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Sorghum (Sorghum bicolor) is an important food and feed crop. Root-lesion nematodes (Pratylenchus spp.) are a group of pathogenic nematodes that cause severe economic losses in various food and cash crops. This study identified diseased sorghum plants with stunted growth and brown, rotting roots in sorghum fields in Shanxi Province, China. A species of root-lesion nematode was isolated by modified Baermann funnel method and named the GL-1 population. Afterward, the GL-1 population of root-lesion nematodes was identified as P. coffeae through a combination of morphological, rDNA-ITS and rDNA-28 S D2-D3 region techniques for molecular biological identification. We also conducted greenhouse experiments to assess the parasitism and pathogenicity of GL-1 and four other P. coffeae populations on sorghum through pot inoculation. At 60 days after inoculation, the results indicated that all five populations of P. coffeae were capable of infecting and causing damage to the sorghum plants. Sorghum is a suitable host for P. coffeae (with a reproduction factor > 1). Moreover, compared with those in the control group, the aboveground fresh weights and root fresh weights of sorghum in the five inoculation groups were significantly lower, and brown spots or even necrotic rot appeared on the roots. All five populations were highly pathogenic to sorghum, but there were significant differences in pathogenicity among the populations. This study provides a scientific basis for identifying and detecting root-lesion nematodes in sorghum., Competing Interests: Declarations. Ethics approval and consent to participate: This article does not contain any studies with human participants or animals performed by any of authors. The diseased sorghum plants were sampled with the permission of the landowner. The authors confirm that all the experimental methods and plants complied with relevant institutional, national, and international guidelines and legislation. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The study assessed Trichoderma spp. as a biocontrol agent for managing wilt-nematode complex in tomato crops, aiming to mitigate yield losses. Fusarium sp. and Meloidogyne sp. were isolated from infected plant samples and confirmed molecularly and morphologically as Fusarium oxysporum f.sp. lycopersici and Meloidogyne incognita. Four Trichoderma spp. procured from the Department of Plant Pathology, Tamil Nadu Agricultural University, India were identified molecularly as Trichoderma asperellum (Tv1), Trichoderma asperelloides (Tasd1), Trichoderma harzianum (Th1), and Trichoderma koningiopsis (Tk1) utilizing ITS and TEF1 primer pairs. Among them, Tv1 effectively inhibited the mycelial growth of pathogen isolates. Furthermore, crude metabolite of Tv1 exhibited similar effects. The mortality rate of M. incognita J2s ranged from 90.48% to 100% after 24-72 h of incubation and inhibition percentage of egg hatching reached 90.20%. The shoot length, root length, fresh weight, and dry weight of the tomato plants treated with Tv1 conidia were increased. In a pot experiment, Tv1 treatment reduced disease incidence by 64.57%, comparable to carbendazim treatment (82.32%). Tv1-treated plants had fewer root galls, egg masses, and J2s per 100 g of roots than the inoculated-untreated control plants. The root-knot index (RKI) was significantly lower in plants treated with carbafuran (1.98 ± 0.047) and Tv1 (3.06 ± 0.086) than in control (4.47 ± 0.109). The bio-control efficiency of Tv1 against M. incognita was 21.04%, and the nematodes' reproductive factor (RF) declined to 0.53 in Tv1 treatment group. Based on the findings above, it was established that Tv1 effectively controlled nematode populations and wilt disease when applied in soil., (© 2024 Wiley‐VCH GmbH.)

The soybean cyst nematode (SCN, Heterodera glycines) is the most yield-limiting pathogen in soybeans worldwide. Using chemical pesticides to control this disease is harmful to human and environment. It is urgent to develop environment-friendly nematicides. The aim of this study was to discover novel biocontrol agents on H. glycines control and soybean growth under greenhouse and field conditions Eight Bacillus strains were isolated from soil rhizosphere soils and the stability and efficiency of H. glycines was assessed in greenhouse and field experiments in 2021 and 2022. In particular, the Ba2-6 strain had the highest potential, because it was a biocontrol agent against H. glycines shown to cause 93.85% juvenile mortality. Furthermore, strains Ba 1-7, Ba2-4, and Ba2-6 effectively reduced the number of females and improved the soybean seed number per plant. Based on their morphological, physiological, biochemical and molecular (16 S rRNA) characteristics, the three strains were identified as B. aryabhattai (Ba1-7), B. megatherium (Ba2-4), and B. halotolerans (Ba2-6). The ability of Ba2-6 to induce systemic resistance to H. glycines in soybeans was investigated by the split-root system and real-time quantitative PCR experiments. The results indicated that the Ba2-6 strain induced systemic resistance to suppress the penetration of H. glycines, and enhanced gene expression of PR1, PR3a, PR5, and NPR1-2, involved in the salicylic acid and jasmonic acid pathways. The study suggests that the strains of B. aryabhattai Ba1-7, B. megatherium Ba2-4, and B. halotolerans Ba2-6 can be considered as effective biocontrol agents to control H. glycines. Further, B. halotolerans Ba2-6 not only promotes soybean growth but also enhances resistance to H. glycines by regulating defense-related gene expression and inducing systemic resistance in soybean., (© 2024. The Author(s).)

Entomopathogenic Photorhabdus bacteria (Enterobacteriaceae: Gamma-proteobacteria), the natural symbionts of Heterorhabditis nematodes, are a rich source for the discovery of biologically active secondary metabolites (SMs). This study describes the isolation of three nematicidal SMs from in vitro culture supernatants of the Arizona-native Photorhabdus luminescens sonorensis strain Caborca by bioactivity-guided fractionation. Nuclear magnetic resonance spectroscopy and comparison to authentic synthetic standards identified these bioactive metabolites as trans -cinnamic acid ( t -CA), (4 E )-5-phenylpent-4-enoic acid (PPA), and indole. PPA and t- CA displayed potent, concentration-dependent nematicidal activities against the root-knot nematode (Meloidogyne incognita) and the citrus nematode ( Tylenchulus semipenetrans ), two economically and globally important plant parasitic nematodes (PPNs) that are ubiquitous in the United States. Southwest. Indole showed potent, concentration-dependent nematistatic activity by inducing the temporary rigid paralysis of the same targeted nematodes. While paralysis was persistent in the presence of indole, the nematodes recovered upon removal of the compound. All three SMs were found to be selective against the tested PPNs, exerting little effects on non-target species such as the bacteria-feeding nematode Caenorhabditis elegans or the entomopathogenic nematodes Steinernema carpocapsae, Heterorhabditis bacteriophora, and Hymenocallis sonorensis . Moreover, none of these SMs showed cytotoxicity against normal or neoplastic human cells. The combination of t- CA + PPA + indole had a synergistic nematicidal effect on both targeted PPNs. Two-component mixtures prepared from these SMs revealed complex, compound-, and nematode species-dependent interactions. These results justify further investigations into the chemical ecology of Photorhabdus SMs, and recommend t- CA, PPA and indole, alone or in combinations, as lead compounds for the development of selective and environmentally benign nematicides against the tested PPNs. IMPORTANCE Two phenylpropanoid and one alkaloid secondary metabolites were isolated and identified from culture filtrates of Photorhabdus l. sonorensis strain Caborca. The three identified metabolites showed selective nematicidal and/or nematistatic activities against two important plant parasitic nematodes, the root-knot nematode (Meloidogyne incognita) and the citrus nematode ( Tylenchulus semipenetrans ). The mixture of all three metabolites had a synergistic nematicidal effect on both targeted nematodes, while other combinations showed compound- and nematode-dependent interactions.

Root-knot nematodes (RKNs) are devastating parasites that invade thousands of plants. In this study, five RKN effectors, which might interact with Prunus sogdiana resistance protein PsoRPM3, were screened and identified. In situ hybridisation results showed that MiCal, MiGST_N_4, MiEFh and MiACPS are expressed in the subventral oesophageal glands (SvG), and MiTSPc hybridization signals are found in the dorsal esophageal gland (DG) of Meloidogyne incognita in the pre-J2. RT-qPCR data indicated that the expression of MiCal , MiGST_N_4 , MiEFh , and MiACPS genes are highly expressed in M. incognita of pra-J2 and J3/J4 stages. The expression of MiTSPc increased significantly in the female stage of M. incognita . Moreover, all effectors found in this study localize in the cytoplasm and nucleus when transiently expressed in plant cells. In addition, MiGST_N_4, MiEFh, MiACPS and MiTSPc can elicit the ROS burst and strong hypersensitive response (HR), as well as significant ion leakage. Our data suggest that MiGST_N_4, MiEFh, MiACPS and MiTSPc effectors may be involved in triggering the immune response of the host plant.

Dicers and dicer-like enzymes play an essential role in small RNA processing in eukaryotes. Nematodes are thought to encode one dicer, DCR-1; only that for Caenorhabditis spp. is well-characterised. Using genomic sequences of eight root-knot nematodes (Meloidogyne spp.), we identified putative coding sequences typical of eukaryotic DICERS. We noted that the primary and secondary structures of DICERS they encode were different for different Meloidogyne species and even for isolates of the same species, suggesting paralogy for the gene. One of the genes for M. incognita (Midcr-1.1) expressed in eggs, juvenile stage 2 and adults, with the highest expression in the adult females. All the Meloidogyne DICERS had seven major domains typical of those for Caenorhabditis spp. and humans with very similar protein folding. RNAi of Midcr-1.1 in J2s using seven dsRNAs, each based on sequences encoding the domains, induced mild paralysis but measurable knockdown was detected in J2s treated with five of the dsRNAs. For four of the dsRNAs, the RNAi effect lasted and reduced the nematode's infectivity. Also, host plant delivery of dsRNAs complementary to coding sequences of the Dicer Dimerisation domain impaired development, reducing nematode infection by 71%. These results confirm the importance of the gene to nematode health.

Sweetpotato, Ipomoea batatas (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes' expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that IbNBS75, IbNBS219, and IbNBS256 respond to stem nematode infection; Ib-NBS240, IbNBS90, and IbNBS80 respond to cold stress, while IbNBS208, IbNBS71, and IbNBS159 respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future., (© 2021 S. Karger AG, Basel.)

Plant parasitic nematodes, including the beet cyst nematode Heterodera schachtii , constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the H. schachtii parasitism of Arabidopsis thaliana . None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on A. thaliana to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated A. thaliana roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant's sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic nematodes.

Soil-borne pathogens and weeds could synergistically affect vegetable growth and result in serious losses. The investigation of antagonistic metabolites from a marine-derived entomopathogenic fungus, Beauveria felina , obtained polyhydroxy steroid ( 1 ), tricyclic diterpenoid ( 2 ), isaridin ( 3 ), and destruxin cyclodepsipeptides ( 4-6 ). The structures and absolute configurations of new 1-3 were elucidated by extensive spectroscopic and X-ray crystallographic analyses, as well as electronic circular dichroism (ECD) calculations. Compounds 1 and 2 showed antifungal activities against carbendazim-resistant strains of Botrytis cinerea, with the minimum inhibitory concentration (MIC) values ranging from 16 to 32 μg/mL, which were significantly better than those of carbendazim (MIC = 256 μg/mL). Compound 5 exhibited significant antagonistic activity against the radicle growth of Amaranthus retroflexus seedlings, which was almost identical to that of the positive control (2,4-dichlorophenoxyacetic acid). The structure-activity differences of 4-6 suggested that the Cl atom in HMPA 1 and β-Me in Pro 2 should be the key factors to their herbicidal activities. Besides, compounds 3-6 showed moderate nematicidal activities against Meloidogyne incognita . These antagonistic effects of 1-6 were first reported and further revealed the synergistically antagonistic potential of B. felina to be developed into the biopesticide.

The cereal cyst nematode, Heterodera avenae is distributed worldwide and causes substantial damage in bread wheat, Triticum aestivum. This nematode is extremely difficult to manage because of its prolonged persistence as unhatched eggs encased in cysts. Due to its sustainable and target-specific nature, RNA interference (RNAi)-based strategy has gained unprecedented importance for pest control. To date, RNAi strategy has not been exploited to manage H. avenae in wheat. In the present study, 40 H. avenae target genes with different molecular function were rationally selected for in vitro soaking analysis in order to assess their susceptibility to RNAi. In contrast to target-specific downregulation of 18 genes, 7 genes were upregulated and 15 genes showed unaltered expression (although combinatorial soaking showed some of these genes are RNAi susceptible), suggesting that a few of the target genes were refractory or recalcitrant to RNAi. However, RNAi of 37 of these genes negatively altered nematode behavior in terms of reduced penetration, development and reproduction in wheat. Subsequently, wheat plants were transformed with seven H. avenae target genes (that showed greatest abrogation of nematode parasitic success) for host-induced gene silencing (HIGS) analysis. Transformed plants were molecularly characterized by PCR, RT-qPCR and Southern hybridization. Production of target gene-specific double- and single-stranded RNA (dsRNA/siRNA) was detected in transformed plants. Transgenic expression of galectin, cathepsin L, vap1, serpin, flp12, RanBPM and chitinase genes conferred 33.24-72.4 % reduction in H. avenae multiplication in T 1 events with single copy ones exhibiting greatest reduction. A similar degree of resistance observed in T 2 plants indicated the consistent HIGS effect in the subsequent generations. Intriguingly, cysts isolated from RNAi plants were of smaller size with translucent cuticle compared to normal size, dark brown control cysts, suggesting H. avenae developmental retardation due to HIGS. Our study reinforces the potential of HIGS to manage nematode problems in crop plant., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The present study reports on the powerful nematicidal activity of a series of electron-deficient alkynes against the root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood. Interestingly, we found that the conjugation of electron-withdrawing carbonyl groups to an alkyne triple bond was extremely proficient in inducing nematode paralysis and death. In particular, dimethylacetylenedicarboxylate ( 10 ), 3-butyn-2-one ( 1 ), and methyl propiolate ( 4 ), with EC 50/48 h of 1.54 ± 0.16, 2.38 ± 0.31, and 2.83 ± 0.28 mg/L, respectively, were shown to be the best tested compounds. Earlier studies reported on the ability of alkynoic esters and alkynones to induce a chemoselective cysteine modification of unprotected peptides. Thus, also following our previous findings on the impairment of vacuolar-type proton translocating ATPase functionality by activated carbonyl derivatives, we speculate that the formation of a vinyl sulfide linkage might be responsible for the nematicidal activity of the presented electron-deficient alkynes.

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 root chemistry is altered by the parasitism of plant-parasitic nematodes (PPN). Here, we investigated the influence of the infective stage juveniles (J2) of Meloidogyne javanica in inducing tomato ( Solanum lycopersicum ) root volatiles and chemotactic effect on conspecifics. In olfactometer assays, J2 avoided the roots of 2-day infected plants but preferred 7-day-infected tomato compared to healthy plants. Chemical analysis showed a 2-7-fold increase in the amounts of monoterpenes emitted from tomato roots infected with M. javanica relative to healthy roots. In further bioassays, the monoterpenes β-pinene, (+)-(2)-carene, α-phellandrene, and β-phellandrene differentially attracted (51-87%) J2 relative to control. Concurrent reduction and increase in the levels of methyl salicylate and ( Z )-methyl dihydrojasmonate, respectively, in the root volatiles reduced J2 responses. These results demonstrate that the host plant can alter its root volatile composition to inhibit PPN attack. The observed plant-produced inhibition of J2 warrants further investigation as a potential management tool for growers.

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-mobile systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. However, in nematodes the mode of action of SPT and its effect on their development have not been studied so far. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids and fatty acid composition. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by SPT, a well-known systemic insecticide for sucking pest control.

Banana plants ( Musa spp.) are susceptible to infection by many plant-parasitic nematodes, including Meloidogyne incognita . In this study, a mixed fermentation broth of chicken manure (CM) and cassava ethanol wastewater (CEW) was used to inhibit M. incognita by reducing egg hatching and by having a lethal effect on second-stage juvenile nematodes (J2s). It also alleviated nematode damage and promoted banana plant growth. Using gas chromatography-mass spectrometry (GC-MS), we identified methyl palmitate and methyl stearate as bioactive compounds. These bioactive compounds repelled J2s and inhibited egg hatching; reduced root galls, egg masses, and nematodes in soil; and downregulated the essential parasitic nematode genes Mi-flp-18 and 16D10 . A Caenorhabditis elegans offspring assay showed that low concentrations of the fermentation broth, methyl palmitate, and methyl stearate were safe for its life cycle. This study explored the effective and environmentally safe strategies for controlling root-knot nematodes.

To date, there has been great demand for ecofriendly nematicides with beneficial properties to the nematode hosting plants. Great efforts are made towards the chemical characterization of botanical extracts exhibiting nematicidal activity against Meloidogyne spp. , but only a small percentage of these data are actually used by the chemical industry in order to develop new formulates. On the other hand, the ready to use farmer produced water extracts based on edible plants could be a sustainable and economic solution for low income countries. Herein, we evaluate the nematicidal potential of Stevia rebaudiana grown in Greece against Meloidogyne incognita and Meloidogyne javanica , two most notorious phytoparasitic nematode species causing great losses in tomato cultivation worldwide. In an effort to recycle the plant's remnants, after leaves selection for commercial use, we use both leaves and wooden stems to test for activity. In vitro tests demonstrate significant paralysis activity of both plant parts' water extracts against the second-stage juvenile (J2) of the parasites; while, in vivo bioassays demonstrated the substantial efficacy of leaves' powder (95% at 1 g kg -1 ) followed by stems. Interestingly, the incorporation of up to 50 g powder/kg of soil is not phytotoxic, which demonstrates the ability to elevate the applied concentration of the nematicidal stevia powder under high inoculum level. Last but not least, the chemical composition analyses using cutting edge analytical methodologies, demonstrated amongst components molecules of already proven nematicidal activity, was exemplified by several flavonoids and essential oil components. Interestingly, and to our knowledge, for the flavonoids, morin and robinin, the anthocyanidin, keracyanin, and a napthalen-2-ol derivative is their first report in Stevia species.

Background: The root-knot nematode Meloidogyne graminicola has become a serious threat to rice production as a result of the cultivation changes from transplanting to direct seeding. The nematicidal activity of Aspergillus welwitschiae have been investigated in vitro, and the disease control efficacy of the active compound has been evaluated under greenhouse and field conditions., Results: The active compound αβ-dehydrocurvularin (αβ-DC), isolated by nematicidal assay-directed fractionation, showed significant nematicidal activity against M. graminicola, with a median lethal concentration (LC 50 ) value of 122.2 μg mL - 1 . αβ-DC effectively decreased the attraction of rice roots to nematodes and the infection of nematodes and also suppressed the development of nematodes under greenhouse conditions. Moreover, αβ-DC efficiently reduced the root gall index under field conditions., Conclusions: To our knowledge, this is the first report to describe the nematicidal activity of αβ-DC against M. graminicola. The results obtained under greenhouse and field conditions provide a basis for developing commercial formulations from αβ-DC to control M. graminicola in the future.

Root-knot nematode diseases cause severe yield and economic losses each year in global agricultural production. Virgibacillus dokdonensis MCCC 1A00493, a deep-sea bacterium, shows a significant nematicidal activity against Meloidogyne incognita in vitro. However, information about the active substances of V. dokdonensis MCCC 1A00493 is limited. In this study, volatile organic compounds (VOCs) from V. dokdonensis MCCC 1A00493 were isolated and analyzed through solid-phase microextraction and gas chromatography-mass spectrometry. Four VOCs, namely, acetaldehyde, dimethyl disulfide, ethylbenzene, and 2-butanone, were identified, and their nematicidal activities were evaluated. The four VOCs had a variety of active modes on M. incognita juveniles. Acetaldehyde had direct contact killing, fumigation, and attraction activities; dimethyl disulfide had direct contact killing and attraction activities; ethylbenzene had an attraction activity; and 2-butanone had a repellent activity. Only acetaldehyde had a fumigant activity to inhibit egg hatching. Combining this fumigant activity against eggs and juveniles could be an effective strategy to control the different developmental stages of M. incognita . The combination of direct contact and attraction activities could also establish trapping and killing strategies against root-knot nematodes. Considering all nematicidal modes or strategies, we could use V. dokdonensis MCCC 1A00493 to set up an integrated strategy to control root-knot nematodes.

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 (LC 50 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.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are an extensive class of small regulatory RNAs. Knowing the specific expression and functions of miRNAs during root-knot nematode (RKN) ( Meloidogyne incognita ) development could provide fundamental information about RKN development as well as a means to design new strategies to control RKN infection, a major problem of many important crops. Employing high throughput deep sequencing, we identified a total of 45 conserved and novel miRNAs from two developmental stages of RKN, eggs and J2 juveniles, during their infection of cotton ( Gossypium hirsutum L. ). Twenty-one of the miRNAs were differentially expressed between the two stages. Compared with their expression in eggs, two miRNAs were upregulated (miR252 and miRN19), whereas 19 miRNAs were downregulated in J2 juveniles. Nine miRNAs were expressed at high levels, with >1000 reads per mapped million (RPM) sequenced reads in both eggs and J2 juveniles (miR1, miR124, miR2-3p, miR252, miR279, miR57-5p, miR7904, miR87, and miR92). Three miRNAs were only expressed in eggs (miR4738, miRN3, and miRN5). These differentially expressed miRNAs may control RKN development by regulating specific protein-coding genes in pathways associated with RKN growth and development.

Soil biota have important effects on crop productivity, but can be difficult to study in situ. Laser ablation tomography (LAT) is a novel method that allows for rapid, three-dimensional quantitative and qualitative analysis of root anatomy, providing new opportunities to investigate interactions between roots and edaphic organisms. LAT was used for analysis of maize roots colonized by arbuscular mycorrhizal fungi, maize roots herbivorized by western corn rootworm, barley roots parasitized by cereal cyst nematode, and common bean roots damaged by Fusarium. UV excitation of root tissues affected by edaphic organisms resulted in differential autofluorescence emission, facilitating the classification of tissues and anatomical features. Samples were spatially resolved in three dimensions, enabling quantification of the volume and distribution of fungal colonization, western corn rootworm damage, nematode feeding sites, tissue compromised by Fusarium, and as well as root anatomical phenotypes. Owing to its capability for high-throughput sample imaging, LAT serves as an excellent tool to conduct large, quantitative screens to characterize genetic control of root anatomy and interactions with edaphic organisms. Additionally, this technology improves interpretation of root-organism interactions in relatively large, opaque root segments, providing opportunities for novel research investigating the effects of root anatomical phenes on associations with edaphic organisms., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Meloidogyne incognita is an economically important plant parasitic nematode. Here we demonstrate substantial variation in the invasiveness of four M. incognita populations relative to tomato. Infective (J2) stage transcriptomes reveal significant variation in the expression of protein-coding and non-coding RNAs between populations. We identify 33 gene expression markers that correlate with invasiveness, and which map to genes with predicted roles in host finding and invasion, including neuropeptides, ion channels, G Protein-Coupled Receptors, cell wall-degrading enzymes and microRNAs. These data demonstrate a surprising diversity in microRNA complements between populations, and identify gene expression markers for invasiveness of M. incognita, to our knowledge for the first time., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Host plant resistance is the most practical approach to control the Southern root-knot nematode (Meloidogyne incognita; RKN), which has emerged as one of the most serious economic pests of Upland cotton (Gossypium hirsutum L.). Previous QTL analyses have identified a resistance locus on chromosome 11 (qMi-C11) affecting galling and another locus on chromosome-14 (qMi-C14) affecting egg production. Although these two QTL regions were fine mapped and candidate genes identified, expression profiling of genes would assist in further narrowing the list of candidate genes in the QTL regions. We applied the comparative transcriptomic approach to compare expression profiles of genes between RKN susceptible and resistance genotypes at an early stage of RKN development that coincides with the establishment of a feeding site and at the late stage of RKN development that coincides with RKN egg production. Sequencing of cDNA libraries produced over 315 million reads of which 240 million reads (76%) were mapped on to the Gossypium hirsutum genome. A total of 3,789 differentially expressed genes (DEGs) were identified which were further grouped into four clusters based on their expression profiles. A large number of DEGs were found to be down regulated in the susceptible genotype at the late stage of RKN development whereas several genes were up regulated in the resistant genotype. Key enriched categories included transcription factor activity, defense response, response to phyto-hormones, cell wall organization, and protein serine/threonine kinase activity. Our results also show that the DEGs in the resistant genotype at qMi-C11 and qMi-C14 loci displayed higher expression of defense response, detoxification and callose deposition genes, than the DEGs in the susceptible genotype., Competing Interests: Co-author RLN is a director of Agricultural Research in Cotton Incorporated, and is involved in the study design and preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A study of the processes of lipid peroxidation and the activity of the peroxidase enzyme, as well as photosynthetic pigments in susceptible tomato plants treated with salicylic acid (SA), during infection with the root-knot nematode Meloidogyneincognita. It was shown that, in the roots of SA-treated plants, the activity of lipid peroxidation is higher compared to the untreated plants, especially in the case of nematode invasion. A significant increase in the activity of lipid peroxidation in SA-treated invasive plants compared with untreated was noted during the transition of larvae to the sedentary stage and the beginning of the formation of feeding areas-giant cells (3-5 days after invasion). This, apparently, contributes to the inhibition of the development of the parasite and the reduction of plant infection and also indicates the involvement of oxidative processes in the mechanism of the induced resistance of plants to root-knot nematodes. In the SA-treated plants, the qualitative and quantitative composition of photosynthetic pigments, disturbed by invasion, was restored and corresponded to the control level.

Nematode diseases have a worldwide importance for several economic agricultural crops, such as soybean. Frequently, new or secondary pathogens arise as emergent diseases due to the intensified use of agricultural lands, causing extensive yield losses. Helicotylenchus dihystera (Hd) and Scutellonema brachyurus (Sb) appear as potential pathogens for soybean in Brazil, since their spread and population densities have been increased on soybean growing areas. Aiming to evaluate the quantitative distribution of Hd and Sb in soybean fields in South Brazil, a survey was conducted during the growing seasons of 2014-2015 and 2015-2016 in which 1,088 soil samples and 1,043 root samples were analyzed. Besides, two greenhouse experiments were conducted to evaluate the pathogenicity of both nematodes to soybean plants, in comparison with P. brachyurus (Pb), a known pathogen of the crop. The survey demonstrated that Pb, Hd and Sb are widely distributed in the States of Paraná, Santa Catarina and Mato Grosso do Sul. Besides, we proved that Hd and Sb multiply and cause root lesions in soybean cv. Potência, since they were found inside roots, and can be considered as potential pathogens for soybean plants., Competing Interests: The authors have declared that no competing interests exist.

Naturally occurring thiophenes possess excellent nematicidal and fungicidal activities. However, thiophenes often have limited application in soil due to their light-dependent toxicity given the living and reproductive condition of soil-borne pathogens. In this study, six new (1-6) and six known thiophenes (7-12) were isolated from Echinops grijsii. Compounds 1-2, 4-5, 8-9, 11 , and 12 showed stronger nematicidal activity against Meloidogyne incognita than commercial nematicide abamectin. 4-10 were demonstrated as nonphototoxic thiophenes. Among these, 4 and 8 were the most potent thiophenes (LC 50 values 2.57 and 0.91 μg/mL in light, 1.80 and 0.86 μg/mL in dark, respectively) against M. incognita. SAR revealed that thiophene skeleton was essential for nematicidal activity, while disubstituted groups were helpful for nonphototoxicity. Although an increased number of acetylenes improved activity, it decreased nonphototoxicity. Acyl groups could suppress the effects of light on activity, with the level of inhibitory effects depending on its number and chain length, while chlorine played important roles in promoting activity. Additionally, compounds 1-2, 4-5, 7, 8, and 10 displayed antifungal activity against six soil-borne fungi in various degrees. The discovery of nonphototoxic thiophenes and elucidation of SAR provide important information for the exploitation and utilization of thiophenes in the integrative management regarding disease complexes caused by the combination of root-knot nematode and soil-borne fungi.

Meloidogyne hapla is the most prevalent plant-parasitic nematode in Washington state wine grape vineyards. Understanding the developmental dynamics of M. hapla can improve the timing of diagnostic sampling and nematicide application. Three Vitis vinifera vineyards in Washington were sampled March 2015 to March 2017 to determine the developmental dynamics of M. hapla by measuring second-stage juveniles (J2) in soil, eggs and adult females in roots, and fine root tips. A model of M. hapla J2 development based on soil growing degree days using a base temperature (T b ) of 0°C (GDD soil ) and a start date of 1 March was developed. This model was validated at two additional vineyards in Washington and was robust with R 2 values > 0.74. M. hapla has one generation per year and overwinters primarily as the J2 infective stage. Juvenile populations declined after 1 March, reaching their lowest density in early July and reaching a maximum density over the winter. M. hapla egg and root tip densities reached a maximum in early August. The number of females per root tip did not vary throughout the year. A single generation with defined peaks in J2 population densities will allow for specific timing of nematicide interventions.

Quantitative trait loci (QTLs) qMi-C11 and qMi-C14 impart a high level of resistance to Meloidogyne incognita in cotton. Breeders had previously backcrossed both QTLs into the susceptible Coker 201 to create the highly resistant M-120 RNR, and we crossed Coker 201 and M-120 RNR to create near-isogenic lines with either qMi-C11 or qMi-C14 . Previous work suggests different modes of action for qMi-C11 and qMi-C14 . To document individual and combined effects of the QTLs on nematode development and reproduction, Coker 201 (neither QTL), M-120 RNR (both QTLs), CH11 near isoline ( qMi-C11 ), and CH14 near isoline ( qMi-C14 ) were inoculated with M. incognita . At 4, 8, 12, 16, 20, 25, and 30 days after inoculation (DAI), roots were stained to observe nematode developmental stages (second-stage juvenile [J2], swollen second-stage juvenile [SJ2], third-stage juvenile [J3], fourth-stage juvenile [J4], and female), and the number of galls was counted. At 20, 25, 30, and 40 DAI, M. incognita eggs were harvested and counted. At 30 DAI, 80% of the nematodes on Coker 201 were female compared with 50, 40, and 33% females on CH14, CH11, and M-120 RNR, respectively, and greater proportions of nematodes remained in J2 in M-120 RNR (41%), CH11 (58%), and CH14 (27%) than in Coker 201 (9%). More nematodes progressed to J3 or J4 on Coker 201 and CH14 than on CH11 or M-120 RNR. Coker 201 and CH14 had more galls than M-120 RNR. Coker 201 had more eggs than the other genotypes at 30 DAI. Nematode development beyond J2 or SJ2 was significantly reduced by qMi-C11 , and development beyond J3 or J4 was significantly reduced by qMi-C14 . This study confirms that qMi-C11 and qMi-C14 act at different times and have different effects on the development of M. incognita , and therefore, they have different modes of action.

The modification of rhizosphere microbial diversity and ecological processes are of rising interest as shifting in microbial community structure impacts the mutual role of host-microbe interactions. Nevertheless, the connection between host-microbial community diversity, their function under biotic stress in addition to their impact on plant performances is poorly understood. The study was designed with the aim to analyze the tripartite interactions among Chitiniphilus sp., Streptomyces sp. and their combination with indigenous rhizospheric microbial population of Bacopa monnieri for enhancing the plant growth and bacoside A content under Meloidogyne incognita stress. Overall, plants treated with the microbial combination recorded enhanced growth as illustrated by significantly higher biomass (2.0 fold), nitrogen uptake (1.8 fold) and bacoside A content (1.3 fold) along with biocontrol efficacy (58.5%) under nematode infected field. The denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S-rDNA revealed that microbial inoculations are major initiators of bacterial community structure in the plant rhizosphere. Additionally, the plants treated with microbial combination showed maximum diversity viz., Shannon's (3.29), Margalef's (4.21), and Simpson's (0.96) indices. Likewise the metabolic profiling data also showed a significant variation among the diversity and evenness indices upon microbial application on the native microflora. We surmise that the application of beneficial microbes in combinational mode not only helped in improving the microbial community structure but also successfully enhanced plant and soil health under biotic stress., (Copyright © 2018. Published by Elsevier GmbH.)

The nematicidal activity of the common plant-pathogenic bacterium Pseudomonas syringae against certain nematodes has been recently identified, but little is known about its virulence factors. In the current study, predictive analysis of nematode-virulent factors in the genome of a P. syringae wild-type strain MB03 revealed a variety of factors with the potential to be pathogenic against nematodes. One of these virulence factors that was predicted with a high score, namely, YqfO03, was a protein with structural domains that are similar to the Nif3 superfamily. This protein was expressed and purified in Escherichia coli , and was investigated for nematicidal properties against the model nematode Caenorhabditis elegans and an agriculturally important pest Meloidogyne incognita . Our results showed that YqfO03 exhibits lethal activity toward C. elegans and M. incognita worms, and it also caused detrimental effects on the growth, brood size, and motility of C. elegans worms. However, C. elegans worms were able to defend themselves against YqfO03 via a physical defense response by avoiding contact with the protein. Discovery of the diverse nematicidal activities of YqfO03 provides new knowledge on the biological function of a bacterial Nif3-family protein and insight into the potential of this protein as a specific means of controlling agricultural nematode pests.

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most important pathogens of soybean. Periodic monitoring of SCN population densities and virulence phenotypes is necessary for developing management strategies utilizing resistant cultivars, the primary strategy used to combat this pest. Therefore, we conducted a statewide survey of Missouri to determine SCN population densities and virulence phenotypes during 2015-2016 and compared these results with a similar survey conducted in 2005. SCN population densities were determined for 393 soil samples representing 74 soybean-producing counties across eight geographical regions of Missouri. Eighty-eight percent of samples tested positive for SCN, up from 50% in 2005, and population densities ranged from 125 to 99,000 eggs per 250 cm 3 of soil. The virulence phenotypes of 48 SCN populations also were determined. For this, female indices (FI) were calculated by dividing the mean number of females that develop on the roots of a set of resistant soybean indicator lines by the mean number of females that develop on the roots of susceptible cultivar Lee74 after 30 days in the greenhouse then multiplying by 100 to obtain a percentage. Notably, all SCN populations evaluated during 2015-2016 had a FI > 10 on PI 88788, the most widely used source of resistance in Missouri, in contrast to 78% in 2005. Moreover, 50% of these populations had a FI > 50 on PI 88788, up from 16% in 2005. Forty-three percent of populations tested also had a FI > 10 on Peking, the second most common source of resistance by farmers. Our results show that over the last decade, SCN has become more prevalent in Missouri fields. Additionally, the percentage of individuals within SCN field populations that are virulent on PI 88788 and Peking has markedly increased. The results stress the importance of rotating cultivars with different types of resistance when using resistant cultivars to manage SCN.

The beet cyst nematode (BCN) Heterodera schachtii causes serious damage and yield losses in numerous important crops worldwide. This study examines the efficacy of three types of transgenic Arabidopsis RNA interference (RNAi) lines to decrease the biological activity of this devastating nematode. The first RNAi construct (E 1 E 2 -RNAi) targets two nematode endoglucanase genes, which are involved in BCN pathogenicity, the second construct (MSP-RNAi) contains a fragment corresponding to the major sperm protein transcript necessary for BCN development and reproduction, and the third construct (E 1 E 2 MSP-RNAi) comprises all three target fragments. Transcript expression profiles of the target genes in all biological stages of the nematode were determined for the initial inoculated population and the resulting progeny. Bioassay data under indoor aseptic cultivation indicated that feeding on these RNAi lines did not affect pathogenic activity and reproductive capacity of the initial population, whereas inoculating the progeny into new transgenic plants corresponding with the lines from which they were recovered reduced the nematode penetration and the number of eggs per cyst. In addition, the male/female ratio increased more than the double, and the effects of RNAi continued in the second generation of the nematodes, because the progeny derived from E 1 E 2 -RNAi and E 1 E 2 MSP-RNAi lines showed an impaired ability to infect wild-type plants.

On invasion of roots, plant-parasitic nematodes secrete effectors to manipulate the cellular regulation of the host to promote parasitism. The root-knot nematode Meloidogyne graminicola is one of the most damaging nematodes of rice. Here, we identified a novel effector of this nematode, named Mg16820, expressed in the nematode subventral glands. We localized the Mg16820 effector in the apoplast during the migration phase of the second-stage juvenile in rice roots. In addition, during early development of the feeding site, Mg16820 was localized in giant cells, where it accumulated in the cytoplasm and the nucleus. Using transient expression in Nicotiana benthamiana leaves, we demonstrated that Mg16820 directed to the apoplast was able to suppress flg22-induced reactive oxygen species production. In addition, expression of Mg16820 in the cytoplasm resulted in the suppression of the R2/Avr2- and Mi-1.2-induced hypersensitive response. A potential target protein of Mg16820 identified with the yeast two-hybrid system was the dehydration stress-inducible protein 1 (DIP1). Bimolecular fluorescence complementation resulted in a strong signal in the nucleus. DIP1 has been described as an abscisic acid (ABA)-responsive gene and ABA is involved in the biotic and abiotic stress response. Our results demonstrate that Mg16820 is able to act in two cellular compartments as an immune suppressor and targets a protein involved in the stress response, therefore indicating an important role for this effector in parasitism., (© 2018 BSPP and John Wiley & Sons Ltd.)

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.)

Analysis of transcriptome sequence data from eggs and second-stage juveniles (J2s) of sugar beet cyst nematode (SBCN, Heterodera schachtii) identified the full-length genome of a positive-sense single-stranded RNA virus, provisionally named sugar beet cyst nematode virus 1 (SBCNV1). The SBCNV1 sequence was detected in both eggs and J2s, indicating its possible vertical transmission. The 9503-nucleotide genome sequence contains a single long open reading frame, which was predicted to encode a polyprotein with conserved domains for picornaviral structural proteins proximal to its amino terminus and RNA helicase, cysteine proteinase and RNA-dependent RNA polymerase (RdRp) conserved domains proximal to its carboxyl terminus, hallmarks of viruses belonging to the order Picornavirales. Phylogenetic analysis of the predicted SBCNV1 RdRp amino acid sequence indicated that the SBCNV1 sequence is most closely related to members of the family Secoviridae, which includes genera of nematode-transmitted plant-infecting viruses. SBCNV1 represents the first fully sequenced viral genome from SBCN.

This study was aimed to investigate the activity of the Asteraceae species Taraxacum officinale against the root-knot nematode Meloidogyne incognita. Leaf and root extracts of T. officinale were tested in vitro at a range of 62.5-1000 and 250-1000 μg mL -1 concentrations on nematode juveniles and eggs, respectively, whereas treatments with 10-40 g kg -1 soil rates of dry leaf and root T. officinale biomass were applied to soil infested by M. incognita in greenhouse experiments on potted tomato. Peak 36 and 50% juvenile mortality and 14.8 and 23.8% egg hatchability reduction were recorded at the maximum concentration of leaf and root extracts, respectively. Soil treatments with T. officinale leaf and root material strongly suppressed nematode multiplication and gall formation on tomato roots and significantly increased plant growth. Chicoric acid and 3-O- and 3,5-di-O-caffeoylquinic acid were found to be the main components of leaf and root extract, respectively, and proved, as the total hydroalcoholic extracts from T. officinale leaf and root material, for an antioxidant activity. Data from this study indicate the suitability of plant materials from T. officinale for a potential formulation of nematicidal products to include in sustainable nematode management strategies.

A series of novel 1,3,4-oxadiazole-cinnamic acid hybrids were synthesized. The bioassays results indicated that compounds 1, 2, 7, and 8 showed excellent nematicidal activities against Tylenchulus semipenetrans with LC 50,48h values of 9.7 ± 1.6, 15.6 ± 2.8, 8.0 ± 0.5, and 19.8 ± 2.9 mg/L, respectively, which were higher than those of avermectin (32.6 ± 4.5 mg/L) and fosthiazate (67.8 ± 1.7 mg/L). Low-toxicity compound 26, with excellent nematicidal activity in vitro (LC 50,48h = 8.2 ± 1.2 mg/L), was designed on the basis of the predictive CoMFA ( q 2 = 0.795, r 2 = 0.921) and CoMSIA ( q 2 = 0.762, r 2 = 0.912) models. The control effect of compound 26 was 69.8% at an effective dose of 1.0 g per plant in a field experiment, which was superior to that of fosthiazate (67.2%). This work indicated that 1,3,4-oxadiazole-cinnamic acid hybrids may be used as potential nematicides.

Root-knot nematodes (RKNs; Meloidogyne spp.) are obligate endoparasites that infect many crops and cause severe yield losses. In this research, we studied the effect of Welsh onion, grown as a companion plant, on the resistance of cucumber plants to RKN infection and analyzed the most abundant components of Welsh onion root exudates. The results showed that, when grown with Welsh onion as a companion plant, cucumber roots had 77.0% fewer root knots and egg masses than the control cucumber roots. Welsh onion root exudates were collected and extracted with chloroform, ethyl ether, n-butanol and ethyl acetate. High concentrations of the extracts from the Welsh onion root exudates decreased the hatchability of RKN eggs. In particular, the inhibitory effect of the n-butanol extract was significant and the hatchability of RKN eggs did not exceed 10%. Gas chromatographic-mass spectrometric analysis revealed that the most abundant component in the n-butanol extract was 4-hydroxy-benzeneethanol. Treatment with 1.2 mM 4-hydroxy-benzeneethanol decreased egg hatchability to 40%, whereas treatment with 9.6 mM or a higher concentration of 4-hydroxy-benzeneethanol decreased egg hatchability to less than 10%. In addition, 1.2 mM or a higher concentration of 4-hydroxy-benzeneethanol decreased the activity of the second-stage juvenile (J2). Higher 4-hydroxy-benzeneethanol concentrations (9.8 and 19.2 mM) were lethal to RKNs to some extent, with death rates greater than 50% at 48 h of treatment. The present results suggest that cultivation with Welsh onion as a companion plant may represent an alternative to the application of synthetic nematicides, with fewer side effects. We confirmed that 4-hydroxy-benzeneethanol is a natural effective nematicide., Competing Interests: The authors have declared that no competing interests exist.

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The efficacy of Verticillium leptobactrum isolate (HR1) was evaluated in the control of root-knot nematode and Fusarium wilt fungus under laboratory and greenhouse conditions. Five concentrations of V. leptobactrum (HR1) isolate were tested for their nematicidal and fungicidal activities against Meloidogyne javanica and Fusarium oxysporum f.sp. lycopersici in vitro. Laboratory trials showed that mycelium growth inhibition of Fusarium wilt fungus was correlated to the increase of the concentration of culture filtrate. All dilutions showed efficiency in reducing the growth of Fusarium oxysporum f.sp. lycopersici. The greatest nematicidal activity was observed at 50, 75, and 100% filtrate dilutions. The egg hatching percentage reached 42%, and the juvenile's corrected mortality registered 90% for the above treatments. In greenhouse experiment, the biocontrol agent fungus enhanced significantly tomato growth components (height and weight of plant and root). The multiplication rate of root-knot nematode and the Fusarium wilt disease incidence declined significantly with soil application of V. leptobactrum as with chemical treatments. The isolate HR1 was efficient to control wilt disease complex caused by M. javanica and Fusarium oxysporum f.sp. lycopersici.

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.

For decades, plant parasitic nematodes have caused serious damage to crop production. Most nematicides are banned because of their negative impacts on the environment and public health. The repeated application of the few commercially available nematicides has caused more incidences of nematicide resistance. To seek novel nematicides, seven linear peptides named rhabdopeptides I-O, 1-7, were isolated from culture broth of Xenorhabdus budapestensis SN84. The structures of the peptides were elucidated on the basis of extensive mass spectrometry (MS), and nuclear magnetic resonance analyses. 3, 4, and 7 were novel compounds. 1, 2, 5, and 6 were isolated and purified for the first time, despite being previously elucidated from an extract mixture based on labeling and MS experiments. All seven compounds were tested for their nematicidal activities against the second-stage juveniles (J2) of Meloidogyne incognita using 24-microwell plates. Rhabdopeptide J, 2, demonstrated strong inhibitory activity with an LC 50 value of 27.8 μg/mL. Rhabdopeptide K, 3, and M, 5, showed moderate inhibitory activity with LC 50 values of 46.3 and 42.4 μg/mL, respectively.

Biofertilizer Ning shield was composed of different strains of plant growth promotion bacteria. In this study, the plant growth promotion and root-knot nematode disease control potential on Trichosanthes kirilowii in the field were evaluated. The application of Ning shield significantly reduced the diseases severity caused by Meloidogyne incognita, the biocontrol efficacy could reached up to 51.08%. Ning shield could also promote the growth of T. kirilowii in the field by increasing seedling emergence, height and the root weight. The results showed that the Ning shield could enhance the production yield up to 36.26%. Ning shield could also promote the plant growth by increasing the contents of available nitrogen, phosphorus, potassium and organic matter, and increasing the contents of leaf chlorophyll and carotenoid pigment. Moreover, Ning shield could efficiently enhance the medicinal compositions of Trichosanthes, referring to the polysaccharides and trichosanthin. Therefore, Ning shield is a promising biofertilizer, which can offer beneficial effects to T. kirilowii growers, including the plant growth promotion, the biological control of root-knot disease and enhancement of the yield and the medicinal quality., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Soybean cyst nematode (SCN) Heterodera glycines is an obligate parasite that relies on the secretion of effector proteins to manipulate host cellular processes that favor the formation of a feeding site within host roots to ensure its survival. The sequence complexity and co-evolutionary forces acting upon these effectors remain unknown. Here we generated a de novo transcriptome assembly representing the early life stages of SCN in both a compatible and an incompatible host interaction to facilitate global effector mining efforts in the absence of an available annotated SCN genome. We then employed a dual effector prediction strategy coupling a newly developed nematode effector prediction tool, N-Preffector, with a traditional secreted protein prediction pipeline to uncover a suite of novel effector candidates. Our analysis distinguished between effectors that co-evolve with the host genotype and those conserved by the pathogen to maintain a core function in parasitism and demonstrated that alternative splicing is one mechanism used to diversify the effector pool. In addition, we confirmed the presence of viral and microbial inhabitants with molecular sequence information. This transcriptome represents the most comprehensive whole-nematode sequence currently available for SCN and can be used as a tool for annotation of expected genome assemblies.

Few molecular details of effectors of Heterodera avenae parasitism are known. We performed a high-throughput sequencing analysis of the H. avenae transcriptome at five developmental stages. A total of 82,549 unigenes were ultimately obtained, and 747 transcripts showed best hits to genes putatively encoding carbohydrate-active enzymes in plant-parasitic nematodes that play an important role in the invasion process. A total of 1,480 unigenes were homologous to known phytonematode effectors, and 63 putative novel effectors were identified in the H. avenae transcriptomes. Twenty-three unigenes were analyzed by qRT-PCR and confirmed to be highly expressed during at least one developmental stage. For in situ hybridization, 17 of the 22 tested putative effectors were specifically expressed and located in the subventral gland cells, and five putative novel effectors were specifically expressed in the dorsal gland. Furthermore, 115 transcripts were found to have putative lethal RNA interference (RNAi) phenotypes. Three target genes with lethal RNAi phenotypes and two of the four tested putative effectors were associated with a decrease in the number of cysts through in vitro RNAi technology. These transcriptomic data lay a foundation for further studies of interactions of H. avenae with cereal and H. avenae parasitic control.

Background: Fluensulfone is a new nematicide with an excellent profile of selective toxicity against plant parasitic nematodes. Here, its effects on the physiology and biochemistry of the potato cyst nematode Globodera pallida have been investigated and comparisons made with its effect on the life-span of the free-living nematode Caenorhabditis elegans to provide insight into its mode of action and its selective toxicity., Results: Fluensulfone exerts acute effects (≤1h; ≥100μM) on stylet thrusting and motility of hatched second stage G. pallida juveniles (J2s). Chronic exposure to lower concentrations of fluensulfone (≥3days; ≤30μM), reveals a slowly developing metabolic insult in which G. pallida J2s sequentially exhibit a reduction in motility, loss of a metabolic marker for cell viability, high lipid content and tissue degeneration prior to death. These effects are absent in adults and dauers of the model genetic nematode Caenorhabditis elegans., Conclusion: The nematicidal action of fluensulfone follows a time-course which progresses from an early impact on motility through to an accumulating metabolic impairment, an inability to access lipid stores and death., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Meloidogyne incognita is a common root-knot nematode with a wide range of plant hosts. We aimed to study the metabolites produced at each stage of the nematode life cycle to understand its development. Metabolites of Meloidogyne incognita were extracted at egg, J2, J3, J4, and female stages and 110 metabolites with available standards were quantified using CE-TOF/MS. Analyses indicated abundance of stage-specific metabolites with the exception of J3 and J4 stages which shared similar metabolic profiles. The egg stage showed increased abundance in glycolysis and energy metabolism related metabolites while the J2 metabolites are associated with tissue formation, motility, and neurotransmission. The J3 and J4 stages indicated amino acid metabolism and urea cycle- related metabolites. The female stage was characterized with polyamine synthesis, antioxidant activity, and synthesis of reproduction related metabolites. Such metabolic profiling helps us understand the dynamic physiological changes related to each developmental stage of the root-knot nematode life cycle., Competing Interests: The authors declare no conflict of interest.

Root-knot nematodes (RKN) represent extensive challenges to Cucurbitaceae crops. However, Cucumis metuliferus (Cm) is known to be resistant to Meloidogyne incognita (Mi) infections. Thus, analysis of differentially expressed genes may lead to a comprehensive gene expression profiling of the incompatible Cm-Mi interaction. In this study, the time-course transcriptome of Cm against Mi infection was monitored using RNA-Seq. More than 170000 transcripts were examined in Cm roots, and 2430 genes were subsequently identified as differentially expressed in response to Mi infection. Based on function annotation and orthologs finding, the potential mechanism of transcriptional factor, cytoskeleton, pathogen-related genes and plant hormone were assessed at the transcription level. A comparison of gene expression levels between Mi-infected Cm and cucumber plants revealed that cytoskeleton-related genes are key regulators of Cm resistance to Mi. We herein discuss the dual nature of cytoskeleton-related genes in the susceptibility and resistance of plant hosts to Mi. Our observations provide novel insights into the responses of Cm to Mi at the transcriptome level. The data generated in this study may be useful for elucidating the mechanism underlying resistance to RKNs in cucurbitaceous crops.