of two years of 2010/11 and 2011/12) weeks arun-2 rampur composite gaurav weeks arun-2 rampur composite gaurav 1 9.2 8.3 9.6 27 50 33.3 38 2 11.4 9.5 11.1 28 44.4 38.9 38.6 3 14.3 15.4 20 29 62.5 36.7 43.8 4 22.7 12 28 30 57.7 40 37.3 5 28 19.2 24 31 48.7 36.8 42.8 6 33.5 24.8 20 32 52.2 33.3 52.7 7 42.8 25 27.3 33 33.3 33.3 36.7 8 38.6 27.5 30.8 34 39.2 37.3 20 9 43.6 35 56.5 35 54.5 35 43.7 10 54.5 36 58 36 47.5 39.7 60 11 57.1 35.4 40 37 36.4 37.6 36.7 12 44 33.8 41.7 38 30 35 33.3 13 38.9 40.9 48 39 34.1 34.7 33.3 14 52.2 36.4 36 40 35.4 32.3 37.5 15 65.2 52.8 64.7 41 34.8 33.3 40 16 56.5 58.6 75.2 42 37.7 35.7 25 17 48 52 63.6 43 35 20.8 25 18 60.6 55.4 57.7 44 30 12.5 30 19 63 37.7 38.6 45 30 25 28.2 20 52.4 36.7 43.7 46 24 16.7 16.7 21 54.5 42.1 40 47 16 13.3 18.3 22 50 48.3 37.5 48 15.8 6.7 12.7 23 53.3 44.5 50 49 16.7 12.7 20 24 42.8 41.7 38.6 50 18.6 6.7 11.1 25 37.7 37.5 40 51 20.8 3.4 14.8 26 32 34.4 33.3 52 8.7 9.1 11.2 mean of 52 weeks 38.9 30.8 35.4 likewise, mean of all parameters mentioned in table 2 indicated that insect rating (3.66 to 3.99 score), percent insect damage (34.3 to 33.9%), tunnel length (11.59 to 16.34 cm) and exit holes number (2.64 to 3.8) were approximately similar in both the tested years. however, average range of score was varied from 1.96 to 6.1, percent infestation (13.2 to 58.2%), tunnel length (4.45 to 22.8 cm) and exit holes number (1.9 to 6.0). moreover, average international journal of applied biology, 2(1), 2018 26 infestation of borer was highest in the month of april (58.2%) followed by may (48.2%) and july (44.9%). the approximately same trend of damage was found in other parameters (score, tunnel length and exit holes) too. during january/february (13.2 to 25.6%) and november/december (16.5 to 13.3%), infestation was low whereas fluctuating infestation was recorded from march to october (29.0 to 58.2%). the environmental conditions in nmrp, rampur, chitwan, nepal is normally favorable for buildup of insect populations large enough to cause serious damage to maize. the highest percentage of damage by stem borer was occurred during rainy or summer season and that was lowest in winter season. the percentage of damage depends on the extent of insect population. during the summer (rainy) season the population of stem borer increased so the damage extent was increased. this result can be deduced from the foregoing results of the locations that temperature plays a significant effect for growth and development of maize stem borer damage which is supported by tamiru et al. (2012). according to him, the physical factors like atmospheric temperature are known to play an important role in the life cycle of insects and adaptability to local climate. mbapila et al. (2002) also targeted temperature as a primary factor for mortality, growth and development lepidopterans. table 2. monthly fluctuation of maize stems borer infestation during two consecutive years of 2010/11 and 2011/12 at nmrp, rampur, chitwan, nepal month score (0-9 scale) mean % damage mean tunnel length (cm) mean exit holes (number) mean 2010/11 2011/12 2010/11 2011/12 2010/11 2011/12 2010/11 2011/12 january 1.33 2.6 1.96 11 15.4 13.2 4.8 9.5 7.15 1.7 2.6 2.1 february 2.3 3.4 2.85 19.9 31.2 25.6 9.5 12.1 10.8 1.6 2.4 2 march 5.1 4.9 5 43.1 42.2 42.7 10.1 18.3 14.2 1.6 2.5 2.1 april 6.1 6.2 6.1 64.8 51.6 58.2 20 18.8 19.4 2.6 2.7 2.6 may 5.5 5.6 5.5 46.9 49.5 48.2 17.2 20.9 19.1 3.4 2.2 2.8 june 5.2 3.3 4.25 46 31.1 38.6 13.1 18.1 15.6 4.4 2.7 3.5 july 4.3 5.2 4.75 39 50.7 44.9 11.5 14.8 13.2 5.1 6.8 6 august 3.7 4.6 4.15 36.9 43 40 18.9 26.7 22.8 3.6 7.9 5.7 september 4.2 4.1 4.15 42.2 37.8 40 13.5 27.3 20.4 3.2 7.3 5.2 october 3.3 2.6 2.95 37 21 29 11.2 18.1 14.7 1.2 3.9 2.5 november 1.6 2.6 2.1 16.2 16.8 16.5 5.4 6.5 5.95 1.5 2.6 2.1 december 1.3 2.8 2.05 9 17.6 13.3 3.9 5 4.45 1.7 2.1 1.9 mean 3.66 3.99 3.82 34.33 33.9 34.2 11.59 16.34 14 2.64 3.8 3.2 table 3. response of maize stem borer damage based on maize growing seasons during two consecutive years of 2010/11 and 2011/12 at nmrp, rampur, chitwan, nepal s.n. winter season /weeks % borer damage temperature (oc) spring season /weeks % borer damage temperature (oc) rainy season /weeks % borer damage temperature (oc) 1 august 35 42.0 29.78 march 10 47.02 25.10 april 14 45.35 26.48 2 36 39.5 29.28 11 42.10 24.86 15 54.93 27.23 3 37 45.2 29.28 12 41.50 26.69 16 62.22 29.18 4 38 39.2 27.92 13 46.77 26.48 17 56.32 29.84 5 39 39.3 28.24 14 45.35 27.23 18 58.12 29.28 6 40 33.5 29.10 15 54.93 29.18 19 53.42 29.11 7 41 34.7 28.49 16 62.22 29.84 20 47.28 28.90 international journal of applied biology, 2(1), 2018 27 8 42 28.8 27.64 17 56.32 29.28 21 47.55 30.22 9 43 27.7 26.33 18 58.12 29.11 22 49.12 29.14 10 44 22.7 23.14 19 53.42 28.90 23 43.85 29.63 11 45 17.8 22.43 20 47.28 30.22 24 47.23 30.46 12 46 17.9 20.45 21 47.55 29.14 25 41.00 30.60 13 47 14.4 19.60 22 49.12 29.63 26 41.30 30.08 14 48 12.9 19.07 23 43.85 30.46 27 40.33 29.44 15 49 12.3 17.70 24 47.23 30.60 28 42.15 29.35 16 50 11.2 16.01 25 41.00 30.08 29 41.52 29.21 17 51 5.5 14.09 26 41.30 29.44 30 41.92 29.24 18 52 4.5 14.77 27 40.33 29.35 31 44.77 29.37 19 1 8.2 13.49 28 42.15 29.21 32 48.77 29.69 20 2 8.0 14.05 29 41.52 29.24 33 41.88 29.79 21 3 13.4 14.16 30 41.92 29.37 august 34 38.65 28.94 22 4 16.6 15.40 august 31 44.77 29.69 23 5 20.2 16.51 24 6 18.7 17.93 25 7 26.1 19.82 26 february 8 25.6 20.73 mean 22.5 21.16 47.08 28.78 47.03 29.2 conclusions the finding of the present experiment showed that rampur composite, a popular and widely adopted maize variety was found more tolerate against maize stem borer infestation as compared to other varieties. moreover, maize stem borer was identified as a key pest infested more in spring and summer season than winter season. vegetative stages (below knee high) are more vulnerable than older stage of crop. the role of temperature at the range of 25-30oc played a significant effect for insects growth and development. references ampofo, j.k.o. & saxena, k. n. 1987. screening methodologies for maize resistance to chilo partellus (lepidoptera: pyralidae). in: toward insect resistant maize for the third world, proceedings of the international symposium on methodologies for developing host plant resistance to maize insects, mexico, d. f. cimmyt, 170-177. dhaliwal, g. s. & arora, r. 2001. integrated pest management concept and approach. kalyani publishers, india. isard, j. 2004. influence of atmospheric condition on high elevation flight of western corn rootworm. entomol. soc. america, 33: 650–656. jaipal, s. & dass, s. 1993. reactions of maize accessions to the stem borer (chillo pertellus). tests agro-chem. cultiv. 14: 152–153. joshi, s. l. 1977. assessment of yield losses due to insect pests occurred in maize in kathmandu, nep, j. agri., 12: 127–134. kumar, h., nyangiri, e.m.o. & asino, g.o. 1993. colonization responses and damage by chilo partellus (lepidoptera:pyralidae) to four variably resistant cultivars of maize. j. econ. entomol. 86(3) 739–746. international journal of applied biology, 2(1), 2018 28 kumar, h. 1993. field resistance in maize cultivars to stem borer chilo partellus. ann. appl. biol. 124: 333–339. mbapila, j.c., overholt, w.a. & kayumbo, h.y. 2002. comparative development and population growth of an exotic stemborer, chilo partellus (swinhoe), and an ecologically similar congener, c.orichalociliellus (strand) (lepidoptera: crambidae). insect science and its application, 22: 21–27. pingali, p. i. 2001. world maize fact and trends. meeting world maize need: technological opportunities and priorities for the public sector, cimmyt, mexico d.f, 57-63. rahman, h., hashmi, a. a. & javed, h. i. 1994. insect pest of maize and sorghum. in: insect pest management: cereal and cash crops, pak. agricl. res. council, 61-85. sekhon, s.s. & kanta, u. 1994. machanisms and bases of resistance in maize to spotted stem borer. reviewed in: j.a. mihm (ed) insect resistant maize recent advances and utilization proceedings of an international symposium held at mexico, d.f.: cimmyt, 106-111. tamiru, e. getu, jembere, b. & bruce, t. (2012). effect of temperature and relative humidity on the development and fecundity of chilo partellus (swinhoe) (lepidoptera: crambidae). bulletin of entomological research, 102(1), 9-15. 2(1)61-65 contact : ashifa cahyani trisnaputri ashifacahyani@gmail.com � international journal of applied biology 61 abstract indonesia is one of the largest producing country of banana in the world, but approximately only 1,5 million tons were consumed. as one of the fruit which contains the highest carbohydrate, the overripe banana with the high contains of sugar has a potential to be developed as the raw material of glucose syrup. according to the research, the banana with a lot of bruise or black mark on the skin has 2,6% strach, 33,6% reduction sugar, and 53,2% sucrose. in the making of banana glucose syrup, it will use α-amylase to hydrolize the strach. based on the reduction sugar analysis there are no significantly different between the addition of 0,25 ml and 0,5 ml α-amylase. the results has qualified the minimum standard of glucose percentage (min. 30%) based on the reference sni 01-2985-1992. the utilization of overripe bananas will greatly improve our glucose syrup production and ensure more of the banana waste can be saved in the future. issn : 2580-2410 eissn : 2580-2119 production banana glucose syrup with the α-amylase supplementation ashifa cahyani trisnaputri1, nani rahayu usman2, muhammad al mustawa3 & abdul mollah jaya1 1 department of agronomy, hasanuddin university, makassar, south sulawesi, indonesia 1 department of food science and technology, hasanuddin university, makassar, south sulawesi, indonesia 3 department of chemistry, hasanuddin university, makassar, south sulawesi, indonesia introduction indonesia is one of the largest producing country of banana in the world. in 2016 indonesia produced approximately 7,45 million tons of bananas, but approximately only 1,5 million tons were consumed (bps, 2016). so, the surplus of banana probably will be wasted. not only in indonesia, but also on a global scale approximately millions of bananas end up or wasted along the supply chain. routinately millions of edible bananas throw away everyday just because it has a minor bruise or black mark on the skin after it’s overripe. in order to reduce the waste, the product which is made from banana as the raw material should be developed. as one of the fruit which contains the highest carbohydrate, the overripe banana with the high contains of sugar has a potential to be developed as the raw material of glucose syrup (high maltose syrup). open access international journal of applied biology keyword α-amylase banana glucose syrup reduction sugar article history received 13 june 2018 accepted 12 july 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 62 bananas has higher carbohydrate content than other fruits and overripe bananas with high sugar content has a potential as an alternative glucose syrup raw material. naturally, total sugars in ripe fruits will increase due to changes in polysaccharides consisting of starch, pectin and hemicellulose into dissolved sugars. according to the research, the banana with a lot of bruise or black mark on the skin has 2,6% strach, 33,6% reduction sugar, and 53,2% sucrose (zhang et al., 2005). glucose syrup are now manufactured and used in many countries through out the world. nowadays, glucose syrup is only produced from a variety of starch raw materials including, such as cassava, sago, rice, sorghum, wheat, corn, etc. (vuilleumier, 1993; e.a. borges da silva, et al., 2006; elkhalifa, et al., 2017). producing glucose syrup by utilizing overripe banana as the raw material will greatly increase our glucose syrup production. the utilization of overripe bananas as a raw material for glucose syrup utilizes enzymatic reactions of α-amylase enzymes used in the fructose syrup manufacturing industry to maximize natural starch in overripe bananas to produce quality high glucose syrup. the purpose of this research is to know the effectiveness of the addition of enzyme α-amylase in the making of banana glucose syrup. materials and methods the materials that were used are banana (musa paradisiaca l.) which have black bruise or mark on its skin (overripe) were used as the raw material. the enzyme used for the hydrolysis of starch was α-amylase (termamyl 120l), provided by novozymes® (denmark). termamyl® 120l is thermostable, produced by a selected strain of bacillus licheniformis. the first step is raw material preparation. peel the banana (500g) and cut it into small pieces. puree the banana using food processor or blender. combine the banana and αamylase in a food processor or blender, and blend it until smooth. after the addition of αamylase a liquefaction process will be happened. liquefaction is a process of dispersion of insoluble starch granules in aqueous solution followed by partial hydrolysis using thermostable amylases (aiyer, 2005). the experimental design that were used is completely randomized design which consist of 1 factor (enzyme concentration) with 2 levels. add 0,25ml (t1) and 0,5ml (t2) α-amylase and put the banana puree at 90°c until the liquefication process is done. the extent of liquefaction was evaluated by the amount of reducing sugars produced, such as glucose, which represents a measure of reducing sugars present in the product as a percentage of the total dry substances (lin et al., 2013). after the liquefaction is done, the next step is purification process. in the purification process, 150 mesh and 400 mesh sieve were used. agitate the sieves to allow the juice to pass through without giving any pressure on the pureed banana to prevent solids pass through the sieve along with the juice (liquid). after that, the liquid will be purified again with using microfiber filter. the banana juice will be evaporated to reduce the moisture content up to 28,5 – 29,5% based on the reference sni 01-2985-1992 (bsn, 1992). after the evaporation process, the viscosity will increase, so the the juice will become a syrup. the optimum temperature to put the syrup is 35°c. in this temperature, the crystallization of dextrose will be prevented. the lower temperature will crystallize the syrup, so it could decrease the quality. before we do the reduction sugar test, the moisture content and total soluble solids must be determined based on the reference sni 01-2985-1992. moisture content of the syrup international journal of applied biology, 2(1), 2018 63 will be determined by using moisture analyzer. moisture analyzer determines the moisture content of a sample with the loss on drying method and consists of a weighing and heating unit (infrared). total soluble solids (% brix) content in the banana juice will be determined by using hand refraktometer. the sample was dripped into the refractometer prism. the temperature and the total soluble solids value will be observed. the total of reduction sugar will be determined based on (sni-01-2892-1992) that use luff schoorl method. in the microbiology test will be focused on escherichia coli dan staphylococcus aureus. the mikrobiology test will use standart plate count method to identify the contaminaton of e.coli and s. aureus. e.coli is one of negative gram bacteria which is classified into bacteria that can cause several digestion diseases, while s. aureus is one of positive gram bacteria that can influence the human antibody and cause several digestion diseases (le loir, 2003). results and discussion moisture is used as a quality factor for sugar syrup that will influence the time of storage. if the sugar syrup contain the high number of moisture content, the time of storage will decrease. based on moisture analysis (table 1) shows that the highest moisture content of syrup is t12 (35,97%) and the lowest moisture content of syrup is t21 (22,47%). both the moisture content value between t1 and t2 are not significantly different and close enough with sni 01-2985-1992 requirement. so, the moisture content has fullfilled the reference sni 01-2985-1992. based on total soluble solids analysis (table 2) shows that, the total soluble solids data (%brix) are inversely proportional with the moisture content. the total soluble solids is the strach that were not hydrolized during the enzymatic process. the highest total soluble solids content is t21 (77,53%) and the lowest total soluble solids content is t12 (64,03%). both the total soluble solids value between t1 and t2 are not significantly different and close enough with sni 01-2985-1992 requirement. so, the moisture content has fullfilled the reference sni 01-2985-1992. table 1. moisture content analysis after adding α-amylase sample code moisture content (%) total soluble solids(%) t11 32,68 67,32 t12 35,97 64,03 t13 23,34 76,66 t21 22,47 77,53 t22 29,68 70,32 t23 29,73 70,27 the reduction sugar analysis will show the potential of overripe banana as the raw material of high glucose syrup. based on the reduction sugar analysis, after the enzymatic reaction by adding α-amylase enzyme, the higest total of reduction sugar is t23 (43,20%) and the lowest total of reduction sugar is t22 (34,99%). so, the are no significantly different between t1 and t2 reduction sugar. in the making of overripe banana glucose syrup, the natural enzymatic reaction during the ripening process that will hydrolyze the strach has to be increased with using α-amylase, so the percentage of reduction sugar could be increased. the ripe banana contain the high number of reduction sugar and sucrose but contain fewer strach. the percentage of strach will decrease during the increasing of the reduction sugar. the decreasing of strach is caused by enzymatic reaction that naturally happened international journal of applied biology, 2(1), 2018 64 during the ripening process (zhang et al., 2005). based on the calories analysis, banana glucose syrup contains 151,43 kcal (table 4). the calories that contain in banana glucose syrup is lower than sugar cane (364 kcal), aren sugar (368 kcal), coconut sugar (386 kcal) and another sweetener such as honey (294 kcal) (rahmawati, 2017). tabel 3. reduction sugar analysis after adding α-amylase sample code carbohydrate (%) glucose (%) t11 34,52 36,34 t12 36,11 38,01 t13 33,89 35,68 t21 34,92 36,76 t22 33,24 34,99 t23 41,04 43,20 table 4. chemical composition of t1 sample (per 100 g) composition amount carbohydrate 36,36% protein 1,25% fat 0,11% calories 151,43 kcal table 5. microbiology test after adding α-amylase sample code e. coli s. aureus requirement t11 3 t12 3 t13 3 t21 3 t22 3 t23 3 3: has fulfilled the health requirement and sni 01-2985-1992 based on the microbiology test, all sample are not contamined by e.coli dan s.aureus (table 5). so, the syrup has fulfilled the health requirement to be consumed. this result shows that there is no relationship between the addition of α-amylase e.coli and s.aureus contamination. the contamination of e.coli and s.aureus will not happened as long as the hygenic of the raw material is kept during the production process. conclusions in this research, overripe banana have a big potential to be the raw material in the making of glucose syrup. based on the reduction sugar analysis are no significantly different between the addition 0,25ml and 0,5 ml α-amylase. the contain of reduction sugar in banana glucose syrup has fulfilled the sni 01-2985-1992 requirement (min. 30%). international journal of applied biology, 2(1), 2018 65 references aiyer, prasanna, v. 2005. amylases and their applications. african journal of biotechnology. 4(13):1525-1529. silva, e.a.b., souza, a.a., souza, s.g., rodrigues, a.e. 2006. analysis of the high-fructose syrup production using reactive smb technology. chemical engineering journal. 118(3): 167-181. elkhalifa, e.a., abdalla, n.k.a., sarah & abdelkareem, a.m. 2017. utilization of sorghum (feterita) starch in production of fructose syrup. international journal of food science and nutrition engineering 2017, 7(4):70–74. le loir y, florence, b. & michel g. 2003. staphylococcus aureus and food poisoning. j gen mol res, 2(1):63–76. lin, q., xiao, h., liu, g. q., liu, z., li, l. & yu, f. 2013. production of maltose syrup by enzymatic conversion of rice starch. food and bioprocess technology, 6(1), 242–248. nurul, r., budiarti, r.s. & harlis. kajian pembuatan gula cair berbahan dasar kulit singkong (manihot utilissima pohl.) dengan pemanfaatan bakteri bacillus licheniformis. universitas jambi, jambi. vuilleumier, s. 1993. worldwide production of high-fructose syrup and crystalline fructose. the american journal of clinical nutrition. 58(1):733s–736s. zhang, p., whistler, r.l., bemiller, j.n. & hamake, b.r. 2005. banana starch: production, physicochemical properties, and digestibility : a review. j carbohy polymers. 59: 443– 458. 1(2)46-57 !!!!!!!!!!!!!!!!!!contact&:&nur&hidayatul&alami&&&&&&&nh_daya@yahoo.com! ©!international!journal!of!applied!biology! 46! abstract& biofertilizers! are! commonly! defined! as! microbial! inoculants! which! are! capable!of!mobilizing!important!nutrient!in!the!soil!from!nonaavailable!to! available!form!for!the!plants!through!their!biological!processes.!hence!to! increase! the!productivity!of!the!soil,! the!use!of!biofertilizer! is!necessary.! candida! yeast! as! a! phosphate! solubilizer! and! decomposer.! azotobacter! bacteria!as!a!nitrogenafixing!bacteria!and!bacillus!as!a!phosphate!solubilizer.! the!combination!of!these!microorganisms!as!biofertilizer!needs!to!be!tested! its! effect! against! mustard! (brassica' juncea! l.)! plant! growth.the! results! showed!that!the!combination!of!bacillus!candida!obtained!the!highest!yield! among!other!biofertilizer!treatments!with!a!value!of!13.6!cm!at!plant!height,! 4.96!grams!in!wet!weight!and!2.29!grams!in!dry!weight.!the!combination!of! azotobacter!candida!showed!the!highest!result!on!the!parameter!of!width! and!number!of!leaves!with!the!values!of!20.3!cm!and!5.3.!combination!of! azotobacter!bacillus!candida!showed!the!highest!yield!on!the!root!length! parameter!with!a!value!of!5.0!cm.!the!addition!of!biofertilizer!is!expected!to! reduce!pollution!on!the!soil!due!to!residues!produced!by!chemical!fertilizers.! issn!:!2580a2410! eissn!:!2580a2119! ! ! ! effect&of&yeast&based&biofertilizer&combined&with&bacteria&on& mustard&plant&growth& ! nur&hidayatul&alami,&silviya&amaliya&trisna&ayu,&nengah&dwianita&kuswytasari,&enny& zulaika,&and&maya&shovitri& & department!of!biology,!institut!teknologi!sepuluh!nopember,!surabaya,!indonesia! ! ! & & & & & & & & & & & & && & & introduction& biofertilizer!application!to!overcome!the!problem!of!land!degradation!due!to!the!use! of!chemical!fertilizers!has!been!the!focus!of!the!study!until!the!last!few!decades.!compared! with!chemical!fertilizers,!biofertilizers!are!shown!to!have!a!better!ability!to!increase!crop! productivity! because! biofertilizers! are! enriched! by! microorganisms! and! plant! growth! promoters! with! a! beneficial! consortium! of! microorganisms! (paszt! et' al.,! 2015).! the! biofertilizeraconsortia!microorganisms!may!include!groups!of!fungi,!bacteria,!or!protozoans! (cavalierasmith,!1998;!lucy!et'al.,!2004;!smith!and!read,!2008;!vessey,!2003)!which,!when! applied,!can!increase!the!availability!of!nutrients!from!biological!activity!and!can!improve!soil! quality.!biofertilizer!is!able!to!provide!elements!of!n,!p!and!k!as!well!as!nutrients!for!plants! that!can!affect!the!growth!and!productivity!of!plants!!(agamy!et'al.,!2013).! study!on!the!production!of!biofertilizers!using!bacteria!and!yeast!has!been!widely! used,!wani!et'al.!(2013)!state!that!azotobacter!bacteria!are!able!to!fix!10!mg!of!nitrogen!from! &&&&&&&&open&access& & & &&&&&&&&&&international&journal&of&applied&biology& keyword! azotobacter' bacillus' biofertilizer' candida' mustard'plant article&history! received!21!november!2017! !accepted!29!december!2017!! international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ! international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international'journal'of'applied'biology,'1(2),'2017' ! 47! oneatenth!of!carbon!sources!conducted!in!vitro!(amprayn!et'al.,!2012).!this!study!uses!a! consortium!of!candida!yeast!and!azotobacter!and!bacillus!bacteria.!some!candida!species! are! able! to! solubilize! phosphate! and! degrade! organic! compounds,! azotobacter! is! a! siderophore!bacteria!and!capable!of!fixing!nitrogen,!and!bacillus!is!able!to!dissolve!phosphate! in!the!soil!(xiao!et'al.,!2013;!new!et'al.,!2013;!fleet,!1992;!ali!and!vidhale,!2013;!maheswar! and!sathiyavani,!2012).!biofertilizers!use!the!molasses!carrier!as!the!main!carbon!source!for! the! growth! of! the! isolates.! molasses! were! selected! because! of! their! effectiveness! and! efficiency!as!carriers!for!controlling!microbial!growth.! plants!that!have!the!potential!to!be!used!as!research!object! is!the!mustard!plant! (brassica'juncea'l.).!mustard!is!an!annual!plant!that!can!grow!in!lowland!to!upland!(devi!et' al.,!2017),!able!to!grow!and!adapt!well!to!almost!all!soil!types,!have!a!short!harvest!age!(35! days),!and!leaves!are!used!by!indonesian!people!as!vegetables!because!of!high!nutritional! value.!based!on!the!ability!of!microorganisms,!it!is!necessary!to!study!more!about!the!effect! of!the!combination!!of!!yeast!and!bacteria!as!biofertilizer!on!mustard!plant!growth.! & materials&and&methods& isolates& isolates!used!in!this!research!were!candida!w3.8!and!candida!g3.2!and!isolates!of! azotobacter!a10!and!bacillus!spp.,!collection!of!microbiology!and!biotechnology!laboratory,! biology!department,!institut!teknologi!sepuluh!nopember!surabaya!(its).! & preparation&of&microbial&suspension& measurements!of!yeast!and!bacteria!growth!were!done!by!adding!each!culture!in! 0.85%! (w/v)! of! physiological! solution,! consisting! of! nacl! and! aquades.! absorbance! was! measured!using!a!uvavis!spectrophotometer!with!od!0.5!at!λ!600!for!yeast!and!od!0.5!at!λ! 620!for!bacteria.!the!value!of!od!is!based!on!the!approximate!number!of!yeast!and!bacterial! cell!densities!(about!107!cfu/ml).! ! growth&of&&yeast&and&bacteria&in&molasses&medium& about! 10%! of! microbial! suspension! from! the! previous! step! then! inoculated! into! molasses!production!medium!and!czapekdox.!molasses!is!a!carrier!medium!which!has!been! known!as!microbial!growth!substrate.!czapekdox!broth!(g!/!l)!consisting!of!sodium!nitrate!(3! g);! dipotassium! phosphate! (1! g);! magnesium! sulphate! (0.5! g);! potassium! chloride! (0.5);! ferrous!sulphate!(feso4),!(0.01!g);!aquadest!(h2o)!(1000!ml).!the!volumes!of!the!inoculum! was!added!to!the!carrier!medium!based!on!the!different!treatment!groups!(table!1.)! ! table&1.&&volumes&of&inoculum& treatment! suspension!volume! treatment!1!!!:! 5!ml!of!candida!w2.8!+!5!ml!of!candida!g3.2!+!90!ml!of!molasses!and!czapek! dox! treatment!2!!!:! 10!ml!of!azotobacter'a10!+!90!ml!of!molasses!and!czapek!dox! treatment!3!!!:!!!! 10!ml!of!bacillus!spp.!+!90!ml!of!molasses!and!czapek!dox! treatment!4!!!:! 3,3!ml!of!candida!w2.8!+!3,3!ml!of!candida!g3.2!+!3,3!ml!of!azotobacter'a10!+! 90!ml!of!molasses!and!czapek!dox! treatment!5!!!:! 3,3!ml!of!candida!w2.8!+!3,3!ml!of!candida'g3.2!+!3,3!ml!of!bacillus!spp.!+!90! ml!of!molasses!and!czapek!dox! treatment!6!!!:! 2,5!ml!of!candida!w2.8!+!2,5!ml!of!candida!g3.2!+!2,5!ml!of!azotobacter'a10!+! 2,5!ml!of!bacillus!spp.!+!90!ml!of!molasses!and!czapek!dox! international'journal'of'applied'biology,'1(2),'2017' ! 48! each! combination! of! biofertilizer! was! homogenized! on! a! rotary! shaker! for! 7! days! (anubrata!and!rajendra,!2014).!measurement!of!growth!was!done!daily!with!total!plate! count! method! to! obtain! maximum! growth! day.! the! best! incubation! time! to! be! used! for! biofertilizer!production!was!based!on!the!total!number!of!inoculums!of!109!cfu/ml!(noel! et.al.,'2016)!in!each!treatment!group.! ! planting&and&maintenance&of&mustard& mustard!seeds!were!soaked!with!water!for!24!hours,!then!seeds!that!were!submerged! in!water!were!selected.!seeds!were!allowed!to!grow!in!seedling!medium!at!least!until!two! leaves!were!formed!or!14!days!after!planting.!application!of!biofertilizer!was!done!in!2!stages,! the!first!given!at!14!days!before!the!seed!was!transferred!to!planting!medium,!and!the!second! at!14!days!after!planting.!biofertilizer!is!composted!by!watering!250!ml!into!polybags.!plants! are!grown!for!4!weeks.!measurements!of!growth!include!plant!height,!leaf!area,!number!of! leaves,!root!length!and!biomass!(dry!weight).! ! plant&height&measurement& plant! height! is! measured! from! ground! level! to! the! highest! leaf.! plant! height! measurement!was!done!once!a!week!for!5!weeks.! ! measurement&of&leaf&area& the! leaf! area! is! calculated! by! millimeter! paper! method! by! putting! the! leaves! on! millimeter!paper!and!leaf!pattern!followed.!the!leaf!area!is!estimated!based!on!the!number! of!squares!contained!in!the!leaf!pattern.!the!measurement!of!leaf!area!is!done!once!a!week! for!5!weeks.! ! number&of&leaf&calculation& the! number! of! leaf! calculation! was! done! on! leaves! that! had! developed! perfectly.! counting!the!number!of!leaves!were!done!once!a!week!for!5!weeks.! ! root&length&measurement& the!measurement!of!root!length!with!destruction!method!was!done!by!removing!the! whole!plant!and!measuring!the!root!length!from!root!base!to!root!tip!by!using!ruler.!root! length!measurements!were!made!at!harvest!time.! & biomass&calculation& biomass!is!obtained!from!the!dry!weight!of!the!plant.!before!weighing!the!plant!is! cleaned!with!water!and!dried!wind.!the!dry!weight!of!the!plant!was!carried!out!by!drying!at! 60!°!c!for!4!days,!then!was!weighed!using!analytical!balance.!biomass!measurements!were! made!during!harvest!time.! ! research&design& the!design!of!this!study!used!a!completely!randomized!design!with!6!treatments!for!5! weeks!and!3!repetitions.!research!design!are!shown!in!the!figure!1.! ! ! ! ! international'journal'of'applied'biology,'1(2),'2017' ! 49! & & & figure&1.&research&design,&cont1:&negative&control&repetition&1;&c1:&biofertilizer&of&candida& repetition&1;&con+&2:&positive&control&repetition&2;&a1:biofertilizer&of&azotobacter& repetition& 1;& ca2:& biofertilizer& of& candida& and& azotobacter& repetition& 2;& cab3:& biofertilizer&of&candida,&azotobacter,&and&bacillus&repetition&3;&cont2:&negative& control&repetition&2;&cb1:&biofertilizer&of&candida&and&bacillus&repetition&1;&con+&1:& positive& control& repetition& 1;& a2:& biofertilizer& of& azotobacter& repetition& 2;& b1:& biofertilizer& of& bacillus& repetition& 1;& cb3:& biofertilizer& of& candida& and& bacillus& repetition&3;&c3:&biofertilizer&of&candida&repetition&3;&b3:&biofertilizer&of&bacillus& repetition& 3;& cb2:& biofertilizer& of& candida& and& bacillus& repetition& 2;& cab2:& biofertilizer& of& candida,& azotobacter,& and& bacillus& repetition& 2;& con+3:& positive& control& repetition& 3;& b2:& biofertilizer& of& bacillus& repetition& 2;& cont3:& negative& control&repetition&3;&ca3:&biofertilizer&of&candida&and&azotobacter&repetition&3;& ca1:&biofertilizer&of&candida&and&azotobacter&repetition&1;&cab1:&biofertilizer&of& candida,&azotobacter,&and&bacillus&repetition&1;&a3:&&biofertilizer&of&azotobacter& repetition&3;&c2:&biofertilizer&of&candida&repetition&2.& ! data&analysis& the!data!analysis!used!anovaaone!way!for!combination!of!yeast!and!bacteria!as! biofertilizer!against!plant!growth.! !! results&and&discussion& the!preparation!of!biofertilizer!should!consider!the!substance!of!carrier!media!and! biological!agents.!carrier!media!should!also!contain!an!important!component!to!support!the! viability!and!growth!of!inoculated!microbes.!one!of!the!carrier!media!for!the!biofertilizer!is! molasses!which!have!been!known!as!the!microbial!growth!substrate.!molasses!were!selected! because!of!their!effectiveness!and!efficiency!as!carriers!for!the!growth!of!biofertilizer!agent.! molasses!can!be!used!as!an!alternative!substrate!because!it!contains!complex!nutrients!that! was!taken!by!microbes!for!metabolism!process,!it!contains!not!less!than!46%!total!sugars! (duraisam!et'al.,!2017),!including!a!water!content,!nitrogen!and!minerals!that!are!used!for! microbial!growth.!& molasses! used! in! this! research! is! a! source! of! carbon! (c)! for! the! microorganism,! molasses!can!be!combined!with!the!medium!czapekdox!broth,!with!a!composition!of!sodium! nitrate! (nano3)! as! a! source! of! protein,! dipotassium! phosphate! (kh2po4),! magnesium! sulphate!(mgso4.7h2o),!potassium!chloride!(kcl),!and!ferrous!sulphate!(feso4)!which!is!a! source!of!salts!and!minerals!for!the!growth!of!biofertilizer!agent.!carrier!media!with!the! cona!1! c!1! con+!2! a!1! cab!3!! cona!2! cb!1! con+!1! b!1! cb!3! c!3! b!3! c!2!a!3!cab!1!ca!1! cona!3!b!2!con+!3!cab!2! ca!3! cb!2! a!2! ca!2! international'journal'of'applied'biology,'1(2),'2017' ! 50! content!of!organic!and!mineral!are!expected!to!make!the!microorganisms!can!compete!with! microorganisms!in!the!soil.!thus!able!to!dominate!around!plant!roots.& the!biofertilizer!should!be!contain!the!minimum!population!of!the!total!bacteria!of!≥! 109!cfu!/!ml!or!9!log!cfu!/!ml!for!the!positive!influence!of!microbes!in!the!biofertilizer!(masso! et'al.,!2015).!therefore,!the!consortium!formulation!of!biological!agents!and!carrier!media!is! necessary!to!enhance!plant!growth!and!productivity.& the!calculation!of!microorganism!was!performed!before!the!biofertilizer!was!applied! to! mustard! for! 7! days.! observations! started! from! day! 0! of! inoculation! to! day! 7! of! the! incubation!period.!the!microbial!growth!results!are!shown!in!figure!2.& & & & & & & & & & & & & & figure&2.&growth&of&yeast&and&bacteria&consortium&on&molasses&medium& & figure!1!shows!that!the!growth!increased!gradually!until!the!7!days!of!incubation!time.! this!suggests!that!yeast!and!bacterial!of!biofertilizer!was!able!to!increase!in!numbers!during! the! application! of! the! soil! so! that! the! microbes! in! the! biofertilizer! potentially! increasing! nutrient!uptake!by!plants!.! the!increase!in!the!population!of!consortium!in!a!test!of!viability!indicates!that!the! agent!biofertilizer!used!consisting!of!a!single!colony!which!include!yeast!candida,!bacteria! azotobacter!and!bacillus,!a!consortium!of!the!yeast!candida!and!azotobacter,!yeast!candida! and!bacillus,!as!well!as!a!consortium!of!yeast!candida,!azotobacter!and!bacillus!able!to!grow.! on!day!0!shows!an!indication!of!metabolic!activity!performed!by!all!inoculants.!yeasts!and! bacteria! utilize! nutrients! in! the! medium! for! growth.! according! to! (brock,! 1991),! in! the! adaptation!phase,!an!increase!in!cell!size,!the!cell!does!not!divide!or!slightly!division.!this! condition,! characterized! by! an! increase! in! the! macromolecular! components,! metabolic! activity,!and!the!number!of!cells!that!slowly!began!to!increase.! in!all!treatments!on!the!first!day!until!the!seventh!day!there!is!an!increase!in!the! number!of!cells.!this!is!because!yeast!and!bacteria!are!in!the!logarithmic!phase.!cells!will! continue! to! multiply! by! utilizing! molasses! as! the! main! source! of! nutrients.! according! to! (paulton,! 1991)! in! the! exponential! or! logarithmic! phase,! the! period! and! volume! of! cells! increase!and!and!the!speed!of!increase!can!be!expressed!with!exponential!function.! n u m b er &o f& c o lo n y& (l o g& c fu /m l) day cab cb ca b a c international'journal'of'applied'biology,'1(2),'2017' ! 51! the!results!of!plant!height!measurements!of!the!mustard!are!shown!in!figure!3.!in! figure!3!it!shows!that!plant!height!increased!every!week!during!the!planting!time!up!to!28! days!after!planting.the!increasing!of!plant!height!can!be!affected!by!the!availability!of!various! nutrients!on!the!soil!that!has!been!provided!by!biofertilizer.!oneaway!anova!results!of!the! week!4!(harvest!time)!show!that!probability!value!is!less!than!0.05!ie!0.001,!so!the!biofertilizer! gives!effect!to!the!height!of!the!mustard!so!that!tukey!test!was!done!at!95%!(α!=!0,05)!using! minitab!software.!tukey!test!results!are!shown!in!table!2.& ! figure&3.&result&of&plant&height& & table&2.&height&of&mustard&plants&at&harvest&time& treatment& plant&high&(cm)& positive!control! 16,167a! bacillus'candida' 13,600ab! bacillus' 12,467abc! azotobacter' 12,100abc! azotobacter'bacillus'candida' 11,833bc! azotobacter'candida' 11,400bc! candida' 11,000bc! negative!control! 8,833c! *figures!followed!by!different!letters!show!significant!differences!based!on!the!tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! based!on!table!2!it!can!be!seen!that!the!influence!of!biofertilizer!on!the!height!of!the! mustard! in! bacillus! candida! treatment! has! the! highest! effect! when! compared! with! the! treatment! of! the! addition! of! another! biofertilizer.! bacillus! is! able! to! synthesize! growth! hormone!iaa,!gibberellins!and!cytokinins!(park!et'al.,!2017),!and!dissolves!phosphates!that! are!not!available!in!the!soil!by!the!mechanism!of!organic!acid!secretion.!phosphate!is!an! important!macronutrient!for!plants!for!high!plant!growth!(wang!et'al.,!2011)!the!ability!of! bacillus!is!combined!with!candida!which!has!the!potential!to!dissolve!organic!and!inorganic! phosphate,!acting!as!a!bioaactivator!for!the!breakdown!of!organic!matter!that!will!contribute! nutrient!supply!for!soil!to!plant!height!.! ! p la n t& h ei gh t& (c m ) treatment week!1 week!2 week!3 week!4 international'journal'of'applied'biology,'1(2),'2017' ! 52! plant!height!in!positive!control!is!highest!because!the!positive!control!(npk!fertilizer)! contains!nutrients!and!minerals!that!can!be!utilized!directly!by!plants,!so!the!plant!can!take! the!nutrition!quickly.!mustard!plants!have!a!short!harvest!age,!thus!the!available!uptake!of! nutrients!is!also!fast.!but!the!continued!use!of!chemical!fertilizers!can!leave!residues!thereby! destroying!soil!and!the!environment.!treatment!of!negative!control!has!the!lowest!effect!on! the!high!of!mustard.!this!is!because!the!negative!controls!in!this!study!only!use!soil!as!a! planting!medium!without!any!addition!of!fertilizers!in!which!contain!more!nutrients!needed! for!plant!growth!height,!so!the!roots!will!only!absorb!the!macro!and!micro!essential!elements! available!in!the!soil.! the!results!of!the!measurements!of!the!leaf!number!and!leaves!area!are!shown!in! figure.!4!and!5.!the!leaf!number!and!leaves!area!has!increased!each!week.!the!result!of!onea way!anova!showed!that!the!probability!value!of!the!area!and!the!number!of!leaves!was!less! than!0.05!ie!0.003!and!0.000!respectively!so!that!the!biofertilizer!had!an!effect!on!the!leaf! area! and! number,! so! that! the! tukey! test! was! done! at! 95%! (α! =! 0.05)! by! using! minitab! software.!tukey!test!results!are!shown!in!table!3.! & figure&4.&result&of&leaf&area& & & & & & & & & & & & & & & & figure&5.&result&of&leaf&number& le af &a re a& (m m 2 ) treatment week!1 week!2 week!3 week!4 n u m b er &o f& le af treatment week!1 week!2 week!3 week!4 international'journal'of'applied'biology,'1(2),'2017' ! 53! table&3.&leaf&area&and&number&of&leaf&at&harvest&time& treatment! leaf&area!(cm)! number&of&leaf! positive&control! 25,100a! 6a! azotobacter'candida' 20,300ab! 5,3b! bacillus'candida' 18,867ab! 5,0b! azotobacter'bacillus'candida' 15,967abc! 5,0b! candida' 14,333!abc! 5,0b! azotobacter' 11,533bc! 5,0b! bacillus' 9,867bc! 5,0b! negative&control! 5,167c! 5,0b! *figures!followed!by!different!letters!show!significant!differences!based!on!the!tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! based!on!table!3!it!can!be!seen!on!the!biofertilizer!treatment!of!azotobacter!and! candida!consortium!showed!the!highest!yield.!the!most!needed!nutrient!in!the!leaf!area!and! the! number! of! leaves! is! the! nitrogen! (ng’etich,! 2013).! the! potential! of! azotobacter! in! nitrogen!fixation!is!combined!with!candida!in!dissolving!phosphate!into!the!forms!available! for!plants.!this!is!consistent!with!(plaster,!2003)!!that!nitrogen!is!more!necessary!in!support! of!vegetative!growth!than!generative!and!important!for!vegetable!crops!consumed!by!the! canopy.! the!positive!control!has!the!highest!result,!this!is!the!same!as!the!previous!parameter! indicating! positive! control! treatment! has! the! highest! value.! this! is! because! the! chemical! fertilizer!(npk!fertilizer)!provided!an!inorganic!nitrogen!element!that!is!ready!for!use!for!leaf! area!and!the!number!of!leaves.!in!the!negative!control!showed!the!lowest!result.!this!is!in! accordance!with!soil!nitrogen!content!test!results!indicating!that!the!nitrogen!content!in!the! soil!with!the!addition!of!npk!fertilizer!amounted!to!0.21%,!in!the!addition!of!biofertilizer!of! 0.14%!and!the!media!of!garden!soil!only!0.02%.! root!length!measurement!results!are!shown!in!figure!6.!based!on!figure!6!it!can!be! seen! that! the! positive! control! effect! on! long! root! growth! with! the! highest! value! when! compared!with!treatment!of!biofertilizer!and!negative!control.!oneaway!anova!results!show! that!the!probability!value!is!less!than!0.05,!so!the!biofertilizer!gives!effect!to!the!root!length! of!the!mustard!so!that!the!tukey!test!was!done!at!95%!(α!=!0.05!)!by!using!minitab!software.! tukey!test!results!are!shown!in!table!4.! ! ! ! ! & & & & & & & & & figure&6.&result&of&root&length& le n gt h &r o o t& (c m ) tratment international'journal'of'applied'biology,'1(2),'2017' ! 54! table&4.&root&length&at&harvest&time& treatment& root&length&(cm)& positive!control! 7,333a! azotobacter'bacillus'candida' 5,000ab! candida' 5,000ab! bacillus'candida' 4,867ab! azotobacter'candida' 4,233ab! bacillus' 4,000ab! azotobacter' 3,333b! negative!control! 2,933b! *figures!followed!by!different!letters!show!significant!differences!based!on!the!tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! based!on!table!4!it!can!be!seen!on!the!biofertilizer!treatment!of!azotobacter!bacillus! candida!consortium!and!single!culture!candida!showed!the!highest!result.!candida!is!able!to! decompose! the! organic! compounds! in! the! soil! and! phosphate! solubilizer,! azotobacter! potential! for! nitrogen! fixation! and! also! have! siderophores,! are! complexing! or! specific! compounds!of!chelating!fe!3+!or!iron!to!bind!elemental!iron!in!the!rhizosphere,!so!it!is!not! available!for!the!development!of!pathogenic!microbes.!bacillus!sp.,!potential!as!a!phosphate! solubilizer!and!root!biological!controller.!it!is!also!appropriate!with!the!test!results!of!the! phosphate!(p2o5)!shows!that!the!content!of!phosphate!in!the!soil!with!the!addition!of!npk! fertilizer!is!0.48%,!biofertilizer!is!0.41%!and!garden!soil!media!is!0.31%.! the!dry!weight!of!mustard!plants!in!this!study!is!indicated!by!weighing!the!total!dry! weight!of!the!harvested!crops!(roots,!stems!and!leaves).!the!results!of!the!measurement!of! the! dry! weight! of! plants! can! be! seen! in! figure! 7.! based! on! the! oneaway! anova! test,! biofertilizer!has!an!effect!on!the!dry!weight!of!the!plant.!this!is!indicated!by!the!value!of!pa value!(p!<0.05)!for!dry!weight!is!0.002.!then!proceed!with!tukey!test!to!know!which!treatment! has!a!significant!influence!on!the!treatments!that!have!been!done.!the!average!result!of!dry! weight!measurement!can!be!seen!in!table!5.! & figure&7.&result&of&biomass& & & & & & b io m as s& (g ra m ) treatment international'journal'of'applied'biology,'1(2),'2017' ! 55! table&5.&biomass&at&harvest&time& treatment& dry&weight&(gram)& positive!control! 3,573a! bacillus'candida' 2,290ab! azotobacter'candida' 2,060b! candida' 1,810b! bacillus' 1,713b! azotobacter'bacillus'candida' 1,766b! azotobacter' 1,463b! negative!control! 1,433b! *figures!followed!by!different!letters!show!significant!differences!based!on!the!tukey!test!at! the!95%!confidence!level!(α!=!0.05).! ! table!5!shows!that!treatment!of!candida'bacillus!consortium!has!the!highest!value!on! the! dry! weight! parameters! when! compared! with! other! biofertilizer! treatment! and! not! significantly!different!from!the!positive!control!treatment!with!the!addition!of!npk.!this!is! because!bacillus!has!the!potential!as!a!phosphate!solubilizer!by!releasing!a!number!of!organic! acids!and!candida!has!the!potential!to!decompose!organic!matter!in!the!soil.! dry!weight!is!also!associated!with!carbon!compounds!that!build!plant!organs.!this! correlates!with!the!content!of!c!in!soil.!the!result!of!the!research!is!in!accordance!with!the! amount!of!soil!c!content!that! indicating!the!content!of!carbon!element!on!soil!with!the! addition!of!npk!fertilizer!is!2.05%,!the!addition!of!biofertilizer!is!1,98%!and!the!garden!soil!is! 1,62%.! & conclusions& the!results!showed!that!all!combination!of!yeast!and!bacteria!as!biofertilizer!effect!on! mustard! growth! when! compared! with! the! treatment! without! biofertilizer.! candida! and! bacillus!consortium!has!the!highest!value!of!13,6!cm!at!plant!height.!the!azotobacter!candida! consortium!on!the!parameters!of!the!leaf!area!and!number!of!leaves!showed!the!highest! yields!with!values!of!20.3!cm!and!5.3,!respectively,!and!the!azotobacter!bacillus!candida! consortium!showed!the!highest!value!of!5.0!cm!on!the!root!length!parameter.!the!bacillus! candida!consortium!showed!!the!highest!value!of!2.29!grams!in!dry!weight.! & acknowledgment&& this!work!was!conducted!as!part!of!“superior!and!commercial!biofertilizer!of!marine! yeast!and!bacteria”.!this!research!was!supported!by!grants!from!pemula!research!2017! awarded!by!its!local!fund!2017.!! & references& agamy,!r.,!hashem,!m.!&!alamri,!s.!2013.!effect!of!soil!amendment!with!yeasts!as!bioa fertilizers!on!the!growth!and!productivity!of!sugar!beet.'african'journal'of'agricultural' research,!8(1).! ali,s.s! &! vidhale,! n.n.! 2013.! bacterial! siderophore! and! their! application:! a! review.! int.j.curr.microbiol.app.sci,!2(12):303a312.! international'journal'of'applied'biology,'1(2),'2017' ! 56! amprayn,!k.o.,!rose,!m.t.,!kecskes,!m.,!pereg,!l.,!nguyen,!h.t.!&!kennedy,!i.r.!2012.!plant! growth! promoting! characteristics! of! soil! yeast! 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strawberry!cultivars.!acta'sci.'pol.'hortorum'cultus,'14(6):83a96.! paulton,!r.j.l.!1991.!the!bacterial!growth!curve.!j'biol'educ,'25:92–93.! plaster,!e.!j.!2003.!soil!science!and!mangement.!delmar!learning!inc.!4th!ed!united!states.! smith,!s.e.!&!read,!d.j.!2008.!mycorrhizal!symbiosis,!3rd!ed.!new!york,!london,!burlington,! san!diego:!elsevier!and!academic.! international'journal'of'applied'biology,'1(2),'2017' ! 57! to&cite&this&article:&& alami,! n.h.,! ayu,! s.a.t.,! kuswytasari,! n.d.,! zulaika,! e! &! shovitri,! m.! 2017.! effect! of! yeast! based! biofertilizer!combined!with!bacteria!on!mustard!plant!growth.!international'journal'of'applied'biology.! 1(2):46a57.! & vessey,!j.k.!2003.!plant!growth!promoting!rhizobacteria!as!biofertilizers.!plant'soil,!255:!571– 86.! wang,!x.,!pan,!q.,!chen,!f.,!yan,!x.!&!liao,!h.!2011.!effects!of!coainoculation!with!arbuscular! mycorrhizal!fungi!and!rhizobia!on!soybean!growth!as!related!to!root!architecture!and! availability!of!!n!and!!p.!mycorrhiza.'21:173–181.! wani,!s.a.,!subhash,!c.!&!tahir,!a.!!2013.!potential!use!of!azotobacter'chroococcum!in!crop! production.!current'agriculture'research'journal,!1(1).! xiao,!c.,!chi,!r.,!pan,!x.,!liu,!f.!&!he,!j.!2013.!rock!phosphate!solubilization!by!four!yeast! strains.'annals'of'microbiology,!63:173a178! ! ! ! ! ! ! ! ! 2(2)27-32 contact : bikram nepali bikramn25@gmail.com © international journal of applied biology 27 abstract pseudomonas fluorescens not only enhances the plant growth but also controls the fungal pathogens by production of anti fungal metabolites. the objective of this experiment was to identify p. fluorescens using different biochemical tests. this research was carried out in plant pathology laboratory at agriculture and forestry university (afu), rampur, chitwan, nepal. the result of this experiment indicated that p. fluorescens gave positive result for catalase test, gelatin liquefaction, fluorescent pigment and oxidase test but negative result for starch hydrolysis test. the colony of p. fluorescens was maximum in maize seed than that of rice seed after two hours of inoculation, whereas higher number of colony was found in rice seed than that of maize seed after twenty four hours of seed inoculation. this findings is useful for identifying colony of p. fluorescens per seed which is necessary for better seedling growth and effective biological control of pathogens. issn : 2580-2410 eissn : 2580-2119 identification of pseudomonas fluorescens using different biochemical tests bikram nepali1, sabin bhattarai1 & jiban shrestha2 1agriculture and forestry university, rampur, chitwan, nepal. 2nepal agricultural research council, national commercial agriculture research program, pakhribas, dhankuta, nepal introduction pseudomonas species encompasses a group of common rod shape, gram negative, one or more polar flagella providing mortality, aerobic in nature, non-spore farming, positive catalase test, positive oxidase test, nonpathogenic saprophytes that colonize soil, water and plant surface environments. it also secretes pyoverdine a fluorescent yellow green siderophore under iron limiting condition. pseudomonas fluorescent also produces additional type of siderophore such as thioquindobactin (scales et al., 2014). it is an obligate aerobe, except for some strains that can utilize no3 as an electron acceptor in place of o2. it is motile by means of multiple polar flagella. pseudomonas fluorescens has simple nutritional requirements and grows well in mineral salts media supplemented with any of a large number of carbon sources. p. fluorescens is saprophytic fluorescent micro-organism, which are very common in soil around the rhizosphere of various plant species that could improve the plant vigour by incorporating free nutrient and plant growth hormone. in case of cereal crop, it help in open access international journal of applied biology keyword inoculation, rice and maize seed, serial dilution, pseudomonas fluorescens article history received 23 october 2018 accepted 23 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 28 increasing crop productivity by reducing soil borne disease and stimulating effect on plant growth. a free living plant growth promoting rhizobacteria (pgpr) pseudomonas fluorescens are able to colonize around plant root and incorporate free atmospheric molecule into plant absorbable nutrient form along with improved plant growth and crop yield. iron chelation, antibiotic production, enhanced nutrient uptake and seedling emergence promotion and by plant phytohormone production. siderophore are iron chelating compounds secreted by bacteria on or around the roots that affect the growth of the plants rhizobacteria. the pgpr, being more potent chelators, starve the deleterious rhizobacteria of their iron nutrient, thus protect the plants from the harmful effects of drb, resulting in better growth and yield (sakthivel et al., 2009). microbial inoculation in seed of crop increases the root biomass production and more colonization of beneficial micro-organism, which are interdependent with these considerations. this micro-organism is natural bio-controllers and improves soil fertility through its own effective bio fertilizer potential pgpr stains. new research found that p. fluorescens predominant inhabitants of soil and aquatic environments. it plays in the very important role in the purification of pollutants of bioremediation from large chemical industry and secondary metabolites decay material and bio-control some harmful pathogen against crop. global food security was threatened by some crop disease and degrading soil fertility. excess use of chemical fertilizer aiming increase crop yield reporting declining soil fertility lead to desert some place in the world. rootassociated soil bacteria would be the potential solution by improving soil condition and promoting defensive mechanism against various pathogens through improving plant growth mechanism. the objective of this research was to identify pseudomonas fluorescens using different biochemical tests and to identify the total number of its colony present in different seed at different time of inoculation. materials and methods experimental location and preparation this experiment was conducted in plant pathology laboratory at agriculture and forestry university (afu), rampur, chitwan, nepal in october 03, 2018. the liquid composition of pseudomonas fluorescens were bring form the agri-care and live-care pvt. ltd., which was examined through different bio-chemical test to assure the liquid component consist of bacteria pseudomonas fluorescens. morphological characterization pure cultures of the selected isolates were streaked on king’s b agar petri plates separately for colony development. biochemical tests for p. fluorescens for the identification of p. fluorescens, certain biochemical tests were conducted according to bergey’s manual for determinative bacteriology. starch hydrolysis filter paper was dipped in a dry old culture suspension and was placed on petri dishes containing starch agar medium and incubated for two days. the plates were than flooded international journal of applied biology, 2(2), 2018 29 with one per cent iodine solution. a colorless halo around the growth and blue color in the rest of the plates showed utilization of starch by the microorganism. gelatin liquefaction filter paper discs were dipped in a day old culture suspension and were placed on petri dishes containing gelatin nutrient agar medium. the petri dishes were incubated at 300c for two days and then flooded with 12.5% hgcl2 solution. the development of yellow halo around the growth indicates utilization of gelatin. fluorescent pigment the test tubes containing sterilized kings b medium were inoculated with the isolate of pseudomonas sp. incubated for five days and observed. yellowish green fluorescent pigment observed under uv light (365 nm) indicated positive results. catalase test capillary tubes (1mm outer isolated by 67mm in length were placed in a 50-ml beaker containing about 10 ml of 3% h2o2. a positive result is the rapid evolution of oxygen by bubbling. this phenomena is due to the breakdown of hydrogen peroxide h2o2 in to water and oxygen. oxidase test saturate whatman no.1 filter papers with a 1% aqueous solution of diemthyl-pphenlyenediamine oxalate. development of red color within a 10 sec indicates a positive reaction. inoculation of pseudomonas into a seed and serial dilution the seed of maize (variety: rampur composite) and rice (variety: masuli) were inoculated with pseudomonas fluorescens at 2.5 ml kg-1 seed in 5% sugar solution. let the seed for two hours soaked and secondly twenty four hours. single seed was dipped into test tube containing a 10 ml of distilled water and shake the tube for one minute. one milliliter of diluted solution from first tube is transfer to second tube containing nine milliliter of distilled water. the process was continue up to six tube and solution made 1/1000000 diluted. material for kings b medium proteose peptone (20g), k2hpo4 (1.5g), mgso4.7h2o4 (1.5g), agar (15g), glycerol (1.5 ml) and water (1 liter) were used in this experiment. results and discussion the entire test showed the presence of pseudomonas fluorescens in the liquid. when the uv is ray was passed into p. fluorescens, a shiny bluish yellow green fluorescent pigment was observed (figure 1). much of the impetus in the study of “unusual” gram-negative bacilli from clinical sources, was derived from king in atlanta. the production of a fluorescent pigment on king’s medium b was characteristic of most isolates of p. putida, p. fluorescens and p. aeruginosa. p. fluorescens perform the best typical gluconate (almost always positive), urea (positive after international journal of applied biology, 2(2), 2018 30 2-3 days), casein (positive), and gelatin (positive) reactions at room temperature (king and phillips, 1978). non-fermenting, catalase-positive gram-negative bacilli that grow on nutrient agar are often isolated in clinical laboratories. biochemical techniques appropriate to a typical clinical microbiology laboratory, and for the most part described in cowan and steel's manual for the identification of medical bacteria. on the basis of the oxidation-fermentation test and further tests for oxidase activity and motility, the organisms were allocated to one of several subgroups on the model of cowan and steel. however, it was found that the production of alkali in the oxidation-fermentation test was a very useful characteristic and had therefore differentiated it from the “no reaction" group of cowan and steel (king and phillips, 1978). solubilization of mineral phosphates and mobilization of other essential nutrients by pgpr also helps in growth improvement of plants. the cell wall of plant cells are mainly composed of cellulose, which was embedded in an amorphous polysaccharide matrix of hemicelluloses, pectin and some glycols and proteins (meyer and abdallah, 1978). also oxidized inositols served as non cellulosic cell wall components. table 1. biochemical characteristics of pseudomonas fluorescens no. biochemical tests identification of p. fluorescens 1 starch hydrolysis negative 2 gelatin liquefaction positive 3 fluorescent pigment positive 4 catalase test positive 5 oxidase test positive pseudomonas fluorescens gave positive result for catalase test, gelatin liquefaction, fluorescent pigment and oxidase test but negative result for starch hydrolysis test. figure 1. fluorescent test under uv ray. colony of p. fluorescens per seed after serial dilution to 106 of individual seed of maize and rice, which was let growth for one day in kings b medium (six media) at 350c. colony of p. fluorescens was found to be international journal of applied biology, 2(2), 2018 31 higher after two hours of inoculation than that of twenty four hours. similarly, the higher colony was found in maize seed than rice after two hours and vice-versa after twenty fours respectively. it was count on an average at two hours of inoculation was 2.17*108(cfu/ml/seed) for rice and 1.81*108 (cfu/ml/seed) for maize, whereas colony was count as 1.35*108 (cfu/ml/seed) and 1.2*108 (cfu/ml/seed) for maize and rice respectively. *i.e. cfu= colony forming units table 2. colony of p. fluorescens per inoculated seed at different time period particulars time maize 2 hrs 24 hrs 1.69 *108 cfu/ml/seed 1.27*108 cfu/ml/seed 1.94*108 cfu/ml/seed 1.53*108 cfu/ml/seed 1.80*108 cfu/ml/seed 1.26*108 cfu/ml/seed rice 2.18*108 cfu/ml/seed 8.9*107 cfu/ml/seed 2.12*108 cfu/ml/seed 1.28*108 cfu/ml/seed 2.21*108 cfu/ml/seed 1.43*108 cfu/ml/seed *cfu=colony forming units. the prevalence of pseudomonas spp. was 68% and 58% with mean count 9.02x104±2.87x104 and 2.43x105±9.32x104 for damietta and kariesh cheese respectively. pseudomonas spp were not detected in feta cheese. the most prevalent spp was p. fluorescens (35.14% and 45.5%) figure 2. colony of p.fluorescens per seed with respect to time. while p. aeruginosa (21.26% and 15.2%); p. putida (27% and 18.2%) and p. stutzeri (16.21%and 21.21%) for damietta and kariesh cheese respectively (el-leboudy et al., 2015). treatments, which were nominal target concentrations of sdp (as active ingredient) of 50, 100, 200, and 300 milligrams per liter (mg/l), were continuously applied for 24 hours (luoma et al., 2015). damietta cheesewere positive samples for pseudomonas spp. with mean count 9.02 x 104±2.8x104. international journal of applied biology, 2(2), 2018 32 conclusions pseudomonas fluorescens a bio-control agent against various plant diseases was most effective after two hours of inoculation in larger size seed than that of small size seed, whereas soaking of inoculated small size seed for twenty four hours result grater colony per seed. aknowledgements the authors were grateful to agriculture and forestry university, rampur, chitwan, nepal for providing pathology lab to conduct this experiment and agricare nepal pvt. ltd. for providing the strain of pseudomonas fluorescens references el-leboudy, a. a., amer, a. a., nasief, m. e. & eltony, s. m. (2015). occurrence and behavior of pseudomonas organisms in white soft cheese. alexandria journal of veterinary sciences. 44(1): 74. https://doi.org/10.5455/ajvs.166387 king, a. & phillips, i. (1978).the identification of pseudomonads and related bacteria in a clinical laboratory. j med microbiol. 11(2): 165-76. luoma, j.a., weber, k.l. & mayer, d.a. (2015). exposure-related effects of pseudomonas fluorescens, strain cl145a, on coldwater, coolwater, and warmwater fish: u.s. geological survey open-file report 2015–1104, 1632 p., http://dx.doi.org/10.3133/ofr20151104. meyer, j. m., & abdallah, m. a. (1978). the fluorescent pigment of pseudomonas fluorescens: biosynthesis, purification and physicochemical properties. journal of general microbiology. 107(2): 319–328. https://doi.org/10.1099/00221287-107-2-319 sakthivel, u., mahalakshmi, s. & karthikeyan, b. (2009). studies on isolation and characterization and its (pseudomonas fluorescens ) on yield of tomato. methods. 1(1): 33–39. scales, b. s., dickson, r. p., lipuma, j. j. & huffnagle, g. b. (2014). microbiology, genomics, and clinical significance of the pseudomonas fluorescens species complex, an unappreciated colonizer of humans. clinical microbiology reviews. 27(4): 927–948. https://doi.org/10.1128/cmr.00044-14. 2(2)20-26 contact : sonny kristianto sonny.kristian@gmail.com © international journal of applied biology 20 abstract this research aims to investigate the accumulation of heavy metals chromium (cr) in the flesh of fish oreochromis mossambicus (oreochromis mossambicus) and find out the feasibility of fish consumption oreochromis mossambicus at floating net on the river krembangan sidoaarjo. this research are observatif by using purposive random sampling techniques. test the content of heavy metal chromium (cr) in the flesh of fish oreochromis mossambicus (oreochromis mossambicus) method using aas (atomic absorbtion spectrofotometry). the data generated in the analysis in quantitative descriptive. in this research show that the heavy metal content of chromium (cr) in the flesh of fish oreochromis mossambicus highs station iii of 0.52 mg/kg; i station of 0.51 mg/kg ii station of 0.48 mg/kg on a sampling sedangkang the morning, at night the highest value on the station iii of 0.50 mg/kg; i station of 0.46 mg/kg and the ii of 0.39 mg/kg. average deposits of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) is still under the raw quality of the already established by the director-general of pom 1989 i.e. amounted to 2.5 mg/kg but fall into the category bioakumulasi medium, so that fish oreochromis mossambicus is still worthy of consumption, but need to watch out for, considering the cr are toxic, carcinogenic, bioakumulatif and biomagnifikasi would potentially interfere with health. issn : 2580-2410 eissn : 2580-2119 bioaccumulation monitoring of heavy metal chromium (cr) in fish oreochromis mossambicus in the floating net cultivation in sidoarjo krembangan river sonny kristianto, dina chamidah & laksitarahmi i pramita biology education department, faculty of language and science, wijaya kusuma university, surabaya, east java, indonesia introduction the condition of bengawan solo river is currently worrying. many people have complained about the water condition of this longest and largest river in the island of java. the results of observations show that, in plain view, the water of bengawan solo river looks turbid mixed with mud in the rainy season; while during the dry season, the water is sometimes slightly dark and smelly. several studies have shown that the water from this river is polluted, and even the content of heavy metals waste is also found. whereas the bengawan solo river is used by some people for their daily needs. the bengawan solo river with an average annual rainfall of 2,100 mm3 is a potential water source for management and development efforts of water resources, to meet various needs and requirements such as for open access international journal of applied biology keyword chromium (cr), oreochromis mossambicus fish, river krembangan sidoarjo article history received 17 september 2018 accepted 12 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 21 domestic consumption, for drinking water and industrial, irrigation, and others (ministry of public works, 2010). the development of solo city into an industrial city has a negative impact on pollution in the river. there are many industries developing in solo and its surrounding areas that dispose their waste into the river. batik (traditional clothing) industry that has become the icon of solo city on the one hand gives positive value for the economy of society but on the other hand also contributes in river pollution. the efforts conducted by the government, social institution, and the society to solve this issue also seem to have been unsuccessful. although with the naked eye, the water of bengawan solo river is polluted but in some parts of this river basin, it is still used by people around for everyday purposes. based on observations to the river, from the city of solo to sragen regency, activities of people who use water from the river such as bathing, washing, fishing, swimming spot for children, and irrigation of agricultural land can be observed. water utilization of the bengawan solo for irrigation occurs in some areas, one of which is in sragen. in dry season, the agricultural land in sragen often suffers from water shortages. people who own rice field near the bengawan solo river usually take water from the river for irrigation. they do not have the awareness and concern about the importance of the quality of water used for irrigation, whereas, this issue is related to the quality of their crops. based on the background, the water condition of bengawan solo river is contaminated with heavy metals waste. but there are still people who use it for daily purposes especially farmers for irrigation. this what prompt the water feasibility study of the bengawan solo river for irrigation is conducted and also to find the solution. materials and methods this research was carried out at the time in the village of krembangan krembangan in june 2016. peneltian is using the method of purposive random sampling to measure the quality of chemical physics krembangan river include temperature, ph, dissolved oxygen (do) and biochemical oxygen demand (bod) by in-situ on 3 stations with three repetitions with use thermometers, ph meter and do meters. test the content of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) method using aas (atomic absorbtion spectrofotometry) with wavelength 357.54 nm conducted in the laboratory of research and standardization of industrial halls and commercial surabaya. figure 1. sampling station international journal of applied biology, 2(2), 2018 22 chemical physics quality data streams krembangan analyzed are descriptive, whereas the determination of the concentration of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) bioconcentration factor values calculation using a calculated based on concentration a compound that is in the organism of the experiment divided by the concentration of compounds of heavy metals contained in the medium of water, the unit kg/l panjaitan (2009). bcf = 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 𝑜𝑓 ℎ𝑒𝑎𝑣𝑦 𝑚𝑒𝑡𝑎𝑙𝑠 (𝐶𝑟) 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑒𝑎𝑡 𝑜𝑓 𝑂𝑟𝑒𝑜𝑐ℎ𝑟𝑜𝑚𝑖𝑠 𝑚𝑜𝑠𝑠𝑎𝑚𝑏𝑖𝑐𝑢𝑠 𝐶𝑜𝑛𝑡𝑒𝑛𝑡 𝑜𝑓 ℎ𝑒𝑎𝑣𝑦 𝑚𝑒𝑡𝑎𝑙𝑠 (𝐶𝑟) 𝑜𝑛 𝑡ℎ𝑒 𝑤𝑎𝑡𝑒𝑟 according to amriani et al (2011) the value of the bioconcentration factor (bcf) has three categories, namely: 1. the value of the bcf > 1000 l/kg, accumulative nature of high category. 2. the value of the bcf = 100 – 1000 l/kg, accumulative nature of the category are. 3. the value of the bcf 100 l/kg <, accumulative nature of low category. the higher the value of the bcf at an organ, indicating the higher the organs of these heavy metals accumulate shindu, (2005). results and discussion quality factor of chemical physics krembangan river the results of the measurement of the quality of chemical physics of the krembangan river include, temperature, ph, bod and do at 3 staiun (table 1) shows the value that fluctuates on each factor measurements of temperature, ph, d.o. and bod on the river krembangan. table 1. the results of the measurement of the physical chemical quality in the krembangan rivers variables station morning afternoon late afternoon temperature (oc) 1 2 3 28.30 28.10 27.90 30.46 30.66 30.50 27.20 26.90 26.80 ph 1 2 3 7.50 7.80 7.90 7.94 8.06 8.03 7.90 8.00 8.00 do (mg/l) 1 2 3 2.80 2.78 2.80 2.79 2.79 2.80 2.79 2.69 2.78 bod (mg/l) 1 2 3 103.85 104.10 104.02 105.36 105.46 105.40 102.90 103.75 103.80 the results of this study (table 1) shows that the average temperature of the krembangan river is different at each time taking morning, noon and afternoon on 3 roaming. the average temperature in the morning between 27.90 28.30 – oc, daytime between 30.46 30.66 oc – and in the evening between 26.20 – 27.20 oc. this is due to the different retrieval time thus affecting the intensity of sunlight that enters into the river krembangan. an increase in temperature will cause an increase in the speed of your metabolism and respiration of international journal of applied biology, 2(2), 2018 23 organisms water resulting in increased consumption of oxygen. rising temperatures have also led to an increase in organic matter decomposition by microbes so that concentrations of bod in water will also increase. the range of optimum temperature for the growth of phytoplankton in waters ranging from 20 oc – 30 oc (effendi, 2003). physics ph factor (table 1) on the river krembangan have difference at any time taking. the average value of ph in the morning between 7.90, 7.50 – during the day between 7.94 – 8.06 and evening between 8.00 – 7.90. this is due to the surrounding communities also use the river as a place of krembangan household waste disposal so that the ph has increased. in addition the value of ph is obtained this is above the normal ph which is owned by surface water, ie 7.0. this can be caused by the presence of deposits of carbonate and bicarbonate dissolved, which is the deciding factor in surface water ph (akoto et al., 2008). most of the aquatic biota are sensitive to changes in ph and ph value of around 7 liked – 8.5 (effendi, 2003). begum, et al. (2009), the high ph in waters can cause the metal content of terendapkan hydroxide precipitates formed. factors (table 1) the average value of a do on river krembangan vary at each station. the average value of do in the morning between 2.78 – 2.80 mg/l, the daytime between 2.79 – 2.80 mg/l and the afternoon between 2.69 – 2.79 mg/l. do on normal dibandingakan river krembangan still raw quality of the ppri no. 82 the year 2001 was 6 mg/l of oxygen concentration. dissolved too low an indication that these waters have been contaminated (azwir 2004). dissolved oxygen levels also fluctuate daily and seasonal basis, depending on the pollution and movement of water masses, photo synthesis, respiration activity of wastewater into a body of water (effendi, 2003). bod factor (table 1) the average value of bod on the river krembangan differently on each station. on the morning of the average value between 103.85 – 104.10 mg/l, the daytime between 105.36 – 105.46 mg/l and the afternoon between 102.90 – 103.80 mg/l. bod on the river krembangan has exceeded the specified threshold the government according to the raw quality of the ppri no. 82-year 2001 is 2mg/l. this indicates the high oxygen consumed by micro-organisms in waters this species to be able to degrade organic waste that goes into the river krembangan. the value of bod are quite high indicating that the river contaminated organic wastes already krembangan, pendegradasian waste is done in aerobic microorganisms, due to the large number of organic wastes are dumped into waterways causes depletion of levels of dissolved oxygen in the waters, if diminishing oxygen could result in death for aquatic plants, plankton, fish and microorganisms. analysis of heavy metal content of cr, biokonsentrasi factor, and category bioakumulasi fish oreochromis mossambicus at krembangan river the results of the analysis of heavy metal cr on the river and fish flesh krembangan oreochromis mossambicus (o. mossambicus) (table 2) method using aas (atomic absorbtion spectrofotometry) with wavelength 357.54 nm which is done in laboratories and standardization research hall industry and trade. table 2. the result analysis of heavy metal cr, bioconcentration factor and bioacumulation ikan mujair in the krembangan rivers time station heavy metal cr in water (mg/l) heavy metal in meat mujair fish (mg/l) bioconcentration faktor (bcf) (l/kg) category morning 1 2 1.17 1.14 0.51 0.48 435.89 421.05 medium medium international journal of applied biology, 2(2), 2018 24 3 1.20 0.52 433.33 medium evening 1 2 3 0.96 1.11 1.03 0.46 0.39 0.50 479,16 351.35 485.43 medium medium medium the results of the analysis of heavy metal cr on krembangan river shows the difference at each station. the average value of the analysis of heavy metal cr on the morning ranged from 1.14 – 1.20 mg/l while the evening ranged from 0.96 – 1.11 mg/l, this is due to the habit of the plant or the person disposing discharge into the river range hours 01.00 – 02.00 wib early days. on the morning of the average value of the results of the analysis of heavy metal cr is higher than the average value of the analysis of heavy metal cr at night because on the morning of the waste has already spread following the flow of the river. heavy metal contamination in rivers can occur due to sidoarjo krembangan disposal of domestic waste and waste industrial result dumped into river krembangan sidoarjo without first being processed or processed but still above the levels of quality raw polutannya the set. the metals in the aquatic environment are generally in the form of ions, there is a free ion, ion pair organic complex ions, and other ions form. although the levels of heavy metals in water is relatively small, but very easily absorbed and accumulated biologically by plants or aquatic animals and will be involved in the system of food webs. this has led to a process of bioakumulasi i.e. heavy metals will be accumulated and increased simply applied in aquatic organisms, including fish oreochromis mossambicus, then through the transformation will happen moving and elevated levels of heavy metals indirectly through the food chain (damandiri 2006). according to the palar (2004) the solubility of elements – elements of heavy metals in water bodies in control by ph, type and concentration of metals and minerals ability, state of khelat teroksida and redox system of berlingkungan. the results of the analysis of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) shows the difference at each station. the results of the analysis of heavy metal cr on fish oreochromis mossambicus which was taken on the morning of between 0.48 – 0.52 mg/l, while in the evening between 0.39 – 0.50 mg/l. value analysis of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) has not exceeded a threshold according to the director-poms no. 03725/b/sk/about the limits of metal impurities on food, namely of 2.5 mg/kg, but although it has yet to go beyond the threshold of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) still need to look out for in the long run because heavy metals are toxic and carcinogenic cr. results of the analysis of the biokonsentrasi factors of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) shows the difference. on the morning of the average value of heavy metal cr biokonsentrasi analysis between 421.05 – 435.89 l/kg, the night between 351.35 – l/kg 485.43. bioakumulasi heavy metal cr on fish oreochromis mossambicus (o. mossambicus) reached bioakumulasi levels are categories, it is because fish oreochromis mossambicus (o. mossambicus) is a predator. in addition the sample fish oreochromis mossambicus (o. mossambicus) taken to be analyzed the content of heavy metals have already entered a period of harvest so that ensured accumulation of heavy metal cr on high, it is seen from the size and weight of fish oreochromis mossambicus (o. mossambicus). age is also one of the factors that influence the length of exposure to toxic substances in the organism, although fish in general have the ability to prevent themselves from the influence of the pollution, but if fish habitats is limited as in floating net will be difficult to prevent themselves from pollution, this has resulted in the presence of elemental accumulation polluters including heavy metals into the body of the fish (dinata 2006). international journal of applied biology, 2(2), 2018 25 generally there are 4 ways of heavy metals accumulate in the body tissues of fish through the water flow in the gills, the process of eating and drinking as well as skin, lasut, (2009). accumulation of heavy metals in fish begins with the process (uptake) through the gills and then absorbed into the body of the fish and the entire network system level excretion of akbar, (2002). from the results of the analysis of the bioakumulasi heavy metal cr on fish oreochromis mossambicus (o. mossambicus) at floating net cultivation in the krembangan fall into the category bioakumulasi are so lacking in long-term consumption is advised. conclusions the quality of chemical physics include temperature, ph, d.o. and bod on the river have entered the category krembangan tainted with high levels of pollution, it is it can be seen from the results of measurements of the physical and chemical quality of rivers krembangan who has beyond the limits set by the government. the content of heavy metal cr on krembangan river have also exceeds the limit set by the government, although the content of heavy metal cr on fish oreochromis mossambicus (o. mossambicus) has not exceeded limits set by bpom amounted to 2.5 mg/kg but to consume fish oreochromis mossambicus in the floating net cultivation in the krembangan in the long term is less recommended, this is due to the level of bioakumulasi belongs to the category bioakumulasi. references ackerley, d.f, gonzales.c.f, park, c.h, blake,r. keyhan,m.& martin,a.2004. chromat reducing properties of soluble flavoprotein from pseudomonas putida and escherichia coli.”applied and environmental biology”. 70.(2): 873-882. akbar hs. 2002. prediction of heavy metal accumulation rates of cd, pb, cu, zn and ni in mussels (penna viridis l) size > 5 cm in the waters of the estuary, the bay of jakarta's kamal [in indonesia] akoto, o., bruce, t. n., darkol, g. 2008, heavy metals pollution profiles in streams serving the owabi reservoir. african journal of environmental science and technology, 2 (11) : 354-359. amriani, hendrarto b & hadiyarto a. 2011. bioaccumulation of heavy metals lead (pb) and zinc (zn) in the blood cockle (anadara granosa l.) and mangrove shells (polymesoda bengalensis l.) in the waters of the gulf of kendari. journal of environmental science, 9 (2): 45-50.[in indonesia] baolin d., ian, l., houston, k., brady, pv.,2003, effect clay minerals on cr (vi) reduction by organic compounds, environmental monitoring and assesment. 84 : 5-18. begum, a., ramaiah, m., harkrishna, s., khan, i., veena, k., 2009b, heavy metal pollution and chemical profile of cauvery river water, e-journal of chemistry, 6 (1) : 47-52. connel, dw and g. j. miller. 1995. ekotoksikologi chemistry and pollution. ui press., jakarta. [in indonesia] connell, d. w. and g.j. miller. 2006. ekotoksikologi chemistry and pollution. jakarta: indonesia university. [in indonesia] effendi, h. 2003. study of water quality for resource management and the aquatic environment. the publisher of canisius. yogyakarta.[in indonesia] the decision of the director general of the food and drug supervision number: 0375/b/sk/vii/89 concerning the limits of maximum metal impurities in food. [in indonesia] international journal of applied biology, 2(2), 2018 26 larashati, s. 2004. the reduction of chromium (cr) in in vitro by a mixed culture of bacteria in isolation from lindi landfill waste (landfill). itb. [in indonesia] mulyani, b. 2004. analysis of the variation of biomass saccharomyces cerevisiae against chrome metal absorption. sain. 2 (4): 1-9. [in indonesia] palar, h. 2008. pollution and toxicology of heavy metals. publisher copyright rineka, jakarta. 152 page. [in indonesia] government regulation of the republic of indonesia number 82 of the year 2001. about water quality management and control of water pollution. [in indonesia] schiavon, m. e. a. h. pilon. smits, m. wirtz, r. hell and m. malagoli. 2008. interactions between chromium and sulfur metabolism in brassica juncea. journal of enviromental quality. 37 : 1536-1545. shindu sf, 2005. heavy metal content of cu, zn and pb in water, fish tilapia (oreochromis niloticus) and carp (cyprinus carpio) in the floating net, reservoirs saguling. institute of holticulture bogor. [in indonesia] the legislation of the republic of indonesia no.32 year 2009 on the protection and management of the environment.[in indonesia] contact : endang triwahyu prasetyawati endang_tp@upnjatim.ac.id 98 abstract red chili (capsicum annum l.) is a horticultural plant classified as an important commodity with high economic value. one of the main problems in the production of red chili is anthracnose disease caused by the colletotrichum capsici pathogen. bacillus spp. expected to have the potential to inhibit the growth of c. capsici and stimulate plant growth. this study aims to determine the potential of bacillus spp. in suppressing the growth of the c. capsici fungus and stimulating the growth of red chili. this study used a completely randomized design (crd) with the treatment without bacillus spp., chemical fungicides, bacillus sp. (ba-6), bacillus sp. (ba-9), bacillus sp. (ba-12), bacillus sp. (ba-15), and bacillus sp. (ba-17). the treatment was repeated four times, and the treatment unit contained five red chili/polybags. the results showed the highest suppression of c. capsici in treating bacillus sp. ba-15 was 6.25% compared to the negative control. bacillus sp is the best bacteria that can stimulate red chili growth. ba-9 has an average plant height of 20.10 cm and an average number of leaves of 10.49. issn : 2580-2410 eissn : 2580-2119 the potential of bacillus spp. in suppressing colletotrichum capsici that causes anthracnose in red chili sasiska rani1, endang triwahyu prasetyawati1*, herry nirwanto1, sri wiyatiningsih1 1faculty of agriculture, upn “veteran” jawa timur, surabaya, indonesia introduction red chili (capsicum annum l.) is one of the important horticultural commodities in indonesia, which has high economic value. the demand for chili products will continue to increase along with the increase in population. national chili demand in one year for big cities is around 800,000 tons/year and will increase by about 10-20% on religious holidays; therefore, this commodity has a good opportunity to be developed (anonim, 2012). chili cultivation is influenced by several factors that can decrease its production. these results caused that the productivity of red chili in indonesia is still far from its potential, which can reach 12-20 tons/ha (syukur et al., 2010). one of the main factors causing the low productivity of chili in indonesia because the pathogens that cause plant diseases. the primary disease in red chili is anthracnose, caused by the colletotrichum capsici pathogenic fungi. anthracnose can cause the chili shoots on ma ture plants to die, followed by infection in the fruit. this problem causes chili productivity to decrease (prasetyo, 2017). anthracnose disease in chili can cause yield losses of up to 90%, especially during the rainy season (badan pusat statistik, 2018). open access international journal of applied biology keyword bacillus spp; colletotricum capsica; anthracnose; red chili. article history received february 1, 2023 accepted june 21, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 7(1), 2023 99 anthracnose is the most common disease and almost occurs in every chili -growing area. pathogen infection can occur from the plant in the field until the plant is harvested because it can reduce production in quality and quantity. a severe level of attack can make the plant die. attacks on fruit can result in fruit damage and a decrease in the quality of chili, so their economic value is also low (nurhayati, 2011). to control anthracnose, we generally still use chemical fungicides because it is considered easy and effective. using chemical fungicides in the long term will harm the environment and humans. an alternative to reduce the use of chemical fungicides is to use biological fungicides (elfina et al., 2015). several studies have been conducted to control anthracnose. one study uses biological fungicides that are more environmentally friendly and harmless to consumers, including bacillus spp. (putro et.al., 2014). bacillus is a biological agent that has the potential to be developed as an antifungal. bacillus has been widely used because it can produce antimicrobials that can suppress the growth of various plant pathogens and belong to the group of pgpr (plant growth promoting rhizobacteria) bacteria that can stimulate plant growth (shafi et al., 2017; fira et al., 2018). bacillus has advantages compared to other bacteria and can produce endospores for surviving structures when environmental conditions are unfavorable (prihatiningsih et al., 2014). this study aims to determine the potential of bacillus spp. to suppress the growth of c. capsici fungi and stimulate the growth of red chili. materials and methods this research was conducted from january to april 2021 in the plant health laboratory and screen house of the faculty of agriculture, university of pembangunan nasional "veteran" jawa timur. the materials used in this study were bacillus spp. from kediri ba-6, ba-9, ba-12, ba15, and ba-17 isolates, a collection of dra. endang triwahyu p., m.si., colletotrichum capsici isolates, na media, pda media, aquades, cotton, tissue, label paper, alvaboard, 70% alcohol, spirtus, aluminum foil, wrap, nb media, garden soil, compost, npk fertilizer, 5% formalin, chemical fungicide active ingredient benomyl, and healthy chili seeds of gada mk variety. this study was arranged in a single, factor completely randomized design (crd) with seven treatments. there are kn (control without treatment), kp (control of chemical fungicides), s1 (bacillus sp. ba-6 isolate), s2 (bacillus sp. ba9 isolate), s3 (bacillus sp. ba-12 isolate), s4 (bacillus sp. ba-15 isolate), s5 (bacillus sp. ba-17 isolate). each treatment was repeated four times, so the total number of experiments was 28 units. each experimental unit consisted of five red chilies. preparation of planting media the planting media used were soil and compost with a ratio of 1:1. the sterilization of the planting media refers to the method (musafa et al., 2015). the media was sterilized using 5% formalin at a dose of 2.5 ml/kg, and this sterilization process lasted for 15 days. then, the sterile media was distributed into polybags. international journal of applied biology, 7(1), 2023 100 production of bacillus spp. suspension bacillus spp. cultures grown on slanted na media were taken ten ose needles and put into an erlenmeyer containing 60 ml of nb media. then homogenize it using a vortex for 30 minutes and incubate it for 20 hours. the bacterial suspension de nsity was 109 cfu/ml (putro et al., 2014). production of c. capsici suspension fungi cultures grown on pda media aged seven days were taken by adding 10 ml of sterile aquades and then rubbed with an ose needle. then put it into an erlenmeyer containing 40 ml of sterile aquades and homogenize it using a vortex for several minutes. the fungi suspension was taken using a pipette, and the amount of conidia density was calculated using a neubauer hemocytometer. the appropriate density for fungal suspension was 106 conidia/ml (putro et al., 2014). testing in vivo bacillus spp. to the c. capsici fungi on red chili this treatment was carried out by soaking the roots of chili seedlings aged 24 days in bacillus spp. suspension in a 109 cfu/ml density for 30 minutes. immersion of red chili seeds in the negative control treatment with sterile aquades, while in the positi ve control, the chili seeds were applied a chemical fungicide by the active ingredient benomyl using the dosage recommended on the label. then, the seeds are planted in polybags containing sterile media. the chili plants were observed for five weeks after planting (wap). watering plants carried out the treatment of plants in the morning by paying attention to soil moisture and fertilizing with 2 grams of npk fertilizer in each plant every two weeks (purwanto, 2020). observation variable the observations were made to determine the growth and resistance of red chili. 1. observations on the growth of red chili included plant height and number of leaves. observations were made starting after inoculation of bacillus spp. up to 35 days at sevenday intervals. 2. observation of plant resistance included incubation period and intensity of disease attack. the disease attack was observed every week from the day after c. capsici inoculation until five weeks. the following formula calculates disease intensity (%): i = σni. vi n . z × 100% information: i = disease intensity ni = the number of each symptom area class (each treatment five chilies) vi = i-th damage category n = number of fruits observed v = highest value of attack category the value of the attack category (score) to anthracnose is based on the scale of plant damage affected by the disease (jauhari and majid, 2019). the value of the attack category international journal of applied biology, 7(1), 2023 101 (scores) is as follows: 0 = no damage 1 = leaf spot 1%-25% 2 = leaf spot 26%-50% 3 = leaf spot 51%-75% 4 = leaf spot 76%-100% the criteria for red chili resistance to anthracnose are as follows (mufida, 2020): 0 % -10 % = very resistant 11 % -20 % = resistant 21% 40% = moderate 41 % 70 % = susceptible x > 70% = very susceptible the data obtained is then analyzed by analysis of variance (anova) to determine whether each treatment has an effect. if it is significantly different, it is continued with the 5% least significance different (lsd) test. results and discussion the growth of red chili red chilies were damaged, so the data presented in plant growth only reached the third observation (3 wap). the results showed that the bacteria bacillus spp. potential to increase plant growth. the treatment of bacillus spp. significantly differed in plant height at the ages of 2 wap and 3 wap (table 1). the treatment of bacillus spp. significantly differed in the number of leaves in red chili at the second observation (2 wap). meanwhile, in the third observation, the treatment of bacillus spp. was not significantly different in the number of leaves in red chili. the average number of leaves in red chili is shown in table 2. table 1. an average of plant height in red chili treatments plant height (cm) 1 wap 2 wap 3 wap kn (control without treatment) 8,00 15,01 d 18,96 abc kp (control of chemical fungicides) 7,03 14,37 cd 18,08 ab bacillus sp. ba-6 isolate 7,85 13,91 bc 18,60 ab bacillus sp. ba-9 isolate 8,38 13,85 bc 20,10 c bacillus sp. ba-12 isolate 7,92 13,02 ab 19,36 bc bacillus sp. ba-15 isolate 7,86 13,17 ab 18,38 ab bacillus sp. ba-17 isolate 8,23 12,57 a 18,01 a lsd 5% nsd 0,97 1,31 note: the numbers followed by the same letter in the same column are not significantly different in the 5% lsd test. nsd = not significantly different international journal of applied biology, 7(1), 2023 102 table 2. an average number of leaves in red chili treatments number of leaves 1 wap 2 wap 3 wap kn (control without treatment) 5.40 8.55 c 10.35 kp (control of chemical fungicides) 5.35 8.25 abc 9.93 bacillus sp. ba-6 isolate 5.55 8.35 bc 10.28 bacillus sp. ba-9 isolate 5.50 8.65 c 10.49 bacillus sp. ba-12 isolate 5.40 7.80 a 10.31 bacillus sp. ba-15 isolate 5.40 8.20 abc 10.08 bacillus sp. ba-17 isolate 5.35 7.90 ab 9.98 lsd 5% nsd 0.49 nsd note: the numbers followed by the same letter in the same column are not significantly different in the 5% lsd test. nsd = not significantly different based on the results obtained regarding the effect of applying bacillus spp. on the growth of red chili, the bacillus spp was shown. isolate had different effects on plant height growth (table 1) and the number of leaves (table 2). it is suspected that thi s is due to bacillus spp.'s ability to develop in different plant tissues. the average plant height and number of leaves in the third week showed that the best result is bacillus sp. ba-9 isolate with an average plant height of 20.10 cm and a number of leaves of 10.49. besides being able to inhibit the growth of pathogens, antagonist bacteria are also widely known as pgpr (plant growth promoting rhizobacteria) (jatnika et al., 2013). this shows that bacillus sp. ba-9 isolate has the potential to stimulate plant growth. previous studies have shown that bacillus subtilis isolate is able to synthesize indole acetic acid (iaa) and gibberellins (yulistiani, 2015). iaa hormone is a hormone that plays a role in plan t growth and development; therefore, certain synthesized bacteria cause increased plant growth (herlina et al., 2016). the ba-9 isolate was suspected that this isolates able to synthesize the highest iaa and gibberellin hormones, so it was the best isolate for increasing the growth of red chili. the resistance of red chili to anthracnose disease bacillus spp. has the potential to increase plant resistance to pathogens because bacillus spp. able to colonize plant tissue (suwarno and masnilah, 2020), so it can enter into plant tissue to infect and minimize pathogens. colonization that occurs in the roots can trigger plants to produce jasmonic acid and plant ethylene to induce plant resistance to pathogens (djaenudin, 2016). based on observations, the incubation period or the appearance of symptoms on the third day after treatment. figure 1 shows a comparison between healthy red chili leaves and those infected with anthracnose. the leaves that are infected by anthracnose symptoms have round spots with a light gray to white center and dark brown edges. international journal of applied biology, 7(1), 2023 103 figure 1. comparison of red chili leaves (a) healthy leaves (b) anthracnose infected leaves based on observations, the incubation period of red chili is relatively fast. the results of previous studies stated that the fastest incubation period of c. capsici in chili was 12 days (herwidyati et al., 2013). it is suspected that this occurs because influenced by environmental conditions there are temperature and humidity that support the growth, reproduction, and development of the pathogen. the development of the pathogen and the severity of the disease are affected by high humidity. the environmenta l conditions during the treatment were suitable and supported the growth of c. capsici. there is an average temperature between 24⁰c–30⁰c with a high relative humidity of 80%–90%. symptoms of anthracnose found in red chili can be seen in figure 2. anthracn ose can cause death in chili shoots which are characterized by dried chili shoots and blackish-brown color (figure 2a). symptoms of anthracnose on red chili leaves include yellowish white leaf spots on the edges, but the deeper the leaves get whiter and lo ok like burning, in the middle of the spots, there are black dots which are the acervulus by c. capsici fungal (figure 2b). furthermore, the symptoms of anthracnose on the stems of red chili are characterized by dry stems, and there is acervulus fungal that look like black bulges (figure 2c). figure 2. symptoms of anthracnose on red chili (a) symptoms on shoots (b) symptoms on leaves (c) symptoms on stems international journal of applied biology, 7(1), 2023 104 the observation of symptoms on stems was carried out further to ensure that symptoms in chili caused due to c. capsici infection. the results showed that on the stems, there were acervulus protrusions which, when observed using a microscope at 10x10 magnification, looks like blackthorns (figure 3a). microscopic observations were continued by making symptomatic preparations on the stems. the results of microscopic observations at 40x10 magnification showed that the conidia were crescent -shaped, not insulated, and there were acervuli which are characteristic of the c. capsici (figure 3b). red chili that shows symptoms of the disease is then re-isolated (figure 3c). figure 3. (a) microscopic direct observations on the stem at 10x10 magnification (b) microscopic observations using a glass object at 40x10 magnification (c) the results of pda media re-isolated in 7 days aged. the next observation of plant resistance was carried out by observing the disease intensity of red chili. observations were made five times every seven days. the results of observations showed that the development of disease intensity (%) of anthracnose on plants of red chili with bacillus spp. was not significantly different (figure 4). figure 4. the histogram of treatment effect on the intensity average of anthracnose disease on plant red chili description: kn=control without treatment, kp=control of chemical fungicides, s1= bacillus sp. ba6 isolate, s2= bacillus sp. ba-9 isolate, s3= bacillus sp. ba-12 isolate, s4= bacillus sp. ba-15 isolate, s5= bacillus sp. ba-17 isolate. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 1 wap 2 wap 3 wap 4 wap 5 wap d is e a se i n te n si ty ( % ) plant age the intensity average of anthracnose disease on plants red chili kn kp s1 s2 s3 s4 s5 c international journal of applied biology, 7(1), 2023 105 the lowest anthracnose disease intensity was found in the bacillus sp. ba-15 isolate. observations in the second week showed that the intensity of anthracnose disease ranges from 21% -40%, which indicated that the plant resistance was moderate. then in the third week, plant resistance to chemical fungicide control treatment, bacillus sp. ba-6 isolate and ba-15 were moderate, with the intensity of anthracnose disease ranging from 21-40%. while in the control without treatment, bacillus sp. ba-9, ba-12, and ba-17 isolates were classified as susceptible because the intensity of anthracnose disease ranges from 41% -70%. the fourth-week observation showed that plant resistance was very susceptible b ecause the intensity of anthracnose disease ranges from >70%, except for the bacillus sp. ba-15 isolate, which has a disease intensity of 40%, is classified as moderate. the fifth week of observation showed that plant resistance in all treatments was very susceptible because the intensity of the disease was >70%. the results of the observation of disease intensity are suspected to indicate that bacillus spp. capable of stimulating the increased resistance of plants in red chili. the observations from the first week to the fifth week showed that the intensity of plant disease given bacillus sp. isolate ba15 treatment was lower than the kn treatment (contr ol without treatment). the mechanism of plant resistance that occurs in red chili is suspected to be resistance induction of isr (induced systemic resistance). according to (prihatiningsih et al., 2017), the tested b. subtilis was able to act as a siderophore producer qualitatively. the ability of bacteria to produce siderophores is an important component in pgpr because siderophores are able to bind iron (fe3+) into siderophores-iron bonds that become available to plants. the siderophores that are produced by microorganisms are beneficial for plants because they can inhibit the growth of pathogens. there is a lack of fe 3+ needed by pathogens because fe3+ is already bound by siderophores (sharma and johri, 2003). iron is an important element in disease development. therefore with the binding of iron by siderophores, the pathogen is less able to infect, thus inhibiting the disease development. the increase in disease intensity from the first week of observation to the fifth week of observation is suspected because of environmental conditions that support the growth of pathogens. in addition, the bacillus spp. bacteria suspected that the colonization in plant tissue had not been maximized; therefore, plant resistance and the antagonistic mechanism of bacillus spp. not maximal. the observation of plant intensity in the third week showed that symptoms of anthracnose began to appear on the stems. as a result, the intensity of anthracnose disease in some red chili has reached 100%. conclusion ba-9 isolate of bacillus spp. bacteria showed the best results in stimulating the growth of chili with the parameters of plant height and the highest number of leaves, with an average plant height of 20.10 cm and an average number of leaves of 10.49. the application of bacillus spp. able to suppress the growth of c. capsici pathogenic fungi in red chili with the best results in the treatment of bacillus sp. ba-15 isolate which able to suppress the growth of c. capsici by 6.25% compared to the negative control. international journal of applied biology, 7(1), 2023 106 references anonim. 2012. production performance and commodity prices of red chili. available online: http://pse.litbang.pertanian.go.id/ind/pdffiles/anjak_2012_10 (accessed on 14 january 2021). badan pusat statistik. 2018. statistics of indonesian vegetables and seasonal fruits 2017. central bureau of statistics. jakarta. djaenudin, n. 2016. interaction of antagonist bacteria with plants: induced resistance in corn plants. crops. 11(2):143-148. elfina, y., m. ali and l. aryanti. 2015. test of several concentrations of forest betel leaf flour extract (piper aduncum l.) to control anthracnose on red chili fruit post harvest. sagu. 14 (2) : 18-27. fira, d., dimkić, i., berić, t., lozo, j. and stanković, s. 2018. biological control of plant pathogens by bacillus species. journal of biotechnology 285 : 44-55. herlina, l., k, kedati, and d, mustikaningtyas. 2016. study of endophytic bacteria producing iaa (indole acetic acid) for plant growth. jurnal sainteknol. 14 (1) : 51-58. herwidyati, kh., suskandini r., and dad r. j. s. 2013. severity of anthracnose disease in chili (capsicum annuum l.) and various types of weeds. journal of research. jatnika, w., a. l. abadi, and l. q. aini. 2013. effect of application of bacillus sp. and pseudomonas sp. on the development of downy mildew caused by the pathogenic fungus peronoslerospora maydis on corn plants. 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annuum l.) thesis. bogor: agriculture faculty, institut pertanian bogor. prihatiningsih, n., djatmiko, h.a. and lestari, p. 2017. siderophore activity of bacillus subtilis as a growth promoter and controller of eggplant plant pathogens. journal of tropical plant pests and diseases. 17(2):170-178. prihatiningsih, n., t. arwiyanto, b. hadisutrisno and j. widada. 2014. selection of antibiotic mutant bacillus subtilis b315 for control of ralstonia solanacearum pr7. journal of agrin. 18(1): 67–79. purwanto, d. 2020. the effect of npk mutiara fertilizer and plant catalyst fertilizer on the growth and yield of curly red chili (capsicum annuum l.) lado f1 variety. agrifor. 19(1):123-134. putro, n.s., aini, l.q. and abadi, a.l. 2014. testing of antagonistic microbial consortium to control anthracnose disease in big red chili (capsicum annuum l.). journal of plant pests and diseases. 2(4):44-53. http://pse.litbang.pertanian.go.id/ind/pdffiles/anjak_2012_10 international journal of applied 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soedirman. purwokerto. 2(1)29-38 contact : fredy leiwakabessy fredyleiwakabessy2017@gmail.com � international journal of applied biology 29 abstract research was conducted to determine the effect of the method and duration of ripening on the content of β-carotene of fe’i banana, as well as determine the method and duration of ripening is effective in maintaining the nutritional value of fe’i banana. through laboratory experimental with randomized complete design consisting of 19 groups of treatments and three repetitions, the data obtained were analyzed using statistical tests anova and continued with duncan test. results of the analysis showed that method ofripening statistically significant difference (p <0,05) on the content of β-carotene in the banana sticks sky. long ripening length statistically significant difference (p <0, 05) on the content of β-carotene in the fe’i banana. grilled ripening method is more effective against βcarotene content in the fe’i banana and takes 10 minutes of ripening length more effectively on the content of β-carotene on a fe’i banana. issn : 2580-2410 eissn : 2580-2119 effect of method and ripening duration on the content of βcarotene of fe’i banana (musa troglodytarum l.) fredy leiwakabessy, giena g. tamtelahitu & maria nindatu biology education departement, faculty of teacher training and education science, pattimura university, ambon, maluku, indonesia introduction one type of banana that is known comes from the moluccas, indonesia is fe’i banana (musa troglodytarum l.). moluccas people call it by the name "pisang tongkat langit" or "banana pointing to the sky", as bunches of fruit that grow upright towards the sky (ploetz et al., in hiariej, 2013). heyne in samson et al. (2013) describe the skin texture of the fe’i banana is reddish with black spots, while orange-yellow flesh. society found that eating fe’i banana to improve kidney function, help reduce fever, as well as enhancing stamina of men, although not yet in clinical trials (samson et al., 2013). in addition, they also believed that fe’i banana is good to be given to infants. bananas are often used as complementary feeding (mp-asi) by the mother because the texture of the fruit is easily digested by the baby just learning to eat solid food. based on the research results, obtained information fe’i banana fruit contain carotenoids, and β-carotene is more dominant (samson et al., 2013). provitamin a and total carotenoids were high at 6360 mg/100 g was found in bananas (englberger in samson et al., 2013). according almatsier, suhardjo and kusnarto in naid et al. (2012), β-carotene is a open access international journal of applied biology keyword β-carotene banana musa troglodytarum article history received 9 april 2018 accepted 28 june 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 30 provitamin a plant pigment and is most important for humans as it can form two molecules of vitamin a. vitamin a is considered to have protective effect in cancer prevention, cardiovascular disease, cataract and macular and neurologic diseases, inflammatory and immune disorders (kidmose et al; arora et al., in fungo & pillay, 2011). for the purposes of consumption, usually people do certain ripening way to food. from a number of ripening techniques that are commonly used, the technical term grilling and steaming techniques. the method is to cook with grilling me is a way of ripening using indirect heat (burns) and the process is faster because the food was burnt near the source of the fire. steaming or usually known as steaming is damp or wet ripening process, using heat from water vapor (mulyatiningsih, 2007). unripe fe’i banana can’t be eaten, because it can cause itching in the throat. for that, usually the banana is processed before being eaten. some way of processing is to be burned, steamed or made into cakes and chips (samson et al., 2013). banana processing cane sky by fire is believed to refine the structure and improve the taste to be eaten. as well as by steaming, because it is not too eliminate the nutritional content of fe’i banana. some studies discusses the influence of the heating process of the carotenoid content of foodstuffs. samson research results, samson et al. (2013) revealed that the duration of heating affect the stability of the carotenoid pigment of fe’i banana. there was an 18% carotenoid degradation while heating the banana sticks between 5 and 10 minutes. effect of steaming food was also put forward by bengtsson et al. in fungo and pillay (2011), where “pengukusan” can markedly decrease the nutrient content of food, the amount of which depends on the type of food by steaming and steamed. the results of the study showed that sweet potato boiling over 20 minute result loss of β-carotene 9 to 16%. in the steaming process, before the carotenoids migrate into water as released from the carotene protein binding it, or the β-carotene protein complex is slightly soluble in water (howard et al., 1999). on the other hand, according to palupi et al. (2007), generally warming will increase the digestibility of foodstuffs that increase the benefits of the nutrients contained therein. acyl studies show that pe cooking methods can increase carotenoid content, as it has been reported that maturation can increase carotenoids bioavailability in carrots, spinach and tomatoes (stahl and sie,; stone et al. in junpatiw et al., 2013). the influence of ripening in bananas also revealed by a study conducted engelberger et al. in fungo & pillay (2011) which showed that carotenoid content was higher found in banana samples cooked in raw banana samples. based on the observation, the ripening process length taken by the community on the banana of sky stick is not known for certain. understanding that developed so far is the fe’i banana burned until the fruit rupture, then it is considered to have matured. in the initial test done, banana sticks to the sky burned his skin broke out are at about 10 minutes, but unknown β-carotene content in it in less than 10 minutes, at 10 minutes, and more than 10 minutes. therefore, it is necessary to study the effect of ripening method (steam and burn) and ripening length in relation to β-carotene content. materials and methods the design of this study used a randomized, fully randomized experimental laboratory design method with a sample of 21 treatment groups and three repetitions. analysis of βcarotene, proximate, and mineral contents was performed on unsweetened skeletons of celestial sticks, burnt banana sticks, and steamed fe’i banana. unbleached skeletons of fe’i international journal of applied biology, 2(1), 2018 31 bananas served as controls, while bananas burned sky sticks and banana sticks of steamed skies with some ripening length as a treatment group. this research conducted in the organic chemistry laboratory of the faculty of mathematics and natural sciences, pattimura university, in march and april 2015. the sample or object in this study is 21 fe’i banana obtained from soya village, district sirimau, ambon. the average weight of a fe’i banana is 260 g, which will be taken from one bunch of fe’i banana. a total of 21 fe’i bananas prepared by the provisions, fe’i banana (to 3 repetitions) did not do any treatment, while the other fe’i banana each ripening fuel treated and steamed with a variety of different length. treatment with a different ripening method in question is as follows: 1. fuel method: three ripes fe’i banana burned near the source of the fire until a different length each 5, 10, and 15 minutes. each length treatment is measured using a thermometer temperature. 2. steamed method: three fe’i banana has not skinned steamed steamed in a pot containing 1500 ml of water that has been boiling up each length 5, 10, and 15 minutes. each length treatment is measured using a thermometer temperature. analysis of β-carotene using uv-vis spectrophotometry the experimental procedure is done adaptation of the working procedure for the analysis of β-carotene by apriantono in musfiroh et al. (2009) and modified according to research needs. stages of experiments were carried out as follows: a) preparation of standard curves β-carotene and wavelength measurement maximum 1. β-carotene pure weighed weighing 50 mg, dissolved up to the mark in a mixture of acetone, n-hexane in a 100 ml flask to obtain a concentration of 500 mg/l. 2. take 20 ml of a solution with a concentration of 500 mg/l, as much as 100 ml to obtain a concentration of 100 mg/l. retrieved 5 ml of solution with concentration of 100 mg/l, diluted to 100 ml to obtain concentration 5 mg/l. take another 10 ml of solution with concentration 5 mg/l, diluted to 50 ml to concentrate to 1 mg/l. 3. perform dilution standard solution with varying concentrations: 0.5, 1.0, 1.5, 2.0, 2.5 mg/l. 4. foreign m each standard solution concentration was measured with a visible spectrophotometer absorbance at maximum wavelength obtained. the maximum wavelength of β-carotene obtained is at 450 nm. 5. standard curve created by the relationship between the absorbance and the concentration of β-carotene. b) determination of β-carotene content 1. samples of the untreated fe’i banana weighed 5 grams, then extracted with a solvent mixture of acetone ml 2 0, 3 0 ml of n-hexane, 0.1 g of magnesium carbonate and stirred for 120 minutes using a magnetic stirrer at room temperature and filtered. 2. the residue was washed with 15 ml of acetone, then washed with 15 ml of nhexane, then washed using 10 ml of distilled water. 3. the crude extract which d i get accommodated (to be established in two phases: an organic phase and water phase). 4. these two phases are separated using a separation funnel. 5. acetone and extract are separated. the aqueous phase is removed, while the organic phase is taken for further analysis. international journal of applied biology, 2(1), 2018 32 6. the organic phase was transferred into a 100 ml flask, vacuum to remove residual solvent at 30 ° c in order to obtain extracts of yellow. 7. column chromatography column is prepared with a length of 40 cm and a diameter of 2.5 cm by 20 cm high alumina adsorbents, the sodium sulfate layer is placed as high as 5 cm above the adsorbent layer. 8. the obtained pigment extract was introduced into the column slowly, eluting with 100 ml acetone hexane solvent, while keeping the top layer always filled with solvent 9. the β-carotene elution results were collected in a 100 ml erlenmeyer and measured volume of the extract produced. samples of burnt and steamed celestial of fe’i banana were also given the same treatment according to the β-carotene measurements on unsweetened skeletons of celestial sticks, each at 5, 10, and 15 minutes of bananas being burned and steamed. this treatment is also done with three repetitions. sample on repetition 1, 2, and 3 in each treatment method of ripening and the ripening length is different measured absorbance using visible spectrophotometer at the wavelength of maximum for β-carotene is 450 nm, is used also blank (acetone: n-hexane 1: 9 v/v). determination of β-carotene content in the sample is based on a standard curve obtained by (y) as the absorbance and (x) as concentration. the absorbance obtained from the sample is fed into the equation in the calibration curve and is obtained: y = ax + b calculation of β-carotene content in the samples using the formula: c reg = regression concentration (mg / l) v = sample volume (l) g = sample weight (kg). results and disscussion the content of β carotene in fe’i banana were measured on samples of fresh fruit as well with steamed ripening method and bake for 5, 10, and 15 minutes. the content of β carotene in bananas fresh sky stick used as a control or comparison with samples made treatment. the measurement results β carotene content in the fe’i banana in this study can be seen in figure 1. figure 1. the content of β carotene fe’i banana 10.3742 11.6136 12.0727 12.4892 14.1872 15.6377 16.6669 0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000 14.0000 16.0000 18.0000 kontrol 5 menit kukus 10 menit 15 menit 5 menit bakar 10 menit 15 menit k an d u n ga n method and duration of ripening β-karoten international journal of applied biology, 2(1), 2018 33 figure 1 shows that the content of β carotene stick banana ripening sky treated steamed or grilled increased when compared to no treatment. nevertheless, the content of β carotene with grilled ripening method is higher than the steamed method. through this picture shows that the highest content of β carotene in the fe’i banana burnt sky for 15 minutes. effect of ripening methods against the content of β-carotene in the fe’i banana (musa troglodytarum l.) based on the results of the analysis of the first hypothesis testing variants in table 1, it appears that in the control group and group ripening methods showed significant differences, where the p value of 0.000 (p <0, 05). this means that there is a significant difference in the mean content of the β-carotene between the control, the ripening method of steam, and the fuel ripening method. table 1. anova test results against effects of ripening methods the content of β-carotene fe’i banana ripening method average ± sd f count value-p control 10,3742 ± 0,20818 124,792 0,000 steamed 12,0585 ± 0.63363 burn 15,4973 ± 1.28132 durationof ripening against the influence of β-carotene content anova statistical test result data for a long length of ripening can be presented in table 2. the results of the analysis in table 2 show a significant difference, with p values of 0.002 (p <0. 05). through this result, it can be concluded that there are significant differences in the content of β-carotene control, with ripening length of 5 minutes, 10 minutes, and 15 minutes. table 2. anova test results effect of duration ripening against β-carotene content of fe’i banana length of ripeninglength average ± sd f count value-p control 10,3742 ± 0,20818 9,964 0.002 5 minutes 12,9004 ± 1.55581 10 minutes 13,8552 ± 2.04878 15 minutes 14,5781 ± 2,36521 ripening methods effective against the content of β carotene based on analysis of the effect of ripening methods that show a significant difference (p <0, 05), then, the analysis continued with duncan's test. duncan's test results further ripening methods influence on the content of β carotene fe’i banana can be seen in table 3. the result of hypothesis test analysis showed significant and very significant difference. duncan test results in table 3 shows that the content of β-carotene control is significantly different from the ripening method and the difference is very evident with the fuel ripening method. while the content of β-carotene by ripening method is different from the real ripening method. if the comparison between the two controls with this ripening international journal of applied biology, 2(1), 2018 34 method can be concluded that the ripening method that is effective against the content of βcarotene is a method of ripening fuel. table 3. the difference duncan test results against ripening methods the content of β carotene fe’i banana ripening method n subset 1 2 3 control 3 10,3742 steamed 9 12,0585 burn 9 15,4973 the effective duration of ripening against the content of β carotene based on the analysis of long influence on the content of the ripening lenght β carotene, the results showed a significant difference (p <0.05), so it can proceed with further test of duncan. duncan further test results are shown in table 4. table 4. duncan test results differences in length of ripening against the content of βcarotene fe’i banana ripening method n subset 1 2 3 control 3 10.3742 5 minutes 6 12.9004 10 minutes 6 13.8552 15 minutes 6 14.5781 the results of this hypothesis test analysis show significant and significant differences. duncan test results in table 4 show that the content of β-carotene is significantly different from the old control ripening length of 5 minutes and highly significant to the long ripening length 10 minutes and 15 minutes. while the content of β-carotene with a ripening length of 5 minutes is significantly different with the ripening length of 10 minutes and 15 minutes. long ripening length 10 minutes is not significantly different from the long ripening length 15 minutes. based on these results, it can be concluded that the length of length that is effective against β-carotene content is 10 minutes. in this research, besides seeing the effect of method and ripening length, we also see the interaction between method and ripening length. the result of the interaction methods with long ripening length on the content of β carotene can be seen in table 5. table 5. results anova interaction method and length of ripeninglength against content of β-carotene fe’i banana source the sum of squares db average squares f sig. (p) mp and wp 1.966 2 0.983 2,305 0.136 description: mp = ripening method; wp = ripeningduration anova test results in table 5 indicate that the interaction between the old method of ripening the ripeninglength does not have significant differences, where the p-value of international journal of applied biology, 2(1), 2018 35 0.136 (p> 0, 05). this means that there is no significant interaction effect of ripening method and ripeninglength to β-carotene content. due to the lack of interaction, then it can’t do a further test or post hoc. interaction methods and duration ripening against the content of β carotene besides seeing the effect of method and ripening length, this research also observed the interaction between method and ripening length. the result of the interaction methods with long ripening length on the content of β carotene can be seen in table 6. table 6. results of anova interaction method and length of ripening length against gynecology β-carotene fe’i banana source the sum of squares db average squares f sig. (p) rm and rd 1.966 2 0.983 2,305 0.136 description: rm = ripening method; rd = ripening duration results of analysis of variance in table 6 shows that the interaction between the old method of ripening the ripening length does not have a significant difference (p> 0, 05). this means that there is no significant interaction effect of ripening method and ripening length to β-carotene content. due to the lack of interaction, then it can’t be tested further. discussion effect of ripening methods against the content of β-carotene in the fe’i banana (musa troglodytarum l.) the content of β-carotene in the fe’i banana controls according image 1 of 10 3742 mg / kg increased after ripening. the content of β-carotene are needed in the body according to the national health interview survey, the average intake for men of 2.9 mg / day, while for women the average adult 2.5 mg / day, and to reduce risk of chronic disease, required 3-6 mg/day. when compared with the results of the study, it can be concluded that in 1 piece of fe’i banana provides β-carotene needed for the availability of body nutrients. ramadhani et al. (2012) describes the function of β-carotene that protects the eyes from cataract attacks. β-carotene and other carotenoids that are not converted into vitamin a, has antioxidant properties, so as to maintain the integrity of the body's cells. based on statistical analysis, a significant difference (p <0, 05) average β-carotene content of the control with the treatment method of ripening. these results explain the benefits of food processing processes disclosed by palupi et al. (2007) process that can be profitable by processing of some components of the nutrients contained in the food material, the change in the levels of nutrient content, improved digestibility and availability of nutrients and reduction of various compounds anti-nutrition contained therein. in this study, the results of a further duncan test shows the difference between ripening method and the method of β-carotene content of fuel is higher. according socaciu in meiliana et al. (2014), processing food causes changes in food matrices that produce a negative influence (β-carotene damage due to isomerization and oxidation) or positive (increase the availability and bioavailability of β-carotene). food processing process in this research yields positive influence with increasing of bioavailability of β-carotene. the positive effects on ripening bananas fruits are also explained by studies conducted engelberger et al. in fungo & pillay (2011) which shows that higher carotenoids content was international journal of applied biology, 2(1), 2018 36 found in samples of the ripe bananas than the unripe bananas. not only on fe’i banana, there is also aresearch revealed that ripening canimprove bioavailability carotenoids in carrots, spinach and tomatoes (stahl & sie; stone et al. in junpatiw et al., 2013). duration of ripening against the influence of β-carotene content in the fe’i banana (musa troglodytarum l.) according to begum in rahayu & pribadi (2012), although heating is the most important way to extend the shelf life of a material, but it can affect the degradation of the food substances contained in the material and this is highly dependent on the weight / duration of the heating process. the effect of heat on nutritional value is not only influenced by the temperature factor alone, but also influenced the duration of heating length. actually there is no difference in the nutritional value of a material due to heating, as long as the heating is applied is not excessive and not too long. any processing method selected, retention carotenoids decreased with process length longer, temperature higher processing and the process of cutting or sublimate. reduce ripening length, temperature, and lag length between peeling, cutting and smooth can improve retention significantly. high temperature / length processing which is short is a good alternative (dutta, et al., 2005). in line with the opinion of the baloch et al. in madalena et al. (2007) describes the longer warming length causing greater thermal isomerization so that the activity of vitamin a activity decreases. in contrast to the results of this study, where the higher temperatures due to increasing ripening length actually increasing the content of βcarotene. this is presumably because the fe’i banana burnt sky without any previous physical treatment such as stripping of the skin of the fruit, so the heat of the treatment methods of ripening (fire and water vapor) does not reduce the content of β-carotene in the fruit. ripening methods effective against the content of β-carotene on fe’i banana (musa troglodytarum l.) ripening is one of the simple and easy process of heat treatment. ripening can be done with a medium of hot water called boiling or steam heat or so-called steaming. steaming will also reduce nutrients but by less than the boiling process. warming up with steaming is some lengths uneven because the ingredients on the edges usually undergo excess steaming, while in the middle it has less steam (rahayu & pribadi, 2012). when compared with the control of bananas that are not given any treatment, steamed banana sky rods have a higher content of β-carotene. thus, the steaming process is known to increase the content of β-carotene, but not as much as the β-carotene content by the fuel method. this is consistent with studies of howard et al. (1999) explains that the steaming process, few carotenoids migrate to the water as released from protein carotene its binding, or protein complex β-carotene slightly soluble in water. therefore the steaming process using steam, then when steaming β-carotene slightly soluble in water vapor so that its contents are not too large compared with the controls. further test results of duncan β-carotene content for the treatment of ripening methods differ significantly with the controls, and differed significantly from the steam method. in simple terms can be explained that the content of β-carotene fuel control with different ripening methods is very significant, whereas the control with steamed ripening method is not too different significantly. in addition, the β-carotene content of the steam method by the fuel method did not differ significantly. this proves that the growing process international journal of applied biology, 2(1), 2018 37 of ripening in the community about the banana of sky stick that is burned for consumption actually increases the content of β-carotene in it. actually, during the ripening length of 15 minutes there is also an increase in the amount of nutrient content, but statistically it is not significantly different with the ripening length of 10 minutes. thus, the effective length of length taken is at 10 minutes. this is in accordance with the habits of people who cultivate bananas sky by burning sticks until his skin broke. in this study, the length duration of 10 minutes of banana skin stick sky began to break. the effective durationof ripening against βcarotene content of fe’i banana (musa troglodytarum l.) in the long ripening length 10 minutes, with decreasing water content, the heat treatment with a long long length increase the content of β carotene fe’i banana. actually, during the ripening length of 15 minutes there is also an increase in the amount of nutrient content, but statistically it is not significantly different with the ripening length of 10 minutes. thus, the effective length of length taken was at 10 minutes. this is in accordance with the habits of people who cultivate bananas sky by burning sticks until his skin broke. in this study, the length duration of 10 minutes of banana skin stick sky began to break. mulyatiningsih (2007) explains that ripening with high temperatures can cause liquids that are in the food not much is lost. the fluid inside is retained by the hardened and caramelized food layer. as with the results of this study, the high temperature is proportional to the length of ripening length. this causes the longer ripening length and the higher the temperature, then the nutrient content in the fe’i banana is not lost. conclusions old ripening method and ripening length are statistically each showed significant differences (p <0, 05) on the content of β-carotene. grilled ripening method and the duration of 10 minutes is more effective against β-carotene content of fe’i banana. acknowledgment thanks submitted to the head of laboratory of chemistry faculty of science, pattimura university and staff; director of the postgraduate pattimura university, chairman of the biology education studies program, with the whole staff lecture as well as all those who helped. references dutta, d., utpal r. c. & runu c. 2005. structure, health benefits, antioxidant property and processing and storage of carotenoids. african journal of biotechnology, 4(13):1510– 1520. fungo, r. & pillay, m. 2011. β-carotene content of selected banana genotypes from uganda. african journal of biotechnology, 10(28): 5423–5430. hiariej, a. & arumingtyas, e.l. 2013. genetic variation analysis of fei banana (musa troglodytarum l.) from the maluku islands based on cpdna rbcl gene sequences. proceedings: international graduate research conference 2013, chiang mai university, thailand. international journal of applied biology, 2(1), 2018 38 howard, l. a., wong, a. d., perry, a. k. & klein, b. p. 1999. β-carotene and ascorbic acid retention in fresh and processed vegetables. journal of food science, 64(5): 341-363. junpatiw, a., lertrat, k., lomthaisong, k. & tangwongchai, r. 2013. effects of steaming, boiling and frozen storage on carotenoid contents of various sweet corn cultivars. international food research journal, 20(5): 2219–2225. madalena., heriyanto., hastutidan, s.p. & limantara, l. 2007. the effect of heating time to the content of pigments and vitamin a in cassava (manihot esculenta crantz) and ceararubber (manihot glaziovii muell. arg) leaves. indo. j. chem. 7(1): 105–110. meiliana., roekistiningsih & sutjiati, e. 2014. effect of processing process leafsingkong (manihot esculenta crantz) with various treatment of β-carotene. indonesian journal of human nutrition,1(1):23–34 [in indonesia] mulyatiningsih, e. 2007. basic cooking technique. yogyakarta state university, yogyakarta. [in indonesia] musfiroh, i., indriyati, w., muchtaridi & setiya, y. 2009. proximate analysis and stipulation of β-carotene content in dutch eggplant jam (cyphomandra betacea sendtn.) with visible spectrophotometric method. padjadjaran university, bandung [in indonesia] naid., tadjuddin., andi muflihunna. & madi, m. i. o.. 2012. analysis of the content of βcarotene in pare (momordica charantia l.) origin of ternate by spectrophotometric uvvis. majalah farmasi dan farmakologi, 16(3):127–130. [in indonesia] palupi, n. s., zakaria, f. r. & prangdimurti, e. 2007. effect of processing on food nutrition value. bogor agricultural institute, bogor [in indonesia] rahayu, e. & pribadi, p. 2012. levels of vitamin and mineral in fresh flesh and wet sweetened karika dieng (carica pubescens lenne & k. koch). biosaintifika, 4(2): 89–97. ramadhani, g. a., izzati, m. & parman, s. 2012. analysis of proximat, antioxidant and preferred cereal foods from flour essentials (zea mays l.) and pumpkin flour (cucurbitamoschata durch). buletin anatomi dan fisiologi, 20(2): 32–39. [in indonesia] samson, e., h. semangun & f. rondonuwu. 2013. analysis of carotenoid content of crude extract of tongkat langit banana fruit (musa troglodytarum) using nir spectroscopy (near infrared). traditional medicine journal, 18(1):17–21. 2(1)40-45 contact : agusrahman ekaputra abas albusrahman@gmail.com � international journal of applied biology 40 abstract the biosynthesis of gold nanoparticles by using algae spirogyra peipingensis was conducted. this research aimed to determine biosynthesis of gold nanoparticles by using algae spirogyra peipingensis with different concentration and incubation time. the synthesis of gold nanoparticles using haucl4 solution with variations of concentration 5 ppm, 10 ppm, 15 ppm and 20 ppm respectively in 1 liter of aquabidest. algae spirogyra peipingensis was grown in haucl4 medium with the addition of 0.2 gr sulfahri-01 nutrient. then each 5 gram spirogyra algae incubated in haucl4 medium with the addition of sulfahri-01 0.2 gram nutrients. 4 hours long incubation under exposure to sunlight. nanoparticle size determination is done by looking at the color that appears in the solution. from the results of the research, it is known that spirogyra peipingensis algae is able to synthesize gold nanoparticles characterized by the color change in algae biomass from green to purple color after being treated and forming gold nanoparticles with size 40-60 nm. the best-used haucl4 consent is 5 ppm with the smallest particle size. issn : 2580-2410 eissn : 2580-2119 the potential of algae spyrogyra peipingensis to produce nanogold in batch culture medium agusrahman ekaputra abas1, chitriani armidha2, arbaina syahdinnur1 1 department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia 2 department of chemistry, faculty of mathematics and natural sciences, hasanuddin university, makassar, indonesia introduction the development of nanotechnology applications has driven the industrial revolution globally. it has been reported that more than 400 companies worldwide have undertaken nanotechnology research and development activities. as a relatively new discipline, globally nanotechnology research and applications are growing very rapidly (hoerudin & irawan, 2015). nanoparticles are a scope of nanotechnology. it is called a nanoparticle when the particle size is in the range of 1-100 nm. with unique properties and sizes, nanoparticles have great potential as a material that is superior in the future. the nanoparticle material can be organic material such as 2.5 nm dna, 50 nm protein, 100 nm virus type (pantidos & horsfall, 2014), or inorganic materials such as semi-conductor open access international journal of applied biology keyword intracelullar, spirogyra peipingensis, haucl4, biosynthesis. article history received 8 june 2018 accepted 14 july 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 41 nanoparticles (titanium oxide and zinc oxide), and precious metal nanoparticles (silver and gold) (shaileyee & c mandal, 2014). according to rohiman et al (2014), in the world of nanotechnology, research on gold nanoparticles has attracted the attention of many world researchers because of its superiority in its various applications. gold nanoparticles (aunps) are gold ions that are reduced to non-charged gold and nano-sized (maruani, 2013). aunps have prominent advantages compared to gold particles, which have optical and electronic properties with low toxicity (rohiman, et al, 2014) in addition, aunps can be used as a water purifier (qian, et al, 2013), carriers of cancer-killing drugs (alhalili, et al, 2017), chemical and biological sensors (qin, et al, 2018), catalysts (amdouni, et al, 2018), biohydrogen production and various biomedical applications (zhang & shen, 2007). the production of aunps can be carried out by various chemical methods, but the production of eco-friendly nanoparticles are being intensively developed (sovawi, et al., 2016) through biosynthetic pathways using biological agents such as yeast, fungi, bacteria, plants, plant extracts and algae (thakkar, 2010). algae are thallophyta organisms that have been known to have a lot of potential. one of the most easily found algae in indonesia is spirogyra peipingensis algae. this algae has the potential to reduce both organic and inorganic compounds. based on the bahri (2017) study, spirogyra pepingensis algae can significantly reduce toxicity, absorbing heavy metals (gupta & rastogi, 2008), and reducing textile waste (özer, et al., 2006) that have mechanisms such as activated carbon. the use of algae as a gold metal bioreductor has previously been done by roychordhury (2016). algae used derived from the group of prokaryotic algae (cyanobacteria) namely anabaena sphaerica and eukaryotic green algae namely chlorococcum infusionum. the alignment of cell components in algae such as carotenoids, polysaccharides, proteins and pigments in chloroplasts / thylakoids has a major role as reducing agents in the biosynthesis of nanoparticle metals (roychordhury, et al., 2016). carotenoids, polysaccharides, proteins and chloroplast pigments are also found in spirogyra algae (tipnee, 2015). therefore, spirogyra peipingensis algae is believed to have the potential to reduce gold particles to nano-sized gold particles. materials and methods algae preparation alga spirogyra peipingensis obtained from the rice field area around the city of makassar. then the algae is cleaned and identifiable under a light microscope before being cultured on growth medium sulfahri-01 (3000 lux, 12:12) which later becomes algae stock during the research phase. preparation of haucl4 solution 0.1 grams of gold metal dissolved into 20 ml aquaregia (3hcl: 1hno3). heat over hot plate until completely dissolved. then dilute it into a 500 ml measuring flask to make a parent solution of 200 ppm. the haucl 4 gold mains solution was diluted with aquabides with variations in dilution concentration of 5 ppm / l, 10 ppm / l, 15 ppm / l, 20 ppm / l in 1000 ml aquarium. international journal of applied biology, 2(1), 2018 42 incubation of algae spirogyra peipingensis into gold medium 5 gr of spirogyra peipingensis biomass were incubated on haucl4 gold medium and 0.2 gr of additional surlfahri-01 nutrient media under exposure to 4 hours of sunlight. the top of the aquarium is covered with a cling wrap that is sufficiently ventilated for air circulation. determines the size of gold nanoparticles after spirogyra peipingensis algae incubation for 4 hours, the yellowish amorphous media solution was changed. the color variations formed can be used to determine the particle size of the synthesized product. this refers to the color guide of the solution to determine the size of the gold nanoparticles published by noutarianni (2013). results and discussion algae preparation fresh algae biomass spirogyra peipingensis taken in the rice fields sudiang, makassar, indonesia. then cleaned and identified under a magnification light microscope 100 times above sedgwick rafter counting chamber (src). determination of spirogyra peipingensis algae type was based on the publication (zarina, et al, 2007) with unbranched filamentous morphology, with vegetative cell width 104-157 μm and length 156-200 μm and 5-7 pyrenoid. after identification, then alga spirogyra peipingensis in culture in 0.2 gr / l medium sulfahri-01 with water height 20 cm at 3000 lux lighting lamp with a duration of 12:12. the composition of the media has been tested based on sulfahri, et al, (2016). from several variations of water height (10,20,30 cm), 20 cm is the most ideal water level with the highest level of productivity. algae incubation 5 gr biomass algae spirogyra peipingensis was incubated in a medium composed of 0.2 gr/l sulfahri-01 in several concentrations of haucl4 with a series of 5 ppm, 10 ppm, 15 ppm and 20 ppm. each solution was placed in a 20 cm glass tank with a capacity of 1000 ml and placed under 4 hours of sun exposure. the sulfahri-01 medium is the most superior medium among several types of mediums compared to having high nitrogen content which may play an important role (sulfahri, et al, 2016) after incubation for 4 hours, a change of color occurs in spirogyra peipingnsis biomass. the algae biomass, which was initially light green, became dark purple and was confirmed to have died from exposure to the metal mixture in its incubation medium (fig.2a,2b). this change indicates that algae spirogyra peipingensis can positively synthesize. this is in accordance with the results obtained by parial, et al (2012) who trace the types of phormidium valderianum algae, p. tenue, microcoleus chthonoplastes, rhizoclonium fontinale and ulva intestinalis. these five algae can positively synthesize intracellular gold nanoparticles. according to parial, et al, (2012) the five algae potentially as "bionanofactories" and this is evident from the visual transformation of the algal thalli from green to deep purple. international journal of applied biology, 2(1), 2018 43 fig. 2a. before incubation fig. 2b. after incubation the ability of algae as a reducing agent to synthesize gold nanoparticles is much in play by the variety of cellular components such as carotenoids, polysaccharides, proteins and complete pigments in chloroplasts / tilakoid (royconduri, et al, 2016). the reduction of the metal ions occurs upon the surface of the algal cell as well as on the cytoplasmic membrane, leading to the formation of gold nanoparticles (senapati, et al, 2012). the exact process of intracellular formation of gold nanoparticles by alga biomass is not yet fully understood (parial, et al, 2012). determines the size of gold nanoparticles fig. 3a. variations colour of media solutions 3b. gnps sizing guide in this way (fig. 3a), it is possible to deduce the size of the au nps produced, ranging from 40-60 nm. this refers to the gnps sizing guide based on the color of the solution (fig.3b) from queensland university of technology (notarriani, 2014). from this research, it can be seen that the bigger haucl4 concentration, then the size of gnps in the file will be greater because the particle size generated in the concentration series 5,10,15,20 ppm respectively contribute the change of color dry awaiting young to blue, which describes the existence the addition of the diameter of the nanoparticles, therefore, the 5 ppm concentration is considered the best as an incubation medium in producing gold nanoparticles. according to parial (2012), all applications of gold (or other) nanoparticles thus demand a well-defined size and accurate measurement of these parameters is correspondingly important. the optical property of surface plasmon resonance is directly international journal of applied biology, 2(1), 2018 44 dependent on the size of the gold nanoparticle: ~20nm particles show an orange-red colour but the colour gradually shifts to blue when particle size increases to ~100 nm. plasmon resonance also changes with aspect ratio. conclusions from the study, it can be concluded that spirogyra peipingensis algae can synthesize gold nanoparticles. incubation of haucl4 medium 5 ppm 0.2 g/l sulfahri-01 best produces gold nanoparticles. from the color of the solution obtained, can also be concluded the smaller the concentration of haucl4 the smaller the size of the particles produced. the intracellular gold nanoparticles biosynthesis has great potential to develop to industrial scale because it is easier, cheaper, and environmentally friendly. acknowledgment the authors gratefully acknowledge financial support from the ministry of research, technology and higher education of indonesia with project pkm (program kreativitas mahasiswa). references alhalili, z., figueroa, d., johnston, m. r., shapter, j., & sanderson, b. 2017. effect of modification protocols on the effectiveness of gold nanoparticles as drug delivery vehicles for killing of breast cancer cells. australian journal of chemistry. 69(12) : 1402-1412. amdouni, s., cherifi, y., coffinier, y., addad, a., zaïbi, m. a., oueslati, m., & boukherroub, r. 2018. gold nanoparticles coated silicon nanowires for efficient catalytic and photocatalytic applications. materials science in semiconductor processing. 75 : 206213. bahri, s., kamsinar, sarioja, edi, t., nurul, f. 2017. application of cigeratte filter from algae spyrogira peipingensis to reduce nicotine, tar, and carbon monoxide. international journal of applied biology, 1(1): 1-8. gupta, v. k., & rastogi, a. 2008. biosorption of lead from aqueous solutions by green algae spirogyra species: kinetics and equilibrium studies. journal of hazardous materials, 152(1), 407-414. hoerudin & irawan b. 2015. the prospect of nanotechnology in building food security. jakarta (id): iaard press. 49-67 [in indonesia] maruani, i. 2013. au-nanoparticle preparation by utilizing green tea leaf extracts for melamin pollution detector. doctoral dissertation, upi [in indonesia] özer, a., akkaya, g., & turabik, m. 2006. biosorption of acid blue 290 (ab 290) and acid blue 324 (ab 324) dyes on spirogyra rhizopus. journal of hazardous materials. 135(1): 355364. pantidos, n., & horsfall, l. e. 2014. biological synthesis of metallic nanoparticles by bacteria, fungi and plants. journal of nanomedicine & nanotechnology, 5(5): 1-9. qian, h., pretzer, l. a., velazquez, j. c., zhao, z., & wong, m. s. 2013. gold nanoparticles for cleaning contaminated water. journal of chemical technology and biotechnology, 88(5), 735-741. international journal of applied biology, 2(1), 2018 45 rohiman, a., buchari, m., juliastuti, e., & idris, i. (2014). synthesis, characterization, and applications of gold nanoparticles (aunps) on the growth of silicon nanowires (sinws). research and development on nanotechnology in indonesia, 1(2), 74. [in indonesia]. senapati, s., syed, a., moeez, s., kumar, a., & ahmad, a. 2012. intracellular synthesis of gold nanoparticles using alga tetraselmis kochinensis. materials letters, 79, 116-118. shaileyee & c mandal, subhash. 2014. synthesis and therapeutic activity of silver nanoparticles using bio-based protocol. the pharma review. sovawi, a. c., harjono, h., & kusuma, s. b. w. (2017). synthesis of gold nanoparticles with bioreductor red guava fruit extract (psidium guajava l.). indonesian journal of chemical science, 5(3), 169-173 [in indonesia]. sulfahri, mohamad amin, sutiman bambang sumitro & murni saptasari. 2016. comparison of biomass production from algae spirogyra hyalina and spirogyra peipingensis, biofuels, 8:3, 359-366. thakkar, k. n., mhatre, s. s., & parikh, r. y. 2010. biological synthesis of metallic nanoparticles. nanomedicine: nanotechnology, biology and medicine, 6 (2), 257-262. tipnee, s., ramaraj, r., & unpaprom, y. 2015. nutritional evaluation of edible fresh water green macroalga spirogyra varians. emer. life sci. res, 1, 1-7. zarina, a., masud-ul-hasan, and mustafa shameel. 2007. diversity of the genus spirogyra (zygnemophyceae shameel) in the north-eastern areas of pakistan. proc. pakistan acad. sci. 44(4): 225-248. zhang, y., & shen, j. 2007. enhancement effect of gold nanoparticles on biohydrogen production from artificial wastewater. international journal of hydrogen energy, 32(1), 17-23. 3(1)1-6 contact : pramita laksitarahmi isrianto laksitarahmi@yahoo.co.id © international journal of applied biology 1 abstract one of the efforts to optimize the utilization of natural materials that is used them as medicinal plants, including dlingu (acorus calamus l.) and garlic (allium sativum) which have the potential as antioxidants. this study aims to determine the effectiveness of a combination extract of acorus calamus l. (dlingu) and allium sativum (garlic) on antioxidant activity. this research is an experimental research which is the test of antioxidant content and antioxidant activity test with 5 series of concentrations and 4 times of repetition. the research materials, namely dlingu stem and single clove garlic tubers were extracted, phytochemical screening was carried out and then antiradical free ic50 was determined. based on the test results of the chemical extract of dligu and single clove garlic, the highest content was alkaloid 5.16%, tannin 4.05%, saponin 3.01%, and flavonoid 2.18%. the test results of the chemical antioxidant activity of extracts dligu and single clove garlic contain ic50 values of 17.062 ppm, including a very strong class of antioxidants. issn : 2580-2410 eissn : 2580-2119 antioxidant activity in combination extract of acorus calamus l. (dlingu) and allium sativum (garlic) pramita laksitarahmi isrianto1 & putu oky ari tania2 1educational program of biology, faculty of language and science, wijaya kusuma university, surabaya, east java, indonesia 2biomedical department and biomolecular research, faculty of medicine, wijaya kusuma university, surabaya, east java, indonesia introduction the development efforts toward natural materials as traditional medicine is conducted immediately to find out the newest medicines in overcoming the various of health problems which is caused by the modern medicine contain chemicals that have side effects either direct effects or indirect accumulated effects (nurhasan, 2015). the utilization of natural materials which is used as medicine rarely causes the side effects. one of the efforts is to optimize the utilization of natural materials which have the potential as medicine, including dlingu (acorus calamus l.) from araceae family. dlingu (acorus calamus l.) is a water plant which is found excessively as the wild plant at the bank, marsh and the flooded material during the year. biological activity that is resulted by dlingu plant is able to produce the compounds particularly from the class of terpen, flavonoid, open access international journal of applied biology keyword antioxidant, dlingu, single clove (male) garlic. article history received 12 december 2018 accepted 10 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 2 alcohol saponin, aldehyde, and phenol such as, karvakrol, eugenol, timol, sinamaldehyde, sinamat acid, and perilaldehyde, volatile oil (nurhasan, 2015). the high and low quality of volatile oil depends on the area of dlingu rhizome itself. according to afifah (2015), combination extract of acorus calamus (l.), curcuma mangga val, and allium sativum (linn) is able to keep down candida albicans. antioxidant can be found from natural materials that were dominated by land plant as medicinal plant such as dlingu and garlic. chemical substances of garlic which have biological activity and the benefits in treatment, namely; the compound of organosulfur (the compound of s-ak(en)-ill-sistein sulfoxide (acsos) such as allicin. the active substances that contain in garlic have the special utility as the medicine of high blood pressure, to assuage a headache, to reduce the cholesterol, as the medicine of stomach disorder, as anti-cancer, and as anti-inflammation (ikhtiarsyah, et al., 2014). moreover, the male species of garlic (allium sativum) is besides as herbal antibiotic, also able to treat the wound, reduce the inflammation and does not cause the scar tissue, accelerate the decreasing of inflammation symptom ( florid symptom) at the wound. it is caused by the content of active substance -allicin which has the function as antiinflammation. garlic is enriched by vitamine b1, b2, b3, folate, vitamine c, calcium, iron, magnesium, mangan, phosphor, calium, natrium and zinc (wadrianto, 2017). based on the background above, the utilization of combination extract of dlingu and garlic has not yet been observed excessively, if it is compared to the only individual plant. therefore, the combination of extract is conducted because on each of plants has a potential as antioxidant, thus it is expected to prevent and treat infection that is caused by the activity of normal floral bacteria, such as s.aureus and candida albicans. later, from the combination extract of both plants is also expected to make the supply like as embrocating oil for antiinflammation in increasing the value of product. based on some previous researches, both plants dlingu and single clove garlic have the potential as antioxidant. the main function of antioxidant is to minimize the occurrence of oxidation process either in the foods or the bodies. besides antioxidant is used in industry of pharmacy, it is also used widely in foods’ industry, petroleum’s industry, rubber’s industry, and so on (sayuti & rina, 2015). in the bodies, antioxidant is also expected able to obstruct the oxidation process. oxidation process which occurs continuously can affect the degenerative disease and aging. this research aims to know the antioxidants’ activity in combination extract of acorus calamus l. (dlingu) and allium sativum (garlic). materials and methods the research was done in laboratory of biology fbs wijaya kusuma university of surabaya and laboratory of research association and industrial consultation (bpki). it was conducted on june until september 2018. the sample that was used is the extract of single clove of garlic (allium sativum) and the extract of dlingu rhizome (acorus calamus l.) by using descriptive qualitative method. tools that were used during the research are the cutter, grinder, oven, big tray, analytical balance, watch glass, erlenmeyer 500 ml, 250 ml, aluminium foil, shaker, graduated glass 100 ml, beaker glass 100 ml, distillated paper, filter buchner, rotary evaporator, spectrophotometer uv-vis, incubator, vortex, autoclaf, disk paper, ose needle, petri cup, reaction tube, spatula. the steps that were conducted in this research are: international journal of applied biology, 3(1), 2019 3 preparation of sample the sample that was used in this research is dlingu rhizome and single clove garlic or is usually called the male garlic which obtained from traditional market buduran sidoarjo. the sample of dligu rhizome and single clove garlic were washed previously until clean, then, dried with the wind. furthermore, was conducted the process of sorting, washing, cutting into the small pieces, drying, grinding and filtering thus dry powder of each plants was obtained. maceration method the sample as much 100 gr was macerated by 70% ethanol solvent and was waited during 1x24 hours, after that, the obtaining extract was filtered by using vacuum filter buchner. then, the obtaining filtrate was putted into the rotary vacuum evaporator during 24 hours until thick extract was obtained. determining photochemistry analysis of alkaloid, tannin, saponin and flavonoid the extract was done in laboratory of research association and industrial consultation (bpki). test of antioxidant’s content for determining the antioxidant’s activity of each concentration, solvent sample was taken 1 ml by using micro pipette, putted into reaction tube, then added 4 ml dpph solvent 50 µm. the mixture was homogenized and left during 30 minutes in the dark place, the absorption was measured by using spectrophotometer uv-vis in wave length 517 nm. the antioxidant’s activity of sample was determined by the large of radical absorption resistance dpph through percentage calculation (%) inhibition of absorption dpph (renhoran, 2012). ic50 value was calculated from regression linear curve between the concentration of various concentration toward percentage (%) of antioxidant’s activity (kuntorini & maria, 2010). results and discussion on this research, the aim is to determine the existence of antioxidant’s activity in combination extract of dlingu and single clove garlic. the sample of this research is dlingu rhizome and single clove garlic or is called the male garlic. previously, the sample was sorted, then, prepared the sample of dlingu stems as much 350 gram and male garlic tubers as much 200 gram. after both sorted samples which were cut into small pieces were blown to bits by using grinder to expand the surface thus to ease the extraction process and to produce the excessive extraction. the method that was used in extracting the sample is maceration method by using ethanol solvent because its characteristics of high polarity, low boiling point, and able to extract the material more excessive than the other organic solvent. based on the result of organoleptic on the table 1, was found the result as follows: table 1. organoleptic of combination extract of dlingu and male garlic no. organoleptic the information of maceration result dlingu male garlic combination 1 form liquid liquid liquid 2 color deep brown (sepia) jet deep brown brown international journal of applied biology, 3(1), 2019 4 the test of photochemistry compound became the first base in detecting the existence of a secondary metabolic compound in the sample. based on the test result, the compound in combination extract of dlingu and garlic can be seen on the table 2. table 2. test of compound in combination extract’s sample of dlingu and male garlic. no. contents percentage (%) 1 alkaloid 5,16 2 tannin 4,05 3 saponin 3,01 4 flavonoid 2,18 the test result of compound in combination extract’s sample of dlingu and single clove garlic on this research showed the excessive contents were alkaloid 5,16 %, tannin 4,05%, saponin 3,01%, and flavonoid 2,18%. the compound of flavonoid, xenolic, tannin are the responsible compound toward the antioxidant activity. the research result of prasanto et al (2017) showed that the single clove garlic has the stronger antioxidant ability rather than the other variety of garlic. alkaloid compound has the great role in antioxidant, particularly indol group because it has the ability to stop the free-radical chained reaction efficiently. indeed, the other alkaloid compounds which have the characteristic as antioxidant are quinolone, caffeine which acted as the reducer of radical hydroxide; and melatonin for radiation and toxicity of medicine (harrizul et al., 2013). based on the observed research, it also showed that the extract of ethanol and chloroform of the garlic extract contained much compounds of alkaloid and triterpenoid (azzahra, 2015). this fact was also showed on silistyorini’s research (2015), suggested that the result test of higher potential antioxidant in ethanol extract of garlic (allium sativum linn) has ic50 value as much 151,1 ppm. the result of activity test in combination extract of dlingu and single clove garlic on this research was categorized strong 17,062 ppm (table 3). from the table 3, it also can be seen that the inhibition % of concentration 50 ppm was as much 14,66, concentration 100 ppm as much 37,45, concentration 150 ppm as much 50,82, concentration 200 ppm as much 68,72, and concentration 250 ppm was as much 83,14. the resulted average was highest on the concentration 250 ppm. on the table 4.4 above, the result of antioxidant activity in combination extract of dlingu and male garlic showed that the strongest antioxidant activity was caused by ic50 value < 50 µg/ml, as cited on phongpainchit et al research (2007) that the antioxidant activity was very strong if ic50 value ≤ 50 µg/ml, it was strong if ic50 value 50-100 µg/ml, it was medium if ic50 value 100-150 µg/ml, it was low if ic50 value 151-200 ppm, meanwhile if ic50 value ≥ 200 ppm then its antioxidant’s activity was very low. based on the research which was conducted by kurniati (2013) explained that the compound of alkaloid, saponin, flavonoid, and phenolic in 70% ethanol fraction of the leaf premna cordifolia linn 3 scent exclusive exclusive smell of garlic exclusive smell international journal of applied biology, 3(1), 2019 5 was responsible compound toward antioxidant activity. some of dligu rhizomes (acorus calamu l.) also emphasized that the rough extract of acorus calamu l. by using dpph method with ethanol solvent showed the medium antioxidant activity with ic50 value 137,7 mg/l (hasan, 2015). table 3. test result of antioxidant’s activity in combination extract of dlingu and male garlic figure 1. linear regression curve of antioxidant test in combination extract of dlingu and single clove garlic by using dpph method based on figure 1 above, it showed that the higher of ppm concentration then % value of inhibition was more increasing, and it showed the existence of relationship between the concentration and % inhibition. antioxidant activity was influenced by compound structure on sample, thus the occurrence of inhibition (%) can be influenced by compound structure on sample. electron relocation of secondary metabolic compound such as alkaloid and phenol occured through the resonance in radical structure of antioxidant, thus prevented the forming of new radical and obstructed the chained reaction of free radical (cholisoh and utami (2008) as cited by sulistyorini (2015). from the result test above, combination extract of dligu rhizome and single clove garlic tuber contained alkaloid, tannin, saponin and flavonoid (table 2) which quite high thus, these compounds were expected in having the potential as anti-inflammation compound. combination extract of both can catch radical with higher extract concentration, thus the result of ic50 value was categorized strong. y = 2.9502x 0.3343 r² = 0.9926 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 80 90 sample concentration (ppm) abs. average % inhibition ic50 (inhibition concentration 50%) antioxidant activity 50 0,734 14,66 17,062 ppm very strong (ic50< 50 ppm) 100 0,538 37,45 150 0,423 50,82 200 0,269 68,72 250 0,145 83,14 international journal of applied biology, 3(1), 2019 6 conclusions combination extract of acorus calamus l. (dlingu) rhizome and allium sativum (single clove garlic) tuber had the very strong influence toward antioxidant activity with antioxidant value as much 17,062µg/ml. references azzahra, velayaty l. 2015. profile of thin layer chromatography in ethanol extract of mango’s ginger (curcuma mangga val), (acorus calamus) rhizome, garlic tuber (allium sativum) and their ingredients. minithesis. biology department, fst. islamic state university of maulana malik ibrahim.malang harrizul, r., ernita w.s. and rusdi.2013. ratio influence of ethanol-water solvent toward the total content of phenolic compound and antioxidant capacity of soursop leaf’s extract (annona muricata l.) .journal of sciences and pharmaceutical technology.page 35-42.issn:1410-0177. hasan, muhammad nur. 2015. the influence of rhizome extract (acorus calamus l) in some of organic solvents toward antioxidant activity and antifungus in vitro. https://www.researchgate.net/publication/308916822. ikhtiarsyah, yusrista gilang, inna a, maria d.s., endang sulistyiorini. 2014. bawang putih/ garlic (allium sativum l.).http://ccrc.farmasi.ugm.ac.id/en/?page_id=441. it was accessed on 2nd march 2018. kuntorini, evi mintowati, maria dewi astuti. 2010. determining antioxidant activity in ethanol extract of eleutherine americana merr.. journal of science and applied chemistry, vol.4, number. 1:15 – 22. kurniati, ruth indah. 2013. antioxidant activity test of (premna cordifolia linn) leaf by using dpph method (2,2-difenil-1-pikrilhidrazil). publication document. pharmacy program. medical faculty. tanjungpura university. pontianak. nurhasan, muhammad.2015. the influence of rhizome extract (acorus calamus l) in some of organic solvents toward antioxidant activity and antifungus in vitro. minithesis. uin syarif hidayatullaaah.jakarta. prasonto,djuned, eriska riyanti, meirina gartika. 2017. antioxidant activity test of garlic extract (allium sativum).odonto dental journal. volume 4(2).2017. renhoran, mawaddah. 2012. antioxidant activity and antimicrobe of extract sargassum polycystum. minithesis. ipb. bogor. sayuti, kesuma, rina yenrina. 2015. natural and synthetic antioxidant andalas university press.padang. international journal of applied biology, 3(1), 2019 7 sulistyorini, a. 2015. antioxidant and antifungus potential of garlic tubers extract (allium sativum l.) in some of organic solvents. minithesis. biology department, science and technology faculty, maulana malik ibrahim islamic state university. malang wadrianto, glori k. 2017. 12 amazing use of garlic. http://lifestyle.kompas.com/read/2017/11/01/110036020/12-manfaatmenakjubkan-dari-bawang-putih. it was accessed on 30th march 2018. contact : alpiani alpiani.pia@gmail.com 123 abstract capture fisheries production in sikka regency is still significant, with substantial pelagic fish such as tuna and skipjack. the advancement of the fisheries sector in the sikka regency is inseparable from the role of pt. karya cipta buana sentosa (kcbs) in its support of supporting facilities, including landing facilities for fishing fleets on the coast of the city of maumere and other facilities that support the fish marketing process to improve the welfare of the people of sikka district, especially people who make a living as fishermen. the flores sea, especially in the waters of maumere, is a tuna fishing area for anglers in the sikka district. the type of tuna that fishers mainly produce in the sikka district is yellowfin tuna. this study is 1) to identify the form of marketing channels in pt. kcbs 2) to analyze the value of tuna marketing margin at pt. kcbs. the method used in this research is a mixedmethod combining qualitative and quantitative methods. this research results in the form of a tuna marketing channel at pt. kcbs starts from fishers who go directly to suppliers, from suppliers to pt. kcbs then to retailers and consumers as end-users, tuna fish supply chain management at pt. kcbs is still not optimal due to uncertain ship berthing schedules and the absence of trust to build longterm cooperation and relationships or partnerships between fishers and suppliers; the tuna fish supply chain is directed at optimizing the management of auction activities in terms of developing information systems, fulfilling more demanding demands. responsive, strengthening sustainable fisherman partnerships whose results will support supply chain performance and the value of tuna marketing margins at pt. karya cipta buana sentosa (kcbs) is rp. 37,000 with a farmer's share value of less than 50%, and it can be concluded that the marketing margin is inefficient. issn : 2580-2410 eissn : 2580-2119 supply chain of tuna fishery resources (thunnus sp) in maumere district, sikka regency, east nusa tenggara province andi nur apung massiseng1, alpiani alpiani1*, andi ummung1, st. zaenab2 1 study program of fisheries agribusiness, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar, indonesia 2 study program of aquaculture, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar, indonesia introduction one factor that plays an essential role in determining the quality control process in the supply chain is distributing goods, starting from fish production on ships until the company receives the product. the handling of the product at each stage is a critical point that will determine the quality of the tuna loin product when the product arrives at the open access international journal of applied biology keyword supply chain; tuna; maumere; sikka district; kcbs article history received june 14, 2022 accepted july 7, 2022 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 6(1), 2022 124 company. a quality sorting (grading) process is carried out (jati et al. 2014). capture fisheries production in sikka regency is still significant, with giant pelagic fish such as tuna and skipjack. tuna fishing fishers are scattered in several places: wiring, nangahure, wolomarang village and hewuli village, west alok district. in maumere, sikka regency, there are around 600 tuna anglers and every year, the government helps 20 units of vessels measuring one to two gross tons (gt) for tuna fishers. one boat can be used by 2 to 3 anglers. for tuna production in 2019 in maumere, sikka regency, as much as 2,500 tons and skipjack tuna as much as 3000 tons out of the total output of 18,000 tons of captured fish. maumere, sikka regency, located in east nusa tenggara (ntt) province, is one of the income sources for capturing fisheries products in eastern indonesia. in 2019, marine fish production in sikka regency reached 19,287.3 tons with a value of rp. 351.8 billion. in addition to being sold in the sikka district, fishery production is also sold to several communities on the island of flores, such as in east flores district, ende district, nagekeo district, ngada district, and even sold to west manggarai district (labuan bajo). tuna fish in 2019 was 1930.5 tons with a value of 86.87 billion, and skipjack fish of 3150.75 tons with a production value of 44.11 billion. especially for tuna and skipjack, the products are sold outside the province of east nusa tenggara (ntt), such as on the islands of bali and java. they are exported abroad to export destination countries, namely japan, south korea, china, vietnam, malaysia and singapore. grdp of sikka regency is 12% of the value of fish produced by fishers in sikka regency, also contributing to pad of around 1.9 billion. as a strategic partner in efforts to improve the community's productive economy, anglers from the sikka district government will always support every step by the company in developing fisheries in the sikka regency area so that with the existence of pt. karya cipta buana sentosa (kcbs) maumere can help improve the economy of fishers in sikka regency. the flores sea, especially in the waters of maumere, is a tuna fishing area for fishers in the sikka district. the type of tuna that fishers mainly produce in the sikka district is yellowfin tuna. materials and methods this study uses a mixed-method, which is a combination of qualitative and quantitative descriptive methods. the qualitative descriptive analysis provides an overview of marketing channels, while quantitative research determines costs, profits, margins and marketing efficiency. data collection technique data collection techniques used by researchers in the research process are: 1. observation observation is a direct visit to the research location, namely at pt. karya cipta buana sentosa (kcbs) maumere sikka regency aims to determine the shape of the tuna marketing channel directly and to determine the factors that influence the amount of tuna marketing margin in sikka regency. 2. interview interviews are used as a data collection technique if the researcher wants to conduct a preliminary study to find problems that must be investigated. in-depth interviews were conducted with key informants, namely people with extensive knowledge of tuna supply chain management at fish auctions, especially at pt. karya cipta buana sentosa (kcbs) maumere. international journal of applied biology, 6(1), 2022 125 3. documentation according to arikunto (2019), the documentation method is looking for data on things or variables in the form of notes, transcripts, books, newspapers, magazines, inscriptions, meeting minutes, research journals and so on related to research. data analysis data analysis is simplifying data into a form that is easier to read and interpret. in this study, researchers used the data analysis method of the miles and huberman model in sugiyono (2016) interactively, which was divided into three steps, namely: data reduction the reduced data include all data regarding research problems. the reduced data will provide a more specific picture and make it easier for researchers to collect further data and look for additional data if needed. the longer the researcher is in the field, the more the amount of data will be, and the more complex and complicated it will be. therefore, data reduction needs to be done so that the data does not accumulate so as not to complicate further analysis. data presentation data presentation can be done through narrative descriptions, charts, relationships between categories and flow charts. the presentation of data in this form makes it easier for researchers to understand what is happening. in this step, the researcher tries to compile relevant data so that the information obtained is concluded and has a specific meaning to answer the research problem. good presentation of data is an essential step toward achieving accurate and reliable qualitative analysis. draw conclusions this stage is the stage of concluding all the data that has been obtained as a result of the research. conclusion drawing or verification attempts to find or understand the meaning, regularities, patterns, explanations, causal paths or propositions. after the verification, conclusions can be drawn based on the research results presented in the form of a narrative. drawing conclusions is the final stage of data analysis activities. drawing this conclusion is the final stage of data processing. the data analysis used is descriptive analysis technique and quantitative analysis. the descriptive analysis provides an overview of marketing channels, while quantitative analysis is used to determine costs, profits, margins and marketing efficiency is as follows: 1. to find out the marketing channels by using descriptive analysis. 2. to find out the costs, benefits and marketing margins of each marketing agency in various marketing channels, namely: a. marketing fee marketing costs are the sum of the marketing costs of each agency. marketing costs include transportation costs, loading and unloading costs, user fees and storage costs. so marketing costs can be calculated using the formula: bp = bp1 + bp2 + ... + ben information : bp = yellowfin tuna marketing costs (rp/head/kg). bp1, bp2,..., ben = marketing costs of each marketing agency (rp/head/kg). international journal of applied biology, 6(1), 2022 126 marketing advantage the advantages of each marketing agency involved in tuna marketing are formulated as follows: kp = mp – bp information : kp = yellowfin tuna marketing profit (rp/head/kg) mp = yellowfin tuna marketing margin (rp/head/kg). bp = yellowfin tuna marketing costs (rp/head/kg). marketing profit is the sum of the profits received by each marketing agency, formulated: kp = kp1 + kp2 + ... + kpn information : kp = yellowfin tuna marketing profit (rp/head/kg) a. kp1+kp2+...+kpn= profit of each yellowfin tuna marketing agency (rp/head/kg). b. marketing margin marketing margin is the difference between the price paid by consumers and the price received by anglers, which can be formulated as follows: mp = pr – pf information : mp = yellowfin tuna marketing margin (rp/head/kg). pr = price at the consumer level (rp/head/kg). pf = price at producer level (rp/head/kg). to determine the economic efficiency of tuna marketing, margin analysis and farmer's share were used. the margin analysis formula has been described above, and for the farmer's share, using the following formula: f = (pf/pr) x 100% information : ø f = share of price received by fishermen (%) pf = price at fisherman level (rp/head/kg). pr = price at the end consumer level (rp/head/kg). provisions: the marketing system is said to be efficient if the farmer's share value is more than 50%, and if the farmer's share value is less than 50%, then it is inefficient. the larger the marketing margin, the more inefficient the marketing system is. results overview of research site pt. karya cipta buana sentosa (kcbs) maumere is located on don slipi street, wailiti village, west alok district, sikka regency. pt. karya cipta buana sentosa (kcbs) maumere began to be built in the 1994 fiscal year by pt. central jakarta kcbs. on the way with domestic shareholders in 2015, it was taken over by foreign capital owners (pma) until now. initially, pmdn (domestic shareholder) kcbs, as a marine product management company, made domestic sales. since being taken over by pma, sales have become 90% of exports. pt. karya cipta buana sentosa (kcbs) maumere is a company processing tun a and skipjack tuna into ready-to-eat products such as sashimi, pocket, katsuobushi, negitoro, and several times sells whole or logs. international journal of applied biology, 6(1), 2022 127 pt. karya cipta buana sentosa (kcbs) maumere is currently led by mrs masayuki takaku, director theodorus e. u. hon, and commissioner yeung oi siong. currently pt. karya cipta buana sentosa (kcbs) maumere exports to several destination countries: japan, korea, vietnam, singapore, malaysia, hong kong and china. there are 114 employees at kcbs, consisting of 50 daily employees, pt. karya cipta buana sentosa (kcbs) maumere employs all local sons and cooperates with nearby smes such as animal feed processing. pt. karya cipta buana sentosa (kcbs) maumere has partner fishermen of 24 pole handline boats and 200 handline fishing boats. kcbs is a member of ap2hi (indonesian pole and handline fisheries association). athaillah et al. (2018) asserted that tuna processing companies are highly dependent on the catches of anglers, while the yields of fishermen themselves are highly dependent on climatic and weather conditions, the success of the net, and the presence of tuna. the company only obtains raw materials for tuna from fishers. description of interview results based on the analytical method used in this study, namely qualitative, quantitative and descriptive techniques, the researcher carried out the stages: data collection, reduction, data display and conclusion drawing. in the research process, the researchers interviewed (data collection) 10 supply chain actors or critical informants consisting of 3 anglers who own large boats, two collectors and five retailers. interviews were conducted by asking several questions that had been systematically arranged. in the end, the answers from each of these informants were processed (data reduction) as a collection of data which were then presented (data display) to describe (conclusion drawing) the tuna supply chain at tpi pt. karya cipta buana sentosa (kcbs) maumere. the information obtained from the informants consisted of several points, including the informants' background, the scope of work, work processes, operating costs and selling prices, and problems in the tuna supply chain at pt. karya cipta buana sentosa (kcbs) maumere. based on the results of interviews with three key informants described previously, it can be seen that the tuna supply chain at pt. karya cipta buana sentosa (kcbs) maumere, namely acceptance or purchase through two doors, namely: fishers directly sell to pt. karya cipta buana sentosa (kcbs) maumere fishers who work on tuna sell the fish they catch directly to pt. karya cipta buana sentosa (kcbs) maumere participates in the auction process because they already have a price agreement. pt. karya cipta buana sentosa (kcbs) maumere bought tuna from fishers at a price of rp. 18.000/kg and then pt. karya cipta buana sentosa (kcbs) maumere packs for export outside the sikka district, such as in japan, korea, vietnam, singapore, malaysia, hong kong, and china, for rp. 35,000 to 50,000/kg. fishermen sell to suppliers from fishers selling to suppliers, suppliers are selling them to retailers, and retailers are selling them to final consumers. the supplier buys tuna from fishers at a price of rp. 20,000 / kg. then the supplier sells to the retailer at a price of rp. 35,500/kg up to rp. 40,000/kg. if there are still remaining sales to retailers, the supplier sells to pt. karya cipta buana sentosa (kcbs) maumere with a price per kilogram of rp. 18,000; in accordance with the price that has been determined/agreed upon. retailers sell them to consumers as endusers at different prices, namely rp. 55.000/kg up to rp. 60,000/kg. international journal of applied biology, 6(1), 2022 128 the supplier will pack the tuna in a vehicle for distribution to retailers; the supplier usually already has a retailer subscription so that the supplier will contact the retailer if the supplier carries out the sales process; the amount of tuna supplied is usually 50 to 100/kg transactions, regular suppliers selling tuna to retailers with an average price of rp. 35,000/kg to rp. 40,000/kg. the supplier's income for a week is around rp. 4,000,000; up to rp. 6,000,000, depending on the number of fish catches purchased from fishers. the problem is that when the price of tuna from fishers rises, it will affect the supplier in terms of initial capital, considering that the supplier has to pay for the labour that helps during the packaging and delivery process to retailers in the market. catching tuna usually requires assistants (fishers) of about 5 to 10 cultivators. the fishing gear used is fads and nets, which are legal fishing gear. the length of the tuna fishing process is usually two days to 3 days, and the longest is up to 5 days with a distance of 5 miles to 10 miles. the acquisition of tuna in one trip is usually 3 tons to 5 tons. the operational costs incurred in the capture process are around rp. 750,000 to rp. 1,000,000, and anglers who own large boats for tuna earn around rp. 1,000,000 to rp. 2,000,000/day, while for small anglers, it is around rp. 250,000 to 400,000 / person. research result based on the results of interviews and direct observations that researchers did during the research process at pt. karya cipta buana sentosa (kcbs) maumere, found the tuna supply chain at pt. the work of cipta buana sentosa (kcbs) maumere can be seen in the following image: figure 1. tuna supply chain at pt. kcbs maumere information : : product flow ... : financial flow .... ..... : information flow supply chain actors include fishers working on tuna, landing and selling tuna to pt. karya cipta buana sentosa (kcbs) maumere, or anglers, sell to tpi suppliers, and then the supplier sells them to pt. karya cipta buana sentosa (kcbs) maumere or suppliers sell them to retailers, then retailers sell them to consumers. there are three types of flow in the supply chain: product flow, financial flow and information flow. product flow product flow in the tuna supply chain at pt. karya cipta buana sentosa (kcbs) maumere is a stream that flows from upstream to downstream and through two doors, namely: suppliers fisherma n pt. kcbs retailer consumer international journal of applied biology, 6(1), 2022 129 1. tuna fish is cultivated by fishers and then landed at tpi for auction to suppliers. the following flow is from suppliers to retailers, suppliers buy tuna fish from fishers and then resell it to retailers in the market, the final product flow from retailers to final consumers, and tuna supplied from suppliers is then sold directly to final consumers come to the market. meanwhile, tuna which the supplier does not sell out can be resold to pt. karya cipta buana sentosa (kcbs) maumere. the first product flow can be described as follows: figure 2. tuna supply chain at tpi maumere information : 2. tuna fish is cultivated by fishers and then landed at kcbs to be sold to pt. karya cipta buana sentosa (kcbs) maumere, sometimes even the sales process takes place in the middle of the sea before landing because at pt. karya cipta buana sentosa (kcbs) maumere has a particular motorboat to buy or pick it up in the middle of the sea before landing at a mutually agreed price. the second product flow can be described as follows: figure 3. tuna supply chain at pt. kcbs maumere keterangan : : product flow ... : financial flow .... ..... : information flow : product flow ... : financial flow .... ..... : information flow suppliers fisherm an pt. kcbs retailer consumer suppliers fisherman pt. kcbs international journal of applied biology, 6(1), 2022 130 financial flow financial flows flow from downstream (downstream) to upstream (upstream). the final consumer buys tuna from retailers for rp. 55,000 up to rp. 60,000/head/kg, then retailers buy tuna from suppliers for rp. 35,000 up to rp. 40,000 / kg/head, and finally, the supplier buys tuna fish products from fishers at a price of rp. 20,000 / kg / head, and pt. karya cipta buana sentosa (kcbs) maumere bought tuna at a price of rp. 18.000/kg total. the financial flow in this supply chain uses cash transactions. information flow information flow in the tuna supply chain at pt. karya cipta buana sentosa (kcbs) maumere is a flow that flows from two directions, namely from upstream to downstream and from downstream to upstream. between supply chain actors, the information provided is information about product prices, product quality, product inventory and the number of purchases. supply chain actors from upstream will provide information about product prices, product quality and product inventory, while supply chain actors from downstream directions will provide feedback such as price negotiations, appreciation or complaints about product quality, number of orders and purchases. supply chain evaluation product development the total production of tuna at pt. karya cipta buana sentosa (kcbs) maumere in 2019 was 24 tons or equal to 24,000 kg. (data source before the 2019 corona virus outbreak). the catch of superior fish landed at pt. karya cipta buana sentosa (kcbs) maumere is tuna and skipjack tuna. tuna fish production during the 2019 coronavirus pandemic decreased by 20 tons or equal to 20,000 kg from the total fishery production of pt. karya cipta buana sentosa (kcbs) maumere in 2019. this happens because there are many fishermen whose primary market for their product, namely tuna, is the local market. many fishing boats are not operating for fear of the 2019 coronavirus outbreak. fishers determine the selling price based on the price of the fish caught the previous day and compare it with the total yield. -the catch of the day of the transaction. if the catch is a lot, the price will go down. on the other hand, if the catch is small, the price of fish will increase. fulfilment of customer aspirations is carried out by maintaining the quality of fish to keep it fresh for consumption and export. procurement fisherman to supplier based on interviews, fishers establish partnership relationships with suppliers, a shortterm transactional relationship. fishers and suppliers do not have definite plans such as schedules and purchase volumes with anglers. due to the uncertain anchoring schedule for ships, suppliers must come directly to tpi to check which vessels. who has landed to make purchases shows that fishers do not carry out intense communication with suppliers? there is no regular discussion about problems in the tuna supply process, such as tuna quality assurance, which must be to the fishers' constraints and guarantee issues—the availability of tuna fish products that are adjusted to the anglers' capacity in producing tuna fish. anglers and suppliers also do not have a policy to minimize the intensity of tuna supply. in terms of solving problems such as the availability and quality of tuna fish products that do not match or meet international journal of applied biology, 6(1), 2022 131 market demands, it only involves one party and is not integrated with the customer. nelayan ke pt. karya cipta buana sentosa (kcbs) maumere based on the interview, the fishers established a partnership relationship with pt. karya cipta buana sentosa (kcbs) maumere, but fishermen are not required to sell their catch to pt. karya cipta buana sentosa (kcbs) maumere and the existing agreement is only regarding the price set by pt. karya cipta buana sentosa (kcbs) maumere so that pt. karya cipta buana sentosa (kcbs) maumere does not participate in the auction process anymore. supplier to retailer the relationship between suppliers and retailers is a short-term transactional relationship; suppliers do not have definite plans with retailers, such as schedules and purchase volumes. besides that, suppliers and retailers also do not regularly discuss what problems occur in the tuna supply, such as quality assurance problems and product availability. tuna that must be by market demand.pengecer ke konsumen akhir. on certain occasions, retailers provide discounts and take simple approaches such as asking for needs and offering tuna fish products to potential buyers who pass through their stalls in an effort to create good relationships with their customers. planning and control based on its shape, tuna is a finished product. based on its function, it is called transit inventory; tuna fish supplies arise because the lead time for shipping from the sea is worked on for 3 to 7 days and then landed at pt. the work of cipta buana sentosa (kcbs) maumere will decrease when it lands and is auctioned to suppliers at tpi. then from tpi, the collectors will make purchases and packaging in approximately 2 to 3 hours and distribute to retailers in about 41 minutes. this inventory will be reduced if the collector prioritizes the supplier of the closer market, taking into account the consequences of postage, price and quality. then, the retailer will carry out the sales process for about 1 to 2 days until the tuna is sold out. based on interviews, anglers do not plan with suppliers, so fishermen cannot control the production of tuna fish products. the planning process that is less collaborative between fishermen, and suppliers, causes fishermen and traders to be unable to control tuna fish production. production operation figure 4. tuna production processes at tpi/pt. kcbs maumere tuna fish production process at pt. the work of cipta buana sentosa (kcbs) maumere starts from the preparation process, namely the preparation of fads, the preparation for capture, and the arrest process. after carrying out the process of catching anglers, they will then carry out the process of unloading the results at the pt. karya cipta buana sentosa fishing in the sea preparation unload the catch tpi / pt. kcbs maumere export international journal of applied biology, 6(1), 2022 132 (kcbs) maumere and the employees will clean and pack for further processing to be exported abroad. as for landing at tpi, fishers land tuna and carry out an auction process at tpi, and then the supplier will carry out the loading process to distribute tuna to retailers in the market for sale to final consumers. shipping/distribution figure 5. distribution of tuna in tpi/pt. kcbs maumere tuna caught by fishers at sea is distributed to tpi/pt. kcbs maumere also goes by sea by passing the fishing zone of pt. kcbs maumere which has been determined, namely in the flores sea, then for collectors in terms of choosing the route the collector will determine the delivery route to the retailer to minimize transportation costs and time because the collectors consolidate demand and delivery, the collectors will prioritize delivery or sell directly to the ship owned by pt. karya cipta buana sentosa (kcbs) maumere, whose sales transaction occurred in the middle of the sea. discussion marketing channel in this study, it is explained that the tuna supply chain network at pt. karya cipta buana sentosa (kcbs) maumere started from fishers directly to pt. karya cipta buana sentosa (kcbs) maumere and from fishers to tpi through suppliers, suppliers to retailers and finally to consumers as end-users. the existing supply chain flow is not too long, so it can provide advantages such as time, cost and effort problems. as in previous research conducted by tivani lowing et al., the skipjack supply chain management analysis at the tumumpa manadopada fish auction is not too long to save time, cost, and effort. zaenab et al. (2020) emphasized that every fishery commodity has a supply chain value. in this study, it was assessed that the tuna supply chain at pt. karya cipta buana sentosa (kcbs) maumere is not optimal or not well integrated because there is no trust between fishermen and suppliers at tpi to establish cooperation and partnership relationships to achieve common goals to improve supply chains and supply chain actors' probability in terms of problem-solving such as quality assurance and product availability assurance only involve one party and are not integrated to the customer so that the supply chain at pt. karya cipta buana sentosa (kcbs) maumere did not show significant development despite having the opportunity to expand the market. the tuna supply chain bottleneck at pt. karya cipta buana sentosa (kcbs) maumere based on observations of phenomena, these obstacles include the management of information flow at pt. the work of cipta buana sentosa (kcbs) maumere is not centralized, and policy support is less than optimal. the solution is obtained through an assessment of the fisherman tpi / pt. kcbs maumere contractor consumer retailer suppliers demand international journal of applied biology, 6(1), 2022 133 details of each activity in the tuna supply chain. prayoga et al. (2018) say that the keys to success include trust-building (trust), coordination and cooperation. information flow (pull) tuna fish stream based on information (pull) figure 6. illustration of tuna supply chain recommendations at pt. kcbs maumere the recommendations for the tuna supply chain include optimizing the role of supply chain actors, centralizing and developing information systems, and good collaboration between parties. business processes in collaborative relationships are directed to become a pull system. the goal is to make service actions more responsive. through clear sales planning and careful production planning so as to reduce operational risks such as product absence, bullwhip, forecasting errors, and market risk. the purpose of supply chain management is to integrate all company operational processes from upstream (upstream) and downstream (downstream) to end-users through the provision of products, information, and services to increase added value for consumers and other stakeholders (setiawan et al., 2019). furthermore, fachry et al. (2021) say that stakeholders have an essential role in the development of a business if it is framed in the form of a partnership. marketing profit and margin marketing fee cost is the value of all predictable economic inputs or sacrifices that can be measured to produce a product. while marketing is a product distribution of goods from producers to consumers. producers are the first link, and consumers are the last link. (fishery business management). marketing costs incurred at pt. karya cipta buana sentosa (kcbs) maumere covers transportation costs, loading and unloading costs, user fees and storage costs. so marketing costs can be calculated using the formula: information : bp = tuna marketing cost (rp/kg/head) bp1, bp2,....bpn = marketing costs per agency (rp/kg/head) so the process of calculating marketing costs using the above formula is as follows: bp = bp1+bp2+........+bpn 146.000 = 18.000+20.000+18.000+35.000+55.000 production collaboration marketing collaboration fisherman suppliers retailer consumer international journal of applied biology, 6(1), 2022 134 profit marketing the profit of each marketing agency involved in tuna marketing can be formulated as follows: information : kp = marketing profit of tuna (rp/kg/head) kp1+kp2+.....kpn = fish marketing profit tuna per institution (rp/kg/head) so the process of calculating marketing profits using the above formula is as follows: margin marketing marketing margin is the difference between the price at the consumer level (pr) and the price at the producer level (pf). tuna marketing can be formulated as follows: information : mp = tuna marketing margin (rp/kg) pr = price at the consumer level (rp/kg pf = price at producer level (rp/kg) so that the marketing margin calculation process using the above formula is as follows: margin analysis and farmer's share to find out the economic efficiency of tuna marketing, margin analysis and farmer's share were used using the following formula: information : f = share of price received by fishermen (%) pf = price at fisherman level (rp/kg/head) pr = price at the final consumer level (rp/kg/head) so the process of calculating the margin and farmer's share using the formula above is as follows: the provisions of the marketing system are said to be efficient if the value of the farmer's share is more than 50%, and if the value of the farmer's share is less than 50%, then it is not efficient. the bigger the marketing margin, the more inefficient the marketing system is. kp = kp1+kp2+..... ken 43.000 = 3.000+5.000+15.000+20.000 mp = pr pf 37.000 = 55.000 18.000 f = (pf / pr) 100 12 % = 18.000/55.000 x 100 international journal of applied biology, 6(1), 2022 135 conclusions based on the results of research at pt. cipta buana sentosa (kcbs) maumere, sikka regency, the following conclusions can be drawn: 1. form a marketing channel for tuna at pt. karya cipta buana sentosa (kcbs) starts from fishers who go directly to suppliers, from suppliers to pt. kcbs then goes to retailers and finally to consumers as end-users. the supplier does not follow the auction process because the supplier has set the selling price—tuna fish supply chain management at pt. karya cipta buana sentosa (kcbs) is still not optimal due to the uncertain anchoring schedule of ships and the lack of trust to build long-term cooperation and relationships or partnerships with fishermen and suppliers at pt. karya cipta buana sentosa (kcbs). the tuna supply chain is directed at optimizing the management of auction activities in terms of developing information systems, meeting demand more responsively, and strengthening sustainable fisher partnerships, which will support supply chain performance. 2. the value of the tuna marketing margin at pt. karya cipta buana sentosa (kcbs) is rp. 37,000 with a farmer's share value of less than 50%, and it can be concluded that the marketing margin is not efficient. international journal of applied biology, 6(1), 2022 136 references arikunto, s. 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https://scholar.google.com/citations?view_op=view_citation&hl=id&user=0phpd2aaaaaj&citation_for_view=0phpd2aaaaaj:5nxa0vek-isc https://scholar.google.com/citations?view_op=view_citation&hl=id&user=0phpd2aaaaaj&citation_for_view=0phpd2aaaaaj:5nxa0vek-isc https://scholar.google.com/citations?view_op=view_citation&hl=id&user=0phpd2aaaaaj&citation_for_view=0phpd2aaaaaj:5nxa0vek-isc contact : inanpi hidayati sumiasih inanpihs@trilogi.ac.id 59 abstract attaqie farm agroedutourism produced 1.5 tons/ha of star fruit waste in 2018, which was wasted because it was not utilized by starfruit farmers. attaqie farm strives to implement the sustainable agriculture system (spt) to support sustainable agriculture. the waste originating from one cultivation can be used as a resource in other forms. the application of integrated and sustainable agriculture is beneficial for farmers to preserve the ecology while maintaining the culture adopted in an area. farmers' objectives for agricultural cul tivation are good, healthy, and maximum yields, therefore the availability of compost is needed. the technology that has developed makes it easier for farmers or researchers to make compost in a short time. the study began with making compost with star fruit waste, namely residual leaves and fruit from thinning. this research was carried out using a randomized block design (rbd). the treatment was replicated 4 times, with 3 experimental units, and 5 dose levels. nutrients in the star fruit waste compost showed good results for compost criteria according to sni 197030-2004 standard. the effective dosage for star fruit compost in mustard cultivation was 20 tons/ha (equivalent to 40 g/polybag). issn : 2580-2410 eissn : 2580-2119 the utilization potential of tasikamdu star fruit composted waste for mustard cultivation (brassica rapa l.) in supporting sustainable agriculture inanpi hidayati sumiasih*, mutiara dewi puspitawati, fayyadh yusuf maulana, yadarabullah faculty of science, technology, and design, trilogi university, jakarta. jl. tmp. kalibata no. 1, jakarta 12760 indonesia introduction attaqie farm agroedutourism produced 1.5 tons/ha of star fruit waste in 2018, which was wasted because it was not utilized by starfruit farmers (siahaan 2021). waste left in the field would reduce the aesthetic aspect of the tourism sector. attaqie farm strives to implement the sustainable agriculture system to support sustainable agriculture, so the waste must be used in another form. the star fruit waste can be utilized as organic fertilizer that would also support the sustainability of the farm. cultivation based on the use of compost, control of plant pest organisms (ppo), and integrated resource management is a proper development concept in the agricultural sector. this is with the aim that the open access international journal of applied biology keyword compost, mustard, star fruit waste, sustainable agriculture article history received january 10, 2023 accepted june 21, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 7(1), 2023 60 management of the agricultural sector is socially, economically, and sustainably feasi ble (wihardjaka 2018). cultivation systems can be developed and combined to be integrated as one system. according to ummah (2017), waste from cultivation can be used as a resource for other cultivation. the application of integrated and sustainable agricu lture is beneficial for farmers to preserve the ecology while maintaining the culture adopted in an area. sustainable agriculture can bring positive impacts such as more accessible information about integrated farming, better knowledge about the agricultural sector, and increasing usage of organic fertilizer by farmers who aim to cultivate food both conventionally and non -conventionally. organic materials such as agricultural waste or household waste can undergo decomposition which resulted in available nutrients for plants. plant cultivation using organic fertilizer or organic based is preferred by the general public because of the notion that it can be more beneficial and healthier for consumers. since more people care about their health, the utilization of organic fertilizer is increasing. a healthy lifestyle has become the basis for abandoning various unhealthy habits such as the use of excess chemicals, the use of artificial pesticides, plant growth regulators, and inorganic fertilizers. mustard is one of the popular vegetables that is favored by the community (fuad 2010) because it is easy to grow and cultivated. the benefits of mustard according to vivonda et al. (2016), are that it can reduce coughs and headaches because mustard has substances and vitamins that are important for human health. farmers prefer the use of chemicals fertilizer due to fast plant growth, high yields, and relatively cheap prices. however, excessive use of chemical fertilizer could damage the soil. to reduce chemical use, organic fertilizers such as compost are needed. farmers' objectives for agricultural cultivation are good, healthy, and maximum yields, therefore the availability of compost is needed. the technology that has developed makes it easier for farmers or researchers to make compost in a short time. this acceleration of composting should still happen naturally to produce good quality compost (murbandono 2010). the composting process consists of three stages, namely intensive decomposition which can produce hot temperatures due to the presence of bacteria from the bio -activator. the second stage is conversion and synthesis for compost maturation. then the third stage is post-maturation. good compost that can be used, can be seen from the standard characteristics of organic compost, which are containing nutrients, brownish-black in color, and crumbles (setyorini et al. 2006). tasikmadu star fruit waste at attaqie farm produces waste such as residual fruit, twigs, and leaves from thinning. with this, it is necessary to use it as compost and to support sustainable agriculture in the ecology. international journal of applied biology, 7(1), 2023 61 materials and methods time and place this research was conducted t attaqie farm, tuban in augustus – november 2021. the analysis of star fruit waste compost content and the soil cont ent were conducted at soil research center, bogor. materials the tools used were: buckets, machetes, branch shears, digital scales, trays, stationery, popsicle sticks, polybags, watering jugs, leaf chopping machines, carts, tarpaulins, and leaf color chart. the materials used were: leave and fruit residue from thinning, em-4, water, molasses, and mustard nauli f1 (east-west seed indonesia) seeds. research method the research began with the production of compost using star fruit waste as material. this research was carried out using a randomized block design (rbd). the treatment was replicated 4 times, with 3 experimental units, and 5 dose levels. the doses given were: d0: 0 ton/ha compost (equivalent to 0 g/polybag) d1: 10 ton/ha compost (equivalent to 20 g/polybag) d2: 20 ton/ha compost (equivalent to 40 g/polybag) d3: 30 ton/ha compost (equivalent to 60 g/polybag) d4: 40 ton/ha compost (equivalent to 80 g/polybag) the compost produced and the soil from attaqie farm were analyzed. the compost was tested 3 times, at 1, 3, and 6 months old. the soil sample was collected from five points sampling from different spots. the data collected were analyzed using anova (analysis of variance) using star (statistical tools for agriculture research) application. if when analyzed there was a significant difference found the data was further analyzed using duncan’s multiple range test with α = 5%. the procedure for producing star fruit waste compost the making of star fruit waste compost started by: 1) pruning of 50 kg star fruit leaves. pruning was carried out for 3 days to collect enough leaves and twigs for materials for making the compost. 2) separation of leaves and twigs. this was done conventionally with a machete. 3) chopping leaves using a leaf chopper machine. this process was repeated 3-4 times so that the organic material reaches a size of 2-3 cm. if the organic material is too fine (<1 cm), it will experience compaction and will damage the process of oxygen availability so that organic matter will be moist and the decomposition process will be hampered. 4) sorting and collecting residual fruit to collect 50 kg star fruit. the fruit to be composted was sorted by criteria: the size of small to medium and yellow-brown star fruit. the yellowbrown star fruit was selected due to the sufficient water content available in the star fruit to make it easier when pounding takes place international journal of applied biology, 7(1), 2023 62 5) pounding of residual star fruit. the fruits collected were pounded until becoming mush so they can decompose properly. 6) preparation of the solution, namely with 5 liters of water, 3 liters of molasses, and 50 ml of em-4 then mixed for the bacterial culture process. the mixture was then fermented for 1x24 hours. 7) accumulation of organic matter. chopped leaves and pou nded fruit were mixed and the prepared solution was administered to the organic material. 8) storage for 4 weeks. the mixture was stirred twice a day while in storage. after that, the ph and temperature were measured. 9) compost was ready to use the application of star fruit compost to mustard cultivation: 1) mustard seeds were planted in a seedling tray with a planting medium with a ratio of 1:1:1. 2) before transplanting, a medium for planting the seedling was prepared. the soil was mixed with star fruit compost with the soil in the polybag is mixed with compost and left for 1 week 3) transplant from tray to polybag when mustard seedlings had 4 leaves 4) observations started after transplanting results and discussion nutrient content of attaqie farm soil the analysis of soil samples from attaqie farm was conducted at soil research center, bogor. the results showed that the soil sample was suitable for cultivation, as attaqie farm was a productive star fruit orchard. table 1 shows the analysis results of the soil sample. the compost contained n, p, k, ca, and mg. the nutrients can affect the growth of cultivated plants. and from the results, it is shown that the n -total of the soil was 0.27% which is categorized as moderate according to soil research cent er (2009). the soil sample also had natural p content in the form of phosphate which involves the synthesis of protoplasm and then decomposed with phosphate bacteria (andraski et al. 1985). the high p content in the soil is not fully absorbed by plant roots. nutrients bound by cations of micronutrients such as ca and mg will be deposited in the soil and cannot be absorbed (hanafiah 2005). according to puspitawati & sumiasih (2021), phosphorus nutrients in the soil with low ph (acidic) cannot be available for plants. the additional compost in the soil can help to make phosphate available for plants and also support microbes in the soil. international journal of applied biology, 7(1), 2023 63 table 1. attaqie farm soil nutrient content soil parameter value unit criteria ph h2o 6.50 slightly acidic kcl 5.92 slightly acidic c-organic 2.00 % moderate n-total 0.27 % moderate p* 59.9 ppm high p 1.045 ppm ca 19.65 cmol(+)/kg high mg 6.30 cmol(+)/kg high k 1.56 cmol(+)/kg very high na 0.89 cmol(+)/kg high ktk 22.74 cmol(+)/kg moderate al tr cmol(+)/kg h 0.69 cmol(+)/kg fe 12.0 ppm high cu 3.8 ppm sufficient zn 3.9 ppm sufficient mn 49.6 ppm high sand 11.44 texture (pipette methods) dust 35.35 texture (pipette methods) loam 53.21 texture (pipette methods) notes: attaqie farm soil sample analysis results from bogor soil research center. criteria based on soil research center (2009). nutrient content of star fruit waste compost nutrient content in compost will affect plant growth from root to shoot. the nutrient content of star fruit compost is shown in table 2. the presence of nutrients showed that there had been a decomposition process by thermophilic bacteria during composting so the organic material turned into available nutrients. c -organic was the parameter to determine the organic matter available in star fruit waste compost. the ratio of c/n in the first month of the composting process had already met the compost sni quality standard. the readiness of organic compost can be seen in table 2, that in the firstand thirdmonth n, p, and k had increased. nitrogen (n) in the compost is needed by the mustard plant, especially in the vegetative period (kurniawan et al. 2017). according to puspitawati & sumiasih (2021), the accumulation of photosynthate in the generative phase of star fruit resulted in a good nutrient amount in the star fruit compost. the addition of molasses and local micro -organism (lmo) in the compost-making process was helpful because the lmo supported b y molasses breaks down organic material. anaerobic bacteria working to decompose the organic matter in a compost pile causes the international journal of applied biology, 7(1), 2023 64 pile to warm up. as the temperature rises, different organisms thrive, such as thermophilic and mesophilic bacteria. all the bacteria are active to continue the organic material degradation process. the making of star fruit compost is also supported by environmental factors. in this study, acidity level (ph) and temperature were observed. when ph reached >8, lime was added and when the temperature reached > 70 the compost pile was mixed thoroughly to reduce the temperature. table 2. nutrient content of star fruit waste compost from attaqie farm parameter compost 1 month compost 3 month compost 6 month quality minimum value c-organik 51.53 49.90 45.61 27 c/n 19 16 37 20 kadar air 68.8 76.7 83.01 50 ph 9.1 9.0 8.8 7.49 hara makro : n 2.78 3.06 1.22 0.40 p 0.88 0.44 0.62 0.10 k 2.70 2.50 4.57 32 ca 2.65 2.36 3.46 25.50 mg 1.34 1.25 1.63 0.60 s 0.01 0.03 0.02 notes: quality minimum from sni no,19-7030-2004. source: soil research institute test result (2021), bogor mustard harvest in this study, mustard was harvested 4 weeks after planting (wap). the criteria for mustard that was ready to be harvested had 8-9 leaves, leaves were fully developed in an oval shape, leaf color was green all around, stalk color was light green, and plant height was adequate. figure 1 showed mustard samples from different compost dosages. there was no significant visual difference among harvested mustard. however, the absorbed nutrient can affect plant growth and the yield of mustard (brassica rapa l.). according to research by habibi et al. (2017), the addition of compost in the soil can support the symbiotic relationship between bacteria and mustard. this symbiosis can improve the absorption of n through the root nodule, to support mustard growth. international journal of applied biology, 7(1), 2023 65 figure 1: comparison of mustard at various doses of starfruit waste compost. d0: control, d1: 10 tons/ha, d2: 20 tons/ha, d3: 30 tons/ha, d4: 40 tons/ha. research parameters results (plant height, leaf width, number of leaves, yield) mustard plants were planted in the soil mixed with star fruit waste compost with different dosage levels. the growth of mustard (brassica rapa l.) was observed for 4 weeks and the results are shown in table 3. the different treatments led to various results in all parameters. the plant height was measured from the ground to the highest shoot of mustard. from the plant height parameter at 4 wap, it is shown that the addition of 40 to/ha (equal to 80 g/polybag) resulted in an average plant height of 14.78 cm. nutrients given to mustard by compost resulted in good growth compared to 0 ton/ha treatment. according to maulana & sumiasih (2022), the use of starfruit waste compost had an effect due to the presence of nitrogen in the compost to increase mus tard growth. furthermore, the compost was mixed with the soil a week before transplanting, so that the soil absorbs nutrients for plants. this is in accordance with ismayanti et al. (2020) leaf width was a parameter to determine growth in leaves. the increase in leaf width is influenced by the absorption of nutrients available in the soil. if the nutrient content is available and adsorbed by the plant, then photosynthate and protein will increase and result in a wider leaf (jatra et al. 2021). the results of the leaf width parameters were significantly different with an error rate of 5%. the dose of starfruit waste compost at 30 tons/ha resulted in a leaf width of 5.42 cm. however, the optimum dosage was 20 tons/ha since the result (5.17 cm) was not significantly different from the 30 tons/ha treatment. if too much compost is applied, it would disturb the growth in the vegetative phase. thus, optimization is needed to avoid nutrient toxicity in plants. nitrogen is responsible for converting carbohydrates into proteins, which are then used as protoplasm to increase leaf growth and widening (handayani & elfarisna 2021). international journal of applied biology, 7(1), 2023 66 table 3. observation results at 4 wap (weeks after planting) treatment plant height (cm) leaf width (cm) number of leaves yield (g) d0 (0 ton/ha) 13.93 4.72 bc 8.50 9.26 d1 (10 ton/ha) 13.48 4.59 c 8.00 7.79 d2 (20 ton/ha) 14.53 5.17 ab 9.00 9.74 d3 (30 ton/ha) 14.37 5.42 a 8.92 10.23 d4 (40 ton/ha) 14.78 5.31 a 9.00 10.09 notes: d1: 10 ton/ha, d2: 20 ton/ha, d3: 30 ton/ha, d4: 40 ton/ha. numbers followed by the same letter showed no significant difference using duncan’s multiple range test (dmrt) with α = 5%. the number of leaves was analyzed using the star application to see the effect of various doses of compost. the observation results at 4 weeks showed that the number of leaves was not significantly different across treatments. this was because the nutrients available in the starfruit waste compost were sufficient for the development of leaves. according to latarang & syakur (2006), the addition of compost helps the plant to be able to get nutrients so that the number of leaves reached optimal results. the formation of leaves in leafy vegetables in the development phase is determined by the number and size of cells which are influenced by the availability of nutrients in the compost. harvested yields in this study were not significantly different across various treatments given. excessive use of compost would result in abnormal conditions such as wetter mustard tissue, delayed production period, and shortened vegetative phase. if these conditions occurred, it would affect the yield of the harvested mustard (roidi 2016). the mechanism of action of compost in plants is by releasing organic substances which are then absorbed by normal root hairs, and accumulation of compost in the rhizosphere (habibi et al. 2017). international journal of applied biology, 7(1), 2023 67 leaf color figure 2: leaf color of mustard with star fruit compost as treatments the measurement for leaf color was conducted using leaf color chart (lcc) to see the function of nitrogen (n) in plant leaves. the use of starfruit waste compost had no significant effect on leaf color, which can be seen in figure 2. for mustard leaves, a score of 3 showed that mustard had sufficient n nutrients and also had no chlorosis symptoms in the leaves. when there is a lack of n nutrients, the chlorophyll content in the leaves would be lost and the leaves would be yellowish green. leaves have a function to carry out photosynthesis to get the energy from the sun (vivonda et al. 2016). root weight the analysis of variance results through the calculation of the star application showed that the highest root weight was at 30 tons/ha compost, although there was no significant effect on the 5% error level. the ability of roots to absorb water is directly related to the value of root weight (salisbury & ross 1995). the appropriate application of compost would make the root grow better, as shown by the various weights of mustard roots with the treatment of starfruit waste compost dosage. the differentiation process in mustard proves that there is a growth process in root weight (munthe et al. 2018). table 4. the effect of compost on mustard root weight treatment root weight (g) dosis pupuk kompos d0 (control) 0.56 d1 (10 tons/ha) 0.59 d2 (20 tons/ha) 0.47 d3 (30 tons/ha) 0.60 d4 (40 tons/ha) 0.48 notes: d1: 10 tons/ha, d2: 20 tons/ha, d3: 30 tons/ha, d4: 40 tons/ha. the results showed no significant difference using duncan’s multiple range test (dmrt) with α = 5%. 0 0.5 1 1.5 2 2.5 3 3.5 1 mst 2 mst 3 mst 4 mst l e a f c o lo r (l c c ) weeks after planting (wap) d0 (kontrol) d1 (10 ton/ha) d2 (20 ton/ha) d3 (30 ton/ha) d4 (40 ton/ha) international journal of applied biology, 7(1), 2023 68 root length plant root length is a parameter to estimate the ability of the root to absorb nutrients from the soil. when plants have longer roots, they will have a better yield. the results from this research showed that there was no significant difference in root length across all star fruit compost treatments. however, the mustard plants had enough nutrients and the roots were well developed. table 5: the effect of compost on mustard root length treatment root length (cm) compost fertilizer dosage d0 (control) 7.76 d1 (10 tons/ha) 8.00 d2 (20 tons/ha) 7.13 d3 (30 tons/ha) 8.23 d4 (40 tons/ha) 7.99 notes: d1: 10 tons/ha, d2: 20 tons/ha, d3: 30 tons/ha, d4: 40 tons/ha. the results showed no significant difference using duncan’s multiple range test (dmrt) with α = 5%. conclusion nutrients in the star fruit waste compost showed good results for compost criteria according to sni 19-7030-2004 standard. the star fruit compost was ready to use and effective in the 3rd month. the results of observations of the potential use of star frui t waste compost for mustard showed that some of the parameters had no significant difference. there was a significant difference in leaf width, with the dose level of 30 tons/ha resulting in the highest leaf width. however, the effective compost dose was 20 tons/ha (equivalent to 40 g/polybag). sustainable agriculture at attaqie farm was fairly well run because attaqie farm was able to utilize waste from one system to another in its ecology. acknowledgment the author would like to express gratitude to the ministry of education and culture through the university excellence applied research grants, trilogi university, and attaqie farm tuban east java which have facilitated the research. international journal of applied biology, 7(1), 2023 69 references fuad a. 2010. budidaya tanaman sawi (brassica juncea l.). [tugas akhir]: surakarta (id) universitas sebelas maret. handayani, i., & elfarisna, e. (2021). efektivitas penggunaan pupuk organik cair kulit pisang kepok terhadap pertumbuhan dan produksi tanaman pakcoy. jurnal agrosains dan teknologi, 6(1), 25-34. habibi z, satriawan h, agusni. 2017. pengaruh dosis pupuk kompos terhadap pertumbuhan tanaman sawi (brassica rapa l.). [jurnal]: agrotropika hayati vol.4. no. 4 (305-313). aceh (id): universitas almuslim hanafiah ka. 2005. dasar-dasar ilmu tanah. jakarta: rajagrafindo persada ismayanti, r. t., fuskhah, e., & sutarno, s. (2020). pengaruh berbagai dosis pupuk kompos eceng gondok dan pupuk hijau azolla microphylla terhadap pertumbuhan dan produksi tanaman pakcoy (brassica rapa l.). buana sains, 20(2), 217-226. jatra at, banu ls, sholihah sm. 2021. pengaruh dosis kompos kulit bawang merah terhadap pertumbuhan sawi samhong (brassica rapa). [jurnal]: ilmiah respati 12(2): 122 -132. kurniawan a, islami t, koesriharti k. (2017). pengaruh aplikasi pupuk n dan k terhadap pertumbuhan dan hasil tanaman pakcoy (brassica rapa var. chinensis) flamingo f1. [jurnal]: produksi tanaman 5(2). latarang b, syakur a. 2006. pertumbuhan dan hasil bawang merah (allium ascalonicum l.) pada berbagai dosis pupuk kandang. [jurnal]: agroland 13(3): 265-269. maulana fy, sumiasih ih. 2022. aplikasi kompos limbah belimbing tasikmadu terhadap pertumbuhan dan panen pakcoy (brassica rapa l.) untuk mendukung pertanian berkelanjutan. [skripsi]: jakarta (id). universitas trilo gi murbandono. 2010. membuat kompos. jakarta (id). peneber swadaya pr. munthe k, pane e, panggabean el. (2018). budidaya tanaman sawi (brassica juncea l.) pada media tanam yang berbeda secara vertikultur. agrotekma: jurnal agroteknologi dan ilmu pertanian 2(2): 138-151. puspitawati, m. d., & sumiasih, i. h. (2021, march). organic fertilizer from starfruit waste sustainable agriculture solution. in iop conference series: earth and environmental science (vol. 709, no. 1, p. 012069). iop publishing. roidi aa. 2016. pengaruh pemberian pupuk cair daun lamtoro (leucaena leucocephalai) terhadap pertumbuhan dan produktivitas tanaman tanaman pakcoy (brassica chinensis l.). [skripsi]. yogyakarta (id): universitas sanata dharma setyorini d, saraswati r, anwar ek. 2006. pupuk organik dan pupuk hayati. bandung (id): balai besar penelitian dan pengembangan sumberdaya lahan pertanian salisbury, f.b. & ross, c.w. 1995. fisiologi tumbuhan jilid 3. terjemahan diah r. lukman dan sumaryono. itb press. bandung. vivonda, t., armaini, dan s. yoseva.2016. optimalisasi pertumbuhan danproduksi tanaman pakcoy (brassica chinensis l.) melalui aplikasi beberapa dosis pupuk bokashi. jom fakultas pertanian, 3(2) : 1-11.[28 november 2020]. wihardjaka a. 2018. penerapan model pertanian ra mah lingkungan sebagai jaminan perbaikan kuantitas dan kualitas hasil tanaman pangan. jurnal pangan. 27(2):1 -10. 3(1)24-29 contact : andi evi erviani evierviani@unhas.ac.id © international journal of applied biology 24 abstract the research about the potential of sea worm p. aibuhitensis extract as antimicrobe toward bacteria s. typhi and fungus c. albicans had been conducted. the aim this research is to know the extract’s concentration of sea worm p. aibuhitensis which is effective to inhibit the growth of bacteria s. typhi and fungus c. albicans. the result of research that was obtained shows that sea worm’s extract which used ethanol solution 96% and was needed in the culture of bacteria s. typhi with the concentration of sea worm’s extract 7.5%, 15%, 30%, and 60% involved the zone of inhibition’s form at the medium with duration incubation 1x24 hours and 2x24 hours. based on the research which was conducted, it can be concluded that the sea worm p. aibuhitensis extract had the potential as anti-microbe toward bacteria s. typhi because can inhibit the growth of that bacteria, but it had no effect as anti-microbe toward fungus c. albicans. issn : 2580-2410 eissn : 2580-2119 the potential of sea worm perinereis aibuhitensis extract as anti-microbe toward bacteria salmonella typhi and fungus candida albicans andi evi erviani, asadi abdullah, nurwahida & febriany tairas departement of biology, faculty mathematic and natural science, hasanuddin university, makassar introduction one of the sea worms has the potential to be cultivated at makassar city that is sea worm p. aibuhitensis, this sea worm was founded in great quantity at reklamasi area of losari beach, makassar city. this worm is only used as fish bait. the potential of this worm is not yet utilized properly because the lack of scientific information about that worm. various scientific researches about sea worm p. aibuhitensis need to be conducted to know the potential of one of that sea biota and its further utilization. activity test as anti-microbe is also not yet conducted, therefore the research about activity test of sea worm p. aibuhitensis extract as anti-microbe needs to be conducted. sea worm contents the high protein but it is not yet utilized optimally in indonesia. sea worm has quite complete nutrition thus can be used as alternative food. the contents of nutrition in the sea worm are; protein, fat, carbohydrate, ashes, fat acid and amino acid, vitamins a, b1, b6, b12, e and chemical elements p, i2, ca, mg, c which are almost equal to the nutrition contents of fish (silaban 2012). the extract of sea worm s. austral had proven in containing 56.35% protein, 15.08% ashes concentration, 9.82% fat concentration, and 5.06% open access international journal of applied biology keyword perinereis aibuhitensis, anti-microbe, salmonella typhi, candida albicans. article history received 07 january 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 25 carbohydrate concentration (nurhikma, et al, 2017). there were also various amino acids in sea worm where amino acid is very important to support the various physiological activities of body. one of amino acids in the sea worm is glutamate acid which has the important role in the metabolism of sugar and fat, in addition, glutamate acid in the animal or plant can be used as medical treatment substance in solving epilepsy disease, mental retardation, dystrophy, abscess, hypoglycemic coma, and also the side effect of insulin treatment for diabetes. based on the information above and the lack of information about the ability of sea worm p. aibuhitensis as anti-microbe, then activity test of sea worm p. aibuhitensis extract as anti-microbe toward bacteria s. typhi and fungus c. albicans is important to be done. materials and methods materials the materials which were used –sea worm p. aibuhitensis which was obtained at reklamasi area of losari beach and the materials for extracting were absolute ethanol, culture of bacteria s. typhi, chloramphenicol, fungus c. albicans, mgcl2 72%, potato dextrose agar (pda), aquades, nacmc and griseofulvin. the extraction of sea worm p. aibuhitensis the material is the worm p. aibuhitensis. the worm p. aibuhitensis were taken around 1000 gram. furthermore, the samples were washed by using flow water. the clean sample of worms were dried, after that pounded by using blender. the pounded worm p. aibuhitensis were used for extraction process (purwaningsih et al. 2008). extraction process was conducted by using 24 hour maseration method. the pounded worm were soaked by using ethanol solution with ratio 1:4 (b:v), were conducted maseration during 24 hours by using orbital shaker 24 hours at room temperature, then were distillated by using filter paper whatman number 42. maseration was conducted in 3x24 hours. the resulted filtrates were separated with its solvent by using rotary vacuum evaporator at temperature 40˚̊c during 6 hours. then, the resulted extracts were weighted. activity test of anti-microbe from sea worm p. aibuhitensis toward bacteria s. typhi the used method for activity test was conducted through measuring the zone of inhibition’s form. na medium which had been sterilized was poured sufficiently into petri cup. after it is dense, the suspense bacteria were scratched into all surfaces of medium with swab technique by using sterilized cotton bud. after that, it was included paper disk which had been soaked by the extract of sea worm p. aibuhitensis in various concentration, those were 7.5%, 15%, 30%, and 60%, chloramphenicol as positive control and nacmc as negative control. petri cup was labeled, then was incubated in incubator at temperature 37˚̊c during 24 hours, and then was observed and measured its zone of inhibition. incubation was continued during 48 hours and was measured again its zone of inhibition which was formed. activity test of anti-microbe from sea worm p. aibuhitensis toward fungus c. albicans medium of potato dextrose agar (pda) which had been cooled was poured into petri cup and was waited until dense. after that, the suspense of fungus was taken by using cotton bud, then was stretched into medium potato dextrose agar (pda) by using swab method. after that, it was included paper disk which had been soaked by the extract of sea worm p. aibuhitensis in various concentration, those were 60%, 30%, 15% and 7.5%, positive control international journal of applied biology, 3(1), 2019 26 (griseofulvin) and negative control (nacmc). petri cup was labeled, then was incubated in incubator at temperature 37˚̊c during 24 hours and 48 hours, then was observed and measured the transparent zone which was formed around paper disk by using ruler. data analysis data which was gained during the observation of resistibility test was analyzed descriptively which was showed in form of figures and histogram. results and discussion activity test of anti-microbe from sea worm p. aibuhitensis toward bacteria s. typhi the observation toward bacteria s. typhi by using four kinds of concentration showed the form of various transparent zones at the time of incubation 1x24 hours. at the concentration 7.5% sea worm p. aibuhitensis extract did not form the zone of inhibition or transparent zone, concentration 15% formed the zone of inhibition or transparent zone around 9 mm which was included on medium category, concentration 30% formed the zone of inhibition or transparent zone around 10 mm which was also included on medium category, and concentration 60% formed the zone of inhibition or transparent zone around 12 mm which was included on strong category, while positive control chloramphenicol which formed the zone of inhibition or transparent zone 14 mm was included on strong category and negative control nacmc did not form zone of inhibition or transparent zone. at the incubation 2x24 hours, the fourth concentrations which were used also showed the existence of the formed transparent zone, those were at the concentration 7.5% did not form the zone of inhibition or transparent zone, concentration 15% (8.5 mm) included on medium category, concentration 30% (10 mm) included also on medium category, concentration 60% (12 mm) included on strong category., while positive control (14 mm) included on strong category and negative control was not formed transparent zone. figure 1 show the zone of inhibition or transparent zone which was formed at incubation 1x24 hours and 2x24 hours. (a) 1x 24 hours (b) 2x24 hours figure 1. the test of resistibility of sea worm p. aibuhitensis extract in inhibiting the growth of bacteria s. typhi note: a : concentration 60% b : concentration 30% c : concentration 15 % d : concentration 7.5% e : negative control f : positive control international journal of applied biology, 3(1), 2019 27 histogram of average diameter’s measurement result of zone of inhibition or transparent zone at incubation 1x24 hours and 2x24 hours can be seen in figure 2. figure 2. the comparison histogram of average diameter’s measurement result of inhibition (mm) sea worm p. aibuhitensis extract in inhibiting the growth of s. typhi at incubation 1x24 hours and 2x24 hours on the resistibility test, concentration 60% of sea worm extract had the higher zone of inhibition rather than other concentrations. it was caused because of the higher concentration of anti-microbial substance which was given then the more excessive antimicrobial substance on the worm extract thus had the bigger effect. ningtyas (2010) explained that the higher concentration of extract than the more excessive active substance of its anti-bacteria. the addition of anti-bacterial substance’s concentration expected can increase the penetration of anti-bacterial substance into microbe’s cell which will damage metabolism system of cell and can cause the death of cell. most of bacteria’s growth will more decreased along with the increasing of additional anti-bacterial concentration. the higher concentration of extract then the number of detached anti-bacterial substances are bigger, thus make easier the penetration of those substances into cell (maleki et al., 2008). nevertheless, if it was compared to positive control with the gain 14 mm showed positive control more effective in inhibiting the growth of bacteria s. typhi. the lysis of bacteria cell was caused by the disability of cell wall to maintain the form and to protect the bacteria which has the high inside osmotic pressure (ajizah et al., 2007). without cell wall, bacteria cannot survive toward the outside influence and will die. activity test of anti-microbe from sea worm p. aibuhitensis toward fungus c. albicans the observation result of zone of inhibition toward fungus c. albicans after incubation during 24 hours until 2x24 hours can be seen in figure 3. international journal of applied biology, 3(1), 2019 28 (a) 24 hours (b) 2x24 hours figure 3. the test of resistibility of sea worm p. aibuhitensis extract in inhibiting the growth of fungus c. albicans note: a : concentration 60% b : concentration 30% c : concentration 15 % d : concentration 7.5% e : negative control f : positive control on the concentration 60%, 30%, 15%, 7.5% and also positive control griseofulvin were not formed transparent zone. thus can be concluded that sea worm p. aibuhitensis extract and griseofulvin cannot inhibit the growth of fungus c. albicans. resistance of anti-fungus is defined as adaptation or adjustment of fungus cell which is stable because of anti-fungus medicines, thus causes the sensitivity toward that anti-fungus decreased rather than the normal condition. generally, fungus can be resistance intrinsically toward anti-fungus medicines (primary resistance) or resistance can occur as the response toward anti-fungus medicine during the treatment (secondary resistance). fungoustatic medicine will more accelerate the resistance rather than fungousidal medicine (apsari and adiguna, 2013) candida as fungus can play the role as commensal or pathogen, where this fungus can change its phenotype randomly and reversible. the change of this phenotype supports adaptation mechanism of c. albicans toward the change from host or all factors which are on the human body that can influence its emergence and also on the way of disease which can be caused by the use of anti-fungus, immune response or physiological change. the effects are morphological appearance, form of cell, virulence and anti-genetic of candida can change (yugo and ridhawati, 2013). according to apsari and adiguna (2013), some of funguses have also biofilm that can cause that fungus susceptible toward anti-fungus medicine. based on nobile and mitchell (2005), the ability of microorganism to affect its environment is based on its ability to form a community. c. albicans forms its community by binding the colony which is called biofilm. according to mukherjee, et al. (2005) biofilm is microbes’ colony (usually the cause of a disease) which forms organic polymer matrix that can be used as the sign of microbes’ growth. that biofilm can be functioned as the protection, thus microbe which formed biofilm usually has the resistance toward the usual anti-microbe or avoid the immunity system of host’s cell. the development of biofilm usually along with the increasing of clinical infection at host’s cell, so this biofilm can be one of the factor of virulence and resistance (kusumaningtyas, 2009) international journal of applied biology, 3(1), 2019 29 according to pramana et. al. (2006), some of researches mentioned that c. albicans had been resistance toward some kinds of anti-fungus, they are; flukonazol, ketonazol, nistatin, and amfoterisin b (hastuti et al, 2013). conclusions the effective concentration of sea worm p. aibuhitensis extract in inhibiting the growth of bacteria s. typhi was on the concentration 60% with forming the diameter of inhibiting zone 12 mm. but, sea worm p. aibuhitensis extract cannot inhibit the growth of fungus c. albicans which was signed by no forming of zone of inhibition. references ajizah, a., thihana and mirhanuddin. 2007. potential of ulus wood extract eusideroxylon zwageri t. et b. journal of bioscientie, 4(1) : 37-42. apsari a.s and adiguna m.s. 2013. antifungal resistance and strategy for overcoming. mdvi 40(2): 90-91. hastusti u.s, ummah y.p.i and khasanah h.n. 2013. power antifungal ethanol extract of piper aduncum and piperomia pellucida leaves against c. albicans growth in invitro. seminar nasional xi pendidikan biologi fkip uns. 87-88. kusumaningtyas e. 2009. mechanism of c. albicans infection on cell surfaces. lokakarya nasional penyakit zoonosis. maleki, s., et all. 2008. antibacterial activity of the fruits of iranian torilis leptophylla against some clinical pathogen. journal of biological science. 11 (9): 1286-1289. mukherjee pk, z hou g, m unyon r and g hannoum ma. 2005. candida biofilm: a welldesigned protected environment. med mycol. 43(3): 191-208 nobile c j and mitchell ap. 2005. regulation of cell-surface genes and biofilm formation by the c. albicans transcription factor bcr1p. curr bio. 15(12): 1150-5 ningtyas, r., 2010. antioxidant test, antbacteria of kecombrang leaf water extract (etlingera elatior (jack) r. m. smith) as a natural preservative against escherichia coli and staphylococcus aureus. skripsi fakultas sains dan teknologi universitas islam negri syarif hidayatullah, jakarta nurhikma, nurhayati t, purwaningsih s. 2017 content of amino acids, fatty acids, and mineral sea worms from southeast sulawesi. jphpi, volume 20, nomor 1. silaban b br.2012. nutritional profile of sipuncula (peanut worm) conservative marine biota on nusalaut island, central maluku.. ambon: laporan hasil penelitian dosen pemula. dibiayai dengan pnbp lembaga penelitian universitas pattimura. purwaningsih s., dkk. 2008. extraction of active components as anticancer in red snails (cerithidea) sustainable cells. jurnal ilmu-ilmu perairan dan perikanan indonesia. 15(2):103-108. yugo m.r dan ridhawati. 2013. antifungal activity of ethanol extract of kunyit leaf curcuma longa linn. against the fungus c. albicans. fakultas kedokteran, universitas indonesia. contact : khusnul khatimah khusnul.khatimah@polipangkep.ac.id 15 abstract increased fishing and agricultural activities trigger eutrophication events in marine waters. excessive availability of nutrients can cause changes in the composition of community structures in marine ecosystems. therefore an effort is needed to mitigate the eutrophication process in coastal and marine areas by increasing the nutrient partition coefficient in compartments that can absorb or consume these nutrients such as macroalgae or seaweed. the aim of this study was to determine the absorption capacity of nutrients (n and p) from gracilaria sp. seaweed as a mitigating agent for eutrophication in waters. this research was conducted in october–november 2018 in a controlled manner at the pui-p2rl wet laboratory, hasanuddin university. seaweed maintenance media is intensive pond wastewater that has gone through a filtering and sterilization process. the results showed that gracilaria sp. able to absorb nutrients in the form of nh 3, no2 , no3 and po4. gracilaria sp. able to reduce no3 by 0.840 ± 0.065 μg/l/day, no2 by 2.100 ± 0.609 μg/l/day, nh3 by 1.506 ± 0.204 μg/l/day and po4 by 8.756 ± 2.785 μg/l/day. issn : 2580-2410 eissn : 2580-2119 the potential of seaweed gracilaria sp. as an organic waste bioremediation agent. khusnul khatimah1*, andi sompa2, haerunnisa2, st. zaenab3 1department of aquaculture technology, pangkajene islands state agricultural polytechnic, pangkep, indonesia. 2department of water resources management, puangrimaggalatung university, wajo, indonesia. 3department of aquaculture, cokroaminoto university, makassar, indonesia. introduction the health of ecosystems and their use in coastal areas is an indicator of water quality, especially in the field of marine conservation and various activities in other fisheries sectors. aquaculture and agricultural activities are a major challenge in maintaining water quality in coastal areas, where increasing concentrations of nutrients (nitrogen, phosphorus and silicon) from these activities can cause water eutrophication which has a negative impact on organisms. this condition has been found in the coastal waters of the west coast of south sulawesi where nutrient enrichment occurs quite large (lukman et al., 2014; nasir et al., 2015) and symptoms of eutrophication have been identified (nurfadillah, 2016). open access international journal of applied biology keyword gracilaria, bioremediation, eutrophication, nutrients, organic waste article history received february 22, 2023 accepted june 14, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:khusnul.khatimah@polipangkep.ac.id international journal of applied biology, 7(1), 2023 16 disposal of aquaculture waste, especially shrimp and fish ponds, and agriculture is the main source of nutrients that cause eutrophication which has an impact on the ecosystem of the west coast of south sulawesi (nasir et al., 2016; hopkins et al., 1995). excessive availability of nutrients can cause changes in the composition of community structures in marine ecosystems such as microorganisms (kegler et al., 2017), plankton (nasir et al., 2015) and coral reef ecosystems (teichberg et al., 2018; edinger et al., 1998). therefore efforts to overcome eutrophication need serious attention. the increase in nutrients in waters, especially in coastal areas, needs to be mitigated by increasing the nutrient partition coefficient in compartments that can absorb or consume these nutrients (carpenter, 2008). on the other hand, seaweed is known to live in waters that have a fairly high concentration of nutrients (kasim, 2016). seaweed utilizes nutrients (n and p) as a food source for its growth and development. nitrogen is needed as a constituent of amino acids, while phosp horus is used as energy in photosynthesis (graham & wilcox, 2000). therefore, seaweed has the potential to become a phytoremediation agent because it is able to adapt to the aquatic environment with extreme nutrient conditions around aquaculture ponds (kom arawidjaja, 2005). this ability to absorb natural nutrients can then be an alternative to mitigating the effects of eutrophication in waters. furthermore, from an economic standpoint, the indonesian government is pushing for increased seaweed cultivation and industrialization in response to the increasing demand for seaweed commodities both on a national and international scale (radiarta et al. 2016). the potential and good quality of indonesian seaweed has made it in demand by various countries in the world. there are more than 550 types of seaweed in indonesia and most of the products from seaweed have been exported as dried seaweed or in processed form. the various opportunities that are owned by one of the biological resources in indonesia make seaweed a superior export product (directorate general of national export development/mjl/ 004/9/2013). rhodophyta and chlorophyta are seaweeds that are widely distributed in sulawesi waters and are found to be quite abundant (admadja et al., 1996). in addition to its relatively easy cultivation techniques and high tolerance for environmental factors, this group of seaweed is known to have good potential in absorbing nutrients that enter the waters. gracilaria sp. is a seaweed that can be used as a phytoremediation agent for organic matter. this type of seaweed has the ability to accumulate organic matter in cells (komarawidjaja, 2005). in addition, gracilaria also has good growth at sufficient nitrogen concentrations. however, comprehensive research on the absorption capacity of seaweed in the context of eutrophication mitigation efforts has not been widely carried out, especially in the south sulawesi region. based on this, research on the absorption capacity of seaweed is important. the capacity of various types of seaweed, which are used economically by the community, in absorbing nutrients as well as in reducing eutrophication in coastal and marine waters needs to be calculated as a reference in developing integrated coastal management based on a international journal of applied biology, 7(1), 2023 17 balance between environmental and economic levels. this research is useful in developing the application of ecosystem-based seaweed cultivation, a sustainable aquaculture system which is becoming a world trend (fao, 2007). this study aims to determine the ability of gracilaria sp seaweed to absorb no3, no2, nh3 and po4 and see the potential of gracilaria sp. in mitigating significantly the excess of nutrients (eutrophication) in the waters. materials and methods this research was carried out at the pui-p2rl wet laboratory at hasanuddin university in october november 2018 which included literature studies, laboratory tests, data collection, sample analysis, data processing, and data analysis. analysis of water samples was carried out at the productivity and water quality laboratory, faculty of marine and fisheries sciences and analysis of seaweed samples was carried out at the animal feed chemistry laboratory, faculty of animal husbandry, hasanuddin university. the tools and materials used in this study were seaweed and int ensive pond wastewater as materials to be tested, chemicals to analyze nutrient parameters (no3, no2, nh3 and po4), as well as instruments to measure water quality (salinity, ph and do). sampling stage gracillaria sp seaweed was taken from the waters of takalar regency. seawater to be used as a medium in a controlled container is taken from the intensive pond disposal area of takalar regency. measurement of water quality parameters measurement of nutrient parameters (no2, no3, po4, nh3) and water quality ( salinity, temperature, ph and do) in the test media was carried out every 10 days (for 1 period/30 days). the methods used in measuring the parameters of water quality and seaweed during rearing and sampling are as follows: table 1. water quality parameters no parameter sample methods/tools 1 p total seaweed hno3 -hclo4 spectrophotometry 2 n-total seaweed kjeldahl 4 growth seaweed analytical balance 6 phosphate (po4) sea water spectrophotometry 7 nitrates (no3) sea water spectrophotometry 8 nitrite (no2) sea water spectrophotometry 9 ammonia (nh3) sea water spectrophotometry 10 light intensity sea water lux-meter 11 temperature sea water thermometer 12 salinity sea water hand refractometer 13 do sea water do meters 14 ph sea water ph meter measurement of nutrient parameters (no2,nh3,no3 and po4) international journal of applied biology, 7(1), 2023 18 measurement of nutrient parameters using the spectrophotometric method and the linear standard curve method following the measurement procedure of grasshoff et al., 1983. nitrate measurement using the cadmium reduction method, nitrite measurement using the sulfanilamide method, ammonia measurement using the ammonium molybate method, and phosphate measurement using the stony chloride method. measurement of the growth rate of seaweed biomass the growth rate of biomass is obtained from the total final biomass growth minus the initial biomass divided by the time required. the formula for calculating the growth rate of biomass (harianto et al., 2012): lpb == bt − b0 t where: lpb : biomass growth rate (gram/day) bt : final biomass (gram) b0 : initial biomass (grams) t : time (days) the rate of reduction of nutrients in water decline rate = = 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐧𝐮𝐭𝐫𝐢𝐞𝐧𝐭 𝐓𝟏− 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 𝐨𝐟 𝐧𝐮𝐭𝐫𝐢𝐞𝐧𝐭 𝐓𝟐 𝐭 where: t1 : initial nutrient concentration t2 : final nutrient concentration t : observation period (days) total n analysis measurement of total n in seaweed was carried out at the beginning and end of rearing. standard measurement of total n in seaweed using the kjeldahl method (aoac, 1980). to calculate the total n, the following formula is used: total n (%) [{0,0007∗×(vb−vs)×f×20 s ×100 where: vs = ml 0.05 n naoh titrant for sample vb = ml of naoh titrant for blank f = correction factor of 0.05 naoh solution s = sample weight * = per ml 0.05 naoh equivalent to 0.0007 g n data analysis research data were processed and analyzed in the form of tables and graphs using microsoft excel 2013 software and the prism application. international journal of applied biology, 7(1), 2023 19 results and discussion nutrient concentrations (ammonia, nitrate, nitrite, and phosphate) in gracillaria sp. figure 1 shows that there was a decrease in the concentration of nutrients in the form of nitrate, nitrite, ammonia and phosphate in the seaweed rearing medium in each measurement period (per 10 days). however, on the last day of the study (30th day) there was an increase in the concentration of nitrite and nitrate in the rearing medium. figure 1. graph of nutrient concentrations in gracillaria sp. nutrient concentrations (ammonia, nitrate, nitrite, and phosphate) in the gracilaria sp. on the 0th, 10th, 20th and 30th day respectively for ammonia (0.047 mg/l ± 0.003 mg/l; 0.014 mg/l ± 0.003 mg/l; 0.002 mg/l ± 0.001 mg/l; 0.001 mg/l ± 0.002 mg/l), nitrate (0.162 mg/l ± 0.004 mg/l; 0.115 mg/l ± 0.009 mg/l; 0.079 mg/l ± 0.019 mg/l; 0.136 mg /l ± 0.001 mg/l), nitrite (0.125 mg/l ± 0.006 mg/l; 0.083 mg/l ± 0.006 mg/l; 0.056 mg/l ± 0.005 mg/l; 0.062 mg/l ± 0.005 mg/l ), and phosphate (0.764 mg/l ± 0.013 mg/l; 0.670 mg/l ± 0.024 mg/l; 0.536 mg/l ± 0.053 mg/l; 0.461 mg/l ± 0.046 mg/l). the absorption of nutrients by seaweed can be calculated from the value of the rate of decrease in nutrient concentrations found in the seaweed rearing medium. this can occur if the decrease in nutrient concentration is not caused by nitrogen transformation factors (nitrification and ammonification). the decrease in n concentration in the rearing water medium was not necessarily caused by the absorption of seaweed. the decrease in nutrient concentration can be caused by the process of ammonia oxidation or nitrification, where in this co ndition there is a decrease in the concentration of ammonia and an increase in the concentration of nitrite, which then nitrite will also undergo oxidation to become nitrate. based on figure 1, there was a decrease in ammonia concentration in the gracilaria sp. caused by absorption by seaweed that occurs in each measurement period (day 0 to day 30). the same pattern of decrease in concentration also occurred in nitrate and nitrite content until the 20th day, but showed the opposite pattern on the 30th day o r the last day of the study, namely an increase in concentration in the gracillaria seaweed rearing medium. figure 1 shows the symptoms of nitrification in the water media for seaweed gracillaria sp. at the end of the study (30th day), where there was a decrease in ammonia and an increase in nitrite and nitrate concentrations at the same time. this is thought to be a symptom of nutrient oxidation in the seaweed rearing medium at the end of the study, but this symptom still needs international journal of applied biology, 7(1), 2023 20 further investigation because the o2 indication did not show a significant change during the trial period. in gracilaria sp. the rate of decrease in the concentration of phosphate in the rearing medium occurred at the beginning of the study until the end of the study (figure 1). in t he gracilaria seaweed rearing medium, the decrease in phosphate concentration was due to absorption by the seaweed which occurred on day 10 to day 30. phosphate has a role as forming cell membranes and energy transfer within cells (pramesti, 2013). phosphate at the end of the study is thought to inhibit metabolic processes because no energy is used for the process so that the growth of seaweed decreases. nutrient depletion rate (ammonia, nitrate, nitrite, and phosphate) table 1. the rate of nutrient reduction (mean ± standard deviation) in each treatment during observation. nutrient type average nutrient depletion rate (µg/l/day) 10th day 1 2 3 ammonia 3.300 ± 0.265 1.133 ± 0.208 0.085 ± 0.139 nitrate 4.633 ± 1.274 3.667 ± 2.434 -5.779 ± 2.487 nitrite 4.167 ± 1.002 2.667 ± 0.709 -0.533 ± 0.115 phosphate 9.367 ± 1.815 13.367 ± 5.008 3.533 ± 1.531 the value of the daily reduction rate of ammonia, nitrate, nitrite, and phosphate concentrations on gracillaria sp. can be seen in table 2. on the maintenance media of gracilaria sp. there was a decrease in nutrient concentrations in the form of ammonia and phosphate until the end of the study, while a decrease in nutrient concentrations in the form of nitrate and nitrite only occurred until the 20th day, after the 30th day it was seen that there was an increase in the concentration of nitrate and nitrite in the rearing medium. the highest rate of decrease in ammonia, nitrate and nitrite concentrations occurred in the first 10 days (0-10 days) and the lowest occurred at the end of the study (second 10 days), in contrast to phosphate concentrations which showed the opposite condition, namely a decrease in phosphate concentrations in the second 10 days. observation is greater than the first 10 days. figure 2. graph of nutrient depletion rate per 10 days value of the average amount of reduction of ammonia, nitrate, nitrite, and phosphate international journal of applied biology, 7(1), 2023 21 in the media maintenance of gracillaria sp. respectively 1.506 ± 0.204 μg/l/day; 0.840 ± 0.065 μg/l/day; 2.100 ± 0.609 μg/l/day; and 8,756 ± 2,785 μg/l/day. the minus value in the figure above shows that there is no rate of decrease in nitrite and nitrate concentrations on the 30th day. this means that there was an increase in nitrite and nitrate indicating the occurrence of nitrification process at that time. total n and total p values of gracillaria sp. figure 3. total n and p total values of gracillaria sp. at the beginning and end of the study in figure 3, the highest ntotal and ptotal values were found at the end of the study (30th day), namely 17.23 ± 0.557% and 0.547 ± 0.039%, while the ntotal and pttotal values for gracillaria sp. at the beginning of the study that is equal to 7.280 ± 0.355% and 0.337 ± 0.014%. the mean difference between the initial (0th day) and final (30th day) ptotal values of maintenance on gracilaria sp was 0.21 ± 0.050% and the ntotal was 9,950 ± 0,911%. this shows that gracilaria sp. has the ability to absorb more nitrogen elements compared to phosphorus elements contained in the rearing medium. based on the results of ntotal analysis on gracilaria sp., there was an increase in ntotal and ptotal at the end of the rearing (figure 3). the results of the analysis showed that the ntotal content was higher than the ptotal in seaweed. this shows that the uptake of nitrogen by grass is higher than the consumption of phosphorus. excess nitrogen available in the waters can stimulate seaweed to consume nitrogen in large quantities, which results in high concentrations of n tissue (gordon et al., 1981). the process of absorption of nutrients in seaweed is carried out by diffusion through all parts of its body (supriyantini et al, 2018). the cell membrane which is the outermost part of the cell after the cell wall acts as a protector of the contents of the cells in the body will regulate nutrients that go out and enter the cell. the amount of nutrients that diffuse into the cell depends on the concentration of nutrients inside and outside the cell. this statement was confirmed by lobban and harrison (1994) that nutrients will increase cell metabolic activity by entering the cell gradually and then developing vacuoles in the cell. where vacuoles play a very important role in life because the survival mechanism of seaweed depends on the ability of vacuoles to maintain the concentration of dissolved substances in its thallus. international journal of applied biology, 7(1), 2023 22 seaweed growth figure 4. the average weight value of seaweed during the study seaweed growth can be seen from the weight added value of the seaweed. the average weight value of gracillaria sp. from the beginning to the end of the study respectively 72.013 ± 0.023 gr, 72.830 ± 0.235 gr, 101.513 ± 1.825, and 20.007 ± 0.076 gr. figure 3 shows that there is an increase in the weight of gracilaria sp. on day 10 to day 20 and then decreased in weight at the end of the study (day 30). the highest weight gain of gracilaria sp. occurred on the 20th day, which was 98.51 gr, then on the 30th day there was a decrease of 67.01 gr. in figure 4, there is an increase in the weight of seaweed gracilaria sp. on day 10 to day 20. the increase in seaweed weight until the 20th day was due to the absorption of nutrients (n, p) at that time. according to gordillo et al., (2002) that the rate of absorption of phosphate and nitrate accordingly has a positive correlation with increasing the rate of growth of seaweed. on the 30th day there was a decrease in the weight of the seaweed which indicated that the ability of the seaweed to absorb nutrients decreased. on the 30th day, some of the seaweed thallus died and rotted, resulting in decomposition and an increase in nutrients. in addition, light penetration in the seaweed rearing medium was less than optimal which caused some of the seaweed thallus to turn white and die. according to atmadja (1996) that the growth of seaweed is closely related to the penetration of light and nutrients found in the environment where the seaweed lives. water quality parameters the results of measuring water quality parameters on gracillaria sp. presented in table 3. table 3. water quality parameter values during the study (mean ± standard deviation) parameters days 0 10 20 30 salinity (ppt) 31 ± 0.58 30 ± 0.24 29 ± 0.38 30 ± 0.15 ph 7.37 ± 0.13 7.77 ± 0.22 8.10 ± 0.06 8.16 ± 0.06 do (ppm) 5.80 ± 0.40 6.67 ± 0.33 6.72 ± 0.19 6.91 ± 0.11 temperature (°c) 29 ± 0.58 29 ± 0.64 30 ± 0.37 30 ± 0.18 international journal of applied biology, 7(1), 2023 23 table 3 shows that the salinity values in the gracillaria sp seaweed rearing media ranged from 29-31 ppt, ph ranged from 7.37-8.16, do ranged from 5.80-6.91 ppm, and temperature ranged from 29-30°c. during the research process, the water quality in the exp erimental pond unit was in a relatively stable condition. the stability of the water quality conditions from the beginning to the end of the experiment can explain that the absorption of nutrients by seaweed is not affected by water quality factors so that the ability to absorb nutrients from seaweed can be identified. conclusion gracilaria sp seaweed is able to absorb nutrients in the form of nh3 and po4 up to 30 days and in the form of no2 and no3 up to 20 days, with the ability to reduce no3 concentrations by 0.840 ± 0.065 μg/l/day, no2 by 2.100 ± 0.609 μg/day l/day, nh3 of 1,506 ± 0,204 μg/l/day and po4 of 8,756 ± 2,785 μg/l/day. international journal of applied biology, 7(1), 2023 24 references association of official analytical chemists (aoac). 1980. official methods of analysis association of official analytical chemist. 13th edition atmadja ws, kadi a, sulistijo, rachmaniar. 1996. introduction to the types of indonesian seaweed. oceanology research and development center. lipi. jakarta. carpenter, s. r. (2008). phosphorus control is critical to mitigating eutrophication. proceedings of the national academy of sciences, 105(32), 11039–11040. https://doi.org/10.1073/pnas.0806112105. directorate general of national export development/mjl/004/9/2013 september. in http://djpen.kemendag.go.id/app_frontend/admin/docs/publication/620139036751 7.pdf accessed in march 2019 edinger, e.n., j. jompa, g.v. limmon, w. widjatmoko, m.j. risk (1998). reef degradation and coral biodiversity in indonesia: effects of land -based pollution, destructive fishing practices and changes over time. marine pollution bulletin 36(8), 617-630. fao. 2011. the cassava transformation in africa. the food and agriculture organization of the united nations (fao). gordillo, f. j., dring, m. j., & savidge, g. 2002. nitrate and phosphate uptake characteristics of three species of brown algae cultured at low salinity. ma rine ecology progress series, 234, 111-118. gordon, d. m., birch, p. b., & mccomb, a. j. 1981. effects of inorganic phosphorus and nitrogen on the growth of an estuarine cladophora in culture. graham, l.e., l. e. wilcox, 2000. algae (chapter 23 – macroalgal and perihyton ecology). prentice-hall, inc. p.s. 619. grasshoff, k., enrhardt, m., kremling, k. 1983. method of seawater analysis. second, revised and extended edition. verlag chemie. germany harianto, e., budiardi, t., & sudrajat, a. o. 2014. growth performance of 7-g anguilla bicolor bicolor at different densities. journal of indonesian aquaculture, 13(2), 120 -131. hopkins, j. s., m. r. de voe, and a. f. holland. 1995. environmental impacts of shrimp farming effluent in the continental united stated. kasim, m. 2016. ecological, biological studies, utilization and cultivation of macroalgae. self help spreader. jakarta. kegler hf, lukman m, teichberg m, plass-johnson j, hassenrück c, wild c and gärdes a., 2017. bacterial community composition and potential driving factors in different reef habitats of the spermonde archipelago, indonesia. front. microbiol. 8:662. doi: 10.3389/fmicb.2017.00662. komarawidjaja, w. 2005. gracilaria sp. seaweed. as phytoremedian organic matter in aquaculture ponds. j.tek. ling. p3tl-bppt, 6 (2): 410 – 415. lobban, c. s., & harrison, p. j. 1994. seaweed ecology and physiology. cambridge universitypress. lukman, m., a. nasir, k. amri, r. tambaru, m. hatta, nurfadilah, r.j. noer, 2014. dissolved silicates in coastal waters of south sulawesi. journal of tropical marine science and technology, 6(2): 461-478. nasir a, lukman m, tuwo a, hatta m, tambaru r and nurfadilah, 2016. the use of c/n ratio in assessing the influence of land-based material in coastal water of south sulawesi and spermonde archipelago, indonesia. front. mar. sci. 3:266. doi: 10.3389/fmars.2016.00266. international journal of applied biology, 7(1), 2023 25 nasir, a., m. lukman, a. tuwo, and nurfadilah, 2015b. ratio of nutrients to diatom dinoflagellate communities in spermonde waters, south sulawesi. journal of tropical marine science and technology, vol. 7, no. 2: 587-601. nasir, a., tuwo, a., lukman, m., usman, h., 2015a. impact of increased nutrient on the variability of chlorophyll-a in the west coast of south sulawesi, indonesia. international journal of scientific & engineering research, 6(5): 821-826. nurfadilah. 2016. comprehensive assessment of eutrophication conditions with primary and secondary symptom approaches in muara sungai pangkep waters. hasanuddin university masters degree, makassar. pramesti, r. 2013. enriched seawater media on the growth rate of seaweed gracilaria lichenoides (l) harvey. marina oceanographic bulletin, 2(1), 66-73. radiarta in, erlania, haryadi j, rosdiana a. 2016. analysis of the development of seaweed cultivation on sebatik island, nunukan regency, north kalimantan. journal of indonesian fisheries policy. 8(1) : 29-40. supriyantini, e., santosa, g. w., & alamanda, l. n. 2018. growth of gracilaria sp. seaweed. in media containing copper (cu) with different concentrations. marina oceanographic bulletin, 7(1), 15. teichberg, m., wild, c., bednarz, v. n., kegler, h. f., lukman, m., gärdes, a. a., ... & plass johnson, j. g. 2018. spatio-temporal patterns in coral reef communities of the spermonde archipelago , 2012–2014, i: comprehensive reef monitoring of water and benthic indicators reflect changes in reef health. frontiers in marine science, 5, 33. 2(1)12-21 contact : setiyo prajoko setiyoprajoko@untidar.ac.id � international journal of applied biology 12 abstract the aims of this study are to determine the feasibility of bengawan solo river water for irrigation of rice field in sragen, central java and to find solution to the problem. the data used in this study were obtained from various literatures related to the issues discussed. the collected data were then selected and analyzed with descriptive argumentative. the result indicates that the water quality of bengawan solo river has dropped below the water quality standard grade iv according to government regulation number 82 year 2001. the use of water from this river has caused the quality of rice to decline below the rice quality standard according to indonesian national standard (sni) 01.6128-2008 due to the bioaccumulation of heavy metals on rice crops, i.e. copper (cu), mercury (hg), cadmium (cd), and chromium (cr) exceeded the threshold. solution proposed to overcome this problem is with counseling programs to farmers conducted by relevant agencies on the use of rice field pollution control technology by physics, chemistry, and biology. issn : 2580-2410 eissn : 2580-2119 water feasibility study of bengawan solo river for irrigation: the need for technology to solve rice field pollution in sragen, indonesia setiyo prajoko & riva ismawati biology education department, faculty of teacher training and education, universitas tidar, magelang, indonesia introduction the condition of bengawan solo river is currently worrying. many people have complained about the water condition of this longest and largest river in the island of java. the results of observations show that, in plain view, the water of bengawan solo river looks turbid mixed with mud in the rainy season; while during the dry season, the water is sometimes slightly dark and smelly. several studies have shown that the water from this river is polluted, and even the content of heavy metals waste is also found. whereas the bengawan solo river is used by some people for their daily needs. the bengawan solo river with an average annual rainfall of 2,100 mm3 is a potential water source for management and development efforts of water resources, to meet various needs and requirements such as for domestic consumption, for drinking water and industrial, irrigation, and others (ministry of public works, 2010). open access international journal of applied biology keyword feasibility bengawan solo heavy metals quality standard solution article history received 12 may 2018 accepted 10 june 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(1), 2018 13 the development of solo city into an industrial city has a negative impact on pollution in the river. there are many industries developing in solo and its surrounding areas that dispose their waste into the river. batik (traditional clothing) industry that has become the icon of solo city on the one hand gives positive value for the economy of society but on the other hand also contributes in river pollution. the efforts conducted by the government, social institution, and the society to solve this issue also seem to have been unsuccessful. although with the naked eye, the water of bengawan solo river is polluted but in some parts of this river basin, it is still used by people around for everyday purposes. based on observations to the river, from the city of solo to sragen regency, activities of people who use water from the river such as bathing, washing, fishing, swimming spot for children, and irrigation of agricultural land can be observed. water utilization of the bengawan solo for irrigation occurs in some areas, one of which is in sragen. in dry season, the agricultural land in sragen often suffers from water shortages. people who own rice field near the bengawan solo river usually take water from the river for irrigation. they do not have the awareness and concern about the importance of the quality of water used for irrigation, whereas, this issue is related to the quality of their crops. based on the background, the water condition of bengawan solo river is contaminated with heavy metals waste. but there are still people who use it for daily purposes especially farmers for irrigation. this what prompt the water feasibility study of the bengawan solo river for irrigation is conducted and also to find the solution. materials and methods this article was written using the literature study method. information is obtained from a variety of literatures and compiled based on the results of the study. writing is arranged in such a way that they are interrelated between each other and in accordance with the topics covered. the data that used in this paper comes from literatures related to the issues discussed. references that used is taken from books, scientific articles, and newspapers. then the data analyzed both qualitative and quantitative. results and discussion pollution in bengawan solo river various studies have concluded that bengawan solo river has been polluted. this river is currently in an unhealthy condition because of the water quality and the environmental support capacity is continuously declining. pollution can also be seen in the bengawan solo river fragment from solo to sragen. utomo et al. (2010) elaborates that the bengawan solo river has been heavily polluted with poor water quality because it contains low oxygen, high co2, high nh3, high cod, high phenol, and high fat oil. in addition, heavy metals concentration in some locations like kampung sewu, bak kramat, and tundungan is quite high at cr 0.180-0.375 mg/l, cu 0.026-0.293 mg/l, and zn 0.515-2.892 mg/l. heavy metals waste in the form of mercury (hg) is also found in this river. riyatun et al. (2004) reveals that mercury level found in the water and also the fish that live freely in bengawan solo river is high (0.38-0.64 ppm). it draws concern to the feasibility of the water designation and the fish consumption. similarly, astirin et al. (2001) also concludes that all rivers in surakarta are polluted based on plankton diversity parameters as the bioindicator. meanwhile, blhd sragen (local environmental agency) (2012) indicates that the level of water pollution at bengawan solo river in sragen city has exceeded the standard limit of international journal of applied biology, 2(1), 2018 14 water quality. it can be seen from the conditions of bo d 13.5 mg/l, cod 40 mg/l , do 0.4 mg/l, phosphate 0.126 mg/l , fe 0.4647 mg/l, mn 0.1523 mg/l), nitrite 0.261 mg/l, total coliform 920.104/ 100 ml, and fecal coliform 170.104/100ml. the data of bengawan solo pollution is presented on table 1. table 1. result of research on pollution of bengawan solo river basin researchers aspects studied information astirin op et al., 2001 plankton diversity mild to severe pollution winarno k. et al., 2000 diversity and wealth community of macrobentos medium-heavy pollution supriyadi ds, 2002 chemical character and physical character fluctuations in chemical and physical contents occur sanaky, 2003 phytoplankton diversity a decline in water quality riyatun et al., 2003 mercury (hg) indicated to contaminate with hg utomo et al., 2010 bod, cod, ph, phenol, oil-fat, ammonia, cd, cr, zn, pb, cu, and zn heavily polluted with poor water quality, contain cr, cu, pb, and zn contents exceeding grade iv of water quality standard yuliastuti., 2011 tss, do, bod, cod, n, p, fe a decline in water quality blhd (local environmental agency), sragen, 2012 temperature, tss, tds, ph, nitrate, ammonia, cd, cr, cu, fe, pb, mn, zn, cl, nitrite, phenol, ni, debit , total coliform and fecal coliform there is a decrease in water quality, some parameters exceed the standard limit of water quality grade iv (total coliform and fecal coliform) astuti et al., 2014 analysis of pb, on white shrimp in bone river (bengawan solo watershed) pb content in white shrimp is found setyarini et al., 2016 cu, cd, pb, on broom fish in solo bengawan river the content of cu, cd, pb on pleco fish exceeds the limit due to the polluted environment. according to the indonesian government regulation no. 82 year 2001, water quality standards classified based on the parameters of physics, chemistry, and biology are as follows: (a) water quality grade 1, water that can be utilized for drinking water service and/or other usages that require similar water quality to the utilization; (b) grade 2, water that may be used for recreational water facilities, cultivation of freshwater fish, livestock, water to irrigate crops and / or other designations that require the same water quality as those usages; (c) grade 3, water which designation may be used for the cultivation of freshwater fish, livestock, water for cropping, and / or other purposes that require the same water quality to those designations; (d) grade 4, water that may be used for irrigation, cropping, and / or other designations that require the same water quality as that purposes. under the government regulation, it is clearly stated that minimum water quality standard for irrigation is grade 4. however, based on research results, especially in 2012, clearly show that the water quality of the solo river is below grade iv of water quality standard based on total coliform, fecal coliform, and cod parameters. in addition, heavy metals waste in the river water that exceeds threshold of water quality standard class iv is international journal of applied biology, 2(1), 2018 15 also discovered. the presence of mercury content that exceeds grade iv of water quality standard threshold. a research by utomo also shows that the content of heavy metals cr, cu, pb, and zn are high enough. thus, water of bengawan solo river is no longer feasible to be used for irrigation. quality of rice harvest from rice fields along bengawan solo river basin in sragen regency the quality of rice crops is important to note, not only the quantity considering the role of this plant as a staple food source. according to the directorate general of processing and marketing of agricultural products on rice quality standards based on sni (indonesia national standard) no.01.6128 -2008, rice is classified into 5 grades of quality; namely i, ii, iii, iv, and v. general terms of rice are: 1) pest, disease free, 2) free of musty odor, sour, or other unknown odors, 3) free of bran mixture, and 4) free of chemical substances that endanger consumers. while, the minimum specific terms rice are included in quality v. the parameters of rice quality v including milling degree of at least 85%, moisture at 15%, rice head size of at least 60%, minimum grains intact 35%, broken grains maximum 5%, groat grains maximum of 3%, yellow/damaged grains maximum 5%, maximum 5% of lime grains, maximum 0.2% of foreign objects, maximum 3 grains/100 gram, and mixture of other varieties of up to 10 %. from the general terms of rice quality standards, rice must be free from chemicals that endanger consumers. unfortunately, the fact found that there is still rice that contains heavy chemical substances that are harmful to consumers. sragen as the second largest rice granary in central java utilizes bengawan solo river for irrigation. sragen has a total area of 40129 hectares of rice fields. the fact shows that most of the agricultural land in sragen is crossed by bengawan solo river. based on survey results, farmers take water from this river for irrigation during the dry season. it impacts on the quality of the crops, whereas, during the dry season, the water volume is small and polluted. it affects the quality of their crops. the results of the laboratory analysis universitas sebelas maret, surakarta in 2003 and 2004 showed that rice yields along the bengawan solo river basin were contaminated with heavy metal of cu 0.2 mg / kg. previously in 2003, a research also found that heavy metals content in rice plants in gesi region (an area crossed by bengawan solo) were in the forms of hg 0.61 mg / kg, cd 5.69 mg / kg, cr 0.67 mg/kg (suara merdeka, 2004). bioaccumulation of heavy metals in agricultural crops according to kurnia et al. (2009), the entry of pollutants on agricultural land is classified into agricultural and non-agricultural activities. a heavy metal contaminant is defined as a type of metal that has a molecular weight and specific gravity greater than 5 g/cm3. types of heavy metals that need special attention are hg, pb, cd, cu, cr, co, mn, and ni. pollutants may come from agricultural activities and non-agricultural such as industries and mining. heavy metals can contaminate rice plants through gradual mechanism. raskin et al. (1997) explains the bioaccumulation in plants by phytoextraction. heavy metals dissolved in water are in the root environment (rhizosphere). these substances will comppete with elements of nutrients for other plants (macro/microelement) absorbed through the roots (rhizofiltration). next, accumulation of heavy metals in the roots through the transport system of symplast and apoplast occurs. then, the heavy metals will spread throughout the other parts of plants. some types of metals can bind to other elements so as to form a complex of compounds with lower toxic effects on plants. furthermore, the complex will be brought to the vacuole (haryanti, 2009). meanwhile, according to grant international journal of applied biology, 2(1), 2018 16 (1998), the mechanism of absorption and accumulation of heavy metals by plants can be divided into three sustainable processes, namely absorption by roots, translocation of heavy metals from roots to other parts of plant, and localizing metals on cells and tissues. it aims to keep the metals from inhibiting plant metabolism. in an attempt to prevent metal poisoning of cells, plants have detoxification mechanisms; for example by hoarding metals in certain organs such as roots. toxicology of heavy metals waste the presence of heavy metals in organisms, both animals and plants that are foods for humans, are very dangerous. the human position at the top of food chain must have become a heavy metals accumulator in large quantities. according to duruibe et a.l (2007), heavy metals can cause biotoxic to humans biochemically and can cause clinical symptoms of heavy metals poisoning. sudarmaji et al. (2006) discloses the clinical implications of heavy metals biotoxic for human health including: pb poisoning can lead to disturbances in the nervous system, kidneys, reproductive system, and circulatory system; mercury poisoning can lead to minamata disease.; and other symptoms such as cerebral palsy, mental disorders, and libido disorders. cadmium poisoning can lead to interference with the kidneys, heart, blood vessels, and bone disorders. cu toxicity can cause disturbances in the respiratory system, skin, kidneys, and blood vessels. while as toxicity can cause disturbances in the eyes, skin, blood, kidneys, liver, respiratory organs, reproductive system, gastrointestinal, and immune system. the clinical symptoms of heavy metals toxicity cannot be directly observed at once. this is because the accumulation process takes a significant time. clinical symptoms will appear when the accumulation in the body has exceeded the threshold. the need for socialization of rice fields pollution control technologies solution offered to overcome the problem of poor water quality of bengawan solo river, that its use is no longer appropriate for irrigation, can be done through preventive and curative actions. preventive action is done by preventing farmers from using the river water to irrigate their fields. while curative measures can be done by socializing the use of rice field pollution control technologies. after recognizing the technology, furthermore , it is expected that the farmers will apply the technology to reduce the heavy metals content in the water of the bengawan solo so that it is safe for irrigation. preventive actions preventive action is a useful action to prevent the use of bengawan solo water contaminated by heavy metals for irrigation. therefore, a regular research on the quality of river water by the authorities is required. such research should be conducted to coincide with the growing season in the dry season. the results of the research should also be published to farmer groups, especially farmers who have rice fields along the bengawan solo. thus, the coordination between the local environmental agency and local agricultural agency which are responsible to provide counseling to farmer groups is needed. farmers need to be given insights through extension activities on the dangers of using bengawan solo water for irrigation; instead the ground water can be use as the replacement (pantek well). pantek well is a drill well to take deep ground water inside. preventive action also needs to be performed in formal education. students who attend schools around bengawan solo river basin are necessary to develop a scientific attitude, especially the attitude of environmental care. prajoko (2017) suggests that international journal of applied biology, 2(1), 2018 17 it is important to develop a scientific attitude at schools in order to form a scientific attitude culture since early age. so, the attitude of environmental care as a manifestation of scientific attitude will be formed early on. thus, it needs to develop teaching instruments that are based on environmental insights, especially pollution at bengawan solo river, to students as early preventive efforts. purnomo et al. (2013) uses a module of research on the pollution of the pepe river (tributary of bengawan solo) as the material source of biology learning on environmental pollution. the results showed that the use of the module is effective for improving biology learning in that specific material. meanwhile, according to putri (2012), the development of biology learning instruments integrated with character is empirically feasible in terms of concept mastery as well as in the improvement of character education of environmental care. curative actions curative action is an action to reduce the heavy metals content contained in the water of the bengawan solo river before being used for irrigation. this action can be done by developing pollution control technologies for rice fields by chemical, physics, and biology which needs to be socialized to the society. chemically, replace the chemical fertilizers with organic fertilizers. natural ingredients such as organic and inorganic fertilizers, manures, and limes can improve the quality of the plant. nurjaya (2003) conducted a research in a greenhouse using zeolite, manure, ash husk, and carbon to absorb more pb and cd from rice fields in tegal regency, central java, which are planted with garlic. the results showed that ash husk was able to absorb pb and cd from the soil with more ameliorant, both by leaves and tubers. according to kurnia (2009), control of agricultural land pollution in chemical is relatively more expensive. the purpose of chemical solving is only to reduce or minimize the impact caused by pollution, because to completely eliminate the pollution impact is difficult. the solving by physics is done in order the influence of heavy metals contained in the soil is reduced. this can be done by heating and absorption techniques using active charcoal, zeolite and bentonite, drainage techniques, and agricultural waste (endrawanto & winarno, 1996). the use of agricultural waste such as rice husk is also effective to absorb heavy metals. according to nurhasni et al. (2010), the optimum conditions of absorption are ph 5 for cd, and ph 6 for cr, the concentration of metal ions 20 mg / l, and the duration of heating 2.5 hours. the efficiency of absorption in the multi components waste water for cd is 70.42%, while for cr is 71 .55%. absorption of cadmium and chromium metal ions under optimum conditions applied to chemical laboratory waste water of syarif hidayatullah islamic state university of jakarta has a respective absorption efficiency of 66 .45% for cd and 49.29% for cr. biologically is by phytoremediation using mendong plant, water hyacinth, and rice field grass, or other plants that have the potential to become hyperacumulator; while by bioremediation using microorganisms. phytoremediation and bioremediation are mechanisms of heavy metals absorption by biological agents of either plants or microorganisms. basically, before the polluted river water is used for irrigation, the heavy metals content should be reduced so that the levels do not exceed the specified threshold. phytoremediation phytoremediation is defined as leaching of pollutant which is mediated by plants, including trees, grasses, and aquatic plants. leaching can be translated as destruction, international journal of applied biology, 2(1), 2018 18 inactivation, or immobilization of pollutants to harmless forms chaney et al. (1995). there are several strategies of plant performing phytoremediation. the strategies are based on the ability to accumulate contaminants (phytoextraction) or on the ability to absorb and transpire water from within the soil (creation of hydraulic barriers). the ability of roots to absorb contaminants from groundwater (rhizofiltration) and the plants’ ability in metabolizing contaminants within the tissues (phytotransformation) are also used in phytoremediation strategies. phytoremediation is also based on the ability of plants to stimulate biodegradation activity by microbes associated with roots (phytostimulation), and immobilization of contaminants in the soil by exudates of roots (phytostabilization), and the ability of plants to absorb large quantities of metals from the soil and is economically used to remediate problematic soil (phytomining) (chaney et al., 1995). in addition to mendong plants, rice field grass, and water hyacinth, the following are plant species that have the potential as a hyperaccumulator. table 2. types of plants potentially as hyperacumulators contaminant type plant zn (zink) cd (cadmium) pb (plumbum) co (cobalt) cu (kuprum) mn (manganese) ni (nickel) cs (cesium) as (arsenic) se (selenium) fe (ferum) hg (merkurium) salinity crude oil thlaspi caerulescens, t. calaminare, sambucus, rumex thla spi caerulescens, sambucus, rumex , mimulus guttatus, lolium miscanthus lolium miscanthus, thlaspi rotundifolium agrotis gigantea, haumani astrum robertii, mimulusguttatus aeolanthus biformifolius, lolium miscanthus alyxia rubricaulis alyssum berkolinii, a. lesbiacum, berkheya coddi, hybanthus flor ibundhus, thlaspi goesingense, t. montanum, senesio coronatus, lol ium miscanthus, phyllanthus serpentinus amaranthus retroflexus reynoutria sachalinensis, clamidomonas sp . astragalus racemosus poaceae arabidopsis thaliana attriplex spp., halosarcia spp., enneapogon spp. euphorbia, cetraria, amaranthus retroflexus. source: hidayati (2005) the heavy metals absorbed by plants will further, the toxic effects, be reduced by the plants. pulford & watson (2002) describe the mechanisms for reducing toxic effects can be done by combining metals absorbed by the plants with substances contained in the plant. then, the result of the merger will be brought into the vacuole in plant cells to be stored. bioremediation bioremediation is the use of microorganisms to reduce pollutant in the environment. when bioremediation is conducting, enzymes produced by microorganisms modify toxic pollutants by altering the chemical structure of the pollutants and this is what so called as biotransformation. in many cases, biotransformation leads to biodegradation where toxic international journal of applied biology, 2(1), 2018 19 pollutants are degraded; the structure becomes no longer complex and thus ultimately becomes harmless and nontoxic metabolites. several types of bacteria, such as pseudomonas, thiobacillus, bacillus, and nitrogenfixing bacteria are reported to be able to accumulate heavy metals. microorganisms have a variety of ways of absorbing toxic metals and several mechanisms are known at the molecular level. resistance to metals is usually encoded by plasmids contained in the bacteria. skladany (1993) reveals that plasmids that become efflux path way for as and cd exists in certain bacteria and is able to differentiate the absorption paths for p and mn, through as and cd which each enters into the cells. some metals and their components are target in the biotransformation that may increase or decrease toxic. the efflux path ways for metals such as cd and ion hg2+ are detoxified by metal translocation penetrating via intracellular reductase. the alkylation process of pb and as components by prokaryotic and eukaryotic microbes may increase the toxic potential. in indonesia, the use of bacteria as bioremediator to accumulate heavy metals bacillus sp., has been tested on rice fields contaminated by industrial waste. kurnia (2009) explains that the success of reduction of heavy metals pollution by microbes accumulating heavy metals is influenced by its superiority in accumulating heavy metals. each type of microbe has different characters to be able to live in the same environment. microorganisms capable of living at relatively higher concentrations of metals than predetermined environmental quality standards may be used as reference in the use of such microbes in reducing pollution, both on farmland and in aquatic body, in accordance with the high content of heavy metals within the contaminated environments. the use of microbes is expected to improve the health of farmers and the benefits of farming, so as to accelerate poverty alleviation. utilization of microbes accumulating heavy metals does not cause soil contamination. conclusions based on the study that has been done, it can be concluded that the water quality of bengawan solo river has dropped below the water standard grade iv especially during the dry season. so it is no longer appropriate allocation for irrigation. the use of water for irrigation from the river is the cause of the declining quality of rice harvest in sragen regency because there is a bioaccumulation of heavy metals on rice grains. preventive action can be done to prevent pollution of rice fields by conducting socialization to farmers group through counseling activities. to develop teaching instruments based on environmental insight is necessary for students as an early socialization effort. curative action can be done with the use of pollution control technology on rice fields either by physics, chemistry, and biology. references astirin, o.p., setyawan, a.d. & harini m. 2001. plankton diversity as a river quality indicator in surakarta city. biodiversitas, 3 (2): 236-241. astuti, t. d., kadir, s., & boekoesoe, l. 2014. analysis of lead metal pollution (pb) on white shrimp (litopenaeus vannamei) in bone river (doctoral dissertation, ung). environment agency of sragen regency. 2012. water laboratory test results of bengawan solo river in sragen. chaney, r. l., angle, j. s., baker, a. j., & li, y. m. 1998. u.s. patent no. 5,711,784. washington, dc: u.s. patent and trademark office. international journal of applied biology, 2(1), 2018 20 duruibe, j. o., ogwuegbu, m. o. c., & egwurugwu, j. n. 2007. heavy metal pollution and human biotoxic effects. international journal of physical sciences, 2(5), 112118.endrawanto dan h. winarno. 1996. process physical and chemical waste processing. proceedings of training and workshop on the role of bioremidiation in environmental management . lipi-bppt-hsf. grant, c.a., w.t. buckley, l.d. bailey, and f. selles, 1998. cadmium accumulation in crops. ca. j. plant sci. 78: 1-17. haryanti, s., nintya setiari, rini budi hastuti, endah dwi hastuti, dan yulita nurchayati. 2009. physiological response and anatomy of water hyacinth (eichornia crassipes) in various irrigated waters. jurnal penelitian sains dan teknologi. vol 10. no.1: 3040 hidayati, n. 2005. phytoremediation and potential hiperakumulator. hayati, 12 (1): 35-40. ministry of public works. 2010. decree of the minister of public workers on the water resources management pattern of the bengawan solo river region. kurnia, u., suganda, h., saraswati, r. & nurjaya. 2009. rice field pollution control technologies. 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(online), (http://suaramerdeka.com), accessed 29 october 2013 [in indonesia]. sudarmaji, mukono, j. & corie i.p. 2006. heavy metal toxicology and its impact on health, 2 (2): 129-142. supriyadi d.s. 2002. conditions of muara water based on physical and chemical parameters in estuary of solo ujung pangkah village of gresik, east java. unpublished thesis. bogor: ipb [in indonesia] utomo, a.d., ridho m.r. edward s. & putranto, d.a. 2010. pollution on the solo river solo between solo and sragen, central java. bawal, 3 (1):25-32 [in indonesia] winarno k., astirin, o.p. & setyawan, d.s. 2000. monitoring of jabung swamp water quality based on bentos community's diversity and wealth. biosmart, 2 (1): 40-46 [in indonesia] yuliastuti, e. 2011. study of water quality of karanganyar river in efforts to control water pollution. thesis not published. semarang: undip [in indonesia]. 3(2)7-14 contact : ardiansyah kurniawan ardian_turen@yahoo.co.id © international journal of applied biology 7 abstract cellulose as an abundant source of glucose in indonesia requires acceleration of decomposition utilizing cellulolytic bacteria. cellulolytic bacteria can be obtained from the isolation of mangrove organic matter, such as sediments. muntok sub-district is one of the regions with the most tin mining in west bangka regency also has mangroves in the coastal area. exploration of cellulolytic bacteria in mangroves with different environmental characteristics encourages researchers to find new bacterial strains that produce cellulase enzymes with new properties. thirteen isolates were successfully isolated from three locations. tembelok mangrove sediments produced seven bacterial isolates, peltim mangrove samples produced three isolates and from sukal mangrove three isolates were obtained. seven isolates showed clear zones in the lugol test and three isolates including were gram-positive bacteria. molecular test with 16s rrna analysis showed tbl1 isolate has 85% similar identity of vibrio parahaemolyticus strain hy3 and tbl2 isolate has 98% similar identity of bacillus amyloliquefaciens strain hs8. bacillus amyloliquefaciens potential to further study as cellulose degrading bacteria for feed ingredients. issn : 2580-2410 eissn : 2580-2119 molecular identification of cellulolytic bacteria from mangrove sediment at tin minning region in west bangka ardiansyah kurniawan1, suci puspita sari1, euis asriani1, andi kurniawan2, abu bakar sambah2 & asep awaludin prihanto2 1faculty of agriculture, fisheries and biology, bangka belitung university, bangka, indonesia 2faculty of fisheries and marine science, brawijaya university, malang, indonesia introduction cellulose is the most common organic polymer especially in the tropics. at the molecular level, cellulose is a linear polymer of glucose composed. it can be a problem and challenges that cellulose needs biodegradation before using an energy source of glucose. animal feed, in addition to ruminants, can be produced with sufficient energy and economical prices through the utilization of cellulose which has been neglected so far. cellulose as an abundant source of glucose in indonesia requires acceleration of decomposition utilizing cellulolytic bacteria. cellulolytic bacteria have the ability to accelerate decomposition of organic material (hapsoh, 2017). andriani et al (2012) added that cellulolytic bacteria which secrete cellulase enzymes are useful as rough degradators of animal feed raw materials. cellulolytic bacteria can be obtained from the isolation of mangrove organic matter, such as sediments open access international journal of applied biology keyword 16s rrna, cellulose, cellulolytic bacteria, mangrove, tin minning article history received 12 april 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 8 (kurniawan et al., 2018a; yahya et al., 2014; chantarasiri, 2015), leaf litter (kurniawan et al., 2018b; ningsih et al., 2014 ; yulma et al., 2017) and weathered wood (kurniawan et al., 2018c; kurniawan et al., 2018d). muntok sub-district is one of the regions with the most tin mining in west bangka regency (susanto, 2015) also has mangroves in the coastal area. the tin smelting industry owned by pt. timah is also concentrated in this area. tin mining provides the economy and development for the people of bangka island but also has a negative impact on the environment (kurniawan, 2012). the existence of cellulolytic bacteria in mangrove can be influenced by human activity in the vicinity, as sari and rosalina’s statement (2014) that unconventional tin mining affects the success of mangrove planting. mixing between mine waste soil can cause a decrease in physical and chemical fertility of native mangrove soil (umroh, 2015). it is necessary to identify cellulolytic bacteria from mangroves on muntok sub-district to complete data on mangrove cellulolytic bacteria resources on bangka island. exploration of cellulolytic bacteria in mangroves with different environmental characteristics encourages researchers to find new bacterial strains that produce cellulase enzymes with new properties. kurniawan and ekowati (2016) show that microorganisms have the ability to adapt to environmental changes due to heavy metal contamination and can reduce the contamination from the environment. the purpose of this study is to isolate endophytic bacteria that produce cellulase enzymes that can be used for animal feed purposes, especially in aquaculture commodities. materials and methods materials and tools the materials used in this study were carboxy methyl cellulose (cmc), mgso4.7h2o, kno3, k2hpo4, cacl2.2h2o, yeast extract, agar, lugol solution (2g of potassium iodine and 1g iodine in 300 ml of aquadest), and congored 0,1% solution (0,1 g congored in 100 ml aquadest). primers for this analysis were 20f (5’ – gtaatcgtcg gccagtagagtttgatcctggctc-3’) and primer reverse 1510r (5’ – caggaaaca gctatgaccg gctaccttgttacgact-3’). tools used in this research is the petri dish, incubator (memmert, germany), refrigerator, pcr machine (biorad, usa), dna extraction kit (promega, usa), bioedit software, and abiprism®310 genetic analyzer (thermo fisher, usa). research sites samples of mangrove soil were taken from sukal mangrove, peltim mangrove and tembelok mangrove in muntok sub-district, west bangka regency, province of bangka belitung archipelago, indonesia. peltim mangroves are the closest mangrove to the tin smelting industry, sukal mangroves represent coastal areas that are used for shellfish cultivation, and tembelok becomes mangroves that have high density and far distance from human activities. location of sampling of mangrove soil is shown in figure 1. bacterial isolation and screening were done in the fisheries microbiology laboratory of brawijaya university, while dna isolation was carried out in the genetics laboratory of maulana malik ibrahim state islamic university, malang. international journal of applied biology, 3(1), 2019 9 figure 1. soil sampling location in west bangka, province of bangka belitung archipelago isolation of cellulolytic bacteria soil sampling is done with a depth of 10-20 cm. all sample stored in the sterile container. each sampling location was taken 2 samples and composite into a combined sample. mangrove soil samples were weighed as much as 1g and diluted to 10-7 dilutions. the resulting of dilution were grown on 1% cmc (carboxy methyl cellulose) enriched medium with a pour plate method and incubated in the incubator for 48 to 72 hours at 30° 37°c temperature. the cultivation results were isolated with the same medium to obtain the pure isolate using to scratch plate method. screening of cellulolytic bacteria selection of cellulolytic bacteria was done by the cellulose hydrolysis test method. pure bacterial isolates were recycled on carboxy methyl cellulose enriched agar medium (cmc) by a streak method. one loop of bacteria is scratched on the medium by forming a line of approximately 1cm. the culture was incubated at 72 hours at 30 ° c. qualitative test uses lugol methods. lugol and congored solution is dripped to cover the entire medium and is allowed to stand for a minute. the clear zone formed around bacterial colonies was observed and identified as isolating of cellulose-degrading bacteria. isolates identified to have cellulolytic activity were selected for further identification. isolates showing the largest clear zone and positive grams were identified by sequencing dna analysis. molecular identification the protocol for the identification of best producer of cellulose, the methods by prihanto et al., (2016) was used with slight modification. dna isolation was performed using the procedure of the dna isolation kit (wizard of genomic dna purification kit from promega). the initial stage of 16s-rrna analysis was done by isolating genomic dna from selected bacteria then followed by amplification using pcr technique. dna sample which was isolated as much as 2 μl was dissolved in the mixture containing 6 μl ddh2o, 10 μl pcr kit gotaq® green master mix (10 x taq polymerase buffer, dntp, mgcl2, primer, taq dna polymerase, ddh2o), 1 μl forward primer, and primary reverse 1 μl. mixture it is then inserted into the pcr machine. the amplification process was conditioned at a pre-denaturation temperature of 95 °c for 5 minutes 1 cycle and denaturation with 30 cycles at 95°c for 30 seconds, (33°c, 31.5°c and 30°c) for 30 seconds, and 72°c for 30 seconds, continued with 1 extension cycle at 72°c international journal of applied biology, 3(1), 2019 10 for 10 minutes and 4°c for 5 minutes. the pcr results of all amplified dna samples were confirmed using horizontal electrophoresis of 0,8% agarose gel. dna bands that form later compared to markers. the next step is purification pcr results using dna fagment extraction kit. pcr results that have been purified added with solution special buffer, hi-ditm formamide (genetic analysis grade-applied biosystem) and sequenced using abiprism® 310 genetic analyzer. dna sequencing uses the services of pt. genetika science indonesia. sequencing results used to find out similarities dna sequences with bacterial dna sequences others in genbank use help with the basic local alignment search program tool (blast) on the site http: //www.ncbi.nlm.nih.gov. bacterial kinship is presented in the shape of the phylogenetic tree image created with the help of fr phylogeny programs. this program is available on the site http: // www.phylogeny.fr. results and discussion the results of the analysis show that cellulolytic bacteria can be isolated from the third sediment sample of mangrove sites. thirteen isolates were successfully isolated from three locations. tembelok mangrove sediments produced seven bacterial isolates (tbl1, tbl2, tbl3, tbl3.7, tbl2.6, tbl1.6, tbl1.5), peltim mangrove samples produced three isolates (ptl1.6, ptl7, ptl5) and from sukal mangrove three isolates were obtained (skl1.6, skl1.5, skl1.7). the ability to grow bacteria on media with 1% cmc means that thirteen isolates have the ability to utilize cellulase, as huang (2012) stated that cellulolytic bacteria are able to live by degrading cellulase. hydrolysis using lugol can ensure that bacterial isolates have the ability to cellulose degradation. the results of qualitative analysis using lugol are presented in figure 2 and table 1. there were seven isolates that showed clear zones in the lugol test with three isolates including gram-positive bacteria. figure 2. qualitative test results of cellulolytic bacteria using lugol method. table 1. results of qualitative tests for cellulolytic bacteria with lugol and congored. isolate code diameter of the clear zone (mm) gram staining lugol congored tbl1 15 1 positive tbl2 6 5 positive tbl3 9 0 negative tbl3.7 0 0 tbl2.6 0 0 ptl1.6 0 0 skl1.6 0 0 tbl1.6 7 0 negative international journal of applied biology, 3(1), 2019 11 skl1.5 13 5 positive skl1.7 0 0 tbl1.5 8 1 negative ptl7 5 1 negative ptl5 0 0 cellulolytic bacterial identified in tembelok mangroves were more number of isolates than other mangrove samples, possibly related to the density and utilization of the mangrove environment. the density of tembelok mangrove trees is still high with various types of mangroves associated, while the mangrove sukal and peltim are avicennia marina homogeneous species with low density. among the different environments, the sediments of mangrove forests are suitable for exploring cellulose-degrading microorganisms because of continuous input of cellulosic carbon in the form of litter which then acts as a substrate for decomposition by microbe (behera et al., 2017). the higher density of mangroves has a positive impact on the addition of the number of mangrove leaf litter and reduce the number of vibrio sp bacteria colonies (arta et al., 2009). tembelok mangroves include mangroves with minimal human activity around them, while the coastal areas around the mangrove sukal are used for cultivation of blood clams and peltim mangroves adjacent to the tin smelting industry. rigonato (2017) shows that pristine mangroves have a pronounced effect on microbial community composition and contaminated mangroves that have different microbial communities at different times. chemical pollution, particularly accumulation and bio-transformation of toxic metals could be a significant factor for reduction of mangrove biodiversity (maiti and chowdhury, 2013). the results of molecular test with 16s rrna analysis are the tbl1 isolate has 85% similar identity of vibrio parahaemolyticus strain hy3 and isolate tbl2 has 98% similar identity of bacillus amyloliquefaciens strain hs8. hence, we named this isolate as vibrio parahaemolyticus_ubb_tbl1 and bacillus amyloliquefaciens_ubb_tbl2. phylogenetic tree of this isolate was depicted in figure 3. figure 3. phylogenetic tree of bacterial isolates the results of the analysis of genetic joining phylogenetic trees (neighbor joining) can be seen in figure 3. the numbers contained in each tree branch show the bootstrap value. based on phylogenetic trees which included several genbank sequence data as a comparison, it showed that vibrio parahaemolyticus_ubb_tbl1 bacteria were in the same branch and international journal of applied biology, 3(1), 2019 12 node (genus) as vibrio parahaemolyticus strain hy3 with boostrap value of 0 and bacillus amyloliquefaciens_ubb_tbl2 with bacillus amyloliquefaciens strain hs8 with boostrap value of 0.96. vibrio parahaemolyticus is a gram-negative halophilic bacterium that is found in estuarine, marine and coastal environments and pathogenic bacterium for humans (letchumanan et al, 2014). this bacterium is also found in shrimp from the north coast of java (shobharani et al, 2013). gao et al. (2011) reported a cellulolytic bacterium from vibrio genus isolated from mangrove soil in xiamen, fujian province of china. bacillus amyloliquefaciens is also identified at the mangrove forests of andaman and nicobar islands (geetha et al., 2011) and kraton mangrove, pasuruan, east java (yahya et al., 2014). bacillus amyloliquefaciens obtained from cassava yeast tape has the ability to degrade flour substrates cassava, rice bran and carboxymethylcellulose (soeka et al., 2015). the potential of bacillus amyloliquefaciens as cellulose degrading bacteria for feed ingredients can be studied further. conclusions thirteen isolates were successfully isolated from three locations. tembelok mangrove sediments produced seven bacterial isolates, peltim mangrove samples produced three isolates and from sukal mangrove three isolates were obtained. seven isolates showed clear zones in the lugol test and three isolates including were gram-positive bacteria. molecular test with 16s rrna analysis showed tbl1 isolate has 85% similar identity of vibrio parahaemolyticus strain hy3 and tbl2 isolate has 98% similar identity of bacillus amyloliquefaciens strain hs8. bacillus amyloliquefaciens potential to further study as cellulose degrading bacteria for feed ingredients. acknowledgment the authors were grateful to the directorate general of research and technology research and development at the ministry of research, technology and higher education for financially supported at university cooperation research between bangka belitung university and brawijaya university in 2017-2018. references andriani, y., sastrawibawa, s., safitri, r. & abun. 2012. isolation and identification of cellulolytic microbes as biodegradators rough fiber in feed materials from agricultural waste. ijas. 2 (3). arta, a.p, maidie, a. & saptiani, g. 2009. the effect of mangrove vegetation treatment on vibrio sp bacterial population in bontang coast. jurnal kehutanan tropika humida. 2 (2): 133-142. behera, b.c., sethi, b.k., mishra, r.r., dutta, s.k. & thatoi, h.n. 2017. microbial cellulases – diversity & biotechnology with reference to mangrove environment: a review. journal of genetic engineering and biotechnology. 15 : 197–210. chantarasiri, a. 2015. aquatic bacillus cereus jd0404 isolated from the muddy sediments of mangrove swamps in thailand and characterization of its cellulolytic activity. egypt j aquat res. 41(3):257–64. international journal of applied biology, 3(1), 2019 13 gao, z.m., xiao, j., wang, x.n., ruan, l.w., chen, x.l. & zhang, y.z. 2011. int. j. syst. evol. microbiol. 62: 1958–1962. geetha, i., manonmani, a.m. & prabakaran, g. 2011. bacillus amyloliquefaciens: a mosquitocidal bacterium from mangrove forests of andaman & nicobar islands, india. acta tropica. 120. (3): 155-159. hapsoh, wawan, dini, i.r. & siregar, j.a.. 2017. compatibility tests of potential cellulolytic bacteria and growth optimization in several organic materials. international journal of science and applied technology. 2 (2). kurniawan, a., sari, s.p., asriani, e., kurniawan, a., sambah, a.b. & prihanto, a.w. 2018b. mangrove leaf litter cellulolytic bacteria on bangka island. samakia. 9 (1). kurniawan, a., sari, s.p., asriani, e., kurniawan, a., sambah, a.b. & prihanto, a.w. 2018c. isolation and identification of cellulose degradation bacteria from tukak sadai mangrove ecosystem, south bangka. jurnal perikanan pantura. vol. 1. no.2 kurniawan, a., sari, s.p., asriani, e., kurniawan, a., sambah, a.b. & prihanto, a.w. 2018d. cellulolytic bacteria in weathered wood in sungailiat mangrove, bangka dan tukak sadai, south bangka. proceedings of the national wetland environment seminar. 3 (1): 301-305. kurniawan, a., sari, s.p., asriani, e., kurniawan, a., sambah, a.b. & prihanto, a.w. 2018a. isolation and identification of cellulolytic bacteria from mangrove sediment in bangka island. iop conf. series: earth and environmental science 137. kurniawan, a. 2012. introduction to post-tin mining aquaculture. ubb press. kurniawan. a, and n. ekowati, 2016. mycoremediation of heavy metal: a review. bioteknologi & biosains indonesia. 3 (1) : 36-45. letchumanan. v, chan, k.g. & lee, l.h. 2014. vibrio parahaemolyticus: a review on the pathogenesis, prevalence and advance molecular identification techniques. frontiers in microbiology. 5 (705) maiti, s.k & chowdhury, a. 2013. effects of anthropogenic pollution on mangrove biodiversity : a review. journal of environmental protection. 4 : 1428-1434. ningsih, r.l., khotimah, s. & lovadi, i. 2014. cellulose degradation bacteria from avicennia alba blume leaf litter in the safety mangrove forest in pontianak regency. protobiont. 3 (1): 34 – 40 prihanto, a.a, & wakayama, m. 2016. adv. food nutr. res. 79 1–25. rigonato, j. 2017. temporal assessment of microbial communities in soils of two contrasting mangroves. braz j microbiol. http://dx.doi.org/10.1016/j.bjm.2017.04.008 sari, s. p. & rosalina, d. 2014. success rate of mangrove planting in post-mining land in south bangka regency. maspari journal. 6 (2). shobharani, p., yogesh, d., halami. p.m. & sachindra. n.m. 2013. potential of cellulase from bacillus megaterium for hydrolysis of sargassum. journal of aquatic food product technology. 22 (5). 520-535. soeka,y.s., rahmansyah, m. & sulistiani. 2015. optimization of α-amylase enzyme from bacillus amyloliquefaciens o1 which are induced by rice bran and international journal of applied biology, 3(1), 2019 14 carboxymethylcellulose. jurnal biologi. 11 (2) : 259-266. susanto. 2015. tin-bottom areas in bangka belitung with spot_6 satellite data. national seminar on science and technology 2015. faculty of engineering, muhammadiyah university, jakarta. umroh. 2015. seeding and planting rhizophora apiculata in post-conventional tin mining (ti) areas in muara kudai, bangka regency.jurnal kelautan. 8 (1). yahya, happy, n., yenny, r. & soemarno. 2014. characteristic of extracelluler metabolic of coastal ecosystem bacteria producing histidine decarboxylase crude. international food research journal. 21 (4) : 1337-1347 yulma, ihsan, b., sunarti, malasari, e., wahyuni, n. & mursyban. 2017. identification of bacteria in decomposed mangrove leaf litter in mangrove and proboscis conservation areas in tarakan city. journal of tropical biodiversity and biotechnology. 2. 28-33. 2(2)44-51 contact : achmad faqih sha’ab a.faqih@mail.ugm.ac.id ó international journal of applied biology 44 abstract the diversity of the butterfly in sebangau national park, particularly in the mangkok resort area has different morphological features in each species. the differences distinguish each species between families. steps to know the diversity species or populations can be measured and identified based on similarities or differences in shannon-wiener's diversity analysis. this research was conducted by random purposive sampling using insect net and food trap. observation and sampling were conducted in the area of natural forest and forest restoration during the dry and rainy seasons. the results showed, the species obtained and identified as many as 25 species from 4 families namely papilionidae, nymphalidae, pieridae and lycaenidae. butterflies obtained from food trap are 24 individuals from 5 species. the level of diversity, evenness and richness of the species of butterflies in each region during the rainy and dry seasons which are the location of the study are included in the "medium" category with the average value of the diversity index, the evenness and the richness of the butterfly species of 2.502. the relative dominance and relative abundance of the butterfly species in the mangkok resort shows no species communities that are extreme dominant from species other. issn : 2580-2410 eissn : 2580-2119 diversity of ordo lepidoptera in mangkok resort, sebangau national park, central kalimantan achmad faqih sha’ab1 and rc hidayat soesilohadi2 1laboratory of entomology, faculty of biology, universitas gadjah mada, indonesia 2laboratory of ecology and biology conservation, faculty of biology, universitas gadjah mada, indonesia introduction kalimantan is home to some of the most complex and diverse ecosystems of forests (myers et al., 2000; sodhi et al., 2004). the rate of loss of peatland and degradation in kalimantan is also very high, as a result of selective logging, fires, and conversion to plantations (harrison et al., 2009). change of one of environmental factor, such as vegetation, will have an impact on the diversity of the insects, especially the butterfly. according to patton, (1963), species of host plants and butterfly larvae feed is different between species of butterflies, because the butterfly needs nutritional content that suitable for the development of the larvae. open access international journal of applied biology keyword diversity butterfly mangkok resort article history received 27 august 2018 accepted 25 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 45 cleary et al, (2006) stated that, about 1,000 species of butterflies had been documented in kalimantan, with 98 of them in sebangau national park. houlihan et al, (2013) say, to date, as many as 48 species have been recorded in the sebangau forest. the list includes 27 species that are interested in fermenting fruit and can be monitored using a fruit trap. houlihan et al., (2013) say, to date, as many as 48 species had been documented in the forests of national park sebangau. the diversity of the butterflies in national park sebangau, especially in the mangkok resorts certainly has different morphological features in each species. differences in the morphology characteristics of each species of course differentiate between the familiy. steps to know the kinship relationships between species or populations can be measured based on the similarity of characters and kinship analysis can be done in various ways, including through the phenetic approach done through grouping based on the similarity of character (terry, 2000). the research conducted at mangkok resort, sebangau national park, is the first step as the first research to obtain data about the diversity of the butterflies, because the information and data of the butterflies species located in the mangkok resort was not yet available and considering resort mangkok is also a field research area (field station), so that research needs to be done both in terms of diversity and kinship relationships of butterflies between species. materials and methods experimental location this study was conducted in june 2017 until march 2018. the study was conducted in the mangkok resort area of sebangau national park in central kalimantan and is at the coordinating point 1140 01’ 11.25” sl – 20 24’ 11.25” el. figure 1. mangkok resort map international journal of applied biology, 2(2), 2018 46 data collection the survey sampling of butterflies was by random sampling purposive sampling. data collected in the study were species data and number of individual butterfly obtained in the field during the study. habitat characteristics data include a biotic component derived from the relative temperature and humidity level data. while the biotic components in this study only inventory vegetation that is suspected of feed, shelter and cover of butterflies. data analysis shanon-winner diversity index (maguran, 1988): h" = −∑ (() ln()) () = -. / evenness index (maguran et al., 2011): 0 = 1" 23 type richness index (margalef, 1958): r = 356 2(/) relative abundance index (helvoort, 1981): (kr) = ∑89 ∑: x 100% domination index (odum, 1971): d = s (ni/n)2 results and discussion the results of the research, sampling locations were conducted in two regions, namely natural forests and restoration forests located at kilometers 8, 10, 12, 14 and 16. the sampling area in each study location was 40x50 m2. total species of butterflies obtained in the sebangau national park mangkok resort area of 25 species from four families, namely papilionidae (8 species), nymphalidae (11 species), pieridae (2 species), and lycaenidae (4 species). based on the observation and sample obtained, the number of observed individuals was 2,493 butterflies. sampling of butterflies using food traps during the dry season in the forest restoration and natural forests is obtained by 16 individuals from 3 species, while in the rainy season the trap that got butterflies only in natural forest is 8 individuals from 2 species. the result of calculation of index of diversity, richness, and evenness of the butterflies in the forest restoration area at each season obtained value of h '= 1,952 which is included in the "low" category, while in dry season has value h' = 2,412 included into the "medium" category. the evenness index of the butterflies species during the rainy and dry seasons has a value of e = 0.848 and 0.940 which is included in the "almost evenly" category. the richness index of the butterfly in the rainy season and in the dry season has dmg (r) = 1,488 and 2,050 which are in the "low" category. the average value obtained from forest restoration area during rainy season and dry season is 1,975 which belong to the "low" category (table 1). the result of calculation of diversity index, richness, and evenness of the butterflies in the natural forest area in each season obtained the value of h '= 2,331 and 2,641 belong to the same category of "medium". the evenness index of the rainy season has a value of e = 0.841 which is included in the "almost evenly" category, while in the dry season the evenness of butterflies species has the value e = 0.975 included in the “almost evenly" category. the richness index of the butterfly in the dry season and the rainy season has a value of dmg (r) = 2,145 and 2,171 belonging to the "low" category. the average value obtained from the international journal of applied biology, 2(2), 2018 47 natural forest area during the rainy season and the dry season is 2,776, which belongs to the "medium" category (table 1). table 1. index of diversity, richness, and evenness of the butterflies habitat / season number of family number of species number of individuals h' dmg e nf/d 4 15 631 2,6 41 2,171 0,975 fr/d 4 13 349 2,4 12 2,050 0,940 nf/r 4 16 1089 2,3 31 2,145 0,841 fr/r 4 10 424 1,9 52 1,488 0,848 information: nf: natural forest; fr: forest restoration; d: dry; r: rainy; h ': index of diversity; dmg (r): the wealth index; e: the evenness index. figure 2. histogram of diversity, richness, and evenness butterflies of the natural forest region and the restoration of dry seasion and rainy seasion. the average value obtained from natural forest areas and forest restoration during the dry and rainy seasons is 2.502. this value shows that the level of diversity, evenness, and species richness fall into the "medium" category. table 2. index of dominance of the butterfly habitat / season dominance index nf/d 0,00498 nf/r 0,00918 rf/d 0,00760 rf/r 0,01754 information: nf: natural forest; fr: forest restoration; d: dry; r: rainy; h ': index of diversity. h’ dmg e h’ dmg e h’ h’ dmg dmg e e in de ks d iv er si ty region-season in de ks d iv er si ty region-season fr/d fr/r nf/d nf/r international journal of applied biology, 2(2), 2018 48 the index values of dominance of the butterflies in the area of the mangkok resort based on simpson criteria are low or good (d <1) ranged from 0.00498 0.01754, and show that the individual abundance of each species in each region of the season seen no dominance phenomenon stand out (table 2). in line with the dominance index, the relative abundance indexes of the most abundant butterflies’ species in this research were found in all regions and in each season by achieving an average percentage of 77.24% of the graphium evemon species. the abundance of butterflies species in the second and third positions is occupied by graphium sarpedon species, and graphium agamemnon which reaches an average percentage of 52.86% and 41.40%. all three species are common species scattered in all areas of the mangkok resort. the greater number of other species and often found in the forest, indicates a preference that the area has been disturbed by proven loss of forest cover on the riverbank adjacent to the restoration forest area. successful vegetation in inventarization at the research sites serving as feed, shelter, and butterflies cover. vegetation from the myrtaceae family is noted more frequently visited by the butterflies of the papilionidae family such as papilio iswara, graphium agamemnon, and graphium evemon. the butterfly of the nymphalidae family such as pandita sinope, mycaelesis horsfieldi, and acraea terpsicore are observed butterflies more frequently visiting plants from the icacinace and myrtacinacae families. table 3. vegetation of feed, shelter, and butterfly cover local name scientific name family function ha hr bati-bati syzygium zeylainca myrtaceae fsc ö ö manggis hutan/idat garcinia sp. clusiaceae fsc ö x butun barringtonia sp. lecythidaceae sc ö x jejambu eugenia cuprea myrtaceae f ö ö jamai rhodamnia cinerea myrtaceae psc ö ö luwari schima wallichii theacea psc ö x pansulan pternandra coerulescens melastomataceae sc ö x pepagar aporosa sp. euphorbiaceae sc ö x puak artocarpus sp. moraceae fsc ö x semonu elaeocorpus microphylum elaeocarpaceae sc ö x ubar syzygium sp myrtaceae f ö ö ubar merah syzygium sp myrtaceae f ö x ubar putih syzygium sp myrtaceae f ö ö ubar samak syzygium sp myrtaceae f ö x nenasi syzygium zeylanicum myrtaceae sc ö ö temboras memecylon sp melastomataceae fsc ö x kremunting kodok melastoma malabathricum melastomataceae f ö x kremunting padang ochtocharis bornensis melastomataceae s ö x koman psychotria viridiflora rubiaceae s ö x asam keranji dialum indum l. fabaceae fsc ö x medang kabui actinodaphne lauraceae fsc ö x international journal of applied biology, 2(2), 2018 49 pasir-pasir stenomurus sp icacinaceae sc ö ö asam-asam dicryoneura acumonata sapindaceae fsc ö ö information: nf: natural forest; fr: forest restoration; ö: exist; x: no; f: feed; s: shelter; c: cover discussion the number of butterfly species found in the mangkok resort of sebangau national park in the area of restoration forest and natural forest amounted to 25 species belonging to 4 families namely papilionidae (8 species), nymphalidae (11 species), pieridae (2 species), and lycaenidae ( 4 species). table 4. presence and number of butterflies in each region for each season no species family ha hr rainy dry number of individuals 1 graphium agamemnom papilionidae 146 105 ö ö 251 2 graphium sarpedon papilionidae 241 108 ö ö 349 3 graphium evemon papilionidae 396 149 ö ö 545 4 graphium antipathies papilionidae 23 10 ö x 33 5 graphium delesserti papilionidae 32 12 ö x 44 6 papilio helenus papilionidae 27 52 x ö 79 7 papilio demolion papilionidae 15 50 x ö 65 8 chilasa paradoxa papilionidae 40 0 x ö 40 9 euploea mulciber nymphalidae 88 0 ö ö 88 10 euploea radamanthus nymphalidae 35 0 ö x 35 11 euploea eyndhovii nymphalidae 37 0 ö x 37 12 euploea crameri nymphalidae 15 9 x ö 24 13 hypolimnas bolina nymphalidae 16 0 ö x 16 14 junonia atlites nymphalidae 27 40 ö x 67 15 agatasa calydonia nymphalidae 62 6 x ö 68 16 pandita sinope nymphalidae 92 39 x ö 131 17 melanitis leda nymphalidae 47 16 x ö 63 18 acraea violae nymphalidae 38 32 x ö 70 19 mycalesis horsfieldi nymphalidae 36 8 x ö 44 20 eurema sarilita pieridae 81 57 ö ö 138 21 eurema andersoni pieridae 38 29 ö x 67 22 arhopala psoudocentaurus lycaenidae 55 28 x ö 83 23 celastrina oreas lycaenidae 42 8 ö x 50 24 rapala manea lycaenidae 44 15 ö x 59 25 caleta roxus lycaenidae 47 0 x x 47 total 4 1720 773 2493 information: nf: natural forest; fr: forest restoration; ö: exist; x: no garcia et al., (2014) suggests that fluctuations in seasonal variation in the availability of fruits during the rainy season and phenological patterns of host plants or in the abundance of international journal of applied biology, 2(2), 2018 50 predators or parasites can lead to changes in species relative abundance and consequently in species. research margaret et al., (2014) shows that, the differences in butterfly communities between restoration sites and primary forest is different in each month. clara et al., (2014) say, in essence, the restored area seems to be going through the process of adding species as the age of restoration increases, the more forest species colonize the older areas. drumbel et al., (2005) say, the composition of the butterfly community in each season greatly affects the presence in each habitat, and the habitat that is degraded or that is in the regeneration will fluctuate according to the degree of disturbance that occurs. therefore, restored forests can be important shelters for forest species and increase the permeability of the landscape by allowing fauna mobility between forest patches. as a result, restoration forests could potentially increase regional biodiversity and restore some ecosystems (clara et al., 2014). each family and species of butterfly has its own preference for the plants and sources of feed. this causes the species of butterfly in natural forests to be more diverse because of the varying tree height resulting in canopy cover is also highly variable. drumbel et al., (2005) reported that selective logging affects vegetation structures and by opening shade areas will produce more homogeneous vegetation structure. houlihan et al., (2013) states, butterflies have different preferences to direct sunlight. the relation to this case vu et al., (2011) suggests that the structural complexity of habitat and the diversity of vegetation forms correlates with the diversity of insect species, which in this case are butterflies. conclusions the level of diversity, evenness and richness of butterfly species in the forest of the restoration during the rainy and dry seasons fall into the "low" category with an average value of the diversity index, the fairness and richness of the butter species of 1.975, while the diversity, fairness and richness of the butterfly species natural forests during the rainy and dry seasons fall into the "medium" category with an average value of diversity index, evenness and richness of the butter species of 2,776. acknowledgments the authors would like to thank sebangau national park central kalimantan for his permission to access the peat swamp forest research station mangkok resort, sebangau, central kalimantan, indonesia. thanks to academic advisers and friends while in the field. references borror, d. j., c. a, triplehorn., & n. f, johnson. 1992. pengenalan pelajaran serangga. edisi keenam. yogyakarta: gadjah mada university press. terjemahan dari: introduction to the study of insect. sixth edition. clara, l. b., s, anna., d. b, ribeiro., l. c, garcia., & a. v. l, freitas. 2014. fruit-feeding butterfly communities are influenced by restoration age in tropical forests. society for ecological restoration 1: 1-6. drumbell, a.j., & j. a, hill. 2005. impacts of selective logging on canopy and ground assemblages of tropical forest butterflies: implications for sampling. biological conservation 125: 123-131. international journal of applied biology, 2(2), 2018 51 garcia, l. c., r. j. hobbs, f. a. m. santos., and r. r. rodrigues. 2014. flower and fruit availability along a forest restoration gradient. biotropica 46: 114–123. harrison, m. e., s. e, page., & s. h, limin. 2009. the global impact of indonesian forest fires. biologist 56: 156 – 163. helvoort, b.v. 1981. bird populations in the rural ecosistems of west java. nature conservation depertment. netrherlands. houlihan, p. r., m. e, harrison., & s. m, cheyne. 2013. impacts of forest gaps on butterfly diversity in a bornean peat-swamp forest. journal of asia-pacific entomolgy 16: 67-73. margaret, n., v, anu., n, philip., & r, heikki. 2014. fruit-feeding communities as indicators of forest restoration in an afro-tropical rainforest. biological conservation 174: 75-83. magurran, a. e. 1988. ecological diversity and its measurement. chapman and hall: usa. myers, n., r. a, mittermeier., c. g, mittermeier., g. a. b, fonseca., j, kent. 2000. biodiversity hotspots for conservation priorities. nature 403: 853–858. odum, e. p. 1971. fundamentals of ecology. toronto: w. b. saunders company. page, s. e., s, siegert., j, rieley., h, boehm., a, jaya., & s. h, limin. 2002. the amount of carbon released from peat and forest fires in indonesia during 1997. nature 420: 61-65. patton, r.l. (1963). introductory insect physiology. w.b. saunders company, philadelphia and london. toppan company limited. tokyo, japan. sodhi, n. s., l.p, koh., b.w, brook., p. k. l, neg. 2004. southeast asian biodiversity: an impending disaster. trends ecology evol 19: 654–660. terry, t.m. 2000. microbial taxonomy and evolution. mcb 229. spring 2000. university of connecticut. department of molecular & cell biology. vu. l. v., & c. q, vu. 2011. diversity pattern of butterfly communities (lepidoptera, papilionidae) in different habitat types in a tropical rain forest of southern vietnam. international scholarly research network isrn zoology 1: 1-8. contact : lukman daris daris.lukman70@gmail.com 35 abstract this study aimed to determine the potential diversity of the coral reef and coral reef fish ecosystems of kodingareng keke island and the development strategies and priorities of kodingareng keke island as a marine tourism destination. this research used purposive sampling with 50 respondents. the coral data collection method was line intercept transect (lit), with a transect line length of 50 meters and depth of 5 meters. the process used to obtain coral reef fish data was the underwater visual census (uvc). the analysis showed that the average coral ranged from 63,6% to 77,8%. the average abundance of fish at 3 observation stations, namely station i (17,65%), station ii (14,78%) and station iii (18,67%), thus it can be concluded that this location is feasible for snorkelling and diving tourism. the priority programs for the development of the marine tourism area of kodingareng keke island include increasing supervision, creating periodical object development master plans (ripo), implementing local regulations on the use of coastal resources, involving local communities, the general public and the government in nature conservation activities, setting labour standards and improving the quality of local human resources through training. issn : 2580-2410 eissn : 2580-2119 the status of diversity of coral reefs and reef fish supporting marine tourism on kodingareng keke island, makassar city. lukman daris1*, andi nur apung massiseng 1, jaya2, wahyuti1, st. zaenab2 1study program of fisheries agrobusiness, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar, indonesia. 2study program of aquaculture, faculty of fisheries, cokroaminoto makassar university, 90245, jalan perintis kemerdekaan km. 11 tamalanrea, kota makassar, indonesia . introduction tourism is one of the essential mainstay sectors that can provide economic improvements for coastal communities, the nation and the state. currently, beach tourism is one of the areas that can develop very quickly in many coastal countries (bergmann et al., 2017). marine and coastal tourism is an inseparable component of the growth of the global tourism industry (leposa, 2020). the provision of community welfare can be obtained from the provision of environmental goods and services originating from marine and coastal ecosystems. community social development, especially in coastal areas, can be encouraged by marine open access international journal of applied biology keyword coral reef; coral reef fish; marine tourism; article history received mei 8, 2023 accepted june 13, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:daris.lukman70@gmail.com international journal of applied biology, 7(1), 2023 36 and coastal ecosystems (hatam et al., 2015; fachry & alpiani, 2021). the international coastal maritime tourism society defines coastal and marine tourism as an entertainment activity travelling from home to tourist attractions. tourism activities focus on marine areas and small islands (orams & lück, 2014). the concept of coastal and marine tourism includes various activities with maritime nuances, such as recreational diving, snorkelling, fishing, swimming and boating. indonesia is known as one of the most maritime coun tries in the world, with an area of 2/3 consisting of water. this condition is undoubtedly one of the supporting factors and directly benefits indonesia in developing coastal tourism potential and small islands. one of the areas in eastern indonesia with a variety of coastal and small island tourism potentials is makassar city. the potential for coastal and small island tourism in this region is very large to be encouraged and developed into coastal, marine and beach tourism. most people who live in coastal areas have a livelihood as fishermen who depend on the sea for their livelihood by fishing to fulfil their daily needs (fisher et al., 2014; roswiyanti et al., 2022). therefore, the community generally inhabits and uses the allotment of marine biological resource wealth, which is relatively high up to the limit of over-exploitation. this has a significant impact, such as a decrease in ecosystems in coastal areas which will ultimately affect the decrease in the income of the fisherman concerned. currently, the environmental conditions of coastal areas can be threatened very quickly because of their dynamic nature as a result of various human activities. this has a significant real impact on the coastal environment in the future. therefore, serious attention is needed to overcome the impacts arising from tourism activities on the sustainability of coastal and marine tourism. sustainable management in tourism development needs support from stakeholders, especially the local government and the community, so that a balance of the three (3) aspects can be achieved, namely economic, social and environmental aspects (wang et al., 2016; putri et al., 2020). marine area development plans must be linked to fundamental interests, namely empowering coastal communities. coastal communities are people who have much knowledge about the objective conditions of their area, therefore in the development of marine tourism areas, an approach to the local community should always be initiated as a model of a participatory planning ap proach that places coastal communities in the possibility of sharing, increasing and analyzing their knowledge about marine and coastal life, plan and act (muhtazib et al, 2022). community-centred development emphasizes empowerment, which views community potential as the leading resource in development and views togetherness as a goal to be achieved in the development process. makassar city has enormous potential to be developed as a marine tourism area. this can be seen from the location and typology of th e area, which is quite supportive. the coastal area has sandy beaches with beautiful natural views that can be enjoyed both during the day and at night. in addition, there are various tourism options for visitors in the form of outbound tours, theme parks, historical sites, and a variety of people's livelihoods with exceptional food that can be developed as a tourist attraction. whereas in small island areas, there are beaches with typical island vegetation, various types of coral reefs and types of reef fish which can be developed as diving and snorkelling tourist attractions. the coastal areas and small islands of makassar city from the supply side are beautiful tourist destinations. in contrast, from the demand side, the potential for tourists to co me is international journal of applied biology, 7(1), 2023 37 quite promising because some tourists can spend quite a lot of money to enjoy the natural panorama of the beach, diving and snorkelling on the coast. and the small islands of makassar city. currently, tourism development is focused only on coastal areas, while on small islands, it has yet to be optimal. this is due to the low attention and seriousness of the makassar city government in utilizing the resources of small islands for tourism utilization. this can be seen from the lack of facilities and infrastructure to support tourism on small islands, the low level of knowledge and welfare of the people in the field of tourism, and the tendency for degradation and functional areas to occur as a result of the low level of public awareness and respect for nature and the dominance of the economic aspect over the biological aspect. and social culture in area management. kodingareng keke island is one of the islands in makassar city which has good potential for marine tourism to be developed. its strategic location, quite close to makassar, can be reached approximately 25 minutes from the kayu bangkoa crossing port, making kodingareng keke island quite attractive to tourists to enjoy snorkelling and diving tours. apart from the superior tourism potential of kodingareng keke island, until now, the management of marine tourism on the island has not been carried out optimally, so the income of the community and makassar city's income from the marine tourism sector has not been optimal. this research was conducted to determine the potential of coastal natural resources and underwater objects and formulate strategies and priorities for developing kodingareng keke island as the leading destination for marine tourism in makassar city. materials and methods the research was conducted from january to march 2022 on kodingareng keke island, makassar city, south sulawesi province (figure 1). figure 1. map of sampling points for research locations international journal of applied biology, 7(1), 2023 38 materials and tools the tools used in this research activity are presented in table 1 below: table 1. research tools no tool's name useful 1. global position system (gps) taking coordinates and tracking coral reef areas 2 underwater digital camera documentation of coral reefs 3 computer processing data 4 roll meter measuring coral reef transects 5 scuba diving equipment doing dives and collecting data on coral reefs 6 steel frame (58x44 cm) observation of coral reefs 7 waterproof paper to write underwater 8 boat field data collection sea transport work procedures research preparation before conducting research activities, tools and materials, as well as supporting references/literature, are prepared in advance to assist in collecting field data, namely collecting data on potential marine resources on kodingareng keke island, collecting data using questionnaires and preparing cameras for documentation. dive survey preparation before diving on kodingareng keke island, prepare a three-seat scuba gear with six tubes, an underwater camera for underwater documentation, a lone para pipe for co ral reef transects, and a 50-meter tape measure. before the dive, the station points will be observed for the condition of the waters. when diving, use a boat to the location of the diving point. retrieval of data on coral reefs and reef fish data collection in the field using the line intercept transect (lit) method with a transect line length of 50 meters and a depth of 5 meters (loya, 1978; sarbini, 2016), reef fish data using the underwater visual census (uvc) method is carried out by under water shooting using a digital camera. a 50-meter-long transect was laid, and framed photos were taken at each meter. shooting was carried out from the 1st meter on the left side of the transect line, followed by taking photos at the 2nd meter on the right . continuous shooting is carried out until the end of the transect. so for odd meters (1st meter, 3rd, 5th,..) is taken on the left, while for even meters (2nd meter, 4th, 6th,..) on the right. shooting starts from the transect of 90 cm in each meter (manuputty, 2006). shooting should be done about 60 cm from the bottom of the substrate and done perpendicularly. taking pictures of coral reefs and fish, assisted by two (2) people with two different roles, namely as a photographer and holding the frames (kuit er & tonozuka, 2001). coral reef photo processing the photos obtained are then analyzed with computer software such as cpce. this analysis aims to obtain quantitative data such as the percentage of coral cover or other international journal of applied biology, 7(1), 2023 39 substrates. the basic operations of cpce consist of defining a digital image, defining frame boundaries, overlaying random points, identifying the coral species and substrate type under each random point, and saving the data to a file. after the images have been processed, the data can be automatically assembled into an excel spreadsheet for statistical analysis. cpce is specifically designed to flow logically from one operation to the next and to simplify and speed up processing efforts (kohler et al., 2005) the results of the analysis of all photos (50 photos) on one transect are then calculated automatically by the software used. then quantitative data can be obtained. data analysis a) swot analysis the method to find strategies in the development of coastal and marine problems uses swot analysis in which there are internal factors (inside) and external factors (strengths, weaknesses, opportunities and threats) (freddy, 2014) presented in table 2. table 2. swot analysis table internal factors external factors strengths weaknesses opportunities so strategy wo strategy threats st strategy wt strategy based on table 2, 4 (four) bases are obtained in determining the strategy, namely: 1) so strategy, used to take advantage of strengths to seize opportunities available in the external environment; 2) wo strategy aims to improve internal weaknesses to seize opportunities from the outside environment; 3) st strategy, used to utilize strength to minimize threats that come from outside; 4) wt strategy, used to reduce weaknesses and overcome external threats. b) priority strategy for marine tourism development on kodingareng keke island prioritizing the strategy for developing marine diving tourism on kodingareng keke island by giving weights based on the degree of importance to each element of strengths, weaknesses, opportunities and threats based on respondents' perceptions in developin g marine tourism diving kodingareng keke island with the following details: 1) very important : 3 2) important : 2 3) less important : 1 international journal of applied biology, 7(1), 2023 40 c) analysis of marine tourism development programs the analysis of the dive marine tourism development program on kodingareng keke island was carried out after prioritization was determined based on the weighting, which was then formulated based on the elements forming the strategy and then used as a policy direction in preparing priority strategies based on the ranking of each strategy produced. from the resulting priority strategy, various development programs for kodingareng keke island as a marine tourism and diving area were born. results and discussion general condition of kodingareng keke island kodingareng keke island is one of the 12 islands which are island and water tourism objects owned by the city of makassar. this island is uninhabited, where at the lowest tide, there is a relatively wide plain composed of coral rubble material, especially in the wate rs to the west, which was formed due to the sedimentation process. on the island's south side, the beach is composed of coral fragments of various sizes, while on the island's north side lies white sand with fine–medium sizes. meanwhile, the east and south waters are shipping lanes in and out of the port of makassar. with clear water conditions without contamination from city waste, kodingareng keke island is an ideal location for marine tourism, especially diving, witnessing the beauty of coral reefs and the diversity of biota on the seabed. while on the beach, white sand stretches and is overgrown with several pine trees and other trees around it. kodingareng keke island is classified as a tourist attraction which is quite crowded with local and foreign tourists. there is no regular transportation to the island of kodingareng keke, so to reach the location of the island is done by renting a 40 pk motorboat (local boat) which is available at the jetty in front of rotterdam at a rate of idr 800,000/day. the t rip to the island takes about 30 minutes. figure 2. kodingareng keke island marine tourism is a tourism activity that utilizes the potential of marine biodiversity as the main attraction. marine tourism activities, in general, aim to obtain economic benefits primarily for the local community, tourism stakeholders and the local government. international journal of applied biology, 7(1), 2023 41 kodingareng keke island has the biophysical carrying capacity of the tourism environment. the availability of natural factors in the form of a white sand e nvironment, coral reef ecosystem, artificial reef atlantis garden and japanese shipwreck is a unique attraction for marine lovers, especially diving. another thing is supported by the availability of local boat facilities every day from makassar to this island, which only takes 40 minutes. tourist visits to kodingareng keke island, both local and foreign tourists, have started to get busy in the last five years reaching 1,135 tourists, consisting of 535 local tourists and 600 foreign tourists. data on tourist visits to kodingareng keke island for the last five years are presented in table 2 below: table 2. data on visits by kodingareng keke island tourists for 2017 -2021 year total of local visit foreign tourist tourist destination 1 2017 165 74 snorkelling, swimming 2 2018 177 88 diving, sunbathe 3 2019 198 96 fishing, jetski 4 2020 233 115 diving, snorkelling 5 2021 256 125 diving, snorkelling source: makassar city tourism office, 2022 the number of tourists visiting kodingareng keke island, both local and foreign, especially from 2017 and 2018, has increased. however, there has been a decrease in visitors from 2019 and 2020 due to covid 19, where there was a travel ban for foreign commu nities. in addition, due to the limited data on the latest underwater objects owned by the city government and the lack of resources that understand the development of diving tourism, the island of kodingareng keke needs to be faster to develop into a lead ing diving tourism object. public facilities and infrastructure to support tourism activities on kodingareng keke island, including the availability of lodging, restaurants, clean water facilities, docks and mooring ports for tourism ships, still need to b e improved in quality and quantity. diversity of coral reef ecosystems for marine tourism a distinctive feature of marine tourism areas is the presence of natural resources of the coast, waters and biota in the area, which are still natural and attractive for tourists. natural beaches can be in the form of white sand beaches that look stunning for visitors. the attractiveness of the waters for tourists can be the depth and clarity of the waters, which make it easier for visitors to swim and snorkel. while t he biota in the area can be coral fish and coral reefs, as well as various other types of aquatic biota. a) the marine natural resource potential of kodingareng keke island is shown from several parameters that support the development of the island as a marin e tourism object, especially diving, including: a) oceanographic parameters, b) parameters of coastal and island typology, and c) biological parameters. these parameters were obtained by the author from secondary data from related agencies and recent surve y data on the condition of the underwater coral reefs and coral fish of kodingareng keke island, as follows: international journal of applied biology, 7(1), 2023 42 7 7 .8 6 3 .6 7 7 0 10 20 30 40 50 60 70 80 90 c o r a l c o v e r % karang hidup sstation 1 sstation 2 sstation 3 b) in terms of physical oceanographic parameters, namely waves, surface currents, water brightness and sea water temperature on kodingareng keke islan d, it shows that the wave height ranges from 10 cm to 15 cm but sometimes reaches 50 cm in the west monsoon. in addition, the speed of surface currents ranges from 1.13 cm/sec to 1.28 cm/sec, and the water brightness reaches 100% at a depth of 15 meters. various marine tourism activities have great potential in this physical oceanographic condition. the marine tourism objects that can be developed according to the physical oceanographic conditions are snorkelling, diving, water skiing, windsurfing and fishi ng which can be done all year round. c) in terms of coastal typology parameters, it shows that kodingareng keke island has a very supportive potential for marine tourism with a sloping sandy beach profile and a slope at sea depth, which are standard criteria for various coastal and marine tourism activities. the types of marine tourism activities that can be developed based on the parameters and typology of the beach are sunbathing, beach volleyball, panoramic photography, swimming and diving. d) in terms of biological parameters, which include the condition of coral reefs, coral fish and also several factors of underwater heritage which are very specific for the sale value of diving tourism, are described as follows: the condition of the coral reef ecosystem and observations of reef fish on kodingareng keke island was carried out by direct observation using the lit (line intercept transect) method with three observation stations at a depth of 5 meters. the results of observations at three different stations on three sides of the island showed that the average live coral cover ranged from 63.6% to 77.8%, so it can be concluded that it is very suitable for snorkelling and diving tourism. data in the following figure: figure 3. percentage of live coral cover live coral international journal of applied biology, 7(1), 2023 43 0.00 5.00 10.00 15.00 20.00 category 1 17.63 14.78 18.67 stasiun 1 stasiun 2 stasiun 3station 3station 2station 1 figure 4. percentage of live coral cover diversity of reef fish for marine tourism the abundance of reef fish species found in the three survey locations of kodingareng keke island is also suitable for snorkelling and diving tourism objects. the existence of reef fish in the waters depends on the coral reefs' health, which is shown in th e percentage of live coral cover. this is possible because live reef fish are associated with the shape and type of coral reefs as a place to live, protect and find food. reef fish are relatively sedentary for most of their lives. the average number of fish abundance at three observation stations, station 1 (17.63%), station 2 (14.78%) and station 3 (18.67%). they are presented in figure 4 above. the suitability of diving tourism on kodingareng keke island is in the very suitable category, with an area (of 5.742 ha) and a very high area of live coral cover (42.88%) (anwar, 2011). the islands in the very suitable category have a relatively wide average of coral reefs on the west, south and north sides. analysis of the development strategy for the marine tourism area of kodingareng keke island the preparation of development strategy through studying the potential of the kodingareng keke island marine tourism area was prepared by examining the poten tial and characteristics in the field in exploring the elements of strengths, weaknesses, opportunities and threats. in addition, it is also necessary to analyze in more depth the swot elements that may or are expected to have the potential to emerge in th e future. thus, the formulated strategy for the development of the kodingareng keke island marine tourism area can be anticipatory towards changes that occur in the future, which are presented in table 3. the factors that become the strengths and weaknesses in preparing the strategy for the development of the kodingareng keke island marine tourism area in an integrated and sustainable manner. based on the results of the identification, there are four factors which are strengths and also four factors which are weaknesses, in preparing the development strategy for the marine tourism area of kodingareng keke island. in terms of opportunity and international journal of applied biology, 7(1), 2023 44 threat factors, there are also four factors that become opportunities and threats in preparing the development strategy for the marine tourism area of kodingareng keke island. strengths and weaknesses are internal factors, and opportunities and threats are external factors. the strengths and opportunities factors have a positive impact. in contrast, the weaknesses and threats factors hurt the development strategy for the marine tourism area of kodingareng keke island (table 3). table 3. identification of strengths, opportunities, weaknesses and threats aspect strengths weakness opportunity threats ecology diversity of coral reefs, various types of reef fish, underwater heritage japanese ww ii), natural white sand beaches environmental sustainability threatened by tourism activities environmental sustainability issues related to damage to coral reefs excessive exploitation of marine resources from within and outside the area by using tourists who are not environmentally friendly economy there are supporting tourist objects and attractions public understanding of the objectives and economic benefits of the tourism industry sector is still weak the tourism sector is now considered the prima donna for national development many depend on foreign aid and investment socio-cultural the diversity of ethnic, cultural and community groups is relatively high the potential for managing human resources is still minimal the government's political will which makes tourism a mainstay in pad revenue socio-cultural changes and cultural influences from foreign tourists law and institutions there are clear rules regarding the development of tourist areas contained in regional regulations. there is no integrated and sustainable tourism planning yet makassar city spatial planning policy which develops kodingareng keke island as a marine tourism area increased crime and drug trafficking source: primary data after processing, 2022 international journal of applied biology, 7(1), 2023 45 there are seven strategies produced in this study, namely: 1) strict maintenance of environmental sustainability and development of tourist areas. 2) dissemination and promotion of marine tourism development ideas. 3) integrated and comprehensive planning across sectors. 4) dissemination of benefits and economic objectives from the existence of marine tourism accompanied by an increase in human resources in the field of marine tourism. 5) law enforcement and regular preparation of tourism development plans. 6) determination of marine tourism area development zones. 7) local culture needs to be developed and preserved. conclusion based on the results of research that has been conducted on tana keke island, makassar city, it can be concluded that: 1. the average number of corals on tana keke island ranges from 63.6% to 77.8%, and the average number of fish abundance at three observation stations, station i (17.65%), station ii (14.78%) and station iii (18.67%) making it suitable for snorkelling and div ing tours. 2. priority programs for the development of the marine tourism area of kodingareng keke island are increased supervision, regular preparation of object development master plans (ripo), implementation of local regulations for the use of coastal reso urces, involvement of local communities, the general public and government in nature conservation activities, setting labour standards, and improving the quality of local human resources through training. acknowledgements we thank the chancellor of cokroaminoto university makassar for the support given to researchers so they can produce works in the form of journals. we also remember to thank all kodingareng keke island 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ó international journal of applied biology 52 abstract tuberculosis caused by mycobacterium tuberculosis is the biggest infectious disease causing human death in the world. the main challenge in controlling tuberculosis is to quickly and accurately diagnose tuberculosis infection. several kits have been produced to diagnose tuberculosis, but have different sensitivity and specificity. this shows that the kit is not yet ideal for diagnosing tuberculosis, so the search for candidates for specific antigens still needs to be done. one potential antigen is the rv 1926c encoding mpt 63 protein. this protein is known to induce th1 cells and produce ifn λ from pbmc cells of patients infected with tuberculosis. the purpose of this study was to clone the rv 1926c from mycobacterium tuberculosis as a tuberculosis immunodiagnostic kit. the method used is isolating rv 1926c with pcr, ligation to pgem-t vector and transformation to e.coli host cell jm 109. clone characterization was carried out by pcr and migration analysis. the results obtained are the recombinant clones obtained have successfully inserted with the rv 1926 c issn : 2580-2410 eissn : 2580-2119 ligation, transformation and characterization of rv 1926c mycobacterium tuberculosis to escherichia coli jm 109 for latent tuberculosis immunodiagnostic rosana agus1, sjafaraenan1 and mochammad hatta2 1department of biology, faculty of life sciences, hasanuddin university, indonesia 2department of microbiology, faculty of medicine, hasanuddin university, indonesia introduction tuberculosis (tb) is the ninth leading cause of death worldwide. in 2016, there were an estimated 1.3 million tb deaths among hiv-negative people and an additional 374.000 deaths among hiv-positive people. in 2016 an estimated 10.4 million people fell ill with tb: 90% were adults, 65% were male, 10% were people living with hiv (74% in africa) and 56% were in five countries: india, indonesia, china, the philippines and pakistan (who, 2017). generally most of the people in indonesia have received the bcg vaccine when they were toddlers. but the effectiveness of this vaccine does not last until adulthood, so it is suspected that everyone can be infected by m. tuberculosis and is latent. latent tb infection open access international journal of applied biology keyword rv 1926c immunodiagnostic e.coli, jm 109 latent mpt 63 article history received 17 november 2018 accepted 25 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 53 has the potential to become active tb and people with active tb can be a source of new infections latent tuberculosis infection (ltbi) is the presence of tuberculosis in the body without symptoms or radiographic evidence or bacteriological examination. it is estimated that up to 13 million people in the united states are latent tb, and 5-10% of infected people will suffer from tb, which is equivalent to 650,000 to 1,300,000 (cdc, 2013). the guidelines recommend that either tuberculin skin test (tst) or interferon gamma release assays (igra) can be use to test for latent tb in in high-income and upper middleincome countries with estimated tb incidence less than 100 per 100 000. consistent with existing who recommendations, the guidelines reiterated that igra should not replace tuberculin skin test in low-income and other middle-income countries (who, 2018). tuberculin skin test (tst), also referred to as the mantoux or purified protein derivative (ppd) test, detects tb exposure through the skin. advantages of tst are simple and easy to administer and can detect tb infection. disadvantages of tst are false negatives: the failure of a bump to develop can be the result of a compromised immune system, false positives: the bacterial particles in the bcg vaccine can stimulate the production of tb antibodies, causing a false positive, sensitivity varies widely across populations and is inconclusive for children, hiv patients with low cd4 counts (tag, 2018). some weaknesses of tst include not being able to distinguish between active and latent tb patients. this test will be positive in people who are bcg vaccinated and who are in contact with other mycobacteria (pai et al., 2016). therefore the specificity of ppd is questionable, especially in endemic areas such as indonesia. due to the limitations of the tst test, the study was directed to find the specific antigen to be used as an immunodiagnostic. especially the availability of tb diagnostic reagents that can identify new and latent infected individuals with high risk that can develop into active tuberculosis. one potential antigen is the rv1926c encoding mpt 63 protein. this protein is known to induce th1 cells and produce ifn λ from pbmc cells of patients infected with tb.the purpose of this study was to clone the rv 1926c from m. tuberculosis to e.coli jm 109 as immunodiagnostic latent tuberculosis materials and methods bacterial strains and plasmids the cloning vectors pgem-t easy and bacterial strain jm 109 (promega) was used. the strains m. tuberculosis was obtained from clinical isolate from indonesia. culture condition bacterial strain jm 109 is a useful host for transformation of pgem-t vector. bacterial were incubate with stirring over night in luriabertani medium in the presence of ampicillin (1μg/ml) at 37°c. the clinical isolate of m. tuberculosis is cultured in the lowenstein-jensen medium. chromosome dna extraction m.tuberculosis chromosome dna extracted using qiagen dneasy kit. dna extraction were measured using a spectrophotometer at absorbance of 260 nm. amplification international journal of applied biology, 2(2), 2018 54 amplification of rv 1926c was carried out by pcr using a specific primer. the primer sequence used in this study according (5) as follow forward: 5'cagcaggatcccgcctatcccatcaccgga-3' and reverse : 5’gcccaagcttcggctcccaaatcagcag-3' .the pcr conditions for amplification were pre denaturation 940c 10 minutes, denaturation 940c 1 minute, annealing 560c 1 minute and elongation 720c 1 minute in 30 cycles. creation of plasmid of the recombinant rv 1926c in e. coli jm 109 fragment of the rv 1926c were obtained by pcr using two primers pair and dna of the m. tuberculosis indonesian strain as a template. the pgem-t vector and pcr product were cut with bamhi and hindiii, mixed, and treated with t4 dna ligase. the resulting recombinant plasmid pgem-t-rv1926 was transformed in e. coli jm 109. transformation isolation of dna and transformation of the e. coli jm 109 cells were performed as described in the guidebook [6], with the following modifications. transformed cells were spread on the appropriate indicator plates containing ampicillin. colonies were scored for phenotype on luria bertani agar plates after 24 hours at 37°c. results and discussion pcr product and purification the results of pcr amplification of rv 1926 observed by electrophoresis agarosa. a band was revealed which corresponded to the gene with the apparent of 412 bp. figure 1. pcr product of rv 1926c, (k-) negative control , (1,2) rv 1926c , (k+) positive control , (m 100) marker 100 bp international journal of applied biology, 2(2), 2018 55 ligation and transformation of the rv 1926c to the pgem-t vector the results of the transformation in figure 1 showed that there were colonies and white colonies in petridish containing lb medium, x-gal, iptg and ampicillin. white colonies indicate that the insertion dna has been successfully inserted into the vector, while the blue colony means that the insertion dna has not been successfully transferred to the vector. white e. coli colonies (transformant cells) showed rv 1926c coding dna successfully ligated in the mcs (multi cloning site) area found in the lacz pgem-t gene. the insertion of this dna fragment will inhibit the lacz gene to encode the subunit of β-galactosidase, so that the enzyme cannot degrade the available galactose substrate. the bacterial colonies are blue, meaning they do not have insertion dna fragments so they can degrade the available galactose substrate (medical biochemistry, 2017). figure 2. recombinant clone pgem-t-rv 1926c characterization of recombinant plasmid of pgem-t-rv 1926c isolation of plasmid were performed according to the procedure instructions (biorad). characterization was done by pcr analysis and migration analysis. migration analysis was done by comparing pgem-t (3000 bp) and pgem-t-rv 1926c (3412 bp). a band shows difference length while pgem-t-rv1926c had slower migration than pgem-t (figure 3). international journal of applied biology, 2(2), 2018 56 figure 3. migration analysis: (1) pgem-t: 3000 bp (2) pgem-t-rv1926: 3412bp this suggests that the plasmid without insert dna (pgem-t) will move faster than the recombinant plasmid (pgem-t-rv 1926c). this means that the dna inserts rv 1926c has been successfully ligation into the vector pgem-t. pcr analysis was done by using exactly the same cycle as it mentioned before for amplified the rv 1926c gene. electrophoresis showed that plasmid recombinant contain the rv 1926c gene as dna insert was 412 bp (figure 4) figure 4. pcr analysis: (a,b,c,d) rv 1926c : 412bp. (m) marker 100 bp conclusions gene of rv 1926c m. tuberculosis indonesian isolates have been successfully ligated to pgem-t vectors and transformed into e. coli jm 109 acknowledgments the authors would like to thank kemenristek dikti which has funded this research through a pdupt 2018 references centers for disease control and prevention national, 2013, health, united states, with special feature on prescription drug medical biochemistry, 2017, blue white screening pai, m. and giovanni sotgiu, 2016, diagnostics for latent tb infection: incremental, not transformative progress, eur respir j 2016; 47: 704–706 | samal, 2012, evaluation of diagnostic potential of recombinant mpt63 protein in bovine tuberculosis, deemed university indian veterinary research institute treatment action group, 2018, module four tb diagnostics, tb/hiv advocacy toolkit international journal of applied biology, 2(2), 2018 57 world health organization, 2017, global tuberculosis report world health organization, 2018, who guidelines on the management of latent tuberculosis infection launched today sambrook, j., fristch, e.f. dan maniatis, t. molecular cloning a laboratory manual. cold spring harbor laboratory press, 1989. treatment action group, 2018, module four tb diagnostics, tb/hiv advocacy toolkit 2(2)1-12 contact : dewi wahyuni k. baderan dewi.baderan@ung.ac.id © international journal of applied biology 1 abstract this study aimed to analyze the structure of vegetation, diversity and patterns of mangrove zonation in the tanjung panjang nature reserve area. data collection of mangrove vegetation structure was carried out by using the line transect method (plot measuring 20 m x 20 m), measurement of environmental parameters that supported mangrove life, and 12 soil samples carried out by laboratory tests. the sample distribution was 18 research sample points. the results showed that mangrove vegetation density both at the level of trees, saplings and seedlings was in the category of total damage with density values at tree level 0.04-0.22 ind/ha), sapling 00.07ind/ha), and seedlings 0.02-0.08 ind/ha). the percentage of closure types ranged from 0% 38%, meaning that the criteria of damage was rare, which was <50%, and some were included in the criteria of total damage, which was 0%. the index of mangrove species diversity was sequential starting from the level of trees, saplings and seedlings, which were 0.37-1.53, 0.00-0.31, and 0.00, where the tree level diversity index was in the medium category, and the sapling and seedling levels were in the low category. issn : 2580-2410 eissn : 2580-2119 vegetation structure, species diversity, and mangrove zonation patterns in the tanjung panjang nature reserve area, gorontalo, indonesia dewi wahyuni k. baderan, ramli utina & nurain lapolo department of biology, faculty of mathematics and natural sciences, universitas negeri gorontalo, gorontalo, indonesia introduction mangrove ecosystem is one of the main potential of coastal areas which plays physical and ecological roles. the physical role is that mangrove protects coastal areas from the threat of erosion, sedimentation and breakwater (zulkarnaini et al. 2017). mangroves also protect coastal areas from natural disasters, such as hurricanes and tsunamis (gedan et al. 2011: kathiresan and rajendran, 2005: kusman et al. 2005: cochard et al, 2008). in addition, mangroves can retain the habitat of biota association to maintain biodiversity, and has economic potential for forest products, fisheries, and ecotourism (suwardi, 2013). mangroves are spread throughout the world, including indonesia, which is one of the countries with the largest mangrove forests in the world. indonesia has the highest level of mangrove diversity in the world with 202 mangrove species. of these 202 species, 43 species (including 33 species of trees and some shrubs) are true mangrove species, and other species are found around mangrove forests as associated mangrove species (noor et al. 2012). the open access international journal of applied biology keyword vegetation, diversity, zonation, mangrove, tanjung panjang nature reserve article history received 14 october 2018 accepted 21 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 2 high level of biodiversity makes mangrove forests an important asset in the perspective of ecological and economic functions (dahuri, 1996). however, mangroves are not currently seen as an important asset that can be maintained, so that mangrove damage increases every year. this happens because mangrove forests in many places become objects of various development activities, especially ponds. as a result, the mangrove ecosystem continues to experience formation changes. the loss of mangrove areas into ponds is also found in the gorontalo region. the tanjung panjang nature reserve is one example of the largest mangrove area in gorontalo that has been converted into pond. it is estimated that an area of 16 ha has been opened since 1994. the pond area increased to 297 ha in 2000 and in 2015 the area increased by 7,129 ha (blue forests, 2017). this illustrates that the designation of mangrove forests into ponds has increased over a period of 15 years from 2000 to 2015 (lapolo et al. 2018). the conversion of mangroves into ponds can cause a decline in the functions and benefits of mangrove forests, especially the loss of mangrove species that will affect coastal productivity and the reduced diversity of species from mangrove ecosystems globally, especially indonesia. the study reported in this article aimed to analyze the structure of vegetation, diversity and patterns of mangrove zonation in the area of tanjung panjang nature reserve. the data collected can be used as one of the supporting data for policy making in reducing mangrove damage in the area. materials and methods research site the present study was conducted in the tanjung panjang nature reserve area located in pohuwato regency, gorontalo, indonesia, which included three villages namely siduwonge village, patuhu village, and limbula village (figure 1). geographically, the site where the study was conducted was located between the coordinates 0025’28,93” 0030’1,93” north latitude and 121044’27,60” 121047’0,44” east longitude. data collection an exploratory survey method was used in the study. data were collected in the form of primary and secondary data. collecting primary data was carried out by identifying all mangrove species using a book (giesen et al. 2006; noor et al. 2012), the level of diversity, and the pattern of mangrove zonation using the line transect method. secondary data collection was from other studies published in journals, pohuwato regency statistics in figures and other supporting data. mangrove vegetation data collection was done by determining the starting point of measurement; a 100 m transect line was drawn from south to north. on each transect line a plot of observations was made at the tree level. on each mangrove zonation along the transect line was placed a plot with a size of 20 m x 20 m with limit signs on every 10 m and alternates to the left and right transects (kusmana, 1997). the plot was made in the size of 10 x 10 m2 for tree groups, 5 x 5 m2 for sapling and 1 x 1 m2 for seedlings (bengen, 2004). each transect line was determined by two sample plots. in each sample plot, there were individual counts and measurements of tree trunk diameter (dbh) or 1.3 m from the soil surface were taken, and the circumference of the dbh and the names of mangrove species found in the tally sheet were recorded. for seedlings, the diameter measurements were carried out under the starting part of the discovery of the branch. international journal of applied biology, 2(2), 2018 3 figure 1. research sitemap within the area of tanjung panjang natural reserve of pohuwato district, gorontalo province, indonesia environmental parameter furthermore, environmental parameters were measured at the same location by taking mangrove vegetation data at each research station. the measurement includes soil ph, soil texture, and soil samples at low tide using core panels made of pipes at 0-30 cm depth (sara et al. 2014). the aim was to examine soil nutrient content (n, p, k), air temperature, air ph, and salinity. results and discussion species which form mangrove vegetation the study found 13 mangrove species, consisting of 11 true mangrove species and 2 associated mangrove species (see table 1). the mangrove families were avicenniaceae, combretaceae, lythraceae, meliaceae, rhizophoraceae, sonneratiaceae, aizoaceae, and convolvulaceae. the true species of mangroves found in the research site were avicennia marina, lumnitzera racemosa, phempis acidula, xylocarpus granatum, xylocarpus moluccensis, bruguiera gymnorrhiza, ceriops tagal, rhizophora apiculata, rhizophora mucronata, rhizophora stylosa, and sonneratia alba. for the mangrove types, the associations at the research site were aturvium portulacastrum, and ipomea pes-caprae. table 1. mangroves species in the research site no family scientific name local name a. true mangroves group 1 avicenniaceae avicennia marina api-api 2 combretaceae lumnitzera racemosa dulu’-dulu’ 3 lythraceae pemphis acidula santigi tanjung panjang nature reserve international journal of applied biology, 2(2), 2018 4 4 meliaceae xylocarpus granatum tambu putih 5 xylocarpus moluccensis tambu hitam 6 rhizophoraceae bruguiera gymnorrhiza sala’ – sala’ 7 ceriops tagal tanggere 8 rhizophora apiculata bangko 9 rhizophora mucronata bangko 10 rhizophora stylosa bangko 11 sonneratiaceae sonneratia alba parappa b. mangrove associates group 12 aizoaceae rhizophora stylosa . 13 convolvulaceae sonneratia alba . mangrove vegetation structure at the observation station based on the standard criteria for mangrove forest destruction of kep-menlh no. 201 (2004) in baderan (2013) was included in the rare damage and full damage criteria (figure 2). this was due to the condition of the mangrove forest with <1000 (ind / ha) and 0 density, and <50% and 0 closing value of species. the density value obtained by each observation station starting from the tree level was 0.04-0.22 ind / ha; the sapling level was 0.00-0.07 ind / ha; and the seedling level was 0.02-0.08 ind / ha. figure 2. mangrove’s density in the in the site area the percentage of the type closure obtained in this study ranged from 0% -38% (table 2). the percentage value of tree level closure was 9% -38%, and the sapling rate ranged from 0% -37%. this showed that the closure of tree-level mangroves was in the criteria of rare damage (<50%), while the closure of the level of mangrove species was total damage (0%). the diversity of mangrove species the diversity index (h’) of mangrove species shown in figure 4 revealed sequential values, starting from the tree level, saplings, and seedlings, which were 0.37-1.53, 0.00-0.31, and 0.00. the graph shows that the diversity index which is in the medium category is only found at the tree level (1.53) station ii zone 3, and the others at the sapling and seedling levels are in the low category. overall, the index of mangrove species diversity in the observation station was in the low category. 0.00 0.05 0.10 0.15 0.20 0.25 zone 1 zone 2 zone 3 zone 1 zone 2 zone 3 station i station ii m an gr ov e´ s d en si ty ( in d/ ha ) research station k tree k sapling k seedling international journal of applied biology, 2(2), 2018 5 figure 4. mangrove´s diversity in the site area table 2. the percentage of mangrove species closure at the research site tree stake no species ci (%) rci (%) species ci (%) rci (%) 1 ceriops tagal 3.96 44.76 ceriops tagal 0.20 79.24 2 rhizophora mucronata 3.61 40.74 rhizophora mucronata 0.01 4.15 3 rhizophora. stylosa 0.44 4.92 rhizophora. stylosa 4 xylocarpus granatum 0.11 1.27 xylocarpus granatum 0.00 0.29 5 xylocarpus mollucensis 0.05 0.56 xylocarpus mollucensis 0.00 1.07 6 bruguiera gymnorrhiza 0.43 4.87 bruguiera gymnorrhiza 7 rhizophora apiculata 0.02 0.21 rhizophora apiculata 8 avicennia marina 0.14 1.53 avicennia marina 0.04 15.25 9 sonneratia alba 0.10 1.13 sonneratia alba total 8.86 100.00 0.25 100.00 mangrove zonation mangrove zonation in the research site grew from the edge of the sea to the land, dominated by ceriops tagal species in each research site (figure 5 and figure 6). the zonation patterns were all in a disturbed condition. this was because the shore and the land had turned into ponds. at station ii of patuhu village, the pond distance from the coastline was only 50 m. this indicated that the conversion of land into ponds in the research site had a negative influence on the formation of mangrove zonation, as well as the physical changes (mangrove habitat). mangrove stands at the research site would find it difficult to withstand any forms of physical changes. the outermost physical seas which protected mangrove ecosystems in the mainland or which had become pond areas would largely lose their physical functions as protection from coastal abrasion, erosion and sedimentation. although there was a new mangrove habitat in the near estuary of tidal flow, changes due to sedimentation rate from the pond may occur. figure 5. true mangrove zonation pattern in the site area (station i) 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 zone 1 zone 2 zone 3 zone 1 zone 2 zone 3 station i station ii m an gr ov e´ s di ve rs it y in de x (h a) research station h' tree h' sapling h' seedling fishpond avicennia marina, ceriops tagal rhizophora mucronata, ceriops tagal, xylocarpus granatum international journal of applied biology, 2(2), 2018 6 figure 6. true mangrove zonation pattern in the site area (station ii) environmental parameters at the research site the environmental parameters measured were mangrove areas and ponds. what was measured was salinity, water ph, temperature, and soil ph (table 3 and table 4). the ph of the water obtained in the mangrove area ranged from 5.6-7.0. this ph was in the neutral category. temperatures ranged from 30.60c-33.30c. stations i and ii zone 1 had soil ph values of 5.2 and 5.6 which were categorized as acids. the ph value of the soil in several other zones was in the range of 6. this showed that the soil ph in the research site was still in the neutral category. the soil texture found were sandy and dusty clay. at stations i and ii zone 3 there was a sandy clay soil texture. this texture had several characteristics: coarse sand, did not stick to each other and was not easily formed so that it could be quickly destroyed. station i and ii zone 1 and 2 had dusty clay soil texture. some characteristics of this texture were slippery soil, easily fused and could be formed but quickly destroyed. the soil at the research site was almost entirely textured with dusty clay. this texture could hold water, was hard and did not break quickly. baderan (2013) contends that the texture of dusty clay soil is suitable for ponds. the soil has high clay content and low sand. high sand content is generally not suitable for ponds, because of its high porosity and low soil binding capacity which can be easily detached when used for building embankments. the highest salinity obtained in the pond area was in station ii zone 3 with a value of 23.6 ppt. the average value of salinity obtained in all stations was 14.0 ppt. the ph of the water in each zone ranged from 6-7 with an average of 6.5. this value was in the neutral category. the highest temperature parameter in the pond area was found at station i zone 3, which was 38.50c. the average temperature was 34.90c. the ph value of the soil ranged from 5-6 with the acid (5) and neutral (6) categories. the average value of soil ph obtained was 5.7 in the acid category. table 3. mangrove environmental parameters in the site area station zone salinity (ppt) water ph temp (0c) soil ph texsture i 1 3.9 7.0 31.6 5.2 dusty clay 2 9.1 5.6 33.3 6.1 dusty clay 3 22.9 6 30.6 7 sandy clay ii 1 5.8 6.0 31.8 5.6 dusty clay 2 11.3 6.0 31.5 6 dusty clay 3 22.8 6.0 32.0 6 sandy clay average 12.6 6.1 33.3 6 fishpond sonneratia, rhizophora spp. ceriops tagal rhizophora mucronata, ceriops tagal international journal of applied biology, 2(2), 2018 7 table 4. pond environmental parameters in the site area station zona salinity (ppt) water ph temp (0c) soil ph texsture i 1 7.0 6.5 31.8 6.1 dusty clay 2 10.0 6.0 35.0 6.1 dusty clay 3 16.0 7.0 38.5 6.2 sandy clay ii 1 5.0 6.7 33.0 6 dusty clay 2 22.6 6.0 34.7 6.1 dusty clay 3 23.6 7.0 36.1 7 sandy clay average 14.0 6.5 34.9 6.25 the results of the analysis of the laboratory of chemistry and soil fertility, hasanuddin university, showed that the percentage value of soil nutrients obtained was higher in the pond area. the highest nutrient was 0.24 which is found in the area of the station 1 zone 3, while the lowest nutrient was in the mangrove area of the station 2 zone 3, which was 0.1. the results of the analysis showed that the soil nutrient content in the form of n, p, and k obtained in the pond area ranged from 0.18-0.24, while in mangrove areas only ranged from 0.09-0.21. discussion mangrove forests in the tanjung panjang nature reserve area from the 1990s until now continue to experience damage caused by mangrove conversion function into massive shrimp and milkfish aquaculture. the damage to mangroves resulted in changes in the area of mangrove forests and reduced biodiversity, including the diversity of mangrove species. as previously reported by damanik and djamaluddin (2012), in pohuwato district, 25 mangrove species were found in popayato, wanggarasi, randangan, marisa and paguat sub-districts. the study found fewer species, namely 13 mangrove species scattered in the research site. the mangrove species found in the research site grew and developed in one zonation so that it looked different from mangrove zonation in general. the number of true mangrove species found in the study area was 11 species that were dominated by the rhizophoraceae family including ceriops tagal and rhizophora mucronata species, and a small portion of others were from the meliaceae and sonneratiaceae families. this amount was less than that in the langge mangrove area, north gorontalo regency which was reported by baderan et al. (2017), namely, 12 species which were also dominated by the rhizophoraceae family. certain mangrove species were increasingly depleted in the tanjung panjang area due to habitat damage. baderan (2017) asserts that such damage impacts on the livelihoods of communities around mangrove areas, and results in the extinction of various species of flora, fauna and certain biota in the world, as well as in the destruction of the mangrove habitat itself. the loss of mangrove stands means that the parent tree producing seeds is lost. the destruction of most mangrove habitats means a decrease in the area suitable for replanting mangroves. the density of mangroves in the research site was included in the criteria for rare and total damage, namely, the density of trees / ha ie <1000 ind / ha and 0 ind / ha. this is in accordance with the standard criteria for mangrove forest destruction stipulated in the kepmenlh no. 201 (2004) as cited in baderan (2013), which stated that the dominant research locations fall into the category of total damage. however, there remained mangrove stands international journal of applied biology, 2(2), 2018 8 which could withstand extreme environmental conditions. these mangroves were included in the criteria of rare damage. the density of mangrove vegetation in most research site both from the level of trees, saplings and seedlings was almost entirely in the category of total damage. this was due to the direct or indirect mangrove damage. directly, this damage was caused by the activities of people living around the area in converting mangrove areas into ponds. indirectly, such damage was caused by changes in environmental factors, namely salinity, tides, and soil texture which were not able to support the survival of mangrove vegetation in the area. mangrove vegetation density is related to each other, both between trees, saplings and seedlings. mangroves in the research site were not productive to meet the needs of natural regeneration. this is presented in figure 3. it can be seen that the fluctuating value of mangrove vegetation density, indicated by one station in zone 3 had no sapling vegetation at al. humaidy (2010) perceived that mangroves in coastal areas that were still productive were characterized by the existence of mangroves in meeting the needs of natural regeneration to meet saplings. natural regeneration is needed to reduce supply or additions from other mangrove broodstock to be used as mangrove seed stock. mangroves at the study site could not naturally increase their area, but with the help of mangrove rehabilitation efforts. however, from the observations presented in figure 3 it can be seen that the density of the stake or seedlings was in the category of total damage. this requires a relatively long time and requires pre-rehabilitation efforts before direct rehabilitation is carried out to restore the ecological function of the mangrove area. environmental parameters also contributed to the level of mangrove damage. salinity obtained was categorized as not in accordance with the sustainability of mangrove ecosystem life. juwita et al. (2015) confirms that the optimum limit of mangrove life is around 15 ppt 25 ppt, while the tolerance limit ranges from 10 ppt 35 ppt. however, some mangroves, such as the rhizophora mucronata species are able to tolerate the extreme surrounding environment, so that at the study site there were still mangrove stands found, such as rhizophora mucronata and ceriops tagal. another factor that influenced low salinity was that land clearing at the research location was very dominating so that mangroves found it difficult to keep growing and developing even in conditions that were suitable for their growth. generally, the level of salinity in the research site did not support the stability of the mangrove ecosystem, because of several factors that also affected the rate of mangrove damage, such as land subsidence, sedimentation, and sea level rise. rahim et al. (2017) also stated that salinity affected the growth and density of mangroves. if the salinity was too high, the growth of mangroves would become stunted. mangroves are not plants that need a lot of salt but mangroves are salt-tolerant plants. as the study reported, the salinity range for the torosiaje jaya region is 20 ppt 22 ppt. the ph of mangrove waters at the research site was in the range of 5.6-7.0. the result showed that station i zone 3 met the criteria for ph which was suitable for the sustainability of mangrove life, namely 7. this is reinforced by zaky et al. (2012), the ph of the water suitable for mangrove life is 7, where the value is the optimal ph for the fulfillment of nutrients in the soil. unlike the case in the pond area, the average ph of water obtained was 6.5, the ph was higher than the ph of the mangrove area. humaidy (2010) asserts that the description of the compound n (nitrogen) can increase the ph of the water so that the ph in the pond area is greater than the mangrove area. the existence of n can improve the sustainability of the cycle n so as to maintain the stability of the ph of the the presence of toxic and metals can reduce international journal of applied biology, 2(2), 2018 9 ph. humaidy (2010) also explains that the presence of water ph and soil ph has values that are not much different, namely 6-7. another thing is also confirmed by zaky et al. (2012), in accordance with decree no. 51 / menklh / 2004, that the ph of the soil suitable for mangrove life was 8 which was the optimal ph for the fulfillment of nutrients in the soil. the ph obtained in mangrove areas ranged from 5.2-6.1, and the pond area ranged from 6-7. that is why the ph of the soil in the mangrove area did not meet the criteria for suitable mangrove living areas. incompatibility of soil ph in the mangrove area was due to the lack of soil organic matter such as n compounds produced from mangrove leaf litter. this is due to the location of the research at each station, and the density of the mangroves included in the criteria of total damage. temperature was also one of the environmental parameters that affected the life of mangrove biota. the highest temperature was found in the pond area which ranged between 31.50c-38.50c, compared to the mangrove area the temperature was lower which was only 30.60c-33.30c. temperature pressure was higher in the pond area, because of the openness of the land and without mangrove vegetation cover, causing it to have direct sunlight, whereas in the mangrove area the temperature was lower because it was blocked by the cover of mangrove canopy. this is consistent with the study of pasongli et al. (2015) which stated that the increase in temperature in the pond area was due to direct sun exposure. the optimum temperature for aquaculture biota was only around 270c-320c with a life tolerance of 160c-360c. while the temperature in the mangrove area was slightly lower than the pond, the temperature was not suitable for mangrove sustainability. juwita et al. (2015) stated that the optimum temperature limit for mangrove growth ranged from 290c300c. this would affect the photosynthesis process and the process of mangrove growth would be hampered. thus, the temperature at the research site was not suitable for mangrove growth. other conditions that affected the high content of soil nutrients in the pond area were the use of phonska fertilizer containing n, p, and k content. the application of n, p, k fertilizer was aimed at increasing the production of ponds. the results of the analysis of laboratory tests showed that soil samples at station i zone 3 had n content of 0.17, p ie 24.3 and k ie 16.52. the content of compounds n, p, k in this pond area sought to support the growth of phytoplankton which was a natural feed from shrimp and milkfish cultivated in the pond. this was confirmed by pamungkas's research (2011) which showed that the use of fertilizers containing n, p and k was very necessary for the growth of phytoplankton. this means that the provision of fertilizer can increase the number of phytoplankton. unlike the case in the mangrove area, the low soil nutrients (n, p, and k) in the region were influenced by vegetation density and cover which were categorized as damaged, resulting in low decomposition of the resulting mangrove leaf litter. this is because most of the research sites located in the tanjung panjang nature reserve area had been damaged, causing the diversity and density of mangroves to be in the low category compared to the condition of the mangrove ecosystem that remains good. other conditions at station ii zone 1 showed higher soil nutrient content compared to zones 2 and 3. this was because soil samples carried out by laboratory tests are from the species of avicennia marina mangroves. it was concluded that mangrove vegetation density both at the level of trees, saplings and seedlings was in the category of total damage at the tree level ranging from 0.04 0.22 ind / ha), saplings ie 0 0.07 ind / ha), and seedlings 0.02 0.08 ind / ha ) the percentage of closing types ranged from 0% 38%. at the tree level it ranged from 9% 38%, included in the criteria for rare damage, namely <50%, while at the sapling level ranged from 0% 37%, international journal of applied biology, 2(2), 2018 10 included in the criteria of total damage ie 0%. the index of mangrove species diversity was sequential starting from the level of trees, saplings and seedlings, which were 0.37 1.53, 0.00 0.31, and 0.00, where the tree level diversity index was in the medium category, and the sapling and seedling levels were in the low category. it is necessary to restore conservative mangrove and restocking habitats in a sustainable manner, as well as the arrangement of mangrove areas in the research site by making efforts to rehabilitate the area (mangrove planting), namely the improvement of green belt from the coastline as far as 200-300 m with the species suitable for zonation, and 100 m on the dominant river bank rhizopora mucronata. conclusion mangrove vegetation density both at the level of trees, saplings and seedlings was in the category of total damage with density values at tree level 0.04-0.22 ind/ha), sapling 00.07ind/ha), and seedlings 0.02-0.08 ind/ha). the percentage of closure types ranged from 0% 38%, meaning that the criteria of damage was rare, which was <50%, and some were included in the criteria of total damage, which was 0%. the index of mangrove species diversity was sequential starting from the level of trees, saplings and seedlings, which were 0.37-1.53, 0.00-0.31, and 0.00, where the tree level diversity index was in the medium category, and the sapling and seedling levels were in the low category. acknowledgements the authors would like to sincerely thank particular individuals and institutions that have contributed to this research, namely, the postgraduate program on population and environment studies, gorontalo state university, which has provided administrative support for this research; center for study of coastal ecology and local wisdom of gorontalo state university; and blue forests which has provided supporting data and references for this research; government officials of siduwonge, patuhu, and limbula villages, randangan district, pohuwato regency, gorontalo province, and bksda; as well as the field research team that has helped collect research data. references baderan, d.w. (2013). economic valuation model as the basis for mangrove forest rehabilitation in coastal areas of kwandang district, north gorontalo province, gorontalo province. dissertation. postgraduate program of the faculty of geography, gajah mada university, yogyakarta. baderan, d.w. (2017). spatial and widespread distribution of mangrove forest damage in the kwandang coastal area north gorontalo district gorontalo province. geoeco. 3(1) : 2460-0768. baderan, d.w., rahim, s., kumaji, s.s (2017). structure of vegetation, biomass, and carbon stock of langge mangrove forest, north gorontalo regency, gorontalo province (research report). australian journal of basic and applied sciences. 11 (14) 48-57. blue forests. 2017. biophysical and socio economic mapping for the restoration of tanjung panjang natural reserve. reports on the mapping of the tanjung panjang natural reserve survey, roam program. supported by iucn, thailand. international journal of applied biology, 2(2), 2018 11 bengen, d.g. (2004). technical guidelines for introduction and management of mangrove ecosystems. bogor coastal and marine resources study center. bogor. brower, j.e., jerrold h.z., car i.n.v.e. (1990). field and laboratory methods for general ecology. third edition. wm.c.brown publisher, usa, new york. cochard, r. ranamukhaarachchi. s. shivakoti. gp, shipin, ov, pj. and seeland. k, (2008). the 2004 tsunami in aceh and southern thailand: a review on coastal ecosytem. wave hazard and vulnerability. perspectives in plant ecology. evolution and systematics. 10: 3-40. dahuri, r., j. rais, s. p. ginting, and m. j. sitepu, (1996). management of integrated coastal and ocean management resources. pt. pradnya paramita, jakarta. damanik, r, and djamaludin r. (2012). atlas mangrove tomini bay. mangrove condition survey gulf of tomini program susclam (sustainable coastal livelihoods and management program). gorontalo. gedan, kb, kirwan. ml, wolanski. e. barbier, eb and silliman. br. (2011). the present and future role of coastal wetland vegetation in protecting shoreline: answering recent challenges to the paradigm. climatic change. 106: 7-29. giesen w., wulffraat s., zieren m., scholten l. (2006). mangrove guidebook for southeast asia. bangkok: rap publication, food and agriculture organization of the united nations regional office for asia and the pacific. humaidy d. (2010). a study on the degradation of mangrove ecosystem for rehabilitation in the coastal area of kasemen subdistrict, serang city. (thesis). department of water resources management, faculty of fisheries and marine sciences, bogor agricultural university, bogor. juwita, e., soewardi, k., yonvitner. (2015). habitat conditions and mangrove ecosystems in simpang pesak district, east belitung for shrimp pond development. journal of humans and the environment. 22(1): 59-65.. kathiresan rajendra k and n, (2005). tsunami coastal mangrove forest mitigated. estuarine. coastal and shelf science. journal of experimental marine biology and ecology, 234: 255-273. krebs, c.j. (1989). ecologycal methodology. harper collins publisher inc, new york. kusmana, c. (1997). suryvey method for vegetation. institute of agriculture, bogor. bogor. kusman c. s. basuni, s. wilarso. ichwandi i., o. haridjaja. a. salah and samsuri, (2005). directives for mangrove forest and coastal forest rehabilitaiton and tsumani in earthquake disaster areas in the provinces of nanggroe aceh darussalam and north sumatra. journal of tropical forest management. 11(2) : 70-84. lapolo, n., utina r, baderan dwk. (2018). diversity and density of crabs in degraded mangrove areas at tanjung panjang nature reserve in gorontalo, indonesia. journal of biodiversity. 19(3) : 1154-1159. noor, y.r., khazali m., suryadiputra i.n.n. (2012). guide to introduction to mangroves in indonesia. third print. wetlands international indonesia program. bogor. pamungkas, n a. (2011). development of phytoplankton abundance with liquid organic fertilizer. periodic fisheries. 39(1): 79-90. pasongli, h., dirawan, g d., and suprapta. (2015). zoning of pond suitability for vaname shrimp cultivation development (penaeus vannamei) on water quality aspects in todowongi village, jailolo district, west halmahera regency. bioeducation journal. 3(2): 324-335. international journal of applied biology, 2(2), 2018 12 rahim, s, baderan, dwk, hamidun, s. (2017). the density, composition and mangrove forest of torosiaje jaya village, gorontalo, indonesia. bonorowo wetlands. 7(1): 38-42. barru south sulawesi. thesis (online). department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar. retrieved on 3 january 2018. zaky, a r., suryono, c a., pribadi, r. (2012). study of mangrove land conditions in bedono village, sayung district, demak regency and mangunharjo village, tugu district, semarang city. journal of marine research. 1(2): 88-97. zulkarnaini, z. saam, v. amrivo, and d. miswadi. (2017). community structure and economic evaluation mangrove village in bengkalis district. international journal of oceans and oceanography issn 0973-2667. 11(1): 63-74. retrieved on 3 january 2018. 3(1)30-35 contact : ajeng dyah pythaloka ajengdyahpythaloka@gmail.com © international journal of applied biology 30 abstract implement health efforts both public health efforts are first rate and first rate individual health efforts are needed health center management be integrated and continuous in order to produce the performance of public health center effective and efficient. this study aims to find out about the picture of the level of achievement of performance results pertaining to quality health center health center services, management of health centers and health care public health center madising na mario pare-pare. this research method is quantitative descriptive method, whereby it purports to describe the level of achievement of the performance results of health centers. from the research achievement of quality performance owned health center services obtained an average value of 9,7 performance with good performance categories. public health center management obtained an average performance score of 10 with a good performance categories. health services obtained the value of the average performance of 70,9% with less performance categories. therefore, efforts to improve the performance of health centers needed professional human resources and improvement of health facilities, especially the budget required. issn : 2580-2410 eissn : 2580-2119 performance assessment of public health center madising na mario parepare, south sulawesi, indonesia ajeng dyah pythaloka, muh. yusri abadi & darmawansyah department of administration and health policy, faculty of public health, hasanuddin university, makassar introduction the public health centers is a regional public service agency (rpsa) which functions to provide health services to the public both in large cities and remote areas. in operational activities, public health centers are entities that do not prioritize profits so they must prioritize the principle of budget efficiency and optimal productivity (mahardika and supadmi, 2014). health efforts are every activity to maintain and improve health, aiming to realize optimal health status for the public. public health centers are basic health care facilities that carry out health care efforts, improve health (promotive), prevent disease (preventive), cure (curative), and restore health (rehabilitative), which are carried out in a comprehensive, integrated and sustainable manner. the concept of the unity of health efforts is a guideline and guideline for all health care facilities in indonesia, including public health centers (ministry of health, 2014). there are six main health service programs in the public health centers, namely treatment programs, health promotion, maternal and child health services, prevention and control of infectious and non-public diseases, and environmental health (azwary, 2013). open access international journal of applied biology keyword achievement, performance, services, health center. article history received 15 january 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 31 implementing health efforts both first-level public health efforts and first-degree individual health efforts are needed by the health center management in an integrated and sustainable manner to produce effective and efficient public health centers performance (republic indonesia ministry of health, 2016). realizing the vision, mission and goals of health centers needed a suitable management model and effective management model for health centers which consists of p1 (planning), p2 (mobilization and implementation), and p3 (monitoring, control and assessment) (republic indonesia ministry of health, 2012). performance assessment is a formal system for assessing and evaluating the performance of an individual or group task (saputri, 2015). evaluate the performance of health centers regularly conducted are crucial to ensure quality of service and supporting the implementation of the functions and organization of basic health measures which are the responsibility of the district / city. based on this, the central government compiled guidelines for evaluating the performance of public health centers, which was an attempt to evaluate the evaluation of the work results or achievements of the health centers. the scope of the assessment of the performance of the public health centers includes evaluating the achievement of the results of the implementation of health services, management of the health center, and quality of service (wijayani, et.al., 2017). madising na mario health center as the spearhead of basic health services in its work area makes every effort to realize healthy indonesia by improving service and developing services and establishing harmonious cross-sector relations to improve the health status of the community in its labukkang and mallusetasi villages in accordance with its vision and the mission of the public health centers (madising na mario health center, 2015). the data from the recapitulation of the calculation of the component coverage of the performance of the 2014 madising na mario health center obtained an average score of 82% (madising na mario health center, 2014). to achieve the expected results, there are three components outlined in the health center performance appraisal, namely the quality service component of the health center, the health center management component and the health service component of the public health centers (hestiwati, 2012). the performance evaluation of the public health centers is expected to provide an overview to each person in charge and executor at the public health centers about the level of achievement of the types of activities that are the responsibility and as an evaluation material / evaluation of his work performance materials and methods this type of research is quantitative descriptive. this research was conducted at the madising na mario health center in parepare city, south sulawesi, indonesia. the population in the study was all the results of the performance of the public health centers with samples of all the results of the achievement of the public health centers performance related to the service quality of the public health centers, management of the public health centers and health services of the public health centers. the analysis used is an analysis of the performance of the public health centers in accordance with the public health centers management guidelines according to the regulation of the minister of health of the republic of indonesia number 44 year 2016. data presentation will be developed in the form of tables and narratives. tables are used for the presentation of the results of categorized data analysis and then accompanied by explanations in the form of narratives. international journal of applied biology, 3(1), 2019 32 results and discussion based on the achievement of the quality service performance of the madising na mario public health center in the municipality of parepare, the average performance value of the public health centers service quality was 9.7 in the good category. the number of indicators that have good performance with a score of 10 are 9 indicators including dropping out of antenatal care services delivery by health workers, handling high obstetric / risk complications, acid resistant basil error rate, adherence to antenatal care standards, adherence to standards for examining pulmonary tuberculosis, the level of patient satisfaction with public health centers services, the availability of public health centers service standards, and the health center quality team. the number of indicators that have sufficient performance achievements with a value of 7 is only one indicator, namely the malaria blood check error rate (table 1). table 1: results of achieving quality services at madising na mario health center in parepare city performance program final score performance category antenatal care drop out service 10 good childbirth by health workers 10 good handling high obstetric / risk complications 10 good error rate exam for acid resistant basil 10 good error rate for malaria blood checks 7 fair compliance with the standard antenatal care 10 good compliance checks tuberculosis 10 good satisfaction of health center services 10 good availability of service operational standards 10 good average health centers performance 9.7 good description: performance of puskesmas service quality: good = ≥ 8.5; enough = 5.5 8.4; less = <5.5 the achievement of the management performance of the madising na mario public health center in the municipality of parepare shows that the average value of the health center management performance is 10 in the good category. all types of management have the same achievement value which is equal to 10 with good categories, including operational management of health centers, equipment and drug management, financial management, and staff management (table 2). table 2: results of achieving management performance at madising na mario health center in parepare city management program final score performance category operational management 10 good tools and medicine management 10 good financial management 10 good labour management 10 good average health centers management 10 good description: public health centers management performance: good = ≥ 8.5; enough = 5.5 8.4; less = <5.5 international journal of applied biology, 3(1), 2019 33 based on the achievement of the health service performance of the madising na mario public health center in the municipality of parepare, it was shown that the average value of health center health service performance was 70.9% with less categories. efforts to achieve performance in a good category, namely efforts to prevent and eradicate infectious diseases (96.3%). efforts to achieve poor performance were health promotion efforts (62.7%), environmental health efforts (53.5%), efforts to improve community nutrition (79.7%), and health development efforts (38.1%) (table 3). table 3: results of achievement of health service performance at madising na mario health center, pare-pare city health efforts program final score performance category health promotion efforts 62 less environmental health efforts 53 less maternal and child health efforts including family planning 83 enough efforts to improve community nutrition 80 less efforts to prevent and eradicate infectious diseases 96 good treatment efforts 82 enough health development efforts 38 less average public health centers efforts 71 less description: efforts of health center service coverage: good = ≥ 91%; enough = 81 90%; less = <80%; the achievement of the quality coverage of health services on average has had a level of achievement with a good category, but from 10 there is one sub-variable / indicator that is still in sufficient category with a value of 7, namely an error rate of malaria blood checks. malaria blood tests are categorized as sufficient because of a lack of budget, this examination is carried out in a large enough scope by the public health centers so that the implementation of the achievement of malaria blood checks is not optimal. wildan, et.al (2016) stating that the structure is the infrastructure, equipment, tools, organization and management, finance, human resources and other funding sources. in other words, the structure is the input of health facilities. if the structure is good, the possibility of quality is also good. whether or not the structure as input to health facilities can be measured by the reasonableness, quantity, cost, quality of the structural components. the management of the public health center obtained by the public health centers is in a good category by optimizing the analysis of the problem, formulating the problem, the objectives of the activities, targets, targets and indicators of the success of the activities optimally. mairizon and kiswanto stated that it had not been done well that monthly miniworkshops and quarterly mini workshops were indicators of the weak implementation of the management functions of the public health centers in the implementation (mairizon and kiswanto, 2013). this causes unclear direction of the goals to be achieved and the division of tasks in achieving these conditions. the research conducted by anton ferri stated that the use of improper funds would result in errors in the preparation and distribution of services for carrying out activities, and would subsequently give a false picture in the financial statements (ferri, 2009). as we know the number of operational funds is closely related to the number of products produced, thus an public health centers institution engaged in health international journal of applied biology, 3(1), 2019 34 services, activity cost reports are used to determine the extent of the role of funds with the level of results of activities that have been carried out. the results of the measurement of health service performance of public health centers which have a performance average value with less categories. the results of observations and research conducted by steven konli at the gunawan village health center show that health services to the community in the area have not been fully implemented (konli, 2014). this is because the service personnel come in less on time, employee discipline is still lacking, the number of health workers is not in accordance with the number of patients who come, and the facilities and infrastructure are lacking so that these conditions make people less satisfied with the services provided. pohan stated that health services that were not in line with patient expectations were expected to be input for health service organizations to try to fulfill them (pohan, 2007). if the performance of health services obtained by patients at a health service facility is in line with their expectations, patients will always come to the health care facility. patients will always seek health services at facilities whose health service performance can meet patient expectations. conclusions the conclusion of this study is that the quality of public health centers services has a good performance, health center management has good performance, and health center health services have less performance. suggestions to the health center and related parties so that it is necessary to prioritize indicators that have not yet been achieved properly in accordance with the target so that future coverage will be better so that it will ensure improved public health and better quality of public health centers services, and maintain public health centers management whose targets have been well achieved. references azwary, b. 2013. the role of medics in improving public health services in kasai village health center, pulau derawan district, berau district. e-journal of government science vol. 1: 385-99 ferri, a. 2009. evaluation of implementation of integrated public health centers record and reporting system in karimun district. yogyakarta: gajah mada university. hestiwati. 2012. analysis of performance evaluation of public health centers in palu city, central sulawesi province. makassar, hasanuddin university. konli, s. 2014. public health services in gunawan village health center, sesayap district, tana tidung regency. e-journal of government science, xi (2). 1925-1936 madising na mario health center. 2014. madising na mario health center performance report”, parepare city, madising na mario health center. madising na mario health center. 2015. profile of madising na mario health center in parepare city in 2015”, parepare city, madising na mario health center. mahardika, k. and supadmi, n.l. 2014. comparative analysis of south denpasar and east denpasar public health centers performance using the balaced scorecard method. ejournal of accounting, vol viii (1): 1-13 mairizon and kiswanto. 2013. implementation of public management functions. journal of public policy, vol. 4. 119-218 international journal of applied biology, 3(1), 2019 35 ministry of health. 2014. republic of indonesia minister of health regulation number 30 year 2014 concerning standard pharmaceutical services at public health centers. jakarta, ministry of health. pohan, i.s. 2007. quality assurance of health services. jakarta: medical book egc republic indonesia ministry of health. 2012. ministry of health performance accountability report 2012. jakarta, ministry of health of the republic of indonesia. republic indonesia ministry of health. 2016. minister of health regulation of the republic of indonesia number 44 year 2016: concerning management guidelines for public health centers. jakarta, ministry of health. saputri, i. 2015. measurement of internal and external performance at manukan kulon health center surabaya. journal of accounting science and research, vol. ii (2): 14-18 wijayani, t.r.a., utami, s., and witcahyo, e. 2017. relationship between organizational culture profiles (using the approach of the competing values framework) with community health organization organizations in jember regency in 2012. repository. wildan, m., yuswadi, h., wahono, p., and puspitaningtyas, z. 2016. measuring quality of public service performance (national health insurance program) with patient satisfaction survey. repository contact : hind zrikem dr.hind.zrikem@gmail.com © international journal of applied biology 21 abstract human diseases due to a. paraphrophilus aren’t usual. the following case report describes the first isolated case of a. paraphrophilus brain abscess in our laboratory. a 9-year-old boy presented to pediatric emergencies for frontal headache, vomiting, blurred vision and left hemiparesis. radiological diagnosis consists with a frontal abscess. gram staining of purulent samples showed abundant neutrophils with gram negative bacilli. culture was made on blood agar, chocolate agar, chapman’s agar and incubated in 5% co2 at 37°c for 24 hours. positive culture was detected only on chocolate agar. it was monomicrobial with small yellowish non-hemolytic colonies. gram stain of colonies showed pleomorphic gram-negative coccobacilli. the strain required v factor for growth. the isolated strain was sensitive to all antibiotics tested. the interest of this case is that it shows the emergence of a. paraphrophilus as a causative agent of brain abscesses on pediatric population without associated congenital heart disease. it may also help identifying risk factors of these infections and how to prevent them. issn : 2580-2410 eissn : 2580-2119 haemophilus paraphrophilus brain abscess in a 9-year-old boy: case report and literature review hind zrikem, loubna ait said, & kawtar zahlane microbiology department, university hospital mohammed vi, marrakech, morocco introduction h. aphrophilus and h. paraphrophilus are now combined as the same species: aggregatibacter aphrophilus, with the v-factor dependence of h. paraphrophilus being considered as a variable phenotypic characteristic [1]. a. aphrophilus belongs to the hacek group, fastidious gram-negative bacilli that are found in the human upper respiratory and genitourinary tracts [1]. a. aphrophilus may cause brain abscess and infective endocarditis and has been isolated from various other body sites including peritoneum, pleura, wound and bone [2]. case report a 9-year-old boy was admitted to pediatric emergencies for frontal headache not relieved with painkillers, vomiting and blurred vision. he had no obvious associated conditions except a sinusitis that took place two weeks ago. on admission, he was afebrile with a stable hemodynamic state. neurological examination showed a glasgow coma scale open access international journal of applied biology keyword child, aggregatibacter paraphrophilus, sinusitis, brain abscess. article history received 25 january 2020 accepted 14 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 22 score of 13, a supple neck and left hemiparesis. the remaining physical examination was normal. ct with contrast demonstrates a ring-enhancing lesion along with midline shift to the left. drainage of the abscess was achieved and empiric parenteral antibiotic therapy was initiated with metronidazole (10mg/kg/8h), and ceftriaxone (100mg/kg/j). other investigations were carried out: ultrasound of liver, chest x-ray and dental examination. these were all negative. blood tests showed leukocytes 14 x 103/ ul with 9,8 x 103/ul neutrophils, hemoglobin 12g/dl and c-reactive protein 2,8 mg/l. gram staining of purulent samples showed abundant neutrophils with gram negative bacilli. a small volume of pus was inoculated onto blood agar, chocolate agar, chapman’s agar and incubated in 5% co2 at 37°c for 24 hours. positive culture was detected only on chocolate agar. it was monomicrobial with small yellowish non-hemolytic colonies (figure 1). gram stain of colonies showed pleomorphic gram-negative coccobacilli. the strain required v, but not x factor for growth, it was oxidase positive and catalase negative. late identification by api nh system revealed haemophilus paraphrophilus (figure 3). antimicrobial susceptibility was detected by the disk diffusion test using an inoculum of 0, 5 mcfarland on m-h base agar supplemented by 5% horse blood and 20mg/l β-nad. the isolates were susceptible to all antibiotics tested according to eucast breakpo int standards (figure 2). normalization of wbc counts was obtained by the 10th postoperative day. after six weeks of intravenous antibiotic therapy, ct scan control showed diminished lesion and neurological manifestations disappeared. figure 1: culture on chocolate agar international journal of applied biology, 4(1), 2020 23 figure 2: antimicrobial susceptibility figure 3: api nh identification of h. paraphrophilus discussion brain abscess is a rare disease in childhood requiring prompt medical and/or surgical treatment [3]. to our knowledge this is the third case report of a. paraphrophilus brain abscess in a child without underlying heart disease. bacteria reach the brain through contiguous spread, hematogenous dissemination from a distant focus or through a head trauma (penetrating injury, post-neurosurgery) [4]. congenital heart disease is the most common underlying condition, and the most common preceding infection is sinusitis [3]. we assume that in the case of our patient, the brain abscess was due to a contiguous spread from the sinusitis that took place 2 weeks earlier. the most frequently isolated microorganisms from brain abscesses in pediatric population are viridans streptococci, staphylococcus aureus and enterobacteriaceae [5]. a. paraphrophilus has emerged as an important cause of brain abscesses [6]. a. paraphrophilus is a small, pleomorphic gram-negative coccobacilli, it’s slow growing requires enriched culture media and increased carbon dioxide tension [7]. non motile, facultatively anaerobic with no dependence to x factor but v factor is required for growth [2]. colonies on chocolate agar are granular, yellowish and opaque [2]. glucose is fermented, onpg is hydrolyzed and oxidase is positive [2]. phenotypic characteristics differentiating a. paraphrophilus from other hacek group species are shown in table 1. fortunately, a. paraphrophilus is sensitive to a wide range of antibiotics [8]. production of international journal of applied biology, 4(1), 2020 24 beta-lactamase should be tested, while third-generation cephalosporins should be considered drug of choice [9]. ciprofloxacin and the newer fluoroquinolones have potent activity against a. paraphrophilus, and can be used as alternatives for penicillin allergic individuals, and for those infected with strains resistant to cephalosporins [10]. our isolated strain was susceptible to all antibiotics tested according to the eucast breakpoint standards [11]. table 1. phenotypic characteristics differentiating a. paraphrophilus from other hacek group species [2][6] x v co2 cat ox nit odc ldc onpg ind glu lac sac a. aphrophilus + + + + + + + + a.paraphrophilus + + + + + + a. actinomycetemcomitans + + + + eikenella corrodens + + + + + cardiobacterium hominis + + + + + kingella kingae + + + haemophilus influenza + + + + + v v v + haemophilus parainfluenzae + v + + v v v + + x: v-factor-dependence; v: v-factor-dependence; co2: capnophile; cat: catalase; ox: oxidase; nit: nitrate; odc: ornithine decarboxylase; ldc: lysine decarboxylase; ind: indole; glu: glucose; lac: lactose; sac: saccharose; v: variable the literature review since 1984 showed only 10 previously reported cases of a. paraphrophilus brain abscess (table 2). two of the 10 cases are children (20%) and six are males (60%). 30% have underlying congenital heart disease while no predisposing factor was identified in two cases. therapy is based on antibiotics combined with surgical drainage in 80% of cases. the mortality rate is 20%. table 2. reported cases of a. paraphrophilus brain abscesses case no. date [r] age (y) / gender predisposing factors therapy outcome 1. 1984 [12] 49 / f none antibiotics (cefotaxime, metronidazole) death 2. 1985 [13] 26 / f congenital heart disease antibiotics (ampicillin, gentamicin) death 3. 1987 [14] 47 / m poor dentition surgical + antibiotics (ampicillin, metronidazole) recovery 4. 1988 [15] 51 / m dental manipulation surgical + antibiotics (ampicillin, ceftriaxone) recovery 5. 1990 [16] 43 / m congenital heart disease tooth extraction surgical + antibiotics (ciprofloxacin, metronidazole) recovery 6. 1996 [17] 66 / f toothache surgical + antibiotics (penicillin g, metronidazole) recovery 7. 1998 [18] 42 / m none surgical + antibiotics (cefotaxime, ciprofloxacin) recovery 8. 2008 [8] 3 / m otitis media surgical + antibiotics (3rd generation cephalosporins, metronidazole) recovery international journal of applied biology, 4(1), 2020 25 9. 2010 [19] 53 / m congenital heart disease dental filling ingestion surgical + antibiotics (meropenem, metronidazole) recovery 10. 2013 [20] 6 / f dental extraction surgical + antibiotics (ceftriaxone, metronidazole) recovery 11. 2019 (present case) 9 / m sinusitis surgical + antibiotics (ceftriaxone, metronidazole) recovery r = reference; m= male; f= female conclusions a. paraphrophilus is emerging as a causative agent of brain abscesses in pediatric population. prevention is therefore crucial and involves good dental hygiene and the treatment of any ent infection. references [1] w. m. janda. 2013. update on the hacek group of fastidious gram-negative bacilli, part i. clin. microbiol. newsl. 35(11): 87–92. [2] n. nørskov-lauritsen and m. kilian. 2006. reclassification of actinobacillus actinomycetemcomitans, haemophilus aphrophilus, haemophilus paraphrophilus and haemophilus segnis as aggregatibacter actinomycetemcomitans gen. nov., comb. nov., aggregatibacter aphrophilus comb. nov. and aggregatibacter. int. j. syst. evol. microbiol. 56(9): 2135–2146. [3] z. sahbudak bal et al. 2018. brain abscess in children: a rare but serious infection. clin. pediatr. (phila). 57(5): 574–579. [4] i. brook. 2017. microbiology and treatment of brain abscess. j. clin. neurosci. 38: 8– 12. [5] m. c. brouwer et al. 2014. clinical characteristics and outcome of brain abscess : systematic review and meta-analysis. neurology. 82(9): 806–813. [6] n. nørskov-lauritsen. 2014. classification, identification, and clinical significance of haemophilus and aggregatibacter species with host specificity for humans. clin. microbiol. 27(2): 214–240. [7] f. j. cooke and m. p. e. slack. 2016. gram-negative coccobacilli, fourth edi. elsevier ltd. [8] j. hoefele et al. 2008. haemophilus paraphrophilus, a rare cause of intracerebral abscess in children. eur. j. pediatr. 167(6): 629–632. [9] a. wanger et al. 2017. overview of bacteria. [10] s. maraki et al. 2016. aggregatibacter aphrophilus brain abscess secondary to primary tooth extraction: case report and literature review. j. microbiol. immunol. infect. 49(1): 119–122. [11] v. janvier et al. 2019. comité de l’antibiogramme de la société française de microbiologie recommandations. france. international journal of applied biology, 4(1), 2020 26 [12] m. habib et al. 1984. metastatic cerebral abscesses due to hemophilus paraphrophilus. arch. neurol. 41(12): 1290–1291. [13] k. t. jensen and t. højbjerg. 1985. meningitis and brain abscess due to haemophilus paraphrophilus. eur. j. clin. microbiol. 4(4): 419–421. [14] c. j. papasian et al. haemophilus paraphrophilus brain abscess. 1987. diagn. microbiol. infect. dis. 7(3): 205–209. [15] a. j. pajeau et al. 1988. haemophilus paraphrophilus frontal lobe abscess: case report. neurosurgery. 23(5): 643–645. [16] j. p. visvanathan k. 1991. ciprofloxacin treatment of haemophilus paraphrophilus brain abscess. j infect. 22(3): 306–307. [17] a. j. h. simpson et al. 1996. polymicrobial brain abscess involving haemophilus paraphrophilus and actinomyces odontolyticus. postgrad. med. j. 72(847) : 297–298. [18] a. j. hart et al. 1998. haemophilus paraphrophilus; a rare cause of intracranial abscess. j. infect. 37(1) : 75–76. [19] a. s. et al. 2010. liver and brain abscess caused by aggregatibacter paraphrophilus in association with a large patent foramen ovale: a case report. j. med. case rep. 4(1) : 2–5. [20] d. a. d.-s. dra. maría eugenia vázquez-toledo, dr. rené farfán-quiroz. 2013. absceso cerebral por haemophilus paraphrophilus posterior a tratamiento dental en una niña. informe de un caso. acta pediatr mex. 34(1) : 33–38. 3(2)15-23 contact : ulfah nur amaliah ulfahamaliah3@gmail.com © international journal of applied biology 15 abstract the use of medicine as oral hypoglycemic and insulin have some limitation, the treatment for diabetics needs more expensive cost relatively because it uses in the long term and can give the unwanted side effect. at the recent time, one of the conducted efforts is by using the plant as alternative medicine. this research aims to know the potential of leaves’ extract and to know the effectiveness of siam weed leaves extract’s dosage which most effectivein lowering the level of blood glucose in mice which is induced by alloxan. on this research, anti-diabetic effect of siam weed leaves was observed to male mice which had been induced by alloxan until the level of blood glucose >200 mg/dl. the given treatment orally was conducted during seven days with three variations of dosage concentration of infused extract, they are 5%, 10% and 20 %. furthermore, it was also used the comparison i.e. infused extract of cinnamon bark with 10 % concentration. data analyse used spss 23.0 program with one way anova then was continued by using duncan test. the research result shows that the siam weed leaves extract with dosage concentration 5%, 10% and 20% can decrease blood glucose level on mice that have suffered hyperglycemia and most effective on 20%. thus, it can be concluded that siam weed leaves extract have the potential in lowering blood glucose. issn : 2580-2410 eissn : 2580-2119 the use extract of siam leaf eupatorium odoratum l. as alternative material in lowering blood glucose ulfah nur amaliah, eva johannes, munif s. hassan & elis tambaru departement of biology, faculty mathematic and natural science, hasanuddin university, makassar introduction hyperglycemia is the condition where the blood glucose level exceeds normal limits. the increasing of glucose level in blood plasma (hyperglycemia) is one of bases in diagnosis of diabetes mellitus. it is caused by the abnormality of metabolism, specifically the abnormality on carbohydrate metabolism (wulandari, 2010). the treatment on people with diabetes mellitus needs relative more expensive cost because it uses in long term and can give unwanted side effect. furthermore, the medicine likes as oral hypoglycemic and insulin have some limitation. oral hypoglycaemic medicine in sulfonylurea group can cause gastrointestinal disorder such as qualm, diarrhea, stomach-ache, and hyper secretion of gastric acid (jung et al. 2006 in sakika et al. 2015; dalimartha and adrian, 2012 in pasaribu et al. 2012). therefore, it needs the effective medicine, the relative low side effect, the relative open access international journal of applied biology keyword anti-diabetic, infused extract, eupatorium odoratum blood glucose article history received 07 january 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 16 cheap and affordable cost. one of conducted efforts is by using plant as alternative medication. it encourages the amount of research about the plants which have the potential to decrease blood glucose level, one of the plant which has the potential as the lowering of blood glucose is siam weed eupatorium odoratum l. siam weed eupatorium odoratum l. is one of the most abundant weed plants. weed is one of plant pest organism which inhibit the growth, development, and productivity of cultivated plants. the existence of weeds at agriculture area generally gives the negative effect toward the plants, because weeds have the high competitive capacity thus enabling light, co2, water, nutrient, and space to grow competition which were used simultaneously (riry, 2008 in palijama et al. 2012), because of its abundant availability, then it is very potential to be utilized. siam weed includes to asteraceae family, traditionally is used to healing the wounds and infection, headache, diarrhea, astringent, antispasmodic, antihypertensive, antiinflammation and diuretic (vital and rivera, 2009 in yenti et al. 2014). siam weed leaves contain some main compounds such as tannin, flavonoid, saponin, and steroid. essentials oil of siam weed leaves has a substance of 𝜶-pinene, cadinene, camphora, limonene, βcaryophylleneand candinol isomer (benjamin, 1987 in yenti et al. 2014). the research of alisi, et al, (2011), in marianne et al, (2014), explained that siam weed has the potential as antioxidant and able to prevent free radicals which is believed as the cause of various degenerative disease and premature aging. and also there was a research which claimed that these siam weed leaves’ extract able to be as anti-diabetic (murthy and kiran, 2012; raman et al. 2012; marianne et al. 2014). based on that, then need to be conducted the research about the potential of siam weed leaves’ extract to know the bioactive compounds which contained in that plant, thus made as one of alternative in lowering the level of blood glucose. materials and methods tools the tools were used knife/hook, animal raising cage, gloves, drinking water place, analytical balance, blender, jar, mess, oral sonde, mortar and pestle, syringe, syringe 1 cc, stirring rod, spatula, wiper, digital scales, hot plate, stirrer, infused pen, buchner funnel, sprayers, thin layer chromatography plate, glucometer (nesco®), measuring cup, surgical scissor, measuring flask, vial bottle, alcohol thermometer 10-110 ºc, and glass tools (phyrex). materials the materials which were used are siam weed leaves eupatorium odoratuml. which were obtained from kassiloe village, labakkang district, pangkep regency. the material were used aquades, dragendorf reagent, chloride acid (hcl), carboximetilcellulosa na (na-cmc), standart mice’ food, glibenclamide5 mg, alloxan monohydrate, cinnamon bark cinnamomunburmanii, glucotest stripe, aluminum foil, picric acid, api (aqua pro injection), filter paper, cling wrap, and label. the used test animals are male mice balb c around 18 mice with the age 3-4 months, the average body weight 20-35 gr, healthy and behave normally, which were obtained from bio pharmacy laboratory of pharmacy faculty, hasanuddin university. before used, the mice were acclimatized during 7 days with laboratory condition. international journal of applied biology, 3(1), 2019 17 ethical clearance this research had been evaluated and gained the ethical approval recommendation of medical research ethical committee, medical faculty, hasanuddin university, no.318/h4.8.4.5.31/pp36-kometik/2018, 23th april 2018. research framework used complete randomized design with the used treatment group namely: k1 : negative control (na-cmc 1%) k2 : positive control (glibenclamide) 0,083 mg/kgbb k3 : comparison group (cinnamon infused extract 10%) k4 : treatment i group (siam weed leaves infused extract 5%) k5 : treatment ii group (siam weed leaves infused extract 10%) k6 : treatment iii group (siam weed leaves infused extract 20%) making infused extract of siam weed leaves siam weed leaves were washed and air dried did not under direct sunlight during 5 days until the leaves become brown. furthermore, the extraction done by using infused method. the process of making extraction that is the sample mashed into powder. after that, it was sifted by using mess in order to gain the powder in the certain subtle degree. after that, it put into the top infused pan then was poured with water as much 100 ml, the bottom of pan was filled with water until full then was heated until the water in the pan boiled. furthermore, the temperature of extract in the pan was measured by using alcohol thermometer until its temperature gained 90ºc. after gained 90ºc temperature, waited during 15 minutes, while occasionally steering. the result of infusion was filter while still hot by using buchner funnel which coated with filter paper until gained the filtrate. the filtrate was the extract of siam weed leaves. the making of infusion’s result with concentration 10% then the powder of leaves were weighed was 10 grams then were added water with volume 100 ml., the it will be gained the concentration 10% (kusumaningrumet al. 2013). to make the infusion result with concentration 5% and 20% were made with similar way, by using 5 grams and 20 grams powder of siam weed leaves (radiansah, 2013). phytochemical test of infused extract of siam weed leaves after gained the infused extract of siam weed leaves, then conducted phytochemical analysis this phytochemical analysis was conducted in order the contained compounds of infused extract in siam weed leaves can be known. phytochemicaltest was based on harborne (1996) in halim et al. (2015), to know the existence of active compounds such as alkaloid, flavonoid, saponin, and tannin. this phytochemical test was conducted by using tube test and thin layer chromatography plate test which was given reagent appropriate with the wanted compounds to be gained then was seen the occurrence of color change. treatment toward animal test by using alloxan induced method the animals which were used as much 18 male mice in where divided into six groups. before conducting the test of anti-diabetic effect, the tested animals were fasted during 8 hours. after fasted, the level of blood glucose of all mice was observed to determine blood glucose level of early fasting (gdp0). after that, induced by alloxan in dosage 3,5 mg/kgbbsubcutaneously. then let it during 3 x 24 hours until occurring the increasing effect of stable blood glucose’ level (marianne et al. 2014). furthermore, was conducted the measurement of fasting blood glucose’ level (gdp1), the mice which had been experienced international journal of applied biology, 3(1), 2019 18 the increasing of blood glucose’ level (>200 mg/dl) were clarified hyperglycemic, in this case, it was ready to be given the treatment. the treatment was given by giving test supply (the infused extract of siam weed leaves) orally (p.o) once per day during 7 days, with the dosage concentration of extract respectively 5%, 10% and 20%. the level of blood glucose (mg/dl) were measured at the early (before treatment), after inducing alloxan (post-alloxan), and the fasting blood glucose’ level after 7 days treatment (gdp2) on each group (marianne et al. 2014). the parameter are the occurrence of decreasing blood glucose’ level after the animals became diabetics, compared to the negative control (na-cmc 1%), positive control (glibenclamide) and comparison (infused extract of cinnamon bark). each of test group used 3 mice. examining the level of blood glucose the level of blood glucose (mg/dl) was determined by using glucometer (nesco® multicheck 1). glucometer was activated, and then was put chip and strip on the tool. the blood of mice were taken from the part of their tails then were dropped on the tip of strip and automatically the level of blood glucose will appear on the monitor screen. data analysis data analysis was processed statistically by using software test spss 23.0 for windows. the measurement result of all parameters in this research were analyzed statistically by using one way anova, was conducted the continued test by using duncan test results and discussion phytochemicaltest of infused extract on siam weed leaves the extract was obtained through infusion method by using water solvent (aquades). the process of extract making observed excessively in temperature and making time to minimalize the damage on the compound which contained in siam weed leaves, specifically on the heating process. phytochemical analysis was conducted qualitatively to know the compound that has the role as anti-diabetic. the result of photochemistry analysis of infused extract can be seen as the following: table 3. result of extract photochemistry analysis note: (+) = contained testing compound (–) = did not contain testing compound numb. the sort of testing reagent result note 1. flavonoid sitroborat form the green colour + 2. saponin the heat water + hcl form the stable foamas high as 1-3 cm + 3. alkaloid dragendorff form the red-brown colour + 4. tanin fecl3 form the blue colour + international journal of applied biology, 3(1), 2019 19 according to prameswari et al. (2014), from the fourth compound, alkaloid and flavonoid are the natural active compound which known have hypoglycemic activity. flavonoid is one of secondary metabolic compoundwhich can be found on the plants tissue, can has the rule as anti-oxidative compound by giving one of its hydrogen atom (redha, 2010). tannin is the component of very complex organics substance, consisted of phenolic compound which hard to be separated and is the secondary metabolite active compound which has some benefits such as astringent, antidiarrheal, antibacterial, and biological antioxidant (malangngi et al. 2012). the function of tannin as astringent can pucker up the epithelium of small intestine’s wall thus decreases the absorption of food essence and inhibits the intake of glucose in blood (prameswari et al. 2014). saponin (triterpenes) based on kirwanto (2014), it works in similar activity with insulin reaction, thus can take blood glucose into cells. moreover, according to prameswari et al. (2014), alkaloids compound works by stimulating hypothalamus to increase the secretion growth hormone releasing hormone (ghrh), thus the secretion of growth hormone (gh) on hypophise increased. the high level of gh can stimulate the liver to secrete insulin-like growth factor-1 (igf-1). igf-1 has the effect in inducing hypoglycemia and decreasing gluconeogenesis thus the level of blood glucose and the need of insulin lowered. the level of blood glucose after treatment table 4. the influence of giving infused extract from siam weed leaves toward lowering the level of blood glucose on male mice numb. treatment n the average level of blood glucose from tested animals (mg/dl) gdp1-gdp2 (mg/dl) gdp0 gdp1 gdp2 1. k1 3 74,6 254 238,6 68 2. k2 3 83,3 213,6 79 134,6 3. k3 3 76,3 426 137 289 4. k4 3 96 486,3 296 190,3 5. k5 3 72,6 222,6 132,3 90,3 6. k6 3 67,6 382,3 137,6 244,6 (a) (b) (c) (d) figure 1. result of extract phytochemical analysis note: (a): test result of alkaloid, (b): test result of flavonoid, (c): test result of tannin, (d): test result of saponin international journal of applied biology, 3(1), 2019 20 0 100 200 300 400 500 600 k1 k2 k3 k4 k5 k6 le ve l o f b lo od g lu co se ( m g/ dl ) treatment group average level of blood glucose gdp0 gdp1 gdp2 note: k1 : negative control (na-cmc 1%) k2 : positive control (glibenclamyde) 0,083 mg/kgbb k3 : comparison group (cinnamon infused extract 10%) k4 : treatment i group (siam weed leaves infused extract 5%) k5 : treatment ii group (siam weed leaves infused extract 10%) k6 : treatment iii group (siam weed leaves infused extract 20%) gdp0 : fasting blood glucose before treatment (mg/dl) gdp1 : the early fasting blood glucose after inducing alloxan (mg/dl) gdp2 : fasting blood glucose after 7 days treatment (mg/dl) gdp1 – gdp2 : the lowering of blood glucose (mg/dl) the result of measuring blood glucose’ level of mice before and after treatment (table 4), can be drawn into histogram which served the average level of blood glucose of mice, before treatment, after inducing alloxan and also after 7 days of given treatment, as following: figure 2. histogram of average level of mice blood glucose the above histogram shows the existence of different in average level of mice blood glucose on the three times of measurement. gdp0 shows the measurement of early fasting blood glucose’ level that is before the treatment, which shows that the average level of fasting blood glucose has enormity which almost similar on each group that still stands at the normal level (<100 mg/dl). the result of measuring gdp1 shows the average level of mice blood glucose begin to increase on each group (>200 mg/dl),which shows the occurrence of hyperglycemic after inducing monohydrate alloxan as diabetogenic agent. the alloxan selectively can damage β cells from langerhans island with secreted insulin hormone. the work mechanism of alloxan is by putting ca ion into mitochondria cell which cause the process of cell oxidation disturbed. damaging β cells of pancreas causes the lowering of insulin secretion (panjuantiningrum, 2009). furthermore, on measurement gdp2 that is after treatment, seen that there are various measurement on each group. the average level of blood glucose on k1 (negative control) still be categorized hyperglycemia. whereas, on k2 (positive control), k3 international journal of applied biology, 3(1), 2019 21 0 50 100 150 200 250 300 350 k1 k2 k3 k4 k5 k6 l ev el o f b lo od g lu co se (m g/ dl ) treatment group decreasing average level of mice glucose gdp1-gdp2 (comparison), the treatment on dosage i, ii and iii of blood glucose’ level had been decreased. the average deviation between gdp1 and gdp2 can be seen on the following histogram : figure 3. histogram of decreasing average level of mice glucose (gdp1-gdp2) based on above histogram, can be seen the enormity of blood glucose decreasing level on each treatment group. on k1 (negative control) can be known that the occurrence of the lowest blood glucose decreasing level compared to all treatment groups. whereas on the given treatment of glibenclamide (k2: positive control), cinnamon extract (k3: comparison), dosage of extract i (k4), dosage ii (k5), and dosage of extract iii (k6) occurred the large enough decreasing of blood glucose level. this research result shows that by giving infused extract of siam weed leaves can decrease the best blood glucose’ level on the concentration 20%. this case can occur because there is antioxidant compound which is had by siam weed leaves. based on the research which conducted by marianne et al. (2014), that siam weed leaves have antioxidant and cytoprotection activity. antioxidant activity can ward off free radicals which occur because of giving alloxan, thus the damage of β cells pancreas can be prevented and can repair the function of β cells pancreas so after 7 days of extract giving occurred the decreasing of blood glucose’ level on each tested animal. in accordance with the research which conducted by vijayaraghavan et al. (2013), said that after doing phytochemicals analysis of this siam weed leaves was known that this plant contains various antioxidant compound such as tannin, saponin, flavonoid, beta cyanins, quinones, glycosides, cardioglycosides, terpenoid, phenol, cumarin, steroid, and alkaloid. on this research also was done thephytochemicals test qualitatively and was obtained 4 compounds such as flavonoid, alkaloid, saponin, and tannin. according to marianne et al. (2014), flavonoid is known as inhibition of hydroxy and superhydroxy radicals which can protect the membrane lipid of β cells pancreas toward damaging reaction. according to widowati (2008), there are some plant mechanisms in lowering blood glucose’ level that is the plant has ability as astringent which can precipitate the protein of intestinal mucous membrane and form a layer which protects the intestine, thus can inhibit the glucose’ supply and increasing rate of glucose in blood is not too high. moreover, there is also plant which decreased blood glucose’ level by accelerating the secretion of glucose from circulation and by accelerating blood circulation that is through the work of heart and excretion of kidney, thus the secretion rate of urine increased. and there is also plants which accelerate the secretion of glucose through increasing of metabolism or entering into fat deposit. this international journal of applied biology, 3(1), 2019 22 process involves pancreas to produce insulin. according to subroto (2006), there are 5 activities of plant in healing diabetes. there is a plant with activity to inhibit alpha-glycosidase (inhibitor), the plant with activity to stimulate insulin secretion, there is also a plant with activity to repair the function of insulin, there is also plant which has double activities such as to inhibit alpha-glycosidase and to repair the function of insulin, and also a plant with complex activities such as to decelerate food digestion, to decrease the absorption rate of carbohydrate into the liver circulation, to influence the transport of glucose which is mediated by nitric oxide (no) and to modulate the insulin secretion which mediated by no. decreasing of blood glucose occurs because of there is antioxidant compound activity which contained in siam weed leaves such as flavonoid, saponin, tannin, and alkaloid. from the normality test is obtained the significance shapiro wilk as much 0,536. this result >0,05, then data distributed normal. moreover, on the homogeneity test used levene test, the data observed was not homogeny, then was transformed thus was obtained significant score as much 0,082 where higher than >0,05 that showed there was homogeny variation, therefore the assumption of anova test’s requirement had been fulfilled. the result anova test gdp1-gdp2 (appendix 5), can be known that the result of anova test was gained p-value > 0,05 is 0,318 then there is no real influence between treatment because pvalue >0,05, thus it cannot be continued with post hoc test ( duncan = dmrt). conclusions from the result of research which have been conducted can be concluded that the infused extract of siam weed leaves eupatorium odoratum l. have potential in lowering the level of blood glucose on male mice mus musculus l. with the most effective dosage of concentration on concentration 20%. references halim m. o., s. w. paini, and t. w. b. dwi. 2015. the powder proportion influence of beluntas leaves (pluchea indica 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surakarta. subroto, m. a. 2006. herbal concoctions for diabetes melitus. jakarta. penebar swadaya. vijayaraghavan, kavitha, s. a. mohamed and r. maruthi. 2013. studies on phytochemical screening and antioxidant activity of chromolaena odorata and annona squamosa. international journal of innovative research in science, engineering and technology. 2(12) : 7315 – 7321. widowati, w. 2008. the potential of antioxidant as antidiabetic. jkm. 7(2) : 1 – 10. wulandari, c. e. 2010. the influence of giving extract red onion (allium ascalonicum) toward decreasing blood glucose level on wistar mice with hypergyclemia. medical faculty. universitas diponegoro. semarang. yenti r., a. ria, and e. p. agustina. 2014. cream formulation from ethanol extract of siam weed leaves (eupatorium odoratum l.) as anti inflamation. journal of scientia. 4(1) : 7 – 11. contact : mahamad sayab miya sachintimilsina66@gmail.com 130 abstract indigenous communities and tribes of nepal have been using medicinal plants for the treatment of several diseases or ailments since their origin. various studies were conducted in the terai region of nepal regarding the pharmacological, ethnobotanical, and medicinal importance of plant species. this study aims to compile information on ethnomedicinal uses of plants by major ethnic groups in the terai districts of nepal. a total of 35 published documents based on ethnomedicinal uses of several medicinal plants in terai of nepal till 2020 a.d. were accessed for the study from online portals like research gate, scopus, and google scholar. a total of 300 plant species from 98 families were documented which are used for the treatment of 120 diseases or ailments by the 8 ethnic groups of 6 terai districts. leaf of plants is found to be used for the treatment of maximum number of diseases (72). plants from families like fabaceae, malvaceae, poaceae, etc. were used for the treatement of diseases like diabetes, jaundice, syphilis, gastritis and other wounds, fractures along mental disorders. indigenous knowledge and ethnomedicinal importance of different plant species must be explored, documented, and passed through different generations which could widen the scope of modern herbal medicine science. issn : 2580-2410 eissn : 2580-2119 ethnomedicinal uses of plants by major ethnic tribes in terai districts of nepal mahamad sayab miya, sachin timilsina 1*, asmit neupane 1 1 tribhuvan university, institute of forestry, pokhara campus, pokhara, 33700, nepal introduction the globally, the use of chemicals for the control of pests has continued to increase (sharma et al., 2019; al-kawaz, 2019). these chemicals are also utilized in agriculture due to losses of around 30% initiated by insect attacks and other pest organisms (das, 2013; kumar et al., 2016). 2, 2-dichlorovinyl dimethyl phosphate (ddvp) is a commonly used organophosphate pesticide (op) in controlling insect pests in residential and agricultural areas; however, its toxicity has gone beyond the target organism, and this chemical is frequently detected in water bodies (ezike, 2017). the use of plants and their products as a major source of medicine started from the beginning of human civilization and is practicing in recent days too (bhattarai et al., 2006; ekor, 2014). although plants are used as medicine from the beginning of human civilization, the earliest evidence is found in sanskrit texts viz. rigveda was written between 4500 and 1600 bc (adhikari et al., 2003). the term “ethnobotany” was first given by john harshberger in 1896 which deals with the study of the relationship between indigenous society and their open access international journal of applied biology keyword diseases; indigenous groups; medicinal plants; treatment article history received 05 october 2021 accepted 30 december 2021 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 5(2), 2021 131 plant environment (schults, 1962). ethno-medicine deals with the traditional medicine used by different aboriginal people living in a society (quinlan, 2011). the world health organization (who) stated that about 25% of modern medicines are manufactured from traditionally used plant parts; and these plants lead to the discovery of 75% herbal drugs (mian-ying et al., 2002). approximately over 21000 plant species are used for medicinal purposes throughout the world (who, 2002). more than 80% of people in developing countries cannot afford the basic medical facilities, drugs, vaccines, and even the richer population in both developed and developing countries depend upon complementary and alternatives medical treatment in the form of herbal medicine. so, its demand is rising day by day (pandey et al., 2013). medicinal plants are mostly used due to their fewer side effects, and better patient compliance (brown et al., 2008). it is estimated that there is one physician for 30000 people but one healer for fewer than 100 people in nepal (gillam, 1989). there are 103 ethnic indigenous groups with more than 63 varieties of dialects in nepal (cbs, 2003). there are about 8.4 million indigenous people of different groups with their own culture, religion, traditional medicine practices. out of 6653 species of angiosperm plants, 1792 to 2331 were recorded as potential medicinal and aromatic plants in nepal (rokaya et al., 2010). out of 1950 species of medicinal plants (ghimire, 2008), 143 species are categorized as commercial medicinal plants (bhattarai & ghimire, 2006) in nepal. in nepal, 679 species of medicinal plants are found in the lower sub-tropical region (1000-1500m) (ghimire et al., 2006). medicinal plants can be used for the treatment of several diseases (cragg & newman, 2003) as they are recognized as key sources of novel biomolecules (heinrich et al., 1998). in nepal, modern medicines are not available for a large population (bhattarai et al., 2009) so, indigenous knowledge about the use of plants as medicine remains the foundation for primary health care in most of the rural areas. this leads to the strong respect of the local population in traditional medicines and traditional medicinal practitioners (bhattarai et al., 2006). it is estimated that only 15-20% of nepal’s population living in and around urban areas have access to modern healthcare facilities while the rest of them depends upon traditional medicines (sharma et al., 2004). ethnobotanical knowledge remains endemic to certain ethnic groups in nepal due to the lack of interaction between the indigenous people and the scientific community (bhattarai et al., 2009). the younger generation has less interest in ethnobotanical knowledge comparing to the old generation that leads to danger to the continued use of local medicinal plants (kargioglu et al., 2008). therefore, it is necessary to preserve the communication between indigenous peoples and local communities (iplcs) and their environment to foster medicinal knowledge for coming generations (ambu et al., 2020). miya et al. (2020) has reviewed ethnomedicinal uses of plants by major ethnic groups in the hilly districts of nepal. however, various studies have been performed regarding ethnomedicinal importance of different plant species in terai districts. as terai districts are habitats for many indigenous ethnic groups. this study aims to compile information on ethnomedicinal uses of plants by major ethnic groups in the terai districts of nepal. it aims to provide a list of plants that have ethnomedicinal value in the terai districts of nepal. materials and methods this paper is entirely based on a desk review of several published sources including research notes and reports, academic papers, journals, and thesis from 1962 to 2020 a.d. international journal of applied biology, 5(2), 2021 132 for the present review, 35 articles (mostly published) based on ethnomedicinal uses of several medicinal plants in the terai of nepal were reviewed by using online search engines like google scholar, scopus, and research gate. we found several papers related to ethnomedicinal uses of plants in the terai region, and ethnomedicinal importance regarding these plants were overview analytically for rational discussion, conclusion, and recommendation. different scholarly works, papers, reports, and other pieces of literature were studied collected, analyzed, reviewed, and arranged logically for the preparation of this manuscript. the whole process is detailly explained in figure 1 with the help of the prisma flowchart. figure 1: prisma flowchart for the studies related to ethnomedicinal uses of plants by major ethnic tribes in terai districts of nepal results and discussion ethnomedicinal knowledge of eight ethnic groups of six terai districts is described in this paper. the ethnic groups, their inhabited districts from where researches were carried out, numbers of plants used for the treatment of various diseases are listed in table 1: table 1. number of plants to treat different diseases in various districts by different ethnic groups of hilly region, nepal s.n. ethnic tribes districts (studied) symbols of districts (for later use) numbers of plants used for medicinal purpose number of diseases cured sources 1. bantar morang m 83 58 (acharya & pokhrel, 2006) 2. chepang chitwan c 124 50 (rijal, 2011) total papers downloaded n=273 total papers included after being reviewed by all authors n=35 papers excluded after reading the whole text n=29 papers as per our objective n=64 papers excluded after reading the title and abstract n= 123 international journal of applied biology, 5(2), 2021 133 34 25 (sharma et al., 2014) 3. darai chitwan c 68 41 (poudel & singh, 2016) 4. kisan jhapa jh 37 33 (rajbanshi & thapa, 2019) 5. magar nawalparasi n 3 3 (nemkul & shrestha, 2018) 6. meche jhapa jh 53 46 (rai, 2004) 7. munda jhapa jh 73 55 (ghimire, 2016) 8. tharu parsa pr 71 50 (singh, 2020) 47 87 (singh, 2017) rupandehi r 26 43 (acharya & acharya, 2009) chitwan c 53 38 (dangol & gurung, 1991) 45 39 (meullerboeker,1993) the plant species with their uses are listed in table 2. abbereviation used on table 2: p.u. = parts used, s= stem, b= bark, f= fruit, l= leaf, r= root, fl= flower, t= twig, rh= rhizome, tu= tuber, w= whole plant, bu= bulb, sd= seed, lt= latex, bd= buds, sp=sap). table 2. plants with their uses s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 1. abelmoschus moschatus medik., bankapas, malvaceae r cuts and wounds tharu (c) (dangol & gurung, 1991) 2. abrus precatorius l., rati gedi, fabaceae sd dog bite tharu (pr) (singh, 2020) female sterility bantar (acharya & pokhrel, 2006) fever, stomach disease, eye disease, asthma, and uteral problem tharu (r) (acharya & acharya, 2009) 3. abutilon indicum (link) sweet, baliyari, malvaceae l & s boils tharu (c) (dangol & gurung, 1991) 4. acacia catechu (l.f.) willd., khayar, fabaceae b fracture and cough chepang (rijal, 2011) night blindness munda (ghimire, 2016) 5. acacia nilotica (l.) delile, babul, fabaceae b diarrhea and dysentery tharu (c) (dangol & gurung, 1991) l veneral diseases bantar (acharya & international journal of applied biology, 5(2), 2021 134 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources pokhrel, 2006) 6. achyranthes aspera l., datiwan, amaranthaceae r relief pain after child birth tharu (pr) (singh, 2020) stop bleeding, fever and headache tharu (c) (dangol & gurung, 1991; muellerboeker, 1993) fever chepang (rijal, 2011) kisan (rajbanshi & thapa, 2019) pneumonia meche (rai, 2004) fl jaundice bantar (acharya & pokhrel, 2006) s toothache darai (poudel & singh, 2016) cough chepang (sharma et al., 2014) 7. acorus calamus l., bojho, acoraceae rh stomach problem tharu (pr) (singh, 2020) cough meche (rai, 2004) tharu (c) (dangol & gurung, 1991) bantar (acharya & pokhrel, 2006) fever, tonsillitis and diarrhea darai (poudel & singh, 2016) chronic cough and sore throat munda (ghimire, 2016) cough, cold, and bronchial problem tharu (r) (acharya & acharya, 2009) 8. adhatoda vasica nees, asuro, acanthaceae l cough and skin problem tharu (pr) (singh, 2020) cough, cold, fever and bronchitis tharu (c) (dangol & gurung, 1991; mueller boeker, 1993) cough and catarrh bantar (acharya & pokhrel, 2006) cough darai (poudel & singh, 2016) international journal of applied biology, 5(2), 2021 135 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 9. adiantum philippense l., walking maidenhair, pteridaceae w night fever tharu (c) (mueller boeker, 1993) 10. aegle marmelos (l.) correa, bel, rutaceae f dysentery, piles, acute and chronic diarrhea tharu (pr) (singh, 2017; singh, 2020) heat sickness and abdominal disorders tharu (c), (mueller boeker, 1993) chepang (rijal, 2011) diabetes and constipation munda (ghimire, 2016) diarrhoea and dysentery tharu (r) (acharya & acharya, 2009) l diarrhea tharu (pr) (singh, 2020) diabetes darai (poudel & singh, 2016) b intermittent fever tharu (pr) (singh, 2017) 11. aeschynanthus parviflorus (d. don) spreng., thirjo, gesneriaceae w wounds chepang (rijal, 2011) 12. aeuria lanata juss., julaf, amaranthaceae l fever tharu (c) (dangol & gurung, 1991) 13. ageratum conyzoides l., seto gandhe, asteraceae l cuts tharu (c) (dangol & gurung, 1991) tharu (pr) (singh, 2020), kisan (rajbanshi & thapa, 2019) bantar (acharya & pokhrel, 2006) chepang (sharma et al., 2014). skin diseases munda (ghimire, 2016) 14. albizia lebbeck (l.) benth., kalo siris, sd eyesight problem and baldness tharu (pr) (singh, 2020) international journal of applied biology, 5(2), 2021 136 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources fabaceae 15. allium cepa l., pyaj, amaryllidaceae bu ear pain munda (ghimire, 2016) w wasp bite tharu (pr) (singh, 2020) 16. allium sativum l., lasun, amaryllidaceae bu fever, cold, flu and nerve problem tharu (pr) (singh, 2020) antiseptic meche (rai, 2004) gastritis munda (ghimire, 2016) tonsillitis darai (poudel & singh, 2016) 17. aloe vera (l.) burm. f., ghiu kumari, liliaceae l burns tharu (pr) (singh, 2020) darai (poudel & singh, 2016) meche (rai, 2004) catarrh and cough bantar (acharya & pokhrel, 2006) syphyllis, diabetes, irregular menstruation and high blood pressure munda (ghimire, 2016) 18. alstonia scholaris (l.) r. br., chattiwan, apocynaceae b malaria fever tharu (pr) (singh, 2020) leanness in man meche (rai, 2004) appetizer and increase lactation bantar (acharya & pokhrel, 2006) lt menopause chepang (rijal, 2011) high fever and backache tharu (c) (mueller boeker, 1993) mumps kisan (rajbanshi & thapa, 2019) 19. alternanthera sessilis (l.) r. br. ex. dc, bhiringi jhar, amaranthaceae w dysentery tharu (pr) (singh, 2020) l increase lactation tharu (pr) (singh, 2020) r stomachache bantar (acharya & pokhrel, 2006) 20. amaranthus viridis l., latte sag, amaranthaceae l eyesight problem and baldness tharu (pr) (singh, 2020) r wound and cold chepang (rijal, 2011) 21. amaranthus r abortion bantar (acharya & international journal of applied biology, 5(2), 2021 137 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources spinosus l., kande lundo, amaranthaceae pokhrel, 2006) sd labour pain darai (poudel & singh, 2016) 22. amorphophallus campanulatus (roxb.) blume ex decne, kaan, araceae bu piles tharu (pr) (singh, 2020) 23. ananas comosus (l.) merr., bhui katahar, bromieliaceae f & r heat sickness chepang (rijal, 2011) 24. andrographis paniculata (burm. f.) nees, kaalmegh, acanthaceae l snake bite and intestinal worm tharu (pr) (singh, 2020) 25. andropogon muricatus retz., khas, graminiae r stomach problem tharu (pr) (singh, 2020) 26. anethum sowa roxb. ex fleming, swop, apiaceae sd lactation darai (poudel & singh, 2016) 27. anisomeles indica (l.) kuntze, rato chaarpate, lamiaceae l stomachache bantar (acharya & pokhrel, 2006) 28. annona squamosa l., sitaphal, annonaceae b fever meche (rai, 2004) headache and fever munda (ghimire, 2016) 29. anthocephalus cadamba (roxb.) miq., kadam, rubiaceae b eye inflammation munda (ghimire, 2016) l stomachache munda (ghimire, 2016) 30. antidesma acidum retz., archal, phyllanthaceae b dysentery chepang (rijal, 2011) 31. apium graveolens l., juwano, apiaceae sd fever kisan (rajbanshi & thapa, 2019) lactation enhancer and menstrual disorders darai (poudel & singh, 2016) 32. areca catechu l., supari, arecaceae f scars darai (poudel & singh, 2016) 33. argemone mexicana l., thakal, f& lt conjunctivitis and scabies bantar (acharya & pokhrel, 2006) international journal of applied biology, 5(2), 2021 138 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources papaveraceae 34. arisaema consanguineum (l.) schott, kal, araceae tu wormicide chepang (rijal, 2011) 35. aristolochia indica l., isharmule, aristolochiaceae l scorpion bite tharu (pr) (singh, 2020) 36. artemisia indica willd., titepati, asteraceae l round worms munda (ghimire, 2016) meche (rai, 2004) cuts and wounds darai (poudel & singh, 2016) r dysentery, wounds and abdominal pain chepang (rijal, 2011) fl appetizer, pregnancy disease and lactation bantar (acharya & pokhrel, 2006) 37. artocarpus heterophyllus lam., katahar, moraceae lt mumps kisan (rajbanshi & thapa, 2019) 38. artocarpus lakoocha roxb., badahar, moraceae b gastritis darai (poudel & singh, 2016) 39. asparagus racemosus willd., kurilo, asparagaceae tu tonic chepang (rijal, 2011) tharu(c) (mueller boeker, 1993) urinary problems, increase lactation and boost immunity kisan (rajbanshi & thapa, 2019) 40. azadirachta indica a. juss., neem, meliaceae l pox, measles and allergy tharu (pr) (singh, 2020) enzema, scabies and blood purifier meche (rai, 2004) wounds tharu (c) (mueller boeker, 1993) darai (poudel & singh, 2016) urinary tract problem, fever and skin problems kisan (rajbanshi & thapa, 2019) anemia, fever, scabies, diabetes, skin allergy and irritation munda (ghimire, 2016) suger tharu (r) (acharya & acharya, 2009) international journal of applied biology, 5(2), 2021 139 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources b antihelmintic and cough tharu (r) (acharya & acharya, 2009) fl scabies and intestinal worms bantar (acharya & pokhrel, 2006) 41. basella alba l., poi saag, basellaceae t insomnia bantar (acharya & pokhrel, 2006) 42. barleria cristata l., bhedekuro, acanthaceae r anaemia darai (poudel & singh, 2016) 43. bauhinia variegata (l.) benth. koiralo, fabaceae b dysentery tharu (r) (acharya & acharya, 2009) 44. benincasa hispida (thunb.) cogn., kuvindo, cucurbitaceae f heat sickness chepang (rijal, 2011) 45. bergenia ciliata (haw.) sternb., pakhanbed, saxifragaceae rh body pain, diarrhoea, sprain and abdominal spasm chepang (rijal, 2011) 46. betula alnoides buch-ham. ex. d. don, saur, betulaceae b wounds chepang (rijal, 2011) 47. biophytum sensitivum (l.) dc., lazaiya jhyang, oxalidaceae w fever tharu (c) (dangol & gurung, 1991) 48. bixa orellana f fracture darai (poudel & singh, 2016) 49. blumea lacera (burm. f.) dc., kukure, asteraceae r cutaneous infection meche (rai, 2004) s gastric and ulcer chepang (sharma et al., 2014) l cuts bantar (acharya & pokhrel, 2006) 50. bombax ceiba l., simal, malvaceae b diarrhea and dysentery meche (rai, 2004) fracture and dysentery bantar (acharya & pokhrel, 2006) urinary disorder, intestine bleeding, and excessive vaginal bleeding tharu (r) (acharya & acharya, 2009) b measles tharu (c) (dangol & gurung, 1991) international journal of applied biology, 5(2), 2021 140 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources fl boils munda (ghimire, 2016) r dysentery and measles darai (poudel & singh, 2016) urinary problems and menstrual disorders munda (ghimire, 2016) lt worms and bleeding tharu (c) (mueller boeker, 1993) 51. bonnaya ciliata link & otto., oonmudia, linderniaceae w cuts and wounds tharu (c) (dangol & gurung, 1991) 52. brassica campestris l., tori, brassicaceae sd indigestion chepang (rijal, 2011) 53. breea arvenis (l.) less, gainda kande, asteraceae fl &r scabies bantar (acharya & pokhrel, 2006) 54. bridelia retusa (l.) a. juss, gayo, phyllanthaceae b fracture chepang (rijal, 2011) 55. bryophyllum pinnatum (lam.) oken, ajambari, crassulaceae l gall bladder stone tharu (pr) (singh, 2020) 56. caesalpinia bonduc (l.) roxb., kande jhang, fabaceae sd malaria fever tharu (pr) (singh, 2020) scalp meche (rai, 2004) l fever and intestinal worms bantar (acharya & pokhrel, 2006) 57. caesulia axillaris roxb., thuk jhar, asteraceae w stop bleeding tharu (c) (mueller boeker, 1993) 58. callicarpa macrophylla vahl, gunelo, lamiaceae l indigestion chepang (rijal, 2011) earache tharu (c) (dangol & gurung, 1991) r fever and gastric chepang (rijal, 2011; sharma et al., 2014) tharu (c) (dangol & gurung, 1991) bronchitis meche (rai, 2004) 59. calotropis gigantea (l.) w.t. aiton, lt sprain, fever, rheumatism and joint pain (rai, 2004) international journal of applied biology, 5(2), 2021 141 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources aank, apocynaceae malaria and asthma bantar (acharya & pokhrel, 2006) wounds and fungal infections tharu (c) (mueller boeker, 1993) sprain and fracture darai (poudel & singh, 2016) rheumatism, joint pain and sprain munda (ghimire, 2016) l earache kisan (rajbanshi & thapa, 2019) 60. camellia sinensis (l.) kuntze, chiya, theaceae fl high blood pressure kisan (rajbanshi & thapa, 2019) 61. cannabis sativa l., gaanja, cannabaceae l cold chepang (rijal, 2011) darai (poudel & singh, 2016) indigestion tharu (c) (dangol & gurung, 1991) piles bantar (acharya & pokhrel, 2006) sd abdominal disorders chepang (rijal, 2011) 62. capsicum annuum l., dalle khursani, solanaceae sd fever and indigestion chepang (rijal, 2011) r typhoid chepang (sharma et al., 2014) 63. carica papaya l., mewa, caricaceae lt ring worms meche (rai, 2004) ringworms and scabies munda (ghimire, 2016) f liver swelling bantar (acharya & pokhrel, 2006) 64. caryopteris bicolor (roxb. ex hardw), balamohani, lamiaceae l cold chepang (rijal, 2011) b scabies and abdominal pain chepang (rijal, 2011) 65. cassia fistula l., raj brikshaya, fabaceae f diarrhea, vomiting, appetite and constipation chepang (rijal, 2011; sharma et al., 2014) whopping cough meche (rai, 2004) headache tharu (c) (dangol & gurung, 1991) sd throat problems chepang (rijal, 2011) 66. cassia occidentalis (l.) link, thulo tapre, fabaceae sd & fl skin infections and inflammation meche (rai, 2004) international journal of applied biology, 5(2), 2021 142 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources r ring worms tharu (c) (dangol & gurung, 1991) l itch and ring worms bantar (acharya & pokhrel, 2006) 67. cassia tora l., sano tapre, fabaceae l& sd itch and ring worms bantar (acharya & pokhrel, 2006) 68. catharanthus roseus (l.) g. don, barhamase ful, apocynaceae fl diabetes meche (rai, 2004) 69. centella asiatica (l.) urb., ghodtapre, apiaceae w heat sickness meche (rai, 2004) urine problems munda (ghimire, 2016) l heat sickness and asthma chepang (rijal, 2011) urinary problem and acidity bantar (acharya & pokhrel, 2006) heartburn tharu(c) (mueller boeker, 1993) diarrhea, tonic, headache and appetizer kisan (rajbanshi & thapa, 2019) jaundice darai (poudel & singh, 2016) indigestion, cuts, wounds, skin allergy and irritation munda (ghimire, 2016) tonic, enhance memory, nerve trouble, and skin disease tharu (r) (acharya & acharya, 2009) fever chepang (sharma et al., 2014) 70. cestrum diurnum l., jasmine, solanaceae l weeping illness in child bantar (acharya & pokhrel, 2006) 71. cheilanthes dalhousiae (hook.), ranisinka, cheilanthoidae l abdominal spasm and wounds chepang (rijal, 2011) 72. chenopodium album l., bethe, chenopodiaceae w joint pains and stomach disorders tharu (c) (dangol & gurung, 1991; mueller boeker, 1993) constipation, back and body pain munda (ghimire, 2016) t constipation bantar (acharya & international journal of applied biology, 5(2), 2021 143 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources pokhrel, 2006) 73. chenopodium murale l., pahadia bethe, chenopodiaceae w indigestion and gastritis tharu (c) (dangol & gurung, 1991) 74. chlorophytum arundinaceum baker, seto musli, asparagaceae r impotency tharu (pr) (singh, 2020) 75. chlorophytum nepalense (lindl.) baker, danti saag, asparagaceae tu hydroccle tharu (c) (dangol & gurung, 1991) 76. cinnamomum tamala (buch-ham) t. nees & eberm., dalchini, lauraceae b cough tharu (pr) (singh, 2020) indigestion and headache chepang (sharma et al., 2014) l throat allergy meche (rai, 2004) 77. cipadessa baccifera (roth) miq., paireti, meliaceae l wounds chepang (rijal, 2011) 78. cissus quadrangularis l., had jor, vitaceae s fracture kisan (rajbanshi & thapa, 2019) 79. citrus limon (l.) osbek, kagati, rutaceae f cholera kisan (rajbanshi & thapa, 2019) acidity, vomiting and indigestion munda (ghimire, 2016) 80. citrus medica l., bimiro, rutaceae r dysentery darai (poudel & singh, 2016) gastric chepang (sharma et al., 2014) 81. cissampelos pareira l., batul pate, menispermaceae s abdominal disorders chepang (rijal, 2011; sharma et al., 2014) worms and cuts darai (poudel & singh, 2016) w fever and induce abortion tharu (c) (dangol & gurung, 1991) 82. cleistocalyx operculatus (roxb.) merr. & l.m., kyamuno, myrtaceae l psynocytis and dysentery chepang (rijal, 2011) international journal of applied biology, 5(2), 2021 144 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 83. cleome viscosa l., ban methi, capparaceae w cut and wounds tharu (c) (dangol & gurung, 1991) 84. clerodendrum indicum (l.) gaertn., rudilo, verbenaceae s fever bantar (acharya & pokhrel, 2006) 85. clerodendrum viscosum vent., bhat, lamiaceae l high blood pressure and asthma tharu (pr) (singh, 2020) antidote of poison meche (rai, 2004) fever tharu (c) (dangol & gurung, 1991; mueller boeker, 1993) intestinal worms bantar (acharya & pokhrel, 2006) 86. coccinia grandis (l.) voigt., kundaru, cucurbitaceae l jaundice bantar (acharya & pokhrel, 2006) 87. cochlianthus gracilis benth., boksi kanda, fabaceae b boils and pimples chepang (rijal, 2011) 88. colebrookea oppositifolia sm., dhursuli, lamiaceae l fever chepang (rijal, 2011) wounds meche (rai, 2004) earache tharu (c) (dangol & gurung, 1991) r typhoid, headache, pneumonia, psynocytis and indigestion chepang (rijal, 2011) 89. colocasia esculenta (l.) schott, karkalo, araceae lt cuts kisan (rajbanshi & thapa, 2019) munda (ghimire, 2016) tu lactation darai (poudel & singh, 2016) 90. combretum roxburghii spreng., (dars) combretaceae b wormicide chepang (rijal, 2011) 91. coriaria nepalensis wall., machhaino, coriariaceae r diarrhoea and dysentery chepang (sharma et al., 2014) 92. costus speciosus rh fever tharu (singh, 2020) international journal of applied biology, 5(2), 2021 145 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources (koeing) sm., betlauri, costaceae (pr) burns chepang (rijal, 2011) s wounds tharu (c) (mueller boeker, 1993) joint pain darai (poudel & singh, 2016) 93. crateva unilocularis buch. –ham., silpigaan, capparaceae l urinary infection darai (poudel & singh, 2016) b liver problem chepang (rijal, 2011) 94. crinum amoenum ker gawl. ex roxb., himalayan crinum lily, amaryllidaceae bu swollen testes tharu (c) (mueller boeker, 1993) 95. crinum asiaticum l., hade phool, amaryllidaceae fl nasal bleeding and diabetes bantar (acharya & pokhrel, 2006) bu wounds and burns kisan (rajbanshi & thapa, 2019) 96. crotalaria albida roth., bhuling, fabaceae s indigestion chepang (rijal, 2011) 97. crotalaria javanica jungh., runche, fabaceae r body swelling meche (rai, 2004) l eczema bantar (acharya & pokhrel, 2006) 98. croton bonplandianum baill., mirchaiya jhar, euphorbiaceae l &r ulcer and cuts bantar (acharya & pokhrel, 2006) 99. cucumis sativus l., kankro, cucurbitaceae sd indigestion chepang (rijal, 2011) heartburn darai (poudel & singh, 2016) tharu(c) (mueller boeker, 1993) 100. cucumis melo l. kharbujo, cucurbitaceae r dysentery tharu (c) (dangol & gurung, 1991) 101. cucurbita maxima duchesne, pharsi, cucurbitaceae) sd indigestion chepang (rijal, 2011) f jaundice darai (poudel & singh, 2016) 102. curcuma longa l. besar, zingiberaceae rh allergy and bone fracture tharu (pr) (singh, 2020) 103. curcuma rh cough tharu (singh, 2020) international journal of applied biology, 5(2), 2021 146 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources angustifolia roxb., kalo haledo, zingiberaceae (pr) cuts and wounds meche (rai, 2004) 104. curcuma caesia roxb., haledo, zingiberaceae rh appetizer munda (ghimire, 2016) 105. cuscuta reflexa roxb., aakash beli, convolvulaceae w jaundice tharu (pr) (singh, 2020) tharu(c) (dangol & gurung, 1991) darai (poudel & singh, 2016) kisan (rajbanshi & thapa, 2019) meche (rai, 2004) munda (ghimire, 2016) tonsillitis and swelling bantar (acharya & pokhrel, 2006) 106. cynodon dactylon (l.) pers., dubo, poaceae w indigestion chepang (rijal, 2011) pneumonia and fever munda (ghimire, 2016) r burns, cuts and wounds bantar (acharya & pokhrel, 2006) kidney problems chepang (sharma et al., 2014) l hair struck on neck darai (poudel & singh, 2016) cuts, wounds, scabies, and haematuria tharu (r) (acharya & acharya, 2009) 107. cyperus compressus l., mothe, cyperaceae w cuts and scabies tharu (c) (dangol & gurung, 1991) 108. cyperus rotundus l., mothe, cyperaceae r stomachache bantar (acharya & pokhrel, 2006) intestinal worms kisan (rajbanshi & thapa, 2019) obesity and rheumatism munda (ghimire, 2016) 109. dalbergia sissoo roxb., sissoo, fabaceae l &b scabies and diarrhea bantar (acharya & pokhrel, 2006) l anemia and gonorrhea munda (ghimire, international journal of applied biology, 5(2), 2021 147 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 2016) 110. datura stramonium l., dhaturo, solanaceae sd scabies munda (ghimire, 2016) meche (rai, 2004) bronchitis tharu(c) (mueller boeker, 1993) burns munda (ghimire, 2016) 111. dendrocalamus hamiltonii gambie, choya bans, poeceae bd purgative tharu (c) (dangol & gurung, 1991) 112. dendrocalamus strictus (roxb.) nees, kaban bans, poaceae sp enuresis chepang (rijal, 2011) 113. desmodium oojeinense (roxb.) h. ohashi, sandan, fabaceae s cuts and wounds chepang (rijal, 2011) 114. desmotrichum plicatile lindl., jiwanti, orchidaceae bu fever chepang (rijal, 2011) 115. dillenia pentagyna roxb., tantari, dilleniaceae b diarrhea and to relief pain after child birth tharu (pr) (singh, 2020) tick bite tharu (c) (mueller boeker, 1993) 116. dioscorea alata l., pangnang, dioscoreaceae tu wormicide chepang (rijal, 2011) 117. dioscorea bulbifera l., githa, dioscoreaceae tu wormicide chepang (rijal, 2011) appetizer and high blood pressure kisan (rajbanshi & thapa, 2019) 118. dioscorea deltoidea wall. ex griseb., bhyakur, dioscoreaceae tu wormicide chepang (rijal, 2011) f stomach pain tharu (r) (acharya & acharya, 2009) 119. dioscorea prazeri prain & burkill, jyar, dioscoreaceae tu wormicide chepang (rijal, 2011) 120. diplocyclos palmatus (l.) c. f tonic chepang (rijal, 2011) international journal of applied biology, 5(2), 2021 148 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources jefferey, saawaa, cucurbitaceae 121. diploknema butyracea (roxb.) h.j. lam, chiuri, sapotaceae sd herpes zoster chepang (rijal, 2011) 122. dolichos lablab (l.) sweet, simi, fabaceae l dandruff kisan (rajbanshi & thapa, 2019) 123. drymaria cordata (l.) willd. ex schult., abhijalo, caryophyllaceae l cutaneous infections meche (rai, 2004) 124. drymeria cordta subsp. diandra (sw.) j.a. duke., abhijalo, caryophyllaceae l wormicide and psynocytis chepang (rijal, 2011) headache and sinusitis kisan (rajbanshi & thapa, 2019) sinusitis, cuts and wounds munda (ghimire, 2016) w gastritis darai (poudel & singh, 2016) 125. dryoathyrium boryanum (willd.) ching, kali neuro, pteridaceae rh abdominal spasm chepang (rijal, 2011) r diarrhoea and dysentery chepang (sharma et al., 2014) 126. duchesnea indica (andrews) teschem., vui kafal, rosaceae l heat sickness, marasmus, kwashiorkor and fever chepang (rijal, 2011) w urinary disorders and fever tharu (c) (dangol & gurung, 1991) 127. eclipta prostrata (l.) l., jire jhaar, asteraceae l fever and indigestion chepang (rijal, 2011) scabies and cuts bantar (acharya & pokhrel, 2006) cuts and wounds darai (poudel & singh, 2016) w eye problem and chapped skin between toe tharu(c) (mueller boeker, 1993) 128. elephantopus scaber l., sahasrabuti, asteraceae l wounds chepang (rijal, 2011) muscular pain meche (rai, 2004) 129. ensete glaucum (roxb.) cheesman, ban kera, musaceae tu heat sickness and urine infections chepang (rijal, 2011) 130. entada f wormicide chepang (rijal, 2011) international journal of applied biology, 5(2), 2021 149 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources phaseoloides (l.) merr., pangra, fabaceae w poison and worms chepang (sharma et al., 2014) 131. equisetum debile roxb. ex vaucher, sime jhar, equisetaceae w fracture meche (rai, 2004) b gastric, cuts and wounds chepang (sharma et al., 2014) 132. erythrina stricta roxb., phaledo, fabaceae b typhoid, heat sickness, abdominal disorder, pneumonia and fever chepang (rijal, 2011) 133. eupatorium adenophorum spreng., banmara, asteraceae l antiseptic, cut and wound meche (rai, 2004) r cuts and wounds chepang (sharma et al., 2014) 134. euphorbia hirta l., dudhe jhaar, euphorbiaceae lt wound, cold and cough chepang (rijal, 2011) pimples and wounds meche (rai, 2004) w piles bantar (acharya & pokhrel, 2006) rheumatism munda (ghimire, 2016) r lactation darai (poudel & singh, 2016) 135. euphorbia royleana boiss., siudi, euphorbiaceae lt swelling meche (rai, 2004) leprosy, whooping cough, irritation and skin allergy munda (ghimire, 2016) l dysentery darai (poudel & singh, 2016) 136. ficus benghalensis l. bar, moraceae l dysentery and skin problem tharu (pr) (singh, 2020) gastritis darai (poudel & singh, 2016) r female sterility tharu (pr) (singh, 2020) diarrhoea munda (ghimire, 2016) lt heel crack, cataract and veneral diseases bantar (acharya & pokhrel, 2006) rheumatism and carbuncles munda (ghimire, 2016) b diarrhoea, dysentery, and diabetes tharu (r) (acharya & acharya, 2009) 137. ficus hispida l., l ear ache and hearing weakness tharu (c) (dangol & international journal of applied biology, 5(2), 2021 150 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources thotne, moraceae gurung, 1991; mueller boeker, 1993) 138. ficus religiosa l., pipal, moraceae b irregular menstuaration and ear ache tharu (c) (dangol & gurung, 1991) l ear ache tharu(c) (mueller boeker, 1993) jaundice tharu (pr) (singh, 2017) f constipation munda (ghimire, 2016) 139. ficus semicordata buch-ham. ex sm. khanyu, moraceae sp heat sickness chepang (rijal, 2011) l earache tharu(c) (mueller boeker, 1993) r headache and fever tharu (r) (acharya & acharya, 2009) s fever and fracture chepang (sharma et al., 2014) 140. fimbristylis aestivalis (retz.) vahl, summer fimbry, cyperaceae tu appetizer bantar (acharya & pokhrel, 2006) 141. fogopyrum esculentum moench, phapar, polygonaceae sd typhoid darai (poudel & singh, 2016) 142. fomitopsis pinicola (sw.) p. karst., jali chyau, fomitopsidaceae w snake bite chepang (rijal, 2011) 143. gardenia jasminoides j. ellis, indrakamal, rubiaceae f blood purifier tharu (pr) (singh, 2020) sd rheumatoid arthritis tharu (pr) (singh, 2017) 144. gaultheria fragrantissima wall., dhasingre, ericaceae l massage to get relief from par of head, hands and legs tharu (r) (acharya & acharya, 2009) 145. girardinia diversifolia (link) friis, chalne sisno, urticaceae r snake bite and tonic chepang (rijal, 2011) s pain chepang (sharma et al., 2014) 146. grewia pumile ham. w dysentery tharu (c) (dangol & international journal of applied biology, 5(2), 2021 151 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources -ham ex d. don, phusre, tiliaceae gurung, 1991) 147. hedyotis corymbosa l., piringo jhar, rubiaceae w acidity bantar (acharya & pokhrel, 2006) 148. helicteres isora l., kapaase, malvaceae r worm infection tharu (c) (mueller boeker, 1993) 149. heliotropium indicum l., hattisude, boraginaceae l cuts and tongue infections bantar (acharya & pokhrel, 2006) 150. hibiscus rosasinensis l., ghanti phool, malvaceae fl high blood pressure tharu (pr) (singh, 2020) liver disorders and high blood pressure tharu (pr) (singh, 2017) b abortion munda (ghimire, 2016) l& fl headache tharu (pr) (singh, 2017) 151. hibiscus sabdariffa l., belchandan, malvaceae l foot wounds caused by muddy water during rainy season meche (rai, 2004) 152. holarrhena pubescens wall. ex g. don, khirro, apocynaceae l dysentery chepang (rijal, 2011) b stomachache and rheumatism tharu(c) (mueller boeker, 1993) heat sicknesss darai (poudel & singh, 2016) dysentery, febrifuge, diarrhoea, worms, and seed-astringent tharu (r) (acharya & acharya, 2009) amobic dysentery, piles, chest infections, and toothache tharu (pr) (singh, 2017) 153. hydrangea anomala d. don, bahuni kath, hydrangeaceae w kidney stone tharu (pr) (singh, 2020) r malaria, kidney and bladder stone tharu (pr) (singh, 2017) b wounds or burns tharu (pr) (singh, 2017) 154. hydrocotyle sibthorpioides lam., tarpurin, araliaceae w urinary disorders and fever tharu (c) (dangol & gurung, 1991) l asthma meche (rai, 2004) 155. hygrophila auriculata r eczema bantar (acharya & pokhrel, 2006) international journal of applied biology, 5(2), 2021 152 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources schumach., tal makhan, acanthaceae 156. hypericum cordifolium choisy, areli, hypericaceae b rheumatism darai (poudel & singh, 2016) 157. imperata cylindrica (l.) p. beauv., siru, poaceae r cholera bantar (acharya & pokhrel, 2006) wounds munda (ghimire, 2016) skin softener tharu (pr) (singh, 2017) 158. inula cappa (buchham. ex d. don) dc, gaitihare, asteraceae r headache, fever, disorder arise due to meat tharu (r) (acharya & acharya, 2009) 159. ipomoea aquatica forssk., karmi sag, convolvulaceae s lactation darai (poudel & singh, 2016) 160. ipomoea batatas (l.) lam., sakharkhand, convolvulaceae t lactation kisan (rajbanshi & thapa, 2019) 161. jatropha curcas l., sajjiwan), euphorbiaceae sp burn chepang (rijal, 2011) s teeth infections bantar (acharya & pokhrel, 2006) teeth infections chepang (rijal, 2011) constipation darai (poudel & singh, 2016) bleeding gums, gingivitis and bad breath kisan (rajbanshi & thapa, 2019) lt toothache and swelling testes meche (rai, 2004) burns, gum infections, chapped skin and wounds tharu (c) (dangol & gurung, 1991; mueller boeker, 1993) sd burn injury and wounds chepang (sharma et al., 2014) 162. juglans regia l., okhar, juglandaceae f typhoid darai (poudel & singh, 2016) 163. lagerstroemia parviflora l., bot l fever chepang (rijal, 2011) l pneumonia and typhoid magar (nemkul & international journal of applied biology, 5(2), 2021 153 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources dhaiyaro, lythraceae &b shrestha, 2018) 164. lantana camara l., banmara, verbenaceae w tetanus munda (ghimire, 2016) l fever munda (ghimire, 2016) 165. lawsonia inermis l., mehandi, lythraceae l fungal infection tharu (pr) (singh, 2020) mud infection and heel cracks bantar (acharya & pokhrel, 2006) wounds munda (ghimire, 2016) mouth ulcer tharu (pr) (singh, 2017) 166. leucas aspera (willd.) link, gummaa, lamiaceae l &r ringworms tharu (c) (dangol & gurung, 1991) 167. leucas cephalotes (roth) spreng., dronpuspi, lamiaceae fl sinusitis tharu (pr) (singh, 2020) sinusitis meche (rai, 2004) ringworms tharu (c) (dangol & gurung, 1991) cough and cold tharu (pr) (singh, 2017) t malarial fever tharu (pr) (singh, 2017) 168. leucas indica (l.) vatke, dulphe jhar, lamiaceae t body pain bantar (acharya & pokhrel, 2006) 169. lindera neesiana (wall. ex nees) kurz, siltimur, lauraceae f cholera, abdominal pain, diarrhea, and vomiting chepang (rijal, 2011) 170. luffa cylindrica m. roem., gheraula, cucurbitaceae sd chicken pox tharu (c) (mueller boeker, 1993) 171. luffa echinata roxb., jungali gheraula, cucurbitaceae sd malaria, headache and sinusitis kisan (rajbanshi & thapa, 2019) 172. lygodium japonicum (thunb.) l ringworms darai (poudel & singh, 2016) international journal of applied biology, 5(2), 2021 154 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources sw., janai lahara, lygodiaceae eczema, ringworm and wounds tharu (pr) (singh, 2017) sd kidney and urinary problems tharu (pr) (singh, 2017) t diuretic tharu (pr) (singh, 2017) 173. madhuca indica j.f. gmel., mahuwa, sapotaceae fl increase milk in lactating mothers tharu (pr) (singh, 2017) sd diabetes, skin disease, rheumatism, headache, arthritis, insulin production and control blood glucose level tharu (pr) (singh, 2017) b bleeding and spongy gum, pharyngitis, and chronic tonsillitis tharu (pr) (singh, 2017) 174. mallotus philippensis (lam.) muell. arg., sindhure, euphorbiaceae s anti-helminthic chepang (rijal, 2011) diarrhea and dysentery chepang (rijal, 2011) gastritis tharu (c) (dangol & gurung, 1991) l dysentery chepang (rijal, 2011) f abdominal spasm chepang (rijal, 2011) b diarrhoea, dysentery, and bronchitis tharu (r) (acharya & acharya, 2009) r &f flatulence, cough, ulcers, wounds, and constipation (singh, 2017). tharu (pr) (singh, 2017) 175. mangifera indica l., aanp, anacardiaceae l burns tharu (pr) (singh, 2020) diabetes munda (ghimire, 2016) b diarrhea chepang (rijal, 2011) dysentery darai (poudel & singh, 2016) dysentery and diarrhea munda (ghimire, 2016) f stomachache meche (rai, 2004) typhoid chepang (sharma et al., 2014) sd hairfall munda (ghimire, 2016) 176. matricaria chamomilla l., fl stomach problem tharu (pr) (singh, 2020) international journal of applied biology, 5(2), 2021 155 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources daminiful, asteraceae sore stomach, laxative, and irritable bowel syndrome tharu (pr) (singh, 2017) 177. matteuccia struthiopteris (l.) tod., neuro, onocleaceae l blood in stool and diarrhoea darai (poudel & singh, 2016) 178. melia azedarach l., bakaino, meliaceae f anthelmintic, vomiting, urinary discharge, and blood impurities tharu (r) (acharya & acharya, 2009) l pyrexia, piles, gonorrhea, and gingivitis tharu (pr) (singh, 2017) 179. melothria heterophylla (lour.) cogn., ban kakro, cucurbitaceae f hydrocele tharu (c) (dangol & gurung, 1991) 180. mentha spicata l., pudina, lamiaceae l vomiting tharu (pr) (singh, 2020) cough meche (rai, 2004) stomachace and gastritis munda (ghimire, 2016) 181. michelia champaca l., champ, magnoliaceae fl eye problem tharu (pr) (singh, 2020) fever, nausea, and dyspepsia tharu (pr) (singh, 2017) b fever tharu (pr) (singh, 2017) 182. mikania micarantha kunth, lahare banmara, asteraceae l cuts and wounds darai (poudel & singh, 2016) 183. mimosa pudica l., lajjawati, fabaceae l hairfall and dandruff tharu (pr) (singh, 2020) gland tumor, sinus disorderneurological problems, diarrhoea, and dysentery tharu (pr) (singh, 2017) indigestion and piles munda (ghimire, 2016) r fever chepang (rijal, 2011) scabies bantar (acharya & pokhrel, 2006) boils and diarrhea kisan (rajbanshi & thapa, 2019) gastritis darai (poudel & international journal of applied biology, 5(2), 2021 156 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources singh, 2016) diabetes and diarrhea munda (ghimire, 2016) cold, cough, and remove stone from different parts of body tharu (pr) (singh, 2017) 184. mimosa rubicaulis lam., bokshi ghas, fabaceae r abdominal spasm, sprain and wound chepang (rijal, 2011) 185. mirabilis jalapa l., malatiphool, nyctaginaceae fl nose bleeding tharu (c) (mueller boeker, 1993) 186. momordica charantia l., tite karela, cucurbitaceae f diabetes tharu (pr) (singh, 2020) piles bantar (acharya & pokhrel, 2006) high blood pressure munda (ghimire, 2016) 187. moringa oleifera lam., shitalchini, moringaceae w dental care tharu (pr) (singh, 2020) b leprosy tharu (pr) (singh, 2020) rheumatism bantar (acharya & pokhrel, 2006) fl high blood pressure meche (rai, 2004) tonic kisan (rajbanshi & thapa, 2019) 188. mucuna monosperma wall., baldhangro, fabaceae f tonic chepang (rijal, 2011) 189. murraya koenigii (l.) spreng., mitho neem, rutaceae l diabetes tharu (pr) (singh, 2020) hairfall and dandruff tharu (pr) (singh, 2020) dysentery and diarrhoea tharu (r) (acharya & acharya, 2009) snake bite, liver, and dysentery tharu (pr) (singh, 2017) r stomach problem tharu (pr) (singh, 2020) 190. musa balbisiana colla, athiya kera, r &f cholera kisan (rajbanshi & thapa, 2019) international journal of applied biology, 5(2), 2021 157 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources musaceae 191. musa paradisiaca l., kera, musaceae fl chest pain darai (poudel & singh, 2016) 192. mussaenda macrophylla wall., dhobini, rubiaceae l indigestion, cold and fever chepang (rijal, 2011) r typhoid and fever chepang (sharma et al., 2014) 193. myrica esculenta buch-ham. ex d. don, kafal, myricaceae b malnourishment chepang (rijal, 2011) f abdominal pain darai (poudel & singh, 2016) 194. nephrolepsis cordifolia (l.) k. presl, pani amala, nephrolepidaceae tu heat sickness chepang (rijal, 2011) stomach ulcer, cough, and intestinal disorders tharu (pr) (singh, 2017) l bleeding tharu (pr) (singh, 2017) 195. nicotiana tobacum l., tambakhu, solanaceae l toothache tharu (pr) (singh, 2020) wormicide chepang (rijal, 2011) remove lice meche (rai, 2004) ear problem bantar (acharya & pokhrel, 2006) 196. nyctanthes arbortristis l. parijat, oleaceae l heat sickness darai (poudel & singh, 2016) digestive, laxative, tonic, diuretic, diaphoretic, antidote to reptile venoms tharu (pr) (singh, 2017) r spleen enlargement tharu (pr) (singh, 2017) s &b expectorant, malaria, and rheumatic joint pain tharu (pr) (singh, 2017) 197. ocimum basilicum l., babari, lamiaceae l fever darai (poudel & singh, 2016) irritation and skin allergy munda (ghimire, 2016) 198. ocimum gratissimum l., bantulsi, lamiaceae l chest pain and respiratory problem meche (rai, 2004) 199. ocimum sanctum l., tulasi, lamiaceae l cough, ring worm infection, bronchial asthma and headache tharu (pr) (singh, 2020) cough and cold chepang (rijal, 2011) skin allergy and irritation meche (rai, 2004) cough and catarrh bantar (acharya & pokhrel, 2006) international journal of applied biology, 5(2), 2021 158 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources fever, cough and tonsilitis kisan (rajbanshi & thapa, 2019) tonsillitis darai (poudel & singh, 2016) cough, cold, dermatitis and sore throat munda (ghimire, 2016) itching, snake bite, rashes in skin, reduce acne, pimples and scars, stress reduce tharu (pr) (singh, 2017) r& fl sexual disorders munda (ghimire, 2016) sd premature ejaculation, increase semen quantity, reduce blood sugar and blood cholesterol tharu (pr) (singh, 2017) fl cough and colds tharu (pr) (singh, 2017) 200. odina wodier roxb., hallunde, anacardiaceae b nose bleeding tharu (c) (dangol & gurung, 1991) 201. opuntia stricta (haw.) haw., paate siudi, cactaceae w burns munda (ghimire, 2016) r obesity and diabetes munda (ghimire, 2016) 202. oroxylum indicum (l.) benth. ex kurz, tatelo, bignoniaceae sd wounds chepang (rijal, 2011) pneumonia meche (rai, 2004) munda (ghimire, 2016) typhoid magar (nemkul & shrestha, 2018) b wounds tharu (c) (mueller boeker, 1993) kisan (rajbanshi & thapa, 2019) jaundice and chronic wounds munda (ghimire, 2016) infective wounds magar (nemkul & shrestha, 2018) tumors, cut injury, and wounds chepang (sharma et al., 2014) l joint pain tharu (pr) (singh, 2017) international journal of applied biology, 5(2), 2021 159 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources r & b mouth ulcer, appetite, intestinal problems, wounds and tonic tharu (pr) (singh, 2017) 203. osbeckia stellata buch-ham. ex k. gawle, angeri, melastomataceae r abdominal disorders chepang (rijal, 2011) 204. oxalis corniculata l., chariamilo, oxalidaceae l stomachace bantar (acharya & pokhrel, 2006) earache darai (poudel & singh, 2016) acidity munda (ghimire, 2016) fever, influenza, diarrhoea, snake bite, hook worms, anthelmintic, and urinary tract infection tharu (pr) (singh, 2017) w fever, brushes and nose bleeding tharu (c) (dangol & gurung, 1991; mueller boeker, 1993) fever, dysentery, and diarrhoea tharu (r) (acharya & acharya, 2009) 205. paederia foetida l., bire lahara, rubiaceae s teeth infection chepang (rijal, 2011) r snake bite chepang (rijal, 2011) 206. pennisetum polystachion (l.) schult., mission grass, poaceae r abdominal pain chepang (rijal, 2011) 207. persicaria barbata (l.) h. hara, bish, polygonaceae l ring worms meche (rai, 2004) w swollen body tharu (c) (dangol & gurung, 1991) 208. phoenix humilis royle. ex becc., thakal, arecaceae sd itch tharu (c) (mueller boeker, 1993) sp stomachache and diarrhoea bantar (acharya & pokhrel, 2006) 209. phyla nodiflora (l.) greene, jal pippali, verbenaceae w ulcers and stomachache tharu (c) (dangol & gurung, 1991) 210. phyllanthus amarus schumach. & thonn., l diabetes tharu (pr) (singh, 2017) r menorrhagia tharu (singh, 2017) international journal of applied biology, 5(2), 2021 160 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources bhuinamala, phyllanthaceae (pr) w wounds, ulcers, and intermittent fever tharu (pr) (singh, 2017) 211. phyllanthus emblica l., amla, phyllanthaceae f anemia tharu (pr) (singh, 2020) cold chepang (rijal, 2011) scalp, dysentery and diarrhea munda (ghimire, 2016) sore throat inflammation tharu (pr) (singh, 2017) fl mental disorders and blood purifier bantar (acharya & pokhrel, 2006) l burns tharu (c) (mueller boeker, 1993) scalp tharu (pr) (singh, 2017) f& sd gastritis, diuretic, cooling, and laxative source of vitamin c tharu (r) (acharya & acharya, 2009) 212. physalis minima l., isamgoli, solanaceae l menstruation disorders and eye infection tharu (c) (dangol & gurung, 1991) 213. physalis peruviana l., rasbhari, solanaceae r jaundice meche (rai, 2004) piles bantar (acharya & pokhrel, 2006) 214. pilea symmeria wedd., aailanata, urticaceae b diarrhea chepang (rijal, 2011) fl wounds chepang (sharma et al., 2014)) 215. piper longum l., pipala, piperaceae l dry cough meche (rai, 2004) mouth refreshment, indigestion, easing urination, cough, and cold tharu (pr) (singh, 2017) f asthma, cough and chest pain chepang (rijal, 2011) tuberculosis kisan (rajbanshi & thapa, 2019) cough and digestion tharu (r) (acharya & acharya, 2009) indigestion chepang (sharma et al., 2014) r pneumonia meche (rai, 2004) 216. piper nigrum l., marich, piperaceae sd cough munda (ghimire, 2016) international journal of applied biology, 5(2), 2021 161 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 217. pistia stratios l., kumbhika, araceae w swelling bantar (acharya & pokhrel, 2006) 218. plumeria alba l., galaichi, apocynaceae fl fever darai (poudel & singh, 2016) 219. plumbago zeylanica l., chitu, plumbaginaceae b baldness tharu (pr) (singh, 2020) baldness tharu (pr) (singh, 2017) r digestion, appetite, ulcer, scabie, and several skin diseases tharu (pr) (singh, 2017) 220. pogostemon benghalensis (burm.f.) kuntz, rudilo, lamiaceae l cold, indgestion, cough, kwashiorkor and marasmus chepang (rijal, 2011) sinusitis and typhoid darai (poudel & singh, 2016) l & r headache and typhoid chepang (sharma et al., 2014) w fever tharu (c) (dangol & gurung, 1991) 221. pouzolzia zeylanica (l.) benn., ban paate, urticaceae w ring worms and wounds of fingers and toes tharu (c) (dangol & gurung, 1991) 222. premna barbata wall. ex schauer, gineri, lamiaceae l urine infection and heat sickness chepang (rijal, 2011) b fever tharu (c) (dangol & gurung, 1991) 223. primula umbellate (lour.) merr., umbelled rock jasmine, primulaceae l & fl insomnia and diarrhea bantar (acharya & pokhrel, 2006) 224. psidium guajava l., amba, myrtaceae l dysentery and diarrhea chepang (rijal, 2011) dysentery and diarrhea tharu (pr) (singh, 2020) urinary problems and mental disorders bantar (acharya & pokhrel, 2006) fever tharu (c) (mueller boeker, 1993) diarrhoea darai (poudel & singh, 2016) sd cough chepang (rijal, 2011) b diarrhoea and dysentery chepang (sharma et al., international journal of applied biology, 5(2), 2021 162 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 2014) diarrhea tharu (c) (mueller boeker, 1993) diarrhea kisan (rajbanshi & thapa, 2019) fl headache munda (ghimire, 2016) f diarrhea and dysentery meche (rai, 2004) 225. pueraria phaseoloides (roxb.) benth., birali lahara, fabaceae sp wounds chepang (rijal, 2011) 226. punica granatum l., anar, punicaceae f eye problem tharu (pr) (singh, 2020) sd & fl piles and tonic bantar (acharya & pokhrel, 2006) 227. rauvolfia serpentina (l.) benth. ex kurz, sarpagandha, apocynaceae r high blood pressure tharu (pr) (singh, 2020) malaria chepang (rijal, 2011) fever and snake bite tharu (c) (dangol & gurung, 1991) stomach problem and fever kisan (rajbanshi & thapa, 2019) cholera, diarrhoea, dysentery, uterine contraction during childbirth, snake bite, and malarial fever tharu (pr) (singh, 2017) l fever chepang (rijal, 2011) pneumonia meche (rai, 2004) mental disorders bantar (acharya & pokhrel, 2006) snake bite darai (poudel & singh, 2016) f reduce blood pressure, dysentery, and hypotonic tharu (r) (acharya & acharya, 2009) 228. rhododendron arboreum sm., laligurans, ericaceae fl dysentery chepang (rijal, 2011) bone prick darai (poudel & singh, 2016) 229. ricinus communis l mumps and increase lactation tharu (singh, 2020) international journal of applied biology, 5(2), 2021 163 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources l., arandi, euphorbiaceae (pr) sd cracking heels meche (rai, 2004) bantar (acharya & pokhrel, 2006) 230. rorippa benghalensis (dc.) h. hara, tori ghas, brassicaceae l malaria and headache bantar (acharya & pokhrel, 2006) 231. rotala rotundifolia (buch-ham. ex roxb.) koehne, sim jhar, lythraceae l hemicranias bantar (acharya & pokhrel, 2006) 232. rubus ellipticus sm., ainselu, rosaceae r dysentery chepang (sharma et al., 2014) 233. rungia parviflora (retz) nees, runchejhar, acanthaceae w cuts and wounds darai (poudel & singh, 2016) 234. saccharum officinarum l., ukhu, poaceae s jaundice darai (poudel & singh, 2016) munda (ghimire, 2016) tharu (pr) (singh, 2020) 235. saccharum spontaneoum l., kans, poaceae fl cuts bantar (acharya & pokhrel, 2006) 236. sansevieria frifasciata prain, snake plant asparagaceae l ulcers tharu (c) (dangol & gurung, 1991) 237. sapindus mukorossi gaertn., ritha, sapindaceae sd tonsillitis tharu (pr) (singh, 2020) 238. saraca indica l., ashoka, fabaceae b irregular menstruation tharu (pr) (singh, 2020) 239. saussurea graminifolia wall. ex dc., saw-wort, asteraceae l cough chepang (rijal, 2011) 240. schefflera venulosa (wight & arn.) hams, kursiulo, araliaceae l irregular menstruation and fever tharu (c) (mueller boeker, 1993) international journal of applied biology, 5(2), 2021 164 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources 241. schima wallichii (dc.) korth., chilaune, theaceae b burn, abdominal pain, taenia pedis, snake bite, wormicide and malnourishment chepang (rijal, 2011) 242. scindapsus officinalis schott, kanchiro, araceae r abortion and fracture chepang (rijal, 2011) s bodyache and backache chepang (sharma et al., 2014) 243. scoparia dulcis l., mitha jhar, scrophulariaceae l cough chepang (rijal, 2011) fever tharu (c) (dangol & gurung, 1991) veneral diseases and stomachache bantar (acharya & pokhrel, 2006) 244. semecarpus anacardium l. f., bhalayo, anacardiaceae f headache meche (rai, 2004) acute rheumatism, asthma, and dysentery tharu (r) (acharya & acharya, 2009) 245. sesamum orientale l., til, pedaliaceae sd sunburn and ringworms meche (rai, 2004) 246. shorea robusta roth, sal, dipterocarpaceae lt & b diarrhea and dysentery chepang (rijal, 2011) b diarrhea and dysentery chepang (sharma et al., 2014) f diarrhea and dysentery munda (ghimire, 2016) wounds munda (ghimire, 2016) t gingivitis kisan (rajbanshi & thapa, 2019) fracture darai (poudel & singh, 2016) appetizer tharu (c) (mueller boeker, 1993) 247. sida cordifolia l., balu, malvaceae l boils kisan (rajbanshi & thapa, 2019) 248. sida rhombifolia l., sano chilya, malvaceae l wounds chepang (rijal, 2011) wounds tharu (c) (mueller boeker, 1993) 249. solanum aculeatissimum jacq., kantakari, solanceae f fever and toothache tharu (c) (dangol & gurung, 1991) sd teeth germs meche (rai, 2004) r scabies bantar (acharya & pokhrel, 2006) 250. solanum anguivi r scabies bantar (acharya & international journal of applied biology, 5(2), 2021 165 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources lam., bihi, solanaceae pokhrel, 2006) 251. solanum indicum l., kande bihi, solanaceae f blood purifier and pneumonia tharu (pr) (singh, 2020) 252. solanum melongena l., bhanta, solanaceae r placenta retention darai (poudel & singh, 2016) 253. solanum nigrum l. jungali bihi, solanaceae r intermittent fever and easy child delivery bantar (acharya & pokhrel, 2006) 254. solanum tuberosum l., aalu, solanaceae tu burns munda (ghimire, 2016) 255. sonchus arvensis l., dudhi, asteraceae r typhoid chepang (sharma et al., 2014) 256. spatholobus parviflorus (dc.) kuntze, sal lahara, fabaceae b diarrhea chepang (rijal, 2011) r menstrual disorders tharu (c) (mueller boeker, 1993) 257. sphaeranthus indicus l., gorakhmundi, asteraceae r stomachache and fever bantar (acharya & pokhrel, 2006) 258. spilanthes calva dc., morathi, asteraceae l cuts and mun infections bantar (acharya & pokhrel, 2006) 259. spondias pinnata l. f. kurz, amaro, anacardiaceae l dysentery, pox and measles tharu (pr) (singh, 2020) b rheumatic arthritis tharu (pr) (singh, 2020) 260. stephania elegans hook. f. & thomson, taro lahara, menispermaceae l blood dysentery and boils bantar (acharya & pokhrel, 2006) 261. stephania japonica (thunb.) miers, badalpaate, menispermaceae r scorpion bite and abdominal spasm chepang (rijal, 2011) gastric and indigestion chepang (sharma et al., 2014) 262. stereospermum cheloniodes dc., patala, bignoniaceae b leprosy tharu (c) (dangol & gurung, 1991) sd migraine headache tharu (c) (mueller boeker, 1993) 263. streblus asper lour., l toothache tharu (singh, 2020) international journal of applied biology, 5(2), 2021 166 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources bedulla, moraceae (pr) 264. swertia angustifolia buch-ham. ex d. don, goru tite, gentianaceae s fever chepang (rijal, 2011) vomiting and stomachache munda (ghimire, 2016) r fever munda (ghimire, 2016) w anthelmintic, diarrhoea, fever, headache and stomach ache newar (balami, 2004) 265. swertia chirayita (roxb. ex fleming) h. karst, chiraito, gentianaceae l& s fever chepang (rijal, 2011) 266. syzygium aromaticum l., lwang, myrtaceae f tonsillitis darai (poudel & singh, 2016) sd & bu toothache munda (ghimire, 2016) 267. syzygium cumini (l.) skeels, jamun, myrtaceae b diabetes tharu (pr) (singh, 2020) dysentery meche (rai, 2004) dysentery and diarrhea munda (ghimire, 2016) f diarrhea chepang (rijal, 2011) diarrhea tharu (c) (mueller boeker, 1993) indigestion bantar (acharya & pokhrel, 2006) constipation and diabetes munda (ghimire, 2016) 268. tagetes erecta l., sayapatri, asteraceae l cut injury tharu (pr) (singh, 2020) cut injury kisan (rajbanshi & thapa, 2019) pneumonia and chest pain meche (rai, 2004) fever, cuts, and wounds munda (ghimire, 2016) 269. tamarindus indica l., imali, fabaceae f high blood pressure tharu (pr) (singh, 2020) sd stomachache meche (rai, 2004) fl piles bantar (acharya & pokhrel, 2006) 270. terminalia belerica (gaertn.) roxb., f bronchial asthma tharu (pr) (singh, 2020) international journal of applied biology, 5(2), 2021 167 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources barro, combretaceae chest pain and cough chepang (rijal, 2011) constipation meche (rai, 2004) gastritis darai (poudel & singh, 2016) cough and eye disease tharu (r) (acharya & acharya, 2009) 271. terminalia catappa l., kathe badam, combretaceae sd anemia bantar (acharya & pokhrel, 2006) 272. terminalia chebula (gaertn.) retz., harro, combretaceae b tonsilities kisan (rajbanshi & thapa, 2019) f chest pain and cough chepang (c) (rijal, 2011) gastric meche (rai, 2004) chest pain and bronchitis tharu (c) (mueller boeker, 1993) gastritis darai (poudel & singh, 2016) gastritis tharu (r) (acharya & acharya, 2009) 273. terminalia elliptica willd., asna, combretaceae b dysentery tharu (pr) (singh, 2020) abdominal pain, fracture, diarrhea, cuts and dysentery chepang (rijal, 2011) s wounds and inflammation chepang (rijal, 2011) 274. thespesia lampas (cav.) dalzell & a. gibson, ban kapas, malvaceae r fever, indigestion, typhoid, dysentery and hangover of alcohol chepang (rijal, 2011) sd constipation chepang (rijal, 2011) 275. thevetia peruviana (pers.) k. schum., kaner, apocynaceae f ear infection tharu (c) (dangol & gurung, 1991) l wounds bantar (acharya & pokhrel, 2006) 276. thysanolaena maxima (roxb.) kuntze, amriso, poaceae r fever and indigestion chepang (rijal, 2011) antihelmintic newar (balami, 2004) tharu (r) (acharya & acharya, 2009) l typhoid chepang (sharma et al., 2014) 277. tinospora cordifolia s diabetes, stomach problem tharu (singh, 2020) international journal of applied biology, 5(2), 2021 168 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources (thunb.) miers, gurjo, menispermaceae (pr) heat sickness chepang (rijal, 2011) veneral diseases bantar (acharya & pokhrel, 2006) earache darai (poudel & singh, 2016) 278. trachyspermum copticum link, jwano, apiaceae w cold, pimples, rashes, sore throat, wounds and pneumonia tharu (c) (mueller boeker, 1993) sd gastritis munda (ghimire, 2016) 279. trewia nudiflora l., bhelar, euphorbiaceae f ringworm kisan (rajbanshi & thapa, 2019) 280. tribulus terrestris l., gokhur, zygophyllaceae r miscarriage and body pain chepang (rijal, 2011) s abdominal spasm chepang (rijal, 2011) 281. trichodesma indicum (l.) r. br., kanike kuro, boraginaceae w headache tharu (c) (dangol & gurung, 1991) 282. trichosanthes dioica roxb., parbal, cucurbitaceae f heat sickness darai (poudel & singh, 2016) 283. tridax procumbens l., kurkure, asteraceae l cuts and wounds bantar (acharya & pokhrel, 2006) 284. trigonella foenum graceum l., methi, fabaceae sd cold and running nose kisan (rajbanshi & thapa, 2019) cough munda (ghimire, 2016) 285. triumfetta rhomboidea jacq, ballu kuro, malvaceae l boils bantar (acharya & pokhrel, 2006) 286. urena lobata l., dalle kuro, malvaceae l eczema and skin infections meche (rai, 2004) boils bantar (acharya & pokhrel, 2006) 287. urtica dioica l., sisnoo, urticaceae r snake and scorpion bite chepang (rijal, 2011) s fever chepang (rijal, 2011) blood pressure darai (poudel & singh, 2016) 288. vicia tetrasperma (l.) schreb., sd mental disorders bantar (acharya & pokhrel, 2006) international journal of applied biology, 5(2), 2021 169 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources aankura, fabaceae 289. viscum album l., hadchur, santalaceae b sprain and fracture chepang (rijal, 2011) cut injury and wounds chepang (sharma et al., 2014) w fracture darai (poudel & singh, 2016) fracture munda (ghimire, 2016) 290. vitex negundo l., simali, verbenaceae l stomach problem tharu (pr) (singh, 2020) constipation bantar (acharya & pokhrel, 2006) r rheumatic arthritis tharu (pr) (singh, 2020) 291. withania somnifera (l.) dunal, aswogandha, solanaceae l abdominal disorders darai (poudel & singh, 2016) 293. woodfordia fruticosa (l.) kurtz, dhiero, lythraceae fl diarrhoea and dysentery chepang (rijal, 2011) b &r diarrhoea chepang (sharma et al., 2014) 294. xanthium strumarium l., bhende kuro, asteraceae l eczema bantar (acharya & pokhrel, 2006) 295. xeromphis spinosa (thunb.) keay, main kanda, rubiaceae f sbdominal spasm chepang (rijal, 2011) b leprosy tharu (c) (dangol & gurung, 1991) 296. xeromphis uliginosa (retz.) maheshw, pidar, rubiaceae f diarrhea tharu (c) (mueller boeker, 1993) 297. youngia japonica (l.) dc., dulla jhar, asteraceae w appetizer, blood purifier, constipation and boils bantar (acharya & pokhrel, 2006) 298. zanthoxylum armatum dc., timur, rutaceae f oedema, abdominal spasm, teeth infection and cholera chepang (rijal, 2011) sd indigestion chepang (sharma et al., 2014) 299. zingiber officinale roscoe, aduwa, zingiberaceae rh cough tharu (pr) (singh, 2020) cold tharu (c) (mueller boeker, 1993) cough, cold, hair fall and kisan (rajbanshi & international journal of applied biology, 5(2), 2021 170 s.n. scientific names, vernacular names and family names p. u. diseases treated ethnic groups sources sinusitis thapa, 2019) vomiting and cough munda (ghimire, 2016) 300. ziziphus mauritiana lam. bayer, rhamnaceae b dysentery and diarrhea tharu (pr) (singh, 2020) sd chicken pox tharu (c) (mueller boeker, 1993) abdominal pain munda (ghimire, 2016) l heart burn tharu (c) (mueller boeker, 1993) f cough darai (poudel & singh, 2016) the figure 2 shows the number of diseases treated by ethnic tribeal groups of terai region using different parts of plant body or whole plant body where it is clear that maximum number of diseases are treated using leaf (72) of plants then, by the use of bark (55) followed by root (48). buds of plant (1) is least used for the treatment of diseases by ethnic tribal people of terai. figure 2. bar graph showing number of diseases treated by using different parts of plant body or whole plant body. 27 55 38 72 48 25 9 12 14 12 29 25 1 5 41 0 10 20 30 40 50 60 70 80 n u m b e r o f d is e a se s t re a te d parts of plant used international journal of applied biology, 5(2), 2021 171 conclusions and recommendation this study provides ethnomedicinal information about the plant species used by major ethnic tribes of the terai region of nepal. a total of 300 plant species from 98 families are documented in this study. the maximum number of plants used by the tribal group of terai is from fabaceae (26.53%) family followed by asteraceae (19.38%), and then lamiaceae (14.29%). these plant species are used in the treatement of 120 types of diseases and ailments by 8 ethnic groups residing in six terai districts, namely chitwan, parsa, nawalparasi, rupandhehi, jhapa, and morang. many parts of a plant or whole plant body are found to be used by the tribal people among which leaf is used for the treatment of a maximun number of diseases (72) and bud is the least used body part (1). many ethnic and indigenous knowledge about plant species, their identification, collection process, and treatment of diseases must be documented and passed on from generation to generation. this continuity of ethnic tribal knowledge on medicinal plants and their pharmacological values could widen the scope and importance of herbal medicine science. moreover, ethnomedicinal knowledge ensures the cure of the diseases and ailments locally which ultimately saves time, money and prevents extreme situations. researches and explorations should be carried out about the phytochemical as well as pharmacological values of medicinal plants and the knowledge must be utilized for future medical endeavors. conflicts of interest the authors declare no conflict of interest. author contribution statements all the authors were involved in concept development, literature review, and analysis. m.s. miya and s. timilsina reviewed the literature, analyzed the quantitative data, and prepared the draft manuscript. 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indigenous health management of tharu tribals in the eastern part of parsa, nepal. journal of pharmacognosy and phytochemistry. 9(3):268-274. https://doi.org/10.22271/phyto.2020.v9.i3d.11277 world health organization (who). 2002. who traditional medicine strategy. who, geneva https://doi.org/10.3126/eco.v24i0.20641 https://doi.org/10.22271/phyto.2020.v9.i3d.11277 3(1)55-61 contact : reni yunus reni_yunus@yahoo.co.id © international journal of applied biology 55 abstract nowadays many plants can be used as traditional medicines to overcome various diseases including infections, because the use of traditional medicines is relatively safer compared to drugs derived from chemicals. one of the local plants in southeast sulawesi that has been empirically used as a medicine for wounds is a race of plants. komba-komba leaves chromolaena odorata containcompounds alkaloid, flavonoids, tannins, phenols, triterpenoids, saponins and steroids which have their respective functions in inhibiting the growth of microorganisms or antibacterial. this study aims to determine the inhibition of komba-komba leaf extract chromolaena odorata on the growth ofbacteria staphylococcus aureus. this type of research is experimental laboratories. the method used was agar diffusion with 5 treatment concentrations, namely komba-sheep leaf extract concentration chromolaena odorata 20%, 40%, 60%, 80% and 100%, positive control (tetracycline) and negative control (aquadest) and repetition . the results showed that there was a inhibition zone of komba-komba leaf extract chromolaena odorata on the growth ofbacteria staphylococcus aureus at a concentration of 20% at 2 mm, a concentration of 40% at 3.7 mm, a concentration of 60% at 4.7 mm, a concentration of 80% 6.2 mm and a concentration of 100% by 8 mm. issn : 2580-2410 eissn : 2580-2119 inhibition of juice komba-komba chromolaena odorata leaf against growth of bacteria staphylococcus aureus reni yunus, muh.hilman & satya darmayani department of health analyst, poltekkes kemenkes kendari introduction indonesia is a tropical country that has high biodiversity, making indonesia has many plants used by the community as traditional medicine. in indonesia there are around 30,000 types of plants and 7,000 of them have medicinal properties. indonesia's diversity of biological resources ranks second after brazil (jumiarni & komalasari, 2017). all types of plants have long been targeted as new drug searches. the development of drug use, especially from plants to help improve public health. one of the benefits of using drugs from these plants is the kirinyuh (plantchromolaena odorata) which is used as an antibacterial (awoyinka, 2007). empirical studies on the local community of the muna tribe show the use of this leaves as a wound healer. kirinyuh leaves chromolaena odorata in the local community of the muna tribe are known as the leaves of "komba-komba" (jumiarni & komalasari, 2017). open access international journal of applied biology international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. keyword chromolaena odorata, staphylococcus aureus, antibacterial activity article history received 13 february 2019 accepted 24 june 2019 international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 56 indonesia is a tropical country so the prevalence of infectious diseases caused by bacteria is still high. one of the bacteria that infects wounds on the skin is thebacteria staphylococcus aureus. bacteria staphylococcus aureus infects wounds, zits, ulcers and others. these wounds are usually treated with antibiotics which can inhibit inflammation and kill bacteria, for example tetracycline, erythromycin, cloromhenicol, doxycycline and clindamycin. however, these drugs have side effects, namely irritating the skin to the wound or to zits, while the use of long-term antibiotics can also cause resistance and can cause organ damage and immuno hypersensitivity (djajadisastra, 2009). the prevalence of skin infections is increasing. based on the results of the nindya nugerahdita 2009 study on the prevalence of skin disease and treatment in the petamburan area of central jakarta, the results showed that the prevalence of skin diseases of (47.57%) of 103 families observed in the petamburan village in central jakarta with the most skin diseases was caused by ( 71.43%) and the remainder is caused by bacteria of (28.57%). in this era, many plants can be used as traditional medicines to treat various diseases including infections, because many people assume that the use of traditional medicines is relatively safer compared to drugs derived from chemicals. one of the plants that can be used as medicine is the komba-komba or kirinyuh plants. leaves of komba-komba or kirinyuh chromolaena odorata contain compounds alkaloid, flavonoids, tannins, phenols, triterpenoids, saponins and steroids which have their respective functions in inhibiting the growth of microorganisms or antibacterials (dalimartha, 2000). previous research has shown that komba-komba leaves extract ointment or at a concentration of 20% has an effect in accelerating wound healing in laying hens (rahman, 2017). in this study we used komba komba leaf extract without the addition of other chemical solvents such as ethanol or methanol to obtain active compounds. the reason researchers chose juice of komba komba leaf, without addition the chemical solvents, is that in its application as a natural antibiotic, people can make it easier. based on the antibiotic content found in komba-komba leaf (choromolaena odorata) which has efficacy as an antibacterial, researchers are interested in conducting research on "inhibition test of juice komba-komba leaf (choromolaena odorata) on the growth of bacteria staphylococcus aureus". the purpose of this study was to determine the inhibition of juice komba-komba leaf chromolaena odorata to the growth of staphylococcus aureus and to determine the inhibition of juice komba-komba leaf chromolaena odorata on the growth ofbacteria staphylococcus aureus at concentrations of 20%, 40%, 60 %, 80% and 100%. materials and methods this study was conducted in march-june 2018 at the microbiology laboratory of the department of health analyst at the poltekkes kendari. the method used is experimental laboratories, using the one-shot case study design, namely research design with treatment ofvariables independent. the inhibitory test of juice komba-komba chromolaena odorata leaf using method agar diffusion. data obtained by measuring the diameter of the inhibitory zone formed. measurements were made twice with positive control tetracycline of 19 mm. international journal of applied biology, 3(1), 2019 57 tools and materials the tools used are digital balance, horn spoon, erlenmeyer, stirring rod, measuring pipette, ball filler, measuring cup, lamp spirits, three feet, wire mesh, incubator, atoclave, porcelain cup, test tube, tube rack, wire ose, tweezers, spray bottle, blender, filter paper, dropper pipette, ruler / ruler, funnel, beaker. the ingredients used were leaves of sheep chromolaena odorata, antibiotics tetracycline,disc paper blank,media nutrient agar (na), aquadest, filter paper, label paper, aluminum foil, pure culture of staphylococcus aureus. pure stock making of staphylococcus aureus bacteria the test bacteria used or bacteria to be purified is staphylococcus aureus. the making of bacterial stock is carried out using ose then planted or inoculated by scraping on themedia na slopingand then incubated in an incubator temperature of 370c for 1 x 24 hours. making test bacterial suspensions. the making of bacterial suspension was taken by test bacteria by using sterile ose wire and then suspended in 2 mlnacl 0.9%in a sterile test tube and homogenized according to the standard mc. farland 0.5 which is characterized by the formation of turbidity after being suspended. sampling and preparation of samples leaves of komba komba chromolaena odorata taken are cleaned in washing with running water then stored at room temperature until dry after that the leaves are weighed as much as 500 grams then in a blender. making juice of komba-komba leaves chromolaena odorata weighed 500 grams of leaves of the sheep chromolaena odorata using digital scales and then blended and squeezed and filtered as much as 150 ml ago made in 5 variations of concentration, namely in variations in concentrations of 20%, 40%, 60%, 80%, and 100%. preparation of tetracycline antibiotic (positive control) tetracycline 250 mg created a concentration of 5% by weighing 0.5 grams of tetracycline then dissolved tests antibacterial activity antibacterial activity tested by usingmethod agar diffusion (disk diffusion method)of the kirby-bauer. themethod is diffusion carried out by means of a 0.1bacterial suspension ml spread on the surface ofmedia nutrient agar (na)by using ski gel then the paper disc is dipped in the leaf extract of the sheep chromolaena odorata at each concentration of 20%, 40 %, 60, 80% and 100% using tweezers stored on the surface ofmedia nutrient agar (na)then labeled using label paper. then a positive control was made using tetracycline paper disc dipped in tetracycline for positive control and planted on the surface of the media nutrient agar (na) and then incubated for 1x24 hours temperature 370c. results and discussion this study shows that the juice of the leaf komba-komba chromolaena odorata has a inhibitory effect on staphylococcus aureus. testing of inhibition was carried out twice repetition, the first major inhibition was found at a concentration of 100% with a diameter of 10.5 mm and the smallest inhibition was found at a concentration of 20% with a diameter of international journal of applied biology, 3(1), 2019 58 1.5 mm while in the repetition of both power the biggest inhibition was at a concentration of 100% with a diameter of 5.5 mm and the smallest inhibition was found at a concentration of 20% with a diameter of 2.5 mm. for the results of research the inhibitory zone can be seen more clearly in figure 1 and table 1 below. figure 1. inhibition zone formed after incubation table 1. results measurement of inhibition zones juice of komba-komba leaf chromolaena odorata on the growth of bacteria staphylococcus aureus no concentration observaton time average diameter of inhibition zone interpretation 1 20 % 24 hours 2 mm resistent 2 40 % 24 hours 3,7 mm resistent 3 60 % 24 hours 4,7 mm resistent 4 80 % 24 hours 6,2 mm resistent 5 100 % 24 hours 8 mm resistent 6 control (+) 24 hours 19 mm sensitive 7 control (-) 24 hours discussion in the test of inhibitory power of juice of komba-komba chromolaena odorata leaf which will be tested on bacteria staphylococcus aureus with using method agar diffusion or scatter method with juice of komba-komba leaf testing chromolaena odorata made in 5 variations of concentration, namely concentrations of 20%, 40%, 60%, 80% and 100% carried out in the laboratory of health polytechnic kendari department of health analyst. the testing of inhibition power of juice komba-komba leaf chromolaena odorata on the growth of bacteria was staphylococcus aureus carried out in several stages, starting from the leaf selection stage to testing the bacterial inhibition. the stage of leaf selection is done by selecting leaves that are still young and taken by manual then carried out until the stage of making the concentration for testing the inhibitory power. testing the inhibition of the growth of staphylococcus aureus by using themethod diffusion to be incubated for 1 x 24 hours in an incubator with inhibition zones was characterized by the formation of clear areas around the paper disc, using drugs tetracycline as a positive control and aquadest as a negative control. the inhibitory power of juice komba-komba chromolaena odorata leaf at a concentration of 20% inhibition zone formed an average of 2 mm. the concentration of 40% of the inhibition zone formed 3.7 mm. the concentration of 60% of the inhibition zone formed 4.7 mm. concentration of 80% inhibition zone 6.2 mm. the concentration of 100% of the inhibition zone formed is 8 mm. so that from the 5 concentrations formed a clear area around the paper disc which is called the inhibitory zone. the inhibitory zone formed is still categorized as resistant (weak) because of the large inhibition zone formed less than 12 mm but at a concentration of 100% in this test although it is included in the category of resistance international journal of applied biology, 3(1), 2019 59 but effective in inhibiting the growth of staphylococcus aureus because it has the greatest inhibitory power testing this obstacle. this is in line with the research of purwati and undri rastuti (2009) explaining ethyl acetate leaf euphatorium odoratum extracts based on screening of secondary metabolites containing flavonoids, with secondary metabolites in extract of ethyl acetate leaves euphatorium odoratum has antioxidant activity with a sequence of inhibitory activities of 0.15 % (b / v). flavonoids denaturate bacterial cell proteins and can damage bacterial cytoplasmic membranes so that they can inhibit the growth of bacteria staphylococcus aureus in testing the inhibitory power. in another study (rahman, 2017) also used chromolaena odorata leaf extract incisive forwound healing in laying hens, which in their study showed that the effective concentration of deep wound healing in laying hens was at a concentration of 20%. the previous research by vital and rivera (2009) in his research carried out a test of the antimicrobial activity ofleaf extract chromolaena odorata, the results showed 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unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. issn : 2580-2410 eissn : 2580-2119 reevaluation of land fitness for soybean plant in kabamatan stabat, langkat district leni handayani1, a rauf2, rahmawaty3, t supriana4 1department of of agricultural science, universitas sumatera utara, indonesia. 2department of agriculture agroecotechnology, universitas sumatera utara, indonesia. 3department of agriculture forestry, universitas sumatera utara, indonesia. 4department agriculture agribusiness, universitas sumatera utara, indonesia. article history received 23 january 2020 accepted 03 march 2020 keywords farming, soybean, agricultural, land suitability. introduction in increasing crop productivity, the government is more focused on doing business related to increasing non-physical production such as expansion of crop areas, providing superior seeds and counseling on eradicating pests. though plant production is not only influenced by non-physical conditions but also must pay attention to other physical conditions. as food, soybean ranks number 3 after rice and corn as the main commodity of food crops in the food and feed industry in indonesia (isnowati, 2014). in indonesia, the total area of soybean plantations has decreased by 41.6% over the last 20 years, namely from 1995 with a planting area of 1,476,284 ha until 2015, which only reached 614,095 ha of planted area (bps, 2016). of the 2015 land area, only 963,183 tons of soybeans were produced contact : leni handayani leni.handayani99@yahoo.com 15 abstract a decrease in the area of soybean farming has an effect on reducing soybean production from year to year so that it has not been able to meet the needs of national soybean consumption. land suitability assessment is an effort to be able to optimize land use. in the process of assessing land suitability manually, it is considered inaccurate. the purpose of this study was to determine the land suitability class for soybean plants. the land suitability classification system used is the fao land suitability classification classified at the sub-class level. land suitability evaluation uses a matching system, as well as comparing the characteristics of land with plant growing community formulated in the technical evaluation of land guidelines for agricultural commodities. in the matching process leibig's minimum law is used to determine the limiting factors that will affect the suitability of the class and sub-class of the land. requirements for growing plants become criteria in conformity evaluation. the results showed that the limiting factors of land suitability for soybean plants that had to be adjusted were temperature, rainfall, soil texture, c-organic, n-total and p available soil. the limiting factor of temperature and soil texture cannot be improved so that the marginal fit class (s3) on actual land suitability remains marginal fit (s3) in terms of potential land suitability. mailto:leni.handayani99@yahoo.com international journal of applied biology, 4(1), 2020 16 the amount of soybean production in 2015 has not been able to meet the needs of national soybean consumption which reached 2.87 million tons / year which will be predicted to continue to increase from year to year due to population growth (global agricultural information network, 2016). the still minimal use of land due to the decrease in soybean land area from year to year has an effect on the production of soybean produced. according to the research of pilvere, et al.,(2014) states that inappropriate land use will have implications for low production which will have an impact on inefficient use of land resources. one effort to increase soybean agricultural production output is to allocate planting in suitable land (widiatmaka, et al.,2016). land use planning in the form of land suitability assessment is important to allocate soybean planting on suitable land to optimize land use (dengiz, et al.,2013) and can also be used as a solution to increase production (zhang, et al.,2015) soybeans to be sufficient national consumption needs. land suitability assessments are important as a prerequisite for sustainable agriculture and play a role in planning for increasing agricultural productivity (baniya, 2008; neupane, et al.,2014). besides aiming to increase agricultural productivity, assessing land suitability also takes into account the element of protection (fao, 1993; wali, et al.,2016). the intended protection is to protect existing natural resources from the dangers of damage such as avoiding land degradation or erosion due to the continuous land management process (fao, 1976; elaalem, et al.,2011). lack of understanding of land suitability will have an impact on environmental disturbances that have consequences on long-term problems that are difficult to change (rodrigo, et al.,2004). materials and methods materials and tools used during research are materials and tools commonly used for soil surveys and laboratory activities. the material used in the form of topographic maps, land maps, land use maps, administrative maps of stabat district and other maps related to research. equipment used both in the field or in the laboratory, namely soil drill to take soil samples and determine the effective depth of field in the field, abney level to determine the slope. gps (global positinoning system) to determine the position of the study area and measure the altitude and a set of tools used for soil analysis in laboratories. the study used a randomized block design (rbd) with 8 factorial x and 4 factorial y. where the soil sample conditions will be seen namely temperature, water, oxygen content, root media, nutrient retention, nutrient availability, erosion, flooding, and land preparation. on land suitability of the 8 factorial x will be given an assessment s1 = very suitable s2 = quite suitable s3 = appropriate and at the management level x2 namely: moderate, low, fertilizer and value acquisition. in factorial y there are 4 divisions namely: 1. actual land suitability 2. the heaviest limiting factor 3. repair and business 4. potential land with the following formula: yij = µ + ti + bj + ε ij; i = 1, 2, 3 ... t j = 1, 2, 3 ... r international journal of applied biology, 4(1), 2020 17 yij = response or observation value from treatment i and repeat test j µ = general midpoint ti = the effect of the i-th treatment bj = jth block's influence ε ij = the effect of experimental error from the i-th and j-replication treatments results and discussion table 1. soybean (glycine max) land suitability assessment in langkat regency temperature regime (tc) average temperature (oc) 25.8 s2 tc s2 s2 water availability (wa) rainfall during growth (mm) average humidity(%) oxygen avaliability (oa) 1805 81 s2 wa s2 s2 d s s2 s1 drainage good rooting media (rc) s1 s3 rc s3 soil texture coarse material(%) land depth(cm) rather rough s3 29 s2 110 s1 nutrition retention (nr) s3 nr s1 cec soil (me/100g) 22.28 (s) base saturation (%) 32.84 s1 s1 ph h2o 6.25 s1 c-organic (%) 0.61 s3 o s avaliability of hara (na) s2 na s1 n-total (%) 0.10 (r) s2 p s p2o5 bray ii (ppm p) 9.47 (s) s2 p s k-exchange (me/ 100 g) 0.625 (t) s1 danger of erotion(eh) s1 slope (%) < 2 s1 danger of erotion sr s1 flood hazard (fh) s1 flood period f0 s1 land preparation (lp) s1 surface rock(%) 0 s1 rock outcrops 0 s1 land suitability value s3 rc, nr s3 rc keterangan : s1 = very appropriate tp = management level o = organic material s2 = sufficiently appropriate s = medium r =low t = high s3 = marginal accordance p = inp fertilization inp = input d = drainage conformity of potential land tp inp repair bussiness heaviest limiting factor value actual land suitability land characteristic international journal of applied biology, 4(1), 2020 18 d factor c factor hara danger of flood hazard land availability erotion preparation nutrition retention b factor water oxygen rooting availability availability media a factor 120 100 80 60 40 20 0 land characteristic figure 1. land characteristic of langkat regency the limiting factors of land suitability for soybean plants that must be improved are temperature, rainfall, soil texture, c-organic, n-total and p-available soil. the limiting factor of temperature and soil texture cannot be improved so that the marginal fit class (s3) on actual land suitability remains marginal fit (s3) in terms of potential land suitability. this is in accordance with the statement of mubekti (2012) which states the land limiting factors consist of two types, namely (1) permanent limiting factors in the sense that it is very difficult to repair if it will be opened for agricultural business, such limiting factors, such as temperature, soil texture , altitude, and (2) limiting factors that can be corrected, for example soil fertility, al toxicity elements, soil acidity. the limiting factor of high annual rainfall (1805 mm) can be done with the management of water systems with moderate to high level of management, namely by making drainage channels so that the class is quite suitable (s2) on the suitability of actual land to be very suitable (s1) in terms of potential land suitability. the limiting factor of n-total soil can be improved with a moderate level of management, namely by fertilizing according to plant needs. estimated total n requirement of soybean plants for very suitable criteria requires 0.51% while the total n availability found in the soil at the time of the study was 0.10% so that an additional 0.40% is needed which is equivalent to 80 kg n / ha or 173.91 kg urea / ha. the total fertilizer needed for soybean plants in the area requires an average increase of 25% to meet the needs of microbes and nutrient loss n so as to need the addition of fertilizer as much as 100 kg n / ha or 217.39 kg urea / ha. that way the suitability for total n becomes very suitable (s1) from the quite appropriate class (s2). the p2o5 limiting factor of the soil can be improved with a low level of management that is by fertilizing according to plant requirements. the need for p2o5 soybean plants according to the criteria is very suitable, it requires 16 ppm while the availability of p2o5 contained in the soil at the time of the research is 9.47 ppm so that an additional 6.53 ppm is international journal of applied biology, 4(1), 2020 19 needed which is equivalent to 130.6 kg p2o5 / ha equivalent to 362.77 kg sp36 / ha. the total fertilizer needed for soybeans in the area required an additional 25% on average to meet microbial needs and phosphate loss so as to need the addition of fertilizer as much as 163.25 kg p2o5 / ha or 453.46 kg sp36 / ha. that way the suitability for pospat becomes very suitable (s1) of the class quite appropriate (s2). the limiting factor of c-organic can be improved with a moderate level of management, namely by the addition of organic matter. estimation of soybean c-organic requirement for very suitable criteria requires 3% while the availability of c-organic in the soil during research is 0.61% so an additional 2.39% is needed which is equivalent to 47.8 tons c / ha or 82.4 tons of material organic / ha. however, the provision of organic material can be adjusted to the ability of farmers and the availability of organic material in the area. according to akil et al (2015) the application of manure, or ashes of rice straw as a cover in the planting hole of 1-3 t / ha is considered optimal. that way the land suitability class criteria for c-organic become very appropriate (s1) from the marginal appropriate class (s3). conclusion the results of this study indicate that the adjustment of land suitability classes in the study area for soybean plants is included in the marginal suit class (s3), so that from the planting of soybean commodities it produces less than maximum production with rainfall limiting factors, nutrient retention. to improve the limiting factor for nutrient retention by adding organic material, the limiting factor for rainfall is quite high by making drainage channels. references central bureau of statistics. 2016. soybean harvested area by province (ha), 1993-2015. jakarta baniya, nabarath. 2008. land suitability evaluation using gis fo vegetable crops in kathmandu valley /nepal. institute of horticutural scienc faculty of agriculture and horticulture, humboldt university zu berlin, berlin, germany. dengiz, orhan, mehmet arif o¨zyazici, and mustafa sag˘lam. 2013. multi-criteria assessment and geostatistical approach for determination of rice growing suitability sites in gokirmak catchment. the international society of paddy and water environment engineering and springer japan. fao, 1976. a framework for land evaluation. soils bulletin 32, food and agriculture organization of the united nations, rome, italy. fao, 1993. guidelines for land-use planning. fao development series 1, vol.8 (96): food and agriculture organization of the united nations, rome, italy. global agricultural information network. 2016. indonesia oilseeds and products annual report 2016. gain report: usda foreign agricultural service. harseno, edy dan vickey igor r tampubolon. 2007. application of geographic information systems in mapping administrative, land, geological, land use, slopes, special region of yogyakarta and watershed areas in central java using arcview gis software. scientific magazine ukrim edisi 1/th xii/2007. isnowati, sri. 2014. factors affecting soybean production in kebonagung village, tegowanu district, grobogan regency. sepa : vol. 10 no.2 februari 2014 : 177 – 185. pilvere i., a. nipers, dan i. upite. 2014. agricultural land utilization efficiency: the case of latvia. international journal of trade, economics and finance, vol. 5, no. 1, february 2014. international journal of applied biology, 4(1), 2020 20 sicat, rodrigo. s, dkk. 2004. fuzzy modeling of farmers’ knowledge for land suitability classification. agricultural systems 83: 49–75. widiatmaka, wiwin ambarwulan, yudi setiawan,and christian walter. 2016. assessing the suitability and availability of land for agriculture in tuban regency, east java, indonesia. applied and environmental soil science volume 2016, article id 7302148, 13 pages. zhang, jiuquan, yirong su, jinshui wu, and hongbo liang. 2015. gis based land suitability assessment for tobacco production using ahp and fuzzy set in shandong province of china. computers and electronics in agriculture 114 leni handayani1, a rauf2, rahmawaty3, t supriana4 introduction materials and methods results and discussion table 1. soybean (glycine max) land suitability assessment in langkat regency land suitability value s3 rc, nr s3 rc conclusion contact : izevbuwa, osazee ekundayo osazee.izevbuwa@iuokada.edu.ng 44 abstract this study was conducted to determine the prevalence of intestinal parasites from volunteered individuals in the study site. a total of 300 stool samples, comprising 150 females and 150 males were collected randomly from the study population in ovia north-east local government area in edo state, aged between 1 and 60 years. the stool samples were aseptically collected and examined using both direct smear method and formol ether sedimentation techniques to determine the individual parasite load of the sampled population. the age group of 16-20 had the highest number of participants 102(34%) while the age group of 6-10, 51-55 and 56-60 had the least number of participants of 3(1%) respectively. out of the 300 stool samples examined 87, (29%) were positive with intestinal parasites while 213(71%) were negative, not having any intestinal parasitic infection however the age group of 11-15(n=10) and 31-35(n=10) had the highest prevalence rate of 11.5%, while the lowest prevalence (1%) was found in individuals of ages 41-60 years. there was no intestinal parasites found among the individuals below11 years of age.. of the 150 male candidates examined, 57 (65.5%) were infected with intestinal parasites. on the other hand, their female counterparts had a total prevalence of 34.5%. a total number of six parasitic species were isolated from the fecal specimens. these were ascaris lumbricoides, trichuris trichiura, strongyloides stercolaris, entamoeba coli, hookworms and entamoeba histolytica. ascaris lumbricoides had the highest prevalence rate (12%), while trichuris trichiura and strongyloides stercolaris had the least prevalence rate (1%) respectively. the intestinal parasite infection and age were negatively correlated and there was a significant difference between the male and female infection rates (p<0.05). issn : 2580-2410 eissn : 2580-2119 prevalence of intestinal parasites amongst selected age groups within okada, south –south nigeria. okafor-elenwo, e. j., izevbuwa, e.o., & akpoka, o. a. department of biological science, college of natural and applied sciences, igbinedion university, okada, nigeria. introduction intestinal parasites are parasites that populate the gastro-intestinal tract, typically protozoa and helminthes which are the two major types of intestinal parasites (udensi, et al., 2015). there are over 65,000 species of protozoa found in nature existing in various forms of open access international journal of applied biology keyword prevalence, intestinal parasites, formol ether, helminthes, protozoans. article history received 03 march 2020 accepted 14 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 45 symbiotic relationships with man and his domestic animal who becomes victims of the parasites (udensi, et al., 2015). parasitic infections of the gastro-intestinal tract of man are amongst the most common infections globally and it is estimated that about 3.5 billion people are affected with children being more susceptible to the infection due to their vulnerability to nutritional deficiencies (udensi, et al., 2015; nxasana, et al., 2013,). the helminthes t. trichiura, ascaris lumbricoides and the hookworm as well as the protozoa entamoeba histolytica have been observed to cause infections of varying degrees in 48 million people worldwide (udensi, et al., 2015; ekpo, et al., 2008; elliott, et al., 2007; alaofe, et al., 2008). intestinal parasitic infections are a serious public health problem because they can cause iron deficiency, anaemia, growth retardation in growing adults and children as well as causing varying physical and mental health conditions (nxasana et al., 2013; esiet, et al., 2017). high prevalence of infections associated with intestinal parasites can be attributed to poverty, poor environmental hygiene and inadequacies in medical services (nxasana et al., 2013; odo, et al., 2016; akinbo, et al., 2011; kwitshana, et al., 2008; ostan, et al., 2007;okyay, et al., 2004). in nigeria, a considerable amount of human and animal wastes are discharged into the soil daily leading to seepage with pathogenic organisms which includes cysts, eggs and larvae of these parasites (udensi, et al., 2015). materials and methods sterile universal specimen bottles (with screw caps) without preservatives were used to collect stool samples from the participants dung. this study’s unique identification numbers were also assigned to the sample bottles matching the biodata of the individual participant provided in a re-filled questionnaire. all participants were asked to transfer an aliquot of their early morning stool samples into the sterile labelled sample bottles. the samples were then received and transported in cooler bags to the microbiology laboratory of igbinedion university, okada within two hours of sample collection. properly collected stool samples were then processed using formalin ethyl-acetate concentration technique as described by nyundo et al., (2017). after concentration, a drop from the sediment was placed onto a sterile, grease-free microscope slide and covered with cover slip. the entire cover slip was examined with 10x objective and 40x objective respectively to observe the presence and morphology of eggs, cysts or larvae of intestinal parasites. the results obtained from each sample were recorded in special record sheets. results the total number of individuals examined was 300. these were grouped into different age categories with an interval of 5 years, for each class boundary. the highest number of individuals was found in the class boundary of 1620 years, with a total number of 102 individuals. the least number of participants (3 each) was obtained from the class boundaries of 6-10, 51-55 and 56-60 years respectively. the variation in the number of participants in different age groups was likened to the availability of the individuals at the time of study. international journal of applied biology, 4(1), 2020 46 the sex related distribution of the participants is shown in figure 2. there were equal number of male and female participants showing a prevalence of 50% each. out of the 300 individuals examined in the various age groups, 87 had intestinal parasites in their stools. prevalence of the infection is 29% while the uninfected had a prevalence of 71%. the age prevalence of the infection is shown in figure 4. the highest prevalence was obtained from the 16-20 years and 31 – 35 years age categories. this was followed by the 1115 years category with a total prevalence of 9%. out of the 87 infected individuals, the males were 57 representing a prevalence of 65.5% while the females were comparatively lower both in the number of individuals (n=30) and the prevalence rate(34.5%). the total number of intestinal parasites obtained from the fecal specimens was 87, out of which, the parasite species were six in number. the prevalence of the individual parasite group showed that a. lumbricoides had the highest prevalence rate compared with the other parasitic species. the lowest occurring parasites were t. trichiura and s. stercoralis, fig. 5 figure 1. age distribution of the subjects frequency; 1-5; 9 frequency; 6-10; 3 frequency; 11-15; 72 frequency; 16-20; 102 frequency; 21-25; 51 frequency; 26-30; 30 frequency; 31-35; 9 frequency; 36-40; 6frequency; 41-45; 6frequency; 46-50; 6 frequency; 51-55; 3frequency; 56-60; 3 1-5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 international journal of applied biology, 4(1), 2020 47 figure 2. sex distribution of subjects figure 3. percentage of subjects with intestinal parasites number of subjects; male ; 150; 50% number of subjects; female; 150; 50% subjects; positive; 87; 29% subjects; negative; 213; 71% international journal of applied biology, 4(1), 2020 48 figure 4. age related prevalence of intestinal parasites figure 5. gender prevalence of positive samples. 1-5 6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 male 65.5% female 34.5% international journal of applied biology, 4(1), 2020 49 figure 6. prevalence of parasites discussion in this study, the prevalence of 29% gastro-intestinal parasites was observed amongst in-patients of igbinedion university teaching hospital, okada, edo state. this result is similar to the report by ede, et al., on the prevalence of intestinal parasites among primary school children in wukari l.g.a of taraba state, nigeria. however, in some rural communities in north – eastern nigeria, a higher prevalence of 80.9% was reported amongst almajiri school children (damen, et al., 2013). similar results of (58.5%) and (51.0%) were also observed in rural communities in benue state, nigeria. similar prevalence rate of intestinal parasites of 29.0% was also recorded among preschool children in some urban communities in benue state, nigeria. the low percentage rate of infection recorded in these studies may be attributed to public enlightenment and sensitization on good environmental and personal hygiene and accessibility to health care facilities. the presence of six (6) species of intestinal parasites in this study area suggests that the prevailing environmental conditions support a wide range of intestinal parasites. among the helminthes observed, the most prevalent was ascaris lumbricoides with 12% occurrence, followed by hookworm with 10%. for the protozoan parasites, e. histolytica had the highest prevalence rate of (3%) while strongyloides stercoralis and trichostrongylus spp both had the least prevalence of 1% each. these patterns observed are in line with the findings of some researchers in parts of nigeria (udensi, et al., 2015; akinbo, et al., 2011 and kpurkpur et al., 2016). according to udensi, et al., 2015, the patterns of intestinal parasites distribution among school age children may be attributed to factors such as poor toilet facilities, children playing in filthy environment, as well as poor supply of portable drinking water. the age groups of 16-20 and 31-35 years had the highest prevalence of (10%) which is not in accordance with reports by akinbo et al., 2011, who recorded higher prevalence of intestinal parasites amongst patients aged 51 years and above. the lower prevalence rate of intestinal parasitic infection among participants aged 41 years and above, as well as children prevalence (%) ; a. lumbricoides ; 12 prevalence (%) ; t. trichiuria ; 1 prevalence (%) ; entamoeba coli; 2 prevalence (%) ; hookworm ; 10 prevalence (%) ; strongyloide stercolaris; 1 prevalence (%) ; entamoeba histolytica; 3 a. lumbricoides t. trichiuria entamoeba coli hookworm strongyloide stercolaris entamoeba histolytica international journal of applied biology, 4(1), 2020 50 aged 1-6 and 6-10 years respectively compared with the participants aged 16-20 and 31 -35 could indicate behavioral patterns observed among families in rural and sub urban settings where the younger ones stay at home and are well catered for, while the older children run most of the errands which includes open water related chores such as fishing, washing and processing of some food products in streams and rivers. the middle aged class is also mostly engaged in agricultural activities and fish farming which constantly expose them to parasites deposited in the soils, water bodies and other reservoirs in their immediate environment. in this study, it was discovered that more males (65%) than females (35%) were infected. this could relate to the fact that males are more outgoing and could be exposed to environmental contaminants than the females who mostly stay at home to attend to most of the domestic chores. the correlation between age and the infection rate of intestinal parasites in the present study is negative. this observation is in line with the pattern of intestinal parasitic infection in most rural study areas. this agrees with the work of udensi et al., (2015) who observed similar results in his study. conclusions from the present study, it was ascertained that the rate of occurrence of intestinal parasitic infection was low probably due to the periurban nature of okada community. the six parasitic species isolated from the fecal specimens were randomly distributed among the various age groups except for the 1-10 age brackets. the prevalence was high among the young adults of ages 11-15, 16-20 and the adult age group of 31-35. the gender prevalence was highest among the males. there was a negative correlation between age and the infection rate. the pattern of infection with intestinal parasites in this study shows that the more active age groups have more parasite loads compared to the others. it is therefore, pertinent to consider this infection pattern while initiating control programs of parasitic infections in rural communities. references akinbo, f.o., omoregie, r., eromowon, r., igbinedion, i.o, and airueghiomon, u. (2011). prevalence of intestinal parasites among patients of a tertiary hospitals in benin city, nigeria. north american journal of medical sciences, 3(10): 462-464. alaofè, h., zee, j., dossa, r., o’brien, h. t.( 2008). intestinal parasitic infections in adolescent girls from two boarding schools in southern benin. transactions of the royal society of tropical medicine and hygiene, 10(2):653–661. damen, j.g., luka, j., biwan, e. i. and lugos, m. (2013). prevalence of intestinal parasites among pupils in rural north-eastern nigeria. journal of the nigeria medical association, 52(1): 4-6. elliott, a. m., kizza, m., quigley, m. a., ndibazza, j., nampijja, m., muhangi, l. (2007).the impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood. clinical trials, 4(1):42–57. international journal of applied biology, 4(1), 2020 51 ekpo, u. f., odoemene, s. n., mafiana, c. f., sam-wobo, s. o.( 2008). helminthiasis and hygiene conditions of schools in ikenne, ogun state, nigeria. plos neglected tropical diseases, 2(1): 146. esiet, u.p. and edet, i.a. (2017). comparative relevance of intestinal parasites among children in public and private schools in calabar south , cross river state, nigeria. american journal of research communication, 5(1): 8097. kpurkpur, t, sani, a. and nongu, c. (2016). prevalence of intestinal parasitic infection and their association with nutritional status of rural and urban pre-school children in benue state, nigeria. international journal of maternal and child health and aids. 5(2):146-152. kwitshana, z. l., tsoka, j. m., mabaso, m. l. (2008). intestinal parasitic infections in adult patients in kwazulu-natal. south african medical journal, 98(9):709–711. nxasana, n., babak, bhat, v.g. and vasaikar, s.d. (2013). prevalence of intestinal parasites in primary school children of mthatha, eastern cape province , south africa. annals of medical and health sciences research, 3(4): 511-516. nyundo, a. a., munisi, d. z. and gesase, a. p. (2017). prevalence and correlates of intestinal parasites among patients admitted to mirembe national mental health hospital dodoma, tanzania. journal of parasitology research, (2017): 6. okyay, p., ertug, s., gultekin, b., onen, o., beser, e. (2004). intestinal parasites prevalence and related factors in school children, a western city sample – turkey. bmc public health. 4(64): 1-6 ostan, i., kilimcioğlu, a. a., girginkardeşler, n., ozyurt, b. c., limoncu, m. e., ok, u.z. ( 2007). health inequities: lower socio-economic conditions and higher incidences of intestinal parasites. bmc public health, 7(3):42. udensi, j.u., mgbemene, i.c., emeka-nwabunnia, i., ugochukwu, m.g. and awurum, i.n. (2015). prevalence of intestinal parasites among primary school children in three geopolitical zones of imo state, nigeria. science journal of public health, 5(3): 2528 contact : muhamad iksan iksanbioumb@gmail.com 57 abstract environmental conditions caused by air pollution are so large that it impacts on changes in the ecosystem that affects all aspects of human life. climate change is caused by increasing greenhouse gases in the earth's atmosphere because the earth's atmosphere receives more carbon than it releases. this study aims to determine the potential of biomass and carbon uptake in mangrove stems in pohorua village, muna regency. the research is quantitative descriptive, sampling using the point center quarted method (pcqm) technique measured around the height of the chest height mangrove tree (dbh). data analysis was performed using an allometric equation in which each mangrove plant has a specific gravity. carbon uptake found in mangroves stored in the roots, stems and leaves of mangrove plants, the results of the study showed that mangroves can absorb carbon quickly. issn : 2580-2410 eissn : 2580-2119 biomass and carbon uptake of mangrove forests pohorua village, muna regency muhamad iksan1, w o d al zarliani2, la nare1 s. hafidhawati1 a, & fitriani baena3 1 study program biology education, faculty of teacher training and education 2 agribusiness study program, faculty of agriculture 3 primary teacher education, faculty of teacher training and education universitas muhammadiyah buton, jln. betoambari no. 36 kota baubau, 97321, southeast sulawesi province, indonesia. introduction management and utilization activities that are not matched by efforts to repair and maintain will result in reduced area of mangrove forests, because mangrove forests have a very important function as carbon sinks and storage. with the ability of mangroves to store carbon, the increase in carbon emissions in nature can be reduced. mangrove forests have a key role in climate change mitigation strategies (eddy et al. 2015) that report that the rate of degradation and loss of mangrove forests in indonesia is classified as high, almost 50% 60% of total mangrove forests in indonesia have been lost due to various anthropogenic activities between others are fisheries, plantations, agriculture, logging, industry, settlement, salt ponds and mining. the east muna area, which is located in pohorua village, has the potential of mangrove forest resources that can be found almost along the coast with a length of ± 450 ha. overall with a small island for a long time, coastal communities have used mangrove forests for various daily needs. this resulted in an increase in the intensity of the exploitation of open access international journal of applied biology keyword biomass, carbon uptake, mangrove forest, pohorua village. article history received 2 november 2019 accepted 29 december 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 3(2), 2019 58 mangrove forests in the form of the use of mangrove wood as fuel resulting in the condition of mangrove forests in some places experiencing a decrease in both quantity and quality. the decline in the quality of mangrove forests in the area is thought to be caused by ongoing forest use activities that are carried out excessively without considering environmental sustainability. one form of utilization is the exploitation of wood as the main product of mangrove forests on a large scale for various purposes of building materials and firewood (rakhfid et al., 2014). calculation of biomass is one important step that must be taken in climate change mitigation activities in the forestry sector to estimate carbon sequestration and reserves in a particular area. therefore, this research is important to do because there are no data on biomass analysis and carbon uptake of mangrove forests in pohurua village, muna regency. the approach and strategy that will be achieved to the maximum to be used as supporting data so that the development and wise use of mangrove forests without damaging areas that are subject to development, such as ecotourism development. materials and methods this study uses quantitative descriptive methods with data collection techniques in the form of primary data and secondary data. primary data collection is done in two ways, namely calculating the density, biomass, carbon content and carbon uptake of mangrove species at the study site using the point center quarter method (pcqm) while secondary data are obtained from previous studies. this method is widely used to analyze forests that have dense densities (indriyanto, 2010). calculating aboveground biomass (rods) is done by a nondestructive sampling method by measuring the diameter of the stem at breast height (dbh 1.3 m) and then entering the stem diameter data into the allometric equation. this method is used to reduce destructive actions during measurement (suryono et al., 2018). data analysis surface biomass (stem) determine the surface biomass (stem) using the allometric equation compiled by komiyama et al. (2008) as follows : information : 𝜌 = wood specific gravity (gram/cm3) d = diameter (cm) calculate the total biomass of all trees by adding up the biomass of all trees by the formula hairiah et al. (2011): information : b1,b2,b3,.............,bn = biomass of each tree carbon in biomass calculate carbon content in biomass using the formula heriyanto et al. (2012): information : 50% = percentage value of carbon content in biomass total biomass of all trees = b1+b2+b3+................+bn carbon content = biomass x 50% bb = 0,251 x 𝝆 d 2.46 international journal of applied biology, 3(2), 2019 59 carbon dioxide uptake (co2) calculate carbon dioxide (co2) absorption using the formula heriyanto et al. (2012): information : co2 = carbon dioxide uptake mr = relative molecular weight co2 (44) ar = relative atomic molecular weight c (12) results and discussion biomass analysis of the biomass potential and carbon uptake of mangrove trees is carried out non destructively by using a breast height (dbh) trunk diameter data approach which is then included in the allometric equation. the biomass value of each species in the study location is presented in table 1. table 1. biomass value of mangrove species at each research station station species of mangrove biomass (kg/ha) bruguiera gymnorhiza 732,25 avicenia alba 755,63 i soneratia alba 2659,9 rhizophora mucronata 14460,13 total 18607,91 bruguiera gymnorhiza 1163,58 avicenia alba 3517,36 ii soneratia alba 2474,01 rhizophora mucronata 3517,36 total 10672,31 avicenia alba 3316,21 iii soneratia alba 2827,42 rhizophora mucronata 5904,15 total 12047,78 biomass is a picture of the total organic material from photosynthesis to grow horizontally and vertically, causing an increase in tree diameter. the greater diameter of the tree is caused by the storage of biomass resulting from the conversion of co2 which increases more in line with the amount of co2 absorbed by the diatmosphere tree (rahim, 2016). based on the analysis of total aboveground biomass potential on the mangrove tree trunk rhizophora mucronata has the highest biomass of 23881.64 kg / m2 and bruguiera gymnorhiza has the lowest biomass value of 1895.83 kg / m2. the estimated biomass value of each research station is station i at 18607.91 kg / m2, station ii at 10672.31 kg / m2 and station iii at 12047.78 kg / m2. station i has the greatest biomass value because the diameter of the tree and the number of individuals is greater than that of station i and ii. another factor that (co2) = mr.co2/ar. c (or 3,67 x carbon content) international journal of applied biology, 3(2), 2019 60 causes the low value of biomass at the study site is the specific gravity of each species of mangrove tree. this is in line with what sianturi (2018) estimates of the amount of biomass on land, especially trees, is influenced by tree diameter, specific gravity, tree height and soil fertility, causing differences in the amount of biomass and carbon stock in a vegetation. rahman (2017) further stated that the biomass value of each mangrove species is different and is influenced by the ability of plant sequestration that can be analyzed based on the value of the density, tree diameter or height. plant biomass increases because plants absorb co2 from the air and convert these substances into organic matter through photosynthesis. according to kedang et al. (2018) said that the biomass content in tree trunks had the biggest proportion compared to other tree organs because the composition of natural chemicals and extractive substances was greater than other tree organs so that the greater the diameter of the tree the value of the biomass content was higher. carbon uptake analysis of carbon uptake by converting carbon dioxide molecules then diverting carbon content in mangrove tree biomass. the carbon uptake values for each research station are presented in table 2. table 2. carbon absorption values for each research station station species of mangrove carbon uptake (kg c/ha) bruguiera gymnorhiza 1343,69 avicenia alba 1386,58 i soneratia alba 3873,67 rhizophora mucronata 25841,75 total 32445,69 bruguiera gymnorhiza 25841,75 avicenia alba 2135,17 ii soneratia alba 4539,82 rhizophora mucronata 6454,35 total 38971,09 avicenia alba 6085,24 iii soneratia alba 5256,8 rhizophora mucronata 10834,11 total 22176,15 carbon content in plants illustrates how much the plant binds co2 from the air. some carbon will be used as energy for plant physiology and enter into plant structures such as roots, stems, twigs and leaves (syukri, 2017). carbon is an element that is absorbed from the atmosphere through the process of photosynthesis and stored in the form of biomass. the level of carbon sequestration in forests is influenced by various factors including climate, topography, land characteristics, age and density of vegetation, species composition and quality of growing sites (manafe, 2016). based on the research results of carbon absorption of bruguiera gymnorhiza species of 27185.44 kg / m2, avicenia alba of 9606.99 kg / m2, soneratia alba of 13670.29 kg / m2, international journal of applied biology, 3(2), 2019 61 rhizophora mucronata of 43130.21 kg / m2 and total absorption of mangrove carbon station i is greater than other stations with a value of 34145.53 kg / m2 while the station that has the lowest total carbon uptake is found at station ii with a value of 19906.16 kg / m2. carbon uptake at station i is greater than at station ii and iii, this is due to the percentage of carbon stock increasing in line with the increase in biomass. the greater the biomass content, the greater the carbon stock will affect carbon sequestration. this is due to several factors that affect the value of carbon stock including physical chemical factors of environment, density and type of substrate. in line with what was stated by manafe (2016) the level of carbon sequestration in forests is influenced by various factors including climate, topography, land characteristics, age and density of vegetation, species composition and quality of growing sites. the difference in the value of biomass and carbon content stored in various ecosystems depends on the diversity and density of plants and management of these ecosystems. according to manafe (2016) the difference in stored carbon stocks is due to differences in the amount of diameter between the stands where the larger the diameter of the trees making up a land, the greater the weight of tree biomass on the land as well. large biomass weight will affect the amount of carbon stock in a land. however, according to yaya (2013) a high density does not guarantee the ability of carbon absorption and storage is high, but the absorption of co2 to become biomass is influenced by the work of enzymes in photodynthesis because each type has a different ability of photosynthesis. species that have a sufficient density and ability to store carbon indicate that they are sufficiently able to adapt to environmental conditions and increase the success of rehabilitation efforts in the interests of climate mitigation. the low carbon content value at station ii is due to local community activities that damage mangrove forests such as clearing land for ponds, roads and excessive mangrove wood harvesting without regard to environmental impacts. this is supported by the opinion of senoaji (2016) that human activities play an important role in terms of increasing or decreasing the carbon content stored in mangrove ecosystems. mangrove planting activities will increase stored carbon content, while cutting mangroves will reduce stored carbon content. rhizophora mucronata type counts only 11 ind / m2 at station ii because it is widely used by local communities for various needs such as the use of wood, natural dyes, medicine and others so that a large influence on stored carbon. this is supported by restuhadi et al. (2013) that rhizophora mucronata is used as fuel and charcoal, tannin from bark is used as a natural coloring agent and is sometimes used as a remedy for hematuria. the following is the physical condition of mangrove forest damage in the village of pohurua, muna regency. conclusions mangrove forest biomass potential in pohurua village muna regency at station i was 18607.91 kg / m2, station ii was 10672.31 kg / m2, station iii was 12047.78 kg / m2 and the total biomass value was 41612.23 kg / m2 mangrove forest carbon uptake in pohurua village muna regency at station i was 32445.69 kg / m2, station ii was 38971.09 kg / m2, station iii was 22176.15 kg / m2 and total carbon uptake was 76358.45 kg / m2. references aida, g., wardiatno, y., fahrudin, a., kamal, m.m. 2014. produksi serasah mangrove di pesisir tangerang, banten. jurnal pertanian indonesia. vol 19 (2) :91-97. international journal of applied biology, 3(2), 2019 62 alongi, d.m. 2014. early growth responses of mangroves to 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irawan, a., abimanyu, h., meirinawati, h., surinati, d., syukri, a.f., yuliana, c.i., yuniati, p.i. 2018. carbon sequestration index as a determinant for climate change mitigation: case study of bintan island. journal earth and enviromental science. 3(1)45-54 contact : inanpi hidayati sumiasih inanpihs@trilogi.ac.id © international journal of applied biology 45 abstract this study aims to investigate effect of several maturity stage at harvest and storage temperature to mangosteen color changes and shelf life during storage. the research used completely randomized design of two factors. the first factor was the fruit maturity stage at harvest consisting of: maturity stage-1 (mostly at 104 days after anthesis day after harvest (daa), fruit skin color was yellowish green with a little bit of red specks), maturity stage-2 (at 106 daa, fruit skin color was reddish yellow with red specks spread evenly near sepals), maturity stage-3 (at 108 daa, fruit skin color was reddish yellow with red specks all over) and maturity stage-4 (at 110 daa, fruit skin color was evenly red). the second factor was storage temperature of 15 oc and room temperature. the result of harvesting mangosteen at maturity stage 1 could maintain skin color longer than at maturity stage 2, 3, and 4. mangosteen that were harvested at maturity stage 1 and 2, combined with storage temperature of 15 oc could maintain fruit quality up to 30 days after harvest and could be used for export market. while harvesting at maturity stage 3 could maintain fruit quality up to 25 days after harvest and maturity stage 4 up to 20 days after harvest. harvesting at maturity stage 4 followed by 15 oc storage temperature and all maturity stages combined with room temperature storage could be used for local market. issn : 2580-2410 eissn : 2580-2119 study of several stages of maturity and storage temperature on color changes and shelf life of mangosteen (garcinia mangostana l.) inanpi hidayati sumiasih1,3, roedhy poerwanto1,2 & darda efendi1,2 1agronomy and horticulture department, faculty of agriculture, bogor agricultural university jl. meranti, ipb darmaga campus, bogor 16680, indonesia 2center for tropical horticulture studies (pkht) baranangsiang campus of ipb, jl. raya pajajaran, bogor 16144 3agrotechnology program, faculty of bioindustry, and trilogi university, jakarta. jl. tmp. kalibata no. 1, jakarta 12760 indonesia introduction fruit freshness during storage is determined by the correct harvest stage and storage condition. the information in the correct harvest stage is needed by mangosteen farmers, local trader and exporter in effort to keep the produce fresh and prolong the shelf life. mangosteen fruit usually is harvested by indonesian farmers in various harvest stage, from when the skin color is greenish yellow with red to blackish purple complexion. after harvested, the fruit color that is purplish red changes quickly into blackish purple. to get the open access international journal of applied biology keyword fruit color, horticultural commodities, queen of tropical fruits, shelf life article history received 25 january 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 46 best quality of mangosteen, harvest should be done in stage when fruit color is randomly bright with pink to red specks all over the fruit. after harvest and in storage, changes will happen in mangosteen in its skin color, sepals and aril. that is due to the fruits still undergo physiological process even after detached from its tree. those changes may deplete mangosteen quality. therefore, there is a need of effort to slow down the physiological process of mangosteen fruit. efendi (2005) stated that the effort to prolong mangosteen storage time could be done by using controlled storage atmosphere, coating materials and low storage temperature. incorrect harvest stage and storage of mangosteen fruit can make the fruit skin harden faster and deplete its quality. manurakchinakorn et al. (2016) stated that mangosteen fruit at ripeness stage 6 (purple black pericarp) has a very limited storage life and deteriorate rapidly. sartika and poerwanto (2010), stated that the problem occurred in mangosteen storage in room temperature for two weeks was hardened fruit skin. prolonging storage time of mangosteen fruit and keep its quality can be done by keeping the fruit in low temperature and harvest the fruit at the right maturity stage. low temperature storage lower respiration and transpiration rate, thus can prolong storage time with minimal weight loss, keep its quality and resulted in higher value in the market. when mangosteen fruit harvested past the maturity stage with incorrect storage, mangosteen fruit can quickly lost quality, i.e. sepals turn brown and hardened fruit skin before it reaches consumers. therefore, research in the correct harvest stage and storage temperature to prolong storage is needed so mangosteen fruit can reaches domestic and international consumers with intact quality. materials and methods mangosteen fruit was from mangosteen plantation in tanggamus regency, lampung. the research was conducted in postharvest laboratory, agronomy and horticulture department, bogor agricultural university. materials used were maturity stage indicators, phenolphthalein indicator (pp), naoh 0.1 n solution and distilled water. tools used in this research were; erlenmeyer flask, analytic scale, cool storage, hand refractometer, pipette, volumetric flask, color reader and thermometer. this research used completely randomized design with two factors. first factor was mangosteen maturity stage at harvest that was consisted of four stages that were: maturity stage-1 (mostly at 104 days after anthesis (daa), fruit skin color was yellowish green with a little bit of red specks), maturity stage-2 (at 106 daa, fruit skin color was reddish yellow with red specks spread evenly near sepals), maturity stage-3 (at 108 daa, fruit skin color was reddish yellow with red specks all over) and maturity stage-4 (at 110 daa, fruit skin color was evenly red). second factor was storage temperature that consisted of two levels that were: storage temperature of 15 oc and room temperature (28 oc). manurakchinakorn el al. (2014) stated that mangosteen is sensitive to low temperature and develops chilling injury (ci) when stored at temperature below 13 oc. if the result of analysis of variance showed significant difference at f-test with alpha of 5% or 1% post-test of duncan multiple range test was conducted. destructive observations were conducted every five days for 30 days after harvest (dah). study by anggraeni (2008) stated that mangosteen fruit that was harvested at mature stage and stored at room temperature could only last for 15 days after harvest, while storage at temperature of 15 oc could make mangosteen fruit lasted for 20 days after harvest. the observations were total titratable acid (tta) (%), fruit skin water content, fruit skin color international journal of applied biology, 3(1), 2019 47 appearance, optimum consumption point, organoleptic test, color scoring, and mangosteen fruit sepal freshness. results and discussion total titratable acid (tta) total titratable acid (tta) content showed not much of a difference in amongst all treatments (showed in table 1). at the early storage time, tta of mangosteen was around 0.60-0.74%. generally, tta value of every treatment was declined especially after 10 dah. the decline of tta was possibly because the organic acids in the fruit was used as substrate in fruit respiration during ripening. wills and tirmazi (1981) stated that organic acids are the energy reserve for fruit and will decrease during increasing metabolic activity at fruit ripening. study by suketi (2011) showed that tta content of papaya was decreased as fruit maturity level was increased, showed by fruit skin that was became yellow. that happened because tta was depleted along with fruit ripening. table 1. tta content of mangosteen at several maturity stage and storage temperature during storage notes: numbers followed by the same letter in the same column showed insignificant difference based on dmrt 1%. – was the fruit that skin hardened with dried aril (rotten). mangosteen aril appearance results of observation on aril appearance started from 0 – 30 dah showed in figure 1 and 2. at harvest (0 dah) at 15 oc, mangosteen that was harvested at maturity stage 1 and 2 had aril that could not be separated with endocarp and had a lot of yellow sap, while at 5 dah mangosteen could already be consumed. at the end of storage, mangosteen that was harvested at maturity stage 3 and 4 had hollow space between aril and endocarp. endocarp color was brownish and aril color was unappealing. while fruit that was harvested at maturity stage 1 and 2 could still able to be consumed in the end of storage and appeared to be in good condition with white aril and fresh endocarp. anjaritha et al. (2019) about mangosteen (garcinia mangostana l.) is a tropical fruit with high market value but has short shelf-life during postharvest handling. at room temperature storage, the limit of consumers acceptability at all harvest maturity stages was 15 dah. at 20 dah all mangosteen from all maturity stages had already rotten and could not be consumed (aril was brown and endocarp was dried and brown). study by sumiasih et al. (2011) showed that mangosteen with 15 oc storage temperature and harvested at maturity stage 1 and 2 had the most favorable taste at 20 dah and could be kept for 30 dah. treatment 0 5 10 15 20 25 30 dah (%) stage 1 0.74 a 0.69 0.68 0.54 0.40 0.24 0.20 stage 2 0.68 ab 0.64 0.66 0.55 0.38 0.24 0.19 stage 3 0.67 ab 0.65 0.65 0.54 0.40 0.24 0.19 stage 4 0.60 b 0.61 0.63 0.54 0.37 0.23 0.14 temperature of 15 oc 0,66 0,66 0.67 a 0.55 0.47 0.48 0.36 room temperature 0,69 0,65 0.64 b 0.54 international journal of applied biology, 3(1), 2019 48 figure 1. the appearance of mangosteen aril from 0 dah to 30 dah: (a) maturity stage 1, (b) maturity stage 2, (c) maturity stage 3, and (d) maturity stage 4 at storage temperature of 15 oc figure 2. the appearance of mangosteen aril from 0 dah to 30 dah: (a) maturity stage 1, (b) maturity stage 2, (c) maturity stage 3, and (d) maturity stage 4 at room temperature storage storage temperature of 15 oc (a) (b) (c) (d) 30 dah 30 dah 0 dah 0 dah 0 dah 0 dah 10 hsp 10 hsp 10 hsp 10 hsp 15 dah 15 dah 15 dah 15 dah 25 dah 25 dah 25 dah 25 dah 30 dah 30 dah room temperature storage (a) (b) (c) (d) 0 hsp 0 hsp 10 dah 10 dah 10 dah 10 dah 15 dah 15 dah 15 dah 15 dah 0 hsp 0 dah 20 dah 20 dah 20 dah 20 dah 0 dah 0 dah 0 dah international journal of applied biology, 3(1), 2019 49 water content percentage (%) the combination of maturity stage and storage temperature gave significant difference on skin water content (table 2). in storage, there was changes in mangosteen fruit skin water content. the percentage of mangosteen fruit skin water content from 0 dah to 13 dah was not significantly different, while at 20 dah and 25 dah showed significant difference between treatments. mangosteen fruit at maturity stage 1 and 2 showed higher skin water content than other treatments. in storage, mangosteen fruit skin water content was decreased, in storage temperature of 15 oc and in room temperature. mangosteen fruit water content generally declined along with longer storage time. this was supported by sumiasih et al. (2016) about star fruit harvested at several maturity stages and kept in different storage temperature, showed that weight loss could be an indicator of declined quality of horticultural products. storage at low temperature was the part of post-harvest treatment that could be used to minimize water content loss in star fruit. weight loss percentage in low temperature storage (18 oc) was slower compared to room temperature storage. mangosteen at storage temperature of 15 oc showed higher water content than at room temperature. this showed that lower storage temperature can supress water loss in fruit. if water loss can be suppressed, it was expected that weight loss also declined and increase mangosteen fruit skin resistance so the freshness and appearance of mangosteen can be maintained. according to kader (1992), water loss content in mangosteen was due to respiration and transpiration process that could be the reason for fruit shrinkage, because not only it affected directly in quantitative loss (weight loss) but also loss in quality and appearance of crispiness and juice content. aizat et al. (2019) about mangosteen have rich in potent bioactive compounds, such as xanthones, and is known to possess pharmacologically important anti-inflammatory and antitumor properties. table 2. mangosteen fruit skin water content at several maturity stages and storage temperature combination of treatments fruit skin water content (%) 0 5 10 15 20 25 30 (dah) stage 1,15 oc 78.02 76.89 70.57 68.19 69.46 a 68. 38 a 68.96 stage 2,15 oc 78.49 75.96 69.82 66.95 66.19 a 67.43 a 68.67 stage 3,15 oc 77.88 75.44 67.18 65.53 62.57 ab 62.51 a 64.63 stage 4,15 oc 78.85 77.76 66.58 64.94 62.43 bc 61.82 ab 60.29 stage 1, room 78.23 75.16 62.02 59.59 58.76 cd 43.36 c 32.00 stage 2, room 77.68 74.48 63.00 60.91 57.20 e 41.39 cd 31.36 stage 3, room 78.10 76.73 61.52 59.35 48.06 f 22.88 e 26.17 stage 4, room 78.55 76.41 62.70 56.79 39.25 g 21.72 e 22.56 note: the numbers followed by the same letter in the same column showed no significant difference based on dmrt at 1%. international journal of applied biology, 3(1), 2019 50 y stadia 1 = 0,013x2 0,5585x + 6,171 r² = 0,8888 y stadia2 = 0,0118x2 0,5323x + 6,0383 r² = 0,8768 y stadia 3 = 0,0126x2 0,546x + 6,0698 r² = 0,886 y stadia 4 = 0,0143x2 0,5743x + 6,1312 r² = 0,9036 0 1 2 3 4 5 6 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 sk or w ar na s ep al lama penyimpanan (hsp) suhu 15 oc y stadia 1 = 0,038x2 0,8646x + 5,5506 r² = 0,9301 y stadai 2 = 0,0389x2 0,8777x + 5,5556 r² = 0,9139 y stadia 3 = 0,0403x2 0,902x + 5,626 r² = 0,9033 y stadia 4 = 0,0402x2 0,8961x + 5,5764 r² = 0,9012 0 1 2 3 4 5 6 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 sk or w ar na s ep al lama penyimpanan (hsp) suhu ruang (28 oc) y stadia 1 = -0,0016x2 0,3003x + 5,577 r² = 0,9186 y stadia 2 = -0,0008x2 0,3109x + 5,5667 r² = 0,9186 y satadia 3 = 0,0013x2 0,3511x + 5,699 r² = 0,9312 y stadia 4 = 0,0012x2 0,344x + 5,6171 r² = 0,9244 0 1 2 3 4 5 6 0 2 4 6 8 1012141618202224262830 sk or k es eg ar an s ep al lama penyimpanan (hsp) suhu 15oc y stadia 1 = 0,0332x2 0,8166x + 5,8192 r² = 0,966 y stadia 2 = 0,0323x2 0,7953x + 5,7032 r² = 0,9628 y stadia 3 = 0,0328x2 0,803x + 5,6996 r² = 0,9671 y stadia 4 = 0,0327x2 0,7971x + 5,65 r² = 0,9645 0 1 2 3 4 5 6 0 2 4 6 8 1012141618202224262830 sk or k es eg ar an s ep al lama penyimpanan (hsp) suhu ruang (28 oc) mangosteen fruit sepal color and freshness scoring the effect of maturity stage and storage temperature to sepal color was shown at figure 3. figure 3. mangosteen sepal color score at several maturity stages and room temperature the effect of maturity stage and storage temperature to mangosteen sepal freshness score was shown at figure 4. figure 4. mangosteen fruit sepal freshness from several maturity stages and storage temperature color and freshness of mangosteen fruit sepal had effect on mangosteen quality during storage. fresh mangosteen fruit had green sepal and then turned into brown, indicating the fruit had lost its freshness. mangosteen fruit that stored at storage temperature of 15 oc had lower decrease in sepal color and freshness score. color and freshness of international journal of applied biology, 3(1), 2019 51 mangosteen fruit sepal at all maturity stages that were kept in 15 oc declined at 12 dah and continue to decline gradually up to 20 dah and until the end of storage. while mangosteen fruit sepal at all maturity stages that were kept at room temperature started to decline at 6 dag and continue to slowly decline up to 12 dah and until the end of storage. study results from suyanti et al. (1999) showed that mangosteen that harvested when the skin color was green with a bit of purple (104 days after anthesis) had sepal that kept its freshness up to 6 dah. ismadi et al. (2010) showed that after storage mangosteen had several changes in appearance. characteristics for fruit that suitable for consumptions are black brownish skin fruit, there is still yellowish green specks at the sepal, has not loss its weight and the skin are not hardened. mangosteen fruit skin appearance observation result of mangosteen fruit skin color started at harvest (0 dah) until the end of storage (30 dah) at 15 oc (figure 5) and at room temperature (figure 6). according to kader 2003, after harvest and during storage, the mangosteen will canges color which is one of the parameters of fruit maturity. figure 5. mangosteen fruit skin appearance frim 0 dah to 30 dah: (a) maturity stage 1, (b) maturity stage 2, (c) maturity stage 3, and (d) maturity stage 4 in storage temperature of 15 oc figure 5 showed that mangosteen at all maturity stages in storage temperature of 15 oc was able to keep its skin color than mangosteen kept in room temperature. according to sumiasih et al. 2017, skin color was one of the external attributes for horticulture products, for example at their research of maintaining citrus skin color. skin color had important role in consumers’ decision making to purchase. 0 dah 0 dah 0 dah 0 dah 12 dah 30 dah 12 dah 12 dah 12 dah 24 dah 24 dah 24 dah 24 dah 30 dah 30 dah 30 dah international journal of applied biology, 3(1), 2019 52 mangosteen that was stored at 15 oc (30 dah) had red brownish skin color for maturity stage 1, red purplish skin color for maturity stage 2 and 3 and black purplish skin color for maturity stage 4. while mangosteen that was stored at room temperature had the same skin color appearance at 6 dah that were black purplish. figure 6. mangosteen fruit skin appearance frim 0 dah to 30 dah: (a) maturity stage 1, (b) maturity stage 2, (c) maturity stage 3, and (d) maturity stage 4 in room temperature storage consumers acceptance limit of mangosteen at maturity stage 1, 2, 3, and 4 in storage temperature of 15 oc were at 30 dah, 30 dah, 25 dah, and 20 dah, respectively, while in room temperature at all maturity stages was 20 dah. the optimum consumption point of mangosteen that was kept at 15 oc was 20 dah (stage 1 and 2) and 10 dah (stage 3 and 4). as for mangosteen that was stored in room temperature at stage 1 was 15 dah and at stage 2, 3, and 4 was 10 dah. conclusions the result showed that harvesting mangosteen at maturity stage 1 could kept skin color, sepal color and freshness longer than at maturity stage 2, 3 and 4. mangosteen that was harvested at maturity stage 1 and 2 with storage temperature of 15 oc could maintain fruit quality (appropriate for consumption) for 30 days after harvest and could be used for export market. mangosteen harvested at maturity stage 3 and maturity stage 4 could be 0 hsp 0 hsp 0 hsp 0 hsp 6 hsp 6 hsp 6 hsp 6 hsp 12 hsp 12 hsp 12 hsp 12 hsp 18 hsp 18 hsp 18 hsp 18 hsp (a) (b) (c) (d) international journal of applied biology, 3(1), 2019 53 stored up to 25 dah and 20 dah, respectively. at maturity stage 4 with storage temperature of 15 oc and at all maturity stages with room temperature was appropriate for local market. castro et al. 2012 about the maximum storage period for mangosteen at 25 °c is two weeks; while storage at13 °c can guarantee the conservation of this fruit for 25 days. references aizat, w.m., ahmad-hashim, f.h. and jaafar, s.n.s., 2019. valorization of mangosteen, “the queen of fruits,” and new advances in postharvest and in food and engineering applications: a review. journal of advanced research. anggraeni, w., 2008. penggunaan bahan pelapis dan plastik kemasan untuk meningkatkan daya simpan buah manggis (garnicia mangostana l.) [skripsi]. agronomy and horticulture department, faculty of agriculture, bogor agricultural university. castro, m.f.p.p.m., anjos, v.d.d.a., rezende, a.c.b., benato, e.a. and valentini, s.r.d.t., 2012. postharvest technologies for mangosteen (garcinia mangostana l.) conservation. food science and technology, 32(4), pp.668-672. efendi, d., 2005. rekayasa genetika untuk mengatasi masalah-masalah pascapanen. bul. agron. 33 (2) 49-56. ismadi., poerwanto, r., efendi, d., bintang, m., muchtadi, d and sutrisno., 2010. studi pengerasan buah manggis selama penyimpanan. national conference on horticulture. kader, a.a., 2003. mangosteen recommendation for maintaining postharvest quality.http//rics.undavis.edu/postharvest2//produce/producefacts/fruitmangostee n.shtml. kader, a.a. 1992., quality and safety factors: definition and evaluation for fresh horticulture crop. university of california division of agriculture and nature resources. oakland. manurakchinakorn, s., nuymak, p. and issarakraisila, m., 2014. enhanced chilling tolerance in heat-treated mangosteen. international food research journal, 21(1). manurakchinakorn, s., chainarong, y. and sawatpadungkit, c., 2016. quality of mangosteen juice colored with mangosteen pericarp. international food research journal, 23(3), p.1033. parijadi, a.a., ridwani, s., dwivany, f.m., putri, s.p. and fukusaki, e., 2019. a metabolomicsbased approach for the evaluation of off-tree ripening conditions and different postharvest treatments in mangosteen (garcinia mangostana). metabolomics, 15(5), p.73. sartika, r and poerwanto, r., 2010. pengaruh suhu dan kelembaban udara terhadap shelf_life dan karakteristik buah manggis (garcinia mangostana l.) selama penyimpanan. agronomy and horticulture department, faculty of agriculture, bogor agricultural university. suketi, k. 2011. studi morfologi bunga, dan perkembangan buah sebagai dasar pengendalian mutu buah pepaya ipb [disertation]. agronomy and horticulture department, faculty of agriculture, bogor agricultural university. sumiasih, i.h., octaviani, l., lestari, d.i. and yunita, e.r., 2016. studi perubahan kualitas pascapanen buah belimbing dengan beberapa pengemasan dan suhu simpan. agrin: jurnal penelitian pertanian, 20(2). sumiasih, i.h., poerwanto, r., efendi, d., agusta, a. and yuliani, s., 2017. the analysis of βcryptoxanthin and zeaxanthin using hplc in the accumulation of orange color on lowland citrus. international journal of applied biology, 1(2), pp.37-45. international journal of applied biology, 3(1), 2019 54 suyanti, s., roosmani, a.b.s.t and sjaifullah., 1999. pengaruh tingkat ketuaan terhadap mutu pascapanen buah manggis selama penyimpanan. j. hort. 1(9):51-58. wills, r.b.h and tirmazi, s.i.h., 1981. inhibition of ripening of avocado with calcium. school of food tecnologi, new south wales university. australia. contact : jaya thakur jayathakur1984@yahoo.com 36 abstract the study was conducted to determine the effect of arbuscular mycorrhizal (am) fungi inoculation on growth of pea grown under water stressed pot culture conditions. water stress was given to the pea plants after 30 days at the interval of 4, 8 and 12 days. the data was collected at an interval of 15 days. three replicates of each set were maintained. . the mixture of am fungi used for current experiment included the species of acaulospora denticulata, a. gerdemannii, glomus macrocarpum, g. maculosum, g. fasciculatum and scutellospora minuta. the mycorrhizal plants have shown more shoot and root length as compared to the control plants. the height of shoot and root was significantly decreased with the increase in drought stress. mycorrhizal plants with low water stress showed enhanced shoot and root length than high water stress. the mycorrhizal plants have shown more number of leaves than control plants during drought stress. the number of leaves significantly reduced with the increase in drought stress. the leaves produced by the control plants were comparatively smaller than those of mycorrhizal plants. the dry weight of root and shoot of both control and mycorrhizal plants decreased with the increase in water stress. mycorrhizal plants showed more dry weight of shoot and root as compared to control plants. plants inoculated with am fungi produce more dry weight than the control plants. the fresh weight of both control and mycorrhizal plants has been decreased with the increase in water stress interval and also the fresh weight of root and shoot was observed higher in mycorrhizal plants as compared to those of control plants. issn : 2580-2410 eissn : 2580-2119 effect of water stress and am fungi on the growth performance of pea plant jaya thakur1 & b.p. shinde2 1 assistant professor, dav college for women, chandigarh 2 principal , vidya prathisthan, baramati, maharashtra p. g. department of botany, fergusson college pune ms india introduction the term “pea” can refer to small spherical seed or to the pod. pea is a crop of high commercial significance. pea belongs to the leguminosae family (genus: pisum, subfamily: faboideae tribe: fabaceae), which has an important ecological advantage because it contributes to the development of low-input farming systems by fixing atmospheric nitrogen and it serves as a break crop which further minimizes the need for external inputs. pea (pisum open access international journal of applied biology keyword am fungi, water stress, acaulospora denticulata, glomus macrocarpum, control, mycorrhizal plants. article history received 21 february 2020 accepted 14 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 37 sativum) is an important pulse crop. pea requires cool growing season with moderate temperature. the best suitable temperature for its germination is about 22°c and 13°c to 18°c for plant growth and development. pea requires less rain and low humidity for proper growth. pea crop can be grown in a variety of soils. a well drained soil with ample moisture retaining capacity such as deep loam soil is the best for its cultivation. light soils like sandy loam, are suitable for early crops especially for green pod production. heavy soils like clay loam are preferred for grain production. the ph range from 6 to 7.5 is the most suitable for this crop. mycorrhizal fungi are ubiquitous, present in all natural ecosystems in most climatic zones throughout the world. the mycorrhizal habitat probably evolved as a survival mechanism for both partners in the association, allowing each to survive in environment of low fertility, drought, disease and temperature extremes where, alone they could not. amf are of ecological and economical important as they can improve pathogen resistance (vigo et al., 2000; de la pena et al., 2006) as well as biomass production (smith et al., 2009) of the host plant. in addition, amf mitigate different kinds of plant stresses such as drought (michelson and rosendahl, 1990; auge et al., 2001; aroca et al., 2007), or heavy metal toxicity (hildebrandt et al., 1999) and protect plants against root herbivores (gange, 2001). amf helps in promoting plant growth and development by increasing nutrient acquisition and alleviating stress conditions of plants (koide and kabir, 2000; koide and mosse, 2004; barea et al., 2005). the present study was conducted to determine the effect am fungi on the various growth parameters of pea plant during drought stress conditions. materials and methods preparation of experimental set up water stress treatment was given at the fergusson college botanical garden. in this experiment, seeds of pea (pisum sativum) were sown in the pots with and without mycorrhiza. fifteen replicates of both control and mycorrhizal plants were maintained during present investigation. these plants were watered with normal water for one month at an interval of 4 days. the mixture of am fungi used for current experiment included the species of acaulospora denticulata, a. gerdemannii, glomus macrocarpum, g. maculosum, g. fasciculatum and scutellospora minuta. the number of am propagules per 100 gm soil was 260. ten gram of mycorrhizal soil was added in the pots at the time of sowing of seeds in mycorrhizal set. the am fungi have been shown to help in retaining moisture of soil and also help in uptake of important nutrients during stress conditions (heikham et al., 2009). the water stress treatment was started after one month old pea seedlings at an interval of 4, 8 and 12 days for next one and a half month. the different parameters studied in mycorrhizal and non-mycorrhizal plants include dry and fresh weight of root and shoot, shoot length, root length and number of leaves. study of growth parameters growth parameters were measured at the interval of 15 days from one month (30 days, 45 days and 60 days). dry and fresh weight of root and shoot, shoot length, root length and number of leaves of mycorrhizal and non mycorrhizal plants were measured to study the comparative account of growth. international journal of applied biology, 4(1), 2020 38 results a study was conducted to determine the effect of arbuscular mycorrhizal (am) fungi inoculation on growth of pea grown under water stressed pot culture conditions. water stress was given to the pea plants after 30 days at the interval of 4, 8 and 12 days. the data was collected at an interval of 15 days. three replicates of each set were maintained. . the mixture of am fungi used for current experiment included the species of acaulospora denticulata, a. gerdemannii, glomus macrocarpum, g. maculosum, g. fasciculatum and scutellospora minuta. effect of water stress and am fungi on the dry weight of root and shoot of pea plant the effect of water stress on the dry weight of pea was studied with and without am fungi at the interval of 4, 8 and 12 days. the dry weight of root and shoot was recorded in control and mycorrhizal plants. the water stress was given at 4, 8 and 12 days interval in mycorrhizal as well as control plants. water stress has caused a decrease in the dry weight of both control and mycorrhizal plants. at the interval of 4 days, in control plants, the dry weight of shoot was 0.223±0.69 g. but it kept on decreasing with the increase in water stress. at the interval of 8 days, it has been found to be decreased up to 0.192±0.74 g. at the interval of 12 days, it has been decreased to 0.140±0.71 g. however, in mycorrhizal plants, at the interval of 4 days, the dry weight of shoot was 0.256 ±0.08 g, at the interval of 8 days, it was 0.230±0.33 g and at the interval of 12 days, dry weight was 0.187±0.15 g. the dry weight of root in control plants at the interval of 4 days was 0.008±0.51 g and it had decreased to 0.003±0.58 g and it declined significantly at the interval of 12 days where it had been found to be 0.001±0.51 g. in mycorrhizal plants, the dry weight of root at the interval of 4 days was 0.013±0.12 g, at the interval of 8 days, it was 0.009±0.85g but at the end of 12 days, it was found to be 0.007±0.73 g. the results were significant at p≤ 0.05 level. table 1. effect of water stress and am fungi on the dry weight of root and shoot of pea plant * results are significant at p ≤ 0.05 level. effect of water stress and am fungi on the fresh weight of root and shoot of pea plant water stress has caused a decrease in the fresh weight of both control and mycorrhizal plants (table 2). at the interval of 4 days, in control plants, the fresh weight of shoot was 1.033±0.21 g. but it kept on decreasing with the increase in water stress. at the interval of 8 days, it has been found to be decreased up to 0.973±0.19 g. at the interval of 12 days, it has been decreased to 0.777±0.41 g. however, in mycorrhizal plants, at the interval of 4 days, the fresh weight of shoot was 2.50±0.93 g, at the interval of 8 days, it was 2.155±0.63 g and at the interval of 12 days, fresh weight was1.823±0.48 g. water stress control (g) mycorrhizal (g) days 4 8 12 4 8 12 shoot 0.223±0.69 0.192±0.74 0.140±0.71 0.256±0.08 0.230±0.33 0.187±0.15 root 0.008±0.51 0.003±0.58 0.001±0.51 0.013±0.12 0.009±0.85 0.007±0.73 international journal of applied biology, 4(1), 2020 39 the fresh weight of root in control plants at the interval of 4 days was 0.113±0.48 g and at the interval of 8 days, it had been found to be 0.096±0.54 g and it had decreased significantly at the interval of 12 days where it had been found to be 0.071±0.64 g. in mycorrhizal plants, the fresh weight of root at the interval of 4 days was 0.231±0.37 g, at the interval of 8 days, it was 0.219±0.58 g but at the end of 12 days, it was found to be 0.198±0.08 g. table 2. effect of water stress on the fresh weight of root and shoot of pea plant. water stress control(g) mycorrhizal (g) days 4 days 8 days 12 days 4 days 8 days 12 days shoot 1.033±0.21 0.973±0.19 0.777±0.41 2.50±0.93 2.155±0.63 1.823±0.48 root 0.113±0.48 0.096±0.54 0.071±0.64 0.231±0.37 0.219±0.58 0.198±0.08 * results differ significantly at p ≤ 0.05 level. the fresh and dry weight of both control and mycorrhizal plants has been decreased with the increase in water stress interval. it has been found that the mycorrhizal plants have shown more fresh and dry weight of root and shoot as compared to those of control plants. this showed that am fungi helped the pea plants during water stress. effect of pea water stress and am fungi on the shoot and root length and the number of leaves of pea plant water stress has a negative effect on the shoot length, root length and leaf number of pea plant. it has been found that shoot length was 20.07±0.21 cm at the interval of 4 days water stress. at the interval of 8 days, shoot length was reduced to 16.21±0.87 cm. at the interval of 12 days, it was 11.01±2.11cm. in case of mycorrhizal plants, shoot length was 23.37±3.43cm at the interval of 4 days, at the interval of 8 days, it was 19.37±2.63 cm and it has been decreased to 14.12±2.41cm at the interval of 12 days. in control plants, root length was 4.50±1.09 cm at the interval of 4 days, root length at the interval of 8 days was recorded 4.32±0.46 cm and has been found to decrease up to 4.1±2.39 cm at the interval of 12 days. in mycorrhizal plants, it was recorded 4.80±1.62 cm at the interval of 4 days, at the interval of 8 days, it was 4.53±2.23 cm and at the interval of 12 days it was reduced to 4.28±3.81cm. leaf number of control plants was 28.71±2.47 at the interval of 4 days, 25.2±0.54 at the interval of 8 days and has been reduced to 21.63±0.87 at the interval of 12 days. in mycorrhizal plants, it has been found to be decreased from 35.92±2.26 (at the interval of 4 days), 31.11±2.69 (at the interval of 8 days) and 25.71±1.89 (at the interval of 12 days). (table3) water stress has caused a decrease in the shoot length, root length and the numbers of leaves at the interval of 4, 8 and 12 days in both control and mycorrhizal pea plants, but all these growth attributes were more in mycorrhizal plants as compared to control plants. the results were significant at p ≤ 0.05 level. international journal of applied biology, 4(1), 2020 40 table 3. effect of water stress and am fungi on the shoot and root length and the number of leaves of pea plant * results differ significantly at p≤0.05 level. discussion effect of water stress and am fungi on growth performance of pea plant the present investigation was conducted to determine the effect of arbuscular mycorrhizal fungi and water stress on growth of pea plant. the data was prepared and collected at the time interval of 15 days. the treatment of water stress was given to the plants after four, eight and twelve days. effect of water stress and am fungi on the shoot and root length and the number of leaves of pea plant the mycorrhizal plants have shown more shoot and root length as compared to the control plants. the height of shoot and root was significantly decreased with the increase in drought stress. mycorrhizal plants with low water stress showed enhanced shoot and root length than high water stress. the results were in agreement with auge and moore (2005) and galvan et al., (2008). kothari et al., (1991) observed that rate of water uptake per unit root length and per unit tissue by am were twice that of control plants. the mycorrhizal plants have shown more number of leaves than control plants during drought stress. the number of leaves significantly reduced with the increase in drought stress. the leaves produced by the control plants were comparatively smaller than those of mycorrhizal plants. the results were significant at p≤0.05 level. effect of water stress and am fungi on the dry weight of root and shoot of pea plant the dry weight of root and shoot was recorded in control and mycorrhizal plants after giving drought stress. the water stress was given at 4, 8 and 12 days interval in mycorrhizal as well as control plants. the dry weight of root and shoot of both control and mycorrhizal plants decreased with the increase in water stress. mycorrhizal plants showed more dry weight of shoot and root as compared to control plants. the results were significant at p≤0.05. plants inoculated with am fungi produce more dry weight than the control plants. these results have also been reported by wu and xia (2004, 2006) and wu et al., (2008). water stress control mycorrhizal days 4 days 8 days 12 days 4 days 8 days 12 days shoot length(cm) 20.07±0.21 16.21±0.87 11.01±2.11 23.37±3.43 19.37±2.63 14.12±2.41 root length(cm) 4.50±1.09 4.32±0.46 4.1±2.39 4.80±1.62 4.53±2.23 4.28±3.81 leaf number 28.71±2.47 25.2±0.54 21.63±0.87 35.92±2.26 31.11±2.69 25.71±1.89 international journal of applied biology, 4(1), 2020 41 effect of water stress and am fungi on the fresh weight of root and shoot of pea plant the fresh weight of both control and mycorrhizal plants has been decreased with the increase in water stress interval. it has been found that the fresh weight of root and shoot was observed higher in mycorrhizal plants as compared to those of control plants. fresh weight of shoot and root was significantly high in those plants which were watered at the interval of 4 days. however, it was least in the plants watered at the interval of 12 days. the results were in agreement with patale and shinde (2012). the results were significant at p≤0.05 level. conclusions effect of water stress and am fungi on growth performance of pea plant water stress treatment was given to the plants after 30 days at the interval four, eight and twelve days. the data was recorded at the interval of 15 days to determine the effect of arbuscular mycorrhizal fungi and water stress on growth of pea plant. effect of water stress and am fungi on the dry weight of root and shoot of pea plant. the water stress was given at the interval of 4, 8 and 12 days in mycorrhizal as well as control plants and the dry weight of root and shoot was recorded in control and mycorrhiza l plants. the dry weight of root and shoot of both control and mycorrhizal plants decreased with the increase in water stress. mycorrhizal plants showed more dry weight of shoot and root as compared to control plants. mycorrhizal plants with minimum water stress interval showed enhanced shoot and root length than maximum water stress interval. effect of water stress and am fungi on the fresh weight of root and shoot of pea plant. the fresh weight of both control and mycorrhizal plants has been decreased with the increase in water stress interval. fresh weight of shoot and root was significantly high in those plants which were watered at the interval of 4 days than those which were watered at 12 days interval. it has been found that the fresh weight of root and shoot was observed higher in mycorrhizal plants as compared to those of control plants. effect of water stress and am fungi on the shoot and root length and the number of leaves of pea plant. the mycorrhizal pea plants were taller than non mycorrhizal plants during water stress. the height of shoot and root was significantly decreased with the increase in drought stress. mycorrhizal plants with low water stress showed enhanced shoot and root length than high water stress. the mycorrhizal plants have shown more number of leaves than control plants during drought stress. there was a significant decrease in the number of leaves with the increase in drought stress. references aroca, r., porcel,r. and ruiz-lozano, j.m. 2007. how does arbuscular mycorrhizal symbiosis regulate root hydraulic properties and plasma membrane aquaporins in phaseolus vulgaris under drought, cold or salinity stresses? new phytologist, 173: 808-816. auge, r.m. and moore, j.l. 2005. arbuscular mycorrhizal symbiosis and plant drought resistance. in: mehrotra vs (ed) mycorrhiza: role and applications. allied publishers limited, new delhi, pp 13. international journal of applied biology, 4(1), 2020 42 auge, r.m., kubikova, e. and j.l. moore 2001. foliar dehydration tolerance of mycorrhizal cowpea, soyabean and bush bean. new phytologist,151(2):535-541. barea, j.m., m.j. pozo, r. azcon and c. azcon-aguilar 2005. microbial cooperation in the rhizosphere. j. exp.bot., 56:1761-1778. de la pena, e., rodriguez, e.s., van der putten, w.h., freitas, h. and moens, m. 2006. mechanism of control of root feeding nematodes by mycorrhizal fungi in the dune grass ammophila arenaria. new phytologist, 169:829-840. galvan, g.a.,k. burger-meijer, th.w. kuyper, c. kik and o.e. scholten. 2008. possibilities fir breeding to improve responsiveness to arbuscular mycorrhizal fungi in onion. 16th ifoam organic world congress. gange, a.c. 2001. species-specific responses of a root and shoot feeding insect to arbuscular mycorrhizal colonisation of its host plant. new phytol, 150: 611-618. heikham, e. kapoor, r. and giri, b. 2009. arbuscular mycorrhizal fungi in alleviation of salt stress: a review. annals of botany, 104(7): 1263-1280. hildebrandt, u., kaldrof, m. and bothe, h. 1999. the zinc violet and its colonisation by arbuscular mycorrhizal fungi. journal of plant physiology, 154: 709-717. koide, r.t. and kabir, z. 2000. extraradical hyphae of the mycorrhizal fungus glomus intraradices can hydrolyse organic phosphate. new phytol, 148:511-517. koide, r.t. and mosse, b. 2004. a history of research on arbuscular mycorrhiza. mycorrhiza, 14: 145-163. kothari, s.k., h. marschnere and v. romheld 1991. effect of arbuscular mycorrhizal fungus and rhizosphere microorganisms on manganese reduction in the rhizosphere and manganese concentration in maize (zea mays l.). new phytol, 117:649-655. michelson, a. and rosendahl, s. 1990. the effect of va mycorrhizal fungi, phosphorus and drought stress on the growth of acacia nilotica and leucaena leucocephala seedlings. plant and soil, 124: 7-13. patale, s. w. and b. p. shinde 2012. studies on am fungi of bt cotton (gossypium hirsutum, l.). ph.d. thesis, university of pune. smith, f.a., grace, e.j. and smith, s.e. 2009. more than a carbon economy: nutrient trade and ecological sustainability in facultative arbuscular mycorrhizal symbiosis. new phytol, 182: 347-358. vigo, c., norman, j.r. and hooker, j.e. 2000. biocontrol of pathogen phytophthora parasitica by arbuscular mycorrhizal fungi is a consequence of effects on infection loci. plant pathology,49: 509-514. wu, q.s. and r. x. xia 2006. arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. j. plant. physiol, 163: 417-425. wu, q.s. and r.x. xia. 2004. effects of arbuscular mycorrhizal fungi on growth and osmotic adjustment matter content of trifoliate orange seedlings under water stress. j. plant physiol. mol. biol., 30: 583-588. international journal of applied biology, 4(1), 2020 43 wu, q.s., r.x. xia and y.n. zou 2008. improved soil structure and citrus growth after inoculation with arbuscular mycorrhizal fungi under drought stress. europ. j. of soil biol., 44:122-128. 3(1)81-87 contact : saroj thapa thapasaroj1998@gmail.com © international journal of applied biology 81 abstract rice (oryza sativa l.) is first staple crop of nepal. the national average yield of rice is less than its potential yield, for which poor agronomic management has been reported as the critical factor. among various agro-management practices seedling age, seeding density and nitrogen rates significantly affect the growth and yield of rice. the lower seeding density produces the taller plant, more effective tiller, lower sterility and higher grain yield. on the other hand, seedling of older age from higher seeding density gives the lowest yield. transplanting younger seedling with low seeding density and application of recommended dose of nitrogen gives a higher yield. this article summarizes various effects of seedling age, seeding density and nitrogen rates on plant height, number of effective tillers, number of filled grains, thousand grain weight and grain yield of rice. this information may be useful for maize growers and researchers. issn : 2580-2410 eissn : 2580-2119 effect of seedling age, seeding density and nitrogen fertilizer on growth and grain yield of rice (oryza sativa l.) saroj thapa1, khagendra thapa1, jiban shrestha2 & amit chaudhary1 1agriculture and forestry university, rampur, chitwan, nepal 2nepal agricultural research council, agriculture botany division, khumaltar, lalitpur, nepal introduction rice (oryza sativa l.) is considered as the most important staple food crop in nepal, followed by maize (zea mays l.) and wheat (triticum aestivum l.) respectively. rice is cultivated in wide range of altitude from plain of terai to jumla (3050 metre above sea level) of high hill region of nepal (narc, 2007). total rice cultivated area in nepal is 1.36 million ha with total production of 4.3 million ton (t) (cbs, 2015-16). the yield of rice in nepal and world context are 3.16 and 4.36 t ha-1 respectively (faostat, 2012). dependency of rainwater, low input use and poor crop management practices are the major causes of low rice yield in nepal (aicc, 2018). nepal has still very low productivity compared to other developed countries though rice is major cereal crop for nepal and its high yielding varieties are already available in global market. there is huge gap between rice productivity at researcher level and farmer’s field. it is so because farmers are still unable to do proper nursery as well stand crop management. padalia (1980) and lal and roy (1996) concluded seedling health determines the final yield of transplanted rice. practices of proper nursery management, integrated nutrient and pest management, maintenance of proper seedling density and timely open access international journal of applied biology keyword rice oryza sativa l. growth, grain yield, nitrogen (n), seedling age, seeding density. article history received 12 may 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 82 transplantation of seedling can improve growth and development of rice plant (naeem sarwar, 2011; subedi, 2013). rainfed rice covers 79% proportion of total rice produced in nepal. among it, distribution of rainfed rice in lowland and upland area are 70 and 9 % respectively (cbs, 2015/16). irregular and insufficient rainfall occurrence has delayed the transplantation date of seedling which has been reducing the final yield of rice (mishra and salokhe, 2008 ) as plant height, tiller production, panicle length, grain formation, grains per panicle, and other yield attributing characters are highly affected due to delayed transplantation of seedling (ali et. al., 1995). reduction of primary tiller occurs due to detoriation of buds in older seedlings as the consequence of delayed transplantation (mobasser, 2007). sufficient nutrient supply in rice nursery helps to enhance the dry weight, height, root density, root length and overall growth as well development of seedling (singh, 2005). proper growth and good final yield is obtained only after proper management of rice seedlings at their young stage and also using of healthy seedlings. reduction in seedling mortality, enhancement in stress tolerating capacity, increased number of effective tillers etc. are possible as the result of optimum application of nitrogenous fertilizers in nursery time and using of good seedlings only. about 50% and 100% more dry matter accumulation have been observed in rice seedlings due to application of nitrogen and phosphorus in nursery (ros et al., 1997). maximum rice productivity (4.9 t ha-1) have been observed after the application of di-ammonium phosphate and single super phosphate @ 50 kg p2o5 ha -1 in nursery. it valued 21% more yield compared to control method (rajagopalan and krishnarajan, 1987). practice of sufficient water supply, labour, weed management including other inputs that determines the optimum age of seedlings to be transplanted for better grain yield. in case of lowland field, seedlings of 25 to 50 days old are used for transplantation by farmers (wagh, 1988). previous researches has shown better yield when seedlings are transplanted before it gets 25 days old (rao, 1987; wagh, 1988). researches has directed towards the yield performance of rice when transplanted under age of 20 days old although findings are still contradictory (narc, 2004). few researchers have studied the synergistic influence of seedbed management on the characteristics of younger/older seedlings and the consequent impact on plant growth and yield after transplanting. thus the objectives of this study were to quantify the distinctive effects of nursery management and seedling age on yield and yield components of fine rice materials and methods relevant literatures from 1980 to 2018 were collected from journal articles, books, dissertations and working papers on effect of seedlings age, seeding density and nitrogen fertilizer on growth and grain yeld of rice. web pages were visited and the relevant information was collected. author's experience in agriculture intensification is also internalized in the paper. finally, information collected from different sources were analyzed and presented. results and discussion plant height both the environment as well as crop management practice effect the height of plant which is one of the major parameter determining growth status of plant. nutrient management, health of young seedlings, crop field environment and genetic characteristics international journal of applied biology, 3(1), 2019 83 also determine height of plant (yoshida, 1981). crop geometry of 20×15 cm found to be significantly increasing tiller number and plant height compared to spacing of 20× 10cm, 20×25 cm or 20×20 cm (nayak et al.,2011). transplantation of the younger seedling of 12 days old showed significantly taller plant height as compared to 30 days older seedlings in case of rice (mishra and salokhe, 2008). similarly, compared to 21 days old seedlings, 7 and 14 days old younger seedlings produced significantly taller plant height of rice (gani et al., 2002). insufficient fertilizer application and maintaining higher seedling density in nursery significantly reduces plant height (naeem sarwar, 2011). seedling health mainly depend upon nutrient management during its early growth stage (mishra and salokhe, 2008). both the process of cell division and elongation are responsible in improvement of plant height. significant increment in dry matter accumulation as the result of higher photo assimilates due to larger leaf area by the action of optimum utilization of nitrogenous fertilizer have been observed in rice plant (chaturvedi, 2005; zhilin et al., 1997). longer root length, higher root density, lesser transplantation shock due to lesser leaf area and good health of younger seedlings are responsible for increasing cell division and elongation process which ultimately increases plant height (sangsu et al., 1999; rahman, 2001). better seedlings used for transplantation showed significant enhancement of plant height (farooq, 2007). effective tillers effective tiller number can be increased by using nitrogenous fertilizer (budhar and palaniappan, 1996). the combined practice of using n fertilizer and low seeding density during nursery management can increase the number of productive tiller of rice seedlings. on other hand, if nitrogenous fertilizer was not applied then the number of effective tiller found to be higher for high seeding density than low seedling density (subedi, 2013). generally, lower seeding density favours the number of effective tillers significantly (sarwar, 2011). it has also been observed that in case of high seeding density, there were no tiller formation occurred as compared to low seeding density when 40 days old seedlings were used for transplantation. the age of seedling also effects the effective tiller number significantly. comparison to younger seedlings, older seedlings have more number of productive tillers (adhikari, 2013). mobasser (2007) found that higher number of effective trillers (355 effective tillers m-2) was produced in case of 45 days older seedlings as compared to younger ones. if young seedlings of 12 days old are transplanted then higher number of effective tillers are produced independent to amount of nitrogenous fertilizer applied and seeding density maintained in nursery (mishra and salokhe, 2008). this result occurs due to proper growth and development of root, increased cell division and enlargement process which are the intermediary consequence of increased photo assimilate. these enhanced processes finally increases the height of plant as well as tiller density in rice (shrirame et al., 2000; vijayakumar et al., 2005; salem et al., 2011). number of filled grains the number of filled grains per panicle is also one of the essential yield attribute. the number of filled grains per panicle has been found to be significantly higher in case of low seeding density compared to medium seeding density but result was not significant compared to high seeding density. there was also non-significant difference between effect of medium and high seeding density on filled grain number per panicle. comparison to transplanted older seedlings, younger seedling led to higher number of filled grains (subedi, 2013). highest number of filled grains produced in case of transplanted 45 days old seedlings among international journal of applied biology, 3(1), 2019 84 transplanted 30, 60 and 75 days old seedlings (khatun, 2002). despite of significant effect of seedling age on filled grain number, there was no significant effect seen on amount of nitrogen fertilizer application done in nursery. whereas, mursal (2008) found that number of tiller per clump, seed weight and number of seed per panicle increased due to application of n fertilizer application. huge or low amount of n application can lead to decreased filled grains number per panicle. the relation observed is that enormous amount of n utilization increases number of filled grains and also reduce number of unfilled grains. thousand grain weight the 1000 grain weight being an essential yield attribute mainly depends upon genetic characteristic of that plant compared to environmental influence (ashraf, 1999). optimum utilization of fertilizer significantly enhances the 1000 grain weight value (panda, 1991). rice crop management with high seeding density, no nitrogen application and use of older seedling significantly reduces 1000 grain weight than compared to optimum management including practices of low seeding density, optimum n application and transplantation of younger seedlings (saha, 1979; sarwar, 2011).the low nitrogen application of @ 150 kg ha-1 produced significantly higher test weight than compared to higher nitrogen level of 200 kg ha-1. it may have happen due to excessive growth of rice plant creating quite large number of spikelet which caused deficiency of carbohydrates require to fill all the empty grains reducing dry matter accumulation. this finally reduced the test weight of rice crop (pramanik and bera, 2013; channabasavanna and setty, 1994; raju and reddy, 1993). furthermore, hasegawa et al. (1994) also supported the result of reduced dry matter accumulation due to application of high amount of nitrogen in rice. grain yield the factors including seeding density, n fertilizer application and seedling transplanting age have shown significant interactive effect on final grain yield of rice. grain yield is reduced when practice of high seeding density and older seedlings transplantation with or without using n fertilizer are used. the amount and quality of grain can also be effected by types of nitrogenous fertilizer used for cultivation (gately and kelly, 1987). urea is considered as cheaper nitrogenous fertilizer that also don’t have detrimental effect on amount and quality of rice grain yield. gately et al. (1988) found that higher grain yield as the positive effect of calcium ammonium nitrate application which also have higher amount of n content compared to urea. ebaid and ghanem (2000) reported that application of nitrogen up to 144 kg ha-1 enhanced the growth of crop as well as its final grain yield too. el-batal et al. (2004) found that plant height, panicle length, no. of filled grains panicle-1and grain yield significantly increased when nitrogen dose increased between 120 to 190 kg ha-1. likewise, yoseftabar (2013) higher plant growth, yield attributes and grain yield obtained as the result of nitrogen application done at the rate of 100, 200 and 300 kg ha-1 respectively. comparison to practice of high seeding density, older seedlings, with or without nitrogen application has produced minimum grain yield (5.74 t ha-1) which is significantly lower compared to yield of 6096 t ha-1 obtained as the result of low seeding density, younger seedlings and with or without n application. result found to be non-significant for low, medium and high seeding density in case of young seedling transplantation and without n application (subedi, 2013). higher competitive condition for plant growth and changes in pattern of growth are caused by late transplantation of seedling (mandal, 1984). higher development of plyllocron and higher effective tiller number per plant which finally increases international journal of applied biology, 3(1), 2019 85 grin yield. this result is obtained by the transplantation as younger seedlings (de datta, 1980). moreover, younger seedlings of 20 days old maintaining low seeding density of 100 g m-2and with n application when transplanted in field produced maximum grain yield of 6.96 t ha-1 (subedi, 2013). conclusions the transplanting younger seedlings produces the maximum yield, irrespective of nursery management. the seedlings obtained from high seeding density should not be transplanted at older age because it reduced the yield of rice drastically. transplanting younger seedling with low seeding density and application recommended dose of nitrogen gives higher yield references adhikari, b., mehera, b. & haefele, s. 2013. impact of rice nursery nutrient management, seeding density and seedling age on yield and yield attributes. american journal of plant 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(2005). effect of nursery fertilization on seedling growth and yield of rice. indian journal of agronomy, 50:187-189. subedi, r. 2013. nursery management influences yield and yield attributes of rainfed lowland rice. journal of sustainable society, 2: 86-91. vijayakumar, m., sundar singh, s.d., prabhakaran, n.k. & thiyagarajan, t.m. 2005. effect of sri (system of rice intensification) practices on the yield attributes, yield and water productivity of rice (oryza satival.). acta agronomica, hungarica, 52(4): 399-408. wagh, r. s. 1988. effect of age of seedlings at transplanting, plant densities and nitrogen fertilization on the yield of rice variety r711. oryzae, 25: 188-190. yoseftabar, s. 2013. effect nitrogen management on panicle structure and yield in rice (oryza sativa l.).intl. j. agri. crop. sci., 5: 1224-1227 yoshida, s. 1981. mineral nutrient of rice: in fundamentals of rice crop science. irri , pp. 111-146. zhilin, l., sarker, r.s., nayak, s.k., ravi, i. & li, j.l. 1997. physiological effect of nitrogen application on aromatic rice. journal of south china agricultural university, 18:13–17 contact : ferissa ablola fbablola@up.edu.ph 123 abstract the robust development of molecular biology and bioinformatics in recent years has given researchers vital tools for addressing long -standing issues in all branches of biology. the process of reconstructing phylogenetic relationships using molecular data is known as "molecular systematics." in this systematic review, the utilization of molecular techniques in systematics was described in the two fields of microbiological research and public health. twenty articles were included in this study, showcasing nine unique molecular methods with distinct advantages and limitations of use. it was revealed that most articles under biological research aim to identify a specific group of microorganisms. at the same time, the determination of phylogenetic relationships and outbreak investigation were the main goals of public health utilization of molecular systematics techniques. issn : 2580-2410 eissn : 2580-2119 the role of molecular systematics in microbiological research and public health: a systematic review ferissa ablola 1*, anna liza bautista 2, gilden maecah migalang3, christa jesusa naval4 1 philippine department of health research institute for tropical medicine 2 unhs-division of laguna region iv-a calabarzon 3 msu – iligan institute of technology 4 university of northern philippines introduction hundreds of millions of people worldwide are now afflicted with illnesses caused by bacteria, viruses, protists, and fungi. according to the most recent study from the who (2022), people are anticipated to live longer and enjoy better health for an increasing number of years. the global healthy life expectancy (hale) improved from 58.3 to 63.7 years. despite this, health disparities continue to take a disproportionate toll on the lives and health of those in areas with fewer resources. the bulk of communicable illnesses, including hiv, t uberculosis (tb), neglected tropical diseases, malaria, hepatitis b, and covid-19, continue to be borne by lowand middle-income nations. despite substantial expenditures and advancements in communicable disease initiatives, these nations continue to shou lder most of this burden. without systematics, which identifies species and explores their evolutionary links, it would be hard to make significant progress against these illnesses. it is improbable that public health open access international journal of applied biology keyword molecular systematics; public health,; bioinformatics; microbiology. article history received may 25, 2023 accepted june 19, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:patienncesunday@gmail.com international journal of applied biology, 7(1), 2023 124 initiatives will be successful if they do not target a particular condition. due to evolution, many species' anatomy, biochemistry, and dna have become more distinct. creatures belonging to one clade may be able. to cause human illnesses, microbes may not belong to some other, closely rela ted clade. in order to correctly identify the underlying cause of disease and study the processes that contribute to its development, it is crucial to have a thorough understanding of the differences within clades. given that just 10–20 percent of species are estimated to have been described, it should not surprise that new disease-causing agents are continuously being identified. in addition, the advent of new techniques has shown that single species of pathogenic organisms sometimes consist of two or more different species. pathogenicity, treatment sensitivity, and reaction to the formulation of a vaccine, for instance, cryptic species, may differ significantly from one another. previously thought of as innocuous microorganisms are now capable of causing sickness in immunocompromised populations, which are rising at an alarming pace. genetic mutations are the underlying cause of the continuing alteration in the genetic composition of organisms. changes in pathogenicity are thus feasible and may be frequent. an increase in the use of chemicals for disease prevention results in the evolution of chemical resistance and the number of genetically different strains of a pathogenic taxon. this article discussed the relevance of molecular systematics in microbiological research and public health by describing several molecular systematic methodologies using genome and non-genome sequencing in microbiology research. in addition, the advantages and limitations of these techniques were also discussed. this article also provided insights on the current status of molecular systematics in the country from the pillar institutions of public health in the country. materials and methods literature search strategy preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines were followed in this review. a total of forty-two (42) full-text studies were collected from various databases, including pubmed, researchgate, sciencedirect, google scholar, oxford academic, wiley online, and ebscohost. the search terms used were as follows: public health, microbiology, systematics, research, and molecular biology . eligibility criteria the titles and summaries of the articles in molecular biology, health sciences, and education were reviewed to include peer-reviewed experimental research articles, literature reviews, and synthesis articles which demonstrated the importance and use of molecular techniques in microbial systematics. only full-text articles written in english and published between 2012 to 2022 were included in this review to ensure updated information on the research topic. international journal of applied biology, 7(1), 2023 125 data extraction studies selected from databases used were then imported to microsoft excel and were tabulated according to the author, year of publication, study objectives, the molecular technique used, organism involved, advantages and limitations of the technique, and its impact on microbial systematics. study selection flowchart figure 1. study selection flowchart results a total of thirty-six full-text research articles were collected from various databases, books, and websites. during the screening, eight duplicate studies were removed. another four studies were excluded since it was published before 2012, and four were also removed since the articles were not written in english. overall, there were twenty research articles analyzed in this systematic review. this review was able to document nine molecular techniques , including 1) 16s rrna gene-based ribotyping/ oligotyping; 2) amplified ribosomal dna restriction analysis (ardra), 3) random. international journal of applied biology, 7(1), 2023 126 table 1. summary of research articles (n=20) analyzed in the systematic review indicating its role in biological research and public health. author and year of publication (2012-2022) role of molecular systematic microbiology (identification/ determination of phylogenetic relationship) microorganism involved molecular technique used cited advantages of the molecular technique cited limitations of the molecular technique impact on microbial systematics microbiological research nunez et al. (2017) determination of phylogenetic relationship among acidithiobacilli species complex acidithiobacillus spp. 16s rrna gene-based ribotyping, oligotyping, and multilocus sequencing analysis (mlsa) oligotyping was used to profile high entropy nucleotide positions and resolve meaningful differences between closely related trains at the 16s rrna gene level. due to oligotyping greater discriminatory power, mlsa was used as a proxy for genome-wide divergence in a smaller but representative set of strains. at least six new lineages or phylotypes, supported by the different methods used in this study, were evident within the acidithiobacillus species complex and were determined. jarocki et al. (2016) identification of the members of the bifidobacterium genus bifidobacterium spp. ardra, rapd-pcr, rep-pcr, and sds-page fingerprinting highly discriminatory, easy-to-handle, and relatively low-cost procedure for rapid differentiation of bifidobacteria at the intra-species level. appears to be more laborious, and timeconsuming compared to other pcrbased methods rapid and accurate identification of these microorganisms at the species, subspecies, and even strain level due to the increasing application of bifidobacteria hwang et al. (2022) for inferring phylogenetic relationships and evolutionary study epilithic diatoms rapd-pcr small subunit (ssu) rrna coding gene is the most widely used and suitable for inferring phylogenetic relationships, and the ribulose1,5bisphosphate carboxylase/oxygenas e large subunit (rbcl) gene appears more suitable for evolutionary study. not mentioned in the article identification of three new species of diatoms gomila et al. (2017) identification of pseudomonas syringae species pseudomonas syringae species group three-gene multilocus sequence analysis (3gene mlsa) and wholegenome comparisons this study was able to circumscribe the p. syringae species complex and classify its strains into species according to the taxonomic rules and thresholds. not mentioned in the article classification of pseudomonas isolates international journal of applied biology, 7(1), 2023 127 author and year of publication (2012-2022) role of molecular systematic microbiology (identification/ determination of phylogenetic relationship) microorganism involved molecular technique used cited advantages of the molecular technique cited limitations of the molecular technique impact on microbial systematics fang et al. (2019) identification of shewanella-type strains shewanella type strains standard multilocus sequence analysis (mlsa) allows researchers to make rapid, economic, and precise identification of shewanella strains not mentioned in the article other strains of shewanella were also discovered in food samples consisting of both marine products and cooked food for sale. klaus et al. (2022) identification of hyperthermophilic ehaeon thermococcus sp. strain hyperthermoph ilic ehaeon thermococcus sp. strain activity-based protein profiling allows researchers to make rapid, economic, and precise identification of thermococcus strains not mentioned in the article abpp, in extremophilic environments, represents an alternative approach to addressing biological diversity and screening for novel biocatalysts. babafemi et al. (2017) detection of mycobacterium tuberculosis in pathological samples: mycobacterium tuberculosis real-time polymerase chain reaction (rt-pcr) based systems rt-pcr assay demonstrated a high degree of sensitivity for pulmonary tb and good sensitivity for extra-pulmonary tb. not mentioned in the article it indicated a high degree of specificity for ruling in tb infection from sampling regimes. ma (2022) identification of coronavirus coronavirus isothermal amplification technologies eliminating the need for expensive equipment such as thermocyclers. conducted at a constant temperature, has mild reaction conditions and low instrument dependence. coertse et al. (2019) identification of rabies virus rabies virus reverse transcription recombinase polymerase amplification assay allowed for very quick and sensitive detection of rabies virus do not cover the full genetic diversity of rabv considered as a supplementary tool where basic laboratory infrastructure is available, wang et al. (2015) identification of listeria monocytogenes listeria monocytogenes multiple inner primers-loopmediated isothermal amplification (mip-lamp) accuracy was shown to be 100% when compared to the culture-biotechnical method. may not detect extremely low levels of microorganism s that are nonculturable cells mip-lamp methodology was demonstrated to be a reliable, sensitive, and specific tool for rapid detection of l. monocytogenes strains. public health srinivasan et al. (2015) identification of most common hospitalassociated bacterial pathogens as well as acinetobacter baumanni; bordetella pertussis; burkholderia non-16s rrna-based molecular identification using naïve non-16s-based identity produced high confidence genus level identification and discordances between clinical and 16s-based identities due wide variety of bacterial species that are relevant to clinical settings can be accurately international journal of applied biology, 7(1), 2023 128 author and year of publication (2012-2022) role of molecular systematic microbiology (identification/ determination of phylogenetic relationship) microorganism involved molecular technique used cited advantages of the molecular technique cited limitations of the molecular technique impact on microbial systematics endemic communityacquire cenocepacia; burkholderia cepacia; burkholderia dolosa; burkholderiamu ltivorans; pyrrocinia; burkholderiasta bilis; vietnamiensis; citrobacter freundii; enterobacter aerogenes; bayes classifier and an alignmentbased approach good species level identification to insufficient representation of the clinical identities, phenotypic misidentificatio n, new taxonomic or phylogenetic placements identified routinely by microbiologists using high-quality sequences produced from 16s rrna gene amplicons. banoon et al. (2019). characterization and construction of phylogenetic relationship of different isolates of bacterial species from different sources in babylon province hospitals staphylococcus aureus random amplified polymorphic dna (rapd) fingerprinting technique rapd markers proved to have fingerprinting and diagnostic potential the technique cannot suggest the possible and frequent occurrence of mutants in s. aureus in different host cells the dna signature assigned to each s. aureus for epidemiological studies, medical diagnostics, and the location of novel pathogenic strains of bacteria is helpful. dallal et al. (2016). identification of s. aureus subtypes isolated from food samples staphylococcus aureus pcr-based coagulase genotyping by rflp analysis (coa-rflp) classification based on the rflp of the coa gene is a simple and accurate method for typing s. aureus isolated from various sources. not mentioned in the article with the aid of this typing approach, phylogenetic relationships or lineages between isolates from various sources can be established. meierkolthoff et al. (2014) characterization of the type of strain (u5/41t) of escherichia coli for subspecies delineation escherichia coli dna: dnahybridization (dddh it yields groups with a similar upper bound of character divergence. it directly addresses how to best cluster the sequences of genomes of interest. inconsistencies can occur when distance or similarity thresholds are used and the underlying distances specifically deviate from ultrametricity. analyses of the genome sequences of a large number of e. coli strains, which in turn suggests the presence of five subspecies within e. coli. ashton et al. (2016) characterization of salmonella for routine public health surveillance salmonella spp. multilocus sequence typing (mlst) the designation accurately depicts the relatedness of the strains, and this method is automatable. in the mlst database, a number of sts (including subspecies ii– iv) lacked a serovar the true phylogenetic relationship between isolates can be revealed using mlst data. serotypes can international journal of applied biology, 7(1), 2023 129 author and year of publication (2012-2022) role of molecular systematic microbiology (identification/ determination of phylogenetic relationship) microorganism involved molecular technique used cited advantages of the molecular technique cited limitations of the molecular technique impact on microbial systematics classification, and a surprisingly high number of unique sts were discovered. correctly be assigned to monophasic strains with imperfect antigenic structures. masim et al. (2021) identification of methicillin resistant staphylococcus aureus and outbreak investigation methicillinresistant staphylococcus aureus whole genome sequencing, multilocus sequence typing (mlst) the entire genome is scrutinized, and in one test, every single variant in the genome is identified; amr and virulence genes were captured either chromosomal or acquired requires highend equipment and technicians required knowledge and skills in bioinformatics genotypic characterization of circulating mrsa strains, with phenotypic and epidemiological data, led to the identification of several global epidemic clones present in the philippines jamoralin et al. (2021) identification of neisseria gonorrhea neisseria gonorrhea whole genome sequencing, multilocus sequence typing (mlst) the entire genome is scrutinized, and in one test, every single variant in the genome is identified; amr and virulence genes were captured either chromosomal or acquired requires highend equipment and technicians required knowledge and skills in bioinformatics genotypic characterization of circulating n. gonorrhea strains, with phenotypic and epidemiological data, led to the identification of several global epidemic clones present in the philippines chilam et al. (2021) identification of pseudomonas aeruginosa and outbreak investigation pseudomonas aeruginosa whole genome sequencing, multilocus sequence typing (mlst) the entire genome is scrutinized, and in one test, every single variant in the genome is identified; amr and virulence genes were captured either chromosomal or acquired requires highend equipment and technicians required knowledge and skills in bioinformatics genotypic characterization of circulating p. aeruginosa strains, with phenotypic and epidemiological data, led to the identification of several global epidemic clones present in the philippines chilam et al. (2021) identification of acinetobacter baumannii and outbreak investigation acinetobacter baumannii whole genome sequencing, multilocus sequence typing (mlst) the entire genome is scrutinized, and in one test, every single variant in the genome is identified; amr and virulence genes were requires highend equipment and technicians required knowledge and genotypic characterization of circulating acinetobacter baumannii strains, with phenotypic international journal of applied biology, 7(1), 2023 130 author and year of publication (2012-2022) role of molecular systematic microbiology (identification/ determination of phylogenetic relationship) microorganism involved molecular technique used cited advantages of the molecular technique cited limitations of the molecular technique impact on microbial systematics captured either chromosomal or acquired skills in bioinformatics and epidemiological data, led to the identification of several global epidemic clones present in the philippines lagrada et al (2022) identification of salmonella sp. and outbreak investigation salmonella sp. whole genome sequencing, multilocus sequence typing (mlst) the entire genome is scrutinized, and in one test, every single variant in the genome is identified; amr and virulence genes were captured either chromosomal or acquired requires highend equipment and technicians required knowledge and skills in bioinformatics genotypic characterization of circulating salmonella strains, with phenotypic and epidemiological data, led to the identification of several global epidemic clones present in the philippines amplified polymorphic dna polymerase chain reaction (rapd-pcr); 4) sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (sds-page); 5) three gene multilocus sequence analysis (3-gene mlsa); 6) genotyping by rflp analysis; 7) dna hybridization; 8) multilocus sequence typing (mlst) and 9) whole genome sequencing (wgs). most of the studies involved in this review utilized these molecular techniques primarily for identification (n=10), determination of phylogenetic relationship (n=5), and outbreak investigation (n=5) (table 1). it can be noted that the aim of most articles under biological research is to identify a certain group of microorganisms, while the determination of phylogenetic relationship and outbreak investigation were the main goals of public health utilization of molecular systematics techniques. discussion microbial systematics in the era of molecular biology one of the many fields of systematics is molecular systematics which makes use of the information contained in molecular data to reconstruct phylogenetic relationships (mauro and agorreta, 2010). systematics plays an important role in the field of biology by providing means for describing the organisms' characteristics and how they can be grouped together. thus, the two main goals of systematics in microbiology include the identification and analysis of relatedness among microorganisms. in phylogeny, the pattern of historical relationships among lineages of elements can provide an understanding of the groups sharing common international journal of applied biology, 7(1), 2023 131 ancestry. historically, the physical characteristics of organisms and classifying species were according to the most common traits they held. unfortunately, this method of identification of microorganisms, plants, and animals assumes that it is because they have common physical traits and that they have a common ancestry. it is, therefore, a challenge for taxonomists to begin using molecular differences to compare species. some morphological characters, such as those based on the shape of a structure, can be less easy to distinguish because of overlaps between different character states (brown, 2002). on the other hand, in molecular systematics, one can determine the similarities and differences among organisms using variations in protein and deoxyribonucleic acid (dna) molecules. for instance, some sources of molecular characteristics that can be used , such as a nuclear genome, nuclear ribosomal dna, mitochondrial genome, and chloroplast genome (onarici & sumer, 2002), can provide a much more accurate taxonomic picture of the relationship among microorganisms. the goal of systematic studies is to provide insight into the history of groups of organisms and the evolutionary processes that create diversity among species. in particular, testing of systematic questions and hypotheses, and the estimation of evolutionary processes and patterns, which includes divergence among taxa, can be provided by the study of molecular systematics. molecular systematics allows the examination of how species have changed over evolutionary time as well as the relationships between species that have no common physical characteristics. moreover, molecular changes can be used to explore and understand how populations are related evolutionarily and genetically. specifically, phylogenetics conveys the degree of changes in the evolutionary lines (hofling et al., 1997). molecular systematics requires phylogenetic analysis as a tool for studying phylogenetic relationships of living organisms. for example, the use of molecular markers allows the detection of variations or polymorphisms that exist among individuals in the population for specific regions of dna. the development of molecular markers has led to an explosion of studies that have used them to answer questions ranging from relatedness among species to the evolutionary history of populations, the amount of genetic va riation within a species, patterns of behavior, how patterns of gene expression can vary among closely related populations, and many other aspects of organismal variation ( monsen-collar & dolcemascolo, 2010). the nuclear ribosomal dna (rdna) and mitochondrial dna (mtdna) that have been most commonly used for phylogenetic research are highly conserved molecular markers and/or gene regions that are useful for investigating phylogenetic relationships at higher categorical levels (hwang & kim, 1999). a review by bhandari et al. (2014), which focuses on two different types of molecular markers, presented a successful understanding of the evolutionary relationships among prokaryotes. furthermore, the molecular markers can provide the identification of different prokaryotic taxa ranging from phyla to genera in clear molecular terms, and evolutionary relationships among them can also be reliably taken. the availability of fast dna sequencing techniques, along with the development of robust statistical analysis method s, provided new momentum in the field of molecular systematics. international journal of applied biology, 7(1), 2023 132 molecular techniques in the field of microbial systematics microbial systematics plays a crucial role in determining the extent of microbial identification and classification applied in numerous research endeavors (i.e., bioremediation). microorganisms are vastly distributed in nature, but inadequate information about their abundance and scope of diversity exists – creating a huge challenge among microbiologists and other biology experts (sangal et al., 2016). however, as technological advancements pave the way for these challenges to be addressed, so is a growing knowledge as well as gaps about microorganisms, including their appropriate identification and classification. many researchers have embraced and advocated for the use of genomics in microbial systematics. for instance, thompson et al. (2015) reiterated that there is a necessity to establish genome-based microbial taxonomy using complete genome sequences. accordingly, ecological and clinical microbiology concerns can be addressed from this molecular approach to microbial identification and classification. there is a growing utilization of 'omics'-based approaches in systematics. however, limitations can ensue when these techniques fail to prioritize major regulatory pathways (keller et al., 2020), i.e., enzymes involved in microbial pathogen -host interactions. chemical techniques are introduced to overcome these limitations, particularly in microbial systems. previously restricted to morphological and biochemical culturing tests alone, the identification and classification of microorganisms can now be carried out utilizing a number of instrumental techniques that have improved efficiency and analytical accuracy. in this section, four molecular approaches in microbial systematics will be described in terms of their application/principle and their advantages and disadvantages over other molecular techniques. activity-based protein profiling (abpp) using chemical probes, this technique directly reads out enzyme activity which is critical in enriching relevant target enzymes in complex and dynamic but regulated systems. this technique targets enzyme identification using its active site for chemical alteration, providing druggable targets for potential minute particle therapeutics (bachovchin & cravatta, 2012). abpp holds promising benefits in microbial systematics. used as a tool for the dynamic profiling of multiple enzymes in an endogenous setting (bachovchin et al., 2010), enzymes that are acquiescent to variation using small-scale particles can be selected. this will, in turn, provide an avenue to identify and filter out discriminatory inhibitors, as well as enzymes with unknown functions and substrates (niphakis & cravatt, 2014). in a rich community of bacteria, such as the gut, abpp positions are advantageous. mayers et al. (2017) successfully enhanced a complicated sample for relevant but rare enzymes. our knowledge of enzyme activity has been enhanced by abpp. most research has used some fundamental techniques, such as click chemistry-abpp (cc-abpp) and competitiveabpp strategies, to increase the specificity and accuracy of this technology. but these techniques also hold limitations. for example, nonspecific binding in competitive -abpp international journal of applied biology, 7(1), 2023 133 strategy is a major limitation for activity-based protein profiling. even at higher concentrations, electrophilic and photoreactive probes would very certainly label proteins in some unspecific ways (wright & sieber, 2016). limitations of bulky prote in groups and cell permeability of the probes also exist. this concern has been addressed through click chemistry-abpp by using a particular probe that can be differentiated with a diversity of identifiers. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) recognized as one of the most effective and adaptable methods for bacterial protein separation and resolution, sds-page may be utilized to identify phenotypic diversity and relationships within species (franco-duarte et al., 2019). this method involves loading protein samples onto polyacrylamide gel with pores such that the protein can move through the pores in reaction to an electric field. sds-page can provide information on protein quantity, size, and subunit composition, as well as purify samples for additional analysis (gallagher, 2012). with an upper stacking gel and a lower resolving gel that have various ph levels and polyacrylamide concentrations, the sds-page system can be described as a fragmented gel. proteins can move swiftly and pile into a tight band in the upper stacking gel owing to its lower polyacrylamide content before moving into the higher polyacrylamide -resolving gel for segregation. using their respective mobilities, the distribution of protein bands along the gel is transformed into numerical values. through the use of specialized software, these values produce a binary data matrix that provides a similarity matrix, which is then transformed into similarity phenograms (deosthali et al., 2021). particularly for the investigations of the classification of biodiversity among microorganisms, proteins have a great deal of promise for typing strains of clinical relevance and for taxonomic purposesfor instance, cellulase was extracted, screened for, and purified from bacteria found in molasses by islam and roy (2018), who also used morphological and biochemical techniques to identify the microorganisms. using sds-page, protein molecular weight was identified, which helped in the identification of three bacterial strains. s ds-page is a powerful system that divides protein molecules exclusively on the basis of mass. this method allows for the better classification of several microbial entities. sds-page is frequently used to separate complicated protein mixtures with great resolution. but the purposeful denaturation of proteins before electrophoresis is an obvious downside of sds-page. enzymatic activity, protein reactions, and other molecular processes are typically impossible to measure on proteins purified by sds-page. to isolate native proteins for studies of structure-function connections, alternative techniques must be utilized. functional characteristics, such as the existence of non-covalently metal ions, are also lost. international journal of applied biology, 7(1), 2023 134 random amplified polymorphism of dna (rapd) markers. random amplified polymorphism of dna (rapd) markers is a technique that is utilized to identify dna-based polymorphism that typically determines the value of dna -based markers. rapd markers are a development of employing markers based on polymerase chain reaction (pcr). the standard rapd method uses pcr to amplify nanogram quantities of whole genomic dna at low annealing temperatures using short artificial oligonucleotides of random values as primers. typically, agarose gels are used to separate amplification products before staining them with ethidium bromide. the detection or classification of several bacterial species has been made possible by markers based on rapd fingerprints. without the need for replicating, scanning, or any other type of molecular characterization of the dna of the target species, rapd analysis acquires a vast range of genetic identifiers that only require modest amounts of dna. due to its speed and user-friendliness, rapd has been widely employed to fingerprint the microbial population (panigrahi, 2019). in microbiology, this technique has been used to identify and analyze genetic variation in bacterial species isolated from different sources. for example, bannon et al. (2019) characterized staphylococcus aureus isolates via rapd analysis, each of which gives a different genetic profile. constructing evolutionary relationships using 8 rapd markers, the entire collection of s. aureus isolates was categorized into one main group and nineteen subgroups. studies involving related species, as well as analyses at the individual level, have both used rapd markers. but like any other molecular technique, this approach comes with a downside. because rapds are so sensitive to the reaction conditions, their main setback is their limited reproducibility; hence, highly standardized experimentation procedures are required. because small random primers are utilized, which have the ability to amplify dna fragments from a range of organisms, caution must be taken to prevent dna samples from becoming contaminated during rapd investigations, which often call for pure, high molecular-weight dna. restriction fragment length polymorphism (rflp) markers acting as a molecular identifier unique to a particular clone/restrictio n enzyme combination, this technique is frequently used to verify the little but noticeable variations in a string of double-stranded dna. the underlying principle behind the method is the enzymatic digestion of the dna by endonucleases, coupled by electro phoresis in an agarose gel and southern hybridization. electrophoretic tracks resulting from enzymatic digestion can be seen at various spots throughout the length of the genome as restriction enzymes split the dna into many fragments. segments of the radioactively tagged dna may combine with homologous sections of the membrane-immobilized dna to form hybrids. once the excess probe that did not undergo hybridization has been removed, exposing the surface to a radiography film will sensitize the film in the areas where the probe settles, producing bands that could be indicative of genetic variation. using the rflp marker, the gene sequence of the area that codes for genetic products can be used to identify genetic variation (mittal, 2013). international journal of applied biology, 7(1), 2023 135 hence, a restriction endonuclease will produce dna fragments of varying lengths depending on the species and the particular organism. being the first dna-based biomarkers for making genetic linkage maps, the number of rflp markers is almost limitless and is higher in allelic polymorphism. when combined with other techniques, such as pcr, rflp allows for increased receptivity and applicability, especially in microbial diversity investigations. yang et al. (2013) enumerated some of the advantages of this technique when coupled with other molecular methods, including high reliability, co-dominance, and selective neutrality. because established restriction enzymes are used to create rflp from particular locations, the results are consistent throughout time and space. for example, successful investigations of the diversity of v. cholerae strains (chowdhury et al., 2010), y. pestis taxonomy (qi et al., 2016), as well as s. aureus coagulase gene diversity (dallal et al., 2016) have been reported. moreover, rflp has also been utilized in phylogenetic studies of microbes to establish phylogenetic characterization, such as the establishment of genetic relationships among clinical strains of aspergillus fumigatus and species identification of methicillin-resistant staphylococci using mitochondrial dna regions (mittal, 2013). rflp has the benefit of being a rapid, easy, and precise molecular technique for population profiling and identification. however, it has less discriminatory strength and costs more to run than many other fingerprinting methods, such as rapd (tabit, 2016). hence, this technique requires a lot of work and time. furthermore, this method can only examine particular mutations at enzyme cut sites, which restricts the ability to identify whole genome variation. finally, the applicability of rflp markers is constrained by the relatively low polymorphism and requirement for radioisotope detection (yang et al. 2013). numerous molecular methods, some of which are presented here, enable the identification and characterization of microorganisms and establish different levels of subspecific and interspecies similarity. from the standpoint of microbial systematics, it is notable that a single approach might not provide all of the necessary information. to determine the levels of similarity rather accurately among microorganisms, it is essential to use many techniques. genome sequencing unfolds the genetic relationship among microorganisms a unique species are currently identified in the taxonomy of bacteria and archaea using the polyphasic approach, which considers an organism's various dimensions, including its phenotypic, genotypic, and chemotaxonomic features (tindall et al., 2010). since genetic data reveals the evolutionary links between various lineages, genotypic characterization is a crucial part of characterizing species in this process (chun & rainey, 2014). genotypic characterization has been greatly influenced by emerging sequencing technologies. new sequencing methods have allowed for faster and more efficient genotyping of the entire organism’s genome and open opportunities to improve many aspects of microbiology research (scheben et al., 2017). international journal of applied biology, 7(1), 2023 136 the sequencing of dna and proteins began in the 1970s with the sequencing of the virus lambda by sanger et al. (1977). around this time, dna sequencing was done for organisms with tiny genomes, such as viruses, but it was not possible to sequence a bacterium completely due to economic and technological constraints. later, however, advances in sequencing technologies and the deciphering of the human genome made it possible to sequence the entire bacterial genome. these techniques, termed next -generation sequencing technologies, have a high throughput, can produce more sequences, and are also less expensive. the technologies used in next-generation sequencing techniques, including single-molecule dna sequencing and sequencing by ligation, apply a sequencing-by-synthesis approach. this technique produces clonal dna colonies by attaching dna molecules and primers on a slide and amplifying them with dna polymerase. several different kinds of fluorescently tagged reversible-terminator nucleotides are introduced, and the incorporated nucleotides are photographed in order to assess the dna sequence. the subsequent cycle can begin after the fluorescent dye with the terminal 3' blocker is chemically removed from the dna. a completely contiguous dna sequence of the microbe's chromosome is what a genome sequencing study aims to achieve. after a microbial genome has been mapped, the next step is to annotate it; genes or proteins can be used to infer structural, physiological, and other biological information. predicted pro tein-coding sequences, also known as open reading frames, are identified in the first step of the annotation process using bioinformatics analysis and tools like blast (basic local alignment search tool). because these species lack introns and have a high gene density, it is much simpler and more precise to identify open reading frames in the genomes of bacteria and other prokaryotic organisms. only a portion of the open reading frames in the genome's sequence actually code for proteins, and the next step in the annotation process is to predict their activities by comparing them to genes with corresponding known functions in databases (donkor, 2013). genome sequences have significantly improved the categorization of species compared to earlier methods because they clarify operational features of taxonomic groups and effectively solve inconsistencies in the phylogeny of upper taxa (whitman, 2015). the study of the epidemiology, pathophysiology, and microbiological characteristics of bacteria is built on the precise species and variant assignment of bacterial isolates, enabling improved worldwide monitoring and comparative genomics. for example, wu et al. (2020) updated the complicated taxonomy of enterobacter species and subspecies based on whole-genome taxonomic analysis. utilizing the genome data comes once a genome has been sequenced and annotated; it provides the fundamental knowledge necessary to comprehend the biology of the organism (donkor, 2013). the entire genome of each microorganism (metagenome) in an environmental sample is used by the most recent genome sequencing technology in microbiology. new species can be identified, and genes essential for the community's survival can be characterized by sequencing every gene in a microbial population. the wa y we research bacteria has been changed by genome sequencing technologies (antoniewicz, 2020). similar to how information technology gave rise to google maps, which allows us to international journal of applied biology, 7(1), 2023 137 find out specifics about locations across the world, genetic information from genome sequencing is utilized to construct maps of various organisms' genomes. studies are no longer restricted to the small percentage of bacteria that will grow in a laboratory because they can be carried out on a lone microbe that has been directly deri ved from its natural home. from a microbiology perspective, genome sequencing still needs a significant amount of dna template, which is acquired from uniform cell cultures. the majority of these species, however, could not be sequenced because it is predicted that the majority of all bacterial species remain uncultivated due to unknown growth requirements. the advent of 16s ribosomal rna gene pcr analysis, which allowed for the amplification and sequencing of an extremely informative gene from the majority of bacterial species, transformed the study of microbial genomics. although phylogenetic trees could be built using the 16s rrna gene sequence, investigations were restricted to this one gene. metagenomics, a technique that involves sequencing all the dna from an environmental sample, made it possible to make yet another significant advancement. the benefit of metagenomics is that it offers sequences for all of the genes present in environmental populations. however, the synthesis of genes and individual genomes continues to be difficult because of the tremendous diversity of several microbial communities. short reads from next-generation sequencing are mostly used in metagenomics. the method becomes constrained in its capacity to precisely identify differences between bacterial strains, such as the existence of novel genes, as the richness of the microbial community rises. roles of molecular systematics in microbiological research molecular systematics has become the primary way to determine evolutionary relationships among microorganisms because morphological and other phenotypic characters are either absent or change too rapidly to be useful for phylogenetic inferences (eisen, 1995). in systematic molecular studies, proteins and dna are the two molecules that have been most studied. these molecules contain phylogenetic information in a linear array. these arrays are informative because they reflect indirectly (in the case of protei ns) or directly (in the case of dna) the heritable developmental program of the organisms (powell, 1991). moreover, systematic molecular studies and the molecule of choice for most such studies of microorganisms have been the small subunit of the rrna (ss-rrna), which revolutionized the understanding of the diversity and phylogenetic relationship, particularly to microorganisms (eisen, 1995). the progress in molecular biological tools such as dna hybridization, polymerase chain reaction-restriction fragment length polymorphisms (pcr-rflp), randomly amplified polymorphic dna (rapd), allozyme data, microsatellite dna provide various molecular data to gather phylogenetic relationships among taxa (hwang & kim, 1999). the study of nuñez et. al., 2017 used 16s rrna gene-based ribotyping, oligotyping, and multilocus sequencing analysis (mlsa) to determine phylogenetic relationships and phylogenetic species boundaries of acidithiobacillus spp. these sulfur-oxidizing acidophilic bacteria thrive in natural environments of low ph, and their properties have long been harnessed for the biotechnological processing of minerals. in another case, the international journal of applied biology, 7(1), 2023 138 characterization of members of the genus bifidobacterium, sequencing of the 16s rrna gene, and some other genes considered to be molecular markers was investigated (sidarenka et al., 2008). in addition, among the tested procedures, rep -pcr proved to be the most effective and reliable, allowing rapid differentiation of bifidobacterium strains (jarocki et al., 2016). furthermore, rep-pcr fingerprinting using the boxa1r primer can be considered a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies, and potentially up to the strain level (masco et al., 2003). the enormous demand for probiotic bacteria and the accurate identification of these specific bacteria to produce high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. in the study of carvajal & galvez (2015), which characterized methylobacterium, the use of the 16s rrna gene sequence was preferred since it provides more accurate, elucidated identification and definition of the difficult bacterial isolates. the study revealed that through the identification of strains of methylobacterium which are mostly found in the leaf surfaces of plants and have been shown to produce phytohormones (i.e., cytokinin and auxins). furthermore, the identification of methylobacterium strains showed that some strains could metabolize long chains of aliphatic hydrocarbons and thrive in polluted environments, therefore its potential as pollution indicators. hwang et al. (2022) studied three species of diatoms from mountain streams in south korea using both morphological (lm and sem) and molecular approaches focused on small subunit (ssu) rrna and ribulose bisphosphate carboxylase (rbcl) genes. the results obtained from the molecular analysis of this study showed that the three species had clear differences in phylogenetic distance. on the other hand, in algae taxonomy, the use of ssu nrdna in establishing relationships between members of gracilariceae was first carried out at the family-to-species level in red algae (maggs et al., 2007). roles of molecular systematics in microbiology in public health the study by allard and brown (2020) stated that the field of epidemiology, public health, and food safety needs more interdisciplinary teams with expertise in molecular systematics. the ten public health articles included in this study revealed that mlst, secondary to whole genome sequencing, is the most utilized molecular technique for health surveillance and outbreak investigation. the studies of chilam et al. (2021) discussed the genomic characterization of the two medically important nosocomial pathogens, pseudomonas aeruginosa and acinetobacter baumannii. whole genome sequencing was performed to determine all genes present in the isolate, including antimicrobial resistance (amr) genes and virulence genes. integrons were also detected, which carry the series of amr genes, which can either be chromosomal in nature or transmitted via plasmids. through mlst analysis, the prevailing clonal systems and sequence types of these two pathogens in the country were assessed. in addition, the results of whole genome sequencing results permit further investigation of the makeup of the organism through bioinformatics analysis. furthermore, the phylogenetic relationship among international journal of applied biology, 7(1), 2023 139 isolates shows strong evidence of p. aeruginosa and a. baumannii outbreak occurrence in multiple hospital sites in the country. the results showed that the isolates were from a common source since the single nucleotide polymorphism (snp) difference is very minimal, which ranges from 0-1. jamoralin et al. (2021) sequenced the whole genomes of 21 n. gonorrhea isolates collected in 2013–2014 by arsp. the multilocus sequence type, multiantigen sequence type, presence of determinants of antimicrobial resistance, and relatedness among the isolates were all derived from the sequence data. their study led to the placement of philippine genomes within the global lineage a and the determination of an international transmission route. moving forward with molecular systematics in microbiology in the philippines the department of healthresearch institute for tropical medicine (ritm) and the department of science and technology – industrial technology development institute were two of the leading government institutions that facilitate public health and biologica l research development. the antimicrobial resistance surveillance reference laboratory (arsrl) is one of the national reference labs situated at ritm and conducts real -time surveillance of medically important pathogens. arsrl is partnered with a sentinel laboratory-based antimicrobial resistance surveillance on aerobic bacteria from clinical specimens. currently participating in the program are 24 sentinel sites and two gonococcal surveillance sites, representing 16 out of the 17 regions of the country. this laboratory utilizes satscan software which performs prospective real-time or time-periodic disease surveillance for the early detection of disease outbreaks. the detected cluster of isolates was sent out to arsrl and was then subjected to whole-genome sequencing. sequence data were used for phylogenetic analysis and computation of snps differences. the results of the bioinformatics analyses were communicated as soon as possible to the infection control team of the hospital for proper management. on the other hand, the industrial fermentation technology section (ifts), a unit under the environment and biotechnology division (ebd) of the dost-itdi mandated to conduct r&d and technical services on biotechnology. one of the main research conducted in this institute is the identification and phylogenetic analysis of actinomycetes from various sources. in addition, ebd also dwells into cataloging possible philippine strains of zika and african swine fever viruses and coconut cadang-cadang viroid through genome sequencing. these developments and programs showcase the utilization and importance of molecular systematics to push forward research and surveillance in the country. international journal of applied biology, 7(1), 2023 140 synthesis the figure above shows that the field of microbiological research and public health both utilizes the techniques of molecular systematics. in congruence with the collected articles in this review, rapid and accurate identification of microorganisms was the primary goal of microbiological research for the utilization of these methods. while it can be noted that, for public health purposes, molecular systematics involves the initial step of identifying the isolates followed by the assessment of the relationship among the group of organisms. then the generated phylogenetic tree and snps matrix would reveal the occurrence of the outbreak in a specific spatiotemporal event. conclusion molecular systematics and its methodologies unleash the full capacity of investigation in the microbiological field and public health setting. the main utilization of molecular systematics in these two areas of study involves identification, assessment of phylogenetic relationships, and outbreak investigation among microbial isolates. references abdulina, d. r., parish, l. m., & iutynska, g. o. 2018. microbial communities and sulphatereducing bacteria in soils near main-gas pipeline. мікробіологічний журнал, (80, № 5), 3-14. adékambi, t., drancourt, m., & raoult, d. 2009. the rpob gene is a tool for clinical microbiologists. trends in microbiology, 17(1), 37–45. https://doi.org/10.1016/j.tim.2008.09.008 amit roy, s. r. 2014. molecular markers in phylogenetic studies -a review. journal of phylogenetics & evolutionary biology, 02(02). https://doi.org/10.4172/23299002.1000131 alverson a. j. 2008. molecular systematics and the diatom species. protist, 159(3), 339 –353. https://doi.org/10.1016/j.protis.2008.04.001 antoniewicz, m. r. 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leng, 2011; romeis, j, 2008; shapiro, 2000). this condition is expected as the impact of using chemical (synthetic) insecticide excessively which is very dangerous and is not recommended by environment observer because can endanger the nontarget organisms (kumar, 2012). to overcome that problem, botanical insecticide becomes one of alternatives in conserving the non-target organism in order to keep the natural balance. the use of natural biopesticide of plant’s extraction gives the secure effect (kandagal, 2011; nathan, 2004; leng, 2011). its application of use is not leave behind the chemical residue which endangers the non-target organism, human being and environment (horne & page, 2008; tanada & kaya, 1993). botanical insecticide which aims as biopesticide for the open access international journal of applied biology keyword biopesticide, mirabilis jalapa, lectin, immunity system, spodoptera litura. article history received 18 february 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 63 insect’s pests, one of them is the use of plant extraction mirabiis jalapa (maulina, 2018a; maulina, 2018b; maulina, 2018c). this plant contains antiviral and antiviroid activity compounds in form of ribosome inactivating protein (rip) which is known as mirabilis antiviral protein (map) (vivanco, 1999). testing of effectiveness and determining concentration of that compound are needed to prevent the resistance of target pests. the result of laboratory test which had been done by m. jalapa as biopesticide has ld50 on concentration 0.8% toward spodoptera (maulina, 2018a). the use of sub lethal concentration of m. jalapa is conducted in order the pest target is not become resistance with it. the main aim on application of biopesticide m. jalapa is to weaken its body immunity. spodoptera litura is one of dangerous pests of agricultural plants. its folifagus characteristic can destruct the plants of yield agriculture thus makes 100% defoliation (suharsono, 2011). resistance of s. litura toward various chemical compound needs to be anticipated because the spreading covers south pacific and asia region (sparck, t.c, 2014; mei, z.x, 2012; scheiner, 2000). this becomes urgent reason to restrain spodoptera pest by using alternative effort of botanical biopesticide. focusing on its restraint, immune system becomes the main thing that needs to be known of its whole mechanisms which occurs in its body. therefore, biopesticide m. jalapa gives chance toward weakening immune system of s. litura. immune system becomes the indicator toward the death of pest. this case occurs because defense mechanism becomes the barrier of its body toward the strange object and its response when its body is getting the attack of strange object. the main task of m. jalapa as biopesticide is to break the defense, thus spodoptera is weakened. generally, insect has cellular and humoral defense. mechanism of humoral and cellular response cannot be separated one another. both of them stimulate each other to do their role (gillot, 2005; chapman, 2009). humoral response becomes the most crucial part because has the role to activate the enzyme and stimulation of strange object’s recognition. in its humoral mechanism, it will stimulate immune system of running cellular. therefore, humoral response becomes one of parameters in immunity sequence which is one of the factors that cause of its death. this research aims to analyze how does the influence of giving leaves extract of m. jalapa toward the defense of humoral immune s. litura. the response of humoral immune which was observed is the expression of lectin protein. lectin is protein, not enzyme or glikoprotein which binds or reacts with carbohydrate of various strange objects (yu, qx, 2000; yu qx, 2005; marmaras, 2009). lectin has very important role in defense mechanism of insect’s body. through the observation with sub-lethal concentration of m. jalapa will be seen how does the response of its humoral run. the final result of this research will determine the potential of m. jalapa as biopesticide toward s. litura pest is observed by defense mechanism of its body. materials and methods sample of research this research used larva s. litura the fourth instar as sample of research. larva s. litura was obtained by balai penelitian tanaman pemanis dan serat (balittas) malang, east java. the process of multiplication and treatment of larva was conducted at laboratory of plant pest with the condition of temperature 25-26˚c and humidity 50-55%. during the process of larva multiplication was given woof diet of darker colored mustard greens and was international journal of applied biology, 3(1), 2019 64 treated inside of plastic topless (volume 5 liter, diameter 12 cm and height 11 cm). each topless contains 50 larva of s. litura and the topless of treatment was cleaned every 12 hours. furthermore, hemolimf of research sample was used as the subject of research for the content of lectin protein. extract of m. jalapa biopesticide the crude extract of m. jalapa was obtained through maceration process which conducted at upt materia medica batu-government health service of east java province. the leaves of m. jalapa were obtained from field searching at lampung province. the leaves of m. jalapa was dried without sunlight. meseration was conducted by using ethanol 96% during 3 days. the result of this maseration collection was done further on evaporation process until it formed the paste. the paste was in concentration 100%. this research was used sub-lethal concentration were 0.1%, 0.2%, 0.4%, 0.8% (w/v), and the control. in this study, % (w/v) is defined as the percent of weight of m. jalapa extract (in gram) in the total volume of solution (100 ml ethanol). m. jalapa extract having certain concetration was sprayed throughout the surface of green mustard feed. after 24 h of exposure, hemolymph of the larvae were taken for the immune system measurements. the treatment was implemented individually on each larva, and was replicated on five different larvae for each condition. lectin analysis hemolimf s. litura was collected in tube eppendorftube which had been filled by crystal phenylthiourea (ptu). the solution was centrifuged during 5 minutes with temperature 4oc; 800g then pellet and supernatant were separated on different epperndorftube. supernatant was used for hemagglutinin-assay (ha) test, pellet was washed by using tbs ph 7.4. then, 50 ul tbs and pellet were suspended with speed 12.000 g during 15 minutes, the result of centrifugal deposits was used for ha. hemolimf 2 ml was homogenized in the pressure 400kp/cm2 during 5 minutes, was continued by centrifuging in the speed 12.000 g during 15 minutes, this supernatant was used as lectin source. hemagglutination assay test was conducted by preparing blood of vertebrate animals with its anticoagulant. wash 3 times tbs ph 7 concentration 2%, prepare test container (titertek) “v”, put in test sample as much 24 µm (centrifugal deposits) in tbs ph 7,4, drip 25µm erythrocyte suspense and was incubated with room temperature during 60 minutes (suryani, 2014). results and discussion immune system of insect is the result sequence of defense body toward specific and non-specific response toward detected strange object of its body. the defense mechanism of insect covers cellular and humoral defense. humoral response holds the main role in the sequence of immune system through various enzymatic and non-enzimatic reaction which has the role in recognizing the body toward strange object and its resistance effort. the mechanism depends on recognition effort of strange object through its receptor. there are 8 receptors which involved in mechanism of humoral immune, namely: immulectins, thioestercontinuing proteins (teps), lps-binding protein, peptidoglican recognitions proteins (pgrps), gram-negative bacteria binding protein (gnbp), β 1,3-glukan recognition protein (βgrp), haemolin (immunoglobulin superfamily) and b. mori multibinding protein (bmmbp). induction of strange object which is received by these receptors will give the impact to the international journal of applied biology, 3(1), 2019 65 action, such as: (1) induction and secretion of anti microbial peptides (amps); (2) mechanism of melanization (marmaras, 2009). receptor becomes the important part in defense mechanism. lectin is protein which acts as recognizing receptor of strange object which binds directly with carbohydrate (yu qx, 2005). its existence becomes the main key to activate phenoloxidation on hemolimf plasma (yu, qx, 2000; yu qx, 2005; marmaras, 2009). immulectin lies in granular and eonocytoid cell also has function in increasing encapsulation (yu qx, 2004). its ability in recognizing strange object as its non-self in form of glycoprotein and glycolipid makes it as the main receptor in mechanism of immune system. lectin that able to induce the sequence of cellular and humoral in defense system becomes the indicator for recognizing strange object and for further signal transduction. m c c a b c d 100 kda 64 kda 52 kda 40 kda figure 1. the profile of spodoptera litura lectin protein result of research showed that lectin protein existed in s. litura when was exposing biopesticide m. jalapa. this is shown by the profile of lectin protein which has molecule weight in 40kda. figure 1 is the result of electrophoresis test of hemolimf s. litura in part of supernatant. the speed of lectin response was measured through the form of hemaglutination assay (ha) measurement ability (titer) which showed different result on each given concentration. test of ha which was conducted observes the lectin response toward its binding with membrane cell of blood of vertebrate animals (carbohydrate). there are 4 concentrations of sub-lethal m. jalapa which were exposed in larva s.litura, namely: 0.8%, 0.4%, 0.2% and 0.1%. test result shows that on concentration 0.2% lectin binding with carbohydrate and erythrocyte cell of vertebrate were formed faster than the control (figure 2). forming of ha measurement ability (titer) on the control occurred in the eightieth minutes while concentration 0.1% and 0,2% occurred faster in the sixtieth minutes (figure 1). the speed of measurement ability (titer) forming was influenced by many hemocyte, because the receptor of immulectin was on the surface of its cell, thus the more number of hemocyte, then the number of lectin receptor will more increase and the activity of lectin binding with strange object will be faster to be recognized. concentration 0.2% is the optimum concentration of immune system’s mechanism which was shown by the increasing number of hemocyte (p<0.05) (maulina, 2014; suryani, 2014). m: marker c: control a: concentration 0.1% of m. jalapa b: concentration 0.2% of m. jalapa c: concentration 0.4% of m. jalapa d: concentration 0.8% of m. jalapa international journal of applied biology, 3(1), 2019 66 giving of m. jalapa on the concentration 0.4% and 0.8% gave longer time response than in the control, they were in the ninetieth minutes and in the hundredth five minutes. this was expected because of more decreasing number of hemocyte. on the higher concentration, hemocyte cells cannot proliferate anymore. toxicity of high concentration causes enzymatic and coordination system disturbance thus cells which induced the cell mitosis are obstructed (maulina, 2013). figure 2. speed of forming hemaglutination in principle, the application of biopesticide m. jalapa is not kill the target pest s. litura totally but its use can induce the reaction of immune system by lowering the physiological function at the whole (maulina, 2014). this condition is intended on the preventive efforts of sustainable resistance of target pest. the prevention of resistance is needed in order to be done easier for the control of this pest. if resistance occurs, resurgence will occur certainly because there is a multiplying use of insecticide doses (spark, 2015; dutcher, 2007). therefore, the use of biopesticide m. jalapa prevents both of cases occur based on the principle recommendation of integrated pest management (ipm) (kumar, 2015; kather, 2012; kumar, 2012; kandagal, 2011; leng, 2011; nathan, 2004) . the result of this research can be used as basic reference in using biopesticide m. jalapa to apply widely in agricultural land. setting the amount of spodoptera pest needs the consideration in the proper concentration in applying on the actual agricultural land. the aim of weakening immune system which was conducted in this research ends on the mortality of target pest thus can be used to control the population of pest. conclusions mirabilis jalapa has the potential as biopesticide, the indicator was seen through mechanism of humoral immune system of larva spodoptera litura body. the result of analysis is seen that the given leaves’ extract of m. jalapa can induce the activity of lectin protein. giving the concentration 0.2% of m. jalapa extract able to induce the speed of lectin forming reaction, this condition stimulates the occurrence of weakening sequence of immunity process of s. litura. 80 75 60 90 105 0 20 40 60 80 100 120 control 0.1% 0.2% 0.4% 0.8% t im e (m in ut e) concentration of mirabilis jalapa extract international journal of applied biology, 3(1), 2019 67 acknowledgement we express our deepest gratitude to directorate of research and community service, directorate general strengthening research and development and ministry of research, technology and higher education who have provided the research grants for this doctoral dissertation research in accordance with the research contract, number: 062/sp2h/lt/drpm/2018. references anggraeni, t., melanie. & putra, r. e. 2011. cellular a humoral immune defenses of oxya japonica (orthoptera: acrididae) to entomophathogenic fungi metharizium anisopliae. enthomological research, 41, 1 6. ashida m & brey p. 1997. recent advances in research on the insect prophenoloxidase cascade. molecular mechanisms of immune responses in insects (ed. by p brey & d hultmark), pp. 135–171. chapman & hall, london, uk. bai, y., yan, r., ke, x., ye,g., huang, f., luo, y., & cheng, j. 2011. effect of transgenic bt rice on growth, reproduction, and superoxide dismutase activity of folsomia candida (collembola: isotomidae) in laboratory studies, journal economic of entomology, 104, 1892 1899. boman, h. g. 1986. antibacterial immune proteins in insects. immune mechanisms in invertebrate vectors (ed. by amlackie), pp. 45–58. symposia of the zoological society, london, uk. cerenius, l & soderhall, k 2004. the prophenoloxidase-activating system in invertebrates. immunological reviews 198: 116–126. chapman, r. f. 2009. the insect structure and function 4thedition. cambrigde university. dutcher, j. d. 2007. a review of resurgence and replacement causing pest outbreaks in ipm. general concept in intergrated and disease management, 27-43. entomology departement, university of georgia, tifton ga, usa. gillot, c. 2005. enthomology third edition. university of saskatchewanspringer. gonzales, i., santoyo, & aguilar, a.c. 2011. prophenoloxidase: a key component of the insect immune system, journal of entomologia experimentalis et applicata, 142, 1 16. horne, p. a. & page, j. 2008. integrated pest management for crops and pastures, victoria: landlink press. kandagal, a. s. & khetagoudar, m. c. 2011. study on lavarsidal activity of weed extract against spodoptera litura. journal of environmental biology. vol. 34, 253-257. kather, h. f. 2012. prospect of biopesticides in insect pest management. journal pharmacologia. vol. 3 (12): 641-656. doi: 10.5567/phrmacologia.2012.641-656. kumar, s. & singh, a. 2015. biopesticide: present status and future prospect. journal fertilizer and pesticide. vol. 6 (2). doi: 10. 4172/jbfbp. 1000e129. kumar, s. 2012. biopesticide: a need for food and evironmental safety. journal biofertilizer and biopesticide. vol. 3 (4). doi: 10.41772/2155-6202. 1000e107. leng, p., zhang, z., pan, g. & zhao, m. 2011. applications and development trens in biopesticide. african journal of biotechnology. vol. 10(86), pp. 19864-19873. marmaras, v.j. & lampropoulou, m. 2009. regulator and signaling in insect haemocyte immunity, departement of biology, university of patras, journal of cell signaling, 17, 8-16. international journal of applied biology, 3(1), 2019 68 maulina, d & anggraeni, t. 2014. the effect of the combination of two biological control agents, mirabilis jalapa and bacillus thuringiensis, to spodoptera exigua’s immune respons and their mortality. aip confference proceeding-international conferences mathematics and sains: bandung. maulina, d., sumitro, s. b., amin, m., lestari, s. r. 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chemistry. vol : 275, 37373-37381. doi: 10.1074/jbc.m003021200. contact : ambeng ambeng@unhas.ac.id 27 abstract community structure analysis is one way to describe and assess the environmental quality of an ecosystem. one part of environmental biotic that establish mangrove ecosystems is bivalves. the mangrove area in the pangkajene river estuary has become one of the bivalve habitats, which has been widely used by the community. research on the bivalve community structure of the mangrove area in the pangkajene river estuary was conducted in april june 2019, which aims to get an overview of bivalve community structure in the mangrove area of pangkajene river estuary. the research area was divided into three estuary, each estuary was divided into two sampling areas. sampling was carried out using a 50 x 50 cm plot method. community structure analysis includes density, diversity index, uniformity, dominance, and distribution patterns. the results showed that the composition of bivalve community consist of 19 species included in 12 families. the results showed that the composition of bivalvia community consist of 19 species included in 12 families with the highest relative density, saccostrea sp. with a density of 15.11 ind/m2 (average relative density 50.77%). diversity index values in each sampling area are low and high, ranging from 0.91 to 1.91 and the value of uniformity varies with no species dominating in all sampling areas. commonly, all species of uniform spread, except saccostrea sp. with clumped distribution patterns. issn : 2580-2410 eissn : 2580-2119 analysis of bivalvia community structure in the pangkajene river estuary, pangkajene dan kepulauan regency ambeng1,5, hazairin zubair2, putu oka ngakan3, & adi tonggiroh4 1department of biology, faculty of mathematics and natural sciences, hasanuddin university 2soil and water conservation, faculty of agriculture, hasanuddin university 3conservation biology, faculty of forestry, hasanuddin university 4department of geological engineering, faculty of technic, hasanuddin university 5graduate student of geoscience and environmental technology, department of geological engineering, hasanuddin university introduction mangrove ecosystems are one of the coastal ecosystems that have high ecological and economic potential (printrakoon & temkin, 2008). one area in south sulawesi province that has natural resource potential for coastal areas is pangkajene dan kepulauan regency, in the form of mangrove ecosystems, fish ponds, coral reefs as well as diversity of fish and other marine biota species (noveria et al., 2008). mangrove ecosystems are dynamic ecosystems and have an important contribution to the availability of organic detritus for living things that open access international journal of applied biology keyword structure, community, bivalvia, pangkajene river. article history received 10 february 2020 accepted 27 march 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 28 live in and around them (karimah, 2017). one of macro invertebrate who has ecological and economic value in the mangrove ecosystem is bivalvia, which can live on the bottom of the waters. bivalvia is one of the members of the mollusks with a shell, which lives and obtains food by filtering (filter feeders) (anonymous, animal diversity web (adw) and can be used as a bioindicator for changes in the quality of the aquatic environment (odum, 1993). an analysis on a substrate or habitat can give a description of the environmental quality of waters (athifah et al., 2019) (wahyuni et al., 2017). based on the description above, this research was conducted on the structure of bivalve communities in the pangkajene river estuary, pangkajene dan kepulauan regency, south sulawesi. materials and methods time and place of research this research was done in april june 2019, in the river estuary area of pangkajene dan kepulauan regency, south sulawesi. data analysis was carried out at the zoology laboratory, department of biology, faculty of mathematics and natural sciences, hasanuddin university, makassar. tools and materials the tools used in this research were global positioning system (gps), plot size of 50x50 cm, meter roll, sample bags, tweezers, trays, writing instruments, cameras, bivalvia identification books. the materials used are samples of bivalves, formaldehyde, alcohol, and tissue. sampling method sampling is done at three estuary, each estuary was divided into two sampling areas. between the coordinates 04 ° 49 '52.8 "ls s. 04 ° 32" 06.5 "ls and 119 ° 29" 51.0 "east 119 ° 30" 23.9 "east with using the plot method (litaay et al., 2014); (fitriana, 2006); (suwondo et al., 2012). 50x50 cm plot size. bivalves contained in the plot were taken and put into sample bags preserved using alcohol and formalin, then labeled plot and station numbers. the sample is then taken to the laboratory for identification. data analysis the resulting bivalvia type of identification data is subsequently obtained at the analysis of density, relative density, diversity index (shanon-wiener index), uniformity index, dominancy index and dissemination analysis (morisita index). species density is the total number of each species in one observation area that can be calculated with the equation (kalidass, 2014): 𝐷𝑖 ∶ 𝑛𝑖 𝐴 information : in : specific density (ind / m2) ni : total number of individual species a : total area of sampling plot (m2) international journal of applied biology, 4(1), 2020 29 the relative density value is the percentage value of the density of a species to the total density of all species, which is calculated using the formula (heip et al., 1998): 𝑅𝐷𝑖 ∶ 𝑛𝑖 𝑁 𝑥100% information : rdi : relative density (%) ni : total number of species (i) n : the total number of individuals of all species a diversity index is used to see the relationship of species abundance with the number of individuals of each species in a community, which is calculated based on the shannon wiener diversity index (hutcheson, 1970); (kassim et al., 2018) h′ = − ∑ ( 𝑛𝑖 𝑁 ) ln ( 𝑛𝑖 𝑁 ) 𝑠 𝑖=1 information : h ' : diversity index (shanon-wiener) ni : total number of species (i) n : the total number of individuals of all species s : number of species found ∑ : number from species 1 to species s the criteria for the shanon-wiener diversity index value is as follows (soeparmo, 1992) h '<1.0 : low diversity of genera or species 1.0 <h '1.5 ك : diversity of genera or species high diversity of genera or species : 2.0 ك’ h ك 1.6 h '> 2 : diversity of genera or species is very high uniformity index is used to decide the balance of species distribution in a community. uniformity values are calculated using the evenness index formula (heip et al., 1998); (okpiliya, 2012): 𝐽′ = 𝐻′ log 𝑆 information j ' : uniformity index h ' : diversity index s : number of species uniformity index values range from 0-1, with the criteria for evaluating the condition of a community as follows: 0 <j 0.5 ك : depressed community 0.5 <j 0.75 ك : unstable community international journal of applied biology, 4(1), 2020 30 0.75 <e ك : stable community dominance index is used to find out species who dominate in a community. analysis of dominance index value refers to the simpson index formula (kerkhoff, 2010); (insafitri, 2010). 𝐷 = ∑ 𝑃𝑖2 information d : dominance index (simpson) pi : ni / n the simpson dominance index value ranges from 0-1, with the criteria for dominance index value as follows (odum, 1993): 0 <c 0.5 ك : no species dominate (stable community) 0.5 <c 1 ك : there is a species that dominates (unstable community) type dispersion index determination of personal distribution patterns of each type in a community refers to the morisita index with the following formula (amaral et al., 2015); (hayes & castillo, 2017): i_ (δ) = q ((x ^ 2x) / ((x) ^ 2 x)) information: i_δ : index morisita q : the total number of plots x : number of species in each plot the criteria for determining species distribution patterns are as follows (silveria et al., 1976): i_ (δ) <1 : uniform distribution pattern i_ (δ) = 1 : random distribution pattern i_ (δ)> 1 : group distribution patterns results and discussion the results of the research on the pangkajene river estuary acquired 12 families bivalvia, consisting of 19 types of bivalvia community. results of bivalve density analysis at the pangkajene river estuary, pangkajene dan kepulauan regency are presented in table 1. international journal of applied biology, 4(1), 2020 31 table 1. species density (ind/m2) and relative density (%) of bivalves at the research sampling area, at pangkajene river estuary, pangkajene dan kepulauan regency. no species absolute density (ind / m2) (d) average average relative density (%) (rd) average north estuary middle estuary south estuary north estuary middle estuary south estuary i ii iii iv v vi i ii iii iv v vi 1 anadara antiquata 0.67 0 0.67 3.33 0 0 0.78 3.57 0 4 5.15 0 0 2.12 2 batissa violaceae 0 0 0 0.67 0 0.67 0.22 0 0 0 1.03 0 2.7 0.62 3 corculum cardissa 0 0 0 1.33 0.67 0 0.33 0 0 0 2.06 2.13 0 0.7 4 chlamys squamosa 0.67 0.67 1.33 0 0 3.33 1 3.57 9.09 8 0 0 13.5 5.7 5 fragum unedo 0 0 0 0.67 0 0 0.11 0 0 0 1.03 0 0 0.17 6 gaffarium tumidium 3.33 0.67 1.33 2.67 0.67 0.67 1.56 17.9 9.09 8 4.12 2.13 2.7 7.32 7 harvella pricularia 0 0 0 0 0.67 0 0.11 0 0 0 0 2.13 0 0.36 8 hiatula chinensis 0 0 0 12 0.67 0 2.11 0 0 0 18.6 2.13 0 3.45 9 mactra maculata 4.67 0 1.33 2.67 0.67 0.67 1.67 25 0 8 4.12 2.13 2.7 6.99 10 marcia upima 0.67 1.33 0.67 0 0 0 0.45 3.57 18.2 4 0 0 0 4.29 11 meretrix meretrix 0.67 1.33 3.33 1.33 0 0 1.11 3.57 18.2 20 2.06 0 0 7.3 12 pitar manillae 0 0.67 0 1.33 0 0 0.33 0 9.09 0 2.06 0 0 1.86 13 pteria penguin 0 0 0 0 0.67 0 0.11 0 0 0 0 2.13 0 0.36 14 saccostrea sp. 5.33 2.67 6 34 25.3 17.3 15.1 28.6 36.4 36 52.6 80.9 70.3 50.8 15 scapharca sp. 0.67 0 0 0.67 0.67 0 0.34 3.57 0 0 1.03 2.13 0 1.12 16 semele crenulata 0.67 0 0 2.67 0 0 0.56 3.57 0 0 4.12 0 0 1.28 17 tellina timorensis 1.33 0 2 0 0.67 1.33 0.89 7.14 0 12 0 2.13 5.41 4.45 18 vepricardium sp. 0 0 0 1.33 0 0 0.22 0 0 0 2.06 0 0 0.34 19 trachycardium subragosum 0 0 0 0 0.67 0.67 0.22 0 0 0 0 2.13 2.7 0.81 total 18.7 7.33 16.7 64.7 31.3 24.7 27.2 100 100 100 100 100 100 100 some species such as gaffarium tumidum, anadara antiquate, fragum unedo, tellina sp., have been commonly found in mangrove areas (riniatsih & widianingsih, 2007); (fauziani, 2017); (islami et al., 2018). some of these bivalves have quite high economic values, which can increase people's income. several types of bivalves have been developed both for consumption, raw materials for jewelry and for raw materials for medicines (islami et al., 2018). the highest density of bivalves in sampling area iv (64.7 ind/m2) and lowest in sampling area ii (7.33 ind/m2). the highest relative density of species is saccostrea sp. in the sampling area v (80.9%) and the lowest in the sampling area i (28.57%). whereas the density of bivalves in the middle estuary sampling area (16.7 64.7 ind/m2) and lowest in the northern estuary sampling area (7.33 18.7 ind/m2). diversity index values (h ') obtained at all sampling area ranged from 0.91 1.91, and for sampling areas i and iv based on diversity index criteria included high diversity criteria, while those in the v-vi sampling area included low to moderate diversity levels. the diversity value of a community describes the level of variation of species that live in an ecosystem. ecosystems tend to be balanced if the diversity index value is high, and vice versa the ecosystem is indicated to be depressed if the diversity index value is low (clark, 1974). uniformity index values close to 1.00 indicate that the number of species in one station are same, or there are no species who dominated, and if the value of j '<0.5 or close to 0, indicates that the uniformity of species is not balanced, or there are species who international journal of applied biology, 4(1), 2020 32 dominated (hartati & awwaluddin, 2007). the index value of the uniformity of the bivalve community in the pangkajene river estuary area varies from 0.39 to 0.92. based on the uniformity index criteria, the sampling areas i, ii, and iii show a stable bivalve community (0.83 0.92). in the sampling areas iv and vi the bivalve community was unstable (0.55 and 0.65), where in the sampling area v the bivalvia community was in a depressed state (0.39). the variation in the diversity index value is thought to be due to the condition of mangroves that have experienced disturbances (pressures) due to land conversion activities being the location of community aquaculture ponds. the result of the analysis of the index of diversity and the uniformity of bivalves community in the estuary area of pangkajene river, pangkep regency is presented in table 2. table 2. diversity index (h') and uniformity index (j') of bivalve communities in the pangkajene river estuary sampling area diversity index (h') uniformity index (j') north estuary i 1.91 0.83 ii 1.64 0.92 middle estuary iii 1.81 0.87 iv 1.66 0.65 south estuary v 0.91 0.39 vi 1.07 0.55 the highest dominance index value was found in saccostrea sp. in the sampling area iii with a dominance index value of 0.135 and the lowest type of anadara antiquate, scapharca sp., semele crenulata, marcia upima, meretrix meretrix, chlamys squamosal with a dominance index value of 0.001. the overall dominance index value in the pangkajene river estuary area ranges from 0.001-0.135, which based on the simpson dominance index criteria (odum, 1993) shows that there are no bivalves that dominates in all sampling areas. a low dominance index value indicates that all bivalves found in the sampling area have good adaptability to live in the area. according to kharisma et al., (2012) that the depth and content of organic matter also affect the presence of bivalves in certain waters or habitats. furthermore, according to islami (2013), environmental factors such as salinity and temperature have an influence on the distribution and physiological functions of bivalves. the results of the calculation of the mean distribution index (id) obtained 18 species of bivalves with the index value of the spread pattern id <1 (table 3), based on index morisita criteria, the distribution pattern is uniform. this shows that the bivalve community can live in all research sampling areas or not be limited by environmental factors. saccostrea sp has a distribution index value of 1.58 (id>1) which indicates that this species has a clumped distribution pattern so that it can only live on certain substrate. table 3. dominance index (d) of species and distribution patterns of bivalve communities in the pangkajene river estuary no species dominance index distribution pattern sampling area i ii iii iv v vi 1 anadara antiquata 0.001 0.017 0.023 0.17 2 batissa violaceae 0.002 0.010 0.11 3 corculum cardissa 0.006 0.007 0.09 international journal of applied biology, 4(1), 2020 33 no species dominance index distribution pattern sampling area i ii iii iv v vi 4 chlamys squamosa 0.001 0.048 0.041 0.073 0.26 5 fragum unedo 0.002 0.06 6 gaffarium tumidium 0.032 0.048 0.041 0.017 0.007 0.010 0.19 7 harvella pricularia 0.007 0.13 8 hiatula chinensis 0.098 0.007 0.14 9 mactra maculata 0.063 0.041 0.017 0.007 0.010 0.14 10 marcia upima 0.001 0.096 0.017 0.28 11 meretrix meretrix 0.001 0.096 0.104 0.006 0.16 12 pitar manillae 0.048 0.006 0.30 13 pteria penguin 0.007 0.13 14 saccostrea sp. 0.082 0.135 0.135 0.114 0.030 0.061 1.58 15 scapharca sp. 0.001 0.002 0.007 0.13 16 semele crenulata 0.001 0.017 0.13 17 tellina timorensis 0.005 0.065 0.007 0.025 0.16 18 trachycardium subragosum 0.007 0.010 0.14 19 vepricardium sp. 0.006 0.06 total 0.189 0.470 0.459 0.319 0.090 0.198 conclusions the results obtained 19 species from 12 families that compose the bivalvia community in the pangkajene river estuary. the highest relative density of species is saccostrea sp. diversity index in each sampling area is low until high with the value of uniformity varies and no species dominates in all sampling areas. in general, all species spread uniformly, except saccostrea sp., with clumped distribution patterns. references amaral, m.k., pellico, n.s., lingnau, c. & figueiredo, f.a., 2015. evaluation of the morisita index for determination of the spatial distribution of species in a fragment of araucaria forest. applied ecology and environtmental research, xiii(2), pp.361 anonim, n.d. animal diversity web (adw). 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perairan bontolebang kabupaten kepulauan selayar sulawesi selatan. semnas mipa, pp.1-8. noveria, m., aswatini, harfina, d. & malamassan, m.a., 2008. kondisi sosial ekonomi masyarakat dilokkasi coremap ii kabupaten pangkajene dan kepulauan. jakarta: cremap-lipi. odum, e.p., 1993. dasar-dasar ekologi. 3rd ed. yogyakarta: gadjah mada university press. okpiliya, f.i., 2012. ecological diversity indices: any hope for one again? journal of environment and earth science, ii(10), pp.45-52. international journal of applied biology, 4(1), 2020 35 printrakoon, c. & temkin, i., 2008. comparative ecology of two parapatric populations of isognomon (bivalvia: isognomonidae) of kungkrabaen bay, thailand. the raffles bulletin of zoology, (18), pp.75-94. riniatsih, i. & widianingsih, 2007. kelimpahan dan pola sebaran kerang-kerangan (bivalve) di ekosistem padang lamun, perairan jepara. ilmu kelautan, xii(1), pp.53-58. silveria, n.s., nakano, o., barbin, d. & villa, n.n., 1976. manual de ecologia dos inseto. brasil: cares piracicaba. p.419. soeparmo, 1992. metode dan teknisk analisis komponen biotik ekosistem darat. surabaya: universitas airlangga. suwondo, ferbrita, e. & siregar, n., 2012. kepadatan dan distribusi bivalvia pada mangrove di pantai cermin kabupaten serdang bedagai provinsi sumatra utara. jurnal biogenesis, ix(1), pp.45-50. wahyuni, i., sari, i.j. & ekanara, b., 2017. biodiversitas mollusca (gastropoda dan bivalvia) sebagai bioindikator kualitas perairan di kawasan pesisir pulau tunda, banten. biodidaktika, xii(2), pp.45-56. contact : jiban shrestha jibshrestha@gmail.com 35 abstract the performance evaluation of maize genotypes at a specific growing region is a key to crop improvement. the objectives of this study were to evaluate grain yield of maize genotypes at bijayanagar (mahatgaun vdc) and tallo lorpa (dillichaur vdc), jumla district of nepal during spring seasons of 2012 and 2013. the maize genotypes were significant (p< 0.05) for grain yield, days to tasseling and silking and disease scoring where as anthesis-silking interval, plant height and ear height were non significant. the pooled analysis of the results showed that the highest grain yield was produced by ganesh-1 (4830 kg/ha) followed by kkt-pop (4408 kg/ha), kkt-14 (3952 kg/ha) and mgu-08 (3913 kg/ha, respectively. from this study, ganesh-1, kkt-pop, kkt-14 and mgu-08 could be preferable choice for cultivation in jumla district of nepal issn : 2580-2410 eissn : 2580-2119 performance evaluation of maize in jumla district of nepal: from yielding perspective jiban shrestha1*, dil bahadur gurung2, keshab babu koirala2, & tirtha raj rijal3 1nepal agricultural research council, agriculture botany division, khumaltar, lalitpur, nepal 2nepal agricultural research council, kathmandu, nepal 3national maize research program, rampur, chitwan, nepal introduction maize (zea mays l.) is one of the most important cereal crops in the world after wheat and rice (golbashy et al., 2010). it is cultivated in a wider range of environments than wheat and rice because of its greater adaptability (koutsika-sotiriou, 1999). maize is second major crop after rice in term of area and production in nepal (moald, 2017). the farm level yield of maize (2.55 t/ha) is not satisfactory as compared to attainable yield (5.7 t/ha) in nepal (moad, 2017; karki et al., 2015). maize production in the area suffers much from low fertility, low management, lack of improved varieties, and very severe infections of foliar diseases like turcicum leaf blight, high infestations of striga and stalk borers (assefa, 1998). as a result, evaluating the performance of hybrid maize genotypes in specific agro ecology on different traits is very crucial.the environmental conditions affect the performance of maize genotypes which requires a breeding program that needs to take into account the consequences of environment and genotype interaction in the selection and release of improved varieties.the karnali region is rich diversity for local maize germplasm. the need of local germplasm collection, their evaluation and utilization for the development of pools populations and varieties is only the means to develop the new opvs for higher grain yield production as well open access international journal of applied biology keyword agronomic, performance, grain yield, maize. article history received 28 october 2019 accepted 29 december 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 3(2), 2019 36 as to fit in the existing cropping systems. jumla, 2,531 square kilometer (sq.km) area with altitude ranging from 915 to 4,679 meters from amsl, is a unique district located in the far north-western part of nepal. the plane lands in jumla district are located at the altitude of about 2,350 meters (shrestha, 2002). the area under maize cultivation in karnali zone represents only 1.16% of the total maize cultivated area and its maize production represents only 0.78% of the total maize production in nepal. the average maize productivity in karnali zone was 1.66 t/ha which was 15% lower as compared to average national productivity of maize (moad, 2012). there is no single maize variety released for karnali zone up to now. therefore, this study was carried out to identify high yielding maize varieties suitable for karnali zone of nepal. materials and methods plant materials the maize genotypes used in this study were dlp-01, dlp-14, dlp-16, dlppop (dlp01, dlp-14, dlp-16 and dlp-18), hml-04, hml-08, hml-28, hml-pop (hml-04, hml-08, hml-28 and hml-05), jml-27, jml-30, jml-32, jml-pop (jml-27, jml-30 and jml32), kkt02, kkt-03, kkt-14, kkt-pop (kkt-02, kkt-03 and kkt-14), mgu-03, mgu-08, mgu-15, mgupop (mgu-03, mgu-08 and mgu-15), dolmu pop (dlp-01, dlp-14 mgu-03 and mgu-14), jumka pop (jml-14, jml-26, kkt-02 and kkt-03), karnali pool yellow (hml-25, kkt-18 and hml-18), karnali pool white (dlp-05, dlp17, hml-11, mgu-14 and mgu-30), ganesh-1 and pool-17 (check variety). all these genotypes were received from national maize research program, rampur, chitwan, nepal. experimental site, crop husbandry and experimental design twenty six maize genotypes (including ganesh-1 and pool-17) during spring seasons of 2012 and 2013 at bijayanagar (mahatgaun vdc) and tallo lorpa (dillichaur vdc), jumla. there were 2 sites i. e. site 1 and site 2 at tallo lorpa, jumla. the longitude of tallo lorpa is 82° 16’, latitude 29° 19’ and altitude 2540 m. similarly, the longitude of bijayanagar is 82° 10’, latitude 29° 17’ and altitude 2580 m. the genotypes were tested in plot size of two rows of three meter length in randomized block design with two replications and plant spacing of 75 × 25 cm2. the plots were fertilized with 120:60:40 n:p2o5:k2o kg/ha in the form of urea, di-amonium phosphate (dap), and murate of potash (mop). entire dose of phosphorus and potash was applied at the time of sowing while urea was added in three split doses and also 15 t/ha farm yard manure (fym) was incorporated in soil at the time of land preparation. irrigation was applied as per the requirement of crop. all the trials received standard cultural practices to control weeds and pests. data recording and measurements plants harvested from central two rows were used on data measurements. days to anthesis were counted from sowing to until 50% of plants had shed their pollen and days to silking were recorded from sowing to 50% plants extruded their silks. anthesis silking interval (asi) was calculated as the difference between silking and anthesis interval. plant height and ear height was measured as the distance from ground to the tip of the tassel and the base of the uppermost ear respectively on five plants per plot. disease scoring was done from 1 to 5 scale (payak & sharma, 1983; shrestha et al., 2019). grain yield (kg/ha) at 15% moisture content was calculated using fresh ear weight with the help of the formula adopted by international journal of applied biology, 3(2), 2019 37 carangal et al. (1971) and shrestha et al. (2018) to adjust the grain yield (kg/ha) at 15% moisture content. data analysis all collected data were entered in microsoft excel 2016 and analyzed by using genstat (version 14th edition; vsn international, hemel hempstead, uk). all the data collected were statistically analyzed using the analysis of variance (anova) procedure described by gomez and gomez (1984) for randomized complete block design (rcbd) experiments. separation of treatment means for significant difference was done by using the fisher least significant difference (f-lsd) procedure at 0.05 probability level (obi, 1986; shrestha, 2019). results and discussion the findings of maize experiments at bijaynagar, jumla showed that the grain yield was highest in kkt-pop (6594 kg/ha), jml-30 (4271 kg/ha) and dlp-14 (3141 kg/ha) respectively. the days to 50% tasseling varied from 98 days (hml-28) to 140 days (kkt-02 and kkt-03) and silking varied from 102 days (hml-04, hml-28) to 145 days (kkt-02 and kkt03). the plant height varied from 102 cm (hml-08) to 157 cm (dlp-16) and ear height from 40 cm (dlp-01) to 74 cm (kkt-pop). the disease score varied from 1.5 (hml04 and kkt-02) to 3 (hml-28 and ganesh-1). the findings of the maize experiments at tallo lorpa, jumla showed that the grain yield was highest in ganesh-1 (6210 kg/ha), jml-30 (6203 kg/ha) and kkt-14 (5466 kg/ha) respectively. the days to 50% tasseling varied from 102 days (mgu-pop and hml-04) to 157 days (kkt-02) and silking varied from 107 days (dlp-pop and mgu-pop) to 153 days (kkt14). the plant height varied from 104 cm (pool-17) to 187 cm (jumka-pop) and ear height from 51 cm (dlp-01) to 104 cm (kkt-14). the disease score varied from 2 (dlp-01, hml-04, hml-pop, jml-27, jml-30, kkt-14, karnali pool yellow and karnali pool white) to 3 (dlp-14). the pooled analysis of maize experiments at tallo lorpa and bijaynagar showed that grain yield ranges from 1776 kg/ha (dlp-01) to 4830 kg/ha (ganesh-1). the grain yield was highest in ganesh-1 followed by kkt-pop (4408 kg/ha), kkt-14 (3952 kg/ha), mgu-08 (3913 kg/ha), karnali pool white (3482 kg/ha), karnali pool yellow (3402 kg/ha) and kkt03 (3392 kg/ha) respectively. table 1. grain yield and agronomic traits of maize genotypes evaluated at bijayanagar, jumla district during spring season of 2012 and 2013 sn genotypes 50% tasseling (days) 50% silking (days) asi (days) plant height (cm) ear height (cm) disease score (1-5) grain yield (kg/ha) 1 kkt-03 140 145 5 153 59 2 2183 2 kkt-14 124 130 6 153 64 2 3018 3 kkt-pop 124 130 6 177 74 2.5 6594 4 mgu-03 116 125 9 123 54 2 2666 5 mgu-08 119 124 5 111 47 3 2634 international journal of applied biology, 3(2), 2019 38 6 mgu-15 119 121 2 118 54 2 1927 7 mgu-pop 107 111 4 149 67 2 2952 8 dolmu pop 106 113 7 145 61 2.5 2733 9 jumka pop 107 111 4 147 73 2 2432 10 karnali 105 109 4 134 71 2 2910 pool yellow 11 karnali pool white 116 122 6 148 77 2.5 3173 12 pool-17 116 122 6 144 54 2.5 2746 13 ganesh-1 116 121 5 215 118 3 5060 14 dlp-01 104 106 2 140 40 2 1555 15 dlp-14 102 108 6 150 55 2 3141 16 hml-pop 104 109 5 148 58 2.5 2615 17 jml-27 114 119 5 130 48 2 2808 18 jml-30 107 111 4 141 61 2 4271 19 jml-32 111 116 5 123 47 2 2779 20 jml-pop 109 114 5 119 51 2 2467 21 kkt-02 140 145 5 104 52 1.5 2891 22 dlp-16 112 116 4 157 68 2 2728 23 dlp-pop 99 104 5 128 73 2 3052 24 hml-04 99 102 3 114 36 1.5 1391 25 hml-08 109 113 4 102 35 2.5 1850 26 hml-28 98 102 4 129 44 3 2336 grand mean 112 117 5 139 59 2.2 2881 cv% 10.45 9.4 24.2 15.5 23. 4 22.4 13.5 f test * * * * ns ns * ns = non-significant at 5% level of significance, * = significant at 5% level of significance international journal of applied biology, 3(2), 2019 39 table 2. grain yield and agronomic traits of maize genotypes evaluated at tallo lorpa (site 1), jumla district during spring season of 2012 and 2013. sn genotypes 50% tasseling (days) 50% silking (days) asi (days) plant height (cm) ear height (cm) disease score (1-5) grain yield (kg/ha) 1 kkt-03 132 136 4 169 77 2.5 5326 2 kkt-14 145 153 8 182 104 2 5466 3 kkt-pop 140 145 5 173 88 2.5 3972 4 mgu-03 122 126 4 164 88 2.5 3322 5 mgu-08 121 125 4 173 68 2 4278 6 mgu-15 125 128 3 153 81 2 1339 7 mgu-pop 102 107 5 145 63 2 4496 8 dolmu pop 107 111 4 145 72 2 3795 9 jumka pop 124 128 4 187 98 2.5 3792 karnali pool 123 127 4 129 71 2 10 yellow 4540 11 karnali pool 126 130 4 128 57 2 4052 white 12 pool-17 122 126 4 104 42 2.5 3235 13 ganesh-1 140 145 5 184 77 2.5 6210 14 dlp-01 104 109 5 111 51 2 1705 15 dlp-14 109 114 5 133 62 3 3942 16 hml-pop 109 114 5 154 82 2 3698 17 jml-27 111 113 2 176 90 2 3171 18 jml-30 109 113 4 173 75 2 6203 19 jml-32 109 113 4 115 46 2.5 2275 20 jml-pop 144 147 3 127 85 2.5 3071 21 kkt-02 157 162 5 178 97 2.5 3088 22 dlp-16 107 113 6 119 57 2.5 3868 23 dlp-pop 102 107 5 130 71 2.5 3760 24 hml-04 102 109 7 120 38 2 2723 25 hml-08 107 113 6 133 70 2.5 4066 international journal of applied biology, 3(2), 2019 40 26 hml-28 107 113 6 135 66 2.5 2131 grand mean 119 124 5 148 72 2.3 3751 cv% 10.7 10.3 26 15.1 22.9 12.9 34.11 f test * * * * ns * * ns = non-significant at 5% level of significance, * = significant at 5% level of significance table 3. grain yield and agronomic traits of maize genotypes evaluated at tallo lorpa (site 2), jumla district during spring season of 2012 and 2013. sn genotypes 50% tasseling (days) 50% silking (days) asi (days) plant height (cm) ear height (cm) disease score (1-5) grain yield (kg/ha) 1 kkt-03 114 119 5 118 65 2 2697 2 kkt-14 109 115 6 136 45 2 3373 3 kkt-pop 117 123 6 146 44 2.5 2659 4 mgu-03 109 117 8 99 43 2 2811 5 mgu-08 111 117 6 122 36 2.5 3535 6 mgu-15 109 115 6 100 38 2 1493 7 mgu-pop 111 116 5 130 44 2 1648 8 dolmu pop 109 115 6 116 44 2 2179 9 jumka pop 109 117 8 134 39 2 3169 10 karnali pool 113 119 6 92 43 2 2563 yellow 11 karnali pool 113 119 6 131 33 2.5 2952 white 12 pool-17 118 123 5 123 32 2.5 2659 13 ganesh-1 115 122 7 148 38 2.5 3221 14 dlp-01 104 111 7 147 53 2 2070 15 dlp-14 109 115 6 149 51 2 1700 16 hml-pop 113 119 6 137 46 2.5 2336 17 jml-27 111 116 5 144 48 2 2666 18 jml-30 113 119 6 144 38 2 1264 19 jml-32 109 117 8 142 47 2 3642 international journal of applied biology, 3(2), 2019 41 20 jml-pop 109 114 5 119 44 2 3388 21 kkt-02 107 113 6 137 46 1.5 2846 22 dlp-16 107 112 5 156 46 2 1755 23 dlp-pop 111 115 4 125 48 2 2400 24 hml-04 109 115 6 122 43 1.5 2438 25 hml-08 111 117 6 136 46 2.5 2207 26 hml-28 109 114 5 131 41 3 2036 grand mean 111 117 6 130 44 2.1 2527 cv% 3.2 1.9 19.7 27.02 11.7 19.3 22.5 f test * * * ns ns * * ns = non-significant at 5% level of significance, * = significant at 5% level of significance table 4. mean of the gain yield and agronomic traits of maize genotypes evaluated at jumla district (bijaynagar and tallo lorpa) during spring season of 2012 and 2013. sn genotypes 50% tasseling (days) 50% silking (days) asi (days) plant height (cm) ear height (cm) disease score (1-5) grain yield (kg/ha) 1 kkt-03 128.67 133.33 4.67 146.7 67 2.16 3392 2 kkt-14 126 132.67 6.67 157 71 2 3952 3 kkt-pop 127 132.67 5.67 165.3 68.7 2.5 4408 4 mgu-03 115.67 122.67 7 128.7 61.7 2.16 2933 5 mgu-08 117 122 5 135.3 50.3 2.5 3913 6 mgu-15 117.67 121.33 3.67 123.7 57.7 2 1587 7 mgu-pop 106.67 111.33 4.67 141.3 58 2 3032 8 dolmu pop 107.33 113 5.67 135.3 59 2.16 2902 9 jumka pop 113.33 118.67 5.33 156 70 2.16 3131 10 karnali pool yellow 113.67 118.33 4.67 118.3 61.7 2 3402 11 karnali international journal of applied biology, 3(2), 2019 42 pool white 118.33 123.67 5.33 135.7 55.7 2.33 3482 12 pool-17 118.67 123.67 5 123.7 42.7 2.5 2880 13 ganesh-1 123.67 129.33 5.67 182.3 77.7 2.66 4830 14 dlp-01 104 108.67 4.67 132.7 48 2 1776 15 dlp-14 106.67 112.33 5.67 144 56 2.33 2928 16 hml-pop 108.67 114 5.33 146.3 62 2.33 2883 17 jml-27 112 116 4 150 62 2 2882 18 jml-30 109.67 114.33 4.67 152.7 58 2 3338 19 jml-32 109.67 115.33 5.67 126.7 46.7 2.16 2899 20 jml-pop 120.67 125 4.33 121.7 60 2.16 2975 21 kkt-02 134.67 140 5.33 139.7 65 1.83 2941 22 dlp-16 108.67 113.67 5 144 57 2.16 2784 23 dlp-pop 104 108.67 4.67 127.7 64 2.16 3071 24 hml-04 103.33 108.67 5.33 118.7 39 1.66 2184 25 hml-08 109 114.33 5.33 123.7 50.3 2.5 2708 26 hml-28 104.67 109.67 5 131.7 50.3 2.83 2168 grand mean 114.21 119.36 5.15 138.8 58.4 2.2 3053 cv,% 7.6 7.3 24.5 14.1 23.5 12.3 28.2 lsd0.05 14.24 14.28 2.06 32.07 22.52 0.445 1412.6 f-test ** ** ns * ns ** * ns = non-significant at 5% level of significance, * = significant at 5% level of significance, ** = significant at 1% level of significance the genotypes were found significant (p< 0.05) for grain yield. these results are in line with those of kunwar and shrestha (2014), shrestha (2016) and prasai et al. (2015) who reported significant differences among maize cultivars for grain yield.other traits namely disease score, days to 50% tasseling and silking were highly significant (p< 0.01) where as plant height significant and ear height and asi were non significant. difference in days to tasseling and silking among maize genotypes were also observed by prasai et al. (2014), dhakal et al. (2017) and kunwar et al. (2014). result of combined analysis over year showed that there was highly significant (p< 0.01) influence of genotypes on days to tasseling and days to silking. hussain et al. (2011) reported differential pattern of maize varieties for plant height. ear height was significant for genotypes that results get sufficient validation from the findings of nazir et al. (2010) and international journal of applied biology, 3(2), 2019 43 ahmad et al. (2011). grain yield was significant and this results was similar to the results were reported by akbar et al. (2009) who evaluated and identified high yielding maize varieties among different genotypes tested. the reason for differences in grain yield among the maize genotypes under different locations was due to their variation in their genetic makeup. the grain yield of maize is the most important and complex quantitative character controlled by numerous genes. the gain yield of maize under different environment conditions may be due to both environmental and genetic effect. different researchers have reported significant amount of variability in different maize populations including top-crosses and open pollinated varieties (sampoux et al., 1989). these results are in line with those of grzesiak (2001), who also observed considerable genotypic variability among various maize genotypes under different location. conclusions the study showed variation for almost all the traits studied among the maize genotypes, which is an indication of the presence of sufficient variability and can be exploited through selection. the highest grain yield was one of the basic criteria for identifying superior varieties. the genotypes kkt-pop, kkt-03, kkt-14, mgu-08, ganesh-1, karnali pool white and karnali pool yellow were found superior in their grain yield potentiality in jumla district. so these maize genotypes were found suitable genotypes for karnali zone of nepal. acknowledgements the authors are thankful to agricultural research station (ars), jumla for providing experimental fields at jumla. nepal agricultural research council was highly acknowledged for research funding. references ahmad, s.q., khan, s., ghaffar, m. & ahmad, f. 2011. genetic diversity analysis for yield and other parameters in maize (zea mays l.) genotypes. asian j. agric. sci., 3(5): 385388. akbar, m., saleem, m., ashraf, m.y., hussain, a., azhar, f.m. & ahmad, r. 2009. combining ability studies for physiological and grain yield traits in maize at two temperatures. pakistan j. bot., 41(4): 1817-1829. assefa, t. 1998. survey of maize diseases in western and northwestern ethiopia. in the sixth eastern and southern africa regional maize conference, cimmyt. addis ababa, ethiopia. pp. 121-124. carangal, v.r., ali, s.m., koble, a.f., rinke, e.h. & sentz, j.c. 1971. comparison of s1 with testcross evaluation for recurrent selection in maize. crop science, 11: 658-661. dhakal, b., shrestha, k. p., joshi, b. p. & shrestha, j. 2017. evaluation of early maize genotypes for grain yield and agromorphological traits. journal of maize research and development, 3(1): 67-76. golbashy, m., ebrahimi, m., khorasani, s.k. & choucan, r. 2010. evaluation of drought tolerance of some corn (zea mays l.) hybrids. iran. afr. j. agric. res., 5 (19): 27142719 international journal of applied biology, 3(2), 2019 44 gomez, k. a. & gomez, a. a. 1984, statistical procedures for agricultural research. 2nd ed. new york . john wesley and sons, 680p. grzesiak, s. 2001. genotypic variation between maize (zea mays l.) single-cross hybrids in response to drought stress. acta physiologiae plantarium, 23(4): 443-456. hussain, n., khan, m. y. & baloch, m.s. 2011. screening of maize varieties for grain yield at dera ismail khan. j. anim. plant sci., 21(3): 626-628. karki, t. b., shrestha, j., achhami, b. b. & kc, g. 2015. status and prospects of maize research in nepal. journal of maize research and development, 1(2): 1-9. koutsika-sotiriou, m. 1999. hybrid seed production in maize. in basra, a. s. (2ed) “heterosis and hybrid seed production in agronomic crops”, food products kunwar, c. b. & shrestha, j. 2014. evaluating performance of maize hybrids in terai region of nepal.world journal of agricultural research, 2 (1): 22-25. kunwar, c.b., bhurer, k.p., paudel, s.p., chhetri, j.b. & shrestha, j. 2014. early and extra early maturity maize variety for terai, inner terai and foot hill of nepal. in: y. p. giri, y.g. khadka, b.n. mahato, b.p. sah, s.p. khatiwada, m.r. bhatta, b.k. chettri, a. k. gautam, d. gauchan, a.r. ansari, j.d. ranjit, r. shrestha, b. sapkota (eds). proceedings of the 27th national summer crops workshop, vol. ii, held on 18-20th april, 2013 at national maize research program, rampur, chitwan, nepal. pp.78-81. moad. 2012. statistical information on nepalese agriculture. government of nepal. ministry of agriculture and cooperatives, agribussiness promotion and statistics, singha durbar, kathmandu, nepal. moald. 2017. statistical information on nepalese agriculture. government of nepal. ministry of agriculture and livestock development, singha durbar, kathmandu, nepal. nazir, h., zaman, q., amjad, m. & nadeeman, a. aziz. 2010. response of maize varieties under agro ecological conditions of dera ismail khan. j. agric. res., 48(1): 59-63. obi, i. u. 1986, statistical methods of detecting differences between treatment means, snaap press limited, enugu, nigeria, 45p. payak, m.m. & sharma, r.c. 1983. disease rating scales in maize in india. in techniques of scoring for resistance to important diseases of maize. all india coordinated maizeimprovement project, indian agricultural research institute, new delhi. pp. 14. prasai, h. k., sharma, s., kushwaha, u.k.s. & joshi, b.p. 2014. varietal improvement of early maize for far western hills of nepal. in: y. p. giri, y. g. khadka, b. n. mahato, b. p. sah, s. p. khatiwada, m. r. bhatta, b. k. chettri, a. k. gautam, d. gauchan, a. r. ansari, j. d. ranjit, r. shrestha, b. sapkota (eds). proceedings of the 27th national summer crops workshop, vol. ii, held on 18-20th april, 2013 at national maize research program, rampur, chitwan, nepal. pp.174-177. international journal of applied biology, 3(2), 2019 45 prasai, h.k., kushwaha, u.k.s., joshi, b.p. & shrestha, j. 2015. performance evaluation of early maize genotypes in far western hills of nepal. journal of maize research and development, 1(1): 106-111. sampoux, j.p., gallais, a. & lefort-buson, m. 1989. s1 value combined with topcross value for forage maize selection. agronomie, 9(5): 511-520. shrestha, g.l. 2002. special techniques of organic rice farming in jumla, nepal a unique rice culture in the high himalayas. 1st rda/arnoa international conference “development of basic standard for organic rice cultivation” 12-15 november 2002, rda and dankook univ. korea (2002). shrestha, j. 2016. evaluation of quality protein maize genotypes for grain yield in mid hill districts of nepal. international journal of life sciences, 10(1): 7-9. shrestha, j. 2019. p-value: a true test of significance in agricultural research. retrieved from https://www.linkedin.com/pulse/p-value-test-significance-agricultural-researchjiban-shrestha/ shrestha, j., subedi, s., timsina, k.p., gairhe, s. kandel, m. & subedi, m. 2019.maize research. new india publishing agency (nipa), new delhi-34, india shrestha, j., yadav, d. n., amgain, l.p. & sharma, j.p. 2018. effects of nitrogen and plant density on maize (zea mays l.) phenology and grain yield. current agriculture research journal, 6(2): 175-182 contact : hari kumar prasai hkprasai60@gmail.com 46 abstract lentil and chickpea are important winter legumes of nepal. the experiments were carried out to evaluate the agronomic performance of lentil and chickpea genotypes at research field of regional agricultural research station (rars), bhagetada, doti, nepal during winter seasons of 2012 and 2013. the 16 lentil genotypes were evaluated in both years whereas 20 and 14 genotypes of chickpea were evaluated in 2012 and 2013 respectively. the genotypes were evaluated in a randomized complete block design with three replications. the results showed that both lentil and chickpea genotypes indicated differences for their morphological traits namely plant height, flowering, maturity, pods/plant and grain yield. the combined analysis of trials over years showed that lentil genotype namely black masuro produced the highest grain yield (790 kg/ha) followed by lg 12 (746 kg/ha) and ill 3111 (747 kg/ha), respectively. similarly, chickpea genotypes namely iccx 84050831 produced the highest grain yield (953 kg/ha) followed by iccx 840508-40 (911 kg/ha) and bg 372 (850 kg/ha), respectively. it is suggested that the superior genotypes derived from these experiments could be further evaluated in farmers’ fields before making recommendation for general cultivation. issn : 2580-2410 eissn : 2580-2119 performance evaluation of lentil and chickpea genotypes in doti district of nepal hari kumar prasai1*, subarna sharma1, ujjwal kumar singh kushwaha2, bishnu prasad joshi1, & jiban shrestha2 1regional agricultural research station, bhagetada, dipayal, doti, nepal. 2agriculture botany division, narc, khumaltar, lalitpur, nepal. introduction grain legumes (pulses) are an important component of cropping system of nepal. in the mountains and hills, grain legumes are primarily for home consumption, while in the terai and also in some warmer valleys, they are grown both for home consumption and market. the bulk of production in the terai and inner terai is from the winter grain legumes such as lentil, chickpea, field peas and grasspea and in the summer from pigeon pea (neupane and shrestha, 2015). lentil locally known as masuro is one of the major pulse crop of nepal accounting 62% area and 64% production of the total legume (moad, 2013). in nepal, during 2016/17 lentil was cultivated in the total area of 206,969 ha and the total production was 254,308 t with productivity of 1,229 kg/ha (moald, 2017). lentil is either grown as relay crop open access international journal of applied biology keyword chickpea, lentil, grain yield. article history received 31 october 2019 accepted 29 december 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 3(2), 2019 47 (no till, seed broadcasted 1-2 weeks prior to paddy to harvest) or after rice harvest and land cultivated (post-rice system), and to a smaller extent grown mixed with wheat, tori, mustard, linseed and field pea. inclusion of lentil in cereal based cropping systems helps in sustainable productivity by improving fertility status of soil, and thereby reducing use of chemical fertilizers and pesticides. lentils are grown in terai, inner terai and mid hills of the country. the cultivation of lentil has been increased because of increasing preference for its internal consumption and potential for export market. nepalese lentils have greater demand in the international market. bangladesh, singapore, srilanka, germany, korea, uk, indonesia are the major importers. lentil is recognized as one of the major agricultural product with high export potential and medium socio-economic impacts by nepal trade integration strategy (ntis) (mocs, 2010). lentil shares about 3.1% of the total lentil export in the world (usaid, 2011). chickpea is an important winter legume grown mainly in the rainfed area of nepal, mainly in the rice or maize based ecosystem either as a sole or mixed crop with other winter crops. it is an important legume to the population, as it is the primary protein source for nearly 2 million nepalese people (pande et al., 2005).chickpea is an excellent source of protein, especially when compared to other legume pulses. it is high in unsaturated fatty acids and minerals, including calcium, magnesium, phosphorus and potassium (jukanti, 2012). chickpea is mainly used for human consumption as well as for feeding animals. it is used as ‘dal’ in split form. whole fried or boiled seeds of chickpea are also eaten. husk and bits of ‘dal’ are used as nutritious feed for animals. green leaves of chickpea are used as vegetable (sag). grains are also used as vegetable (chhole). flour is used in the preparation of various sweets. it is also mixed with wheat flour for ‘chapati’ making. chickpea can also be used as green fodder. its straw is an excellent fodder for animals.in nepal, during 2016/17 chickpea was cultivated in the total area of 9,933 ha and total production was 10,969 t with productivity of 1,104 kg/ha (moald, 2017). far western hills have only 10 ha cultivated area of chickpea with the average productivity of 453 kg/ha which is 54.28% lower than the national average productivity. the information on varietal suitability of lentil and chickpea for far western hills of nepal was not sufficient so with the objective of identifying high yielding and agronomically superior lentil and chickpea genotypes for this regions, these experiments were carried out at rars, bhagetada, doti, nepal. materials and methods description of the experimental site these experiments were carried out in the research field of regional agricultural research station (rars), bhagetada, dipayal, doti district during winter seasons of 2012 and 2013. the site is located at the latitude of n 29°15'16.4" and longitude of e 80°55'59.3". this research station is situated at the bank of seti river with the altitude of 546 meter above sea level. (prasai et al., 2018). the soil was light texture, low organic matter (1-2%) and acidic in nature containing ph 6 (rars, 2015). plant materials total sixteen lentil genotypes namely ill 7715, ill 6467, lg 124, ill 7537, ill 1, pl-4, ill 3111, ill 6256, ill 3490, ill7979, wbl 77, hg 57, ill9926, black masuro, shital and sagun were included in these experiments for 2012. similarly, sixteen genotypes namely ill international journal of applied biology, 3(2), 2019 48 6467,lg 12, ln 0136, ill 7715, ill 7163, pl 4, rl 4, ill 6819, wbl 77, ill 3111, ill 3490, ill 7979, black masuro, hg 57, shital and sagun were included in the experiments for 2013. twenty genotypes of chickpea, namely iccx 840508-40, iccx 840508-44, iccx 840508-33, iccx 840508-38, iccv 98933, iccx 840508-41, kwr 108, bg 1206, kpg-59, iccx 840508-32, iccv 98909, bg 372, iccv 98937, iccx 840508-31, iccx 840508-21, kpg 173-4, iccv 87312, iccx 840508-36, tara and dhanush were included in the experiment for 2012. similarly, fourteen genotypes namely iccv 87312, iccv 98937, iccx 840508-31, iccx 840508-38, iccx 840508-40, iccx 840508-41, iccx 840508-44, kwr 108, iccv 98933, iccv 98909,kpg 59, bg 372, tara and dhanush were included in the experiments for 2013. experimental design and field management the experiments were laid out in randomized complete block design with three replications in both years. for lentil the plot size of 4.5 m2 was maintained in both years. for chickpwa, plot size was of 7.2 m2 in both the years. the chemical fertilizers was applied by the rate of 20:40:20 npk kg/ha. the cultural practices and plant protection measures were applied as per recommendations made by grain legume research program, khajura, banke. data analysis the traits namely days to flowering, days to maturity, plant height, pods/plant, seed/pod, and grain yield were recorded. analysis of variance for agronomic traits of grain legumes were analyzed with mstatc software (russel & eisensmith, 1983). genotypes were compared using the f-test. the treatment means were compared by the least significant difference (lsd) test at 5% level (gomez & gomez, 1984; shrestha, 2019). results and discussion results lentil among the lentil genotypes evaluated in 2012, ill-3111 produced the highest grain yield (764 kg/ha) followed by lg 124 (688 kg/ha) and ill 6467 (626 kg/ha), respectively. statistically, grain yield and days to flowering showed significant differences among the genotypes (table 1). the genotypes ill-3111, lg 124 and ill 6467 produced 46.08, 31.54 and 19.69% higher grain yield than the standard check variety (shital). total 16 genotypes of lentil were included in the experiments in 2013. among them, lg 12 producedd the highest grain yield (1050 kg/ha) followed by black masuro (983 kg/ha) and il 3111 (879 kg/ha), respectively. differences for flowering days and maturity was found highly significant and grain yield was found significant among genotypes (table 2). table 1. gain yield and growth traits of lentil genotypes at rars bhagetada, doti during winter season of 2012 s. n. genotype flowering day maturity day plant height (cm) pods/plant grain yield (kg/ha) 1 ill 7715 104 137 25.00 79 593 2 ill 6467 103 138 22.25 93 626 3 lg 124 103 137 23.50 71 688 international journal of applied biology, 3(2), 2019 49 4 ill 7537 105 138 23.00 68 420 5 ill-1 101 149 22.75 68 595 6 pl-4 104 139 23.50 67 484 7 ill-3111 100 137 26.50 87 764 8 ill 6256 106 138 18.50 58 372 9 ill 3490 104 137 22.50 78 604 10 ill 7979 105 138 21.75 60 428 11 wbl-77 104 139 22.75 74 432 12 hg 57 104 138 22.75 79 578 13 ill 9926 101 134 21.75 57 386 14 black masuro 107 139 23.00 87 484 15 shital 102 134 24.50 65 523 16 sagun 105 138 24.00 77 542 f-test ** ns ns ns * cv % 2.16 5.82 13.12 14.62 20.97 sem (±) 0.52 0.829 0.43 2.67 27.9 *,**, significant at 0.05 and 0.01 probability level respectively. ns, non-significant table 2. gain yield and growth traits of lentil genotypes evaluated at rars bhagetada, doti during winter season of 2013 s. n genotype flowering day maturity day plant height (cm) pods/plant seeds/pod grain yield (kg/ha) 1 ill 6467 102 146 21.73 28 1 509 2 lg 12 101 146 22.27 37 2 1050 3 ln 0136 105 148 20.20 32 2 799 4 ill 7715 104 147 19.60 28 2 754 5 ill 7163 101 146 26.80 51 2 637 6 pl 4 106 149 24.00 31 2 638 7 rl 4 106 148 22.27 39 2 727 8 ill 6819 100 145 21.20 33 2 675 9 wbl 77 107 149 23.20 28 2 697 10 ill 3111 104 147 25.00 56 2 879 11 ill 3490 101 145 22.03 33 2 633 international journal of applied biology, 3(2), 2019 50 12 ill 7979 104 147 26.07 26 2 550 13 black 106 149 21.87 54 2 983 masuro 14 hg 57 104 147 25.27 44 2 824 15 shital 102 146 25.47 32 2 593 16 sagun 104 147 25.67 34 2 718 f-test ** ** ns ns ns * cv% 0.66 0.35 17.71 26.23 16.13 20.43 sem (±) 0.541 0.314 0.553 2.4 0.0441 37.2 *,**, significant at 0.05 and 0.01 probability level respectively. ns, non-significant the genotypes lg 12, black masuro and il 3111 produced 77.06, 65.76 and 48.22% higher grain yield respectively than standard check variety (shital). combined analysis over years (2012 and 2013) showed that the genotypes namely black masuro (789 kg/ha), lg 12 (746 kg/ha) and ill 3111(747 kg/ha) were found high yielding genotypes that produced 53.20, 44.85 and 45.04% higher grain yield, respectively than check variety (shital). statistically, genotypic variance and variance due to interaction between genotype and year (g ×y) for grain yield were found significant. flowering days and maturity also revealed highly significant differences among the genotypes (table 3). chickpea a total of 20 genotypes of chickpea were included in experiments in 2012.among them, kpg 173-4 produced the highest grain yield (687 kg/ha) followed by iccx 840508-40 (514 kg/ha) and bg 1206 (501 kg/ha), respectively. the genotypes kpg 173-4 produced the 29.37% higher grain yield than check variety (tara). all the characters except plant height and grain yield showed non-significant differences due to genotypes. the genotypic variance was significant for grain yield and highly significant for plant height (table 4). similarly, 14 genotypes of chickpea were included experiments in 2013. among them, iccx 840508-31 produced the highest grain yield (1463 kg/ha) followed by bg 372 (1208 kg/ha) and iccv 87312 (1197 kg/ha) respectively. the genotypes iccx 840508-31, bg 372 and iccv 87312 produced 70.31, 40.62 and 39.34% higher yield respectivley than check variety (tara). statistically, days to flowering and maturity showed highly significant difference due to genotypes, whereas grain yield due to genotypes was found significant (table 5). the chickpea genotypes namely iccx 840508-31 (953 kg/ha), iccx 840508-40 (911 kg/ha) and bg 372 (850 kg/ha) found high yielding genotypes from combined analysis over years (2012-2013). these promising namely iccx 840508-31, iccx 840508-40 and bg 372 produced 37.12, 31.07 and 22.30% higher yield than the check variety (tara).statistically, effect of the genotypes for grain yield was significant. similarly the interaction between genotypes and years (g×y) for grain yield was also found highly significant (table 6). international journal of applied biology, 3(2), 2019 51 table 3. combined analysis (over years; 2012 and 2013) of lentil genotypes for grain yield and growth traits at rars, bhagetada, doti during winter seasons s. n. genotype flowering day maturity day plant height (cm) grain yield (kg/ha) 1 ill 3464 103 142 20.76 474 2 lg 12 103 142 21.47 746 3 il 7715 104 142 21.80 632 4 pl-4 105 144 24.10 545 5 wbl 77 106 145 21.93 465 6 ill 3111 103 142 24.33 747 7 ill 3490 103 141 22.02 516 8 ill 7979 105 143 23.37 591 9 black masuro 107 144 22.10 789 10 hg 57 105 143 23.30 646 11 shital 102 141 24.73 515 12 sagun 104 143 24.83 619 f-test genotype (g) ** ** ns * year (y) ns ** ns ** g ×y ns ns ns ns cv % 1.56 1.18 16.19 20.97 sem (±) 0.423 0.355 0.400 31.746 *,**, significant at 0.05 and 0.01 probability level respectively. ns, non-significant table 4. gain yield and growth traits of chickpea genotypes at rars bhagetada, doti during winter season of 2012 s. n. genotype flowering day maturity day plant height (cm) pods/plant grain yield (kg/ha) 1 iccx 840508-40 109 146 37.67 26 514 2 iccx 840508-44 108 152 33.00 24 463 3 iccx 840508-33 108 152 43.33 23 464 4 iccx 840508-38 108 155 46.00 26 321 5 iccv 98933 108 152 42.00 25 456 6 iccx 840508-41 108 162 37.33 21 383 international journal of applied biology, 3(2), 2019 52 7 kwr 108 108 153 40.00 21 488 8 bg 1206 108 151 46.00 24 501 9 kpg-59 109 151 41.00 16 418 10 iccx 840508-32 109 149 38.67 25 342 11 iccv 98909 108 150 43.67 25 396 12 bg 372 109 150 38.00 30 459 13 iccv 98437 108 149 39.67 22 439 14 iccx 840508-31 109 151 35.00 14 442 15 iccx 840508-21 112 152 31.33 27 341 16 kg 173-4 111 151 47.00 23 687 17 iccv 87312 108 153 34.67 27 394 18 iccx 840508-36 109 152 37.67 17 374 19 tara 104 150 32.33 21 531 20 dhanush 106 150 32.33 28 453 f-test ns ns ** ns * cv % 2.1 3.14 10.82 17.46 19.05 sem (±) 0.338 0.709 1.08 0.918 19.4 *,**, significant at 0.05 and 0.01 probability level respectively. ns, non-significant table 5. gain yield and growth traits of chickpea genotypes at rars bhagetada, doti during winter season of 2013 s. n. genotype flowering day maturity day plant height (cm) pods/plant seeds/pod grain yield (kg/ha) 1 iccv 87312 105 159 43.6 35 2 1197 2 iccv 98937 108 159 50.73 41 2 921 3 iccx 840508-31 105 159 49.6 28 2 1463 4 iccx 840508-38 101 154 53.2 39 2 1124 5 iccx 840508-40 102 155 49.4 46 2 1359 6 iccx 840508-41 100 153 49.2 33 1 864 7 iccx 840508-44 101 152 50 26 2 611 8 kwr 108 102 154 51.6 32 2 1062 9 iccv 98933 100 153 53.53 24 2 833 10 iccv 98909 101 151 47.07 36 2 1088 international journal of applied biology, 3(2), 2019 53 11 kpg 59 105 156 48.73 42 2 661 12 bg 372 97 153 49.8 28 2 1208 13 tara 104 156 51.73 35 2 859 14 dhanush 103 154 38.87 33 2 1187 f-test ** ** ns ns ns * cv% 2.6 0.28 7.85 18.74 20.24 24.24 sem (±) 0.711 0.702 1.03 1.71 0.034 66.8 *,**, significant at 0.05 and 0.01 probability level respectively. ns, non-significant table 6. combined analysis (over years; 2012 and 2013) of chickpea genotypes for grain yield and growth traits at rars bhagetada, doti during winter seasons s. n. genotype flowering day maturity day plant height (cm) pods/plant seeds/pod grain yield (kg/ha) 1 iccv 87312 107 156 39.13 29 2 795 2 iccv 98937 108 154 45.20 31 1 679 3 iccx 840508-31 107 155 42.30 25 2 953 4 iccx 840508-38 105 155 48.77 32 1 722 5 iccx 840508-40 105 154 41.20 35 1 911 6 iccx 840508-41 104 153 43.27 27 1 622 7 iccx 840508-44 105 153 43.87 26 2 462 8 kwr 108 105 154 45.80 27 2 775 9 iccv 98933 104 153 47.77 23 1 644 10 iccv 98909 104 151 45.37 30 2 741 11 kpg-59 107 153 45.27 28 2 465 12 bg 372 103 152 43.90 29 2 850 13 tara 104 153 42.03 28 1 695 14 dhanush 104 152 35.60 30 2 820 f-test genotype (g) ** ** ** ns ns * year (y) ** ** ** ** ** ** g × y ** ** ns ns ns ** cv% 0.86 0.6 10.4 11.05 18.42 20.1 international journal of applied biology, 3(2), 2019 54 sem (±) 0.372 0.383 0.913 0.847 0.028 38.9 *,**, significant at 0.05 and 0.01 probability level respectively. ns, non-significant discussion in these experiments, variation was observed in agromorphological traits among lentil and chickpea genotypes in both years. the variation on days to flowering in lentil was reported by neupane (2013), dugassa et al. (2014), yadav et al. (2016) and darai et al. (2017). the researchers singh et al. (2006), yadav et al. (2016) and darai et al. (2017) have reported significant difference on this trait among evaluated lentil genotypes. these results showed the presence of inherent genetic variability among the genotypes which gives an opportunity for legume breeders to select most promising genotypes or improve those traits for variety development through selection and hybridization. significant difference on lentil yield and yield attributing traits were also reported by neupane (2013), dugassa et al.(2014), nath et al. (2014), and kumar et al. (2016). a wide range of variation was observed for all traits under study suggesting variability among the cultivars for these traits. results of the present experiments are in conformity with ramanappa et al. (2013). the estimates of variability revealed that genetic variability was significant among the cultivars under study. the present findings were in accordance with parameshwarappa et al. (2012) for plant height and days to fifty percent flowering. therefore selection has to be precisely made based on the perse performance of the cultivars under replicated trails (akanksha et al., 2016). similarly, previous studies on chickpea landraces indicated significant variations for traits like plant height, days to flowering, days to maturity, number of pods per plant, and grain yield (tesfamickael et al., 2014). conclusions the lentil genotypes namely black masuro, lg 12 and ill 3111 produced 53.20, 44.85 and 45.04% higher yield than the check variety (shital). similarly chickpea genotypes namely iccx 840508-31, iccx 840508-40 and bg 372 were identified as high yielding genotypes that produced 37.12, 31.07 and 22.30% higher yield respectively than the check variety (tara) at bhagetada, doti, nepal. so, these research findings suggest that these genotypes were promising genotypes for far western region of nepal. references akanksha, t., anita, b. & namita, p. 2016. genetic variability, correlation and path analysis in yield and yield components in chickpea (cicer arietinum l.) cultivars under late sown condition. international journal of agriculture science, 54(8): 2884-2886. darai, r., sarker, a., sah, r. p., pokhrel, k. & chaudhary, r. 2017. ammi biplot analysis for genotype x environment interaction on yield trait of high fe content lentil genotypes in terai and mid-hill environment of nepal. ann. agric. crop sci., 2 (1): 1026-1030. doi: 10.1017/s0021859600085543 dugassa, a., legesse, h. & geleta, n. 2014. genetic variability, yield and yield associations of lentil (lens culinaris medik.) genotypes grown at gitilo najo, western ethiopia. sci. technol. arts res. j., 3 (4): 10-18. doi: http://dx.doi.org/10.4314/star.v3i4.2 http://dx.doi.org/10.4314/star.v3i4.2 international journal of applied biology, 3(2), 2019 55 gomez, k. & gomez, a.a. 1984. statistical procedures for agricultural research. 2nd edition. john wiley and sons inc, new york, usa. 680 p. jukanti, a. k. 2012. "nutritional quality and health benefits of chickpea". british journal of nutrition, 108: s11–s26. doi:10.1017/s0007114512000797 kumar, p., vimal, s. c. & kumar, a. 2017. study of simple correlation coefficients for yield and its component traits in lentil (lens culinaris medik.). int. j. curr. microbiol. app. sci., 6 (9): 3260-3265. moad.2013. statistical information on nepalese agriculture 2012/13. agri-business promotionand statistics division, ministry of agriculture development, kathmandu, nepal. moald. 2017. statistical information on nepalese agriculture 2016/2017. government of nepal, ministry of agriculture development. agri business promotion and statistics division. singh durbar, kathmandu, nepal. mocs.2010. nepal trade integration strategy 2010. government of nepal. ministry of commerce and supplies. kathmandu, nepal. nath, u.k., rani, s., poul, m. r., alam, m. n. & horneburg, b. 2014. selection of superior lentil (lens culinaris medik.) genotypes by assessing character association and genetic diversity. the scientific world journal. volume, 2014. retrieved from http://dx.doi.org/10.1155/2014/372405 neupane, r. 2013. varietal investivation on lentil for mid hills. in. giri y. p., khatiwoda, s.p., mahato, b.n., gautam, a.k., bhatta, m. r., ranjit, j. d., chettri, b.k., paneru, r.b. and sapkota, b (eds.). proceedings of 28th national winter crops workshop, held on 9-10th march, 2011 at rars, lumle. nepal agricultural research council, 11421. neupane, b. p. & shrestha, j. 2015. scenario of entomological research in legume crops in nepal. international journal of applied sciences and biotechnology, 3(3): 367-372 pande, s., stevenson, p., rao, j. narayana, neupane, r. k., chaudhary, r. n., grzywacz, d., bourai, v. a. & kishore, g. krishna. 2005. "reviving chickpea production in nepal through integrated crop management, with emphasis on botrytis gray mold". plant disease, 89: 1252–1262. parameshwarappa, s.g., salimath, p.m., upadhaya, h.d., patil, s.s. & kajjidoni, s.t. 2012. genetic variability studies in minicore collection of chickpea (cicer arietinum l) under different environments. karnataka journal of agricultuaral sciences, 25(3): 305-308. prasai, h.k., sah, s. k., gautam, a.k. & regmi, a. p. 2018. conservation agriculture for productivity and profitability of wheat and lentil in maize based cropping system in far western nepal. j bangladesh agril univ, 16(3): 403–410 ramanappa, t.m. chandrashekara, k. & nuthan, d. 2013. analysis of variability for economically important traits in chickpea (cicer arietinum l.). international journal of research in applied, natural and social science, 1(3): 133-140 rars. 2015. research achievement in regional agricultural research station (rars), doti, nepal; 2015. http://narc.gov.np/org/ars_doti.php https://en.wikipedia.org/wiki/digital_object_identifier https://doi.org/10.1017%2fs0007114512000797 http://dx.doi.org/10.1155/2014/372405 international journal of applied biology, 3(2), 2019 56 russel, f. & eisensmith, s.p. 1983. mstat-c. crop and soil sci. dept. michigan state univ. usa; 1983. shrestha, j. 2019. p-value: a true test of significance in agricultural research. retrieved from https://www.linkedin.com/pulse/p-value-test-significance-agriculturalresearch-jiban-shrestha/ singh, g., hafiz, m. & manzar, a. 2006. genetic variability for economic traits in lentil (lens culinaris medik). new botanist, vol. xxxiii : 117-22 tesfamichael, s.m., githiri, s.m., nyende, a.b., rao, g.n.v.p.r., odeny, d.a., rathore, a. & kumar, a. 2014. assessment of genetic variation and heritability of agronomic traits in chickpea (cicer arietinum l.). inter j agro agri res, 5: 76-88 usaid. 2011.value chain/market analysis of the lentil sub-sector in nepal. united states agency for international development, general development office, kathmandu, nepal; 2011. yadav, n.k., ghimire, s. k., sah, b. p., sarker, a. shrestha, s. m. & sah, s. k. 2016. genotype x environment interaction and stability analysis in lentil (lens culinaris medik.) international journal of environment, agriculture and biotechnology, 1(3): 354-61. https://www.linkedin.com/pulse/p-value-test-significance-agricultural-research-jiban-shrestha/?lipi=urn%3ali%3apage%3ad_flagship3_pulse_read%3birmagrtwtcq6iejqbiyoya%3d%3d https://www.linkedin.com/pulse/p-value-test-significance-agricultural-research-jiban-shrestha/?lipi=urn%3ali%3apage%3ad_flagship3_pulse_read%3birmagrtwtcq6iejqbiyoya%3d%3d 3(1)36-44 contact : muhamad iksan iksanbioumb@gmail.com © international journal of applied biology 36 abstract sustainable development can be caused by more human’s activity. this activity, it will have an impact on the surrounding environment which can disrupt the stability of the ecosystem, one of which is the river water ecosystem. the research aims to determine the ability of ferns in accumulating heavy metals so that they can be used as indicators of the depletion of river waters ecosystem environments and can be used as fitoremediant areas of waters which accumulate heavy metals. this research is descriptive quantitative. retrieval of initial data or sample in this study used survey methods and random sampling techniques, testing samples by using the ranger x-ray flourenciece (xrf) method. based on the results of the study that, ferns can accumulate heavy metals, the parts that accumulate are located below the root surface and above the soil surface, namely stems and leaves. in the roots have more amount in accumulating heavy metals than in the stem and leaves, this is because the metal is indicated on the soil. from these results, it can be concluded that ferns can be used as water purification plants in river ecosystems that are indicated by heavy metals. issn : 2580-2410 eissn : 2580-2119 the ability of ferns to accumulate heavy metals (hg, pb and cd) in the waters of the gorontalo river muhamad iksan, la aba & kusrini study program biology education, faculty of teacher training and education, universitas muhammadiyah buton, jln. betoambari no. 36 kota baubau, 97321, southeast sulawesi province, indonesia. introduction bone bolango regency is a division of gorontalo regency which is in one unit of gorontalo province. bone bolango regency was formed based on law number 6 of 2003 and was officially established on may 16, 2003. bone bolango regency has an area of 1,984.54 km² or 16.24% of the total area of gorontalo province. the area of bone bolango regency is located between 09⁰ 01⁰15 ’north latitude and 121⁰84 123⁰26’ east longitude. bone bolango regency has a mineral potential that is traditionally managed by the general public. mining activities in the bone bolango district are estimated to be around 2,112 miners. in addition, the mining results obtained are still in the form of a mixture of materials and then use drum for the process of destruction of the material. this process is to facilitate the processing of gold which is still integrated with the material. to separate the gold, the material inserted into the drum is mixed with mercury which works as a gold binder. tromol is placed very close to the river so that the waste from the drum goes open access international journal of applied biology keyword heavy metal (hg, pb and cd), ferns (pteridophyta) article history received 15 january 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 37 directly to the river body. this waste discharge causes pollution of heavy metals into the aquatic environment. in addition to gold processing there are several other activities that cause polluted aquatic ecosystems such as workshops, agriculture and household activities as well as the use of pesticides and herbicides in the process of eradicating pests, this can result in the deposition of heavy metals into sediments and then into the aquatic environment through infiltration or percolation. in these processing and implementing activities of environmental quality standards are absolutely necessary to ensure the stability of the river ecosystem. disposal of special gold mining processing wastes that are managed traditionally contain heavy metal elements such as (hg, pb and cd) which can pollute the river environment and ultimately affect the life of flora and fauna, threatening the diversity of species and their ecosystems. the river is a living environment that provides important life for living things, which have high diversity and resources. the river resources will continue to be maintained and stable if there is no ecological pressure in them, which is caused by human activities that use resources excessively which results in changes in the river's ecological system. river pollution, which is one form of pressure from human activities that is directly carried out in the river, as well as human activities on land against the river environment and the resources therein can cause harm to natural systems (ecosystems) that have been arranged previously and for humans that are part of the system natural. human activities in utilizing resources often produce pollutant waste which can endanger life in the river flow. increasing human activities around river flows can cause changes in these aquatic ecosystems. on the other hand, the threat of the preservation of biodiversity, in the form of a decline in population and plant species continues, which is caused by human activities. (irawanto, 2014). these activities can trigger changes in aquatic ecosystems because they can produce heavy metals such as mercury (hg), lead (pb) and cadmium (cd) which are not needed by organisms. the presence of heavy metals such as mercury (hg) into the aquatic environment does not interfere with these aquatic ecosystems, but it is very dangerous if the heavy metals enter the plant's body. the entry of heavy metals into plant growth will disrupt all plant tissue systems so that it will cause khorosis and necrosis even death. according to (forstner and wittman, 2001). heavy metals in the food chain system cause bioaccumulation of heavy metals in the organism's body in the food chain system. bioaccumulation is the process of increasing the concentration of heavy metals in the body of living things according to the level of the food pyramid and the higher the tropical structure of one type of organism in the food chain the greater the level of heavy metals contained in the organism's body. one of the biotic components in the food chain in river ecosystems is ferns (pteridophyta) which are biotic components in the food chain in river ecosystems that are very tolerant of water pollution. it is known that there are types of ferns which are used as ingredients of vegetables consumed by the general public of gorontalo, namely types of pteridium aquilinum. anthony et al. (2007) suggested that there were 10 types of ferns that could be used as hyperaccumulators, namely adiantum aethiopicum, blechnum cartilagineum, blechnum nudum, cheilanthes nitida, dennstaedtia davallioides, doodia aspera, nephrolepis multiflora, pellaea falcata, pteridium aquilinum and arsenic (as) hyperaccumulating pteris vittata. the ability of ferns to accumulate heavy metals, significantly more accumulated to the roots of leaves, shows limited mobility and heavy translocations absorbed by ferns. absorption of heavy metals causes plants to continue international journal of applied biology, 3(1), 2019 38 to grow unhindered and is an important means of heavy metal tolerance (ernst etal. 1992 ), but can cause death if the toxicity has crossed the threshold (baker et.al. 2000). materials and methods this research is descriptive quantitative. the method used in this study is the roaming method. this is intended to facilitate data collection of ferns (pteridophyta) found in the vicinity of the bone river and the tombulilato river stream. the way of sampling plants, carried out by using hands and shovels, to maintain the authenticity and quality of plant samples stored in thick plastic, then the sample is taken to a biology botanical laboratory for preparation of plant samples, after that, the samples are washed until the water has not changed and finally washed by using distilled water. processing of plant samples is carried out by each plant species so that it can be distinguished, then cut into small pieces and wrapped in allium foil and labeled on each organ of the species then put into the oven at 400c until it gets a constant weight. then weighed again the dry weight of the plant and then mashed facilitate analysis and then to examine the content of heavy metals using the x-ray flourenciece (xrf) s2 ranger data analysis identification of ferns is carried out in a biology laboratory by describing the morphological characteristics of plant species obtained from two river streams. each type of plant found is described in its taxonomic hierarchy, identification of ferns is carried out at the biological laboratory of gorontalo state university, using key books such as stenis (2008), hidayat (2011) and p.s. short & d.j. dixon, (2011). analysis of levels of heavy metal mercury (hg) in ferns (pteridophyta) was carried out at the gorontalo state physics laboratory using x-ray flourenceiece (xrf) s2 ranger. the results of the analysis are obtained in table form. results and discussion based on the results of the study found 5 types of ferns (pteridophyta) found around the river flow namely pteridium revolutum, cyclosorus interruptus, pityrogramma calomelanos, cheilanthes nitidad and nephrolepis multiflora. table 1. fern species around the river basin. no type ferns (pteridophyta) bone river tombulilato river st 1 st 2 st 3 st 1 st 2 st 3 1 pteridium revolutum √ √ √ √ √ √ 2 cyclosorus interruptus √ √ √ 3 pityrogramma calomelanos √ √ √ √ √ √ 4 cheilanthes nitida √ √ √ √ √ √ 5 nephrolepis multiflora √ √ √ √ √ √ international journal of applied biology, 3(1), 2019 39 table 2. the content of heavy metals (hg, pb and cd) contained in the organs of ferns (pteridophyta), at station i of the river bone, and the river tombulilato. no location type ferns (pteridophyta) the average content of heavy metal in ferns (pteridophyta) 1 bone river root (ppm) stem (ppm) leaf (ppm) hg pb cd hg pb cd hg pb cd pteridium revolutum 0.30 246 160 0.30 250 235 0.23 270 247 cyclosorus interruptus 0.31 158 143 0.28 145 122 0.20 142 135 pityrogramma calomelanos 0.26 157 147 0.21 156 139 0.18 168 154 cheilanthes nitida 0.33 352 276 0.31 243 356 0.30 266 342 nephrolepis multiflora 0.32 123 223 0.26 223 212 0.22 243 345 2 tombulila to river pteridium revolutum 0.05 134 124 0.04 141 132 0.02 121 154 pityrogramma calomelanos 0.05 189 179 0.04 111 98 0.03 114 111 cheilanthes nitida 0.20 178 230 0.15 199 265 0.12 173 278 cyclosorus interruptus 0.04 156 189 0.02 103 165 0.03 150 245 table 3. the content of heavy metals (hg, pb and cd) contained in the organs of ferns (pteridophyta), at station ii of the river bone, and the river tombulilato. no location type ferns (pteridophyta) the average content of heavy metal in ferns (pteridophyta) 1 bone river root (ppm) stem (ppm) leaf (ppm) hg pb cd hg pb cd hg pb cd pteridium revolutum 0.35 359 165 0.31 368 265 0.33 387 127 cyclosorus interruptus 0.30 368 345 0.32 477 387 0.36 327 399 pityrogramma calomelanos 0.32 365 178 0.34 334 343 0.30 311 122 cheilanthes nitida 0.31 434 346 0.36 376 389 0.30 387 392 nephrolepis multiflora 0.32 267 227 0.25 235 233 0.24 245 367 2 tombulila to river pteridium revolutum 0.18 247 189 0.14 198 100 0.11 256 121 pityrogramma calomelanos 0.17 200 120 0.12 290 156 0.15 256 179 cheilanthes nitida 0.14 298 189 0.13 367 180 0.11 293 104 cyclosorus interruptus 0.06 202 126 0.03 278 179 0.05 208 137 international journal of applied biology, 3(1), 2019 40 table 4. the content of heavy metals (hg, pb and cd) contained in the organs of ferns (pteridophyta), at station iii of the river bone, and the river tombulilato. no location type ferns (pteridophyta the average content of heavy metal in ferns (pteridophyta) 1 bone river root (ppm) stem (ppm) leaf (ppm) hg pb cd hg pb cd hg pb cd pteridium revolutum 0.33 423 465 0.30 469 352 0.34 326 433 cyclosorus interruptus 0.30 398 322 0.32 377 367 0.37 427 387 pityrogramma calomelanos 0.31 475 376 0.32 344 337 0.32 411 310 cheilanthes nitida 0.31 483 323 0.27 445 321 0.30 455 356 nephrolepis multiflora 0.32 377 267 0.30 398 241 0.34 340 360 2 tombulila to river pteridium revolutum 0.07 251 289 0.03 276 276 0.05 260 210 pityrogramma calomelanos 0.08 176 230 0.06 387 278 0.06 241 220 cheilanthes nitida 0.11 293 181 0.10 243 251 0.09 276 203 cyclosorus interruptus 0.07 193 220 0.04 214 292 0.05 190 125 heavy metal content in nail plants (pteridophyta) in the bone river region and tombulilato river based on the results of the analysis carried out at the gorontalo state physics laboratory, using the ranger s2 flourenciece x-ray (xrf) testing method. the samples analyzed were ferns (pteridophyta) which were taken directly around the bone river and tombulilato river bone bolango district. based on the results of the analysis show that the river is polluted by heavy metals (hg, pb, cd) as seen in the heavy metal content table. from the results of the research, there were 5 types of ferns (pteridophyta) which could accumulate heavy metals (hg, pb and cd), namely pteridium revolutum, cyclosorus interruptus, pityrogramma calomelanos, cheilanthes nitidad and nephrolepis multiflora. based on the results of analysis of heavy metal levels in ferns (pteridophyta) that river waters are indicated by heavy metals. the presence of heavy metals around river waters cannot be avoided because it is a place for community activities ranging from gold processing, agriculture and household activities. these activities cause the presence of heavy metals in river waters. as one example, gold processing around the river uses mercury in the amalgamation process. activities that cause heavy metals in river waters due to direct waste from drum and river then settle into sediments. heavy metals found in sediments greatly influence the heavy metal content in plants that grow above them, so that high or low metal content in the sediment will reflect metal content in plants, darmono (1995). the heavy metal content in plants is also influenced by sedimentation (palar, 2012). according to hernandez international journal of applied biology, 3(1), 2019 41 et al (2010) waste originating from mining extracts that enter into soil containing pb and cd heavy metals is more than 1500 mg/kg-1. malar et al (2014) suggested that contaminated wasting water containing heavy metal pb around 1000 mg / l can be seen if plants have shown symptoms of chlorosis. according to cheng (2003) when plants show chlorosis, the plant has accumulated cd and pb heavy metals in excess of 800 mg / kg. then confirmed by malar at al (2014) plants experiencing symptoms of chlorosis have accumulated heavy metals cd and pb 100-1000 mg/l. in addition, the high content of heavy metal lead (pb) and cadmium (cd) detected in ferns, due to corrosive from rock excavations in the mining area so that heavy metals in the rocks are carried away by the rain and head to the river. according to palar (2004) heavy metals can occur due to rock blasting which is then carried by rain to the river through a run-off process. another factor is the result of the use of pesticides and herbicides by farmers as pest control. according to tangahu et al (2011), that the content of heavy metal lead (pb) found on herbicides and pesticides reaches 1100 mg/m3. then this is reaffirmed by malar et al (2014) that pesticides can cause high accumulation of heavy metals into the sediment. apart from the sources discussed earlier, motorized vehicles are also one of the main sources of pollution because they contain various pollutants which are harmful to humans, animals, plants. according to fergusson (1990) pollutants (pollutants) originating from motor’ vehicle gases are generally in the form of combustion gases and heavy metal particles such as lead (pb). black lead (pb) released from motorized vehicles averages 0.02-0.05 µm in size. the smaller the particle size the longer it stays. if it is observed along river waters starting from the eastern suwawa sub-district to the botupingge sub-district, the highway is close to the bone river. the amount of heavy metals in the environment is strongly influenced by the volume or density of traffic, distance from the highway, and engine acceleration and wind speed. the presence of heavy metals in the environment is strongly influenced by environmental factors such as acidity, current velocity, turbidity, dissolved oxygen and salinity. according to palar (2004) the normal acidity (ph) of water is between 6-8. if the ph value is below or above the normal value, the environmental conditions of the waters have been polluted, the lower the ph value, the greater the accumulated heavy metal content or vice versa the higher the ph value, the greater the heavy metal content. based on the measurement results of dixolved oxygen (do) then, the bone river and the tombulilato river are in the moderate polluted category. according to supardi, (1984) water pollution is divided into 3 parts, namely lightly polluted if do levels = 5 mg/l, medium polluted if do levels are between 2-5 mg / l and heavily polluted if do levels are between 0, 1 2 mg/l. based on data from (baliristi gorontalo province, 2014) that the bone river has been moderately polluted in the upstream part (suwawa timur district, dumbaya bulan village) and lightly polluted in the lower part (bendungan, suwawa tengah district). the existence of heavy metals in the environment will endanger the life of living things and be more dangerous if they enter into the metabolic system of living things in excess of the boundary limit. plants that are able to accumulate heavy metals in their bodies are called accumulator plants. hernandez et al. (2010) that plants that are hyperaccumulators if they are able to accumulate heavy metals more than 1000 mg/kg. then confirmed by landis, et al. (2011) if the accumulation ability is as much as 100 ppm or more than 1000 mg / kg dry weight is considered to be a hyperaccumulator plant. draghiceanu et al. (2014) my plants are hyperaccumulators if they are able to accumulate more than 1,000 mg/kg cadmium (cd) and international journal of applied biology, 3(1), 2019 42 10,000 mg/kg lead (pb). so that ferns are potential plants as hyperaccumulators. as with other aquatic plants used in phytoremediation (tangahu, 2011). conclusions from the results of the discussion, it can be concluded that there are 5 types of ferns on the bone river, namely pteridium revolutum, cyclosorus interruptus, pityrogramma calomelanos, cheilanthes nit and nephrolepis multiflora, while the river tombulylate has 4 types of ferns namely pteridium revolutum, pityrogramma calomelanos, cheilanthes nitidad and nephrolepis multiflora. the presence of ferns in each region is strongly influenced by climatic conditions, thus affecting the accumulation of heavy metals in ferns. heavy metal levels (hg, pb, cd) found in ferns (pteridophyta) in river waters are very dangerous. this can disrupt the ecosystem inside and can break the food chain in the ecosystem. these activities are in the form of gold processing which the waste is discharged into the river, the use of herbicides and pesticides in eradicating pests that settle into sediments and into the river through infiltration, workshop which removes the remaining oil into the ditch / ditch which then goes to rivers and discarded household waste to the river. all of these activities are the main sources of heavy metals which can pollute the river environment including living organisms in the waters and people who use the river as a livelihood. therefore the right solution to remediate land contaminated with heavy metals due to human activities can use ferns as fitoremediant. references allen, h.e; garrison, a.w; and luther iii, gw. 1998. industrial discharges of metals to waters dalam buku metals in surface waters. sleeping bear press inc. ann arbor press. michigan.usa.262p allowey, b.j. & d.c. ayres. 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diversity between maize germplasm (zea mays l.) is useful for their systematic and efficient use in breeding programs. fourteen early maize genotypes were studied to assess their performance and genotypic diversity at doti, nepal in 2015. days to tasseling, days to silking, plant height, ear height, ear length, ear diameter and grain yield were significant among genotypes. genotype so3tey-pobm, compol-niobp and across-99402 were found higher yielder with earlier maturity. days to tasseling (0.85), days to silking (0.82), plant height (0.79), ear length (0.71) and ear diameter (0.66) were found highly heritable traits. grain yield (0.39) and ear height (0.47) medium and remaining traits showed low heritability. high pcv was observed for grain yield (35.10%), number of plants/plot (34.46%), tesseling silking interval (26.85%), harvested ears/plot (24.45%) and husk cover rating (22.85%) where other traits showed medium to low pcv. grain yield showed high gcv (21.96%), ear height and husk cover had medium and remaining traits showed low gcv (<10%). plant height (r₌0.498), harvested plants/plot (r₌0.412), harvested ear/plot (r₌0.762), ear length (r₌0.472) and ear diameter (r₌0.470) showed significant positive correlation with grain yield. the yield can be improved if selection applied in favor of those yield components. issn : 2580-2410 eissn : 2580-2119 agronomic performance and genotypic diversity for morphological traits among early maize genotypes bhim nath adhikari1, jiban shrestha2, bishal dhakal1, bishnu prasad joshi1, & naba raj bhatta1 1regional agriculture research station (rars), dipal, doti, narc, nepal 2national commercial agriculture research program, pakhribas, dhankuta, nepal introduction maize (zea mays l.) is second important crop after rice in nepal. it is cultivated in 891583 ha with 2503 kg/ha national productivity (moad, 2017). the majority of the maize area lies in the mid hills across the country where it is the first principal staple food and feed crop and produce about 70% of national maize production. therefore maize can be considered as source of livelihood for hilly farmers. it is a traditional crop cultivated on slopping upland terraces (bariland) in the hills under rainfed condition during the summer on which fingermillet is relayed in majority, however sole maize was dominantly followed by wheat. hilly areas of western nepal is mostly food deficit, drought prone areas where annual open access international journal of applied biology keyword maize variability heritability genetic advance correlation article history received 30 october 2018 accepted 25 december 2018 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 2(2), 2018 34 average rainfall is about 1000 mm and rainfall distribution is erratic and unpredictable. lower productivity of maize is associated with lack of suitable varieties for stress environments, inadequate variety in the existing system, lack of agricultural inputs like irrigation, fertilizers etc. early maturing maize will also ensure for timely wheat sowing in maize-wheat system which may lead proper germination and initial growth of wheat because wheat is grown on residual moisture and if sown late germination and growth may be adversely affected due to lack of moisture in bariland farming. in addition early maturing maize also compete less with fingermillet for sunlight, nutrients and moisture than full season maize in maize/millet relay cropping with ultimately support to increase fingermillet yield. thus improved maize genotypes with early maturity and high yield, tolerant to drought and better fit into existing system will increase and stabilize yield of maize, fingermillet and wheat on bariland farming system of hilly areas. variability is the differences between individuals in a population due to genetic composition and growing environment (sumanth et al., 2017). the existence of variability is utmost role as success of any plant breeding program depends on the genetic variability and selection skill of plant breeder (adhikari et al., 2018). selection is only effective if the parent population have significant amount of variability. phenotypic coefficient of variation (pcv) and genotypic coefficient of variation (gcv) provides insight information on magnitude of variability in a population and heritability shows the component of a character transmitted to future generations (pal et al., 2016; girma et al., 2018) further, heritability coupled with genetic advance shows the expected genetic gain in next generation (shukla et al., 2006). in addition, correlation between yield and yield attributing traits are prime important for indirect selection for those traits which have high heritability and closely associated to contributes to improve yield (aditya & bhartiya, 2013). therefore, the study was under taken to identify suitable early maturing genotypes and to know the information on variability, heritability, genetic advance and traits correlation on tested materials to utilize it on future maize breeding activity which aim to develop suitable genotype for rainfed bariland farming of mid hills areas of nepal. materials and methods experimental location the experiment was conducted at regional agricultural research station, dipayal, doti. geographically, it is situated at 80° 55' east longitude and 29° 15' north latitude and the altitude of this station is 510 masl (rars, 2015). the climate is sub tropical, generally monsoon starts late in july and it is erratic with average annual rainfall of about 900mm. experimental field soil was shallow in depth and porous, sandy loam, slightly acidic with ph 5.5-6.0, low in nitrogen and organic matter i.e. 1-2% and 0.6 % respectively and the water holding capacity of the soil is very low (rars, 2015). genotypes the experiment was carried out with fourteen different early maize genotypes in which 13 were received from national maize research program (nmrp), rampur and farmer’s variety was collected from dipayal, doti. the tested genotype were: earlymid katamani, rajahar local, s97teyghayb(3), pop-445/pop-446, compol-nibp, rc/pool-17, s03tey/lm, arun-4 (standard check variety), farmer’s variety (local check variety), zm621/pool-15, eeyc1, so3tey-ln/pp, so3tey-po-bm and across-99402. international journal of applied biology, 2(2), 2018 35 experimental design and cultural practices the experiment was planted on 2nd week of june in randomized complete block design (rcbd) with three replications. along with fym @ 10 t/ha chemical fertilizers at the rate of 60:60:40 n p2o5 k2o kg/ha in the form of urea, dap and murate of potash were applied during final land preparation. top dressing was done by 60 kg n/ha in two splits, i.e. 30 kg n/ha at knee high stage and 30 kg n/ha at just before tasseling stage. individual plot size was 3m × 3m (4 rows of 3m long). seed was shown @ 2 seeds/hills in 75 cm apart rows by maintaining 25 cm between hill to hill in a row. thinning was done to maintain plant population after 3 weeks of germination. intercultural operations were done as per recommendation of national maize research program. data measurement days to tasseling, days to silking, number of plants harvested, number of cob harvested, field weight were taken from central 2 rows from each plot. five plants form central rows randomly selected and plant height, ear height, ear length, ear diameter recorded and mean values was taken for analysis. husk cover rating was taken as 1-5 scale where 1 is good and 5 is poor. after taking field weight, composite grains from five randomly selected ears prepared and moisture reading taken for thrice and mean was used for yield estimation. grain yield was estimated at 15% moisture and 80% shelling coefficient by below formula which was also adopted by carangal et al. (1971) and shrestha et al. (2018). grain yield ( kg ha ) = f. w. ( kgplot) × (100 − hmp) × s × 10000 (100 − dmp) × npa where, f.w. = fresh weight of ear in kg per plot at harvest hmp = grain moisture percentage at harvest dmp = desired moisture percentage, i.e. 15% npa = net harvest plot area, m2 s = shelling coefficient, i.e. 0.8 data analysis analysis of variance and correlation among traits were done by using genstat 18th edition at 0.05 level of significance. likewise significance of correlation coefficient was done as proposed by kothari (2004). the phenotypic and genotypic variances were computed according to falconer (1981). broad sense heritability was calculated using formula suggested by allard (1960) and categorized as low (0-0.3), moderate (0.30-0.6) and high (>0.6). the estimates of genetic advance were obtained at 5% selection intensity and categorized as high (>20%), medium (10-20%) and low (<10%) as suggested by johnson et al. (1955). coefficient of genotypic and phenotypic variation were also categorized as high (>20%), medium (1020%) and low (<10%) as proposed by sivasubramanian and madhavamenon (1973). international journal of applied biology, 2(2), 2018 36 results and discussion analysis of variance the analysis of variance for 10 quantitative characters showed significant differences among the evaluated genotypes for days to tasseling, days to silking, plant height, ear height, ear length, ear diameter and grain yield. however, differences were not significant for tasseling silking interval days, number of harvested plants/plot, number of harvested cobs/plot and husk cover rating (table 1 and 2). statistical traits indicated the existence of inherent genetic variation among tested genotypes for those traits. thus there is scope to enhance genetic yield potential through selection and hybridization. existence of significant genetic variability among maize genotypes for yield and major yield attributing traits were reported by vashistha et al. (2013), ogunniyan and olakojo (2014), pahadi and sapkota (2016), bhusal et al. (2017), kandel et al. (2018) and sharma et al., (2018). prasai et al. (2015) also reported genetic variability on early maize. similarly, shrestha (2014) observed morphological variation in maize inbred lines in chitwan, nepal. agronomic performance agronomic performance observed on yield and yield attributing characters were summarized and presented in table 1 and 2. days to tasseling, silking and tasseling silking interval genotypic differences were observed significant for both days to silking and tasseling but the interval between silking and tasseling remained statistically non-significant. the silking tasseling intervals remained 2-3 days for all tested genotypes. farmers variety observed earliest in tasseling and silking took 37 and 40 days respectively which was followed by s97teyghayb(3) and arun-4 both took 45 and 48 days respectively for tasseling and silking. genotype s03tey/lm observed late took 51 days for tasseling and 53 days for silking. this observation clearly indicated that farmer’s variety is extra early and could be one of the parents for maize hybridization program to develop early maturing variety. significant difference among tested maize genotypes for days to tasseling and silking were previously reported by (vashistha et al., 2013; prasai et al., 2015; sharma et al., 2018; bhusal et al., 2017). plant and ear height significant differences for plant and ear height observed indicated the existence of genetic variability among genotypes kept under study. rajahar local was the tallest genotype in plant height (287 cm) and ear height (126 cm) whereas farmers' variety was observed as dwarf genotypes. standard check variety (arun-4) and zm621/pool-15 were found medium in plant and ear height. in favor of present study, genotypic variation on plant and ear height were previously reported by (ogunniyan and olakojo, 2014; parsai et al., 2015; sharma et al., 2018). plant and ear harvested tested genotypes were not differed statistically for number of plants and ear harvested per unit area. mean harvested plants and ear were found 14 and 16 respectively. similar result on harvested plant and ears on early maturing maize were reported by katuwal et al. (2017). international journal of applied biology, 2(2), 2018 37 ear length, diameter and husk cover rating genotypic differences were observed for ear length and diameter but rating based on husk cover remained at par between genotypes. across-99402 was observed highest ear length and farmer’s variety was shortest ear length. likewise, genotype zm 621/pool-15 was found having highest ear diameter while farmer’s variety was lowest. husk cover rating was observed 2-3 for all genotypes. genetic difference on ear length and diameter were also reported by (vashistha et al., 2013; pahadi & sapkota, 2016). grain yield the grain yield was found statistically significant indicating the existence of genetic variability on yield potential on the tested genotypes. higher yielder genotypes observed were so3tey-po-bm, compol-niobp, across-99402 could be selected as promising genotypes for early maturing groups or may be used as a parent in maize breeding program to develop early maturing maize varieties. likewise, farmer’s variety was poor yielder but due to its early maturing character it can also be used as a parent for the purpose. genetic variability on maize genotypes was reported by many authors including (vashistha et al., 2013; bhusal et al., 2017; katuwal et al., 2017). genetic parameter estimation estimated genetic parameter such as the phenotypic coefficient of variation (pcv), the genotypic coefficient of variation (gcv), broad sense heritability, and genetic advance as percent of means are presented in table 3. yield and yield attributing traits under study showed higher phenotypic coefficient of variance than genotypic coefficient of variance indicated high environmental influence on the expression of these trait, however, as compared with others, less environmental influence was observed for days to silking, tasseling and plant height. similar results were presented by (pahadi and sapkota, 2017; bisen et al., 2018; sharma et al., 2018). high pcv was observed for grain yield, number of plants/plot, tesseling silking interval, harvested ears/plot and husk cover rating where as remaining traits showed medium to low pcv. similarly high gcv was estimated for grain yield, medium for ear height and husk cover rating and remaining traits showed low gcv. in line with this findings high pcv and gcv for grain yield was also reported by (ogunniyan and olakayo, 2014; bhusal et al., 2017; bisen et al., 2018; sharma et al., 2018). table 1. mean performance of yield attributing traits on maize sn genotype dt ds tsi ph eh 1 early mid katamani 50fg 52def 2.33 250def 100abcd 2 rajahar local variety 48bcdf 50bcde 2.67 287g 126d 3 s97teyghayb(3) 45b 48b 3.00 211ab 75a 4 pop-445/pop-446 46bcde 49bcd 3.67 222bc 78ab 5 compol-nibp 50fg 53ef 3.00 232bcd 77ab 6 rc/pool-17 47bcdef 50bcd 2.33 251def 111cd 7 s03tey/lm 51g 53f 2.33 256ef 107cd 8 arun-4 45bc 48bc 3.00 245cde 106cd 9 farmer’s variety 37a 40a 2.67 195a 75a 10 zm621/pool-15 48cef 51cdef 3.00 241cde 97abc 11 eeyc1 45bcd 48b 2.67 271fg 101abcd 12 so3tey-ln/pp 49fg 52def 2.33 253def 96abc international journal of applied biology, 2(2), 2018 38 13 so3tey-po-bm 49fg 52def 3.00 273fg 102bcd 14 across-99402 49fg 51def 2.33 233bcd 90abc mean 47 50 2.74 244 96 sem 1.17 1.24 0.57 9.97 11.14 p value <.001 <.001 0.534 <.001 0.002 cv% 3.0 3.1 25.9 5.0 14.2 lsd (0.05) 2.4 2.56 20.48 22.9 means followed by a superscripted common letter with in a column are not significantly different from each other at p ≤ 0.05. dt= days to tasseling, ds= days to silking, tsi= tasseling silking interval, ph= plant height (cm), eh=ear height (cm) due to the lower differences estimated between pcv and gcv selection based on phenotypic performance for days to tasseling, silking and plant height would be effective to bring about considerable genetic improvement on the tested maize population for these traits but agronomic performance can’t improve by providing favorable environment without selection. traits showing higher difference between gcv and pcv indicated that these traits are more influenced by growing environment thus selection is not effective on those traits, can be maintained by providing optimum growing environment. table 2. mean performance of grain yield and yield attributing traits on maize sn genotypes p/p e/p el ed hc gy 1 early mid katamani 14 16 14.00bc 4.20bc 3.00 2945abcd 2 rajahar local variety 18 17 13.33b 4.17b 2.67 3013abcd 3 s97teyghayb(3) 19 17 14.33bcd 4.37bcde 2.67 3109bcd 4 pop-445/pop-446 11 13 14.00bc 4.40bcde 2.67 2225ab 5 compol-nibp 14 18 14.67bcd 4.60ef 2.33 4179d 6 rc/pool-17 15 15 13.67b 4.40bcde 3.00 2299ab 7 s03tey/lm 9 17 14.67bcd 4.53ef 3.00 3409bcd 8 arun-4 15 14 14.00bc 4.20bcd 2.67 2834abcd 9 farmer’s variety 10 11 10.00a 3.87a 2.00 1409a 10 zm621/pool-15 14 11 14.33bcd 4.77f 2.33 2392abc 11 eeyc1 18 18 14.33bcd 4.50cef 2.00 3880bcd 12 so3tey-ln/pp 14 18 14.00bc 4.37bcde 2.00 3522bcd 13 so3tey-po-bm 14 17 15.33cd 4.60ef 2.00 4333d 14 across-99402 18 18 15.67d 4.63ef 2.33 3976cd mean 14 16 14.02 4.40 2.48 3108.93 sem 3.91 2.97 0.64 0.13 0.41 695.1 p value 0.314 0.273 <.001 <0.001 0.108 0.01 cv% 33.2 23.3 5.6 3.6 20.4 27.4 lsd (0.05) 1.32 0.26 1428.8 means followed by a superscripted common letter with in a column are not significantly different from each other at p ≤ 0.05. p/p= number of harvested plants/plot, e/p= number of harvested ears/plot, el= ear length (cm), ed= ear diameter (cm) hc= husk cover rating in 1-5 scale, gy= grain yield kg/ha among the studied traits broad sense heritability ranged from 0.04 (tsi) to 0.85 days to tasseling. heritability is grouped as low (<0.3), moderate (0.3-0.6) and high (>0.6). based on this category days to tasseling, days to silking, plant height, ear length and ear diameter were found highly heritable traits. grain yield and ear height medium and remaining traits showed low heritability. high heritability for days to tasseling, days to silking, plant height, international journal of applied biology, 2(2), 2018 39 ear length and ear diameter and high to medium for grain yield were also reported by (vasistha et al., 2013; bhusal et al., 2017; sharma et al., 2018). medium heritability for grain yield may be due to the influence of the environment on the yield as it is polygenic trait. low heritability estimates might be due to the variation of environmental component involved for those traits and vice versa. high heritability estimated traits indicated a high response to selection for particular traits. estimated heritability is not very much useful because it includes the effect of both additive and non additive gene. the genetic advance with heritability is therefore a useful indicator to achieve expected result on the trait of interest of a population after selection. further, genetic advance in percentage of mean give more precise result in comparison to only genetic advance. genetic advance as percent mean was categorized as low (0-10%), moderate (10-20%) and high (≥20%). in the present study high genetic advance as percent of mean was estimated for grain yield. days to tasseling, days to silking, plant height, ear height and ear length showed medium and remaining traits showed low genetic advance as percent of mean. in conformity to this findings, high genetic advance for grain yield and medium to low for other traits also reported by (vasistha et al., 2013; ogunniyan and olakayo, 2014; sharma et al., 2018). high to medium heritability coupled with medium to low genetic gain indicate observed characters among tested genotypes governed by non-additive gene action and thus heterosis breeding, family selection and progeny testing methods is used for improvement on such traits. table 3. phenotypic (vp) and genotypic variance (vg), phenotypic coefficient (pcv) and genotypic coefficient of variance (gcv), heritability (broad sense), genetic advance (ga) and genetic advance as percent of mean (gam) for observed traits sn traits treatment error vg vp hbs gcv pcv ga gam 1 dt 36.26 2.06 11.40 13.46 0.85 7.17 7.79 6.40 13.60 2 ds 34.65 2.33 10.77 13.11 0.82 6.59 7.27 5.28 10.60 3 tsi 0.57 0.50 0.02 0.53 0.04 5.52 26.85 0.05 2.01 4 ph 1830.05 148.96 560.36 709.32 0.79 9.69 10.90 37.35 15.29 5 eh 690.50 186.18 168.11 354.29 0.47 13.53 19.65 15.85 16.55 6 p/p 28.34 23.01 1.77 24.79 0.07 9.22 34.46 0.63 4.38 7 e/p 17.31 13.28 1.34 14.62 0.09 7.41 24.45 0.62 3.98 10 el 5.15 0.62 1.51 2.13 0.71 8.76 10.42 1.83 13.08 11 ed 0.17 0.02 0.05 0.07 0.66 4.92 6.08 0.31 7.08 12 hc 0.45 0.25 0.06 0.32 0.20 10.23 22.85 0.20 8.13 15 gy 2123268 724770 466166 1190936 0.39 21.96 35.10 758.22 24.39 dt= days to tasseling, ds= days to silking, tsi= tasseling silking interval, ph= plant height (cm), eh=ear height (cm), p/p= number of harvested plants/plot, e/p= number of harvested ears/plot, el= ear length (cm), ed= ear diameter (cm), hc= husk cover rating in 1-5 scale, gy= grain yield kg/ha, vg=genotypic variance, vp=phenotypic variance, hbs= heritability broad sense, gcv= genotypic coefficient of variation, pcv= phenotypic coefficient of variation, ga= genetic advance, gam= genetic advance as percent of mean. correlation study days to tasseling and silking sowed significant positive correlation. these traits both showed significant positive association plant height, ear length and ear diameter indicated that plant height, ear length and ear diameter can be simultaneously increase while selecting relatively late plants. in line with this findings (ogunniyan and olakaago, 2014; bhusal et al., 2017) reported significant positive correlation between days to tasseling, silking and plant international journal of applied biology, 2(2), 2018 40 height. further, in conformity to present study (pahadi and sapkota, 2016; khan et al., 2018) also reported significant positive correlation for days to silking with ear length and diameter, however khan et al. (2018) reported non-significant positive relationship between days to tasseling with ear length and diameter. similarly, significant positive relationship with days to siliking and non significant positive with days to tasseling towards ear length and ear diamenter was also reported by sharma et al. (2018). further plant height have shown significant positive correlation with ear height indicated that ear height can be altered simultaneously while selecting plants on the basis of height. in addition, plant height has shown significant positive association with number of ear harvested showed that ear prolificacy is higher in tall plants. very similar findings on relationship between plant height, ear height and number of harvested plants were also reoprted by (ogunniyan and olakaago, 2014; bhusal et al., 2017; sharma et al., 2018). harvested plants/plot showed significant association with harvested ear/plot. likewise number of harvested ear showed significant positive association with ear length and ear diameter. significant positive relation was also observed between ear length and ear diameter. these findings were also partially supported by (pahadi and sapkota, 2016; bhusal et al., 2017; sharma et al., 2018). in the present study non significant positive correlation observed between plant height and ear length (r₌0.304), plant height and ear diameter (r₌0.285), plant height and number of plants/plot (r₌0.267). similarly ear height showed non significant positive correlation with plants/plot (r₌0.227) and husk cover rating (r₌0.22). further both silking and tasseling days shown non significant positive association with husk cover rating and number of ears/plot. number of harvested pods/plant showed non significant positive correlation with ear length (r₌0.285) and ear diameter (r₌0.304). on the other side, non significant negative correlation observed between husk cover and ear diameter, husk cover and number of harvested ear/plot, husk cover and number of harvest plants/plot and both days to tasseling and silking with harvested plants/plot. table 4. phenotypic correlation coefficient of grain yield and yield attributing traits on early maize genotypes. traits dt ds ph eh p/p e/p hc el ed dt 1 ds 0.981** 1 ph 0.355* 0.351* 1 eh 0.162 0.165 0.733** 1 p/p -0.078 -0.038 0.267 0.227 1 e/p 0.282 0.257 0.378* 0.146 0.587** 1 hc 0.225 0.219 0.018 0.220 -0.108 -0.147 1 el 0.701** 0.718** 0.304 0.071 0.285 0.360* 0.108 1 ed 0.467** 0.492** 0.285 0.037 0.304 0.316* -0.150 0.684** 1 gy 0.300 0.274 0.498** 0.158 0.412** 0.762** -0.264 0.472** 0.470** * and ** significant at p₌0.05 and p₌ 0.01 level of significance respectively. dt= days to tasseling, ds= days to silking, tsi= tasseling silking interval, ph= plant height (cm), eh=ear height (cm), p/p= number of harvested plants/plot, e/p= number of harvested ears/plot, el= ear length (cm), ed= ear diameter (cm), hc= husk cover rating in 1-5 scale, gy= grain yield kg/ha grain yield had shown significant positive association for plant height, number of harvested plants/plot, number of harvested ears/plot, ear length and ear diameter indicated that selection in favour of these traits will improve grain yield on early maturing maize international journal of applied biology, 2(2), 2018 41 genotypes. these findings were in conformity with previous reporting by (bhusal et al., 2017; ogunniyan and olakaago, 2014; khan et al., 2018; sharma et al., 2018). in addition non significant correlation was observed for days to tasseling (r₌0.3), days to silking (r₌0.274), ear height (r₌0.158) and husk cover rating (r₌-0.264)with grain yield. conclusions this study identified maize genotypes so3tey-po-bm, compol-niobp and across99402 suitable for rainfed bariland farming based on the maturity and yield performance. dipal local was found extra early, thus it could be one of the parents for maize breeding program for developing early maturing maize varieties. this study indicated the genetic variability on the studied genotypes mostly inherited by non additive gene action and direct selection is not fruitful, so hybridization followed by selection is recommended for improvement. as plant height, number of harvested pods/plot, number of harvested ears/plot, ear length and ear diameter were found significantly positively correlated with maize grain yield thus selection applied in favour these traits will simultaneously improve maze yield of selected plants. acknowledgments the authors would like to thank national maize research program, rampur for providing testing genotypes and technical support. we would like to thank technical officer mr. mb chodaro and technical assistant staffs of regional agricultural research station, doti, nepal for their support on conducting research activities. references adhikari, b.n., pokhrel, b.b. & shrestha, j. 2018. evaluation and development of fingermillet (eleusine coracana l.) genotypes for cultivation in high hills of nepal. fmg. & mngmt., 3(1): 37-46. aditya, j.p. & bhartiya, a. 2013. genetic variability, correlation and path analysis for quantitative characters in rainfed upland rice of uttarakhand hills. journal of rice research, 6(2): 24-34. allard, r.w. 1960. principles of plant breeding. 1st edn., john wiley and sons inc., new york. bhusal, t., lal, g.m., marker, s. & synrem, g.j. 2017. genetic variability and traits association in maize (zea mays l.) genotypes. annals of plants and soil research, 19(1): 59-65. bisen, n., rahangdale, c.p. & sahu, r.p. 2018. genetic variability and correlation studies of yield and yield component in maize hybrids (zea mays l.) under kymore plateau and satpura hill region of madhya pradesh. international journal of agriculture, environment and biotechnology, 11(1): 71-77. doi: 10.30954/09741712.2018.00178.9 carangal, v.r., ali, s.m., koble, a.f., rinke, e.h. & sentz, j.c. 1971. comparison of s1 with testcross evaluation for recurrent selection in maize. crop science, 11: 658-661. falconer, d.s. 1964. introduction to quantitative genetics. oliver and boyd ltd, welbeck street, london. girma, b.t., kitil, m.a., banje, d.g., biru, h.m. & serbessa, t.b. 2018. genetic variability study of yield and yield related traits in rice (oryza sativa l.) genotypes. adv. crop. sci. tech., 6 (4): 381. doi:10.4172/2329-8863.1000381 johnson, h.n., robinson, h.f. & comstock, r.e. 1955. estimate of genetic and environmental international journal of applied biology, 2(2), 2018 42 variability in soybean. agronomy journal, 27: 314-318. kandel, m., ghimire, s. k., ojha, b. r. & shrestha, j. 2018. correlation and path analysis for grain yield and its attributing traits of maize inbred lines (zea mays l.) under heat stress condition. international journal of agriculture, environment and food sciences, 2(4): 124-130. katuwal, r.b., chaudhary, r.n., yadav, j.k., yadav, m., yadav, d., yadav, b. & niraula, b. 2017. performance evaluation of early, full season and quality protein maize varieties in the eastern hills of nepal. in: giri y.p, mahato, b.n., gautam, a.k., khatiwoda, s.p., shrestha, r., joshi, b.k., khatri, b.b., rai, s.k., ghimire, y.n., luitel, b.p., upreti, h.k., sah, k, sharma, p.n., bajracharya, a.s.r., baidhya, s., shrestha, j. & manandhar, s. 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165-168. contact : david christian ferdinand davidchristianferdinand@gmail.com 70 abstract sars-cov-2 virus can cause coronavirus disease 2019 (covid-19). the interaction between spike protein and ace2 receptor causes virus entry into the cells. the aim of this study was to review the utilization of nutraceutical and phytochemical agents to inhi bit the interaction of spike protein envelope sars-cov-2 virus and ace2 receptor. the data literature was retrieved from several databases such as google scholar, science direct, and pubmed. this study was conducted from september-october 2020. the review process was conducted based on prisma guidelines.the results showed that nutraceuticals such as tuna peptides (eeaggataaqiem), nisin, teicoplanin, zinc, propolis, ganoderma lucidum, brown algae, and lectin have the potential to inhibit the interaction of s pike protein sars-cov-2 virus and ace2 receptor. phytochemical compounds such as curcumin, luteolin, egcg, hesperidin, resveratrol, saikosaponin, nicotianamine, procyanidin and the others also have the same. from this study, it can be concluded that nutraceuticals and phytochemical agents have potential benefits in covid-19 treatment based on in silico study. however, we need further studies based on in vitro experiments and in vivo to ensure the effectivity of those nutraceutical agents. issn : 2580-2410 eissn : 2580-2119 the utilization of nutraceuticals and phytochemical compounds to inhibit the interaction of spike-protein sars-cov-2 virus and ace-2 receptor for covid-19 therapy (literature review) david christian ferdinand *, & lanny hartanti widya mandala catholic university, surabaya, indonesia introduction sars-cov-2 is a new virus that was discovered in december 2020 (huang et al., 2020). the first infection case was reported in several patients in wuhan, hubei province, china with symptoms like pneumonia. the sars-cov-2 virus can spread rapidly (helmy et al., 2020). the transmission of sars-cov-2 virus into human body can cause coronavirus diseases 2019 or covid-19 (bourgonje et al., 2020). the common symptoms of covid-19 are cough, fever, shortness of breath and also death caused by pneumonia (tosepu et al., 2020). nowadays, more than 200 countries were infected by the sars-cov-2 virus (helmy et al., 2020). due to these problems, covid-19 has become a global pandemic cases (asyary and veruswati, 2020). open access international journal of applied biology keyword sars-cov-2, ace2, spike protein, nutraceutical, phytochemical article history received february 1, 2023 accepted june 21, 2023 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 7(1), 2023 71 there is no specific antiviral for sars-cov-2 virus. so, the drug development that specific to this virus is urgently needed to overcome this pandemic (ansori et al., 2020). in order to develop covid-19 drugs, we must understand the life cycles of sars-cov2 virus (saxena, 2020). the initial process of sars-cov-2 virus infection is the interaction between spike protein and ace2 receptor that make this virus enter the cells (jeong et al., 2020). the interaction affinity between the sars-cov-2 virus spike protein and the ace2 receptor is very high (cohen et al., 2020). thus, sars-cov-2 virus spike protein is a major key in the development of vaccines and antiviral drugs (ansori et al., 2020). unfortunately, sarscov-2 virus is composed of a single stranded rna that makes the process of rna -proof reading occur incompletely that led to the mutation (helmy et al., 2020). based on the article published by ecdc (2020), it was explained that the presence of the sars-cov-2 virus mutation in the united kingdom could affect the protein spike. the mutation process occurs due to deletions in amino acids p681h, a570d, s982a, d1118h, t7161, 114, n501y, d614g, and deletions in amino acids 69-70 can increase the transmission power of the virus up to 70% that cause the cellular infection process also increases. based on previous research conducted by khan et al (2020), there are 13 different sars-cov-2 mutant viruses in several countries including italy, australia, finland, south korea, china, nepal, vietnam, brazil, japan, india, us, taiwan and sweden. that study found that the mutation process can cause changes to the sars-cov-2 spike protein, especially in virus mutants originating from australia (247 position, serine → arginine), south korea (221 position, serine → tryptophan), india (408 position, arginine → isoleucine), finla nd (49 position, histidine → tyrosine), and sweden (797 position, phenylalanine → cysteine). the presence of mutations can increase the stability of the rbd-spike protein. ansori et al (2020), in their research carried out the process of identifying the genome sequences of each type of mutant sars-cov-2 virus in indonesia by accessing the genbank database and gisaid epicov. the results of the identification of genomic sequences showed that 10 mutant types of sars-cov-2 virus were found in indonesia including the main virus (wuhan-hu-1), jkt-eijk0141, jkt-eijk0317, jkt-eijk2444, ej-itd853sp, ej-itd3590nt, jkt eijk01, jkt-eijk02, jkt-eijk03, and jkt-eijk04. the results of each viral genome sequence will determine the site of mutation both in the nucleotide a nd amino acid sections. the results showed that each type of virus mutant had a similarity level of 99.9% -100%. the study explained that the mutation did not significantly affect the spike protein activity so that the protein spike was the main target site in the development of vaccines and drugs for covid19. therefore, spike protein was chosen as the target site because the mutation only affects the stability of the rbd-spike and does not significantly affect changes in the target site of the vaccine or drug candidate. several of the drugs that are used for covid-19 therapy are chloroquine (cq) and hydroxychloroquine (hcq). these drugs can inhibit the interaction between spike protein and ace2 receptors (sinha and balayla, 2020). the mechanism of action of cq and hcq is increasing the ph (alkalization) of the endosomes and lysosomes of the cells so that the sars international journal of applied biology, 7(1), 2023 72 cov-2 virus cannot fuse into the cell membrane. in addition, cq can also interfere with the glycosylation process of cell receptors (drozdal et al., 2020). unfortunately, cq and its derivatives have side effects such as nausea, vomiting, diarrhea, headaches, dizziness, seizures, disturbances of taste and smell, and tinnitus. in long-term use, cq and hcq can cause retinopathy and cardiopathy due to the toxic effects of these drugs (zou et al., 2020). therefore, it is necessary to have an alternative therapy. nutraceutical is a type of therapy used to prevent and treat chronic diseases such as diabetes, heart disease, neurodegenerative, respiratory di sorders, and so on. nutraceutical is a product developed from a variety of processed ingredients including minerals, foodstuffs, herbs, and natural derivatives based on very strict specification requirements (cgmp) to ensure the effectiveness of therapy (pathak, 2010). the advantages of nutraceuticals are relatively more economical and safer costs due to its lower side effects compared to synthetic drugs (gil et al., 2016). in addition, the use of nutraceuticals during a pandemic also has an important role in maintaining body health and preventing the transmission of the sars-cov2 virus (matteo et al., 2020). nutraceuticals also contains phytochemical components that can significantly provide benefits for the health of the body (sharma, prakash, and gupta, 2014). therefore, this research will conduct a literature review process related to the use of nutraceuticals and phytochemical components that can inhibit the interaction between the sars-cov-2 virus spike-protein envelope and the ace2 receptor for covid-19 therapy. materials and methods literature search in this study, the literature search was conducted by using several search engines: google scholar (https://scholar.google.com), science direct (http://www.sciencedirect.com/), and pubmed (http://www.ncbi.nlm.nih.gov/pubmed). the literature search process was done using several of keywords combination: [“covid-19”, “sars-cov-2”, “ace2”, “spike protein”, “nutraceutical”] and [“covid-19”, “sars-cov-2”, “ace2”, “spike protein”, “plant”] and [“covid-19”, “sars-cov-2”, “ace2”, “spike protein”, “food”]. the search process was occurred in september and october 2020. the filters of literature search were: years (2020) and language (english). screening literature the literature for searching process was then screened to eliminate duplication. then, the literature was screened through several criteria of inclusion: international journal; english language; and article types (research or review). also, this study used the exclusion criter ia: indonesian language, article type: commentary, news, opinion, or letter. the results of all these processes were documented carefully both its included and excluded data. eligibility study the eligibility study was conducted by screening the article d eeply from its abstract, methodology, and results. then, the literature was screened based on the relevance of data article and research topic. the excluded articles are documented with a reasons. based on article by (sharma, prakash, and gupta, 2014), it is explained that nutraceutical is a food https://scholar.google.com/ http://www.sciencedirect.com/ http://www.ncbi.nlm.nih.gov/pubmed international journal of applied biology, 7(1), 2023 73 component that used to prevent and treat various kinds of chronic diseases. but, generally food ingredients also contain phytochemical compounds that significantly provide health benefits. therefore, the process of inclusion data also carried both on nutraceutical and phytochemical with the consideration that covid-19 is an urgent case. inclusion data the inclusion data from every literature process was reported briefly into a prisma diagram. then the final data was also reported into table of summary. data articles with a year of publication less than 2020 were also be used for supporting reference in the discussion section. results and discussion the results of the literature review process are systematically reported in the prisma flowchart and the number of articles accepted and rejected is recorded. the presentation of article data from a systematic study can be seen in figure 1. figure 1. the results of literature review reported on prisma diagram from the results of the literature review, it was found that various types of nutraceuticals and phytochemicals that can be used for covid-19 therapy can be explained as follows. phytochemical alkaloids based on in silico research conducted by gyebi et al (2020), the following results were obtained: cryptopyrolepine (-10.7 kcal/mol), 10-hydroxysambaresine (-10.4 kcal/mol), and strychnopentamine (-9,9 kcal/mol). besides, the study also explained that three alkaloid components were effective in binding ace-rbd with their respective bond energies: -10.7 international journal of applied biology, 7(1), 2023 74 kcal/mol; -10.5 kcal/mol; and -10.5 kcal/mol (gyebi et al., 2020). other components such as bis-benzylisoquinoline alkaloids-tetrandine from the stephaniae tetrandae radix plant can also suppress spike protein (xian et al., 2020). amarogentin based on research conducted by maurya et al. (2020), it was found that ag compounds can bind to spike glycoproteins very well seen from the dockin g score value of 149.76 kcal/mol where this component can form 9 hydrogen bonds in the residue amino acids gln314, ser735, val736, cys738, thr739, arg765, and thr768. andrographolide (agp) andrographolide (agp) is a type of phytochemical component commonl y found in andrographis paniculata plants (murugan et al., 2020). agp has a broad spectrum antiviral effect because it can inhibit spike protein (-6.1 kcal/mol) and ace2 (-6.8 kcal/mol) from the sars-cov-2 virus (huang et al., 2020). artemisinin and its derivatives the molecular docking test of the artemisinin compound and its derivatives against the sars-cov-2 virus-rbd spike compared to the hcq drug was carried out using autodock vina software. the results explained that artemisinin and its derivatives a re more effective when compared to the hcq drug, which has a vina score of -5.5 kcal/mol (sehailia and chemat, 2020). baicalin and scutellarin based on a review article by muchtaridi et al (2020) and verma et al (2020), it is explained that baicalin is a phytochemical component contained in the scutellaria baicalensis which has an ic50 value of ace2 activity of 2.24 mm. the inhibition of ace2 by baicalin and scutellarin can prevent the sars-cov-2 virus infection process (yang et al., 2020). a review article by huang et al (2020) explained that baicalin could inhibit the activity of the sars-cov virus at ec50 by 12.5 μg/ml. baicalin effectively inhibits ace2 (in vitro study) seen from the ic50 parameter of 2.24 mm. in a review article by khare et al (2020), it is also stated that baicalin and scutellarin can inhibit ace2 as seen from the docking score parameter in the in silico test results. berbamine berbamine is an alkaloid component that is found in many berberis amurensis plants (cao et al., 2017). based on the latest research conducted by balmeh et al (2020), a molecular docking test process was carried out using autodock vina software from 30 phytochemical components against the target protein of the sars-cov-2 virus. phytochemical components that have the potential to inhibit ace2 activity: berbamine (-12.3 kcal/mol), naringin (-8.6 kcal/mol), and officinatrione (-8.4 kcal/mol). berbamine is the most effective compound, can penetrate bbb, and is non-carcinogenic with the recommended dose for humans of 0.0064 mmol/kgbw/day carbazole, murrayquinnon, and murrayyanine carbazole is an alkaloid class compound that can bind to various receptors on viral international journal of applied biology, 7(1), 2023 75 enzymes, including hiv, hsv, hcv, hpv, and hcmv (caruso et al., 2019). a review article by gupta et al (2020) explained that the carbazole component (-5.11 kcal/mol) had better spike protein inhibition effectiveness than the murrayquinone-a component (-4.696 kcal/mol), and murrayanine (-4.502 kcal/mol). emodin emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a phytochemical compound in the antraquinone class. emodin has many properties such as antiviral, antibacterial, anti inflammatory, and anticancer (dellafiora et al., 2020). based on previous resear ch, it was found that emodin was effective in inhibiting the interaction between the sars -cov virus spike protein and the ace2 receptor (xian et al., 2020; fuzimoto and isidoro, 2020; mirzaie et al., 2020; yang et al., 2020; jahan and onay, 2020; mckee et al., 2020; silveira et al., 2020). based on the in vitro test results of the rheum sp. and polygonum sp. using the luciferase assay method with extract concentrations of 0, 10, 50, 100, 200, and 400 μm respectively showed that emodin compounds had the effectiveness of inhibiting spike protein interactions with ace2 in sars-cov virus with ic50 = 200 μm (bernarba and pandiella, 2020). meanwhile, based on an article review by muchtaridi et al (2020), it was also explained that the emodin contained in rheum officinale and reynoutria multiflora had effectiveness against ace2 seen from in vitro test results with ic 50 parameters of 1-10 μm / ml and 1-10 μm / ml, respectively. therefore, it is necessary to have further research on the potential test of emodin against the sars-cov-2 virus. epigallocatechin-gallate (egcg), epicatechin gallate (ecg), and 3-galloylcatechin epigallocatechin-gallate (egcg) is a component of the significant polyphenol class compounds in the camellia sinensis (l.) (mhatre et al., 2020). based on the in silico test results, epigallocatechin gallate effectively inhibits the activity of the sars-cov-2 virus spike protein from preventing virus entry (jahan and onay, 2020). compound (-)-epicatechin gallate has a high affinity for spike-rbd sars-cov-2 virus. compound (-)-epicatechin gallate can interact with the amino acid residues of phe486 and leu455 in spike-rbd (weak interaction). meanwhile, a strong interaction is formed on the amino acid arg403 residue. ( -)-epicatechin gallate has an ld50 value of 2.558 mol/kgbw (rat) so it is relatively safe for consumption (istifli et al., 2020). based on research conducted by subbaiyan et al. (2020) by conducting molecular docking testing of components of several types of phytochemicals against the sars -cov-2 virus spike protein using the igemdock version 2.1 analysis software, it was found that egcg was the most effective component (total bond energy = -130.556 kcal/mol) compared to the other components such as: curcumin (total bond energy = -115.198 kcal/mol), ajoene (total bond energy = -74.2819 kcal/mol), allicin (total bond energy = -62.4326 kcal/mol), aloe emedin (total bond energy = -69.2503 kcal/mol), apigenin (total bond energy = -108.614 kcal/mol), chrysophanol (total bond energy = -107.385 kcal/mol), diallyltrisulfide (total bond energy = -53.2872 kcal/mol), ursolic acid (total bond energy = -89.9499 kcal/mol), and zingerone (total bond energy = -102.184 kcal/mol). international journal of applied biology, 7(1), 2023 76 fisetin fisetin (3,3', 4', 7-tetrahydroxyflavone) is a flavonoid class compound found in various types of vegetables and fruits with 2-160 μg/g (khan et al., 2013). based on research conducted by pandey et al (2020), it was found that fisetin (-8.5 kcal/mol) is the most effective compared to other components such as pterostilbene (-6.7 kcal/mol), isorhamnetin (-8.3 kcal/mol), genistein (-8.2 kcal/mol), luteolin (-8.2 kcal/mol), apigenin (-7.7 kcal/mol), and hcq drug (-5.6 kcal/mol). fisetin can interact with the s1 and s2 domains on the sars-cov-2 virus spike protein and potentially inhibits cell entry (oladele et al., 2020). gingerenone and zingiberene based on previous research by chakotiya a nd sharma (2020), the results of the molecular docking test between the ginger bioactive compounds against the spike protein are as follows: gingerenone (-4.21 kcal/mol), zingiberene (-6.23 kcal/mol), zingerone (-5.96 kcal/mol), shoagol (-3.93 kcal/mol), 1-dehydro6gingerdione (-6.02 kcal/mol), and gingerole (5.16 kcal/mol). from the results of this study, it was explained that the bioactive components of gingerenone and zingiberene were very effective in inhibiting the interaction between spike protein and ace2 in the cell entry process. besides being able to inhibit the cell entry process, ginger can also boost the human immune system. therefore, ginger ( zingiber officinale) is highly recommended for covid-19 therapy (chakotiya and sharma, 2020). hesperidin and its derivatives hesperidin (hesperetin 7-routineoside) is a glycoside compound composed of hesperetin (aglycone) and routineose (glycone). based on the latest research, it was found that the components of the hesperidin compound and several types of citrus flavonoids can bind to the sars-cov-2 virus protein. the presence of an aglycone group, hesperetin, causes this compound to be able to bind to the ace2 receptor (verma et al., 2020; yang et al., 2020; meneguzzo et al., 2020). hesperidin is potential to inhibit h-ace2 with a docking score of 8,111 kcal/mol. hesperidin can form hydrogen bonds in gln102, asp350, asp382, tyr385, leu391, ala484, lys562 and asp206, glu398, lys562 on ace2 protein homologs (gupta et al., 2020). based on some research literatures, it was explained that naringin, hesperitin, and naringenin compounds can strongly bind rbd-ace2 receptors. naringin is a flavonoid class compound found in tcm from exocarpium citri grandis (meneguzzo et al., 2020). based on in silico research conducted by basu et al (2020), using the cluspro software program, it was found that hesperidin (∆g = -8.99 kcal/mol) was the most effective component compared to emodin (∆g = -6.19 kcal/mol), anthraquinone (∆g = -6.15 kcal/mol), and rhein (∆g = -8.73 kcal/mol), chrysin (∆g = -6.87 kcal/mol). in addition, it was also explained that hesperidin was more effective than hcq (∆g = -7.82 kcal/mol). kaempferol kaemferol compounds can be found in brassica oleraceavar. kaemferol has good oral bioavailability and excellent binding capacity to spike proteins (wondkum and mohhamed, 2020). kaempferol was able to cause tmprss2 to downregulate (49.14-79.48%) at concentrations of 5 and 15 μm. tmprss2 is a transmembrane serine enzyme that plays a role international journal of applied biology, 7(1), 2023 77 in the interaction process between spike protein and ace2 (bernarba and pandiella, 2020). besides, kaempferol is also able to bind the c-terminal atom in subunit 1 (s1) of the sarscov-2 virus spike protein with a binding energy of -7.4 kcal/mol better than hydroxychloroquine (-5.6 kcal/mol) (pandey et al., 2020). curcumin curcumin is a polyphenol compound that is found in many curcuma longa (turmeric) (babaei et al., 2020). based on research conducted by emirik (2020), it was explained that the bioactive compounds of turmeric are effective in inhibiting the activity of sars-cov-2 spike protein. silveira et al (2020) also explained that curcumin could increase ace2 expression. curcumin can inhibit the virus entry process by changing the surface of the viral envelope protein structure so that the entry process is blocked (zahedipour et al., 2020). a review article by paraiso et al (2020) explained that the polyphenolic compounds from curcuma spp. (curcumin and its derivatives) are effective in inhibiting the spike protein sars -cov-2 compared to the drug nafamostat. curcumin (-7.8 kcal/mol) can also bind h-ace2 with the type of interaction π → σ. a review article by soni et al (2020) explained that curcumin could bind to the sars-cov-2 virus spike protein, especially in subunit 1. essential oils essential oils are volatile components from natural ingredients that are lipophilic ( asif et al., 2020). based on in silico research, the essential oil components contained in the ammoides verticillata plant are known to have effectiveness against ace2 seen from the docking score: isothymol (-4.4067 kcal/mol), limonene (-4.4067 kcal/mol), p-cymene (-4.3630 kcal/mol), and γ-terpinene (-4.2320 kcal/mol). the isothymol component has the best effect than other essential oil components (abdelli et al., 2020). besides, according to reserach conducted by boukhatem and zerser (2020), it was explained that aromatic plants that can be utilized for covid-19 therapy because they have inhibitory power against ace2 activity include: lycoris radiate (ec50 = 2.4 ± 0.2 μg/ml), artemisia annua (ec50 = 34.5 ± 2.6 μg/ml), pyrrosia lingua (ec50 = 43.2 ± 14.1 μg/ml), lindera aggregate, saikosaponins b2 (ec50 = 1.7 ± 0.1 μm/l), rheum officinale (ec50 = 1-10 μg/ml), and polygonum multiforum (ec50 = 1-10 μg/ml). kulkarni et al (2020) carried out a molecular docking test by using the autodock vina. the results indicate that bioactive compounds such as as anathole (-5.0 kcal/mol), cinnamaldehyde (-4.3 kcal/mol), carvacrol (-5.2 kcal/mol), geraniol (-5.0 kcal/mol), cinnamyl acetate (-5.2 kcal/mol), l-4-terpineol (-5.1 kcal/mol), thymol (-5.4 kcal/mol) and pulegone (5.4 kcal/mol) are effective in inhibiting the sars-cov virus spike protein. nicotianamine nicotianamine (na) is a type of non-peptide trimer amino acid that is found in many higher plants (takada et al., 2019). na has a vital role in maintaining the body's balance system (homeostasis) by binding to divalent metal ions such as fe 2+, cu2+, ni2+, mn2+, and zn2+ (bonneau et al., 2016). based on a review article by verma et al (2020) and yang et al (2020), nicotianamine components have the potential to bind ace2 in the sars -cov-2 virus. luteolin international journal of applied biology, 7(1), 2023 78 luteolin (3,4,5,7-tetrahydroxy flavone) is a component of the flavonoid class of compounds which are found in leeks, carrots, celery, parsley, broccoli, and chrysanthemums (imran et al., 2019). luteolin can bind to the surface of the sars-cov virus spike protein (fuzimoto and isidoro, 2020; yang et al., 2020; singh et al., 2020). luteolin is effective in inhibiting the sars-cov virus spike protein with an ec50 of 10 μm (paraiso et al., 2020). a review article by oladele et al (2020) also explained that the luteolin component can interact with the sars-cov-2 virus spike protein, especially in the s1 and s2 domains so that it has the potential to inhibit the cell entry process. proanthocyanidin proanthocyanidin is a type of secondary metabolite composition of the condensed tannin class which is found in many fruits, nuts, flowers and seeds (rauf et al., 2019). based on research conducted by maroli et al (2020) who tested the procyanidin -a component against ace2 and the sars-cov-2 virus spike protein, it was found that the energy of the procyanidin binding to ace2 and spike protein were respectively: -50.21 ± 6, 3 kcal/mol and 23.06 ± 4.39 kcal/mol. the presence of a bond between procyanidin and the sars-cov-2 virus protein can cause changes in the virus structure so that the infection cycle is disrupted. based on research conducted by iheagwam and rotimi (2020), it was fo und that the proanthocyanidin b1 component had the binding power to spike-rbd (-6.4 kcal/mol); ace2 (9.7 kcal/mol). quercetin quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one) is a flavonoid class compound that is found in many vegetables and fruits (batiha et al., 2020). based on research conducted by pandey et al (2020), it was found that quercetin compounds were effective in binding the sars-cov-2 virus spike protein, especially in the subunit 2 (s2) section with a bond energy of -8.5 kcal/mol. the energy of the quercetin bond is better than that of hcq at -5.6 kcal/mol (pandey et al., 2020). quercetin can bind to the spike protein and ace2 in the sars-cov-2 virus (mrityunjaya et al., 2020). based on in silico research by sen et al (2020), the results showed that the quercetin component had bond energy of -5.88 kcal/mol and affinity energy of -6.4 kcal/mol and was more effective than the drug arbidol. vijayakumar et al (2020) carried out an in silico testing process of flavonoid compounds against the sars-cov-2 spike protein. these studies indicate that the compound quercetin (7.8 kcal/mol) is the most potent component. quercetin can bind the residue chains of gly496, asn501, tyr505, and tyr453 from the sars-cov-2 spike protein. based on the docking score results, this study recommends that quercetin be subjected to further testing both in vitro and in vivo. kiran et al (2020) carried out a molecular docking testing and the results of this study indicate that quercetin (lf score = -10,227) is the most effective compound when compared to chrysoeriol (lf score = -9.035), luteolin (lf score = -9.228). these three components are useful in binding the sars-cov-2 virus spike protein. resveratrol resveratrol (3,4',5-trihydroxystilbene) is a polyphenol compound in the stilbenoid international journal of applied biology, 7(1), 2023 79 group (shaito et al., 2020). resveratrol mechanism is prevent the sars-cov-2 virus from entering the body's cells is a natural compound in the polyphenol group (ferreira et al., 2020). based on research by pandey et al (2020), the results showed that resveratrol was effective in binding the sars-cov-2 virus spike protein, especially in the subunit 2 (s2) section and the effectiveness of the bond energy (-7.9 kcal/mol) was better than hcq (-5.6 kcal/mol). a review article by quiles et al (2020) explained that resveratrol at a dose of 50 mg/kgbw can increase ace2 levels so that it actively competes with sars-cov-2 in occupying the receptors. resveratrol can interact with s1 and s2 domains on the sars-cov-2 virus spike protein and has the potential to inhibit cell entry (oladele et al., 2020). saikosaponin saikosaponin is one type of bioactive component found in bupleurum spp., scrophularia scorodonia, and heteromorpha spp. (sinha et al., 2020b). saikosaponin b2 effectively inhibits viral attachment and penetration by interfering with the spike protein activity (xian et al., 2020). sinha et al (2020b), conducted a molecular docking test of the saikosaponin against the sars-cov-2. the results showed that 5 types of saikosaponin were selected with the highest docking score: saikosaponin v (-8.299 kcal/mol); saikosaponin u (8.429 kcal/mol); saikosaponin c (-7.274 kcal/mol); saikosaponin k (-6.251 kcal/mol); and saikosaponin 1b (-6.195 kcal/mol). saikosaponin u and v are the most recommended because they have the best effectiveness in the sars-cov-2 spike protein. terpenes based on research conducted by muhseen et al (2002), showed that terpene compounds such as: npact01552, npact01557 and glycyrrhizin had bond energies: -11 kcal/mol, 10.3 kcal/mol, and -9.5 kcal/mol (the three components with the best docking results). the three components are components of the terpenes found in trevesia palmata, aralia dasyphylla, and glycyrrizha glabra. these results explained that the three components could inhibit the spike protein interaction with ace2. theaflavine (tf) a review article by mhatre et al (2020) explained that theaflavins (tf) and theaflavine3 (tf3) were effective in inhibiting the interaction between the sars-cov-2 virus spike protein and the ace2 receptor seen from the results of a literature review in an in silico study. theaflavins (tf) has hydrophobic interactions and binds to the residues of arg454, phe456, asn460, cys480, gln493, asn501, and val503 with ∆g values of -8.53 kcal/mol. bioactive components in the asparagus racemosus plant based on research by chikhale et al (2020b), the molecular docking test was carried out to see the interaction between the phytochemical components of the asparagus racemosus (willd.) plant against the sars-cov-2 virus spike protein. the results showed that the components of asparoside-c (-7.165 kcal/mol), asparoside-d (-6.445 kcal/mol), and asparoside-f (-6.615 kcal/mol) were the most effective in inhibiting rbd protein spi kes seen from the lowest docking score. international journal of applied biology, 7(1), 2023 80 bioactive components in solanum tuberosum and brassica juncea plants based on research conducted by dave et al (2020), by testing the interaction between the phytochemical components contained in the solanum tuberosum and brassica juncea plants in silico. the results was found that only three phytochemical components had the potential to bind the sars virus spike protein : curcumenol (∆g = -6.43 kcal/mol), ndesmethylselegiline (∆g = -6.28 kcal/mol), and phentermine (∆g = -6.25 kcal/mol). bioactive components in cinnamomum sp. in silico test results of the cinnamon component against the sars-cov-2 virus spike protein found that the pavetannin c1 component (-11.1 kcal/mol) has the potential to inhibit spike protein (prasanth et al., 2020). bioactive components in clerodendrum sp. based on the research conducted by kar et al (2020), showed that the taraxerol (rbdspike = -45.19 kcal/mol;) is the most potent when compared to the friedelin (rbd-spike = 42.22 kcal/mol) and stigmasterol (rbd-spike = -41.25 kcal/mol). bioactive components in the glycyrrhiza glabra plant the molecular docking test was carried out by sinha et al., 2020a to see any interactions between the bioactive components against the target protein. the results showed that glycyrrhizic acid is the best component in in hibiting the sars-cov-2 virus spike protein (sinha et al., 2020a). based on research conducted by vardhan and sahoo (2020), molecular docking results: maslinic acid (-9.3 kcal/mol), glycyrrhizic acid (-9.3 kcal/mol), corosolic acid (-9.4 kcal/mol), 2-hydroxyseneganolide (-9.2 kcal/mol), and oleanane (-9.0 kcal/mol) are useful in binding the sars-cov-2 virus spike protein. bioactive components from ipomoea obscura (l.) the docking test results of the phytochemical components of the ipomoea obscura (l.) showed that the deoxycholic acid was very effective in inhibiting ace2 (poochi et al., 2020). bioactive components from nigella sativa l. based on research, it was explained that phytochemical components such as nigellidine have a high affinity for the sars-cov-2 virus spike protein. meanwhile, phytochemical components such as hederagenin, thymoquinone (essential oil), thymohydroquinone have a high affinity for ace2 (kosha k and koshak, 2020). bioactive components from uncaria tomentosa (cat's claw) based on the research conducted by perez et al (2020), the best docking results from these study: proanthocyanidin c1 (-8.6 kcal/mol), qag-2 (-8.2 kcal / mol), 3isodihydrocadambine (-7.6 kcal / mol), uncarine f (-7.1 kcal / mol), and uncaric acid (-7.0 kcal / mol). in this study, it can be explained that the components of proanthocyanidin c1 are the most effective (perez et al., 2020). bioactive components from withania somnifera a research by chikhale et al (2020a), the results showed that the phytochemical components: qgrg (-9.25 kcal/mol), withanoside x (-7.07 kcal/mol), ashwagandanolide (international journal of applied biology, 7(1), 2023 81 6.50 kcal/mol), dihydrowithaferin a (-2.82 kcal/mol), and withanolide n (-0.57 kcal/mol) have the potential to inhibit the sars-cov-2 virus spike protein. based on a review article by straughn and kakar (2020), information was also presented that withaferin -a (wfa) is a lactone steroid class compound which is very useful in inhibiting the interaction between spike protein and ace2. nutraceuticals bioactive components in fungi ganoderma lucidum is a type of fungus widely used in the tcm treatment system and is commonly known as ling zhi (teekachunhatean et al., 2012). based on an article review by chinsembu (2020), it was explained that one type of fungus (basidiomycetes) that has the potential to inhibit spike protein interactions with ace2 is ganoderma lucidum (curtis) karst. these fungi contain bioactive components in the form of ganoderic acid f (triterpene compound) with an ic50 of 4.7 × 10 -6 m. lectin lectin can inhibit interactions with the ace2 receptor. lectins are found in many plants: (1) allium porrum l., urtica dioca l. and nicotiana tabacum l. (chinsembu, 2020). brown algae brown algae (ecklonia cava) contains phlorotannins, which are useful for inhib iting the sars-cov virus spike protein and isolates from these algae have received distribution authorization from the fda (chinsembu, 2020). nisin nisin is a food preservative product obtained from lactic acid in bacteria. based on the research conducted by bhattacharrya et al (2020), the values of ∆g nisin z (-10.8 kcal/mol) and nisin h (-11.3 kcal/mol) were higher than those of rbd (-11 kcal/mol). nisin h is more hydrophilic than nisin z. nisin z and nisin h are very potent in interacting with h -ace2. nisin h competitively occupies the ace2 receptor. lactoferrin lactoferrin can reduce il-6, tnf-α, and ferritin. it is also useful in inhibiting the viral entry and replication process (mrityunjaya et al., 2020). zinc supplement zinc supplement (100 μm) can decrease hace2 activity in rat lungs. the recommended dosage for zinc supplements is 20-92 mg/week (mrityunjaya et al., 2020). teicoplanin based on a review article by wang et al (2020), teicoplanin is a natural compound in the lipoglycopeptide antibiotic class isolated from the fermentation of actinoplanes teichomycetius. teicoplanin has the effect of inhibiting the virus entry process in binding to the sars-cov-2 spike protein on hek293t and huh7 cells (ic 50 = 1.66 μm). hek293t and huh7 are cell types that can express ace2. tuna peptides a research conducted by yu et al (2020) focuses on in silico testing of natural peptide international journal of applied biology, 7(1), 2023 82 components contained in tuna against ace2 enzymes in the life cycle of the sars-cov-2 virus. the results of molecular docking show that only one peptide can bind ace2, namely eeaggataaqiem (cdocker value = 144 kcal/mol). eeaggataaqiem peptide can inhibit the viral attachment process by forming six hydrogen bonds and six carbon bonds on ace2 protein residues and changing the charge by electrostatic interactions. propolis a review article by berretta et al (2020) explained that bioactive components contained in propolis such as myricetin, caffeic acid phenethyl ether, hesperetin and pinocembrin were useful in binding ace2 seen from the docki ng score. based on the in silico test results, it was found that propolis has a% inhibitory activity against ace2 by 90% with a strong bond. the best% inhibition of ace2 was found in catechins and p -coumaric acid. conclusion the utilization of nutraceutical agents can be used for covid-19 therapy as seen from the in silico test results. phytochemical components such as curcumin, emodin, quercetin, hesperidin, resveratrol, andrographolide, epigallocatechin-gallate and so on can inhibit the interaction between the sars-cov-2 virus spike protein and the ace2 receptor based on the in silico test results. other components such as nisin, peptides from tuna, propolis, and so on can also be used to inhibit the interaction between the sars-cov-2 virus spike protein and the 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the leaves of seagrass enhalus acoroides in three different coastal waters. the sampling of seagrass leaves was carried out in three locations in the coastal waters of ambon island, namely the coastal waters of galala, rutong, and waai. the levels of flavonoid were identified using microscopic-microchemical methods. the test results of the flavonoid levels were analyzed descriptively. the results of this research showed that samples of seagrass leaves from the coastal waters of galala, rutong, and waai, after added with naoh, alcl3, and nh3 reagents showed a color change. it means that the samples from the three coastal waters were positive to contain flavonoids. therefore, the analysis was continued to determine the average levels of flavonoids of e. acoroides. the average levels of flavonoid in the three coastal waters of galala, rutong, and waai were 0.0192%, 0.1475%, and 3.5697% respectively. the environmental conditions and substrate of seagrass e. acoroides in the coastal waters of rutong and waai caused higher levels of flavonoids than the levels of flavonoids in the coastal waters of galala. issn : 2580-2410 eissn : 2580-2119 analysis of flavonoid levels of enhalus acoroides in different coastal waters in ambon island, indonesia prelly m.j. tuapattinaya & dominggus rumahlatu study program of biology education, faculty of teacher training and education, pattimura university, ambon, indonesia introduction seagrass is classified into angiosperms plants which grow in marine environment (borum and greve, 2004; subhashini et al., 2013; immaculate et al., 2018 ) the diversity of seagrass species in the world reaches 70 species, 12 of which are found in indonesian waters, including cymodocea serrulata, c. rotundata, enhalus acoroides, halodule uninervis, h. pinifolia, halophilia minor, h. ovalis, h. decipiens, h. spinulosa, thalassia hemprichii, syringodium isoetifolium and thalassodendron ciliatum spreading in java, sumatra, bali, kalimantan, sulawesi, maluku, east nusa tenggara and papua (tuahatu et al., 2016; subhashini et al., 2013; tuhumury, 2008; nontji, 2005 ) on the waters of kumbang island, karimunjawa islands are found 6 species of seagrass, namely t. hemprichii, c. rotundata, c. serrulata, h pinifolia, h. uninervis and h. ovali (hartati et al., 2012). in maluku waters, 10 seagrass species were found, namely c. rotundata, c. serrulata, h. pinifolia, h. uninervis, s. open access international journal of applied biology keyword levels of flavonoids, the leaves of seagrass enhalus acoroides, different coastal waters article history received 23 may 2019 accepted 24 june 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, p-issn : 2580-2410 e-issn : 2580-2119. journal homepage : http://journal.unhas.ac.id/index.php/ijoab international journal of applied biology, 3(1), 2019 71 isoetifolium, t. ciliatum, e. acoroides, h. minor, h. ovalis, and t. hemprichii and spread on the ambon island seram island, aru islands, buru island and saparua island (irawan, 2017). ecologically, seagrass is different from terrestrial plants including the compounds they contain. different environments cause biochemical adaptations by seagrasses to produce certain compounds called bioactive compounds or secondary metabolites (owolabi et al., 2018; subhashini et al., 2013; mansson et al., 2011). the research conducted by santoso et al. (2012) found secondary metabolites of saponins, triterpene and sterols in 80% ethanol extract in 7 seagrass species. mani et al. (2012), reported that secondary metabolites was found in methanol extract of seagrass syringodium isoetifolium, such as saponins, phenols and alkaloids. many secondary metabolites from seagrass have been known to be biologically active and important biomedicine and can be used as potential drugs. the roots of e. acoroides are used as medicine for stings of various types of stingrays and scorpions. halophila sp. is a powerful drug against malaria, skin diseases, and it is found to be very effective in the early stages of leprosy (mani et al., 2012). riniatsih and setyati (2009) utilized simplicia extracts and powder of e. acoroides and t. hemprichii as agents to control vibrio bacteria. in addition, kannan et al. (2010a) reported that there were antimicrobial activities from the extracts of seagrass h. stipulacea, c. serrulata, and h. pinifolia. one of the secondary metabolites found in all parts of seagrass plants is flavonoids (baby et al., 2017; zidorn, 2016; subhashini et al., 2013). baby et al. (2017) and gustavina et al. (2018) confirmed that in addition to flavonoids, enhalus acoroides contains the compounds of triterpenoid, steroids, saponins, tannins. qi et al. (2008) explains that enhalus acoroides contains 11 pure compounds classified as flavonoids and steroids. enhalus acoroides is also reported to contain phenolic bioactive compounds which tend to be potential antioxidants (kannan et al., 2010b). based on the survey observed on the coastal waters of ambon islands, namely on the rutong, waai, and galala beaches, it was found that enhalus acoroides was used as vegetables and herbal medicines by the community. moreover, it is also known that seagrass enhalus acoroides on the rutong and waai coastal waters can grow well. this is because the coastal waters in rutong and waai are still natural and have not been polluted with harmful substances or metals. on the other hand, the number of seagrass enhalus acoroides in the coastal waters of galala is relatively small, because the waters have been polluted by factory waste and household waste from the community (rijal et al., 2014). materials and methods the collection of enhalus acoroides samples and measurement of waters condition factors the sample collection of the leaf of seagrass e. acoroides and the measurement of water condition factors (temperature, salinity, and substrate) were simultaneously carried out in three locations in the coastal waters of ambon island, namely galala, rutong, and waai coastal waters (figure 1). the identification and analysis of the flavonoids levels of the leaves of seagrass e. acoroides were carried out at the laboratorium kimia dasar (basic chemistry laboratory), faculty of mathematics and natural sciences (fmipa), pattimura university. the seagrass leave samples were intact leaves (not damaged nor defective). the extraction process of the leaves of enhalus acoroides the leaf samples of e. acoroides were cleaned from sand and salt impurities attached to the leaves using distilled water. the seagrass leaves were then dried and mashed. 70 g international journal of applied biology, 3(1), 2019 72 powder of leaf samples of e. acoroides was extracted using 400 ml of methanol 90%. the extraction process (maceration) lasted for 24 hours, which was then evaporated using vacuum rotary evaporator at a temperature of 55oc to produce a viscous extract, then followed by drying process in the desiccator until the extract in the form of paste was obtained. the identification of flavonoid levels the identification of flavonoid levels was qualitatively carried out using microscopicmicrochemical methods (mulyani and laksana, 2011). methanol extract of e. acoroides was inserted into the test tube and then identified with the addition of reagents and without reagents. the identification without reagent was carried out by adding 5 drops of aquedes to the test tube, while the identification process with reagents was carried out by adding several reagents, namely nh3 25%, naoh 40%, and alcl3 5% (5 drops each), into the tube. the use of reagents in this study with concentration nh3 25%, naoh 40%, and alcl3 5% based on the reason that these reagents can provide color when seagrass leaf extract contains flavonoids. according to mabry et al. (1970) in chang et al. (2002) that the principle of the calorimetry method is that reactants will form stable complexes with hydroxyl groups from flavones and flavonols. color changes in extracts were observed in each treatment (with and without reagents). the color before and after the reagents were given was compared, and the color changes obtained were observed. the results of the flavonoid level test were analyzed descriptively. the descriptive analysis was used to compare the results of flavonoid levels based on the three coastal waters, namely the coastal waters of galala, rutong, and waai. the quantification of flavonoid levels of the sample e. acoroides was carried out by using spectrophotometric method with quercetin as standard. the standard curve was obtained by measuring the absorbance of the standard quercetin solution at concentrations of 5, 10, 15, 20, 25 ppm. the maximum wave length determined for absorbance was 438 nm. figure 1. location for collecting samples of enhalus acoroides results and discussion the results of flavonoids identification of the leaves of seagrass enhalus acoroides the results of the identification of flavonoid content in the leaves of seagrass e. acoroides in the coastal waters of galala, rutong, and waai (table 1) showed that the extract international journal of applied biology, 3(1), 2019 73 test with aquades reagents on the three coastal water locations did not show any color changes. while the extract test with the three other types of reagents namely naoh, alcl3, and nh3 indicated color changes. table 1. results of identification of flavonoids in the leaves of seagrass enhalus enhalus sample reagent aquades naoh 40% alcl3 5% nh3 25% leaves of e. acoroides of galela coastal waters + + + leaves of e. acoroides of rutong coastal waters + + + leaves of e. acoroides of waai coastal waters + + + description: (-) = no change in color after given reagent, does not contain flavonoids (+) = there is a change in color after given reagent, containing flavonoids the reaction on extract of the leaves of seagrass e. acoroides added with naoh, alcl3 and nh3 reagents produced green changes to yellow on each test tube. thus, it could be concluded that they positively contained flavonoids. the color change in the flavonoids color test results with various reagents was caused by the effect of reactant concentration (the level of polarity of reagents / solvents) and surface area, where the concentration and surface area are related to the frequency of collisions. the greater the concentration, the greater the possibility of particles colliding with each other, so that the reaction increases faster. similarly, the wider the surface area (the size of fine pieces), the more the collision, and the faster the reaction rate. color changes that occured in the reagent treatment indicated that the samples positively contained flavonoids. the indication of the color change that occurred in this test was caused by the reaction between reagent (alcl3) and the carbonyl and hydroxyl groups on flavones and flavonols which produced yellow to brownish color, while ammonia and naoh with flavonoids would form a red quinoid compound (mulyani and laksana, 2011; popova et al., 2004; robinson, 1995). the presence of flavonoids in the test material can also be visualized by adding concentrated mg and hcl powder into the alcohol extract. it will become orange to red when it contains flavones, red to dark red (flavanols), dark red to magenta (flavonone) (fannsworth, 1966). flavonoids detected from leaf samples of e. acoroides are very important as antioxidant compounds. this is in accordance with the statement by redha (2010); santoso et al. (2012); subhashini et al. (2013), that flavonoids as a group of phenolic compounds are mostly found in plant tissues both roots, stems, and leaves, and they can act as antioxidants. in addition, the extract of e. acoroides contains bioactive compounds from the triterpenoids, steroids, saponins, tannins types (dewi et al., 2012; baby et al., 2017; gustavina et al., 2018). flavonoid levels of the seagrass leaf enhalus acoroides the flavonoid levels of the seagrass leaf enhalus acoroides (quantity) were determined by using a spectrophotometric method with quercetin as standard. the data on the standard solutions (quercetin) and the absorbance are presented in table 2. international journal of applied biology, 3(1), 2019 74 table 2. absorbance of standard quercetin solutions no concentration (ppm) absorbance (nm) 1 5 0.207 2 10 0.396 3 15 0.613 4 20 0.811 5 25 1.091 after the value of the absorbance and the standard solution was obtained, the data were visualized in the form of a graph of the quercetin standard solution concentration and the absorbance (figure 2). figure 2. quercetin standard curve; standard concentration on standard absorbance the graph of the quercetin standard curve (figure 2) shows a linear correlation between absorbance and concentration. this shows that the greater the concentration, the greater the absorbance value. after that, the results of the flavonoid level analysis of the seagrass leaf enhalus enhalus in coastal waters of galala, rutong and waai were calculated based on the linearity correlation on the standard curve and are presented in table 3. table 3. the results of the flavonoid level analysis of the seagrass leaf enhalus enhalus sample code repetition sampel weight (mg) flavonoid weight (mg) flavonoid levels (%) average levels of flavonoid (%) galala 1 70.0312 36.25 0.0192 0.0192 2 70.0617 38.75 0.0193 3 70.0615 35.75 0.0191 rutong 1 70.3821 40.00 0.0198 0.1475 2 70.5984 47.50 0.2116 3 70.5985 41.50 0.2110 waai 1 10.0516 352.5 3.5069 3.5697 2 10.0417 360.0 3.5850 3 10.0516 352.5 3.5069 the results of the highest average levels of flavonoids of seagrass leaf e. acoroides were at the coastal waters of waai, which was 3.5697, while the lowest average levels of flavonoid were at the coastal waters of galala, which was 0.0192%. this occured due to the differences in the substrate that became the growing place of seagrass on the three beaches. the substrate in the waai and rutong beaches was in the form of sandy mud, while the substrate in the galala beach was coarse sand and household waste from the community that y = 0.0437x 0.0313 r² = 0.9948 0 0.2 0.4 0.6 0.8 1 1.2 0 5 10 15 20 25 30 a bs or ba nc e concentration international journal of applied biology, 3(1), 2019 75 affected the growth rate of seagrass e. acoroides. this is in accordance with the research results by rahman et al. (2016) that the water conditions which has the most significant effect on the growth rarte of seagrass leaf were sandy mud substrates. leaves with a good growth rate will affect the compounds contained in the leaves. flavonoids have varying forms and are in free form (aglycone). they are also bound as glycosides and generally have bonds with sugar groups, so that flavonoids are easily soluble in polar solvents or water (corradini et al., 2011). the presence of flavonoid chemical compounds in the extract of seagrass leaf e. acoroides in this research shows that seagrass e. acoroides has the potential to be a natural chemical antifouling, antibacterial, antifungal, and other pharmaceutical raw materials (dewi et al., 2018). table 4. results of the measurements of water condition factors sampel code repetition water condition factors temperature (0c) salinity (%o) substrate galala 1 28.29 28 coarse sand 2 28.31 28 coarse sand 3 28.30 28 coarse sand average 28.30 28 rutong 1 29.67 30 muddy sand 2 29.67 30 muddy sand 3 29.66 30 muddy sand average 29.67 30 waai 1 29.66 29 muddy sand 2 29.67 29 muddy sand 3 29.67 29 muddy sand average 29.67 29 the results of the measurements of the physical condition of the coastal waters (table 4) of galala, rutong and waai covering temperature and salinity show an average range of 28.30oc-29.67oc and 28-30%o. it is a condition that seagrass e. acoroides can tolerate for life. according to erftemeijer and middelburg (1993), e. acoroides lives at a temperature of 26.5oc320c in shallow waters and can even tolerate temperatures up to 380c during low tide during the day. hutomo (1999) revealed that the optimum range of tolerance to sea water salinity was 35%o. the results of this research (table 3) show that the highest levels of flavonoids of the seagrass leaf e. acoroides were in waai coastal waters, which was 3.5697%, while the lowest levels of flavonoids of the seagrass leaf were at galala coastal waters of 0.0193%. differences in flavonoid levels in plants can be caused by differences in environmental conditions where these plants grow, temperature, ultraviolet light, nutrient availability, water and co2 levels in the atmosphere (rohaeti et al., 2011). the results of measurements of water condition factors (table 4) show that the environmental conditions where seagrass e. acoroides grows in rutong and waai villages are almost the same. the environmental conditions are muddy sand substrate, and the water has not been polluted by human activities. however, the environmental conditions in the galala village are different, coarse sand substrate with a mixture of waste and the water conditions with high human activities, namely the activities of boats and factories operating around the waters, waste from ships, factories and communities. international journal of applied biology, 3(1), 2019 76 rumahlatu et al. (2018) reported that the levels of heavy metal cd in the coastal waters of galala village ranged from 0.35-1.15 ppm for sediments and 0.10-0.35 ppm for water, while the levels of heavy metal pb in the coastal waters of galala village ranged from 1.05-1.27 ppm for sediment and 0.01 ppm for water. in addition, rizal (2010) reported that the highest levels of bioaccumulation of lead (pb) and cadmium (cd) were found in seagrass leaves. alkaline pb and cd have an effect on the flavonoid levels. this is due to the chemical nature of flavonoids which are acidic, so they can dissolve in bases (singh et al., 2003). the high content of pb and cd found in the leaves of seagrass enhalus acoroides causes the flavonoids content to be dissolved in bases thereby reducing flavonoid levels in the leaves of seagrass enhalus acoroides which grow in the coastal waters of galala village. the environmental conditions and substrate of seagrass enhalus acoroides in the coastal waters of rutong and waai caused flavonoid levels to be higher than those in the coastal waters of galala. muddy sand substrate in both waters is a suitable substrate for the growth of seagrass enhalus acoroides. erftemeijer and middelburg (1993) explain that the rates of the leaf growth and the production of seagrass enhalus acoroides are higher on muddy sand substrates (terigenous sediments) than on other types of substrate, because muddy sand substrates generally have higher n and p nutrient availability. even though the conditions of the water and the growing substrate of seagrass enhalus acoroides in the coastal waters of rutong and waai were almost the same, the results of flavonoid level teasting on the leaves of seagrass enhalus acoroides from the coastal waters of waai were higher than that in the coastal waters of rutong. this is influenced by the availability of nutrition in the two waters. the research conducted by soedradjad & syamsunihar (2014) reported that the phenolic and flavonoids content of plants increase along with the increasing doses of organic fertilizer in the growing media. high fertilizer dosages will support the availability and absorption of more nitrogen elements, so that the phenolic and flavonoids content of plants can increase. the important elements needed by seagrass are nitrogen (n), phosphate (p) and organic c. nitrate (no3) is the main form of nitrogen in natural waters and is the main nutrient in seagrass ecosystems (kilminster et al., 2006; effendi, 2003). amri et al. (2011) affirm that the nutrient composition of n, p, and s of a waters is strongly influenced by the anthropogenic activity. according to philips and menez in badria (2007), seagrass growth comes from recycling nitrogen in sediments and water columns. dead rizoma and seagrass roots add nitrate in the sendimen. the differences in the flavonoid levels in the coastal waters of waai and rutong were due to the differences in nitrogen levels. conclusions enhalus acoroides in the coastal waters of galala, rutong, and waai contain flavonoids. the average flavonoid levels in the three coastal waters were 0.0192%, 0.1475%, and 3.5697% respectively. in addition, the levels of flavonoids in the enhalus acoroides in the research location were influenced by the differences in environmental conditions and water substrate. the average levels of condition factors of temperature and salinity were in the range of 28.30oc-29.67oc dan 28-30%o with the coarse sand substrate conditions in the coastal waters of galala, while muddy sand substrate condition in the coastal waters of rutong and waai. the differences in environmental conditions and substrate conditions for the life of enhalus acoroides in the coastal waters of rutong and waai cause the flavonoid levels to be higher than the flavonoid levels in the coastal waters of galala. international journal of applied biology, 3(1), 2019 77 acknowledgement the author would like to thank the dean faculty of teacher training and education, pattimura university, which has provided research funding through grants for study programs, year 2018. references amri, k., setiadi, d., qayim, i., & djokosetiyanto, d. 2012. nutrient content of seagrasss enhalus acoroides leaves in barranglompo and bonebatang islands: implication to increased antrhropogenic pressure. indonesian journal of marine sciences, 16(4): 181186. doi: 10.14710/ik.ijms.16.4.181-186. baby, l., sankar, t.v., & chandramohanakumar, n. 2017. changes in phenolic compounds in seagrasses against changes in the ecosystem. journal of pharmacognosy and phytochemistry, 6(3): 742-747. badria, s. 2007. laju pertumbuhan daun lamun enhalus acoroides pada dua substrat berbeda di teluk banten [the growth rate of seagrass leaf enhalus acoroides in two different substrates in banten bay]. thesis. marine science and technology study program. fishery and marine science faculty. institut pertanian bogor. 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(2016), consumers reject star-fruits that were kept at room temperature after ten days due to a decline in quality and freshness. the innovation of star fruit products is developed to extend star fruit shelf life and retain star fruit freshness as well as increasing added value. sorbet can be used as one alternative of star fruit innovation product because sorbet is a processed product that is kept in low temperature, thus can extend shelf life. according open access international journal of applied biology keyword antioxidant activity, hedonic test, organoleptic test. article history received 24 january 2020 accepted 24 february 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:inanpihs@trilogi.ac.id international journal of applied biology, 4(1), 2020 2 to sayuti and yenrina (2015), vitamin c oxidation would be limited when vitamin c is kept in cold condition or at low temperature, around 8 oc. sorbet has a similar texture to ice cream, and this can increase its appeal to consumers to buy star fruit products. sorbet also has advantages such as low-fat content, contains natural fruit fiber, and high in vitamin c (sakawulan et al. 2014). the advantage of sorbet also opens up opportunities for sorbet to become functional food that relatively inexpensive and tasty (silalahi et al. 2014). natural additional ingredients that can be used to improve star fruit sorbet taste and add acidity are sugar and lime juice. lime juice addition is a right combination for star fruit sorbet because lime has a refreshing sour taste that can improve the overall taste as well as increase its nutrition value. vitamin c content of lime is quite high, around 29.1 mg/ 100 g fruit (usda 2018). according to febrianti (2010), besides having high vitamin c content, lime also contains citric acid that can replace the use of commercial citric acid. a combination of fruits in producing sorbet had developed to improve or repair the quality of produced sorbet. a study conducted by claudia et al. (2016) showed that the addition of pineapple in yellow pumpkin sorbet could improve freshness and reduce the pungent smell. the use of sugar in sorbet is not only to add flavor but also to form a balance between sour and bitter taste in a product (putri 2016). the addition of lime and sugar can affect the physical, chemical, and organoleptic properties of star fruit sorbet. organoleptic tests using hedonic tests can represent how the product accepted by consumers. physical and chemical analysis conducted to determine and to attain the quality parameter of the sorbet. materials and methods time and place this research was conducted on june to july 2018. the research was conducted at the food process engineering laboratory of trilogi university, saraswati indo genetech (sig) laboratory, and garda dharma technology laboratory, bogor. materials the tools used in this research were oxone ox-315 scale, analytical balance kern pl420-3f, knife, spatula, oxone ox-863, sieve, gas stove rinnay exotic, ice cream maker cuisineart ice-100bchk, beaker glass iwaki cte33, ph meter phep h198107, refractometer atc, spectrophotometer shiamdzu uv 1700, and other laboratory equipment. the materials used in the production of star fruit sorbet were star fruit, cmc (carboxyl methyl cellulose), lime, sucrose sugar, dpph solution, and other materials. research method the research consisted of two phases, the first phase was the production of lime juice and the second phase was production of star fruit sorbet as the main study. the experimental design used was completely randomized factorial design (crfd) with two factors, namely concentration of sugar and the concentration of lime juice. each factor consisted of three levels of treatments, which 15 %, 20 %, and 25 % of sugar and 5 %, 10 %, and 15 % of the lime juice ratio of fruit and water. international journal of applied biology, 4(1), 2020 3 production of lime juice production of lime juice based on method developed by jurianti et al. (2010) with some modification. lime fruits were sorted and then washed with water until clean. lime fruits were cut into two parts transversely using a knife and then squeezed using a citrus juice extractor. the juice then strained using sieve cloth. lime juice then stored in a bottle. the modification of the process made in the lime had not turned into syrup and only extracting the juice. production of star fruit sorbet production of star fruit sorbet based on method developed by rahmawati et al. (2010) with some modification. star-fruits were sorted following criteria and washed until clean and then blanched. blanching done to activate enzyme by steaming star-fruits for 5 – 10 minutes at 70 – 80 oc and then directly followed by cooling star-fruits in the refrigerator to bring the temperature down. star-fruits then peeled and its pulp extracted, and then blended with water with 1:1 ratio of pulp: water. star fruit pulp mixture then mixed with stabilizer, sugar, and lime juice according to treatments. the next step was to freeze the mixture using an ice cream maker until sorbet formed, marked by the formation of soft ice crystal inside the mixture. star fruit sorbet then stored in the freezer to attain better texture. the modifications of the process made in the steaming time that was 3 – 5 in previous research and also in straining the pulp, which not conducted in this research. other modifications were in the aging process that was conducted in the production of star fruit sorbet and in the mixing process which was without heating the mixture. the production of star fruit sorbet in this research used star-fruits, sugar, cmc stabilizer, and lime juice. the production of sorbet was using ten formulations, and one of them was control, which without treatment factors. the formulations of star fruit sorbet shown in table 1. table 1. star fruit sorbet formulation ingredients composition (g) s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 star fruit pulp 300 300 300 300 300 300 300 300 300 300 water 300 300 300 300 300 300 300 300 300 300 sucrose sugar 0 90 120 150 90 120 150 90 120 150 cmc 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 lime juice 0 30 30 30 60 60 60 90 90 90 results and discussion organoleptic test the organoleptic test was conducted to determine panelist predilection and acceptance level on star fruit sorbet. parameters tested were color, taste, aroma, texture, and overall on star fruit sorbet products. scoring scale used in this hedonic test was 1 (one) to 5 (five). the scoring or value given by panelists captures how much the panelists like the product. the higher the score the panelists give, the higher the acceptance level of the international journal of applied biology, 4(1), 2020 4 product (setyaningsih et al. 2010). based on results from conducted hedonic test, there were three formulations with highest overall score from the panelists, which were s3 (150 g sugar + 30 g lime juice), s4 (90 g sugar + 60 gr lime juice), and s5 (120 g sugar + 60 g lime juice) with average of 3.60, 3.41, and 3.53 respectively, that were in neutral scale (table 2 and figure 1). the three best formulations then further tested against overrun, ph, sugar content, melting time, vitamin c and antioxidants, and compared to s0 as control. table 2. scoring results from the hedonic test of star fruit sorbet formulation color teste flavour texture 0verall s0 (g 0 g + jn 0 g) 3.32ab±1.24 1.76ef±0.77 2.72bc±0.84 3.16a±1.21 2.44cd±1.04 s1 (g 90 g + jn 30 g) 2.96bc±0.84 3.72a±1.02 3.68a±0.90 3.00a±0.81 3.36ab±0.81 s2 (g 120 g + jn 30 g) 2.24cd±1.12 3.88a±1.09 3.36ab±0.95 3.12a±0.78 3.40ab±0.86 s3 (g 150 g + jn 30 g) 3.27ab±1.10 3.88a±1.05 3.52a±0.77 3.36a±0.81 4.00a±0.70 s4 (g 90 g + jn 60 g) 3.88a±0.92 3.08bc±1.18 3.44ab±0.71 3.16a±0.89 3.52ab±0.91 s5 (g 120 g + jn 60 g) 3.96a±0.84 3.40ab±1.22 3.52a±0.87 3.24a±0.77 3.52ab±0.82 s6 (g 150 g + jn 60 g) 4.12a±0.88 3.36ab±1.25 3.40ab±0.81 3.24a±0.92 3.44ab±0.91 s7 (g 90 g + jn 90 g) 3.92a±1.07 2.32de±1.06 3.28ab±0.93 3.20a±0.91 2.84bc±0.85 s8 (g 120 g + jn 90 g) 3.72ab±0.93 2.32de±1.18 3.08ab±0.70 2.96a±1.13 2.84bc±1.02 s9 (g 150 g + jn 90 g) 3.27ab±1.06 2.60cd±1.25 3.40ab±0.86 2.84a±1.02 2.92bc±1.18 note: s: sugar, lj: lime juice, (1) dislike it very much, (2) dislike it a little, (3) neutral, (4) like it a little, (5) like it very much. number followed by the same notation showed no significant difference at α= 5% in tukey’s range test. figure 1. spider web of the organoleptic test of star fruit sorbet international journal of applied biology, 4(1), 2020 5 color the color of a product can affect the consumers’ acceptance level of that product. ways to retain the natural color of star fruit are by doing blanching star fruit before processing and also by adding lime juice. lime juice acts as vitamin c and citric acid source (febrianti 2010). according to wulandari (2016), citric acid can help reduce the browning reactions. scoring on panelists’ predilection of color is conducted by observing the color, and the highest score captures the color preferred by panelists (setyaningsih et al. 2010). based on color scoring for star fruit sorbet (figure 2), there was an increase of preference score in products with added lime juice compared to control. analysis of variance on color scores showed significant difference (p< 0.05), meaning that the addition of sugar and lime juice gave a significant effect in every formulation. panelists level of predilection on color parameter ranged from 2.24 to 4.12 scale of dislike a little to like a little. the most preferred formulation with a score of 4.12 was the s6 formulation with 150 g sugar + 60 g lime juice, which resulted in a bright yellow color. formulation s2 with 120 g sugar + 30 g lime juice was the formulation with the lowest score of 2.24 which resulted in a dark yellow color. according to kusumawati’s study (2008) that the higher the amount of added citric acid in star fruit juice, the brighter yellow the result is. lime juice contains around 7 % of citric acid (prastiwi & ferdiansyah 2017). formulation s6 score (table 2) followed by the same notation with control, s3, s4, s5, s7, s8, and s9, which means they were not significantly different. figure 2. the scoring result of star fruit sorbet color taste taste is a factor that can affect consumers’ predilection and acceptance level of a product. scoring results from panelists on star fruit sorbet taste shown in figure 3. addition of lime juice and sugar in star fruit sorbet affected panelists’ predilection level shown by the higher taste scores compared to control (s0). according to hidayat et al. (2017) on the making of pumpkin syrup, the addition of lime juice improves the panelists’ preference, on the syrup compared to control which without lime juice. the higher amount of added lime juice, the lower panelists’ predilection level because additional lime juice can increase sourness in star fruit product. while the addition 3.32±1.24 2.96±0.84 2.24±1.32 3.27±1.10 3.88±0.92 3.96±0.84 4.12±0.88 3.92±1.07 3.72±0.93 3.27±1.06 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 c o lo u r sc o re formulation s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 (jn 0 + g 0) (jn 90 + g 30) (jn 120 + g 30) (jn 150 + g 30) (jn 90 + g 60) (jn 120 + g 60) (jn 150 + g 60) (jn 90 + g 90) (jn 120 + g 90) (jn 150 + g 90) international journal of applied biology, 4(1), 2020 6 of sugar showed that the more sugar added, the higher panelists’ score in taste attribute. according to putri (2016) besides for adding flavor, sugar can also form a balance in taste with sourness and bitterness from a product. analysis of variance on taste scores showed a significant difference (p< 0.05), meaning that the addition of sugar and lime juice gave significant effect in every formulation. panelists’ scores on taste parameter ranged from 1.76 to 3.88, which was in the scale of dislike a little to neutral. hedonic test score result showed the highest taste score was formulation s2 (120 g sugar + 30 g lime juice) and s3 (150 g sugar + 30 g lime juice) both had a score of 3.88 which was neutral. formulation s2 and s3 scores followed with the same notation (table 2) as formulation s1, s5, and s6, which means they were not significantly different. figure 3. the scoring result of star fruit sorbet taste aroma aroma is one of the parameters that can determine consumers’ level of acceptance of product quality. aroma is usually the determining factor of the deliciousness of food products (hidayat et al. 2017). the scoring result of aroma attribute on star fruit sorbet shown in figure 4. the graph showed that aroma of star fruit sorbet score in this study increased with added lime juice. aroma of sorbet with added lime juice was distinct lime aroma which gave fresh notes. analysis of variance on aroma scores showed significant difference (p< 0.05), meaning that addition of sugar and lime juice gave significant effect in every formulation. panelists’ score on aroma ranged from 2.72 to 3.68 that was in scale of dislike a little to neutral. the score on control was the lowest which was 2.72 compared to other formulations with added lime juice. the highest score was formulation s1 (90 g sugar + 30 g lime juice) with score of 3.68. formulation s1 had the same notation with s2, s3, s4, s5, s6, s7, s8, and s9 which means they were not significantly different. this result was similar with previous research conducted by hidayat et al. (2017) on pumpkin syrup, where addition of lime juice made pumpkin syrup aroma preferred by panelists compared to without added lime juice. according to study by febrianti (2010), panelists preferred aroma of papaya jam with additional sugar and lime juice. addition of sugar concentration in this study was not affecting aroma of star fruit sorbet sugar aroma was masked by lime juice aroma. 1.76±0.77 3.72±1.02 3.88±1.09 3.88±1.05 3.08±1.18 3.40±1.22 3.36±1.25 2.32±1.06 2.32±1.18 2.60±1.25 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 t a st e sc o re formulation s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 (jn 0 + g 0) (jn 90 + g 30) (jn 120 + g 30) (jn 150 + g 30) (jn 90 + g 60) (jn 120 + g 60) (jn 150 + g 60) (jn 90 + g 90) (jn 120 + g 90) (jn 150 + g 90) international journal of applied biology, 4(1), 2020 7 figure 4. scoring result of star fruit sorbet aroma texture the scoring results of star fruit sorbet texture shown in figure 5. according to dewi (2010), besides act as sweetener, the addition of sugar can improve the texture of frozen products because sugar can deter the formation of big ice crystals in freezing process. hence, the texture of the product is softer and preferred by panelists. according to rahmawati (2017), a stabilizer is one of determining factors of sorbet texture, that the higher the stabilizer concentration, the softer the sorbet texture, and the more preferred by the panelists. in this study, stabilizer used was cmc with the same concentration for all formulations, which was 1.8 g. novianti (2014) stated that cms in the production of ice cream function as a binder, prevent crystallization, stabilizer, gel formation, added viscosity, and to improve texture. analysis of variance on texture scores showed no significant difference (p> 0.05), meaning that the addition of sugar and lime juice gave no significant effect in texture among every formulation. scores from panelists ranged from 2.84 to 3.36, which was in the scale of dislike a little to neutral. the highest score was formulation s3 (150 g sugar + 30 g lime juice) with score of 3.36 or neutral. formulation s3 score was followed by the same notation with all other formulations which mean they were not significantly different or had the same texture. 2.72±0.84 3.68±0.90 3.36±0.95 3.52±0.77 3.44±0.71 3.52±0.87 3.40±0.81 3.28±0.93 3.08±0.70 3.40±0.86 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 a ro m a s c o re formulation s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 (jn 0 + g 0) (jn 90 + g 30) (jn 120 + g 30) (jn 150 + g 30) (jn 90 + g 60) (jn 120 + g 60) (jn 150 + g 60) (jn 90 + g 90) (jn 120 + g 90) (jn 150 + g 90) international journal of applied biology, 4(1), 2020 8 figure 5. the scoring result of star fruit sorbet texture overall the overall score included aspects such as color, taste, aroma, and texture from star fruit sorbet, which scored in unity by panelists. scoring results from panelists (figure 6) showed that addition of sugar and lime juice could increase panelists’ predilection and preferences of star fruit sorbet in the overall aspect. this was because the lowest score was formulation s0 (control), which without additional sugar and lime juice. however, too much of lime juice caused overall score decline as sorbet taste became sourer. and in formulation with the addition of 90 g lime juice, there was some bitter aftertaste. analysis of variance on overall scores showed a significant difference (p< 0.05), meaning that addition of sugar and lime juice gave significant effect in every formulation. from all tested attributes such as color, taste, aroma, and texture, only taste attribute was affecting the overall score of star fruit sorbet. according to hidayat et al. (2017), a food product that has right color, aroma, visual, and texture, will not be accepted by panelists or consumers if the taste of the product is not tasty or not preferred. the highest overall score was formulation s3 (150 g sugar + 30 g lime juice) with a score of 4.00 or like a little. formulation s3 had the same notation with formulation s1, s2, s4, s5, and s6 meaning they were not significantly different. 3.16±1.21 3.00±0.81 3.12±0.78 3.36±0.81 3.16±0.89 3.24±0.77 3.24±0.92 3.20±0.91 2.96±1.13 2.84±1.02 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 t e x tu re s c o re formulation s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 (jn 0 + g 0) (jn 90 + g 30) (jn 120 + g 30) (jn 150 + g 30) (jn 90 + g 60) (jn 120 + g 60) (jn 150 + g 60) (jn 90 + g 90) (jn 120 + g 90) (jn 150 + g 90) international journal of applied biology, 4(1), 2020 9 figure 6. the overall scoring result of star fruit sorbet overrun overrun defined as the volume of sorbet or ice cream obtained in excess of the volume of the mix and is one of parameters in ice cream or sorbet industry. the higher overrun score means the better produced (rahmawati 2017). overrun test result for star fruit sorbet (figure 7) showed score between 23.53 – 29.16 %. the lowest overrun score was formulation s3, which had highest sugar content of 150 g and 30 g lime juice. according to maria & zubaidah (2014), the addition of sucrose in high concentration can increase mixture thickness causing higher viscosity and make mixture harder to rise, in other words, has a low overrun. formulation with the highest overrun score of 29.15 % was formulation s0 (control). because in control formulation, there was no additional sugar and so the mixture has a low viscosity. according to kesuma (2011), when mixture thickness is increasing, overrun will decrease. aside from sugar, sorbet viscosity also affected by the use of a stabilizer. in this research, cmc stabilizer used with the same concentration in all treatments, which was 1.8 g, so it was not affecting obtained overrun score. figure 7. overrun score result of star fruit sorbet 2.44±1.04 3.36±0.81 3.40±0.86 4.00±0.70 3.52±0.91 3.52±0.82 3.44±0.91 2.84±0.85 2.84±1.02 2.92±1.18 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 o v e ra ll s c o re formulation s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 29.16 23.52 27.38 27.38 0 5 10 15 20 25 30 35 s0 s3 s4 s5 o v e rr u n s c o re ( % ) formulation (jn 0 + g 0) (jn 90 + g 30) (jn 120 + g 30) (jn 150 + g 30) (jn 90 + g 60) (jn 120 + g 60) (jn 150 + g 60) (jn 90 + g 90) (jn 120 + g 90) (jn 150 + g 90) international journal of applied biology, 4(1), 2020 10 ph the acidity of sorbet products is affected by the ingredients. sorbet in this research were made from star-fruits with additional sugar and lime juice in every formulation. the result showed that the addition of lime juice affected the ph of the resulted star fruit sorbet. figure 8 showed that the more lime juice added, the lower the ph of star fruit sorbet because lime juice contains citric acid and amino acids. formulation s0 (control) had a higher ph of 6.3 because there was no additional lime juice in the making of it. the lowest ph was in the formulation s4 that had an additional 90 g sugar and 60 g lime juice, with ph of 3.8. this due to lime juice contains citric acid that can affect the ph to be more acidic. febrianti (2010), stated that additional lime juice significantly lowers the ph of papaya jam. according to hidayat et al. (2017), the more lime juice added in pumpkin syrup product means, the lower the ph, because lime juice contains a lot of organic acids such as citric acid. figure 8. ph score result of star fruit sorbet sugar content the scoring results of star fruit sorbet texture shown in figure 5. according to dewi the formulation of star fruit sorbet in this research had a different concentration of sugar and lime juice. results of sugar content analysis of star fruit sorbet shown in figure 9. the lowest sugar content was formulation s0 (control) with a value of 5.95 obrix because there was no additional sugar in it. the highest sugar content was formulation s3 with 26.85 obrix because of the addition of 150 g sugar and 30 g lime juice. the result showed that the higher the sugar concentration used in the formulation, the higher the sugar content score of star fruit sorbet. according to maria & zubaidah (2014), the increase of sucrose concentration affects product sugar content. addition of lime juice in sorbet was not significantly affect sugar content score result because lime had low sugar content. 6.3 4.2 3.8 3.9 0 1 2 3 4 5 6 7 8 s0 s3 s4 s5 p h s c o re formulation international journal of applied biology, 4(1), 2020 11 figure 9. sugar content score result of star fruit sorbet melting time formulation with the shortest melting time was s0 (control) because control was made without additional sugar. formulation with the highest melting time was s3 with additional 150 g sugar and 30 g lime juice. results of the melting time testing of star fruit sorbet is shown in figure 10. according to research by maria & zubaidah (2014), the higher sucrose content means, the longer melting time because sucrose can bind water in a product. therefore, the higher sugar concentration, the more water-bound. another factor that can affect the melting time in the frozen products is the amount of stabilizer used. in this research, a cmc stabilizer used with the same concentration in all treatments, which was 1.8 g. the addition of stabilizers can increase viscosity and increase water binding ability so the product would not melt as quickly (claudia et al. 2016). figure 10. the melting time score result of star fruit sorbet vitamin c vitamin c is a very sensitive vitamin that can be easily broken down by heat and also a vitamin soluble in water. the addition of sugar and lime juice in star fruit sorbet affected vitamin c content. vitamin c content in star fruit was in the range of 0.986 – 1.1144 mg. the highest vitamin c content of 1.144 mg was in formulation s4, which had an additional 90 g sugar and 60 g lime juice. while the lowest vitamin c of 0.968 mg was in s3 with 150 g sugar 5.95 26.85 18.30 21.7 0 5 10 15 20 25 30 35 s0 s3 s4 s5 s u g a r c o n te n t sc o re ( o b ri x ) formulation 41.39 43.16 42.25 42.72 39.5 40 40.5 41 41.5 42 42.5 43 43.5 44 s0 s3 s4 s5 m e lt in g t im e s c o re ( m in u te s) formulation international journal of applied biology, 4(1), 2020 12 and 30 gr lime juice (figure 11). many factors can lower vitamin c in star fruit sorbet. according to rahmawati (2017), a higher amount of added sugar can lower vitamin c content because vitamin c is oxidized. according to sayuti & yenrina (2015), vitamin c can also be lost in processing, such as blanching, washing, cutting, and blending, even in frozen storage. the addition of lime can contribute to increasing vitamin c content in star fruit sorbet because lime juice has a high vitamin c content of 29.10 mg/100 g (usda 2018). according to rachmayati et al. (2017), ripeness of star fruit as the ingredient can also affect vitamin c content because the riper the fruit means the lower vitamin c content. figure 11. vitamin c content result of star fruit sorbet antioxidant the addition of lime juice and sugar in star fruit sorbet affected the antioxidant content of star fruit sorbet. antioxidant activity measured using the dpph method and grouped based on its ic50, where the smaller value means the higher its antioxidant activity. the half-maximal inhibitory concentration (ic50) is a measure of the effectiveness of a tested sample in inhibiting 50 % dpph free radicals. ic50 in star fruit sorbet ranged from 228.81 to 314.84 ppm. the antioxidant activity test result shown in figure 12. the highest antioxidant activity was in formulation s4 (90 g sugar + 60 g lime juice) with ic50 of 228.81 ppm. while the lowest antioxidant activity was in formulation s1 with ic50 of 320.86 ppm. according to elon & polancos (2015), lime juice beside high in vitamin c also contains flavonoid that contributes to antioxidant effect. according to rachmayati et al. (2017), aside from ripeness of the fruit, the addition of sugar also affected the antioxidant content of a product. the more sugar added means the lower its antioxidant content because sugar causing vitamin c as one of the antioxidant sources to be oxidized. the result showed that with the same lime concentration, different sugar concentration led to higher antioxidant activity in formulation with lower sugar that was s4 (90 d sugar + 60 g lime juice) with 228.81 ppm compared to s5 (120 g sugar + 60 g lime juice) with 255.4 ppm. star fruit sorbet with the addition of lime juice had a higher antioxidant activity of 228.81 ppm, compared to mulberry sorbet in a study by rahmawati (2017), which was 1657.540 ppm. 1.056 0.968 1.144 0.968 0 0.2 0.4 0.6 0.8 1 1.2 1.4 s0 s3 s4 s5 v it a m in c s c o re ( m g ) formulation international journal of applied biology, 4(1), 2020 13 figure 12. antioxidant activity result of star fruit sorbet conclusions the best formulations selected based on panelists’ predilection in the organoleptic tests were the formulation of 150 g sugar + 20 g lime, 90 g sugar + 60 g lime, and 120 g sugar + 60 g lime. the best formulation from all test results was formulation with 150 g sugar + 30 g lime juice with overrun of 23.52%, ph of 4.20, sugar content of 26.85 obrix, melting time of 43.16 minutes, vitamin c of 0.968 mg and antioxidant activity of 320.86 ppm. analysis of variance showed a significant difference level (p<0.05). there were treatments with a significant difference in color, taste, aroma, and overall, but no significant difference in texture of star fruit sorbet. the physical appearance of formulation of 150 g sugar + 30 mg lime juice had soft texture and bright yellow color. acknowladgement the author would like to express gratitude to attaqie farm tuban east java which have facilitated the research. references [bps] badan pusat statistik. 2017. jumlah tanaman, produksi dan harga tanaman belimbing. tuban. claudia, n.b., rusmarilin, h., limbong, l.n. 2016. pengaruh perbandingan sari labu kuning dengan sari nenas dan penambahan gelatin terhadap mutu sorbet air kelapa. jurnal rekayasa pangan dan pertanian. 4(4): 500-507. dewi, r.k. 2010. stabilizer concentration and sucrose to the velva tomato fruit quality. jurnal teknik kimia. 4(2): 330-334. elon, y., polancos, j. 2015. manfaat jeruk nipis (citrus aurantifolia) dan olahraga untuk menurunkan koresterol total klien dewasa. jurnal skolastik keprawatan. 1(2): 148 155. febrianti, t. 2010. pengaruh penambahan sari buah jeruk nipis terhadap nilai ph dan kandungan vitamin c pada proses pembuatan selai papaya [skripsi]. samarinda (id): politeknik pertanian negeri samarinda. hidayat, m.a., herawati, n., johan, v.s. 2017. penambahan sari jeruk nipis terhadap karakteristik sirup labu siam. jurnal online mahasiswa fakultas pertanian. 4(2): 115. 314.84 320.86 228.81 255.41 0 50 100 150 200 250 300 350 400 s0 s3 s4 s5 a n ti o x id a n t sc o re ( p p m ) formulation international journal of applied biology, 4(1), 2020 14 kesuma, t.i. 2011. pengaruh jenis dan konsentrasi pati terhadap karakteristik tepung nanas (ananas comocus (l) merr) dan pengaruh cmc terhadap karakteristik velva berbahan dasar tepung nanas [skripsi]. bogor (id): institut pertanian bogor. kusumawati, r.p. 2008. pengaruh penambahan asam sitrat dan pewarna alami kayu secang (caesalpinia sappan l) terhadap stabilitas warna sari buah belimbing manis (averrhoa carambola l) [skripsi]. bogor (id): institut pertanian bogor. maria. d.n., zubaidah, e. 2014. pembuatan velva jambu biji probiotik (lactobacillus acidophilus) kajian persentase penambahan sukrosa dan cmc. jurnal pangan dan agroindustri. 2(4):18-28. novianti, i.w. 2014. pengaruh penambahan puree tape sukun (artocarpus communis forst) dan cmc (carboxy methyl cellulose) terhadap sifat organoleptik es krim. e-journal boga. 3(1): 54-64. prastiwi, s.s., ferdiansyah, f. 2017. kandungan dan aktifitas farmakologi jeruk nipis (citrus aurantifolia s.). jurnal farmaka. 15(2): 1-8. putri, r.a. 2016. pengaruh proporsi gula pasir terhadap sifat organoleptik sirup belimbing wuluh. e-journal boga. 5(3): 73-82. rachmayati, h., susanto, w.h., maligan, j.m. 2017. pengaruh tingkat kematangan buah belimbing (averrhoa carambola l.) dan proporsi penambahan gula terhadap karakteristik fisik, kimia dan organoleptik jelly drink mengandung karaginan. jurnal pangan dan agroindustri. 5(1): 49-60. rahmawati. p.,s. 2017. penambahan konsentrasi bahan penstabil dan konsentrasi sukrosa terhadap karakteristik sorbet murbei hitam (morus nigra sp.) 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[skripsi]. bandar lampung (id): universitas lampung. contact : harem othman smail harem.othman@koyauniversity.org 52 abstract the main aims of this review were to understand the roles of evolutionary process in human disease. the suffering of human from disease may be millions years ago and until now are continuing and the human disease can be classified into many types based on their sources such as bacterial, genetics and viral. for the past sixty years the scientist carried out high number of experiment to understand and the decision of the evolutionary process impact of the human disease. the main example of effect of evolution on the human health are using overuse of antibiotics against bacterial infection and the results to the speedy evolution of bacteria that are resistant to multiple antibiotics such that even vancomycin. the process of natural selection which is proposed by charles darwin play vital roles in biological and medical process and also helps to predict and find the relationship between natural selection process of evolution and phenotypical traits. understanding the developmental and genetic underpinnings of unique evolutionary changes have been hindered by way of insufficient databases of evolutionary anatomy and through the lack of a computational method to become aware of underlying candidate genes and regulators to the developing o the process of the evolution with helps of other branches of modern sciences such as genetics, bioinformatics, epidemiology, ecology, microbiology, molecular biology and biochemistry. issn : 2580-2410 eissn : 2580-2119 evolution of human diseases harem othman smail department of biology, faculty of science and health, koya university koya koy45, kurdistan region-f. r. iraq introduction for tens of millions of years, human beings and their ancestors suffered from diseases -each the variety prompted with the aid of infectious pathogens (e.g., bacteria, viruses, parasites) and the form precipitated through our own our bodies as they age and degenerate (armelagos et al., 1996). in two randomly selected human genomes, 99.9% of the dna sequence are identical (das et al., 2009). the recent speedy accumulation of functionalgenomics and proteomics statistics provides perception into establishing the evolutionary relationship between the genotypes and phenotypes of human diseases (park et al., 2012). the latest extent in genomics statistics is revealing a sudden point of view of gene loss as a pervasive source of genetic variation that can purpose adaptive phenotypic diversity (albalat and cañestro 2016). open access international journal of applied biology keyword gene conversion, drug-resistant, natural ecosystems, evolution, charles darwin, genetic variation. article history received 05 may 2020 accepted 16 june 2020 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 4(1), 2020 53 gene conversion, one amongst the two mechanisms of homologous recombination, involves the unifacial transfer of genetic artifact from a 'donor's sequence to an amazingly homologous 'acceptor (chen et al., 2007.). genetic and mutational statistics on a growing, wide variety of issues have illustrated how phenotypic effects can end result from the mixed action of alleles in many genes (badano and katsanis 2002). comparative analysis across ethnically various human populations and across human and dehumanised primate species is vital for reconstructing human organic process history and for perception the genetic foundation of the human ailment (tishkoff and verrelli 2003). one central motive of genome sequencing initiatives is to impact a better understanding of the genetics of disease and furnish assistance with the identification of disease-associated genes (miller and kumar 2001). most genes worried in fundamental cell approaches have already developed in the unicellular ancestor of eukaryotes. other massive agencies of genes, most exceedingly those concerned in signaling processes, can be traced back to multicellular metazoan evolution (domazet-lošo and two tautz 2008). identifying the causes of similarities and variations in genetic ailment prevalence amongst humans is central to the perception sickness etiology. while current citizenry aren't powerfully differentiated, brobdingnagian amounts of genomic information currently build it viable to find out regarding refined patterns of genetic variation (prohaska et al., 2019.). advances in genetic science and computing unit of measurement reworking the aptitude for the characterization of organic systems and researchers area unit currently poised for a precision-focused transformation within the approach they put together for and reply to, infectious diseases (ladner et al., 2019.). sociocultural transitions and clinical advancements will disrupt biological process equilibriums underlying trendy human anatomy, physiology and existence history (mitteroecker 2019). infectious diseases of domesticated animals affect human well-being through food insecurity, loss of livelihoods, and human infections (farrell and davies 2019). evolution of bacterial disease the great social and financial have an effect on of bacterial pathogens, from drugresistant infections in hospitals to the devastation of agricultural resources, has resulted in main funding to understand the causes and penalties of pathogen evolution (jackson et al., 2011). pathogenic microorganism utilizes a number of mechanisms to cause sickness in human hosts. bacterial pathogens categorical a large variety of molecules that bind host cell targets to facilitate a variety of special host responses. the molecular strategies used by using micro organism to interact with the host can be special to particular pathogens or conserved across quite a few one of a kind species (wilson et al., 2002). recent advances in metagenomics lookup have generated a bounty of data that gives a perception into the dynamic genetic trade happening between bacteriophage (phage) and their bacterial hosts. metagenomic researchers of the microbiomes from a range of environments have proven that many of the genes sequenced are of phage origin. among these genes are phageencoded exotoxin genes (casas and maloy 2011). the cell-surface proteins of the infecting microorganism streptococcus and staphylococcus are probably involved in the process of injection. these proteins share many points which include secretion signal peptides, cell-wall spanning regions, membrane anchor domains and repeated domains of a variety of functions. these common elements can also have advanced with the aid of gene duplication and swapping of gene fragments (goward et al., 1993). the essential microbial ailments affecting marine cultured bivalves have been international journal of applied biology, 4(1), 2020 54 revised on the groundwork of the etiologic agents, pathogenesis and pathogenicity. several current bivalve-interaction models area unit studied, at the side of cellulose larvae-vibrio pectinicida, brown brown rot, juvenile oyster unwellness, pacific oyster nocardiosis and summer mortalities of oysters (paillard et al., 2004). many of the foremost virulent microorganism pathogens square measure genetically monomorphic, and perception their organic process and phylogeographic patterns can assist our understanding of the outcomes of communicable disease on human records (achtman 2008). relationship between evolution of bacterial disease and antibiotics when seen from an evolutionary perspective, manifestations of infectious diseases can be categorized as (1) variations of the host to counteract damaging components of the disease, (2) variant of the pathogen to manipulate the host, or (3) “side effects” of the sickness that do now not serve adaptive features for either the host or the pathogen. although the features of most manifestations are not known, help or rejection of these hypotheses ought to be effectively derivable in many cases from analyses of existing records and tremendously simple experiments (ewald 1980).bacterial adaptation to antibiotics has been very profitable and over the past decade the expand in antibiotic resistance has generated large medical issues (andersson two 2003).in addition to sterilisation international ecology, science and human public boom conjointly have an effect on organic process trajectories, dramatically fast organic process alternate in alternative species, above all in commercially vital, pest, and illness organisms. such adjustments are apparent in antibiotic and human immunodeficiency virus (hiv) resistance to drugs, plant and insect resistance to pesticides, fast changes in invasive species, life-history trade in industrial fisheries, and pest adaptation to biological engineering merchandise (palumbi 2001). since their introduction for human remedy 60 years ago, antibiotics have shown to be a fantastic success and represent one of the most applicable scientific inventions for reducing human morbidity and mortality. unfortunately, the intensive use and misuse of antibiotics have resulted in antibiotic resistance amongst several human pathogens, decreasing the possibilities for infections' treatment and jeopardizing clinical procedures, such as organ transplantations or implants of prostheses, where infective problems are frequent and antibiotic remedy is wished to forestall or deal with these infections. there are two important mechanisms concerned with the improvement of antibiotic resistance, particularly mutation two and acquisition of resistance genes two by way of horizontal gene switch (hgt). given that human pathogens had been inclined to antibiotics earlier than the use of these capsules for the therapy of infections, the beginning of antibiotic resistance determinants obtained through hgt must necessarily lay in the non-pathogenic microbiosphere (martinez 2009). the remedy of infectious disorder is compromised by using the development of antibiotic-resistant lines of microbial pathogens. a range of biochemical methods is concerned that might also maintain antibiotics out of the cell, alter the target of the drug, or disable the antibiotic. studies have proven that resistance determinants occur by means of either of two genetic mechanisms: mutation and acquisition (mazel 1999). natural ecosystems incorporate a large quantity of possible resistance genes; nevertheless, just a few of them are currently existing in gene‐transfer units and disseminated among pathogens. along the review, the tactics implied in this situation and the penalties for the future evolution of resistance and the environmental microbiota are discussed two (baquero et al., 2009).for the past 60 years or so, we have carried out at an international experiment in evolutionary decision pressure by means of making use of tonnes of antibiotics international journal of applied biology, 4(1), 2020 55 to the planet, to treat patients and to promote growth in animals used for meals production. the consequences are solely too depressing apparent—widespread antibiotic resistance in pathogens. this manner is darwinian “natural” selection, at the sharp end. each year, thousands of people die from hospital-acquired bacterial infection, a whole lot of which is multi-drug resistant. this disaster is pushed by means of overuse of antibiotics and our incapability to manage the dissemination of bacteria and their drug-resistance genes (salmond and welch 2008). the therapy of bacterial infections is increasingly intricate due to the fact microorganisms can develop resistance to antimicrobial marketers (martínez 2007). overuse of antibiotics in humans and farm animals has led to the speedy evolution of bacteria that are resistant to multiple tablets such that even vancomycin, the drug of last resort, is no longer fantastic in opposition to some strains. apart from the discovery and exploitation of the herbal peptide antimicrobial dealers that structure part of the innate immune system of plant life and animals, there have been few new antibiotics developed in latest (tan et al., 2000). understanding the stipulations that favor the evolution and maintenance of antibiotic resistance is the central goal of epidemiology. a imperative function explaining the adaptation to harsh, or ‘sink’, environments is the supply of advisable mutations by way of migration from a ‘source’ population. given that antibiotic resistance is frequently related to opposing pleiotropic health costs, the expanded migration fee is estimated not only to enlarge the fee of resistance evolution, but additionally to amplify the likelihood of fixation of resistance mutations with minimal fitness fees (perron et al., 2007). antibiotic resistance is one of the greatest challenges of the twenty-first century. however, the growing grasp of bacterial pathogenesis and intracellular verbal exchange has printed many plausible techniques to increase novel tablets to deal with bacteria-mediated disorder (rasko and sperandio 2010). bacteria can gather target-mediated antibiotic resistance in three ways. the first is the accumulation of factor mutations in the chromosomal gene encoding the goal (maiden 1998). the biofilm mode of life gives benefits to microorganisms, such as improved resistance towards environmental stresses, consisting of antibiotic challenge. the neighborhood level resistance furnished by means of biofuels is distinct from resistance mechanisms that function at a cellular level, and can't be disregarded in the development of novel strategies to fight infectious diseases. the evaluate compares the mechanisms of antibiotic resistance at mobile and community ranges in the mild of past and present antibiotic discovery efforts. future views on novel strategies for therapy of biofilm-related infectious illnesses are discover (penesyan et al., 20150. the most normal micro organism had been coagulasenegative staphylococci (cns) (35•1%), escherichia coli (11•4%), staphylococcus aureus (9•9%), enterococcus spp. (8•2%), and pseudomonas aeruginosa (7•5%). the susceptibility of cns to oxacillin decreased from 67–44% over six years, whilst that of enterobacteriaceae to amoxicillin and piperacillin was once decreased via about 50%. p. aeruginosa susceptibility to ceftazidime remained remarkably stable at round 90%, in spite of massive empirical use ( durand-gasselin et al ., 1995). many doctors will suggest ameliorations to a way of life and increasing fiber consumption. empirical antibiotics continue to be the mainstay of therapy for sufferers with diverticular ailment and rifaximin seems to be the high-quality choice. in extreme or relapsing disease, surgical intervention is regularly the only remaining therapy option. although novel remedy picks are but to end up available, the addition of treatment options primarily based on mesalazine (mesalamine) and probiotics may additionally beautify treatment efficacy (tursi and papagrigoriadis 2009). evolution of micro organism closer to resistance to antimicrobial agents, inclusive of multidrug resistance, is unavoidable because international journal of applied biology, 4(1), 2020 56 it represents a particular aspect of the commonplace evolution of microorganism that is unstoppable. therefore, the sole capacity of dealing with this situation is to delay the emergence and subsequent dissemination of resistant bacteria or resistance genes (courvalin 2016). yersinia pestis, the causative agent of plague, looks to have developed from a gastrointestinal pathogen, yersinia pseudotuberculosis, in simply 1,500–20,000 years — an 'eye blink' in evolutionary time. the third pathogenic yersinia, yersinia enterocolitica, also causes gastroenteritis however, is distantly associated with you. pasties' and y. pseudotuberculosis (wren 2003). the shared evolutionary destiny of humans and their symbiotic microorganism has chosen for mutualistic interactions that are crucial for human health, and ecological or genetic adjustments that uncouple this shared fate can result in disease. in this way, searching to ecological and evolutionary principles may provide new techniques for restoring and keeping human health (dethlefsen et al., 2007). the discovering that horizontal gene transfer and genome decay have key roles in the evolution of bacterial pathogens was in particularly surprising. it has also turned out to be evident that even the definitions for 'pathogen' and 'virulence factor' want to be re-evaluated (pallen and wren 2007). genetics and evolution over 200 years ago, erasmus darwin famously argued that the price of what is acknowledged these days as an evolutionary strategy would be to “unravel the principle of diseases”. charles darwin noticed hereditary sickness as proof of inheritance of version. from the book of on the origin of species (1859) to the 1940s, darwinism played an important function in biological, medical, and social sciences alike (gluckman et al., 2011). the organic process history of a factor helps predict its operate and relationship to phenotypical traits. although sequence conservation is frequently wont to decipher factor operate and verify medical connection, ways for helpful logical thinking from comparative expression records square measure missing (chen et al., 2019). it appears clear that the evolution of the ancestors of chimpanzees and hominins separated 7–9 million years ago with some migration out of africa with the aid of the before hominins; homo sapiens slowly emerged as climate change resulted in drier, much less forested african conditions. the african populations accelerated and evolved in many extraordinary prerequisites with slow mutation and selection rates in the human genome, but with an awful lot extra fast mutation going on in mitochondrial dna (james et al., 2019). historical elements of this evolution will be discussed. evidence from a variety of sources shows that the human lifespan is increasing, and may nicely continue to extend to tiers that are hard to predict (kyriazis 2019). the coding regions of the duplicates were consequently diverged from every different through nucleotide substitutions, earlier than being similarly expanded via duplications of short repeats (djian 1998). however, the effect of the secretion on evolutionary quotes is countered through tissue-specific constraints that have been held steady over the past 75 million years (winter et al., 2004). understanding the developmental and genetic underpinnings of unique evolutionary modifications has been hindered by way of insufficient databases of evolutionary ana tomy and through the lack of a computational method to become aware of underlying candidate genes and regulators (mabee et al., 2007).the suitability of the fugu genome to facilitate the identification of candidate human sickness genes the usage of comparative positional cloning is dependent upon the extent to which synteny and gene order are conserved between the international journal of applied biology, 4(1), 2020 57 two species (gilley and fried 1999 ). epidemiological observations have led to the hypothesis that the danger of creating some persistent noncommunicable ailments in maturity is influenced no longer only by way of genetic and person lifestyle elements however also with the aid of environmental elements appearing in early life. research in evolutionary biology, developmental biology, and animal and human physiology affords assist for this notion (gluckman and hanson 2004 ). single ester polymorphism (snp) technologies are often accustomed recognize disease-causing genes in humans and to apprehend the interindividual variation in drug response. these areas of lookup have predominant scientific advantages (shastry 2007). for most of human history, the environmental demands of survival necessitated prodigious amounts of physical exertion. the avoidance of predators, hunting, gathering, and the literal “chopping wood and carrying water” of day by day existence supplied a healthful dose of bodily endeavor that obviated the need for deliberate exercise. nevertheless, 21st century people are now immersed within an surroundings explicitly designed to do away with bodily labor (archer and blair 2011). in most cases, the growing organism responds to an environmental cue with the aid of producing a selectively and at once fantastic phenotype. one subset of phenotypic responses to environmental stimuli, however, does no longer necessarily provide an instant selective advantage. rather, these sorts of responses, which we call ‘predictive adaptive responses’ (pars), act primarily to improve health at a later stage of development (gluckman et al., 2005). humans have evolved an awful lot longer lifespan than the wonderful apes, which rarely exceed 50 years. since 1800, lifespans have doubled again, mostly due to upgrades in environment, food, and medicinal drug that minimized mortality at previous ages. infections reason most mortality in wild chimpanzees and in standard forager-farmers with confined get admission to two cutting-edge medicine (finch 2010). the controller is crucial for the suitable segregation and inheritance of genetic information. neocentromeres are ectopic controllers that originate every so often from noncentromeric areas of chromosomes. despite the entire absence of everyday centromeric α-satellite dna, human neocentromeres are capable to form a major constriction and gather a functional kinetochore. since the invention and characterization of the primary case of somebody's neocentromere in our laboratory a decade past, 60 examples of constitutional human neocentromeres dispensed extensively throughout the genome have been described (amor and choo 2002). the sec7 area adp-ribosylation issue (arf) guanine nucleotide alternate elements (gefs) are determined in all eukaryotes, and are concerned with membrane redesigning tactics for the duration of the cell (bui et al., 2002). multiple gene duplication and deletion events were identified in abc genes in special lineages indicating that the method of gene evolution is nevertheless ongoing. gene duplication resulting in both gene delivery or gene death plays an important position in the evolution of the vertebrate abc genes (moitra and dean 2011). the forked field (fox) household of transcription factors, which originated in unicellular eukaryotes, has extended over time through more than one duplication events, and occasionally through gene loss, to over forty individuals in mammals. fox genes have advanced to accumulate a specialized characteristic in many key organic processes. mutations in fox genes have a profound result on human sickness, causing phenotypes as diverse as cancer, glaucoma and language issues (hannenhalli and kaestner2009). recent work in the mouse suggests that the tail bud regulatory network relies on the interconnected activities of the lin28/let-7 axis and the hox13 genes. as this community is probably to be conserved in other mammals, it is feasible that the last length and the anatomical international journal of applied biology, 4(1), 2020 58 composition of the adult tail end result from the balance between the progenitor-promoting and -repressing things to do furnished through those genes (mallo 2019). the diverse liquid proteins, sanctioning the survival of distinctive polar fishes in physical change seas supply uncomparable vistas into the breadth of genetic sources and mechanisms that turn out important new functions. although most new genes evolved from preexisting genes ancestors, some are deemed to have arisen from noncoding dna. however, the pertinent mechanisms, functions, and selective forces stay unsure (zhuang two et al 2019). reduced evolutionary conservation of tissue-specific genes might also characterize a bottleneck for drug projects, prompting improvement of novel fashions with a smaller evolutionary gap to humans (ryaboshapkina and hammar 2019). relationship between viral disease in human and evolutionary a key priority for infectious disorder search is to create clear however infective agent genetic variation, modulated via host immunity, transmission bottlenecks, and epidemic dynamics, determines the big style of infective agent phylogenies observed at scales that vary from person host to populace (grenfell et al., 2004). the recent appearance of extreme acute respiratory syndrome coronavirus (sars–cov) highlights the persistent risk to human health posed by way of rising viruses. however, the central procedures in the evolution of emerging viruses are unclear, specifically the selection pressures confronted by viruses in new host species (holmes and rambaut two 2004). while most trees confirmed evolutionary relationships steady with present day antigenic complexes and species, several adjustments to the modern classification are proposed (powers et al., 2001). in latest years, population and evolutionary biologists have puzzled the ordinary view that parasite-mediated morbidity and mortality¿virulence¿is a primitive personality and an artifact of latest associations between parasites and their hosts. a range of hypotheses has been proposed that favor virulence and advocate that it will be maintained by way of herbal decision (levin 1996). understanding the evolutionary basis for virus emergence is consequently a key research goal and many of the debates in this region can be considered within the rigorous theoretical framework set up through evolutionary genetics. in particular, the respective roles played by using natural decision and genetic flow in shaping genetic variation are also of quintessential importance of perception the nature of viral emergence (holmes and drummond two 2007 ). in particular, current studies have published a greater complex relationship between antigenic evolution, natural decision and resentment than earlier realized (nelson and holmes 2007). evolutionary medication is defined as the utility of concepts of evolutionary theory to medical exercise and research. among its proponents are organic anthropologists who provide a quantity of views on human evolutionary approaches and present day health challenges. examples consist of improving the grasp of sickness resistance afforded via overuse of antibiotics and distinguishing the variations between “defenses” (healthful bodily reactions) and “defects” (unhealthful reactions) (trevathan 2018). recent studies have known new ways in which within which infective agent and microorganism exposures in early lifestyles engage with host genetic background/variants to change the threat for growing respiratory illness and allergic diseases. recent research suggests that hrv-c is the important pathogenic agent related to infant wheeze, hospitalizations and likely the subsequent development of bronchial asthma (daley 2014). the evolutionary records of ebola virus stays unclear. in this study, 27 ebola virus traces with complete glycoprotein genes, which include 5 species (zaire, sudan, reston, tai forest, bundibugyo), have been analyzed. here, we propose a speculation of the evolutionary international journal of applied biology, 4(1), 2020 59 records of ebola virus which will be useful to look into the molecular evolution of these viruses (li and chen 2014). published ebov makona genomes from medical samples obtained early in the outbreak in guinea (three patients) and sierra leone (78 patients) (baize et al., 2014, gire et al., 2014) established that near-real-time sequencing could provide treasured information to researchers concerned with the international outbreak response (park et al., 2015). ebola outbreak in the world has ever witnessed, with over 28,000 instances and o ver 11,000 deaths (de la two et al., 2015). early theories of virulence cautioned that pathogens would evolve to avirulent commensals given that harming the host would be a negative longterm survival strategy (bull and lauring 2014). zika virus (zikv), located in 1947, had precipitated sporadic ailment at some point of africa and asia till the 2007 micronesia and 2013 french polynesia outbreaks. the rapid expansion of geographic variation and make bigger in severe pathogenicity first cited in the 2015–2016 brazilian outbreak has raised questions involving the molecular evolution of this virus (wang et al., 2016). evolution of hiv viruses the fundamental purpose of receiving immune deficiency syndrome (aids) is human immunodeficiency virus type 1 (hiv-1). we have been the use of evolutionary comparisons to hint (i) the origin(s) of hiv-1 and (ii) the origin(s) of aids. the closest loved ones of hiv-1 are simian immunodeficiency viruses (sivs) infecting wild-living chimpanzees (pan troglodytes troglodytes) and gorillas (gorilla gorilla gorilla) in west central africa (sharp, and hahn 2010). nine countries in southern africa account for much less than 2% of the world's population, but now they characterize about one third of international hiv infections. where normally enforced, hiv screening of donated blood and antiretroviral treatment (art) of pregnant women have been notably superb in preventing transfusion-associated and perinatally acquired hiv, respectively (de cock et al., 2012). although several researchers have characterized the intrapatient evolution of viral sequences all through hiv-1 infection, potential studies inspecting intrapatient evolution throughout hiv-2 infection have been restrained (macneil et al., 2007). hiv‐1 is swiftly evolving with growing range of international strains. in individuals, hiv‐1 infection is usually initiated by one or a few transmitted/founder (tf) viruses, and within each infected person, evolves to terrific range shaped through antibody and t cell responses. moreover, virus integration happens early on in infection, earlier than a protective antibody or t cells response can take place (bonsignori et al., 2017). early prediction of hiv-1 composition evolution could in addition be useful for clinical observation and treatment of well contamination (groenink et al., 1993). molecular phylogenetics has revolutionized the determine concerning by not solely evolution however conjointly disparate fields like genetics, bioinformatics, epidemiology, ecology, microbiology, molecular biology and biochemistry (castro-nallar et al., 2012). the unique milieu of the cns fosters viral compartmentalization as well as the evolution of viral sequences, permitting for new cell types, such as macrophages and microglia, to be contaminated (bednar et al., 2015). often, pathogens have to adapt to correctly infect a novel host, for instance by evolving to use specific mobile surface receptors, to break out the immune response, or to ensure they are transmitted by means of the new host. in viruses there are regularly limited molecular options to attain this, and the equal sequence modifications are regularly considered every time a virus infects a particular host two (longdon et al., 2014). recent tendencies contain prediction of reproducible patterns in parallel evolution experiments, forecasting the future of characterizing populations the usage of records from their past, and controlled manipulation of evolutionary dynamics (lässig et al., 2017). killing international journal of applied biology, 4(1), 2020 60 of hiv-infected cells through cd8+ t-cells imposes strong choice stress on the virus to escape (kløverpris et al., 2016). hiv testing is the indispensable entry point for each remedy and prevention. the need to pick out acute hiv contamination (the length without delay after hiv acquisition, when folks are most infectious) and hiv-2 infection, which does no longer reply to many first-line antiretroviral agents, poses challenges for the standard algorithm of western blot confirmation after an over and over reactive antibody screening take a look at (branson 2010). analysis of enormous sequence and neutralization data units showed the 332 glycan to be significantly under-represented in transmission subtype c viruses compared to continual viruses, with the absence of this glycan corresponding with resistance to pgt128 (moore et al., 2012). viral sequence evolution also printed speedy and extraordinarily reproducible escape from these responses, mirroring the diversifications to host immune pressures found all through natural hiv-1 contamination (dudek et al., 2012). the virus’ within-host evolutionary rates have been argued to be a lot higher than its between-host evolutionary rates. however, this conclusion depends on analyses of a quick component of the virus envelope gene (alizon and fraser 2013). the tree structure is presently the generic paradigm to characterize evolutionary relationships between organisms, species or different taxa. however, horizontal, or reticulate, genomic exchanges are pervasive in nature and confound characterization of phylogenetic trees. drawing from algebraic topology, our existing a special evolutionary framework that comprehensively captures both clonal and reticulate evolution (chan et al., 2013). because viruses will evolve quicker than their hosts, the innate immune system of modern vertebrates is for the most phase optimized to defend towards historical viruses, alternatively than more recent viral threats. thus, the evolutionary records of restriction factors might, in part, provide an explanation for why human beings are susceptible or resistant to the viruses existing in the modern-day world (duggal et al., 2012). hiv evolution differs inside and among hosts and on the role played by using superb decision (rambaut et al., 2004). large-scale empirical analyses of the biological process dynamics of important microorganisms ar currently possible as a result of the growing convenience of pathogen sequence records and therefore the development of latest process and statistical methods of analysis (pybus and rambaut 2009 ). the rapidity of sequence change in rna viruses capacity that they are useful experimental models for the learn about of evolution in ordinary and it enables us to watch them trade in 'real time', and retrace the unfold through populations with molecular phylogenies (moya et al., 2004). early prediction of hiv-1 composition evolution could in addition be useful for clinical observation and treatment of well contamination (groenink et al., 1993). molecular phylogenetics has revolutionized the verify relating to by not solely evolution however along disparate fields like bioscience, bioinformatics, epidemiology, ecology, microbiology, molecular biology and biochemistry (castro-nallar et al., 2012). the unique milieu of the cns fosters viral compartmentalization as well as the evolution of viral sequences, permitting for new mobile phone types, such as macrophages and microglia, to be contaminated (bednar et al., 2015). often, pathogens have to adapt to correctly infect a novel host, for instance by evolving to use specific mobile surface receptors, to break out the immune response, or to ensure they are transmitted by means of the new host. in viruses there are regularly limited molecular options to attain this, and the equal sequence modifications are regularly considered every time a virus infects a particular host two (longdon et al., 2014). recent tendencies contains prediction of duplicable patterns in parallel evolution experiments, forecasting the future of characterizing populations the usage of records from international journal of applied biology, 4(1), 2020 61 their past, and controlled manipulation of evolutionary dynamics (lässig et al., 2017). killing of hiv-infected cells through cd8+ t-cells imposes strong choice stress on the virus to escape (kløverpris et al., 2016). hiv testing is the indispensable entry point for each remedy and prevention. the need to pick out acute hiv contamination (the length without delay after hiv acquisition, when folks are most infectious) and hiv-2 infection, which does no longer reply to many first-line antiretroviral agents, poses challenges for the standard algorithm of western blot confirmation after an over and over reactive antibody screening take a look at (branson 2010). analysis of big sequence and neutralization data units showed the 332 glycan to be significantly under-represented in transmission subtype c viruses compared to continual viruses, with the absence of this glycan corresponding with resistance to pgt128 (moore et al., 2012). viral sequence evolution also printed speedy and extraordinarily reproducible escape from these responses, mirroring the diversifications to host immune pressures found all through natural hiv-1 contamination (dudek et al., 2012). the virus’ within-host evolutionary rates have been argued to be a lot higher than its between-host evolutionary rates. however, this conclusion depends on analyses of a quick component of the virus envelope gene (alizon and fraser 2013). the tree structure is presently the generic paradigm to characterize evolutionary relationships between organisms, species or different taxa. however, horizontal, or reticulate, genomic exchanges square measure pervasive in nature and confound characterization of organic process trees. drawing from algebraic topology, our existing a special evolutionary framework that comprehensively captures both clonal and reticulate evolution (chan et al., 2013). because viruses will evolve quicker than their hosts, the innate immune system of modern vertebrates is for the most phase optimized to defend towards historical viruses, alternatively than more recent viral threats. thus, the evolutionary records of restriction factors might, in part, provide an explanation for why human beings are susceptible or resistant to the viruses existing in the modern-day world (duggal et al., 2012). hiv evolution differs inside and among hosts and on the role played by using superb decision (rambaut et al., 2004). large -scale empirical analyses of the biological process dynamics of important microorganisms are currently possible as a result of the growing convenience of pathogen sequence records and therefore the development of latest process and statistical methods of analysis (pybus and rambaut 2009 ). the rapidity of sequence change in rna viruses capacity that they are useful experimental models for the learn about of evolution in ordinary and it enables us to watch them trade in 'real time', and retrace the unfold through populations with molecular phylogenies (moya et al., 2004). conclusions from this review i reached the following conclusions: there are no doubt the evolutionary process is raised millions years ago and has 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considered the genetic inheritance of the new improved lines and their interactions. pedigree breeding method was used to develop two single, double and three-way (and reciprocal) crosses through marker-assisted selection. southern blot analysis was used to determine success of introgression of resistance/tolerance genes/qtls and selection, also validated by phenotyped results. agro-morphological and yield parameters of the various populations were analysed. the results indicated levels of significant differences amongst and between treatments for nondrought stress (ns) and reproductive drought stress (rs) and their interactions. there were significant variation among parents and improved lines on some traits in ns treatment, but rs significantly affected parameters of df, ffg, ym and most especially the susceptible parent, while the improved lines were tolerant. significant interactions was recorded (p≤0.05) between treatment and variety (trt*var.) on pl, t, ffg and glw. cluster analysis and pca of relationship among the 9 traits in the two treatments revealed that each of single, double and three-way (and reciprocal) crosses had good lines either under ns and rs. issn : 2580-2410 eissn : 2580-2119 genetic inheritance of multiple traits of blast, bacteria leaf blight resistant and drought tolerant rice lines ibrahim silas akos1,2, mohd rafii yusop*1,3, mohd razi ismail1,3, shairul izan ramlee3, noraziyah abd aziz shamsudin4, asfaliza binti ramli5, jalloh momodu1, jamilu halidu1, & senesie swaray1 1laboratory of climate-smart food crop production, institute of tropical agriculture and food security, universiti putra malaysia, 43400 upm serdang, selangor, malaysia. 2department of crop science, faculty of agriculture, kaduna state university, kafanchan campus, nigeria. 3department of crop science, faculty of agriculture, universiti putra malaysia, 43400 upm serdang, selangor, malaysia. 4school of environmental and natural resource sciences, faculty of science and technology, universiti kebangsaan malaysia. 5malaysian agricultural research and development institute, rice research centre, persiaran mardi-upm, 43400, serdang, selangor, malaysia. open access international journal of applied biology keyword rice, genetic, inheritance, resistant, tolerance. article history received 25 september 2019 accepted 29 december 2019 international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. international journal of applied biology, 3(2), 2019 2 introduction rice, a multi-environmental (rain-fed lowland, upland and deep water) (ou, 1985, latif et al., 2011) cereal and staple food crop of the world, it is one of the most cultivated crops along with wheat and corn. it provides nutrient and meets the economic need of the farming populace. it is consumed by over 50% of the world’s population as their major source of calorie (luo et al., 1998). blast and bacteria leaf blight are two major important diseases of rice that causes significant yield loss to farmers (asghar et al., 2007, jia et al., 2000, zhang et al., 2015). the management practice for the disease can be fungicide, biotechnological methods, agronomic practices and cultivation of resistant cultivars (ribot et al., 2008). the use of the resistant variety is the most environmental and economically friendly approach to blast threat (castano et al., 1990, saifullah et al., 1995, khan et al., 2001, haq et al., 2002). the challenge is often that due to the changeful nature of the virulent races, the resistance traits may be lost in the cause of time. the most effective strategy to minimize yield losses due to blast, bacteria leaf blight and drought stress is through the development of durable, broadspectrum resistant varieties (jena and mackill, 2008; kumar et al., 2014; sundaram et al., 2014). sundaram et al. (2014) reported that atleast 40 genes that confer bacteria blight resistance have been identified, while many have been fine-mapped and cloned (natrajkumar et al., 2012). 101 blast resistant (r) genes (rajashekara et al., 2014) and 350 qtls have been identified (sharma et al., 2012). closely linked markers available for many blast resistance and bacteria blight genes have been identified (sundaram et al., 2014). drought stress is a condition of water availability deficit, it could be water deficit condition at any of the stages of rice development which has the potential of affecting yield. drought stress is increasingly becoming a challenge to farming communities today with a global scale, it affects over 23million hectares of farming areas in asia (bray et al., 2000; kumbhar et al., 2015). the increasing human population with approximated 10billion by the year 2050 also requires 50% increase to current global rice production to meet the food projection demand (maclean et al., 2002, bourman et al., 2007). this places a demand for strategy to increase yield production in the phase of climate change to develop rice adapted to drought stress (pandey and sukla, 2015). understanding plant diversity in relation to behaviour and adaptation of drought-prone environment is important (alonso-blanco et al., 2009), the ability to design an effective strategy of phenotyping requires good understanding of plant survival mechanism under drought stress. understanding of plant diversity is relevant to assessment of genotypic variability under different water deficit condition as an important pre-condition for a successful drought tolerance breeding programme (sarkar et al., 2013, abenavoli et al., 2016, anower et al., 2017). blast, bacteria leaf blight (blb) and drought stress could affect rice drastically, and result showed that high incidences caused yield losses of 100% (zhai and zhu, 1999), 1-50% (scardaci et al., 2003) and 100% depending on the stage of rice development and duration respectively. 50% yield loss was recorded for abiotic stress (bray et al., 2000, iqbal et al., 2013, li et al., 2014). the parameters of panicle length, effective tillers, grain length and width ratio, 100 grain weight, fully grain weight were amongst those considered important agronomic trait which has correlation with increased yield potential in rice, considered as quantitative traits which could be affected by the influence of environment (han et al., 2004, guo et al., 2003, han et al., 2006, irri-ses, 2013, taglea et al., 2016, chang et al., 2016).new improved lines has been developed with three resistance/tolerant traits of blast (magnaporthe grisea) international journal of applied biology, 3(2), 2019 3 bacteria leaf blight (xanthomonas oryzae) and drought tolerance (mr219) through pyramiding of marker-assisted selection. this research considered the genetic inheritance of the target genes and their interactions. materials and methods plant material and breeding design plant materials used were new developed lines from three parents with resistances to blast, m. grisea anomorph p. oryzae, a popular malaysia high yielding rice variety known as putra1. bacteria leaf blight resistance variety known as irbb60, and a drought tolerant malaysia variety referred to as mr219 (ir99784-156-137-1-3 drought tolerance). these varieties were developed as single crosses, double cross and three-way cross with reciprocal cross as well. these new lines were developed in a glass house at rice research centre and laboratory of climate and smart-food crop production, universiti putra malaysia. dna extraction and nanodrop spectrophotometry three-four weeks old leaves samples were collected and ground using mortar and pestle in liquid nitrogen. the procedure was according to doyle and doyle (1987) cetyltrimethylammonium bromide ctab method with modification following the protocol of mccouch et al (1988). the dna pellet was diluted in 50µl te buffer, 2µl was pipetted on nanodrop spectrophotometry machine to measure the quantity and purity of the dna samples. this formed the basis for the constitution of the dna working solution for polymerized chain reaction (pcr) amplification. molecular markers simple sequence repeat (ssr) markers polymorphic and linked to the genes/qtls for the three parental varieties were purchased. putra1, a blast resistance variety had two polymorphic markers that were also linked to the genes of resistance (r), mr219 (ir 99784156-137-1-3) drought tolerance variety used had three qdty and irbb60 had four r genes (pinta et al. 2013, miah et al. 2016, kumar et al. 2014, khan et al. 2015, pradhan et al. 2015, shamsudin et al. 2016, www.gramene.org). polymerized chain reaction and electrophoresis the pairs of primers for the various genes/qtls were optimized to amplify simple sequence repeat loci for polymerase chain reaction (pcr). survey of the three parental varieties was carried out to identify ssr markers polymorphic to each of them. total pcr reaction of 15μl which contained 70ng dna template had 7.5μl master mix (thermo scientific, waltham, ma, usa), 4.5μl nucleaus free water and 1.0 μmol l-1 concentration of each primer (forward and reverse). the pcr amplification was conducted in a thermocycler (t100tm, bio-rad, hercules, ca, usa) following the touchdown protocol with the lid temperature of 1050c. the initial denaturation, annealing and extension temperatures were thus; 940c for 3mins followed by 940c for 30sec, a temperature of 620c for 1min., +10c per cycle followed by 720c for 30sec., then a returned to step 2, 9× followed by 940c, 30sec., 520c, 1min, 720c, 2mins., another return to step 6, 29×., and finally 720c, 10mins. followed by rapid cooling to 40cꝏ prior to analysis. http://www.gramene.org/ international journal of applied biology, 3(2), 2019 4 southern blot analysis using gel electrophoresis was carried out. 2.0% metaphortm agarose (lonza) gel containing 5μl midori green in 1× tbe buffer (0.05 mol l−1 tris, 0.05 mol l−1 boric acid, 1 mmoll−1 edta, ph 8.0) was run with 5μl of pcr product mixed with loading dye. the gel run was at a constant voltage of 80v for 45-60 minutes. molecular imager system (geldoctm xr, biorad) was used to analyzed band pattern under uv light for amplified products. identification of polymorphic markers and progeny selection the basis for a successful marker-assisted selection was the identification of the polymorphic markers for each of the parent trait. blast resistance had rm6836 and rm8225, irbb60 had xa13 prom, rm122, rg136, pta248 and rm224 while mr219 ir99784-156-1371-3 drought tolerance had rm511, rm1261 and rm520 polymorphic to the drought tolerance qtls. two f1s with double bands corresponding with the parent plants were selected, that was in a cross between a common recipient parent putra 1 and donor irbb60, putra1 and mr219. the selected progenies formed the bases for a double cross and three-way and reciprocal crosses. the selection of the subsequent generations of f1(2) and f1 three-way crosses were based on southern blot analysis of the bands which coincide with the first recipient and the introgressed variety. those were selected and advanced until a non-segregating f3(2) double, f4 single, and f3 three-way crosses pure-line generation was reached. fungal, bacteria culture and inoculation most virulent strains of fungus magnaporthe grisea pathotype p7.2 and bacterium xanthomonas oryzae pv oryzae mx01552 were obtained from malaysia agricultural research and development institute (mardi). they were sub-cultured in potato dextrose agar (pda) for 14 days in 250c and nutrient agar (na) for two days in 300c respectively (suresh et al. 2013, mahdieh et al. 2013). the m. grisea mycelia culture was prepared in sterile distilled water suspension at concentration of 1.9×106 conidia ml-1. the xoo culture was also prepared in a suspension concentration of 109. the fungus suspension was sprayed on young leaves of 2-3 weeks old at relative humidity >90% for 48hours for disease infection and after 7 days disease scoring was carried out according to the irri-ses, (2013) glass house scoring, thus; from 0-2, resistant (r), 3 considered as moderately resistant (mr) and score 4-6 as ms, while 7-9 as s. the xoo pathogen was clip inoculated on 3-4 weeks old leaves at 1-2cm from the tip of the leaves. the scoring was by measuring the length of infected area with meter rule in centimeter according to irri-ses, 2013. glass house scale (banito et al. 2012) and modification according to amante-bordeos et al. (1992), thus; 0-5 considered as r, >5-10 mr, >10-15 ms, while >15 were considered as s. drought stress imposition reproductive drought stress was imposed on the rice lines carrying drought tolerance qtls. according to irri-ses (2013) one week of water deficit stress for glass house experiment is enough to affect yield. the reproductive stage was between 70-90 days after sowing. stress was imposed for >2weeks with leaves turned from u-shaped to 0-shaped as criterion for international journal of applied biology, 3(2), 2019 5 scoring drought (irri-ses, 2013, kadioglu and terzi, 2007), dried soil and soil moisture meter with >15cm depth measured dried. experimental layout and cultivation the experiment was set out in a glass house at rice research centre, universiti putra malaysia with 3 plants each per bucket and label accordingly. the leaves of the parents to their progenies were collected and genotyped using southern blot analysis to determined the ideal plants for selection until line stability of the progenies as non segregating generation reached. the non-segregating (pure-lines) and parent plants were laid in rows according to their genotypes. data collection nine quantitative traits were measured and data collected from the 3 parents and their progenies (improved lines) in single trial under non drought stress (ns) and reproductive drought stress (rs) treatment in five (5) replications in around 120 days after seeds sowing. these quantitative traits included; days to 50% flowering (df), height of plant (hp), panicle length (pl), effective tillers (et), tillers produced (t), fully filled grain (ffg), 100-grain weight (100-gw), grain length and width ratio (glw) and yield maturity(ym) as indicated in table 4. for ns and table 7. for rs treatments. statistical analysis all evaluated data were subjected to analysis of variance (anova) using statistical analysis software (sas) version 9.4 in complete randomized design (crd). the expressed results were in mean, mean square, correlation coefficient (cv) and standard deviation to set the relationship among the parameters (traits). to determine the genetic variability among the 9 quantitative traits, cluster analysis was employed. the genetic relationship among the parent varieties and improved rice lines was determined in conformity with unweighted pair group method using arithmetic average (upgma) algorithm and sequential, agglomerative, hierarchic and non-overlapping (sahn) method using numerical taxonomy multivariate analysis system, exeter software, setauket, ny, usa software (ntsys v2.1). table 1. description of major symbols symbol description pb pd pbd pdb dpb df hp pl et t ffg glw 100gw cross between putra1 and irbb60 cross between putra1 and mr219 three-way cross between putar1 and irbb60(f1) and mr219 drought tolerant double cross(from two f1s; p×d and p×b three-way reciprocal cross (between mr219 drought tolerant and f1 putra1×irbb60) days to 50% flowering height of plant measured from the base to the tip of panicle panicle length measured in centimetre effective tiller refers to those that had gains total number of tillers produced fully filled grain obtained by counting the number of grains produced per panicle grain length and width ratio of a grain 100grain weight is a measure of 100 grains in grams international journal of applied biology, 3(2), 2019 6 results and discussion genotypic and phenotypic selection of improved lines electrophoresis analysis (gel scoring) results with bands showing alignment to the recipient parents were selected as pure and non-segregating lines in the f4 single crosses for pd and pb, f3 three-way and reciprocal crosses (pbd, dpb) and f3(2) (double) cross (pdb) as shown in the figures 1, 2, 3, 4 and 5 below. challenging the plants with the disease pathogens and subjection to water deficit stress for the improved lines and susceptible varieties was undertaken. the results showed that the improved lines were resistant and moderately resistant to blast and bacterial leaf blight and as well tolerant to drought stress according to acceptable yield percentage for tolerant rice (miah et al. 2016, ashkani et al. 2011, yambao and ingram, 1988). the susceptible varieties in each case were truly susceptible to the disease pathogens and drought stress. figure 1. f3(2) cross for improved lines(pdb) figure 2. f3 three-way reciprocal cross of improved lines(dpb) figure 3. f3 three-way cross of improved lines pbd ym ds ns yield maturity is the number of days from sowing to harvest. reproductive drought stress non-drought stress international journal of applied biology, 3(2), 2019 7 figure 4. f4 single cross improved lines(pd) figure 5. f4 single cross for pb improved lines agronomic trait assessment of improved lines these are also considered as agronomic traits; days to 50% flowering, height of plant, panicle length, number of tillers, effective tillers, fully filled grain, 100-grain weight, grain length and width ratio, yield maturity. the effect of blast, bacteria leaf blight and drought on any of these parameters could result in poor yield and quality. improved lines were evaluated under two treatments condition, namely; non-drought stress (ns) and reproductive drought stress (rs) condition. under ns the data of both parents and improved lines were analysed, whereas in rs treatment, a susceptible and improved lines were analysed. they were to further validate the results of the southern blot analysis (genotyping) and the phenotyping results that showed resistances and tolerance to blast, bacteria leaf blight (blb) and drought. 8 table 2. polymorphic, linked/quantitative trait loci and flanking simple sequence repeat (ssr) markers for genes/qtls used. variety ssr markers genes/qtls chromosom e position expected base pair size description putra1 blast resistance rm6836 piz, pi2, pi9 6 240 polymorphic/linked rm8225 piz 6 221 polymorphic/linked irbb60 bacteria leaf blight resistance rm224 xa4 11 157 polymorphic/linked rm122 rm153 rm13 xa5 xa5 xa5 5 5 5 227 201 141 polymorphic/linked linked polymorphic/linked rg136 xa13prom xa13 xa13 8 8 246 polymorphic/linked polymorphic/linked rm21 pta248 xa-21 xa-21 11 11 157 polymorphic/linked polymorphic/linked mr219 ir99784-156-137-1-3 drought tolerance rm511 qdty12.1 12 130 polymorphic/linked rm1261 qdty12.1 12 167 polymorphic/flanking marker rm28099 qdty12.1 12 120 flanking marker rm28076 qdty12.1 12 287 flanking marker rm520 qdty3.2 3 247 polymorphic/linked rm236 qdty2.2 2 174 linked rm276 qdty2.2,3.1 6 149 flanking marker askani et al., 2011, miah et al., 2016, shamsudin et al., 2016, www.gramene.org, he et al, 2006, khan et al., 2015, pradhan et al., 2015 http://www.gramene.org/ international journal of applied biology, 3(2), 2019 9 table 3. anova for the parameters showing level of significance for non-drought stress (ns) source df df(no) hp(cm) pl(cm) et(no) t(no) ffg(no) 100gw(no) glw(cm) ym(days) var 13 6.15* 73.9** 33.39* 13.73ns 16.90** 336.66* 0.02** 0.61** 3.48* rep. 4 3.31ns 50.4ns 0.28ns 2.13ns 5.32ns 239.19ns 0.02ns 0.07ns 2.19ns error 52 3.13ns 24.6ns 1.04ns 5.31ns 5.33ns 117.92ns 0.009ns 0.1ns 1.52ns c. total 69 ∗significant at p≤0.05, ∗∗highly significant at p≤0.01, ns: non-significant p> 0.05, df. (degree of freedom), var. (variety), rep. (replications), c. total (corrected total), df (days to 50% flowering), hp (cm) (height of plant measured in centimetres), pl (panicle length in centimetres), et (effective tillers), tillers (total number of tillers), ffg (fully filled grains), 100-gw (100 grain weight in grams) international journal of applied biology, 3(2), 2019 10 figure 6. comparison of agro-morphogical and yield parameters of parent plants and their progenies (improved lines) in nondrought stress condition 0 20 40 60 80 100 120 140 160 180 200 m e a n parent plant and progenies mean comparison of agro-morphogical and yield parametersbetween parents and progenies in nondrought stress condition df hp pl et t ffg 100gw glw ym international journal of applied biology, 3(2), 2019 11 table 4. mean squares, least significant differences, coefficient of variations, standard deviation and broad sense heritability of vegetative traits and yield components of different rice varieties (genotypes) for reproductive drought stress and non drought stress treatments. varieties df(no) rs ns hp(cm) rs ns pl(cm) ns rs et(no) ns rs t(no) ns rs ffg ns rs putra1 89a 103.19c 25.03a b 10.2bcd 10.6bcde 172.8ab drought 88ab 100.3c 25.9ab 13.6a 13.8a 168.4abc irbb60 87.8abc 96.6a 103.61c 97.52a 25.06b 20.68c 7.8de 9ab 8.8de 9.6ab 172.4ab 26.4c pb12 87.6abc 104.34c 24.9b 9.4bcde 9.6cde 177.6a pb15 86.8abcd 102.64c 25.9ab 9cde 9.2de 166abcd pd14 85.8bcd 91.8d 102.56c 96.6a 25.28b 23.7ab 10.6bcd 11.4a 11abcd 11.4ab 178.4a 52.4ab pd15 87.4abcd 92.8cd 104.54c 97.32a 24.66b 22.6ab 9.6bcde 8b 11abcd 8.4b 157.2cd 49.8ab pdb3 87.4abcd 92.8 104.06c 100.6a 24.88b 24.1a 11.2abc 10ab 13ab 10ab 153.4d 48.2ab pbd1 87.6abc 94bc 104.72c 100.5a 24.9b 22.1bc 11abc 11.6a 12.4abc 11.6a 170.6abc 45.4b pbd3 88ab 94bc 99.9c 96.22a 22.86c 23.3ab 12ab 11.6ab 13.2ab 10.6ab 168.4abc 59.4a dpb7 85.2d 91.8d 113.44a 102.1a 25.04b 24a 9.4bcde 11.8a 9.8cde 11.8a 161.4bcd 53.4ab dpb12 86bcd 92.8cd 106.1bc 99.68a 25.04b 23.4ab 7.2e 9.2ab 7.8e 9.4ab 152.4d 52.2ab dpb13 86.2bcd 93cd 103.6c 98.44a 25.18b 23.4ab 9.4bcde 10.2ab 9.6cde 11.4ab 161bcd 43.76b dpb20 85.6cd 95b 112.3ab 96.3a 26.58a 23.7ab 9cde 10.2ab 9.2de 10.4ab 165.8abd 46.2b lsd 2.25 1.56 6.29 11.82 1.3 1.74 2.92 3.33 2.93 3.07 13.78 11.83 cv 2.03 1.3 4.74 9.35 4.07 5.87 23.13 25.43 21.69 22.86 6.54 19.34 mean 87.03 93.46 104.67 98.54 25.09 23.09 9.96 10.2 10.64 10.46 166.13 47.72 std dev. h2 b (%) 1.93 16.09 1.79 56.18 5.95 28.63 8.51 -17.54 1.20 31.13 1.55 28.40 2.59 24.03 2.65 2.18 2.74 2.43 30.24 -0.93 12.89 27.06 11.85 41.43 heritability percentage categorised as low (0-30%), moderate (30-60%), and high (≥60%) 12 table 5. continued varieties 100gw(g) ns rs glw(cm) ns rs ym(days) ns rs putra1 2.41de 4.97bc 117.4ab drought 2.47cde 4.98b 117.4ab irbb60 2.35e 2.43a 5.61a 4.42a 116.8b 133.2a pb12 2.55abc 4.96bc 118.2a pb15 2.49cde 4.81bcd 117.6ab pd14 2.55abc 2.34a 4.86bcd 4.49a 117.4ab 128.4b pd15 2.52abcd 2.4a 4.86bcd 4.49a 117.6ab 127.6b pdb3 2.47cde 2.38a 4.53d 4.5a 117ab 127.8b pbd1 2.63a 2.42a 4.59bcd 4.51a 117.6ab 127.8b pbd3 2.52abcd 2.38a 4.63bcd 4.47a 117.6ab 129b dpb7 2.52abcd 2.32a 4.51cd 4.57ab 117ab 127.4b dpb12 2.52abcd 2.35a 4.57cd 4.75a 117.6ab 128.2b dpb13 2.6ab 2.39a 4.51d 4.57ab 117ab 128.2b dpb20 2.56abc 2.43a 4.08e 4.53 117.8ab 127.6b lsd 0.12 0.11 0.4 0.2 1.31 1.8 cv 3.83 3.75 6.63 3.5 0.88 1.09 mean 2.51 2.39 4.75 4.53 117.43 128.52 std dev. h2 b (%) 0.11 16.67 0.09 0.5 0.44 50.50 0.17 6.25 1.02 -7.27 2.05 56.22 var. (variety), irbb60 (b) (bacteria leaf blight resistance), drought (d) (mr219 drought tolerance), reciprocal cross with mr219 drought, putra 1 and irbb60 (dpb), putra1, irbb60 and mr219 drought tolerance (pbd), putra1, mr219 drought tolerance and irbb60 (pdb), putra1 and mr219 drought tolerance (pd), putra1 and irbb60 (pb), lsd(least significant difference), cv(coefficient of variation), mean(average), df(days to 50% flowering), hp(cm)(height of plant measured in centimeters), pl(panicle length in centimeters)et(effective tillers), tillers (total number of tillers), ffg(fully filled grains), 100-gwt(100 grain weight in grams), ym (yield maturity counted on number of days from sowing to physiological maturity). agro-morphological and yield traits data for single cross (f4), double cross (f3(2)), and threeway cross (f3) for non-drought stress. some parameters of pure-line agro-morphological and yield traits were observed and measured to ascertain the differences between the various rice lines and the parent plants. table 3. shows an analysis of variance (anova) of 9 traits, the varieties were all significant with the exception of effective tillers (et). days to 50% flowering (df), panicle length (pl) and yield maturity (ym) were significant at 5% level of probability (p≤0.05), while height of plant (hp), tillers (t), fully filled grain (ffg), 100gw, grain length and width ratio (glw) were significant at 1% level of probability (p≤0.01). no significant differences among the replications were recorded in all the morphological and yield traits under non drought stress. comparison of parameters of non-drought stress (ns) and reproductive drought stress (rs). table 4. and table 5. compares nine (9) mean data of the two treatments of nondrought stress (ns) and reproductive drought stress (rs). the comparison was between improved lines from the three crossed methods. even though there were 14 lines in all on ns and 10 on rs because, non drought stress had the three (3) parent plants and two (2) single cross lines pb12, pb15 which did not have drought tolerance qtls to be subjected in to international journal of applied biology, 3(2), 2019 13 reproductive drought stress treatment. now, the parameters of days to 50% flowering (df), height of plants (hp), fully filled grain (ffg), yield maturity (ym) showed that the means of reproductive drought stress (rs) was higher on all the improved lines and each compared to their non-drought stress lines. while days to 50 % flowering on ns has the highest and lowest number of days as 89 and 86 days respectively. rs had 91.8 and 96 days for lowest and highest respectively. height of plants (hp) for ns measured 102.3-112.3 cm and rs 96.3-102.1 cm for lowest and highest respectively. the length of panicle for ns was 22.86 cm-26.5 cm and rs was 22.6 cm-24.1 cm for lowest and highest respectively. there was an exceptional difference between the line pbd3, which showed that the rs measured 23.2 cm in length whereas the ns was 22.86 cm in length. the mean number of fully filled grain on ns lines had lowest mean as 152.2 and highest 178, and rs was 43.7 and 59.4 for lowest and highest count respectively. the parameter of 100-grains weigh measurement taken indicated that the lowest weight for ns was 2.52g and the highest weight was 2.63g, for the rs it was 2.32-2.43g, lowest and highest respectively. there were slight variations between the two treatments among some lines on the parameters of grain length and weight ratio. even though, the ns ratio was higher than rs on five (5) improved lines, four (4) of the rs and reciprocal lines of dpb7, dpb12, dpb13, dpb20 were higher than their counterpart lines. effective tillers (et) and number of tiller (t) didn’t show any clear difference between the two treatments. it was reported that water deficit stress affect these quantitative traits which was also confirmed in this study (latiffe et al., 2004; atlin et al., 2006; barnabas et al., 2008; garrity and o’toole, 1994). there were alternating variation among them. this was because the treatment for drought was at the reproductive stage when the tillers were matured and could not be affected by just about two weeks of water deficit stress. record has it that drought affect tillers at vegetative stage (cruz et al., 1986). in consideration of non drought stress (ns) in the table 4. and table 5. with 14 lines (parental and improved lines), it indicated the mean days to 50% flowering of putra1 was 89 days. this was also corroborated with the findings of miah et al. (2015) on the same trait of the same variety (putra 1) and significantly different from other traits, but all other traits were not significantly different from each other. dpb7 was significantly different on height of plant, while the other 8 traits were not different from each other. they tally with their parent plants, which indicated their was no variation. dpb20 and pbd3 were significantly different from each other and the other genotypes on parameter of panicle length. the other genotypes including the parent plants were not different from each other. all other parameters did not show difference between parents and progenies except the parameters of effective tillers, tillers and grain length and width ratio showing significant difference between irbb60 and others, although with some similarities with other genotypes. considering the means of reproductive drought stress (rs), it has indicated that the improved lines were different from the susceptible (control) variety because, the improved lines were introgressed with drought tolerance qtls (qdty) which conferred on it ability to adjust to some level of water deficit stress as against the control check that had no drought tolerance qdty. field evaluation of mr219 carrying these three qdty2.2, qdty3.2, qdty12.1, confirmed tolerance to reproductive drought stress with grain yield of 756-2521 kg ha-1 for 2013 and 903-2523 kg ha-1 in the year 2014, while the susceptible variety had grain yield of 13 kg ha-1 in 2013 and didn’t flower in 2014 (shamsuddin et al., 2016). venuprasad et al. (2009) and swamy et al. (2013) reported tolerance ability of qdty3.1 and qdty2.2 respectively. mishra et al. (2013) reported that twenty one (21) experiments conducted in eastern india international journal of applied biology, 3(2), 2019 14 and irri confirmed that qdty12.1 has an effect that increases with increasing severity of water deficit stress. these confirms the reason the improved lines performed better compared with its susceptible check. heritability estimate on non drought stress (ns) and reproductive drought stress (rs) treatments. broad sense heritability is the total ratio of genetic variance to phenotypic variance. in order words, the proportion of the parental gene inherited in the progenies that are influenced by the environment and expressed in the phenotypic traits. the percentage ratio is expressed as low, medium and high represented as 0-30%, 30-60% and ≥60% respectively. the broad sense heritability of characters studied for non drought stress (ns) and reproductive drought stress (rs) treatments (table 4 and table 5) were all within the range of 0 to 56.22, which was within low to moderate heritability. these indicated the magnitude of heritability that was influenced by environmental factors. low percentage is higher environmentally influenced and moderate heritability is lesser. climatic factors which are environmental in nature presents a great challenge to rice plant, there are various optimum temperature requirements for various growth phases and stages, outside the range it affects that phase of development (tashiro and wardlaw, 1989, baker and allen, 1993, singh et al., 1996, http:ricepedia.org). control experiment and field experiment often presents a variation in response to environmental factors. for instance, irri-ses (2013) categorised scoring and protocols for diseases and tolerance evaluation for green or glass house experiment differently from field. the low and medium heritability may not present a true genotypic content of the improved lines in respect of the treatment since heritability estimate in this case is not restrictive and does not consider experimental environment. the varieties used as recipient parents were all high yielding, resistant and tolerant. and confirmed to be so resistant and tolerant by the genotyped and phenotyped results. the magnitude of heritability among the parameters of non drought stress (ns) showed that all characters were within the lowest percentage of heritability which suggests environmental influence, except days to 50% flowering (df), fully filled grains (ffg) and yield maturity (ym) which were medium ranged as; 56.18%, 41.43% and 56.22% respectively. reproductive drought stage (rs) treatment showed characters that were also low ranged with exception of traits of panicle length 31.13%, tillers (t), 30.24% and grain length and width ratio (glw), 50.50% as medium heritability, indicated as having lesser environmental influence compared to low heritability. all ranges of heritability percentage estimates were also reported by oladosu et al. (2014), meena et al. (2016), ridzuan et al. (2018). the low heritability may not present the true nature of the inherited genes, because controlled environment of research may present a variation different than when in a field environment. international journal of applied biology, 3(2), 2019 15 table 6. estimates of correlation coefficients on the phenotype among 9 traits in rice lines for non drought stress ∗significant at p≤ 0.05, ∗∗highly significant at p≤ 0.01, ns: non-significant p> 0.0 df(no) hp(cm) pl(cm) et(no) t(no) ffg(no) 100gw(g) glw(cm) ym(days) df(n0) 1 hp(cm) -0.26246* 1 pl(cm) -0.2019 0.30623** 1 et(no) 0.00606 -0.07271 -0.11752 1 t(no) 0.00196 -0.12024 -0.24529* 0.92492** 1 ffg(no) 0.00394 -0.13601 0.06923 0.08221 -0.00771 1 100gw(g) -0.2765* 0.07116 0.06867 0.01494 -0.03721 0.13656 1 glw(cm) 0.34151** -0.26206* -0.11788 0.01078 0.00824 0.09208 -0.29898 1 ym(days) -0.01376 0.1382 0.05447 0.11179 0.04018 0.23267 0.17624 -0.12587 1 16 correlation coefficient relationship for non-drought stress the r-values and test of significance provided by proc corr in sas program (table 6) showed days to 50% flowering (df) and height of plant (hp), 100-grain weight were significantly different (p≤0.05), while with grain length and width ratio showed high significant difference (p≤0.01). all the traits were low and negatively correlated. height of plant (hp) with panicle length (pl) and grain length and width ratio (glw) were significant at (p≤0.05) and (p≤0.01) respectively. both were similarly low and negatively correlated. panicle length (pl) and effective tillers (et) were jointly significant to tillers (t) at (p≤0.05) and (p≤0.01). while the former is low and negatively correlated, the later is high and positively correlated. notable high correlated relationship was observed between height of plant (hp), effective tillers and 100gw. the relationship with et though both were high but it was strongly negative, while with 100gw was strong positive linear relationship. ridzuan et al. (2019) and oladosu et al. (2018) also used phenotypic traits to determine relationships. karl pearson’s correlation coefficient r-value helps to identify association that exists between two unique traits, even though it is not able to measure the magnitude (extent) of association but gives a clue as to the relationship. the interpretation of correlation coefficient was given, but ratner (2009) provides an accepted standard guideline. the r-value could indicate no linear relationship, positive linear relationship and negatively linear relationship represented by 0, +1, and -1 respectively. low, moderate and strong positive linear relationships are represented with values ranging from 0-0.3, 0.3-0.7 and 0.7-1, respectively while 0 to -0.3, -0.3 to -0.7, and -0.7 to -1 would indicate low, moderate and strong negative linear relationship respectively. international journal of applied biology, 3(2), 2019 17 table 7. anova for the parameters showing level of significance for reproductive stage drought stress (rs) ∗significant at p ≤ 0.05, ∗∗highly significant at p ≤ 0.01, ns: non-significant p > 0.05, df. (degree of freedom), rep.(replications), cor. total (corrected total), df(days to 50% flowering), hp(cm)(height of plant measured in centimetres), pl(panicle length in centimetres), et(effective tillers),tillers (total number of tillers), ffg(fully filled grains), 100-gw(100 grain weight in grams) source df df(day) hp(cm) pl(cm) et(no) t(no) ffg(no) 100gw(g) glw(cm) ym(days) var 9 11.02** 21.53ns 5.47** 7.47ns 6.22ns 384.90** 0.01ns 0.04ns 14.63** rep. 4 0.93ns 74.74ns 0.62ns 8.65ns 7.13ns 86.59ns 0.01ns 0.02ns 0.97ns error 36 1.49ns 84.94ns 1.84ns 6.73ns 5.72ns 84.83ns 0.01ns 0.03ns 1.97ns cor. total 49 18 table 8. estimates of correlation coefficients on the phenotype among 9 traits in rice varieties for reproductive drought stress df(days) hp(cm) pl(cm) et(no) t(no) ffg(no) 100gw(g) glw(cm) ym(days) df(days) 1 hp(cm) -0.11295 1 pl(cm) -0.34917* 0.04025 1 et(no) -0.1276 0.02553 0.06464 1 t(no) -0.11063 0.02957 0.05282 0.98204** 1 ffg(no) -0.35762* -0.06439 0.24756 0.1528 0.1108 1 100gw(g) 0.26915 -0.0041 -0.18723 -0.13435 -0.13343 -0.11789 1 glw(cm) -0.21732 0.08631 0.17222 0.07339 0.0711 0.2032 -0.00037 1 ym(days) 0.59004** -0.11094 -0.53245** -0.0833 -0.06109 -0.4332* 0.13789 -0.22637 1 19 anova for agro-morphological and yield traits data for single cross (f4) double cross f3(2), and three-way cross and reciprocal (f3) under reproductive drought stress (rs). phenotyping for reproductive drought tolerance was carried out to determine the level of improved lines tolerance to water deficit condition. this is very importance due to challenges of climate change and irregular rainfall for rainfed rice and also for irregular irrigation water supply. the anova table 7. showed highly significance difference (p≤0.01) among varieties (improved lines) on the parameters for df, pl, ffg, ym, while there was non among the replicates. this clearly indicated the effect of drought on these very important parameters (quantitative traits) whose effect affect yield drastically (taglea et al., 2016, chang et al., 2016). correlation coefficient relationship for non-drought stress in table 8 which estimated the relationship among phenotypes of the reproductive drought stress traits indicated that days to 50% flowering (df) had a significant relationship with panicle length (pl) and fully filled grain (ffg) at (p≤ 0.05) although negative and moderate but yield maturity (ym) at (p≤0.01), it was positive and moderate correlated relationship. panicle length (pl) and yield maturity (ym), effective tillers (et) and fully filled grain (ffg) were both significant at p≤0.01. while the former is negative and moderately correlated the later was low but positively correlated. the strongest positively and high correlated relationship but not significant was between effective tillers (et) and total number of tillers per plant (t). it is because most of the tillers were effective despite the drought stress. stress was introduced at the reproductive stage when tillers were already matured and it could not affect it. international journal of applied biology, 3(2), 2019 20 table 9. anova for the parameters showing interaction levels of significance ∗significant at p ≤ 0.05, ∗∗highly significant at p ≤ 0.01, ns: non-significant p > 0.05, df. (degree of freedom), rep.(replications), cor.. total (corrected total), df(days to 50% flowering), hp(cm)(height of plant measured in centimetres), pl(panicle length in centimetres), et (effective tillers),tillers (total number of tillers), ffg(fully filled grains), 100-gw(100 grain weight in grams), ym (yield maturity counted on number of days from sowing to physiological maturity). sov df df(days) hp(cm) pl(cm) et(no) t(no) ffg(no) 100gw(g) glw(cm) ym(days) treatment 1 1020.17* * 1091.15* * 69.17** 7.04ns 0.02ns 325366.67* * 0.60** 0.18ns 2733.4** varieties 10 6.22* 67.75ns 3.70** 10.30* 11.87* * 307.53** 0.01ns 0.19** 1.09ns replications 4 0.89ns 89.62ns 1.89ns 10.26ns 8.51ns 165.90ns 0.01ns 0.08ns 1.92ns trt*var 8 3.6ns 42.50ns 2.59* 5.43 10.66* 211.25* 0.01ns 0.17* 0.75ns error total 76 99 2.52 53.68 1.13 4.33 4.40 90.70 0.01 0.06 1.25 international journal of applied biology, 3(2), 2019 21 table 10. correlation coefficient of interaction of non drought stress (ns) and reproductive drought stress (rs) treatments. df(days) hp(cm) pl(cm) et(no) t(no) ffg(no) 100gw(g) glw(cm) ym(days) df(days) 1 hp(cm) -0.43695** 1 pl(cm) -0.5883** 0.39373** 1 et(no) 0.12171ns -0.00503ns -0.22585* 1 t(no) 0.02853ns -0.00643ns -0.23369* 0.92342** 1 ffg(no) -0.87446** 0.37901** 0.55552** -0.09132ns 0.0005ns 1 100gw(g) -0.60745** 0.30235** 0.35449** -0.1395ns -0.0953ns 0.68357** 1 glw(cm) -0.00568ns -0.05578ns -0.02718ns 0.10715ns 0.11803ns 0.15551ns 0.11917ns 1 ym(days) 0.87083** -0.41243** -0.56762** 0.13997ns 0.01887ns -0.95227** -0.64997** -0.14577ns 1 ∗significant at p ≤ 0.05, ∗∗highly significant at p ≤ 0.01, ns: non-significant p ≤ 0.05, df. (degree of freedom), rep.(replications), cor..total (corrected total), df(days to 50% flowering), hp(cm)(height of plant measured in centimeters), pl(panicle length in centimeters), et(effective tillers),tillers (total number of tillers), ffg(fully filled grains), 100-gw(100 grain weight in grams), ym (yield maturity counted on number of days from sowing to physiological maturity). international journal of applied biology, 3(2), 2019 22 interactions of reproductive drought stress (rs) and non drought stress (ns). there were interactions between treatment and varieties (trt*var.) in table 9. panicle length (pl), tillers (t), fully filled grain (ffg) and grain length and width ratio (glw) interacted at p≤0.05. while the other parameters were not significant. the significant interactions was because of the variation in treatments (reproductive drought stress (rs) and non drought stress (ns)) while the varieties comprised of data from the improved lines (progenies) subjected to stress and non stress conditions. shortage of water was responsible because of its dire need for living cells functioning and turgidity (sukhla, 2012). the parameters affected are yield borne especially fully filled grains (ffg). juraimi et al. (2009), sikuku et al. (2009), masitah, (2018) reported that days to flowering, yield maturity, fully filled grain, panicle length are affected by drought stress. variation of significance on major yield component was also attributed to biochemical, physiological, morphological and anatomical effect of water deficit condition (serraj et al., 2009). reproductive stage of rice is a very sensitive stage and responded to drought stress in affecting flowering and heading as also confirmed by davatgar et al. (2009). drought stress often results in low tissue water potential for rice (sikuku et al., 2010). generally, low yield underscore the importance of water (juraimi et al., 2009). correlation coefficient of interaction the interaction correlation coefficient table 10. showed highly significant interaction between days to 50% flowering (df) with height of plant (hp), panicle length (pl), fully filled grain (ffg), 100 grain weight (100gw) and yield maturity (ym). all correlation coefficient relationships were either negatively medium or high, with the exception of yield maturity which was both strong and positively correlated relationship. this clearly indicated the influence of days to 50% flowering to yield maturity due basically to water stress treatment which delayed maturity because of distortion of the normal physiological and biochemical processes of the rice plant (serraj, 2009; juraimi, et al., 2009). for height of plant (hp), there were highly significant (p≤0.01), negatively medium correlation relationship with panicle length (pl), effective tillers (et), 100grain weight (100gw) and yield maturity (ym). the relationship between effective tillers (et) and tillers (t) was significant p≤0.01 but with strong negative correlation. fully filled grains (ffg) was also highly significant (p≤0.01) with 100 grain weight (100gw) and yield maturity (ym) at medium and strongly negative correlated relationship respectively. the parameter of 100 grain weight (100gw) was highly significant (p≤0.01), medium and negatively correlated. clustering and principal component analysis the analysis of genetic variability is one important criterion for parental selection by the estimate of the extend of variation that existed among the genotypes. the specific information on the nature and degree of genetic variability is critical for selection of ideal parent so as to minimize the number of crosses that would have been required. (guerra et al., 1999, yatung et al., 2014). international journal of applied biology, 3(2), 2019 23 non drought stress the study considered 14 genotypes which comprised of the 3 parents and their progenies (improved lines). they were clustered into four main groups based on their quantitative characteristics at genetic similarity of 0.122 in order of distance to the various genotypes in the population. clustering of 39 genotypes according to morphological characteristics majorly on the size of fruit was reported by geleta et al., 2005. the genetic distance dendrogram was constructed in accordance to the upgma method using ntsys-pc version 2.1. the figure 7. showed the clustered genotypes into four groups. groups iii and iv comprised of two genotypes each namely dpb7, dpb20 and pb15, pbd3 respectively. group i and ii comprised of 6 and 4 genotypes respectively. while putra 1, irbb60, pbd1, drought (mr219 drought tolerance), pb12,pd14 were clustered in group i, pd15, pbd3, dpb13 and dpb12 were clustered in group ii. there parental plants and were clustered along with the first two crossed generations that produced the three-way and double crosses. the pca results are indicated in figure 8. the farthest genotypes from the centroid were pbd3. pb15, pd14, pb12, dpb7, dpb12, while the closest to the centroid were dpb13, pd15, pdb3. three genotypes were intermediary between the farthest and closest, which included; putra1, irbb60 and pbd1. international journal of applied biology, 3(2), 2019 24 figure 7. clustering pattern of the agro-morphological and yield based on 14 trait at dissimilarity coefficient of 0.122 coefficient 0.050.230.410.590.77 drought putra1 irbb60 pbd1 drought pb12 pd14 pd15 pdb3 dpb13 dpb12 dpb7 dpb20 pb15 pbd3 group i group iv group iii group ii 0.122 25 figure 8. principal component analysis (pca) of relationship among fourteen (14) rice lines in a 2-dimentional graph pc1 pc2 26 reproductive drought stress in this study, 10 genotypes (lines) were clustered together into six groups based on nine quantitative traits at the reproductive stage drought stress (rs) to separate different genotypes in the population. upgma method using ntsys-pv v2.1 was similarly used to construct the genetic distance dendrogram. the genotypes here were clustered into 6-group at 0.094 genetic similarity as shown in figure 9. group i, iii, iv and vi comprised of only one line (genotype) each such as control, dpb7, pdb3 and pbd3 respectively. while group ii and v each comprised of three genotypes; pd14, pd15, dpb12 and pbd1, dpb13, dpb20 genotypes respectively. figure 10. showed the result of pca. the genotypes distanced away from the centroid in the order of distance comprised of control, pbd3, dpb7, and three genotypes were intermediary such as pbd1, dpb20, pd14, while the others were closest to the centroid, and they included pd15, dpb13, dpb12 and pdb3. group i stood out as the lowest with yield since it was the control and was susceptible to rs. table 11. grouping of 14 and 10 improved and selected lines for non drought stress (ns) and reproductive drought stress (rs) respectively for cluster analysis treatment cluster number number of genotypes genotypes ns i 6 putra1, irbb60, pbd1, drought, pb12, pd14 ii 4 pd15, pdb3, dpb13, dpb12 iii 2 dpb7, dpb20 iv 2 pb15, pbd3 rs i 1 control ii 3 pd14, pd15, dpb12 iii 1 dpb7 iv 1 pdb3 v 3 pbd1, dpb13, dpb20 vi 1 pbd3 international journal of applied biology, 3(2), 2019 27 figure 9. relationship among the 10 developed and selected lines (genotypes) based on 9 characteristics (traits) using sahn clustering of upgma method coefficient 0.050.160.260.360.47 control control pd14 pd15 dpb12 dpb7 pdb3 pbd1 dpb13 dpb20 pbd3 0.094 group i group iv group v group vi group ii group iii international journal of applied biology, 3(2), 2019 28 figure 10. principal component analysis of relationship amongst 10 improved lines and selected lines in two dimensional graph pc1 pc2 29 conclusion the two recipient parents (putra1 and mr219 drought tolerant) used in the different crossing methods were high yielding, resistant and tolerant to blast (magnaporthe grisea) and drought respectively. and a donor irbb60 resistant to bacteria leaf blight (xanthomonas oryzae). the yield parameters showed that there was no yield loss on non-drought stress treatment on improved lines except on reproductive drought stress treatment. the interactions showed that reproductive drought stress affected days to flowering, fully filled grain and yield maturity significantly. the profitability of these parameters are often dependent on the reproductive stage, but shortage of water tempered with their due performances. even though there was reduction in yield, but it was still within average yield performance of drought tolerant variety due to the presence of introgressed genes/qtls compared with the susceptible variety in reproductive drought stress (rs) treatment. prior knowledge of the rice genotype is important in making informed decision on selection of the best cultivars for variety improvement. genetic variation is an indication of the possibility of diverse source of origin and the result gave a clue as to the genetic/qtl variance of the rice populations. in consideration of the variation pattern and other agro-morphological and yield performance, the improved and selected lines pb12, pbd1, and pd14 in group i, pdb3 in group ii, dpb20 in group iii and pb15, pbd3 in group iv could be considered as better lines on good yield under non drought stress (ns) treatment. in reproductive drought stress (rs) treatment, pd14, dpb12 in group ii, dpb7 in group iii and pdb3in group iv. while in groups v and vi were pbd1, dpb20 and pbd3 respectively. generally, the yield performances of the different lines for both ns and rs were all within considerable high yielding range, with high potentials for increased yield with different environmental trials since the qtls could be influenced by 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xa-4, rm122;xa-5, rg136; xa-13, rm21;xa-21) and drought tolerance (rm236;qdty2.2, rm520;qdty3.1, rm511;qdty12.1) in pedigree, backcross and recurrent selection breeding methods. the objectives are to create awareness on the environmental safety of host-resistance, significance of single multiline resistance variety, effect of the interaction of stress conditions and associated simple sequence repeat (ssr) linked markers. issn : 2580-2410 eissn : 2580-2119 a review on gene pyramiding of agronomic, biotic and abiotic traits in rice variety development ibrahim silas akos1,2, mohd rafii yusop*1,3, mohd razi ismail1,3, shairul izan ramlee3, noraziyah abd aziz shamsudin4, asfaliza binti ramli5, bello sani haliru1, muhammad ismai’la1, & samuel chibuike chukwu1 1laboratory of climate-smart food crop production, institute of tropical agriculture and food security, universiti putra malaysia, 43400 upm serdang, selangor, malaysia. 2department of crop science, faculty of agriculture, kaduna state university, kafanchan campus, nigeria. 3department of crop science, faculty of agriculture, universiti putra malaysia, 43400 upm serdang, selangor, malaysia. 4school of environmental and natural resource sciences, faculty of science and technology, universiti kebangsaan malaysia. 5malaysian agricultural research and development institute, rice research centre, persiaran mardi-upm, 43400, serdang, selangor, malaysia. open access international journal of applied biology international journal of applied biology is licensed under a creative commons attribution 4.0 international license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. keyword agronomic, abiotic and biotic, gene pyramiding, rice, traits. article history received 2 october 2019 accepted 29 december 2019 66 introduction rice is an important staple food crop, which forms a major part of the human diet and a good source of carbohydrate for over half of the world’s human population. asia alone produces 90% of the world produced rice, with irrigated lowland providing 75% of the world’s production in about 79 million ha of arable land (bourman et al. 2007; maclean et al. 2002) in the year 2010, global statistics showed that, harvested rice was from an estimated cultivated land area covering 154 million ha. 137 million ha of the cultivated land representing 88% was from asia, while southeast asia alone, cultivated 48 million ha, representing 31% production area from the world’s harvested areas. rain-fed lowland accounted for more than 55-60% of the total rice-cultivated land (redfern et al. 2012; toojinda et al. 2005). however, by the year 2030 minimum of 40% additional production, from the total global estimate is required to meet the need of the ever increasing population, most especially in asia (khush 2005). the region is always faced with abiotic and biotic stresses that hinder yield and quality of grains of its high yielding varieties. to sustain production, irrigated rice cultivation should be explored, because it offers a better option, as a result of current challenges and effect of climate change, resulting in severe water deficits in the rain-fed lowlands and also frequent flooding. these form part of the major agricultural constraints in these regions since they are unpredictable (dey and upadhyaya 1996). abiotic conditions include; salt, extreme temperature, drought, submergence, mineral deficiency, wounding and oxidative stress among others, while biotic stresses could include, and not restricted to fungal pathogens of blast, bacteria leaf blight and insects, viruses, false smut, sheath blight. these stress conditions affect leaf length and width (flag-surface area), tillering, length of panicle, filled grains number/panicle, 1000 grain weight (seed quality), thereby resulting in low yield. the simultaneous assemblage of agronomic traits of yield, biotic and abiotic resistance and tolerance respectively are through pyramiding approach, resulting in development of a multi-line resistance variety. this process employs marker-assisted selection, through; pedigree, backcrossing or recurrent selection breeding method. this is carried out by introgressing the desired multiple quantitative trait loci (qtls) into a desired background high yielding variety of rice, in the case of pedigree method (servin et al. 2004). pradhan et al (2015) reported that, pyramiding of multi-resistance genes into single line often confers wider spectrum of resistance and durability. the objectives aress to raise awareness on the cost-effect and environmental safety associated to development of host resistance variety of rice, to inform breeders of the significance of multiple disease resistance to blast, bacteria leaf blight diseases (biotic) and drought tolerance (abiotic) conditions, the effect of interaction of these stresses on plant development, and the ssr markers linked to blast, blb and drought for marker-assisted selection. agronomic traits of rice seeds are the most valuable component of the agronomic traits of rice, and high yield is the prime goal of crop production. therefore, to determine rice yield; culm length (cl), leaf length (ll), days to heading (dth), leaf width (lw), spikelet number per panicle (sn), panicle number per plant (pn), 1000-grain weight (gw), panicle length (pl), fully filled grain (ffg) and flowering time are important parameters for consideration and improvement (fujita et al. 2014, xue et al. 2008). these will however, result in increased yield even in limited arable 67 land, with capacity to meet the food demand of the ever increasing world population. reduction in rice yield is usually connected to increase in the percentage of spikelet sterility and number of spikelets per panicle (shamsudin et al. 2016). this spikelet encloses the rice grain, borne at its terminal ends. these yield and their related traits are complex and controlled by many quantitative trait loci (qtls). leaves formation is crucial as it forms an integral part of the agronomic trait, it occurs in two positions on the culm, each one at a node. it is made up of sheaths and blades, which basically form the surface area for photosynthetic activities, for manufacture of food substances (rice) borne on the panicles. the wider the surface area (flag leaf surface) and presence of chlorophyll pigmentation (with absence of diseases) on leaves the better, a good trait and sign for higher grain formation. the vegetative and floral organs are often the target of disease pathogens. abiotic stresses in rice these are non-component part of the environment which exerts stress to the rice plant, thereby affecting its productivity. many of these stress factors have been identified, among which are; mineral deficiency, salinity, drought, wounding, submergence, temperature extremes, oxidative stress and nutrient deficiency. these have quite a lot of impact on agricultural practices globally, with an estimated average yield reduction of major crop plants to more than 50% (wang et al. 2003). drought it is an important economic challenge for sustainable rice production, and yield in mainly rain-fed growing regions in countries of asia. drought stress imposes a great threat on rice cultivation, with recorded effect on over two million (2,000,000) hectares in asia (bray et al. 2000, kumbhar et al. 2015). in almost every cropping year, severe drought affects production of rice in south, southeast asia and africa (luo and zhang 2001). 45% of approximate rice growing areas of asia do not have stable access to irrigation sources and are often subjected to drought (crosson 1995). all stages of rice growth with even early reproductive stage could be adversely affected by drought, leading to drastic yield reduction. at the meiotic stage, some cultivated variety’s yield is reduced (swamy and kumar 2013), likewise in cereal crops, like wheat and barley (barnabas et al. 2008; boyer and westgate 2004). the resultant effect of drought to rice yield is often attributed to increased percentage sterility of spikelet (fukai et al. 1999; jondee et al. 2002; liu et al. 2006) and number of spikelets per panicle (boonjung and fukai 1996). the time and duration of stress on rice also determines the level of seed yield potential (garrity and o’toole 1994). the unpredicted and unsustainability of rainfall pattern due to climate change, is responsible for severity of drought being experienced, along with source of irrigation, type of soil, availability of water all through and out of seasons, and crop’s growth stage (swamy and kumar 2013). fewer water supplies for farming globally informed the need to improve adaptability to drought, and the search for resistance varieties as a crucial necessity (pandey and sukla 2015). many of the genes involved for response of plant to drought stress have been identified (zhang et al. 2012). this drought stress response result to changes in metabolism, physiology and reduction in growth (sairam and srivastava, 2001), it ultimately alters physiological process thereby reducing germination, photosynthesis and seedling vigour (pandey and sukla, 2015). 68 iron toxicity this is mostly associated to presence of soils of adjacent slopes, or iron in parent rock from which ground-water flows into lowland as a result of poor drainage (warda, 1988). fetoxicity can be traced to wide range of soils, such as; peat soils, valley-bottom soils and acidclay soils receiving interflow water from adjacent slopes and acid sulfate soils (becker and asch 2005). plants are able to take up fe in ferocious form, under anaerobic and acidic conditions (ph˂5) of water logged soils. rice is sensitive to fe toxicity immediately after trans-planting to tillering and also during heading/flowering (prade et al. 1990). average yield loss of 30% has been recorded, although it varies depending on variety and intensity of toxicity (abifarin 1989; masajo et al. 1986; warda 1997). salinity salinity occurs due to poor irrigation practices and lack of efficient drainage system, which leads to an upsurge in water table and rise in soil salinity and sodium/alkalinity (bertrand et al. 1993, miezan and dingkuhn 2001). salinity poses stress to rice, which is most sensitive at seedling and reproductive stage (gregrio et al. 2002). it is more severe in arid climates than humid, which implied environmental variance. 50% yield loss was recorded in humid tropics, at an electric conductivity (ec) of 9.5ms/cm (flower and yeo 1981), while at arid, the equivalent yield loss of 50% was recorded at ec of 3.5ms/cm (dingkuhn et al. 1993). these conditions affect rice yield significantly and therefore, combining tolerant traits to salinity at both seedlings and reproductive stage is essential to develop salt tolerant variety. phosphorus deficiency weathered soils of humid have a major limitation in crop production due to (p) deficiency. optimum soil p is generally low, with a total available p of only 2-4% for plants utilization, due to high phosphorus fixing potential of fine textured soils of sub-humid and humid zones (abekoe and sahrawat 2001). an estimated 5.7 billion ha globally is deficient of available plant phosphorus (batjes 1997), while 50% of prospective arable land globally has acid soils, with sub-saharan africa being most widely distributed. fertilizer application is a corrective measure, and more so, when soluble and absorbable by plant. excess water this could be categorized into two; flash flood of transient nature or submergence that keeps the crop under the water for a short period (≤ 2 weeks), and longer period of flooding, with stagnated water of different depths for up to few months. depth levels are; partial/stagnant; 30-40cm, semi-deep; ˃100cm, deep-water; up to 3m, very deep; ≥4m (mackill et al. 2012). although rice adapt to water logged conditions, but a situation of complete submergence for a longer than necessary period could be of bad effect. this largely affect usual gas exchange and interception of light, because of flood duration, temperature, turbidity level, flood water turbulence, its depth, although varying in seasons and locations (das et al. 2009). extreme temperature rice responds to temperature changes, although its response or sensitivity is dependent on the stage of development; stage of flowering, and 9 to 11 days before flowering are quite sensitive to extreme temperatures of hot or very cold weather, which results in high 69 spikelet sterility (andaya and mackill 2003a, manneh et al. 2007, yoshida et al. 1981, zenna et al. 2010). rice responds to temperatures of less than 150c. this implies that crop failure is seen at the onset of low temperatures at different stages of growth like, germination, seedlings, vegetative and reproductive, and to maturity of grains (andaya and mackill 2003a). this level of failure depends on water temperature or ambient air, growth stage, air, cropping pattern and variety (zenna et al. 2010). air temperature exceeding 350c causes heat injuries to exposed rice plant. 10c rise in minimum temperature during the dry season, causes grain yield decline by 10% (peng et al. 2004), while an insignificant crop yield was recorded, when affected by maximum temperature. increasing night temperature at a day temperature of 330c led to significant decline in grain-filling and gain yield, while at constant day temperature of 290c, rising night temperature had not significantly affected growth or yield. biotic stresses in rice when pathogenic agents of rice feed on plant parts, it does that primarily to obtain its’ nourishment for survival. unfortunately, the impact due to feeding becomes deleterious to host plants. these disease agents have quite a wide range; fungi, bacteria, viruses, insects, false smut, sheath blight, etc. leaf blast blast is an important fungal pathogen (magnaporthe grisea anamorph; pyricularia oryzae) with deleterious multi-diversity of effect on host plant, whose effect usually leads to poor yield. the infection was noticed and first reported in asia for more than 300 years ago, and presently wide spread in at least 85 countries of the world. it is able to adapt to several environmental and field conditions of irrigated, low-land rain-fed, upland or deep water rice field (latif et al. 2011a, ou 1985). rice blast pathogen causes significant yield losses in the whole of southeast asia, south america and all major rice producing areas of the world. its occurrence and severity vary, either by its location, year or and even within field, depending on the condition of the environment and crop management practices (latif et al. 2011). the pathogen’s long years of existence resulted to adaptability, with sudden changes that are prevalent in the virulence characteristics of the population. the diseases pathogen could not be permanently brought under control, being as a result of either controlled or partial resistance by a single dominant gene, which is mostly not stable to an onslaught by a genetically variable pathogen (mccough et al. 1995). the effect of blast pathogens in malaysia has been quite alarming. in 2006, an outbreak in kemubu agricultural development authority area in pasit puteh, kelantan, malaysia, was estimated to have destroyed 60% of 4000 ha of cultivated rice field (rahim 2010). the pathogen’s effect on rice crop is at all growth stages (sharma and bambawale 2008), and also, has the ability to attack more than fifty other species of grasses (ou 1985). the world’s estimated devastation of the disease resulted in yield losses ranged from 1-50% (scardaci et al. 2003). annual yield loss of 10-30% has also been recorded due to blast fungus m. oryzae (skamnioti and gurr 2009). xiao et al. (2016) reported frequent and severe incidences of blast on rice in the tropical province of china due to high temperature and abundant rainfall during the growing season, resulting in damage of over 1500 ha of cultivated hybrid rice in 2008. out of this, 250 ha was totally yield-less due to severe devastation of the panicles. 70 magnaporthe grisea affect rice leaves (fig.1b), the symptoms appear smallish brown or greenish dots. these dots develop to about 1.5cm long and 0.3-0.5cm wide, diamondshaped lesions with a white or grey center after 2-3 days of infestation. it also affects the panicles (fig.1d), collar and seeds (fig.1a, c), and when the collar is unable to endure the weight of the panicle, it eventually leads to the fall of the panicle, as a result, translocation and development is hindered, causing immatured drying, while the effect on the seeds lead to non-viability of seeds as planting material and reduced quality for cooking. using durable resistance genes for breeding resistance variety are currently the major and economical methods being used to control the disease. it is also, environmentally safe (hirano 1994). figure 1: rice blast symptoms on neck, leaf, seeds and panicle. [source: tebeest et al. 2012] a. neck and node blast; the effect of this leads to fall of panicle, and poor seed set or non at all, depending on the development stage of rice in which infection occurs (5.5cm(h)×3.1(w)). b. leaf blast; formation of lesions on leaves, which increseases from dots to expanded patches, turning leaves from brownish to dark brown colour. this affects the surface area for photosynthesis (5.5cm (h)×3.0cm (w)). c. seeds blast; brownish spots and blotches are symptoms, and the effect is on the pedicels which renders the seed inviable (5.5 cm (h)×3.4 cm (w)). d. infected panicles; spikelet are formed on the panicles, this results in poor seed set due to poor translocation of nutrients to other parts of plant (5.5 cm (h) ×4.8 cm (w)). bacteria leaf blight bacteria leaf blight (blb) (xanthomonas oryzae pv oryzae) is also important, and one of the oldest known bacterial diseases of rice in asia (hassan-naqvi et al. 2010), and also, one of the most serious bacterial diseases in many of the rice growing regions of the world (xu et al. 2010). it was first observed in japanese farms in 1884 (tagami and mizukami 1965). it is a rod–shaped bacterium, with leaf blight symptoms which occurs at all growth stages of rice (fig. 2) plant, with yield losses ranging from 20-30%, and as high as 80% depending on the stage of growth where attack or infestation occurs, the geographic location or seasonal condition (ou 1985; singh et al. 1977). zhai and zhu (1999) reported 100% loss in severe incidence of blb infection, it affects both inbred and hybrid rice (mew 1987). like blast, the b d a . c 71 bacteria races are controlled by artificial and natural selection of genes resistant to the blb pathogens. it is quite necessary to identify the new resistant genes to control the changeful races (xia et al. 2012), and also utilize pyramiding method, for wider spectrum of host resistance, since thirty five (shang et al. 2009) blb resistance genes have been identified, in both cultivated rice and their wild relatives (nino-lui et al. 2006; singh et al. 2007; wang et al. 2009). figure 2: leaves of rice infected with bacterial leaf blight [source: http://www.knowledgebank.irri.org] the diagram shows the stages of infection of rice leaves by bacteria leaf blight from early signs to dry leaves; a. oozes; these are early symptoms of infection. on young plants, milky-like dew drop are observed usually in the morning (5.5 cm (h)× 4.8 cm (w)). b. dry oozes; the oozes dry sticking on the leaves (5.5 cm (h)× 3.8cm (w)). c. dry leaves; the dry oozes eventually leads to drying of leaves (5.5 cm (h)× 3.0 cm (w)). virus-resistance the historical view of the effect of viruses on rice spanned several decades, and the devastation impact cut across the entire world with significant yield losses in severe infection. rice stripe virus (rsv) and rice dwarf virus (rdv) caused greater losses to yield of rice in the 1960s than other viruses (toriyama 2010). the environmental and cost effect of the use of insecticide in controlling the vector insect, necessitated the need for the development of genetic resistant variety against rice viruses generally or their insect vectors. resistant genes against rice plant viruses of rdv (sasaya et al. 2013; shimizu et al. 2009) and rsv (xiong et al. 2009), have been developed and evaluated accordingly. tungro disease of rice is a dangerous viral disease, which is caused when infected by rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). green leafhopper (nephotettix virescens) is the vector of rtsv, which helps to transmit the virus. beside the use of marker-assisted selection, a transformation approach has led to the use of coat protein mediated resistance strategy. transgenic rice plant having rtvs replicate gene produced by huet et al. (1999) showed moderate resistance to rtsv. c b a 72 figure 3: rice plants infected with tungro insect-resistance rice has been devastated by insects. genetically modified rice with expressed gene for bacillus thuringiensis (bt rice) (high et al. 2004; tu et al. 2000; wang et al. 2014) produced resistance to insect attack. transformed transgenic rice with a synthetic cry1ab gene was significantly tolerant to eight lepidopteran insect along with striped stem borer (ssb; chilo suppressalis) and yellow stem borer (ysb; scipophaga incertulas). field evaluation of improved insect-resistant rice showed high tolerance to leaf folder (rlf; cnaphalocrocis medinalis) and (ysb; scipophaga incertulas) in china (chen et al. 2011; deka and barthakur 2010; tu et al. 2000), likewise in pakistan (mahmood-ur-rahman et al. 2007), the mediterranean region (breitler et al. 2004) and in india (ramesh et al. 2004). other categories of insects that infest rice also includes these two species of planthopper; brown planthopper (bph), nilaparvata lugens (stal) and whitebacked planthopper (wbph), sogatella furcifera (horvath). it infests rice in rainfed and irrigated wetland environments. continuously submerged field, high shade, and humid conditions form a good habitat for it, other thriving conditions are; excessive use of nitrogen, early season insecticide spraying encourages insects to develop, densely seeded crops and closed canopy of rice plant (www.knowlegdebankirri.org). figure 4: rice insect (a)rice brown planthopper (b) rice striped stem borer false smut false smut is a disease of rice caused by villosiclava virens and has recorded effect of reducing grain yield and quality globally (yang et al. 2012; osada 1995). increasing incidences of rice false smut has been reported, causing great concerns to rice production. the asian v. virens strains produces mycotoxin which are poisonous to humans and animals (zhou et al. a b 73 2012; koiso et al. 1994; nakamura, et al. 1994). v. virens is a fungus classified as ascomycete which produces asexual chlamydospores (ustilaginoidea virens) and sexual ascospores (tanaka et al. 2008). both have the potential of germination and production of conidia (wang 1988; wang 1995). the v. virens has an optimum temperature range of 200c-300c upon which the spores germinate in potato-sugar-agar (psa). it is quite slow in growth, taking 14 days to achieve about 37mm of colony diameter of mycelium growth (fu et al. 2013). rice spikelets are infected by v. virens during booting (fan et al. 2015; tang et al. 2013), and the pattern of infection is in two stages: (a) epiphytic stage. it is a stage where it spores in to an emerging panicle and plunge on the surface of the spikelets, and germinates with the hyphae stretching inside the space of spikelet through the tiny openings in between the lemma and palea (ashizawa et al. 2012; song et al. 2016). this phase does not show sites of any infection, probably as a result of epiphytic growth (fan et al. 2015; fan et al. 2014; tanaka et al. 2016). (b) biotrophic stage. haphae reaches the inner floral organs at this phase, it primarily infect stamen filaments, stigmata and styles, with occasional infection on the lodicules and ovaries (tang et al. 2013; song et al. 2016; yong et al. 2016. the fungal mycelia of the v. virens attaches to the floral organs of rice and extract so much nourishment to develop the false smut ball, maybe by stimulating a system of grain filling intercepting nutrient store (fan et al. 2015; song et al. 2016). the false smut disease produces visible false smut ball which is often larger in size to a matured grain of rice, probably so much nutrient required. it could also be formed by utilizing the rice flowers because it has plenty of sugar; glucose, sucrose, oligosaccharides, etc. figure 5: false smut infected rice: spore balls turn greenish black when mature from initially orange colour sheath blight sheath blight of rice (shb) is caused by rhizoctonia solani kuhn (teleomorph: thanatephorus cucumeris (frank) donk), it is destructive and causes significant yield losses and affects quality (lee and rush 1983; nagarajkumar et al. 2004). it also has the potential of affecting plants like lettuce, tomatoes, maize, barley and sorghum (zhang et al. 2009). yield losses ranging from 10-30% (xie et al. 2008) to even 50% in high incidences has been recorded (meng et al. 2001). r. solani is a global soil saprophytic and plant parasite that manifest when triggered (anees et al. 2010; sumner 1996). the pathogen survives in unfavorable condition by forming dormant mycelia or sclerotia because of restricted movement due to absence of spores (anees at al. 2010; summer 1996). sclerotia have the potential of surviving in soil for up to two years, and could be dispersed at the time of field preparation and on flood irrigation of fields (webster and gunnell 1992; brooks 2007). the sclerotia could move on continuously flooded irrigation to adjoining fields and attach to available plants, thereby causing shb disease. and favorable conditions enhance the pathogen’s spread. early symptoms are found on leaf sheath close to water-line as water-soaked lesions. advance infection occurs when 74 hyphae grow upwards to parts of plant not earlier infected, thereby developing more lesions and sclerotia on leaf sheath for the cycle to be completed (webster and gunnell 1992; brooks 2007). the flowering stage favours disease development when canopy of rice is dense, creating a micro-climate conducive for pathogen growth and spread (brooks 2007). the pathogen also infects seed to matured plant, resulting in average to high losses of yield, depending on part of plant infected. observable symptoms are lesions formation, plant lodging, and lack of grain formation. extended lesions on lower leaves of rice infected may lead to softened stem, resulting in lodging (wu et al. 2012). this lodging temper with the normal design of rice canopy, it affects the potential for photosynthesis and overall production of biomass (wu et al. 2012; hitaka 1969). the effect on flowering or during panicle initiation leads to loss in total seed weight as a result of low percentage of filled spikelets, which eventually leads to significant yield losses (nagarajkumar et al. 2004). severe lodging may result during rice sheath blight epidemic thereby obstructing the transport of nutrients, water, and carbohydrates assimilate via the phloem channels, affecting grain filling (wu et al. 2012). other factors that promotes shb are double cropping, application of high rate of nitrogenous (n) fertilizer, dwarf build, early maturing, high plant density, high tillering, morphological traits of rice plant cultivars due to the height of the plant, because it affects micro-climate and transmission of light due to canopy, supporting increase in disease (han et al. 2003; tang et al. 2007) and susceptible variety has been revealed to support shb severity in many world rice growing environment (lee and rush, 1983; nagarajkumar et al. 2004). figure 6: sheath blight infected rice interaction of stress conditions the simultaneous occurrence of abiotic and biotic stresses is quite complex, hence their response to either stress conditions are mostly controlled by different hormone, whose pathway could interact and inhibit each other (anderson et al. 2004; asselbergh et al. 2008b). most times, when plants are exposed to pest or pathogens, drought stress effect increases (audebert et al. 2000; englishloeb et al. 1997; khan and khan 1996; smit and vamerali 1998). increased abiotic stress is able to reduce the defenses of plants, and thereby strengthen susceptibility to disease (amtmann et al. 2008; goel et al. 2008; mittler and blumwald 2010). when stress factors occur simultaneously together, it may be regarded to be additive or interactive (niinemets 2010). a stress factor can be said to interact, when a stress factor alters or affect the normal response of the other as a result of acclimation response. the effect of interaction between two abiotic stresses on crop was studied, and mittler and blumwald (2010) reviewed it, and the stresses were observed to be deleterious. the 75 combination of drought stress and heat in particular, is capable of causing high damage to crops in comparison with each individual stress (barnabas et al. 2008; keles and oncel 2002; mittler 2006, rizhsky et al. 2002). consequently, observations on the simultaneous effect of a biotic impact of herbivore or pathogen and an abiotic stress, were positive and or negative interactions based on the nature, time period and extend of damage of the different stress. rise in temperature is capable of creating a negative interaction effect, by reducing the strength of resistance to pathogens of viruses, fungi, nematodes and bacteria. six years experiments on wheat, with higher mean temperature were correlated under the period with increased susceptibility to cochliobolus sativus fungus (sharma et al. 2007). plant resistance to pathogen can be affected by drought stress. date palms and parthenocissus quinquefolia vine suffers wide spread infestation of bacterial and fungal leaf scorch symptoms, as a result of water deficit (mcelrone et al. 2001; suleman et al. 2001). in contrast, positive interaction occurs between abiotic and pathogens stress. many types of bacteria and arbuscular mycorrhizal fungi, have been identified to support stress tolerance in some crop species via production of suppressing ethylene, antioxidants, stabilizing soil structure, improving abscisic acid (aba) regulation and increasing osmolyte production amongst others (aroca et al. 2008; grover et al. 2011; kohler et al. 2008; saravanakumar and samiyappan, 2007). infection of soil-borne plant parasitic nematodes worsens or prevents the effect of abiotic stress on plants. plant water relations are severely disrupted due to their parasitism in roots (haverkort et al. 1991; smit and vamerali 1998). the presence of nematodes ameliorates the intense effect of water deficit. the nematode heterodera sacchari caused increased water deficit related losses in upland variety of rice in ivory coast, by supporting the reduction of leaf water potential, leaf and dry weight and stomatal conductance (audebert et al. 2000). a good knowledge of the diversity of plant, helps in assessing the behavior of plant and its’ adaptation to areas prone to drought (alonso-blanco et al. 2009). and also, the knowledge of plant survival, under stress is important in developing an effective strategy for phenotyping (sarkar et al. 2013), and even in successfully breeding drought tolerance variety. an assessment of genotypic variability of rice under different water deficit conditions is also necessary (abenavoli et al. 2016; anower et al. 2017). gene pyramiding this is a breeding method, for the assemblage of gene of desired traits, from different sources into a single elite cultivar. this in turn exhibits the different traits as a single multiple gene variety. it is a host resistant/tolerant variety, with wider broad-spectrum resistance and high yielding potential (pradhan et al. 2015). three methods have been identified; pedigree, recurrent selection and backcross methods (ruengphayak et al. 2015). gene pyramiding has been applied to enhance resistance to biotic and abiotic stresses, by selecting for two or more genes at a time. in rice, such approach (pyramiding) has been used to develop a single variety resistance against blast and bacteria blight (huang et al. 1997, singh 2001). the use of markers allows selecting for qtl-allele-linked markers that have the same phenotypic effect. for the enhancement and improvement of a quantitatively inherited trait in plant breeding, pyramiding of multiple genes or qtls is recommended as a potential strategy (richardson et al. 2006). an appropriate breeding scheme for marker-assisted gene pyramiding (magp) depends on the number of genes/qtls, the heritability of traits of interest, and other factors (richardson et al. 2006). 76 xu et al. (2012) stated that to improve elite variety of rice, pyramiding approach was used to introgress different strains of desired characters of plants, which were successful in exhibiting wider broad spectrum resistance against the invasion of disease agents or tolerance of abiotic conditions. he reported that the introgression of bacteria blight resistance genes, xa7 and xa21 conferred on the variety the ability to withstand all the seven xoo strains. blast resistance genes pi2 and pi54 and, bacterial blight resistance genes xa13 and xa21, were introgressed into pusa basmatic 6 and were found to be superior hybrids to parents in terms of yield, early maturing and better quality of parameters. the improved varieties conferred resistance to magnaporthe oryzae in south, east and north-west of india (ellur et al. 2016). pyramiding backcross method was carried out by introgressing pi1; piz-5 and pita genes to improve resistivity of blast m. grisea to leaf blast attack, which were successful and more resistant on trial evaluation carried out in the philippines and india (hitalmani et al. 2000). huang et al. (1997) reported pyramid of four resistant genes of bacteria blight xa-4, xa-5, xa-13 and xa21 and were listed for resistance against xanthomonas oryzae pv. oryzae and found to have higher level of resistance with wide broad spectrum. three resistance genes xa5+xa13+xa21 were introgressed, using backcross method into susceptible deep water rice variety popular with indian farmers, referred to as jalmagna, from swarna bacteria blight pyramid line, which produced better yield and characters (pradhan et al. 2015). other multi-traits varieties are; lac 23, xiangzi 3150, gumei 2 (carrying minimum of two r genes each) (huang et al. 2011; mackill and bonman 1992; wu et al. 2005), digu, ir64 and sanhuangzhan 2 (carrying minimum of three r genes each) (chen et al. 2004, liu et al. 2004; sallaud et al. 2003, shang et al. 2009), tetep and moroberekan (carrying a minimum of four r genes each) (barman et al. 2004; chen et al. 1999). pita and pi46 were transferred into an elite restorer line hh179 through marker-assisted backcross breeding (mabb). three categories of improved lines were obtained (i) only harboring pita, (ii) only harboring pi46 and (iii) harboring both pita and pi46. this pyramiding approach strategy should be consistently employed for durable resistance (jia et al. 2003; tabien et al. 2002), because the emergence of virulent races has overcome the resistivity of varieties carrying single gene (koide et al. 2010). the concurrent presence of introgressed multiple major resistant genes often confers lasting broad spectrum resistance (xiao et al. 2015), and confirmable genotypically through marker-assisted selections. conventional gene pyramiding takes longer time, and resources to develop and technical expertise in transfer of pollen grains from anthers to stigmas. length of time or generations is for evaluation trial, to ascertain the success of gene introgression through continued phenotyping, until non-segregating and multiple gene variety exhibiting all the introgressed genes are present in a single line (miah et al. 2013). pedigree method it is a breeding method that is associated with self-pollinating plants, which mostly have trait for high yield, but lacking in biotic or abiotic traits. it begins with the crossing of two genotypes, with each having desirable traits not present in the other. if the two original parents are not able to provide all desired characters required, a third parent would be introgressed into the hybrid progeny of the first generation (f1) (fig. 3). in successive generations, the superior types are selected for and a record is kept of parent to progeny relation (ruengphayak et al. 2015). 77 crossing the progeny of the f1 individuals which produces f2 generation, usually offers the opportunity for first selection in pedigree programs. the emphasis here (f2 generation) is, selection of individuals that carry undesirable major genes for elimination. in successive generations of hybrid, selection leading to natural selection as self-pollinated, pure breeding lines is produced. the families of the hybrid f2 plant express unique characters. usually, fewer superior varieties of plant are selected in each of the unique superior family of the hybrid generations. progressively, at f5 generation, homozygosity would have been attained and the emphasis shift almost completely to select within families. the pedigree record helps greatly in removal or eliminations of non-superior families with undesirable traits. now, at this point, any family selected is harvested in mass, so as to obtain larger quantity of seeds required in evaluating families for quantitative traits. most preferable evaluation, which should encourage or stimulate planting activities for commercial purposes, should be grown in plots within close range. at either f7 or f8 generation, usually it is required that visual selection is needed, to reduce the number of families to manageable proportion and for accurate evaluation, for yield performance level and quality (jiang 2013). to conclude evaluation of good quality strains, it requires; observation study on multilocational and time period (years), precise or definite yield testing and, quality testing in multiple locations and over a period of time after which new variety is release for commercial cultivation or production. 78 putra-karah mr219 irbb60; bacteria high yield (blast) × leaf blight (blb) (high yielding) mas (marker-assisted selection) f1 putra-karah(mr219) blast+blb phenotyping × mr219 drought tolerance (high yielding) mas putra-karah (mr219) (blast+blb+drought t.) f1 phenotyping mas f2 phenotyping f3 phenotyping f4 putra-karah (mr219) blast+blb+drought tolerant symbol of selfing or self-pollination f1, f2, f3, f4 filial generations figure 7: pedigree, three-way cross pyramid chart of biotic and abiotic stresses figure above shows, a schematic pedigree breeding chart of crosses that leads to development of a single multiline blast and bacteria leaf blight resistance and drought tolerance rice variety, with confirmatory tests through genotyping and phenotyping recurrent selection the selection of certain traits generation after generation in self-pollinated crop plants, the inter-breeding of reselected plants allows the breeder access favourable 79 recombination, as well as stabilized traits within the gene pool. ideotype in each interbreeding line (ibl) should be selected, but total reliant on the phenotype should be done with aught most caution because, it is not always an indication of actual genotype. yield and quality trial with test crosses and selection of best ten lines should be done, intercross and repeat (vales 2010). after the completion of recurrent selection, new selection of individuals to be the new parents of interbreeding lines (ibl). these are the recurrent selection which has been done in two separate programs. hybrid (f1) single cross of the progeny is used as visual indicators or morphological markers of the combining ability that lies in the saved seeds. these specific inbred parental lines are kept in reserve, until the progeny testing of the different (a×b) hybrids have shown which has better selection for combining ability, and will make better hybrid. this strategy is complicated, therefore, good note taking and organization is of immense importance, it includes the following types for cross-pollinated crops; simple recurrent selection (srs), recurrent selection for general combining ability (rsgca), recurrent selection for specific combining ability (rssca), reciprocal recurrent selection (rrs) (singh, 2012, allard, 2010). backcross method most times a peculiar variety of plant is improved, by introgressing to it some specific desirable traits that it lacks. the carrier of the trait is required, and then mating the progeny back to a plant having the genotype of the superior parent, a process referred to as backcrossing. after a number of backcrosses, say five or six, in case of conventional approach, the progeny will be hybrid of the character being introgressed (fig.4), but like the superior parent for all other gene traits. when the last backcross generation is selfed alongside selection, it will give rise to some progeny pure breeding for the genes being introgressed. backcross methods have predictable outcomes, rapidity and require small number of plants. conversely, the procedure does not provide opportunity for the occurrence of chance combinations of genes, which could lead to striking improvements in performance (vales, 2010). marker assisted backcross has shown that less time was required to recover genes. suh et al (2013) stated that at least three backcrosses are needed to recover the phenotype of the recurrent parent. while miah etal (2016) recovered at bc2f2 92.7% and 97.7% of lowest and highest recurrent genome recovery respectively, broad spectrum of blast resistance genes into cultivated mr219 rice variety. 80 x f1 seed elite alleles (50%) x f2 seed elite alleles (75%) x f3 seed elite alleles (87.5%) x f4 seed elite alleles (93.75%) x f5 seed elite alleles (96.875%) figure 8: backcross method of breeding this is a schematic diagram of a backcross, which is basically recovery of gene of interest from a recurrent parent into a recipient parent lacking in a specific desired trait. the progeny of each successive generation is crossed back to a recipient parent, generation after generation until the trait is fully recovered. letters: “b” donor parent carrying trait of interest “a”, a desired parent lacking in trait present in b parent “ ‘c’, ‘d’, ‘e’, ‘f’, ‘g’ ” are transgenic elite lines in progression. this is a schematic diagram of a backcross, which is basically recovery of gene of interest from a recurrent parent into a recipient parent lacking in a specific desired trait. the progeny of each successive generation is crossed back to a recipient parent, generation after generation until the trait is fully recovered. letters: “b” donor parent carrying trait of interest “a”, a desired parent lacking in trait present in b parent “ ‘c’, ‘d’, ‘e’, ‘f’, ‘g’ ” are transgenic elite lines in progression. a b c b d b e b f b g 81 table 1. some selected ssr (microsatellite) polymorphic and tightly linked markers for blast, bacteria leaf blight resistance and drought tolerant rice variety genes primer sequences (5′ –3′) chr. position exp. size references ssr linked marker forward primer reverse primer putra 1(blast resistance) rm6836 piz, pi2,pi9 tgttgcatatggtgctatttga gatacggcttctaggccaaa 6 240 askani et al.,2011 m8225 piz atgcgtgttcagaaattagg tgttgtatacctcatcgacag 6 221 miah, et al., 2016 mr219 drought tolerance rm511 qdty12.1 cttcgatccggtgacgac aacgaaagcgaagctgtctc 12 130 shamsudin et al., 2016, www.gramene.com rm520 qdty3.1 aggagcaagaaaagttcccc gccaatgtgtgacgcaatag 3 247 rm236 qdty2.2 gcgctggtggaaaatgag ggcatccctctttgattcctc 2 174 rm276 qdty2.2,3.1 ctcaacgttgacacctcgtg tcctccatcgagcagtatca 6 149 rm1261 qdty12.1 gtccatgcccaagacacaac gttacatcatgggtgacccc 12 167 irbb60 (bact. leaf blight) rm224 xa-4 atcgatcgatcttcacgagg tgctataaaaggcattcggg 11 157 he et al., 2006 khan et al., 2015 rm122 xa-5 gagtcgatgtaatgtcatcagtgc gaaggaggtatcgctttgttggac 5 227 rm13 xa-5 tccaacatggcaagagagag ggtggcattcgattccag 5 141 rg136 xa-13 tcttgcccgtcactgcagatatcc gcagccctaatgctacaattcttc 8 246 pradhan et al., 2015 chen et al., 1997 xa13prom xa13 gccatggctcagtgtttat gagctccagctctccaaatg 8 rm21 xa-21 acagtattccgtaggcacgg gctccatgagggtggtagag 11 157 pta248 xa-21 agacgcggaagggtggttcccgga agacgcggtaatcgaagatgaaa 11 (askani et al. 2011, miah et al. 2016, shamsudin et al. 2016, he et al.2006, khan et al. 2015, pradhan et al. 2016, www,gramene.com) 82 molecular markers in plant breeding these are biological features determined by the forms of genetic loci or alleles, and are transmittable through generations. they serve the purpose of experimental tags, to keep record of cell, chromosome or a gene, individual tissue and nucleus. this is classified into: classical markers; morphological, cytological and biochemical markers (collard et al. 2005) and dna markers (xu et al. 2010). morphological markers these are simply tools that assist in selection of desired traits carrying plants, which is used right from ancient times in plant breeding. these markers are visible characters of plants such as shape of leaf, colours of flower, pubescence, pod, seed, hilium, flesh, seed shape, fruit shape, awn length and type, exocarp (rind) stripe and colour, length of stem. generally, these stands for genetic polymorphisms that are easily identified and altered or manipulated, and mostly used to build up of linkage maps by 2 or 3 points classical test. agronomic traits are linked with these markers which are in use, as criteria in practical breeding for indirect selection. during green revolution, the choice of semi-dwarf variety of rice was one important selection, which led to the much success of high-yielding cultivars (liu 1991). these morphological markers have limitations, which are in some situations not linked to traits of economic importance (e.g., yield and quality), which could lead to ineffective and undesirable crop plant growth and development. cytological markers also known as cell chromosome karyotype and bond, which are invariably the structural features of chromosome. these patterns of banding display of colour, position and width order, divulge the different euchromatin and heterchromatin distributions. for example q and g bands are products of quinacrine hydrochloride and giemsa respectively, and the reverse g bands are r bands. these landmarks of chromosomes are useful in normal chromosomes characterization, and detection of chromosomal aberration or mutation, as well as in physical mapping and linkage group identification. using cytological markers directly restrict genetic mapping and breeding, as such, cytological along with physical maps of morphological markers, lay a foundation for gene linked map through molecular techniques. biochemical markers it refers to as protein markers and could be categorized into molecular markers or dna markers. a substitute structural variant of protein or enzyme, with variation in weight of molecules and electrophoretic mobility, but having same function (catalysis) is also referred to as isozymes (xu et al. 2010). dna markers they are dna fragment showing mutation that can be used in detection of polymorphism between genes (alleles) or different genotypes, for certain sequence of dna in a gene pool or population. in order words, it is little portion of dna sequence that reveals polymorphism with different individuals (xu et al. 2010). in the detection of polymorphism, two important methods in use includes; southern blotting, also referred to as nuclear acid hybridization technique (southern 1975), and polymerized chain reaction (pcr) technique. some commonly used dna markers for plant 83 breeding include; microsatellite or simple sequence repeat (ssr), restrictive fragment length polymorphism (rflp), random amplified polymorphism dna (rapd), single nucleotide polymorphism (snp) and amplified fragment length polymorphism (aflp) (farooq and azam 2002, semagn et al. 2006) (table 2). table 2. composition of dna marker system most widely used in plants feature and description ssr snp aflp rapd relp types of primers sequence specific allelic specific pcr primers bp specific sequence 10 random nucleotide s low copy dna cdna clones genome coverage complete genomes complete genomes complete genomes complete genomes low copy coding region level of ease of automation high high moderatehigh moderate low loci number high(1,000s) very high moderate(1 000s) small(˂1,0 00) small(˂1,00 0) ratio of effective complex high moderatehigh high moderate low polymorphism level high high high high moderate primary applicability all purposes multipurpo se diversity and genetics diversity genetics sequencing and/or cloning yes yes none none yes amenable to automation high high moderate moderate low radio-active detection usually none none yes or none none yes, usually reliability/reprodu cibility high high high low high cost/analysis low low moderate low high polymorphism type changes in single length of repeat single base changes indels single base changes indels single base changes indels single base changes indels genotyping through-put high high high low low amount of dna required small(0.050.12ug) small(≥0.05 ug) moderate(0. 5-1.0ug) small(0.01 -0.1ug) large(550ug) quality of dna required moderatehigh high high moderate high technical demand low high moderate low moderate 84 source: modified (collard et al. 2010; korzum 2003; semagn et al. 2006; xu et al. 2010) conclusion the economic value of rice as a source of income and staple food crop is of great importance because, increasing human population which invariably requires increase production to meet the projected global ratio, places a demand (khush 1999) irrespective of devastation by various biotic and abiotic stresses, which could singly or simultaneously affect rice production. this therefore, justifies the review effort towards development of a single multipline resistance and tolerance rice variety through one of the approaches termed pyramiding, and via marker-assisted selection as a check for sustainable yield (pradhan et al. 2015), as well as, for ease of selection of rice carrying the whole genes introgressed respectively. this approach is proven to confer broad spectrum of resistance and tolerance on crop plant, which is safer compared to chemical sprays which possess a great threat to environment and other important insects and microorganism. development of multiline host disease resistance and or non-disease stress variety of rice is always an effort to create a broad spectrum of resistance/tolerance against concurrent attack by stresses of abiotic and biotic, depending on the genes present in a particular plant, and also against the failure of single gene of stress condition, with capacity of susceptibility because of the pathogens evolving nature. this way, yield value is sustained, without cause to environmental pollution via chemical sprays which destroys and contaminates relevant fauna and flora, and aquatic environment. the interaction of abiotic and biotic resistance and or tolerance genes could hamper or strengthen expression at the site of attack. it therefore implies that, lack of expression of a certain gene may not necessarily be the absence of that gene in the plant, so long as markerassisted selection was employed in determining the presence of the genes. it simply means a stress condition suppresses the expression. for instance, increased abiotic stress is a potential reducer of the defenses of plants, and thereby strengthens susceptibility to disease (amtmann et al. 2008; goel et al. 2008; mittler and blumwald 2010). marker-assisted selection is a tool that is able to distinctly reveal more than two genes present in a particular plant. this saves times and creates precision in selection of rice lines time demand/required low low moderate low moderate level of ease of use easy easy moderate easy not easy required dna(g) 0.05 0.05 0.5-1.0 0.02 10 inheritance/expres sion codominant codominan nt dominant and codominant dominant codominant start-up cost/development high high moderate low moderatehigh polymorphic loci per analysis(number) 1.0-3.0 1 20-100 1.5-5.0 1.0-3.0 marker index high moderate moderatehigh moderate low pcr-based yes yes yes yes usually not genome abundance moderatehigh very high high high high 85 of choice, either having abiotic, biotic stress resistance/tolerance or both, so long the linked primers are accurately determined. references abenavoli, m.r., leone, m., sunseri, f., bacchi, m.& sorgona a. 2016. root phenotyping for drought tolerance in bean landraces from calabria (italy). j. agron. crop sci. 202:1– 12. abekoe, m.k. 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