peruvian journal of agronomy 3(1): 1-5 (2019) issn: 2616-4477 (versión electrónica) doi: ttp://dx.doi.org/10.21704/pja.v3i1.1132 © the authors. published by universidad nacional agraria la molina received for publication: 29 january 2019 accepted for publication: 12 february 2019 life cycle of the diamondback moth plutella xylostella l. (lepidoptera: plutellidae), in broccoli and cauliflower under laboratory conditions ciclo biológico de la palomilla dorso de diamante plutella xylostella l., en brócoli y coliflor bajo condiciones de laboratorio huaripata, c.1*; sánchez, g.1 *corresponding author: cesar.huaripata@gmail.com abstract this research aimed to determine the biological cycle of plutella xylostella (dbm), raised in broccoli var. calabrese and cauliflower var. snowball in laboratory conditions averaging 76.1 ° f and 65% rh. the mating of the moths was carried out in one-liter plastic containers and the postures were incubated inside 200 ml polypropylene cups, both covered with nets. to provide suitable food for larvae the plants were cultivated under cover to avoid infestation with pests. fresh leaves were used to feed the larvae until pupation. the results were analyzed with the nonparametric statistical test of kruskal-wallis. the following biological parameters were obtained, reared with broccoli and cauliflower leaves respectively: incubation period 3 days; larval period 9.76 and 9.69 days; pupal period 5.1 and 5.3 days; biological cycle 19.5 and 19.9 days and 175 and 187 eggs as the capacity of oviposition of mated females. these results do not show significant statistical differences of the moth’s biological cycle between the two host plants. key words: plutella xylostella, dbm, life cycle, brassicaceae, broccoli, cauliflower. resumen esta investigación tuvo como objetivo determinar el ciclo biológico de plutella xylostella (pdd), criada en brócoli var. calabrese y coliflor var. snowball bajo condiciones de laboratorio promediando 76.1 ° f y 65% de hr. el apareamiento de las polillas se realizó en recipientes de plástico de un litro y las posturas se incubaron dentro de vasos de polipropileno de 200 ml, ambos cubiertos con redes. para proporcionar alimento adecuado para las larvas, las plantas se cultivaron bajo cubierta para evitar la infestación con plagas. se usaron hojas frescas para alimentar a las larvas hasta la pupación. los resultados se analizaron con la prueba estadística no paramétrica de kruskal-wallis. se obtuvieron los siguientes parámetros biológicos de pdd, criadas con brócoli y hojas de coliflor respectivamente: período de incubación 3 días; período larval 9,76 y 9,69 días; periodo pupal 5.1 y 5.3 días; ciclo biológico 19.5 y 19.9 días; 175 y 187 huevos como la capacidad de oviposición de hembras apareadas. estos resultados no muestran diferencias estadísticas significativas del ciclo biológico de la polilla entre las dos plantas hospedadoras. palabras clave: plutella xylostella, pdd, ciclo biológico, brassicaceae, brócoli, coliflor. 1klaus raven büller entomological museum (krbem), universidad nacional agraria la molina (unalm) introduction vegetables, and within them brassicaceae family, are very appreciated in human feeding due to its high nutritional and medicinal value, mainly for its properties in cancer prevention (cohen, kristal & stanford, 2000; keck & finley, 2004). the chemo-protective effect of these vegetables is due to their high content of glucosinolates, anticancer substances that are responsible for detoxification (lampe & peterson, 2002). climatic conditions in peru allow the growth of several species in the brassicaceae; all of them are attacked by insect pests., (sánchez & vergara, 2014) cite brevicoryne brassicae l., hellula phidilealis walk., leptophobia aripa boisduval, and plutella xylostella l. the last one is considered the main pest; it is known as “diamondback moth”, a cosmopolitan species with mediterranean origin life cycle of the diamondback moth plutella xylostella l. (lepidoptera: plutellidae), in broccoli and cauliflower under laboratory conditions january april 2019 2 (lingappa, basavanagoud, kulkarni, patil, & kambrekar, 2004) p. xylostella, is the key pest in tropical areas, given the numerous generations per year and the absence of effective natural enemies (garcía-morató, 2000); this species reduces yields and product quality, especially in summer due to high infestation (sánchez & vergara, 2014). due to the importance of this pest, the objective was to determine the biological cycle of plutella xylostella l., in broccoli (brassica oleraceae var. italika) plenck, and in cauliflower (brassica oleraceae var. botrytis) l. the results will provide important data for a better integrated pest management of diamondback moth in peru. materials and methods this study was conducted in the laboratory of the entomology museum “klaus raven büller” (mekrb) of the universidad nacional agraria la molina (unalm), from march to august 2013. all the rearing work was conducted under controlled conditions, averaged at 76.1 °f of temperature and 65.1% rh. in order to obtain more reliable data, the rearing was carried out until reaching three generations of p. xylostella. outside mekrb, an area covered with canvas and tulle was installed, inside which pots were placed with broccoli (brassica oleracea var. italika, calabrese variety) and cauliflower (brassica oleracea var. botrytis, snowball variety), in order to provide fresh leaves, free of insect’s pest and (figure 1a and 1b). for each variety, twenty clay pots of four kg capacity were used, the pots were filled with 50% agricultural soil and 50% compost. to ensure the fertility and good development of the plants, two plants were placed per pot. fifty adults of p. xylostella were collected from an agricultural field located in the district of carabayllo (lima, peru). these specimens were taken to the mekrb laboratory where they were conditioned in two five-liter glass jars, on the bottom of which paper towel and fresh “broccoli” leaves were placed, as an oviposition substrate. twenty moths were placed in each bottle; to ensure a proper gas exchange, the opening of these containers was covered with nets secured with rubber bands. for the mass rearing, the leaves containing eggs of p. xylostella were extracted, and then transferred to petri dishes of 15 cm in diameter x 1.5 cm in height. the larvae that emerged were fed broccoli leaves until their pupal stage. the pupae were placed individually in polypropylene containers of seven cm in diameter, five cm in diameter and four cm in height. once the adults emerged, they were used as breeding stock for this research. adult breeding and egg production for mating, twenty polypropylene containers of one-liter capacity were used (twelve cm in their highest diameter, nine cm in their lowest diameter and thirteen cm in height) a c brócoli figure 1. a) plant material under cover (nursery and transplant); b) plants of broccoli (calabrese variety) and cauliflower (var. snowball); c) mating containers; d) rearing containers. b d cauliflower huaripata, c.; sánchez, g. peruvian journal of agronomy 3 (1): 1-5 (2019) 3 (figure 1c). each container was covered with net to favor gas exchange. at the bottom of each container was placed paper towel, a container of two cm in diameter x one cm high, with diluted honey for adult feeding, and broccoli or cauliflower leaves for oviposition; a pair of adults of p. xylostella was placed inside each container. of these containers, ten were used for the study in broccoli and ten for cauliflower. individual breeding of eggs the eggs were removed from the mating containers, and these with the help of the marten hair brush, were individually placed in polypropylene rearing containers of seven cm in their highest diameter, five cm in their lowest diameter and four cm in height and covered with net at the top (figure 1d). each rearing container was conditioned with paper towel moistened in the bottom and a fraction of leaf of broccoli or cauliflower according to the case for the hatching and feeding of the larva. sixty rearing containers were used for broccoli research and sixty for cauliflower, material that was used to determine the biological parameters of p. xylostella. once the rearing was started, eggs, larvae, and pupae were observed daily until adults were obtained. the incubation, larval, pupal, biological cycle length and oviposition capacity were recorded. the results were analyzed using the nonparametric statistical test of kruskall-wallis, statistical analyzes were performed using the spss 19 statistics software. results in broccoli leaves, p. xylostella recorded an incubation period of 3 days, incubation period did not have significant differences between the three generations (kruskal wallis test: x2 = 0.00, with gl = 2 and p ≥ 0.05); the larval stage was 9.76 days with a range of 8 to 11 days, the duration of this period fed broccoli leaves had significant differences between the three generations (kruskal wallis test: x2 = 9.56, with gl = 2 and p ≥ 0.05); the pupal stage was: 5.13 days, with a range of 4 to 7 days, had significant differences between the three generations (kruskal-wallis test: x2 = 7.19, with gl = 2 and p ≥ 0.05); the biological cycle was 19.4 days; with a range of 16.8 to 21.8 days, there were no significant differences between the three generations (kruskal-wallis test: x2 = 0.96, with gl = 2 and p ≥ 0.05). the oviposition capacity was 187 eggs, with a range of 53 to 336 eggs, according to the analysis, there were no significant differences between the three generations (kruskal-wallis test: x2 = 2.98, with gl = 2 and p ≥ 0.05) (table 1). in leaves of cauliflower, p. xylostella recorded an incubation period of 3 days, incubation period did not have significant differences between the three generations (kruskal wallis test: x2 = 0.00, with gl = 2 and p ≥ 0.05); the larval stage was 9.69 days with a range of 8 to 12 days, there were significant differences between the three generations (kruskal-wallis test: x2 = 8.39, with gl = 2 and p ≥ 0.05); the pupal stage was 5.29 days with a range of 4 to 7 days, there were significant differences between the three generations (kruskal-wallis test: x2 = 21.45, with cauliflower incubation period larval period pupal period biological period oviposition capacity g i 3a 9.62a 5.38ab 19.87a 160.1a g ii 3a 9.92b 4.94a 19.8a 173.5a g iii 3a 9.52a 5.58ab 19.97a 191.4a average 3 9.69 5.3 19.88 175 table 2. average of biological parameters of plutella xylostella l., during three successive generations (g) in cauliflower, under laboratory conditions. march august 2013. la molina, lima peru. broccoli incubation larval pupal period biological period oviposition capacityperiod period g i 3a 9.62a 5.28a 19.48a 171.7a g ii 3a 9.64a 4.98ab 19.38a 171.3a g iii 3a 10.02b 5.14a 19.63a 216.7a average 3 9.76 5.13 19.49 187 table 1. average of biological parameters of plutella xylostella l., during three successive generations (g) in broccoli, under laboratory conditions. march august 2013. la molina, lima peru. life cycle of the diamondback moth plutella xylostella l. (lepidoptera: plutellidae), in broccoli and cauliflower under laboratory conditions january april 2019 4 gl = 2 and p <0.05); the biological cycle was 19.8 days with a range of 17.8 to 22.2 days, there were no significant differences between the three generations (kruskal-wallis test: x2 = 0.9, with gl = 2 and p ≥ 0.05). the oviposition capacity was 175 eggs with a range of 72 to 286 eggs, according to the analysis, there were no significant differences between the three generations (kruskal-wallis test: x2 = 1.26, with gl = 2 and p ≥ 0.05) (table 2). discussion in this study, the incubation period was three days, both in broccoli and in cauliflower. these results are similar to those obtained by (hasanshahi et al., 2014) in three varieties of cauliflower; also, (fernández & alvarez, 1988) recorded 2.9 days in cabbage (brassica oleracea var. capitata) and (ebrahimi, talebi, fathipour, & zamani, 2008) recorded 3 days in rapeseed cultivars (brassica napus l.) the larval period showed similar results for both cauliflower and broccoli (9.76 and 9.69 days respectively). these results coincide with those obtained by (niu, li, li, & liu, 2013), 9.6 days in cauliflower, however, other researchers recorded different values for this parameter. these values range from 6.64 days (25 °c) (saeed, sayyed, shad, & zaka, 2010) to 11.09 days (23.5 °c) (sarnthoy, keinmeesuke, sinchaisri, & nakasuji, 1989). this variation may be due to differences in many factors under which these studies were carried out, such as the cultivar used and the strain of p. xylostella used, which, according to (sarnthoy et al., 1989), shows variable results. the pupal period recorded in broccoli and cauliflower (5.13 and 5.3 days respectively) was very similar for both varieties. the range of values recorded by other researchers varies from 3.86 in cauliflower (golizadeh, kamali, fathipour, & abbasipour, 2009) to 5.89 in broccoli and 6.0 days in cauliflower (syed & abro, 2003). this variation would be a function of the same variables that affect the duration of the larval status mentioned above. in broccoli the biological cycle of p. xylostella was 19.49 days and in cauliflower it was 19.88 days. these slight differences are not significant, since in all the previous parameters there were no wide differences. in broccoli, the oviposition capacity of p. xylostella was 187 eggs, and in cauliflower it was 175 eggs. these values do not differ significantly. conclusions in broccoli leaves, the duration of the biological cycle of p. xylostella was 19.4 days, the egg state lasted 3 days, the larva 9.76 days, the pupa 5.13 days and the oviposition capacity was 187 eggs on average. in cauliflower leaves, the duration of the biological cycle of p. xylostella was 19.8 days, the egg state lasted 3 days, the larva 9.69 days, pupa 5. 3 days and the capacity of oviposition was 175 eggs on average. there are no significant differences between the biological parameters of p. xylostella bred in both broccoli and cauliflower. in peru, it is the first study of p. xylostella in these species broccoli and cauliflower. acknowledgements my deep gratitude to professor clorinda vergara, for allowing me to use the facilities and equipment of the entomology museum klaus raven büller and professor yony callohuari, for his invaluable support in this manuscript. to professor agustín martos, for providing me with materials to elaborate the nursery of this study and for his friendship. references cohen, j. h., kristal, a. r., & stanford, j. l. 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(2014). plagas de hortalizas (cuarta edición). lima perú: departamento de entomología. universidad nacional agraria la molina, 172 p. sarnthoy, o., keinmeesuke, p., sinchaisri, n., & nakasuji, f. (1989). development and reproductive rate of the diamondback moth plutella xylostella from thailand. applied entomology and zoology, 24(2), 202–208. https://doi.org/10.1303/aez.24.202 syed, t. s., & abro, g. h. (2003). effect of brassica vegetable hosts on biology and life table parameters of plutella xylostella under laboratory conditions. pakistan journal of biological sciences, 6(22), 1891– 1896. https://doi.org/10.3923/pjbs.2003.1891.1896 peruvian journal of agronomy 1 (1): 1-7 (2017) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v1i1.1005 © the authors. published by universidad nacional agraria la molina received for publication: 2 september 2017 accepted for publication: 2 october 2017 recovering degraded lands in the peruvian amazon by cover crops and sustainable agroforestry systems recuperación de tierras degradadas en la amazonía peruana por cultivos de cobertura y sistemas agroforestales sostenibles alegre, j.1*; lao, c.1; silva, c.1; schrevens, e.2 *corresponding author: jalegre@lamolina.edu.pe abstract this long term research is being carried out in degraded lands of low fertility conditions in the humid tropics of the peruvian amazon. the objective is to recover degraded lands for sustainable agriculture that was affected by shifting agriculture and overgrazed pastures. the recovering trials in three farms are fertilized cover crops with the legume centrosema (centrosema macrocarpum), and the establishment of th prototype agroforestry systems are based on a) woody trees with swietenia macrophylla, guazuma crinita (gc), calycophylum spruceanum (cs) and simarouba amara (sa), b) woody-fruit trees with cedrelinga cateniformis, gc, sa, inga edulis and bactris gasipaes, and a c) silvopastoral system with centrosema and woody trees with gc, sa and cs. initial degraded compacted soils were covered with degraded grass (brachiaria brizantha) and weeds, and the soil were very acid (80% of al saturation) with 4 ppm of p and low soil organic matter and cation exchange capacity. soil was weeded and fertilized with a combined fertilizer based on rock phosphate (40 kg/ha), and then centrosema was planted followed by the plantation of trees with localized fertilization application. in three months we had 100% cover and weeds were controlled. average centrosema biomass in 8 months was 8.12 t/ha, and while the different trees were growing with 55 to 89 percentage of survival due to water stress, centrosema recovered the soil compaction up to 20 cm depth. biomass can be used as forage for small animals and to enrich soil. mean total nitrogen accumulation in biomass was 232 kg/ha. keywords: sustainable systems, change land use, agroforestry, cover crop. resumen esta investigación a largo plazo se está llevando a cabo en tierras degradadas de baja fertilidad en el trópico húmedo de la amazonía peruana. el objetivo es recuperar tierras degradadas para una agricultura sostenible que fue afectada por la agricultura migratoria y los pastos sobre pastoreados. los ensayos de recuperación en tres campos agrícolas son cultivos de cobertura fertilizados con la leguminosa centrosema (centrosema macrocarpum), y el establecimiento de 3 sistemas agroforestales prototipo se basan en a) árboles leñosos con swietenia macrophylla, guazuma crinita (gc), calycophylum spruceanum (cs) y simarouba amara (sa), b) árboles maderables-frutales con cedrelinga cateniformis, gc, sa, inga edulis y bactris gasipaes, y c) sistema silvopastoril con centrosema y árboles leñosos con gc, sa y cs. los suelos compactados degradados iniciales fueron cubiertos con hierba degradada (brachiaria brizantha) y malezas, y el suelo fue muy ácido (80% de saturación de al) con 4 ppm de p y baja materia orgánica del suelo y capacidad de intercambio catiónico. se deshierbó la tierra y se fertilizó con un fertilizante combinado a base de fosfato de roca (40 kg/ha), luego se plantó el centrosema seguido de la plantación de árboles con aplicación de fertilización localizada. en tres meses tuvimos un 100% de cobertura y las malezas fueron controladas. el promedio de la biomasa centrosema en 8 meses fue de 8.12 t/ha, y mientras que los diferentes árboles crecieron con 55 a 89 por ciento de supervivencia debido al estrés hídrico, centrosema recuperó la compactación del suelo hasta 20 cm de profundidad. la biomasa puede utilizarse como forraje para animales pequeños y para enriquecer el suelo. la acumulación media total de nitrógeno en la biomasa fue de 232 kg/ha. palabras clave: sistemas sostenibles, cambio en el uso de la tierra, agroforestería, cultivo de cobertura. 1 departamento de suelos, facultad de agronomía, universidad nacional agraria la molina, av. la molina s/n, lima 12, perú 2 department of biosystems, faculty of bioscience engineering, katholieke universiteit leuven, geo-institute, celestijnenlaan 200 e, 3001 heverlee, belgium recovering degraded lands in the peruvian amazon by cover crops and sustainable agroforestry systems junio-setiembre 2017 2 a b c introduction the peruvian amazon had a reserve of 78.9 million hectares of natural forest, comprising 58.8% of the total peruvian territory. the high rate of deforestation of 150,000 hectares per year in the peruvian amazon due to slash and burn and other inadequate land uses has reached the loss of forest of 7,172,554 has by year 2000 (cabrera et al., 2005). in the last 30 years, studies of soil degradation in the amazon region have been documented, indicating that soil degradation by erosion is still the main factor of soil depletion in latin america (montanarella et al., 2015). besides the physical factors, several other causes affect the degradation of the amazon; the main driving forces of deforestation are the selective extraction of wood, new roads and land tenure (kaimowitz and angelsen, 1998). several technical options to recover degraded land have been studied to recover compacted soils by overgrazed pastures or intensive mechanization (alegre et al., 1986; lal, 2015). some high and low input technologies in long term trials have been tested in the humid acid tropical soil of yurimaguas, peru and one of the main options was the agroforestry systems (palm, 1995; alegre, 2015). the introduction of perennial cropping systems with coffee or cacao managed with cover crops also offers an alternative to recover degraded land in the amazon (puertas et al., 2008; arévalo-gardini et al., 2016). the potential of trees and cover crops to contribute to the maintenance and rehabilitation of the soil’s physical characteristics such as better bulk density, mechanical resistance and soil aggregation has been well established, including an improvement on productivity (alegre and rao, 1996; rao et al., 1998; alegre et al., 2005). there is evidence that nutrient cycling of litterfall from shallow or deep roots of trees and cover crops can capture nutrients from topsoil or the subsoil. therefore, crop production can be improved (nair et al., 1999). crops can also force associated trees to take up a great part of nutrient from deep or laterally distant soils, and can deplete these nutrients when they are released from decomposing tree litter (schroth et al., 2001). the objective of the present work is to recover degraded lands in the peruvian amazon for sustainable agriculture that was affected by shifting agriculture and overgrazed pastures. materials and methods this study was located in the santo tomas community in yurimaguas, loreto, perú. a total of three farmers with extensive degraded ultisol lands (tyler et al., 1978) with overgrazing brachiaria (brachiaria brizantha) pastures were selected. the degraded pasture was sprayed with contact herbicides. three plots of 70x40 m with degraded pasture for each of the three farmers were set up (fig. 1) and each plot was divided in 3 subplots and monitored initially with replicated soil physical (bulk density and mechanical resistance) and chemical soil properties (organic matter, available phosphorus and potassium, cation exchange capacity and aluminum saturation) that were measured from 0-15, and >15 to 30 cm depth. the recovering trials in three farms were fertilized with 40 kg of p/ha and planted by hand, cover crops with legume centrosema macrocarpum (5 kg/ha of centrosema seed) and after a month, three prototype agroforestry systems (afs) were established based on a) afs 1: multistrata system 1 with woody of fast, medium and slow growing trees species with swietenia macrophylla, guazuma crinita (gc) , calycophylum spruceanum (cs) and simarouba amara (sa), b) afs 2: multistrata system 2 with fast, medium and slow growing woody-fruit trees species with cedrelinga cateniformis, gc, sa, inga edulis and bactris gasipaes, and c) afs 3: silvopastoral system with centrosema and fast and medium woody trees with gc, sa and cs (fig. 2). each of the three farmer’s sites was considered as a block (three replications) and the three treatments were the three afs described above. for the statistical analysis, we used lsd test in the r statistical software (r core team, 2016). figure 1. picture of a) initial degraded pasture with brachiaria, b) after burning with herbicide, and c) planting cover crop with centrosema. alegre, j.; lao, c.; silva, c.; schrevens, e. peruvian journal of agronomy 1(1):1-7 3 figure 2. prototype of three agroforestry systems of different tree species in santo tomas, yurimaguas. recovering degraded lands in the peruvian amazon by cover crops and sustainable agroforestry systems junio-setiembre 2017 4 all nursery trees during plantation were fertilized with a tropical fertiphos fertilizer (rock phosphate fertilizer enriched with calcium and magnesium, and some organic matter components) at the rate of 200 g/plant in a 20x30 cm hole. results and discussion initial degraded compacted soils were covered with degraded grass (brachiaria brizantha) and weeds, and the topsoil was very acid with a ph ranging from 3.8 to 4.8 with low organic matter and very low phosphorus level (less than 3 ppm) and medium level of potassium, and low cec with aluminum saturation fluctuating from low (8.77%) to high (72.44%) levels as shown in table 1. table 1. initial level of soil nutrients at 0 15 cm depth in the three farmers sites under overgrazing pastures in santo tomas, yurimaguas. farmers sites ph om (%) available p (ppm) available k (ppm) cec meq.100/g al saturation (%) washington 4.16 0.96 3.0 62 5.94 18.80 roberto 4.80 1.81 2.6 44 9.35 8.77 clais 3.82 1.18 2.3 50 11.38 72.44 rainfall distribution during 2016 was very variable. for some months, rainfall was less than the average for the area (figs. 3 and 4). figure 3. annual rainfall distribution from 2007 to 2016 in santo tomas, yurimaguas. figure 4. monthly rainfall distribution during 2016 in santo tomas, yurimaguas. three months after planting the cover crops, we had 100% of soil surface covered with centrosema at two farmer’s site, and weeds were controlled. total average of centrosema dry biomass of each farmer site ranged from 7 to 8.25 t/ha (fig. 5). the farmers site “w” had the highest centrosema biomass production because the farmer planted centrosema before and it got adapted, responding more efficiently to the p application. figure 5. eight month year old dry biomass in the three farms in santo tomas, yurimaguas (w, r and c are washington, roberto and clais farmers, respectively). there were significant differences between afs for the centrosema biomass production between farmer’s sites as shown in figure 6. agroforestry system 1 produced 9.77 t/ha of dry biomass which was significantly higher than the other two afs with 7.99 and 7.2 for afs 2 and afs 3, respectively. also afs 1 had more tree species and some of the fast growing species were self-pruned, consequently, the litterfall was increased. (figs. 7a,b). figure 6. biomass production of centrosema covers for each of the agroforestry systems established in santo tomas yurimaguas. * means fallowed with the same letter are not significant different at 0.05 probability afs 1 is a multistrata system with fast and slow growing woody tree species afs 2 is a multistrata system with slow and fast growing woody and fruit species afs 3 is a silvopastoral system with fast growing woody species. alegre, j.; lao, c.; silva, c.; schrevens, e. peruvian journal of agronomy 1(1):1-7 5 a b figure 7. recovered degraded pasture with centrosema (a), and trees (b) in agroforestry forestry systems. trees suffered water stress during planting time on january 2016 because of an unusual reduction of rainfall with less than 120 mm during few rainfall events. normally by this time of the year it rains between 250-300 mm. (figs. 3 and 4). the fast growing tree species presented more susceptibility to water stress, and simarouba amara presented the highest percentage of mortality with 43% followed by calycophylum spruceanum with 24% of mortality. for the rest of trees the percentage of mortality was less than 11% (fig. 8). figure 8. average percentage of mortality of trees planted for each species in the three farmer’s sites in santo tomas, yurimaguas. the cutting of centrosema around trees during the growing phase of the perennial trees was used in some cases as forage for small animals, and in other cases it was left as mulch to enrich soil with nitrogen (n). results showed an average biomass accumulation of 215-250 kg/ ha of n (fig. 9), which is more than the average of 150 kg/ha that was found in non-fertilized plots in yurimaguas (palm, 1995; alegre et al., 2005). there was also a significant increase in potassium and calcium levels in the centrosema biomass due to recycling biomass and lower recycling levels in phosphorus. figure 9. mean content of n, p, k and ca in the centrosema biomass in three farmer’s sites in santo tomas, yurimaguas. soil strength (ss) is shown in figure 10 and there was compaction of up to 20 cm in depth with ss higher than 100 kpa, ranging from 117 to 204 kpa. after a year of recovering with centrosema cover and trees, these values of mr were reduced to less than 100 kpa with ranges from 25 to 85 kpa. bulk density followed the same trends. figure 10. soil strength at different depths before and one year after establishing the cover crops and trees in three farmers sites in santo tomas, yurimaguas. recovering degraded lands in the peruvian amazon by cover crops and sustainable agroforestry systems junio-setiembre 2017 6 soil nutrients improvement in 8 months were only for organic matter (om) and available potassium (k) as shown in figures 11 and 12 due to higher centrosema litterfall production and fast decomposition rates that incorporate good quality organic matter to the soil in the short time. for the other nutrients, there were no differences, and it is expected that trees will build organic matter in the medium to long term with the continuous addition of litterfall. figure 11. soil organic matter content after eight months of cover crops establishment and trees planted in three farmers sites in santo tomas, yurimaguas. figure 12. soil available potassium content eight months after one year of cover crops establishment and trees planted in three farmers sites in santo tomas, yurimaguas. conclusions in three months we had 100% cover with centrosema and weeds were controlled. total average centrosema biomass in 8 months was 8.12 t/ha. the multistrata agroforestry system 1 with more planted species produced 9.77 t/ha of dry biomass which was significantly higher than the other two afs. while the trees were growing, there was a 55 to 80% of survival due to water stress; centrosema can be used as forage for small animals and to enrich soil. nitrogen accumulation in biomass was 232 t/ha. soil compaction was reduced at 20 cm depth and organic matter and potassium level were increased in the short time after cover crops and trees establishment. acknowledgments the authors are grateful to vlir (belgium flemish universities) for financial support from the belgium government, and the soil science department, faculty of agronomy, unalm for the technical and laboratory facilities. references alegre, j.c., d.k. cassel, and d.e. bandy. 1986. reclamation of an ultisol damaged by mechanical land clearing. soil science society of america journal 50: 1026-1031. alegre, j.c., and m.r. rao. 1996. soil and water conservation by counter hedging in the humid tropics of peru. agriculture, ecosystems and environment 57: 17-25. alegre, j.c., m.r. rao, l.a. arevalo, w. guzman, and m.d. faminow. 2005. planted tree fallows for improving land productivity and reducing deforestation in the humid tropics of peru. agriculture, ecosystems and environment 110: 104-117. alegre, j. 2015. sistema agroforestal de multiestratos: recuperación de suelos degradados en la amazonia. leisa 31: 28-30. arévalo-gardini, e., m. canto, j. alegre, o. loli, a. julca, and v. baligar. 2016. changes in soil physical and chemical properties in long term improved natural and traditional agroforestry management system of cacao genotypes in peruvian amazon. plos one 10: e0132147. cabrera, m., r. soto, t. lapeyre, h. portuguez, p. huerta, c. sifuentes, p. del pozo, v. gabriel, et al. 2005. mapa de deforestación de la amazonia peruana 2000. memoria descriptiva. retrieved from sinia.minam. gob.pe/download/file/fid/39519 kaimowitz, d., and a. angelsen. 1998. economic models of tropical deforestation: a review. retrieved from: http://www.cifor.org/publications/pdf_files/books/ model.pdf lal, r. 2015. restoring soil quality to mitigate soil degradation. sustainability 7: 5875-5895. montanarella, l., d.j. pennock, n. mckenzie, m. badraoui, v. chude, i. baptista, t. mamo, m. yemefack, et al. 2016. world´s soils are under threat. soil 2:79-82. nair, p.k.r., r.j. buresh, d.n. mugendi, and c.r. latt. 1999. nutrient cycling in tropical agroforestry systems: myths and science. in l.e. buck, j.p. lassoie, and e.c.m. fernandes [eds.], agroforestry in sustainable agricultural systems, 14-43. cr press, boca raton, florida, usa. palm, c.a. 1995. contribution of agroforestry trees to nutrient requirement of intercropped plants. agroforestry systems 30: 105-124. puertas f., e. arévalo, l. zúñiga, j. alegre, o. loli, h. alegre, j.; lao, c.; silva, c.; schrevens, e. peruvian journal of agronomy 1(1):1-7 7 soplin, and v. baligar. 2008. establecimiento de cultivos de cobertura y extracción total de nutrientes en un suelo de trópico húmedo en la amazonía peruana. ecología aplicada 7: 23-28. r core team. 2016. r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. rao, m.r., p.k.r nair, and c.k. ong. 1998. biophysical interactions in tropical agroforestry systems. agroforestry systems 38: 3-50. schroth, g., j. lehmann, m.r.l. rodrigues, e. barros, and j.l.v. macêdo. 2001. plant-soil interactions in multistrata agroforestry in the humid tropics. agroforestry systems 53: 85-102. tyler, e.j., s.w. buol, and p.a. sanchez. 1978. genetic association of properties of soils an area in the upper amazon basin of peru. soil science society of america journal 42: 771-776. peruvian journal of agronomy 2 (1): 9 14 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i1.1127 © the authors. published by universidad nacional agraria la molina received for publication: 30 january 2018 accepted for publication: 30 march 2018 pre-basic seed potato (solanum tuberosum l.) production using temporary immersion bioreactors producción de semilla pre-básica de papa (solanum tuberosum l.) mediante biorreactores de inmersión temporal tapia, m.de l.*1, arbizu, c.*2, beraún, f. 3, lorenzo, j.4, escalona, m.5 *corresponding author: ltapia@lamolina.edu.pe / carbizu@lamolina.edu.pe abstract potato is an important food and cash crop in perú. its production is limited due to seed quality, and other biotic and abiotic factors. here we explore a new alternative for potato pre-basic seed production named temporary immersion bioreactor (tib). potato microtubers production using tib comprises two phases: multiplication and microtuberization. during the multiplication phase, we used 50 segments of four nodes from a semi-solid medium with an immersion and frequency time of five minutes and three hours, respectively. in addition, a photoperiod of 16 hours of light and eight hours of darkness during 28 days were employed. for the microtuberization phase and under darkness conditions during 60 days, we used 80 g/l of sucrose and 30 ml/explant as a volume for the medium. then, it was possible to obtain five, six, and 2.5 microtubers per explant of potato varieties “canchan inia”, “capiro”, and “papa 3”, respectively. keywords: microtubers, temporary immersion bioreactor, pre-basic seed, microtuberization. resumen la papa es un alimento importante y cultivo de alta importancia económica en perú. su producción es limitada debido a la calidad de las semillas y otros factores bióticos y abióticos. aquí exploramos una nueva alternativa para la producción de semilla pre-básica de papa llamada biorreactor de inmersión temporal (bit). la producción de microtubérculos de papa utilizando bit comprende dos fases: multiplicación y microtuberización. durante la fase de multiplicación, se utilizaron 50 segmentos de cuatro nudos de un medio semisólido con un tiempo de inmersión y frecuencia de cinco minutos y tres horas, respectivamente. además, se empleó un fotoperiodo de 16 horas de luz y ocho horas de oscuridad durante 28 días. para la fase de microtuberización y en condiciones de oscuridad durante 60 días, se utilizaron 80 g/l de sacarosa y 30 ml/explante como volúmen para el medio. luego fue posible obtener cinco, seis y 2.5 microtubérculos por explante de las variedades de papa “canchan inia”, “capiro” y “papa 3”, respectivamente. palabras clave: microtuberculos, biorreactor de inmersión temporal, semilla prebásica, microtuberización 1instituto de biotecnología-cultivo de tejidos, facultad de agronomía, universidad nacional agraria la molina, av. la molina s/n, lima 12, perú. 2instituto de biotecnología-genómica y bioinformática, facultad de agronomía, universidad nacional agraria la molina, av. la molina s/n, lima 12, perú. 3departamento de contabilidad, facultad de ciencias contables, universidad nacional del callao, av. juan pablo ii 306, callao 2, perú.. 4laboratory for plant breeding, centro de bioplantas, universidad de ciego de ávila, carretera morón km 9 ½, ciego de ávila, cuba. 5laboratory for plant cell and tissue culture, centro de bioplantas, universidad de ciego de ávila, carretera morón km 9 ½, ciego de ávila, cuba. introduction micropropagation is a widely used technique to obtain plants free of diseases that may be used for the production of minitubers and microtubers throughout the year, becoming the first generation of pre-basic seed (ranalli 2007). the production of potato microtubers represents a potentially novel method for seed production compared to some limitations present with the use of minitubers, which are difficult to store, delicate, difficult to handle and require an additional acclimatization process (coleman et al. 2001). in some potato varieties, the use of nodal segments with two nodes (4 buds) as an explant produced in vitro seedlings with greater vigor compared to the use of nodal segments with a node (venkatasalam et al. 2012). larger microtubers are sowed directly into the field. on the other hand, in vitro plants that are obtained from minitubers require acclimatization (donnely et al. 2003), resulting in a not very economical method. radouani and lauer (2015) compared yields of tubers produced from microtubers and minitubers of two potato varieties “nicola” and “russet burbank”. microtubers had a small weight of 0.2-1.5 g, medium weight of more than 1.5 to 3 g, and large ones greater than 3 g. small minitubers weighed 15-20 g, the medium ones more than 20 to 40 g, and the big ones greater than 40 g. both varieties produced more tubers from microtubers than from minitubers, 1,033,333 vs pre-basic seed potato (solanum tuberosum l.) production using temporary immersion bioreactors january april 2018 10 568,750 tubers/ha for “nicola” and 605,417 vs 482,291 tubers/ha for “russet burbank”, respectively. in addition, large mini and microtubers had the highest yield with a total of 49 and 40 t/ha, respectively. the physiologically older microtubers were more prolific than the younger ones; this was particularly found for late cultivars (ranalli et al. 1994). they found that the yield of potato obtained from microtubers had lower yields compared to minitubers and conventional seed. on the other hand, it was observed that microtubers had longer storage ability (7 months) and also better performance than those with shorter storage time (3 months) (désiré et al. 1995). direct planting in the microtuber field can affect the growth and development of the plant. greater numbers of tubers per plant were recorded in plants from physiologically older microtubers. potato cultivation represents 10% of the peruvian agricultural gross domestic product, and it is cultivated in 19 out of the 24 departments of perú. however, potato farmers use only about 0.2% (1,145 tons) of certified seed (inei, 2012). therefore, the present study aims to compare the production of microtubers of three potato varieties “canchan inia”, “capiro” and “papa 3” using temporary immersion bioreactors. the temporary inversion system consisted of two 4 l polypropylene containers, silicone hoses, 0.2 mn hydrophobic filters, electrovalves and an air compressor; one container is used for the explants and the other container is used for the culture medium; both bottles are connected through hoses, the frequency and time of investment is controlled through an automated system materials and methods in vitro potato plants (solanum tuberosum l.) of three potato varieties “canchan inia”, “capiro” and “papa 3” were used. these three potato varieties were acquired from the international potato center (cip for its acronym in spanish) germplasm bank which were obtained from the cultivation of meristems seeded in a starting medium composed of ms salt (murashige and skoog 1962). tests that were carried out at cip were: nash for the detection of potato spindle tuber viroid (pstvd), serological test of elisa, and test of indicator plant. all of these tests were performed over a period of 2 months. the basal composition of the murashigue & skoog medium (1962) modified by espinoza (1991) was used the modification consisted of the use of ammonium nitrate (1,750 mgl), potassium nitrate (2,000 mg/l), calcium chloride (450 mg/l) and phosphate (175 mg/l) thiamin (0.4 mg/l) glycine (2.0 mg/l), nicotimic acid (0.5 mg/l), pyridoxine (0.5 mg/l) calcium pantothenate (2 mg/l) and arginine (4 mg/l). the ph of the culture medium was adjusted to 5.8 before steam sterilization in the autoclave. the time and frequency of immersion were 4 minutes every three hours. the culture conditions were 16 hours of light, and 8 hours of darkness; fluorescent white light lamps were used to guarantee a flow of photosynthetic photons of 40 μmol.m-2.s-1. we used a relative humidity between 60-70% during the phase of multiplication, and in the microtuberization phase the sucrose concentration was increased. incubation conditions were performed under dark conditions. for inoculation in the temporary immersion bioreactors (tib), segments with 2 nodes (4 axillary buds) were used from a third subculture in semi-solid medium. polypropylene containers of 4-liter capacity were used. the culture medium was the ms (murashige and skoog 1962) modified by espinoza et al. (1986). for the tib multiplication phase, 50 explants/tib, 15 ml medium/explant volume, time and immersion frequency of 4 minutes every 3 hours for 28 days were used with a photoperiod of 16 hours of light and 8 hours of darkness. after this time, the microtuberization was induced in the tib; the flask containing the culture medium was replaced by another with fresh medium of the same composition but, supplemented with 80 g/l of sucrose without renewal of the medium. the volume of the medium was 1.5 liters (30 ml/explant), and the immersion time and frequency were 4 minutes every 3 hours (8f/day), culture time of 60 days under darkness at a temperature of 22 celsius. three tibs were used per treatment (fig. 1). in order to harvest the microtubers, the container was opened and carefully separated from the plant and root residues. then, microtubers were rinsed with tap water to remove the culture medium, and were placed in trays on filter paper to remove moisture. the total number of microtubers per bioreactor was evaluated, as well as the number of microtubers per shoot. in addition, fresh mass (g) was evaluated using a sartorius analytical weight. statistical analyses statistical analyses were performed using the spss software version 11.5. the analyses for the different variables were performed through a one-way anova, bifactorial anova or a t-test, as parametric analyses. one-way anova and bifactorial anova were followed by a tukey test. previously, normal distribution and homogeneity of variance were also demonstrated according to the kolmogorov-smirnov and levene tests, respectively. the data of discrete variables were transformed with the equation y’ = (y+0.5) x 0.5, and the percentage variables according to the equation y’ = 2arcosen ((y/100) x 0.5). results protocol of microtuberización for obtaining pre-basic seed in three potato varieties. figure 2 shows the results of the protocol used in the present study for the formation of microtubers in three potato varieties. no significant differences were found in the number of microtubers per bioreactor between the “canchan inia” and “capiro” varieties but with “papa 3” variety. varieties “canchan inia” and “capiro” produced tapia, m.de l., arbizu, c., beraún, f., lorenzo, j., escalona, m. peruvian journal of agronomy 2 (1): 9 14 (2018) 11 figure 1. protocol for the production of potato microtubers using temporary immersion bioreactors (tib) a total of 250 and 270 microtubers, respectively. on the other hand, “papa 3” produced only 100 microtubers (fig. 2a). as for the number of microtubers, the “canchan inia” and “capiro” potato varieties produced a significant average of 5 and 6 microtubers per explant, respectively. variety “papa 3” had a lower average (2.5 microtubers per explant) (fig. 2b). no statistical difference was observed between the three potato varieties for fresh mass of the microtubers (fig. 2c). potato variety “papa 3” produced microtubers of 1.2 cm, resulting significantly different compared to the other two varieties (1 cm) (fig. 2d). by analyzing the quality of the microtubers that were formed in the bioreactor and calculating the percentage of microtubers (pre-basic seed) of the three potato varieties with suitable characteristics for direct seeding in the field (greater than 0.5 g), we found significant differences between the three potato varieties. the percentage of microtubers of variety “papa3” was significantly higher (90%) while varieties “canchan inia” and “capiro” averaged 50 and 36 percent, respectively (fig. 3). discussion the dependence of potato genotype for the formation of microtubers is well documented. gopal et al. (1998) evaluated the formation of microtubers in semi-solid medium of 22 potato genotypes (solanum tuberosum l.) under six in vitro culture conditions. cultures in short photoperiod (10 h, 6-12 μmol.m-2.s-1) and low temperatures (day: 20°c and night: 18°c) had higher yield (225 mg/ plant) and higher number of microtubers (2/plants) than those maintained under long-day conditions (16 h, 38pre-basic seed potato (solanum tuberosum l.) production using temporary immersion bioreactors january april 2018 12 50 μmol.m-2.s-1) combined with high temperatures (day: 28°c and night: 25°c) which yielded 207 mg/plant, and the number of microtubers was 0.9/plants. the mean of the 12 genotypes that tuberized in all treatments varied in the following values, yield of microtubers was 207-644 mg/ plant), number of microtubers was 0.91-2.05, fresh mass of microtubers ranged from 114 to 263 mg, and number of eyes was 3.48-5.96. the interaction genotype by culture conditions was significant indicating the importance of developing specific protocols specific for each genotype to maximize microtuberization. temporal immersion bioreactors are also reported on differences in the performance of microtubers associated with the genotype. kämäräinen-karppinen et al (2010) evaluated four genotypes and found that the number of microtubers/bioreactors ranged from 30 in “asterix” to 75 in “velox”. the latter cultivar yielded a greater number of microtubers with crop capacity than the rest. teisson and alvard (1999) also found differences in the microtuberization of three potato cultivars in liquid medium using temporary immersion. the number of microtubers/ bioreactor was in the range of 48 in the “desirée”, 52 and 68 for “bintje” and “ostara”, respectively. variety “ostara” achieved a greater number of microtubers/internode (2.3) and fresh mass varied from 20.6 g for “bintje”, 35.5 g figure 2. pre-basic seed in the number of microtubers per bioreactor (a), number of microtubules per explant (b), fresh mass of microtubers (c), and diameter of the microtubers (d). in each figure, means with equal letters do not have statistically significant differences (monofactorial anova, tukey p> 0.05). for statistical processing, the number of microtubers was transformed according to y = (y + 0.5) x 0.5. data represent the mean of three bit / treatment. for “ostara” and 39.9 g in “desirée”. the microtubers of the latter cultivar were very large, some of them with mass of 6 g. in addition, jiménez et al. (1999) evaluated the microtuberization of two cultivars using tib and the number of microtubers/single node was 3.1 and 2.8 for “desirée” and “atlantic”, respectively. the validation of the proposed protocol here for three potato varieties showed a different behavior in the number of microtubers/bioreactor and number of microtubers/ explant. for both parameters, “papa 3” had the lowest response level (100 microtubers/tib and 2 microtubers/ explant). regarding fresh mass of the microtubers, there were no differences between the three potato varieties. this may have been influenced by the favorable cultivation conditions and nutrient level that is achieved in the tib. however, microtubers of “papa 3” had a larger diameter influenced perhaps by lower number of microtubers. also, 90% of microtubers of this variety had a mass between 0.53.0 g and, were therefore suitable for direct seeding in the field. the response to microtuberization of seven potato cultivars of different genetic origin and maturity group was investigated under different conditions of photoperiod and intensities of light in a system free of plant growth regulators. multivariate analysis showed that the genotype was the factor that most affected the variable response in tapia, m.de l., arbizu, c., beraún, f., lorenzo, j., escalona, m. peruvian journal of agronomy 2 (1): 9 14 (2018) 13 figure 3. sixty-day old microtubers of potatoes of the following three varieties used in the present study, “canchan inia” (a), “capiro” (b) and “papa 3” (c). microtuberization, indicating that the genetic origin of the clone plays a basic role in tuberization under in vitro conditions (dobranszki et al. 2008). conclusions response of “canchan inia”, “capiro” and “papa 3” potato varieties was compared to the protocol for microtuberization of potato in the tib that includes two phases, one of multiplication and growth of plants in vitro, and another one of formation of microtubers in the bioreactor. the evaluation of the microtubers/explant is different for each variety, “canchan inia” (5), “capiro” (6), “papa 3” (2.5). we conclude that there are differences due to the genetics of each variety. pre-basic potato seed was obtained for the three peruvian commercial varieties using temporary immersion bioreactors, demonstrating that microtubers can be used as planting material for direct sowing in the field. yields were heavily influenced by the potato variety. references coleman w.k., donnelly, d.j. and coleman s.e. 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(2012). effect of carbon sources and explants on in vitro multiplication of potato. potato journal 39(2): 166-172. peruvian journal of agronomy 3(1): 24 28 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i1.1281 © the authors. published by universidad nacional agraria la molina received for publication: 11 january 2019 accepted for publication: 30 march 2019 yield of five potato varieties in temporary immersion bioreactors (tib) rendimiento de 5 variedades de papa en biorreactores de inmersión temporal (bit) carrión, a.1*; tapia, m.del.1,2 *corresponding author: acarrione.6@gmail.com 1 instituto de biotecnología, universidad nacional agraria la molina, av. la universidad s/n, lima, perú 2 profesor principal, departamento de fitotecnia, universidad nacional agraria la molina, av. la universidad s/n, lima, perú abstract the potato (solanum tuberosum l.) is important as both a food and a source of economic activity in peru. however, potato production is limited by seed quality and other biotic and abiotic factors. we explore a new alternative method for producing prebasic seeds of peruvian potato varieties known as temporary immersion bioreactors (bit). the process of producing potato microtubers using bit consists of two phases: proliferation and microtuberization. during the proliferation phase, we seeded six nodal segments of three nodes in a liquid culture medium with 30 g of sucrose under a photoperiod of 16 light hours and eight dark hours. this phase also included an irrigation cycle of five minutes every three hours for 30 days. during the microtuberization phase, the conditions were changed to a medium with 80 g of sucrose in darkness, and the same irrigation cycle was used for 60 days. under these conditions we obtained 20, 18.4, 13.4, 13.4, and 4.6 microtubers of the varieties peruanita, canchan, capiro, unica, and yungay, respectively. key words: microtubers, prebasic seed, temporary immersion bioreactors. resumen la papa (solanum tuberosum l.) es un alimento importante y cultivo de alta importancia económica en perú. su producción es limitada debido a la calidad de las semillas y otros factores bióticos y abióticos. aquí exploramos una nueva alternativa para la producción de semilla prebásica de variedades peruanas de papa llamada biorreactores de inmersión temporal (bit). la producción de microtubérculos de papa utilizando bit comprende dos fases: proliferación y microtuberización. durante la fase de proliferación se utilizó 6 segmentos nodales de 3 nudos sembrados en un medio de cultivo líquido con 30 g de sacarosa bajo un fotoperiodo de 16 horas luz y 8 de oscuridad, además de tener un ciclo de riego de 5 minutos cada 3 horas por 30 días; para la fase de microtuberización se cambió a un medio de 80 g de sacarosa, en oscuridad y con el mismo ciclo de riego por 60 días. bajo estas condiciones fue posible obtener 20, 18.4, 13.4, 13.4 y 4.6 microtubérculos por biorreactor de las variedades “peruanita,” “canchan,” “capiro,” “unica” y “yungay,” respectivamente. palabras clave: microtubérculos, semilla prebásica, biorreactores de inmersión temporal. introduction potatoes are the world’s most important food crop after wheat and rice and are a staple food for 1.3 billion people, with increasing popularity in the developing world. (stokstad, 2019). in peru, growers cultivate four potato species: solanum tuberosum along with three other species exclusive to the andes. pinstituto de biotecnología, universidad nacional agraria la molina, av. la universidad s/n, lima, perú. mountains and on the coast (egúsquiza, 2014). peru ranks 18th among main consumer countries, with an annual consumption of 78.4 kg per capita. additionally, potato production has increased from 4,704,987 tons in 2004 to 7,704,987 tons in 2014 (faostat, 2015). potato cultivation accounts for 25% of peru’s agricultural gross domestic product and is grown in 19 of the country’s 24 departments. however, certified seed only accounts for 0.2% (1,145 tons) of seed farmers use. in other words, small producers continue to plant seed potatoes acquired through “informal seed systems” that often have a poor sanitary status, leading to significant yield reductions (egúsquiza, 2014). low potato yields in peru and other countries in the region can be attributed to the use of low-quality seeds and recycling of tubers. therefore, there is consensus on the need to incorporate effective and efficient seed production technologies that are also consistent with carrión, a.; tapia, m.del. peruvian journal of agronomy 3 (1): 24-28 (2019) 25 the reality of potato cultivation in latin america. (del rio et al., 2017). among these technologies are certified seed tubers, which allow doubling of yields, although its availability is weatherand season-dependent. another is the use of potato seedlings in vitro; however, this can be costly for farmers because of the high mortality of plants during the acclimatization phase, a critical stage of in vitro plant production stage, as well as the production of tuberseeds, which is dependent on weather and season. another technology that could help overcome these problems is the use of microtubers, which are easy to handle, store, and transport and can be produced throughout the year (dobránszki, magyar-tábori, & hudák, 2008) temporary immersion bioreactors have been used for efficient production of potato microtubers (rokka, kämäräinen-karppinen, virtanen, & pirttilä, 2013; elaleem, modawi, & khalafalla, 2015; pumisutapon & topoonyanont, 2015; rahman, shahinul islam, chowdhury, & sreeramanan subramaniam, 2015; tapia, lorenzo, mosqueda, & escalona, 2017; ali, khan, nouroz, erum, & nasim, 2018; naik & buckseth, 2018; tapia, arbizu, beraún, lorenzo, & escalona, 2018). in peru the canchan, capiro, unica, yungay, and peruanita cultivars stand out for their culinary quality in frying or dry matter content in the tubers. however, there is no history of obtaining microtubers of the last three of these varieties. our intent is to evaluate the production of peruvian potato variety microtubers using temporary immersion bioreactors. materials and methods experimental place our experimental work was carried out in the institute of biotechnology (ibt) facilities, area cultivation of fabrics of the national agrarian university la molina (238 masl). plant material plant material used as the mother material was in vitro potato seedlings of the canchan, unica, yungay, capiro, and peruanita varieties from the international potato center. culture medium the culture medium’s basal composition was based on the work of murashige and skoog (1962). espinoza et al. (1992) modified the medium with ammonium nitrate (1750 mg l-1), potassium nitrate (2000 mg l−1), calcium chloride (450 mg l−1), phosphate (175 mg l−1), thiamine (0.4 mg l−1), glycine (2.0 mg l−1), nicotinic acid (0.5 mg l−1), pyridoxine (0.5 mg l−1), calcium pantothenate (2 mg l−1), acid folic acid (1 mg l−1), and arginine (4 mg l−1). we also added myo-inositol (100 mg l−1) to the culture medium and adjusted the ph to 5.7 using sodium hydroxide or 1 n hydrochloric acid. to prepare the proliferation medium we added 30 g of sucrose and added 80 g of sucrose per liter of medium to the tubers. the medium was sterilized in an autoclave at 121°c and 1.2 kg cm−2 for 25 minutes. temporary immersion bioreactors two 500 ml vessels were used following the model proposed by escalona et al. (1999). installation of the five varieties to the temporary immersion system we used three to four knot stem segments of canchan, capiro, yungay, unica, and peruanita. we seeded six segments in a vessel containing 100 ml of proliferation culture medium, sealed and installed the system on the shelves, and set an immersion time and frequency of 4 min every 3 hours for 28 days with a 16 h light/8 darkness photoperiod using fluorescent lamps (25–30 μmol m-2 s−1). after four weeks, the proliferation medium in the laminar flow chamber was changed to the tuberization medium. after this, the system was sealed and the vessel containing the seedlings was covered with a bag to place the specimens in darkness. we then installed the containers on shelves and set the immersion time and frequency to four minutes every three hours (8f/day). the culture time we set to 60 days in the dark at 22 ± 0.2°c and the specimens were incubated at 24°c for eight weeks. after the tuberization process was completed, we carefully separated the microtubers from the plant and root residues, rinsed them with running water to remove the culture medium, and placed them in trays on filter paper to remove moisture. evaluation system after harvesting the microtubers we evaluated the number of microtubers obtained per experimental unit, the fresh weight of each microtuber, and the yield per experimental unit. experimental design and statistical analysis we performed a completely randomized design with three replications and processed the resulting data in minitab 17. we conducted a comparison of means using the tukey test to observe if there were significant differences between varieties. results behavior of the five varieties to the temporary immersion system figure 1 indicates no significant differences between means of average weight of five varieties of microtubers produced in the bioreactor using the tukey test with a 5% significance level; however, the yungay variety obtained an average weight of 0.528 g, which slightly surpasses the minimum weight that microtubers must have for greenhouse development. yield of five potato varieties in temporary immersion bioreactors (tib) january april 2019 26 figure 1. comparison test of means of average microtuber weight produced by each variety in the temporary immersion system (tukey α = 0.05) figure 2 shows a comparison of the number of microtubers of the five varieties studied produced by the bioreactor and whether the tukey test with a level of significance of 5% found significant differences between their means. the figure highlights the variety “peruanita” having the highest yields with 20 microtubers per bioreactor on average; however, this is not significant in comparison with values obtained for varieties canchan, capiro, and unica. in contrast, the yungay variety produced the lowest number of microtubers with 4.6 microtubers per bioreactor. figure 2. comparison of microtuber yield means for each variety in the temporary immersion system (tukey α = 0.05) discussion with respect to average tuber weight, the yungay variety exceeded the minimum weight needed to be considered as a prebasic seed; however, this weight is lower than the weight that yu, joyce, cameron, and mccown, (2000) obtained for the russet burbank cultivar, (1,216 g), that pérez et al. (2007) obtained for the cv. atlantic (2,745 g), that castro (2011) obtained for the capiro (1.00 g) and canchan (0.97 g) varieties, and that araque et al. (2018) obtained for the capiro variety (123.90 mg). park et al. (2009) classified microtubers of the superior potato cultivar in three categories: small (4.0–6.0 mm and 0.18 g), medium (6.1–8.0 mm and 0.29 g), and large (>8.1 mm and >0.54 g). based on this categorization, the microtubers produced in the bioreactors would mostly be categorized as small to medium. khalil, el aal, and samy (2017) demonstrated that using only sucrose to induce tuberization was favorable for producing unica variety microtubers, obtaining 89.5 mg per microtuber. the unica variety produced fewer tubers per bioreactor than the other varieties evaluated, yielding 0.7 microtubers per explant sown; however, this number is higher than that yu et al. (2000) obtained for the russet burbank cultivar (175 microtubers and 50 stem segments), that pérez et al. (2007) obtained for the cv. atlantic cultivar (186 microtubers and 60 stem segments in 4l flasks), that mani, mhamdi, bettaieb, and hannachi (2014) obtained from seven stem segments in 50 ml of medium with alaska (3.7 ± 1.1), safran (6.4 ± 2.1), and spuntia (13.83 ± 3.8) cultivars, that elaleem et al. (2015) obtained for the almera (6.0 ± 0.5) and diamant (3.0 ± 0.0) cultivars grown in 25 ml of culture medium, and that castro (2011) obtained in the capiro (70.5) and canchan (69) varieties, with 50 stem segments already obtained by khalil et al. (2017) in the unica variety (6.4) from five explants. in contrast, areque et al. (2018) obtained better production with the capiro variety (49 tubers from 12 explants) and tapia et al. (2018) obtained better yields with the canchan (250) and capiro (270) varieties from 50 explants these varying potato variety responses are due to plant regeneration in vitro in temporary immersion systems depends on abiotic (e.g., temperature and photoperiod) and biotic (e.g., manipulation of organic and inorganic medium constituents, explant type and species, and nutritional characteristics) factors. additionally, morphological, organogenic, and nutritional characteristics are determined by genotype, making it possible to improve in vitro investigations. when using the most organogenic genotypes, genotypic variability is used to induce in vitro organogenesis (casas et al., 1993). complete regeneration from the explant is often specific to the species, variety, or introduced genotype, which determines its morphogenic expression capacity (ziv, 2005). sucrose concentration in the tuberization medium considerably influences microtuberization, with an optimal concentration of 8% (80 gr.l−1) as demonstrated by mani et al. (2014), elaleem et al. (2015), salem and hassanein (2016), islam, roni, jamal, and shimasaki (2017), khalil et al. (2017), and ali et al. (2018). garcía and azofeifa (2017) showed that exposure to light during the tuberization phase is favorable to microtuber production and size, although in our case light exposure inhibited production and induced budding in the microtubers formed. carrión, a.; tapia, m.del. peruvian journal of agronomy 3 (1): 24-28 (2019) 27 acknowledgments thanks to the team of the biotechnology institute of the universidad nacional agraria la molina, for the support given during the realization of this investigation, and the international center of the potato to provide the vegetal material and to allow the search of bibliographical references. conclusions producing microtubers in temporary immersion bioreactors is influenced by genotype. additionally, we observed that native potato varieties such as peruanita adapt very well to this technique of prebasic seed production. references ali, s., khan, n., nouroz, f., erum, s., & nasim, w. 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(2015). efficient microtuber production of potato in modified nutrient spray bioreactor system. scientia horticulturae, 192, 369–374. https://doi.org/10.1016/j. scienta.2015.06.014 rokka, v.m., kämäräinen-karppinen, t., virtanen, e., & pirttilä, a.m. (2013). bioreactor technologies for mass propagation of potato: future prospects. in k.g. ramawat, & j.m. mérillon (eds.), bulbous plants: biotechnology. crc press. salem, j. & hassanein, a. (2017). in vitro propagation, microtuberization, and molecular characterization of three potato cultivars. biologia plantarum, 61(3), 427–437. stokstad, e. (2019). the new potato. science 363(6427), 574–577. tapia, m. de l., lorenzo, j. c., mosqueda, o. & escalona, m. (2017). obtención de microtubérculos y minitubérculos como semilla pre-básica en tres cultivares peruanos de papa. biotecnología vegetal, 17(3), 153-159 https://revista.ibp.co.cu/index.php/ bv/article/view/554/html tapia, m. de l., arbizu, c., beraún, f., lorenzo, j. & escalona, m. (2018). pre-basic seed potato (solanum tuberosum l.) production using temporary immersion bioreactors. peruvian journal of agronomy, 2, 9–14. yu, w., joyce, p., cameron, d. & mccown, b. (2000). sucrose utilization during potato microtuber growth in bioreactors. plant cell reports, 19, 407–413. https://doi.org/10.1007/s002990050748 ziv, m. (2005). simple bioreactors for mass propagation of plants. plant cell, tissue and organ culture, 81(3), 277–285. peruvian journal of agronomy 2 (1): 15 19 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i1.1129 © the authors. published by universidad nacional agraria la molina received for publication: 04 october 2017 accepted for publication: 30 march 2018 garden pea used as a temporary crop to establish grass and legumes pastures: effect on weed botanical composition la arveja como cultivo temporal para el establecimiento de pasturas de gramíneas y leguminosas: efecto en la composición botánica de las malezas rojas, j.*1, soplín, h.2, bojórquez, c1. and ordoñez, h.1 *corresponding autor: d.rojas.unmsm@gmail.com abstract this research was carried out at ivita’s research center of the universidad nacional mayor de san marcos in the department of junín, peru. the main objective was to determine the effect of garden pea plants (pisum sativum), seeded by broadcast to establish a temporary crop, on the abundance and botanical composition of weed species during the establishment of a pasture mixture of grasses and legumes in the mantaro valley. the following treatments were evaluated: t1, mixed pasture with manual weeding, without garden pea. t2, mixed pasture without weeding and garden pea. t3, mixed pasture with garden pea and without weeding. a completely randomized block design with three replications was used for statistical analysis of the experiment. all plots were broadcasted with, 10 kg ha-1 of lolium multiflorum lam. ‘tama’, 10 kg/ha of lolium boucheanum k. ‘belinda’, 5 kg/ha of medicago sativa l. ‘sw8210’ and 3 kg/ha of trifolium pratense l. ‘quiñequeli’. an additional 50 kg/ha-1 of pisum sativum l. ‘inia 103 remate’ was broadcasted first on treatment t3. weed botanical composition was evaluated on t2 and t3 treatments. the asteraceae and poaceae weed families recor ded greater number of species while the brassicaceae family recorded the greatest abundance, during pasture establisment in the mantaro valley. the most important species according to the importance value index (ivi) were: brassica rapa subsp. campestris, avena sativa and medicago polymorpha. key words: weeds, garden pea, pasture. resumen esta investigación se condujo en el centro de investigación del ivita mantaro, de la universidad nacional mayor de san marcos, en el departamento de junín, perú. el objetivo principal fue evaluar el efecto de la arveja (pisum sativum) sembrada al voleo como cultivo temporal, sobre la abundancia y composición botánica de las especies de malezas, durante el establecimiento de una pastura asociada (gramíneas con leguminosas), en el valle del mantaro. los tratamientos fueron: t1, establecimiento de la pastura con deshierbo manual, sin arveja; t2, establecimiento de la pastura sin deshierbo y sin arveja y t3, establecimiento de la pastura con arveja y sin deshierbo. los tratamientos se dispusieron en un diseño de bloques completos al azar, con tres repeticiones. en cada una de las parcelas se sembraron al voleo, la pastura (gramíneas y leguminosas), en cantidades de 10 kg/ha de lolium multiflorum lam. ‘tama’, 10 kg/ha de lolium x boucheanum k. ‘belinda’, 5 kg/ha de medicago sativa l. ‘sw8210’ y 3 kg/ha de trifolium pratense l. ‘quiñequeli’. en el tratamiento t3, previo a la siembra de la pastura, se sembró al voleo 50 kg/ha de pisum sativum l. ‘inia 103 remate’. se evaluó la composición botánica de las malezas en los tratamientos t2 y t3. las familias asteraceae y poaceae de malezas registraron el mayor número y variedad de especies, mientras que la familia brassicaceae registró la más alta abundancia, durante el establecimiento de la pastura en el valle del mantaro. las especies más importantes, según el índice de valor de importancia fueron: brassica rapa subsp. campestris, avena sativa y medicago polymorpha. palabras clave: malezas, arveja, pastura. 1universidad nacional mayor de san marcos, facultad de medicina veterinaria, ivita mantaro, unidad de investigación de pastos y forrajes. jauja, perú. 2universidad nacional agraria la molina, facultad de agronomía, departamento académico de fitotecnia, lima, perú. introduction in the mantaro valley, 60 kg/ha of seed is used for sowing garden pea, and its cultivation involves cultural practices (sowing, weeding, tillage, hilling, pest management and control, etc.). as early varieties of garden pea with erect growth and high demand as vegetables are available, some studies showed the possibility of planting them as a temporary crop during pasture establishment (ordóñez et al., 1999; ordóñez and bojórquez, 2001). the role of garden pea is not limited to protecting pasture from adverse climatic factors and providing dry matter in total forage production, but also to provide ecological and economic pea as a temporal crop during the establishment of a pasture of grassess and legumes – i. effect on weed botanical composition january april 2018 16 services with the sale of green pods. temporary crops have certain benefits such as: rapid growth and cover of the soil when forage grasses and legumes are in the early stages of development, weeds are supressed, soil erosion by water is reduced, fodder production is ensured and competition is minimized. forage crop (ordoñez and bojórquez, 2011). the establishment of pastures in the valley, takes an average of 120 days, during which a great diversity of weeds, emerge together with the components of the pastures. apparently, forage mixtures planted in the mantaro valley are characterized by a tolerance to competition produced by weeds and by having a subsequent recovery in vigour and dry matter production (ordóñez and bojórquez, 2011). in the central highlands there is no research on weed diversity nor on their effect on pasture establishment (rojas et al., 2011, 2012, 2013a, 2014a) garden pea and pastures are usually planted alone, and each has its own management technology. however, better soil utilization is garden pea sowing as a temporary crop (ordóñez et al., 1999; ordóñez and bojórquez, 2001), where light and soil moisture and nutrients are better utilized (rojas et al., 2010). on the other hand, it is possible that garden pea could counteract the negative effect of weeds on the establishment of mixed pasture. at the ivita mantaro research center, herbicides are not used in pasture establishment and it is preferred to let mixed pastures to establish by themselves for approximately 120 days (rojas et al., 2014a). pastures are usually seeded on land where potatoes have been harvested and annual broadleaf weeds appear (rojas et al., 2013b and 2013a). a viable alternative with probable biological and economic benefits could be the simultaneous sowing of mixed pastures with garden pea, as a temporary crop, where the garden pea would replace, at least in part the weeds, producing greater benefits. temporary crops must have rapid growth and cover the soil when forage legumes are still in the early stages of development. they also suppress weeds by competition, reduce water soil erosion, ensure forage production, while producing minimal competition to forage crops (ordoñez and bojórquez, 2011). therefore, the following research question was formulated: what is the effect of broadcasted garden pea plants, as a temporary crop, on weed diversity, during the establish ment of a mixed pasture (grasses with legumes) in the mantaro valley?, and as a null hypothesis: sowing pea as a temporary crop does not influence the diversity or botani cal composition of weeds during the establishment of a mixed pasture (grasses with legumes) in the mantaro va lley. the main objective of this study was to determine the effect of a garden pea crop broadcast sown for the establishment of a temporary crop, on the abundance and botanical composition of weed species during the establishment of a mixed pasture. materials and methods the study was carried out at the ivita mantaro research center of the universidad nacional mayor de san marcos, located in the mantaro valley, province of jauja, in the junín region, at 3320 masl, with an average annual precipitation of 750 mm, annual mean temperature 11 °c, on a sandy-clay loam soil poor in nitrogen, medium in phosphorus and high in potassium. the preparation of the soil was conventional, with two disc plow passes, two disk dredge passes, after which 3 m wide beds were constructed with a leveling blade. a mixture of four forage species was sowed, at the density recorded in table 1. the sowing consisted in passing a rigid tip harrow to remove the soil, then garden pea seed cv. ‘inia 103 remate’ was broadcasted at 50 kg/ha, followed by a disc harrow pass to cover the seed. the pasture mixture was seeded at a density of 28 kg/ha, with the grasses accounting for 71 percent of the mixture and the legumes 29 percent. treatments evaluated are shown in table 2. table 1. amount, species and cultivars present in the grasslegume mixed pasture sown in the mantaro valley, junín, 2012 common name scientific name cultivar kg ha-1 italian rye grass lolium multiflorum tama 10 hybrid rye grass lolium x boucheanum belinda 10 alfalfa medicago sativa sw8210 5 red clover trifolium pratense quiñequeli 3 table 2. treatments evaluated in this study treatments description t1 mixed pasture establishment with manual weeding without pea. t2 mixed pasture establishment without weeding nor pea t3 mixed pasture establishment, with pea, without weeding. a completely randomized block design with three repli cations was used. at 120 days after sowing (das) weed diversity was evaluated in treatments t2 and t3, in four quadrants of 0.5 m x 0.5 m (0.25 m2 in area), making one square meter per treatment. in such quadrants weeds were counted by species and number of individuals were registered. with this data the density and frequency were determined and, using their relative value, the importance value index (ivi) was obtained (matteucci and colman, 1982; mostacedo and fredericksen, 2000). the importance value index is calculated as the sum from (i) the relative frequency; (ii) the relative density; and (iii) the relative dominance. the frequency is calculated as the number of plots where a specie is observed divided by the total number of survey plots. relative frequency is calculated by dividing the frequency by the sum of the frequencies of all species, multiplied by 100 (to obtain a percentage). density is calculated as the total number of individuals of a species. relative density is calculated by dividing the density by the sum of the densities of all rojas, j., soplín, h., bojórquez, c. and ordoñez, h. peruvian journal of agronomy 2 (1): 15 19 (2018) 17 species, multiplied by 100. dominance is calculated as the total basal area of a species. relative dominance is calcula ted by dividing the dominance by the sum of the dominance of all species, multiplied by 100. the botanical composition of the mixed pasture was evaluated according to t’mannetje and haydock (1963) and haydock and shaw (1975), in four quadrants of 0.25 m2, making one square meter in each treatment. (ordoñez and bojórquez, 2011). for the taxonomic identifications of the weed diversity we used those determined by rojas et al. (2010). for the taxonomic location, the classification system apg ii and iii which is considered, the most modern system for the classification of angiosperms according to phylogenetic criteria (apg, 2009). results and discussion a total of 11 weed species belonging to seven families were recorded (table 3). the asteraceae and poaceae families accounted for 27.27 percent each of the total number of genera and species respectively (table 4). the most representative families in terms of number of species were: asteraceae and poaceae, which together registered 54.55 percent. of the remaining families, five were represented by a single species and constituted 45.45 percent (table 4). data in table 3 indicate that there was no greater effect on weed diversity due to garden pea sowing, since in t2 and t3 treatments, weed species were the same, with the exception of veronica persica and bromus catharticus that disappeared with the sowing of pea. however, data in table 5 shows that in treatment 3, sowing of garden pea caused a significant reduction in plant density per m2 of tagetes multiflora, fuertesimalva limensis, erodium cicutarium and to a lesser extent of medicago polymorpha, with no major effects on brassica rapa subsp. campestris or avena sativa. table 3. diversity of weeds recorded at 120 days after seeding of the mixed pasture sown in the mantaro valley, junín. february 2013. plant family plant species t1 * t2 t3 brassicaceae brassica rapa subsp. campestris x x malvaceae fuertesimalva limensis x x fabaceae medicago polymorpha x x geraniaceae erodium cicutarium x x plantaginaceae veronica persica x asteraceae galinsoga parviflora x x tagetes multiflora x x sonchus oleraceus x x poaceae avena sativa x x bromus catharticus x pennisetum clandestinum x x *t1 (mixed pasture establishment with manual weeding, without garden pea; t2 (mixed pasture establishment without weeding nor garden pea), and t3 (mixed pasture establishment with garden pea and without weeding. table 4. families with the highest number of genera and species recorded during the establishment of the mixed pasture. mantaro valley, junín. february 2013. plant family genus % plant species % asteraceae 3 27.27 3 27.27 poaceae 3 27.27 3 27.27 otras 5 45.45 5 45.45 total 11 100 11 100 in treatment 2, (table 5) the species presenting important value index (ivi) equal to or greater than 10 percent were five: brassica rapa subsp. campestris with 33.8 percent, fuertesimalva limensis, with 12.0 percent, avena sativa with 11.2 percent, medicago polymorpha with 10.9 percent, and erodium cicutarium with 10.0 percent. two species had higher density (>50 individuals/m): brassica rapa subsp. campestris and avena sativa with 209.3 and 40.7, respectively. in treatment 3, the species with hi gher ivi (>10%) were three: brassica rapa subsp. campestris with 40.0 percent, avena sativa with 14.1 percent and medicago polymorpha with 11.4 percent. a single species recorded higher density (>50 individuals m-2); this was brassica rapa subsp. campestris with 202.67 plants m-2 (table 5)). brassica rapa subsp. campestris is an important weed in the peruvian sierra, as it is found as an invasive species in many crops (flores and malpartida, 1987, villagomez 1988, monsalve and cano, 2005). tabla 5. density and importance value index of weed diversity during the establishment of the mixed pasture. mantaro valley, junín. february 2013. species t2 * t3 plant density plants m-2 % ivi plant density plants m-2 % ivi brassica rapa subsp. campestris 209.3 33.8 202.7 40.0 fuertesimalva limensis 37.3 12.0 19.3 9.6 medicago polymorpha 28.7 10.9 22.0 11.4 erodium cicutarium 21.3 10.0 12.0 9.8 veronica persica 2.7 1.6 0.0 0.0 galinsoga parviflora 3.3 2.9 2.7 3.1 tagetes multiflora 36.7 9.5 13.3 6.1 sonchus oleraceus 0.7 1.3 1.3 2.8 avena sativa 40.7 11.2 39.3 14.1 bromus catharticus 2.7 2.8 0.0 0.0 pennisetum clandestinum 12.0 4.0 3.3 3.2 total 395.3 100.0 316.0 100.0 ivi = ímportance value index t2 (mixed pasture establishment without weeding nor garden pea); t3 (mixed pasture establishment with garden pea and without weeding). pea as a temporal crop during the establishment of a pasture of grassess and legumes – i. effect on weed botanical composition january april 2018 18 brassica rapa subsp. campestris, fuertesimalva limensis and tagetes multiflora, are the most important ecolo gical weeds in the establishment of pastures grown in the mantaro valley (rojas et al., 2011, 2012, 2013b, 2014a, 2014b). the interaction “cultivated grass-weeds”, during the 120 days after sowing, does not seem to affect the popu lation and final production of the forage (ordóñez and bojórquez, 2011; rojas et al., 2014a, bojórquez et al., 2015). brassica rapa subsp. campestris recorded the highest importance value index values in treatments 2 and 3 with 33.8 and 40.0 percent respectively. brassica rapa subsp. campestris produces biocidal compounds such as glucosinolates, which by hydrolysis give rise to substances such as isothiocyanates. both products have been considered as products toxic to pest and diseases (brown and morra 1997, kirkegaard and sarwar, 1998). the amount of glucosinolates is greater during flowering and serve to keep weeds at bay. conclusions the asteraceae and poaceae families recorded more species, while the brassicacea family showed higher abundance during the mixed pasture establishment in the mantaro valley. the most important species according to the importance value index were: brassica rapa subsp. cam pestris, avena sativa and medicago polymorpha. it is hypotesized that brassica rapa could act as a weed control agent during mixed pasture establishment. references bojórquez, c., rojas, j. and ordóñez, h. (2015). pastos cultivados en el valle del mantaro. lima, perú: fondo editorial universidad nacional mayor de san marcos. cepredim-unmsm. brown, p. d. and morra, m. j. (1997). control of soilborne plant pests using glucosinlate containing plants. advances in agronomy, 61(c), 167-231. doi: 10.1016/ s0065-2113(08)60664-1 flores, a. and malpartida, e. (1987). manejo de praderas nativas y pasturas en la región altoandina del perú, volume ii. perú: banco agrario. haydock, k. and shaw, n. (1975). the comparative yield method for estimating dry matter yield of pasture. australian journal of experimental agriculture, 15(6), 663-670. doi: 10.1071/ea9750663 kirkegaard, j. a. and m. sarwar. (1998). biofumigation potential of brassicas: i. variation in glucosinolate profiles of diverse field-grown brassicas. plant and soil, 201(1), 71-89. mannetje, lt. and haydock, k. (1963). the dry – weight – rank method for the botanical analysis of pasture. grass and forage science, 18(4), 268-275. https://doi. org/10.1111/j.1365-2494.1963.tb00362.x matteucci, d. and colman, a. (1982). metodología para el estudio de la vegetación, issue 22 of serie de biología. washington, d. c.: secretaria general de la organización de los estados americanos (programa regional de desarrollo científico y tecnológico). monsalve, c., and cano, a. (2005). avances en el conocimiento de la diversidad de la familia brassicaceae en ancash, perú. revista peruana de biología 12(1): 107-124. doi: http://dx.doi.org/10.15381/rpb. v12i1.2365 mostacedo, b. and fredericksen, s. (2000). manual de métodos básicos de muestreo y análisis en ecología vegetal. bolivia: proyecto de manejo forestal sostenible (bolfor). ordóñez, h. and bojórquez, c. (2001). composición botánica y contribución biológica y económica de la siembra de pastura asociada con arveja en la sierra central. in: xxiv reunión científica anual del appa. lima, perú. ordóñez, h., bojórquez c. and pinillos, o. (1999). arveja como cultivo financiador en el establecimiento de pasturas asociadas en el valle del mantaro. in: xxii reunión científica anual del appa. huancavelica, perú. ordóñez, j. and bojórquez, c. (2011). manejo del establecimiento de pasturas para zonas alto andinas del perú. perú, huancayo: editorial concytec rojas, j., bojórquez, c. and ordóñez, h. (2011). evaluación de malezas en el establecimiento de pasturas cultivadas en la sierra central del perú. in: xxxiv reunión científica anual del appa. trujillo, perú. rojas, j., bojórquez, c., ordóñez, h. and rojas, e. (2012). diversidad de malezas en el establecimiento de pastos cultivados en el valle del mantaro, junín. in: xxxv reunión científica anual del appa. puno, perú. rojas, j., bojórquez, c., ordóñez, h., noli, c. and rojas, e. (2014a). malezas tóxicas para el ganado vacuno en el valle del mantaro, junín. in: xxxvii reunión científica anual del appa. abancay, perú. rojas, j., kroschel, j., cañedo, v. and zuñiga, d. (2010). malezas en dos zonas agroecológicas del cultivo de papa en la sierra central del perú. in: xiii congreso nacional de botánica. tingo maría, perú. rojas, j., ordóñez, h., bojórquez, c. and rojas, e. (2013a). invasión de kikuyu (pennisetum clandestinum) en camellones de melga de pastos cultivados en el valle del mantaro, junín. in: xxxvi reunión científica anual del appa. lima, perú. rojas, j., ordóñez, h., bojórquez, c. and rojas, e. (2014b). diversidad de malezas en el establecimiento de pastos cultivados en el valle del mantaro, junín. agro aportes. rev. fac. agron. uncp, 8, 17. rojas, j., ordóñez, h., bojórquez, c., rojas, e. and noli, c. (2013b). diversidad de malezas en el cultivo de avena forrajera (avena sativa), en la sierra central del perú. in: xxxvi reunión científica anual del appa. rojas, j., soplín, h., bojórquez, c. and ordoñez, h. peruvian journal of agronomy 2 (1): 15 19 (2018) 19 lima, perú. the angiosperm phylogeny group, apg. (2009). an update of the angiosperm phylogeny group classification for the orders and families of flowering plants: apg iii. botanical journal of the linnean society, 161(2), 105-121. https://doi.org/10.1111/ j.1095-8339.2009.00996.x villagomez, v. (1988). informe sobre los cultivos de papa. in: informe del taller de expertos en manejo mejorado de malezas en los países andinos. lima, perú: fao/rlac 16. programa de protección de cultivos. peruvian journal of agronomy 2 (2): 15 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i2.1133 © the authors. published by universidad nacional agraria la molina received for publication: 15 may 2018 accepted for publication: 07 june 2018 allometric models for non-destructive leaf area estimation in eugenia uniflora (l.) modelos alométricos para la estimación no destructiva del área foliar en eugenia uniflora (l.) introduction leaf area (la) is one of the six most important traits that drive plant form and function (díaz et al., 2016). this descriptor has been widely used to describe a range of variables including growth, productivity, photosynthetic efficiency, soil characteristics including salinity and acidity, transfer and exchange of heat, carbon, nutrients and water, which in turn affect plant yield (cristofori et al., 2007; pompelli et al., 2012). thus, a correct determination of the la becomes even more important in crop species, since the leaf is the organ of greater influence with the environment and it is through this that the agronomic studies are based on the important decision making. directly measure of la of individuals is both, laborious, expensive as well as a time consuming task and often constrained by logistical factors. leaf area is traditionally quantified by direct methods, which are destructively or obtained through high-cost equipment, such as am350 portable leaf area meter (adc bioscientific ltd., hoddesdon, uk). with the intensification of modeling techniques, numerous studies have proposed allometric models to predict the la of different species (blanco and folegatti, 2005; antunes et al., 2008; pompelli et al., 2012; keramatlou et al., 2015; liu et al., 2017). hence, pompelli, m. f.1*, figueirôa, j. m.2, lozano-isla, f1. * corresponding author. marcelo.pompelli@ufpe.br abstract we aimed to propose a reliable and accurate model using non-destructive measurements of leaf length (l) and/or width (w) for estimating leaf area (la) of surinam cherry (eugenia uniflora l.). for model construction, 560 leaves were randomly sampled from different levels of the tree canopies and encompassed the full spectrum of measurable leaf sizes. power models better fit e. uniflora leaf area than linear models; but, among of then, the best fit were made when product of the l and w (lw) were used. to validate these models, independent data set of 156 leaves were used. thus, we developed a single power model (yi = β0x β1) [la = 0.685 (lw)0.989; standard errors: β0 = 0.014, β1 = 0.005; r 2 a = 0.997] with high precision and accuracy, random dispersal pattern of residuals and unbiased. a simpler linear model [la = 0.094 + (lw * 0.655); standard errors: β0 = 0.025, β 1 = 0.001; r 2 a = 0.998] also described here to estimate leaf area of e. uniflora, which are as good as the first. the simplicity of the latter model may be relevant in field studies, as it does not demand high precision or expensive instruments. keywords: surinam cherry; estimate model; leaf length; leaf width resumen nuestro objetivo fue proponer un modelo confiable y preciso utilizando mediciones no destructivas de la longitud de la hoja (l) y / o el ancho (w) para estimar el área foliar (la) de la cereza de surinam (eugenia uniflora l.). para la construcción del modelo, se tomaron 560 hojas al azar de diferentes niveles de las copas de los árboles y abarcaron todo el espectro de tamaños de hojas medibles. los modelos exponenciales se ajustan mejor al área foliar de e. uniflora que los modelos lineales; el mejor ajuste se realizó cuando se usaron productos de l y w (lw). para validar estos modelos, se utilizaron conjuntos de datos independientes de 156 hojas. por lo tanto, desarrollamos un único modelo de potencia (yi = β0x β1) [la = 0.685 (lw)0.989; error estándar: β0 = 0.014, β1 = 0.005; r 2 a = 0.997] con alta precisión y exactitud, patrón de dispersión aleatorio de residuales e imparcial. un modelo lineal más simple [la = 0.094 + (lw * 0.655); error estándar: β0 = 0.025, β 1 = 0.001; r 2 a = 0.998] también se describe aquí para estimar el área foliar de e. uniflora, que es tan buena como la primera. la simplicidad de este último modelo puede ser relevante en los estudios de campo, ya que no exige instrumentos costosos o de alta precisión. palabras llave: cereza de surinam; modelo de estimación; longitud de la hoja; ancho de la hoja 1plant ecophysiology laboratory, federal university of pernambuco, department of botany, ccb, recife, pe, brazil, 50670901 2secretaria de meio ambiente do estado de pernambuco, recife, pernambuco, brazil allometric models for non-destructive leaf area estimation in eugenia uniflora (l.) mayaugust 2018 2 simple linear measurements like leaf length (l), leaf width (w) are used in allometric equations to predict the leaf area (peksen, 2007). given the fact that eugenia uniflora is a commercial crop species, non-destructive methods such as allometry are best suited for measure leaf area because preserve the leaf on the plant instead destructive methods (cristofori et al., 2007). this paper describes, for first time allometric equations to predict leaf area of eugenia uniflora checking the accuracy and perform an unbiased tool for use in land and agronomic studies. material and methods for model construction, 560 healthy leaves were collected at least 20 healthy plants naturally grown at caetés ecological station, paulista, pernambuco, brazil (7º55’28”s; 34º56’02”o; 88 m.a.s.l.) in april 2018 (end of the growing season). to validate the model, an independent data set of 156 leaves were sampled randomly from different levels of the tree canopy, removed from the branches and taken to the laboratory. the maximum leaf length (l) (from lamina tip to the point of the petiole intersection to the midrib) and leaf width (w) (the widest linear length perpendicular to the midrib) were measured to the nearest of 0.001 cm. the leaves were scanned using a scanner (epson 1200 x 1200 dpi) and images were analyzed using the image-pro® plus software (2001). the leaves encompassed the broadest range as possible. the minimum leaf area sampled was 0.17 cm2 and maximum was 72.40 cm2 (table 1). nine theoretical models (more widely used in the literature) were tested, based on different combinations between the components of la (dependent variable) and respective values of l and w (independent variables). the equations were deduced by the principle of parsimony (steel and penny, 2000), and thus from the “simplest”, or an “optimal” description of the data. all equations were adjusted following the linear simple, modified linear (from exclude β0) and power models (more information, see table 2). all parameters of each model were obtained using datafit version 8.0.32 (oakdale engineering, 2002). the statistical criteria used to select the models were based on (i) the analysis of variance (f test, p < 0.001), (ii) adjusted coefficient of determination (r²a), (iii) mean squared error (mse), (iv) student’s t-test (p < 0.001) for absolute mean of errors with confidence intervals (cumming et al., 2007), (v) dispersion pattern of residuals in percentage terms (%) and the best relationship (major r2a) between observed leaf area and estimated leaf area of the independent data set used to validating equations. the dispersion of the residues were observed in the total sample set, both in small leaves and in larger leaves. the hypothesis of normality of the errors were evaluated, so that heteroscedasticity was considered a reason for model disqualification. these procedures allowed us to assess the occurrence of bias and accuracy in all proposed models (walther and moore, 2005). results and discussion all nine developed equations (table 3) presented good predictors of the e. uniflora leaf area, since r2a was always higher than 0.8; coefficient higher than those proposed to some crop plants (cristofori et al., 2007; peksen, 2007; kumar, 2009; souza and amaral, 2015) and within the range of those proposed by others (blanco and folegatti, 2005; antunes et al., 2008; demirsoy, 2009; pompelli et al., 2012; shabani and sepaskhah, 2017). thus, at first glance, all proposed equations should be able to predict with accuracy the leaf area of e. uniflora. however, when we analyzed the deviation between estimated leaf area table 1. means ± standard deviations (sd), minimum (min), maximum (max) values for the leaf length (l), width (w), and leaf area (la) of the eugenia uniflora l. l (cm) w (cm) la (cm2) mean ± sd min max mean ± d min max mean ± sd min max 3.55 ± 2.32 0.72 14.31 1.97 ±1.42 0.27 7.62 5.83 ± 10.73 0.17 72.40 table 2. statistical models and equations to predict leaf area as a function of linear dimensions of leaves (model #1) linear yi = β0 + β1 * length + εi (model #2) linear yi = β0 + β1 * width + εi (model #3) linear yi = β0 + β1 * (length* width) + εi (model #4) linear without intercept yi = β1 * length + εi (model #5) linear without intercept yi = β1 * width + εi (model #6) linear without intercept yi = β1 *(length* width) + εi (model #7) power yi = β0 * length β1 + εi (model #8) power yi = β0 * width β1 + εi (model #9) power yi = β0 * (length*width) β1 + εi yi = leaf area, β0 and β1 = model coefficients and = random error and observed leaf area (fig. 1), we demonstrate that equations #4, #5, and #8 were biased, because a significant underestimation of la. in this case, the underestimation can lead for 53.1% of the estimated leaf area (using equation #8) to 93.2% of the estimated leaf area (using equation #4). in similar manner, the equation #6 can lead to overestimation leaf area in about 19.1% of the leaves. all these equations had an estimated significant difference from zero (biased) and were excluded pompelli, m. f., figueirôa, j. m., lozano-isla, f. peruvian journal of agronomy 2 (2): 1-5 (2018) 3 from the further analysis as recommended by antunes et al. (2008) for coffee trees, by pompelli et al. (2012) for purging-nut trees and by yadav et al. (2007); all agronomic species. with the disqualification of equations #4, #5, #6 and #8, five equations remained to be analyzed in more details. thus, a deep analysis of relationship between estimated leaf area and dispersal pattern of residuals, revealed that equations #1 lead to an overestimation of 34.5% and #2 to an underestimation of 36.2% of the leaves (when considering relative errors ≥ 40%). a possible source of error must be due to negative values of β0, that in these equations were bigger than -7.4. higher values of β0 were previously (blanco and folegatti, 2005; cristofori et al., 2007; antunes et al., 2008; pompelli et al., 2012; schmildt et al., 2015) used to disqualification the allometric equations, because such equations return negative values of la (i.e., invalid biological condition). the heteroscedasticity of residuals of model #7, was used to disqualify it, because this model lead to overestimation or underestimation on 21.2% of the leaves (when considering relative errors ≥ 20%). the biased pattern of residuals showed in equations #1, #2 and #7 are as large as the smaller sampled leaves. so, we can only recommended the use of these equations when a stratification of the leaf size classes is performed, simultaneously checking the dispersion pattern of the residues in all classes of leaves, as suggested by others (walther and moore, 2005; antunes et al., 2008; zuur et al., 2010; pompelli et al., 2012). however, this “solution”, sometimes becomes laborious and impractical, despite the ease of adjustment and operation of this type of model. from nine initial proposed models, only two models (#3 and #9) were entirety approved. as shows in fig. 2, the equations #3 lead to overestimation some leaf areas. therefore, this overestimation is lower than 4%, which cannot invalidate this equation. in this issue, we argue that the best equation is equation #9, made with power model. even if the model #7 has been previously disqualified, we can verify that it presents an excellent fit curve (r2a = 0.985) between linear dimensions of leaves and observed leaf area (fig. 3b). models using single leaf dimension power model incorporating l or w may be an interesting option because table 3. statistical models, regression coefficients (β0, β1), degrees of freedom of residuals (r-d.f.), mean squared error (mse), coefficients of determination adjusted for the degrees of freedom (r2adj) and p value as a function of linear dimensions of leaves. models coefficients r-d.f. mse r2adj p β0 β1 #1 -7.641 3.854 352 9.311 0.884 <0.001 #2 -7.418 6.834 352 10.207 0.873 <0.001 #3 0.094 0.655 352 0.141 0.998 <0.001 #4 2.353 353 23.516 0.819 <0.001 #5 4.221 353 23.770 0.818 <0.001 #6 0.658 353 0.146 0.996 <0.001 #7 0.407 1.944 352 1.166 0.985 <0.001 #8 2.343 1.591 352 6.198 0.923 <0.001 #9 0.685 0.989 352 0.136 0.997 <0.001 figure 1. statistical analysis of the deviation of the estimated area from the observed area for an individual leaf. leaf area for eugenia uniflora was estimated using several models in which β0 and β1 are coefficients. vertical bars denote means and spreads denote 99% confidence intervals of the difference. numbers below the graph denote model numbers (see further details in the table 1). asterisks in the bars denote a biased model. it requires measurement of only one leaf dimension, thus simplifying measurement procedures (blanco and folegatti, 2005; cristofori et al., 2007; antunes et al., 2008; pompelli et al., 2012). because of this, we prefer to keep this equation to next step of validation. when we pooled the relationship between observed leaf area and linear estimated leaf area of the independent sample leaves (fig. 3, right panel), we verified that equations #3, #7, and #9 returns good fit curves, showing the coefficient of determination above of 0.960. however, as suggested above, the equation #7 returns the lesser determination coefficient of all, besides being present the lesser p value (p = 0.186) than others (p ≥ 0.674). finally, we argue that from nine initial proposed models, only two models (model #3 and #9) can provide an unbiased estimation of leaf area using the linear dimensions of leaves. these models were approved in all statistical analysis and then are able to use without errors, both in field and in greenhouses evaluations. however, among then, the allometric models for non-destructive leaf area estimation in eugenia uniflora (l.) mayaugust 2018 4 equation #3 is simpler than equation #9, because it does not require more complex calculations. in the other hand, if the researcher has notion of the error merged in equation #7 and this may lead to an overestimation of approximately 21% of the estimated leaf area, this equation could also be an interesting option because it requires measurement of only one leaf dimension, simplifying measurement procedures (blanco and folegatti, 2005; pompelli et al., 2012), an important aspect specially in the field when a large number of leaves has to be monitored. conclusion in this study, we developed a reliable and accurate equation to estimate the leaf area or eugenia uniflora using nondestructive method. a power equation, type yi = β0x β1 [la = 0.685 (lw)0.989] was made. this equation may estimate figure 2. the relationship between estimated leaf area and dispersal pattern of residuals to each selected equations. red oval shape denote strong underestimated (a, b) or overestimated (c) leaf area. red arrows denote strong biased estimated leaf areas, mainly in the smaller leaves, while black arrows denote slightly skewed estimated leaf areas. see further details in the text. figure 3. the relationship between observed leaf area and linear dimensions of leaf (a, b and c) or between estimated leaf area (d, e and f) for model #3 (a and d), model #7 (b and e) and model #9 (c and f). l, leaf length; lw, product of leaf length and leaf width; ns, not significant; ra 2, coefficients of determination adjusted for the degrees of freedom. the leaf area with 99.7% of accuracy. the simplification of this equation could be done using a linear equation [la = 0.094 + (lw * 0.655) without loss of accuracy. this procedure should be less laborious because use a linear equation instead a power equation. this is the first study that describes with great accuracy an allometric equation to estimate the leaf area of eugenia uniflora, showing all common mistakes in allometric equations published until now. acknowledgements the authors thank to secretaria de meio ambiente do estado de pernambuco (grants 03/2013) and the pompelli, m. f., figueirôa, j. m., lozano-isla, f. peruvian journal of agronomy 2 (2): 1-5 (2018) 5 foundation for science and technology of pernambuco, facepe (grants apq-0239-2.03/15) for financially supporting this research. references antunes, w.c., pompelli, m.f., carretero, d.m. and damatta, f.m. (2008). allometric models for nondestructive leaf area estimation in coffee (coffea arabica and coffea canephora). annals of applied biology, 153(1), 33-40. https://doi.org/10.1111/j.17447348.2008.00235.x blanco, f.f. and folegatti, m.v. (2005). estimation of leaf area for greenhouse cucumber by linear measurements under salinity and grafting. scientia agricola, 62(4), 305-309. http://dx.doi.org/10.1590/s010390162005000400001 cristofori, v., rouphael, y., mendoza-de gyves, e. and bignami, c. (2007). a simple model for estimating leaf area of hazelnut from linear measurements. scientia horticulturae, 113(2), 221-225. https://doi. org/10.1016/j.scienta.2007.02.006 cumming, g., fidler, f. and vaux, d.l. (2007). error bars in experimental biology. journal of cell biology 177(1), 7-11. doi: 10.1083/jcb.200611141 datafit version 8.032. (2002). oakdale enginering, oakdale, ca, usa demirsoy, h. (2009). leaf area estimation in some species of fruit tree by using models as a non-destructive method. fruits, 64(1), 45-51. https://doi.org/10.1051/ fruits/2008049 díaz, s., kattge, j., cornelissen, j.h.c., wright, i.j., lavorel, s., dray, s., reu, b., kleyer, m., wirth, c., colin prentice, i., garnier, e., bönisch, g., westoby, m., poorter, h., reich, p.b., moles, a.t., dickie, j., gillison, a.n., zanne, a.e., chave, j., joseph wright, s., sheremet’ev, s.n., jactel, h., baraloto, c., cerabolini, b., pierce, s., shipley, b., kirkup, d., casanoves, f., joswig, j.s., günther, a., falczuk, v., rüger, n., mahecha, m.d. and gorné, l.d. (2016). the global spectrum of plant form and function. nature, 529, 167-171. https://doi.org/10.1038/nature16489 image pro plus version 4.5.029. (2001). media cybernetics inc. rockville, md, usa keramatlou, i., sharifani, m., sabouri, h., alizadeh, m. and kamkar, b. 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(2000). parsimony, likelihood, and the role of models in molecular phylogenetics. molecular biology and evolution, 17(6), 839-850. doi: 10.1093/oxfordjournals.molbev.a026364 walther, b.a. and moore, j.l. (2005). the concepts of bias, precision and accuracy, and their use in testing the performance of species richness estimators, with a literature review of estimator performance. ecography, 28(6), 815-829. https://doi.org/10.1111/j.2005.09067590.04112.x yadav, s.k., mishra, y.d. and singh, r.k. (2007). total leaf area estimation of flemingia semialata roxb. by linear regression. agricultural science digest 27(1), 44-46. zuur, a.f., ieno, e.n. and elphick, c.s. (2010). a protocol for data exploration to avoid common statistical problems. methods in ecology and evolution 1(1), 3-14. https://doi.org/10.1111/j.2041-210x.2009.00001.x peruvian journal of agronomy 2 (3): 54 59 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i1.1198 © the authors. published by universidad nacional agraria la molina received for publication: 04 april 2018 accepted for publication: 30 april 2018 falling of asparagus flowers (asparagus officinalis linneo.) for the handling of prodiplosis longifila gagné in the irrigation of the chavimochic project caída de flores del espárrago (asparagus officinalis linneo.) para el manejo de prodiplosis longifila gagné en la irrigación chavimochic prado, s.1*, castillo, j.2*, rodriguez, s.3. * corresponding author: joracava@lamolina.edu.pe abstract the asparagus, a dioecious plant, usually includes plants of both sexes, though also hermaphrodite ones. the male inflorescences are frequented by adult prodiplosis longifila, who lay such large amounts of eggs on them that its populations can reach millions of individuals over a lapse of 20 to 30 days, which is the plant regular flowering period. the field investigation took place in two locations: (i) the irrigation research unit of the universidad nacional agraria la molina (unalm) and (ii) properties of the agricultural society of virú (savsa). the number of fallen flowers and larvae per bud after applying multiple treatments on asparagus plots of the two locations was counted to determine the best concentration and application time of various fertilizers to induce the highest percentage of flower fall to manage p. longifila populations. in the unalm the fertilizer b dose 2 generated the highest percentage of fallen flowers, 82.1 % ± 14.85 %, and the fertilizer c dose 1 caused 74.94 % ± 13.78 %. in savsa the fertilizer b dose 3 made a percentage of fallen flowers of 50.6 % ± 21.56 % and fertilizer b dose 2, 49.85 % ± 16.28 %. the average number of p. longifila 2nd-stage larvae, evaluated on fifteen flowers and in three moments was 3.9 larvae. foliar fertilizers applications affected both male and female flowers almost equally in all treatments, percentage-wise. foliar fertilizers promote asparagus flowers falling, consequently contribute to a significative reduction of p. longifila populations, due to a larvae exposition to rapid desiccation under the sun. key words: management, prodiplosis, asparagus resumen el espárrago, planta dioica, generalmente comprende plantas para ambos sexos, aunque también plantas hermafroditas. las influorescencias masculinas son frecuentadas por adultos de prodiplosis longifila oviponiendo en las mismas, poblaciones que pueden alcanzar a millones de individuos en un lapso de tiempo de 20 a 30 días, período regular que dura la floración. la investigación se realizó en condiciones de campo en dos localidades, en la (i) unidad de investigación en riegos de la universidad nacional agraria la molina (unalm) y en el (ii) fundo sociedad agrícola virú (savsa) para determinar niveles y momentos de aplicación de diversos fertilizantes foliares para generar el mayor porcentaje de caída de flores para el manejo de poblaciones de p. longifila; se determinó el promedio de larvas por cada flor. en la unalm el fertilizante b dosis 2 generó el mayor porcentaje de caída de flores de 82.1% ± 14.85 y el fertilizante c dosis 1 generó un porcentaje de caída de flores de 74.94% ± 13.78 en savsa el fertilizante b dosis 3 generó un porcentaje de caída de flores de 50.6% ± 21.56 y el fertilizante b dosis 2 generó una caída de flores de 49.85 % ± 16.28. el promedio de larvas de ii estadio de p. longifila, evaluadas en quince flores y tres momentos fue de 3.9 larvas. tanto las flores masculinas y femeninas son afectadas por la aplicación de los fertilizantes foliares casi en el mismo porcentaje en todos los tratamientos. se concluye que la aplicación de fertilizantes foliares promueve la caída de flores del espárrago y con ello una significativa reducción de las poblaciones de p. longifila, debido a que las larvas quedan expuestas a la desecación o al sol y rápidamente muere. palabras claves: manejo, prodiplosis, espárrago 1thesis to opt for the degree of agricultural engineer of the faculty of agronomy. universidad nacional agraria la molina. 2professor of the entomology department of the faculty of agronomy. universidad nacional agraria la molina. 3professor of the plant breeding department of the faculty of agronomy. universidad nacional agraria la molina prado, s., castillo, j., rodriguez, s. peruvian journal of agronomy 2 (3): 54-59 (2018) 55 introduction prodiplosis longifila gagné is the most significant pest in asparagus cultivation (asparagus officinalis linneo), specifically in northern peru; the larvae of this dipteran are located in the tender sprouts and apexes of green shoots causing injuries and malformations, which can lead to the sprout death. (castillo, 2006; delgado de la flor et al., 1993). the asparagus is a dioecious plant, though there also exist hermaphrodite plants. the inflorescences are frequented by adult female p. longifila, which lay eggs on them, thus generating populations of adult insects that can reach millions of individuals in a time lapse of 20 to 30 days, flowering period of the plants. this pest population originated in flowering crops, can have three destinations: 1st, settle on the same field, attacking sprouts of the second sprouting; 2nd, invade neighboring fields in their first sprouting; and 3rd, attack fields during harvest, these attacks are so aggressive that they raise productions costs considerably. in all cases, there is a gradual carbohydrate reduction in the crown, only perceived over time. (castillo, 2006). velasco (2015) carried out a study of prodiplosis longifila populations on a molecular level based on altitude distribution, finding that there are not any groups attributable to high or low regions, there is no populational differentiation, populations can maintain a genetic flow despite environmental variation, this partly explains the wide range of geographic distribution. a way to handle populations of this insect is by inducing a premature falling of the asparagus flowers, avoiding the adult females laying eggs on them, and thus decreasing the larvae number that could develop on the inflorescences and consequently reduce the emerging adult number. the present investigation studied the flowers falling due to foliar fertilization with different macro and microelements. foliar fertilization based on the absorption of nutrients through the foliar tissue is more effective when the solution stays more time in the form of a thin layer on the foliar surface, however, if salt accumulates on the foliar surface without being absorbed, could cause burning and wrinkling. consequently, foliar spraying should be applied in doses from 2 to 5 percent. (mengel & kirkby, 2000). the investigation establishes several objectives: to determine the effects of foliar fertilizers on the falling of asparagus flowers to handle p. longifila; to find which fertilizer produces the highest percentage of fall of flowers, and to establish the average number of larvae per flower. materials and methods for the present investigation, we used commercial products, as shown in table 1 with their respective concentrations. the field work was carried in two localities: lima and la libertad. in lima, it took place in the irrigation research unit of the universidad nacional agraria la molina (unalm) (november and december) and in la libertad, in the district of virú in properties of the agricultural society of virú (savsa) (april and may). in both cases, we applied one-way tests with factorial arrangements, where the factors were the levels of fertilization and the moments of application. la molina, lima-perú. (unalm). we used the asparagus variety atlas, in its ninth campaign, with thirteen treatments; each treatment had three plots, and the experimental unit was an asparagus plant, with twelve repetitions. the total area of the experiment was 485.44 m2. for each treatment we made, three applications of fertilizers in the same doses, with the following phenologies: 30% of phylloclade opening (30th of november), 20% of flowering (7th of december) and 50% of flowering (15th of december). before applying the fertilizers, we chose the sample plants per each plot, and marked their 10th branch, starting to count from the first inferior branch of the plant. we used four fertilizer products in three different doses, as shown in table 2, with their respective concentrations in table 3. the fertilizers were applied with the help of a backpack sprayer. table 1. fertilizers used and their components in percentage. fertilizers concentration of elements (%) mo mn mg fe cu co zn b s n a 3 4 3 3 0.65 b 0.05 3 2 4 0.5 0.005 4 2.8 c 46 d 0.1 9 traces 7.2 0.3 0.6 0.4 e 0.3 3.5 7.5 0.28 0.7 0.65 table 2. fertilizers and their respective doses in the unalm – la molina. lima-perú, november -december. fertilizers doses (kg/cil or lt/cil*) d1** d2 d3 a 1 0.5 1.5 b 1 0.5 1.5 c 2 4 6 d 0.25 0.35 0.5 control 0 0 0 * cylinder of 200 lt ** doses falling of asparagus flowers (asparagus officinalis linneo.) for the handling of prodiplosis longifila gagné in the irrigation of the chavimochic project september december 2018 56 table 3. fertilizers used with their respective levels in savsa. virú, la libertad perú, may june. fertilizers doses (kg/cil or lt/cil*) d1** d2 d3 a 0.4 0.5 0.6 b 0.5 0.6 0.7 c 2 2.5 3 d 0.5 0.6 0.7 e 0.5 0.6 0.7 control 0 0 0 * cylinder of 200 lt ** doses virú, la libertadperú. savsa. for savsa we used the asparagus variety uc-157 f1 in its ninth campaign, with sixteen treatments. each treatment had two different plots, and the experimental unit was an asparagus plant with ten repetitions. the total area of the experiment was 5832 m2. for each treatment, we made four applications in the same doses to the following phenologies: 50% of ramification (30th of april), 20% of phylloclade opening (05th of may), 20% of flowering (9th of may) and 30% of flowering (15th of may). before the application, we chose the plants per each plot and marked the 12th branch, starting to count from the first inferior branch of the plant. we applied the fertilizers by addition of surfactants and adherents to the solution, to favor the use of the foliar fertilizer (leece, 1976). this way, the amount of falling flowers is increased. we used five different products in three separate doses along watered controls as shown in table 3. to determine the percentage of fallen flowers per plant, we compared the number of flower buds and flowers of the first and last evaluations; we also counted the number of larvae present on the flower. one hundred flowers were evaluated at three times. from 10 rows, one meter/row and 10 male flowers/meter were evaluated and collected in the field. only the second larval stage was considered since the first stage is difficult to see and susceptible to dry, the third stage falls off the flower to the ground. results experimental work in unalm significative statistical differences of the percentage of fallen flowers were observed for the parameters levels of fertilization and application moments of foliar fertilizers (p < 0.001); but not in their interaction. the treatments which caused the highest falling of flowers were treatment b dose 2 with 82.11 % + 14.85 % and c with 79.94 % + 13.78 % of fallen flowers; being both treatments statistically equal. (p < 0 .05). (figure 1). experimental work in savsa statistical differences were observed concerning the percentage of fallen flowers for the factors levels of fertilization and moments of application and with their interaction (p < 0.001). the treatments which caused the highest falling of flowers were b dose 3 (50.6 % ± 21.56 %), b dose 2 (49.85 % ± 16.28 %), e dose 1 (48.60 % ± 20.98 %), e dose d3 (47.81 % ± 1.72 %) and e dose 2 (45.09 % ± 23.42 %). all these treatments were statistically equal (p < 0.05). all the treatments had numerically more falling flowers than those of the control (figure 2). falling of flowers in female and male plants after comparing the average percentage of fallen flowers for male plants, all treatments produced more falling of flowers than the absolute control, with treatments b and e being the ones to stand out with 48.60 % ± 7.97 % and 46.56 % ± 8.63 % respectively, and treatments b, e, c, d, and a being statistically equal. (p < .05). (figure 3). after comparing the average percentage of fallen flowers for female plants, all treatments produced more falling flowers than the absolute control, with treatments b and e being the ones to stand out with 54.03 % ± 5.97 % and 48.1 % ± 6.1 %, and treatments b, e, c being statistically equal. (p < 0.05) (figure 4). the average number of p. longifila larvae per affected flower was 3.9 ± 1.68, with infestations from 1 to 7 larvae per flower. figure 5. discussion from the experiment that took place in la molina, treatments b dose 2 and treatment c dose 1 were the ones that had the highest percentages of fallen flowers with 82.11 % + 14.85 % and 79.94 % + 13.78 %, respectively; unlike in virú, the most top records treatments were b dose 3 and b dose 2 with 50.6 % ± 21.56 % and 49.85 % ± 16.28 %, respectively. (figures 1 and 2). these differences could be explained by various factors like the varietal and locality aspect, opportunity of application and others, since in la molina the atlas variety was used and in savsa the uc-157 f1 variety; the localities are 500 km apart and each one of them has unique climatic variables. additionally, the fertilizers were applied differently. all these observed differences could interfere with the results. as fuentes (1999), and trinidad & aguilar (2000) explain, the factors which influence foliar fertilization are the plant (species, stage of cultivation, state of nutrition, prado, s., castillo, j., rodriguez, s. peruvian journal of agronomy 2 (3): 54-59 (2018) 57 figure 4. the average percentage of fallen flowers in female plants, standard error and tukey test for the different treatments. virú. la libertad. perú, may-june. 2008. figure 1. average percentage of fallen flowers, standard error and tukey test for the different treatments. la molina, lima-perú. 2008. figure 2. average percentage of fallen flowers, standard error and tukey test for the different treatments. virú. la libertad. perú, mayjune. 2008. figure 3. average percentage of fallen flowers in male plants, standard error and tukey test for the different treatments. virú. la libertad. perú, may-june. 2008. etc.), the solution used (concentration, ph, synergistic or antagonistic, hygroscopicity, dose, etc.) and environmental conditions (temperature, light, humidity, luminosity, etc.). observations of claudinei de almeida et al. (2000) reinforce this assessment, in a study of the foliar application of urea on bean crops, where variations depended on the time of the day the application took place. the foliar applications of fertilizers were able to produce the falling of flowers in varying degrees, depending on their concentrations. approximately, about the 50 % of flowers fell in both types of flowers, without differences between them. all treatments caused premature flowers falling, higher than the control. (figures 3 and 4). flowers falling is caused by dehydration due to the concentration of soluble salts on its external surface, which leads to the decompensation of nutrient concentration with osmotic effects (marschner, 1997). application of foliar nutrients can produce levels of salt on leaves higher than those found in the soil; which is why biggest concern of foliar spraying is the leaves sensitivity to high concentrations of salt. the salts act in a corrosive manner on a foliar level, since they provoke the extraction of water (alcanter & trejotrellez, 2007). in the same way mengel & kirkby (2000) state that on warm days when evaporation is high, the water from a foliar pulverization can evaporate rapidly with the salts staying on the foliar surface without being absorbed, which can lead to burning and wrinkling of the leaves. the insect could infest some male flowers. in the present experiment, p. longifila adults plagued 35% of the total male flowers. likewise, in the field flowers infested with p. longifila 2nd-stage larvae, stayed closed, with a falling of asparagus flowers (asparagus officinalis linneo.) for the handling of prodiplosis longifila gagné in the irrigation of the chavimochic project september december 2018 58 red-violet coloration, generating a humid microclimate that facilitated the survival of the larvae. the larvae scraped internal surfaces while feeding, damages similar to those described by rodríguez (1992) of p. longifila larvae in tomato crops. once the larva hatches from the egg, needs space for developing, and the male plants equipped only with stamens have enough space to accommodate them. in the case of female flowers is reduced their available area when is fertilized the ovary and fruit starts developing, and the corolla opens, showing conditions not ideal for the larval development. when is exposed the larva and the nutrients to the sun both present desiccation and the larva dries and dies quickly, as described by castillo (2006) in a study on p. longifila damages in asparagus. during asparagus flowering, the p. longifila adults lay eggs on the flowers. when the percentage of flowering decreases, the emergence of adults starts in high numbers (garcía, 2006). according to the average number of larvae per flower (3.9) and percentages of male inflorescences infested (32.2% and 37.8%, respectively) has been calculated that adult populations can reach over 70 million of individuals per hectare during a flowering period over a period of 20 to 25 days. (castillo, 2006). the effect of foliar fertilizers application produces the falling of flowers of asparagus plants and, at the same time, a significative reduction of the insect population, since the larvae do not complete their biological cycle. the early fall of flowers in the furrows, where there is no shadow, dries the flower; consequently, the larvae die, or it is unable to leave the bud and find shelter under the plant to complete the larval stage. cuya (2011) concludes that within the components of integrated management of p. longifila, cultural control is essential as well as handling of irrigation and fertilization, but to the soil and not directly onto the foliage. the treatments applied during this experiment were made to cause an early fall of the inflorescent since they are the favorite places of p. longifila to lay their eggs and where their larvae can complete a full cycle and generate new populations that infest other fields. conclusions under the conditions the experiment was conducted, can be concluded that: the foliar fertilizer application on asparagus plants produced a higher fall of flowers than in controls. the different responses in the two localities can be due to various causes, environmental, moments and number of applications or composition of the foliar fertilizers. in the unalm (la molina, lima-perú), the fertilizer that generated the highest number of fallen flowers in the atlas variety, was the fertilizer b, with a dose of 0.5 kg/200 liters of water, accumulating 1.5 kg on the three applications and reaching an average percentage of fallen flowers of 82.1 %. in savsa (virú, la libertad-perú), the fertilizer that generated the highest number of falling flowers in the uc-157 f1 variety was the fertilizer b, with a dose of 0.6 kg/200 liters of water (d3), accumulating 2.4kg on 4 opportunities and reaching an average percentage of fallen flowers of 50.6 %. the percentage of infested male flowers by prodiplosis longifila was 35 %. the average number of 2nd-stage larvae of prodiplosis longifila evaluated on affected flowers was 3.9. by generating a premature falling of the male flowers, the larvae did not reach adulthood, and thus the number of individuals in the prodiplosis longifila populations was reduced. references alcanter, g. & trejo -trellez, l. (2007). nutrición de cultivos. mundi prensa. méxico. almeida, c. de, carvalho, m., arf, o., sá, m. de, & buzetti, s. (2000). uréia em cobertura e via foliar em feijoeiro. scientia agricola, 57(2), 293-298. https:// dx.doi.org/10.1590/s0103-90162000000200016 castillo, j. (2006). prodiplosis longifila gagné en la irrigación chavimochic, la libertad. trujillo, perú. arenagro. cultivando el desierto, revista institucional de la asociación de agricultores agroexportadores propietarios de terrenos de chavimochic, aptch, 2(2), 11–19. cuya, c. (2011) prodiplosis longifila gagné (diptera: figure 5. the average number of larvae of prodiplosis longifila (in 2nd-stage) evaluated in male asparagus flowers. virú, la libertad perú, may-june. 2008. prado, s., castillo, j., rodriguez, s. peruvian journal of agronomy 2 (3): 54-59 (2018) 59 cecidomyiidae) en el cultivo de espárrago en la empresa savsa. monographic work to opt for the degree of ingeniero agrónomo. universidad nacional agraria la molina. delgado de la flor, f., montauban, r. & hurtado, f. (1993). cultivo del espárrago. proyecto tta. lima, perú. 122 p. fuentes, j. (1999). el suelo y los fertilizantes. mundi prensa. españa. garcía, m. (2006). manejo de poblaciones de prodiplosis longifila. la libertad. trujillo, perú. arenagro. cultivando el desierto, revista institucional de la asociación de agricultores a g ro e x p o r t a d o re s propietarios de terrenos de chavimochic aptch, 2(2), 24–26. leece, d. (1976). composition and ultrastructure of leaf cuticles from fruit trees, relative to differential foliar absorption. plant physiology, 3, 833-847. marschner, h. (1997). mineral nutrition of higher plants. germany. institute of plant nutrition university of hohenheim. mengel, k. & kirkby, e. (2000). principios de nutrición vegetal. switzerland. international potash institute. rodríguez, s. (1992). estudio biológico y morfotaxonómico de prodiplosis longifila gagné en tomate (lycopersicon sculentum l.). thesis. universidad nacional pedro ruiz gallo. lambayeque, perú. trinidad, a. & aguilar, d. (2000). fertilización foliar, un respaldo importante en el rendimiento de los cultivos. instituto de recursos naturales (irenat) méxico. velasco, s. (2015). análisis de poblaciones de prodiplosis longifila (diptera: cecidomyiidae) separadas altitudinalmente, mediante secuencias de adn mitocondrial. thesis to opt for the degree of doctor. universidad del valle. colombia. peruvian journal of agronomy 3(2): 68-73 (2019) issn: 2616-4477 (versión electrónica) http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 05 july 2019 accepted for publication: 30 july 2019 effects of foliar extracts of guaba (inga edulis) and cadaghi (corymbia torrelliana) on coffee (coffea arabica ‘caturra roja’) under nursery conditions in chanchamayo, peru efecto de extractos foliares de inga edulis y corymbia torreliana sobre café (coffea arabica ‘caturra roja’) en vivero (chanchamayo, perú) ojeda, w.1; borjas, r.1; alvarado, l.1; castro-cepero, v. 1; julca-otiniano, a.1* *corresponding author: ajo@lamolina.edu.pe abstract the objective of this study was to determine the allelopathic effects of leaf extracts of the forest species guaba (inga edulis) and cadaghi (corymbia torrelliana) on coffee (coffea arabica ‘caturra roja’) plants grown under nursery conditions in chanchamayo, peru. different doses of fresh leaf extracts were applied to coffee plants using a completely randomized design, with four treatments for each forest species (t1 = 0‰, t2 = 10‰, t3 = 20‰, and t4 = 30‰) and 12 replicate plants per treatment. it was found that none of the foliar extracts had an allelopathic effect on coffee plants in terms of height, stem diameter, number of leaves and dry weight; however fresh weight was significantly altered following “cadaghi” treatment. key words: allelopathy, forest species, coffee, plant extracts. resumen este trabajo se realizó con el objetivo de determinar el efecto alelopático en vivero de extractos foliares de guaba (inga edulis) y cadagui (corymbia torreliana) sobre café (coffea arabica “caturra roja”) en chanchamayo (perú). se emplearon diferentes dosis de extractos de hojas frescas, bajo un diseño completamente al azar con 4 tratamientos (t1=0, t2=10, t3=20 y t4=30%0) y 12 repeticiones (1 planta=1 repetición). se encontró que ninguno de los extractos foliares tuvo un efecto alelopático en las plantas de café en términos de altura, diámetro del tallo, número de hojas y peso seco, sin embargo para la variable peso fresco se encontró diferencias significativas en el tratamiento de “cadaghi”. palabras clave: alelopatía, especies forestales, café, extractos vegetales. 1 grupo de investigación agricultura y desarrollo sustentable en el trópico peruano. facultad de agronomía. departamento de fitotecnia. universidad nacional agraria. la molina. lima. perú. introduction a well-designed agroforestry system can be both ecologically attractive and economically viable for farmers. however, the presence of trees can also have associated issues, such as competition for nutrients, light and water, increased evapotranspiration, and, in some cases, an increase in work (benzing, 2001). trees that are grown in association with coffee (coffea spp.) plantations provide shade and reduce environmental stress for the crop but also modify the growing environment via their roots, branches, and leaves, which can have negative effects, such as the accumulation of toxic substances in the soil (allelopathy). several studies have investigated the allelopathic potential of various plant species, such as forest trees (ballester, arias, cobián, lópez & vieitez, 1992; da silva, bomfim, almeida, & borges, 2012; sousa de oliveira et al., 2005), fruit trees (inoue et al., 2010), shrubs (márquez & monteiro, 2001), forage (souza, rodrigues & rodrigues, 1997), weeds (goncalves, tonet & stofell, 2015; rawat, narwal, kadiyan, maikhuri, & negi, 2012; souza et al., 2011) and even cultivated plants such as rice (oryza spp.) (amb & ahluwalia, 2016). in peru, coffee plantations occupy more than 420,000 ha, and nearly all of this cultivated land is in agroforestry systems or under shade trees. according to julca et al. (2010), more than 48% of plantations use only trees in the genus inga, 26% use inga spp. mixed with other plant species, such as banana (musa spp.), and the remaining 26% use a mixture of different forest species as shade, such as cedar (cedrus spp.), mahogany (swietenia spp.), and eucalypts (eucalypteae). peruvian coffee farmers usually select the tree species they use based on the likely economic benefit they will obtain doi: http://dx.doi.org/10.21704/pja.v3i2.1316 ojeda, w.; borjas, r.; alvarado, l.; castro-cepero, v.; julca-otiniano, a. peruvian journal of agronomy 3(2): 68-73 (2019) 69 (wood, firewood, and/or fruit) and their availability in the production area. however, they do not have an adequate knowledge of the effects of the various tree species on the crop. for example, the allelopathic effect of some forest species on coffee has not been documented, despite the suspicion in recent years that this phenomenon is occurring in some coffee-producing areas of peru, such as tingo maría and chanchamayo, and is a reason for concern among coffee farmers – and this concern is even greater if one considers that the trees are used as permanent shade and the coffee plantations remain in the field for a long time. therefore, the aim of this research was to investigate the allelopathic effects of the foliar extracts of guaba (inga edulis) and cadaghi (corymbia torrelliana) on the coffee (coffea arabica) cultivar ‘caturra roja’ under nursery conditions in chanchamayo, peru. materials and methods the trials were carried out in annex 14 of the san ramón district, chanchamayo province, department of junín, which is 850 meters above sea level. plant propagation. germination. coffee (c. arabica ‘caturra roja’) seeds were disinfected with the fungicide homai wp (2 g/100 seeds) and then broadcast outdoors at a density of 1 kg/ m2 in a germination bed that was 2 m long, 1 m wide, and approximately 25 cm high and contained washed, fine river sand as a substrate. after sowing, the seeds were covered with a thin layer of sand and the entire bed was covered with a jute blanket (which was later removed when the seedlings started to emerge). a 1.5-m-high shed was installed over the bed, which was constructed from wooden poles and had a raschel mesh roof. the seedlings remained in the bed until they reached the “butterfly state,” at which time they were transplanted into bags. seedling growth. seedlings were grown in black polyethylene bags with a capacity of 1 kg containing farm soil that had been sterilized with hot water (80 ºc for 1 h) to avoid the presence of phytopathogens. a hole was made in the center of each bag and one seedling was carefully placed in this, ensuring that the root was straight. the bags were then placed on a metal mesh table (3 m × 1 m) inside a 2.5-m-high shed built from wooden posts and with a raschel mesh roof that was covered with transparent plastic to maintain 40% shade and prevent the entry of rain. irrigation. a watering can was used for irrigation, and an attempt was made to always maintain the substrate at field capacity. due to the climatic conditions during the development of the trial, irrigation was carried out every other day throughout the experimental period. pest and disease control. the coffee leaf miner leucoptera coffeella and the pathogenic fungus cercospora coffeicola were not controlled during the trial to avoid any interference with the results of the study. weed control. manual weed control was performed continuously throughout the trial to avoid any issues with weed invasion. preparation of plant extracts. extracts were obtained from the fresh leaves of two forest species that are used as shade in coffee plantations by farmers in different coffeegrowing regions in peru (table 1). table 1. forest species used in the trials to obtain the plant extracts, leaves were taken from the middle third of the crown of each tree species and processed following the methodology of cazón, de viana, and gianello (2000). briefly, the leaf material (50 g) was air dried at room temperature and ground, following which 400 ml of distilled water was added and the mixture was left for 12 h. then, 100 ml of distilled water and 250 ml of methyl alcohol (sufficient volumes to reach a final ratio of 1:5:10 of plant tissue:methanol:water) were added and left to macerate at room temperature for 48 h. the resulting hydroalcoholic solution was filtered and concentrated in a rotary vacuum evaporator to reduce the initial volume to approximately 250 ml, from which aliquots were taken for use in the assays. this procedure was carried out in the q6 laboratory of the department of chemistry of the faculty of sciences of the national agrarian university – la molina (unalm), peru. the extracts were added to the irrigation water (250 ml/bag) and applied to the coffee plants at a range of concentrations at 30 and 60 days after transplant (dat). experimental design. the study involved two trials (one trial per forest species), each of which had a completely randomized design with four treatments and 12 replicates per treatment, where each replicate corresponded to a single plant/bag. the details of the treatments are presented in table 2. duncan test (α=0.05) was used to analyze the data. table 2. dosages used to determine the effect of each foliar extract on coffea arabica ‘caturra roja’. scientific name family common name code inga edulis fabaceae guaba, pacae evie corymbia torrelliana myrtaceae cadaghi evet treatment plant extract concentration amount (ml) t1 control 0.0 t2 10‰ 2.5 t3 20‰ 5.0 t4 30‰ 7.5 effects of foliar extracts of guaba (inga edulis) and cadaghi (corymbia torrelliana) on coffee (coffea arabica ‘caturra roja’) under nursery conditions in chanchamayo, peru may august 2019 70 plant measurements. the following measurements were made on each coffee plant: plant height (cm) from the base of the plant to the apex of the terminal bud at 0, 30, 60, 90, and 120 dat. stem diameter (mm) 2 cm from the base of the plant at 0, 30, 60, 90, and 120 dat. number of real and fully developed leaves at 0, 30, 60, 90, and 120 dat. fresh weight (g) of the total plant (aerial part and roots) at the end of the trial. dry weight (g) of the total plant at the end of the trial, which was determined by kiln drying the materials at 60 °c for 72 h. results plant height. there were no significant differences (duncan test, α=0.05) in the heights of the coffee plants between the treatments in which the foliar extracts were applied and the control treatment for either of the forest species evaluated. in the guaba trial, the greatest height was reached with the control treatment (t1; 16.38 cm), followed by t3, t4, and t2. in the cadaghi trial, the greatest height was reached with the control treatment and t3 (17.21 cm), followed by t2 and t4. thus, none of the treatments had a negative effect on the longitudinal growth of the plants, with the height increasing over time in all cases (figure 1). stem diameter. there were no significant differences (duncan test, α=0.05) in the stem diameters of the coffee plants between the treatments in which foliar extracts were applied and the control for either of the forest species evaluated. in the guaba trial, the largest diameter corresponded to the control, followed by t2, t4 and t3, whereas in the cadaghi trial, the largest value corresponded to t2, followed by t1 = t4 and t3. thus, none of the treatments had a negative effect on the diametral growth of the plants, with the diameter increasing with time in all cases (figure 1). number of leaves. there were no significant differences (duncan test, α=0.05) in the numbers of leaves on the coffee plants between the treatments in which the foliar extracts were applied and the control treatment for either of the forest species evaluated. in the guaba trial, the highest number of leaves corresponded to t2, followed by t3, t1, and t4, whereas in the cadaghi trial, the highest value corresponded to t2, followed by t1, t3, and t4. thus, none of the treatments had a negative effect on the rate of leaf production, with the number of leaves increasing over time in all cases (figure 2). figure 1. heights (cm) and diameters (mm) of coffee (coffea arabica ‘caturra roja’) plants treated with foliar extracts of guaba (inga edulis) and cadaghi (corymbia torrelliana) under nursery conditions in chanchamayo, peru. dat, days after transplant. see table 2 for a description of the treatments.9 ojeda, w.; borjas, r.; alvarado, l.; castro-cepero, v.; julca-otiniano, a. peruvian journal of agronomy 3(2): 68-73 (2019) 71 fresh and dry weights. there was a significant difference (duncan test, α = 0.05) in the fresh weights of coffee plants between treatments in the cadaghi test, with the control plants having a significantly greater weight than those in the t2, t3, and t4 treatment groups. however, there were no significant differences between the treatments in terms of dry and fresh weight (figure 2). discussion cadaghi is recommended for use in agroforestry systems with a wide range of crops (grisi, anair souto, passos da silva, venturim, & nogueira, 2011), while guaba is the preferred shade species in coffee plantations (villagaray & bautista, 2011). however, there have been no previous studies on the allelopathic effects of either of these forest species on cultivated plants. in the present study, the leaf extracts from these species had no significant allelopathic effects on coffee plants under nursery conditions, which contrasts with the findings of several other studies on closely related forest species. for example, benzing (2001) reported that eucalyptus (eucalyptus spp.) have negative allelopathic effects on many other plant species via their roots and leaves. however, it has also been shown that the allelopathic potential of eucalyptus can be more pronounced in areas with low and erratic rainfall due to a lack of dilution of the phytotoxic substances excreted by these trees, with e. camaldulensis appearing to have a particularly high allelopathic potential, resulting in it not being recommended for use in mixed plantations under low and/or erratic rainfall conditions (may & ash, 1990; lisanework &michelsen, 1993; as cited by ceccon & martínez-ramos, 1999). in terms of other forest species, singh et al. (1998), as cited by benzing (2001) found a very clear negative relationship between the distance from poplar (populus deltoides) trees, the phenol content in the soil, and wheat (triticum aestivum) yield. in addition, the aqueous leaf litter extract of alnus nepalensis produces allelopathic effects on the germination percentage of species such as wheat, millet (eleusine coracana), mustard (brassica nigra), and pea (pisum sativum) (uniyal & chhetri, 2010, as cited by varela, 2017) these effects on both the germination and root growth of pea were confirmed, as well as on the root growth of rice (oryza sativa) and beans (phaseolus vulgaris) (kumar et al. , 2006, as cited by varela, 2017). furthermore, lines and fournier (1979) concluded that both aqueous extracts and essential oils of the vegetative and reproductive parts of cypress (cupressus lusitanica) have an allelopathic effect on the germination of the herbaceous species lepidium costaricensis, bidens pilosa, and rumex crispus under laboratory conditions. souto, gonzales and reigosa (1993) examined how the phytotoxicity produced by the aerial parts of four forest corymbia torreliana corymbia torreliana inga edulis inga edulis figure 2. numbers of leaves and fresh and dry weights (g) of coffee (coffea arabica ‘caturra roja’) plants treated with foliar extracts of guaba (inga edulis) and cadaghi (corymbia torrelliana) under nursery conditions in chanchamayo, peru. dat, days after transplant; fw, fresh weight; dw, dry weight. see table 2 for a description of the treatments. effects of foliar extracts of guaba (inga edulis) and cadaghi (corymbia torrelliana) on coffee (coffea arabica ‘caturra roja’) under nursery conditions in chanchamayo, peru may august 2019 72 species, eucalyptus (eucalyptus globulus), acacia (acacia melanoxylon), oak (quercus robus), and pine (pinus radiata), changed during the decomposition process in four different soils by measuring the effects of soil extracts on the germination and growth of lettuce (lactuca sativa) seeds under laboratory conditions. their results indicated that allelopathic effects may occur during the decomposition of residues from the four forest species, with eucalyptus and acacia having the largest effects and oak and pine having more moderate effects. pérez et al. (2011) investigated the allelopathic potential of the leaves of three exotic tree species (alianthus altisima, robinia pseudoacacia, and ulmus punila) and one autochthonous species (populus alba) in henares, spain on the germination of four native herbaceous understory species (chenopodium album, dactylis glomerata, brachypodium phoenicoides, and brachypodium silvaticum) under laboratory conditions, and reported that leaf litter extracts from all of the tree species tended to reduce the speed and, to a lesser extent, the germination rate of the four herbaceous species, with no consistent effect of leaf extracts from exotic trees on native herbaceous plants. in a study on the retardant effects of fresh eucalyptus (eucalyptus robusta) leaf extract on the growth of bean, corn (zea mays), lettuce, pea, rice, and sorghum (sorghum vulgare) seeds under laboratory conditions in pereira, colombia, ávila et al. (2007) observed that the extract had a greater effect on the monocotyledons (corn, rice, and sorghum) than on the dicotyledons (peas, beans, and lettuce), indicating that this could form a basis for the development of grass control products. the results of the present study indicated that any allelopathic substances that are present in the plant extracts obtained from the two forest species investigated would not accumulate at the concentrations required to cause an effect (be it positive or negative) on coffee plants under nursery conditions. however, it has been shown that allelopathic activity depends on several factors, such as the sensitivity of the receptor species, release of the toxin to the environment, biotic and abiotic activity, and factors that interact with the toxin in the soil (microorganisms, temperature, ph, etc.) (blum et al., 1992, cited by blanco, 2006). therefore, it is also possible that the environmental conditions and test time were not optimal for observing the activities of these substances. conclusions the foliar extracts of guaba and cadaghi did not have any allelopathic effects on the coffee cultivar ‘caturra roja’ under nursery conditions in chanchamayo, peru. references amb, m.k. & ahluwalia, a.s. 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(2011). sistemas agroforestales con tecnología limpia en los suelos del vraem, perú. acta nova, 5 (2), 289-311. varela, g. 2017. efectos alelopáticos de alnus nepalensis d. don en cuatro cultivos agrícolas de importancia socioeconómica en la zona de intag, noroccidente del ecuador. trabajo de titulación ing. forestal. universidad técnica del norte. ibarra. ecuador. 128 pp. retrieved from http://repositorio.utn.edu. ec/handle/123456789/6875 peruvian journal of agronomy 2 (1): 20 27 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i1.1130 © the authors. published by universidad nacional agraria la molina received for publication: 30 january 2018 accepted for publication: 30 march 2018 characterization of pineapple farms (ananas comosus var. comosus) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) caracterización de fincas productoras de piña (ananas comosus var. comosus) en la microcuenca cuyani, distrito de pichanaki, provincia de chanchamayo (junín, perú) maraví, j.1, buendía, o.1, alvarado, l.1 , borjas, r.1, castro-cepero, v.1, julca, a.1 *corresponding author: ajo@lamolina.edu.pe abstract the study was carried out in the cuyani microbasin in the district of pichanaki (peru), with the main goal of describing pineapple farms. a sample of 31 producers out of a total of 45 was selected, using the “proportions method”. in addition, a survey was conducted with questions related to economic, social, environmental aspects. it was found that the person in charge of the farm is mostly male, above 33 years of age and most of them with full high school education. the farm has a total extension between 1 to 10 ha, of which only 0.25 to 1.5 ha is planted with pineapple, mainly under a conventional production system, with yields ranging from 50 to 80 t / ha in varieties such as hawaiian and md2 (golden) and consider that weeds and diseases are the most important limiting factors of the crop. the conglomerate analysis by the ward method with a square euclidean distance of 1500 brought the farms together into five groups, the largest group comprising 38.7% of farms. keywords: systems, farms, characterization, pineapple, survey. resumen el estudio se realizó en la microcuenca cuyani en el distrito de pichanaki (perú), con el objetivo de caracterizar fincas productoras de piña. se trabajó con una organización que cuenta con 45 productores de los cuales se tomó una muestra de 31 productores a través del “método de proporciones” y se realizó una encuesta con preguntas relacionadas a los aspectos económicos, sociales, ambientales. se encontró que el responsable de la finca en su mayoría es varón, por encima de los 33 años y la mayor parte con estudios de secundaria completa. tienen una finca de 1 a 10 ha; pero solo sólo siembran de 0.25 a 1.5 ha con piña, predomina el sistema de producción convencional, con rendimientos que van de 50 a 80 t/ha en variedades como hawaiana y md2 (golden) y consideran que las malezas y enfermedades son los factores limitantes más importantes del cultivo. el análisis de conglomerado por el método de ward con una distancia euclidiana cuadrada de 1500, reunió las fincas en cinco grupos, el más grande reúne al 38.7% de fincas. palabras claves: sistemas, fincas, caracterización, piña, encuesta. 1grupo investigación: agricultura y desarrollo sustentable en el trópico peruano. facultad de agronomía. departamento de fitotecnia. universidad nacional agraria la molina. lima (perú). introduction the pineapple (ananas comosus (l.) merr. var. comosus) is an herbaceous species distributed in the tropics, originating in tropical america. according to data from the integrated system of agricultural statistics (siea), in 2014 the area harvested in perú was 15,917 ha. junín was the main supplier of the national market, which concentrates most of the land used for pineapple production (mendieta, 2015); in 2015, it reached 6,200 ha in production, which represents more than 70% of national production accor ding to the central reserve bank of peru huancayo branch (2016). the districts of chanchamayo and satipo are the ones with the largest area of cultivation in the central rainforest. the pineapple culture in peru has become an activity of great socioeconomic importance, with a great demand due to the nutritional properties such as vitamins, minerals, fibers and enzymes that contribute to a balanced nutrition. it is the fourth fruit with the highest national production, after banana, mango and grape with 124,700 mt, in the first quarter of 2017 (siea, 2017). in recent years, the pine apple production chain has generated employment and economic growth in the peruvian rainforest, from the sale of seeds, to labor for various tasks, the use of machinery, marketing, etc. (munive, 2015). the cultivars most commonly used in the central forest are ‘samba’, ‘hawaiana’, ‘cayena lisa’ and ‘md2’. the latter is also known as “golden” and is aimed at fresh and industrial consumption and is widely accepted in the international market as being of high internal quality (pac, 2005 quoted by munive 2015). on the other hand, agriculture in pichanaki, as in most of the country, is an activity of small producers, that is, the agricultural units or farms are small. but the farms are very diverse and complex so it is necessary to make maraví, j., buendía, o., alvarado, l., borjas, r., castro-cepero, v., julca, a. peruvian journal of agronomy 2 (1): 20 27 (2018) 21 a characterization as a previous step for any subsequent project (santistevan et al., 2014); for malagon and praguer (2001), the characterization is a determining stage for the development of research in production systems. it consists of determining a set of variables that distinguish a particular production area or unit and that makes it different from others. among other things, it seeks to distinguish the outstanding aspects for research in the selected area, identify the prevailing systems and identify the limiting factors. in this sense, by grouping the farms according to their main differences and relationships, the aim is to maximize homogeneity within the group and heterogeneity among them (cabrera et al., 2004). therefore, this work was carried out with the objective of characterizing the pineapple farms (ananas comosus var. comosus) in the cuyani microbasin, pichanaki district (chanchamayo, junín, peru). materials and methods the investigation was carried out in the junín region, chanchamayo province, pichanaki district, in the cuyani microbasin. it limits on the west with bella vista; on the east with pichiquiari and on the southeast with alto pichanaki (figure 1). for this particular investigation, we worked with the most representative organization of the study area, which has 45 pineapple producers from which a sample of 31 producers was selected. the “method of proportions”, already used in other similar researches, was used (merma and julca, 2012); the method has the following formula: figure 1. map of the pichanaki district and the cuyani microbasin study area, chanchamayo province, junín region, perú (adapted from: mdp, 2015) donde: n: sample size n: target population (universe) p: success probability 0.5 (this value is usually assumed) q: error probability 0.5 d: % error the data collection was carried out through semistructured surveys, related to economic, social, environmental and technical management aspects for agricultural production. the surveys were carried out in the field, taking advantage of the meetings held by the community members, and to complete the information, they were surveyed on their own farms. finally, a clustering analysis was performed by the ward method, with a euclidean square distance of 1,500. results and discussion characteristic of the pineapple producer. figure 2 shows that the person responsible for the farm is mostly male (84%) and only 16% of the farms are managed by women. these results corroborate data from the national agricultural census (inei, 2012), which showed that of every 10 farmers, about 8 are men and 2 are women in the selva region. the age of the farmers is between 33 to 45 years (45%), 20 to 32 years (29%), 46 to 58 years (16%) and those over 59 years (10%). likewise, the census data are consistent with the data obtained, as they indicate that the highest percentage of producers in the agricultural sector according to their age is registered in the group from 35 to 44 years old with 24.4%. the data obtained conclude that the largest group of pineapple producers are men between the ages of 30 and 45. the educational level of the pineapple farmers shows that 65% of the ha most of them have completed high school education, 26% elemetary school education, 6% have not had any educational formation and 3% have a technical career at the pichanaki institute. compared to the data of the 2007 national census, there is a similarity, where 40% of the total population of the pichanaki district are people who have completed elementary school education, characterization of pineapple farms (ananas comosus var. comosus) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) january april 2018 22 32% are people who have high school education and 16% do not have any educational level (pichanaki district municipality, 2016). farmers who live on their own farms or in neighboring towns, build them with mud (10%) and wood (35%); those who reside in pichanaki and cuyani center, build them of noble material (55%). this is corroborated with the data of the housing census vi of 2007 that shows that the housing construction material that predominated was of noble material with 70.05% by the urban sector of pichanaki, followed by 22.20% of wooden houses that represent the majority the rural sector and a minimum percentage of quincha and tapia (district municipality of pichanaki, 2016). the coverage of basic services for farmers, such as water supply, drainage and electricity, is incomplete and inadequate in most rural areas, unlike in the urban sector. forty-two percent of the producers who live in the towns and communities have water supplied by rivers and irrigation ditches, as well as electricity. unlike the pineapple farmers who live in pichanaki or centro cuyani, who have full basic services (58%). data from the 2007 census indicate that 46.13% of the houses in the district have access to drinking water service, either through the public water network, direct connection, common pylon, truck, cistern, among others, and 53.88% are supplied by rivers, springs or irrigation ditches (pichanaki district municipality, 2016). the difference in results is due to the fact that the largest number of pineapple farmers surveyed reside in pichanaki, so they have full basic services. according to the research carried out in the cuyani microbasin by the pichanaki district municipality in 2015, the main activity is agriculture. in the study area, it was found that 90% of the farmers depend on this activity and 10% have other incomes, as they work in state institutions and in the commercial sector. regarding the type of breeding, 55% breed poultry, 13% breed birds and guinea pigs, 3% breed birds and pigs, 3% only breed guinea pigs, and 26% do not manage any type of breeding, as they are farmers that reside in pichanaki (figure 2). figure 2 shows that 48% of farmers do not belong to an organization, 42% indicated that they were members of the native alto cuyani or pampa arecha community; that is, farmers who rent fields from the communities indicated that they also actively participate in community meetings; and 10% of them belong to a coffee cooperative. likewise, this organization is only just being established, so they are in the process of being legalized. characteristics of the pineapple farm: of the farmers surveyed, 39% have between 6 and 10 ha of total land, 32% between 1 and 5 ha, 16% between 16 and 20 ha and 13% between 11 and 15 ha (figure 3). however, 52% of producers also manage other traditional crops such as coffee (42%), bananas (35%), cassava (26%) and kion and corn (16%). according to the results obtained, they show that producers who do not have other crops handle more than 2 ha of pineapple, so they are completely dedicated to this crop. the pichanaki district had 340.85 hectares of pineapple in 2012, according to cenagro (riabm, 2015). plantations increased in this area, as it is a profitable crop and due to the coffee crisis in 2013. as a result, 48% of those surveyed have little experience, so they manage an area between 0.25 to 1.5 ha of pineapple, 29% between 2 to 3.5 ha, 13% between 4 to 5.5 ha and a smaller group (10%) that have between 6 to 7 ha (figure 3). those who have plantations of 2 ha to more, are producers who have many years of experience in the management of this crop. the ministry of agriculture estimated in 2008 that there are about 14,000 hectares of pineapple with an average yield of 15 t/ha (p.e.p.p. 2010), but in 2015 the average yield in chanchamayo was 36 t/ha and in satipo 81 t/ha (d.e.i.a, 2016). in the present study, 39% obtained a yield of 70 to 80 t/ha per hectare, higher than the average yield in chanchamayo, which is due to the fact that these farmers have experience in this crop; 22% obtained yields between 50 to 60 t/ha, which is considered an acceptable production; finally, 39% indicated that this was their first time managing the crop, so they do not know how much their average yield will be (figure 3). the investment made by a producer includes everything from renting the field, buying the seed, slashing, burning and cleaning, preparing the soil, disinfecting the seed, sowing, weeding, fertilization, sanitary control, induction of flowering, equipment and tools to be used. these are important tasks that must be performed for the management of this crop. in figure 3, it is shown that 42% mentioned that their investment per ha varies between s/. 12,000 to 16,000, 23% invested between s/. 17,000 to 21,000, 6% between s/. 9,000 to 11,000, another 6% between s/. 21,000 to 30,000 and 23% do not know how much their investment will be, because the installation of the plantation is recent. the highest production costs occur in farmers who grow the md2 (golden) cultivar, since the management is more laborious than the hawaiian cultivar. according to data from the regional directorate of agriculture of junín, to produce golden pineapple in a hectare of satipo land, the farmer invests approximately s/. 30,000 with a profit of s/. 150,000 with good agricultural management (inforegión, 2010). the pichis palcazu special project (2010) indicates that the calculation of the production cost for the pineapple case was not easy to determine, considering the different existing practices in each of the producing areas; in the case of the samba cultivar, which is traditional in chanchamayo, the investment is s/. 18,000 per ha, without technological package. in figure 3, it is shown that 35% of the farms have planted in a small to medium secondary forest; a secondary forest is forest growth, after a disturbance to the ecosystem that can be naturally or man-made (giacomotti, 2016). the producers mentioned that their agricultural lands were previously coffee plantations affected by yellow coffee rust (hemileia vastatrix), so they were abandoned for more maraví, j., buendía, o., alvarado, l., borjas, r., castro-cepero, v., julca, a. peruvian journal of agronomy 2 (1): 20 27 (2018) 23 figure 2. socio-economic status of the pineapple producers in the cuyani microbasin, pichanaki district, chanchamayo province. peru characterization of pineapple farms (ananas comosus var. comosus) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) january april 2018 24 figure 3. economic and environmental characteristics of the pineapple producing farm in the cuyani microcuenca, pichanaki district, chanchamayo province. peru. maraví, j., buendía, o., alvarado, l., borjas, r., castro-cepero, v., julca, a. peruvian journal of agronomy 2 (1): 20 27 (2018) 25 than 3 years. this type of vegetation has a successional character, that is to say, it begins with the development of a vegetation dominated by herbs, to give way to a tree vegetation that, over time, will assume a structure and floristic composition similar to the original forest, which will depend on the duration and intensity of previous use (giacomotti, 2016). sixty-five percent of farmers plant the same field where pineapple was previously planted. these are producers who have from 2 to more hectares and have experience with this crop. however, if plantations increase and there is no advice or adequate planning and control by public institutions, there may be negative impacts in the future. for example, costa rica is a large-scale pineapple producing country and due to agricultural expansion and the monoculture system, environmental and social problems have arisen. in the year 2015 the extension of the amazon rainforests was 68’188.726 ha which represent 53.06% of the national surface and correspond to 94.06% of the total forests. but the area lost is significant because in 2014 the accumulated loss of rainforest was 7.3 million ha (minam, 2016). seventy-one percent of those surveyed mentioned that they do not own timber trees on all their agricultural property, as these fields are dedicated only to pineapple cultivation. also, for the habilitation of the land, it is slash and burned, so that the wooded areas are lost. twenty-nine percent of the respondents indicated that they did own timber trees, because they have other land without exploitation or because they manage shade crops, such as coffee (figure 3). factors directly affecting production efficiency are involved in agricultural production; for 52% of producers, the presence of weeds is the biggest problem for their production due to the long growing cycle. on the other hand, when the field is not weeded in time, there is an increase in labor costs or the use of herbicides must be required to clean the field, putting production at risk. likewise, weeds are alternate hosts of diseases and pests. for 19% of producers, the presence of diseases is the limiting factor for higher yields even in the case of the hawaiian cultivar, which is tolerant. a 10% group indicated that drought and climatic instability was the main factor affecting the crop, as this affects the planting schedule and alters the crop cycle. another 10% indicated that the natural flowering of the pineapple in the months of may to august causes losses in production, because it affects the harvest and sale of fruit. six percent indicated pest damage as the main factor and 3% indicated disease and weed presence as the main factors (figure 3). currently, the towns of chanchamayo and satipo use better technology than other areas of the country (p.e.p.p.p. 2010). training in pre-harvest and post-harvest management by different companies, state institutions and ngos such as caritas, has enabled pineapple producer associations and small farmers to develop in this crop. pineapple cultivation is very extractive, so the production system applied by the farmer will determine whether or not the soil is vulnerable to nutrient washing and erosion. according to the results obtained in this study, 10% of the producers surveyed in the cuyani microbasin received the intervention of senasa. therefore, more institutional support is needed for small farmers to manage this intensive crop properly. productivity is a function of the amount of resources used (fuente, 2012). of the farmers surveyed, 58% figure 4. cluster of pineapple producing farms in the alto cuyani microbasin, pichanaki district, chanchamayo province. characterization of pineapple farms (ananas comosus var. comosus) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) january april 2018 26 indicated that they were satisfied with the productivity of their farm, as they obtained acceptable production and recovered their investment. 13% indicated that they are very satisfied, since last season production was profitable; a smaller group (6%) indicated that they were dissatisfied because their merchandise was sold in the pichanaki market, with very low sales prices since they could not enter the lima market because it was saturated, therefore, they did not make a profit. the last group of producers (23%), who are planting this crop for the first time, are not sure whether their production will be satisfactory. cluster analysis allows to group farms with similar characteristics. grouping is important because future actions could be carried out for each group of farms and no longer on an individual basis. in this study, cluster analysis by the ward method and with a square euclidean distance of 1000, classified the farms into five groups. the largest group (38.7%) is made up of farms: 1, 19, 10, 18, 11, 7, 13, 9, 15, 16, 21 and 14 (figure 4). this group is characterized by the fact that the people in charge of the farms are men, their production system is conventional, they plant the hawaiian cultivar and sell it in the wholesale market of lima. they are not trained and consider weeds and diseases to be the main limiting factors of production. cluster analysis has been used by other researchers in studies with other crops. for example, to characterize coffee farms in ecuador (santistevan et al., 2014) and peru to characterize citrus producing farms in cañete (collantes, 2016) and cocoa farms in san martín (tuesta et al., 2014). conclusions the pineapple producing farms in the cuyani micro-basin are very diverse. in general, a deficit of basic services is reported, a problem that should be addressed as soon as possible. the most widely planted variety of pineapple is hawaiian and there is a lack of training, especially in the area of pests and diseases. this task should be assumed by the state with the aim of achieving the sustainability of the crop. additionally, the result of the cluster analysis will be a very useful tool for decision-making and technology transfer in the future. acknowledgements the national fund for scientific, technological and technological innovation development (fondecyt), an initiative of the national council for science, technology and technological innovation (concytec), through the project “strategy and mechanisms for the governance of natural resources in the pichanaki model forest of central peru” (grant agreement no. 123-2015-fondecyt). literature cited banco central de reserva del perú. 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(2014). tipología de fincas cacaoteras en la subcuenca media del río huayabamba, distrito de huicungo (san martín, perú). ecología aplicada, 13(2), 71-78. peruvian journal of agronomy 3(2): 74-80 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i2.1317 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 12 july 2019 accepted for publication: 30 july 2019 gamma radiosensitivity of coffee (coffea arabica l. var. typica) estudio de la radiosensibilidad gamma del café (coffea arabica l. var. typica) quintana, v.1, alvarado, l.1, saravia, d.1, borjas, r. 1, castro-cepero, v.1, julca-otiniano, a. 1* gómez, l.2 *corresponding author: ajo@lamolina.edu.pe abstract the effects of gamma radiation on the germination, survival, and morphological damage in characteristics of the m1 generation of coffee (coffea arabica l. var. typica) plants was evaluated using seeds collected from santa teresa and chaupimayo, peru. under net house, the percentage of germination was 68%, 35%, 10%, and 0% for the santa teresa seeds and 75%, 49%, 17% and 0% for the chaupimayo seeds with irradiation treatments of 0, 50, 100 and 150 gy, respectively, whereas under laboratory conditions, germination levels were between 70% and 94% across all treatments. the survival rate also decreased with increasing radiation levels, with values of 45%, 32%, 28%, and 10% in the laboratory and 29%, 9%, 6%, and 0% in the net house for the santa teresa seeds and 58%, 45%, 38%, and 8% in the laboratory and 42%, 15%, 7%, and 0% in the net house for the chaupimayo seeds with irradiation treatments of 0, 50, 100, and 150 gy, respectively. morphological changes were observed in the plants that survived irradiation at a dose of 100 gy in terms of the leaf shape, leaf apex shape, young leaf color, plant height, stem diameter, number of leaves per plant, leaf length, leaf width, and distance from the cotyledon to the first node. key words: coffee, radiosensitivity, gamma ray, germination, survival, morphology. resumen se evaluó los efectos de la radiación gamma sobre la germinación, la supervivencia y características morfológico de plantas de café de generación m1 (coffea arabica l. var. typica), utilizando semillas colectadas de santa teresa y chaupimayo, perú. en casa malla, el porcentaje de germinación fue del 68%, 35%, 10% y 0% para las semillas de santa teresa y 75%, 49%, 17% y 0% para las semillas de chaupimayo con tratamientos de irradiación de 0, 50, 100 y 150 gy, respectivamente, mientras que en condiciones de laboratorio, los niveles de germinación fueron entre 70% y 94% en todos los tratamientos. el porcentaje de supervivencia también disminuyó con el aumento de los niveles de radiación, con valores de 45%, 32%, 28% y 10% en el laboratorio y 29%, 9%, 6% y 0% en la casa malla para las semillas de santa teresa y 58 %, 45%, 38% y 8% en el laboratorio y 42%, 15%, 7% y 0% en la casa malla para las semillas de chaupimayo con tratamientos de irradiación de 0, 50, 100 y 150 gy, respectivamente. se observaron cambios morfológicos en las plantas que sobrevivieron a la irradiación a una dosis de 100 gy en características como forma y ápice de la hoja, color de la hoja joven, altura de la planta, diámetro del tallo, número de hojas por planta, longitud de la hoja, ancho de la hoja y distancia del cotiledón al primer nudo. palabras clave: café, radiosensibilidad, radiación gamma, germinación, supervivencia, morfología. 1 research group on agriculture and sustainable development in the peruvian tropic. agronomy faculty. department of plant production, universidad nacional agraria la molina. lima perú. 2 cereals and native grains research program. universidad nacional agraria la molina. lima perú. introduction coffee (coffea spp.) is reported to support the livelihoods of millions of small-scale farmers around the world (bacon, 2004), and i s an important economic crop for many tropical countries. in peru, more than 100 000 families that have settled in the jungle depend on activities associated with coffee production. however, the recent epidemic of coffee rust ( h e m i l e i a v a s t a t r i x ) from mexico to peru has been particularly severe, with some reports anticipating a reduction in yield (40%–50%) over the entire region, which will potentially affect many coffee production nations in latin america (cressey, 2013). for example, vandermeer, jackson, and perfecto (2014) reported that over 60% of the plants in an experimental plot i n m e x i c o experienced >80% defoliation and nearly 9% died, and similar patterns have been reported quintana, v., alvarado, l., saravia, d., borjas, r., castro-cepero, v., julca-otiniano, a., gómez, l. peruvian journal of agronomy 3(2): 74-80 (2019) 75 anecdotally from across the region (cressey, 2013). despite the success of rust breeding programs for coffee in colombia and brazil (van der vossen, 2001), there is evidence that some improved commercial varieties derived from t h e timor hybrid (c. canephora × c. arabica) have lost their resistance, possibly due to the emergence of new virulent races of the pathogen (alvarado, 2005; várzea & marques, 2005). variability in the virulence of h. vastatrix appears to be due to natural mutation processes, but could also arise from other mechanisms, such as cryptic sex, the hidden sexual reproduction of the pathogen (carvalho et al., 2011). therefore, breeding programs have focused on efforts to broaden the genetic base of commercial coffee varieties (castro-caicedo, cortina-guerrero, roux & wingfield, 2013). mutation induction using different mutagenic agents has been acknowledged reliable method for breeding plants with improved characteristics in many crops [international atomic energy agency (iaea) database, https://www.iaea.org]. therefore, the objectives of this study were to determine the germination, survival and morphological characteristics of coffee seedlings derived from irradiated seeds. the sensitivity of plant materials to various mutagens has been investigated and summarized for many plant species. however, these should be considered as a guide, only rather than being treated as fixed numbers, particularly for those plant species that have not been extensively studied, as there is significant variation in the sensitivity of plants to mutagenic treatments both at the species level and within genotypes (shu, 2009). the radiosensitivity of the irradiated genotypes is determined by exposing the material to a range of radiation intensities and selecting those doses that allow visible effects of the radiation, to be observed while maintaining the survival of the tissues (tullmann-neto, 1997). most of the effects observed in the m1 generation will be physiological, with the lesions that appear on the m1 plants being indicative of the degree of mutagenic effects in the plants. these effects can be quantified in a number of ways and can be used to establish dose threshold values of mutagens that cause the mutation required. materials and methods the effect of gamma radiation was examined in coffee (c. arabica l. var. typica) seeds that had been collected from two locations: santa teresa and chaupimayo, peru. dry seeds of coffee with endocarps (parchments) were irradiated with gamma rays at doses of 50, 100, 150, 200 and 300 gy at the peruvian energy nuclear institute using a gammacell 220. germination and survival tests in the laboratory, the irradiated seeds from all the treatment groups were germinated in a pol-eko-aparatura thermostatic cabinet (poland) that was set to 30ºc and a photoperiod of 12 h of illumination and 12 h of darkness. all of the seeds with endocarps, were first disinfected with homai® wp fungicide ( thiophanate-methyl + thiram) washed 2% detergent, followed by 2% bleach, and rinsed with plenty of distilled water. the seeds were then hydrated by immersing them under water in glass containers for a period of 24 hours at room temperature, after which they were placed on paper towel in the growth chamber. after sowing, the fungicide benomyl was applied to the paper towel with a spray. the net house had the same environmental conditions as the laboratory, i.e., a temperature of 30° c and a photoperiod of 12 h of light and 12 h of darkness. irradiated seeds from each treatment group had the endocarp removed. then, these seeds were disinfected, laid on top of sand in a tray, and kept hydrated with distilled water the germination process. the seedlings that survived, in each treatment group were transplanted in the nursery at “genova farm” at the regional development institute (ird) of the national agrarian university la molina (unalm) in san ramon district, chanchamayo province, department of junín, peru, for further plant growth and development. recorded data. the following data were recorded during the study: percentage of germination, percentage of survival and a morphological description of the seedlings, which included the seedling height, number of true leaves, stem diameter, distance from the cotyledon to the first node, leaf shape, leaf color, stipule shape, leaf apex shape, leaf length and width leaf). results and discussion germination according to rosa et al. (2010), the germination stage sensu stricto begins approximately 7 days, after sowing in coffee plants and is completed when the radicle penetrates the outer layer of the endosperm. in the laboratory experiment, the percentage of sensu estrictu was evaluated 62 days after sowing. seeds from santa teresa, that were irradiated with 0 gy and 50 gy exhibited 90% germination, whereas those that were irradiated with 100 gy and 150 gy had 84% and 70% germination, respectively (figure 1). by contrast, seeds from chaupimayo exhibited 94%, 88%, and 78% germination following irradiation with 0, 50, 100, 150 gy, respectively. thus, the percentage of germination was higher than 70% for both types of seeds across all treatment groups, but the seeds from chaupimayo obtained higher values than those from santa teresa. since the optimum gamma radiosensitivity of coffee (coffea arabica l. var. typica) may august 2019 76 temperature range for coffee seed germination is between 28°c and 30°c (wellman, 1961; huxley, 1964) and the growth chamber had a temperature of 30°c, this difference in the germination percentage may have resulted from differences in the quality of the seeds. in the net house, experiment the percentage of germination was evaluated at 75 days after sowing. the seeds from santa exhibited germination levels of 68%, 35%, 10%, and 0%, while those from chaupimayo, had germination levels of 75%, 49%, 17%, and 0%, following irradiation with 0, 50, 100 and 150 gy, respectively (figure 2). similarly, kumar, nepolean & gopalan (2003) found that the survival and germination, of phaseolus lunatus seeds decreased with increasing gamma radiation over a range of 200–1000 gy, and cheema and atta (2003) showed that seed germination in the m1 generation of three different varieties of rice (oryza sativa) decreased with an increased radiation dose under field conditions. survival in the laboratory, seedlings that germinated from santa teresa seeds exhibited survival rates of 45%, 32%, 28%, and 10% at 22 days after germination, while those that germinated from the chaupimayo seeds had survival rates of 58%, 45%, 38%, and 8% in the 0, 50, 100, and 150 gy treatment groups, respectively (figure 3). no published information is available on the effects of gamma radiation on the survival rates of coffee, but it has been shown that increasing doses of gamma radiation to the seeds, decreases the number of plants surviving for jatropha curcas (sonsgri et al., 2011) and rye (secale cereale l.) “sorom” (soraluz, 2015). in the net house, seedlings that germinated from santa teresa seeds had survival rates of 29%, 9%, 6%, and 0% at 112 days after germination, while those that germinated from the chaupimayo seeds had survival rates of 42%, 15%, 7%, and 0% in the 0, 50, 100 and 150 gy treatment groups, respectively (figure 4). rate of plant growth and development based on the germination and survival results, only the m1 generation of plants in the 100 gy treatment group were transplanted to the nursery for comparison of growth and development. in general, the m1 plants that originated from the irradiated seeds had slower development than the control (0 gy treatment) plants. in the control group, 3% of plants were in the matchstick (initial) stage, 6% were in the butterfly stage, and 91% were in the true leaf formation (figure 5). similarly, kiong, lai, hussein, and harun (2008) found that mutagenic treatments resulted in a lower germination rate and slower development, and vargas (2016), found that 0 10 20 30 40 50 60 70 80 90 100 0 gy 50 gy 100 gy 150 gy g er m in at io n (% ) sta. teresa chaupimayo figure 1. percentage of germination of coffee (coffea arabica l. var. typica) seeds originating from santa teresa and chaupimayo under laboratory conditions at 62 days after sowing following irradiation with gamma rays at 0 (control), 50, 100, and 150 gy. 0 10 20 30 40 50 60 70 80 0 gy 50 gy 100 gy 150 gy g er m in at io n (% ) sta. teresa chaupimayo figure 2. percentage of germination of coffee (coffea arabica l. var. typica) seeds originating from santa teresa and chaupimayo under net house conditions at 75 days after sowing following irradiation with gamma rays at 0 (control), 50, 100, and 150 gy. 0 10 20 30 40 50 60 70 0 gy 50 gy 100 gy 150 gy g er m in at io n (% ) sta. teresa chaupimayo figure 3. percentage of germination of coffee (coffea arabica l. var. typica) seeds originating from santa teresa and chaupimayo under laboratory conditions at 84 days after sowing following irradiation with gamma rays at 0 (control), 50, 100, and 150 gy. 0 5 10 15 20 25 30 35 40 45 0 gy 50 gy 100 gy 150 gy su rv iv al ( % ) sta. teresa chaupimayo figure 4. percentage of survival of germinated coffee (coffea arabica l. var. typica) seeds originating from santa teresa and chaupimayo under net house conditions at 112 days after sowing following irradiation with gamma rays at 0 (control), 50, 100, and 150 gy. quintana, v., alvarado, l., saravia, d., borjas, r., castro-cepero, v., julca-otiniano, a., gómez, l. peruvian journal of agronomy 3(2): 74-80 (2019) 77 increased sodium azide dose increased the time to true leaf generation in coffee plants. furthermore, al-salhi et al. (2004) and hameed et al. (2008), reported that exposure to high doses of gamma radiation affect protein synthesis, the hormonal equilibrium, the foliar interchange of gases, and enzymatic activity in a range of crop, all of which are related to plant growth. morphological characteristics stem: the plant height, stem diameter, and length of the first internode, had mean values of 22 cm, 2.7 cm, and 1.01 cm; respectively, in control plants and 12 cm, 2.2 cm, and 0.15 cm, respectively, in m1 plants in the 100 gy treatment group (figure 6). canul et al. (2012) similarly reported irradiated euphorbia pulcherrima plants had a reduced plant height, while vargas (2016) showed that increased doses of sodium azide reduced the height of coffee plants. leaves: the leaf length, leaf width, and number of leaves had mean values of 6.7 cm, 3.2 cm and 10 leaves, respectively, in the control plants and 5.5 cm, 2.2 cm, and 7 leaves, respectively, in m1 plants in the 100 gy treatment group (figure 7). thus, m1 plants irradiated with 100 gy showed lower values for all characters than the unirradiated control plants. leaf shape: all of the control plants had a lanceolate leaf shape, compared with only 54% of the m1 plants in the 100 gy treatment group (figure 8). among the remaining m1 plants, 18% exhibited an ovate leaf shape and 28% had irregular modifications (figure 9). similarly, moh and orbegozo (1960) and moh (1961) reported that the irradiation of coffee seeds with x rays induced a high frequency of morphological variation in the leaves, of m1 plants, particularly the formation of narrow leaves, which, according to mendes (1955), could be the result of aneuploidy. there was also some modification of the leaf apex, which was acuminate shaped in all of the control plants but only 66% of the m1 plants from the 100 gy treatment group, with the remainder being apiculate shaped (6%) or having other modifications (28%) (figure 10). the stipule shape was ovate in all control plants. however, only 76% of the m1 plants exhibited this shape, with the 24% showing modification of the stipule without a defined form. leaf color: all of the control plants had green leaves, whereas 2% of the m1 plants exhibited some other color (figure 11), ranging from yellow-green, through to white with yellow stripes or sports. 0 20 40 60 80 100 matchstick butterfly true leaf s ur vi va l (% ) 0 gy 100 gy figure 5. effect of 100 gy gamma irradiation on the grade of development of m1 coffee (coffea arabica l. var. typica) plants grown under nursery conditions “genova farm” san ramon district, department of junín, peru. 0 5 10 15 20 25 plant height stem diameter lenght internode 1 a ve ra ge (c m ) 0 gy 100 gy figure 6.effects of 100 gy gamma irradiation on the stem characteristics of the m1 generation of coffee (coffea arabica l. var. typica) plants grown under nursery conditions on “genova farm san ramon district, department of junín, peru. 0 2 4 6 8 10 12 leaf length (cm) leaf width (cm) number of leaves a v e ra g e 0 gy 100 gy figure 7.effects of 100 gy gamma irradiation on the leaf characteristics of the m1 generation of coffee (coffea arabica l. var. typica) plants grown under nursery conditions on “genova farm,” san ramon district, department of junín, peru. 0 10 20 30 40 50 60 70 80 90 100 other-modification ovate lanceolate pl an ts ( % ) 0 gy 100 gy figure 8. effects of 100 gy irradiation on the leaf morphology of the m1 generation of coffee (coffea arabica l. var. typica) plants grown under nursery conditions on “genova farm,” san ramon district, department of junín, peru. gamma radiosensitivity of coffee (coffea arabica l. var. typica) may august 2019 78 figure 9. morphological modifications in the leaves of the m1 generation of coffee(coffea arabica l. var. typica) plants grown under nursery conditions on “genova farm,” san ramon district, department of junín, peru. quintana, v., alvarado, l., saravia, d., borjas, r., castro-cepero, v., julca-otiniano, a., gómez, l. peruvian journal of agronomy 3(2): 74-80 (2019) 79 it was observed in 2 % of the m1 plants (figure 11). some plants had yellow-green color and white and yellow stripes or spots. conclusions gamma radiation affected the percentage of seed germination, the survival rate, the growth, and the duration of the phenological phases in the typica variety of coffee, with these negative effects increasing with an increased dose; under laboratory, growth chamber, and nursery conditions. among the doses used in the present study, a fairly high survival of seedlings was observed with 100 gy. however, 100 gy of gamma radiation caused somatic changes in the m1 population, with modifications in the shape, apex type, and color of the leaves. acknowledgments supported by the international atomic energy agency (iaea) under the research contract n° 19292: screening for resistance to coffee rust (hemileia vastatrix) in peruvian conditions. references al-salhi, m., ghannam, m., al-ayed, m., el-kameesy, s., & roshdy, s. 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(1997). utilización de radiaciones gamma en el mejoramiento genético de plantas autógamas. in curso mutaciones inducidas en el mejoramiento de plantas. aiea-udo, núcleo monagas. reference material. van der vossen, a.m. (2001). coffee breeding practices. in: r.j. clarke & o.g. vitzthum, (eds.). coffee recent developments: agronomy. (pp. 184–200). london uk. blackwell. vargas, l. (2016). efecto de la azida sódica (nan3) en la germinación y morfología post germinación en café (coffea sp). thesis biól. lima, perú. universidad nacional agraria la molina (unalm). várzea, v.m. & marques, d.v. (2005). population variability of hemileia vastatrix vs. coffee durable resistance. in: l. zambolin, e.m. zambolim, & v.m. várzea (eds.). durable resistance to coffee leaf rust. (pp. 53–74). viçosa mg, brazil. editora ufv. wellman, l. (1961). coffee. botany, cultivation and utilization. world crops books. london. inglaterra. 488 p. cressey, d. (2013). coffee rust regains foothold: researchers marshal technonology in bid to thwart fungal outbreak in central america. nature, 493(7434), 587–588 vandermeer, j., jackson, d., & perfecto, i. (2014). qualitative dynamics of the coffee rust epidemic: educating intuition with theoretical ecology. bioscience, 64(3), 210–218 shu, q. y. (2009). turning plant mutation breeding into a new era: molecular mutation breeding. in q.y. shu (ed.), induced plant mutations in the genomics era. (pp. 425–427) food and agriculture organization of the united nations, rome. rosa, v.f., mcdonald, m.b., veiga, a.d., vilela, f.l., & ferreira, i.a. (2010). staging coffee seedling growth: a rationale for shortening the coffee seed germination test. seed science and technology, 38(2), 421–431. kiong, a.l., lai, a.g., hussein, s., & harun, a.r. (2008). physiological responses of orthosiphon stamineus plantlets to gamma irradiation. american-eurasian journal of sustainable agriculture, 2, 135–149. peruvian journal of agronomy 2 (2): 22 26 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i2.1201 © the authors. published by universidad nacional agraria la molina received for publication: 21 august 2018 accepted for publication: 22 september 2018 application of nitrogen fertilizer in maize in southern asia: a review aplicación de fertilizantes nitrogenados al maíz en asia meridional: una revisión jiban shrestha1*, amit chaudhary2 and dipesh pokhrel1 * corresponding author. jibshrestha@gmail.com abstract the rate, time and method of nitrogen (n) fertilizer application are strongly related to growth, development, and yield of the crop. this study principally focuses on the role of the nitrogen in growth, development, and production of the maize, emphasizing time and methods of fertilizer application and their suitable rates. the review shows that crop yield increases up to certain limit and declines if applied in an excess amount of nitrogen. nitrogen affects various physiological and biochemical processes in plant cells that ultimately affect the growth and development of the plant. nitrogen response by maize differs due to growth stages, environment and genotype of maize. hybrid and improved maize varieties are more nitrogen-responsive than local varieties of maize. proper nitrogen applications as basal doses at planting stage, split doses at critical growth stages namely knee high, and flowering stages are necessary for higher grain yield. this review serves as a useful tool to maize researchers and growers for making the right decision on nitrogen application on maize. keywords: maize; nitrogen fertilization; crop growth; yield; grain nitrogen content. resumen la tasa, tiempo y método de aplicación de fertilizantes nitrogenados (n) están fuertemente relacionados con el crecimiento, desarrollo y rendimiento del cultivo. este estudio se centra principalmente en el papel del nitrógeno en el crecimiento, desarrollo y producción del maíz, enfatizando el tiempo y los métodos de aplicación de fertilizantes y sus tasas adecuadas. la investigación muestra que el rendimiento de los cultivos aumenta hasta cierto límite y disminuye si se aplica una cantidad excesiva de nitrógeno. el nitrógeno afecta varios procesos fisiológicos y bioquímicos en las células de la planta que finalmente afectan el crecimiento y desarrollo de la planta. la respuesta del nitrógeno del maíz difiere debido a las etapas de crecimiento, el medio ambiente y el genotipo del maíz. las variedades híbridas y mejoradas de maíz son más sensibles al nitrógeno que las variedades locales de maíz. las aplicaciones apropiadas de nitrógeno como dosis basales en la etapa de siembra, dosis divididas en las etapas críticas de crecimiento, es decir, en las etapas de crecimiento a la altura de la rodilla y de floración, son necesarias para un mayor rendimiento de grano. esta revisión sirve como una herramienta útil para los investigadores y cultivadores de maíz para tomar la decisión correcta sobre la aplicación de nitrógeno en el maíz. palabras clave: maiz; fertilización de nitrógeno; crecimiento del cultivo; rendimiento; contenido de nitrógeno en grano. 1 nepal agricultural research council, national commercial agriculture research program, pakhribas, dhankuta, nepal 2 tribhuvan university, institute of agriculture and animal sciences, lamjung campus, lamjung, nepal role of nitrogen in maize maize (zea mays l.) is the third most important crop worldwide following rice (oryza sativa l.) and wheat (triticum aestivum l.). the maize kernel is composed of approximately 72% starch, 10% protein, 5% oil, 2% sugar, and 1% ash with the remainder being water (perry, 1988). the use of nitrogen fertilizers results in higher biomass and protein yield and increases the concentration of protein in the plant tissue. as the protein concentration of corn grain increases, zein makes up an increasing proportion of the protein (tsai et al., 1992). nitrogen often affects the amino acid composition of the protein, and thus the quality of nutrients. in cereals, an abundant supply of nitrogen reduces the relative contribution of lysine and threonine, thus reducing the biological value of the protein. increased nitrogen supply often leads to kernel integrity and strength, resulting in better milling properties of the grain (blumenthal et al., 2008). nitrogen regulates the efficiency of the use of nutrients in the plant. the nitrogen affects various physiological and biochemical processes in plant cells and, ultimately, affects growth and development (brady, 1990). in proteins, alkaloids, nucleic acids, coenzymes, porphyrins, nitrogen is the main ingredient. the porphyrins are responsible for the inheritance, metabolic process and growth of plants. porphyrins are the main component of cytochrome and chlorophyll (jain, 2000). the protoplast of plant cells contains mainly nitrogen. it plays an essential role in the growth and proper development of the plant. the lack of nitrogen reduces the growth of the plants and lower yields. shrestha, j.; chaudhary, a.; pokhrel, d. peruvian journal of agronomy 2 (2): 22 26 (2018) 23 on the contrary, when optimum levels of nitrogen are used, higher yields are produced due to the larger leaf area and the proper growth of the plant. in addition to its function in the creation of green tissue, nitrogen plays a very important role in the development of the ear and kernel. studies on nitrogen translocation in the plant showed that nitrogen appears to move from other plant tissues to the ear before silking, apparently in the case of the nitrogen-intense process of kernel embryo formation (ciampitti and vyn, 2010). time and methods of nitrogen application in maize the timing of nitrogen application affects the yield (bhattarai et al., 2004). in two split dose methods, half of the nitrogen used as the basal dose and other parts of the plant during the knee high stage are inevitable. of course, there is a need for half of the total nitrogen remaining at knee high and tasseling stage as equal split doses are necessary. crop productivity and the availability of nitrogen are unfavorable when used very late in the growing season. when a small amount of nitrogen is used in the initial stages of growth, the rate of nitrogen absorption is greatly reduced by the plant. during central of vegetative growth, maize plant starts to uptake nitrogen at a faster rate and continues at silking stage to be at peak point (hanway, 1963). use of nitrogen in early plant growth improves yield and improves vegetative growth and development, and when is given in later stages of plant growth, late maturity and maturation cannot adequately promote the final yield. although the level of nitrogen in the grain increases, it is used in later growth stages (sankaran and subbiah mudaliar, 1997). therefore, an excellent method of fulfilling a higher demand for nitrogen could be its application during tasseling and silking stages. maximum grain and better biomass yield observed after the application of nitrogen @ 80 kg/ha in two split doses ; half part applied at sowing as basal and remaining half during knee-high stage as side dress respectively. nurudeen et al. (2015) found that the application of nitrogen fertilizer @ 80 kg n/ha gave higher benefit cost ratio (1.5). bhattarai et al. (2004) found that during the sowing, earthing up and silking stages, the nitrogen applied at 3 equal divided doses to 60 kg/ha, maximized the yield of corn grain. in comparison to traditional broadcast fertilization, fertilization in rows or fertilization in rows combined partly with top dressing increased the values of a percentage of fertilizer nitrogen in the total nitrogen uptake as well as the agricultural and physiological effectiveness of the nitrogen use (szulc et al., 2016). effect of nitrogen on growth of maize maize is a plant that requires a high quantity of nutrients due to its enormous nutrient utilizing capacity. a higher volume of nitrogen is required for higher yield. nitrogen is required in a more significant amount than other nutrients. low yields occur when less nitrogen is used during the stages of tasseling and silking stages, but factors such as nutrient in soil, relief, variety and the expected value of maize yield influence the need. the general nutritional recommendation for corn crops is 120: 60: 40 kg/ha, n, p, and k respectively (singh and singh, 2002). the rate of application of nitrogen affects the yield of corn (abebe and feyisa, 2017). the excessive use of nitrogen in the plants causes continued growth and results in late maturation period. similarly, lodging occurs due to weak stems with reduced cell walls (produced by overuse of nitrogen). resistance to pests and diseases of the plants also decreased due to high nitrogen consumption. the capacity of the crop to absorb and use nutrients in the soil depends on the presence of nitrogen in the plants. therefore, maize yield is reduced due to lack and excess of nitrogen, so the use of nitrogen fertilizers plays an important role in better yields (goydani and singh, 1968). the efficient use of nitrogen varies from 150 to 200 kg/ha (mkhabela and palishikhulu, 2001). diallo et al. (1997) found that leaf senescence got increased due to smaller leaf area and less photosynthesis due to nitrogen deficiency. when nitrogen consumption increased from 0 to 150 kg/ha, the height of the plant extended between 164 and 209 cm (sharma, 1973). the leaf area index was also significantly higher in all phases of plant growth when nitrogen was used in the range of 60 to 180 kg/ha. thakur et al. (1998) found that increment of amount of nitrogen applied results more leaf number and thicker stem. leaf area and leaf number significantly increased by both rate and application of nitrogen (hati and panda, 1970). nitrogen applied to range from 0 to 150 kg/ha significantly increased dry matter at every crop growth stages (terman and allen, 1974). shrestha (2015) found early tasseling and silking stages in maize occurred as result of higher nitrogen application at 200kg n/ha. rai (1961) reported that application of nitrogen as well as increase in its rate induced earliness both in tasseling and silking stages. yadav (1990) also found earlier silking occurred due to a higher percentage of nitrogen applied. effect of nitrogen on yield and yield components of maize shrestha (2015) found that the application of higher nitrogen dose (200 kg n/ha) gave the highest number of cobs/plant (1.09), cob length (15.90 cm), cob diameter (5.10), number of grains/grain row (32.54,the number of grain rows/cob (14.11), number of grains/cob (459.9) and the greatest test weight (318.2g) and shelling recovery (72.14%). also, thicker cob diameter was observed (hati and panda, 1970). singh (1973) found that a higher level of nitrogen markedly increased grain number per cob and grain weight per cob. application of 210 kg n/ha maximize ear length to 15.64 cm (pokhrel et al., 2009). likewise, nitrogen applied to range 60 to 240 kg/ha effectively improved stover yield (krishnamoorthy et al., application of nitrogen fertilizer in maize in southern asia: a review may august 2018 24 1974). yadav (1990) found that in comparison to 0 and 30 kg n/ha applied, grain:stover ratio for 90 and 60 kg n/ha were significantly improved. whereas, grain yield for 80, 120 and 160 kg n/ha noted to be increased by 114.59%, 160.8%, and 58.3% respectively compared to 0 kg n/ha used (verma and singh 1976). deficiency of nitrogen showed an increased number of barren crop plants (singh, 1988). kamprath et al. (1982) noted that barrenness in plant increased when nitrogen applied to range above 168 to 280 kg/ha but significantly reduced when used between 56 to 168 kg/ha. a higher level of nitrogen applied enhanced test grain weight (gokmen et al., 2001; wajid et al., 2007). gungula et al. (2007) and dawadi and sah (2012) found a higher application rate of nitrogen effectively increased kernel number per ear and kernel rows number per cob. a higher level of nitrogen (180 kg n/ha) improved seed yield to 2.85 t/ha of inbred (nml-1) maize (adhikary and adhikary, 2013). grain nitrogen uptake the presence of nitrogen (n) in maize and genotype are two main factors affecting grain yield (gy) and grain nitrogen content (gnc). the high gy is also based on longevity of leaves, which depends on the balance between post-silking n uptake and remobilization of vegetative n. due to complex interactions between n uptake, n-remobilization, dry matter production (dm), gy and gnc, selection of high gnc hybrids as a single target often led to low gy (uribelarrea et al., 2007). nitrogen uptake by the maize plant increases its concentration in both the plant or in the grain due to the higher total dry matter content. the nitrogen applied from 0 to 150 kg/ha showed a proportional enhancement in grain yield and nitrogen concentration in the various parts of the plant (ram and thakur, 1966). also, nitrogen used up to 160 kg/ha was useful for the nitrogen concentration in the grain, higher nitrogen levels increased both nitrogen absorption and nitrogen content in maize (ahlawat et al., 1981). in the same way pokhrel et al. (2009) found that more nitrogen applied produces a larger nitrogen intake per grain due to higher dry matter yield. dry matter yield positively correlated with the absorption of nitrogen by maize plant (lian, 1991). factors affecting nitrogen requirement in maize the rate of nitrogen fertilizer needed to be supplied to a maize monocrop is influenced by the soil nitrogen content (carefoot et al., 1989), n source (bache and heathcoat, 1969; jones, 1976), soil reaction (riley and barber, 1971; jones, 1976), rainfall (dennett, 1984), maize maturity period (mackay and barber, 1986) and yield level desired (arnon, 1975). martins et al. (2008) reported that higher soil nitrogen resulted in higher nitrogen absorption of plants, influencing the physiological, biochemical and agronomic traits. improved maize cultivars could be more efficient in using nitrogen fertilizer than the local varieties (sallah and twumasi-afriyie, 1999). hybrid maize is a heavy feeder and more responsive to nutrients (sarkar et al., 2000). muza et al. (2004) found that the commercial maize hybrids require high nitrogen levels and fertile soils and hybrids are more responsive to nitrogen fertilizer. plant population ranging between 69,000 and 81,000 plants/ha, showed a significantly higher uptake of nitrogen than the 57,000 plant population/ha observed during 12 leaf and tasseling stages (al-kaisi and yin, 2003). adhikari et al. (2016) and sherchan et al. (2004) also reported that response of nitrogen and its application time to maize differs due to genetic characters, growing season (winter, spring, and summer), maturity period (early and full season), and growing domain (mountain/hill and terai). losses of n takes place due to several reasons like leaching as well as volatilization from most of the soils, only one nitrogen management strategy may not be reliable, because the loss occurs in various ways. if nitrogen supplied is under the demand of the plants, the efficiency of use of nitrogen increases (keeney, 1982). conclusion the nitrogen is essential for physiological and biochemical processes that ultimately affects growth and development. the application of nitrogen up to 200 kg n/ha increased the growth traits, yield and yield attributing traits. the nitrogen uptake in grain increases with application of increased level of nitrogen up to 150 kg n/ha applied in soil. the amount of nitrogen fertilizer varies with soil and environmental condition as well as genetic architecture of plants. this study suggests that recommended nitrogen application as basal dose at planting stage, split doses at critical growth stages namely knee high, and flowering stages should be applied for enhancing maize production. literature cited abebe, z., & feyisa, h. 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(2007). divergent selection for grain protein affects nitrogen use in maize hybrids. field crops research, 100(1), 82–90. https://doi.org/10.1016/j.fcr.2006.05.008 verma, b. s., & singh, r. r. (1976). effect of nitrogen, moisture regime and plant density on grain yield and quality of hybrid maize. india, no. 77-212400. cimmyt. wajid, a., ghaffar, a., maqsood, m., hussain, k., & nasim, w. (2007). yield response of maize hybrids to varying nitrogen rates. pakistan journal of agricultural sciences, 44(2), 217-220. yadav, d. n. (1990). growth and productivity of maize under different crop sequences and nitrogen rates. doctoral dissertation, ph. d. thesis, gb pant univ. of agric. and tech., pantanagar, india. peruvian journal of agronomy 3(3): 120–125 (2019) issn: 2616–4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1209 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 26 september 2019 accepted for publication: 22 october 2019 physiological and agronomic characterisation of six cassava clones (manihot esculenta crantz) caracterización fisiológica y agronómica de seis clones de yuca (manihot esculenta crantz) eguiluz, a. (1)*; pinedo, r. (1); figueroa, c.(1) *corresponding author: aeguiluz@lamolina.edu.pe abstract to evaluate the behaviour of cassava clones (manihot esculenta crantz), the physiological quality of the propagation material was assessed, and an agronomic characterisation was conducted; this was essential for the generation of information on the clones’ productive potential. the trial was conducted in the experimental field of the roots and tubers programme (pips-rt) of the national agrarian university–la molina. following a completely randomised block design with four replications, clones from the pips-rt were evaluated. variables were assessed to determine the clones’ physiological quality in terms of the number of viable stakes, normal seedlings, abnormal seedlings and percentage of stake survival. the agronomic characteristics evaluated were plant height, stem diameter and yield. the morocha clone had the highest percentage of viable stakes and normal seedlings, i.e. 80.95% and 73.81%, respectively. the highest yield was also produced by the morocha clones (13 t/ha) followed by the donoso 2 (7.20 t/ha) and blanca cajamarca (6.80 t/ha) varieties. keywords: characterisation, yield, seedling, productive potential resumen con la finalidad de evaluar el comportamiento seis clones de yuca (manihot esculenta crantz), se realizaron evaluaciones relacionadas a la calidad fisiológica del material de propagación y caracterización agronómica, requerida para generar información sobre la potencialidad de los clones. el ensayo se llevó a cabo en el campo experimental de programa de raíces y tuberosas de la unalm (pips–rt), bajo un diseño de bloques completamente al azar con cuatro repeticiones, se evaluaron clones provenientes de una colecta del pips-rt. las variables en estudio para determinar la calidad fisiológica de los clones fueron número de estacas viables, plántulas normales, plántulas anormales y el porcentaje de supervivencia de estacas; mientras que para características agronómicas fueron altura de la planta, diámetro del tallo y rendimiento. el clon morocha presentó el mayor porcentaje de estacas viables y plántulas normales con 80.95% y 73.81% respectivamente. asimismo, el mayor rendimiento se halló con los clones morocha (13 t/ha) seguido de donoso 2 (7.20 t/ha) y blanca cajamarca (6.80 t/ha). palabras clave: caracterización, rendimiento, plántula, potencial productivo 1 universidad nacional agraria la molina (unalm). facultad de agronomía, departamento académico de fitotecnia. lima, perú. introduction currently, plant genetic resources are essential in sustainable agricultural production, and their conservation and effective use are fundamental for global food security (reveles & velasquez, 2017). therefore, a continuous flow of improved crops and varieties adapted to particular local conditions will be necessary (fao, 2017). there is a positive outlook for production in countries with food diversification programmes or in those that intend to limit the imports of staple foods, especially wheat and rice (fao, 2017). cassava (manihot esculenta crantz) cultivation has high economic and nutritional importance. globally, it is considered as the fourth most important commodity following rice, wheat and corn (mejía, elias-da-silva, mejía, & garcía-dávila, 2015). in addition to direct consumption, it is also used as a raw material in the preparation of concentrates for animals, paper and textiles, biofuels and starch for the food and pharmaceutical industries (beovides et al., 2014; fao, 2013). cassava is a species of the euphorbiaceae family, which is distributed in tropical and subtropical areas of the eguiluz, a.; pinedo, r.; figueroa, c. peruvian journal of agronomy 3(3): 120–125 (2019) 121 world with the greatest proportion in mexico and brazil (fao, 2013). the morphological and genetic diversity of this species has been characterised in terms of cultivated varieties with around 7000 varieties distributed worldwide (inta, 2016). these are mostly morphotypes and are maintained by traditional farmers. cassava is native to the american tropics (inta, 2016). there is a great diversity of ecotypes, which must be collected, protected, characterised and evaluated because they are the basis for the development of improved varieties to ensure the production of food and other requirements (agre et al., 2015; fao; 2017; reveles & velásquez, 2017). in peru, cassava cultivation provides one of the main energy sources and is part of the subsistence strategies of families. a large number of local varieties are distinguished, identified by farmers by a common or singular name based on morphological characteristics, place of origin or names of people who introduced the variety to the community (inta, 2016). morphological characteristics are used to study genetic variability, to identify plants and to conserve genetic resources (fao, 2013; inta, 2016). therefore, characterisation is the first step to improving crops and conservation programmes. in addition to the influence of the particular genotype, the characteristics are strongly influenced by the environment, such as the amount of rainfall, soil fertility and damage due to pests and diseases (inta, 2016). in areas of prolonged drought (which is the major constraint to the success of food crops), cassava (manihot esculenta crantz) is a viable crop choice. however, despite this crop’s rusticity, it is affected by extreme environments (león et al., 2014). the vigour of the cassava stakes as a means of propagation is dependent on genetic, physical, and physiological characteristics (teichert, amaral, & meneghello, 2012). therefore, the viability level of the stakes and their ability to thrive once the roots and leaves have formed are an expression of the clone’s physiological qualities. under this consideration, the potential for yield, stability and pest resistance along with the physiological quality of the mother plant should be taken into account in the selection of stakes. an otherwise perfect genotype cannot express its full potential if a physiologically deteriorated propagation material is used (clayuca, 2006). regarding the agronomic character of yield per unit area and with particular respect to the performance of tuberous roots, peso and vasquez (1995) believe that not only is performance the most important characteristic, but it also constitutes a clonal characteristic, although it is influenced by environmental and cultural factors. currently, manuals are available for cassava characterisation, such as the descriptors published by the international board for plant genetic resources in 1983, the brazilian company of agricultural research in 1998 and the international institute of tropical agriculture in 1998. most cassava research prioritises peruvian jungle agroecosystems and is focused on studies with dualpurpose clones (roots and fodder); however, in this paper, the research focuses on the identification of promising high yield clones for coastal conditions. according to minagri (2018), the departments of amazonas, junín, loreto, san martín and ucayali account for more than 70% of peruvian cassava production. however, the highest yields are obtained in the coastal departments of lima (34.5 t/ha), arequipa (15.04 t/ha), ancash (20.3 t/ha) and ica (25 t/ha) compared with the national yield of 12 t/ha in recent years. cassava can withstand prolonged periods of drought in which most other food plants perish (clayuca, 2006). this makes it very useful in regions where annual rainfall is low or where there is an irregular seasonal distribution of rainfall (grace, 1977). in addition, it has enormous potential for productivity in poor soils owing to its plasticity and tolerance against pests and diseases (fao, 2013; inta, 2016). therefore, the main objective of this research was to evaluate the physiological and agronomical performance of cassava clones in the national agrarian university–la molina (unalm) roots and tubers programme (pipsrt) with high yield potential and quality root production. materials and methods the study was conducted between october 2016 and october 2017 in the pips-rt of the unalm located in la molina, lima, at 241 m above sea level (the geographic coordinates are 12º05. 099 ’s, 76º56.974’ so). the plant material under investigation is presented in table 1. the material was selected due to the use of the main clones in the peruvian coastal area. the characteristics of the experimental area were as follows: code name origin vegetative period (months) t1 rosado cañete cañete 9–10 months t2 donoso 1 lima. pips-rt late, older than 12 months t3 donoso 2 lima. pips-rt late, older than 12 months t4 morocha lima. pips-rt semi-early 7–8 months t5 negra mochera lima. pips-rt semi-early 9–10 months t6 blanca cajamarca cajamarca semi-early 9–10 months. table 1. code, name, origin and vegetative period of the genetic material used in the experiment. physiological and agronomic characterisation of six cassava clones (manihot esculenta crantz) september december 2019 122 groove length (6 m), distance between rows (1.2 m), distance between strokes (1 m), number of stokes by groove (5), number of stakes per stroke (1), number of grooves by plot (4), plot area (28.8 m2), street width (1.50 m) and total experimental area (705.6 m2). the genetic material under study is part of the collection of cassava varieties and morphotypes of the pips-rt of the unalm. through previous characterisation work conducted by the pips-rt, the best-performing material clones were screened in terms of yield, physiological quality of stakes for propagation in crop fields and resistance to biotic and adverse abiotic factors. the selected clones were characterised by the presentation in the roots of the following pulp colours: rosé cañete (yellow group 8d), donoso 1 (white group 155d), donoso 2 (white group 155d), morocha (yellow–orange group 19c), black mochera (white group 155d) and blanca cajamarca (white group 155d) (table 1). agronomic husbandry the vegetative propagation used fresh stakes from mature plants (villagómez & rodríguez, 1993). the stake diameter was related to the diameters of the central cylinder and the stem; the stakes had a diameter between 1 and 1.5 cm (inia, 2014) and a length of 8–10 cm, and to ensure rooting, they had 3–5 buds located in the nodes. the stake was inclined for planting; this encouraged horizontal root development to prevent damage while developing. hilling was conducted 4 months after planting so that the reserve roots could develop a favourable shape. at the time of hilling, approximately 50 g/stroke of fertiliser was applied at a dose of 20-20-20 npk, according to the recommendation of león et al. (2014). cassava is recognised for its high rusticity and tolerance to biotic and abiotic stresses, including low soil fertility (alzate, 2009; jaramillo, 2002). therefore, it was considered appropriate to use a low level of fertilisation to observe the expression of the clones under these conditions. weeding was performed manually according to requirements. the first weeding took place 30 days after planting. a gravity irrigation system was used depending on the availability of water and the crop’s requirements. in the first stage, the crops were watered twice a week and from the third month once a week or depending on the conditions. the first irrigation was at approximately 15 days. cassava is not very demanding on soil moisture content, but a prolonged moisture deficit has a negative impact on growth and yield (inta, 2016). although cassava is tolerant to pest attack compared with other crops, its production does not occur in critical periods; therefore, evaluations of pests and diseases were conducted once a week with remedial applications made according to the incidence. after 9 months, the storage root was harvested. one of the last activities in cassava production consists of collecting the useful part of the plant, in this case the reserve root. the optimal degree of humidity to facilitate the extraction of the tuberous root (while avoiding tearing the bark) was taken into consideration during this stage (inta, 2016). some characteristics indicative of maturity were also observed, such as the fall of the basal leaves and the cracking of the soil at the base of the plant stem. the crop was harvested manually depending on the maturity of the clone, and prior to harvesting, the crop was watered. evaluation variables physiological quality variables the percentage of stakes installed in the experiment was evaluated and then analysed. the number of viable stakes, number of normal seedlings and stake survival percentage of the six clones were evaluated. the criteria for the evaluation of any abnormal plants were size, colour and leaf shape, as well as deformations compared with normally developed plants in each block according to the cassava descriptor (biodiversity international, 2011). additionally, some evaluations that are not included in the descriptors were used. agronomic variables the variables that were evaluated were plant height, stem diameter and yield. the physiological and agronomic variables used in the evaluation of the experiment allowed the best clones to be defined. noerwijatia and budionob (2015) applied stress tolerance and susceptibility indices and average productivity and performance stability indices as selection criteria for identifying the best clone at different heights. data analysis an analysis of variance was conducted using infostat (2017 version) statistical software. duncan’s multiple range test was employed to compare the means of the treatments at a significance level of 5%. results seedling emergence in the analysis of variance, no significant statistical differences were found in the variables for the treatments for the viable stakes and normal seedlings (table 2). twenty-two days after the start of the experiment, an average of 63% sprouting had occurred. the results indicate that 14% of the plants were abnormal. by comparing the mean variables of the viable stakes via duncan’s multiple range test, the best response was obtained from the morocha clone, which had 73.81% viable stakes when forming rootlets and primary leaves; the lowest percentage of viable stakes was with the donoso eguiluz, a.; pinedo, r.; figueroa, c. peruvian journal of agronomy 3(3): 120–125 (2019) 123 2 clone at just 52.38%. the morocha, blanca cajamarca, donoso 1 and rosado cañete clones were statistically identical; however, they are statistically different of the negra mochera and donoso 2 clones (table 3). of a total of 168 stakes planted, 71.4% exhibited good development and reached a height of between 70 and 100 cm. figure 1 shows that the percentage of plants in vegetative growth was between 58.2% in the negra mochera genotype and 83.2% in the donoso 1 genotype. the analysis of variance for yield reveals significant differences between the treatments, whereas for plant height, no significant statistical differences were found. however, highly significant statistical differences were found for stem thickness. the coefficients of variation for the three variables were 32,019, 13,095 and 5,661, respectively (table 4). duncan’s multiple range test for the comparison of means for yield (table 5). there were significant differences between the clones; the morocha clone produced a higher average yield that reached 13.00 t/ha, which was superior to the others. clones t3 and t6 were not significantly different with yields of 7.20 and 6.80 but were better with t2, t5 and t1 treatments. treatments t2, t5 and t1 with 5.93, 5.50 y 5.03 t/ha respectably, did not result in significant differences, but they were less than other treatments. the highest plant height was demonstrated with t5 followed by t4 and t3; however, they were not statistically different from the other treatments. with respect to stem diameter, the highest mean was obtained from the t5 treatment followed by t3 and t1. this was statistically different from the other treatments. at 63 days after sowing (dds), a 60% yield was achieved along with the emergence of stems. in this regard, the inta (2016) mentions that this stage includes the time from sowing until 60 dds. figure 1. percentage of stake survival of cassava clones in the final field sources of variation mean squares gl viable sprouting (%) normal plantlets (%) treatment 5 188.21 162.28 block 2 79.37 121.88 error 10 70.01 62.36 total 17 cv % 11.28 12.1 table 2. analysis of variance of physiological variables in the percentage of viable stakes and normal seedlings. * 0.05 probability significance, ** 0.01 probability significance table 3. duncan’s multiple range test for viable stakes (%) and normal seedling (%) clones viable sprouting normal plantlets morocha 80.95 a 73.81 a blanca cajamarca 79.76 a 70.24 a donoso 1 77.38 a 66.67 a rosado cañete 76.19 a 65.48 a negra mochera 71.43 ab 63.10 ab donoso 2 59.52 b 52.38 b note: different letters indicate statistically significant differences, according to duncan’s multiple range test (p ≤ 0.05). table 4. analysis of variance for yield (t/ha), plant height (cm) and stem diameter (cm) f.v. gl means squares yield plant height stem diameter block 2 1.694 279.243 0.024 treatment 5 25.786* 636.23 0.302 ** error 10 5.381 1209.315 0.025 total 17 cv % 32.019 13.095 5.661 * 0.05 probability significance, ** 0.01 probability significance. table 5. duncan’s multiple range test for yield, plant height and stem diameter t yield t/ha t plant height (cm) t stem diameter (cm) t4 13.00 a t5 282.500 a t5 3.233 a t3 7.20 ab t4 278.967 a t3 3.033 ab t6 6.80 ab t3 263.993 a t1 2.967 abc t2 5.93 b t6 263.433 a t4 2.633 b cd t5 5.50 b t1 263.067 a t6 2.533 cd t1 5.03 b t2 241.467 a t2 2.433 d means with a common letter are not significantly different (p > 0.05) t = treatment physiological and agronomic characterisation of six cassava clones (manihot esculenta crantz) september december 2019 124 clones t4, t5 and t6 have promising potential for future improvement or in propagation plans for commercial purposes because they did not exhibit brittle roots, and they responded with yields above the national average under conditions of low fertilisation. these results could be useful in the implementation of sustainable agronomic management of cassava on the peruvian coast. references agre, a.p., dansi, a., rabbi, i.y., battachargee, r., dansi, m., melaku, g., augusto, b., sanni, a., akouegninou, a., & akpagana, k. (2015). agromorphological characterization of elite cassava (manihot esculenta crantz) cultivars collected in benin. international journal of current research in biosciences and plant biology, 2(2): 1–14. retrieved from https://core.ac.uk/download/ pdf/132668705.pdf alzate, a. (2009). variabilidad genética y grado de adopción de la yuca (manihot esculenta crantz) cultivada por pequeños agricultores de la costa atlántica colombiana. universidad nacional de colombia facultad de ciencias agropecuarias, palmira colombia. 63 p. beovides, y., milián, m., coto, o., rayas, a., basail, m., santos a., lópez, j., medero, v., cruz, j., ruíz, e., & rodríguez, d. (2014). caracterización morfológica y agronómica de cultivares cubanos de yuca (manihot esculenta crantz). cultivos tropicales, 35(2), 43-50. retrieved from http:// www.redalyc.org/articulo.oa?id=193230070006 clayuca (consorcio latinoamericano y del caribe de apoyo a la investigación y al desarrollo de la yuca–clayuca). (2006). aspecto tecnológicos sobre producción de yuca. ciat. colombia. 28 p. fao (organización de las naciones unidas para la alimentación y la agricultura). (2013). ahorrar para crecer: la yuca, guía para la intensificación sostenible de su cultivo. retrieved from http:// www.fao.org/ag/save-and-grow/cassava/pdf/faoyuca.pdf fao (organización de las naciones unidas para la alimentación y la agricultura). (2017). perspectivas alimentarias, resumen de mercado octubre de 2016. roma-italia. p. 7. retrieved from http://www.fao.org/publications/card/es/ c/1bbcf185-eff5-4647-9379-97799849d912/ grace, m.r. (1977). colección fao: producción y protección vegetal. n°3. elaboración de la yuca. roma, italia. p. 11. inga, h., & lópez, j. (2001). diversidad de yuca (manihot discussion cassava stakes were used as a means of propagation due to the species’ allogamous condition and its heterozygous genetic constitution (jaramillo, 2002). a 60% yield and the emergence of stems were achieved at 63 dds. according to the inta (2016), this stage includes the time from sowing until 60 dds; the stakes when sprouting first form roots (5–7 dds), and then the stems and leaves develop. cassava tolerates a wide temperature range; however, this factor may affect sprouting (inta, 2016). stakes cut from healthy stems that are free of pests and diseases have higher sprouting and root production rates (fao, 2013). the tangible result of the physiological quality of the stake is in its ability to sprout and produce a vigorous plant (clayuca, 2006). in cassava, the height and stem diameter are important characteristics because they are the media used for vegetative multiplication. in the experiment, height varied from 241.67 to 282.50 m, and clone t4 achieved the best expression in this variable. cassava is a perennial shrub with a sympodial branching habit whose height can range from 3 to 5 m (the average is 3 m). it is characterised by the sprouting of the stakes, which first form roots (5–7 dds) before the stems and leaves develop. the morocha clone (t4) achieved the best yield (13 t/ha). it should be noted that the national average yield from the morocha clone does not exceed 12 t/ha, which could prove interesting for future work. in this regard, when evaluating the agronomic behaviour of eight clones, peso and vasquez (1995) found a yield that varied between 13.85 and 26.76 t/ha, whereas perdomo et al. (2008) identified potential root yields for fresh consumption of between 14.00 and 18.00 t/ha. the best-performing clone may have a promising future since it is within the global average of performance. according to fao (2017), the average global yield has increased by almost 1.8% per year during the last decade, reaching 12.8 t/ha. regarding plant height in the test, clones t4 and t5 reached 278.97–282.50 cm, which was a better performance than the other clones; however, they were not statistically different. when evaluating plant height, león et al. (2014) revealed significant differences (p < 0.05) among clones (2.17–2.62 m). the cassava shrub can measure 1.5–4.0 m high (inta, 2016); however, under the conditions in loreto, the cassava plant reaches 2–3 m in height (inga & lópez, 2001). conclusions the morocha clone demonstrated the highest percentage of sprouting behaviour and normal plantlets at 80.95% and 73.81%, respectively. the highest yield was also found with the morocha clone (13 t/ha) followed by the donoso 2 (7.20 t/ha) and the blanca cajamarca (6.80 t/ha) varieties. eguiluz, a.; pinedo, r.; figueroa, c. peruvian journal of agronomy 3(3): 120–125 (2019) 125 esculenta crantz) en jenaro herrera, loreto–perú. instituto de investigaciones de la amazonía peruana (iiap), loreto, perú. 47 p. retrieved from http:// www.iiap.org.pe/upload/publicacion/st028.pdf inia (instituto nacional de investigación agraria). (2014). tecnología de producción de yuca: selección, preparación y desinfección de estacas de yuca. hoja divulgativa n°16.iquitos. retrieved from https:// www.inia.gob.pe/ inta (instituto nacional de innovación y transferencia en tecnología agropecuaria). (2016). cultivo de yuca (manihot esculenta crantz) en costa rica. san josé, c.r. 87 p. león, r., pérez, m., gutiérrez, m., rodríguez, a., fuenmayor, f., & marín, c. (2014). caracterización ecofisiológica de cuatro clones de yuca del banco de germoplasma del inia-ceniap. agronomía tropical, 64, 97–105 mejía j., elias-da-silva r., mejía k., & garcía-dávila c. (2015). caracterización morfológica y genética de etnovariedades de la yuca manihot esculenta cratnz, en seis localidades de la cuenca baja del río ucayali–perú. folia amazónica, 24, 71–82. minagri (ministerio de agricultura y riego). (2018). boletín estadístico mensual: el agro en cifras noviembre 2018 (online). sistema integrado de estadística agraria (siea). retrieved from h t t p : / / s i e a . m i n a g r i . g o b . p e / s i e a / ? q = n o t i c i a s / boletin-estadistico-mensual-el-agro-en-cifrasnoviembre-2018 noerwijatia, k., & budionob, r. (2015). yield and yield components evaluation of cassava (manihot esculenta crantz) clones in different altitudes. energy procedia, 155–161. jaramillo, g.. (2002). recursos genéticos de manihot en el centro internacional de agricultura tropical (ciat). in b. ospina & h. ceballos (eds.), la yuca en el tercer milenio: sistemas modernos de producción, procesamiento, utilización y comercialización (pp. 271–294). cali, colombia: centro internacional de agricultura tropical (ciat). https://hdl.handle.net/10568/54117 peso, n., & vásquez, w. (1995). comparativo de rendimiento de ocho clones de yuca (manihot esculenta, crantz). folia amazónica, 7 (1-2)–1995 reveles, l.r. & velásquez, r. (2017). patrimonio fitogenético: banco de germoplasma de semillas ortodoxas del campo experimental zacatecas. folleto técnico núm 81. campo experimental zacatecas. cirnoc–inifap, 44 páginas. teichert, s., amaral, f., & meneghello, g.e. (2012). sementes: fundamentos científicos e tecnológicos. tercera edición. pelotas r.s. brasil. 527 p. villagómez v. & rodríguez g. (1993). el cultivo de la yuca (manihot esculenta). ediciones unalm. impresión comercial grafica jara s.r.l. limaperú. p. 5, 30, 36, 42, 66. peruvian journal of agronomy 2 (2): 14 21 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i2.1202 © the authors. published by universidad nacional agraria la molina received for publication: 15 july 2018 accepted for publication: 17 september 2018 morphological and molecular identification of phytophthora cinnamomi rands as a causal agent of crown and root rot in blueberry (vaccinium corymbosum) in peru identificación morfológica y molecular de phytophthora cinnamomi rands como agente causal de la pudrición de corona y raíces en arándanos (vaccinium corymborum) en perú. huarhua, m.; flores, j.; acuña, r.; apaza w.1* * corresponding author. wapaza@lamolina.edu.pe abstract the blueberry production in peru has had rapid growth in the last five years, from having no area of more than 3050 hectares at present. as a result of this increase, many pathologic plant problems have appeared among them phytophthora sp. causing plants with symptoms of wilt, reddish of lower leaves, regressive death, rotting of roots and crown of different production areas of peru. however, there is no identification reported in peru. therefore, this study was conducted in the plant pathology clinic of the universidad nacional agraria la molina with the objective of to identify the species of phytophthora causing crown and root rot isolated from blueberry in peru. four isolates from the blueberry´s field were identified by morphological, molecular and pathogenically features. all the isolates formed corralled-type colonies with hyphal swelling on pda and v-8 agar and produced obpyriform or ellipsoidal sporangia and globes chlamydospores. the internal transcribed spacer (its) of rdna was amplified and sequenced with primer its1/its4 to confirm the identification, all the its nucleotide sequences from isolated obtained were submitted at genbank of ncbi (number accession mh777152 to isolate ar-1, mh777151 to isolate ar-2, mh777150 to isolate ar-4, and mh777149 to isolate ar-3). each one of the sequences showed 100% of homology with sequences of reference of p. cinnamomi in genbank. pathogenicity tests showed symptoms of redness and yellowing and root and crown rot on blueberry cv. biloxi after 30 days of inoculation. finally, concluded that p. cinnamomi is the species causing crown and root rot in blueberry in peru. to our knowledge, this is the first report of p. cinnamomi on blueberry in peru. keywords: phytophthora cinnamomi, blueberries, root rot, crown rot, molecular identification. resumen la producción de arándanos en el perú ha tenido un rápido crecimiento en los últimos cinco años, de no tener área alguna a 3050 hectáreas al presente. como resultado de este crecimiento, han aparecido muchos problemas fitopatológicos entre ellos el ataque de phytophthora sp. causando los síntomas de marchitez, enrojecimiento de hojas basales, muerte regresiva, pudrición de raíces y corona en las diferentes áreas de producción en el perú. si embargo, no existen reportes de la identificación de estos problemas en el perú. por lo tanto, este estudio fue realizado en la clínica de enfermedades de plantas de la universidad nacional agraria la molina con el objetivo de identificar las especies de phytophthora que causan la pudrición de corona y raíces del arándano en el perú. cuatro aislamientos de campos de arándano fueron identificados en forma morfológica y molecular, así como su característica de patogenicidad. todos los aislamientos formaron colonias de tipo coraloide con hinchamientos hifales en medio pda y agar v-8 y produjeron esporangios piriformes o elipsoides y clamidosporas globosas. los espacios intergénicos (its) de rdna fueron amplificados y secuenciados con los iniciadores its1/its4 para confirmar la identificación. todas las secuencias nucleotidas its de los aislamientos fueron enviados al genbank de ncbi (número de accesión mh777152 para el aislamiento ar-1, mh777151 para el aislamiento ar-2, mh777150 para el aislamiento ar-4, y mh777149 para el aislamiento ar-3). cada una de las secuencias mostraron 100% de homología con las secuencias de referencias de p. cinnamomi en el genbank. la prueba de patogenicidad mostro síntomas de enrojecimiento, amarillamiento y pudrición de raíces y corona en plantas de arándano cultivar biloxi después de 30 días de inoculado. finalmente, se concluye que p. cinnamomi es la especie que causa la pudrición de corona y raíces de arándano en el perú. este es el primer reporte de p. cinnamomi en el arándano en el perú. palabra clave: phytophthora cinnamomi, arándanos, pudrición de raíces, pudrición de corona, identificación molecular 1clínica de diagnosis de fitopatología de la universidad nacional agraria la molina, apartado postal 12-056 la molina, lima – perú. huarhua, m.; flores, j.; acuña, r.; apaza w. peruvian journal of agronomy 2 (2): 14 21 (2018) 15 introduction the blueberry belongs to the family of berries and has high growth prospects in the international market, due to its nutritional characteristics, because it contains an essential number of antioxidants. in peru, blueberry start to be cultivated between 2007-2008. by the year 2016, the cultivated blueberry area was estimated at 3050 hectares, with a production of 26.6 thousand tons, which is almost wholly exported (minagri, 2016). the implementation of more areas of blueberry crops is required to satisfy the national and international demand. however, one of the factors that limit this objective is the development of diseases that negatively affect the production. the most important disease is the root rot caused by oomycetes of the genus phytophthora, it produces a gradual decay that eventually, lead to death all the prostrate shrubs (toapanta-gallegos et al., 2017). since 2011, to the plant pathology clinic of the universidad nacional agraria la molina, have arrived blueberry plants of the biloxi variety with symptoms of root rot of different production areas on the coast of peru: chavimochic, lambayeque, chongoyape, cañete, lunahuana, chincha, ica and pisco. symptoms include loss of vigor, yellowing and foliar redness, defoliation and root rot. in the plants with advanced symptoms of the disease, there is rottenness of the neck, regressive death and finally the death of the plants. reports from other countries indicate that this disease caused by phytophthora cinnamomi (polashock et al., 2017). the objective of this research is to identify phytophthora species that affect blueberry crops from different regions of peru, for which it is necessary to perform morphological, molecular and pathogenicity tests to confirm its presence. material and methods a) isolates the isolates of phytophthora were obtained from roots of blueberry plants collected from commercial fields located in cañete, pisco, lima, chavimochic (table 1). the plants showed leaf chlorosis, foliage reddening, dieback, root and crown rot (figure 1). the samples were processed in the plant pathology clinic lab of the universidad nacional agraria la molina. the root and crown were washed thoroughly under abundant tap water. portions of roots and crown with lesion were cut into small pieces, superficial tissue was disinfected in 70% ethanol for 5 seconds after that, each sample was disinfected with 1% sodium hypochlorite solution for 5 minutes and then washed for twice in sterile distilled water (sdw) (dhingra et al., 1995), then dried on sterile paper. crown and root pieces, about 410 mm-wide at the junction of the healthy and necrotic tissue were placed on parb (10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin, and 20 mg benomyl) selective medium. the plates were incubated at 24°c for three days in darkness (erwin and ribeiro, 1996). the pure cultures of the fungus were obtained from a hyphal tip. b) morphological and cultural characterization each isolate on parb was transferred on potato dextrose agar (pda) and v8 juice agar (v-8) to evaluate the growth of characters. petri plates with 20 ml of each medium were inoculated with 7 mm disc cut from mycelium pieces taken from an actively growing colony on parb and incubated at 24 °c for 7 days. each treatment was replicated four times. observations cultural colony pattern were recorded at seven days after inoculation following the descriptions of erwin and ribeiro (1996). sporangia were produced by cutting 5-mm-diameter disks from the advancing margin of a colony grown on pda and v8, floating these disks on 10 ml of 1% non-sterile soil extract solution (w/v) for 4–5 days at 24 °c under continuous light from fluorescent lamps (erwin and ribeiro, 1996). after 72 h, hyphal portions were mounted in koh at 3% and observed microscopically at 400 x magnification (leica dm 1000 led microscope). for each isolate: shape, length (l) and breadth (b) of 50 sporangia were measured, and was calculated length to breadth ratio (l/b). additionally, were taken sporangiophore breadth, chlamydospore diameter, and swelling hyphal measurements. c) pathogenicity test the pathogenicity method was for figure 1. a: blueberry plants with symptoms of yellowing and foliar redness. b: blueberry plants with advanced symptoms of root and neck rot. table 1. isolates of phytophthora sp. obtained from blueberry (vaccinium sp.) from peru isolate source locality ar-1 crown/ roots limacañete ar-2 crown/ roots ica –pisco ar-3 crown/ roots limacañete ar-4 crown/ roots la libertad chavimochic morphological and molecular identification of phytophthora cinnamomi rands as a causal agent of crown and root rot in blueberry (vaccinium corymbosum) in peru may august 2018 16 inoculation of soil. this method is convenient for species of phytophthora, which do not readily sporulate such as phytophthora cinnamomi (drenth and barbara, 2001). for the production of inoculum was boiled a natural substrate of wheat in distilled water for 8 minutes. then the excess water was removed and left to dry then it was bagged 200 gr in polypropylene bag for autoclaving at 121°c for 50 minutes on two consecutive days. after that, the bags were inoculated with two small discs of agar containing mycelium and incubate at 24°c for three weeks. the mixture was shaking every 2 days to obtain an even spread of phytophthora mycelium throughout the substrate. two-month-old blueberry plants were transplanted to a plastic ball with 1 kg of an autoclaved soil mixture of sand, rice husk and peat (in relation 1:1:1). one month later, the plants were inoculated with 20 wheat seeds with mycelium, it was added on the substrate around to the neck into each plant and was covered by the same substrate at 1cm, immediately was put sterile water to the inoculation and was maintained the humidity of the substrate. c) molecular characterization to extract genomic dna the oomycete was cultivated on potato dextrose agar for five days at 25 ° c. the extraction of genomic dna was carried out in triplicate for each evaluated isolation, following the protocol of zelayamolina et al. (2011) with some modifications. a mycelium block about 5 mm in diameter was cut and placed in 1.5 ml eppendorf tubes. five hundred microliters of lysis buffer (100 mm tris hcl, 10 mm ethylenediaminetetraacetic acid [edta], 1m kcl; ph 8.0) was added and mix well with the pestle. then, chloroform/isoamyl alcohol (24:1, v/v) were added, the tube was vortexed until the two phases formed an emulsion and centrifuged for 10 min at 12000 rpm. the supernatant was transferred to a 1.5 ml tube. to precipitate the dna, 1 ml isopropanol was added. the tube was inverted five times and held at −20°c for 15 min. a dna pellet was formed by centrifugation for 10 min at 12000rpm. the supernatant was carefully discarded, and the pellet was washed with 1 ml of 70% (v/v) ethanol. the tube was then placed upside down over a paper towel for 2 hours or until the pellet was dried entirely. the pellet was resuspended in 30 μl of te buffer (100 mm tris/hcl, ph 8.0, 10 mm edta) and stored at -20°c. the dna concentrations were quantitatively determined using the nanodrop spectrophotometer (nanodrop 2000). also, it was qualitatively determined by performing electrophoresis for 30 minutes at 90 v on the agarose gel stained with hydragreen. for the development of the polymerase chain reaction, the universal primers its 1 (tccgtaggtgaacctgcgg) and its 4 (tcctccgcttattgatatgc) were used to amplify the rdna nuclear region of internal transcribed spacers (its), including 5,8s rdna, as described by white et al. (1990). the total 25ul reaction mixture contained 1 ul of genomic dna of 50 ng, 4 ul of 10x pcr buffer (actaq ™), 2 ul of each 2.5 mm dntps (actaq ™), 0.125 ul (5 u) of taq enzyme dna polymerase (actaq ™) and 0.5 ul of each primer of 20 mm. the amplifications were carried out with a thermocycler (thermo scientific) with the following parameters: initial denaturation of 94 °c during 2 min; followed by 35 cycles of denaturation at 98 °c for 1 min, annealing at 58 °c for 1 min, and elongation at 72 °c for 2 min, and a final extension at 72 °c for 10 min. the pcr products have a size of 850 bp. these were separated by electrophoresis with 0.5x tae buffer in 2% agarose gels for 30 min at 90v and stained with hydragreen. the observation of the fragments was using a transilluminator of ultraviolet light (uvp brand). 100bp dna ladder (promega) was used as a reference. fragments amplified by pcr were sequenced in both directions in macrogen-korea and compared with the gene bank database of the national center for biotechnology information (ncbi). d) analysis of sequences and phylogenetic analyses the sequences obtained were assembled, aligned and edited using codon code aligner 8.0.2 program (codoncode aligner, usa). all sequences obtained in this study were compared with those on genbank using “blastn” (http://www.ncbi.nlm.nih.gov/blast/). clustalw nucleotide sequence alignments (thompson et al., 1994) and neighbor joining (nj) clustering analysis were performed using mega5. nj trees employed the kimura-2-parameter (k2p) distance model (kimura, 1980) with pairwise deletion of missing sites and nodal support was estimated using 500 bootstrap replicates. some sequences of genus phytophthora obtained from the genbank were considered in the phylogenetic analysis: genbank ay995407.1 of phytophthora megasperma, genbank kp676165.1 of phytophthora citrophthora and genbank mf536298.1, genbank kf271786.1, genbank kc478665.1, genbank ef055303.1, genbank ay302149.1, genbank mg560191.1, genbank kc478663.1, genbank kc184904.1, genbank kf559323.1, genbank dq173250.1 of phytophthora cinnamomi. fusarium oxysporum (genbank ay387701.1) was used as an outgroup to phylogenetic analysis. results and discussion morphological and cultural characterization based on cultural characteristics of the colony and morphometric evaluation of asexual reproduction structures. phytophthora cinnamomi was identified in the four isolates. cultural patterns of ar-1, ar-2, ar-3, ar-4 isolates obtained on pda medium formed colonies with a cottonlike aspect, white colored and growth pattern rose-type. in the v-8 medium, the colonies were similar to those obtained in pda medium, but with the less marked rosette growth pattern (figure 2). this typical growth is described huarhua, m.; flores, j.; acuña, r.; apaza w. peruvian journal of agronomy 2 (2): 14 21 (2018) 17 by mehrlich (1935), who mentions that the colony of phytophthora cinnamomi in pda has a pattern similar to the petals of a rose or camellia flower, further references that this morphology feature is unique for this species. thus, this characteristic of rose -type growth is typical of p. cinnamomi according to erwin and ribeiro (1996). according to the morphological characteristics in pda and v-8 media, the growth of coenocytic hyphae typical of pseudo-fungi of the family pythiaceae was evidenced. besides, a coralloid mycelium, with abundant hyphal swellings and rounded chlamydospores with a thin wall forming clusters. likewise, in the soil solution, the formation of numerous zoosporangia was observed from an obpyriform to an ellipsoid shape with presence of an a b figure 2. phytophthora cinnamomi isolates exhibiting rosaceous colony morphology on potato dextrose agar (a) and v-8 medium (b). figure 3. morphological feature dyed with cotton blue of phytophthora cinnamomi isolated from blueberry. obpyriform to ellipsoids sporangia without papillae with long pedicels (a-b), coralloid type mycelium with hyphal swellings (c), chlamydospores (d). a b c d apical pore, a wide pore without papilla formation, which at the time of maturity released numerous zoospores, the thin, long and generally simple sporangiophore (no branching), no oospore formation was observed (figure 3). these characteristics are similar in the four isolates and are related to phytophthora cinnamomi (erwin and ribeiro, 1996; huaman et al., 2015; drenth and barbara, 2001; ho and zentmyer, 1977; larach et al., 2009). regarding the characteristics in measure in the four isolations were the following: the zoosporangia oscillated 49-57 x 35-40 um in length and width respectively, the ratio length/width (l/a) varied between 1.36 to 1.43, with a pedicel thickness from 3 to 3.5 um, chlamydospores were between 40-43 um in diameter and hyphal swellings (hh) from 22 to 28 um in each of the isolates (table 2). due to the characteristics of the asexual structures of these fungi, it was shown that they were phytophthora cinnamomi, which conform to those described by erwin and ribeiro (1996), gallegly and chuanxue (2008), waterhouse and waterston (1996) and polashock et al. (2017) for the species phytophthora cinnamomi. pathogenicity test all the isolates identified as phytophthora cinnamomi in the morphological and molecular tests turned out to be pathogenic in the blueberry culture. likewise, in the parb medium, it was possible to re-isolate the pathogen under study from the diseased tissues, which shows the success of koch’s postulate. the symptoms observed in the inoculated plants began to be found after 30 days with foliar yellowing after 30 days, and foliar redness after 45 days; also, rot symptoms were observed at the root and neck levels of the plant (figure 4). on the other hand, the control plants did not show any symptoms in the foliar system, at the level of the neck or roots. these symptoms coincide with the symptoms developed in blueberry fields in the evaluated areas of peru, as well as with the symptoms described for phytophthora cinnamomi in different parts of the world (lan et al., 2016; shands et al., 2016). molecular identification before the revolution of molecular biology, fungi and oomycetes were identified morphologically. nowadays with the advance of technology, the study of microorganisms through their dna has many applications including the identification of species, the observation of the relationships between groups of organisms, even the detection of pathogens in water and soil without resorting to isolation in culture medium (cooke et al., 2000). morphological and molecular identification of phytophthora cinnamomi rands as a causal agent of crown and root rot in blueberry (vaccinium corymbosum) in peru may august 2018 18 figura 4. pathogenicity test on blueberry plants inoculated with phytophthora cinnamomi. chlorosis (a), foliar reddening (b), crown and root rot (c) and root system without inoculation (d). table 2. morphometric characteristics of 4 isolates of phytophthora cinnamomi isolated from blueberry plants with symptoms of root rot. isolates sporangia (um) l x b ratio l/b zoosporangia breadth (um) chlamydospore (um) sh (um) colony diameter (mm) a 3 days-24ºc pda v-8 ar-1 57 x 40 (38-75 x 28-50) 1.43 3.1 40 (30 50) 28 39 45 ar-2 49 x 35 (43-63 x 28-40) 1.40 3.3 40 (33 -60) 22 38 43 ar-3 50 x 37 (43-55 x 33-40) 1.36 3.0 43 (30 – 53) 22 37 42 ar-4 52 x 38 (38-60 x 25-48) 1.37 3.5 40 (35 – 50) 23 39 44 l/b =length/breadth zoosporangia sh = swelling hyphal the extraction of genomic dna from the ar-1, ar2, ar-3, and ar-4 isolates, morphologically identified as phytophthora cinnamomi, had a yield higher than 50 ng for each sample and the quality was more significant than 1.9 in the 260/230 ratio. the quality of the extraction can be observed in figure 5. the polymerase chain reaction (pcr) technique is a system that allows obtaining several million copies of dna sequence to facilitate the diagnosis of a particular species (rodríguez and barrera, 2004). with the use of its primers, a 900 bp fragment was amplified in the four isolates (figure 6). they were achieved without the presence of nonspecific bands. so, they were sent directly to sequencing. the nucleotide sequences were edited using codon code aligner. the sequences edited were aligned by blast with the data of p. cinnamomi sequences stored in genbank. the percentage of similarity was at 100% to p. cinnamomi to each one of the sequences ar-1, ar-2, ar3, and ar-4. by comparing and aligning the sequences of the ar-1, ar-2, ar-3 and ar-4 isolates with other p. cinnamomi sequences from different cultures, from different regions, and with other species of the genus phytophthora, a phylogenetic tree was created (figure 7). the tree was inferred using the maximum likelihood method based on the jukes and cantor (1969) model). the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved 21 nucleotide sequences. the ar-1, ar-2, ar-3 and ar-4 sequences (shaded in gray) in figure 6 were grouped in the same clade with the p. sequences from different cultures. likewise, the sequences of the isolates ar-1, ar-2, ar-3, and ar-4 were separated in the distance from p. megasperma, p. infestans, p. citrophthora and had greater separation with fusarium oxysporum as an external group (figure 7). these results corroborate the identification of the isolates as part of the species p. cinnamomi. when comparing the its nucleotide sequences region of the four blueberries phytophthora cinnamomi isolates (ar-1, ar-2, ar-3, and ar-4), they all showed the same nucleotide sequence; therefore, we can infer that these isolates corresponded to clones of the same isolation which huarhua, m.; flores, j.; acuña, r.; apaza w. peruvian journal of agronomy 2 (2): 14 21 (2018) 19 figure 5. agarose gel electrophoresis results of genomic dna extracted from blueberry samples. m: molecular -weight markers (high ranger 1kb dna ladder, norgen), ar-1, ar-2, ar-3 y ar-4 isolates from cañete, pisco, cañete and chavimochic, respectively. figure 6. result of gel electrophoresis of pcr product using its 1/its4 primers. m: molecular -weight markers (100bp dna ladder, promega), ar-1, ar-2, ar-3 y ar-4 isolates from cañete, pisco, cañete and chavimochic, respectively. is present in the four provinces sampled (cañete, pisco, lima, and chavimochic). the sequence was deposited in the genbank gene bank with accession number mh777152 to isolate ar-1, mh777151 to isolate ar-2, mh777150 to isolate ar-4, and mh777149 to isolate ar-3. similarly, according to the grouping arrangement in the tree, it is concluded that there are no genetic differences between the isolates of other p. cinnamomi from peru for different populations of p. cinnamomi from other countries. this result corroborates the highly conserved nature of the its region for the species p. cinnamomi, and the low efficiency of the endpoint pcr technique to discriminate different populations of the same species to establish patterns that explain processes of diversification (garzon, 2013). conclusions. in conclusion, from morphological, cultural, molecular and pathological analyses, the causal agent of root and crown rot causing wilting and decline of blueberry trees was identified as phytophthora cinnamomi. to our knowledge, this is the first report of p. cinnamomi on blueberry in peru. acknowledgements dr. blas and joel flores of the institute of biotechnology (ibt) of the national agrarian university la molina. references cooke, d. e., drenth, a., duncan, j. m., wagels, g. morphological and molecular identification of phytophthora cinnamomi rands as a causal agent of crown and root rot in blueberry (vaccinium corymbosum) in peru may august 2018 20 figure 7. molecular phylogenetic analysis by maximum likelihood method of ar-1, ar-2, ar-3 and ar-4 isolates belong to phytophthora cinnamomi group. and brasier, c. m. 2000. a molecular phylogeny of phytophthora and related oomycetes. fungal genetics and biology, 30(1): 17-32. https://doi.org/10.1006/ fgbi.2000.1202 dhingra, o. and sinclair, j. 1995. basic plant pathology methods, crc press, u.s.a. second edition. 435 p. drenth, a. and barbara, s. 2001. practical guide to detection and identification of phytophthora. crc for tropical plant protection 1:1-41. erwin, d. c. and ribeiro, o. 1996. phytophthora diseases worldwide, aps press, u.s.a. 562 p. gallegly, m. e. and chuanxue hong. 2008. phytophthora identifying species by morphology and dna fingerprints. aps press, u.s.a. 158 p. garzon, n. 2013. caracterización e identificación molecular de hongos de suelo aislados de los páramos de guasca y cruz verde, cundinamarca – colombia. pontificia universidad javeriana. tesis de pregrado. ho, h., and zentmyer, g. 1977. morphology of phytophthora cinnamomi. mycologia 69(1):701-713. doi: 10.2307/3758860 huaman, n., valeriano, j., and granados, e. 2015. huarhua, m.; 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(113):1-2. drenth, a. and barbara, s. 2001. practical guide to detection and identification of phytophthora. tropical plant protection, 1-41. zelaya-molina, l. x., ortega, m. a. and dorrance, a. e. 2011. easy and efficient protocol for oomycete dna extraction suitable for population genetic analysis. biotechnology letters, 33(4), 715-720. https://doi. org/10.1007/s10529-010-0478-3 peruvian journal of agronomy 3(3): 112–119 (2019) issn: 2616–4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1251 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 06 november 2018 accepted for publication: 15 november 2019 genetic variation of maiden lily (lilium rubellum baker) within and among three natural populations in fukushima prefecture in japan variación genética del lirio rosa (lilium rubellum baker) dentro y entre tres poblaciones naturales de la prefectura de fukushima en japón castro, m.(1)*; nishikawa, t.(2) *corresponding author: rcastro@lamolina.edu.pe abstract lily (genus lilium) is one of the most important flowers for bulb and cut flower production in the world. lilium rubellum baker is a native species of japan that exhibits early flowering, is pink coloured, and possesses a pleasant fragrance, which makes it an important genetic resource for breeding lilies. in this work, the genetic diversity of l. rubellum was studied among three natural populations located in fukushima prefecture, japan, which have been designated as the mt. azuma, nango and atsushio-kano populations. a total of 31 accessions collected in 1997 were analysed using random amplified polymorphic dna (rapd) markers. eighteen decamer primers produced 98% polymorphic rapd bands; furthermore, 11 of the 18 primers produced 10 or more polymorphic bands with a mean per primer polymorphic information content of 0.382. among the three populations, the mean shannon index, nei´s gene diversity and percentage of polymorphic loci were 0.2749, 0.4099 and 76.7%, respectively. these data revealed that there is a high genetic diversity within all the populations. analysis of the three genetic diversity indices within populations showed that the nango population had the highest genetic diversity, whereas the atsushio-kano population had the lowest. an unweighted pair group method with arithmetic mean dendrogram based on jaccard´s similarity coefficient was constructed, and the three populations of l. rubellum collected in the fukushima prefecture were clearly differentiated at 0.61 similarity index. the nango and mt. azuma populations were genetically closer than the atsushio-kano population. the analysis of molecular variance showed a 29.53% variance among the populations. this study revealed that there is high genetic diversity within populations and moderate genetic diversity among the three natural populations of l. rubellum baker in the fukushima prefecture of japan. keywords: genetic diversity, rapd marker, lilium rubellum resumen el lirio (género lilium) es una de las flores bulbosas de corte más importantes en el mundo. lilium rubellum baker es una especie nativa de japón, produce una floración muy temprana de color rosado, y una agradable fragancia, por lo que esta especie es un importante recurso genético para el mejoramiento genético de los lirios. en este trabajo se estudió la diversidad genética de l. rubellum entre tres poblaciones naturales de la prefectura de fukushima en japón: mt. azuma, nango y atsushio-kano. se analizaron un total de 31 accesiones mediante la prueba de adn polimórfico amplificado aleatorio (rapd). 18 decámeros cebadores produjeron 98% de bandas polimórficas, 11 de estos 18 cebadores producen 10 o más bandas polimórficas con una media por cebador de contenido de información polimórfica (pic) = 0.382. la media del índice de sahnnon ho = 0.2749, de la diversidad genética de nei he = 0.4099 y el porcentaje de loci polimórficos = 76.7 revelaron que existe una alta diversidad genética dentro de todas estas poblaciones. los análisis de los tres índices de diversidad genética intrapoblacional, mostraron que la población de nango tenía la mayor diversidad genética y la población de atsushio-kano la más baja. se construyó un dendrograma upgma basado en el coeficiente de similitud de jaccard y con un índice de similitud de 0.61, las tres poblaciones de l. rubellum recolectadas en la prefectura de fukushima fueron claramente diferenciadas. las poblaciones de nango y de monte azuma estaban más cercanamente relacionadas genéticamente que con la población de atsushio-kano. con el análisis de la varianza molecular (amova) se observó que existe un 29,53% variación interpoblacional. este estudio reveló que existe una alta diversidad genética dentro de las poblaciones y una moderada diversidad genética entre las tres poblaciones naturales de l. rubellum baker en la prefectura de fukushima en japón. palabras clave: diversidad genética, marcador rapd, lilium rubellum 1 universidad nacional agraria la molina (unalm). jardín botánico “octavio velarde núñez”. departamento académico de biología. av. la molina s/n, lima 12, perú. apartado postal: 12-056, perú. e-mail: rcastro@lamolina.edu.pe 2 genebank national institute of agrobiological sciences 2-1-2 kannondai, ibaraki, 305-8602, japan. e-mail: tomotaro@affrc.go.jp castro, m.; nishikawa, t. peruvian journal of agronomy 3(3): 112–119 (2019) 113 introduction ornamental plants are used by humans because of their beauty, symbolic significance (mendonca de carvalho, 2011), colour (kaufman & lohr, 2008), fragrance and therapeutic (matsuo, takaesu, & asano, 2008) and emotional value. the lily has become one of the most important ornamental plants grown for its flower bulbs and cut flowers worldwide over the last 50 years (van tuyl & arens, 2011). furthermore, lilium species can have pharmaceutical applications since they contain steroidal saponins, glycoalkaloids and phenolic glycerides as well as antioxidants (mammadov, deniz, rakhimzhanova, kılınçarslan, & mammadov, 2017). the centre of origin of the lilium genus is the himalayan region in asia (de jong, 1974). this genus has about 100 species grouped into seven sections distributed across the northern hemisphere extending as far south as the southern asia (van tuyl et al., 2011). japan is known to be rich in lilium species. fifteen species are distributed across all prefectures of japan, and japanese lilium species have played an important role in the development of major important cultivars of eastern lily, asiatic hybrids and oriental hybrids (okasaki, asano, & oosawa, 1994). however, lilium species are also under threat of genetic erosion, requiring increased attention and efforts to conserve lilium germplasm (yi, lee, chung, lee, & lim, 2013). lilium rubellum baker grows wild in the mountainous region of northeastern honshu, japan (mcrae, 1998; shimizu, 1971). it flowers early and has an attractive pink colour and pleasant fragrance (lim & van tuyl, 2007). it is easily propagated by seed and is cultivated in the fukushima prefecture (okasaki et al., 1994). the desirable quality of this delicate, pink-coloured lily is that it can hybridise with other lilies of the section archelirion, and their offspring are referred to as oriental hybrids, one of the popular variety groups of lilies (okasaki et al., 1994). understandably, l. rubellum is an important germplasm resource for breeding lilies, and evaluating its genetic diversity will provide valuable understanding of distinct populations. only seven lilium species have had their genetic diversity assessed using molecular techniques (arzatefernández, miwa, shimada, yonekura, & ogawa, 2005; huang et al., 2009; ikinci & oberprieler, 2010; persson, lundquist, & nybom, 1998; wen & hsiao, 2001; wu et al., 2015). one of the first techniques used in order to assess genetic diversity was random amplified polymorphic dna (rapd) markers (williams, kubelik, livak, rafalski, & tingey, 1990). this technique is based on the amplification of discrete dna segments by polymerase chain reaction (pcr) using small size oligonucleotide primers of random design (halward, stalker, larue, & kochert, 1992; sharma, dawson, & waugh, 1995) currently, there are many other, more modern molecular techniques, but rapd markers are still useful in large-scale genetic studies of diverse populations (velascoramírez, torres-morán, molina-moret, de jesús sánchezgonzález, & santacruz-ruvalcaba, 2014). despite its low resolving power, the simplicity and low cost of agarose gel electrophoresis has made the use of rapd markers prevalent. the markers are quick, easily generated by pcr, and do not require prior sequence information (idrees & irshad, 2015). furthermore, their effectiveness in lilium was established by yamagishi (1995), who developed rapd markers for 13 species. in this study, rapd markers were used (i) to assess the genetic differentiation among populations of l. rubellum in the prefecture of fukushima, japan, and (ii) to estimate the levels of genetic diversity within l. rubellum populations. this information will be useful in planning conservation and breeding programmes of l. rubellum. materials and methods plant materials leaf samples of 31 individuals of l. rubellum were collected from three different sites in fukushima prefecture (figure 1) located in the north central region of honshu island, japan: 1) mt. azuma-kofuji, fukushima city (altitude 1,300 masl; figure 2); 2) atsushio-kano village (currently kitakata city; altitude, 500 masl; figure 3); and 3) nango village, figure 4, which consisted of aburakubo (altitude, 700 masl), shimoyama (altitude, 600 masl), misawa (altitude, 600 masl) and nagachisawaguchi (currently minamiaizu town; altitude, 700 masl). the collections were made in june and july of 1997 (table 1). the leaves were stored at −80 °c until dna extraction. figure 1. collection cities in fuskushima prefecture genetic variation of maiden lily (lilium rubellum baker) within and among three natural populations in fukushima prefecture in japan september december 2019 114 dna extraction total dna was extracted by the ctab method (murray & thompson, 1980). approximately 1 g of frozen leaves was ground to a fine powder with liquid nitrogen in a mortar with a pestle. the powder was transferred to a beaker containing 5 ml of 2× ctab buffer (2% ctab, 100 mm tris-hcl [ph 8.0], 20 mm edta and 1.4 m nacl) and immediately mixed. the mixture was transferred to a centrifuge tube and incubated for 10 min at 65 °c. an equal volume of chloroform/isoamyl alcohol (24:1) was added and mixed gently for 30 min at room temperature. five millilitres of 1× ctab buffer (1% ctab, 50 mm tris-hcl [ph 8.0], 10 mm edta and 0.7 m nacl) was added to the mixture. after centrifugation at 4,000 rpm for 15 min, the supernatant was transferred to a new tube by decanting. an equal volume of chloroform/isoamyl alcohol (24:1) was added and mixed gently for 10 min, then centrifuge again at 4,000 rpm for 15 min. the supernatant figure 2. rose lily in mt. azuma figure 3. rose lily in atsushio-kano figure 4. rose lily in nango population accesion number sample name 1. mt azuma 1 azuma a 8 2 azuma a 16 3 azuma b 14 4 azuma b 11 5 azuma b 5 6 azuma a 18 7 azuma c 13 8 azuma c 17 9 azuma c11 17 azuma a 7 18 azuma a 6 19 azuma b 4 20 azuma b 20 21 azuma c 16 22 azuma c 19 2. atsushiokano 27 atsushio-kano village1 28 atsushio-kano village 3 29 atsushio-kano village 6 30 atsushio-kano village 10 31 atsushio-kano village 12 3. nango 10 nango misawa 5 11 nango misawa 1 12 nango nagachisawaguchi 2 13 nango nagachisawaguchi 13 14 nango aburakubo b1 15 nango aburakubo 18 16 nango shimoyama 1 23 nango misawa 6 24 nango nagachisawaguchi 18 25 nango aburakubo 9 26 nango aburakubo b8 table 1. sample name collected in fukushima prefecture castro, m.; nishikawa, t. peruvian journal of agronomy 3(3): 112–119 (2019) 115 was transferred to a new tube and 0.1 vol of 10% ctab (10% ctab and 0.7 m nacl) was added to obtain a ctab–nucleic acid precipitate. after gentle inversion, the mixture was left for 30 min at room temperature. the precipitate was recovered by centrifuging at 4,000 rpm for 15 min and removing the supernatant. the precipitate was dissolved in 5 ml of hs-te (10 mm tris-hcl [ph 8.0], 1 mm edta and 1 m nacl) containing 5 µl of rnase and incubated at 55 °c until completely dissolved. five millilitres of isopropanol was added to the solution to remove the ctab. after gentle inversion, the solution was centrifuge at 4,000 rpm for 10 min and the supernatant was removed. the precipitate was washed with 5 ml of 70% ethanol and dissolved in 3 ml of te (10 mm trishcl [ph 8.0] and 1 mm edta). amplification by rapd procedures a total of 31 accessions from three natural populations of this prefecture were analysed using 18 random decamer primers. for dna amplification, 10 µl of the reaction mixture was prepared, containing 10 ng of dna, 1 µl pcr 10× buffer (100 mm tris-hcl [ph 8.3], 500 mm kcl), 1 µl of dntps (2.0 mm each), 1.2 µl of mgcl2 (25 mm), 0.5 units of dna polymerase (amplitaq gold), 0.1 µl of a decamer primer (20 pmol/µl) and sterilised, distilled water to a final volume of 10 µl. amplification was performed in a ptc-100 programmable thermal controller (mj research, inc.) as follows: first cycle at 95 °c for 10 min; followed by 50 cycles at 94 °c for 1 min, 35 °c for 1 min and at 72 °c for 2 min; and 1 cycle at 94 °c for 1 min; 35 °c for 1 min at 72 °c for 5 min; and a final hold at 4 °c. for each rapd assay, primers of the pc, ra, opa, opb and opc series were randomly selected (table 2). the amplified dna segments were separated in 1.5% agarose gels (20 × 15 cm) prepared in tbe buffer (sambrook, fritsch, & maniatis, 1989). electrophoresis was performed at 80 v for 4 h. then the gel was stained with ethidium bromide (0.5 µg/ml) and the bands were visualised using ultraviolet light. data analysis scoring of polymorphic dna fragments was done using 1 or 0 representing the presence or absence of a polymorphic band, respectively. from these data, the polymorphic information content (pic) per primer and the genetic similarity between genotypes of the three sampled populations were estimated using the jaccard similarity coefficient, which was calculated using ntsys-pc software version 2.2 (rohlf, 1992; van tuyl & arens, 2011; van tuyl et al., 2011). based on the resulting similarity matrix, a grouping analysis was performed with the unweighted pair-group method using arithmetic averages (upgma). for assessing genetic variation within populations, the programme genalex 6.5 (peakall & smouse, 2012) was used. nei’s gene diversity (1973) (he) and shannon index (ho) were also estimated. analyses of molecular variance (amova) between and within populations were calculated using the arlequin software version 3.5 (excoffier & lischer, 2010) primer code sequence number of amplified bands number of polymorphic bands % gc pic opa-08 5´-gtgacgtagg-3´ 13 13 60 0.344 opa-09 5´-gggtaacgcc-3´ 14 13 70 0.414 opa-10 5´-gtgatcgcag-3´ 9 9 60 0.413 opb-01 5´gtttcgctcc-3´ 14 14 60 0.457 opb-08 5´-gtccacacgg-3´ 11 11 70 0.380 opb-17 5´-agggaacgag-3´ 14 12 60 0.283 opc-01 5´-ttcgagccag-3´ 9 8 60 0.377 opc-08 5´-tggaccggtg-3´ 12 12 70 0.364 opc-09 5´-ctcaccgtcc-3´ 8 8 70 0.391 opc-15 5´-gacggatcag-3´ 7 7 60 0.358 opc-04 5´-ccgcatctac-3´ 16 16 60 0.428 pc 3 5´-aaccgcgctc-3´ 14 14 70 0.382 pc 7 5´-cgcggacgat-3´ 11 11 70 0.432 ra 3 5´-cgatcgagga-3´ 9 9 60 0.431 ra5 5´-aagcagcaag-3´ 10 10 50 0.341 ra35 5´-aagctccccg-3´ 9 9 70 0.456 ra37 5´-tgtggccggt-3´ 11 11 70 0.378 ra41 5´-gagtgcgcag-3´ 9 9 70 0.317 total number of bands 200 196 table 2. primers and polymorphic information content genetic variation of maiden lily (lilium rubellum baker) within and among three natural populations in fukushima prefecture in japan september december 2019 116 genetic diversity in this study, the nei´s gene diversity or expected heterozygosity he within populations ranged from 0.244 (atsushio-kano population) to 0.296 (nango population) as shown in table 3. the shannon index ho ranged from 0.365 (atsushio-kano population) to 0.442 (nango population). it is important to note that the shannon index ranges from 0 to 1, and the closer the value is to one, the higher the diversity. the percentage of polymorphic loci ranged from 68.5% (atsushio-kano population) to 83% (nango population). the nei´s gene diversity he, the shannon index ho, and the percentage of polymorphic loci were 0.275, 0.409 and 76.7%, respectively; this showed a relatively high genetic diversity within the populations of l. rubellum. genetic structure the similarity matrix calculated using the jaccard coefficient between genotypes based on rapd markers exhibited coefficients of similarity between pairs of genotypes that ranged from 0.54 to 0.85 (figure 5). the greatest similarity was found between the individuals from c 13 and c 17 from mt. azuma (0.85). in the dendrogram at 0.61 similarity index, the three populations of l. rubellum collected in fukushima prefecture were clearly differentiated. population 1, made up of 15 samples taken in mt. azuma-fukushima city, had a percentage of similarity with a range of 0.66%–0.85%; population 2, made up of five samples taken in atsushio-kano village, had a percentage of similarity with a range of 0.62%– 0.74%; and population 3, made up of 11 samples taken in results degree of polymorphism a total of 200 bands were amplified using 18 primers, and 196 were polymorphic (98%; table 2). they ranged in size from 0.20 to 2.24 kb. the number of bands per primer varied from 7 to 16 with a mean of 11.1. in this study, 100% of the primers used produced polymorphic bands. populations he ho % polymorphic loci mt. azuma 0.284103443 0.421575859 78.5 atsushiokano 0.24402529 0.365970296 68.5 nango 0.296599687 0.442330341 83 mean 0.274909473 0.409958832 76.67 table 3. genetic diversity indices of three populations of lilium rubellum he, genetic diversity of nei; ho, shannon index. figure 5. upgma dendrogram based on jaccardæs similarity coefficients source of variation sum of squares variance components percentage variation among populations 314.52 13.21 29.52 within populations 883.03 31.54 70.46 total 1197.55 44.75 table 4. summary of analysis of molecular variance for the three populations of lilium rubellum fst = 0.2952 castro, m.; nishikawa, t. peruvian journal of agronomy 3(3): 112–119 (2019) 117 the nango villages, had a percentage of similarity with a range of 0.63%–0.76% (table 1; figure 5). the nango and mt. azuma populations were closer genetically than the atsushio-kano population. as shown in table 4, amova analysis revealed that of the total variation, 29.52% occurred among the populations and the remainder within the populations. discussion three natural populations of l. rubellum in fukushima prefecture, japan, were analysed by rapd marker analysis with 18 primers to produce bands, 98% of which were polymorphic. eleven of these primers showed ≥10 polymorphic bands (opa-08, opa-09, opb-01, opb-08, opb-17, opc-08, opc-04, pc 3, pc 7, ra5 and ra37) with a pic mean of 76.7%. in future analyses, primers that produce ≥10 polymorphic bands would be useful in evaluating the genetic variability in l. rubellum. the genetic diversity mean of l. rubellum (ho = 0.2749, he = 0.4099, and percentage of polymorphic loci = 76.7%) compared with other narrowly distributed species of the genus lilium, was moderately high (guo et al., 2011) and very similar to the genetic diversity of l. regale (ho = 0.2750). of the three populations analysed, the nango population showed the highest genetic diversity and the atsushio-kano population showed the lowest genetic diversity within the respective populations. the mt. azuma population had intermediate values of the three indices analysed. since these samples were collected near the alpine belt (approximately 1,300 masl), the population was expected to have greater variation because subalpine species have been shown to harbour greater variation in the highlands of central japan (ohsawa & ide, 2011). the relatively high genetic diversity of l. rubellum may be attributable to its potential mixed mating system and longrange pollen and seed dispersal, which is also common in other lilium species like l. regale (wu et al., 2015) in the upgma dendrogram at 0.61 similarity index, the three populations of l. rubellum collected in fukushima prefecture were clearly differentiated. the analysis of interpopulation variation through amova showed that there was 29.52% of variation among populations. moreover, nango is located at the southern edge of the distribution of l. rubellum, and it is closer geographically to the atsushio-kano village (outside kitakata town) than to mt. azuma, fukushima city (figure 1). despite geographic distances, as can be seen in the dendrogram (figure 5), the populations of mt. azuma and nango were in the same cluster (0.55), indicating that they are closely related compared to the atsushio-kano population. however, the number of samples collected representing the atsushio-kano population was quite small. in future analysis, a higher number of samples collected in this village should be taken in order to validate the accuracy of population clustering. conclusions the assessment of genetic diversity is of great importance to the sustainability of plant populations (wang et al., 2007). the evolutionary potential of a species largely depends on its level of genetic variability, and, for plants, the overall level of genetic variability in a population is often correlated with the geographical distribution range of the species (hamrick & godt, 1996). the wide separation between populations found based on results of the amova analysis may result in increased genetic diversity due to the fact that there may be little cross pollination among the three populations included in this study. the populations sampled in this study were separated by many kilometres, and distance could be a significant factor accounting for the relatively high genetic variation among populations. we hope that the genetic diversity evaluated in germplasm collected over 20 years ago will serve to compare with the genetic diversity that can be found using other molecular markers. the l. rubellum populations reported here offer important information on the conservation strategies for this endangered species and may be of value to breeding programmes. acknowledgements we would like to thank the japan international cooperation agency (jica) for their financial support, dr. t. nagamine for facilities usage, and ms. n. nakamaru for her technical assistance. we also would like to acknowledge mr. y. ohno, mr. y. meguro and mr. m. sugano for their supply of materials and cooperation in collecting materials. references arzate-fernández, a. m., miwa, m., shimada, t., yonekura, t., & ogawa, k. 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(1995). detection of section-specific random amplified polymorphic dna (rapd) markers in lilium. theoretical and applied genetics, 91(6–7), 830–835. https://doi.org/10.1007/bf00223888 yi, j. y., lee, g. a., chung, j. w., lee, s. y., & lim, k. b. (2013). efficient cryopreservation of lilium spp. shoot tips using droplet-vitrification. y, 1(2), plant breeding and biotechnology, 131–136. peruvian journal of agronomy 2 (2): 6 13 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i2.1200 © the authors. published by universidad nacional agraria la molina received for publication: 15 july 2018 accepted for publication: 17 september 2018 characterization of banana farms (musa spp.) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) caracterización de fincas bananeras (musa spp.) en la microcuenca de cuyani, distrito de pichanaki, provincia de chanchamayo (junín, perú) introduction banana (musa spp.) is native to southeast asia, from where it passed to india and africa. the europeans introduced it to america and the antilles and today it is a widely distributed crop, both in the tropics and subtropics. however, the largest commercial plantations are found in the humid tropics (rodríguez and guerrero, 2002). according to the special project huallaga central and bajo mayo-pehcbm (2016), banana production is concentrated in africa, latin america and the caribbean with a world production in 2011 of 145 million mt. in peru, banana cultivation is expanding rapidly in the jungle and the north of the country and, according to the ministry of agriculture (minagri), in 2016, reached 169,646 ha harvested (agencia agraria noticias, 2018). in the central forest there were about 19,000 ha in 2015 (agencia agraria noticias, 2015) and in the district of pichanaki, 1,595 ha (inei, 2012). jazmín maraví loyola1, omar buendía martínez1, leonel alvarado huamán1, ricardo borjas ventura1, viviana castro-cepero1, alberto julca otiniano1* * corresponding author. ajo@lamolina.edu.pe abstract the objective of this study was to characterize banana farms in the cuyani microbasin in the district of pichanaki, chanchamayo province (junín, peru). we worked with a producer organization that has 400 partners, of which a sample was taken (n = 80) using the proportions method. it was found that the banana farms are very diverse, the person in charge is mostly a man, but there is an interesting percentage of the female gender in the administration of the farms. in general, producers have a basic educational level. the production system is traditional, the banana is grown alone or associated with other crops, such as coffee. most producers consider that pests and diseases are the main factors limiting production, since they reduce yields and increase production costs. the farms were classified in five groups, the most important of which was 45% of the farms whose owner is a male, between 44 and 56 years old, with secondary education, who lives on the same farm. they do not have a property title, the house does not have basic services, and they have poultry and guinea pigs. they have 2 to 3 hectares of banana (cultivars: island and palillo, 600 to 1000 plants/ha). its production costs are between 2000 and 3600 soles and a yield of 6.5 to 7.5 t/ha. the cultivar isla is the most common but also the cultivars palillo, morado and biscocho are planted. keywords: banana; characterization; cluster; farms; survey. resumen este estudio se realizó con el objetivo de caracterizar fincas productoras de plátano en la microcuenca cuyani en el distrito de pichanaki, provincia de chanchamayo (junín, perú). se trabajó con una organización de productores que cuenta con 400 socios, de los cuales se tomó una muestra (n = 80) usando el método de proporciones. se encontró que las fincas productoras de plátano son muy diversas, el responsable es mayoritariamente un hombre, pero hay un porcentaje interesante del género femenino en la administración de los fundos. en general, los productores tienen un nivel educativo básico. el sistema de producción es tradicional, el plátano se cultiva solo o asociado con otros cultivos, como el café. la mayoría de los productores consideran que las plagas y enfermedades son los principales factores limitantes de la producción, ya que reducen los rendimientos y aumentan los costos de producción. las fincas se clasificaron en cinco grupos, el más importante reunió al 45% de las fincas y se caracterizó por tener como responsable de la finca a hombres de 44 a 56 años, con educación secundaria, que viven en la misma finca. no tienen título de propiedad, la vivienda no tiene los servicios básicos y crían aves y cuyes. tienen de 2 a 3 ha de plátano (cultivares: isla y palillo, 600 a 1000 plantas/ha). sus costos de producción están entre 2000 y 3600 soles y un rendimiento de 6.5 a 7.5 t/ha. el plátano isla es el más cultivado; pero también se siembran los cultivares palillo, morado y biscocho. palabras claves: plátano, caracterización, conglomerado, fincas, encuesta. 1grupo investigación agricultura y desarrollo sustentable en el trópico peruano. facultad de agronomía. departamento de fitotecnia. universidad nacional agraria la molina. lima (perú). jazmín maraví loyola, omar buendía martínez, leonel alvarado huamán, ricardo borjas ventura, viviana castro-cepero, alberto julca otiniano peruvian journal of agronomy 2 (2): 6 13 (2018) 7 banana has generated a significant and growing interest in the consumer and is one of the freshest and most widely marketed products locally and nationally. it is a basic food for the population, especially in the diet of the inhabitants of the jungle. the production systems are mostly traditional, both in monoculture, and associated mostly with coffee and cocoa (pehcbm, 2016). on the other hand, agriculture in pichanaki, as in most of the country, is an activity of small producers, that is, the agricultural units or farms are small. but the farms are very diverse and complex so it is necessary to make a characterization as a previous step for any subsequent project (santistevan et al., 2014). for malagon and praguer (2001) the characterization is a determining stage for the development of research in production systems. it consists of determining a set of variables that distinguish a particular production area or unit and that makes it different from others. among other things, it seeks to distinguish the outstanding aspects for research in the selected area, identify the prevailing systems and identify the limiting factors. in this sense, by grouping the farms according to their main differences and relationships, the aim is to maximize homogeneity within the group and heterogeneity among them (cabrera et al., 2004). therefore, this work was carried out with the objective of characterizing the banana farms (musa spp.) in the cuyani microbasin, pichanaki district (chanchamayo, junín, peru). materials and methods the investigation was carried out in the junín region, chanchamayo province, pichanaki district, in the cuyani microbasin. it limits on the west with bella vista; on the east with pichiquiari and on the southeast with alto pichanaki (figure 1). for this particular investigation, we worked with the most representative organization of the study area, which has 400 banana producers from which a sample of 80 producers was selected. the “method of proportions”, already used in other similar researches, was used (merma and julca, 2012); the method has the following formula: 1 1 4 4 2 2 + − = n d pq d pq n where: n: sample size n: target population (universe) p: success probability 0.5 (value assumed) q: error probability 0.5 d: % error the data collection was carried out through semistructured surveys, related to economic, social, environmental and technical management aspects for agricultural production. the surveys were carried out in the field, taking advantage of the meetings held by the community members, and to complete the information, they were surveyed on their own farms. finally, a clustering analysis was performed by the ward method, with a euclidean square distance of 1200. results and discussion characteristics of the banana producer: figure 2 shows that the person responsible for the farm is mostly male (77%) and 23% of the farms are managed by women. these results corroborate data from the national agricultural census (inei, 2012), which showed that of every 10 farmers, about 8 are men and 2 are women in the selva region. the participation of women in the administration of agricultural farms is important for the financing of future projects, due to the gender approach. the most frequent age ranges of producers are from 31 to 43 years (40%), 44 to 56 years (26%), showing the presence of a young sector, which would indicate the sustainability in the management of the crop in the following decades. figure 1. map of the pichanaki district and the cuyani micro-basin study area, chanchamayo province, junín region, peru (adapted from: mdp, 2015). the study did not consider the town of valle hermoso characterization of banana farms (musa spp.) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) may august 2018 8 the educational level of banana farmers shows that 51% of them have completed elementary education, 44% have high school education, 3% have not had any educational formation, 1% have a technical career and 1% have university education (figure 2). compared to the data of the 2007 national census, there is a similarity, where 40% of the total population of the pichanaki district are people who have completed elementary education, 32% are people who have high school education and 16% do not have any educational level (pichanaki district municipality, 2016). according to these data, the educative level of the producers is basic, aspect that has an effect in the technified handling of the crop, as well as the access to projects or programs of technological improvement. the coverage of basic services for farmers, such as water supply, drainage and electricity, is incomplete and inadequate in most rural areas, unlike in the urban sector. forty-nine percent of the producers who live in the towns and communities have water supplied by rivers and irrigation ditches, as well as electricity. farmers who live in pichanaki or centro cuyani, have full basic services (45%) and a group of less than 6% have only water, but it is not from a potable source (figure 2). according to the research carried out in the cuyani microbasin by the pichanaki district municipality (2015), the main economic activity is agriculture. in the study area, it was found that 72% of the farmers depend on this activity and 28% have other incomes, as they work in state institutions and in the commercial and transport sectors. most of banana producers raise animals, 65% have poultry and 19% poultry + guinea pigs; only 13% do not raise animals, since they are farmers residing in pichanaki (figure 2). the possession of animals promotes that the producer resides in the same farm and not in the city. this is important because it is an indicator of the degree of control that the producer has with respect to his agricultural unit (caballero, 2002). during the investigation of the banana farms, it was found that the producers have their own fields (95%), rented (2.5%) and both own and rented fields (2.5%). of the producers who have their own fields, 42% have a land title and 54% have a certificate of ownership. in the group of those with own and rented fields, 3% have land title and lease contracts, and 1% have lease contracts. land tenure is one of the main problems of peruvian agriculture; according to the national agricultural census of 2012, of every 100 producers, 45.4% have a land title, that is, the property is registered in public registries or in the process of registration; the remaining 54.6% of producers do not have a land title. in the case of banana producers, most have a certificate of ownership, which puts agricultural activity at risk, as the legalization of the land is an important step towards improving its management. in figure 2, it is shown that 60% of the producers do not belong to any organization and 25% belong to a coffee cooperative. but most producers indicated that this organization is just being formed and is in the process of legalization. this aspect is important, because only legally constituted organizations can access state support programs. characteristics of the banana farm: of the farmers surveyed, 55% have between 1 and 5 ha of total land, 26% between 6 and 10 ha, 8% between 16 and 20 ha, 7% between 11 and 15 ha and 4% between 21 and 40 ha (figure 3). banana producers also manage other agricultural crops, such as coffee (71%), cocoa (7%), pineapple (9%), corn (6%), fruit trees and kion (4%). in the cuyani microbasin, the area of the farm planted with bananas is small, since 62% of those surveyed have between 0.75 and 1.5 ha, 29% have between 2 and 3 ha and only 9% have between 4 and 6 ha (figure 3). the producers who manage more than 2 ha were mainly found in cumbre barinetti, where bananas are the main crop and therefore the basis of their economy. in other population centers and assessed communities, banana cultivation is managed in small agricultural areas, so farmers depend on other crops. production systems are mostly traditional and often occur in associations with other types of agricultural products, such as coffee, cocoa, among others, and in some countries are managed under a monoculture system (pehcbm, 2016). figure 3 shows that 64% of the producers surveyed associate bananas with other crops, 22% manage them as monocultures and 14% have agricultural land where they work with both production systems. the producers who associate the crop with coffee are 76%; in the cuyani microbasin, the association of coffee with bananas is common, as a temporary shade for the coffee tree to manage the luminous intensity. likewise, 13% associate it with corn, 6% with fruit trees and 5% with cocoa. the number of banana cultivars handled is approximately 300, of which half are primary clones and half are somatic mutants. the existing banana cultivars in peru and america have originated from musa acuminata and through the formation of hybrids with musa balbisiana (figueroa and wilson, 1992). this has resulted in fruits that are grouped into: plantain type and banana type. the plantain type (aab group) is the one that can be eaten cooked, fried, green or ripe. the main commercial varieties include ‘bellaco’ and ‘inguiri’. the banana type (aaa group) is the one that is consumed as table fruit, and here the commercial varieties ‘seda’ (gros michell), ‘cavendish’, ‘isla’, ‘moquicho or biscochito’ and ‘capirona’ stand out (pehcbm, 2016). the main cultivars managed by the producer are isla, palillo, bellaco, bizcocho, morado and largo, in order of importance. the ‘isla’ cultivar is the most used by the producer with 71%. in the case of ‘isla’ cultivar, 50% of producers received a sales price of 0.6 soles per kg, 16% received 0.7 soles per kg and another 16% received a price of 0.7 to 1.0 soles per kg. the remaining 18% have just installed the crop. in jazmín maraví loyola, omar buendía martínez, leonel alvarado huamán, ricardo borjas ventura, viviana castro-cepero, alberto julca otiniano peruvian journal of agronomy 2 (2): 6 13 (2018) 9 figure 2. socio-economic status of banana producers in the cuyani microbasin, pichanaki district, chanchamayo province. peru the case of the ‘palillo’ cultivar, 70% of farmers received a price of 0.7 to 0.9 soles per kg and 7% received between 1.0 and 1.5 soles per kg; the remaining 23% have recently been installed. according to these data, the price of ‘palillo’ cultivar is higher than that of ‘isla’. figueroa and wilson (1992), mention that one of the disadvantages of isla cultivar is that it is very susceptible to banana weevils. in 2000, bananas reached a price of 0.32 soles per kg, while in 2016 it reached 0.58 soles per kg (dgesep, 2017). the highest banana yield in peru was reported in the piura region in 2013, with 22.7 t/ha, followed by the san martín region with 13.1 t/ha and junín with 11.4 t/ha. in this study (figure 3), a yield of 6.5 to 7.5 t/ha (43% of producers), 7.6 to 8.6 t/ha (30%), 5 to 6 t/ha (21%) and between 9.7 to 12 t/ha (6%) was reported. the low yields are explained by a traditional management of the farm, most of the producers that allow a high incidence of phytosanitary problems such as the “panama disease” and the “banana weevil”. ninety-five percent of those surveyed indicated that they sell their product in the pichanaki market because they manage small agricultural plots and are not competitive to enter the lima market. four percent indicated that their production was only for self-consumption, due to their low characterization of banana farms (musa spp.) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) may august 2018 10 production and 1% do not know which market they will sell their production to. the low prices in the local market, is explained by a lower demand in the quality of the product, in addition that the farmers do not have a harvest plan and therefore do not make an adequate management of the fruit to offer. regarding the cost of production, 47% of farmers have a production cost per hectare ranging from 2000 to 3600 soles, 14% invest from 3700 to 5000 soles and these are producers who invest a little more in phytosanitary management, fertilization; finally, 39% invest from 1500 to 2000 soles, are producers who manage bananas at low planting densities and work in a traditional way (figure 3). according to the national agricultural census (2012), the lack of water is one of the major problems for the development of the agricultural sector in peru. ninetynine percent of the banana producers surveyed do not have irrigation water and are dependent on rainfall. this is confirmed by census data indicating that 64.8% of the country’s agricultural land is not irrigated and is cultivated using only rainfall. in the jungle region, 94.7% is rainfed agriculture. only 1% of the water is available for irrigation through the sprinkler system with the help of a motor pump that is supplied by streams. about diseases, 50% of banana producers mentioned that “panama disease”, “yellow sigatoka” and “black sigatoka” are the diseases that most affect production. nineteen percent indicated only “panama disease” and 7% to “yellow sigatoka” and “black sigatoka”. about 10% of producers reported other unknown diseases (figure 3). but the “panama disease” caused by fusarium oxysporum f. sp. cubense is the disease that generates the greatest economic losses in this crop (figueroa and wilson, 1992). on the other hand, 64% of the producers indicated that the most important pest is the “banana weevil” (cosmopolites sordidus); this pest drills the base of the pseudo stem and the plant bulb, causing the progressive decay of the bud and shoots. in the study, 19% of the producers reported weevil damage and unknown pests, 6% indicated that they had recently installed their field, so there are no pests present, another 6% have no pests present in their plantation and 5% indicated the presence of unknown pests (figure 3). regarding crop rotation, producers indicated that at the end of the season they would grow coffee and those who had associated coffee with bananas would only keep the coffee (40%); others (cumbre barinetti) mentioned that they would continue growing bananas (20%); another group indicated that they did not know whether they would rotate or continue growing bananas (14%). also, 10% indicated that they will plant fruit trees, achiote (5%), cassava (4%) and pineapple (1%). agricultural production is influenced by factors that directly affect the efficiency of production. for 44% of producers, the presence of diseases is the biggest problem for production, especially due to the incidence of “panama disease”. for 28% of producers, pests and diseases are the main problem; 15% consider that the main problem is the presence of weeds, as they consider that if they do not remove the weeds in time there is a delay in the growth of the crop. a smaller group of 6% reported only pest damage and 5% lack of fertilizer (figure 3). according to the national agricultural census (2012), of the total number of farmers who received training or technical advice, 72.2% were trained in agricultural crops, 32.2% in livestock and 11% in management, conservation and processing. in the jungle region, 88.9% of agricultural producers received training in agricultural crops. figure 3 shows that 80% of the producers indicate that they have not received training, 9% of producers indicate that they have received training, which was provided by the pichis palcazú project and the main topic was cultural work; 6% of producers indicated that they were trained by servicio nacional de sanidad agraria (senasa). the producers indicated that the institutions come to carry out training, but there is no follow-up to the talks given by the specialists. the use of conglomerates has been reported in ecuador to group coffee farms (santistevan et al., 2014) and cattle farms (vargas et al., 2012). in peru, for the analysis of farms with prevalent crops in cusco (merma and julca, 2012), also mandarin farms in cañete (collantes et al., 2015) and cocoa farms in san martín (tuesta et al., 2014). in colombia, for typification studies in citrus and guava production systems (cleves & jarma, 2014). in mexico, also to classify guava producers (borja et al., 2018) and in costa rica to classify dairy herds (vargas et al., 2013). the grouping of farms is important because future actions could be carried out for each group and no longer individually (criollo et al., 2016), given that groups are formed by similarity between the elements that compose it and it could be assumed that those farms that are grouped, alone present significant differences with the rest (castro et al., 2012). in this study, conglomerate analysis determined the presence of five groups of farms, groups 1 and 2, were the most numerous with 45 and 31. 2% of the farms, respectively (figure 4). there are important differences between groups of farms (table 1); for example, group 1 is characterized by having men between the ages of 44 and 56, with secondary education, who live on the same farm. they do not have property titles, the house does not have basic services, and they have poultry and guinea pigs. they have 2 to 3 ha of banana with the cultivars isla and palillo with a density of 600 to 1000 plants/ha. their production costs are between 2000 and 3600 soles (1us$ = 3.30 soles) and they have an average yield of 6.5 to 7.5 t/ha. group 5 has the youngest farm managers residing in the town of pichanaki and their housing is of noble material with basic services. they have from 4 to 6 ha of banana cv. palillo, the density of plantation and the costs of production are similar to the first group; but its yield of banana is greater and is between 7.6 to 8.6 t/ha. table 1 also shows that, in all groups, diseases are the main limiting factor in production and the cultivar isla banana is the most used but the other cultivars such as palillo, morado and biscocho are also planted. jazmín maraví loyola, omar buendía martínez, leonel alvarado huamán, ricardo borjas ventura, viviana castro-cepero, alberto julca otiniano peruvian journal of agronomy 2 (2): 6 13 (2018) 11 figure 3. characteristics of the production of the banana farm in the cuyani microbasin, pichanaki district, chanchamayo province. peru conclusions in the cuyani microbasin, the banana farms are very diverse. the person responsible for the farm is mostly male, but there is an interesting percentage of the female gender in the administration of the farms. in general, the producers have a basic level of education, the production system is traditional, the banana is grown alone or in association with other crops. most producers consider pests and diseases to be the main limiting factors of production, as they reduce yields and increase production costs. animal husbandry encourages the producer to reside on the farm and not in the city and this is important because it is an indicator of the degree of control that the producer has over his agricultural unit. the farms were classified into five groups, the most important being 45% of the farms and is characterized by having men between the ages of 44 and 56, with secondary education and who lives on the same farm. they do not have a property title, the house does not have basic services, and they have poultry and guinea pigs. they have 2 to 3 ha of banana with the cultivars isla and palillo with a density of 600 to 1000 plants/ha. their production costs are between 2000 and 3600 soles (1us$ = 3.30 soles) and characterization of banana farms (musa spp.) in cuyani microbasin, pichanaki district, chanchamayo province (junín, perú) may august 2018 12 figure 4. dendrogram of the banana farms in the cuyani microbasin, district of pichanaki, chanchamayo province variables group 1 group 2 group 3 group 4 group 5 age of the farm responsible (years) 44 to 56 31 to 43 31 to 43 44 to 56 18 to 30 level of education high school basic school high school basic school basic school residency farm farm farm farm pichanaki housing material wood wood noble wood noble animals poultry/guinea pigs poultry poultry poultry none basic services non drinking water non drinking water non drinking water non drinking water all property of the land certificate of ownership property title certificate of ownership certificate of ownership property title area with banana (ha) 2.0 – 3.0 0.25 1.5 0.25 1.5 0.25 1.5 4.0 – 6.0 cultivars isla/ palillo isla bizcocho isla/morado palillo density (plants /ha) 600 1000 600 1000 200 600 200 600 600 1000 factors that limit production diseases diseases/pests diseases diseases diseases market pichanaki pichanaki pichanaki pichanaki pichanaki production system tradicional tradicional tradicional tradicional tradicional production costs (s/. x ha) 2000 3600 2000 3600 1000 2000 2000 3600 2000 3600 yield (t/ ha) 6.5 7.5 7.6 8.6 5.0 – 6.0 5.0 6.0 7.6 8.6 table 1. main characteristics of the groups of banana farms in the cuyani microbasin, district of pichanaki, province of chanchamayo, peru have an average yield of 6.5 to 7.5 t/ha. all groups consider that diseases are the main limiting factor of production and the cultivar isla is the most common, in three of the five groups. all the groups consider that diseases are the main limitation of production. other cultivars such as palillo, morado and biscocho are also used. acknowledgements the national fund for scientific, technological and technological innovation development (fondecyt), an initiative of the national council for science, technology and technological innovation (concytec), through the project “strategy and mechanisms for the governance 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ecuador). ecología aplicada 13(2):187–192. http://www.lamolina.edu.pe/ecolapl/ articulo%2020%20no%202%20vol%2013.pdf vargas, j., benítez, d. and torres, a. 2012. tipificación de fincas ganaderas en el piedemonte tropical de las provincias cotopaxi y los ríos, ecuador. revista amazónica ciencia y tecnología 1 (1): 3 -10. http:// revistas.proeditio.com/revistamazonica/article/ view/150 vargas, b., solís, o., sáenz, f. and león, h. 2013. caracterización y clasificación de hatos lecheros en costa rica mediante análisis multivariado. agronomía mesoamericana. 24(2): 257275. https://www.medvet.una.ac.cr/posgrado/gen/invest/ articagronmesanalmultiv.pdf peruvian journal of agronomy 3(1): 16-23 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i1.1279 © the authors. published by universidad nacional agraria la molina received for publication: 12 march 2019 accepted for publication: 12 april 2019 soil insects associated with lucumo (pouteria lucuma l.) trees in la molina, lima, peru insectos de suelo asociados con árboles de lúcumo (pouteria lucuma l.) en la molina, lima, perú livia, c.1*; sánchez, g.2 *corresponding author: 20080931@lamolina.edu.pe abstract this study aimed to determine the composition and relative abundance of soil insects associated with lucumo (pouteria lucuma l.) trees growing in the agricultural fields of the universidad nacional agraria la molina in lima, peru. in total, 12 pitfall traps were installed in a 5-ha lucumo field and samples were collected on a weekly basis for approximately 4 months (march–june). insects in each sample were separated into morphotypes, and the number of individuals in each morphotype and date of collection were recorded. the collected insects were then identified at the entomology museum laboratory in lima through comparison with museum samples and with the help of identification keys. individuals in the orders orthoptera (families acrididae and gryllidae), dermaptera (anisolabididae), coleoptera (carabidae, staphylinidae, tenebrionidae, scarabaeidae and elateridae), blattodea (blatellidae) and hymenoptera (formicidae) were identified. among the coleoptera, tetracha chilensis (laporte, 1834) (carabidae) was the most abundant species; furthermore, staphylinidae of the subfamily oxytelinae and the families elateridae (conoderus spp.), tenebrionidae (epitragopsis sp.) and scarabaeidae (ataenius sp.) were identified. additional taxa identified were as follow: gryllus assimilis (fabricius, 1775) (gryllidae) in orthoptera, euborellia annulipes (lucas, 1847) (anisolabididae) in dermaptera and linepithema spp. in hymenoptera. keywords: insect, soil, lucumo, peru resumen con la finalidad de determinar la composición y abundancia relativa de los insectos de suelo en el cultivo de lúcumo en el área agrícola de la universidad nacional agraria la molina, se instalaron 12 trampas de caída en un campo de lúcumo (banco de germoplasma de lúcumo del programa de investigación en árboles frutales), de 5 has. se realizaron evaluaciones semanales durante aproximadamente 4 meses (marzo-junio). las muestras colectadas se procesaron separando los individuos por morfotipos, registrando la cantidad y fecha de colección. finalmente, se identificaron los individuos en el laboratorio del museo de entomología comparando con las muestras del museo y con ayuda de llaves de identificación. se identificaron individuos de las órdenes orthoptera (fam. acrididae y gryllidae); dermaptera (fam. anisolabididae); coleoptera (fam. carabidae, staphylinidae, tenebrionidae, scarabaeidae y elateridae); blattodea (fam. blatellidae); e hymenoptera (fam. formicidae). del orden coleoptera, se identificaron a las familias: carabidae teniendo como especie más abundante a tetracha chilensis (laporte, 1834); staphylinidae, los individuos de la subfamilia oxytelinae; elateridae, conoderus sp.; tenebrionidae, epitragopsis sp.; y scarabaeidae, ataenius sp. de coleoptera, la especie más abundante fue tetracha chilensis (carabidae). del orden orthoptera, gryllus assimilis fabricios (gryllidae). dermaptera, euborellia annulipes lucas (anisolabididae). del orden hymenoptera, el género linepithema sp. fue el más abundante. palabras clave: insectos, suelo, lúcumo, perú. 1 estudiante de la facultad de agronomía, universidad nacional agraria la molina 2 laboratorio del museo de entomología klaus raven büller, departamento de entomología de la facultad de agronomía, universidad nacional agraria la molina. introduction various perennial crops, primarily fruit trees, are grown in peru, particularly along the coast. one such tree is the lucumo (pouteria lucuma l.), which is very important in this region, although it is not sown at a large scale on the coast. peru is considered to be the primary producer of lucuma in the international market, with lima, piura, cajamarca, la libertad, ica and ayacucho being the regions with the highest production (lavado, yenque, & livia, c.; sánchez, g. peruvian journal of agronomy 3(1): 16-23 (2019) 17 robles, 2012). of these, the valleys of cañete, huaral and huacho of the lima region and the chincha valley of the ica region produce higher quality fruits for export purposes (siicex 2015). studies have been conducted on insects that affect this crop; these insects are mostly found in the canopy of trees. these studies have enabled the determination of many pests that are known today. on the contrary, limited information is available regarding the insects that inhabit the soils in which this crop is grown; among these, potential pests, or beneficial insects can be found to the crop. five orders of insects are believed to use soils as their main habitat: blattodea, dermaptera, orthoptera, coleoptera and hymenoptera. within the order blattodea, the families blattidae, blatellidae and blaberidae are found most frequently in soil. in dermaptera, members of the families anisolabididae and labiduridae inhabit soil. among these, the species euborellia annulipes (lucas, 1847) (anisolabididae) is considered as a predator species, particularly of eggs and larval stages of some insects. however, rondón (1999) analysed the stomach content of the aforementioned species and determined that these may primarily be phytophagous species and may secondarily feed on some soil arthropods; these findings suggest that e. annulipes is not a predator species. most soil-dwelling orthoptera belong to the family acrididae, with approximately 84 species recorded in peru (lieberman, 1963, as cited by beingolea, 1990). however, gryllidae have been recorded in soil, including gryllus assimilis (fabricius, 1775), which is considered to be a potential pest of several crops (sánchez & vergara, 2014). among the coleoptera, members of the family carabidae are generally considered to use soil as their main habitat. this family comprises >40,000 species worldwide (lovëi & sunderland, 1996); most of these are generalist predators, and some are important in the natural control of agricultural pests (vélez-azañero & lizárraga, 2013). for example, blennidus peruvianus (dejean, 1828), which is one of the most abundant soil predators in fields of various crops, including sweet potato, asparagus, beans, corn, potato and tomato (vergara & amaya de guerra, 1978; velapatiño, 1997; schuller & sánchez, 2003; rondón & vergara, 2004), along with tetracha chilensis (laporte, 1834). however, other species in this family, such as notiobia peruviana dejean, are phytophagous and prefer to eat seeds (lietti, montero, faccini, & nisensohn, 2000, as cited by castañeda, sánchez, & arellano, 2007). similarly, although most members of the family staphylinidae are predators of insects and other invertebrates, some species feed on fungi or decomposing organic matter (arnett & thomas, 2001). the order coleoptera includes the family scarabaeidae, including some species of economic importance because they can cause considerable damage by defoliation as adults or feeding on the roots as larvae (arnett & thomas, 2002). within this family, anomala testaceipennis (blanchard, 1850) and paranomala undulata (melsheimer, 1845) are particularly important pests of various crops, such as potato, strawberry, cotton, sugar cane, beans, lima beans, asparagus and corn (alata, 1973, as cited by gonzalesbustamente, 1994; vergara & amaya de guerra, 1978; raven, 1988). some species in the family elateridae are pests of crops, with adults feeding on nectar, pollen, floral parts, fungi and extrafloral nectaries, with most insects being diurnal and some, particularly in the neotropics, being crepuscular or nocturnal (johnson & quartone, 2004, as cited by aguirre-tapiero, 2009). in contrast, members of the family tenebrionidae are mainly scavengers (van et al., 2000) that can additionally feed on wood, although they often emerge nocturnally to feed on lichens, fungi or some other plant material. finally, in the hymenoptera, members of the family formicidae mainly inhabit the soil. therefore, the objective of this research was to determine the composition and relative abundance of insects inhabiting the soils in which lucumo trees are grown in la molina, lima, peru. materials and methods weekly evaluations of the soil invertebrate fauna in a field of the germplasm bank of lucumo of the research program in fruit trees at the universidad nacional agraria la molina in lima, peru, were conducted between 9th march and 8th june 2013 (n = 13 samples). lucumo trees were planted over 5 ha in these fields, and soil insects were collected using pitfall traps. field work installation of pitfall traps quadrat sampling was used to distribute the pitfall traps as uniformly as possible across the lucumo field. the field was divided into three sectors, each of which had an irregular shape, rendering it impossible to determine the specific area. therefore, the relative size of each sector was used to determine the distribution of pitfall traps, resulting in the installation of 3, 4 and 5 pitfall traps in the first, second and third sectors, respectively, with 12 traps installed in total. the pitfall traps were poly-wrap polyethylene soil insects associated with lucumo (pouteria lucuma l.) trees in la molina, lima, peru january april 2019 18 containers that were 15 cm in height and 10 cm in diameter. they were filled with 250 ml of a solution that was approximately 9 parts water to 1 part formaldehyde with 4–5 g of detergent. the formaldehyde was added to prevent the decomposition of the collected insects, whereas the detergent was used to break the surface tension of the solution. to install the pitfall traps, a hole was dug in the ground using a small hoe such that the mouth of the container was flush with the surface of the soil, and the soil was then flattened to create a uniform surface. the pitfall traps were randomly distributed through the lucumo field to cover the largest possible area. lucumo trees were planted at a spacing of 4 m × 4 m, and the pitfall traps were placed between the trees at a distance of 2 m from each tree and the edge of the irrigation channel. a brightly coloured stake was then placed alongside each trap to enable easy identification. no pitfall traps were installed within 10 m of the ends of the field to avoid any edge effects. sample collection the pitfall traps were removed on a weekly basis and their contents were poured into hermetically sealed polyethylene cups, 7 cm in height and 5 cm in diameter, marked with the trap number and date of evaluation using indelible ink. the pitfall trap containers were washed, cleaned, refilled with 250 ml of solution (as mentioned above) and reinstalled. sample processing in the laboratory the collected samples were transported to the research laboratory of the entomology museum of the universidad nacional agraria la molina for processing. each sample was sieved through an organza cloth strainer and rinsed with running water until it was clean and free from formalin odours. the sieved sample was then returned to the original polyethylene cup, which was washed and filled with clean water to facilitate the separation of the captured insects and prevent them from being damaged. the samples were placed in petri dishes and separated and grouped into morphotypes based on their morphological characteristics (oliver & beattie, 1993, 1996a, b) using knife and fine forceps. further, the number of individuals in each morphotype was counted, and the invertebrates were placed in small 10-ml bottles containing 70% alcohol. these bottles were labelled with a code that consisted of the first letter of the crop, the first three letters of the order to which the invertebrates belonged and the morphotype number, for example, lucumo and order hymenoptera samples were coded as ‘lhym1’, ‘lhym2’ and ‘lhym3’. a sample of each species that was collected during the evaluation was mounted on entomological pins and/ or placed in alcohol, labelled and registered in the entomology museum of the universidad nacional agraria la molina. taxonomic identification the collected individuals were identified by specialists in the laboratory of the klaus raven büller entomology museum of the universidad nacional agraria la molina using identification keys and comparing them with samples in the museum collection. results in total, 66,901 insects were collected during the study period, with 96.6% of these species developing in and mainly inhabiting the soil. these soil insects included members of the orthoptera, blattodea, dermaptera, coleoptera and hymenoptera (fig. 1). the order hymenoptera was the most predominant, comprising 97.66% of the total soil insects collected (63,087 individuals), followed by the orders coleoptera (1.95%, 1,261 individuals), orthoptera (0.30%, 191 individuals), dermaptera (0.08%, 55 individuals) and blattodea (0.01%, 6 individuals). on the first collection date (16 march 2013), 19,881 insects were captured, most of which belonged to the family formicidae (fig. 2). an overall decrease in the number of insects was observed until 13th april, when 719 individuals were captured, with a slight increase recorded on 6th april (2,573 individuals) because a large number of ants were captured in one of the traps. the second highest number of insects was captured on 20th april (18,649 individuals), with 3,000–9,000 individuals of the formicidae family recorded in three pitfall traps. figure 1. insects that were captured in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru between march and june 2013. livia, c.; sánchez, g. peruvian journal of agronomy 3(1): 16-23 (2019) 19 however, the number then decreased again from 27th april to 25th may, when the lowest number of insects was captured throughout the entire study period (486 individuals). a slight increase in the number was observed in the last two evaluations (1,315 individuals on 1st june and 1,567 individuals on 8th june). order orthoptera members of the families acrididae and gryllidae were recorded in the samples. a low abundance of acrididae was observed among the samples, with only two individuals in the nymphal stage being identified as ‘gen sp1’. generally, adults of this family are found to be feeding on the leaves of plants. however, most nymphs are found in the ground, which explains why only this stage was captured in the pitfall traps. the family grylidae was represented by gryllus assimilis (fabricius, 1775), with 189 individuals captured in total. particularly high numbers of this species were captured on 16th march (24 individuals), 6th april (25 individuals) and 18th may (19 individuals; fig. 3). order dermaptera euborellia annulipes (lucas, 1847) in the family anisolabididae was the only representative of the order dermaptera that was recorded in the samples. in total, 55 individuals were collected, with high numbers occurring on 16th march (7 individuals), 23th march (8 individuals), 27th april (8 individuals), 4th may (7 individuals) and 11th may (7 individuals; fig. 4). order blattodea six individuals belonging to the family blatellidae were collected; however, they could not be identified and were recorded as gen sp1. order coleoptera five families were recorded in the order coleoptera: carabidae, staphylinidae, scarabaeidae, elateridae and tenebrionidae. among these, members of the family carabidae were most abundant (76.6%, 966 individuals), figure 2. changes in the number of soil insects captured in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru between march and june 2013. figure 3. incidence of gryllus assimilis (fabricius, 1775) in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru in march–june 2013. figure 4. incidence of euborellia annulipes (lucas, hf, 1847) in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru in march– june 2013. soil insects associated with lucumo (pouteria lucuma l.) trees in la molina, lima, peru january april 2019 20 figure 7. incidence of conoderus sp. in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peruin march–june 2013. followed by tenebrionidae (11.82%, 149 individuals), elateridae (9.99%, 126 individuals), staphylinidae (1.1%, 14 individuals) and scarabaeidae (0.5%, 6 individuals; fig. 5). although insects belonging to other families were identified, these were not included in this study because they are not considered to be soil invertebrates. family carabidae five species of carabidae were recorded: tetracha chilensis (laporte, 1834) (653 individuals), blennidus peruvianus (dejean, 1828) (293 individuals), notiobia peruviana (dejean, 1829) (15 individuals), tetragonoderus sp. (3 individuals) and gen sp. (harpalini) (2 individuals). the two individuals of gen sp. were captured on 20th april and 4th may, whereas tetragonoderus sp. was only collected on 25th may. in contrast, n. peruviana was collected on 10 of the 13 collection dates, with a maximum of three individuals recorded in a collection. similarly, b. peruvianus was captured throughout the study period, with a maximum of 80 individuals captured on 25th may and a minimum of 10 individuals captured on 24th april (fig. 6). finally, a high number of tetracha chilensis was captured on 16th march (295 individuals), 23th march (141 individuals) and 30th march (78 individuals). however, the population subsequently decreased, with no individuals captured on the last three collection dates (fig. 6). family staphylinidae in the family staphylinidae, two individuals of the species platydracus notatus (solsky, 1872), two morphotypes of the subfamily oxytelinae recorded as gen sp1. (1 individual) and gen sp2. (8 individuals), two morphotypes of the subfamily aleocharinae recorded as gen sp3. (1 individual) and gen sp4. (1 individual) and one morphotype of the subfamily staphylininae recorded as gen sp5. (1 individual) were collected. family scarabaeidae two species of scarabaeidae were collected: paranomala undulata (melsheimer, 1845) (2 individuals) and ataenius sp. (4 individuals). paranomala undulata was collected on 23th march and 20th march, whereas ataenius sp. was recorded on 30th march, 11th may, 25th may and 1st june (1 individual on each date). family elateridae two species of elateridae were collected: conoderus sp. (116 individuals) and horistonotus sp. (10 individuals). horistonotus sp. was collected on five dates: 16th march (4 individuals), 23th march (2 individuals), 6th april (1 individual), 20th april (2 individuals) and 18th may (1 individual). in contrast, conoderus sp. had a more constant presence, although the highest numbers were recorded at the beginning of the study period on 16th march (25 individuals) and 23th march (24 individuals), with numbers gradually decreasing until no individuals were recorded on 25th may (fig. 7). family tenebrionidae four species of tenebrionidae were recorded: epitragopsis figure 6. incidences of tetracha chilensis (laporte, 1834) and blennidus peruvianus (dejean, 1828) in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru in march–june 2013. figure 5. families of soil insects in the order coleoptera that were captured in pitfall traps in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru in march–june 2013. livia, c.; sánchez, g. peruvian journal of agronomy 3(1): 16-23 (2019) 21 sp. (74 individuals), gondwanocrypticus sp. (24 individuals), blapstinus holosericius (laporte, 1840) (50 individuals) and hipalmus costatus (guérin-méneville, 1831) (1 individual). the largest number of epitragopsis sp. was recorded on the first collection date (16th march), with 16 individuals captured (fig. 8). subsequently, the population decreased, with no individuals recorded on two occasions. high numbers of gondwanocrypticus sp. were recorded on the second collection date (8 individuals), following which few or no individuals were collected in the subsequent evaluations (fig. 8). blapstinus holosericius showed peaks on 20th april (8 individuals) and 27th april (7 individuals) and was not recorded on 16th march (fig. 8). finally, one individual of hipalmus costatus was recorded on 20th april. order hymenoptera several taxa in the family formicidae were recorded during the study period, including linepithema sp. (60,342 individuals), tetramorium sp. (1,185 individuals), brachymyrmex sp. (1,377 individuals), brachymyrmex sp1. (9 individuals), three morphotypes of the subfamily formicinae recorded as gen sp1. (25 individuals), gen sp2. (138 individuals) and gen sp3. (5 individuals) and two taxa recorded only as gen sp4. and gen sp5. linepithema sp. was the most abundant taxon, with 19,130 individuals recorded on the first collection date, following which the population decreased remarkably (fig. 9). further, a second peak was recorded on 20th april (18,463 individuals), whereas the lowest collection was recorded on 25th may (384 individuals). the highest number of tetramorium sp. was obtained on the first collection date (173 individuals), following which the number of individuals gradually decreased, approaching the lowest record of only 2 individuals on 25th may (fig. 10). in contrast, the highest number of brachymyrmex sp. was collected on the last collection date (633 individuals), whereas only 6 individuals were collected on 25th may (fig. 10). discussion orthoptera the number of g. assimilis captured generally increased on the dates when irrigation occurred (fig. 3). the abundance of litter on the field beneath the lucumo trees provided a refuge for these insects, with individuals being captured in the pitfall traps while they were moving. unlike acrididae adults, gryllidae adults are not very commonly found on trees, resulting in the traps collecting both first stage and adult individuals. this species is considered to be a potential pest of crops such as corn (sánchez & vergara, 2014). dermaptera low numbers of individuals were captured between the third and fifth collection dates and during the final collection. this may have occurred because of irrigation during the study period, which displaced individuals that were present in the irrigation furrows. on the dates with figure 8. incidences of epitragopsis sp., gondwanocrypticus sp. and blapstinus holosericius (laporte, 1840) in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru in march–june 2013. figure 9. incidence of linepithema sp. in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru in march–june 2013. figure 10. incidences of tetramorium sp. and brachymyrmex sp. in pitfall traps installed in a field of lucumo (pouteria lucuma l.) in la molina, lima, peru, in march–june 2013. soil insects associated with lucumo (pouteria lucuma l.) trees in la molina, lima, peru january april 2019 22 no risks, individuals may have accidentally fallen into the pitfall traps. blattodea members of the order blattodea are known to be omnivores; therefore, their presence may have been supported by the refuge areas and diverse food sources that were available. coleoptera among the members of the family carabidae, tetracha chilensis and blennidus peruvianus were the most abundant. a high abundance of the former species can be explained by the large number of refuge areas that were observed in lucumo fields; as a result, a wide variety of arthropods were available in the soil as a food source for this species. b. peruvianus has been reported to be one of the most abundant soil predators in the fields of several crops such as sweet potato, asparagus, bean, potato and tomato (vergara & amaya de guerra, 1978; velapatiño, 1997; schuller & sánchez, 2003; rondón & vergara, 2004). among the least abundant species, notiobia peruviana is considered to be a highly polyphagous species; however, it appears to primarily feed on the seeds of various plants (nisensohn, faccini, montero, & lietti, 1999; lietti, et al., 2000; arndt & kirmse, 2002, as cited by yábar, castro, melo, & gianoli, 2006). staphylinidae staphylinidae is the family that showed the highest diversity of species with regard to the others families. most staphylinidae are predators of insects and other invertebrates; however, some feed on fungi or decomposing organic matter and most live among the leaf litter of forests and mosses (arnett & thomas, 2001), which resembles the habitat observed in lucumo fields. scarabaidae members of this family are usually found in places with abundant decomposing organic matter. consequently, fruit fields, particularly perennial crops, represent a favourable agro-ecosystem for these insects because of the large number of leaves and fruits that fall on the ground. elateridae the decrease in the number of the members of this family during the last evaluations coincided with the end of the harvest period in the field. probably, conditions in this period were not favorable these insects. moreover, rondón & vergara (2004) identified conoderus sp. in a sweet potato field in cañete, lima, supporting its presence in this region. tenebrionidae no specific information could be obtained regarding the behaviours or characteristics of gondwanocrypticus sp., b. holosericius and h. costatus. however, epitragopsis olivaceus (erichson, 1847) was previously recorded in a sweet potato field in cañete (rondón & vergara, 2004), although this may have been a different species from that observed here. tenebrionidae are known to be saprophagous and generally feed on a various decaying plants and fungi (van et al., 2000). therefore, these insects may have fallen into the traps while searching for shelter and/or food because these conditions are common in lucumo fields. hymenoptera formicidae are known to inhabit the soil and litter (fernández, 2003), which commonly occurs in lucumo fields. furthermore, the large distance between plants enabled these insects to establish their nests throughout the entire field. several nests were located on the edges of the water channels and were disturbed by the irrigation, which may have displaced the ants and resulted in their capture. conclusions soil insects belonging to the orders orthoptera, dermaptera, blattodea, coleoptera and hymenoptera were captured in pitfall traps installed in a lucumo field in lima, peru between march and june 2013. members of the order hymenoptera were most abundant, followed by coleoptera, orthoptera, dermaptera and blattodea. in the order orthoptera, members of the family gryllidae were identified, with gryllus assimilis (fabricius, 1775) being the most abundant. in the order dermaptera, euborellia annulipes (lucas, 1847) in the family anisolabididae was recorded, whereas in the order blattodea, members of the family blatellidae were collected. four families in the order coleoptera were recorded, among which carabidae was the most abundant, with tetracha chilensis (laporte, 1834) being predominant. in the family staphylinidae, the subfamily oxytelinae was the most abundant, whereas ataenius sp. (scarabaeidae), conoderus sp. (elateridae) and epitragopsis sp. (tenebrionidae) were additionally present. finally, three species were identified in the order hymenoptera, belonging to the family formicidae: linepithema sp., tetramorium sp. and brachymyrmex sp.; linepithema sp. was found to be the most abundant. references aguirre-tapiero, m. (2009). clave de identificación de géneros conocidos y esperados de elateridae leach (coleoptera: elateroidea) en colombia. boletín del museo de entomología de la universidad del valle. colombia, 10(2), 25–35. arnett, r. and thomas, m. (2001). american beetles – archostemata, myxophaga, adephaga, polyphaga: staphyliniformia. washington, usa: crc press llc. livia, c.; sánchez, g. peruvian journal of agronomy 3(1): 16-23 (2019) 23 arnett, r. and thomas, m. (2002). american beetles polyphaga: scarabaeoidea through curculionoidea. washington, usa: crc press llc. beingolea, o. 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(1996a). designing a costeffective invertebrate survey: a test of methods for rapid assessment of biodiversity. ecological applications, 6, 594–607. https://doi.org/10.2307/2269394 oliver, i. and beattie, a.j. (1996b). invertebrate morphospecies as surrogates for species: a case study. conservation biology, 10, 99–109. https://doi. org/10.1046/j.1523-1739.1996.10010099.x raven, k. (1988). orden coleoptera. lima, perú: universidad nacional agraria la molina, departamento de entomología. rondón, s. (1999). artrópodos de suelo en los cultivos de camote y algodonero en la costa central del perú. tesis para optar el grado de magister scientiae en entomología. lima, perú: universidad nacional agraria la molina, escuela de posgrado. rondón, s. and vergara, c. (2004). diversidad de artrópodos del suelo en cuatro cultivares de camote ipomoea batatas (l.) lam. (convolvulaceae) en el valle de cañete, lima, perú. revista peruana de entomología, 44, 73–80. sánchez, g. and vergara, c. (2014). plagas de hortalizas. lima, perú: universidad nacional agraria la molina, departamento de entomología. schuller, s. and sánchez, g. (2003). los artrópodos de suelo depredadores en agroecosistemas de maíz en el valle de chancay, lima, perú. revista peruana de entomología, 43, 47–57. siicex (sistema integrado de información de comercio exterior). (2015). lúcumo. consultado 14 abr. 2015. available in: http://www.siicex.gob.pe/siicex/ resources/fichaproducto/115pdf2015feb10.pdf van, k., aspôk, h., aspôk, u., balderson, j., britton, e., byers, g., … murray, m. (2000). the insects of australia. australia: melbourne university press. velapatiño, j. (1997). algunos artrópodos presentes en el suelo del área agrícola de la universidad nacional agraria la molina. revista peruana de entomología, 40, 89–90. vélez-azañero, a. and lizárraga, a. (2013). diversidad de carabidae (coleoptera) asociados a la cuenca baja del río lurín. lima-perú. the biologist (lima), 11(1), 97–106. vergara, c. and amaya de guerra, j. (1978). apreciaciones sobre la fluctuación de una comunidad insectil en el cultivo de papa en chimbote (santa). revista peruana de entomología, 21(1), 57–59. yábar, e., castro, e., melo, l. and gianoli, e. (2006). predación de bembidion sp., notiobia peruviana (dejean) y metius sp. (coleóptera: carabidae) sobre huevos de premnotrypes latithorax (pierce) (coleóptera: curculionidae) en condiciones de laboratorio. revista peruana de entomología, 45, 91–94. peruvian journal of agronomy 2 (1): 18 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i1.1126 © the authors. published by universidad nacional agraria la molina received for publication: 30 january 2018 accepted for publication: 30 march 2018 antifungal activity of five chemical and two biological fungicides for the management of botrytis cinerea, causal agent of gray mold in strawberry actividad antifúngica de cinco fungicidas químicos y dos biológicos para el manejo de botrytis cinerea agente causal del moho gris en fresa llanos, a.1*; apaza, w.1 *corresponding author: alejandro.llanosm@hotmail.com abstract gray mold, caused by botrytis cinerea, is one of the most important plant diseases in strawberry in peru. because of its high variability and its pathogenic characteristics, proper fungicides use is essential to control this disease. the aim of this study was to evaluate the effect of five chemical fungicides: tryfloxistrobin (0.0625%), pyrimethanil + fluopyram (0.1%), pyrimethanil (0.1%), fenhexamid (0.135%) and carbendazim (0.125%), and two biological fungicides: bacillus subtilis qst713 (0.75%) and bacillus pumilus qst2808 (0.75%) in laboratory and field conditions. in the laboratory phase, the inhibition of mycelial growth was measured by evaluating diameter growth at 4 dai (p≤0.05). furthermore, conidial germination inhibition was evaluated in two different ways. first, one hundred conidia were placed in sterile water with fungicide and evaluated at 24 hai (p≤0.05). second, the number of cfus was counted in poisoned pda medium at 3 ddi. the doses 1x106 and 125 conidia/ml were used for each test respectively. the field experiment was in aucallama, huaral. chemical and biological fungicides were sprayed in a strawberry cultivar aromas. twelve harvests were done. the results showed that chemical fungicides; trifloxystrobin, pyrimethanil + fluopyram, pyrimethanil, fenhexamid and the biological fungicide bacillus subtilis qst713 had considerable activity against botrytis cinerea in most of the variables assessed. these results will contribute to the evaluation of the potential of each one of these fungicides for the management of botrytis cinerea. keywords. botrytis cinerea, gray mold, fragaria x annanasa, fungicides, strawberry. resumen el moho gris, causado por botrytis cinerea, es una de las enfermedades más importantes de las plantas de fresa en el perú. debido a su alta variabilidad y sus características patogénicas, el uso adecuado de fungicidas es esencial para controlar esta enfermedad. el objetivo de este estudio fue evaluar el efecto de cinco fungicidas químicos: tryfloxistrobin (0.0625%), pyrimethanil + fluopyram (0.1%), pyrimethanil (0.1%), fenhexamid (0.135%) y carbendazim (0.125%) y dos fungicidas biológicos bacillus subtilis qst713 (0.75%) y bacillus pumilus qst2808 (0.75%) en condiciones de laboratorio y de campo. en la fase de laboratorio, la inhibición del crecimiento micelial se midió evaluando el crecimiento del diámetro a 4 dai (p≤0.05). además, la inhibición de la germinación conidial se evaluó de dos maneras diferentes. primero, se colocaron cien conidios en agua estéril con fungicida y se evaluaron a 24 hai (p≤0.05). en segundo lugar, el número de ufc se contó en el medio de pda envenenado a 3 ddi. las dosis 1x106 y 125 conidia/ml se utilizaron para cada prueba respectivamente. el experimento de campo fue en aucallama, huaral. se aplicaron fungicidas químicos y biológicos en un cultivar de fresa aromas. se hicieron doce cosechas. los resultados mostraron que los fungicidas químicos trifloxystrobin, pyrimethanil + fluopyram, pyrimethanil, fenhexamid y el fungicida biológico bacillus subtilis qst713 tuvieron una actividad considerable contra botrytis cinerea en la mayoría de las variables evaluadas. estos resultados contribuirán a la evaluación del potencial de cada uno de estos fungicidas para el manejo de botrytis cinerea. palabra clave. botrytis cinerea, moho gris, fragaria x annanasa, fungicidas, fresa. 1universidad nacional agraria la molina. departamento de fitopatología. facultad de agronomía. lima. perú. introduction strawberry is a fruit with a high content of flavonoids, anthocyanins, and phenolic compounds. this fruit contains vitamins a and c (cano, 2013). strawberry is a worldwideproduced crop. furthermore, it has a high demand due to its fresh consumption. this fruit is widely used for making desserts because its qualities such as color, aromas, and acidity (chaves and wang, 2004). apart from its commercial interest, this crop has a great social value due to the high demand of workers required for its production as well as in post-harvest and in the industry (cano, 2013). this crop is susceptible to several pathogens which antifungal activity of five chemical and two biological fungicides for the management of botrytis cinerea, causal agent of gray mold in strawberry january april 2018 2 can infect at both levels, field and postharvest. among the most common diseases are: anthracnose, caused by the fungus colletotrichum spp. leaf spots, caused by mycosphaerella fragariae; fruit rot, caused by the fungus gnomonia co-mari; powdery mildew, caused by the fungus sphaerotheca macularis; wilting, caused by the fungus fusarium oxysporum; phytophthora crown rot (root rot), caused by phytophthora nicotianae and fruit rot (gray mold), caused by the fungus botrytis cinerea (koike et al., 2016). the latter is considered the main pathogen because it can infect any state of the plant (álvarez, 2012). botrytis cinerea pers.: fr [teleomorph: botryotinia fuckeliana (de bary) whetzel], infects more than 200 different plant species including fruit trees and vegetables. it causes serious economic losses before and after harvesting (benito et al., 2000; rebollar 2011). around 20% of the world crop is affected by b. cinerea causing an investment of billions of euros per year for its control (genoscope, 2005). there are numerous stu dies related to this fungus due to the considerable incidence of it and the economic repercussions that have on important crops such as vine, tomato, strawbe rry, and ornamental crops (benito et al., 2000). in strawberry c u l t i v a t i o n , botrytis cinerea causes large economic losses whose estimates are around 30% of the total production. in highhumidity conditions, this pathogen could cause economic losses between 40-50 %. even in post-harvest, it is more aggressive, affecting 95% of the fruits 48 after harvest (chaves and wang, 2004). infected fruits initially maintain their original shape. at the beginning, the rotten parts are soft and pulpy. then, a mass of mycelium and gray-brown conidia are produced on the surface, making the fruit to get dry. direct infection of the fruit can also occur if the fruits are exposed to water film on the surface. these infections develop in the same way as fruits whose flowers were infected. however, these la tter infections differ in that multiple initial lesions can appear on any part of the fruit’s surface (koike et al., 2016). control of botrytis cinerea is not easy to manage for several reasons: i) it is capable of attacking crops at any stage of development, even post-harvest, ii) it infects any vegetable organ, iii) it is able to grow at very low storage temperatures, and iv) it is genetic and morphologically heterogeneous organism, which allows it to grow and develop in different crop conditions. although there are several types of control strategies that play an important role in mitigating the development of this pathogen, traditionally the most used practice has been chemical fungicides (espinosa, 2006). nevertheless, excessive use of these fungicides leads to the development of resistant strains, environmental contamination, and even health problems in humans, leading to a high cost production in the farming industry (ghorbani et al., 2004; molina et al. 2006). currently, the use of biological fungicides has emerged as an important tool for controlling diseases in agriculture. the aims of this study were: to evaluate the effect of different chemical and biological fungicides in mycelial growth and conidial germination of botrytis cinerea under in vitro conditions and to measure the effect at fruit level in the harvest under field conditions. materials and methods the research was developed in two phases. the first, laboratory phase, consisted of three in vitro tests. the second phase or field phase was carried out in a commercial field evaluating various parameters in the harvest. table 1. fungicides evaluated for the control of botrytis cinerea in the laboratory and field phase treatment fungicide a.i. product frac group* evaluated dose 1 none unsprayed control none none 2 trifloxystrobin flint 50 wg 11 0.063% 3 pyrimethanil + fluopyram luna tranquility 500 sc 9 + 7 0.100% 4 pyrimethanil scala 40 sc 9 0.100% 5 fenhexamid teldor sc 17 0.135% 6 bacillus subtilis strain qst713 serenade aso 44 0.750% 7 bacillus pumilus strain qst2808 sonata aso 0.750% 8 carbendazim fordazim 5 fw 1 0.125% *fungicide resistance action committee fungicides the fungicides evaluated in both phases were: trifloxystrobin (flint 75wg, 0.0625%), pyrimethanil + fluopyram (luna tranquility 500 sc, 0.1%), pyrimethanil (scala 40 sc, 0.1%), fenhexamid (teldor sc, 0.135%), carbendazim (fordazim 5 fw, 0.125%), bacillus subtilis strain qst713 (serenade aso, 0.75%) and bacillus pumilus strain qst2808 (sonata aso, 0.75%). the characteristics of these fungicides are described in table 1. laboratory phase this phase was carried out in the plant pathology laboratory of the universidad nacional agraria la molina. evaluation in mycelial growth botrytis cinerea was isolated from fruits of strawberry cv. aromas that presented typical signs and symptoms in huaral, aucallama. this fungus was placed on papa dextrose agar (pda) and stored at 25 ° c for four days. mycelial plugs were obtained from the growing part of the fungus to obtain a pure culture. the fungicides were mixed with pda culture medium at the indicated concentrations (table 1). each treatment llanos, a; apaza, w. peruvian journal of agronomy 2 (1): 1-8 (2018) 3 consisted of four 8.4 cm petri dishes which were filled with 25 cc of the mix solution. 0.6-cm-diameter mycelium plugs of b. cinerea were placed in the central zone of each petri dish. treatments were incubated at 25 ± 1 cº. mycelial growth was measured every 24 h until the control covered the surface of the medium. the percentage of inhibition mycelial growth was determined by the following equation: eq. (1) img =inhibition of mycelial growth mgf = mycelial growth with fungicide treatment mgc = mycelial growth with the control or unsprayed treatment evaluation of conidial germination to evaluate the effect of fungicides on conidial germination, two tests were established. in the first test, a solution of conidia at a concentration of 1x106 conidia/ ml was prepared. a drop of the obtained concentration (0.2 ml) was placed on the slide. subsequently, a drop of fungicide solution with the established dose was placed on top of the first drop. four repetitions were established per treatment. these slides were placed in humid chambers at room temperature to conserve moisture. germination was defined when the germ tube reached half the length of the coni dium (jackson et al., 1985). this test was evaluated every 4 h for a period of 24 h, taking 100 conidia per repetition. in the second test, a concentration of 125 conidia/ ml was prepared. these conidia were placed in petri dishes with pda poisoned except for the control. the conidia were scattered with the help of drigalski triangle throughout the culture medium. finally, these conidia were incubated at 25 c ° +/1. the percentage of inhibition of conidial germination was determined by the following equation: eq. (2) icg = inhibition of conidial germination gf = germination with the fungicide treatment gc = germination with the control or unsprayed treatment field phase features of the experimental field the field trial was carried out in huaral, lima. the crop had been established approximately two and a half months before the installation of the experiment. the strawberry cultivar was aromas. the field was a furrow irrigation. the experiment was installed the beginning of flowering stage. by the time of installing the experiment, no application of fungicides had been sprayed. the field had disea-ses attacks in previous seasons. in addition, due to the high relative humidity, the field presented necessary conditions for the development of the pathogen. the experimental design was a randomized complete block design (rcb). four blocks and thirty-two plots were established (figure 1). the field showed an average temperature of 15.65 ° c with a maximum of 25 ° c and a minimum of 11.9 ° c. finally, the average relative humidity of the field was 94.94% during the trial. plant categorization due to variability of plants within the plots, a categorization was established. to obtain a more precise weight and number of fruits in the evaluations. the plants were separated into 4 categories due to their size: category 1 (large), category 2 (medium), category 3 (small) category 4 (very small). because the size of the plants had a direct effect on production, plants of category 4 were excluded. these plants did not show a normal development. they had growth, flowering, and production problems. figure 1a. figure 1b figure 1a layout of plots in the experimental area; figure 1b. distribution of assessed plants (e) and no-assessed plants (x) in the plot. antifungal activity of five chemical and two biological fungicides for the management of botrytis cinerea, causal agent of gray mold in strawberry january april 2018 4 treatment application chemical fungicides were sprayed every other week while biological fungicides were sprayed weekly. in total, 6 applications of chemical fungicides and 3 applications of biological products were established. a 20l-capacity backpack was used. each fungicide treatment was applied with its respective commercial dose. all treatments were mixed with kinetic (adjuvant) at 0.0125% concentration. the a pplications presented an average water expenditure of 550 l/ha. the first application started once the presence of the pathogen was confirmed. assessment evaluation the total number of evaluations (harvests) was twelve. they were conducted twice a week (monday and friday). the evaluation was carried out in the two central rows of each plot. every plot consisted of four rows, each row was formed by 2-line of strawberry plants (double row so-wing). (figure 1). the fruits were evaluated in three cate-gories. i) fruits of the first category: fruits of great size and good conformation (>3.5 cm). ii) fruits of second category: small fruits and/or fruits with some deformations (<3.5 cm) iii) fruits with the presence of botrytis cinerea: fruits with symptoms or signs of the pathogen. data of yield and number of fruits were taken in every plot. the fruits of the first and second category were classified a ccording to the commercial standards required by the bu-yer. once harvested, the fruits were counted, weighed and separated by the aforementioned categories. area under disease progress curve (audpc) the area under the disease progress curve (audpc) was calculated through the number of fruits with the presence of botrytis cinerea. the incidence of each harvest was obtained by dividing the number of strawberry fruits accumulated at the moment of the harvest between the number of fruits obtained during the development of the research (total number of fruits with bc from harvest 1 to 12) this value was obtained by means of the following equation: σ = summation of n observations n = i-th observation yi = proportion of disease (incidence) affected in the i-th observation. ti = time (days) after the i-th observation adjusting yield and number of fruits. to estimate the real values in each category, the data obtained by the central rows were adjusted by the following procedure: the weight and the number of fruits obtained by evaluation (2-rows area) were divided among the number of plants considered productive. this procedure was necessary to obtain the average weight (gr) and number adjusted of fruits per plant. then, to obtain the yield per plot the adjusted average weight obtained per plant was multiplied by the average number of plants in the central furrows (64 plants). finally, to calculate the yield and the number of fruits per ha. the values were taken from the area of the two central furrows (9 m2). statistical analisys the data obtained were evaluated by using the statistical software sas 9.0 (statistical analysis system) (p≤0.05). figure 2. effect on mycelial growth of chemical and biological fungicides using the poisoned culture technique 4 days after placing in the petri dish. mycelial disc of botrytis cinerea was used as inoculum. llanos, a; apaza, w. peruvian journal of agronomy 2 (1): 1-8 (2018) 5 analysis of variance (anova) was performed under a completely randomized design (crd) in the laboratory phase and a randomized complete block design (rcb) in the field phase. in all cases, four repetitions were established. the statistical analysis of means was by the tukey test (p≤0.05). results and discussions laboratory phase effect of fungicides on mycelial growth the results are shown in table 2. the analysis of variance in mycelial growth showed significant differences (p≤0.05) those that presented an excellent control in the mycelial development of botrytis cinerea were fenhexamid, bacillus subtilis strain qst713, pyrimethanil + fluopyram and pyrimethanil (table 2, figure 2). the effect of these active ingredients on botrytis cinerea has also been mentioned at 4 days after inoculation with the mycelial plug. at that moment the control occupied the entire area of the culture medium, coinciding with that reported by plascenciatenorio et al. (2012). of all evaluated treatments, figure 3. effect of chemical and biological fungicides on conidial germination after 24 hours. conidial solution on water was used. la molina, 2015.. individually by elad et al. (2007); vidhyasekaran (2004); veloukas and karaoglanidis (2012); vitale et al. (2016) and nguyen et al. (2005). effect of fungicides on conidial germination the germination results of conidia in sterile water and pda medium are shown in table 2. these results showed highly significant differences (p≤0.05) after 24 hours in the percentage of germination. initial germination could be observed after 4 hours coinciding by la torre & rioja (2002). in these two in vitro tests, the active ingredients that showed an inhibition greater than 90% were the treatments pyrimethanil + fluopyram, pyrimethanil, fenhexamid and trifloxystrobin as chemical fungicides, and bacillus subtilis as a biological fungicide. these former treatments did not show statistical differences among them (p>0.05) (table 2, figure 3). these results coincide with rebollar table 2. effect of chemical and biological fungicides in in vitro tests for mycelial growth and conidial germination of botrytis cinerea. n° fungicide a.i. product mycelial growth conidial germination steril water conidial germination pda diam. growth pimg (%) germination (%) picg (%) cfu picg (%) 1 none unsprayed control 8.40 a 0.00 37.75 a 0.00 137.5 a 0.00 2 trifloxystrobin flint 50 wg 5.68 c 32.14 0.75 dc 98.01 1.75 d 98.73 3 pyrimethanil + fluopyram luna tranquility 500 sc 0.00 e 100.00 0.00 d 100.00 1.50 d 98.91 4 pyrimethanil scala 40 sc 0.00 e 100.00 0.00 d 100.00 2.00 d 98.55 5 fenhexamid teldor sc 0.00 e 100.00 0.50 dc 98.68 1.75 d 98.73 6 bacillus subtilis strain qst713 serenade aso 0.00 e 100.00 2.25 c 94.04 5.25 cd 96.18 7 bacillus pumilus strain qst2808 sonata aso 6.89 b 18.50 31.25 a 17.22 56.00 b 59.27 8 carbendazim fordazim 5 fw 1.60 d 80.95 22.75 b 39.74 16.50 c 88.00 c.v.(%) 3.5 14.8 19.82 (2011), elad et al. (2007), veloukas and karaoglanidis (2012) and lisboa (2003) who evaluated these active ingredients at this level. regarding trifloxystrobin treatment, its good control at this level coincides with myresiotis et al. (2007) who also states that this type means with the same letters do not present statistical differences (α = 0.05), pimg: percentage of inhibition of mycelial growth picg: percentage of inhibition of conidial germination, cfu: colony forming units antifungal activity of five chemical and two biological fungicides for the management of botrytis cinerea, causal agent of gray mold in strawberry january april 2018 6 of fungicide does not show a dominant effect on the inhibition during the mycelium stage. additionally, elad et al. (2007), mentioned that this behavior could be related to the fact that terminal oxidase (aox), a component present in the mitochondria of botrytis cinerea, allows the flow of electrons avoiding the blocking of cytochrome metabolism caused by strobilurins. however, the chemical fungicide that showed the lowest control in both tests was carbendazim coinciding with elad et al. (2007) who mentioned that this class of fungicides does not prevent the germination of conidia in botrytis cinerea. field phase area under disease progress curve (audpc) according to the analysis of variance in the audpc during the experiment, highly significant differences between treatments were found (p≤0.05). bacillus subtilis, fenhexamid, pyrimethanil, pyrimethanil + fluopyram and trifloxystrobin obtained 131.80, 123.48, 119.58, 107.84 and 95.38 respectively, no showing significant differences between them (p>0.05) (table 3). these treatments had significant differences (p≤0.05) with the control treatment, whose value was the highest (202.65). finally, these results coincide with rosslenbroich and stuebler (2000) who mentioned the effect of some these active ingredients against bc (table 3). effect of fungicides on fruit yield highly significant differences were found in the analysis of variance with the category-1 fruit (p≤0.05). the treatments with the highest value were treated with pyrimethanil and pyrimethanil + fluopyram whose values were 7,685.2 and 7,679.0 kg/ha respectively. these treatments did not present statistical differences between them (p>0.05). these results coincide with wedge (2007) who mentioned the e -ffect of pyrimethanil in the control of bc in strawberry cv. camarosa. in addition, bacillus subtilis showed the highest value of the group of biological fungicides in the performance of category 1. neither the group of chemical treatments nor control presented significant differences with this biological treatment (p>0.05). finally, no significant differences were found table 4. effect of chemical and biological fungicides on the number of strawberry cv. aromas and area under the botrytis cinerea progress curve in field conditions n° ia number of fruits abcpe category 1 category 2 infected fruits * infected fruit 1 none 270 932.0 a 164 662.0 a 52 117.0 a 10.78 a 202.65 a 2 trifloxystrobin 298 616.0 a 154 150.0 a 22 139.0 c 4.72 d 95.38 c 3 pyrimethanil + fluopyram 298 185.0 a 145 664.0 a 24 487.0 c 5.17 d 107.84 bc 4 pyrimethanil 325 907.0 a 176 047.0 a 27 595.0 c 5.24 d 119.58 bc 5 fenhexamid 268 450.0 a 167 682.0 a 25 996.0 c 5.63 dc 123.48 bc 6 bacillus subtilis strain qst713 306 820.0 a 165 800.0 a 39 750.0 b 7.81 bc 131.80 bc 7 bacillus pumilus strain qst2808 259 907.0 a 155 061.0 a 41 581.0 ab 9.14 ab 153.77 ab 8 carbendazim 319 190.0 a 165 016.0 a 43 423.0 ab 8.28 ab 151.67 ab c.v.(%) 12.05 11.35 13.31 15.20 16.07 means with the same letters do not present statistical differences (α = 0.05) * percentage of number of infected fruit in comparison with the total number during the experiment (cat.1 + cat.2 + infected fruit) table 3. effect of chemical and biological fungicides on strawberry cv. aromas yield under field conditions. n° i.a. cat. 1 cat. 2 infected fruit *infected fruit (%) 1 none 5 034.4 b 1 347.7 a 391.8 a 5.97 a 2 trifloxystrobin 6 615.8 ab 1 326.5 a 152.6 bc 1.88 bc 3 pyrimethanil + fluopyram 7 679.0 a 1 227.5 a 152.7 bc 1.69 c 4 pyrimethanil 7 685.2 a 1 484.1 a 119.4 c 1.28 c 5 fenhexamid 5 924.0 ab 1 350.2 a 136.0 c 1.85 bc 6 bacillus subtilis strain qst713 6 401.1 ab 1 347.3 a 321.5 a 3.96 ab 7 bacillus pumilus strain qst2808 5 424.7 b 1 279.9 a 271.3 ab 3.97 ab 8 carbendazim 6 461.4 ab 1 327.7 a 398.0 a 4.90 a c.v.(%) 12.26 13.80 22.05 29.95 means with the same letters do not present statistical differences (α = 0.05) cat. 1: category 1 cat. 2: category 2 * percentage of the yield of infected fruit in comparison with the total yield during the experiment (cat.1+cat.2+infected fruit) llanos, a; apaza, w. peruvian journal of agronomy 2 (1): 1-8 (2018) 7 with category-2 fruit (p>0.05). regarding the number of fruits infected with botrytis cinerea, highly significant differences (p≤0.05) were found in this category. the treatments pyrimethanil + fluopyram with 152.7, trifloxystrobin with 152.6 kg/ ha, fenhexamid with 136 kg/ha and pyrimethanil with 119.4 kg/ha presented the lowest values. these treatments did not present significant differences between them. on the other hand, carbendazim, the control, bacillus subtilis strain qst713, bacillus pumilus strain qst2808 presented the highest values in the yield of fruits infected with botrytis cinerea with 398, 391.8, 321.5 and 271.3 kg/ ha respectively. the control treatment presented the highest fruit percentage of bc with a value of 5.97%. the treatments that presented significant differences (p≤0.05) at this level with the control treatment were trifloxystrobin with 1.88%, fenhexamid with 1.85%, pyrimethanil + fluopyram with 1.69% and pyrimethanil with 1.28%. they did not present statistical differences between them (p>0.05) (table 3). effect of fungicides on the number of fruits. the results did not show significant differences in category 1 and category 2 (p>0.05) (table 4).on the other hand, the analysis of variance in the number of fruits with the presence of botrytis cinerea obtained highly significant differences (p≤0.05).the control treatment presented the highest value, 52 117 fruits/ha. the lowest values were obtained with the treatments pyrimethanil, fenhexamid, pyrimethanil and trifloxystrobin whose values were 27,595; 25,966; 24,487; 22,139 fruits/ha respectively. they did not show statistical differences between them (p>0.05). the analysis of variance showed highly significant differences in the percentage of fruits with the presence of botrytis cinerea (p≤0.05). in this analysis, the control treatment obtained the highest percentage with a value of 10.78%. on the other hand, the treatments that presented the lowest values were: fenhexamid with 5.63%, pyrimethanil with 5.24%, pyrimethanil + fluopyram with 5.17% and trifloxystrobin with 4.72%. these treatments did not show significant differences between them (p>0.05). the effect of fenhexamid on the incidence of affected fruits coincide with sallato et al. (2007) and bay et al. (2010), who found low values of the disease after applications of fenhexamid. however, veloukas and karaoglanidis (2012) found good control of fluopyram against botrytis cinerea in strawberry fruits cv. camarosa. furthermore, wilcox (2007) observed a good effect of trifloxystrobin against this pathogen. regarding biological fungicide bacillus subtilis, elad et al. (2007) and lisboa (2003) stated that this biological fungicide has an effect against botrytis cinerea. conclusion the fungicides that showed high mycelial inhibition were pyrimethanil, pyrimethanil + fluopyram, fenhexamid and the biological fungicide bacillus subtilis strain qst713. regarding conidial inhibition, the fungicides described above and the fungicide trifloxystrobin showed excellent control at this level. under field conditions, the chemical fungicides trifloxystrobin, fenhexamid, fluopyram + pyrimethanil, pyrimethanil and the biological fungicide bacillus subtilis showed a high activity against botrytis cinerea. this study, which show the performance of chemical and biological fungicides, is a baseline to help strawberry growers to develop effective fungicide spray programs, for gray mold in agreement with integrated pest management (ipm). literature cited álvarez, t. (2013). biocontrol de botrytis cinerea a partir de extractos fenólicos de fresa. thesis to opt for the degree of maestro en ciencia en producción agrícola sustentable, instituto politécnico nacional, méxico. http://repositoriodigital.ipn.mx/ handle/123456789/16418 bay, i., eynard, j. and gubler, w. (2010). evaluation of fungicide programs for management of botrytis bunch rot of grapes: 2010 field trial. [on line] california: university of california, davis. retrieved from: https:// escholarship.org/uc/item/8gs9k471 [2017, march 12] benito, e.p., arranz, m. and eslava a. (2000). factores de patogenicidad de botrytis cinerea. revista iberoamericana de micología 17, s43-s46. cano, a. (2013). estrategias biológicas para el manejo de enfermedades en el cultivo de fresa (fragaria spp). revista colombiana de ciencias hortícolas, 7(2), 263276. chaves, n. and wang, a. 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(2003). efectividad de bacillus subtilis y de una cepa nativa de trichoderma harzianum sobre la incidencia y severidad de pudrición gris (botrytis cinerea) en vid vinífera. thesis to opt for the degree of ingeniero agrónomo. universidad de talca, chile. http://dspace.utalca.cl/handle/1950/919 molina, g., zaldúa, s., gonzález, g. and sanfuentes, e. (2006). selección de hongos antagonistas para el control biológico de botrytis cinerea en viveros forestales en chile. bosque, 27(2), 126-134 myresiotis, c.k., karaoglanidis, g.s. and tzavellaklonaris, k. (2007). resistance of botrytis cinerea isolates from vegetables crops to anilipyrimidine, phenylpyrrole, hydroxyanilide, benzimidazole, and dicarboximide fungicides. plant disease, 91(4), 407413. https://doi.org/10.1094/pdis-91-4-0407 nguyen, h., soon-ok, o., gyoung, k., jae-seoun, h., jaeseoun, h. and young, k. (2005). bacillus subtilis s10210 as a biocontrol agent against botrytis cinerea in strawberries. the plant pathology journal, 21(1), 59 – 63. http://dx.doi.org/10.5423/ppj.2005.21.1.059 plascencia-tenorio, r., olalde-portugal, v., menaviolante, h.g., ceja-torres, f., venegas-gonzales, j., oyoque-salcedo, g. and angoa-perez. m.v. (2012). antagonismo in vitro de aislados bacterianos de fresa comercial y silvestre vs botrytis cinerea y rhizopus stolonifer. ra ximhai, 8(3), 103-110. rossleinbroich, h.j. and stuebler, d. (2000). botrytis cinerea – history of chemical control and novel fungicide for its management. crop protection, 19(8), 557-561. https://doi.org/10.1016/s0261-2194(00)00072-7 sallato. b.v., torres, r., zoffoli, p. and la torre, b.a. (2007). effect of boscalid on postharvest decay of strawberry caused by botrytis cinerea and rhizopus stolonifer. spanish journal of agriculture research, 5(1), 67-78. doi: 10.5424/sjar/2007051-224 rebollar, a. (2011). manejo del mildiu y el moho gris de la zarzamora en michoacán (1 ed.) méxico: universidad autónoma chapingo. veloukas, t. and karaoglanidis. g.s. (2012). biological activity of the succinate dehydrogenase inhibitor fluopyram against botrytis cinerea and fungal baseline sensitivity. pest management science, 68(6), 858-864. https://doi.org/10.1002/ps.3241 vidhyasekaran, p. (2004). concise encyclopedia of plant pathology. new york, usa: the haworth press, inc. vitale, a., panebianco, a. and polizzi, g. (2016). baseline sensitivity and efficacy of fluopyram against botrytis cinerea from table grape in italy. annals of applied biology, 169(1), 36-45. https://doi.org/10.1111/ aab.12277 wedge, d., smith, b., quebedeaux, j. and constantin, r. (2007). fungicide management strategies for control of strawberry fruit rot diseases in lousiana and mississippi. crop protection, 26(9), 1449-1458. https://doi.org/10.1016/j.cropro.2006.12.007 peruvian journal of agronomy 3(3): 126–133 (2019) issn: 2616–4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1206 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 01 september 2019 accepted for publication: 20 september 2019 peruvian botanical biopesticides for sustainable development and protection of the environment biopesticidas botánicos de origen peruano para el desarrollo sostenible y la protección del ambiente bracho-pérez, j. (1); tacza-valverde, i. (1); vásquez-castro, j. (2)* *corresponding author: jaque@lamolina.edu.pe abstract daphnia magna is proposed as a bioindicator to establish the minimum concentration capable of controlling pests before performing toxicity tests. this study uses the proposed pest control extracts of two peruvian species, clibadium peruvianum poepp. (seeds) and petiveria alliacea l. (leaves). the toxicological effects of the plant extracts were evaluated with d. magna, using five neonates over a period of 24–48 h. a lack of mobility or the absence of heart rhythm for 15 s under a stereomicroscope was considered to indicate mortality. organic extracts were discarded due to their higher toxicity when compared with the aqueous extracts of c. peruvianum and p. alliacea, which had lc50 = 460.74 mg/l and lc50 = 711.18 mg l −1 at a concentration of 10 mg l−1, respectively. using this daphnia-safe concentration, toxicity tests were performed on the third instar larvae of musca domestica (housefly). higher activity was observed with an aqueous extract of seeds of c. peruvianum and a leaf aqueous extract of p. alliacea, showing 58.33% and 56.7% mortality against m. domestica, respectively. both extracts induced abnormal changes in the development of the housefly, causing deformation, burns, and dehydration of tissues in the larvae. it is evident that using d. magna as a preliminary toxicological test allows the determination of concentrations that are safer to use while maintaining the activity of the extracts as a botanical biopesticide, thus posing the lowest risk to the environment, ecosystems, their species, and human health. key words: botanical biopesticides, clibadium peruvianum, daphnia magna, housefly, musca domestica, petiveria alliacea. resumen se propuso el uso de daphnia magna como bioindicador para establecer la concentración mínima capaz de controlar las plagas antes de realizar pruebas de toxicidad. este estudio consistió en el uso de dos especies peruanas, clibadium peruvianum poepp (semillas) y petiveria alliacea l. (hojas). los efectos toxicológicos de los extractos de plantas se evaluaron con d. magna, utilizando cinco neonatos en un período de 24–48 h. la falta de movilidad o ausencia de ritmo cardíaco durante 15 s bajo microscopio estereoscópico se utilizó como un indicador de mortalidad. los extractos orgánicos se descartaron debido a su mayor toxicidad en comparación con los extractos acuosos de c. peruvianum y p. alliacea, que tenían cl50 = 460.74 mg l −1 y cl50 = 711.18 mg l −1 a 10 mg l−1 de concentración, respectivamente. usando esta concentración segura, se realizaron pruebas de toxicidad en larvas de musca domestica de tercer estadio. se observó una mayor actividad con extracto acuoso de semilla de c. peruvianum 58,33% y extracto acuoso de hoja de p. alliacea 56,7% de mortalidad contra m. domestica. ambos extractos indujeron cambios anormales en el desarrollo de la mosca común, causando deformación, quemaduras y deshidratación de los tejidos de las larvas. es evidente que el uso de d. magna como prueba toxicológica preliminar permite el uso de concentraciones más seguras, manteniendo la actividad de los extractos como un bioplaguicida botánico y presentando el menor riesgo para el medio ambiente, los ecosistemas, sus especies y la atención de la salud humana. palabras clave: bioplaguicida botánico, clibadium peruvianum, daphnia magna, mosca común, musca domestica, petiveria alliacea. 1 universidad nacional tecnológica de lima sur. facultad de ingeniería y gestión. campus sector 3 grupo 1a 03 cercado (av. central y av. bolívar) villa el salvador, lima 42, perú 2 universidad nacional agraria la molina (unalm), departamento de entomología, facultad de agronomía, av. la molina s/n, lima 12, perú introduction one of the organisms most used in toxicological studies with pesticides is the crustacean daphnia magna straus (cladocera: daphniidae). because d. magna is easy to rear in the laboratory, and has advantages such as short life cycle and high susceptibility to pesticides and other environmental pollutants, it has become a worldwide reference species for this type of study (mansour et al., 2015; qi et al., 2018). bracho-pérez, j.; tacza-valverde, i.; vásquez-castro, j. peruvian journal of agronomy 3(3): 126–133 (2019) 127 the housefly, musca domestica l. (diptera: muscidae), is considered a vector of viruses, bacteria, and protozoa that cause many diseases in humans and other mammals and has become an important public health concern (urzúa et al., 2010a; sripongpun, 2008). management of m. domestica has been carried out through the application and extensive use of synthetic insecticides, which in turn have caused serious environmental problems, such as the development of pest resistance, ecological damage, and irreversible damage to human health (vásquez, de baptista, trevizan, & gadanha, 2008; ahmed, zain, & irfanullah, 2004; nivsarkar, cherian, & padh, 2001; kristensen & jespersen, 2003; taşkin, kence, & göçmen, 2004; khalaf, hussein, & shoukry, 2009). a vast number of studies conducted worldwide have focused on the insect-control uses of insect growth regulators (igrs) and plant secondary metabolites (céspedes, salazar, martínez, & aranda, 2005; torres et al., 2003; magalhães et al., 2010; pohlit, rezende, lopes, lopes, & neto, 2011; urzúa et al., 2010b). plant-derived compounds that are rich sources of bioactive chemicals provide promising alternatives to the current use of chemical pesticides for insect control (khalaf et al., 2009). the asteraceae family constitutes an important source of secondary metabolites, such as monoterpenes, sesquiterpenes, sesquiterpene lactones, diterpenes, triterpenes, flavonoids, coumarins, polyacetylenes, and benzofurans (raal et al., 2011; alvarenga, ferreira, emerenciano & cabrol-bass, 2001; ferreira, brant, alvarenga, & emerenciano, 2005). the asteraceae family is widely distributed in perú, where there are almost 250 genera and 1590 species (beltrán et al., 2006). one species of this family, clibadium peruvianum poepp. ex dc. (asterales: asteraceae), is known as “huaca” in the rainforest of perú. rainforest natives have long used leaves from c. peruvianum as a fishing aid (arriagada, 1995; arriagada, 2003; pérez, muñoz, noyola, & garcía, 2006; bohm & stuessy, 1981; bohm & stuessy, 1985; czerson, bohlmann, stuessy, & fischer, 1979). the use of piscicidal extracts in fishing is considered favorable because these extracts are not persistent chemicals. furthermore, they may represent a good pest control alternative that avoids the use of synthetic and non-biodegradable products. for instance, rotenone is a piscicide that is also currently used as a botanical control in agriculture (harada, 1996; gabriel & okey, 2009). petiveria alliacea l. (phytolaccaceae) is a perennial herbaceous plant native to the amazon rainforest and widely distributed in other areas, including tropical and central america, africa, sri lanka, and the southeastern united states. it is known as “mucura.” preparations of this plant have been widely used in the traditional medicine of south and central america to treat many disorders. reports indicate that it has anti-inflammatory, antimicrobial, anticancer, and stimulant effects, among others. most studies of this plant have focused on its roots and on medical–pharmacological applications (kubec, kim, & musah, 2002; williams, rosner, levy, & barton, 2007; luz et al., 2016). however, some studies have shown that this plant also possesses great potential in controlling pests and that it is not necessary to depredate (uproot or destroy the plants) it for this purpose, since an effective preparation does not require the use of the plant’s roots. in this sense, several studies of leaf and stem extracts have demonstrated their ability to control pests, such as via acaricidal and insecticidal action against larvae and adults of the cattle tick rhipicephalus (boophilus) and whitefly (bemisia tabaci genn.), respectively (rosado et al., 2010; cruz, gamboa, borges, & ruiz, 2013). this study aimed to evaluate the toxicological effects of hexane, ethanol, and aqueous extracts of c. peruvianum and p. alliacea on d. magna in order to determine the extract with the lowest mortality risk and then to carry out toxicity tests on the third instar larvae of m. domestica. materials and methods material two peruvian species, c. peruvianum poepp. (seeds) and p. alliacea l. (leaves), were collected from both tingo maría city, rupa-rupa district, situated in the huánuco department (09°24´ s, 75°58´ w), and castillo district, leoncio prado province, located in the huánuco department (09°16´ s, 76°00´ w), in august 2016. the weberbauer herbarium, universidad nacional agraria la molina, then confirmed the identification of both plants when voucher specimens were deposited there. the plants were dried in an oven at 40 °c for 48 h and pulverized, and then 10 g of each dried plant material part was extracted with ethanol, hexane, or distilled water using an ultrasound bath at 48 °c for 2 h. the extracts were filtered, and the organic extracts were dried on a rotary evaporator under reduced pressure at 38 °c and dissolved in propylene glycol and distilled water. preparation of extracts dry ground seeds (3 × 10 g) and leaves (3 × 10 g) were extracted with 100 ml of 96% ethanol, hexane, or distilled water by ultrasonication at 48 °c for 2 h and then filtered. the organic extracts were dried on a rotary evaporator under reduced pressure at 38 °c and dissolved in propylene glycol. test organism experiments were conducted with strains of d. magna. cultures of d. magna were maintained at a temperature of 20 °c ± 2 °c in water at ph 7.14, with conductivity of 10 µs cm−1, alkalinity of 93.5 mg caco3 l −1, and total hardness of 45.6 mg caco3 l −1. the d. magna were fed peruvian botanical biopesticides for sustainable development and protection of the environment september december 2019 128 with the algal species pseudokirchneriella subcapitata according to iso 6341 (2012), oecd 202 (2004), and usepa 2021.0 (2002) methods. acute ecotoxicity a reference test with sodium chloride (nacl) was also conducted to test the sensitivity of d. magna (24-h old). the static toxicity test was performed in plastic vessels containing 4 ml of test medium. for each concentration, five neonates were exposed to five test concentrations of dried extracts per liter from 1% (1000 mg l−1) to 0.01 mg l−1 (10 mg l−1), without feeding during the test, for 24 and 48 h. for each concentration, three replicates were used. plastic vessels were incubated in darkness at 20°c ± 2°c. the immobilization of treated organisms or the absence of heart rhythm for 15 s under a stereomicroscope was used to indicate mortality. low environmental risk decision taking into account the results obtained from the acute ecotoxicity test, the vegetable extracts and respective concentrations that exhibited toxicity against d. magna were discarded. the remaining extracts were used to carry out the control bioassays for the selected pest, m. domestica. residual contact toxicity bioassays were performed in order to demonstrate the insecticidal activity of each extract against m. domestica. for this purpose, each of the selected extracts from both plant types was mixed with an increasing amount of distilled water to obtain four solutions with dilutions ranging from 100% (undiluted) to 10% (100, 80, 50, and 10). an aliquot of 1 ml of each extract was applied to each of four petri dishes. in each petri dish, the applied liquid was spread over the entire treatment area to form a thin film, which was evaporated under the environmental chemistry laboratory conditions (25 °c ± 2 °c and 65% ± 2% relative humidity [rh]) for 10 min. once the treatment area was dry, 40 third instar larvae of m. domestica were placed in each petri dish, and the dishes were covered and sealed with parafilm. the same process was performed with distilled water as a control. four replicates per treatment were used in the experiment. for evaluation, the adults that emerged after 15 days were counted (pérez, bracho, & vásquez, 2012). data analysis taking into account the data related to acute ecotoxicity, the lc50 (concentration lethal to 50% of the population) was determined by applying the probit analysis as part of an appropriate statistical program (ibm spss statistics 22). student’s t-test was used to examine the hypothesis of possible differences between the mean results from the two treatments. the efficacy of treatments (percentage of mortality at different concentrations) was evaluated through a one-way analysis of variance (anova), with a significance level of p = 0.05 after transformation of the data to square root of the arcsine to ensure a homogeneity of variances, using ibm spss statistics (zar, 1996). results and discussion acute ecotoxicity clibadium peruvianum poepp. (huaca) the seeds of c. peruvianum were selected as the organ of interest for this plant, based on the study of pérez et al. (2012), which demonstrated that the alcoholic extract of its seeds had higher insecticidal activity compared with the other organs of the plant. studies of ecotoxicity using d. magna have demonstrated that at 48 h of exposure, the lethal average concentration reached its highest value (lc50 = 460.74 mg l−1) for a concentration of 10 mg l−1 (0.01%), demonstrating that the aqueous extract of the seed possesses a minor toxic effect, unlike the more powerful effects of the alcoholic extracts (lc50 = 164.36 mg l −1) and hexanic extracts (lc50 = 371.47 mg l −1). as a result, the aqueous extract of the c. peruvianum became the only extract considered for further studies of biopesticide activity and development of future formulations for pest control (table 1). petiveria alliacea l. (mucura) the acute toxicity results of the extracts drawn from the leaves of the mucura plant demonstrated that the aqueous extract also exhibited the highest lethal concentration (lc50 = 711.18 mg l−1) compared with those of the alcoholic extract (lc50 = 255.50 mg l −1) and hexanic extract (lc50 = 65.36 mg l−1); the hexanic extract showed mortality in a concentration range of 10-100 mg l−1, equivalent to 0.1%0.01% (table 2). the results obtained indicate that the aqueous extract of the p. alliacea has the lowest acute toxicity, even lower than the aqueous extract of c. peruvianum. however, both aqueous extracts must be considered since they both generate the lowest toxic effect to m. daphnia, which is the selected bioindicator. this implies that the other extracts should no longer be considered in order to reduce future environmental toxicity risks, and 10 mg l−1 (0.01%) of dried extract per liter was the most appropriate concentration for the development of biopesticide activity studies. insecticidal activity the results of the insecticidal activity reveal that both the aqueous extracts prepared caused mortality higher than 50%, specifically, 58.33% for c. peruvianum (huaca) and bracho-pérez, j.; tacza-valverde, i.; vásquez-castro, j. peruvian journal of agronomy 3(3): 126–133 (2019) 129 56.70% for p. alliacea (mucura). this indicates that huaca has a greater tendency to maintain a higher insecticidal effect (table 3). however, the means of observed mortality are not homogeneous. by applying student’s t-test, it can be determined that the hypothesis of the difference between the means of the two determinations is not confirmed. the level of significance (p) of this test is lower than that of levene’s statistic, f = 0.05, for huaca compared with mucura, obtaining p = 0.262 and p = 0.167, respectively. this means that the equality of the means was rejected. therefore, the means obtained must be transformed by applying the square root of the arccosine and then by table 1. toxic effect of clibadium peruvianum poepp. (huaca) extracts in terms of mortality of daphnia magna during 24 and 48 h of exposure. extract concentration (mg l−1)1 number of individuals of d. magna casualties 24 h 48 h mortality (%) casualties mortality (%) c lib ad iu m p er uv ia nu m p oe pp . ( hu ac a) aqueous 10 15 0 0.00 1 6.67 100 15 1 6.67 3 20.00 500 15 7 46.67 9 60.00 1000 15 8 53.33 10 66.67 10000 15 15 100.00 15 100.00 2lc50 674.93 460.74 ethanolic 10 15 3 20.00 4 26.67 100 15 5 33.33 7 46.67 500 15 11 73.33 14 93.33 1000 15 14 93.33 15 100.00 10000 15 15 100.00 15 100.00 lc50 295.84 164.35 hexanic 10 15 2 13.33 5 33.33 100 15 4 26.67 8 53.33 500 15 6 40.00 10 66.67 1000 15 9 60.00 13 86.67 10000 15 15 100.00 15 100.00 lc50 942.24 371.47 1mg l−1 = mg of dry aqueous extract per liter, 2lc50 is the concentration of c. peruvianum extracts that kill 50% of the test population of daphnia magna within a designated period. extract concentration (mg l−1)1 number of individuals of d. magna casualties 24 h 48 h mortality (%) casualties mortality (%) p et iv er ia a lli ac ea l . ( m uc ur a) aqueous 10 15 0 0.00 0 0.00 100 15 0 0.00 0 0.00 500 15 0 0.00 5 33.33 1000 15 8 53.33 10 66.67 10000 15 15 100.00 15 100.00 2lc50 973.45 711.18 ethanolic 10 15 0 0.00 1 6.67 100 15 0 0.00 4 26.67 500 15 9 60.00 10 66.67 1000 15 14 93.33 15 100.00 10000 15 15 100.00 15 100.00 lc50 446.09 255.50 hexanic 10 15 3 20.00 5 33.33 100 15 9 60.00 11 73.33 500 15 13 86.67 13 86.67 1000 15 15 100.00 15 100.00 10000 15 15 100.00 15 100.00 lc50 104.78 65.35 table 2. toxic effect of petiveria alliacea l. (mucura) extracts in terms of mortality of daphnia magna during 24 and 48 h of exposure. 1mg l−1 = mg of dry aqueous extract per liter,2lc50 is the concentration of p. alliacea extracts that kills 50% of the test population of daphnia magna within a designated period. peruvian botanical biopesticides for sustainable development and protection of the environment september december 2019 130 related to the effect of inhibiting the fly growth process by substances known as igrs, a consideration that has been supported by other authors (khalaf et al., 2009; youssef, 1997; wang, li, & lei, 2005). on the other hand, because the insecticidal activity considered in this study was found in extracts of the seeds and leaves, it is not necessary to kill the plants to obtain the needed raw material for these insecticides. the plants can continue to grow, thus preserving the species. it is very remarkable that the dilution chosen for the aqueous extract of c. peruvianum showed a pattern of biological activity similar to previous studies on this species, but at a much lower concentration. both aqueous extracts caused deformations in the larvae, but to a lower extent. it is evident that using a bioindicator, such as d. magna, as part of the preliminary toxicological tests allows the choosing of the safest concentrations of a biopesticide while maintaining biological activity. finally, emphasis should be placed on the importance of continuing the chemical composition studies to establish the relationship between chemical composition and biological activity. this will enable the design of increasingly environment-friendly formulations of pest control, capable of protecting pest ecosystems, the species that inhabit them, and human health. conclusions daphnia magna can be used as a bioindicator to determine the minimum concentration capable of controlling pests before performing toxicity tests on pest species. toxicological evaluation with d. magna demonstrated that alcoholic and hexanic extracts of c. peruvianum and p. alliacea are more toxic to d. magna than the aqueous extract, evidencing the greater environmental impact of organic extracts. the aqueous extracts of c. peruvianum and p. alliacea are environment-friendly, as measured by their relatively low toxicity to d. magna, and the appropriate extract that should be used for the control of m. domestica. finally, the toxicity tests carried out on musca domestica demonstrated that both aqueous extracts of c. peruvianum and p. alliacea on d. magna have great potential for insect pest control. references ahmed, s., abdin, z., & irfan, m. 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(1997). toxic and synergistic properties of several volatile oils against larvae of the house fly, musca domestica vicina maquart (diptera: muscidae). journal of the egyptian-german society of zoology, 22, 131–149. zar, j. h. 1996. biostatistical analysis. prentice-hall. inc. upper saddle river. new jersey, usa. peruvian journal of agronomy 1 (1): 1420 (2017) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v1i1.1063 © the authors. published by universidad nacional agraria la molina received for publication: 16 october 2017 accepted for publication: 2 2 december 2017 development of advanced mutant lines of barley with higher mineral concentrations through radiation-induced mutagenesis in perú desarrollo de líneas mutantes avanzadas de cebada con mayores concentraciones de minerales a través de mutagénesis inducida por radiación en perú gomez, l.1*; aldaba, g.1; ibañez, m.1; aguilar, e.1 *corresponding author: luzgomez@lamolina.edu.pe abstract in peru it is very important to increase food quality and production in the rural areas where a high poverty and malnutrition problems are found. mutation induction is used to improve well adapted cultivars, by upgrading one or two characteristics, while retaining all its original attributes. hordeum vulgare mutant lines were developed from the cultivar unalm 96, following irradiation at 200 and 300 gray. mutant lines were selected in the m8 generation with higher agronomic performance and nutritive quality. they were adapted to the highlands with grain yield within the range of 5, 100 and 8,731 kg/ha, over the value of the parental material (4,246 kg/ha) and showed improved contents of p (131 mg/g dry weight-dw), zn (66 mg/g dw), mn (55 mg/g dw), fe (57 mg/g dw) and cu (63 µg/g dw). keywords: barley, mutation, gamma irradiation, quality resumen en perú es muy importante aumentar la calidad de los alimentos y la producción en las zonas rurales donde se encuentran altos problemas de pobreza y malnutrición. la inducción de la mutación se utiliza para mejorar los cultivares bien adaptados, mediante la mejora de una o dos características, y conservando todos sus otros atributos. las líneas mutantes de hordeum vulgare se desarrollaron a partir del cultivar unalm 96, después de la irradiación a 200 y 300 gray. las líneas mutantes se seleccionaron en la generación m8 con mayor rendimiento agronómico y calidad nutritiva. se adaptaron a las tierras altas con un rendimiento de grano dentro del rango de 5, 100 y 8,731 kg/ha sobre el valor del material parental (4,246 kg/ha) y mostraron mejores contenidos de p (131 mg/g de peso seco-dw), zn (66 mg/g dw), mn (55 mg/g dw), fe (57 mg/g dw) y cu (63 μg/g dw). palabras clave: cebada, mutación, irradiación gamma, calidad 1cereals and native grains research program, universidad nacional agraria la molina, av. la molina s/n, lima 12, perú introduction agriculture in the high andean region of peru is done under adverse conditions, with frequent drought and frost and in impoverished soils and mostly oriented to auto subsistence production. above 3000 masl, the rural population uses mostly potatoes, barley, wheat, quinoa, beans and peas as staple food, and at lower altitudes maize, amaranth, among other crops. diets consumed in the highland usually contain various sources of dietary protein, among them a mixture of cereals (barley, wheat and maize) with native grains (quinoa and amaranth). this mixture has a high biological value because amino acids and other nutritive compounds are provided by cereals (jood and singh, 2001; mckevith, 2004; mahesh et al., 2010). barley (hordeum vulgare) is an important introduced food crop in the highlands, and 70% of barley are used for human consumption as pearled grains, flakes and flour (gomez-pando et al., 2005). it could be a main source of minerals for low income rural population because it is a staple food in the peruvian andean region. it has agronomic value because its adaptability to marginal soils and climates. it has nutritious and medicinal value because it contributes with carbohydrates (~80%), proteins (6.9 to 25.0%), lipids (0.99 to 3.2%), minerals (~2 to 4%), fiber and other compounds to the diet (kent, 1971; newman and mcguire, 1985; ulrich, 2001). in terms of minerals, barley is a good source of potassium, magnesium and phosphorus, iron, sulfur, copper, zinc, manganese, chromium, selenium, iodine and molybdenum. it is considered an excellent source of vitamins b (villacres, 1996) partly due to its content of soluble fiber (beta glucans), it protects the intestinal mucous membranes and reduces blood sugar levels (fastnaught, 2002; gordon, 2002; finocchiaro et al., 2005). national yield average of barley in peru is relatively low, it is about 1,499.7 kg / ha (faostat, 2016). the production does not satisfy domestic demand so it is important to increase the quantity and quality of barley production in order to improve the nutrition and profitability of farmers in the andean region. gomez, l.; aldaba, g.; ibañez, m.; aguilar, e. peruvian journal of agronomy 1(1):14-20 15 there are various plant breeding tools for obtaining increased productivity and quality, e.g., mutation induction. mutation breeding has been used for improving various crops (micke, 1998; jain et al., 1998; jamil and khan, 2002; muthusamy et al., 2003; chopra, 2005; gnanamurthy et al., 2012; kurowska et al., 2012; mehlo et al., 2013; tshilenge-lukanda et al., 2013; oladosu et al., 2016, raina et al., 2016, gozukirmizi and karlik, 2017). mutations can improve agronomic traits and genetic quality while conserving valuable traits of existing varieties, including adaptation, quality, etc. mutations are random and the values of the mutations depend on changes in the morphology and physiology of plants that impact the agronomic and quality performance. the present investigation was undertaken to induce useful mutations in barley using gamma irradiation. the present investigation was undertaken to induce useful mutations in barley using gamma irradiation. materials and methods genetic materials—the experimental material was the barley commercial cultivar unalm96. management of mutant population—barley dry seeds were gamma irradiated at doses of 200 and 300 gray. treated seeds of each dose along with an equal number of control (untreated) seeds were grown. all surviving plants were harvested individually to the m2 generation and radio sensitivity was evaluated through germination, seedling survival, stem length and root length. the m2 population was screened, the spectrum and frequency of mutations was established and chlorophyll mutations were identified using gustaffson’s classification (gustaffson, 1947). in the m3 generation progeny, tests were conducted to determine the inheritance of changes or likely mutations in the m2 generation. during the m4 to m8 generations, agronomic and quality characters were evaluated. field control practices were based on standard managements for barley. determination of quality traits— determination of physical characteristics—thousand kernel weight and test weight with the protocol of icarda (1988). determination of chemical characteristics—grain protein content was made following the protocol of asbc (1992). mineral grain content—mg 24, p 131, s 34, k139, zn 66, ca 44, mn 55, fe 57 and cu 63 was made by james hutton laboratories in the uk in dundee, using icp-ms – jat technology. evaluation of agronomic traits—yield (kg/ha), life cycle (days), plant height (cm) and disease response were recorded. general descriptive statistics—for each characteristic, data matrix was constructed using microsoft office 2007. the analysis started with basic descriptive statistics: mean standard deviation (sd) and coefficient of variation (cv). selection of mutant lines for quantitative traits—to select the mutant lines, a range of theoretical values were established with values in most cases greater than or less than 10 20 percent from those mean values of the parental material. yield trials were conducted using a randomized block design with three replications results and discussion somatic effects—in the m1 generation, general reductions of germination, survival, length of seedlings height, length of seedlings root and fertility with the increment of the doses of gamma ray were observed. these somatic effects were reported in other crops with different treatments and mutagens (ciftci et al., 2006; albokari et al., 2012; scaldaferro et al., 2013; arisha et al., 2014). chlorophyll mutation—genetic differences in chlorophyll mutation have been observed. in a population of 342 958 plants, several types of chlorophyll mutants were identified. in six row spike plants, there were albino (0.09%), alboviridis (0.0003%), striata (1.7%), alboxantha (0.0005%), xanthalba (0.0011%), chlorine (0.15%), lutescens (0.005%), tigrina (0,25%) and xantha (0.03%). in two row spike plants, there were tigrina (0,006%) and chlorine (0.02%). in figure 1, some of these mutants are presented. a b a b c d figure 1. chlorophyll mutation types in barley cv unalm 96 irradiated with 200 gy of gamma ray at m2 generation (a: chlorine, b: albino, c: tigrina, d: striata). la molina peru chlorophyll mutations are important markers for the evaluation of genetic effects of mutagenic treatments and have been reported in various crops (gomez et al., 2009; bhosale and hallale, 2011; ugorjiet al, 2012; gnanamurthy development of advanced mutant lines of barley with higher mineral concentrations through radiation-induced mutagenesis in perú junio-setiembre 2017 16 et al., 2012; tshilenge-lukanda et al., 2013; gomez-pando and eguiluz-de la barra, 2013). morphological characteristics mutations—in the m2 and following generations, a wide mutation spectrum was identified. the population that developed with the dose of 200 gray was studied and the following modifications were found: stem color (1.68%), auricle leaf color (3.3%), spike morphology (4.78%), awn color (2%), awn –light curly (0.43%), awnleted (5%), elevated hood (0.18%), sessile hood (0.19%) and naked grain (0.70%). some of these spike morphology mutations are presented in figure 2. figure 2. spike morphology mutation types in barley cv unalm 96 irradiated with 200 gy of gamma ray at m2 generation (a:6 row with different types of awn, b:two row with different types of awn. la molinaperu morphological mutations were reported for barley and other crops (gomez et al., 2009; gnanamurthyet al., 2012; dyulgerova, b. 2012; jagajanantham et al., 2013). some of the morphological mutations had economic importance such as naked grains. barley is used at the highland roasted so naked grains are more easily processed. agronomic traits—in the m3 and following generations, plant height reduction (2.12%) and earliness (0.19%) were observed and the flowering periods were significantly reduced from 10 to 11 days compared to the control. dockter and hansson (2015) identified more than 1000 different short-culm barley mutants. in the m8 generation, 64 lines were selected with higher agronomic performances adapted to the highlands with grain yield within the range of 5100 – 8731 kg/ha, compared with the parental material with 4246 kg/ha (table 1). table 1.grain yield of selected barley mutants of unalm 96 number of lines range of yield (kg/ha) 1 5250 10 5125 7731 11 5100 6863 37 5131 8731 1 5294 2 5106 5856 1 5675 1 5550 unalm 96 4246 improvement of agronomic characteristics using gamma irradiation has been reported in barley and in other crops (gustafsson et al., 1971; khan et al., 2005; gomez et al., 2009; mudibu et al., 2010; muthusamy et al., 2011; kurowska et al., 2012; ugorji et al., 2012; tshilengelukanda et al., 2013; gomez-pando and eguiluz-de la barra, 2013; udensiand ntui, 2013; gomez-pando, 2014; arisha et al., 2014; badr et al., 2014, obare et al., 2014). quality traits—six hundreds and 65 advanced mutant lines in the m8 generation from the variety unalm 96, with different morphological and agronomic types of variation, were evaluated for their grain carbohydrate content and chemical characteristics, e.g., protein and mineral contents. increase and decrease in all the characteristics evaluated were observed in the lines relative to the parental material. almost all evaluated traits were improved and lines were selected with values of more than 20% over parentalal unalm 96. for physical grain traits, 12 mutant lines were identified with improved thousand kernel weight within the range of 70.2 and 78.9 g which was greater than the value of parental material (56 g). for protein content, 21 mutant lines were identified within the range of 12.9 to 14.1%; the parental material had 10.5% protein content. for mineral components, we identified many mutant lines. for mg24, 50 mutants within the range of 1.44 to 4.04 mg/g dw were selected (parent material had 1.167 mg/g of mg 24 (mg/g dw)). for p131, 54 mutant lines ranging from 5.27 7.59 mg/g dw were superior to the parent with 3.980 mg/g dw. for s34, 63 mutant lines ranging from 1.61 – 2.05 mg/g dw of s34 were superior to the parent with 1.28 mg/g dw. for zn66, 67 mutant lines were selected ranging from 0.0574 to 0.0649 mg/g dw, which was superior to the parent with 0.0466 mg/g dw. for ca 44, 32 mutant lines a b gomez, l.; aldaba, g.; ibañez, m.; aguilar, e. peruvian journal of agronomy 1(1):14-20 17 were selected ranging from 0.3976 to 0.4855 mg/g dw, which was superior to the parent with 0.3258 mg/g. for mn 55, 67 mutant lines were selected ranging from 0.0193 – 0.0322 mg/g dw, more than the parent with 0.0158 mg/g dw. for fe57, 16 mutant lines were selected ranging from 0.11 – 0.4761 mg/g dw, which is better than the parent with 0.0417 mg/g dw. for cu63, 72 mutant lines were identified ranging from 0.0099 to 0.0210 µg /g dw, better than the parent with 0.0067 µg /g dw. some mutant lines showed improved resistance to leaf rust, lodging and grain content of p-131 from 2.88 to 6.16 mg/g dw, zn66 from 0.044 to 0.0586 mg/g dw, mn55 from 0.0142 to 0.0382 mg/g dw, fe57 from 0.0436 to 0.1396 mg/g dw and cu63 from 0.0.006 to 0.0142 µg/g dw (table 2). genetic variability for quality characters can be induced successfully through mutations. similar results were reported for barley (muthusamy et al., 2002; gómez et al., 2009; dyulgerova, 2012; mehlo et al., 2013; gómez, 2014), quinoa (gomez-pando, 2014) and other crops (mehlo et al., 2013; muthusamy et al., 2002). weight of 1000 seeds (g) and test weight (kg/hl-1) were improved and are related to the kernel plumpness that are associated with endosperm size and starch content. in general there were values of tkw over and below the value of the parental material (56.8 g). for this character, 12 lines with higher value were selected (65 to 78.9 g). the mass of a thousand grains is a good indicator of quality of malting and feed barley; the amount of malt extract, is positively correlated with grain size (madić et al., 2005; madić et al., 2006). significant breeding progress was observed for 1000-grain weight (ferrio et al., 2004). this trait was enhanced in 106 spring barley accessions of slovak and former czechoslovakia (žakóvá et al., 2006). grain test weight is strongly related to the degree of grain quality (grain size, shape, amount of starch, etc.), the parental material had an average value of 66 kg/hl and naked mutant lines with higher values for this characteristic were identified within the range of 71.1 to 80.3 kg/hl. it mainly depended on the degree of filling, and shape and grain moisture (madić et al., 2005; madić et al., 2006). it is also an indirect indicator of malt extract and a variety with malting quality must have a value >66 kg/hl. the parental material unalm 96 had 10.5% protein content and a group of mutant lines with values over and below the value of the parental material were identified. globally there is evidence of improvement in food and malting barley quality (swanston et al., 2002; tamm, 2003). the use of barley is determined mainly by the protein. from the point of view of the brewery, barley should have a high content of starch as a source of fermentable sugars and protein content between 10 and 12% for good enzymatic activity. from a nutritional standpoint, the barley must have a good carbohydrate and high protein content, and other nutrients such as minerals (emebiri et al, 2003; emebiri et al, 2004; emebiri et al, 2005; iqbalet al., 2007). crops can be enriched with micronutrients using plant breeding and/ or transgenic strategies, because micronutrient enrichment traits exist within their genomes that can be modified for substantially increasing micronutrient levels without negatively impacting crop productivity (welchand graham, 2004). gregorio et al. (2000), white and broadley (2009) and mulualem (2015) indicated that the increment of bioavailable mineral elements in food crops through plant breeding is one important way to contribute to the solution of mineral deficiencies in human population and that can be combined with agronomic characteristics improvement also. velu et al. (2012) and velu et al. (2014) mentioned that is very important to consider the association between micronutrients and agronomic traits such as grain yield, plant height, grain size and quality traits. conclusions the dose applied of 200 and 300 gray induced genetic variability for several studied traits when compared to the parental material. in addition, high doses of gamma irradiation in barley (300 gray) caused severe somatic effects. it was possible to identify improved or novel phenotypes that can be used as cultivars or exploited as source of desirable characters in conventional breeding programs of barley. these improved mutant lines are free of the regulatory restrictions imposed on genetically modified organism. in some advanced mutant lines the change was in one or two desired characters without changing the rest of the genotype. acknowledgements the authors would like to thank the iaea for providing table 2. agronomic and quality characteristics of selected advanced mutant lines of barley from cultivar unalm 96 nº origin m7 lm 09 yield (kg/ha) p131 8 (mg/g dw) zn66 (mg/g dw) mn55 (mg/g dw) fe57 (mg/g dw) cu63 (ug/g dw) cm6h-546 7075 3.93 0.0586 0.0187 0.062 0.0142 cm6h-718 6863 3.52 0.0555 0.0185 0.0671 0.0099 cm6h-658 6656 6.14 0.0516 0.0186 0.0629 0.0097 cm6h-722 6494 3.55 0.0543 0.0179 0.0622 0.0091 cm6h-721 6019 3.75 0.0513 0.0155 0.0548 0.0095 cm6h-542 8731 3.55 0.0535 0.0158 0.0436 0.0096 cm6h-717 6769 3.78 0.0581 0.0282 0.1396 0.0098 cm6h-26 6294 2.88 0.044 0.0142 0.0473 0.006 cm6h-543 7888 3.94 0.0556 0.0169 0.0541 0.0127 unalm 96 4246 4.31 0.0479 0.0162 0.0813 0.0076 development of advanced mutant lines of barley with higher mineral concentrations through radiation-induced mutagenesis in perú junio-setiembre 2017 18 funding for these research projects through iaea’s coordinated research project (crp) “improving nutritional quality by altering concentrations of enhancing factors using induced mutation and biotechnology in crops. references albokari, m.m.a., s.m. alzahrani, and a.s. alsalman. 2012. radio sensitivity of some local cultivars of wheat (triticum aestivum l.) to gamma irradiation. bangladesh journal of botany 41: 1-5. arisha, m.h., b.k. liang, s.n. muhammad shah, z.h. gong, and d.w. li. 2014. kill curve analysis and response of first generation capsicum annuum l. b12 cultivar to ethyl methane sulfonate. genetics and molecular research 13: 10049-10061. american society of brewing chemists. 1992. barley. american society of brewing chemists, st. paul, minnesota. usa. badr, a., h.i. sayed ahmed, m. hamouda, m. halawa, and m. elhiti. 2014. variation in growth, yield and molecular genetic diversity of m2 plants of cowpea following exposure to gamma radiation. life science journal 11: 10-19. bhosale, u.p., and b.v. hallale. 2011. gamma radiation induced mutations in black gram (vigna mungo (l.) hepper). asian journal of plant science and research 1: 96-100. ciftci, c.y., türkan, a.d., khawar, k.m., atak, m. and özcan, s. use of gamma rays to induce mutations in four pea (pisumsativum l.) cultivars. turk. j. biol., 30: 29-37. 2006. chopra, v.l., mutagenesis: investigating the process and processing the outcome for crop improvement. curr. sci.; 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cakmak, i.; hao, y.; singh, r.p., biofortification strategies to increase grain zinc and iron concentrations in wheat.journal of cereal science 59: 365-372. 2014 welch, r.m. and graham, r.d., breeding for micronutrients in staple food crops from a human nutrition perspective. j. exp. bot., 55, 353-364, 2004. white, p. j., and broadley, m. r., biofortification of crops with seven mineral elements often lacking in human diets—iron, zinc, copper, calcium, magnesium, selenium and iodine. new phytol. 182:49-84. 2009. žakóvá, m. and benková, m., characterization of spring barley accessions based on multivariate analysis. comm. biometry crop sci. 1:124-134, 2006. peruvian journal of agronomy 2(3): 44-53 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i3.1210 © the authors. published by universidad nacional agraria la molina received for publication: 15 november 2018 accepted for publication: 30 december 2018 sustainability of lemon (citrus aurantifolia swingle) farms in the province of santa elena, ecuador sustentabilidad de fincas productoras de limon (citrus aurantifolia swingle) en la provincia de santa elena, ecuador santistevan-mendez, m.1, borjas-ventura, r. 1, alvarado-huaman, l. 1, anzules-toala, v. 1, castro-cepero, v. 1, julcaotiniano, a. 1* *corresponding author: ajo@lamolina.edu.pe abstract the objective was to evaluate the sustainability of lemon farms (citrus aurantifolia swingle) in santa elena, ecuador. surveys were applied to a sample of 83 lemon producers from this province, with structured questions about economic, ecological and socio-cultural dimensions. the sustainability was determined with techniques of multicriteria analysis to calculate the economic indicator (ik), ecological indicator (ie) and socio-cultural indicator (isc), to estimate the general sustainability indicator (is gen) of each farm. the 25.3% of the farms had an ik> 2; 60.24% an ie> 2 and 55.4% an isc> 2. the 74.7% of the farms had an is gen <2, which indicates that the farms are not sustainable. key words: dimensions, survey, multicriteria, ecological. resumen el objetivo fue evaluar la sustentabilidad de fincas productoras de limón (citrus aurantifolia swingle) en santa elena, ecuador. se aplicaron encuestas a una muestra de 83 productores de limón de esta provincia, con preguntas estructuradas sobre dimensiones económicas, ecológicas y socio culturales. la sustentabilidad se determinó con técnicas de análisis multicriterio y se calcularon el indicador económico (ik), indicador ecológico (ie) e indicador socio cultural (isc), para estimar el indicador de sustentabilidad general (is gen) de cada finca. el 25.3 % de las fincas tuvieron un ik > 2; el 60.24 % un ie > 2 y el 55.4 % un isc > 2. el 74.7 % del sistema de producción de limón en santa elena tuvieron un is gen < 2, lo que indica que las fincas no son sustentables. palabras clave: dimensiones, encuesta, multicriterio, ecológico. 1 grupo de investigación agricultura y desarrollo sustentable en el trópico peruano. facultad de agronomía. departamento de fitotecnia. universidad nacional agraria la molina. lima (perú). introduction lemons represent approximately 10% of the world citrus production with more than 120 billion tons per year (usda, 2018). ecuador produced 28 000 t annual (fao, 2018), with some provinces as the economic base for the production and commercialization of citrus, especially citrus aurantifolia swingle, known as “lime” or “key lime” or “lemon”. this lemon is the most cultivated species and, with the lemon tahiti (citrus latifolia tan), it amounts to approximately 4 965 ha. in santa elena province, there are 500 hectares of lemon, cultivated by approximately 400 farmers (pidaasse, 2011). deficiencies in management and technological improvement are crucial for the development of the citrus sector, which must be considered with sustainability criteria (santistevan et al., 2017). gómez-limón & arriaza (2011), define sustainable agriculture as one that promotes food sufficiency, conserves natural resources, protects the environment and is economically viable. in addition, agricultural farms should not only generate an economic rent, should also maintain the appropriate conditions of the environment so that the crop develops successfully, minimizing disturbances to the natural environment, but above all that allows to improve the quality of life of the agricultural producers. in ecuador, in recent years, there have been some studies related to the evaluation of sustainability in different agricultural production systems (garcía, 2015; santistevan-mendez, m., borjas-ventura, r. , alvarado-huaman, l. , anzules-toala, v., castro-cepero, v., julca-otiniano, a. peruvian journal of agronomy 2 (3): 44-53 (2018) 45 palomeque, 2016; reina, 2016; santistevan et al., 2016), using the multicriteria analysis with indicators (sarandón & flores, 2009; sarandón et al., 2016). these studies suggest a different and innovative vision in the analysis of agricultural systems and also in the design of future plans for the agricultural development of this country. in this sense, the present study had the objective of evaluate the sustainability of lemon farms (citrus aurantifolia) in the province of santa elena, ecuador. materials and methods the study was carried out in two rural parroquia of the santa elena canton of the province of santa elena, which is located in the litoral region, in the extreme west of the ecuadorian territory (figure 1) at 2°13’36’’ls and 80°51’29’’lo, with an area of 3,762.8 km2, which represents approximately 1.46% of the ecuadorian territory (sambito, 2014). from a finite population of 400 lemon producers a random sample of 83 was taken (scheaffer et al., 1987). to evaluate sustainability, a multicriteria analysis method proposed by sarandón et al. (2006) was used; the indicators were selected and constructed according to the methodology and conceptual framework proposed by sarandón (2002) and sarandón & flores (2009) adapted for a perennial crop such as lemon. these were integrated by indicators and sub-indicators and whose definition was consulted with technicians and farmers in the area of study (márquez & julca, 2015). the information was obtained from a structured survey, which considered closed questions of the dimensions: economic, sociocultural and environmental. then we made the comparisons between farms, the data obtained for each variable was standardized on a scale of 0 to 4. the value 4 represents the highest sustainability and 0 the lowest sustainability (table 1). the values obtained for each subindicator were weighted multiplying it by a coefficient according to the relative importance of each variable with respect to sustainability. the weighting of sub-indicators and variables was valided with technicians and farmers in the area (márquez & julca, 2015). the formulas used to calculate the indicators of sustainability were: economic indicator (ik) = ecological indicator (ie) = sociocultural indicator (isc) = with economic (ik), environmental (ia) and social (isc) indicators values found, the general sustainability index (isgen) was calculated, weighting the three dimensions equally, according to the definition of sustainability. it was considered that to be sustainable a farm must have an isgen greater to 2 and, in addition, none of the three dimensions individually, should have an indicator with a value less to 2. figure 1. location of the province of santa elena in ecuador sustainability of lemon (citrus aurantifolia swingle) farms in the province of santa elena, ecuador september december 2018 46 economic dimensión profitability of the farm (a) net income (b) economic risk (c) value a1 a2 a3 a4 a5 b c1 c2 c3 4 >20 >60 <5 100 340-350 >610 >4 0-20 4 3 15-20 60-50 5.1-10 75 330-339 550-600 4 21-40 3 2 11-14 45-49 10.1-15 50 320-329 500-549 3 41-60 2 1 9-10 40-44 15.1-25 25 310-319 450-499 2 61-80 1 0 <9 <40 >25 <25 <310 <450 1 81-100 0 ecological dimensión soil life conservation (a) risk of soil erosion (b) management of the bd (c) value a1 a2 b1 b2 azc 4 100 total 0-5 b2-1 >1 3 75-99 alta 6-15 b2-2 0.5-1 2 50-74 media 16-30 b2-3 0.25-0.5 1 25-49 baja 31-45 b2-4 <0.25 0 <25 mnc >45 b2-5 0 sociocultural dimensión value satisfaction of basic needs (a) social integration (b) c&ce (c) a1 a2 a3 a4 b c 4 a1-1 a2-1 a3-1 a4-1 very high c1-1 3 a1-2 a2-2 a3-2 a4-2 high c1-2 2 a1-3 a2-3 a3-3 a4-3 media c1-3 1 a1-4 a2-4 a3-4 a4-4 low c1-4 0 a1-5 a2-5 a3-5 a4-5 null c1-5 legend: in economic dimension: a1: productivity t ha-1; a2: physical quality of the lemon (mm); a3: incidence of pests and diseases (%); a4: use of rootstock for lemon subtle (%); a5: density of plantation (plants ha-1). b: net income (dollars); c: economic risk; c1: diversification in production (number of products); c2: dependence of external inputs (%); c3: numbers of commercialization routes for lemon. in ecological dimension: a1: management of vegetation cover (%); a2: crop diversification; mnc: monoculture; b: risk of soil erosion; b1: predominant slope (%); b2: soil conservation; b2-1: level curves or terraces; b2-2: live and dead barriers; b2-3: borderlines; b2-4: staggered against the slope; b2-5: sowing in favor of the slope; c: biodiversity management (bd); azc: areas of conservation area (ha). in sociocultural dimension: a1: housing (material); a1-1: concrete; a1-2: mixed; a1-3: wood; a1-4: reed; a1-5: do not have your own home. a2: access to education. a2-1: university or institute. a2-2: high school. a2-3: elementary and secondary school. a2-4: elementary and secondary school. a2-5: without access to education. a3: access to health and health coverage. a3-1: adequate infrastructure and permanence of doctors. a3-2: medically equipped and temporary doctors. a3-3: badly equipped and temporary doctors. a3-4: badly equipped and without qualified personnel. a3-5: without health center. a4: basic services. a4-1: installation of all services. a4-2: installation of water and energy. a4-3: installation of light and well water. a4-4: no installation of light and well water. a4-5: without light and without water. b: social integration; c: ecological knowledge and awareness (c & ce). c1-1: wide view on ecology, beyond your farm. c1-2: knowledge of ecology from everyday practice. no use of agrochemicals, conservation practices. c1-3: partialized view of ecology. awareness about some practices that affect the environment. c1-4: they do not present an ecological knowledge. but it uses low input practices. c1-5: no kind of ecological awareness. table 1.sub-indicators and indicators used to evaluate the sustainability of lemon producing farms in santa elena, ecuador. the mathematic formula to calculate the isgen was: general sustainability index (isgen) = the sustainability analysis was carried out individually for the 86 farms. cluster multivariate analisis technique was used to select four “type farms” and the critical points of the three dimensions of sustainability to be compared were analysed. the “type farms” are those that represent a group of farmers or production systems with similar characteristics (salazar, 2012; tuesta et al., 2014; santistevan et al., 2015; collantes, 2016). santistevan-mendez, m., borjas-ventura, r. , alvarado-huaman, l. , anzules-toala, v., castro-cepero, v., julca-otiniano, a. peruvian journal of agronomy 2 (3): 44-53 (2018) 47 table 2.most important characteristics of “type farms” producing lemon in santa elena, ecuador (santistevan et al., 2015). table 3.scores obtained in each of the sub-indicators in the evaluation of the sustainability of lemon producing farms, in santa elena, ecuador. characteristic type i type ii type iii type iv number of farms (%) 44.6 15.7 26.5 13.3 average total area of farmland (ha) 2.3 4.3 4.5 16.5 average area with lemon (ha) 1.2 1.2 2.5 4.5 average n° plants/ha 250 275 260 280 investment/ha/year (us$) 325 400 625 1450 monthly income (us$) 450 540 675 700 location manglaralto colonche colonche manglaralto economic dimension profitability of the farm (a) net income (b) economic risk (c) a1 a2 a3 a4 a5 b c1 c2 c3 value 1.6 1.9 1.1 3.2 2 0.8 0.8 1.2 0.7 ecological dimension soil life conservation (a) risk of soil erosion (b) management of the bd (c) a1 a2 b1 b2 azc value 1.4 0.6 3.6 3.2 1.6 sociocultural dimension satisfaction of basic needs (a) social integration (b) knowledge and ec (c) a1 a2 a3 a4 b c value 3.6 1.8 1.9 2.7 1.4 1.9 results the lemon farms in santa elena can be organized into four groups (santistevan et al., 2015) and the most important characteristics of the “type farms” of each group are presented in table 2. table 3 shows that when assessing economic sustainability, for most variables, lemon farms had average values far from four, ranging from zero to four. for example, in the sub-indicator of profitability sub-indicator (a), the variable use of rootstock for lemon (a4) had a value close to four, with the mandarin cleopatra as the most frequent rootstock; the variables a1, a2, a3 and a5 values less than 2. the indicator of net income (b) the value was 0.8, i.e. between 200 and 300 us$/month. the sub-indicator economic risk (c) has the highest value in dependence on external inputs (c2=1.2); but in all cases the values were less than two. the economic indicator (ik) was greater than two in only 25.30% of the farms evaluated, a result that indicates that most of the farms were not economically sustainable (figure 2). table 3 also presents the results of the ecological sustainability assessment. for the sub-indicator conservation of soil life (a), variables management of vegetation cover (a1) and crop diversification (a2) have values less than two. for risk of soil erosion (b), subindicators predominant slope variable (b1) and soil conservation (b2) haved values greater than two, the farmes use land with slopes less than 5% and the crop is installed using contour lines or terraces, repectively. the legend: in economic dimension: a1: productivity; a2: physical quality of the lemon; a3: incidence of pests and diseases; a4: use of rootstock for lemon subtle; a5: planting density. b: net income: c1: diversification in production; c2: dependence of external inputs; c3: numbers of commercialization routes for lemon. in ecological dimension: a1: management of vegetation cover; a2: crop diversification; cd: monoculture; b1: predominant slope; b2: soil conservation. management of bd: management of biodiversity. mb: areas of conservation. in sociocultural dimension: a1: housing (type of material); a2: access to education; a3: access to health and health coverage; a4: basic services; b: social integration; c: knowledge and ecological conscience sustainability of lemon (citrus aurantifolia swingle) farms in the province of santa elena, ecuador september december 2018 48 biodiversity management sub-indicator has the variables areas of conservation zones (azc) with a value less than two. the ecological indicator (ie) is greater than two in the 60.24% of the farms evaluated, a result that indicates that most of the farms are ecologically sustainable (figure 2). as for socio-cultural sustainability, table 3 shows that, for most variables, lemon farms had average values of less than four also ranging from zero to four. for the subindicator satisfaction of basic needs (a), both variables housing material (a1) and basic services (a4) had values greater than two, i.e. the houses are made of concrete and have water, drainage and telephone installations. the subindicator social integration (b) had a value less than two and the same was found for the sub-indicator ecological knowledge and awareness (c). the sociocultural indicator (isc) was greater than two in 55.4% of the farms evaluated, a result that indicates that most of the farms were socioculturally sustainable (figure 2). in general, 74.7% of the farms evaluated had an isgen less than 2 (figure 2), i.e. only 25.3% of lemon farms are currently sustainable. discussion dimensions of sustainability economic sustainability is understood as the set of strategies that allow the agricultural business to remain financially viable over time and provide an acceptable livelihood for the rural family (lien et al., 2007). in this study, only 25.3% of farms are economically sustainable (figure 2), while 74.7% do not, because lemon production is affecte by low productivity and low fruit quality. the incidence of pests and diseases is high, and although the use of patterns for lemon cultivation is an adequate and widespread technique among producers in this area, it is not enough to obtain high yields or to improve the quality of lemon (table 3). the values of productivity and plantation density indicate that there is potential for increasing productivity, with densities of 312 plants ha-1, productivity values of up to 42.1 t ha-1 have been reported (medina et al., 2004). likewise, there may be other factors that would affect the production of citrus aurantifolia such as the inappropriate use of fertilizers, use of unimproved cultivars, lack of weed control and others. this would indicate the need for comprehensive improvement in crop management, including agronomic practices such as pest, disease and weed management (lardizabal & medlicott, 2013). the monthly net income is low, below $300, which is related to low productivity and fruit quality. in addition, having only one marketing channel and a high dependence on external inputs shows the high financial risk assumed by lemon farmers in the santa elena area. however, santa elena farmers have chosen to specialize in this crop; this is probably because the farmer cannot diversify or because alternative crops that can be grown in their area do not have the profitability they seek. a high dependence on external inputs carries a danger for farmers as they can increase in price regardless of the price of the product (in this case the lemon). this would cause an increase in production costs and a crisis of economic resources that would result in this small producer being expelled from the market (altieri figure 2. evaluation of sociocultural sustainability (isc), ecological (ie), economic (ik) and general sustainability index (isg) of lemon producing farms, in santa elena, ecuador. santistevan-mendez, m., borjas-ventura, r. , alvarado-huaman, l. , anzules-toala, v., castro-cepero, v., julca-otiniano, a. peruvian journal of agronomy 2 (3): 44-53 (2018) 49 & nicholls, 2010). the low social integration (table 3) shows that small farmers are not organized, do not have bargaining power and are highly dependent on wholesalers who determine the final price of the product. ecological sustainability seeks to improve the wellbeing of the farmer by protecting the resources used to meet human needs and by ensuring that the production of waste does not cause harm to the farmer himself (goodland, 1995). only 60.24% of lemon farms are ecologically sustainable, while 39.76% did not reach acceptable levels of sustainability (figure 2); producers who achieved positive levels of sustainability implement practices that reduce the risk of soil erosion, such as the use of soils with little slope and soil conservation practices (table 3). in this regard, peña et al., 2016; sanclemente & patiño, 2015 and murillo et al., 2016, indicate that the good vegetation cover not only conserves the soil, but also improves its characteristics which in turn can have positive effects on the growth and development of the cultivated plants. although the implementation of this agricultural practice should be well studied, otherwise it could cause a decrease in crop yields (salazar et al., 2012). on the other hand, crop diversification not only helps to increase the conservation of tropical soils, but also has an important effect on the biology of soils, favoring the growth and development of different species within the agroecosystem and generating extra income for farmers (fernández et al., 2015). work to improve ecological sustainability must be done on the management of low vegetation cover and diversification, for example, with the implementation of other crops or other citrus species as oranges or mandarins. the management of biodiversity on lemon farms can bring great benefits to farmers, such as a decrease in the use of external inputs (blanco, 2016; paleologos et al., 2017). socio-cultural sustainability refers to agriculture that provides meaningful and equitable employment for farmers, workers and their families and produces food for a broad group of consumers (pilgeram, 2011). in this study, 55.4% of farms are socio-culturally sustainable (figure 2). in santa elena, a majority group of lemon farmers live in houses built with concrete material or are of mixed type, have basic education or have no formal education and the community has a medical center that has neither the adequate infrastructure nor the trained personnel to maintain the health of lemon farmers. on the other hand, the farmers in this area have electricity in their homes and get their water from wells. various authors use the education factor as a socioeconomic index within sustainable agriculture (machado & ríos, 2016) and access to drinking water as a key indicator of environmental sustainability (fausto & justo, 2006). obtaining water from wells can be a risk for the inhabitants of santa elena, since it can have high concentrations of microorganisms and other agents harmful to human health (méndez et al., 2015; zegarra, 2017), so more in-depth studies are needed on the type of water that is consumed by these populations in order to ensure that it meets all standards for consumption. the low access to education and health services (table 3), shows that the work of the government in the study area is deficient and that it is necessary to reverse this situation to improve the socio-cultural sustainability of lemon production in this area. while low social integration and ecological knowledge and awareness, suggest the need for greater dissemination of the advantages that have the producer organization and ecological awareness for the improvement of this dimension of sustainability. critical points of sustainability the concept of sustainability is complex because it implies simultaneously fulfilling several objectives: productives, ecologicals or environmentals, social, culturals, economics and temporal (sarandón & flores, 2009). therefore, the “critical points” can change from one production system to another and it is logical to find some differences between the four types of farms evaluated (table 2). the analysis of the economic dimension (figure 3) shows that, in the short term, pest and disease control should be improved, which, added to an increase in plantation density, will have a favorable impact on higher farm productivity and an improvement in the quality of the lemon, which would improve family income. in the medium term, access to new markets should be explored, which would help to improve family income. in the environmental dimension (figure 3, da), in the short term, the management of vegetation cover and the increase in the area dedicated to conservation zones could be improved. in the medium term, the task could be achieving a greater diversification of crops, focusing on associated crops accompanied by natural vegetation which would favor the beginning of certification processes for the lemon as organic, sustainable production and others. the certification of agricultural sustainability of lemon (citrus aurantifolia swingle) farms in the province of santa elena, ecuador september december 2018 50 figure 3. critical points of sustainability in the economic (ik), environmental (ie) and sociocultural (isc) dimensions in “lemon-producing” type farms in santa elena, ecuador. santistevan-mendez, m., borjas-ventura, r. , alvarado-huaman, l. , anzules-toala, v., castro-cepero, v., julca-otiniano, a. peruvian journal of agronomy 2 (3): 44-53 (2018) 51 products allows to enter other types of market that in some cases could mean improvements in price up to 30%. in the analysis of the socio-cultural dimension (figure 3) the need for improvements in the variables of access to education, social integration and technological knowledge and ecological awareness is evident. finally, for a farm to be considered sustainable, the general index (is gen) must be greater than two and none of the three indicators must have a value less than two (sarandón et al., 2006). with these criteria, only 25.3% of lemon farms are currently sustainable. these results show that achieving sustainability of agricultural production systems is a complex task, where improvement is not always the same in each of the three dimensions of sustainability. the results obtained in this study are consistent with other studies carried out in ecuador in recent years, where sustainability has been evaluated in various agricultural production systems (santistevan et al., 2015; garcía, 2015; palomeque, 2016; reina, 2016), which suggest a different vision for the future design of agricultural development plans for the country. however, at the present time a low number of sustainable farms is observed, which is mainly explained by the results obtained in the variables related to the economic and sociocultural dimensions, which shows the low level and quality of life of agricultural producers. changing this situation is not always in the hands of the farmers because it depends mainly on other instances such as local, regional and national government that must invest in infrastructure and services to improve the competitiveness of this productive sector (santistevan et al., 2016). conclusion the majority (74.7%) of the lemon producing farms in santa elena (ecuador) are not sustainable, however adequate techniques are presented such as soil conservation, predominant slope, use of rootstock and planting density that could be replicated in other systems of production. references altieri, m. & nicholls, c. 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(2017). evaluación de agua no tratada de pozos, en la zona sur de la ciudad de juliaca, 2017. revista científica investigación andina, 17(1), 207-216. http://revistas.uancv.edu.pe/index.php/ rcia/article/viewfile/325/269 peruvian journal of agronomy 3(3): 104–111 (2019) issn: 2616–4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i3.1229 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 05 november 2019 accepted for publication: 29 november 2019 fertilisation methods for commercial yield in three garlic cultivars (allium sativum l.) métodos de fertilización para rendimiento comercial en tres cultivares de ajo (allium sativum l.) condor, j.(1); olivera, j.(2)*; pinedo, r.(3) *corresponding author: agols4407@gmail.com abstract garlic (allium sativum l.) is an important crop for domestic consumption in peru. however, there is insufficient information available on crop management, particularly on fertiliser application to local cultivars. in order to evaluate the response of three garlic cultivars to three fertilisation methods, an experiment was conducted at the donoso experimental station in huaral district, a province of lima. three garlic cultivars were used as experimental materials: ‘cincomesino’, ‘arequipeño 14’ and ‘margosino’. three methods of fertilisation were applied as treatments: broadcast application before furrowing (m1), fertilisation in a superficial groove or false furrow (m2), and fertilisation in the lateral furrows, or band application (m3). the experiments were installed in three parcels for each cultivar, with a randomised complete block design for each parcel and four replications. in general, localised fertilisation methods showed the best performance for the broadcast method. regarding total yield, fertilisation at the sides of the furrow (m3) for ‘cincomesino’ reached 13.08 t/ha. the highest yield for the ‘arequipeño 14’ cultivar (12.25 t/ha) was achieved using fertilisation with a surface groove or false furrow (m2). for the ‘margosino’ cultivar, fertilisation on the sides of the furrow was ideal, and the yield was 10.95 t/ha. keywords: broadcast application, crop yield, false furrow, ‘margosino’, nitrogen resumen el ajo (allium sativum l.), es un cultivo de importancia para el consumo interno en el perú. no existe suficiente información disponible sobre el manejo de cultivo, principalmente la fertilización aplicada a los cultivares de uso local. con el objetivo de evaluar la respuesta del rendimiento comercial de ajo con tres métodos de fertilización, se realizó un experimento en la estación experimental donoso (inia), ubicada en la provincia de huaral, región lima. como material experimental se emplearon tres cultivares de ajo: ‘cincomesino’, ‘arequipeño 14’ y ‘margosino’ y tres métodos de fertilización: fertilización al voleo antes del surcado (m1), fertilización en surco superficial o falso surco (m2) y fertilización en los laterales del surco o aplicación en banda (m3). los tratamientos fueron instalados en tres parcelas, una para cada cultivar, bajo un diseño de bloques completos al azar, con cuatro repeticiones. los resultados mostraron que los métodos de fertilización localizados destacan sobre el método de fertilización al voleo. en relación con el rendimiento total, para el cultivar ‘cincomesino’ con el método m3 alcanzó el mejor rendimiento de 13.08 t/ha, seguido del cultivar ‘arequipeño 14’, que alcanzó el mayor rendimiento de 12.25 t/ha con m2 y el cultivar ‘margosino’ el mayor rendimiento resultó 10.95 t/ha con el tratamiento m3. palabras clave: falso surco, ‘margosino’, nitrógeno, rendimiento de cultivo, voleo 1 instituto nacional de innovación agraria (inia) estación experimental agraria (eea) donoso–huaral. lima, perú. 2 instituto nacional de innovación agraria (inia) estación experimental agraria (eea) donoso–huaral, consultor del programa de hortalizas. lima, perú. 3 universidad nacional agraria la molina (unalm). facultad de agronomía. docente del departamento de fitotecnia. lima, perú. introduction garlic is native to central and southern asia, from where it spread to the mediterranean and then to the rest of the world. by around 3000 b.c.e., it was being consumed in india and egypt (garcía, 1990). by the end of the 15th century, the spaniards had introduced it to the americas (campelo, arboleya, maeso, paullier & giménez, 2016). garlic is one of the oldest herbaceous crops (salomon, 2002). in peru, the most widespread garlic cultivars are susceptible to diseases caused by viruses and other pathogens as well as to shortages of quality seed (nicho & cóndor, 2012). their production is intended for both internal consumption and export as fresh and processed condor, j.; olivera, j.; pinedo, r. peruvian journal of agronomy 3(3): 104–111 (2019) 105 products. in the second quarter of 2017, the area of this crop sown nationwide was 7,600 ha. in the fourth quarter of 2017, the national yield was 10.8 t/ha. in the arequipa region, the average yield was 12.3 t/ha (sifuentes, albújar, & cajas, 2017; sifuentes, albújar, & cajas, 2018). small and medium farms are the primary producers of this crop and most of the national production is destined for the domestic market. the use of pathogen-free seed allows an increase in the yield of this crop, in some cases exceeding 50% (olivera, 2009; salomon, 2002). the wide gap between the current low yields and the potential yield reveals a series of factors that limit garlic production. some of these limiting factors are environmental, while others are related to agronomic or cultural practices (usman, fagam, dayi, & isah, 2016). timely availability of nutrients is an important factor affecting the quantity and quality of garlic crops (burba, 2003). proper management of the nutrition of the different garlic cultivars through the timely application of fertilisers is part of the production process which, in combination with other factors, promotes an increase in the yield and quality of crops. in addition, given the increasing price of fertilisers and the environmental costs associated with their excessive use, it is necessary to increase the efficiency of pesticide use (harold & reetz, 2016; lipinski & gaviola, 2006; mirzaei, liaghati, & damghani, 2007). the production of horticultural plants requires multiple applications of fertilisers (ugás, siura, delgado, casas & toledo, 2000). fertilisers that are added to the soil undergo transformations that can modify their availability. their application methods are directly related to the plant’s use of nutrients and the changes these nutrients undergo in the soil. to the extent possible, the application methods used should be economical, accurate and effective (california fertiliser association, 1995; harold & reetz, 2016). a fertilisation plan must take into account the variability of environmental and management factors such as irrigation and tillage in order to distribute nitrogen with flexibility, according to the specific conditions of each campaign. a reduction in yield can be avoided by improving the efficiency of nitrogen fertilisation, incidentally mitigating the negative environmental impacts resulting from excess application (harold & reetz, 2016; quemada, delgado, mateos, & villalobos, 2017). fertiliser (organic or mineral) application methods are essential components of good agricultural practices. the amount and regulation of absorption depend on several factors, such as crop variety, planting date, crop rotation, soil conditions and weather. in efficient agricultural practices, the farmer selects the quantity and opportunity over time, so that plants use the nutrients as efficiently as possible (asociación internacional de la industria de los fertilizantes [aiif], 2002). recent soil analyses have shown that most soils are deficient in nitrogen (about 100%) and phosphorous (about 90%); therefore, proper management of these two elements is critical for good crop production. the proper management of fertilisers should focus on the maximum use of the applied p and k. for this purpose, alternatives to pre-sowing application over the entire surface, such as localised applications or application in bands (delgado, quemada, villalobos & mateos, 2017), must be taken into account. similarly, the dose of fertilisers should be based on the results of soil analysis, taking as reference a fertiliser dose of 220-100-200 npk. it is also recommended to perform the first fertilisation 15 d after planting, with 1/3 of the nitrogen and all phosphorus and potassium, followed by a second nitrogen fertilisation at 30 d and a third at 60 d after the initial application (nicho & cóndor, 2012). there are various methods of application, such as spreading fertiliser uniformly over the entire surface of the land, or broadcasting and modified broadcasting, which is similar, except that fertiliser is applied only on the bed or in thin bands along the bed (sierra, simonne, & treadwell, 2007). another system involves localised placement at the base of the plant. in this case, the fertiliser must be located under the teeth or next to them in order to avoid failures in sprouting (garcía, 1990). nitrogen is usually the only nutrient with insufficient availability for the cultivation of garlic, and the use of this element in three applications during cultivation is common practice. according to burba (2003), the first application should be 30% of the dose, the second 35% and the third 35%, while aguado, portela, and lipinski (2014) recommends 25%-35%-40%, starting 50 or 60 d after the beginning of leaf emergence. broadcast fertilisation (that is, application to the surface of a crop field) is mainly used in dense crops not sown in rows or sown in dense rows (small grains) or in meadows. it is also used when fertilisers need to be incorporated into the soil after application (phosphate fertilisers) or to prevent losses from the evaporation of nitrogen (urea, diammonium phosphate). incorporation by tillage or plowing is also recommended to increase fertility of the entire plow layer. if the fertiliser is spread by surface broadcasting by hand or with fertiliser distribution equipment, the spreading should be as uniform as possible (aiif, 2002). in canada, on clay soils, fertiliser can be broadcast and incorporated prior to planting white garlic to bring soil fertility to 80 kg ha−1 n and 33 kg ha−1 p (bandara, krieger, slinkard, & tanino, 2000). when fertiliser application is localised (placed only in selected places in the field), it is concentrated in specific parts of the soil during planting, either in bands, in a strip below the soil surface, or next to and under the seed. this process can be carried out by hand or by means of special sowing equipment and/or equipment for fertiliser application (seed sowing and fertiliser) (aiif, 2002). in the furrow irrigation system, the most appropriate form of nitrogen fertiliser application is burying it next to the plant line. when adequate machinery is not available, the fertiliser can be placed in the furrow immediately fertilisation methods for commercial yield in three garlic cultivars (allium sativum l.) september december 2019 106 after irrigation so that it dissolves and is incorporated (lipinski, 2015). to reduce fertiliser runoff, nitrogen applications should be split: the first application should be by broadcasting and the second and third application in bands (nicho & cóndor, 2012). the growth of the garlic plant is slow during the first 100 d; therefore, there is low absorption of nutrients. during that time, the plant partially uses reserve substances in the cloves. this absorption becomes more intense from 100 d on, extending to the end of the crop development (campillo & toro, 2002). after sowing, both the number of leaves and plant height increase. the onset of bulb formation is characterised by an increase in the ratio of bulb diameter to neck diameter. in this phase, the number of teeth per plant increases rapidly, leaf growth rate decreases and finally, the increase in plant height stops (brewster & rabinowitch, 2002). starting with the hypothesis that there are differences in fertiliser application methods that affect the yield of local garlic cultivars, the objective of the present study was to determine the most appropriate method of fertilisation for local garlic cultivars. materials and methods the experiment was conducted between july 2017 and january 2018. the plots were located in the experimental agricultural station donoso-huaral, 5.6 km from the city of huaral, in the region of lima at a south latitude 11°14′ and 180 masl. the experiment was arranged in a random complete block design and three treatments were installed in each plot corresponding to each cultivar, in four distributed random blocks. four furrows were installed in each experimental unit. the average temperature of the huaral valley (data obtained from the weather station of the eea donoso-huaral, corresponding to the year 2017) during the months of crop growth was 17.3 °c in july and 21.6 °c in december, while the average relative humidity reached 58.5% in july. the three experimental plots were located in a sandy strip, according to the analysis of soils with low levels of salts (electrical conductivity: 0.52 ms / cm), moderately alkaline ph (7.71) and low organic matter content ( 0.7%), typical of coastal soils (valladolid, 2001); also high in phosphorus (43 ppm) and potassium (234 ppm), low in calcium carbonate (10.56%) and medium cation exchange capacity (cec) with 10.86. the cultivars under study were ‘cincomesino’ (c1), ‘arequipeño 14’ (c2) and ‘margosino’ (c3). the genetic material was provided by the national vegetable research programme of the inia and the treatments for each cultivar included three methods of fertilisation: method 1 (m1), broadcast application before furrowing; method 2 (m2), fertilisation in a surface groove or false furrow; and method 3 (m3), lateral furrow or band fertilisation. figure 1 shows the bulbs and cloves of cultivars ‘cincomesino’, ‘arequipeño 14’ and ‘margosino’. according to loayza, nicho, cahuas, and cosme (2005), the early garlic cultivars are those in which the beginning of gradual foliar drying from the basal part to the apical part of the plant is carried out from the fourth figure 1. bulbs and cloves of three garlic cultivars, first calibre: (a) ‘cincomesino’, (b) ‘arequipeño 14’ and (c) ‘margosino’. condor, j.; olivera, j.; pinedo, r. peruvian journal of agronomy 3(3): 104–111 (2019) 107 month to the fifth month, 15 days. this group includes the cultivar ‘cincomesino’, which is widespread in peru, with a vegetative period of 5.0 to 5.5 months (early) and ‘arequipeño 14’; both belong to the napurí garlic group. the ‘margosino’ cultivar belongs to the late cultivar group, for which the beginning of gradual foliar drying from the basal to the apical part occurs from the fifth to the sixth month (loayza et al., 2005). based on the results of the soil analysis, we used ammonium sulfate, diammonium phosphate and potassium sulfate at a fertilisation level of 250-80-100 npk, taking into account the recommendations proposed by the vegetable programme of the national agrarian university (ugás et al., 2000) of 200-80-100 npk. however, fertilisation levels differed among experiments. the experimental data were statistically analysed for each plot corresponding to a different cultivar by analysis of variance using the statistical software sas at p ˂ 0.05, and the means were compared using the duncan test. manual sowing was carried out at 10 cm between plants in double rows with a 60 cm groove, as recommended by ugás et al. (2000). with regard to insect pests, the presence of ‘thrips’ was evaluated, and foliar diseases such as botrytis caused by botrytis porri and rust caused by puccinia allii were also evaluated. control was performed for both pathogens using insecticides and fungicides. surface irrigation was carried out weekly based on the physical characteristics of the soil using gravity and the slope of the land. the bulbs were harvested when 70% of the foliage was dry (145 d after planting), and there was a shortening of the vegetative period due to weather conditions during the experiment. vegetative parameters were determined for the three cultivars. plant height was collected by measuring from the soil surface to the top of the longest leaf of 10 randomly selected plants using a ruler at physiological maturity of the crop, and the mean values in cm were computed for further analysis. to determine the neck thickness of the bulbs using a caliper, 10 random plants were evaluated at the neck level of the plant. number of leaves per plant was recorded by counting the number of leaves of 10 randomly selected plants per plot at physiological maturity, and the mean value was recorded. regarding yield parameters, three variables were evaluated. for bulb weight, 10 random bulbs were evaluated and the average bulb weight was determined. average bulb size was recorded by measuring the diameter at the middle (equatorial diameter) of 10 randomly selected bulbs in each plot using a caliper, and from this parameter, the shape of the bulb was determined. with regard to the polar diameter of bulbs, 10 randomly selected bulbs were evaluated with the help of a caliper– an evaluation that helped us determine the bulb shape. bulbs were sorted by calibre in accordance with the classifications used in the internal market, such as the wholesale market of lima, although there is also an export classification mentioned in loayza et al. (2005) and nicho and cóndor (2012). among the harvested bulbs, first-calibre bulbs with 6 to 8 cm equatorial bulb diameter were classified then weighed to determine the bulb weight. the variable yield of second-calibre bulbs included those whose equatorial diameter was in the range of 4 to 6 cm, and the third-calibre yield included those with an equatorial diameter of 2 to 4 cm. to define the total yield/ha, all commercial bulbs were weighed to calculate the yield in t/ha. results the experiment was conducted with slightly increased mean temperatures compared to previous campaigns; this climatic variation showed some minor changes in the vegetative period of culture and in the process of clove differentiation, in both cases shortening the process. likewise, conditions were favourable for the presence of pests and diseases, which had an impact on crop production cost (nicho & cóndor, 2012). table 1 shows no statistical difference in plant heights of the early garlic cultivar ‘cincomesino’. m1 showed the best results but did not significantly differ from the other treatments. for neck thickness, no statistical difference between treatments was found, although m1 had the best results. the same occurred with the number of leaves, with a slight increase in m1. for bulb polar diameter, there was a statistical difference between treatments. m1 had the highest value, followed by similar means of m3 and m2 (figure 2). for equatorial bulb diameter, there was no statistically significant difference between treatments, since all treatments resulted in the same mean diameter (figure 2). for the yield variable (first calibre) of cv. ‘cincomesino’ (table 2), there was no significant statistical difference between treatments, while m3 had the highest value. for second-calibre yield, there was no significant difference between treatments; m2 had the highest value, similar to that of m1. in the third-calibre yield, there was no significant difference between treatments, and m3 had the highest value. for total yield, the treatments showed no significant statistical difference, while m3 had the highest value (table 2). treatments plant height (cm) neck thickness (mm) leaf number m1 (1)72.1 (3)9.12 (1)11.9 m2 (3)72.0 (1)8.72 (2)11.8 m3 (2)70.3 (2)8.47 (3)11.7 cv% 2.12 5.71 1.65 table 1. effect of fertilisation methods on vegetative garlic growth in cv. ‘cincomesino’ fertilisation methods for commercial yield in three garlic cultivars (allium sativum l.) september december 2019 108 between m1 and m2, but they were comparable to that of m3. for total yield at the winter planting (july), there were no significant differences, but m3 stood out at 11.0 t/ha, followed by m1 and m2 at 10.9 t/ha. plant height (table 3) showed no significant difference between treatments in cv. ‘arequipeño 14’. m1 reached a greater height than m2 and m3, which were 69.1 and 68.4 cm, respectively. for the neck thickness variable, m1 was 10.0 mm, followed by m2 (in a superficial groove or false furrow), with m3 (fertilisation in the lateral furrows or band application) in third place. for leaf number, no significant difference was found between treatments; the results were similar for m1 and m2, with the same number of leaves and both values slightly greater than those of the m3 treatment. the duncan test for polar diameter showed a statistical difference. m3 treatment had the highest value (figure 3), but the values were similar in the other treatments. for equatorial diameter, m3 had the highest value, showing a greater difference compared to m1 and m2.in cv. ‘arequipeño 14’ (table 2), the first-calibre yield showed no significant differences, while in the secondcalibre yield, m2 was higher than m1 and m3. for the third-calibre yield, m2 showed the highest value. for total yield, m2 displayed the highest value. for cv. ‘margosino’ (table 2), the first-calibre yield showed no significant difference between treatments, but in the m2 yield, it was superior and similar to that of ‘arequipeño 14’; however, it only reached 4.0 t/ha in m2. for the second-calibre yield, there were no significant differences either, and in terms of yield in t/ha, m1 and m2 were at 4.67 t/ha, followed by m3 at 4.02 t/ha. for the third-calibre yield, there were no significant differences figure 2. effects of different methods of fertilisation on garlic bulb size (mm) in cv. ‘cincomesino’. all the values are means ± se (n = 10). table 2. effects of different methods of fertilisation on the yield (t/ha) of three garlic cultivars: ‘cincomesino’, ‘arequipeño 14’ and ‘margosino’ cultivar t yield 1st yield 2nd yield 3rd total yield ‘cincomesino’ m1 4.67±1.19 a 5.17±0.33 a 2.42±1.11 a 12.25±0.31 a m2 5.00±1.89 a 5.17±1.02 a 2.17±0.82 a 12.33±0.60 a m3 5.33±1.51 a 5.08±1.23 a 2.67±1.07 a 13.08±0.31 a c.v. % 25.13 11.18 17.35 3.98 ‘arequipeño 14’ m1 4.83±1.35 a 4.58±1.20 a 2.75±0.67 a 11.83±0.57 a m2 5.17±1.72 a 5.00±1.25 a 2.83±0.57 a 12.25±1.40 a m3 4.83±0.63 a 4.58±0.56 a 2.17±0,63 a 11.58±1.98 a c.v. % 25.42 17.25 19.59 14.74 ‘margosino’ m1 3.92±1.93 a 4.67±1.41 a 2.33±1.13 b 10.92±1.34 a m2 4.00±0.75 a 4.67±1.40 a 2.21±1.01 b 10.88±1.48 a m3 3.58±1.49 a 4.02±0.02 a 3.38±0.68 a 10.96±1.48 a c.v. % 31.8 22.8 23.3 15.52 t (treatment) 1 values expressed by mean ± se (n = 10) 2 different letters indicate statistically significant differences according to duncan’s test (p ≤ 0.05). treatments plant height (cm) neck thickness (mm) leaf number m1 (3)69.2 (3)10.0 (1)12 m2 (2)69.1 (1)9.90 (2)12 m3 (1)68.4 (2)9.70 (3)12 cv % 2.81 8.34 3.13 table 3. effect of fertilisation methods on the garlic vegetative growth of cv. ‘arequipeño-14’ condor, j.; olivera, j.; pinedo, r. peruvian journal of agronomy 3(3): 104–111 (2019) 109 for plant height in cv. ‘margosino’ (table 4), m1 had the highest value compared to the other cultivars under study. for neck thickness, m1 also had the highest value compared to the other treatments; treatments m1 and m3 had the same number of leaves. m3 had the largest bulb polar diameter, followed by m2 and m3 (figure 4). for bulb equatorial diameter, a similar relationship was found: m3 in first place, followed closely by m1, then m2. figure 5 shows the three cultivars and three methods of fertilisation. the percentages of garlic bulbs of first (6–8 cm) calibre reached on average 30% of the total production; however, for the case of ‘cincomesino’ and ‘arequipeño-14’, m3 (lateral groove fertilisation) had the highest result, and in the case of ‘margosino’, m2 (false groove fertilisation) was highest. among the secondcalibre garlic yield, 40% of the total production belonged to this group. according to table 1, results similar to those of the plant height experiment were recorded by loayza et al. (2005): 72.6 cm in the autumn planting season and 75.5 cm in the winter planting season for cv. ‘cincomesino’. however, reports from other latitudes, in experiments with local cultivars and different levels of nitrogen fertilisation, recorded a plant height maximum of 56.0 cm (naruka & dhaka, 2001). for neck thickness, similar results were obtained by loayza et al. (2005) at 0.87 cm and 1.19 cm in the autumn and winter planting season, respectively. for leaf number, the results obtained were in the range reported by loayza et al. (2005) for this cultivar. with regard to the plant height of cv. ‘arequipeño 14’, m1 reached 69.2 cm, compared to m2 and m3, which reached 69.1 and 68.4 cm, respectively–values that approximate the results of loayza et al. (2005) of 70.9 cm in an autumn planting of the same cultivar. for the neck thickness variable, m1 was 10.0 mm, the same as the results of loayza et al. (2005) for an autumn planting (9.9 mm). for leaf number, no significant difference was found between treatments, with a mean of 12.1 leaves. loayza et al. (2005) reported a lower number of leaves in the autumn planting and a similar number of leaves in a winter planting. figure 3. effect of fertilisation method on garlic bulb size (mm) in cv. ‘arequipeño-14’. all the values are means ± se (n = 10). treatments plant height (cm) neck thickness (mm) leaf number m1 (1)68,0 (1)8,88 (3)12,0 m2 (2)67,6 (3)8,50 (1)12,0 m3 (3)66,8 (2)7,85 (2)11,8 cv % 3,41 16,79 325 figure 4. effect of fertilisation method on garlic bulb size (mm) in cv. ‘margosino’. all values are means ± se (n = 10). 4.67 5.00 5.33 4.83 5.17 4.83 3.92 4.00 3.58 5.17 5.17 5.08 4.58 5.00 4.58 4.67 4.67 4.02 2.42 2.17 2.67 2.75 2.83 2.17 2.33 2.21 3.38 0% 20% 40% 60% 80% 100% m1 m2 m3 m1 m2 m3 m1 m2 m3 'cincomesino' 'arequipeño 14' 'margosino' p e r c e n ta g e w e ig h t (% ) rdt o 1ª rdt o 2ª rdt o 3ª figure 5. percentage distribution of bulb weights: first, second and third calibre of three garlic cultivars for different fertilisation methods table 4 effect of fertilisation methods on the garlic vegetative growth in cv ‘margosino’ fertilisation methods for commercial yield in three garlic cultivars (allium sativum l.) september december 2019 110 regarding cv. ‘margosino’, plant height values reached 68.0 cm compared to 75.6 in loayza et al. (2005) for a winter planting. in m1 for neck thickness, 8.88 mm was the highest value; loayza et al. (2005) found a larger neck diameter in both the autumn (11.9 mm) and winter plantings (13.1 mm). this difference was possibly caused by the weather conditions during the study, which were not favourable for greater thickening of the neck; these can be correlated with the lower plant height obtained. all three treatments showed similar results for number of leaves, with means of 12.0. loayza et al. (2005) found 12.7 leaves in autumn and 11.6 leaves in winter. although the plants had a similar number of leaves in terms of neck diameter and plant height, they showed greater plant development. total yield in cv. ‘cincomesino’ reached 10.4 t/ha in the winter planting in loayza et al. (2005), and in the may planting, it reached 14 t/ha compared to the 13.1 t/ ha obtained in the m3 treatment in the present study. in cv. ‘arequipeño’, the total yield obtained in m2 was 12.25 t/ha. loayza et al. (2005) mentions total yields of 9.3 t/ha for the june planting and 13.3 t/ha for the may planting. a similar yield was obtained for white garlic in mexico, where pérez, garcía, ramírez, and barrera (2003) obtained 12.2 t/ha in cv. ‘cristal’. for cv. ‘margosino’, loayza et al. (2005) found a similar yield of 10.9 t/ha for the june planting (winter) and 12.0 t/ha for the may planting (autumn), corroborating that the high yields are proportional to the length of the growth period (between sprouting and bulbification), as pointed out by burba (2003). plant height of cv. ‘arequipeño 14’ in treatment m1 reached a high of 69.2 cm –a value that approximates the results of loayza et al. (2005) in the autumn planting of 70.9 cm. for the neck thickness variable, the m1 result was superior, with the 10.0 mm result similar to that of 9.9 mm obtained in the autumn planting by loayza et al. (2005). for leaf number, no significant difference was found between treatments, with an average of 12.0 leaves. loayza et al. (2005) reported 11.4 in autumn plantings and 12.7 in winter. plant height in cv. ‘margosino’ (table 4) reached a maximum of 68.0 cm in m1 –less than those recorded by loayza et al. (2005) of 75.6 cm in the autumn and 75.7 cm in the winter planting. for neck thickness, 8.88 mm was the highest value; loayza et al. (2005) obtained a larger neck diameter in autumn plantings (11.9 mm) and in winter plantings (13.1 mm). this difference was possibly caused by the weather conditions during the study, which were not favourable for thickening of the neck and could be correlated with the lower plant height obtained. regarding the number of leaves, loayza et al. (2005) found 12.7 leaves in autumn and 11.6 leaves in winter. although they had a similar number of leaves, in terms of neck diameter and plant height, the plants showed greater development. conclusions the vegetative development variables were higher for the broadcast fertilisation method, while the yield and bulb quality variables showed better results for lateral fertilisation in cultivars ‘cincomesino’ and ‘margosino’. thus, this method of fertilisation might be the most suitable depending on the crop yield, owing to the response of the plant to the location of the fertiliser and improved absorption and translocation of nutrients by the plant. the highest yield for ‘arequipeño-14’ was obtained by applying the superficial groove or false furrow fertilisation method, which allowed better uptake owing to its proximity to the roots. acknowledgements the authors wish to thank inia and the experimental agricultural station donoso-huaral for the technological and logistical support received, as well as the technical staff and field personnel of the national vegetable programme, who made this study possible. references aguado, g., portela, j. & lipinski, v. 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(2018). producción agrícola. en boletín estadístico de producción agrícola y ganadera. iv trimestre 2017. ministerio de agricultura y riego. retrieved from http://siea. minagri.gob.pe/siea/sites/default/files/produccionagricola-ganadera-ivtrimestre2017_220318_0.pdf ugás, r., siura, s., delgado, f., casas, a., & toledo, j. (2000). hortalizas. datos básicos. lima, perú: programa de investigación en hortalizas, unalm. usman, m., fagam, a., dayi, r., & isah, z. (2016). phenotypic response of two garlic varieties to different nitrogen fertilization grown under irrigation in sudan savannah ecological zone of nigeria. international journal of agronomy, 2016, 1–10. http:// dx.doi.org/10.1155/2016/2495828 valladolid, a. (2001). el cultivo del frijol (phaseolus vulgaris l.) en la costa del perú. lima, perú. inia. retrieved from http:// repositorio.inia.gob. pe/ handle/ inia/ 860 peruvian journal of agronomy 2 (3): 35-43 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i3.1231 © the authors. published by universidad nacional agraria la molina received for publication: 30 november 2018 accepted for publication: 28 december 2018 pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru pseudomonas de la rizosfera del palto (persea americana mill.) con actividad de biocontrol de phytophthora cinnamomi rands aisladas en la costa central del perú j. mamani; l. aragón1* *corresponding author: lili@lamolina.edu.pe abstract in the rhizosphere, as the zone of biological activity, diversity of microorganisms can be found like bacteria of the genus pseudomonas. they are characterized for controlling pathogens like phytophthora cinnamomi, also to be promoters of growth. for this reason, in the present study, bacteria of the genus pseudomonas were isolated from the avocado rhizosphere of the provinces of casma, huaral, and lima. a total of six strains (r2, r5, r7, r10, s10 and s6) were selected for evaluating their biocontrol capacity against p. cinnamomi under in vitro and greenhouse conditions. in the in vitro test, strains s6 and s10 controlled 30.3 and 44 %, respectively. under greenhouse conditions, pseudomonas strains were inoculated on 4-month-old avocado cv. “zutano” plants. five months later, we evaluated the following variables: severity in roots, height increase, fresh root and leaf weight, and percentage of root and leaf dry matter. in greenhouse, the best strains in the control of p. cinnamomi were s6, r2, r7 and r10, controlling 55.2, 39.5, 33.7 and 31.0 %, respectively. in the increase of height, the strains s6, r2, r7 and r10 reached 11.4, 9.3, 7.6 and 5.1 cm, respectively. the percentage of dry matter of roots, strains s10, r10 and r7 obtained 29.6, 27.5 and 27.9 %, respectively. in this study, it was observed that although the application of pseudomonas controls p. cinnamomi, it also induces the root and apical growth of avocado. keywords: avocado, promoter of growth, pseudomonas, rhizosphere. resumen en la rizósfera como zona de actividad biológica, se pueden encontrar diversidad de microorganismos como bacterias del género pseudomonas que se caracterizan por controlar patógenos como phytophthora cinnamomi, también por ser promotores de crecimiento (pgpr). por esta razón en la presente investigación se aislaron bacterias del género pseudomonas de la rizósfera de palto de las provincias de lima, huaral y casma. se seleccionaron 6 cepas (r2, r5, r7, r10, s10 y s6) con las que se realizaron pruebas para evaluar su capacidad biocontroladora frente a p. cinnamomi in vitro e invernadero. en la prueba in vitro las cepas s6 y s10 controlaron un 30.3 y 44 %; respectivamente. en condiciones de invernadero, se inocularon cepas de pseudomonas en plantones de palto cv. zutano de 4 meses de edad; transcurridos cinco meses, se evaluó variables como severidad en raíces, incremento de altura, peso fresco radicular y foliar y porcentaje de materia seca radicular y foliar. en invernadero, las mejores cepas en el control de p. cinnamomi fueron s6, r2, r7 y r10 que controlaron un 55.2, 39.5, 33.7 y 31.0 %; respectivamente. en el incremento de altura, las cepas s6, r2, r7 y r10 alcanzaron 11.4, 9.3, 7.6 y 5.1cm; respectivamente. el porcentaje de materia seca de raíces, las cepas s10, r10 y r7 obtuvieron 29.6, 27.5 y 27.9 %; respectivamente. en este estudio se observó que si bien la aplicación de pseudomonas ejerce un control sobre p. cinnamomi también induce el crecimiento radicular y apical de la planta. palabras claves: palto, pgpr, pseudomonas, rizósfera. 1 department of plant pathology, faculty of agronomy, universidad nacional agraria la molina, av. la molina s/n, lima 12. perú. introduction peru has an avocado producing area of around 30,320 hectares with a total production of 349,317 tons. the coastal departments of ancash, ica, la libertad, and lima have the biggest cultivated area. (minagri, 2014). peru is the second exporter of avocado worldwide, with 175.6 million kilos exported (arteaga, 2016). therefore, avocado production in peru will continue to be an attractive business due to the opening of new markets and new consumers who value their nutritional properties. the production of the avocado depends on climatic, edaphic, nutritional and sanitary factors. within the sanitary aspect, diseases are one of the factors that augment the production costs of the fruit. among the most important, is “root rot” caused by phytophthora cinnamomi (chromista, heterokontophyta). this pathogen limits tree development, reduces fruit production and quality, directly affecting profitability. currently, this oomycete is controlled by chemical products, such as fosetil-al and metalaxyl (mora, 2007), pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru september december 2018 36 but the constant use of chemical products diminishes its effectiveness over time due to the resistance that this phytopathogen develops towards the fungicide. for these reasons, biological control is considered to take advantage of soil microorganisms with antifungal activity, including bacteria of the genus pseudomonas, responsible for the suppression of some soil pathogens (raaijmakers et al., 2002). bacteria of the genus pseudomonas have been widely studied as biological controllers for their ability to colonize the root, compete aggressively with other microorganisms, adapt to different situations of environmental stress, synthesize antibiotics and enzymes, and activate systemic resistance in plants (weller et al., 2006). for this reason, in the present work, we evaluated under in vitro conditions the antagonistic effect of strains of pseudomonas spp. isolated from the rhizosphere of avocado on p. cinnamomi by scoring the progress of the pathogen in centimeters. in addition, in the greenhouse; the severity of the infection of p. cinnamomi in avocado seedlings previously inoculated with the strains of pseudomonas spp. was measured by the percentage of lesions in roots, and the effect of these strains on the growth of the avocado seedlings was measured by the increase of height of the stem in centimeters. materials and methods field work. the sampling was carried out in commercial avocado fields that reported root rot problems in casma, huaral and lima. in each field, 10 avocado trees were randomly selected. roots were collected from each tree with a volume of rhizospheric soi from four cardinal points, with a total equivalent of 250g of soil. these samples were transported and kept in cold conditions by using gel packs to maintain their temperature and humidity. laboratory work. the laboratory work (in vitro isolation) was carried out in the facilities of the laboratory of the department of phytopathology of the national agrarian university of la molina (unalm for its acronym in spanish), located in the district of la molina, province and department of lima, at 12° 05’ 06” s of latitude, 76° 57’ 07” wg of longitude and 243.7 m.a.s.l. sample treatment. the microflora of both the rhizosphere and the soil was analyzed separately. under aseptic conditions, 3 g of root of each sample was placed in test tubes with 9 ml of peptonated water in continuous agitation, performing 7 serial dilutions (10-1, 10-2, 10-3, 104, 10-5, 10-6 and 10-7), adapted from andres (1991). for the soil sample near the root, 10 g were placed in 90 ml of peptone water (ratio 1: 9), following the same previous procedure. count of pseudomonas spp. in regard to the quantification of pseudomonas spp., 1 ml of the dilution (10-2 to 10-5) was seeded in triplicate in tubes containing 9 ml of asparagine broth. these tubes were incubated for four days at 37°c (burges, 1960). this procedure was performed for both root and soil. isolation and identification of pseudomonas spp. the tubes that were positive (fluorescent) to the asparagine broth culture were striated in the king-b culture medium and incubated for 48 hours, and fluorescence was evaluated in ultraviolet light at 366 nm (schaad, 2001) together with the morphology of the colonies (palleroni, 2005). the colonies that were positive for fluorescence were then selected. from each isolate, a colony was selected, which was striated in the tryptone soy agar (tsa) culture medium to ensure the purity of each isolate and to be stored in tsa agar wedges at 4°c for the next tests (martínez, 2010). pathogen isolation (phytophthora cinnamomi rands). rootlets of 1-3 millimeters in diameter that presented the typical symptomatology corresponding to a black and firm rot that originated from the zone of elongation were selected. they were washed with distilled water and segments of about 1 cm long that presented the area of pathogen progress were cut. then, they were immersed for a few seconds in a 70% alcohol solution in order to prevent possible contamination (zentmyer, 1980). these pieces of 1 to 2 mm2 of diseased and asymptomatic tissue were seeded in selective culture medium cma (corn meal agar) with parb (pimaricinampicillin-rifampicin-benomyl) (erwin & ribeiro, 1996). they were incubated at 22°c for seven days, and the growth rate of the colony and its characteristics were observed (alvarado-rosales et al., 2007). each pure colony in cma was transferred in v8 juice agar medium for four days at 21°c. from slices of grown mycelium in v8 juice agar that was introduced in petri dishes containing 1% soil solution, structures of the pathogen were visualized as sporangia. the identification of p. cinnamomi was conducted by following the dichotomous key to taiwan species of phytophthora (ho, 1992). antagonism test against phytophthora cinnamomi. from each previous isolate, the strains that controlled phytophthora cinnamomi were identified. these strains were used in the challenge test in nutrient agar culture medium. it was necessary to prepare culture medium in which the corresponding reagents were included because the results of this test were not separately obtained. in each plate containing medium pda + king-b + nutrient agar a controller was included (pseudomonas spp. strain) and a pathogen disk. this methodology was carried out with the objective of identifying strains of pseudomonas spp. with greater control over p. cinnamomi, demonstrating a higher percentage of inhibition of the radial mycelial growth of the pathogen (% i). j. mamani; l. aragón peruvian journal of agronomy 2 (3): 35-43 (2018) 37 greenhouse phase. the greenhouse phase was carried out in the greenhouse research station of the department of phytopathology of unalm. vegetal material. we used for four-month old plants of avocado var. ‘zutano’ which were treated before to the germination (immersed in homai fungicide solution in 5 g/l dose of product), according to the disinfection protocol of the nursery of the fruit trees research program of national agrarian university la molina. the transplant to bags was made in january 2016 to be taken to the greenhouse. preparation of the substrate. the substrate mixture consisted of 50% soil, 25% river sand and 25% organic matter (herrera & narrea, 2011). then, it was sterilized at 121°c for 15 minutes. the substrate was used in black polyethylene bags measuring 8x16x2.2 cm3 (5.5 l), where the sterile soil was placed. then, we proceeded to transplant the avocado. preparation, density and inoculation of the controller. selected strains of pseudomonas were seeded in peptone water at 27°c and 150 rpm (revolutions per minute) until reaching a population of 6x108 (colony forming units) cfu/ml (martínez, 2010). pathogen inoculation. according to drenth & sendall (2001), it is recommended to use mycelium developed in wheat previously sterilized at 121°c and a pressure of 1.1 bar (15 lb/in2 or 15 psi) for 30 minutes for two consecutive times. the inoculation was done in five-month-old avocado plants. the application was made around the root system of the avocado at a depth of 5 cm (the dose of inoculum was 2.5 g of wheat with mycelial development/kg of soil) (zentmyer & richards, 1952). the humidity of the substrate was maintained with periodic irrigation to favor the development of the pathogen. the mycelium of phytophthora cinnamomi was inoculated ten days after the inoculation of the controller, according to martínez (2010). evaluated parameters. disease severity and root length. the severity and root length were measured by processing digital photographs of avocado roots using assess software (lamari, 2002). plant height. we used a millimeter ruler. measurement (length of the main stem) was taken from the neck of the plant to the terminal bud at the time of inoculation of the biocontroller and at the end of the experiment. the results were expressed in centimeters. fresh weight, and dry root and foliar matter. to obtain the percentage of dry matter, firstable the roots and leaves of the plant were placed in paper bags to be weighed; then they were taken to the stove at a temperature of 70°c for 48 hours to obtain the dry weight. the weights were expressed in grams using electronic balance. biochemical characterization of pseudomonas spp. the following biochemical tests were carried out, according to palleroni (2005): (i) production of fluorescent pigments, (ii) resistance to high and low temperatures, (iii) and gelatin liquefaction, in order to characterize strains of non-pathogenic fluorescent pseudomonas from the rest of species fluorescence in king-b culture medium. the culture medium of king-b is used for the detection of fluorescein, a green or blue fluorescent soluble in water. after growth of 24-48 hours at 27°c, the colonies, previously striated in king-b, were observed with an ultraviolet lamp (366 nm). growth at 4 and 41°c. strains of pseudomonas spp. were planted in tubes containing tsa culture medium, which were incubated at 4 and 41°c. jellied liquefaction. this test is used to determine the capacity of a microorganism to produce enzymes of the proteolytic type (gelatinases) that liquefy the gelatin. it is positive for pseudomonas aeruginosa (pathogenic) (mac faddin, 1980). the tubes containing nutrient gelatin were prepared considering three replications per treatment. the strains of pseudomonas spp. were then inoculated by puncture and incubated at 37°c for 48 hours. finally, they were placed in the refrigerator at 4°c for 2 hours. the test was positive if the inoculated medium became liquid, and the test was negative if the inoculated medium maintained its characteristics (mac faddin, 1980). experimental design. the statistical design used in the antagonism test against phytophthora cinnamomi was a completely randomized statistical design (crd), with ten treatments of strains isolated from pseudomonas spp., four dishes per treatment and control treatment of p. cinnamomi pathogen (table 1). the parameter that was evaluated in this test was the percentage of inhibition of grown radial mycelial, and the differences between the average of cultures analyzed out through the tukey test (p = 0.05). in greenhouse, the statistical analysis of variance (anova) and tukey test with a level of significance at 5% were conducted to identify whether or not there are significant differences between the results obtained with each evaluated strain. all statistical analyses tools were applied with minimum confidence of 95% and sas 9.2 software (sas, 2009). pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru september december 2018 38 table 1. treatments used in the present study. treatment description t1 p. cinnamomi + strain of pseudomonas spp. (r2) t2 p. cinnamomi + strain of pseudomonas spp. (r5) t3 p. cinnamomi + strain of pseudomonas spp. (r7) t4 p. cinnamomi + strain of pseudomonas spp. (r10) t5 p. cinnamomi + strain of pseudomonas spp. (s10) t6 p. cinnamomi + strain of pseudomonas spp. (s6) t7 control treatment: p. cinnamomi (phy) t8 absolute control treatment (t) results isolation, purification and identification of pseudomonas spp. a bacterial density of pseudomonas spp. was obtained from the sampled areas which varied between 105 and 108 nmp/g of roots and dry soil. in table 2, it can be observed that the casma area presented the highest levels of bacterial density because the sampled farm promotes the microbial flora with applications of organic matter. we worked with casma strains because they had a higher inoculum potential compared to the other areas sampled. table 2. count of pseudomonas spp. (nmp/g) isolated from the central coast of peru. location nmp/g lima soil 2.3x106 organisms /g of soil root 2.3x106 organisms/g of root huaral soil 9.3x105 organisms /g of soil root 4.3x105 organisms/g of root casma soil ˃1.1x108 organisms /g of soil root ˃1.1x108 organisms/g of root the isolates that grew and emitted fluorescence in asparagine broth were striated in king-b culture medium (also called pseudomonas agar f) for the isolation of all pseudomonas spp. (schaad, 2001), thus differentiating them from other gram-negative microorganisms that do not emit fluorescence. a total of 14 positive isolates were obtained for this test. antagonic effect in vitro test. figure 1 shows that the isolates from soil s7, s10, s2, s9, s4, s1, s6 and s3 inhibit the advance of p. cinnamomi; being constant in time, while in isolations from roots (r7, r10, r4 and r5) there is a strong inhibition at 4 das (days after sowing), but it is lost quickly over time. all the evaluated strains reported some control varying the percentages of inhibition between 28.3 to 47% (figure 2). the isolates r2, r5, r7, r10, s6 and s10 showed growths above the mycelium of p. cinnamomi, being reduced to a yellow gelatinous mass. from there, samples were taken to be observed in a microscope. in isolates from the confrontation p. cinnamomi and pseudomonas spp. fluorescence observed in a microscope (40x), the disintegration of the cytoplasmic content of the mycelium of p. cinnamomi was observed. fig. 1. advance of p. cinnamomi (in centimeters) against soil and root isolates of pseudomonas spp. for 7 days after seed in vitro test. in the biochemical characterization tests, according to the bergey manual (palleroni, 2005), carried out on strains of pseudomonas spp. selected (r2, r5, r7, r10, s6 and s10) the results were obtained (table 3) in which it is determined that the strains evaluated do not correspond to pseudomonas aeruginosa (pathogenic to man). table 3. reaction of different strains of pseudomonas spp. to the biochemical tests. fluorescentpigments grows at 4°c grows at 41°c gelatin liquefaction p. fluorescens + + +/ p. putida + + p. aeruginosa + + + strain r2 + + strain r5 + + strain r7 + + strain r10 + + strain s10 + + strain s6 + + greenhouse evaluation. evaluation of the severity of avocado roots. treatments r5, s10, r10, r7 and r2 (60.4, 66.2, 69.0, 77.5, 78.4 and 90.7%, respectively) show percentage of severity that is not significantly different from the treatment inoculated with the pathogen. on the other hand, the s6 treatment has a significant difference with the control treatment and the treatment inoculated with p. cinnamomi with only 44.8% of severity in avocado roots (figs. 2 and 3, table 4). figure 2, strains r5 and s10 showed severity values (90.7 and 78.4%, respectively) above the control inoculated with p. cinnamomi (77.4%), this loss of antagonistic capacity could be due to population decline of these strains (van et al., 1997). j. mamani; l. aragón peruvian journal of agronomy 2 (3): 35-43 (2018) 39 fig. 2. measurement of severity of avocado roots (%), avocado root length, height increase of avocado plant (cm), fresh radicular weight (g), fresh leaf weight (g) and dry matter radicular (%) of treatments inoculated with strains pseudomonas spp. against p. cinnamomi, under greenhouse conditions. pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru september december 2018 40 it is also observed that the best treatment in the control of p. cinnamomi in greenhouse was t6 (inoculated with the s6 strain of pseudomonas spp.) which showed 59.0% of control. evaluation of the height increase of avocado plant. inoculation of strains s6 and r2 showed a greater increase in height, 11.4 and 9.3 cm respectively, compared to the control treatment (fig. 2). there is no significant difference between treatments s6 and r2, according to the tukey test at 95% (table 4). figures 2 and 3 show all treatments with inoculation of pseudomonas spp. have values that are above the control inoculated with p. cinnnamomi (2.1 cm). the treatments inoculated with strains of pseudomonas spp. present greater amplitude of the interval of variance of their repetitions. evaluation of fresh radicular weight. regarding fresh radicular weight, treatments s10 and s6 (45.6 and 45.2 g) show a significant difference compared to the treatment inoculated with p. cinnamomi (30.8 g), but lower than the absolute control treatment (148.6 g), as observed in figs. 2 and 3. for this variable, the repetitions of each treatment were close values, thus a shorter interval of variance. evaluation of the dry matter radicular. on the percentage of dry matter of roots, there is a significant difference between the treatments. treatment s10 with 29.0% of dry matter of roots is the best compared to the control inoculated with p. cinnamomi (18.6%). table 4. measurement of avocado variables with the comparison test of means (tukey) of treatments inoculated with strains pseudomonas spp. against p. cinnamomi, under greenhouse conditions. treatm ents strains severitya (%) increase in heightb (cm) foliar biomassc (g) root lengthd (cm) biomass of rootse (g) root dry matterf (%) t1 r2 60.462 ab 9.3 ab 74.86 bcd 989.1 b 33.320 bc 24.200 abcd t2 r5 90.720 a 4.0 d 66.70 cd 160.6 c 17.340 c 25.780 abc t3 r7 66.274 ab 7.6 bc 110.38 b 672.2 bc 31.500 bc 27.960 ab t4 r10 69.034 ab 5.1 cd 69.04 cd 640.5 bc 31.500 bc 27.500 ab t5 s10 78.456 a 3.1 d 81.68 bcd 841.1 b 45.680 b 29.620 a t6 s6 44.848 b 11.4 a 91.76 bc 1142.2 b 45.220 b 22.180 bcd t7 phy 77.466 a 2.1 d 48.68 d 648.1 bc 30.880 bc 18.680 d t8 t 6.578 c 4.5 d 154.68 a 3924.6 a 148.640 a 20.360 cd coefficient of variation (alpha 0.05): 24.86a, 25.17b, 22.07c, 26.17d, 17.55e, 13.98f. avocado root length evaluation. the effect of pseudomonas sp. on the length of avocado roots showed a significant difference between treatments. in all treatments, shorter root length was observed compared to the control treatment (figs. 2 and 3, table 4). therefore, inoculated pseudomonas strains influenced root growth but did not achieve significant difference with respect to the control. treatments s6, r2 and s10 presented longer root lengths (1142.2, 841.1 and 989.1, respectively) than the control inoculated with the pathogen (648.1). fig. 3. photograph of foliar and root area of avocado cv. ‘zutano’, six months after inoculated with soil and root isolates of pseudomonas sp. and p. cinnamomi, under greenhouse conditions. j. mamani; l. aragón peruvian journal of agronomy 2 (3): 35-43 (2018) 41 evaluation of fresh leaf weight. treatments r7 and s6 (110.4 and 91.8 g) show a significant difference compared to the treatment inoculated with p. cinnamomi (48.7 g), but lower than the absolute control treatment (156.6 g), as observed in figs. 2 and 3. dry leaf matter. figure 2 shows there is no significant difference among treatments, and that the repetitions of treatments t2 (r5), t3 (r7) and t5 (s10) present higher values than the absolute control (t). discussion this study shows the inhibition of phytophthora cinnamomi against pseudomonas in vitro and greenhouse. villa et al. (2005) found that strains of pseudomonas sp. inhibited the growth of sclerotium rolfsii between 60 and 90%. other studies also report that pseudomonas cepacia and p. fluorescens were significant for suppressing p. cinnnamomi that grew in vitro and in vivo (yang et al., 2001). the coagulation of the cytoplasm was observed; this same partial degradation or coagulation of the cytoplasmic content was observed in cultures of phytophthora capsici with pseudomonas fluorescens in which diby et al. (2005) attributes it to hydrolytic enzymes. broadbent & baker (1974) also demonstrated that p. putida and p. fluorescens isolated from avocado soils caused massive mycelia lysis of p. cinnamomi in vitro. the strains of isolated pseudomonas were not pathogenic for humans according to rodríguez et al. (2005) who demonstrated that to isolate pseudomonas aeruginosa (opportunistic pathogen in humans) the bacteria should be cultured in a gelatin culture medium, thus demonstrating the presence of gelatinases. the promising strains of pseudomonas gave a negative result to this test. therefore, they are not pathogenic to man. pseudomonas putida and pseudomonas fluorescens are unable to grow at 42°c and do not produce pyocyanin unlike p. aeruginosa, corroborated by the uk standards for microbiology investigations (2015). several possible mechanisms have been described by which soil pseudomonas suppresses conditions related to pathogenicity. de la fuente et al. (2000) identified three native strains of fluorescent pseudomonas (up61, up143 and up148) producing hcn, fluorescent siderophores such as pyoverdine proteases and antibiotics [2, 4-diacetylchloroglucinol (dapg), plt (piolterine), prn (pyrrolnitrine) and phenazine derivatives] with antifungal activity (julisch et al., 2001). thomashow and weller (1996) also described the production of antibiotics by fluorescent pseudomonas. therefore, the control effect of treatment t6 (s6) could be explained by mechanisms such as competition for iron, competition for colonization sites and nutrients exuded from the root, as well as the induction of plant defense mechanisms (van weels et al. 1997). another mechanism of control is the production of extracellular enzymes, such as chitinases, laminases and glucanases that can degrade the walls of fungal cells. van weels et al. (1997) isolated a strain of pseudomonas stutzeri that produced extracellular chitinase and laminase, and found that these enzymes digest and lyse the mycelium of fusarium solani. marques et al. (2010) who mentioned that the production of iaa by pgpr generally causes the elongation and accumulation of p and n in the tissues of the plant would explain the increase in root length. in the root system, it has been seen that high levels of iaa increase the formation of lateral and adventitious roots, but inhibits the growth of the primary root. this improvement in growth was also observed in the 1970s when some fluorescent pseudomonas strains improved the growth of potatoes and sugarcane when applied to the seeds (schroth & hancock, 1982). the increase in height is explained because pseudomonas can act as promoters of plant growth in two ways: directly by suppression of pathogens or indirectly through the secretion of phytohormones and vitamins, or by increasing the absorption of minerals per plant. glick (1995) proposes that pseudomonas can manifest its growth by promoting effects indirectly, stimulating the beneficial actions of other microorganisms associated with the roots, such as mycorrhizae. when the stimulation of plant growth occurs in the absence of other microorganisms, it has been attributed to the increase in the availability of mineral nutrients, such as phosphate or nitrogen, due to the production of phytohormones stimulating plant growth or to the degradation of ethylene precursors in the root by these bacteria. faggioli et al. (2007) also observed that the inoculation of corn plants with pgpr does not significantly influence the height but the percentage of dry matter (fig. 2). conclusions the soil sampled from casma area had a higher quantity of pseudomonas spp. organisms. the pseudomonas spp. isolated from soil showed greater controlling effect on phytophthora cinnamomi compared to isolates from roots, evaluated in vitro. the strain of pseudomonas spp. s6 (t6) showed greater control of p. cinnamomi in the greenhouse and the strain of pseudomonas spp. s10 (t5) showed greater root system development in the greenhouse. it was ruled out that all pseudomonas spp. inoculated were pathogenic to humans (pseudomonas aeruginosa). acknowledgments this research was funded by sociedad agrícola virú s.a. (virú) company which grant me the first place in the contest “premio virú 20 años”. we thank dr. c. arbizu berrocal for assistance. references arteaga, w. 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(1952). pathogenicity of phytophthora cinnamomi to avocado trees and the effects of irrigation on disease development. phytopathology,42,35-37. peruvian journal of agronomy 3(1): 6-15 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i3.1228 © the authors. published by universidad nacional agraria la molina received for publication: 7 november 2018 accepted for publication: 15 january 2019 reproductive development of lemon (citrus aurantifolia swingle) under different soil moisture levels desarrollo reproductivo del limón (citrus aurantifolia swingle) bajo diferentes niveles de humedad del suelo caballero, m.*1; caballero, h.2; cobeña, g.3; solórzano, c.4 *corresponding author: 20181511@lamolina.edu.pe abstract according to the food and agriculture organization of the united nations, citrus fruits dominate the worldwide production of all fruits. because of its geographical position, ecuador has favourable growing conditions for citrus fruit production and most of the country has favourable conditions for plants and their relationships with environmental conditions. the objective of the present research was to determine the reproductive phenology of lemon sutil (citrus aurantifolia swingle) under varying soil moisture levels. a database configuration assistant) using a randomized complete block design as applied and four treatments and six repetitions were distributed as follows: treatment 1 [crop coefficient (kc) 0.3], treatment 2 (kc 0.5), treatment 3 (kc 0.7) and treatment 4 (kc 0.9). fruit quality, skin and pulp weight, seed quantity, juice content, degree brix and polar and equatorial diameter were evaluated, and the phenology was adjusted to bbch scale coding. no statistically significant difference was found during the study that resulted from rains that homogenised the entire substrate and maintained soil moisture. we established that from the phenological phase of primordia to fruit harvest, there was an interval of 138–140 d wherein the average weight of the fruit (42.62 g) fluctuated according to the weight of the skin (7.65 g), weight of the pulp (34.73 g), number of seeds (5.05), amount of juice (14.36 ml), degrees brix (5.5), polar and equatorial diameters (44.32 and 42.12 mm, respectively) and the titratable acidity (6.54%). we concluded that the kcs proposed in the present research should be evaluated during the dry season because, in this investigation, irrigation was induced by kc for only 2 months after the rains. keywords: production, quality, primordium, phases, fruit. resumen de acuerdo con una investigación de la organización de las naciones unidas para la alimentación y la agricultura, los cítricos dominan el primer lugar en la producción mundial de los frutos. ecuador, debido a su posición geográfica, tiene condiciones favorables para obtener una buena producción de limón, de la misma manera, la mayor parte del país tiene condiciones climáticas favorables para las plantas y su relación con las condiciones ambientales. el objetivo de la investigación fue determinar la fenología reproductiva de limón (citrus aurantifolia swing) sometida a diferentes niveles de humedad en el suelo. se utilizó un dbca (diseño de bloques aleatorios completos); cuatro tratamientos y seis repeticiones se distribuyeron de la siguiente manera: tratamiento 1 (kc 0,3), tratamiento 2 (kc 0,5), tratamiento 3 (kc 0,7) y tratamiento 4 (kc 0,9). asimismo, se evaluaron parámetros de calidad como el peso de la fruta, piel y pulpa, cantidad de semilla, contenido de jugo, grados brix, diámetro polar y ecuatorial; la fenología se ajustó a la escala bbch (codificación). no se presentaron diferencias estadísticamente significativas durante el estudio debido a la presencia de lluvias que homogeneizaron todo el sustrato y mantuvieron la humedad del suelo. se estableció que desde la fase fenológica del primordio hasta la cosecha de fruta hay un intervalo de 138-140 días y el peso promedio de la fruta fluctúa entre 42.62 g, peso de la piel 7.65 g, peso de la pulpa 34.73 g, número de semillas 5.05, cantidad de jugo 14.36 ml, grados brix 5.5, diámetros polar y ecuatorial de 44.32 mm y 42.12 mm respectivamente, acidez titulable de 6.54%. se llegó a la conclusión de que los coeficientes de cultivo propuestos en la investigación deberían evaluarse durante la estación seca, ya que en esta investigación el kc solo indujo el riego durante dos meses porque hubo un período posterior a las lluvias. palabras clave: producción, calidad, primordio, fases, fruto. 1 estudiante de posgrado. universidad nacional agraria la molina (unalm). facultad de agronomía. lima perú 2 universidad técnica de manabí, utm. docente de la facultad de filosofía, letras y ciencias de la educación. portoviejo ecuador. 3 universidad técnica de manabí, utm. médico veterinario zootecnista (mvz). facultad de medicina veterinaria. portoviejo ecuador. 4 universidad técnica de manabí, utm. docente del instituto de ciencias básicas (icb), carrera de pedagogía química y biología e ingeniería química. portoviejo ecuador. caballero, m.; caballero, h.; cobeña, g.; solórzano, c. peruvian journal of agronomy 3 (1): 6-15 (2019) 7 introduction citrus fruits dominate the production of all fruits worldwide. according to research by the food and agriculture organization of the united nations (fao), the total production of lemons was 173 mt. the leading lemon-producing countries are mexico, india, argentina, spain, the united states, iran and italy (faostat, 2016). according to utepi (2006), the geographic location and growing conditions in ecuador are suitable for cultivating lemons that are destined for the fresh fruit markets for domestic consumption and for export. this indicates that there is no real citrus-processing industry in the country. at one time, the main citrus-producing provinces were pichincha, manabí and guayas; therefore , this changed, and by 2011, the manabí region became the largest planted area (1200 ha) for citrus (el comercio, 2012). precipitation is the main climatic factor that influences the growth and development of citrus plants. in tropical climate, soil moisture comes from precipitation, and this humidity (or relative humidity [rh]) can be modified using irrigation (davies & albrigo, 1994). the largest planted area in ecuador comprises citrus aurantifolia swingle followed by the tahiti lime (c. latifolia), with both varieties occupying ~4400 ha. lemons in manabí are one of the main products that are a source of income for the grower; therefore, it is known as a main source of income for the people inhabiting southeast of portoviejo. (iniap, 2016). the phenological behaviour and annual yield of citrus crops vary for each production cycle, especially when there is precipitation during the dry season after floral induction, which is the process before flowering under conditions of water deficit, and this subsequently causes extremely high losses in reproductive structures and a low annual harvest (mateus, pulido, gutiérrez, & orduz, 2010). knowing the stages of growth helps in compiling information on the beginning, culmination, conclusion and duration of each stage and to correlate this information with environmental factors and elements (heuveldop, tasis, quiros, & espinoza, 1986). the study of plant parts during their growth and development, such as sprouting, flowering, fruiting and fruit maturity, is known as ‘phenology’. these structures are primarily the result of environmental settings, and the phenological results can be achieved from the plant stages according to the climate and microclimate in which they are evaluated. these phenological observations provide information on the behaviour of different varieties of plants within the specific territories in which they are being developed (pérez, romero, navarro, & botía, 2008). the water requirements for lemons is fundamental and its availability is affected by different environmental scenarios, such as temperature, humidity, lighting, wind speed and the basic characteristics of the plant (leaf area and stomatal regulation of the leaves). the quantity of water necessary for the crops is estimated from the temperature, environmental humidity, soil moisture and evapotranspiration (et). maintaining adequate and constant humidity in the soil during cultivation, followed by providing sources of high nutrition, guarantee high production and quality, even in subtropical areas with high rainfall (enciso, sauls, wiendenfeld, & nelson, 2008). the aim of the present study was to compile information on the reproductive behaviour of the limón sutil (c. aurantifolia swingle) under various humidity levels for determining its development within the manabí area under its climatic conditions and for assessing the phenology of this species in winter. materials and methods the study was conducted in colon parish , portoviejo, manabí province, ecuador, located 1°05’ 19.9” s latitude and 80º 24’ 18.6” longitude w at an altitude of 60 masl (fig. 1). figure 1. study location. source: google earth ec, 2018. reproductive development of lemon (citrus aurantifolia swingle) under different soil moisture levels january april 2019 8 and plants 6 × 6 m apart on flat topography using a drip irrigation system. the experimental design (table 1) was a randomized complete block (rcb) using a database configuration assistant (dbca) with four treatments and six repetitions. the plants to be evaluated were marked with a different coloured tape for each experimental unit. for this study, physical–chemical and foliar analyses were conducted in the plants, and a chemical analysis of the soil was performed for assessing the nutritional content in both the crop and soil. five soil samples were obtained from each hole in the ground. the variables to be analysed were based on the biologische bundesanstalt, bundessortenamt und chemische industrie (bbch)-scale proposed by agustí, martínez, mariano and almela (1987). the author created this scale table 1. field experimental design. source: own elaboration. caption: borders experimental unit a b c d e f g h figure 2. graphic illustration of the reproductive stages of limón. source: images. caballero mario, 2018.pale green– white closed button (a); open button (b); open flower (c); senescent (dry) phase; falling petals (d); appearance of new fruits (e); fruiting (f); background colour (g) and appearance of coating colour (h). graphs based on bbch scale of citrus (agustí, 2003). the study was conducted in a 15-year-old in a commercial plantation of lemon (c. aurantifolia swing.) grafted onto mandarin cleopatra (c. reshni) and planted in rows caballero, m.; caballero, h.; cobeña, g.; solórzano, c. peruvian journal of agronomy 3 (1): 6-15 (2019) 9 to categorise phenological descriptions using codes and stages in plant development under the environmental conditions found in manabí province. the analysed variables were as follows: primordium growth, fruit growth, days to flower opening and fruit quality. for determining plant phenology, two branches were selected from each cardinal point (n, s, e and w) of the tree. using the methods by garrán, ragone, and ciuccio, (1993), two trees were marked as references for each treatment, two branches were selected and the floral primordia in the green state (smaller primordium) were chosen. the trees were evaluated every 3 day from initial primordium until flower development, and every 8 d when the fruit was tied, depending on fruit growth. the considerations of data collection were obtained from the initial baseline to the harvested fruit using the visual criteria of physiological maturity illustrated in fig. 2. the bbch extended scale is a system of uniform coding for the phenological identification of the growth stages for all species of monoand dicotyledonous plants. it is the result of a working group formed by the federal center for biological research for agriculture and forestry of the german federal republic, the federal institute of varieties of the federal republic of germany, the german association of agrochemicals and the institute for horticulture and floriculture in grossbeeren/erfurt, germany. the decimal code is divided primarily between the main and secondary growth stages and is based on the well-known codes developed by zadoks, chang, and konzak, (1974) to provide greater detail to the phenological keys. the data collected were classified and coded as follows: primordium (pr), intermediate button (bi), inflated button (bh), open flower (fa), fruit1 (f1), peach fruit (fr.a), fruit chickpea (fr. g), fruit marble (fr.c), fruit pin-pon (fr.p), fruit tennis (fr.t), fruit developed (fr.d) and fruit collected (fr.). these terms of identification are placed taking into account the growth and morphology of the lemon, as a part of the field visual observations. general water requirement for citrus crops we used the methods proposed by fao (2006), which defines specific coefficients for each crop (kc) and then calculates the evapotranspiration of the crop (etc) using the following formula: etc = kc × eto the kc suggested by fao—0.75 for rainy months and 0.80 for dry months—was used in this study. for calculating the reference et (eto), the penman–monteith equation described by fao (1998) was used with data obtained from the meteorological station in lodana parish processed with cropwat version 8.0 in 2016. for determining when and how much to water, the following water balance method was used (sokolov, 1981): δ ɵ = (p + r + ac) – (et + es + pp), where, δɵ = the change in water content, p = precipitation, r = irrigation, ac = capillary contribution, et = evapotranspiration, it = runoff and pp = deep percolation. irrigation in lemon cultivation for determining the spacing of the irrigation rings, a search was conducted for the effective plant roots, which for the subtle lemon were at a depth of 0.25–0.30 cm and 1.0–1.70 m from the base of the stem to the horizon. using these criteria, irrigation rings were placed and the marriotte bottle technique was used for determining the number of droppers per ring (fig. 3). figure 3. (search for effective roots) for guaranteeing that all netafim self-compensating drippers (fig. 4) produced the same water outlet, 15 drippers were placed with the two rings, one with five drippers located near the stem and the other with 10 drippers located along the outer perimeter. each dripper had a flow rate of 5 l water/h. figure 4. netafim self-compensating drippers. testing the amount water used was calculated on the basis of the water demand of the crop, its water behaviour and the climatic conditions of the area. initially, the irrigation frequency was arbitrary and soil moisture was determined once a week using the gravimetric method to provide an adequate supply of water to the crop (et). the samples were weighed when wet, dried in a stove at 105°c to a constant weight and reweighed. the difference between the weight of the wet and dry sample represented the moisture content in the plant at the time of sampling (burgos, perdomo, morales, & cayón, 1998). reproductive development of lemon (citrus aurantifolia swingle) under different soil moisture levels january april 2019 10 graphic date code registred name day code bbch temp. hum precip helio description of the scale 01/06/2018 1 primordio 6 55 27.4 75 0 1.5 reproductive shoot, its colour is green in the form of a button many times isolated, or in clusters, it is usually found with or without leaves 01/12/2018 2 botón inter-medio 4 56 26.8 73 0 9.5 the growth of the floral button, where the petals grow pass to white colour the sepals that wrap the crown of the floral button become visible 01/14/2018 3 botón hin-chado 2 59 26.6 81 8.7 1.4 with great relevance appear floral buds ready to burst, fully developed petals appear 01/17/2018 4 flor abierta 2 60 27.2 75 0.9 6.1 opened flower in its entirety, and more than 50% open where its reproductive structures are appreciated 01/23/2018 5 fruto 1 6 72 26 83 0.3 2.9 after flowering, you see a fruit already formed; there are falling petals and stamens 02/03/2018 6 fruto ‘arverja’ 11 74 26.1 88 1.4 0.3 fruits in the growth process, the growth is slow even having the right conditions for its development 02/17/2018 7 fruto ‘garban-zo’ 14 25.5 93 5.6 0.1 03/09/2018 8 fruto ‘canica’ 22 27.6 82 0 4.6 03/29/2018 9 fruto ‘pin-pon’ 20 26.1 86 7.2 0.2 04/24/2018 10 fruto ‘tenis’ 26 79 27.6 77 0.1 7.5 05/08/2018 11 fruto ‘desar-rollado’ 14 81 26.8 76 0 4.9 it begins to have indexes in its change of colouration, and its softer shell, and its ease to detach itself from the branch when touching it 05/14/2018 12 fruto ‘co-sechado’ 6 83 25.6 87 0 0.6 fruit in 80% to collect, in this phase it turns a yellowish green colour which is a criterion of harvest in the farmer table 2. reproductive phenology scale of the subtle lemon. source: own elaboration, author photos, caballero, mario, 2018. taken as an example of the bbch scale de (agustí, 2003). data from weather station lodana-manabí-ecuador, 2018 caballero, m.; caballero, h.; cobeña, g.; solórzano, c. peruvian journal of agronomy 3 (1): 6-15 (2019) 11 fruit quality the fruit was harvested at physiological maturity using the grower’s criteria and the keys by which the grower determined the quality of the fruit. the following variables were measured: fruit, skin and pulp weight; number of seeds and amount of juice. a brix american-made atc range e-line brand brixometer was used for determining degrees brix. results and discussion the growth of floral primordium into an initial bud was between 4 and 6 d at 27.5°c. growth from the initial button into the intermediate button as 6 d at 27°c and from the intermediate button into a swollen bud was 4 d at 26°c and 3 d at 25.5°c. the time between primordium to open flower fluctuated between 12 and 14 d. the time range from floral primordium to harvested fruit varied between 138 and 140 d and depended on environmental conditions, crop management and plant genetics. the reproductive phenology scale of the subtle lemon was based on the bbch scale proposed by agustí, almela, aznar, juan, and eres (1995) (table 2) for which the code and name are of the authorship based on the morphological characteristics. figure 5 shows how the growth of the lemon fluctuates as a function of temperature. the growth trend is directly proportional to temperature and when the temperature increases, the phenological phase of ‘fruit type marble’ benefits. in addition, temperatures <24ºc inhibit the growth of the lemon fruit. valiente and albrigo (2000) have reported that temperature has a more significant influence on lemon growth and flowering than rainfall in florida. according to hardy and sanderson (2008), the duration of the phenological stages can vary on the basis of different climatic conditions, particularly temperature, in different years. the value of the sum of grades dais (level of winter rigor in a locality) must remain constant with the temperature among locations or years, this being the most important factor in determining the phenological stages. stenzel, neves, marur, scholz, & gomes, (2006) have reported that there are other variables (solar radiation, soil temperature, water availability, atmospheric humidity, winds, nutrition and health status) that affect the metabolism of plants and influence the development processes of citrus. it can be postulated that within the study location, a temperature range that fluctuates between 25°c and 30ºc, such as that during the months evaluated in this study, is optimal for vegetative development, fruit set, growth and quality of the fruit; however, a temperature of >30ºc reduces the metabolic activity of the plant. this may be because when temperatures are >30ºc, the carbohydrate reserves in the fruit reduce. figure 6 shows that the humidity is constant during the evaluation dates with an outstanding peak in the phenological phase of ‘tennis-type fruit’. most citrus fruits adapt well to different moisture levmonth temp. rh % precip. (mm) heliofo. (h) november 27.23 87.4 0.0 1.25 december 27.90 87.2 0.2 1.82 january 27.00 2.4 4.0 4.63 february 25.80 90.0 3.5 0.20 march 26.85 90.0 3.5 0.20 april 27.60 77.0 0.1 7.50 may 26.20 81.5 0.0 2.75 table 3. meteorological data on temperature, relative humidity, heliophane and precipitation during the evaluation months. figure 5. fluctuation from initial primordia into fruit harvested as a function of temperature in lemon (citrus aurantifolia swingle). figure 6. fluctuation of subtle lemon (citrus aurantifolia swingle) from primordium to fruit harvested as a function of humidity. source: inamhi. instituto nacional de meteorología e hidrología del ecuador. quito. datos tomado de estación meteorológica la teodomira, lodana manabi ecuador. lodana-manabí-ecuador, 2018. reproductive development of lemon (citrus aurantifolia swingle) under different soil moisture levels january april 2019 12 els, such as those in tropical regions where during the period of vegetative development, the rh almost never drops below 70% during the day and reaches saturation at night. for fruit to set, it must have moderate environmental humidity. changes in environmental rh causes physiological changes in the timing and amount of fruit fall, and the lower the rh, the greater the fruit fall (albornoz, 1992). in tangerines and ‘valencia’ oranges, for example, changing rh values during the night reduce fruit growth. when there are fluctuations in environmental factors, such as rain, temperature, rh and soil moisture, the fruit is damaged, making it less desirable in the market. changes in rh also affect the shape of the fruit in tropical and subtropical climates, often making them ovoid instead of round. figure 7 shows that when there is precipitation during the primordial phenological phase, the rains affect the timing and amount of fruit falls; however, rainfall does not affect fruit mooring during the developing fruit phase as much as during the initial stage of fruit formation. one of the factors that most influences fruit flowering in tropical climates is water stress. when there is drought followed by high amounts of rain, there is a large effect on the ability of the tree to flower. the fruit stomatal opening and transpiration are significantly reduced under drought conditions, which delays fruit development and substantially reduces its final size. notably, the rains also enhance the shape of the fruit and the thickness of the shell of the fruit. water stress reduces its consistency and turgor, which makes the fruit more vulnerable to handling and transport. agustí (2003) has indicated that the harvest and the quality of the fruits tend to be better during the rainy season. figure 8 shows how soil moisture influences flowering and fruiting. exogenous factors such as soil moisture are associated with the fall of flower, and citrus fruits can grow and fructify under very diverse environmental conditions, from subtropical climates to tropical zones, there does not appear to be a common climatic characteristic that can act as an essential factor for inducing flowering; therefore, citrus fruits have been considered to be self-inductive species. carbohydrates levels, hormones, temperature, mineral nutrition and water balance are other factors that influence flowering behaviour. under tropical conditions, the dominant force on floral induction is water stress. the fruits and roots during development act as negative moderating factors because of their participation in the synthesis of gibberellins, which are the main inhibitors of floral induction. blooms can present continuously throughout the year, but they peak more after the beginning of a rainy season that is preceded by a dry period. the intensity of flowering and the curdling behaviour are determined by the availability of water. rebolledo (2012) indicates that in the tropics water stress is the main factor affecting flowering, while in the subtropics, low winter temperatures concentrate flowering in spring. it is known that a prolonged drought or soil temperatures <12ºc cause the initiation of bud dormancy. the increase in soil temperature or the initiation of periods of rainfall increases the percentage of sprouted nodes by figure 7. fluctuations of subtle lemon (citrus aurantifolia swingle) from primordium to fruit harvested as a function of precipitation. figure 8. percentage of flowering and fruiting in relation to soil moisture. analysis of variance/variables* p. de fruit (g) p. of the rind (g) p. of pulp (g) n. of the seed juice (ml) º brix d. polar (mm) d. ecuatorial (mm) % acidity titulable cv. 20.3 24.07 20.45 37.26 32.2 11.12 9.09 7.28 18.33 p-value 0.0003 0.0021 0.0004 0.261 0.0076 0.0001 0.0013 0.0049 0.8986 media 42.62 7.65 34.73 5.05 14.36 7.5 44.32 42.12 6.54 experimental error 25.87 3.39 22.38 3.55 21.4 0.7 16.25 9.4 1.44 table 4. (fruit quality) *analysis of variance with the infostat student statistical package, in addition to the tukey’s a multiple range test = 0.05 for comparison of means. caballero, m.; caballero, h.; cobeña, g.; solórzano, c. peruvian journal of agronomy 3 (1): 6-15 (2019) 13 modifying the balance in the synthesis and/or transport of hormones from the root to the shoot. summary analysis of variance of fruit quality to fully investigate fruit production, the biometric characteristics of limón sutil were determined and are presented in table 4. the variance analysis of the quality of the subtle lemon fruit in table 4 shows that the average total weight of the fruit is 42.62 g, average weight of the shell is 7.65 g, average weight of the pulp is 34.73 g, average number of seeds is 5.05, average percentage of the juice is 14.36 ml, average degrees brix is 7.5º, average polar diameter is 44.32 mm, average equatorial diameter 42.12 mm and average titratable acidity is 6.54%. the fruit was weighed 1 d after the harvest. these data on fruit quality agree with those by orozco (2014) in his study on the application of four vegetable regulators in the productive potential of subtle lemon in the canton grit. the results his study are associated with greater leaf area of the reproductive shoots, which provides for increased photosynthetic activity in the plant, resulting in more and larger fruit and better nutritional reserves. these data coincide with those of the study by puente (2006) that was conducted for determining the physical and chemical characteristics of the subtle lemon. further, these data also agree with those in the study by olazabal, bravo and hernández (2005). comparing data of the studies mentioned with those of the present investigation, brix degrees and titratable acidity are below those detailed by these authors, and these variations may be due to the harvest season, the type of soil and climatic factors that influence the yields and biometric characteristics of lemon. temperature is the most influential factor in the amount of fruit acidity. the higher the day/night thermal regime, the lower the acid concentration. the fruit yield in the 3456 m2 study area was 122.7 kg/ha. figure 9 indicates that the larger the fruit, the greater the juice content. water is the main component of the fruit, representing between 85% and 90% of its total weight; consequently, under drought conditions or very prolonged summers, citrus orchards suffer water stress from low water availability. less water available to meet their physiological needs causes delays in plant growth and, if the fruits have already developed, their growth and quality are reduced from low juice content and lower acidity. in areas with high temperatures, the fruits show more rapid metabolic and morphological development, reaching a good size, high acidity and pleasant aroma; however, they are also more prone to rapid degradation resulting from a higher rate of respiration and, thus, a shorter storage life compared with fruits from areas with lower temperatures. in general, because a fruit tree needs a lot of water, irrigation influences the amount of juice in the fruit; however, the juice content and quality of fruits from the same tree are not always homogeneous. this may be the result of competition among the fruits for resources or the lack of photoassimilates resulting from a tree crown that was not adequately controlled by pruning. in general, the size or weight parameter of the fruit is associated with equatorial diameter and volume (bain, 1985). in the early stages, total soluble solids (tss) increases with the increase in the size of the fruit (agustí, 2003). hardy and sanderson (2010) mentioned that the content of soluble solids increases mainly from the accumulation of sucrose during the maturation phase. the same behaviour was reported by agustí (2003), who pointed out that in varieties that mature early, the sucrose content rapidly increases and that the fruits continue to mature when the temperature decreases (in subtropical regions); however, in varieties that mature later, fruits ripen when the temperature increases and the sucrose content in the fruit increases relatively little (agustí, 2003). conclusions the kcs proposed in the present investigation should be evaluated during the dry season because, in this study, irrigation was induced by the kc for only 2 months before the rainy season. for the phenological evaluation, some primordia were observed, which, as a result of rainfall, in drip irrigation, the roots of the plants were concentrated and developed only within the moist areas of soil; however, under ecuadorian coastal conditions, where there are two distinct periods (dry and rainy), lemon roots extended much farther than they did in areas with less rainfall. our limited knowledge on the ecophysiological parameters that determine the growth and development of the fruit in each area makes the technological options more non-specific. the technological development achieved under subtropical conditions serves as a starting point for adjusting the methods and techniques that allow the creation figure 9. relationship between fruit weight and juice content. reproductive development of lemon (citrus aurantifolia swingle) under different soil moisture levels january april 2019 14 of a management platform for the species grown in the ecuadorian tropics. the subtle lemon has high acidity, which helps preserve food and keep it fresh; therefore, it is more commercialised and very desirable in the culinary industry. references agustí, m. (2003). citricultura, 2ª edición. ed. mundi-prensa. madrid, españa. 422 pp. agustí, m., almela, v., aznar, m., juan, m., & eres, v. (1995). desarrollo y tamaño final del fruto en los agrios. valencia: generalitat valenciana. agustí, m., martínez, a., mariano, m., & almela, v. (1987). cuajado y desarrollo de los frutos críticos. escala bbch. instituto agroforestal mediterráneo, universidad politécnico de valencia, españa. albornoz, g. (1992). el tomate de árbol en el ecuador. (cyphomandra betacea sendt). universidad central del ecuador. facultad de ciencias agrícolas. quito, ecuador. pp. 35-66. bain j, (1985), morphological, anatomical and physiological changes in the developing fruit of the valencia orange, citrus sinensis l. osbeck. australian journal of botanic, 6, 1-24. burgos, c., perdomo, r., morales, c., & cayón, d. 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(2014). aplicación de cuatro reguladores vegetales, en la potencialidad productiva del limón sutil en la cooperativa los guayacanes, cantón arenillas. trabajo de titulación. utmach, unidad académica de ciencias agropecuarias, machala, ecuador. olazabal, a., bravo, j.e., & hernández. (2005). fisiología poscosecha de frutas y hortalizas. facultad de ingeniería, universidad nacional de colombia, bogotá. pp. 5-32. pérez, j., romero, p., navarro, j., & botía, p. (2008). p. response of sweet orange cv ‘lane late’ to deficit irrigation in two rootstocks. in: water relations, leaf gas exchange and vegetative growth. irrigation science; 26(5): 415-425. puente, h.c.j. (2006). determinación de las características físicas y químicas del limón sutil (citrus aurantifolia swingle). thesis. universidad técnica del norte, facultad de ingeniería en ciencias agropecuarias y ambientales. 142 pp. rebolledo roa, a. (2012). fisiología de la floración y fructificación en los cítricos. corporación universitaria lasallista. zadoks, j.c., chang, t.t., & konzak, c.f. (1974) a decimal code for the growth stages of cereals. weed research, 14, 415–421 and eucarpia bulletin, 7, 49–52. sokolov, a.a. (1981). métodos de cálculo del balance hídrico guía internacional de investigación y métodos (no. c 25357). instituto de hidrología de españa, madrid unesco, parís (francia). stenzel, n.m.c., neves, c.s.v.j., marur, c.j., scholz, m.b.s., & gomes, j.c. (2006). maturation curves and degree-days accumulation for fruits of folha murcha orange trees. scientia agricola, 63 (3), 219-225. http://dx.doi.org/10.1590/s010390162006000300002 utepi, (2006). unidad técnica de estudios para la incaballero, m.; caballero, h.; cobeña, g.; solórzano, c. peruvian journal of agronomy 3 (1): 6-15 (2019) 15 dustria lima y limón. estudio agroindustrial en el ecuador. competitividad de la cadena de valor y perspectiva de mercado. 81p. [29 april 2018]. retrieved from https://www.unido.org/researchers/ publications valiente, j.l., & albrigo, g.l. (2000). modeling flowering date ofsweet orange treesin central florida based on historical weather. in: proceedings of the international society of citriculture, 1, 296-299.3 peruvian journal of agronomy 1 (1): 32-38 (2017) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v1i1.1065 © the authors. published by universidad nacional agraria la molina received for publication: 19 november 2017 accepted for publication: 22 december 2017 germination behavior of jatropha curcas l. after different imbibition times comportamiento de germinación de jatropha curcas l. después de diferentes tiempos de imbibición lozano, f.1; miranda,p.1; pompelli, m.1* *corresponding author: marcelo.pompelli@ufpe.br abstract jatropha curcas is an important specie for production of biofuel. the specie can survive and produce fruits and seeds even in drought condition. for an adequate establishment in the field is necessary that seeds have a good quality in vigor and viability. in this study, we evaluated the seed water relation with different imbibition times, in deionized water, from 0 to 24 hours. imbibed seeds were sown in polyethylene trays with 1,200 g of river sand. the germination was recorded every day for 25 days. seeds with at least 10 mm radicle on the soil surface was considered as germinated. to determinate seed water content (swc), 10 seeds were weighed in fresh (sfw), turgid (stw) and dry weight (sdw) at 105°c for 24 hours. after 24 hours of imbibition our results show a decrease in the germination rate from 85% to 47%, and an increase of the mean germination time from 4.8 to 7.1 days. the initial moisture of the seed used in this experiment was about 8% and after 24 hours of imbibition, the swc was around 60%. the initial low moisture in the seeds produce imbibition damage because the tissue hydration takes place in a not controlled way so that the reconstruction of internal structures of the cells and organelles were affected. according to the pca analysis the seed germination had a negative correlation with the imbibition time (r = -0.72, p < 0.05) and with the electrical conductive (r = -0.88, p < 0.05), variables related to the seed vigor. this study suggests that electrical conductivity may be useful in j. curcas for vigor test and their seeds do not need previously water imbibition to improve germination from seeds with initial moisture less than 8%. key words: biofuel, seed water content, seed moisture, germinability resumen jatropha curcas es una especie importante para la producción de biocombustible. la especie puede sobrevivir y producir frutos y semillas incluso en condiciones de sequía. para un establecimiento adecuado en el campo es necesario que las semillas tengan una buena calidad en vigor y viabilidad. en este estudio, evaluamos la relación agua de la semilla con diferentes tiempos de imbibición, en agua desionizada, de 0 a 24 horas. las semillas embebidas se sembraron en bandejas de polietileno con 1,200 g de arena de río. la germinación se registró todos los días durante 25 días. las semillas con al menos 10 mm de radícula en la superficie del suelo se consideraron germinadas. para determinar el contenido de agua de la semilla (swc), se pesaron 10 semillas en fresco (sfw), turgentes (stw) y peso seco (sdw) a 105°c durante 24 horas. después de 24 horas de imbibición, nuestros resultados muestran una disminución en la tasa de germinación de 85% a 47%, y un aumento del tiempo de germinación promedio de 4.8 a 7.1 días. la humedad inicial de la semilla utilizada en este experimento fue de aproximadamente 8% y después de 24 horas de imbibición, el swc fue de alrededor del 60%. la baja humedad inicial en las semillas produce daño por imbibición debido a que la hidratación del tejido tiene lugar de una manera no controlada, de modo que la reconstrucción de las estructuras internas de las células y los orgánulos se vieron afectadas. de acuerdo con el análisis de pca, la germinación de la semilla tuvo una correlación negativa con el tiempo de imbibición (r = -0.72, p <0.05) y con la conductiva eléctrica (r = -0.88, p <0.05), variables relacionadas con el vigor de la semilla. este estudio sugiere que la conductividad eléctrica puede ser útil en j. curcas para la prueba de vigor y sus semillas no necesitan previamente imbibición de agua para mejorar la germinación de las semillas con una humedad inicial inferior al 8%. palabras clave: biocombustible, contenido de agua de la semilla, humedad de la semilla, germinabilidad 1plant physiology laboratory, federal university of pernambuco, department of botany, recife, pe, brazil, 50670901 introduction jatropha curcas l. belongs to the family euphorbiaceae, is native from the american tropics (abhilash et al., 2010). this species looks like a small tree with 6 m in height (sunil et al., 2013). j. curcas is a seed-bearing plant and can produce from 1,500 to 2,000 kg of seed per hectare/ year or 540 to 680 liters of biofuel per hectare, considering that j. curcas seeds contain about 40% to 58% of oil (pandey et al., 2012; marcelo francisco pompelli et al., 2010). moreover, j. curcas is a non-edible, eco-friendly, non-toxic, biodegradable fuel-producing plant that has attracted worldwide attention as an alternative sustainable lozano, f.; miranda,p.; pompelli, m. peruvian journal of agronomy 1(1): 32-38 33 energy source for the future (dharma et al., 2017). this species can be cultivated on marginal and salt affected areas, without competing with crop food production (elhag and gafar, 2014). in general, more than 95% of the oil produced for biodiesel purposes comes from edible oils, like corn and soy, which can have a negative impact on food production (khan et al., 2014). thus, j. curcas seeds seems as a good source of oil and it has great economic potential as an alternative biofuel (berchmans and hirata, 2008; chen et al., 2008). the seeds of j. curcas have a short viability period and they are more sensitive to salinity at germination (elhag and gafar, 2014; moncaleano-escandon et al., 2013). j. curcas is drought tolerant (arcoverde et al., 2011; marcelo f. pompelli et al., 2010) and probably also has salinity tolerant (lozano-isla, in press). seed deterioration is a natural and irreversible process, even under ideal storage conditions (castellión et al., 2010; copeland and mcdonald, 1999; marcos-filho, 1998; moncaleano-escandon et al., 2013). while deterioration is both irreversible and inevitable, the speed of the process can be controlled with appropriate harvesting, drying and storage techniques. there are several factors that are known to influence the progress of deterioration during seed storage. both high temperatures and humidity during storage increase the deterioration speed of seeds (copeland and mcdonald, 1999; pukacka et al., 2009), and decreasing either of these factors significantly increases the storage life of seeds (castellión et al., 2010). dry seeds suffer a variety of biochemical and metabolic changes, including lipid peroxidation, enzyme inactivation and rupture of cellular membranes (alencar et al., 2015; moncaleanoescandon et al., 2013). in another way, seed imbibition is an important process in the plant life cycle and determines whether seed germination and plant growth will be successful or not (ribeiro et al., 2015). in arid environments, the water needed for germination is available for only short periods and consequently, successful crop establishment depends not only on rapid and uniform germination of the seed, but also on the ability of the seed to germinate under low water availability (windauer et al., 2007). the speed and uniformity of seed germination are prominent parameters especially for field crop seeds to compete with weed seeds (ruttanaruangboworn et al., 2017). water uptake is the fundamental requirement for the initiation and completion of seed germination (koornneef et al., 2002). studies on germination and seedling establishment, which are the critical stages in the plant life cycle and in j. curcas, have not been conducted. knowledge of the capacity of the species to complete this stage successfully is fundamental for crop production (windauer et al., 2011). considerable variation was registered in j. curcas for seed germination, seedling growth and biomass parameters. the small value of error or environmental variances of the seedling growth traits suggest that the majority of characters are under genetic control (ginwal et al., 2005). based on these, the main objective of this study was to evaluate the behavior of j. curcas seeds under different imbibition time, seed water relation and aspects about germination. materials and methods plant material—the experiment was carried out with 2 kg of jatropha curcas seeds its where collected in a commercial plantation from the atlantic rain forest region (09°28’s; 35°51’w m.a.s.l.). the plantation consisted of plants that were at least 8 years of age, and the spacing between plants was 2 m × 2 m. fruits of j. curcas were randomly collected during the rainy season from 2015. the seeds presented 72% viability and stored as recommended by moncaleano-escandon et al. (2013). seed imbibiton test and water relation—the seeds were distributed in 52 frasks (400 ml) with 25 seed for each replication treatment in a controlled room chamber at 25°c. for each frask, 100 ml deionized water was applied and the seeds were soaked according to the imbibition treatments (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours). the ph (w3b, bel engineering, italy) and electrical conductivity (cd-4306, lutron, taiwan) were evaluated with 20 ml of soaking solution for each imbibition treatment. for seed water content, 50 ml deionized water was applied in frasks of 100 ml and were added 10 seed previously weighted in an analytic scale, according to different imbibition time. after each treatment was take the seeds imbibition weight and putted in papers bags for oven at 105°c for 24 hours and determinate seeds dry weight. the water relation variables were calculated according the following formulas: smt(%)=1-(sdw/sfw)*100 and swc(%)=(stwsfw)/sfw*100. where smt(%), seed moisture; swc(%) seed water content; sdw(g), seed dry weight; sfw(g), seed fresh weight and stw(g), seed turgid weight. germination test—for each replication treatment, 25 seeds were uniformly distributed in polyethylene trays content 1000 g of river sand and covered with 200 g of the substrate. the germination experiment was carried out in greenhouse condition with average temperature of 27.5°c and 78% relative humidity. seed germination was evaluated daily according to agronomic criteria consider germinated seed when the radicle had emerged about 10 mm above the soil surface. when no additional germination was observed in all treatments at least in five consecutive days, the germination was considered completed (moncaleanoescandon et al., 2013). data analysis—the experiment was carried out in a completely randomized design with 13 treatments of imbibition times (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours) with four replications with seeds of j. curcas. statistical analysis were performed in the statistical software r (r core team, 2017). the analysis of variance germination behavior of jatropha curcas l. after different imbibition times junio-setiembre 2017 34 (anova) was performed to evaluate the differences between the factors and the comparison of the means with the student-newman-keuls test (p < 0.05) (de mendiburu, 2017). the germination variables and graphs were performed according the germinar r package (lozano isla et al., 2017). for the multivariate analysis, correlation analysis (de mendiburu, 2017; wei and simko, 2017) and principal component analysis were conducted (husson et al., 2017). results electrical conductivity and ph evaluation—the water solution from the soaked seeds showed variation under the imbibition time for electrical conductivity (ec) and ph, figure 1 a-b. the ph range from 7.7 to 5.1 showing difference between the different imbibition time with a reduction of the ph in the time (r = -0.88, p<0.05), figure 1 a. while the ec show an increase in relation to imbibition time (r = 0.80, p<0.05) with value ranges from 0.021 to 0.69 ds m−1, figure 1 b. according the correlation analysis exits a negative correlation between ec and ph (r = -0.74, p<0.05). seed water relation—the initial seed moisture (smt) was 7.9% and after 24 hours of imbibition was 9.5%, figure 1 c. the smt show a strong positive correlation with the imbibition time (r = 0.89, p < 0.05). while the seed water content (swc) show a fast increase reaching up to 25.7% until the first two hour of imbibition; afterwards, the swc increases continuously to about 59.2% in 24 hours shown an increase around 6.5 times from initial values. figure 1 d; this results is supported for a high correlation between imbibition time with the swc (r = 0.93, p < 0.05). also exist a high correlation (r = 0.96, p < 0.05) between smt and swc. seed germination analysis—initial germinability of 85% of the seeds of j. curcas decreases significantly after 2 hours of imbibition. after that, the range of germinability was 68% to 44% from 2 to 24 hour of imbibition (figure 2 a), showing a strong negative correlation with imbibition time (r = -0.72, p < 0.05). the mean germination time from seed without imbibition has a value at 4.8 day in comparison with the other treatments with values around 5.9 to 7.1 day for germination for 2 to 24 hours of imbibition (figure 2 b). seed germinability presented a negative strong correlation with a mean germination time (r = −0.88, p < 0.05). the germination synchrony did not show difference between the imbibition times (figure 2 c). for the experiment, the maximum value for the uncertainty is 4.64 bits and the values ranged from 1.86 to 2.34 bits without difference between treatments and the results didn’t show any trend with the imbibition time. while the germination synchrony show a high correlation (r = −0.92, p < 0.05) with the germination uncertainty. multivariate analysis—the principal component analysis according to the studied variables explain 75.03% of the variance between the first and second dimension. in the first dimension there is a high positive correlation between elc (r = 0.97, p<0.05), swc (r = 0.96, p<0.05), smt (r = 0.91, p<0.05), stw (r = 0.91, p<0.05), ibth (r = 0.87, p<0.05), mgt (r = 0.82, p<0.05) with a negative correlation with the grp (r = -0.92, p<0.05). while in the second dimension sfw (r = 0.77, p<0.05), sdw (r = 0.71, p<0.05) present positive correlation in contrast with hpt (r = -0.75, p<0.05) with negative correlation (figure 3). figura 1. response of jatropha curcas seeds after different imbibition time. (a) ph; (b) electrical conductivity; (c) seed moisture and (d) seed water content. the letter represent the mean difference with student-newmankeuls test (p = 0.05). means are represent with (±se). n = 4. lozano, f.; miranda,p.; pompelli, m. peruvian journal of agronomy 1(1): 32-38 35 figure 2 germination response in jatropha curcas seeds after different imbibition times. (a) germination (%); (b) mean germination time (days); (c) germination synchrony and (d) germination uncertainty (bits). the letter represent the mean difference with student-newman-keuls test (p = 0.05). means are represent with (±se). n = 4. discussion the water content in seeds with impermeable seed coats has important implications for germination, because impermeable coats prevent germination until environmental conditions promote water absorption by seeds followed by germination (kestring et al., 2009; ribeiro et al., 2015). this study found a reduction in the seed germination of j. curcas, according to seed water figure 3. principal component analysis from the variables in jatropha curcas seeds after different imbibition times. where: ibth, imbibition time; grp, germination percentage; mgt, mean germination time; syn, germination synchrony; unc, germination uncertainty; ph, potential of hydrogen; ec, electrical conductivity; swc, seed water content; smt, seed moisture; sdw, seed dry weight; sfw, seed fresh weight; stw, seed turgid weight. imbibition. it is supposed that seeds need a small amount of water for promote the germination because the water imbibition had linearly decrease the germinability and increase its mean germination time, two parameters related to the seed vigor. this phenomena was previously reported in other species, like corn (matthews and hosseini, 2006), rice (ruttanaruangboworn et al., 2017) castor oil (ribeiro et al., 2015) and astrophytum species (sánchez-salas et al., 2012); however, in contrast of mimosa bimucronata (kestring et al., 2009) a floodplain species, where the water uptake sharply increases the seed germination. also, it was observed during the time line of the experiment there was an increase in ec that reflect in lost the seed germinability from seed steeping in water from 2 to 24 hours. the seeds used in this experiment were stored in dry environments and hence had very low levels of metabolism. we argue that, during seed imbibition, they swell and metabolic activity increases. hydration of tissue components during imbibition takes place in a not controlled way so that the reconstruction of internal structures of the cells and organelles were affected. therefore, leakage of stored components and enzymes, coloring, cracking or absence of cotyledons, and overall damage to the hypocotyl may occur during germination (hobbs and obendorf, 1972; pollock et al., 1969). the amount of the constituents of the leaked depended unequivocally on the initial water content of seeds; the lower moisture in seed at the initial water content show more leakage occurring in seeds with low water contents, below 10% in soybeans seeds (ishida et al., 1988). this damage takes place in the early stages of germination behavior of jatropha curcas l. after different imbibition times junio-setiembre 2017 36 imbibition (parrish and leopold, 1977). this indicates that membrane functions are restored, even though the activities of respiration and metabolism are restricted. water molecules are semi-bound and mobile water necessary for metabolism is deficient for moisture contents between 12-24% (koizumi et al., 2008). according to these, the loss of viability can be explained base on the initial seed water content of the seeds used in the experiment because they had an initial moisture around 8%, that is low value compared with the moisture at harvest that is around 18% (marcelo francisco pompelli et al., 2010). a possible explanation could be the loss of water by storage condition of the seed for the experiment. in other crops like soybean seeds, water content is usually 10 to 20% at harvest and falls further during storage, seed water contents below 10% were shown to be desirable for long period storage because seeds stop their biological activities and the stored materials are consumed at a minimum level (windauer et al., 2007). j. curcas seeds after 24 hour of imbibition increases 6.5 times its initial moisture as reported in soybean seeds (ishida et al., 1988). dried seeds can raise their water content to a certain level, two or three times the dry weight, and this rapid increase of water is often accompanied by some deterioration of the tissues, called imbibitional damage. this damage is expressed as a reduced rate of germination and reduced yield of surviving plants (ishida et al., 1988). it can be the reason in decrease in the germination percentage in this research. it was reported that soybean seeds with the water content below 13% suffered seriously from imbibitional damage while those above 17% did not, where respiration and metabolic activity rapidly increase with the increase of moisture content (ishida et al., 1988; vertucci and leopold, 1984). imbibition damage results from the rapid entry of water into the cotyledons during imbibition, leading to cell death and high solute leakage from the seeds (powell et al., 1986) and the extensive loss of cellular material and enzymes from the seeds (duke and kakefuda, 1981; powell and matthews, 1981) indicates extensive membrane disruption. the electrical conductivity was related to seed water content and the germination for this reason ec tests has been applied to detect vigor differences in many other grain legumes and indeed some other species (hampton and tekrony, 1995; moncaleano-escandon et al., 2013). the conductivity will increase as the laboratory germination falls, in addition to the reduced ability of germination seeds to retain cell contents (matthews and hosseini, 2006). reports on pea lots, the ec readings for lots have been found to relate significantly to field emergence (powell and matthews, 1981; thornton et al., 1990). to alleviate the effects of imbibition damage as a result of the increase in the water content of seeds, a slow and controlled hydration is essential as the first step in the reactivation of metabolic processes in dry seed (vertucci and leopold, 1984) leading an increase in the germination and growth ability. the ec vigor test would be developed and standardized for these species (abdullah et al., 1991; powell, 1986; yaklich and kulik, 1979). furthermore, it was reported than the relationship between field emergence and ec turned out to be not only interesting, but useful in practical seed technology (matthews and powell, 2006) as present in these work for j. curcas. conclusions the initial water content in j. curcas seeds should be consider at germination because it will alter seed germinability according to the imbibition time. the ec measurement could have a role such us ageing based vigor test or controlled deterioration test, by giving a measure of viability in 24 hours in place of a normal germination test that takes around 15 days or longer in j. curcas. acknowledgments the authors thanks the national council for scientific and technological development (cnpq grants 404357/20130) for financial support to the work execution. the first author thanks the coordination of improvement of higher education personnel (capes) for the scholarship. the authors would also like to thanks miriam vieira de albuquerque for her support during the experiment and noellle b. vanderlei dos santos for her kind revisions of this manuscript. references abdullah, w.d., powell, a.a. and matthews, s. 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(1979). evaluation of vigor tests in soybean seeds: relationship of the standard germination test, seedling vigor classification, seedling length, and tetrazolium staining to field performance1. crop science 19(2), 247-252 https://doi.org/10.2135/cr opsci1979.0011183x001900020019x peruvian journal of agronomy 3(2): 57-67 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i2.1315 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 02 november 2018 accepted for publication: 12 june 2019 characterization and control of the causal agent of leaf spot in strawberry (fragaria ananassa) under greenhouse conditions caracterización y control del agente causal de la mancha foliar en fresa (fragaria ananassa) en invernadero orellana, c.1*; mattos, l.2 *corresponding author: corellanau81@gmail.com abstract strawberry (fragaria ananassa) plants showing symptoms of leaf spot were found in the producing areas of manchay bajo, pachacamac district, lima, peru. the responsible pathogen was isolated and observed under a microscope to examine its morphological characteristics and the size of its structures, resulting in its identification as the fungus corynespora cassiicola. healthy strawberry plants that had been inoculated with c. cassiicola in a greenhouse developed lesions after 3 days and exhibited symptoms consisting of dark brown spots that had light brown centers and were surrounded by yellow halos, which coalesced and subsequently caused necrosis of the whole leaf. in vitro tests demonstrated that the fungus trichoderma harzianum strain t-22 showed antagonistic activity against c. cassiicola, causing 41.8% mycelial growth inhibition, and the fungicides fosetyl-aluminum (aliette®) and thiabendazole (mertect®a) inhibited mycelial growth by 100.00% and 96.32%, respectively. however, in vivo, 0.75 g/l of the fungicide azoxystrobim (stronsil®) gave the best control of c. cassiicola and the lowest area under the disease progress curve (audpc), followed by 1.5 ml/l of thiabendazole (mertect) under greenhouse conditions. the isolate was found to be pathogenic, and its morphological identification was confirmed. key words: leaf spot, corynespora cassiicola, strawberry, fragaria ananassa resumen plantas de fresa (fragaria ananassa) mostrando síntomas de mancha foliar fueron encontradas en las áreas productoras de manchay bajo, distrito de pachacamac, lima. después del aislamiento, observaciones bajo el microscopio de sus características morfológicas y tamaño de sus estructuras, permitieron identificar al patógeno como el hongo corynespora cassiicola. las plantas de fresa sanas inoculadas en invernadero, produjeron lesiones después de tres días. los síntomas consistieron en manchas de color marrón oscuro con el centro de marrón claro, rodeados por un halo amarillo, que coalescen y posteriormente causaron la necrosis de toda la hoja. el hongo trichoderma harzianum strain t-22 mostró una actividad antagónica contra c. cassiicola causando una inhibición de micelio en un 41.8 %. en los ensayos «in vitro», los fungicidas fosetil aluminio (aliette) y tiabendazol (mertect) inhibieron el crecimiento micelial de c. cassiicola en un 100 y 96.32 %, respectivamente. en invernadero, el fungicida azosystrobim (stronsil) a una concentración de 0.75 g/l tuvo el mejor control sobre c. cassiicola y el menor audpc (área bajo la curva de progreso de enfermedad), seguido de tiabendazol (mertect) a una concentración de 1.5 ml/l. palabras clave: mancha foliar, corynespora cassiicola, cultivo de fresa (fragaria ananassa). introduction strawberry (fragaria ananassa) plants are grown in pachacamac district, peru, due to the favorable environmental conditions in this region. however, plants that grow here are also prone to a disease that is characterized by the presence of necrotic spots on the leaves, with brownish tones being acquired quickly under favorable conditions. these leaf spots appear as small, circular, dark brown areas that have a light brown center and are surrounded by a purple or reddish halo, and infected plants also develop black lesions on the stems that are elongated and sunken in shape and surrounded by a reddish halo. the disease can affect any phenological stage of the plant and can eventually destroy the crop if a suitable control strategy is not carried out in time. during the summer of 2015 in the area of manchay bajo, 1 mg. sc. phytopathology, of the universidad nacional agraria la molina. 2 senior professor of the dept. of phytopathology, faculty of agronomy of the universidad nacional agraria la molina http://revistas.lamolina.edu.pe/index.php/jpagronomy/index mailto:corellanau81@gmail.com characterization and control of the causal agent of leaf spot in strawberry (fragaria ananassa) under greenhouse conditions may august 2019 58 pachacamac district, it was found that strawberry plants were dying from some unknown agent that caused purpleedged foliar spots. therefore, the aims of this study were to identify the causal agent of leaf spot in strawberry and to determine the best biological or chemical control agent against this disease under greenhouse conditions. materials and methods they were collected from random portions of leaves and petioles of five strawberry plants (cultivar ‘camino real’) with symptoms of leaf spot from a cultivation area located in manchay low, pachacamac district. samples of leaves and petioles were placed in polyethylene bags duly identified with place and date of sampling, which were immediately transported to the laboratory. the laboratory and greenhouse phase were conducted from january to april 2016 and october 2016 to january 2017 at the department of phytopathology, national agrarian university la molina of lima, peru. isolation and identification of the pathogen the leaves and petioles of diseased strawberry plants were washed with a continuous stream of water followed by three washes with sterile distilled water. the samples were placed inside an aseptic chamber and immersed in a 0.5% sodium hypochlorite solution for 3 minutes. the disinfected materials were then cut into small pieces taking into account the margins of advancement of the lesions, placed on sterile filter paper, and left to dry until they were dehydrated. the dehydrated materials were placed on plates containing a previously prepared potato dextrose agar (pda) medium, which were sealed with parafilm and incubated at 28 °c–30 °c for 15 days. once mycelium development of the causative agent of the disease had occurred, peals were made until a pure culture was obtained. identification of the pathogen was carried out in three stages. first, the pathogen was identified to the genus level using the key of barnett and hunter (1972). second, it was identified to species level based on the morphological characteristics of its fruiting structures using the key of ellis (1971). to determine the dimensions of sporebearing structures, the length, diameter, and number of proliferations were measured in 50 conidiophores, and the length, diameter, truncated base diameter, and number of pseudosepta were measured in 50 conidia. third, the identity of the pathogen was confirmed by biomolecular dna analysis at the institute of molecular biotechnology of the national agrarian university la molina according to the protocol developed by aamir, sutar, singh, and baghela (2015) with some modifications. molecular identification the species-specific molecular identification of the strain was based on an analysis of the internal transcribed spacer (its) 1-5.8s-its2 region of the rdna (white, bruns, lee, and taylor 1990). for the extraction of dna, the mycelium of a fungus that had been grown in a liquid culture based on potato dextrose for 5 days at 28 °c was collected and the mycelial mass was recovered by filtration through gauze. the dna was then obtained following the methodology of aamir et al. (2015). the dna integrity was verified by electrophoresis in 1% (w/v) agarose gel. the internal regions its1 and its2 between ribosomal genes (rdna) 18s-5.8s and 28s (white et al., 1990) were amplified by polymerase chain reaction (pcr) with the primers its1 (5’-ccgtaggtgaacctgcgg-3’) and its4 (5’-tcctc¬cgcttattgatatgc-3’). the reaction mixture consisted of pcr buffer (1x), the its1 and its4 primers (1 mm), mgcl2 (3 mm), deoxyribonucleotide triphosphate (dntp; 0.2 mm), taq dna polymerase (2.5 units), and dna (100 ng) to give a final volume of 50 ml. the pcr amplification was carried out in a thermal cycler (techne®-312, minnesota, usa) under the following conditions: initial denaturation at 95 °c for 2 min, followed by 30 cycles of denaturation at 94 °c for 1 min, alignment at 54 °c for 30 s, and extension at 72 °c for 1 min, with a final extension at 72 °c for 5 min (aamir et al., 2015). the amplification products were visualized by electrophoresis using 2% (w/v) agarose gel (sigma®), and the amplified pcr products were sequenced in the laboratory psomagen inc: humanizing phenomics 120 (www.macrogenusa.com). the consensus sequence was constructed using bioedit sequence alignment editor version 7.2.5 (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). then, this sequence was compared using the genbank database of the national center for biotechnology information (http://blast.ncbi.nlm.gov) through the blast program, and these are presented as percent identity in this study. then, the sequences were aligned using mega program version 7.0 (https://www. megasoftware.net/) with the help of the mrbayes version 3.2.6 program (http://mrbayes.sourceforge.net.) and they were analyzed by bayesian inference after obtaining information on distribution and arrangement using a phylogenetic tree, which was built using figtree version 1.4.3 (https://beast.community/figtree). pathogenicity test and inoculation of strawberry plants for the greenhouse experiment a pathogenicity test and inoculations were carried out on 5-week-old strawberry plants of the ‘camino real’ cultivar under greenhouse conditions. for the pathogenicity test, five plants were inoculated and five were left as uninoculated controls, while for the greenhouse experiment, 120 plants were inoculated to test the effects of nine treatments compared with an untreated control. in all cases, the plants were inoculated by sprinkling a spore suspension that contained 1.8 × 104 conidia/ml in a volume of 8–10 cc/plant over the entire leaf area of each plant. for all uninoculated control plants, only water was sprayed on the leaves. subsequently, each of the plants was covered with orellana, c.; mattos, l. peruvian journal of agronomy 3(2): 57-67 (2019) 59 a polypropylene bag containing holes for 2 days to provide the necessary humidity for developing disease symptoms. reisolation of the pathogen samples were taken from inoculated plants that showed symptoms of leaf spot. these samples were obtained from the border of the necrotic and healthy tissue on leaves with lesions and petioles with symptoms of canker. the samples were disinfected, dehydrated, and planted in apg medium, following which mycelium purification was performed and the developed colony was placed under alternating periods of 12 hours of artificial light and 12 hours of darkness to induce sporulation. in vitro evaluation of the effectiveness of biocontrol agents the biocontrol agents used included bacillus subtilis (5 g/l), pseudomonas fluorescens (2.5 g/l), and trichoderma harzianum strain t-22 (5 g/l). the effectiveness of biocontrol agents was examined using the dual sowing method: bacteria b. subtilis and p. fluorescens as well as fungus t. harzianum strain t-22. each biocontrol agent was sown on four plates facing the isolated pathogen, using agar, potato, sugar, peptone as the culture medium for the bacteria and pda for the fungus. all of the confrontation plates were sealed with parafilm and incubated at 28 °c–30 °c. during sowing for the biocontrol bacteria and biocontrol fungus, pathogen colonies were observed; daily measurements of mycelium were performed and compared with the data obtained for controls. the effectiveness of each biocontrol agent was then determined by calculating the percentage of mycelial inhibition (pi) using the following equation of pandey et al. (jaramillo, barrezueta, luna, & castillo, 2017): pi = ((dc − dt) / dt) × 100 where dc is the average diameter of the control colony and dt is the average diameter of the treated colony. the average growth rate was determined by dividing the radial mycelial growth by the number of days elapsed. the significance of any differences in growth between treatments was analyzed using analysis of variance (anova) and tukey’s multiple comparison test with a significance level of α = 0.05. in vitro evaluation of the effectiveness of fungicides the active ingredients of fungicides used were azoxystrobin (0.75 g/l), chlorothalonil (1.5 ml/l), difenoconazole (0.15 ml/l), thiabendazole (1.5 ml/l), and fosetyl-aluminum (3 g/l). the efficacy of fungicides was evaluated using the poisoned food test. four plates containing pda medium were used with the corresponding fungicide and isolated pathogen undergoing active growth and for the controls, whereas 4 plates containing pda medium were used without fungicides but with the isolated pathogen. all plates were sealed with parafilm and incubated at 28 °c–30 °c. the average rate of growth was then determined and the efficacy of each fungicide was assessed by calculating the pi. the significance of any differences in growth between treatments were analyzed using anova and tukey’s multiple comparison test with a significance level of α = 0.05. in vivo evaluation of the effectiveness of biocontrol agents and fungicides strawberry seedlings were moved from a field in the lower manchay area to the greenhouse in october 2016 when they were 3 weeks old and were planted in a mixture of compost, sand, and farmland soil at a ratio of 1:1:1. a total of 130 seedlings were transplanted, 10 of which were used for the pathogenicity test (five for inoculation and five for the uninoculated control). the remaining 120 plants were used to test the effectiveness of the biocontrol agents and fungicides in vivo. a completely randomized experimental design was used, with a total of nine treatments plus a control, four replicates per treatment, and three subsamples per replicate (10 × 4 × 3 = 120 plants). the same biocontrol agents and fungicides were tested as in the in vitro tests outlined above. the biocontrol agents and fungicides were applied every 7 days in the afternoon when the daytime temperatures were lower. before each application, the progression of the disease was evaluated using the following scale: 0% = healthy plant, 0%–5% = appearance of small spots, 5%– 25% = spots covering up to 25% of the plant; 25%–50% = spots covering up to 50% of the plant; 50%–75% = spots covering up to 75% of the plant, and 75%–100% = spots affecting the entire plant. at the start of the experiment, all plants had 0% infection and the percentages were recorded as foliar disease progressed. the area under the disease progress curve (audpc) was then calculated for each treatment using the formula described by pedrozasandoval and samaniego (2009). the significance of differences between treatments was assessed using anova followed by tukey’s multiple comparison test in the sas 8.0 statistical program with a significance level of α = 0.05. results isolation and identification of the pathogen after 15 days of incubation, the development of a mycelium was observed on each tissue sample exhibiting leaf spot symptoms. each mycelium produced solitary, monopodial conidiophores bearing conidia that were solitary or in chains, which together formed a fluffy layer that covered the entire leaf spot area (figure 1). the colony had a large cotton-like mycelium that was light green in color at 4 days after sowing in pda medium and developed a cottony, dark brown mycelium at 13 days after sowing (figure 2). characterization and control of the causal agent of leaf spot in strawberry (fragaria ananassa) under greenhouse conditions may august 2019 60 a b c d figure 1. leaf spot in strawberry (fragaria ananassa) plants. a) strawberry field infected with leaf spot. b) leaves exhibiting symptoms of leaf spot. c) symptoms of leaf spot after 15 days of incubation in a humid chamber. d) pathogen structures developed on an area of leaf showing symptoms of leaf spot. figure 2. leaf samples sown in potato dextrose agar (pda) medium. a) fungal mycelia isolated 4 days after sowing. b) pure culture in pda medium 13 days after sowing. figure 3. fruiting structures of c. cassiicola observed under a microscope: a) conidiophore and conidia in formation. b) conidia with transverse septa. appendix. the conidia emerged from an apical pore on the conidiophore either individually or in short chains and developed acropetally. based on these characteristics and the key of barnett and hunter (1972), the fungus was identified as belonging to the genus corynespora. based on these dimensions (table 1), the macroscopic and microscopic characteristics described above, and the observation of the pathogen under a compound microscope showed the presence of a mycelium, conidiophores, and conidia with dark pigmentation. each cylindrical conidiophore was simple, short or long, and bore exogenous, straight or curved conidia that were ellipsoidal or obclavate, fragmospore type, solitary with a thick wall, and had a rounded apex without an apical a b a b orellana, c.; mattos, l. peruvian journal of agronomy 3(2): 57-67 (2019) 61 value conidiophore conidia length diameter no. length diameter diameter (μ) no. (μ) (μ) proliferations (μ) (μ) truncate base pseudosepta minimum 111 5 3 41 9 4 4 maximum 363 11 9 116 12 5 8 average 164 6 5 59 10 5 5 published range, (ellis, 1971) 110–850 4–11 2–9 40–20 9–22 4–8 4–20 table 1. dimensions of the conidiophore and conidia of the isolated corynespora sp. obtained from leaf spots on strawberry (fragaria ananassa) leaves. code of strain morphological identification reported country molecular identification homology identity (%) accession number aee (peru) c. cassiicola argentina c. cassiicola 100 kx786348 florida (usa) c. cassiicola 100 ku593529 india c. cassiicola 100 km458795 china c. cassiicola 100 kt002182 mexico c. cassiicola 100 gu461298 table 2. percentage identity of the isolated strain with sequences of corynespora cassiicola accessions from different parts of the world. figure 4. phylogenetic tree constructed by bayesian inference wherein it shows the relationships between the isolate under study corynespora cassiicola aee (perú) and corynespora cassiicola species. characterization and control of the causal agent of leaf spot in strawberry (fragaria ananassa) under greenhouse conditions may august 2019 62 identification keys of barnett and hunter (1972) and ellis (1971), it was confirmed that the fungus belonged to the genus corynespora, with similar characteristics to the species corynespora cassiicola (figure 3). molecular identification comparative analysis of the sequence using genbank allowed the identification of the strain with 100% identity indexes with the compared species, reported in different parts of the world (table 2). phylogenetic analysis of the sequence of c. cassiicola strains isolated from strawberry and other 15 sequences of corynespora derived from genbank generated a tree by bayesian inference, which revealed two major groups (i and ii) formed by subgroups (bootstrap values : 51–83). in group i, two subgroups (c1 and c2) were grouped. subgroup c1 included the isolate fj852737 corresponding to the sequences of c. citrícola (bootstrap value: 56). subgroup c2 included the isolates fj852739 and fj852740, with the former corresponding to the sequences of c. proliferata and latter to those of c. smithii (bootstrap value: 51). in group ii, six subgroups were grouped (c3, c4, c5, c6, c7, and c8). subgroup c3 included the isolates fj852594 and fj852593 corresponding to the sequences of c. citrícola (bootstrap value: 82). subgroup c4 included the isolates jq717031, ky588854, fj852596, fj852882, and ky305472 corresponding to the sequences of c. citrícola, c. proliferate, c. proliferate, c. proliferate, and c. smithii, respectively (bootstrap value: 82). subgroup c5 included the isolate ab539438 corresponding to the sequence of c. smithii (bootstrap value: 83). subgroup c6 included the isolate mh484001 corresponding to the sequence of c. smithii (bootstrap value: 80). subgroup c7 included the isolate under study, i.e., aee of c. cassiicola aligned with the ailamientos mk530180, mk392012, and mh255527 corresponding to the sequences of c. cassiicola isolated from mexico and china (bootstrap value: 80). the alignment of the isolate aee among the species of c. cassiicola within subgroup c7 showed the probability of it belonging to this species. subgroup c8 included the isolate dq369804 corresponding to the sequence of rhizoctonia solani, which was only used to anchor the phylogenetic tree (figure 4). pathogenicity test strawberry plants began to show symptoms of leaf spot on the upper and lower sides of young and mature leaves 3 days after being inoculated with c. cassiicola, whereas sporadic petioles showed signs of infection 4–5 days after inoculation. both young and mature leaves exhibited spots and circular patches of brown that were 2–6 mm in diameter and had a light brown circle in the center. as time progressed, these spots enlarged until they coalesced with each other. in addition, a thick, yellow halo appeared around the spots, which expanded once they had joined together and subsequently caused necrosis of the leaf (figure 5a). on the petiole, the disease appeared as a small black spot surrounded by a reddish halo, which subsequently enlarged to become a canker (figure 5b). the control plants did not show any symptoms of leaf spot (figure 5c). reisolation of the pathogen the mycelium that developed from the leaf spot and canker sores were similar to those found in the pathogen isolate. both developed light green-gray cotton mycelium, and the mycelium gradually acquired a dark brown tone. subsequently, measurements of the conidiophore and conidia were performed, and the results are shown in table 3. in vitro evaluation of the biocontrol agents b. subtilis (biosplent) exhibited 24.60% mycelial inhibition against the pathogen, while p. fluorescens (biodelta) achieved 12.00% inhibition compared with 0.00% inhibition for the control (pda). anova indicated that there was a significant difference between treatments, with a coefficient of variability of 7.57% (table 4), and tukey’s multiple comparison test showed that b. subtilis had a significantly higher percentage inhibition against figure 5. disease symptoms in strawberry (fragaria ananassa) plants. a) strawberry plant showing necrotic spots and yellowing at 11 days after inoculation with corynespora. cassiicola. b) symptoms of leaf spot on the petiole. c) uninoculated control plant. a b c orellana, c.; mattos, l. peruvian journal of agronomy 3(2): 57-67 (2019) 63 c. cassiicola than the other treatments at the 0.05 level (table 5). t. harzianum biocontrol strain t-22 inhibited the growth of c. cassiicola mycelia by 41.8% compared with 0.0% for the control. anova indicated that there was a significant difference between treatments, with a coefficient of variability of 9.32% (table 6), and tukey’s multiple comparison test showed that t. harzianum strain t-22 significantly inhibited the growth of c. cassiicola compared with the control at the 0.05 level (table 7). in vitro evaluation of the fungicides the fungicides tiabendazole (mertect®; t4) and fosetyl-aluminum (aliette®; t5) achieved the greatest percentage inhibition of c. cassiicola mycelia, with rates of 96% and 100%, respectively, while the fungicides azoxystrobin (stronsil®; t1), chlorothalonil (zetron® 720; t2), and difenoconazole (difeconasil®; t3) inhibited the development of the colony by 31%, 51%, and 68%, respectively. anova indicated that there was a significant difference between treatments, with a coefficient of variability of 4.69% (table 8), and tukey’s multiple comparison test showed that fosetyl-aluminum and tiabendazol achieved significantly greater levels of inhibition than the other treatments at the 0.05 level (table 9). value conidiophore conidia long length (μ) diameter (μ) n°proliferations long (μ) diameter (μ) diameter (μ) truncate base no. pseudosepta minimum 118.58 4.84 2 43.56 6.66 3.63 3 maximum 219.01 6.05 6 82.28 10.89 4.84 7 average 167.22 5.28 4.16 57.79 9.32 4.65 4.28 published range (ellis, 1971) 110–850 4–11 2–9 40–220 9–22 4–8 4–20 table 3. average measurements of the conidiophore and conidia that were reisolated from strawberry (fragaria ananassa) plants in the pathogenicity test. source of variability degrees of freedom sum of squares mean square f p treatment 2 0.12005000 0.06002500 697.06 <0.0001 ** error 9 0.00077500 0.00008611 total 11 0.12082500 c.v. = 7.57% α = 0.05 ** = highly significant table 4. results of the analysis of variance for the percentage mycelial inhibition of bacillus subtilis and pseudomonas fluorescens against corynespora cassiicola. treatment description average level of significance* t2 b. subtilis against c. cassiicola 0.245000 a t1 p. fluorescens against c. cassiicola 0.122500 b t0 control 0.000000 c table 5. results of the tukey’s multiple comparison test for the percentage mycelial inhibition of bacillus subtilis and pseudomonas fluorescens against corynespora cassiicola. *rows with different letters are significantly different (p < 0.05) source of variability degrees of freedom sum of squares mean square f p treatment 1 0.34861250 0.34861250 919.42 <0.0001** error 6 0.00227500 0.00037917 total 7 0.35088750 c.v. = 9.32 α = 0.05 ** = highly significant table 6. results of the analysis of variance for the percentage mycelial inhibition of trichoderma harzianum strain t-22 against corynespora cassiicola. treatment description average level of significance t1 trichoderma harzianum strain t-22 against c. cassiicola. 0.41750 a t0 c. cassiicola (control) 0.00000 b table 7. results of the tukey’s multiple comparison test for the percentage mycelial inhibition of trichoderma harzianum strain t-22 against corynespora cassiicola. *rows with different letters are significantly different (p < 0.05) characterization and control of the causal agent of leaf spot in strawberry (fragaria ananassa) under greenhouse conditions may august 2019 64 in vivo evaluation of the biocontrol agents and fungicides strawberry plants that were cultivated under greenhouse conditions exhibited the lowest leaf spot severity when treated with azoxystrobin (stronsil), which had a lower audpc value compared with the other treatments (table 10 and figure 6). anova indicated that there was a significant difference between treatments, with a variability coefficient of 14.34% (table 11), and tukey’s multiple comparison test showed that azoxystrobin resulted in a significantly lower audpc than the other treatments at the 0.05 level (table 12). discussion the morphological characteristics of the fungus that was isolated from strawberry plants showing symptoms of leaf spot, such as the color of the colony and the characteristics of the conidiophore (cylindrical, simple, short or long) and conidia (exogenous, straight or curved, ellipsoidal or obclaved, fragmospore type, individual or in short chains, and acropetal formation) coincided with reports by barnett and hunter (1972) and jayasuriya and thennakoon (2007) for the genus corynespora. the microscopic characteristics of this fungus, such as the dimensions of the conidiophores and conidia, together with the macroscopic characteristics resembled the species c. cassiicola (barnett and hunter, 1972; ellis, 1971). ‘camino real’ strawberry plants developed disease symptoms on the leaves and petioles 3 days after inoculation with isolated c. cassicola under greenhouse conditions. similarly, breton et al. and lamotte (déon et al., 2012) stated that c. cassiicola infection occurs 1–2 days after inoculation due to secretion of the toxin cassiicolina, which is a glycosylated protein of 27 amino acids. the symptoms of leaf spot in the inoculated leaves were characterized by dark brown spots with a light brown center surrounded by a yellow halo. this matches the description of coelho, pino, fonseca and muniz (2013) who reported that the disease originating from c. cassiicola causes lesions on the leaves that initially have a yellow halo and then progress to develop a beige or light brown area in the center. in addition, they mention that the spores of this pathogen are mainly dispersed by wind, irrigation water, sprinkler systems, and rain. tan (manju, 2011) indicates that symptoms of leaf spot can occur on leaves, petioles, and twigs throughout the year. infected leaves of strawberry plants that were collected from the field exhibited a reddish halo around the leaf spots, whereas those that were inoculated in the greenhouse source of variability degrees of freedom sum of squares mean square f p treatment 5 30429.66337 608.593.267 810.57 <0001 ** error 18 135.14753 750.820 total 23 30564.81090 c.v. = 4.69% α = 0.05 ** = highly significant table 8. results of the analysis of variance for the in vitro evaluation of fungicides against corynespora cassiicola. treatment commercial fungicide active ingredient average level of significance t5 aliette® fosetyl-aluminum 100.000 a t4 mertect® tiabendazol 97.893 a t3 difeconasil® difeconazol 68.405 b t2 zetron® 720 clorotalonil 53.053 c t1 stronsil® azoxystrobin 30.508 d t6 pda (control) potato, dextrose, agar 0.000 e table 9. results of tukey’s multiple comparison test for the in vitro evaluation of fungicides against corynespora cassiicola. treatment active ingredient audpc t1 bacillus subtilis 487.02 t2 trichoderma harzianum strain t-22 490.61 t3 pseudomonas fluorescens 383.09 t4 azoxystrobin 194.10 t5 clorotalonil 543.82 t6 difenoconazol 409.05 t7 tiabendazol 358.48 t8 fosetyl-aluminum 494.39 t9 inoculation 555.68 t10 without inoculation 0.00 table 10. average area under the disease progress curve (audpc) for leaf spot in strawberry (fragaria ananassa) plants following the application of nine treatments and in an uninoculated control under greenhouse conditions. orellana, c.; mattos, l. peruvian journal of agronomy 3(2): 57-67 (2019) 65 did not. it is important to note that the plants from which leaves were collected in the field were around 16 months of age, whereas those that were used in the pathogenicity test were only 5 weeks old. in addition, the field crop was grown under a temperature range of 15.9 to 25.1 °c with a relative humidity of 87.3%, whereas the greenhouse had a temperature range of 23.7 to 38.7 °c and a relative humidity of 78%. jacob (manju, 2011) previously reported that the symptoms originating from c. cassiicola infection vary widely between different clones, localities, and leaf stages at the time the infection established. furthermore, miller and alfieri (1974) found that the symptoms that developed on variegated and nonvariegated ligustrum sinense plants following inoculation with c. cassiicola isolated from variegated plants were indistinguishable from those found in naturally infected plants, with tiny reddish, circular spots that enlarged into light brown lesions with purple margins being formed in the variegated plants, and brown lesions with yellow halos being produced in the non-variegated plants. therefore, it is very likely that age and environmental conditions influenced the expression of the symptoms in the present figure 6. average area under the disease progress curve (audpc) for different treatments used to control leaf spot in strawberry (fragaria ananassa) under greenhouse conditions.. source of variability degrees of freedom sum of squares mean square f p treatment 9 1093589.571 121509.952 38.52 <0001 ** error 30 94627.650 3154.255 total 39 1188217.220 c.v. = 14.34% α = 0.05 ** = highly significant table 11. results of the analysis of variance for the average values of the area under the disease progress curve (audpc) for leaf spot in strawberry (fragaria ananassa) plants with different treatments under greenhouse conditions.. table 12. results of the tukey’s multiple comparison test for the average values of the area under the disease progress curve (audpc) for leaf spot in strawberry (fragaria ananassa) plants with different treatments under greenhouse conditions. treatment active ingredient fungicide average level of significance t9 -----inoculation 555.69 a t5 clorotalonil zetron® 720 543.82 a b t8 fosetyl-aluminum aliette® 494.39 a b c t2 trichoderma harzianum strain t-22 t-22 490.61 a b c d t1 bacillus subtilis biosplent 487.02 a b c d t6 difenoconazolof difeconasil® 409.05 b c d t3 pseudomonas fluorescens biodelta 383.09 c d t7 tiabendazol mertect® 358.49 d t4 azoxystrobin stronsil® 194.1 e t10 ----no inoculation 0 f characterization and control of the causal agent of leaf spot in strawberry (fragaria ananassa) under greenhouse conditions may august 2019 66 study. the experimental inoculations gave positive results and the final characteristics of the fungus that was isolated from leaves and petioles exhibiting symptoms were the same as those of the pathogen that was initially isolated, fulfilling koch’s postulates. consequently, it is considered that c. cassiicola is the causal agent of leaf spot in strawberry. the fungus t. harzianum strain t-22 was able to control c. cassiicola growth in vitro, achieving 41.8% mycelial inhibition. leal (2000; lorenzo, 2001) indicated that trichoderma species have a direct action against phytopathogenic fungi and use different mechanisms to regulate their development, with the main mechanisms being competition for space and nutrients, mycoparasitism, and antibiosis. among the fungicides that were tested, fosetyl-aluminum (aliette) had the greatest efficacy against c. cassiicola in vitro, inhibiting mycelial growth by 100%. this inhibition was caused by the acidic environment it created (ph 2.3) when mixed with the culture medium. the fungicide tiabendazol (mertect) showed the second highest level of control in vitro, achieving 96.32% inhibition of the mycelium. by contrast, azoxystrobin achieved the greatest control of leaf spot disease in strawberry plants under greenhouse conditions, with the lowest audpc value. similarly, carmona, gally, sautua, abello, and lopez (2011) found that the application of azoxystrobin to soybean (glyxine max) exhibiting ringed spots originating from c. cassiicola controlled the disease and increased yield, the 1000-seed weight, and, in some cases, the number of grains per square meter. conclusions the causal agent of leaf spot in strawberry was identified morphologically and biomolecularly as c. cassiicola. the biological control agent t. harzianum strain t-22 achieved significant control of c. cassiicola in vitro, with 41.8% mycelial inhibition, compared with 24.6% for the bacterium b. subtilis (biosplent) and 12.0% for the bacterium p. fluorescens (biodelta). in addition, the fungicides tiabendazol (mertect) and fosetyl-aluminum (aliette) achieved control levels of 96.32% and 100.00%, respectively, in vitro. however, the fungicide azoxystrobin (stronsil) was the best treatment for strawberry leaf spot in vivo under greenhouse conditions, with fosetyl-aluminum (aliette) having no effect on this disease under the same conditions. 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o f _ f u n g a l _ r i b o s o m a l _ r n a _ g e n e s _ f o r _ phylogenetics peruvian journal of agronomy 2 (2): 27 33 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i2.1199 © the authors. published by universidad nacional agraria la molina received for publication: 12 july 2018 accepted for publication: 30 october 2018 insect pests associated with andean lupin (lupinus mutabilis sweet) and their parasitoids in peruvian central coast – (lima, la molina) insectos plaga asociados al cultivo del tarwi (lupinus mutabilis sweet) y sus parasitoides en costa central del perú– (lima, la molina) yony t. callohuari1*, clorinda e. vergara1, jorge e. jiménez2 * corresponding author. ycallohuari@lamolina.edu.pe abstract this survey aimed to determine the insect pests associated with andean lupin (lupinus mutabilis sweet) and its parasitoids, under peruvian central coast conditions. sampling was made every seven days with the following methods: linear meter evaluations, plant organ evaluations, pitfall traps, and beating trays. infested plant organs were taken to the laboratory for adult insect recovery (phytophagous or parasitoids). pitfall traps and beating tray samples were also processed and stored. during the survey, 14,051 individuals belonging to 11 orders and 91 families were recorded. insects that infested andean lupin were melanagromyza lini spencer (stem borer), liriomyza huidobrensis (blanchard) (leafminer), crocidosema aporema (walsingham) (budborer), grammopsoides tenuicornis (casey) (stem borer) and frankliniella occidentalis (pergande) (bud and flower thrips). diglyphus websteri (crawford), d. begini (ashmead), chrysocharis flacilla (walker), c. caribea boucek, chrysocharis sp. and halticoptera arduine (walker) were recorded as parasitoids of l. huidobrensis, whereas carcelia sp. was recorded for c. aporema. key words: lupinus mutabilis, adean lupin, liriomyza huidobrensis, melanagromyza lini, insect fauna. resumen el objetivo de esta investigación fue determinar los insectos plaga asociados al cultivo de tarwi (lupinus mutabilis sweet) y sus parasitoides, en condiciones de la costa central. las evaluaciones se realizaron cada siete días y se emplearon las siguientes metodologías: evaluación por metro lineal, evaluación por órganos de la planta, trampas de caída y sacudida de plantas. se llevaron al laboratorio órganos infestados de la planta para la recuperación de fitófagos adultos y/o parasitoides, las muestras provenientes de las trampas de caída y de sacudida de plantas, para ser procesados. durante el periodo de las evaluaciones se registraron 14 051 individuos distribuidos en 11 órdenes y 91 familias. las especies que infestaron al cultivo fueron: melanagromyza lini spencer (barrenador de tallos), liriomyza huidobrensis (blanchard) (minador de hojas), crocidosema aporema (walsingham) (barrenador de brotes), grammopsoides tenuicornis (casey) (barrenador de tallos) y frankliniella occidentalis (pergande) (picador-chupador, raspador de brotes y flores). se registraron los siguientes parasitoides de l. huidobrensis: diglyphus websteri (crawford), d. begini (ashmead), chrysocharis flacilla (walker), c. caribea boucek, chrysocharis sp. y halticoptera arduine (walker), mientras que para c. aporema se registró a carcelia sp. palabras clave: lupinus mutabilis, tarwi, liriomyza huidobrensis, melanagromyza lini, entomofauna. 1 klaus raven büller entomological museum, universidad nacional agraria la molina (unalm), lima, peru. 2 cereals and native grains program, universidad nacional agraria la molina (unalm), lima, peru. introduction andean lupin (lupinus mutabilis sweet) (“tarwi,” “tarhui,” “chocho”) is an andean grain, used mainly as part of traditional cuisine. nutritional value of this grain has been addressed in numerous publications (jacobsen & mujica, 2006), as well as its potential to prevent and support the treatment of degenerative diseases (biesalski et al., 2009, fornasini et al., 2012; kris-etherton et al., 2002). in recent years, interest in andean native crops has increased due to its nutritional and therapeutic qualities, which increases its demand and leads, as in the case of quinoa (cruces et al., 2016), to be produced in large extensions. this increment implies a higher risk in crop health. it is also known that when a crop is introduced to areas that are different from its place of origin, its phenology is modified and it can sometimes become host of new pests and diseases (white, 1984; cammell & knight, 1992; kiritani, 2007; sigsgaard, jacobsen, & christiansen, 2008). in peru, agricultural activity at high altitudes involves productive risks due to unfavorable climatic conditions and incipient productive technology. the opposite scenario is seen on the coast, where the accelerated phenology of this andean crops, available technology and climatic stability insect pests associated with andean lupin (lupinus mutabilis sweet) and their parasitoids in peruvian central coast – (lima, la molina) may august 2018 28 results in better harvests. publications about andean lupin pests are relatively scarce and developed on high altitude agricultural ecosystems. alata-cóndor (1973) made first publications about insects associated with andean lupin, showing empoasca sp. and metascarta impressifrons muir as harmful insects. frankliniella auripes hood, frankliniella regia hood, frankliniella regina hood and flankliniella alonsoae hood, were recorded associated with another plant of the genus lupinus by ortiz (1973, 1977). later yabar & baca (1981) made observations on insects infesting andean lupin in cusco and recorded feltia spp., copitarsia incommoda (walker), plusia chilensis btlr, heliothis titicacae hardwick (noctuidae), colias sp. (pieridae), leptotes callangae dyar (lycaenidae). they also mentioned astylus sp., thrips sp., agromyza sp., melanagromyza sp., apion sp., epicauta sp., liriomyza sp., crocidosema sp., metascarta impressifrons signoret, empoasca sp., agrotis spp., puranius sp. and two unknown species, anthomyiidae and another belonging to tenebrionoidea superfamily. in the book “plagas de cultivos andinos”, sánchez & vergara (1991) included a list of 22 species of pest insects associated with andean lupin. carrasco (1995) mentions eight species of insects infesting andean lupin fields in cusco: apion (trichapion) lupini (coleoptera: curculionidae), “black beetle of tarhui”; liriomyza andina (malloch) (diptera: agromyzidae), “fly borer of branches and stem”, liriomyza sp. “leafminer fly”; colias lesbia (fabricius) and c. euxanthe feld (lepidoptera: pieridae), “leaf and pod eaters”; gnorimoschema sp. (lepidoptera: gelechiidae), “leafminer moth”; hylemya santi-jacobi (diptera: anthomyiidae) and diabrotica decempunctata (latreille) (coleoptera: chrysomelidae). recently, camarena-mayta et al. (2012) published extensive information about the center of origin, physiology, wild relatives, nutritional values and culinary recipes of andean lupin, with a brief chapter dedicated to pests, mentioning cutworms as copitarsia turbata herrich & schäffer, feltia spp. and prodenia spp; also dr. camarena considered as secondary pests to frankliniella alonsoae hood, f. regia hood and liriomyza sp; additionally, empoasca sp. as an occasional pest. taking this into account, the objective of this research was studying insects associated with andean lupin in central coast conditions, determining the main phytophagous species, the behavior of their populations, and predators and parasitoids of harmful species. this study will allow a comparative analysis between the harmful and the beneficial insect communities among the andean and coastal agroecosystems. this information will help to establish baselines for integrated pest management strategies for this crop on the peruvian coast. materials and methods this survey was carried out in a field of andean lupin in “guayabo 01” farm, within the campus of universidad nacional agraria la molina (unalm), district of la molina, province of lima, lima region, located at 12 ° 05’06 “s, 76 ° 57’06 “w and 251 meters above sea level, from june 30, 2013 to december 24, 2013. l. mutabilis seed from inia (instituto nacional de innovación agraria) of ‘andenes’ variety was used. during growing season no application of insecticides was carried out; however, a fungicide was applied at the beginning to prevent fungi borne diseases in addition to herbicide 45 days after sowing. survey of the insects was carried out in an area of 2500 m2 and was divided into five sectors, as recommended by sarmiento & sánchez (2012). evaluations began on july 30, 2013, finished on december 24, 2013, and were made every seven days in the morning from 6 a.m. to 12 p.m. five techniques were used: direct search at ground level, plant organ evaluation, beating tray, pitfall traps and in situ counting. temperature and relative humidity were recorded. a) field work. direct search at ground level. two linear meters at ground level were evaluated in each sector. presence of cut seedlings or plant cutworms on each side of the linear meters was observed during the first four evaluations. plant organ evaluation a total of 20 plants in the field were evaluated, four by sector. one terminal shoot (terminal bud and 2-3 first leaves) per plant was observed during vegetative development. plants were divided by thirds for evaluation from the beginning of floral bud formation until the end of the growing season. beating tray this procedure was done in a plastic tray (30 cm x 40 cm) with water and detergent to prevent insects from escaping. four plants for each sector were tilted towards the tray and shaken vigorously, trying that all arthropods fall on it, then the content of the tray was transferred into hermetic containers and was labeled indicating the corresponding sector. pitfall traps a fall trap was installed per sector. the traps consisted of cylindrical containers of polypropylene of 400 ml capacity, placed at ground level, with a mixture of coolant and water (1:1), as well as a detergent. the coolant was used to prevent the decomposition of the collected arthropods, and the detergent was used to break the surface tension. in situ counting this method consisted in counting by direct observation of the insects perched on the plants. yony t. callohuari, clorinda e. vergara & jorge e. jiménez peruvian journal of agronomy 2 (2): 27 33 (2018) 29 b) laboratory procedures screening and preservation of samples of pitfall traps and beating tray. the contents of each container were sifted; water was applied carefully to clean the dirt. each sieved sample was moved to another bowl with 75% alcohol. sorting, coding, counting, recording and preserving of morphotypes sorting each sample was transferred to a 6 mm diameter petri dish. each plate was examined through a karl zeiss jena stereoscope with 10-100x magnification, where morphotypes were separated. according to oliver & beattie (1993), a morphotype is a group of specimens provisionally equivalent to species, based on conspicuous differences in their external morphology. coding each morphotype was provisionally placed in a taxonomic category and assigned a code using the first 3 or 4 letters of the taxon in which it was classified, followed by a number. the provisional determination was preferably family, example: “dol01”, if the specimen was provisionally determined as a dolichopodidae; “dip01”, if the specimen was determined only up to its order level, in this case, diptera case and so on. to the identified and encoded morphotype, at least one photograph was taken as reference for subsequent samples. the code of the picture was added to the morphotype code, in such a way that each container had the morphotype code and the code of the corresponding photograph annotated to help the comparison. count and record once the morphotype was established and a code assigned, all individuals corresponding to said morphotype were counted by sector and by week. it was recorded in a physical record and later this information was transferred to a digital spreadsheet. in the case of very abundant morphotypes such as some of collembola, thysanoptera, eulophidae, and cicadellidae, a manual hand counter was used. preserving each morphotype was preserved in a glass container of 2 cm in diameter x 5 cm in height with a rubber cap and ordered alphabetically, considering its code. all samples were stored in museo de entomología klaus raven büller (mekrb) at unalm. procedure for adults and / or parasitoids recovery leaves and stems infested with immature phytophagous insects were taken to the laboratory and were conditioned in 400 ml polypropylene containers covered with organza fabric to allow perspiration and a double layer of paper towel on the base to avoid excess moisture. it was observed periodically until adults or parasitoids were obtained. location in the corresponding taxonomic category of specimens by morphotype collected all morphotypes were determined at a family level, some at a genus level and others at a species level, depending on their abundance or importance in the crop. the determination at the family level in most cases was made without the need of mounting; however, in the case of small or tiny specimens, it was necessary to make a tip or slide mounting before clarification treatment according to the corresponding taxonomic group. aphididae and thysanoptera mounting the procedure suggested by voegtlin et al. (2003) for aphididae was used. procedure for mounting diptera preserved in 75 ° alcohol specimens were placed in containers with 96° alcohol for 24 hours, then transferred to containers with ethyl acetate for 48 hours and then mounted in entomological tips. microhymenoptera mounting mounting was carried out according to steps suggested by pitkin (2003). taxonomic keys used families − insecta (triplehorn & johnson, 2005) − hymenoptera (fernandez & sharkey, 2006) genera − tachinidae (wood & zumbado, 2010) species − thysanoptera (mound & kibby, 2005) − agromyzidae (korytkowski, 2014; spencer, 1973) − halticoptera (de santis, 1987) − diglyphus (gordh & hendrickson, 1979) and dr. mujica (pers. comm.) − chrysocharis (hansson, 1987) results and discussion a total of 14,051 specimens were recorded in 11 orders and 91 families (table 1). the most abundant insect families were: thripidae (3626 specimens), order thysanoptera; eulophidae (1713), scelionidae (672) and formicidae (523), order hymenoptera; cicadellidae (1347) and aphididae (632), order hemiptera; sciaridae (511), agromyzidae (244), phoridae (231), sphaeroceridae (229) and dolichopodidae (207), order diptera; and latridiidae (348), order coleoptera. insect pests associated with andean lupin (lupinus mutabilis sweet) and their parasitoids in peruvian central coast – (lima, la molina) may august 2018 30 table 1. orders, number of speciemens and number of families families of insects collected from july 30 to december 24, 2013. la molina, lima peru order number of specimens number of families thysanoptera 5191 1 hymenoptera 3200 23 hemiptera 2264 12 diptera 2155 29 coleoptera 841 15 psocoptera 193 2 orthoptera 90 1 lepidoptera 65 4 neuroptera 42 2 dermaptera 7 1 dyctioptera 3 1 total 14,051 91 insect pests associated with andean lupin in la molina. melanagromyza lini spencer, 1963 (diptera: agromyzidae). this is a neotropical species, registered in colombia (ruiz & checa, 1990), in chile cited as m. aguilerai by spencer (1982) (sasakawa, 1994), peru (korytkowski, 2014; spencer, 1963) and argentina (sasakawa, 1992). m. lini is a stemborer species and has been reported on linum usitatissimum l. (spencer, 1963), on vicia faba l. (ruiz & checa, 1990), phaseolus vulgaris and chenopodium pallidicaule (korytkowski, 2014). in andean lupin, the larvae of this species came to infest 100% of the plants evaluated and caused the weakening of the stems and subsequent entry of pathogens. no parasitoids were recovered, so biological control of this species is probably very limited, a fact that could not be confirmed due to the scarce information about the biology of this species. liriomyza huidobrensis blanchard, 1926 (diptera: agromyzidae) this is a polyphagous species (spencer, 1973), benavent et al. (2004) indicate that it has 109 host species distributed in 33 families. in peru korytkowski (2014) records species from amaranthaceae, asteraceae, brassicaceae, cucurbitaceae, fabaceae, malvaceae, solanaceae, apiaceae and liliaceae families, as hosts of l. huidobrensis. in this study, l. huidobrensis has been present during the whole period of the crop, infesting up to 98% of the leaves of the lower third, 85% of the middle third and 26% of the upper third. this preference for the lower leaves has been described in other crops such as potatoes (solanum tuberosum l.) and broad bean (vicia faba l.) (galantini & redolfi 1993, neder et al., 1993). gonzales-bustamante (1995) mentions the phenomenon of “extrusion” of the eggs of l. huidobrensis as one of the causes for the lower infestation of the upper third. the adult females performed feeding punctures in the leaves, so the presence of these punctures would constitute evidence of a future attack of this species, even before the mines made by the larvae become evident. according to martin et al. (2005), these punctures also helps the female to detect if the plant is suitable for oviposition. crocidosema aporema (walsingham, 1914) (lepidoptera: tortricidae) this tortricid moth is an oligophagous species of neotropical origin that colonized soybean when this crop was introduced into the new world (sánchez & pereyra, 2008). small larvae feed on growing buds and occasionally tunnel into leaf petioles (turnipseed & kogan, 1976). c. aporema also feeds on shoots of many other cultivated legumes such as peanuts, clover, alfalfa, lotus, melilotus, lupin, broad bean, beans and others (sánchez & pereyra, 2008). in this study, c. aporema showed a preference for the upper third, due to the architecture of andean lupin, since it presents most of its buds in the upper third. another detail to mention is that due to the continuous sprouting of l. mutabilis, the damage of this species occurred from four weeks before flowering to the end of the growing season, unlike soybeans, where flowering periods and therefore of infestation, are shorter (correa, 1980). grammopsoides tenuicornis (casey, 1913) (coleoptera: cerambycidae) this longhorn beetle is a species registered in panama, ecuador, colombia (monné 2014) and peru (vergaracobián & amaya, 1978). it has been previously registered as grammopsoides rufipes breuning in peru by (vergaracobián & amaya, 1978) in a potato crop in chimbote, later by campbell (1980) and the international soybean program (1980) in soybeans. in andean lupin, this species infested the stems, maintaining a level of infestation of 30%, not as high as in the case of m. lini, however, it was observed that a single larva of g. tenuicornis, due to its size, caused a significant weakening of the stem. frankliniella occidentalis (pergande, 1895) (thysanoptera: thripidae) f. occidentalis is a widely distributed (kirk & terry, 2003) and highly polyphagous species, since it feeds more than 250 crops from more than 60 botanical families (reitz, 2009); however, a considerable degree of specificity has been found towards some hosts in certain populations (mound & marullo, 1996). f. occidentalis was by far the most numerous species of phytophagous insect collected in andean lupin. the number of specimens recorded by evaluation was increasing, starting with 15 individuals three weeks after flowering until reaching 1296 individuals at harvest period. this species infested the buds and flowers of andean lupin, causing deformations in the growing leaves and feeding on the petals, however, direct damage to the pods was not observed. according to van yony t. callohuari, clorinda e. vergara & jorge e. jiménez peruvian journal of agronomy 2 (2): 27 33 (2018) 31 emden (2013), the highest damage they can cause in other legumes is the non-formation of the pods. this damage is of particular importance in ‘determinate’ legume varieties. the latter is not the case of andean lupin, which would be able to compensate the damage due to its continuous flowering behavior. b) parasitoids of insect pests associated with tarwi culture in la molina in this survey was registered parasitoidism for liriomyza huidobrensis and crocidosema aporema, both species of insect pests associated with andean lupin. chrysocharis caribea boucek, c. flacilla (walker), chrysocharis sp., diglyphus begini (ashmead), d. websteri (crawford) (hymenoptera: eulophidae) and halticoptera arduine (walker) (hymenoptera: pteromalidae) were recorded in this research as parasitoids for l. huidobrensis. according to the literature, in peru, l. huidobrensis has a considerable amount of parasitoid insects, 16 species of the eulophidae (sánchez & redolfi, 1987, 1989, cisneros & mujica, 1998, delgado & aguilar, 1980; mujica & kroschel, 2011), a species of pteromalidae (delgado & aguilar, 1980, cisneros & mujica, 1998, arellano & redolfi, 1989, mujica & kroschel, 2011); a species braconidae (delgado & aguilar, 1980, sánchez & redolfi, 1989, cisneros & mujica, 1998) and a species of the eucoilidae (sánchez & redolfi, 1989, 1987, cisneros & mujica, 1998, mujica & kroschel , 2011). as parasitoid of crocidosema aporema was recorded in this survey carcelia sp. (diptera: tachinidae) in peru, trichogramma bactrae nagaraja (trichogrammatidae) has been registered as an egg parasitoid of c. aporema (whu & valdivieso, 1999); however, researchers from argentina, uruguay and chile have registered different parasitoids from the families braconidae, ichneumonidae, aphelinidae, trichogrammatidae and tachinidae (liljesthröm & rojasfajardo, 2005, ribeiro et al., 2015, rojas, 1968, sánchez & pereyra, 2008). conclusions species that could be considered pests in andean lupin in this survey for its damage and abundance are: melanagromyza lini spencer, liriomyza huidobrensis (blanchard), crocidosema aporema (walsingham), frankliniella occidentalis (pergande) and grammopsoides tenuicornis (casey). diglyphus websteri (crawford), d. begini (ashmead), chrysocharis flacilla (walker), c. caribea boucek, chrysocharis sp., closterocerus sp. 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(1981). algunos lepidópteros que atacan al “tarhui” (lupinus mutabilis) en el cusco. revista peruana de entomología, 24(1), 81–85. http:// sisbib.unmsm.edu.pe/bvrevistas/entomologia/v24/ pdf/a09v24.pdf peruvian journal of agronomy 3(3): 91-103 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1342 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 13 october 2019 accepted for publication: 11 november 2019 characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru caracterización de fundos productores de palto y espárrago en la irrigación chavimochic, en la libertad, perú apaza, w.(1)*; quiroz, p.(2); julca-otiniano, a.(3) *corresponding author: wapaza@lamolina.edu.pe abstract the chavimochic irrigation project is one of the leading projects in peru. in this project, irrigation water is derived from the santa river to irrigate the valleys of chao, viru, and moche, including desert zones between valleys. in this study, we aimed to characterise the hass avocado and asparagus farms in desert areas of the chavimochic irrigation project. social, economic and environmental information was collected through structured surveys of 12 asparagus and 17 avocado farms. four groups of asparagus farms and three groups of avocado farms were identified based on principal component and cluster analyses. profitability of the avocado crop was higher than that of the asparagus crop, and irrigation caused more significant variability in the crop yield of avocado than that of asparagus. from the social perspective, the income of farm personnel was related to the level of education, and female personnel showed more participation in the chavimochic irrigation project than in other agroecosystems of peru. keywords: avocado, asparagus, characterisation, farms, chavimochic irrigation resumen la irrigación de chavimochic es uno de los proyectos más importantes realizados en el perú. esta irrigación usa el agua derivada del rio santa para irrigar los valles de chao, virú y moche. incorporando las zonas de desierto entre valles. el presente trabajo tuvo como objetivos caracterizar los fundos productores de palto hass y espárrago en las zonas de desierto de la irrigación de chavimochic. información social, económica y ambiental fue recolectada mediante encuestas estructuradas realizadas a los 12 fundos de espárrago y 17 fundos de palto. mediante la técnica de análisis de componentes principales y de agrupamientos de conglomerados se identificó 4 grupos de fundos de espárrago y tres grupos de fundos de palto. la rentabilidad del cultivo de palto es mayor que la de cultivo de espárrago, el uso del recurso hídrico por los fundos tiene mayor variabilidad en el cultivo de palto que el espárrago. en la parte social el nivel de ingresos del personal de los fundos tiene relación con el grado de instrucción, la participación del género femenino es mayor que en otros agroecosistemas del perú. palabras claves: palto, espárrago, caracterización, fundos, irrigación chavimochic 1 universidad nacional agraria la molina (unalm). phd student of sustainable agriculture. lima, perú. 2 universidad nacional agraria la molina (unalm). faculty of agronomy. lima, perú. 3 universidad nacional agraria. la molina (unalm). grupo de investigación agricultura y desarrollo sustentable en el trópico peruano. facultad de agronomía. departamento de fitotecnia. lima, perú. introduction the chavimochic irrigation project consists of the derivation of water from the santa river for irrigating the valleys of chao, virú, moche, and chicama, including the desert zones between each valley. this irrigation has made possible to incorporate desert areas into agriculture. in this area, intensive agriculture has been developed under pressurised irrigation with agro-export crops. crops grown in this area include avocado (8,402 ha), asparagus (5,528 ha), sugarcane (3,183 ha), blueberries (3,270 ha), and others, adding up to a total area of 21,297 ha (junta de riego presurizado de chao, virú y moche, 2016). chavimochic is one of the leading centres of agroexports of peru and generates substantial incomes for the development of la libertad and the country in general. recently, there has been a major change in the crop cultivation scheme in chavimochic. ten years ago, the main crop was asparagus (two types: white and green) reaching an area of 11,000 ha. most of this area has subsequently been replaced by avocado, which is now the characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru september december 2019 92 most important crop, with an area of 8,400 ha. thus, with the increase in avocado cultivation area, the asparagus cultivation area is decreasing, leading to the decline of yield and profitability. currently, avocado and asparagus are the most important crops in chavimochic; therefore, the impact of chavimochic on the agroecosystem is significant. multivariate statistical analysis is used in the typification of producer farms. this exploratory analysis makes it possible to classify, summarise and order all of the variables measured. these variables allow the farms with common characteristics to be grouped into sets (escobar & berdegué, 1990; ortuño & coronel de renolfi, 2005). the objective of this study was to characterise avocado and asparagus farms of the chavimochic irrigation project in la libertad, peru. materials and methods this research was carried out in the chavimochic irrigation project located in la libertad along the northwestern coast of peru. avocado and asparagus farms are located in the desert areas in this region. crops are produced using a pressurised irrigation system, with the intention of agroexport. all farms have a social name and are grouped in the pressurised irrigation board of chao, virú and moche. this irrigation board administers and charges the consumption of irrigation water in the chavimochic irrigation project. the farms have a high technological production level. this research was carried out in all avocado (17) and asparagus farms (12) of the chavimochic irrigation project. to characterise the avocado and asparagus farms, data were collected on several by handing out structured surveys to managers or heads of avocado and asparagus farms and to the workers on these farms. soil samples were also collected to analyse soil microbiota. additionally, data were collected on water consumption and area of the pressurised irrigation board of chao, virú and moche via in-situ visits. structured surveys visits were made to all producer farms, and structured surveys were carried out at different levels: managers or heads of farms and workers on each farm. managers or heads of farms are the people who make the technical and administrative management decisions for the avocado and asparagus crops. managers of all 17 avocado farms and 12 asparagus farms were included in the survey. additionally, 10 workers were randomly selected from each farm, totalling 170 surveys on avocado farms and 120 surveys on asparagus farms. all surveys focused on social, economic and environmental variables. soil samples soil samples were taken from all avocado and asparagus production fields. a representative plot was selected at each farm, and 20 sub-samples were collected from each plot. each sample was composed of 2 kg of soil. a total of 29 samples (17 avocado and 12 asparagus) were processed. all samples were collected from the root zone and used to analyse soil characteristics. soil characterisation samples were sent to the soil laboratory of the universidad nacional agraria la molina, and soil organic matter content, acidity, electrical conductivity, n, p2o5, and k2o contents and cation exchange capacity were quantified. analysis of fungal flora fungal flora in soil samples were analysed in the laboratory of the clínica de diagnosis de fitopatología. six serial dilutions of soil samples were prepared using sterile water in test tubes and streaked in petri dishes containing potato dextrose agar medium with oxytetracycline, as described previously (french & torres, 1980). the petri dishes were incubated at 25°c for 7 days, and the growing fungi were quantified and identified. the identification of fungi was performed according to the keys of barnett and hunter (1998), which differentiates among fungal genera based on morphological features. to perform diversity analysis, the number of identified species per gram of soil was determined. in-situ visits in-situ visits were made to all avocado and asparagus farms to validate the data generated in the surveys and to collect additional information on crop management practices from the farm managers or production chiefs. the data collected during the in-situ visits included vegetable crop coverage and diversity per farm, conservation zone percentage in the farms, and incidence of pests per crop and farm. data analysis data collected from the surveys (total of 65 variables per avocado and asparagus field), soil sample analysis, in-situ visits, and primary sources of the pressurised irrigation board were analysed by multivariate analysis based on principal component and cluster analyses. variables with less than 30% variation coefficient were discarded, as these were not discriminatory, according to escobar and berdegué (1990). the results of cluster analysis were displayed in a dendrogram (valerio et al., 2004; escobar & berdegué, 1990). distance between the farms was expressed as euclidean distance squared and using the ward method, the most recommended combination for perennial fruit trees (miranda & carranza, 2013). results and discussion characterisation of asparagus and avocado production farms farms in the chavimochic irrigation project investigated in apaza, w.; quiroz, p.; julca-otiniano, a. peruvian journal of agronomy 3(3): 91-103 (2019) 93 this study are listed in table 1. asparagus was cultivated over an area of 3,688.7 ha in 12 farms, while avocado was cultivated on an area of 8,515.2 ha in 17 farms. all farms were irrigated via drip irrigation. all avocados and asparagus produced on these farms are exported. thus, intensive farming systems are employed with high technological level. table 1. production areas of asparagus and avocado fields of the chavimochic irrigation project in december 2017. characterisation of asparagus farms principal component analysis separated the 29 farms into four groups (figure 1). the results of cluster analysis (ward-euclidian distance) also revealed the same grouping of farms (figure 2). the first group comprised farms not linked to the market (milag, scarlor, iansac, upao, and mauel), i.e. companies that do not directly export their products but sell it to other companies that export asparagus. the companies that constitute this group showed lower yield and usually smaller area of cultivation than companies in other groups. the second group comprised farms that produce asparagus both for canning and fresh export (alima, green, talsa and danper). these companies process their produce and export it directly to different markets. these companies also grow other crops in addition to asparagus, such as avocado and blueberry, and have different quality certifications and good agronomic practices. the third group comprised companies of medium size (yugos and morav). these companies produce fresh green asparagus, which is used for processing and export. these companies also produce white asparagus. the fourth group comprised a large canning company (savsa), with diverse crops, various certifications and quality assurance. savsa is the largest cannery in the chavimochic irrigation project and is currently diversifying its production with the avocado crop. figure 3 shows the relationship between variables used for principal component analysis. the variables were as follows: red label pesticides (pestroj), number of applications (nroapplica), exportable, organic matter (mo), microorganisms (microorg), another crops (otroscul), processing (procesa), biodiversity (biodiver), incomes (ingresos), yield (rto), certifications (certificaciones), area (area) and blanco (blanco). the analysis suggests that companies with the highest asparagus production area, most considerable number of certifications, yields, asparagus farm areas avocado farm areas farm code white asparagus (ha) green asparagus (ha) farm code avocado (ha) scarlos 57.00 0.00 alpam 410.93 alima 553.41 0.00 beggie 1120.34 yugosl 0.00 172.00 simon 567.94 danpe 399.20 598.80 arena 68.71 mauel 50.00 50.15 agron 14.00 milag 35.00 0.00 lima 150.00 green 884.71 0.00 arato 604.93 insac 55.08 0.00 yugos 360 morav 0.00 66.00 avop 512.76 talsa 480.00 0.00 campo 2523.61 savsa 1003.47 0.00 deshi 149.20 upao 170.91 0.00 green 216.91 hass 331.76 inver 18 norte 56.20 talsa 60 savsa 1350 12 farms 3688.78 886.95 17 farms 8515.29 source: junta de riego presurizado de chao, virú y moche. figure 1. biplot graph of the main components of the asparagus fields of the chavimochic irrigation project in 2017. figure 2. dendrogram showing the similarity among asparagus producing farms of the chavimochic irrigation project in 2017. characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru september december 2019 94 incomes, and highest biodiversity are those that apply the least number of applications and do not make use of highly toxic pesticides. it is important to note that a number of the smaller companies that do not process their production use highly toxic pesticides (red label) because they do not have certifications that limit the use of these products. farms with extensive areas are highly likely to leave areas for increased biodiversity. characterisation of avocado farms principal component analysis of 17 avocado farms is shown in figure 4. in general, the analysis of the relation between the variables was not as defined as in the case of asparagus; therefore, component 1 explained 32.1% of the variability, and component 2 explained only 16.9% of the variability, i.e. between the two reaches 43.7%. the results of cluster analysis of these 17 avocado farms are shown in figure 5. the grouping of avocado farms showed higher dispersion than asparagus farms, indicating greater variability in each of the components evaluated in the estates than in asparagus. three large groups were differentiated using the two methods. group 1 comprised simon, agron and alp farms, which sell their fruit to third parties, do not have many quality certifications, and apply high levels of pesticides, especially those with red labels. the second group comprised norte, talsa, inver, yogos. desh, and aren farms with medium-sized areas that sell their produce to third parties, do not have a processing plant, and require third party service. the third group comprised large companies, such as hass, arato, beggie, avop, campo and savsa, with high fruit processing capacity that export their produce directly to different markets and have the highest number of quality certifications. figure 6 shows the quantity of red label pesticide applications plotted against the number of certifications, biodiversity and farm area. the higher the area of the avocado plantations, the smaller the use of red label figure 3. variables analysed by principal component analysis of the asparagus farms. figure 4. biplot graph of the main components of the avocado plantation of the chavimochic irrigation project in 2017. figure 5. dendrogram showing the similarity among avocado plantations of the chavimochic irrigation project in 2017. figure 6. variables analysed by main principal component analysis of the avocado plantations. apaza, w.; quiroz, p.; julca-otiniano, a. peruvian journal of agronomy 3(3): 91-103 (2019) 95 more than 25 years in asparagus cultivation in that area, unlike avocado, whose intensive cultivation dates back to only 12 years. over time, the water consumption of the avocado crop has decreased. in both crops, all farms use at least one method for calculating the irrigation requirement, with the largest farms using a greater number of methods for calculating the irrigation requirements. in the chavimochic irrigation project, the pressurised irrigation board of chao, virú and moche manages the pesticides. thus, large farms have less environmental impact, as their certifications are very restrictive in the use of red label pesticides (li, 2018; ambrus & yang, 2015). analysis of the dimensions of characterisation environmental dimension tables 2 and 3 show the environmental indicators for the asparagus and avocado farms. one of the critical indicators for arid agroecosystems, as in the case of the chavimochic irrigation project, is the use of water resources. asparagus requires an average of 9,433.3 m3 of water per hectare per year, while avocado crop requires 16,941.2 m3 water. the average water requirement of asparagus in chavimochic is generally lower than that reported previously by other authors (salazar, 2012; muñoz, 2016) in other agroecosystems such as ica, where the expenditure reaches 15,000 m3/year. in the region of ica, evapotranspiration is low, and the area of asparagus is exclusively used for fresh green production, which has higher water requirement than white asparagus, which is mostly produced in chavimochic. figure 7 shows the water requirement of avocado and asparagus farms. the volume of water required for avocado cultivation per hectare per year showed greater variability between farms [standard deviation (sd) of 2,236.3] compared with asparagus whose variability was lower (sd of 290 for green asparagus and 568.9 for white asparagus). this is because the farmers who manage asparagus cultivation have a significant experience of table 2. environmental indicators at asparagus farms of the chavimochic irrigation project in december 2017. farm code soil life conservation water resource use diversity management contamination plant cover (%) organic matter use (tm/ ha) water use (m3/ ha/ año) irrigation calculation methods (number) number soil fungi species 1 plant diversity 2 conservation areas 3 number of pesticide applications number of red label pesticides biological control method uses (n° of applications) number of certifications scarlos 0 0 8000 2 1 0 2 26 5 0 1 alima 25 0 9000 3 3 0 2 19 4 1 2 yugosl 0 25 10400 2 4 0 1 18 4 0 3 danpe 25 0 11000 4 3 0 2 17 3 2 6 mauel 0 0 10400 3 2 0 0 25 5 0 1 milag 0 10 8400 2 2 0 0 19 6 0 1 green 25 0 8700 3 1 0 1 17 3 1 4 insac 0 0 9000 2 3 0 0 18 6 0 2 morav 0 15 10900 2 3 0 1 15 4 1 2 talsa 25 30 9000 3 4 0 2 22 3 2 6 savsa 0 10 10000 4 3 0 2 23 3 1 6 upao 25 0 8400 3 1 0 1 29 6 1 1 average 10.4 7.5 9433.3 2.8 2.5 0.0 1.2 20.7 4.3 0.8 2.9 notes 1soil fungi diversity scale: 0, less than 2 species; 1, 2–4 species; 2, 5–7 species; 3, 8–10species; 4, more than 10 species. 2plant cover scale: 0, monoculture; 1, low; 2, medium; 3, high; 4, total. 3farm areas for conservation scale: 0, none; 1, 0.1–0.5% of the total farm area; 2, 0.51–1%, 3, 1.1–2.5%; 4, more than 2.5%. figure 7. water consumption (m3 ha−1 year−1) in avocado and asparagus farms of the chavimochic irrigation project in december 2017. characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru september december 2019 96 distribution and cost of water used, based on the volume (m3) of water used. the tariff is 0.114 peruvian nuevos soles per m3, if the water expense is between 10,000 and 13,000 m3; and 0.218 peruvian nuevos soles per m3, if the water expense is higher than 13,000 m3. therefore, if an avocado plantation spends 16,000 m3 ha−1 year−1, the cost is s/.3488 peruvian nuevos soles. thus, companies try to use the water resource as efficiently as possible. the use of water resources is one of the main factors affecting the sustainability of arid agroecosystems. regarding the production diversity, asparagus is grown in a monoculture system, which does not allow other plant species to prosper. cultivation of white asparagus involves tasks such as hilling and deshelling, which prevent the development of other plant species. in the case of avocado, several companies have managed to place crops such as legumes (beans) or gramineae (barley) in the root development zone because the avocado crop requires no soil removal below the top of the plant. the use of associated crops is very low compared with other agroecosystems such as in the mountains (pinedo, gómez & julca, 2018) and jungle (tuesta, julca, borja, rodríguez, & santistevan, 2014), where the association with other crops is widespread because the production system is more familiar and extensive, whereas the avocado and asparagus farms are intensive systems that do not allow this type of management. the relationship between yield and water consumption (m3 ha−1 year−1) in the avocado crop was tested via regression analysis, and the results were non-significant, implying that higher water consumption does not equate to a higher yield. there are other factors, besides water, that influence the yield of avocado (teliz, 2006). the number of fungal species was higher in asparagus farms than in avocado farms. however, this indicator is quite relative, as the high biomass that has the asparagus in the soil allows the proliferation of fungi that develop on the crown (delgado de la flor, montauban, & hurtado, 1987), whereas in avocado, the highest biomass of the crop is in the aerial part. the application of pesticides was more frequent in the asparagus crop (average 20.7 times) than in the avocado crop (12.8 applications). one of the major insect pests in the farms is prodiplodis longifilia, which affects not only the yield of asparagus because of its effect on shoots but also the quality of green asparagus (castillo, table 3. environmental indicators of avocado farms of the chavimochic irrigation project in december 2017. farm code soil life conservation water resource use diversity management contamination plant cover (%) organic matter use (tm/ ha) water use (m3/ ha/año) irrigation calculation methods (number) number of soil fungi species1 plant diversity 2 conservation areas3 number of pesticide applications number of red label pesticides biological control method uses (n° of applications) number of certifications alpam 25 0 18000 1 1 1 1 15 5 2 2 beggie 25 0 14500 4 1 2 2 14 2 4 7 simon 5 0 18000 1 0 0 0 13 5 0 2 arena 25 0 15000 3 1 1 0 9 4 2 2 agron 7 0 18200 3 1 1 1 15 6 2 1 lima 35 0 16000 2 1 0 2 13 4 2 3 arato 35 5 16500 4 1 1 2 15 2 4 7 yugos 7 15 15200 2 2 2 1 12 3 2 2 avop 35 15 17000 3 2 3 4 14 2 4 5 campo 25 0 16500 3 1 3 3 13 1 4 8 deshi 0 10 14500 2 1 1 2 13 4 2 2 green 10 0 20000 3 2 1 1 13 3 2 2 hass 35 0 20000 2 1 2 3 10 3 3 4 inver 5 15 15200 3 1 1 0 13 5 2 2 norte 5 0 15000 2 2 1 1 14 6 3 1 talsa 40 0 16000 3 1 2 2 10 4 4 2 savsa 5 0 22400 3 1 2 2 12 2 3 7 average 19.1 3.5 16941.2 2.6 1.2 1.4 1.6 12.8 3.6 2.6 3.5 notes 1soil fungi diversity scale: 0, less than 2 species; 1, 2–4 species; 2, 5–7 species; 3, 8–10 species; 4, more than 10 species. 2plant cover scale: 0, monoculture; 1, low; 2, medium; 3, high; 4, total. 3farm areas for conservation scale: 0, none; 1, 0.1–0.5% of the total farm area; 2, 0.51–1%, 3, 1.1–2.5%; 4, more than 2.5%. apaza, w.; quiroz, p.; julca-otiniano, a. peruvian journal of agronomy 3(3): 91-103 (2019) 97 2006). the use of red label pesticides is higher in the cultivation of asparagus than in avocado, and methomyl is the most highly used red label pesticide. figure 8 shows the results of the assessment of different diseases encountered during the cultivation of asparagus and avocado. in asparagus cultivation, prodiplosis was the biggest problem, followed by foliar spot caused by stemphylium vesicarium and lepidoptera because these disease problems can directly affect the commercial product as well as have a high impact on the harvest. therefore, these are the two main phytosanitary problems accounting for most of the pesticide applications, consistent with castillo (2019) and delgado (2016). in the avocado crop, scales is the most important disease that needs to be controlled. although this pest does not affect avocado yield, it limits access to markets in united states and china, where tolerance levels are very low (senasa, 2014). mites are the second most crucial pest for avocado producers that account for most of the pesticide application. these two pests are widespread in arid agroecosystems lacking rainfall, which facilitates their development. all avocado and asparagus production farms have at least one certification. larger farms have more certifications because of their connection with the market and the demand of their buyers. many of the certifications require less use of pesticides, especially red label pesticides (rain forest, tesco and fair for life). in all cases, the high requirement of food safety implies that no residues are permitted in the destination markets to comply with the indications on labels, and a maximum limit of residues is compulsory for the farms of both crops. regression analysis of the percentage of red label pesticides and number of certifications of each farm revealed a significant relationship in avocado farms (r = 0.754). however, in both crops, an inverse relationship was detected between the number of certifications and use of red label pesticides (figure 9). figure 8. main sanitary problems of the asparagus and avocado fields in 2017. figure 9. relationship between the percentage of red label pesticides and number of certifications used in asparagus and avocado farms of the chavimochic irrigation project in 2017. characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru september december 2019 98 sarandón and flores (2014) reported that modern agriculture is based on the intensive use of pesticides, resulting in numerous problems of contamination and insecticide resistance in pests. farm certifications and market restrictions with respect to the use of certain pesticides and their maximum residue limits exert pressure on the use of pesticides by the avocado and asparagus farms. greater care is taken in avocado than in asparagus because the avocado fruit is directly exposed to pesticides, and the high fat content of avocadoes retains lipophilic pesticides (gilbert, garcía, & molina, 2009). economic dimension analysis tables 4 and 5 show the economic indicators of the asparagus farms. variation was observed in the yield of asparagus because the farms had different ages of asparagus and different technical management, and several farms were dedicated to green and white asparagus. the cost of cultivation also varied between farms because some farms produce fresh white asparagus, which increases their cost, table 4. economic indicators of asparagus farms of the chavimochic irrigation project in december 2017. farm code yield (kg/ ha/year) cost (usd/ ha/year) gross income (usd/ha/year) cost per kg net income (usd/ha/ año) export quality (%) other crops1 pest incidence (%) scarlos 7500 5250 8325 0.70 3075 76 0 18 alima 8200 6150 13120 0.75 6970 79 1 15 yugosl 9000 7830 12150 0.87 4320 76 1 22 danpe 13200 8400 12880 0.64 4480 80 3 14 mauel 11200 4410 5175 0.39 765 84 0 14 milag 7800 4960 6882 0.64 1922 80 0 18 green 4500 6650 12825 1.48 6175 79 2 14 insac 6200 5780 7480 0.93 1700 80 1 13 morav 9500 8500 13500 0.89 5000 75 2 22 talsa 7500 8250 15400 1.10 7150 80 2 21 savsa 10000 7350 11550 0.74 4200 82 1 20 upao 11000 5823 7603.5 0.53 1781 83 0 23 average 8800.0 6612.7 10574.2 0.75 3961.5 79.5 1.1 17.8 note 1only asparagus (0), other crop (1), two crops (2), three crops (3). table 5. economic indicators of the avocado farms of the chavimochic irrigation project in december 2017. farm code yield (kg/ ha/year) cost (usd/ha/ year) gross income (usd/ha/ year) cost per kg net income (usd/ha/ año) export quality (%) other crops1 pest incidence (%) alpam 14000 4000 24750 0.29 20750 95 0 9 beggie 16700 6000 27720 0.36 21720 96 1 5 simon 10000 6000 16500 0.60 10500 88 1 5 arena 19500 7200 32175 0.37 24975 96 0 3 agron 12500 6000 20625 0.48 14625 85 1 7 lima 13300 7000 21945 0.53 14945 91 0 8 arato 16200 6000 26730 0.37 20730 98 1 10 yugos 15500 6500 24750 0.42 18250 90 0 10 avop 15000 6100 24750 0.41 18650 92 1 8 campo 17000 6000 28050 0.35 22050 98 1 5 deshi 16500 7000 27225 0.42 20225 93 0 10 green 18000 5000 29700 0.28 24700 88 1 5 hass 15000 7500 24750 0.50 17250 94 1 8 inver 16000 6500 26400 0.41 19900 90 0 8 norte 14000 6500 23100 0.46 16600 94 0 7 talsa 15200 6200 23430 0.41 17230 90 0 10 savsa 16500 7200 27225 0.44 20025 92 0 8 average 15347.1 6276.5 25283.8 0.41 19007 92.4 0.5 7.4 note 1only avocados (0), other crop (1), two crops (2), three crops (3). apaza, w.; quiroz, p.; julca-otiniano, a. peruvian journal of agronomy 3(3): 91-103 (2019) 99 especially during harvest, but leads to higher incomes. figure 10 shows comparative yields (kg ha−1) of both avocado and asparagus; although avocado showed higher yield, it also showed greater dispersion between farms. the dispersion of avocado and asparagus yield was very similar (figure 10), with avocado showing higher biomass yield than asparagus. therefore, the production of avocado (kg ha−1) was substantially higher than that of asparagus. larger farms showed higher net incomes, mainly because of higher returns owing to connections with the external market (figure 11). additionally, fresh white asparagus was exported, which is highly profitable, and the returns are greater than those obtained from white asparagus used for canning. small companies do not have the logistics or connections to directly enter into exportation. ortiz (2018) alluded to the reconfiguration of the asparagus market: fresh green asparagus companies in peru maintain intense competition with producers in mexico (with cheaper costs and no restrictions on quarantine and pest fumigation). this has decreased the commercial window of peru, thus reducing prices and consequently the area of cultivation in peru. this is why large companies that export directly have higher incomes, while companies with smaller areas suffer losses. in recent years, increase in the cost of production and reduction in prices obtained in the market have created economic problems for small companies with high costs for the cultivation of asparagus. the average incidence of pests is high, reaching 17.5%. this is mainly due to the attack of p. longifilia, which affects the crop and the harvest of green asparagus (castillo, 2019). this pest causes an average reduction of 79.5% in export. figure 11 compares the net income of all asparagus and avocado farms. the profitability of asparagus farms was higher in the larger farms. although avocado farms showed a similar tendency, it was not as marked; the smaller avocado farms showed high income. tables 4 and 5 show that the profitability of avocado was higher than that of asparagus. in contrast to asparagus, avocado growers have not diversified their crops, which explains why the vast majority of grower only produce avocado. pest incidence was lower in avocado (table 5) (7.4%), being the pests of more significant presence the scales which do not affect the yield, but the quality of exportable. markets such as the united states and recently china require that pest damage showed be less than 5%. social dimension analysis the gender distribution and ages of workers in the asparagus and avocado fields of the chavimochic irrigation project are shown in figure 12. males showed more participation (61%) than females (39%). however, the participation of females in the chavimochic irrigation project is higher than that reported; for example, 13% in cañete in crops such as citrus and avocado (collantes & rodríguez, 2015) and 20% in chanchamayo in the pineapple crop (maraví et al., 2018). relatively young adults of both genders (26– 40 years old) showed greater participation than other age groups. figure 10. yield (kg ha−1 year−1) of avocado and asparagus farms in 2017. figure 11. net income per hectare per year from the asparagus and avocado farms in 2017. (a, b) net income from the asparagus farms (a) and avocado farms (b). characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru september december 2019 100 figure 13 shows that 44% of the families of farmworkers comprised 3–4 members, while 34% of the farmers comprised 1–2 members. this shows that families of farmworkers were small in size in comparison with families in other areas such as jungles and mountains, where the families are larger in size with more than 5 members. figure 14 shows that everyone had access to health services, mainly in the nearest cities of chao, virú and moche, which have social security health services. additionally, water, electricity, and sewage were available to 75% of the workers. most of the workers had their own houses. figure 15 shows the educational level of the personnel working on asparagus farms. approximately 65% of the managerial personnel and farm chiefs had a master’s degree, while the remaining 35% were engineers. at the plot chief level, 50% were technicians, and 34% had a professional title. among the specialised workers, 61% were technicians. among the workers, 71% possessed secondary education. this indicated that the higher the level of responsibility, the higher the level of education. the level of education was also related to the level of income. figure 16 shows the wage scale of farmworkers. workers at a higher position earned higher income, and farm managers and personnel with specialised jobs, who possessed a higher level of education, earned the highest income. thus, at the farms, the level of income of farmworkers depends on the level of their education. figure 12. gender and age distribution of workers in asparagus and avocado farms in 2017. (a) percentage of workers by gender. (b) gender and age of workers on asparagus and avocado farms. figure 13. family size of avocado and asparagus farmworkers of the chavimochic irrigation project in 2017. figure 14. access of farmworkers of avocado and asparagus farms to various services. (a) access to health. (b) access to services at the place of residence. (c) home ownership of workers. apaza, w.; quiroz, p.; julca-otiniano, a. peruvian journal of agronomy 3(3): 91-103 (2019) 101 conclusions in the chavimochic irrigation project, four groups of asparagus farms were identified, which showed a strong correlation with the type of processing, access to market, handling of pesticides and quality certifications. additionally, three groups of avocado farms were identified, where farm size, quality certifications and crop management were the most critical factors. the income level of farmworkers showed a direct correlation figure 15. level of education of the different types of workers of the asparagus and avocado fields of the chavimochic irrigation project in 2017. figure 16. income level in peruvian nuevos soles of the different types of workers of asparagus and avocado farms of the chavimochic irrigation project in 2017. characterisation of avocado and asparagus farms in the chavimochic irrigation project in la libertad, peru september december 2019 102 with the education level. the participation of females in asparagus and avocado farms was higher than that in other agricultural areas. acknowledgements we want to thank junta de riego presurizado de chao viru and moche for the support provided in the collection of information and data; farms of asparagus and avocados in the chavimochic irrigation project, and minedu grants for laboratory analysis. references ambrus, á., & yang, y. z. 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(2006). el aguacate y su manejo integrado. mundi prensa. mexico. 219 pp. peruvian journal of agronomy 3(3): 81-90 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1339 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 01 june 2019 accepted for publication: 30 july 2019 effect of the applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa ‘century’ efecto de las aplicaciones de un formulado biológico (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) en el rendimiento de allium cepa ‘century’ mamani, e. (1); acosta, m. (2); gonzales, m. (3)* *corresponding author: mariacgonzales@hotmail.com; mcgonzales@lamolina.edu.pe abstract this research was conducted from september to december 2016 on an agricultural farm in the district of santa rita de siguas, arequipa province, peru. the concentrations used were 2, 4, and 6 kg ha−1 with 4 applications by means of impregnation to the transplant and via drench every 15 days. the statistical design was through a dbca with factorial arrangement randomly distributed in four blocks. the tests used were the orthogonal contrast test and duncan’s multiple comparison test (α = 0.05). the findings showed that the treatment concentrations of 4 kg ha−1 and 6 kg ha−1 resulted in a lower incidence of fusarium oxysporum, at 6.71% and 9.36%, an abcpe of 637.78 and 950.14 units, and an exportable yield of 66.34 t ha−1 and 65.42 t ha−1, respectively, displaying significant differences from the control. the first application was statistically significant to the treatments with the highest number of applications, showing a greater exportable yield of 67.54 t ha−1. the best interactions between concentrations and applications were 6 kg ha−1 with 1 application, 4 kg ha−1 with 2 applications, and 4 kg ha−1 with 1 application, with exportable yields of 70.66, 69.61, and 69.11 t ha−1, respectively. keywords: biological formulation, azotobacter salinestris, bacillus amyloliquefaciens, and rhizophagus intraradices. resumen la investigación se realizó entre los meses de setiembre y diciembre del 2016, en la zona de santa rita de siguas – arequipa. las dosis usadas fueron dos, cuatro y seis kg ha-1 con cuatro aplicaciones mediante impregnación al transplante y vía drench cada 15 días. el diseño estadístico fue un dbca con arreglo factorial repartidos aleatoriamente en 4 bloques. se utilizó la prueba de contrastes ortogonales y la prueba de comparación múltiple de medias duncan (α = 0,05). en los resultados las dosis de los tratamientos de 4 kg ha-1 y 6 kg ha-1 presentaron una menor incidencia a fusarium oxysporum con 6,71 % y 9,36%, un abcpe de 637,78 y 950,14 unidades y un rendimiento exportable de 66,34 t ha-1 y 65,42 t ha-1 respectivamente mostrando diferencias significativas respecto al testigo. la primera aplicación fue estadísticamente significativa a los tratamientos con más número de aplicaciones mostrando un mayor rendimiento exportable de 67,54 t ha-1. las mejores interacciones entre dosis y aplicaciones fueron 6 kg ha-1 con 1 aplicación, 4 kg ha-1 con 2 aplicaciones y 4 kg ha-1 con 1 aplicación, con rendimientos exportables de 70,66, 69,61 y 69,11 t ha-1 respectivamente. palabras clave: formulado biológico, azotobacter salinestris, bacillus amyloliquefaciens y rhizophagus intraradices. 1 ing. agrónomo. facultad de agronomía–universidad nacional san agustín de arequipa (unsa) 2 gerente general de la empresa novagri sac 3 docente departamento académico de fitopatología, facultad de agronomía–universidad nacional agraria la molina (unalm) introduction in peru, 19 thousand hectares of land is cultivated with onions, representing 0.4% of the global harvested area, and 748 thousand tons of onions are produced, which represents 0.9% of the global production (apcho, caballero, & miranda, 2017).the highest production of all peruvian departments is reported from arequipa, where there are 438,323 tons of produce and 9,164 hectares of planted area, representing 58% of the national production. this is followed by ica, where 154,111 tons are produced and 2,423 hectares are planted, representing 20% of national production. in addition to these coastal locations, onions are produced in lima, la libertad, tacna, and lambayeque, and also in ayacucho and junín, in the mountains (apcho et al., 2017). currently, excessive amounts of agrochemicals are used in producing yellow onions, and this is causing serious environmental and economic problems. this is exemplified in the progressive decay of the quality of the soils used for agricultural effect of the applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa cv. “century” september december 2019 82 production, largely due to excessive application of pesticides and chemical fertilizers. it is also worth noting that the indiscriminate use of pesticides is having a negative impact on yellow onion exports to the international market, in which the tendency is to consume organic products because of their superior nutraceutical properties. one of the chief alternatives to application of agrochemicals is the application of biological formulations, prepared from beneficial microorganisms, which, according to research findings, have positive effects both on the agronomic performance of the cultivated plants and on the economic returns. nonetheless, biological formulations are used rarely in producing yellow onions, if at all, because there are no biological formulations in our market whose effectiveness and efficiency in producing yellow onions have been demonstrated through research. given this problem, this study was conducted to evaluate the effect of different concentrations and applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa ‘century’ under the edaphoclimatic conditions of santa rita de siguas. material and methods duration and location of the experiment: onion sets were planted on september 9th 2016, and the onions were harvested on december 16th 2016. the experiment took place in the field called fundo samuel zegarra, located in santa rita de siguas district, in the province and region of arequipa at latitude 16° 29’ south, longitude 72° 11’ west, and at 1,277 meters above sea level. biological material: we used the product biokit, formulated as 1 kg of dry powder. it is a biological formula containing strains of the bacterial a. salinestris and b. amyloliquefaciens and the arbuscular mycorrhiza r. intraradices in the following composition: a. salinestris ufc ….….…………….0.035% b. amyloliquefaciens ufc …………. 0.775% r. intraradices …..……..................... more than 90 imp inerts ...........….….….….….….……. 99.19% total …............. … …………………..100% edaphic characteristics of the experimental area: analysis of a representative soil sample revealed a strongly alkaline soil, with a high organic matter content, high to very high phosphorus and potassium contents, and a sandy loam texture class. land preparation: the soil was milled using a polydis, and stones that make farming difficult were removed from the soil surface. the floor was leveled using a disc and rail plow, and the beds were marked with a roller, with two lines of drip irrigators placed per bed. transplantation: transplantation was conducted when the seedlings were 15 cm high and 6 mm in diameter. four rows of seedlings were placed in each bed for each drip tape. molasses that served as a food source for the inoculated microorganisms were applied to the drip irrigation system by injection. plant material: seedlings of yellow onion (a. cepa ‘century’). treatments: the treatments using the biological product are shown in table 1. the first application of the biological product was by impregnation and before transplantation. this application was made in all treatments except for the conventional control, in which the seedlings were disinfected with a chemical fungicide based on carbendazim. the second application, by drenching, was conducted 15 days after the first application. a washer was used for this application, which was made in all treatments except for the t1 (control), t2 (2 kg ha−1), t6 (4 kg ha−1), and t10 (6 kg ha−1) treatments, as these treatments only required 1 application per impregnation. the third application, by drenching using a backpack, was conducted 15 days after the second application for the treatments t4 (2 kg ha−1), t5 (2 kg ha−1), t8 (4 kg ha−1), t9 (4 kg ha−1), t12 (6 kg ha−1), and t13 (6 kg ha−1). there was no third application to t1 and the other treatments as they involved either one or a maximum of 2 applications. the fourth application, by drenching, was conducted 15 days after the third application for the treatments t5 (2 kg ha−1), t9 (4 kg ha−1), and t13 (6 kg ha−1) and was not conducted for t1 and the other treatments that received only up to 3 applications. fertilization: fertilizers were applied by fertigation. the fertilizer levels were lower than those applied ordinarily at the farm in different crop seasons; it is important because n and k2o levels were reduced to one-third and p2o5 levels by 25 units less than the levels applied normally. (table 1) irrigation: an irrigation program was established, taking into account the crop coefficient (kc), according to the stages of development of the onion crop, and the daily evapotranspiration recorded by the fundo américa weather station. the experimental field was drip irrigated every other day. harvest: the field was undercut 99 days after transplant, when the onion plants reached 60% of bland necks. levels n p2o5 k2o cao mg zn b2o4 fundo américa. (2015) 219.1 76.7 270.8 34.3 23.2 2.2 0.8 experimental area. (2016) 40 50 80 32 2 2.6 2 table 1. fertilization levels (kg ha−1) used in the farm and the experimental area mamani, e.; acosta, m.; gonzales, m. peruvian journal of agronomy 3(3): 81-90 (2019) 83 experimental design: we used the completely randomized blocks design, in a 3 × 4 factorial arrangement, plus 1 conventional control; factor 1: n = concentration of the biological formulation (2, 4, and 6 kg ha−1), and factor 2: a = number of applications (1 application, 2 applications, 3 applications, and four applications), totaling 13 treatments with four repetitions (table 2). the results were compared using the analysis of variance (anova). the betweentreatment differences were determined using the orthogonal contrasts test, at the level of α = 0.05, and the duncan’s multiple comparison test (α = 0.05). evaluated variables surviving plants after transplanting: two evaluations were performed, at 17 and 35 days after transplantation (ddt), respectively. the percentage of plants surviving in 3 linear meters of bed was calculated (out of a total of approximately 83 plants). incidence of fusarium oxysporum f.sp cepae: the number of plants damaged by f. oxysporum in 3 meters of bed were counted at 28, 42, 56, and 70 ddt, when the first symptoms of the disease appeared. two sampling points measuring 1 linear meter each were taken for each experimental unit. the disease symptoms were basal plate and root rot. area under the disease progress curve (audpc) of f. oxysporum f.sp cepae: based on the incidence values (proportion of disease) of f. oxysporum recorded in time (days) the area under the progress curve of the disease (audpc), determining the accumulation of disease for each treatment. campbell &madden, 1990. plant height: we took 10 plants at random from the central lines and measured their height from the neck of the plant to the apex of the longest leaf. this was evaluated after transplantation, with a frequency of 21 days. number of leaves: we counted the number of leaves at 6, 21, 42, 63, and 84 days after transplantation, selecting 10 plants at random from each experimental unit. diameter of the bulb: the diameters of 20 bulbs chosen at random after harvest were measured using a digital verrnier. colonization by r. intraradices (%): samples were taken at random from the roots of three onion plants for each experimental unit, at 93 ddt. we used the methodology of philips and hayman (1970), which consists of the staining of roots with trypan blue for microscopic observation. spore frequency: soil samples weighing approximately 2 kg were taken from each experimental unit after harvest. we used the modified wet sieving and decantation protocol proposed by gerdemann and nicolson (1963), followed by centrifugation in saccharose proposed by walker and mizecw, 1982. number symbol concentration application number (days after trans-plant) application form t1 t0 0 any t2 n1a0 2 kg ha−1 1 application (at zero ddt) impregnation t3 n1a1 2 kg ha−1 2 applications (at 0 and 15 ddt) impregnation and drench t4 n1a2 2 kg ha−1 3 applications (at 0, 15, and 30 ddt) impregnation and drench t5 n1a3 2 kg ha−1 4 applications (at 0, 15, 30, and 45 ddt) impregnation and drench t6 n2a0 4 kg ha−1 1 application to (0 ddt) impregnation t7 n2a1 4 kg ha−1 2 applications (at 0 and 15 ddt) impregnationand drench t8 n2a2 4 kg ha−1 3 applications (at 0, 15, and 30 ddt) impregnationand drench t9 n2a3 4 kg ha−1 4 applications (at 0, 15, 30, and 45 ddt) impregnationand drench t10 n3a0 6 kg ha−1 1 application to (0 ddt) impregnation t11 n3a1 6 kg ha−1 2 applications (at 0 and 15 ddt) impregnation and drench t12 n3a2 6 kg ha−1 3 applications (at 0, 15, and 30 ddt) impregnation and drench t13 n3a3 6 kg ha−1 4 applications (at 0, 15, 30, and 45 ddt) impregnation and drench table 2. description of the treatments, irrigation of santa rita de siguas–arequipa, 2016 where: n: is the application level or concentration (n1, n2, and n3) or (2, 4, and 6 kg ha−1) a: number of applications (a1, a2, a3, and a4) or (1 application, 2 applications, 3 applications, and 4 applications). effect of the applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa cv. “century” september december 2019 84 total yield: immediately after harvest, all the onion bulbs were weighed separately according to each experimental unit. the yield is expressed in . the bulbs were then classified according to the categories of the american export market. economic analysis: to determine the profitability of each of the treatments under study, economic analysis was carried out, taking into account the variable costs (such as the costs of applying the treatments) and the fixed costs. results survival (%): the treatments with the lowest percentages of survival at 35 ddt were t5 (2 kg ha−1, with four applications) and t13 (6 kg ha−1, with four applications) at 82.33% and 87.45%, respectively, where as the treatments with the highest percentages of arrest were t6 (4 kg ha−1, with one application) and t8 (4 kg ha−1with three applications), at 95.55% and 94.92%, respectively. for the statistical analysis, the original data were transformed using the formula (arcsen √x 10) * 180 π, where x represented each of the obtained data. according to anova, we determined that there were no significant differences between treatments, with a coefficient of variability of 10.22 % (table 3). incidence: the treatments t4 (2 kg ha−1, with three applications) and t5 (2 kg ha−1, with four applications) had the highest rates of incidence of f. oxysporum, with incidences of 19.57% and 14.39%, respectively. meanwhile, the treatments with the lowest percentages of incidence of f. oxysporum were t6 (4 kg ha−1, with 1 application) and t8 (4 kg ha−1, with three applications) with incidences of 4.17% and 4.61%, respectively. the anova of the incidence of f. oxysporum determined that there are significant differences for treatments (table 4). duncan’s multiple comparison test (α = 0.05) revealed significant differences in the main dose factor, with doses of 4 and 6 kg ha−1 for the factor number of applications presented significant differences being the first application that had a treatments survival (%) number of leaves plant height (cm) bulb diameter (mm) colonization rhizophagus intraradices (%) spore frequency (%) t1 (control) 87.96 a 8.64 a 72,48 a 80.01 a 27.50 a 9.75 a t2 (2 kg ha−1 with one application) 92.92 a 8.77 a 72,78 a 79.79 a 52.50 a 17.25 a t3 (2 kg ha−1 with two applications) 87.57 a 8.88 a 72,29 a 80.59 a 50.00 a 14.75 a t4 (2 kg ha−1 with three applications) 89.12 a 8.68 a 73,25 a 81.41 a 35.00 a 9.50 a t5 (2 kg ha−1 with four applications) 82.33 a 8.58 a 74,20 a 79.75 a 45.00 a 12.75 a t6 (4 kg ha−1 with one applications) 95.55 a 8.62 a 74,43 a 81.16 a 55.00 a 15.75 a t7 (4 kg ha−1 with two applications) 89.70 a 8.50 a 73,58 a 80.83 a 40.00 a 14.25 a t8 (4 kg ha−1 with three applications) 94.92 a 8.69 a 73,62 a 80.07 a 45.00 a 12.50 a t9 (4 kg ha−1 with four applications) 90.55 a 8.84 a 73,84 a 82.71 a 52.50 a 17.25 a t10 (6 kg ha−1 with one applications) 90.19 a 8.68 a 75,22 a 83.18 a 50.00 a 18.75 a t11 (6 kg ha−1 with two applications) 89.07 a 8.70 a 73,36 a 81.53 a 32.50 a 13.75 a t12 (6 kg ha−1 with three applications) 87.96 a 8.87 a 72,35 a 81.40 a 22.50 a 11.25 a t13 (6 kg ha−1 with four applications) 87.45 a 8.77 a 72.35 a 81.54 a 40.00 a 12.25 a c.v 10.22% 3.97%, 4.38% 4.24% 29.09% 18.54% table 3. results of the variables evaluated and comparison test of means duncan (α = 0.05) mamani, e.; acosta, m.; gonzales, m. peruvian journal of agronomy 3(3): 81-90 (2019) 85 with treatments t8 (4 kg ha−1, with three applications) and t6 (4 kg ha−1, with one application) with 512.01 units and 564.22 units of f. oxysporum, respectively. the original data were transformed using the formula √ x for the anova, which showed that there is no significance between treatments, with a coefficient of variability of 27.67%. when conducting the duncan’s multiple comparison test (α = 0.05), the main factor “dose” showed the lowest audpc of f. oxysporum with the dose of 4 kg ha−1 (637.78 units) (table 5). number of leaves: the plants inoculated with the microorganisms had practically the same number of leaves as did the control. the anova did not reveal significant differences between treatments, with the coefficient of variability being 3.97% (table 3). lower percentage of incidence of f. oxysporum. as for the simple effect of the number of applications in doses, lower incidences of f. oxysporum (figure 1) were observed with the n1 concentration (2 kg ha−1), with two applications and one application of the biological formulation; the n2 concentration (4 kg ha−1), with three applications, 1 application, and four applications of the biological formulation; and the n3 concentration (6 kg ha−1), with one application, 2 applications, and three applications of the biological formulation (table 5). area under the curve of disease progress (audpc): a higher audpc was observed with the t4 (2 kg ha−1, with three applications) treatment and the control, with 1524.09 units and 1101.67 units of f. oxysporum, respectively, whereas a lower audpc was observed treatments incidence of fusarium oxysporum (%) audpc yield (t ha−1) exportable discard total t10 (6 kg ha−1 with one applications) 5.53 bcd 679.79 ab 70.66 a 10.23 a 80.89 a t7 (4 kg ha−1 with two applications) 11.44 abc 722.02 ab 69.61 a 12.78 a 82.39 a t6 (4 kg ha−1 with one applications) 4.61 cd 564.22 ab 69.11 a 9.54 a 78.65 ab t13 (6 kg ha−1 with four applications) 11.90 abc 1042.05 ab 67.18 ab 8.19 a 75.37 ab t8 (4 kg ha−1 with three applications) 4.17 d 512.01 b 67.14 ab 14.37 a 81.51 a t11 (6 kg ha−1 with two applications) 9.45 abcd 1093.98 ab 66.50 ab 11.86 a 78.36 ab t5 (2 kg ha−1 with four applications) 14.39 ab 1063.11 ab 65.41 ab 10.32 a 75.73 ab t2 (2 kg ha−1 with one applications) 10.48 abcd 1010.30 ab 62.84 abc 13.23 a 76.05 ab t1 (control) 12.59 abc 1101.67 ab 62.46 abc 9.60 a 72.06 bc t9 (4 kg ha−1 with four applications) 6.63 bcd 752.89 ab 59.52 bc 12.13 a 71.65 bc t12 (6 kg ha−1 with three applications) 10.58 abcd 984.73 ab 57.33 c 13.53 a 70.86 bc t4 (2 kg ha−1 with three applications) 19.56 a 1524.09 a 55.40 c 10.08 a 65.48 c t3 (2 kg ha−1 with two applications) 5.67 bcd 776.18 ab 55.10 c 11.87 a 66.97 c c.v 27.00% 27.67% 7.82% 7.45% 6.59% table 4. results of the variables evaluated and comparison test of means; duncan (α = 0.05) effect of the applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa cv. “century” september december 2019 86 plant height: the treatments with the highest plants at 84 ddt were t10 (6 kg ha−1, with one application), t6 (4 kg ha−1, with one application), t5 (2 kg ha−1, with four applications) with plant heights measuring 75.22, 74.43, and 74.20 cm, respectively. treatments with low plant height included t3 (2 kg ha−1, with two applications) t13 (6 kg ha−1, with four applications), with plant heights of 72.29 cm and 72.35 cm, respectively. the anova revealed significant differences, and the coefficient of variability was 4.38% (table 3). bulb diameter: bulb diameter was greatest in the treatments with the highest concentrations of the biological formulation; t10 (6 kg ha−1, with one application) showed the largest average diameter (83.18 mm) with respect to other treatments. the bulb diameters for t5 (2 kg ha−1, with four applications), t2 (2 kg ha−1, with one application), and the control were 79.75, 79.79, and 80.01 mm, respectively; these treatments resulted in smaller equatorial bulb diameters. the statistical analysis revealed no significant differences for blocks and treatments, and the coefficient of variability was 4.24% (table 3). rate of colonization by r. intraradices (%): the highest rate of settlement by r. intraradices (55%) was with treatment t6, in which 4 kg ha−1 was in a single application. this was followed by treatments t2 (4 kg ha−1, with four applications), and t9 (2 kg ha−1, with one application), with a rate of 52.5% for each, and the colonization rate of the control was 27.50%. for the statistical analysis, the original data were transformed using the formula (arcsen √x 10) * 180 π. the anova revealed significant differences between blocks, but no significant differences were found in terms of treatments. the coefficient of variability was 29.09%, (table 3). number of r. intraradices spores: the spore count revealed that r. intraradices spores were present in all treatments, including control. the treatment with the highest number of spores was t10 (6 kg ha−1, with one application), which had an average of 18.75 spores per gram of soil, and the treatments with the least number of spores were the control and t4 (2 kg ha−1, with three applications), with an average count of 9.75 and 9.50 spores per gram of soil, respectively. the anova revealed significant differences between blocks but no significant differences among treatments (table 3). yield: we weighed all the bulbs harvested from each experimental unit, and the weights obtained were projected to tons per hectare (t ha−1). the best total yields were t7 (4 kg ha−1, with two applications), with 82.39 ha−1; t8 (4 kg ha−1, with three applications), with 81.50 t ha−1; and t10 (6 kg ha−1, with one application), with 80.90 t ha−1. the lowest total yields were found in t3 (2 kg ha−1, with two applications), with 66.97 t ha−1 and t4 (2 kg ha−1, with three applications), with 65.48 t ha−1. the best treatments for exportable yield were t10 (6 kg ha−1, with 1 application), t7 (4 kg ha−1, with two applications), and t6 (4 0.00 5.00 10.00 15.00 20.00 a3 a4 a1 a2 a2 a4 a1 a3 a4 a3 a2 a1 n1 n2 n3 19.57 14.39 10.48 5.67 11.44 6.63 4.61 4.17 11.90 10.58 9.45 5.53 applications ii nc id en ce (% ) concentration incidence of f. oxysporum (%) figure 1. simple effects on incidence of fusarium oxysporum (%) at 70 days after transplant, irrigation of santa rita–arequipa, 2016 concentration (kg ha−1) incidence of f. oxysporum (%) audpc number of applications incidence of f. oxysporum (%) 0 (control) 12.59 a 1101.67 a none (control) 12.59 a 2 12.53 a 1093.42 ab 3 applications 11.44 ab 6 9.36 ab 950.14 ab 4 applications 10.97 ab 4 6.71 b 637.78 b 2 applications 8.51 ab 1 application 6.87 b table 5. simple and main effect of concentration and number of applications on the incidence and audpc of fusarium oxysporum (%) at 70 (ddt), irrigation of santa rita–arequipa, 2016 mamani, e.; acosta, m.; gonzales, m. peruvian journal of agronomy 3(3): 81-90 (2019) 87 kg ha−1, with one application), with 70.66, 69.61, and 69.11 t ha−1, respectively. the lowest exportable yields were the treatments t4 (2 kg ha−1, with three applications), with 55.40 t ha−1; t12 (6 kg ha−1, with three applications), with 57.33 t ha−1; and t3 (2 kg ha−1, with two applications), with 55.10 t ha−1. figure 2. the anova for exportable yield and for total yield revealed significant differences between treatments, concentration and number of applications, factors of the biological formula, and the interactions of both factors. the coefficient of variability for exportable yield was 7.82% and that for total yield was 6.59% (table 4). the anova of orthogonal contrasts for total yield revealed that treatments t10 (6 kg ha−1, with one application), t11 (6 kg ha−1, with two applications), and t13 (6 kg ha−1, with 4 applications) were statistically different from the control. the first application resulted in the highest performance, and the highest yields were obtained with dose concentrations of 4 kg ha−1 and 6 kg ha−1, and these were statistically different from the concentration of 2 kg ha−1 and the control (table 6). evaluation of the main factor concentration showed that the concentrations of the biological formula with the greatest effects on yield were of 4 kg ha−1 and 6 kg ha−1. regarding the number of applications of the main factor, the first application had the greatest effect on yield. discussion survival: as prehension of the onion seedlings was affected by the presence of fusarium in all the experimental units, we inferred that the biological formula does not have a significant effect on the onion seedlings when there are high rates of incidence of f. oxysporum. the development of the disease was promoted by the warm temperatures, with maximum temperatures of 28.57 °c, which, according to koike, gladders, and paulus (2007), is the optimal temperature for development of the disease. in addition to being present in the field of commercial onion growing, there is a record of the presence of the disease in the previous seasons, which reaffirms the ability of the disease to survive for decades in the form of chlamydospores, with a resistance structure that allows it to continue even in the absence of the host crop or in unfavorable environmental conditions (ploetz, 2015). biometric variables: with respect to the biometric variables we considered in the investigation, such as the number of leaves, the height of the plant, and the diameter of the bulb, there were no significant differences found in the results of concentration (kg ha -1 ) yield (t ha -1 ) n. of a.* yield (t ha -1 ) expor-table total ex-por-table total 4 66.34 a 78.55 a 1 67.54 a 78.53 a 6 65.42 a 76.37 ab 4 64.03 ab 74.25 ab 0 62.46 ab 72.06 b 2 63.73 ab 75.91 ab 2 59.68 b 71.06 b 0 62.46 ab 72.06 b 3 59.96 b 72.62 b table 6.concentration and number of applications of the variable total yield (t ha −1) of the treatments under study, irrigation of santa rita–arequipa, 2016 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 a1 a4 a2 a3 a2 a3 a1 a4 a1 a2 a4 a3 n1 n2 n3 76.06 75.73 66.96 65.47 82.39 81.51 78.64 71.64 80.89 78.36 75.37 70.86 applications yi eld (t h a-1 ) concentration yield(t ha-1) figure 2. main effects of treatments under study on yield (t ha-1), irrigation of santa rita–arequipa, 2016 *n. of a.: number of applications effect of the applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa cv. “century” september december 2019 88 any treatment in relation to the control treatment, for this reason, it is considered that this complex of rhizobacterial microorganisms (pgpr) that promote plant growth has not shown any influence on the development of onion plants, although it is recognized that the use of the biological formulation has the potential to improve both production and yields in the final results. this can be supported by the authors cakmakçi, dönmez, aydın and şahin. (2006), who mention the conditions by which pgpr strains have limits on their ability to colonize the rhizosphere, these conditions include soil type, temperature, moisture content, organic matter and ph. banerjee, yesmin and vessey (2006); and bly, woodard and gelderman (2009) have reported that the reason why the use of pgpr is not yet a widespread practice is largely due to the inconsistency in the promotion of plant growth by most of the pgpr strains in different field conditions. . these finding agree with those of sánchez, ley, ricardo, and collazo (2015), who evaluated the effect of the arbuscular mycorrhizal fungi (amf) glomus spp., glomus clarum, and glomus intraradices in the tomato crop (solanum lycopersicum l. var., amalia) and found no significant differences in the number of leaves in the tomato plants inoculated with the amf and the control plants. incidence: in the treatments in which the biological formulation was applied, there was a lower incidence of f. oxysporum with respect to the control; this observation is relevant, as the control treatment seedlings were disinfected with a chemical fungicide based on carbendazim before transplantation. this would seem to demonstrate that the biological formula plays an important role in inhibiting the development of phytopathogens of the root system, as a chemical fungicide would do, which coincides with the findings by hsiang, jamie, and mcdonald (2008), who inoculated onion plants with biological formulations based on g. intraradices and reduced the incidence of f. oxysporum. furthermore, application of the biological formula will have a greater effect if it is done at the time of either sowing or transplantation (bashan, de-bashan, prabhu, & hernandez, 2014). this was corroborated by qingxiao (2014), who performed an assay to ascertain the effect in the biological control of streptomyces spp. in radish plants and reported that the disease was suppressed completely by application before planting. the audpc was lower with a dose of 4 kg ha−1, which corroborates the results obtained previously in the incidence evaluation, the dose of 4 kg.ha-1 could have a favorable effect in reducing the development of the disease caused by f. oxysporum, possibly by the induction of systemic resistance or competition for nutrients of the rhizosphere microorganisms (nadeem, zahir, naveed, & ashraf, 2010). according to alarcón, ferrera, gonzález and villegas (2000), in addition to the nutritional benefit of the symbiosis, the arbuscular mycorrhizal fungi also act as agents of biological control of the phytopathogens present in the ground. the colonization percentages reached during the trial did not differ statistically between the inoculated treatments, and the conventional control may likely have influenced the high phosphorus content in the experimental area. authors such as rodríguez et al. (2010) mention that mycorrhization is generally inhibited in soils with a high phosphorus content, as high phosphorus concentrations determine the permeability of cell membranes and the radical exudation of carbohydrates and amino acids available as metabolites for the fungus [kurle and pfleguer (1994) cited by regalado, (2002)]. the spore count revealed the presence of r. intraradices spores in all treatments, including control. this would seem to corroborate the existence of native rhizophagus spp. spores in the soils of the cultivated fields in the irrigation of santa rita that would also explain the colonization observed in the control treatment. fernández (2006) suggests that amf form symbiotic relationships with the underground organs of most higher plants, enabling the survival of the spores of rhizophagus spp. in the soils of the cultivated fields. yield: the effect of the biological formulation was important in increasing the yield of yellow onions. the possible direct mechanisms by which pgpr microorganisms increased the yields of this crop include its control effect on the incidence of plants infected with f. oxysporum, due to their antagonistic action against pathogenic microorganisms present in the soil, in addition to their components antibiotics and siderophores production. according to soesanto et al. (2010), each microbial antagonist, including pgpr, has its own mechanism of inhibition in control of plant pathogens and may have more than one mechanism. it is likely that the yields obtained from the treatments are due not only to a lower incidence of f. oxysporum. they may also result from the action or effect of the mycorrhiza and the rhizobacteria either interacting with each other or independently producing changes in the chemical composition in the rhizosphere and its interaction with the plant roots, given the ability of pgpr to increase the availability of nutrients for the plant by eliminating the chemical phosphate bonds to become soluble phosphate and be absorbed by the roots, reaching optimal yields similar to high levels of nitrogen, phosphorus and potassium fertilization.. finally, the pgpr of the biological formulation may have increased the yield of the yellow onions because bacillus spp. solubilize the inorganic phosphorus present in the soil, thereby enhancing the entry of this macronutrient to the plant, which results in a larger amount of biomass (rajankar, tambekar & wate, 2007). conclusions a biological formula consisting of the microorganisms azotobacter salinestris, bacillus amyloliquefaciens, and mamani, e.; acosta, m.; gonzales, m. peruvian journal of agronomy 3(3): 81-90 (2019) 89 rhizophagus intraradices had a positive effect on the yield of the yellow onion in the spring–summer season under the edaphoclimatic conditions of the santa rita of siguas– arequipa irrigation. the concentrations of the biological formula with the most significant effect on the onion yield were 4 kg ha−1 and 6 kg ha−1, producing exportable and total yields of 66.34 t ha−1 and 65.42 t ha−1, respectively, which were superior to those obtained using other concentrations. moreover, the two concentrations also resulted in a lower incidence of fusarium oxysporum disease and a smaller audpc of the disease. the first application of the biological formula had the greatest effect on the yield of the yellow onion crop. the yield obtained with this application was 67.54 t ha−1, which was superior to those obtained with other applications. this corroborated the theory that applying the biological formula before transplantation has a greater effect than the later applications via drenching. regarding the best combination of concentration and number of applications, t6 treatments (4 kg ha−1, with one application), t7 (4 kg ha−1, with two applications) and t10 (6 kg ha−1, with one application) treatments stand out, with exportable yields of 69.11, 69.61, and 70.66 t ha−1, respectively. these combinations produced higher exportable yields and a lower incidence and audpc of f. oxysporum. references alarcón, a., ferrera-cerrato, r., gonzález-chávez, m. c., & villegas-monter a. (2000). arbuscular mycorrhizal fungi in the dynamics of appearance of stolons and nutrition of strawberry plants cv. fern obtained by in vitro culture. terra latinoamericana 18: 4–8. apcho e., caballero m. & miranda r. (2017). strategic planning of the onion in peru to 2027. master’s thesis. pontifical catholic university of peru. lima peru. banerjee m. r., yesmin l., & vessey j. k. (2006) plantgrowth-promoting rhizobacteria as biofertilizers and biopesticides. in m.k. rai (ed.). handbook of microbial biofertilizers. (pp. 137−181). new york, ny, usa: food products press. bashan, y., de-bashan, l., prabhu, s.r., & hernandez, j.p. (2014). advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013). plant and soil, 378(1–2), 1– 33. bly u., woodard h., & gelderman r. (2009) biological inoculants and other products for soybeans during 2009 (44309 and 44409). soil/water research. south dakota state university, 2009 research progress report. retrieved from http://extension.agron.iastate. edu/compendium/compendiumpdfs/pr09-13.pdf cakmakçi r., dönmez f., aydın a., & şahin f. (2006) growth promotion of plants by plant growth-promoting rhizobacteria under greenhouse and two different field soil conditions. soil biology and biochemistry 3 8 ( 6 ) : 1 4 8 2 – 1 4 8 7 . h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / j . soilbio.2005.09.019 campbell cl and madden lv (1990) introduction to plant disease epidemiology. john wiley & sons, new york fernández, a. (2006). micorriza una simbiosis vital en la naturaleza, [october, 31, 2017]. retrieved from https://www.consumer.es/medio-ambiente/micorrizauna-simbiosis-vital-en-la-naturaleza.html hsiang, t., jamie, m.d.l.a., & mcdonald, m.r. (2008) effects of glomus intraradices and onion cultivar on allium white rot development in organic soils in ontario. canadian journal of plant pathology, 30(4), 543-553. retrieved from https://atrium.lib.uoguelph. ca/xmlui/handle/10214/2379?show=full koike, s. t., gladders, p. & paulus a. o. (2007). vegetable diseases: a color handbook, gulf professional publishing. 448p. nadeem, s. m, zahir z. a., naveed, m. & ashraf, m. (2010). microbial accdeaminase: prospects and applications for inducing salt tolerance in plants. critical reviews in plant sciences, 29(6), 360–393. qingxiao, m. (2014). characterization of bacillus amyloliquefaciens strain bac03 in disease control and plant growth promotion. ph.d. thesis. michigan state university. ee.uu. ploetz, r. c. (2015). management of fusarium wilt of banana: a review with special reference to tropical race 4. crop protection, 73, 7–15. phillips, j.m. and hayman, d.s. (1970) improved procedures for clearing roots and staining parasitic vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. transactions of the british mycological society, 55, 158-161. http://dx.doi. org/10.1016/s0007-1536(70)80110-3 rajankar, p. n., tambekar, d. h. & wate, s. r. (2007). study of phosphate solubilization efficiences of fungi and bacteria isolated from saline belt of purna river basin. research journal of agriculture and biological sciences, 3(6), 701-703. regalado, f. (2002). incremento en la eficiencia de la fertilización de portainjertos de aguacate mediante la inoculación con micorriza vesiculo arbuscular. thesis. zamorano. honduras. effect of the applications of a biological formulation (azotobacter salinestris, bacillus amyloliquefaciens, rhizophagus intraradices) on the yield of allium cepa cv. “century” september december 2019 90 rodríguez, r., alcantar, e., covarrubias, g., zamora, f., garcía, l., lópez, r., & salcedo, e. (2010). caracterización física y química de sustratos agrícolas a partir de bagazo de agave tequilero. interciencia, 35(7), 515-520. sánchez j. a., ley-rivas j. f., ricardo n. e., & collazo e. (2015). efecto de cuatro especies de hongos micorrizógenos arbusculares en la producción de frutos de tomate. agronomía costarricense. (1): 47–59. soesanto l, mugiastuti l, & ruth fr. (2010). kajian mekanisme antagonis pseudomonas fluorescens p60 terhadap fusarium oxysporum f.sp. lycopersici pada tanaman tomat in vivo. j. hama dan penyakit tumbuhan tropika 7(1): 53–61. walker, c. y mizecw, m. (1982). populations of endogonaceous fungi at two locations in central iowa. canadian journal of botany 60: 2518-2529. peruvian journal of agronomy 3(3): 134–143 (2019) issn: 2616–4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1207 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 29 september 2019 accepted for publication: 01 october 2019 floristic composition and diversity in plots of early post-agricultural succession in four agroecosystems in the district of cajatambo, lima composición florística y diversidad en parcelas de sucesión post-agrícola temprana en cuatro agroecosistemas en el distrito de cajatambo, lima castro-cepero, v. (1)* *corresponding author: vcastro@lamolina.edu.pe abstract in this paper, the floristic composition is studied in plots of early post-agricultural succession induced in four agroecosystems (ocopata, rancas, tupicocha and ucupi) in the district of cajatambo, lima. the plots are located between 2,992 masl and 4,220 masl in three plant formations (agriculture, scrubland and grassland), and the description of the floristic composition was made before and after inducing the succession between 2015 and 2017. the number of determined plants is 78, all of which belong to 30 botanical families, of which the asteraceae (16 species), fabaceae (11 species) and poaceae (8 species) families stand out. indeed, the only common species among the four agroecosystems is medicago polymorpha; moreover, five species appear in three of the four agroecosystems: brassica rapa subsp. campestris and verbena litoralis (ocopata, tupicocha and rancas), oenothera rosea and trifolium repens (ocopata, rancas and ucupi) and bidens andicola (tupicocha, rancas and ucupi). keywords: vegetation, floristic composition, succession, agriculture, plant diversity resumen se estudió la composición florística en parcelas de sucesión post-agrícola temprana inducida en cuatro agroecosistemas (ocopata, rancas, tupicocha y ucupi) en el distrito de cajatambo, lima. las parcelas se ubicaron entre 2 992 y 4 220 metros sobre el nivel del mar en tres formaciones vegetales (agricultura, matorral y pastizal), y la descripción de la composición florística se hizo antes y después de inducir la sucesión entre los años 2015 y 2017. el número de plantas determinadas fue de 78, pertenecientes a 30 familias botánicas de las cuales destacan las familias asteraceae (16 especies), fabaceae (11 especies) y poaceae (8 especies). se encontró que la única especie común entre los cuatro agroecosistemas fue medicago polymorpha y que cinco especies aparecieron en tres de los cuatro agroecosistemas: brassica rapa subsp. campestris y verbena litoralis (ocopata, tupicocha y rancas); oenothera rosea y trifolium repens (ocopata, rancas y ucupi) y bidens andicola (tupicocha, rancas y ucupi). palabras clave: vegetación, composición florística, sucesión, agricultura, diversidad vegetal 1 universidad nacional agraria la molina, (unalm). programa de doctorado en agricultura sustentable, escuela de posgrado, av la molina s/n, lima-perú introduction ecological succession is a key concept for the sustainable management of agroecosystems, but little is known about its specific role in agricultural systems, particularly in mountain ecosystems (sarandón & flores, 2014). it is defined as the process of directed temporal change (linear or cyclic) of vegetation during a specific ecological time, where, usually after a disturbance, changes are observed in the taxonomic composition, three-dimensional structure of the cover and functions of the system. it is also defined as the occupation of an area by organisms involved in an incessant process of action and reaction, which, over time, leads to changes in both the environment and the community (krebs, 1985; pickett & cadenasso, 2005; pickett & white, 1985). agricultural activity takes advantage of these changes to maintain agroecosystems in the initial stages of ecological succession. therefore, the said system can produce large quantities of harvested material, albeit with unpredictable effects on development and stability processes. by maintaining a mosaic of cultivated and resting plots, farmers have captured the essence of the ecological succession process, which, in essence, is a regeneration process (gutiérrez, aguilera & gonzález, 2008). alternatively, plant succession provides ecological resources and services of interest, such as grazing areas and castro-cepero, v. peruvian journal of agronomy 3(3): 134–143 (2019) 135 soil fertility, which can be used to implement ecologically sustainable management systems (espinoza-bretado & návar, 2005). one of the key principles of sustainability is to integrate biological and ecological processes into food production. in this sense, biological diversity is an instrument that, from an ecological management perspective, ensures the design of sustainable agricultural systems, which includes mechanisms for system regulation and recovery. indeed, this requires knowing the relevant components of the system as well as how they articulate with the aforementioned processes (pretty, 2008; sarandón & flores, 2014). according to gliessman (2002), although diversity has been interpreted as the number of different species that make up a community in a given place, the concept of diversity is multidimensional, ranging from genes and species to spatial (horizontal and vertical), functional and temporal diversity. agricultural activity simplifies ecosystems by replacing multidimensional diversity with minimal species and varieties, which are simultaneously and homogeneously managed (sarandón & flores, 2014). in this sense, biological diversity is vital insofar that it provides services that go beyond the production of food, fibre and fuels (altieri & nicholls, 2012; gliessman, 2002). these services include the following: succession, biotic regulation, nutrient recycling, energy flow and water regulation (sarandón & flores, 2014). the biotic composition of an ecosystem reflects the climate, the nature of the soil, the availability of water and nutrients and anthropic and biotic factors. vegetation and floristic composition, as the structural basis of the community, are repeated in different areas with similar physical and biological contexts. in spite of this, no two spaces are occupied by identical plant communities, since vegetation type results in different communities of birds, arthropods, microorganisms and other organisms (matteucci & colma, 1982; ramos, castro & sánchez, 2015). therefore, monitoring the dynamics of succession in abandoned plots allows us to obtain information that can be used to design sustainable systems. farm spaces represent a special type of secondary succession, as they are continuously disturbed and allow a certain level of regeneration during inactive periods. this favours weeds, which, in natural conditions, are classified as pioneers of succession; they occupy the habitats of those that are normally eradicated, and, therefore, they are considered as invasive (caamal-maldonado & armendarizyañez, 2002). thus, early post-agricultural succession is defined as a form of secondary succession that takes place in fields where agricultural activity has altered original vegetation. when agricultural activity is interrupted or ceases, weeds are ubiquitous and may dominate fields during early succession (omacini, tognetti, trebino & chaneton, 2005). post-agricultural succession studies are indispensable for the success of agroecosystem management activities, as they provide information on how different geographical and climatic areas function and regenerate (myster, 2008; sarandón & flores, 2014). in the andean region, omacini et al. (2005) identified the sustained dominance of exotic species 20 years after the interruption of agriculture in the pampeana region (argentine); in the same way, ortuño, beck and sarmiento (2006) found minimal changes in the richness and diversity of post-agricultural plots in central altiplano (bolivia), which can possibly be attributed to the species adapting to harsh climate conditions, restricted soil conditions and anthropogenic influence. based on the little information available on succession in high andean mountain environments, it is necessary to characterise the process in these environments (krebs, 1985). for example, in the district of cajatambo, a space in the mountains of the department of lima that presents a process of abandonment of hillside agriculture and that has a vacuum of biological information (walsh perú, 2011). this research was carried out within the framework of the project, determination of criteria for the establishment of environmental quality standards for biological diversity. case study: cajatambo district, financed by the fondo para la innovación, ciencia y tecnología (fincyt), with the objective of characterising floristic composition and diversity in early post-agricultural succession plots in four agroecosystems of the cajatambo district, lima. materials and methods the study area was located in the district of cajatambo, in the province of the same name, to the north–east of the department of lima in the western part of the andes, at 10° 28′ 16.65″ sl and 76° 59′ 35.91″ wl, limited by the departments of ancash, huánuco and pasco. the district is located between 2,600 masl and 4,800 masl, and the capital, cajatambo (figure 1), is located 3,376 masl (quinteros, 2009). the dry season runs from may to october, wherein the days are mostly clear, and the rainy season runs from november to april, with storms usually occurring in the afternoon. establishment of permanent plots for monitoringinduced post-agricultural succession a permanent or long-term plot is one that, once installed, is measured at least three consecutive times. in this type of plot, different variables are measured repeatedly, thereby obtaining a series of data (one in each plot) for the construction of growth models and community dynamics (gadow, rojo, álvarez & rodríguez, 1999). three plant formations were selected (agriculture, shrubland and grassland) using an accessibility criterion, that is, plant formations in which the establishment of the plot could be guaranteed in the long term. therefore, four sites were floristic composition and diversity in plots of early post-agricultural succession in four agroecosystems in the district of cajatambo, lima september december 2019 136 selected: ocopata (ocp), corresponding to agriculture; tupicocha (tpc) and rancas (rnc), corresponding to shrublands; and ucupi (ucp), corresponding to grasslands (figure 2). three 5 x 5 m plots were installed in each locality and were monitored three times a year during 2015–2017. the ocp, tpc and rnc plots were installed in june 2015, and the ucp plots were installed six months later. the first record of floristic composition was made in june 2015, and, after making the necessary measurements, aerial vegetation coverage was eliminated figure 1: province and district of cajatambo. source: google map data (2017). figure 2. long-term plots for the monitoring of induced post-agricultural succession. castro-cepero, v. peruvian journal of agronomy 3(3): 134–143 (2019) 137 in the 5 x 5 m squares; in this way, succession was induced. the development of the vegetation was monitored by observing the floristic composition and the number of species in september and december of 2015; april, july and october of 2016; and january, may and october of 2017, thereby covering the dry, rainy and intermediate seasons. characterisation of the floristic composition and plant diversity in permanent plots for the monitoring of induced post-agricultural succession the botanical material was collected in the long-term plots according to the direct collection technique (inbio, 2008; mesa & bernal, 2005; cámara & díaz, 2013); processing was carried out at the collection site and at the augusto weberbauer herbarium of la molina national agrarian university. the classification system of the flowering plants proposed by angiosperm phylogeny group iv (apg et al., 2016) was used. the scientific names follow the guidelines of the catalogue of gymnosperms and angiosperms of the peruvian flora (brako & zarucchi, 1993) and the tropicos database of the missouri botanical garden (www.tropicos.org). using species richness data for each agroecosystem, a general description of alpha diversity and its changes over time was made through the shannon– weaver and simpson indices, using past 3.25 software (hammer, harper & ryan, 2001). with this information, a list of species, botanical families and life forms was created for plants that are registered in the four agroecosystems represented by the long-term exclusion plots. results and discussion characterisation of permanent plots for the monitoring of induced postagricultural succession according to tovar (2007) and quinteros (2009), as well as the altitude of the plots, the plot of tpc corresponds to the lower part of the basin, between 2,600 masl and 3,000 masl; the climate is dry and semicold, with scarce precipitations, and the average temperature is between 12°c and 16°c. the plot of ocp, between 3,200 masl and 3,600 masl, corresponds to a semi-humid and semi-cold climate, with moderate rainfall in summer and moderate drought throughout the rest of the year as well as temperatures ranging between 8°c and 12°c. the rnc plot, between 3,600 masl and 3,900 masl, presents a slightly humid and semi-cold climate, with temperatures below 10°c and the presence of frost between june and august. finally, the plot of ucp corresponds to heights above 3,900 masl, where the climate is moderately humid and cold, with abundant rainfall in summer, mild drought the rest of the year and temperatures between 5°c and 8°c, falling to 0°c at night. according to the information provided by the owners of the plots, three of the four selected agroecosystems have exhibited agricultural activity that subsequently (for various reasons) ceased. in ocp, the main crop was maize, which was rotated with beans, ollucos and tarwi during breaks, the last of which was prolonged and thus allowed shrub development of the tarwi and favouring the entrance of the kikuyo. this is the reason why it remained in rest 8 years ago; currently, it is used for the grazing of cattle since it is near a water canal. in tpc, the main crop was maize; however, due to its remoteness and the lack of water irrigation, agricultural activity was abandoned 20 years ago. in rnc, the main crop was potato; however, due to the reduction of agricultural activities in the area and the change to livestock, agricultural activity was abandoned 10 years ago. in ucp, the main activity was sheep grazing, which is still being used today. the soil texture varies from sandy clay loam (ocp), to sandy loam (tpc and rnc) and loam (ucp). ocp has the highest organic matter content, as shown in table 1. precipitation data obtained from the cajatambo conventional weather station (cws) (figure 3) for 2015– 2017 indicates that the rainy months were concentrated between december and april, with maximum values reached in february; the dry months were between june and august. indeed, sporadic rain occurred throughout the year, which characterises them as intermediate months. precipitation for 2015 was 560 mm, 317 mm for 2016 and 770 mm for 2017 (figure 4). plot ocopata (ocp) tupicocha (tpc) rancas (rnc) ucupi (ucp) plant formation agriculture shrubland shrubland grassland geographic coordinates 77°00’2.5” w.l 10°28’39.4” s.l 77°01’27.9” w.l 10°27’25.3” s.l 77°00’21.8” w.l 10°25’42.4” s.l 76°56’36.5” w.l 10°29’54.3” s.l height (masl) 3,505 2,992 3,635 4,220 climate semi-humid and semi-cold dry and semi-cold wet and semicold wet and cold temperature (°c) 8 to 12 12 to 16 <10 5 to 8 slope (°) 40 30 30 20 orientation n n ne s soil texture sandy clay loam sandy loam sandy loam loam o.m. (%) 10 5 8 8 last sowing 12 years 20 years 15 years table 1. main characteristics of the four agroecosystems evaluated in cajatambo, lima. floristic composition and diversity in plots of early post-agricultural succession in four agroecosystems in the district of cajatambo, lima september december 2019 138 the total diversity of identified families and the total abundance of species for the main families in the four agroecosystems evaluated between 2015 and 2017 are consistent with previous studies conducted in the valleys of the lima region, where the asteraceae, fabaceae and poaceae families are dominant. according to huamán, chávez, arias and zegarra (2007), 68 families are registered for the province of barranca (0 masl to 484 masl), including poaceae (37 species) and fabaceae (32 species); 56 families are registered for quinteros (2009) in the district of cajatambo (2,600 masl to 4,800 masl), including asteraceae (55 species) and solanaceae (18 species). according to paulino, la torre and huamán (2015) for the province of oyón (2,500 masl to 4,300 masl), 40 families are registered, of which asteraceae (65 species) and poaceae (23 species) stand out. moreover, according to gonzáles, navarro, la torre and cano (2015), 77 families are registered for the province of canta floristic composition and plant diversity in long-term plots for the monitoring of induced post-agricultural succession initial richness is defined as the number of different species present in a defined geographical space (colín, maeda & muñoz, 2006). in the four agroecosystems evaluated, a total richness of 78 species belonging to 30 botanical families was determined, of which the asteraceae (16 species), fabaceae (11 species), poaceae (8 species), geraniaceae (3 species), lamiaceae (3 species) and rosaceae (3 species) stood out (figure 5). the list of 78 species, their form of life, botanical family and agroecosystems are shown in table 2. figure 3. evolution of average monthly precipitation (mm) from january 2015 to december 2017. source: cajatambo cws, senamhi. figure 4. comparison of accumulated monthly precipitation (mm) from 2015 to 2017. source: cajatambo cws, senamhi. figure 5. main plant families and their richness recorded in the plots of early post-agricultural succession in cajatambo, lima (2015 to 2017). castro-cepero, v. peruvian journal of agronomy 3(3): 134–143 (2019) 139 n° scientific name families life form location ocp tpc rnc ucp 1 achyrocline alata dc. asteraceae herbaceous broadleaf 0 1 0 0 2 agave cordillerensis lodé & pino agavaceae leaf succulent 1 1 0 0 3 ageratina sternbergiana (dc.) r.m. king & h. rob. asteraceae herbaceous broadleaf 0 1 1 0 4 aldama helianthoides (rich.) e.e.schill. & panero asteraceae herbaceous broadleaf 0 1 0 0 5 ambrosia arborescens mill. asteraceae perennial broadleaf 0 1 1 0 6 astragalus garbancillo cav. fabaceae herbaceous broadleaf 0 0 0 1 7 austrocylindropuntia floccosa (salm-dyck) f. ritter cactaceae stem succulent 0 0 0 1 8 austrocylindropuntia subulata (muehlenpf.) backeb. cactaceae stem succulent 1 0 0 0 9 avena sterilis l. poaceae grass 1 1 0 0 10 baccharis pentlandii dc. asteraceae perennial broadleaf 0 0 1 0 11 berberis lutea ruiz & pav. berberidaceae perennial broadleaf 0 0 1 0 12 bidens andicola kunth asteraceae herbaceous broadleaf 0 1 1 1 13 bidens pilosa l. asteraceae herbaceous broadleaf 1 1 0 0 14 bomarea dulcis (hook.) beauverd alstroemeriaceae herbaceous broadleaf 0 1 0 0 15 brassica rapa subsp. campestris (l.) clapham brassicaceae herbaceous broadleaf 1 1 1 0 16 bromus catharticus vahl poaceae herbaceous broadleaf 0 0 1 0 17 bromus sp. poaceae grass 0 1 0 1 18 caiophora cirsiifolia c. presl loasaceae herbaceous broadleaf 0 0 1 0 19 calamagrostis vicunarum (wedd.) pilg. poaceae grass 0 0 0 1 20 calceolaria cuneiformis ruiz & pav. calceolariaceae herbaceous broadleaf 0 0 1 0 21 cestrum auriculatum l’hér. solanaceae perennial broadleaf 0 1 0 0 22 chuquiraga spinosa less. asteraceae perennial broadleaf 0 0 0 1 23 citharexylum cf. dentatum tafalla ex d. don verbenaceae deciduous broadleaf 0 1 0 0 24 croton ruizianus müll. arg. euphorbiaceae perennial broadleaf 0 1 0 0 25 cuscuta odorata ruiz & pav. convolvulaceae parasite 1 0 0 0 26 dalea exilis dc fabaceae herbaceous broadleaf 0 1 0 0 27 desmodium molliculum kunth fabaceae herbaceous broadleaf 1 0 0 0 28 dichondra microcalyx (hallier f.) fabris convolvulaceae herbaceous broadleaf 0 0 1 0 29 dicliptera cf. montana lindau acanthaceae herbaceous broadleaf 0 1 0 0 30 eragrostis sp. poaceae grass 0 1 1 0 31 erodium cicutarium (l.) l’hér. ex aiton geraniaceae herbaceous broadleaf 0 1 1 0 32 festuca sp. poaceae grass 0 1 0 0 33 fuertesimalva peruviana (l.) fryxell malvaceae herbaceous broadleaf 0 1 0 0 34 galium sp. rubiaceae herbaceous broadleaf 0 0 1 0 35 geranium matucanense r. knuth geraniaceae herbaceous broadleaf 0 1 0 0 36 geranium sessiliflorum cav. geraniaceae herbaceous broadleaf 0 0 1 1 37 gomphrena globosa l. amaranthaceae herbaceous broadleaf 0 0 1 0 38 hesperomeles cuneata lindl. rosaceae perennial broadleaf 0 0 1 0 39 hypochaeris brevicaulis phil. asteraceae herbaceous broadleaf 0 0 1 1 40 lachemilla pinnata (ruiz & pav.) rothm. rosaceae herbaceous broadleaf 0 0 1 1 41 lepidium bipinnatifidum desv. brassicaceae herbaceous broadleaf 0 0 1 0 42 lupinus ballianus c.p.sm. fabaceae perennial broadleaf 1 0 1 0 table 2. list of 78 species found indicating their presence (1) or absence (0) at ocp, tpc, rnc and ucp in cajatambo, lima during 2015–2017. floristic composition and diversity in plots of early post-agricultural succession in four agroecosystems in the district of cajatambo, lima september december 2019 140 (550 masl to 3,200 masl), of which asteraceae (66 species) and poaceae (41 species) stand out. according to gonzáles (2016), the asteraceae family has a great richness of species within peruvian flora, being these plants often dominant elements that give the physiognomic aspect to several plant formations in the region of lima. eight life forms were found using the modified küchler and zonneveld classification (1988) (figure 6). plant habit, also known as plant life form, is the characteristic appearance of a plant species, which includes growth size, configuration and orientation. it develops from specific genetic growth patterns in combination with environmental factors. according to matteucci and colma (1982), life forms have an adaptive connotation as species adapt to physical parameters and the biota within the community; therefore, natural selection favours the development of specialised characteristics that allow the species to n° scientific name families life form location ocp tpc rnc ucp 43 lupinus microphyllus desvaux fabaceae perennial broadleaf 0 0 0 1 44 marrubium vulgare l. lamiaceae herbaceous broadleaf 0 0 1 0 45 medicago polymorpha l. fabaceae herbaceous broadleaf 1 1 1 1 46 medicago sativa l. fabaceae herbaceous broadleaf 1 0 0 0 47 melilotus sp. fabaceae herbaceous broadleaf 0 1 0 0 48 minthostachys mollis griseb. lamiaceae perennial broadleaf 0 1 1 0 49 muehlenbeckia volcanica (benth.) endl. polygonaceae herbaceous broadleaf 1 0 0 0 50 oenothera rosea l’hér. ex aiton onagraceae herbaceous broadleaf 1 0 1 1 51 ophryosporus peruvianus (j. gmel.) r.m. & h. rob. asteraceae herbaceous broadleaf 0 1 0 0 52 otholobium pubescens (poir.) j.w.grimes fabaceae perennial broadleaf 1 0 0 0 53 oxalis peduncularis kunth oxalidaceae herbaceous broadleaf 0 0 1 0 54 paranephelius uniflorus poepp. & endl. asteraceae herbaceous broadleaf 0 0 0 1 55 paronychia andina a. gray caryophyllaceae herbaceous broadleaf 0 0 0 1 56 paspalum sp. poaceae grass 1 0 1 0 57 passiflora trifoliata cav. passifloraceae climbing 0 0 1 0 58 passiflora tripartita var. mollissima (kunth) holm-niels. & jørgensen passifloraceae climbing 0 1 0 0 59 pennisetum clandestinum hochst. ex chiov. poaceae grass 1 1 0 0 60 perezia coerulescens wedd. asteraceae herbaceous broadleaf 0 0 0 1 61 plantago lanceolata l. plantaginaceae herbaceous broadleaf 1 0 1 0 62 ranunculus praeomorsus kunth ex dc. ranunculaceae perennial broadleaf 0 0 1 0 63 salvia sagittata ruiz & pav. lamiaceae herbaceous broadleaf 1 0 0 0 64 senecio nutans sch. bip. asteraceae herbaceous broadleaf 0 0 1 0 65 siphocampylus tupaeformis zahlbr. campanulaceae herbaceous broadleaf 0 0 1 0 66 solanum acaule bitter solanaceae herbaceous broadleaf 0 0 1 1 67 stellaria cf. weddellii pedersen caryophyllaceae herbaceous broadleaf 0 0 0 1 68 tagetes multiflora kunth asteraceae herbaceous broadleaf 1 1 0 0 69 taraxacum officinale weber in wigg. asteraceae herbaceous broadleaf 1 0 1 0 70 tecoma stans (l.) juss. ex kunth bignoniaceae perennial broadleaf 0 1 0 0 71 tetraglochin cristatum (britton) rothm. rosaceae perennial broadleaf 0 0 0 1 72 trifolium repens l. fabaceae herbaceous broadleaf 1 0 1 1 73 ullucus tuberosus caldas basellaceae herbaceous broadleaf 0 0 1 0 74 urtica leptophylla kunth urticaceae herbaceous broadleaf 0 1 1 0 75 verbena litoralis kunth verbenaceae herbaceous broadleaf 1 1 1 0 76 veronica persica poir. plantaginaceae herbaceous broadleaf 1 0 1 0 77 vicia andicola kunth fabaceae herbaceous broadleaf 0 0 1 0 78 werneria pygmaea gillies ex hook. & arn. asteraceae herbaceous broadleaf 0 0 0 1 castro-cepero, v. peruvian journal of agronomy 3(3): 134–143 (2019) 141 uniquely exploit the environment and evolve over time. the analysis of life forms suggests a general tendency in the four agroecosystems to maintain the broadleaf herbaceous weed lifestyle. however, each agroecosystem has a different dynamic due to the predominance of certain species. of the 78 species that were recorded in the four agroecosystems, 50 were broadleaf herbaceous, which, in this study, is the most dominant species. finally, using initial richness data from each agroecosystem, a general description of alpha diversity (and its changes over time) was obtained using the shannon–weaver and simpson indices. herein, diversity is defined as ‘the relationship between the number of species and the relative abundance of each of them in a given community’ (margalef, 1980). among the levels of diversity, alpha diversity (or diversity within a habitat) is determined by the ecological interactions, evolutionary history and biogeographic of the area in question (halffter & moreno, 2005). this can be measured through several diversity indices. the shannon–weaver index is widely used, which is derived from information theory and provides a measure of structure as a result of ecosystem interactions. this is expressed in bits/individual, and its values increase as the richness and uniformity of the community increases (margalef, 1980; magurran, 2004). in addition, the simpson index is often used, which is sensitive to the abundance of the most dominant species a community; accordingly, it is a measure of dominance, varies from zero to one and lacks units (magurran, 2004). table 3 shows that, in the ocp plot, the initial richness is nine species, which decreases to two species affected after the induction of the succession. in 2016, a richness value of eight species was reached, and the sampling ends with ten species. in this temporal evolution, it is evident that, on average, diversity is 1.87 bits/individual; moreover, there is no dominance in any season. in tpc, the initial richness is 28 species, which decreases to one species after the induction of the succession. in 2016, the richness recovers, reaching 31 species in april and july, but then decreases to 10 species. the average diversity is 2.13 bits/individual, and the dominance is low for all sampling seasons. in rnc, the initial richness was 30 species, which decreases to four species after the induction of succession. the richness increases to 12 species by 2016, with the sampling ending at 11 species. diversity on average is 2.65 bits/individual, and dominance, as in tpc, is low for all sampling seasons. finally, in ucp, the initial richness was seven species. contrastingly, after induction of succession, the richness increased to 12, suggesting that induction disturbed the dominant structure, which, in turn, allowed other species to enter and establish themselves—species that were either latent in the soil or else introduced by the wind. the sampling concludes with a richness of four species, figure 6. number of species according to the life forms recorded in the plots of early post-agricultural succession in cajatambo, lima (2015 to 2017). location parameters 2015 2016 2017 jun. set. dec. apr. jul. oct. jan. may oct. ocopata richness (s) 9 2 4 8 5 6 6 9 10 diversity (h) 2.4930 0.6501 1.9058 2.0934 1.3804 2.1410 1.8178 2.3285 2.0934 dominance (d) 0.2206 0.7222 0.2813 0.3021 0.5191 0.27 0.378 0.2477 0.3126 tupicocha richness (s) 28 1 1 31 31 8 9 w.d. 10 diversity (h) 4.3901 0.0000 0.0000 2.9287 2.6127 2.5348 2.0097 w.d. 2.594 dominance (d) 0.0562 1.0000 1.0000 0.0531 0.0548 0.1540 0.1614 w.d. 0.1536 rancas richness (s) 30 4 2 12 10 6 4 19 11 diversity (h) 4.3093 1.9751 0.9545 2.7642 2.9114 2.3718 1.9072 3.7193 2.9546 dominance (d) 0.0636 0.2593 0.5313 0.2296 0.1663 0.2099 0.2778 0.0934 0.1754 ucupi richness (s) w.d. w.d. 7 12 6 7 2 16 4 diversity (h) w.d. w.d. 1.8943 3.1653 1.9736 2.5781 0.9494 3.0874 1.3388 dominance (d) w.d. w.d. 0.3633 0.1352 0.3238 0.1852 0.5346 0.1679 0.5018 table 3. values of species richness, shannon–weaver diversity index and dominance for each agroecosystem and monitoring date (w.d., without data). floristic composition and diversity in plots of early post-agricultural succession in four agroecosystems in the district of cajatambo, lima september december 2019 142 which supports the assumption of initial dominance. the mean diversity is 2 bits/individual, and the dominance is medium, both at the beginning and end of the test. based on the floristic composition of the plots, the only common species among the four agroecosystems is medicago polymorpha, which is associated with the landscape in question: periodically visited by cattle and exposed to excessive sun, seasonal rain and frost. five species appear in three of the four agroecosystems: brassica rapa subsp. campestris and verbena litoralis (ocp, tpc and rnc), oenothera rosea and trifolium repens (ocp, rnc and ucp) and bidens andicola (tpc, rnc and ucp). the description of the plant community composition is essential to observe spatial and temporal changes in parameters of ecological importance. indeed, the analysis of communities allows to monitor the variations in the agroecosystem; therefore, vegetation is an adequate indicator since its structure and composition reflect the vital ecosystem factors (matteucci & colma, 1982). conclusion the early post-agricultural succession of the four agroecosystems evaluated in cajatambo presents a floristic composition represented by 78 species belonging to 30 botanical families, of which the asteraceae (16 species), 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(2011). estudio de impacto ambiental de la central hidroeléctrica rapay salto 1 y salto 2. snpower. lima. 892 p. peruvian journal of agronomy 4(3): 93–103 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i3.1341 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 14 october 2020 accepted for publication: 20 november 2020 collection and morphological characterization of 149 accessions of achiote (bixa orellana l.) from seven departments in perú colecta y caracterización morfológica de 149 accesiones de achiote (bixa orellana l.) provenientes de siete departamentos del perú juan nolasco-chumpitaz1,2*; paul ccoyllo-llacsa1; gabriela koc-sanchez1; percy medina-morales1 *corresponding author: juanluisnolasco@outlook.com https://orcid.org/0000-0001-6358-5497 abstract the aim of this study was to characterize and identify groups of achiote (bixa orellana l.) with characteristics related to increased yield and bixin content. a total of 149 achiote accessions were collected from the departments of loreto, san martin, junín, pasco, huánuco, ucayali, and cusco, in perú. these were then evaluated using ten quantitative and three qualitative capsule and seed descriptors. undesirable characteristics such as high spinosity and dehiscence predominated, while none of the quantitative descriptors correlated significantly with bixin content. principal component analysis indicates that the quantitative descriptors (except for bixin content) are statistically significant, forming four clusters. notably, one of the clusters included accessions characterized by heavy seeds, and another cluster included accessions with high number of seeds and bixin content. keywords: achiote, annatto, bixin, morphological characterization, perú resumen el objetivo de este estudio fue caracterizar e identificar grupos de achiote (bixa orellana l.) con características relacionadas con mayor rendimiento y contenido de bixina. se recolectaron un total de 149 accesiones de achiote de los departamentos de loreto, san martín, junín, pasco, huánuco, ucayali y cusco, en perú. luego se evaluaron utilizando diez descriptores de cápsulas y semillas cuantitativos y tres cualitativos. predominaron características indeseables como alta espinosidad y dehiscencia, mientras que ninguno de los descriptores cuantitativos se correlacionó significativamente con el contenido de bixina. el análisis de componentes principales indica que los descriptores cuantitativos (excepto el contenido de bixina) son estadísticamente significativos, formando cuatro grupos. notablemente, uno de los grupos incluye accesiones caracterizadas por semillas pesadas, y otro grupo incluye accesiones con alto número de semillas y contenido de bixina. palabras clave: achiote, annatto, bixina, descriptores morfológicos, perú. 1 instituto nacional de innovación agraria. sub-dirección de productos agrarios, dirección de desarrollo tecnológico agrario, av. la molina 1981 la molina, lima, perú. 2 universidad nacional agraria la molina, facultad de agronomía. lima, perú. cite this article: nolasco-chumpitaz, j., ccoyllo-llacsa, p., koc-sanchez, g., & medina-morales, p. (2020). collection and morphological characterization of 149 accessions of achiote (bixa orellana l.) from seven departments in perú. peruvian journal of agronomy, 4(3), 93–103. http://dx.doi.org/10.21704/pja.v4i3.1341 introduction achiote (bixa orellana l.), a shrubby plant standing from 3 to 10 m tall, is native to the american tropics, and bears globose to ovoid fruits with colors such as red, green, yellow, or shades of these. it has historically been used as a food, medicine, and colorant (camacaro et al., 2018; raddatz-mota et al., 2017; valério et al., 2015). the use of achiote as a colorant in the food, textile, pharmaceutical, and cosmetic industries is due to the carotenoid compounds present in the seeds, especially the apocarotenoid bixin (alcázar et al., 2017; habibi-najafi et al., 2018; viuda-martos et al., 2012). bixin is the second most economically important natural colorant in the world and 60% of the world’s supply comes from latin america, mainly perú, brazil, and mexico (raddatz-mota collection and morphological characterization of 149 accessions of achiote (bixa orellana l.) from seven departments in perú september december 2020 94 et al., 2017; rajagopal et al., 2016; yolmeh et al., 2015). moreover, perú is recognized as one of the main achiote seed exporters in the world (stringheta et al., 2018). according to the peruvian ministerio de agricultura y riego [minagri] (2019), in 2018, achiote was cultivated in 11,635 ha of land. the departments with the largest areas of achiote cultivation were cusco (6,205 ha), pasco (4,390 ha), huánuco (414 ha), junín (203 ha), and ucayali (185 ha). the national average yield was 601 kg ha−1, producing 6,988 t of grain. other countries such as mexico and colombia had annual yields of 1.2 and 1.13 t ha−1, respectively, for the year 2017 (servicio de información agroalimentaria y pesquera [siap], 2019; minagricultura, 2020). currently, the bixin content of achiote seeds varies from 1% to 6%, with brazil reporting a national average of 3.5% and a maximum of 5% in the state of sao paulo (albuquerque & meireles, 2011). brazilian varieties such as embrapa 37 have bixin contents that exceed 5%, which is relevant because the market requires contents greater than 4% for export (dias et al., 2017). in perú, promising accessions with bixin contents of 3.55% to 4.55% have been reported in the national germplasm bank (instituto nacional de investigación agraria [inia], 2009). aside from this report, there is no official report or census on the bixin contents of peruvian achiote, although several companies own accessions with bixin contents lower than the minimum percentage required for export in brazil. despite being an important agro-industrial crop in perú, local achiote production is characterized by low yields, with lower bixin contents compared to those of other countries. this may be partly attributed to the absence of an established variety or ecotype. little is known about the characteristics of local achiote plants and there are also very few published studies on the characterization of peruvian material. such information is necessary to improve this crop genetically. the objectives of this research are to analyze and classify, using morphological descriptors, achiote accessions from seven departments in perú in order to identify accessions with great potential to increase yield and bixin content. methodology germplasm collection achiote capsules (fruits) were collected between may and november 2019. a total of 149 achiote accessions were collected from the departments of loreto, san martín, junín, pasco, huánuco, ucayali, and cusco (25, 24, 25, 23, 4, 24, and 24 accessions, respectively). each accession was provided by farmers, and was derived from plants that grew from seeds. the majority of plants were older than 3 years and less than 2.5 m tall. the location of each collection site was mapped by gps (fig. 1 and mean gps data on table 1). prior to sampling, we verified, together with the farmer that the plant was a different type. samples consisted of two panicles collected at the stage of commercial maturity (capsules and seeds in good quality). the panicles were transported to the laboratory of the la molina experimental center of the instituto nacional de innovación agraria (inia), where the fruits were characterized. the passport data for each sample were compiled in accordance with directive no. 001-2005-iniea-dgiasudirgeb of inia-perú, specifically, within the “norms that define the standardized use of formats for the documentation of the data of passport in the sudirgeb ex situ germplasm bank.” (instituto nacional de investigación y extensión agraria [iniea], 2006). each figure 1. sites (red dots) where the 149 achiote accessions were collected. department number of accessions utm* latitude utm* length elevation (m.a.s.l.) loreto (l) 25 683192 18m 9563896 102 san martín (sm) 24 275200 18m 9326970 708 junín (j) 25 522614 18l 8780152 659 pasco (p) 23 502876 18l 8864447 282 huánuco (h) 4 474506 18l 8932754 288 ucayali (u) 24 515248 18l 9039663 222 cusco (c) 24 745212 18l 8609063 793 table 1. average locations of collection points of achiote accessions by department. *universal transverse mercator j. nolasco-chumpitaz; p. ccoyllo-llacsa; g. koc-sanchez; p. medina-morales peruvian journal of agronomy 4(3): 93–103 (2020) 95 of the samples was admitted into the national achiote germplasm bank of the el porvenir-san martin agrarian experimental station-inia as new accessions. the conditions of the plants from which samples were collected varied greatly. for example, they differed in management, age (from 3 years to older than 15 years), soil, and other characteristics. the data of yield per plant (which is linked to descriptors such as number of capsules per panicle, number of panicles per plant, among others) was not considered in our analysis. the accessions were coded with the initials of the department from where they were collected followed by a correlative number (for example, code l-020 corresponds to accession number 20 collected in loreto). characterization of the fruits and seeds the samples were cleaned, and then dried in an oven at 40℃ for 2 days (arce, 1999) prior to evaluating the morphological descriptors of the fruits. the seeds were extracted from each capsule and they were stored under controlled conditions (seeds were packed in trilaminate aluminum envelopes and placed in a sealed container without light, with a temperature of 2°c–8°c and relative humidity of 50%–60%) for a maximum of 3 days before determining the bixin content. quantitative (table 2) and qualitative (table 3) descriptors reported by arce (1999) were used to characterize achiote fruit and seeds. these descriptors have been used previously in similar studies (valdez-ojeda et al., 2008). in addition, we also used other descriptors (lópez et al., 2018) such as capsule thickness, weight of seeds per capsule, seed length, and seed diameter. the lengths, widths, and thicknesses of 20 randomly selected capsules were measured from each accession using a digital vernier according to the methodology of arce (1999). seed weight was determined with an analytical balance and the number of seeds per capsule was counted manually. the lengths and diameters of the seeds were measured using dinocapture 2.0 (2019) software after photographing 20 randomly selected seeds from each accession using a digital microscope (dino-lite, new taipei city, taiwan). the qualitative characteristics (degree of spinosity, length of spines, and dehiscence), were scored by visual observation and compared to the graphs of arce (1999). determination of bixin content the bixin content of achiote seeds was determined using standard methods described by the food and agriculture organization [fao] (1982); and seeds were extracted using peruvian standard methods for the extraction of bixin and norbixin, as reported in ntp 209.256: 1991 (instituto de investigación tecnológica industrial y de normas técnicas [initec], 1991). the bixin extraction step was performed exhaustively on the seeds using chloroform until complete discoloration. the resulting extracts were further diluted with chloroform and the absorbance’s of the diluted samples were measured at 470 nm using a uv-vis spectrophotometer (thermo scientific, genesys 10s). absorbance values were converted to the equivalent g of bixin 100 g−1 sample, using a standard curve of known bixin (chemical purity 99.3%) concentrations (i.e., from 0.25 to 3 mg l−1). bixin concentrations were calculated using the following equation: y = 0.406x − 0.005 (r2 = 0.9998), where y = bixin content in mg l−1; and x = absorbance. organ descriptor unit corresponding abbreviation used in text capsule length cm lc width cm ac thickness cm gc length-width ratio rla number of seeds per capsule ns seed weight per capsule g psc seed weight of 100 seeds g pp100s seed length mm ls seed diameter mm ds bixin content g 100g−1 bix table 2. quantitative descriptors used to characterize achiote (bixa orellana l.) accessions. descriptor score description corresponding abbreviation used in text capsule spinosity 0 no spines ec 1 very low number 3 low number 7 high number 9 very high number spine length 1 very short le 3 short 7 long 9 very long capsule dehiscence 0 non-dehiscent deh 1 dehiscent table 3. qualitative descriptors used to characterize achiote (bixa orellana l.) fruits. collection and morphological characterization of 149 accessions of achiote (bixa orellana l.) from seven departments in perú september december 2020 96 statistical analysis data were analyzed using sas (2020) software. pearson coefficients and the descriptive statistics were generated using the corr function. principal component analysis (pca) (only quantitative descriptors) and cluster analysis (quantitative and qualitative descriptors) were performed using rstudio (2020) software. specifically, we implemented the prcomp function within the package factoextra (kassambara & mundt, 2020), and the functions daisy and hclust within the cluster package (maechler et al., 2019), to perform pca and cluster analysis, respectively. we determined the number of clusters using the function kgs within the package maptree (white & gramacy, 2012). pca was performed by first standardizing the data using the scale function within r. for the analysis, we employed the criteria of cliff and kaiser as described by franco & hidalgo (2003). according to these criteria, the analysis should only consider the components that, when added together, contribute 70% or more of the variance and whose eigenvalues are greater than or equal to one. in the cluster analysis, a dendrogram based on both quantitative and qualitative descriptors was constructed. the distance matrix was based on gower distances, which is recommended for mixed data (franco & hidalgo, 2003). groupings are based on ward’s hierarchical method, and the number of clusters was estimated using the kelleygardner-sutcliffe penalty function as described by grum & atieno (2007). results morphological variability the means of the ten quantitative characteristics measured in the 149 achiote accessions, collected in seven departments of perú, are shown in table 4. the results indicate a moderate level of variation as evidenced by high coefficients of variability (7.87% to 30.54%), with the highest value observed for bixin content. this is reflected by the wide range of bixin contents among the accessions, with the highest and lowest values observed for accessions l-021(3.997 g 100 gr−1) and j-061 (0.895 g 100 gr−1). table 5 shows the frequencies of the grades of each qualitative descriptor. high and very high grades (quantity of spines) of capsule spinosity (ec) were observed in 71.2% of all observations. furthermore, long and very long spines (le) were observed in 67.8% of the accessions. three accessions (p-081, p-082, and p-083) have no spines. the distribution of dehiscent and non-dehiscent fruits among the accessions is roughly equal (54.4% and 45.6%, respectively). correlation between the descriptors the pairwise pearson correlation coefficients between the ten quantitative descriptors are shown in table 6. most of the correlations (36) between descriptors were significant (p < 0.05), and only nine were not significant. among the morphological descriptors of the capsule, significant correlations were found between capsule length (lc) and length-width ratio (rla) (0.726), capsule width (ac) and capsule thickness (gc) (0.637), and capsule width (ac) and rla (−0.655). this means that capsule length is directly correlated with the length/width ratio, lc* ac gc rla ns psc pp100s ls ds bix population (n = 149) average 4.72 2.97 2.15 1.64 39.55 1.15 2.93 4.87 3.97 2.27 s.d. 0.91 0.53 0.48 0.42 8.86 0.34 0.73 0.43 0.31 0.69 c.v. 19.29 17.78 22.38 25.76 22.39 29.86 24.98 8.73 7.87 30.54 minimum value 2.54 1.75 1.15 0.86 18.60 0.50 1.16 3.43 3.23 0.895 accession j-055 sm-047 j-051 u-107 u-125 u-123 u-123 u-123 l-024 j-061 maximum value 7.17 4.81 3.48 2.74 69.90 2.42 5.14 6.09 4.67 3.997 accession j-072 j-061 l-002 j-060 l-008 j-072 c-128 j-072 c-129 l-021 table 4. statistical values of ten quantitative descriptors used to describe 149 achiote accessions and the identities of the accessions having the minimum and maximum values. s.d.: standard deviation; c.v.: variability coefficient (%). *lc: capsule length (cm); ac: capsule width (cm); gc: capsule thickness (cm); rla: capsule length-width ratio; ns: number of seeds per capsule; psc: seed weight per capsule (g); pp100s: weight of 100 seeds (g); ls: seed length (mm); ds: seed diameter (mm); bix: seed bixin content (g 100g−1) descriptor grade frequency number percentage ec* 0 3 2.0 1 11 7.4 3 29 19.5 7 60 40.3 9 46 30.9 le 1 15 10.1 3 33 22.1 7 48 32.2 9 53 35.6 deh 0 68 45.6 1 81 54.4 table 5. frequencies of the grades of the qualitative descriptors used to characterize 149 achiote accessions. ec: capsule spinosity; le: spine length; deh: capsule dehiscence j. nolasco-chumpitaz; p. ccoyllo-llacsa; g. koc-sanchez; p. medina-morales peruvian journal of agronomy 4(3): 93–103 (2020) 97 while width is negatively correlated to capsule length. rla and gc are also negatively correlated (−0.482). among the seed-related descriptors, the number of seeds per capsule (ns) is correlated (0.459 and 0.506) to gc and seed weight per capsule (psc), respectively. moreover, the weight of the seeds (psc) is significantly correlated with lc (0.474) and weight of 100 seeds (pp100s) (0.702). lastly, pp100s is positively correlated to lc (0.501). thus, accessions with high seed weight per capsule tend to have longer capsules and heavier individual seeds. capsule length is correlated with seed length (ls) (0.450), rla (0.509), psc (0.480), and pp100s (0.620), suggesting that accession with elongated seeds tend to have elongated capsules and heavier seeds. furthermore, seed diameter (ds) is also positively correlated with pp100s (0.644) and ls (0.634). bixin content (bix) is significantly positively correlated (albeit at low coefficients) to ac, gc, and ns and negative correlated to rla, pp100s, ls, and ds. these results indicate that, among accessions, bixin content tends to be higher with the more oval-shaped fruits and with fruits with high number of seeds (ns) per capsule. principal component analysis the first three principal components (pc) contribute a total of 72.3% of the variance; and their corresponding eigenvalues are all greater than 1, as shown in table 7. along the axis of component 1, (which contributes 39.1% to the variance), the descriptors rla, pp100s, lc, ls, ds stand out, followed by psc, ac, and gc. ns and bix are less relevant. component 2 contributes 22.3% of the morphological variance, and along this axis, the descriptors ns, psc, ac, and gc stand out. component 3 contributes 10.9% of the variance, and along this axis, the descriptors rla, ns, and ac stand out. the rest of the descriptors have low discriminatory power. a graph of the first two pcs (fig. 2) shows a somewhat positive correlation between ns, gc, and ac; these descriptors are correlated to a lesser extent with bix. conversely, the fruit descriptors rla, lc, psc, pp100s are more or less correlated to seed descriptors ls and ds due to the narrow angle between them. gc and ac are most closely related, as indicated by the smallest angle between the descriptors in this graphic. whereas, bix and rla are furthest related, as indicated by the near horizontally opposite angle. overall, the results show that lc* ac gc rla ns psc pp100s ls ds ac 0.012 gc −0.063 0.637*** rla 0.726*** −0.655*** −0.482*** ns 0.059 0.329*** 0.459*** −0.168* psc 0.474*** 0.089 0.152 0.270*** 0.506*** pp100s 0.501*** −0.153 −0.191* 0.441*** −0.233** 0.702*** ls 0.450*** −0.280*** −0.291*** 0.509*** −0.135 0.480*** 0.620*** ds 0.407*** −0.213** −0.254** 0.422*** −0.323*** 0.350*** 0.644*** 0.634*** bix −0.076 0.166* 0.193* −0.186* 0.174* −0.037 −0.202* −0.178* −0.206* *, **, *** indicates statistical significance at p-value of 0.05, 0.01, and 0.001, respectively. table 6. pearson correlation coefficient matrix of the quantitative descriptors used to characterize 149 achiote accessions. *lc: capsule length (cm); ac: capsule width (cm); gc: capsule thickness (cm); rla: capsule length-width ratio; ns: number of seeds per capsule; psc: seed weight per capsule (g); pp100s: weight of 100 seeds (g); ls: seed length (mm); ds: seed diameter (mm); bix: seed bixin content (g 100g−1) component 1 2 3 eigen value 3.9099 2.2292 1.0889 variance percent 39.099 22.292 10.889 cumulative variance percent 39.099 61.392 72.281 descriptors correlation coefficients lc* −0.667 0.383 −0.259 ac 0.498 0.624 0.395 gc 0.496 0.666 0.167 rla −0.816 −0.150 −0.478 ns 0.283 0.712 −0.459 psc −0.515 0.761 −0.070 pp100s −0.804 0.286 0.298 ls −0.799 0.139 0.119 ds −0.760 0.049 0.391 bix 0.313 0.191 −0.372 table 7. pca output of the quantitative descriptors used to characterize achiote accessions. *lc: capsule length (cm); ac: capsule width (cm); gc: capsule thickness (cm); rla: capsule length-width ratio; ns: number of seeds per capsule; psc: seed weight per capsule (g); pp100s: weight of 100 seeds (g); ls: seed length (mm); ds: seed diameter (mm); bix: seed bixin content (g 100g−1) collection and morphological characterization of 149 accessions of achiote (bixa orellana l.) from seven departments in perú september december 2020 98 figure 2. contributions of the achiote descriptors* as determined by the two principal component axes. *lc: capsule length (cm); ac: capsule width (cm); gc: capsule thickness (cm); rla: capsule length-width ratio; ns: number of seeds per capsule; psc: seed weight per capsule (g); pp100s: weight of 100 seeds (g); ls: seed length (mm); ds: seed diameter (mm); bix: seed bixin content (g 100g−1) figure 3. distribution of the 149 achiote accessions along the two principal components. the different clusters (in graphic named groups) are indicated by a color and symbol (cluster 1: red dot, cluster 2: green triangle, cluster 3: blue square, and cluster 4: purple plus sign). j. nolasco-chumpitaz; p. ccoyllo-llacsa; g. koc-sanchez; p. medina-morales peruvian journal of agronomy 4(3): 93–103 (2020) 99 figure 4. dendrogram of 149 achiote accessions generated using cluster analysis by processing 13 quantitative and qualitative morphological descriptors. collection and morphological characterization of 149 accessions of achiote (bixa orellana l.) from seven departments in perú september december 2020 100 accessions with higher bixin contents tend to have wide or rounded capsules and high numbers of seeds. the pca plot of all the accessions (fig. 3) was constructed based on the first two components. while the plot shows no clear clusters, we nevertheless identify the clusters that were generated by cluster analysis. cluster analysis four clusters were identified at a gower distance of 0.75 (fig. 4). the features of each cluster are summarized below. cluster 1 contains 26 accessions from loreto, san martín, junín, pasco, and ucayali departments. this group is characterized by the absence of capsule dehiscence and high spinosity (ec) and long spines (le). the values for all descriptors (including bix) were generally average, except for ns and lc, which were less than average. cluster 2 contains 42 accessions from junín, pasco, huánuco, ucayali, and cusco departments. this group is characterized by non-dehiscent capsules, low degree of capsule spinosity, and short spines. in addition, the accessions in this cluster have the lowest ac, gc, and bix values, while having the highest lc, rla, psc, pp100s, ls, and ds values. furthermore, the highest seed weights were observed in this cluster, which suggests that it has good yield potential, however, such yields will have low bixin contents. cluster 3 is made up of 37 accessions from loreto, san martín, and ucayali departments. it is characterized by dehiscent capsules with a very high degree of spinosity and very long spines. on average, this cluster has highest bix, ns, ac, and gc values, while its rla, psc, pp100s, ls, and ds values are the lowest. moreover, accessions in this cluster tend to have higher bix values. cluster 4 is made up of 44 accessions from loreto, san martin, junín, pasco, huánuco, ucayali, and cusco. it is characterized by dehiscent capsules with high degree of spinosity and long spines. most of the descriptors (including bix) tend to have average values, except for psc values, which tend to be lower than the general average. figure 4 shows 2 major groups, which differ mainly by the non-dehiscent capsules of one group (which include clusters 1 and 2) and the dehiscent capsules of the other group (which contain clusters 3 and 4). discussion the ranges of values observed for lc (7.17 to 2.54 cm), ac (4.81 to 1.75 cm) and gc (3.48 to 1.15 cm) (table 4) agree to some extent with those obtained from india, perú, and venezuela by akshatha et al. (2011), lópez et al. (2018), and mazzani et al. (2000), respectively. these authors reported capsule dimensions ranging from 5.7 to 3.0 cm long and from 4.8 to 2.0 cm wide. we found rla values ranging from 0.86 to 2.74 (average 1.64), which are higher than those (0.75 to 1.5) reported by valdez-ojeda et al. (2008) for mexican accessions. rla values can also be used to classify capsules as oval, elongated, or spherical (akshatha et al., 2011). our findings indicate that our accessions are predominantly elongated. the ns values of our accessions range from 18.6 to 69.9 seeds per fruit, which are higher than those (30 to 60 seeds per fruit) reported by other authors (akshatha et al., 2011; moreira et al., 2015; rivera-madrid et al., 2006). the average psc of our accessions (1.15 g) is lower than that reported by lópez et al. (2018) (1.55 ± 0.29 g). the pp100s values of our accessions (5.14 to 1.16 g) are generally higher than the reference values of 3.8 to 1.10 g reported previously (akshatha et al., 2011; mantovani et al., 2013). the ls (6.09 to 3.43 mm) and ds (4.67 to 3.23 mm) values of our accessions are slightly higher than the previously reported length (0.5 ± 0.02 cm) and width (0.4 ± 0.02 cm) of seeds in perú (lópez et al., 2018). comparing our results to previous studies, the extreme values we found for various descriptors suggest that the achiote populations are diverging in terms of capsule and seed characteristics. bixin contents in our accessions range from 0.895 (j-061) to 3.997 g 100 g−1 (l-021), which is consistent with previously reported values (0.49 to 2.65 g 100 g−1) generated by similar extraction methods (akshatha et al., 2011; rivera-madrid et al., 2006; valdez-ojeda et al., 2008; viuda-martos et al., 2012). other authors, using exhaustive methods such as supercritical fluid extraction, have obtained higher bix values, i.e., from 4.90 to 7.58 g 100 g−1 (albuquerque & meireles, 2012; rodrigues et al., 2014). meanwhile, extractions using alkaline solvents result in bix contents ranging from 1.66 to 5.05 g 100 g−1 (dias et al., 2017; mantovani et al., 2013). in both the cases of exhaustive methods (super critical fluid extraction and alkaline solvents), brazilian achiote seeds were used. we suggest that commonly used standard methods of bixin extraction often underestimate the bixin content of the accessions. therefore, in the future, we intend to use exhaustive methods to better extract pure bixin. in our study, only two accessions have bixin contents that are close to the 4% bixin requirement for export; l-021 and l-024 produce achiote seeds with 3.997% and 3.934% bixin, respectively. bixin is degraded by light, high temperatures (arce, 1999), and high environmental humidity (biego et al., 2013). dehiscent fruits tend to expose seeds to such unfavorable environmental conditions, generating lowquality seed (medina et al., 2001). furthermore, capsules with little or no spines are preferable because they make the fruit harvesting easy. the frequencies of these desirable characteristics (low spinosity and no dehiscence) in our j. nolasco-chumpitaz; p. ccoyllo-llacsa; g. koc-sanchez; p. medina-morales peruvian journal of agronomy 4(3): 93–103 (2020) 101 accessions were low to medium (table 5), indicating that selection may be an effective genetic improvement tool. the low pearson coefficients (table 6) indicate little correlation between the descriptors, especially for bix. this may be related to the differences in location, climate, age, and management among the accessions. most likely, however, this is because the accessions are biologically independent from each other. therefore, future characterization studies of achiote should focus on accessions of the same age, planted in the same plot, and managed under controlled conditions. the pca results (table 7) indicate that most of the descriptors are relevant, that is, they are indeed important in describing the accessions. the exception is bix, which is of little relevance in discriminating between accessions. this agrees with the pearson correlation analysis and can be explained by the fact that bix is unrelated to other traits studied. analyzing achiote accessions using pca and morphological descriptors such as those evaluated in the present study has been done previously (mazzani et al., 2000). these authors obtained three main components that explained 83.8% of the variance, however, they did not evaluate bixin contents. the results of correlation analysis and pca are somewhat concordant with the clusters generated by cluster analysis (both graphically represented in fig. 3). for example, cluster 3 includes accessions with high values of ns, ac, and bix. in clusters 2 and 3 include accessions with high values in descriptors related to yield (pp100s and psc) and quality (ns and bix), respectively. therefore, these clusters may identify potential candidate accessions for subsequent genetic improvement work. the results of cluster analysis show that the departmental origin of the accessions is not related to their clustering, which indicates that the achiote plants in each department are highly diverse, based on the descriptors used in this study. future work should include establishing the progeny of the accessions used in this study in a germplasm bank to monitor their morphological descriptors in order to confirm the stabilities of these characteristics and the clusters obtained in this study through time. we have also identified accessions with potential for genetically improving this crop; these accessions with the highest bixin contents (3.997 to 3.761 g 100 g−1 bixin, top 5th percentile) are l-021, l-024, c-130, l-27, and l-22. conclusion we characterized 149 achiote (bixa orellana l.) accessions from the departments of loreto, san martin, junín, pasco, huánuco, ucayali, and cusco. various fruit and seed traits are correlated. a few accessions have high values for characteristics related to crop yield and bixin content such accessions may have potentially important roles to play both commercially and as materials for future achiote breeding programs. acknowledgements to the staff of the inia agrarian experimental stations, for their invaluable support in the collections, as well as to the programa nacional de innovación agraria (pnia) for financing through the pnia 187_pi project. references akshatha, v., giridhar, p., & ravishankar, g.a. 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(2012). maptree: mapping, pruning, and graphing tree models. r package version 1.4-7. https://cran.r-project.org/ package=maptree yolmeh, m., habibi-najafi, m.b., shakouri, s., & hosseini, f. (2015). comparing antibacterial and antioxidant activity of annatto dye extracted by conventional and ultrasound-assisted methods. zahedan journal of research in medical sciences, 17(7). https://doi. org/10.17795/zjrms1020 peruvian journal of agronomy 1 (1): 21-31 (2017) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v1i1.1064 © the authors. published by universidad nacional agraria la molina received for publication: 16 july 2017 accepted for publication: 2 2 december 2017 ethno-botany and in-situ conservation of the genetic diversity of arracacha (arracacia xanthorrhiza bancroft), yacon (smallanthus sonchifolious h.robinson), and wild relatives etnobotánica y conservación in-situ de la diversidad genética de la arracacha (arracacia xanthorrhiza bancroft), yacón (smallanthus sonchifolius h. robinson), y sus parientes silvestres ignacio, s.1; camarena, f.2*; baudoin, j.3; blas, r.2 *corresponding author: camafe@lamolina.edu.pe abstract the study was conducted in the districts of huacrachuco and san pedro de cholon, province of marañon and huanuco region. the aim of the study was to describe the in-situ conservation and ethno-botanical indicators of the phenotypic variability of arracacha (arracacia xanthorrhiza bancroft), yacon (smallanthus sonchifolius h. robinson) and wild relatives. work was done in the farmers’ plots where they preserved from one to three morphotypes of a. xanthorrhiza and s. sonchifolius, and farmer communities where preserved wild plant relatives. a total of thirteen farmers from three farmer communities in the huarachuco district, and nine from two farmer communities in the district of san pedro de cholon were selected. six in-situ conservation and three ethno-botanical indicators were described. by using morphological descriptors 23 samples of grown arracacha and 34 of grown yacon, three samples each of wild arracacha and yacon were characterized. based on this characterization, results were as follow: three different morphotypes of grown arracacha for 21 descriptors, four different morphotypes of grown yacon for 18 descriptors, two ecotypes of wild arracacha (the low field ecotype located in a range of altitude of 2500 to 2750 meters above sea level and the height field ecotype located above 3530 m.a.s.l.) and two ecotypes of wild yacon (yellow and red ecotypes). keywords: arracacia, smallanthus, in situ conservation, ethno-botanical, ecotype, morphotype. resumen el estudio fue realizado en los distritos de huacrachuco y san pedro de cholón, provincia de marañón, huánuco. el objetivo fue describir los indicadores de conservación in situ y etnobotánicos de la variabilidad fenotípica de arracacha (arracacia xanthorrhiza bancroft), yacón (smallanthus sonchifolius h. robinson) y sus parientes silvestres. se trabajó en parcelas de agricultores donde conservan de uno a tres morfotipos de a. xanthorrhiza y s. sonchifolius, y en comunidades donde conservan los parientes silvestres. se seleccionaron un total de 13 agricultores de tres comunidades del primer distrito y nueve de dos comunidades del segundo distrito. se describieron seis indicadores de conservación in situ y tres etnobotánicos. utilizando descriptores morfológicos se caracterizaron 23 muestras de arracacha cultivada y 34 de yacón cultivado, tres muestras de arracacha silvestre y tres de yacón silvestre. con base a esta caracterización se encontraron: tres morfotipos diferentes de arracacha cultivada para 21 descriptores, cuatro morfotipos de yacón cultivado para 18 descriptores, dos ecotipos silvestres de arracacha (uno de la zona baja ubicada de 2500 a 2750 m.s.n.m. y otro de la zona alta ubicada a 3530 m.s.n.m.) y dos ecotipos de yacón silvestre (amarrillo y rojo). palabras claves: arracacia, smallanthus, conservación in situ, etnobotánica, ecotipo, morfotipo. 1profesor de la escuela académico profesional de agronomía, facultad de ciencias agrarias-universidad nacional hermilio valdizán, perú. 2profesor principal del departamento de fitotecnia de la facultad de agronomíauniversidad nacional agraria la molina, perú 3profesor principal de faculté universitaire des sciences agronomiques de gembloux, bélgica introduction in peru there are 1 060 species of pteridophytas, 24 gymnosperms and 17 119 angiosperms (zúñiga 2004). these species would have originated about 80 thousand years ago, since then they are in permanent evolution (tapia, 2001). this natural and cultivated plant wealth is the “national patrimony”, fundamental for the biosphere to continue producing ecological goods and services (food, recreation, genes, education, etc.) for the wellbeing and survival of man, it is a source for development socioeconomic status of families, communities and nations. as mentioned in seminar et al. (2003), andean roots and tubers (artc) such as the achira, the ahipa, the arracacha, the maca, the mauca, the mashua, the oca, the ulluco and the yacon, are important for the food and culture of the andean rural people. ethno-botany and in-situ conservation of the genetic diversity of arracacha (arracacia xanthorrhiza bancroft), yacon (smallanthus sonchifolious h.robinson), and wild relatives junio-setiembre 2017 22 the genetic erosion of local varieties is due to factors, such as population increase, soil degradation, environmental changes, the introduction of modern varieties, migration, deforestation, globalization and ethnic acculturation, oil exploitation (ipgri, 1999) and the loss of knowledge of the peasants. according to the cbd (2000), around 34,000 species of plants and 5,200 animal species (including one of every eight species of birds in the world) would be in danger of extinction, 45% of natural forests have disappeared, 30% of races of the main domestic animal species are exposed to a high risk of extinction and some species disappear at a rate between 50 to 100 times higher than the natural rate. peasants are left without resources and the productivity of agroecosystems decreases in time and space, compromising food security and sovereignty in rural and urban areas. this context, motivated the creation of a conservation program of these resources for future generations (brush, 2003). according to sevilla and holle (1995), the best way to conserve biodiversity is to keep the cultivated species and their wild relatives in the place where they have originated, evolved and are currently produced, considering traditional logistical and logistical strategies as indispensable in logistic and rational strategies. this article describes the in situ and ethnobotanical conservation indicators of the phenotypic variability of the arracacha, the yacon and its wild relatives from the province of huacrachuco, huánuco. materials and methods the communities of the district of huacrachuco are located from 8º24’38 ‘’ to 8º54’29 ‘’ south latitude and from 77º1’05 ‘’ to 77º44’24 ‘’ west longitude, with altitudes of 2,500 m.a.s.l. at more than 3,500 m.a.s.l. .; the communities of the district of san pedro de cholón from 8º3’47 ‘’ to 8º45’53 ‘’ south latitude and from 77º20’24 ‘’ to 77º50’52 ‘’ west longitude, with altitudes of 2 400 m.a.s.l. to more than 2 800 m.a.s.l. huacrachuco presents two climate stations, warm-temperate from june to september and cold from january to march, an average temperature of 16º c and an annual precipitation of 600 mm. san pedro de chonta presents a more homogeneous climate, with an average temperature of 20º c and an annual rainfall that exceeds 900 mm. the method used in the study was descriptiveexplanatory. the methodology consisted of three procedures (adapted from paz, 1997): i) participatory planning, the scope of the study was socialized in each district through workshops with authorities and leading farmers of the conservationist communities (cc); then, in focus groups by communities, preliminary information on in situ, ethnobotanical conservation and wild ecotypes of arracacha and yacon was collected (madriz, 2000). based on this information, the scope of work, the road map, communities and conservationist farmers, and farmers with ethnobotanical knowledge were identified. the cc were selected based on the number of farmers who mentioned conserving from one to three arracacha and yacon morphotypes in their plots (according to seminario et al., 1999, “a morphotype is an intraspecific population that presents the general and typical morphology of the species but presents certain qualitative characteristics that differentiate it from other morphotypes “) and according to the information about the existence of wild ecotypes in the community environment. the conservationist farmers (agco) were selected according to the number of morphotypes (from one to three), of the species under study preserved in their plots, corroborated through on-site visits. farmers with ethnobotanical knowledge were identified according to the information they provided. ii) participatory research, through semi-structured surveys with farmers organized in focus groups (whyte, 1989) and (geilfus, 1997), the indicators of in situ conservation, ethnobotanical and socioeconomic variables of the communities were described. then, the indicators and variables that were indicated by 80% or more of the participants were described. iii) case study, through semi-structured interviews and open dialogues with 22 agco, in situ and ethnobotanical conservation indicators were described (williams, s. a.); and the geographic and ecological parameters of the habitats of the wild ecotypes were measured in their habitats (eisenhardt, 1989, tellis, 1997, neale et al., 2006). the characterization of s. sonchifolius varieties was carried out in farmers’ plots, using 20 standardized morphological descriptors proposed by arbizu et al. (2001) and a. xanthorrhiza with 22 descriptors 2003 version of the prta-unc, adapted by seminario (2006) and tapia (2007). the wild arracacha and yacón ecotypes were characterized in their natural habitats, by means of characteristics of plants that the farmers use to differentiate the variation, only in the case of the arracacha of puna was it supplemented in plants reproduced in flowerpots. for wild arracachas, habitat, plant size and root conformation were used; and for wild yacon the color of the flower, size of the reserve root, shape of the reserve root and archetype of the plant.la unidad de muestreo para la caracterización de las accesiones cultivadas fue la planta en estado de madurez fisiológica, todas las características fueron evaluadas en cinco muestras, excepto en el caso de las flores y frutos del yacón se muestreó según la disponibilidad del material, y la caracterización de los ecotipos silvestres se realizó con dos o tres plantas, según la abundancia. se caracterizó 14 muestras de a. xanthorrhiza en el distrito de huacrachuco y nueve en san pedro de chonta, 22 de s. sonchifolius en el distrito de huacrachuco y 12 en san pedro de chonta, tres muestras de arracacha silvestre y tres de yacón silvestre en el distrito de huacrachuco. statistical analyzes were carried out with the infostat / professional program, version 2007p. the indicators were analyzed through descriptive statistics techniques. the phenotypic grouping of the arracacha and yacón variability was performed by means of a hierarchical conglomerate analysis (ac) for qualitative data, using the ward algorithm ignacio, s.; camarena, f.; baudoin, j.; blas, r. peruvian journal of agronomy 1(1):21-31 23 and the gower distance, with fashion data of the described characteristics (infostat, 2004). the phenotypic groups were corroborated with the prediction ellipses technique (95% confidence) of the discriminant analysis (da), using these groups as classification variables. the most important discriminatory characteristics within the groups were identified through the linear da method (hidalgo, 2003). the identification of the wild arracacha and yacón ecotypes was made based on the information collected in the field. figure 1. conservation communities according to the percentage of farmers who conserve between one and three morphs of arracacha (a. xanthorrhiza) and yacon (s. sonchifolius) in the districts of huacrachuco (a) and san pedro de cholón (b). identification of conservationist communities and farmers—a total of 52 people, authorities and producer leaders participated in the planning phase (38 in huacrachuco and 14 in cholón). based on the number of farmers who reported conserving between one to three morphs of yacón and arracacha, three ccs (out of a total of eight) were selected in the district of huacrachuco: marcopata, quillabamba and nuevo chavín (figure 1a) and two (of a total of four) in the district of san pedro de cholón: cholón and chogopillo (figure 1b). the community of huanchay was not considered because only two farmers participated in the planning and communities of huacrachuco and yamos because half or more participating farmers indicated not to keep the species under study in their plots. the farmers of marcopata and quillabamba asserted the existence of two climatologically differentiated zones where the wild arracacha and yacon ecotypes are developed: 1) temperate zone or low zone, located from 2,500 to 2,750 m.a.s.l. and 2) zone of puna or high zone, located to more than 3000 m.a.s.l. in the district of huacrachuco, 23 farmers reported conserving in their plots one to three arracacha morphotypes, 21 indicated the same number of yacon morphotypes and only one farmer indicated to conserve more than three yacon morphotypes (figure 2a). in the district of san pedro de cholón, nine farmers mentioned maintaining one to three arracacha morphotypes, 12 said that they conserve this same number of yacon morphotypes and only one farmer indicated that he conserves more than three arracacha varieties in his plot (figure 2b ). farmers’ information showed the situation of loss of arracacha and yacón cultivated varieties in family agroecosystems, for example, in huacrachuco 15 farmers mentioned that they do not sow arracacha morphotypes and 16 do not sow yacon; in san pedro de cholón, four farmers said that they do not sow arracacha and two do not sow yacon. for the characterization work of arracacha and yacon morphotypes, 13 agco were selected from the three selected communities in huacrachuco and nine from the two communities of san pedro de cholón. figure 2. percentage of farmers surveyed in the districts of huacrachuco (a) and san pedro de cholón (b) according to the number of arracacha (a. xanthorrhiza) and yacón (s. sonchifolius) morphotypes conserved in their plots. a b a a b ethno-botany and in-situ conservation of the genetic diversity of arracacha (arracacia xanthorrhiza bancroft), yacon (smallanthus sonchifolious h.robinson), and wild relatives junio-setiembre 2017 24 sociocultural aspect of conservationist communities—it was found that the communities have active comuneros (when they live in the localities) and passive (people who are registered but live in other localities). nuevo chavín, nueva esperanza, san cristóbal, chogopillo, san vicente and ututo have the status of a hamlet; while huanchay, marcopata, quillabamba and yamos are peasant communities (cc). a hamlet has its own authorities but they fulfill the mandates of the cc to which it belongs, a cc is made up of several villages whose maximum authority is the president, elected by democratic vote for a period of two years. in the district of huacrachuco, 22 farmers mentioned that they speak quechua but in huanchay, nueva esperanza, san cristóbal and nuevo chavín they speak quechua and spanish; in quillabamba, marcopata and yamos they speak only spanish. in the district of san pedro de cholón, only two growers mentioned that they speak quechua and are migrants from the huacrachuco district or from other localities. 20 farmers in huacrachuco and six in san pedro de cholón stated that they only have incomplete primary education, seven in huacrachuco and three in san pedro de cholón studied complete primary education, 11 in huacrachuco and five in san pedro de cholón did not have access to education and no one mentioned that he had access to secondary school. it was observed that the older the producers, the less educated they are. the main source of income for peasant families is agriculture and family upbringing. monthly monetary income was low (s /. 170.00 in huacrachuco and s /. 196.25 in san pedro de chonta), according to the poverty map prepared by foncodes (2006) the communities are in the category of poor because said income is lower to the minimum living wage. this situation hinders the conservation of arracacha and yacón germplasm because the heads of households temporarily abandon the plots to work in other paid activities. the decision on the use and use of resources is made in a general assembly and the person in charge of enforcing them are the presidents in the cc and the lieutenant governor in the hamlets. according to ruiz and lapeña (2003), the conservation and sustainable use of agrobiodiversity and its components must be at the community level. the language and level of education are related to the practices and customs of the conservation of agrobiodiversity because its use and use obey a culture of traditional knowledge and linguistics. the loss of the quechua language and the lack of access by farmers to formal education are threats to the conservation of the arracacha and yacón germplasm, despite the fact that these plant genetic resources are indispensable to ensure the food sovereignty and monetary income of rural societies ( ruiz and lapeña 2003). for pia (2005) culture can not survive for long without a basis of sustainable agriculture, an ethic of land use and the adequate use of agrobiodiversity and natural resources existing in the farms. phenotypic classification of native varieties of the arracacha and yacon the 23 arracacha samples (a. xanthorrhiza) characterized based on 21 qualitative descriptors (annex 1), were grouped into three different phenotypic groups (gf), with a cofeetic correlation value of 0.67 (figure 3a). the characteristic color of the edge of the leaflets showed no variability in the samples. the prediction ellipses corroborated the classification of the phenotypic groups identified (figure 3b). figure 3. dendogram (ward method, gower distance) (a) and prediction ellipses (b) for the phenotypic classification of 23 arracacha samples (a. xanthorrhiza) grown in the province of marañón. the gf were nominated based on the most discriminating characteristics within each group of easy b a ignacio, s.; camarena, f.; baudoin, j.; blas, r. peruvian journal of agronomy 1(1):21-31 25 observation in the plants (secondary color of the petiole of the leaves) and the name of the district of origin of the samples: green and without secondary color in the petiolescholón (gf 1), green and grayish purple-huacrachuco (gf 2), green-huacrachuco (gf 3). in the ad, the percentage of the eigenvalue associated with the first discriminant function with standardized data explained 88.25% of the variability observed for the 21 characteristics (table 1) and separated two characteristics of the leaf as the most discriminatory: secondary color of the underside and the secondary color of the petiole; secondarily, it discriminated the predominant color of the petiole, the edge of the leaflets and the secondary color of the surface of the reserve root. the misclassification rate was zero, which indicates that the 23 accessions are classified into three different gfs with high similarity within each group. the importance of these characteristics shows that the producers keep the accessions mainly by the secondary color of the surface of the reserve root. according to the color of the pulp there are four types of arracacha: white, yellow, white with purplish and yellow pigmentation with purplish pigmentation (tapia and fries, 2007). the 34 accessions of yacon (s. shonchifolius), characterized on the basis of 17 qualitative descriptors (annex 2), were grouped into four different gfs, with a cofenetic correlation of 0.62 (figure 4a). the branching of the stems and the leaf apex showed no variability. the prediction ellipses, with a confidence of 90%, clearly corroborate the classification of the phenotypic groups identified (figure 4b). the groups were nominated based on the color of the root of the reserve root and the name of the district of origin of the samples: light yellow and orange-gray huacrachuco (gf 1), whitish yellow-huacrachuco (gf 2), yellow whitish-cholón (gf 3) and amarillo claro-huacrachuco (gf 4). in the ad, the percentage of eigenvalue associated with the first discriminant function with standardized data explained 91.19% of the observed variability (table 2) and separated a characteristic of the leaf (color from the edge of the sheet of the leaf) and a of the root (color of the pulp of the reserve root) as the most discriminating; secondarily, they discriminated the number of teeth of the flower, the color of the surface of the reserve root and the color of table 1. qualitative characteristics that discriminate the three phenotypic groups of 23 arracacha samples (a. xanthorrhiza) grown in the province of marañón. qualitative characteristic discriminant axis 1 predominant foliage color 1.26 predominant color of the underside -0.97 secondary color of underside 4.24 distribution of the secondary color of the underside -1.74 predominant color of the petiole -2.68 secondary color of the petiole -4.32 distribution of the secondary color of the petiole 0.69 petiole serosity -0.29 leaflets edge -2.36 acumen of terminal leaflet -1.16 dissection of the terminal leaflet -1.13 predominant color of the pulp of the colinos 0.04 secondary color of the pulp of the colinos 0.46 distribution of the secondary color of the pulp of the colinos 0.46 predominant surface color of the reserve root 0.04 secondary color of the surface of the reserve root 2.93 distribution of the secondary color of the surface of the reserve root -1.26 form of the reserve root 1.56 predominant color of the reserve root pulp 0.68 secondary color of the reserve root pulp 0.21 distribution of the secondary color of the pulp of the reserve root 0.21 table 2: phenotypic characteristics that discriminate four phenotypic groups of the 34 yacon samples (s. sonchifolius) in the province of marañón qualitative characteristics dicriminant axis 1 predominant color of stems -0.29 secondary color of stems and their distribution -0.22 foliage color 0.38 beam film color -0.02 rib color -0.17 overlapping wings 0.00 plate shape 0.38 foil base -0.11 sheet edge 1.16 bloom habit -0.24 color of ligulate flowers -0.65 flower shape 0.17 number of flower teeth -0.83 seed production 0.10 surface color of the reserve root 0.95 color of the reserve root pulp 1.17 slit in the reserve roots 0.07 color of the propagules 0.78 ethno-botany and in-situ conservation of the genetic diversity of arracacha (arracacia xanthorrhiza bancroft), yacon (smallanthus sonchifolious h.robinson), and wild relatives junio-setiembre 2017 26 the propagules. the misclassification rate was zero, which indicates that within the four different gfs there is a high similarity within each one. the gf classification of this species is more related to the classification of the producers, who classify by the color of the reserve roots. figure 4. dendogram (ward method, gower distance) and prediction ellipses for the phenotypic classification of 34 yacon samples (s. sonchifolius) grown in the province of marañón. mansilla et al. (2006), based on a molecular study, found greater diversity of yacones in the accessions from the central zone of peru. seminar et al. (2003) report, for the northern zone (cajamarca, amazonas, lambayeque, piura and la libertad), seven yacon morphotypes, four of them of greater distribution and cultivation: purple (distinguished by the pulp color of the root), yellow and white (distinguished by the pulp color of the root), and purple green (distinguished by the color of the foliage). tapia and fries (2007) also report eight morphotypes, cultivated in the north of peru, whose differential characteristics are the external color of the root, main and secondary color of the pulp, color of the stem, hue in the color of the flower, form of the leaf, shoot color, branch of the stem and vegetative cycle. the majority of qualitative characteristics showed variation within the samples, which indicates the existence of different morphotypes in the native botanical varieties of a. xanthorrhiza and s. sonchifolius, because these characteristics are little affected by the environment (vásquez et al., 2004). description of the wild arracacha and yacon ecotypes— the farmers of huacrachuco distinguish two ecotypes of wild arracacha: tempera and puna. two tempering samples were described, whose populations develop in the lower part of the communities of marcopata and quillabamba, of 2,500 m.a.s.l. at 2750 m.a.s.l., on the right bank of the rio grande de huacrachuco; they inhabit in pircas, grasslands, forests, dry hillsides; the soils are light brown and with abundant stoniness. it has the following morphological characteristics: stems higher than 1.5 m. of height, smaller leaves than of the cultivated varieties, distribution of the branches are similar to these, internodes of the stem more elongated, reserve roots thinner and smaller than of the cultivated ones (figure 5a); after the physiological maturity all the foliage dries and only the roots and colines are buried for the regeneration of the new plants. according to the habitats described by blas et al. (2008a, b) for the arracachas in peru, it would belong to the species a. incisa. another sample of puna arracacha was described, whose population develops in the pishtaj ravine (upper part of the community of marcopata) at 3530 m.a.s.l. it inhabits within an abundant population of pajonales (ichu sp.); the soil presents a high slope, open texture and dark coloration (figure 5b). the plants reach heights between 0.60 m-0.90 m, the reserve roots are thicker in relation to the tempering ecotype; the branching is more leafy, they have internodes and smaller leaves in relation to the cultivated varieties. according to the habitat described by blas et al. (2008a, b) and the morphological characteristics that it presents would also be a species of a. incise. according to the producers, the wild yacon accessions evaluated were also classified into two ecotypes: yellow and red. the yellow ecotype (figure 6b) develops in the forests of the llapshagaga creek (nuevo chavín) and has the following characteristics: plant height between 0.50 m. more than 1 m., inflorescence chapters smaller than the cultivated varieties, they do not present ramifications, the leaves are horned and smaller than the cultivated ones, small mature roots (average of 9 cm long and 3.5 cm. diameter) and excrete white latex; at the end of the vegetative cycle, the entire leaf part is dried and only the reserve roots and the structures of the regeneration strains are buried. this ecotype also develops in the pishtaj stream b a ignacio, s.; camarena, f.; baudoin, j.; blas, r. peruvian journal of agronomy 1(1):21-31 27 (marcopata). according to the characteristics described by grau and rhea (1997) for wild yacon species, this ecotype would belong to the species smallanthus jelksii. the red ecotype (figure 6a) develops on the slopes adjacent to the huacrachuco-marcopata road; the plants present red flowers, average height of the stem of 35 cm., beam of the pubescent leaves, smaller leaves than of the cultivated varieties, the reserve roots measure 6 cm. of length and 1 cm. in diameter on average. the arracacha and yacón ecotypes show morphological differences due to the adaptation conditions to different habitats (tempera or low zone and puna or high zone) or speciation differences. in the district of san pedro de chonta there were no arracachas or wild yacon; the probable loss of this genetic material can be due to two factors: 1) the degradation of natural habitats due to anthropogenic interventions resulting from agricultural activity and the extraction of wood and firewood from low forests, 2) biophysical barriers that determine the adaptation to particular ecological floors. figure 5. wild arracacha arracacha, grandfather arracacha of tempera in their natural habitat (a. incisa) (a) and arracacha of puna grandfather reproduced in pot (a. incisa) (b). figure 6. figure 6: yacon wild ecotypes: sacha yacón rojo (s.i.) (a) and sacha yacon amarillo (s. jelksii) (b). indicators of in situ conservation of arracacha, yacon and their wild relatives-six in situ conservation indicators were described for arracacha and yacon: agroclimatic conditions of conservation communities, climatic conditions of wild ecotype habitats, traditional practices of conservation, biotic factors, traditional classification of the phenotypic variability of the arracacha and yacon cultivated, and commercialization of the roots of the arracacha and the yacon. the communities of the huacrachuco district are located in the montano sub tropical life zone and the communities of the san pedro de cholón district in the montano cálido life zone. altitude, ecological levels, life zones, temperature variation and relative humidity are biophysical variables that influence the morphotype variability of arracacha and yacon. the agro ecosystems of these crops are located from 2500 m.a.s.l. up to more than 3000 m.a.s.l., in ecologic levels quechua semárida (communities of the district of huacrachuco) and quechua semihúmeda (communities of the district of san pedro de cholón) (tapia and fries, 2007). the wild ecotypes are developed in the quechua and suni flats, in natural ecosystems (slopes with thickets, grasslands, pyrrhus, forests, etc.) where the pressures of natural selection are present. in the low zones or tempering of the communities of marcopata and quillabamba the wild ecotypes of the arracacha are developed and in the high areas or puna both ecotypes are wild. b a b a ethno-botany and in-situ conservation of the genetic diversity of arracacha (arracacia xanthorrhiza bancroft), yacon (smallanthus sonchifolious h.robinson), and wild relatives junio-setiembre 2017 28 local practices related to the conservation of arracacha and yacon morphotypes are: i) seed management, the arracacha seed colonies are resting for a week under shade or exposed to the sun, then before sowing, women perform a cut in v in the base of the seed to stimulate the rooted, a better development of the plant and the production of roots reservantes of good quality. the producers of lauricocha (ayacucho), pariahuanca (junín) and santa teresa (cusco) make a round cut at the base of the seed, in addition to four types of cuts: round, triangle, oblique round and capshaped (calua, 2006). in the case of yacon, the parent or daughter strains are rested in shade for one or two weeks before isolating the propágalos, some farmers leave the strains buried or discovered at the harvesting site until planting. the farmers of lauricocha (ayacucho) place the selected hives in the shade of the fruit trees or bury them in the farm, in order to avoid dehydration (calua, 2006); ii) land preparation, are similar for arracacha and yacon; in huacrachuco they use barretillas for the fallow and pillories for the desterronado (or pestle); in cholón and nuevo chavín, some make the possession, which consists of building pools of 20x20x20 cm. using the barretilla, the same day of the sowing; iii) sowing and management of plantations, sowing is done using peaks and lampas. the arracacha corms or the yacon strains are divided into propagules, before sowing. these crops are usually planted on the edges of fields of corn, potatoes, etc., in some cases in small family gardens, around the house and rarely in monoculture. in the case of the arracacha, this practice would be related to the demands of the crop, which according to tapia and fries, (2007) prefers low to medium soils in n and high in p and k; iv) weeding and hilling, the weeding is carried out according to the presence of the weeds in the agros ecosystems and the hilling is carried out once or twice during the campaign; v) harvest, the time that lasts from sowing to harvest varies according to the agroclimatic conditions of the communities. in huacrachuco, the arracacha and yacon harvest takes place 10-12 months after sowing and in san pedro de chonta the arracacha is harvested from six months to 10 months after sowing; yacon is usually harvested at eight months. the main harvesting practices of the arracacha and the yacón are the removal of the plants with barretilla or the cutting of the foliage prior to harvesting. the biotic factors that influence the evolution of morphotypes and ecotypes of arracacha and yacon are pests and diseases, although their incidence is low in huacrachuco and san pedro de chonta. the most frequent pests that attack the arracacha are the plant cutters or shiuri (agrotis sp.) and the fly miner (s. i.); in cajamarca and piura the main pests of the arracacha are the white worm (s. i.), the chanso (bothinus maimon), the cuso (a white larva-s.i.) and the aphid (myzus sp.); among the diseases, the leaf spot and the rotting of the reserve roots stand out (seminario, 2004). the potential pests of yacon are the plant cutters or shiuri (agrotis sp.), the diabrotica (diabrotica sp.) and the slug (salix sp.) that attacks leaves and roots in the district of hucrachuco. the potential importance of yacon pests may be due to the existence of two defense systems presented by plants (tapia and fries, 2007): abundance of hairs on the surface of the leaves that are barriers for insect access and the production of toxic for insects by the glands of the base of the leaves. the traditional classification of arracacha and yacon variability is based on the color of the flesh, flavor and color of the skin of the reserve roots. in case of yacon, the farmers relate the color of the skin and the pulp of the reserve roots with the archetype of the plant and the coloration of the stems; for example, the varieties whose reserve roots are purple pulp have a higher leaf volume, are more leafy and the leaves are darker in relation to white varieties; in case of the arracacha also relate the coloration of the pulp and the skin of the storage roots with the coloring of the young leaves and the production, for example, the dark green color of the young leaves is related to the purple color of the pulp, the yellow varieties are more productive, the yellow and white varieties are less astringent with respect to the purple ones. holle (2006) points out that peasant knowledge must be incorporated into conventional descriptors, when the assumption that the characters have a certain heritability is met, for example, a high correspondence between the common name and the morphotypes found for 25 samples of goose evidence relevance of peasant knowledge. to the wild ecotypes of the arracacha and yacon, the farmers of the different communities generally classify by the color of the flower, the use they give it and the habitat where they develop. the arracacha and yacon reserve roots are generally used for human consumption, 25% of respondents indicated that they give to the neighbor and 30% make barter. in times of harvest, families consume arracacha four to five times a week on average, in different forms: parboiled in place of potatoes or cassava, in soups in the form of slices. for the yacon, only 20% of the producers mentioned selling the reserve roots in the local market and only in times of harvest do families consume up to five times per week. ethnobotanical indicators—three ethnobotanical indicators have been described: linguistics, customs of use and exploitation of the agrobiodiversity of the arracacha and yacon, and the contribution of these resources to the family economy. the common name used by farmers for arracacha cultivated is rich; the wild ecotypes are known as field rich because they consider that they are plants of the grandfather apus or ricahca because they believe that they were domesticated by the auquillos (pre-inca settlers); in the north of peru (cajamarca and piura), wild arracachas are known as arracacha “de zorro”, “de monte”, “de gentile”, “de cerro”, “de jalca”, purunracacha and arracachilla (seminario, 2004 ). the cultivated yacon is commonly known as llacón and the wild ecotypes as field llacón because they believe that these plants are owned by the apus, llaconcito or llaconcillo because they give a connotation of an infra-specific group in relation to the cultivated ones, for example, stems and smaller storage ignacio, s.; camarena, f.; baudoin, j.; blas, r. peruvian journal of agronomy 1(1):21-31 29 roots. most producers know the nutritional benefits of cultivated morphotypes. the nutritional value of the arracacha is given by the quality of its starch and its high content of carotene, calcium, phosphorus, iron and niacin (jiménez, 2005, amaya and julca, 2006), and by the calcium and ascorbic acid content of reserve roots superior to potatoes and iron and niacin superior to cassava (jiménez, 2005). the healing benefits of the wild ecotypes are known only by farmers called healers who use the foliar part of the mature plants to cure: i) the scare or poor field (charm of the hill or aukillo), in the communities of marcopata and quillabamba use grandfather’s grandfather combined with other herbs, such as the frame (ambrosia sp.), the chincho (tagetes elliptica), culantro de perro (coriandrum sativum); heat in a pot macerating with kerosene, alcohol or human urine until the branches become flaccid and secrete aqueous substances; then perform the rubbing or “shocpi” the naked patient and finally the remains are thrown to the hill where there is little frequency of passers-by and pay coca and cigar to the aukillos; ii) childbirth and childbirth, with the young leaves of the cultivated and / or wild arracacha an infusion is prepared and then the patient drinks as tea water; the pucha or puchita is also prepared by rubbing the leaves between the fingers until they become flaccid and then a rub is performed to locate the patient on the affected or painful part; iii) cephalalgias, colds and headache, with the leaves of the cultivated or wild arracachas an infusion is prepared for a time of approximately 10 minutes, then they give the patient to drink; the treatment is repeated for three or four days. tapia and fries (2007), indicate that the cultivated arracacha is used as an infusion to calm stomach pains, regulate menstrual disorders and facilitate recovery after delivery. another form of use is rubbing the leaves with your hands until they take a yellowish color and then with them rub the body. from the cultivated yacon the reserve roots are consumed in the form of fresh, to quench the thirst after the days of work in the farm, as revitalizing in the long walks (12 hours or more) but this practice has been lost in recent years. the roots of the wild yellow ecotype are used as fresh by the shepherd children and for the feeding of pigs by grazing in the open field, the leaves are used to feed guinea pigs; 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oscar ortiz-oblitas2 *corresponding author: jspacheco@uce.edu.ec abstract the objective of this research was to determine the consequences of the adoption of agroecological production systems in the parish of toacaso in terms of social, environmental, economic, and general sustainability. considering the economic, social, cultural, and environmental dimensions, the methodology proposed by sarandón (2002) was used to determine the indicators. the methodology proposed by ortiz and pradel (2009) was adopted in the evaluation of impacts in integrated pest management programs. surveys with questions related to the social, economic, and environmental consequences were conducted on 44 agroecological farmers and 44 conventional producers in the parish of toacaso. additionally, a sample of 44 conventional producers from the parish of mulaló was identified as “control” treatment, which allowed to perform a comparison with and without the adoption of agroecological practices. the 27.27% of the 44 productive units that implemented the agroecological production system achieved general sustainability, the average general sustainability index was 2.16, where 86.36% achieved environmental sustainability, 47.72% economic sustainability, and 47.73% social sustainability. keywords: sustainability, environmental sustainability, economic sustainability, social sustainability, adoption, agroecology. resumen el objetivo de esta investigación fue determinar las consecuencias de la adopción en términos de sustentabilidad social, ambiental, económica y general de sistemas de producción agroecológicos de la parroquia de toacaso. para determinar los indicadores se utilizó la metodología propuesta por sarandón (2002), considerando las dimensiones económica, social, cultural y ambiental y se adaptó la metodología propuesta por ortiz y pradel (2009) en la evaluación de impactos en programas de manejo integrado de plagas, se realizó encuestas con preguntas alusivas a las consecuencias sociales, económicas y ambientales, dirigida a 44 agricultores agroecológicos y 44 a productores convencionales en la parroquia toacaso. adicionalmente, se identificó como tratamiento “control” 1 universidad central del ecuador, av. universitaria, quito 170129, ecuador 2 universidad nacional agraria la molina, av. la molina s/n, lima-perú how to cite this article: pacheco-jiménez, j., ortiz-oblitas, o. (2022). sustainability of agroecological farms in toacaso, cotopaxi-ecuador. peruvian journal of agronomy, 6(1), 103-113. https://doi.org/10.21704/pja.v6i1.1766 sustainability of agroecological farms in toacaso, cotopaxi-ecuador january april 2022 104 a una muestra de 44 productores convencionales tomados en la parroquia mulaló, lo que permitió realizar una comparación con y sin adopción de prácticas agroecológicas. de las 44 unidades productivas que implementaron el sistema de producción agroecológica el 27,27% alcanzaron sustentabilidad general, el índice promedio de sustentabilidad general fue de 2,16, 86,36% alcanzo sustentabilidad ambiental, el 47,72% sustentabilidad económica, y el 47,73% tienen sustentabilidad social. palabras clave: sustentabilidad, sustentabilidad ambiental, sustentabilidad económica, sustentabilidad social, adopción, agroecología. introduction the current challenge of agriculture is to produce enough food for a growing population, which is estimated to reach 9.1 billion people by 2050 (pérez et al. 2018). according to the projections of the instituto nacional de estadística y censos [inec] (2010) ecuador will reach 23.4 million inhabitants. the challenge is even greater because more nutritious, healthy and innocuous food needs to be produced (hunter et al. 2017) to ensure the food safety of people; while conventional agriculture has resulted in increased food production, it has also generated environmental problems due to the inappropriate use of agrochemicals. for this reason, sustainable agriculture is proposed to achieve these objectives with minimal environmental impact, according to (pérez et al. 2018) it is necessary not only to produce more food but also to ensure sufficient resources such as clean water, agricultural land, energy, and labor. agroecology is a holistic approach based on the practice of ecological principles that promote the efficient use of energy to produce food with little dependence on external inputs, in diverse and socially equitable agro-systems (altieri 2018; gliessman 1998) this form of production is an alternative for family farming, traditionally devalued (heifer foundation, 2014) and ignored by public policies that favor commercial agriculture (export, industry, and markets) (idrovo, 2016). the consequences of intensive production, with high dependence on external inputs, have been evidenced in the deterioration of the production systems of farmers. although agrochemicals have supported food production, they have also had negative effects on the environment and human health, it is estimated that every year around 25 million farmers worldwide have involuntary pesticide poisoning (carvalho, 2017). and that some 1.8 billion farmers worldwide use pesticides to protect their crops, the mechanisms of action of pesticides are not limited to pests, but also have negative effects on biodiversity, ecosystems, and health (carvalho, 2017). mclaughlin et al. (2014) report case studies on the association between breast cancer and agriculture, and the relationship between pesticide exposure and miscarriage. zúñiga et al. (2021) report the evidence generated from the study of seven agricultural regions of chile on the exposure to pesticides in children, the general population, and agricultural workers, with negative effects on cognitive functioning, nervous system, reproductive system, genotoxic and carcinogenic. on the other hand, meeker & boas (2011) remark evidence of impaired thyroid function with exposure to pesticides. similarly, everett & matheson (2019) notes the association of herbicide and insecticide use with gestational diabetes in women exposed to agriculture. idrovo (2016) reports an increase in the import of fertilizers and pesticides; in ecuador increased by 69% in the period 2005 to 2015 with an increasing trend to industrialization and agricultural exports indicating also greater investment of production units. naranjo (2017) mentions that in ecuador crops such as corn, potato and tomato are among the crops with the highest use of pesticides, in the same way, the case of vegetables whose seeds are imported. in addition, it is common practice among farmers to apply higher amounts of pesticides when insect resistance to certain compounds increases, usually by increasing the frequency of application or using products with more toxic active ingredients. given the high risk of intensive agriculture using external resources, and the existence of pacheco-jiménez, j., ortiz-oblitas, o. peruvian journal of agronomy 6(1): 103-113 (2022) https://doi.org/10.21704/pja.v6i1.1766 105 experiences of adoption of agro-ecological farming; it is necessary to study the consequences of the adoption of agro-ecological methods in terms of economic and general environmental sustainability. this analysis will allow drawing lessons on how to solve the problem of family agriculture in ecuador and contribute to mitigating the impacts of conventional agriculture on the environment and the health of producers and consumers. the objective of this research was to determine the consequences of the adoption of agroecological methods for the cultivation of vegetables in toacaso in terms of economic, social, and environmental sustainability. materials and methods methodology this study was conducted in the andean region of ecuador, in toacaso parish located in the canton latacunga, cotopaxi province. three stages of adoption of agro-ecological practices were identified in farmers: a consolidated stage that shows the adoption of efficient practices in the management of soil, water, crops, animals, and commercialization, which have between 13 and 22 years of having adopted agroecological practices; the second stage of transition in which farmers who adopted this production system after 13 years and the initial stage to which belong those farmers who have taken more than 20 years to adopt this production system. the framework for the evaluation of natural resource management systems incorporating sustainability indicators (mesmis) was used to determine the consequences of adoption in terms of, social, economic, and environmental sustainability. the indicators were determined using the methodology proposed by sarandón (2002), considering the economic, social, cultural, and environmental dimensions. the methodology proposed by ortiz and pradel (2009) was adopted in the evaluation of impacts in integrated pest management (ipm) programs, using surveys with questions that investigate the social, economic, and environmental consequences. the mesmis methodology was applied in 7 stages: 1) characterization of productive systems through surveys on farmers, 2) identify critical points of production systems of the environmental, economic, social, and technical types that can affect the stability of the productive system, 3) build indicators, 4) measure each indicator, 5) obtain the index of economic, social, environmental sustainability, 6) analysis of results 7) comparison of results of agroecological production system with conventional production systems. (masera et al., 2000) the questionnaire addressed 44 agroecological producers and 44 conventional producers (nonagroecological) in the parish of toacaso was used as an instrument. additionally, a sample of 44 conventional producers (non-agroecological) taken in the parish of mulaló, which has similar agroecological conditions to the parish of toacaso, was identified as a “control” treatment. the indicators to determine the social, environmental, and economic sustainability index were constructed using the methodology proposed by sarandón (2002). the data were standardized by converting to a scale from 0 to 4, with 4 being the highest value of sustainability and 0 being the lowest value. some indicators were estimated by multiplying the value of the scale by a coefficient in function of the importance of each variable regarding sustainability, as observed in the formulas used to obtain the index of social, environmental, and economic sustainability, respectively. formula to determine the sociocultural sustainability index: . [1] sustainability of agroecological farms in toacaso, cotopaxi-ecuador january april 2022 106 formula to determine the environmental sustainability index: [2] formula to determine the economic sustainability index: [3] general sustainability index to determine the general sustainability index (gen si), economic (ki), environmental (ei) and sociocultural (sci) indicators were used. a productive system is sustainable if the general sustainability index is greater than two (gen si ˃ 2) and if none of the three dimensions has a value less than two (sarandón, 2002). the formula for determining the overall sustainability index: gen si =(ki+ei+sci)/3 [4] socio-cultural, environmental and economic sub-indicators, and their evaluation are presented in the following tables. the sociocultural scale and sub-indicators for satisfying basic needs are shown in table 1, and the indicator scale for acceptability of the productive system, social integration, and ecological awareness is shown in table 2. the environmental scale and sub-indicators of soil conservation are shown in table 3, the indicator scale for risk of erosion is shown in table 4, and the indicator scale for management of biodiversity is shown in table 5. the economic indicators food self-suffiency scale and sub-indicators are shown in table 6, the indicator scale for adequate income for a family is shown in table 7, and the scale and sub-indicators of economic risk are shown in table 8. results and discussions general sustainability it was found that only 27.27% of the 44 production systems that adopted agroecological production methods achieved general sustainability, the overall average sustainability index was 2.16. general sustainability was observed in 6 productive units of the consolidated agroecological production systems corresponding to 46.15%, in 5 productive units of the production systems in the transitional stage representing 35.71%, and in 1 productive unit of the production systems in the initial stage of adoption of agroecology equivalent to 5.88%. all economic, environmental, and social indicators were higher for the consolidated table 1. scale and sub-indicators for meeting basic needs. indicator: a. meeting basic needs sub-indicators: scale a1. land tenure and type of housing a2. access to education a3. access to health a4. access to basic services 4 own brick or mixed dwelling farmers or their children have access to university public hospital electricity, water, phone, cell phone, internet 3 own block housing farmers or their children have access to technological studies public health center electricity, water, cell phone, internet 2 borrowed brick or mixed housing farmers or their children have access to high school private medical coverage electricity, water, phone or cell phone 1 borrowed block house farmers or their children have access to primary school folk healer electricity, water 0 block leased house farmers and their children have no access to education. no access no access pacheco-jiménez, j., ortiz-oblitas, o. peruvian journal of agronomy 6(1): 103-113 (2022) https://doi.org/10.21704/pja.v6i1.1766 107 table 2. indicator scale: acceptability of the productive system, social integration and ecological awareness. indicators: scale b. acceptability of the production system c. social integration d. ecological awareness and healthy eating 4 it has more healthy food and improves its nutrition, found no disadvantages. very high (4: partnerships, links with non-agricultural enterprises, links with the private sector and ngos) broad vision: environmental care and health. 3 sometimes it has more food and would like to grow larger area. high (link to 3 of 4) it reduces to no use of agrochemicals. 2 it has more food, but there are no markets and prices are low. middle (2 of 4) it perceives that it consumes healthy products that improve its nutrition. 1 it has more food, but it loses a lot (economic loss), it is not profitable, it produces less. low (link 1 of 4) it has a slight ecological knowledge (some practices) 0 there are no advantages to the production system. null no awareness of food and ecology. table 3. scale and sub-indicators of soil conservation. indicator: a. conservation of soil sub-indicators: scale a1. crop rotation with legumes a2. crop diversification a3. organic matter in soil a4. type of tillage 4 permanent rotation with legumes more than 20 species grown in association more than 6% om in soil zero tillage 3 eventual rotation with legumes 6 to 20 species cultivated in association 3 to 6% om in soil manual tillage 2 rotation with corn and potato 1 to 15 species grown in association 1 to 2.9% om in soil tillage with yunta 1 eventual rotation with other crops from 6 to 10 species grown in association less than 1% om in soil mixed tillage 0 does not rotate up to 5 cultivated species does not incorporate om mechanical tillage table 4. indicator scale: risk of erosion. indicator: b. risk of erosion sub-indicators: scale b1. pending b2. irrigation b3. use of insecticides and fungicides 4 0 to 5% drip not applicable 3 5 to 15% spraying 1 application 2 16 to 30% mixed 2 applications 1 31 to 45% gravity 3 applications 0 less than 45% rainfed 4 o more table 5. scale of indicators: management of biodiversity. indicator: c. management of biodiversity sub-indicators: scale c1. crop diversity c2. diversity of animals c3. undertakes practices of: seed production, use of living barriers, crop rotation, crop association 4 more than 20 9 or more performs all four practices 3 16 to 20 6 to 8 performs three practices 2 11 to 15 3 to 5 performs two practices 1 6 to 10 1 to 2 performs a practice 0 5 or less 0 no practice sustainability of agroecological farms in toacaso, cotopaxi-ecuador january april 2022 108 table 6. food self-sufficiency scale and sub-indicators. indicator: a. food self-sufficiency sub-indicators: scale a1. diversification of production a2. area dedicated to agro-ecological production 4 more than 20 products more than 1.5 ha 3 16 to 20 products 1 to 1.5 ha 2 11 to 15 products 0.51 to 0.99 ha 1 6 to 10 products 0.1 to 0.5 ha 0 5 or fewer products less than 0.1 ha table 7. indicator scale adequate income for family. indicator: b. adequate income for the family sub-indicators: scale b 4 meets the following conditions: a) does not work in other activities outside of the productive unit, b) expresses it has more food, c) states that the money he receives for agriculture supports its family d) has access to credit 3 complies with 3 conditions of income for the family 2 complies with 2 conditions of income for the family 1 complies with 1 condition of income for the family 0 it does not meet any sufficient income conditions for the family table 8. scale and sub-indicators of economic risk. indicator: c. economic risk sub-indicators: scale c1. diversification for sale c2. number of commercialization channels c3. dependence on external inputs for production 4 more than 20 non-value-added products, or 16 or more value-added products 5 does not use fertilizers, fungicides, insecticides or herbicides 3 16-20 non-value-added products, or 1115 value-added products 4 uses fertilizers only 2 11 to 15 non-value-added products, or 6 to 10 value-added products 3 uses pesticides 1 to 2 applications 1 to 10 non-value-added products, or 5 value-added products 2 uses pesticides 3 or 4 applications 0 5 or less non-value-added products, or less than 5 value-added products 1 uses pesticides more than 4 applications stage, followed by the transition stage and finally the initial stage of adoption, a trend that was maintained in the general sustainability index where the systems in the consolidated stage had an average gen si of 2.49, in the systems in transition stage the gen si was 2.12 and, in the systems, in the initial stage of adoption the gen si was 1.87. this can be seen in table 9, table 10, and figure 1. among the factors that affected sustainability are the diversification of production, the area designated for agro-ecological production, diversification for sale, and the number of commercialization channels that affected the economic index, others like the diversity of associated crops, the type of tillage, and the diversity of cultivated species and animals affected the environmental index, access to education and the acceptability of the production system, social interaction, knowledge, and ecological awareness affected the sociocultural index. in the conventional productive systems of toacaso, it is observed that, on average, the social sustainability index was 1.65 and in mulaló 1.40, the sociocultural factors were determinants for this result: access to education, basic services, acceptability of the productive system, social integration and knowledge and ecological awareness. for the agroecological system was 2.06. this can be seen in table 11 and figure 2. the environmental sustainability index reached an average value of 1.63 in the conventional systems of toacaso and 1.36 in pacheco-jiménez, j., ortiz-oblitas, o. peruvian journal of agronomy 6(1): 103-113 (2022) https://doi.org/10.21704/pja.v6i1.1766 109 table 9. gen si agroecological production systems in consolidated, initial and transitional stage (ki: economic sustainability index; ei: environmental sustainability index; sci: sociocultural sustainability index; si: overall sustainability index) stage of adoption ki ei sci gen si consolidated 2.32 2.69 2.45 2.49 transition 1.90 2.41 2.05 2.12 initial 1.65 2.26 1.69 1.87 average 1.96 2.45 2.06 2.16 table 10. general sustainability of agroecological production systems by adoption stages (ki. index of economic sustainability, ei. environmental sustainability index, sci. sociocultural sustainability index, gen si. general sustainability index). stage of adoption value ki ei sci gen si sustainability consolidated ˃ a 2 69.23 % 84.61 % 18.18 % 46.15 % yes ˂ a 2 30.77 % 15.39 % 81.82 % 53.85 % no transition ˃ a 2 64.28 % 100 % 22.72 % 35.71 % yes ˂ a 2 35.72 % 0.00 % 77.28 % 64.29 % no initial ˃ a 2 17.64 % 76.47 % 6.81 % 5.88 % yes ˂ a 2 82.36 % 23.53 % 93.19 % 94.12 % no table 11. gen si of the agroecological and conventional production systems of toacaso and mulaló. production system ki ei sci gen si agroecological toacaso 1.96 2.45 2.06 2.16 conventional toacaso 1.14 1.63 1.65 1.475 mulalo 1.02 1.36 1.40 1.259 (ki. index of economic sustainability, ei. environmental sustainability index, sci. sociocultural sustainability index, gen si. general sustainability index) figure 1. overall sustainability of agro-ecological production systems by adoption stages sustainability of agroecological farms in toacaso, cotopaxi-ecuador january april 2022 110 mulaló, in this case, crop rotation, crop diversity, organic matter in the soil, type of tillage, diversity of cultivated species, and animal diversity were found as determining factors, in the case of mulaló in addition to those aforementioned; water management and use of practices that favor biodiversity were added. in the agroecological system, the average environmental sustainability index was 2.45 (table 11 and figure 2). studies conducted by sanjinez (2019) in rice crop report as factors that affected the environmental index the lack of crop rotation, lack of crop diversification, and poor management of biodiversity, anzules (2019) mentions among other limiting factors of this indicator in the cocoa crop, crop diversity, and biodiversity, coaquira (2020) states that among the determining factors in environmental sustainability in potato crop were the use of machinery and the irrigation system. the average value of the economic sustainability index was 1.14 for the conventional system of toacaso, 1.02 for mulaló, and 1.96 for the agroecological system of toacaso. these values were influenced by the diversity of production, the area used for cultivation, sufficient income for the family, diversification for sale, and the number of commercialization channels (table 11 and figure 2). other studies such as marquez (2015) in coffee cultivation found diversity for sale as a determining factor figure 2. gen si of the agroecological and conventional production systems of toacaso and mulaló. scale: 0= lower sustainability value, 4= higher sustainability value of the economic index, aliaga (2019) in chili supano cultivation found diversification of sales as determining factor, anzules (2019) in cocoa cultivation found a diversity of production as limiting factor. overall sustainability analysis of the production systems found that 46.15% of the farms which adopted agroecological production are sustainable, compared to 9.09% of the conventional farms of toacaso and no farms of mulaló, as shown in table 12 and figure 3. similar values were obtained by anzules (2019) with 48% of sustainable farms assessing the sustainability of cocoa in santo domingo de los tsáchilas, ecuador, and marquez (2015) with 4.92% sustainable conventional farms and 39.34% sustainable organic farms in the coffee study in cusco, peru. conclusions the largest number of productive units that achieved general sustainability, out of the three surveyed production systems, was presented in those that have implemented agroecological practices, presenting greater sustainability in the farms in the consolidated stage followed by those in transition and initial stage respectively. environmental determinants of sustainability pacheco-jiménez, j., ortiz-oblitas, o. peruvian journal of agronomy 6(1): 103-113 (2022) https://doi.org/10.21704/pja.v6i1.1766 111 table 12. sustainability of agro-ecological and conventional production systems in toacaso and mulaló. production system value ki ei sci gen si sustainability agroecological toacazo ˃ a 2 69.23 84.61 47.73 46.15 yes ˂ a 2 30.77 15.39 52.27 53.85 no conventional toacazo ˃ a 2 2.32 18.18 22.73 9.09 yes ˂ a 2 97.73 81.82 77.27 90.91 no mulalo ˃ a 2 2.27 0 6.82 0 yes ˂ a 2 97.73 100 93.18 100 no (ki. index of economic sustainability, ei. environmental sustainability index, sci. sociocultural sustainability index, gen si. general sustainability index) figure 3. sustainability of the agroecological and conventional production systems of toacaso and mulaló. were crop rotation, crop diversity, soil organic matter, type of tillage, diversity of cultivated species, and animal diversity, in the case of mulaló; in addition to the aforementioned, water management and use of practices that favor biodiversity. the sociocultural factors that influenced sustainability were added: access to education, basic services, acceptability of the productive system, social integration and knowledge, and ecological awareness. the economic factors that influenced sustainability were the diversity of production, the area allocated for cultivation, sufficient income for the family, diversification for sale, and the number of commercialization channels. the adoption of agroecological systems has generated positive environmental, sociocultural, and economic consequences, their strengthening and planning will favor sustainable development in the toacaso parish. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. sustainability of agroecological farms in toacaso, cotopaxi-ecuador january april 2022 112 orcid and e-mail j. pacheco-jiménez jspacheco@uce.edu.ec https://orcid.org/0000-0002-2158-8523 o. ortiz-oblitas o.ortiz@cgiar.org https://orcid.org/0000-0001-8941-2957 references aliaga, j. 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(2002). la agricultura como actividad transformadora del ambiente. el impacto de la agricultura intensiva de la revolución verde. in s. sarandón (ed.), agroecología: el camino hacia una agricultura sustentable. ediciones científicas americanas, la plata, argentina. sanjinez, f. (2019). sustentabilidad del agroecosistema del cultivo de arroz (oryza sativa l.) en tumbes, perú. [doctoris philosophiae thesis, universidad nacional agraria la molina]. unalm repository. https://repositorio.lamolina.edu.pe/ handle/20.500.12996/4083 zúñiga, l., zúñiga, z., saracini, c., pancetti, f., teresa, m., muñoz, m., lucero, b., foerster, c., & cortés, s. (2021). exposición a plaguicidas en chile y salud poblacional: urgencia para la toma de decisiones. gaceta sanitaria, 35(5), 480–487. https://doi. org/10.1016/j.gaceta.2020.04.020 404 not found peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index review article https://doi.org/10.21704/pja.v6i2.1767 received for publication: 30 january 2021 accepted for publication: 28 august 2022 published: 30 august 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by use of biorational insecticides for the management of storage insect pests: a review uso de insecticidas biorracionales para el manejo de plagas de insectos de almacenamiento: una revisión sovit parajuli1*, aavash adhikari1, sandip paudel1, dipesh oli1, sagar bhandari2, jiban shrestha3 *corresponding author: parajulisovit52@gmail.com *https://orcid.org/0000-0001-8068-7487 abstract pests of various species cause havoc on storage grains, resulting in both qualitative and quantitative grain losses. insect pests feed stored grains and reduce the weight, nutritional content, and germination of these grains. contamination, odor, mold, and heat damage are also caused by infestations, decreasing the grain’s quality and rendering it unfit for human or animal consumption. commercial grain buyers might choose between refusing to accept insect-infested grain or paying a reduced price for it. various pest management practices have been tried. the emergence of insecticidal and fungicidal resistance, as well as damage to nontarget organisms and acute and chronic effects on humans and the environment have necessitated the use of biorational methods over chemical control of storage-product pests. the term biorational refers to several products that are relatively non-toxic and have few environmental adverse effects. biorational techniques, such as the use of microbials, pheromones, and food attractants, natural enemies, botanicals, and biological control, were used as alternatives to chemical pesticides for suppressing and controlling storage-product pests. to ensure food security and agricultural sustainability, the use of such biorational chemicals is unavoidable. keywords: grain infestation, microbials, pheromones, botanicals, natural enemies resumen las plagas de varias especies causan estragos en los granos almacenados, lo que resulta en pérdidas de granos tanto cualitativas como cuantitativas. las plagas de insectos alimentan los granos almacenados y reducen el peso, el contenido nutricional y la germinación de estos granos. la contaminación, el olor, el moho y el daño por calor también son causados por infestaciones, lo que disminuye la calidad del grano y lo vuelve inadecuado para el consumo humano o animal. los compradores comerciales de granos pueden elegir entre negarse a aceptar granos infestados de insectos o pagar un precio reducido por ellos. se han probado varias prácticas de manejo de plagas. la aparición de resistencia a insecticidas y fungicidas, así como el daño a organismos no objetivo y los efectos agudos y crónicos en humanos y el medio ambiente han requerido el uso de métodos biorracionales sobre el control químico de plagas de productos almacenados. el término biorracional se refiere a varios productos que son relativamente no tóxicos y tienen pocos efectos ambientales 1 faculty of agriculture, agriculture and forestry university (afu), rampur, chitwan, nepal 2 institute of agriculture and animal science, campus of live sciences, dang, nepal 3 nepal agricultural research council, national plant breeding and genetics research centre, khumaltar, lalitpur, nepal how to cite this article: parajuli, s., adhikari, a., paudel, s., oli, d., bhandari, s., & shrestha, j. (2022). use of biorational insecticides for the management of storage insect pests: a review. peruvian journal of agronomy, 6(2), 132-146. https://doi.org/10.21704/ pja.v6i2.1767 parajuli, s., adhikari, a., paudel, s., oli, d., bhandari, s., & shrestha, j. peruvian journal of agronomy 6(2): 132-146 (2022) https://doi.org/10.21704/pja.v6i2.1767 133 adversos. las técnicas biorracionales, como el uso de microbios, feromonas y atrayentes alimentarios, enemigos naturales, productos botánicos y control biológico, se utilizaron como alternativas a los plaguicidas químicos para suprimir y controlar las plagas de productos almacenados. para garantizar la seguridad alimentaria y la sostenibilidad agrícola, el uso de estos productos químicos biorracionales es inevitable. palabras clave: infestación de granos, microbios, feromonas, botánicos, enemigos naturales introduction agro-based enterprises support the most of the world’s population, both directly and indirectly. pests attack crops at all stages of development, from early vegetative to flowering, fruiting, and harvest. even after harvest, a large range of pest groups, including bacteria, fungus, insects, rodents, and birds, continues to damage these commodities. storage pests are pests that cause damage to stored commodities, lowering their nutritional and economic worth, and more than 20,000 species of field and storage pests harm around one-third of worldwide food production worth more than $100 billion per year (shukla & toke, 2013). according to yankanchi et al. (2014), tropical storage-grain insects cause 2030% qualitative and quantitative grain loss, while temperate storage-grain insects cause 5-10%. several of pest management methods have been tried, with some issues regarding their effectiveness and effects rose each time. in comparison to other pest categories, cereals have a greater interaction with insect pests. chemical insecticides have been used to control a number of stored products (arthur & rogers, 2003), but due to scale limitations or lack of appropriate chemical control, interest in biological control techniques is growing, for instance, the parasitoid beetles are used to manage stored product beetles (schöller et al., 2006). on a commercial basis, several biological pest management techniques are available and the biocontrol measures used are in stored grains and goods. storage-product pests have caused damage to stored products, not only by destroying the product and rendering it unfit for consumption but also by causing a significant economic loss. according to hagstrum & flinn (1995), such insect pests can have a significant economic impact on stored grains and processed products. these insects do not require a significant amount of food, since a food accumulation on cracks and crevices, inside machinery, and under the floor is sufficient (campbell et al., 2004). it is also challenging for humans to clean and handle such waste. chemical pesticides can serve as a check, but they are not a long-term solution because various insect pests may develop resistance to them (subramanyam & hagstrum, 1995). pesticide use in agriculture has caused health issues, environmental issues, yield loss owing to non-target chemical application, which has resulted in pesticide-induced pest resurgence, and finally a financial load on farmers (koirala et al., 2009). farmers are affected by headache, eye burning, skin irritation, teary eyes, weakness, and other ailments and discomforts because of pesticide application (atreya, 2012). pesticide exposure also causes acute diseases such headaches, skin irritation, respiratory and throat discomfort (yassin, 2002). long-term pesticide exposure causes major health problems like cancer, endocrine disruption, and neurological consequences (reigart & roberts, 1999). similarly, long-term low-dose pesticide exposure has been associated with health problems such as immune suppression, hormone disruption, decreased memory, reproductive abnormalities, and cancer (gupta, 2004). storage-grain pests cause economic losses as well as a reduction in the nutritional value of stored products, rendering them unfit for consumption (padin et al., 2002). pest-related losses have been estimated to reach around 9% in developed countries and up to 20% in developing countries (phillips & throne, 2010). developed countries have various infrastructures, a scientific environment for evaluation, and systematic policies in place to deal with the damage that such pests can cause. for pests of stored products, biological control measures may be more effective than chemical pesticides because they can be hidden inside the storage room behind sacs, machinery, holes, and other places difficult to access, and chemical control is difficult to implement. many biological control techniques use of biorational insecticides for the management of storage insect pests: a review may august 2022 134 have been used to control stored bulk grains. the ability of parasitic wasps such as theocolax elegans and anisopteromalus calandrae to effectively suppress and control various pest populations in bulk grain storage is one example (scholler & flinn, 2000). species such as beauveria bassiana, nosema spp. vuillemin, mattesia spp., and metarhizium anisopliae have been examined and evaluated. however, only limited testing of such organisms has been done (brower et al., 1995). similarly, the bacterium bacillus thuringiensis (bt.) has been employed as a grainprotectant in the united states, and it has been used to suppress indian meal moth larvae in india (brower et al., 1995). stored grain pests face similar issues from lepidopteran pests, which have been targeted with different biocontrol agents. the bacterium b. thuringiensis is useful in controlling lepidopteran larvae that have been attacking stored grains, but resistance has also been reported (mcgaughey & beeman, 1988). granulosis virus, which is available on a commercial scale in the market, has been used to control the moth on stored products such as nuts and fruits, particularly dry fruits (vail, 1991). physical control (inert dust, ionizing radiation, light and sound), thermal control (low temperature control, high temperature disinfection), ionization, and fumigation are the methods used to control storage-grain pests in addition to bio control approaches. in north america and africa, inert dusts have been proven to be efficient in controlling various storage insects (fields & muir, 1996). calcium was added to diatomaceous earth (de), which is made up of the fossilized remains of diatoms. using ionizing radiation to control storage-grain pests can be an effective and environmentally benign method. low temperatures also reduce feeding behavior and fertility, as well as storage pest survival (longstaf & evans, 1983). phosphine and methyl bromide are two typical fumigants used for storage pest management, according to rajendran & sriranjini (2008). poor countries are still falling behind due to a lack of awareness and concern about the storage grain pests and the extent of their damage. synthetic insecticides and fumigants are important chemical control strategies for stored-grain insects (zettler & arthur, 2000; benhalima et al., 2004). however, the usage of such chemical pesticides harms consumers’ health and the environment while also increasing insecticidal resistance. the use of biological approaches poses little to no risk to the environment (edde, 2012). the objective of this review paper was to gather information on biorational insecticides for managing the storage insect pests. storage pests major insect pests of stored grains include lesser grain borer (rhyzopertha dominica), rice weevil (sitophilus oryzae), khapra beetle (trogoderma granarium), rust red flour beetle (tribolium castaneum), long headed flour beetle (latheticus oryzae), saw toothed beetle (oryzaephilus surinamensis), rice moth (corcyra cephalonica), almond moth (cadra cautella), angoumois grain moth (sitotroga cerealella), pulse beetles (callosobruchus chinensis, c. maculates, c. analis) (ahmad et al., 2021). the non-insect pests include rodents, mites and fungi. aspergillus and penicillium are common fungi that cause damage to crops in both the storage and field. according to the hypothesis, during storage and microbial growth, the microbes use the carbohydrate and oil deposits, causing a loss of those elements and negatively affecting germination capacity (mohapatra et al., 2017). fungi can damage grain quality in various ways. according to (multon, 1988), such fungi are responsible for lowering the baking quality of wheat grain. also, fungi produce the toxic metabolites, known as mycotoxins, such as aflatoxins, fusariotoxins, ochratoxins, fumonisins, trichothecenes, deoxynivalenol zearalenone, citrinin, patulin, alternariatoxins and moniliformin (mohapatra et al., 2017). the cumulative effect of all storage pests is what causes damage to the stored grains and other products. insects are the most serious hazard to stored products, mainly grains. indeed, they interact and associate with the crop at an early stage, causing damage both during crop standing and storage. hundreds of bug species are responsible for the infestation of stored products. some of the storage grains pests along with their hosts are listed in table 1. parajuli, s., adhikari, a., paudel, s., oli, d., bhandari, s., & shrestha, j. peruvian journal of agronomy 6(2): 132-146 (2022) https://doi.org/10.21704/pja.v6i2.1767 135 table 1: list of pests of stored grains common name pest host rice moth corcyra cephalonica n/a lesser grain borer rhyzopertha dominica n/a pulse beetle callosobruchus chinensisc. maculates pulses, bean and grain tamarind/groundnut bruchid caryedon serratus groundnut, tamarind and other legumes rice weevil sitophilus oryzae, s. zeamais, s. granaries rice, wheat, sorghum, maize, barley khapra beetle trichoderma granarium cereals, groundnut and pulses angoumois grain moth rhyzopertha dominica paddy, maize and wheat grain moth sitotroga cerealella rice, wheat and maize potato tuber moth phthorimaea operculella potato red flour beetle tribolium castaneum, t. confusum broken grains, damaged grains, milled products, machinery long-headed flour beetle latheticus oryzae n/a cigarette beetle lasioderma serricorne wheat flour, cereal bran, groundnut, chillies, cocoa beans, spices, turmeric drug store beetle stegobium paniceum turmeric, coriander, ginger, dryvegetable and animal matter sweet potato weevil cylas formicarius sweet potato saw toothed grain beetle cryptolestus minutas , laemophloeus pusillus dry fruits, maize, cereals and oil seeds red rust grain beetle cryptolestes ferrugineus n/a flat grain beetle cryptolestes pusillus n/a source: ahmed (1983) rodents, particularly mice and rats, have a greater impact on the quality of stored grains. storage structures, electrical installations, and a water pipe have all been reported to be damaged (smith, 1995). house mice (mus musculus), brown rats (rattus norvegicus), and black rats (rattus rattus) affect field and stored-grain in many ways (table 2). the nature, behavior, and habitat of these rats have all been widely investigated (lund, 1994). citellus spp., tamias spp., xerus spp., funiscurus spp., and halosciurus spp. are some of the other ground or tree squirrels that eat stored grains (smith, 1995). table 2: important rodent species damaging stored grains rodent species common name habitat rattus rattus house rat rural and urban residential places bandicota indica large bandicoot rat rural environment rattus meltada soft furred field cat crop fields and grassland mus musculus house mouse warehouse and godowns mus booduga field mouse crop fields bandicota bengalensis indian mole rat stores, warehouses and crop fields tatera indica indian gerbil crop fields and grassland source: (indian grain storage and management and research institute, 2021; tobin & fall, 2004) loss assessment several storage pests, including insects, mites, fungi, and rodents, can cause grain loss. insect consumption of grains or products includes not only direct kernel consumption but also the accumulation of detritus, leaving the product unfit for human consumption. they also cause harm to grains by poking holes in them for oviposition. insect pests caused a 12% loss before harvest and 36% loss after the harvest of grains. insect-related losses have been estimated to be between 5 and 10%, with up to 30% in the tropics (ahmad et al., use of biorational insecticides for the management of storage insect pests: a review may august 2022 136 2021). this has resulted in an annual loss of $200 million in net storage crop value in the united states (weaver & petroff, 2004). the pulse beetle (c. chinensis l.) has caused massive qualitative and quantitative losses. pest damage caused a quantitative loss (a decrease in the weight of stored grains), a qualitative loss (a decrease in size, an unappealing shape, and the accumulation of pest wastes), and a reduction in seed viability. psocid pests are annoyance and a concern for godowns and storage facilities (kleih & pike, 1995). they have been found to cause an apparent grain degradation and loss (about 3% in a storage period of 6 months). prostephanus truncatus is a severe corn pest in east and west africa, causing more damage to unhusked corn (group for assistance on systems relating to grains after harvest, 1987). mite is a lesser-known but more serious issue to storage products. they feed on germ section of stored grains and release storage fungus and bacteria, according to fleurat-lessard (1988). aspergillus halophilicus, a. restrictus, a. glaucus, a. candidus, a. ochraceus, and a. flavus are some fungi that damage the grain germ and cause discoloration (hall & harman, 1991). c. maculatus (fab.) (coleoptera: bruchidae) is a significant pest of cowpea that causes greater damage. the indication of insectinfested stored products is given in table 3. biorational management microbials various types of microbials or microbial pesticides have been employed to reduce the effect of different storage grain pests, mainly insects. spinosad, a popular bacterial pesticide, is made from the metabolites of an actinomycete bacterium called saccharopolyspora spinosa. the bacterial pesticide was used to effectively manage stored wheat grains that lost weight owing to insects (flinn et al., 2004). when exposed to uv rays from the sun, spinosad loses its insecticidal properties within a week, making it difficult to use in open fields. however, it has been reported that it can retain its insecticidal activity for up to 12 months when stored away from sunlight, making it beneficial in the control of the smaller grain borer (r. dominica f.) and the red flour beetle (t. castaneum herbst). the application of 1mg of spinosad per kg of wheat under stored conditions provided complete control and progeny suppression of the f1 in grain beetle (cryptolestes pusillus schonherr), the confused flour beetle (t. confusum), and the rusty grain beetle (cryptolestes ferrugineus stephens) (fang et al., 2002). pheromones pheromones are chemicals released or excreted by individual organisms that cause responses in other members of the same species. mass trapping and mating disruption techniques and pheromones have been successfully used in pest management. in fields, many pheromone baited traps can be employed to catch newly emerged moth males and reduce the number of males available for mating. pheromones are typically seen to be safer and more environmentally friendly than conventional pesticides because table 3: indication of insect-infested stored products indication commodities insects pupal cases sticking to shells and gunny bags groundnuts in shells caryedon serratus eggs on grain surface pulses (whole) callosobruchus spp. webbing or silken strands whole and milled cereals corcyra cephalonica, plodia interpunctella oilseed/oilcakes ephestia cautella, plodia interpunctella. dry fruits e. cautella tree nuts e. cautella exit holes wheat, rice, maize rhyzopertha dominica, sitophilus spp. paddy sitotroga cerealella pulses(whole) callosobruchus spp. source: rajendran (1999) parajuli, s., adhikari, a., paudel, s., oli, d., bhandari, s., & shrestha, j. peruvian journal of agronomy 6(2): 132-146 (2022) https://doi.org/10.21704/pja.v6i2.1767 137 they exist naturally, may target specific pest species, have low acute toxicity to vertebrates, and are usually volatile compounds that do not leave harmful residues (dang et al., 2016). pheromone monitoring traps help focus and narrow down the investigation to certain locations. we can detect and locate storedproduct pests (spps) population via pheromone monitoring before they can cause major product damage. they function as hormones outside the body of the creature that secretes them, influencing the organism that receives them (trematerra, 2011). according to phillips et al. (2000), about 20 different species of stored-product insects have slow-release formulations of pheromone lures in monitoring traps. pheromones are used by the indian meal moth (plodia interpunctella), the cigarette beetle (lasioderma serricorne f.; coleoptera: anobiidae), the red and confused flour beetles (t. castaneum and t. confusum jacquelin du val, respectively), and the warehouse beetle (trogoderma variabile ballion; coleoptera: dermestidae). the placement of the pheromones is critical for them to act efficiently. in an experiment, responding beetles were suffocated after passing through a corrugation tunnel to the cup of oil (barak & burkholder, 1985). according to nansen et al. (2004), male p. interpunctella were attracted to pheromone-baited traps on flat landing sites. natural enemies for stored-product pest the application or release of any natural enemy depends on the history of the pest and background knowledge. before releasing any natural enemy for control, important aspects such as understanding of its lifecycle, behavior, feeding mode, etc, are critical. some natural enemies or predators kill their prey right after an attack, whereas others require a close biosystemic connection to do so (hagstrum & subramanyam, 2006). the former is classified as a generalist, whereas the latter is classified as a specialist. the pirate warehouse bug (xylocoris flavipes) is a generalist that feeds on the eggs and larvae of insects that feed on stored products (arbogast, 1975). the histerid beetle (teretriosoma nigrescens), which feeds on beetles, is another example (rees, 1985). such beetles feed on various families of hazardous beetles, protect stored grains and products from pest damage. egg parasitoids of trichogramma spp. westwood are one of the possible generalist parasitoids employed widely in the investigation of its potential on field crops (wajnberg & hassan, 1994). the use of the parasitoid theocolax elegans, and other different pests such as the weevil (sitophilus spp.), lesser grain borer (r. dominica), drugstore beetle (stegobium paniceum), cowpea weevil (callosobruchus spp.), and angoumois grain moth (s. cerealella) have been effectively suppressed (flinn & hagstrum, 2001; flinn, 1998). the type of natural enemy to be used, as well as how it should be handled and implemented, is determined in part by the storage situation because the pest complex for numerous species is encountered while considering storage pest control (press et al., 1982). botanicals botanicals are plant-derived chemical compounds that are employed as insect repellents, deterrents for feeding and oviposition, and disruptions of insect pest biochemistry, physiology, and behavior. several spice crops, such as chilli, garlic, turmeric, ginger, and botanicals, such as neem, bakaino, century plant, undi, china berry, and lac tree, have been used successfully to control insect pests (shukla et al., 2007). according to srinivasan (2008), several plant items have been successfully evaluated with satisfactory results as preventatives against various stored-grain insect pests. pigeon pea was tested for 8 months against pulse beetle damage with neem seed oil at 10 ml/kg, mahua oil at 10 ml/kg and a mass fraction of 4 % of neem seed kernel, all of which were found to be repellent and powerful oviposition inhibitors (singal & chouhan, 1997). for the control of coleopteran insect pests on stored grains, essential oils were proven to be a satisfactory alternative to chemical insecticides (pérez et al., 2010). kiruba et al. (2008) reported that c. chinensis (l.) oviposition and f1 emergence were suppressed on red gram treated with 2 ml/kg ginger grass oil. similarly, when essential oils of cymbopogon citratus use of biorational insecticides for the management of storage insect pests: a review may august 2022 138 (stapf) and cymbopogon nardus (rendle) were applied to rice grains (oryza sativa l.) for the control of cowpea bruchid (c. maculatus (f.)), oviposition and f1 emergence were delayed compared to a control study (paranagama et al., 2003). garlic (allium sativum) has been found to resist t. castaneum, and its oil proved effective in killing t. castaneum and s. zeamais (ho, 1995). turmeric (curcuma longa) repels various insects, when 2 % turmeric powder is added to rice and wheat, it kills a variety of storage pests (jilani & su, 1983). neem (azadirachta indica) has pesticide properties in almost every part of the plant, with the seed kernel being the most potent. t. granarium can be controlled with neem products, while the pulse weevil can be controlled using 0.5 % neem oil as a surface protectant (ketkar, 1987). the oil of the lac tree (schleichera trijuga) is used as a surface protectant against the pulse weevil, and the extract is used as a repellent and insecticidal against s. zeamais adults and t. castaneum eggs (ketkar, 1987). teotia & tewari (1971) suggested that china berry (melia azedarach) leaf and drupe powders (1 % and 4 %), respectively, protect wheat against s. cerealella. the list of spices and botanicals used to control storage product pests is given in table 4. biological control biological control of storage-product pests includes a variety of entomopathogenic fungi, nematodes, bacteria, predators, parasitoids, and wasps. these biological control agents are commercially available only when scaled up for organic synthesis, according to weaver & petroff (2004). entomopathogenic fungi (epf) are a viable commercial treatment against a range of insects in open field conditions, such as termites, among other biological control approaches (rath, 2000). unfortunately, little progress has been made in terms of commercial availability while in storage. conidia of the entomopathogenic fungi have been used in dry or mixed form (with rice grain) against stored-grain insects (kaur et al., 2014). sedehi et al. (2014) conducted an experiment with different isolates that resulted in moderate to high mortality at various phases (immature stage and adult stage). the conidia of b. bassiana were suspended in a mixture of corn oil and mineral oil and applied to s. zeamais, with the oil suspension formulation having more efficacy among the three formulations. according to batta et al. (2010), liquid formulations of entomopathogenic fungus were successful in controlling s. granarius, r. dominica, s. oryzae, table 4: list of spices and botanicals used to control storage pests scientific name common name effect on storage pest references spices syzygium aromaticum clove tree repels t. castaneum. repels a number of grain insects grainge & ahmed (1988) zingiber officinale ginger causes adult mortality in c. chinensis and repels t. castaneum ho (1995) piper nigrum black pepper inhibit development of f1 of callosobruchus chinensis morallo-rejesus et al. (1990) allium sativum garlic repels tribolium castaneum. oil kills t. castaneum and sitophilus zeamais strong repellent for t. castaneum and s.zeamais ho (1995) curcuma longa turmeric repels a number of stored insects. a 2% powder mix with rice and wheat can protect from storage pests jilani & su, (1983) botanicals calophyllum inophyllum undi oil used as surface protectant against pulse weevils ketkar, (1987) vitex negundo indian privet leaves have insecticidal property against stored-grain pests ahmed & koppel (1987) agave americana century plant, leaves are used against stored pests grainge & ahmed (1988) parajuli, s., adhikari, a., paudel, s., oli, d., bhandari, s., & shrestha, j. peruvian journal of agronomy 6(2): 132-146 (2022) https://doi.org/10.21704/pja.v6i2.1767 139 and t. molitor. entomopathogenic nematodes acts as endoparasites of storage pests (gaugler et al., 2002) that were found to be a successful biocontrol option (kaya & gaugler, 1993). though entomopathogenic nematodes have not been studied much for storage pest management, they do have some qualities that make them a good biocontrol option. they are not harmful to vertebrates and several species are commercially available (bathon, 1996; grewal, 2002). tolerance to various pesticides (koppenhöfer et al., 2000), a wide host range (capinera & epsky, 1992), and active host finding capacity are all important characteristics of such nematodes (lewis & campbell, 2002). they have been used as biocontrol agents against a variety of storagegrain insects. they are used to control pyralidae and curculionidae (shannag & capinera, 2000) and storage insects (shapiro & mccoy, 2000). morris (1985) proved that nematodes can effectively suppress storage product insects such as t. molitor l. and ephestia kuehniella zeller. anthocorid bugs have efficiently controlled c. cephalonica as well as other insect pests such as thrips, aphids, and mealy bugs, in cropping systems in sub-saharan africa and the mediterranean region (zhang et al., 2012; efe & cakmak, 2013; wang et al., 2014). a predatory bug, x. flavipes, is one of the most important biological control agents used against storedgrain pests such as moths, mites, and bruchids (rahman et al., 2009). blaptostethus pallescens, a biocontrol agent, has efficiently reduced the eggs and larvae of lepidopteran pests, sucking pests such as mealy bugs, aphids, thrips, mites, and stored insect pests (kaur et al., 2019). the combo of b. bassiana arsef 5500 and m. anisopliae arsef 2974 isolates had the highest mortality rate (51.66 %) among rice weevil (s. oryzae l.) (dal bello et al., 2000). monitoring of stored grain pests monitoring of stored-grain pests aids in the identification and isolation of pest populations and prevents both qualitative and quantitative losses in stored commodities. it also advises the practitioners to learn about the efficacy of a specific integrated pest management (ipm) strategy for a specific storage-product pest (campbell et al., 2002). the type and nature of the pest, commodity, and storage methods all influence the pest monitoring technique. some monitoring systems and techniques include bulk commodity storage, pheromone attractant, food attractant, and white painted bins. additionally, new monitoring techniques include parasitoid stored product detection using near infrared spectroscopy and grain rotting detection using electronic nose technology. food attractants for flour beetles (tribolium spp.) as well as attractiveness to attagenus, trogoderma, and anthrenus larvae can improve the effectiveness of pheromone traps (burkholder & ma, 1985). it has been used to distinguish between infested and non-infested wheat kernels in order to identify wheat internal insect pests (dowell et al., 1998). it is also been employed in wheat to determine the difference between unparasitized and parasitized weevil larvae by wasps (burks et al., 2000). electronic nose technology has been employed recently for the rapid detection of grain quality by taking grain attributes such as odor and volatility into account (magan and evans, 2000). conclusion biorational pesticides can be an environmentallyfriendly and long-term alternative to chemical pesticides in various ways. natural enemies, such as predators and parasitoids, can be employed to control pests. however, prior to releasing any natural enemy, critical considerations such as understanding of its life cycle, behavior, and style of feeding must be made. various types of microbials or microbial pesticides have been employed to reduce the impact of a variety of storage grain pests, especially insects. botanicals, or chemical compounds produced from plants, such as a. sativum, c. longa, m. azedarach, a. indica, and others, can be employed as insect repellents, deterrents for feeding and oviposition, and disruptors of insect pests’ biochemistry, physiology, and behavior. pheromones, as biorational chemicals, can be an effective pest control tool for a variety of storage grain pests, use of biorational insecticides for the management of storage insect pests: a review may august 2022 140 particularly insects. they function as hormones outside of the body of the creature that secretes them, having an influence on the organism that receives them. entomopathogenic fungi and nematodes can also be used as biological controls to counteract the use of chemicals. biorational methods should be promoted, scaled up, and commercialized so that they may effectively control diseases without acquiring resistance on the one hand and recover the environment on the other. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. orcid and e-mail sandip paudel mrsandippaudel@gmail.com dipesh oli olidipesh872@gmail.com sagar bhandari bhandarisagar09@gmail.com jiban shrestha jibshrestha@gmail.com references ahmad, r., hassan, s., ahmad, s., nighat, s., devi, y. k., javeed, k., usmani, s., ansari, m. j., erturk, s., alkan, m., & hussain, b. 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(2012). survival and developmental characteristics of the predatory bug orius similis (hemiptera: anthocoridae) fed on tetranychus cinnabarinus (acari: tetranychidae) at three constant temperatures. european journal of entomology, 109(4), 503. 404 not found peruvian journal of agronomy 4(1): 35-39 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i1.1464 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 10 april 2020 accepted for publication: 30 april 2020 spray penetration into asparagus (asparagus officinalis l.) canopy using different nozzle inclinations and application rates penetración de la pulverización dentro del dosel del espárrago (asparagus officinalis l.) usando diferentes inclinaciones de boquilla y tasas de aplicación vásquez-castro, j. (1)*; ancco, a. (2); la torre, b. (3) *corresponding author: jaque@lamolina.edu.pe https://orcid.org/0000-0003-3407-6938 abstract asparagus (asparagus officinalis l.) is a difficult crop to treat with spraying pesticides because its architecture makes it difficult for droplets to penetrate inside of the canopy, where its pests are located. this study aims to examine the influence of operational parameters, such as nozzle inclination and application rates on the inner side of the canopy. we installed nozzles in the spray boom and its droplegs in three different inclinations: 0° with the plant, 30° in the direction of movement of the tractor-sprayer assembly (+30°), and 30° in the opposite direction (−30°). we applied the mixture in application rates of 600, 900, and 1,200 l ha−1. more so, the regulation used by the farmer of 0° and 800 l ha−1 was applied as a control. copper (cu) was used as a tracer for the mixture in a dose of 13.5 g ha−1. also, non-plasticized polyvinyl chloride grooved tubes (pvc) were installed inside the asparagus canopy and polyethylene sheets were placed on the pipes at different heights from the ground. later, we analyzed the sheets with atomic absorption spectrophotometry. results showed that the nozzle inclination and sampling height had notable effects on the copper deposit. conversely, different application rates showed no varied effects significantly. the greatest copper deposition in the asparagus canopy was achieved with a nozzle inclination of +30° with any application rate. finally, we recommend regulating the sprayers with a nozzle inclination of +30° and an application rate of 600 l ha−1 as the most effective adjustment for the distribution of pesticides and represent the lowest cost of operation since there is no influence of the application rate. key words: sprayer, nozzle, pesticide, application technology, deposition resumen el espárrago (asparagus officinalis l.) es un cultivo muy difícil de tratar con plaguicidas vía aspersión, pues la arquitectura de la planta dificulta la penetración de las gotas en el dosel, en cuyo interior se localizan las principales plagas del cultivo. el objetivo del presente trabajo fue evaluar la influencia de los parámetros operacionales, tales como, el volumen de aplicación e inclinación de las boquillas sobre el depósito de la pulverización al interior del dosel del espárrago. las boquillas fueron instaladas en la barra de pulverización y en los bajantes en una de las tres inclinaciones estudiadas, 0° en relación a la planta, 30º en sentido al desplazamiento del conjunto tractor-pulverizador (+30º) y 30º en sentido opuesto (−30º). las tasas de aplicación estudiadas fueron 600, 900 y 1 200 l ha−1. además de esos tratamientos, se estudió la regulación empleada por el productor de 0° y 800 l ha−1. para el tratamiento del cultivo fue usado el cobre (cu) como trazador, en la dosis de 13,5 g ha−1. tubos ranurados de policloruro de vinilo no plastificado (pvc) fueron instalados al interior del dosel de la planta. láminas de polietileno fueron acondicionadas sobre los tubos de pvc a diferentes alturas en relación al suelo. después de la aplicación, las láminas de polietileno fueron analizadas mediante técnica de espectrofotometría de absorción atómica. hubo efecto significativo de la inclinación de boquilla y de la altura de muestreo sobre el depósito de cobre. por otro lado, no hubo efecto significativo del volumen de aplicación sobre el depósito. la mayor deposición de cobre al interior del dosel de la planta se consiguió con la inclinación de boquillas de +30º en cualquiera de los volúmenes de aplicación. al no existir influencia del volumen de aplicación en el depósito de la pulverización al interior del dosel del espárrago, se recomienda regular los pulverizadores con inclinación de boquillas de +30º y volumen de aplicación de 600 l ha−1 por ser la regulación más eficaz en la distribución de los plaguicidas y que a su vez representa el menor costo de operación. palabras clave: pulverizador, boquilla, pesticida, tecnología de aplicación, deposición 1 universidad nacional agraria la molina, faculty of agronomy. entomology department, av. la molina s/n, lima 12, peru. 2 universidad nacional agraria la molina, faculty of agronomy. lima, peru. 3 universidad nacional agraria la molina, faculty of agronomy. soils department. lima, perú. spray penetration into asparagus (asparagus officinalis l.) canopy using different nozzle inclinations and application rates january april 2020 36 introduction peru is one of the main asparagus (asparagus officinalis l.) exporters of the world, reaching a planted area of 33,870 ha in 2015 (ministerio de agricultura y riego [minagri], 2020). the coastal agroecosystem and the efficient management of this crop have allowed high yields in the past years (camborda et al., 2015). however, in recent years, the yield has reduced considerably among other factors due to severe pest attacks and flaws in control strategy, especially in chemical control (ortega et al., 2014). this scenario has led to the reduction of crop areas in the country (apaza et al., 2019). the effectiveness of the restraint of phytosanitary problems of asparagus, such as prodiplosis longifila gagné (diptera: cecidomyiidae) and stemphilium spp, depends on choosing the adequate product, time application, and the distribution of the spray on the plant. the use of pesticides as the only containment measure and the inadequate regulation of sprayers have caused serious problems to the asparagus production. due to the flaws in pest control, producers have increased the dose and number of applications per season, causing problems related to resistance and pesticide residues (vásquez-castro, 2012, 2013). asparagus is generally sown in lines separated by 1.5 m, and at their greatest development can grow up to 1.6 m in height, 1.3 m in width, and creates a dense foliage that is a favorable microclimate to pests. consequently, this hinders the spray penetration to the asparagus canopy. more so, horizontal spray boom with vertical section adaptations (droplegs) is recommended for herbaceous crops of notable height, as this equipment manages to spray the top and sides of the asparagus (knott, 1987). nevertheless, a low spray penetration rate is seen in the asparagus canopy, mainly in the lower third of the plant, preferred place of p. longifila and stemphilium spp. (scodellaro, 1995; castillo, 2019). conversely, high application rates are widely used to enhance the application quality for pest control; but the results have not been satisfactory. good results have been obtained in other crops when nozzles were fitted with inclination angles over the spray boom (panisson et al., 2004; foqué & nuyttens, 2011). nevertheless, negative results are known (zhu et al., 2002). however, some reports in international literature showed that low application rates result in higher pesticide deposits and lower operation costs (véliz et al., 2010; sánchez-hermosilla et al., 2013; cunha, victor, & sales, 2018). there is little scientific information about application technology in asparagus crops. thus, in this study, we evaluate the influence of some operational parameters, nozzle inclination, and application rates on the pesticide deposit in the asparagus canopy. materials and methods the fieldwork was done in a commercial production farm of green asparagus, variety uc-157, located at the ica valley (south coast of peru). the plants were at the second sprouting stage (1.6 m of height, 1.3 m canopy-width and dense foliage). a mounted sprayer (jacto brand, condor 600® model) was used for the application of the mixture. the sprayer was equipped in a 13.5 m long horizontal spray boom, eight droplegs at 1.5 m apart, and 59 empty conejet nozzles (teejet brand, txvk-10® model) (figure 1). to avoid the pressure being an influencing variable for the nozzles drops spectrum, all the treatments were performed at a working pressure of 1,000 kpa and this resulted in an average flow of 1.17 l min−1. this way, the different application rates used (600, 800, 900, and 1,200 l ha−1) were obtained through travel speed regulation of the tractor-sprayer assembly, keeping the tractor engine rotation at 1,700 rpm, providing 540 rpm in the power takeoff. the travel speeds were 5.1, 3.8, 3.4, and 2.6 km h−1 for the application rates of 600, 800, 900, and 1,200 l ha−1, respectively. the nozzles were installed in the horizontal spray boom and the droplegs in one of the following three inclination angles: 0° with the plant, 30° in the direction of displacement from the tractor-sprayer assembly (+30°), and 30° in the opposite direction (−30°). in addition to those treatments, we applied the regulation used by the farmer, nozzle on 0°, and an application rate of 800 l ha−1, as control. more so, we used copper as a tracer with a dose of 13.5 g ha−1 for the crop treatment. for the evaluation of the mixture deposit in the asparagus canopy, we used the methodology proposed by travis et al. (1985), and pergher et al. (1999) with some modifications. pvc tubes of ¾″ diameter and 1.68 m long were installed inside the canopy, simulating the stem of the plants. polyethylene sheets of 0.14 × 0.16 m were placed above the tubes at different heights from the ground (0.04–0.18; 0.26–0.40; 0.48–0.62; 0.70–0.84; 0.92–1.06; 1.14–1.28; 1.36–1.50 m). these sheets served as collectors of surface droplets containing copper (figure 2). the sheets were collected and examined by atomic absorption spectrophotometry to quantify the copper deposition (ppm) after the application. in total, we applied ten treatments that corresponded to three nozzles inclination with three application rates each and one control treatment (regulation used by the farmer). figure 1. the scheme of the tractor-sprayer assembly used in the experiment. notice the horizontal spray boom, the droplegs, and the nozzles distribution. vásquez-castro, j.; ancco, a.; la torre, b. peruvian journal of agronomy 4(1): 35-39 (2020) 37 furthermore, for each treatment, we installed nine pvc tubes distributed in different parts of the experimental plot, which had an area of 810 m2 (60 m × 13.5 m). we analyzed the deposition data with repeated measures design by considering the treatment effects, the sampling height, and the interaction between them. we also applied an f-test for orthogonal contrasts by considering that the treatments were arranged in the 3 × 3 factorial scheme with an additional treatment and the t-test for the contrasts in which we observed a notable effect at 5% level of the f-test. finally, using the mixed process of sas (1999) software, we analyzed the data. results and discussion the variance analysis (f-test) showed a notable effect related to the treatment and sampling height (table 1). the four orthogonal contrasts used for the splitting of the treatment were also significant, including the interaction of nozzle tilt angle × application rate. the interaction between treatment and sampling height was not noticeable, which indicated that the effects of the treatments were independent of the sampling height. however, we decided to detail the analysis of these two factors because the effects of the treatments and sampling height were significant (table 2). the results showed in table 2 demonstrated that the treatments with +30° nozzle tilt angle had higher deposits in the inner part of the canopy, followed by the −30° nozzle tilt angle treatment with an application rate of 900 l ha−1. the smallest deposits occurred generally in the treatments with 0° nozzle tilt angle. the regulation used by the farmer (0° y 800 l ha−1) had a very low copper deposit values in all the sampling heights. the quantity of copper deposited in the inner canopy with the regulation used by the farmer was seven times lower than the treatment of 600 l ha−1 of application rate and +30° nozzle tilt angle. these results explained the flaws found in the control of key pests, such as p. longifila and stemphylium spp., that in later years have caused huge economic loses. these pests show colonizing preference for the inner third-inferior asparagus canopy, which is the place of difficult access to spraying. the +30° nozzle tilt angle probably increased the area of exposition of the jet to the plant surface, which together with the turbulence generated by the air resistance to the trajectory of the droplets favored their penetration, mainly for the thirdinferior part of the plant. the travel speed of the tractor-spray assembly did not influence figure 2. the pvc tube installed inside the asparagus canopy. notice the polyethylene sheets placed at different heights from the ground (h1-h7). table 1. analysis of variance of copper deposit by a mixed model for repeated measurements. origin of variation d.f. f pr> f treatment 9 51,32 <0,0001 control × other treatments 1 66,39 <0,0001 nozzle tilt angle 2 178,49 <0,0001 application rate 2 8,02 0,0007 nozzle tilt angle × application rate 4 5,61 0,0005 sampling height 6 29,30 <0,0001 treatment × sampling height 54 1,14 0,2346 table 2. copper deposit means (ppm) in function to treatments and sampling height. nozzle tilt angle application rate (lha-1) height (h) h1 h2 h3 h4 h5 h6 h7 0° 600 0,124d 0,096bc 0,048cd 0,038c 0,039c 0,041c 0,043cd 0° 800 0,078d 0,062c 0,024d 0,016c 0,007c 0,010c 0,010d 0° 900 0,110d 0,059c 0,034d 0,022 c 0,026 c 0,022c 0,028cd 0° 1200 0,098d 0,092bc 0,043cd 0,011 c 0,006 c 0,014c 0,023cd -30° 600 0,152cd 0,063c 0,046cd 0,042 c 0,042 c 0,048bc 0,103bc -30° 900 0,320b 0,172b 0,126bc 0,132ab 0,136ab 0,134ab 0,136ab -30° 1200 0,229c 0,143bc 0,103bcd 0,066bc 0,080bc 0,086abc 0,100bc +30° 600 0,360b 0,334a 0,187ab 0,159a 0,133ab 0,140a 0,207a +30° 900 0,476a 0,318a 0,237a 0,217a 0,211a 0,161a 0,159ab +30° 1200 0,324b 0,297a 0,229a 0,168a 0,198a 0,161a 0,174ab different letters in columns represent significant differences in a t-test (p ≤ 0.05). spray penetration into asparagus (asparagus officinalis l.) canopy using different nozzle inclinations and application rates january april 2020 38 the results. as per each nozzle inclination, no statistical differences among the different application rates obtained with different travel speeds were seen. similarly, da cunha et al. (2018) showed that the travel speed did not influence the deposit of pesticides in soybean crops. flaws in the control of other pests of asparagus and problems related to their pesticide resistance in peru were known in the literature (bustillo, 2009; vásquez-castro, 2012). an inadequate regulation of the sprayers likely increased the incidence of these problems. generally, there were no notable differences in the deposition of the pesticide in different application rates. so, for pest control, it is indifferent to apply 600, 800, 900, and 1,200 l ha−1; however, the greatest economic benefit for the farmer will be achieved with the lower application rates, where lower operational cost in oil and labor is considered. contrastingly, the timing of the pest control plays an important role as per large plantations. therefore, the faster the application, the greater the probability of success in controlling the pest, which can be achieved with lower application rates. interestingly, this study is the first to be performed in the field of pesticide application technology in peru and one of the few studies related to asparagus crop worldwide. consequently, we advise for more research as per finding the most efficient pesticide applying method for a crop as important and complex as asparagus. conclusions the nozzle tilt angle of 30° in the direction of movement of the tractor-spray assembly provides a large deposit of the pesticide inside the asparagus canopy. the application rate does not influence the pesticide deposit. finally, increasing the volume of water for the pulverizations does not enhance the coverage or the deposition of substances in the asparagus crops. references apaza, w., quiroz, p., & julca-otiniano, a. 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(2002). influence of plant structure, orifice size, and nozzle inclination on spray penetration into peanut canopy. transactions of the asae, 45(5), 1295–1301. http://doi. org/10.13031/2013.11058 peruvian journal of agronomy 4(1): 10-16 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i1.1451 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 10 january 2020 accepted for publication: 18 march 2020 pollinating insects of cherimoya (annona cherimola miller) in la molina, lima, peru insectos polinizadores del chirimoyo (annona cherimola miller) en la molina, lima-perú morales, b.(1)*; bautista, j.(2); vergara, c.(3) *corresponding author: 20140959@lamolina.edu.pe https://orcid.org/0000-0002-8879-3118 abstract this survey aimed to determine the pollinating insects of the flowers of the cherimoya crop (annona cherimola mill.) located at the program of research and social projection in fruit trees (pips-frutales) at the universidad nacional agraria la molina. fifteen trees were selected randomly, and evaluations were performed from 31 october to 15 november 2018. three replicates were performed, each replicate was formed by a set of five trees and were evaluated in three different weeks. each tree was evaluated in four quadrants, according to the location of the flowers (external or internal) and the floral phases of the crop (female or male). the collection of the insects from the cherimoya flowers was performed using an aspirator, then the insect morphotypes were determined and classified according to order, family, genus and/or species through the use of taxonomic dichotomous keys and the support of specialists. the collected insects belonged to the orders coleoptera, hymenoptera, and hemiptera, with coleoptera having the highest diversity (17 morphospecies) and quantity (242 specimens). the genus europs (monotomidae) proved to be the most abundant group of coleoptera during the assessment period, followed by morphospecies of the families nitidulidae, anthicidae, staphylinidae, scarabaeidae, anobiidae, and buprestidae. the pollinating insects present in the cherimoya crop were satisfactorily classified into genera and species. keywords: annona cherimola, europs sp., floral phase, monotomidae, pollinator resumen el objetivo de esta investigación fue determinar la presencia de insectos polinizadores de las flores del cultivo de chirimoyo (annona cherimola mill.), ubicado en el programa de investigación y proyección social en frutales (pipsfrutales) de la universidad nacional agraria la molina. se seleccionaron 15 árboles al azar y las evaluaciones se realizaron desde el 31 de octubre hasta el 15 de noviembre del 2018. se realizaron tres repeticiones, cada repetición fue formada por un conjunto de cinco árboles y fueron evaluadas en tres semanas diferentes. cada árbol fue evaluado en cuatro cuadrantes según la ubicación de las flores (externa o interna) y las fases florales del cultivo (hembra y macho). se realizó la colecta de los insectos en las flores del chirimoyo empleando un aspirador, luego se determinaron los morfotipos de insectos y fueron clasificados de acuerdo al orden, familia, género y/o especie mediante el uso de llaves dicotómicas taxonómicas y el apoyo de especialistas. los insectos colectados pertenecieron a las ordenes coleoptera, hymenoptera y hemiptera, siendo el primero el predominante en diversidad (17 morfoespecies) y cantidad (242 individuos). el género europs (monotomidae) con una sola morfoespecie, resultó ser el grupo más constante y abundante de coleoptera durante el período de evaluación, seguidos de morfoespecies de las familias nitidulidae, anthicidae, staphylinidae, scarabaeidae, anobiidae y buprestidae. los insectos polinizadores presentes en el cultivo del chirimoyo fueron clasificados satisfactoriamente en géneros y especies. palabras claves: annona cherimola, europs sp., fase floral, monotomidae, polinizador 1 universidad nacional agraria la molina. student of the faculty of agronomy. lima, perú. 2 universidad nacional agraria la molina. agronomist engineer. lima, perú. 3 universidad nacional agraria la molina. faculty of agronomy. department of entomology, lima, perú. introduction the cherimoya (annona cherimola miller) is native to subtropical areas of the ecuadorian and peruvian andes (gonzález, 2013). it belongs to the genus annona, which comprises approximately 120 species, and is of great commercial importance (garcía et al., 2009). there is also growing interest in the commercialization of cherimoya, due to its pleasant flavor and aroma, and good acceptance in the market as an exotic fruit (gonzález, 2013). morales, b.; bautista, j.; vergara, c. peruvian journal of agronomy 4(1): 10-16 (2020) 11 the expansion of the cultivation of cherimoya outside of the area of its native range has been limited by its floral behavior, called dicogamia protoginea (guirado et al., 2001). this behavior consists of the early maturation of pistils wherein they are only receptive before the stamens of the same flower releases pollen (franciosi, 1992). this characteristic of cherimoya flowers makes crosspollination necessary through the participation of natural pollinators (ortiz-sánchez & cabello-garcía, 1991). the flowers of the cherimoya tree go through a cycle of floral opening that takes place in three consecutive phases, called the pre-female, female, and male phases (guirado et al., 2001). in the pre-female phase, the flower is receptive, but it is not possible for it to be pollinated due to the reduced opening of the petals (farré et al, 1999). generally, beetles are introduced to the flowers in the female phase and remain inactive at the base of the petals or by walking on the stamens and stigmas. in the male phase, the beetles are dragged out of the flower due to falling petals and thus disperse to new flowers in the female state, covered with viable pollen for 24 hours (gonzález et al., 2007). in annonaceae, natural pollination generally occurs by the action of insects, which are determined by the floral characteristics of the crop. the flowers of the cherimoya have a small floral chamber, which is why they are pollinated by small beetles belonging to the families nitidulidae, curculionidae, and chrysomelidae (gottsberger, 1999). the beetles are enticed to enter the flowers by the characteristic aroma of fermented fruit that the flowers release through osmophores located on the internal base of the petals (gonzález et al., 2007). in addition, the fleshy petals serve as a food source for the insects (meyer, 2003). in italy, studies on natural pollination in cherimoya have identified orius laevigatus fieber (hemipteraanthocoridae) as the most important pollinator of annona spp. (caleca et al., 2002). in florida, beetles of the family nitidulidae are indicated as the main pollinators of fruit species of the genus annona spp. (george et al., 1989). recently, in the caribbean islands, the species europs fervidus blatchley was noted as the main pollinating agent of the atemoya fruit tree (annona x atemoya) (jenkins et al., 2015). in peru, no research has been reported yet focused on studying the pollinating insects of this crop. therefore, it is necessary to determine these insects to establish an alternative, efficient system of pollination using insect species in the production area of this crop. taking this into account, the objective of this investigation was to determine the presence of insects associated with the pollination of cherimoya at la molina, peru. this information will allow future research to be carried out to determine the efficiency of these pollinating insects and to achieve better results in the production of this crop. materials and methods this research was carried out in the cherimoya plantation located in the fields at the universidad nacional agraria la molina (unalm), la molina district, lima region, peru, geographically located at latitude 12°4’58.51 “s, longitude 76°56’51.93 “w, and 236 m above sea level. the field evaluations were carried out in seven-day intervals, beginning on october 31, 2018, and ending on november 15, 2018. fifteen trees were randomly selected and grouped in blocks, with five trees in each block. this was done to evaluate one block per week, that is, the evaluations were done on different trees weekly. two hemispheres were evaluated in each tree: north and south. each of these was divided into two strata (lower and upper half) and in each stratum four flowers were selected at random, of which two flowers were selected from the outside of the crown and two flowers from the inside of the crown. field work the assessments were conducted during the period of greatest flower presence on the trees. two floral stages of the crop were evaluated (female and male phases) and each evaluation was conducted over two continuous days. determination of the duration of the opening cycle of the flowers five trees were selected at random. in each tree four oneyear-old wooden branches and two flowers per branch were selected. these flowers were monitored from the prefemale phase to the fall of the petals (male phase). this was done to determine the duration and timing of each flower phase. this assessment showed that the period of flower opening was approximately two days. guirado et al. (2001) also pointed out that once the flower bud reaches its final size, the opening cycle takes place in two days. based on the data obtained, the times of collection of the insects were determined. collecting the insects the collections were made using an oral aspirator that included a collection bottle (30 ml) that was introduced directly into the flower. on the first day, evaluations were made on flowers that were in the female phase, from 11:00 a.m. to 2:30 p.m. on the second day, on flowers in the male phase, from 3:00 to 6:30 p.m. the meteorological data of temperature (°c) and relative humidity (%) were obtained from the “von humboldt” meteorological station located at the universidad nacional agraria la molina. laboratory procedure processing and checking the samples of the collecting vials the insects collected in the field were taken to the insect preparation room of the klaus raven büller entomological pollinating insects of cherimoya (annona cherimola miller) in la molina, lima, peru january april 2020 12 museum at the universidad nacional agraria la molina. in the room they were subjected to freezing for five hours to ensure death and to avoid deterioration of the insects, such as breaking legs and antennae. classification each sample was placed in a 6 mm diameter petri dish and examined through a motic smz -168 stereoscope, where the insects were separated into morphotypes. for this purpose, the following were considered: body coloration, elytra, pronotum, type of antenna, and shape and structure of the body. coding the vials were labeled with the morphotype data and according to the taxonomic category in which they were provisionally classified. each morphotype was coded using the first three letters of the taxon to which it belonged, at the order or family level, followed by a number (e.g. colmorfo-1). counting and recording specimens classified in morphotypes and coded were counted by week and sector evaluated. the data obtained were recorded in a digital spreadsheet in microsoft excel. assembly and preservation three copies of each morphotype were assembled. insects smaller than 15 mm were mounted on canson cardboard points using gum arabic. those with a length greater than 15 mm were mounted directly with an entomological pin. in this case, the pin went through the upper internal margin of the right elitre of the insect. subsequently, the insects were labeled with the collection data. the insects of each morphotype that were not mounted were placed in 15 ml glass vials with 75% alcohol. taxonomy determination in the taxonomic determination of the specimens, dichotomous keys of bibliographic sources were used, and specialists were consulted. morphotypes were determined at the family level, some at the genus level, and others at the species level. subsequently, the specimens were coded according to the standards followed in the insect collection of the museo entomológico klaus raven büller of the universidad nacional agraria la molina, and were deposited in that collection. photographs of each morphotype were also recorded using a leica s8 apo stereomicroscope connected to a canon eos 5d mark iii camera. results a total of 373 specimens were recorded on cherimoya flowers corresponding to 17 morphotypes. the collected insects were distributed in three orders: coleoptera, hymenoptera, and hemiptera, with order coleoptera having the highest number of specimens (table 1). the most abundant insect families were monotomidae and nitidulidae, order coleoptera; and formicidae, order hymenoptera. insects collected on cherimoya flowers there were 177 specimens in the female phase of the flowers and 196 in the male phase (table 1), with no significant difference between the two phases, according to tukey’s mean comparison test (α=0.05). the lowest number of specimens was recorded in the first two weeks of collections with a total of 106 and 119 specimens respectively, with the female phase showing the highest number, while in the third week 148 specimens were recorded, with the male phase showing the highest number. order coleoptera in the order coleoptera, the presence of a species to be determined from the genus europs (fig. 1), belonging to the family monotomidae, was repeatedly recorded. this species was dominant in all evaluations but was present to a greater extent in the male phase of the flowers. the family nitidulidae presented the highest diversity of specimens, with species from the genera colopterus, urophorus, and carpophilus. the genus colopterus turned out to be the most diverse group with four species, while the genera urophorus (fig. 2) and carpophilus presented one species each, with the species carpophilus hemipterus (linnaeus, 1758) being determined for carpophilus (fig.  3). figure 1. adult europs sp. (order coleoptera, family monotomidae). morales, b.; bautista, j.; vergara, c. peruvian journal of agronomy 4(1): 10-16 (2020) 13 order family genus/species number of specimens by phase total female male hemiptera aphididae aphis spiraecola 9 10 19 greenidia ficicola 3 0 3 pseudococcidae planococcus citri 8 3 11 coleoptera anobiidae unidentified 0 2 2 anthicidae anthicus sp. 5 3 8 buprestidae chrysobothris sp. 1 0 1 monotomidae europs sp. 78 128 206 nitidulidae urophorus sp. 3 1 4 colopterus sp.1 0 1 1 colopterus sp.2 2 8 10 colopterus sp.3 1 0 1 colopterus sp.4 2 1 3 carpophilus hemipterus 3 0 3 scarabaeidae paranomala undulata peruviana 2 0 2 staphylinidae unidentified 0 1 1 hymenoptera formicidae linepithema humile 59 38 97 tapinoma melanocephalum 1 0 1 total 177 196 table 1. order, family, genus, species, and number of specimens collected in the female and male phase of cherimoya flowers from october 31 to november 15, 2018, la molina, lima, peru figure 2. adult urophorus sp. (order coleoptera, family nitidulidae). figure 3. adult carpophilus hemipterus. (order coleoptera, family nitidulidae). pollinating insects of cherimoya (annona cherimola miller) in la molina, lima, peru january april 2020 14 in the collection, the species paranomala undulata peruviana (guérin-méneville, 1831) (family scarabaeidae), species from the genera anthicus (anthicidae) and chrysobothris (buprestidae), and the families staphylinidae and anobiidae were also recorded. all of these were collected from the flowers in a smaller proportion than the other families. order hymenoptera the order hymenoptera was the group with the second highest number of specimens, with the presence of species linepithema humile (mayr, 1868) and tapinoma melanocephalum (fabricius, 1793) from the family formicidae. both species occurred in greater numbers in the female phase. order hemiptera the aphididae family was recorded, represented by the species aphis spiraecola (patch, 1914) and greenidia ficicola (takahashi, 1921). the pseudococcidae family was recorded, represented by the species planococcus citri (risso, 1813). discussion number of insects collected according to the flower phase of the crop during the investigation, a greater number of insects were obtained in the male phase of the flowers compared to the female phase, showing a significant difference in the number of specimens captured in both phases. this is contrary to the findings of tsukada et al. (2005), who studied cherimoya flower visitors in italy and found no significant difference in the composition of flower visitors between the male and female phases. the greater number of insects collected during the male phase could be related to the arrangement of the flower, since according to gonzález et al. (2007) during this phase there is an opening at the base of the petals which allows insects to enter in search of food or shelter. order coleoptera among the groups of insects found, the order coleoptera was predominant, similar to results by tsukada et al. (2005) who found 20 species from that order on cherimoya flowers in japan, and caleca et al. (2002), who obtained 24 species of coleoptera out of the 49 species collected on flowers of annona sp. and certain hybrids in experimental orchards in sicily. according to stefanescu et al. (2018), the biology of the larval stages of coleoptera is usually different from the adult stage, with adults visiting flowers for reasons related to obtaining food (from pollen, nectar, and/or floral tissues), mating, and/or protection from predators. therefore, the higher incidence of insects of the order coleoptera in flowers may be related to the type of diet they require (in this case pollen), and to fulfill their life cycle satisfactorily. in addition, they possess morphological adaptations, both oral system and of the integument with its hairy covering, in which pollen grains may adhere and then pass to other flowers (krenn et al., 2005). within this order, the presence of a species (still to be determined) from the genus europs (family monotomidae) was consistently recorded. this species turned out to be the predominant species, which coincides with other research on annonaceae flowers, where it is cited as the most common visitor, followed by loberus testaceus reitter (erotylidae) (jenkins et al., 2013). among families collected in this investigation, the family nitidulidae was the most diverse, with six species within the genera colopterus, urophorus, and carpophilus. this family was also cited among the flower visitors in japan by tsukada et al. (2005). order hymenoptera the incidence of linepithema humile and tapinoma melanocephalum in flowers may be related to the presence of insects of the order hemiptera. according to vergara et al. (2007), a trophobiotic relationship between formicidae and hemiptera exists in which the ants feed on the honeydew excreted by the members of the coccoidea superfamily. this coincides with what was found in the evaluated flowers, since in addition to ants, aphis spiraecola and planococcus citri (hemiptera) were found. order hemiptera the presence of aphis spiraecola and greenidia ficicola in cherimoya flowers may be related to their feeding habit, according to rosales-lópez et al. (2013), who pointed out that aphids are polyphagous insects that inhabit different plant organs and therefore can be found in flowers. on the other hand, the presence of planococcus citri in flowers is related to their feeding habits. according to rao et al. (2006), this species is polyphagous and cosmopolitan, and may be found in shoots, fruits, and flowers. conclusions in the flowers of the cherimoya crop in the agricultural field of la molina, 17 morphotypes of insects belonging to the orders coleoptera, hymenoptera, and hemiptera were found. the order coleoptera was the most abundant, with the family monotomidae best represented in abundance throughout the collection period, as well as one species in the genus europs. the order hymenoptera was the second most abundant group, represented only by the family formicidae, morales, b.; bautista, j.; vergara, c. peruvian journal of agronomy 4(1): 10-16 (2020) 15 with the species linepithema humile and tapinoma melanocephalum. to a lesser extent, the order hemiptera, with the family aphididae represented by the species aphis spiraecola and greenidia ficicola, and the family pseudococcidae represented by the species planococcus citri were present. these species are not considered pollinating insects; however, they are of agricultural importance since they are considered pests. acknowledgements we thank the ministerio de educación del perú for funding this research. we are grateful to the researchers alfredo giraldo, walter díaz, nidia elguera, laura cruz, and carmen livia for their support in managing and determining the specimens collected, and for guidance in processing the data. we would like to thank the program of research and social projection in fruit trees (pipsfrutales) at the universidad nacional agraria la molina for granting permission to carry out the collections in the cherimoya fields. references caleca, v., lo verde, g., salvatore, r., & haralabos, t. 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(2005). flower visitors to cherimoya, annona pollinating insects of cherimoya (annona cherimola miller) in la molina, lima, peru january april 2020 16 cherimola (magnoliales: annonaceae) in japan. applied entomology and zoolog, 40(2), 317–324. https://doi.org/10.1303/aez.2005.317 vergara, e.v., echavarría, h., & serna, f.j. (2007). hormigas (hymenoptera: formicidae) asociadas al arboretum de la universidad nacional de colombia, sede medellín. sociedad entomológica aragonesa, 40, 497−505. peruvian journal of agronomy 3(2): 39-46 (2019) issn: 2616-4477 (versión electrónica) http://revistas.lamolina.edu.pe/index.php/jpagronomy doi: http://dx.doi.org/10.21704/pja.v3i2.1313 © the authors. published by universidad nacional agraria la molina received for publication: 28 may 2019 accepted for publication: 15 june 2019 can salvia splendens ‘red vista’ be used in association with the mycorrhizal fungus glomus intraradices for the assisted phytoextraction of lead from the soil in lima, peru? puede salvia splendens ‘red vista’ ser usada en asociación con el hongo micorrítico glomus intraradices para la fitoextracción asistida de plomo del suelo en lima, perú? vicente, e.(1)*, tello, l.(2) *corresponding author: eduardovicente9293@gmail.com abstract in peru, gasoline containing lead as a main component was used in vehicles up until 2006, when the government banned its sale. however, since this was preceded by a century of constant use, there is a high chance that most urban soils are polluted with this heavy metal. hyperaccumulator plants that take up heavy metals from the soil and store them in their tissues without showing any symptoms of toxicity may be the solution to this problem, as the use of these plants for phytoremediation does not require large amounts of money, can be carried out in situ, and is environmentally friendly, making it one of the best options for urban areas. however, unfortunately, there are only a few known species of hyperaccumulator plants that can be grown in urban environments. therefore, we conducted a bioassay at the soil fertility laboratory of the agronomy faculty at the national agrarian university la molina, peru, over a period of 4 months to determine the hyperaccumulation capacity of salvia splendens ‘red vista’ and to examine whether the mycorrhizal fungus glomus intraradices can enhance the extraction of lead from the soil. after harvest, the plants were divided into three parts (roots, leaves, and inflorescences) to determine the concentrations of lead in the various tissues. we found that s. splendens ‘red vista’ did not accumulate high amounts of lead in its tissues even when it was associated with g. intraradices and consequently should not be considered for use in phytoextraction. key words: salvia splendens; glomus intraradices; phytoremediation resumen en perú, la gasolina usada en vehículos motorizados contuvo plomo como uno de sus principales componentes por mucho tiempo, esto solo cambio luego del año 2006 cuando el gobierno prohibió la comercialización de gasolina con plomo en su composición. esto sucedió hace solo unos años y luego de poco más de un siglo de constante actividad del parque automotor, de esta forma consideramos que existe una gran posibilidad de que la mayoría de suelos urbanos estén contaminados con este metal pesado. las plantas hiperacumuladoras podrían ser la solución a este problema; estas plantas extraen y acumulan metales pesados del suelo en su tejido sin mostrar signos de toxicidad. el uso de plantas hiperacumuladoras en estrategias de fitorremediación no necesita de mucho dinero; es realizado in situ y es amigable con el ambiente, debido a esto es probablemente una de las mejores opciones para las áreas urbanas. desafortunadamente no hay muchas especies conocidas de plantas hiperacumuladoras para áreas urbanas. realizamos este bioensayo para determinar la capacidad de fitoextracción de plomo del suelo de salvia splendens var. ‘red vista’. el bioensayo fue establecido en las instalaciones del laboratorio de fertilidad de suelos de la universidad nacional agraria la molina, duró cuatro meses y también se usó el hongo micorrítico glomus intraradices en algunos tratamientos para probar la eficiencia de las micorrizas en la extracción de plomo en el suelo. las plantas fueron cosechadas y divididas en tres partes: raíces, tallos y hojas, e inflorescencias para observar la dinámica de plomo en la planta. los resultados finales mostraron que salvia splendens var. ‘red vista’ no acumula grandes cantidades de plomo en su tejido aun cuando está asociado con glomus intraradices y debido a esto no debe ser considerado para técnicas de fitoextracción de plomo. palabras clave: salvia splendens, glomus intraradices, fitorremediacion 1 bachelor of agronomy at universidad nacional agraria la molina. lima, peru. 2 principal professor at universidad nacional agraria la molina. lima, peru. http://revistas.lamolina.edu.pe/index.php/jpagronomy mailto:eduardovicente9293@gmail.com can salvia splendens ‘red vista’ be used in association with the mycorrhizal fungus glomus intraradices for the assisted phytoextraction of lead from the soil in lima, peru? may august 2019 40 introduction the use of gasoline containing lead was banned on january 1, 2005 in peru (organismo supervisor de la inversión en energía y minería, 1998; tamayo, vásquez, & de la cruz, 2015). however, the parks and other green areas in this country were exposed to the gaseous byproducts of the combustion of this fuel for a long period prior to this and lead tends to remain in the soil for many years, making it highly probable that they are contaminated with lead and are now the main source of lead pollution in peru. lead is an extremely toxic element that can have terrible consequences. the world health organization stated that there is no amount of lead in the blood that can be considered safe, with levels as low as 5 μg/dl being capable of causing permanent damage (word health organization, 2014). most of the time, the symptoms of lead poisoning go unnoticed – for example, it can result in the cognitive development of children being severely delayed, as well as behavioral disorders and even hypertension. however, it can also cause more obvious symptoms, such as anemia, fatigue, insomnia, and gastrointestinal disturbances (word health organization, 2014; londoño, londoño, & muñoz, 2017). the use of hyperaccumulator plants for phytoremediation is probably the best strategy to decontaminate urban soils. compared with other remediation strategies, it can be carried out in situ, is not disruptive, and does not require huge amounts of money. however, because of an urban environment, it is necessary to use only ornamental plants. few ornamental plant species are currently registered as being capable of extracting heavy metals from the soil. however, nowak (2007) determined that the ornamental plant salvia splendens ‘sello torreador’ has the ability to accumulate up to 300 ppm of lead in its tissue without showing any symptoms of lead toxicity. consequently, we considered that s. splendens ‘red vista’ may have the same ability. therefore, in the present study, we investigated the ability of ‘red vista’ to extract lead from the soil, as well as the effects that mycorrhizae have on the phytoextraction of this heavy metal. materials and methods as indicated in the guide for soil sampling of the minister of the environment (ministerio del ambiente, 2014), we first performed identification sampling; this type of sampling is performed to identify if the soil is contaminated. soil samples were collected from two of the main parks in lima, peru [campo de marte (total area, 38 ha; n = 42 soil samples) and kennedy park (total area, 2.5 ha; n = 19 soil samples)] following the guide for soil sampling of the minister of the environment to determine the average concentration of lead in the soil and the sites with the highest concentrations of lead. identification sampling showed that 38 of the 42 samples obtained from campo de marte and four of the 19 samples obtained from kennedy park had concentrations of lead that exceeded the limits set by the environmental quality standards for the soil of parks and residential areas (140 ppm). detailed sampling was the second type of sampling performed, which was performed to identify the area of soil impacted by contamination. it was performed near the high-concentration sites in each park (campo de marte, n = 64 soil samples; kennedy park, n = 13 soil samples) identified after the identification sampling. during both sets of sampling, the soil samples were taken from the top 10 cm of the soil and were sent to the soil, plant, water and fertilizer analysis laboratory of the national agrarian university la molina, lima, peru (12°4ʹ24ʺs, 76°56ʹ10ʺw; utm 18l 0288166 8663907; 244 meters above sea level) for analysis. in addition, 100 kg of soil was taken from the top 10 cm of the soil profile in each park (in the case of campo de marte, only from sites with lead concentrations >140 ppm), which was the soil used in the bioassay. samples from high-concentration sites at campo de marte were used to represent urban soils with high lead concentrations, whereas samples from kennedy park were used to represent urban soils with low lead concentrations. the soil characteristics and the levels of other heavy metals of the soil obtained in each park are shown in tables 1 and 2. two types of soil (contaminated soil from campo de marte and uncontaminated soil from kennedy park), three types of water (deionized water, deionized water with 0.1 ppm table 1. characteristics of the soils obtained from campo de marte and kennedy park mechanical analysis cec exchangeable cations sum of cations source ph (1:1) e.c. (1:1) (ds/m) caco3 (%) m.o. % p (ppm) k (ppm) sand loam clay textural classification ca+2 mg+2 k+ na+ al+3 + h+ % % % meq/100 g kennedy park 7.49 0.51 1 2.67 48.5 137 75 14 11 sandy loam 15.5 13.33 1.67 0.34 0.18 0 15.52 campo de marte 7.3 0.7 0.3 9.47 21 236 59 20 21 clay sandy loam 27 23.9 1.82 0.56 0.27 0 26.56 vicente, e., tello, l. peruvian journal of agronomy 3 (2): 39-46 (2019) 41 table 2. analysis of the metal concentrations (ppm) in soil samples obtained from campo de marte and kennedy park. source cu fe zn mn cd cr pb kennedy park 16.60 45.10 23.70 4.50 1.46 24.7 71.61 campo de marte 296.00 82.00 255.00 2.50 9.36 91.49 200.86 of lead, and regular irrigation water with 0.1 ppm of lead), and two levels of mycorrhizae (0 and 2.5 g/kg of soil) were tested in the bioassay. to produce lead-contaminated water, lead acetate was added to the deionized water, which contained 0 ppm of lead, and the irrigation water, which contained 0.035 ppm of lead (see table 3), to give a final concentration of 0.1 ppm of lead. table 3: analysis of the regular irrigation water that was used in the bioassay analysis of the regular irrigation water that was used in the bioassay ph 7.41 e.c. ds/m 0.67 ca meq/l 4.87 mg meq/l 0.56 k meq/l 0.14 na meq/l 1.25 sum of cations 6.82 nitrates meq/l 0.01 carbonates meq/l 0 bicarbonates meq/l 2.95 sulfates meq/l 1.29 chlorides meq/l 2.5 sum of anions 6.75 na % 18.34 sar 0.76 b ppm 0.24 classification c2-s1 cu ppm 0.001 zn ppm 0.078 mn ppm 0.002 fe ppm 0.001 pb ppm 0.035 cd ppm 0.029 cr ppm 0 salvia splendens ‘red vista’ seeds were placed individually in the cells of seedbed trays that contained a mixture of sand and moss (1:4, v:v) as a substrate. in the case of the mycorrhizal treatment, this substrate was mixed with the product mycosym triton (active component: mycorrhizal fungus g. intraradices; inert substrate: lightweight expanded clay aggregate) at a concentration of 2.5 g per kilogram of substrate. the s. splendens seeds were planted in wet substrate and were watered with deionized water while they were in the seedbed trays. after 26 days, each seedling had at least two true leaves, at which time 36 of the plants were transplanted into separate pots (4 kg capacity; n = 3 pots per treatment). the bioassay was carried out in the facilities of the soil fertility laboratory of the national agrarian university la molina from october 22, 2016 to january 20, 2017. the plants were watered every monday, wednesday, and friday; and were measured at the same time to determine the lengths of the stems and inflorescences. each plant was then harvested 90 days after sowing in the seedbed trays and separated into three parts: inflorescence, stem and leaves, and roots. each part of the plant was weighed and washed with deionized water and then placed in a laboratory oven at 70°c for 48 hours, after which the weight was remeasured. each part was then ground and put in an erlenmeyer flask for acid digestion. the atomic absorption spectrophotometry technique was used to measure the lead concentration in each plant tissue. a completely randomized experimental design was used with a 3×2 factorial arrangement and three replications for each type of soil, giving a total of 18 experimental units in soil obtained from campo de marte and kennedy park, respectively (see table 4). table 4: description of the treatments soil treatment quality of water mycorrhizae t1 deionized no mycorrhizae t2 deionized mycorrhizae (2.5 g/kg) campo de marte t3 deionized + pb no mycorrhizae t4 deionized + pb mycorrhizae (2.5 g/kg) t5 irrigation + pb no mycorrhizae t6 irrigation + pb mycorrhizae (2.5 g/kg) t7 deionized no mycorrhizae t8 deionized mycorrhizae (2.5 g/kg) kennedy park t9 deionized + pb no mycorrhizae t10 deionized + pb mycorrhizae (2.5 g/kg) t11 irrigation + pb no mycorrhizae t12 irrigation + pb mycorrhizae (2.5 g/kg) a homogeneity of variances test and normality test were initially performed on the data. we performed analysis of variance (anova) to determine the influence of the variables on lead concentrations in the tissues and to determine the influence of the interaction between these variables on lead concentrations in the tissues. it is considered that in the case that the p value is greater than 0.05, the null hypothesis cannot be rejected; in other words, there are no significant differences in the treatments can salvia splendens ‘red vista’ be used in association with the mycorrhizal fungus glomus intraradices for the assisted phytoextraction of lead from the soil in lima, peru? may august 2019 42 analyzed. the initial and final concentrations of lead in the soil were then compared using anova and the student’s one-sample t-test. all statistical analyses were performed in spss (ibm spss statistics 23) using a significance level of p < 0.05. results campo de marte concentration of lead in the root tissue an anova test was performed to determine the influensce of each variable. the variable water (p value: 0.084) and the interaction between variable water and variable mycorrhizae (p value: 0.054) did not have a significant influence on the concentration of lead in the root tissue. however, plants that were grown in association with the mycorrhizal fungus g. intraradices (p value: 0.027) had a significantly higher concentration of lead in their root tissues (53.97 ppm) than those that were grown without mycorrhizae (39.41 ppm) (table 5). table 5. average concentrations of lead (ppm) in the root tissues of plants grown in soil obtained from campo de marte under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 29.59 55.34 33.30 39.41 c a m p o de marte mycorrhizae 66.33 53.41 42.17 53.97 avg* 47.96 54.37 37.73 46.69 *avg: average concentration of lead in the stem and leaf tissues an anova test was performed to determine the influence of each variable. the variable water (p value: 0.889 > 0.05), the variable mycorrhizae (p value: 0.123 > 0.05), and the interaction between variable water and variable mycorrhizae (p value: 0.661 >0.05) did not have a significant influence on the concentration of lead in the stem and leaf tissues. table 6. average concentrations of lead (ppm) in the stem and leaf tissues of plants grown in soil obtained from campo de marte under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized campo de marte no mycorrhizae 11.86 10.71 12.49 11.69 mycorrhizae 10.36 10.17 9.5 10.01 avg* 11.11 10.44 10.997 10.85 *avg: average plants that were grown in soil obtained from campo de marte did not translocate a significant amount of lead to the stem and leaves under any of the water or mycorrhizal conditions, with no significant difference between any of the treatments (table 6). concentration of lead in the inflorescence tissue an anova test was performed to determine the influence of each variable. the variable water (p value: 0.256), the variable mycorrhizae (p value: 0.123), and the interaction between variable water and variable mycorrhizae (p value: 0.874) did not have a significant influence on the concentration of lead in the inflorescence tissue. plants that were grown in soil obtained from campo de marte did not translocate a significant amount of lead to the inflorescence under any of the water or mycorrhizal conditions, with no significant difference between any of the treatments (table 7). table 7. average concentrations of lead (ppm) in the inflorescence tissues of plants grown in soil obtained from campo de marte under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 16.12 14.80 13.07 14.66 campo de marte mycorrhizae 13.14 13.03 9.06 11.75 avg* 14.63 13.91 11.07 13.20 *avg: average length of the stem an anova test was performed to determine the influence of each variable. the variable water (p value: 0.187), the variable mycorrhizae (p value: 0.255), and the interaction between variable water and variable mycorrhizae (p value: 0.981) did not have a significant influence on the length of the stem. the presence of lead in the soil obtained from campo de marte stunted the growth of the aerial parts of the plants, with no significant difference in the length of the stem between any of the treatments (table 8). table 8. average lengths (cm) of the stems of plants grown in soil obtained from campo de marte under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 10.77 9.5 11.23 10.5 campo de marte mycorrhizae 9.77 8.83 10.4 9.67 avg* 10.27 9.165 10.815 10.08 *avg: average vicente, e., tello, l. peruvian journal of agronomy 3 (2): 39-46 (2019) 43 length of the inflorescence an anova test was performed to determine the influence of each variable. the variable water (p value: 0.899), the variable mycorrhizae (p value: 0.369), and the interaction between variable water and variable mycorrhizae (p value: 0.222) did not have a significant influence on the length of the stem. the presence of lead in the soil obtained from campo de marte also had a stunting effect on the length of the inflorescence for all treatments (table 9), decreasing the ornamental value of the plants. table 9. average lengths (cm) of the inflorescences of plants grown in soil obtained from campo de marte under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 14.20 17.67 13.87 15.24 campo de marte mycorrhizae 13.70 12.00 15.43 13.71 avg* 13.95 14.83 14.65 14.48 *avg: average kennedy park concentration of lead in the root tissue an anova test was performed to determine the influence of each variable. the variable water (p value: 0.002), the variable mycorrhizae (p value: 0.045), and the interaction between variable water and variable mycorrhizae (p value: 0.010) did have a significant influence on the concentration of lead in the root tissue. plants that were grown in association with g. intraradices accumulated more lead in their roots, as did plants that were watered with contaminated water, with plants that received both treatments accumulating the most lead (table 10). table 10. average concentrations of lead (ppm) in the root tissues of plants grown in soil obtained from kennedy park under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 18.53 26.02 21.76 22.10 kennedy park mycorrhizae 17.823 25.185 34.925 25.98 avg* 18.18 25.60 28.34 24.04 *avg: average concentration of lead in the stem and leaf tissues an anova test was performed to determine the influence of each variable. the variable water (p value: 0.121) and the interaction between variable water and variable mycorrhizae (p value: 0.946) did not have a significant influence on the concentration of lead in the stem and leaf tissues; however, the variable mycorrhizae (p value: 0.012) did have a significant influence on the concentration of lead in the stem and leaf tissues. plants that were grown in association with g. intraradices accumulated significantly more lead than those that were grown without mycorrhizae. (table 11) table 11. average concentrations of lead (ppm) in the stem and leaf tissues of plants grown in soil obtained from kennedy park under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 7.44 10.75 9.82 9.34 kennedy park mycorrhizae 11.02 15.4 14.35 13.59 avg* 9.23 13.08 12.08 11.46 *avg: average concentration of lead in the inflorescence tissue an anova test was performed to determine the influence of each variable. the variable water (p value: 0.135), the variable mycorrhizae (p value: 0.107), and the interaction between variable water and variable mycorrhizae (p value: 0.068) did not have a significant influence on the concentration of lead in the inflorescence tissue. plants that were grown in soil obtained from kennedy park did not translocate a significant amount of lead to their inflorescences, with no significant differences between any of the treatments (table 12). table 12. average concentrations of lead (ppm) in the inflorescence tissues of plants grown in soil obtained from kennedy park under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 10.52 10.02 10.34 10.29 kennedy park mycorrhizae 9.36 11.91 12.54 11.27 avg* 9.94 10.97 11.44 10.78 *avg: average length of the stem an anova test was performed to determine the influence of each variable. the variable water (p value: 0.683), the variable mycorrhizae (p value: 0.187), and the interaction between variable water and variable mycorrhizae (p value: 0.287) did not have a significant influence on the length of the stem. the length of the stem was similar for all of the plants that were grown in soil obtained from kennedy park, with no significant differences between any of the treatments (table 13). can salvia splendens ‘red vista’ be used in association with the mycorrhizal fungus glomus intraradices for the assisted phytoextraction of lead from the soil in lima, peru? may august 2019 44 table 13. average lengths (cm) of the stems of plants grown in soil obtained from kennedy park under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 10.33 10.53 11.47 10.78 kennedy park mycorrhizae 11.03 9 7.6 9.21 avg* 10.68 9.77 9.54 9.99 *avg: average length of the inflorescence an anova test was performed to determine the influence of each variable. the variable water (p value: 0.126) and the interaction between variable water and variable mycorrhizae (p value: 0.583) did not have a significant influence on the length of the inflorescence; however, the variable mycorrhizae (p value: 0.47) did have a significant influence on the length of the inflorescence. plants that were grown in association with g. intraradices had a lower average inflorescence length than plants that were grown without mycorrhizae and showed symptoms of lead toxicity that were similar to the plants that were grown in contaminated soil obtained from campo de marte. (table 14). table 14. average lengths (cm) of the inflorescences of plants grown in soil obtained from kennedy park under different water and mycorrhizal regimes mycorrhizae water deionized + pb irrigation + pb avg* deionized no mycorrhizae 22.47 19.07 18.90 20.14 kennedy park mycorrhizae 19.07 16.37 11.00 15.48 avg* 20.77 17.72 14.95 17.81 *avg: average discussion the concentration of lead in soil obtained from campo de marte did not affect the growth of s. splendens ‘red vista’ plants (figure 1) but did affect other characteristics associated with their ornamental value (i.e., the volume and number of secondary inflorescences). salvia splendens ‘red vista’ plants growing in soil obtained from campo de marte accumulated an average of 46.69 ppm of lead in the roots, 10.85 ppm of lead in the stems and leaves, and 13.20 ppm of lead in the inflorescences. the concentration of lead in the soil increased from 200.86 ppm to 230.40 ppm during the bioassay. the results of the student’s one-sample t-test indicated that there was a significant difference between the initial concentration of lead in the soil and the final concentration of lead in the soil (p value: 0.000; degrees of freedom: 17. the p value was less than 0.05; therefore, there was a statistically significant difference), indicating that most of the lead added through contaminated water stayed in the soil, which suggests that only a very small amount of lead was available for the plant and most of it was retained in the root tissue. the soil at campo de marte contained 9.47% organic matter and had a cation exchange capacity of 26.56 cmol+/kg and a ph of 7.3. lead is a heavy metal that has a high affinity to form stable compounds with organic matter that cannot be taken up by plants, and at a ph of 4.0 or higher, lead tends to be strongly retained by the humic matter (steinnes, 2013). furthermore, this soil also contained a high concentration of copper (296 ppm), which tends to precipitate with lead to form very stable compounds. therefore, since s. splendens ‘red vista’ did not have high concentrations of lead in its tissues and exhibited symptoms of lead toxicity that were similar to those described by nowak (2007), we should assume that this variety cannot be considered a phytoextractor plant and should not be considered for use in phytoremediation strategies at least under similar conditions to the current bioassay. figure 1. growth of plants in soil obtained from campo de marte the concentration of lead in the soil obtained from kennedy park did not affect the growth (figure 2) or ornamental value of the s. splendens ‘red vista’ plants except in treatments t10 and t12 (treatments with mycorrhizae and water contaminated with lead), in which case they exhibited very similar symptoms to the plants that were grown in soil obtained from campo de marte. salvia splendens ‘red vista’ plants that were grown in soil obtained from kennedy park accumulated an average of 24.04 ppm of lead in their roots, 11.46 ppm of lead in their stems and leaves, and 10.78 ppm of lead in their inflorescences. the concentration of lead in the soil decreased from 71.61 ppm to 68.795 ppm during the bioassay, and the results of the student’s one-sample t-test indicated that there was no significant difference between the initial concentration of lead in the soil and the final concentration of lead in the soil (p value: 0.054; degrees of vicente, e., tello, l. peruvian journal of agronomy 3 (2): 39-46 (2019) 45 freedom: 17. the p value was higher than 0.05; therefore, there is no statistically significant difference), indicating that most of the lead added through contaminated water did not stay in the soil and most of it was not taken by the plant).the kennedy park soil contained 2.67% organic matter, had a sandy loam texture, and did not contain high concentrations of any element that would precipitate with lead to form very stable compounds. consequently, the lead that was added to the water was not strongly absorbed by the soil, which contained only a low concentration of lead, but rather was quickly washed out of the soil by water used for irrigation or taken up by the plants, sometimes causing them to show symptoms of lead toxicity that were similar to plants that were grown in contaminated soil (observed in t10 and t12). figure 2. growth of plants in soil obtained from kennedy park although the average length of the stem was similar across all treatments, the final average length of the inflorescence was lower in plants that were grown in association with mycorrhizae (15.48 cm) than in those that were grown without mycorrhizae (20.14 cm). mycorrhizal fungi, in this case g. intraradices, allow plants to explore a greater volume of soil and increase the amount of nutrients and other elements the plant can extract. the plants did not translocate a significant amount of lead to their inflorescences but the presence of mycorrhizae helped them to accumulate lead in their roots, which probably explains why the translocation of valuable nutrients decreased, leading to a shorter inflorescence. when lead was added to the water (treatments t10 and t12), a reduction in the ornamental value was observed in addition to the reduction in inflorescence length. thus, the mycorrhizae had a negative effect on the plants, which was best observed when these plants were watered with lead-contaminated water. translocation and bioconcentration factors the translocation factor was calculated by dividing the concentration of lead in the aerial parts of the plant by that in the roots, while the bioconcentration factor was calculated by dividing the concentration of lead in the roots by that in the soil (tu, ma, & bondada, 2003; rizzi, petruzzelli, poggio & vigna guidi, 2004; maldonado, favela, rivera & volke 2011). the translocation factor was 0.26 for plants grown in soil from campo de marte and 0.46 for plants grown in soil from kennedy park, while the bioconcentration factor was 0.21 for plants grown in soil from campo de marte and 0.34 for plants grown in soil from kennedy park. therefore, since values of at least one are required to consider a species as having some kind of phytoextraction ability, these values indicate that s. splendens ‘red vista’ is unable to extract high amounts of lead from the soil and thus cannot be considered a hyperaccumulator species. conclusion the ornamental plant s. splendens ‘red vista’ was unable to extract high amounts of lead from the soil even when it was grown in association with the mycorrhizal fungus g. intraradices, demonstrating that under the tested conditions it does not behave as a hyperaccumulator plant. references steinnes, e. (2013). lead. in b. alloway (ed.) heavy metals in soils. (pp. 395-411) usa: springer. londoño, l., londoño, p., & muñoz, f. (2017) risks of heavy metals in human andanimal health. biotecnologia en el sector agropecuario y agroindustrial, 14(2), 145 153. doi:10.18684/bsaa(14)145-153 maldonado m.a., favela t.e., rivera c.f. & volke s.t.l. (2011). lead bioaccumulation in acacia farnesiana and its effect on lipid peroxidation and glutathione production. plant and soil, 339, 377-389. ministerio del ambiente. (2014). guía para el muestreo de suelos. perú: mavet impresiones e.i.r.l. nowak, j. (2007). effects of cadmium and lead concentrations and arbuscular mycorrhiza on growth, flowering, and heavy metal accumulation in scarlet sage (salvia splendens sello ‘torreador’). acta agrobotanica, 60, 79-83. organismo supervisor de la inversión en energía y minería. (1998). aprueban el reglamento de infracciones y sanciones al d.s.no.019-98-mtc que dispuso eliminar del mercado la oferta de gasolina 95ron con plomo. resolución 405-98-os/cd. in el peruano. [november 4, 1998]. rizzi l., petruzzelli g., poggio g. & vigna guidi g. (2004). soil physical changes and plant availability of zn and pb in a treatability test of phytostabilization. chemosphere. 57, 1039-1046. can salvia splendens ‘red vista’ be used in association with the mycorrhizal fungus glomus intraradices for the assisted phytoextraction of lead from the soil in lima, peru? may august 2019 46 tamayo, j., vásquez, a., & de la cruz, r. (2015). la industria de los hidrocarburos en el perú: 20 años de aporte al desarrollo del país. perú. osinergmin. tu c., ma l.q. & bondada b. (2003). arsenic accumulation in the hyperaccumulator chinese brake and its utilization potential for phytoremediation. journal of environmental quality. 31, 1671-1675. world health organization (2014). semana de acción internacional para prevenir el envenenamiento por plomo: del 19 al 25 de octubre de 2014, organización mundial de la salud. retrieved from https://www.who. int/phe/health_topics/faq_lead_poisoning_prevention_ campaign_es.pdf peruvian journal of agronomy 2 (3): 2734 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i3.1230 © the authors. published by universidad nacional agraria la molina received for publication: 15 november 2018 accepted for publication: 10 december 2018 cultural, biological and chemical control of the white rot fungus (sclerotium cepivorum, berk) in onions (allium cepa) in arequipa´s countryside control químico, biológico y cultural de la pudrición blanca (sclerotium cepivorum, berk) de la cebolla (allium cepa) en la campiña de arequipa m. gonzales1*; l. mattos2 *corresponding author: mariacgonzales@hotmail.com abstract this experiment was carried out at the province of arequipa, to determine broccoli residual effects on the sclerotia population of sclerotium cepivorum on the soil and determine the best fungicides and biocontrol agents on the biggest red onion production. in two field areas infected with sclerotium cepivorum, soil samples were carried out to determine the number of sclerotia at the beginning and the end of the trial. one area received broccoli residues before treatment installation. the experiment had a laboratory and a field phase. the block design was used completely randomized (dbca), using seven fungicide treatments (t1) control, iprodione (t2), thiabendazole (t3), boscalid (t4), carbendazim (t5), t harzianum (t6), bacillus subtilis (t7). both areas received the same treatments. the density of sclerotia in the soil was not significant between these areas. however, the addition of broccoli had lower percentages of incidence and severity and higher yields in all treatments. at the area that received broccoli, the boscalid (t4) and the iprodine (t2) fungicide were highlighted with 43.33 t/ha and 28.33 t/ha, respectively; the area without broccoli, the t4 with 38.33 t/ha and t2 with 25.56 t/ha. t5 (carbendazim) had the lowest yield: 15.00 t/ha without broccoli and 19.58 t/ha with broccoli. keywords: biocontrol agent, sclerotia, incidence, severity, pathogen. resumen el experimento fue llevado a cabo en la provincia de arequipa, con el objetivo de determinar el efecto de residuos de brócoli sobre la población de esclerotes de sclerotium cepivorum en el suelo y determinar los mejores fungicidas y biocontroladores en la mayor producción de la cebolla roja. en dos áreas de un campo infestado con sclerotium cepivorum se realizaron muestreos de suelo para determinar el número de esclerotes al inicio y al término del ensayo. un área recibió residuos de brócoli antes de instalar los tratamientos. el experimento tuvo una fase de laboratorio, y una fase de campo. se utilizó el diseño de bloques completamente al azar (dbca), con 7 tratamientos fungicidas (t1) control, iprodione (t2), thiabendazole (t3), boscalid (t4), carbendazim (t5), t harzianum ( t6), bacillus subtilis (t7). ambas áreas recibieron los mismos tratamientos. la densidad de esclerotes presentes en el suelo no fue significativa entre las áreas; sin embargo, la incorporación de brócoli favoreció a menores porcentajes de incidencia y severidad, y mayores rendimientos en todos los tratamientos. en el área incorporada con brócoli, destacaron boscalid (t4) e iprodione (t2) con 43.33 t/ha y 28.33 t/ha respectivamente; el área sin brócoli el t4 con 38.33 t/ha y t2 con 25.56 t/ha. el rendimiento más bajo lo tuvo el t5 (carbendazima): 15.00 t/ha sin brócoli y 19.58 t/ha con brócoli. palabras claves: biocontrolares, esclerotes, incidencia, severidad, patógeno. 1 docente auxiliar. facultad de agronomía-universidad nacional san agustín de arequipa (unsa). egresada de la maestría en fitopatología de la universidad nacional agraria la molina. 2 docente principal. departamento académico de fitopatología, facultad de agronomía, -universidad nacional agraria la molina (unalm). introduction red onion is a vegetable of great importance and with the highest internal consumption nationwide, which is grown intensely in different areas. it also has a great social relevance since its production creates a permanent source of labor and favors, indirectly, the economic family income. national onion production is aimed to mainly cover the internal market, being red onion, the primary type of onions produced, which is focused, mostly, in arequipa, department taking part and generating relevant amounts of production volumes, making more than the 70% of the national production (siea, 2012). production is influenced by many factors that determine the growing yields and final quality of harvests. it should be noted within these factors the negative effect diseases caused. species from the allium kind, are widely damaged by the sclerotium cepivorum berk fungus (schwartz &mohan, 2008). in arequipa, onion growing is at high risk of being affected by the white rot, directly damaging the commercial product. this pathogen causes significant losses that might rise to the 100% since it is difficult to control and because control strategies used cultural, biological and chemical control of the white rot fungus (sclerotium cepivorum, berk) in onions (allium cepa) in arequipa´s countryside september december 2018 28 are poorly efficient, and so, many farmers have changed crops definitely or keep cultivating in completely infested grounds, which represents high production costs and considerable crop losses. taking into consideration the great economic importance of this growing and the inefficiency of traditional control and preventive methods this research paper attempted to find feasible alternatives regarding the phytopathogen control that causes white rot, by means of biological and chemical controls and the addition of organic residues. objectives • determine the effect of broccoli residue addition on the sclerotia density found in the soil. • determine the best fungicides and biocontrol agents that allow better quality and higher production of red onions. materials and methods experiment location the experiment was carried out in two phases, one in the laboratory and the other at field conditions. the laboratory phase was developed at the laboratorio del departamento de fitopatología (department of phytopathology´s laboratory) from the universidad nacional agraria la molina. and the field phase was carried out at the chilina valley, district of cayma, province and department of arequipa, located at 16°28`south latitude, 71°27`west latitude and 2,300 m.a.s.l. sampling collection infested onion bulbs collected were those that showed symptoms (yellowish color on outer leaves, wilt) and signs (sclerotia and mycelium) of the sclerotium cepivorum berk pathogen. collected samples were placed in paper bags and taken to the laboratory for pathogen isolation. isolation of sclerotium cepivorum small portions of the infected tissue were cut, covering the disease growing area and survival structures. these pieces were disinfected using sodium hypochlorite at 1% (during 5´ for the affected tissue and 10´ for sclerotia); then they were grown in petri dishes with potato dextrose agar oxytetracycline (pdao), incubated at 20ºc. once fungi colony was developed, subcultures were made until obtaining pure cultivation. laboratory phase a) in vitro biological and chemical control test: the potato dextrose agar (pda) medium was prepared to carry out the poisoned food test. in an erlenmeyer containing the selective medium (45ºc), a fungicide treatment was added along with the mixture homogenization and this was plated inside an aseptic chamber, using a 0.5 cm diameter punch, small areas of sclerotium cepivorum mycelium were cut and placed in the middle of the surface of the medium contained in the plate. the procedure from the biological control test was the same used for the chemical control. commercial and biological fungicide treatments used for this test are shown in table 1. the design was completely randomized (dca, by its spanish acronym), having four repetitions per treatment. the evaluation consisted of making daily entries of the radial growth of the fungi colony developed in the plate containing poisoned food, and these were compared to the reference samples. the experiment concluded when the reference sample showed fungi increase along the entire surface of the medium; the most efficient fungicides where those that did not allow the pathogen growth. table 1: in vitro treatments for the sclerotium cepivorum control treatments active ingredients commercial name dose (g,ml/100l) type of product t1 t2 t3 t4 t5 t6 t7 control iprodione thiabendazole boscalid carbendazim t harzianum bacillus subtilis rovral 50% pm mertect 500 sc cantus 500 wg protexin 500fw tricho-d serenade 0.0 g 0.2 g 0.125ml 0.2 g 0.25 ml 0.15g 0.5 ml none contact systemic systemic systemic biocontrol ag. biocontrol ag. field phase a) ground preparation and delimitation of experiment area: experiment was located at an agricultural land for commercial onion production having a white rot (sclerotium cepivorum berk) presence background. b) experiment field characteristics: this study was carried out in a total area of 450 m2, which was divided into two equal sections of 225 m2 and located at each one blocks and patches of 6m2. in one of the experiment area sections, broccoli residues were added in a quantity of 5 kg/m2, and it was covered with a transparent polyethylene fabric, and later, weekly irrigation was performed to make the residue decomposition process easier. after 30 days, furrow mapping and onion seedling plantation on the definite field at a 250,000 plant/ha density was carried out. the experiment was made under the completely randomized block design (dbca) for the effect of fungicides and biocontrol agents in front of the pathogen, with 21 experimental units, three blocks with seven randomly distributed treatments per each block, both for the experiment area with broccoli addition and for the experiment area without broccoli addition. an individual analysis was carried out for each patch (with and without broccoli) and also, a homogeneity test of variances so to later proceed to make the combined analysis, where the brassica factor was included to analyze its effect. the statistical test f was performed for the different mean squares and the turkey test at 0.05 of significance to compare averages of m. gonzales; l. mattos peruvian journal of agronomy 2 (3): 27-34 (2018) 29 treatments. treatments (fungicides and biocontrol agents) used for this phase were the same used for the in vitro test, for both experimental field sections (table 2). these treatments have been conducted in two experiments: • experiment 1: soil with no brassica addition. • experiment 2: soil with brassicas. experimental field sections was determined by quantifying the final population (pf) by the end of the campaign. disease incidence: the percentage of healthy and diseased plants was determined by the symptoms at the foliar part (wilt), every 15 days and by taking 30 plants as a sample, which was located at the middle of each patch. epidemics were compared by estimating the area under the disease progress curve (audpc), calculations were made using the sas statistical package, and a variance analysis to compare the audpc based on the tukey test (p<0.05). severity: by damage in harvested bulbs, examining a 30-plant sample, roots and bulbs with pathogen signs and according to the affected area percentage in degrees. the assessment scale used was a variation to the scale proposed by arenas, 1997. yield: the production by kg from each treatment and each repetition was evaluated. results in vitro test. isolation of sclerotium cepivorum: colonies of sclerotium cepivorum isolated in pdao showed the following characteristics: white colony, during the entire mycelial growth, very fine cottony mycelium adhered to the substrate, radial growth colony. throughout the colony growth, small conglomerations of mycelium were formed or formation of sphere-shaped black sclerotia. in vitro biological and chemical control of sclerotium cepivorum: chemical products tested “in vitro” inhibited the development of sclerotium cepivorum as well as biological products. chemical products that completely inhibited pathogen growth were iprodione (t2) and carbendazim (t5); therefore, they the most effective products; meanwhile, fungicides thiabendazole (t3) and boscalid (t4) allowed the development of the pathogen before completely inhibit it. on the other hand, biocontrol agents also effectively inhibited pathogen growth. bacillus subtilis inhibits the development of s. cepivorum more rapidly than t. harzianum. boscalid and t. harzianum products in the anva test and statistical significance turned out to be highly significant regarding the other treatments and at the tukey multiple comparison test (p<0.05) it could be observed that in thianendazol, iprodione, b. subtilis, carbendazim products there are not statistically significant differences among them. finally, there is a highly significant difference of the reference in regards of treatments, since its growth was satisfactory and completed its growth upon four days. in table 3, results from the variance analysis and the statistical significance from the sclerotium cepivorum growth inhibition by fungicide and biocontrol agent inhibition are shown. table 2. treatments for the control of sclerotium cepivorum on field for both experimental field areas. treatments active ingredients commercial name dose (g,ml/200l) type of product t1 control none 0.0 g none t2 iprodione rovral 50% pm 400 g contact t3 thiabendazole mertect 500 sc 250 ml systemic t4 boscalid cantus 500 wg 400 g systemic t5 carbendazim protexin 500fw 500 ml systemic t6 t harzianum tricho-d 300 g biocontrol ag. t7 bacillus subtilis serenade 1000 ml biocontrol ag. c) sclerotia count of sclerotium cepivorum berk.: the initial population (pi, by its spanish acronym) of sclerotia for both experimental field sections and the final population (pf, by its spanish acronym) was determined by the end of the experiment. samples of 1kg of soil per patch were taken and as of treatment, considering each point from each experimental unit. collected samples were placed in a paper bag and taken to a 60°c stove for five days. the sucrose flotation technique (utkhede & rahe, 1979) was to determine the sclerotia population, and they were put under the stereoscope to count the amount of sclerotia present. a homogeneity test of variances was made between brassica patches and without brassica patches, where it was determined that there is enough homogeneity to be able of performing a combined analysis. d) biological and chemical control test on field: applications of fungicides and biocontrol agents were made in two instances: first application: after transplantation, a total of 150 plants were treated by root immersion for 15 minutes, in a fungicide solution or biocontrol agents according to the treatment and appointed doses. second application: after the appearance of first symptoms, which were presented upon 60 days after the transplantation (ddt). the application was made on the plant stem, using a 20-litre bag at a continuous stream. third application: during the crop´s critical time, which corresponds to the formation and thickening of the bulb. application was done to the plant stem upon 105 (ddt), using a 20-liter bag at a continuous stream. e) variables to be evaluated: initial population (pi) and final population (pf) of sclerotia: the initial population (pi) of sclerotia from both cultural, biological and chemical control of the white rot fungus (sclerotium cepivorum, berk) in onions (allium cepa) in arequipa´s countryside september december 2018 30 table 3. mycelial growth (mm) of sclerotinia cepivorum in in vitro test treatments evaluated products radial growth (mm) inhibition (%) tukey ∞0.05 t1 control 43.44 0.00 a t2 iprodione 0.00 100.00 c t3 thiabendazole 1.50 96.50 c t4 boscalid 15.33 64.70 b t5 carbendazim 0.00 100.00 c t6 t.harzianum 13.44 69.10 b t7 b. subtilis 0.44 99.00 c field results experiment with no broccoli addition final and initial sclerotia population: the number of sclerotia in treatments and repetitions was variable; however, the variance analysis (anva) shows that there are no significant statistical differences between blocks and between treatments, with a coefficient of variation (cv) of 30.56%. the initial inoculum of sclerotium cepivorum was increased in treatments; however, there are no significant statistical differences between blocks and between treatments for final sclerotia population, with a coefficient of variation (cv) of 28.38 %. incidence: in the variance analysis there are no statistical differences between blocks, but in fact, significant differences between treatments have been observed; there are differences between fungicides applied for controlling the sclerotium cepivorum. when performing the comparison test as of tukey, the t1 (reference), is statistically equal to treatments with fungicide: t5 (carbendazim), and biocontrol agents: t7 (b. subtilis) and t6 (t. harzianum), recording the biggest incidences, and therefore, the pathogen behavior was not affected by the effect of fungicides and biocontrol agents. the t4 (boscalid) showed a better fungicidal effect against the pathogen since it showed a lower incidence percentage of 17.78%, table 4. comparison test of tukey, the t1 and t5, there are no significant differences and the disease development was statistically higher in audpc values; while at treatment 4 (boscalid), there are significant differences between treatments, since it shows a lower value of audpc. table 4. the efficiency treatment percentage, with a higher rate of s. cepivorum control, was, t4 with 70.82%, followed by t2 with 41.64%. severity: the variance analysis, anva, determined that there are significant differences between blocks and treatments, with a c.v of 11.33 %, severity in the bulb was highly significant between treatments, and during treatments, it was shown that they had the highest incidence percentage. the t4 presented highly significant differences among them, and also shown the lowest severity levels, with a percentage of 11.67 %, which corresponds to degree 3, with the presence of pathogen´s plant structures in roots and the outer part of the bulb, which allow classifying these bulbs as commercial ones. the reference treatment presented the highest severity levels with 54.63 % of severity, which corresponds to a degree 5, with the presence of non-usable or non-commercial bulbs. table 5 shows tukey´s comparison test and the severity percentage in treatments. yields: the harvested commercial bulb yield was classified by first quality, second quality and total yield; variance analysis anva were carried out for each one, where for first yields highly significant differences were shown between blocks and treatments with a coefficient of variation of 22.03%. this shows that yields both from blocks and treatments were variable, presenting certain difference due to the high sclerotia density and high disease incidence, and as a consequence, different severity degrees were shown in the several phenological stages of the growing, impacting yields as a result. the variance analysis in the second quality yield does not show significant differences between blocks and treatments, with a coefficient of variation of 24.80 %. as for the total yield, there are no differences between blocks, but they are between treatments. first quality yield, the t4 (boscalid) showed the highest values with 28.06 t/ha, and it was significantly different in regards of the other treatments, lowest levels of yield were for the reference and t5 (carbendazim) with 4.31 and 8.06 t/ ha, respectively. as for the second quality yield, no significant differences between treatments are shown. finally, as for the total yield, treatment 4 had the highest yield with 38.33 t/ha, and with a lower incidence percentage and lower degree of severity of bulbs, followed by treatment 2, (iprodione) with 25.56 t/ha. the lowest yield was shown in treatment 5 (carbendazim) with 15 t/ ha. table 5. table 4. percentage of disease incidence, area under disease progress curve (audpc), severity and control treatment percentage, on field without broccoli. (alfa=0.05) treatment incidence sig. % of disease % of control audpc sig. severity sig. t1 (control) 70.0 a 100.0 0.0 2941.7 a 54.6 a t2 (iprodione) 52.2 a b 58.4 41.6 1716.7 a 32.6 b t3 (thiabendazole) 48.9 a b 64.3 35.7 1891.7 a b 38.0 a b t4 (boscalid) 17.8 b 29.2 70.8 858.3 a b 11.7 c t5 (carbendazim) 74.4 a 100.0 0.0 2941.7 b 50.9 a b t6 (t.harzianum) 56.7 a 68.8 31.2 2025.0 b c 43.9 a b t7 (b. subtilis) 63.3 a 89.0 11.1 2616.7 c 39.5 a b a comparison of epidemics was carried out using the area under the disease progress curve (audpc). in the m. gonzales; l. mattos peruvian journal of agronomy 2 (3): 27-34 (2018) 31 table 5. test for first and second yields and total yield with different fungicide treatments with no broccoli addition. (alfa=0.05). treatment first yield t/ha second yield t/ha total yield t/ha mean sig. mean mean sig. t1 (control) 4.31 b 11.67 15 b t2 (iprodione) 11.06 b 13.89 25.56 ab t3 (thiabendazole) 9.72 b 15 24.72 ab t4 (boscalid) 28.06 a 10.28 38.33 a t5 (carbendazim) 8.06 b 6.94 15.97 b t6 (t.harzianum) 8.94 b 13.89 22.83 ab t7 (b. subtilis) 8.19 b 12.78 20.97 b experiment with broccoli addition initial and final sclerotia population: the number of sclerotia in treatments and blocks was variable, as a result, there several sources of initial inoculum. however, the variance analysis performed (anva) shows that there are no significant statistical differences between blocks and treatments, with a coefficient of variation (cv) of 29.42; the variance analysis performed (anva) showed that the final population of sclerotia in the experimental field does not present significant statistical differences between treatments and between blocks, with a coefficient of variation (cv) of 29.40 %. incidence: there are statistical differences between blocks and highly significant differences between treatments with a coefficient of variation of 9.11%. at the comparison test of tukey, the t1 (reference) and t5 (carbendazim) are statistically equal and presented the highest percentages of final incidence; while t3 (thiabendazole), t6 (t. harzianum) and t7 (b. subtilis) presented an effect on the pathogen statistically equal, since there are no differences between these treatments. lowest final incidence percentages and best fungicidal effect against the pathogen was presented in t4 (boscalid). table 6. table 6: percentage test for disease incidence, audpc, severity and control percentage of treatments, on field with broccoli addition. (alfa=0.05). treatment incidence sig. audpc sig. % of disease % of control severity sig. t1 (control) 68.9 a 2700.0 a 100.0 0.0 48.9 a t2 (iprodione) 45.6 b 1525.0 a b 56.5 43.5 34.7 a t3 thiabendazole) 53.3 a b 1650.0 b 61.1 38.9 35.7 a t4 (boscalid) 11.1 c 566.7 c 21.0 79.0 7.0 b t5 (carbendazim) 67.8 a 2366.7 c 87.7 12.4 39.9 a t6 (t.harzianum) 55.6 a b 1766.7 c 65.4 34.6 40.9 a t7 (b. subtilis) 54.4 a b 2183.3 d 80.9 19.1 37.9 a at the area under the disease progress curve (audpc) there are no significant differences between blocks, but there are highly significant differences between treatments, and thus the epidemiological development in treatments had a different behavior among them. in the comparison test between t1 (reference) and 5 (carbendazim), there are no significant differences. the t4 (boscalid) shows a lower value of audpc, and therefore, there is a highly significant difference between treatments. as for the coefficient of variation, the highest percentage of control for s cepivorum with t4 is 79.01%, followed by t2 with 43.52%, the lowest percentage of control was obtained from the treatment 5 with a percentage of 12.35%. table 6. severity: the variance analysis anva determined that there are no statistical differences between blocks but there are significant differences between treatments with a c.v of 9.40 %, the biggest damage in bulbs was presented in t1 (reference) with a 48.89 %, which corresponds to a degree 5, according to the assessment scale, where plant and conservation structures from the pathogen and nonprofitable bulbs are shown. the t4 (boscalid) with a severity percentage of 7.04 % corresponding to a degree 3 of severity, where harvested bulbs can be considered as profitable, or with low severity levels; therefore, t4 in regards of the other treatments, presented highly significant differences, likewise, incidence levels of the disease were lower in this treatment, increasing upon 105 days, bulb filling stage, and remaining constant throughout time. table 6. yield: variance analysis for first and second quality bulbs shows significant statistical differences between blocks and treatments, with a coefficient of variation of 23.43 % and 31.17 % respectively. the total yield shows that there are no differences between blocks; however, there were differences between treatments with a coefficient of variation of 15.72 %. in the comparison test for first quality yields, the t4 (boscalid) showed the highest values with 38.33 t/ha, and it was significantly different regarding the other treatments, including the reference treatment, which showed the lowest levels of yield along with treatment 5 (carbendazim) with 5.19 and 8.28 t/ha, respectively. the biggest second quality yield is shown in the reference treatment with 20.83 t/ha, with a tendency of having a yield similar to the other treatments, excepting for the t4 (boscalid). the highest total yield was presented by t4 with 43.33 t/ha, with significant differences between treatments using fungicides and biocontrol agents. table 7. cultural, biological and chemical control of the white rot fungus (sclerotium cepivorum, berk) in onions (allium cepa) in arequipa´s countryside september december 2018 32 table 7: test for first and second quality yields and total yield with different fungicide treatments with broccoli addition. (alfa=0.05). treatment first yield second yield total yield t/ha t/ha t/ha media sig. media sig. media sig. t1 (control) 5.3 b 5.0 b 19.6 b t2 (iprodione) 12.8 b 18.1 ab 30.1 b t3 (thiabendazole) 12.1 b 15.8 ab 28.3 b t4 (boscalid) 38.3 a 20.8 a 43.3 a t5 (carbendazim) 8.2 b 11.4 ab 22.5 b t6 (t.harzianum) 11.7 b 15.6 ab 27.5 b t7 (b. subtilis) 9.7 b 12.8 ab 26.1 b combined analysis of the two experiments with broccoli and without broccoli final and initial sclerotia population: the combined analysis in its variation block source within brassicas; treatment brassicas and brassica treatment interaction does not show a significant statistical difference. the comparison test of brassicas factor in the two experimental fields (with and without broccoli) did not show significant differences in the final and initial sclerotia population. incidence: the combined analysis does not show statistical differences between the final incidence and audpc, so to determine the difference between these two experimental fields and final incidence treatments and audpc, a tukey´s multiple comparison tests were performed. where fields with an addition of broccoli and with no broccoli show no significant differences, thus, brassica effect did not impact on the pathogen behavior; however, audpc and final incidence averages for the field with addition of broccoli were lower in final incidence percentage with 50.95 % and audpc with 1822.6. the comparison test of the combined final incidence analysis shows statistical differences between chemical and biological treatments. the t4 showed highly significant differences with a lower final incidence percentage of 14.4 %. t1 and t5 are the treatments with the highest values on the development of the disease progression curve audpc, there were no significant differences with treatment 7, but there were statistical differences regarding the other treatments. treatment 4 showed significant differences between treatments using biocontrol agents and treatments using fungicides, with a lower level in audpc of 712.50. fig.1. the best treatment was boscalid; treatments thiabendazole and carbendazim had a poor effect on the pathogen, which did not stop the disease development. product efficiency decreases, and more applications will be needed to reach the same control percentage since control effect from fungicides is partial. as for the control effect from biocontrol agents, fighting capacity depends mainly on environmental conditions, soil type, formulation, pathogen and pathogen-host relation. figure 1: combined analysis of audpc between study treatments severity: the variance homogeneity test between patches with broccoli and without broccoli showed that there is homogeneity to perform the combined analysis. there are significant differences too between brassica block factors and treatment factors. there are no significant differences in the brassica treatment factor because brassica effect was a determining factor for the disease development and in the manifestation of symptoms and signs. the tukey´s comparison test 0.05, showed that the brassica factor did not present significant differences between them, the severity percentage for both fields was statistically the same in regards of its severity manifestation in harvested bulbs. t1 (reference) and t5 (carbendazim) are statistically equal and with high severity percentages of 51.76% and 45.41% respectively. t4 (boscalid) showed a lower severity percentage regarding the other treatments with 9.35%, is statistically different. yields: a variance homogeneity test was performed on the patches with broccoli and with no broccoli, which showed that there is homogeneity was done a combined analysis, the coefficient of variation was 44.32%. the brassica factor (with broccoli and no broccoli) in the tukey´s comparison test, 0.05, presented significant differences between the addition of brassicas and with no addition of brassicas, therefore, this effect influences treatments and bulb production (t/ha). the first quality yield of treatments t5 (carbendazim) and t1 (reference), showed the lowest values of yield, being statistically different regarding treatment t4 (boscalid), which showed the highest value of yield with 33.20 t/ha. the second quality yield showed significant differences between treatments; thus, the brassica factor influences the yield. the total yield in the comparison tests showed significant differences and a higher yield in treatment 4 (boscalid) with de 40.83 t/ ha. table 8. profitability analysis net profitability analysis reaches its highest value in treatment t4 (boscalid) with 0.68 with no addition of broccoli, and profitability of 1.00 in the area treated with the addition of broccoli. also, it should be noted that among m. gonzales; l. mattos peruvian journal of agronomy 2 (3): 27-34 (2018) 33 treatments being studied, in the area with brassica residues is seen three treatments showing positive profitability and unlike the untreated area, they contributed directly in obtaining a better production. the rest of treatments showed negative profitabilities in this trial. therefore, from this point of view, the best treatment was treatment 4. table 8: tukey´s comparison test for first and second quality yield and total yield in treatments. treatment first yield second yield total yield t/ha t/ha t/ha media sig. media sig. media sig. t1 (control) 8.1 b 16.3 a 23.5 b c t2 (iprodione) 11.9 b 14.7 a 27.0 b t3 (thiabendazole) 10.9 b 16.5 a 27.4 b t4 (boscalid) 33.2 a 7.6 b 40.8 a t5 (carbendazim) 4.8 b 9.2 a b 17.3 c t6 (t.harzianum) 10.3 b 14.9 a 25.2 b c t7 (b. subtilis) 9.0 b 12.8 a b 19.2 b c discussion in vitro biological and chemical control of sclerotium cepivorum: similar results are reported by oliveira et al (1982), who performed fungicide tests with five isolations from different locations of sclerotium rolfsii (fungus similar to s. cepivorum), where he found that the in vitro sensitivity effect from different fungicides on the radial mycelium growth and the growing tendency of the sclerotium rolfsii fungus was effective. he also mentions that the thiabendazole and iprodione presented fungicidal effects, delaying the mycelial growth. it could be assumed that the same behavior would be present for another type of fungicide, such as the boscalid, and that its fungicidal effect could vary according to the different fungus isolations. contrasting results had already been noted by other researchers for this fungus, which previous works showed variation in reactions of the mycelial growth from different fungus isolation, using fungicides such as utkhede and rahe (1983), proved the effective control in vitro of s cepivorum with four isolations of the b subtilis bacteria. regarding t. harzianum, a pathogen growth inhibition occurred, with a radial growth of 13,44 mm, which represents a 69.1%. these results also agree with reports from oliveira et al. (1982), arenas (1997), obregón (2001); who obtained similar controls of s. cepivorum, by using trichoderma harzianum. final and initial sclerotia population with no broccoli addition: according to what was reported and the number of initial sclerotia in treatments with densities of 0.45 to densities of 0.77 sclerotia per soil gram, are capable of producing very high incidences of disease on field, and their behavior regarding the disease development will depend on favorable conditions and control measures performed to inhibit or delay their infection. rosas et al. (2010), point out that there is only enough one sclerotium per soil gran, this is equal to 1,000 sclerotia/kg of soil, to produce the death of 100% of onion plants. initial and final sclerotia population with broccoli addition: the initial population of sclerotium cepivorum regarding the final population was increased in treatments in an aggregate way, coley smith et al. (1990), mentions that survival structures in s cepivorum are very resistant against adverse conditions and its survival is kept above the 92%, and feasibility can reach up to 96% between 5 and 10 years that remain in the ground. incidence with broccoli and without broccoli: the lowest control percentage was obtained from t5 and presented a behavior equal to the reference treatment. in the case of biocontrol agents, t. harzianum was more effective than b. subtilis. these results coincide with results obtained by rosas et al. (2010), where t. harzianum´s efficiency was proved in a higher percentage than bacillus subtilis for the control of s cepivorum. table 4. according to these incidence results and disease progression, coley smith et al. (1990), mentions that the low efficiency of some fungicides (thiabendazole, carbendazim, iprodione) is due to the microbial degradation, and also adds that, the control is more limited as sclerotia population increases in the soil. combined analysis of the two experiments with broccoli and without broccoli final and initial sclerotia population: sclerotia density at the area treated with residues was reduced; however, there no significant differences between both experimental areas, villar et al. (1990) mentions that when adding cabbage or broccoli residues on the soil infested with s. cepivorum, this significantly reduces the number of dead plants and the disease incidence. conclusions fungicides iprodione and carbendazim were the ones that completely inhibited the growth of the in vitro pathogen, which showed significant differences regarding the reference treatment. the best product for the in vitro biological control of sclerotium cepivorum was bacillus subtilis, which inhibited the fungus growth a lot faster than trichoderma harzianum that also achieved a good control but with a slower inhibition. field conditions with broccoli residues and without broccoli residues, fungicides that had a better control effect were boscalid with 79.01% and 70.82% and iprodione with 43.52% and 41.64% against sclerotium cepivorum and also having a lower incidence percentage and a lower severity level in bulbs. in field conditions with broccoli residues and without broccoli residues, trichoderma harzianum had a better control effect with 34.57% and 31.16% against sclerotium cepivorum, unlike bacillus subtilis with 19.14% and 11.05%. cultural, biological and chemical control of the white rot fungus (sclerotium cepivorum, berk) in onions (allium cepa) in arequipa´s countryside september december 2018 34 the effect of adding broccoli residues on the sclerotia density on the soil was not statistically significant, but with the addition of broccoli residues, there were significant statistical differences for incidence, severity and yields, both for patches with chemicals, biocontrol agents and on the reference one. also, lower percentages of incidence, severity and higher yields were obtained, unlike the case with no addition of broccoli residues. treatments that showed a higher yield in red onion production was treatment 4 (boscalid) with yields of 43.33 t/ha for the area with the addition of broccoli, and 38.33 t/ha for the area with no addition of broccoli; followed by treatment 2 (iprodione), where yield on the area with addition of broccoli was 28.33 t/ha and on the area with no addition of broccoli was 25.56 t/ha. literature cited arenas, m.a. (1997). biocontroladores de sclerotium cepivorum, berk. en cebolla cv. roja arequipeña. thesis to opt for the degree of ingeniero agrónomo. universidad nacional de san agustín arequipa – perú. 115 p. coley-smith, j.r., mitchell, c.m. & sansford, c.e. (1990). long-term survival of sclerotia of sclerotium cepivorum and stromatinia gladioli. plant pathology, 39, 58-69. https://doi.org/10.1111/j.1365-3059.1990. tb02476.x obregón, m. (2001). evaluación in vitro del poder antagónico de trichoderma harzianum rifai con respecto al hongo fitopatógeno sclerotium cepivorum berkeley causante de la enfermedad “torbó en cebolla”. in: xlvii reunión anual del pccmca resúmenes (19 p). san josé, costa rica. oliveira, v.l., belle, m., menezes, j. & ferreira, f. (1982). ação antagônica de trichoderma harzianum, paecilomyces lilacinus e penicillium sp. sobre sclerotium cepivorum, causador da podridão branca do alho. fitop. bras., 7(3), 531. rosas, v., ulacio, d., jimenez, m., perdomo, w. & pardo, a. (2010). análisis epidemiológico y control de sclerotium cepivorum berk. y la pudrición blanca en ajo. bioagro, 22(3), 185-192. https://www.redalyc.org/ html/857/85717010003/ sistema integrado de estadística agrícola (siea), (2012). indicadores productivos. editorial oa-minag. lima perú. schwartz, h.f. & mohan, s. (2008). compendium of onion and garlic disease. american phytopathological society (aps). st paul, minnesota. 127 p. utkhede, r.s. & rahe, j.e. (1979). wet sieving floatation technique for isolation of sclerotia of sclerotium cepivorum from muck soil. phytopathology 69:295297. utkhede, r.s. & rahe, j.e. (1983). interactions of antagonist and pathogen in biological control of onion white rot. phytopathology, 73, 890-893. doi: 10.1094/ phyto-73-890. villar, a.c., zavaleta, e. & garcia, r. (1990). efecto de la incorporación de residuos de crucíferas (brassicae) sobre fitopatógenos de suelo. ii efecto de la incorporación de col y brócoli sobre la pudrición blanca (sclerotium cepivorum berk.) de la cebolla, bajo condiciones de invernadero. revista mexicana de fitopatología, 8, 160-165. peruvian journal of agronomy 2 (3): 35-43 (2018) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v2i3.1231 © universidad nacional agraria la molina, lima perú received for publication: 30 november 2018 accepted for publication: 28 december 2018 pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru pseudomonas de la rizosfera del palto (persea americana mill.) con actividad de biocontrol de phytophthora cinnamomi rands aisladas en la costa central del perú j. mamani; l. aragón1* *corresponding author: lili@lamolina.edu.pe abstract in the rhizosphere, as the zone of biological activity, diversity of microorganisms can be found like bacteria of the genus pseudomonas. they are characterized for controlling pathogens like phytophthora cinnamomi, also to be promoters of growth. for this reason, in the present study, bacteria of the genus pseudomonas were isolated from the avocado rhizosphere of the provinces of casma, huaral, and lima. a total of six strains (r2, r5, r7, r10, s10 and s6) were selected for evaluating their biocontrol capacity against p. cinnamomi under in vitro and greenhouse conditions. in the in vitro test, strains s6 and s10 controlled 30.3 and 44 %, respectively. under greenhouse conditions, pseudomonas strains were inoculated on 4-month-old avocado cv. “zutano” plants. five months later, we evaluated the following variables: severity in roots, height increase, fresh root and leaf weight, and percentage of root and leaf dry matter. in greenhouse, the best strains in the control of p. cinnamomi were s6, r2, r7 and r10, controlling 55.2, 39.5, 33.7 and 31.0 %, respectively. in the increase of height, the strains s6, r2, r7 and r10 reached 11.4, 9.3, 7.6 and 5.1 cm, respectively. the percentage of dry matter of roots, strains s10, r10 and r7 obtained 29.6, 27.5 and 27.9 %, respectively. in this study, it was observed that although the application of pseudomonas controls p. cinnamomi, it also induces the root and apical growth of avocado. keywords: avocado, promoter of growth, pseudomonas, rhizosphere. resumen en la rizósfera como zona de actividad biológica, se pueden encontrar diversidad de microorganismos como bacterias del género pseudomonas que se caracterizan por controlar patógenos como phytophthora cinnamomi, también por ser promotores de crecimiento (pgpr). por esta razón en la presente investigación se aislaron bacterias del género pseudomonas de la rizósfera de palto de las provincias de lima, huaral y casma. se seleccionaron 6 cepas (r2, r5, r7, r10, s10 y s6) con las que se realizaron pruebas para evaluar su capacidad biocontroladora frente a p. cinnamomi in vitro e invernadero. en la prueba in vitro las cepas s6 y s10 controlaron un 30.3 y 44 %; respectivamente. en condiciones de invernadero, se inocularon cepas de pseudomonas en plantones de palto cv. zutano de 4 meses de edad; transcurridos cinco meses, se evaluó variables como severidad en raíces, incremento de altura, peso fresco radicular y foliar y porcentaje de materia seca radicular y foliar. en invernadero, las mejores cepas en el control de p. cinnamomi fueron s6, r2, r7 y r10 que controlaron un 55.2, 39.5, 33.7 y 31.0 %; respectivamente. en el incremento de altura, las cepas s6, r2, r7 y r10 alcanzaron 11.4, 9.3, 7.6 y 5.1cm; respectivamente. el porcentaje de materia seca de raíces, las cepas s10, r10 y r7 obtuvieron 29.6, 27.5 y 27.9 %; respectivamente. en este estudio se observó que si bien la aplicación de pseudomonas ejerce un control sobre p. cinnamomi también induce el crecimiento radicular y apical de la planta. palabras claves: palto, pgpr, pseudomonas, rizósfera. 1 department of plant pathology, faculty of agronomy, universidad nacional agraria la molina, av. la molina s/n, lima 12. perú. introduction peru has an avocado producing area of around 30,320 hectares with a total production of 349,317 tons. the coastal departments of ancash, ica, la libertad, and lima have the biggest cultivated area. (minagri, 2014). peru is the second exporter of avocado worldwide, with 175.6 million kilos exported (arteaga, 2016). therefore, avocado production in peru will continue to be an attractive business due to the opening of new markets and new consumers who value their nutritional properties. the production of the avocado depends on climatic, edaphic, nutritional and sanitary factors. within the sanitary aspect, diseases are one of the factors that augment the production costs of the fruit. among the most important, is “root rot” caused by phytophthora cinnamomi (chromista, heterokontophyta). this pathogen limits tree development, reduces fruit production and quality, directly affecting profitability. currently, this oomycete is controlled by chemical products, such as fosetil-al and metalaxyl (mora, 2007), pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru september december 2018 36 but the constant use of chemical products diminishes its effectiveness over time due to the resistance that this phytopathogen develops towards the fungicide. for these reasons, biological control is considered to take advantage of soil microorganisms with antifungal activity, including bacteria of the genus pseudomonas, responsible for the suppression of some soil pathogens (raaijmakers et al., 2002). bacteria of the genus pseudomonas have been widely studied as biological controllers for their ability to colonize the root, compete aggressively with other microorganisms, adapt to different situations of environmental stress, synthesize antibiotics and enzymes, and activate systemic resistance in plants (weller et al., 2006). for this reason, in the present work, we evaluated under in vitro conditions the antagonistic effect of strains of pseudomonas spp. isolated from the rhizosphere of avocado on p. cinnamomi by scoring the progress of the pathogen in centimeters. in addition, in the greenhouse; the severity of the infection of p. cinnamomi in avocado seedlings previously inoculated with the strains of pseudomonas spp. was measured by the percentage of lesions in roots, and the effect of these strains on the growth of the avocado seedlings was measured by the increase of height of the stem in centimeters. materials and methods field work. the sampling was carried out in commercial avocado fields that reported root rot problems in casma, huaral and lima. in each field, 10 avocado trees were randomly selected. roots were collected from each tree with a volume of rhizospheric soi from four cardinal points, with a total equivalent of 250g of soil. these samples were transported and kept in cold conditions by using gel packs to maintain their temperature and humidity. laboratory work. the laboratory work (in vitro isolation) was carried out in the facilities of the laboratory of the department of phytopathology of the national agrarian university of la molina (unalm for its acronym in spanish), located in the district of la molina, province and department of lima, at 12° 05’ 06” s of latitude, 76° 57’ 07” wg of longitude and 243.7 m.a.s.l. sample treatment. the microflora of both the rhizosphere and the soil was analyzed separately. under aseptic conditions, 3 g of root of each sample was placed in test tubes with 9 ml of peptonated water in continuous agitation, performing 7 serial dilutions (10-1, 10-2, 10-3, 104, 10-5, 10-6 and 10-7), adapted from andres (1991). for the soil sample near the root, 10 g were placed in 90 ml of peptone water (ratio 1: 9), following the same previous procedure. count of pseudomonas spp. in regard to the quantification of pseudomonas spp., 1 ml of the dilution (10-2 to 10-5) was seeded in triplicate in tubes containing 9 ml of asparagine broth. these tubes were incubated for four days at 37°c (burges, 1960). this procedure was performed for both root and soil. isolation and identification of pseudomonas spp. the tubes that were positive (fluorescent) to the asparagine broth culture were striated in the king-b culture medium and incubated for 48 hours, and fluorescence was evaluated in ultraviolet light at 366 nm (schaad, 2001) together with the morphology of the colonies (palleroni, 2005). the colonies that were positive for fluorescence were then selected. from each isolate, a colony was selected, which was striated in the tryptone soy agar (tsa) culture medium to ensure the purity of each isolate and to be stored in tsa agar wedges at 4°c for the next tests (martínez, 2010). pathogen isolation (phytophthora cinnamomi rands). rootlets of 1-3 millimeters in diameter that presented the typical symptomatology corresponding to a black and firm rot that originated from the zone of elongation were selected. they were washed with distilled water and segments of about 1 cm long that presented the area of pathogen progress were cut. then, they were immersed for a few seconds in a 70% alcohol solution in order to prevent possible contamination (zentmyer, 1980). these pieces of 1 to 2 mm2 of diseased and asymptomatic tissue were seeded in selective culture medium cma (corn meal agar) with parb (pimaricinampicillin-rifampicin-benomyl) (erwin & ribeiro, 1996). they were incubated at 22°c for seven days, and the growth rate of the colony and its characteristics were observed (alvarado-rosales et al., 2007). each pure colony in cma was transferred in v8 juice agar medium for four days at 21°c. from slices of grown mycelium in v8 juice agar that was introduced in petri dishes containing 1% soil solution, structures of the pathogen were visualized as sporangia. the identification of p. cinnamomi was conducted by following the dichotomous key to taiwan species of phytophthora (ho, 1992). antagonism test against phytophthora cinnamomi. from each previous isolate, the strains that controlled phytophthora cinnamomi were identified. these strains were used in the challenge test in nutrient agar culture medium. it was necessary to prepare culture medium in which the corresponding reagents were included because the results of this test were not separately obtained. in each plate containing medium pda + king-b + nutrient agar a controller was included (pseudomonas spp. strain) and a pathogen disk. this methodology was carried out with the objective of identifying strains of pseudomonas spp. with greater control over p. cinnamomi, demonstrating a higher percentage of inhibition of the radial mycelial growth of the pathogen (% i). j. mamani; l. aragón peruvian journal of agronomy 2 (3): 35-43 (2018) 37 greenhouse phase. the greenhouse phase was carried out in the greenhouse research station of the department of phytopathology of unalm. vegetal material. we used for four-month old plants of avocado var. ‘zutano’ which were treated before to the germination (immersed in homai fungicide solution in 5 g/l dose of product), according to the disinfection protocol of the nursery of the fruit trees research program of national agrarian university la molina. the transplant to bags was made in january 2016 to be taken to the greenhouse. preparation of the substrate. the substrate mixture consisted of 50% soil, 25% river sand and 25% organic matter (herrera & narrea, 2011). then, it was sterilized at 121°c for 15 minutes. the substrate was used in black polyethylene bags measuring 8x16x2.2 cm3 (5.5 l), where the sterile soil was placed. then, we proceeded to transplant the avocado. preparation, density and inoculation of the controller. selected strains of pseudomonas were seeded in peptone water at 27°c and 150 rpm (revolutions per minute) until reaching a population of 6x108 (colony forming units) cfu/ml (martínez, 2010). pathogen inoculation. according to drenth & sendall (2001), it is recommended to use mycelium developed in wheat previously sterilized at 121°c and a pressure of 1.1 bar (15 lb/in2 or 15 psi) for 30 minutes for two consecutive times. the inoculation was done in five-month-old avocado plants. the application was made around the root system of the avocado at a depth of 5 cm (the dose of inoculum was 2.5 g of wheat with mycelial development/kg of soil) (zentmyer & richards, 1952). the humidity of the substrate was maintained with periodic irrigation to favor the development of the pathogen. the mycelium of phytophthora cinnamomi was inoculated ten days after the inoculation of the controller, according to martínez (2010). evaluated parameters. disease severity and root length. the severity and root length were measured by processing digital photographs of avocado roots using assess software (lamari, 2002). plant height. we used a millimeter ruler. measurement (length of the main stem) was taken from the neck of the plant to the terminal bud at the time of inoculation of the biocontroller and at the end of the experiment. the results were expressed in centimeters. fresh weight, and dry root and foliar matter. to obtain the percentage of dry matter, firstable the roots and leaves of the plant were placed in paper bags to be weighed; then they were taken to the stove at a temperature of 70°c for 48 hours to obtain the dry weight. the weights were expressed in grams using electronic balance. biochemical characterization of pseudomonas spp. the following biochemical tests were carried out, according to palleroni (2005): (i) production of fluorescent pigments, (ii) resistance to high and low temperatures, (iii) and gelatin liquefaction, in order to characterize strains of non-pathogenic fluorescent pseudomonas from the rest of species fluorescence in king-b culture medium. the culture medium of king-b is used for the detection of fluorescein, a green or blue fluorescent soluble in water. after growth of 24-48 hours at 27°c, the colonies, previously striated in king-b, were observed with an ultraviolet lamp (366 nm). growth at 4 and 41°c. strains of pseudomonas spp. were planted in tubes containing tsa culture medium, which were incubated at 4 and 41°c. jellied liquefaction. this test is used to determine the capacity of a microorganism to produce enzymes of the proteolytic type (gelatinases) that liquefy the gelatin. it is positive for pseudomonas aeruginosa (pathogenic) (mac faddin, 1980). the tubes containing nutrient gelatin were prepared considering three replications per treatment. the strains of pseudomonas spp. were then inoculated by puncture and incubated at 37°c for 48 hours. finally, they were placed in the refrigerator at 4°c for 2 hours. the test was positive if the inoculated medium became liquid, and the test was negative if the inoculated medium maintained its characteristics (mac faddin, 1980). experimental design. the statistical design used in the antagonism test against phytophthora cinnamomi was a completely randomized statistical design (crd), with ten treatments of strains isolated from pseudomonas spp., four dishes per treatment and control treatment of p. cinnamomi pathogen (table 1). the parameter that was evaluated in this test was the percentage of inhibition of grown radial mycelial, and the differences between the average of cultures analyzed out through the tukey test (p = 0.05). in greenhouse, the statistical analysis of variance (anova) and tukey test with a level of significance at 5% were conducted to identify whether or not there are significant differences between the results obtained with each evaluated strain. all statistical analyses tools were applied with minimum confidence of 95% and sas 9.2 software (sas, 2009). pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru september december 2018 38 table 1. treatments used in the present study. treatment description t1 p. cinnamomi + strain of pseudomonas spp. (r2) t2 p. cinnamomi + strain of pseudomonas spp. (r5) t3 p. cinnamomi + strain of pseudomonas spp. (r7) t4 p. cinnamomi + strain of pseudomonas spp. (r10) t5 p. cinnamomi + strain of pseudomonas spp. (s10) t6 p. cinnamomi + strain of pseudomonas spp. (s6) t7 control treatment: p. cinnamomi (phy) t8 absolute control treatment (t) results isolation, purification and identification of pseudomonas spp. a bacterial density of pseudomonas spp. was obtained from the sampled areas which varied between 105 and 108 nmp/g of roots and dry soil. in table 2, it can be observed that the casma area presented the highest levels of bacterial density because the sampled farm promotes the microbial flora with applications of organic matter. we worked with casma strains because they had a higher inoculum potential compared to the other areas sampled. table 2. count of pseudomonas spp. (nmp/g) isolated from the central coast of peru. location nmp/g lima soil 2.3x106 organisms /g of soil root 2.3x106 organisms/g of root huaral soil 9.3x105 organisms /g of soil root 4.3x105 organisms/g of root casma soil ˃1.1x108 organisms /g of soil root ˃1.1x108 organisms/g of root the isolates that grew and emitted fluorescence in asparagine broth were striated in king-b culture medium (also called pseudomonas agar f) for the isolation of all pseudomonas spp. (schaad, 2001), thus differentiating them from other gram-negative microorganisms that do not emit fluorescence. a total of 14 positive isolates were obtained for this test. antagonic effect in vitro test. figure 1 shows that the isolates from soil s7, s10, s2, s9, s4, s1, s6 and s3 inhibit the advance of p. cinnamomi; being constant in time, while in isolations from roots (r7, r10, r4 and r5) there is a strong inhibition at 4 das (days after sowing), but it is lost quickly over time. all the evaluated strains reported some control varying the percentages of inhibition between 28.3 to 47% (figure 2). the isolates r2, r5, r7, r10, s6 and s10 showed growths above the mycelium of p. cinnamomi, being reduced to a yellow gelatinous mass. from there, samples were taken to be observed in a microscope. in isolates from the confrontation p. cinnamomi and pseudomonas spp. fluorescence observed in a microscope (40x), the disintegration of the cytoplasmic content of the mycelium of p. cinnamomi was observed. fig. 1. advance of p. cinnamomi (in centimeters) against soil and root isolates of pseudomonas spp. for 7 days after seed in vitro test. in the biochemical characterization tests, according to the bergey manual (palleroni, 2005), carried out on strains of pseudomonas spp. selected (r2, r5, r7, r10, s6 and s10) the results were obtained (table 3) in which it is determined that the strains evaluated do not correspond to pseudomonas aeruginosa (pathogenic to man). table 3. reaction of different strains of pseudomonas spp. to the biochemical tests. fluorescentpigments grows at 4°c grows at 41°c gelatin liquefaction p. fluorescens + + +/ p. putida + + p. aeruginosa + + + strain r2 + + strain r5 + + strain r7 + + strain r10 + + strain s10 + + strain s6 + + greenhouse evaluation. evaluation of the severity of avocado roots. treatments r5, s10, r10, r7 and r2 (60.4, 66.2, 69.0, 77.5, 78.4 and 90.7%, respectively) show percentage of severity that is not significantly different from the treatment inoculated with the pathogen. on the other hand, the s6 treatment has a significant difference with the control treatment and the treatment inoculated with p. cinnamomi with only 44.8% of severity in avocado roots (figs. 2 and 3, table 4). figure 2, strains r5 and s10 showed severity values (90.7 and 78.4%, respectively) above the control inoculated with p. cinnamomi (77.4%), this loss of antagonistic capacity could be due to population decline of these strains (van et al., 1997). j. mamani; l. aragón peruvian journal of agronomy 2 (3): 35-43 (2018) 39 fig. 2. measurement of severity of avocado roots (%), avocado root length, height increase of avocado plant (cm), fresh radicular weight (g), fresh leaf weight (g) and dry matter radicular (%) of treatments inoculated with strains pseudomonas spp. against p. cinnamomi, under greenhouse conditions. pseudomonas of the rhizosphere of avocado (persea americana mill.) with biocontrol activity of phytophthora cinnamomi rands isolated in the central coast of peru september december 2018 40 it is also observed that the best treatment in the control of p. cinnamomi in greenhouse was t6 (inoculated with the s6 strain of pseudomonas spp.) which showed 59.0% of control. evaluation of the height increase of avocado plant. inoculation of strains s6 and r2 showed a greater increase in height, 11.4 and 9.3 cm respectively, compared to the control treatment (fig. 2). there is no significant difference between treatments s6 and r2, according to the tukey test at 95% (table 4). figures 2 and 3 show all treatments with inoculation of pseudomonas spp. have values that are above the control inoculated with p. cinnnamomi (2.1 cm). the treatments inoculated with strains of pseudomonas spp. present greater amplitude of the interval of variance of their repetitions. evaluation of fresh radicular weight. regarding fresh radicular weight, treatments s10 and s6 (45.6 and 45.2 g) show a significant difference compared to the treatment inoculated with p. cinnamomi (30.8 g), but lower than the absolute control treatment (148.6 g), as observed in figs. 2 and 3. for this variable, the repetitions of each treatment were close values, thus a shorter interval of variance. evaluation of the dry matter radicular. on the percentage of dry matter of roots, there is a significant difference between the treatments. treatment s10 with 29.0% of dry matter of roots is the best compared to the control inoculated with p. cinnamomi (18.6%). table 4. measurement of avocado variables with the comparison test of means (tukey) of treatments inoculated with strains pseudomonas spp. against p. cinnamomi, under greenhouse conditions. treatm ents strains severitya (%) increase in heightb (cm) foliar biomassc (g) root lengthd (cm) biomass of rootse (g) root dry matterf (%) t1 r2 60.462 ab 9.3 ab 74.86 bcd 989.1 b 33.320 bc 24.200 abcd t2 r5 90.720 a 4.0 d 66.70 cd 160.6 c 17.340 c 25.780 abc t3 r7 66.274 ab 7.6 bc 110.38 b 672.2 bc 31.500 bc 27.960 ab t4 r10 69.034 ab 5.1 cd 69.04 cd 640.5 bc 31.500 bc 27.500 ab t5 s10 78.456 a 3.1 d 81.68 bcd 841.1 b 45.680 b 29.620 a t6 s6 44.848 b 11.4 a 91.76 bc 1142.2 b 45.220 b 22.180 bcd t7 phy 77.466 a 2.1 d 48.68 d 648.1 bc 30.880 bc 18.680 d t8 t 6.578 c 4.5 d 154.68 a 3924.6 a 148.640 a 20.360 cd coefficient of variation (alpha 0.05): 24.86a, 25.17b, 22.07c, 26.17d, 17.55e, 13.98f. avocado root length evaluation. the effect of pseudomonas sp. on the length of avocado roots showed a significant difference between treatments. in all treatments, shorter root length was observed compared to the control treatment (figs. 2 and 3, table 4). therefore, inoculated pseudomonas strains influenced root growth but did not achieve significant difference with respect to the control. treatments s6, r2 and s10 presented longer root lengths (1142.2, 841.1 and 989.1, respectively) than the control inoculated with the pathogen (648.1). fig. 3. photograph of foliar and root area of avocado cv. ‘zutano’, six months after inoculated with soil and root isolates of pseudomonas sp. and p. cinnamomi, under greenhouse conditions. j. mamani; l. aragón peruvian journal of agronomy 2 (3): 35-43 (2018) 41 evaluation of fresh leaf weight. treatments r7 and s6 (110.4 and 91.8 g) show a significant difference compared to the treatment inoculated with p. cinnamomi (48.7 g), but lower than the absolute control treatment (156.6 g), as observed in figs. 2 and 3. dry leaf matter. figure 2 shows there is no significant difference among treatments, and that the repetitions of treatments t2 (r5), t3 (r7) and t5 (s10) present higher values than the absolute control (t). discussion this study shows the inhibition of phytophthora cinnamomi against pseudomonas in vitro and greenhouse. villa et al. (2005) found that strains of pseudomonas sp. inhibited the growth of sclerotium rolfsii between 60 and 90%. other studies also report that pseudomonas cepacia and p. fluorescens were significant for suppressing p. cinnnamomi that grew in vitro and in vivo (yang et al., 2001). the coagulation of the cytoplasm was observed; this same partial degradation or coagulation of the cytoplasmic content was observed in cultures of phytophthora capsici with pseudomonas fluorescens in which diby et al. (2005) attributes it to hydrolytic enzymes. broadbent & baker (1974) also demonstrated that p. putida and p. fluorescens isolated from avocado soils caused massive mycelia lysis of p. cinnamomi in vitro. the strains of isolated pseudomonas were not pathogenic for humans according to rodríguez et al. (2005) who demonstrated that to isolate pseudomonas aeruginosa (opportunistic pathogen in humans) the bacteria should be cultured in a gelatin culture medium, thus demonstrating the presence of gelatinases. the promising strains of pseudomonas gave a negative result to this test. therefore, they are not pathogenic to man. pseudomonas putida and pseudomonas fluorescens are unable to grow at 42°c and do not produce pyocyanin unlike p. aeruginosa, corroborated by the uk standards for microbiology investigations (2015). several possible mechanisms have been described by which soil pseudomonas suppresses conditions related to pathogenicity. de la fuente et al. (2000) identified three native strains of fluorescent pseudomonas (up61, up143 and up148) producing hcn, fluorescent siderophores such as pyoverdine proteases and antibiotics [2, 4-diacetylchloroglucinol (dapg), plt (piolterine), prn (pyrrolnitrine) and phenazine derivatives] with antifungal activity (julisch et al., 2001). thomashow and weller (1996) also described the production of antibiotics by fluorescent pseudomonas. therefore, the control effect of treatment t6 (s6) could be explained by mechanisms such as competition for iron, competition for colonization sites and nutrients exuded from the root, as well as the induction of plant defense mechanisms (van weels et al. 1997). another mechanism of control is the production of extracellular enzymes, such as chitinases, laminases and glucanases that can degrade the walls of fungal cells. van weels et al. (1997) isolated a strain of pseudomonas stutzeri that produced extracellular chitinase and laminase, and found that these enzymes digest and lyse the mycelium of fusarium solani. marques et al. (2010) who mentioned that the production of iaa by pgpr generally causes the elongation and accumulation of p and n in the tissues of the plant would explain the increase in root length. in the root system, it has been seen that high levels of iaa increase the formation of lateral and adventitious roots, but inhibits the growth of the primary root. this improvement in growth was also observed in the 1970s when some fluorescent pseudomonas strains improved the growth of potatoes and sugarcane when applied to the seeds (schroth & hancock, 1982). the increase in height is explained because pseudomonas can act as promoters of plant growth in two ways: directly by suppression of pathogens or indirectly through the secretion of phytohormones and vitamins, or by increasing the absorption of minerals per plant. glick (1995) proposes that pseudomonas can manifest its growth by promoting effects indirectly, stimulating the beneficial actions of other microorganisms associated with the roots, such as mycorrhizae. when the stimulation of plant growth occurs in the absence of other microorganisms, it has been attributed to the increase in the availability of mineral nutrients, such as phosphate or nitrogen, due to the production of phytohormones stimulating plant growth or to the degradation of ethylene precursors in the root by these bacteria. faggioli et al. (2007) also observed that the inoculation of corn plants with pgpr does not significantly influence the height but the percentage of dry matter (fig. 2). conclusions the soil sampled from casma area had a higher quantity of pseudomonas spp. organisms. the pseudomonas spp. isolated from soil showed greater controlling effect on phytophthora cinnamomi compared to isolates from roots, evaluated in vitro. the strain of pseudomonas spp. s6 (t6) showed greater control of p. cinnamomi in the greenhouse and the strain of pseudomonas spp. s10 (t5) showed greater root system development in the greenhouse. it was ruled out that all pseudomonas spp. inoculated were pathogenic to humans (pseudomonas aeruginosa). acknowledgments this research was funded by sociedad agrícola virú s.a. (virú) company which grant me the first place in the contest “premio virú 20 años”. we thank dr. c. arbizu berrocal for assistance. references arteaga, w. 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(2001). 16s rdna fingerprinting of rhizosphere bacterial communities associated with healthy and phytophthora infected avocado roots. fems microbiology ecology, 35(2), 129-136; 20 zentmyer, a. (1980). phytophthora cinnamomi and diseases it causes. phytopathological monograph. phytopathology. soc. 96 pp. zentmyer, g. & richards, s. (1952). pathogenicity of phytophthora cinnamomi to avocado trees and the effects of irrigation on disease development. phytopathology,42,35-37. peruvian journal of agronomy 4(1): 1-9 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i1.1438 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 13 november 2019 accepted for publication: 20 january 2020 soil arthropods associated with sweetpotato crop (ipomoea batata l.) in la molina, lima, peru artrópodos de suelo asociado al cultivo de camote (ipomoea batata l.) en la molina, lima, perú livia-tacza, c.(1)*; sánchez, g. (1) *corresponding author: clivia@lamolina.edu.pe https://orcid.org/0000-0003-4456-322x abstract sweetpotato (ipomoea batata l.) is an economically important crop grown in peru. the present study aimed to determine the soil arthropods associated with sweetpotato and its relative abundance in sweetpotato production fields in la molina, lima, peru. evaluations were conducted at universidad nacional agraria la molina, from february to august 2017. samples were collected using pitfall traps and examined at the klaus raven büller entomology museum. soil morphospecies were extracted and separated from collected samples and then counted, recorded and identified. they were also admitted to the entomology museum collection. eighty morphospecies in the classes collembola, insecta, malacostraca, arachnida and chilopoda of phylum arthropoda were recorded. within the collembola class, entomobryidae was the most abundant family; within insecta, gryllus spp. (gryllidae); within malacostraca, porcellio laevis (porcellionidae); within arachnida, theridion volubile (theridiidae) of the order aranea; and within chilopoda, the order lithobiomorpha. by understanding the diversity of arthropods, it may inform future measures and improve control of pests by taking into consideration the soil arthropods among which important beneficial natural enemies could be found. keywords: arthropods, sweetpotato, soil, pitfall traps resumen en perú, el camote es un cultivo económicamente importante. el presente trabajo se realizó con el fin de determinar la asociación de los artrópodos de suelo con el cultivo de camote y su abundancia relativa en los campos de producción de camote en la molina, lima, perú. se evaluó en el área agrícola de la universidad nacional agraria la molina, lote ‘pancal’, del 3 de febrero al 11 de agosto, 2017. las muestras colectadas fueron examinadas en los laboratorios del museo de entomología klaus raven büller. se instalaron seis trampas de caída las cuales se evaluaron semanalmente. de las muestras colectadas se extrajeron y separaron las morfoespecies de suelo, se contabilizaron, registraron e identificaron. así mismo, fueron ingresadas a la colección del museo de entomología. se registraron a las clases collembola, insecta, malacostraca, arachnida y chilopoda del phyllum arthropoda. de la clase collembola, la más abundante fue la familia entomobryidae; de insecta, gryllus spp. (gryllidae); malacostraca, porcellio laevis (porcellionidae); arachnida, theridion volubile (theridiidae) del orden aranea; y chilopoda, el orden lithobiomorpha. se registraron 79 morfoespecies. palabras clave: artrópodos, camote, suelo, trampas de caída 1 universidad nacional agraria la molina. faculty of agronomy. department of entomology. lima, perú. introduction sweetpotato (ipomoea batata l.) is one of the most widely sown crops in the world (martin 1988), and it typically performs well in warm valleys. native to central america (ishida et al., 2000), it has been cultivated in peru since pre-incan times (fonseca, et al., 2002). in peru, it is sown in coastal valleys (at sea level), inter-andean valleys and jungle regions (500 to 2000 m altitude) (fonseca et al., 2002). between 2017 and 2018, a total of 6,000 hectares (ha) of sweetpotato were registered nationwide, 2,400 ha being grown in the lima region, followed by lambayeque (700 ha), ica (700 ha), and cajamarca (600 ha), reaching yields of up to 17.8 tonnes/ha (sistema integrado de estadística agraria, siea, 2017). soil arthropods associated with sweetpotato crop (ipomoea batata l.) in la molina, lima, peru january april 2020 2 the physical-chemical nature, structure and sources of organic matter in the soil allow for the development of a wide diversity of flora and fauna, which interact with and maintain functioning ecosystems through processes such as nutrient cycle, decomposition of organic matter, emission of greenhouse gases, modification of soil structure, water treatment and erosion (cortet et al., 1999). soil arthropods are individuals that play an important role in soil ecosystems. behan-pelletier (1993) considered the study of soil arthropods difficult to establish and define since most arthropods, with some exceptions, spend a certain stage of their life in the ground. in sweetpotato, robles (2002) recorded the orders dermaptera and coleoptera of the class insecta and the class arachnida. schuller (2003) documented the class insecta with the families staphylinidae and carabidae of the order coleoptera and the class arachnida in chancay near lima in tomato (solanum lycopersicum l.). in maize (zea mays l.), the order dermaptera occurs most abundantly, followed by the coleoptera, which is dominated by the families staphylinidae and carabidae, and the class arachnida. in artichoke (cynara scolymus l.) grown in huaura (larco, 2018), three orders have been reported: coleoptera with the family carabidae as the most abundant, followed by the families linyphiidae and theridiidae within the order aranea and the families anisolabididae and labiduridae within dermaptera. in avocado (persea americana mill.), the families sicariidae, lycosidae and dysderidae within the order aranea have been observed as well as the orders coleoptera and dermaptera. pyle et al. (1981) points out that agriculture is the main factor that disturbs the agroecosystem of soil arthropods. however, altieri (1999) emphasises that the disturbance in the diversity and abundance of these systems will depend on the type, age, diversity, structure and management of the crop. for example, sweetpotato (ipomoea batatas l.) requires less input and labour than other crops such as corn, and it tolerates marginal growth conditions (international potato center, 2020). generally, it does not require many intensive agricultural practices, which has led to it becoming a refuge for many species of arthropods due to the favourable conditions for survival. for this reason, the objective was to determine the relative diversity and abundance of the arthropod inhabitants of the soil in the sweetpotato crop in la molina, lima, peru. materials and methods weekly collections of soil arthropods were made, by means of pitfall traps, of a 1.1 ha sweetpotato field planted with the variety ‘jonathan’ that had previously been planted with maize. the field was located in a sector of the fundo, called ‘pancal’, in the agricultural area of the universidad nacional agraria la molina, located in the district of la molina, st. la molina sn (lima, peru). the evaluation periods were from february 3rd to august 11th, 2017, and a total of 27 samples were collected. typical cultural practices were performed during the collection period, including fertilisation, irrigation and weeding. at 36 days after sowing, cultivation and pounding were performed. no insecticide applications were made. field work installation of pitfall traps six pitfall traps consisting of 12.5-cm-high and 12-cmdiameter polyethylene poly-rap containers were installed. they were filled weekly with approximately 250 ml of a solution of water and formaldehyde and 2 ml of liquid soap. formaldehyde was used to prevent the decomposition of the collected insects and liquid soap to break the surface tension. the field was divided into six sectors, and a pitfall trap was placed in each sector. they were located at the back of the furrow. a hole was excavated to a depth where the mouth of the container was flush with the surface of the soil. the location of each pitfall trap was flagged. each time a cultural practice was performed in either field, the pitfall traps were removed and subsequently replaced. traps were placed at least 5 m from the field perimeter so as to minimise edge effects. sample collection on each evaluation date, the pitfall traps were removed, and the contents were emptied into airtight 6 × 4 cm polyethylene containers previously marked with indelible ink with the trap number and date. then, the traps were washed and again filled with the solution (water, formaldehyde and detergent) and reinstalled. the pitfall traps were removed once to prevent them from being damaged due to the hilling and subsequently relocated to the same place. when trap containers were damaged, they were changed and reinstalled. sample processing in the laboratory taxonomic separation and counting of the collected soil arthropods was performed in the research laboratory at the klauss raven büller entomology museum of the entomology department of the universidad nacional agraria la molina. the contents of the 6 x 4 cm polyethylene container were screened with an organza cloth placed in a strainer, and the sample was washed with tap water and then with distilled water. the washed sample was placed in 25-cm-diameter glass petri dishes. the separation and grouping of soil arthropods based on morphological characteristics, called morphospecies (oliver & beattie, 1993, 1996a, 1996b), was done using fine tweezers. selected individuals were counted and kept in small glass bottles with 30 ml of 75% alcohol and labelled with their corresponding code. the code consisted of 1) the first letter of the crop, 2) the abbreviation of the order of the sample and 3) the number of the sample. livia-tacza, c.; sánchez, g. peruvian journal of agronomy 4(1): 1-9 (2020) 3 for example, a recorded individual belonging to the order hymenoptera (hym) and being the first identified individual ‘1’ was coded as hym1, the second individual of the same order as ‘hym2’ and so on. those samples where an order could not be identified were classified as morpho1, morpho2, etc. one or a group of individuals of each species, depending on the number of individuals collected during the evaluation, were mounted on entomological pins and/or stored in alcohol, labelled and registered in the entomology museum of the universidad nacional agraria la molina. taxonomic identification collected individuals were identified in the laboratory using identification keys, with the support of specialists and by comparison with samples from the collection of the klaus raven büller entomology museum of the universidad nacional agraria la molina. these were counted and evaluated for class, order, family, genus and/ or, in some cases, due to its abundance and/or importance, even species. results during the entire evaluation period, 22,822 individuals were recorded in 5 classes, 15 orders and 28 families. the arthropods identified and counted are shown in table 1. classes represented included chilopoda, arachnida, malacostraca, collembola and insecta. in descending order of number of individuals counted, the class collembola was the most abundant (18,592), followed by insecta (2,161), malacostraca, arachnida and chilopoda. with respect to the number of morphospecies, the class insecta was the most predominant, followed by the classes arachnida, collembola, chilopoda and malacostraca (fig. 1). class insecta 2161 class collembola 18592 order coleoptera 357 order entomobryomorpha 18499 family carabidae 210 family entomobryidae 18499 blennidus peruvianus (dejean, 1828) 190 seira sp. calleida sp. 1 lepidocyrtus sp. carab1. sp. 1 entomobrya sp. carab2. sp. 5 order poduromorpha 53 tetracha chilensis (laporte, 1834) 4 family brachystomellidae 53 tetragonoderus sp2. 9 brachystomella sp. family elateridae 3 order symphypleona 40 conoderus sp. 1 family bourletiellidae 40 heteroderes sp. 2 rastriopes sp. family scarabaeidae 8 aphodius sp. 3 ataenius sp. 2 class malacostraca 1124 paranomala undulata (melsheimer, 1845) 3 order isopoda 1124 family staphylinidae 54 family porcellionidae 1124 subfamily aleocharinae 35 porcellio laevis (latreille 1804) (adults) 377 aleoc. sp1. 25 porcellio laevis (latreille 1804) (nymphs) 747 aleoc. sp2. 1 aleoc. sp3. 7 table 1: morphospecies of soil arthropods captured with pitfall traps in sweetpotato from february to august 2017 figure 1. number of individuals and morphospecies (percentages) by arthropod class in sweetpotato from february to august 2017, in lima, peru chilopoda arachnida malacostraca collembola insecta % individuals 0.07 4.06 4.93 81.47 9.47 % morphospecies 3.75 33.75 1.25 3.75 57.5 0 10 20 30 40 50 60 70 80 90 p e r c e n t a g e ( % ) soil arthropods associated with sweetpotato crop (ipomoea batata l.) in la molina, lima, peru january april 2020 4 aleoc. sp4. 1 class arachnida 928 aleoc. sp5. 1 subclass scorpiones 5 subfamily oxytelinae 1 subclass acari 267 oxyt. sp1. 1 order mesostigmata 1 subfamily paederinae 6 mesostigmata1 1 paederus sp. 4 order oribatida 100 paederi. sp1. 2 oribatida1 100 subfamily staphylininae 11 order prostigmata 166 platydracus notatus 5 prostigmata1 77 staphy. sp1. 2 prostigmata2 89 staphy. sp2. 4 order aranea 656 staphylinidae1 1 family gnaphosidae 216 family tenebrionidae 82 family linyphiidae 71 blapstinus cisteloides (erichson, 1847) 17 family lycosidae 54 blapstinus holosericius (laporte, 1840) 48 family mysmenidae 1 epitragopsis olivaceus (erichson, 1847) 2 family oxyopidae 7 parepitragus pulverulentus (erichson, 1847) 5 family salticidae 1 order hymenoptera 383 family scytodidae 11 family formicidae 119 family sicariidae 1 dolichoderinae 34 family theridiidae 289 linepithema sp. 34 family thomisidae 3 formicinae 18 family trachelidae 2 brachymyrmex sp. 18 myrmicinae 12 solenopsis sp. 12 class chilopoda 17 morfol1 sp. 1 order geophilomorpha 6 morfol2 sp. 1 family geophilidae 6 morfol3 sp. 2 geophilidae1 3 morfol4 sp. 1 geophilidae2 3 morfol5 sp. 3 order lithobiomorpha 11 morfol6 sp. 43 family henicopidae 11 morfol7 sp. 2 morfol8 sp. 2 family scelionidae 264 baeus sp. 251 trimorus sp. 13 order orthoptera 1396 family acrididae 3 family gryllidae 1393 gryllus sp. adult 564 gryllus sp. nymph 829 order dermaptera 24 family anisolabididae 24 euborellia annulipes (lucas, hf, 1847) 24 order blattodea 1 family blattellidae 1 total 22822 livia-tacza, c.; sánchez, g. peruvian journal of agronomy 4(1): 1-9 (2020) 5 classes of arthropods that were recorded consistently throughout the evaluation period were collembola, insecta, malacostraca and arachnida. class collembola a total of 18,592 individuals were recorded throughout the evaluation belonging to the orders entomobryomorpha, poduromorpha and symphypleona according to the taxonomic classification of bellinger et al. (2012). entomobryomorpha was the most abundant with 99.5%, followed by poduromorpha with 0.3% and symphypleona with 0.2%. from the order entomobryomorpha, 18,499 individuals of the family entomobryidae were identified, among which were the genera seira sp., lepidocyrtus sp. and entomobrya sp.; from the order poduromorpha, 53 individuals of the genus brachystomella sp. of the family brachystomellidae; and from the order symphypleona, 40 individuals of the genus rastriopes sp. of the family bourletiellidae. the bourletiellidae (symphypleona) and brachystomellidae (poduromorpha) were collected on 10 evaluation dates. however, neither family was consistently captured, and on several evaluation dates, they were not recorded at all. conversely, the entomobryidae (entomobryomorpha) were the most abundant and constant throughout the evaluation period. figure 2 illustrates that during the period of vegetative development fewer individuals were collected compared to the period of root filling. irrigation and weeding may have influenced the decrease in the number of individuals registered in june (928 individuals) and july (642 individuals), respectively. the highest number of recorded individuals occurred on may 12th (1,699) and june 23rd (1,702). class insecta of this class, 2,161 individuals were collected throughout the evaluation, among which individuals of the order orthoptera (64.6%), coleoptera (17.72%), hymenoptera (16.52%), dermaptera and blattodea were identified, the first three being more abundant (fig. 3). from the order orthoptera, gryllus spp. was the morphospecies recorded in greater abundance at both adult (564 individuals) and nymphal (829) forms. figure 4 illustrates, in adults, a decrease in the number of individuals recorded in the evaluation of may 26th, which likely may have been due to irrigation realised on may 21st. likewise, weeding and irrigation could have affected the capture of these individuals during subsequent evaluations. with respect to the nymphs, 135 individuals were collected on may 19th, and irrigation occurred on may 21st. furthermore, adult individuals collected likely decreased close to harvest. given the previous results with the adults collected, the number of individuals was expected to decrease in the subsequent evaluation. however, agricultural practices performed before the evaluation of june 16th (121 individuals) did not seem to have much influence. it could not be determined if weeding influenced the number of nymphs collected. from the order coleoptera, the families carabidae, elateridae, scarabaeidae, staphylinidae and tenebrionidae were recorded. the family carabidae was the most figure 2: seasonal occurrence of entomobryidae (order entomobryomorpha) captured in sweetpotato from february to august 2017, in lima, peru. 64.6 1.11 0.05 16.52 17.72 orthoptera dermaptera blattodea coleoptera hymenoptera figure 3: percentage of orders of the class insecta registered in the sweetpotato from february to august 2017, in lima, peru. figure 4: seasonal occurrence of gryllus spp. (orthoptera: gryllidae) captured in sweetpotato from february to august 2017, in lima, peru.. soil arthropods associated with sweetpotato crop (ipomoea batata l.) in la molina, lima, peru january april 2020 6 abundant with 210 individuals in total, followed by tenebrionidae (82), staphylinidae (54), scarabaeidae (8) and elateridae with 3 individuals (fig. 5). however, with respect to the number of morphospecies registered, the family staphylinidae was predominant. blennidus peruvianus (family carabidae) was the most abundant species, and its presence was almost constant throughout the evaluation period. figure 6 illustrates how the different agricultural practices influence collection rate. within the order hymenoptera, baeus sp. was the most abundant genus. in figure 7, the ascending presence can be observed from june 9th (1 individual) to the last evaluation date, having the highest record on july 28th with 78 individuals. class malacostraca throughout the evaluation, 1,124 individuals belonging to the porcellio laevis latreille 1804 species (order isopoda: family porcellionidae) were counted. individuals in adult and juvenile stages were registered with juveniles (747 individuals) more abundant than adults (377 individuals). figure 8 shows the increase in the population of porcellio adults during the first three evaluations. as with other species, the cultivation practices and field management seemed to have strong influence on the number of individuals captured during each collection. regarding juveniles, the juvenile curve had a tendency almost similar to that of adults; however, the number of juveniles captured in a given evaluation was usually higher. class arachnida of this class, 928 individuals were registered throughout the evaluation, among which subclasses acari and scorpiones from the order aranea were identified. the order aranea was the most abundant with 656 individuals and, in turn, the one that presented the highest number of morphospecies (fig. 9). within the order aranea, 11 families were identified: gnaphosidae, linyphiidae, lycosidae, mysmenidae, oxyopidae, salticidae, scytodidae, sicariidae, theridiidae, thomisidae and trachelidae. the most abundant families were gnaphosidae and theridiidae. in the subclass acari, the order oribatida predominates. figure 5: percentage of individuals and morphospecies of the families within the order coleoptera registered in sweetpotato from february to august 2017, in lima, peru. figure 6: seasonal occurrence of blennidus peruvianus (dejean, 1828) (coleoptera: carabidae) in sweetpotato from february to august 2017, in lima, peru. figure 7: seasonal occurrence of baeus sp. (hymenoptera: scelionidae) in sweetpotato from february to august 2017, in lima, peru. figure 8: seasonal occurrence of porcellio laevis latreille 1804 (porcellionidae) captured in sweetpotato from february to august 2017, in lima, peru.. 0 10 20 30 40 50 60 70 carabidae elateridae scarabaeidae staphylinidae tenebrionidae p e rc e n ta g e ( % ) families of order coleoptera % individuals % morphospecies 0 20 40 60 80 100 120 3 -f 1 0 -f 1 7 -f 2 4 -f 3 -m 1 0 -m 1 7 -m 3 1 -m 7 -a 1 4 -a 2 1 -a 2 8 -a 5 -m y 1 2 -m y 1 9 -m y 2 6 -m y 2 -j n 9 -j n 1 6 -j n 2 3 -j n 3 0 -j n 7 -j l 1 4 -j l 2 1 -j l 2 8 -j l 4 -a g 1 1 -a g n º in d iv id u a ls / 6 p it f a ll t r a p s evaluation date adult nymph vegetative development root filling period beginning ending irrigation fertilization, earth up weeded livia-tacza, c.; sánchez, g. peruvian journal of agronomy 4(1): 1-9 (2020) 7 discussion some of the agricultural practices performed in the sweetpotato crop likely influenced the dynamics of the different arthropods identified due to the alteration of habitats that it generates. the field was subjected to practices such as fertilisation, cultivation, harvesting, hilling, irrigation and manual weeding, but not pesticide applications which should allow for a large number of arthropods to establish in the crop. class collembola individuals within the class collembola (entomobryidae) were the most abundant. in a study carried out in cañete, another town in the region costa, rondón (1999) recorded a large number of individuals of this class, registering 38,197 individuals, in sweetpotato. in the present investigation of the three families identified, entomobryidae predominated, with the genera seira sp., lepidocyrtus sp. and entomobrya sp. during the period of crop vegetative growth, a small number of individuals were recorded. in this period, the soil was more exposed to the environment. during the period of starch accumulation in the root closer to harvest, the plants almost entirely covered the soil, and the number of individuals captured began to increase. based on capture data, lush vegetation conditions generate a favourable microclimate for the establishment of individuals in this group. class insecta of this class, only morphospecies predominantly living in the soil were considered. hence, within this group, the families gryllidae and acrididae of the order orthoptera; anisolabididae of dermaptera; blattellidae of blattodea; carabidae, staphylinidae, elateridae, scarabaeidae and tenebrionidae of coleoptera; and formicidae and scelionidae of hymenoptera were registered. from the order orthoptera, more nymphs than adults were captured. the evaluation period was carried out between the months of february and july, months that covered the entire summer, autumn and early winter seasons at the evaluation site. the time of the year was likely favourable for the development of gryllus spp., and the number of individuals recorded increased until the onset of autumn and early winter. furthermore, minimum soil disturbance from reduced agricultural practices and not applying pesticides likely allowed for a greater number of individuals to establish. it should also be noted that the largest record of individuals, both adult and nymphal, occurred when the plants almost completely covered the soil, which provided refuge area, food sources and favourable oviposition conditions. due to the greater number of individuals in the nymphal state registered in this period, conditions favouring reproduction likely occurred, especially for oviposition, which permits a greater number of generations to establish. from hymenoptera, members of the family formicidae were considered due to their characteristic of mainly establishing their nests in different types of soil (kaspari 2003). in addition, individuals from the family scelionidae were also considered due to the behaviour of the identified morphospecies. the most representative and abundant individuals were females of the genus baeus sp. and endoparasitoids of spider ootheques (araujo et al., 2013). baeus females are small ‘wingless’ wasps with a highly fusiform body (stevens & austin, 2007). because of these characteristics, they are frequently found in the soil. loiácono and margaría (2004) indicate that they parasitise eggs of some spider species of the family theridiidae and araneidae. the fact that there is a great abundance and diversity of spiders in this crop probably allowed baeus sp. to become well established. of coleoptera, b. peruvianus (family carabidae) was the most abundant. the number of individuals captured markedly increased during the latter half of the crop production. plants should reach an adequate size to generate a favourable microclimate and facilitate the establishment of a greater number of arthropods, which then provides ample food source for b. peruvianus. furthermore, the microclimate may have also been favourable for its establishment. however, research on the developmental cycle and/or any description on a favourable temperature of this species is lacking. class malacostraca porcellio laevis was registered at both adult and juvenile stages, and the juveniles were more abundant than the adults. the conditions of the sweetpotato agroecosystem were likely favourable, allowing its establishment. the presence of these individuals is important, because, as noted by leistikow and wägele (1999), they are involved in the synthesis of nutrients and in maintaining energy flow in the soil (biochemical cycles). order aranea subclass acari subclass scorpiones % individuals 69.57 29.9 0.53 % morphospecies 77.8 14.8 7.4 0 10 20 30 40 50 60 70 80 90 p e rc e n ta g e ( % ) figure 9: percentage of orders of the class arachnida registered in the sweetpotato from february to august 2017, in lima, peru. soil arthropods associated with sweetpotato crop (ipomoea batata l.) in la molina, lima, peru january april 2020 8 class arachnida the order aranea was abundant and diverse with zelotes laetus (family gnaphosidae) and theridion volubile (family theridiidae) as the most frequently occurring species; individuals of the subclass scorpiones and acari were also recorded. coddington et al. (2004) noted that it is the only taxon entirely composed of predators. as with the other groups of arthropods, the field conditions with reduced agricultural practices and lack of pesticide application likely allowed for the survival of a large number and diversity of individuals of this class. class chilopoda chilopoda are considered important terrestrial predators (prado-sepúlveda et al., 2016). however, the number of individuals captured was the lowest with respect to the other arthropods. it is unknown whether conditions were favourable or not for these individuals, but it is likely that there are other more efficient capture methods for these individuals. conclusion in this study, the classes collembola, insecta, malacostraca, arachnida and chilopoda were recorded. the class collembola had the greatest number of individuals, and the class insecta had the most identified morphospecies. seira sp., lepidocyrtus sp. and entomobrya sp. (order entomobryomorpha: family entomobryidae) were the genera belonging to the most abundant family registered in the class collembola. gryllus sp. (orthoptera: gryllidae), baeus sp. (hymenoptera: scelionidae) and b. peruvianus (coleoptera: carabidae) were the most abundant morphospecies of the class insecta. in malacostraca, p. laevis (isopoda: porcellionidae) was the only registered species. zelotes laetus (family gnaphosidae) and t. volubile (family theridiidae) were the most abundant morphospecies in the class arachnida. geophilidae (order geophilomorpha) and henicopidae (order lithobiomorpha) were the only families registered in the class chilopoda. through capturing, identifying and cataloguing the rich diversity of soil arthropods that occur in sweetpotato agroecosystems of peru, fundamental knowledge is established. the specimens collected and preserved may be of use for future research not only taxonomically but also as a snapshot of the number of species occurring in this region at this time. acknowledgments the authors would like to thank dr. josé palacios vargas, for the identification of the morphospecies of the class collembola. to bach. manuel andia navarro, for the identification of spider morphospecies. to the biol., mg. sc. clorinda vergara cobian for her support in the identification of morphospecies of the class insecta. to biol., mg. sc. javier huanca, for the identification of the individuals of the subclass acari. references altieri, m. 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(2017). boletín estadístico de producción agrícola y ganadera. iv trimestre. ministerio de agricultura y riego. http://www.minagri.gob.pe/portal/download/pdf/ herramientas/boletines/prod-agricola-ganadera/ prod-agricola-ganadera-iv-trimestre2017_020318. pdf stevens, n. & austin, a. (2007). systematics, distribution and biology of the australian ‘microflea’ wasps, baeus spp. (hymenoptera: scelionidae): parasitoids of spider eggs. zootaxa, 1499, 1–45. peruvian journal of agronomy 4(1): 27-34 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i1.1462 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 24 december 2019 accepted for publication: 18 march 2020 lead phyto extraction in the huachipa zoological park through the use of tagetes erecta associated with a mycorrhiza and edta fitoextracción de plomo en el parque zoológico de huachipa mediante el uso de tagetes erecta asociado a micorrizas y edta carrera, o.(1)*; tello, l. (2) *corresponding author: ocarrera.romero@gmail.com https://orcid.org/0000-0002-4309-7522 abstract phytoremediation is a potentially less costly approach to treat contaminated soil. here, the phytoremediation capacity of marigold (tagetes erecta) in conjunction with edta and mycorrhizae was analyzed. specifically, the ability of the marigold to extract lead from contaminated soils in the bird forest zone of the huachipa zoological park (pzh) was evaluated. marigold was planted in main areas of interest within the pzh. plants were treated with different concentrations of mycorrhizae (0 and 0.5 g/kg) and edta (0, 0.5 and 1 mg/kg) and later analyzed by atomic absorption spectrophotometry for lead concentrations in the aerial parts and roots of the plants at the end of the experiment. we show that marigold has the ability to extract lead; and under the conditions of the experiment, mycorrhizae did not improve the extraction efficiency. however, edta did increase the amount of lead extracted, thus affecting the physiological development of the plant. keywords: phytoextraction, mycorrhizae, edta, lead, recreational parks resumen. el uso de la fitorremediación para solucionar problemas de contaminación de suelos ha aparecido como una alternativa de menor costo a los tratamientos convencionales. en base a esto, se analizó la capacidad fitorremediadora del marigold (tagetes erecta) asistida con edta y micorrizas. específicamente se evaluó la capacidad del marigold para extraer plomo de los suelos contaminados en la zona del bosque de aves del parque zoológico de huachipa (pzh). para esto se realizó la plantación de marigold en las zonas de mayor interés dentro del pzh, con tratamientos de diferentes concentraciones de micorrizas (0 y 0,5 g/kg) y edta (0, 0,5 y 1 mg/kg), para posteriormente ser analizadas mediante espectrofotometría de absorción atómica y analizar la concentración de plomo en las partes aéreas y radiculares de las plantas al final del experimento. con esta investigación se demostró que el marigold es una planta con capacidad de extracción de plomo, y que si bien, bajo las condiciones del experimento, las micorrizas no presentaron una mejora en la eficiencia de extracción, el edta si aumentó la cantidad de plomo extraído, por lo que el desarrollo fisiológico de la planta se ve afectado. palabras claves: fitoextracción, especie acumuladora, micorrizas, edta, plomo, parques recreacionales 1 universidad nacional agraria la molina. lima, perú: *ocarrera.romero@gmail.com https://orcid.org/0000-0002-4309-7522 2 universidad nacional agraria la molina. lima, perú. ltello@lamolina.edu.pe https://orcid.org/0000-0002-1335-062x. introduction soil contamination is defined as the presence of contaminants in soil at concentrations high enough to cause threats to ecological quality and / or present risks to human health (duarte et al., 2017). one of the most common soil contaminants is lead, which is toxic in small doses, and can affect almost all organs and systems in the human body, especially the nervous system. its main route of bioavailability is through the soil and dust (corzo & velásquez, 2014). in recent years, phytoremediation has emerged as an approach that takes advantage of the ability of certain plants to absorb, accumulate, metabolize, volatilize, or stabilize contaminants such as heavy metals, organic compounds, and others that are present in the soil, air, water, or sediments (delgadillo-lópez et al., 2011). marigold (tagetes erecta) can extract metals from soil, and thus it can be used for phytoremediation (castillo et al., 2011; shah et al., 2017). this plant is well-adapted lead phyto extraction in the huachipa zoological park through the use of tagetes erecta associated with a mycorrhiza and edta january april 2020 28 to the coastal climatic conditions of peru, its transplants produce high seedling yields, and its harvest yields are high (mendez, 2009). to improve the efficiency of phytoremediation, other elements can be added, such as arbuscular mycorrhizae, which mitigate stress and increase plant growth in sites heavily contaminated with heavy metals (duñabeitia et al., 2004; gaur & adholeya, 2004; amna et al., 2015). likewise, edta is a chelating agent that can be used to solubilize different metals in soils and enhance their absorption and translocation in plants with high rates of biomass production (huang et al., 1997; lombi et al., 2001). the present study aimed to determine the efficiency of lead extraction of marigold (tagetes erecta) associated with mycorrhizae and/or edta in the soil of the bird forest zone of the huachipa zoo. materials and methods study area previous research has demonstrated soil contamination in the so-called “bosque de aves” and its surroundings in the zoological park of huachipa (tello et al., 2018), district of ate, province of lima, department of lima (12° 0’51.88”s, 76°53’44. 36”w) (figure 1). the study area is close to the bed of the rímac river, which is contaminated due to the various activities that take place around it. for example, juárez (2006) mentioned that the rímac river basin supports intense mining activity in the middle and upper part of the basin, resulting in a high risk of contamination by heavy metals and posing a negative impact on the environment and human health. soil sampling an initial sampling was carried to corroborate the presence of lead in the soil within the study site. twelve 10-cm deep core soil samples were collected to determine the lead content of the soil and to confirm previous data. the sampling procedure is as follows: the surface vegetation was cleared and approximately 0.5 kg (weight required for heavy metal analysis) of samples were collected and stored in properly labeled ziploc bags. we followed sampling guidelines published in the “guía para muestreo de suelos” of minam (2014), which considers sampling depths between 0 and 10 cm as those most relevant for oral or dermal contact of contaminants. thus, we sampled soil at a depth of 10 cm. determination of metals in the soil the soil samples were sent to the laboratorio de suelos, plantas, aguas y fertilizantes de la unalm (laspaf-unalm) for lead analysis using atomic absorption spectrophotometry (perkinelmer analyst 200 spectrophotometer). it should be noted that this laboratory follows a method of analysis established for peruvian soil by the eqs (supreme decree n° 011-2017-minam) and which follows the epa 3050 and epa 3051 guidelines. preparation of seedlings of tagetes erecta in may, tagetes erecta was sown in a greenhouse located within the unalm and then transported to the study area. one thousand seeds of yellow tagetes erecta were germinated in trays with 72 to 200 holes containing a mixture of river sand and sifted moss in a 1:3 ratio. the germination percentage of the seeds was 80%. seeds were planted at the rate of 1 seed/hole in the trays, being careful not to place them too deep or too close figure 1. map of the project location carrera, o.; tello, l. peruvian journal of agronomy 4(1): 27-34 (2020) 29 to the surface, either of which could affect germination success. the trays were kept inside the laboratorio de fertilidad de la universidad nacional agraria la molina, and were watered 3 times a week until the appearance of the first pair of true leaves. transfer to bags and application of mycorrhizae after 2 weeks or after the development of first pair of true leaves, seedlings were transferred into 6 × 6-inch low-density polyethylene bags from plastperu in which holes were punched to allow aeration and drainage of the substrate. the substrate consisted of a mixture of compost, previously sifted moss, and farm soil in a 4:3:3 ratio. each culture bag was filled with approximately half a kilogram of substrate. in some of the bags, glomus intaradices was added according to the quantities specified in table 1. the seedlings were then carefully removed from the germination tray and transplanted to the culture bags. in this manner, the mycorrhizae were in contact with the roots of the plants. plants were irrigated 3 times a week until the development of the second pair of true leaves, at which point they were ready for field transfer. transfer of plants to the field in june, after all plants had developed the second pair of true leaves, they were transported to the study site at the zoological park of huachipa before transplanting, samples were randomly collected from each of the 3 areas of interest and composited into 1 kg of soil that was stored in a ziploc bag. this randomized, composite sample was analyzed to obtain the general physicochemical characteristics of the study area. the experiment was arranged in a completely randomized block with two factors (amount of mycorrhizae and amount of edta added). the treatments and their respective distribution in the areas of the park are shown in table 2, each treatment comprised 20 plants, distributed in 5 columns and 4 rows, spaced 20 cm apart. therefore, each experimental unit is approximately 0.8 m2. all plants were transplanted with a hand shovel to a depth of approximately 10 cm. each plot was irrigated with 4l of well water per session, with a total of 4 irrigation sessions per week. application of edta at the third irrigation session, the plants were considered to have already adapted to their new environment. at this point, edta was added according to the treatments and quantities specified in table 1. however, because high doses can negatively affect the growth of plants, edta was added in small doses. initially, edta was added at a rate of 22.4 mg per 0.8m2 of each treatment, which was subsequently reduced to 11.2 mg per irrigation session. however, all doses were not fully aplied because the field plants had to be removed weeks before due to loss of samples. before and after each irrigation session, soil conductivity was measured using a conductometer from maruplast, to measure any changes in soil salinity. treatment number amount of edta (g/kg) amount of mycorrhiza (glomus intaradices) (g) 1 0 0 2 0 1.5 3 0.5 0 4 0.5 1.5 5 1 0 6 1 1.5 table 1: treatments applied treatment replicate coordinates treatment replicate coordinates south west south west 1 a 12° 0.843’s 76° 53.690’w 1 c 12° 0.824’s 76° 53.676’w 2 2 3 3 4 4 5 5 c 12° 0.823’s 76° 53.680’w 6 6 1 b 12° 0.827’s 76° 53.668’w 1 d 12° 0.832’s 76° 53.697’w2 2 3 3 4 b 12° 0.827’s 76° 53.678’w 4 d 12° 0.838’s 76° 53.697’w5 5 6 6 table 2: location of treatments lead phyto extraction in the huachipa zoological park through the use of tagetes erecta associated with a mycorrhiza and edta january april 2020 30 pest control to control pests, fuerza 200 sc insecticide (concentrated suspension) made by point andina s.a was used. the active ingredient of this insecticide is fipronil, which attacks the central nervous system of insects. however, the mining fly (liriomyza huidobrensis) persisted in attacking the experimental plants, affecting their development. plant harvest and post-harvest soil sampling two months after transplanting, the plants were harvested using landscaping scissors. plants were cut above the root. both the aerial parts and the roots were stored in ziploc bags. all transplanted plants were harvested because of losses due to pests. after harvesting, soil samples were collected from each treatment and then stored in labeled ziploc bags. all samples were transported to the national agrarian university of la molina. soil and plant samples were sent to the laboratorio de suelos, plantas, agua y fertilizantes, and to the laboratorio de fertilidad, respectively. determination of lead content in plants after harvesting, plants were separated and the lengths of the aerial parts and roots, and number of leaves were measured. to determine the lead content in the plant, we followed the epa 3050 methodology. briefly, the aerial parts and roots of each sample were carefully washed, first with tap water and then with deionized water. then the aerial parts and roots of each treatment were placed in their respective paper bags and then heated in an oven for 48 h at 70 ° c. the dry weight of each sample was recorded and dried samples were ground using a mortar and pestle. two grams of ground material were placed inside flasks. in treatments that yielded insufficient biomass (< 2 g) all the dust resulting from the grinding was placed. the samples were placed in an oven (70 ° c) again for approximately 8 hours. subsequently, 25 ml of solution of 1:1 hno3 and 1:1 h2so4 was added to each of the flasks; in the case of flasks containing samples that weighed less than 2 g, only 10 ml of solution was added. next, the flasks were heated on a hot plate at 170 ° c to continue the digestion process. the samples were digested until it all visible particles had dissolved into solution. subsequently, the flasks were removed from the plate, allowed to cool, and then filtered through a whatman no. 41 filter paper. filtrates were transferred to centrifuge tubes, where they were stored and until subsequent lead analysis by atomic absorption spectrophotometry. determination of translocation factors the plants were appraised for their ability to translocate heavy metals from the roots to the aerial parts (mattina et al., 2003, cited by marchiol et al., 2004). the following formula was used to determine the translocation factor: statistical design the data on lead concentrations in the plant, roots, and aerial parts, and plant height were analyzed via normality test and equality of varianeces to evaluate the assumptions of normality and homogeneity, respectively. both assumptions were met. a p-value of 0.05 was considered significant. all statistical tests were carried out using minitab 2019 software from minitab inc. developer. results and discussion initial lead content in the study area the results of lead analysis are shown in table 3. in all areas, spots with high lead content were observed. code zone coordinates pb ppm latitude length za-1 behind the atmosphere of the aliblanca turkey 12°00,825 76°53,672 348.92 za-2 behind the horned owl environment 12°00,823 76°53,672 336.79 za-3 between the atmosphere of the spectacled owl and blue-headed macaw 12°00,826 76°53,665 357.93 za-4 next to the environment of cucacu 12°00,822 76°53,673 393.96 zba-1 pacarana environment 12°04,937’ 76°56,866’ 41.84 zba-2 exterior of the environment of pacarana 12°00,828’ 76°53,702’ 307.91 zba-3 facing the environment of pacarana 12°00,844’ 76°53,702 235.35 zba-4 macaw atmosphere 12°00,845’ 76°53,696 269.39 zba-5 near the entrance right side 12°00,854’ 76°53,703 480.55 ze-1 in front of the camel quarantine zone 12°00,853 76°53,682 562.05 ze-2 in front of the environment of smaller felines 12°00,857 76°53,682 179.29 ze-3 to the left side of smaller felines 12°00,861’ 76°53,691’ 27.49 table 3: initial lead content in the study area carrera, o.; tello, l. peruvian journal of agronomy 4(1): 27-34 (2020) 31 specifically, ze-1, zba-5, za-4 had lead concentrations of 562.05, 480.55, and 393.96 ppm, respectively, which were the highest measurements we obtained for soil in the study area. most of these values are above the environmental quality standard established for park floors (i.e., 140 ppm). these results confirm findings of a previous research carried out in the zoological park of huachipa, by tello et al. (2018) who observed that soils in the park, specifically in the area of the bird forest, are contaminated by lead. this is likely due to sediments left by waters of the rímac river, which tends to overflow and flood this area (tello et al., 2018). physical-chemical characterization of the treated soil table 4 shows the characteristics of the soil of the zoological park of huachipa. the soil texture is frank, with a neutral ph, slight salinity, and with a high level of organic matter (> 4%). analyses of other metals such as cd and cr show generally low concentrations that do not exceed the values established by the eca (cd: 10 ppm and cr: 400 ppm). table 5 shows the concentrations of lead in the treated soils before planting. the average values vary between 167.83 and 219.27 ppm, which are close to the value of lead concentration in the composite sample (176.21 ppm). of the 24 soil samples evaluated, 21 have lead concentrations that exceed the eca for park soils (d.s. no. 011-2017-minam). the three samples, whose lead concentrations are below the rct, correspond to samples collected from the bosque de aves. table 6 shows the concentrations of lead in treated soils after harvest. in the majority of cases, the concentration of lead in the soil increased, contradicting previous research on phytoremediation that report a decrease in the concentration of lead in the soil post-remediation. a possible explanation for our findings lies in the use of water from the rímac river by the park managers to irrigate the plantations around the bird forest on 4 occasions. because, the sediments of the rímac river are contaminated by lead, using the river water for irrigation could have contaminated our study site as suggested by tello et al. (2018). moreover, the damping effect of soils and its high organic matter content (4.89%) may also contribute to higher lead concentrations. the rímac river water quality reports (sedapal, 2009) show that in months such as january and february, lead concentrations increase above the eca values in stations close to the pzh; however, in other months, the lead concentrations remain below the threshold limit. these observations suggest that lead concentrations in the rímac river increase periodically, probably due to the effluents of the industries that are close to the rímac river basin. tagetes erecta development morphological parameters figure 2 shows that treatment 1 and 2 produced the tallest plants, while treatments 3 and 4 produced plants that were slightly shorter than treatment 5 and 6 plants. parameter units value ph (1:1) 7.03 c.e. (1:1) ds/m 2.92 caco3 % % 1.3 m.o. % % 4.89 p ppm ppm 50.3 k ppm ppm 432 mechanical analysis sand % 51 silt % 34 clay % 15 clase textural fr. cic meq/100g 14.08 changeable cations ca+2 meq/100g 11.09 mg+2 meq/100g 1.53 k+ meq/100g 0.88 na+ meq/100g 0.57 al+3+h+ meq/100g 0 sum of cations meq/100g 14.08 sum of bases meq/100g 14.08 % sat. of bases % 100 fe ppm 50.4 cu ppm 34.4 mn ppm 4.24 zn ppm 44.8 b ppm 3.8 pb ppm 176.21 cd ppm 6.47 cr ppm 17.59 table 4: characteristics of the soil of huachipa zoological park figure 2. plant heights in the different treatments. m, mycorrhizae; e, edta t6 m: 0.5 g e: 1 g/kg t5 m: 0 g e: 1 g/kg t4 m: 0.5 g e: 0.5 g/kg t3 m: 0 g e: 0.5 g/kg t2 m: 0.5 g e: 0 g/kg t1 m: 0 g e: 0 g/kg 20 15 10 5 0 treatment h ei g h t (c m ) lead phyto extraction in the huachipa zoological park through the use of tagetes erecta associated with a mycorrhiza and edta january april 2020 32 similarly, figure 3 shows that as in the case with plant heights, treatments 1 and 2 produced the longest roots while treatments 3 and 4 produced the shortest roots. however, for the same concentration of edta, the longest roots occurred in the presence of mycorrhizae. statistical analysis indicates that neither mycorrhizae concentration (0 and 1.5 g / kg of soil) nor edta x mycorrhiza interaction significantly influenced plant height and root length. however, significant differences were found between plant height and root length in treatments with different concentrations of edta (0, 0.5 and 1 g /kg of soil). in the case of root lengths, significant differences were found between the factors (mycorrhizae and edta) and their interaction, indicating that mycorrhizae and edta affect root length. in cases where different concentrations of mycorrhizae produced no effect on root length, this may indicate that fungi did not colonize plant roots under the soil conditions presented in table 4. treatments 3, 4, 5, and 6 produced shorter plants; this may be due to the presence of edta, which enabled the plants to extract more lead from the soil, leading to a higher concentration of the contaminant in the plant. thus, the physiological development of the plants in these treatments was negatively affected. this confirms previous research carried out by sinhal et al. (2009), who reported that marigold plants treated with edta accumulated more metals but also showed a great reduction in growth. in general, treatments with edta showed reduced growth in both the aerial and belowground parts of the plant. this may be because chelating agents usually produce symptoms of severe phytotoxicity in plants (ebbs & kochian, cited by diez, 2008), since they increase the extraction of lead, causing stunted growth. lead extraction in tagetes erecta the concentrations of lead in the aerial parts of the plants in each treatment are presented in figure 4. treatments 1 and 2 produced the least amounts of lead accumulation in the aerial parts, while treatments 3 and 4 produced the highest concentrations of lead. in the case of the roots (figure 5), treatments 1 and 2 again produced the least amount of lead accumulation, while treatments 3 and 4 produced the highest concentrations of lead. statistical analysis indicates that neither mycorrhizae concentration (0 and 1.5 g / kg of soil) nor the edta x treatments edta (mg) mycorrhiza (mg/kg soil) repetitions averagea b c d pb (ppm) 1 0 0 279.56 214.44 217.00 121.06 208.02 2 0 0.5 180.88 248.63 231.63 114.25 193.85 3 0.5 0 206.38 235.75 178.75 231.63 213.13 4 0.5 0.5 150.25 154.56 177.25 189.25 167.83 5 1 0 228.13 193.5 174.19 217.5 203.33 6 1 0.5 346.13 194.19 199.63 137.13 219.27 table 5: initial concentrations of lead in the treated soils treatments edta (mg) mycorrhiza (mg/kg soil) repetitions averagea b c d pb (ppm) 1 0 0 385.88 153.63 235.75 78.81 213.52 2 0 0.5 384.25 167 253 174.63 244.72 3 0.5 0 227.13 139.63 199.25 307.56 218.39 4 0.5 0.5 166.19 141.38 248.63 258.69 203.72 5 1 0 124.56 312 179.63 172.38 197.14 6 1 0.5 119.5 163 276.25 173.56 183.08 table 6: final concentrations of lead in the treated soils t6 m: 0.5 g e: 1 g/kg t5 m: 0 g e: 1 g/kg t4 m: 0.5 g e: 0.5 g/kg t3 m: 0 g e: 0.5 g/kg t2 m: 0.5 g e: 0 g/kg t1 m: 0 g e: 0 g/kg 30 25 20 15 10 5 0 treatment le n g th ( cm ) figure 3. root lengths of plants in the different treatments. m, mycorrhizae; e, edta; the asterisk indicates values that are far from normal distribution. carrera, o.; tello, l. peruvian journal of agronomy 4(1): 27-34 (2020) 33 mycorrhiza interaction produced significant differences in lead concentrations within the plant (both aerial parts and roots). determination of translocation factors table 7 shows the translocation factors associated with each treatment in the experiment. the results show that the translocation factor never exceeds the value of 1. a plant is considered hyperaccumulating when its translocation factor exceeds 1, meaning the plant possesses a great capacity to transport metals from the roots to the aerial parts (zhao, 2002). conclusions according to the results obtained in the present investigation, we can draw the following conclusions: -marigold has the ability to extract lead from soil; however, none of the treatments had a translocation factor greater than 1; therefore, this species is not considered as hyperaccumulative under the conditions of the experiment. -the association of marigold with arbuscular mycorrhizae, under the conditions of the experiment, did not present significant differences with respect to the total lead extraction carried out by the plant without inoculating. -the association of marigold with edta did show significant differences with respect to the total lead extraction carried out by the plant alone. the plants to which edta was added extracted higher amounts of lead; however, due to the high concentrations of lead in the organism, they presented deficiencies in their physiological development. -the association of marigold with arbuscular mycorrhizae and edta, under the conditions of the experiment, did not present significant differences with respect to the total lead extraction carried out. references amna, masood, s., syed, j.h., munis, m.f.h., & chaudhary, h.j. (2015). phyto-extraction of nickel by linum usitatissimum in association with glomus intaradices. international journal of phytoremediation, 17(10), 981–987. https://doi.org/ 10.1080/15226514.2014.989311 castillo, o.s., dasgupta-schubert, n., alvarado, c.j., zaragoza, e.m., & villegas, h.j. (2011). the effect of the symbiosis between tagetes erecta l. (marigold) and glomus intraradices in the uptake of copper(ii) and its implications for phytoremediation. t6 m: 0.5 mg e: 1 mg/kg t5 m: 0 mg e: 1 mg/kg t4 m: 0.5 mg e: 0.5 mg/kg t3 m: 0 mg e: 0.5 mg/kg t2 m: 0.5 mg e: 0 mg/kg t1 m: 0 mg e: 0 mg/kg 200 150 100 50 0 treatment le ad c o n ce n tr at io n ( p p m ) figure 4. lead concentrations in the aerial parts of the treated plants t6 m: 0.5 g e: 1 g/kg t5 m: 0 g e: 1 g/kg t4 m: 0.5 g e: 0.5 g/kg t3 m: 0 g e: 0.5 g/kg t2 m: 0.5 g e: 0 g/kg t1 m: 0 g e: 0 g/kg 800 600 400 200 0 treatment le ad c o n ce n tr at io n ( p p m ) figure 5. lead concentrations in the roots of the treated plants treatment mycorrhizae edta repetition translocation factor ave rage t1 0 0 a 0.19 0.49b 0.3 c 0.99 t2 0 0.5 a 0.91 0.50b 0.26 c 0.34 t3 0 1 a 0.51 0.47b 0.35 c 0.55 t4 0.5 0 a 0.19 0.16 c 0.13 t5 0.5 0.5 a 0.08 0.17b 0.17 c 0.27 t6 0.5 1 a 0.39 0.43b 0.46 c -table 7: translocation factors in each treatment. lead phyto extraction in the huachipa zoological park through the use of tagetes erecta associated with a mycorrhiza and edta january april 2020 34 new biotechnology, 29(1), 156–164. https://doi. org/10.1016/j.nbt.2011.05.009 corzo, i., & velásquez, m. (2014). el plomo y sus efectos en la salud. acta médica del centro, 8(3), 141–148. delgadillo-lópez, a.e., gonzález-ramírez, c.a., prietogarcía, f., villagómez-ibarra, j., & acevedosandoval, o. (2011). fitorremediación: una alternativa para eliminar la contaminación. tropical and subtropical agroecosystems, 14(2), 597–612. diez, l. (2008). fitocorrección de suelos contaminados con metales pesados: evaluación de plantas tolerantes y optimización del proceso mediante prácticas agronómicas. [doctoral thesis, universidad de santiago de compostela]. duarte, a., cachada, a., & rocha-santos, t. (2017). soil pollution—from monitoring to remediation. cambridge: academic press. duñabeitia, m., rodríguez, n., salcedo, i., & sarrionandia, e. (2004). field mycorrhization and its influence on the establishment and development of the seedlings in a broadleaf plantation in the basque country. forest ecology and management, 195(1–2), 129– 139. https://doi.org/10.1016/j.foreco.2004.02.038 gaur, a., & adholeya, a. (2004). prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. current science, 86(4), 528–534. https://www.jstor.org/stable/24107905 huang, j.w., chen, j., berti, w.r., & cunningham, s.d. (1997). phytoremediation of lead-contaminated soils: role of synthetic chelates in lead phytoextraction. environmental science and technology, 31(3), 800–805. https://doi.org/10.1021/ es9604828 juárez, h. (2006). contaminación del río rímac por metales pesados y efecto en la agricultura en el cono este de lima metropolitana. [master’s thesis, universidad nacional agraria la molina] lombi, e., zhao, f.j., dunham, s.j., & mcgrath, s.p. (2001). phytoremediation of heavy metal contaminated soils: natural hyperaccumulation versus chemically enhanced phytoextraction. journal of environmental quality, 30(6), 1919– 1926. https://doi.org/10.2134/jeq2001.1919 marchiol, l.; sacco, p.; assolari, s.; zerbi, g. (2004). reclamation of polluted soil phytoremediation potential. water, air, and soil pollution, 158(1), 345–356. mendez garcía, e. f. (2009). el cultivo de marigold (tagetes erecta l.) en el perú: presente y futuro. [master’s thesis, universidad nacional agraria la molina] servicio de agua potable y alcantarillado de lima sedapal (2009) convenio de cooperación institucional no. 002-2009 / minsa-sedapal shah, k., mankad, a.u., & reddy, m.n. (2017). lead accumulation and its effects on growth and biochemical parameters in tagetes erecta l. int. j. life. sci. scienti. res., 3(4), 1142–1147. https://doi. org/10.21276/ijlssr.2017.3.4.7 sinhal, v.k., srivastava, a., & singh, v.p. (2010). edta and citric acid mediated phytoextraction of zn, cu, pb and cd through marigold (tagetes erecta). journal of environmental biology, 31(3), 255–259. tello, l., jave, j., & guerrero, j. (2018). análisis de cuantificación de plomo en suelos de parques recreacionales de la ciudad de lima—perú. ecología aplicada, 17(1). https://doi.org/10.21704/ rea.v17i1.1168 zhao, f.j., hamon, r.e., lombi, e., mclaughlin, m.j., & mcgrath, s.p. (2002). characteristics of cadmium uptake in two contrasting ecotypes of the hyperaccumulator thlaspi caerulescens. journal of experimental botany, 53(368), 535–543. https://doi. org/10.1093/jexbot/53.368.535 peruvian journal of agronomy 4(3): 88–92 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i3.1650 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 02 january 2020 accepted for publication: 20 november 2020 response of five eco-physiological parameters, to the application of potassium in sunflower (asteraceae), under semi-arid climate respuesta de cinco parámetros ecofisiológicos, a la aplicación de potasio en girasol (asteraceae), bajo clima semiárido ernesto díaz-lópez1* *corresponding author: ernesto.lopez@uttehuacan.edu.mx https://orcid.org/0000-0001-5623-8470 abstract in order to know the effect of three levels of potassium on five eco-physiological parameters in sunflower, open-pollinated achenes victory cultivar, were sown at a density of 11.08 plants m-2 with a fertilization of 100n-50p kg ha-1 n=nitrogen, p= phosphorous, k= potassium. traits evaluated were: agronomic yield, leaf area index, light attenuation coefficient and intercepted radiation. the treatments consisted of three potassium levels: 0, 50 and 100 kg ha-1 (k2o) and four repetitions (3x4) resulting in 12 experimental units, which were evaluated under a randomized complete block design. the results indicate that the application of 50 and 100 kg ha-1 of potassium increase the agronomic yield, leaf area index, intercepted radiation as well as the light attenuation coefficient. from this investigation it can be concluded, that potassium is a very important nutrient for sunflower when it is sown in dry climates such as the tehuacan valley, puebla. key words: agronomic yield, light attenuation coefficient, solar radiation. resumen con el objetivo de conocer el efecto de tres niveles de potasio, sobre cinco parámetros ecofisiológicos en girasol, aquenios de polinización libre del cultivar victoria fueron sembrados a una densidad de 11.08 plantas m-2, con una fertilización de 100-50 kg ha-1 de np. las características a evaluar: fueron rendimiento agronómico, índice de área foliar, coeficiente de atenuación de luz y radiación interceptada. los tratamientos consistieron de tres niveles de potasio: 0, 50 y 100 kg ha-1 (k2o) y cuatro repeticiones (3x4)=12 unidades experimentales, los cuales fueron evaluados bajo un diseño de bloques completos al azar. los resultados indican que la aplicación de 50 y 100 kg ha-1 de potasio, incrementan el rendimiento agronómico, índice de área foliar, radiación interceptada así como el coeficiente de atenuación de luz. de esta investigación se puede concluir, que el potasio es un nutrimento de suma importancia para el girasol cuando es sembrado en climas secos como del valle de tehuacán, puebla. palabras clave: rendimiento agronómico, coeficiente de atenuación de luz, radiación solar. 1 universidad tecnológica de tehuacán. ingeniería en agricultura sustentable y protegida. prolongación de la 1 sur 1101, san pablo tepetzingo, tehuacán, puebla, méxico. c.p. 75859. cite this article: díaz-lópez, e. (2020). response of five eco-physiological parameters, to the application of potassium in sunflower (asteraceae), under semi-arid climate. peruvian journal of agronomy, 4(3), 88–92. http://dx.doi.org/10.21704/pja. v4i3.1650 introduction sunflower (helianthus annuus l.), is an oilseed plant that belongs to the asteraceae family (redonda & villaseñor, 2011). for many years it has been subject to the extraction of fatty acids, for human consumption and industrial use, due to the high quality of the oil extracted from its achenes. this cultivation plant is currently used as an ornamental, due to the large range of shades of its flowers linked to the newly created hybrids, although free-pollinated materials can also be exploited as ornamentals (ávila, 2009). among other uses, sunflower can be cited as livestock feed, in arid and semi-arid areas of northern mexico. for instance, it is important to carry out studies of this species in arid and semi-arid areas, such as the tehuacán valley, in the state of puebla, where it can be a viable alternative to obtain foreign e. díaz-lópez peruvian journal of agronomy 4(3): 88–92 (2020) 89 exchange, and thus, become an option to monoculture corn. in relation to potassium, this together with nitrogen and phosphorus, are considered as indispensable macroelements in plant nutrition, due to the high amounts that crops require. thus, under this trend, potassium is involved in physiological processes of great importance in plants, among them we can mention: stomatal opening and closing mejía et al. (2008), as well as in nautical movements such as thigmotropism, which present some species like mimosa pudica l. (salisbury & ross, 1998). another aspect of great importance in which potassium intervenes, is directly in the cellular water balance, maintaining the turgidity of the protoplasm and, consequently, it has a very important role in the postharvest quality of fruits and flowers, as well as the structural component of the sclerenchematic system, so it avoids stemming in crops such as corn (maya & ramírez, 2002). despite the previous arguments about this nutrient, in mexico the importance that this element deserves in plant nutrition of extensive crops has not been given. thus, for the foregoing, the objective of the present investigation was: to evaluate the effect of three potassium levels, on five eco-physiological parameters in sunflower, when it is sown under a semi-arid climate. the hypothesis was: the application of high potassium levels will increase the leaf area index, intercepted radiation, as well as the sunflower’s agronomic yield, when it is sown under arid weather conditions. materials and methods the present investigation was carried out in the experimental field of the universidad tecnológica de tehuacan, located at 18° 24´51´´ north latitude, 97° 20´ 00´´ west longitude and 1409 masl. the climate of the region is semi-arid whose family is: bs1eg, which corresponds to a semi-arid climate, whose annual average temperature is greater than 18 °c and less than 27 °c. the rainfall regime runs from may to september, with the total rainfall being greater than 400 and less than 600 mm. the temperature oscillation between the warmest month and the coldest month is greater than 7 °c and less than 14 °c, respectively. the warmest month, occurs before the summer solstice (garcía, 2005). the genetic material consisted of open pollinated sunflower achenes cv. victory of, which were sown in beds 25 m long, 1.50 m wide and 0.25 m high. the topological arrangement was (0.30 x 0.30), resulting in a population density of 11.08 plants m-2, distributed in three rows. the soil corresponds to an endoleptic lithosol, which is why the planting beds were made. to know the initial soil conditions, a composite sample was taken, the analysis data are presented in table 1. the above parameters were carried out using the following methodology: ph, in water by the potentiometric method with a soil water ratio (1:2) (w/v); organic matter by the method of walkley & walk (2005); saturated bases, by extraction of ammonium acetate and neutral ph; texture, by means of the bouyoucos hydrometer using sodium hexametaphosphate as dispersant (loeza et al., 2016; secretaría del medio ambiente y recursos naturales, 2000). the entire experiment was fertilized with 100 kg ha-1 of nitrogen and 50 kg ha-1 of phosphorus, whose sources were urea (46% n) and triple calcium superphosphate (46% p2o5), applied at the time of planting respectively. weed control was carried out manually as they appeared within the crop. in the same way, as a preventive way to attack cutting worms and whiteflies, cypermethrin was applied at 30 days. the treatments consisted of three levels of potassium 0, 50 and 100 kg ha-1 whose source was potassium chloride (60% k2o), applied at the time of planting and four replications (3x4), for a total of 12 experimental units. the treatments were evaluated under a randomized complete block design, using the mathematical model yij = µ + ƭi + βj + εij where: yij, is the variable response of the i-th level of potassium in the j-th block; µ, is the true general mean; ƭi, is the effect of the i-th level of potassium; βj, is the effect of the j-th block and εij, is the experimental error of the i-th level of potassium in the j-th block (cochran & cox, 2008). the experimental unit consists of three grooves, within which the central groove served as a useful plot. the response variables were: agronomic performance and total weight of three-chapter achenes with a pce-ls model analytical balance. the corresponding means for each treatment were calculated. for the leaf area index, destructive sampling was performed at 30, 60, 90 and 120 days after sowing (das), to obtain the values and use the equation lai = ((la) (pd)) / 10000 where: lai, is the leaf area index; la, leaf area determined by the ratio la = (l) (w) (0.70), so la, is the leaf area in centimeters; l and w, are the length and width of the true leaf, respectively (díaz et al., 2015; escalante & kohashi, 1993); is the population density. radiation intercepted, measured directly from the culture, with the help of an accupar lp-80 model ceptometer (díaz et al., 2013). light attenuation coefficient, determined by beer’s law physical properties chemical properties apparent density 1.75 g cm-3 nitrogen 5.3 mg kg-1 kjeldahl texture clay-loam phosphorus 3.3 mg kg-1 olsen colour ph 7.5 dry 10 yr 7/4 e. c. 4.5 ds m-1 damp 10 yr 4/4 saturated bases cmol(+) kg-1 na+ 0.3 k+ 0.8 ca++ 3.7 mg++ 0.9 o. m. 1.5 % table 1. physical and chemical properties of an endoleptic lithosol soil. universidad tecnológica de tehuacán. summer of 2017. e. c.: electric conductivity; o. m.: organic matter. response of five eco-physiological parameters, to the application of potassium in sunflower (asteraceae), under semi-arid climate september december 2020 90 f = 1-exp (-klai), where: f, is the radiation intercepted in percentage; k, is the light attenuation coefficient and lai, is the leaf area index (morales et al., 2014; díaz et al, 2011). evaporation was calculated with the help of an “a” type tank evaporimeter to express the results in mm. when the response variables are significant, the tukey multiple comparison test (p≤0.05) was applied, using the sas proc glm statistical package (sas institute, 2004). results and discussion the agronomic yield presented significant differences, so the treatments where 50 and 100 kg ha-1 of potassium were applied, exceeded the control with 41.11 and 46.05 g plant-1 respectively. in relation to control, this just presented, 33.12 g plant-1, that is, 19.43% lower yield than the treatment with the application of 50 kg ha-1 of potassium and 28.07% less than the application of 100 kg of potassium (figure 1). these results differ with those reported by (maya & ramírez, 2002), who mention that the application of 0, 120 and 240 kg ha-1 of potassium in the crop of corn, had no significant effect on grain yield, when applying, high doses of potassium. this difference might be caused by the different species used in their study compared to our study. in addition, the phenotypic plasticity for the absorption of nutrients such as potassium, due to the large radical system that sunflower presents and its ability to absorb alkali metals such as potassium (escalante et al., 2017; díaz et al., 2017) might be causing a different response. the leaf area index for all treatments, was adjusted to a third-grade model, with a high coefficient of determination 0.99 and highly significant (p≤0.05), which indicates that 99 % of the lai, is a function of the days after planting. the treatments in question indicate an average increases between 30 and 90 days and then decrease due to the senescence of the leaves at 120 days. it is evident that the application of potassium in 50 and 100 kg ha-1, produced a positive effect on the lai, increasing this ecophysiological index compared to the control (figure 2). this behavior has been corroborated by aguilar et al. (2005), shipley (2002) who report that the lai in sunflower at 90 das, reaches its maximum expansion, and then decreases, due to the abscission of leaves at a density of 7.5 plants m-2, coinciding with the present investigation under a similar climate. the radiation intercepted by the culture was adjusted to logarithmic models in all treatments (figure 3). it can be seen that the coefficient of determination was highly significant for all treatments. thus, the highest amount of intercepted radiation was for treatments 50 and 100 kg ha-1 of potassium, with 80 and 83% of intercepted radiation, while the control only reached 75% of radiation. this greater amount of radiation was reached at 120 das. this response has been corroborated by díaz et al. (2010), who worked with a combined sunflower-bean agro-ecosystem and mentioned that when the sunflower inflorescence is cut, the system only intercepts 80% of the radiation, thus coinciding with the present study. figure 1. sunflower agronomic yield (helianthus annuus l.), under three levels of potassium. universidad tecnológica de tehuacán. summer, 2017. hsd, honest significant difference. 33.12 b 41.11 a 46.05 a 0 5 10 15 20 25 30 35 40 45 50 control 50 100 a g r o n o m ic y ie ld ( g p la n t1 ) treatments kg ha-1 (k2o) yield hsd= 6.91* figure 3. radiation intercepted by sunflower (helianthus annuus l.), as a function of three levels of potassium 0, 50 and 100 kg ha-1. universidad tecnológica de tehuacán. summer, 2017. das, days after sowing. lai(witness) = -0.00001das3 + 0.002das2 0.1129das + 1.92 r² = 0.99** lai(50) = -0.00001das3 + 0.0025das2 0.1333das + 2.2 r² = 0.99** lai(100) = -0.00001das3 + 0.0027das2 0.1349das + 2.2 r² = 0.99** 0 0.5 1 1.5 2 2.5 3 0 20 40 60 80 100 120 l e a f a r e a i n d e x ( l a i) days after sowing (das) control 50 100 figure 2. dynamics of the sunflower leaf area index (helianthus annuus l.) at 30, 60, 90 and 120 das, under three levels of potassium. universidad tecnológica de tehuacán. summer, 2017. ir(control) = 50.62ln(das) 171.42 r² = 0.97** ir(50) = 52.466ln(das) 173.63 r² = 0.99** ir(100) = 54.025ln(das) 177.93 r² = 0.99** 0 10 20 30 40 50 60 70 80 90 0 20 40 60 80 100 120 in te r c e p te d r a d ia ti o n ( % ) days after sowin (das) control 50 100 e. díaz-lópez peruvian journal of agronomy 4(3): 88–92 (2020) 91 regarding the light extinction coefficient, the adjustment models k vs time (days), in all cases they were adjusted to quadratic models, with highly significant determination coefficients for 50 and 100 kg ha-1 of potassium, while the control it only presented a significant coefficient of determination. under this tenor, the values of potassium for 50 and 100 kg ha-1 of potassium, ranged between 0.47 and 0.70 at the beginning and end of the ontogenic cycle, indicating that only 53% of the intercepted radiation passed to the lower stratum of the canopy, while at the end of the crop cycle, only 30% managed to reach the lower stratum (figure 4). this result might be explained by an increase in the index of foliar area of the crop that produces a greater interception of the light favoring a greater amount of radiation intercepted by the upper canopy and allowing to pass only a small fraction of radiation (díaz et al., 2010; rodríguez et al., 2004) . the total accumulated evaporation of the culture, during its ontogenic cycle was 598 mm, of which the greatest evaporation occurred at 30 das, with 162.5 mm, to subsequently decrease progressively at 120 das with 130.4 mm. this evaporation behavior is explained by the foliar area index, because when it increases, it intercepts a greater amount of radiation, preventing it from reaching the ground, and per se prevents soil water from evaporating (figure 5). thus, the adjustment model for this variable was linear, with a significant coefficient of determination. the negative slope of the model indicates that evaporation decreases by 0.308 mm every day of the crop cycle. conclusions the main conclusions of our study were: i) the highest index of foliar area and intercepted radiation were obtained with the high level of potassium; ii) similarly, the highest agronomic yield was obtained with the application of 100 kg ha-1 of potassium; iii) the greatest extinction of light caused by the vegetable canopy was presented with the application of potassium at levels of 50 and 100 kg ha-1; and the ontogenetic development of the crop increased the canopy causing a decrease of the evaporation of the crop as it reached physiological maturity; iv) potassium is an essential nutrient for the growth and yield of sunflower; v) this study shows that fertilization with potassium is necessary for the crop of sunflower, when it is planted in the valley of tehuacan, puebla. references aguilar, g. l., escalante, e. j. a., ficikovsky, z. l. tijerina, ch. l., & mark, e. e. 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(2017). potential absorption of calcium in sunflower (helianthus annuus l.) and its effect on ph and electrical conductivity. international journal of current microbiology and applied sciences, 6(6), 3299–3304. figure 4. light attenuation coefficient (k), in sunflower (helianthus annuus l.) under three potassium levels at 30, 60, 90 and 120 days after sowing. universidad tecnológica de tehuacán. summer, 2017. das, days after sowing. k(witness) = -0.00007das2 + 0.0138das + 1e-15 r² = 0.96** k(50) = -0.00006das2 + 0.0108das + 0.2125 r² = 0.99** k(100) = -0.00005das2 + 0.0079das + 0.4025 r² = 0.87* 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 20 40 60 80 100 120 140 li g th a tt e n u a ti o n c o e ff ic ie n t ( k ) days after sowing (das) witness 50 100 figure 5. evaporation of water during the sunflower cultivation cycle (helianthus annuus l.), at 30, 60, 90 and 120 das. universidad tecnológica de tehuacán. summer, 2017. ev = -0.308das + 172.6 r² = 0.86* 100 110 120 130 140 150 160 170 180 0 20 40 60 80 100 120 140 e v a p o ra ti o n ( m m ) days after sowing (das) response of five eco-physiological parameters, to the application of potassium in sunflower (asteraceae), under semi-arid climate september december 2020 92 díaz, l. e., morales, r. a., vargas, r. e. j., hernández, h. p., reséndiz, m. r. c., brena, h. i., & loeza, c. j. m. 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(2004). sas/ stat user´s guide, version 8.02. sas institute, inc. cary nc, usa. peruvian journal of agronomy 4(3): 82–87 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i3.1649 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 22 june 2020 accepted for publication: 20 november 2020 reaction to drought: a case study of native potatoes (solanum spp.) cultivated in huánuco, perú reacción a sequía: un caso de estudio de papas nativas (solanum spp.) cultivadas en huánuco, perú rolando egúsquiza bayona1*; jesús salinas f.2; mario vidal f.3 *corresponding author: pegusquiza@lamolina.edu.pe https://orcid.org/0000-0001-6113-5927 abstract climate change is an obvious threat to agriculture, food security and conservation of plant genetic resources. potato is a globally important food. in peru, there is high variability and diversity of wild and cultivated species, such that they are considered as one of the region’s most vulnerable to the impact of climate change. with these considerations, we report the results of an investigation in which a preliminary screening of the reaction to drought of 67 native potato morphotypes was conducted to verify for differences in morphological indicators of growth, development, and production of tubers in response to continuous irrigation (ci) and restricted irrigation (ri) treatments. in the course of this study, 21 irrigations were applied to plants by ci (every 1-2 days) and, in the same period, 7 irrigations were applied to plants by ri (every 5-6 days). comparison of characteristics average in all the morphotypes with plants under ci and ri, indicated that ri did not show differences in height of plants, length of internodes, or leaf and terminal leaflet indexes, and that ri reduced the stem thickness and dry weight of foliage. in this study, we highlight the morphotypes whose plant characteristics present higher averages with respect to their clonal counterparts that were treated with ci. the positive relationship between the number and weight of tubers harvested, as well as the significant reduction of both components of low ri yield is confirmed. in addition, we also highlight the response of some morphotypes that present higher tuber yield under ri. the native morphotypes that were identified as tolerant (according their responses in the characteristics of their plants and yield of tubers under ri) are as follows: bgr 19 (“rayhuana”); bgr 99 (“yuracc ñahui hualash”); bgr 170 (“yana utcush”); and bgr 238 (“muru huayro”). key words: potato, drought, native morphotypes, morphological indicators, irrigation. resumen el cambio climático es una amenaza evidente para la agricultura, alimentación y conservación de los recursos genéticos vegetales. la papa es un alimento de importancia mundial y en el perú se encuentra la mayor variabilidad y diversidad de especies silvestres y cultivadas donde, al mismo tiempo, se considera que es una de las regiones de mayor vulnerabilidad a los impactos del cambio climático. con estas consideraciones, el presente trabajo da cuenta de los resultados de una investigación en la que se realizó un tamizado preliminar de la reacción a sequía de 67 morfotipos de papas nativas en el que se evaluaron diferencias en indicadores morfológicos del crecimiento, desarrollo y producción de tubérculos en respuesta a tratamientos de riego continuo (rc) y riego restringido (rr). durante el periodo del estudio se realizaron 21 riegos a plantas bajo rc (cada 1-2 días) y, en el mismo periodo, se aplicaron 7 riegos a plantas con rr (cada 5-6 días). la comparación de promedios de las características en todos los morfotipos con plantas bajo rc y rr, indicó que el rr no produjo diferencias en la altura de plantas, longitud de entrenudos ni en los índices de hoja y del foliolo terminal y que el rr redujo el grosor de tallos y el peso seco del follaje. sin embargo, se destacan los resultados en los que bajo 1 universidad nacional agraria la molina. facultad de agronomía, dpto. académico de fitotecnia. lima, perú. pegusquiza@lamolina.edu.pe 2 universidad nacional agraria la molina. facultad de economía y planificación, dpto. de estadística e informática. lima, perú. jsalinas@lamolina.edu.pe. 3 universidad nacional agraria la molina. facultad de agronomía. lima, perú. 20110997@lamolina.edu.pe. cite this article: egúsquiza, r., salinas, j., & vidal, m. (2020). reaction to drought: a case study of native potatoes (solanum spp.) cultivated in huánuco, perú. peruvian journal of agronomy, 4(3), 82–87. http://dx.doi.org/10.21704/pja.v4i3.1649 r. egúsquiza; j. salinas; m. vidal peruvian journal of agronomy 4(3): 82–87 (2020) 83 introduction as in many regions of the world, in peru, potato cultivation is of a huge nutritional and economic importance. moreover, peru has largest surface and annual production of potatoes in latin america (instituto nacional de estadística e informática [inei], 2013); ministerio de agricultura [minag], 2019); as it is produced in 11 mountain agro-ecosystems (tapia, 1990), that are above 3000 masl, mostly under rainfed and highly dependent on regularity of rains and where 90% of the national production is harvested. another characteristic of the potato in the andes of peru has to do with the presence of not less than 3,000 native varieties that represent a gene reserve of enormous importance, which is due to its great variability and diversity. the genetic resources contained in the native potatoes of peru require further research to minimize the current threats that put their conservation at risk. on the other hand, changes in the water regime, as a consequence of climate change, are an obvious threat that puts crop production at risk. in ifpri report, o´toole and chang (1979) indicated that the yield of wheat and rice under irrigation will be the most affected and that changes in rainfall regimes increased the probability of reduced production. ministerio del ambiente (minam, 2010) indicated that peru is a country that is highly vulnerable to climate change; however. according to harris (1978), potato production can increase up to two tons, per centimeter of water applied in irrigation or rain, and that regional differences in annual yields are due to differences in water supply. mendoza and estrada (1979) indicated that in the peruvian highlands, water extremes are permanent physical threats to better potato production. egúsquiza (2014) indicated that potato cultivation is highly sensitive to lack of water, and that under mountain conditions, potato cultivation requires approximately 600 to 1000 mm of rain per hectare. however, below this range, it will be under a water deficit regime. mamani (1993) reported that the emergence and onset of stolonization of potato plants were the growth period most susceptible to drought. similarly, it was found that an early drought (pre-tuberization drought) yielded smaller tubers in both sensitive and tolerant varieties in comparison with a late drought. in this regard, we evaluated the changes in morphology of plant and yield of morphotypes of native potatoes subjected to periods of irrigation restriction to identify their levels of vulnerability, register differences in their responses, and identify morphotypes with greater tolerance to drought. materials and methods at the regional institute of development (ird, acronym from spanish) of sierra of the universidad nacional agraria la molina (unalm, 11°50′48″ s, 75°23′14″ w; 3322 masl), reaction to drought cycles was evaluated in plants from sprout cuttings of 67 morphotypes from the regional germplasm bank (bgr) of huánuco. the plants were installed under a three-pound moonroof environment with soil mix, compost, and rice husk substrate (v/v 1:1:0.5). the chemical characteristics of the substrate were: ph=6.65; ce=1.21; caco3=1.60; mo=15.72; p=99.6 ppm; and k=78 ppm. average temperature fluctuated between 11.18°c and 2.06°c, with a maximum of 20.3°c and minimum of 2.06°c. the treatments were continuous irrigation (ci) and restricted irrigation (ri); which were started when the plants reached a general average of 40 cm, such that, at the same time, the sliding roof was closed. three plants of each morphotype were watered every 1–2 days (ci) and another three of the same clone, were watered with the same amount of water per pot every 5-6 days, when they showed symptoms of drought stress (ri). seven cycles of ci and ri were performed until the senescence of the plants is triggered. during the treatment period, humidity of available samples in the pots were conducted and volumetric humidity percentage of substrate was recorded. between 60 and 70 days after commencement of treatments, when majority of the plants were in a phonological state of full growth, random samples of morphotypes were investigated and the plants’ height, internode length, stem thickness, leaf index, and terminal leaflet index were recorded. in preharvest, the foliage was separated and the dry weight was recorded, whereas at harvest, which was carried 120 days after transplantation, weight and number of tubers were recorded. in each characteristic, the morphotypes were classified according to the magnitude of differences, due to the effect of ci and ri. morphotypes that showed lower condiciones de rr se encuentran morfotipos cuyas características de plantas presentan mayores promedios respecto a sus contrapartes clonales en condiciones de rc. se confirma la relación positiva entre el número y peso de tubérculos cosechados y la significativa reducción de ambos componentes del rendimiento bajo rr, pero, igualmente, se destaca la respuesta de algunos morfotipos que presentan mayor rendimiento de tubérculos en condiciones de rr. los morfotipos nativos identificados como tolerantes por sus respuestas en las características de sus plantas y en el rendimiento de tubérculos bajo condiciones de rr, fueron el bgr 19 (´rayhuana´); el bgr 99 (´yuracc ñahui hualash´); el bgr 170 (´yana utcush´); y el bgr 238 (´muru huayro´). palabras claves: papa, sequía, morfotipos nativos, indicadores morfológicos, riego. reaction to drought: a case study of native potatoes (solanum spp.) cultivated in huánuco, perú september december 2020 84 values under ri conditions are considered susceptible, and tolerant to those whose averages were similar or higher under ri. statistical tests were done by using a descriptive and exploratory analysis of the data (cobo et al., 2007). subsequently, in each characteristic, paired student´s t test was performed on paired samples to compare differences due to the treatments effect (clifford & taylor, 2008; gutiérrez & de la vara, 2008). finally, pearson correlation was done between the characteristics under ci and ri. results and discussion from the beginning of treatments and until senescence of the plants is reached, seven cycles of continuous and restricted irrigation were applied. in total, each plant with ci received 21 liters, while, in this same period, each plant with ri (drought) received 7 liters. the volumetric humidity in substrate of plants under ci varied between 18 and 20% and remained within this range, which corresponds to its field capacity. the substrate of plants ri varied between 8 and 17% indicating that the availability of water was reduced (figure 1). effects on plant characteristics generally, the conditions of available moisture deficit are expected to affect the physiological functions of plants and reduce the values of morphological characteristics. according to the obtained results (table 1), when comparing the averages of characteristics of all the native morphotypes, humidity stress reduced the thickness of stems and dry weight of the plants. the results show statistically non-significant effects on plant size, internode length, leaf index, and terminal leaflet index, indicating that drought did not modify these characteristics; however, among the morphotypes, different responses were attributed to genetic differences, such that 45.4, 22.2, 44.4, 44.0, 51.8, and 28.1% of low ri morphotypes yielded plants with higher averages of plant height, stem thickness, internode length, leaf index, terminal leaflet index, and dry weight of foliage, respectively. pearson’s correlation coefficients between all the characteristics of the plants under ci (table 2) and ri (table 3) were highly significant between height and stem thickness and between number of tubers and weight of tubers. according to the results, it is possible to affirm that the significant relationship between plants with greater size and thicker stems are due to morphological adaptations that keep the plant upright. in the same way, due to the wide variation in the characteristics present within the total morphotypes, it is possible to affirm that table 1. averages and statistical significance of the differences in characteristics of native potato plants grown in continuous irrigation (ci) and restricted irrigation (ri) conditions characteristics morphotypes n averages significance ci1 ri2 plants height (cm) all 44 55.06 54.24 ns susceptibles 16 61.59 55.15 ** tolerants 20 51.08 54.43 ** stem thickness (cm) all 27 0.74 0.69 ** susceptibles 21 0.75 0.67 ** tolerants 6 0.72 0.77 * internode length (cm) all 27 5.85 5.66 ns susceptibles 15 6.27 5.57 ** tolerants 12 5.31 5.77 ** leaf index all 25 2.03 2.03 ns susceptibles 14 2.15 1.99 ** tolerants 11 1.89 2.11 ** terminal leaflet index all 27 2.13 2.14 ns susceptibles 13 2.23 2.06 ** tolerants 14 2.05 2.22 ** foliage dry weight (g) all 64 30.10 26.28 ** susceptibles 27 37.25 22.73 ** tolerants 18 24.57 30.85 ** (1) ci =continuous irrigation (2) ri = restricted irrigation ns = non-significant correlations 18.35 20.35 18.17 19.60 18.70 17.13 15.22 12.28 10.33 8.73 0.00 5.00 10.00 15.00 20.00 25.00 7 9 8 0 8 1 8 2 8 3 v o lu m e tr ic h u m id it y (% ) days after the transplant con riego sin riego figure 1: variation of volumetric humidity of substrate during the fourth cycle of humidity control r. egúsquiza; j. salinas; m. vidal peruvian journal of agronomy 4(3): 82–87 (2020) 85 greater size of the plants is not always due to greater length of internodes and that greater size of plants is not always due to higher dry weight of foliage. the non-significant correlations between the six characteristics of the plants with the two characteristics of the yield (number and weight of tubers), confirm the evidences that suggest that, in conditions of abiotic stresses, the morphological and physiological responses that interpose the foliage of the plants are different from those of tuberization. the stem thickness-internode length and leaf index-number of tubers correlations that were significant only in plants under rc require further biological verification. by supplying a lower volume of water, a lesser acceleration is expected in the growth and development of the plant, due to a lower rate of division, cell expansion (martínez, 1988), lower pressure of cell turgor (taiz & zeiger, 1998), and changes in stomatal conductance (león, 2019). the tolerance of the morphotypes is attributed to resistance mechanisms of evasion, through which plants interpose a defense barrier, thus reducing stress damage (sevilla & holle, 2004; gonzales, 1999). in addition, given that at the end of an ri cycle, watering of the plants under stress was done, repair and recovery mechanisms would have been expressed as proposed by ekanayake (1994). effects on tuber yield the average number of tubers per plant of all the morphotypes evaluated under ci and ri conditions (table 4) indicate non-statistically significant difference; however, morphotypes presented under ci conditions, had a greater number. on the other hand, under ri conditions, the average yield of tubers per plant of all the morphotypes is 25.5% lower as a result of the drought. however, 17 morphotypes (28.3%) showed higher performance under ri compared to their clonal counterparts under ci. potato growers recognize that the final yield of the potato crop is highly dependent on water and that the plants height stem thickness internode length leaf index terminal leaflet index foliage dry weight tuber weight stem thickness 0.55 (**) internode length 0.25 (ns) 0.48 (*) leaf index 0.10 (ns) 0.13 (ns) 0.01 (ns) terminal leaflet index. 0.19 (ns) 0.30 (ns) 0.32 (ns) 0.26 (ns) foliage dry weight 0.29 (ns) 0.12 (ns) 0.04 (ns) 0.11 (ns) 0.08 (ns) tuber weight 0.16 (ns) 0.26 (ns) 0.27 (ns) 0.06 (ns) 0.21 (ns) 0.34 (ns) n° of tubers 0.14 (ns) 0.06 (ns) 0.20 (ns) 0.49 (*) 0.05 (ns) 0.01 (ns) 0.59 (**) table 2: correlations between characteristics of morphotypes under continuous irrigation (ci) plants height stem thickness internode length leaf index terminal leaflet index foliage dry weight tuber weight stem thickness 0.55 (**) internode length 0.16 (ns) 0.18 (ns) leaf index 0.16 (ns) 0.28 (ns) 0.20 (ns) terminal leaflet index 0.04 (ns) 0.17 (ns) 0.38 (ns) 0.03 (ns) foliage dry weight 0.04 (ns) 0.19 (ns) 0.05 (ns) 0.06 (ns) 0.06 (ns) tuber weight 0.06 (ns) 0.34 (ns) 0.16 (ns) 0.27 (ns) 0.22 (ns) 0.32 (ns) n° of tubers 0.06 (ns) 0.41 (ns) 0.08 (ns) 0.21 (ns) 0.05 (ns) 0.35 (ns) 0.88 (**) table 3: correlations between characteristics of morphotypes under restricted irrigation (ri) ns = non-significant correlations ns = non-significant correlations reaction to drought: a case study of native potatoes (solanum spp.) cultivated in huánuco, perú september december 2020 86 frequency of irrigation should be shorter compared to those other crops. the sensitivity of the potato plant is explained by the weak nature of the roots that overcome the physical barriers of the soil. similarly, it is explained by the lower depth and lower absorption efficiency of the deep roots. potato plant is sensitive to water deficit because its roots unable to overcome small retention tension at lower soil depths compared to the roots of other cultivated plants. plants are known to oppose certain reactions in conditions of water deficit and the immediate response is closure of the stomata. prolonged closure of the stomata reduces the entry of co2, thus reducing photosynthetic activity, which ultimately reduces the tuber yield. the adaptation of native potato morphotypes to high andean pluvial conditions, (a probable susceptibility to excess irrigation water in the morphotypes with lower yield under ci conditions), and physiological compensation mechanisms may explain that some native morphotypes are capable of exhibiting higher performance even under ri. among the group of native potato morphotypes considered tolerant for presenting a greater number and weight of tubers under ri conditions, bgr 19 (“rayhuana”) stands out and also did not reduce size, leaf index, terminal leaflet and, foliage dry weight; bgr 238 (“muru huayro”) did not reduce its leaf indexes or dry foliage weight; bgr 170 (“yana utcush”) did not reduce its stem thickness or length of internodes, and bgr 99 (“yuracc ñahui hualash”), did not reduce its bearing or dry foliage weight. conclusions the comparison of averages in morphotype characteristics that add up to a broad genetic base, demonstrate that the conditions of drought does not affect the bearing of plants, length of internodes, leaf index, terminal leaflet or the number of tubers per plant. stem thickness, dry weight of foliage, and fresh weight of tubers can be significantly decreased even when comparisons are done between native morphotypes that add broad genetic base. in all the characteristics of the plant and tuber yield, it is possible to find native morphotypes that demonstrate a drought stress tolerance reaction. “rayhuana”, “muru huayro”, “yana utcush” and “yuracc ñahui hualash” morphotypes, cultivated in the huánuco region, peru, presented responses in plants and tubers that identify them as drought tolerant and promising genetic resources for future further studies. acknowledgments the authors acknowledge to pnia/inia project for their economic collaboration and of all employees from regional institute of development (ird) of sierra of the agrarian national university la molina (unalm) for their valuable help during the experimental stages of the study. references clifford, r., & taylor, r. 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(2008). análisis y diseño de experimentos. segunda edición. ed. mcgrawhill interamericana. ciudad de méxico, méxico. characteristics morphotypes n averages significance ci1 ri2 number of tubers all 60 11.93 10.39 ns susceptibles 28 15.53 8.77 ** tolerants 17 8.06 13.80 ** tuber weight all 60 115.18 85.77 ** susceptibles 39 143.00 78.30 ** tolerants 17 63.5 107.7 ** table 04. averages and statistical significance of the differences in number and weight of tubers per plant of native grown potatoes under conditions of continuous irrigation (ci) and restricted irrigation (ri) (1) ci =continuous irrigation (2) ri = restricted irrigation ns = non-significant correlations r. egúsquiza; j. salinas; m. vidal peruvian journal of agronomy 4(3): 82–87 (2020) 87 harris, p.m. (1978). water. in p.m. harris (ed.) the potato crop (pp. 245–278). chapman & hall, london. instituto nacional de estadística e informática (2013). resultados definitivos. iv censo nacional agropecuario 2012. 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(1990). zonificación agroecológica y ecodesarrollo en la sierra. segundo encuentro de agricultura ecológica. cajamarca–perú. peruvian journal of agronomy 3(3): 144–152 (2019) issn: 2616–4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i3.1367 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 08 december 2019 accepted for publication: 31 december 2019 first report of enterobacter cloacae as a causative agent of soft rot disease in dragon fruit (hylocereus undatus) stems in peru primer reporte de enterobacter cloacae como causante de la pudricion blanda en tallos de pitahaya (hylocereus undatus) en perú soto, j.(1)*; cadenas, c.(1); mattos, l.(1); trigoso, c.(1) *corresponding author: jmsoto@lamolina.edu.pe abstract the objective of the present research was to identify the causal agent of soft rot disease in the stems of pitahaya plants (hylocereus undatus (haw.) britton & rose), also known as dragon fruit, in two production areas of peru. typical symptoms observed include rotting and soft consistency in stems, as well as yellowish colourations, which usually begin at the tips and outer edges of the stems and extend until they are completely decomposed. symptomatic samples of pitahaya stalks were collected from two commercial fields, the first from the district of independencia, pisco, ica, and the second from the district of naranjos, rioja, san martin. the collected samples were transferred to the phytopathology laboratory of the national agrarian university – la molina where 19 bacterial colonies isolated from symptomatic stem tissue were processed. nine bacterial colonies were selected from the initial 19 for further analysis. the nine selected colonies were gram negative, positive for the catalase enzyme, negative for the enzyme oxidase, and positive for pectinase production, which causes soft rot in potato tubers. these results indicated that these isolates correspond to the enterobacteriaceae family. according to molecular tests and analysis of the 16s region of ribosomal dna, all bacteria corresponded to a single taxonomic genus, enterobacter. this bacterium presented a 99.85% homology with enterobacter cloacae (accession number mh788982.1). the sequences from the nine selected isolates were entered into genbank under accession number mn784371. in the pathogenicity test, 100% infection was obtained in the pitahaya stems and plants inoculated with e. cloacae, and the symptomatology that developed was the same as that observed in the two fields from where the samples were collected. given these results, it can be concluded that e. cloacae is a causative agent of soft rot disease of the stems of h. undatus in the districts of independencia and naranjos, and this is also the first report of this bacterium as a pathogen of pitahaya in peru. keywords: soft rot disease, pitahaya, dragon fruit, hylocereus, enterobacter resumen el objetivo del presente trabajo de investigación fue identificar el agente causal de la pudrición blanda de tallos de plantas de pitahaya (hylocereus undatus) en dos zonas productoras en el perú. la sintomatología observada son pudriciones de consistencia blanda en tallos y con coloraciones amarillentas, las que por lo general inician en las puntas y en los bordes externos de los cladiolos y se van extendiendo hasta la descomposición total de los mismos. se recolectaron muestras de tallos de dos campos comerciales del cultivo, la primera muestra fue del distrito de independencia, en pisco, ica y la segunda muestra del distrito de naranjos, en rioja, san martin. las muestras colectadas se trasladaron al laboratorio de fitopatología de la universidad nacional agraria la molina donde se procesaron y aislaron 19 colonias bacterianas de los tallos con los síntomas de pudrición blanda. se seleccionaron nueve colonias bacterianas de las 19 iniciales. las nueve seleccionadas fueron las gram negativas, las cuales resultaron positivas a la enzima catalasa, negativas a la enzima oxidasa y positivas a la producción de pectinasas ocasionando también pudriciones blandas en tubérculos de papa, las que demostraron que estos aislamientos corresponden a la familia enterobacteriaceae. según las pruebas moleculares con el análisis de la región 16s de adn ribosomal, todas las bacterias corresponden a un solo género taxonómico, enterobacter. esta bacteria presentó un 99.85 % de homología con enterobacter cloacae (número de accesión mh788982.1). las secuencias provenientes de los nueve aislamientos seleccionados fueron introducidas en el genbank con el número de accesión mn784371. en la prueba de patogenicidad se obtuvo un 100 % de infección en los tallos y plantas inoculadas con e. cloacae, y la sintomatología desarrollada fue igual a la observada en los dos campos de donde se procedieron las muestras. se concluye que enterobacter cloacae como causante de la pudrición blanda de los tallos de h. undatus en los distritos de independencia y naranjos, siendo este el primer reporte de esta bacteria como patógeno de pitahaya en el perú. palabras clave: pudrición blanda, pitahaya, fruta de dragon, hylocereus, enterobacter 1 universidad nacional agraria la molina, apartado postal 12-056 la molina, lima – perú. soto, j.; cadenas, c.; mattos, l.; trigoso, c. peruvian journal of agronomy 3(3): 144–152 (2019) 145 introduction pitahaya or pitaya is the common name given to several species of plants in the genera hylocereus, selenicereus, cereus, leptocereus, escontria, myrtilloactos, stenocereus, and opuntia within the cactaceae family. they are native to central and south america, and there are about 35 species. several of the species can be consumed as fruits or vegetables or used as forage (esquivel, 2004; mizrahi, mouyal, nerd, & sitrit., 2004; legaria, alvarado, & hernández, 2005; tel, abbo, bar, & mizrahi, 2004). the genus hylocereus, which has 16 recognised species, is the most widely distributed worldwide. the genome is highly polymorphic, which implies a great deal of variation (legaria et al., 2005). the species hylocereus undatus, h. polyrhizus, h. costaricensis, h. triangularis, and h. purpusii, traditionally known as red pitahayas and referred to as dragon fruit in english, are grown mainly in central america and israel (esquivel, 2004). the yellow pitahayas, which correspond to 20 species of the genus selenicereus, are distributed across bolivia, peru, ecuador, colombia, and venezuela (tel et al., 2004). pitahaya is mainly consumed as fresh fruit, but it also contains watersoluble nitrogen pigments called betalains. furthermore, its broad range of colours, red-violet to yellow, and ability for the natural pigment to remain stable in a wider ph range compared with other natural dyes, such as anthocyanins, make them great potential natural dyes (stintzing, schieber, & carle, 2001; stintzing, schieber, & reinhold, 2002; strack, vogt, & schliemann, 2003; wybraniec et al., 2001). the consumption of flowers and young shoots as fresh vegetables has also been reported (cálix de dios, 2004). the seeds are used as probiotics owing to their oligosaccharide content and are an important ingredient in health foods and nutraceutical products (wichienchot, jatupornpipat, & rastall, 2010). the growing demand for pitahaya in peruvian national markets and its potential as fresh fruit to the international market is spurring na increase in planted production acreage, especially along the coast and in the highlands and jungle of peru. however, as production has increased, several phytosanitary issues have become evident, including those caused by pathogens, and there are no official reports of the causative agents of the diseases that are being observed in the cultivation of pitahaya in peru. worldwide, there have been reports that the stems and fruits of pitahaya are affected by bacteria, such as pectobacterium carotovorum, xanthomonas campestris, and enterobacter spp.; by fungi, such as colletotrichum gloeosporioides, botryosphaeria dothidea, fusarium oxysporum, neoscytalidium dimidiatum, curvularia lunata, fusicoccum sp.; and other pathogens, such as cactus virus x (valencia, sandoval, & cardenas, 2004; wright, rivera, ghirlanda, & lori, 2007; palmateer, ploetz van santen, & correll, 2007; masyahit, sijam, awan & ghazali, 2009; mohd, salleh, & zakaria, 2013; retana, castro, blanco, & quesada, 2019). in mexico, the united states of america, and costa rica, there have been reports of the identification of new species of enterobacteria that cause soft rot on the stems of pitahaya in commercial fields (valencia, cruz, & rodríguez, 2003; retana et al., 2019). the same rotting symptom has been observed in several production areas of pitahaya in peru, and the disease is negatively impacting crop production during some seasons of the year. for these reasons, the present research was conducted with the objective of identifying the causative agent of the soft rot of pitahaya stems in two production areas of peru. materials and methods sample selection and collection pitayaha samples (h. undatus) were collected from two production areas of peru, one sample in the district of independencia, pisco, ica, and another in the district of los naranjos, rioja, san martin. at the commercial plantations, pitahaya plants whose stems exhibited symptoms of soft rot at different states were located, and a description of the symptoms was taken. fifteen symptomatic stem samples were collected from each plantation, and the material was transferred to the phytopathology laboratory of the universidad nacional agraria la molina. isolation of the causative agent to isolate the causative agent, the methodology described by french and hebert (1980) was used to isolate both fungi and bacteria. the pitahaya stems were washed with clean, running water to remove any impurities from the field and then cut into small pieces, which were placed in a biosecurity booth ii biobase. inside the biosecurity cabin, the stem pieces were immersed in a solution of 1% sodium hypochlorite for 3 min, then rinsed with sterile distilled water and allowed to air-dry for 15 min on sterile paper towel in order to remove excess water. segments of approximately 0.5 cm were removed from the internal part of the stem pieces that covered both diseased and healthy tissues. five tissue segments were placed in various petri dishes containing the na, potato dextrose agar (pda), and corn meal agar with antibiotics and benomyl (cmaparb) media. another isolation methodology used was to macerate small portions of symptomatic tissue with 10 ml of sterile, distilled water in ceramic mortars using a pestle. aliquots of the macerated juice were extracted and distributed into petri dishes containing either na, pda, or cmaparb. all petri dishes were placed in a thermo scientific incubator for 48 h at a temperature of 30 °c. once the bacterial isolates were obtained, visually distinct colonies were transferred to individual petri dishes with na media using a sewing needle. a drop of sterile, distilled water was added to the sample to facilitate streaking of the plate. plates of the selected and isolated colonies were incubated first report of enterobacter cloacae as a causative agent of soft rot disease in dragon fruit (hylocereus undatus) stems in peru september december 2019 146 for 24 h at 30 °c. these purified colonies were used to perform the corresponding identification tests. phenotypic identification tests gram staining the gram staining test was conducted in order to discriminate gram-positive from gram-negative bacteria. it was conducted using the methodology described by schaad (2001). after staining on slides, the preparations were observed on a leica dm500 compound microscope at a magnification of 1000× per immersion to determine, by colouring, whether the isolates were gram positive or gram negative. additionally, the shapes of the bacterial cells were observed (garcia de la guarda & alvarado, 2012) oxidase enzyme test to determine the presence of the cytochrome c oxidase enzyme, each isolated bacterial colony was subsampled and placed on filter paper with tetramethyl-pphenylenediamine. whether or not the colour of the filter paper changed from white to purplish blue was observed, which is indicative of the presence of enzyme (schaad, 2001). catalase enzyme tests to determine the presence of the catalase enzyme, the methodology described by rodríguez, gamboa, hernández, and garcía (2005) was used. each isolated bacterial colony was sub-sampled and placed on a slide with a drop of 3% hydrogen peroxide (h2o2). oxygen release was evaluated by the production of effervescence (schaad, 2001). pectinase test to determine the presence of pectinase enzymes, isolated and selected colonies were carried onto crystal violet pectate (cvp) media, which was used to inoculate slices of potato (solanum tuberosum) tubers (schaad, 2001). planting in half cvp to determine the activity of the pectinmethylgalacturonase and/or pectinliase enzymes, the selected bacterial isolates were placed at five points equidistant from each other on a petri plate containing cvp media. the inoculated plates were incubated for 48 h at 30 °c, after which a hole formation in the middle of the points was observed (schaad, 2001). sliced potato tuber inoculation selected bacterial isolates were inoculated into slices of disinfected potato tubers and placed into humidity chambers. for inoculation, an aliquot of bacterial suspension from each of them was placed onto the slices. the inoculated slices were incubated for 3 days at 30 °c and then observed for soft rot symptom development (schaad, 2001). molecular identification of bacterial isolates dna extraction the method used was adapted from ramos and barboza (2016). for dna extraction from each of the selected bacterial isolates, tubes containing 2 ml of the nutrient broth medium were prepared, and the isolates were added individually using a small pestle. the tubes were then incubated for 48 h at 30 °c. after the incubation time, a 1 ml aliquot was extracted from each tube and transferred to 1.5 ml eppendorf vial tubes. the following reagents were then added to each sample: 558 µl of te buffer, 30 µl of 10% sds, 6 µl of proteinase k, 6 µl of rnase, 100 µl of 5 m sodium chloride, 80 µl of 2% ctab buffer (10 mm tris hcl, 20 mm edta, 0.7 m nacl), and 6 µl of beta mercaptoethanol. the samples were incubated for 15 min at 65 °c in a thermo-agitator, and then 800 µl of isoamyl alcohol chloroform was added (24:1 v/v) and vortexed for 10 s. the samples were allowed to stand for 5 min in the biofreezer at −20 °c, after which 500 µl of isopropanol was added and centrifuged for 7 min at 12000 rpm at 4 °c. after centrifugation, the supernatant was removed from each tube, and 700 µl of 80% ethanol was added. the samples were centrifuged again for 5 min at 12,000 rpm at 4 °c. the supernatant was removed, and the tubes with dna pellets were allowed to dry inclined overnight. the next day, the dna pellets were resuspended in te buffer. to quantitatively determine the concentrations of dna extracted from each selected isolation, a nanodrop 2000 spectrophotometer was used, and gel electrophoresis on a 1% agarose gel stained with hydragreen run at 80 volts for 30 min was performed (ramos & barboza, 2016). amplification of dna by polymerase chain reaction (pcr) the pcr was performed with primers 27f (5´-agagtttgatcmtggctcag-3´) and 1492r (5´ tacggytaccttgttacgactt-3´) described by lane et al. (1985), which amplify the total rna sequence of the 16s ribosomal gene. the pcrs were performed according to the aime protocol (2006) with some modifications. the stock solution or master mix was prepared in 25 µl reaction volumes with 5 µl of 5x green gotaq® flexi buffer (promega), 2 µl of 25 mm magnesium chloride solution (promega), 0.5 µl of 2.5 mm triphosphate dinucleotides, 0.5 µl of each of the primers at 20 µm (forward and reverse), 0.125 µl of mix gotaq® flexi dna polymerase (promega), and 1 µl of dna at a concentration of 30 to 90 ng/µl. a pcr without dna was used as a negative control. the amplification reaction was performed starting with an initial pre-denaturation of the dna at 96 °c for 2 min, followed by 35 cycles of denaturation at 96 °c for 1 min, annealing at 56 °c for 1 min, and extension at 72 °c for 2 min, followed by a final extension at 72 °c for 10 min. soto, j.; cadenas, c.; mattos, l.; trigoso, c. peruvian journal of agronomy 3(3): 144–152 (2019) 147 the pcr products were separated by electrophoresis with 0.5x tae buffer in 2% agarose gels for 30 min at 90 v and stained with hydragreen ™ safe dna dye using a 100 bp dna size reference ladder (promega). fragments were observed using an ultraviolet light transilluminator. the pcr amplified fragments were sequenced in both directions at the macrogen company in korea and compared with the national biotechnology information center (ncbi) gene bank database (genbank) (ramos & barboza, 2016). phylogenetic analysis first, nucleotide sequences were edited to eliminate unclear sequences in the electropherogram, and the 5 ’and 3’ ends of the dna strands obtained were cut. the edited dna strand sequences were then aligned to determine the quality of the nucleotide sequences of selected bacterial isolate dna samples. bidirectional alignment and comparison with chromatograms were performed using the bioedit programme (hall, 1999). the phylogenetic analysis was conducted using the mega 7.0 software in which the “closest neighbour” and “maximum likelihood” tests were performed with 1000 bootstraps. consensus sequences of the aligned isolates were also determined using muscle and used to query the ncbi genbank using the basic local alignment search tool (blast). pathogenicity test the stems of pitahaya plants (h. undatus) were planted in propagation bags with sterile substrate and allowed to root, after which they were grown for 60 days. pathogenicity tests were conducted on healthy pitahaya plants under two different conditions: 1) in excised stems in humidity chambers or 2) in plants in bags in the greenhouse. for the first set of conditions, healthy stems of pitahaya plants were cut and transferred to the phytopathology laboratory, washed with clean water, disinfected in 1% sodium hypochlorite, and then placed in the humidity chambers. stems were inoculated in a biosafety cabin ii with sterile toothpicks. bacteria were collected from each of the isolated colony plates with individual toothpicks, and several parts of the pitahaya stems were perforated to inoculate the stems. the control stems were punctured with the tip of toothpicks only containing half na. the humidity chambers were sealed and set at 30 °c. for the pathogenicity test under greenhouse conditions, pure bacterial isolates grown in petri dishes with na medium were taken to the phytopathology greenhouse. healthy pitahaya plant stems were inoculated using the same methodology as in the laboratory using sterile toothpicks. the inoculated plants were covered with plastic sheets to maintain a warm, humid environment for 20 days. results symptomology observed in the field symptomatic stems at different stages of development were observed at the commercial pitahaya plantations starting with small, wet, yellow spots that later developed into soft, watery rot with a yellowish, pre-necrotic margin. the rot typically starts at the tips or on the protruding edges of the stems and extends towards the stem center (fig. 1a, b, and c). figure 1. symptoms at different stages of soft rot caused by enterobacter cloacae in pitahaya isolations obtained and selected first report of enterobacter cloacae as a causative agent of soft rot disease in dragon fruit (hylocereus undatus) stems in peru september december 2019 148 molecular identification of the nine selected bacterial isolates the results of the amplifications of the ribosomal dnas of the 16s region using primers 27f and 1492r generated an amplicon of 1500 bp and are presented in figure 3. after performing a blast search with the 16s region to genbank, all nine bacterial isolates selected corresponded all to a single taxonomic genus, enterobacter. these isolates shared a 99.85% homology with enterobacter cloacae (accession number mh788982.1). the sequences from our selected isolates were introduced into genbank under accession number mn784371 (fig. 4). the results of the phylogenetic analysis using the sequences of the selected isolates show clusters corresponding to e. cloacae (red and purple squares) and distant relationship with e. hormaechei (green triangles). agrobacterium tumefaciens is included as an outgroup species. pathogenicity test in the pathogenicity testing conducted inside a humidity chamber, e. cloacae developed symptoms of soft rot in all inoculated stems. after 24 h, small, yellow, wet spots of approximately 5 mm in diameter were observed from the point of inoculation, which extended to sizes of 10 cm after ~8 days, whereas no symptoms were observed in the uninoculated control (fig. 5a and b). the plants in some stems, the rot appears restricted or limited in sectors, whereas complete rots occur in others. when the rot is restricted, the decomposed tissue detaches from the healthy tissue by its own weight, leaving its respective epidermis and vascular ducts attached to the rest of the plant’s healthy stem. the remaining epidermis, when dried, has a soft, sometimes crusty and whitish, texture and can be easily removed when manipulated (fig. 1d). nineteen isolated bacterial colonies obtained appeared to differ morphologically from each other. following gram staining tests, nine gram-negative bacterial colonies were selected. the nine gram-negative colonies selected also tested positive for 3% koh, and all had coccobacillus forms and were catalase positive and oxidase negative (fig. 2a, b, and c). the nine selected colonies also tested positive for pectinase enzymatic activity, which was confirmed by observing the formation of shallow holes on inoculated tissue. the nine bacterial colonies also caused an aqueous or soft rot in the potato tubers where they were inoculated (fig. 2d, e and table 1). of the nine bacterial isolates selected, five (sot1, sot2, sot3, sot 4, and sot5) were from independencia in the district of pisco, ica, and four (sot6, sot7, sot8, and sot9) were from naranjos in the district of rioja, san martin. figure 2. a. results of phenotypic tests on bacteria with reddish colour. gram negative. b. positive to 3% potassium hydroxide. c. positive catalase. d. degradation of pectin in cpv medium. e. sliced potato rot figure 3 amplifications of ribosomal dna regions with primers 27f and 1492r. (1) white sequence (2. sot 1, 3. sot 2, 4. sot3, 5. sot4, 6. sot5, 7. sot6, 8. sot7, 9. sot8, 10. sot9) sequences (m) ladder 1 kb prueba resultado colour and shape of the colony light beige, small round colonies gram staining reddish 3% koh positive catalase positive oxidase negative violet pectate crystal (cpv) smooth hole formation in the middle potato tuber rot positive table 1. phenotypic test results soto, j.; cadenas, c.; mattos, l.; trigoso, c. peruvian journal of agronomy 3(3): 144–152 (2019) 149 figure 4 phylogenetic relationship between the sequences of bacteria obtained from h. undatus with other nearby species of the enterobacter genus. figura 5. pathogenicity test. a and b stem inculation in wet chamber, c and d stems incised in stems in greenhouse first report of enterobacter cloacae as a causative agent of soft rot disease in dragon fruit (hylocereus undatus) stems in peru september december 2019 150 inoculated in the greenhouse also developed symptoms of soft rot with the same characteristics observed in stems in the humidity chambers (fig. 5c and d) discussion the samples were collected from two different agroecological zones, the first, in the district of independencia in pisco, is located along the peruvian coast, which is a desert, agricultural area dependent on irrigation for the growth and development of crops. from july to september, relative humidity is high, and, considering that the sampling was carried out in the middle of september, the high relative humidity of the early hours and heat in the afternoons favoured the appearance of bacterial diseases in the cultivation of pitahaya. the district of naranjos de rioja is located in a jungle region, where rainfall is abundant from the months of september to march and the crops are mostly grown in dry land. the high temperature of this area, along with rainfall, favours the presence of bacterial diseases during these times. the sampling in naranjos was carried out in the month of january. other factors that favour the presence of diseases caused by enterobacter in pitahaya are management practices that include pruning and pest damage of the epidermis that acts as an entry point for bacteria and other organisms (zimmerman & granata, 2002). the symptoms occurring at both sampling sites are quite similar, making it difficult to describe any site-specific characteristics. when the progress of the enterobacter rot symptoms was restricted, it was likely due to a defence response of the plant generated, thanks to the high-calcium content, which the plant is able to store in the form of oxalate in its cells (franceschi & horner 1980; webb,1999; faheed, mazen, & abd, 2012). in the presence of a phytopathogen, the oxalate calcium migrates to the middle lamina of the tissue cells, binds with the pectins, and gives rise to the calcium polypectates, which are more difficult to degrade by the pectic enzymes of the bacteria. depending on the calcium content, a plant can restrict disease progression and, in the case of pitahayas, cause the detachment and separation of the infected tissue from the healthy tissues as observed. the nine gram-negative bacterial colonies were selected for traits, including the ability to degrade pectin compounds, which was determined by the formation of holes in the cvp medium. however, these holes were not as deep as those produced by the pectobacterium genus (schaad, 2001), which could indicate that the isolated and selected bacteria have less capacity to produce pectinolytic enzymes than those of the genus pectobacterium. the nine bacterial colonies also caused symptoms of soft rot in the inoculated potato slices, which would corroborate the secretion of pectinolytic enzymes, and these results are consistent with those published by other researchers who report that e. cloacae also causes soft rot in potato tubers (abd, alkhazindar, & sayed, 2018). some enterobacteria that cause soft rot are characterised within the entire group of phytopathogens by their high pectinolytic activity, which gives them the property of producing the symptoms of soft rot in reserve organs (husain & kelman, 1956). it has also been documented that many of the enterobacter species have a strong glucose dehydrogenase enzymatic activity, which oxidises d-glucose to d-gluconate without the presence of pyrroloquinoline quinone (grimont & grimont, 2006). the results of the identification of the isolated and selected bacterial colonies showed that e. cloacae has a high percentage of identity, but the literature mentions an enterobacter cloacae complex, which is composed of up to six enterobacter species: e. asburiae, e. cloacae, e. hormaechei, e. kobei, e. ludwigii, and e. nimipressuralis. these species share close genetic relationships and belong to conserved regions; however, polymorphisms allow for them to be molecularly differentiated. phenotypically, they can be distinguished by biochemical tests, such as the biolog, vitek, and tsi, among others (o’hara et al., 1989; brenner, mcwhorter, kai, steigerwalt, & farmer,1986; hoffmann & roggenkamp, 2003). the 16s rdna region was used to construct phylogenetic relationships because it is a highly repeated genome fragment that has remained fairly uniform throughout evolution. genetic differences in the sequences of the rdna of e. cloacae in the 16s region do not allow for the formation of a homogeneous taxonomic group, demonstrated by an irregular phylogenetic tree in which the strains of e. cloacae are strongly interrelated with other species of this genus, such as e. hormaechei, e. aerogenes, and other enterobacteria, including escherichia coli. this illustrates the genetic heterogeneity of the species, which can hinder their systematic identification (tang et al., 1998; hoffmann & roggenkamp, 2003). it is important to mention that e. cloacae is a commensalistic bacterium that inhabits water, sewage, soil, and meat and is present in hospital environments and the digestive tract of humans and animals (grimont & grimont, 2006). there are also several reports of these enterobacteria affecting various plant species, such as pitahayas in costa rica by e. hormaechei (retana et al., 2019) and in malaysia by e. cloacae (masyahit et al., 2009); papaya plants (carica papaya l.) in hawaii; onion bulbs (allium cepa l.) in california and colorado, usa (bishop & davis 1990); and ginger (zingiber officinale) in hawaii (nishijima et al., 2004). these reports indicate that e. cloacae has a polyphagous character, causing diseases in different plant species. this is the first official report of e. cloacae in pitahaya in peru. the pathogenicity test, which was performed by reproducing the same symptoms observed in the fields that were sampled and fulfilling koch’s postulates, revealed that e. cloacae is a causative agent of soft rot disease of pitahaya in the districts of independencia, pisco, and soto, j.; cadenas, c.; mattos, l.; trigoso, c. peruvian journal of agronomy 3(3): 144–152 (2019) 151 naranjos, rioja. however, other species of enterobacteria cause similar symptomatology in other parts of the world. continued sampling in other production areas of this crop should be performed to determine if there are other species associated with this disease. conclusion according to the results of this research work it is concluded that enterobacter cloacae is the causative agent of the soft rot of pitahaya cladioli in the districts of independencia (in pisco) and naranjos (in rioja); 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(2002). insect pests and diseases. in: p.s. nobel (ed.), cacti: biology and uses (pp. 235–254). los angeles. university of california press. peruvian journal of agronomy 4(3): 75–81 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i3.1645 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 16 august 2020 accepted for publication: 20 november 2020 prodiplosis longifila gagné (diptera: cecidomyiidae) in asparagus (asparagus officinalis) crop in chavimochic irrigation project prodiplosis longifila gagné (diptera: cecidomyiidae) en el cultivo de espárrago (asparagus officinalis) en el proyecto de irrigación de chavimochic jorge ramón castillo valiente1*; susana patricia rodríguez quispe2; walter eduardo apaza tapia3; alberto marcial julca-otiniano4; manuel antonio canto saenz5; teresa rosales sanchez6 *corresponding author: joracava@lamolina.edu.pe https://orcid.org/0000-0003-3561-2253 abstract the present study was conducted in the fields of the chavimochic irrigation, located in la libertad region-peru. prodiplosis longifila gagné (diptera cecidomyiidae) is considered the key pest of asparagus and other vegetables cultivation. this study aimed to characterize the presence of this insect pest in asparagus crop under drip irrigation conditions, in order to find the best alternatives for its control. the cultivar most planted in the chavimochic irrigation is cultivar uc 157 and that the meteorological conditions of the area are optimal for the production of asparagus; but also for the establishment and reproduction of the insect. the morphology of asparagus is favorable for reproduction of the insect, from larva to adult, since it frequently bracts and flowers at larval stage and establishs itself at the adult stage; at the base of the plant. all experiments conducted, had at least three repetitions and the results are given on average. the insect can have many hosts and an average biological cycle of 11 days, with the highest populations in the months of may and november. throughout the day; the adult populations are very active from 10 pm to 4 am. the drip irrigation system provides better conditions for the establishment of the insect and its reproduction in the fields, as well as the design of double row sowing so water management becomes an important factor for managing its populations. from the 100% puparium that fell to the ground, only 37.95% emerged as adults. the percentage of damaged stems per linear meter can be 70.46 and healthy stems 29.54. the number of larvae per shoot can be from 68 to 489 while number of larvae per male flower can be from 2 to 7 individuals. there is a relationship between flowering and the number or pre-pupae fallen to the ground, but as the crop develops, the adult´s number decrease, but nevertheless is still aggressive and permanent. key words: prodiplosis, asparagus, chavimochic resumen el presente trabajo se desarrolló en los campos de la irrigación de chavimochic, ubicada en la región la libertad perú. prodiplosis longifila gagné (diptera cecidomyiidae) es considerada plaga clave del cultivo de esparrago y de otras hortalizas. el objetivo del presente trabajo fue determinar el comportamiento del insecto a nivel de campo, en el cultivo de espárrago, bajo condiciones de riego por goteo, para buscar las mejores alternativas de su control. el cultivar mayormente sembrado en la irrigación chavimochic es el cultivar uc 157 y que las condiciones meteorológicas de la zona son óptimas para la producción de espárrago; pero también para el establecimiento y reproducción del insecto. la morfología del espárrago es favorable para la reproducción del insecto, desde larva hasta adulto, ya que puede frecuentar brácteas y flores al estado larval y establecerse al estado adulto, en la base de la planta. el insecto tiene muchos hospederos y un ciclo biológico promedio de 11 días, siendo los meses de mayo y noviembre donde desarrolla las más altas poblaciones. a lo largo del día se determinó que las poblaciones de adultos son muy activas desde las 10 1 universidad nacional agraria la molina. docente. lima, perú. joracava@lamolina.edu.pe 2 universidad nacional agraria la molina. docente. lima, perú. srodriguez@lamolina.edu.pe 3 universidad nacional agraria la molina. docente. lima, perú. wapaza@lamolina.edu.pe 4 universidad nacional agraria la molina. docente. lima, perú. ajo@lamolina.edu.pe 5 universidad nacional agraria la molina. docente. lima, perú. mcanto@lamolina.edu.pe 6 junta de usuarios de riego presurizado jurp. la libertad. perú. t.rosales@jriegopresurizado.org.pe cite this article: castillo, j., rodríguez, s., apaza-tapia, w., julca-otiniano, a., canto, m. & rosales, t. (2020). prodiplosis longifila gagné (diptera: cecidomyiidae) in asparagus (asparagus officinalis) crop in chavimochic irrigation project. peruvian journal of agronomy, 4(3), 75–81. http://dx.doi.org/10.21704/pja.v4i3.1645 prodiplosis longifila gagné (diptera: cecidomyiidae) in asparagus (asparagus officinalis) crop in chavimochic irrigation project september december 2020 76 introduction the chavimochic irrigation project is located in an arid ecosystem where an export agriculture has been established with a drip irrigation system, in the region of la libertad, northern peru. in this region asparagus crop reached its maximum planted area in 2008 with 11,000 ha planted (gobierno regional la libertad, 2010). uc157 f1 is the predominant cultivar, with approximately 90% of the asparagus cultivated area being a monoculture, thus causing the presence of many pests (cisneros, 1995) (figure 1). prodiplosis longifila gagné is considered among the main entomological problems in the chavimochic irrigation project (sánchez & apaza, 2000), as a result of their damages to asparagus crop, as well as other crops in the area. nine species of the prodiplosis genus are recognized worldwide: p. platani gagné, p. myricae (beutenmüller), p. vaccini (felt), p. citrulli (felt), p. floricola (felt), p. morrisis gagné, p. violicola (coquillett), p. falcata gagné, and p. longifila gagné. the last among the list has been reported for peru (gagné, 1986). the aggressiveness of p. longifila is related to its multivoltine nature, that is, it produces many generations per year and has a wide range of hosts. the hosts reported for the united states are “lime” (citrus aurentifolia) and “wild cotton” (gossypium spp.). for colombia and peru this insect pest has been reported for tomatoes, potatoes, and alfalfa, among other crops (peña et al., 1989). chenopodium ambrosoides, medicago sativa, solanum spp., phaseolus spp., and ricinus comunis are hosts reported for peru (gagné, 1986). at present, this insect has been documented as a pest of asparagus offinialis, tagetes spp., allium spp., persea spp., cynara scolyumus, capsicum spp., cucumis spp., citrullus spp. and brassica oleraceae among other most important crops. among weed hosts, we can find chenopodium murale, amaranthus spp. and nicandria physalodes. in asparagus crops, this pest generates many populations during most of its phenology, causing great infestations that can have negative consequences in production of the crop and neighboring ones. this study aimed to characterize the presence of this insect pest in asparagus crop in chavimochic irrigation project and analyze its behavior in order to understand why it has become a key pest of asparagus and other vegetables. materials and methods we began the study by reviewing the information provided by “crop protection committee of the asociación de agricultores agroexportadores propietarios de terrenos de chavimochic – aptch” (association of agricultural exporters and owners of land of chavimochic crop protection committee) and “junta de usuarios de riego presurizado del distrito de riego moche virú chao” – (jurp.) (pressurized irrigation users board). field observations, interviews of field consultant (entomologists), agronomists, and field technicians related to pest management of different agro-industrial companies engaged in asparagus production were conducted. most of the information collected consisted of results from field experiments conducted with aptch crop protection committee and pest management technicians from different agro-industrial companies. in all of experiments carried out in the field, at least, three repetitions were made and the results are given on average. results and discussions due to the climatic conditions of peru, especially in the northern zone, the asparagus crop is able to grow and produce twice a year, thus designing two growing season, one in summer autumn and another in winter spring. the winter – spring season generally has the best yields, p.m. hasta las 4 a.m. el número de larvas por turión puede ser de 68 hasta 489 y el número de lavas por flor masculina de 2 a 7 individuos. el porcentaje de tallos dañados por metro lineal pueden ser del 70.46 y de tallos sanos del 29.54. el sistema de riego por goteo provee mejores condiciones para su establecimiento y reproducción en los campos, así como el diseño de siembra a doble hilera. el sistema de riego por goteo provee mejores condiciones para su establecimiento y reproducción, así como el diseño de siembra a doble hilera, por lo que el manejo del agua se convierte en un factor importante para el manejo de sus poblaciones. palabras claves: prodiplosis, esparrago, chavimochic figure 1. asparagus plantation in the chavimochic irrigation project. j. castillo; s. rodríguez; w. apaza-tapia; a. julca-otiniano; m. canto; t. rosales peruvian journal of agronomy 4(3): 75–81 (2020) 77 since it occurs in the mediterranean area, which is the place of origin of the asparagus plant, given that in this season, the crop has the necessary requirements to transfer photosynthates, (such as fructooligosaccharides), from the aerial to root part (benson, 1980). this optimal condition enables asparagus crops to produce all year round, thus implying that the plant is always active and, as such, produces sprouts continuously in different fields. therefore, pests such as prodiplosis longifila or others can damage soft asparagus tissues, generating several populations up to millions of insects per season. during crop management, six (female plants) or four (male plants) developmental stages are considered: spear emergence, lateral shoot growth, phylloclades appearance, flowering, fruit development (only in female plants) and fruit maturation (only in female plants). in general, shoots are generated twice per growing season, either in summer or winter (figure 2). of the six stages that can be controlled with great efforts the first three are most susceptible to insect damages; however, in flowering stages, the populations of the insect are so high the extent that it is impossible to control the damages caused by the insect (castillo, 2006). the insect damages are related to morphology of the asparagus plant, whose large amount of foliage helps the insect, at adult stage, to look for shelter in basal part of the plant, initially ovipositing in the emergent spears and later in flowers, thus generating large larvae populations that will later turn into adults. these populations can be recycled in the same field, since another generation can be obtained from new young shoots; however, those populations can be a source of adult infestation to neighboring fields. from experiments conducted in the chavimochic irrigation project, the biological cycle of p. longifila gagné was determined to be 16 days under field conditions, between the months of may and june 2014 in first sprouting fields under 21.6°c, 82.07% hr. environmental conditions. (table 1), which coincides with data obtained by rodríguez (1992), in which 17.54 days, was determined under spring conditions and 12.71 determined under summer conditions in tomato crop. according to pest management specialists of the chavimochic area, temperature is a key factor in the presence of p. longifila gagné, among the environmental factors. results of field evaluations along the peruvian coast show that zones with temperatures above 30°c and below 11°c, as well as those where day/night differential temperatures are greater than 10°c, are not adequate for propagation of the pest. according to the studies conducted with this insect by valarezo et al. (2003) in tomato crop under ecuadorian conditions, it was found that when there was an increase in temperature (23.13 to 27.92°c), relative humidity (71.19 to 86.47%) and precipitation (0.16 to 108.5 mm.), the population of this insect decreased, inferring that these abiotic factors have a direct effect on the populations. in colombia, hernández (2014) characterized the damage and distribution of the pest in “tahiti” lemons and solanaceous crops, finding prodiplosis in an altitude between 739 and 2168 masl, with temperatures between 22°c and 38.7°c and relative humidity levels between 37 % and 80%. velasco (2015), conducted a molecular study on prodiplosis longifla populations based on geographic altitude distribution, and found that there is no population differentiation, since no groups were attributable to high or low regions, indicating that a population can maintain gene flow despite environmental variation, which partially explains its wide geographic distribution. by observations of the author, along the peruvian coast, this insect pest is distributed along the coast, precisely between the regions of lambayeque and ica. currently, there are reports about the presence this insect in the region of piura, but with different behavior; however, as reported in the aforementioned studies, this insect may likely colonize other areas. in a study regarding population dynamics of the insect under light stimulation that was reported by aptch plant health committee, it was found that the highest activity of prodiplosis longifila gagné adult was between 10 pm and 4 am of the next day (figure 3). duration in days stage minimum maximum average egg 1.5 1.8 1.6 larvae 4.3 5.2 4.7 pre-pupae 9.2 11.2 10.2 development cycle male 15.1 17.2 16.4 female 15.4 17.7 16.7 figure 2. asparagus growth stages during the first growing season in the chavimochic irrigation project. 0 5 10 15 20 25 30 35 40 45 50 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 n u m b e r o f st e m s assessment days spear emergence lateral shoot growth phylloclades appearance flowering fruit maturation table 1. duration of the biological cycle of prodiplosis longifila gagné, under field conditions in the asparagus crop in the chavimochic irrigation project. 2014. prodiplosis longifila gagné (diptera: cecidomyiidae) in asparagus (asparagus officinalis) crop in chavimochic irrigation project september december 2020 78 based on population dynamics studies conducted in the irrigation project, two months were recognized when the insect increases its population and reaches its highest population throughout the year: may and november (figure 4). drip irrigation system in asparagus has been applied since 1987; however, its massive implementation started at the beginning of year 2000 (instituto peruano del espárrago y hortalizas [ipeh], 2013). this irrigation system optimizes the use of water and fertilizer, and maybe of benefit to the adult insect, which prefers soil surfaces of constantly humid and protected areas the moistened soil folloging the drippers provides humid conditions for the pupae to survive and grow to the adult stage. based on data obtained from observations and field experiments conducted by entomologist of the crop protection committee, through collection of preprepuae and emerged adults in containers, it was found that from the highest peak of infestation of 100% puparium that fell to the ground, only 37.95 % emerged as adults (figure 5). however, the population generated per hectare in this proportion is still high enough to produce a heavy infestation in the asparagus crop. asparagus are planted in single or double rows within a drip line. under double row crops, the insect finds the protective conditions it requires to reach the adult stage, establishing itself in the basal part of the plants and waiting for the emerging shoots to infest them and perform several cycles under similar conditions (figure 6). the number of larvae that may infest an asparagus spear can be few (1–10) or more (250 – 400); however, damages in the fern are the same since they cause fern misshaping, thus causing fern deterioration lack of leaf area, and rottening (figures 7 and 8). 0 10 20 30 40 50 60 6-8 pm 8-10 pm 10-12 pm 12-2 am 2-4 am 4-6 am n u m b e r o f in d iv id u a ls time period figure 3. number of prodiplosis longifila gagné adults captured per night in 100 cm2 light traps with a sticky surface panel. (2014). 0 100 200 300 400 500 600 700 800 900 n u m b er o f ad u lt s month of the year 2014 2015 figure 4. number of prodiplosis longfila gagné adults in sticky surface panel traps throughout the year (2014 and 2015). 0 200 400 600 800 1000 1200 1400 1600 1800 2000 n u m b e r o f in d iv id u a ls date pre pupae adults figure 5. number of prodiplosis longfila gagné pre-pupae collected on the ground and adults emerged per square meter. figure 6. double row asparagus crop with drip irrigation system between rows. 0 100 200 300 400 500 600 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 n u m b e r o f in d iv id u a ls spear number figure 7. average number of larvae per infested spear j. castillo; s. rodríguez; w. apaza-tapia; a. julca-otiniano; m. canto; t. rosales peruvian journal of agronomy 4(3): 75–81 (2020) 79 in underground crown or rhizome of the asparagus plant, there are bud groups forming structures called bud clusters. these buds will either emerge to form an aerial foliage or will be harvested as a young stem or spear. emerging buds will delay the emergence of adjacent buds in the same cluster. removal of emerging spears during harvest reduces auxin level and growth of the next bud will consequently occur. according to benson (1980), as the shoots emerge, reserves of the plant are depleted, producing thinner spears or shoots, and at this moment, harvest time should be completed. insects can also exhaust the carbohydrate reserves by destroying emerging shoots or spears, which stimulates another underground bud to form a new shoot or spear; however, if the infestation is constant or persistent, it causes exhausts the crown, thus affecting the next harvest as well as lifespan of the asparagus crop. this has been observed by evaluating the number of healthy (32, 19, and 32) and affected stems (84, 56, and 58) per meter in the uc 157 f1 cultivar, where 29.54% of the stems were healthy, while 70.46% of the stems were affected by the insect (figures 9 and 10). infestations during flowering stage are caused by larvae that develop in male flowers, and not in female flowers. these infested flowers offer more space for development of the larvae. the proportion of male flowers infested with prodiplosis longifila larvae can be as high as 40% and the average number of larvae per flower can be 3.7 (prado, 2008). (figures 11 and 12) figure 8. asparagus spear infested by a large number of prodiplosis larvae 0 10 20 30 40 50 60 70 80 90 1 2 3 n u m b e r o f st e m s replications affected stems healthy stems figure 9. number of stems infested with prodiplosis longifila and healthy stems in the uc-157-f1 cultivar asparagus field. figure 10. number of emerging shoots infested and damaged by prodiplosis per meter. 0 1 2 3 4 5 6 7 8 1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 n u m b e r o f in d iv id u la ls number of flower figure 11. number of larvae in male asparagus flowers figure 12. number of prodiplosis longifila larvae in male asparagus flower. prodiplosis longifila gagné (diptera: cecidomyiidae) in asparagus (asparagus officinalis) crop in chavimochic irrigation project september december 2020 80 this infestation often goes unnoticed by growers; however, the number of insects produced in this phenology stage is high, producing 34.22 pre-pupae per square meter at its peak. (figure 13). adults from infested fields may reinfest the same or neighboring fields. populations that flourish from active shoots or by infested male flowers per hectare can be counted as millions, according to the crop’s amount of foliage. therefore, the success of this insect pest management is based on combination of various factors. it was observed that as the crop develops over time the number of adults generated decreases; however, the population still remains a permanent source of infestation for the crop (figure 14). conclusions uc 157 is the predominant asparagus cultivar grown in the chavimochic irrigation project the agroecological conditions of the chavimochic irrigation project are optimal to produce asparagus all year round, given the fact that prodiplosis is a constant pest. the plant morphology is favorable for the reproduction of prodiplosis longifila since its larvae infest the bracts of emerging shoots and male flowers. the phenology most attacked by the insect is the spear emergence. the biological cycle of the insect is approximately of 16.4 days for males and 16.7 for females. the presence of the adult insect throughout the day increases from 10 p.m. to 4 a.m. of the next day. may and november are the months with the highest infestations by prodiplosis longifila . double row crops provide conditions for the insect to protect itself in the basal part of the plant. the drip irrigation system maintains the soil humidity, providing optimal conditions for the insect pest pupae to survive and emerge as adult. from the 100% puparium that fell to the ground, only 37.95% emerged as adults. the healthy stems were 29.54% while 70.46% of the stems were affected the number of larvae per shoot can be from 68 to 489 individuals the number of lavas per male flower can be from 2 to 7 individuals. there is a high relationship between flowering and the number of prepupae fallen to the ground. as the crop develops over time the number of adults generated decreases, but still that population is a permanent source of infestation for the crop acknowledgment we are very thankful to the “crop protection commitee” of the asociación de agricultores agroexportadores propietarios de terrenos de chavimochic – aptch” (association of growers, exporters and land owners of chavimochic – crop protection committee) and the “junta de usuarios de riego presurizado del distrito de riego moche virú chao” jurp. (pressurized irrigation users board). references benson, b. (1980). asparagus physiology. california asparagus seed and transplants, inc. davis, california u.s.a. castillo, j. (2006). prodiplosis longifila gagné en la irrigación chavimochic-la libertad. arenagro, 2, 11–19. 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 70 1 6 11 15 18 22 25 29 n ° p re p u p a e /m ² f lo w e ri n g p e rc e n tg e days flowering % nº prepupae/m² figure 13. flowering percentage and number of fallen prodiplosis longifila pre-pupae per square meter. 0 10 20 30 40 50 60 70 80 90 100 110 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 n u m b e r o f a d u lt s sampling days crop maturity number of adults/100 cm² figure 14. number of prodiplosis longifila adults per 100 cm2 in light traps with sticky surface panel throughout the crop phenology. j. castillo; s. rodríguez; w. apaza-tapia; a. julca-otiniano; m. canto; t. rosales peruvian journal of agronomy 4(3): 75–81 (2020) 81 cisneros, f. (1995). control de plagas agrícolas. segunda edición. 313 p. gagné, r. (1986). revison of prodiplosis (diptera: cecidomyiidae) with descriptions of three new species. annals of the entomological society of america, 79 (1), 235–245. gobierno regional la libertad. (2010). proyecto especial chavimochic en cifras 2000-2010. hernández, l. (2014). caracterización del daño y distribución geográfica de cecidomyiidae (diptera) y sus parasitoides asociados a solanáceas y limón thaiti en colombia. [master’s thesis, universidad nacional de colombia]. instituto peruano del espárrago y hortalizas (2013). consejo directivo ipeh 2011–2013. peña, j., gagne, r., & duncan, r. (1989). biology and characterization of prodiplosis longfila (diptera: cecidomyiidae) on lime in florida. florida entomologist, 72, 444–450 prado, s. (2008). efecto de la aplicación de fertilizantes foliares en la caída de flores del esparrago (asparagus officinalis l.) para el manejo de prodiplosis longifila gagne. [thesis, universidad nacional agraria la molina] rodríguez, s. (1992). biología y morfo-taxonomía de la “caracha” (díptera:cecidomyiidae) en tomate (lycopersicon sculentum mill). c.v. río grande. [thesis, universidad nacional pedro ruiz gallo]. sánchez,g. & apaza,w. (2000). plagas y enfermedades del espárrago en el perú. 140 p. valarezo, o., cañarte, e., navarrete, b., & arias, m. (2003). prodiplosis longifila (diptera:cecidomyiidae) principal plaga del tomate en ecuador. instituto autónomo de investigaciones agropecuarias. estación experimental portoviejo. ecuador. velasco, s. (2015). análisis de poblaciones de prodiplosis longifila (diptera: cecidomyiidae) separadas altitudinalmente, mediante secuencias de adn mitocondrial. [doctoral dissertation, universidad del valle de colombia] peruvian journal of agronomy 4(2): 48-54 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i2.1533 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 07 april 2020 accepted for publication: 09 june 2020 inoculation effect of azospirillum sp. and two levels of nitrogen on the performance of the hybrid corn ‘insignia 800’ efecto de la inoculación de azospirillum sp. y de dos niveles de nitrógeno sobre el rendimiento de maíz híbrido ‘insignia 800’ sergio eduardo contreras-liza1*; eroncio mendoza-nieto1; jean pierre quiliche durán1; cecilia maura mejíadomínguez2; betty martha palaciosrodriguez2; julia delia velasquez gamarra2 *corresponding author: scontreras@unjfsc.edu.pe https://orcid.org/0000-0002-6895-4332 abstract the objective of this investigation was to evaluate the effect of inoculating azospirillum sp. and two levels of nitrogen on the productive characteristics of hard yellow corn variety ‘insignia 800’ under the conditions of nuevo imperial, cañete (central coast of peru). a randomized complete block design with five treatments and four replications was used in a field experiment using the commercial corn hybrid ‘insignia 800’. the treatments were two levels of nitrogen (90 and 180 kg n ha-1), plus one or two applications to the foliage of azospirillum sp. (1.08 x 107 cfu per plant), and a control treatment (without inoculation). ten agronomic variables related to productive traits were evaluated. treatment comparison was executed with the scott knott test at the 5% significance level using the infostat program. inoculation with azospirillum sp. did not significantly affect the grain yield per hectare, grain weight per plot, shelling percentage, grain depth, number of ears per plant and diameter of the cob, but it influenced significantly some productive traits such as cob weight per plot, grain weight per plant, cob diameter and ear length, as well as a high benefit-cost ratio due to inoculation. inoculating the plant foliage with azospirillum sp. had a significant effect and greater economic efficiency for some productive characteristics of the hard yellow corn cv. ´insignia 800´, and did not significantly affect grain yield. keywords: yellow dent corn, azospirillum, agronomic characters, nitrogen dose resumen el objetivo de esta investigación fue evaluar el efecto de inocular azospirillum sp. y dos niveles de nitrógeno sobre las características productivas del maíz amarillo duro variedad ‘insignia 800’ en las condiciones de nuevo imperial, cañete (costa central del perú). se utilizó un diseño de bloques completos al azar con cinco tratamientos y cuatro repeticiones en un experimento de campo utilizando el híbrido de maíz comercial ‘insignia 800’. los tratamientos fueron dos niveles de nitrógeno (90 y 180 kg n ha-1), más una o dos aplicaciones al follaje de azospirillum sp. (1.08 x 107 ufc por planta), y un tratamiento control (sin inoculación). se evaluaron diez variables agronómicas relacionadas con rasgos productivos. la comparación de tratamientos se realizó con la prueba de scott knott al nivel de significancia del 5% utilizando el programa infostat. la inoculación con azospirillum sp. no afectó significativamente el rendimiento de grano por hectárea, peso de grano por parcela, porcentaje de desgrane, profundidad del grano, número de mazorcas por planta y diámetro de la mazorca, pero influyó significativamente en algunos rasgos productivos como el peso de mazorca por parcela, peso de grano por planta, diámetro de la mazorca y longitud de la mazorca, así como también se obtuvo una alta relación beneficio-costo por la inoculación. la inoculación al follaje de la planta con azospirillum sp. tuvo efectos significativos en algunas características productivas del maíz amarillo duro cv. ´insignia 800´ y una mayor eficiencia económica, pero no afectó significativamente el rendimiento de grano. palabras clave: maíz amarillo duro, azospirillum, caracteres agronómicos, dosis de nitrógeno 1 universidad nacional josé faustino sánchez carrión. departamento de agronomía. av. mercedes indacochea 609, huacho, perú. 2 universidad nacional josé faustino sánchez carrión. departamento de bromatología y nutrición. av. mercedes indacochea 609, huacho, perú. cite this article: contreras-liza, s., mendoza-nieto, e., quiliche, j.p., mejía-domínguez, c.m., palaciosrodriguez, b.m., & velasquez, j.d. (2020). inoculation effect of azospirillum sp. and two levels of nitrogen on the performance of the hybrid corn ‘insignia 800’. peruvian journal of agronomy, 4(2), 48-54. http://dx.doi.org/10.21704/pja.v4i2.1533 s. contreras-liza, e. mendoza-nieto, j.p. quiliche, c.m. mejía-domínguez, b.m. palaciosrodriguez & j.d. velasquez peruvian journal of agronomy 4(2): 48-54 (2020) 49 introduction the use of plant growth-promoting bacteria (pgpr) for the formulation of biofertilizers has become one of the most promising clean technologies for the development of sustainable agriculture. among these bacteria, the ones that stand out the most, are those of the azospirillum genus. these bacteria have the capacity to fix nitrogen, solubilize phosphorus, produce cytokinins, gibberellins and auxins, and reduce nitrates, making them useful as bio fertilizers that improve agricultural product quality without generating consequences to the environment (bashan et al., 2013; fibach-paldi et al., 2012). maize (zea mays) is one of the most important agricultural products in peru, with the area under cultivation exceeding half a million hectares nationwide, of which 265,000 are planted with the hard yellow corn (ministerio de agricultura y riego [minagri], 2017) and this is a product in high demand in the agricultural and industrial market, mostly on the coast of peru. hard yellow corn is sustained, thanks to the high demand of the poultry industry that require twice the volume produced in the country (eguren, 2003). according to walters et al. (2018), some corn landraces grown under traditional agricultural practices with little or no fertilizer could have developed strategies to improve yield in conditions of low nitrogen content in the soil, and in these landraces, from 29 % -82% of assimilated nitrogen was derived from atmospheric nitrogen (n2). rangel-lucio et al. (2011), found some degree of affinity or effect of a homologous strain, of azospirillum sp., obtained from the traditional varieties h-28 and chalqueño maize, re-inoculated in these same varieties and the subsequent recognition of the strain in modern varieties. likewise, rangel-lucio et al., (2014) found that the bio fertilization of azospirillum sp. has a potential benefit in the production of sorghum, and in particular, the strains of a. brasilense vs-7 and vs-9, presenting an effect on yield of 55% and 49% higher than the control mean fertilized with nitrogen; their results also demonstrated the existing affinity between strain and plant genotype. sangoquiza et al. (2018) investigated the biological response of azospirillum sp. against different types of stress, for which they performed the characterization of the isolates as well as their biological response to stress conditions due to temperature, salinity, ph. their results demonstrated that lyophilized azospirillum isolates grow best at temperatures of 28° c to 38° c and at a ph of 7 to 8. piscoya & ugaz (2016) demonstrated that an application of diazotrophic bacteria (azotobacter sp., 22%, azospirillum sp., 49% and enterobacter sp., 16%) and 50% of chemical fertilizer (240 kg of nitrogen per hectare in the form of urea) increases plant height, number of leaves and stem diameter, as well as grain yield in hard yellow corn. according to the research by zambonin et al. (2019), no interaction between hybrids and inoculation was found for any phenotypic variable studied, and the specificity between hybrids and inoculation was not verified. likewise, they found that inoculation with a. brasilense did not interfere with grain yield and corn yield components. alvarado et al. (2018), found that the combined use of synthetic fertilizer and inoculants, maintains and increases grain size and maintains yield similar to that obtained with just synthetic fertilization in 18 varieties of corn. according to munaretto et al. (2019), the foliar application of a. brasilense, whether alone or combined with seed treatment, increased grain yield and yield components of wheat cultivars. martínez et al. (2018) concluded that the use of the biofertilizer azospirillum brasilense represented an increase in grain yield in corn of 28%. the objective of the work was to evaluate the effect of azospirillum sp. and two doses of nitrogen on yield components of hard yellow corn ´insignia 800´ under the conditions of nuevo imperial, cañete (central coast of peru). materials and methods the research was carried out in nuevo imperial, cañete (central coast of peru), located at the geographical coordinates of 13°4′34.39 ″ s, 76°19′4.04 ″ w, altitude 205 masl. the research was carried out from july to december. the average environmental temperature fluctuated between 17 and 23° c. a soil characterization analysis was carried out and the results showed low in salts, very low organic matter, medium available phosphorus, low available potassium and sandy loam texture, according to standards (prialé, 2016; sono, 2018) (table 1). chemical characterization physical characterization textural class ph e.c.(1:1) 1 caco3 o.m. 2 p k sand lime clay texture ( 1:1 ) ds/m % % ppm ppm % % % 7,80 0,28 2,90 0,83 9,1 86 75 15 10 fr.a. table 1. soil characterization analysis at nuevo imperial, cañete 1 ce= electric conductivity 2 o.m. = soil organic matter content, % inoculation effect of azospirillum sp. and two levels of nitrogen on the performance of the hybrid corn ‘insignia 800’ may august 2020 50 according to the soil analysis, a general fertilizer formula of 180-100-150 kg ha-1 of n-p2o5-k2o was determining, which was applied according to the treatments proposed in the experiment, considering the levels of phosphorus and potassium as fixed factors. nitrogen levels were dosed according to table 2, in each of the experimental units. the combinations of nitrogen (n) doses were in the form of urea (46% n) and one or two of foliar applications of azospirillum sp. the treatments were: 180 n without inoculating (control), 180 n + a1 (t1), 180 n + a1 + a2 (t2), 90 n + a1 (t3) and 90 n + a1 + a2 (t4) (table 2). the nitrogen doses were applied in two equal parts, 50% at the first hilling two weeks after sowing, together with all the phosphorus and potassium doses, which were constant for all treatments in amounts of 100 and 150 kg ha-1 of p2o5 and k2o respectively, in the form of triple calcium superphosphate (46% p2o5) and potassium sulfate (50% k2o). the remaining 50% nitrogen was incorporated into the second hilling, about seven weeks after sowing. ´insignia 800´ is a simple hybrid of corn, tolerant to black spot (phyllachora maydis), according to information from the supplying company (interoc sa) and was used in the field experiment, at a planting density of 65 000 plants per hectare (0.90 m x 0.40 m). furrow irrigation was supplied to field experiments with a weekly frequency, from emergence to harvest maturity. the bacterial strain was isolated from the rhizosphere in maize fields of the central coast of peru and was identified as azospirillum sp. in the laboratorio de biotecnología de la producción -universidad nacional josé faustino sánchez carrión (huacho), by morphological characterization and biochemical profile (catalase test, urease reaction, motility test and oxidase test). catalase test: it was carried out on a slide adding 2 drops of 30% hydrogen peroxide and it was put in contact with the biomass of the bacterium, observing the presence of bubbles indicating the release of oxygen. urease test: the microorganism was cultured in christensen’s urea agar, incubating at 37ºc for 24 hours, and the colour variation of the culture medium from yellow to red was observed, due to the variation of ph by the phenol red indicator. motility test: the semi-solid mobility mannitol medium was used in a tube in a straight position and it was seeded by sting, incubating at 37 ºc for 24 hours, producing cloudiness and elongation in the line of the sting. oxidase test: it was carried out with a filter paper and 3 drops of kovacs’s reagent were added to the centre of the paper, spreading the seeding loop with bacterial biomass, resulting in a positive reaction with a colour change to purple-black. the increment of the azospirillum strain was carried out in nutrient broth culture medium and diluted at a concentration of 1.08 x 107 colony-forming unit (cfu) per plant. then foliage and surrounding soil were sprayed after the emergence of the plants (15 days after planting) and hilling (45 days after planting), according to the indicated treatments. herbicides were not applied to avoid interactions in the soil with the inoculating agent. the variables related to maize production that were submitted to evaluation were: yield per hectare (kg), grain weight per plot (kg), shelling percentage (%), grain depth (cm), ear weight per plot (kg), grain weight per plant (kg), ear diameter (cm), ear length (cm), number of ears per plant and cob diameter (cm). random samples of 10 plants were evaluated for each variable, except for grain yield, in which two central rows of each experimental unit were harvested. a benefit-cost (b/c) analysis was performed on the treatments under evaluation. for this, the information based on escalante (2018) was obtained to calculate production costs, the gross value of production (gvp) and profitability rate (b/c) for each treatment, in order to compare the level of economic efficiency. the design used was randomized complete blocks with four replications per treatment; the experimental units were randomized for each inoculation treatment and had a dimension of 6 rows of 4 meters in length; for the evaluations, only the two central rows were taken into account. the data were processed in the infostat program (2017 version). the analysis of variance (anova) was done to test for the effects of the variables. also, the scott-knott test (bhering et al., 2008) was executed because of its unambiguous mean grouping to compare the treatment efficacy, at a significance level of 5%. results it is shown that the inoculation treatments did not significantly affect the yield per hectare, grain weight per plot, shelling percentage, grain depth, number of ears per plant and ear diameter (table 3); the yield of inoculated plots, were not statistically different from the control plots, but it can be noted that the inoculation treatments with code treatments (kg n ha-1)1 control 180 n (no inoculation) t1 180 n + a1 t2 180 n + a1+ a2 t3 90 n + a1 t4 90 n + a1 +a2 table 2. nitrogen levels and inoculation treatments in the experiment 1a1, the first application of azospirillum sp. to the foliage of the plants 15 days after sowing, a2, the second application of azospirillum sp. to the foliage of the plants 45 days after planting s. contreras-liza, e. mendoza-nieto, j.p. quiliche, c.m. mejía-domínguez, b.m. palaciosrodriguez & j.d. velasquez peruvian journal of agronomy 4(2): 48-54 (2020) 51 azospirillum sp. influenced some product characteristics such as ear weight, grain weight per plant, cob diameter and ear length (table 3). in the variables in which statistical significance was found, the best inoculation treatment found was t2 (180 n ha-1 + two applications of azospirillum sp., about 15 and 45 days after sowing), affecting weight and number of ears per plot, as well as cob diameter and ear length, both characters correlated to grain yield (hallauer et al., 2010); however, in the present experiment the inoculation with azospirillum sp., did not affect these grain yield components significantly (table 3). the number of ears per plant and ear diameter (table 3) were characters that were not significant due to the effect of inoculation treatments in relation to the control, in which a commercial dose of nitrogen (180 kg n per hectare) was applied. likewise, respect to cob weight and cob diameter (table 3), with the application of two doses of inoculation with azospirillum sp. in addition to the full dose of nitrogen (t2), both characters improved significantly; there are not many studies related to these variables and their relationship to grain yield per hectare it can be distinguished that ear length (table 3) was significantly greater with the application of the t2 treatment, this trait is considered to be correlated to grain yield in hard yellow corn (hallauer et al., 2010; chura & tejada, 2014). it is also important that in the characters affected by azospirillum inoculation, the number of applications seems to influence significantly, this could be observed in the treatments with two applications (a2), differing from the inoculation with a single application (a1) at 15 days after sowing (table 3). in table 4 shows the economic analysis of the treatments evaluated in the research and it is noted that in the treatments with the complete dosage of nitrogen (180 n) or with half the dose of nitrogen (90 n), and received one or two doses of azospirillum sp., the highest economic profit rate per hectare was obtained with the highest benefit-cost rate (b/c). the control treatment (180 n, without inoculating) showed a low b/c rate. discussion the results obtained by some authors show that when inoculants are used, there is an increase in corn yield due to the application of beneficial microorganisms (martínez et al., 2018; alvarado et al., 2018), which coincides in part with the findings in this study, although not specifically for grain yield. as proposed by zambonin et 1means with the same letter do not differ significantly p>0,05; % cv is the coefficient of variability expressed as a percentage. the yield was projected as a density of 65 000 plants per ha table 3. grain yield components and azospirillum sp. inoculation in hybrid corn ‘insignia 800’ treatment grain yield grain weight per plant grain depth shelling percentage ear number per plant ear diameter cob weight per plot cob diameter ear length kg ha-1 kg1 cm.1 % n plant1 cm.1 kg plot-1 cm.1 cm.1 control 6 250,50 a 1,08 b 1,77 a 82 a 0,86 a 4,08 a 0,22 b 2,30 b 14,53 b t1 6 756,50 a 1,08 b 1,79 a 83 a 0,88 a 4,16 a 0,22 b 2,31 b 14,59 b t2 7 786,75 a 1,28 a 1,92 a 82 a 1,00 a 4,32 a 0,27 a 2,47 a 15,74 a t3 7 301,50 a 1,09 b 1,85 a 82 a 0,89 a 4,22 a 0,23 b 2,37 b 14,86 b t4 7 351,75 a 1,10 b 1,91 a 83 a 0,94 a 4,29 a 0,24 b 2,38 b 14,76 b %cv 21,55 7,68 6,52 0.98 17,82 3,16 7,62 2,89 4,11 code treatment yield 1 gvp 2 cost 3 b/c4 t2 180 n + a1+ a2 7 788 7 632,24 6 100 0,2512 t4 90 n + a1+ a2 7 352 7 204,96 5 785 0,2455 t3 90 n + a1 7 302 7 155,96 5 735 0,2478 t1 180 n + a1 6 757 6 621,86 6 050 0,0945 control 180 n (no inoculated) 6 251 6 125,98 6 000 0,0210 table 4. grain yield and benefit-cost ratio per hectare (soles) 1 grain yield, kg per ha., 2 gross value of production, soles per ha-1, 3 production cost, soles per ha-1, 4 benefit/cost ratio inoculation effect of azospirillum sp. and two levels of nitrogen on the performance of the hybrid corn ‘insignia 800’ may august 2020 52 al. (2019), in the case of inoculation with a. brasilense, the inoculation treatment did not interfere with grain yield and corn yield components; in the present investigation a growth-promoting effect was shown by the inoculation of azospirillum sp. in the hard yellow corn hybrid but without significantly affecting the grain yield under field conditions in cañete. these results are also similar to those found by rockenbach et al. (2017), regarding ear diameter but they differ in relation to grain yield since this author found no significant differences in hybrid pioneer 30k73 for inoculating a. brasiliense. the present results coincide with the references of various authors, regarding the use of azospirillum spp. and nitrogen fertilization in corn. the inoculation with a. brasilense increased nitrogen use efficiency (nue), maize grain yield and agronomic characteristic in the as1572 hybrid (skonieski et al., 2019) but there was no response in another hybrid; the use of this diazotrophic bacterium is viable even when high rates of nitrogen in the form of urea applied to the soil (galindo et al., 2016). however, schaefer et al. (2019) consider that azospirillum brasilense helps plant growth and yield but does not replace the effect of n fertilization. interestingly, we found no differences in grain yield for control (180 kg n, no inoculation) or the half n dose per hectare (90 kg n plus azospirillum inoculation) in hybrid corn cv. ́ insignia 800´. some characters are yield components in maize hybrids (chura & tejada, 2014), so there is consistency with the findings of the present experiment and are similar to piscoya & ugaz (2016) and alvarado et al. (2018), regarding the promotion of growth by azospirillum sp. inoculation for traits that are components of maize yield. the varietal factor could be a cause to which this difference or lack of effects of the inoculation on the yield is attributed, as suggested by rangel et al. (2014). this is an aspect that has not been developed in the present investigation where a simple hybrid (a single genotype) of hard yellow corn was used. regarding the number of applications of azospirillum, we found that two applications are the best treatment for maize inoculation; for de souza et al. (2019), the application of 30 kg ha-1 of n at sowing and a single application of 150 kg ha-1, or two applications of 75 kg ha-1 in topdressing, inoculated with azospirillum brasilense provided better nutrition and development, with a positive effect for the mass of 100 grains which reflects an increase in grain yield, similarly as we found in the research. also, the application dose of azospirillum was important as found by numoto et al. (2019) who suggested that the dose of inoculant that provided the best agronomic result was 100 ml ha–1 in conjunction with the application of n at sowing or top dressing and as we found, in relation to azospirillum application, that best performed when it considered two inoculations to maize foliage, for some characteristics such as ear weight, grain weight and ear length per plant. conclusions a significant effect of azospirillum sp. was found in some characteristics in hybrid corn cv. ´insignia 800´, but no significant effect was detected for grain yield. the recommended number of applications in this cultivar was two foliar applications at 15 and 45 days after planting plus a commercial dose of nitrogen in order to obtain a greater economic efficiency at nuevo imperial, cañete. acknowledgements the authors acknowledge the participation of the following agronomy graduates in our research: julio ayllón (cañete), elin teodoro and juan pacheco (huacho). likewise, we thank graduate student pedro rodríguez of the laboratorio de biotecnología de la producción, department of agronomy universidad nacional josé faustino sánchez carrión for his collaboration in the isolation, formulation, and inoculum preparation of bacterial strains. references alvarado, r., aceves, e., guerrero, j., olvera, j.i., bustamante, a., vargas s., & hernández, j. h. 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[thesis, universidad de piura] inoculation effect of azospirillum sp. and two levels of nitrogen on the performance of the hybrid corn ‘insignia 800’ may august 2020 54 walters, w.a., jin, z., youngblut, n., wallace, j. g., sutter, j., zhang, w., gonzález-peña, a., peiffer, j., koren, o., qiaoyuan, s., knight, r., glavina del río, t., tringe, s.g., buckler, s., dangl, j.l. & ley, r. (2018). large-scale replicated field study of maize rhizosphere identifies heritable microbes. proceedings of the national academy of sciences, 115(28), 7368–7373. https://doi.org/10.1073/ pnas.1800918115 zambonin, g., pacentchuk, f., lima, f. n., huzarnovakowiski, j., & sandini, i. e. (2019). response of maize crop hybrids, with different transgenic events, to inoculation with azospirillum brasilense. applied research & agrotechnology, 12 (1), 33– 40. https://revistas.unicentro.br/index.php/repaa/ article/view/5613 peruvian journal of agronomy 4(2): 6167 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i2.1546 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 03 june 2020 accepted for publication: 25 august 2020 susceptibility to spirodiclofen in field populations of tetranychus urticae (acari: tetranychidae) from strawberry plantations in lima region, peru susceptibilidad al spirodiclofen en poblaciones de campo de tetranychus urticae (acari: tetranychidae) procedentes de plantaciones de fresa de la región lima, perú juan flores-bernedo1; javier vásquez-castro2* *corresponding author: jaque@lamolina.edu.pe https://orcid.org/0000-0003-3407-6938 abstract the use of inhibitors of lipids synthesis is among the available strategies for the control of tetranychus urticae (acari: tetranychidae) in strawberry plantations. the study aimed to monitor the susceptibility to spirodiclofen in field populations of t. urticae from strawberry plantations in lima region, peru. adult t. urticae were collected from strawberry plantations in districts of aucallama, chancay, huaral, and santa rosa de quives in lima region, peru. a population collected from acalypha wilkesiana in campus of the universidad nacional agraria la molina (unalm), where chemical pesticide has never been applied, was used as a susceptible reference line. this reference line was subjected to toxicological bioassays to estimate the lc50 and lc95. afterward, bioassays were conducted on the population using the lc50 and lc95 estimated for the susceptible reference line. the obtained data was subjected to one-way analysis of variance and mean comparison was done by tukey test (α = 0.05). there were no significant differences between the four populations’ lc50 and lc95, with respect to the estimated susceptible reference line. this implies that the four studied populations of t. urticae are susceptible to spirodiclofen, with no evident resistance problems. therefore, spirodiclofen is a useful tool for the control of t. urticae form strawberry plantations in the studied location. the factors that influenced the results are discussed in the present work. keywords: resistance, bioassay, diagnostic dose, lc50, lc95 resumen el uso de inhibidores de la síntesis de lípidos es una de las estrategias disponibles para el control de tetranychus urticae (acari: tetranychidae) en el cultivo de fresa. el objetivo de la presente investigación fue monitorear la susceptibilidad al spirodiclofen en poblaciones de t. urticae procedentes de campos de fresa de la región lima. se colectaron individuos adultos de t. urticae en campos de fresa de los distritos de aucallama, chancay, huaral y santa rosa de quives en la región lima. se utilizó como línea susceptible de referencia una población procedente del campus de la universidad nacional agraria la molina (unalm), colectada sobre plantas de acalypha wilkesiana, en donde nunca se han realizado aplicaciones de plaguicidas químicos. la línea susceptible de referencia fue sometida a bioensayos toxicológicos con el fin de estimar la cl50 y la cl95. luego, se realizaron los bioensayos con las poblaciones de campo, utilizando la cl50 y cl95 encontrada para la línea susceptible de referencia. posteriormente, los resultados fueron sometidos al análisis de varianza y comparación de medias por la prueba de tukey (α=0.05). las cuatro poblaciones de campo no mostraron diferencias estadísticamente significativas frente a la cl50 y cl95 estimadas para la línea susceptible de referencia. esto significa que las cuatro poblaciones estudiadas de t. urticae son susceptibles al spirodiclofen, no evidenciándose problemas de resistencia. así, el spirodiclofen es una herramienta valiosa para el control de t. urticae en el cultivo de fresa en las localidades estudiadas. los factores que influyeron en los resultados son discutidos en el presente trabajo. palabras clave: resistencia, bioensayo, dosis diagnóstica, cl50, cl95. 1 universidad nacional agraria la molina, faculty of agronomy, av. la molina s/n, lima 12, peru.. 2 universidad nacional agraria la molina, entomology department, faculty of agronomy, av. la molina s/n, lima 12, peru. cite this article: flores-bernedo, j., & vásquez-castro, j. (2020). susceptibility to spirodiclofen in field populations of tetranychus urticae (acari: tetranychidae) from strawberry plantations in lima region, peru. peruvian journal of agronomy, 4(2), 6167. http://dx.doi.org/10.21704/pja.v4i2.1546 susceptibility to spirodiclofen in field populations of tetranychus urticae (acari: tetranychidae) from strawberry plantations in lima region, peru may august 2020 62 introduction peru registers 2,000 ha of strawberry (fragaria x ananassa duch.); of these registers, approximately 1,200 is located in the lima region, where the valleys of chancay, huaral, and huaura stand out, with more than 50% of the national production area (olivera, 2012). this crop is mainly cultivated by peasant farmers, with little technical and financial assistance (redagrícola, 2017). the two-spotted spider mite (t. urticae) is a polyphagous pest that affects more than 1,000 vegetable species of 140 different families (grbić et al., 2011) and is regarded as the most important pest of strawberry (gonzález-domínguez et al., 2015; karlec et al., 2017), causing the death of the plant (hoy, 2011). the chemical method is an important measure for controlling t. urticae; therefore, most strawberry producers employ this method as the only strategy to control this pest (bernardi et al., 2013; lópez et al., 2014). however, this strategy becomes less efficient as a result of the ease in which this pest develops resistance to chemical acaricides. this is explained by the mite’s short life cycle, abundant progeny and arrhenotokous parthenogenesis, characteristics that favored the evolution of resistance, and reasons why this pest has become very difficult to control in many regions in the world (bi et al., 2016; van leeuwen et al., 2010; yalçin et al., 2018). spirodiclofen is a relatively new acaricide that is registered for mite control in a variety of crops. this molecule interferes with formation of fatty acids, acting on acetyl-coa carboxylase enzyme (accase), a key enzyme in synthesis and metabolism of lipids (cheng et al., 2013; lümmen et al., 2014; sparks & nauen, 2015). on the other hand, there are many reports of resistance of t. urticae to spirodiclofen in literature (demaeght et al., 2013; farahani et al., 2018; ferreira et al., 2015; mohammadzadeh et al., 2014). however, in peru, there are no scientific reports in this regard. therefore, the study aimed to monitor the susceptibility to spirodiclofen in field populations of t. urticae from strawberry plantations in north coast of lima region, peru. materials and methods characterization of susceptibility of t. urticae to spirodiclofen the susceptible reference line was collected in acaliph plantation (acalypha wilkesiana müll. arg.) at the universidad nacional agraria la molina (unalm), lima region, peru. for identification of the specie, male mites were mounted on slide sheets and identified using dichotomous keys for tetranychidae family of species (north american plant protection organisation [nappo], 2014; seeman & beard, 2011). the mites were transferred to unalm pesticide toxicology laboratory for breeding, according to the method proposed by bustos et al. (2009). the formulation used for the bioassays was that of spirosil® 250 sc (spirodiclofen), which is registered for the control of t. urticae in strawberry (fragaria x ananassa) (servicio nacional de sanidad agraria [senasa], 2020). the bioassay method described by sato et al. (2005) was adopted with some modifications. beans leaflets of approximately 30 days old were cut into a circular shape (50 mm diameter) and then individually conditioned with the underside facing up in a petri dish (90 mm diameter × 9 mm height) using a sponge moistened with distilled water. strips of moistened paper towel were put over the edges of the leaf disc to act as a physical barrier to prevent mite leakage. later, the leaves were sprayed with the acaricidal solution using an electric sprayer (national, electric sprayer ms-10®). once the surface of the leaves was dry, 10 adult females (aged between 5 and 10 days) were placed on them and this was repeated five times. the bioassays were initially conducted with the susceptible reference line, using four doses equivalent to 20, 10, 1, and 0.1% of the commercial product label dosage. this exploratory test was conducted to estimate the highest and lowest doses at which mortality rates of 99% and 1% are obtained, respectively (biological activity window). afterward, we calculated seven logarithmic doses that would cover the mortality range. then, the bioassay was conducted using 50 individuals for each dose and control, which was sprayed distilled water. the mites were kept in a bioclimatic chamber (climacell®) at 27°c, 65 ± 5% of relative humidity and photoperiod of 14 h:10 h light:dark cycle for 72 h (time at which mortality was evaluated), confirming death for individuals unable to move when stimulated by the touch of a brush. to obtain the lc50 and lc95, we applied the probit analysis using the polosuite software (leora software, 2016). monitoring the resistance of t. urticae to spirodiclofen once the bioassays of the susceptible reference line were completed, we collected field populations from strawberry plantations of the san andreas variety in four districts of the provinces of huaral and canta: aucallama district (145 masl; 11°34’44.7”s, 77°11’40.7”w), chancay district (44 masl; 11°32’42.1”s, 77°16’40.5”w), huaral district (188 masl; 11°33’08.6”s, 77°12’06.9”w), and santa rosa de quives district (940 masl; 11°42’11.0”s, 76°56’23.7”w). the collected mites were transferred to the laboratory for breeding until production of f1, which was used for the bioassays. we used adult females aged between 5 and 10 days, which were exposed to lc50 and lc95 obtained for the susceptible reference line, using the same procedure described above. the experimental design was completely randomized with five repetitions. we subjected the mortality data obtained for each population to one-way analysis of variance and mean comparison by tukey test at the significance level of 5 %. in additionally, we conducted interviews with the producers that own the j. flores-bernedo; j. vásquez-castro peruvian journal of agronomy 4(2): 61-67 (2020) 63 strawberry plantations from which t. urticae populations were collected to better understand the results obtained. results and discussions characterization of t. urticae susceptibility to spirodiclofen table 1 shows the mortality percentage obtained from the exploratory test for four concentrations of the commercial product whose active ingredient is spirodiclofen. this exploratory test aimed to find the minimum concentration that causes the lowest mortality above 0 % and maximum concentration that causes the highest mortality below 100 %. these concentrations represented 0.1, 1, 10, and 20% of the label dosage equivalent to 0.0005, 0.005, 0.05, and 0.1 ml.l−1, respectively. the label dosage indicated in the fact sheet of spirosil 250 sc® for t. urticae control in strawberry plantations is 50 ml.hl−1 or its 0.5 ml.l−1 equivalent. the chosen concentrations to be applied in characterization of susceptibility of t. urticae to spirodiclofen were 0.0005 and 0.1 ml.l−1, which yielded the mortality of 12% and 90 %, respectively. this allowed other equidistant concentrations to be calculated in a logarithmic scale in order to carry out the bioassay to estimate lc50 and lc95. the seven concentrations obtained for characterization of susceptibility of t. urticae to spirodiclofen were 0.0005, 0.001, 0.003, 0.007, 0.02, 0.04, and 0.1 ml.l−1. the mortality of t. urticae due to application of the aforementioned pesticide concentrations are presented in table 2. the results obtained from table 2 were subjected to probit analysis using polo suite software (leora software, 2016). table 3 presents the probit analysis results. the lc50 was 0.0056 ml.l −1, while the 95% confidence interval was between 0.004 and 0.008 ml.l−1, indicating that there is a 95 % probability that the lc50 falls between both values, which are very close to the lc50 estimated by the probit analysis. the lc95 was 0.1008 ml.l−1, while the 95% confidence interval was between 0.057 and 0.227 ml.l−1, indicating that there is a 95% probability that the lc95 falls between both values, which are more distant than the lc95 estimated by the probit analysis. these results are common in toxicological experiments, where confidence intervals draw hyperbolic curves with respect to dose-mortality regression line, thus approaching lc50 and distancing at ends of the curve (for instance, lc10 or lc95). the angular coefficient was 11.2, indicating that the doses used had a significant effect on mortality. when the angular coefficient of dose-mortality regression line is greater than 1.96, the regression is significant; therefore, our results are highly reliable. the calculated and tabulated chi-square values (x2) were of 4.0943 and 11.1, respectively, indicating that data from the experiment fits the probit model satisfactorily. the heterogeneity, which results from dividing the calculated chi-square by the degrees of freedom (5), resulted in a value of 0.8189. in arthropod bioassays, it is recommended that the heterogeneity value does not exceed 4 (robertson et al., 2017), as in the present study. the statistical results of the probit analysis proved the suitability of the experimental method. figure 1 presents the dose/mortality regression curve of the spirodiclofen-susceptible reference line of t. dosage (ml.l−1) number of individuals number of deaths mortality (%) control 50 2 4 0.0005 50 6 12 0.005 50 33 66 0.05 50 42 84 0.1 50 45 90 table 1: mortality of susceptible reference line treated with spirodiclofen acaricide (bioassay to find biological window). dosage (ml.l−1) number of individuals number of deaths mortality (%) control 50 1 2 0.0005 50 5 10 0.001 50 6 12 0.003 50 16 32 0.007 50 32 64 0.02 50 40 80 0.04 50 43 86 0.1 50 46 92 table 3: probit analysis to characterize the basic line of susceptibility of t. urticae to spirodiclofen. population lc50 (ci 95%) lc95 (ci 95%) angular coefficient ± se x 2 d.f. heterogeneity susceptible 0.0056 (0.004–0.008) 0.1008 (0.057–0.227) 11.2 ± 0.12 4.0943 5 0.8189 lc50: mean lethal concentration (ml.l −1); lc95: lethal concentration 95 (ml.l −1); ci 95%: confidence interval at 95 %; se: standard error of mean; x2: chi-square; d.f.: degrees of freedom. table 2: mortality of susceptible reference line exposed to spirodiclofen. susceptibility to spirodiclofen in field populations of tetranychus urticae (acari: tetranychidae) from strawberry plantations in lima region, peru may august 2020 64 urticae. it can be noted that a slight increase in the acaricide concentration can result in a significant mortality response. this curve shows the high sensitivity and efficiency of the bioassay method in assessment of susceptibility of t. urticae to spirodiclofen. monitoring of t. urticae resistance to spirodiclofen the results of mortality and mean comparison by tukey test (α = 0.05) are presented in table 4 and 5. the lc50 and lc95 of the susceptible reference line caused the same mortality in all the studied populations; that is, the populations of t. urticae from aucallama, chancay, huaral, and santa rosa de quives were susceptible to spirodiclofen. these results confirm the susceptibility of the four studied populations to spirodiclofen. however, it is important to consider the interviews with the strawberry producers of the four localities, which are essential to understand the factors that influenced the susceptibility of the pest to spirodiclofen. huaral population the determining factor in this locality was crop management. for several years, the producer has been running a product rotation program with different modes of action. in june-november 2019 season, the producer applied 10 pesticides for the control of t. urticae in the following sequence: bifenazate, spirodiclofen, abamectin, etoxazole, cyflumetofen, abamectin, etoxazole, bifenazate, abamectin, and spiromesifen. the producer avoided the sequential use of two active ingredients that possess the same mode of action. marčić (2012) presumed that delay of resistance includes actions such as rotation and mixing of compounds of different modes of action. rotation between products of different modes of action is a necessary strategy for management of resistance and the producer in this locality is clearly aware of this. aucallama population the factor that influenced the preservation of susceptibility of t. urticae to spirodiclofen was that the producer was unaware of the existence of this acaricide under any trade name, despite the fact that different formulations that contain the active ingredient are sold in pesticide stores in the locality. the strategy for control of t. urticae used by the producer in aucallama was the exclusive use of abamectin, without rotation with other molecules. chancay population the factor that influenced the susceptibility of t. urticae to spirodiclofen in the chancay valley was the commercial destination of the strawberry, in which usa and japan are the main market. for japan, maximum residue limit (mrl) is 2 mg.kg−1; however, the same does not apply for usa. in the latter country, spirodiclofen does not have an mrl for strawberry crops; therefore, it cannot be used. as chancay production is indistinctly shipped to japan and usa, the exporting companies in the area have decided not to use spirodiclofen in their plantations. the companies (arcor de perú s.a., gofresh s.a.c, and agro laure s.a.c) are in charge of collecting and exporting strawberry from chancay valley, demanding the farmers’ compliance to a series of regulations, among which is the use of authorized pesticides in target market. in this way, the use of spirodiclofen is not allowed for the farmers that supply strawberries to exporting companies. therefore, the population of t. urticae remains susceptible figure 1. dose/mortality regression curve of spirodiclofen over the susceptible reference line t. urticae population n mortality (%) significance s.e. susceptible 50 50 a 1.32 huaral 50 49 a 1.32 aucallama 50 49 a 1.32 santa rosa de quives 50 48 a 1.32 chancay 50 46 a 1.32 table 4: mortality (%) of four field populations of t. urticae due to lc50 of spirodiclofen (0.0056 ml.l−1) obtained for the susceptible reference line. note: n: number of adult females treated; means comparison –tukey test at 5% level of significance. means with the same letter are not significantly different (p > 0.05). s.e.: standard error of mean. table 5: mortality (%) of four field populations of t. urticae due to lc95 of spirodiclofen (0.1008 ml.l−1) obtained for the susceptible reference line. population n mortality (%) significance s.e. susceptible 50 95 a 1.83 huaral 50 97 a 1.83 aucallama 50 97 a 1.83 santa rosa de quives 50 96 a 1.83 chancay 50 94 a 1.83 note: n: number of adult females treated; means comparison –tukey test at 5% level of significance. means with the same letter are not significantly different (p > 0.05). s.e.: standard error of mean. j. flores-bernedo; j. vásquez-castro peruvian journal of agronomy 4(2): 61-67 (2020) 65 to spirodiclofen due to absence of selective pressure by this acaricide. however, other active ingredients (such as abamectin, etoxazole, bifenazate, and spiromesifen) are used. notably, spiromesifen has the same mode of action as spirodiclofen. santa rosa de quives population in this locality, the producer was unaware of any commercial product that contains spirodiclofen. on the other hand, the pesticide stores in santa rosa de quives do not sell any formulation containing spirodiclofen. the most widely used acaricide for control of t. urticae in this locality is abamectin. the producer indicated that he had used spiromesifen a few times. in huaral, chancay and santa rosa de quives, spiromesifen (which has the same mode of action as spirodiclofen) is used. apparently, there is no cross resistance between these two acaricides, despite having the same mode of action (ferreira et al., 2015). the mechanism of resistance to spirodiclofen provides only low to no cross-resistance to spiromesifen (kramer & nauen, 2011). therefore, the multiple use of the three commercially available lipid biosynthesis inhibitors (irac group 23) is recommended (elbert et al., 2008). this may partially explain the high susceptibility of t. urticae to spirodiclofen in the studied valleys. spirodiclofen is a useful tool for control of t. urticae in strawberry plantations in huaral, aucallama, chancay, and santa rosa de quives valleys and, therefore, its inclusion in the acaricide rotation program for the control of mites is recommended. however, given that spirodiclofen is a contact acaricide (nauen et al., 2003) and that t. urticae resides mainly on the underside of leaves (renkena et al., 2020), it is important to improve the application method to achieve an adequate coverage for effective pest control, with the least possible environmental contamination. conclusion populations of t. urticae from strawberry plantations of the provinces of aucallama, chancay, huaral, and santa rosa de quives are highly susceptible to spirodiclofen. the sporadic use of this molecule by farmers and the apparent no cross-resistance between spirodiclofen and spiromesifen have contributed to maintaining the susceptibility of the populations studied. thus, spirodiclofen is a useful tool in t. urticae control programs in strawberry cultivation in the lima region. references bernardi, d., botton, m., silva, u., bernardi, o., malausa, t., garcia, m., & nava, d. 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(2018). acaricide resistance in tetranychus urticae red form (acari: tetranychidae) collected from strawberry in southern j. flores-bernedo; j. vásquez-castro peruvian journal of agronomy 4(2): 61-67 (2020) 67 turkey: bioassay and biochemical studies. systematic and applied acarology, 23(12), 2279–2287. https:// doi.org/10.11158/saa.23.12.1 peruvian journal of agronomy 4(2): 68–74 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i2.1571 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 26 april 2020 accepted for publication: 20 august 2020 effects of nitrogen fertilization on the photosynthesis and biomass distribution in a potato crop efectos de la fertilización nitrogenada sobre la fotosíntesis y distribución de biomasa en un cultivo de papa josé e. salas-rosales1; pedro manuel villa1,2*; alice c. rodrigues2; fermín rada3 *corresponding author: pedro.villa@ufv.br https://orcid.org/0000-0003-4826-3187 abstract nitrogen fertilization has positive effects on growth and production of potato crop. the objective of this study was to assess the differences of the photosynthetic response and biomass partitioning patterns during the main phenophases of the potato cultivar capiro under different nitrogen nutrition fertilization treatments in the tropical andes of mérida, venezuela. plots of 40 m2 (2,625 plants m-2) were established under a random block design with three replications by nitrogen fertilization treatment: 0, 100, 200, and 300 kg. n ha-1. photosynthesis and biomass were measured in the different organs in the main phenological stages of the crop. the results indicate that photosynthesis tends to increase slightly with the nitrogen supply; although the differences were not always significant and, decreases during crop growth. tubers yield it was markedly influenced by the nitrogen fertilization. the total biomass production, as well as biomass allocation in different organs showed differences between treatments, maintaining the following order: 300-n> 200-n> 100-n> 0-n. when analyzing the biomass accumulation curves, it is estimated that the application of 250 kg n ha-1 as mineral fertilizer is enough to reach optimal production yields. keywords: dry weight accumulation; nitrogen supply, solanum tuberosum, tuber yield resumen la fertilización nitrogenada tiene efectos positivos sobre el crecimiento y la producción del cultivo de papa. el objetivo de este estudio fue evaluar las diferencias de la respuesta fotosintética y los patrones de partición de biomasa durante las principales fenofases del cultivo de papa (cultivar capiro) bajo diferentes tratamientos de fertilización con nitrógeno en los andes tropicales de mérida, venezuela. se establecieron parcelas de 40 m2 (2.625 plantas m-2) bajo un diseño de bloques al azar con tres repeticiones por tratamiento de fertilización nitrogenada: 0, 100, 200 y 300 kg n ha-1. la fotosíntesis y la biomasa se midieron en los diferentes órganos en las principales etapas fenológicas del cultivo. los resultados indican que la fotosíntesis tiende a aumentar ligeramente con el suministro de nitrógeno; aunque las diferencias no siempre fueron significativas y disminuye durante el crecimiento del cultivo. el rendimiento de los tubérculos estuvo marcadamente influenciado por la fertilización nitrogenada. la producción total de biomasa, así como la asignación de biomasa en diferentes órganos mostraron diferencias entre los tratamientos, manteniendo el siguiente orden: 300-n> 200-n> 100-n> 0-n. al analizar las curvas de acumulación de biomasa, se estima que la aplicación de 250 kg n ha-1 como fertilizante mineral es suficiente para alcanzar rendimientos de producción óptimos. palabras clave: acumulación de peso seco; suministro de nitrógeno, solanum tuberosum, rendimiento 1 instituto nacional de investigaciones agrícolas (inia), 5101, mérida, estado mérida, venezuela.. 2 universidade federal de viçosa, programa de pós-graduação em botânica, viçosa, minas gerais, brasil. 3 universidad de los andes (ula), instituto de ciencias ambientales y ecológicas (icae), 5101, mérida, venezuela. cite this article: salas-rosales, j.e., villa, p.m., rodrigues, a.c., & rada, f. (2020). effects of nitrogen fertilization on the photosynthesis and biomass distribution in a potato crop. peruvian journal of agronomy, 4(2), 68–74. http://dx.doi.org/10.21704/pja. v4i2.1571 j.e. salas-rosales; p.m. villa; a.c. rodrigues; f. rada peruvian journal of agronomy 4(2): 68-74 (2020) 69 introduction the response of plants to different types of environmental stress involves structural and functional changes ruled by processes linked to the carbon balance (lambers, 1998; lambers et al., 1998; schurr et al., 2006). plants may respond to environmental heterogeneity through physiological and morphological plasticity that allows them to optimize resources (lambers et al., 1998). additionally, the morphological plasticity may also be a consequence of higher physiological plasticity (lambers, 1998; lambers et al. 1998; schurr et al., 2006). thus, the change in the biomass partitioning patterns throughout the organs may constitute an adaptation or acclimation mechanism in response to the environmental pressures (osone & tateno, 2005; schurr et al., 2006). this ecophysiological approach may serve as a fundamental instrument for addressing crop yields, given its socio-economic and environmental relevance. however, studies with this approach are still lacking for several crops, including superfoods playing a central role in global food security. potato is one of the main agricultural products contributing to global food security, given its high-yield production per unit area (devaux et al., 2014). potato is originally from the andes, where it remains one of the main crops for its economic relevance (villa & sarmiento, 2009; machado & sarmiento, 2012). the diacol capiro cultivar has been farmed due to its high productivity, edaphoclimatic adaptability, and favorable properties for industrial processing (for example, potato chips). this potato cultivar has a vegetative cycle, which ranges from 130 and 150 days, with up to 35 t ha-1 crop yields in optimal growth conditions. nevertheless, there are still not enough ecophysiological data about this cultivar showing the effects of different growing conditions (i.e., nutrients availability) on the biomass and tuber growth, production, and accumulation. tubers are the organs of main economic interest in the case of potato crop, and they are notable for their high assimilation and nutrient accumulation throughout the crop cycle (e.g., van delden, 2001; machado & sarmiento, 2012; qiqige et al., 2017). various studies have determined the role of nitrogen as an essential element to attain high productivity levels of potato crop (biemond & vos, 1992; machado & sarmiento, 2012; silva et al., 2013; qiqige et al., 2017). similarly, reduced levels of nitrogen may lead to considerable reductions of production yields and to a reduced biomass distribution (van delden, 2001; alva et al., 2002; qiqige et al., 2017). this response of the crop has been connected to the influence of leaf nitrogen on photosynthesis (evans & poorter, 2001), which varies according to the growth stage and to the nitrogen demand (lambers et al., 1998; poorter & nagel, 2000). one method of analysis of the efficiency of nitrogen fertilization in agricultural crops has been the examination of the photosynthesis dynamics and biomass distribution and accumulation patterns throughout the different organs (biemond & vos, 1992; alva et al., 2002; qiqige et al., 2017). the biomass accumulation in potato crop may vary a lot throughout its phenological stages, yet with unchanged fertilization rates and growth conditions (biemond & vos, 1992; van delden, 2001). on the other hand, nitrogen availability also determines the photosynthetic response within the crops, particularly in the case of leaf nitrogen destined to protein regeneration (evans & poorter, 2001). therefore, the proportion between the nitrogen fertilization and the carbon gains considerably influences the growth and production yields (lambers et al., 1998; poorter & nagel, 2000). in this context, there is a need to examine nitrogen fertilization alternatives for the potato crop by analyzing the implications for the production. thus, the objective of this study was to assess the differences of the photosynthetic response and biomass partitioning patterns during the main phenostages of the potato crop cultivar capiro under different nitrogen nutrition fertilization treatments in the tropical andes of mérida, venezuela. material and methods study area and experimental design the field experiment was carried out in mucuchíes, in the municipality of rangel, state of mérida, venezuela (8°46’94’’n 70°55’47’’w, 3,200 masl) from april through july 2005. the diacol-capiro cultivar was selected due to its socio-economic importance in the region and earliness (development cycle of 4 to 4½ months). mucuchíes has a mean annual temperature of 11.5 °c, and mean annual precipitation of 800 mm. the soil was characterized (0-30 cm) by a loamy-sandy texture, total nitrogen of 0.03%, available phosphorus of 0.8 ppm, 1.0 meq 100 g interchangeable potassium, and ph of 5.5, and bulk density of 0.74 g cm-3. twelve plots (each 5 m × 8 m = 40m2) were established with a planting density of 2.625 plants per m-2 (0.25 m between plants, and 1.5 m between rows), with randomized block design, four different nitrogen fertilization treatment and three replications. each plot contained five rows 8 m long, with two outer rows acting as barriers, and each row totaled 32 plants. the treatments used were as follows: 0, 100, 200, and 300 kg n ha-1. ammonium nitrate ((nh₄) no3) was the source of inorganic nitrogen used. inorganic fertilization source with ammonium nitrate (33.5% n) was supplied. potassium sulfate (17% assimilable k2o) and phosphate rock (25% assimilable p2o5) were applied to all plots. a total 20 kg n ha-1 were applied in the 100-n treatment, 37.5 kg n ha-1 in the 200-n treatment, and 75 kg n ha-1 in the 400-n treatment during sowing. during sowing, we also applied 100 kg ha-1 p2o5 in the form of phosphate rock (400 kg ha-1) and 200 kg ha-1 k2o in the effects of nitrogen fertilization on the photosynthesis and biomass distribution in a potato crop may august 2020 70 form of potassium sulfate (476 kg ha-1) to all experimental plots. data collection in each plot the growth parameters such as, number of stem, plant height (in cm), and biomass (g. plant) of the different organs was measured along the days after sowing (dap). the samplings were taken during the main phenological stages of the crop: near the moment of emergence, on the 36th day after sowing (dap), at the beginning of tuberization (60 dap), at the time of maximum leaf expansion (105 dap), and at harvest (127 dap). thus, number of stem, plant height and biomass was determined in each phenological stage from six plants selected in each plot, with the exception of the last sampling where ten plants per plot were harvested, given the significance of the crop yield from the agronomic point of view (villa et al., 2020). samples of biomass were dried at 70°c until they reached a constant weight; subsequently, these samples were weighted and milled to further obtain composite samples per plot and sampling session. gas exchange measurements were conducted in fully expanded leaf blade of three individual in each plot during the emergence (near 36 dap), tuberization (64 dap), maximum leaf expansion (105 dap), and harvest (127 dap). three of the fully mature and expanded top leaves were randomly obtained per plot (36 leaves for each treatment). gas exchange measurements were conducted using portable gas exchange system (li-cor 6400; li-cor, lincoln, nebraska, usa) measuring leaf photosynthesis for amplitude of photosynthetically active radiation (par) from 25 μmol m-2 s-1 up to 2500 μmol m-2 s-1. data analysis all analyses were carried out in r environment (r core team 2018). variation in the biomass in different organs, number of stem and plant height, were compared between all treatments by one-way analysis of variance (anova; for normally distributed data) followed by a post hoc tukey’s test (p < 0.05) using the car package (fox et al., 2017). the different treatments were the factors and the block design effects were key aspects to take into account. this test is suitable for randomized block design and nonnormal distribution data (crawley, 2013). we adjusted the response curves of assimilation based on gas exchange measurements. the non-rectangular hyperbola equation is commonly used to describe the response of foliar photosynthesis to radiation (xu et al., 2019). the r software served to estimate the parameters of the non-rectangular hyperbola following the same equation: where pn is the co2 exchange rate (net photosynthesis rate), pb is the gross photosynthesis, and resp is the leaf respiration in the dark. with respect to the non-rectangular hyperbola, rad is the radiation incident on the leaves, m is the initial slope of the light response curve, pmax is the rate of gross photosynthesis when light saturation is reached, q is the curvature parameter (table 1). finally, tuber and total accumulated biomass along the dap were adjusted with gompertz functions (y= a*expb*exp-c*t), where y is the biomass, t the time, a, b and c are parameters obtained by adjusting the experimental data. results and discussion overall, the results showed that photosynthesis tends to increase with nitrogen supply. this is the case even for noncontrasting differences, mainly at 64 dap. photosynthesis tends to drop substantially, from emergence until harvest time (figure 1). in this study, photosynthesis per unit of leaf area was found to be slightly different (~15 %) between contrasting treatments, but it was consistent in all the samples, following the order: 300-n > 200-n > 100-n > 0-n (figure 1). in contrast, other studies have shown that nitrogen limitation has a striking effect in the reduction of leaf photosynthesis for different species (lambers, 1998; de groot et al., 2003). for instance, de groot et al. (2003), conclude that the limitation of photosynthesis of pn = 1/2q [(mrad + pmax) √ (mrad + pmax)2 – 4mqpmaxrad] – resp equation 1 pn = pb – resp equation 2 dap treatments parameters of the nonrectangular hyperbola pmax m a resp r2 36 0-n 12.85 0.0034 0.060 2.49 0.92 100-n 13.06 0.0039 0.080 2.47 0.90 200-n 14.52 0.023 0.048 0.99 0.96 300-n 15.22 0.025 0.045 1.55 0.95 64 0-n 11.76 0.010 0.023 1.29 0.91 100-n 12.05 0.020 0.027 1.03 0.92 200-n 12.96 0.025 0.037 0.89 0.90 300-n 13.25 0.027 0.047 0.99 0.93 105 0-n 6.08 0.012 0.020 1.12 0.96 100-n 7.35 0.019 0.024 1.03 0.94 200-n 8.12 0.021 0.025 0.90 0.92 300-n 9.27 0.023 0.027 0.78 0.90 127 0-n 3.90 0.009 0.019 1.05 0.89 100-n 4.10 0.016 0.020 0.90 0.87 200-n 5.86 0.025 0.022 0.72 0.86 300-n 6.09 0.029 0.030 0.60 0.90 table 1. parameters of the non-rectangular hyperbola during days after planting (dap) in the different treatments of nitrogen fertilization: 0, 100, 200 and 300 kg n ha-1. the coefficient of determination (r2) is indicated. j.e. salas-rosales; p.m. villa; a.c. rodrigues; f. rada peruvian journal of agronomy 4(2): 68-74 (2020) 71 lycopersicon esculentum under nitrogen deficit is due to the excessive production of assimilates that do not have a direct drain or a strong drain, which leads to a significant reduction of photosynthesis, compared to the treatments without nitrogen deficit. in this regard, allegedly, the small differences of photosynthesis between nitrogen deficit conditions and those of fertilization, are due to the tubers’ demand of a large proportion of available assimilates, which increases with the time until the final harvest and avoids an accumulation of assimilates in the leaves to take place and, consequently, avoids limited photosynthesis. nitrogen fertilization had significant effects on the production of tubers and total biomass, following this order: 300-n > 200-n > 100-n > 0-n (figure 2), which increased as nitrogen fertilization was higher, 0-n (30.87 ± 4.5 mg ha-1) <100-n (35.01 ± 2.23 mg ha-1) <200n (36.41 ± 1.37 mg ha-1) <300-n (38.61 ± 3.37 mg ha1). however, there was only a 25% difference in tuber production between the 0-n and the 300-n treatments: 36g plant-1, and 49g plant-1, respectively. furthermore, there was a small difference (4%) between the 200-n and the 300-n treatments, without having significant differences (p > 0.05). these results are consistent with the ones found in other studies, where, as observed, biomass accumulation in potato crop grew with nitrogen supply (biemond & vos 1992; alva et al., 2002; machado & sarmiento, 2012; qiqige et al., 2017). these results suggest that, from an agricultural and ecological point of view, there is no justification for applying measures above 300 kg n ha-1 figure 1. leaf photosynthesisphotosynthetically active radiation (par) response curves. a is the net carbon assimilation rate during days after planting (dap) in the different treatments of nitrogen fertilization: (●) 0, (∆) 100, (◊) 200 and (□) 300 kg n ha-1. figure 2. pattern of total dry biomass accumulation in tubers (a) and total dry biomass (b) during days after planting (dap) in the different treatments of nitrogen fertilization: 0, 100, 200 and 300 kg n ha-1. the curves correspond to the adjustments gompertz functions. effects of nitrogen fertilization on the photosynthesis and biomass distribution in a potato crop may august 2020 72 in the form of mineral fertilizer, and that it would not be financially recommended, as there is no relevant increase of production when plants cannot keep the nitrogen surplus. the dynamics of biomass accumulation tend to increase up until the 64th dap and stays stable until its maximum leaf expansion; then, there is a reduction with the onset of senescence, after the 105th dap (figure 3). the pattern of biomass partitioning to the roots and stolons was not very different between the treatments (figure 3). the plants are capable of responding to the heterogeneity of the environment (e.g. availability of nutrients used for growth) through their physiological or morphological plasticity, by which they can optimize the use of resources and the changes in the patterns of biomass partitioning throughout the organs of the plant (lambers et al., 1998; schurr et al., 2006; qiqige et al., 2017). this can be crucial for agricultural production and financial ends. to this day, there are a relevant number of studies on the effects of nitrogen nutrition on the production of potato tubers. conversely, a reduced number of studies deal with accumulation and partitioning of biomass to the different organs, under contrasting doses of nitrogen fertilization, during the growth of the crop. even so, a few of them have limited their aim to examine partitioning of biomass to the stem and tubers, though leaving behind additional considerations regarding partitioning to stolon’s and roots. furthermore, these studies have, in a lesser extent, examined the phenomena under nitrogen deficit conditions. the results suggest that the changes in the patterns of biomass partitioning throughout the plant depend mainly on the strength of the tubers as sinks of assimilated of photosynthesis, despite the nitrogen deficit conditions, maintain a reasonable production capacity from an agroecological perspective. the result showed significant differences in other growth parameters (i.e. number of stems and height of plants) in the different growth stages before senescence (table 2). concerning biomass partitioning, this study found the greatest values of stem biomass during the periods of maximum leaf expansion. in this sense, alva et al. (2002) found that leaf and stem weights were 60g and 30g in each plant, respectively, for russet burbank, and between 50 and 20g in each plant for hilite russet, respectively. on the other hand, biemond & vos (1992) found that, despite the great differences in the total weight of the stems, nitrogen treatments only offered a mild effect (n1= 2.5; n2 = 8 and n3 = 16 g n per plant) on the distribution of the dry weight of the stems throughout the leaves and the stems. the results showed that there is a higher leaves dry weight compared to stems in all treatments. however, near the 44 dap, leaves dry weight with respect to the stems dry weight was between 60% and 65% for n-100 and n-300, respectively. the effects of nitrogen nutrition on belowground biomass compartments, like the roots and stem, include more biomass accumulation in these organs under nitrogen deficit conditions, mainly during the first stages of development. it was not the case with the aerial organs. these results lead to conclude that the changes in assimilates distribution patterns of potato crop constitute figure 3. pattern of dry weight accumulation of different organs during days after planting (dap) for in the different treatments of nitrogen fertilization: 0, 100, 200 and 300 kg n ha-1. the different letters indicate that there are significant differences between treatments per sampling period (p <0.05). j.e. salas-rosales; p.m. villa; a.c. rodrigues; f. rada peruvian journal of agronomy 4(2): 68-74 (2020) 73 a plastic response of acclimation to stress due to n deficit (villa et al., 2020). additionally, these results are very consistent with previous results provided by several review and research works dealing with other vegetal species (lambers et al., 1998; forde, 2002). according to other studies, fast-growing species tend to be more plastic than those growing more slowly, in terms of biomass partition as their response to the different levels of nitrogen availability or supply in the soil (lambers, 1998; lambers et al., 1998). conclusions this research showed that photosynthesis and growth parameters (i.e. biomass, number of stem, and plant height) tend to increase with the supply of nitrogen. thus, this result reveals the high potential of the tubers to store assimilated of photosynthesis under conditions of nitrogen deficit. however, results allow us to conclude that the application of 300 kg n ha-1 as mineral fertilizer is not justified from a socioeconomic and environmental perspective, considering the little existing differences in tuber production with respect to the 200-n treatment. thus, we recommend research that demonstrates the efficiency of nitrogen fertilization close to 200 kg, and even less using combinations with organic fertilizers under different environmental conditions (i.e. topography, climate) and agronomic management (i.e. irrigation). finally, in this study, the whole harvest did not attain its maximum productive potential. the suggestion is to combine mineral fertilizers with organic sources in order to guarantee and synchronize the nitrogen availability, in accordance with the actual demand of the crop during each phenological stage. acknowledgments the authors acknowledge the collaboration of all employees from the institute of environmental and ecological sciences (instituto de ciencias ambientales y ecológicas – icae) of the universidad de los andes (ula) for their unconditional help during the experimental stages of the study. we specially acknowledge johnny marques, francis guillen, zulay méndez, luis cedeño, kleira quintero, and wilmer espinosa for their important technical support in the field and laboratory. we also thank luis castillo for providing certified seeds. the author wishes to thank the institute of international education’s scholar rescue fund for their support to fermín rada during the past couple of years than has been participating in this research. references alva, a.k., hodges, t., boydston, r.a. & collins, h.p. 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(2019). a general non-rectangular hyperbola equation for photosynthetic light response curve of rice at various leaf ages. scientific reports, 9, 9909. https://doi.org/10.1038/s41598-019-46248-y peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 23 september 2020 accepted for publication: 20 january 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina doi: http://dx.doi.org/10.21704/pja.v5i1.1680 favorable morphoclimatic factors for the preservation of wetting organic carbon in mountain soils factores morfoclimáticos favorables para la preservación del carbono orgánico humificado en suelos de montaña sandro sardón nina1*; raúl d. zapata hernández2; luis a. arias lópez2 *corresponding author: ssardonnina@gmail.com *https://orcid.org/0000-0002-1999-1655 abstract humic substances (hs) are the main component of soil organic matter (som), a product of the pedogenetic process. in this study, the morphometric factors and climatic variable that condition the degree of humification, the organic carbon content of humic acids (ha) fulvic acids (fa) of 42 soil samples are related through the functional equation of factors of state of the soil proposed by jenny. the degree of humification was determined by the nagoya method proposed by kumada. the quantification of organic carbon was determined using the method by walkley and black. the morphometric parameters of the relief were obtained from the digital elevation model (dem) and the climate parameter of the modis sensor. the results show that the relief factor conditions the degree of humification and the climate factor conditions the organic carbon content of humic acids (ha) and fulvic acids (fa). keywords: humic acids, fulvic acids, humification, morphometry, humic substances. resumen las sustancias húmicas (sh) son el componente principal de la materia orgánica del suelo (mos), producto del proceso pedogenético. en este estudio, se relacionan los factores morfométricos y la variable climática que condicionan el grado de humificación, el contenido de carbono orgánico de los ácidos húmicos (ah) y ácidos fúlvicos (af) de 42 muestras de suelos mediante la ecuación funcional de factores de estado del suelo propuesto por jenny. el grado de humificación se determinó por el método nagoya propuesto por kumada. la cuantificación del carbono orgánico se determinó mediante el método de walkley y black. los parámetros morfométricos del relieve se obtuvieron a partir del modelo digital de elevación (mde) y el parámetro climático del sensor modis. los resultados muestran que el factor relieve condiciona al grado de humificación y el factor clima condiciona al contenido de carbono orgánico de los ácidos húmicos (ah) y ácidos fúlvicos (af). palabras clave: ácidos húmicos, ácidos fúlvicos, humificación, morfometría, sustancias húmicas. 1 universidad nacional del altiplano, puno, perú 2 universidad nacional de colombia, medellin, colombia how to cite this article: sardón, s., zapata, r., & arias, l. (2021). favorable morphoclimatic factors for the preservation of wetting organic carbon in mountain soils. peruvian journal of agronomy, 5(1), 35–43. http://dx.doi.org/10.21704/pja.v5i1.1680 patricia sello favorable morphoclimatic factors for the preservation of wetting organic carbon in mountain soils january april 2021 36 introduction soil organic matter (som) is the most complex and least-understood component of soil science. som is a mixture of residues of plants, microbes, and animals at various stages of decomposition and heterogeneous organic substances closely associated with the mineral fraction (kononova, 1975; christensen, 1992; zaccone et al., 2018; osorio, 2018; gallardo, 2016). som is composed of humic substances, which are a series of substances with a relatively high molecular weight that varies from a few hundred for fulvic acids to more than 300,000 for humic acids, presenting a color range from yellow to black, formed by secondary synthesis reactions (stevenson, 1994). according to kumada (1987), humification is a set of processes that transform organic matter into compounds that have high visible light absorption capacity and high contents of organic groups such as carbonyl and carboxyl. kumada (1987) was able to obtain information from ha molecules, such as: functional groups, elemental composition and degree of humification. for the degree of humification, it proposes to distinguish four types of humic acids a, b, rp and p. this can be known through the formation of humic acids, in which the start of the humification process begins with the rp type (first humification states of organic matter), evolving into type b and finally type a (each type exhibits a relatively stable form). in strongly acidic soils, the rp type can be replaced by p-type soils. soil and som is the product of “soil formation state factors” and are expressed by the following functional equation (jenny, 1941, 1980): soil and som = f (cl, o, r, p, t …) these state factors correspond to climate (cl), organisms (o), topography, or relief (r), parental material (p), and time (t). the ellipsis in the equation indicates that, in addition to the five factors mentioned above, other variables can be included. these soil-forming factors are independent variables that define the soil system. in this interpretation, the soil properties and the humification process become dependent variables and can be expressed based on soil-forming state factors. scientists in the soil area have different interpretations of the importance of forming factors concerning organic carbon content and degree of humification. this research was developed to obtain quantitative relationships that condition the content of organic carbon in humic acids (ha), in fulvic acids (fa) and degree of humification with climateand reliefindependent variables. figure 1: location of the research area. s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 35–43 (2021) 37 materials and methods description of the study area the study was conducted in the san rafael district of ambo province, located in the huanuco department, peru. this place covers an area of 44,189.73 ha., located between the coordinates: lower-right end is 10’27’41.83’’ s and 76’1’32.14’’ o; and the topleft end is 10’12’17.43’’ s and 76’15’16.33’’ o (figure 1). the 42 samples were analyzed in the soil laboratory of the school of geosciences of the faculty of sciences of the universidad nacional de colombia, medellin. soil sample land order (soil survey staff) south latitude west length m1 aridisol 10°17’32.7’’ 76°9’32.6’’ m2 10°16’57.7’’ 76°9’38.7’’ m3 10°17’18.1’’ 76°9’32.9’’ m4 10°17’49.6’’ 76°9’54.1’’ m5 entisol 10°18’5.6’’ 76°9’42.4’’ m6 10°14’51.9’’ 76°7’34.9’’ m7 10°21’44.3’’ 76°11’58.8’’ m8 10°20’10.3’’ 76°11’9.8’’ m9 10°14’8.7’’ 76°10’3.7’’ m10 10°14’4.7’’ 76°10’15.7’’ m11 10°14’12.8’’ 76°11’30.4’’ m12 10°14’20.3’’ 76°5’50.2’’ m13 10°20’50.7’’ 76°13’21.9’’ m14 10°19’23.8’’ 76°11’56.5’’ m15 10°14’47.1’’ 76°7’52.3’’ m16 10°16’3.0’’ 76°7’40.6’’ m17 10°18’11.9’’ 76°6’49.9’’ m18 histosol 10°14’26.9’’ 76°5’31.5’’ m19 10°14’34.9’’ 76°5’20.0’’ m20 10°18’44.6’’ 76°14’33.4’’ m21 10°19’49.5’’ 76°3’2.7’’ m22 10°19’50.3’’ 76°2’30.9’’ m23 10°20’1.1’’ 76°4’38.6’’ m24 inceptisol 10°15’14.8’’ 76°9’19.9’’ m25 10°18’26.4’’ 76°9’52.8’’ m26 10°13’33.0’’ 76°6’37.8’’ m27 10°19’1.1’’ 76°11’21.3’’ m28 10°15’25.4’’ 76°7’39.9’’ m29 10°14’19.8’’ 76°6’18.4’’ m30 10°14’36.7’’ 76°6’46.8’’ m31 10°18’10.4’’ 76°6’53.1’’ m32 10°21’32.9’’ 76°13’7.7’’ m33 10°20’24.1’’ 76°7’22.6’’ m34 10°21’36.4’’ 76°6’56.9’’ m35 10°23’0.5’’ 76°4’50.1’’ m36 10°23’15.7’’ 76°6’48.3’’ m37 mollisol 10°20’49.6’’ 76°13’57.8’’ m38 10°20’59.3’’ 76°14’43.3’’ m39 10°19’47.3’’ 76°15’11.4’’ m40 10°19’21.7’’ 76°14’48.8’’ m41 10°19’31.4’’ 76°14’42.0’’ m42 10°19’45.7’’ 76°14’40.2’’ table 1: location of the sampled points favorable morphoclimatic factors for the preservation of wetting organic carbon in mountain soils january april 2021 38 ∆log k = log k400 – log k600 rf = k600 x 1,000/c where: log k400 and log k600 is the optical density of an ha solution at 400 nm and 600 nm., both spectrum ranges are obtained with a genesys visible spectrophotometer™ 20. c = volume in ml of 0.1n of kmno4 consumed by 30 ml of ha solution used to determine the absorption spectrum. in this investigation, kmno4 was replaced by 0.1n of k2cr2o7. quantification of organic carbon the organic carbon (oc) content in humic and fulvic acids was determined using the wet combustion method by walkley and black (1934). the organic forms of soil c oxidize in the presence of excess dichromate in the middle of a strong acid. after the oxidation stage of c, at the reaction temperature for a certain period, the non-reduced cr +6 added in excess was valued with ferrous sulfate fe+2. the difference between these two states of cr oxidation is equal to the organic carbon content of the sample (allison et al., 1965; walkley, 1947). sampling forty-two simple samples were collected in the different soil orders (soil survey staff, 2014) (table 1). sampling points were randomly distributed based on previous soil organic matter results. the samples were taken from the epipedon, their morphological characteristics were described and those of the surrounding relief in field, also, have been georeferenced with gps, coordinate reference system and datum wgs 84. extraction and fractionation of organic matter from the soil the extraction and fractionation of organic matter from soil were carried out by the nagoya method, described by kumada (1987) according to figure 2. chemical characterization of humic acids the characterization of humic acids was performed according to kumada (1987). this system group has into four types: a, b, p, and rp, according to their position in the orthogonal axis diagram whose coordinates correspond to the rf and ∆log k parameters. these parameters are obtained by the following expression: som supernatant fulvic acids extraction with 0.1n naoh + na4p2o7 boil at 100°c for 30min. add 1g. by na2so4 cool in a water bath plus ice centrifuge at 11000 rpm for 15 min. wash the soil residue twice with 20 ml of extractant containing na2so4 by centrifugation solid waste supernatant humic acids humines ha + fa acidify the extract with a concentration of h2so4 (1ml:100ml) and let it rest for 30min. filter the extracts in an erlenmeyer flask and dissolve the ha with 0.1n naoh in another erlenmeyer residue figure 2: extraction and fractionation of soil organic matter (kumada, 1987). s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 35–43 (2021) 39 obtaining morphoclimatic factors the morphometric parameters of the relief were obtained from the digital elevation model (dem) of the radarsat-2 satellite in masl. the spatial resolution of 6 meters of the dem was resampled to 30 meters, and the following morphometric parameters were obtained: terrain slope, slope orientation (aspect), the curvature of the slope (profile and flat curvature), and topographic wetness indexes (twi). this extraction of morphometric parameters was performed using geographic information system (gis) techniques using grass gis version 7.4.1 software. the next factor of the state is the climate. the most important variables are humidity (h) and temperature (t). this research worked with a single climate component, which is the annual temperature of the earth’s surface. the temperature subfactor was obtained from the modis sensor in degrees celsius. this modis product (mod11a2, version 6) provides a land surface temperature (lst) every 8 days of day and night data per pixel at a spatial resolution of 1 km (wan et al., 2015). the data were obtained from earthdata (national aeronautics and space administration-nasa) from 2008 to 2018, and the annual temperature calculation was subsequently performed. statistical analysis the relationships between the morphoclimatic parameters and the degree of humification were performed by ordinal logistic regression analysis. simple linear regression and multiple linear regression were used to understand the relationship of morphoclimatic parameters to the contents of ha and fa. these tests used a significance level of (p < 0.05). for the relief factor, the quantitative relationships of organic carbon from humic acids (ha), fulvic acids (fa), and the degree of humification require compliance with the following equation: oc = f(relief) cl, o, p, t … all factors, except relief, must be kept constant. for the climate factor, the quantitative relationships of organic carbon in ha, in fa, and degree of humification, the following equation is required to be met: oc = f(temperature) o, r, p, t… all factors, except temperature, must be kept constant. results and discussion degree of humification and state factor that conditions the humification process the degree of humification in mountain soils show the predominance of humic acid types in the following order: p > b > a > rp (see table 2 and figure 3). this codification of kumada (1987) proposes the theory for the formation of ha. the start of the humification process begins with the rp type (first humification states of organic matter), evolving to type b, and finally, type a (each type exhibits a relatively stable form). in strongly acidic soils, type p replaces the rp type. the mountain soils of the order histosol are classified as type rp (2) and type p (first humification states), the order inceptisol as type p, b, and a (immature to mature state), the order mollisol as type p and a (immature and mature state) and the soils of the order aridisol and entisol as type p and b (immature state and evolved to the mature form). in establishing the relationship of morphoclimatic factors with the degree of humification, the relief factor was identified as the condition for the humification process to occur. an ordinal logistic regression analysis, only the slope profile curvature parameter or subfactor presents a significant relationship (p < 0.05), while the slope subfactors, slope orientation (aspect), flat curvature, topographical index of humidity, and the land surface temperature did not present a significant relationship. figure 3. classification of types of humic acids in mountain soils. favorable morphoclimatic factors for the preservation of wetting organic carbon in mountain soils january april 2021 40 profile curvature measures the rate of change of the slope with changes in the distance; this parameter relates to the flow velocity and the processes of transporting particles on the slope. on the slopes of the mountains, it is common to find sigmoidal profiles, i.e. profiles consisting of a convex upper segment, a straight intermediate segment, and a concave lower segment (derruau, 1966; ruhe, 1975). figure 4 shows the profile curvature segments that condition the degree of humification. in the convex segment, divergent flow and erosion are the soil sample soil order (soil survey staff) organic carbon in ha organic carbon in fa rf ∆log k type of ha (g 100 g-1) (g 100 g-1) m1 aridisol 0.05 0.05 75 0.57 b m2 0.15 0.10 23 0.67 p m3 0.15 0.05 57 0.68 b m4 0.15 0.20 34 0.56 p m5 entisol 0.15 0.15 42 0.64 p m6 0.20 0.35 60 0.67 b m7 0.10 0.05 45 0.55 p m8 0.15 0.05 26 0.65 p m9 0.15 0.10 41 0.66 p m10 0.05 0.05 62 0.55 p m11 0.10 0.05 70 0.63 b m12 1.00 1.50 53 0.45 p m13 0.15 0.05 28 0.70 p m14 0.15 0.10 66 0.54 p m15 0.75 0.10 18 0.55 p m16 0.15 0.10 61 0.62 b m17 0.45 0.20 24 0.61 p m18 histosol 6.00 1.75 29 0.55 p m19 3.75 1.25 20 0.54 p m20 2.10 0.30 26 0.57 p m21 11.00 3.50 10 0.96 rp (2) m22 5.25 1.00 29 0.51 p m23 5.25 0.75 23 0.50 p m24 inceptisol 0.20 0.20 35 0.63 p m25 0.15 0.30 73 0.69 p m26 1.20 0.10 15 0.58 p m27 0.05 0.05 17 0.63 p m28 0.90 0.25 17 0.57 p m29 0.25 0.30 80 0.58 b m30 2.10 0.50 26 0.59 p m31 0.15 0.20 123 0.62 a m32 0.15 0.05 77 0.56 b m33 0.60 0.05 20 0.62 p m34 0.90 0.40 29 0.53 p m35 1.00 1.00 76 0.52 p m36 4.50 1.00 23 0.62 p m37 mollisol 0.15 0.35 104 0.64 a m38 2.50 0.75 27 0.53 p m39 4.50 0.50 13 0.57 p m40 1.50 0.50 51 0.50 p m41 3.00 0.75 20 0.54 p m42 5.25 1.50 29 0.50 p table 2: organic carbon content from humic and fulvic acids, and the degree of humification in different soil orders. s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 35–43 (2021) 41 dominant processes (hall, 1983; schaetzl, 2013). in this segment, the most likely degree of humification is type p (immature state), due to the low persistence of water in materials, faster flow, and more intense erosion. these conditions are not conducive to the development and stability of the humus. the straight segment occupies the middle part of the slope and is characterized by a very homogeneous inclination in its route. the dominant process in this location is a transit condition, for water and materials, without significant accumulations or removals of materials (schaetzl, 2013). in this position, the degree of humification varies from type p, b, and a (immature to mature state). this variability also occurs in the development of soil diagnostic horizons (hall, 1983). type p is in the transition to the convex segment, while types a and b are likely to be in the concavity. the concave segment, located at the lower part of the slope, acts as a recipient of sediment deposition and concentration of runoff waters, subsurface flow, and part of the water table. likewise, the persistence of moisture in the materials underlying this segment is greater than in the segments of the middle and upper part (hall, 1983; schaetzl, 2013). the degree of humification is typed a and b (corresponds to the most evolved grade, but each type exhibits a relatively stable form). in this position of the slope, organic matter increases by favorable water retention conditions (hall, 1983). finally, the flat bottoms, located above 3900 masl have an acidic moisture regime. in this sector, the degree of humification is of type rp and corresponds to the beginning of the humification process in one of the soils of the order histosol. these soils develop under conditions of restricted drainage and nearordinal logistic regression (relief factor) degree of humification of ha coefficients p-value slope of the terrain 0.010 0.71 ns slope orientation (aspect) 0.002 0.45 ns profile curvature 0.060 *0.04 flat curvature -0.003 0.82 ns topographical moisture index 0.542 0.09 ns ordinal logistic regression (climate factor) degree of humification of ha coefficient p-value land surface temperature -0.348 0.08 ns table 3: coefficients between the morphoclimatic variables and the degree of humification (p is the level of significance). note: ns: not significant. *p < 0.05 figure 4: degree of humification in the profile curvature of the slope. favorable morphoclimatic factors for the preservation of wetting organic carbon in mountain soils january april 2021 42 continuous water saturation over time. the difficulty of oxygen circulation in these conditions prevents the breakdown of plant remains and thus allows their accumulation as plant materials in different states of decomposition (stevenson, 1994; buol et al., 2011). state factor that conditions the organic carbon content in humic acids (ha) and fulvic acids (fa) in table 4, it can be observed that the variable land surface temperature (climate factor) acts as an important factor that expresses effectiveness in the organic carbon content in the fraction of ha and fa since the coefficient presents a steep slope. the topographic moisture index, a morphoclimatic index, exerts low effectiveness in the organic carbon content of humic acids. figure 5 shows the inverse relationship between the land surface temperature and the oc content in humic acids and fulvic acids, i.e. an increase in temperature decreases the co content in ha and fa in different soils. jenny et al., (1948) also found an inverse relationship between organic matter and temperature in colombia. in some soils in venezuela, the organic carbon content was found to increase with altitude and was associated with a decrease in temperature, mainly for regions located above 3000 masl (ochoa et al., 1981). oc in ha and fa fractions decrease to temperate areas greater than 22 °c (figure 4). these very high-temperature conditions have a significant influence on maximum mineralization well above multiple linear regression (relief factor) co in the ha co in the fa coefficients p value coefficients p value intercept -0.870 -0.174 slope of the terrain -0.025 0.24 ns -0.005 0.39 ns slope orientation (aspect) 0.002 0.33 ns 0 0.41 ns profile curvature 0.026 0.32 ns 0 0.97 ns flat curvature 0.010 0.54 ns 0.001 0.69 ns topographical moisture index 0.443 0.06 ns 0.109 0.11 ns simple linear regression (climate factor) ha organic carbon fa organic carbon coefficients p value coefficients p value intercept 18.513 5.09 land surface temperature -0.774 * 1.18 x 10-7 -0.21 * 5.64 x 10-7 table 4: coefficients between morphoclimatic variables and organic carbon content in ha and fa (p is significance level). note: ns: not significant. *p < 0.05 figure 5: relationship between land surface temperature (lst) and organic carbon contents. a) lst with oc in humic acids and b) tst with oc fulvic acids. s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 35–43 (2021) 43 humification (bartholomew & clark, 1965). therefore, at temperatures below 22 °c organic matter accumulation and increased wetting rate occur. conclusion the research carried out states that the degree of humification is conditioned by the relief factor and the organic carbon content in ha and fa is conditioned by the climate factor. the morphoclimatic parameters that influence these relationships are the profile curvature of the slope and the temperature of the terrain surface according to multiple linear regression. references allison, l. e., bollen, w. b., & moodie, c. d. 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(1947). a critical examination of a rapid method for determining organic carbon in soils-effect of variations in digestion conditions and of inorganic soil constituents. soil science, 63(4), 251–264. wan, z., hook, s., & hulley, g. (2015). mod11a2 modis/ terra land surface temperature/emissivity 8-day l3 global 1km sin grid v006 [data set]. nasa eosdis lp daac. https://doi: 10.5067/modis/mod11a2.006 zaccone, c., plaza, c., ciavatta, c., miano, t. m., & shotyk, w. (2018). advances in the determination of humification degree in peat since: applications in geochemical and paleoenvironmental studies. earth-science reviews, 185, 163–178. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 30 september 2020 accepted for publication: 20 january 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina doi: http://dx.doi.org/10.21704/pja.v5i1.1670 control of botrytis cinerea pers. in ‘okitsu’ satsuma mandarin by biological and chemical fungicides in huaura, peru control de botrytis cinerea pers. en mandarina satsuma cv. ‘okitsu’ mediante fungicidas biológicos y químicos en huaura, perú denis paolo cáceres candia1; alejandro risco mendoza1; patricia elena quiroz-delgado1; walter eduardo apaza-tapia1* *corresponding author: wapaza@lamolina.edu.pe *https://orcid.org/0000-0001-7510-8866 abstract the flowers blight caused by botrytis cinerea pers. is among the most important citrus diseases, especially in cultivars whose flowering coincides with the humid seasons of the year. as a result of the pathogenic features of this fungus and the complex nature of its control, it is necessary to establish a correct plan for the usage of highly efficient fungicides. the study aimed to evaluate the effect of four chemical fungicides, such as captan (0.25%), propineb (0.25%), fludioxonil + cyprodinil (0.05%), and iprodione (0.15%); as well as the effect of a biological fungicide, such as melaleuca alternifolia extract (0.1%), on botrytis cinerea pers. two phases were established: the first, under laboratory conditions of department of plant pathology of national agrarian university-la molina (unalm), evaluated the effect on mycelial inhibition at 1, 3, and 7 days after inoculation with poisoned potato dextrose agar medium. the second, under field conditions (sayan huaura), evaluated the effect on incidence of the disease in flowers. in the field condition, two applications, incidence, and humid chambers were evaluated. the yield was estimated by counting the fruits. the results showed that, under laboratory conditions, captan, fludioxonil + cyprodinil, and iprodione exhibited high efficacy in the control of b. cinerea. however, under field conditions, fludioxonil + cyprodinil and iprodione exhibited a significant control of b. cinerea. a similar trend was obtained for the yield estimates. key words: flowers blight, botrytis cinerea, mandarin, satsuma okitsu, fungicides. resumen el tizón de las flores causado por botrytis cinerea pers. es una de las enfermedades más importantes en el cultivo de cítricos, especialmente en aquellos cultivares cuya floración coincide con épocas húmedas del año. debido a las características patogénicas de este hongo y a la complejidad de su control, resulta de suma importancia establecer un correcto plan de uso de fungicidas de alta eficacia. el presente trabajo de investigación tuvo como objetivo evaluar el efecto de cuatro fungicidas de origen químico; captan (0,25%), propineb (0,25%), fludioxonil + cyprodinil (0,05%) e iprodione (0,15%), y uno de origen biológico; extracto de melaleuca alternifolia (0,1%). se establecieron dos fases; la primera bajo condiciones de laboratorio (unalm) evaluando el efecto en la inhibición micelial a los 1,3 y 7 ddi con la metodología de medio pda envenenado; y la segunda bajo condiciones de campo (sayán-huaura) evaluando el efecto en la incidencia de la enfermedad en flores. para esta segunda fase se determinó realizar dos aplicaciones y se evaluó la incidencia en campo mismo y en cámaras húmedas. por último, se estimó la producción bajo la metodología de conteo 1 universidad nacional agraria la molina. facultad de agronomía, la molina, lima, perú. email: denispcc123@gmail.com, alejandrorisco@lamolina.edu.pe, patriciaquiroz@lamolina.edu.pe, wapaza@lamolina.edu.pe how to cite this article: cáceres, d., risco, a., quiroz-delgado, p., & apaza-tapia, w. (2021). control of botrytis cinerea pers. in ‘okitsu’ satsuma mandarin by biological and chemical fungicides in huaura, peru. peruvian journal of agronomy, 5(1), 18–24. http://dx.doi.org/10.21704/pja.v5i1.1670 patricia sello d. cáceres; a. risco; p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(1): 18–24 (2021) 19 introduction citrus fruits is among the main agricultural export products in peru and the world demand for citrus fruits has increased in recent years. in 2017, the export of citrus fruits in peru was approximately 143,000 tons, thus positioning peru as the seventh worldwide exporter of citrus fruits. in addition, we consolidated our position as the leading citrus fruit exporter in america by surpassing countries such as chile, united states of america, and argentina. (ministerio de comercio exterior y turismo [mincetur], 2018). the weather condition of the peruvian coast is highly temperate and relatively humid. as a consequence of this condition, citrus fruits are affected by pests and diseases throughout their crop cycle. for this reason, in the main citrus production, chemical control method is the most adopted method for better pest control, with less time and high efficiency (colonia, 2013). during the flowering season of citrus and many other crops, “gray mold” or “flower blight,” caused the fungus botrytis cinerea pers., is the most important disease. this fungus infection causes petals necrosis which are covered with mycelium and gray spores under wet conditions (benito et al., 2000). infected flowers often induce fruit dropping and injury to developing fruit which reduces the yield and diminishes the fruit quality (castro et al., 2000). for the control of this disease, different fungicides are applied, among which some are no longer effective (llanos & apaza, 2018; pappas and elena, 1992). in the province of huaura, fungicides with different active principles are used to control gray mold disease in citrus fruits, among them are carbendazim, captan, or propineb. the study aimed to determine the effect of different chemical fungicides and one biological fungicide on the development of b. cinerea under field and laboratory conditions. furthermore, profitability was evaluated during a production campaign through the use of these proposed fungicides. materials and methods the present research was carried out in two phases: (i) the laboratory phase was performed in the diagnosis clinic of agronomy faculty, department of plant pathology of national agrarian university la molina (unalm), lima state of peru; (ii) the field condition phase was performed in the citrus production fields of “el chilco” farm located in santa rosa town, sayan district, huaura province, lima state, peru. to evaluate the effect of the treatments on incidence of b. cinerea, the products were applied to ‘okitsu’ satsuma mandarin plants. tangerine flowers with symptoms of b. cinerea were collected and, using a hypodermic syringe, structures of the fungus (mycelium and spores) were collected and placed in sterilized petri dishes containing potato dextrose agar (pda) medium. the plates were incubated at 25°c for four days. when the pathogen colony attained a growth greater than 50% of the plate diameter, it was visualized with a microscope to identify its morphology. from the same plates, some portion of pda medium with structures of the fungus was transferred to another plate containing a sterile culture medium in order to obtain a pure colony of b. cinerea. the fungicide test was performed using the poisoned food technique. in brief, pda culture medium was prepared and poured into erlenmeyer flasks in similar quantity (100 ml) and then sterilized in an autoclave. six experimental treatments were performed in this study, which correspond to evaluation of four chemical fungicides, one biological fungicide, and one control (control without fungicide) (table 1). the culture medium was poisoned before it reached an approximate temperature of 45°c. it was incubated at room temperature until it solidified. each treatment performed in four replicates (4 plates). a portion of 0.6 cm diameter of pda medium extracted from the growth zone of the pure colony was placed in the central part of each plate containing solidified poisoned pda medium. for control, only portions of de frutos. los resultados demostraron que a nivel in vitro los fungicidas químicos; captan, fludioxonil + cyprodinil e iprodione presentaron alta eficacia en el control de b. cinerea; sin embargo, bajo condiciones de campo solo los fungicidas; fludioxonil + cyprodinil e iprodione tuvieron un porcentaje de control importante frente a b. cinerea. resultados similares se obtuvieron en el estimado de producción. palabras clave: tizón de las flores, botrytis cinerea, mandarina, satsuma okitsu, fungicidas. control of botrytis cinerea pers. in ‘okitsu’ satsuma mandarin by biological and chemical fungicides in huaura, peru january april 2021 20 pda medium without a colony of the fungus were used following the same procedure above. the seeded plates were conditioned in an incubator at 25°c. mycelial growth was measured at 1, 3, and 7 days after inoculation (dai). growth of the fungus colony was measured through the distance from end of the initial inoculum disk. average diameter of mycelium growth (mm) in the petri dish with poisoned pda and percentage of growth inhibition (pic) for each treatment were determined following standard procedures. the experimental design was completely randomized with four replicates. normality analysis, variance homogeneity, and tukey’s mean comparison test at 95% confidence level (α = 0.05) were performed using minitab statistical program v18. the second phase, under field condition, was performed in a commercial ‘okitsu’ satsuma mandarin (citrus reticulata) field grafted on citrumelo rootstock, which is four years old, during the flowering and fruit set season of 2019 campaign. this field was under unconventional agronomic management, with advancement of the phenological stages to obtain early harvests and flowering that coincided with the coldest and wettest times of the year. two applications were made at an interval of 10 days (6th and 16th of may, 2019) using a 20 l capacity sprayer backpack. 5 l of water was used for the application of the treatments for each plant. the fungicides used in this phase are shown in table 1. the treatments, except timorex gold and control, were mixed with aquacid (ph regulator) at a dose of 0.1 ml/l. the trees were spaced by 6 meters between lines and 4 meters between plants. a tree of ‘okitsu’ satsuma mandarin was considered as a sampling unit. each treatment consisted of four trees (four replicates). in each tree, 25 inflorescences were evaluated per quadrant of the tree, while five flowers per tree quadrant were evaluated in a humid chamber. incidence (%) of b. cinerea was measured in citrus fields and humid chambers. with the incidence data, area under the disease progress curve (audpc) and relative area under the disease progress curve (audpcr) were calculated. audpcr was calculated by dividing abcpe by number of days of the experiment and then multiplied by 100 (campbell & madden, 1990). audpc was calculated as follows: ∑ = summation of n observations n = i-th observation yi = proportion of disease (incidence) affected in the i-th observation ti = time (days) after the i-th observation. for crop yield estimation, the curdled fruit was counted and relationship between the number of fruits per plant and yield per plant was used. for this estimation, the formula proposed by otero (2004) for satsuma mandarin was applied. the experimental design adopted was randomized complete block design (rbcd), with six treatments and four replicates. for the evaluated variables, normality, analysis of variance homogeneity, and tukey’s mean comparison test were performed at 95% confidence level (α = 0.05) using minitab statistical program v18. results and discussions the highest pic values (%) at 7 dai were obtained by captan (100%) and iprodione (100%) treatments, followed by fludioxonil + cyprodinil (94.1%) treatment. however, propineb (0%), tea tree extract (0%), and control (0%) treatments were statistically equal at 1, 3, and 7 dai (table 2). the result at 7 dai under laboratory condition is shown in figure 1 for each treatment. treatment active ingredient product doses t0 control ------t1 captan forsem 80 pm 0.25 % t2 propineb syl 70 pm 0.25 % t3 fludioxonil + cyrprodinil switch 62.5 wg 0.05 % t4 iprodione fobos 50 pm 0.15 % t5 tea tree extract timorex gold (20 %) 0.1 % table 1. treatment with fungicides doses (%), in vitro test, and control test for the control of botrytis cinerea in citrus. d. cáceres; a. risco; p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(1): 18–24 (2021) 21 the best treatments in the poisoned pda medium test were captan and iprodione treatments, both of which achieved a 100% pic. however, in figure 1, it can be seen that b. cinerea managed to infect the initial mycelial disc following treatment with captan. therefore, it can be concluded that iprodione treatment was the best, since it completely inhibited the growth of b. cinerea. this latter event can be explained by the fact that iprodione exhibits a local and contact systemic action, unlike captan which exhibits only a contact action (williamson et al., 2007). ortiz (2009) evaluated (via in vitro conditions) different fungicides for the of control b. cinerea isolated from artichoke heads and demonstrated that mycelial growth rate was 0 cm in petri dishes using iprodione treatment and 0.37 cm using propineb treatment. this result is similar to the result obtained in this study. however, in the same experiment, a growth rate of 0.58 cm was observed following captan treatment, which differs from the result of the present study. similar results were observed in the in vitro efficacy evaluation of fungicides for the control of b. cinerea isolated from rose cultivation, in which treatment with captan allowed mycelial growth of 2.5 cm, while propineb allowed a mycelial growth of 8.3 cm at 7 dai in petri dishes with artificial culture medium (restrepo, 2010). panebianco et al. (2005) conducted a poisoned pda medium test using 302 isolates of b. cinerea from grapes fruits and found 10.3% specific resistance to iprodione and no isolate resistant to fludioxonil. treatment with melaleuca alternifolia extract achieved a pic of approximately 35% for the first two evaluations. antonov et al. (1997) evaluated the inhibition of conidia germination and mycelial growth of b. cinerea with natural products and found that, at a concentration of 1%, tea tree oil achieved complete inhibition of conidia germination and growth of germinative tube by 92%. yu et al. (2015) mentioned that the fungicidal action of tea tree oil against b. cinerea is mainly due to two terpenes (terpinen-4ol and 1,8-cineole) which act by affecting the cell membrane and organelles of the fungus, respectively. for the second phase, under field condition, incidence of ‘okitsu’ satsuma mandarin flowers in field and humid chambers was evaluated. figure 2 relates the incidence data obtained during assessments to the relative humidity (rh) data of the area during the assessments. this figure clearly treatments evaluation dates 1 dai 3 dai 7 dai t0 control 0.0% d 0.0% d 0.0% c t1 captan 100.0% a 100.0% a 100.0% a t2 propineb 59.6% b 60.0% b 0.0% c t3 fludioxonil + cyprodinil 100.0% a 100.0% a 94.1% b t4 iprodione 100.0% a 100.0% a 100.0% a t5 tea tree extract 31.6% c 36.5% c 0.0% c α: 0.05 cv: 2.35% cv: 2.26% cv: 3.29% table 2. percentage growth inhibition (pic) of botrytis cinerea by treatment under laboratory condition. note: dai: days after inoculation; equal letters have no statistical difference in tukey (α: 0.05); cv: coefficient of variability. figure 1. mycelial growth (mm) of botrytis cinerea from each treatment in poisoned pda medium at 7 days after inoculation. control of botrytis cinerea pers. in ‘okitsu’ satsuma mandarin by biological and chemical fungicides in huaura, peru january april 2021 22 shows the difference between the curve drawn by control and other treatments. in addition, how the trend line of rh increased according to the passage of time was clearly illustrated and this coincides precisely with increase in incidence of the disease in each of the treatments. at very humid climate condition, b. cinerea was able to establish itself on citrus flower petals and stamens. this disease can kill single flowers, entire inflorescences, or initiate a regressive death that advances by several centimeters at the peduncle (mooney, 2001). in the analysis of the test under field condition, a relationship between rh and incidence of b. cinerea in ‘okitsu’ satsuma mandarin flowers was determined. it was observed that the incidence of the pathogen increased considerably when rh is approximately 90%. this coincides with la torre and rioja (2002), who report that the optimal conditions for b. cinerea infection in many crops is 20°c and rh above 90%. castro et al. (2000) mentioned that, for citrus cultivation, flower buds or recently curdled fruits are the most susceptible to attack by b. cinerea and that the attack occurs only under very humid condition and cold temperature (18°c). to determine the effectiveness of each treatment, audpc and audpcr were determined. the results obtained are shown in table 3. based on audpcr, the best treatment was fludioxonil + cyprodinil, with 2.9% audpcr, followed iprodione, with 4.24% audpcr. however, based on the statistical test applied, these results were non-significantly. the control treatment presented the highest percentage audpcr (13.32%), which turned out to be significantly different from the other treatments. eyzaguirre (1972) compared seven fungicides for the control of b. cinerea in open flowers of valencia orange and found that the disease control is better achieved with systemic/curative action products than with contact/preventive action products. this coincides with our evaluation under field conditions, since fludioxonil + cyprodinil had systemic and curative action, unlike the other treatments that presented mainly contact fungicide characteristics. treatment with captan under field condition (table 3), unlike under laboratory condition, did not achieve such a high percentage of control; however, it maintained a significant difference with control when evaluated in terms of audpc. captan, like most old fungicides, has maintained its effectiveness in spite its intensive usage. this is due to the fact that it has multiple mode of actions and hardly generates resistance. however, the control achieved by captan is not totally efficient, since it has low biochemical specificity and is limited to a protective action on the applied crop surface (mondino, 2002). propineb, like captan, is among the first groups of fungicides in the market, which are characterized by multiple sites of action. these two treatments are highly recommended fungicides for the control of gray rot in crops such as grapevines (trimmer et al., 2003) and are still recommended for this purpose till date, although only for preventive purpose and within a plan of rotating applications. figure 2. progress of the incidence of b. cinerea in satsuma var. okitsu mandarin flowers for each treatment during the evaluation period and its relationship with relative humidity of the area. d. cáceres; a. risco; p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(1): 18–24 (2021) 23 in this study, tea tree oil treatment showed a deficient control under field condition. in the technical data sheet of products authorized for our country, it does not register a recommended dose for citrus fruits; however, 200 ml/cil was determined due to water used in the area for fungicide application, which is 2000 liters per hectare, as a dose of 2 liters per hectare. the latter would be used if the cost, in comparison with other alternatives, is higher. based on personal experience, in blueberry crop, the optimum dose for the control of b. cinerea is 750 ml / cil, which is remarkably high compared to that of the present study. as at the time, it was determined that the possible cause of the low control was due to the low dose applied. the incidence of b. cinerea in humid chambers was also evaluated for three opportunities during the flowering season, which occurred throughout the month of may 2019. the condition of these humid chambers was of a rh percentage close to 100%. incidence results of the three evaluations performed are shown in table 3. in the three evaluations performed, fluidoxonil + cyprodinil is the only treatment that presents a significant difference with control. for the second evaluation performed on may 22nd iprodione treatment presents a significant difference with control. finally, estimated average yield per treatment and approximate profitability of each treatment were determined according to the formula proposed by otero (2004) and number of trees per hectare in the field tested were 416 in total. the results are shown in table 4. by analyzing table 4, it was determined that, for all treatments, it is cost effective to make two applications for the control of b. cinerea during the flowering season. it is important to mention that the present investigation was performed in an advanced field of ‘okitsu’ satsuma mandarin, where the beginning of harvest is in the middle of december. based on this, the flowering coincided with cold and humid climates optimal for the development of b. cinerea, for which treatments audpc audpcr incidence of flowers with botrytis cinerea in humid chambers 2020, may 13 2020, may 22 2020, may 29 t0 control 3.86 a 13.32% 51.25% b 90.00% a 56.25% ab t1 captan 2.11 b 7.26% 46.25% ab 86.25% a 47.50% abc t2 propineb 1.78 bc 6.13% 33.75% bc 80.00% a 57.50% a t3 fludioxonil + cyprodinil 0.84 d 0.029 18.75% c 18.75% b 21.25% c t4 iprodione 1.23 cd 4.24% 33.75% bc 37.50% b 30.00% bc t5 tea tree oil 1.94 bc 0.0667 60.00% ab 90.00% a 50.00% ab α: 0.05 cv:17.77% cv: 24.6% cv: 16.8% cv: 27.2% table 3. audpc, adpcer, and incidence of citrus flowers in humid chambers with botrytis cinerea in satsuma mandarin treated with different fungicides in field condition. note: means with different letters have statistical difference (tukey α: 0.05); audpc: area under disease progress curve; audpcr: area under disease progress curve relative. treatment field (tn/ ha) cost (us$) doses xha application cost (us$/ha) profitability (us$/ha) testigo 34.65 $34,650.00 captan 37.14 $14.93 5.0 $74.65 $36,990.70 propineb 34.8 $10.45 5.0 $52.25 $34,695.50 fludioxonil + cyprodinil 40.59 $223.88 1.0 $223.88 $40,142.24 iprodione 39.99 $41.79 3.0 $125.37 $39,739.26 extracto de árbol del té 36.08 $73.13 2.0 $146.26 $35,787.48 table 4. economic analysis of mandarin satsuma for each treatment according to average yield data. control of botrytis cinerea pers. in ‘okitsu’ satsuma mandarin by biological and chemical fungicides in huaura, peru january april 2021 24 it is determined that the use of specific fungicides in this particular case is justified to obtain greater profitability. conclusions products of chemical origin (iprodione and fludioxonil + cyprodinil) are the best for the control of b. cinerea in ‘okitsu’ satsuma mandarin based on the parameters evaluated in this study: mycelial pic, field and wet chamber incidence, and economic analysis. on the other hand, the product of biological origin used in this study does not exhibit a good control of the disease. references antonov, a., stewart, a., & walter, m. 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(2015). antifungal modes of action of tea tree oil and its two characteristic components against botrytis cinerea. journal of applied microbiology, 119(5), 1253–1262. https://doi.org/10.1111/ jam.12939 peruvian journal of agronomy 3(2): 47-56 (2019) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v3i2.1314 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 02 may 2019 accepted for publication: 16 july 2019 a simple and rapid algorithm for predicting froghopper (aeneolamia spp.) population increase in sugarcane fields based on temperature and relative humidity un algoritmo simple y rápido para predecir el aumento de la población de mosca pinta en los campos de caña de azúcar, en términos de temperatura y humedad relativa.condiciones de laboratorio martinez-martinez, c.1, somoza-vargas, c.1* *corresponding author: carlos.somoza1@catolica.edu.sv abstract in integrated pest management practices, knowledge from multiple disciplines is incorporated to facilitate the understanding of a problem and the development a practical, feasible, and ecologically sustainable solution. a froghopper (aeneolamia spp.) plague can trigger major economic losses in sugarcane plantations in countries such as el salvador and others in latin america. losses are often due to a lack of understanding of the life cycle of a pest and the underestimation of its annual reproductive potential. an algorithm was developed to model the most relevant aspects of froghopper reproduction and its interactions with the environment, to facilitate the prediction of potential increases in adult populations and its propagation in fields. data on several biological variables were collected as numerical measures and used to perform calculations based on a mathematical model designed particularly to simulate the reproduction of the pest, its economic threshold, and potential losses due to major natural events, with the aim of developing a tool that could support decision-making. the predictions of the tool were consistent with the findings of other studies in the field. the software and its installation instructions can be downloaded for free from https://drive.google.com/file/d/1ouwttbi lwmhofuth4wcktuzjfwdd89/view key words: pest management, applied software, population prediction, entomology resumen en las prácticas de manejo integrado de plagas, el conocimiento de múltiples disciplinas coopera para ayudar a entender el problema y, por lo tanto, para deducir una solución práctica, viable y ecológicamente sostenible. la plaga de la mosca pinta (aeneolamia spp.) es capaz de producir importantes pérdidas económicas en los cultivos de caña de azúcar en países como el salvador y el resto de américa latina. las causas a menudo se deben a la falta de conocimiento sobre el ciclo de vida de la plaga y la subestimación de su potencial reproductivo de un año a otro. por este motivo, se desarrolló un algoritmo para modelar los aspectos más relevantes sobre la reproducción de mosca pinta y su interacción con el entorno, a fin de predecir el posible aumento de la población adulta y su propagación en un área de campo. los datos de varias variables biológicas se almacenaron como medidas numéricas y sirvieron para realizar cálculos en base a un modelo matemático especialmente diseñado para simular la reproducción de la plaga, su umbral económico y posibles pérdidas debido a importantes causas naturales, para constituir una herramienta de software que puede respaldar la toma de decisiones cuyos resultados concuerdan con las medidas encontradas en la literatura. el software y sus instrucciones de instalación se pueden descargar libremente desde https://drive.google.com/file/d/1ouwttbi lwmhofuth4wcktuzjfwdd89/view palabras clave: manejo de plagas, software aplicado, predicción poblacional, entomología. introduction froghopper (aeneolamia spp.) is an insect in the family cercopidae. it is a homopterous insect that affects the production of sugarcane and other grasses in tropical and subtropical countries. its life cycle involves several stages including egg, nymph, and adult (peck, pérez, & medina, 2002; valbuena, 2006; cruz-zapata et al., 2016). the oviposition of the insect is performed in the vicinity of the host-plant roots, up to depths of 2 cm; while other species may perform oviposition on plant sections at lower depths (peck et al., 2002; cruz-zapata et al., 2016). aeneolamia spp. commonly produces diapausal eggs, a characteristic that facilitates their persistence under 1faculty of engineering and architecture, catholic university of el salvador, el salvador, central america. by pass metapan y carretera antigua a san salvador. carlos.martinez@catolica.edu.sv. (503) 7315 6546, (503) 2484 0613, (503) 7859 8618 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index mailto:carlos.somoza1@catolica.edu.sv mailto:carlos.martinez@catolica.edu.sv a simple and rapid algorithm for predicting froghopper (aeneolamia spp.) population increase in sugarcane fields based on temperature and relative humidity may august 2019 48 adverse conditions until conditions improve so that they can resume development and hatch (peck et al., 2002). the capacity of aeneolamia spp. to produce diapausal eggs makes the plague particularly challenging to control. when environmental conditions are suitable, the eggs hatch within 12 to 18 days of oviposition (thompson & león, 2005); however, it has been reported that they can maintain the diapausal state for several months to hatch under suitable conditions. female individuals often lay between 20 and 50 eggs per posture (peck et al., 2002) and between 50 and 100 eggs per posture under optimum conditions (thompson & león, 2005). diapause of the egg stage is important because more than 98% of the eggs are laid in the upper 2-cm soil layer during the dry season (fewkes, 1963), and only hatch when environmental conditions are more favorable. up to 85% of the eggs wait for the rainy season of the following year to hatch, 48 hours after the first heavy rainfall and adults emerge after 27 to 34 days (king, 1975). morales (1993) observed three distinct types of diapause under field and laboratory conditions, including a “short” diapause, with hatching a month after oviposition, a “moderate” diapause, with hatching three months after oviposition, and a “long” diapause, with hatching after six months. in the same study, morales (1993) observed that different generations of aeneolamia varia overlapped resulting in periods where plagues displayed population peaks, an observation that was also made by valbuena (2006). following emergence from eggs, the nymphs undergo five different stages where they parasitize and feed on root systems initially to when they emerge on the soil surface later in the last two larval stages, producing foamy secretions that facilitate tolerance against adverse environmental conditions and offer protection against predators until the sclerotization of their exoskeleton is complete and adult life begins (lópez-collado, pérezaguilar, & villanueva-jiménez, 2012; cruz-zapata et al., 2016; peck et al., 2002). the nymph cycle is complete within 25 to 35 days, going through the five instars. in their adult stages, aeneolamia spp. display yellow and red stripes on a black background, exhibiting limited flying capacity, although the can jump during feeding. they suck sap directly from the plant and introduce enzymes and divers compounds that damage and kill plant tissue (thompson & león, 2005; lópez-collardo et al., 2012). in addition, adults have a five–eight day lifespan (peck et al., 2002). aeneolamia spp. have a sucking mouth and feed by sucking sap directly from plant, through which they are also able to introduce diverse toxic compounds and enzymes into the foliar tissues that cause yellow and white spots and the leaves gradually dry out. when the populations of insects increase, the damages observed on foliar systems are considerable, which in turn impairs the photosynthetic capacity of the foliar systems greatly, resulting in a substantial decrease in the contents of sugars in plants such as sugarcane at the time of harvesting (thompson & león, 2005; lópez-collardo et al., 2012; cruz-zapata et al., 2016). severe infestations in sugarcane plantations could decrease production in terms of both raw tonnage and sucrose yield. mendoza (2001) and mendoca (2001) reported sucrose losses of 34% in ecuador and losses of 60% in brazil, respectively. according to fewkes (1969), pest levels lower than 0.5 adults per plant do not cause significant damage within a maximum of two weeks; however, with an increase population level or time, yield could decline by 20%, which is equivalent to 0.8 tons of sugarcane per hectare. a key factor to consider is that humidity and temperature levels influence the severity of the effects of the pest on sugarcane plant. in addition, temperature and humidity influence sugarcane growth and development. guagliumi (1972) reported that severe infestation of young sugarcane plantations could lead to losses equivalent to a 40% decrease in sugarcane tonnage per hectare, which is a major negative setback for farmers since their profits reduce considerably. cultural control refers to all the strategies aimed at providing plants with optimum conditions for development and enhancing resistance to infestation by pests, in addition to the creation of conditions unfavorable conditions for the proliferation of insects (sotelo & cardona, 2002). for field sampling activities, gómez (2007) proposed the placing of two sticky yellow traps per hectare, which has to be complemented with the inspection and counting of nymphs and adults per unit land area. similarly, the author recommended the use of 25 traps per hectare to reduce aeneolamia spp. populations. field preparation activities could decrease the incidence of the pests since they expose the eggs to harsh environmental conditions, in addition to natural predators and enemies. soil tillage has been demonstrated to reduce pest populations significantly compared to the case in uncultivated soils (guitiérrez & gómez, 2009). considering its high economic costs, soil tillage should be considered under severe cases (ruedaramírez, torrado-león & becerra, 2013). constant monitoring of crops is required to facilitate the timely detection of the pests. in addition, the conditions and microclimates within crop systems should be monitored since they could favor the growth and development of the pests (gómez, 2007). finally, gómez (2007) recommended the management of host weeds, particularly grasses, since they provide excellent host on which aeneolamia spp. complete their life cycles. natural enemies such as salpingogaster nigri have been applied as alternative biological control methods (guitiérrez & gómez, 2009) since two or three generations of aeneolamia spp. insects may overlap during a life cycle, a biological control method would be appropriate. although salpingogaster nigri exhibit high reproduction rates in the field, granobles et al. (2012) observed that large-scale production of the parasitic fly under laboratory conditions was a major challenge. regarding the use of entomopathogenic fungi as biological pesticides, the application of metarhizium anisopliae is a good alternative martinez-martinez, c., somoza-vargas, c. peruvian journal of agronomy 3 (2): 47-56 (2019) 49 for the control of aeneolamia spp. nymphs and adults. the adhesion of the entomopathogenic fungus to the exoskeletons of the insects lead heir degradation by the action of various enzymes and chemical compounds, facilitating penetration into the insects, where the fungi multiply and colonize the internal organs and utilize them as sources of nutrition during growth and development. following the death of the insects, and if the environmental conditions including humidity and temperature are favorable, the fungi emerge from the insects and release conidia, which infect other individuals (somoza-vargas et al., 2018). matabanchoy, bustillo, castro, mesa, & moreno (2012) tested the efficiency of m. anisopliae as a biological control method and obtaining promising results. the fungus can be propagated relatively easily under laboratory conditions, where it is placed in aqueous solution and sprayed on the plants to be treated. favorable environmental conditions such as high temperature and high relative humidity are required for its proper development and efficiency in the control of aeneolamia spp. (sotelo, 1984). other natural enemies that could be used in the biological control of aeneolamia spp. are nematodes in the heterorhabditis and steinernema genera (sendoya, et al., 2012). moreno, bustillo, lópez, castro, & ramírez (2012) reported that the use of heterorhabditis bacteriophora could result in mortality rates of up to 48% for aeneolamia spp. nymphs in sugar cane. chemical control of aeneolamia spp. is recommended when their populations exceed the economic threshold to minimize production costs and economic losses, and after considering the potential effectiveness of cultural and biological control methods (gómez, 2007; guitiérrez & gómez, 2009). the persistent application of chemical pesticides can lead to resistance against the active ingredients in subsequent pest generations, which, in turn, would prompt a farmer to increase doses or mix different agrochemicals, which could also have adverse impacts on beneficial insect species (sotelo & cardona, 2002). imidacloprid, endosulfan, and pyrethroids have been applied extensively due to their rapid and effective insecticidal effects. population dynamics can be defined as the changes exhibited by biological communities in addition to the various mechanisms that influence and regulate such changes and mechanisms (vargas & rodríguez, 2008), in addition to how such factors interact. population dynamics are influenced by three types of distribution, including uniform (individuals are dispersed with similar distances among them, so that intraspecific relationships may not seem ideal), random (in the case of species that do not seek coexistence in groups), and aggregated (individuals seek to exist in different large groups that are separated and most agricultural pests display such distribution since it maximizes multiplication and survival rates of groups) (vargas & rodríguez, 2008). among the external factors that influence population dynamics, temperature is one of the most influential factors influencing key aspects of insect development, including larvae, average life span of adults and female oviposition capacity. to determine how influenced the insect development can be by the environment conditions, degrees days and growing degree days (gdd) are the most common measures used to evaluate how environmental conditions influence insect development. the measures, which are based on temperature, facilitate the prediction pest activity based on environmental conditions, and could facilitate early planning for effective control measures (adams, 2014; vargas & rodríguez, 2008). other environmental variables that influence the population dynamics of aeneolamia spp. are relative humidity and photoperiod. due to the need for increased food production to sustain growing populations, modern agricultural activities have led to irreversible damage in ecosystems. the application of insecticides for crop protection is widespread among farmers because of its low cost and high level of effectiveness in the short-term. in the case of the control of froghopper in sugarcane plantations in el salvador, unanticipated challenges have emerged due to the intensive application of insecticides such as pyrethroids, endosulfan, and counter®, which have polluted shallow waters, deteriorated ecosystems, and decreased the populations of natural controlling agents, in addition to enhancing the resistance of pests to pesticides overall and ushering in other complications associated with a poor understanding of key biological and ecological aspects of the pest, including its reproductive capacity and its natural enemies (gómez, 2007; cruz-zapata et al., 2016). in integrated pest management control, the single action of spraying agrochemicals should not be considered adequate to prevent losses. indeed, chemical control should be the last strategy after attempting cultural and biological control methods (gómez, 2007). to facilitate the addressing of the challenge of aeneolamia spp. in sugarcane production, the present paper describe the development and testing of a simple software tool that has been demonstrated to predict increases in froghopper populations that could infest a sugarcane plantation under field conditions accurately based on the two most critical environment variables including temperature and relative humidity. the development of the tool required an extensive literature review on the biology of aeneolamia spp., particularly their life cycle and diapause capacity (morales, 1993). the information was combined with agronomic knowledge on sugarcane cultivation, in addition to biological insights and observations following the treatment of aeneolamia spp. with numerous natural enemies (sendoya et al., 2012; moreno et al., 2012; matabanchoy et al., 2012). the studies above were carried out to present simulation of froghopper reproduction and decline due to environmental and biological factors that influence their infestation capacity. a project of this nature could facilitate the understanding of the challenge and decision-making processes for its effective management, while integrating knowledge from diverse disciplines such as biology, agronomy, and bioinformatics. the algorithm a simple and rapid algorithm for predicting froghopper (aeneolamia spp.) population increase in sugarcane fields based on temperature and relative humidity may august 2019 50 was executed in a front-end desktop application compatible with the most common operative systems available to computer users, using a programming language. methodology 1) design of the algorithm the first step in the design of the simulating algorithm was understanding the life cycle of the pest and obtaining scientific data for developing a data model stored in arrays categorized for based on periods such as months and quarters in a year. the environmental and pest variables that were considered for the algorithm included monthly average temperature in the site where the sugarcane is planted (expressed in celsius), monthly average relative humidity, the threshold temperature at which plant and pest biological activity is halted, the theoretical rate of population increase observed after every generation, the proportion of the insect population that is female, the number of eggs laid per female, the proportion of eggs that would be damaged at a given relative humidity range, and diapause levels in froghopper species can have three potential outcomes: (1) instant hatching, (2) short diapause where eggs hatch two months after oviposition and (3) long diapause with hatching after six months. other key variables were obtained from literature and are appropriate methods for evaluating populations based on temperature, for example, the number of nymphs and adults present per square meter per each gdd, above the threshold temperature. all the input variables integrated into the algorithm are listed in the second frame of figure 1. two sets of values per variable are embedded within the figure 1: simplified flowchart of the algorithm, with emphasis on the input data required for the numeric simulation. the clause [3] refers to each iteration for the three known diapauses, and [12] is the number of monthly iterations and data used for calculations; the arrays modeling diapause are bi-dimensional because three ratios of diapause are present for different periods throughout the year. martinez-martinez, c., somoza-vargas, c. peruvian journal of agronomy 3 (2): 47-56 (2019) 51 source code and can be loaded by a user to simulate low or high population conditions. in every instance, a user could modify the values using data based on assumptions or applied research. the flowchart in figure 1 represents a tool required for software development, which in the present case was used to define the data requirements of the code routines that estimated the pest populations. 2) growing degree days equation the gdd was considered a key factor influencing pest reproduction, and indices for high and low populations or nymphs and adults were formulated to simulate the presence of individuals in sugarcane plantations based on temperature and area per unit square meter. the data were based on wiedijk (1982) and olán-hernández, sánchezsoto, bautista-martínez, zaldívar-cruz, & cortezmadrigal (2016). therefore, the potential presence of the insects was calculated by multiplying the indices obtained from literature by the gdd, as presented in equations 1 and 2. all the gdd calculations were performed with a base temperature of 15°c; however, in the front-end interface of the software, the variable could be modified freely by a user based on their requirements. in addition, the egg diapause rates were based on the results reported by morales (1993), and the amounts of fertile eggs oviposited by the female individuals were based on chaves et al. (2014). equation number 3 was formulated to model the phenomena of diapause. the constant of adult population variation per generation “kp” is a rate for measuring the increase in adult froghoppers after each generation based on data from valbuena (2006), while offering a user the possibility to alter the value. where, where, 3)diapause consideration and equations the three types of diapause identified for the pest, implied that there was a need to model three equations to predict population in the short, medium, and long term, for one (equation 4), three (equation 5), and six (equation 6) months, respectively. this would allow the determination of the extent of overlap across the different generations. however, since external factors such as relative humidity and natural enemies could influence the increase in populations of froghopper species, the factors were modeled into each equation as the percentage of the population that would survive at any given conditions. the percentage of the population surviving under different levels of relative humidity could be entered manually by the user in the front-end interface of the software; however, sample data and routine tests were based on information presented by suiji, alice, fontes, pires, & o’neil (2002) and wiedijk (1982). reduction in pest populations due to biological control were incorporated into the equations for the fungus metarhizium anisopliae and the nematode heterorhabditis bacteriophor, and both integrated into a single factor for biological control that represented the proportion of the froghopper population capable of surviving such an exposure. where, : agdd = i ad gdd ngdd = i n gdd dm= k m pfem efem k p (1) (2) :adult population at field conditions based on given gdd. :nymph population at field conditions based on given gdd. :growing degree days, using celsius scale to measure temperature units. :rate of increase in adult population per gdd. :rate of increase in nymph population per gdd. (3) p1m= (ngdd + a' gdd ds k p)f rh fbio (4) p3 m= (ngdd + a' gdd ds k p + a''gdd dm k p)f rh f bio (5) p6 m= (ngdd + a' gdd ds k p + a''gdd dm k p+ a'''gdd dl k p)f rh f bio (6) dm agdd ngdd gdd i ad i n :diapause rate for a given month, which is the amount of eggs that may hatch immediately at a particular moment based on some population parameters. k m :the theoretical proportion of eggs that hatch per month. pfem :proportion of the adult population that are reproductive females. efem :the amount of viable eggs laid per female. k p :constant of adult population variation per generation p1m :predicted population within one month. p3m :predicted population within three months. p6m :predicted population within six months. ngdd :nymph population in based on given gdd. a 'gdd :adult population of the past month in based on given gdd. a ' 'gdd :adult population of the past three months based on given gdd. a ' ' 'gdd :adult population of the past six months based on given gdd. a simple and rapid algorithm for predicting froghopper (aeneolamia spp.) population increase in sugarcane fields based on temperature and relative humidity may august 2019 52 considering a future estimate of froghopper population within a year is essential for taking appropriate preventive action, equation 7 was developed for making predictions based on eggs laid and their respective diapause levels that are consistent with diapause indices as demonstrated in equation 3. future populations within six, three, and one month were estimated, considering all the potential generational overlaps within each period. for the long term diapause, only one generation was considered; however, for the middle-term diapause, it was assumed that one generation could persist for two months while another could persist for a month, so that in equation 7, it was necessary to integrate two diapause indices and two generational constants in equation 7. for the population with a short-term diapause, two generations of one month, one generation spanning two months, and one generation spanning six months were considered, since it was assumed that the most critical part of the population could be observed one year in the future, which is why all the diapause indices and the four generational constants were multiplied. equation 8 is a factorized form of the previous formula. once the future froghopper population was obtained suing the algorithm and stored as the p12 m array, it was calculated the intercrossed addition to population between future months, as presented in equation 9. where, p9 m : 4) biocontrol estimates the impact over of the fungus metarhizium anisopliae on the froghopper population was based on data published by matabanchoy, bustillo, castro, mesa & moreno (2012). the results of the study were tabulated and a regression equation (10) obtained to determine the effects of the treatment based on the amount of conidia applied in a field. a similar procedure was applied with the data of salguero et al (2012) with regard to treatments with the nematode heterorhabditis bacteriophor; however, in the case of the nematode, two different trends for froghopper control were examined under low dosage (equation 11) and under high dosage (12). where, since the algorithm was intended to be rapid and simple, it was based only on the major environmental variables and froghopper population dynamics parameters, assuming that other circumstances had limited influence on ds :short diapause of eggs that would hatch soon after being laid. dm :middle diapause of eggs that would hatch approximately two months after being laid. dl :long diapause of eggs that would hatch in approximately five months after being laid. f rh :fraction of the population that would survive due to relative humidity (rh). fbio :fraction of the population that would survive due to biological control. k p :constant of adult population variation per generation. p12m= (p6 m dl k p + p3m d s dm k p2 + p1md s2 dm dl k p4)k p f rh (7) p12m= (p6m dl +(p3m+ p1m ds dl k p2 )d sd m k p)k p f rh (8) pi 12m= p12m + ( p11 m ds+ p9m dm+ p6 m dl ) k p f rh (9) p12m :predicted population within 12 months.. p11m :predicted population within 11 months based on short diapause individuals. :predicted population within 9 months based on middle diapause individual. p6 m :predicted population within 6 months based on long diapause individuals. pi 12m :predicted population within 12 months, including new generations that would be produced in the following year. :the value of calculated p12m corresponding to one month in the past. n3m n6m :the value of calculated p12m corresponding to three months in the past. n1m :the value of calculated p12m corresponding to six months in the past. sm= 26.61+18.88 ln(con) (10) s(l− n)= 4. 9e 0 . 15i (11) s(h− n)= 39.01ln(i )+10.4 (12) sm :proportion of the population surviving following metarhizium application.. s(t − n) :proportion of population surviving when the nematode h. bacteriophora is applied at relatively low doses.. s(h− n) :proportion of population surviving when the nematode h. bacteriophora is applied at relatively high doses.. con :conidia per hectare, in the order of magnitude of 1012. i :infective juveniles (individuals) per hectare, in the order of magnitude of 1010.. martinez-martinez, c., somoza-vargas, c. peruvian journal of agronomy 3 (2): 47-56 (2019) 53 population prediction. some assumptions were required to simplify the data processing and the amount and complexity of input variables to be entered by the user. some of the considerations included instead of considering the direct effects of harvest, the algorithm was based on indices that described the rate of nymphs and adults per gdd in each month. another consideration was that environmental variables such as average temperature and rh remained constant throughout the months from the hypothetical year prior to the running of the simulation run and the following predicted year. other parameters associated with insect biology were also considered constant, such as the monthly oviposition rate, the proportion of females within the population, the responses of individuals to incidental heavy rains, among others. results and discussion using the temperature and the humidity levels presented in a report on the environmental profiles of the coastal zone of el salvador (table 1), a prediction sequence was run in the software, using a high population density based on relatively high indices of nymph and adult existence per gdd (figure 2, upper part) without any control to produce a numeric simulation (figure 2, lower part), which illustrated the trends in the reproduction of the pest without any corrective strategies. the results (figure 3a) are consistent with those of olán-hernández et al., (2016), which identified population peaks of aeneolamia contigua (walker) adults in the months preceding months with higher records of rh and precipitation, in addition to being consistent with the effects of the three types of diapause defined by morales (1993) and the influence of temperature on mortality as described by suiji et al. (2002). in a second run, a biological control month average temperature °c average relative humidity % january 30.6 68 february 31.4 75 march 32.5 78 april 32.3 80 may 30.9 83 jun 28.9 86 july 30.2 68 august 29.8 88 september 28.7 84 october 28.8 86 november 28.8 72 december 29.5 69 table 1. monthly average temperatures and relative humidity in the coastal zone of el salvador. source: national service of territorial studies of el salvador (snet), http://www.snet.gob.sv/meteorologia/perfiles.pdf figure 2. upper part: froghopper population calculation software, this image illustrates the user interface where population rates of nymphs and adults per gdd can be input. lower section. froghopper calc software, this image exhibits the user interface where output of the simulation is presented. figure 3. a) line graph illustrating the population dynamics predicted by the software for one year in the future without any control strategy applied for the pest. b) line graph illustrating the population dynamics predicted by the software for a year in the future taking into account the application of 5 × 1013 conidia.ha-1 of m. anisopliae in april. a decrease in the population can be observed in the following month. c) line graph illustrating the population dynamics predicted by the software for one year in the future taking into account the application of 15 × 1010 ij.ha−1 of h. bacteriophora d) line graph illustrating the population dynamics of aeneolamia spp. in sugarcane following the application of a first biological control of 5 × 1013 conidia.ha−1 of m. anisopliae strategy in april followed by the application of a biological control strategy using 15 × 1010 ij.ha−1 of h. bacteriophora in may. a simple and rapid algorithm for predicting froghopper (aeneolamia spp.) population increase in sugarcane fields based on temperature and relative humidity may august 2019 54 of 5 × 1013 conidia/ha of m. anisopliae was applied in april, and a reducing effect of the population in the month following the application was observed (figure 3b), directly affecting the nymphs with a short diapause and reducing the rate of population increase in may. these results are consistent with those presented by matabanchoy et al. (2012), who reported mortality ranging from 62.5 to 71.4% with a similar concentration of m. anisopliae conidia following application to control aeneolamia varia in sugarcane. however, in the software prediction, although only two applications of the treatment were considered, it is safe to say that an increase in the number of applications increase the effectiveness of the biological control method. a third run with a biological control of 15 × 1010 ij.ha−1 of h. bacteriophora (moreno et al., 2012), with may as the time of the application, was predicted. although the results predicted an almost complete decrease of the adults in june based on the estimate of a single control event, generational overlap allowed the pest to be present normally again from the month of july and in the following months (figure 3c). in a last simulation, the two previously defined biological control strategies were applied simultaneously (figure 3d). the combined effects of the fungus and the nematodes caused the disappearance of the population peak in and a minimum value for the adult population reached was observed in june. the results of the study reinforced the need to adopt integrated pest management systems to maximize the effectiveness of each strategy. notably, the software predicted the expected results of control strategies against the pest accurately, which could facilitate the minimization of the negative economic impacts associated with the pest. similarly, it is critical to note that it is possible to reduce the incidence of the pest in the month of june, when dam levels of up to 86% are reported, which offer ideal conditions for the recovery of the insect populations. the spatial distribution of adults (figure 4) was consistent with an aggregation pattern based on the oviposition sites, as observed by figueredo, andrade, niño, quintero, & azad (2011). they observed that the infestation began from aggregation centers, and spread across entire plantations. the sofwtare accurately predicted such a trend, with 30 m2 infested in may without the application of any control strategies. in a year, the plague would have reproduced adequately to infest 9404 m2. however, such values may change in the field due to the ever-changing conditions that could influence pest growth and development. conclusion the software was developed based on an elaborate mathematical model that predicted the trends of a pest in a crop of interest. using global and regional data, it is possible to identify the appropriate periods for implementing corrective actions using biological control strategies reduce the need for the application of agrochemicals. adopting biological control strategies could minimize the potential negative environmental and health impacts of such agrochemicals. rh and temperature are the most influential environmental factors influencing the development of aeneolamia spp., and they can be used to simulate potential increases in pest populations, with results similar to those reported by other studies based on open-field experiments. tools such as the one presented in the present paper facilitate the formulation of action plans to mitigate the effects of pests on crops using alternatives that are more environmentally friendly. references adams, n. e., (2014). using growing degree days for insect management. new england: university of new hampshire, u.s. department of agriculture and n.h. counties cooperating. chaves, v. d., pimentel, g. v., valverde, a. p., silva, l. a., barbosa, m. p., & peternelli, l. a., (2014). biology and preferred oviposition site of the mahanarva indentata froghopper (hemiptera: cercopidae) on sugarcane. florida entomologist, 97(1), 73-79. cruz-zapata, g., garcía-lópez, e., sánchez-soto, s., bautista-martínez, n., ortíz días, j. j., & osorio-osorio, r., (2016). identity of spittlebugs (hemiptera: cercopidae) and their hosts in sugarcane fields in cardenas, tabasco, mexico. southwestern entomologist, 41(1), 145-151. http://dx.doi.org/ 10.3958/059.041.0116 fewkes, d. w., (1963). the effect of exposure to dry conditions on the eggs of aeneolamia varia saccharina (homoptera: cercopidae). annals of the entomological society of america, 56(5), 719-720. fewkes, d.w. 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de baptista, g.(2); gadanha-junior, c.(3); bracho-pérez, j.(4) *corresponding author: jaque@lamolina.edu.pe https://orcid.org/0000-0003-3407-6938 abstract chemical insecticides are commonly used to control insect pests in stored wheat. however, the presence of insecticide residues in food may endanger consumers. we studied the degradation and persistence of two insecticides, fenitrothion and esfenvalerate, in wheat grain, bran, and flour. the application system was calibrated to treat grain at theoretical concentrations of 10 and 0.5 mg kg−1 of fenitrothion and esfenvalerate, respectively. samples treated with the insecticide mixture were collected at 0, 15, 30, 60, 90, 120, 180, 240, and 360 days after treatment. samples were analyzed quantitatively by gas chromatography with an electron capture detector (ecd, ni63). the experimental design was completely randomized with three replicates. esfenvalerate was more persistent than fenitrothion, with the residues of both insecticides concentrated mainly in the bran, and with least amounts in the flour. the concentrations of fenitrothion residues during the 120-day preharvest interval exceeded the maximum residue limit (mrl) of 1 mg kg−1 set by brazilian legislation. we discuss the factors that influence the degradation/persistence of fenitrothion and esfenvalerate. key words: degradation, persistence, chromatography, maximum residue limit, preharvest interval. resumen debido a los problemas causados por los insectos en el trigo almacenado, insecticidas químicos son utilizados para el control de esas plagas, existiendo la posibilidad de colocar en riesgo la salud de los consumidores debido a los residuos de estas substancias en los alimentos. el objetivo de este trabajo fue estudiar la degradación/persistencia de los insecticidas fenitrotion y esfenvalerato en granos de trigo y en algunos de sus derivados (salvado y harina). el sistema de aplicación fue calibrado para el tratamiento de los granos en las concentraciones teóricas de 10 y 0,5 mg kg-1 de fenitrotion y esfenvalerato, respectivamente. las muestras tratadas con la mezcla insecticida fueron colectadas a los cero, 15, 30, 60, 90, 120, 180, 240 y 360 días después del tratamiento. la determinación cuantitativa se hizo mediante técnica de cromatografía en fase gaseosa, utilizándose cromatógrafo equipado con detector de captura de electrones (ecd, ni63). el diseño experimental fue enteramente al azar, con tres repeticiones. el esfenvalerato fue más persistente que el fenitrotion. los residuos de ambos insecticidas en los granos de trigo se concentraron principalmente en el salvado y en menores cantidades en la harina. el residuo de fenitrotion en el periodo de carencia de 120 días fue superior al límite máximo de residuo (lmr) de 1 mg kg-1 permitido por la legislación brasileña, la misma que resultó inadecuada en la reglamentación de ese insecticida. los factores que influyeron en la degradación/persistencia del fenitrotion y esfenvalerato son discutidos en el presente trabajo. palabras clave: degradación, persistencia, cromatografía, limite máximo de residuo, periodo de carencia. 1 universidad nacional agraria la molina, departamento de entomología, facultad de agronomía, av. la molina s/n, lima 12, peru. 2 universidade de são paulo (usp), departamento de entomologia e acarologia, escola superior de agricultura “luiz de queiroz”, av. pádua dias 11, são paulo, brazil. 3 universidade de são paulo (usp), departamento de engenharia de biossistemas, escola superior de agricultura “luiz de queiroz”, av. pádua dias 11, são paulo, brazil.. 4 universidad nacional tecnológica de lima sur. facultad de ingeniería y gestión. campus sector 3 grupo 1a 03 cercado (av. central y av. bolívar) villa el salvador, lima 42, peru. introduction chemical insecticides are one of the most important inputs in modern agricultural production systems, as they are the principal method of pest control. thus, to avoid consuming food with dangerous levels of these substances, every registered insecticide has a maximum residue limit (mrl) and a preharvest interval allowed by law. however, the improper use of the insecticides in stored products may change the residue levels so that they surpass their mrl, vásquez-castro, j.; de baptista, g.; gadanha-junior, c.; bracho-pérez, j. peruvian journal of agronomy 4(2): 40-47 (2020) 41 thus putting consumers’ health at risk (dordevic & durovicpejcev, 2016; watanabe et al., 2018). the persistence of the insecticide in stored products depends on several factors, including the physico-chemical characteristics of the pesticides, and the environmental conditions (e.g., humidity and temperature) of the warehouse in which the grain is stored (farha et al., 2016). in general, pyrethroid insecticides are much more persistent than the organophosphorus ones (afridi et al., 2001), although both groups of insecticides are more stable at lower humidity levels (samson et al., 1988). with regards to warehouse temperature, insecticides generally degrade faster at higher temperatures (arthur et al., 1991). insecticides residues may be present not only in stored grains, but also in their by-products, although insecticide residue concentrations generally diminish relative to that in the whole grain (barbosa, 2004). therefore, it is important to measure the changes in residues levels that occur during the processing of grains, and these changes must be considered in determining the mrl and preharvest interval for each substrate. in brazil, the list of preventive insecticides currently registered for wheat grain treatment in warehouses is as follows: fenitrothion, malathion, pirimiphos-methyl, bifenthrin, deltamethrin, esfenvalerate, and permethrin (agência nacional de vigilância sanitária [anvisa], 2019). it should be noted that ministerial resolution no. 165 of the 29th of august 2003, which regulates the use of fenitrothion, has been modified by resolution no 347 of the 22nd of november 2004 (resolução re n0 347, 2004). in this resolution, the mrl and preharvest interval values for fenitrothion on stored wheat grain were modified from 10 mg kg−1 and 14 days, respectively, to 1 mg kg−1 and 120 days, respectively. there are efforts worldwide to eliminate highly toxic insecticides on stored grains. however, the alimentarius codex of the food and agriculture organization of the united nations (fao) and who does not have registry entries for fenitrothion or esfenvalerate applied to stored wheat; there is only an mrl for esfenvalerate used for preharvest treatments: 0.05 mg kg−1 (fao, 2019). similarly, an mrl for fenitrothion on wheat gluten has been registered with the us environmental protection agency (epa, 2012). the presence of insecticide residues in foods constitutes a serious risk for consumers, but the scientific evidence regarding this subject is shallow (valcke et al. 2017). moreover, there are some serious inconsistencies between the brazilian legislation and insecticide manufacturers; some of the mrls issued by the former are higher than what would be expected based on the dose recommended by the latter, thus allowing the immediate consumption of grain after treatment (trevizan & baptista, 2000). on an international level, legislation on pesticide residues in foods has sometimes been used as a mechanism to raise trade barriers. therefore, it is important to study the fate of insecticide residues in stored grains under brazilian conditions to provide reliable information in crafting adequate legislation. the objective of this work is to study the dissipation of fenitrothion and esfenvalerate in wheat grains, flour, and bran. materials and methods the research was conducted at the pesticide residues analysis laboratory (larp) of the agricultural superior school “luiz de queiroz” at the são paulo university (usp), piracicaba, são paulo (22°42′47′′ s, 47°37′40′′ o), brazil. treatment of the grains the wheat cultivar “brs 208”, developed by the brazilian agricultural research corporation (embrapa), was used. to treat the grains, a mobile application system equipped with a double-jet hydraulic fan nozzle (model tj8002evs, spraying systems co., illinois, usa) was used. the nozzle was operated at 200 kpa and installed 0.5 m above the grain mass. the application system was calibrated to treat the grains at theoretical concentrations of 10 mg kg−1 of fenitrothion and 0.5 mg kg−1 of esfenvalerate. the commercial product sumigranplus® (containing 500 g l−1 of fenitrothion + 25 g l−1 of esfenvalerate) was used. the mobile system moved at a speed of 2.2 km h−1 over the mass of grains. under these operational conditions, the volume of mix equivalent to 5 l of insecticide emulsion was applied per ton of grain (0.4% of commercial product). for the control, the same procedure was performed, except only water was used. the experiment consisted of three replicates. the temperature and relative humidity during treatment applications ranged 16°c –18°c and 72%– 79%, respectively. after applications, the grains were placed in plastic bags and stored in the laboratory, where temperature and relative humidity were not controlled. the moisture content of the grains during the storage period was measured by the oven drying method at 105 ± 3°c for 24 h, according to the rules for seed analysis–ras (brazil, 1992). sampling the grain samples were collected at 0, 15, 30, 60, 90, 120, 180, 240, and 360 days after the treatments. wheat grain processing to produce flour and bran was done at the quality control laboratory of the cargill alimentos company, located in the municipality of tatuí, são paulo, brazil. analytical procedure the analytical method of vásquez-castro et al. (2007) was used to analyze the residues on the grains and their by-products. the procedure starts with the extraction of the residues with ethyl acetate, followed by separation by dissipation of fenitrothion and esfenvalerate in wheat grains, bran and flour may august 2020 42 silica gel-column chromatography, and then quantitative determination through gas chromatography (gc) with an electron capture detector (ecd, ni63), thermoquest trace model (milan, italy). analytical method validation the analytical method was validated by spiking samples (grain, bran and flour) with 0.05 and 0.5 mg kg−1 of fenitrothion and esfenvalerate, respectively. three replicates were performed for each dose. recovery rates of between 70%–120% were considered acceptable. statistical analysis the data for each insecticide and substrate were analyzed by linear regression, using the following model: y = α + βx + e, where “y” is the natural logarithm of the observed value of the insecticide residue; “α” is the logarithm of the insecticide initial value; “β” is the degradation constant; “x” is the time after the insecticide application; and “e” is the random error, which is assumed to be independent and randomly distributed. the above model requires values of the half-life of each insecticide in each substrate, which were calculated using the following formula: half-life = −ln(2) = −0.693147 β β linear regressions were analyzed using the general linear models test in sas software (1999), raleigh, usa. results and discussion the recovery rates of the insecticides in different substrates ranged between 70% and 120%, thus validating the analytical method. in the control, none of the insecticides were recovered, that is, all substrates were free of contamination by these compounds. the amounts of fenitrothion and esfenvalerate recovered on day 0 were 6.24 and 0.35 mg kg−1, respectively (tables 1 and 2), which represent 62% and 70% of the theoretical concentrations of the organophosphorus and pyrethroid insecticide, respectively. several studies, conducted under both laboratory and storage conditions, have reported on the dissipation of insecticides from treated grains, with a large degrees of variation in the amounts of insecticide lost over the storage period (arthur et al., 1991; sgarbiero et al., 2003). warehouses usually use conical-jet nozzles to apply residual insecticides because they are easier to operate, in contrast to fan-jet nozzles (miike et al., 2002). however, conical-jet nozzles require a high level of hydraulic pressure to produce droplets and operating them at such high pressures produce fine drops (sumner, 2012). in brazil, the high interior temperature of warehouses may cause the evaporation of fine drops, thus leading to the loss of insecticides during their application. in contrast, continuous flow fan-jet nozzles operate at relatively low pressures, generating larger drops, and distribute droplets in a more uniform, transverse pattern, compared to their conical counterparts. therefore, continuous flow fan-jet nozzles have several advantages for the treatment of stored grains. however, this type of nozzle requires greater care in their calibration (vásquez-castro, et al., 2012) although grain should be treated uniformly, some studies have shown the efficacy of insecticides in controlling pests when applied non-uniformly (arthur, 1992). for example, minett & williams (1976) proposed mixing non-treated grains with overly treated grains as a method for controlling insect pests. however, this method is legally problematic; for example, the highly inconsistent distribution of insecticide residues in the grain mass may include areas with higher residue concentrations than that allowed by the mrls, resulting in consequential economic impacts to the producer. daa residues (mean ± sd) degradation index accumulated degradation index 0 6.24 ± 0.10 15 3.89 ± 0.26 1.60 1.60 30 4.08 ±0.26 0.95 1.53 60 3.65 ± 0.05 1.12 1.71 90 3.34 ± 0.10 1.09 1.87 120 3.19 ± 0.16 1.05 1.96 180 0.88 ± 0.02 3.64 7.11 240 0.76 ± 0.03 1.15 8.21 360 0.54 ± 0.02 1.40 11.48 table 1. fenitrothion residues (mg kg−1) in wheat grains treated with the insecticide mixture fenitrothion + esfenvalerate. daa: days after application. daa residues (mean ± sd) degradation index accumulated degradation index 0 0.35 ± 0.04 15 0.31 ± 0.02 1.12 1.12 30 0.37 ± 0.01 0.85 0.95 60 0.26 ± 0.02 1.41 1.33 90 0.26 ± 0.04 1.00 1.33 120 0.28 ± 0.04 0.93 1.24 180 0.23 ± 0.01 1.23 1.52 240 0.25 ± 0.02 0.91 1.38 360 0.25 ± 0.02 1.00 1.38 daa: days after application. table 2. esfenvalerate residues (mg kg−1) in wheat grains treated with the insecticide mixture fenitrothion + esfenvalerate. vásquez-castro, j.; de baptista, g.; gadanha-junior, c.; bracho-pérez, j. peruvian journal of agronomy 4(2): 40-47 (2020) 43 the degradation index values presented in the tables are indicators of the amount of the reduction in insecticide residues during the period between successive measurements. that is, these values show the degradation of the insecticides in different time intervals. the accumulated degradation index represents the degradation of insecticides throughout the evaluation period. the moisture content of the grains during the experiment ranged between 10% and 11%, that is, the storage conditions were optimal for the preservation of the grains. the values and adjusted regressions of fenitrothion and esfenvalerate residues in grains treated with the sumigranplus® formulation are shown in figs. 1 and 2. the regression of fenitrothion follows a steeper slope, indicating a higher degradation rate in contrast to that of esfenvalerate. table 3 shows the insecticide residues, β parameter estimates, descriptive level of the probability values of the f-test of the regression analysis, and the estimated half-life of the insecticides. the insecticide deposit was calculated as the exponent of α. even though there were some low coefficients of determination, the p-values (p < 0.05) of the linear regressions indicate significantly linear relationships, mainly for esfenvalerate. we also calculated the standard deviation from regressions, and results indicate that no condition affected the regressions significantly (p > 0.05). the degradation rate of esfenvalerate was low, as reflected by a half-life of 747 days in wheat grains and wide confidence intervals. hence, longer-term experiments that evaluate esfenvalerate for more than 360 days should be performed to corroborate our results. in the case of fenitrothion, the half-life values range from 98 days for grain, to 226 days for bran, confirming the longer persistence of this insecticide in bran. the stability of esfenvalerate and fenitrothion depends on the following physico-chemical factors: vapor pressure (with values of 2.0 10−7 and 1.8 10−2 pa, respectively), n-octanol-water partition coefficient (kow) (with logarithmic values of 6.22 and 3.43), solubility in water (with values of 0.002 and 21 mg l−1, respectively), and molecular weight (with values of 419.9 and 277.2, respectively) (tomlin, 1995). the pesticides become more volatile as the vapor pressure increase, which explains the higher amount of fenitrothion loss during the storage of grains. lower log kow values indicate a higher degree of hydrophilicity, and the more water-soluble insecticide penetrates grains more easily, thus increasing the degradation rate. in terms of molecular weight, the general rule is that big molecules are less soluble than smaller ones (seiber, 1999). thus, fenitrothion is more soluble than esfenvalerate. figure 1. mean values of fenitrothion residues through time after treating wheat grains with the sumigranplus® formulation. values have been logarithmically transformed and the regression has been adjusted. figure 2. mean values of esfenvalerate residues through time after treating wheat grains with the sumigranplus® formulation. the values have been logarithmically transformed and the regression has been adjusted. y = -0.0071x + 1.6625 r² = 0.8999 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 0 60 120 180 240 300 360 fe ni tr ot hi on re si du es l n (m g kg -1 ) time after application (days) y = -0.0009x 1.151 r² = 0.4772 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 0 60 120 180 240 300 360 e sf en va le ra te re si du es l n (m g kg -1 ) time after application (days) table 3. fenitrothion and esfenvalerate residues in treated wheat grains, bran, and flour, estimates of the parameter β of the degradation regression, and the mean half-life of the active ingredient based on β. fenitrothion grain 5.272 7.05 <0.0001 98 87–113 bran 8.478 3.06 <0.0001 226 186–288 flour 2.650 6.52 <0.0001 106 98–116 esfenvalerate grain 0.316 0.92 <0.0001 747 499–1482 bran 0.547 1.87 <0.0001 369 242–774 flour 0.256 1.40 0.0002 494 334–953 substrate initial residue (mg kg−1) × 103 linear regression (pr > f) halflife (days) confidence interval (95%) of half-life β̂ dissipation of fenitrothion and esfenvalerate in wheat grains, bran and flour may august 2020 44 various studies have shown that pyrethroids are more stable than organophosphorus in liquid applications (afridi et al., 2001). however, in solid formulations that do not require dilution in water (dust formulations), the two groups of insecticides have similar dissipation rates (yu et al., 2014). among the organophosphorus insecticides used to protect stored grains, fenitrothion is considered highly unstable (gragasin, et al., 1994; rumbos et al., 2018), whereas among pyrethroids, esfenvalerate is considered more persistent (joia et al., 1985). the processes of volatilization, hydrolysis, and oxidation are known to assist in the postharvest degradation of insecticides (holland et al., 1994). in particular, volatilization and photolytic decomposition are the most important mechanisms involved in the degradation of the organophosphorus components of insecticides (gragasin et al., 1994; gonzález-curbelo et al., 2017). the results of the analysis of wheat bran and flour are shown in tables 4 and 5. insecticide residues were not recovered from the control samples, confirming that the materials we used were insecticide-free. the degradation rates of the insecticides varied among the different substrates. the cumulative degradation indexes of fenitrothion in wheat grains, flour, and bran are 11.48 (table 1), 9.59, and 4.10 (table 4), respectively. these results indicate that fenitrothion is most persistent in bran and least persistent in grains. for esfenvalerate, the degradation indexes in wheat grains, flour and bran are 1.38 (table 2), 1.60, and 3.51 (table 5), respectively, indicating that esfenvalerate is most persistent in grains and least persistent in bran. in contrast to our observations, papadopoulou-mourkidou and tomazou (1991) reported that the pyrethroid permethrin is more persistent in bran than in grains. moreover, at the end of our experiment (360 days after treatments), of the original residues present in wheat grains, flour, and bran, 9%, 11%, and 24% of the fenitrothion residues remained, respectively; whereas 71%, 61%, and 28% of the esfenvalerate residues remained, respectively. these results demonstrate that esfenvalerate is more stable than fenitrothion. both insecticides tended to concentrate in the pericarp and embryo, which are external and oily parts of the grain; and to a lesser extent in the amylaceous endosperm. the quantity of insecticide residue varied according to the type of substrate, i.e., the highest levels were in bran, followed by grains, then flour. similar results have been reported in other studies conducted in different countries (trevizan & baptista, 2000; sgarbiero et al., 2003; bajwa & sandhu, 2014). typically, a grain of wheat is composed of 82% endosperm, 3% embryo, and 15% pericarp (rowlands, 1971). therefore, insecticides concentrate in a small portion of the grain, which explains the presence of high and low quantities of residues in the bran and flour, respectively. an insecticide’s penetration rate affects its metabolic route and its persistence, that is, insecticides that penetrate the grain quickly will also be degraded rapidly. in general, hydrophilic compounds have a higher rate of penetration than lipophilic compounds, although the latter penetrates grains more easily as the water content decreases (rowlands, 1967). there is limited information on the fate of esfenvalerate in storage grains, and only a few studies have been conducted on its predecessor, fenvalerate (joia et al., 1985). the amount of fenitrothion residues found in wheat grains with a preharvest interval of 120 days was three times higher than the mrl (1 mg kg−1) set by brazilian legislation, despite having been applied at less than (i.e., 62%) the manufacturer’s recommended dose of 10 mg kg−1. even in the wheat by-products from grains with a preharvest interval of 120 days, the levels of fenitrothion residues were higher than the mrl established for grains. among these by-products, bran deserves special attention because of its high affinity for pesticides due to its high oil content; consequently, after 120 days of treatment, the level of fenitrothion residues on bran was 450% higher than the mrl for grains. many people believe that wheat bran is a healthy food, without knowing about the health risks involved in the consuming bran that may not meet the required standards. the amount of insecticide residues found on wheat grains treated with 0.5 mg kg−1 of esfenvalerate was clearly lower than the mrl of 1 mg kg−1 for stored grains. if the only basis for evaluation is the comparison between the agronomic value (0.5 mg kg−1) and the mrl (1 mg kg−1), then grains may be immediately consumed after insecticide application. therefore, it is necessary to modify legislation to be more consistent with the appropriate use of insecticides. this issue was raised by trevizan and baptista (2000) in relation to deltamethrin, and sgarbiero et al. (2003) in relation to pirimiphosmethyl. additionally, the alimentarius codex of the fao has established an mrl of 0.5 mg kg−1 for esfenvalerate in wheat grains (fao, 2019). thus, the preharvest interval for this insecticide should last more than one year, making its postharvest use questionable. these results reveal the importance of conducting studies on pesticide dissipation to establish mrl values consistent with how these substances are actually used. this study demonstrates the necessity of conducting further studies on pesticide dissipation, to provide the regulators with the relevant information to craft suitable pesticide legislation that is appropriate for each country, with the goal of protecting the health of consumers. based on our findings, we recommend modifying the preharvest interval for fenitrothion to 180 days, to be consistent with the mrl of 1 mg kg−1. likewise, we vásquez-castro, j.; de baptista, g.; gadanha-junior, c.; bracho-pérez, j. peruvian journal of agronomy 4(2): 40-47 (2020) 45 table 4. fenitrothion residues (mg kg−1) in wheat flour and bran. substrate daa residues (mean ± sd) degradation index accumulated degradation index flour 0 2.65 ± 0.34 15 2.65 ± 0.48 1.00 1.00 30 1.89 ± 0.28 1.40 1.41 60 2.08 ± 0.33 0.91 1.28 90 1.47 ± 0.10 1.41 1.80 120 1.37 ± 0.06 1.07 1.93 180 0.63 ± 0.05 2.17 4.19 240 0.54 ± 0.03 1.17 4.88 360 0.28 ± 0.02 1.96 9.59 bran 0 12.34 ± 2.29 15 7.93 ± 0.96 1.56 1.56 30 6.92 ± 0.34 1.14 1.78 60 6.60 ± 0.54 1.05 1.87 90 6.35 ± 0.59 1.04 1.94 120 4.52 ± 0.34 1.41 2.73 180 4.60 ± 0.71 0.98 2.68 240 4.62 ± 0.30 1.00 2.67 360 3.01 ± 0.36 1.53 4.10 table 5. esfenvalerate residues (mg kg−1) in wheat flour and bran. daa: days after application. substrate daa residues (mean ± sd) degradation index accumulated degradation index flour 0 0.23 ± 0.03 15 0.23 ± 0.05 0.99 0.99 30 0.21 ± 0.03 1.11 1.10 60 0.33 ± 0.12 0.64 0.70 90 0.24 ± 0.02 1.36 0.96 120 0.25 ± 0.01 0.97 0.93 180 0.19 ± 0.01 1.30 1.21 240 0.19 ± 0.02 1.00 1.21 360 0.14 ± 0.02 1.33 1.60 bran 0 1.10 ± 0.14 15 0.46 ± 0.03 2.41 2.41 30 0.45 ± 0.01 1.01 2.43 60 0.36 ± 0.02 1.26 3.06 90 0.42 ± 0.04 0.85 2.60 120 0.38 ± 0.01 1.11 2.89 180 0.37 ± 0.06 1.04 3.00 240 0.38 ± 0.00 0.96 2.89 360 0.31 ± 0.03 1.21 3.51 daa: days after application. dissipation of fenitrothion and esfenvalerate in wheat grains, bran and flour may august 2020 46 recommend modifying the mrl of esfenvalerate to 0.35 mg kg−1, which is consistent with the required preharvest interval of 15 days. conclusions esfenvalerate is more persistent than fenitrothion in wheat grain, flour, and bran. maximum and minimum concentrations of both insecticides occur in bran and flour, respectively. current brazilian regulations of the usage of fenitrothion and esfenvalerate in stored wheat are not consistent with measured dissipation rates. references afridi, i.a.k., parveen, z. & masud, s.z. 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(2012). insecticidal effect and residual action of fenitrothion and esfenvalerate on sitophilus oryzae and s. zeamais (coleoptera: curculionidae) in stored maize and wheat. isrn agronomy, 2012, 1–11. watanabe, m., noguchi, m., hashimoto, t. & yoshida, s. (2018). chlorpyriphos-methyl, pirimiphos-metyl and fenitrothion residues in commercial wheat products. food hygiene and safety science, 59, 228–233. http://doi.org/10.3358/shokueishi.59.228 yu, ch., li, y., zhang, q., zou, n., gu, k., li, x. & pan, c. (2014). decrease of pirimiphos-methyl and deltamethrin residues in stored rice with post-harvest treatment. international journal of environmental research and public health, 11, 5372–5381. http:// doi.org/10.3390/ijerph110505372 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index obituary https://doi.org/10.21704/pja.v7i1.1865 received for publication: 27 april 2023 accepted for publication: 28 april 2023 published: 30 april 2023 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by miguel holle (1937-2023) roger chetelat1, carlos f. quiros1, eduardo vallejos2 *corresponding author: cfquiros@ucdavis.edu geneticist, plant explorer, germplasm conservationist and educator miguel holle passed away surrounded by his family in lima peru on april 11, 2023. he did his undergraduate work in horticulture at colorado state university, fort collins and obtained his phd at iowa state university under lincoln peirce. he started his academic career at universidad agraria la molina in lima peru in the early 1960’s where he taught courses in horticulture and vegetable breeding. miguel provided a nurturing environment for his students encouraging them to be critical thinkers and self-reliant. together with alfredo montes and gunter bushbek they organized a progressive research program on vegetable crops production, genetics and breeding and post-harvest at el huerto (the orchard) at la molina. his plant exploration days started in 1970 when charley rick asked him to organize an expedition to collect wild tomatoes in peru. this began a long friendship and collaboration between the two scientists spanning four decades, to collect and study the wild tomato relatives throughout south america. in his memoirs rick described their meeting as “we had fallen in with the most wonderful person, who was to be our friend, colleague, mentor, and most delightful travel companion, not only for these three months, but for many more trips and visits both in south america and in the us, and prospects for future adventures continuing. thus, by affiliating with miguel, we may have experienced the luckiest break of our entire tomato chasing experiences!”. miguel knew his country particularly well and was intimately familiar with the ecology and distribution of the wild tomato relatives, allowing the collection of natural populations from the coastal zone to the andean regions of peru, as well as chile and ecuador. as dr. holle’s former graduate students, we (cfq, ev) were able to participate in those collecting trips on more than one occasion. his participation in these numerous forays resulted in the collection of over 720 accessions, representing a large share of the wild material maintained by the cm rick tomato genetics resource center at uc davis. this national and international germplasm bank serves tomato researchers and breeders throughout the world. his work led to numerous research articles. in 1975 miguel spent a sabbatical year at the department of vegetable crops, at uc davis in the laboratory of dr. rick. after his return, he found the political situation in peru unfavorable for research and academic activities at the university, prompting him to start his international career. miguel first worked at catie in turrialba, costa rica for several years on tropical horticulture, and then moved to ciat in colombia to work on genetic resources. miguel returned to peru in the 1980’s to work in the city puno promoting the conservation and cultivation of native andean crops, in an internationally sponsored program. finally, he joined the international potato center (cip) where he managed the condesan program (consorcio para el desarrollo sostenible de la ecoregion andina) which provided research grants to researchers working on andean crops in peru, ecuador. and bolivia. at the end of his career, he co-authored a book with ricardo sevilla on conservation of genetic resources entitled “recursos geneticos vegetales.” miguel made an indelible impression on those with whom he interacted for his dedication to research and conservation of genetic resources, and foremost for his passion for teaching. he will be remembered as an excellent and charismatic teacher who was always willing to help his students but letting them find their own ways to attain their independence. we were extremely lucky to have had him as a mentor and later as a good friend and colleague. 1 dept of plant science*s, uc davis. 2 horticultural sciences department, university of florida, gainesville. how to cite this obituary: chetelat, r., quiroz, c., vallejos, e. (2023). miguel holle (1937-2023) obituary. peruvian journal of agronomy, 7(1), 82. https://doi.org/10.21704/pja.v7i1.1865 peruvian journal of agronomy 4(2): 55-60 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i2.1541 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 01 december 2019 accepted for publication: 18 july 2020 physicochemical and microbiological quality of honeys produced by stingless bees scaptotrigona polysticta, melipona illota and tetragonisca angustula (apidae: meliponini) in san martín, peru calidad físico química y microbiológica de las mieles producidas por las abejas sin aguijón scaptotrigona polysticta, melipona illota y tetragonisca angustula (apidae: meliponini) en san martín, perú marilena marconi1*; javier ormeño luna2; carlos daniel vecco giove1 *corresponding author: marilena.marconi@gmail.com https://orcid.org/0000-0001-6691-1921 abstract the honeys from stingless bees (hymenoptera: apidae: meliponini) are recognized for their medicinal properties. they are commonly used by many indigenous groups around the world. however, in peru stingless beekeeping is practiced in an artisanal way and the honeys remain products whose qualities are still little studied. the objective of this work was to analyze physicochemical characteristics and microbiological quality of honeys produced by scaptotrigona polysticta moure, melipona illota cockerell and tetragonisca angustula latreille in apiaries of department of san martin, peru. in june and november 2019, 30 honey samples were collected from 24 colonies housed in two apiaries in the towns of tarapoto and chasuta. significant differences (p <0,05) were found for ph (3,8 ± 0,6; 3,5 ± 0,6; 4,7 ± 0,6) and the percentage of total solutes (70,4 ± 2,5%; 61,2 ± 3,2%; 73,0 ± 2,7%), among the honey samples of s. polysticta, m. illota. and t. angustula, respectively. the color of s. polysticta honey was variable, from light amber to dark yellow (115 ± 23 mm pfund), while t. angustula honey was darker yellow (178 ± 33 mm pfund). values for mesophilic and anaerobic sulfite-reducing microorganisms were under the level of 1 cfu / ml for all species. fungi and yeasts exceeded the allowed value (> 10 cfu / ml) according peruvian sanitary norm № 071-minsa/digesa-v.01, in 30 and 50% of the samples of s. polysticta and t. angustula, respectively. these results contribute to the knowledge of the stingless bee honeys of peru providing important references for the quality standard setting process and the advancement of stingless beekeeping. keywords: meliponini, honeys, peruvian amazon, tarapoto, meliponiculture. resumen las mieles de abejas sin aguijón (hymenoptera: apidae: meliponini) son reconocidas por sus propiedades medicinales y comúnmente utilizadas por muchos grupos indígenas de todo el mundo. sin embargo, la crianza de abejas sin aguijón en el perú se practica de forma artesanal y las mieles son productos de cualidades aún poco estudiadas. el objetivo de este trabajo fue analizar las características fisicoquímicas y la calidad microbiológica de las mieles producidas por scaptotrigona polysticta moure, melipona illota cockerell y tetragonisca angustula latreille en apiarios del departamento de san martín, perú. en junio y noviembre de 2019 se recolectaron 30 muestras de miel de 24 colonias alojadas en dos colmenares en las localidades de tarapoto y chasuta. se encontraron diferencias significativas (p <0,05) para el ph (3,8 ± 0,6; 3,5 ± 0,6; 4,7 ± 0,6) y el porcentaje de solutos totales (70,4 ± 2,5%; 61,2 ± 3,2%; 73,0 ± 2,7%), entre las muestras de miel de s. polysticta, m. illota. y t. angustula, respectivamente. el color de la miel de s. polysticta varió de ámbar claro a amarillo oscuro (115 ± 23 mm pfund), mientras que lo de la miel de t. angustula se presentó amarillo 1 urku estudios amazónicos. tarapoto, perú. e-mail: marilena.marconi@gmail.com; https://orcid.org/0000-0001-6691-1921 e-mail: carvec2@gmail.com; https://orcid.org/0000-0003-2705-823x 2 universidad nacional de san martín, laboratorio de apicultura. tarapoto, perú. e-mail: javierol@unsm.edu.pe; https://orcid.org/0000-0003-3584-1451 cite this article: marconi, m., ormeño, j., & vecco, d. (2020). physicochemical and microbiological quality of honeys produced by stingless bees scaptotrigona polysticta, melipona illota and tetragonisca angustula (apidae: meliponini) in san martín, peru. peruvian journal of agronomy, 4(2):55-60. http://dx.doi.org/10.21704/pja.v4i2.1541 physicochemical and microbiological quality of honeys produced by stingless bees scaptotrigona polysticta, melipona illota and tetragonisca angustula (apidae: meliponini) in san martín, peru may august 2020 56 más oscuro (178 ± 33 mm pfund). los valores de microorganismos mesófilos y anaerobios sulfitos reductores estuvieron por debajo del nivel de 1 ufc / ml para todas las mieles. los hongos y levaduras excedieron el valor permitido (> 10 ufc / ml) según la norma sanitaria peruana № 071-minsa/digesa-v.01, en 30 y 50% de las muestras de s. polysticta y t. angustula, respectivamente. estos resultados contribuyen al conocimiento de las mieles de abejas sin aguijón del perú, al proporcionar referencias para establecer estándares de calidad que promuevan el avance de la meliponicultura. introduction meliponini (hymenoptera: apidae), commonly called stingless bees, are important pollinators that inhabit the tropical and subtropical areas of the world (yáñez-ordóñez et al., 2008). in the amazon region, these insects would pollinate 38% of all plant species (kerr et al., 2001). since the time of the maya, the meliponini were managed for production of honey, whose antioxidative (cauich et al., 2015), antiseptic, antimicrobial, anticancer and anti-inflammatory properties have been proven in several studies (alvarez-suárez et al., 2012; silva et al., 2013). nowadays, stingless beekeeping or “meliponiculture” remains a widespread practice in many latin american countries (nates-parra & rosso-londoño, 2013; paris et al., 2018). despite the importance attached to their honeys, little has been done to value the resource (de oliveira, 2013). the introduction of stingless bee honey to the market is limited by the low knowledge of the characteristics that define the quality, as well as the absence of regulations and technical procedures that define the standards. in peru, about 170 species of stingless bees have been reported (rasmussen & gonzalez, 2009) and meliponiculture is practiced in an artisanal way by native peoples. melipona illota cockerell, tetragonisca angustula latreille and scaptotrigona spp. moure are commonly raised (rasmussen & castillo, 2003). the quality standards for honey of exotic honeybee (apis mellifera linnaeus 1758) have been normed in peru (ntp 209.168) (instituto nacional de defensa de la competencia y de la protección de la propiedad intelectual [indecopi], 2014), while stingless bee honey remains an unknown product. to date, there is hardly a single scientific work to characterize it (rodríguez-malaver et al., 2009; red de seguridad alimentaria consejo nacional de investigaciones científicas y técnicas [rsa-conicet], 2018). the purpose of this study was to determine the physicochemical characteristics and microbiological quality of the honeys produced by scaptotrigona polysticta, melipona illota and tetragonisca angustula in apiaries of san martin, peruvian amazon. materials and methods procedure in the apiaries. the study was carried out in two apiaries in the localities of tarapoto and chasuta, a transitional area of the tropical dry-humid forest in huallaga river basin of san martin, peru (figures 1-2). in june and november 2019, 30 honey samples of s. polysticta figure 1. location of the apiaries. 2a 2b figure 2. 2.a meliponario chasuta. 2.b meliponario tarapoto. (n1 = 15), m. illota (n2 = 4) and t. angustula (n3 = 11) were collected from 24 colonies (table 1) reared in modern hives. sterile syringes and a basic glove, lab coat and mask were used to extract the honey. each sample consisted of the total amount of honey extracted from ten ripe pots randomly chosen within the colonies. the samples were m. marconi; j. ormeño; d. vecco peruvian journal of agronomy 4(2): 55-60 (2020) 57 tarapoto chasuta s. polysticta 15 0 m. illota 4 0 t. angustula 6 5 table 1. number of honey samples collected by apiary. stored at a temperature of 4 °c. in the apiary of chasuta were present only beehives of the species tetragonisca angustula. chemical-physical analysis. the analyzes were carried out in the takiwasi laboratory, in tarapoto (http://www. laboratorio.takiwasi.com/). hydrogen potential (ph) was measured with a ph meter (hanna brand, model hi 8424); the total polyphenol content with a spectrophotometer (unico brand), according to the procedure described by meda et al. (2005); and the color, determined according to instituto argentino de normalización y certificación (iram, 2007), by means of a pfund kohler colorimeter and a spectrophotometer (unico ®). the standards of the united states department of agriculture (usda, 1985) were used to establish the correspondence among mm pfund values and color. a field brixometer (0 90% brix) (texim ®, model tr-099) was used to measure the total solutes. color and total polyphenols of m. illota honey have not been determined due to insufficient sample volume for this analysis. microbiological analysis. the colony-forming units (cfu / ml) were counted in plates for: a) aerobic mesophilic organisms, b) anaerobic sulfite-reducing agents (cf. clostridium perfringens), c) molds and yeasts, according to the following standards from international organization for standardization (iso): 4833-1 (iso, 2019), 15213 (iso, 2003) and 7937 (iso, 2004), respectively. the results were compared with the maximum values allowed by the peruvian sanitary technical standard nts № 071-minsa/ digesa-v.01 (ministerio de salud, [minsa], 2008). data analysis the data are expressed as mean ± standard deviation. a t-student test was applied for comparison of means. the statistical analyses were performed using analysis toolpak for microsoft excel 2019 (microsoft corporation, redmond, usa). a p-value <0,05 was considered significant. results chemical-physical quality. the honeys showed differences in the parameters ph, percentage of total solutes, color, and total polyphenols (table 2). t. angustula honey presented the highest ph value (4,7 ± 0,6) and a high total solute content (73,0 ± 2,7%), compared to the other species. the s. polysticta honeys showed a strong variation of color: from light amber to dark yellow, while the samples of t. angustula were all dark yellow (table 3). total polyphenols were higher in t. angustula honeys compared to that observed for s. polysticta samples (table 4). microbiological quality. honey samples from s. polysticta, m. illota and t. angustula were free of bacterial contamination; but 30% and 50% of the samples of s. polysticta and t. angustula, respectively, exceeded the maximum threshold allowed by the peruvian sanitary standard for molds and yeasts (table 5). discussion the results of this study are consistent with those obtained by other authors (souza et al., 2006; fuenmayor et al., 2012; vit et al., 2016; lemos et al., 2017; rsa-conicet, 2018; grajales-conesa et al., 2018) in terms of acidity and total solute content. however, the methods of analysis used in the different scientific papers could be distinct, so it would be difficult to make a comparison among our results and another one. in general, the honeys of stingless bees have been characterized by low ph, because the presence of different acids (gluconic, acetic, lactic, citric, succinic, formic, malic, maleic and oxalic), and a reduced total solute content, if compared with apis mellifera honey (vit et al., 1998). n ph % total solutes s. polysticta 15 3,8 ± 0,6 b 70,4 ± 2,5 b m. illota 4 3,5 ± 0,6 c 61,2 ± 3,2 c t. angustula 11 4,7 ± 0,6 a 73,0 ± 2,7 a note: different letters indicate significance for p <0,05. note: different letters indicate significance for p <0,05. species n mm pfund range color (spectrum) s. polysticta 6 115 ± 23 ª 77 141 light ambera m b e r d a r k yellow t. angustula 5 178 ± 33 ᵇ 147 216 dark yellow species n tpc (mg/100g) s. polysticta 6 8,9 ± 0,8 ª t. angustula 5 12,2 ± 2,8 ᵇ note: different letters indicate significance for p <0,05. table 2. main chemical parameters of samples (n) of honey from s. polysticta, m. illota. and t. angustula (one day after harvest; conservation at 4 ° c). table 3. values of mm pfund and color of samples (n) of honeys of s. polysticta and t. angustula (seven days after harvest; conservation at 4 ° c). table 4. total polyphenol content (tpc) in mg / 100 g for samples (n) of s. polysticta and t. angustula honeys (seven days after harvest; storage at 4 ° c). physicochemical and microbiological quality of honeys produced by stingless bees scaptotrigona polysticta, melipona illota and tetragonisca angustula (apidae: meliponini) in san martín, peru may august 2020 58 the color of honey, which can usually vary from practically transparent to almost black, reflects the amount of carotenoid, chlorophyll, and xanthophyll pigments (suescún & vit, 2008). the darkest hue of the honeys of t. angustula could be related to the major presence of minerals and phenolic compounds (instituto nacional de tecnología agropecuaria [inta], 2019), as well as calcium and iron phosphate; while the light colored honeys of s. polysticta would prove to be richer in vitamin a (zandamela, 2008). phenolic compounds are not only a marker of the floral origin of the honey, but also potential indicators of their biological activity (quiñones et al., 2012). in this study, the total polyphenols were much lower than those recorded for the same bee species by rodríguez-malaver et al. (2009). however, due to the lack of information about the area of origin and the season of extraction of the samples in this investigation, it was not possible to speculate on the causes of these differences. microorganisms in the honey can originate by primary sources, when they come from the nectar or for influence of the bees themselves, and inoculate by secondary sources, when operating conditions influence the collection, transport and storage of honey (pucciarelli et al., 2014). absence of mesophiles and sulfite-reducing anaerobic bacteria in all the analyzed samples would indicate the optimal microbiological quality of the honeys. although the high water content of stingless bees honeys could favor the growth of molds and yeasts (gil, 2010), the causes of this presence in the samples of s. polysticta and t. angustula deserve to be studied in depth, without ignoring presence of yeasts in the “bees bread” (portillo, 2016), the conservation and sanitation conditions of inert laboratory surfaces (sarmiento et al., 2014) in storage (ascencio, 2014) and harvest protocols (rsa-conicet, 2018). it assumes that several causes could have concurred to observe significant differences among physicochemical parameters of honeys from s. polysticta, m. illota and t. angustula: changes in floral origin of the honey, the kinds of materials used by different species, the processes of production and storage used for each species, would be considered as the main factors to define the quality of the honey (de oliveira & santos, 2011). anyhow, these aspects were not part of our study, being a preliminary analysis on the quality of the honeys of some stingless bee species commonly reared in the san martín region. we believe it should be the following step to deepen the knowledge on the nature of honeys from peruvian stingless bees. conclusions the results of this research agree with those obtained in other countries and the absence of bacteria in all the samples analyzed would indicate an optimal microbiological quality. however, the presence of mold and yeast in the s. polysticta and t. angustula samples deserves to be investigated. the characterization of physicochemical parameters and the microbiological quality are essential to define for the process of setting quality standards of the honeys, providing the reference values. due to the scant knowledge about the stingless bee honeys in peru, this study represents a starting point for new research aimed at enhancing stingless bee honeys and boosting the meliponiculture in the country. acknowledgments to the research and development institute (iid) of the universidad nacional de san martín (unsm), for financing the research. to the cooperative mushuk runa ltda. and their meliponicultures. to basilia miriam fernández argudín, for the review of the article. to the takiwasi laboratory for the analysis of honey samples. to the team of estudios amazónicos and césar guerra saldaña. references alvarez-suarez, j. m., giampieri, f., gonzalez-paramas, a. m., damiani, e., astolfi, p., & martínez–sánchez, g. 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[doctoral dissertation, universidad autónoma de barcelona]. uab repository. https://www.tdx.cat/ handle/10803/5701 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 23 september 2020 accepted for publication: 20 january 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina doi: http://dx.doi.org/10.21704/pja.v5i1.1676 comparison of organic carbon from humic and fulvic acids and the degree of humification in five soil orders comparación del carbono orgánico proveniente de los ácidos húmicos, ácidos fúlvicos y el grado de humificación en cinco órdenes de suelos sandro sardón nina1*; raúl d. zapata hernández2; luis a. arias lópez2 *corresponding author: ssardonnina@gmail.com *https://orcid.org/0000-0002-1999-1655 abstract this study compared the organic carbon (oc) content in fractions of humic acids (ha) and fulvic acids (fa) in five soil orders (aridisol, entisol, histosol, inceptisol and mollisol) and know their association with the degree of humification. extraction and fractionation, as well as the degree of humification was carried out by the nagoya method proposed by kumada. oc quantification was determined by the walkley and black method. the results are: the average oc content of has of the order aridisol differs from that of histosols, inceptisols, and mollisols. the order entisol presented differences with the histosols and mollisols, the soils of the order inceptisol presented differences with the aridisols and histosols and those of the order histosol differed from the aridisols, entisols and inceptisols. similarly, those of the mollisol order differed with the aridisols, entisols and inceptisols. in the fraction of fa the average oc content of the order aridisol deferred from that found in histosols, inceptisols and mollisols. the entisol order differed from the mollisols; likewise, the inceptisol order differed from the aridisols and mollisols and the histosol order differed from the aridisols. finally, the order mollisol was also different from the aridisols, entisols, and inceptisols. soil types do not show wetting patterns, because they are not based on pedogenetic processes and these have a wide range of characteristics in surface horizons. keywords: humus fractions, soils orders, humic substances. resumen en este estudio se realizaron las comparaciones del contenido de carbono orgánico (co) en las fracciones de los ácidos húmicos (ah) y los ácidos fúlvicos (af) en cinco órdenes de suelos (aridisol, entisol, histosol, inceptisol y mollisol) y conocer su asociación con el grado de humificación. la extracción y fraccionamiento, así como el grado de humificación se realizó por el método nagoya propuesta por kumada. la cuantificación del co se determinó por el método walkley y black. los resultados son: el contenido promedio de co de los ah del orden aridisol difiere del de los histosoles, inceptisoles y mollisoles. el orden entisol presentó diferencias con los histosoles y mollisoles, los suelos del orden inceptisol presentaron diferencias con los aridisoles e histosoles y los del orden histosol se diferenciaron de los aridisoles, entisoles e inceptisoles. de la misma forma, los del orden mollisol difirieron con los aridisoles, entisoles e inceptisoles. en la fracción de los af el contenido promedio de co del orden aridisol difirió del encontrado en los histosoles, inceptisoles y mollisoles. el orden entisol se diferenció de los mollisoles; así mismo, el orden inceptisol difirió de los aridisoles y mollisoles y el orden histosol se diferenció de los aridisoles. por último, el orden mollisol se 1 universidad nacional del altiplano, puno, perú 2 universidad nacional de colombia, medellin, colombia how to cite this article: sardón, s., zapata, r., & arias, l. (2021). comparison of organic carbon from humic and fulvic acids and the degree of humification in five soil orders. peruvian journal of agronomy, 5(1), 25–34. http://dx.doi.org/10.21704/pja.v5i1.1676 patricia sello comparison of organic carbon from humic and fulvic acids and the degree of humification in five soil orders january april 2021 26 introduction the soil organic matter (som) comprises a mixture of residues of plants, microbes, and animals at various stages of decomposition and heterogeneous organic substances closely associated with the mineral fraction (kononova, 1975; christensen, 1992; zaccone et al., 2018; osorio, 2018; gallardo, 2016). according to kononova (1966), this complex process involves two groups: 1. the decomposition of the original components of the tissues and their conversion by microorganisms into simpler chemical compounds identifiable by organic chemistry (proteins and amino acids, simple carbohydrates and compounds, resins, fats, lignins, and others) and partially into products of complete mineralization (co2 production by breath, no2, no3, nh3, ch4, h2o and others). 2. synthesis of organic compounds with the formation of high molecular weight humic substances. this set of processes are called humification and produces a mixture of substances that have high resistance to subsequent microbial attack and are completely different from any vegetable or animal substance (duchaufour, 1987). in this way, the soil organic carbon (soc) content is the component of som. on earth, the soc content of the upper horizons of soils (epipedons) contains between 1200 pg to 1500 pg of c (1 pg = 1015 g = 1012 kg = 109 t) (gallardo & merino, 2007). although the organic carbon of humic substances or humus accounts for 60 % to 80 % of soc (piccolo et al., 2018; ismailmeyer, 2018). kumada (1987) was able to obtain information from ha molecules, such as functional groups, elemental composition, and degree of humification. for the degree of humification, he proposes to distinguish four types of humic acids a, b, rp, and p. this can be known through the formation of humic acids, in which the start of the humification process begins with the type rp (first humification states of organic material), evolving to type b and finally to type a (each type exhibits a relatively stable form). in strongly acidic soils, the rp type can be replaced by the p type. the brown to black component (humic substances) refers to humic acids (ha), fulvic acids (fa) and humins (hm) which represent the most recalcitrant and stable organic carbon reservoir from the microbial point of view (el-metwally et al., 2014; duchaufour, 1987). humus or humic substances are the product of the pedogenetic process (simonson, 1959; fanning & fanning, 1989; zapata, 2001, 2006, 2014; buol et al., 2011). a slight change in the recalcitrant soil organic carbon reserve will have a major impact on the concentration of atmospheric carbon dioxide (co2) (li et al., 2019). the objective of this research was to compare the composition of organic carbon content in the fractions of humic acids (ha) and fulvic acids (fa). also, to know if the degree of humification (a, b, p and rp) in soils is related to soil orders (aridisol, entisol, histosol, inceptisol, and mollisol). materials and methods description of the study area the study was conducted in the district of san rafael, ambo province, huanuco department of peru. this area covers an area of 44,189.73 ha, located between the coordinates: lower-right end is 10’27’41.83’’ s and 76’1’32.14’’ o; and the top-left end is 10’12’17.43’’ s and 76’15’16.33’’ o, datum wgs-84 (figure 1). the 42 simple samples were analyzed in the soil laboratory of the school of geosciences of the faculty of sciences of the universidad nacional de colombia, medellin. the study area is located between 2100 masl and 4890 masl. the average monthly air temperature in the low and medium area (2100 masl to 3500 masl) ranges from 12 °c to 20 °c, in the high areas (3,500 masl to 4,200 masl) it ranges from 8 °c to 16 °c, and in areas above 4,200 masl the temperature ranges from 4 °c to 10 °c (ministerio de agricultura y riego [minagri], 2016). soils were classified according to the keys to soil taxonomy (soil survey staff, 2014). in the san rafael district, the orders aridisol, entisol, histosol, inceptisol, and mollisol were identified (figure 2). diferenció también de los aridisoles, entisoles e inceptisoles. los tipos de suelo no muestran patrones de humificación, debido a que no se basan en los procesos pedogenéticos y éstos presentan una amplia gama de características en los horizontes superficiales. palabras clave: fracciones del humus, ordenes de suelos, sustancias húmicas. s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 25–34 (2021) 27 these soils developed from weathering profiles with highly variable evolution, from incipient to evolved, formed from sedimentary rocks (sandstones, lutites, limestones, and clusters), metamorphic rocks (shale), plutonic rocks (diorite), and from deposits of alluvial, colluvial, and glacial materials. the district presents a mountainous relief of slopes or long and inclined slopes with narrow valleys that present flat accumulation bottoms. sampling forty-two simple samples were collected in the different soil orders (table 1). the sampling points were distributed in a random way supported by the previous results of soil organic matter and according to the area that corresponds to it. the samples were taken from the first horizon, their morphological characteristics were described and those of the surrounding relief, also have been georeferenced with a gps navigator. figure 1: location of the research area. aridisol entisol histosol inceptisol mollisol figure 2: soil orders in the san rafael district. comparison of organic carbon from humic and fulvic acids and the degree of humification in five soil orders january april 2021 28 of naoh + 0.1m na2p2o7.10h2o (soil to extracting ratio, 1:300, w/v), this ratio depends on the organic carbon content in the soil. it is then boiled (100 oc) for 30 min by shaking the erlenmeyer several times. after boiling 1 g of na2so4 is added as a coagulating agent, after cooling in a water bath plus ice, it was centrifuged at 11,000 rpm by 15 min. extraction and fractionation of organic matter from the soil the extraction and fractionation of organic matter from the soil were carried out by the nagoya method, described by kumada (1987). soil samples dried outdoors and sifted. mos fractionation was performed by mixed extraction with two basic solutions at 0.1n soil sample land order (soil survey staff) south latitude west length m1 aridisol 10°17’32.7’’ 76°9’32.6’’ m2 10°16’57.7’’ 76°9’38.7’’ m3 10°17’18.1’’ 76°9’32.9’’ m4 10°17’49.6’’ 76°9’54.1’’ m5 entisol 10°18’5.6’’ 76°9’42.4’’ m6 10°14’51.9’’ 76°7’34.9’’ m7 10°21’44.3’’ 76°11’58.8’’ m8 10°20’10.3’’ 76°11’9.8’’ m9 10°14’8.7’’ 76°10’3.7’’ m10 10°14’4.7’’ 76°10’15.7’’ m11 10°14’12.8’’ 76°11’30.4’’ m12 10°14’20.3’’ 76°5’50.2’’ m13 10°20’50.7’’ 76°13’21.9’’ m14 10°19’23.8’’ 76°11’56.5’’ m15 10°14’47.1’’ 76°7’52.3’’ m16 10°16’3.0’’ 76°7’40.6’’ m17 10°18’11.9’’ 76°6’49.9’’ m18 histosol 10°14’26.9’’ 76°5’31.5’’ m19 10°14’34.9’’ 76°5’20.0’’ m20 10°18’44.6’’ 76°14’33.4’’ m21 10°19’49.5’’ 76°3’2.7’’ m22 10°19’50.3’’ 76°2’30.9’’ m23 10°20’1.1’’ 76°4’38.6’’ m24 inceptisol 10°15’14.8’’ 76°9’19.9’’ m25 10°18’26.4’’ 76°9’52.8’’ m26 10°13’33.0’’ 76°6’37.8’’ m27 10°19’1.1’’ 76°11’21.3’’ m28 10°15’25.4’’ 76°7’39.9’’ m29 10°14’19.8’’ 76°6’18.4’’ m30 10°14’36.7’’ 76°6’46.8’’ m31 10°18’10.4’’ 76°6’53.1’’ m32 10°21’32.9’’ 76°13’7.7’’ m33 10°20’24.1’’ 76°7’22.6’’ m34 10°21’36.4’’ 76°6’56.9’’ m35 10°23’0.5’’ 76°4’50.1’’ m36 10°23’15.7’’ 76°6’48.3’’ m37 mollisol 10°20’49.6’’ 76°13’57.8’’ m38 10°20’59.3’’ 76°14’43.3’’ m39 10°19’47.3’’ 76°15’11.4’’ m40 10°19’21.7’’ 76°14’48.8’’ m41 10°19’31.4’’ 76°14’42.0’’ m42 10°19’45.7’’ 76°14’40.2’’ table 1: location of the sampled points. s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 25–34 (2021) 29 the alkaline extract was then decayed and the soil residue was washed twice with 20 ml of extracting agent containing na2so4 by centrifugation, as in the previous step. once the soluble extract and washes were combined, the extract was acidified with a concentration of h2so4 (1 ml:100 ml) and left to rest for 30 min. subsequently, acidified extracts were filtered through the filter (toyo roshi no. 5c) into a 100 ml volumetric erlenmeyer, then the precipitate is washed with h2so4 (ratio, acid to water, 1 ml:100 ml) and the filtered volume (fa) to 100 ml is completed. the precipitated ha (on the filter paper) is dissolved with 0.1n naoh, the solution is collected in an erlenmeyer of 100 ml or 250 ml volume (depending on the ha content). chemical characterization of humic acids the characterization of humic acids was performed according to kumada (1987). this system groups has into four types: a, b, p, and rp, according to their position in the orthogonal axis diagram whose coordinates correspond to the rf and ∆log k parameters. these parameters are obtained by the following expression: ∆log k = log k400 – log k600 rf = k600 x 1,000/c where: log k400 and log k600 is the optical density of an ha solution at 400 nm and 600 nm, both spectrum ranges are obtained with a genesys visible spectrophotometer™ 20. c = volume in ml of 0.1n of kmno4 consumed by 30 ml of ha solution used to determine the absorption spectrum. in this investigation, kmno4 was replaced by 0.1n of k2cr2o7. the humification process begins with the rp type (first humification states of organic matter), evolving to type b, and finally to type a (each type exhibits a relatively stable form). in strongly acidic soils, the rp type can be replaced by p type soils. quantification of organic carbon the organic carbon (oc) content in humic and fulvic acids was determined by the wet combustion method by walkley and black (1934). the organic forms of soil c oxidize in the presence of excess dichromate in the middle of a strong acid. after the c oxidation step, at the reaction temperature for a certain amount of time, the non-reduced cr+6, added in excess, was titrated with ferrous sulfate fe+2. the difference between these two states of cr oxidation is equal to the organic carbon content of the sample (allison et al., 1965; walkley, 1947). statistical analysis the organic carbon results in the ha and fa were determined by mean comparison analysis with the t student test. to know the differences in the predominance of the degree of humification in the soils were calculated by pearson’s chi-square independence test. these tests used a significance level of (p < 0.05) according to r software. results and discussion the results of the quantification of organic carbon from humic acids (ha) and fulvic acids (fa), and the determination of the degree of humification in five soil orders, are summarized in table 2. comparison of the organic carbon content of ha and fa in soils the dispersion or variability of organic carbon content in ha and fa fractions in the soils studied are presented in boxplot (figure 3). in the fraction of the has (figure 3a), the order histosol and mollisol show greater dispersion of the values, while in the fraction of the fa (figure 3b), the order histosol has greater dispersion. the soils of the order mollisol have a dark horizon (epipedon) and without diagnostic sub horizon (endopedon) expressed in an ap/ac/c type profile located on the slopes or mountain slopes, while the order histosol presents a histic epipedon that is expressed with highly decomposed surface horizons and with ustic, udic, and acuic moisture regimes; developed from morrenic deposits, metamorphic rocks, plutonic rocks, and organic materials. both soils are located in very diverse contexts that include the life zones of very humid paramo and pluvial paramo (minagri, 2016). according to the soil survey staff (1999) each order is differentiated by the presence or absence of diagnostic horizons or characteristics that reflect the formation processes, thus being very heterogeneous with respect to properties. therefore, the organic matter content and nature of soils is highly variable (stevenson, 1994; lindsay, 1979; spain et al., 1983). comparison of organic carbon from humic and fulvic acids and the degree of humification in five soil orders january april 2021 30 the average content of oc in the ha in the soils of the aridisol order is 0.13 g 100 g-1, in entisol 0.27 g 100 g-1, in histosol 5 .56 g 100 g-1, in inceptisol 0.93 g 100 g-1 and mollisol is 2.82 g 100 g-1. the average content of oc in the fa is slightly lower than that of has (figure 4). the order aridisol is 0.10 g 100 g-1, in entisol 0.22 g 100 g-1, in histosol 1.43 g 100 g-1, in inceptisol 0.34 g 100 g-1 and in mollisol is 0.73 g 100 g-1. previous studies, conducted by several authors, have emphasized that prairie soils (mollisol) and table 2: organic carbon content from humic and fulvic acids, and the degree of humification in different soil orders. soil sample land order (soil survey staff) organic carbon in the ha organic carbon in fa rf ∆log k type of ha (g 100 g-1) (g 100 g-1) m1 aridisol 0.05 0.05 75 0.57 b m2 0.15 0.10 23 0.67 p m3 0.15 0.05 57 0.68 b m4 0.15 0.20 34 0.56 p m5 entisol 0.15 0.15 42 0.64 p m6 0.20 0.35 60 0.67 b m7 0.10 0.05 45 0.55 p m8 0.15 0.05 26 0.65 p m9 0.15 0.10 41 0.66 p m10 0.05 0.05 62 0.55 p m11 0.10 0.05 70 0.63 b m12 1.00 1.50 53 0.45 p m13 0.15 0.05 28 0.70 p m14 0.15 0.10 66 0.54 p m15 0.75 0.10 18 0.55 p m16 0.15 0.10 61 0.62 b m17 0.45 0.20 24 0.61 p m18 histosol 6.00 1.75 29 0.55 p m19 3.75 1.25 20 0.54 p m20 2.10 0.30 26 0.57 p m21 11.00 3.50 10 0.96 rp(2) m22 5.25 1.00 29 0.51 p m23 5.25 0.75 23 0.50 p m24 inceptisol 0.20 0.20 35 0.63 p m25 0.15 0.30 73 0.69 p m26 1.20 0.10 15 0.58 p m27 0.05 0.05 17 0.63 p m28 0.90 0.25 17 0.57 p m29 0.25 0.30 80 0.58 b m30 2.10 0.50 26 0.59 p m31 0.15 0.20 123 0.62 a m32 0.15 0.05 77 0.56 b m33 0.60 0.05 20 0.62 p m34 0.90 0.40 29 0.53 p m35 1.00 1.00 76 0.52 p m36 4.50 1.00 23 0.62 p m37 mollisol 0.15 0.35 104 0.64 a m38 2.50 0.75 27 0.53 p m39 4.50 0.50 13 0.57 p m40 1.50 0.50 51 0.50 p m41 3.00 0.75 20 0.54 p m42 5.25 1.50 29 0.50 p s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 25–34 (2021) 31 peat soils (histosol) have higher ha content than fa content (kononova, 1966; gallardo, 2016). also, stevenson (1994) and zamboni et al. (2006) found that mollisol soils have high ha content than other soil types. in peru, león (2016) reports higher organic carbon content in humic substances in six high andean wetland soils (histosol) located in the departments of ancash, huancavelica, arequipa, and puno. the high content oc in soils does not guarantee the quality of humus or soil organic matter. when comparing the oc content in the ha and fa fractions (table 3 and table 4), the aridisol and entisol soils have no significant difference. aridisol soils do not have water available for most of the time so they are warm enough (soil survey staff, 1999, 2014; buol et al., 2011; jaramillo, 2014), these soils have an ochric epipedon and are located in the mountainous steppe life zona – tropical low montane and evapotranspiration exceeds precipitation for most of the year (minagri, 2016). generally, in these soils, microbial populations are scarce, due to the limited amounts of water available in the soil profile, the reactions are relatively less intense (buol et al., 2011). the soils of the order entisol can be presented in a wide diversity of climates, on very variable slopes, some of them very pronounced and associated with very diverse natural plant cover. the absence of characteristic features of the different pedogenetic processes is expressed in these soils (soil survey staff, 1999) and presents an ochric epipedon (minagri, 2016). these conditions of variability can influence the organic carbon content as well, presenting minimum carbon content in the ha and fa fractions. while the order aridisol presents differences with the orders of histosol, inceptisol, and mollisol. figure 3: box plots of carbon content from humic acids (a) and fulvic acids (b) in five soil orders. figure 4: distribution of fractions of humic acids (ha) and fulvic acids (fa). a) oc content in ha b) oc content in fa comparison of organic carbon from humic and fulvic acids and the degree of humification in five soil orders january april 2021 32 the contents of the fraction of the ha in the entisol and inceptisol orders are similar. this may respond that both orders have a wide range of characteristics such as one or more pedogenetic horizons and develop over a wide range of climates (soil survey staff, 1999). comparison of the degree of humification in soils according to table 2 and figure 5, two ha samples are type p and two ha samples are type b from the arid sun order, being the degree of immature humification and evolving into the mature form. the mature form in aridisol occurs in many semi-arid regions, which have lower concentration values of c, mos is highly transformed and resilient to possible climate change (gallardo, 2016). in the inceptisol order, ten ha samples are type p, two ha samples are type b and one ha sample is type a, their degree of maturation being immature to mature. this immature to mature state may coincide with soil development. the mollisol order consists of five type p ha samples and one type a ha sample. the degree of humification corresponds to the immature and mature states. the immature state corresponds to anthropically intervened soils, which have superficial horizons of ap. the type a humification grade is presented in soils with horizon a (soils in grasslands), without anthropic intervention. in these soils of the order mollisol, humic acids are often preserved for relatively long periods (>3000 years), linked to clayhumus complexes (tsutsuki et al., 1988; bockheim, 2014). in these soils, it is possible to detect highly moistened humified humic acids in the outer horizons. when performing pearson’s chi-square independence test, the soil variables and degree of humification are independent or not associated (p = 0.266, being non-significant). conclusion the work carried out, establishes the comparison of the organic carbon content in the fractions of humic acids (ha), fulvic acids (fa) and the degree of humification for five soil orders in the region of san rafael (peru). the most significant results are as follows: 1) in the order aridisol, the low availability of water for most of the time and where evapotranspiration exceeds precipitation, constitute conditions very unfavorable to the development of pedogenetic processes. therefore, it is inferred that minimum organic carbon content is presented in ha entisol histosol inceptisol mollisol aridisol p 0.09 ns p 0.01 * p 0.03 * p 0.02 * entisol p 0.01 * p 0.08 ns p 0.02 * histosol p 0.01 * p 0.08 ns inceptisol p 0.02 * table 3: comparison of means of organic carbon in humic acids (ha), in the five soil orders. note: ns: not significant; * p < 0.05 table 4: comparison of means of organic carbon in fulvic acids (fa), in the five soil orders entisol histosol inceptisol mollisol aridisol p 0.32 ns p 0.03 * p 0.03 * p 0.01 * entisol p 0.05 ns p 0.40 ns p 0.02 * histosol p 0.06 ns p 0.20 ns inceptisol p 0.04 * note: ns: not significant; * p < 0.05 figure 5: distribution of the degree of humification in the different soil orders. s. sardón; r. zapata; l. arias peruvian journal of agronomy 5(1): 25–34 (2021) 33 and fa fractions and differs from other soil orders. 2) in the soils of the order entisol and inceptisol, it has characteristics of a wide diversity of climates, inclined slopes, various types of vegetation that lead to the incipient formation of pedogenetic horizons. therefore, they have a minimal carbon content in humic fractions. 3) in the order, mollisol, located under a cover of pastures that annually provide a large amount of organic matter, forming clay-humus complexes, leads to the development of highly moistened humified humic acids and high carbon content in humic fractions. 4) in the order histosol, due to continuous input, accumulation of organic matter, and cold climates delay the humiliation process. therefore, they have a high carbon content in the humic fraction and the recent start of the humification process. finally, soil types are not associated or do not show wetting patterns, which may be because the distinctions made when classifying them are not based on pedogenetic processes and these have a wide range of characteristics in surface horizons; 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(2014). los procesos químicos del suelo. universidad nacional de colombia, medellín, colombia. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 24 february 2021 accepted for publication: 28 november 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina https://doi.org/10.21704/pja.v5i3.1845 effects of seedling thickness on the production and quality of onion (allium cepa l.) ‘santa rita’ in arequipa, peru efecto del grosor de las plántulas en la producción y calidad de la cebolla (allium cepa l.) cv. santa rita en arequipa, perú diego almeyda carbajal1; andrés virgilio casas díaz1*; mirna zuzunaga bedón1 *corresponding author: cda@lamolina.edu.pe *https://orcid.org/0000-0001-7461-3924 abstract onion crop begins with seedling preparation and finishes with transplanting. in some peruvian onionproductive areas, it is assumed that seedling thickness is important to have a better yield. four different seedling thickness of red onion (allium cepa l.) were evaluated between february and june 2017 in santa rita de siguas, arequipa, peru. the seedling thicknesses evaluated were very thin (2.00 mm – 3.49 mm), thin (3.50 mm – 4.99 mm), standard (5.00 mm – 6.49 mm) and thick (6.50 mm – 7.99 mm). the plant density was 340 000 plants ha−1. the experimental design was a randomized complete block with four treatments and five replications. the variables evaluated were plant height (cm), leaf number, stemlike diameter (mm), bulb diameter (mm), total yield (t ha−1), and marketable yield categories (t ha−1). the leaf number, plant height, and stemlike diameter among treatments were significantly different, with higher values in the “standard” and “thick” treatments up to 60 days after transplanting. the harvest was earlier in the “standard” and “thick” treatments. the “very thin” and “thin” treatments needed more days to harvest than the others. the “thin” treatment showed the highest total yield. there were no significant differences between marketable yield categories in all treatments. it was concluded that seedling thickness upon transplanting influences the yield under the conditions in this study. keywords: onion, seedling, thickness, yield, quality resumen el cultivo de la cebolla comienza con la preparación de las plántulas y termina con el trasplante. en algunas zonas peruanas productoras de cebolla, se asume que el grosor de las plántulas es importante para tener un mejor rendimiento. se evaluaron cuatro diferentes espesores de plántulas de cebolla roja (allium cepa l.) entre febrero y junio de 2017 en santa rita de siguas, arequipa, perú. los espesores de plántulas evaluados fueron muy fino (2.00 mm – 3.49 mm), fino (3.50 mm – 4.99 mm), estándar (5.00 mm – 6.49 mm) y grueso (6.50 mm – 7.99 mm). la densidad de plantas fue de 340 000 plantas ha-1. el diseño experimental fue un bloque completo al azar con cuatro tratamientos y cinco repeticiones. las variables evaluadas fueron altura de la planta (cm), número de hojas, diámetro del tallo (mm), diámetro del bulbo (mm), rendimiento total (t ha-1) y categorías de rendimiento comercial (t ha-1). el número de hojas, la altura de la planta y el diámetro del tallo entre los tratamientos fueron significativamente diferentes, con valores más altos en los tratamientos 1 universidad nacional agraria la molina, facultad de agronomía, lima, perú. how to cite this article: almeyda, d., casas, a., & zuzunaga, m. (2021). effects of seedling thickness on the production and quality of onion (allium cepa l.) ‘santa rita’ in arequipa, peru. peruvian journal of agronomy, 5(3), 71–77. https://doi.org/10.21704/ pja.v5i3.1845 effects of seedling thickness on the production and quality of onion (allium cepa l.) ‘santa rita’ in arequipa, peru september december 2021 72 “estándar” y “grueso” hasta 60 días después del trasplante. la cosecha se adelantó en los tratamientos “estándar” y “grueso”. los tratamientos “muy fino” y “fino” necesitaron más días para la cosecha que los demás. el tratamiento “fino” mostró el mayor rendimiento total. no hubo diferencias significativas entre las categorías de rendimiento comercial en todos los tratamientos. se concluyó que el grosor de la plántula al trasplantar influye en el rendimiento bajo las condiciones de este estudio. palabras clave: cebolla, plántula, grosor, rendimiento, calidad introduction in peru, various domesticated allium species are cultivated, including allium cepa l. this vegetable possibly originated from the mountainous regions of central asia, where inhabitants of some valleys would initiate the domestication process (brewster, 2001). after many years, its arrival in peru took place through the travel of european settlers. in perú, arequipa has the highest regional production and the largest onion cultivation area (ministerio de agricultura y riego [minagri], 2019). the red onion allium cepa ‘santa rita’ comes from the continuous mass selection of the ‘americana’ variety. this led to an adaptation of the late onion plantings in santa rita de siguas to adverse weather conditions. this cultivar allows onion production in an important commercial window for arequipa (sales on april, may, and june), which is why this cultivar was used in this study. according to guillén (2012), the methods used for sowing in peru are direct sowing and transplantation. direct sowing is minimal due to its high cost, so seedling or bulb transplantation are currently used. plants grown from transplants show better performance compared to those grown via direct seeding. in lincolnshire, transplanted onions produce uniform bulb yields of 45 t ha−1, and these mature two weeks earlier than directly seeded plants, resulting in a higher production (currah et al., 1990 cited by brewster, 2001). instituto nacional de investigaciones agropecuarias (inia, 2012) indicates that seedlings for transplanting have a minimum quality standard (<6.0 mm in diameter at the neck level). if this parameter is not met, there is a great disadvantage in achieving a rapid and vigorous recovery after transplanting. therefore, this study aimed to evaluate the yield, growth, development, and quality of the cultivar ‘santa rita’ based on the thickness of the seedlings before transplanting. we evaluated the effects of the seedling diameters between 20 and 80 days after transplanting on biometric parameters (height, number of leaves, diameter of false stem, bulb diameter, and bulbification index), yield, and production quality. materials and methods this study was done in fundo america located in the district of santa rita de siguas in arequipa province. plants ready for transplant were two months old. the definitive field period was from february to june 2017. the plant density was 10 cm between plants and 60 cm between lines. a) treatments the four different levels of seedling thickness for transplanting were very thin (2.00 mm – 3.49 mm), thin (3.50 mm – 4.99 mm), standard (5.00 mm – 6.49 mm), and thick (6.50 mm – 7.99 mm). these sizes are normally used by farmers who grow onions using the seedling method. b) experimental design a completely randomized block design was used, with four treatments and five blocks. the statistical tests performed were analysis of variance and tukey’s test at 5 % significance level for mean comparisons. c) evaluations all samples were selected from the central lines of each experimental unit. each sample consisted of five randomly chosen plants that were used for all the evaluations indicated below. harvest in all plots was done 130 days after planting. d. almeyda; a. casas; m. zuzunaga peruvian journal of agronomy 5(3): 71–77 (2021) 73 d) morphological variables all data were collected 80 days after transplanting. plant height: plant height was measured starting from the neck of the plant to the apex of the largest leaf. number of leaves: photosynthetically active leaves were counted. photosynthetically active leaves are those that did not have any damaged tissues due to a plague, disease, or senescence. false stem diameter: this was evaluated by measuring the base diameter of the false stem. bulb diameter: this was evaluated by measuring the equatorial diameter of the bulb. bulbification index: the bulb diameter/false stem diameter relationship was calculated, which is an index that indicates the beginning and development of the bulb. e) production and commercial quality. total yield: all bulbs from the central lines of each plot were harvested. the total yield represented the total weight of the bulbs after neck cutting and curing. grade 1: bulbs that are healthy, compact, and dry, with thin necks and an equatorial diameter of 7 cm to 10 cm. grade 2: bulbs with similar characteristics as the first ones, but with an equatorial diameter between 5 cm to 7 cm. discard: diseased, elongated, split, or double bulbs. results and discussion plant height table 1 shows the height of each transplanted seedlings from 20 to 80 days after transplanting. the “standard” and “thick” treatments had the tallest height in 40 days after transplanting, but only the “standard” treatment continued to lengthen until 80 days; then, the “very thin” and “thin” treatments surpassed it. the “very thin” treatment was shorter until 50 days after transplanting; however, after 70 days, it was superior to all the others, with the highest plant table 2. number of leaves of onion ‘santa rita’ seedlings with four different thicknesses treatment 20 dat 30 dat 40 dat 50 dat 60 dat 70 dat 80 dat very thin 3.44 b 4.88 b 6.52 c 7.68 b 9.04 b 9.96 a 10.72 a thin 3.72 ab 5.72 a 7.60 b 8.32 b 9.60 ab 10.44 a 10.68 a standard 3.84 a 6.16 a 8.24 a 9.60 a 10.20 a 10.44 a 10.60 a thick 4.04 a 5.96 a 8.08 ab 9.04 a 9.92 a 10.24 a 10.40 a cv (%) 8.20 % 10.05 % 9.74 % 9.58 % 5.82 % 4.95 % 7.72 % statistical sig * * * * * n.s. n.s. dat: days after transplanting table 1. onion (a. cepa) plant height (cm) cv. ‘santa rita’ using seedlings with four different thicknesses treatment 20 dat 30 dat 40 dat 50 dat 60 dat 70 dat 80 dat very thin 13.40 b 19.43 b 36.73 b 48.13 c 62.42 ab 66.05 a 70.88 a thin 15.34 a 23.56 ab 41.90 a 52.69 b 62.09 ab 67.31 a 67.94 ab standard 16.93 a 26.44 a 44.47 a 58.05 a 64.63 a 66.25 a 65.72 b thick 16.51 a 26.38 a 42.61 a 54.54 b 60.42 b 61.96 b 64.07 b cv (%) 10.77 % 14.97 % 8.25 % 7.35 % 4.07 % 4.29 % 4.92 % statistical sig * * * * * * * dat: days after transplanting effects of seedling thickness on the production and quality of onion (allium cepa l.) ‘santa rita’ in arequipa, peru september december 2021 74 days after transplanting, there were no significant differences in the number of leaves. poma (2013) evaluated the number of leaves of the cultivar ‘roja de camaná’ according to the level of fertilization and planting system, obtaining plants with eight leaves on average at 71 days after transplanting under the conditions in majes irrigation. this indicates that the average number of leaves in the different treatments was within the range and was even higher. according to guzmán, cited by chimborazo (2015), a foliage grows until the external conditions favor bulb formation. these conditions are usually the decrease in temperature and the change in photoperiod. these conditions varied during april, having a different impact on each treatment, starting with the “standard” and “thick” treatments. false stem diameter table 3 shows the results of the false stem diameter variable from 20 to 80 days after transplanting. the analysis of variance shows that the seedling thickness factor generated significant differences in the diameter of the false stem throughout the experiment, except at 60 days after transplanting. the “standard” and “thick” treatments had a similar behavior; however, the “standard” treatment stood out, with a higher false stem diameter up to 60 days after transplanting. at 60 days after transplanting, the “very thin” and “thin” treatments were not significantly different from the “standard” and “thick” treatments because the false stem diameter at this growth stage coincides. the “very thin” treatment obtained the largest false stem diameter at 80 days after transplanting, with a measurement of 17.43 mm. according to brewster (2001), the necks lose turgor and soften due to senescence. this indicates the final stage of the growth of height of 70.88 cm after transplanting. guzmán, cited by chimborazo (2015), indicates that the length of the leaves ranges from 40 cm to 65 cm, which is similar to the results obtained in this study. the results are also similar to those obtained in national studies, such as amaya (2011) in which the cultivar ‘roja arequipeña’ reached 50 cm at 90 days after transplanting under laredo conditions. on the other hand, under conditions similar to this study, poma (2013) described the 60-cm heights of the cultivar ‘roja de camaná’ at 70 days after transplanting in majes irrigation. as shown in table 1, the trend of the height increase in all treatments was similar, and the “very thin” treatment had the lowest height until 60 days after transplanting. when the growth in other treatments decelerated, this growth of this one continued to accelerate. according to brewster (2001), the height variable is determined by cultivar factors, planting density, and climate. based on the experiment, seedling thickness should also be considered. number of leaves table 2 shows the number of leaves in each treatment 20 to 80 days after transplanting (dat). based on the results of the analysis of variance in each evaluation date, there are significant differences in the number of leaves up to 60 dat based on the seedling thickness factor; there were no significant differences between the thickness of the seedlings afterwards. the “standard” treatment had more leaves up to 60 dat, however all treatments were similar in this characteristic after 70 dat. the “very thin” and “thin” treatments had fewer leaves on average (except at 30 days after transplanting, where the “thin” treatment was similar to the “standard” and “thick” treatments). despite these differences, 70 table 3. false stem diameter (mm) of onion ‘santa rita’ seedlings with four different thicknesses treatment 20 dat 30 dat 40 dat 50 dat 60 dat 70 dat 80 dat very thin 3.33 b 4.92 c 8.41 c 11.61 c 15.97 a 16.99 a 17.43 a thin 4.22 a 6.48 b 10.24 b 12.68 bc 15.71 a 16.91 a 16.36 ab standard 4.60 a 7.49 a 11.44 a 14.85 a 15.84 a 15.22 ab 14.76 b thick 4.59 a 7.38 ab 11.06 ab 13.78 ab 14.93 a 14.42 b 14.70 b cv (%) 14.73 % 17.41 % 12.96 % 10.55 % 5.78 % 10.06 % 11.09 % statistical sig * * * * n.s. * * dat: days after transplanting d. almeyda; a. casas; m. zuzunaga peruvian journal of agronomy 5(3): 71–77 (2021) 75 bulbs, concluding with the fall of aerial parts. the “standard” and “thick” treatments showed this characteristic at 60 days after transplanting when they reached their maximum false stem diameter, which coincided with their slow growth (height and number of leaves). the “thin” treatment reached the maximum false neck diameter first at 70 days after transplanting, followed by the “very thin” treatment at 80 days. the differences between each treatment were related to the days to harvest. bulb diameter the bulb diameters in each treatment 60, 70, and 80 days after transplanting are shown in table 4. the bulb diameters of the “standard” and “thick” treatments were statistically higher up to 70 days after transplanting. after 80 days, the “thick” and “thin” treatments did not have significant differences, while the standard treatment had the highest bulb diameter. the “very thin” treatment had the lowest bulb diameter in all evaluations. bulb index table 4 shows the bulb indices in all treatments 60, 70, and 80 days after transplanting. since the false stem diameter and bulb diameter are inversely proportional during bulb formation, this relationship allows a more precise identification of the time of harvest. this parameter determines the start of bulb formation when the ratio is 2. during the evaluations, all values were >2, which indicates that all treatments started bulb formation. yield table 5 summarizes the total yield (t ha−1), their qualities (t ha−1), and the percentage (%) that each one represents with respect to the total yield. the analysis of variance showed that the thickness of the seedlings generates significant differences in the total yield and on grade 1 onions. the analysis of variance of the other qualities were not significantly different, including the percentage that each represents with respect to the total yield. according to the tukey’s test, the highest yield was obtained by the “thin” treatment (29.24 t ha−1), while the lowest yield was obtained by the “thick” treatment (25.01 t ha−1). the yield in the “very thin” and “standard” treatments was not significantly different, but it was significantly higher than that of the “thick” treatment. these yields were lower than those reported by gonzáles (2003), poma (2013), león (2015), and minagri (2019) because this study was initiated at the beginning of the year, when the conditions accelerate onion development, preventing the yields from reaching their peaks. in addition, the atypical rains caused by the “niño costero” phenomenon, which favored the development of fusarium, reduced the initial transplanted population. the results of this study were similar to those of riekels et al. (1976), who reported that the purpose of producing seedlings is to obtain seedlings with a thickness of 3.5 mm to guarantee good yields. however, inia (2012) mentioned that thicknesses >6 mm are recommended to obtain a better exportable onion performance table 4. bulb diameter (mm) and bulb index of onion ‘santa rita’ seedlings with four different thicknesses bulb diameter bulb index treatment 60 dat 70 dat 80 dat 60 dat 70 dat 80 dat very thin 32.48 c 46.49 c 63.62 b 2.05 c 2.79 b 3.7 b thin 38.33 b 53.44 b 65.59 ab 2.45 b 3.25 b 4.15 ab standard 46.85 a 59.99 a 71.54 a 2.99 a 4.02 a 4.97 ab thick 48.68 a 59.03 a 68.69 ab 3.32 a 4.19 a 4.74 a cv (%) 16.95 % 11.10 % 6.85 % 19.89 % 18.39 % 16.25 % statistical sig * * * * * * dat: days after transplanting effects of seedling thickness on the production and quality of onion (allium cepa l.) ‘santa rita’ in arequipa, peru september december 2021 76 under the conditions in chile. this was different from the results obtained in this study because thicker plants had a lower performance. figure 1 shows that the “standard” treatment had the highest first-grade quality (58.48 %), while the “thick” treatment had the lowest percentage (47.47 %). regarding the second-grade quality, the “thick” treatment had the highest percentage (41.64 %), while the “standard” treatment had the lowest percentage (34.21 %). the discard quality was the highest in the “thick” treatment (10.90 %), while the “standard” treatment had the lowest. based on the descriptive statistics, it is evident that the “thick” treatment had the lowest first-grade quality, with an increased second-grade and discard qualities. this suggests that it is the least recommended thickness. according to the graph, it can also be seen that the most recommended treatment is the one with the highest first-grade quality and with the table 5. total yield (t ha−1) and quality (%) of onion ‘santa rita’ seedlings with four different thicknesses treatment total yield t ha−1 1 grade 2 grade discard t ha−1 % t ha−1 % t ha−1 % very thin (2.00–3.49 mm) 26.16 ab 14.48 a 55.36 a 9.03 a 34.54 a 2.64 a 10.10 a thin (3.50–4.99 mm) 29.24 a 15.80 a 54.04 a 11.03 a 37.73 a 2.41 a 8.23 a standard (5.00–6.49 mm) 27.05 ab 15.82 a 58.48 a 9.25 a 34.21 a 1.98 a 7.31 a thick (6.50–7.99 mm) 25.01 b 11.87 a 47.47 a 10.41 a 41.64 a 2.73 a 10.90 a c.v (%) 9.69 % 22.06 % 18.46 % 22.77 % 22.77 % 33.71% 33.71 % statistical sig * * n.s. n.s. n.s. n.s. n.s. 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% v er y th in th in st a n d a rd th ic k v er y th in th in st a n d a rd th ic k v er y th in th in st a n d a rd th ic k 1 grade 2 grade discard 55.36%54.04% 58.48% 47.47% 34.54% 37.73% 34.21% 41.64% 10.10% 8.23% 7.31% 10.90% q u a li ty p er ce n ta g e (% ) figure 1. quality (%) of onion ‘santa rita’ seedlings with four different thicknesses with respect to the total yield d. almeyda; a. casas; m. zuzunaga peruvian journal of agronomy 5(3): 71–77 (2021) 77 lowest second-grade and discard qualities. these observations were not valid based on analytical statistics. conclusions seedling thickness significantly affected the height, number of leaves, false stem diameter, and bulb diameter of onion ‘santa rita.’ it also significantly affected the bulbification, as those with seedling thicknesses of ≤ 4.99 mm had a greater bulbification. it also significantly affected the total yield, where the “thin” treatment (3.50 mm – 4.99 mm) had the highest performance under the test conditions. however, the seedling thickness did not significantly affect the grade 1, grade 2, and discard quality of onion ‘santa rita.’ references amaya, j. (2011). respuesta de niveles de npk en el crecimiento y producción de cebolla (allium cepa l.) var. “roja arequipeña” en pampas de san juan, laredo. [thesis, universidad nacional de trujillo]. la libertad, perú. brewster, j. (2001). las cebollas y otros alliums. zaragoza, españa. 253 pp. chimborazo, d. (2015). evaluación del rendimiento de cebolla de bulbo (allium cepa l.) var. ‘red nice’ a partir de plántulas obtenidas mediante la poda de sus hojas. [thesis, universidad técnica de ambato]. cevallos, ecuador. gonzáles, e. 2003. efecto de la fertilización nitrogenada y de la aplicación de manganeso y zinc, bajo dos modalidades: al suelo vía fertirrigación y a la planta via aspersión foliar; en el rendimiento del cultivo de cebolla (allium cepa l.) cv. roja arequipeña. [master’s thesis, universidad nacional agraria la molina]. lima, peru guillén, l. (2012). manejo y producción de hortalizas. universidad católica santa maría. arequipa, perú. 557 pp. instituto nacional de investigaciones agropecuarias. (2002). informativo la platina n° 17: tamaño o grosor de los almácigos de cebollas y sus efectos en el trasplante y cosecha final. santiago, chile. 102 pp. león, s. 2015. niveles de nitrógeno y fitorreguladores en el bulbeo de la cebolla (allium cepa l.) cultivar ‘roja ilabaya’. [thesis, universidad nacional jorge basadre grohmann]. tacna, perú ministerio de agricultura y riego. (2019). serie de estadísticas de producción agrícola [on line]. https://guiastematicas. biblioteca.pucp.edu.pe/estadisticas-peruanas/agriculturariego poma, r. (2013). tres sistemas de plantación y tres niveles de fertilización en la producción de cebolla (allium cepa l.) cv. ‘roja de camaná’ bajo riego a goteo en zonas áridas. [thesis, universidad nacional san agustín]. arequipa, perú. riekels, j., tiessen, h., & nonnecke, i. (1976). onions. ontario, canada. 32 pp. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 09 july 2021 accepted for publication: 28 august 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina https://doi.org/10.21704/pja.v5i2.1771 distribution of stem-end rot on the canopy in ‘hass’ avocado trees in two coastal areas in peru distribución de la pudrición peduncular en la canopia de arboles de palto cv. ‘hass’ en dos áreas costeras del perú a.k. llanos1; w.e. apaza1 *corresponding author: allanos@lamolina.edu.pe *https://orcid.org/0000-0002-6032-4141 abstract stem-end rot (ser) of avocado is caused by several fungal species, and it is presented worldwide. this plant disease currently affects several avocado producer regions in peru, causing fruit rot, impacting the industry negatively. research about ser distribution in the canopy of avocado trees is limited. thus, the present study aimed to compare which areas in the canopy are prone to have more ser in ‘hass’ avocado harvested fruit in two different coastal areas in peru. the experiment was conducted in the northern (barranca) and southern (cañete) of lima. ‘hass’avocado fruits from both producer areas were collected to identify the causal agent; lasiodiplodia theobromae was isolated from infected fruits. identification was conducted based on morphological features and a partial dna sequence of the translation elongation factor 1-α gene (tef1-α). the results showed that fruits inside the tree canopy were prone to have a higher disease incidence than the fruits located in the external site (p<0.001). besides, internal-site fruits displayed a higher percentage of infected fruit for each grade disease (p<0.001) than external-site fruits, except for grade 0 (fruits without symptoms) and grade 1. finally, the results suggested that the altitude where the fruit is positioned on the canopy could influence the incidence of ser, where fruits located in the high part revealed less incidence than the low section. the results are valuable for enhancing management strategies and avoiding postharvest loss of avocado fruits in our region. keywords: lasiodiplodia theobromae, stem-end rot, avocado, canopy, ser resumen la pudrición peduncular del palto (ser por sus siglas en inglés) es causada por varias especies de hongos, y se presenta a nivel mundial. esta enfermedad afecta actualmente a varias regiones productoras de palta en el perú, causando la pudrición de la fruta, impactando negativamente a la industria. la investigación sobre la distribución del ser en la copa de los árboles de palta es escasa. por ello, el presente estudio tuvo como objetivo comparar qué zonas de la copa son propensas a tener más ser en la fruta cosechada de palta ‘hass’ en dos zonas costeras diferentes del perú. el experimento se realizó en el norte (barranca) y en el sur (cañete) de lima. se recolectaron frutos de palta ‘hass’ de ambas zonas productoras para identificar el agente causal; se aisló lasiodiplodia theobromae de los frutos infectados. la identificación se realizó en base a las características morfológicas y a una secuencia parcial de adn del gen del factor de elongación de traducción 1-α (tef1-α). los resultados mostraron que los frutos dentro de la copa del árbol fueron propensos a tener una mayor incidencia de la enfermedad que los frutos situados en la parte externa (p<0,001). además, 1 universidad nacional agraria la molina, facultad de agronomía, departamento académico de fitopatología.. how to cite this article: llanos, a. k., apaza, w. e. (2021). distribution of stem-end rot on the canopy in ‘hass’ avocado trees in two coastal areas in peru. peruvian journal of agronomy, 5(2), 60–70. https://doi.org/10.21704/pja.v5i2.1771 user sello a. k. llanos; w. e. apaza peruvian journal of agronomy 5(2): 60–70 (2021) 61 los frutos situados en el interior mostraron un mayor porcentaje de frutos infectados para cada grado de la enfermedad (p<0,001) que los frutos situados en el exterior, excepto para el grado 0 (frutos sin síntomas) y el grado 1. finalmente, los resultados sugirieron que la altitud en la que se encuentra el fruto en la canopia podría influir en la incidencia del ser, donde los frutos situados en la parte alta revelaron menor incidencia que la sección baja. estos resultados son valiosos para mejorar las estrategias de manejo y evitar la pérdida poscosecha de los frutos de aguacate en nuestra región. palabras clave: lasiodiplodia theobromae, pudrición peduncular, aguacate, canopia, ser introduction avocado (persea americana mill.) is a fruittree crop cultivated merely in tropical and subtropical areas worldwide because of climatic requirements such as temperature and rainfall (food and agriculture organization of the united nations [fao], 2020; ministerio de agricultura y riego [minagri], 2008). this crop has experienced the fastest production growth, and currently, its global production climbed to 6.3 million tons in 2018 (altendorf, 2019). the americas are the biggest producers globally, where more than 70.0 % of the production came from this area (fao, 2020). in peru, avocado is an economically significant fruit, and it is considered one of the biggest producers worldwide. peru is ranked as the third leading producer, followed by mexico and the dominican republic, and the second avocado exporter worldwide (fao, 2020; altendorf, 2019; centre de cooperation international en recherche agronomique pour le développement [cirad], 2019). in 2019, peru registered more than 500 thousand tons over an expansion of 31 000 ha with a wholesale of 720 million usd (cirad, 2019). due to a high-tech production system and climatic conditions, the coastal region is the most important area in peru (cirad, 2019; minagri, 2008). as a result, the planted area in peru has been increasing rapidly, becoming avocado a valuable crop. among the existing cultivars are hass, fuerte, ettinger, zutano, and bacon. however, the cultivar hass is the most extended one produced for the international market (cirad, 2019; minagri, 2008). like other big-extended crops, avocado faces limitation factors in its production; the phytosanitary aspect is considered one of the main ones. one of the plant diseases that are infecting avocados is stem-end rot (ser), affecting these fruits after harvest. ser is a postharvest disease that infects this fruit and others, including mango and citrus. (diskin et al., 2017; zhang, 2014; zhang & swingle, 2005). this plant disease lives endophytically until favorable conditions occur with fruit ripening (johnson et al., 1992). this plant disease starts in the stem end, a section attached to the fruit, showing a shriveling. after that, a decay that appears in this zone produces a dark discoloration and softening of the pulp. this disease advances through vascular bounds, sometimes showing from dark to brown color. as the avocado ripens, these lesions expand to the whole pulp, eventually showing a complete decayed fruit (guarnaccia et al., 2016; madhupani & adikaram, 2017; twizeyimana et al., 2013). many species of the botryosphaeria family cause ser. among the species reported is lasiodiplodia theobromae, a worldwidedistributed plant pathogen infecting more tropical and subtropical areas (punithalingam, 1976; voorhees, 1942). l. theobromae has been identified as infecting over 500 host plants, including fruit trees, vegetables, and ornamental plants (punithalingam, 1980). this fungus has been reported infecting other fruit trees such as peach, mango, and grapevine (li et al., 1995; khanzada et al., 2004; úrbez-torres et al., 2008). also, among the symptoms described in fruit-crop diseases are sunken necrotic lesions, gummosis, earlier defoliation, twig dieback, reduced vigor, and as a consequence, a lower production (li et al., 1995; khanzada et al., 2004). due to its infection features, l. theobromae rarely takes place when the fruits remain in the tree. this pathogen remains latent in the fruit tissue, and symptoms are expressed until harvest; at this point, the plant pathogen develops the infection in this plant tissue. distribution of stem-end rot on the canopy in ‘hass’ avocado trees in two coastal areas in peru may august 2021 62 some management approaches to control ser have been described to reduce the presence of this plant disease during the postharvest time. among them are using ripening inhibitors, harvest practice, pre and postharvest chemical and biological control, application of plant extracts, and physical control (galsurker et al., 2018). even though several articles have focused on the biology and management of this plant disease, information about the distribution of ser on avocado trees in the canopy is still scarce. thus, this study aimed to understand which canopy areas in the ‘hass’ avocado tree are more prevalent to this plant disease. the results will help enhance the management of this pathogen and select which areas in the canopy must be fully and well protected when strategy management measures are applied on the field. materials and methods location and data collection the experiments were conducted in two different ‘hass’ avocado commercial plots, located in the northern (barranca) and southern (cañete) regions of lima. these places were located in the coastal area of peru. the maximum and minimum were recorded; barranca showed values from 14 °c to 28 °c, and cañete from 15 °c to 29 °c. in addition, relative humidity (hr) for barranca and cañete displayed values between 85 % to 100 % and 80 % to 98 %, respectively. the locations were chosen because of historical disease presence. plant and fruit materials the evaluated plant materials were 5-years-old ‘hass’ avocado trees cultivar hass on zutano rootstock with a height between 6 m to 7 m and natural leaf mulch in both areas. in addition, both sites were conducted under exportable conditions, with mechanical (barranca) and manual (cañete) pruning conducted annually after harvest in august, ‘hass’ avocado fruits were usually harvested with short pedicel using secateurs. no fungicides applications were applied to managed ser directly; however, fungicides to control lasidioplodia were applied during the season (thiabendazole and copper sulfate pentahydrate). in the northern-studied area (barranca), the planted distance was 2 m x 6 m with a density of 1000 tree per ha with a drip irrigation system. the experimental plot in cañete had a planted distance of 7 m x 3.5 m with a density of 400 tree per ha irrigated by microsprinkler. ‘hass’ avocado trees were selected arbitrarily on the plot assigned. the avocado fruits were collected during a commercial harvest season from june to august. collection and isolation ‘hass’ avocado fruits were collected from the evaluated area (barranca). they were transported immediately to universidad nacional agraria la molina (unalm). isolation procedures were carried out in the plant disease clinic of the plant pathology department at unalm. the pathogen’s isolation was performed by washing with clean tap water and immersing the fruits with sodium hypochlorite at 1 % for one minute and air-dried. then they were rinsed in distilled water for two minutes and air-dried to avoid contamination. fruits were placed in a moist chamber at 25 °c. fruits with initial typical ser symptoms were selected for isolation. pieces of the fruit stem-end were cut into small sections (from 2 mm to 3 mm) with a sterile scalpel. the disinfected pieces were placed in potato dextrose agar with oxitetraciclina (pda+) and stored at 25 °c for four days in dark conditions. once the mycelium was visible, they were transferred to pda+ again to get pure culture. morphological and molecular characterization morphological identification was made by taxonomical fungi keys elaborated by barnett & hunter (2006). also, morphological structures such as pycnidia and conidia were examined using a compound microscope (dl1000 led leica, wetzlar, germany). for molecular characterization, dna extraction was performed following the method described by saitoh et al. (2006) and balogun et al. (2008). three-day older cultures were used. a 5-mm diameter plug of active mycelia growth extracted from the culture was placed in a 1.5-ml sterile eppendorf tube. a total of a. k. llanos; w. e. apaza peruvian journal of agronomy 5(2): 60–70 (2021) 63 (5x), 2 µl of mgcl2, 0.5 µl of dntp, 0.5 µl for each primer (forward and reverse), 0.1 µl taqpolymerase and 1 µl of gdna. the amplification reactions were conducted in a simpliamptm thermal cycler (thermo fisher scientific, singapore) with the following protocol: 5 min at 94 °c; 40 cycles of 1 min at 94 °c, 1 min at 58.1 °c, 1 min at 74 °c, and a final extension of 7 min at 74 °c. pcr products were sent for sequencing to the university of california riverside (ucr). the tef1-α gene sequences of isolates of l. theobromae from previous studies were used to compare homology by using blast (altschup et al., 1990). this procedure was used for each isolate to identify the percentage of homology with l. theobromae. finally, sequences of tef1-α gen of lasiodiplodia spp. and diplodia seriata (table 2) were obtained from genbank, and they were used for phylogenetic analysis. alignment and phylogenetic analyses were performed by molecular evolutionary genetics analysis (mega-x v 10.2,2) using five isolates (table 3). maximum likelihood analysis and bootstrap values were calculated using 1000 replicates. the evolution model for the analysis was t92: tamura 3-parameter. 500 μl of lysis buffer (100 mm tris-hcl, 50 mm ethylenediaminetetraacetic acid [edta], 1m kcl; ph 8.0) was added to the tube, and the mycelium was dispersed with a sterile toothpick. after 10 minutes at room temperature, 300 μl of phenol: chloroform: isoamyl alcohol in the following volume rate 25:24:1 was added. the mixture was centrifuged for 10 minutes at 1200 rpm. a supernatant of 300 μl was transferred to a new 1.5-ml eppendorf tube and was stored at 37 °c for 30 minutes (thermo mixer eppendorf). a 300 μl of isopropanol was added, and it was stored at -20 °c for 15 minutes. finally, this was centrifuged at 1200 rpm for 10 minutes at room temperature, and the supernatant was discarded. dna was washed with 1 ml of ethanol at 70 % by centrifugation at 1200 rpm for 5 minutes, and the ethanol was discarded. dna pellets were air-dried, and 30 μl of nuclease-free water was added. the mixture was stored at -30 °c. for identification at the species level, a couple of primers previously developed (ef1-728f and ef1986r) were used to amplify translation elongation factor 1-alpha (tef1-α) (carbone & kohn, 1999) (table 1). the amplification of the tef1-α was carried out in a 25.0 µl reaction, using 15.4 µl of hplc-grade water, 5 µl of buffer table 1. primers used to amplify gdna of efα gene of lasiodiplodia spp. target gene primer direction sequence (5’-3’) cite ef-α ef1728f forward catcgagaagttcgagaagg (carbone & kohn, 1999) ef1986r reverse tacttgaaggaacccttacc table 2. isolates used in the present study isolate species host origin collector genbank accession no. ( ef1-α) cmw9074 lasiodiplodia theobromae pinus sp mexico b. slippers ay236901 cmw10130 l. theobromae vitis donniana uganda j. roux ay236900 cbs115812 l. gonubiensis syzygium cordatum south africa d. pavlic dq458877 cf/uenf427 l. theobromae persea americana brazil p. santos ky223707 cbs 164-96 l. theobromae fruit on coral reef coast new guinea a. aptroot ay640258 cmw8230 diplodia seriata picea glauca canada j. reid dq280418 cmw8230 d. seriata malus domestica south africa w. a. smith dq280419 distribution of stem-end rot on the canopy in ‘hass’ avocado trees in two coastal areas in peru may august 2021 64 distribution of stem-end rot in the canopy of ‘hass’avocado fruit two experiments were conducted in barranca to analyze the distribution of ser in the internal and external part of the canopy in ‘hass’ avocado fruits after being harvested. a total of 400 avocado fruits were collected from 40 trees; for each tree, ten avocados were selected, five from each part of the canopy. the collected avocado fruits had an exportable fruit size, categorized as number 16, whose dry matter value was between 23 % and 24 %. avocado fruits were placed in a plastic container (40 cm x 30 cm x 40 cm) as a moist chamber with a humid paper towel at the bottom, and each fruit was placed above a petri dish to avoid contact with the paper towel. the avocado fruits were stored at 22 °c in dark conditions, and after 14 days, they were analyzed by cutting the fruit down the middle lengthwise. typical symptoms in the avocado fruit were counted as infected ones. evaluation of the severity of the plant disease in avocado fruit was classified in five grades (g) depending on the percentage of damage: g0= 0 %, g1 = 1 % to 5 %, g2 = 6 % to 25 %, g3 = 26 % to 50 % and g4 = >50 %. additionally, three experiments were conducted to understand the influence of the fruit position where the canopy was divided in three sections in the presence of ser in cañete. the avocado tree canopy was divided into three sections: high (h), middle (m), and low (l). four avocado trees were selected arbitrarily for the experiment, and four avocado fruits were collected for each section of each tree. the size and storage of the collected fruit and the evaluation of the incidence of ser were as it was described previously. statistical analysis the data obtained from the evaluation of ser on ‘hass’ avocado fruit were recorded and tabulated in an excell spreadsheet document. this data was analyzed by sas (statistical analysis system version 9.4, cary, nc). the percentage of avocado stem-end rot data obtained in the experiment was tested by one-way analysis (anova) with the proc glm command. in addition, the means were compared with tukey analysis with a significant level of 0.05. homogeneity and normality were assessed and satisfied. results and discussion development of avocado stem-end rot on ‘hass’avocado fruits dark necrosis in the peduncle area developed once the plant disease started developing in the ‘hass’ avocado fruit. the fungus colonization was usually initiated from the stem-end of the fruit to the whole fruit, and it was faster in the core than the rind. ser was capable of developing soft brown to black decay symptoms in the entire fruit (figure 1). morphological and molecular characterization a total of 5 isolates were obtained from barranca. these isolates were used for morphological characterization. lasiodiplodia was identified following a taxonomical key made by barnett & table 3. list of lasiodiplodia theobromae isolates from ‘hass’ avocado fruit isolate speciea host (persea americana mill.) origin (dep-prov)b 1bar_ef l. theobromae ‘hass’ lima barranca 2bar_ef l. theobromae ‘hass’ lima barranca 3bar_ef l. theobromae ‘hass’ lima barranca 4bar_ef l. theobromae ‘hass’ lima barranca 5bar_ef l. theobromae ‘hass’ lima barranca a l. theobromae from avocado tree were determined based on morphology and phylogenetic analyses. b dep = department and prov = province a. k. llanos; w. e. apaza peruvian journal of agronomy 5(2): 60–70 (2021) 65 figure 1. a. initial symptoms and b. advanced infection of ser in ‘hass’ avocado fruits. figure 2. phylogeny of lasiodiplodia theobromae based on analysis of partial sequences of translation elongation factor 1-alpha (ef-1α). support bootstrap values were obtained by using 1000 replicates generated in mega-x v.10.2.2. the phylogeny was constructed using the genus diplodia as the outgroup. distribution of stem-end rot on the canopy in ‘hass’ avocado trees in two coastal areas in peru may august 2021 66 hunter (2006). besides, the isolate had similar features to the description that was made by punithalingam (1976) in terms of the appearance of the colony and the conidia. in addition, we compared the results with other previous studies (pereira et al., 2009; úrbez-torres et al., 2006). furthermore, in this study, five genomic dna from five isolates were examined. the sequence was compared in genbank, and these isolates were assigned to lasiodiplodia theobromae, matching morphological features. the phylogenetic tree included two clades notoriously different (figure 2). l. theobromae isolates showed a bootstrap support value of 100%. lasiodiplodia isolates collected in barranca were put together in the same group with isolates that were collected in mexico, uganda, brazil, and new guinea. l. gonubiensis and diplodia seriata were classified in a different group. ser of avocado constitutes a severe problem in the industry because it restricts its commercialization after harvesting. this disease was reported caused by several fungal species, mainly of the botryosphaeriaceae family (twizeyimana et al., 2013; wanjiku et al., 2020). fungal species population studies of this plant disease in our region is scarce, and to our knowledge, this is the first attempt to identify the causal agent. the isolation of l. theobromae from avocado fruits in the study with ser symptoms coincides with other reports where this pathogen was reported (darvas & kotze, 1987; menge & ploetz, 2003). l. theobromae, an ascomycete, is prevalent in tropical and subtropical zones, infecting more than 500 species worldwide (punithalingam, 1976, 1980). besides, the presence of this fungus on fruits concurs with other reports of l. theobromae such as citrus fruit (zhang, 2014), blueberry (xu et al., 2015), mamey zapote (tovar-pedraza et al., 2012), and mango (munirah, 2017). the explanation for identifying l. theobromae in the isolates studied could be that this fungal pathogen could be prevalent in our country, as reported in others such as israel (menge & ploetz, 2003). nevertheless, l. theobromae could be absent in other regions, including the us (menge & ploetz, 2003; twizeyimana et al., 2013). thus, further analysis with a significant number of isolates needs to be done to identify and understand the fungal population species related to stem-end rot in avocado fruits in peruvian conditions. distribution of stem-end rot in the canopy of ‘hass’ avocado trees evaluation of the incidence of ser in the canopy of ‘hass’ avocado fruit showed a statistical difference between internal and external positions (p = <0.001). the highest incidence was shown in the interior area, where the mean incidence of ser was 37.25 % ± 6.5 %; a lower value was obtained in the external site whose mean value was 13.75 % ± 3.5 % (figure 3a). these results figure 3. a. effect of the position of ‘hass’avocado fruit in the incidence and b. severity grade of ser on the canopy of ‘hass’ avocado fruit. i = internal, e = external, g = severity grade of ser. the experiments were conducted in the northern region of lima (barranca). lines in the graphs show standard deviations. a. k. llanos; w. e. apaza peruvian journal of agronomy 5(2): 60–70 (2021) 67 showed that the position of the fruit on the canopy influences the incidence of this pathogen. an additional analysis was conducted where the grade of the damage of this pathogen in the fruit was measured (figure 3b). the percentage of fruit with grade 0 (fruits without symptoms) was higher in the external than the internal part of the canopy (p = <0.001). conversely, evaluation of the severity of ser showed that mean frequency where higher in all the grades evaluated (g1, g2, g3, g4), showing a statistical difference in all of them except for g1. additionally, three experiments were conducted to study the influence of fruitposition height on the percentage of incidence of ser. in the first and second experiments, the highest incidence value of ser was obtained at the low section of the canopy (figure 4a and 4b). nevertheless, only one experiment showed a statistical difference between the low and high sections (p = 0.03). besides, in neither of the cases, the middle and low positions showed a statistical difference. the third experiment was performed to evaluate the grade of severity of this pathogen at different sections (figure 4c). the percentage of avocado fruits without symptoms (g0) decreased from high to low section. no difference was found evaluating the grade (g1, g2, g3, and g4) of the severity of ser in different sections. interestingly avocado fruit at middle and low section only showed a grade of severity g3 and g4 for each section respectively. in the study, fruits on the internal part of the canopy showed a statistical difference with fruits that are located on the external site. in addition, the severity grade of ser was higher in the internal part than the external for all the categories, except grade 0 (g0). these results could be supported by a previous study where it was found that the firmness in some cultivars was higher in avocado fruits exposed to the sun than the shaded fruit (woolf et al., 2000). also, the concentration of antifungal compounds diene after harvesting could affect the infection of ser in the flesh fruit. it was reported that, even though the initial content of diene was similar in shade and sun fruit, the concentration of diene in the fruit flesh decreased faster in shade fruit than fruits exposed to the sun when they were stored at 20 °c. however, diene concentration in the harvested fruit peel does not vary seven days after being harvested. after this period of time, it follows a different pattern; this compound increases in sun fruits at a double rate than shaded fruit (woolf et al., 2000). additionally, similar findings concur with an experiment conducted in mango where fruits exposed to sunlight, whose figure 4: a. and b. effect of the position of the ‘hass’ avocado fruit in the incidence in two experiments and c. severity grade of ‘hass’ avocado fruits on different fruit-position height of the canopy. h= high; m= middle and l=low. the experiment was conducted in the southern region of lima (cañete). lines in the graphs show standard deviations. distribution of stem-end rot on the canopy in ‘hass’ avocado trees in two coastal areas in peru may august 2021 68 level of anthocyanin was higher, presented less fungal development than fruits located in the internal part of the canopy (diskin et al., 2017; sivankalyani et al., 2016). regarding the influence of the fruit-position fruit on ser incidence, there was no statistical difference between the middle and low section in the plant’s canopy. interestingly, fruits located at high section showed the lowest ser incidence compared to the low and middle section. however, the high section only did show statistical differences with the low section in one experiment. because of logistics, we were not able to collect more fruits per plant. that is why for further research, we suggest increasing the number of avocado fruits in future experiments to get stronger statistical results for this evaluation. a popular explanation that could explain this is that conidia from branches cannot reach the high part of the canopy. an additional study evaluating conidia at different sections of the canopy should be performed to corroborate this hypothesis. as mentioned previously, the exposition of fruits to the sunlight could have been influenced the results; fruits located at the low section could have been receiving less sunlight than the middle and high sections, affecting the presence of ser. pruning has been considered good practice to reduce the impact of this disease in harvest conditions (galsurker et al., 2018). as described, the lack of adequate pruning practice on the avocado experiment areas could have reduced the exposition of fruits for internal and low sections of the canopy, triggering feasible conditions for ser. consequently, inadequate pruning could negatively impact the effectiveness of chemical applications if this practice wants to be integrated to manage this pathogen because not a good application coverage could be reachable under these circumstances. a variation of the avocado tree could have influenced our data. it is reported that the age of the tree and holding time influence the prevalence of ser on other crops (brown & miller, 1999; zhang, 2014). conclusions our study identifies lasiodiplodia theobromae as potential pathogens causing ser in ‘hass’ avocado fruit. besides, our findings demonstrate that fruits located in the internal and low part of the canopy had less presence of ser than hass avocado fruits positioned in the external, middle, and high parts, respectively. these findings help understand the distribution of ser on the canopy of avocado trees, giving valuable information to enhance strategies management by providing information about which canopy areas must be fully and well protected. these future strategies management could diminish the damage of this fungal pathogen whose infection affects the commercial product during postharvest, causing significant economic losses in agriculture. orcid and e-mail a. k. llanos allanos@lamolina.edu.pe https://orcid.org/0000-0002-6032-4141 w. e. apaza wapaza@lamolina.edu.pe https://orcid.org/0000-0001-7510-8866 references altendorf, s. 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(2014). lasiodiplodia theobromae in citrus fruit (diplodia stem-end rot). in s. bautista (ed.), postharvest decay: control strategies (pp. 309–331). elsevier. https:// doi.org/10.1016/b978-0-12-411552-1.00010-7 zhang, j., & swingle, p. p. (2005). effects of curing on green mold and stem-end rot of citrus fruit and its potential application under florida packing system. plant disease, 89(8), 834–840. https://doi.org/10.1094/pd-89-0834 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i1.1733 received for publication: 02 august 2021 accepted for publication: 02 march 2022 published: 30 april 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by incidence of leaf diseases in the agroforestry systems at yurimaguas, peru incidencia de las enfermedades foliares en los sistemas agroforestales de yurimaguas, perú l. aragón1*; h. huarhua1; m. cerna1; j. flores1; f. dueñas2; c.p. lao3; r. solis4; j. alegre5 *corresponding author: lili@lamolina.edu.pe abstract an agroforestry system (saf) is characterized by having a diversity of components, such as timber and nontimber forest species and crops, pastures, or a livestock production system. this diversity of components in the system reduces the intensity of diseases, e.g. foliar diseases. this study aimed to detect the fungal microorganisms associated with the leaf spots of plant species that are part of the agroforestry production systems of the peruvian farmers from yurimaguas and to determine the level of incidence of the leaf spots in the systems of agroforestry production. different land cropping systems were implemented in the farms, such as palm (bactris gasipaes) to produce palm hearts, cocoa (theobroma cacao), and plantain (mussa sp.). also, silvopastoral systems with fast and slow growing timber species and cattle with pastures for grazing and reforestation in areas of secondary forests in a state of degradation. prospecting, collecting, and determining the incidence of diseases in each farm were carried out and later they were identified with molecular methods using the primers its 1 and its 4. the symptoms predominantly observed were, leaf spots in cocoa (rootstock), palm, and plantain. symptoms like wilting, decline, or rot were not observed. the incidence was evaluated in two collection periods (2018 and 2019). the fungi isolated from the leaf spots were pestalotiopsis sydowiana and colletotrichum siamense as causative agents of leaf spots on palm and cocoa, and mycosphaerella fijiensis on plantain. when determining the incidence from april 2018 to october 2019, a decrease in this parameter (incidence) was observed for farms with palm, especially in those where the production system was improved by the use of fertilizants as a requirement of the crop. it was concluded that the highest intensity of foliar diseases occurred in agricultural systems with monoculture of palm with 100 % at the beginning of the evaluation, and for agroforestry systems in the silvopastoral prototype, it was only detected in a range of 0 % to 25 %. keywords: agroforestry systems, leaf spots, palm peri resumen un sistema agroforestal (saf) se caracteriza por tener una diversidad de componentes como especies forestales maderables y no maderables, así como cultivos, pastos o un sistema de producción ganadera. esta diversidad de componentes en el sistema reduce la intensidad de las enfermedades, por ejemplo, las foliares. 1 universidad nacional agraria la molina, facultad de agronomía, departamento académico de fitopatología, lima, peru. 2 universidad nacional agraria la molina, facultad de agronomía, departamento académico de horticultura, docente, lima, perú. 3 universidad nacional agraria de la selva, área de suelos, facultad de agronomía, tingo maría. huánuco, perú. 4 instituto de investigación peruano de la amazonía, iquitos, perú. 5 universidad nacional agraria la molina, facultad de agronomía, departamento académico de suelos, docente, lima, perú. how to cite this article: aragón, l, huarhua, h., cerna, m., flores, j., dueñas, f., lao, c., solis, r., alegre, j. (2022). incidence of leaf diseases in the agroforestry systems at yurimaguas, peru. peruvian journal of agronomy, 6(1), 93-102. https://doi.org/10.21704/pja.v6i1.1733 incidence of leaf diseases in the agroforestry systems at yurimaguas, peru january april 2022 94 los objetivos fueron detectar los microorganismos fúngicos asociados a las manchas foliares de las especies vegetales que forman parte de los sistemas de producción agroforestal de los agricultores de yurimaguas (loreto) que participaron en el sla y determinar el nivel de incidencia de las manchas foliares en los sistemas de producción agroforestal. en estas fincas se implementaron diferentes sistemas de cultivo de la tierra, como la palma (bactris gasipaes) para producir palmitos y cacao (theobroma cacao), incluyendo el plátano (mussa sp.). también, sistemas silvopastoriles con especies maderables de crecimiento rápido y lento y ganado con pastos para el pastoreo y la reforestación en áreas de bosques secundarios en estado de degradación. se realizó la prospección, recolección y determinación de la incidencia de las enfermedades en cada finca, y posteriormente se identificaron con métodos moleculares utilizando los cebadores its 1 e its 4. los síntomas que se observaron, predominantemente, fueron manchas foliares en cacao (patrón), palma y plátano. no se observaron síntomas de marchitamiento, decaimiento o podredumbre. la incidencia se evaluó en dos periodos de recolección (2018 y 2019). los hongos aislados de las manchas foliares fueron pestalotiopsis sydowiana y colletotrichum siamense como agentes causantes de las manchas foliares en palma y cacao, y mycosphaerella fijiensis en plátano. al determinar la incidencia desde abril de 2018 hasta octubre de 2019, se observó una disminución de este parámetro para las fincas con palma, especialmente en aquellas donde la implementación consistió en mejorar el sistema de producción a través de la fertilización con base en los requerimientos del cultivo. se concluyó que la mayor intensidad de enfermedades foliares se presentó en los sistemas agrícolas con monocultivo de palma con un 100 % al inicio de la evaluación, y para los sistemas agroforestales en el prototipo silvopastoril, solo se detectó en un rango de 0 % a 25 %. palabras clave: sistemas agroforestales, manchas foliares, palma 1. introduction an agroforestry system is production in which, unlike a monoculture, at least two or more plant species coexist; or plants and animals; but one of them must be a perennial tree species (nair, 2014). this greater diversity of species makes the existing ecology more complex than if it were a monoculture. from a phytopathological point of view, their development and productivity can affect commercial production fields. in addition, phytopathogens can affect the quality of the harvested product generating economic losses. these phytopathogens can be fungi, bacteria, viruses, or nematodes, among others. favorable conditions for the pathogen growth, such as temperature, relative humidity, optimal rainfall, etc. together with the host susceptibility reaction, produce high levels of disease intensity, generating epidemics (agrios 2005). greater activity of these behaviors is observed in intensive monoculture systems. but, under an agroforestry system, in which there are different plant species, the behavior of phytopathogens is expected to be different because of the coexistence of a greater diversity of plant species. banerjee et al (2015) found that agroforestry systems were characterized by having a greater diversity of bacterial communities in their biofilm, such as bacteria of the genera arthrobacter, acidobacteria_gp16, burkholderia, rhodanobacter, and rhizobium at the level of the rhizosphere. müller et al (2006) reported the role of microorganisms in the phyllosphere of plant species in forests, a role related to nutrient transformation processes. additionally, the action of these microorganisms is attributed to changes in environmental biotic behavior, such as the increase in the population density of certain groups of microorganisms, which would impact their metabolic activity. díddier and castro (2017) reported that, in the experience of the organic banana agroforestry system in costa rica, there is coexistence with sigatoka (whose causal agent is mycosphaerella fijiensis); under a condition of enough leaves and clusters; with fruits that satisfy the quality standards for the national and export markets. they also showed that nematicide applications were not carried out because the nematode populations were below the damage thresholds, and the increase in biodiversity in the system explained this. mosquera-mena (2013) mentioned that small producers in the urabá region from antioquia, colombia, reported a favorable phytosanitary balance in agroforestry systems because these systems favored a greater diversity of microorganisms. aragón, l, huarhua, h., cerna, m., flores, j., dueñas, f., lao, c., solis, r., alegre, j. peruvian journal of agronomy 6(1): 93-102 (2022) https://doi.org/10.21704/pja.v6i1.1733 95 according to the experience of the project to recover natural ecosystems in the caquetaño foothills in ecuador (1998), one advantage of agroforestry systems was the reduction of pest and disease problems through sanitary pruning practices. montagnini et al. (2015) analyzed the effect of output under shade in the decrease or increase of pests and diseases in the agroforestry system of coffee production. for example, in colletotrichum kahawai, the presence of trees reduced the spread of pathogen propagules by reducing the impact of rain. under a shady environment, the activity of biocontrol fungi, such as beauveria bassiana and lecanicillium lecanii on cbb and rust, respectively, were favored. based on these behaviors in the different multifunctional systems, the following objectives were proposed in the present work; detect fungal microorganisms associated with leaf diseases such as leaf spots of plant species that are part of the agroforestry production systems of farmers from yurimaguas (loreto) who participated in the sustainable landscapes for the amazon project, and determine the level of incidence of leaf diseases in agroforestry production systems as a comparative parameter between the different multifunctional systems. 2. material and methods location the study was carried out at the yurimaguas district, alto amazonas province, loreto region, perú (figure 1). this zone was the development area of the “sustainable landscapes for the amazon project” funded by ciat (centro internacional de agricultura tropical) – unalm (universidad nacional agraria la molina). eighteen farmers participated in the project committed certain areas for implementation or conservation purposes (table 1). in this study, the farmers expressed their interest in two types of strategies for the sustainable use of the forest. on the one hand, there is the implementation of systems with different components, among which the agro-forestry systems (saf) stand out, followed by the silvopastoral systems (ssp) with natural pastures or paddocks; forest enrichment systems (sef), and finally reforestation (r). there are different saf´s: palm heart implementation, cocoa implementation, palm crop improvement, and cocoa crop improvement. forest enrichment (sef) corresponds to forest areas with more tree species installed. in reforestation (r), there are pasture areas with tree species established (table 1). detection of fungal microorganisms associated with leaf spots of plant species in the agroforestry production systems phytopathological sampling a disease evaluation was made for each agroforestry system (18 in total), after the diseased plants be collected, following the methodology reported by french & hebert (1982). plant organs showing any disease symptom were collected in propylene plastic bags, at least ten units per type of symptom, and were kept under refrigeration conditions, because of the high temperatures in yurimaguas, to later be transported to the unalm plant pathology laboratory (french & herbert 1982). isolation and maintenance the samples were analyzed at the plant pathology laboratory from the unalm. the collected samples were washed with plain water to remove traces of soil or dust. they were then disinfected with ethanol (70%) for one minute and finally rinsed with sterilized distilled water for the same period. the diseased tissue sections of the disinfected samples were placed in petri dishes containing pdaa (acidified potato dextrose agar) culture medium. the plates were incubated at 25 °c in the dark. after five days, the developed cultures were checked (french & hebert, 1982). the purified isolates were kept in tubes containing pda medium under refrigeration (10 °c). incidence of leaf diseases in the agroforestry systems at yurimaguas, peru january april 2022 96 location of soil monitoring point map figure 1. location map of the farms in the district of yurimaguas (loreto) that participated in the sustainable landscapes for the amazon project, according to geo-referencing (elaboration: bach. andré mauricio valderrama espinoza). identification of isolates morphological identification. the preparations of the fungal structures were made on slides and coverslips. the propagative structures were visualized through the compound microscope. barnett’s (1999) identification key was used for identification at the gender level. molecular identification. the fungi were cultivated in pda for 3 to 5 days at 25 °c to obtain the extraction material. the dna extraction methodology was carried out from agar described by saitoh et al. (2006) modified by huarhua et al. (2020). a 5 to 10 mm mycelium block was used with agar, which was placed in a 1.5 ml eppendorf tube to which 500 microliters of lysis buffer (100 mm tris hcl, 10 mm ethylene diamine tetraacetic acid [edta], 1m kcl; ph 8.0). the mixture was kept at room temperature for 10 minutes, then it was triturated with micropistils, then phenol-chloroform isoamyl-alcohol (24: 24: 1) was applied, and it was centrifuged for 10 minutes at 12000 rpm, it was recovered the upper aqueous phase, and a similar volume was added to the iso-propanol collection to be incubated at -20 °c for 15 minutes. after this time, it was centrifuged at 12000 rpm for 10 minutes at room temperature, and the supernatant was discarded; then, a wash was carried out with 1 ml of 70% ethanol and centrifuged for 5 minutes. after that time, the ethanol was removed, and it was left to dry for 2 hours. finally, the dna was resuspended in 30 µl of ultra-pure water and stored at -20 °c. the concentration and quality of dna were determined using the nanodrop 2000 spectrophotometer and by 1% agarose gel electrophoresis, which was stained with hydragreen and run at 90 v for 30 minutes. genomic dna was used as the template strand for pcr to amplify the internal transcribed space (its) of the ribosomal dna region, including 5.8s rdna and partial regions of the 18s and 25s ribosomal subunits for which the primers were used. its 1 (tccgtaggtgaacctgcgg) and its 4 (tcctccgcttattgatatgc) as described by white et al. (1990). aragón, l, huarhua, h., cerna, m., flores, j., dueñas, f., lao, c., solis, r., alegre, j. peruvian journal of agronomy 6(1): 93-102 (2022) https://doi.org/10.21704/pja.v6i1.1733 97 table 1. result of the phytopathological analyzes and molecular identification, as well as the incidence observed in the 2018 and 2019 sampling of the farms (each one corresponds to a farmer) of yurimaguas (loreto) of the sustainable landscapes project for the amazon. id_soils implementation of: area (ha) symptons pathogen incidence (april, 2018) incidence (october, 2019) y04 palm heart improvement 0.6 leaf spots colletotrichum siamense, neopestalotiopsis foedans 100% 60% implementation of cocoa in agroforestry 2.1 leaf spots cercospora sp 20% no y07 palm heart implementation 1 leaf spots neopestalotiopsis foedans colletotrichum siamense 100% 90% reforestation 1 without leaf spots no no y08 improvement palm heart 2.9 leaf spots colletotrichum siamense, neopestalotiopsis foedans 50% 10% y13 reforestation 2.7 without symptons no no y19 silvopastoral systems 3 without symptons no no reforestation 1.9 without symptons no no y26 cocoa improvement in agroforesty 1.4 sigatoka paracercospora fijiensis 80% 70% reforestation 2.3 without leaf spots no no y36 silvopastoral systems 2.6 without symptons no no reforestation 2.7 without symptons no no y37 silvopastoral systems 4.2 leaf spots in paliperro cercospora sp 10% 5% y38 implementation of cocoa in agroforestry 0.9 without symptons no no reforestation 1.3 without symptons no no y39 silvopastoral systems 3 without symptons no no y40 reforestation 2.5 without symptons no no y41 implementation of cocoa in agroforestry 1.8 sigatoka (banano); leaf spot (cocoa) paracercospora fijiensis (banano); colletotrichum siamense (cacao) 80% 25% 80% 25% forest enrichment 2 without symptons no no y43 cocoa improvement in agroforesty 1.5 die-back lasiodiplodia theobromae 20% 20% implementation of cocoa in agroforestry 2 without symptons no no y45 implementation of cocoa in agroforestry 1 poor development (cocoa) sigatoka (plantain) abiotic, unsuitable weather (cocoa) paracercospora fijiensis 100% 100% 100% 100% silvopastoral systems 2.7 without symptons no no y47 implementation of cocoa in agroforestry 1.25 without symptons no no reforestation 0.8 without symptons no no y48 palm heart implementation 1.4 leaf spots colletotrichum siamense, neopestalotiopsis foedans 60% 40% implementation of cocoa in agroforestry 0.9 without symptons no no reforestation 2.2 without symptons no no y50 silvopastoral systems 2.8 without symptons no no forest enrichment 3 without symptons no no y51 silvopastoral systems 3.6 without symptons no no the total pcr reaction mix (25 µl) contained 1 µl of genomic dna (50 to 100 ng / µl), 4 µl of 10x pcr buffer (actaq ™), 2 out of 2.5 mm dntps (actaq ™), 0.125 µl (5 u) of the enzyme taq dna polymerase (actaqtm) and 0.5 µl of each primer at a concentration of 20 mm. the amplification was carried out with a thermal cycler (thermo scientific), following the next reaction cycles: initial denaturation of 94 °c for 2 minutes; followed by 35 cycles of denaturation at 98 °c for 1 min, a hybridization temperature at 58 °c for 1 min, elongation of 72 incidence of leaf diseases in the agroforestry systems at yurimaguas, peru january april 2022 98 figure 2. leaf spots on palm heart. the small, blackish spots correspond to colletotrichum siamense and the straw-colored spots to neopestalotiopsis foedans. figure 3. banana leaf spots caused by mycosphaerella fijiensis; the causative agent of black leaf streak disease. °c for 2 min, and final elongation temperature of 72 °c for 10 min (ferrer et al., 2001; kumar & shukla, 2005; huarhua et al., 2018; rep et al., 2004; inami et al., 2014). the pcr products were separated by 2% agarose gel electrophoresis (0.5 x tae buffer) containing hidragreen and run at 90v for 30 minutes. for the visualization of the fragments, the ultraviolet light transilluminator (uvp brand) was used, the 100bp ladder (promega) was used as a marker. the pcr amplified fragments were sequenced in both directions. for sequencing, the eluted products were sent to macrogen korea for processing and delivery of the chromatograms, and these were “cleaned” of indeterminacies with the mega 7 program (kumar et al., 2016), thus obtaining the sequences that were compared using blast (basic local alignment search tool; https://blast.ncbi.nlm.nih.gov/blast.cgi) to determine similarities in genbank. determination of the level of incidence of leaf spots in agroforestry production systems incidence assessment two visits were made to assess the incidence of diseases in the plant species in each of the agroforestry systems. incidence assessment was calculated by the percentage of affected plants concerning the total, one for each year during the duration of the project (2018, 2019) (french & hebert, 1982). it should be noted that in 2016 a preliminary survey trip was made and in 2017 the evaluation of diseases in each agroforestry production system was done. 3. results and discussion between 2018 and 2019, the fields of the 18 farmers who participated in the sustainable landscapes for the amazon project (ciat unalm) were visited. the production systems were characterized by having different agroforestry systems: cocoa implementation systems, palm implementation to produce palm heart, cocoa maintenance, palm maintenance, silvopastoral design, forest enrichment, and reforestation (table 1). symptoms of leaf spots were observed predominantly in systems with less diversity of species such as those for the implementation or maintenance of palm, and only in an agroforestry system for implementing cocoa (it was found in the installation of the rootstocks): as well as in plantain as part of the cocoa implementation system. lasiodiplodia theobromae, which was causing regressive death in a cocoa holding system, was isolated but it was a rare case. this fungus behaves as an endophyte, and under stress conditions, its behavior turns as a phytopathogen; therefore, it aragón, l, huarhua, h., cerna, m., flores, j., dueñas, f., lao, c., solis, r., alegre, j. peruvian journal of agronomy 6(1): 93-102 (2022) https://doi.org/10.21704/pja.v6i1.1733 99 is an indicator of a stress condition in plants. the farmer reported that the focus of plants in which this phytopathogen was detected corresponded to a flood zone accentuated with problems in fertilization. detection of fungal microorganisms associated with leaf spots of plant species in the agroforestry production systems table 1 shows the results of the phytopathological analyzes of each of the farms in which leaf spots were detected. the species identified morphologically and molecularly corresponded to the species pestalotiopsis sydowiana and colletotrichum siamense as causal agents of leaf spots in palm and cocoa; and mycosphaerella fijiensis on plantain. figures 2 and 3 show the characteristic symptoms and those caused by the isolated pathogens. no necrotic lesions were detected in pastures or tree species located between farms, nor in the forest species situated in the reforestation or forest strengthening systems in the silvopastoral systems. molecular identification the method used to get the genomic dna of the leaf spot isolates from the farms is described by saitoh et al. (2006). genomic dna was evaluated to determine purity and sufficient concentration to perform the polymerase chain reaction (pcr). the concentration of the evaluated samples was found between 90.1 ng / µl and 428 ng / µl. the samples’ pcr amplification for the its region of rdna, generated products of 500 to 600 base pairs (bp) with the primers its 1 and its 4. the analysis of the sequences compared with the ncbi database -blast showed a percent identity of 100%. the identification results were: pestalotiopsis sydowiana (y04, y07, y08, y41, and y48), lasiodiplodia theobromae (y43). in the case of other isolates from the same farms y04, y07, y08, y41, and y48, it was possible to determine based on the its region of the isolates that the colletotrichum species had 100% similarity of their sequences with the colletotrichum siamense, colletotrichum gloesporioides, colletotrichum karsti (table 2). belisário et al. (2020) reported n. foedans as a causal agent of leaf spots in licuala grandis (totuma, ornamental palm). maharachchikumbura et al. (2016) identified the new genera neopestalotiopsis and pseudopestalotiopsis (from the genus pestalotiopsis) based on the regions of the genome that encode the internal transcribed space (its), partial β-tubulin (tub ), and partial translation of elongation factor 1 alpha (tef); such new genera do not show differences with the morphological structures of the genus pestalotiopsis; this is also reported by norphanphoun et al. (2019) for which pestalotiopsis sp is recognized as a cryptic species. morsbach et al. (1998) said colletotrichum is a causal agent of necrotic lesions in the nursery stage or the first phenological stages of the palm crop. arroyo et al. (2004) and peña (1996) reported colletotrichum sp as a causal agent of leaf spots, which they call black leaf spot; which appear as small black spots surrounded by a table 2. results of molecular id of the isolates (from the plant samples with symptoms of leaf spots and regressive death) compared with the ncbi. mu species max score query cover % identity id ncbi y48 pestalotiopsis sydowiana 963 100% 100% mn856236.1 y04 pestalotiopsis sydowiana 965 100% 100% mn856236.1 y48 colletotrichum siamense / colletotrichum gloeosporioides 961 100% 100% mk184442.1 mn548460.1 y04 colletotrichum siamense/ colletotrichum karsti / colletotrichum gloeosporioides 1007 100% 100% mn635698.1 mn486559.1 mh700456.1 y43 lasiodiplodia theobromae 950 100% 100% mt103324.1 incidence of leaf diseases in the agroforestry systems at yurimaguas, peru january april 2022 100 small chlorotic circular halo; as it was observed in the palm plants of the farms of yurimaguas. weir et al. (2012) determined colletotrichum gloeosporioides as a species complex and, due to the identification of its genes, several species of colletotrichum (such as c. siamense) are included. james et al. (2014) reported colletotrichum siamense in cacao, as it was found in the molecular identification of isolates from cacao from the yurimaguas farms. determination of the incidence of leaf spots in agroforestry production systems table 1 also shows the incidence percentages determined during the visits to the farms. a decrease in the incidence from 2018 to 2019 is distinguished for the improvement of cocoa, cocoa implementation, implementation of palm, and improvement of palm plantations. in plantain, that were part of the cocoa implementation and improvement systems, the reduction was minimal, or there was no incidence decrease. the causative agents of leaf spots have a necrotrophic behavior; this means that, according to their physiology of parasitism, they cause the death of plant living tissue mainly through toxins. the pathogen feeds on dead tissue as a consequence of the action of these metabolites. with plants stressed by low fertilization levels or extreme cases of fertilizer deficiencies, they become more susceptible to the action of necrotrophic pathogens. this is supported by pornsuriya et al (2020), who showed that neopestalotiopsis species (pestalotiopsis) are weak and secondary pathogens. alfenas et al., 2009, also mentioned the opportunistic behavior of pestalotiopsis spp agent, the causant of eucalyptus leaf spots, as well as the fact of infecting physiologically weak plants. when determining the incidence from april 2018 to october 2019, a decrease in this parameter could be observed for farms with palm, especially in those which the typology of the palm trees was improved through an increase in the fertilization of the farms according to the crop. by having plants with adequate nutrient requirements, susceptibility to necrotrophs was reduced. bovi (1993), cited by morsbach et al. (1998), also refers to the fact that the action of colletotrichum can be minimized with good nutrition. arroyo et al. (2004) show that colletotrichum sp occurs mainly during the first six months of the crop or in the first two-thirds of the leaves that differentiate leaflets; as it could be seen in the plantations of the yurimaguas farms, especially those in which the fertilization levels were not adequate. a constant harvest characterized the palm heart crop, therefore fertilization of macroelements and microelements is highly required. in the case of banana, based on the concept of the disease triangle; the banana variety was susceptible to sigatoka, the environmental conditions favored that pathogen, and the inoculum source existed in the production system, so the disease development was clear. in this case, no incidence reduction was observed, because no palliative measurements had been implemented, since the aim of the plantain installation was to generate shade for the cacao rootstocks that were later grafted with a commercial variety, so there was no requirement for the bananas crop fertilization. although the fertilization practice was improved by reducing susceptibility of the palm trees in the systems implementation and maintenance, the influence of the diversity of microorganisms action, existing in the biofilm of the phyllosphere, was not ruled out (müller et al, 2006). it could be corroborated through the microbiome study which it was not considered as part of the ciat project. this reduction can also be explained by the induction of systemic resistance generated by the microbial load existing in the rhizosphere (banerjee et al, 2015). 4. conclusions the fungi pestalotiopsis sydowiana, colletotrichum siamense, and mycosphaerella fijiensis were isolated and identified as the causative agents of leaf spots on palm, cocoa, and plantain leaves, respectively. they reported the lowest diseases incidence in the silvopastoral systems, and the highest occurred in monocultures. therefore, a greater diversity of plant species within a production aragón, l, huarhua, h., cerna, m., flores, j., dueñas, f., lao, c., solis, r., alegre, j. peruvian journal of agronomy 6(1): 93-102 (2022) https://doi.org/10.21704/pja.v6i1.1733 101 system under the yurimaguas’ farms conditions leads to a reduce development of leaf diseases. acknowledgments the development of this research was possible thanks to the funds granted by the ciat-unalm project, vlir unalm, and the diagnosis of phytopathology clinic laboratory. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. orcid and e-mail l. aragón lili@lamolina.edu.pe 0000-0003-0312-5020 h. huarhua medalihuarhua@lamolina.edu.pe 0000-0002-1482-8170 m. cerna mcerna@lamolina.edu.pe 0000 0003 1182 872x j. flores gerardo@lamolina.edu.pe 0000 002 2651 5446 f. dueñas fduenas@lamolina.edu.pe 0000-0002-6149-3334 c.p. lao ceila.lao@unas.edu.pe 0000-0002-0125-2133 r. solis reynaldosolisleyva@gmail.com 0000-0002-5905-4922 j. alegre jalegre@lamolina.edu.pe 0000-0002-7282-045x 5. references agrios, g. 1995. fitopatología. editorial limusa s.a. méxico. 838 p. alfenas, a. c., zauza, e. a. v., mafia, r. g., & assis, t. f. 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(pp. 315–322). academic, new york, peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 05 july 2021 accepted for publication: 28 november 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina https://doi.org/ 10.21704/pja.v5i3.1846 control of avocado root rot caused by phytophthora cinnamomi with different trichoderma strains at chavimochic irrigation project control de la pudrición de la raíz del palto causada por phytophthora cinnamomi con diferentes cepas de trichoderma en la irrigación de chavimochic paul gastañadui1, rocio moreno2, patricia quiroz-delgado1, walter apazatapia1* *corresponding author: wapaza@lamolina.edu.pe *https://orcid.org/0000-0001-7510-8866 abstract avocado root rot caused by phytophthora cinnamomi is one of the main problems affecting avocado (persea americana) cultivation in peru, especially at the chavimochic irrigation project. the objective of this study was to evaluate the effect of different trichoderma strains on the control of phytophthora cinnamomi in zutano rootstock under greenhouse conditions. five isolates of trichoderma were tested: trichoderma sp. (chav01); trichoderma harzianum (chavo2); trichoderma harzianum (unalm01); trichoderma viride (unalm02); and a commercial strain of trichoderma sp. evaluations were performed at 30, 45, and 60 days. all isolates colonized the rhizosphere of the avocado. no relation was found between the formation of more trichoderma colonies and phytophthora improved control. all strains controlled the root rot, but chav01 and chav02 showed the greatest diameter of stem, dry matter in the root, and percentage of healthy root in comparison with unalm01, unalm02, and the commercial strain. thus, the native isolates of trichoderma from the chavimochic area can be added to the list of potential new trichoderma species to control phytophthora cinnamomi. keywords: trichoderma, avocado, phytophthora cinnamomi, biological control, root rot resumen la pudrición de la raíz del palto causada por phytophthora cinnamomi es uno de los principales problemas que afectan al cultivo de la palta (persea americana) en el perú, especialmente en el proyecto de irrigación chavimochic. el objetivo de este estudio fue evaluar el efecto de diferentes cepas de trichoderma en el control de phytophthora cinnamomi en portainjertos de zutano bajo condiciones de invernadero. se probaron cinco aislamientos de trichoderma: trichoderma sp. (chav01); trichoderma harzianum (chavo2); trichoderma harzianum (unalm01); trichoderma viride (unalm02); y una cepa comercial de trichoderma sp. las evaluaciones se realizaron a los 30, 45 y 60 días. todos los aislados colonizaron la rizosfera del aguacate. 1 universidad nacional agraria la molina, facultad de agronomía, lima, perú. 2 arato peru s.a. trujillo, perú, lima, perú. how to cite this article: gastañadui, p., moreno, r., quiroz-delgado, p., & apaza-tapia, w. (2021). control of avocado root rot caused by phytophthora cinnamomi with different trichoderma strains at chavimochic irrigation project. peruvian journal of agronomy, 5(3), 78–86. https://doi.org/10.21704/pja.v5i3.1846 p. gastañadui; r. moreno, p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(3): 78–86 (2021) 79 no se encontró relación entre la formación de más colonias de trichoderma y la mejora del control de phytophthora. todas las cepas controlaron la podredumbre de la raíz, pero chav01 y chav02 mostraron el mayor diámetro de tallo, materia seca en la raíz y porcentaje de raíz sana en comparación con unalm01, unalm02 y la cepa comercial. por lo tanto, los aislados nativos de trichoderma de la zona de chavimochic pueden ser añadidos a la lista de nuevas especies potenciales de trichoderma para controlar phytophthora cinnamomi. palabras clave: trichoderma, aguacate, phytophthora cinnamomi, control biológico, pudrición de la raíz introduction root rot caused by phytophthora cinnamomi is one of the most destructive diseases in global avocado cultivation. it attacks trees of all ages, including those cultivated under greenhouse conditions, and causes significant economic losses in avocado crops (hardham et al., 2018; coffey, 1987; ploetz, 2013). p. cinnamomi was first described in 1922 by rands as the causal agent of stem canker in cinnamon trees in sumatra, and was first discovered on avocado trees (persea americana) in puerto rico in 1929, where it caused severe root rot. since then, p. cinnamomi has been reported in over 70 countries, with a wide host range (zentmeyer, 1985). the use of fungicides for the control of phytopathogenic fungi diseases has limitations, making biological control methods more appealing. the biological control of p. cinnamomi through the incorporation of different agents into the soil has been investigated by different authors, who have shown that bacteria present in the soil, such as pseudomonas spp. and streptomyces spp. inhibit the in vitro growth of p. cinnamomi (mass & kotzé, 1990; finlay & mccracken, 1991; stirling et al., 1992). likewise, there are a large number of fungal species that show antagonistic effects toward p. cinnamomi, such as trichoderma spp., myrothecium roridum, aspergillus spp., or paecilomyces spp. (reeves, 1975; gees & coffey, 1989; casale, 1990; finlay & mccracken, 1991; duvenhage & kotzé, 1993; mcleod et al., 1995). soil suppressiveness is one of the main factors inhibiting the development of phytophthora cinnamomi. this suppressiveness can be increased with the inoculation of trichoderma, gliocladium, bacillus, and others (erwin & ribeiro, 1996). in peru, chavimochic irrigation is one of the main avocado cultivation zones, with an area of 7500 ha. p. cinnamomi, is one of the main pathogens causing avocado root rot in this region. it is estimated that 10% of plantations are affected by this disease (villavicencio, 2018). the main source of inoculum are the chlamydospores brought in by irrigation water from the santa river (ancash, peru). this situation lead us to investigate the effectiveness of trichoderma strains by incorporating them into the soil for the control and isolation of p. cinnamomi, which was obtained from avocado plantations in the chavimochic valley. the objectives of this research were: i) to determine the best trichoderma strain for the control of p. cinnamomi in avocado plantations; and ii) to evaluate the effect of the different strains on the biometry of the avocado zutano rootstock inoculated with p. cinnamomi. materials and methods this experiment was performed in the greenhouse facilities at the arato perú s.a. company, in the province of virú, department of la libertad, peru from september to december (2015) with temperatures fluctuating between 17 ℃ and 25 ℃. plant material zutano variety avocado seeds were disinfected by immersion for 10 minutes in a 0.1% methyl thiophanate + thiram (homai wp) solution. the seeds were pregerminated. once they presented a radicle of 3 cm, they were sown in 8 liter polypropylene bags containing a mixture of sterile sand plus earthworm humus in the ratio of 3:1. once the plants had a growth of 60 cm and 8 formed leaves, they were inoculated with p. cinnamomi. control of avocado root rot caused by phytophthora cinnamomi with different trichoderma strains at chavimochic irrigation project september december 2021 80 inoculation method plant roots with wilt symptoms and regressive death were planted on a selective corn meal agar (nutriselect® basic, merck) with the antibiotic pimaricin ampicillin rifampicin benomyl. once a pure isolation of p. cinnamomi was obtained, it was increased on plates with papa dextrose agar-difco medium. slices of 3 cm in diameter with mycelial growth were then extracted from the medium and placed in 200 g bags with sterile wheat. these bags were incubated at 25℃ for 21 days until p. cinnamomi completed growth throughout the bag. for phytophthora inoculation, 90-day-old avocado seedlings were placed on containers with water for 24 hours. once the substrate was saturated, 35 g of wheat with mycelium of p. cinnamomi was placed around the neck of each plant and covered with the same saturated substrate. treatments with trichoderma the different trichoderma treatments used are shown in table 1. the trichoderma isolates (cha01 and cha02) were obtained from soil collected from the rhizosphere of healthy avocado plants from the chavimochic irrigation project. the isolate chav01 was obtained in the chao area from the rhizosphere soil of hass avocado rootstock grafted on zutano rootstock. the isolation of t. harzianum was isolated from the virú area in the rhizosphere soil of hass avocado rootstock grafted on lula rootstock. the isolation was carried out in papa dextrose agar oxytetracycline (pdao) medium through serial dilutions. the other isolates were provided by the institutions indicated in table 1. identification to genus and species level was carried out at the phytopathology diagnostic clinic of the universidad nacional agraria la molina (lima, perú). the inoculations with the antagonist trichoderma was performed as follows. for the first inoculation, each trichoderma isolate was inoculated in bags with sterile corn. in the sowing stage of the zutano avocado seeds in substrate, 20 g of corn with each strain of trichoderma growth were used per 8 liter substrate bag per plant. for the second inoculation, the trichoderma strains were extracted from the bags of wheat with a trichoderma suspension of 1 × 108 colony-forming units (cfu) per milliliter of sterile water solution. this was applied directly to the roots using 200 cm3 of the solution per plant 30 days after the avocado seeds were sown in the substrate. for the third inoculation, the same procedure was carried out 30 days after the second trichoderma inoculation. evaluation three evaluations were performed at 30, 45, and 60 days after inoculation. for each evaluation there were 10 replicates in a completely randomized design. for the evaluation, the plants were extracted from the bags and the table 1. trichoderma strains used for the control of p. cinnamomi in avocado under greenhouse conditions. chavimochic trujillo. trial strains of trichoderma code place of origin t1 trichoderma sp. chav01 chavimochic irrigation project t2 trichoderma harzianum chav02 chavimochic irrigation project t3 trichoderma harzianum unalm01 universidad nacional agraria la molina(a) t4 trichoderma viride unalm02 universidad nacional agraria la molina(a) t5 trichoderma sp. trichomax sol solagro s.a.c.(b) t6 inoculated control (phytophthora cinnamomi) chavimochic irrigation project t7 absolute control (a) isolate from fungi collection of phytopathology department of universidad nacional agraria la molina. (b) isolate from solagro sac company, marketed under the name of trichomax p. gastañadui; r. moreno, p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(3): 78–86 (2021) 81 results and discussion biometric parameters the results are shown in table 2 and fig. 2. after 60 days, the stem diameters in chav01 and chav02 were statistically different than the inoculated control. the stem diameter of the rest of the treatments did not differ from the control. the dry weight of the roots from chav01 and chav02 also showed differences to the p. cinnamomi-inoculated control, but chav01 did not show any differences to the control that was not inoculated. no differences were observed among the different treatments in total root length, but statistical differences were observed between the treated samples and both controls. of these parameters, dry weight best differentiates and evaluates the effects of the different trichoderma strains on p. cinnamomi due to the fact that roots affected by p. cinnamomi normally undergo a process of root necrosis and tissue death that significantly reduces their weight. sid ahmed et al. (1999) found that the control of phytophthora capsica in peppers using trichoderma harzianum was best indicated by the dry matter weight results. root systems were washed, then the biometric parameters were evaluated, including stem diameter, dry weight, total root length, and the percentage of healthy root. stem diameter was measured using a vernier by measuring the neck of the plant 2 cm from the end of the seed. the dry weight of each root was determined by allowing the fresh roots to air dry for 3 days, then drying the roots in a paper bag in the oven for another 3–4 days at an average temperature of 70 ℃. the total root length was measured by photographing each experimental unit, then processing the photos with the asses 2.0 program (lamari, 2008). the percentage of healthy root was determined visually by visual estimation using the graphic scale shown in fig. 1 after the fresh roots were washed. this scale had 11 classes ranging from 0 % to 100 % healthy root. trichoderma colony-forming units rhizosphere samples were taken from each evaluation unit 45 and 60 days after inoculation with p. cinnamomi. these samples were diluted in distilled water, then sown in pdao culture medium to quantify the trichoderma colonies. 1: 100 % healthy root 2: 95 % to 99 % healthy root 3: 85 % to 94 % healthy root 4: 75 % to 84 % healthy root 5: 74 % to 65 % healthy root 6: 64 % to 55 % healthy root 7: 45 % to 54 % healthy root 8: 44 % to 35 % healthy root 9: 34 % to 25 % healthy root 10: 24 % a 15 % healthy root 11: less than 14 % healthy root healthy root percentage evaluation scale fig. 1. pictographic scale used to evaluate the percentage of healthy avocado roots inoculated with phytophthora cinnamomi. control of avocado root rot caused by phytophthora cinnamomi with different trichoderma strains at chavimochic irrigation project september december 2021 82 fig. 2. stem diameter (cm), root dry weight (g), and root length (cm) of avocado seedlings inoculated with phytophthora cinnamomi and treated with different trichoderma isolates for 60 days p. gastañadui; r. moreno, p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(3): 78–86 (2021) 83 healthy root percentage the healthy root percentage results are shown in table 3, and figs. 3 and 4. the healthy root percentage and incidence of symptoms in the aerial part of the plants made the differences between the different trichoderma isolates clearer. it was observed that the best treatments were chav01 and chav02, which both showed statistical differences to the inoculated control and the rest of the treatments. these two isolates were obtained from the rhizospheres of healthy plants from the chavimochic irrigation project, so their adaptations to the soil conditions and the irrigation environment was better than that of the isolates from the universidad nacional agraria la molina, which were obtained from capsicum and tomato plants. differences between strains is a characteristic of trichoderma as antagonists (bae et al., 2011). the commercial strain trichomax generally has a lower control efficiency than the table 2. biometric parameters: stem diameter (cm), root dry weight (g) and root length (cm) in avocado seedlings inoculated with phytophthora cinnamomi treated with different trichoderma strains stem diameter (cm)(a) root dry weight (g) (a) root length (cm) (a) treat. antagonist strain 1° eval. 2° eval. 3° eval. 1° eval. 2° eval. 3° eval. 1° eval. 2° eval. 3° eval. 1 trichoderma sp. (chav01) 7.01 ab 8.04 bc 9.67 ab 1.56 b 2.17 bc 2.75 ab 15.4 b 25.4 abc 28.0 b 2 t. harzianum (chav02) 6.76 b 8.62 ab 9.48 bc 1.51 b 2.37 ab 2.43 bc 14.6 b 27.4 ab 27.6 b 3 t. harzianum (unalm01) 7.18 ab 6.38 d 7.80 d 1.57 b 1.98 bc 2.11 cd 16.0 b 17.8 cd 27.8 b 4 t. viride (unalm01) 6.81 b 8.04 bc 8.56 bcd 1.52 b 2.12 bc 2.19 c 15.9 b 25.2 ab 25.4 b 5 trichomax solagro 7.38 ab 7.04 cd 7.61 d 1.54 b 1.72 c 1.94 cd 17.6 b 22.0 bcd 25.6 b 6 inoculated control 6.56 b 7.19 bcd 8.07 cd 1.45 b 1.70 c 1.65 d 17.7 b 16.8 d 13.5 c 7 control no inoculated 8.25 a 9.59 a 11.12 a 2.13 a 2.79 a 3.17 a 25.6 a 31.8 a 37.8 a variability coefficient (%) 13.6% 14.9% 12.98% 11.4% 17.2% 17.0% 18.7% 24.7% 27.8% p value 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 (a) tukey with alfa = 0.05. equal letter has no statistical differences table 3. percentage of healthy roots in avocado seedlings inoculated with phytophthora cinnamomi treated with different trichoderma strains treat. antagonist strain percentage of healthy root (%)(a) 1° eval. (30 days) 2° eval. (45 days) 3° eval. (60 days) 1 trichoderma sp (chav01) 14 b 68 b 82 b 2 t. harzianum (chav02) 16 b 50 c 64 c 3 t. harzianum (unalm01) 14 b 28 e 50 cd 4 t. viride (unalm02) 14 b 40 d 60 c 5 trichomax solagro 12 b 20 f 42 d 6 inoculated control 14 b 16 f 20 e 7 control no inoculated 98 a 100 a 100 a variability coefficient (%) 22.5% 13.2% 18.1% value p > alfa 0.05 0.05 0.05 (a)tukey with alfa = 0.05. equal letter has no statistical differences. fig. 3. percentage of healthy roots treated with different trichoderma strains in avocado seedlings inoculated with phytophthora cinnamomi control of avocado root rot caused by phytophthora cinnamomi with different trichoderma strains at chavimochic irrigation project september december 2021 84 fig. 4. roots of zutano avocado inoculated with phytophthora cinnamomi and treated with different trichoderma strains p. gastañadui; r. moreno, p. quiroz-delgado; w. apaza-tapia peruvian journal of agronomy 5(3): 78–86 (2021) 85 rest of the isolates. these results corroborate findings from other authors that antagonist adaptation to the agroecosystem conditions is a determining factor in their biological control of root pathogens (benítez et al., 2004; samuels, 2006). pathogen control mechanisms occur mainly via antibiosis through metabolites, which inhibits pathogen development (vinale, 2008; bae et al., 2016). colony-forming units of trichoderma when the colony-forming units (cfu) of all the isolates were quantified, it was observed that the values 45 days after inoculation with p. cinnamomi were higher compared to after 60 days (table 4). this is due to the fact that trichoderma populations generally tend to decrease with time after being applied to the soil (finlay & mccracken, 1991). in fact, trichoderma was found in the rhizosphere zone, which is where the samples were extracted, indicating that all the isolates were able to colonize the rhizosphere of the avocado. no relationship was found between a greater quantity of trichoderma cfu and better control. this can be explained by the fact that the presence of trichoderma in the root already exerts control via antibiosis and competition, which is probably the most important characteristic with respect to its control. the population dynamics of trichoderma are highly variable in the soil due to several factors, including temperature, humidity, roots, and the presence of organic matter, among others. in our study, trichoderma colonies were detected in all soil treatments applied around the roots of the avocado seedlings. conclusions all trichoderma treatments controlled the avocado root rot caused by p. cinnamomi. significant statistical differences with respect to the inoculated control were found for the percentage of healthy roots 60 days post inoculation. chav01 best controlled the avocado root rot. chav02 displayed the best performance for the percentage of healthy root and the incidence of plants with aerial symptoms. overall, the native isolates from the chavimochic region better controlled phytophthora cinnamomi than the isolates from unalm and the commercial strain trichomax. no direct relationship was found between the number of colonies forming units of the different trichoderma strains and the control effect on phytophthora cinnamomi. all trichoderma isolates were able to colonize the rhizosphere of the avocado. acknowledgments we would like to thank innovate peru for financing of this research work. references bae, h., roberts, d. p., lim, h. s., strem, m. d., park, s. c., ryu, c. m., melnick, r. l., & bailey, b. a. (2011). endophytic trichoderma isolates from tropical environments delay disease onset and induce resistance against phytophthora capsici in hot pepper using multiple mechanisms. molecular plant–microbe interactions, 24(3), 336–351. https://doi.org/10.1094/mpmi-09-10-0221 bae, s.-j., mohanta, t. k., chung, j. y., ryu, m., park, g., shim, s., hong, s.-b., seo, h., bae, d.-w., bae, i., kim, j.-j., & bae, h. (2016). trichoderma metabolites as biological control agents against phytophthora pathogens. biological control, 92, 128–138. http://doi. org/10.1016/j.biocontrol.2015.10.005 benítez, t., rincón, a. m., limón, m. c., & codón, a. c. (2004). biocontrol mechanisms of trichoderma strains. international microbiology, 7(4), 249–260. table 4. colony-forming units of different trichoderma strains in roots of zutano avocado inoculated with phytophthora cinnamomi treat. antagonist strain 1st sample (a)(b) (45 days) 2nd sample (a)(b) (60 days) (ufc × g soil) (ufc × g soil) 1 trichoderma sp (chav01) 2.0 × 105 bc 1.2 × 103 b 2 t. harzianum (chav02) 2.0 × 105 bc 2.2 x102 c 3 t. harzianum (unalm01) 3.7 × 105 a 2.0 × 103 b 4 t. viride (unalm02) 2.7 × 105 ab 3.3 × 103 a 5 trichomax solagro 1.0 × 105 c 2.2 × 103 ab 6 inoculated control 0 d 0 d 7 control non inoculated 0 d 0 d variability coefficient (%) 27.14% 25.22% (a) transformed data to root of x. (b) tukey with alfa = 0.05. equal letter has no statistical differences control of avocado root rot caused by phytophthora cinnamomi with different trichoderma strains at chavimochic irrigation project september december 2021 86 casale, w. l. 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(1989). evaluation of strain of myrothecium roridum as a potential biocontrol agent against phytophthora cinnamomi. phytopathology, 79(10), 1079–1084. https://doi.org/10.1094/ phyto-79-1079 hardham, a. r., & blackman, l. m. (2018). phytophthora cinnamomi. molecular plant pathology, 19(2), 260–285. https://doi.org/10.1111/mpp.12568 lamari, l. (2008). assess 2.0 image analysis software for plant disease quantification. american phytopathological society, st. paul. mass, e. m. c., & kotzé, j. m. (1990). the effect of bacteria on root severity caused by phytophthora cinnamomi. south african avocado growers’ association yearbook, 13, 65–66. mcleod, a., labuschagne, n., & kotzé, j. m. (1995). evaluation of trichoderma for biological control of avocado root rot in bark medium artificially infested with phytophthora cinnamomi. south african avocado growers’ association yearbook, 18, 32–37. ploetz, r. c. (2013). phytophthora root rot of avocado. in k. lamour (ed.), phytophthora: a global perspective (pp. 197–203). cabi plant protection series. cabi publishing. reeves, r. j. (1975). behaviour of phytophthora cinnamomi rands in different soils and water regimes. soil biology and biochemistry, 7(1), 19–24. https://doi. org/10.1016/0038-0717(75)90025-5 samuels, g. j. (2006). trichoderma: systematics, the sexual state, and ecology. phytopathology, 96(2), 195–206. https://doi.org/10.1094/phyto-96-0195 sid ahmed, a., pérez‐sánchez, c., egea, c., & candela, m. e. (1999). evaluation of trichoderma harzianum for controlling root rot caused by phytophthora capsici in pepper plants. plant pathology, 48(1), 58–65. https://doi. org/10.1046/j.1365-3059.1999.00317.x stirling, a. m., hayward, a. c., & pegg, k. g. (1992). evaluation of the biological control potential of bacteria isolated from soil suppressive to phytophthora cinnamomi. australasian plant pathology, 21(4), 133–142. https:// doi.org/10.1071/app9920133 villavicencio, y. (2018). comportamiento de cinco patrones de palto (persea americana mill.) a phytophthora cinnamomi rands en chavimochic en invernadero [thesis, universidad nacional agraria la molina]. unalm repository. https://repositorio.lamolina.edu.pe/ handle/20.500.12996/3308 vinale, f., sivasithamparam, k., ghisalberti, e. l., marra, r., woo, s. l., & lorito, m. (2008). trichoderma–plant–pathogen interactions. soil biology and biochemistry, 40(1), 1–10. https://doi.org/10.1016/j.soilbio.2007.07.002 zemtmeyer, g. a. (1985). origen and distribution of phytophthora cinnamomi. california avocado society 1985 yearbook, 69, 89–94. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i2.1930 received for publication: 14 setember 2021 accepted for publication: 27 june 2022 published: 30 august 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by effects of salinity on three mandarin cultivars grafted on two different rootstocks efectos de salinidad de tres cultivares de mandarina en dos patrones diferentes velásquez, r.1* ; burga, c.2; vargas, l.3 *corresponding author: rvelasquez@inia.gob.pe *https://orcid.org/0000-0002-9951-0136 abstract citrus, one of the most important fruit crops in the world and also they are sensitive to salt stress. the negative effects of stresses often lead to reductions in fruit yield and quality. to assess the effects of salinity on some growth traits, a greenhouse test was performed with the cultivars ‘mihowase’, ‘primosole’ and ‘w. murcott’ as grafted on ‘cleopatra’ and ‘swingle citrumelo’ as rootstocks. the experiment was conducted at the agrarian experimental station of national institution for agricultural innovation in donoso-huaral, ubicated 90 km north of lima. the plants were irrigated with water plus nacl with an electrical conductivity of 0.5 or 4.5 ds/m as salt stress. the variables under evaluation were leave losses, fresh and dry weight of stem, leaves and roots as well as relative water content in the plants. the results showed that the rootstocks ‘cleopatra’ was more tolerant than ‘swingle citrumelo’. the cultivars used as scions affected both rootstocks in all the evaluated traits being more notorious in the amount of feeding roots. selection of mandarin trees for production shout take in consideration the combination scion/rootstock. keywords: mandarin, salt stress, rootstocks, ‘cleopatra’, ‘swingle citrumelo’ resumen cítricos, es uno de los más importantes cultivos de frutales en el mundo y son susceptibles a sales. los efectos negativos de las sales generalmente reducen producción y calidad de fruta. para evaluar los efectos de la salinidad en algunos parámetros de crecimiento, un experimento en invernadero fue conducido con los cultivares ‘mihowase’, ‘primosole’ y ‘w. murcott’ en los patrones ‘cleopatra’ y ‘swingle citrumelo’ el experimento fue conducido en la estación experimental agraria (eea) del instituto nacional de innovación agraria (inia), huaral-donoso a 90 km al norte de lima. las plantas fueron irrigadas con una solución salina clna y con una conductividad eléctrica de 0.5 o 4.5 ds/m. las variables evaluadas fueron caídas de hojas, peso fresco y seco de tallos, hojas y raíces y contenido de agua por las plantas. los resultados mostraron que el patrón ‘cleopatra’ es más tolerante que ‘swingle citrumelo’. las variedades afectaron todas las variables 1 national institute of agricultural innovation, donoso huaral agricultural experiment station, huaral chancay highway km 5 ½ huaral, perú. 2 external consultant. 3 josé faustino sánchez carrión university, huacho-perú. how to cite this article: velásquez, r., burga, c., vargas, l. (2022). effects of salinity on three mandarin cultivars grafted on two different rootstocks. peruvian journal of agronomy, 6(2), 114–122. https://doi.org/10.21704/pja.v6i2.1930 https://orcid.org/0000-0002-9951-0136 https://orcid.org/0000-0002-9951-0136 velásquez, r., burga, c., vargas, l. peruvian journal of agronomy 6(2): 114–122(2022) https://doi.org/10.21704/pja.v6i2.1930 115 de los dos patrones, siendo más notorio en la cantidad de pelos absorbentes de las raíces. la selección de plantas de mandarinas para producción debería considerar combinación variedad/patrón. palabras clave: mandarina, estrés salino, patrón, ‘cleopatra’, “citrumelo swigle” introduction high salt concentration either in the soil of water affects growth, yield and quality of mayor fruit crops. salinity effects are well known under arid and semiarid conditions due to lack of sufficient water and higher evapotranspiration rates (zahra et al., 2020). high levels of salts affect biochemical and physiological processes in the cells. these effects are observed initially on osmosis regulation fallow by ion toxicity and imbalance in nutrient absorption. during the initial state of salt stress, the plants present reduced capacity to absorb water by the roots, cell membrane damages, reduced ability to detoxify oxygen radicals, reduced photosynthesis and stomata aperture (siddiqui et al., 2018; acosta et al., 2017). high levels of b⁺, cl⁻ and na⁺ ions on leaves present symptoms of toxicity as yellowing of the borders and under the present of high temperatures and winds, loss of older leaves is very common. absorption of a specific ion, or combination with others, might induce imbalance in plant nutrition, as have been observed in citrus (etehadpour et al., 2020). the citrus plants are considered sensitive to the salts present in the soil or irrigation water. levels of salts in the soil over 2.0 ds/m or 1.0 ds/m in the irrigation water might reduce yield by 13.0 % to 13.5 % for an increase of 1.0 ds/m (khoshbakht et al., 2018). the ions b⁺, na⁺ and cl⁻ when present in the citrus leaves at the levels of 0.005 % to 0.17 % of dry matter, 0.04 % and 0.7 % respectively are considered toxic (farhangi & torabian, 2017). toxicity symptoms are observed when the levels of cl⁻ ion in the leaves are 1.0 % dry weight and 0.1 % to 0.25 % for na⁺. it will induce yield reduction when the cl⁻ in leaves is 0.2 % dry weight (syverten et al., 1988). most fruit crops are grafted and are available different combinations of scion and rootstocks to improve fruit quality, early production or to overcome environmental and biological constrains. citrus rootstocks are available for many years like sweet orange, sour orange and “lima rangpur” (citrus reticulate var. austera x citrus limon), ‘cleopatra’ (citrus reticulate) and new ones like ‘swingle citrumelo’ or cpb 445 (duncan´grapefuit macfad x poncirus trifoliata l.), c 22, c 35 and hrs 942 from specific breeding programs in usa, spain and brazil (gonzales, 2017). citrus rootstocks present mechanisms of salt tolerance in the process of absorption and translocations of cl⁻ and na⁺ ions present in the soil to the pattern. “lima rangpur” exclude cl⁻ and sequester na⁺ in the roots, ‘cleopatra’ mandarin excludes both ions and trifoliate orange (poncirus trifolata) exclude na⁺ and translocate cl⁻ to the pattern (khoshbakht et al., 2018). the patter might affect the absorption and translocations of ions by the rootstocks (hasanuzzaman et al., 2021; vardi et al., 1988). new citrus cultivars are available as scion and rootstocks and their combinations are under continuing evaluation. the purpose of this study was to evaluate under salt stress the early cultivars ‘mihowase’ and ‘primosole’ as scions and the late cultivar ‘w. murcott’, that have not been previously evaluated, on the two most commonly used rootstocks: ‘cleopatra’ y ‘swingle citrumelo’. these new grafted mandarin combinations might extend the window of supply for export and local market. materials and methods the experiment was conducted under greenhouse condition, from october to december of 2019, at the agricultural experimental station in donosohuaral, 90 km north of lima at 11˚ 31̔̕ 17” s and 77˚ 14̕ 6” w and 130 masl. the average temperature was 25.5º c, relative humidity 88.5 % and natural light conditions. the plants were provided by the tree nursery agroviperos located in the sta. rosa valley near the agricultural experimental station. the plants were grown for 9 months in a greenhouse in 6.0 kg plastic effects of salinity on three mandarin cultivars grafted on two different rootstocks may august 2022 116 bag container with a substrate with ec = 0.05 ds/m, ph = 7.7, om = 10.5 % and cec = 12.68. the scions were: ‘mihowase’ (c. unshiu milho), ‘primosole’ (c. ushiu milho x c. reticulata) and “w. murcot” (c.reticulata x c. sinensis) grated on the rootstocks ‘cleopatra’ (c. reshni) and ‘swingle citrumelo’ (c. paradise x poncirus trifoliata l. raf). the treatments were allocated in a complete block design with 3 replications and 3 plant per replication. it was evaluated the number of leaves loss at 45 and 90 days, fresh and dry weight of roots, stem and leaves, and water loss at 90 days. plant water content was estimated by the differences between fresh weight less dry weight. statistical analysis of the data was done with the spss v.22 statistic program and the means were compared with the duncan test (p < 0.05). results leaf loss ‘cleopatra’ rootstocks showed less leaves loss (18.13 %) when it is compared at the low and high salt treatments; however, ‘swingle citrumelo’ showed higher leave loss (31.65 %). more leaf loss was observed at the second period of evaluation, 45 to 90 days, in both rootstocks. the cultivars used as scions affected the leaf loss of both rootstocks. ‘cleopatra’ rootstocks were not affected by the ‘mihowase’ scion but presented more leaf loss with the ‘primosole’ and ‘w. murcott’ scion. ‘swingle citrumelo’ roots showed less leaf loss with ‘mihowase’ scion but similar with ‘primosole’ and ‘w. murcott’ (table 1 and figure 1). similar results have been obtained when engrafted and ungrafted rootstocks respond different to salt stress, where engrafted are more susceptible. plant dry weight ‘cleopatra’ rootstock showed 18.13 % plant dry weight loss and ‘swingle citrumelo’ 9.52 % under 4.5 ds/m salt concentration. they also observed the dry weight loss in stem + leaves and in the roots of both rootstocks with more weight loss in roots. the tree cultivars used as scions reduced dry weight loss of both rootstocks but ‘w. murcott’ increased the weight loss of stem + leaves and roots on ‘swingle citrumelo’ (table 2, figure 2, figura 3, and figura 4). plant water loss plant water content was estimated by the difference between fresh weigth less dry weight. the plants of ‘cleopatra’ rootstock losses 26.7 % and ‘swingle citrumelo’ 5.59 % of water, respectevely. the three cultivars used as scion reduce this water loss in ‘cleopatra’ but incresed on ‘swingle citrumelo’ when compared to the rootstocks alone (table 2 and figure 5). table 1. duncan test of the effects of salts stress on the leaves loss of each scion/rootstock combination a 45 and 90 days. number of days 0-90 0-45 45-90 electric conductivity (ds/m) 0.5 4.5 0.5 4.5 0.5 4.5 v ar ie ty /p at te rn cleopatra 14.67 a 17.33 ab 8.00 d 8.67 d 6.67 abc 8.67 abcd mihowase/cleopatra 17.33 ab 16.33 a 0.33 a 1.67 ab 14.67 def 17.00 def primosole/cleopatra 21.33 ab 26.33 ab 3.33 bc 4.00 bc 17.33 def 23.00 f w. murcott/cleopatra 19.00 ab 23.99 ab 4.00 bc 10.00 de 9.67 abcde 19.00 ef s. citrumelo 20.00 ab 26.33 ab 9.67 d 11.33 ef 10.33 bcde 15.00 cdef mihowase/s. citrumelo 17.33 ab 17.00 ab 4.67 c 3.00 bc 12.67 cde 14.00 cdef primosole/s. citrumelo 15.33 a 19.00 ab 15.00 g 17.33 h 0.33 a 1.67 ab w. murcott/s. citrumelo 20.33 ab 26.67 ab 8.00 d 12.67 f 12.67 cde 14.00 cdef coefficient of variation (cv) 22.65 11.4 25.2 *means with the same letter are not statistically different with the duncan test (p<0.05) velásquez, r., burga, c., vargas, l. peruvian journal of agronomy 6(2): 114–122(2022) https://doi.org/10.21704/pja.v6i2.1930 117 table 2. effects of salt stress on plant dry weight and plant water content for each scion/rootstock combination. weight part of the plant (g) total plant stem+leaves roots stem+leaves/ roots water content in plant electric conductivity (ds/m) 0.5 4.5 0.5 4.5 0.5 4.5 0.5 4.5 0.5 4.5 v ar ie ty /p at te rn cleopatra 20.8 ab 17.87 a 16.0 a 11.23 a 6.27 a 4.77 ab 2.79 e 2.33 d 18.7 ab 13.7 abcd mihowase/ cleopatra 61.4 f 61.83 f 44.5 gh 17.0 hi 25.17 ijklm 19.13 fghi 1.51 abc 1.47 abc 33.33 fghij 30.00 abcd primosole/ cleopatra 47.43 de 45.2 de 41.73 gh 18.0 fg 22.77 ghijklm 22.0 ghijkl 1.13 a 1.14 a 24.4 bcdef 23.63 cdefg w.murcott/ cleopatra 65.77 fg 61.97 f 60.13 jkl 19.0 jk 28.93 mno 27.27 klm 1.29 ab 1.27 ab 36.23 ijk 34.37 ghij s. citrumelo 25.83 abc 23.27 ab 25.87 cd 20.0 bc 11.83 bcd 10.27 abcd 1.29 a 1.19 ab 19.67 abcd 18.57 abcd mihowase/s. citrumelo 45.43 de 41.93 cde 35.87 de 21.0 de 18.43 efgh 16.53 defg 1.56 abc 1.51 abc 24.37 cdefg 18.57 abd primosole/s. citrumelo 70.47 g 62.47 f 63.17 kl 22.0 no 34.27 nop 27.2 klm 1.26 a 1.12 ab 35.10 hij 30.37 fghi w. murcott/s. citrumelo 64.33 fg 49.87 de 41.6 fg 23.0 j 28.2 ghijkl 22.1 klmn 1.31 ab 1.26 ab 33.9 fghij 25.87 defgh coefficient of variation (cv) 19.3 8.8 16.5 22.2 19.01 *means with the same letter are not statistical different at the duncan test (p<0.05) figure 1. effects of salt stress on leaves loss / plant of each scion/rootstock combination at 90 days. *cle (‘cleopatra’), mio (‘mihowase’), pri (‘primosole’), wmu (‘w. murcott’), cit (‘swingle citrumelo’). effects of salinity on three mandarin cultivars grafted on two different rootstocks may august 2022 118 figure 2. effects of salt stress on plant dry weight (g) for each scion/rootstock combination at 90 days. *cle (‘cleopatra’), mio (‘mihowase’), pri (‘primosole’), wmu (‘w. murcott’), cit (‘swingle citrumelo’). *cle (‘cleopatra’), mio (‘mihowase’), pri (‘primosole’), wmu (‘w. murcott’), cit (‘swingle citrumelo’) figure 3. effects of salt stress on roots for each scion/rootstock combination at 90 days. velásquez, r., burga, c., vargas, l. peruvian journal of agronomy 6(2): 114–122(2022) https://doi.org/10.21704/pja.v6i2.1930 119 *cle (‘cleopatra’), mio (‘mihowase’), pri (‘primosole’), wmu (‘w. murcott’), cit (‘swingle citrumelo’) figure 4. effects of salt stress on roots for each scion/rootstock combination at 90 days. figure 5. effects of salt stress on plant water content (g) for each scion/rootstock combination at 90 days. *cle (cleopatra), mio (mihowase), pri (primosole), wmu (w. murcott), cit (swingle citrumelo). effects of salinity on three mandarin cultivars grafted on two different rootstocks may august 2022 120 discussion leaf loss the results on leaf defoliation showed that the ‘cleopatra’ rootstock and the three scions grafted in this rootstock presented less leaves loss than in ‘swingle citrumelo’ rootstock and the three scions in this rootstock. “rangpur lime” and ‘cleopatra’ mandarin are mentioned as the most salt resistance and ‘swingle citrumelo’ and trifoliate orange as the most susceptible. ‘cleopatra’ resistance to salts is associated with its capacity to limit the accumulation of cl⁻ in leaves. this response of leaves loss was most significant at the first 45 days treatment. similar results have been observed when the plants were exposed to the salinity treatment from the first day and they were not applied gradually to allow plant adaptation to the new conditions (simpson et al., 2014). the cultivar ‘mihowase’ as scion grafted on both rootstocks reduced more leaves loss than the other two cultivar used as scions. similar results have been reported where the effects of the scion grafted on a rootstock can be linear of quadratic pending in the rootstock (brito, 2014). the defoliation observed on the older leaves, more than 2 months old, did not show symptoms of injury like leaf bronzing and/ or leaf tip yellowing probable because the leaves had less than 0.7 % cl⁻ dry weight; minimum level of cl⁻ in leaves to showed up visible symptoms (ferguson & grattan, 2005). plant dry weight the presence of salts reduced total plant dry weight in all treatments and this reduction on plant biomass was less on the rootstocks ‘cleopatra’ and its scion combinations than on the rootstock ‘swingle citrumelo’ and its scion combinations. this effect of biomass reduction was higher on leaves and stems than on roots. both rootstocks and their scion combinations presented similar leaves and stem dry weight loss, however the rootstock ‘cleopatra’ and its scion combination presented less mass loss than the rootstock ‘swingle citrumelo’ and lower leaves plus stem/root ratio. the decrease of shoot/root ratio is a common response to salt stress, related to factors associated with water stress (osmotic effect) and constitute a typical mechanism of plant resistance under saline conditions. similar results have been reported about the effects of salinity on plant mass reduction on different scion/rootstocks combination on orange or grapefruit (brito et al. 2014; garcia et al. 2016) but there are lack of information on mandarin. much attention has been dedicated to understand adverse effects of na⁺ and cl⁻ on morphological, physiological, and biochemical processes on citrus and how these ions contribute to plant growth inhibition. chlorophyll content, net co₂ assimilation rate, transpiration and stomata conductance significantly decrease in response to salinity. the salt–tolerant ‘cleopatra’ was less affected on these physiological parameters than the salt-sensitive ‘swingle citrumelo’ (mahmoud et al., 2020). ‘cleopatra’ mandarin is using a donor of salt tolerance in traditional breeding programs, because it possesses the three mechanism of salt tolerance in citricus; chloride exclusion, water saving and accumulation of soluble solids (garcia et al., 2016; rodrigues et al., 2019; mahmoud et al., 2020). plant water loss the two rootstocks, ‘cleopatra’ mandarin, ‘swingle citrumelo’, and the scions grafted in these rootstocks presented water loss at 90 days. the ‘swingle citrumelo’ and the scions grafted on this rootstock showed 48.2 % more water loss than the ‘cleopatra’ rootstock and the scions grafted in it. similar results have been presented in previous research on orange (navarro, 2010) but there are still missing information on plant dehydration on mandarin cultivars and interaction with different rootstocks combinations. plants dehydration under salinity stress presented lower root hydraulic conductance, leaf and stem water potential, decrease in stomata conductance, leaf ultrastructure disorganization, and photosynthesis decline, due to more difficulty in taking up water from the soil and salt accumulation (acosta et al., 2017; simpson et al., 2014). specific mechanism for salt avoidance, has been suggested for salt – tolerant ‘cleopatra’ mandarin, like minimization of salt entry in the plant, decrease velásquez, r., burga, c., vargas, l. peruvian journal of agronomy 6(2): 114–122(2022) https://doi.org/10.21704/pja.v6i2.1930 121 of salt concentration in the cytoplast of the cell (compartmentalization in vacuoles), and accumulation of proline, organic and inorganic solutes that reduces cellular osmotic potential (ziogas et al., 2021). proline is widely used in traditional breeding programs to transfer salt tolerant from ‘cleopatra’. conclusions the combination of mandarin scion/rootstocks showed similar growth and biomass responses to water salinity as previously reported on orange and grapefruit. ‘cleopatra’ mandarin and the scions grafted in it presented more salt tolerant than ‘swingle citrumelo’ in all traits under evaluation like: leaves loss, plant dry weight loss, canopy/root ratio decreases and plant water loss. the response to salinity of the scion/rootstocks combinations were also influenced by the scion with being more notorious in the amount of feeding roots. mandarin agronomic practices should take in consideration the combination scion/rootstock. acknowledgements this work was financed by the national program agrarian innovation (project 191_pi). the authors want to thanks inia donoso – huaral for its support. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. orcid and e-mail velásquez, r. rvelasquez@inia.gob.pe https://orcid.org/0000-0002-9951-0136 burga, c. caburga00@gmail.com https://orcid.org/0000-0002-7186-6288 vargas, l. yems1718@gmail.com 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(2020). fall armyworm (spodoptera frugiperda): a threat in crop production in africa and asia. peruvian journal of agronomy, 4(3), 121–133. http://dx.doi.org/10.21704/pja.v4i3.1495 introduction maize (zea mays) is one of the world’s major cereal crops because of its high importance as a staple food and its stover need for animal feed and fuel (abebe et al., 2017). maize is also called as a queen of cereals and miracle crops because of its immense potential (jeyaraman, 2017). the crop is rich in nutrients containing approximately 72% starch, 10% protein, and 4% fat, supplying an energy density of 365 kcal/100 g (ranum et al., 2014). in recent years, the productivity of maize is getting lower than its potential due to many biotic and environmental constraints. prominent among such constraints are pests and disease which reduces the production and yield of the crop. many insects fall armyworm (spodoptera frugiperda): a threat in crop production in africa and asia september december 2020 122 are directly responsible for the damage and reduction in yield of maize. fall armyworm (faw) is considered as the most important and devastating insect pest causing severe damage to maize crops (ayala et al., 2013). it is a noctuid moth pest native to america’s tropical and subtropical areas (luginbill, 1928; pogue, 2002; early et al., 2018). the spread of the pest then began rapidly and almost all sub saharan african countries were invaded by 2017 (day et al., 2017). faw moth performs migratory habit as well as localized dispersal habit. until oviposition, the moth will migrate over 500 km (300 miles) (prasanna et al., 2018). the strong-flying ability of adults (100 km per night) and high reproduction rate has spread its population rapidly, hence causes serious consequences (food and agriculture organization [fao], 2019). faw is a polyphagous insect with several hosts species (roger et al., 2017) and has the ability to cause severe famine since its hosts are the main staple food crops (daily nation, 2018). it results in loss of photosynthetic area, slower or impaired reproduction, grain damage, structural damages, and lodging of the maize plant (chimweta et al., 2019). two strains of faw are mostly prevalent namely the rice and the maize strains (adamczyk et al., 1997). among these, the maize strain is most prevalent and causes serious damage to maize. since maize is the staple food for many people across the world, proper control and management of faw are required before it does great damage to the crop fields. despite heavy concerns of scientists, fall armyworm is spreading rapidly in many countries all over the world for the last two years (fao, 2018). thus, proper knowledge of insect biology, host range and management practices are needed to control the insect (rwomushana et al., 2018). therefore, the main aim of this review is to highlight the status and control measures in maize field of this devastating and challenging pest. distribution of faw fall armyworm is a threatening pest and a major concern for the researchers worldwide. the global distribution of fall armyworm in different continents is given is table 1. being native to america, faw was first recorded in early 2016 in central and western africa (goergen et al., 2016). in asia, indian state of karnataka at college of agriculture, recorded the new invasive pest faw in may, 2018 (kalleshwaraswamy et al., 2018). in july 2018, 70% of maize fields in chikkabalpura district of karnataka were affected with faw as reported by the indian council of agricultural research (icar). the infestation was seen in maize and sugarcane in other five states namely: tamil nadu, telangana, andra pradesh, maharashtra and west bengal within less than five months of infestation in karnataka (bhosale, 2018). in june, 2018 faw larvae were first identified in srilanka in damana area of ampara district of eastern province. in bangladesh the faw larvae were firstly recorded for in different districts in two different crops cabbage and maize by the bangladesh agriculture research institute. in nepal, the first case of fall armyworm was documented from nawalparasi district on 9 may, 2019 and then it was seen in the neighboring district chitwan (bajracharya & bhat, 2019). after a few months the infestation was also seen in the districts like kavre, sarlahi, sindupalchowk, and bhojpur. by early 2019, it subsequently appeared in additional asian countries: myanmar, thailand, china, vietnam, malaysia, japan and indonesia. japan reported the presence of faw in july 2019 (fao, 2019). faw hosts a huge number of cultivated plant species are threatened by spodoptera frugiperda (casmuz et al., 2010). severe infestation is caused on its primary hosts, maize and sorghum and other monoculture crops like soybean and cotton (pitre & hogg, 1983; bueno et al., 2011). serious invasion is caused on the cereals and forages and faw caterpillars feed on about 186 plant species from 42 diverse families (casmuz et al., 2010). it causes severe damage to maize (zea mays l.), sorghum (sorghum bicolor l., moench), rice (oryza sativa), cotton (gossypium hirsutum l.), potato (solanum tuberosum l.), vegetables, as well as other cultivated and wild plant species (goergen et al., 2016). the favourite hosts of faw are bermuda grass (cynodon dactylon l., pers.) and peanut (arachis hypogea l.) (sparks, 1979) (table 2). biology of faw spodoptera frugiperda is a holometabolous insect with a high rate of reproduction. warm, humid temperature with heavy rainfall is suitable for survival and multiplication as the insect cannot develop below 10°c. its life-cycle composes four advancement stages viz. egg, six larval instars, pupae and adult, and is completed within about a month in summer, but 2 months in the spring and autumn, and about 3 months in the winter (reinert & engelke, 2010). faw is reproductively active in tropical, where the insect can produce 10 generations per year than temperate where the generation per year is two or fewer (chhetri & acharya, 2019; metcalf et al., 1965). the life cycle of faw begins when the adult female lays eggs on the upper or underside of leaves, more commonly continent total number of countries per continent no of countries (incidence of faw) africa 54 47 asia 48 17 europe 44 4 north america 23 23 oceania 14 1 south america 13 13 table 1. global distribution of faw. source: cabi, 2020 k. adhikari; s. bhandari; l. dhakal; j. shrestha peruvian journal of agronomy 4(3): 121–133 (2020) 123 near the base junction of leaf and stem on the underside of leaves (jarrod et al., 2015). the number of laid eggs per mass can vary between 150 to 200 (prasanna et al., 2018). a single female can lay from 1500 to over 2000 (maximum) eggs (igyuve et al., 2018). the newly deposited eggs are dome shaped with flattened base measuring 0.4mm in diameter and 0.3 mm in height (jarrod et al., 2015). eggs are pale yellow or creamy white in color then turn to light brown before hatching where female covers the egg mass with downy materials (a layer of scales) that gives common name scientific name family references peanut arachis hypogea l. fabaceae sparks (1979); yu (1982) bermuda grass cynodon dactylon (l.) pers. poaceae sparks (1979) maize zea mays l. poaceae paulillo et al. (2000); fao (2018) sweet corn zea mays saccharata (l.) sturt poaceae meagher et al. (2016); seal (2018); jeger et al. (2018); chormule et al. (2019) sugarcane saccharaum officinarum l. poaceae chormule et al. (2019) sorghum sorghum bicolor (l.) moench poaceae fuller et al. (1997); chormule et al. (2019) rice oryza sativa l. poaceae stout et al. (2009); whitford et al. (2015), fao (2017) wheat triticum aestivum l. poaceae murúa et al. (2008); pitre et al. (1983) millet pennisatum americanum l. poaceae ríos-díez and saldamando-benjumea (2011), fao (2017) oat avena sativa l. poaceae silva et al. (2017) barley hordeum vulgare l. poaceae alfonso et al. (1997) rye grass lolium perenne l. poaceae pitre and hogg (1983) para grass brachiaria mutica l. poaceae ashley et al. (2006); de sa et al. (2009) cotton gossypium hirsutum l. malvaceae hardke et al. (2015) cowpea vigna unguiculata (l.)walp fabaceae yu (1982) soybean glycine max (l.) merril fabaceae yu (1982) chickpea cicer arietinum l. fabaceae montezano et al. (2018) potato solanum tuberosum l. solanaceae yu (1982) cucumber cucumis sativus l. cucurbitaceae yu (1982); montezano et al. (2018) cabbage brassica oleracea var. capitata l. brassicaceae montezano et al. (2018) mustard brassica juncea l. brassicaceae yu (1982) turnip brassica rapa l. brassicaceae yu (1982) onion allium cepa l. amaryllidaceae fernandes et al. (2012) beet beta vulagris var. vulagaris l. amaranthaceae montezano et al. (2018) sweet potato ipomoea batatas (l.) lam. convolvulaceae montezano et al. (2018) alfalfa medicago sativa l. fabaceae murúa et al. (2008) tobacco nicotiana tabacum solanaceae leal-bertioli et al. (2003); martinelli et al. (2007) tomato solanum lycopersicum (l.) mill. solanaceae rojas et al. (2003) table 2: common feeding host of spodoptera frugiperda. fall armyworm (spodoptera frugiperda): a threat in crop production in africa and asia september december 2020 124 moldy appearance (kalleshwaraswamy et al., 2018) the pre-oviposition period of this insect ranges from 3-4 days depending on temperature. the incubation period of the egg was found to be only 2 days at 30°c (garcia et al., 2017). depending upon temperature and climatic conditions, during the summer, larva completes its stage in about 14 days and 30 days during winter (capinera, 2002). young larvae are generally greenish with black head turning to brownish body with reddish brown head of the sixth instars. the mature larvae also contain a white inverted ‘y’shaped mark in the face (oliver & chapin,1981). rearing the larvae at 25°c, mean development of instar from first to sixth stage takes 3.3, 1.7, 1.5, 2.0 and 3.7 days respectively (pitre & hogg, 1983). pupation typically occurs at a depth 2 to 8 cm below the soil (capinera, 2002) but also can take place in the reproductive part of maize such as mature maize ears. the larvae create a cocoon, oval in form and length of 20-30mm, by adding silk to soil particles after eating voraciously for 2-3 weeks. a reddish brown pupae of 14 to 18 mm length and 4.5 mm width dwells inside the cocoon (igyuve et al., 2018). the pupal stage is completed in 9-12 days (rwomushana et al., 2018). debora et al. (2017) reported the pupal period of s. frugiperda on maize was found to be 8.54 days. adult female is larger than male with a body length of 1.7 cm a wingspan of 3.8 cm. the males have the body length of 1.6 cm and 3.7 cm wingspan. the male forewing is mottled with a discal cell having straw color on three quarters and dark brown on one quarter of the area having triangular white spots at the tip and near the middle of the wing (bhushal & chapagain, 2020). the females forewing are less distinctly marked, ranging from a uniform greyish brown to a little grey brown mottling. both male and female contain hindwing with straw colour having dark brown margin (igyuve et al., 2018). economic importance and damage of faw spodoptera frugiperda, being a major pest of the maize field, have been responsible for 40-70% of overall maize yield loss in africa (day et al., 2017). late planted fields and later maturing hybrids are more likely to become infested. the favorable environment condition with the constant fecundity of the pest leads to the severe damage of the crop (goergen et al., 2016). larvae are voracious feeders and feed whorls of young leaves, ears and tassels, depending on the growth stages, causing a huge damage to the host (sarmento et al., 2002). when the larval population rises; they defoliate every plant that comes on their way (bhushal & chapagain, 2020). typical damage signs of faw larvae are the presence of holes in the maize leaves due to the feeding of epidermal tissues (sisay et al., 2019). larger larvae entirely section the stem seedlings (goergen et al., 2016) and feed on the kernels and cob decreasing the yield and quality of the maize grains (capinera, 2017). the outcome recorded by hruska and gould (1997) revealed that 55-100% s. frugiperda infestation during the mid-to-late corn stage resulted in 15-73 percent yield losses nicaragua. over 12 african countries reported the loss of 8.3-20.6 million tons of maize annually due to faw infestation (day et al., 2017). similarly, america revealed about 39% yield loss in maize due to faw damage (cruz et al., 2012). studies have shown that less than 1% of the total area planted with maize had been affected in china but there is an increasing risk that faw could spread to various parts of china (fao, 2019). in india, infestation level has been documented upto 49.2% on maize (chormule et al., 2019; deole & paul, 2018; dhar et al., 2019) except 100% infestation has been reported from karnataka (mallapur et al., 2018). in srilanka, 50% of the maize-grown area was affected and 10% of originally anticipated production was lost due to faw infestation (fao, 2019). in nepal, loss by fall armyworm had reached up to 70% without control measures and 20-30% with control measures (national plant protection organization [nppo], 2019). any official estimation of losses due to faw in many south asian countries are not currently available. integrated pest management of faw ipm (integrated pest management) is an integrated pest control strategy for preventing insect pests and their damage. practice of ipm mainly incorporate the use of biological, cultural physical methods of pest control (prasanna et al., 2018). ipm highlights the growth of healthy crop with the minimum damage to agro-ecosystems and encourages various mechanisms for natural pest control. ipm is the best option for faw management (day et al., 2017). the devastating loss caused by faw forced farmers to use chemicals directly rather than the ipm methods in nepal (bhushal & chapagain, 2020). however, this pest can be managed in the incidence phase by the use of ipm practices. a technical guide for ipm was published in africa by international maize and wheat improvement center (cimmyt, 2018). cultural practices for managing faw cultural practices are most efficient for controlling faw infestation. avoiding late season planting, avoiding staggered planting, planting early maturing variety reduces the infestation as the maize are severely affected by faw during late season. ffs (farmer field study) in kenya revealed high infestation of faw during late planting maize compared to earlier planted crops, in january 2018 (pechan et al., 2000). invasion of faw can also be minimized by maize intercropping and crop rotation with non-host crops like bean and sunflower (fao, 2018). 10 days ahead of the maize plantation, the planting of the trap crops such as beans at the boundary of maize can attract faw towards the bean and thus the main crop will be protected. the main cultural practices include: ploughing properly, maintenance of good soil health and adequate moisture, clean cultivation, proper weeding. use of push-pull strategy has been proven best k. adhikari; s. bhandari; l. dhakal; j. shrestha peruvian journal of agronomy 4(3): 121–133 (2020) 125 that controls the pest population. intercropping of maize with pest-repellent “push crop (desmodium uncinatum and desmodium intortum) and planting pest-attractive “pull crop” (pennisetum purpureum or brachiaria spp) around it comes under this strategy (dively, 2018). some of the farmers in kenya, tanzania, uganda, adopted climatesmart-push-pull technique which showed the depletion of faw larvae by 82.7% per plant and subsequently plant damage reduced by 86.7% per plot (khan et al., 2018). pest monitoring for managing faw the effective implementation of ipm also depends upon the pest monitoring. most effective monitoring tools of faw management are pheromones and light traps. mating disruption is possible (shorey et al., 1994) by using sex pheromone for s. frugiperda which includes (z)-9-tetradecenyl acetate (z-9-14: oaca) that is similar to trichoplusia ni, s. exigua and agrotis ipsilon exigua (klun et al., 1996). sex pheromones attract the males as the pheromones are similar to the chemicals produced by females and aggregation pheromones are also used in monitoring of s. frugiperda (prasanna et al., 2018). since adult faw show nocturnal behaviour, it is easier to control them through black light traps (hunt et al., 2001; qureshi et al., 2006). however, for detecting and monitoring adult faw, pheromone traps were found more effective than black light traps (starratt & mcleod, 1982). during regular monitoring, hand collection of the egg masses and destroying under small scale can be effective. this method was found to be ‘somewhat successful’ by the majority of farmers (rwomushana et al., 2018). for the management of s. frugiperda in ethiopia, 15 percent farmers practice handpicking (kumela et al., 2019). in addition to handpicking, destruction of eggs and larvae, and keeping the mixture of sand with lime or ash in the whorl of the attacked maize can kill the larvae in ethiopia (gebreziher, 2020). biological measures for managing faw biological control method is the alternative pest control measures providing environmentally safe and sustainable plant protection by the use of natural enemies of the pest (bhushal & chapagain, 2020) . in this method of pest management, the population of faw is decreased by the use of antagonist natural enemies [parasitoids (table 3), predators (table 4) and pathogens (table 5)]. the most important biological control agent for to control faw in maize and vegetables is telenomus remus (pomari et al., 2012). as a part of ipm programme, t. remus had been released to the maize field in venezuela that has caused the faw parasitism up to 90% showing the high biocontrol capability for s. frudiperda (ferrer, 2001). various predators like calosoma granalatum (prasanna et al., 2018), assasin and flower bug (van waddill & whitcomb, 1982), earwigs (romero-sueldo et al., 2014; silva et al., 2018), ladybird beetle, ant etc. attack the faw at various stages. the enthomopathogen like spodoptera frugiperda, nucleo polyhidroxy virus parasitic natural enemy host stage attacked crop references archytas incertus larva maize virla et al. (1999) archytas marmoratus larva/pupae maize/sorghum van waddill and whitcomb (1982) charops ater larvae maize sisay et al. (2018) chelonus curvimaculatus eggs/larva maize valicente and barreto (1999) chelonus insularis eggs/larva maize/sorghum virla et al. (1999); bahena and garcía (1991) coccygidium luteum larvae maize sisay et al. (2018) cotesia icipe larva maize sisay et al. (2018) cotesia marginiventris larva maize alam (1978) cotesia ruficrus larva maize yaseen (1979) euplectrus platyhypenae larva maize alam (1978) palexorista zonata larvae maize sisay et al. (2018) telenomus remus nixon egg maize/vegetables kenis et al. (2019) trichogramma rojasi egg maize behle & popham (2012) trichogramma spp egg maize luginbill (1928) table 3: parasitoid natural enemies of fall armyworm natural enemy life stage calleida decora larva calosoma alternans larva calosoma sayi larva carabidae larva/pupa table 4: predators of faw larva and pupa in maize source: cabi, 2020 fall armyworm (spodoptera frugiperda): a threat in crop production in africa and asia september december 2020 126 (sf npv), metarhizium anisopilae, beauveria bassiana, and bacillus thuringiensis are useful to decrease faw population (molina-ochoa et al., 2003). fao (2018) suggests options such as spraying sugar water to attract and maintain populations of ants and other natural enemies. it also recommends “recycling pathogens” by collecting caterpillars killed by disease, and using them to prepare a solution for spraying on plants. technique of biological control not only controls the pest population but also benefits the environment and human health (parra, 2010). botanicals for managing faw the plant extracts of azadirachta indica, nicotina tabacum, chrysanthemum cinerariifollium, tephrosia vogeli are used for controlling faw population. botanicals like azadirachta indica, phytolacca dodecandra, and schinnus molle were found most effective against faw larvae causing the highest mortality (96%) (sisay et al., 2019). 5% neem seed kernel emulsion was found to have repelling property against faw (lamsal et al., 2020). research in feeding bioassay revealed the highest faw larval mortality by the use of l. javanica (62%) and n. tabacum (60%) with highest concentration evaluated at 10% w/v (phambala et al., 2020). similarly, tobacco leaf extracts at 50% concentration are found effective against faw (sakadzo et al., 2020). methanolic extracts of melia azedarach seed (1% and 10%) caused faw larval mortality because of decline in feeding (bullangpoti et al., 2012). the use of ethanolic extracts of argemone ochroleuca also reduced larval feeding activity (martínez et al., 2017). also, biopesticides from the extracts of weed like water lettuce reduced the survival rate of faw by 14% while hydrilla and duckweed caused 11% and 9% reduction of faw growth, respectively (fu et al., 2020). zingiber officinale and malva sylvestris were effective causing mortality for caterpillars of faw (rioba & stevenson, 2020). biopesticides for managing faw biopesticides are based on naturally occurring substances or organisms that kill pests. they are the formulations obtained from particular bacteria, fungal or viral strains (prasanna et al., 2018) various biopesticides like beauveria bassiana strain r444, baculo virus, bacillus thuringiensis sub-species kurstaki strain sa-11 (table 6), cowurine, natural enemy host stage attacked bacillus cereus larvae bacillus thuringiensis larvae bacillus thuringiensis alesti larvae bacillus thuringiensis darmstadiensis larvae bacillus thuringiensis thuringiensis larvae bacillus thuringiensis kurstaki larvae beauveria bassiana eggs/larvae granulosis virus larvae metarhizium anisopliae eggs/larvae nucleopolyhedrosis virus larvae spodoptera frugiperda multiple nucleopolyhedrovirus larvae table 5: pathogenic natural enemy of fall armyworm source: cabi, 2020 active substance target crops countries registered beauveria bassiana strain r444 lepidoptera, including spodoptera frugiperda barley, brassica, maize, sweetcorn, sorghum, tomato, wheat south africa (emergency approval in 2017). bacillus thuringiensis subspecies kurstaki strain sa11 lepidoptera, including spodoptera frugiperda maize, sweetcorn, sorghum, wheat south africa (emergency approval in 2017) baculovirus spodoptera frugiperda unspecified pending, brazil s f m n p v b a c u l o v í r u s spodoptera frugiperda spodoptera frugiperda cereals, cotton, sweetcorn, sorghum, turf brazil (monograph b51), usa table 6. biopesticides registered to control spodoptera frugiperda. source: fao, 2018 k. adhikari; s. bhandari; l. dhakal; j. shrestha peruvian journal of agronomy 4(3): 121–133 (2020) 127 bacterial fermentation products like spinetoram and spinosad are helpful to control faw (prasanna et al., 2018). baculovirus is extremely host-specific, non-pathogenic for beneficial insects and other non-target organisms, and best suited for integrated pest management. neem-based biopesticides are similarly effective as insecticides to fight against fall armyworm (cabi, 2020). chemical control for managing faw synthetic pesticides are hazardous in sustainable agriculture and use of the pesticides are not considered good in ipm technology. however, chemicals are used under the economic threshold level in severe condition (khan et al., 2018). chemicals like cypermethrin, lamda, cyhalothrin, bifenthrin, beta cyfluthrin, deltamethrin were effective in reducing the populations of faw worldwide (fao, 2018). icar-indian institute of maize research has recommended some pesticides like emamectin benzoate, spinosad and chlorantraniliprole having reducing property against faw (lamsal et al., 2020). furthermore,chemicals like radient, tracer, karate, ampligoposses were found to have efficacy causing 90% mortality of faw larvae at 72 h of application (sisay et al., 2019). the different insecticides used for control of fall armyworm is given in table 7. conclusion faw (spodoptera frugiperda) infestation causes the greatest economic loss to a top rank cereal, maize and affects millions of people worldwide. it has the ability to breed rapidly and migrate to new territory due to its strong-flying nature. it possesses the potential of causing approximately 100% crop loss in maize if not managed in time. faw was reported firstly in central and western africa in 2016 followed by five asian countries possessing threat to south america in the last 3-4 years. if the necessary precautions are not taken with the ongoing pest distribution, it can active ingredient chemical class irac classification (mode of action) benfuracarb/fenvalerate carbamate/pyrethroid 1a/3a acetylcholinesterase (ache) inhibitors/ sodium channel modulators carbosulfan carbamate 1a acetylcholinesterase (ache) inhibitors chlorantraniliprole diamides 28 ryanodine receptor modulators chlorpyrifos organophosphate 1b acetylcholinesterase (ache) inhibitors c h l o r o p y r i f o s / cypermethrin o r g a n o p h o s p h a t e / pyrethroid 1b/3a acetylcholinesterase (ache) inhibitors/sodium channel modulators c h l o r a n t r a n i l i p r o l e / lamda-cyhalothrin diamides/pyrethroid 28/3a ryanodine receptor modulators/sodium channel modulators emamectin benzoate avermectin 6 glutamate-gated chloride channel (glucl) allosteric modulators flubendiamide diamides 28 ryanodine receptor modulators indoxacarb oxadiazine 22a voltage-dependent sodium channel blockers lufenuron benzoylureas 15 inhibitors of chitin biosynthesis, type 0 mercaptothion organophosphates 1b acetylcholinesterase (ache) inhibitors methomy carbamate 1a acetylcholinesterase (ache) inhibitors novaluron/indoxacarb b e n z o y l u r e a s / oxadiazine 15/22a inhibitors of chitin biosynthesis, type 0/ voltagedependent sodium channel blockers profenofos organophosphates 1b acetylcholinesterase (ache) inhibitors pyridalyl dichloropropene derivative pyridalyl un compounds of unknown or uncertain moa s p i n e t o r a m / methoxyfenozide spinosyns / diacylhydrazines 5/18 nicotinic acetylcholine receptor (nachr) allosteric modulators/ ecdysone receptor agonists spinetoram spinosyns 5 nicotinic acetylcholine receptor (nachr) allosteric modulators table. 7. different modes of action on the insecticides registered for the control of fall armyworm on maize. source: bezuidenhout and nunkumar, 2017 fall armyworm (spodoptera frugiperda): a threat in crop production in africa and asia september december 2020 128 cause major threats to agricultural crops worldwide challenging food security. starting from the larval stage it voraciously feeds on maize cob and whorls, continuing towards stem destruction on its adult stage causing tons of damage to the crop. so necessary control strategies should be implemented to avoid the huge amount of loss. the use of pheromone traps, light traps as mechanical control, biological control and botanical control reduces the outbreak of faw populations. being eco friendly, these measures preserve the soil as well as environmental health protecting the crop. chemical measures should be avoided as much as possible until and unless the infestation exceeds 50%. with the best possible strategies and measures, faw infestation can be reduced to an extreme limit encouraging the better production of crops. references abebe, z., dabala, c., & birhanu, t. 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(1982). host plant induction of glutathione s-transferase in the fall armyworm. pesticide biochemistry and physiology, 18(1), 101–106. https:// doi.org/10.1016/0048-3575(82)90092-x peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i2.1933 received for publication: 06 june 2022 accepted for publication: 31 july 2022 published: 30 august 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by impact of two tillage practices on selected soil properties, growth and yield of maize on an ultisol impacto de dos prácticas de labranza en determinadas propiedades del suelo, crecimiento y rendimiento del maíz en un ultisol de nigeria oluwatosin komolafe1* *corresponding author: komolafeolaoluwa@ymail.com *https://orcid.org/0000-0002-6777-5631 abstract this study investigated the effect of two tillage practices on maize yield and growth, and selected soil properties of an ultisol of osun state in nigeria. this was aimed at selecting an appropriate tillage practice for crop growth and soil maintenance. the study was divided into two experimental plots during the early and late cropping season in 2014. the first plot was manually cleared to have zero tillage while the second plot was plowed twice and harrowed once for conventional tillage. each plot had three blocks (23.0 m x 2.5 m) with an alley of 1.0 m between blocks and 1.0 m within plots. three seeds of the test crop were a distance of 75 cm x 50 cm per hill and each plot weeded manually at two weeks intervals till harvest. the selected soil physical and chemical properties and plant growth parameters were collected and determined using standard method after each cropping season. at the end of the experiment zero tillage, had the highest plant height and soil values compared to conventional tillage. zero tillage also had higher soil chemical values when compared to conventional tillage. the grain yield showed a significant difference between the tillage practices. zero tillage had a higher yield (1.71 t/ha) when compared with conventional tillage (0.97 t/ha). the study concluded that zero tillage was a better alternative for crop growth and soil maintenance of an ultisol. keywords: conventional tillage, maize crop, plow, harrowed, ultisol. resumen este estudio investigó el efecto de dos prácticas de labranza sobre el rendimiento y el crecimiento del maíz, y propiedades seleccionadas del suelo de un ultisol del estado de osun en nigeria. esto tuvo como objetivo seleccionar una práctica de labranza adecuada para el crecimiento de los cultivos y el mantenimiento del suelo. el estudio se dividió en dos parcelas experimentales durante la temporada de cultivo temprana y tardía en 2014. la primera parcela se desbrozó manualmente para tener labranza cero, mientras que la segunda parcela se aró dos veces y se rastrilló una vez para labranza convencional. cada parcela tenía tres bloques (23.0 m x 2.5 m) con un callejón de 1.0 m entre bloques y 1.0 m dentro de las parcelas. tres semillas del cultivo de prueba se colocaron a una distancia de 75 cm x 50 cm por montículo y cada parcela se desyerbó manualmente a intervalos de dos semanas hasta la cosecha. las propiedades físicas y químicas del suelo seleccionadas y los parámetros de crecimiento de las plantas se recolectaron y determinaron utilizando el método estándar después de cada 1 1obafemi awolowo university, institute of ecology, ile-ife, nigeria. how to cite this article: oluwatosin komolafe (2022). impact of two tillage practices on selected soil properties, growth and yield of maize on an ultisol. peruvian journal of agronomy, 6(2), 123-131. https://doi.org/10.21704/pja.v6i2.1933 mailto:komolafeolaoluwa@ymail.com https://orcid.org/0000-0002-6777-5631 impact of two tillage practices on selected soil properties, growth and yield of maize on an ultisol may august 2022 124 temporada de cultivo. al final del experimento, tuvo los valores más altos de altura de planta y suelo en comparación con la labranza convencional. la labranza cero también tuvo valores químicos del suelo más altos en comparación con la labranza convencional. el rendimiento de grano mostró una diferencia significativa entre las prácticas de labranza. la labranza cero tuvo un mayor rendimiento (1.71 t/ ha) en comparación con la labranza convencional (0.97 t/ha). el estudio concluyó que la labranza cero era una mejor alternativa para el crecimiento de los cultivos y el mantenimiento del suelo de un ultisol. palabras clave: labranza convencional, cultivo de maíz, arado, grada, ultisol. 1. introduction soil nutrient depletion has been an environmental challenge in sub-saharan africa for centuries. the depletion of nutrients in these soils is caused by unfavorable changes to its properties. this comportment could be attributed to an inappropriate soil management practices (alam et al., 2014). such practices lead to the reduction in organic matter, increase soil acidity and encourage soil erosion. examples of such practices are inappropriate soil fertility and tillage management practices (mohanty et al., 2007). tillage practices have been an age long practice in farming. according to cookson et al. (2008), tillage is a practice used to stir the soil for crop production. it helps to manipulate and loosen the soil for the cultivation. for this reason, its effect on the soil properties and growth of crops has been debated over the years (reference). tillage impacts the soil chemical, physical and biological properties either positively or negatively. the advantages of proper tillage practice include the stimulation of soil nutrients by incorporating crop residues and pest management, and the seed bed formation. however, inappropriate tillage practices cause soil compaction, increase soil erosion and may lead to loss of soil cover in a long term (hamza & anderson, 2005). this indicates that selection of an appropriate tillage practice is essential for reducing soil nutrient depletion and optimum crop productivity. tillage is categorized into conventional and zero tillage (or no-till) practices. conventional tillage involves tillage practices that leave less than 15 % of crop residue on the soil, while zero tillage is the process of retaining most crop residue on the soil surface (singh et al., 2018). each has advantages and disadvantages, for instance, conventional tillageloosens the soil, control weeds and integrates organic matter into the soil (ram et al., 2018). though its disadvantages include soil erosion, loss of soil cover and disruption of soil microbial activities. in contrast, zero tillage reduces soil disturbance and increases biological activities in the soil (crittenden et al., 2015). however, its disadvantages include increased use of herbicides, and often it may be inappropriate for all soil types (soane et al., 2012). hence, the selection of a suitable tillage system should be based on different factors that include climatic factor and type of crops. crops such as maize require a suitable environment to achieve optimum growth and yield. maize is an important cereal crop that can be cropped and produced all year. this crop also possesses vitamins (such as a and e) and mineral salts (rouf shah et al., 2016), therefore, it is ranked as the most important cereal crops, before rice and wheat (faostat, 2017). in nigeria, maize could be used as a medicinal and food crop, or even as a raw material in industries, since it could be converted to starch, cornflakes and flour (ali et al., 2018). the maize has increased its market demand and the need to increase its yield. nevertheless, in many parts of africa, its production has been lower than the population growth, hence the need for increased maize production (santpoort, 2020). to increase and ensure maximum yield, suitable soil management practices, such as manure application and selection of appropriate tillage, are necessary (adedokun et al., 2018). although, many factors such as pest infestation, and post-harvest losses may contribute to low yield, the most important factor is the type of tillage practice on a particular soil type (aikins & afuakwa, 2012). according to rasmussen (1999), different rates of crop productivity could be observed in different soil types depending on the type of tillage practice. however, there oluwatosin komolafe. peruvian journal of agronomy 6(2): 123-131(2022) https://doi.org/10.21704/pja.v6i2.1933 125 is a lack of information on the effect of tillage practices on soil properties and crop responses in different soil types in sub-saharan africa. this study aimed to assess the impact of two different tillage practices on the growth of maize and its effects on soil properties of an ultisol. 7º30’0” 2. materials and methods the study was carried out at the teaching and research farm, obafemi awolowo university, (oau), ile-ife, osun state, nigeria in the early (april july) and late (august november) seasons of 2014. the research farm was located at latitude 7º30’0” n and longitude 4º30’0” e at an elevation of 268 m above mean sea level. the experimental sites had a rainfall pattern in 2014 that ranged between 26.1 mm and 224.0 mm, and soil temperature ranged between 33.9 0c and 39.1 0c (figure 1 and 2). the site was fallowed for seven years. ja n fe b m ar ap r m ay ju n ju ly au g se p oc t no v de c 0 50 100 150 200 250 2014 months r a in fa ll ( m m ) figure 1: monthly rainfall pattern for the study area 2014 source: oau teaching and research farm meteorological station the experiment was conducted on two experimental plots. the first plot was manually cleared (zero tillage) while the second plot was plowed twice at 25 cm depth and harrowed once (conventional tillage). three pre-soil samples were collected and mixed into composite sample for analysis using a soil auger. each experimental plot had three blocks (23.0 m x 2.5 m) with an alley of 1.0 m between blocks and 1.0 m within plots. each experimental plot was replicated thrice. seeds of the test crop maize variety ‘dtsyn-8w’ were obtained from the institute of agricultural research and training (iar and t), ibadan. all plots were manually weeded using hoe at two weeks intervals till harvest. the test crop was sown at three seeds per hill using 75 cm x 50 cm planting distance. cow dung compost was applied on each experimental plots at 3 t/ ha after two weeks of sowing. thinning to two seeds per hill was also done after two weeks of sowing. the experiment was laid out in a randomized complete block design (rcbd) with three replications. ja n fe b m ar ap r m ay ju n ju ly au g se p oc t no v de c 30 35 40 45 2014 months so il t em pe ra tu re (0 c ) figure 2: monthly soil temperature of the area of study 2013-2014 source: oau teaching and research farm meteorological station. during the wet and dry seasons, three post-soil samples per experimental plot were collected before and after each experiment (six samples per season in total). the total of postsoil samples was 6. the depth of soil sampling was 6 inches. these samples were air-dried, crushed, and sieved using a 2 mm mesh before laboratory analysis. soil the ph of the soil was determined in a 1:1 soil to water suspension using the dwyer model wph1 waterproof ph tester (model wph1 waterproof, dwyer). the particle size distribution was determined using the hydrometer method (bouyoucos, 1951). the organic carbon was determined using following the method described by walkley and impact of two tillage practices on selected soil properties, growth and yield of maize on an ultisol may august 2022 126 black method (walkley & black, 1934). the exchangeable cations were determined using atomic absorption spectrophotometer (aas). the bulk density was determined using the core method. finally, the base saturation was determined according to the equation 1. base saturation = exchangeable bases x 100 eq1 cec collection of data on growth parameters commenced at 2 weeks after planting (wap) and continued till 10 wap when the maize plant had attained maturity. the growth parameters data collected were plant height, number of leaves, and stem girth using a tape rule, direct counting, and vernier caliper, respectively. the grain yield was determined by shelling a sample of ears (n = 5) from each plot and applying the shelling percentage to the entire experimental plot. the formula used to convert grain yield to grain moisture-standardized yield is shown in equation 2:7.5. yield (at 12.5% grain moisture) = grain yield×(100– actual grain moisture %) eq2 87.5 data were analyzed using anova (analysis of variance) and their treatment means were adjudged by duncan’s multiple range test (p < 0.05) method using sas 9.0 using the statistical software sas 9.0, while graphs were plotted using graphpad prism 5. 3.1 results and discussion 3.1 pre-soil properties table 1 shows the soil properties before planting. the pre-soil properties of the experimental site indicated its ph (7.86) was slightly alkaline and its textural class was sandy loam. the organic carbon was high which could be attributed to the soil being fallow for seven years. the base saturation was high while the bulk density was moderate. the carbon and nitrogen ratio and the cation exchange capacity in the soil were also moderate. table 1: pre-soil physical and chemical properties properties values ph (1 : 1 soil-water) 7.86 organic carbon (%) 2.251 exchangeable acidity (meq/100 gr) 0.4 c/n 10.61 cec (meq/100 gr) 21.01 base saturation (%) 98.13 bulk density (g/cm3) 1.55 sand (%) 79.2 clay (%) 11.4 silt (%) 9.4 textural class sandy loam 3.2 growth parameters the effect of tillage practices on growth of maize and some selected soil properties of an ultisol were investigated in this study. figure 3 and figure 4 show the growth parameters during early and late planting seasons under different tillage practices. all growth parameters were higher under conventional tillage when compared to zero tillage during the early planting season. conventional tillage when used appropriately is known to increase soil porosity. this ensures a better soil aeration and subsequently leads to better plant growth. this corresponds with the findings of guan et al. (2015) who observed that conventional tillage practices such as rotary and plow tillage during an early planting season, improved growth parameters such as root length and root weight density when compared to zero tillage. better performances, however, were observed in zero tillage soils during the late planting season compared to conventional tillage. this could be attributed to improved water retention and increased organic matter observed in soils with zero tillage (alam et al., 2014)namely, zero tillage (zt. this agrees with the works of ram et al., (2018) who also observed increased crop growth in triticum aestivum under zero tillage practice when compared to conventional tillage in long term field experimentation. oluwatosin komolafe. peruvian journal of agronomy 6(2): 123-131(2022) https://doi.org/10.21704/pja.v6i2.1933 127 plant height 2 4 6 8 10 0 50 100 150 200 conventional tillage zero tillage number of weeks h ei gh t (c m ) stem girth 2 4 6 8 10 0 2 4 6 8 conventional tillage zero tillage number of weeks g ir th ( cm ) number of leaves 2 4 6 8 10 0 5 10 15 conventional tillage zero tillage number of w eeks n um be r of l ea ve s figure 3: growth parameters under different tillage practices during early season plant height 2 4 6 8 10 0 50 100 150 200 conventional tillage zero tillage number of weeks h ei g h t (c m ) stem girth 2 4 6 8 10 0 2 4 6 8 conventional tillage zero tillage number of weeks g ir th ( cm ) number of leaves 2 4 6 8 10 0 5 10 15 conventional tillage zero tillage number of weeks n u m b er o f l ea ve s figure 4: growth parameters under different tillage practices during late season impact of two tillage practices on selected soil properties, growth and yield of maize on an ultisol may august 2022 128 3.3 selected soil properties during the early season (table 2) conventional tillage had a higher ph (7.7) compared to zero tillage (7.5). however, the study observed that zero tillage (table 3) had a higher soil ph (1:1 soil to water) compared to conventional tillage. this could be due to an increased crop residue observed in zero tillage soils in comparison to conventional tillage. this agrees with the observations of lal (1997). after two cropping seasons, he observed that soils with zero tillage had higher ph values when compared to conventional tillage. cookson et al. (2008) also reported in his study that continuous conventional tillage practice reduces soil ph at a 0 cm to 5 cm depth. at the end of the late season (table 3), soil organic carbon for zero tillage soils was higher in comparison to conventional tillage. this could be due to the fast decomposition of crop residues observed in conventionally tilled soils. this agrees with the study of haddaway et al. (2017). he concluded in his study that soils under no-till (zero tillage) had a higher soil organic carbon when compared to intensive tillage (conventional tillage). mathew et al. (2012) also reported in a two year study, that a higher soil organic carbon was recorded in notill soils when compared to conventional tillage. this study observed a significant difference between the cation exchangeable acidity for both tillage practices. cation exchange capacity in conventionally tilled soils was lower (7.39 cmol/ kg) compared to zero tillage (8.30 cmol/kg) at the end of the experiment (table 3). increased cec recorded in zero tillage could be due to increased organic matter observed in zero tillage. increased organic matter is responsible for increased negative charges which results into a higher soil cec. this agrees with the observations of thomas et al. (2007) and dorneles et al. (2015). they stated that zero tillage had values of cec surpassing recorded values of ct (conventional tillage). the lower bulk density observed in conventional tillage could be ascribed to the continuous use of farm machineries on the soil. this agrees with the work of osunbitan et al. (2005) who stated that conventionally tilled soils have lower bulk density when compared with zero tillage. however, this study disagrees with basamba et al. (2006). in his study of the effect tillage practices on soil physical properties of an acidsavanna oxisol, he recorded a lower bulk density for zero tillage soils when compared with conventionally tilled soils. table 2: soil properties during early season properties ct zt ph (1 : 1 soil-water) 7.7a 7.5b organic carbon (%) 2.47a 2.01b exchangeable acidity ( meq/100 gr) 0.5a 0.5a cec ( meq/100 gr) 30.83a 15.58b base saturation (%) 98.3a 96.8b bulk density (g/cm3) 1.43a 1.47b sand (%) 81.2a 78.66b clay (%) 8.4b 9.5a silt (%) 10.40b 11.83a means of the same letter are not significantly different legend: ct = conventional tillage, zt = zero tillage table 3: soil properties during late season properties ct zt ph (1 : 1 soil-water) 7.63b 7.70a organic carbon (%) 3.06b 3.49a exchangeable acidity ( meq/100 gr ) 0.93b 1.80a cec ( meq/100 gr) 7.39b 8.30a base saturation (%) 78.30b 87.40a bulk density (g/cm3) 1.35a 1.38b sand (%) 85.33b 90.0a clay (%) 5.93a 4.60b silt (%) 8.73a 5.40b means of the same letter are not significantly different legend: ct = conventional tillage, zt = zero tillage base saturation in conventional tillage was lower when compared with zero tillage at the end of the experiment (table 3). the increased base saturation observed in zero tillage could be due to higher organic matter and base cations present in zero tillage soils. this disagrees with the work of tarkalson et al. (2006). in a study which spanned a 27 years period, he concluded that base saturation was lower in zero tillage practice when compared to conventional tillage. this oluwatosin komolafe. peruvian journal of agronomy 6(2): 123-131(2022) https://doi.org/10.21704/pja.v6i2.1933 129 study, however, agrees with the reports of omeke (2017) who stated that conventional tillage lowers the values of base saturation in soils. table 4: mean grain yield of crops under conventionally tilled and no-till during early and late seasons season ct( t ha-1) zt early 0.79 0.78 dry 0.97 1.71 percentage increase (%) 18% 19.78 legend: ct = conventional tillage, zt = zero tillage 3.4 grain yield a similar yield was observed when conventional tillage was compared to zero tillage during the early season. the yield during the early season indicated no significant difference (p > 0.05) between conventional tillage (0.79 t/ha) and zero tillage (0.78 t/ha). however, during the late season, significant difference was observed between both tillage practices. zero tillage had a higher grain yield with 1.71 t/ha compared to conventional tillage with 0.97 t/ha. it could be attributed to the increased soil organic carbon observed in zero tillage. zero tillage is known to have a slower organic matter decomposition compared conventional tillage. this could lead to increased yield in subsequent seasons (cooper et al., 2021). conclusion selected soil physical and chemical properties and plant growth parameters were examined under two tillage practices on an ultisol. this was in a view of selecting an appropriate tillage practice for continuous cultivation. soil properties under zero tillage were more improved at the end of the second planting season. the organic carbon and base saturation were higher in zero tillage soils when compared with conventional tillage. however, an increased bulk density was observed in conventionally tilled soils. although growth parameters were higher in conventional tillage in the first season, it was observed that plant growth under zero tillage during the second season was higher when compared to conventional tillage. this implies that adoption of zero tillage practice would be necessary for crop growth, especially for maize cultivation, zero tillage should be considered as the preferred tillage practice. author contributions conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. orcid and e-mail oluwatosin komolafe komolafeolaoluwa@ymail.com https://orcid.org/0000-0002-6777-5631 references alam, md. k., islam, md. m., salahin, n., & hasanuzzaman, m. 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(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, 29–38. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i1.1863 received for publication: 09 june 2021 accepted for publication: 27 february 2022 published: 30 april 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by modification of the flowering dynamics of pineapple (ananas comosus l.) cv. ‘md2’ using aviglycine in the central jungle of perú modificación de la dinámica de la flora ción en piña (ananas comosus l.) cv. ‘md2’ por la acción de la aviglicina en la selva central del perú segundo bello-amez1; ricardo borjas-ventura1; leonel alvarado-huamán1*; noel bello-medina1; diana rebaza-fernández2; viviana castro-cepero1; alberto julca-otiniano1 *corresponding author: lealvarado@lamolina.edu.pe *https://orcid.org/0000-0002-2121-2454 abstract pineapple (ananas comosus) is a widely cultivated fruit in tropical countries. however, its natural flowering (nf) is a physiological event that can make harvesting difficult, bringing with it financial problems. therefore, this study aimed to determine the effect of aviglycine (avg) on the flowering of pineapple cv. ‘md2’ in the central jungle of peru. three doses (150 mg/l, 250 mg/l, and 350 mg/l) of avg (commercial product retain 15%) were studied with six and eleven applications. the results confirmed the appearance of nf under satipo conditions. the high doses of avg (250 mg/l and 350 mg/l) inhibited the appearance of inflorescences in almost all evaluations and particularly with eleven applications compared with the treatment without avg and flower induction (nf). the nf presented three periods with different relative rates of inflorescence appearance (rria), where the first period had the highest rria and longest duration (42 d). in conclusion, the application of avg had a significant effect in delaying the appearance of inflorescences; thus, its application in pineapple cultivation is recommended. however, more studies are needed to further deepen the knowledge on the management of this growth regulator. keywords: aviglycine, bloom retardant, growth regulators, natural bloom resumen la piña (ananas comosus) es una especie muy cultivada en diferentes países tropicales. sin embargo, su floración natural es un evento fisiológico que puede dificultar la labor de cosecha, trayendo consigo problemas económicos. en tal sentido, este trabajo tuvo como objetivo determinar el efecto de la aviglicina sobre la floración de piña cv. ‘md2’ en la selva central del perú. se estudiaron tres dosis (150 mg · l-1, 1 grupo de investigación agricultura y desarrollo sustentable en el trópico peruano, departamento de fitotecnia, facultad de agronomía, universidad nacional agraria la molina, av. la molina s/n, la molina, lima-perú. 2 departamento de estadística e informática, facultad de economía y planificación, universidad nacional agraria la molina, av. la molina s/n, la molina, lima-perú. how to cite this article: bello-amez, s., borjas-ventura, r., alvarado-huamán, l., bello-medina, n., rebaza-fernández, d., castro-cepero, v., julca-otiniano, a. (2022). modification of the flowering dynamics of pineapple (ananas comosus l.) cv. ‘md2’ using aviglycine in the central jungle of perú. peruvian journal of agronomy, 6(1), 1–12. https://doi.org/10.21704/pja. v6i1.1863 modification of the flowering dynamics of pineapple (ananas comosus l.) cv. ‘md2’ using aviglycine in the central jungle of perú january april 2022 2 250 mg · l-1 y 350 mg · l-1) de aviglicina (avg) (producto comercial retain 15 %), con 6 y 11 aplicaciones. en general, los resultados confirman la aparición de floración natural (fn) en condiciones de satipo. las altas dosis de avg (250 mg · l-1 y 350 mg · l-1) inhibieron la aparición de las inflorescencias durante casi todas las evaluaciones, especialmente con once aplicaciones comparadas con el tratamiento sin avg ni tif (tratamiento de inducción floral) (fn). asimismo, fn presentó tres periodos con tasas relativa de aparición de inflorescencias (trai) diferentes, siendo la primera la que tuvo mayor trai y mayor duración (42 días). finalmente, se concluye que la aplicación de avg tuvo un efecto significativo en el retardo de la aparición de las inflorescencias, motivo por el cual se sugiere su aplicación en el cultivo de piña. sin embargo, son necesarios más estudios para seguir profundizando el conocimiento sobre el manejo de este regulador de crecimiento. palabras clave: avg, floración natural, reguladores de crecimiento, retardante de floración. introduction pineapple (ananas comosus) is the third most cultivated fruit globally and is native to tropical america (centre for agricultural bioscience international [cabi], 2021). this crop plays a significant role in the economy of small farmers in tropical developing countries (hossain, 2016; rahim & othman, 2019). in peru, 15,901 ha of this tropical fruit are cultivated (ministerio de agricultura [minagri], 2021). although it does not represent a large extension, it has great potential due to its properties and nutritional content (hossain, 2016) that could be exploited to increase the cultivated area and its export. the ‘md2’ stands out among other pineapple cultivars planted in peru due to its high organoleptic quality compared with ‘cayena lisa’ (neri et al., 2021). one of the critical factors in pineapple management is flowering, which is related to the environmental factors of the production site (cunha, 2005; food and agriculture organization [fao], 2021). however, natural flowering (nf) is a major problem that occurs during some months of the year. it affects harvesting programs, decreases harvesting efficiency, increases costs, and decreases the price of the fruit, making it difficult to invest in controlling pests and diseases (kuan et al., 2005; martin-prevel et al., 1993; bello & julca, 1994, 1995). several environmental factors affect nf, such as decreased sunshine hours (short days), low temperatures (mainly at night), declined solar radiation due to high cloud cover, extreme relative humidity (low and high), and altitude (masl) (gowing, 1961; aubert et al., 1973; friend & lydon, 1979; reinhardt et al., 1986; bello, 1991; cunha et al., 1999; cunha, 2009; maruthasalam et al., 2009; bartholomew, 2014). based on this evidence, nf is a “stressinduced flowering.” ethylene (c2h4) is responsible for nf in pineapple, where its biosynthesis occurs in almost all plant tissues, particularly in the meristematic regions. ethylene production also increases markedly in leaf abscission, fruit ripening, and senescence (kende, 1993). in addition, some external stimuli such as drought, cold, and wounds can induce its synthesis (ecker & davis, 1987; ohme-takagi & shinshi, 1995). various strategies have been employed to reduce and avoid nf, such as the use of smaller planting material (py, 1960; bello & julca 1994, 1995), to maintain adequate moisture in the soil (irrigation), ensure constant foliar fertilization with nitrogen, and apply chemical inhibitors of floral differentiation such as aviglycine (avg), which is a potent inhibitor of ethylene biosynthesis that hinders the conversion of s-adenosyl methionine to 1-aminocyclopropane1-carboxylic acid (yang & hoffman, 1984). this compound is widely used pre-and postharvest to improve the quality attributes of climacteric fruits (romani et al., 1983; starrett & laties, 1991; ju et al., 1999; manriquez et al., 1999; shellie, 1999; clayton et al., 2000; amarante et al., 2002). since 2005, several studies have been conduced that used avg in pineapple (kuang et al., 2005; wang et al., 2007). however, its commercial use in pineapple is scant, specifically under peruvian conditions whose climate particularities could affect the efficacy of this growth regulator. thus, the objective of this study was to determine the effect of avg on the dynamics of the flowering of pineapple (ananas comosus cv. ‘md2’) in the central jungle of perú. bello-amez, s.; borjas-ventura, r.; alvarado-huamán, l.; bello-medina, n.; rebaza-fernández, d.; castro-cepero, v.; julca-otiniano, a. peruvian journal of agronomy 6(1): 1–12(2022) https://doi.org/10.21704/pja.v6i1.1863 3 materials and methods plant materials and study area eight-month-old pineapple cultivar ‘md2’ was employed in this study. the pineapples were planted in the district of río negro, province of satipo, junín region, located at 720 masl (fig. 1) with a plantation density of 50,000 plant per hectare. the climate in this region is characterized by a gradual increase in rainfall from september to march, whereas rainfall decreases significantly from april to august (marca-huamancha et al., 2018). pineapple suckers of 400 g were planted on september 20, 2017 (fig. 2). the treatment was performed eight months after transplanting (may 5, 2018), and the plants were harvested from november 10 to 20, 2018. experimental plots were fertilized with nitrogen, phosphorus, potassium, and magnesium in the concentrations of 10, 2, 12, and 1 g per plant, respectively. treatments retain® 15 %, the active ingredient in avg (150 g/kg), was used. six treatments composed of various doses of retain® 15 % (150 mg/l, 250 mg/l, and 350 mg/l) were applied every 7 d figure 1. location of the district of río negro in the province of satipo in the department of junín, peru. figure 2. key dates in the management of the md2 pineapple experiment in satipo. mat, months after transplant. modification of the flowering dynamics of pineapple (ananas comosus l.) cv. ‘md2’ using aviglycine in the central jungle of perú january april 2022 4 and 14 d, resulting in eleven and six applications, respectively. the avg application was done in the morning (8 am – 10 am), and it was directed toward the heart of the plants (without adjuvant agrochemicals). two treatments without avg were also included: with and without flower induction treatment (fit) as controls. the applications were performed from april 2 to june 18, 2018 (table 1). the fit was prepared using calcium carbide and water (2.5 g/l). the fit solution of 40 ml was applied at the heart of pineapple plants in the afternoon (from 4.30 pm). data collection the number of inflorescences was counted at each sampling time. it is important to note that treatment one (t1) represented nf (table 1). the data collected were used to construct inflorescence appearance curves with their respective equations (independent variable: days; dependent variable: number of inflorescences). the curves were divided into periods. in each period (based on the slope of the curve), the relative rates of inflorescence appearance (rria) were determined using the formula: rria = (lnff − lnfi)/(ti − tf), where, ln: natural logarithm, ff: number of inflorescences at the end of each period, fi: number of inflorescences at the beginning of each period, ti: initial time of each period, tf: final time of each period (beadle, 1993). experimental design and data analysis the experimental design used was a randomized complete block design with four replications. each experimental unit consisted of 40 plants, with 25 central plants per replicate that were evaluated at harvest time. the data were analyzed using a two-way analysis of variance followed by the tukey test (p < 0.05). results table 2 shows that the number of inflorescences increased over time. the appearance of inflorescences in plants treated with 0 mg avg l-1 without and with the fit (t1 and t8) was earlier than in the avg-treated plants. in addition, both treatments resulted in more plants with inflorescence than others in almost all evaluations (p ≤ 0.05). the plant treated with t8 showed a significantly higher number of inflorescences than t1 in all evaluations except for the last four. besides that, the avg-treated plant with six applications showed a higher number of inflorescences than eleven applications throughout sampling time (p ≤ 0.05). in the final sampling, the t4-treated plants had the least inflorescence. flower appearance in pineapple cv. ‘md2’ planted under satipo’s (peru) conditions followed polynomial equations of the second, third, and fourth degree (table 3) as a function of the treatment received. in the case of nf (0 mg avg · l-1 without fit; t1), flower emergence table 1. treatments, doses, application frequency, and date of aviglycine applications on pineapple cv. ‘md2’. treat. avg (mg/l) application frequency total number of applications start of application for all treatments end of application t1 0 (without fit) ------------------------------------------t2 150 (without fit) every 7 d 11 april 2 june 18 t3 250 (without fit) every 7 d 11 april 2 june 18 t4 350 (without fit) every 7 d 11 april 2 june 18 t5 150 (without fit) every 14 d 6 april 2 june 18 t6 250 (without fit) every 14 d 6 april 2 june 18 t7 350 (without fit) every 14 d 6 april 2 june 18 t8 0 (with fit) -----------1 may 21 may 21 note: treat., treatments; fit, flower induction treatment; avg, aviglycine. bello-amez, s.; borjas-ventura, r.; alvarado-huamán, l.; bello-medina, n.; rebaza-fernández, d.; castro-cepero, v.; julca-otiniano, a. peruvian journal of agronomy 6(1): 1–12(2022) https://doi.org/10.21704/pja.v6i1.1863 5 followed a second-degree polynomial equation. in comparison, the flower emergence followed a fourth-degree polynomial equation for the plants that received only fit (0 mg avg · l-1 with fit; t8). in both cases, the slope was noticeable from the first evaluation. however, the fit treatment had the greatest slope and reached stability in the shortest time (fig. 3). in the treatments where avg was applied eleven times, it was observed that the dose used modified the degree of the polynomial equations (table 3). furthermore, in the high doses of 250 and 350 mg/l of avg (t3 and t4), the slope was visible from the evaluation carried out on august 28/18 (fig. 3). those plants that received six applications of avg (t5, t6, and t7) showed an increase in flowering by following a third-degree equation, and the slope was detected from the third or fourth evaluation (july 9–23/18) (table 3, fig. 3). the inflorescence emergence curves generally had three periods, except in the t8, which had only two periods (fig. 3). in addition, the duration of each period was related to the treatments applied (table 4, fig. 3). in the treatment with nf (t1), the first period was 42 d with an rria of 0.058 (day-1), and the rate decreased in the second and third periods (both were 28 d). for the t8, the first period was 14 d with an rria of 0.137, stabilizing in the second period that lasted until the end of the experiment. table 2. number of inflorescences observed in response to aviglycine application on pineapple cv. ‘md2’. number of inflorescences ----------------------------------------sampling dates----------------------------------------------t avg (mg/l) appl. 6.25.18 7.9.18 7.23.18 8.6.18 8.20.18 9.3.18 9.17.18 10.1.18 t1 0 −fit 1.75 b 8.25 b 13.5 b 19.00 b 22.25 a 23.75 ab 25.00 a 25.00 a t2 150 11 – fit 0.00 c 0.00 c 1.75 c 2.00 de 4.50 c 20.50 bc 22.50 b 24.25 a t3 250 11 − fit 0.00 c 0.00 c 0.00 d 0.00 e 0.75 d 11.50 d 19.75 c 23.25 a t4 350 11 – fit 0.00 c 0.00 c 0.25 d 0.00 e 0.00 d 3.00 e 10.50 d 20.00 b t5 150 6 − fit 0.00 c 0.00 c 1.25 cd 3.50 d 11.00 b 18.75 c 19.25 c 22.75 a t6 250 6 − fit 0.25 c 0.25 c 2.50 c 7.00 c 13.75 b 21.25 abc 24.25 ab 25.00 a t7 350 6 − fit 0.75 bc 0.00 c 2.00 c 6.50 c 13.50 b 20.50 bc 23.50 ab 24.50 a t8 0 +fit 3.75 a 25.00 a 25.00 a 25.00 a 25.00 a 25.00 a 25.00 a 25.00 a cv 56.17 9.2 9.73 11.17 13.25 9.1 4.5 4.1 note: t, treatments; fit, flower induction treatment; −fit, without fit; +fit, with fit; appl., number of applications. different letters within each column indicate statistical differences using tukey test at p < 0.05. table 3. equations of the curves of inflorescence appearance in the experiment with pineapple cv. ‘md2’ in satipo. treatments equation r² t1 0 −fit y = −0.003x 2 + 256.81x − 6*106 0.9985 t2 150 11/−fit y = −3*10 -6x4 + 0.5595x3 − 36359x2 + 109x − 1013 0.9606 t3 250 11/−fit y = 0.0044x 2 − 379.34x + 8*106 0.950 t4 350 11/−fit y = 6*10 -5x3 − 8.1594x2 + 353319x − 5*109 0.9961 t5 150 6/−fit y = -8*10 -5x3 + 9.8097x2 − 425066x + 6*109 0.9807 t6 250 6/−fit y = -9*10 -5x3 + 11.753x2 − 509226x + 7*109 0.9968 t7 350 6/−fit y = -9*10 -5x3 + 12.172x2 − 527381x + 8*109 0.9977 t8 0 +fit y = -4*10 -6x4 + 0.6356x3 − 41314x2 + 109x − 1013 0.9697 note: fit, flower induction treatment; −fit, without fit; +fit, with fit. variable x = number of days. modification of the flowering dynamics of pineapple (ananas comosus l.) cv. ‘md2’ using aviglycine in the central jungle of perú january april 2022 6 figure 3. curves of inflorescence number vs. time in the experiment with pineapple cv.‘md2’ in satipo. fit, flower induction treatment; t1, avg 0 mg/l and without fit; t2, avg 150 mg/l and without fit (11 applications); t3, avg 250 mg/l and without fit (11 applications); t4, avg 350 mg/l and without fit (11 applications); t5, avg 150 mg/l and without fit (6 applications); t6, avg 250 mg/l and without fit (6 applications); t7, avg 350 mg/l and without fit (6 applications); t8, avg 0 mg/l and with fit. bello-amez, s.; borjas-ventura, r.; alvarado-huamán, l.; bello-medina, n.; rebaza-fernández, d.; castro-cepero, v.; julca-otiniano, a. peruvian journal of agronomy 6(1): 1–12(2022) https://doi.org/10.21704/pja.v6i1.1863 7 the plants subjected to eleven applications of avg (t2, t3, and t4) (fig. 3, table 4) exhibited a significant increase in the rria in the second period (compared with the first), followed by a significant decrease in the second or third period. for t2 and t4, the duration of the first, second, and third periods were 56, 14, and 28 d, respectively, whereas for t3, they were 56, 28, and 14 d, respectively. in the treatments with six applications of avg (t5 and t6) (fig. 3, table 4), a high rria was observed in the first period (lasting 28 d), and it dropped in the second and third periods, which lasted for 42 and 28 d, respectively. in the first period, the plants with six applications had higher rria than those with eleven applications. on the contrary, in the second period, the plants with eleven applications showed higher values than those with six. discussion the physiological phenomenon of nf affects pineapple production all over the world (mendez, 2010), interrupting harvesting programs, decreasing harvesting efficiency, increasing costs, decreasing fruit prices, and making pest and disease control more difficult (kuan et al., 2005; martin-prevel et al., 1993; bello & julca, 1994, 1995). in short, np endangers the sustainability of the production of this crop. this study found that the emergence of inflorescences had gradually increased from the first to the last evaluation in treatment t1 (nf; 0 mg/l avg without fit) (table 2), which is directly related to the climatic conditions of the area. although we did not have the climactic data, marcahuamancha et al. (2018) reported that satipo has low temperature and water deficit conditions, table 4. relative rate and periods of inflorescence emergence in the pineapple cv. ‘md2’ experiment in satipo in 2018. treatment period avg (mg/l) appl. i ii iii t1 0 −fit 0.058* 25/6–06/8** (d = 42) 0.008 06/8–03/9 (d = 28) 0.002 03/9–01/10 (d = 28) t2 150 11 0.067 25/6–20/8 (d = 56) 0.110 20/8–03/9 (d = 14) 0.007 03/9–01/10 (d = 28) t3 250 11 0.031 25/6–20/8 (d = 56) 0.127 20/8–17/9 (d = 28) 0.012 17/9–01/10 (d = 14) t4 350 11 0.000 25/6–20/8 (d = 56) 0.240 20/8–03/9 (d = 14) 0.069 03/9–01/10 (d = 28) t5 150 6 0.088 25/6–23/7 (d = 28) 0.066 23/7–03/9 (d = 42) 0.007 03/9–01/10 (d = 28) t6 250 6 0.094 25/6–23/7 (d = 28) 0.051 23/7–03/9 (d = 42) 0.006 03/9–01/10 (d = 28) t7 350 6 0.043 25/6–23/7 (d = 28) 0.057 23/7–03/9 d = 42) 0.006 03/9–01/10 (d = 28) t8 0 +fit 0.137 25/6–09/7 (d = 14) 0.000 09/7–01/10 (d = 84) --------------------------note: fit, flower induction treatment; −fit, without fit; +fit, with fit; appl., number of applications; d, number of days. treatments 2–7 did not include fit (−fit). *relative rate of inflorescence appearance (rria). **dates. modification of the flowering dynamics of pineapple (ananas comosus l.) cv. ‘md2’ using aviglycine in the central jungle of perú january april 2022 8 which stimulate nf (cunha, 2005; fao, 2021) by triggering the synthesis of ethylene. the application of fit, a common practice in pineapple-producing areas, was also evaluated (t8; 0 mg/l avg with fit). as expected, fit significantly increased the number of inflorescences from the first evaluation, which was higher than the other treatments (p ≤ 0.05) (table 2). this practice stimulates flowering using growth regulators (collazos et al., 2017) that favor ethylene synthesis. the practice seeks to homogenize flowering and harvest (fao, 2021). likewise, the possible negative effects of nf have inspired research with the aim to delay the uncontrolled emergence of inflorescences without affecting yield. among this group of trials are those aimed at reducing nf using ethylene synthesis inhibitors such as avg (kuan et al., 2005; wang et al., 2007). ethylene biosynthesis pathway is composed, essentially, of three phases, starting with the formation of s-adenosyl-l-methionine (sam; phase 1), followed by the transformation of sam into 1-aminocyclopropane-1-carboxylic acid (acc; phase 2), which serves as a substrate for ethylene formation (phase 3) (borjas-ventura et al., 2020). the avg inhibit the conversion of sam into acc in phase 2 (khan ali, 2018). nevertheless, as expected, the effects of avg depend on the dosage and its frequency, as well as on the cultivar under study (arruda, 2017; loría, 2016). in this study, the results showed that high doses of avg (250 mg/l and 350 mg/l) with an application frequency of eleven times (t3 and t4) suppressed (p ≤ 0.05) the inflorescences’ emergence until almost the last evaluation compared with treatments that received only six applications (t5, t6, and t7) or without avg (table 2). other researchers found that high doses of avg inhibit the presence of inflorescences compared with low doses and a control (kuan et al., 2005). our results suggest that the activity of avg is dose-dependent because of the slow penetration of this product into the plant (kuan et al., 2005). on the other hand, the dynamics of inflorescence emergence in pineapple cv ‘md2’ have not been extensively studied. the patterns of inflorescence emergence changed depending on the treatments applied. plants treated with t1 (nf) and t8 (0 mg/l avg with fit) showed different curves in inflorescence emergence compared with avg treatments (fig. 3). these results showed the plasticity of this pineapple cultivar in response to the imposed conditions (dávila-velderrain et al., 2016). by evaluating the appearance of inflorescences over time, a series of data were obtained that allowed a functional analysis of the appearance of inflorescences. in this research, the equation that best represented inflorescence formation at t1 (nf; 0 mg/l avg without fit) was a seconddegree polynomial equation, also known as a quadratic equation. in the remaining treatments, third and fourth-degree polynomial equations were obtained (table 3). similar equations have already been used to describe flowering in other species (sun & frelich, 2011; chen et al., 2003). likewise, the curves of inflorescence appearance in the treatments had, in general, three periods, except for t8 (0 mg/l avg with fit) that showed only two (fig. 3, table 4), the first one being of short duration and with greater inflorescence appearance, whereas in the second period, there was no more inflorescence appearance. in addition, each period had a different duration and rria (table 4). for t1 (nf) (0 mg/l avg without fit) and the treatment with six applications of avg, the first phase showed high rria; rria had dropped in the second period and stabilized in the third period. however, treatments that received eleven applications, particularly 250 and 350 mg/l avg, showed a prolonged first phase (56 days) with low rria, followed by an increase and then a drastic drop in rria. these results indicated the strong inflorescence-suppressing capacity of avg (kuan et al., 2005; wang et al., 2007). conclusions based on the results obtained, high avg doses (at 250 mg/l and 350 mg/l avg, eleven applications) inhibited nf in pineapple cv ‘md2’. bello-amez, s.; borjas-ventura, r.; alvarado-huamán, l.; bello-medina, n.; rebaza-fernández, d.; castro-cepero, v.; julca-otiniano, a. peruvian journal of agronomy 6(1): 1–12(2022) https://doi.org/10.21704/pja.v6i1.1863 9 the treatment delayed or completely controlled early nf and losses due to excess fruit production at unscheduled times. in addition, this study has established nf under satipo conditions, which has three periods with different appearance rates, where the first period was the longest (42 d). further studies to evaluate avg application in other cultivars and regions are required to deepen the knowledge on the management of this growth regulator. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. author contributions elaboration and execution, development of methodology, conception and design; 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(1984). ethylene biosynthesis and its regulation in higher plants. annual review of plant physiology, 35, 155–189. https://doi.org/10.1146/ annurev.pp.35.060184.001103 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i3.1974 received for publication: 27 september 2022 accepted for publication: 30 november 2022 published: 31december 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by eclidean distance can recognize the best stevia genotype and environment to produce rebaudioside and stevioside under controlled conditions la distancia euclidiana puede reconocer el mejor genotipo y entorno de stevia para producir rebaudiósido y esteviósido en condiciones controladas maría de lourdes tapia y figueroa1*;luz r. gómez pando1 *corresponding author:ltapia@lamolina.edu.pe *https://orcid.org/0000-0002-5000-3504 abstract stevia rebaudiana is considered an important medicinal plant possessing low-calorie glucoside sweeteners. the present work describes the comparison of three stevia genotypes (ibt 1, ibt 2 and ibt 3) in two contrasting environments simulated under controlled conditions: sullana in peru; and misiones in paraguay (regarded as the center of origin of stevia). in the study, we explored the euclidean distance as an integrating indicator for simultaneous selection of several stevia traits. plant scientists often record multiple morphological, physiological and biochemical indicators in their experiments. common statistical data evaluations involve univariate analyses such as t-test, mann-whitney and analysis of variance followed by tukey hsd. however, these analyses do not evaluate integrally the effects of the experimental treatments because each indicator is analyzed independently. euclidean distance from each treatment combination to the ideal phenotype of the stevia plantlets was calculated. ibt 2 grown in sullana environmental conditions showed the best integral results, while ibt 1 displayed the worst results. esponse parameters to different contrasting environments. the analysis shown here indicates that the use of the euclidean distance could contribute to establishing a more integrated evaluation of the contrasting stevia genotypes. on the other hand, the euclidean distance, as a non-dimensional indicator, can help to compare different phenotype traits. keywords: biostatistics, research methods, glucoside, phenotypic traits, sweet grass. resumen stevia rebaudiana se considera una importante planta medicinal que posee edulcorantes glucósidos bajos en calorías. el presente trabajo describe la comparación de tres genotipos de stevia (ibt 1, ibt 2 e ibt 3) en dos ambientes contrastantes simulados bajo condiciones controladas: sullana en perú; y misiones en paraguay (considerado como el centro de origen de la stevia). exploramos la distancia euclidiana como un indicador integrador para la selección simultánea de varios rasgos de stevia. los científicos de plantas suelen registrar múltiples indicadores morfológicos, fisiológicos y bioquímicos en sus experimentos. las 1 universidad nacional agraria la molina, av. la molina s/n, lima, perú how to cite this article: tapia, m., & gómez, l. (2022). euclidean distance can recognize the best stevia genotype and environment to produce rebaudioside and stevioside under controlled conditions. peruvian journal of agronomy, 6(3), 222–228. https://doi. org/10.21704/pja.v6i3.1974 tapia, m., & gómez, l. peruvian journal of agronomy, 6(3), 222–228 (2022) https://doi.org/10.21704/pja.v6i3.1974 223 evaluaciones de datos estadísticos comunes implican análisis univariados como la prueba t, mann-whitney y el análisis de varianza seguido de tukey hsd. sin embargo, dichos análisis no evalúan integralmente los efectos de los tratamientos experimentales porque cada indicador se analiza de forma independiente. se calculó la distancia euclidiana de cada combinación de tratamientos al fenotipo ideal de las plántulas de stevia. ibt 2 cultivado en condiciones ambientales de sullana presentó los mejores resultados integrales, mientras que ibt 1 mostró los peores resultados. el análisis indica que el uso de la distancia euclidiana podría contribuir a establecer una evaluación más integrada de los genotipos contrastantes de stevia. así también, puede ayudar a comparar diferentes rasgos fenotípicos. palabras clave: bioestadística, métodos de búsqueda, glucósidos, rasgos fenotípicos, hierba dulce. introduction stevia rebaudiana (asteraceae; sweet grass) is considered an important medicinal plant as it possesses many beneficial effects on type ii diabetes, possessing low-calorie glucoside sweeteners (brahmachari et al., 2011). also, the plant contains important natural antioxidants such as flavonoids, phenols, tannin and essential oils (christaki et al., 2013). it is a native species of the tropical region of south america; and is still found in the wild in paraguay (giuffre et al., 2013; gusmaini et al., 2022). the bushes of this species are perennial and reach 0.9 m height. its leaves, lanceolate or elliptic and toothed, are alternate, simple, of a shiny dark green color and a rough surface, sometimes somewhat hairy, up to 5 cm long by 2 cm wide. the stems, pubescent and straight, only branch after the first vegetative cycle, with a tendency to lean. the roots are mostly superficial, although a thickened section sinks deeper; fibrous, threadlike and perennial, they are the only part of the plant in which steviosides it is not founded (ijaz et al., 2015; hossain et al., 2017). as for their flowers, they are dioecious plants that at the beginning of spring present small, tubular and white flowers, without perceptible fragrance, in corymboid panicles formed by small axillary chapters; they take more than a month to produce all the flowers (büyük et al., 2022; gusmaini et al., 2022). in nature they are pollinated by bees. the fruits are achenes endowed with a hairy pappus that facilitates their transport by the wind. the yield difference in steviosides and rebaudiosides is very pronounced between the different genotypes. currently, the best quality and most profitable is the “paraguayan stevia”, with up to 4 to 5 annual harvests (giuffre et al., 2013; hossain et al., 2017). the present work describes the comparison of three new stevia genotypes (ibt 1, ibt 2 and ibt 3) in two contrasting environments simulated under controlled conditions: sullana in peru; and misiones in paraguay (considered the center of origin of stevia). in this study we explore euclidean distance as an integrating indicator for simultaneous selection of several stevia traits. plant scientists usually record multiple morphological, physiological and biochemical indicators in their experiments. the common statistical data evaluations involve univariate analyses such as t-test, mann-whitney and analysis of variance (anova) followed by tukey hsd (lorenzo et al., 2015). however, such analyses do not evaluate integrally the effects of the experimental treatments because each indicator has been analyzed independently. for this reason, in this study we explore euclidean distance combined with the stevia phenotypic traits as an integrating indicator. euclidean distance has been widely used in many scientific fields e.g. in context of pattern recognition (ichino, 1988), bioinformatics (tavazoie et al., 1999), intelligent control systems (jafar & zilouchian, 2001; fliege et al., 2019), spectral identification (granahan & sweet 2001), information retrieval (kogan, 2007), simultaneous selection of several agricultural traits (gomez-pando et al., 2009; haque & haque, 2018; angassa & mohammed, 2022), and plant in vitro culture experiments (lorenzo et al. 2013; gómez et al., 2018; villalobos-olivera et al., 2019). however, as far as we know, the statistical management reported here has not been frequently utilized in studies of interactions of stevia with different environments. materials and methods three stevia genotypes (ibt1, ibt2 and ibt3) were grown under controlled conditions, simulating two contrasting environments: euclidean distance can recognize the best stevia genotype and environment to produce rebaudioside and stevioside under controlled conditions september december 2022 224 sullana in peru; and misiones in paraguay that is regarded as the center of origin of stevia (table 1). ibt1 is the morita ii variety, developed in japan by toyoshigue morita and introduced to peru. ibt2 is the miskibamba variety derived from the morita ii variety; and ibt3 is a mutant line derived from the miskibamba variety using gamma radiation at a dose of 20 gy in the laboratory of the institute of biotechnology (ibt) of universidad nacional agraria la molina. thirty-day plantlets used in the study were obtained under in vitro conditions in ms (murashige – skoog) culture medium following established protocols for stevia. acclimatization was carried out for one month in a greenhouse using the sterilized premix 3 substrate (500 g sterile-premix soil per plantlet). all data of this study were statistically evaluated using spss (version 8.0 for windows, spss inc., new york, ny) to perform twoway analysis of variance (anova) and student – newman keuls tests (p=0.05). the overall coefficients of variation (ocv) were used to assess sensitivity and they were calculated as follows: (standard desviation/average)*100 (1) in this formula, we considered the average values of the six combinations compared (2 environments and 3 stevia genotypes) to calculate the standard deviation and average. therefore, the higher the difference between the six treatments compared, the higher is the ocv (lorenzo et al., 2015). the ocvs were categorized as follows: low from 12.37 % to 26.99 %, medium from 26.99 % to 41.61 % and high from 41.61 % to 56.2 3%. ranges for ocv classification as high, medium and low depend on ocv values recorded in each experiment (lorenzo et al., 2015). in addition, to identify the most significant integral effect of the six combinations studied on stevia phenotype, the euclidean distances to the expert criteria were calculated. as the ideal expert criteria, the maximum values of plant height, numbers of leaves and shoots per plant, root length, plant fresh weight, and rebaudioside and stevioside content were considered. an excel sheet (microsoft office) was used. original data were standardized with 0 to 1 by the min-max normalization (kantardzic, 2003). min and max values of each variable were identified and then the following formula was used to standardize data: (value to be normalized-min.value observed in the experiment)/(max.value observed in the experiment – min.value observed in the experiment) (2) after standardization of all treatment data, the euclidian distance to the best expert criteria was calculated according to equation: euclidean distance (3) to the expert criteria n = number of dependent variables evaluated q = indicators evaluated p= indicators described as expert criteria: maximum values of the seven dependent variables described i = indicators evaluated table 1. environments compared under control conditions time (h) temperature (oc) relative humidity (%) radiation time (h) temperature (oc) relative humidity (%) radiation (w/m²) 0-0 20.0 80.0 0.0 0-0 21.0 76.0 0.0 0-6 19.7 81.6 0.0 0-6 18.8 83.3 0.0 6-11 20.6 77.7 100.0 6-11 24.4 58.0 200.0 12-5 28.1 52.7 300.0 11-5 35.0 22.5 300.0 6-6 28.7 50.9 100.0 5-8 28.7 34.3 200.0 7-12 23.8 66.6 0.0 8-12 26.3 36.0 0.0 tapia, m., & gómez, l. peruvian journal of agronomy, 6(3), 222–228 (2022) https://doi.org/10.21704/pja.v6i3.1974 225 results and discussion several statistically significant differences among the six combinations studied were recorded (table 2). however, high ocvs were only noted in the number of leaves per plant, and in the plant fresh weight. the highest values were recorded in ibt 2 grown in the chamber that simulates the environment of sullana (peru). on the other hand, medium ocvs were observed in the number of shoots per plant and in the root length (table 2). the ibt 2 grown in sullana showed the highest values of shoots per plant. this genotype, grown in misiones showed the longest roots. the calculation of the euclidean distance from treatment combination to the ideal phenotype of the stevia plantlets is shown figure 1. the ibt 2 grown in sullana showed the best integral results, while ibt 1 displayed the worst results. a comprehensive picture of the effects of stevia genotypes and environments on the plant phenotypes is shown in table 2. each dependent variable was analyzed separately, giving a broad insight into the physiological changes induced by the treatment. however, experimental noise and sensitivity of individual measurements make many of these parameters insufficient to accurately rank the treatments in terms of severity. therefore, we combined all measurements by calculating euclidean distances to integrate results. before calculating the euclidean distances, standardization of variables is important to prevent certain features from dominating distance determinations, merely because they have large numerical values (duda et al., 2001; kantardzic, 2003). the following information was considered as the ideal phenotype of stevia to calculate the euclidean distances: maximum values of plant height; number of leaves per plant; number of shoots per plant; root length; plant fresh weight; rebaudioside content; and stevioside content. table 2. phenotypes of stevia materials in two contrasting environmental conditions simulated under controlled conditions (averages ± se). experimental location simulated stevia genotype plant height (cm) number of leaves per plant number of shoots per plant root length (cm) plant fresh weight (g) rebaudioside content (g/100 g dw) stevioside content (g/100g dw) sullana (peru) ibt 1 9.33 ± 0.81 b 34.01 ± 2.94 c 3.66 ± 0.26 c 11.25 ± 1.23 cd 2.91 ± 0.15 c 10.10 ± 1.03 f 3.44 ± 0.26 f ibt 2 13.37 ± 1.25 a 57.66 ± 3.87 a 7.00 ± 0.54 a 13.66 ± 1.56 b 7.04 ± 0.64 a 16.05 ± 1.68 e 4.59 ± 0.36 a ibt 3 13.37 ± 0.92 a 45.00 ± 3.56 b 5.00 ± 0.48 b 12.33 ± 1.13 bc 3.94 ± 0.26 b 18.48 ± 1.54 c 4.58 ± 0.34 b misiones (paraguay, center of origin of stevia) ibt 1 12.66 ± 1.14 a 20.31 ± 1.96 d 2.00 ± 0.19 d 10.00 ± 0.98 d 2.48 ± 0.23 cd 16.46 ± 1.13 d 3.61 ± 0.28 e ibt 2 12.75 ± 1.11 a 12.50 ± 1.12 d 5.00 ± 0.33 b 26.33 ± 1.35 a 1.73 ± 0.14 d 19.66 ± 1.59 b 4.53 ± 0.34 c ibt 3 11.50 ± 1.15 a 37.00 ± 2.98 c 4.50 ± 0.34 bc 14.00 ± 1.38 b 2.39 ± 0.12 cd 20.21 ± 1.96 a 4.27 ± 0.31 d ocv (%)* 12.72 47.54 36.57 40.69 56.23 21.95 12.37 classification of ocv** low high medium medium high low low note. results with the same letter are not statistically different (two-way anova, student-newman-keuls, p>0.05). for statistical analysis only, the numbers of leaves and shoots were transformed according to y´=y0.5. * overall coefficient of variation = (standard deviation/average)*100. to calculate this coefficient, average values were considered. the higher the difference among the six treatments compared, the higher the overall coefficient of variation. ocvs were calculated for those indicators with statistically significant difference according to two-way anova and student-newman-keuls tests. ** low from 12.37 % to 26.99 %, medium from 26.99 % to 41.61 % and high from 41.61 % to 56.23 %. figure 1. euclidean distance from each environment simulated and stevia genotype to the expert criteria. results with the same letter are not statistically different (two-way anova, student-newman-keuls, p>0.05), vertical bars represent se. euclidean distance can recognize the best stevia genotype and environment to produce rebaudioside and stevioside under controlled conditions september december 2022 226 the equation (3) means that the euclidian distance tends to increase with increase in the number of parameters. the number of recorded variables should be the same for all treatments; and traits must be quantitative. theoretically, the number of variables evaluated is limitless. we would like to mention other methods available, such as the biplot metrics or nmds (nondimensional scaling), to visualize the distances between the treatments. a biplot is a graphical representation of multivariate data, where the elements of a data matrix are represented according to dots and vectors associated with the rows and columns of the matrix. on the other hand, the goal of nmds is to represent the original position of data in multidimensional space as accurately as possible using a reduced number of dimensions that can be easily plotted and visualized. nmds relies on rank orders (distances) for ordination (i.e. non-metric). the use of distances omits some of the issues associated with using predictor variables alone (e.g. sensitivity to transformation). the nmds allows for much more flexible technique that accepts a variety of data types (chapman et al., 2001; gabriel, 2002; krzanowski, 2004; faria & demetrio, 2008; blasius et al., 2009). conclusions as discussed earlier, the euclidean distance has been widely used in other scientific fields, but to our knowledge it has not been used to integrate stevia plantlet response parameters to different contrasting environments. the analysis shown here indicates that the use of the euclidean distance could contribute to establishing a more integrated evaluation of the contrasting stevia genotypes. on the other hand, the euclidean distance, as a non-dimensional indicator, can help to compare different phenotype traits. acknowledgements this research was supported by the universidad nacional agraria la molina, lima, peru. orcid and e-mail tapia, l. ltapia@lamolina.edu.pe https://orcid.org/0000-0002-5000-3504 gómez luzgomez@lamolina.edu.pe https://orcid.org/0000-0001-5536-5179 references angassa, d., & mohammed, j. 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(2015). coefficient of variation can identify the most important effects of experimental treatments. not bot horti agrobo cluj-nap,43(1), 287–291. https://doi.org/10.15835/nbha4319881 tavazoie, s., hughes, j. campbell, m., cho, r., & church, g. (1999). systematic determination of genetic network architecture. nat genet, 22, 281–285. https://doi.org/10.1038/10343 villalobos-olivera, a., hernández, l., martínez, j., quintana, n., zevallos, b. e., yabor, l., martínez-montero, m. e., gonzálezolmedo, j., sershen, j. c. l. (2019) euclidean distance can recognize the biojas® concentration that produces the ideal physiological status of pineapple in vitro-plantlets. in vitro cell.dev.biol.plant, 56, 259–263 (2020). https://doi. org/10.1007/s11627-019-10023-5 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i3.1980 received for publication: 27 september 2022 accepted for publication: 30 november 2022 published: 31 december 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by clonal propagation of stevia (stevia rebaudiana bertoni) advanced lines in a temporary immersion bioreactor system propagación clonal de líneas avanzadas de stevia (stevia rebaudiana bertoni) en un sistema de biorreactores de inmersión temporal maría de lourdes tapia y figueroa1*; luz r. gómez pando1 *corresponding author: ltapia@lamolina.edu.pe *https://orcid.org/0000-0002-5000-3504 abstract stevia (stevia rebaudiana a bertoni), is a native species from paraguay and brazil, which is used as a natural sweetener with medicinal value in the preparation of beverages, sweets and others. its propagation through sexual seeds is limited by their poor quality, for this reason its asexual propagation is promoted through different in vitro techniques. in the present investigation, the response of three genotypes of stevia to propagation in temporary immersion bioreactor (tib) system was studied with four different media as a treatments. the results showed that the treatment m2 (ms + 1 mg/l bap + 0.1mg/l naa + 30 g sucrose) gave the best results for all evaluated characteristics with the exception of the number of rootlets/seedling and being the treatment m1 (ms without growth regulators + 30 g of sucrose) achieved the highest number of roots and good values for the other characteristics. high quality plants were achieved and the genotype that responded best was ibt-1, and in the acclimatization phase a high survival rate was obtained. keywords: sweetener, in vitro, bioreactor, genotype, stevia. resumen la stevia (stevia rebaudiana bertoni), es una especie originaria de paraguay y brasil, la cual se utiliza como edulcorante natural con valor medicinal en la preparación de bebidas, dulces y otros. su propagación a través de semillas sexuales está limitada por la mala calidad de las mismas, por lo que se promueve su propagación asexual mediante diferentes técnicas in vitro. en la presente investigación se estudió la respuesta de tres líneas avanzadas de stevia a la propagación en biorreactores de inmersión temporal con cuatro tratamientos diferentes. el tratamiento m2 (ms + 1 mg/l bap + 0.1 mg/l naa + 30 g sacarosa) dio los mejores resultados para todas las características evaluadas con excepción del número de raicillas/plántula y siendo el tratamiento m1 (ms sin reguladores de crecimiento + 30 g de sacarosa) con la que se logró el mayor número, de raíces y buenos valores para las demás características. se lograron plantas de alta calidad y el genotipo que mejor respondió fue ibt-1 y en la fase de aclimatación se obtuvo una alta tasa de supervivencia. palabras clave: edulcorante, in vitro, biorreactor, genotipo, stevia. 1 universidad nacional agraria la molina, av. la molina s/n, lima, perú how to cite this article: tapia, m., & gómez, l. (2022). clonal propagation of stevia (stevia rebaudiana bertoni) advanced lines in a temporary immersion bioreactor system. peruvian journal of agronomy, 6(3), 256–262. https://doi.org/10.21704/pja.v6i3.1980 mailto:ltapia@lamolina.edu.pe tapia, m., & gómez, l. peruvian journal of agronomy, 6(3), 256–262 (2022) https://doi.org/10.21704/pja.v6i3.1980 257 introduction stevia (stevia rebaudiana bertoni), is a specie used as a natural sweetener in the preparation of beverages. it has medicinal value due to its ability to reduce glycemia and hypertension problems. hossain et al. (2017), points out the value in human health of steviol, which is a substance synthesized by the plant and is the one that provides benefits such as regulation of blood pressure, control of blood glucose levels, as well as inducing the pancreas to insulin production, also has antibacterial and antifungal effect. yadav et al. (2011), mentions that stevia is a species belonging to the asteraceae family. it can be propagated by cuttings, seeds, or tissue culture, through the use of explants such as leaves, axillary shoots, root shoots, or internodal explants. the use of temporary immersion bioreactors (tib) is an alternative to achieve numerous, uniforms, vigorous plants, with larger size and numerous leaves and a better root system. the use of tib in stevia micropropagation was reported by akita et al. (1994) and acquired greater development of shoots with a total mass of 64.6 kg. likewise, alvarenga & salazar (2015), indicated a greater number of vigorous shoots with better morphological development of stevia in bioreactors in a liquid medium, much higher than that obtained in semisolid media. plants under temporary immersion show better development at the level of buds, leaves and stems, due to the conditions provided by the temporary immersion bioreactor system (vives et al., 2017). on the other hand, rosales et al. (2018) points out that temporary immersion media are more effective for micropropagation, because they provide more space for seedling development . in addition, melviana et al. (2021), indicates that the availability of seedlings to absorb nutrients is related to the immersion period, which is favorable for plant growth. although there are studies of the favorable response of stevia to propagation in bioreactors, it is important to evaluate the response of new advanced lines of this species in a tib system. the present work aims to study the response of new advanced lines of stevia to in vitro culture in a tib system. materials and methods experiment location this research work was carried out in the in vitro tissue culture laboratory of the institute of biotechnology ibt, at the la molina national agrarian university, located in la molina, lima province, lima department. peru. vegetal material three genotypes were used. advanced lines, ibt-1, ibt-2 and ibt-3. ibt – 1: variety morita ii, was developed by toyoshigue morita. ibt – 2: variety miskibamba, developed from morita ii by mass selection. ibt – 3: clone derived from variety miskibamba by gamma radiation. culture media it was used following four culture media m1: (ms without growth regulators + 30 g of sucrose) m2: (ms +1 mg/l benzyl amino purine (bap) + 0.1 mg/l naphthaleneacetic acid (naa) + 30 g sucrose) m3: (ms+ 1.5 mg/l benzyl amino purine (bap) + 0.1 mg/l naphthaleneacetic acid (naa) + 30 g of sucrose) m4: (ms+ 2.5 mg/l benzyl amino purine (bap) + 0 naphthaleneacetic acid (naa) + 30 g sucrose) clonal propagation of stevia (stevia rebaudiana bertoni) advanced lines in a temporary immersion bioreactor system september december 2022 258 methodology 1.explants (internode with four buds) come from mother plants of advanced lines ibt1, ibt2 and ibt3 cultivated in vitro, which are free of diseases. 2.preparation and sterilization of four liquid culture media. 3.for the bioreactors, two twin 500 ml transparent erlenmeyer flasks were used. 4.20 cuttings with four buds were planted in one erlenmeyer. in other erlenmeyer, the liquid culture medium was placed. both were interconnected by silicone hoses and hermetically sealed with rubber lids. a fiveminute immersion was used in intervals of three hours, regulated by the timer (micro pc-moller r) that at the same time controls the photoperiod. the protocol established by escalona et al. (1999) was used in this experiment (figure 1a). 5.after 30 days, the plants were removed, incubated at a temperature of 22 °c and 16 hours of light and 8 hours of darkness (figure 1b) . evaluations height plant, number of stems, number of leaves, fresh weight and number of roots per seedling were measured following established procedure for this type of studies. the experiments were organized in a completely randomized design due to the use controlled parameter in the incubation chamber. table 1. mean squares of the anva of seedling height, number of stems/seedlings, number of leaves/seedlings, weight (g) and number of roots/seedlings of stevia (stevia rebaudinaa bertoni) propagated in temporary immersion bioreactors in four different culture media. la molina 2021. source of variation degree free seedling height number of stems/seedling number of leaves/ seedling weight number of roots/seedling treatment 3 55.84 *** 265.22 *** 1966.2 *** 2.811 *** 40.18 *** residuals 12 0.64 1.02 14.1 0.028 0.10 c.v (%) 11.652 14.501 12.417 12.059 15.983 media 6.858 6.967 30.213 1.373 2.017 note. “***” α = 0.001, there are highly significant differences between treatments table 1. clonal micropropagation of stevia. (a) tib system showing both erlenmeyers with plantlet and culture media, (b) plantlets removed after 30 days incubation. tapia, m., & gómez, l. peruvian journal of agronomy, 6(3), 256–262 (2022) https://doi.org/10.21704/pja.v6i3.1980 259 the data obtained in the evaluations were processed using the statistical program r study version 3.6.3. results and discussion determination of the effect of different culture media the results of the analysis of variance are presented in table 1 and highly significant differences can be seen in treatments for all the variables evaluated. the coefficient of variation for seedling height was 11.66 %, number of stems per seedling 14.50 %, number of leaves/ seedling 12.41 %, fresh weight/seedling 12.05 %, while for the number of rootlets/seedling it was equal to 15.98 %. a general mean of 6.85 cm for seedling height, 6.96 stems/seedling, 30.21 leaves/seedling, 1.37 g fresh weight/seedling and 2.07 rootlets/seedling were observed. table 2 shows the mean values for seedling height, number of stems per seedling, number of leaves/seedlings, fresh weight/seedling and number of rootlets/seedlings. significant differences (tukey significance test α= 0.05) were observed for all the characteristics. a range of 1.90 cm to 9.88 cm for seedling height were founded, and it can be seen that the lowest value was obtained with the m4 medium (ms + 2.5 mg/l bap + 0 naa +30 g sucrose) and the highest in the m2 medium (ms +1 mg/l bap + 0.1 mg/l naa + 30 g sucrose). for the number of stems/seedlings, the range was of 1 to 19 stems, the lowest value was found in the medium m4 and the highest with the m2. for the number of leaves/seedlings, the observed values ranged from 7 to 59.46 leaves, with the lowest value in medium m4 and the highest in medium m2. for fresh weight/seedling the range varied from 0.27 g to 2.27 g and the lowest value was founded in medium m4 and the highest in medium m2. for number of rootlets/seedlings the observed values ranged from 0 to 6.66 rootlets and no rootlets were formed in the m3 (ms + 1.5 mg/l bap + 0.1 mg/l naa+ 30 g of sucrose) and m4 media and the highest number of rootlets was detected in the m1 media (ms without growth regulators + 30 g of sucrose) (table 2). in the present research, the best values were observed in the m2 medium (ms + 1 mg/l bap + 0.1 mg/l naa + 30 g sucrose) and the lowest in the m4 medium (ms + 2.5 mg/l bap + 0 naa + 30 g sucrose) for all the characteristics evaluated with the exception of rootlets formation. alvarenga & salazar (2015), using stevia micropropagation with temporary immersion bioreactors, observed seedlings with 23.40 leaves on average and a height range of 3.7 cm to 6.00 cm and absence of roots. villamarin et al. (2020) points out that the addition of iba (0.37 mg. l-1) promoted the development of more shoots, larger size, more leaves, and root formation, in the tib system. according to aguilar et al. (2019), the high concentration of 6-benzyl amino purine can act negatively on the development of stevia plants, suggesting the use of low concentrations, such as 0.5 mg. l-1, complement with auxins or use culture media without growth regulators. alexander et al. (2016) pointed out that the addition of 2 ppm of kinetin to the culture medium generated a greater number of shoots. on the other hand, exposure to red light promotes a greater development of shoots; also stimulating the accumulation of steviosides. table 2. mean values of seedling height, number of stems/seedlings, number of leaves/seedlings, weight (g) and number of roots/seedlings of stevia (stevia rebaudinaa bertoni) in temporary immersion bioreactors in four different culture media. la molina 2021. treatment seedling height (cm) number of stems/ seedling number of leaves/ seedling weight (g) number of roots/ seedling m1 9.600 a 4.167 b 32.750 b 1.675 b 6.667 a m2 9.880 a 19.000 a 59.467 a 2.270 a 1.400 b m3 6.053 b 3.700 b 21.633 c 1.270 c 0.000 c m4 1.900 c 1.000 c 7.000 d 0.277 d 0.000 c note. means with the same letter are not significantly different, according to tukey test (α = 0.05) clonal propagation of stevia (stevia rebaudiana bertoni) advanced lines in a temporary immersion bioreactor system september december 2022 260 evaluation of the performance of three genotypes of stevia in tib system the results of the analysis of variance are presented in table 3, and highly significant differences can be seen in treatments for all the variables evaluated. a coefficient of variation was found for seedling height, number of stems per seedling, number of leaves/seedlings, fresh weight/seedling and number of rootlets/seedling equal to 11.02 %, 10.72 %, 12.62 %, 8.01 % and 9.846 %, respectively. a general mean of 9.10 cm for seedling height, 3.93 stems/seedling, 29.94 leaves/seedling, 1.65 g fresh weight/seedling and 6.74 rootlets/seedling was found. table 4 shows the mean values for seedling height, number of stems per seedling, number of leaves/seedlings, fresh weight/seedling and number of rootlets/seedling and the tukey significance test (α= 0.05) and significant differences are observed for seedling height and number of leaves/seedlings. a range of 8.13 cm to 9.90 cm were observed for seedlings height, and the lowest seedling height corresponded to the ibt3 genotype and the highest value to ibt1 genotype. the number of stems/seedlings had a range of 3.80 to 4.17 stems and the lowest value corresponded to the ibt3 genotype and the highest value to ibt1 genotype. for number of leaves/seedlings the corresponded values ranged from 21.50 to 36.50 leaves and the lowest value was found in the ibt3 genotype and the highest value in ibt1 genotype. for fresh weight/ seedling, the observed range varied from 1.52 g to 1.73 g, with the lowest value corresponding to the ibt3 genotype and the highest to ibt1 genotype. for the number of rootlets/seedlings, the range was 6.26 to 6.80 rootlets and the lowest number was found in ibt3 genotype and the highest in ibt1 genotype and ibt2 genotype. among the genotypes studied, ibt1 line stands out, presenting the highest values of seedling height, number of stems per seedling, number of leaves per seedling, fresh weight per seedling and number of rootlets per seedling. in some characters slightly higher values than ibt2 genotype were observed in ibt1; however, these two genotypes presented values higher than ibt3 genotype for the characters evaluated. oviedo (2017), who worked with progenies 7 and 3, reports that there are significant differences between progenies in plant size, number of leaves and shoots. this coincides with the results obtained with the ibt1 and ibt3 genotypes which responses were also different in the condition of this experiment. bayraktar (2019) and melviana et al. (2021b) indicate that immersion periods of more than 10 seconds are detrimental to seedlings, which become chlorotic. in the present work, the immersion was 4 seconds and the seedlings did not present negative effects. vives et al. (2017) and ramírez et al. (2016), reported that in micropropagation in temporary immersion bioreactors, seedlings with better development at the level of shoots, leaves and stems are achieved, due to continuous gas exchange which favors better plant nutrition. in the present research work, in similar way, seedling height of 9.90 and 36.50 number of leaves in the ibt1 genotype were founded. table 3. mean squares of anva of seedling height, number of stems, number of leaves/seedlings, fresh weight/seedling and number of roots/seedlings of three genotypes of stevia (svevia rebaudinaa bertoni) propagated in temporary immersion bioreactors. la molina 2021. source of variation degree free seedling height number of stems/ seedling number of leaves/seedling weight number of roots/ seedling treatment 2 4.053 * 0.208 283.52 *** 0.070 * 0.505 residuals 12 1.006 0.178 13.76 0.018 0.4406 c.v (%) 11.02 10.72 12.62 8.01 9.85 media 9.10 3.93 29.34 1.65 6.74 note. “***” α = 0.001, there are highly significant differences between treatments tapia, m., & gómez, l. peruvian journal of agronomy, 6(3), 256–262 (2022) https://doi.org/10.21704/pja.v6i3.1980 261 conclusions good quality in vitro plants were obtained in the three advanced stevia lines, in the m2 culture medium (ms + 1 mg/l bap + 0.1 mg/l naa + 30 g sucrose) in temporary immersion bioreactors system. among the genotypes, ibt1 stands out with the best performance and the least response in the treatments studied was observed in ibt3 genotype. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. id orcid and e-mails tapia, m. ltapia@lamolina.edu.pe https://orcid.org/0000-0002-5000-3504 gómez, l. luzgomez@lamolina.edu.pe https://orcid.org/0000-0001-5536-5179 references aguilar, d., rodríguez, j. l., piña j., & silva, v. 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(2020). trichoderma asperellum, an inoculant for the production os steviol glycosides in stevia rebaudinaa bertoni plants micropropagated in a temporary immersion bioreactor. revista mexicana de ingeniería química, 19(3), 1153–1161. https://doi.org/10.24275/rmiq/bio947 https://doi.org/10.4141/cjps10086 peruvian journal of agronomy 1 (1): 8-13 (2017) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v1i1.1062 © the authors. published by universidad nacional agraria la molina received for publication: 4 october 2017 accepted for publication: 2 december 2017 sustainability of cacao farms in the district of huicungo (san martín, perú) with the “rapid agroecological method” sustentabilidad de fincas cacaoteras en el distrito de huicungo (san martín, perú) con el “método agroecológico rápido” tuesta, o.1; santistevan, m.1; borjas, r.1; castro, v.2; julca, a.1* *corresponding author: ajo@lamolina.edu.pe abstract this work was carried out with the objective to determine the sustainability of cocoa farms in the district of huicungo (san martín, perú) by using the “rapid agroecological method”. the study was conducted in the san martín region, province of mariscal cáceres, district of huicungo. the zone has an annual precipitation of 1, 200 mm and an annual temperature of 30ºc. for the sustainability analysis, we selected “type farms” from each of the three groups found in the area. in each of these, indicators of soil quality and crop health were evaluated using values ranging from 1 to 10 (1: less sustainable, 10: more sustainable). with the collected data, duncan test (p ≤ 0.05) was performed to determine the statistical differences between the “type farms”. results showed the evaluated cacao farms had values greater than five, therefore, they are sustainable. there were no statistical differences between the three “type farms” studied here. keywords: sustainability, farms, soil, health, cultivation. resumen este trabajo se llevó a cabo con el objetivo de determinar la sostenibilidad de las fincas de cacao en el distrito de huicungo (san martín, perú) utilizando el “método agroecológico rápido”. el estudio se realizó en la región de san martín, provincia de mariscal cáceres, distrito de huicungo. la zona tiene una precipitación anual de 1,200 mm y una temperatura anual de 30ºc. para el análisis de sostenibilidad, seleccionamos “fincas tipo” de cada uno de los tres grupos encontrados en el área. en cada uno de estos, los indicadores de la calidad del suelo y la salud del cultivo se evaluaron utilizando valores que van de 1 a 10 (1: menos sostenible, 10: más sostenible). con los datos recopilados, se realizó la prueba de duncan (p ≤ 0.05) para determinar las diferencias estadísticas entre las “fincas tipo”. los resultados mostraron que las fincas de cacao evaluadas tenían valores superiores a cinco, por lo tanto, son sostenibles. no hubo diferencias estadísticas entre las tres “fincas tipo” estudiadas aquí. palabras claves: sustentabilidad, fincas, calidad, suelo, salud, cultivo. 1departamento de fitotecnia, facultad de agronomía, universidad nacional agraria la molina, av. la molina s/n, lima 12, perú. 2facultad de ciencias, universidad nacional agraria la molina, av. la molina s/n, lima 12, perú. introduction the sustainability of agriculture can be defined as the capacity of an agroecosystem to maintain the quality and quantity of natural resources in the medium and long term, reconciling agricultural productivity with the reduction of impacts on the environment and considering social needs of rural communities (brown, 1987 cited by zinck et al., 2004). other authors, cited by gómez-limón et al. (2011), define sustainable agriculture as the one that promotes food security, conserves natural resources, protects the environment and is economically viable. to evaluate sustainability, the use of mathematical models, time series, indicators, among others have been suggested (gómez-limón et al., 2011). according to de muner (2011), sustainability evaluation studies of production systems that use indicators turned out to be an effective tool for evaluating the ecological, economic and social sustainability of agricultural production systems, as in the case of the family production system of arabica coffee in espírito santo in brazil. meza and julca (2015) have also used indicators to evaluate cassava cropping systems in cusco, perú. recently, they have also used indicators in ecuador to evaluate productive units in the amazon (bravo-medina et al., 2017). but there are also authors who indicate that sustainability indicators, in general, cannot be considered universal, and due to the way of inferring the conditions of an agroecosystem, they are not commonly used by farmers (gómez et al., 1996, masera, 1999). there are other methodologies that allow the comparison of farms sustainability independently tuesta, o.; santistevan, m.; borjas, r.; castro, v.; julca, a. peruvian journal of agronomy 1(1):8-13 9 from the existing situations. for example, altieri and nicholls (2002), proposed a rapid agroecological method for sustainability evaluation of coffee plantations and has been used by merma and julca (2012) to evaluate the sustainability of diverse production systems in cusco (perú). however, this method only evaluates the quality of the soil and the health of the crop, and it does not consider the three dimensions of sustainability (márquez and julca, 2015). but it is a relatively simple tool, its use can be extended and its practicity widespread between farmers and field technicians, especially for preliminary studies of sustainability of agricultural production systems. in peru, cacao (theobroma cacao) is a crop of great importance and there are several regions that have edaphoclimatic conditions that favor the development of this crop, as is the case of san martín (arévalo et al., 2004). the crop is grown under shade in agroforestry systems, associated almost exclusively with small producers and has always been used in alternative development programs in coca-growing areas. this work was carried out with the objective of knowing the sustainability of cacao farms in the district of huicungo (san martín, peru) with the “rapid agro-ecological method”. materials and methods this study was carried out in the middle part of the subbasin of the huayabamba river, located in the san martín region, province of mariscal cáceres, huicungo district. the area is a tropical humid forest (bh-t), with an average annual rainfall of 1,200 mm, an average annual temperature of 30°c, a maximum annual average of 35°c, and the minimum annual average is 23.2°c. for the sustainability analysis, we selected three “type farms” from each of the three groups that exist in the area. group i (68% of the farms), is characterized by developing other activities parallel to the cultivation of cacao, has a conventional production system, with polyclonal plantations and a yield of 963 kg/ha. group ii (13% of farms) basically has organic production farms, where hybrid plantations associated with ccn51 predominate, with a yield of 623 kg/ha. in group iii (19% of the farms), monoclonal plantations exist using only ccn-51, with a yield of 933 kg/ha (tuesta et al., 2014). studies using “type farms” have been carried out by salazar (2012), collantes (2016) and santistevan et al. (2017). indicators of soil quality and health of the cacao crop were evaluated in each of the “type farms”. each indicator can have values ranging from 1 to 10, with 1 being the least sustainable value and 10 the most sustainable value. the valuation given to each indicator was validated with producers and professionals with experience with this crop. the indicators used in the present study were obtained from the proposal of altieri and nicholls (2002) and araujo et al. (2008). details are shown in table 1. with the collected data, a duncan test (p ≤ 0.05) was performed to determine the statistical differences between the “type farms”. subsequently, amoeba graphics were made for soil quality and crop health. results and discussion soil quality—in general terms, a different behavior of soil quality was found in each “type farm”; but type ii, had higher values for half of the indicators evaluated as cs3 (color, smell and organic matter), which logically is related to cs4 (moisture retention), cs6 (lower risk of erosion), cs7 ( structure) and cs10 (microbial activity). this is important, because the physical properties of the soil and the organic matter content of the soils are the variables with the greatest contribution to the integral fertility, suggesting an adequate physical and biological condition, which helps the penetration and development of roots. hence, they favor the use of nutrients and water in the soil (power, 2010, viana et al., 2014, bravo et al., 2015, cited by bravomedina et al., 2017). the structure and moisture retention are favored by the presence of organic matter (julca et al., 2006). soil quality had average values greater than five, although practically similar to each other (6.99, 7.04 and 6.97, for “type farms” i, ii and iii, respectively), so there were no statistical differences between them, as shown in table 2 and illustrated in figure 1. these results are explained as a response to the fact that cacao cultivation is mainly developed under an agroforestry system, being the one that most resembles a natural forest (müller, 2006). somarriba (2006), points out that agroforestry systems promote the conservation and fertility of soils, besides being the best form of land use in tropical climates (lobão et al., 2004) and recommended for organic production. other authors state that organic production has a positive impact on soil quality, on the amount of microorganisms (hole et al., 2005) and can reduce soil erosion (arnhold et al., 2014). larrea (2007) and araujo et al. (2008), report a partial sustainability in agroforestry systems with cocoa using indicators for the soil. crop health—it was different in each “type farm”. farm type i, had higher values for most of the indicators studied, with the exception of sc3 (damage incidence) and sc7 (management system), which was better for type iii and ii, respectively. the average values were greater than five, although the “farm types” i (6.56) and iii (6.28) were statistically superior to type ii (5.76). but, there are still some aspects that can be improved by performing some cultural activities that could improve the performance of the crop in general, as shown in figure 1. for example, pria and camargo (1997), point out that pruning is one of the ways to control many diseases in the cultivation of cacao. in other crops of the tropics, such as coffee, its effect has also been demonstrated to reduce the incidence levels of rust (rafael-rutte et al., 2014). the development of suitable technological practices and their application by the grower helps to improve yield and quality of cacao (proamazonía, 2003). the potential of cacao will be exhibited by using profitable and sustainable technologies to guarantee the livelihood of the grower now and in the future (iica, 2006). sustainability of cacao farms in the district of huicungo (san martín, perú) with the “rapid agroecological method” junio-setiembre 2017 10 tabla 1. indicators for the rapid evaluation of cacao farms in huicungo (san martín, perú)* considering soil quality and crop health soil quality (sq) indicator established value characteristics sq1. compaction 1 compacted soil 5 thin compacted layer 10 no compaction sq2. soil depth 1 exposed subsoil 5 thin superficial soil 10 superficial soil >1m sq3. organic matter color 1 pale, no presence of humus 5 light brown, some presence of humus 10 dark brown, abundant humus sq4. moisture retention 1 dry soil, does not hold water 5 limited moisture level available or short time 10 reasonable moisture level for a reasonable period of time sq5. soil cover 1 no cover, 100% exposed 5 less than 50% soil covered by residues 10 more than 50% soil covered by residues sq6. erosion 1 severe erosion 5 low erosion signs 10 no visible signs of erosion sq7. structure 1 loose, powdery soil without visible aggregates 5 few aggregates that break with little pressure 10 well-formed aggregates – difficult to break sq8. status of residues 1 slowly decomposing organic residues 5 presence of last year’s decomposing residues 10 most residues well-decomposed sq9. presence of invertebrates 1 no signs of invertebrate presence or activity 5 a few earthworms and arthropods present 10 abundant presence of invertebrate organisms sq10. microbiologic activity 1 very little effervescence after application of water peroxide 5 light to medium effervescence 10 abundant effervescence crop health (ch) ch1. appearance of the crop 1 chlorotic, discolored foliage with deficiency signs 5 light green foliage with some discoloring 10 dark green foliage, no signs of deficiency ch2. crop growth 1 uneven stand; short and thin branches; limited new growth 5 denser. but not uniform stand; thicker branches; some new growth 10 abundant branches and foliage; vigorous growth ch3. damage incidence 1 susceptible, more than 50% of plants with damaged leaves and/or fruits 5 between 20-45% plants with damage 10 resistant, with less than 20% of plants with light damage ch4. insect pest incidence 1 more than 15 leafhopper nymphs per leaf, or more than 85% damaged leaves 5 between 5–14 leafhopper nymphs per leaf, or 30–40% damaged leaves 10 less than 5 leafhopper nymphs per leaf, and less than 30% damaged leave ch5. actual or potential yield 1 low in relation to local average 5 medium, acceptable 10 high, above average ch6. vegetational diversity 1 monoculture 5 2-3 clones with low number of tree species 10 more than 5 clones and more than 10 tree species tuesta, o.; santistevan, m.; borjas, r.; castro, v.; julca, a. peruvian journal of agronomy 1(1):8-13 11 ch7. management system 1 conventional 5 in transition to organic with ipm or input substitution 10 organic, diversified with low external biological inputs ch8. harvest residue 1 not using them, put them where harvest ocurred 5 residues are collected, but not use them 10 uses all residues, humus is produced *adapted from de araujo et al., (2008), and altieri and nicholls (2002) continuation table 1 table 2. soil quality of “type farms” with cacao in huicungo (san martín) indicators type farms i type farms ii type farms iii compaction 7.80 7.40 7.20 soil depth 6.40 6.20 6.60 color, odor, ad organic matter 5.50 6.20 5.80 moisture retention 8.60 9.10 8.80 soil cover 7.20 6.50 7.10 erosion 5.20 6.20 5.80 structure 6.50 7.40 7.00 status of residues 7.40 6.10 7.20 presence of invertebrates 7.80 7.10 7.20 microbiological activity 7.50 8.20 7.00 average soil quality 6.99 a 7.04 a 6.97 a same letter indicate significative difference, according to duncan test (p≤0.05) table 3. crop health of “type farms” with cacao in huicungo (san martín) indicators type farms i type farms ii type farms iii appearance 6.2 6 6 crop growth 7.8 7.1 7.8 disease incidence 8.5 7.5 8.9 insect pest incidence 7.4 5.2 7 actual or potential yield 9.2 6 9 vegetational diversity 5.4 4 3.5 management system 3 6.7 3 harvest residues 5 3.6 5 average crop health 6.56 a 5.76 b 6.28 a same letter indicate significative difference, according to duncan test (p≤0.05) if we do the exercise of obtaining the average for each farm [(cs + sc) / 2], we obtain values of 6.8, 6.4 and 6.6 for farms type i, ii and iii, respectively. the three groups exceed the value of five, considered as the “threshold of sustainability” (altieri and nicholls, 2002), meaning that all cacao farms evaluated with the “rapid agro-ecological method” can be considered sustainable. this can be explained due to the work conducted for years in the san martin region with the cultivation of cacao since the development of sustainable production systems implies a continuous adaptation to local or regional socioeconomic and ecological circumstances (nieto and caicedo, 2012). the method used in the present study is relatively simple and can be used by farmers and field technicians, especially for preliminary sustainability studies of agricultural production systems. because sustainability studies should have a broader criterion always considering the three dimensions of sustainability (márquez and julca, 2015). conclusions using the “rapid agro-ecological method”, the cacao farms of huicungo (san martín) are sustainable. the method is relatively simple and can be used especially for preliminary sustainability studies of agricultural production systems. references altieri, m.; 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berroterán, j.l.; farshad, a.; mamen, a.; wokabi, s.; van ranst, e. 2004. approaches to assessing sustainable agriculture of sustainable agriculture, 23(4): 87-109. tuesta, o.; santistevan, m.; borjas, r.; castro, v.; julca, a. peruvian journal of agronomy 1(1):8-13 13 figure 1. soil quality (top) and crop health (bottom) of cacao farms in huicungo (san martín) peruvian journal of agronomy 4(3): 104–120 (2020) issn: 2616-4477 (versión electrónica) doi: http://dx.doi.org/10.21704/pja.v4i3.1490 http://revistas.lamolina.edu.pe/index.php/jpagronomy/index © the authors. published by universidad nacional agraria la molina received for publication: 19 august 2020 accepted for publication: 20 november 2020 marker-assisted selection:a smart biotechnological strategy for modern plant breeding selección asistida por marcadores: una estrategia biotecnológica inteligente para el fitomejoramiento moderno shruti shrestha1*; sudeep subedi2; jiban shrestha2 *corresponding author: shrutis2903@gmail.com https://orcid.org/0000-0002-8954-5538 abstract plant breeders and geneticists use molecular marker-assisted selection also called as mas as a useful approach for breeding of plant to make selection more efficient and speed up the breeding cycle. mas can be more efficient, effective, and reliable than phenotypic selection. molecular markers are useful to identify the economically important traits in the breeding population for further manipulation in a short time. due to the applicability of markers at the seedling stage ensuring high precision at the reduced level of cost, marker-assisted selection offer the chances to improve responses from selection. the mas using dna level polymorphism accelerate the pace of selection. the main marker technologies applied are chiefly co-dominant markers i.e. microsatellite markers/ssr (simple sequence repeats) marker, rflp (restriction fragment length polymorphism) marker and snps (single nucleotide polymorphisms). this review overviews the various mas technologies and their applications in crop improvement programs. keywords: breeding, marker assisted selection (mas), single nucleotide polymorphisms (snps) resumen los fitomejoradores y genetistas utilizan la selección asistida por marcadores moleculares, también denominada mas (por sus siglas en inglés), como un enfoque útil para la reproducción de plantas para hacer la selección más eficiente y acelerar el ciclo de reproducción. mas puede ser más eficiente, eficaz y confiable que la selección fenotípica. los marcadores moleculares son útiles para identificar los rasgos económicamente importantes en la población reproductora para su posterior manipulación en poco tiempo. debido a la aplicabilidad de los marcadores en la etapa de plántula, lo que garantiza una alta precisión a un nivel de costo reducido, la selección asistida por marcadores ofrece la oportunidad de mejorar las respuestas de la selección. el mas que usa polimorfismo a nivel de adn acelera el ritmo de selección. las principales tecnologías de marcadores aplicadas son principalmente marcadores codominantes, es decir, marcadores de microsatélites / marcador ssr (repeticiones de secuencia simple), marcador rflp (polimorfismo de longitud de fragmentos de restricción) y snp (polimorfismos de un solo nucleótido). esta revisión describe las diversas tecnologías mas y sus aplicaciones en programas de mejora de cultivos. palabras clave: reproducción, selección asistida por marcadores, polimorfismos de nucleótido único (snp) 1 agriculture and forestry university, rampur, chitwan, nepal 2 nepal agricultural research council, national plant breeding and genetics research centre, khumaltar, lalitpur, nepal cite this review: shrestha, s., subedi, s., & shrestha, j. (2020). marker-assisted selection: a smart biotechnological strategy for modern plant breeding. peruvian journal of agronomy, 4(3), 104–120. http://dx.doi.org/10.21704/pja.v4i3.1490 introduction to mas agricultural researches are being carried out with the primary aim of improving different crop species keeping in mind the desirable traits. although there are several revolution and more sophisticated process, there is the need of introducing new molecular technology in our breeding scheme like marker assisted selection which is more efficient than conventional breeding schemes (lema, 2018). detectable differences are seen due to the presence of markers’ specific biomolecules which contain proteins among various species. a molecular marker, used based on naturally occurring dna polymorphism is a sequence of dna that can be identified easily. the ideal marker s. shrestha; s. subedi; j. shrestha peruvian journal of agronomy 4(3): 104–120 (2020) 105 must be easily reproducible, polymorphic, easy, readily and cheaply detected and must have even distribution throughout the genome (nadeem et al., 2018). molecular marker is a powerful tool found in quantitative trait loci (qtl) that helps in detection of the genes carrying desirable traits. it consists of a specific molecule that helps to identify different species. a short dna sequence, like a sequence that surrounds a single base-pair change (single nucleotide polymorphism, snp), or like mini and microsatellites which are long one (alsamarai & al-kazaz, 2015). within genome there exist many regions that contain genes that are associated with a quantitative trait like yield, height, and is known as quantitative trait loci (qtls). the progress of dna markers in the 1980s resulted in the selection of qtls that helps in the representation of quantitative traits (collard et al., 2005). in agriculture, for the formation of linkage map dna markers are mainly used for diverse crop species and this linkage map is utilized for determining chromosomal regions that contain genes that control simple traits and quantitative traits using qtl 170 analysis (mohan et al., 1997). linkage maps construction and undergoing qtl analysis which helps in defining particular genomic regions that is associated with particular traits is known as qtl /genetic/gene/genome mapping (mccough & doerge, 1995; mohan et al., 1997). the process of selecting genes using such markers is referred to as marker-assisted selection (mas) and is relatively a new discipline of molecular breeding. there are various types of markers available and the use of particular marker depends upon its availability, objectives of the project, required quantity and quality of dna, level of polymorphism detecting efficiency, the required time for conducting analysis, cost per unit information, genetic diversity of species under consideration and their utility across the population. like, for self-pollinated, rapd (random-amplified polymorphic dna) markers are more useful than rflps for polymorphism detection within a gene pool. for characterizing other species, rflps that is mapped in one population can be used as heterozygous probes. markers have been elaborated and used for enhancing global food production monitoring its economically important traits. the use of molecular markers has led to the improvement of important crop like rice (mackill et al., 1999). it has been used for example, in the enhancement of heterosis for the grain yield in the b73xmo17 elite single cross hybrid maize and also we can find successful example of mabc (marker assisted back crossing) and forward crossing in maize (abler et al., 1991). even in wheat, multiparent advanced generation intercross (magic) approach is being used in uk and australia to develop multi-parent recombinant inbred lines (rils). for whole genome profiling as well as for background screening, single nucleotide polymorphisms (snps), and diversity array technology (dart) have also been used widely (gupta et al., 2010). the improvement has also been made using barley (thomas, 2003), oilseed (snowdon & friedt, 2004), horticultural crops (mehlenbacher, 1995), and pulses (kelly et al., 2003). rflp markers for the cereal cyst nematode have been used in the selection of cre1 resistance gene in wheat (ogbonnaya et al., 2001). this review will give important information and a clear concept about the newly emerging biotechnological interventions using markers. the rice crop is used as an example to show recent advances in mas. characteristics of markers in mas co-dominant markers provide more information than dominant markers as there will be no masking action. so, markers should be co-dominant in mas approaches. marker loci should be extensively and evenly distributed so that it can show all resistant genes present of the concerned traits in the chromosome. the detection work of markers should be rapid, easy, and simple and this detection system should be cost-effective and amenable to automation. markers should be highly reproducible in all cells. the marker system should be highly polymorphic to show differences between genotypes that contain and that do not the target gene. the marker should be reliable in nature which map close to the target gene. closer the marker to the target gene, lower will be the recombination frequency. also, rather than using a marker if two markers are used flanking the target gene, there will be higher accuracy of marker assisted selection. types of markers techniques different kinds of dna markers have been used based on different polymorphism detecting techniques (southern blotting, northern blotting, pcr – polymerase chain reaction, and dna sequencing) (collard et al., 2005). the different molecular marker techniques are given in table 1. procedure of mas the general procedure of mas is given in figure 1 (rana et al., 2019). marker-assisted selection involves the following major methods: (1) screening of populations (e.g., f2, f3, recombinant inbred lines, double haploids, etc.) for genotypes of interest based on molecular markers, (2) marker-assisted backcross, where one or more genes per qtls of interest are transferred from a donor parent to a recipient parent by repeated backcrossing to improve the target trait, (3) gene pyramiding schemes, where genes (two or more) identified in multiple lines/parents are accumulated into a single genotype, (4) marker-based recurrent selection, a complex scheme used for more loci involving several generations of selection and random mating of selected individuals, (5) selection based on an marker-assisted selection: a smart biotechnological strategy for modern plant breeding. september december 2020 106 sn techniques references 1 aflp (amplified fragment length polymorphism) used for dna fingerprinting (vuylsteke et al., 2007) 2 ap-pcr (arbitrarily primed pcr) used for genomic fingerprinting (welsh & mcclelland, 1991) 3 as-pcr (allele-specific pcr) used for detection of mutations, polymorphisms, and haplotypes (bottema et al., 1993) 4 asap (allele-specific associated primers) used for developing resistance in pisum sativum against bean yellow mosaic virus (yu et al., 1996) 5 caps (cleaved amplified polymorphic sequences) used for preparation of genetic map (shavrukov, 2016) 6 daf (dna amplification fingerprinting) used for producing a characteristic spectrum of short dna products useful for detecting genetic differences (caetano-anollés et al., 1991) 7 isa (inter-ssr amplification) used for genome fingerprinting (zietkiewicz et al., 1994) 8 rapd (random-amplified polymorphic dna) used for comparing dna sequences (kumar & gurusubramanian, 2011) 9 rflp (restriction fragment length polymorphism) used to characterize the microbial communities (schütte et al., 2008) 10 sap (specific amplicon polymorphism) for analysis of pcr products amplified from mapped loci of rice genomic dna (williams et al., 1991) 11 scar (sequence characterized amplified region) used for bdv2 gene’s molecular confirmation in wheat germplasm and assessment for resistance against barely yellow dwarf viruses (kausar et al., 2015) 12 spar (single primer amplification reactions) for the assessment of diversity in jatropha curcas l. (ranade et al., 2008) 13 sslp (microsatellite simple sequence length polymorphism) for its characterization in rice (panaud et al., 1996) 14 ssr (simple sequence repeats) for analysis of its polymorphism between n22 and uma rice varieties (waghmare et al., 2018) 15 sts (sequence tagged sites) for its generation and validation from diverse genotypes of dioecious jojoba (heikrujam et al., 2014) table 1 different molecular marker techniques marker-assisted selection figure 1. basic procedure for marker-assisted selection (rana et al., 2019) s. shrestha; s. subedi; j. shrestha peruvian journal of agronomy 4(3): 104–120 (2020) 107 index combining molecular and phenotypic data, and (6) genomic selection, in which genomic estimated breeding value is obtained using information from genome-wide markers. mas in gene pyramiding gene pyramiding refers to the incorporation of a desirable or resistant gene which has known effects on the target trait from multiple parents to develop superior cultivars. the more resistant gene present, the more challenging it becomes to break the resistance of the plant. like if one plant has only one resistant gene then it may only survive for 1-2 years but with the application of gene pyramiding it may survive for many years because pathogen requires double or multiple mutation to break resistant in cultivars. three bacterial resistance genes (xa5, xa13, and xa21) were introgressed in a rice cultivar samba masuri which proved to have durable resistance in rice with no yield penalty (kottapalli et al., 2010). pyramiding is very precise as it includes one gene only at one time. some important things that are to be considered while selecting such genes are the pathogens should be avirulent to the resistant gene i.e. allele frequency of corresponding avr gene must be 1 and this is how cultivar remains durable (joshi & nayak, 2010). cultivar with durable and broad spectrum resistance is desired and can be achieved by combing different resistance genes through marker assisted gene pyramiding (liu et al., 2000). the main advantages of using it are it helps to develop durable resistance, eliminates extensive phenotyping, control linkage drag and breeding duration are reduced. some examples of application of mas for gene pyramiding in various crops are presented in table 2. when the markers are tightly linked to resistant gene, with the help of marker phenotype numbers of the resistant gene carried by progeny can be identified indirectly. it has been found that through the incorporation of multiple genes, durable (broad spectrum) resistance against certain pathogens can be obtained (kloppers & pretorius, 1997; shanti et al., 2001). when qualitative resistance fails, quantitative resistance can assist as an insurance policy as in the single stripe rust gene and two qtls pyramiding (castro et al., 2003). we can undergo pyramiding using multiple parents and their number of genes as pyramiding into indica rice cultivar pr106 with the use of three bacterial blight resistance genes (singh et al., 2001). pyramiding of genes like xa1, xa5, xa13, xa21, xa26 and xa27 in rice (oryza sativa l.) for resistance to bacterial leaf blight disease has also been reported (chu et al., 2006; chukwu et al., 2019; sun et al., 2004). marker assisted gene pyramiding has also been done for bacterial blight and blast resistance with the use of marker-assisted backcrossing strategy and pyramiding two bacterial blight resistance genes (xa21 and xa13) and two major blast resistance genes (pi54 and pi1) into mega rice variety “tellahamsa” (jamaloddin et al., 2020). mas in back crossing molecular markers are broadly used in improving efficiency of backcrossing to develop high yielding superior cultivars that contributes to the higher yield. in this backcrossing process, the donor’s genetic background is removed and that of a recurrent parent is recovered. this process takes longer time and is unreliable and therefore mas in back crossing aids in transferring the beneficial gene to the recurrent parent determining young plants containing preferred trait and removing all the stray donor genes. effectiveness of marker-assisted backcrossing depends on each backcross generation population, a distance between the target locus and marker, and the numbers of background markers in use (hasan et al., 2015). effective marker backcrossing occurs in three ways (collard & mackill, 2008; holland, 2004). firstly, foreground selection which refers to the using of markers that control the gene of interest for its selection used for such qualities having tough or long phenotypic screening procedures, for knowing about the plants’ reproductive performance in the early stage of its growth and also for selecting recessive alleles. secondly, a recombinant selection signifies the selection of progeny from backcross containing the gene of interest and linked flanking markers which help in reducing the undesirable gene containing in the chromosome segment of the donor and thus helps in minimizing linkage drag. thirdly, background selection denotes the selection of the progeny from backcross that contains recurrent parent’s genome that is not linked to the target locus. this helps to recover recurrent parent with less backcrossing (even maybe in bc2) with an additional gene which is called complete line conversion. as a combination of methods, marker assisted backcross-based gene pyramiding can be accomplished in three schemes (servin et al., 2004) and (malav & chandrawat, 2016). different schemes of gene pyramiding are given in figure 2. in the first scheme, f1 hybrid is produced from the cross between recurrent parent and donor parent which then gives improved recurrent parent when f1 hybrid is backcrossed up to third generation. then, with the crossing between improved recurrent parent and other donor parent yields pyramid multiple genes. however, this is less acknowledged because it is time-consuming. in the second scheme, f1 hybrid is produced with the crossing between the recurrent parent and donor parents. then, improved f1 is produced through intercrossing which then gives improved recurrent parent when backcrossing of improved f1 is done with the recurrent parent that may results in the loss of pyramided gene. the third scheme is the combination of the first and second scheme in which instantaneous crossing between the recurrent parent and number of donor parents takes place and the result from this cross is allowed to backcross up to the third generation and finally yields pyramided lines when intercrossed marker-assisted selection: a smart biotechnological strategy for modern plant breeding. september december 2020 108 applications of mas in rice breeding mas in rice breeding for bacterial leaf blight bacterial blight (bb) is one of the most destructive rice. twenty-eight genes conferring resistance to bacterial leaf blight (bb) have been reported in rice (nino-liu et al., 2006). several genes have been associated with tightly linked dna markers, and some of them have been cloned (xa1, xa5, xa13, xa21, xa26, xa27) and used for breeding bb-resistant rice cultivars. with the exception of xa5 and xa13, the bb resistance genes are dominant in nature and the markers are developed from the sequencing information of these genes, which are widely used in mas (chu et al., 2006). the resistance genes xa5, xa13, and xa21 have been pyramided into an indica rice cultivar (pr106) using mas that expressed strong resistance to bb races of india (singh et al., 2001). mas in rice breeding for blast disease blast disease is one of the most serious diseases of rice. blast resistance is governed by a specific interaction of a particular resistance (r) gene in rice with a particular avirulence gene in the pathogen. since the initial definition of the plant resistance (r) genes by flor (1942), many r genes have been identified. the vast majority of the known r genes is composed of proteins carrying nucleotidebinding sites and leucine-rich repeat motifs (nbs-lrr) (jones & dangl, 2006). many r genes have been identified in rice and most code for nbs-lrr genes. about 40 major blast genes have been identified, about 30 genes have been mapped on different rice chromosomes, and tightly linked dna markers have been developed. the dna markers have been used effectively to identify resistance genes, and mas has been applied for integrating different resistance genes into rice cultivars lacking the desired traits. the pcr-based allele-specific and indel marker sets are available for nine blast resistance genes, and they provide an efficient marker system for mas for blast resistance breeding (hayashi et al., 2006). the breeding works in rice using mas is given in table 2. application of mas in other various crops the efforts and mechanism of mas in plant breeding in various crops are given in table 3. mas vs conventional breeding conventional breeding is the traditional types of breeding which involve the production of cultivars using old tools and techniques and not as sophisticated as modern breeding technology. marker-assisted selection makes phenotypic evaluation in laboratory relatively easy than conventional breeding. it is very hard to achieve pyramiding with the conventional methods (collard & mackill, 2008). the difference between mas and conventional breeding is given in table 4. figure 2: different schemes of gene pyramiding. rp= recurrent parent; dp= donor parent; bc= backcross; irp= improved recurrent parent. a. stepwise transfer; b. simultaneous transfer; c. simultaneous and stepwise transfer. (adopted from malav & chandrawat, 2016). s. shrestha; s. subedi; j. shrestha peruvian journal of agronomy 4(3): 104–120 (2020) 109 crop target trait(s) (target gene) and marker type reference (a) marker-assisted gene pyramiding rice bacterial leaf blight resistance (xa1, xa5, xa13, xa21, xa26 and xa27); pcr (chu et al., 2006; chukwu et al., 2019; sun et al., 2004) two bacterial blight resistance genes and two blast resistance genes into mega rice variety “tellahamsa” (xa21, xa13 and pi54, pi1); pta248 (xa21), xa13prom (xa13), pi54mas (pi54) and rm224 (pi1) (jamaloddin et al., 2020) bacterial blight and blast resistance into indian rice variety mtu1010 (xa21, xa13 and pi54); ssr (arunakumari et al., 2016) bacterial leaf blight resistance (xa21 and xa13); (xa7 and xa14); r gene pyramid of (xa4, xa5 and xa21) (arshad et al., 2016) blast resistance (pi1+piz-5+pita); rflp, pcrbased sap (pi-tq5, pi-tq1, pi-tq6, pi-lm2); rflp (hittalmani et al., 2000; tabien et al., 2000) brown plant hopper resistance (bph14 and bph15); ssr and indel markers (hu et al., 2012) gall midge resistance and bacterial blight to rphr-1005 (gm4, gm8 and xa21); ssr (kumar et al., 2017) blast resistance and bacterial blight resistance in gz63s (pi9 and xa23); scar blast resistance genes in swarna-sub1 (pi1, pi2, and pi54) (patroti et al., 2019) stripe disease resistance and eating quality of wuyujing 3 (stv-bi and wx-mq) ; pcr (tao et al., 2016) (b) marker assisted backcrossing rice high-yielding drought-tolerant nils of sabitri 2 qtls (qdty3.2 and qdty12.1) (dixit et al., 2017) bacterial blight resistance (xa5, xa13, and xa21); ssr (ramalingam et al., 2017) resistance to blast, gall midge, submergence, and salinity in a released rice variety crmas2621-7-1 blast (pi2, pi9), gall midge (gm1, gm4), submergence (sub1), and salinity (saltol); ssr (das & rao, 2015) bacterial blight resistance (xa23) (ji et al., 2014) bacterial blight resistance in deepwater rice variety, jalmagna (xa5 + xa13 + xa21); sts (pradhan et al., 2015) brown plant hopper resistance (bph14 and bph15); ssr and sts (xu, 2013) bacterial blight resistance in improved samba mahsuri (xa38); ssr (yugander et al., 2018) blast resistance (pi54, pi1 and pita); ssr and sts (khan et al., 2018) bacterial blight and blast resistance gene into jgl1798 (xa21,xa13 and pi54); ssr (swathi et al., 2019) blast resistance in variety adt43 (pi1, pi2 and pi33); ssr (divya et al., 2014) cooking and eating quality (waxy gene region); aflps (zhou et al., 2003) bacterial blight and blast resistance into rphr-1005 (xa21 and pi54); ssr (kumar et al., 2016) (c) marker-assisted validation rice bacterial blight resistance (xa39); ssr (zhang et al., 2015) bacterial blight resistance (xa40); rm27320 and id55 (kim et al., 2015) heat resistance (qhtsf4.1); m4 (nogoy et al., 2016; ye et al., 2015) deep roots (qtls on 1, 2, 7 and 9 chromosomes); rflp and ssr (hasan et al., 2015) heading date (qtls hd1,hd4, hd5, or hd6); rflp, sts, ssr, caps, dcaps (hasan et al., 2015) quality (waxy); rflp (hasan et al., 2015) brown plant hopper (bph25, bph26) rm6273, rm6775 (kurokawa et al., 2016) table 2. breeding works in rice using mas marker-assisted selection: a smart biotechnological strategy for modern plant breeding. september december 2020 110 crop target trait(s) (target gene) and marker type reference (a) marker-assisted gene pyramiding wheat powdery mildew resistance (pm2+pm4a; pm2+pm21; pm4a+pm21) ; combinations rflp (liu et al., 2000) leaf rust resistance (lr19 and lr24);ssr and scar (singh et al., 2017) leaf rust resistance (lr19 and lr24); sts (singh et al., 2004) fhb resistance (3 qtl); ssr (miedaner et al., 2006) fhb resistance and don content (3 qtl); ssr (wilde et al., 2007) cereal cyst nematode resistance (crex and crey); scar (barloy et al., 2007) fhb resistance (3 qtl); ssr (wilde et al., 2008) maize enrichment of lysine and tryptophan (opaque2 and novel opaque16); umc1066, umc1141 and umc1149 sarika et al. (2018) broccoli diamondback moths resistance (cry1ac+cry1c) (cao et al., 2002) soybean lepidopteron resistance (cry1ac+corn earworm qtl) (walker et al., 2002) soybean mosaic virus resistance (rsc4, rsc8, and rsc14q); ssr (wang et al., 2017) soybean mosaic virus resistance (rsv1, rsv3, and rsv4); ssr markers (sat_154 and satt510) and a genespecific marker (rsv1-f/r) (shi et al., 2009) soybean rust resistance (rpp2, rpp3 and rpp4); markers satt460 and af162283 maphosa et al. (2012) pea powdery mildew resistance (er1,er2 and er3) rflp, rapd/ scar and ssr (ghafoor & mcphee, 2012) mung bean powdery mildew resistance (pmr1, pmr2); rflp, aflp (chaitieng et al., 2002; humphry et al., 2003; miyagi et al., 2004) apple apple scab resistant (rvi2, rvi4, rvi5, rvi6, rvi11, rvi12, rvi13, rvi14 and rvi15); ssr and scar (patocchi et al., 2009) (b) marker assisted backcrossing wheat hmw-glutenins (glu a1 and glu-d1 genes); as-pcr (de bustos et al., 2001) fusarium head blight (fhb), orange blossom wheat midge, leaf rust resistance 8 qtl and sm1 and lr21 (somers et al., 2005) powdery mildew (pm1c, pm2, pm4b, pm12, pm13,pm16, pm20, pm21, pm23, and 13 undocumented genes); aflp (zhou et al., 2005) stripe rust (1 qtl); ssr (chhuneja et al., 2008) maize southwestern corn borer resistance (3 qtl); rflp (willcox et al., 2002) proa enhancement in sweet corn (lcye); ssr (yang et al., 2018) barley yellow dwarf virus resistance (yd2); pcr based marker (jefferies et al., 2003) (c) marker-assisted validation wheat fhb resistance (1 qtl);ssr (pumphrey et al., 2007) scab resistance (1 qtl); ssr (zhou et al., 2003) powdery mildew resistance (3 qtl); ssr (tucker et al., 2006) leaf rust resistance (lr1, lr9, lr24,lr47); sts, scar, caps (nocente et al., 2007) (d) others tomato septoria leaf spot resistance (2 inbred lines nc 85l-1w (2007) and nc 839-2(2007)-1); rapd (joshi et al., 2015) tomato mosaic virus (tm-1, tm-2, and tm-22); pcrbased markers (osei et al., 2019) powdery mildew resistance (ol-2 gene); rapd, aflp (de giovanni et al., 2004) potato potato virus y resistance (ryadg gene); rflp (hämäläinen et al., 1997) late blight of potato resistance (r1 gene); rflp and aflp (meksem et al., 1995) rose powdery mildew resistance (single gene rpp1); aflps, rgas (linde & debener, 2003; linde et al., 2006) barley powdery mildew resistance (mlg resistance locus); rflp (kurth et al., 2001) common bean anthracnose resistance (gene are); scar, rapd and rflp (adam-blondon et al., 1994) apple powdery mildew resistance (single gene pl-w); isozymes, scar, ssr, aflp, rapd (batlle & alston, 1996; evans & james, 2003; hemmat et al., 1994; liebhard et al., 2002) table 3. breeding works on various crops using mas s. shrestha; s. subedi; j. shrestha peruvian journal of agronomy 4(3): 104–120 (2020) 111 mas conventional breeding marker assisted selection aids in determining specific plants with all the resistance alleles imparting more durable multi-genic resistance. different allele separately imparts same resistance to particular disease which creates confusion in the multi-genic resistance phenotypic selection. marker assisted breeding provides an opportunity to discard the plants without desirable allele or alleles in the early stage of their growth by observing the banding pattern after running the gel electrophoresis. conventional breeding cannot be used to detect desirable trait until the plants’ area well established in the field. banding patterns can be evaluated for screening the alleles presence that is linked to all those traits. for the improvement of the plant to make it stress tolerance, insect and disease resistance, screening for each trait must be done through separate trials. with the aid of markers, the presence or the absence of concerned allele/alleles can be screened without any particular seasonal consideration. conventional breeders can improve their cultivar making it cold tolerance only by screening in the cold season that results in lower breeding rate. it is possible to determine allele or alleles associated with the resistance to a specific pest with the use of the markers. conventional breeding does not allow the screening for the resistance of parasitic pest of another country in own country. molecular markers aid in undergoing the process of line conversion faster than that of conventional breeding. the process of line conversion is lagged as conventional breeding is incapable in passing recessive desired alleles into subsequent generation as quickly as with the aid of markers since breeders cannot identify heterozygous plant phenotypically and should undergo selfing several times for its accomplishment. table 4. differences between mas and conventional breeding (dreher et al., 2002) figure 3: comparison between conventional backcrossing and background selection during marker assisted backcrossing (rani et al., 2014) conventional back crossing marker-assisted back crossing marker-assisted selection: a smart biotechnological strategy for modern plant breeding. september december 2020 112 the comparison between conventional backcrossing and background selection during marker assisted backcrossing is given in figure 3. the advantages and disadvantages of mas and conventional breeding is given in table 5. importance of mas it makes efficient use of glasshouse or nursery making the selection possible in the seedling stage as several lines can be discarded early in the breeding scheme which is non-profitable. mas allows a single selection of plants as screening is carried out using markers that eliminate error due to environmental factors. mas is not affected by environmental factors and allows for the determination of certain traits (resistance to disease, insect, abiotic stress) independent of the environment as indirect selection of traits is done with the use of markers (osei et al., 2019). it can even save breeders time, resources, and effort. it also aids in the enhancement of the heterosis, highdensity linkage maps construction. genetic contribution of each parent to its each progeny can also be determined with the aid of marker assisted selection and enables the effective selection for horizontal resistance. rflp and ssr/microsatellites are co-dominant markers which are technically simple, reliable, robust, and transferable between populations (kochert, 1994; mccouch et al., 1997; tanksley et al., 1989). rapd and aflp are dominant markers which are quick, simple, a small amount of dna required and have possibility of multiple loci and generation of the high level of polymorphism respectively (vos et al., 1995; welsh & mcclelland, 1990; williams et al., 1991). marker assisted selection helps in genes pyramiding and also makes backcrossing more efficient. it helps in visualizing the loci for quantitative resistance and compilation of qtls from different donors into one genotype to promote the level of quantitative resistance. desirable allele can be recognized in the initial stage as reported for qpm; mutant opaque2 allele can be spotted with the increase in the level of lysine and tryptophan in the kernel in the initial stage of plant growth before the visibility of its reproductive life that will ultimately be economic (dreher et al., 2002). stress resistant and quality of rice can be obtained with the aid of marker assisted selection through gene stacking (das et al., 2017). when recessive alleles governs the trait of our interest but it’s challenging enough to detect that alleles from phenotypic evaluation of heterozygous plant and from the traditional backcrossing method as it turns out to be time and resource consuming; mas makes our work much easier for the detection of recessive alleles with the application of the markers linked with them. advantages disadvantages (a) marker assisted selection (mas) it involves genotypic selection of traits of interest of plants through the use of molecular markers. this method is expensive in genotyping large number of plants. it helps to maintain high level of genetic purity through cultural identification. there may be low level of recombination between marker and qtl resulting in the need of flanking markers. it is useful for genetic diversity assessment and selection of parents. it is still not widely used due to less researches, published papers, knowledge gap and limited polymorphic markers. marker assisted backcrossing helps to reduce linkage drag and parent’s genotype can be reconstructed in three generations. marker assisted backcrossing is capable of refining only the existing elite genotypes of plants. pyramiding of desirable genes is easy, fast and an early stage screening is possible. there may arise the problem in the exact determination of position and effect of qtl. poor heritability and environmental factors do not create problem. it cannot predict phenotype with 100 percent reliability. (b) conventional breeding it is being used widely in the development of cultivars. its phenotypic selection resulting in longer time to develop superior variety. it is simple and easy as there is no need of consideration of qtl and target gene. it requires to undergo ‘grow-out tests’ for the assessment of purity. publications of researches based on it are easily available. it doesn’t deal with the genetic diversity and it is difficult to distinguish homozygous and heterozygous plants just from the phenotype. it is breeder-friendly. recurrent parent genotype reconstruction takes more than six generations. it is cheap and more reliable method. time consuming and hard to test phenotypically the presence of more than one gene. table 5. advantages and disadvantages of mas and conventional breeding (lema, 2018) s. shrestha; s. subedi; j. shrestha peruvian journal of agronomy 4(3): 104–120 (2020) 113 limitations of mas markers may not be useful for every trait as an effective phenotyping method already exist which is less expensive than mas. the type of information that is required for conducting qtl validation and mapping has a limited number of published reports. it is an expensive method as it includes large start-up expenses, licensing costs, maintenance costs, etc. there are limited markers with limited polymorphism. like, the ssr marker in wheat was utilized for indication of the sr2 gene responsible for stem rust resistance for all except for four australian cultivars which is susceptible to it (spielmeyer et al., 2003). sometimes there is insufficient linkage between markers and genes. recombination events may occur between the gene of our interest and marker used which may lead to false positive. while conducting mas, the interaction between quantitative trait loci and environmental effects are not considered. markers that are developed for mas may be valid for one population and may not be valid for the other. knowledge gap between molecular biology experts and the breeder creates the problem in the understanding of the concepts and the language used by the expert (collard et al., 2005). there is also inadequate coordination between different researchers and plant breeders. conclusion the scope of marker assisted selection is going to be wider as more and more genes are identified and their functions and interactions are annotated. mas is used to accelerate the recurrent parents’ retrieval with the aid of molecular backcrossing. the use of markers that flank a target gene can minimize the number of backcross generations. mas technology has been successfully utilized for the breeding of disease-resistant crops. rice yield is subjected to severe losses due to adverse effect of a number of stress factors; utilization of tolerant/resistant cultivars is the most effective method of controlling reduced crop production. through the process of gene pyramiding, multiple stress resistant genes could be incorporated into a single rice variety in order to develop a rice variety with high yield, biotic stress resistance and abiotic stress tolerance along with enhanced nutritional quality. references abler, b. s. b., edwards, m. d., & stuber, c. w. 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(1994). genome fingerprinting by simple sequence repeat (ssr)anchored polymerase chain reaction amplification. genomics, 20(2), 176–183. https://doi.org/10.1006/ geno.1994.1151 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i3.1966 received for publication: 04 august 2022 accepted for publication: 30 november 2022 published: 31december 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by cost-effectiveness of agriculture risk management strategies for resilience to climate shocks in niger state, nigeria rentabilidad de las estrategias de gestión de riesgos agrícolas para la resiliencia a los choques climáticos en el estado de níger, nigeria adedayo olufemi adekunle1*;raphael olanrewaju babatunde1; segun bamidele fakayode2; morolake bolaji3 *corresponding author: dayo.olufemi@gmail.com *https://orcid.org/0000-0002-9940-5889 abstract the ability of a system, community or society exposed to hazards to resist, absorb, to accommodate and recover from the effects of a hazard in a timely and efficient manner is termed resilience. in this study we used a recent cross-sectional survey data of 293 farming households in niger state, nigeria to examine the cost effectiveness of risk management strategies for resilience to climate shocks. benefit-cost analysis of mitigation strategies was examined where benefit-cost ratio (bcr), and net present value (npv) were used as decision rules. the mitigation strategies mostly used were drought tolerant crops (62.8 %), crop diversification (60.8 %), buffer stock (54.6 %) and dry season farming (41.3 %). the result showed that about 52 % of the sampled households were food secure while 48 % of them were food insecure. the result showed that dry season rice farming had the highest net benefit (nb) and npv ($ 199.98) while rain fed maize ($ 35.01) and rice had the least npv; the result showed the npv for drought tolerant cropscassava ($ 170.25), millets ($ 101.58) and sorghum ($ 96.43). the study recommends that households should take up dry season rice farming and grow drought tolerant crops such as cassava, millets and sorghum. governments should invest in and research and breeding of drought tolerant crops in order to improve food security and household resilience. keywords: benefit-cost, risk management, resilience, food security, irrigation farming. resumen la capacidad de un sistema, comunidad o sociedad expuesta a peligros para resistir, absorber, adaptarse y recuperarse de los efectos de un peligro de manera oportuna y eficiente se denomina resiliencia. en este estudio, utilizamos datos de una encuesta transversal reciente de 293 hogares agrícolas en el estado de níger, nigeria, para examinar la rentabilidad de las estrategias de gestión de riesgos para la resiliencia a los choques climáticos. se examinó el análisis de beneficio-costo de las estrategias de mitigación donde se utilizaron como reglas de decisión la relación costo-beneficio (bcr) y el valor actual neto (van). las estrategias de mitigación más utilizadas fueron los cultivos tolerantes a la sequía (62.8 %), la diversificación de cultivos (60.8 %), 1 agricultural economics and farm management department, university of ilorin, nigeria. 2 agricultural economics and extension department, federal university of oye ekiti, nigeria 3 nigerian institute of social and economic research (niser), ibadan, nigeria. how to cite this article: adekunle, a.o., babatunde, r.o., fakayode, s.b., & bolaji, m. (2022). cost-effectiveness of agriculture risk management strategies for resilience to climate shocks in niger state, nigeria. peruvian journal of agronomy, 6(3), 200–209. https://doi.org/10.21704/pja.v6i3.1966 adekunle, a.o.; babatunde, r.o.; fakayode, s.b.; bolaji, m. peruvian journal of agronomy 6(3): 200– 209(2022) https://doi.org/10.21704/pja.v6i3.1966 201 las existencias reguladoras (54.6 %) y la agricultura de estación seca (41.3 %). el resultado mostró que alrededor del 52 % de los hogares muestreados tenían seguridad alimentaria, mientras que el 48 % de ellos tenían inseguridad alimentaria. el resultado mostró que el cultivo de arroz de estación seca tuvo el mayor beneficio neto (bn) y vpn ($ 199.98) mientras que el maíz de secano ($ 35.01) y el arroz 0 tuvieron el menor vpn; el resultado mostró el vpn para cultivos tolerantes a la sequía: yuca ($ 170.25), mijo ($ 101.58) y sorgo ($ 96.43). el estudio recomienda que los hogares se dediquen al cultivo de arroz en la estación seca y cultiven cultivos tolerantes a la sequía, como la yuca, el mijo y el sorgo. los gobiernos deberían invertir, investigar y mejorar cultivos tolerantes a la sequía para mejorar la seguridad alimentaria y la resiliencia de los hogares. palabras clave: beneficio-costo, gestión de riesgos, resiliencia, seguridad alimentaria, agricultura de riego. introduction agriculture is facing several risks in nigeria like any other sub-saharan african countries which occur at different rate. an increased agricultural production in nigeria is recognized as an important factor for growth in the economic progress of the country and food and nutrition security for the rapidly increasing population (akanbi et al., 2022). risk management challenges in agriculture are very many, most especially in nigeria where agricultural systems are becoming more sensitive to risks that are already known to agriculture such political environment, demography and economic changes, and climatic factors and the emerging ones like price volatility, supply chains, and zoonoses (obiri et al., 2017). the known livestock and crops varieties are already exposed to climate changes that impact their biochemical properties and physiology resulting to bad productivity. there is a strong inverse correlation between feed intake of poultry, pigs and cow and heat stress as reported in studies (baiyeri & aba, 2017). existing cultivars of crop and animals can no longer be resilient as they were before the era of climate variability. drought is one of the anomalies that have plagued the northern part of nigeria since the beginning of the 20th century. it is the inability of rainfall to meet the evapotranspiration demands of crops resulting in general water stress and crop failures (abubakar & yamusa, 2013). they suggested that the probability of drought at the on-set and towards the end of the rainy season is usually very high in northern nigeria. dry spells at the beginning of the season usually result in multiple plantings and low or no yields leading to low food security index. in the same vein, drought that occur at the end of season could bring about water stress at critical periods of need, especially during the reproductive stage of most crops and result in crop failures and shrinking of yields (abubakar & yamusa, 2013). typically, floods are outcome of extreme weather events such as precipitation e.g. prolongs rainfall and melting snow from snowfall, which are exacerbated by the geographical location and human activities of a place. areas liable to flooding are low-lying areas, but the southern parts of nigeria are more vulnerable due to the double maxima rainfall experienced for a prolonged period, usually between marchoctober and as early as february-november in some southern states like cross river and rivers states (atu & okon, 2018). flood over the years has caused severe damage to property, infrastructure, crops and deaths across the country, and has been considered as a source of increased risks to disease and hunger, damage to property, loss of life, contamination, and spoiling of agricultural land (umoh, 2008). in nigeria farmers are using climate-resilient adaptation measures. the major agricultural ecosystems and the broad adaptation areas are: crop farming (improved soil and land management, crop-specific innovation, water management practices, climate information services and education, access to finance, and off-farm diversification), livestock farming (improved livestock management systems, improved breeding strategies, sustainable health improvement, proper feed formulation early maturing and heat-resistant bird varieties), and fish farming (water harvesting measures, organic material, quick-maturing varieties) (onyeneke et al., 2019). cost-effectiveness of agriculture risk management strategies for resilience to climate shocks in niger state, nigeria september december 2022 202 drought as a recurring event causes widespread yield declining as well as crop failure in some occasion, in nigeria agriculture. adapting to such event is very critical to ensure national food security and economic growth. one main adaptation technique is the development of drought tolerant crop varieties (lunduka et al., 2019). a drought tolerant crop variety is a variety that has ability to produce almost 30 % of its probable yield (1 t.ha−1 to 3 t.ha−1) when suffering insufficient water supply for six weeks before or during flowering stage and grain formation (magorokosho et al., 2009). in controlled experimental study, drought tolerant crops are high yielding more than several commercial hybrid varieties (international maize and wheat improvement center [cimmyt], 2013). moreover, as a matter of policy knowing the cost efficiency of climate-coping strategies before applying them is very important. understanding the cost associated with climate change adaptation interventions in agriculture is important for mobilizing institutional support and providing timely resources to improve resilience and adaptive capacities (sova et al., 2012). households, communities and government agencies will need to enact adaptive initiatives in order to cope with expected and unexpected climate change effects. doing so will bear a cost. developing countries that are already inundated with development deficits often lack the ability to meet the additional costs of adapting to climate change (stern, 2006). benefit-cost analysis aims to compare what would happen in the absence of a project (also called a baseline) with what happens after the project has been implemented (the project outcome). the costs of the project are weighed against the benefits that have accrued during this period of project implementation (buckley & peterson, 2015). smallholder farmers in nigeria are facing the challenge of low agricultural productivity due to several factors including climate change. thus majority of rural households in nigeria are engaged in crop and livestock production as their main source of livelihood, they are making efforts to sustain their productions and improve their welfare. most times, the benefits of the objectives of self-sufficiency and the results of farmers’ efforts in the production of livestock and crop products are eroded by natural events such as floods, drought and pest infestation as well as economic failure. in spite of applying different various adaptation strategies many of the farming households are still living in suffering. the issues might be that, these farmers are not using economic strategies or they are not using these strategies the right way. the high poverty among farmers may imply that cost constraints would severely limit climate adaptation, and hence, the benefits from climate adaptation (buckley & peterson, 2015). although there are empirical studies on climate change adaptation strategies among farmers in nigeria, few studies have conducted bca of climate change adaptation strategies and none has conducted studies on the adoptions of drought tolerant crops and dry season irrigation farming in nigeria. many of the available studies have analyzed the benefit cost of climate change adaptation strategies on agricultural production and have shown the efficient strategies to farming households, none of the studies delved into the drought tolerant crops and dry season irrigation farming as coping strategies applied by farming households. such studies include alves (2015) analyses benefit-cost of climate change adaptation: the use of participatory methodologies. henri-ukoha (2020) assesses the cost-benefits of climate change adaptation strategies of cassava-based farmers in southern nigeria. adzawla et al. (2020) presents the benefit-cost analysis of on-farm climate change adaptation strategies in ghana. considering the economic implications of climate adaptation, there is a need to understand the costs and benefits of climate adaptation strategies; it is also important to understand the welfare benefits of these adaptation strategies. this would help farming households to properly identify and utilize the effective strategies and influence policy formulation and funding of adaptation strategies. generally, studies on the cost-benefits of climate change adaptation strategies and their welfare impacts are very scarce. these studies are lacking in nigeria, and hence there is need for this study, as this would help farming households to know the income benefits of the major adaptation strategies. adekunle, a.o.; babatunde, r.o.; fakayode, s.b.; bolaji, m. peruvian journal of agronomy 6(3): 200– 209(2022) https://doi.org/10.21704/pja.v6i3.1966 203 the main objective of this paper is to examine the cost effectiveness of ex-ante (i.e. action taken before a negative event to prevent the effects of that event) risk management strategies for resilience to climate shocks in niger state, nigeria. the specific objectives are to: (i) identify the risk management strategies employed by farming households; and (ii) examine the benefitcost analysis of dry season irrigated farming and drought tolerant crops in the study area. we used survey data collected in 2020 from 30 villages in niger state to examine the cost effectiveness of climate shock mitigation strategies. this present study fills the gap of previous studies by focusing on the determining factors of adaptation strategies on household in nigeria. the result of this study is useful for policy formulation by government at all levels. it will serve also as a reference material to researchers and students alike. materials and methods study area niger state lies on latitude 8o to 11o30’ north and longitude 3o30’ to 07o40’ east. the state is bordered to the north by zamfara state, west by kebbi state, south by kogi state, south west by kwara state, north-east by kaduna state and south east by federal capital territory. the state also has an international boundary with the republic of benin along agwara and borgu lgas to the north west. the state covers a land area of 76 469.90 square kilometres, which is about 10 % of the total land area of nigeria out of which about 85 % is arable. the 2006 population and housing census put the state’s population at 3 950 249 (niger state bureau of statistic [nsbs], 2012). the population projection was 5 556 200 at a growth rate of 3.5 % in 2019 (city population, 2020). niger state shares in all three dams of the niger dams project, including one at shiroro gorge on the kaduna river and one at jebba in kwara state, the reservoir of which lies partly in niger state. when excess water is from these dams and reservoir during the raining season nearby villages and farms are usually flooded. it has a large hectare of inland water and vast arable land, majority of the population are farmers. niger state experiences distinct dry and wet seasons with annual rain fall varying from 1 100 mm in the southern parts to 1 600 mm in the northern parts. the maximum temperature (usually not more than 34 oc) is recorded between march and june, while the minimum is usually between december and january. the rainy seasons last for about 120 days in the northern parts to about 150 days in the southern parts of the state. generally, the fertile soil and hydrology of the state permits the cultivation of most of nigeria’s staple crops and still allows sufficient opportunities for grazing, fresh water fishing and forestry development (nsbs, 2012). data a three-stage sampling technique was used to select the sample of households for this study. in the 1st stage all the three agricultural zones in niger state were purposively selected and the study area was stratified into two according to prevailing climatic shock experiences that is droughts prevailing or vulnerable villages and floods vulnerable villages, these villages were identified with the use of digital elevation map (dem) as shown in figure 1 below. in stage 2, 15 villages were randomly selected from drought affected area which is the 1st stratum and 15 villages were equally randomly selected from floods affected area which is the 2nd stratum. in the last stage, in each village 10 farming households were selected with simple random technique and a total of 300 respondents were selected for the study but 293 had adequate information fit for analysis. primary and secondary data were used for the study. the primary data was collected through questionnaire by the use of interview schedule method. information was collected on the socio-economic characteristics of farming households in the study area, sources of livelihood available to households, household total income; expenditure of farming households. analytical technique descriptive statistics which include frequency table, graph and percentages were used to cost-effectiveness of agriculture risk management strategies for resilience to climate shocks in niger state, nigeria september december 2022 204 analyze socioeconomic characteristics of the respondents, mitigation strategies applied by the households and cost-benefit analysis of the most applied strategies. model specification the models used to achieve the objectives of the study are given below: benefit-cost analysis (bca) in estimating the cost effectiveness of dry season irrigated farming and drought tolerant crops, bca was employed. for each adaptation strategy, the total costs incurred when using those strategies and benefits were identified and to compute the net benefit for that particular adaptation strategy equation 1 was used. nbi=∑tb-∑tc (1) where; nbi = the net benefits tb = the total benefits tc = the total costs. the bcr for each adaptation strategy was subsequently computed following the equation below: ∑bcri =[∑bt⁄(1+r) t]/[∑ct⁄(1+r) t] (2) where; bcri = benefit cost ratio of the i th strategy bt = total benefits at year t, ct = total costs at year t, r = discount rate (1 + r)t = discount factor at year t. the net present value was estimated as: the net present value (npv) = ∑(bt-ct)⁄(1+r) t (3) where; figure 1: digital evaluation model (map) of niger state. source: modified of ikusemoran et al. (2014) adekunle, a.o.; babatunde, r.o.; fakayode, s.b.; bolaji, m. peruvian journal of agronomy 6(3): 200– 209(2022) https://doi.org/10.21704/pja.v6i3.1966 205 bt = total benefits in year t ct = total costs in year t r = discount rate (1+r) t = discount factor for year t decision was made and conclusions drawn on each adaptation strategy. generally, the higher the bcr, the better the strategy while the lower the bcr, the less economically viable the practice. the adaptation strategy with a positive and highest npv is the most economic and efficient. sensitivity test was carried out, where the net benefit was discounted at 5 %, 10 % and 15 %. the npv was computed based on per hectare average returns. the climate adaptation strategies that were practiced were recorded for one-year period, adoption with immediate costs and benefits, t, was assumed to be 1 year in this study. results and discussion employed risk management strategies figure 2 shows the major mitigation strategies applied by the households, it was observed that many households applied more than one strategies. over 62 % of the farmers applied drought-tolerant crops while 61 % of the farmers reported the adoption of diversification of crop, plot and livestock. more than 40 % of the farmers adopted dry seasons farming to mitigate the impacts of climate and other shocks. about 23 % said to be doing nothing to mitigate the impact of climate shocks in the study area. it was observed that application of some of the ex-ante coping strategies was determined by the recourses available to the farming households in the study area. benefit cost analysis (bca) of mitigation strategies farmers in niger state experience multiple climate stresses and shocks in different seasons throughout the year. these climate stresses affect the livelihood sectors including agriculture, fisheries, and livestock in different ways. climate extremes distress the agriculture, farming system, land use pattern, crop productivity and livelihood activities. as presented in figure 2 there are good numbers of ex-ante risk management strategies practiced by people in the area. to protect the crop from extreme weather events and to sustain food security the farmers in the area practiced irrigated farming during the figure 2: risk management strategy employed by farming households cost-effectiveness of agriculture risk management strategies for resilience to climate shocks in niger state, nigeria september december 2022 206 dry season the major crop planted during this period was rice. table 1 below shows that rice covered the mean area of 1.8 ha and total area of land among the respondents was 222.4 ha. this indicates that majority of those who practiced this strategy are small holder farmers. in the rainy season farming, rice covered the mean area of 1.65 ha and a total of 204.08 ha. in order to smoothing food consumption and income, majority of the farmers used drought tolerant crops; the most common drought tolerant crops used were millets, sorghum and cassava (okogbenin et al., 2013). cassava covered the largest total farm size of 205.60 ha and average cassava farm size was 2.36 ha this also indicates that cassava farms were dominated by small scale farming. millets covered 148.68 ha of land and the average farm size was 1.47 ha which. the farmers also applied sorghum to prevent the effect of climate shocks in the area; this crop covered the total area of 119.80 ha and the average of 1.62 ha among the respondents. in considering the benefit cost ratio (bcr), sorghum had the highest bcr (1.96), and it had the least average total cost of adoption ($ 54.43) among the respondents, this could make it accessible for a resource poor farmer. thus, the net benefit ($ 106.07) of adopting sorghum was the smallest among the drought tolerant crops. the drought tolerant crop that had the highest benefit was cassava with $ 187.27 and the cost of adoption was $ 116.33 and had the bcr of 1.60, and millets had the smallest bcr which was 1.3. to compared rain fed rice and maize with crops planted in other strategies, all the drought tolerant crops and irrigated rice had higher net benefit, benefit cost ratio, and net present values except maize with bcr which is higher than 1.30. in the last season many of the farmers produced rain fed rice at lost due to extreme weather events, the net benefits of production among the respondents were negative ($ 15.18) which suggested that total cost of production was higher than total revenue; and had the bcr of 0.88 which is far below the acceptable bcr which is 1. this implies that rain fed rice farming is risky in terms of investment (table 2). however, the dry season irrigated rice farming had the net benefit ($ 219.98) which was the highest, but the cost of adoption ($ 166.51) was the highest as well, this could make it discouraging for the majority of farming households who are resource poor to adopt. it had the least bcr (1.30) which made it to be the least to be adopted when they are considered according to bcr (table 2). the result of further estimations shows the strategy that had the highest npv was dry season rice farming and it implies that when planting rice in the dry season with irrigation, the benefit would be more than the cost by $ 199.98 for each household. this is line with the findings of adzawla et al., (2020) that discovered that irrigation farming has the highest npv among many adaptation techniques in ghana. millets and sorghum would give $ 101.58 and $ 96.43 respectively on average. compared with other crops (drought tolerant crops) cassava had the highest npv which was $ 170.25, this supports the result of henri-ukoha (2020). this implies that irrigated rice farming is more beneficial than cassava, than millets and sorghum in the area and it should be considered as the mitigation strategy against climate shocks. the sensitivity analysis shows that even if the discounts rate can change to 5 % or 15 % the npv for the different crops would be positive. however, the npv of dry season irrigated rice is the highest and cassava is followed which is a drought tolerant crop; this shows that their total revenues are more than the costs of production and their net benefits are the table 1: area of land used for different adaptation strategies by the respondents strategy crop mean farm size (ha) average output (kg) dry season irrigation rice 1.83 1281 rainy season farming rice 1.65 577.5 maize 1.54 924.0 drought tolerant crops millets 1.47 705.6 sorghum 1.62 777.6 cassava 2.36 4720 adekunle, a.o.; babatunde, r.o.; fakayode, s.b.; bolaji, m. peruvian journal of agronomy 6(3): 200– 209(2022) https://doi.org/10.21704/pja.v6i3.1966 207 highest therefore, they are more beneficial than millets and sorghum (table 2). conclusion the study has found that: majority of the farming households are small-scale farmers, climate change and variability risks are prominent among the risks faced by farmers, and both drought tolerant crops and dry season farming are cost effective among the adaptation techniques. dry season rice farming has the highest net benefit (nb). the following conclusions have been drawn on the findings; over 62 % of the farmers applied drought-tolerant crops while more than 41 % of the farmers adopted dry seasons farming to mitigate the impacts of climate and other shocks. it is observed that, dry season rice farming has the highest nb. however, according to the findings of this study, the following recommendations are outlined to improve the resilience of farming households in the country; since agriculture is the main source of rural livelihood, any improvement of incomes and food supply would be an increase of agricultural production and the main aim should be to improve productivity. a developmental strategy aims at improve agricultural productivity will improve the rural household’s resilience. among the adaptation methods found, dry season farming and use of drought tolerant crops should be encouraged among the farming households and they need to be motivated to adopt these strategies. government at all levels should rehabilitate the irrigation facilities as well as funding the breeding of drought tolerant crops. all these strategies are to ensure all year-round food production and improved households’ resilience capacity and to alleviate food insecurity. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. orcid and e-mail adedayo olufemi adekunle dayo.olufemi@gmail.com https://orcid.org/0000-0002-9940-5889 raphael olanrewaju babatunde ralphag20@yahoo.com https://orcid.org/0000-0002-3438-1054 segun bamidele fakayode segunfakayode@gmail.com https://orcid.org/0000-0002-1641-7260 morolake bolaji rollyeve@gmail.com https://orcid.org/0000-0001-9792-0150 table 2: benefit cost ratio, net present value and internal rate of returns for irrigated and drought tolerant crops crop total variable cost total revenue net benefit bcr npv 5% npv 10% npv 15% ($) ($) ($) ($) ($) ($) dry season irrigation rice 166.51 386.49 219.98 1.30 209.42 199.98 191.38 rainy season farming rice 126.33 111.16 -15.18 0.88 maize 119.54 158.06 38.52 1.32 36.67 35.01 33.51 drought tolerant crops millets 87.25 199.00 111.74 1.30 106.38 101.58 97.22 sorghum 54.42 160.50 106.07 1.96 100.98 96.43 92.28 cassava 116.33 303.67 187.27 1.60 178.29 170.25 162.93 cost-effectiveness of agriculture risk management strategies for resilience to climate shocks in niger state, nigeria september december 2022 208 references abubakar, i. u., & yamusa, m. a. 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(2008). programming risks in wetland farming: evidence from nigerian flood plains. journal of human ecology, 24(2), 85–92. https://doi.org/10.1080/0970 9274.2008.11906104 https://cgspace.cgiar.org/bitstream/handle/10568/21076/ccafs_wp_16.pdf?sequence=1&isallowed=y https://cgspace.cgiar.org/bitstream/handle/10568/21076/ccafs_wp_16.pdf?sequence=1&isallowed=y https://cgspace.cgiar.org/bitstream/handle/10568/21076/ccafs_wp_16.pdf?sequence=1&isallowed=y peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v7i1.1999 received for publication: 19 july 2022 accepted for publication: 10 april 2023 published: 15 april 2023 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by optimizing the use of biochar in okra (abelmoschus esculentus l.) production in nigeria optimizando el uso del biocarbón en la producción de okra (abelmoschus esculentus l.) en nigeria mercy funke salami1*; miracle mark2; olasumbo ibitomi3; kehinde kikelomo osasona1; victor adeniyi1; shakirat salami1; hamdalat sulaiman1 *corresponding author: markmercy12@gmail.com *https://orcid.org/0000-0003-0236-0985 abstract soil fertility has been a challenge worldwide and increasing crop productivity is essential for food security. consequently, to ameliorate this problem of low soil fertility, biochar is used. however, farmers are unaware of the optimal amount of okra biochar dosage required, especially in nigeria. this study, therefore, looked at the amount of biochar required to increase production and examined the effect of biochar on okra growth in a completely randomized design (crd) experiment. treatments used were biochar at two different levels: 50 g.kg-1 and 100 g.kg-1 of soil (treatment 1 and treatment 2), npk at the rate of 0.08929 g.kg-1 of soil (treatment 3) and a control. the result showed that okra planted with biochar grew significantly in height, weight, and number of fruits compared to those treated with npk and control with treatment 2 giving the best yield. we conclude that biochar contributes significantly to okra growth and that the optimal amount required is 50 g·kg1 of soil, we recommend that farmers use this dose to maximize the benefit of biochar. key word: biochar, okra, production, soil fertility, nigeria resumen la fertilidad del suelo ha sido un reto en todo el mundo y el aumento de la productividad de los cultivos es esencial para la seguridad alimentaria. en consecuencia, para mejorar este problema de baja fertilidad del suelo, se utiliza el biocarbón. sin embargo, los agricultores desconocen la cantidad óptima de dosis de biocarbón de okra necesaria, especialmente en nigeria. este estudio, por lo tanto, analizó la cantidad de biocarbón necesaria para aumentar la producción y examinó el efecto del biocarbón en el crecimiento de okra en un experimento de diseño completamente aleatorizado (dca). los tratamientos utilizados fueron biochar en dos niveles diferentes: 50 g·kg-1 y 100 g·kg-1 de suelo (tratamiento 1 y tratamiento 2), npk a razón de 0,08929 g·kg-1 de suelo (tratamiento 3) y un control. el resultado mostró que la okra sembrada en biocarbón creció significativamente en altura, peso y número de frutos en comparación con las tratadas con npk y control, siendo el tratamiento 2 el que dio el mejor rendimiento. concluimos que el biocarbón contribuye significativamente al crecimiento de okra y que la cantidad óptima requerida es de 50 g·kg-1 de suelo, recomendamos que los agricultores utilicen esta dosis para maximizar el beneficio del biocarbón. palabra clave: biocarbón, okra, producción, fertilidad del suelo, nigeria 1 university of ilorin, faculty of agriculture, department of agricultural economics and farm management, p.m.b.1515, ilorin, nigeria 2 university of ilorin, faculty of social sciences, department of agricultural geography and environmental management, p.m.b.1515, ilorin, nigeria 3 federal polytechnic offa, faculty of science, department of science laboratory science, offa, nigeria how to cite this article: salami, m. f., mark, m., ibitomi, o., osasona, k. k., adeniyi, v., salami, s., & sulaiman, h. (2023). optimizing the use of biochar in okra (abelmoschus esculentus l.) production in nigeria. peruvian journal of agronomy, 7(1), 2026. https://doi.org/10.21704/pja.v7i1.1999 salami, m. f., mark, m., ibitomi, o., osasona, k. k., adeniyi, v., salami, s., & sulaiman, h. peruvian journal of agronomy, 7(1), 20-26 (2023) https://doi.org/10.21704/pja.v7i1.1999 21 introduction given that nearly all the world’s arable land is presently cultivated, future food and fiber demand will have to be satisfied by increasing plant yield per unit of land area. anthropogenic land degradation challenges our ability to meet food and fiber demand in the twenty-first century, with over 50 % of global soils deteriorating and certain locations approaching 70 % (gomiero, 2016; willet et al., 2019). only roughly 11 % of the world’s land area is classified as class i-iii arable land, which needs support a 50 % increase in agricultural production to feed 9.5 billion people by 2050 (zilberman et al., 2013). both onsite (e.g., erosion) and offsite (sediment deposition) factors contribute to land deterioration. physical (crusting, compaction, erosion, desertification), chemical (acidification, leaching, salinization, fertility depletion), and biological (carbon oxidation/loss, microbial biodiversity) activities all have an impact on agricultural output on-site (european comision [ec], 2020). surface water eutrophication, groundwater contamination, and trace gas emissions (co2, ch4, n2o, ) to the atmosphere are all offsite impacts. many of the same biological, chemical, and physical soil qualities that are changed by onsite soil degrading processes also have an impact on soil fertility and plant nutrient availability. soil fertility has been improved, primarily through the application of fertilizers, the most important of which are inorganic fertilizers. however, inorganic fertilizer use is frowned upon since it poses a number of health and environmental risks (cui et al., 2018). inorganic fertilizers pollute groundwater are not environmentally beneficial (shukla & saxena, 2018; zhang et al., 2013; savci, 2012). plant tissues absorb heavy metals more frequently as a result of continuous and consistent use of inorganic fertilizers, lowering crop nutritional and grain quality (lenart-boro & boro, 2014; abdiani et al., 2019; maqbool et al., 2020). overuse of inorganic fertilizers, as a result of nutrient leaching, deterioration of soil physical properties, the buildup of harmful compounds in water bodies, and other factors, has resulted in soil, air, and water pollution, as well as serious health, environmental problems and biodiversity loss (cui et al., 2018; sharma & singhvi, 2017). organic fertilizer, particularly biochar, is a better eco-friendly choice (abukari et al., 2021). biochar is a carbon-rich, highly porous substance made from organic biomass after it has been pyrolyzed (tomczyk et al., 2020). biochar production is a long-term solution for trash and disease management (oni et al., 2019). it retains 50 % of the original carbon, which is extremely recalcitrant in nature; as a result, its synthesis aids in carbon sequestration by trapping carbon in plant biomass (tomczyk et al., 2020). the temperature, heating rate, and residence time maintained during biochar formation have a strong correlation with the elemental makeup and structural structure of the material (zhao et al. 2018). in addition to biochar, some biooil and gases are created, which can be used to generate energy and various compounds (zhao et al., 2018). jeffery et al. (2011) provided a general estimate of a 10 % increase in crop yield with biochar soil amendment in a meta-analysis of data from 16 articles that were accessible up to march 2010. additionally, it was found that adding biochar to soil increased crop productivity significantly, with an 8.4 % increase in crop production and a 12.5 % increase in aboveground biomass, in 103 research published before april 1st 2013 (liu et al., 2013). additionally, a considerable rise in crop output and aboveground biomass was noted by biederman & harpole (2013) after adding biochar to the soil. recent studies have revealed that the reactions of soil and plants to the application of biochar can be either positive or negative (gravel et al., 2013), or neutral (ali jaaf et al., 2022), depending on the type of feedstock, the temperature at which it is pyrolyzed, the application rate and method, the type of crop and soil, and the environmental circumstances (joseph et. al., 2021; rivelli et al., 2022). the lack of understanding about the proper proportion of biochar to use, particularly in africa, is a challenge. furthermore, because there is less research on the ideal amount of biochar necessary, we undertook an experiment to determine the best quantity. as a result, the specific objectives of the study were to: 1) examine the effect of biochar on the physical features (plant height, number of fruit, fruit optimizing the use of biochar in okra (abelmoschus esculentus l.) production in nigeria january to april 2023 22 weight, and fruit length ) of okra; 2) examine the effect of biochar on the yield of okra; and 3) determine the optimum biochar dose required per kg of soil. material and methods the study area the experiment was carried out at the faculty of agriculture screen house which is part of the university of ilorin teaching and research farm, university of ilorin, ilorin, kwara state. the university of ilorin is located in the southern guinea savanna ecological zone of nigeria, which lies on latitude and longitude (n 80 28’ 53.3’’ e 40 40’ 28.9’’). it is categorized under the bimodal rainfall pattern, with high rainfall in june and september, and a break between midjuly and august. this city has a tropical climate. the annual rainfall in the area is about 1200 mm, and the least amount of rainfall occurs in january. the average in this month is 10 mm. most of the precipitation here falls in september, averaging 232 mm and temperature varies between 33 0c and 34 0c during the year, with an average temperature of 24.5 0c. the temperature is the highest on average in march, at around 29 0c. august is the coldest month, with the temperature averaging 24.5 0c with a distinct dry season from december to march. throughout the year, the temperature varies by 4.5 0c. collection and preparation of materials the biochar was gotten from hill crest agroallied industry, offa, kwara state. the biochar was produced with rice husk. viable seeds of okra were obtained from a store at the fate junction, ilorin. the npk (15:15:15) was bought from an agrochemicals store at fate junction, ilorin. the soil was collected from the faculty of agriculture nursery area. the soil collected was sterilized by heating in a drum to minimize the incidence of spores and disease-causing organisms and then allowed to cool before use. the sterilized soil was measured at the rate of 5 kg/bucket. the soil has a definite sandy-loamy textural characteristic and a ph of 6.5, which is mildly acidic. field experimental layout, land preparation, and crop in this study, the experiment was carried out using a completely randomised design (crd). treatments used were biochar at two different levels, and npk was applied at the rate of 0.08929 g.kg-1 of soil. biochar was hand applied into the soil using two rates: 50 g.kg-1 and 100 g.kg-1 of soil; npk 15:15:15 and a control (soil that received no treatment). each treatment was replicated seven times as well as the control making a total of 28 treatments. the treatments were labeled thus: treatment 1: biochar application rates at 50 g.kg-1 of soil; b1r1, b1r2, b1r3, b1r4, b1r5, b1r6 and b1r7 treatment 2: biochar application rates at 100 g.kg-1 of soil ; b2r1, b2r2, b2r3, b2r4, b2r5, b2r6, b2r7 treatment 3: npk treatment; nr1, nr2, nr3, nr4, nr5, nr6, nr7 treatment 4: control; cr1, cr2, cr3, cr4, cr5, cr6, cr7 weed control, manual thinning, and watering of the plants manual thinning that is, removal of excess vegetable was carried out to avoid overcrowding and competition for photosynthesis, water and nutrient and this help the viable plant to grow perfectly. weeding was done manually by hand-pulling occasionally till harvesting time. watering can was used to irrigate the plants very early in the morning on daily basis. data collection data collection commenced one week after planting and was done weekly. the following parameters was collected. plant height plant height was recorded for each plant in the bucket of okra using a ruler. the measurements were taken from the soil level to the highest point of the stem apex and the mean recorded. salami, m. f., mark, m., ibitomi, o., osasona, k. k., adeniyi, v., salami, s., & sulaiman, h. peruvian journal of agronomy, 7(1), 20-26 (2023) https://doi.org/10.21704/pja.v7i1.1999 23 number of fruit total number of fruits on each selected plant of okra of each bucket was counted and recorded. yield of the plants after harvesting, the yield was measured using a weighing balance. data analysis data collected, such as plant height, number of fruits and fruit length collected, were subjected to analysis of variance (anova) using gensat 17th edition. the mean was separated using least significant difference (lsd) at 5 % probability level. also, profitability ratios i.e gross margin, operating ratio and rate of returns to investment was used. analysis of variance analysis of variance (anova) was used to analyze objectives 1 and 2 which are: to know the effects of biochar usage on the physical characteristics of okra and to examine the effect of biochar on the yield of okra using the mean average of the sampled plant. results and discussion effect of biochar and npk fertilizer on the height of okra plant the effects of biochar and npk fertilizer on the growth of okra plant height are shown in table 1. the treatments significantly affect the growth of the plant height. throughout the weeks of data collection, there was no significant difference between the two applications rate of biochar. this is due to the fact that the micro and macronutrients found in biochar are slowly released into the soil and taken up by plants, increasing plant productivity and yield (thomas et al., 2013; hammer et al., 2014; drake et al., 2016; kim et al., 2016). as a result, the beneficial and actual effect of biochar could be clearly observed in long-term experiments. nevertheless, their effects significantly differ from the effects of npk fertilizer and the control except at the fifth and eighth week after planting, where their effects were significantly the same with the control. plants treated with npk fertilizer had the shortest plant height throughout the weeks while biochar had the highest effect in increasing the height of the okra plant. this result is in tandem with the works of williams & qureshi (2015), who reported a significant effect of biochar in increasing the plant height growth of the okra plant. table 1. effect of biochar and npk fertilizer on the growth of okra plant height (cm). trts 1wap 2wap 3wap 4wap 5wap 6wap 7wap 8wap b1 8.15a 13.11a 18.61a 23.07a 24.82a 25.68a 25.96a 26.03a b2 8.38a 13.04a 18.89a 22.86a 24.86a 25.71a 26.00a 26.07a npk 5.36c 8.32c 12.10c 15.33c 16.64b 17.14c 17.36c 17.62b con 7.48b 10.93b 16.75b 21.07b 23.11a 23.75b 23.79b 23.84a lsd (0.05) 0.42 0.53 0.60 0.89 3.53 0.92 0.91 2.96 key notes: mean with same letter in a column are significantly the same, b1 = 50g of biochar/kg of soil, b2 = 100g of biochar/kg of soil, con = control, wap = week after planting, lsd(0.05) = least significant difference at 5 % of significance, ns = not significant effect of biochar and npk fertilizer on the yield performance of okra plant table 2 shows the effects of biochar and npk fertilizer on the number of fruit, the fruit’s weight, and the average fruit length of the okra plant. the treatments significantly affect the number of fruits, the fruit’s weight, and the fruit length. plants treated with 50 g of biochar per kilogram of soil had the highest number of fruits with the highest fruit weight, while plants treated with 100 g of biochar per kilogram of soil and those treated with npk fertilizer significantly had the same number of fruit and fruit weight. the least number of fruit and smallest fruit weight were obtained from the control plants. fruits harvested from a plant treated with biochar at 50 g.kg-1 of soil had the same length as those treated with biochar at 50 g.kg-1 of soil this is probably due to the duration of the experiment optimizing the use of biochar in okra (abelmoschus esculentus l.) production in nigeria january to april 2023 24 and it is consistent with observation of glaser et al., 2002 that biochar performs better in the second and third years of use than it does in the first. however, the length of fruits harvested from biochar-treated pots significantly differs from the fruit length of the control plants and plants treated with npk fertilizer. this finding is in tandem with the report of farias et al., 2020, who reported a significant effect of biochar in increasing the yield of okra plants. acknowledgments the authors of this manuscript declare that there was no external grant recieved for the implementation of the research. author contributions mf-s: conceptualization of the work, experimental design, review of statistical analysis of results, discussion of results, drafting table 2. effect of biochar and npk fertilizer on the yield of okra plant (kg). trts number of fruit fruit weight (g) average fruit length (cm) b1 5.00a 26.37a 5.06a b2 3.71b 21.09b 4.99ab npk 3.86b 17.37b 4.13bc control 2.43c 12.16c 4.49c lsd(0.05) 0.59 4.12 0.54 key notes: mean with same letter in a column are significantly the same, b1 = 50 g of biochar/kg of soil, b2 = 100 g of biochar/kg of soil, wap = week after planting, lsd(0.05) = least significant difference at 5 % of significance, ns = not significant conclusion this study shows that the use of biochar as a soil fertility improver was able to increase both the vegetative growth and reproductive performance of the okra plant. the two application rates of biochar significantly increased the height of the okra plant throughout the weeks of data collection except for the fifth and eighth weeks, where their effect was not manifested. the effects of both application rates on plant height and average fruit length were the same however, their effects on the number of fruit and fruit’s weight differs. 50 g of biochar per kilogram of soil significantly had the highest number of fruit and the highest fruit weight. its yield significantly differs from the yield of 100 g of biochar per kilogram of soil, npk fertilizer, and control. npk fertilizer and 100 g of biochar per kilogram of soil had the same effects on yield, but their effects significantly differ from the control, which had the least performance. 50 g of biochar per kilogram of soil having the least operational ratio to produce okra had the highest return on investment and is therefore recommended as the optimum required for farmers. of the manuscript, revision of the manuscript, and supervision of the study m-m: revision of the manuscript, support, logistics management o-i: support, logistics management kk-o: revision of the manuscript, support, logistics management v-a: experimental design, execution of fieldwork, statistical analysis of results, discussion of results s-s: experimental design, execution of fieldwork, statistical analysis of results, discussion of results h-s: support, logistics management conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript salami, m. f., mark, m., ibitomi, o., osasona, k. k., adeniyi, v., salami, s., & sulaiman, h. peruvian journal of agronomy, 7(1), 20-26 (2023) https://doi.org/10.21704/pja.v7i1.1999 25 id orcid and emails: mercy funke salami markmercy12@gmail.com https://orcid.org/0000-0003-0236-0985 miracle mark markmarcus149@gmail.com https://orcid.org/0000-0002-4482-7152 olasumbo ibitomi ibitomioluyemiolasumbo@gmail.com https://orcid.org/0000-0003-2486-5036 kehinde osasona okennieegreat@gmail.com https://orcid.org/0000-0001-5652-4514 victor adeniyi adeniyivictor117@gmail.com https://orcid.org/0000-0001-8861-4901 shakirat salami salamishakirat214@gmail.com https://orcid.org/0000-0003-1614-9769 hamdalat sulaiman 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(2013). food, fuel, and plant nutrient use in the future. council for agricultural science and technology, 51, 1–24 https://doi.org/10.3390/agronomy12092089 https://doi.org/10.3390/agronomy12092089 https://doi.org/10.1007/s11157-020-09523-3 https://doi.org/10.1007/s11157-020-09523-3 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i3.1975 received for publication: 17 july 2021 accepted for publication: 30 november 2022 published: 31december 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by efficiency of trichoderma viride as a biocontrol agent for phytophthora capsici in pepper (capsicum annuum l.) eficiencia de trichoderma viride como un agente biocontrolador para phytophthora capsici en pimiento (capsicum annuum l.) romero, v.1*; aragón, l.1; casas, a.1; apaza, w.1 *corresponding author:vromerou@gmail.com *https://orcid.org/0000-0003-3486-7780 abstract phytophthora capsici is one of the most devastating pathogens that limits the production of paprika (capsicum annuum l.) worldwide. likewise, trichoderma viride stands out as a biological agent due to its antagonistic effect, resistance inducer, growth stimulator, etc. the present work evaluated the effectiveness of t. viride as a biocontrol agent against p. capsici in paprika using three growth methods (direct seeding, plantlet and bare root). twelve treatments were developed under greenhouse conditions, including a control (without inoculum) and a completely randomized design with a factorial arrangement. t. viride inoculation was carried out 40 days after sowing at a concentration of 106 conidia ml-1 while p. capsici was inoculated 50 days after sowing using three colonized wheat grains per plant. the inoculation method of the controlling agent in the direct seeding and plantlet was given by drench, and in the bare root was carried out by immersing of the seedling for 5 minutes prior to the transplant. then, the correlation between plant growth method and p. capsici, and the interaction between t. viride and the plant growth method were made. the results showed that the highest efficacy of t. viride as a p. capsici biocontrol agent was in the method of the plantlet and bare root. the correlation between the method of growing crop and root rot was lower in bare root (74 % severity). in the other two treatments (direct seedling and plantlet) 100 % of plants were dead; finally, the effect of t. viride as a growth inducer was not evidenced in any of the treatments. regarding audpc, the direct seeding method showed a higher incidence. the bare root planting method obtained the lowest value of the t. viride and p. capsici interaction. keywords: biocontrol, capsicum annuum l, phytophthora capsici, trichoderma viride, growth methods. resumen phytophthora capsici es uno de los patógenos más devastadores que limita la producción de paprika (capsicum annuum l.) en el mundo. asimismo, trichoderma destaca como agente biocontrolador por su efecto antagonista, inductor de resistencia, estimulador de crecimiento, etc. el presente trabajo evaluó la eficacia de t. viride como controlador biológico para p. capsici en páprika bajo tres métodos de siembra (directa, plantín y raíz desnuda). se instalaron doce tratamientos bajo condiciones de invernadero, incluyendo un testigo (sin inóculo) y se empleó un diseño completamente al azar con arreglo factorial. la inoculación de t. viride se 1 universidad nacional agraria la molina, av. la molina s/n, lima, perú. how to cite this article: romero, v., aragón, l., casas, a., & apaza, w. (2022). efficiency of trichoderma viride as a biocontrol agent for phytophthora capsici in pepper (capsicum annuum l.). peruvian journal of agronomy, 6(3), 229–238. https://doi. org/10.21704/pja.v6i3.1975 efficiency of trichoderma viride as a biocontrol agent for phytophthora capsici in pepper (capsicum annuum l.) september december 2022 230 llevó a cabo a los 40 días posterior a la siembra a una concentración de 106 conidias ml-1, mientras que la inoculación de p. capsici se realizó a los 50 días posterior a la siembra empleándose tres granos de trigo colonizados por planta. el método de inoculación de t. viride en la siembra directa y plantín se hizo vía drench y en la siembra a raíz desnuda se realizó por inmersión de la plántula durante 5 minutos previo al transplante. luego se realizó la correlación entre los métodos de siembra y p. capsici, y la interacción entre t. viride y los métodos de siembra. los resultados mostraron que la mayor eficacia de t. viride como de p. capsici se registró en los métodos de siembra plantín y raíz desnuda; la correlación entre el método de siembra y la pudrición radicular fue menor en la siembra a raíz desnuda (74 % severidad). en los otros tratamientos (directa y plantín) el 100 % de plantas murieron; finalmente, el efecto de t. viride como inductor de crecimiento no se evidenció en ninguno de los tratamientos. con respecto al abcpe, el método de siembra directa mostró mayor incidencia; el menor valor de la interacción t. viride y p. capsici fue obtenido en el método de siembra raíz desnuda. palabras claves: biocontrolador, capsicum annuum l, phytophthora capsici, trichoderma viride, growth methods. introduction chili pepper (capsicum annuum l.), one of the most widely grown vegetables worldwide, native from tropical and subtropical regions of america, cultivated approximately 3.8 million ha, and the harvested crops produce around of 41 million tons annually, representing 3.76 % of the global horticulture crop production and 6.51 % of global horticulture crop area (food and agriculture organization [fao], 2020b). by 2018, approximately 70 % of the global area of pepper crops was in asia, 18 % in africa, 7 % in america and 5 % in mediterranean countries (fao, 2020b). peru is a great exporter of dry pepper, located in 2017 as the world’s fourth largest exporter of dry pepper, totaling 32 million tons (food and agriculture organization [fao], 2020a). according to ministerio de agricultura y riego (minagri, 2019), the sowing projection for the 2019-2020 farming season adds up to 6.1 million ha, meaning a growth of 45.4 % with respect to the 2018-2019 season, reporting as main producing areas barranca (32.4 %), arequipa (22.2 %), ancash (17.2 %), piura (9.9 %), ica (9.3 %) and lambayeque (6 %). this crop, grown in greenhouses and fields, is attacked for several diseases responsible for economic losses. one of the most destructive diseases is root rot, caused by the oomycete pathogen p. capsici (tomah et al., 2020). this soil-borne pathogen is widespread throughout the world and can infect and cause severe losses in several crops, with a host range of over 15 plant families (barchenger et al., 2018). most of its host plants come from solanaceae, cucurbitaceae and fabeaceae. symptoms vary significantly according to the host, plant part infected and environmental conditions. the disease may manifest in a plant’s underground parts, involving the root and crown rot of pepper (lamour et al., 2012). during high disease pressure, pathogen dispersal causes aerial blight of leaves, fruit and stems (callaghan et al., 2016). for an integrated pest and disease management scheme, cultural and chemical methods, such as crop rotation, resistant varieties, and fungicides, are the most commonly used. nevertheless, some chemicals employed to manage this pathogen usually fail due to the development of fungicide resistance (silvar et al., 2006) or variable efficacy against the diverse propagules of the pathogen (stasz & martin, 1988). likewise, highly resistant pepper cultivars showed susceptibility or moderate resistance when the plants were inoculated with p. capsici (dunn & smart, 2015) due to many pathogen physiological races (monroy-barbosa & bosland, 2011). hence, biological control employing fungal antagonists can be a robust and sustainable disease management strategy. in this sense, the trichoderma genus is the most used for the biocontrol of soil fungus pathogens. these genera have also been shown to be particularly effective in managing p. capsici (tomah et al., 2020). they inhabit diverse environments, undergo various interactions with other organisms, live in soil and saprophytically grow on wood, bark and many other substrates, and interact with animals and plants (zeilinger, 2016). trichoderma spp. possess many antagonistic mechanisms against pathogens of crops, such as lytic enzymes, romero, v., aragón, l., casas, a., & apaza, w. peruvian journal of agronomy, 6(3), 229–238 (2022) https://doi.org/10.21704/pja.v6i3.1975 231 mycoparasitism, and competition for nutrients and space for their successful colonization (harman, 2006; mukherjee et al., 2012). besides, the feature endophytic of this genus, which produces a broad spectrum of secondary metabolites, often results in improving assimilation of micro and macronutrients that, in some cases, they are not available to plants; increased root growth and development; enhanced productivity and, improved tolerance to various stresses including diseases, there are numerous reports of various trichoderma spp. induced resistance against different plant pathogens (siddaiah et al., 2017). t. viride is a mycoparasite that produces a lot of hydrolytic enzymes against the pathogenic crop. moreover, in response to the high ability of biomass transformation is of great interest research fields and biological control industries. this fungus is considered one of the best biocontrol agents, because of its ability to produce many enzymes, such as chitinases, glucanases and proteases. furthermore, t. viride produces glycosyl hydrolases, such as xylanases, cellulases and mannanases under suitable conditions (elgorban, 2016). the first goal of this study was to evaluate the efficiency of trichoderma viride as a biocontroller for p. capsici under three methods of growing crops. the second aim was to determine the correlation between the methods of growing crops with p. capsici, and the last one was assessment the interaction between t. viride and the methods of growing crops. these results will provide a more effective biocontrol of t. viride against p. capsici in pepper. also, it will contribute to a reduction in pesticide residues in the fruit and the environment. material and methods p. capsici inoculum: the isolate was obtained from p. capsici collection of the unalm u. tennessee project; plants infected with inoculum were collected from pepper fields of viru. the most colonized petri plates with mycelium were chosen to be cultivated in parb medium and incubated for seven days for the growth of the colony (ocampo, 2003). for mycelium multiplication, medium parb fragments colonized were replicated to petri plates containing agar v8 medium (ocampo, 2003). pathogen inoculum used in treatments was prepared over cooked wheat and passed through cooking (1 kilogram per 1.5 liter of water) over low heat for approximately ten minutes. then, it was left to stand, drain and air. subsequently, filled the bags with this wheat, leaving the upper third empty, were provisionally covered with cotton, tied with a garter without leaving air inside, and finally sterilized in the autoclave. after sterilization, the pure culture obtained in the v8 medium was seeded into the bags and incubated at 24 °c until the mycelium completely invaded the wheat. trichoderma viride isolation: t. viride strain from the mycoteca of the plant pathology department of the universidad nacional agraria la molina. the multiplication was carried out from the test tube containing the mycelium grown in pda (potato dextrose agar) medium, from where they were transferred to petri dishes containing pda with oxytetracycline and then incubated at a 24 ºc until the growth of the colony covered the entire surface of the medium. for the treatment with t. viride on the plants the most colonized petri plates were selected, 25 ml of deionized water was added, and rubbed with a triangle previously sterilized in order to homogenize each suspension. the suspensions were diluted to a concentration of 106 conidia ml-1. the conidia count was performed with help of the spencer neubauer striped hematocimeter. (french & hebert, 1980). plant production: the sowing of the paprika cultivar was carried out on three different methods (direct seeding, plantlet and bare root) at the phytopathology greenhouse of unalm. in direct seeding was used polypropylene bags containing 1000 g of sterile soil. for plantlet production was employed trays contained imported peat sunshine, and for bare root sowing, the seeds were sown in a row with a density of 50 seeds per 0.16 square meter (5 kg pot). they were kept under these conditions for 40 days until t. viride was inoculated. afterward, plantlets placed on trays and plots were transplanted into polypropylene bags. https://www.sciencedirect.com/science/article/pii/s1049964419305663 efficiency of trichoderma viride as a biocontrol agent for phytophthora capsici in pepper (capsicum annuum l.) september december 2022 232 controller inoculation (t. viride): it was performed at 40 days in the three seeding methods after the zero-day (sowing) at a concentration of 106 conidia ml-1. a. direct sowing: 10 ml was applied by drenching at the base of the plant. b. plantlet: after five days of inoculation, 1 ml of controller solution per plant by drench. the plantlets were transplanted into a polypropylene bag containing 1 kg of sterile soil. c. bare root: the plants were immersed in the controller inoculum solution for five minutes, then transplanted into a polypropylene bag containing 1 kg sterile soil. pathogen inoculation (p. capsici): inoculation was previously tested with five colonized wheat grains per plant. however, given that the disease symptoms shown per plant were very aggressive, the dose was reduced to three grains per plant. in this way the inoculation de p. capsici was carried out 50 days after sowing in each method, inoculating three colonized wheat grains per plant (ocampo, 2003). evaluation of disease in the greenhouse, disease severity was evaluated seven days after inoculation using a symptom scale: 0, healthy plant; 1, leaf epinasties; 2, pronounced epinasties; 3, epinasties plus leaf fall star; 4, severe loss of leaves; and 5, dead plant. the symptom scale data and disease incidence were recorded per plant on nine evaluations starting seven days after pathogen inoculation and ending with the death of the control inoculated with p. capsici at the 4-day interval in all treatments. the area under the disease progress curve (audpc) was calculated for each treatment according to shaner & finney (1977). in addition, at the end of the experiment, the root length, root dry weight, foliage dry weight, and plant height of each plant were determined. experimental design: the experiment was organized in a completely randomized design with a 3x4 factorial arrangement (12 treatments, with ten repetitions), where firts factor (m) corresponded to sowing methods and second factor (p) to p. capsici. the variables studied were disease severity, root length, root dry weight, foliage dry weight and plant height. the recorded data were subjected to an analysis of variance (anova) followed by tukey’s comparison of means, with a significance level of α = 0.05 probability, the package statistical statistical analysis system (sas) version 8. results and discussion the parameters were evaluated by comparing trials with their controls respectively inside each method of growing crops. the result of treatments on morphological parameters is shown in table 1. interaction between p. capsici, t. viride and method growing crop: treatments inoculated with t. viride (t3, t7, t11) resulted in increases of most growth parameters assessed in comparison to the experimental control (t2, t6, t10), which correspond a treatment inoculated only with p. capsici. greater values for the parameters as root length and foliage dry weight were registered on the plantlet method; and root dry weight on the bare root method. all treatments showed significant statistical differences in favor of the effect of t. viride (α = 0.05) (table 1, figures 1 and 2). the values achieved in the plantlet and bare root method respond to its greater absorption capacity of controlling inoculum, directly influenced by the inoculum method. in both cases, the contact of the roots with the controlling inoculum is significatively compared to the direct seeding method, where the application was by drench. then, due to having a more compact soil surface, only some of the solution is absorbed by the roots due to losses that can occur for percolation and/or leaching. according to pineda-insuausti et al. (2017), the plantlet method is favored by the positive interaction that occurs between t. viride and plantlet substrate, based on the high enzymatic capacity that t. viride possesses to degrade substrates, and thus increase the efficiency of assimilation of nutrients by the plant concerning the bare root planting method. the result achieved is also favored by an increase in the absorption capacity of the root due to the effect of root breakage at the time of transplanting (harman, 2004). romero, v., aragón, l., casas, a., & apaza, w. peruvian journal of agronomy, 6(3), 229–238 (2022) https://doi.org/10.21704/pja.v6i3.1975 233 in concern to plant height, treatments did not show significant statistical differences with their controls (figure 3). interaction between the method of growing crop and root rot (p. capsici): the treatments inoculated with p. capsici (t2, t6, t10) showed vast differences in values with their respective controls (t1, t5, t9 / without inoculum). table 1 shows the significant difference in values in the parameters root length, root dry weight and foliate dry weight obtained in the bare root method, indicating that they were the most affected plants. however, the plants most affected by their control table 1. average of evaluated parameters for the efficiency test of t. viride as a bio controller for p. capsici under three sowing methods in pepper. treat. cod root length (mm) root dry weight (g) foliage dry weight (g) plant height (cm) severity (scale) 1 ds(t) 242.8 a b 0.43 a b 0.95 b 22.6 a b 0 a b 2 ds (pc) 95,2 b c 0,16 b c 1,04 b c 28,89 a c 5,0 b 3 ds (pc + tv) 117,3 b 0,19 b 1,12 b 30,17 a c 5,0 b 4 ds (tv) 347,4 a b 0,62 a b 1,93 a b 37,80 a 0 a b 5 p (t) 285,3 a b 0,52 a b 1,16 b 34,10 a b 0 a b 6 p (pc) 23,4 b c 0,10 b c 0,37 b c 24,80 a c 5,0 b 7 p (pc + tv) 177,2 b 0,32 b 1,07 b 24,70 a c 4,5 b 8 p (tv) 371,2 a b 0,67 a b 2,14 a b 31,80 a 0 a b 9 br (t) 497,1 a 0,90 a 2,05 a b 33,90 a b 0 a 10 br (pc) 122,0 a c 0,22 a c 1,15 a c 21,70 a c 3,7 a b 11 br (pc + tv) 250,4 a b 0,45 a b 1,83 a b 26,50 a c 2,5 a b 12 br (tv) 360,8 a 0,65 a 2,03 31,80 a 0 a cv % 36,27% 37,14% 44,48% 17,91% 109,89% data is average of ten repetition. different letters indicate significance for p <0.05. ds.: direct seeding, p: plantlet, br: bare root. figure 1: root length (mm), according to control treatments of p. capsici on t. viride under three sowing methods (α = 0.05). were confirmed in direct seeding; conversely, the least affected plants to their control were reported in the bare root method. likewise, all treatments showed significant statistical differences in favor of the control without inoculum (figures 1 and 2). this result would respond to the volume of radical mass developed by the plant at the time of pathogen inoculation. however, it was inoculated on the 50th day after sowing in the three growing methods. growing a crop that develops the lowest root mass is the bare root, resulting from the high density shown in the plot. unlike the plantlet and direct seeding method, under this same criterion, the result observed efficiency of trichoderma viride as a biocontrol agent for phytophthora capsici in pepper (capsicum annuum l.) september december 2022 234 figure 2. foliage dry weight (g), according control treatments of p. capsici on t. viride under three sowing methods (α = 0.05). figure 3: plant height (cm), according to control treatments of p. capsici on t. viride under three methods (α = 0.05). in direct seeding responds to a higher root mass at the time of inoculation of the pathogen. this effect is supported by what has been verified by hickman (1970) about the accumulation of p. capsici zoospores around the apices of the pepper roots claiming the theory of an attraction of the same by chemotaxis mechanism from root exudates after being managing to reproduce the phenomenon using capillaries that incorporated exudates or root extracts. likewise, specific chemoreceptors are pointed out on the surface of zoospores that even motivate the orientation of the germ tube during their germination (apaza et al., 1996). comparing in treatments the interaction between planting methods and root rot (t2, t6, t10 inoculated only with p. capsici). table 1 shows that the significant value in the parameters root length, root dry weight and foliage dry weight was reached by the bare root method (t10: 122 mm, 0.22 g, 1.55 g, respectively); which showed significant statistical differences with the other treatments (figure 1 and 2). the results obtained in the bare root method could be supported in that after starting the plants from an area of high density to another of much lower density, it positively influences the morphological development of the plant (ritchie & dunlap, 1980). romero, v., aragón, l., casas, a., & apaza, w. peruvian journal of agronomy, 6(3), 229–238 (2022) https://doi.org/10.21704/pja.v6i3.1975 235 interaction method of growing crop and t. viride: treatments inoculated only with t. viride (t4, t8, t12) showed light differences with their respective controls (t1, t5, t9 without inoculum). therefore, the direct seeding method obtained the most significant difference in values in the parameters root length, root dry weight and foliage dry weight. likewise, all results showed significant statistical differences in favor of the control without inoculum (table 1, figures 1 and 2). the result would show that the radicalinducing effect of t. viride was not observed, and the highest value obtained in direct seeding would fall more to an effect given by the method of growing crop in which the development of the root is not interrupted and has more space for its development. the interaction between t. viride and the different growing methods (t4, t8, t12 inoculated only with t. viride) were compared. it is observed in table 1 that the highest value for the parameters root length, root dry weight and foliage dry weight was reached by the plantlet method (t8: 371.2 mm, 0.66 g., 2.14 g, respectively). despite this, the statistic did not show significant differences between the plantlet and direct seeding, while the bare root showed a significant statistical difference. none of the three methods showed significant statistical differences with their control (α = 0.05). the significant interaction between the t. viride and plantlet method would be influenced by the substrate that supports the seedling, which is based on the trichoderma mechanism to solubilize nutritional elements that are unavailable in their original form to plants (harman, 2003). it is also attributed to the high enzymatic capacity of t. viride to degrade substrates (infante et al., 2009). in this way, it increases the efficiency of the assimilation of nutrients by the plant. disease development disease severity: when the treatment inoculated only with p. capsici (t2, t6 y t10) were compared, the lowest severity was observed in the bare root method (t10: scale 3.7), which shows a significant statistical difference with other sowing methods. the severity observed in figure 4. paprika plants 76 days after sowing: a. direct seeding (t1, t2, t3, t4); b. plantlet (t5, t6, t7, t8) y c. bare root (t9, t10, t11, t12). efficiency of trichoderma viride as a biocontrol agent for phytophthora capsici in pepper (capsicum annuum l.) september december 2022 236 direct seeding and plantlet methods were scale 5 (100% dead plants) (table 1, figure 4). this result could respond to the positive changes already mentioned in the root morphology due to root-break and transplant given in this sowing method. on the other way, the best effectiveness of t. viride over p. capsici was obtained in the bare root sowing method (t11: scale 2.5) (table 1), which showed a significant statistical difference with the other sowing methods. both the sowing methods as t. viride inoculation method, influence this result. in such a way, the break of roots in the extraction increases the absorption capacity by the root and the immersion given corresponds to the inoculation method more efficiently than the drench used to inoculate the plantlet and direct sowing method. from this perspective, the result is supported by the t. viride mechanism of inducing the plant’s physiological and biochemical defense mechanisms, such as the activation of resistance-related compounds (resistance induction) (harman, 2004). audpc figure 5 shows the audpc values for the different treatments evaluated, where it is observed that, in the direct sowing method, the treatment inoculated with both p. capsici and t. viride (t3: 29) and the treatment inoculated only with p. capsici (t2: 30.55) show similar values. in the plantlet sowing method, the lowest value was observed in the treatment inoculated only with p. capsici (t6: 12). concerning the treatment inoculated with both p. capsici and t. viride (t7: 18.15), this result would be influenced by the fact that, although it had a higher value in the treatment 7, it showed a lower degree of severity than the t6 treatment. in the bare root sowing method, the major value was reached by the treatment inoculated only with p. capsici (t10: 14.4) with respect to the treatment inoculated with both p. capsici and t. viride (t11: 9.75). likewise, less degree of severity was observed in the t11 treatment. conclusions the highest efficiency of t. viride as a bio controller of p. capsici was obtained in the bore root sowing method (32 % effectiveness), followed by plantlet (10 % effectiveness) and in sowing direct were obtained 100 % of died plants. the best relation between the sowing method and p. capsici was obtained in the bore root figure 5. area averages under the disease progress curve (audpc) for the nine evaluations according to treatment in the efficiency test of t. viride as a bio controller for p. capsici under three sowing methods in the pepper crop (capsicum annuum) romero, v., aragón, l., casas, a., & apaza, w. peruvian journal of agronomy, 6(3), 229–238 (2022) https://doi.org/10.21704/pja.v6i3.1975 237 sowing method (74 % severity). the other two sowing methods show 100 % severity (100 % died plants). the best interaction between the sowing method and t. viride was obtained in the plantlet sowing method. the parameters assessment that best show the effect of t. viride are: root length, root dry weight, foliage dry weigh and disease severity. orcid and e-mail romero, v. vromerou@gmail.com https://orcid.org/0000-0003-3486-7780 aragon, l. l.lili@lamolina.edu.pe https://orcid.org/0000-0003-0312-5020 casas, a. l.cda@lamolina.edu.pe https://orcid.org/0000-0001-7461-3924 apaza, w wapaza@lamolina.edu.pe https://orcid.org/0000-0001-7510-8866 references apaza, w., armas, g., baker, r., & carlili, m. j. 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(2016). secondary metabolism in trichoderma–chemistry meets genomics. fungal biology reviews, 30(2), 74–90. https://doi.org/10.1016/j. fbr.2016.05.001 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article received for publication: 30 october 2020 accepted for publication: 20 january 2021 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina doi: http://dx.doi.org/10.21704/pja.v5i1.1656 genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions comportamiento genético del rendimiento de semillas y componentes del rendimiento en sésamo (sesamum indicum l.) en condiciones normales de riego y estrés hídrico suzan abd ellatif kamel ibrahim1; mohamed ali abdelsatar1*; mohamed abd el-raheem ahmed1; magdy m. niazy2 *corresponding author: mohamedtemraz1@yahoo.com *https://orcid.org/0000-0003-0780-1444 abstract six divergent genotypes of sesame (sesamum indicum l.) were crossed using a half diallel excluding reciprocal crosses, to estimate heterosis, combining ability and nature of gene action for studied traits under two irrigation regimes. the two irrigation regimes were normal irrigation conditions with amount of applied water 5952.38 m3/ha and water stress conditions with amount of applied water 2976.19 m3/ha at etay-el-baroud agricultural research station, behaira governorate, agricultural research center, egypt during 2019 summer season. a randomized complete block design with three replications was used for each irrigation regimes. the variation of genotypes and their components from parents, crosses and parents versus crosses were highly significant for all studied traits under both irrigation regimes and their interactions with irrigation. variation attributable to general and specific combining ability was highly significant for seed yield and yield components under both irrigation regimes. the parents l92 and l110 were the best combiners for seed weight per plant and most of its components under both irrigation regimes. the best f1 cross combination was l92 × l110 in specific combining ability and heterotic effects over midand better-parents under both irrigation regimes for seed weight per plant and most of its attributes. the preponderance of additive gene action in the inheritance of most studied traits was observed, that further confirmed by its significance and the value of average degree of dominance exceeding the unity. narrow-sense heritability varied from 0.19 for number of branches per plant to 0.47 for 1000 seed weight under normal irrigation, whereas, under water stress conditions, it ranged from 0.14 for number of branches per plant to 0.42 for fruiting zone length. parents l95 and l93 under normal irrigation and l93 and l110 under water stress conditions carried mostly genes with dominant effects for seed weight per plant, in contrary, l92 and l110 under normal irrigation and l92 and l12 under water stress conditions carried mostly recessive alleles for seed weight per plant. hence, the results will be used to develop a sesame breeding scheme at etay-el-baroud agricultural research station. key words: sesame, sesamum indicum, half diallel analysis, heterosis, combining ability, gene action, irrigation. resumen se cruzaron seis genotipos divergentes de sésamo (sesamum indicum l.) utilizando un medio dialélico excluyendo cruces recíprocos, para estimar la heterosis, combinando la capacidad y la naturaleza de la acción de los genes para los rasgos estudiados bajo dos regímenes de riego. los dos regímenes de riego fueron condiciones de riego normales con una cantidad de agua aplicada de 5952,38 m3/ha y condiciones de estrés hídrico con una cantidad de agua aplicada de 2976,19 m3/ha en la estación de investigación agrícola de etay-el-baroud, gobernación de behaira, centro de investigación agrícola, egipto durante 2019 temporada de verano. se utilizó un diseño de bloques completos al azar con tres repeticiones para cada régimen de riego. la variación de genotipos y sus componentes de padres, cruces y padres versus cruces fue altamente significativa para todos los rasgos estudiados bajo ambos regímenes de riego y sus interacciones con el riego. la variación atribuible a la capacidad de combinación general y específica fue muy significativa para el rendimiento de semillas y los componentes del rendimiento en ambos regímenes de riego. los padres 1 oil crops research department, field crops research institute, agricultural research center, giza, egypt. 2 soils, water and environment research institute, arc, giza, egypt how to cite this article: ibrahim suzan a. k., abdelsatar, m. a., ahmed, m. a., & niazy, m. m. (2021). genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions. peruvian journal of agronomy, 5(1), 1–17. http://dx.doi.org/10.21704/pja.v5i1.1656 patricia sello genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 2 introduction sesame is one of the important oil crops in egypt. it plays a vital and effective role in covering the requirements of oilseeds. to increase the productivity of its seeds, increased genetic diversity is necessary. therefore, the ultimate goal of sesame breeder is to collect information on heterotic effects, per se performance, general and specific combining ability effects of parents along with genetic components of variance for yield and associated traits. the estimation of heterosis either of mid or better parents heterosis played a major role in identifies the desirable cross combinations in preferred direction. moreover, combining ability analysis is a common biometrical tool in half diallel analysis to identify the best general combiners and the desirable specific cross combinations. moreover, another method of half diallel analysis as hayman (1954a, 1954b, and 1960) and jinks (1954) provides the breeder with the detailed information on nature gene action and the genetic control in the studied traits. this information helps breeder in selection of preferred parents for future crosses at breeding programs to improve the sesame yield and related traits. for achieving this objective, the efforts in this study aimed to estimate: heterotic effects, combining ability effects, nature of gene action controlling genetic expression of the studied traits under both normal irrigation and water stress conditions. materials and methods plant materials set of six parental sesame genotypes that had genetically distinct in their morphological traits were mated by a half-diallel mating design (excluding reciprocal crosses) during the 2018 summer season under etay-el-baroud/ behaira agricultural research station, agricultural research center (arc), egypt, to create 15 single-cross combinations. these parental sesame genotypes were labeled as l95 (p1), l92 (p2), l28 (p3), l12 (p4), l93 (p5) and l110 (p6) during the hybridization program by a half-diallel mating design (table 1). the source of these breeding materials was the oil crops research department, field crops research institute, arc, egypt. the compositions and chemical properties of soil site samples were analyzed according to jackson (1973) and are shown in table 2. the wheat crop was sown in the field in the previous campaign. our experiment was sown in the summer season 15th june 2019. experimental design and cultural practices the field evaluation trial of the parents and their f1 crosses was conducted in the summer season of 2019 under normal irrigation conditions (ni) with applied water amount of 5952.38 m3/ha and water stress conditions l92 y l110 fueron los mejores combinadores para peso de semilla por planta y la mayoría de sus componentes bajo ambos regímenes de riego. la mejor combinación cruzada f1 fue l92 × l110 en capacidad de combinación específica y efectos heteróticos sobre padres medios y mejores bajo ambos regímenes de riego para el peso de semilla por planta y la mayoría de sus atributos. se observó la preponderancia de la acción aditiva de genes en la herencia de los rasgos más estudiados, lo que se confirmó además por su importancia y el valor del grado medio de dominancia superior a la unidad. la heredabilidad en sentido estricto varió de 0,19 para el número de ramas por planta a 0,47 para el peso de 1000 semillas bajo riego normal, mientras que, en condiciones de estrés hídrico, varió de 0,14 para el número de ramas por planta a 0,42 para la longitud de la zona de fructificación. los padres l95 y l93 bajo riego normal y l93 y l110 bajo condiciones de estrés hídrico portaban principalmente genes con efectos dominantes para el peso de semilla por planta, por el contrario, l92 y l110 bajo riego normal y l92 y l12 bajo condiciones de estrés hídrico portaban principalmente alelos recesivos para semilla peso por planta. por lo tanto, los resultados se utilizarán para desarrollar un programa de mejoramiento de sésamo en la estación de investigación agrícola de etay-el-baroud. palabras clave: sésamo, sesamum indicum, análisis medio dialélico, heterosis, habilidad combinatoria, acción genética, irrigación. parents name pedigree origin l95 introduce 432 unknown usa 1976 l92 b-42 unknown egypt l28 hybrid 133 na272 × giza 32 egypt l12 hybrid 102 na217 × giza 25 egypt l93 introduce 588 unknown mexico 1986 l110 introduce 304 unknown usa 1975 table 1. name, pedigree and origin of sesame parental genotypes soil properties value composition sand (%) 16.75 silt (%) 34.85 clay (%) 48.40 soil texture clay chemical analysis value concentration of n (mg kg-1) 156 concentration of p (mg kg-1) 7.89 concentration of k (mg kg-1) 374 electrical conductivity (ds/m) 1.59 ph 7.46 table 2. soil composition and chemical properties of the upper 30 cm of the experimental soil for etay-el-baroud station a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 3 (wsc) with applied water amount of 2976.19 m3/ha using a randomized complete block design with three replicates for each irrigation regimes. surface irrigation system was used through siphon pip with the rate of water flow (3 l/s) to regulate and calculate the amount of applied water for each irrigation treatment. the distribution of the amount of applied water was based on the irrigation time taken for each treatment. where, the irrigation time under wsc was half that of ni. four irrigations were applied for each treatment. the experimental plots consisted of two ridges, 5 m long and 60 cm wide, with a spacing of 10 cm between individual plants. seeds of parents and their f1 crosses were hand-planted in adjacent plots. the sesame seedlings were thinned to keep one plant per hill on one side of the ridge. the other cultural practices were done as recommended by oil crops research department, field crops research institute, arc, egypt. data collection ten plants were randomly taken from each plot to measure the average of plant height (cm), fruiting zone length (cm), capsules length (cm), number of branches per plant, 1000-seed weight (g) and seed weight per plant (g), which was adjusted to 15.5 % seed moisture. days to 50 % flowering was recorded on a plot basis. seed oil content (%) was determined, after drying seed at 70°c for 48 h, by the soxhlet extraction technique, using diethyl ether (association of official analytical chemists [aoac], 1990). statistical analysis combined analysis according to steel et al. (1997) was performed for identifying irrigation effects on genetic variance and its components and the interaction between them, that after confirmed of homogeneity from the error variance. combining ability analysis for all studied traits was done according to method 2 model 1 of griffing (1956) for each irrigation regimes. moreover, analysis of jones (1965) was performed for partitioning of dominance genetic effects (b) into three effects as b1 which refers to test of mean deviation of f1 from their mid-parental values, b2 which refers to test of whether mean dominance deviation of the f1 from their midparental values within each array differs over arrays, and b3 which refers to test of dominance deviation that is unique to each f1, for each irrigation regimes. to judge the gene action controlling all studied traits, baker ratio was done according to baker (1978) as follow: 2msgca/(2msgca+ mssca). heterosis was determined for individual crosses as the percentage deviation of f1 means performance from either mid parents or better parents values at both irrigation regimes. to determine the nature of dominance, the potence ratio as according to wigan (1944) and mather and jinks (1971) was computed by the formulae: potence ratio (p) =f1-mp/ hp-mp. where p refers to relative potency of the gene set and f1, mp and hp are the means of the f1 generations, mid parents, and the higher parents, respectively. complete dominance was indicated when p = +1; while partial dominance is indicated when “p” is between (−1 and +1), except the value zero, which indicates the absence of dominance. it is considered overdominance when potence ratio exceeds ±1. the positive and negative signs indicate the direction of the dominance of either parent. hayman’s diallel analysis (hayman, 1954b) was performed to determine the genetic components and related genetic parameters. the adequacy of simple additivedominance model, and the proportion of dominant and recessive alleles in the sesame parental genotypes, along with degree of dominance under two irrigation regimes were determined by covariance (wr)/variance (vr) graph according to jinks (1954), hayman (1954a), and mather and jinks (1982). in addition, the adequacy of simple additive-dominance model was judged using three scaling tests: uniformity of wr and vr (t2-test), joint regression analysis, and variance analysis of (wr + vr) and (wr – vr) for all studied traits under two irrigation regimes. when the previous three tests are failure or inadequacy, the additivedominance model is completely invalid. however, the additive-dominance model is partially adequate, if one of these tests achieves the assumptions. accordingly, partially adequate or completely adequate types can be considered as guide for extra analysis to determine the genetic components of variation (johnson & askel, 1964; wilson et al., 1978). ms-excel (2007) with spreadsheet formula commands was used to conduct the statistical analyses and figures were elaborated in excel, too in this study. results and discussion analysis of variance highly significant genetic variability in respect to genotypes and its components of parents and their f1 crosses were detected for all studied traits under both contrasting irrigation regimes (tables 3 and 4) and their combined analysis. this indicated that existence of significant genetic variability among these populations, and hence increasing the chances of improving these traits. similar results were obtained by ramesh et al. (2014), fahmy et al. (2015), abd el-satar et al. (2016), ismail et al. (2020) and jeeva et al. (2020). the considerable heterosis among created cross combinations was detected for all studied traits under both contrasting irrigation regimes as verified by highly significant parents vs. f1 crosses, as presented by jones genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 4 (1965) where b1 refers to mean deviation of f1’s from the mid-parents. similarly, the high significance of b2 values were obtained for all studied traits under both contrasting irrigation regimes, indicating asymmetry of gene distribution among parents used. moreover, item b3 was highly significant for all studied traits under contrasting irrigation regimes indicating interactions of allelic and non-allelic were inconsistent (kearsy, 1965; mather & jinks, 1971). irrigation regimes and their interaction with genotypes, parents, crosses and parents versus crosses in the combined analysis (table 4) had highly significant effects on performance of all studied traits, indicating that irrigations had valuable environmental variability, which lead to difference of all population performance ranking from one irrigation to another. this could be attributed to providing plant with proper irrigation will help nutrients absorption from the soil which lead to increased vegetative growth and thereby increase metabolic rate and thus increase yield components, and consequently increasing seed weight per plant with compared to water stress conditions. mean performance mean performance of six parental sesame genotypes and their respective 15 f1 crosses for all studied traits are presented in table 5a and 5b. in comparison with their parents, all cross combinations were earliness in flowering by 6.11 % under ni and 5.86 % under wsc and shorter in plant height by 8.29 % at ni and 8.50 % under wsc. conversely, in comparison with their parents, the progress increase was observed in fruiting zone length by 40.99 % under ni and 30.76 % under wsc, capsules length by 18.91 % under ni and 17.51 % under wsc, number of branches per plant by 75.85 % under ni and 77.89 % under wsc, 1000-seed weight by 27.36 % under ni and 26.68 % under wsc, seed weight per plant by 16.55 % under ni and 30.48 % under wsc and seed oil content by 2.51 % under ni and 4.95 % under wsc. this could be attributed to water stress source of variance df days to 50%flowering plant height fruiting zone length capsules length n s n s n s n s replications 2 0.33 0.11 9.35 13.29 1.53 1.33 0.00 0.02 genotypes 20 65.71** 42.31** 677.52** 420.75** 525.35** 227.34** 0.56** 0.28** parents (p) 5 25.43** 22.09** 832.67** 483.39** 267.44** 64.43** 0.30** 0.15** crosses (c) 14 70.82** 45.36** 545.02** 350.52** 353.38** 186.05** 0.47** 0.21** p v c 1 195.56** 100.80** 1756.67** 1090.86** 4222.48** 1620.01** 3.23** 1.94** error 40 0.50 0.76 5.13 4.45 2.72 3.85 0.02 0.01 source of variance df number of branches per plant 1000-seed weight seed weight per plant seed oil content n s n s n s n s replications 2 0.30 0.05 0.07 0.01 4.025 13.765 1.11 0.60 genotypes 20 5.58** 3.04** 1.28** 0.63** 82.764** 87.773** 9.24** 15.83** parents (p) 5 3.39** 1.56** 0.39** 0.17** 70.809** 58.285** 6.72** 10.36** crosses (c) 14 2.18** 1.31** 1.09** 0.47** 65.251** 61.070** 9.64** 13.60** p v c 1 64.13** 34.77** 8.37** 5.14** 387.720** 609.037** 16.26** 74.47** error 40 0.33 0.30 0.02 0.02 7.904 10.738 0.96 1.01 source of variance d.f days to 50% flowering plant height fruiting zone length capsules length number of branches per plant 1000-seed weight seed weight per plant seed oil content irrigation (i) 1 7344.79** 29962.29** 5034.01** 8.26** 49.53** 15.65** 2911.590** 697.90** reps within i 4 0.22 11.32 1.43 0.01 0.17 0.04 8.895 0.85 entries (e) 20 22.31** 268.75** 175.53** 0.20** 1.98** 0.44** 39.642** 4.73** e × i 20 85.71** 829.52** 577.17** 0.65** 6.65** 1.47** 130.894** 20.34** parents (p) 5 9.09** 317.66** 67.08** 0.10** 0.82* 0.13** 29.598* 2.51* p × i 5 38.43** 998.40** 264.78** 0.34** 4.12** 0.43** 99.496** 14.57** crosses (c) 14 23.47** 220.33** 127.93** 0.15** 0.80** 0.35** 28.484** 4.43** c × i 14 92.71** 675.21** 411.50** 0.52** 2.69** 1.22** 97.837** 18.80** p vs c 1 72.14** 702.02** 1384.17** 1.27** 24.17** 3.33** 246.079** 20.04** p × f1× i 1 224.21** 2145.52** 4458.32** 3.90** 74.73** 10.18** 750.678** 70.69** error 80 0.63 4.79 3.28 0.02 0.32 0.02 9.321 0.99 table 3. mean squares of the six sesame parents and their f1 crosses for earliness, yield and its attributes under normal irrigation(n) and water stress conditions (s) in 2019 season note: *, ** refers to significant at 5% and highly significant at 1%, respectively table 4. mean squares of the six sesame parents and their f1 crosses for earliness, yield and its attributes traits across irrigation regimes in 2019 season note: *, ** refers to significant at 5% and highly significant at 1%, respectively a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 5 led to a decrease in the rate of vegetative growth and thus a decrease in the rates of metabolism, thus reducing the movement of photosynthesis products from the leaves as a source to the seed as a sink, and this will negatively affect the yield components and thus seed weight per plant. the earliest sesame parents in flowering were l12 (60.67 day) and l92 (61.67 day) under ni and l110 (44.00) and l12 (45.33) under wsc and their respective crosses l92 × l110 (51.33 day) and l12 × l110 (51.67 day) under ni and l92 × l110 (40.33 day) and l12 × l110 (40.67 day) under wsc. this indicated that genes responsible for earliness were passed to their f1 crosses. as for plant height, the dwarfest the parents were l93 (124.00 cm) and l28 (125.67 cm) under ni and l28 (96.00 cm) and l93 (97.00 cm) under wsc and their f1 crosses l93 × l110 (109.00) under ni and l93 × l110 (84.33 cm) under wsc, again, genes of dwarfness in the parents passed into their f1 crosses. in case of fruiting zone length, the promising parents were l95 (57.27 cm and 42.57 cm) and l92 (53.00 and 37.52 cm) under both ni and wsc, respectively and their f1 cross combinations l95 × l92 (82.67 cm) and l95 × l28 (82.67 cm) under ni and l95 × l92 (64.27 cm) and l95 × l28 (59.07 cm) under wsc. this may be attributed to genes with positive effect on fruiting zone length transformed to their respective f1 crosses. the largest capsules length was detected in the parents l95 (3.03 cm) and l92 (2.83 cm) under ni and l95 (2.45 cm) and l12 (2.39 cm) under wsc and their genes with positive effect passed to the f1 crosses l95 × l93 (3.83 cm and 3.24 cm) and l95 × l28 (3.77 cm and 3.11 cm) under ni and wsc, respectively. the genes responsible for more branches/ plant passed from the parents as l92 (4.33 branch) and l12 (3.67 branch) under ni and l28 (3.00 branch) and l110 (2.67 branch) under wsc to their f1 crosses as l95 × l110 (7.00 branch) and l12 × l110 (6.00 branch) under ni and l95 × l110 (5.33 branch) and l95 × l12 (4.67 branch) under wsc. the positive effect of genes towards increasing 1000-seed weight transformed from the parents as l95 (3.47 g and 2.72 g) and l92 (3.26 g and 2.58 g) under ni and wsc, respectively to their f1 crosses as l95 × l92 (4.72 g) and l92 × l28 (4.61 g) under ni and l95 × l12 (3.80 g) and l95 × l110 (3.67 g) under wsc. the desirable genes effect on seed weight per plant passed from parents as l92 (41.54 g and 28.57 g) and l110 (35.85 g and 25.17 g) under ni and wsc, respectively to l92 × l110 (51.27 g and 37.53 g) and l28 × l12 (45.16 g and 34.69 g) under ni and wsc. the highest proportion of seed oil content was detected in the parents l110 (46.72 %) and l95 (45.84 %) under ni and l95 (52.32%) and l12 (48.31%) under wsc, which passed to their f1 crosses l92 × l12 (48.79 %) and l95 × l110 (48.36 %) under ni and l95 × l92 (55.50 %) and l92 × l12 (52.90 %) under wsc. this result corroborates with the findings of fahmy et al. (2015), abd el-satar et al. (2016), ismail et al. (2020) and jeeva et al. (2020), who observed significant genetic variations among parents and their f1 crosses for all studied traits. genotypes days to 50%flowering plant height fruiting zone length capsules length n s n s n s n s l95 65.67 49.33 165.67 128.67 57.27 42.57 3.03 2.45 l92 61.67 49.00 132.33 105.67 53.00 37.52 2.83 2.37 l28 68.33 51.33 125.67 96.00 38.00 37.41 2.17 1.88 l12 60.67 45.33 144.33 105.00 46.00 37.12 2.77 2.39 l93 62.00 47.67 124.00 97.00 37.00 36.12 2.70 2.14 l110 64.67 44.00 154.00 118.00 34.00 28.24 2.40 2.08 lsd 5% 0.30 0.37 0.97 0.90 0.70 0.84 0.06 0.04 lsd 1% 0.40 0.50 1.29 1.20 0.94 1.12 0.08 0.06 l95 × l92 61.33 42.67 139.00 107.00 82.67 64.27 3.37 2.53 l95 × l28 60.00 45.67 157.33 120.00 82.67 59.07 3.77 3.11 l95 × l12 68.33 52.67 122.67 94.33 75.67 56.75 3.47 2.74 l95 × l93 63.33 48.33 145.33 114.00 62.33 44.79 3.83 3.24 l95 × l110 57.33 42.00 125.00 93.67 64.00 52.10 3.23 2.58 l92 × l28 63.00 48.33 130.67 101.67 63.00 53.68 2.80 2.41 l92 × l12 64.00 47.00 122.33 93.33 59.33 42.92 3.33 2.67 l92 × l93 56.67 42.00 135.67 103.33 50.33 37.90 3.20 2.65 l92 × l110 51.33 40.33 120.00 89.67 49.00 37.84 2.87 2.50 l28 × l12 64.67 42.00 132.33 103.33 62.00 46.30 2.83 2.54 l28 × l93 64.33 46.00 122.00 92.00 63.67 45.55 2.83 2.54 l28 × l110 57.67 51.33 146.00 113.00 53.67 42.08 2.43 2.30 l12 × l93 56.33 44.00 122.00 93.00 54.33 45.07 2.73 2.30 l12 × l110 51.67 40.67 110.33 85.00 49.33 40.12 3.20 2.41 l93 × l110 59.00 41.67 109.00 84.33 63.00 47.37 3.37 2.60 lsd 5% 0.48 0.59 1.53 1.42 1.11 1.32 0.10 0.07 lsd 1% 0.64 0.79 2.04 1.90 1.49 1.77 0.13 0.09 table 5a. mean performance of six parental sesame genotypes and their 15 f1 cross combinations for days to 50% flowering, plant height, fruiting zone length and capsules length under normal irrigation (n) and water stress conditions (s) in 2019 season genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 6 combining ability types of combining ability either general (gca) or specific (sca) and their respective symbol in jones (1965) as additive gene action (a) and non-additive (b) were highly significant in respect to all studied traits under both contrasting irrigation regimes (table 6) and their combined analysis (table 7). this indicated that both types of genes either additive or non-additive played a major role in the inheritance of all studied traits. whereas, the additive gene action was preponderant in the gene expression of most studied traits under both irrigation regimes and their combined analysis as verified by baker ratio and (a/b) ratio of jones method. moreover, interaction of irrigation with both types of combining ability was highly significant for all studied traits, indicating that types of gene action had highly significant response to environmental variability. in the approach used by jones (1965), directional dominance (b1) was significant under both irrigation regimes and their combined analysis, indicating that the existence of directional dominance with the preponderance of additive gene action (tables 6 and 7). the significance of distribution of dominant and recessive alleles in parents (b2) for all studied traits was observed under both irrigation regimes and their combined analysis. this showed the presence of asymmetrical distribution of dominant and recessive genes in parents. specific gene interactions or epistasis was detected for all studied traits under both irrigation regimes and their combined analysis, as revealed by the significance of dominance deviation unique to f1s (b3). similar results were reported by fahmy et al. (2015), abd el-satar et al. (2016) and jeeva et al. (2020), they stated that additive gene action had greater contribution in the inheritance of seed yield and its contributing traits. combining ability effects: general combining ability effects general combining ability (gca) effects were estimated for the parental sesame genotypes and specific combining ability (sca) effects for f1 crosses are presented in table 8. the best combiners with highly significant and negative estimates of gca effects for earliness in flowering were exhibited by l110 and l92 under ni and l110 and l12 under wsc. moreover, l93 (-5.82 and -4.00) and l12 (-3.78 and -4.21) in both ni and wsc proved to be good combiners, respectively, again as desirable direction, with highly significant and negative gca effects towards dwarfness. these parents were considered as good general combiners for these traits and could be used in breeding programs to develop earliness in flowering and short stature varieties. genotypes number of branches per plant 1000-seed weight seed weight per plant seed oil content n s n s n s n s l95 1.33 1.00 3.47 2.72 29.80 17.67 45.84 52.32 l92 4.33 1.67 3.26 2.58 41.54 28.57 42.69 48.01 l28 2.67 3.00 2.70 2.27 28.49 22.06 44.36 48.26 l12 3.67 2.33 2.89 2.28 30.43 17.45 43.73 48.31 l93 2.33 2.00 2.49 2.06 33.00 24.55 45.63 47.23 l110 3.33 2.67 2.89 2.31 35.85 25.17 46.72 47.59 lsd 5% 0.24 0.23 0.06 0.06 1.20 1.40 0.42 0.43 lsd 1% 0.33 0.31 0.08 0.08 1.60 1.87 0.56 0.57 l95 × l92 5.33 3.67 4.72 3.10 34.04 19.00 44.15 55.50 l95 × l28 3.33 3.00 3.40 2.55 36.68 28.25 43.99 48.48 l95 × l12 5.67 4.67 4.57 3.80 35.00 25.82 47.61 51.65 l95 × l93 4.33 3.33 3.64 2.92 32.72 27.22 45.82 50.83 l95 × l110 7.00 5.33 4.54 3.67 38.00 27.48 48.36 52.90 l92 × l28 5.00 3.67 4.61 3.60 38.13 28.56 44.92 51.77 l92 × l12 5.67 4.00 3.80 2.85 39.98 32.02 48.79 52.90 l92 × l93 4.33 3.00 3.60 2.87 36.75 33.50 47.22 51.77 l92 × l110 5.33 3.67 3.71 3.20 51.27 37.53 46.73 51.77 l28 × l12 4.67 3.33 3.38 2.71 45.16 34.69 43.03 48.65 l28 × l93 5.33 3.67 2.52 2.60 42.12 33.91 46.02 50.28 l28 × l110 4.67 3.33 3.50 2.73 38.85 31.37 47.19 51.43 l12 × l93 5.33 3.33 3.50 2.70 37.39 27.39 44.23 47.94 l12 × l110 6.00 4.67 3.46 2.81 35.58 26.91 44.09 47.66 l93 × l110 5.67 3.67 3.39 2.89 38.48 28.24 47.14 51.87 lsd 5% 0.39 0.37 0.10 0.10 1.89 2.21 0.66 0.68 lsd 1% 0.52 0.49 0.13 0.13 2.53 2.95 0.88 0.91 table 5b. mean performance of six parental sesame genotypes and their 15 f1 cross combinations for number of branches per plant, 1000-seed weight, seed weight per plant and seed oil content under normal irrigation (n) and water stress conditions (s) in 2019 season a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 7 the positive direction of gca effects for seed weight per plant and its components is desirable. the best gca effects was detected in l95 and l92 under ni and l95 and l28 under wsc for fruiting zone length, l95 and l93 under both irrigation regimes for capsules length, l110 and l12 under both irrigation regimes for branches number per plant, l95 and l92 under both irrigation regimes for 1000seed weight, l92 and l110 under both irrigation regimes for seed weight per plant and l110 and l93 under ni and l95 and l92 under wsc for seed oil content. this result corroborates with the findings of fahmy et al. (2015), abd el-satar et al. (2016), ismail et al. (2020) and jeeva et al. (2020), they created gca effects in the desired direction for seed yield and yield components. specific combining ability effects the created combinations showed a large variation in the sca effects (tables 9a, 9b) either in the desired or undesired direction under normal irrigation and water stress conditions. accordingly, the possibility of selection for the desired effects, where a negative effect on days to 50 % flowering and plant height is desirable, but a positive effect on seed yield and its components is desirable. sov df days to 50%flowering plant height fruiting zone length capsules length griffing jones n s n s n s n s gca a 5 27.02** 15.74** 301.41** 195.81** 267.95** 107.65** 0.32** 0.10** b1 1 65.19** 33.60** 585.56** 363.62** 1407.49** 540.00** 1.08** 0.65** b2 5 19.82** 2.77** 296.50** 164.43** 63.32** 28.02** 0.05** 0.05** b3 9 15.41** 17.32** 104.63** 71.13** 48.72** 33.03** 0.09** 0.05** sca b 15 20.20** 13.56** 200.65** 121.73** 144.17** 65.16** 0.14** 0.09** total 20 21.90** 14.10** 225.84** 140.25** 175.12** 75.78** 0.19** 0.09** error 40 0.17 0.25 1.71 1.48 0.91 1.28 0.01 0.004 baker ratio 0.73 0.70 0.75 0.76 0.79 0.77 0.82 0.70 sov df number of branches per plant 1000-seed weight seed weight per plant seed oil content griffing jones n s n s n s n s gca a 5 1.39** 0.49** 0.70** 0.20** 34.76** 35.44** 3.00** 7.36** b1 1 21.38** 11.59** 2.79** 1.71** 129.24** 203.01** 5.42** 24.82** b2 5 0.69* 0.70** 0.11** 0.02** 13.99** 9.45* 1.44** 3.99** b3 9 0.60* 0.30* 0.19** 0.15** 19.87** 17.53** 3.77** 2.66** sca b 15 2.02** 1.19** 0.34** 0.21** 25.20** 27.20** 3.11** 4.58** total 20 1.86** 1.01** 0.43** 0.21** 27.59** 29.26** 3.08** 5.28** error 40 0.11 0.10 0.01 0.01 2.63 3.58 0.32 0.34 baker ratio 0.58 0.45 0.81 0.65 0.73 0.72 0.66 0.76 table 6. half diallel’s analyses with griffing method 2 model 1 (1956) and jones (1965) for all studied traits across normal irrigation (n) and water stress conditions (s) in 2019 season note: “gca and sca of griffing (1956)” and “a=additive effects, b=total non-additive (dominance) effects, b1=mean deviation of f1’s from their mid-parents, b2=test if there is equal or unequal distribution among parents and b3=detect existence of unique dominance of each f1, i.e., presence of considerable amount of heterotic effect specific to some crosses of jones (1965) modification”;*, ** significant at 0.05 and 0.01 level of probability, respectively source d.f days to 50% flowering plant height fruiting zone length capsules length number of branches per plant 1000-seed weight seed weight per plant seed oil contentgriffing jones gca a 4 41.60** 488.90** 354.88** 0.39** 1.41** 0.82** 64.87** 6.28** b1 1 96.19** 936.02** 1845.56** 1.70** 32.22** 4.44** 328.11** 26.72** b2 4 14.15** 446.52** 77.10** 0.08** 1.18** 0.10** 22.13** 4.72** b3 5 24.43** 172.61** 75.04** 0.13** 0.84** 0.30** 32.67** 4.94** sca b 10 25.79** 314.81** 193.76** 0.22** 3.04** 0.51** 48.85** 6.32** a*i 4 1.16** 8.32** 20.72** 0.03** 0.47** 0.08** 5.33 4.08** b1*i 1 2.59** 13.16** 101.94** 0.03* 0.74** 0.07** 4.15 3.52** b2*i 4 8.44** 14.41** 14.24** 0.01 0.21 0.03** 1.31 0.71 b3*i 5 8.30** 3.15 6.71** 0.01* 0.07 0.04** 4.72 1.49** sca × i b × i 10 7.97** 7.57** 15.57** 0.01** 0.16 0.04** 3.54 1.37** error 80 0.21 1.60 1.09 0.01 0.11 0.01 3.11 0.33 table 7. half diallel’s analyses with griffing method 2 model 1 (1956) and jones (1965) for all studied traits across irrigation regimes in 2019 season note: i=irrigation regimes; “gca and sca of griffing (1956)” and “a=additive effects, b=total non-additive (dominance) effects, b1=mean deviation of f1’s from their mid-parents, b2=test if there is equal or unequal distribution among parents and b3=detect existence of unique dominance of each f1, i.e., presence of considerable amount of heterotic effect specific to some crosses of jones (1965) modification”; *, ** significant at 0.05 and 0.01 level of probability, respectively genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 8 table 8. general combining ability effects of six parental sesame genotypes for all studied traits under normal irrigation (n) and water stress conditions (s) in 2019 season parents days to 50%flowering plant height fruiting zone length capsules length n s n s n s n s l95 1.79** 1.19** 11.51** 9.21** 10.22** 6.32** 0.33** 0.20** l92 -0.96** -0.26 -2.03** -0.79 1.28** 0.01 0.02 0.01 l28 2.38** 1.94** 1.39** 1.17** 0.11 1.24** -0.26** -0.10** l12 -0.13 -0.43* -3.78** -4.21** -0.93** -0.78* 0.01 -0.01 l93 -0.46** -0.39* -5.82** -4.00** -4.05** -2.34** 0.04 0.02 l110 -2.62** -2.06** -1.28** -1.37** -6.64** -4.45** -0.14** -0.12** lsd gi 5% 0.27 0.33 0.85 0.79 0.62 0.74 0.05 0.04 lsd gi 1% 0.36 0.44 1.14 1.06 0.83 0.99 0.07 0.06 lsd gi-gj 5% 0.73 0.90 2.34 2.18 1.71 2.03 0.15 0.12 lsd gi-gj 1% 0.98 1.21 3.14 2.92 2.28 2.72 0.20 0.16 parents number of branches per plant 1000-seed weight seed weight per plant seed oil content n s n s n s n s l95 -0.43** -0.13 0.39** 0.22** -2.96** -3.67** 0.27 1.45** l92 0.32** -0.21* 0.29** 0.13** 2.94** 1.91** -0.28 0.92** l28 -0.43** 0.00 -0.23** -0.13** -0.23 1.06 -0.69** -0.65** l12 0.36** 0.21* -0.02 -0.04 -0.72 -1.34* -0.53** -0.87** l93 -0.26* -0.25* -0.38** -0.21** -0.79 0.86 0.28 -0.65** l110 0.44** 0.38** -0.04 0.02 1.76** 1.18 0.94** -0.20 lsd gi 5% 0.22 0.21 0.06 0.06 1.06 1.23 0.37 0.38 lsd gi 1% 0.29 0.27 0.08 0.08 1.42 1.65 0.49 0.51 lsd gi-gj 5% 0.60 0.56 0.16 0.16 2.91 3.39 1.01 1.04 lsd gi-gj 1% 0.80 0.76 0.21 0.22 3.89 4.54 1.36 1.39 note: *, ** refers to significant at 5% and highly significant at 1%, respectively table 9a. specific combining ability effects of 15 f1 cross combinations for days to 50% flowering, plant height, fruiting zone length and capsules length under normal irrigation (n) and water stress conditions (s) in 2019 season. crosses days to 50%flowering plant height fruiting zone length capsules length n s n s n s n s l95 × l92 -0.55 -4.04** -3.14** -3.23** 14.01** 13.43** 0.00 -0.17** l95 × l28 -5.21** -3.25** 11.78** 7.82** 15.18** 7.00** 0.68** 0.51** l95 × l12 5.62** 6.13** -17.72** -12.48** 9.22** 6.70** 0.12 0.04 l95 × l93 0.95** 1.75** 6.99** 6.98** -0.99 -3.70** 0.45** 0.53** l95 × l110 -2.88** -2.92** -17.89** -15.98** 3.26** 5.72** 0.04 -0.01 l92 × l28 0.54 0.88* -1.35 -0.52 4.45** 7.92** 0.03 0.01 l92 × l12 4.04** 1.92** -4.51** -3.48** 1.83* -0.82 0.30** 0.18** l92 × l93 -2.96** -3.13** 10.86** 6.32** -4.05** -4.29** 0.13* 0.13** l92 × l110 -6.13** -3.13** -9.35** -9.98** -2.80** -2.23* -0.02 0.12* l28 × l12 1.37** -5.29** 2.07* 4.57** 5.66** 1.32 0.08 0.15** l28 × l93 1.37** -1.33** -6.22** -6.98** 10.45** 2.13* 0.04 0.13* l28 × l110 -3.13** 5.67** 13.24** 11.40** 3.03** 0.78 -0.17** 0.02 l12 × l93 -4.13** -0.96* -1.05 -0.60 2.16** 3.67** -0.32** -0.21** l12 × l110 -6.63** -2.63** -17.26** -11.23** -0.26 0.83 0.33** 0.03 l93 × l110 1.04** -1.67** -16.55** -12.10** 16.53** 9.64** 0.46** 0.20** lsd sij 5% 0.60 0.75 1.93 1.80 1.41 1.68 0.12 0.10 lsd sij 1% 0.81 1.00 2.59 2.41 1.88 2.24 0.17 0.13 lsd sij-sik 5% 1.09 1.35 3.50 3.26 2.55 3.03 0.22 0.18 lsd sij-sik 1% 1.46 1.80 4.68 4.36 3.41 4.05 0.30 0.24 lsd sij-skl 5% 1.01 1.25 3.24 3.02 2.36 2.80 0.21 0.17 lsd sij-skl 1% 1.35 1.67 4.33 4.04 3.15 3.75 0.28 0.22 note: *, ** refers to significant at 5% and highly significant at 1%, respectively high and negative sca effects was observed in l12 × l110, and l92 × l110 under ni and l28 × l12 and l95 × l92 under wsc for earliness in flowering and l95 × l110 and l95 × l12 under both irrigation regimes for plant height. conversely, high and positive sca effects were detected in l93 × l110 and l95 × l28 under ni and l95 × l92 and l93 × l110 under wsc for fruiting zone length, the cross combinations l95 × l28 and l93 × l110 a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 9 under ni and l95 × l93 and l95 × l28 under wsc had high and positive sca effects for capsules length. in case of number of branches per plant, l95 × l110 and l28 × l93 under ni and l95 × l110 and l95 × l12 under wsc possessed high and positive sca effects. the promising created combinations for 1000-seed weight was detected in l92 × l28 and l95 × l12 under ni and l95 × l12 and l92 × l28 under wsc. the best sca effects for seed weight per plant were observed in l92 × l110 and l28 × l12 under ni and l28 × l12 and l92 × l110 under wsc. the good cross combinations for seed oil content were shown by l92 × l12 and l95 × l12 under ni and l95 × l92 and l92 × l12 under wsc. this result corroborates with the findings of fahmy et al. (2015), abd el-satar et al. (2016), ismail et al. (2020) and jeeva et al. (2020), found that the genetic divergence among the parental genotypes had the largest effect on generated cross combinations, especially in estimation of sca effects for seed yield and yield components. heterotic effects two types of heterosis i.e., mid parents heterosis (mph) and better parents heterosis (bph) were estimated to identify the best cross combinations (tables 10a, 10b, 10c, 10d). three types of cross combinations i.e. high × high, high × low or low × high and low × low were detected in created cross combinations, where high (h) refer to significant gca effects in the desired direction and low (l) refers to non-significant gca effects either in positive or negative direction. in case of high × high cross combinations, additive and additive × additive gene actions were governed in the inheritance of traits. additive gene action predominated in the good combiner (high) and a complementary epistatic effect governed in the poor combiner (low) as high × low, so these two gene actions involved in gene expression of cross combination as described by salimath and bahl (1985). the cross combinations had low × low general combiners, indicating that non-additive gene action played a prominent role in the inheritance of seed yield traits as presented by bhutia et al. (2014). the desirable target was negative direction of for mph and bph for the earliness in flowering to produce high yield in a short period and short statured plant suitable for mechanical harvesting and resistant to lodging. accordingly, the best cross combinations with h × h were detected in l92 × l110 over mph and bph under both irrigation regimes for earliness in flowering and l12 × l93 over mph under both irrigation regimes for short statured plants and more crosses with h × l and l × h over mph and bph with negative potence ratio exceeding unity under both irrigation regimes. conversely, the cross combinations with positive direction are desirable for seed weight per plant and its table 9b. specific combining ability effects of 15 f1 cross combinations for number of branches per plant, 1000-seed weight, seed weight per plant and seed oil content under normal irrigation (n) and water stress conditions (s) in 2019 season crosses number of branches per plant 1000-seed weight seed weight per plant seed oil content n s n s n s n s l95 × l92 0.90** 0.71** 0.52** -0.07 -3.04* -6.74** -1.48** 2.79** l95 × l28 -0.35 -0.16 -0.28** -0.36** 2.76* 3.37* -1.22** -2.66** l95 × l12 1.20** 1.30** 0.67** 0.80** 1.58 3.34* 2.23** 0.73 l95 × l93 0.49 0.42 0.10 0.09 -0.64 2.53 -0.37 -0.31 l95 × l110 2.45** 1.80** 0.66** 0.61** 2.09 2.47 1.52** 1.30** l92 × l28 0.57* 0.59* 1.03** 0.77** -1.69 -1.90 0.26 1.16** l92 × l12 0.45 0.71** 0.01 -0.06 0.66 3.96** 3.96** 2.51** l92 × l93 -0.26 0.17 0.17* 0.13 -2.51* 3.24* 1.59** 1.16** l92 × l110 0.03 0.21 -0.06 0.23** 9.46** 6.95** 0.43 0.71 l28 × l12 0.20 -0.16 0.11 0.06 9.01** 7.48** -1.38** -0.17 l28 × l93 1.49** 0.63** -0.39** 0.11 6.03** 4.50** 0.80 1.25** l28 × l110 0.11 -0.33 0.24** 0.01 0.20 1.65 1.31** 1.94** l12 × l93 0.70** 0.09 0.38** 0.12 1.79 0.38 -1.16** -0.88* l12 × l110 0.65* 0.80** -0.01 0.01 -2.57* -0.42 -1.96** -1.61** l93 × l110 0.95** 0.26* 0.28** 0.25** 0.40** -1.29 0.29** 2.38** lsd sij 5% 0.49 0.47 0.13 0.13 2.40 2.80 0.84 0.86 lsd sij 1% 0.66 0.62 0.18 0.18 3.21 3.74 1.12 1.15 lsd sij-sik 5% 0.89 0.84 0.24 0.24 4.34 5.06 1.51 1.55 lsd sij-sik 1% 1.20 1.13 0.32 0.32 5.81 6.77 2.02 2.08 lsd sij-skl 5% 0.83 0.78 0.22 0.22 4.02 4.68 1.40 1.44 lsd sij-skl 1% 1.11 1.04 0.29 0.30 5.38 6.27 1.87 1.92 note: *, ** refers to significant at 5% and highly significant at 1%, respectively genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 10 components. consequently, the best cross combinations with h × h were detected in l95 × l92 under normal irrigation (ni) and l95 × l28 under water stress conditions (wsc) over mph and bph for fruiting zone length, l95 × l93 over mph and bph under both irrigation regimes for capsules length, l12 × l110 over mph and bph under both irrigation regimes for number of branches per plant, l95 × l92 over mph and bph under both irrigation regimes for 1000-seed weight, l92 × l110 in mph and bph under both irrigation regimes for seed weight per plant, l93 × l110 over mph and bph under ni and l95 × l92 over mph and bph under wsc for seed oil content as well as more crosses with h × l and l × h over mph and bph with positive potence ratio exceeding unity under both irrigation regimes for all studied traits. similar these findings were reported by azeez and morakinyo (2014), ramesh et al. (2014), fahmy et al. (2015), abd el-satar et al. (2016), ismail et al. (2020), and jeeva et al. (2020), they found that high heterotic effects significantly affected by genetic variability of parental genotypes. crosses days to 50%flowering plant height n s n s mp p bp mp p bp mp p bp mp p bp l95 × l92 -3.66** 1.17 -0.54 -13.22** 39.00 -12.93** -6.71** 0.60 5.04** -8.68** 0.88 1.26 l95 × l28 -10.45** 5.25 -8.63** -9.27** 4.67 -7.43** 8.01** -0.58 25.20** 6.82** -0.47 25.00** l95 × l12 8.18** -2.07 12.64** 11.27** -2.67 16.18** -20.86** 3.03 -15.01** -19.26** 1.90 -10.16** l95 × l93 -0.78 0.27 2.15** -0.34 0.20 1.40 0.35 -0.02 17.20** 1.03 -0.07 17.53** l95 × l110 -12.02** 15.67 -11.34** -10.00** 1.75 -4.55** -21.79** 5.97 -18.83** -24.05** 5.56 -20.62** l92 × l28 -3.08** 0.60 2.16** -3.65** 1.57 -1.36 1.29 -0.50 3.98* 0.83 -0.17 5.90** l92 × l12 4.63** -5.67 5.49** -0.35 0.09 3.68** -11.57** 2.67 -7.56** -11.39** 36.00 -11.11** l92 × l93 -8.36** 31.00 -8.11** -13.10** 9.50 -11.89** 5.85** -1.80 9.41** 1.97 -0.46 6.53** l92 × l110 -18.73** 7.89 -16.76** -13.26** 2.47 -8.33** -16.18** 2.14 -9.32** -19.82** 3.59 -15.14** l28 × l12 0.26 -0.04 6.59** -13.10** 2.11 -7.35** -1.98 0.29 5.31** 2.82 -0.63 7.64** l28 × l93 -1.28* 0.26 3.76** -7.07** 1.91 -3.50** -2.27 3.40 -1.61 -4.66** 9.00 -4.17* l28 × l110 -13.28** 4.82 -10.82** 7.69** -1.00 16.67** 4.41** -0.44 16.18** 5.61** -0.55 17.71** l12 × l93 -8.15** 7.50 -7.14** -5.38** 2.14 -2.94** -9.07** 1.20 -1.61 -7.92** 2.00 -4.12* l12 × l110 -17.55** 5.50 -14.84** -8.96** 6.00 -7.58** -26.03** 8.03 -23.56** -23.77** 4.08 -19.05** l93 × l110 -6.84** 3.25 -4.84** -9.09** 2.27 -5.30** -21.58** 2.00 -12.10** -21.55** 2.21 -13.06** lsd 5% 1.01 1.17 1.25 1.44 3.24 3.74 3.02 3.48 lsd 1% 1.35 1.56 1.67 1.93 4.33 5.00 4.04 4.66 table 10a. relative heterosis (mp) and heterbeltiosis (bp) as well as potence ratio (p) of 15 f1 cross combinations for days to flowering and plant height under normal irrigation (n) and water stress conditions (s) in 2019 season. note: p refers to relative potency of the gene set; *, ** refers to significant at 5% and highly significant at 1%, respectively crosses fruiting zone length capsules length n s n s mp p bp mp p bp mp p bp mp p bp l95 × l92 49.94** 12.91 44.35** 60.50** 9.60 50.99** 14.77** 4.33 10.99** 4.98** 3.27 3.41** l95 × l28 73.55** 3.64 44.35** 47.72** 7.41 38.78** 44.87** 2.69 24.18** 43.72** 3.32 26.98** l95 × l12 46.55** 4.27 32.13** 42.44** 6.20 33.32** 19.54** 4.25 14.29** 13.24** 10.67 11.85** l95 × l93 32.25** 1.50 8.85** 13.86** 1.69 5.23** 33.72** 5.80 26.37** 41.32** 6.25 32.56** l95 × l110 40.25** 1.58 11.76** 47.15** 2.33 22.40** 19.02** 1.63 6.59** 13.76** 1.72 5.31** l92 × l28 38.46** 2.33 18.87** 43.27** 304.00 43.07** 12.00** 0.90 -1.18** 13.25** 1.13 1.40** l92 × l12 19.87** 2.81 11.95** 15.02** 27.79 14.40** 19.05** 16.00 17.65** 12.18** 43.50 11.87** l92 × l93 11.85** 0.67 -5.03** 2.93* 1.54 1.00 15.66** 6.50 12.94** 17.49** 3.43 11.80** l92 × l110 12.64** 0.58 -7.55** 15.07** 1.07 0.84 9.55** 1.15 1.18** 12.34** 1.90 5.48** l28 × l12 47.62** 5.00 34.78** 24.25** 60.93 23.76** 14.86** 1.22 2.41** 19.16** 1.60 6.42** l28 × l93 69.78** 52.33 67.54** 23.90** 13.55 21.75** 16.44** 1.50 4.94** 26.20** 3.95 18.35** l28 × l110 49.07** 8.83 41.23** 28.19** 2.02 12.48** 6.57** 1.29 1.39** 15.99** 3.06 10.24** l12 × l93 30.92** 2.85 18.12** 23.09** 16.91 21.43** 0.00 0.00 -1.20** 1.40** 0.26 -3.77** l12 × l110 23.33** 1.56 7.25** 22.76** 1.68 8.08** 23.87** 3.36 15.66** 7.68** 1.13 0.84** l93 × l110 77.46** 18.33 70.27** 47.20** 3.86 31.16** 32.03** 5.44 24.69** 22.87** 16.11 21.15** lsd 5% 2.36 2.72 2.80 3.24 0.21 0.24 0.17 0.19 lsd 1% 3.15 3.64 3.75 4.33 0.28 0.32 0.22 0.26 table 10b. relative heterosis (mp) and heterbeltiosis (bp) as well as potence ratio (p) of 15 f1 cross combinations for fruiting zone length and capsules length under normal irrigation (n) and water stress conditions (s) in 2019 season. note: p refers to relative potency of the gene set; *, ** refers to significant at 5% and highly significant at 1%, respectively a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 11 3-hayman numerical and graphical analysis 3-1-adequacy of simple additive-dominance model the validity of the additive-dominance model was discovered through three tests, which differed from partially to full adequate (table 11), hence that emphasized the need to complete the identification of genetic components and their ratios according to hayman (1954b). 3-2-genetic components and their ratios a graphical analysis (figures 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b) revealed the regression line intercepted the covariance axis below the point of origin in fruiting zone length, number of branches per plant and seed weight per plant under both irrigation regimes, capsule length under ni, and seed oil content under wsc indicating that over-dominance was governed in the inheritance of crosses number of branches per plant 1000-seed weight n s n s mp p bp mp p bp mp p bp mp p bp l95 × l92 88.24** 1.67 23.08** 175.00** 7.00 120.00** 40.17** 13.30 36.06** 17.13** 6.48 14.11** l95 × l28 66.67** 2.00 25.00** 50.00** 1.00 0.00 10.44** 0.84 -1.83** 2.27** 0.25 -6.13** l95 × l12 126.67** 2.71 54.55** 180.00** 4.50 100.00** 43.61** 4.84 31.73** 52.23** 5.98 40.00** l95 × l93 136.36** 5.00 85.71** 122.22** 3.67 66.67** 22.17** 1.35 4.90** 22.21** 1.61 7.36** l95 × l110 200.00** 4.67 110.00** 190.91** 4.20 100.00** 42.92** 4.70 30.96** 46.06** 5.74 35.21** l92 × l28 42.86** 1.80 15.38** 57.14** 2.00 22.22** 54.59** 5.74 41.16** 48.42** 7.65 39.59** l92 × l12 41.67** 5.00 30.77** 100.00** 6.00 71.43** 23.55** 3.92 16.55** 17.36** 2.84 10.61** l92 × l93 30.00** 1.00 0.00 63.64** 7.00 50.00** 25.22** 1.87 10.32** 24.03** 2.14 11.51** l92 × l110 39.13** 3.00 23.08** 69.23** 3.00 37.50** 20.65** 3.37 13.69** 31.02** 5.76 24.32** l28 × l12 47.37** 3.00 27.27** 25.00** 2.00 11.11** 21.05** 5.98 16.94** 19.27** 87.67 19.01** l28 × l93 113.33** 17.00 100.00** 46.67** 2.33 22.22** -2.64** -0.65 -6.43** 20.18** 4.09 14.54** l28 × l110 55.56** 5.00 40.00** 17.65** 3.00 11.11** 25.25** 7.42 21.13** 19.13** 20.23 18.01** l12 × l93 77.78** 3.50 45.45** 53.85** 7.00 42.86** 30.24** 4.00 21.08** 24.52** 4.76 18.42** l12 × l110 71.43** 15.00 63.64** 86.67** 13.00 75.00** 19.61** 170.00 19.47** 22.50** 31.00 21.61** l93 × l110 100.00** 5.67 70.00** 57.14** 4.00 37.50** 26.18** 3.52 17.44** 32.27** 5.49 24.93** lsd 5% 0.83 0.96 0.78 0.90 0.22 0.25 0.22 0.26 lsd 1% 1.11 1.28 1.04 1.20 0.29 0.34 0.30 0.34 table 10c. relative heterosis (mp) and heterbeltiosis (bp) as well as potence ratio (p) of 15 f1 cross combinations for number of branches per plant and 1000-seed weight under normal irrigation (n) and water stress conditions (s) in 2019 season note: p refers to relative potency of the gene set; *, ** refers to significant at 5% and highly significant at 1%, respectively crosses seed weight per plant seed oil content n s n s mp p bp mp p bp mp p bp mp p bp l95 × l92 -4.57* -0.28 -18.05** -17.83** -0.76 -33.50** -0.26 -0.07 -3.69** 10.64** 2.48 6.08** l95 × l28 25.84** 11.50 23.08** 42.21** 3.82 28.06** -2.46** -1.49 -4.04** -3.60** -0.89 -7.33** l95 × l12 16.23** 15.52 15.03** 47.07** 74.01 46.14** 6.31** 2.68 3.87** 2.66** 0.67 -1.27 l95 × l93 4.19* 0.82 -0.87 28.97** 1.78 10.90** 0.18 0.78 -0.05 2.12** 0.42 -2.84** l95 × l110 15.76** 1.71 6.00* 28.28** 1.62 9.16** 4.51** 4.76 3.52** 5.89** 1.25 1.11 l92 × l28 8.89** 0.48 -8.22** 12.81** 1.00 -0.04 3.20** 1.67 1.26 7.54** 28.30 7.26** l92 × l12 11.11** 0.72 -3.75 39.19** 1.62 12.10** 12.90** 10.72 11.56** 9.85** 31.62 9.51** l92 × l93 -1.40 -0.12 -11.53** 26.16** 3.46 17.28** 6.94** 2.09 3.50** 8.71** 10.68 7.83** l92 × l110 32.50** 4.42 23.42** 39.67** 6.28 31.37** 4.52** 1.00 0.02 8.30** 19.05 7.83** l28 × l12 53.30** 16.19 48.42** 75.58** 6.47 57.21** -2.31** -3.27 -3.00** 0.76 16.85 0.71 l28 × l93 36.98** 5.04 27.61** 45.52** 8.54 38.16** 2.29** 1.62 0.87 5.31** 4.91 4.19** l28 × l110 20.76** 1.82 8.37** 32.84** 4.99 24.65** 3.62** 1.40 1.01 7.30** 10.40 6.55** l12 × l93 17.90** 4.41 13.30** 30.47** 1.80 11.60** -1.01 -0.48 -3.07** 0.36 0.32 -0.76 l12 × l110 7.37** 0.90 -0.74 26.27** 1.45 6.90* -2.51** -0.76 -5.62** -0.59 -0.80 -1.33 l93 × l110 11.79** 2.85 7.36** 13.60** 10.85 12.20** 2.10** 1.78 0.91 9.40** 24.76 8.99** lsd 5% 4.02 4.64 4.68 5.41 1.40 1.62 1.44 1.66 lsd 1% 5.38 6.21 6.27 7.24 1.87 2.16 1.92 2.22 table 10d. relative heterosis (mp) and heterbeltiosis (bp) as well as potence ratio (p) of 15 f1 cross combinations for seed weight per plant and seed oil content under normal irrigation (n) and water stress conditions (s) in 2019 season note: p refers to relative potency of the gene set; *, ** refers to significant at 5% and highly significant at 1%, respectively genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 12 these traits. this was referred through the average degree of dominance (h1/d) 0.5 (table 12), which had values more than unity for these traits. however, the other cases take the opposite direction. this result corroborates with the findings of fahmy et al. (2015) and abd el-satar et al. (2016), who found that over-dominance had the major role in the inheritance of most studied traits. the significance of additive (a) and non-additive (b) effects in jones (1965) analysis (tables 6 and 7) and additive (d) and two dominance types in hayman (1954b) analysis (table 12) indicated the both additive and non-additive genetic components played a major role in the inheritance of all studied traits under both irrigation regimes. however, the additive gene action was preponderant in the gene expression of most studied traits under both irrigation regimes and their combined analysis as verified by baker ratio and (a/b) ratio of jones method. this was further verified through medium narrowsense heritability (table 12) for most studied traits under both irrigation regimes. these finding are in line with the results of fahmy et al. (2015) and abd el-satar et al. (2016), they found that narrow sense heritability was moderate for seed yield and most yield component traits. hence, maximizing improvement of most studied traits can be achieved through selection of promising segregates in early generation. h1 and h2 dominance types considerable differed (table 12) for all studied traits under both irrigation regimes. this difference was a result of an unequal sharing of dominant and recessive genes in the used parents, which was also proved by the h2/4h1 ratio that was less than 0.25 (table 12) for all studied traits. the f value (table 12) had positive sign for most studied traits, indicated that the occurrence of recessive alleles was lesser than that of the dominant alleles in the parents, and this was further verified through the ratio of kd/kr, which was more than unity under both irrigation regimes for most cases. the ratio of h2/h2 (table 12) ranged from one to two pairs, which refers to the number of gene pairs governing in the inheritance of studied traits, confirmed that one to two pairs of dominant genes controlling the inheritance of all studied traits under both irrigation regimes. same results were obtained by fahmy et al. (2015). 5-distribution of dominant and recessive genes among the parents parental sesame genotypes widely scattered in the wr-vr graphical analysis (figures 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b) for all studied traits under both irrigation regimes, this confirmed their genetic diversity. the most dominant genes were detected in the parents l93, l28 and l95 under ni and l93 under wsc for days to 50% flowering, l92 and l12 under both irrigation regimes for plant height, l93 and l12 under both irrigation regimes for fruiting zone length, l92 and l95 under ni and l92 and l12 under wsc for capsules length, l92 and l12 under ni and l28 and l93 under wsc for number of branches per plant, l93 and l110 under ni and l93 and l92 under wsc for 1000-seed weight, l95 and l93 under ni and l93 and l110 under wsc for seed weight per plant and l93 and l110 under ni and l28 and l93 under wsc, as they located closed to the origin of regression graph. however, the recessive genes were observed in l12 under both irrigation regimes for days to 50% flowering, l110 under both irrigation regimes for plant height, l28 under ni and l92 under wsc for fruiting zone length, l28 under both irrigation regimes for capsules length, l95 under both irrigation regimes traits i joint regression analysis analysis of variance of array fitness t2 (b) ± se b=0 b=1 wr+vr wr-vr days to 50%flowering n ns 0.12±0.27 ns * ** ** partially adequate mode s ns -0.09±0.28 ns * ** ** partially adequate mode plant height n ns 0.17±0.42 ns ns ** ** partially adequate mode s ns 0.20±0.41 ns ns ** ** partially adequate mode fruiting zone length n ns 0.77±0.31 ns ns ** ** partially adequate mode s ns 0.33±0.22 ns * ** ** partially adequate mode capsules length n ns 0.61±0.27 ns ns ** ** partially adequate mode s ns 0.21±0.30 ns ns ** ** partially adequate mode number of branches per plant n ns 0.50±0.19 ns ns ** ** partially adequate mode s ns 0.32±0.23 ns * ** ** partially adequate mode 1000-seed weight n ns 0.18±0.32 ns ns ** ** partially adequate mode s ns 0.06±0.36 ns ns ** ** partially adequate mode seed weight per plant n ns 0.59±0.24 ns ns ** ** partially adequate mode s ns 0.43±0.28 ns ns ns ns fully adequate model seed oil content n ns -0.24±0.25 ns ** ** ** partially adequate mode s ns 1.06±0.38 * ns ** ** partially adequate mode table 11. adequacy test of the data for additive-dominance model for all studied traits under normal irrigation (n) and water stress conditions (s) in 2019 season note: i: irrigation regimes; b: regression-coefficient; wr: covariance; vr: variance.*, ** refers to significant at 5% and highly significant at 1%, respectively a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 13 for number of branches per plant, l28 under ni and l12 and l95 under wsc for 1000-seed weight, l92 and l110 under ni and l92 and l12 under wsc for seed weight per plant and l12 under ni and l92 under wsc for seed oil content, as they located farthest from the origin of regression graph. this result corroborates with the findings of fahmy et al. (2015), who found that asymmetrical distribution of dominant and recessive genes in parental genotypes. parameter i days to 50% flowering plant height fruiting zone length capsules length number of branches per plant 1000-seed weight seed weight per plant seed oil content d n 8.31 275.78** 88.26** 0.09* 1.02 0.12 21.03** 1.92 s 7.12* 159.51** 20.23 0.04 0.42 0.05 15.80** 3.12** f n 6.29 364.43 -18.74 -0.06 0.89 -0.23 14.19 1.52 s 0.85 191.75 -26.11 0.02 0.71 -0.05 3.74 1.92 h1 n 81.92** 864.57** 446.07** 0.45** 5.70** 1.06** 85.79** 11.60** s 51.08** 515.27** 209.77** 0.30* 3.53** 0.65** 80.76** 16.47** h2 n 64.42** 602.20** 390.38** 0.42** 5.16** 0.97** 75.07** 10.54** s 48.78** 370.19** 185.69** 0.26* 2.97** 0.64** 74.78** 13.14** h2 n 42.16** 378.54* 911.77** 0.69** 13.79** 1.80** 82.34** 3.33 s 21.64** 234.78* 349.31** 0.42** 7.46** 1.11** 129.57** 15.91** (h1/d) 0.5 n 3.14 1.77 2.25 2.22 2.37 2.96 2.02 2.46 s 2.68 1.80 3.22 2.59 2.89 3.67 2.26 2.30 h2/4h1 n 0.20 0.17 0.22 0.23 0.23 0.23 0.22 0.23 s 0.24 0.18 0.22 0.22 0.21 0.24 0.23 0.20 kd/kr n 1.27 2.19 0.91 0.74 1.45 0.51 1.40 1.38 s 1.05 2.00 0.67 1.22 1.81 0.74 1.11 1.31 h2/h2 n 0.65 0.63 2.34 1.66 2.67 1.86 1.10 0.32 s 0.44 0.63 1.88 1.63 2.51 1.73 1.73 1.21 h2 (n.s) n 0.38 0.36 0.45 0.46 0.19 0.47 0.29 0.20 s 0.26 0.37 0.42 0.31 0.14 0.26 0.29 0.39 h2 (b.s) n 0.99 0.99 1.00 0.97 0.94 0.98 0.91 0.91 s 0.99 0.99 0.99 0.95 0.90 0.97 0.88 0.94 table 12. components of the genetic variance and their ratios (hayman 1954b) for all studied traits under normal irrigation (n) and water stress conditions (s) in 2019 season note: i, irrigation regimes; *, significant when it exceeds 1.96 after dividing it by its standard error; **, is tested by t test at n−2 degrees of freedom after dividing it by its standard error. d = additive variance, f= relative frequency of dominant and recessive allels, h1= dominance variance, h2= dominance variance, h2= square of difference p vs. all, (h1/d) 0.5= average degree of dominance, h2/4h1= proportion of dominance and recessive genes, kd/kr= proportion between dominant and recessive genes in all parents, h 2/h2= number of effective factors, h 2 (n.s) = narrow-sense heritability, h2 (b.s) = broad-sense heritability fig. 1a: wr/vr graphs for days to 50% flowering under ni fig. 1b: wr/vr graphs for days to 50% flowering under wsc genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 14 fig. 2a: wr/vr graphs for plant height under ni fig. 2b: wr/vr graphs for plant height under wsc fig. 3a: wr/vr graphs for fruiting zone length under ni fig. 3b: wr/vr graphs for fruiting zone length under wsc fig. 4a: wr/vr graphs for capsule length under ni fig. 4b: wr/vr graphs for capsule length under wsc a. k. ibrahim suzan; m. a. abdelsatar; m. a. ahmed; m. m. niazy peruvian journal of agronomy 5(1): 1–17 (2021) 15 fig. 5a: wr/vr graphs for number of branches per plant under ni fig. 5b: wr/vr graphs for number of branches per plant under wsc fig. 6a: wr/vr graphs for 1000-seed weight under ni fig. 6b: wr/vr graphs for 1000-seed weight under wsc fig. 7a: wr/vr graphs forseed weight per plant under ni fig. 7b: wr/vr graphs forseed weight per plant under wsc genetic behavior for seed yield and yield components in sesame (sesamum indicum l.) under normal irrigation and water stress conditions january april 2021 16 conclusion in conclusion, the differences due to parents and their f1 crosses were highly significant for seed yield and yield components under both irrigation regimes and their interactions with irrigation. mean squares of two types of combining ability were highly significant for seed yield and yield components regimes and their interactions with irrigation. the best combiners for seed yield per plant and most of its component were observed in parents l92, l110 and their combination under both irrigation regimes. the additive gene action makes a greater contribution in the inheritance of most studied traits, that further confirmed by its significance and the value of average degree of dominance exceeding the unity. narrow-sense heritability was high for most studied traits under both irrigation regimes. the most dominant genes carried on l95 and l93 under normal irrigation and l93 and l110 under water stress conditions for seed weight per plant, in contrary l92 and l110 under normal irrigation and l92 and l12 under water stress conditions for seed weight per plant possessed high concentration of recessive genes. references abd el-satar, m. a., ahmed fadia, h. a., & elnenny, e. m. m. 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(1944). balance and potence in natural populations. journal of genetic, 46, 150–60. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index review article https://doi.org/10.21704/pja.v6i3.1973 received for publication: 22 march 2022 accepted for publication: 30 november 2022 published: 31december 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by pest management of sesame in ethiopia: a review manejo de plagas de sésamo en etiopía: una revisión zemedkun alemu1*;workishet taye2 *corresponding author: zalemu56@gmail.com *https://orcid.org/0000-0002-8764-4823 abstract sesame is ethiopia’s most significant oil crop, especially tigray, amhara, and some lowland oromia, somalia, and the gambella region. consequently, the crop is exposed to a wide range of insect pests feeding on leaves, flowers, pods, and seeds affecting sesame yields. this article review provides information on the biology, nature of the damage, and management methods of economically important sesame pests. sesame webworm, antigastra catalaunalis (duponchel) is the most common and frequently encountered pre-harvest pest of sesame. sesame seed bugs, elasmolmus sordidus (fabricus) is also the most serious under field and storage conditions. gall fly, asphondylia sesami (felt) could become a severe insect issue because of sesame gall formation, and indian meal moth, plodia interpunctella (hubner) is a critical stored pest and a significant challenging of crop sesame in ethiopia. reports on minor pests are also listed. this paper summarizes current knowledge on pest management strategies, including cultural, biological, and botanical methods, and pesticide applications. the information gathered here indicates that the bioecology, host range, host plant resistance, the occurrence of insecticide resistance, and the development of integrated pest management methods for economical insect pests need to be addressed. keywords: gall fly, indian meal moth, infestation, seed bug, webworm, pest management. resumen el sésamo es el cultivo oleaginoso más importante de etiopia, especialmente en tigray, amhara, y alguanas tierras bajas de oromia, somalia, and the gambella region. en consecuencia, el cultivo está expuesto a una amplia gama de plagas de insectos que se alimentan de hojas, flores, vainas y semillas afectando los rendimientos de sésamo.la revisión de este artículo brinda información sobre la biología, la naturaleza del daño y los métodos de manejo de plagas de sésamo económicamente importantes. el gusano tejedor del sésamo, antigastra catalaunalis (duponchel), es la plaga más común y frecuente durante la pre-cosecha del sésamo. el chinche de sésamo, elasmolmus sordidus (fabricus) es la más grave en condiciones de campo y almacenamiento. la mosca de las agallas, asphondylia sesame (felt) puede convertirse en un grave problema debido a la formación de agallas, y la polilla india de la harina, plodia interpunctella (hubner) es una plaga crítica de almacén y un desafío para el cultivo de sésamo en etiopía. este documento resume el conocimiento actual sobre las estrategias de manejo de plagas, incluidos los métodos culturales, biológicos y botánicos, y las aplicaciones de pesticidas. la información recopilada indica que es necesario abordar la bioecología, el rango de hospedantes, la resistencia de la planta hospedante, la aparición de resistencia a los insecticidas y el desarrollo de métodos de manejo integrado para plagas de importancia económica. palabras clave: mosca de las agallas, polilla india de la harina, infestación, chinche de las semillas, gusano tejedor, manejo de plagas. 1 ethiopian institute of agricultural research, werer agricultural research center, werer, ethiopia 2 ethiopian institute of agricultural research, melkassa agricultural research center, melkassa, ethiopia how to cite this article: zemedkun, a., & workishet, t. (2022). pest management of sesame in ethiopia: a review. peruvian journal of agronomy, 6(3), 210–221. https://doi.org/10.21704/pja.v6i3.1973 mailto:zalemu56@gmail.com https://orcid.org/0000-0002-8764-4823 zemedkun, a., & workishet, t. peruvian journal of agronomy, 6(3), 210–221 (2022) https://doi.org/10.21704/pja.v6i3.1973 211 introduction sesame (sesamum indicum l.) is a centuries-old culinary oil crop that is mostly grown in any part of the world for its oil-rich seeds (pandey et al., 2018). it plays a significant role in the agricultural economy of developing countries, like ethiopia. in ethiopia, it is one of the foremost important and economically vital oil crops (fiseha & muez, 2019), and it is also an export crop after coffee (coffea arabica l.) (desawi et al., 2021). it’s grown within the northern part mainly in tigray (humara, welkayit, and the tahtay adiabo), amhara (wollo and metema), benshangul, and gambella regions with annual rainfall, of 600 mm to 1100 mm, and temperatures of 27 °c (geremew et al., 2012; zerihun, 2012; abadi, 2018). ethiopian sesame is among the best quality in the world, this might be due to the higher quality of seeds in terms of color, taste, and nutty aroma (taghouti et al., 2017). ethiopia is one of the major global producers and exporters of sesame seeds (teshome & esubalew, 2022). currently, it is grown on 520 000 hectares of land which contributes to 255 000 metric tons of sesame seed for the world’s production as a source of income and foreign currency (moti, 2021). however, the national average yield of the crop is poor (0.6 t/ha). (desawi et al., 2021). besides the low yield of the crop obtained by the farmer, other various factors endanger crop production and productivity such as the lack of improved varieties, low yield of landrace cultivars, indeterminate flowering nature, shattering of capsules at maturity, drought, mono-cropping system, weeds, insect pests, and diseases (daniel, 2017). many insect pests can attack sesame, and the number and importance of these insect pests have grown and varied from place to place, time to time, and season to season. among them, sesame webworms, seed bugs, gall flies, and meal moth are the common insect pest that damages crops both during field and storage (kinati, 2017). in this regard, we suppose that quantifying the economic importance of insect pests and the potential effectiveness of current control strategies can provide the necessary to develop improved pest management and control systems. it can also help policymakers prioritize their resource allocations for the effective management of sesame pests. therefore, in this paper, the major insect pests of sesame and their management methods are reviewed. this review paper is based on systematic review techniques and a thorough search of research findings across many databases to ensure comprehensive article retrieval. insect pests of sesame sesame has been attacked worldwide by more than 200 insect pest species belonging to 55 genera (dilipsundar et al., 2019). of those, india has reported 55 species of arthropods (thangjam & vastrad, 2018), bangladesh 29 insect pests (biswas et al., 2001), uganda 38 insect pests (ssekabembe et al., 2006), and nigeria 16 insect pests (zakka et al., 2018), were identified as pests species of sesame. sesame webworm, antigastra catalaunalis (duponchel) (lepidoptera: pyralidae), gall fly, asphondylia sesami (felt) (diptera: cecidomyiidae), seed bug, elasmolomus sordidus (fabricus) (heteroptera: lygaeidae), and meal moth, plodia interpunctella (hubner) (lepidoptera: noctuidae), have been identified as major insect pests in ethiopia (assefa et al., 2020). additionally, termites (termitidae: isoptera) are major pests of sesame, causing damage from seedlings to harvesting and shocks (kinati, 2017). other notable storage pests include the red flour beetle, tribolium confusum (herbst) (tenebrionidae: coleoptera) and also the rice moth, corcyra cephalonica (hubner) (lepidoptera: noctuidae) (negash, 2015). the mealybug, phenacoccus solenopsis (tinsley) (homoptera: pseudococcidae) is a recently introduced invasive sesame pest (zenawi, 2018). to protect the crop from pest damage, different pest management strategies have been used in ethiopia to assist farmers in raising production and productivity, as well as to influence the national economy (geremew et al., 2012). pest management of sesame in ethiopia: a review september december 2022 212 sesame webworm, antigastra catalaunali sesame webworms are the most economically important insect pests found wherever sesame is cultivated (zerabruk & ferdu, 2020). it is a holometabolous insect whose developmental stages have the following characteristics: eggs are laid singly, minutely, conical in shape, and white in color (pandey et al., 2018). the size of eggs is between 0.4 mm to 0.5 mm in length and 0.2 mm to 0.3 mm in width (pandey et al., 2018). the egg development period lasts 2.5 days (pandey et al., 2018) and records 73.3 % to 90 % egg viability (table 1). larvae take 9.9 days to 13.1 days and include five distinct instars (table 1). the first instar larva period is 4.0 days to 5.5 days, the second is 1.5 days to 2.0 days, the third is also 1.5 days to 2.0 days, the fourth is 1.0 day, and the fifth instar larva is 2.0 days to 3.0 days (pandey et al., 2018). pupae first emerge as green and then turn brown. it happened inside a silken cocoon that is transparent and pale white. the pupa’s length and width are 7.4 mm and 1.4 mm, respectively. the pupal period varies from 5.3 days to 7.3 days with 76 % ± 6.5 % adult emergence (pandey et al., 2018). suliman et al. (2013) reported a pupal period of 4.9 days ± 0.21 days. it’s found both in soil and on webbed leaves when the feeding period of the larva is completed (suliman et al., 2013). adults are light reddish-brown to dark reddish-brown. males have less wing expanse and a slender abdomen, while females have more wing expanse and a broader abdomen. males are 8 mm to 8.5 mm in length and 22 mm to 24 mm in breadth with expanded wings. females are longer compared to males, 11.0 mm to 12.0 mm in length and 25.5 mm to 28 mm in width (pandey et al., 2018). a female may produce a maximum of 53 eggs to 92 eggs (pandey et al., 2018). males complete their life cycle within 22 days to 26.3 days, whereas females take 23.8 days to 28.1 days in environmental conditions 30.5 °c ± 2.3 °c and 67.2 % ± 1.8 % rh. (pandey et al., 2018) (table 1). the developmental stages from eggs to adults take 22 days to 39 days (ahirwar et al., 2010). nature of the damage sesame webworm is occurring and infests the crop from seedling to maturity stages (kinati, 2017). the insect is destructive in the larval stages (zenawi et al., 2016); causing about 25 % (tadele, 2005), 25 % to 35 % (geremew et al., 2012), and 17 % to 42 % reduction of yield (zerabruk & ferdu, 2020) in different agroecology of ethiopia. larvae frequently showed cryptic behavior in the narrow gap between the shoot and the capsules (pandey et al., 2018). the first instars have seen the top leaves of the plants, where they first fed on the epidermis of the leaves. the second instar was seen to damage capsules and leaves (simoglou et al., 2017) (figure 1 c & d). young larvae mine young leaves and shoot tips; they bind leaves and shoots together and eat inside (geremew et al., 2012) (figure 1 a & d). the late instars were boring tunnels in the main stem’s mesophyll of the plant. on the sesame, the larva spins leaf silk during feeding time (suliman et al., 2013). moreover, the larvae may multiply quickly and in dense numbers, and they eat aggressively the sesame plant. it was observed that the second stage damaged capsules and buds. then the larva continuously feeding on the capsule. table 1. duration of developmental stages of antigastra catalaunalis (modified of pandey et al., 2018) developmental stage duration (days) egg total 2.3-2.4 larva 1st instar 4 – 5.5 2nd instar 1.25 – 2.0 3rd instar 1.5 – 2.0 4th instar 1.0 5th instar 2.0 – 3.0 total 9.9 –13.1 pupa 5.3 – 7.3 adult males 22.0 – 26.3 females 23.8 – 28.1 total period (egg-adult) 24.1 – 26.0 zemedkun, a., & workishet, t. peruvian journal of agronomy, 6(3), 210–221 (2022) https://doi.org/10.21704/pja.v6i3.1973 213 management strategies many efforts are being made in different regions to enhance productivity and increase the income of farmers. culturally, the pest that has been managed by adjusting planting time can reduce injuries from webworm infestation (ali & jan, 2014). similarly, intercropping of sesame with other crops like black gram, green gram, cluster bean, sorghum, and pearl millet may also have a significant impact in reducing this infestation (uddin & adewale, 2014). limited studies have reported resistance varieties for this pest, but the genotypes es 22, si 250, and uma were found to be unfavorable for the pest genotypes (karuppaiah & nadarajan, 2013). hemipimpla sp. and mermithid nematode are natural enemies that have been used for controlling sesame webworms (egonyu et al., 2009). spraying extract from datura, neem, and henna plants was effective for larvae control (suliman et al., 2013). in ethiopia, several broadspectrum insecticides have been employed under smallholder conditions to reduce the damage caused by a. catalaunali. the application of a repeated single insecticide group is a common practice by farmers (geremew et al., 2012), and the application of diazinon 60 ec three times on the different phonological stages of the crop (zenawi et al., 2016b). also integrating early sowing with two times dimethoate 40 ec during seedling and flowering (zenawi, 2018) or integrating early planting within two and four weeks after the emergence application of figure 1. damage symptom of a. catalaunalis. a) larva feeding the leaf, b) webbed tender shoot (geremew et al., 2012), c & d) damanged symptom on leaf and capsule (simoglou et al., 2017) cypermethrine 720 ec, resulted effective in controlling the pest (egonyu et al., 2009). sesame seed bug, elasmolmus sordidus sesame seed bug is the major pest targeting sesame in both fields and storage facilities (muez & berhanu, 2016). this insect is understood within the area as “stayto”, which suggests it consumes an infinite amount of sesame oil (muez et al., 2008). it is a hemimetabolous insect pest (selemun, 2011) (figure 2), whose developmental stages have the following characteristics: eggs’ color changes from yellow to pale yellow, pink, and eventually full red. its period is 4.5 days ± 0.2 days (osman, 2009) or 5.4 days ± 0.1 days (berhe et al., 2017). nymph color is light red, dark brown, and finally black (berhe et al., 2017). nymphal periods are complete through six distinct instars. the time required to end all nymphal stages is 18.4 days ± 0.1 days (osman, 2009). consistent with berhe et al. (2017), the whole nymphal period is 18.4 days ± 0.1 days. female adults have average fertility of 71.8 eggs, (8.4 ± 0.3) eggs per female per day. adult longevity is 20.2 days ± 0.2 days. for adult females and males, their lifetime is taking 19.2 days ± 0.4 days and 9.9 days ± 0.4 days, respectively. the reproductive period of female continue laying eggs regularly was 7.88 ± 0.26 days. the sex ratio of males and females is 1:1.2 and thus the oviposition period is 7.1 days ± 0.3 pest management of sesame in ethiopia: a review september december 2022 214 days in the environmental condition of 28 °c to 33 °c and 50 % to 65 % rh . the egg to the adult stage takes 32 days to 54 days (berhe et al., 2017) (table 2). table 2. duration of developmental period of elasmolmus sordidus (modified of berhe et al., 2017) developmental stage duration (days) egg 5 – 7 nymphal stage (1-6 instars) 16 – 20 adult longivity 13 – 27 female reproductive period 7.88 ± 0.26 total period (egg-adult) 32 – 54 the pest attacks a wide range of host plants (palanisamy & kalaiyarasan, 2002). sesame, humeray, shico sar, driya/hareg, mashila, hiletay, chomer, wariat, adar wild, teneg, demayto, papaya, neem, groundnut, tomato, and unidentified local name of chiwchiwit, topas, and shewit-hagay are the alternate host of the pest (berhe et al., 2017). nature of the damage both the nymph and the adult infest sesame within the field during drying and in warehouses. the pest affects the nymph and adult life stages. the ideal climate for the survival of the sesame seed bugs is about 30 oc, with cloudy weather conditions and moist soil. the degree of infestation and severity varies not only from one to another year but also between the seasons of the year (berhe et al., 2017). both the nymphs and adults suck the seed oil and its contents, causing qualitative and quantitative losses. in ethiopia, the pest caused 50 % to 100 % seed weight loss (geremew et al., 2012; muez & berhanu, 2016) weight losses, in sudan 1 % to figure 2. schematic representation life cycle of e. sordidus. a) mating of ssb b) egg of ssb c) nymph of ssb d) adult sesame seed bug (selemun, 2011) . zemedkun, a., & workishet, t. peruvian journal of agronomy, 6(3), 210–221 (2022) https://doi.org/10.21704/pja.v6i3.1973 215 2.2 % weight loss and also 11 % to 15.0 % oil content loss (abdelmanan et al., 2015). management strategies for many years different management practices have been done to alleviate the crop from infestation. culturally, the pests are often managed by stalk removal after harvest, plowing, or alternate host destruction around fields. other management is to harvest early when pods/stems are yellow and thresh/shake as early as possible when pods open fully (geremew et al., 2012). ants of varied species, spiders, wasps, egg parasite grionini sp., and lizards predators for seed bug nymphs. extracts of neem and birbira seed powder, pyrethrum flower dust, and nimbicidine are effective in controlling sesame seed bugs in storage (palanisamy & kalaiyarasan, 2002; selemun, 2011). application of insecticides such as malathion 50 % ec, endosulfan 35% ec, and carbaryl 85 % wp at the stalk, and around the soil of the sesame stalk three times a common practice in ethiopia (mandefro et al., 2009). but, this method is ineffective due to re-infestation of the pest within a few days after the pesticide application (muez & berhanu, 2016). however, integrating the use of cultural and chemical control methods is effective in controlling the pest (geremew et al., 2012). gall fly or midge, asphondylia sesame sesamum gall flies are one of the foremost important pests of sesame (chopada et al., 2018; adam et al., 2020). the pest is common in countries like nigeria (philips, 1977), uganda (ubor et al., 2015), sudan (adam et al., 2020), india (chopada et al., 2018), and ethiopia (mandefro et al., 2009). weather factors like high relative humidity, frequent rain, and high temperatures are favorable for the buildup of the epidemic (assefa et al., 2020). the pest is a holometabolous insect pest whose developmental stages have the following characteristics: the egg period is 2 days to 4 days, the larval lasts for 14 days to 21 days. the pupal stages last between 7 days to 12 days and the adult stages last 23 days to 27 days (baskaran, et. al. 1997). nature of damage a female insect lays their eggs on sesame flowers and buds. the hatched larva starts feeding inside the floral buds and young capsule resulting in the formation of gall (assefa et al., 2020; adam et al., 2020) (figure 3). the pest inflicts about 29 % to 34.3 % seed yield reduction (egonyu et al., 2005); 100 % in susceptible genotypes (mehalingam, 2012); 30 % in grain yield decrease (geremew et al., 2012). management methods efforts have been made to reduce the damage to increase the farmer’s income and increase productivity. timely planting, crop rotation, and intercropping with maize, sorghum, and finger millet reduce the severity of the pest (assefa et al., 2020). alternatively, the application of dimethoate and diazinon are insecticides (geremew et al., 2012), and chloranthriprole 18.5 % sc effectively controls the pest (chopada et al., 2018). indian meal moth, plodia interpunctella indian meal moth is a polyphagous species that evolved in response to a significant economic impact (filip & snezana, 2012; campos & phillips, 2013). it is found in africa, asia, europe, america, and oceania (mohandass et al., 2007). this pest is one of the critical stored product moths with a large range of feeding preferences (athanassiou & arthur, 2018). the pest is a holometabolous insect pest with the following characteristics: eggs are grayish-white and have a length between 0.3 mm and 0.5 mm (anonymous. 2023) (figure 4 b). the developmental period is 4.7 days ± 0.8 days (pérez-mendoza & aguilerapeña, 2006). pest management of sesame in ethiopia: a review september december 2022 216 larvae color can vary considerably because of diet content (thomas & marle, 2015). the larvae have five to seven instars (thomas & marle, 2015). the pupa is 6 mm to 11 mm with a pale brown color and takes fifteen to twenty days at 20 °c to 30 °c (thomas & marle, 2015). an adult female produces an average of 212 eggs ± 34 eggs and a maximum of 400 eggs (pérez-mendoza & aguilera-peña, 2006). the whole life cycle is completed within 27 days to 305 days, depending on temperature conditions (william, 2006; thomas & marle, 2015) (figure 4). nature of the damage the hatched larvae start feeding on grain and become large, spin webs, and leave silk threads in their path (william, 2006) (figure 5). the pest damages the grain by making impurities in foodstuffs with its frass and webbing character (athanassiou & arthur, 2018). it inflicts a weight loss of 17.8 % in stored sesame grains (zenawi, 2017), and 9.4 % weight loss and 5.7 % oil loss when packed in polypropylene bags (kumera et al., 2020). management methods few activities were made for controlling the pest such as creating adverse circumstances and disrupting its breeding grounds (william, 2006). matthew and paul (2006) found hymenopteran parasitoids of eggs, and larvae are good biological control methods for this pest. conclusions and recommendations sesame is a vital agricultural crop in ethiopia but is significantly hampered by insect pests. the review focuses on the sesame webworm, seed bug, gall fly, and indian meal moth are major sesame pests causing a huge seed yield reduction in the country. other pests of sesame included in the paper were termites, green peach aphids, cotton whitefly, and mealybug. knowledge of the developmental characteristics, feeding behavior, and nature damage to the crop are essential before effective control strategies are developed. to reduce the economic losses due to insect pests, various control measures such as cultural, biological, botanical, and use of synthetic pesticides have been suggested under this review. application of malathion 50 % ec, endosulfan 35 %, carbaryl 85 % wp, and dimethoate and figure 3. damaged symptom of gall midge on sesame plant (assefa et al.,2020) zemedkun, a., & workishet, t. peruvian journal of agronomy, 6(3), 210–221 (2022) https://doi.org/10.21704/pja.v6i3.1973 217 figure 4. life cycle of indian meal moth, p. interpunctella. a) adult b) egg c) larva d) pupa of indian meal moth. a, c & d of thomas & marle (2015); b of sarefo (2008). figure 5. damaged and webbed sesame grains by p. interpunctella (zenawi, 2017). diazinon 60 % ec are primary means of pest management in the country. farmers frequently get caught in a cycle of increasing the quantity and/or frequency of pesticide applications, which is frequently unsuitable and harmful. sesame producers need effective alternative pest management techniques to escape this pesticide treadmill. even though insect pests provide the greatest threat, there has been insufficient research on pest management methods. this would entail pest management of sesame in ethiopia: a review september december 2022 218 improving the abilities required to survey fields, and appropriately mix and apply insecticides. therefore, in the future to spice up sesame production and productivity research thinking of the biology and host range, insecticide resistance, and developing cultural, selective insecticide control alternatives is urgent. a longer-term solution to insect pest problems in sesame must develop integrated pest management solutions. ethics approval and consent to participate not applicable. human and animal rights no humans or animals were used in this research. availability of data and materials the data that support the findings of this study are available from the corresponding author. funding ethiopian institute of agricultural research conflict of interest the authors declare that they have no conflict of interest. acknowledgments the authors acknowledge the ethiopian institute of agricultural research for providing research review facilities and financial support. orcid and e-mail zemedkun alemu zalemu56@gmail.com https://orcid.org/0000-0002-8764-4823 workishet taye workishet@gmail.com references abadi, b. g. 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(2020). infestation and yield losses due to sesame webworm, antigastra catalaunalis, (duponchel) on different sesame varieties in western tigray, northern ethiopia. journal of agriculture and ecology research international, 21(3), 2 https://doi.org/10.1155/2018/3531495 https://doi.org/10.1155/2018/3531495 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v7i1.1957 received for publication: 03 january 2023 accepted for publication: 24 april 2023 published: 27 april 2023 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease detección del virus de la raya necrótica del fique en semillas de fique (furcraea macrophylla baker) para prevenir la dispersión de la enfermedad de la macana maira gamero1, daniel ortiz2, gloria barrera1* *corresponding author: mgamero@agrosavia.co *https://orcid.org/0000-0003-2069-1868 abstract the fique crop has great potential for the development of the natural fiber market as a beneficial alternative for the protection of the environment. in colombia, one of the main limitations of fiber production in fique plants is the viral disease macana caused by furcraea necrotic streak virus (fnsv). this work aimed to validate the detection of fnsv in asexual planting material from one of the main producing areas and thus contribute to preventing the spread of the disease in the country. the analysis of plants from different geographic altitudes in cauca, colombia, showed a positive correlation with the prevalence of macana disease (being more significant at higher altitudes) but not with the severity of the symptoms. the detection of fnsv on seeds by dot blot immunobinding assay (dbia) using a polyclonal antibody igy showed sensitivity (79 %) and specificity (80 %) when sprouts were analyzed, at the same time, for bulbils, the sensitivity was higher (100 %). moreover, when sprouts were analyzed by the rt-pcr based on fnsv movement protein and polymerase-associated proteins, the sensitivity and specificity were 94 % and 50 %, respectively, in contrast, in the case of bulbils, the specificity was higher (100 %). additionally, the results showed no uniformity in the distribution of the viral particles on vegetal tissue of infected plants, which is necessary to use the largest amount of tissue possible to perform the detection. as part of the optimization of the techniques, it was shown that plant tissue samples could be collected, transported, and stored on filter paper, allowing the detection of the virus 60 days after collection. keywords: macana, viral diagnosis, rt-pcr, dbia, fique, fnsv, natural fiber, validation. resumen el cultivo de fique tiene un gran potencial para el desarrollo del mercado de la fibra natural, como alternativa beneficiosa para la protección del medio ambiente. una de las principales limitaciones en la producción de fibra de fique en colombia es la enfermedad viral macana, causada por furcraea necrotic streak virus. el objetivo de este trabajo fue validar la detección del fnsv en material de siembra asexual procedente de una de las principales zonas productoras, con el fin de contribuir a la prevención de la enfermedad en el país. el análisis de las plantas provenientes de diferentes altitudes geográficas en cauca, colombia, mostró una relación positiva 1 centro de investigación tibaitatá, mosquera, colombia. 2 centro de investigación palmira, popayán, colombia. how to cite this article: gamero, m., ortiz, d., barrera, g. (2023). detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease. peruvian journal of agronomy, 7(1), 69-81 https://doi. org/10.21704/pja.v7i1.1957 detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease january to april 2023 70 con la prevalencia de la enfermedad de la macana (a mayor altitud, mayor prevalencia), pero no hubo relación con la severidad de los síntomas. la detección del fnsv en semillas mediante inmunodetección por dot blot (dbia) usando un anticuerpo polyclonal igy, mostró una sensibilidad de 79% y una especificidad del 80%, en el caso de hijuelos, mientras que, para bulbillos la sensibilidad fue mayor (100%). además, cuando los hijuelos fueron analizados por rt-pcr de dos regiones de genes específicos de proteína de movimiento y proteinas asociadas a la polimerasa del virus, la sensibilidad y especificidad fueron de 94 y 50%, respectivamente, mientras que, en bulbillos la especificidad fue más alta (100%). adicionalmente, los resultados mostraron que no hay uniformidad en la distribución de las partículas virales en el tejido de plantas infectadas, siendo necesario el uso de gran cantidad de tejido para realizar la detección. como parte de la optimización de las técnicas, se demostró que las muestras de tejido vegetal pueden ser recolectadas, transportadas y almacenadas en papel de filtro, lo que permite la detección del virus 60 días después de la recolección. palabras clave: macana, diagnóstico viral, rt-pcr, dbia, fique, fnsv, fibra natural, validación. introduction the global demand for natural fibers (nf) has grown in recent years in response to environmental concerns and the depletion of petroleum resources (safaripour et al., 2021). nf has a long history of serving humanity in a wide range of applications, and its importance was recognized by the united nations and the food and agriculture organization of the united nations, declaring 2009 as the international year of natural fibers. nf are sustainable materials characterized by biodegradability, low cost, and lightness. their resources include lignocellulosiclike seed, bast, leaves, grass, bark, and proteins (kozłowski et al., 2020). the fique (furcraea macrophylla baker), cultivated in the andean region of colombia, produces nf from leaves that are classified as hard fibers (echeverri et al., 2015). the global market of natural fibers has shown significant growth with several commercial and industrial applications, including building materials, interiors of automobiles, medicine, and cosmetics, among others (data bridge market research, 2020). currently, the fique fibers in colombia are used for packaging agricultural products, agromantles, geotextiles, strings, ropes, and handicrafts (ministerio de agricultura y desarrollo rural [madr], 2021). to properly exploit the potential of the fique crop, it is necessary to overcome some limitations, including some associated with phytosanitary problems such as “macana” or necrotic streak disease of fique, which is caused by the positive rna virus furcraea necrotic streak virus fnsv (family: tombusviridae, genus: macanavirus) (adams et al., 2013). the macana disease reduces fiber quality by progressive necrosis on tissue leaves, causing losses of up to 30 % of production (flórez et al., 2013), and in advanced infections can lead to complete loss of the plant, increasing the shortage, which raises approximately 10 000 tons of fiber per year (madr, 2021; ortizgonzález et al., 2021). viral phytopathogens are a significant threat to agricultural production systems worldwide and are difficult to manage due to the evolutionary capacity and adaptation of viruses to climate change, which has affected their geographic distribution and that of their vectors (sarwar et al., 2020). strategies to control viral diseases in plants are based on preventive measures for their dispersion, which include vector control and eradication of infected plants, among others (rubio et al., 2020). the use of virus-free seed is recommended to control the spread of the disease; however, infected plants with fnsv can be asymptomatic, which is necessary to diagnose with laboratory methods (toloza-moreno et al., 2022). for virus detection, the serological techniques are based on the specificity of the antigen-antibody interaction, where the most used methodologies are elisa (enzyme-linked immunosorbent assay), dbia (dot blot immunoassay), and tbia (tissue blot immunoassay) (abd el-aziz, 2019). these techniques have advantages that include sensitivity, simplicity, speed, and low cost, allowing their wide use in phytopathological diagnosis (hsu, 2009; boonham et al., 2014; singh et al., 2021). with the advancement in the knowledge of macana disease and its causal agent, fnsv detection tests have recently been developed for the detection of fnsv in root gamero, m., ortiz, d., barrera, g. peruvian journal of agronomy, 7(1), 6981 (2023) https://doi.org/10.21704/pja.v7i1.1957 71 and leaf extracts of infected plants, including anti-fnsv igy polyclonal antibodies and rtpcr (reverse transcription polymerase chain reaction) (toloza-moreno et al., 2022). in order to validate the usefulness of these diagnostic techniques and prevent the spread of the disease through infected seeds, it is necessary to determine the feasibility of the method on the planting material, optimize the protocols, as well as to establish the diagnostic parameters and their limitations (jacobson, 1998). among the most critical diagnostic validation parameters are specificity, sensitivity, positive and negative predictive values, and prevalence which guarantee the reproducibility of the techniques and the certainty of predicting the presence or absence of the pathogen to be diagnosed (jacobson, 1998). therefore, this work aimed to study the prevalence and severity of the disease in one of the main fique-producing areas of colombia and to validate the methods for the phytosanitary diagnosis of fnsv in asexual fique seed (bulbs and sprouts). it is expected to be able to contribute to the selection of seeds with phytosanitary quality to avoid the spread of the disease. materials and methods disease prevalence and severity evaluation the disease prevalence and severity evaluation were carried out in cauca which is the second higher fique producer department of colombia. for prevalence analysis, 9 stablish fique crops were sampled in totoró and silvia municipalities (between 2 060 and 2 578 meters above sea level (m.a.s.l.), taking 95 adult fique plants (f. macrophylla) by using random and zigzag sampling. the height above sea level and the geographic coordinates were recorded at each sampling site. disease prevalence was determined by the ratio of the number of plants with symptoms and the total number of plants evaluated. a scale was designed to determine the disease severity according to the leaf area affected (table 1). correlation analyses were performed using the linear model y= mx+b with the statgraphics centurion version xvi® program. seed collection for seed collection, fique plants with asexual seeds (bulbils and sprouts) were selected from sampling mentioned above and were used for validation of the diagnostic methods. the samples included 26 plants with symptoms of infection on their leaves, and 19 without symptoms. the tools were disinfected with 5% hypochlorite and 10% alcohol before the collection procedure. the material was labeled and sent to the molecular biology laboratory of tibaitatá investigation center for storage at -80ºc and subsequent processing. table 1. scale for assessing the severity caused by the macana virus in fique leaves. grade 1 2 3 4 5 6 severity 0 % 1 % – 10 % 11 % – 25 % 26 % – 50 % 51 % – 75 % 76 % – 100 % description no visible disease symptoms such as chlorotic or necrotic lesions. chlorotic, necrotic spots or interveinal chlorosis cover up to 10 % of the leaf area. necrotic spots joined together to form necrotic streaks covering 10 % to 25 % of the leaf area. necrotic streaks cover 26 % to 50 % of the leaf area. necrotic streaks cover 51 % to 75 % of the leaf area. necrotic streaks range from 76 % to leaf death. detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease january to april 2023 72 asexual seed preparation for diagnostic each seed was weighed and divided into four segments of equivalent size to estimate the pathogen distribution. each piece was macerated in a mortar by adding liquid nitrogen, and two aliquots were used: one for dbia analysis and another for rna extraction using the commercial rnaqueous® phenol-free total rna isolation kit (thermo fisher scientific), according to the manufacturer’s instructions. rt-pcr we used two pairs of primers previously designed for barrera & belaich (2015): mp-f -5’-atatctacatgcggccttgc -3’ and mp-r-5’gtttgggtttcagcgatgtt-3’, which amplify a fragment of the gene coding for the movement protein (mp) of the fnsv, with an expected size of 492 base pairs (bp) and pol-f 5’agccagctataccacacaacc-3’ and pol-r 5’-taccaccaagcggttagctt, which amplify a segment of polymeraseassociated proteins (pol) with an amplicon of 513 bp. according to the manufacturer’s instructions, retro transcription was performed with the onestep rt-pcr kit (qiagen). the annealing temperature of the primers was 55° c. the products obtained were separated by electrophoresis on 1 % agarose gels in 1x tae buffer and stained with sybrsafe® (invitrogen) for 60 minutes at 80 v. the size of the amplimers was estimated by comparison with a commercial molecular weight marker. dot blot immunobinding assay (dbia) the macerate of each seed was suspended in sterile water. aliquots of 2µl of antigens was spotted onto a nitrocellulose membrane by triplicate and allowed to dry for 5 minutes at 37°c. subsequently, the immunological test was performed according to the protocol by tolozamoreno et al., (2022) with some modifications: decreasing the concentration of the primary antibody to 5 µg/ml, using pbs 1x with 0.05 % tween 20 as washing solution and using a commercial startingblock (pbs) blocking buffer (thermo scientific) with 0.05 % tween 20. the membrane was washed with distilled water and allowed to dry at room temperature. calculations of diagnostic parameters (statistical analysis) to calculate the diagnostic validation parameters, a contingency table was used for the variables: infected plants (symptomatic/asymptomatic) and uninfected plants and results of the diagnostic technique (jacobson, 1998) (table 2). since it has been shown that rt-pcr and dbia techniques can detect asymptomatic infections (barrera & belaich, 2015, barrera et al., 2015), positive plants with both techniques in the absence of symptoms were taken as true positives. the confidence interval (95 % ci) for each parameter was estimated according to the exact or clopper -pearson method (brown et al., 2001). the results were evaluated using the kappa index to determine the concordance between the two techniques, using the formula: p0 is the proportion of observed concordance, and pe is the proportion of expected concordance due to chance (gómez et al., 2007). the index was categorized according to the parameters of landis & koch (1977) where the concordance is very weak if it presents values lower than 0.20, weak if it is between 0.21 and 0.40; moderate between 0.41 and 0.60; good between 0.61 and 0.80 and very good if it is higher than 0.80. printing foliar samples on filter paper cards. the usefulness of filter paper cards was evaluated to optimize protocols and validate an effective transportation costs. prints were made from various parts of a diseased fique leaf on a paper card and allowed to dry at room temperature. then, they were cut and suspended in sterile distilled water. this suspension was used for dbia analysis at days 0, 2, 8, 15, 22 and 40 gamero, m., ortiz, d., barrera, g. peruvian journal of agronomy, 7(1), 6981 (2023) https://doi.org/10.21704/pja.v7i1.1957 73 days after application and viral rna extraction and rt-pcr at days 0, 2, 8, 16 and 60 days after application. results and discussion disease prevalence and severity evaluation from total analyzed plants, 51 showed typical symptoms of the macana virus, characterized by necrotic lines on the upper and lower sides of the leaf because of cell death in the vascular bundles (morales et al., 1992). the prevalence of the disease ranged from 5 % to 41 %, and according to regression analysis, showed a positive linear correlation with altitude, r2=0.8164 (figure 1a), indicating that sites at higher altitudes favor the presence of the disease. this could be related with the low temperatures presented at higher altitudes, which could generate a greater interaction between the vector and the virus promoting the spread of the pathogen. additionally, the plant´s defense mechanisms could be affected by environmental conditions being more susceptible to the infection. these results are consistent with those reported by aguilar et al. (2015), who found that the prevalence of potato virus x is reduced in conditions associated with high temperatures. considering that the temperatures in sampling sites ranged between 8 °c – 18 °c this could suggest that the fnsv is expressed in a wide temperature range. in the context of climate change, the temperature increase may likely affect the prevalence, persistence, and distribution of plant viral infections (rodriguez, 2012; hull, 2014) including the macana disease. on the other hand, disease severity showed a low correlation with altitude (r2=0.0652), and values ranged from 9 % to 55 % from 2000 to 2600 m.a.s.l. (figure 1b). these results differ from that of kubota & ng (2016), who found higher severity of a viral disease at a higher temperature. detection of fnsv in asexual seed the infection was detected in sprouts and bulbils. the macana is a systemic disease which distribution depend on viral capacity to move long-distance through the phloem (kappagantu et al., 2020), suggesting more successful infection in closer tissue to the pathogen entrance in the roots. in this case, the bulbils are born in the inflorescence of the plant, found in the upper part of it, while the sprouts are born at the base of the plant very close to the ground, being more possible found virus presence on it. viral detection in plants with macana disease using both techniques dbia and rt-pcr and using fragments of whole seed (4/4) showed that the virus is not homogeneously distributed since only 28 % of the seeds were positive in the four segments analyzed by rt-pcr (figure 2a) and 55 % were positive in the four fragments by dbia (figure 2b). in any case, a sample was considered positive if it had at least one of the 4 positive segments with both techniques. due to the differences in the distribution of the pathogen in the seeds, it is necessary to process the whole plant material to increase the probability of virus detection. y = 0,0552x 104,52 r² = 0,8164 0 10 20 30 40 50 2000 2200 2400 2600 pr ev al en ce o f m ac an a (% ) altitude (m.a.s.l) y = 0,0185x 15,376 r² = 0,0652 0 10 20 30 40 50 60 2000 2200 2400 2600 se ve ri ty o f m ac an a (% ) altitude (m.a.s.l) a b figure 1. correlation between altitude and macana disease. (a) prevalence (b) severity detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease january to april 2023 74 figure 2. positive segments (s) for viral detection by rt-pcr (a) and dbia (b). the numbers indicate the segments of asexual seed analyzed, since it was divided into 4 parts. validation of diagnostic techniques of the virus in asexual seeds suitable material for validation purposes of diagnostic techniques for plant pathogens should include one of the following matrices: purified virus, plant material naturally or artificially infected with the microorganism, or plant material enriched with the microorganism (van der vlught et al. 2007), so for this work, vegetative seeds from plants with visible symptoms of necrotic streak disease and purified viruses were used as positive controls. also, since there is no gold standard for diagnosing macana and both techniques have previously been shown to be capable of detecting asymptomatic infections (toloza-moreno et al., 2022), positive plants with both techniques were taken as true positives (tp) as infected plants, and negative plants with both methodologies were taken as true negatives (tn) as uninfected plants. the detection of viral presence in bulbils showed that dbia technique was positive in 16 bulbils which 10 were tp, and 5 samples were tn, corresponding to uninfected plants (table 3, appendix s1). on the other hand, with rt-pcr, 10 bulbils analyzed were true positive amplifying a fragment of mp, all corresponding to infected plants. the negative samples (11) presented only 5 true negatives (table 3, appendix s2). in the case of sprouts, 16 were positive with dbia, 15 infected and one uninfected, while the test was negative in 8 plants, 4 infected and 4 uninfected (table 3). the rt-pcr technique detected the virus in 16 plants, 15 of which were infected, while the test was negative in 5 plants, 1 infected and 4 non-infected (table 3). both tests detected the virus in 9 asymptomatic plants, confirming that the phytopathological diagnosis of the macana virus should not be based solely on the presence of symptoms since these are not observed with the naked eye in the early stages of the disease. they can also be confused with symptoms associated with other diseases or abiotic stress (jeong et al., 2014). similarly, other factors influence the appearance of symptoms or not, such as genotypic variability and intrinsic components of the virus, environmental conditions such as light intensity, temperature, and altitude, and nutritional conditions of the plant (van der want & dijkstra, 2006). on the other hand, 11.5 % of the plants with symptomatology were negative with both methodologies, which can be attributed to an error in symptom identification, problems that could be related to the tissue type analyzed, or the technique used. there are several critical factors inherent to the plant tissue that can alter the specificity of an assay; these can be enzyme inhibitors, degrading agents, factors that cause nonspecific binding, and substances previously applied to plants, such as pesticides and growth regulators, among others (cardwell et al., 2018). in the case of rt-pcr, this can be affected by the presence of rnases, polysaccharides, tannins, or plant polyphenols released during cell lysis which decreases the quality and yield of nucleic acid extraction. also, some reagents used for rna extraction can inhibit enzymatic reactions carried out in retro transcription (lacroix et al., 2016). while with dbia, crossreactions with proteins from other pathogens may occur (seida, 2017), generating false positives, so it is necessary to test with opportunistic microorganisms or environmental contaminants gamero, m., ortiz, d., barrera, g. peruvian journal of agronomy, 7(1), 6981 (2023) https://doi.org/10.21704/pja.v7i1.1957 75 table 2. diagnostic test parameters for a detection technique and contingency table for their assessment. test result infected plants healthy plants total symptomatics asymptomatics positive tp tp fp tp+tp+fp negative fn fn tn fn+fn+tn total tp+fn fp+tn n notes: sensitivity (se): tp/ (tp + fn), specificity (sp): tn/ (tn + fp), positive predictive value (ppv): tp/ (tp + fp), false predictive value (fpv): tn/ (tn + fn), prevalence (p): (tp+fn) /n. tp: true positives; fp: false positives; fn: false negatives; tn: true negatives; n: sample size. table 3. contingency tables for bulbils (a and b) and sprouts (c and d) with each diagnostic technique performed. below are the diagnostic parameters obtained from each table. bulbils sprouts (a) infected plants uninfected plants (c) infected plants uninfected plants dbia + total dbia + total + 10 6 16 + 15 1 16 0 5 5 4 4 8 total 10 11 21 total 19 5 24 (b) infected plants uninfected plants (d) infected plants uninfected plants rt-pcr+ total rt-pcr+ total + 10 0 10 + 15 4 19 6 5 11 1 4 5 total 16 5 21 total 16 8 24 diagnostics parameters (a) (95 % ci) (b) (95 % ci) (c) (95 % ci) (d) (95 % ci) sensitivity 100 % (65.5-99.1) 62.5 % (35.8-83.7) 79 % (53,9-93,0) 94% (67.7-99.6) specificity 45 % (18.1-75.4) 100 % (46.2-98.1) 80 % (29.8-98.9) 50% (17.4-82.5) positive predictive value 63 % (35.9-83.7) 100 % (65.5-99.1) 93.75 % (67.7-99.6) 79% (53.9-93.0) negative predictive value 100 % (46.2-98.1) 45.4 % (18.4-75.4) 50 % (17.4-82.5) 80% (29.8-98.9) prevalence 48 % (26.4-69.7) 76.2 % (52.4-91) 79.2 % (57.2-92) 67% (44.6-83.5) note. 95% ci: 95% confidence interval closely related to the pathogen to be detected (cardwell, et al., 2018). all these factors may explain the discrepancy obtained in the results (25.5 %), which is also confirmed by the kappa index that shows a moderate agreement between both methodologies: 0.44 in the case of bulbils and 0.48 in sprouts. the estimated diagnostic indicators for sensitivity, specificity, positive predictive value, negative predictive value, and prevalence are shown in table 2. the most sensitive technique for bulbils was dbia, with a sensitivity of 100 % compared to rt-pcr, which obtained 63 %. in contrast, the most specific technique was rtpcr, with 100 %, whereas dbia obtained 45 %. for dbia, the ppv and npv values were 63 % and 100 %, respectively, while for rt-pcr, the results were 100 % and 45 %, respectively. in the case of sprouts, rt-pcr showed the highest diagnostic sensitivity with 94 % compared to dbia (79 %). the most specific test was dbia (80 %), while rt-pcr showed 50 %. for dbia, the ppv and npv values were 94 % and 50 %, while, with rt-pcr, these parameters obtained similar results, 79 % and 80 %, respectively. detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease january to april 2023 76 the primary method of multiplication of fique is through asexual seeds, with bulbils being the most abundant planting material since one plant can produce up to 3000 seeds, which are used for planting in new areas. in contrast, sprouts are mainly used for replanting established crops, and one plant can produce between 30 and 35 seeds, which is insufficient for planting large areas of crops (cadena de producción del fique [cadefique], 2006). bearing in mind that bulbils are the most used seeds for sowing new crops, the rt-pcr technique is recommended for viral detection in this type of propagule since it has a sensitivity of 62.5 % and a specificity of 100 %, which guarantees that a healthy plant is correctly identified, since it will not show false positives, thus allowing the selection of seeds with high phytosanitary quality for sowing. likewise, this technique has a ppv of 100 % and a high associated prevalence (76.2 %), which ensures the conditional probability that plants with a positive test have the disease. in this sense, specificity and sensitivity allow measuring the validity of a diagnostic test and do not vary with prevalence, i.e., they are inherent characteristics of the test, whereas predictive values indicate the probability that the test provides a correct diagnosis and depend on the prevalence of the disease in the population where the test is applied (pallás & villa, 2019). therefore, if the prevalence is high, a positive result tends to confirm the presence of the disease, whereas, if the prevalence is low, a positive result will not allow affirming its existence. in the case of sprouts, although the rt-pcr technique has a high sensitivity (94 %), its specificity is low (50 %); therefore, it has a high probability of detecting a high number of false positives, which would lead to discard many healthy seed and increase the number of plants to be tested, resulting in high costs and effort for the producer. for this reason, the dbia technique is more appropriate because it presents high percentages of both sensitivity and specificity (79 % and 80 %, respectively), which translates into a high probability of detecting diseased and healthy plants, in addition to being a more economical technique compared to the molecular technique. similarly, the observed prevalence was 79.2 %, increasing the positive test’s predictive capacity to 94 %. printing of foliar samples on filter paper cards tests were conducted to determine the usefulness of printing diseased leaves on chemically treated filter paper to facilitate the shipment of samples from the field to the laboratory, avoid the use of expensive refrigeration equipment, and ensure the viability of the virus on the matrix. viral detection in diseased foliar prints on filter paper cards was maintained until day 60 post application at room temperature using rt-pcr (appendix s3). in the case of dbia, the results were positive up to 40 days after the prints on the card of a diseased leaf sample from cauca (appendix s4). the cards are composed of chaotropic agents and other substances that lyse the cells and immobilize the genetic material inhibiting its degradation and preventing the proliferation of contaminating bacteria, which allows the collection and storage of different types of biological matrices for long periods at room temperature and constitute an effective way of sending samples to the laboratory for diagnosis (burgoyne, 1996; cardona-ospina et al., 2019). the cards have been employed on a wide range of viruses transported at room temperature and characterized by molecular methods in the laboratory, ranging from several days to several weeks (thorne & mcelhinney, 2017), which is confirmed in this work. conclusions the prevalence of the macana disease in fique plants in the department of cauca is related to the altitude, suggesting that environmental conditions associated with higher altitude favor the disease. in contrast, the severity of the disease was not dependent on altitude, which indicates that the virus has a wide adaptation to the climate and can severely affect crops from 2000 m.a.s.l to 2600 m.a.s.l. because some infected plants may not show symptoms, and the virus distribution is not homogeneous in whole tissue plant, it is gamero, m., ortiz, d., barrera, g. peruvian journal of agronomy, 7(1), 6981 (2023) https://doi.org/10.21704/pja.v7i1.1957 77 necessary to send asexual seeds from healthy and diseased plants to the laboratory and process them thoroughly to ensure the detection of the pathogen. the diagnostic techniques analyzed in this work are beneficial as a preventive measure since there is currently no effective diagnostic method to prevent viral spread from planting material. the rt-pcr technique is recommended for bulbils and dbia for sprouts, and the suspected plant material can be printed on filter paper, which maintains the viability of the virus for long periods until its detection in the laboratory. aknowledgements we are grateful to the corporación colombiana de investigación agropecuaria (agrosavia) for providing physical and human resources. this work was supported by grants from the colombian ministry of agriculture and rural development (madr), through the administrative code number tv18. author contributions mg: laboratory sample processing, execution experimental design, review of statistical analysis of results, revision of the manuscript, discussion of results. do: field work carried out, execution experimental design, review of statistical analysis of results, discussion of results, manuscript review. gb: conceptualization of the work, experimental design, statistical analysis of results, discussion of results, support, and supervision of the study. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. id orcid and e-mails gamero, m. mgamero@agrosavia.co https://orcid.org/0000-0003-2069-1868 ortiz, d. dfortiz@agrosavia.co https://orcid.org/0000-0001-6743-9374 barrera, g. gbarrera@agrosavia.co https://orcid.org/0000-0001-82454388 references abd el-aziz, m. 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(2007). explanatory document for the validation of detection methods for plant pests. plant research international (b.v. report 135en). https:// l i b r a r y. w u r. n l / we b q u e r y / w u r p u b s / fulltext/301443 https://sioc.minagricultura.gov.co/fique/documentos/2021-03-31%20cifras%20sectoriales.pdf https://sioc.minagricultura.gov.co/fique/documentos/2021-03-31%20cifras%20sectoriales.pdf https://sioc.minagricultura.gov.co/fique/documentos/2021-03-31%20cifras%20sectoriales.pdf https://sioc.minagricultura.gov.co/fique/documentos/2021-03-31%20cifras%20sectoriales.pdf https://doi.org/10.1111/j.1439-0434.1992.tb01233.x https://doi.org/10.1111/j.1439-0434.1992.tb01233.x https://doi.org/10.1111/j.1439-0434.1992.tb01233.x http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=s0253-57852021000100005&lng=es&tlng= http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=s0253-57852021000100005&lng=es&tlng= http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=s0253-57852021000100005&lng=es&tlng= https://doi.org/10.3389/fpls.2020.01092 https://doi.org/10.3389/fpls.2020.01092 https://doi.org/10.1016/b978-0-12-818654-1.00050-5 https://doi.org/10.1016/b978-0-12-818654-1.00050-5 https://library.wur.nl/webquery/wurpubs/fulltext/301443 https://library.wur.nl/webquery/wurpubs/fulltext/301443 https://library.wur.nl/webquery/wurpubs/fulltext/301443 detection of furcraea necrotic streak virus (fnsv) in fique seed (furcraea macrophylla baker) to prevent the spread of the macana disease january to april 2023 80 appendix appendix s1. dbia using anti-fnsv igy against macana virus. positive (a) and negative (b) results of the dbia test on 5 sprouts (s) and 5 bulbils (b). each row corresponds to a fragment analyzed from the same sample. c+: positive control (purified “macana” virus); c-: negative control (water). appendix s2. detection of fnsv by rt-pcr of a region of the movement protein (mp) gene in symptomatic plants. s: sprout; b: bulbil; f: fraction; c+: positive control; c-: negative control. molecular weight marker: 1 kb (invitrogen™). gamero, m., ortiz, d., barrera, g. peruvian journal of agronomy, 7(1), 6981 (2023) https://doi.org/10.21704/pja.v7i1.1957 81 appendix s3. detection of fnsv in diseased foliar prints on filter paper cards by amplifying of a region of the polymerase-associated proteins gene (pol). times (t) 0, 2, 4, 8, 16, and 60 days. r1, r2, and r3: replicates of the same sample. c+: positive control (purified viral); .c-: negative control (ultrapure water). molecular weight marker: 1 kb (invitrogen™). appendix s4. dbia for the detecting of fnsv in diseased leaves prints on filter paper cards at times (t) 0, 2, 8,15,22, and 40 days post-application. rows correspond to replicates. c+: positive control (purified viral) c-: negative control (water). peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v7i1.2001 received for publication: 06 november 2022 accepted for publication: 20 april 2023 published: 22 april 2023 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by grasshopper (zonocerus variegatus l) infestation and root dry matter content of cassava as influenced by planting date and cassava genotypes infestación de saltamontes (zonocerus variegatus l) y contenido de materia seca en raíces de yuca influenciados por la fecha de siembra y los genotipos de yuca saffea joseph torto1, alusaine edward samura1, abu james sundufu1, david dan quee1, dennis peter musa2, sheku alfred kanu3, sahr ngoba fomba1, prince emmanuel norman4 *corresponding author: norman.prince64@gmail.com *https://orcid.org/0000-0002-0150-8610 abstract dearth of information exists on the effects of planting dates and genotypes on grasshopper (zonocerus variegatus l.) infestation and dry matter accumulation in the root organ of cassava. an experiment was conducted at the school of agriculture and food sciences experimental site of njala university during 2007/2008 and 2015/2016 cropping seasons. this study assessed the effects of planting date and cassava genotypes on grasshopper infestation and root dry matter accumulation in cassava. the experiment utilized four cassava genotypes (cocoa, slicass 1, slicass 4 and slicass 6) and three planting dates (june, september and december) laid out in a randomized complete block design (rcbd) with three replications. findings showed that the incidence and severity of grasshopper attacks, as well as root dry matter contents were significantly (p<0.05) different between variety and planting dates. the grasshopper severity scores increased from 3 months after planting (map) (1.6) to 9 map (2.6). similarly, the incidence increased from 3 map (23.0 %) to 9 map (29.7 %). the lowest percent incidence of grasshoppers was observed in june (24.8%), followed by september (26.0 %), whilst december recorded the highest (30.7 %). similar trends were exhibited for severity score for june (1.5), september (2.2) and december (2.5) planting dates. genotype slicass 6 (42.21 %) had the highest root dry matter content, followed by slicass 1 (42.17 %), while cocoa and slicass 4 had the lowest of 41.90 %. the protected plots recorded higher root dry matter content (44.0 %) than the unprotected plots (40.9 %). identification of tolerant cassava variety, protection of cassava field from grasshopper infestation and harvesting cassava in december could be exploited for selection and production of tolerant cassava variety with desired processing traits such as high root dry matter content. key word: grasshoppers, planting dates, management, root dry matter, cassava 1 department of crop protection, school of agriculture and food sciences, njala university, njala campus, sierra leone. 2 department of horticulture, school of natural resources management, njala university, njala campus, sierra leone. 3 department of crop science, school of agriculture and food sciences, njala university, njala campus, sierra leone. 4 sierra leone agricultural research institute (slari), sierra leone. how to cite this article: torto, s., samura, a., sundufu, a., quee, d., musa, d., kanu, s., fomba, s., norman, p. (2023). grasshopper (zonocerus variegatus l) infestation and root dry matter content of cassava as influenced by planting date and cassava genotypes. peruvian journal of agronomy, 7(1), 42-50. https://doi.org/10.21704/pja.v7i1.2001 mailto:norman.prince64@gmail.com https://orcid.org/0000-0002-0150-8610 torto, s., samura, a., sundufu, a., quee, d., musa, d., kanu, s., fomba, s., norman, p. peruvian journal of agronomy, 7(1), 42-50 (2023) https://doi.org/10.21704/pja.v7i1.2001 43 resumen existe poca información sobre los efectos de las fechas de siembra y los genotipos sobre la infestación de saltamontes (zonocerus variegatus l.) y la acumulación de materia seca en el órgano radicular de la yuca. se realizó un experimento en el sitio experimental de la escuela de agricultura y ciencias de la alimentación, de la universidad de njala durante las temporadas de cultivo 2007/2008 y 2015/2016. este estudio evaluó los efectos de la fecha de siembra y los genotipos de yuca sobre la infestación de saltamontes y la acumulación de materia seca en la raíz de la yuca. el experimento utilizó cuatro genotipos de mandioca (cacao, slicass 1, slicass 4 y slicass 6) y tres fechas de siembra (junio, septiembre y diciembre) dispuestos en un diseño de bloques completos aleatorizados (rcbd) con tres repeticiones. los resultados mostraron que la incidencia y la gravedad de los ataques de saltamontes, así como el contenido de materia seca radicular, fueron significativamente diferentes (p<0.05) entre la variedad y las fechas de plantación. las puntuaciones de severidad del saltamontes aumentaron 3 meses después de la plantación (map) (1.6) a 9 map (2.6). del mismo modo, la incidencia aumentó de 3 map (23 %) a 9 map (29.7 %). el porcentaje más bajo de incidencia de saltamontes se observó en junio (24.8 %), seguido de septiembre (26 %), mientras que diciembre registró el más alto (30.7 %). se observaron tendencias similares para la puntuación de severidad en las fechas de plantación de junio (1.5), septiembre (2.2) y diciembre (2.5). el genotipo slicass 6 (42.21 %) tuvo el mayor contenido de materia seca radicular, seguido de slicass 1 (42.17 %), mientras que cacao y slicass 4 tuvieron el más bajo de 41.90 %. las parcelas protegidas registraron un mayor contenido de materia seca radicular (44 %) que las parcelas no protegidas (40.9 %). la identificación de la variedad de yuca tolerante, la protección del campo de yuca contra la infestación de saltamontes y la cosecha de yuca en diciembre podrían aprovecharse para la selección y producción de la variedad de yuca tolerante con rasgos de procesamiento deseados, tales como un alto contenido de materia seca de la raíz. palabras clave: saltamontes, fechas de plantación, manejo, materia seca radicular, yuca introduction cassava (manihot esculenta crantz) is an important staple food security crop consumed by over 800 million individuals of the global populace (esuma et al., 2019). the crop is utilized as food, feed and different industrial applications that support many livelihoods globally. in sierra leone, cassava is ranked as the second staple food after rice. the fresh storage roots contain mainly starch and other food nutrients such as calcium (0.16 g/kg), phosphorus (0.27 g/kg), vitamin c (0.206 g/kg), and minute quantities of protein and other nutrients (united states department of agriculture [usda], 2016). moreover, the leaves of cassava are consumed as vegetables since they contain protein such as lysine, but lack the amino acid methionine and possibly tryptophan (nweke et al., 2002; chipeta et al., 2013). other cassava products utilized in the country are cassava pellets for animal feed, cassava starch for sweeteners, thickeners and textile paper industry (chipeta et al., 2013). despite the enormous importance of the crop, a number of biotic and abiotic factors (kintché et al., 2017) limits increased cassava productivity. among biotic factors, some of the key insect pests that affect the economic yield of the crop include variegated grasshopper (zonocerus variegatus l.), cassava green mite, and cassava mealy bug. the variegated grasshopper belongs to the same order (orthoptera) as locusts (britton, 2020). this insect defoliates and destroys the stem bark of food crops at the end of the dry season. the feeding behaviour of this pest also results in the reduction in cassava tuber yield and quality and the destruction of cassava cuttings (brima et al., 2000). although often viewed as a polyphagous pest, grasshoppers are selective to some degree, exhibiting definite plant preferences (mansaray et al., 2012). studies carried out by song (2010) have shown that grasshoppers could be conveniently classified as grass-feeders (graminivorous), forb–feeders (forbivorous) or a mixture of the two (ambivorous or mixed feeders). also, picaud et al. (2003) have grasshopper (zonocerus variegatus l) infestation and root dry matter content of cassava as influenced by planting date and cassava genotypes january to april 2023 44 attributed the chemical composition of plants as the main factor in the food selection behaviour of grasshoppers. the pest defoliates and destroys the stem bark of food crops at the end of the dry season (mansaray et al., 2012). moreover, the feeding behaviour of the pest reduces the storage root quality and destroys the cassava stem cutting planting materials (mansaray et al., 2012). the cassava green mite infestation was reported to cause about 15 % and 73 % storage root yield losses in resistant and susceptible genotypes of cassava, respectively (bellotti, 2002). cassava mealy bug infestation causes about 88 % root yield loss in susceptible genotypes of cassava (bellotti et al., 1987). low storage root yields of cassava are opined to be caused by low yielding genotypes, environmental variability and poor environmental management or utilization of elite agronomic packages (karim et al., 2020). research has shown that early nymphs of grasshoppers are deterred from feeding on cassava due to the presence of cyanogenic glucosides (brima et al., 2000). the concentration of this plant secondary compound varies among cassava clones ranging from 80 mg to 167 mg cn per 100 g of fresh leaf (isley, 1944). control of the variegated grasshopper has generally involved knock off chemical pesticides. however, because of the increasing concern of its effect on non-target organisms, prohibitive cost and persistence in the environment, there is the need for environmentally friendly alternative. in this regard, host plant resistance is strongly advocated for the control of pests and diseases than the continual utilization of pesticides due to its adverse environmental effects on the ecosystem and unsustainability for low-income small-scale farmers (bellotti, 2002). host plant resistance can be described as any reduction in the population growth of a target pest as influenced by inheritable characteristic of the host plant compared to a standard variety (bellotti & arias, 2001). host plant resistance is as a result of the presence of secondary compounds found in plants; the nature and concentration of these compounds varies in time and space and also among and within plant varieties (erb & kliebenstein, 2020). end-use quality traits significantly affect the acceptance of cassava genotypes by producers, processors and consumers (fomba et al., 2012). in effect, the success of newly developed cassava genotypes depends not only on their agronomic attributes but also on their acceptability by targeted end-users. cassava genotypes with good food qualities possess high dry matter as one of the key traits. the research questions of the present study included the following: (i) is the effect of planting date on grasshopper infestation and root dry matter content in cassava significant? and (ii) is the effect of cassava genotypes on grasshopper infestation and root dry matter content in cassava significant? the aim of the present study was to assess the effects of planting date and cassava genotypes on grasshopper infestation and root dry matter accumulation in cassava. the results of the study will contribute to solving in-field grasshopper infestation through identification of appropriate planting date, selection and production of tolerant cassava variety with desired processing traits such as high root dry matter content. materials and methods description of field trials the trial was carried out at the school of agriculture farm site, njala university, njala, kori chiefdom, moyamba district, southernsierra leone. njala is located at an elevation of approximately 54 m above sea level on latitudes 8 °06ʹ n and on longitude 12 °06ʹ w. the trial site has vegetation that is mostly of elephant grass (andropogon sp.) and spear grass (imperata cylindrica). the experiment was conducted both in rainy and dry seasons. during the dry season, overhead irrigation was done. there are two distinct seasons, the wet season (may-october) and the dry season (november –april). the mean annual rainfall at njala is 2 525 mm with a mean monthly maximum air temperature ranging from 29 °c to 33 °c. relative humidity is very high often close to 100 % for the greater part of the day and at night especially during the rainy season. during the dry season potential evapotranspiration exceeds rainfall, while during the rainy season precipitation exceeds evapotranspiration. the predominant vegetation at njala is secondary bush and the soil belongs to the njala series (orthoxic palihumult). textures torto, s., samura, a., sundufu, a., quee, d., musa, d., kanu, s., fomba, s., norman, p. peruvian journal of agronomy, 7(1), 42-50 (2023) https://doi.org/10.21704/pja.v7i1.2001 45 are usually gravelly clay loam in the surface and gravelly clay loam to gravelly clay in the subsoil. the soils are low in soil moisture and have a very low nutrient status and are slightly acidic with ph ranging from 5.5 to 6.0. experimental materials and design the trial was laid out in a 3 × 4 factorial arrangement in a randomized complete block design (rcbd) with 3 replications. the two factors included three levels of planting date: june, september and december and four genotypes: cocoa cassava (local check variety), sierra leone improved cassava (slicass) 1, slicass 4 and slicass 6. the origins, potential yields and disease tolerance attributes of the four genotypes are presented in table 1. stem cuttings of each genotype measuring 30 cm in length each were planted on the crest of ridges at 1 m × 1 m spatial arrangement giving a plant population of 10 000 plants ha–1. a total of 40 cuttings were planted per plot. the treatments were established in protected and unprotected experimental blocks. block a (protected plots) was located in an area that was less prone to grasshoppers, whereas block b (unprotected plots) was located closer to the source of grasshopper infestation. the distance between the two blocks was 200 m and the sites were separated by a small stream. the protected block was sprayed periodically with chlorpyrifos to prevent grasshoppers’ infestation. hand picking was also done throughout the growing season to remove any hoppers that entered the protected plots. no fertilizer was applied in both the unprotected and protected plots or sites. hand weeding was done when necessary. data collection and analysis evaluation of the genotypes for susceptibility to grasshopper in the field was based on the injury done to each genotype by the pest. severity was expressed as the total area of the cassava plant tissue affected over the total area of the plant tissue. grasshopper severity was estimated using the visual rating scale of 1 to 5 per individual plant; where: 1 = 0 % to 20% of foliage damaged, 2 = 21 % to 40% of foliage damaged, 3 = 41 % to 60% of foliage damaged, 4 = 61 % to 80% of foliage damaged and 5 = 81 % to 100% of foliage damaged following the procedure by capinera (1993). pest assessments were done at 3, 6, 9 and 12 months after planting (map) which coincided with the rainy season, dry season and dry-rainy season transition periods. percentage incidence was expressed, as the number of infected cassava plants over the total number of cassava stands planted expressed as percentage. data on plant damage was collected on the two middle rows per plot, which at maturity was used for yield estimation. root dry matter content (rdmc) determination was done at harvest (12 map) by selecting three representative roots from the bulk of roots harvested from 5 plants. cassava roots were washed and shredded into pieces. a standard measure of 100 g weight of the fresh samples was taken and oven dried with forced drought oven. samples were reweighed again to obtain a constant weight after 72 h at 65 °c –70 °c (fukuda et al., 2010). all data collected were pooled and subjected to factorial anova and means were separated using the student newman keuls test (snk) at 0.05 level of significance. results and discussion percent incidence analysis of the data using anova shows significant differences (p<0.05) in the percent incidences of grasshoppers with respect to the planting time. the percent incidence of table 1. origin, status and attributes of manihot esculenta genotypes used in the study genotype origin status attributes cocoa sierra leone landrace susceptible to cassava mosaic disease (cmd), low root yielding (7 t.ha-1) slicass 1 nigeria improved resistant to cmd, medium yielding (15 t.ha-1-20 t.ha-1) slicass 4 nigeria improved resistant to cmd, high yielding (20 t.ha-1-30 t.ha-1) slicass 6 nigeria improved resistant to cmd, high yielding (20 t.ha-1-30 t.ha-1) grasshopper (zonocerus variegatus l) infestation and root dry matter content of cassava as influenced by planting date and cassava genotypes january to april 2023 46 grasshoppers was observed to be lowest in june (24.8 %), followed by september (26.0 %), whilst december recorded the highest (30.7 %) across the four cassava varieties (table 2). these findings show that grasshopper attack increases with time from the rainy season to the dry season. the lowest incidence of the pest observed on the field in june could probably be attributed to the heavy rains that serves as a mortality factor of grasshoppers. as the intensity of rainfall decreased between september and december, the mean incidence of grasshoppers significantly increased. this observation concurs with mansaray et al. (2012) who reported that grasshoppers cannot withstand heavy rains, as rain is one of the natural factors responsible for the high mortality rate of the pest in the field. with regards the time of observation, mean incidence was significantly different (p=0.05) during the observation periods. the highest grasshopper incidence was observed at 9 map (29.7 %), closely followed by 6 map (23.9 %), 3 map (23.0 %) and the least was recorded by 12 map (18.6 %), respectively across the four cassava varieties (table 3). the higher incidence at 6 map and 9 map could probably be related to the life cycle of the grasshoppers. this observation is in agreement with the findings of brima et al. (2000) who reported that grasshoppers damage is more common in older than younger cassava plants, and is more severe in the dry season than in the wet season. mansaray et al. (2012) reported similar findings on grasshopper incidence in cassava fields at njala agricultural research centre (narc) experimental site, njala, southern sierra leone. severity score significant (p<0.05) differences were also recorded for grasshopper severity scores with respect to planting time. the highest score was observed in december (2.5), followed by september (2.2) and the least severity score in june (1.5) (table 4). the increased severity score for december and september planting time compared to june could probably be due to the stage of plant growth at the time of infestation by grasshoppers. international institute of tropical agriculture (iita, 2000) reported that young cassava plants suffer more from pest attack than older plants. at 3 4 months after planting, the roots of the cassava plants start to heave as a result of photosynthate translocation from the leaves to storage root organ development. at 7 months after planting, the total number of storage roots could have been formed. beyond this time, the storage root number does not increase, but the storage roots continue to grow in size until they are harvested. therefore, cassava farms attacked by pests at lesser than or 7 months after planting, incur more yield losses than if older plants are attacked. table 2. mean percentage incidence of grasshoppers with respect to planting time time of planting incidence score (%) f p june 24.8 c 5.0 0.014 september 26.0 b december 30.8 a means in column with the same letter are not significantly different at p< 0.05 (snk) table 3. mean percent incidence of grasshoppers with respect to observation time sampling regime (map) incidence score (%) f p 3 23.0 c 518.84 0.0001 6 23.9 b 9 29.7 a 12 18.6 d means in column with the same letter are not significantly different at p< 0.05 (snk) torto, s., samura, a., sundufu, a., quee, d., musa, d., kanu, s., fomba, s., norman, p. peruvian journal of agronomy, 7(1), 42-50 (2023) https://doi.org/10.21704/pja.v7i1.2001 47 with respect to observation time, significant (p < 0.05) differences were also recorded in severity scores across the four cassava varieties. the highest severity score was recorded at 9 map (2.6), followed by 6 map (2.5), 3 map (1.6), while the lowest severity score was recorded at 12 map (1.5) (table 5). the reason for the slightly high severity at 6 map and 9 map could be related to the fact that at this stage of development of the plant; the grasshoppers would have developed to the late instars or adult stage which causes most destruction on cassava plants in the field compared to when the cassava plant is young (bernays et al., 1975). at the younger stage lesser than 4 months after planting, the cassava plants possess higher hcn compound that deters the young instars (1 3) from feeding on the crop, rather they will prefer to die. the above observation is in accordance with brima et al. (2000) who reported that the older nymphs and adults cause more damage to cassava than the young nymphs. moreover, the authors opined that cassava plants that are 3 4 months or lesser than 7 months old are not fed on by nymphs of grasshopper. the low severity impact reported at 3 map could be due to the same observation. these findings also support the view that the first and second instars of grasshoppers prefer to die rather than feed on cassava (bernays et al., 1977). in addition, most of the early nymphs were found on green vegetables known as alternate hosts within or outside the cassava field. the root dry matter contents with respect to variety and planting dates were significantly (p<0.05) different (tables 6 and 7). the highest root dry matter content was recorded in slicass 6 (42.21 %), followed by slicass 1 (42.17 %), while cocoa (41.9 0%) and slicass 4 (41.90 %) had the least root dry matter content (table 6). the slight differences in root dry matter content among the varieties could probably be due to their inherent genetic variation. the highest root dry matter content was recorded in december (43.7 %), followed by september (40.1 %) and the least of 39.8 % was recorded in june (table 7). the variation in dry matter accumulation in the cassava root organ could be attributable to the dry matter reversion in the cassava physiology caused by seasonal variation. the results indicate that harvesting of cassava roots in december or in the dry season exhibits the highest dry matter accumulation useful for processing of the crop compared to harvesting done in the wet season period such as june and september. findings are consistent with sagrilo et al. (2008) who assessed the performance of cassava cultivars in the northwest paraná at different harvest times and concluded that, for those conditions, the physiological rest period at the end of the second crop cycle was more indicated for harvest, due to the higher production of the fresh storage root dry matter content and starch. the root dry matter content significantly (p<0.05) differed with management (table 8). the protected plots recorded higher mean root dry matter content of 44.0 %, while the table 4. mean severity score of grasshoppers with respect to planting time time of planting severity score f p june 1.5 c 18.95 0.0001 september 2.2 b december 2.5 a means in column with the same letter are not significantly different at p< 0.05 (snk) table 5. mean severity score of grasshoppers with respect to sampling time sampling time (map) severity score f p 3 6 9 12 1.6 c 2.5 b 2.6 a 1.5 d 21.37 0.0001 means in column with the same letter are not significantly different at p< 0.05 (snk); severity score: 1= 0 %-20 % of foliage consumed, 2= 21 % 40 % of foliage consumed, 3= 41 %-60 % of foliage consumed, 4= 61 %-80 % of foliage consumed and 5= 81 % 100 % of foliage consumed (capinera, 1993). grasshopper (zonocerus variegatus l) infestation and root dry matter content of cassava as influenced by planting date and cassava genotypes january to april 2023 48 unprotected plots had mean root dry matter of 40.9 %. the difference in mean root dry matter contents between the protected and the unprotected management strategies could probably be due to high degree of feeding of grasshoppers on the cassava varieties on the unprotected plots compared to the protected plots. this is in agreement with the findings of mansaray et al. (2012) who opined that cassava plants in unprotected plots are more vulnerable to grasshoppers than those in protected plots and thus account for dry matter differences. conclusion this study demonstrates that increasing root dry matter content and reducing grasshopper infestation require use of improved farming practices such as protected production environment, adequate planting date and cassava genotypes by farmers. findings established a significant influence of planting date and cassava genotypes on selected agronomic traits and root dry matter content in cassava that could be exploited for increased productivity of the crop in protected and non-protected cropping systems. identification of tolerant cassava variety, protection of cassava field from grasshopper infestation and harvesting cassava in december could be exploited for selection and production of tolerant cassava variety with desired processing traits such as high root dry matter content. the protection of cassava field from grasshopper infestation, early planting in june and harvesting in december contribute to high root dry matter content and invariably the production of good quality planting material that is resistant to grasshopper attack. acknowledgements the authors are thankfully grateful to the research teams at the sierra leone agricultural research institute (slari) and njala university. authors’ contributions tsj, fsn: conceptualization of the work, experimental design and revision of the manuscript. tsj, aes, ajs, ddq, fsn, ksa, mdp, pen: discussion of results, support and supervision of the study. tsj, aes, ajs, table 6. mean root dry matter content with respect to variety variety root dry matter content (%) f p cocoa 41.90 c 38.5 0.000418 slicass 1 42.17 b slicass 4 slicass 6 41.90 c 42.21 a means in column with the same letter are not significantly different at p< 0.05 (snk) table 7. mean root dry matter content with respect to planting time time of planting root dry matter content (%) f p june 39.8 c 547 0.0001 september 40.1 b december 43.7 a means in column with the same letter are not significantly different at p< 0.05 (snk) table 8. mean root dry matter content with respect to treatment treatment root dry matter content (%) f p protected 44.0 a 94.08 0.0003 unprotected 40.9 b means in column with the same letter are not significantly different at p< 0.05 (snk) torto, s., samura, a., sundufu, a., quee, d., musa, d., kanu, s., fomba, s., norman, p. peruvian journal of agronomy, 7(1), 42-50 (2023) https://doi.org/10.21704/pja.v7i1.2001 49 ddq, fsn, ksa, mdp, pen: fieldwork supervision, manuscript review. tsj, aes, mdp, fsn: support and supervision in field work, manuscript revision. tsj, ajs, fsn, ksa, mdp, pen: experimental design, statistical analysis of results. tsj, ajs, pen: field work carried out, review of statistical analysis of results. tsj, fsn, aes: logistics management, execution of field work. conflict of interests the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. id orcid and emails: saffea joseph torto sjtorto7@gmail.com / sjtorto@njala.edu.sl https://orcid.org/0009-0004-7595-7939 alusaine edward samura: alusaine.sam@njala.edu.sl abu james sundufu david dan quee dennis peter musa sheku alfred kanu sahr ngoba fomba prince emmanuel norman https://orcid.org/0000-0003-1259-796x abuj.sundufu@njala.edu.sl https://orcid.org/0000-0003-4027-0083 dandavidquee@yahoo.com ttps://orcid.org/0000-0001-8040-3229 pdmusa@njal.edu.sl https://orcid.org/0000-0002-9763-7613 kanusa@njala.edu.sl https://orcid.org/0000-0003-3300-6332 snfomba@njala.edu.sl norman.prince64@gmail.com https://orcid.org/0000-0002-0150-8610 references bellotti, a. c. 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(orthoptera: acrididae): comparison between a specialist species on a shrubby legume and three graminivorous species. environmental entomology, 32(3), 680–688. https://doi. org/10.1603/0046-225x-32.3.680 sagrilo, e., filho, p. s. v., pequeno, m. g., gonçalves-vidigal, m. c., & kvitschal, m. v. (2008). dry matter production and distribution in three cassava (manihot esculenta crantz) cultivars during the second vegetative plant cycle. brazilian archives of biology and technology, 51(6), 1079–1087. song, h. (2010). grasshopper systematic past, present and future. journal of orthoptera research, 19(1), 57–86. https://doi. org/10.1665/034.019.0112 united states department of agriculture (2016). cassava, raw. (basic. report: 11134). national nutrient database for standard reference release 28. agricultural research service, us department of agriculture. http://faostat.fao.org/site/368/default.aspx http://faostat.fao.org/site/368/default.aspx https://doi.org/10.1016/j.eja.2017.06.011 https://doi.org/10.1016/j.eja.2017.06.011 https://doi.org/10.5923/j.ijaf.20120206.05 https://doi.org/10.5923/j.ijaf.20120206.05 http://dx.doi.org/10.1603/0046-225x-32.3.680 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i2.1964 received for publication: 14 june 2022 accepted for publication: 30 july 2022 published: 30 august 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru identificación de genotipos de trigo harinero triticum aestivum l.) con tolerancia a condiciones de sequía en la costa central del perú abstract wheat is sown mostly in peru, in areas above 3000 m altitude, under rainfed conditions and frequent drought problems during the crop cycle. it is a cereal used as a staple food by the families of small-scale farmers who are dedicated to their cultivation, which is why it is necessary to develop varieties with drought tolerance. this investigation had as objectives (1) to determine the yield potential of wheat genotypes under drought stress conditions, (2) to determine the susceptibility indices and drought tolerance, and (3) to identify drought tolerant genotypes. nine genotypes introduced from cimmyt and the commercial variety “centenario” wheat flour (triticum aestivum l.) were studied in an environment with complete irrigation during the life cycle and another environment with terminal drought stress or deficit irrigation applied in the boot phase (z4.0). a random complete blocks design was used with three repetitions. agronomic characteristics, quality evaluations were carried out following the established protocols for each characteristic evaluated and the stress tolerance indices (sti), mean productivity (mp), geometric mean productivity (gmp), tolerance index (tol), and stress susceptibility index (ssi), were determined. the reduction in the grain yield varied from 17.95 % to 33.27 % mainly due to drought. the ssi ranged from 0.65 (g-3) to 1.21 (g-6 y g-9), meanwhile the tol ranged from 1 316.8 (g-3) to 3 142.68 (g-7). the mp, sti and gmp indexes allowed the identification of genotypes with the greatest tolerance to irrigation and stress conditions of the 5 genotypes: g1, g-2, g-7, g-8 and g10. these results are important for developing new varieties that adapt to drought conditions and to face climate change in the andean region. keywords: bread wheat, drought, tolerance index, genotypes. resumen el trigo se siembra mayormente en el perú, en zonas sobre los 3000 m de altitud, bajo condiciones de secano y problemas de sequias frecuentes durante el ciclo de cultivo. es un cereal empleado como alimento básico por las familias de los agricultores de pequeña escala que se dedican a su cultivo, razón por la que se requiere desarrollar variedades con tolerancia a la sequía. esta investigación tuvo como objetivos (1) determinar el 1 facultad de agronomía, universidad nacional agraria la molina. 2 lancaster environment centre, lancaster university, lancaster la14yq, uk. *corresponding author: luzgomez@lamolina.edu.pe *https://orcid.org/0000-0001-5536-5179 l. gomez-pando1, ian dodd2, diego zamudio ayala1, denisse deza montoya1, ana eguiluz de la barra1. how to cite this article: gomez-pando, l., dodd, i., zamudio, d., deza, d., & eguiluz de la barra, a. (2022). identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru. peruvian journal of agronomy, 6(2), 175-190. https://doi.org/10.21704/pja.v6i2.1964 identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru may august 2022 176 potencial de rendimiento de genotipos de trigo en condiciones de estrés de sequía, (2) determinar los índices de susceptibilidad y tolerancia a la sequía y (3) identifi ar genotipos tolerantes a la sequía. se estudiaron nueve genotipos introducidos del cimmyt y la variedad comercial “centenario” de trigo harinero (triticum aestivum l.) en un ambiente con irrigación completa durante el ciclo de vida y otro ambiente con estrés de sequía terminal o riego deficitar o aplicado en la fase de bota (z4.0). se empleó un diseño de bloques completos al azar con tres repeticiones. se realizaron evaluaciones de características agronómicas y de calidad siguiendo los protocolos establecidos para cada característica evaluada y se determinó los índices de tolerancia al estrés (ite), productividad media (pm), = producción media geométrica (pmg), índice de tolerancia (tol) y el índice de susceptibilidad al estrés (ssi). la reducción en el rendimiento de granos varió de 17.95 % al 33.27 %, principalmente por efecto de la sequía. el ssi varió de 0.65 (g-3) a 1.21 (g-6 y g-9), mientras que el tol varió de 1 316.8 (g-3) a 3 142.68 (g-7). los índices mp, sti y gmp permitieron identificar a 5 genotipos con mayor tolerancia a condiciones de riego y de estrés: g-1, g-2, g-7, g-8 y g-10. estos resultados son importantes para el desarrollo de nuevas variedades que se adapten a condiciones de sequía y enfrentar el cambio climático en la región andina. palabras claves: trigo harinero, sequia, índice de tolerancia, genotipos. introduction wheat is one of the staple foods of peru and around 80 % of its demand is satisfied mainly through imports. almost all of the cultivated area of wheat is found in the andean region above 3,000 m altitude, wheat cultivation is carried out by small-scale farmers, using low to medium technology and mainly allocating production to family consumption. agriculture in the high andean region is generally carried out under rainfed conditions or a rain regime characterized by its erratic distribution, affecting the crop at different stages of development with periods of different drought durations and which are exacerbated by the effect of climate change. farmers in marginal areas with adverse climate problems must have wheat varieties with greater tolerance to drought to ensure their harvest in the high mountains of peru. drought affects wheat yield and quality because it has negative effects on photosynthesis, chlorophyll content, plant height, yield, and yield components (rivero et al. 2007; sallam et al. 2015; sallam et al., 2018). according to bidinger et al. (1987) yield under water stress generally depends on three factors: potential yield, flowering date and stress tolerance, and the magnitude of the effect of each of these factors on yield under stress is associated with the species and its varieties. bauder (2001) reported for wheat that drought stress at maturity decreases yield by 10 %, but moderate stress during early vegetative growth has no effect on yield. genetic improvement of drought tolerance is difficul due to its quantitative nature and the number of interacting traits. the level or severity of drought stress and the phenological state of the crop must be considered in the selection process. drought can occur at any stage of crop development with different degrees of intensity. in most of the studies carried out, the effect of drought in the final phase of cultivation is measured, in which many yield components are defined. the effect of drought should also be considered in the growth phase of the crop considering the photosynthetic reserves accumulated in the stem before flowering and which contribute to the final yield of the crop (gallagher et al., 1976; abid et al., 2016). many investigations related to the response and determination of tolerance levels of wheat genotypes to drought (manes et al., 2012; khan et al., 2013; aktas, 2016; mwadzingeni et al., 2016; patel et al., 2017; sallam et al., 2018; eid & sabry, 2019). kiliç & yağbasanlar (2010) point out that a selection strategy should consider factors such as early flowering, grain filling period, late maturation, number of grains per spike, spike weight and length spike, to increase the yields under drought conditions. many approaches have been established to identify and select genotypes with the highest drought tolerance. one of the most widely used is the determination of the yield of genotypes in environments with drought problems and environments with optimal humidity to identify and select genotypes that have a high yield in both environments, since a genotype with high yield https://doi.org/10.21704/pja.v6i2.1964 177 potential will work well in most environments; however, this method has limitations since it does not consider the concept of yield stability and adaptation to a stressful environments (ceccarelli & grando, 1991; blum, 2005; thiry et al., 2016; patel et al., 2017). the other approach is the use of indices that measure drought susceptibility and tolerance (fischer & maurer, 1978; rosielle & hamblin, 1981; fernández, 1992; thiry et al., 2016; patel et al., 2017; khayatnezhad & gholamin, 2018; patel et al. 2019, mohammadi, 2019). many of them are efficien in identifying high-yield genotypes under conditions of moisture stress (talebi et al., 2009). patel et al. (2019) in a study carried out with 20 wheat genotypes and 13 different indices used, found highly significant differences for potential yield and yield under stress conditions (yp and ys) and all drought tolerance indices, except tol, indicated that the genotypes under study have different genes for the characteristics used in the determination of the yield and drought tolerance indices. this study was conducted to (1) determine the yield potential of wheat genotypes under drought stress conditions, (2) determine the drought susceptibility and tolerance indices, and (3) identify tolerant genotypes to drought. material and methods wheat genotypes nine genotypes and the “centenario” commercial field experiment the experiments were conducted from july to december 2019 in the research program in cereals and native grains of the national agrarian university la molina, located in lima, peru. the irrigation water used was from the river and distributed by gravity using the furrow of the field. two experiments were raised, one of them was the control, which was watered throughout the life cycle considering the requirements of the crop and the soil moisture that was permanently monitored. in the second experiment, a terminal drought stress was applied in the booting phenological phase (z4.0). the surface of each experiment was 108 m2, made up of 30 experimental plots of 9.6 m2 each one. the experiment followed the protocols of a commercial wheat field. the sowing was carried out by hand and with a continuous stream, using a dose of 200 kg of seed per hectare. the n-p-k fertilization dose used in both environments was 100-60-00 kg/ha. data collection agronomic traits days to flowering, days to maturity and plant height were determined. yield and yield components grain yield (kg/ha), number of spikes per square meter, the number of grains per spike and thousand grains weight (g). table 1. wheat genotypes (triticum aestivum l.) studied in the experiment established in la molina, lima, peru. id genetic material g-1 babax/lr42//babax*2/4/sin/trap#1/3/kauz g-2 kamb1/mnnk1//wbll1 g-3 hd2281/yaco/3/kauz*2/trap//kauz g-4 sitta/prinia//frtl g-5 temporalera m 87*2/konk g-6 centenario g-7 babax/lr42//babax*2/4/sni/trap#1/3/ kauz*2/trap//kauz g-8 babax/lr42//babax*2/3/pavon 7s3,+lr47 g-9 pvn//car422/ana/5/bow/crow//buc/pvn/3/ yr/4/trap#1 g-10 babax/lr42//babax*2/4/sni/trap#1/3/ kauz*2/trap//kauz gomez-pando, l, dodd, i., zamudio, d., deza, d., & eguiluz de la barra, a. peruvian journal of agronomy 6(2): 175-190 (2022) variety of bread wheat (triticum aestivum ssp. aestivum) were studied (table 1). the genotypes were selected under marginal conditions in the central highlands for their good yield performance and their resistance to yellow rust disease or puccinia striformis f. sp. tritici. these were introduced from the international breeding center of wheat and maize (cimmyt). identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru may august 2022 178 quality data protein content and hectoliter weight; following the established protocols for evaluating these characteristics. tolerance stress indices statistical analysis a randomized complete block design with three replications was used. for each experiment the analysis of variance (anova) of the evaluated characteristics was made and then for the combined anova the homogeneity of variance was determined using the barlett test (p = 0.05). the mean of the treatments was compared using the duncan test (p = 0.05). range varied from 100 cm to 108.3 cm, the lowest value corresponded to genotypes 6 and 8, and the highest value for genotype 5 and the mean value was equal to 103.3 cm. differed significantly between them (duncan’s test α = 0.05). the grain yield had an average of 8683.1 kg/ha and the range varied from 7335.3 kg/ha to 9614.6 kg/ha; genotype 3 had the lowest value and genotype 10 had the highest value. for the number of spikes per square meter, an average equal to 335.2 spikes per square meter and a range of 300 spikes per square meter to 374.8 spikes per square meter were observed; genotype 7 was the one with the lowest number of spikes per square meter and genotype 5 was the one with the highest table 2. tolerance stress indices and their formulas indices formulas reference mean productivity (mp) mp= (ys+yp)/2 stress tolerance index (sti) sti= (yp x ys)/yp 2 geometric mean productivity (gmp) gmp=√(ys x yp) stress susceptibility index (ssi) ssi= (1-ys/yp)/si stress intensity (si) si= [1-ys/yp] tolerance index (tol) tol= yp-ys rosielle & hamblin (1981) fernández (1992) fernández (1992) and schneider et al. (1997) fischer & maurer (1978) fischer & maurer (1978) rosielle & hamblin (1981) ys= yield under drought conditions yp= yield potential results agronomic efficienc experiment of bread wheat control treatment (aeew-ct) variation for yield, number of spikes per square meter, number of grains per spike, thousand grains weight, days to maturity, plant height, protein content of the grain and hectolitic weight were equal to 7.99 %, 11.20 %, 9.92 %, 3.37 %, 3.59 %, 6.20 %, 3.96 %, and 2.74 %; respectively. drought the formulas in table 3 shows the results of the mean squares of the analysis of variance of the aeew-ct experiment carried out for grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight, days to maturity, plant height, grain protein and hectolitric weight. at the block level, there was a highly significant variation for yield, number of spikes per square meter, and significant differences in the number of spikes per square meter, days to maturity and protein content of the grain. similarly, highly significant differences were observed at the genotype level for yield, number of grains per spike and days to maturity. the coefficien of the yield data and yield components are presented in table 4 and it can be seen that the genotype values resistance indices were calculated using table 2 the results of the evaluations carried out for agronomic characteristics are presented in table 4. it could be seen that all the characters differe significantly except for plant height (duncan’s test, α = 0.05). for days at the heading stage, an average equal to 71.6 days and a range varying from 66 days to 76 days were observed, with the lowest value corresponding to genotype 3 and the highest to genotype 9. for days to maturity, the mean was equal to 119.7 days and the range varied from 111.67 days to 130.0 days, the genotype 6 had the lowest value and the highest value was for genotype 4. for plant height, the https://doi.org/10.21704/pja.v6i2.1964 179 number of spikes per square meter. the number of grains per spike in the experiment presented a range that varied from 47.07 number of grains per spike to 72.07 number of grains per spike, the lowest value corresponded to genotype 5 and the highest to genotype 8 and the mean value was equal to 55.38 number of grains per spike. for the thousand grains weight, a range of 50.89 g to 61.98 g was observed, the lowest value corresponded to genotype 8 and the highest to genotype 2 and the average value was equal to 57.43 g. variation source df yield (kg/ha) number of spikes per square meter number of grains per spike thousand grains weight (g) days to maturity plant height (cm) protein content (%) hectolitric weight (kg/ hl) block 2 4106985** 13163.7** 167.517* 2.053 100.833* 5.833 0.25105 23.1868* genotypes 9 1808957** 1692.1 159.512** 47.711 112.593*** 24.074 0.42645 9.8199 error 18 482042 1410 30.173 3.756 18.426 41.019 0.2551 4.7067 total 29 cv (%) 7.9959 11.201 9.9187 3.3746 3.5871 6.198 3.9591 2.7365 mean 8683.143 335.2381 55.38 57.431 119.6667 103.3333 12.7573 79.2787 significance level: 0 ‘*** 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1. significantly (duncan’s test, α = 0.05). for grain protein, the range varied from 12.21 % to 13.42 %; genotype 1 appeared with the lowest value and genotype 4 had the highest value and the average was equal to 12.8 %. for the hectolitric weight or specific weight (kg/hl), an average value equal to 79.3 kg/hl and a range from 76.42 kg/hl to 82.89 kg/hl was observed. agronomic efficienc experiment of bread wheat drought terminal treatment (aeewdt) = 0.05) showed that there were differences for the evaluated characteristics except for plant height. for days to maturity, the range varied from 111.7 days to 130 days, the lowest value corresponded to genotype 1 and the highest to genotype 7 and the mean value was equal to 119.67 days. the plant height had a range that varied from 96.67 cm to 108.33 cm, the lowest value corresponded to genotypes 8 and 5 and the highest to genotype 9 and the mean value was equal to 102.8 cm. the grain yield and the yield components presented values that differed significantly kg/ha and a range that varied from 577 677.08 kg/ha to 7 097.84 kg/ha; with the lowest value for genotype 4 and the highest to genotype 2. the number of spikes per square meter varied from 251.91 to 355.71 spikes per square meter; the genotype 9 had the gomez-pando, l, dodd, i., zamudio, d., deza, d., & eguiluz de la barra, a. peruvian journal of agronomy 6(2): 175-190 (2022) table 4 shows the quality data: grain protein and hectolitric weight. the results differed table 3. mean squares of the analysis of variance of grain yield, number of spikes per square meter, number of grains per spikes, thousand grains weight (g), days to maturity, plant height (cm), protein of grains (%) and hectolitric weight (kg/hl) of the control experiment of the assessment of tolerant wheat genotypes (triticum aestivum l.) to drought under conditions of la molina lm 2019b. variation for yield and significant differences for number of grains per spikes. likewise, highly significant differences were observed at the genotype level in yield and number of grains per spike and significant differences for days to maturity. the coefficien of variation for yield, number of spikes per square meter, number of grains per spike, thousand grains weight, days to maturity, plant height, protein content of grains and hectolitic weight were equal to 5.46 %, 13.53 %, 7.11 %, 7.19 %, 2.98 %, 6.24 %, 3.29 %, and 3.25 %, respectively. table 6 presents the results of the evaluations of the agronomic characters and the duncan test (α table 5 showed the results of the mean squares of the analysis of variance of the aeew-dt experiment carried out for grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight, days to maturity, plant height, grain protein and hectolitric weight. at the block level, there was a highly significant (duncan test, α = 0.05) and are presented in table 6. in grain yield, it was observed an average value equal to 6 290.1 identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru may august 2022 180 lowest number of spikes per square meter and genotype 5 had the highest number of spikes per square meter and the average value was equal to 305.91 spikes per square meter. for the number of grains per spike, was observed an average value equal to 53.4 number of grains per spike and a range that varied from 45.77 number of grains per spike to 73.27 number of grains per spike, the lowest value corresponded to genotype 2 and the highest to genotype 8. for the thousand grains weight (g) the range varied from 44.31g to 63.21 g, the lowest value corresponded to genotype 8 and the highest to genotype 2 and the average value was equal to 51.9 g. amado (2016) evaluated the effect of the hydric deficit from the heading stage to maturity, in 15 bread wheat genotypes, under controlled conditions, a reduction in the grain yield in a range of 35 % to 68 % and among yield components a maximum reduction in the number of grains per spike of 47 %, in the weight of grains of 68 % and in the harvest index of 42 % were observed. ayed et table 4. mean values of grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight (g), days to maturity, plant height (cm), grain protein (%) and hectolitric weight of the control experiments of the assessment of tolerant wheat genotypes (triticum aestivum l) to drought under conditions of la molina lm 2019b. genotypes genetic material yield (kg/ha) number of spikes per square meter number of grains per spike thousand grains weight (g) days to maturity plant height (cm) protein content (%) hectolitric weight (kg/hl) 1 babax/lr42//babax*2/4/sni/trap#1/3/ kauz*2/trap//kauz 9052.33ab 323.33ab 59.53b 58.83ab 115cd 101.67a 12.21b 79.19abc 2 kamb1/mnnk1//wbll1 8688.64ab 344.76ab 49.63bc 61.98a 121.67bc 101.67a 12.42b 76.42c 3 hd2281/yaco/3/kauz*2/trap//kauz 7335.28c 307.14ab 52.4bc 50.90d 115cd 101.67a 12.88ab 78.01bc 4 sitta/prinia//frtl 7796.42bc 335.71ab 47.57c 59.04ab 130a 106.67a 13.42a 80abc 5 temporalera m 87*2/konk 7900.50bc 374.76a 47.07c 60.07ab 123.33ab 108.33a 13.17ab 77.35bc 6 centenario 8688.64ab 368.57ab 54.73bc 54.42c 111.67d 100a 13.04ab 79.56abc 7 babax/lr42//babax*2/4/sni/trap#1/3/ kauz*2/trap//kauz 9541.90a 300b 58.33b 59.46ab 123.33ab 105a 12.62ab 79.30abc 8 babax/lr42// babax*2/3/pavon 7s3,+lr47 9351.90a 330ab 72.07a 50.89d 115cd 100a 12.38b 80.81ab 9 pvn//car422/ana/5/ bow/crow//buc/ pvn/3/yr/4/trap#1 8832.28ab 326.67ab 56.53bc 60.91ab 126.67ab 105a 12.76ab 79.27abc 10 babax/lr42//babax*2/4/sni/trap#1/3/ kauz*2/trap//kauz 9614.59a 341.43ab 55.93bc 57.80b 115cd 103.33a 12.66ab 82.89a al. (2017) studied the response to water stress of three varieties of durum wheat (mâali, nasr and salim) and two varieties of bread wheat (tahent and utique), observing that the components plants per square meter, spikes per square meter, grains per spike and thousand grains weight were significantly affected by water stress table 6 also shows the results of the grains protein content and the hectolitric weight or specific weight (kg/hl). duncan’s test (α = 0.05) showed that there were significant differences in the results. for grain protein, it was obtained a range that varied from 11.4 % to 12.36 %; genotype 7 appeared with the lowest value and genotype 9 had the highest value and the average value was equal to 11.91 %. for the hectolitric weight the range varied from 76.84 kg/hl to 82.80 kg/hl; genotype 2 had the lowest value and genotype 4 had the highest hectolitric weight and the mean value was equal to 79.82 kg/hl. https://doi.org/10.21704/pja.v6i2.1964 181 table 5. mean squares of the analysis of variance of grain yield, number of spikes per square meter, number of grains per spikes, thousand grains weight (g), days to maturity, plant height (cm), protein of grains (%) and hectolitric weight (kg/hl) of the drought experiment of the assessment of tolerant wheat genotypes (triticum aestivum l.) to drought under conditions of la molina lm 2019b. variation source df yield(kg/ha) number of spikes per square meter number of grains per spike thousand grains weight (g) days to maturity plant height (cm) protein content (%) hectolitric weight (kg/ hl) block 2 784287** 470.3 68.48* 0.43 10.83 39.7 0.03 1.27 genotypes 9 567592** 3294.5 190.63*** 83.95*** 121.83*** 51.13 0.28 8.05 error 18 118124 1712.3 14.4 13.91 12.69 41.18 0.15 6.74 total 29 cv (%) 5.46 13.53 7.11 7.19 2.98 6.24 3.29 3.25 mean 6290.09 305.95 53.37 51.90 119.67 102.8 11.91 79.82 significance level: 0 ‘*** 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1. table 6. mean values of grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight (g), days to maturity, plant height (cm), grain protein (%) and hectolitric weight of the drought experiments of the assessment of tolerant wheat genotypes (triticum aestivum l) to drought under conditions of la molina lm 2019b. genotypes genetic material yield (kg/ha) number of spike per square meter number of grains per spike thousand grains weight (g) days to maturity plant height (cm) protein content (%) hectolitric weight (kg/ hl) 1 babax/lr42// babax*2/4/ sni/trap#1/3/ kauz*2/trap// kauz 6346.70bc 316.67abc 51.63bcd 50.48bcd 111.67f 101.67a 11.91abc 79.18ab 2 kamb1/mnnk1//wbll1 7097.84a 299.52abc 45.77d 63.21a 123.33bcd 103.33a 11.97abc 76.84b 3 hd2281/yaco/3/ kauz*2/trap// kauz 6018.48c 342.86ab 56.8b 45.62d 113.33f 98.33a 11.9abc 81.19ab 4 sitta/prinia//frtl 5776.77c 291.43abc 47.67cd 55.58b 126.67ab 106.67a 12.29ab 82.80a 5 temporalera m 87*2/konk 6344.45bc 355.71a 45.8d 50.76bcd 120cde 106.67a 11.79abc 79.83ab 6 centenario 5814.08c 288.10abc 50.47bcd 49.74bcd 115ef 101.67a 12.14abc 79.03ab 7 babax/lr42// babax*2/4/ sni/trap#1/3/ kauz*2/trap// kauz 6399.23bc 314.29abc 52.63bcd 52.49bc 130a 106.33a 11.40c 79.59ab 8 babax/ lr42//babax*2/3/pavon 7s3,+lr47 6368.0bc 265.71bc 73.27a 44.31d 118.33def 96.67a 11.53bc 81.43ab 9 pvn//car422/ ana/5/bow/ crow//buc/ pvn/3/yr/4/ trap#1 5893.20c 251.91c 54.5bc 53.93b 125abc 108.33a 12.36a 79.30ab 10 babax/lr42// babax*2/4/ sni/trap#1/3/ kauz*2/trap// kauz 6842.17ab 333.33ab 55.2b 52.88b 113.33f 98.33a 11.79abc 79.05ab gomez-pando, l, dodd, i., zamudio, d., deza, d., & eguiluz de la barra, a. peruvian journal of agronomy 6(2): 175-190 (2022) identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru may august 2022 182 a combined analysis of the control experiment (complete irrigation) and drought treatment (deficit irrigation) the results of the mean squares of the combined analysis of variance of the control experiment (complete irrigation) and drought treatment (deficit irrigation) carried out for grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight, days to maturity, plant height, grain protein, and hectolitric weight are shown in table 7. it was appreciated at the treatment level (control and drought) that there was a high y significant variation in yield, thousand grains weight and grain protein and significant variation for number of spikes per square meter. at the block level, highly significant differences were observed for yield and significant differences for number of spikes per square. at the genotype level, highly significant differences were obtained for yield, days to maturity and thousand grains weight and significant differences in protein content. for the genotype x treatment interaction, significant differences were observed for grain yield. the coefficien of variation for grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight, days to maturity, grain protein, and hectolitric weight were 7.54 %, 13.07 %, 10.56 %, 3.63 %, 6.19 %, 5.31 %, 3.65 % and 3.08 %, respectively. the results of the evaluations carried out for the agronomic traits of the genotypes are shown in table 8. duncan’s test (α = 0.05) showed no significan differences for days to maturity. however, there were significant differences for plant height. for days to maturity, it was observed that both treatments reach the maturity stage at 119.67 days. for plant height the value in the control-complete irrigation treatment was equal to 103.3 cm and that in the terminal droughtdeficit irrigation treatment was 102.8 cm terminal drought-irrigation deficit treatment was equal to 6 290.09 kg/ha. for the number of spikes per square meter, a value of 335.24 spikes per square meter for the control-complete irrigation treatment and a result of 305.95 spikes per square meter for the terminal drought-deficit irrigation treatment was found. the number of grains per spike, in the control-complete irrigation treatment was equal to 55.38 number of grains per spike and in the terminal drought-deficit irrigation treatment was equal to 53.37 number of grains per spike. for the thousand grains weight (g), the value in the control-complete irrigation treatment was equal to 57.43 g and that in the terminal drought-deficit irrigation treatment was 51.9 g. abayomi & wright (1999) point out that drought stress can reduce all yield components, especially the number of fertile spikes per unit area and the number of grains per spike. kiliç & yağbasanlar (2010) in a study of 14 wheat genotypes (triticum turgidum ssp. durum) under conditions with and without drought stress and found that drought stress reduced the number of days of heading, grain filling period, the number of days to maturity, plant height, spike number per square meter, peduncle length, ear length, number of grains per spike, thousand grains weight, whereas the chlorophyl content increased, the content of grain protein and sds sedimentation. spikelets per spike were not affected by drought stress, and they point out that the differenti l response of the genotypes shows the different levels of drought tolerance capacity of the wheat genotypes studied. askary et al. (2018) studied the effect of drought stress on the yield and some physiological characteristics of six wheat cultivars, and point out that drought stress decreased grain yield. cultivars ‘alvand’ and ‘chamran’ showed the highest level of photosystem ii (psii) photochemical efficien , membrane stability and grain yield under drought stress and were considered the most drought stress tolerant cultivars in their investigation. mohammadi (2019), indicated a wide variation in the yield of the wheat genotypes studied under rainfed conditions (642 to 5603) kg/ha and supplementary irrigation conditions (931–6389 kg/ha) with an average of 2686 kg/ha and 3516 kg/ha, respectively, over four years and showing a 24 % increase in yield productivity under irrigation compared to rainfed conditions. for yield and yield components, the average results of all genotypes showed significant differences for yield, number of spikes per square meter and thousand weight grains (duncan test α = 0.05) (table 8). it could be seen that the grain yield in the control-complete irrigation treatment was equal to 8683.14 kg/ha and that in the https://doi.org/10.21704/pja.v6i2.1964 183 table 7. mean squares of the combined analysis of variance of grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight (g), days to maturity, plant height (cm), protein of grains (%) and hectolitric weight (kg/hl) of the control experiment (complete irrigation) and drought treatment (deficit irrigation) of tolerant wheat genotypes (triticum aestivum l.) to drought under conditions of la molina lm 2019b. variation source df yield (kg/ha) number of spikes per square meter number of grains per spike days to maturity plant height (cm) thousand grains weight (g) proteíns content (%) hectolitric weight (kg/ hl) treatment 1 85900314*** 12864.8* 60.4 0 4.27 459.01*** 10.83*** 4.47 block 2 4235628*** 8376* 10.98 32.92 11.52 1.13 0.11 13.29 genotypes 9 1651012*** 2722.9 331*** 215.93*** 64.27 118.57*** 0.51* 11.75 trat x gen 9 725537* 2263.7 19.15 18.512 10.93 13.09 0.19 6.12 error 38 318797 1755.7 32.96 18.88 40.73 8.44 0.20 6.01 total 59 cv (%) 7.54 13.07 10.56 3.63 6.19 5.31 3.65 3.08 mean 7486.62 320.6 54.38 119.67 103.07 54.67 12.33 79.55 significance level: 0 ‘*** 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1. table 8. mean values of grain yield, number of spikes per square meter, number of grains per spike, thousand grains weight (g), days to maturity, plant height (cm), protein of grains (%) and hectolitric weight (kg/hl) of the control experiment (complete irrigation) and drought treatment (deficit irrigation) of tolerant wheat genotypes (triticum aestivum l.) to drought under conditions of la molina lm 2019b. treatment yield (kg/ ha) number of spikes per square meter number of grains per spike thousand grains weight (g) days to maturity plant height (cm) proteíns content (%) hectolitric weight (kg/hl) control 8683.14a 335.24a 55.38a 57.43a 119.67a 103.33a 12.7a 79.82a drought 6290.09b 305.95b 53.37a 51.90b 119.67a 102.8b 11.91b 79.28a comparing the values obtained in the different characters evaluated in the control-complete irrigation treatments and the terminal droughtdeficit irrigation treatment, a greater reduction in grain yield, number of spikes per square meter, number of grains per spike and thousand grains weight equal to 27.6 %, 8.7 %, 3.6 %, and 9.6 %, respectively. determination of stress indices to drought considering the stress indices yield losses percentage (yl), tolerance index (tol), and stress susceptibility index (ssi), the genotypes (g-2), (g-3) and (g-5), reached the lowest values for these indices so they would be the ones with the highest tolerance to water stress under the conditions of the experiment. gomez-pando, l, dodd, i., zamudio, d., deza, d., & eguiluz de la barra, a. peruvian journal of agronomy 6(2): 175-190 (2022) the stress intensity (si) equal to 0.3 for all genotypes studied are shown in table 9. this value can be classified as moderate stress compared to that reported by mohammadi (2019), who studied three levels of drought: si<0.4: low 0.4<si<0.7: moderate and si>0.7: severe stress. patel et al. (2019) recommended the use of moderate drought stress environments to detect drought tolerant genotypes rather than severe drought stress environments. duncan’s test (α = 0.05) also showed that there were significant difference for grain protein (%) (table 8). the protein content of the grains in the control-complete irrigation treatment presented a value of 12.7 % and that in the terminal droughtdeficit irrigation treatment was 11.91 %. for the hectolitric weight, the value for the controlcomplete irrigation treatment was equal to 79.28 kg/hl and for terminal drought-deficit irrigation, it was equal to 79.82 kg/hl. identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru may august 2022 184 the highest values for the mean productivity (mp) corresponded to the genotypes (g-2), (g7), (g-8) and (g-10). for geometric mean productivity (gmp), the highest values were observed in the genotypes (g-2), (g-7) and (g-10). similarly, the highest values of the stress tolerance index (sti) corresponded to the genotypes (g2), (g-7) and (g-10). among the genotypes that showed better behavior under stress conditions, genotype g-2 stands out, which had the highest value of mp, gmp and sti and low values of yl, tol and ssi. the grains yield of genotype g-2 under control conditionscomplete irrigation was equal to 8 688.64 kg/ha and that under stress-deficit irrigation conditions was equal to 7 097.84 kg/ha. talebi et al. (2009) suggested that the selection of drought tolerance in wheat could be carried out by selecting for high mp, bpm and sti under stress and without stress, and concluded that among the genotypes studied, gw 173, gw 487, gw 488 and gw 477 could be considered as superior wheat genotypes with higher stress resistance and better yield potential under irrigation and stress conditions. mohammadi (2019) pointed out that a good yield of genotypes under irrigation and in drought conditions leads to high values of sti, mp, gmp, ysi and yi; and low values of tol and ssi. alternatively, the tol and ssi indices presented significant positive correlation values 0.831 and 0.703; respectively, for the control experiment; while for the drought treatment the correlation coefficient were negative and not significant (-0.116 and -0.310), for this reason the use of these indices in the identification of genotypes tolerant to stress conditions would be limited, since when obtaining significant coefficient of correlation only with one environment (without stress) would imply that the selected genotypes would have an optimal behavior only under irrigation conditions. golabaldi et al. (2006) in their study with durum wheat genotypes found similar results when correlating the mentioned indices with the average yields of the environment with stress and without stress. using the sti, mp indices, the correlation coefficien showed that the sti, gmp and mp indices were the ones that best correlated with grain yield in both environments, presenting positive highly significant values (table 10); as reported by zebarjadi et al. (2012) in a study conducted with 20 wheat genotypes. according to the correlation coefficient obtained in table 10, the sti, gmp and mp indices were the ones that helped distinguish genotypes with a high productive capacity and tolerant to drought conditions of this experiment. khodarahmpour et al. (2011); mohammadi et al. (2011); sareen et al. (2012) and thiry et al. (2016) postulated that these indices are adequate for selecting genotypes with high performance in environments with and without stress. similarly, pourdad (2008) and golabadi et al. (2006) pointed out that sti and gmp indices are good for selecting genotypes with stress tolerance. however, khayatnezhad et al. (2010), indicated that these indices may not be accurate to identify high performing genotypes in environments with stress and without stress. ayed et al. (2017) reported a positive and significant correlation coefficien between ys (grain yield in stress) and yp (yield potential) with the sti and mp indices, and with this result he could detect that the ‘salim’ variety was the genotype most susceptible to drought, while the ‘nasr’ variety was the most drought tolerant genotype and therefore the most suitable for cultivation in semi-arid regions. gmp and mohammadi (2019) identified the g-2 genotype (new cultivar) and the improved g-8 line as the most drought stress tolerant genotypes, implying that the indices used were useful to identify genotypes that perform well without stress and relatively well under severe stress and he could select the g-4 and g-7 genotypes with good yield under stress using the tol and ssi indices. https://doi.org/10.21704/pja.v6i2.1964 185 table 9. grain yield values under total irrigation conditions (yp), deficit irrigation (ys), percentage of yield loss due to drought stress (yl), stress susceptibility index (ssi), stress intensity (si), tolerance index, (tol) mean productivity (mp), geometric mean productivity (gmp), stress tolerance index (sti) of the assessment of tolerant wheat genotypes (triticum aestivum l.) to drought in conditions of la molina lm 2019b. genotipes genetic material ys (kg/ha) yp (kg/ha) yl (%) si mp sti gmp tol ssi 1 babax/lr42//babax*2/4/ sni/trap#1/3/kauz*2/trap// kauz 9052.33 6346.70 29.9 0.3 7,699.52 1.45 7,579.74 2,705.63 1.08 2 kamb1/mnnk1//wbll1 8688.64 7097.84 18.3 0.3 7,893.24 1.56 7,853.06 1,590.79 0.66 3 h d 2 2 8 1 / ya c o / 3 / k a u z * 2 /trap//kauz 7335.28 6018.48 18.0 0.3 6,676.88 1.12 6,644.34 1,316.80 0.65 4 sitta/prinia//frtl 7796.42 5776.77 25.9 0.3 6,786.59 1.14 6,711.04 2,019.65 0.94 5 temporalera m 87*2/konk 7900.50 6344.45 19.7 0.3 7,122.48 1.27 7,079.85 1,556.05 0.71 6 centenario 8688.64 5814.08 33.1 0.3 7,266.13 1.28 7,119.56 2,904.10 1.21 7 babax/lr42//babax*2/4/ sni/trap#1/3/kauz*2/trap// kauz 9541.90 6399.23 32.9 0.3 7,970.57 1.54 7,814.14 3,142.68 1.20 8 babax/lr42//babax*2/3/pavon 7s3,+lr47 9351.90 6368.0 31.9 0.3 7,859.66 1.51 7,716.81 2,983.31 1.16 9 pvn//car422/ana/5/bow/ c r o w / / b u c / p v n / 3 / y r / 4 / trap#1 8832.28 5893.20 33.3 0.3 7,362.74 1.32 7,214.60 2,939.08 1.21 10 babax/lr42//babax*2/4/ sni/trap#1/3/kauz*2/trap// kauz 9614.59 6842.17 28.8 0.3 8,228.38 1.66 8,110.78 2,772.42 1.05 table 10. correlations among different drought stress indices and grain yield under irrigated conditions and drought stress conditions or deficit irrigation of the assessment of tolerant wheat genotypes (triticum aestivum l.) to drought in conditions of la molina lm 2019b. ys yp sti mp gmp tol ssi ys 1 yp 0.46 1 sti 0.889*** 0.813** 1 mp 0.930*** 0.753* 0.995*** 1 gmp 0.894*** 0.807** 0.999*** 0.996*** 1 tol 0.831** -0.116 0.484 0.567 0.494 1 ssi 0.703* -0.310 0.300 0.392 0.311 0.978*** 1 significance level: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1; yp = grains yield under irrigated conditions, ys = grain yield under deficient irrigation; sti = stress tolerance index; mp = mean productivity; gmp = geometric mean production; tol = tolerance index; ssi = stress susceptibility index. indentification of tolerants genotypes to drought conditions with high yield in ambient without stress. the g-5 genotype was considered in group c for a good grain yield under stress conditions. the g-3 and g-4 genotypes were grouped in group d that groups the genotypes with low grain yield in enviroments under stress and without stress. mohammadi (2019) pointed out that under mild stress there is no response with a clear tendency to drought stress between the improved lines and the local varieties: noting that the modern cultivar saji in group a was very sensitive in both rainfed gomez-pando, l, dodd, i., zamudio, d., deza, d., & eguiluz de la barra, a. peruvian journal of agronomy 6(2): 175-190 (2022) the classification of the genotypes studied according to fernández (1999) is presented in figure 1 and table 11. it could be seen that the genotypes g-1, g-2, g-7, g-8, and g-10 were classified in group a, which groups those with high yield under stress and without stress conditions. the g-6 and g-9 genotypes were classified in group b, which considers genotypes identification of bread wheat genotypes (triticum aestivum l.) with tolerance to drought conditions at the central coast of peru may august 2022 186 and irrigated conditions. under mild stress, no significant correlation was found between the rainfed and irrigated plots. under moderate stress, the local varieties were separated from the breeding lines. most of the improvement lines were grouped in group b and the local varieties in group c. table 11. classification of genotypes based on their grain yield in a complete control-irrigation enviroment and in a deficit-stress-irrigation ambient of the assessment of tolerant wheat genotypes (triticum aestivum l.) to drought in conditions of la molina lm 2019b. group* n° of genotype genetic material ys (kg/ha) yp (kg/ha) group a 1 babax/lr42//babax*2/4/sni/trap#1/3/kauz*2/trap//kauz 9052.33 6346.7 2 kamb1/mnnk1//wbll1 8688.64 7097.84 7 babax/lr42//babax*2/4/sni/trap#1/3/kauz*2/trap//kauz 9541.9 6399.23 8 babax/lr42//babax*2/3/pavon 7s3,+lr47 9351.9 6368 10 babax/lr42//babax*2/4/sni/trap#1/3/kauz*2/trap//kauz 9614.59 6842.17 group b 6 centenario 8688.64 5814.08 9 pvn//car422/ana/5/bow/crow//buc/pvn/3/yr/4/trap#1 8832.28 5893.2 group c 5 temporalera m 87*2/konk 7900.5 6344.45 group d 3 hd2281/yaco/3/kauz*2/trap//kauz 7335.28 6018.48 4 sitta/prinia//frtl 7796.42 5776.77 group a: genotypes expressing uniform superiority in both stress and non-stress conditions. group b: genotypes expressing good performance only in yield potential conditions and not under stress conditions. group c: genotypes presenting a relatively higher yield only under stress. group d: genotypes with poor yield performance in both environments. figure 1. wheat genotypes (triticum aestivum l.) classified based on the average yield under irrigation conditions and under water stress conditions, in groups a, b, c and d (fernández, 1992). conclusions drought stress affects agronomic traits significantly in different degrees in the genotypes studied, the grain yield varied from 5 776.77 kg/ha (g-4) to 7 097.84 kg/ha (g-2).the susceptibility indices variation (ssi) ranged from 0.65 (ghttps://doi.org/10.21704/pja.v6i2.1964 187 3) to 1.21 (g-6 y g-9), meanwhile the drought tolerance indices (tol) ranged from 1 316.8 (g-3) to 3 142.68 (g-7).the mp, sti and gmp indices allowed to identify the genotypes with the highest tolerance to stress conditions among them babax / lr42 // babax * 2/4 / sni / trap # 1/3 / kauz * 2 / trap // kauz (g1), kamb1 / mnnk1 // wbll1 (g-2), babax / lr42 // babax * 2/4 / sni / trap # 1/3 / kauz * 2 / trap // kauz (g-7), babax / lr42 // babax * 2/3 / pavon 7s3, + lr47 (g-8) and babax / lr42 // babax * 2/4 / sni / trap # 1/3 / kauz * 2 / trap // kauz (g 10).these results are important for developing new varieties that adapt to drought conditions and to face climate change in the andean region, which will improve the food security for the small farmers and promote a sustainable development of agriculture in the peruvian highlands. acknowledgement to the research project: contract nº 221 2018, field based phytohormone phenotyping to select climate resilient cereals. fondo nacional de desarrollo científico, tecnológico y de innovación tecnológica (fondecyt) newton paulet fund. author contributions elaboration and execution, development of methodology, conception and design; 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(2012). assessment of drought tolerance in some bread wheat genotypes using drought resistance indices. biharean biologist 6(2), 94–98. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index review https://doi.org/10.21704/pja.v6i1.1893 received for publication: 02 march 2021 accepted for publication: 02 march 2022 published: 30 april 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review manejo de chloridea virescens (noctuidae) en arándanos (vaccinium corymbosum l.) para promover su cultivo sostenible en perú: una revisión mónica narrea cango1; elías huanuqueño coca2; josué otoniel dilas-jiménez3*; jhon anthony vergara copacondori4 *corresponding author: abstract a review of current and specific literature was carried out in order to elaborate a proposal for the management of chloridea virescens in the cultivation of blueberry (vaccinium corymbosum l.), developing strategies in each component of integrated pest management (ipm), including cultural control, ethological control, biological control, and chemical control (pbua and pqua). likewise, steps in the genetic improvement for quantitative resistance of the blueberry to this pest (lepidoptera: noctuidae) using wild relatives of this crop as a source of resistance genes are proposed. keywords: quantitative resistance, wild blueberry, assisted selection, genetic improvement, chloridea virescens. resumen se realizó una revisión de la literatura actual y específica para elaborar una propuesta de manejo de chloridea virescens en el cultivo del arándano (vaccinium corymbosum l.), desarrollando estrategias en cada componente del manejo integrado de plagas (mip), incluyendo el control cultural, el control etológico, el control biológico y el control químico (pbua y pqua). asimismo, se proponen pasos en el mejoramiento genético para la resistencia cuantitativa del arándano a esta plaga (lepidoptera: noctuidae) utilizando parientes silvestres de este cultivo como fuente de genes de resistencia. palabras clave: resistencia cuantitativa, arándano silvestre, selección asistida, mejora genética, chloridea virescens. 1 universidad nacional agraria la molina, lima, perú. 2universidad nacional agraria la molina, lima, perú. 3 universidad nacional autónoma de tayacaja “daniel hernández morillo”, pampas, huancavelica, perú. 4 universidad nacional de cajamarca, cajamarca, perú. how to cite this article: narrea, m., huanuqueño, e., dilas-jiménez, j. & vergara, j. (2022). management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review. peruvian journal of agronomy, 6(1), 78-92. https://doi.org/10.21704/pja.v6i1.1893 narrea, m.; huanuqueño, e.; otoniel, j.; vergara, j. peruvian journal of agronomy 6(1): 78-92 (2022) https://doi.org/10.21704/pja.v6i1.1893 79 introduction the blueberry is a shrub of the ericaceous family, belonging to the genus vaccinium, which constitutes a group of species widely distributed throughout the northern hemisphere, basically in north america, central europe, and eurasia, also found in south america, and a few in africa and madagascar (garcía et al. 2018). of the 30 species that make up the genus, only a few are commercially important, with v. corymbosum l., which represents approximately 80% of the total cultivated area in the world, standing out, followed in importance by v. ashei reade, with approximately 15%. among the remaining 5%, v. angustifolium aiton and some hybrids of v. angustifolium x v. corymbosum stand out (garcía et al. 2018). these varieties are differentiated by the number of chilling hours they need to break the dormant or resting period of the plant and are grouped as follows: lowbush blueberry (v. angustifolium), rabbit-eye blueberry (v. ashei), and highbush blueberry (v. corymbosum) (buzeta, 1997). the cultivars of “highbush” are separated into “northern” and “southern” depending on the requirements of chilling hours and winter hardiness (hancock, 2009). in peru, in recent years, v. corymbosum has sustained growth in both area and yield, sown mainly on the country’s northern coast. until 2011, peru had not joined the international union for the protection of new varieties of plants (upov); this conditioned that the initial development was carried out with released varieties: biloxi, legacy, misty, o’neal, duke, and brigitta, among others. biloxi has been the most successful variety in the low-lying areas, estimated to have covered between 80% and 90% of the productive coast area. in the heights of the mountains, on the other hand, good results have been obtained with legacy (febres, 2013). for sierra exportadora (2011), the biloxi, misty, and legacy varieties are the ones that best adapt to our country, but we can find other patented varieties with different costs and production management. these varieties, such as ventura, millennial, emerald, susy blue, windsor, springhigh, star, and jewel arrive in peru through fall creek far & nursery, an american company that manages, sells, and reproduces the patents obtained by the universities of florida and georgia (gargurevich, 2017). the international market promotes crop expansion in our country due to the optimal climatic conditions for its development. the main exploitable commercial window is between the end of september and all of october, where there is competition with argentina and uruguay. in november and december, chile is the main competitor with south africa and oceania. given the countries above’ unpredictable frost and rain conditions, peru can take advantage of better international prices (sierra exportadorapcm, 2012; ministerio de desarrollo agrario y riego [midagri], 2016). this crop is potential and strategic to be financed by the various competitive funds that peru has implemented for years such as innóvate perú (formerly fincyt), fondecyt, and pnia, among others, just as coffee cultivation has been financed (dilas-jiménez & cernaqué 2017). according to agrodata peru (2020a), in 2019, our country surpassed chile for the first time and was crowned as the main exporter of blueberries worldwide, with profits of around the us $810 million (the united states, the netherlands, and china are the main destinations of the prized bluish berries). thus, blueberries have climbed to second place in the list of peruvian fruits for export, surpassed by grapes in the first place and above avocados. in the 2019 campaign, the usa was the primary destination with us $458 million (57% of the total), followed by the netherlands with us $179 million (22%), and china rose to us $70 million (9%) (agrodata peru, 2020a). of the five regions of peru where blueberries are most cultivated, la libertad represents the significant growing region, concentrating more than 60% of production. the rest is distributed between ica, lima, ancash, and lambayeque (agrodata peru, 2020b). according to gestion (2019), in 2015 the national average yield was 9 tons/ha. on the other hand, in 2018 it reached 15 t/ha due to the improvement in the sowing and harvesting technique. la libertad and lambayeque regions have the highest yields, which in 2018, reached management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review january april 2022 80 17 and 15 t/ha, respectively. on the contrary, the region of arequipa registers the lowest yields. in the 2019 campaign, camposol sa led exports with us $173 million, followed by hortifrut perú sac with us $101 million, and hortifrut tal with us $68 million (agrodata peru, 2020a). it is expected that in 2020, the blueberry growing areas in peru will reach approximately 12,500 hectares; for its part, the pro arandanos union calculated that the figure would be around 14,000 hectares next year. “this year we are sure that the blueberry will exceed us$1,000 million in exports and will be the first peruvian agroexport product,” they assure from pro arándanos (agronegociosperú, 2020). among the critical pests of the blueberry crop, chloridea virescens is one of the most important ones because it causes damage to the shoots, leaves, inflorescences, and fruits, generating reductions in crop yield. in addition, there are strategies for controlling h. virescens, such as cultural, ethological, biological, and chemical control; however, these strategies must be used within integrated management. in recent years, there have been limitations in chemical control since there is a restriction on the use of active ingredients for the cultivation of blueberries, whose production is destined for the international markets of europe, the united states, and asia. for this reason, new mechanisms should be used within the chemical control, such as biological products based on bacillus thuringiensis, or other strategies such as genetic improvement of the blueberry crop to reduce the susceptibility to this pest (contreraspérez et al., 2019). on the other hand, there is genetic improvement in this crop, for example, the united states department of agriculture (usda)-agricultural research service (ars)national clonal germplasm repository (ncgr) in corvallis – oregon, has performed the genotyping of 367 blueberry samples vaccinium spp. detecting 54 cultivars makes up important germplasm for future genetic improvement studies (bassil et al., 2020). based on all those mentioned above, this study aims to identify scientific and technological advances that allow the generation of an integrated management proposal of chloridea virescens for the sustainable cultivation of blueberries in peru. for this article, an exhaustive review of relevant information related to integrated pest management (ipm) was carried out, emphasizing the cultivation of blueberries, specifically in the management of chloridea virescens. research articles in journals in the last ten years were preferably consulted. scientific information databases were consulted, such as scielo, sciencedirect, and springer, among others, and in a complementary way, other sources of information provided information about blueberries in peru. the keywords used, individually or conjugated, were “blueberry, blueberries, chloridea virescens, integrated management, crispr, cry proteins, resistance, wild cultivars, and peruvian.” the focus of the organization of the information was on the ipm for the blueberry, the same that gave rise to two management proposals: (1) a specific proposal of integrated management of chloridea virescens in the cultivation of blackberries in peru, based on practices of cultural control, ethological control, biological control, and chemical control (pqua and pbua). this proposal is also supported by the results of field tests on chemical control pbua of chloridea virescens in blueberry, carried out in 2018 by the first author of this article, given its implication in the proposed ipm; (2) a proposal for genetic improvement in cultivated varieties of blueberry in peru, to allow sustainable cultivation of this crop. development strengths and opportunities for blueberries in peru according to guo et al., 2019 and meiners (2007), the strengths and opportunities are summarized in: (1) ability to transport high volumes by sea. in 2019, 5 million kg per week were exported; (2) capacity to produce ten months a year; (3) the us will remain attractive; it is more stable and can absorb large volumes; (4) the asian market has the possibility of growth and is demanding narrea, m.; huanuqueño, e.; otoniel, j.; vergara, j. peruvian journal of agronomy 6(1): 78-92 (2022) https://doi.org/10.21704/pja.v6i1.1893 81 in terms of presentation, fiber, and sweetness, for which it is expected that “genetics” will help; (5) we have achieved yields of up to 16.8 t/ha even being on a learning curve; (6) there is potential for its reproduction via tissue culture (42 days to 75 days, 75% to 100% rooting). phenology and pests of blueberry phenology requires to be based on the location of the fruiting stage, that is, on the harvest, to obtain the highest economic returns thanks to the commercial window there is between september and october; for this, pruning is the fundamental activity since it is the starting point in the calendar (maticorena, 2017). changing the phenology and forcing not to harvest in other months often implies a more significant presence of pests, and for this, a good ipm program must be designed. the plant’s growth is divided into two phases or stages: vegetative and reproductive. four stages of vegetative growth are specified, where the first is the vegetative bud, the second is the shoot characterized by short internodes, the third is the lengthening of the internodes and the expansion of the leaves, and the fourth is a new branch made up of fully extended leaves and long internodes (rivadeneira & carlazara, 2011). there are six stages of reproductive growth: first, there is a swollen bud that will give rise to the flowers, and later the bud will open, initiating flowering. the third is flower buds with the closed corolla, the fourth is the flower in full bloom with the open corolla, the fifth is the drop of the corolla and fruit set, and the sixth is green fruit (meyer & prinsloo, 2003). cisternas (2013) mentions the following pests in chile: white worms (hylamorpha elegans, sericoides spp., s. viridis, s. obesa, brachysternus prasinus, b. spectabilis, phytholaema herrmanni, p. dilutipes and tomarus villosus); burritos (aegorhinus superciliosus, aegorhinus nodipennis, aegorhinus phaleratus, otiorhynchus sulcatus, otiorhynchus rugosostriatus, naupactus xanthographus, graphognatus leucoloma and naupactus cervinus); thrips (frankliniella occidentalis, thrips tabaci, and frankliniella australis), cuncunillas negra (dalaca pallens and d. variabilis); cutworms (agrotis ipsilon and peridroma saucia), aphids (aphis gossypii and macrosiphum spp.); leafroller (proeulia spp.); white piglets (pseudococcus viburni, p. calceolariae, p. longispinus and p. cribata); brown fruit bug (leptoglossus chilensis) and shoot borer wasp (ametastegia glabrata). integrated pest management (ipm) in blueberries since the 1940s, entomologists began work related to integrated pest management (ipm) as a “supervised control”. the concept of ipm has been changing over time. however, it is still conceived as a crop protection system that integrates pest management techniques (cultural, ethological, biological, chemical, and genetic practices) (deguine et al., 2021). ipm combines different management strategies and practices to grow and maintain healthy crops while minimizing the use of pesticides. as the cornerstone of sustainable agriculture, it aims to improve farmers’ practices to support increased incomes while improving the conservation and management of natural resources and the health of rural communities and consumers. ipm emphasizes the growth of a healthy crop with the least possible disturbance of agroecosystems and encourages natural pest control mechanisms (food and agriculture organization [fao], 2014). among the components of ipm, we have mainly: cultural control, ethological control, biological control, chemical control, and genetic control. below we detail information about the uses and scientific advances of these components: cultural control: tasks such as irrigation, fertilization, weed control, and pruning can be cited among the most important. proper fertilization and irrigation management are essential to ensure high yields and good quality characteristics in the fruit (baiano et al. 2011). in blueberries, factors included are choosing an appropriate variety for the area, checking the plants in the nursery to avoid weeds, adequate fertilization and irrigation, planting at the correct time, good quality of plants to ensure a good population, a vigorous initial growth, etc. management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review january april 2022 82 (morales, 2017). ethological control: this control method includes physical (light and color) and chemical (semiochemical) factors intended to modify the insect’s behavior. the behavior of insects is determined by their response to the presence or occurrence of stimuli of a chemical nature, although they also respond to physical and mechanical stimuli (care, 2006). for example, acoustic communication in insects has recently been studied with ethological control applications (eskov, 2017). biologic control: since the 19th century, 6227 biological control agents (bca) have been registered, of which 686 have not yet been identified at the species level, that is, there is a knowledge gap for research (cock, 2019). a series of habitat modifications are included to create conditions to achieve biological control that favors the survival, fertility, longevity, and action of natural enemies and improve their colonization of the crop (landis et al., 2000). in this context, biological corridors would act as a refuge area for beneficial insects (pollinators and biological controllers) when the conditions in the crop become harsh or deadly due to the applications programmed for pest control (landis et al. 2000; gurr et al. 2004). chemical control: they provide quick action and grant a wide range of uses and forms of application (fao 2002). among the pesticides for agricultural use, we find chemical pesticides for agricultural use (pqua) and biological pesticides for agricultural use (pbua). we consider the latter within a chemical control because they come in formulations that include other components in addition to the biological components. for example,in the case of pquas, the term includes substances or mixtures of substances applied to crops before or after harvest to protect the product against deterioration during storage and transportation. on the other hand, pbuas are all the substances of a biological nature: microorganisms or products derived from their metabolism; bacteria, fungi, etc. likewise, products derived directly from vegetables, which are not chemically synthesized such as: strychnine, nicotine, pyrethrins, rotenone, and garlic, among others, which alone or in combination with adjuvants, are used to prevent, repel, combat, and destroy insects, mites, pathogens, nematodes, weeds, rodents, or other biological organisms harmful to plants, their products, and derivatives (supreme decree no. 001-2015-minagri). both treatments are highly effective in-field pest control (llanos & apaza, 2018). within the group of bacteria, there are formulations based on bacillus thuringiensis a facultative anaerobic microorganism, chemoorganotrophic, and with catalase activity (zhou et al., 2020). they can ferment glucose, fructose, trehalose, maltose, and ribose, and hydrolyze gelatin, starch, glycogen, esculin, and n-acetyl-glucosamine (sauka & benintende, 2008). however, the main characteristic of b. thuringiensis is that during the sporulation process, it produces a parasporal inclusion formed by one or more crystalline bodies of protein nature that are toxic to different invertebrates, especially insect larvae. these proteins are called cry (from crystal) and constitute the basis of the most widespread biological insecticide worldwide (schnepf et al., 1998; liu et al., 2018). bt toxins began to be used commercially in france in 1938; by 1958, their use had spread to the united states. starting in the 80s, bt became a pesticide of worldwide interest (feitelson et al., 1992). commercialized products of b. thuringiensis mainly consist of spore and crystal preparations, activated or not, which are sprayed on crops as if they were conventional insecticides. these preparations generally come from the subspecies kurstaki (btk) (kamatham et al., 2021). b. thuringiensis is classified into 84 serovars identified by flagellar antigen h serology (sauka & benintende, 2008). since its discovery, some subspecies active, mainly bt subsp. kurstaki (btk), bt subsp. thuringiensis (btt) y bt subsp. galleriae (btg) against pest invertebrates have been reported (rashki et al., 2021) based on biochemical and morphological characteristics and flagellar antigens (schnepf et al., 1998). crystalline toxins exist in a variety of forms: bipyramidal, spherical, rhomboid, cuboidal, narrea, m.; huanuqueño, e.; otoniel, j.; vergara, j. peruvian journal of agronomy 6(1): 78-92 (2022) https://doi.org/10.21704/pja.v6i1.1893 83 elliptical and irregular, among others, and are active against a large number of groups of insects mainly, as well as nematodes and protozoa (rashki et al., 2021; jurat-fuentes & crickmore, 2017). the genes that encode these proteins reside in conjugable megaplasmids (feitelson et al. 1992) were named cry, and their encoded proteins were designated cry δ-endotoxins (ochoa & arrivillaga, 2009). cry proteins are synthesized as inactive protoxins ingested by larvae when feeding. the inclusions are solubilized under the alkaline conditions of the larva’s digestive tract and are converted by insect proteases into active peptides (schnepf et al., 1998; feitelson et al., 1992). the active toxin is recognized by a specific receptor and is inserted into the membrane of the brush border of microvilli of the digestive tract of the insect (gerber & shai, 2000). an oligomerization occurs, resulting in the formation of cation channels of 0.5 to 1 nm in diameter (gerber & shai, 2000). these pores cause a nonspecific influx of ions, mainly k+ ions, which dissipates ionic gradients and lowers the ph of the medium, causing osmotic cell lysis that leaves the larva unable to feed (schnepf et al. 1998). on the other hand, the tissue destruction allows the mixture of the contents of the digestive tract with the hemolymph, which, together with the low ph, favors the germination of bacterial spores, leading to septicemia and the death of the larva a few days after ingestion of crystals (schnepf et al. 1998). when the insect consumes the cry protein, it presents cessation of ingestion, intestinal paralysis, vomiting, diarrhea, osmotic decompensation, total paralysis, and death (vachon et al., 2012; bravo et al., 2007). in the case of lepidopterans such as h. virescens, the proteins considered toxic are those of the cry1 class. cry proteins generally show reduced activity spectra and are often limited to a few species of insects belonging to the same order. however, the toxicity of cry1 proteins is not restricted to lepidopterans (sauka & benintende, 2008). for h. virescens we have the following toxic proteins: cry1aa, cry1ab, cry1ac, cry1ae, cry1be, cry1ca, cry1fa, cry1if, cry1ja, cry1jc, cry2aa, cry2ab, cry2ac, cry2ae, cry9a, cry9ca, and vip3 (sauka & benintende, 2008). b. thuringiensis var. kurstaki hd-1 is one of the strains of b. thuringiensis best studied and is characterized by carrying the following cry antilepidopteran genes: cry1aa, cry1ab, cry1ac, cry2aa, cry2ab and cry1ia (sauka, 2007). b. thuringiensis var. kurstaki hd-1 is par excellence the strain usedto control of lepidopteran insects, agricultural pests, and forest pests. this strain was initially isolated by dulmage in 1970, constituting a milestone in the history of the use of b. thuringiensis as a larvicide since it was responsible for b. thuringiensis-based products can compete with chemical insecticides in terms of efficiency. this strain was up to 200 times more toxic for some species of lepidoptera than other strains used in the products of that time (sauka & benintende, 2008). these products, formulated based on bts, are used mainly to control lepidoptera pests in corn, wheat, cotton, and fruit crops. formulations can be made from b. thuriengensis isolated from peruvian agroecosystems and the evaluation of their bioinsecticidal potential. flores et al. (2011), managed to isolate 54 strains of bacillus thuringiensis from 385 samples of rhizosphere, plant material, and dead insects from central peru; the identification of the isolated strains was carried out by observation in phase-contrast microscopy according to the culture microscopic characteristics and differential biochemical tests. isolated strains were compared with b. thuringiensis hd-11, b. thuringiensis var kurstaki hd-342, and b. thuringiensis aizawai nrrl-hd-130 to evaluate the entomotoxic effect. the bt-unmsm-42 strain was the one that presented higher toxicity than the rest of the isolated strains, with mean mortality of 39.73% with 50 μg/ml and 71.93% for 250 μg/ml, with a standard deviation of 11.30 and 9.98, respectively; however, it did not outperform the reference strains b. thuringiensis hd-11 and b. thuringiensis var kurstaki hd342, which reached mean mortality of 86.5% and 82.5% respectively at a dose of 250 μg/ml. according to sauka & benintende (2008), genetic engineering developed many species of plants that express cry genes from b. thuringiensis and thus turned them into “insecticidal plants’’. these plants are commonly referred to as “bt plants or crops” (e.g., bt corn, bt cotton, etc.). tobacco management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review january april 2022 84 plants (nicotiana tabacum) that produced sufficient amounts of cry protein to control first instar larvae of manduca sexta were developed. since then, at least ten different types of cry genes have been introduced into 26 plant species: cry1aa, cry1ab, cry1ac, cry1ba, cry1ca, cry1h, cry2aa, cry3a, cry6a, and cry9c (sauka & benintende, 2008). genetic control: varietal resistance is an integrated pest management (ipm) strategy that has been considered an alternative that can be ecological since it can reduce dependence on the use of synthetic insecticides and is compatible with other control methods (vallejo & estrada, 2002). according to jiménez (2009), genetic control of pests has been used in two ways: (1) the crop can be genetically manipulated to increase its resistance to attack by pests, and (2) pests can be subject to genetic intervention with the introduction of masses of individuals with a selected genotype. over the years, varieties of insect-resistant crops have been developed, most notably alfalfa, corn, cotton, beans, cassava, vegetables, rice, sorghum, soybeans, and wheat. within integrated pest management, the genetic resistance of a plant to insect attack is a component that can be managed through conventional genetic improvement programs, which in turn are associated with desirable agronomic characteristics (deguine et al., 2021). these characteristics can be found in the wild varieties of many species, such as the blueberry, where rodríguez-saona et al. (2019) found that d. suzukii prefers cultivated fruits for oviposition and better hosts for their offspring than wild fruits. the cultivated fruits were also two times larger, 47% firmer, 14% less acidic, and had lower amounts of brix, phenolic, and anthocyanin per mass than wild fruits. the review of the scientific literature presented above suggests that through a genetic improvement process we can select those resistance characteristics of wild cultivars to cultivated ones while maintaining other desirable agronomic characteristics directly related to higher crop yield and resistance to plagues and diseases. integrated management proposal the proposal developed by the authors regarding the management of chloridea virescens in blueberries is the following: cultural control: the implementation of 6 cultural practices is recommended: (1) weed control: blueberry leaves are small and few, so the damage of h. virescens is significant in the production of this crop. that is why weeds that harbor h. virescens egg-laying should be avoided such as trifolium repens (fabaeae), species of the genus geranium (geraniaceae), and others (blanco et al., 2008). in addition, geomembranes should be placed throughout the soil of the entire crop area before the blueberry plants are installed to prevent and control the weeds from sprouting and developing. (2) management of the environment or field edges: this is where the pest can be harbored; management must be done with evaluations and applications of low impact products on beneficial fauna; (3) use of windbreaker curtains: for both horizontal and vertical crop management. the wind brings thrips and sand that causes stress to the plantation, making it susceptible to pests and diseases; (4) management of planting density: in recent years we have gone from sowing 5000 pl/ha to 10,000 pl/ha. therefore, it is essential to handle high pruning well to avoid ambush of the blueberry, in addition to more control in the evaluations, evaluating a more significant number of plants due to the high density; (5) use of nets: to prevent the entry of h. virescens in the field. the use of nets reduces the entry of 80% of lepidopterans (6) know the neighboring crops: to project the influence and management in the field. ethological control: the following are recommended: (1) use of molasses and light traps: for h. virescens, ten molasses traps should be used per hectare; (2) pheromone use: the dose for h. virescens is 10 to 15 pheromones per ha. they must be placed from the beginning to the end of the campaign. the pest population must be evaluated weekly and correlated with the captures in pheromones. biological control: at least four techniques are narrea, m.; huanuqueño, e.; otoniel, j.; vergara, j. peruvian journal of agronomy 6(1): 78-92 (2022) https://doi.org/10.21704/pja.v6i1.1893 85 recommended: (1) biological corridors: planting the biological corridors around the field and changing them 3 to 4 times a year. the goal is to have flowers throughout the year because they are a food source of pollen and nectar for predators such as chrysopids and ladybugs, species like fennel, yarrow, sage, alder, buttercup, and sunflower; (2) bedbugs: concerning predatory bedbugs, biological corridors will be planted to maintain them and guarantee their action of preying eggs and larvae of h. virescens; (3) chrysopids: as for chrysopids, a minimum of 30 thousand per hectare should be released every 15 days and 2 to 3 releases should be made in a row, always with prior evaluation. the larvae need prey which are the eggs and larvae of h. virescens. adults need nectar and pollen; for this reason, it is crucial to plant biological corridors; (4) pollinating bees: since the crop needs pollinators, it is essential to use low-impact products for predators and pollinators. chemical control: application of pquas and pbuas as follows: pqua: use contact products such as emamectin benzoate or chitin synthesis inhibitors as a last resort. pbua: use nuclear polyhedrosis virus in small larvae and on well-wet foliage, preferably in the afternoon or at night. for this, the use of bts products is known; the most pathogenic commercial strains must be chosen, with doses ranging from 300 to 700 gr per cyl. like viruses, they must be applied in the afternoon or at night and are effective in stage iii larvae. the larvae are affected after feeding on the first day and will die on the 4th-6th day. bt continues to be a powerful tool for controlling h. virescens larvae as there is no resistance. this proposal is based on organic agriculture’s technological and profitability challenges (dilasjiménez et al., 2020). proposal for genetic improvement for durable or quantitative resistance in cultivated varieties of blueberries the genetics of resistance to pests by crops is under the control of two types of genes: (1) vertical resistance, of a specific race, of significant effects and not durable; it is controlled by one or a few genes, and its inheritance is qualitative; (2) horizontal resistance, of non-specific race, of minor genes and long-lasting; it is controlled by multiple genes, each of which contributes to resistance and its inheritance is quantitative. quantitative resistance is the one we choose in this work for being durable and can be used by traditional methods with the support of modern selection techniques. however, before developing the genetic improvement proposal for quantitative resistance in blueberries, we believe it is necessary to point out the characteristics of the techniques that we will propose to use, and we will explain why we will not use other techniques even though it is believed “that they are the most indicated.” this proposal follows these steps: identification of resistance genes it is known that all cultivated varieties of blueberries are susceptible to damage by h. virescens; therefore, a collection of wild plant material related to the cultivated blueberry will be carried out; it will then be evaluated for resistance, and those with good response will be selected. native varieties of wild relatives are a pool of genes from which economic resources are generated by developing improved varieties. some of the phenotypic characteristics that should be evaluated are (1) hardness of the leaf in view that in the field, it is seen that they are slightly attacked by h. virescens, (2) concentration of the fruit in the upper part so that this pest does not have many organs at disposals such as buds and flowers, in addition to being able to program today’s scarce personnel in the fields, (3) the distance between the buds so that h. virescens does not have many microclimates below the middle third of the plant. experience: the united states, through the united states department of agriculture (usda), the agricultural research service (ars), and the national clonal germplasm repository (ncgr) maintain a gene bank in oregon with more than 1800 accessions of vaccinium spp. which come from 34 countries (bassil et al., 2020). management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review january april 2022 86 incorporation of genes from the wild relative to the cultivated varieties the samples (accessions) selected in the previous step will be hybridized with the cultivated ones. experience: allotetraploid hybrids derived from the crossing of vaccinium uliginosum and vaccinium vitis-idaea are fertile, thus offering genetic variability from which to select many characteristics, such as yield, fruit quality, and adaptation to variable ecological conditions in the breeding of v. vitis-idaea (morozov, 2007). selection backcross selection, assisted by modern techniques in the hybrids derived from the previous cross, the plants (f1) that have genes with quantitative effects will be selected through modern techniques, which will be backcrossed (f1 x cultivated) with the cultivated varieties to recover the fruit quality genes of the cultivated ones and will be re-selected in the progeny of the backcross until identifying plants with resistant quantitative trait qtls genes. conventional genetic improvement for quantitative resistance has given good results (kolmer, 1996); however, it has drawbacks. first, an extensive group of individuals needs to be evaluated to find the quantitative resistance genes together; in addition, hundreds of plants have to be inoculated or infested. these are delicate activities, and it is not always possible to inoculate or infest homogeneously in the field. modern techniques appear as a powerful tool in enhancing the selection of these types of characters. experience: the use of molecular markers in the genetic improvement of plants is proven. garkava-gustavsson et al. (2005) used rapid and issr markers to assess the genetic diversity of 15 mountain cranberry vaccinium vitis-idaea populations, 13 from sweden, finland, norway, estonia, and russia, and two populations of v. minus from japan and canada. genetic differentiation between accessions can be exploited in hybridization programs of this species (garkava-gustavsson et al. 2005). marker-assisted selection (mas) -for quantitative inheritance traitsis being applied in breeding programs and directed pyramiding in different crops (liu et al. 2004; asea et al. 2009; moloney et al. 2010; singh et al. 2005). single nucleotide polymorphisms (snp) generated by genotyping by sequencing (gbs) allowed identifying qtls of additive effect for resistance to fusarium in wheat (zhang et al., 2020). in cotton, with the same technology, 3187 polymorphic markers were developed, which allowed the identification of 17 quantitative trait loci (qtl) for the height of the plant, the height of the first fruiting branch node and the number of vegetative shoots (qi et al. 2017). similarly, four qtls for resistance and one qtl for susceptibility to leaf rust in alfalfa were identified in the genetic map in an alfalfa mapping population (adhikari & missaoui, 2019). clonal propagation (asexual) of the selected plants plants selected by modern techniques will be multiplied asexually; they will be taken to field trials with large plots. experience: one of the most significant advantages of the genus vaccinium is that it responds well to asexual propagation both by in vitro methods and cuttings.blueberry species such as v. corymbosum, v. virgatum and v. macrocarpon, they root up to 76% in medium without growth regulators (tetsumura et al. 2017 and debnath and mcrae 2011 cited by erst et al. 2018). in turn, erst et al. 2018 specify that most plants require specific chemicals for the initiation of cell differentiation and the formation of meristems, which is why, in its study by in vitro methods, a rooting of up to 100% in blueberry variety “golubaya rossyp” was achieved. guo et al. (2019), using a rooting bag method, obtained 97.7% rooting after using the blueberry cultivar ‘ozarkblue’ in a culture medium for woody plant supplemented with 0.1 mg/l of iba; likewise, they achieved densities of 1600 seedlings per m² compared to the traditional rooting method that manages to put 270 to 420 seedlings per m² (guo et al. 2019). in micropropagation in vitro, transgenic plants of v. corymbosum and v. vitisidaea had a better response in the regeneration of shoots using zeatin at a concentration of 20μm due to its effect on the induction of regeneration of adventitious shoots from cut leaves. it is also specified that in vitro they can be easily rooted using iba or ex vitro in a humidity chamber without hormonal treatment (meiners et al. 2007). narrea, m.; huanuqueño, e.; otoniel, j.; vergara, j. peruvian journal of agronomy 6(1): 78-92 (2022) https://doi.org/10.21704/pja.v6i1.1893 87 identification of the best clone with quantitative resistance after vegetative multiplication, the plants selected by modern techniques will be taken to the field with natural and artificial inoculation; at the end of the trial, the best material will have been identified, which would be the improved variety. why did we not choose crispr-cas?: to apply this technique, it is required that the quantitative resistant genes have been identified first; likewise, hundreds of genes would have to be edited at the same time, which makes it almost impossible use of this technique nowadays in the case of genetic improvement in blueberry; also, it is still in the process of refining, so it is not safe. experience: the crispr-cas system, discovered as an immune system acquired by certain bacteria, seems to have notable advantages in gene editing over zfn and talen as they are potent tools (ran et al. 2013; chen et al. 2019). in the yeast pichia pastoris, a crispr/cas9 system was developed with episomal sgrna plasmid and 100% genome editing efficiency was obtained, as well as high multicopy gene editing and stable multigene editing without a substantial decrease caused by multi-sgrna (yang et al. 2020). however, off-target dna cleavage remains one of the major imperfections of the system, including sequence mutation, rearrangement, activation, and cell death during genome editing (chen et al. 2020). why do we not recommend bt transgenic with cry proteins? effects of resistance to bt transgenics: the main characteristic of bt is the production of protein crystals containing toxins with specific activity against many pests, including dipteran, lepidopteran and coleopteran insects, as well as nematodes, protozoa, trematodes, and mites (adalat et al. 2020). the gene variants of cry toxins obtained from the bacterium b. thuringiensis (bt), due to their insecticidal effect, have become an alternative to chemical insecticides in agriculture (zhou et al. 2020; grove et al. 2001; zhang et al. 2018; zhang et al. 2020, because they control pests of lepidoptera (moths) and coleoptera (beetles) that feed on plants. two bacterial isolates –variants of cry toxins-, controlled the cotton leaf worm, spodoptera littoralis (boisd.) (lepidoptera: noctuidae), with mortality rates of 100 and 96.6% (abo-bakr et al., 2020). another chimeric protein toxin involving cryia residues 450-612, demonstrated 30 times more activity against h. virescens than the native parental toxin, indicating that this region plays an essential role in the specificity of h. virescens (ge et al. 2020). however, they are ineffective for sap-sucking insects (hemiptera) (liu et al. 2020). susceptible insects acquire resistance in a few years, and many new strains of bt have been isolated to avoid resistance to pests (zhou et al., 2020). the widespread cultivation of transgenic soybeans has caused significant changes in the spectrum of lepidopteran larvae, both in the number of species and their densities in the field. transgenic crops that produce insecticidal toxins from b. thuringiensis (bt) have successfully reduced the incidence of the most common caterpillars that infest soybeans, such as anticarsia gemmatalis (lepidoptera: erebidae) and chrysodeixis includens (lepidoptera: noctuidae). however, lepidopteran species not previously registered have been found in cultivation due to the possibility of adaptation to genetically modified cultivars. for example, the appearance of peridroma saucia hübner (lepidoptera: noctuidae) is described for the first time in brazil, feeding on genetically modified soybean cultivars (takahashi et al. 2019). after five years of research , downes et al. (2010), , found a significant exponential increase in the frequency of alleles that confer resistance to cry2ab in australian field populations of helicoverpa punctigera, since the adoption of a bt cotton; in addition, the frequency of alleles of resistance to the cry2ab protein in populations from cultivation areas is eight times higher than those found for populations from regions not cultivated with bt; a similar result was found for diatraea saccharalis fabricius (grimi et al. 2018). the development of resistance among lepidopterans is a common phenomenon, and a repertoire of resistance mechanisms to various cry toxins has been identified from a laboratory, greenhouse, and field studies in this insect management of chloridea virescens (noctuidae) in blueberries (vaccinium corymbosum l.) to promote sustainable cultivation in peru: a review january april 2022 88 (peterson et al. 2017). cases of pest resistance to crystal proteins bt (cry) produced by transgenic crops increased from 3 in 2005 to 16 in 2016 (tabashnik et al., 2017). gassmann (2016) found that in laboratory selection experiments, the western corn rootworm could develop resistance to all types of bt corn after three to seven generations of selection. the “pyramids” of transgenic crops that produce two or more toxins of b. thuringiensis (bt) active against the same pest are used to delay the evolution of resistance in insect pest populations (santos-amaya et al., 2015). however, this strategy could fail if a single gene in a pest confers resistance to many toxins, as happened with the cp73 strain of the cotton pest h. virescens (f.), which is resistant to the cry1ac and cry2aa toxins of bt (gahan et al. 2005). all the blueberry varieties grown in peru are introduced. unfortunately, no work has been done to evaluate resistance to chloridea virecens, and it seems that they are susceptible since this pest has been found in all of them; however, resistance genes will be donated by wild relatives collected in peru, if a genetic improvement program is developed. in 2005, seven years after releasing a transgenic bt variety of maize resistant to busseola fusca (lepidoptera noctuidae), significant levels of pest survival were observed (van den berg et al. 2013). under laboratory conditions, gassman (2016), showed that between three and seven generations of selection, the pest diabrotica virgifera could generate resistance to all types of cry proteins. in australia, a population of helicoverpa armigera developed resistance to the cry1ac toxin from b. thuringiensis because around 70% of resistant larvae h. armigera were able to survive on cry1ac transgenic cotton (gunning, 2005). final comments the integrated management of blueberries, especially the pbua chemical control with products based on bt proteins cry, and the biological control are highly explicitly recommended for controlling chloridea virescens. the proposal for genetic improvement of varieties of good yield and acceptable quality but susceptible constitutes a good strategy in the medium and long term. using native varieties would allow the possibility of accumulating genes of lasting resistance for local pests in the susceptible ones. with modern selection techniques, this activity would be more efficient and results in a shorter time than what would be obtained with the traditional method alone. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. orcid and e-mail m. narrea mnarrea@lamolina.edu.pe https://orcid.org/0000-0002-5565-746x e. huanuqueño ehh.coca@lamolina.edu.pe https://orcid.org/0000-0002-9118-0662 j. dilas-jiménez jdilas@unat.edu.pe https://orcid.org/0000-0003-4256-8393 j. vergara javergara@unc.edu.pe https://orcid.org/0000-0002-8583-4404 references abo-bakr, a., fahmy, e. m., badawy, f., abd el-latif, a. o., & moussa, s. 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(2020). bacillus thuringiensis novel toxin epp is toxic to mosquitoes and prodenia litura larvae. brazilian journal of microbiology, 1–9. peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v7i1.1979 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal rendimiento de la variedad de papa rolpa local (solanum tuberosum l.) bajo diferentes condiciones de cobertura y niveles de zinc en rolpa, nepal muna aryal1, krishna raj pandey1*, suman dhakal2, sanjeev tumbapo1, yagyaraj joshi1 *corresponding author: krishnapandey2055@gmail.com *https://orcid.org/0000-0001-6862-3421 abstract use of the right mulching techniques is a crucial first step in replacing the issues with irrigation and weed infestation in potato production. to assess the performance of potatoes at two doses of zinc under different mulching conditions, a field experiment was laid out in a two-factorial, randomized complete block design (rcbd) comprising eight treatments with three replications each. the variety used was rolpa local. the first factor consisted of three mulching materials: plant residue (m1), black plastic (m2), silver on black plastic (m3), and a control plot (m0), whereas the second factor compromised two levels of zinc (0 kg/ha and 4 kg/ha). both plastic mulches were found to have a significant influence on germination rate during the field research; however, all other observations for growth and yield parameters, such as plant height, tuber number, tuber weight, and yield per plant, were significantly superior in the silver on black plastic. the highest germination percentage was observed in black plastic mulch (94.67 %). however, all other observations for growth and yield parameters were found to be significantly superior in the silver on black plastic mulch condition, with plant height (87.28 cm), leaf count (145.70), number of tubers per plant (22.16), and a total yield of 21.83 t/ha. similarly, all the yield-attributing characters were found to be significantly different among zinc levels, with the highest total yield of 17.70 t/ha at 4 kg/ha of zinc. silver on black plastic mulch and 4 kg/ha of zinc level resulted in the highest benefit-cost (b:c) ratios of 3.18 and 2.23, respectively. the results showed that using silver on black plastic mulch with a zinc level of 4 kg/ ha improved both vegetative growth and potato yield, with favorable b:c ratios resulting in higher profitability. thus, to maximize profit, it would be advantageous to utilize silver on black plastic mulch with 4 kg/ha of zinc. key word: b:c ratio; canopy diameter; germination percentage; mulching; silver on black plastic; tuber yield; zinc 1 faculty of agriculture, agriculture and forestry university, rampur, chitwan, nepal. 2 assistant professor, department of agronomy, agriculture and forestry university, nepal how to cite this article: aryal, m., pandey, k. r., dhakal, s., tumbapo, s., & joshi. y. (2023). performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal. peruvian journal of agronomy, 7(1), 27-41. https://doi.org/10.21704/pja.v7i1.1979 received for publication: 06 february 2023 accepted for publication: 10 april 2023 published: 18 april 2023 mailto:krishnapandey2055@gmail.com https://orcid.org/0000-0001-6862-3421 performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal january to april 2023 28 resumen el uso de las técnicas de acolchado adecuadas es un primer paso crucial para solucionar los problemas de riego y de infestación de malas hierbas en la producción de patatas. para evaluar el rendimiento de las patatas con dos dosis de zinc en diferentes condiciones de acolchado, se realizó un experimento de campo en un diseño de bloques completos aleatorizados (dcca) bifactorial que comprendía ocho tratamientos con tres repeticiones cada uno. la variedad utilizada fue rolpa local. el primer factor consistió en tres materiales de acolchado: residuos vegetales (m1), plástico negro (m2), plata sobre plástico negro (m3), y una parcela control (m0), mientras que el segundo factor comprometió dos niveles de zinc (0 kg/ha y 4 kg/ha). ambos acolchados plásticos influyeron significativamente en el porcentaje de germinación durante la investigación de campo; sin embargo, todas las demás observaciones de los parámetros de crecimiento y rendimiento, como la altura de la planta, el número de tubérculos, el peso de los tubérculos y el rendimiento por planta, fueron significativamente superiores en el plástico plata sobre negro. el mayor porcentaje de germinación se observó en el acolchado de plástico negro (94.67 %). sin embargo, todas las demás observaciones para los parámetros de crecimiento y rendimiento resultaron ser significativamente superiores en la condición plata sobre acolchado de plástico negro, con altura de planta (87.28 cm), recuento de hojas (145.70), número de tubérculos por planta (22.16), y un rendimiento total de 21.83 t/ha. del mismo modo, todos los caracteres atributivos del rendimiento resultaron ser significativamente diferentes entre los niveles de zinc, con el mayor rendimiento total de 17.70 t/ha a 4 kg/ha de zinc. la plata sobre acolchado de plástico negro y el nivel de zinc de 4 kg/ha dieron lugar a las relaciones beneficio-coste (b:c) más elevadas, de 3.18 y 2.23, respectivamente. los resultados mostraron que el uso de plata sobre acolchado de plástico negro con un nivel de zinc de 4 kg/ha mejoró tanto el crecimiento vegetativo como el rendimiento de la patata, con ratios b:c favorables que resultaron en una mayor rentabilidad. así pues, para maximizar el beneficio, sería ventajoso utilizar plata sobre acolchado de plástico negro con 4 kg/ha de zinc. palabras clave: relación b:c; diámetro del dosel; porcentaje de germinación; acolchado; plata sobre plástico negro; rendimiento de tubérculos; aplicación de zinc introduction the potato (solanum tuberosum l.) is the fourthlargest crop grown in the world after maize, wheat, and rice and the third-most significant crop in terms of human consumption (yuan & sun, 2022). potatoes improve the livelihood and food security of impoverished farmers, and hundreds of millions of people in developing nations rely on potatoes as their primary source of food (international potato center (cip, 2022); devaux et al., 2014). it is one of nepal’s most significant staple crops (timsina et al., 2011). it is considered a key cash crop for alleviating poverty and tackling food insecurity among smallholder farmers in developing nations like nepal (bista et al., 2013). at present, the area under potato cultivation in nepal is about 198,788 ha, with a productivity of 16.73 t/ha (ministry of agriculture & livestock development [moald], 2021). potato is essential in the nepalese context, as it is utilized as a secondary food as part of a vegetable in the terai region and as a staple food in the upper physiographic regions of the country. however, many factors impede potato production, such as lack of quality seeds and fertilizers, improper storage facilities, labor shortages, undulated topography affecting transportation and marketing, lack of pest and disease management, and crop-weed competition for sunlight, nutrients, soil moisture, and space (maldonado et al., 1998; yadav et al., 2015). potato growth and yield are related to soil quality and environment, which are influenced by various factors, including complex soil physical and chemical properties, climatic conditions, enzyme activities, microbial abundance, and diverse production strategies (fan et al., 2019; qin et al., 2017). surface mulch is one of the most economical and important factors of soil protection technology because it affects soil temperature and water potential, which can increase the performance of crops by lowering aryal, m., pandey, k. r., dhakal, s., tumbapo, s., & joshi. y. peruvian journal of agronomy, 7(1), 2741 (2023) https://doi.org/10.21704/pja.v7i1.1979 29 surface evaporation and preserving soil moisture (dvořák et al., 2012; qin et al., 2018; shelton et al., 1995). mulching reduces the problem of weed infestation as it prevents the photosynthesis of weeds (ranaivoson et al., 2018) and minimizes the leaching loss of nutrients (dong et al., 2019). mulching helps in maintaining a better growing environment like soil, temperature, and moisture, which ultimately leads to a higher yield (kader et al., 2017). using the right mulching techniques is a crucial first step in addressing the issues with irrigation and weed infestation in potato production. hatfield et al. (2001) observed a reduction in soil water evaporation of up to 50% due to mulching, which limited the requirement for crop irrigation. memon et al. (2017) reported the improvement of soil biodiversity and environmental advantages due to mulching, as it increased soil water infiltration and crop water availability. the hydrothermal conditions of the soil are influenced by plastic mulching, which raises soil warmth and reduces soil water evaporation (wang et al., 2005). mulching resulted in a higher germination rate and stronger seedlings, which raised the number of stems and branches per plant, resulting in a higher number of tubers at tuber initiation (ahmed et al., 2017). mulching with black plastic has been shown to improve potato stem number, plant height, and yield (bharati et al., 2020). black polyethylene mulch can increase soil temperature up to 9 °c, which stimulates quicker germination (kumari, 2012). black and silver-colored plastic mulches are reported to improve crop growth, yield, and quality more than other colored mulches (amare & desta, 2021). black plastic mulches were reported to have increased soil temperature, reduced weed competition, improved nutrient uptake, and improved soil moisture regimes, which resulted in more large-sized tubers being produced (ibarra-jiménez et al., 2011). silver plastic promoted the optimal root temperature for plant growth with the greatest efficiency of nitrogen utilization and the greatest tuber yield, whereas higher temperatures in black plastic depressed nitrogen metabolism as well as yield (ruiz et al., 1999). compared to black and white plastic mulch, silver plastic mulch had a greater par (photosynthetically active radiation) reflectance, and such increased par reflection by silver plastic mulches lowered root zone temperature, resulting in optimum soil temperature and reducing water loss (amare & desta, 2021). singh et al. (2015) reported that using plant residue as mulching material improved soil moisture by reducing soil water evaporation as a result of less solar radiation reaching the soil surface. further, crop residues (or plant litter) on the soil surface reduced soil erosion, improved soil quality, boosted water infiltration, and lowered the quantity of nutrients and pesticides that reached streams and rivers (delgado, 2010). similarly, zinc (zn) is an important plant mineral whose lack not only limits crop productivity but also has implications for human nutrition and health. zinc plays numerous key roles in plant growth, and a consistent and continuous supply is required for optimum growth and output. zinc deficiency impacts root growth, resulting in decreased water and nutrient absorption from the soil, resulting in reduced plant growth, nutrient composition, and yield (hacisalihoglu et al., 2004). it is essential for enhancing the quantity and quality of potato tubers. an all-india coordinated study on potato and micronutrient response showed that the yield response of potatoes to applied zn fertilizer varies with the soil type, variety, quantity of other major nutrients applied to the soil, and finally the method of application (mondal et al., 2015). according to sarkar et al. (2018), zinc loading in potatoes, both foliar and soil-applied, boosts zn content in potato tubers by 3–4 times, which is much greater than most generally recognized crops. zinc is critical for potato physiology, productivity, and postharvest quality, and its application in potatoes increased yield, average tuber weight, number of tubers, quality indices, and post-harvest indices (banerjee et al., 2017). zn fertilization has been shown to increase ascorbic acid concentration while decreasing tyrosine and total phenol levels in tubers, improving processing quality (sarkar et al., 2018). over-use of macronutrients (n, p, and k) in potato production results in serious depletion of different micronutrients in soil reserves (banerjee et al., 2017). also, diseases, insects, performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal january to april 2023 30 weeds, and other pests cause significant losses in agricultural yield and quality each year, which can be reduced by using suitable mulching and field management techniques, including appropriate fertilization (bharati et al., 2020). the combination of zinc sulfate and mulching may be the most effective strategy for conserving water and increasing vegetable yield (singh et al., 2019). improved growth characteristics as a result of zinc soil application and mulching could be attributed to increased photosynthetic and other metabolic activity, which leads to a rise in numerous plant metabolites responsible for cell division and elongation (hatwar et al., 2003). a lot of independent studies have been done to study the effects of n, p, and k fertilizer and mulching on potato production and quality in different areas of nepal. however, there haven’t been many studies attempting to determine the appropriate zn dose for potato cultivation under various mulch conditions. therefore, figuring out the right zn dosage for potato production under different mulching treatments is a crucial aspect of improving potato production in nepal. materials and methods description of the experimental site a field experiment was conducted during february to july, 2022 in rolpa municipality-4, liwang which is geographically located at 28.3000° n latitude and 82.6333° e longitude. the elevation of the study area was 1896 masl. geographically, the experimental location falls in the mid-hill region of lumbini province. in rolpa, area under cultivation is 1280 ha with production of 20 860 t and yield of 16.30 t/ha. physico-chemical characteristics of the experimental soil representative soil samples were taken at a depth of 15 cm to 25 cm from different parts of experimental fields using a soil sampling auger, and analysis of the sample soil was performed. the soil type was found to be silt-loam. chemical investigation revealed that the ph of the soil was acidic (6.4), the organic matter content was 2.1 % (moderate), the available nitrogen content was 181.3 kg/ha, the phosphorus content was 17.6 kg/ha, and the potassium content was found to be 126.4 kg/ha. the soil analysis indicated that zn was slightly deficient at 1.16 mg/kg. the details of the physico-chemical analysis of the research plot’s soil are depicted in table 1. weather condition during course of experiment fortnight interval average data on different weather parameters i.e., maximum and minimum temperatures, precipitation and relative humidity recorded during the potato growing season is presented in figure 1. experimental details the experiment was conducted in open field conditions using the variety rolpa local. the intention of using local variety was to promote local and to assess the compatibility of local variety with modern technologies of mulching and zinc application. table 1. soil chemical properties of experimental site at liwang, rolpa, 2022 soil parameter value methodology reference textural class silt loam soil textural triangle (usda) ph 6.4 glass-calomel electrode ph meter method (1:2 soil water ratio) (cottenie et al.,1982) organic carbon (%) 2.1 wet oxidation method walkley and black (1934) available n (kg/ha) 181.3 hot alkaline kmno4 method subbiah and asija (1956) available p (kg/ha) 17.6 0.5 m nahco3 extract olsen et al. (1954) available k (kg/ha) 126.4 neutral n nh4oh extract hanway and heidel (1952) available zn (mg/kg) 1.16 dtpa-tea (ph 6.4) extraction lindsay and norvell (1978) aryal, m., pandey, k. r., dhakal, s., tumbapo, s., & joshi. y. peruvian journal of agronomy, 7(1), 2741 (2023) https://doi.org/10.21704/pja.v7i1.1979 31 each plot accommodated 5 rows of plants, and 5 plants were present in each row of the plot with 45 cm × 20 cm of spacing, where the 1st and 5th rows were considered border rows and the middle 3 rows were taken as net plots. five plants were taken as sample plants randomly from the middle row and were tagged for biometrical and phenological observations, and four random plants from the net plot were selected for harvesting data. figure 1. weather condition during the course of experiment (nasa power, 2022) experimental design design of the experiment was two factorial rcbd consisting of 3 replications and 8 treatments with well-spaced 45 cm × 20 cm between row to row and plant to plant respectively. individual plot was of size 2.25 m2 × 1 m2 and the distance between two plots was 25 cm (figure 2) . figure 2. layout of experimental field and individual plot performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal january to april 2023 32 treatment details two factor are used to generate treatment combination (table 2, figure 3). table 2. treatment combination s.n. treatment(t) treatment combination 1 treatment 1(t1) no mulching and no zinc 2 treatment 2(t2) no mulching and 4 kg/ha of zinc 3 treatment 3(t3) plant residue and no zinc 4 treatment 4(t4) plant residue and 4 kg/ha of zinc 5 treatment 5(t5) black plastic and no zinc 6 treatment 6(t6) black plastic and 4 kg/ha of zinc 7 treatment 7(t7) silver on black plastic and no zinc 8 treatment 8(t8) silver on black plastic and 4 kg/ha of zinc factor 1: mulching m0: no mulching m1: plant residue m2: black plastic m3: silver on black plastic factor 2: zinc z0: 0 kg/ha z1: 4 kg/ha zinc (for zinc: zinc sulphate was used) cultural practices field preparation the soil was harrowed until completely free of weed residue. two plowings along with harrowing were done to make the soil soft, fine, well-drained, and well-aerated. manually, stubbles of previous crops and weeds were removed prior to sowing potato tubers. the layout of the field was done by making 24 plots manually using tapes and spades. plant population each plot consisted of 25 plants, and there were 600 plants in total in the whole experimental field. figure 3. establishment of experimental plots: (m0) no mulching, (m1) plant residue, (m2) black plastic and (m3) silver on black plastic fertilizer application urea, dap (di-ammonium phosphate), and mop (muriate of potash) were used as per the national recommended dose, i.e., 140:220:100 kg/ha. a full dose of phosphorus, potassium, and half aryal, m., pandey, k. r., dhakal, s., tumbapo, s., & joshi. y. peruvian journal of agronomy, 7(1), 2741 (2023) https://doi.org/10.21704/pja.v7i1.1979 33 the amount of required nitrogen and zinc (zinc sulfate) at 4 kg/ha were applied as basal doses, whereas the remaining half of the nitrogen was applied 45 days after planting. each plot was fed with 48.75 g of nitrogen, 48.75 g of phosphorus, and 22.5 g of potassium. the plot treated with zinc was fed with 2.75 g of zinc sulfate. seed rate and sowing healthy tubers of a local variety (rolpa) were selected for the experiment. one tuber per hill was sown manually in all rows. on march 3, 2022, tubers were planted. seed tubers were planted on raised beds. irrigation the frequency of irrigation depends on the type of soil and the season’s rainfall. irrigation was applied as needed, and the requirement was fully maintained. earthing up and weeding as the research was based on mulching materials, no earthing up or weeding was done. control practices cutworm infestations were occasionally observed in the research field. the physical handpicking method was used to control cutworms. they were manually pulled out from the soil surface just below the base of the stem and destroyed. data observation recorded in potato phenological traits such as germination percentage, plant height, number of leaves and canopy diameter and yield attributing characters such as total tuber number per hill, tuber weight, tuber diameter, length and circumference were recorded. economic analysis total cost of cultivation was calculated on the basis of local charges for different agro inputs viz. labor, fertilizer, mulching materials, seed tubers and other necessary materials. economic yield of potato was expressed into gross returns (nrs/ha) on the basis of local market price and net return was calculated by deducting the cost of cultivation from the gross returns and expressed in nrs/ha. and then, the benefit-cost (b:c) ratio was calculated by using following formula: benefit cost ratio (b:c) = gross returns/total cost of cultivation statistical analysis the collected data was processed by ms excel and analyzed by using r-studio. all the recorded data were subjected to analysis of variance (anova) and duncan’s multiple range test (dmrt) for mean separation. the significance differences among the means were tested using least significant difference (lsd) at 5 % level of significance (gomez & gomez, 1984). results and discussion morphological parameters germination the analyzed data revealed that the germination percentage at 30 dap was significantly higher in the plot with mulching in comparison to the control. the highest germination percentage was observed in black plastic (94.67%), which was statistically similar to silver on black plastic (94.000%), while the germination percentage in plant residues (86.00%) was observed to be superior to the control (75.33%) plot. the higher germination in the mulch condition might be due to an increase in soil temperature as compared to the control condition. the increased soil temperature caused by the application of various plastic mulches resulted in faster germination, flowering, and physiological maturity in mulched conditions. black plastic mulch can raise soil temperatures by up to 9 degrees celsius, promoting faster germination (bhatta et al., 2020; kumari, 2012). mulching has a more positive effect on germination at 30 dap than the control condition (mahmood et al., 2002). these results are also correlated with the findings of ping et al. performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal january to april 2023 34 (1994), who observed a faster germination rate in the plot with mulching. number of leaves from the table of data analyzed, it shows that at 45 dap, the highest number of leaves was found on silver on black plastic (34.29), which was statistically similar to black plastic (32.33) as compared to the control plot, which remained the same at 60 dap. at 75 dap, all the mulching material has a higher number of leaves as compared to the control plot. the highest number of leaves was found in silver on black plastic (145.70), which was statistically similar to black plastic (129.95) as compared to the control plot (76.62). throughout all days of data collection, it was observed that the number of leaves in the plots with plant residues was greater than in the control plots (table 3). ruíz-machuca et al. (2015) discovered similar results, confirming that black and silver plastics are effective in increasing leaf area when compared to bare land. the positive impact of these plastic mulches in enhancing leaf area would increase the rate of photosynthesis, plant yield, and quality. table 3. number of leaves of potato as influenced by different mulching materials and zinc level at liwang, rolpa, 2022 treatment germination percentage number of leaves plant height 45 dap 60 dap 75 dap 45dap 60 dap 75 dap mulching silver on black plastic 94.00a 34.29a 57.41a 145.70a 43.20a 64.35a 87.28a black plastic 94.67a 32.33a 50.95a 129.95a 40.29ab 56.71b 79.71b plant residue 86.00b 25.41b 36.04b 107.12b 38.08b 53.58c 74.42b control 75.33c 23.50b 30.58b 76.62c 33.04c 50.20d 64.86c sem (+-) 0.49 0.34 0.56 1.38 0.21 0.23 0.43 lsd 5.90*** 3.64*** 6.70*** 16.62*** 3.85*** 2.82*** 5.32*** cv 5.45 11.09 12.45 11.76 5.23 4.05 5.61 zinc 4 kg/ha 88.33a 29.29a 45.10a 119.45a 40.63a 57.08a 78.22a 0kg/ha 86.67a 28.47a 42.39a 110.25a 39.92a 55.34a 71.67a sem (+-) 0.69 0.46 0.78 1.95 0.30 0.32 0.62 lsd 4.17ns 2.78ns 4.74ns 11.75ns 1.84ns 1.99ns 3.76ns cv 5.45 11.09 12.45 11.76 5.23 4.05 5.61 grand mean 87.5 28.88 43.75 114.85 40.28 55.67 76.57 note: lsd: least significant differences, sem (±): standard error of mean, cv: coefficient of variation, means with different letters in columns are significantly different at p <0.05 level by dmrt. *significant at p <0.05 and ** significant at p<0.01 level. ***significant at p<0.001. ns: nonsignificant. plant height the result of the statistical analysis showed that there was a significant effect of mulching materials on plant height. on evaluating the plant height at different daps, the highest plant height was observed in both plastic mulches as compared to the rest of the treatments. at 45, 60, and 75 dap, the plant height observed in silver on black plastic (43.20 cm, 63.22 cm, and 84.00 cm, respectively) was significantly higher than that of the control plot at 45 dap, 60 dap, and 75 dap (33.04 cm, 48.37 cm, and 61.62 cm, respectively). similarly, higher plant height was obtained in plots treated with black plastic and plant residues than in control plots. the growth rate was better and huge in all treatments where mulching was applied (mahmood et al., 2002). the higher soil temperature under plastic mulches leads to an active metabolism in the plants, with an increase in the amount of nutrients absorbed and an enhancement of growth parameters. the enhanced plant height in mulched plants could be attributed to the mulches’ improved availability of soil moisture and optimum soil temperature (joshi et al., 2020). changes in the plant height of potatoes have been observed when using aryal, m., pandey, k. r., dhakal, s., tumbapo, s., & joshi. y. peruvian journal of agronomy, 7(1), 2741 (2023) https://doi.org/10.21704/pja.v7i1.1979 35 different mulches, and plastic mulch increased the plant height more than other mulches. canopy diameter at 45 dap and 60 dap, canopy diameter was found to be widest in silver on black plastic (21.06 cm and 29.77 cm, respectively), which was statistically similar with black plastic (19.78 cm and 28.43 cm, respectively) and has a significantly wider canopy diameter as compared to the control plot (17.41 cm and 24.23 cm, respectively). at 75 dap, the widest canopy diameter was found in silver on black plastic (32.93), which was statistically similar to black plastic (30.91) and plant residue (30.17), and the lowest canopy diameter was found in the control plot (27.18). in all observations, all the mulching materials have shown a wider canopy diameter as compared to the control plot (table 4). table 4. canopy diameter of potato as influenced by different mulching materials and zinc level at liwang, rolpa, 2022 treatment canopy diameter 45 dap 60 dap 75 dap mulching silver on black plastic 18.77a 29.77a 32.93a black plastic 17.43ab 28.43ab 30.91a plant residue 15.44bc 26.52bc 30.17a control 13.73c 24.23c 27.18b sem (+-) 0.23 0.25 0.22 lsd 2.82** 3.02** 2.69** cv 13.94 8.97 7.17 zinc 4 kg/ha 16.96a 27.71a 30.44a 0kg/ha 15.73a 26.77a 30.15a sem (+-) 0.32 0.35 0.31 lsd 1.99ns 2.14ns 1.90ns cv 13.94 8.97 7.17 grand mean 16.34 27.24 30.30 note: lsd: least significant differences, sem (±): standard error of mean, cv: coefficient of variation, means with different letters in columns are significantly different at p <0.05 level by dmrt. *significant at p <0.05 and ** significant at p<0.01 level. ***significant at p<0.001. ns: nonsignificant. under all observations, germination percentages, number of leaves, plant height, and canopy diameter were found to be higher in plots with 4 kg/ha zinc in comparison to control plots; however, the difference was not significant. zinc immobility may be the cause of the plant’s inability to respond in its early stages. similar results were reported by bari et al. (2001); sarkar et al. (2018) and islam et al. (2021). tuber characteristics the circumference, length, and diameter of potatoes were found to be significantly higher in silver on black plastic (15.35 cm, 6.01 cm, and 4.74 cm, respectively), and the lowest circumference was found in the control plot (12.16 cm , 4.56 cm, and 3.68 cm, respectively). there was no significant difference between plant residue and control plots in case of tuber characteristics (table 5). potatoes are sensitive to high soil temperatures and need optimum soil temperatures for tuber initiation and bulking (dvořák et al., 2012). silver plastic mulches are better at reflecting par (photosynthetically active radiation) than black and white plastic mulches. such higher reflection of par by silver plastic mulches reduces root zone temperature, which creates optimum soil temperature and reduces water loss (amare & desta, 2021). the circumference, length, and diameter of potatoes were found to be significantly greater in the zinc-treated plot (14 cm, 5.46 cm, and 4.25 cm, respectively) than in the control plot performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal january to april 2023 36 (13.19 cm, 5.07 cm, and 4.02 cm, respectively). the translocation of assimilates becomes higher in later stages (i.e., in developing tubers) by the application of zinc, which ultimately leads to an increase in tuber size (sarkar et al., 2018). tuber number when compared to the control plot, there were more tubers after mulching. a significantly higher number of tubers per plant was found in the silver on black plastic plot (22.16), and the lowest number of tubers per plant was found in the control plot (11.70). as compared to the control plot, the tuber number in plot treated with plant residue was higher.tuber number was reported to be higher in mulched conditions than in non-mulched conditions because mulching helps to regulate temperature and maintain adequate environmental conditions, which is in accordance with the findings of sekhon et al. (2020). application of zinc at a level of 4 kg/ha has shown a significantly higher tuber number (18.39) as compared to the control plot (15.95). zinc fertilization may have enhanced cytokinin concentration as well as the rate of photosynthesis and respiration, which in turn eliminated apical dominance and resulted in more tubers per plant (sarkar et al., 2018). yield attributed characters the analysis of interaction effect of mulching and zinc on yield per plant is not significant (table 6), so we analyze main effects. a plot with mulching has shown significantly higher tuber weight, yield per plant, and total yield. the highest tuber weight and yield per plant were obtained in silver on black plastic (75.93 g and 465.86 g, respectively), and the lowest tuber weight and yield per plant were recorded in the control plot (39.01 g and 244.82 g, respectively). similarly, a significantly higher total yield was obtained in silver on black plastic (21.83 t/ha), and the lowest yield was found in the control plot (11.72 t/ha). the yield attributed characters i.e. tuber weight, yield per plant and total yield was higher in black plastic and plant residue as compared to control plots (table 7). the lower yields in black plastic compared to silver on black plastic may be due to excessive rainfall during the research and higher temperatures during tuberization and tuber growth (kapoor, 2012). it was also reported that silver and white colored plastic mulches outperformed black colored plastic mulch in tuber yield of potatoes. yield in plastic mulch might be due to maximum temperature, maximum water retention in the soil, and minimum growth of the weed, which table 5. tuber characteristics of potato plant as influenced by different mulching materials and zinc level at liwang, rolpa, 2022 treatment tuber data number of tubers per plantcircumference length diameter mulching silver on black plastic 15.35a 6.01a 4.74a 22.16a black plastic 13.96b 5.55b 4.25b 18.62b plant residue 12.91c 4.95c 3.87c 16.20c control 12.16c 4.56c 3.68c 11.70d sem (+-) 0.063 0.028 0.017 0.19 lsd 0.76*** 0.34*** 0.20*** 2.37*** cv 4.72 5.22 4.084 11.15 zinc 4 kg/ha 14.00a 5.46a 4.25a 18.39a 0kg/ha 13.19b 5.07b 4.02b 15.95b sem (+-) 0.089 0.039 0.024 0.27 lsd 0.54** 0.24** 0.14** 1.67** cv 4.55 5.22 4.084 11.15 grand mean 13.6 5.27 4.13 17.17 note: lsd: least significant differences, sem (±): standard error of mean, cv: coefficient of variation, means with different letters in columns are significantly different at p <0.05 level by dmrt. *significant at p <0.05 and ** significant at p<0.01 level. ***significant at p<0.001. ns: nonsignificant. aryal, m., pandey, k. r., dhakal, s., tumbapo, s., & joshi. y. peruvian journal of agronomy, 7(1), 2741 (2023) https://doi.org/10.21704/pja.v7i1.1979 37 coincides with the findings of ahmed et al., (2017). ruíz-machuca et al. (2015) also found a significant effect on yield for silver on black, followed by white on black mulching. table 6. analysis of variance of yield attributing characters of potato plant as influenced by different mulching materials and zinc level at liwang, rolpa, 2022 source yield attributes df tuber weight yield per plant total yield replication 2 83.10 2975* 3.819 mulch(a) 3 1618.87*** 52227*** 108.842*** zinc(b) 1 321.42** 7930** 15.990** a*b (mulch: zinc) 3 29.71 490 0.133 error 14 28.93 591 1.403 note: *significant at p <0.05, ** significant at p<0.01 level, ***significant at p<0.001, ns: nonsignificant. table 7. yield attributing characters of potato plant as influenced by different mulching materials and zinc level at liwang, rolpa, 2022 treatment yield attributing characters benefit-cost ratio tuber weight (g) yield per plant (g) total yield (t/ha) mulching silver on black plastic 75.93a 465.86a 21.83a 3.18a black plastic 60.51b 376.48b 18.27b 2.28b plant residue 45.73c 319.40c 15.71c 1.75c control 39.01d 244.82d 11.72d 1.13d sem (+-) 0.54 2.48 0.12 0.019 lsd 6.66*** 30.10*** 1.46*** 0.23*** cv 9.72 6.91 7.01 9.16 zinc 4 kg/ha 58.95a 369.82a 17.70a 2.26a 0kg/ha 51.63b 333.46b 15.66b 1.91b sem (+-) 0.77 3.50 0.17 0.027 lsd 4.70** 21.28** 1.03** 0.16*** cv 9.72 6.91 7.01 9.16 grand mean 55.29 351.64 16.88 2.09 note: lsd: least significant differences, sem (±): standard error of mean, cv: coefficient of variation, means with different letters in columns are significantly different at p <0.05 level by dmrt. *significant at p <0.05 and ** significant at p<0.01 level. ***significant at p<0.001. ns: nonsignificant. similarly, application of zinc at a level of 4 kg/ha has shown significantly higher tuber weight (58.95 g), yield per plant (369.82 g), and total yield (17.70 t/ha) as compared to the control plot (51.63 g, 333.46 g, and 15.66 t/ha, respectively). in an earlier study, kumar et al. (2008a) meticulously recorded the favorable effect of znso4 on crop development, increased synthesis, and food material translocation to developing tubers. later research revealed that using zn aided in increasing the average weight of individual tubers from small to medium and medium to large size (kumar et al., 2008b). the increased tuber yield of potatoes might be attributed to the beneficial effect on tuberization as a result of zn application (mondal et al., 2015; singh et al., 2009). benefit-cost ratio the highest b:c ratio was found in plots with silver on black plastic (3.18), followed by black plastic (2.28), plant residue (1.75), and the lowest on a control plot (1.13). similarly, the b:c ratio was found higher in plots with a 4 kg/ha level of zinc (2.26) than control plots (1.91). chaudhary et al. (2022) also reported the highest gross return and a promising b:c ratio of 2.75 on silver on black plastic mulch conditions in comparison to other mulch conditions, which is in accordance with the findings of this study. performance of potato variety rolpa local (solanum tuberosum l.) under different mulching conditions and zinc levels at rolpa, nepal january to april 2023 38 conclusion mulching activities have a positive impact on microclimates and contribute to the maintenance of better growing environments, which is imperative for increasing crop productivity. the use of appropriate mulching practices is a necessary precursor to addressing the irrigation and weed infestation problems in potato production. zinc application further enhances potato tuber quantity and quality. the study concluded that mulching was effective in enhancing the vegetative growth as well as the overall yield of potatoes. among the various mulching materials tested, silver on black plastic and black plastic were the most effective in increasing tuber yield. the interaction effect of mulching and zinc on growth and yield parameters was found to be non-significant. despite having a non-significant effect, it leaned towards the positive effect of using them together. no such declination was seen in the yield or any other parameters. consequently, to maximize profit, it would be advantageous to use silver on black plastic mulch as the best mulching material and 4 kg of zinc per hectare to augment the yield. furthermore, as the research was carried out in only one season at a particular location, future studies should emphasize the investigation of different mulching materials in different agroecological zones for validation. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. id orcid and emails: muna aryal arylmuna93@gmail.com https://orcid.org/0000-0001-7436-4964 krishna raj pandey krishnapandey2055@gmail.com https://orcid.org/0000-0001-6862-3421 suman dhakal sdhakal@afu.edu.np sanjeev tumbapo limboosanjeev51@gmail.com yagya raj joshi yagyarajjoshi9335@gmail.com https://orcid.org/0000-0002-9421-4346 acknowledgments we sincerely acknowledge mr. mitralal paudel, senior agricultural officer, pmamp, rukum east, mr. bharat bhandari, laxmi magar, and the entire staff of pmamp, rolpa, for their great support, inspiration, supervision, and suggestions throughout the entire research period. also, we would like to express our gratitude to mr. bharat mani pokharel, mr. obilal thapa, mr. krishna pd. pokharel, mr. prabin chaudhary, and the entire staff of the agricultural knowledge centre in rolpa for their continuous guidance and support. we owe a great deal of gratitude to numerous solicitous hands, whose contributions have indeed been crucial. references ahmed, n. u., mahmud, n. u., hossain, a., zaman, a. u., & halder, s. c. 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(2022). bibliometric analysis of potato research publications from agronomy category based on web of science from 2000 to 2021. potato research, 65(2), 233–253. https://doi. org/10.1007/s11540-021-09521-0 https://doi.org/10.1016/j.fcr.2017.11.027 https://doi.org/10.1016/j.fcr.2017.11.027 https://doi.org/10.1021/jf981314x https://doi.org/10.1021/jf981314x https://doi.org/10.1080/09064710.2014.960888 https://doi.org/10.1080/09064710.2014.960888 https://doi.org/10.31018/jans.v10i3.1863 https://doi.org/10.31018/jans.v10i3.1863 https://doi.org/10.1016/j.fcr.2019.06.001 https://doi.org/10.1016/j.fcr.2019.06.001 https://doi.org/10.1007/s11540-015-9298-0 https://doi.org/10.1007/s11540-015-9298-0 https://doi.org/10.56093/ijas.v89i2.87088 https://doi.org/10.56093/ijas.v89i2.87088 https://doi.org/10.3126/ajn.v2i0.7533 https://doi.org/10.1016/j.agwat.2005.02.006 https://doi.org/10.1016/j.agwat.2005.02.006 https://doi.org/10.1007/s11540-021-09521-0 https://doi.org/10.1007/s11540-021-09521-0 peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i3.1976 received for publication: 05 october 2022 accepted for publication: 30 november 2022 published: 31december 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by sustainability of amazonian fruit trees plots in loreto, peru sustentabilidad de parcelas con frutales amazónicos en loreto, perú aldi alida guerra-teixeira1*; julio césar alegre-orihuela2; alida ivelice vásquez-guerra3 *corresponding author:aldiguerra@gmail.com *https://orcid.org/0000-0003-3070-2807 abstract this research aimed to: a) evaluate the vegetation indicators of amazonian fruit trees with the diversity of weeds and soil with the number of arthropods in the plots of farmers that were established by inia and iiap in the area of influence of the road iquitos nauta from the loreto state, perú, and b) evaluate the carbon sequestration, and the socio-economical and ecological sustainability of the plots with amazonian fruit trees in the area of influence., thirty-seven farmers were surveyed. the alpha indicator of vegetation diversity reached an index value of 2.07 and the arthropod index was 1.91. the highest carbon (c) value in the biomass was found in the guava fruit tree (inga edulis mart.) in plot 17, with a total carbon stock of 90 t·ha-1 and a co2 flux value of 22 t·ha -1·a-1. additionally, uvilla (pouroma cecropiifolia mart.) with pijuayo (bactris gasipaes kunth) reached a total c uptake of 117.19 t·ha-1 with a co2 flow of 33.42 t·ha -1·a-1. the highest accumulation of c in the soil was in plot 23, with 66.5 t·ha-1, reducing co2 emission by 243.84 t·ha -1·a-1. in the sustainability evaluations, it was found that the economical, ecological, and socio-cultural dimensions reached values of 3.20, 3.33, and 2.04, respectively. it is concluded that amazonian fruit trees are sustainable in the economic and ecological dimensions. however, in the sociocultural dimension, sustainability is weak. key words: sustainability, biodiversity, arthropods, carbon, amazonian fruit trees. resumen la investigación tuvo por objetivos: a) determinar los indicadores de diversidad en vegetación de los frutales amazónicos y diversidad de artrópodos del suelo en las parcelas de agricultores que fueron instalados por el inia e iiap en el área de influencia de la carretera iquitos – nauta, loreto. b) determinar el secuestro de carbono en parcelas con frutales amazónicos en el área de influencia de la carretera iquitos – nauta, loreto. c) evaluar la sustentabilidad económica, ecológica y social de las parcelas con frutales amazónicos en el área de influencia de la carretera iquitos – nauta, loreto. el área de estudio en la región loreto – perú, se encuestó a 37 agricultores. el indicador alfa de diversidad en la vegetación, alcanzó el valor del índice 2.07 y el índice de artrópodos fue de 1.91. los mayores valores de carbono (c) en la biomasa se encontró en el frutal de guaba (inga edulis mart.) en la parcela 17, con una reserva total de carbono de 90 t·ha-1 y con valor de flujo de co2 de 22 t·ha-1·a-1. la asociación de uvilla (pouroma cecropiifolia mart.) con pijuayo (bactris gasipaes kunth) captó en total 117.19 t·ha-1 con un flujo de co2 de 33.42 t·ha -1·a-1. la mayor acumulación 1 universidad nacional de la amazonía peruana. academic department of soils and crops. 2 universidad nacional agraria la molina. faculty of agronomy, academic department of soils, la molina, perú 3 universidad nacional de la amazonía peruana. how to cite this article: guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. (2022). sustainability of amazonian fruit trees plots in loreto, peru. peruvian journal of agronomy, 6(3), 239–255. https://doi.org/10.21704/pja.v6i3.1976 sustainability of amazonian fruit trees plots in loreto, peru september december 2022 240 de c en el suelo fue en la parcela 23 con 66.5 t·ha-1, reduciendo la emisión de co2 en 243.84 t·ha-1·a-1. las evaluaciones de sustentabilidad en la dimensión económica alcanzaron el valor de 3.20, la dimensión ecológica alcanzó el valor de 3.33 y la dimensión sociocultural alcanzó el valor de 2.04. se concluye que los frutales amazónicos son sustentables en la dimensión económica y ecológica. sin embargo, en la dimensión sociocultural presenta sustentabilidad débil. palabras clave: sustentabilidad, biodiversidad, artrópodos, carbono, frutales amazónicos. i. introduction amazonian fruit trees are considered promising because of their great nutritional potential, and have suitable characteristics to be transformed into sustainable crops since they can be used in conservation and ecosystem restoration practices, especially as part of agroforestry systems in the amazon where, in a more orderly manner, they are established in association with other agricultural and forestry crops (gonzález, 2013). plots with amazonian fruit trees are intended to satisfy food needs and generate income for producers through their commercialization (jadán, 2012). these species are high in carbohydrates, vitamins and minerals, which help prevent many diseases (gonzales & torres, 2011). in the area of the iquitos nauta road, in the loreto region, amazonian fruit trees are not only of great economic and nutritional importance (araujo et al., 2021), but also environmentally important. in these plots, crop and space rotation is practiced to give soil periodic fallow, which allows the growth of secondary forests, with the consequent recovery of nutrients and the diversity of arthropods and other components of the soil macrofauna. in this sense, it is necessary to study these production units with sustainability criteria, i.e., considering the social, environmental and economic dimensions. the sustainability of agricultural production units presents different methodologies, one of the most used is the “multi-criteria analysis” proposed by sarandón et al. (2006). in peru, this methodology has been used to study the sustainability of agricultural farms in the amazonian region, such as oil palm farms from the shanusi valley in loreto (leveau, 2018), coffee farms from san martin (rojas et al., 2021) and granadilla farms from oxapampa (romero, 2018). according to pinedo et al. (2021), the “multi-criteria analysis” is the most widespread and applied methodology in the evaluation of sustainability because it is low cost, easy to apply and adaptable to the evaluation of agricultural production systems in latin american countries. agriculture as a sustainable economic activity must: achieve efficient crops, in order to be more productive and profitable so as to ensure future viability; recover more arable land, preserving the quality of the natural resources it uses (water, soil, soil slope, etc.) by minimizing its impact on nature (erosion, pollution, desertification, etc.) and extending biodiversity; train farmers in good practices and safe use of technologies, provide a transfer of appropriate technological knowledge to achieve increasingly sustainable agriculture (syngenta, 2021). sustainable agriculture seeks to improve the quality of the environment; in other words, it is directly related to the protection of natural resources, for food and energy security, and is conditioned by the provision of natural resources and environmental services in a geographical area (duran, 2010). on the other hand, karamelikli (2016), analyzes that, sociocultural sustainability is in relation to environmental sustainability, because it has a harmonious relationship between living organisms and the natural environment, likewise, it has to be sufficiently productive to meet the basic needs and reasonable desires of the entire world population, to enhance the occupation of the countryside and rural development, to be compatible with the preservation of cultural diversity, to promote social equity, etc. likewise, in the plots, with amazonian fruit trees, there are diverse species of fauna, flora and varied ecosystems. preserving this biodiversity is important because it guarantees the sustainability of all forms of life and contributes to the stabilization of other ecosystems in the region. regarding diversity studies, lingmann & jankowski (2014), have described the scale of local spatial analysis and multicriteria spatiotemporal sensitivity to assess the suitability of land by weights that express probability distributions of habitat characteristics to define the guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. peruvian journal of agronomy, 6(3), 239–255 (2022) https://doi.org/10.21704/pja.v6i3.1976 241 inclusion and exclusion of areas with suitability in land use. dyer et al. (2017) stated that within the studies of biodiversity in agriculture, the appropriate methodology is the measurement of alpha diversity, which studies the diversity within the same community at the same habitat, and the shannon index (hʹ) that is the way to calculate the proportion of coverage from each species in a given area. the study of arthropods is important in ecosystem services since it has the function of maintaining the balance in ecosystems (cerna et al., 2015). iannacone et al. (2000), stated that earthworms are sensitive to many agricultural practices, so they can be considered indicators for measure soil fertility. soil functions are affected in agroecosystems, such as water and air dynamics. biogeochemical processes, microorganisms and soil itself define soil quality (dexter, 2004). hernández & vargas (2005) indicate that there are international mechanisms for different countries to buy and sell co2 absorption services, which is an important source of financing to protect and conserve resources and the environment, while at the same time improving the economy. changes in soil quality can be monitored with easy and accurate indicators determined by reproducible methods (moebius et al. 2007). additionally, it is important to develop and promote appropriate cultural practices in amazonian fruit orchards to improve the livelihoods of farmers, but also to reduce co2 emissions and retaining carbon, thus providing important environmental services to the society (food and agriculture organization, 2003). on the other hand, conservación internacional (2021) indicates that companies should reduce their carbon footprint and offer offsets to reduce carbon emissions. this is an opportunity for the amazonian fruit tree production plots to venture into these offers to improve their economic income. to estimate the amount of carbon sequestration in the biomass and soil, there are several allometric models, where it is related to the dry mass of a component or the entire tree, with variables of tree size, such as diameter at breast height, height, basal area and volumen (alegre et al., 2000; arévalo et al., 2002). iiap (2002), conducted a study on the evaluation of carbon in the nanay river basin, in which it reported a value of 104.03 tons/ ha in ‘varillales’ ecosystems and 226.19 t/ha in ‘aguajales’ ecosystems garcía et al., (2012), studied the total soil organic carbon stock (cos), aerial biomass and necrosome in the aguajales (mauritia flexuosa l.) of the lower and upper part of the aguaytía river basin, in the department of ucayali, peru. they found that the upper zone presented less total carbon (3.78 t/ha) compared to the lower zone, which showed significantly higher values (197.86 t/ha). in addition, aerial biomass showed significantly similar data for both zones, being 96.33 t/ha for the low zone and 51.28 t/ha in the high zone. values for necrosome were lower in both zones. they also determined the amount of carbon accumulated in the aerial biomass, with a result of 96.33 t/ha. in economic sustainability evaluation, tongo & soplin (2022) indicates that the economic dimension indicator was lower than environmental dimension and social dimension indicator but was lower variable too, in livestock production systems in the high jungle of peru. therefore, the objective of this study was to evaluate the sustainability of amazonian fruit tree plots in production located along the iquitos nauta highway, loreto, as an alternative to migratory agriculture that preys on the amazon. for this is necessary analyze economic, social and environmental dimension of the agroecosystem. ii. material and methods the study was conducted in a humid tropical zone the province of maynas in the department of loreto, located in northwestern peru, in the area of influence of the iquitos nauta road, which extends from the district of san juan bautista to the jurisdiction of the city of nauta, covering approximately 96 km. it has altitude of 121 m.a.s.l., average annual temperature of 27.3 ºc, average annual relative humidity of 91.6 % and average annual precipitation of 3 282.2 mm·a-1 (holdrige et al.,1971). for the research, the population centers with the largest number of inhabitants were chosen, settled on the left margin of the iquitos nauta road and the right margin of the itaya river, for which a qualitative study of 37 cases was carried out. the information on sustainability was obtained through a questionnaire with structured questions on technical and socioeconomic sustainability of amazonian fruit trees plots in loreto, peru september december 2022 242 aspects, considering the indicators proposed by sarandón et al. (2006). this scale ranges from 0 to 4, where 0 is considered less sustainable and 4 more sustainable. the selection of subindicators, variables and the weighting of each one of these, was carried out jointly with those responsible for the plots under study and the support of technicians and professionals from the agrarian agency of the province of maynas. sustainability data was analyzed through the economic, ecological and sociocultural formulas proposed by sarandón (2006). the sub-indicators, variables and valuation scales to evaluate the economic sustainability of the plots producing amazonian fruit trees on the iquitos nauta highway can be seen in the table 1. on the other hand, table 2 shows the sub-indicators, variables and valuation scales to evaluate the ecological sustainability of the plots producing amazonian fruit trees on the iquitos nauta highway. for its part, table 3 shows the sub-indicators, variables and valuation scales to assess the sociocultural sustainability of the plots producing amazonian fruit trees on the iquitos-nauta highway. finally, following the criteria of sarandón et al. (2006), who considers that, by replacing the values of the indicators in the respective formulas, the general index of sustainability is determined, considering sustainable when its indicators and the general index of sustainability are higher of 2. likewise, it should be noted that none of the three dimensions evaluated should have an indicator with a value less than 2. general sustainability index was calculate with the economic (ik), environmental (ia) and sociocultural (isc) indexes, whose formulas are shown in table 4. the alpha diversity of vegetation and soil arthropods surrounding the amazonian fruit trees was also evaluated using the formula proposed by shannon weaver. for vegetation, samples were extracted using the wood quadrant method with dimensions of one square meter (1 m2) and five points were randomly distributed internally, each point was considered weed subsamples totaling 25 subsamples. the samples were introduced in paper bags and then in plastic bags, duly labeled to later be transferred to the laboratory of the herbarium amazonense of the national university of the peruvian amazon (unap), where the identification of the vegetations found was carried out. to evaluate the macrofauna of the soil surface of the surrounding environment of the amazonian fruit trees, the sampling was systematic and consisted of delineating a quadrant of 25 m2 around the base of the fruit trees. the arthropods were collected with the help of the entomological mesh and support of the hand. the specimens were placed in a glass jar with 70 % alcohol, then labeled and taken to the plant health laboratory of the faculty of agronomy unap, where they were separated by counting the total number of individuals per species and identified by table 1: sub-indicators, variables and valuation scales to assess the economic sustainability of plots producing amazonian fruit trees on the iquitos nauta highway economic dimension assessment scales 0 1 2 3 4 a: economic income from the plots a1: diversification of amazon fruit trees has one fruit tree has 2 fruit trees has 3 fruit trees has 4 fruit trees has more than 4 fruit trees a2: self-consumption production area lower than 0,5 hectare 0,60 ha to 1 ha 1.1 ha to 1,5 ha 1.5 ha to 2,0 ha more than 2 hectares b: monthly net income per producer self-consumption lower than 1000 soles / hectare 1001 to 1500 soles/hectare 1501 to 2000 soles/hectare more than 2000 soles/hectare c: economic risk c1: diversification for sale one fruit tree 2 fruit trees 3 fruit trees 4 fruit trees more than 4 fruit trees c2: number of marketing channels for sale no way of marketing one avenue of marketing 2 avenues of marketing 3 avenues of marketing more than 3 avenues of marketing c3: dependence on external inputs 76 % to 100% 51 % to 75 % 26 % to 50 % 1 % to 26 % 0 % source: adapted from sarandon (2006) guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. peruvian journal of agronomy, 6(3), 239–255 (2022) https://doi.org/10.21704/pja.v6i3.1976 243 table 2: sub-indicators, variables and valuation scales to evaluate the ecological sustainability of plots producing amazonian fruit trees on the iquitos nauta highway ecological dimension assessment scales 0 1 2 3 4 a: soil life conservation a1: organic remains on the ground more than 25 % coverage 49 % to 25 % coverage 75 % to 50 % coverage 99 % to 74 % coverage 100 % coverage a:2 vegetated ground cover time no permanent coverage eventually covered three months of coverage six months of coverage with permanent coverage a3: diversity of amazon fruit trees monoculture association a fruit tree with one crop association of 2 fruit trees with a crop association of 2 fruit trees with 2 crops association of more than 2 fruit trees with more than 2 crops b: erosion risk b1: predominant slope more than 45 % 30 % to 45 % 15 % to 30 % 5 % to 15 % 0 % to 5 % b2: vegetal cover more than 45 % 46 % to 25 % 74 % to 47 % 99 % to 75 % 100 % c: biodiversity management c1: spatial biodiversity without diversity little diversity moderate di-versity high diversity very high diversity c2: temporary biodiversity negligible biological activity little biological activity moderate biological activity high biological activity very high biological activity source: adapted from sarandon (2006) table 3: subindicadores, variables y escalas de valorización para evaluar la sustentabilidad sociocultural de las parcelas productoras de frutales amazónicos en la carretera iquitos -nauta sociocultural dimension assessment scales 0 1 2 3 4 a: satisfaction of basic needs a1: access to health no access with post access a2: access to education no access with access to initial education with access to primary education with access to secondary education a3: living place no access with access a4: services no access access a service access two services access three services access to more than three services b: production system acceptability b1: degree of satisfaction to the needs dissatisfied indifferent to satisfaction a bit satisfied satisfied very satisfied b2: youth desertion greater than 50 % 50 % desertion 25 % desertion 15 % desertion 0 % desertion c: social integration to organizational systems c1: associativity no association associated with an institution associated with two institutions associated with three institutions associated with more than three associations c2: active participation no participation little participation moderate participation high participation very high participation c3: voting equality there is no equality little practice of equality moderate equality practice high equality practice very high equality practice c4: political integration without integration little integration moderate integration high integration very high integration c5: knowledge acquisition communal ignorance little communal knowledge moderate communal knowledge high communal knowledge very high communal knowledge c6: community agenda control ignorance of the communal plan little knowledge of communal plan moderate knowledge of communal plan high knowledge of communal plan very high knowledge of communal plan d: knowledge and ecological awareness unknown residual management little residual management moderate residual management high residual management very high residual management source: adapted from sarandon (2006) and dahl (2008). sustainability of amazonian fruit trees plots in loreto, peru september december 2022 244 families. the diversity index in species richness, both of the vegetation and of soil arthropods, was used the formula proposed by shannon-weaver (1949) which is presented below: s = number of species (richness) pi = proportion of individuals of the species/ respect to the total number of individuals ni/n ni = number of individuals of the species i n = number of all individuals of all species the carbon sequestration in biomass and soil also was determined in the study area using the allometric equations proposed by arévalo et al. (2002) and rueda (2014) for biomass, and the proposed by walkley (1947) for soil. for the evaluation of the plant biomass, the presence of litter at the base of the amazonian fruit trees was calculated, adding to the calculation of the tree biomass. for the calculation of litter biomass, 1 square meter grid was used, randomly placed at the base of the fruit trees, the total fresh litter matter contained in the grid was weighed, and from this total a sample was extracted. fresh sub-sample of approximately 300 g which was labeled to be sent to the laboratory, dried in a recirculating air oven at 70 °c for 48 hours and thus the weight of dry matter of the subsamples was obtained. likewise, the tree biomass was calculated, using allometric equations, proposed by arévalo et al. (2002). the allometric equation for litter biomass was: bh(t/ha) = ((psm/pfm) x pft) x 0.04 bh = litter biomass in dry matter psm = dry weight of the sample collected g pfm = fresh weight of the sample collected g pft = total fresh weight per 0,25 m2 0.04 = conversion factor and for biomass of living trees that do not have an allometric equation, the following formula was used: ba= (0,1184 dap2,53) x 0.01 ba = living tree biomass 0,1184 = constant dap = diameter at chest height 2,53 = exponential constant table 4: sustainability calculation formulas and general index indicators formula economic (ik) ecological (ie) sociocultural (isc) general index (ig) source: adapted from sarandon (2006). guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. peruvian journal of agronomy, 6(3), 239–255 (2022) https://doi.org/10.21704/pja.v6i3.1976 245 iii. results and discussions 3.1. evaluation of sustainability 3.1.1. economic sustainability from the plots evaluated, 86.5 % were economically sustainable, 55 % of the plots diversify into different fruit and agricultural crops (a1=3). 15 % grow plants in areas larger than 1.5 ha (a2=4) and 63 % have a net income higher than 1 200 soles per month (b=4). 68 % have two or three amazonian fruit trees for sale, 86 % have one sales route (c1=2) and 51 % depend from 1 % to 25 % on external inputs (c3=3) (table 5). the respondents stated that one of the major limitations for sales is the number of marketing routes and this is a critical point for the economic risk. that 66.7 % of orange farms are economically sustainable. nevertheless, they reported as critical points the lack of marketing routes and the high dependence on external inputs. 3.1.2. ecological sustainability the ecological sustainability was 94.7 % for the total plots evaluated, the criteria are detailed in table 3, where 43 % of the plots have soil cover (a1=4), 57 % are permanently covered by weeds (a2=2). 46 % are diversified with medium association of fruit trees and other crops (a3=4), 62 % have a slope of 0 % to 3 % (b1=4), 43 % have vegetation cover between 75 % and 99 % (b2=4), 41 % have rainfall greater than (110 to 124) mm per month (b3=3), the plots are diversified by abundance of arthropods and other plants (c1=3), and 46 % of the plots are diversified by 25 % association of fruit trees and other crops (c2=4). amazonian fruit trees are ecologically sustainable reaching a value of 3.33 (table 6). the biodiversity is the indicator that needs to be addressed, precipitation is one of the abiotic factors of greatest risk for soil erosion. altieri (2012) and foley et al. (2011) considered the ecological sustainability very important, due to the spatial variability of biodiversity, which has a serie of processes that interact at different temporal and spatial scales. saynes et al. (2016) considered that maintaining cover the entire vegetation of the plots in a natural way is important in any agricultural system. likewise, in fruit tree plots, the protection of natural resources should be promoted. food security is conditioned by the provision of natural resources and environmental services in a geographical space and for the magnitude that nature recompose itself from anthropic influences (duran, 2010). agroforestry systems provide greater ecological sustainability, due to increased diversification of production systems, and soil moisture conservation (arévalo et al., 2002). mata et al. (2018) considers agroforestry systems to optimize production per unit area and make sustainable the production system while conserving the ecosystem. table 5: evaluation results of economic sustainability sustainability tests respondent (%)subindicators variables code value food safety (a) amazonian fruit trees diversity a1 3 55 selfconsumption production area a2 4 15 gross income by producer (b) gross income by producer b 4 63 economic risk (c) diversification for sale c1 2 68 marketing routes for sale c2 2 86 dependency on external supplies c3 3 51 economic indicator 3,20 amazonian fruit trees are economically sustainable with a value of 3.2, despite it was not applied techniques in their management and not being dependent on external inputs. this is in agreement with sarandón et al. (2006), who considered that a system with high dependence on inputs is not sustainable over time. valarezo et al. (2020) reported that 70 % of lemon farms form portoviejo, ecuador are sustainable in the economic dimension, and one of their recommendations is to improve the diversification of production and marketing canals. additionally, ruíz et al. (2018) reported sustainability of amazonian fruit trees plots in loreto, peru september december 2022 246 3.1.3. sociocultural sustainability from the plots evaluated, 35.13 % were sociocultural sustainable, and the criteria is detailed in table 4. only a 79 % had access to housing with regular unfinished structure and others presented deteriorated structures (a1=2), 100 % had access to primary and secondary school without restriction (a2=2), 74 % did not have access to health centers, 42 % had electricity and cell-phone (a4=0), 55 % did not accept the production system due to the lack of attention to the agrarian sector and, therefore, they felt disillusioned (b1=2), 65 % of the young people preferred to stay in the area, they preferred to study to become an authority (b2=4), 39 % were not part of any organization (c1=1), 45 % did not participate actively in the community (c2=1). 55 % elected their authorities by an agreement in a communal assembly (c3=1), 24 % did not follow communal coexistence agreements (c4=1), 37 % considered that the leaders lead according to their interests and not for the benefit of the community (c5=2) and 32 % conceived ecological aspects with a broad vision (d=2) (table 7). it could be observed that the satisfaction indicator of basic needs (b1) was the lowest and the systems were quite fragile, mainly due to deficiencies in access to education (a2) and health (a4). altieri and nicholls (2000) indicated that social sustainability in basic needs such as education (a2), housing (a3), health (a4) among others is a priority. sepúlveda (2008) considered that social agents play a very important role in achieving sustainable development. moreover, aquino et al. (2018) pointed out that social indicators are oriented to evaluate producer satisfaction, quality of life and social integration. being important the participation of related institutions and organized society in the construction of acceptable social conditions, focused on the availability of basic services and capacity building of human resources. the methodology proposed by sarandón et al. (2006) has been easy to understand and adaptable, as well as low cost, to assess sustainability in amazonian fruit tree production plots. the results of the sustainability of the amazonian fruit trees reached a value of 2.86 (table 8), indicating that the amazonian fruit trees are sustainable (sarandón et al., 2006). the plots producing amazonian fruit trees have critical points in sociocultural sustainability. table 6: evaluation results of ecological sustainability sustainability tests respondent (%)subindicators variables code value conservation of life soil (a) organic residue in the soil a1 4 43 time in cover soil a2 2 57 amazonian fruit trees diversity a3 4 46 erosion risk (b) prevailing slope b1 4 62 vegetable cover b2 4 43 precipitation b3 0 100 biodiversity managment (c) spatial biodiversity c1 3 41 temporary biodiversity c2 4 46 ecological indicator 3,33 table 7: evaluation results of sociocultural sustainability sustainability test respondents (%) subindicators variables code value satisfaction to basic needs (a) access to housing a1 2 79 education access a2 2 100 access to health and health coverage a3 1 74 access to services a4 0 42 aceptability of production system (b) aceptability of production system b1 2 55 abandonment of young people b2 4 65 social integration into organizational systems (c) associativity c1 1 39 active participation c2 1 45 voting equality c3 3 55 knowledge adquisition (election of authorities) c4 1 24 community diary control c5 2 37 ecological knowledge and awareness d 2 32 sociocultural indicator 2,04 guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. peruvian journal of agronomy, 6(3), 239–255 (2022) https://doi.org/10.21704/pja.v6i3.1976 247 table 8: general results of sustainability evaluation ik ie isc ig 3,20 3,33 2,04 2,86 the figure 1 shows the critical points on which it should be done a work to reduce them. arnés and astier (2003) considered a challenge for agriculture to address the high climate variability by designing highly efficient production systems in terms of water and energy management with low levels of environmental degradation and pollution. figure 1: tests points in sustainability of the producing plots in amazonian fruit trees along iquitos – nauta, road in loreto 3.2. identification of amazonian fruit trees table 9 shows the list of amazonian fruit trees most frequently mentioned by those responsible for the plots; ten amazonian fruit trees belonging to eight botanical families were identified, located in an area of 69 hectares. amazonian fruit trees are characterized by their varied nutritional composition, seasonality in production, i.e., they only produce fruit during one period of the year, presenting an alternative for human and animal consumption (velazco et al., 2020). in addition, gonzales & torres (2011) consider that there are 193 species of fruit trees in loreto, of which 139 are harvested from natural areas for consumption by the rural population and commercialized in sustainability of amazonian fruit trees plots in loreto, peru september december 2022 248 amazonian cities. they also indicate that they are semi-domesticated in the different farms and orchards of producers where selection and domestication continue. meanwhile, mostacero et al. (2017), reports for northern peru 45 species of fruit trees; which are distributed in 18 families. mitjans et al. (2019) also identified 17 species of native fruit trees, distributed in 14 families and 17 genera. 3.3. assessment of vegetation diversity and soil arthropods 3.3.1. determination in the vegetation. figure 2 shows the alpha diversity of vegetation diversity, whose value is 2.074, indicating that table 9: list of amazonian fruit trees with most frequency mentioned in iquitos – nauta road in loreto family common name scientific name arecaceae aguaje, achual mauritia flexuosa l. pijuayo bactris gasipaes h.b.k. ungurahui oenocarpus bataua mart. bombacaceae zapote matisia cordata humb. y bon-pl. fabaceae guaba inga edulis mart. icacinaceae umarí poraquiba quadrangularis l. motaceae uvilla pourouma cecropiifolia mart. sapotaceae caimito pouteria caimito (ruiz y pav.) radlk myrtaceae pomarrosa syzygium jambos (l.) alston moraceae pan del árbol artocarpus altilis (parkinson) foseberg the study area is within the normal range in species equity and is emerging as an ecologically sustainable agroecosystem. these data indicate that, the study area is naturally diversified, since the farmer for lack of budget or ignorance does not provide maintenance to their plots. castro et al. (2019) reported a diversity value closer to that found in this study (2.06). those authors observed that the greatest number of species belong to the families asteraceae (12) and poaceae (5) in junin state, being the district of villa rica the most diverse. on the other hand, vásquez et al. (2016) recorded 148 species, of which 129 corresponded to weed species, classified into 33 botanical families in the amazon region. 3.3.2. determination of soil arthropods figure 2 shows the index of arthropod diversity in the soil, which reached 1.9, indicating low biodiversity in terms of the equity of arthropod species. the index shows that, the area under study has a large number of individuals, but with low diversity. the “termites” (nasutitermes sp.) that were found in the barks of amazonian fruit trees were the most abundant species (1 200 individuals per 25 square meter) and the “bug” of the genus reduvius has been the least abundant (2 individuals per 25 square meter). table 10 shows the calculation of the alpha diversity of the arthropods found in the study area. according to chao and jost, (2012) arthropod diversity is related to soil conservation status. ramírez et al. figure 2: shannon–weaver index in vegetation and anthropods soil in amazonian fruit producing plots in iquitos – nauta highway in loreto guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. peruvian journal of agronomy, 6(3), 239–255 (2022) https://doi.org/10.21704/pja.v6i3.1976 249 table 10: calculation of alpha diversity for arthropods in plots with amazonian fruit trees on the iquitos-nauta highway in loreto scientific name avg/kind 25 m2 pi lnpi pi*lnpi h´ lumbricus terrestris linnaeus 1758 93 0,034 -3,46 -0,1 1,91 lycosa erythrognatha lucas 1836 41 0,02 -4,2 -0,1 thomisis sp. sundevall 1883 27 0,01 -4,6 -0,04 rhynchonphorus sp. linnaeus 1758 430 0,2 -1,8 -0,3 nasutitermes sp. 1200 0,5 -0,8 -0,4 gryllus sp. 8 0,003 -5,8 -0,02 mantis sp. 8 0,003 -5,8 -0,02 rasahus sp. 5 0,002 -6,3 -0,01 reduvius sp. 2 0,001 -7,2 -0,005 zelus sp. 5 0,002 -6,3 -0,01 zoreva sp. 15 0,001 -5,2 -0,03 aphis craccivora 50 0,02 -4 -0,1 cerotoma sp. 22 0,01 -4,8 -0,04 colaspis sp. 34 0,013 -4,4 -0,05 diabrotica sp. 42 0,01 -4,2 -0,1 morpho menelaus 3 0,01 -6,8 -0,01 m. rhetenor 5 0,001 -6,3 -0,01 anastrepha spp. 13 0,005 -5,3 -0,03 ecyton sp. 130 0,05 -3,02 -0,14 ectotomma sp. 230 0,08 -2,4 -0,2 atta sp. 270 0,1 -2,3 -0,2 polistes sp, 14 0,01 -5,2 -0,02 vespa sp. 18 0,01 -5 -0,03 total (n) 2665 -1,91 (2019), found diversity with maximum of 1.58 and minimum of 1.44. iannacone et al. (2000), a mean diversity of 2.33. de la cruz et al. (2003) found high heterogeneity in the distribution according to plant formations present in the habitats sampled. 3.4. carbon sequestration 3.4.1. biomass carbon in the fruits figure 3 shows that 15 years old guaba (inga edulis) trees (13 plants/ha) from plot 17 were the species that fix more carbon individually with uptake fluxes of 22.02 t·ha-1·a-1. the 12 years old pijuayo (bactris gasipaes h.b.k.) trees (20 plants/ha) from plot 32 produced a flux of 18.88 t·ha-1·a-1, the 14 years old uvilla (pouroma cecropiifolia mart.) trees (6 plants/ha) from plot 32 produced a flux of 14.54 t·ha-1·a-1 and 18 years old pan del árbol (artocarpus altilis (parkinson) foseberg) from plot 22 which captured 18,8 t·ha1·a-1. figure 4 shows the carbon sequestration in fruit species from plot 32, the association of the 12 years pijuayo trees (20 plants/ha) and 14 years old uvilla (pouroma cecropiifolia mart.) trees (6 plants/ha), sequester 117.19 t·ha-1 and total flows of 33.42 t·ha-1·a-1. it was followed by the figure 3: carbon sequestration in biomass in amazonian fruit trees in producing plots of amazonian fruit trees in iquitos nauta road, loreto sustainability of amazonian fruit trees plots in loreto, peru september december 2022 250 association of four (04) species in plot 24, pijuayo (bactris gasipaes h.b.k.), uvilla (pouroma cecropiifolia mart.), caimito (pouteria caimito) and aguaje (mauritia flexuosa l.) can capture as a total reserve up to 99 t·ha-1·a-1 and total fluxes of 28.78 t·ha-1·a-1. the highest annual co2 fluxes were observed in plots 32 and 22, which have higher diversity of species and number of plants per hectare. the lowest co2 fluxes were noticed in plots 10 and 36. minimun tillage, pruning, organic fertilization maintained in these plots will be easily incorporated into the ecosystem services, thus allowing the area under study to be resilient to climate change. amazonian forests are recognized for their importance in climate change mitigation, attributing to them the potential for carbon storage in biomass and soil, as well as the removal of greenhouse gases from the atmosphere (baker et al., 2019 and vicuña et al., 2019). garcía et al. (2012), evaluating trees and palms with dbh ≥ 10 cm in forests, found 88.60 t·ha-1 in total carbon sequestered, suggesting that the results are influenced by the age, density and extension of the fruit trees. additionally, they found 51.28 t·ha-1 for the biomass of aguaje in high areas. rueda (2014) found in plantations of guaba (around 200 has) a total of 6 180.21 t·ha-1 of carbon, in the biomass the value of 3 090.10 t·ha1and 11 330.30 t·ha-1 stored in the topsoil. alegre et al. (2017) considered that systems covered permanently of centrosema macrocarpum supply litter and therefore a rapid availability of nutrients and a strong defense against erosion. however, as total biomass they are much lower than trees, since they did not sequester more than 6 t·ha-1. 3.4.2. soil carbon soil c stock for the 0 cm to 30 cm depth ranged between 24.64 t·ha-1 and 66.5 t·ha-1 (figure 5). the highest c storage was showed by 13 years old caimito (pouteria caimito) specie (6 plants/ha) that captured 66.5 t·ha-1 in plot 23. the lowest storage were observed for the 15 years old umari (poraquiba quadrangularis l.) specie (13 plants/ha) in plot 37 that captured 24.64 t·ha1, and for the guaba and caimito association in plot 13, which captured a total c stock of 50.32 t·ha-1. these fluctuations in soil c stocks are probably due to the different processes of soil organic matter decomposition in which climatic factors intervene, being the most important the air temperature and humidity, especially when the soil surfaces are exposed for planting fruit trees or other activities. figure 4: carbon sequestration in biomass of fruit trees association in the producing plots of amazonian fruit trees in iquitos – nauta road in loreto guerra-teixeira, a., alegre-orihuela, j., & vásquez-guerra, a. peruvian journal of agronomy, 6(3), 239–255 (2022) https://doi.org/10.21704/pja.v6i3.1976 251 soil functions are affected in agroecosystems, such as water and air dynamics. biogeochemical processes, microorganisms and soil itself define soil quality (dexter, 2004). hernández & vargas (2005) indicate that there are international mechanisms for different countries to buy and sell co2 absorption services, which is an important source of financing to protect and conserve resources and the environment, while at the same time improving the economy. changes in soil quality can be monitored with easy and accurate indicators determined by reproducible methods (moebius et al. 2007). garcia et al. (2012), found values of 3.78 t·ha-1 of stored carbon and rueda (2014) in 200 has of plants of the genus inga, found 11 330.30 t·ha-1. the values found, is influenced by factors such as extension and type of plantations, plantation density, age, soil type, quality and environmental change, among others. garcía et al. (2012) considers that aguajes grow in hydromorphic soils and sequester large amounts of carbon greater than terrestrial ecosystems. they also indicate that, at a depth of 0 cm to 50 cm, they found carbon stocks that fluctuated from 51.28 to 193.9 t·ha-1, considering aguajales a great potential for greenhouse gas emissions. figure 5: soil carbon reserves at the depth of 0 – 30 cm in the producing plots of amazonian fruit trees in iquitos – nauta road in loreto conclusions the diversity index in vegetation reached a value of 2.74; kudzu (pueraria phaseoloides) was predominant in the study area, while, for soil arthropods, it reached an index of 1.91 with a relatively low population of termites (nasutitermes sp.). four species of the amazonian fruit tree producing areas were the ones that captured the highest values of carbon in biomass. these were guaba (inga edulis), pijuayo (bactris gasipaes h. b.k.), uvilla (pouroma cecropiifolia mart.) and pan del árbol (artocarpus altilis (parkinson) foseberg). in associations of several fruit species, pijuayo with uvilla association sequester more co2 ha·a -1 and present more total fluxes than the association of four species such as pijuayo, uvilla , caimito (pouteria caimito) and aguaje (mauritia flexuosa l.). in the soil, the highest carbon stock at the depth of 0 cm to 30 cm was 66.5 t·ha-1, reducing co2 emissions by 243.84 t·ha -1·a-1. in the sustainability evaluations, amazonian fruit trees are sustainable in the economic dimension (3.20) and ecological dimension (3.33). however, sustainability is weak in the sociocultural dimension (2.04). sustainability of amazonian fruit trees plots in loreto, peru september december 2022 252 author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. orcid and e-mail guerra-teixeira. a. aldiguerra@gmail.com https://orcid.org/0000-0003-3070-2807 alegre-orihuela, j. jalegre@lamolina.edu.pe https://orcid.org/0000-0002-7282-045x vásquez-guerra, a. warmy19@gmail.com https://orcid.org/0000-0002-4283-1123 references alegre, j., lao, p., silva, c., & schrevens, e. 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(1947). a critical examination of a rapid method for determining organic carbon in soils-effect of variations in digestion conditions and of inorganic soil constituents. soil sci., 63(4), 251–264. https://www.syngenta.es/agricultura-responsable/agricultura-sostenible https://www.syngenta.es/agricultura-responsable/agricultura-sostenible https://www.syngenta.es/agricultura-responsable/agricultura-sostenible peruvian journal of agronomy http://revistas.lamolina.edu.pe/index.php/jpagronomy/index research article https://doi.org/10.21704/pja.v6i1.1864 received for publication: 1 november 2021 accepted for publication: 27 february 2021 published: 30 april 2022 issn: 2616-4477 © the authors. published by universidad nacional agraria la molina this is an open access article under the cc by application of biofertilizers in crop production: a review aplicación de biofertilizantes en la producción de cultivos: una revisión barsha sharma1; laxmeshwar yadav2; meena pandey2; jiban shrestha3 *corresponding author: agribarsha77@gmail.com *https://orcid.org/0000-0002-2061-3811 abstract nutritious foods are needed for the continuously growing population together with the nutrient for plant growth and production. inorganic chemical-based fertilizers have been base and are used heavily in today’s soil management procedures, posing serious health and environmental concern. biofertilizer has been recognized as a reasonable solution for improving soil fertility and crop output in sustainable farming. the use of beneficial microorganisms as biofertilizers has escalated its importance in the agricultural industry due to its potential significance in food safety and sustainable crop production. biofertilizers can be a valuable component of a comprehensive nutrient management strategy. overall, nitrogen fixers (n-fixers), potassium and phosphorus solubilizers, growth-promoting rhizobacteria (pgpr), endo and ectomycorrhizal fungi, cyanobacteria, and other beneficial microscopic organisms are incorporated into biofertilizers. utilizing bio-fertilizers, enhance nutrient and water uptake, plant development, and plant tolerance to abiotic and biotic impacts. these prospective biological fertilizers would play a essential role in soil production and sustainability and also in environmental protection, being eco-friendly and cost-effective inputs for farmers. keywords: biofertilizer, crop production, sustainability. resumen los alimentos nutritivos son necesarios para la población en continuo crecimiento junto con los nutrientes para el crecimiento y la producción de las plantas. los fertilizantes químicos inorgánicos han sido la base y se utilizan en gran medida en los procedimientos actuales de gestión del suelo, lo que supone una grave preocupación para la salud y el medio ambiente. los biofertilizantes han sido reconocidos como una solución razonable para mejorar la fertilidad del suelo y la producción de los cultivos en la agricultura sostenible. el uso de microorganismos beneficiosos como biofertilizantes ha aumentado su importancia en la industria agrícola debido a su potencial importancia en la seguridad alimentaria y la producción de cultivos sostenibles. los biofertilizantes pueden ser un valioso componente de una estrategia integral de gestión de nutrientes. en general, los biofertilizantes incorporan fijadores de nitrógeno (fijadores de n), solubilizadores de potasio y fósforo, rizobacterias promotoras del crecimiento (pgpr), hongos endo y ectomicorrícicos, cianobacterias y otros organismos microscópicos beneficiosos. la utilización de biofertilizantes mejora la absorción de 1 institute of agriculture and animal science, tribhuvan university, kritipur, kathmandu, nepal. 2 paklihawa campus, institute of agriculture and animal science, tribhuvan university, bhairahawa, rupandehi, nepal. 3 nepal agricultural research council, national plant breeding and genetics research centre, khumaltar, lalitpur, nepal. how to cite this article: sharma, b., yadav, l., pandey, m., shrestha, j. (2022). application of biofertilizers in crop production: a review. peruvian journal of agronomy, 6(1), 13–31. https://doi.org/10.21704/pja.v6i1.1864. application of biofertilizers in crop production: a review. january april 2022 14 nutrientes y agua, el desarrollo de las plantas y su tolerancia a los impactos abióticos y bióticos. estos futuros fertilizantes biológicos desempeñarían un papel esencial en la producción y la sostenibilidad del suelo y también en la protección del medio ambiente, siendo insumos ecológicos y rentables para los agricultores. palabras clave: biofertilizante, producción de cultivos, sostenibilidad. introduction biofertilizers are soil microbe cultures that can be employed as microbial or soil inoculants to increase plant and soil fertility and productivity. biofertilizers are economical and sustainable plant nutrition sources. a biofertilizer is simply a product that contains living microorganisms that colonize the rhizosphere and stimulate development by increasing the supply or availability of nutrients to the host plant when applied to the soil, seed, or plant surface. some of the most often used biofertilizers include nitrogen-fixing soil bacteria (azotobacter, rhizobium), nitrogen-fixing cyanobacteria (anabaena), phosphate-solubilizing bacteria (pseudomonas sp.), and arbuscular mycorrhiza (am) fungus. phytohormone (auxin)-producing bacteria and cellulolytic microorganisms are also included in biofertilizer formulations. by increasing specific microbial processes, these microbial formulations are used to increase the availability of nutrients in a form that can be digested by plants. a biofertilizer is an organic fertilizer supplemented with beneficial microorganisms. biofertilizers are all organic resources for plant growth that are transformed into a useable state for plant uptake by microorganisms, plant associations, or interactions. biofertilizers are carrier or liquid based products constituting living or dormant microbes (i.e., bacteria, fungi, algae, actinomycetes) single or in combination, which assist in fixing atmospheric-n2 or solubilizers of different soil nutrients together with production of growth promoting substances for promoting crop growth and yield (dineshkumar et al., 2018). biofertilizers contain microorganisms with peculiar functions, such as azospirillum, which fixes nitrogen, and p solubilizing bacteria, which solubilizes p from soil and fertilizer to be available to plants (saraswati & sumarno, 2008). biofertilizers are becoming more essential as a means of achieving high-quality harvests while reducing pollution. in agriculture, the application of both nitrogen and phosphate fertilizer is critical for achieving the best seed yield. azimi et al. (2013) discovered that when applying super nitro plass biofertilizer with phosphate bar var2, the pishtaz cultivar (wheat crop) has the highest seed yield (7.6 t/ha), whereas the non-application of biofertilizers treatment has the lowest seed yield (6.3 t/ha). they claimed that using biofertilizers increased grain yield and biomass yield, which is a substantial benefit, resulting in reduced production inputs due to lower costs of chemical fertilizer and higher yield (beyranvand, 2013). trichoderma viride and pseudomonas fluorescens, fertilized with a half dose (50 %) of recommended fertilizers (120:60:40 npk kg/ ha) boost growth and yield of maize crop and are demonstrated as prospective biofertilizers (nepali et al., 2020). biofertilizers once isolated can be readily propagated economically unlike chemical fertilizers and are fuel independent, lucartive, and easily accessible (umesha et al., 2018). combing biofertilizers and chemical can minimize rate of recommended synthetic fertilizers and can conserve soil productivity and sustainable rice production (ghimire et al., 2021). biofertilizers are formulated in an easy-touse, low-cost carrier substance that can increase plant availability and/or mineral nutrient uptake (malusa et al., 2012). a biofertilizer, according to vessey (2003), is a substance which when applied to seed, plant surfaces (leaves), roots, or soil and promote growth through several approaches and that helps to elevate the supply or accessibility of primary nutrients to the host plant containing living microorganisms that colonize the rhizosphere or the interior of the plant. biofertilizers should not be confused with green manure, manure, intercrop, or organic supplemented chemical fertilizer (bhattacharyya & jha, 2012; halpern et al., 2015, although sharma, b.; yadav, l.; pandey, m.; shrestha, j. peruvian journal of agronomy 6(1): 13–31 (2022) https://doi.org/10.21704/pja.v6i1.1864 15 they are also named as microbial inoculants or bio formulations (arora et al., 2011)). it was lately suggested that pgpr classification be limited to microbial strains that satisfy minimum two of three standards, combining aggressive colonization, plant growth stimulation, and biocontrol (bhattacharyya & jha, 2012). the objective of this review paper was to gather information on types of biofertilizer and their role in crop production. types of biofertilizers nitrogen fixing biofertilizers nitrogen is the nutrient that limits plant growth (gupta et al., 2012). even though the atmosphere contains around 80 % nitrogen in its free form, most plants are unable to utilize it. to fix this nitrogen and make it available to the plant, certain group of bacteria are required. biological nitrogen fixers (bnfs) are microorganisms that fix nitrogen in the environment. they convert inert n2 into an organic form that plants can utilize (reed et al., 2011). nitrogen fixation can yield 300–400 kg nitrogen per hectare per year and accounts for up to 25 % of total nitrogen in plants by increasing agricultural production by 10 % – 50 %. nitrogen fixing bacteria are categorized into free-living bacteria (azotobacter and azo spirillium), blue-green algae, and symbionts, such as rhizobium, frankia, and azolla. the n2-fixing bacteria associated with legumes include rhizobium, azorhizobium, mesorhizobium, sinorhizobium, , allorhizobium and bradyrhizobium and those with nonlegumes include achromobacter, alcaligenes, arthrobacter, acetobacter, azomonas, beijerinckia, clostridium, bacillus, enterobacter, erwinia, desulfovibrio, derxia, corynebacterium, campylobacter, herbaspirillum, klebsiella, lignobacter, mycobacterium, rhodospirillum, rhodopseudomonas, xanthobacter, mycobacterium, and methylosinus (meena et al., 2017). . they can efficiently replace chemical fertilizers in a variety of ways like reducing the chemical load on the environment. in the rhizosphere, plant roots emit compounds that induce bacterial colonization and nitrogen fixation.although numerous genera have been identified from the rhizosphere, members of the azotobacter and azospirillum genera have been thorougly studied in the field to enhance legume and cereal yields (bhat et al., 2015). phosphate solubilizing and mobilizing biofertilizers phosphorus which accounts about 0.2 percent of the plant’s dry weight is an indispensable nutrient for plant growth and development. in contrast to other macronutrients, phosphorus is the lowest transportable macronutrient available to plants in many soil conditions. insoluble forms of phosphate are transferred into to soluble forms by microorganisms (kalayu, 2019; prabhu et al., 2019). several bacteria and fungus species participate in phosphate solubilizing process (antoun, 2012). phosphate-solubilizing bacteria (psb) make use of a several processes to convert insoluble phosphates like hpo4 and h2po4 into soluble forms, along with that psb help in formation of organic acids, chelation, and ion exchange reactions. psb constitute 1–50% of microbial populations, while fungi account for only 0.1–0.5% of phosphate-solubilizing activity (sharma et al., 2013). psb can prepare metabolites when interact with hydroxyl (gluconic) and carboxyl (ketogluconic) groups that chelate the phosphate cation and convert it into a soluble form that plants can take. plants are able to utilize nutrient when bonded phosphorous was dissolved because acidic soil and soil ph decreased as a result of acids prepared by psb (itelima et al., 2018). microorganisms use the proton-extrusion mechanism to solubilize phosphate in addition to the organic technique (park et al., 2009; patel & goswami, 2020). not only phosphate, but also other trace elements like fe and zn, are supplied by the psb, which facilitate the plant to flourish. they also produce the enzyme that kills the pathogen, ensuring that the plant is disease-free. phosphate solubilizers incorporates bacillus, application of biofertilizers in crop production: a review. january april 2022 16 rhizobium, pseudomonas and enterobacter bacteria, as well as penicillium and aspergillus fungi (anand et al., 2016). in cameroon’s acidic soils, the use of the pseudomonas fluorescence strain considerably raises maize shoot length, plant dry weight, grain yield, and seed phosphorus content (henri et al., 2008). similarly, in recent study, it was observed that the phosphorus solubilizer aspergillus niger remarkably elevated plant height, fruit size, leaf length/width, and fruit numbers per plant as compared to control plants. also, it was noticed that combined use of phosphorus-solubilizing (aspergillus niger) and nitrogen-fixing (aspergillus niger) biofertilizers in plant as an inoculant assisted plant to perform better than those treated with single type biofertilizer (din et al., 2019). the immobile forms of phosphorous can be mobilized by phosphorus-mobilizing bacteria (suthar et al., 2017). they transport and mobilize insoluble phosphate from the soil layers to the root cortex. phosphate-mobilizing fungi, such as arbuscular mycorrhiza, penetrate the roots and increase their surface area, activate metabolic processes, and absorb nutrients. phosphatesolubilizing bacteria are said to act as phosphate mobilizers on occasion (chang & yang, 2009). they have the capability to solubilize/mobilize roughly 30–50 kg p2o5/ha under appropriate conditions, as a result it can increase crop output by10 % – 20 % (asoegwu et al., 2020). potassium solubilizing and mobilizing biofertilizers potassium (k) is the ranked as the second most common and important nutrient in plants after nitrogen and phosphorus. 1 % – 2 % of phosphorous is only available to the plants even if it is available in surplus amount in soil and the remainder is present as mineral k, which plants cannot absorb. as a result, soil solution k must be replenished regularly (park et al., 2009; meena et al., 2014). a different bacterial and fungal strains have been identified that use variety of process to solubilize insoluble into soluble form, together with acid generation, chelation, acidolysis, complexolysis, and exchange processes (etesami et al, 2017; sindhu et al., 2016; ahmad et al., 2016). a recent investigation discovered that elevation of potassium availability raised k uptake in tea plants in mica waste-treated soil employing the bacillus pseudomycoides, potassium-solubilizing strain (pramanik et al., 2019). likewise, another strain bacillus cereus encourage plant height, shoot dry weight, and branch number by 15 %, 26 %, and 27 %, respectively as compared to control (ali et al., 2021). few fungi such as, aspergillus niger and penicillium niger, may solubilize and mobilize k including from organic and inorganic sources (xiafang & weiyi, 2002). consequently, k solubilizers has a vital role to ascertain those agricultural plants can receive a regular supply of k. these have a favorable effect on the accessibility of other essential nutrients in the soil and hence play a crucial role in soil sustainability (bahadur et al., 2016). sulfur oxidizing biofertilizers sulfur (s) is required as a micronutrient by plants as well. sulfur has been demonstrated to help in the enhancement of biological and physical qualities of soil. the potential of sulfur to buffer soil ph is well-known. sulfur also improves the efficacy of nitrogen and phosphate fertilizers, as well as the ability of plants to absorb micronutrients, according to previous research (el-halfawi et al., 2010). most agricultural soils contain some microorganisms that can oxidize sulphur. however, the most important microorganisms belong to a group of bacteria of thiobacillus genus named as sulfur oxidizing bacteria (soxb). thiobacillus sp. is an example of a sulfuroxidizing bacterium; thiobacillus thioparous and thiobacillus thioxidans may oxidize sulfur to plant-usable sulfates, which aid in plant nutrition (riaz et al., 2020; vidyalakshmi et al., 2009). inoculating thiobacillus with elemental sulfur improves elemental sulfur oxidation, resulting in increased nutrient availability in soil and, as a result, increased plant development, according to a recent study (pourbabaee et al., 2020). sulfur compounds are exceptionally polluting environment when they present in reduced forms. sulfur oxidizing bacteria by naturally sharma, b.; yadav, l.; pandey, m.; shrestha, j. peruvian journal of agronomy 6(1): 13–31 (2022) https://doi.org/10.21704/pja.v6i1.1864 17 eliminating sulfur pollution provide environment protection (pokorna & zabranska, 2015). zinc solubilizing biofertilizers zinc is an essential element that plants require in their tissues in relatively small concentrations (5 mg/kg – 100 mg/kg) for growth and reproduction. zinc deficiency is common in soil, and it’s caused by improper fertilizer application, intensive agriculture, and poor soil health. zinc insufficiency is anticipated to rise from 42 % to 63 % by 2025 if the underlying issues are neglected. zinc is necessary for growth hormone synthesis. zinc deficiency in plants causes stunted shoot growth, compromised membrane integrity, smaller leaves, chlorosis, and increased vulnerability to light, heat, and fungal infections, as well as affecting grain yield, root development, pollen formation, and water intake and transport (kamran et al., 2017; tavallali et al., 2010). zinc deficiency in wheat can cause yellowing of the leaves and limited growth. zinc insufficiency in humans can be caused by eating zinc-deficient wheat (kamran et al., 2017). zinc insufficiency is the sixth biggest cause of death in less developed countries. as a result, among other minor nutrients, addressing the zn deficiency in agriculture is given top priority (graham, 2008; kumar et al., 2019; hussain et al., 2018). microbial inoculants have been found to solubilize the complex form of zinc in soil. (naz et al., 2016). variety of rhizobacteria taxa, such as pseudomonas sp. and bacillus sp., and both mycorrhiza, saccharomyces sp. have been reported to increase zn availability in soil. inc solubilization of zinc take place when these microorganisms utilize chelated ligands and oxidoreductive systems. (kamran et al., 2017). these bacteria manufacture antibiotics, phytochromes, antifungal chemicals and vitamins, which assist the plant collectively (goteti et al., 2013). inoculation of rice plant with a appropriate combination of zn solubilizing bacterial strains (burkholderia sp. and acinetobacter sp.) advance growth characteristics and rice yield and were shown to be more efficient in obtaining zn from the soil than non-inoculated plants, according to a study (vaid et al., 2014). maize productivity has been observed to be boosted by biofertilizers containing zn solubilizing bacteria (hussain et al., 2020). plant growth promoting rhizobacteria plant growth-promoting rhizobacteria (pgpr) are bacteria that invade plant roots and stimulate plant growth (beneduzi et al., 2012). they perform their role as fertilizers by fostering plant growth and development, enabling for biotic and abiotic stress resilient, and facilitating soil mineralization through the breakdown of organic matter. the plant benefits from pgpr inoculation in a variety of ways. they improve plant tolerance to drought (timmusk et al., 2014; vurukonda et al., 2016; niu et al., 2018; ilya et al., 2020), salinity (mayak et al., 2004; bharti et al., 2013), and biotic stress (timmusk et al., 2014; vurukonda et al., 2016; de vasconcellos & cardoso, 2009; verma et al., 2016). they improve seed germination (almaghrabi et al., 2014) and soil fertility (islam et al., 2016; jang et al., 2017) as well as increase growth by releasing phytohormones such as auxins, indol acetic acid, ethylene, gibberellin, and others (kumar et al., 2016; barnawal et al., 2017; tahir et al., 2017). plant secondary metabolites, as well as heavy metal and pollution bioremediation, are all affected by them (sayyed et al., 2015; ordookhani et al., 2011). the two most common types of pgpr are extracellular plant growth-promoting rhizobacteria (epgpr) and intracellular plant growth-promoting rhizobacteria (ipgpr). epgpr are present either in the rhizosphere (on the rhizoplane) or in between the cells of the root cortex, while ipgpr are established essentially inside the peculiar nodular structures of root cells. the bacterial genera included as epgpr are azotobacter, serratia, azospirillum, bacillus, caulobacte, chromobacterium, agrobacterium, erwinia, flavobacterium, arthrobacter, micrococcous, pseudomonas and burkholderia. endophytic microorganisms such as allorhizobium, bradyrhizobium, mesorhizobium, and rhizobium, including frankia species, can application of biofertilizers in crop production: a review. january april 2022 18 fix atmospheric nitrogen especially for higher plants (bhattacharyya & jha, 2012). plant roots and soil microorganism interaction plant-associated microorganisms exchange carbon from plants for minerals accumulated in the soil. mycorrhizaassisted plant uptake include availability of low diffusion rate nutrient like phosphorus (p) and zinc (zn), sulfur (s) to plant assisted by bacteria and mycorrhizal fungus, and transfer of atmospheric nitrogen n2 pool to soil and then to plant (smith & smith, 2012; gahan & schmalenberger, 2014). not only increment of limiting nutrient like nitrogen and phosphorus assisting plant growth, enzymes and hormone engender root development, enhance nutrient use efficiency. the mechanisms involved in the improvement of plant nutrient absorption by several biostimulants were reviewed by halpern et al. (2015). biostimulants are compounds or materials that, when applied to seeds or plants, can influence physiological processes in plants, potentially improving growth, development, or stress response, however nutrients and herbicides are excluded. this group comprise plant growth promoting rhizobacteria (pgpr), which aid in atmospheric n2 fixation, p and iron solubilization, and root morphological changes. plant disease incidence has also been shown to be reduced by microbes acting as root pathogen antagonists or generating systemic resistance in plants (choudhary et al., 2011). when pathogens or insects attack, plants can recruit defensive microorganisms and increase microbial activity to avoid diseases in the rhizosphere. yang et al. (2009) found that pgpr can generate “induced systemic tolerance” to abiotic stresses such as salinity and drought, as well as providing protection for plant health. other mechanisms may be implicated in the pgpr–plant interactions, although they have yet to be discovered. palacios et al. (2014) recently raised the possibility of vitamins playing a role in plant-pgpr interactions, which would require further investigation. according to amin and hamidreaza (2015), various n and p biofertilizers had substantial effects on maize yield and yield components. the capacity of biofertilizers to increase phosphorus and other nutrient availability may be beneficial, especially when the soil is calcareous, which lowers nutrient availability. the grouping of biofertilizers based on their functions is given in table 1. role of biofertilizers in crop production an ecofriendly way to replace chemical fertilizers through nitrogen fixation, phosphate, and potassium solubilization or mineralization, the release of plant growth regulating substances, the production of antibiotics, and the biodegradation of organic matter in the soil, biofertilizers keep the soil environment rich in all kinds of microand macronutrients (bhardwaj et al., 2014). by incorporating artificially multiplied cultures of beneficial microorganisms in the form of biofertilizers, the lost biological activity in the soil due to excessive use of chemical fertilizers can be slowly restored. biofertilizers minimize the ecological disturbance. the use of biofertilizers replace the application of chemical fertilizers; chemical fertilizers are hazardous to environment. biofertilizers are getting popular as a viable alternative to hazardous chemical fertilizers in the pursuit of sustainable agriculture (nosheen et al., 2021). microbes in biofertilizers deliver atmospheric nitrogen to plants directly. the importance of nitrogen-fixing bacteria in soil nitrogen fixation is demonstrated by their contribution to the nitrogen cycle. no2 emissions are increased when nitrogen fertilizer is used. biofertilizer can lessen global warming as a result of this (naher et al., 2015). enhancement on plant tolerance to environmental stress the utilization of biofertilizers has shown to exhibit the improve tolerance capacity of plant to environmental problems. abiotic and biotic variables are the most significant constraints on agricultural productivity. many modern scientific methodologies for crop development sharma, b.; yadav, l.; pandey, m.; shrestha, j. peruvian journal of agronomy 6(1): 13–31 (2022) https://doi.org/10.21704/pja.v6i1.1864 19 under stress have been widely used, with the role of pgprs as a bio-protectant becoming increasingly essential. biofertilizers also helps plants to withstand salinity and drought stress, for example pseudomonas aeruginosa has been shown to be able to resist both biotic and abiotic stress and pseudomonas putida rs-198 enhance germination rate and different growth parameters such as plant height, dry weight, and fresh weight of cotton under alkaline and high salt conditions by accelerating the rate of uptake of k+, mg2+, and ca2+ while declining the rate of absorption of na+. moreover, some strains of pseudomonas used 2, 4-diacetylphloroglucinol to cause plant to resist high temperatures, while salinity stress tolerance are assigned by mycobacterium phlei. ansari et al. (2013) found that inoculating plants with arbuscular mycorrhiza fungus promotes plant development under salt stress as well. ansari et al. (2013) also note that the root endophytic fungus piriformospora indica was discovered to protect the host plant from salt stress. a combination of the am fungus and nitrogen-fixing bacteria benefits drought-resistant legume plants. during water scarcity, pseudomonas sp. increases antioxidant and photosynthetic pigment content of basal plants. rice plants that were subjected to drought was looked for increased photosynthetic efficiency and anti-oxidative response after inoculation with arbuscular mycorrhiza in both drought and saline environments to observe the beneficial effects of mycorrhizae ruiz-sanchez et al. (2010). pgprs have been demonstrated to be a viable biological alternative to chemical agents for conferring disease resistance in a variety of disorders. in addition to functioning as growth promoters, they can offer disease resistance. bacillus subtilis gbo can activate defense mechanisms. bacillus subtillisn11 along with mature compost was observed to control fusarium infestation on banana roots. also, in general pgprs have been highly effective to be control useful spotted wilt viruses in cucumber mosaic, tomatoes and pepper virus, and banana bunchy top virus. mycorrhizae have been proven to provide resistance to fungal infections as well as inhibit the growth of various root diseases such as r. solani and pythium sp. in rare cases. table 1. categorization of biofertilizers according on their functions groups examples n2-fixing biofertilizers free-living clostridium, klebsiella, anabaena, azotobacter, beijerinkia, and nostoc symbiotic frankia, rhizobiumand anabaena azollae associate symbiotic azospirillum p-solubilizing biofertilizers bacteria bacillus subtilis, bacillus circulans, and pseudomonas striata bacillus megaterium var. phosphaticum, fungi penicillium sp. and aspergillus awamori p-mobilizing biofertilizers arbuscular mycorrhiza acaulospora sp., scutellospora sp., and sclerocystis sp.glomus sp., gigaspora sp., ectomycorrhiza pisolithus sp., laccaria sp., and boletus sp. ericoid mycorrhizae pezizellaericae orchid mycorrhiza rhizoctonia solani biofertilizers for micronutrients silicate and zinc solubilizers bacillus sp. plant-growth-promoting rhizobacteria pseudomonas pseudomonas fluroscence source: umesha et al., 2018; singh et al., 2014a; singh et al., 2014b. application of biofertilizers in crop production: a review. january april 2022 20 enhancement on soil fertility the amount of nutrient fixed in soil by biofertilizers in crops is given in table 2. the combination of n fertilizer and a. brasilense increased grain yield by up to 29 percent when compared to n fertilization alone. observation made on rhizobium strain give an idea about complexities about plant-microbe interaction and plant development and grain yield that are affected due to many elements. these studies show that mixing rhizobia secondary metabolites with biofertilizers can help grain crops grow and produce more. inoculating maize plants with a p-solubilizing pseudomonas fluorescens strain, help it to grew faster and had much greater (amf) infection rates than non-inoculated control plants as maintained by krey et al. (2013). two p-solubilizing bacterial strains that have an essential role in plant growth promotion and soil fertility improvement in various agroclimatic areas was found out by kaur and reddy (2014). owen et al. (2015) examined the terminology, composition, and roles of p solubilizers, as well as the many variables that influence their efficacy in increasing p availability in various soil and plant settings. amf has long been thought to be advantageous to plant p nutrition. smith and smith (2012) emphasized their ability to efficiently absorb p from the soil and make it available to the plant they are linked with. soil characteristics and environmental conditions such as temperature and soil moisture, as well as the availability of other nutrients like nitrogen, will impact the outcome of this beneficial interaction (johnson et al., 2015). beneficial dual inoculation (amf and rhizobia, for example) has been successfully tested in the field to boost woody legume growth (lesueur et al., 2001; lesueur & duponnois, 2005). despite this, due to production constraints and a lack of applied research, commercial biofertilizers of this sort are rarely available on international markets. improvement on nitrogen and phosphorus use efficiency the efficacy of arbuscular mycorrhizal fungi (amf), p solubilizers, and n fixatives was boosted when there were higher amounts of p in the soil. with decreased organic matter concentration and a neutral ph, meta-regression revealed that successful amf inoculation was more likely.our comprehensive study serves as the foundation and guidelines for properly selecting and applying biofertilizers. nitrogen and phosphorus are the most important elements, but they are also the most restrictive minerals for living things. most agricultural soils include plenty of organic and inorganic p, but most of it is unavailable to plants. as phosphorus is easily immobilized, less soluble, and less accessible to plants, plants may not have enough phosphorus even after applying phosphorus fertilizer to the soil. the world’s principal source of phosphorus is phosphate ore, a nonrenewable resource, and table 2: amount of nutrients fixed in soil by biofertilizers in crops biofertilizers nutrient fixed (kg/ha/year) crops blue green algae 25 kg n /ha rice, banana azolla 900kg n /ha rice phosphate solubilizing bacteria and fungi solubilize about 50-60%of them fixed phosphorus in the soil all crops rhizobium 50 to 300 kg n/ ha green-gram, black-gram, lentil, cowpea, bengalgram andfodder legumes azotobacter 0.026 to 20 kg n/ha cotton, vegetables, plantation crop, rice, wheat, barley, ragi, mustard, safflower, niger, sunflower, tobacco, fruit, spices, condiment, ornamental flower azospirillum 10-20 kg n /ha sugarcane, vegetables, maize, pearl millet,rice, wheat, fodders, oil (source: sahu et al., 2012) sharma, b.; yadav, l.; pandey, m.; shrestha, j. peruvian journal of agronomy 6(1): 13–31 (2022) https://doi.org/10.21704/pja.v6i1.1864 21 phosphorus mining and trade contribute to global energy consumption that is both environmentally harmful and wasteful. as cheap and high-quality deposits become increasingly scarce, the quality of rock phosphate is rapidly worsening, notably in terms of cadmium pollution evidence on the bio-fertilizers application in crop production increments in grain yields of crops by biofertilizers is given in table 3. the use of microbial inoculants to inoculate legumes with rhizobia is the most well-known success story. it has largely revolutionized legume production methods, reducing their need for nitrogen fertilizer for high yields. alves et al. (2003) and melchiorre et al. (2011) found that using bnf and legume inoculants containing both rhizobia and active compounds such as lipochito oligosaccharides (lcos), which signal the symbiosis of rhizobia with legumes and the formation of nitrogen-fixing root nodules, grain yields in brazil, argentina, and the usa. for decades, free-living diazotrophs have been utilized to promote endophytic and associative nitrogen fixation in cereals and grasses, most notably azotobacter, azospirillum, and more recently herbaspirillum, gluconaceto bacter, and burkholderia. azospirillum bacteria promote plant growth predominantly through the production of phytohormones, particularly indole-3-acetic acid (iaa) (halpern et al., 2015). inoculants employed in the experiments included liquid and peat-based formulations of several azospirillum species and strains applied to both seeds and soil, and soil and plant development stimulation was generally detected only once inoculum potential and proper formulation were considered. other field tests (diaz–zorita & fernandez–canigia, 2009) have shown similar rates of improved yield. ferreira et al. (2013), under two different soil clay and sandy, observed the reaction of maize to azospirillum brasilense inoculation and nutrient (macronutrients and micronutrients) treatments in the brazilian cerrado. the reaction was considerable when maize was infected with azospirillum brasilense, but it differed depending on the soil type. the combination of n fertilizer and a. brasilense increased grain yield by up to 29 % when compared to n fertilization alone. all these observations on azospirillum strains inoculation in the field demonstrate the complexities of plantmicrobe interactions in the field, as well as the many elements that influence plant development and grain yield. marks et al. (2013) effectively used rhizobial signaling molecules to inoculate soybean and maize fields with bradyrhizobium sp. and azospirillum brasilense, respectively. these studies show that mixing rhizobia secondary metabolites with biofertilizers can help grain crops grow and produce more. according to saikia et al. (2010), azospirillum research should focus on a deeper fundamental knowledge of the system’s encompassing essential components other than broad field studies to fully exploit the potential. bacterial biofertilizers may greatly rise the yield of various crops by improving p uptake is indisputable (hinsinger et al., 2009). however, field results are frequently uneven, despite some recent favorable field inoculation research. for example, maize response with increased growth rate and greater arbuscular mycorrhizal fungi (amf) infection rates than non-inoculated control plants when it was inoculated with a p-solubilizing pseudomonas fluorescens strain (krey et al., 2013). in distinct agroclimatic areas, kaur and reddy (2014) identified two p-solubilizing bacterial strains that fulfil a vital role in plant growth increasement and soil fertility amelioration. owen et al. (2015) looked examined the terminology, composition, and roles of p solubilizers, as well as the many variables that influence their efficacy in increasing p availability in various soil and plant environments. these researchers concluded that weak quality control standards and a lack of understanding of the underlying principles confuse the efficiency of commercial biofertilizers, resulting in inconsistent field performance results. amf has long been thought to be advantageous to plant phosphorous nutrition. smith and smith (2012) illuminated their capaability to adequately absorb p from the soil and make it accessible to the plant. the outcome of this beneficial interaction between amf and plant root is impacted by soil properties, environmental conditions such as temperature and soil moisture and as well application of biofertilizers in crop production: a review. january april 2022 22 as the availability of other nutrients such as nitrogen, (johnson et al., 2015). beneficial dual inoculation (amf and rhizobia, for example) has been successfully tested in the field to increase woody legume growth (lesueur et al., 2001; lesueur & duponnois, 2005; mortimer et al., 2013). despite this, due to production constraints and a lack of applied research, commercial biofertilizers of this sort are rarely available on foreign markets. challenges in the use of biofertilizers the main constraints of biofertilizers are the poor availability of carriers; it affected the shelf life. one of the major limitations of biofertilizers is mutation during fermentation (singh et al., 2016). the main challenges with the usage and development of bio-fertilizers include reliability, inappropriate formulations, high levels of contamination, low quality, and consistency of these inoculants in field conditions. biofertilizers that perform well in the lab and greenhouse may not work as well in the field, because it is sensitive to biotic and abiotic stress. crops are grown under a wide range of conditions, including temperature, rainfall, soil type, biodiversity, and crop variety. as a result of these differences, biofertilizer efficacy is inconsistent. furthermore, because the inoculum must build up its concentration and colonize the root, biofertilizers take longer to act than synthetic fertilizers (roy, 2021). to prevent these difficulties, suitable isolates should be chosen based on their performance in the field, with a variety of crops, and in a variety of soil types and environmental circumstances (basu et al., 2021). biofertilizers should also not be used in place of other fertilizers; rather, they should be used in conjunction with them to reduce the amount of fertilizer used (roy, 2021). the shelf life of a biofertilizer is another important consideration throughout its development and commercialization. live microbial cells in biofertilizers have a short shelf life (about 6 months at 20 °c 25 °c), which demands extra care and attention during storage and transportation, raising the product›s cost (mitter et al., 2021). product registration and patent application are examples of constraints. lack of a clear legal and regulatory definition for “plant biofertilizer” or “plant biostimulant.” is the reason behind the absence of worldwide coordinated and harmonious strategy. in maximum places of the world, the process of registering biofertilizers table 3: increments in grain yields of crops by biofertilizers bio-fertilizers crops increment in grain yield (%) references azotobacter cabbage 24.3 verma et al. (1991) garlic 14.8 wange (1995) onion 18 joi and shinde (1976) azospirillum cabbage 7 jeevajyothi et al. (1993) chilli 26.7 paramaguru and natrajan (1993) knolkhol 14.9 chatto et al. (1997) onion 9.6 thiiakavathy and ramaswamy (1999) okra 9 subhiah (1991) radish 9 sundaravelu and mutukrishna (1993) sweet potato 8.5 desmond and walter (1990) rhizobium chickpea 27.9 gupta and namdeo (1996) cowpea 4.09 mishra and solanki (1996) pea 13.38 kanaujia et al. (1999) psm onion 9.6 thiiakavathy and ramaswamy (1999) potato 30.50 guar (1985) pumpkin 51.0 karuthamani et al. (1995) (source: kashyap et al., 2017). sharma, b.; yadav, l.; pandey, m.; shrestha, j. peruvian journal of agronomy 6(1): 13–31 (2022) https://doi.org/10.21704/pja.v6i1.1864 23 is confusing or troublesome, also lengthy, and complex (basu et al., 2021). conclusion biofertilizer is a product that constitute microorganisms which colonize the rhizosphere and encourage development by increasing the availability of nutrients to the host plant. bacteria, fungi, and blue green algae are commonly utilized as biofertilizers. these organisms are available in the plant’s rhizosphere to increase their activity in the soil. they aid plants indirectly by boosting nitrogen fixation and nutrient availability in the soil. crop production must expand into new frontiers while not depleting natural resources or degrading environmental quality. in conclusion by minimizing the usage of chemical fertilizers and enhancing plant nutrient use efficiency, biofertilizers are playing an increasingly important role in improving agricultural production in environment friendly manner. to support the development of organic agriculture, sustainable agriculture, green agriculture, and pollution-free agriculture, biologically based products are the most essential and they are the most advanced biotechnology. biofertilizers restore the soil›s natural fertility and protect it from drought and soil diseases, and thereby stimulating plant growth. conflicts of interest the signing authors of this research work declare that they have no potential conflict of personal or economic interest with other people or organizations that could unduly influence this manuscript. author contributions elaboration and execution, development of methodology, conception and design; editing of articles and supervision of the study have involved all authors. orcid and e-mail barsha sharma agribarsha77@gmail.com https://orcid.org/0000-0002-2061-3811 laxmeshwar yadav laxmeshwaryadav@gmail.com meena pandey pandeymeena999@gmail.com jiban shrestha jibshrestha@gmail.com references ahmad, m., nadeem, s. m., naveed, m., & zahir, z. a. 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