Incognita Second-stage Juveniles Research Articles

Τhe Nematicidal Potential of Bioactive Streptomyces Strains Isolated from Greek Rhizosphere Soils Tested on Arabidopsis Plants of Varying Susceptibility to Meloidogyne spp.

A total of 461 indigenous Streptomycetes strains recovered from various Greek rhizosphere habitats were tested for their bioactivity. All isolates were examined for their ability to suppress the growth of 12 specific target microorganisms. Twenty-six were found to exert antimicrobial activity and were screened for potential nematicidal action. S. monomycini ATHUBA 220, S. colombiensis ATHUBA 438, S. colombiensis ATHUBA 431, and S. youssoufensis ATHUBA 546 were proved to have a nematicidal effect and thus were further sequenced. Batch culture supernatants and solvent extracts were assessed for paralysis on Meloidogyne javanica and Meloidogyne incognita second-stage juveniles (J2). The solvent extracts of S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 had the highest paralysis rates, so these Streptomycetes strains were further on tested for nematodes’ biological cycle arrest on two Arabidopsis thaliana plants; the wild type (Col-0) and the katanin mutant fra2, which is susceptible to M. incognita. Interestingly, S. monomycini ATHUBA 220 and S. colombiensis ATHUBA 438 were able to negatively affect the M. incognita biological cycle in Col-0 and fra2 respectively, and increased growth in Col-0 upon M. incognita infection. However, they were ineffective against M. javanica. Fra2 plants were also proved susceptible to M. javanica infestation, with a reduced growth upon treatments with the Streptomyces strains. The nematicidal action and the plant-growth modulating abilities of the selected Streptomycetes strains are discussed.

Synergism of citral from Cymbopogon citratus essential oil and undecan-2-one against Meloidogyne incognita

Summary The nematicidal activity of the essential oil of Cymbopogon citratus and its main components were studied. During in vitro assays with Meloidogyne incognita second-stage juveniles (J2), the oil showed an LC50 of 166 μg ml−1. In vivo assays that exposed J2 to the LC50 oil concentration significantly reduced galls and eggs on tomato roots. According to gas chromatography-mass spectrometry analyses, the main components of the essential oil were geranial (42.7%) and neral (33.0%). The combination of these two isomers, known as citral, had an enhanced nematicidal activity at low concentrations as shown by in vitro tests through a synergistic interaction with undecan-2-one. In assays with tomato plants inoculated with M. incognita J2, the combination of citral (600 μg ml−1) and undecan-2-one (300 μg ml−1) showed a high reduction in the number of galls (63.4%) and eggs (56.3%) in relation to Tween 80® control. The citral/undecan-2-one combination showed a synergistic nematicidal activity against M. incognita during in vitro and in vivo assays.

Effects of yeast fermentation broths on the Meloidogyne incognita population in soybean

Root-knot nematodes are pathogenic to a wide range of plants worldwide and can reduce the yield potential of important crops. The objective of this study was to investigate the potential effects of yeast fermentation broths (YFBs) on the hatching of Meloidogyne incognita second-stage juveniles (J2s) in vitro. In addition, application methods of the yeasts for the control of the nematode in soybean were evaluated. Fermentation broths (FBs) of Pichia manshurica CCMA0762, Pichia caribbica CCMA0759, Lachancea thermotolerans CCMA0763, Hanseniaspora opuntiae CCMA0760 and Kodamaea (Pichia) ohmeri CCMA0758 were evaluated in the hatching test in vitro. The same treatments were separately applied to the soil, soil+shoot and shoots of plants inoculated with M. incognita, and the results were compared with those of the control and those from a yeast-based commercial product. L. thermotolerans CCMA0763 and P. caribbica CCMA0759 FBs were selected for evaluation of nematode infection. All YFBs inhibited J2 hatching. The L. thermotolerans FB reduced the number of nematodes in one experiment when applied to soil. The most effective application mode differed by FB type. L. thermotolerans and P. caribbica FBs, when applied to shoots, decreased the number of J2s, sausage-shaped juveniles, adult females and total nematodes at different evaluation times.

Activity of papaya seeds (Carica papaya) against Meloidogyne incognita as a soil biofumigant

Seeds of papaya fruit (Carica papaya) are usually unused for the industry, but they have been previously reported to be anthelmintic. However, the effects and mechanisms involved in papaya seeds against root-knot nematodes (RKN) are poorly studied in soil incorporation. Here, we investigated soil biofumigation with papaya seeds against the RKN Meloidogyne incognita and the effect of their volatile organic compounds (VOCs) separately from non-VOCs. Soil biofumigation reduced the number of galls and eggs of RKN about 100% in tomato roots, by using seeds macerates at 4.0 g 100 mL−1, which is a feasible amount for field applications. The VOCs emitted by the seeds killed 80% of M. incognita second-stage juveniles after a few hours of exposure. Furthermore, the whole volatilome of the papaya seed was identified by gas chromatography. Some of the compounds have already been studied against RKN. Therefore, we examined the efficacy of all nonstudied compounds against M. incognita. The most promising compounds were vinyl acetate, phenylacetaldehyde and benzylacetonitrile with lethal concentrations (LC50) of 61.5, 101.3 and 14.4 μg mL−1, respectively. When vinyl acetate and phenylacetaldehyde were applied to the soil at the recommended commercial nematicide dose (396 μg mL−1), the effect on M. incognita was better than the commercial nematicide (Carbofuran), reducing 70% of the number of galls and eggs. However, benzylacetonitrile reduced galls and eggs only by 25%. These results provide evidence for biofumigation with papaya seeds as a sustainable approach to control RKN and the potential use of their VOCs as nematicidal compounds.

Toxicities of 4,5-Dihydroisoxazoles Against Root-Knot Nematodes and in Silico Studies of Their Modes of Action.

The present work sought to contribute to the development of new nematicides. Benzaldehydes were initially converted to nitrile oxides that underwent 1,3-dipolar cycloaddition reactions with methyl acrylate to generate 4,5-dihydroisoxazoles. In in vitro tests, methyl 3-phenyl-4,5-dihydroisoxazole-5-carboxylate (1) and methyl 3-(4-chlorophenyl)-4,5-dihydroisoxazole-5-carboxylate (4) increased the mortality of Meloidogyne exigua and Meloidogyne incognita second-stage juveniles (J2). Compounds 1 and 4 presented necessary concentrations of 398 and 501 μg mL-1, respectively, to kill 50% of M. incognita J2 (LC50 values), while the value for carbofuran (positive control) was 168 μg mL-1. In in vivo tests, compounds 1 and 4 reduced the number of M. incognita galls in tomato roots by 70 and 40%, respectively, and the number of eggs by 89 and 44%. Using an in silico approach, we showed that compounds 1 and 4 were toxic to the nematodes by binding to the allosteric binding sites of the agonist-binding domains of the nematode nicotinic acetylcholine receptors. These results opened up possibilities for further investigations aimed at developing novel commercial nematicides.

Identification and Nematicidal Characterization of Proteases Secreted by Endophytic Bacteria Bacillus cereus BCM2.

The endophytic bacterium Bacillus cereus BCM2 has shown great potential as a biocontrol organism against Meloidogyne incognita, which causes severe root-knot diseases in crops. In our previous study, the metabolite of BCM2 showed high nematicidal activity against the M. incognita second-stage juveniles. However, the mechanism employed by endophytic bacteria to infect and kill nematodes is still unclear. Here, we investigate both the endophytic bacterial extracellular proteins with nematicidal activity and their mechanism of killing nematodes. The first step was detecting the nematicidal activities of crude proteins. The results show that the nematode mortality rate reached 100% within 72 h, and the crude proteins damaged both the cuticle and eggshell, before finally destroying the targets. This suggests possible proteinaceous pathogeny in BCM2. Throughout the process, the fine-detail changes in the nematode cuticle and the intestinal structure were observed using scanning electron microscopy and transmission electron microscopy. These images show that BCM2 extracellular proteins did not damage the internal organization of the nematode but did severely damage its cuticle, which led to content leakage. From the crude proteins, chitosanase, alkaline serine protease, and neutral protease were purified and identified. The M. incognita-B. cereus BCM2 microenvironment simulation demonstrates that BCM2 adheres to the surface of nematodes and helps the metabolites that were produced by BCM2 to rapidly recognize and kill M. incognita. This relationship between plants, endophytic bacteria, and nematodes offers insight into the biological mechanisms that can be utilized for of nematode management.

Short-Term Effects of Sublethal Doses of Nematicides on Meloidogyne incognita.

Meloidogyne species are one of the most important groups of plant-parasitic nematodes globally because of their ability to damage most cultivated plants. Although they are widespread and economically important, there are limited control measures to combat these nematodes. New nonfumigant nematicides have been discovered that have the potential to be widely utilized for the management of plant-parasitic nematodes. Because of the longer half-lives in soil and lower toxicity of new nematicides compared with traditional fumigant and nonfumigant nematicides, understanding how nematodes respond to sublethal doses of nematicides is imperative to understanding whether nematicide resistance has the potential to develop. Characterizing responses of nematodes to sublethal doses will provide the foundation for future work, such as gene expression studies. In this study, the nematicides oxamyl (Vydate), fluazaindolizine (Salibro), fluensulfone (Nimitz), and fluopyram (Velum), were evaluated to understand how sublethal doses affect the fecundity and mobility of Meloidogyne incognita second-stage juveniles (J2). Using a microwell assay system, dose-response curves for each nematicide were established for M. incognita J2. Fluopyram was the most toxic nematicide, with effective doses up to 230 times lower than that of other nematicides. The other nematicides had predicted ED50 values (effective doses that resulted in 50% of the population becoming inactive) of 89.4, 131.7, and 180.6 ppm for oxamyl, fluensulfone, and fluazaindolizine, respectively. The 24-h ED50 of each nematicide was then used in both motility and infectivity assays. The motility and activity of M. incognita J2 exposed to ED50 doses of fluazaindolizine and fluensulfone was significantly reduced, with nematodes initially being motile but eventually becoming inactive. However, the motility of M. incognita J2 exposed to ED50 doses of fluopyram and oxamyl was not different from a water control. In a pot assay, M. incognita J2 exposed to ED50 doses of fluazaindolizine, oxamyl, and fluensulfone were unable to reproduce on tomato, with reproduction factors (RF = final population density/initial population density) of 0 to 0.03. Fluopyram did not reduce reproduction of M. incognita, with a mean RF of 38.7 ± 4.5, which was similar to the RF of 46.3 ± 4.6 for the water control. This study is the first comprehensive evaluation of M. incognita activity, motility, and fecundity after exposure to the traditional nematicide, oxamyl, as well as three new nematicides, fluazaindolizine, fluopyram, and fluensulfone.

Effects of the Endophytic Bacteria Bacillus cereus BCM2 on Tomato Root Exudates and Meloidogyne incognita Infection.

Root-knot nematodes (Meloidogyne spp.) cause serious crop losses worldwide. The colonization of tomato roots by endophytic bacteria Bacillus cereus BCM2 can greatly reduce Meloidogyne incognita damage, and tomato roots carrying BCM2 were repellent to M. incognita second-stage juveniles (J2). Here, the effects of BCM2 colonization on the composition of tomato root exudates was evaluated and potential mechanisms for BCM2-mediated M. incognita control explored using a linked twin-pot assay and GC-MS. On water agar plates, J2 preferentially avoided filter paper treated with tomato root exudates (organic phase only) from plants inoculated with BCM2, visiting these 67.1% less than controls. In a linked twin-pot assay, BCM2 treatment resulted in a 42.0% reduction in the number of nematodes in the soil, a 43.3% reduction in the number of galls and a 47.7% decrease in the density of M. incognita in root tissues. Analysis of root exudate composition revealed that BCM2 inoculation increased the number of components in exudates. Among these, 2,4-di-tert-butylphenol, 3,3-dimethyloctane, and n-tridecane secretions markedly increased. In repellency trials on water agar plates, J2 avoided 2,4-di-tert-butylphenol, n-tridecane, and 3,3-dimethyloctane at concentrations of 4 mmol/liter. In a linked twin-pot assay, inoculation with 2,4-di-tert-butylphenol or 3,3-dimethyloctane reduced the number of nematodes in the soil (by 54.9 and 70.6%, respectively), the number of galls (by 53.7 and 52.4%), and the number of M. incognita in root tissues (by 67.5 and 36.3%). BCM2 colonization in tomato roots affected the composition of root exudates, increasing the secretion of substances that appear to be repellent, thus decreasing M. incognita J2 infection of roots.

Effects of cucurbitacin A on mobility of Meloidogyne incognita second-stage juveniles

Registration authorities of botanical inputs in crude extract form demand that the active ingredients be isolated, identified and efficacy tests on target pests be investigated. Nemarioc-AL phytonematicide, with active ingredient cucurbitacin A, consistently suppress bioactivities of root-knot (Meloidogyne species) nematode, with limited information on the efficacy of its purified active ingredient. The objective of this study was to investigate the effects of cucurbitacin A on mobility of M. incognita second-stage juveniles (J2). Approximately, 450 freshly hatched J2 were exposed to pure cucurbitacin A concentration at 0.00, 0.25, 0.50, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 and 2.50 μg/ml distilled water, arranged in a completely randomized design, with three replicates. Experimental units were placed in an incubator at 25±2°C for 12-, 24-, 48-and 72-h. After each duration, mobile and immobile J2 were each counted using a stereomicroscope. Cucurbitacin A concentration effects were highly significant on J2 immobility at all four exposure periods, contributing 84, 99, 99 and 99% in total treatment variation of the variable at the respective periods. Relative to untreated control, J2 immobility increased with increasing concentrations in density-dependent growth (DDG) patterns. In conclusion, cucurbitacin A had significant bioactivity effects on mobility of M. incognita J2 and could be one of the bioactivities through which Nemarioc-AL phytonematicide suppressed nematode population densities.

Volatile organic compounds produced by castor bean cake incorporated into the soil exhibit toxic activity against Meloidogyne incognita.

This study showed, for the first time, the effect of volatile organic compounds (VOCs) emitted by castor bean cake added to soil on the plant-parasitic nematode Meloidogyne incognita. Identification of nematotoxic volatiles increases the chance of identifying molecules for use in commercial nematicides. VOCs produced after the incorporation of castor bean cake in the soil resulted in immobility (>97.3%) and death (>96.9%) of M. incognita second-stage juveniles (J2) and reduction in M. incognita egg-hatch (>74.3%) at all concentrations of cake tested (1.5-6.0%). A similar reduction in M. incognita infectivity and reproduction was observed when nematodes were exposed to these VOCs and inoculated into tomato plants or exposed directly upon contact with the soil. We identified 32 VOCs produced by castor bean cake when incorporated into soil by using solid phase micro-extraction gas chromatography coupled with mass spectrometry (SPME-GC-MS). Four of the most intense peaks in the chromatogram represented the compounds phenol, 4-methylphenol, γ-decalactone, and skatole. When M. incognita J2 were exposed to these compounds, all of the compounds demonstrated nematicidal activity with low median lethal concentration (LC50 ) values. Castor bean cake incorporated into the soil produces volatile compounds which are toxic to M. incognita. These substances show potential for application in the nematicide-producing industry. © 2018 Society of Chemical Industry.

Plant Volatiles Reduce the Viability of the Root-Knot NematodeMeloidogyne incognitaEither Directly or When Retained in Water

Volatile organic compounds (VOC) produced by green residues for the management of plant-parasitic nematodes are poorly studied for oilseed plants and some Brassica spp. To investigate the activity of VOC in vitro and as biofumigants, dry and aqueous macerates of broccoli (Brassica oleracea var. italica) shoots and sunflower (Helianthus annuus) seed were used against the root-knot nematode Meloidogyne incognita. VOC produced by sunflower seed caused higher mortality of M. incognita second-stage juveniles (J2) than VOC produced by broccoli shoots but both plant species were equally effective in decreasing the infectivity and reproduction of this nematode. The number of galls and eggs produced by the nematode in tomato roots was reduced by 89 and 95%, respectively, on average, at the highest concentrations of broccoli and sunflower seed macerates tested as biofumigants. When nematodes were placed in water exposed to broccoli VOC, J2 immobility increased and the number of galls and eggs produced by the nematode in tomato roots decreased 80 and 96%, respectively. Water exposed to sunflower seed VOC had no effect on the viability of the nematode. Gas chromatography was used to identify five and six chemical groups in broccoli and in sunflower seed macerates, respectively, but only alcohols, sulfurated VOC, and terpenes were detected in the water exposed to these plant macerates. Sulfurated VOC from the water exposed to broccoli macerates were found to be involved in its activity against M. incognita. The purified VOC dimethyl disulfide (DMDS) and 3-pentanol were tested directly against J2 and showed a lethal concentration of 176 and 918 µg/ml (ppm), respectively, whereas dimethyl sulfide had no effect against M. incognita. Furthermore, DMDS and 3-pentanol retained in water killed J2 and reduced gall formation and the number of eggs of M. incognita on tomato roots. Both these plant species produced toxic VOC to M. incognita, whereas only VOC retained in water exposed to broccoli had activity against M. incognita.

A novel in vitro chemotaxis bioassay to assess the response of Meloidogyne incognita towards various test compounds.

Abstract Plant-parasitic, root-knot nematodes (Meloidogyne spp.) are a serious problem in agri- and horticultural crops worldwide. Understanding their complex host recognition process is essential for devising efficient and environmental-friendly management tactics. In this study, the authors report a new, simple, inexpensive, efficient, and quantitative method to analyze the chemotaxis of M. incognita second-stage juveniles (J2s) using a combination of pluronic gel and agar in a petri dish. The authors quantitatively defined the concentration gradient formation of acid fuchsin on the assay plate. Using this novel assay method, the authors have accurately measured the nematode response (attraction or repulsion) to various volatile (isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol) and non-volatile (root exudates of tomato, tobacco, and marigold) compounds. Isoamyl alcohol, 1-butanol, and 2-butanone were attractive to J2s through a broad range of concentrations. On the contrary, J2s were repelled when exposed to various concentrations of 1-octanol. Despite being attractive at lower concentrations, undiluted benzaldehyde was repulsive to J2s. Tomato and tobacco root exudates were attractive to J2s while marigold root exudates repelled J2s. The present quantitative assay method could be used as a reference to screen and identify new candidate molecules that attract or repel nematodes.

Characterisation of the effects on proteases of Heterodera glycines and Meloidogyne incognita second-stage juveniles by inhibitors obtained from cysts of H. glycines

SummaryThe protease inhibitor component ofHeterodera glycinescyst contents was explored using a battery of peptide substrates andH. glycinesandMeloidogyne incognitasecond-stage juveniles as enzyme sources. Protease inhibitors were prepared by heat-denaturingH. glycinescyst-egg extract (hHglCE), which was used in all inhibition exploration. Eight substrates targeting four endoprotease groups (aspartic, cysteine, metallo- and serine proteases) revealed that protease inhibition by hHglCE varied significantly betweenH. glycinesandM. incognitawith seven of the eight substrates. Only cysteine protease activity was inhibited equally betweenH. glycinesandM. incognita. Aspartic protease activity was inhibited more strongly inH. glycinesand serine protease activity was inhibited more strongly inM. incognita. Digestion of five matrix metalloprotease (MMP) substrates was inhibited more strongly inH. glycines(two substrates) andM. incognita(three substrates). These variations were particularly intriguing given the potential association of MMP proteases with developing embryos. Inhibition of digestion of nematode FMRFamide-like peptides (FLPs) showed less variation between nematode species than the targeted substrates, but inhibition did vary significantly across substrates within each species. Digestion of FLP-6 was the least affected by hHglCE but was inhibited significantly more inM. incognitathan inH. glycines. Residue differences between two FLP-14 sequences significantly affected inhibition of FLP-14 digestion in bothH. glycinesandM. incognita. RP-HPLC fractionation of hHglCE clearly demonstrated the presence of high (Fr No.5) and low (Fr No.14) polarity inhibitor components. Potency of inhibition ofM. incognitaserine protease activity, based upon IC50values (1.68 and 2.78 hHglCEeq reaction−1for Fr No.5 and Fr No.14, respectively), was reduced significantly from unfractionated hHglCE (IC50 = 0.61), suggesting inhibitor dilution, loss of component synergy, or both, due to fractionation.

Confirmation of bioactivities of active ingredients of nemarioc-AL and nemafric-BL phytonematicides

ABSTRACTNemarioc-AL and Nemafric-BL phytonematicides consistently reduced populations of plant-parasitic nematodes. The contribution of juvenile hatch inhibition to the overall reduction of the nematode numbers by the two phytonematicides, with cucurbitacin A and B as active ingredients, respectively, remains undocumented. The objectives of this study were to examine (i) the response of Meloidogyne incognita second-stage juveniles (J2) hatch to increasing concentration of cucurbitacins A and B, (ii) the potential saturation of J2 hatch when exposed to cucurbitacins for extended incubation periods, (iii) the minimum inhibition concentration for J2 hatch and (iv) the reversibility of J2 hatch inhibition.. Eggs of M. incognita were exposed to a series of purified cucurbitacin A and B concentrations over five incubation periods of 24, 48, 72 h and extended incubation periods of 7 and 10 days. Methanol-dissolved cucurbitacin A and B were each diluted and pipetted into well-plates making 11 concentrations, ranging from 0.0 to 2.5 µg.ml−1 water solvent. Juvenile counts were made after 24, 48 and 72 h, with those for saturation assessed at 7 and 10 days. Thereafter, treatments were diluted five times, incubated again for 5 days and counted to establish reversibility of J2 hatch inhibition. In all incubation periods, treatment effects were highly significant (P ≤ 0.01), with J2 hatch and cucurbitacin concentrations exhibiting quadratic relations. Minimum inhibition concentrations of the two cucurbitacins were between 1.13 and 1.40 µg.ml−1. Treatment effects for reversibility to J2 hatch inhibition were not significant (P > 0.05). In conclusion, J2 hatch inhibition could be one of the waysthrough which the two phytonematicides reduced population densities of Meloidogyne species.

Meloydogine incognita (Nematoda) reproduction affected by plants overexpressing a steroid inducible gene-expression system

This study reports the impact of an integral glucocorticoid expression system (GR-system) on plant and plant-nematode reproduction. Seedlings expressing a GR-system (transGR+) show affections of growth and development. It is well-known that plant-hormones such as gibberellins, auxins and steroids are essential for developmental and physiological processes, in biotic/abiotic stresses. Here is shown some phenotypic affections reported by tobacco expressing a whole mammalian GR-system, how those effects are rescued by external hormonal treatments and the influence on plant-nematode fecundity. Results demonstrate that: i) GR-system is constitutively expressed in plants and its functionality proved by the activation of the reporter-gene transcription (uidA) after induction with different steroid hormones, dexamethasone (DEX) and progesterone (PRO) as well; ii) transGR + growth affected at hypocotyls and roots development result mitigated by exogenous hormones applications; iii) no differential expression in the expansin-gene TExp4 assayed; iv) Significant reductions of J2-larvae assessed in the egg-masses and soil during the generations on transformed plants. Although the molecular mechanism behind these results is still unknown, all together these results suggest a role of GR as reflected by the impact on the plant development and reduction of Meloydogine incognita (Nematoda) second-stage juveniles in egg-masses and soil.

Sensitivities of nematode-inoculated tomato plants to Nemarioc-AL phytonematicide from fresh Cucumis myriocarpus fruit

Costs related to temperature-dependent drying and grinding equipment of Cucumis myriocarpus fruit for preparation of Nemarioc-AL phytonematicide could inherently restrict on-farm preparation of this product, particularly in resource-poor smallholder farming systems. The objective of this study was to determine the overall sensitivities of tomato (Solanum lycopersicon L.) plants and nematodes to Nemarioc-AL phytonematicide produced using fermented crude extracts of C. myriocarpus fresh fruit thereby establishing the feasibility of producing this product on-farm. Uniform five-week-old tomato cv. ‘Floradade’ seedlings were transplanted into plastic pots containing a growing mixture, with treatments 0, 2, 4, 8, 16, 32 and 64% Nemarioc-AL phytonematicide applied weekly on plants inoculated with 3000 Meloidogyne incognita eggs and second-stage juveniles (J2). At 56 days after initiating the treatments, plant and nematode variables were subjected to analysis of variance. Nematode variables over increasing log2 2-transformed concentrations were subjected to lines of the best fit. Nematodes versus increasing concentrations of phytonematicide exhibited negative quadratic relations, with the model for eggs and J2 in roots and soil being explained by 90 and 95% relations, respectively. Plant variables were subjected to the Curve-fitting Allelochemical Response Data (CARD) computer-based model to generate biological indices. The mean concentration stimulation point (MCSP = Dm + Rh/2) was computed 2.79%, with the overall sensitivity (Σk) of four units. In conclusion, Nemarioc-AL phytonematicide from fermented fresh fruit of C. myriocarpus could be used in managing population densities of Meloidogyne species in tomato production without inducing phytotoxity.

In vitro characterization of Meloidogyne incognita juvenile egress and mortality under Nemarioc-AL phytonematicide

Pure (98%) cucurbitacin A, which is active ingredient of Nemarioc-AL phytonematicide, reduced egress and increased mortality of Meloidoygne incognita secondstage juveniles (J2). However, in vitro information on how J2 egress and mortality would respond to Nemarioc-AL phytonematicide had not been documented. The objective of this study was to determine in vitro the response of M. incognita J2 egress and mortality to increasing concentrations of Nemarioc-AL phytonematicide, maximum inhibition concentration and possible reversibility of J2 egress inhibition and mortality when the phytonematicide was diluted. Eggs and freshly hatched J2 of M. incognita were exposed to phytonematicide concentrations of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0% in distilled water. Three independent experiments with three replications each were conducted in an incubator set at 25±3°C and counts were made at 24, 48 and 72 h exposure periods. Relative to untreated control, J2 egress inhibition and mortality increased with increase in phytonematicide concentration. Relative impact values of J2 egress inhibition and mortality when plotted against increasing phytonematicide concentrations exhibited density-dependent growth (DDG) patterns with the inhibition effects being irreversible. Maximum inhibition concentration for J2 egress could not be established, whereas for J2 mortality it was 3.49%. In conclusion, the irreversible suppressiveness of the product on both nematode developmental stages makes it an ideal alternative in nematode management.

Endophytic Bacillus cereus Effectively Controls Meloidogyne incognita on Tomato Plants Through Rapid Rhizosphere Occupation and Repellent Action.

Root-knot nematodes (Meloidogyne spp.), which cause severe global agricultural losses, can establish a special niche in the root vascular cylinder of crops, making them difficult to control. Endophytic bacteria have great potential as biocontrol organisms against Meloidogyne incognita. Three endophytic bacteria were isolated from plant tissues and showed high nematicidal activity against M. incognita second-stage juveniles (J2) in vitro. The gyrB gene sequence amplification results indicated that the three isolates were Bacillus cereus BCM2, B. cereus SZ5, and B. altitudinis CCM7. The isolates colonized tomato roots rapidly and stably during the colonization dynamic experiment. Three pot experiments were designed to determine the potential of three endophytic bacterial isolates on control of root-knot nematodes. The results showed that the preinoculated B. cereus BCM2 experiment significantly reduced gall and egg mass indexes. The inhibition ratio of gall and egg mass was up to 81.2 and 75.6% on tomato roots and significantly enhanced shoot length and fresh weight. The other two experiments with inoculated endophytic bacteria and M. incognita at the same time or after morbidity had lower inhibition ratios compared with the preinoculated endophytic bacteria experiment. The confocal laser-scanning microscopy method was used to further study the possible mechanism of endophytic bacteria in the biocontrol process. The results showed the localization pattern of the endophytic bacteria B. cereus BCM2-(str')-pBCgfp-1 in tomato root tissues. Root tissue colonized by endophytic bacteria repelled M. incognita J2 infection compared with the untreated control in a repellence experiment. We isolated an endophytic B. cereus strain that stably colonized tomato and controlled M. incognita effectively. This strain has potential for plant growth promotion, successful ecological niche occupation, and M. incognita J2 repellent action induction. It plays an important role in endophytic bacteria against root-knot nematodes.

In vitro evaluations to determine the effect of Bacillus firmus strains on the motility of Meloidogyne incognita second-stage juveniles

Eco-friendly control strategies, such as biological agents containing Bacillus firmus, are known to adversely impact the biology of Meloidogyne incognita and other nematode pests that damage agricultural crops. The efficacy of two isolated strains as well as one reference strain was evaluated for their effect on M. incognita second-stage juvenile (J2) motility. All three B. firmus strains successfully reduced J2 motility, with the reference strain being superior. Non-cultured cell-free products of the isolated strains inhibited J2 motility only between 3 and 7 % compared to the tap-water controls, respectively, and compared to up to 42 % by their cultured, cell-free water filtrate counterparts. The latter range was also recorded for the 100 % cultured, cell-free suspension concentration of the reference strain. The efficacy of cell suspensions of the two isolated strains were low (7-19 %) compared to the 100 % achieved by the reference strain. Although secondary compounds of the two isolated B. firmus strains may contribute towards disrupting the movement and host finding abilities of J2, they had no pronounced effects on J2 motility inhibition.

The effects of stirring, population levels and a potential anti-nematodal product on the respiration of second-stage juveniles of Meloidogyne incognita measured with different technology

The oxygen consumption rate (OCR) ofMeloidogyne incognitasecond-stage juveniles (J2) was measured using polarographic oxygen sensor (POS) and fibre-optic oxygen sensor (FOS) technology. The effect of stirring speed on the OCR of J2 suspended in tap water was determined using POS. Next, the OCR of different J2 numbers was measured using FOS. Also, the OCR of J2 in tap water using POS and FOS was recorded, and also the effect of SoilBioMuti (SBM) on J2 OCR using FOS. Stirring speed had no pronounced effect on the OCR of J2. FOS recorded the OCR of as few as five J2. The OCR of J2 in sterile tap water was significantly lower for POS than FOS. Using FOS, non-filtered (NF) SBM without J2 had the highest OCR, being 42% higher than that of NF SBM with J2. The two filtered SBM (with and without J2) treatments had the lowest OCR values.