Impaginato 357 Adv. Hort. Sci., 2020 34(4): 357­372 DOI: 10.13128/ahsc­9425 Management of root knot nematodes (Meloidogyne sp.) and enhancing growth yield of greenhouse produced tomatoes by using fresh plant derived soil amendments P.C. Otieno (*), R.M.S. Mulwa, J. Otieno Ogweno Egerton University, Department of Crops, Horticulture and Soils, P.O. Box 536, 20115 Egerton, Kenya. Key words: Lippia kituensis, Meloidogyne sp., Ocimum gratissimum, organic amendments, tomato yield. Abstract: Production of greenhouse tomato is hampered by myriad of chal­ lenges emanating from growth medium in the sub­Saharan Africa (SSA), which has led to instability in the production trend. A greenhouse experiment was conducted to study the effect of soil amendment with fresh plant biomass from Lippia kituensis Vatke and Ocimum gratissimum L. aimed at managing root knot nematodes (RKN) and enhancing tomatoes yield. The amendments were applied at 0 (soil negative control), Lippia and Ocimum, each at 200 g, 400 g % and 800 g in 10 kg potted soil mixes, singly and in all possible combinations. Azadirachtin (0.3 w/w) was also used as a positive control. The mixtures were treated inoculums carrying 1000 second instar Meloidogyne sp. juveniles. An unbalanced factorial in a Randomized Complete Block Design with 3 replica­ tions was used. The parameters measured were nematode populations, root gall numbers, galling index, tomato growth, development and yield. Results indicated that interactive effect of soil amendment at 800 g of both Lippia and Ocimum, significantly (p<0.05) reduced the RKN population by 82.1% compared to the non­amended soil. At same rates, galls were reduced by 95.5% while galling index by 83.3%, compared to non­amended treatment. In plant develop­ ment same amendment rates demonstrated higher vegetative growth. For fruit number and marketable yield, 76.7% and 82.2% more fruits per plant were recorded from 800 g LK+ OG at 800 g and Azadirachtin respectively, compared to non­amended soil. Based on the results, Lippia and Ocimum may be poten­ tial sources for nematicidal plant products for greenhouse tomato production. 1. Introduction Tomato is among the leading greenhouse vegetable crops grown in Kenya in both soil and soilless media (HCD, 2012). Tomato constitutes 7% of the total horticultural produce in Kenya and 14% of all the entire veg­ etable produce (Ochilo et al., 2019). In terms of production in the year (*) Corresponding author: pcotieno@egerton.ac.ke Citation: OTIENO P.C., MULWA R.M.S., OTIENO OGWENO J., 2020 ­ Management of root knot nematodes (Meloidogyne sp.) and enhancing growth yield of greenhouse produced tomatoes by using fresh plant derived soil amendments. ­ Adv. Hort. Sci., 34(4): 357­372 Copyright: © 2020 Otieno P.C., Mulwa R.M.S., Otieno Ogweno J. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 14 July 2020 Accepted for publication 28 September 2020 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(4): 357­372 358 2018, Kenya was rated 7th amongst the leading coun­ tries in sub­Saharan Africa with 599,458 tonnes per year, though the trend has not been stable (FAO­ STAT, 2018). To stabilize the trend of production, greenhouse technology is seen as one of the solu­ tions in tomato production, due to high levels of effi­ ciency and the potential to support sustainable socio­ economic development. However, greenhouse toma­ to production has not been vibrant in Kenya as it should be in the tropical humid region (Sanzua et al., 2018). This is because under soil­based production, there is nutrient depletion through plant uptake and leaching beyond the root­zone of vegetable crops (Kirimi et al., 2011), resulting in reduced yield in the following season. Besides, the continuous growing of tomato in the greenhouse soil leads to accumulation of soil borne pests and pathogens especially bacterial wilt Ralstonia solanacearum, Fusarium wilt, and Nematodes (Meloidogyne sp.), which has forced most farmers to abandon greenhouse tomato pro­ duction (HCD, 2017). Studies have shown that soil based media with appropriate amendments combined with other addi­ tives may effectively manage soil­born pests and dis­ eases (McSorley, 2011). Among the pests Root­knot nematodes (RKN) Meloidogyne spp are the most damaging nematodes in tomatoes grown in the trop­ ics (Walker, 2007). Infection of roots with Meloidogyne initiates a series of events that changes the entire physiology of the host plant. Root galls result when nematodes penetrate the cells of the cortex and pericycle the endodermis and reach the stale. About 5­7 cells surrounding the nematode’s head enlarged to become a specialized giant cells, much larger than others. The thick nuclei enlarge, become polyploidy and undergo series of synchro­ nized division, (Mai and Mullin, 1996). Root symp­ toms may appear as root knots (root galls), root lesions, excessive root branching (Ogallo et al., 1997) as root tips are injured and roots rot when nematode is accompanied by plant pathogenic or saprophytic bacteria of fungi (Cerkaukas, 2004). These nema­ todes are prevalent in the greenhouse condition and invade almost all vegetable crops resulting in sub­ stantial yield losses (Stirling and Stirling, 2003). Bekal and Becker (2000) observed that at peak, 100 cm3 of soil contained 1000 nematodes. This declines with weather variation to approximately 50 nematodes per 100 cm3 of soil. According to Stirling and Kopittke (2000), the economic threshold of RKN on most crops range between 2 and 10. On yield various authors have reported on reduction in tomato due to RKN Meloidogyne spp ranging from 28% to 68% (Safiuddin Shahab et al., 2012). Under soil based greenhouse tomato production, nematode infestations are a serious constraint leading to a yield reduction (Pakeerathan et al., 2009). Suppressed plant growth and yield has been observed in nematode infested fields (Vovlas et al., 2005). Many crops grown as vegetables are sus­ ceptible to M. incognita and M. javanica particularly tomato, aubergine, okra, cucumber, melon, carrot, gourds, lettuce and peppers (Varela et al., 2003). It has been suggested by McSorley (2011) that reduction of nematodes in fields treated with plant biomass wastes results in improved soil structure, fertility and improvement of plant resistance from nematode toxins. Similarly, it may increase fungal and bacterial parasites population in the soil or other nematode antagonistic agents. Apart from nematode control, these plant based biomass also provide essential nutrients (such as N and P), which help to rebuild soil organic matter contents, and aid in the re­establishment of beneficial microbial populations (Suresh et al., 2004; Allen et al., 2007; Dauda et al., 2008). Additionally, higher organic matter content increases soil water holding capacity and supports thriving communities of decomposers and predators in the soil system. The nematicidal properties of plants may be a contribution from extracted biomol­ ecules resident in the plant bodies. The major classes of compounds with proven nematicidal activity include alkaloids, fatty acids, glucosinolates, isothio­ cyanates, phenols, diterpenes and a variety of essen­ tial oils (Chitwood, 2002). Neem cake, known to be rich in Azaderachtin is also associated with strong nematicidal activity (Abbasi et al., 2005). Laboratory extracted essential oils have also been reported to affect development of nematode eggs and second juvenile stage (J2) under in vitro conditions (Onifade, 2007). Other extracts with strong pesticidal proper­ ties include rotenone, nicotine and pyrethrins (Berger, 1994). In Verbenaceae, Lippia is among the genus of aro­ matic plants due to their essential oils (Kosgei et al., 2014). They are shrubs or woody herbs, leaves oppo­ site or verticilate, glandular. Flower is pedunculate, crowded spikes; corolla obscurely 2­ lipped, fruits are 2 dry mericarps each with very small seed. Various species in Kenya include; L. dauensis (Chiov.) Chiov, L. grandifolia A. Rich, L. javanica (Birm. F.) Spreng, L. kituensis Vatke, L. somalensis Vatke (Beentje, 1994). Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 359 The species L. kituensis Vatke has opposite leaves, ovate or elliptic; flowers white with yellow throat. In a previous study by (Kosgei et al., 2014), phytochemi­ cal analysis from methanol extracts of Lippia kituen‐ sis Vatke were done and reported to possess monoterpenes, Sesquiterpenes, diterpenes and other essential oil which were found to be effective against larvae of Rhipicephalus appendiculatus. Some of the these essential oils included Alpha­pinene (­)­, Camphene, Sabinene, beta­myrcene, l­Phellandrene, Dl­limonene, Gamma­terpinene, Trans­sabinene hydrate, Alpha­terpinolene, Neo­allo­ocimene, Camphor (1S 4S)­(­)­, Camphore, borneol (=endo­bor­ neol), 4­methyl­1­(1­methylethyl)­ 3­Cyclohexen­1­ol, 4­terpineol. In the same extract, Sesquiterpenes yielded were; Beta­bourbonene, isopropyl­5­methyl­ 9­methylene­ Bicyclo[4.4.0]dec­1­ene, Germacrene D, Gamma­Cadinene, 2­isopropyl­5­methyl­9­methyl­ ene­ Bicyclo[4.4.0]dec­1­ ene. Duschatzky et al. (2004) reported that nematicidal activity of the essential oils isolated from Lippia juneliana and L. turbinata, which were evaluated using in vitro experiments. In another study, the oils of L. juneliana and L. turbinata showed the highest nematicidal activity among the tested oils, killing more than 80% of the juveniles of the Meloidogyne sp. Analysis of the oils revealed that L. juneliana contain; piperitenone oxide (36.5%), limonene (23.1%), camphor (8%) and spathulenol (6.5%) and L. turbinata has l imonene (43.3­60.6%) and piperitenone oxide (39.3­ 17.8%). The genus Ocimum L. (Lamiaceae) comprises 30­ 160 annual and perennial herbs and shrubs, collec­ tively called basil. Species of this genus are popular sources of essential oils and aromatic compounds, of condiments, and ornamental plants (Nagai et al., 2011). The most cultivated species worldwide are O. africanum Lour. O. americanum L., O. basilicum L., O. gratissimum L., O. minimum L. and O. tenuiflorum L., mainly due to their economic and medical impor­ tance (Carović­Stank et al., 2010). Essential oils in Ocimum incude Linalool, Eugenol, Methyleugenol, Trans­α­bergamotene, p­Cresol, 2, 6­di­tert­butyl, δ­ Cadinene and (Z, E)­α­Farnesene (Nagai et al., 2011). Sifola and Barbieri (2006) reported that O. basilicum essential oil is constituted of phenylpropanoids, like eugenol, chavicol and its derivatives, and terpenoids; limonene, linalool and methyl cinnamate. Masi et al. (2006), studying nine different cultivars of O. basilicum commonly utilized, were able to identify five distinct chemotypes based on the main essential oil constituent; Iso­pinocamphone (35.1%) and car­ vone (39.7%) which were the predominant compo­ nents of the essential oil in cultivated O. basilicum. This was also confirmed in another study by Almeida et al. (2010). Several species of Ocimum have been reported to yield oils of diverse nature (Matasyoh et al., 2007; Ogendo et al., 2008). Other studies have also shown that the leaf extract of Ocimum gratissimum contain potential bioactive components of essential oils. These are made up of eugenol, citrol linalol, charvi­ col, thymol, gerianol, triterpenoids saponins and alkaloids (Atuboyedia et al., 2010). It was also report­ ed by Echeverrigaray et al. (2010) that monoter­ penoids significantly reduce the hatching of eggs and mobility of J2 of Meloidoyne spp. Onifade (2007), in the study to find the effect of essential oils from five Ocimum sp. on the pathogenicity of Pratylenchus brachyurus (Godfrey) in tomato, reported that in vitro at 25­100 µg mL­1, the oils of O. gratissimum and O. basillicum completely inhibited egg hatching and larval survival of nematodes after 24 hours. More study is still needed to explore the potential of essen­ tials and other compunds involved in the nematode control in the two plant families Verbenaceae and Lamiaceae. The current work was therfore focused on investigating the potency of Lippia kituensis Vatke and Ocimum gratissimum L. on the management of root knot nematodes Meloidogyne spp. In this study we report results of a greenhouse pot experiment to evaluate management of root knot nematodes using fresh plant derived soil amendments with Lippia kituensis Vatke. and Ocimum gratissimum L. and their effects on tomato yield. 2. Materials and Methods Experimental site The study was conducted in two growing seasons at the Horticulture Research and Teaching Field, Egerton University, Kenya between July 2015 and May 2016. The site received a mean rainfall of 1012 mm with a mean day temperature of 22°C and night ranges of 5­10°C (Jaetzold et al., 2012). The pot experiment was conducted in a polytunnel greenhouse measuring 8 m wide × 60 m length and 3 m height, covered with UV stabilized polythene sheet gauge 200 μm. Plant materials Leafy twigs of L. kituensis Vatke. and O. gratissi‐ mum L. were collected in the wild around Egerton University at flowering stage, when essential oil was Adv. Hort. Sci., 2020 34(4): 357­372 360 at its peak in the plants in the month of July 2015 (Fig. 1). These two shrubs flower all the year around visited by bees hovering over the flowers to collect nectar from scent produced by the plants. The mate­ rials were chopped into aggregates approx. 0.5 cm; the aggregates were incorporated in various propor­ tions (0 ­negative control, 200 g, 400 g and 800 g) in 10 kg of potted solarized forest soil, singly and in all possible combinations. Crop establishment The planting material used in the study was toma­ to seedlings ‘Rio Grande’ Rio Grande is a determinate tomato cultivar with a high yielding potential thus preferred by many farmers. This vigorous variety is well adapted to extreme temperatures. Due to the potential of heavy crops and to keep the fruit clean and easy to pick, it is recommended to support plants with stakes or cages. Seeds used to raise the tomato seedlings were obtained from Simlaws Seed Company in Nakuru (Kenya) and established in a nursery for 5 weeks before transplanting. Five weeks old tomato seedling were transferred to the sub­ strates in the pots in the greenhouse to develop (Fig. 2). When plants reached a height of 30 cm, they were supported using sisal twines tied on a binding wire trellis at a height of 150 cm above the bed. Calcium ammonium nitrate (CAN, 26% N) was applied at the rate of 10 g per pot 21 days after transplanting (DAT) to maintain growth. Plants were pruned to maintain two stems per plant and water­ ing was done continuously during the growing period with rates being adjusted according to plant growth phases. In the first 30 DAT, 2 l of water was applied per plant per day and thereafter, the rate was increased to 4 l to 42 DAT per day. From 43 DAT water supply was increased to 5 l per day as the plants developed, according to the work by Pires et al. (2011). Nematode augmentation, extraction and inoculum preparation Nematodes were collected from a field previously grown with infested tomatoes and augmented on two weeks old potted tomato seedlings established in a greenhouse following the method of Siddiqui and Akhtar (2007). Specifically, Galls were extracted from the roots of infested tomatoes, chopped and mixed with the native soil. The mixture was added to pots planted with 2 week old tomato seedlings and the inoculum allowed to infest and multiply for 8 weeks. After augmentation period, nematode egg masses were extracted from the heavily galled tomato roots by chopping the roots to lengths of 0.5 cm and mac­ erating the tissues to release egg masses. These were placed in 15 cm diameter sieves of 1 mm pore size, lined with cross­layered tissue papers and incubated at 27°C to hatching in glass petri­dishes containing distilled water. After hatching, the second instar juve­ niles (J2) were transferred into 2 L conical flasks. Quantification of juveniles was done under a stereo­ scope with gridded petri dishes. Ten 1 ml replicate samples were drawn from the well mixed suspen­ sions to establish the average number of juveniles per ml. The determined quantity was 20 juveniles per ml. Finally, the nematode inoculum suspension sam­ ples were adjusted to contain approx. 1000 juveniles in 50 ml of distilled water. Nematode inoculation and determination of infesta‐ tion parameters The 50 ml J2s inoculum suspensions were added to pots containing the various plant biomass amend­ ed media planted with 28 day old tomato trans­ plants. The inoculum was allowed to develop under normal tomato culture conditions in a polytunnel greenhouse. Destructive sampling of tomato plants to determine nematode infestation was conducted 100 DAT. Four plants from each replicate were sam­ Fig. 1 ­ A. Lippia kituensis Vatke and B. Ocimum gratissimum L.; C. Chopping of the fresh Ocimum gratissimum plants into smaller pieces; D. Potting of the mixture (soil amend­ ment process). Fig. 2 ­ Transplants on treatment pots in the greenhouse. Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 361 pled at the peak of flowering. Determination of macro nutrient from the tomato leaves Nitrogen (Kjeldahl) and Phosphorus. Tomato leaves tissue were analyzed at 49 DAT for macro ele­ ments N, P, K, Mg and Ca. from the top of the plant, leaves were taken from the third and fourth leaflet per treatment. The sample materials were chopped into aggregates approx. 0.5 cm and oven dried at 70°C until constant weight. The oven dry plant sam­ ples were ground and wet digested by a sulfuric ­ percloric acid mixture as described by Cottenie et al. (1982). Nitrogen and phosphorus contents of vegeta­ tive samples were measured in the digesting extract according to the methods of AOAC (2012). Calcium and potassium content was determined in vegetative sample by ashing dry sample as described by Chapman and Pratt (1978) extract method. Analysis of Potassium, Calcium and Magnesium. A substrate sample weighing 0.3 g was digested in digestion tubes using a digestion mixture comprising of HCl, HNO3, HF and H3 BO3. The temperatures in the block was maintained at 360°C for two hours then samples cooled and transferred to 50 ml volumetric flasks and volume made to the mark. Calibration was done for each element using certified standards. Samples were analyzed using Atomic Absorption Spectrophotometer (AAS), Varian spectra AA10 AAS machine. The determination of these elements in the substrate was done using double acid method of Table 1 ­ Treatment combinations and description of soil amendment rates used in nematode management OG= Ocimum gratissimum L. (g/10 kg soil); LK = Lippia kituensis Vatke (g/10 kg soil), are soil organic amendments; 0 = no amendment; AZAD = positive control of commercially know pesticide Azadirachtin. extraction. AAS was used for estimation of these available elements in the tested substrate. This fol­ lowed the procedure of Okalebo et al. (2002). Experimental design and treatment layout The experimental design used was a factorial embedded in a Randomized Complete Block Design and in total there were 17 treatments. There were three blocks and pots arranged in rows spaced at 0.6 m between and 0.4 m within the rows. There were 4 levels (0 g, 200 g, 400 g, and 800 g of organic amend­ ments from each of the plant species, replicated 3 times. A negative control of solarized non­amended soil and a positive control of 0.3% w/w Azadirachtin, a farmer’s standard commercially known organic based Neem extract were included. In total there were 17 treatment plots of 6 potted tomato plants in each block (Table 1). Nematodes evaluation Nematodes population. To determine the nema­ tode population in the biomass amended pot soil treatments, second stage juveniles (J2) were extract­ ed from 100 cm3 composite sample of soil from each replicate, using the method described by Kimenju et al. (2010). Specifically, at 100 DAT, the soils from each of the four pots were sampled by taking 100 cm3 of sample. The samples were placed in 9 cm diameter sieves with pore diameters of 1 mm lined with double layered tissue paper. The sieves were half immersed in metallic troughs containing 250 ml Soil Amendments treatments (g/pot) Description of treatments 0 0% w/w Lippia kituensis and 0% w/w Ocimum gratissimum (control) 200 OG 2% w/w Ocimum gratissimum 400 OG 4% w/w Ocimum gratissimum 800 OG 8% w/w Ocimum gratissimum 400 LK 4% w/w Lippia kituensis 200 LK +200 OG 2% w/w Lippia kituensis and 2% w/w Ocimum gratissimum 200 LK +400 OG 2% w/w Lippia kituensis and 4% w/w Ocimum gratissimum 200 LK +800G 2% w/w Lippia kituensis and 8% w/w Ocimum gratissimum 400 LK 4% w/w Lippia kituensis 400 LK+ 200 OG 4% w/w Lippia kituensis and 2% w/w Ocimum gratissimum 400 LK+ 400 OG 4% w/w Lippia kituensis and 4% w/w Ocimum gratissimum 400 LK+ 800 OG 4% w/w Lippia kituensis and 8% w/w Ocimum gratissimum 800 LK 8% w/w Lippia kituensis 800 LK +200 OG 8% w/w Lippia kituensis and 2% w/w Ocimum gratissimum 800 LK +400 OG 8% w/w Lippia kituensis and 4% w/w Ocimum gratissimum 800 LK +4 OG 8% w/w Lippia kituensis and 8% w/w Ocimum gratissimum AZAD Commercial (0.3% Azadirachtin) control 362 Adv. Hort. Sci., 2020 34(4): 357­372 of distilled water to allow nematode migration into the water underneath for 24 hours. Nematode counts were determined in 10 replicate samples of 1 ml for each soil sample as previously described. Gall number and galling index. For gall assess­ ments, plants were gently uprooted and their roots thoroughly washed under tap water to remove all the adhering soil. Galling was determined by counts of galls size 1 mm diameter and above by a light microscope using the lowest objective lens x 4. Galled roots were spread in on plastic petri dish made of 1 cm2 grids, numbered at the base. The galls were scored from each square to get the summation per plant. The galling index was scored on a scale of 1­10, where 0= no gall, 1= 1­50 galls, 2= 51­100 galls, 3= 101­150 galls, 4= 151­200 galls, 5= 201­250 galls, 6= 251­300 galls, 7= 301­350 galls, 8= 351­400, 9= 401­450 and 10= 451 and above (Kimenju et al., 2010). The scores were converted into numerical entries and their means worked out for analysis of variance. Tomato growth evaluation Number of leaves. Leaf count data was collected from 4 plants in each plot. Leaf count data collection was commenced 21 DAT and continued at intervals of 14 days up to 91 DAT. At each instance of data col­ lection the mean number of leaves per plant from each replicate was computed. The mean number of leaves per treatment was determined by computing the means. Plant height. Plant height data was collected from 4 plants in each plot. This started at 21 DAT and con­ tinued at 14 days interval up to 77 DAT. At each instance of data collection the mean height per plant from each replicate was computed. The mean height per treatment was determined by computing the means. Root volume. Root volume data was determined by carefully removing the plants from the pot, shak­ ing off the soil, and washed in running water on the trough at 100 DAT. Root volume was determined water displacement method in a one liter plastic measuring cylinder. The cylinder was filled to 500 ml mark, then the roots dipped carefully until the water just covered all the roots on the 4 plants used for root length determination and means computed to get the means. Shoot and root dry weights. From the 4 plants used for root length and volume determination, shoots were separated from the roots at the collar. These were individually placed in kaki paper bags and dried in an oven at 70°C to constant weights. Both parts were weighed separately and means of the weights computed as above. Physiological parameter Stomatal conductance and chlorophyll content. Leaf stomatal conductance (mmol·m­2·s­1) was mea­ sured on four tomato tagged plants from each treat­ ment. Using a steady state leaf porometer (SC­1, Decagon Devices, Pullman, WA), stomatal conduc­ tance was measured on a 2 weeks interval from 21 days after transplanting (DAT). Since tomato plants are hypostomatous, stomatal conductance was mea­ sured only on the abaxial leaf surface on 3 leaves on the upper parts of the plant and the average was computed. Leaf chlorophyll content was taken from the same leaves used for stomatal conductance. The instrument used was a chlorophyll content meter (CCM­200 plus, Opti­Sciences, Tyngsboro, MA) and measurement in chlorophyll concentration index units (CCIs), as an estimate of chlorophyll content on leaves. Yield Number of fruits per plant. Weekly piece meal harvesting of pink stage tomato fruits from the 4 tagged plants in each treatment was done. At each harvest, the number and weight of fruits were recorded for each treatment. Marketable and Non‐marketable fruit yield. Physiologically mature fruits (at pink stage) were har­ vested from the 4 tagged plants in each treatment. Harvesting was piece meal on weekly basis. At each harvest, fruits were sorted into marketable and non­ marketable (kg/plant) separately and their weight determined and recorded. Fruit weight was taken from mature marketable fruits (at pink stage), har­ vested from the 4 tagged tomato plants from each treatment. These were weighed using a spring bal­ ance (ATZ, Shangai Precision and Scientific Instrument Co., Shangai, China) at each harvest and later summed up to give the total marketable weight (kg/plant). Data analysis Data for the two trials were pooled since there was no statistical difference between them. It was then subjected to analysis of variance (ANOVA) and means separated by the Tukey’s HSD using The SAS statistical package version 9. The model fitted for this experiment was; Yijk= μ + βi + αj + γk + αγjk + εijk where yijkl = tomato response, μ = overall mean, βi = effect of the ith block, αj = effect of the jth level of Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 363 Lippia kituensis Vatke ,γk = effect of the kth level of Ocimum gratissimum L., αγjk = interaction effect of the jth level of Lippia kituensis Vatke and kth level of Ocimum gratissimum L. εijk = random error compo­ nent term which are normally and independently dis­ tributed about zero means with a common variance σ2. 3. Results Effect of soil organic amendment on nematode popu‐ lation, gall numbers and galling index The different levels of amendments with Lippia kituensis Vatke and Ocimum gratissimum L. biomass and their combinations significantly (P<0.05) influ­ enced nematode populations in the treatments (Table 2). Various rates of treatment reduced the juvenile populations when compared with the con­ trol. In single treatments of Lippia at 200 g, 400 g and 800 g, nematode numbers was reduced to 38.88 (25.9%), 19.29 (63.3%), and 20.58 (60.8%) respective­ ly, compared to the non­amended treatments. Ocimum had similar trend with reduction to 47.38 (9.75%), 34.58 (34.1%) and 30.63 (41.7%), respective­ ly (Table 2). However, the interactive effect of the two plant biomass from 400 g and above produced better nematode reduction than single treatments alone compared to soil. Gall numbers and galling index were determined 100 DAT and were significantly P<0.05) influenced by organic amendments (Table 2). There was a general decrease of gall numbers in roots of tomato plants with increased levels of Lippia and Ocimum biomass in the potting soil. Application of Lippia singly at rates of 200 g, 400 g, and 800 g per pot reduced gall num­ bers to 190 (66.9%), 155 (73.0%) and 125.60 (78.1%) respectively compared to soil with Ocimum following the same trend with reduced galls numbers to 470.67 (18.1%), 422.5 (26.5%) and 175.3 (69.5%) respective­ ly. Interactive effect of the two plant biomass above at the rates of 200 g was better reduction of gall numbers up to 25.17 galls (95.5%) and no significant differences (P<0.05) were evident among the various combinations. The efficacy of the fresh plant biomass materials in managing nematode proliferation on tomato roots was also evident in the galling index scores from the various treatments. In general the gall index showed a reducing trend with increasing levels of Lippia and Ocimum biomass amendments. The interactive effect of the two plant species both at 800 g produced tomatoes with vigorous, fibrous root system with very few galls only observed under light microscope. Table 2 ­ Effect of fresh organic amendments on nematode population, gall number and galling index OG = Ocimum gratissimum L.; LK = Lippia kituensis Vatke; 0 = no amendment (soil). * Means followed by the same letter series within a column are not significantly different according to Tukey’s honestly significant diffe­ rence (THSD) at P≤0.05. Amendments g/10 kg) No. of nematodes (per 100 cm3 soil) No. of galls/plant Galling index 0 52.50 a * 574.67 a 10.00 a 200 OG 47.38 b 470.67 b 10.00 a 400 OG 34.58 d 422.50 c 9.00 a 800 OG 30.63 ef 175.30 de 5.00 c 200 LK 38.88 c 190.00 de 7.30 b 400 LK 19.29 g 155.00 defgh 5.70 c 800 LK 20.58 g 125.60 defgh 3.00 de 200 LK + 200 OG 38.21 c 211.00 cd 4.67 c 200 LK + 400 OG 33.29 de 197.90 de 5.00 c 200 LK + 800 OG 28.33 f 123.40 defgh 3.67 cd 400 LK + 200 OG 17.50 gh 155.60 defgh 4.50 c 400 LK + 400 OG 12.25 i 130.17 defgh 3.00 de 400 LK + 800 OG 10.38 i 85.00 fgh 2.00 ef 800 LK + 200 OG 12.25 i 100.00 fgh 3.67 cd 800 LK + 400 OG 9.38 i 80.67 fgh 2.00 ef 800 LK + 800 OG 9.42 i 25.67 gh 1.67 ef Azadirachtin 4.17 j 13.17 h 1.00 f Adv. Hort. Sci., 2020 34(4): 357­372 364 Comparatively, those roots of plants grown in non­ amended treatments (soil) had numerous galled roots which were less fibrous (Fig. 3). Effect of fresh plant organic amendments on tissue of greenhouse tomato The different levels of amendments of Lippia and Ocimum soil amendments had significant (P<0.05) influence on tomato plant tissue nutrients. The plant macro nutrient analyzed included N, P, K, Ca and Mg, which are the most essential element in tomato pro­ duction. The interactive effect of the Lippia and Ocimum biomass were significantly higher in the tomato leaf tissues from both amendment rates above 400 g/10 kg of the substrate (Table 3). In single state, both plant biomass registered significantly higher N content at 800 g only. Number of leaves Tomato leaf numbers were significantly influ­ enced by the use Lippia and Ocimum as soil organic amendments (Table 4). There were significantly (P<0.05) higher leaf number at 21 DAT in soil alone and 200g pots of both Lippia and Ocimum levels than 400g and above. However trend changed as from 35 DAT as from 35 DAT on 400 g of both species having the highest leaf numbers. At 49 DAT there were no significant difference (P<0.05 on treatment of both species from rates above as single or in combination 4 Non­amended soil had the least number of leave except for 21 DAT. Compared to the positive con­ trols, Azadirachtin treated had relatively lower num­ ber leaves than 800 g of both Lippia and Ocimum combined, however this was higher than the un­ amended soil. Plant height The result showed that the height of tomato plant was significantly (P<0.05) influenced by the Lippia and Ocimum levels (Table 5). Plants amended with 400 g of Lippia or Ocimum were taller than those of the lower levels of amendment and Azadairachtin. As in the leaf numbers plant height differences were observed 49 DAT were no difference were from each treatment up to the highest rate. Root volume The organic amendments significantly influenced the development of the total root volume of the tomatoes grown in the pots during the production seasons (Table 6). In root volume interactive effect Fig. 3 ­ The effect of Lippia and Ocimum on the tomato root system. A) LK+ OG at 800 g show fibrous root system, B) LK+OG at 200 g with fewer galls, and C) Non­amended soil high nematodes infestation. Table 3 ­ Effect of fresh plant organic amendments on tissue analysis of greenhouse tomato OG = Ocimum gratissimum L.; LK = Lippia kituensis Vatke; 0 = no amendment (soil). * Means followed by the same letter series within a column are not significantly different according to Tukey’s honestly significant diffe­ rence (THSD) at P≤0.05. Amendments (g/10 kg) N (%) P (%) K (%) Ca (%) Mg (%) 0 2.95 d * 0.23 e 2.82 g 0.68 l 0.28 f 200 OG 3.35 bcd 0.33 cd 3.12 ef 0.98 k 0.33 ef 400 OG 3.45 abc 0.30 cde 2.92 fg 1.18 i 0.32 ef 800 OG 3.35 bcd 0.32 cd 3.12 ef 1.68 f 0.36 de 200 LK 3.55 ab 0.33 cd 3.32 de 1.08 j 0.42 cd 400 LK 3.75 ab 0.43 b 3.42 cd 1.58 g 0.31 ef 800 LK 3.85 a 0.56 a 3.72 ab 2.08 b 0.49 abc 200 LK + 200 OG 3.35 bcd 0.34 cd 3.32 de 1.88 d 0.36 de 200 LK + 400 OG 3.75 ab 0.35 bc 3.72 ab 1.38 h 0.36 de 200 LK + 800 OG 3.55 ab 0.60 a 3.12 ef 1.78 e 0.38 de 400 LK + 200 OG 3.85 a 0.38 bc 3.52 bcd 1.88 d 0.35 def 400 LK + 400 OG 3.65 ab 0.37 bc 3.82 a 1.98 c 0.37 de 400 LK + 800 OG 3.65 ab 0.55 a 3.72 ab 2.38 a 0.42 cd 800 LK + 200 OG 3.65 ab 0.61 a 3.62 abc 2.38 a 0.46 bc 800 LK + 400 OG 3.55 ab 0.59 a 3.72 ab 2.08 b 0.52 ab 800 LK + 800 OG 3.65 ab 0.58 a 3.82 a 2.38 a 0.54 a Azadirachtin 3.05 cd 0.26 de 2.92 fg 1.08 j 0.28 f Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 365 was observe in the amendment at 400 g of both species with highest of 312.3 cm3. Azadirachtin treat­ ed soils recorded relatively lower with root volume of 89.00 cm3, which was not significantly different from the non­amended soil. Root and shoot dry weight As observed in the root volume, root and shoot dry weight showed effect by Lippia and Ocimum lev­ els in the treatments for the two seasons in a similar trend (Table 6). Interactive effect of the two plant Table 4 ­ Effect of fresh plant biomass on tomato leaf numbers OG = Ocimum gratissimum L.; LK = Lippia kituensis Vatke; 0 = no amendment (soil). DAT = Days after transplanting. * Means followed by the same letter series within a column are not significantly different according to Tukey’s honestly significant diffe­ rence (THSD) at P≤0.05. Amendments g/10 kg) Leaf numbers 21 DAT 35 DAT 49 DAT 63 DAT 77 DAT 0 12.00 ab * 13.06 gh 20.83 cd 21.83 fg 22.67 k 200 OG 11.39 abc 12.61 h 20.94 bcd 22.67 ef 23.67 j 400 OG 10.28 cdef 14.61 cde 21.94 abcd 25.61 cd 26.20 fg 800 OG 0.28 cdef 14.78 cd 25.39 abc 27.33 abc 27.73 de 200 LK 12.21 a 13.28 fgh 21.83 abcd 26.61 c 27.66 de 400 LK 10.06 defg 13.5 efgh 16.94 d 20.11 g 24.67 i 800 LK 11.22 abc 14.28 cdef 24.89 abc 22.67 ef 27.20 e 200 LK + 200 OG 9.50 efgh 14.28 cdef 23.28 abc 26.83 bc 27.32 de 200 LK + 400 OG 11.22 abc 14.94 bcd 24.39 abc 25.79 cd 27.33 de 200 LK + 800 OG 10.94 bcd 15.22 abc 23.39 abc 26.72 c 27.27 e 400 LK + 200 OG 10.28 cdef 15.22 abc 24.11 abc 29.28 ab 29.53 ab 400 LK + 400 OG 9.28 fgh 14.06 defg 21.17 abcd 23.83 de 26.53 ef 400 LK + 800 OG 10.78 cd 16.21 a 23.50 abc 27.11 bc 29.60 a 800 LK + 200 OG 10.61 cde 15.39 abc 24.22 abc 26.18 cd 28.60 bc 800 LK + 400 OG 8.61 h 16.00 ab 24.56 abc 26.33 cd 29.73 a 800 LK + 800 OG 9.06 gh 16.33 a 27.39 a 29.83 a 29.90 a Azadirachtin 12.33 a 13.56 efgh 23.89 abc 27.17 bc 28.13 cd Table 5 ­ Effect of fresh plant biomass from Lippia kituensis Vatke and Ocimum gratissimum L. on tomato plant height OG = Ocimum gratissimum L.; LK = Lippia kituensis Vatke; 0 = no amendment (soil). DAT= Days after transplanting. * Means followed by the same letter series within a column are not significantly different according to Tukey’s honestly significant diffe­ rence (THSD) at P≤0.05. Amendments g/10kg) Plant height 49 DAT 63 DAT 77 DAT 0 48.78 fg * 91.56 f 91.56 f 200 OG 49.33 f 94.00 ef 94.00 ef 400 OG 71.72 ab 99.00 cde 99.00 cde 800 OG 70.11 abc 98.93 cde 98.93 cde 200 LK 58.22 def 98.60 cde 98.60 cde 400 LK 60.06 cde 102.80 abcd 102.80 abcd 800 LK 73.67 a 108.40 a 108.40 a 200 LK + 200 OG 66.61 abcd 97.47 de 97.47 de 200 LK + 400 OG 72.83 a 99.73 bcde 99.73 bcde 200 LK + 800 OG 67.06 abcd 97.80 de 97.80 de 400 LK + 200 OG 62.22 bcde 101.27 abcde 101.27 abcde 400 LK + 400 OG 72.83 a 105.73 abcd 105.73 abcd 400 LK + 800 OG 69.67 abc 108.27 ab 108.27 ab 800 LK + 200 OG 65.78 abcde 100.13 abcde 100.13 abcde 800 LK + 400 OG 66.50 abcd 106.53 abc 106.53 abc 800 LK + 800 OG 76.00 a 105.47 abcd 105.47 abcd Azadirachtin 69.28 abc 88.40f 88.40 f Adv. Hort. Sci., 2020 34(4): 357­372 366 were significantly higher weight as from 400 g and above. As was observed in the root volume, highest root dry weight of 53.1 g was not significantly differ­ ent from Azadirachtin treated soils 51.7 g but signifi­ cantly different from the control soil 24.1 g. similarly interactive effect of LK and OG registered 101 g, sig­ nificantly higher than Azadirachtin treated soils 86.2 g and soil 46.1 g in LK 800 g combined with 800 g OG, Azadirachtin treated soils and soil alone respectively. Effect of organic amendments on physiology response of tomatoes Chlorophyll content and stomata conductance Organic amendments levels influenced chloro­ phyll content in the tomato plant positively as shown in figure 4. For both season, interactive effect of both plant species biomass were significantly higer than most of the single rates. Soil had the least chlorophyll content compared to the treated soils. The stomatal conductance of the leaves from the rates mentioned were affected in similar manner (Fig. 5). However Azadirachtin treated soil was not significantly differ­ ent from highest combination of the biomass in stomatal conductance. Generally single treatments showed lower physiological process in the leaves. Number of fruits per plant Lippia and Ocimum, significantly (P<0.05) influ­ enced fruit number per plant (Fig. 6). Interactive effect of the two plants had mean of 62.72 fruits, Fig. 4 ­ Effect of fresh plant organic amendments on tomato plant chlorophyll content taken after two week interval. Means followed by the same letter in a letter series within a column per season are not significantly different according to Tukey’s honestly significant difference (THSD) at P≤0.05. OG =Ocimum gratissimum L. and LK = Lippia kituensis Vatke, are soil organic amendments in g/10 Kg soil, 0 = no amendment and AZAD = positive con­ trol of commercially know pesticide Azadirachtin. Fig. 5 ­ Effect of fresh plant organic amendments on tomato plant stomatal conductance taken after two week inter­ val. Means followed by the same letter in a letter series within a column per season are not significantly different according to Tukey’s honestly significant difference (THSD) at P ≤ 0.05. OG =Ocimum gratissimum L. and LK = Lippia kituensis Vatke, are Soil organic amendments in g/10Kg soil, 0= no amendment (soil), AZAD= Azadirachtin. Table 6 ­ Effect of fresh plant biomass on tomato leaf numbers OG = Ocimum gratissimum L.; LK = Lippia kituensis Vatke; 0 = no amendment (soil). * Means followed by the same letter series within a column are not significantly different according to Tukey’s honestly significant diffe­ rence (THSD) at P≤0.05. Amendments (g/10 kg) Root volume (cm­3) Root dry weight (g) Shoot dry weight (g) 0 69.33 i * 24.17 h 46.12 j 200 OG 135.33 h 28.50 h 57.92 i 400 OG 84.6 7i 44.17 cdef 73.86 fg 800 OG 281.00 d 39.83 defg 69.95 fgh 200 LK 132.00 h 39.67 defg 71.42 fg 400 LK 249.67 e 39.58 efg 77.01 e 800 LK 282.33 cd 38.58 fg 86.56 c 200 LK + 200 OG 286.67 bcd 38.00 g 66.47 h 200 LK + 400 OG 229.33 e 43.00 cdefg 66.53 h 200 LK + 800 OG 279.67 d 46.50 bc 75.66 ef 400 LK + 200 OG 194.67 f 45.17 cde 86.10 c 400 LK + 400 OG 165.67 g 45.25 cd 96.68 b 400 LK + 800 OG 312.00 a 53.08 a 99.78 ab 800 LK + 200 OG 248.67 e 56.08 a 97.67 b 800 LK + 400 OG 310.00 ab 53.17 a 98.78 b 800 LK + 800 OG 306.00 abc 53.00 a 100.85 a Azadirachtin 89.00 i 51.75 ab 86.23 cd Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 367 while non­amended control had 22.24 tomatoes per plant for season 1 and 2. Azadirachtin had 32.50 tomatoes per plant. Effect of fresh plant biomass on marketable yield and non‐marketable yield There was a general increase of marketable toma­ to fruits with the increase of the rates of plant organ­ ic amendment (Fig. 7A). The interaction between the especially above in Lippia or Ocimum 400 g produced tomato with higher t/ha than single treatments and soil media. Azadirachtin treated soil had better yield though significantly lower than those in 800 g of both species, which were rated as marketable.. Non­mar­ ketable showed a reverse trend on the fruit weight (Fig. 7). There was an opposite trend in the effect of the amendment from that of marketable. Any combi­ nation biomass lower than 400 g did not produce marketable but poor qualities fruits represented as blossom end rot, blotch ripening, puffiness, gold flex, car face, sunscald and very small size stony fruits, which rendered them non­marketable. (Fig. 7B1, 7B2, 7B3). 4. Discussion and Conclusions The practice of adding organic matter to soil for management of soil pest and increase yield is as old as the agriculture (Akhtar and Alam, 1993) and this has been successfully explored to control some plant parasitic nematodes (Ferraz and Freitas, 2004; Lopes, 2011). This study revealed positive interactive effects of Lippia kituensis Vatke and Ocimum gratissimum L. as fresh biomass for the control of nematodes (Meloidogyne spp.) in greenhouse tomatoes. In over­ all, the results indicated effective nematode control in the tomato crop treated with the plant biomass compared to the control treatments where no amendments were applied. Additionally, it was gen­ erally observed that interactive effect of biomass treatments of the two species at the higher rates (400 g and 800 g) was more effective than the single treatments. In line with this study, Oka et al. (2007) reported sensitivity of plant­parasitic nematodes to plant derived amendments, however, they indicated that the effect varied with the nematodes species targeted and the rates applied. Our results further revealed that the second instar juveniles (J2) of Meloidogyne spp were more susceptible to higher rates of these treatments and effectively suppressed the nematodes in the media. Several postulations on the mechanisms of action of these fresh biomass materials have been put for­ ward. It has been reported that during the decompo­ sition of these organic materials, volatile fatty acids, ammonia and hydrogen sulphide gases are released (McSorley, 2011) and these may enhance nematode control. Alternatively, other authors have explained the mechanisms of nematode population reduction by soil amendments with organic matter to involve Fig. 6 ­ Effect of fresh plant biomass from the plant species Lippia kituensis and Ocimum gratissimum. on tomato plant fruit per plant. Means followed by the same letter in a letter series within a column per season are not significantly different according to Tukey’s honestly signi­ ficant difference (THSD) at P ≤ 0.05. OG =Ocimum gratis‐ simum L. and LK = Lippia kituensis Vatke, are Soil organic amendments in g/10 Kg soil, 0 = no amendment and AZAD = positive. Fig. 7 ­ A. Effect of fresh plant biomass on marketable yield and fruit Quality. Means followed by the same letter in a let­ ter series within a variable are not significantly different according to Tukey’s honestly significant difference (THSD) at P≤0.05. OG =Ocimum gratissimum L. and LK = Lippia kituensis Vatke, are Soil organic amendments, 0 = no amendment and AZAD = Azadirachtin. (2% OG= 200 g OG, 4% OG= 400 g OG 8% OG= 800 g OG; 2% LK= 200g LK, 4% LK= 400 g LK, 8% LK= 800 g LK /10 Kg). B. The picture shows difference in the quality of tomato at different rates of Lippia and Ocimum amendments B1= LK+OG at 800 g; B2= 0 LK+200 g OG; B3= Non­ amended soil. Adv. Hort. Sci., 2020 34(4): 357­372 368 stimulation of antagonistic microorganisms, libera­ tion of secondary volatile or nonvolatile phytochemi­ cals with nematicidal properties (Lopes et al., 2011). As earlier reported by Chavarría­Carvajal and Rodríguez­Kábana (1998), the amendments may improve the growth of the plants and hence increase the tolerance and plant resistance to nematodes. In this study L. kituensis and O. gratissimum biomass additions to soil probably proved toxic to Meloidogyne spp. under greenhouse conditions, even at low rates of two species when combined at 200 g per pot of 10 kg of soil (Table 2). These results concur with the study of Lopes et al. (2011), who reported that soil amendment with the aerial portion of cer­ tain plant species has nematicidal properties. Similar results have been reported by Kagai et al. (2012), working with selected plant biofumigants in the man­ agement of plant parasitic nematodes in Asclepias tubaerosa L. In line with this study, Onifade (2007) earlier reported that essential oils from basil (Ocimum basillicum) had nematicidal effect on para­ sitic nematodes, especially Meloidogyne spp and Pratylenchus penetrance which is root lesion nema­ tode. The second possible mechanisms for nematode suppression by these organic amendments could be direct inhibition or reduced infectivity of nematodes on the plant host. This may also be speculated that the use of Lippia and Ocimum as fresh soil organic amendment enhances antagonism in the soil mixes by increasing the abundance of other competing ben­ eficial organisms, thus reduces the chances RKN sur­ vival. These results are in concurrence with a study by Claudius­Cole et al. (2010), which reported reduc­ tion of Meloidogyne incognita on cow pea Vigna unguiculata (L) Walp using plant extract. Besides, in agreeement with the present study Hasabo and Noweer (2005), earlier reported that the extract of Ocimum reduced nematode population on eggplant, and increased resultant fruit yields. In the present study, different rates of amend­ ments with L. kituensis Vatke and O. gratissimum L. biomass significantly influenced gall numbers and galling index in the treatments especially when the two plant biomass interacted (Table 2). This was demonstrated when galling index was drastically reduced. Various studies have shown similar observa­ tions in using organic amendments to control RKN. Breakdown of plant organic material releases nemati­ cidal substances that may contribute to nematode control (Chen et al., 2000). Akhtar and Malik (2000) also reported that crops and weeds release biochem­ icals that counteract the activities of nematodes. This has also been confirmed by McSorley (2011) that nematicidal compounds released from decomposing materials can stimulate the natural enemies of nema­ todes and improving plant tolerance. In line with the present study, Lippia and Ocimum have been report­ ed to yield essential oils of diverse nature (Atuboyedia et al., 2010). Laboratory analysis of Ocimum yielded eugenol, citrol linalol, charvicol, thy­ mol, gerianol, triterpenoids, saponins and alkaloids (Matasyoh et al., 2007; Ogendo et al., 2008). Based on the findings of the present investigation, it is plau­ sible to suggest that these biomolecules extracted during decomposition of the plants biomasses helped to inhibit nematode activity in the amended soil, leading to low galling index. It also concur with observations by Onifade (2007) indicated that use of essential oils of O. gratissimum and O. basillicum in vitro at rates which completely inhibited egg hatch­ ing and larval survival of nematodes and this proba­ bly caused reduction of gall number. From the present results on macro element analy­ sis of the amended media, it is clear that besides act­ ing as a nematicide for the management of RKN, Lippia and Ocimum also acted as plant nutrient source for tomato growth and yield. Mostly these element were significantly higher in the both amend­ ment rates above 400g and above per 10 kg of the substrate (Table 3). Nitrogen, P, K Mg and Ca are essential macro element, important in entire plant growth and development. In particular, nitrogen is mostly required by plants to achieve high rates of growth and yield of tomato. The presence of these elements may promote physical and physiological changes in the plant and mostly related to photosyn­ thesis, whereas Mg also plays a big role in chlorophyll structure (Taiz and Zieger, 2002). Nitrogen is a criti­ cal macronutrient influencing processes growth and development directly on source­sink relations, alter­ ing the distribution of assimilates between the vege­ tative and the reproductive part resulting into yield (Zuba et al., 2011). Phosphorus is for root develop­ ment, flower initiation, seed and fruit development. Unlike N and P, K does not form any vital organic compounds in the plant, however, its presence is vital for plant growth, being known to be an enzyme activator that promotes metabolism (Silva and Uchida, 2000). The effect of Lippia and Ocimum rates and their interaction on vegetative phase, increased leave number and height was revealed in this study (Table 4). With increased rates of organic amendment in the combination, it is probable that NPK levels in the soil Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 369 may also enhance growth, leading to the increase in leaf number. Leaf number is a function of N in plant (Otieno et al., 2019) and this is very key for the high­ er number of leave observed at the rates of 800 g Lippia and 800 g of Ocimum combined. At DAT 21 there were more leaves in the non­amended soils compared to amended soil in seasons 1 and 2 (Table 4). This was probably due to the loss of nutrient especially N from the decomposing fresh organic amendments by microorganisms involved. The microorganisms involved in the decomposition possi­ bly out­competed the tomatoes in the used the N available for the plant and this may have led to reduction in growth rate. However, this trend was changed as from 35 DAT upwards indicating that both Lippia and Ocimum had started releasing nutri­ ent from decomposition process for tomato use. This is in conformity with observation made by Pakeerathan et al. (2009) in the management of Meloidogyne incognita using different green leaf manures on tomato under field conditions, where N contributed more toward the vegetative components (leaves and stems) of the plant than reproductive components. The height of tomato was also influenced by the higher rates Lippia and Ocimum (Table 5) and proba­ bly this was a function of K in the organic amend­ ments rates applied as observed in table 3. This is in consistence with El­Nemr et al. (2012) who reported Potassium (K) concentration as among the plant macronutrients that affected these growth parame­ ters. In another study, Faruk et al. (2011) reported similar observation on the effect of poultry organic amendment on root knot nematode management and its influence on the height of greenhouse grown tomatoes. The ability of plants to obtain water and mineral nutrients from the soil is related to their capacity to develop extensive roots and root hairs (Taiz and Zieger, 2002). As in Table 6, there was a significant increase in root volume with increased rates of Lippia and Ocimum especially when the two species were combined above 400 g, compared single rates and the controls. For root growth and development, organic amendments in the soil has been known to increase the bulk density of the growing media (Otieno et al., 2020), giving room for root system to explore wider range of the soil environment for more nutrients (Faruk et al., 2011; Otieno et al., 2019). At lower amendment level, fewer root were observed, resulting in low root volume. In contrary, roots pro­ duced from 800 g of Lippia and Ocimum produced higher volume of fibrous roots. From this study it may be speculated that the root system of the toma­ toes from highly amended media probably were affected in two ways; either by enhancing soil struc­ ture in favour of the roots growth (Otieno et al., 2019), or reduction of nematodes population in the soil or both. Increasing amendments to the soil may alter many factors that affect root development in the rhizosphere. These include soil structure, particle aggregation, pH, salinity, level of Carbon dioxide, Oxygen and other chemicals (Akhtar and Malik, 2000). In this way, this probably increased the roots’ ability to increase in dry weight and subsequently shoot dry weight. Similarly, the current result concurs Yadessa et al. (2010), whose findings showed that 10% of FYM produced significantly higher shoot and root dry weight compared to non­amended. Leaf chlorophyll content (CCL) and stomatal con­ ductance responded positively to increasing rates of these organic amendments. At 49 DAT, it was marked with maximum physiological processes in the crop (Figs. 4 and 5), indicating higher formation of chloro­ phyll content on the tomato crop at higher rates of Lippia and Ocimum. Nitrogen is an essential nutrient for normal growth and development of a plant as it is an integral part of the chlorophyll molecule (Kitonga­ Mwanza, 2011), together with Mg; the principle site of light absorption necessary for photosynthesis. Stomatal conductance on the other hand was similar­ ly influenced by both Lippia and Ocimum rates in the organic amendments (Fig. 4). Mostly K is involved in stomata closing and opening, therefore when galls have interfered with root system, the stomatal func­ tion may have been negatively affected at lower rate of amendments showing lower rate of stomatal con­ ductance. This suggests that apart from the influence of K in the organic amendments, nematodes also played a part by the interference of the plant root system (Mai and Mullin, 1996), reducing the flow of water and minerals upward the plant. From the current study, amendments rates influ­ enced yield differently among the individual rates (Fig. 6). Highest yield in terms of fruit numbers regis­ tered per plant was observed in those with 400 g and above in rates of Lippia and Ocimum amendments, indicating that the production of fruits was probably from primary plant nutrients as reflected in tomato tissue analysis (Table 3). This observation is in agree­ ment with that made by Walker (2007), where, the effect of organic amendments, fertilizers and fenamiphos was reported on reduction of parasitic and free­ living nematodes as well as increased yield Adv. Hort. Sci., 2020 34(4): 357­372 370 of tomato. A number of authors have reported that adequate K nutrition is linked with increased yields (Kanai et al., 2007; Afzal et al., 2015), which further confirm the current findings plant derived organic amendments and their impacts on tomato produc­ tion. Finally, the marketable yield of tomato is basically dependent on regular nutrient and moisture avail­ ability in growing media for plant use. Moisture in particular is essential for nutrient movement, where­ as its irregular flow in the plant system may cause blossom end rot of (BER) in tomato. This scenario is more pronounced irregular Calcium mobility in both growing media and the plant system. As for the cur­ rent study, application of both Lippia and Ocimum at 800 g level per 10 kg pot seemed to have increased water holding capacity of the amended soil since organic matter enhances the moisture availability in the media leading to more nutrient availabilty hence higher marketable yield. In concurrence to the pre­ sent study, Akhtar and Malik (2000) reported higher marketable yield following the application of organic matter to the soil. The beneficial effects of organic amendments are generally assumed to be due to the provision to the crops, extra nutrients. The current study therefore emphasizes that yield obtained from high level amendment of Lippia and Ocimum were generally higher in marketable quality compared to non­amended soils. Conclusively, both plant organic amendments played an important role as both biopesticides and organic soil fertility for crop growth and development. Acknowledgements Sincere gratitude to Egerton University, Department of Crops, Horticulture for hosting the project and National Council for Science, Technology and Innovation (Kenya) for the grant that supported implementation of the work. References ABBASI P.A., RIGA E., CONN K.L., LAZAROVITS G., 2005 ­ Effects of neem cake soil amendment on reduction of damping‐off severity and population densities of plant parasitic nematodes and soil borne plant pathogens. ­ Can. J. Plant Path., 27: 38­45. AFZAL I., HUSSAIN B., BASRA S.M.A., ULLAH S.H., SHAKEEL Q., KAMRAN M., 2015 ­ Foliar application of potassium improves fruit quality and yield of tomato plants. ­ Acta Sci. Pol., Hort. Cul., 14: 1­13. AKHTAR M., ALAM M.M., 1993 ­ Utilization of waste mate‐ rials in nematode control: a review. ­ Biore. Tech., 45: 1­7. AKHTAR M., MALIK A., 2000 ­ Roles of organic soil amend‐ ments and soil organisms in the biological control of plant parasitic nematode. ­ Biore. Tech. Rev., 74: 35­47. ALLEN H.L. IV, CHANEY R., DANIELS W.L., HENRY C.L., NEU­ MAN D.R., RUBIN E., RYAN J., TOFFEY W., 2007 ­ The use of soil amendments for remediation, revitalization, and reuse. ­ Environmental Protection Agency, Document No. 542­R­07­013. ALMEIDA L.F.R., FREI F., MANCINI E., MARTINO L.D., FEO V.D., 2010 ­ Phytotoxic activities of Mediterranean essential oils. ­ Molecules, 15: 4309­4323. AOAC, 2012 ­ Official methods of analysis. 19th edition. 2nd revision. ­ Association of Official Analytical Chemists, Gaithersburg, MD, USA. ATUBOYEDIA W.O., APRIOKU J.S., ESOMONU C.T.O., 2010 ­ Antifertility effect of aqueous crude extracts of Ocimum gratissimum L. leaves in male mice. ­ J. Med. Plant Res., 4: 809­816. BEENTJE H.J., 1994 ­ Kenya trees shrubs and lianas. ­ Natural museums of Kenya, Nairobi, pp. 635. BEKAL S., BECKER J.O., 2000 ­ Population dynamics of sting nematodes in California turfgrass. ­ Plant Disease, 84(10): 1081­1084. BERGER A., 1994 ­ Use of natural pesticides, current and future prospects. A report for the plant protection improvement programme in Botswana, Zambia and Tanzania. ­ Agris FAO, Rome, Italy. CAROVIĆ­STANK O.K., LIBER Z., BESENDORFER V., JAVORNIK B., BOHANEC B., KOLAK I., SATOVIC Z., 2010 ­ Genetic relations among basil taxa (Ocimum L.) based on molecular markers, nuclear DNA content, and chro‐ mosome number. ­ Pl. Syst. Evol., 285: 13­22. CERKAUSKAS R., 2004 ­ Asian Vegetable Research Development Centre (VRDC) tomato disease root knot nematodes. ­ AVRDC, Publication no. 04­603. CHAPMAN H.D., PRATT P.F., 1978 ­ Methods of analysis for soils, plants and waters. ‐ Univ. California Div. Agric. Sci. Priced Publication, Oakland, USA. CHAVARRÍA­CARVAJA J.A., RODRÍGUEZ­KÁBANA R., 1998 ­ Changes in soil enzymatic activity and control of Meloidogyne incognita using four organic amend‐ ments. ­ Nematropica, 28: 7­18. CHEN J., ABAWI G., ZUCKERMAN S., 2000 ­ Efficacy of Bacillus thuringiensis, Paecilomyces marquandii, and Sreptomyces costaricanus with and without organic amendments against Meloidogyne halpa infecting let‐ tuce. ­ J. Nematol., 32: 70­77. CHITWOOD D.J., 2002 ­ Phytochemical based strategies for nematode control. ­ Annual Rev. Phytopath., 40: 221­ 249. CLAUDIUS­COLE A.O., AMINU A.E., FAWOLE B., 2010 ­ http://faculty.washington.edu/slb/docs/slb_EPA07.pdf http://faculty.washington.edu/slb/docs/slb_EPA07.pdf http://faculty.washington.edu/slb/docs/slb_EPA07.pdf http://faculty.washington.edu/slb/docs/slb_EPA07.pdf http://faculty.washington.edu/slb/docs/slb_EPA07.pdf Otieno et al. ‐ Management of Meloidogyne in tomato by soil amendments 371 Evaluation of plant extracts in the management of root knot nematode, Meloidogyne incognita, on cow pea Vigna unguiculata (L) Walp. ­ Mycopath., 8(2): 53­60. COTTENIE A.M., VERIOO KIEKENS L., VEIGH G., CAMERLYNCK R., 1982 ­ Chemical analysis of plants and soils. ­ State Univ. Ghent, Belgium, 63. DAUDA S.N., AJAYI F.A., NDOR E., 2008 ­ Growth and yield of water melon (Citrullus lanatus) as affected by poultry manure application. ­ J. Agric. Soc. Sci., 4: 121­134. DUSCHATZKY C.B., MARTINEZ A.N., ALMEIDA N.V., BOLIVARDO S.L., 2004 ­ The nematicidal activity of the essential oils isolated from Aloysia triphylla, A. polystachya, A. gratissima, Lippia argentina Plants against the Root‐Knot Nematode. ­ J. Essent. Oils Res., 16(6): 626­628. ECHEVERRIGARAY S., ZACARIA J., BELTRÃO R., 2010 ­ Nematicidal activity of monoterpenoids against the root knot nematode Meloidogyne incognita. ­ Phytopathol., 100(2): 199­203. EL­NEMR M.A., ABD EL­BAKY M.M.H., SALMAN S.R., EL­ TOHAMY W.A., 2012 ­ Effect of Different Potassium lev‐ els on the growth, yield and quality of tomato grown in sand‐ponic culture. ­ Austr. J. Basic Appl. Sci., 6(3): 779­ 784. FAOSTAT, 2018 ­ Food and agriculture organisation of the United Nations, Faostat . Stat. ­ http://www.fao.org/faostat/en/#data/QC. FARUK M.I., RAHMAN M.L., ALI M.R., RAHMAN M.M., MUSTAFA M.M.H., 2011 ‐ Efficacy of the two organic amendments and a nematicide to manage root‐knot Nematode (Meloidogyne incognita of Tomato (Lycopersicon esculentum L.). ­ Bangladesh J. Agric Res., 36: 477­486. FERRAZ S., FREITAS L.G., 2004 ‐ Use of antagonistic plants and natural products, pp. 931­978. ‐ In: CHEN Z.X, S.Y. CHEN and D.W. DICKSON (eds.). Nematology. Advances and perspectives. Volume II. Nematode management and utilization. CABI, Wallingford, UK, pp. 1234. HASABO S.A., NOWEER E.M.A., 2005 ­ Management of Root knot nematodes Meloidogyne incognita on Eggplant with some plant extracts. ­ Egypt. J. Phytopathol., 33: 65­72. HCD, 2012 ­ Horticulture Validated Report 2010‐2012. ­ Hort. Inform. Res. Centre, Nairobi, Kenya, pp. 118. HCD, 2017 ­ Horticulture validated report 2015‐2016. ­ HCD, Horticultural Crops Directorate KNBS, Kenya National Bureau of Statistics, Kenya, pp. 60. JAETZOLD R., SCHMIDT H., HORNETZ B., SHISANYA C., 2012 ‐ Farm management handbook of Kenya . ­ http://www.fao.org. KAGAI K.K., AGUYOH J.N., TUNYA G.O., 2012 ­ Efficacy of selected plant biofumigants in the management of plant parasitic nematodes in Asclepias (Asclepias tuberosa L.). ­ Int. J. Sci. Nat., 3: 728­734. KANAI S., OHKURA K., ADU­GYAMFI J.J., MOHAPATRA P.K., NGUYEN N.T., SANEOKA H., FUJITA K., 2007 ­ Depression of sink activity precedes the inhibition of biomass production in tomato plants subjected to potassium deficiency stress. ­ J. Exp. Bot., 58: 2917­ 2928. KIMENJU J.W., MWEKE A.N., MUTITU E.W., MUTUA G.K., 2010 ­ Poor hosts of root knot nematodes and their application as rotation crops in Okra production. ­ African J. Hort. Sci., 3: 63­71. KIRIMI J.K., ITULYA F.M., MWAJA V.N., 2011 ­ Effects of nitrogen and soaking on fruit yield of tomatoes. ­ Afr. J. Hort. Sci., 5: 50­60. KITONGA­MWANZA L.M., SWIADER J., MULWA R.M.S., 2011 ­ Evaluation of SPAD chlorophyll Flouoresence for on‐site nitrogen assessment in drip fertigated sweet corn. ­ J. App. Hort., 13(1): 13­13. KOSGEI C.J., MATASYOH J.C., MWENDIA C.M., KARIUKI S.T., GULIYE A.Y., 2014 ­ Chemical composition and lar‐ vicidal activity of essential oil of Lippia kituiensis against larvae of Rhipicephalus appendiculatus. ‐ Int. J. Biol. Chem. Sci., 8(4): 1938­1947. LOPES E.A., FERRAZ S., PAULO AFONSO FERREIRA P.A., DE FREITASII L.G., DALLEMOLE­GIARETTA R., 2011 ‐ Soil amendment with chopped or ground dry leaves of six species of plants for the control of Meloidogyne javani­ ca in tomato under greenhouse conditions. ­ Ciência Rural, Sant. Maria, 41(6): 935­938. MAI W.F., MULLIN P.G., 1996 ­ Plant parasitic nematodes. A pictorial key to genera 5th edition. ­ Comstock Publishing Associates, Cornell University Press, pp. 56. MASI L.D., SIVIERO P., ESPOSITO C., CASTALDO D., SIANO F., LARATTA B., 2006 ­ Assessment of agronomic, chem‐ ical and genetic variability in common basil (Ocimum basilicum L.). ­ European Food Research and Technology, 223: 273­281. MATASYOH L.G., MATASYOH J.C., WACHIRA F.N., MUNGAI M.G., THAIRU A.W., MAKIAMA T.K., 2007 ­ Chemical composition and antibacterial activity of Ocimum gratissimum L. growing in the Eastern Kenya. ­ Afr. J. Biotech., 6: 760­765. MCSORLEY R., 2011 ­ Overview of organic amendments for management of plant parasitic nematodes with case studies from Florida. ­ J. Nematol., 43: 69­81. NAGAI A., DUARTE L.M.L., SANTOS D.Y.A.C., ­ 2011 ­ Influence of viral infection on essential oil composition of Ocimum basilicum (Lamiaceae). ­ Nat. Prod. Comm., 6(8): 1189­1192. OCHILO W.N., GIDEON N., NYAMASYO G.N., KILALO D., OTIENO W., OTIPA M., CHEGE F., KARANJA T., LINGEERA E.K., 2019 ­ Characteristics and production constraints of smallholder tomato production in Kenya. ­ Sci. Afr., 2: e0 0 014. OGALLO J.L., GOODELL P.B., ECKERT J., ROBERTS P.A., 1997 ­ Evaluation of Nemx, a new cultivar of cotton with high resistance to Meloidogyne incognita. ­ J. Nematol., 29(4): 531­537. OGENDO J.O., KOSTYUKOVSKY M., RAVID U., MATASYOH https://apsjournals.apsnet.org/doi/10.1094/PHYTO-100-2-0199 Adv. Hort. Sci., 2020 34(4): 357­372 372 J.C., DENG A.L., OMOLO E.O., KARIUKI S.T., SHAAYA E., 2008 ­ Bioactivity of Ocimum gratissimum oil and two constituents against five insect pest attacking stored food products. ­ J. Stored Prod. Res., 44: 328­334. OKA Y., TKACHI SHUKER N.S., YERUMIYAHU U., 2007 ­ Enhanced nematicidal activity of organic and in organic Azadirachta indica extracts. ­ J. Nematol., 39: 9­16. OKALEBO J.R., GATHUA K.W., WOOMER P.L., 2002 ­ Laboratory methods of soil and plant analysis: A work‐ ing manual. ­ Marvel EPZ (Kenya) LDT, Nairobi, Kenya. ONIFADE A.K., 2007 ­ Effect of essential oils from five Ocimum sp. on the pathogenicity of Pratylenchus bahyurus (Godfrey) in tomato. ­ Agric. J., 2: 185­191. OTIENO P.C., NYALALA S., WOLUKAU J., 2019 ­ Suitability of biosolids from university sewage ponds as a sub‐ strate for crop production. ­ Afr. J. Agric. Res., 14(35): 2062­2074. OTIENO P.C., NYALALA S., WOLUKAU J., 2020 ­ Optimization of biosolids as a substrate for tomato transplant production. ­ Adv. Hort. Sci., 34(2): 313­323. PAKEERATHAN K., MIKUNTHAN G., THARSHANI N., 2009 ­ Effect of different animal manures on Meloidogyne incognita (kofoid and white) on tomato. ­ World J. Agric. Sci., 5: 432­435. PIRES R.C., FURLANI P.R., RIBEIRO R.V., JUNIOR D.B., SAKAI E., LOURENÇÃO A.L., NETO A.T., 2011 ­ Irrigation fre‐ quency and substrate volume effects in the growth and yield of tomato plants under greenhouse conditions. ­ Scientia Agricola, 68(4):400­405. SAFIUDDIN SHAHAB S., MAZID M., AHMED D., 2012 ­ Comparative study of Fusarium oxysporum f sp. lycop­ ersici and Meliodogyne incognita race‐2 on plant growth parameters. ­ J. Agric. Sci., 3: 844­847. SANZUA L.J., MUIGAI E.N., NDUNGU C.K., MWAFAIDA J., 2018 ­ Evaluation of salinity tolerance of water melon (Citrullus lanatus thanb) in kilifi, Kenya. ­ Afr. J. Hort. Sci., 13: 1­12. SIDDIQUI Z.A., AKHTAR M.S., 2007 ­ Biolcontrol of chickpea root‐rot disease complex with phosphate‐ solubilizing microorganisms. ­ J. Plant Pathol., 89: 67­77. SIFOLA M.I., BARBIERI G., 2006 ­ Growth, yield and essen‐ tial oil content of three cultivars of basil grown under different levels of nitrogen in the field. ­ Sci. Hort., 108: 408­413. SILVA J.A., UCHIDA R., 2000 ­ Plant nutrient management in Hawaii’s soils, approaches for tropical and subtropi‐ cal agriculture. ­ Coll. Trop. Agric. and Human Res., Univ. Hawaii at Manoa, pp. 1­6. STIRLING G.R., KOPITTKE R., 2000 ­ Sampling procedures and damage threshold for root knot nematodes (Meloidogyne javanica) on pineapple. ­ Austr. J. Exp. Agric., 40: 1003­1010. STIRLING G.R., STIRLING A.M., 2003 ­ The potential of Brassica green manure crops for controlling root knot nematodes (Meloidogyne javanica) on horticultural crops in subtropical environment. ­ Austr. J. Exp. Agric., 43(6): 623­630. SURESH K.D., SNEH G., KRISHN K.K., MOOL C.M., 2004 ­ Microbial biomass carbon and microbial activities of soils receiving chemical fertilizers and organic amend‐ ments. ­ Arch. Agron. Soil Sci., 50: 7­641. TAIZ L., ZEIGER E., 2002 ­ Plant physiology, 3rd edition. ­ Sinauer Associates, Sunderland, MA, USA, pp. 690. VARELA A.M., SEIF A., LOHR B., 2003 ­ A guide to IPM in tomato production in Eastern and Southern Africa. ‐ ICIPE, Kenya, pp. 200. VOVLAS N., RAPOPORT H.F., JIMÉNEZ­DÍAZ R.M., CASTILLO P., 2005 ­ Differences in feeding sites induced by root knot nematodes, Meloidogyne spp. in chickpea. ­ Phytopathol., 95: 368­375. WALKER G.E., 2007 ­ Effect of organic amendments, fertil‐ izers and fenamiphos on parasitic and free‐living nema‐ todes, tomato growth and yield. ­ Nematol. Medit., 35: 131­136. YADESSA G.B., VAN BRUGGEN A.H., COCHO F.L., 2010 ­ Effect of different soil amendments on bacterial wilt caused by Ralstonia solanacearum and on the yield of tomato. ­ J. Plant Pathol., 92(2): 439­450. ZUBA S.N., NOGUEIRA W.C.L., FERNANDES L.A., SAMPAIO R.A., COSTA C.A., 2011 ­ Yield and nutrition of tomato using different nutrient sources. ­ Horticultura Brasileira, 29: 50­56.