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Root-knot nematodes (RKNs) are distributed globally, including in agricultural fields contaminated by heavy metals (HM), and can cause serious crop damages. Having a method that could control RKNs in HM-contaminated soil while limit HM accumulation in crops could provide significant benefits to both farmers and consumers. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum YMF1.683 exhibited a high nematocidal activity against the RKN Meloidogyne incognita and a high tolerance to CdCl 2 . Comparing to the P. lavendulum YMF1.838 which showed low tolerance to Cd 2+ , strain YMF1.683 effectively suppressed M. incognita infection and significantly reduced the Cd 2+ uptake in tomato root and fruit in soils contaminated by 100 mg/kg Cd 2+ . Transcriptome analyses and validation of gene expression by RT-PCR revealed that the mechanisms contributed to high Cd-resistance in YMF1.683 mainly included activating autophagy pathway, increasing exosome secretion of Cd 2+ , and activating antioxidation systems. The exosomal secretory inhibitor GW4869 reduced the tolerance of YMF1.683 to Cd 2+ , which firstly demonstrated that fungal exosome was involved in HM tolerance. The up-regulation of glutathione synthesis pathway, increasing enzyme activities of both catalase and superoxide dismutase also played important roles in Cd 2+ tolerance of YMF1.683. In Cd 2+ -contaminated soil, YMF1.683 limited Cd 2+ -uptake in tomato by up-regulating the genes of ABCC family in favor of HM sequestration in plant, and down-regulating the genes of ZIP, HMA, NRAMP, YSL families associated with HM absorption, transport, and uptake in plant. Our results demonstrated that YMF1.683 could be a promising bio-agent in eco-friendly management of M. incognita in Cd 2+ contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950's the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans . A new Velcro- like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells., Competing Interests: KD is Director of his company KG Davies Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Davies, Mohan, Phani and Srivastava.)

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 using quantitative 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., (© 2023 Wiley-VCH GmbH.)

We investigated if activity of the pre-infective juveniles (J2s) of root-knot nematodes is linked to the recruitment of a specific microbiome on the nematode surface and/or to the composition of the surrounding microbiota. For this, we determined the J2 activity (active vs. non-motile, which referred to dead and immobile J2s) upon a 3-day incubation in soil suspensions and studied the composition of bacteria, protists, and fungi present on the nematode surface and in the suspensions using amplicon sequencing of the 16S/18S rRNA genes, and ITS region. We also amended suspensions with Pseudomonas protegens strain CHA0 to study its effects on J2 activity and microbial composition. The J2 activity was suppressed in soil suspensions, but increased when suspensions were amended with P. protegens CHA0. The active and non-motile J2s differed in the composition of surface-attached bacteria, which was altered by the presence of P. protegens CHA0 in the soil suspensions. The bacterial genera Algoriphagus, Pedobacter, and Bdellovibrio were enriched on active J2s and may have protected the J2s against antagonists. The incubation time appeared short for attachment of fungi and protists. Altogether, our study is a step forward in disentangling the complex nematode-microbe interactions in soil for more successful nematode control., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Intermingled uninfected and root-knot nematode-infected tomato plants are commonly observed under protected cultivation. To understand the role of rhizobacteria underlying the susceptibility to nematode infectivity in these tomato plants, 36 rhizobacteria (18 from each type) with morphologically distinct colony characteristics were isolated from the rhizosphere of uninfected and root-knot nematode-infected tomato plants. The in vitro nematicidal potential of rhizobacteria from the uninfected rhizosphere was significantly higher than that from the infested rhizosphere. The three most effective antagonists were identified as Microbacterium laevaniformans, Staphylococcus kloosii, Priestia aryabhattai from root-knot-nematode-infected tomato rhizosphere and Staphylococcus sciuri, Bacillus pumilus, and Priestia megaterium from the rhizosphere of uninfected tomato. Volatile organic compounds from these rhizobacteria were characterized. Except for S. kloosi, the soil drenching with other rhizobacteria significantly reduced juvenile penetration (>60%) in tomato roots. Furthermore, the application of a single or consortium of these rhizobacteria affected nematode reproduction in tomato. Four consortia of rhizobacteria (S. sciuri + B. pumilus + P. megaterium), (B. pumilus + P. megaterium), (S. sciuri + B. pumilus), and (S. sciuri + P. megaterium) from uninfested rhizosphere and two consortia (M. laevaniformans + P. aryabhattai), (M. laevaniformans + S. kloosii + P. aryabhattai) from infested rhizosphere (IRh) effectively reduced M. incognita reproduction and considerably enhanced plant growth and yield in tomato. The nematicidal efficacy, however, decreased when S. kloosii was applied in the consortium. These distinctive effects illustrate how the plant susceptibility to nematode infectivity is modulated under natural conditions., (© 2023 Wiley-VCH GmbH.)

Background: In our continuing search for biologically active natural enemies from North of Africa with special reference to Tunisian fungi, our teamwork screened fungi from different ecological habitats in Tunisia. Our previous study on the comparative effectiveness of filamentous fungi in the biocontrol of Meloidogyne javanica, a taxon (Lecanicillium) showed high potentiality against M. javanica. We undertook the present study to evaluate the ability and understand the mechanism of this fungal parasite as a biological control candidate against the root-knot nematode M. javanica. This study used in vitro bioassays with fungal filtrate cultures, scanning electron microscopy (SEM) observation, and isobaric tag for relative and absolute quantitation (iTRAQ) methodology to characterize the biological and molecular features of this fungus., Results: The microscopic and SEM observation revealed that Lecanicillium sp. exhibited exceptional hyperparasitism against M. javanica eggs. The hyphae of this fungi penetrated the eggs, causing destructive damage to the outer eggshell. The exposure to five concentrations of Lecanicillium sp. filtrate cultures showed high inhibition of egg hatching, which increases depending on the exposure time; the best results are recorded at 50%, 75%, and 100% dilutions after seven days of exposure. The SEM observation of nematode-parasitized eggs and juveniles suggests that the production of lytic enzymes degrades the egg cuticle and fungal hyphae penetrate unhatched M.javanica juveniles. Forty-seven unique proteins were identified from the Lecanicillium sp. isolate. These proteins have signalling and stress response functions, bioenergy, metabolism, and protein synthesis and degradation., Conclusion: Collectively, Lecanicillium sp. had ovicidal potentiality proved by SEM and proteomic analysis against root-knot nematode' eggs. This study recommended applying this biological control candidate as a bio-agent on vegetable crops grown in situ., (© 2023. The Author(s).)

Pochonia chlamydosporia is a soil-dwelling fungus and biological control agent of nematodes, active ingredient in commercial bionematicides. The fungus is also endophytically associated with the roots of several plant species, promoting their growth and inducing systemic resistance. In this study, different pathways and tomato defense metabolites were studied to identify mechanisms induced by P. chlamydosporia that contribute to the control of Meloidogyne javanica, at early and late developmental stages. Some defense responses activated by the fungus appeared related to the nematode life cycle. Among the evaluated biochemical analysis, root colonization of P. chlamydosporia showed an increase in the concentration of phenolic compounds, such as chlorogenic acid. In addition, the expression of some host plant genes was also modified. The interaction of the fungus with roots parasitized by M. javanica resulted in the highest expression of Phenylalanine Ammonia-Lyase (PAL), Chalcone synthase (LECHS 2), and Protease Inhibitor (PI1) genes at 24 days post-inoculation. At the second sampling time (44 days), there was an increase in the expression of the Respiratory Burst Oxidase Homolog (RBOH) gene. Fungus reduced the expression of the ACC-oxidase and Pathogenesis-Related Proteins 1 (PR-1) genes in roots. Moreover, P. chlamydosporia inoculation changed metabolites and phytohormone profiles of the gall formed by M. javanica. Plant defense response appeared to involve the jasmonic acid and phytosphingosine cascades. With this analysis, it was possible to propose new molecular mechanisms induced by the fungus that contribute to the control of M. javanica., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Plant-parasitic nematodes are an important group of pests causing economic losses in agriculture worldwide. Among the plant-parasitic nematodes, the root-knot ( Meloidogyne spp.) and root-lesion nematodes ( Pratylenchus spp.) are considered the two most important ones affecting soybeans. In general, they damage soybean roots, causing a reduction of about one-third in productivity. The soil microbial community can exert a suppressive effect on the parasitism of plant-parasitic nematodes. Here, we investigated the effects of soil bacterial diversity on Meloidogyne javanica ( Meloidogyne -assay) and Pratylenchus brachyurus ( Pratylenchus -assay) suppression by manipulating microbial diversity using the dilution-to-extinction approach in two independent experiments under controlled conditions. Furthermore, we recorded the changes in the soil microbial community induced by plant-parasitic nematode infection. In Meloidogyne -assay, microbial diversity reduced the population density of M. javanica and improved plant performance. In Pratylenchus -assay, microbial diversity sustained the performance of soybean plants even at high levels of P. brachyurus parasitism. Each nematode population affected the relative abundance of different bacterial genera and altered the core microbiome of key groups within the bacterial community. Our findings provide fundamental insights into the interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants. IMPORTANCE Root-knot and root-lesion nematodes cause losses of billions of dollars every year to agriculture worldwide. Traditionally, they are controlled by using chemical nematicides, which in general have a negative impact on the environment and human health. Fortunately, the soil microbial community may suppress these pests, acting as an environmentally friendly alternative to control nematodes. However, the effects of soil microbial diversity on the parasitism of plant-parasitic nematodes still poorly understood. In this study, we provide fundamental insight into the interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants, which may be useful for the development of new strategies to control these phytopathogens.

Biocontrol of the nematode Meloidogyne javanica was studied using the Argentinean strains Pseudomonas fluorescens MME3, TAE4, TAR5 and ZME4 and Bacillus sp. B7S, B9T and B19S. Pseudomonas protegens CHA0 was used as a positive control. Egg hatching and juvenile mortality were evaluated in vitro by exposure of nematodes to bacterial suspensions or their cell-free supernatants (CFS). The effect of bacteria on nematode infestation of lettuce was also studied. results showed that most of the tested strains and CFS reduced egg hatching and juvenile survival in vitro. The bacterial suspension of Bacillus sp. B9T produced the lowest hatching of eggs. Juvenile mortality was higher when M. javanica was exposed to Bacillus sp. than to Pseudomonas spp. suspensions. Except for CFS of B9T, all filtrates inhibited hatching at levels similar to or higher than the biocontrol strain P. protegens CHA0. The CFS of CHA0 showed the highest level of juvenile mortality followed by Bacillus sp. strains and P. fluorescens TAE4. None of the inoculated rhizobacteria reverted the negative effect of infestation on the aerial dry weight of lettuce plants. However, inoculation impacted on reproduction of M. javanica by reducing the development of galls and egg masses on roots and diminishing the number of individuals both on roots and in the substrate, as well as the reproduction factor. These results show that most of the analyzed native strains can control the nematode M. javanica. Among them, P. fluorescens TAE4 and Bacillus sp. B9T showed the most promising performances for the biocontrol of this pathogen and have a potential use in the formulation of commercial products., (Copyright © 2021 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 μg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 μg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Background: Root-knot nematode is one of the most significant diseases of vegetable crops in the world. Biological control with microbial antagonists has been emerged as a promising and eco-friendly treatment to control pathogens. The aim of this study was to screen and identify novel biocontrol agents against root-knot nematode, Meloidogyne incognita., Results: A total of 890 fungal isolates were obtained from rhizosphere soil of different crops and screened by nematicidal activity assays. Snef1910 strain showed high virulence against second stage juveniles (J2s) of M. incognita and identified as Trichoderma citrinoviride by morphology analysis and biomolecular assay. Furthermore, T. citrinoviride Snef1910 significantly inhibited egg hatching with the hatching inhibition percentages of 90.27, 77.50, and 67.06% at 48, 72, and 96 h after the treatment, respectively. The results of pot experiment showed that the metabolites of T. citrinoviride Snef1910 significantly decreased the number of root galls, J2s, and nematode egg masses and J2s population density in soil and significantly promoted the growth of tomato plants. In the field experiment, the biocontrol application showed that the control efficacy of T. citrinoviride Snef1910 against root-knot nematode was more than 50%. Meanwhile, T. citrinoviride Snef1910 increased the tomato plant biomass., Conclusions: T. citrinoviride strain Snef1910 could be used as a potential biological control agent against root-knot nematode, M. incognita.

Plant parasitic nematodes such as Meloidogyne incognita have a complex life cycle, occurring sequentially in various niches of the root and rhizosphere. They are known to form a range of interactions with bacteria and other microorganisms that can affect their densities and virulence. High-throughput sequencing can reveal these interactions in high temporal and geographic resolutions, although thus far we have only scratched the surface. In this study, we have carried out a longitudinal sampling scheme, repeatedly collecting rhizosphere soil, roots, galls, and second-stage juveniles from 20 plants to provide a high-resolution view of bacterial succession in these niches, using 16S rRNA metabarcoding. Our findings indicate that a structured community develops in the root, in which gall communities diverge from root segments lacking a gall, and that this structure is maintained throughout the crop season. We describe the successional process leading toward this structure, which is driven by interactions with the nematode and later by an increase in bacteria often found in hypoxic and anaerobic environments. We present evidence that this structure may play a role in the nematode's chemotaxis toward uninfected root segments. Finally, we describe the J2 epibiotic microenvironment as ecologically deterministic, in part, due to the active bacterial attraction of second-stage juveniles. IMPORTANCE The study of high-resolution successional processes within tightly linked microniches is rare. Using the power and relatively low cost of metabarcoding, we describe the bacterial succession and community structure in roots infected with root-knot nematodes and in the nematodes themselves. We reveal separate successional processes in galls and adjacent non-gall root sections, which are driven by the nematode's life cycle and the progression of the crop season. With their relatively low genetic diversity, large geographic range, spatially complex life cycle, and the simplified agricultural ecosystems they occupy, root-knot nematodes can serve as a model organism for terrestrial holobiont ecology. This perspective can improve our understanding of the temporal and spatial aspects of biological control efficacy., (Copyright © 2020 Yergaliyev et al.)

Root-knot nematode (Meloidogyne incognita) is chief plant parasitic nematode of various crops globally. Meanwhile, the negative side effects on human health and environmental concerns associated with haphazard uses of chemical nematicides. Hence, the search for a safe and effective approach is more relevant. The present study was aimed to evaluate the nematicidal potential of Snef1216 (Penicillium chrysogenum) against M. incognita at different concentrations (5%, 10%, 25%, 50%, 75% and 100%) and with the nutritious medium. The egg hatching inhibition and mortality of second stage juveniles of M. incognita were assessed after 6, 12, 24, 48 and 72 h exposure. Results revealed that egg hatching inhibition and percent mortality of M. incognita increased with increasing concentration and exposure time. The highest mortality of juveniles was recorded at 100% conc. i.e., 24.20%, 36%, 66%, 78% and 97.8% at 6, 12, 24, 48 and 72 h, respectively. The highest ovicidal activity was recorded at 100% concentration with 5.20% of eggs hatching. The outcome suggested that Snef1216 (P. chrysogenum) resulted in the lowest LC 50 value was recorded as 3718.496 at 6 h exposure period followed by 10479.87, 11186.682, 14838.58 and 24001.430 at 72, 12, 48 and 24 h respectively via ovicidal assay. Whereas, in the larvicidal assay, the lowest LC 50 value demonstrated at 72 h being 17.628% exposure period followed by 28.345, 50.490, 215.710 and 482.595% at 48, 24, 12 and 6 h respectively. It is concluded that Snef1216 has potential being used as a biocontrol agent against M. incognita and can serve as a source of a novel nematicidal agent of fungal origin.

The root-knot nematode Meloidogyne incognita has a wide host range and it is one of the most economically important crop parasites worldwide. Biological control has been a good approach for reducing M. incognita infection, for which many nematophagous fungi are reportedly applicable. However, the controlling effects of Phanerochaete chrysosporium strain B-22 are still unclear. In the present study we characterized the parasitism of this strain on M. incognita eggs, second-stage juveniles (J2), and adult females. The highest corrected mortality was 71.9% at 3 × 108 colony forming units (CFU) mL-1 and the estimated median lethal concentration of the fungus was 0.96 × 108 CFU mL-1. Two days after treatment with Phanerochaete chrysosporium strain B-22 eggshells were dissolved. A strong lethal effect was noted against J2, as the fungal spores developed in their body walls, germinated, and the resulting hyphae crossed the juvenile cuticle to dissolve it, thereby causing shrinkage and deformation of the juvenile body wall. The spores and hyphae also attacked adult females, causing the shrinkage and dissolution of their bodies and leakage of contents after five days. Greenhouse experiments revealed that different concentrations of the fungal spores effectively controlled M. incognita. In the roots, the highest inhibition rate for adult females, juveniles, egg mass, and gall index was 84.61%, 78.91%, 84.25%, and 79.48%, respectively. The highest juvenile inhibition rate was 89.18% in the soil. Phanerochaete chrysosporium strain B-22 also improved tomato plant growth, therefore being safe for tomato plants while effectively parasitizing M. incognita. This strain is thus a promising biocontrol agent against M. incognita., Competing Interests: The authors declare no conflict of interests.

Background: The soybean cyst nematode Heterodera glycines (Ichinohe) is the most devastating pathogen affecting soybean production worldwide. Biocontrol agents have become eco-friendly candidates to control pathogens. The aim of this study was to discover novel biocontrol agents against H. glycines., Results: Microbacterium maritypicum Sneb159, screened from 804 strains, effectively reduced the number of females in field experiments conducted in 2014 and 2015. The stability and efficiency of H. glycines control by Sneb159 was further assessed in growth chamber and field experiments. Sneb159 decreased H. glycines population densities, especially the number of females by 43.9%-67.7%. To confirm Sneb159 induced plant resistance, a split-root assay was conducted. Sneb159 induced local and systemic resistance to suppress the penetration and development of H. glycines, and enhanced the gene expression of PR2, PR3b, and JAZ1, involved in the salicylic acid and jasmonic acid pathways., Conclusion: This is the first report of M. maritypicum Sneb159 suppressing H. glycines infection. This effect may be the result of Sneb159-induced resistance. Our study indicates that M. maritypicum Sneb159 is a promising biocontrol agent against H. glycines. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Plant-parasitic nematodes are associated with specifically attached soil bacteria. To investigate these bacteria, we employed culture-dependent methods to isolate a representative set of strains from the cuticle of the infective stage (J2) of the root-knot nematode Meloidogyne hapla in different soils. The bacteria with the highest affinity to attach to J2 belonged to the genera Microbacterium, Sphingopyxis, Brevundimonas, Acinetobacter, and Micrococcus as revealed by 16S rRNA gene sequencing. Dynamics of the attachment of two strains showed fast adhesion in less than two hours, and interspecific competition for attachment sites. Isolates from the cuticle of M. hapla J2 attached to the lesion nematode Pratylenchus penetrans, and vice versa, suggesting similar attachment sites on both species. Removal of the surface coat by treatment of J2 with the cationic detergent CTAB reduced bacterial attachment, but did not prevent it. Some of the best attaching bacteria impaired M. hapla performance in vitro by significantly affecting J2 mortality, J2 motility and egg hatch. Most of the tested bacterial attachers significantly reduced the invasion of J2 into tomato roots, suggesting their beneficial role in soil suppressiveness against M. hapla.

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.

Root-knot nematodes (RKN) such as Meloidogyne spp. are among the most detrimental pests in agriculture affecting several crops. New methodologies to manage RKN are needed such as efficient discovery of nematophagous microbes. In this study, we developed an in vitro high-throughput method relying on the free-living nematode Caenorhabditis elegans and the infection of those nematodes with a soil slurry containing a microbiome likely to house nematophagous microbes. Nematodes were monitored for presence of infection and sub-cultured repeatedly for the purpose of isolating pure cultures of the microbe responsible for conferring the nematicidal activity. Once soil microbes were confirmed to be antagonistic to C. elegans, they were tested for pathogenicity against Meloidogyne chitwoodi. Using this methodology, the fungal isolate Mortierella globalpina was confirmed to be pathogenic in vitro against M. chitwoodi by nematode trapping via hyphal adhesion to the cuticle layer, penetration of the cuticle layer, and subsequently digestion of its cellular contents. M. globalpina was also observed to reduce disease symptomology of RKNs in vivo via significant reduction of root-galls on tomato (Solanum lycopersicum var. Rutgers).

The objective of this study was to determine the effects of ILeVO (fluopyram) and VOTiVO (Bacillus firmus I-1582) seed treatments on Heterodera glycines second-stage juvenile (J2) root penetration and behavior. In a growth chamber experiment, roots of soybeans grown from treated or untreated seeds were inoculated with H. glycines J2s at soil depths of 2.5, 5, or 7.5 cm. ILeVO significantly reduced H. glycines root penetration compared with the untreated control, but only when J2s were inoculated at a soil depth of 2.5 cm, which was near the seed. Changes in nematode behavior were assessed by collecting 60-s videos of J2s after 2 h of exposure to exudates from treated seeds or radicles from treated seeds or from soil leachates in which treated seeds were planted. X- and y-coordinates of each of the 13 reference points were recorded every hour for 24 h. A custom program analyzed and transformed the coordinates into nematode motion parameters (speed and total change in curvature). ILeVO, but not VOTiVO, seed exudates significantly reduced J2 speed relative to the untreated control. Soil leachates from ILeVO or VOTiVO treatments had no consistent effect on H. glycines speed or total change in curvature compared with the untreated control. In another experiment, treated or untreated seeds were incubated in wells of 6-well tissue culture plates containing 11.5% Pluronic gel. Seeds were removed after 2 h, and approximately 50 J2s then were pipetted into each well. The plates were scanned every 60 min for 24 h, and the number of J2s in each well that moved a minimum distance of ≥300 µm was determined using another custom software program. ILeVO, but not VOTiVO, significantly reduced the movement of J2 populations relative to control wells in which no seeds were added. And wells that had seeds, treated or not, yielded significantly less J2 movement compared with the no-seed control. The results of these experiments indicate that ILeVO reduces activity on H. glycines J2s but may not affect nematodes beyond a limited area surrounding the treated seed.

Pasteuria penetrans is an endospore forming hyperparasitic bacterium of the plant-pathogenic root-knot nematode, Meloidogyne incognita. For successful parasitization, the first step is adherence of bacterial endospores onto the cuticle surface of nematode juveniles. The knowledge of molecular intricacies involved during this adherence is sparse. Here, we identified a M. incognita selenium-binding protein (Mi-SeBP-1) differentially expressed during the initial interaction of M. incognita and P. penetrans, and show that it is involved in modulating parasitic adhesion of bacterial endospores onto nematode cuticle. Selenium-binding proteins (SeBPs) are selenium associated proteins important for growth regulation, tumor prevention and modulation of oxidation/reduction in cells. Although reported to be present in several nematodes, the function of SeBPs is not known in Phylum Nematoda. In situ hybridization assay localized the Mi-SeBP-1 mRNA to the hypodermal cells. RNAi-mediated silencing of Mi-SeBP-1 significantly increased the adherence of P. penetrans endospores to the nematode juvenile cuticle. Silencing of Mi-SeBP-1 did not change the nematode's ability to parasitize plants and reproduction potential within the host. These results suggest that M. incognita Mi-SeBP-1 might be involved in altering the attachment of microbial pathogens on the nematode cuticle, but is not involved in nematode-host plant interaction. This is the first report for a function of SeBP in Phylum Nematoda., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment., Results: A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle., Conclusions: Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.

Mucins are highly glycosylated polypeptides involved in many host-parasite interactions, but their function in plant-parasitic nematodes is still unknown. In this study, a mucin-like gene was cloned from Meloidogyne incognita (Mi-muc-1, 1125 bp) and characterized. The protein was found to be rich in serine and threonine with numerous O-glycosylation sites in the sequence. Quantitative real-time polymerase chain reaction (qRT-PCR) showed the highest expression in the adult female and in situ hybridization revealed the localization of Mi-muc-1 mRNA expression in the tail area in the region of the phasmid. Knockdown of Mi-muc-1 revealed a dual role: (1) immunologically, there was a significant decrease in attachment of Pasteuria penetrans endospores and a reduction in binding assays with human red blood cells (RBCs), suggesting that Mi-MUC-1 is a glycoprotein present on the surface coat of infective second-stage juveniles (J2s) and is involved in cellular adhesion to the cuticle of infective J2s; pretreatment of J2s with different carbohydrates indicated that the RBCs bind to J2 cuticle receptors different from those involved in the interaction of Pasteuria endospores with Mi-MUC-1; (2) the long-term effect of RNA interference (RNAi)-mediated knockdown of Mi-muc-1 led to a significant reduction in nematode fecundity, suggesting a possible function for this mucin as a mediator in the interaction between the nematode and the host plant., (© 2018 The Authors Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

In the course of screening for microbes with nematicidal activity, we found that Enterobacter asburiae HK169 displayed promising nematicidal activity against the root-knot nematode Meloidogyne incognita , along with plant growth-promoting properties. Soil drenching of a culture of HK169 reduced gall formation by 66% while also increasing root and shoot weights by 251% and 160%, respectively, compared with an untreated control. The cell-free culture filtrate of the HK169 culture killed all juveniles of M. incognita within 48 h. In addition, the nematicidal activity of the culture filtrate was dramatically reduced by a protease inhibitor, suggesting that proteolytic enzymes contribute to the nematicidal activity of HK169. In order to obtain genomic information about the HK169 isolate related to its nematicidal and plant growth-promoting activities, we sequenced and analyzed the whole genome of the HK169 isolate, and the resulting information provided evidence that the HK169 isolate has nematicidal and plant growth-promoting activities. Taken together, these observations enable the future application of E. asburiae HK169 as a biocontrol agent for nematode control and promote our understanding of the beneficial interactions between E. asburiae HK169 and plants.

The entomopathogenic fungus Lecanicillium muscarium (Petch) Zare and Gams is currently being developed as a biocontrol agent against insect pests, as well as some plant-pathogenic fungi and bacteria. Data about its activity against plant-parasitic nematodes exist, but are relatively limited. To expand this understanding, we investigated the biocontrol efficiency of three isolates of L. muscarium (Lm) against the root knot nematode, Meloidogyne incognita, in both in vitro and in vivo conditions. In our experiments, the maximum number of nematode eggs, juveniles (J2s), females, and egg masses that were parasitized were quantified after a 72-h exposure to the fungus. The isolate Lm1 was designated as the best biocontrol agent against nematode eggs as well as J2s. It showed the highest colonization of eggs and significantly decreased egg hatching events. The results from two additional isolates, Lm2 and Lm3, were also significant (P = 0.05) but less pronounced than those observed with Lm1. L. muscarium treatments had significant (P = 0.05) positive effects on plant shoot and root growth compared with the growth of control plants. These results suggest the effectiveness of the fungus may be due to either the infection of eggs and J2s, or the production of secondary metabolites that induced plant defense mechanisms and lead to systemic resistance. Our study demonstrates that L. muscarium could be used as a potential biocontrol agent against root knot nematodes.

Fungal root endophytes, including the common group of dark septate endophytes (DSEs), represent different taxonomic groups and potentially diverse life strategies. In this study, we investigated two unidentified helotialean lineages found previously in a study of DSE fungi of semiarid grasslands, from several other sites, and collected recently from a pezizalean truffle ascoma and eggs of the cereal cyst nematode Heterodera filipjevi. The taxonomic positions and phylogenetic relationships of 21 isolates with different hosts and geographic origins were studied in detail. Four loci, namely, nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]), partial 28S nuc rDNA (28S), partial 18S nuc rDNA (18S), and partial RNA polymerase II second-largest subunit (RPB2), were amplified and sequenced for molecular phylogenetic analyses. Analyses of similar ITS sequences from public databases revealed two globally distributed lineages detected in several biomes from different geographic regions. The host interaction of isolates from nematodes was examined using in vitro bioassays, which revealed that the fungi could penetrate nematode cysts and colonize eggs of H. filipjevi, confirming observations from field-collected samples. This is the first report of a DSE, and we are not aware of other helotialean fungal species colonizing the eggs of a plant-parasitic nematode. Neither conidiomata and conidia nor ascomata formation was detected in any of the isolates. Based on molecular phylogenetic analyses, these isolates represent a distinct lineage within the Helotiales in the Hyaloscyphaceae. For this lineage, we propose here the new genus Polyphilus represented by two new species, P. sieberi and P. frankenii.

Plant Growth Promoting Rhizobacteria (PGPR) have different mechanisms of action in the development of plants, such as growth promotion, production of phytohormones and antibiotic substances and changes in root exudates. These help to control plant diseases. In order to evaluate the potential of microorganisms in the control of Meloidogyne javanica and Ditylenchus spp., five rhizobacteria isolated from rhizosphere of garlic cultivated in the Curitibanos (SC) region were tested. Hatching chambers were set on Petri dishes, in which were added 10mL of bacterial suspension and 1mL of M. javanica eggs suspension, at the rate of 4500, on the filter paper of each chamber. The same procedure was performed with 300 juvenile Ditylenchus spp. The experimental design was completely randomized, with four replications. The evaluations were performed every 72h for nine days. The antagonized population of nematodes was determined in Peters counting chamber, determining the percentage hatching (for M. javanica) and motility (for Ditylenchus spp). Isolates CBSAL02 and CBSAL05 significantly reduced the hatching of M. javanica eggs (74% and 54.77%, respectively) and the motility of Ditylenchus spp. (55.19% and 53.53%, respectively) in vitro. Isolates were identified as belonging to the genera Pseudomonas (CBSAL05) and Bacillus (CBSAL02)., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Chitin is the second most abundant biopolymer after cellulose and virtually unexplored as raw material for bioethanol production. In this paper, we investigate chitosan, the deacetylated form of chitin which is the main component of shellfish waste, as substrate for bioethanol production by fungi. Fungal parasites of invertebrates such as the nematophagous Pochonia chlamydosporia (Pc) or the entomopathogens Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) are biocontrol agents of plant parasitic nematodes (eg. Meloidogyne spp.) or insect pests such as the red palm weevil (Rhynchophorus ferrugineus). These fungi degrade chitin-rich barriers for host penetration. We have therefore tested the chitin/chitosanolytic capabilities of Pc, Bb and Ma for generating reducing sugars using chitosan as only nutrient. Among the microorganisms used in this study, Pc is the best chitosan degrader, even under anaerobic conditions. These fungi have alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) encoding genes in their genomes. We have therefore analyzed their ethanol production under anaerobic conditions using chitosan as raw material. P. chlamydosporia is the largest ethanol producer from chitosan. Our studies are a starting point to develop chitin-chitosan based biofuels., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Heterodera glycines, the soybean cyst nematode, is the most economically important plant-parasitic nematode on soybean production in the U.S. The objectives of this study were to evaluate the potential of plant growth-promoting rhizobacteria (PGPR) strains for mortality of H. glycines J2 in vitro and for reducing nematode population density on soybean in greenhouse, microplot, and field trials. The major group causing mortality to H. glycines in vitro was the genus Bacillus that consisted of 92.6% of the total 663 PGPR strains evaluated. The subsequent greenhouse, microplot, and field trials indicated that B. velezensis strain Bve2 consistently reduced H. glycines cyst population density at 60 DAP. Bacillus mojavensis strain Bmo3 suppressed H. glycines cyst and total H. glycines population density under greenhouse conditions. Bacillus safensis strain Bsa27 and Mixture 1 (Bve2 + Bal13) reduced H. glycines cyst population density at 60 DAP in the field trials. Bacillus subtilis subsp. subtilis strains Bsssu2 and Bsssu3, and B. velezensis strain Bve12 increased early soybean growth including plant height and plant biomass in the greenhouse trials. Bacillus altitudinis strain Bal13 increased early plant growth on soybean in the greenhouse and microplot trials. Mixture 2 (Abamectin + Bve2 + Bal13) increased early plant growth in the microplot trials at 60 DAP, and also enhanced soybean yield at harvest in the field trials. These results demonstrated that individual PGPR strains and mixtures can reduce H. glycines population density in the greenhouse, microplot, and field conditions, and increased yield of soybean.

Cyst nematodes are globally important pathogens in agriculture. Their sedentary lifestyle and long-term association with the roots of host plants render cyst nematodes especially good targets for attack by parasitic fungi. In this context fungi were specifically isolated from nematode eggs of the cereal cyst nematode Heterodera filipjevi. Here, Ijuhya vitellina (Ascomycota, Hypocreales, Bionectriaceae), encountered in wheat fields in Turkey, is newly described on the basis of phylogenetic analyses, morphological characters and life-style related inferences. The species destructively parasitises eggs inside cysts of H. filipjevi. The parasitism was reproduced in in vitro studies. Infected eggs were found to harbour microsclerotia produced by I. vitellina that resemble long-term survival structures also known from other ascomycetes. Microsclerotia were also formed by this species in pure cultures obtained from both, solitarily isolated infected eggs obtained from fields and artificially infected eggs. Hyphae penetrating the eggshell colonised the interior of eggs and became transformed into multicellular, chlamydospore-like structures that developed into microsclerotia. When isolated on artificial media, microsclerotia germinated to produce multiple emerging hyphae. The specific nature of morphological structures produced by I. vitellina inside nematode eggs is interpreted as a unique mode of interaction allowing long-term survival of the fungus inside nematode cysts that are known to survive periods of drought or other harsh environmental conditions. Generic classification of the new species is based on molecular phylogenetic inferences using five different gene regions. I. vitellina is the only species of the genus known to parasitise nematodes and produce microsclerotia. Metabolomic analyses revealed that within the Ijuhya species studied here, only I. vitellina produces chaetoglobosin A and its derivate 19-O-acetylchaetoglobosin A. Nematicidal and nematode-inhibiting activities of these compounds have been demonstrated suggesting that the production of these compounds may represent an adaptation to nematode parasitism.

Microbial interference plays an imperative role in plant development and response to various stresses. However, its involvement in mitigation of oxidative stress generated by plant parasitic nematode in plants remains elusive. In the present investigation, the efficacy of microbe's viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 single and in combinations was examined to mitigate oxidative stress generated by M. incognita in medicinal plant, Bacopa monnieri. Microbial combination with and without pathogen also enhanced the growth parameters along with secondary metabolites (bacoside) of B. monnieri than the pathogen inoculated control. The study showed that initially the production of hydrogen peroxide (H 2 O 2 ) was higher in dual microbes infected with pathogen which further declined over M. incognita inoculated control plants. Superoxide dismutase and free radical scavenging activity were also highest in the same treatment which was linearly related with least lipid peroxidation and root gall formation in B. monnieri under the biotic stress. Microscopic visualization of total reactive oxygen species (ROS), H 2 O 2 , superoxide radical and programmed cell death in host plant further extended our knowledge and corroborated well with the above findings. Furthermore, scanning electron microscopy confirmed good microbial colonization on the host root surface around nematode penetration sites in plants treated with dual microbes under pathogenic stress. The findings offer novel insight into the mechanism adopted by the synergistic microbial strains in mitigating oxidative stress and simultaneously stimulating bacoside production under pathogenic stress., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Seven filamentous fungal species were isolated from individual eggs of Globodera pallida cysts collected from infested fields in Shelley Idaho, USA and identified as Chaetomium globosum, Fusarium oxysporum, Fusarium solani, Fusarium tricinctum, Microdochium bolleyi, Purpureocillium lilacinum, and Plectosphaerella cucumerina. Their ability to reduce infection by G. pallida in planta were assessed in simple, reproducible micro-rhizosphere chambers (micro-ROCs). All fungi reduced G. pallida infection in potato, but greatest reduction was observed with C. globosum at an average reduction of 76%. Further non-destructive methods were developed to rapidly assess biological control potential of putative fungal strains by staining the infectious second stage juveniles of G. pallida with the live fluorescent stain PKH26. In comparisons between the standard, invasive acid fuchsin method and use of the live stain PKH26, no significant difference in infection level of G. pallida was observed whether roots were stained with PKH26 or acid fuchsin. For both methods, a similar reduction (77% for acid fuchsin, and 78% for PKH26 stain) in invasion of infectious stage of G. pallida was observed when potato plants were inoculated with C. globosum compared to non-inoculated potato., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: To generate single spore lines of a population of bacterial parasite of root-knot nematode (RKN), Pasteuria penetrans, isolated from Florida and examine genotypic variation and virulence characteristics exist within the population., Methods and Results: Six single spore lines (SSP), 16SSP, 17SSP, 18SSP, 25SSP, 26SSP and 30SSP were generated. Genetic variability was evaluated by comparing single-nucleotide polymorphisms (SNPs) in six protein-coding genes and the 16S rRNA gene. An average of one SNP was observed for every 69 bp in the 16S rRNA, whereas no SNPs were observed in the protein-coding sequences. Hierarchical cluster analysis of 16S rRNA sequences placed the clones into three distinct clades. Bio-efficacy analysis revealed significant heterogeneity in the level virulence and host specificity between the individual clones., Conclusions: The SNP markers developed to the 5' hypervariable region of the 16S rRNA gene may be useful in biotype differentiation within a population of P. penetrans., Significance and Impact of the Study: This study demonstrates an efficient method for generating single spore lines of P. penetrans and gives a deep insight into genetic heterogeneity and varying level of virulence exists within a population parasitizing a specific Meloidogyne sp. host. The results also suggest that the application of generalist spore lines in nematode management may achieve broad RKN control., (© 2016 The Society for Applied Microbiology.)

Pochonia chlamydosporia is a soil fungus with a multitrophic lifestyle combining endophytic and saprophytic behaviors, in addition to a nematophagous activity directed against eggs of root-knot and other plant parasitic nematodes. The carbohydrate-active enzymes encoded by the genome of P. chlamydosporia suggest that the endophytic and saprophytic lifestyles make use of a plant cell wall polysaccharide degradation machinery that can target cellulose, xylan and, to a lesser extent, pectin. This enzymatic machinery is completed by a chitin breakdown system that involves not only chitinases, but also chitin deacetylases and a large number of chitosanases. P. chlamydosporia can degrade and grow on chitin and is particularly efficient on chitosan. The relevance of chitosan breakdown during nematode egg infection is supported by the immunolocalization of chitosan in Meloidogyne javanica eggs infected by P. chlamydosporia and by the fact that the fungus expresses chitosanase and chitin deacetylase genes during egg infection. This suggests that these enzymes are important for the nematophagous activity of the fungus and they are targets for improving the capabilities of P. chlamydosporia as a biocontrol agent in agriculture., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The present work demonstrated the nematicidal potential of Streptomyces hydrogenans strain DH16 (a strain with strong antagonism against fungal phytopathogens and insect pest) against Meloidogyne incognita. The culture supernatant and solvent extract significantly inhibited egg hatching (almost 100%) along with J2 mortality of more than 95% after 96h. The nematicidal activity of 10-(2,2-dimethyl-cyclohexyl)-6,9-dihydroxy-4,9-dimethyl-dec-2-enoic acid methyl ester (SH2; a new antifungal compound) purified from this streptomycete was also evaluated using different concentrations. The juvenile mortality of the nematode increased with increasing concentration and exposure time and reached the maximum (95%) after 96h at concentration of 100μg/ml. After 160h of incubation, egg hatch of 16% was observed at concentration of 100μg/ml as compared to control where 100% egg hatching was achieved. However, at the highest concentration of the compound (200μg/ml), 100% J2 mortality and 0% egg hatching were observed after 72 and 160h of incubation, respectively. In vivo pot experiments further revealed the nematicidal potential of S. hydrogenans where soil drenching with its culture supernatant and cells effectively controlled root galls, egg masses in nematode infested tomato plants and at the same time promoted the growth of tomato plants. Additionally, in the absence of nematodes, soil drenching with culture supernatant and cells significantly enhanced the various agronomic traits of plants as compared to control plants. Thus, the outcomes of the current study endorse the potential of S. hydrogenans strain DH16 and its metabolites to be developed as safe nematicidal and plant growth promoting agents., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Hirsutella rhossiliensis is a parasite of juvenile nematodes, effective against a diversity of plant-parasitic nematodes. Its global distribution on various nematode hosts and its genetic variation for several geographic regions have been reported, while the global population genetic structure and factors underlying patterns of genetic variation of H. rhossiliensis are unclear. In this study, 87 H. rhossiliensis strains from five nematode species (Globodera sp., Criconemella xenoplax, Rotylenchus robustus, Heterodera schachtii, and Heterodera glycines) in Europe, the United States, and China were investigated by multilocus sequence analyses. A total of 280 variable sites (frequency, 0.6%) at eight loci and six clustering in high accordance with geographic populations or host nematode-associated populations were identified. Although H. rhossiliensis is currently recognized as an asexual fungus, recombination events were frequently detected. In addition, significant genetic isolation by geography and nematode hosts was revealed. Overall, our analyses showed that recombination, geographic isolation, and nematode host adaptation have played significant roles in the evolutionary history of H. rhossiliensis IMPORTANCE: H. rhossiliensis has great potential for use as a biocontrol agent to control nematodes in a sustainable manner as an endoparasitic fungus. Therefore, this study has important implications for the use of H. rhossiliensis as a biocontrol agent and provides interesting insights into the biology of this species., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Plant-parasitic nematodes cause serious crop losses worldwidely. This study intended to discover the antagonistic mechanism of Bacillus cereus strain S2 against Meloidogyne incognita. Treatment with B. cereus strain S2 resulted in a mortality of 77.89% to Caenorhabditis elegans (a model organism) and 90.96% to M. incognita. In pot experiment, control efficiency of B. cereus S2 culture or supernatants were 81.36% and 67.42% towards M. incognita, respectively. In field experiment, control efficiency was 58.97% towards M. incognita. Nematicidal substances were isolated from culture supernatant of B. cereus S2 by polarity gradient extraction, silica gel column chromatography and HPLC. Two nematicidal compounds were identified as C16 sphingosine and phytosphingosine by LC-MS. The median lethal concentration of sphingosine was determined as 0.64 μg/ml. Sphingosine could obviously inhibit reproduction of C. elegans, with an inhibition rate of 42.72% for 24 h. After treatment with sphingosine, ROS was induced in intestinal tract, and genital area disappeared in nematode. Furthermore, B. cereus S2 could induce systemic resistance in tomato, and enhance activity of defense-related enzymes for biocontrol of M. incognita. This study demonstrates the nematicidal activity of B. cereus and its product sphingosine, as well provides a possibility for biocontrol of M. incognita.

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The root knot nematode (RKN), Meloidogyne incognita, belongs to the most damaging plant pathogens worldwide, and is able to infect almost all cultivated plants, like tomato. Recent research supports the hypothesis that bacteria often associated with plant-parasitic nematodes, function as nematode parasites, symbionts, or commensal organisms etc. In this study, we explored the bacterial consortia associated with M. incognita at different developmental stages, including egg mass, adult female and second-stage juvenile using the pyrosequencing approach. The results showed that Proteobacteria, with a proportion of 71-84%, is the most abundant phylum associated with M. incognita in infected tomato roots, followed by Actinobacteria, Bacteroidetes, Firmicutes etc. Egg mass, female and second-stage juvenile of M. incognita harbored a core microbiome with minor difference in communities and diversities. Several bacteria genera identified in M. incognita are recognized cellulosic microorganisms, pathogenic bacteria, nitrogen-fixing bacteria and antagonists to M. incognita. Some genera previously identified in other plant-parasitic nematodes were also found in tomato RKNs. The potential biological control microorganisms, including the known bacterial pathogens and nematode antagonists, such as Actinomycetes and Pseudomonas, showed the largest diversity and proportion in egg mass, and dramatically decreased in second-stage juvenile and female of M. incognita. This is the first comprehensive report of bacterial flora associated with the RKN identified by pyrosequencing-based analysis. The results provide valuable information for understanding nematode-microbiota interactions and may be helpful in the development of novel nematode-control strategies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, understanding the role of phospholipases on the virulence of the filamentous fungus Purpureocillium lilacinum has been still rather limited. In this study, pld gene was characterized. It encodes the protein phospholipase D (PLD) in P. lilacinum. This gene, 3303 bp open reading frame fragment (ORF), encodes a protein of 1100 amino acids with high similarity to the same gene from Penicillium oxalicum and Aspergillus fumigatus. Secondary structure prediction showed two PLD phosphodiesterase domains (437-464 bp and 885-912 bp). The pld gene was significantly regulated during infection of Meloidogyne incognita eggs by P. lilacinum. The expression of pld gene using RT-PCR was the highest at 36 and 48 h, which introduce evidence that the presence of M. incognita may induce the expression of the pld gene in P. lilacinum. In addition, maltose and l-alanine were found to increase the expression of pld gene. An acidic environment (pH 3.0-4.0) and moderate temperatures (27-29 °C) are favorable for pld expression in P. lilacinum., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A strain of marine bacterium Bacillus firmus YBf-10 with nematicidal activity was originally isolated by our group. In the present study, the systemic nematicidal activity and biocontrol efficacy in pot experiment of B. firmus YBf-10 were investigated. Our results showed that YBf-10 exhibits systemic nematicidal activity against Meloidogyne incognita, including lethal activity, inhibition of egg hatch and motility. Pot experiment suggested that soil drenching with YBf-10 efficiently reduced damage of M. incognita to tomato plants, such as reduction of galls, egg masses on roots, and final nematode population in soil; and moreover, YBf-10 significantly promoted host plant growth. In addition, our results also indicated that the systemic nematicidal activity is likely attributed to the secondary metabolites produced by YBf-10. The obtained results of the current study confirmed that B. firmus YBf-10 is a promising nematicidal agent, and has great potential in plant-parasitic nematicidal management.

Acremonium implicatum is an endophytic fungus with biocontrol potential against Meloidogyne incognita based on its opportunistic egg-parasitic, hatching inhibition, and toxic properties. To understand its mode of plant endophytism and opportunistic egg parasitism, GFP-tagged A. implicatum was constructed by PEG-mediated protoplast transformation. By laser scanning confocal microscopy (LSCM), we evaluated the endophytism and opportunistic egg parasitism of a stable gfp transformant (Acr-1). Acr-1 could colonize epidermal tissue, cortical tissue, and xylem of roots and form a mutualistic symbiosis with tomato host plants. LSCM of Acr-1 infecting M. incognita eggs revealed that hyphae penetrated the shell and grew inside eggs to form trophic hyphae. A large number of hyphae enveloped parasitized eggs. In addition, the egg shell integrity was destroyed by fungal penetration. The percentage of egg parasitism was 33.8 %. There were no marked differences between the wild type and mutant in nematode second-stage juvenile mortality and egg hatching and in fungal control efficiency in a pot experiment. In conclusion, gfp-transformation did not change the nematicidal activity of A. implicatum and is a tool to examine the mode of plant endophytism and opportunistic egg parasitism of A. implicatum.

The feasibility of converting food waste into nematocidal biofertilizer by nematophagous fungus Paecilomyces lilacinus (P. lilacinus) was investigated. The culture conditions of P. lilacinus were optimized through response surface methodology. Results showed that fermentation time, the amount of food waste, initial pH and temperature were most important factors for P. lilacinus production. The P. lilacinus production under optimized conditions was 10(9.6 ± 0.3) conidia mL⁻¹. After fermentation, the chemical oxygen demand concentration of food waste was efficiently decreased by 81.92%. Moreover, the property evaluation of the resultant food waste as biofertilizer indicates its high quality with reference to the standard released by the Chinese Ministry of Agriculture. The protease activity and nematocidal ability of P. lilacinus cultured by food waste were 10.8% and 27% higher than those by potato dextrose agar, respectively.

Lysobacter capsici YS1215 isolated from soil previously showed nematicidal potential for biological control of the root-knot nematode. In this study, lactic acid, a nematicidal compound, was isolated from culture filtrate of YS1215, and its ovicidal activity was investigated. Purification and identification of lactic acid were performed by a series of column chromatographies and identified by (1)H and (13)C NMR spectra and GC-MS analysis. Our results showed that bacterial culture filtrate containing lactic acid significantly inhibited egg hatching. The lowest egg hatch rate (5.9%) was found at a high concentration (25 μl/ml) of lactic acid at 5 days after incubation, followed by 20 (15.2%), 15 (23.7%), 10 (29.8%), and 5 (36.4%) μl/ml, while egg hatching in the control (sterile distilled water) was 44.5%. This is the first report of lactic acid as an ovicidal compound, and it may be considered as an alternative of chemical pesticide against root-knot nematodes.

Objective: To evaluate the potential of Trichoderma longibrachiatum spore suspension against Heterodera avenae., Methods: The parasitic and lethal effects of T. longibrachiatum spore suspension against the cysts of H. avenae were studied in vitro and observed under microscope., Results: Microscopic observation showed that the spore suspension of T. longibrachiatum parasitized on the cyst surface, germinated a large number of hyphae, and grew on the surface of the cyst at the initial stage. Later, the cysts were completely surrounded by dense mycelium, and the contents of digestion in cysts was lysed, even some cysts produced vacuoles, and some were split up and finally the cyst was dissolved by the metabolite of T. longibrachiatum. In vitro studies showed that high concentrations of T. longibrachiatum spores had strong parasitic and lethal effects on the cysts of H. avenae, and the probable mechanism of parasitic and lethal effects of T. longibrachiatum against H. avenae were mainly by inducing and increasing chitinase, glucanase and caseinase activity. The cysts were parasitized by 93.3% at 18 days, the hatching of cysts were inhibited by 93.6% at 10 days when treated with the concentrations (1.5 x 10(8) CFU/mL) of T. longibrachiatum., Conclusion: Trichoderma longibrachiatum had strong parasitic and lethal effects on the cysts of H. avenae, and has the potential as a new biocontrol agent.

The lethal effect of the conidia suspension of Trichoderma longibrachiatum against second stage juveniles of Heterodera avenae was observed under microscopic conditions and studied in vitro to preliminarily determine the potential and mechanism of the conidia suspension of T. longibrachiatum against H. avenae. Microscopic observation results showed that the conidia suspension of T. longibrachiatum adhered to or parasitized on the surface of second stage juveniles, even some parasitized nematodes were deformed at the initial stage. Later, the second stage juveniles were wrapped and the integuments were penetrated by a large number of hyphae germinated from the conidia suspension of T. longibrachiatum. Even the body of dead second stage juveniles was completely lysed. In vitro studies showed that different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum (conidia suspension) had strong lethal and parasitic effects on the second stage juveniles of H. avenae, and significant differences existed among the treatments with different concentrations of T. longibrachiatum. In addition, the lethal and parasitic effects increased with increasing the T. longibrachiatum concentration. The mortality and corrected mortality of the second stage juveniles treated with the concentrations of 1.5 x 10(7) cfu x mL(-1) were 91.3% and 90.4% after 72 hours, and the second stage juveniles were parasitized by 88.7% after 14 days. Therefore, the conidia suspension of T. longibrachiatum had a great lethal effect on H. avenae, and the strain could be considered as a potential bio-control agent against H. avenae.

Understanding the interactions of plant-parasitic nematodes with antagonistic soil microbes could provide opportunities for novel crop protection strategies. Three arable soils were investigated for their suppressiveness against the root knot nematode Meloidogyne hapla. For all three soils, M. hapla developed significantly fewer galls, egg masses, and eggs on tomato plants in unsterilized than in sterilized infested soil. Egg numbers were reduced by up to 93%. This suggested suppression by soil microbial communities. The soils significantly differed in the composition of microbial communities and in the suppressiveness to M. hapla. To identify microorganisms interacting with M. hapla in soil, second-stage juveniles (J2) baited in the test soil were cultivation independently analyzed for attached microbes. PCR-denaturing gradient gel electrophoresis of fungal ITS or 16S rRNA genes of bacteria and bacterial groups from nematode and soil samples was performed, and DNA sequences from J2-associated bands were determined. The fingerprints showed many species that were abundant on J2 but not in the surrounding soil, especially in fungal profiles. Fungi associated with J2 from all three soils were related to the genera Davidiella and Rhizophydium, while the genera Eurotium, Ganoderma, and Cylindrocarpon were specific for the most suppressive soil. Among the 20 highly abundant operational taxonomic units of bacteria specific for J2 in suppressive soil, six were closely related to infectious species such as Shigella spp., whereas the most abundant were Malikia spinosa and Rothia amarae, as determined by 16S rRNA amplicon pyrosequencing. In conclusion, a diverse microflora specifically adhered to J2 of M. hapla in soil and presumably affected female fecundity.

A laboratory experiment was conducted to study the parasitic and lethal effects of Trichoderma longibrachiatum conidia suspension on Heterodera avenae cysts. Different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum conidia suspension had strong parasitic and lethal effects on H. avenae cysts, and the effects differed significantly among the different concentrations. When treated with the T. longibrachiatum conidia suspension at a concentration of 1.5 x 10(7) cfu x mL(-1), 96.7% of the H. avenae cysts were parasitized by the conidia at the 18th day, and the hatching rate of the cysts was inhibited by 91.2% at the 22nd day. The microscopic observation showed that at the initial parasitic stage, T. longibrachiatum conidia suspension adhered or parasitized on the cyst surface, germinated a large number of hyphae, and grew on the cyst surface, making the development of cyst embryo stopped and the contents in cysts flocculated, and even, some cysts started to deform, and small dark brown vacuoles formed on the cyst surface. At the later parasitic stage, the cysts were penetrated by dense mycelium, cysts were broken, their contents exosmosed, and the mycelium on the integument of some cysts produced conidiophores, on which, conidium were adhered or parasitized. It was considered that T. longibrachiatum could be used as a potential high-efficient bioagent to control the occurrence and damage of H. avenae.

The fungi Hirsutella rhossiliensis and Hirsutella minnesotensis generally parasitize only plant-parasitic nematodes in nature but parasitize the bacterivorous nematode Caenorhabditis elegans on agar plates. To establish a model system for studying the interaction between fungi and nematodes, we compared the parasitism of the first- to fourth-stage larvae (L1-L4) of C. elegans and second-stage juvenile (J2) of Heterodera glycines by twenty isolates of Hirsutella spp. Although parasitism differed substantially among isolates, both H. minnesotensis and H. rhossiliensis parasitized a higher percentage of H. glycines J2s than of C. elegans larvae. Parasitism of C. elegans L1s was correlated with parasitism of H. glycines J2s. Parasitism of C. elegans by H. rhossiliensis and H. minnesotensis was negatively correlated with larva size and motility, i.e., parasitism was higher for the younger stages. The C. elegans L1 is recommended for studying parasitism of nematodes by H. rhossiliensis and H. minnesotensis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The beneficial endophytic fungus Piriformospora indica colonizes the roots of many plant species, including the model plant Arabidopsis thaliana. Its colonization promotes plant growth, development, and seed production as well as resistance to various biotic and abiotic stresses. In the present work, P. indica was tested as potential antagonist of the sedentary plant-parasitic nematode Heterodera schachtii. This biotrophic cyst-forming nematode induces severe host plant damage by changing the morphogenesis and physiology of infected roots. Here it is shown that P. indica colonization, as well as the application of fungal exudates and cell-wall extracts, significantly affects the vitality, infectivity, development, and reproduction of H. schachtii.

The study of plant parasitic nematodes such as Meloidogyne spp. and their interactions with phytopathogenic bacteria remains underexplored. One of the challenges towards establishing such interactions is the dependence on symptom development as a measure of interaction. In this study, mCherry was employed as a reporter protein to investigate the interaction between the soft rot Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb) and root-knot nematode (M. incognita). Pectobacterium carotovorum subsp. brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This strain was shown to attach to the surface coat of M.incognita J2 at the optimum temperature of 28°C. This suggests that RKN juveniles may play a role in disseminating Pcb in soils that are heavily infested with Pcb. The presence of RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato tubers potentially acting as a source of latent tuber infections., (© 2013 The Society for Applied Microbiology.)