Nematode Multiplication Research Articles

Management of Root-Knot Nematode, Meloidogyne incognita Infecting Tuberose by Using Bioformulations under Field Condition

Aims: To evaluate the effectiveness of different bioformulations for management of root-knot nematode, Meloidogyne incognita infecting tuberose under field condition. Study Design: Randomized Block Design. Place and Duration of Study: Biswanath College of Agriculture, AAU, Biswanath Chariali, Assam during summer season 2024. Methodology: Bulbs of tuberose cultivar Prajwal were planted in root-knot nematode infested sick plot. In this experiment seven treatments were considered and the treatments were replicated thrice. Bioformulations viz., Neem cake, Neem leaf power, Biover, Biozium, Biomonas and AAU-Bioguard were applied treatment wise and mixed with soil before planting of bulbs. Observations on plant height and floral characteristics viz. spike length, rachis length, spike weight, number of floret per spike, floret length and nematode multiplication parameters like number of galls, egg masses per root system and final nematode population in soil were recorded. Results: Upon observation results showed that all the treatments were significantly effective in increasing the plant height, floral characteristic and reducing the number of galls, eggmasses and final nematode population in soil as compared to that of untreated control. Highest plant height(46.50cm), spike length (66.33cm), spike weight (37.33g), rachis length(32cm), number of florets per spike(25.26) and floret length(4.66cm) was recorded in the treatment with Biover@5kg enriched with FYM 5t/ha, which was followed by the treatment with Biozium @5kg enriched with FYM 5t/ha and neem cake @ 1t/ha. Maximum reduction of number of galls (35.18%), eggmasses (31.79%) and final nematode population in soil (39.65%) was recorded in the treatment with Biover@5kg enriched with FYM 5t/ha. Conclusion: From this experiment it can be concluded that all the bioformulations were found to be effective for increasing plant height, floral characteristic and reducing the nematode multiplication and these can be incorporated in root-knot nematode management programme. Among the bioformulations Biover was found to be most effective.

Prevalence and Distribution of Rice Root Knot Nematode in Rice Nurseries in Chitwan and Lamjung Districts of Nepal

To determine the natural infection of the rice root-knot nematode (Meloidogyne graminicola Golden & Brichfield) in rice nurseries, a survey was carried out in Chitwan and Lamjung in June and July of 2022. Rice nurseries were randomly selected based on geographical location, agro-ecological region, land type, cropping practice, and soil type. Forty rice nurseries were surveyed i.e. twenty from each district and 100 seedlings from each nursery as the sample for the study using the simple random method of sampling were evaluated. Field survey revealed that M. graminicola was widely distributed in most rice growing areas of Chitwan and Lamjung districts. Most of the rice nurseries in Lamjung district were found to be infested with M. graminicola as compared to that of the Chitwan district. Disease severity was reported up to 23.26% in Lamjung and 20.2% in Chitwan district. Additionally, the study showed that dry bed conditions were more likely to be affected by rice root-knot disease than wet bed conditions. The majority of farmers planted seedlings on upland (dry) soil, where second-stage juvenile (J2) populations and rice root-knot disease were more common. Most farmers (87.5%) have limited knowledge about rice root knot nematodes and their control methods. This suggested a significant loss in rice output and a high danger of nematode multiplication. This information will provide knowledge about the prevalence of nematode and enabling rice growers in this region to design and implement an appropriate control strategy for managing M. graminicola.

New insights into synergistic interactions of root-knot nematode and wilt causing fungi on pigeon pea in sandy loam soil

AbstractA study under pot conditions was undertaken to know the relationship between plant biomass and inoculum densities of Meloidogyne incognita. Similarly, plant biomass—Fusarium udum relationship was also observed. Results revealed that increased inoculum densities resulted in corresponding decrease in plant biomass markers. Compared to control plants, 2000 nematode inoculation caused significant damage in phytobiomass (23.07–26.32%) of pigeon pea. 2.0 g F. udum inoculation caused significant impairment in different plant growth characteristics (22.96–26.02%) over control plants at P < 0.05. The interactive study revealed that concomitant, M. incognita + F. udum inoculation caused the maximum reduction (33.68–56.66%) in plant biomass. Next to this, M. incognita inoculation 15 days prior to F. udum and F. udum inoculation 15 days prior to M. incognita caused an impairment of (28.55–50.11%) and (23.99–43.33%), respectively. However, they were statistically either at par or non-significant over each other. Disease assay revealed that M. incognita multiplication rate was correspondingly augmented upon the rise of nematode concentration, conversely, the reproduction factor was progressively decreased. Pathogens interactive facet revealed that M. incognita inoculated plants showed a higher root galls (99.40), eggs (79.20), fecundity (199.80), and nematode population (13,785.60). The presence of F. udum suppressed nematode multiplication ability. However, concomitant inoculation of M. incognita and F. udum aggravated percent wilt severity (65.48%) thus exhibiting a synergistic interaction between the vascular pathogens.

CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita

BackgroundPlant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately.ResultsIn the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the β-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The ‘Cas9-free’ homozygous T3 line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility.ConclusionThe present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

Economical factitious diet for mass rearing of greater wax moth, Galleria mellonella (Lepidoptera: pyralidae), a promising host for entomopathogenic nematodes

BackgroundGreater wax moth (Galleria mellonella L.) (Lepidoptera: Pyralidae) is most commonly used for mass multiplication of entomopathogenic nematodes. An investigation on economical modification and comparative analysis of factitious diets of G. mellonella was carried out under laboratory conditions. Different diets were assessed in comparison with standard diet.ResultsModifications based on quantity change of dietary components (wheat flour, maize flour, dry milk, dry yeast, honey and glycerin), addition of vitamin E and vitamin-B complexes, elimination of dry milk and replacement of glycerin with sorbitol showed reduction in the cost of production and increase in food conversion efficiency in G. mellonella. The weight of larva in diet III (303.40 mg) was highest as compared to the larval weight in control diet (280.00mg). The conversion of ingested and digested food into biomass of G. mellonella was the highest for factitious diet III. Also, the highest number of Steinernema abbasi was produced from G. mellonella larvae reared on diet III (25418.43IJs) followed by diet I (21805.31IJs). The economics of the diets also showed that the percent cost reduction in factitious diet III and diet I was 48.86 and 28.85%, respectively. Overall, the results suggested that factitious diet III was superior to diet I followed by diet II.ConclusionAddition of supplements of vitamin E and B-complexes enhances the biological activity of the insect, thereby increasing its food conversion efficiencies. Factitious diet III can be successfully used for mass multiplication of the G. mellonella.

Pomegranate (Punica granatum L.) growth and biochemical alterations in response to meloidogyne incognita infection, minerals, and nano-fertilizers

The effects of four inoculum levels (500, 1,000, 2,000, or 4,000 second-stage juveniles (J2) per plant) of the root-knot nematode, Meloidogyne incognita, on two pomegranate cultivars (‘Manfalouty’ and ‘Wonderful’) were investigated under greenhouse conditions in response to plant inorganic and organic chemical concentrations. Furthermore, the effects of six commercial chemical fertilizers (inorganic fertilizers and nano-fertilizers) on plant growth and nematode reproduction were also studied. Both cultivars recorded the highest gall formation, embedded stages, and final nematode population at the inoculum dosage of 2,000 J2/plant. The highest reproductive rate was achieved with 500 J2/plant, while the lowest rate was observed with 4,000 J2/plant on both cultivars. ‘Wonderful’, at all M. incognita inoculum levels, was more sensitive to nematode infestation than ‘Manfalouty’. ‘Wonderful’ showed a greater reduction in fresh and dry plant weights than ‘Manfalouty’ at 2,000 and 4,000 J2/plant. In both cultivars, the concentrations of N, P, and K decreased with increasing nematode inoculum levels. This decline was more pronounced in ‘Manfalouty’ than in ‘Wonderful’. The nano-fertilizers, Hyper Feed®, and Hyper Feed Solo® reduced all nematode parameters but only in ‘Wonderful’. Treatment with Hyper Feed® resulted in the highest percentage increases in all plant growth parameters in ‘Wonderful’. Total carbohydrate concentration was increased in ‘Manfalouty’ treated with the nano-fertilizers, especially with Hyper Feed®. Also, concentrations of total phenols and tannins increased in ‘Wonderful’ when treated with either nano-fertilizer. Generally, both nano-fertilizers showed an increase in plant N levels. We recommend using nano-fertilizers in integrated pest management (IPM) programs on pomegranate where they improved plant growth parameters and reduced nematode multiplication parameters more markedly than inorganic fertilizers.

An Improved Phenotyping Method for Evaluation of Yam (Dioscorea spp.) Resistance to Nematodes Belonging to the Genera Meloidogyne and Scutellonema.

Phenotyping yam (Dioscorea spp.) germplasm for resistance to parasitic nematodes is hampered by the lack of an efficient screening method. In this study, we developed a new method using rooted yam vine cuttings and yam plantlets generated from semi-autotrophic hydroponics (SAHs) propagation for phenotyping yam genotypes for nematode resistance. The method was evaluated using 26 genotypes of D. rotundata for their reaction to Scutellonema bradys and four root-knot nematode species, Meloidogyne arenaria, M. enterolobii, M. incognita, and M. javanica. Yam plantlets established in nursery bags filled with steam-sterilized soil were used for screening against single nematode species. Plants were inoculated four weeks after planting and assessed for nematode damage eight weeks later. A severity rating scale was used to classify genotypes as resistant, tolerant, or susceptible determine based on the nematode feeding damage on tubers and the rate of nematode multiplication in the roots of inoculated plants. The results demonstrated putative resistance and tolerance against S. bradys in 58% of the genotypes and 88%, 65%, 65%, and 58% against M. arenaria, M. javanica, M. incognita, and M. enterolobii, respectively. The method is rapid, flexible, and seasonally independent, permitting year-round screening under controlled conditions. This method increases the throughput and speed of phenotyping and improves the selection process.

Plant parasitic nematodes on soybean (Glycine max (L) Merr.) in the Metekel zone (Ethiopia) and varietal resistance evaluation to Meloidogyne javanica

AbstractPlant parasitic nematodes cause severe damage to soybean crops, leading to substantial losses in yield and quality. However, more information is needed regarding the occurrence, distribution and damage status of soybean PPNs in Ethiopia. A total of 180 samples of rhizosphere and roots were collected randomly from three major soybean‐producing districts of Metekel Zone. Nematodes were extracted and identified at the genus level based on their morphological characteristics, except Meloidogyne javanica, the deadly species, which was identified using morphological and molecular techniques. Seven nematode genera were recovered: Meloidogyne, Scutellonema, Pratylenchus, Helicotylenchus, Rotylenchulus, Hemicycliophora and Criconema. The genera Meloidogyne, Scutellonema and Pratylenchus were dominant and had FO% of 98.3%, 97.5% and 81.8%, respectively. The host status of 10 soybean varieties to M. javanica infection was determined. Data on the shoot height, root length, fresh shoot and root weight, gall number and nematode density were estimated 3 months after inoculation. The results revealed that Jalele variety suppresses M. javanica multiplication while the other nine supported it. Comparatively, Pawe‐3, Awassa‐95 and Koreme varieties supported the highest nematode multiplication 24.4, 16.2 and 15.3 reproductive factors, respectively. Nova experienced the highest shoot and root relative biomass losses (84%) and roots (63%). In contrast, Jalele did not experience any loss in all parameters. However, Jalele variety resisted M. javanica, making it a promising approach towards soybean breeding programs for nematode management.

Biocidal Efficacy of a New and Native Species of Entomopathogenic Nematode against Gram Pod Borer, Helicoverpa armigera (Hubner)

Efficacy of new and native species of entomopathogenic nematode (EPN), Heterorhabditis casmirica SKUAST-K 104 was evaluated against gram pod borer, Helicoverpa armigera in laboratory conditions. Larval mortality was directly proportional to time period as well as time period but inversely proportional to larval size. H. casmirica SKUAST-K 104 applied @ 50, 100, 150 and 200 IJs per 2nd instar larva resulted in pest mortality by 0.00, 8.33, 16.66, and 25.00 per cent, respectively at 24 hours and they were statistically significant (p ≤ 0.05) from each other. At 200 IJs inoculum level, 8.33, 16.66, 25.0, 41.66 and 50 per cent mortality of 5th instar larva was recorded at 24, 48, 72, 96 and 120 hours post inoculation, respectively. LC50 values was directly proportional to the size of larva but inversely proportional to size of nematode inoculum level. On the other hand, LT50 values was directly proportional to the size of larva but inversely proportional to size of nematode inoculum level. LC50 values calculated at 24 hours for 2nd, 3rd, 4th and 5th instar larvae was 256.88, 277.24, 326.25 and 384.25, respectively, whereas at 120 hours it was 126.11, 160.22, 184.36 and 219.14, respectively. Similarly, LT50 values calculated at inoculum level of 50 IJs per 2nd, 3rd, 4th and 5th instar larvae were 105.0, 113, 122 and 131 hours, respectively but at highest inoculum level of 200 IJs, it was 75, 89, 94 and 100 hours, respectively. Nematode multiplication rate within the host cadaver was directly proportional to the size of the host. Minimum and maximum number of IJs/ larva was 2.72 x 105 and 1.03 x 105, obtained from 2nd and 5th instar larva, respectively.

Densidade inicial de inóculo, época de avaliação e reprodução de Meloidogyne enterolobii em goiabeira 'Paluma'

ABSTRACT Guava decline is a complex disease caused by the interaction between Meloidogyne enterolobii and Neocosmospora falciformis (Syn.: Fusarium solani). Thus, selecting M. enterolobii-resistant genotypes within the genus Psidium is essential for controlling this disease, and developing a resistant cultivar of Psidium guajava could significantly impact this issue. Thus, the objective of this study was to assess the response of the guava plants of the cultivar Paluma to different densities of M. enterolobii inoculum. Guava seedlings were inoculated with 500, 1,000, 5,000, 10,000, and 20,000 eggs + second-stage juveniles (J2) of M. enterolobii per plant. Root and shoot fresh weights, shoot dry weight, root length, plant height, and stem base diameter were evaluated at 70 and 135 days after inoculation (DAI). Total number of M. enterolobii eggs + J2 in the root system and nematode reproduction factor nematode were assessed. Nematode multiplication in roots was not proportional to increases in initial inoculum density; thus, the best plant responses to nematode multiplication in the evaluated cultivar were found for the lowest tested densities. The reproduction factor decreased as the inoculum density was increased, at both evaluations (70 and 135 DAI).

Plant growth promoting Bacillus species elicit defense against Meloidogyne incognita infecting tomato in polyhouse.

The effects of four nematicidal rhizobacterial isolates; Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, and Bacillus cereus on infection and multiplication of root-knot nematode, Meloidogyne incognita on tomato were compared with the application of a chemical nematicide, fluopyram 34.48% SC (Velum Prime). The bio-efficacy trial conducted in pots preinoculated with the above isolates followed by M. incognita inoculation resulted in a significant reduction in percent root galling viz. 91.95 in B. subtilis, 84.21 in B. pumilus, 83.70 in B. megaterium, and 81.8 in B. cereus, at 75 days after inoculation (DAI). The reproduction factor of the nematode was the lowest (15.83) in B. subtilis, followed by B. pumilus (21.00), compared with 48.16 in control, with enhanced photosynthetic and transpiration rates. The mechanism of induced resistance was assessed usingquantitative reverse-transcription polymerase chain reaction(qRT-PCR) for quantification of three key defense genes (PR-1b, JERF3, and CAT) at 0,2,4,8 and16 days DAI. The defence genes, PR-1b, JERF3, and CAT were expressed at 2.5-7.5-folds in rhizobacterialtreated plants, but not in nematicide treatment. The defense enzymes viz., super oxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) when quantified (μmol/mg protein) showed an increase from 1.5 to 17.5 for SOD, 2.1 to 7.8 in PPO, 1.8 to 10.2 in PO, and 1.8 to 8.7 in PAL during 0 to 16 DAI, in rhizobacteria-treated plants.

Transgenic East African Highland Banana Plants Are Protected against Radopholus similis through Host-Delivered RNAi.

The burrowing nematode Radopholus similis is considered a major problem of intensive banana cultivation. It can cause extensive root damage resulting in the toppling disease of banana, which means that plants fall to the ground. Soaking R. similis in double-stranded (ds) RNA of the nematode genes Rps13, chitin synthase (Chs-2), Unc-87, Pat-10 or beta-1,4-endoglucanase (Eng1a) suppressed reproduction on carrot discs, from 2.8-fold (Chs-2) to 7-fold (Rps13). The East African Highland Banana cultivar Nakitembe was then transformed with constructs for expression of dsRNA against the same genes, and for each construct, 30 independent transformants were tested with nematode infection. Four months after transfer from in vitro culture to the greenhouse, the banana plants were transferred to a screenhouse and inoculated with 2000 nematodes per plant, and thirteen weeks later, they were analyzed for several parameters including plant growth, root necrosis and final nematode population. Plants with dsRNA constructs against the nematode genes were on average showing lower nematode multiplication and root damage than the nontransformed controls or the banana plants expressing dsRNA against the nonendogenous gene. In conclusion, RNAi seems to efficiently protect banana against damage caused by R. similis, opening perspectives to control this pest.

Axenic mass culture of soil-borne nematodes using artificial egg yolk medium

ABSTRACT The amenability to axenic culture in vitro on solid medium including MacConkey, soybean casein, nutrient broth and egg yolk medium of Oscheius tipulae, Caenorhabditis elegans, Acrobeloides tricornis, Acrobeloides bodenheimeri and Teratorhabditis synpapillata were tested using Petri dishes. These nematodes were incapable of growing in MacConkey, soybean casein and nutrient broth, except for C. elegans which was able to multiply in soybean casein medium. While all nematodes used were successfully mass cultured using egg yolk medium at 25°C, results revealed that the highest yield of nematodes was observed with O. tipulae (TC2 and OC2), attaining 176 and 150 k nematodes per plate, respectively, after 10 days post-inoculation. In contrast, the lowest yield was recorded for T. synpapillata. The medium was optimised using different egg yolk concentrations (0, 5, 10, 20 and 40 g/L) and was tested for four nematode species O. tipulae, A. bodenheimeri, A. tricornis and T. synpapillata. The results showed that the egg yolk concentrations and the kinetic of nematode multiplication were positively correlated. Additionally, egg yolk liquid medium was applied to the mass culture of nematodes. Oscheius tipulae, A. bodenheimeri and A. tricornis were capable of reproduction in Petri dishes containing egg yolk liquid medium, at 25°C. Contrarily, T. synpapillata was unable to grow on the same liquid medium. The highest nematode yields of O. tipulae and A. bodenheimeri were attained 10,266 and 4110 nematodes in average, respectively, at 16 days post-inoculation. The maximum multiplication of A. tricornis was recorded at 20 days post-inoculation with 8430 nematodes.

Bazı yerel sarımsak genotip ve köy çeşitlerinin soğan sak nematoduna, Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936 (Rhabditida: Anguinidae) karşı dayanıklılıklarının belirlenmesi

Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936 (Rhabditida: Anguinidae) is one of the destructive agents of garlic and reduces yield and market value. One of the most practical and eco-friendly methods for nematode management is using resistant varieties. In the study, two endemic garlic species, Allium tuncelianum (Kolman) Ozhatay, Mathew & Siraneci and Allium macrochaetum subsp. macrochaetum Boiss. & Hausskn. (Alliaceae: Amaryllidaceae), 10 mutant and 32 landraces garlic genotypes, Allium sativum L. (Alliaceae: Amaryllidaceae) were investigated for their resistance reactions to D. dipsaci and effect of D. dipsaci on some plant growth parameters. All experiments were conducted at Atatürk Horticultural Central Research Institute in 2019-2020. None of the genotypes was found resistant to D. dipsaci, and reproduction factors, which ranged from 2.6 to 12.7, were grouped from susceptible to highly susceptible. The Tunceli garlic genotype had the lowest reproduction factor (2.6), 36.6% less than the highly susceptible Muğla6 genotype. Alata1, Muğla1, Muğla7 and Kula genotypes had the lowest decrease rate with nematode treatment at least in one of the plant growth parameters. The genotypes that had lower nematode multiplication and displayed better development under nematode infestation in this study are recommended for the field infested with D. dipsaci as sources for garlic breeding.

In planta transformation of Polianthes tuberosa for concomitant knockdown of flp-1, flp-12 and flp-18 genes induced root-knot nematode resistance

Polianthes tuberosa is a lucrative crop owing to its huge demand in floriculture, perfume and pharmaceutical industry. Root-knot nematodes, especially Meloidogyne incognita poses a major impediment on tuberose yield. Currently nematode management relies on the use of hazardous nematicides and other inefficacious means viz. crop rotation and soil solarization. The ongoing search for an ecologically sustainable and target specific approach, led to the progression of genetic modification based techniques to target genes critical for nematode survival. FMRFamide like peptide (FLPs) genes have drawn interest as prospective targets because of their diverse physiological roles in nematodes. Here, the effect of concomitant silencing of three vital nematode FLP genes (Mi-flp-1, Mi-flp-12 and Mi-flp-18) using host induced RNAi in tuberose was assessed against M. incognita. Firstly, the fusion (flp-1, flp-12 and flp-18) gene cassette was functionally validated using host-delivered RNAi in tobacco model system. Transgenic tobacco expressing dsRNA of fusion gene significantly reduced the nematode multiplication factor (MF) by 82.6%. Since, genetic transformation of tuberose has been neglected so far, and transgenic development is an inevitable step for host-generated RNAi, an Agrobacterium mediated transformation protocol was optimized in tuberose. Tuberose tubers were pricked at the apical meristem of the axis and at the tiny white meristematic buds emerging from them, followed by infection with Agrobacterium hosting pCAMBIA 3301 vector. Maximum transformation efficiency was achieved with 200 μM acetosyringone as observed from the GUS staining. For HIGS, fusion transgenic events were developed in tuberose and molecular analysis confirmed the presence and integration of fusion transgene using PCR, qRT-PCR and Southern blot analysis. Bioefficacy analysis of transgenic plants against M. incognita resulted in 73.75% reduction in nematode fecundity, thereby demonstrating the effectiveness of the three flp genes for nematode management in tuberose. This study established first ever report of in planta Agrobacterium-mediated transformation of tuberose for host delivered RNAi for M. incognita resistance.

A novel tasi RNA-based micro RNA-induced gene silencing strategy to tackle multiple pests and pathogens in cotton (Gossypium hirsutum L.).

This study demonstrates the combinatorial management of multiple pests through atrans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy. Transgenic cotton events demonstrated improved efficacy against cotton leaf curl disease, cotton leaf hopper and root-knot nematode. Cotton (Gossypium hirsutum L.), an important commercial crop grown worldwide is confronted by several pests and pathogens, thus reiterating interventions for their management. In this study, we report, the utility of a novel Arabidopsis miRNA173-directed trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy for the simultaneous management of cotton leaf curl disease (CLCuD), cotton leaf hopper (CLH; Amrasca biguttula biguttula) and root-knot nematode (RKN, Meloidogyne incognita). Cotton transgenics were developed with the MIGS construct targeting a total of 7 genes by an apical meristem-targeted in planta transformation strategy. Stable transgenics were selected using stringent selection pressure, molecular characterization and stress-specific bio-efficacy studies. We identified 8 superior events with 50-100% resistance against CLCuD, while reduction in the root-knot nematode multiplication factor in the range of 35-75% confirmed resistance to RKN. These transgenic cotton events were also detrimental to the growth and development of CLH, as only 43.3-62.5% of nymphs could survive. Based on the corroborating evidences obtained by all the bioefficacy analyses, 3 events viz., L-75-1, E-27-11, E-27-7 were found to be consistent in tackling the target pests. To the best of our knowledge, this report is the first of its kind demonstrating the possibility of combinatorial management of pests/diseases in cotton using MIGS approach. These identified events demonstrate immense utility of the strategy towards combinatorial stress management in cotton improvement programs.

Evaluation for resistance of some spinach (Spinacia oleracea L.) cultivars against root-knot nematode, Meloidogyne javanica

Aim: The present study was carried out to screen ten cultivars of spinach (Spinach oleracea L.) for their response to Meloidogyne javanica under greenhouse conditions with the aim to exploit them for nematode management programme. Methodology: Ten commonly grown spinach cultivars were tested for their reaction to Meloidogyne javanica by inoculating 2000 second-stage juveniles in pots. Five seeds of each cultivar were surface sterilized with 1.0% NaOCl and sown in sterilized clay pots, placed in a completely randomized design. After three months the experiment was terminated and the plant growth, physiological and nematode infested parameters were evaluated. Results: None of the tested cultivar was immune, highly resistant or resistant to nematode infestation. Cultivars Pusa Bharti and Pahari were moderately resistant whereas Giant, All green and Kaveri were highly susceptible to nematode infestation. Five cultivars Pure green, Solan Harit, Green flavor, Super green and Kantedar were found to be susceptible. Interpretation: Growing moderately resistant cultivar in severely infested field with M. incognita may assist to minimize the root-knot nematode multiplication and thereby, reduce the crop damage below the threshold level. Thus using resistant cultivars against M. incognita can prove to be an environmentally friendly and cost-effective management approach. Key words: Meloidogyne javanica, Root-knot nematode, Spinach cultivars

Improved methodology of in vivo mass multiplication of entomopathogenic nematodes: A remarkable bio agent for soil arthropod pests management

In the process of in vivo multiplication of EPN, the Infective Juvenile (IJs) enters into the body of host insect; Galleria mellonella (wax moth) larvae through natural openings and cuticle. They release symbiotic bacteria Photorhabdus luminescens in insect body. The bacteria multiply in hemocoel, cause septicemia disease and kills insect within 48 hours, multiplies and complete 2-3 generations inside host insect body. With the proven effectiveness demonstrated in recent years, the demand of EPNs is increasing day by day. The production of EPNs has been mainly restricted up to laboratory level only due to the low recovery rats of IJs in the existing method. Recovery of EPN by the existing method is labor intensive, time consuming and inconvenient for mass multiplication of EPN at large scale. The standardizations of technology with equipment modifications have been optimized for multiplication of EPN at small scale entrepreneurs. The higher recoveries 241424 IJs/GC in it as compared to existing white trap methodology 197445 IJs/GC. This is the first scalable system in India for mass production entomopathogenic nematode; In vivo methodology based on LOTEK. In present study we describe an improved version of it in which bulk conditioning, harvesting, separations are automated to reduce labor costs.

Ajuste de níveis de inóculo para avaliação da resistência de genótipos de cenoura a Meloidogyne incognita

ABSTRACT One of the main phytopathogens that cause enormous economic losses in several agricultural species, including carrots, are the root-knot nematodes belonging to the genus Meloidogyne. Thus, in order to identify the best population level for inoculum of the root-knot nematode (Meloidogyne incognita) for maximum expression of symptoms of this nematode attack on carrots, an experiment was carried out to evaluate the carrot cultivar Brasília (CBR) and the population Pop-750 (CRO). The experiment was carried out in a greenhouse at Embrapa Vegetable, Brasília-DF, in 5 L pots, with six replications, in a 4x2 factorial scheme in a completely randomized design, in the dosages of 0, 1, 5 and 10 thousand eggs and occasional juveniles of 2nd stage (E+J2R). ‘Rutgers’ susceptible tomato cultivar was used as control to verify inoculum efficiency. Inoculation was carried out 30 days after sowing and evaluation 60 days after inoculation. Gall index (GI), egg mass index (EMI), number of eggs plus occasional second stage juveniles per gram of root (E+J2R) and reproduction factor (RF) were performed. There were differences between genotypes and between inoculum levels for all variables evaluated. For CRO, inoculum levels of M. incognita from 1,000 E+J2 the plants already manifested symptoms and changes in all evaluated variables, with ideal levels around 5 to 7 thousand E+J2R, above 7 thousand E+J2R nematode multiplication to express symptoms decreased. For CBR the response variables E+J2 at root and RF inoculum levels close to 5 thousand E+J2R also present the best results, but when the characterization is based on the evaluation of GI and EMI, suitable inoculum levels would be close to 9 and 12 thousand E+J2R.

Bio-management of rice root-knot nematode, M. graminicola by using various organic amendments and bio-control agents on rice nursery

A screen house experiment was conducted during kharif season, 2020-21 to know the effect of organic amendments and bio-agents (alone and in combination) in comparison with chemical on the population of rice root-knot nematode (Meloidogyne graminicola) in rice. Nursery was grown in 5 kg soil capacity earthen pan filled with infested soil having initial nematode population 285 J2/200 cc soil. Seeds of the rice (Variety- Pusa 1121) were soaked in tap water for 24 h and the sprouted seeds were sown in pots. Hundred seeds of rice were sown in each pot and each treatment was replicated four times. Organic amendments (neem cake and FYM) and bio-agents (Pseudomonas fluorescens, Purpureocillium lilacinum and Trichoderma viride) were added 10 days before sowing. Weighted amount of bio-agents was mixed in neem cake and FYM for enrichment with 7-10 days waiting period. Carbofuran (Furadan 3G) was added at time of sowing. The earthen pots without organic amendments, bio-agents and chemical were treated as control. Observations on plant growth parameters (seedling length in cm fresh and dry seedling weight in g) and nematode multiplication (number of galls/20 seedling, number of eggs/20 seedling and final nematode population in the soil/200 cc soil) were made at transplanting time. The plant parameters were maximum and significantly highest in treated nursery (neem cake @ 50 g/pot + Pseudomonas fluorescens @ 50 g/pot) under screen house conditions. Nematode reproduction and multiplication parameters such as number of galls/seedling, number of eggs/seedling and final nematode population were significantly reduced in neem cake @ 50 g/pot + Pseudomonas fluorescens @ 50 g/pot.