Search

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).) more...

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).) more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

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.) more...

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. more...

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. more...

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. more...

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. more...

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.) more...

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. more...

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. more...

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. more...

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.) more...

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.) more...

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. more...

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. more...

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.) more...

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. more...

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.) more...

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. more...

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. more...

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. more...

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. more...

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. more...

The increased incidence of the crown gall disease caused by Agrobacterium tumefaciens has long been associated with activities of root-knot nematodes (Meloidogyne spp.). Pot experiments on tomato were designed to assess plant vitality, nematode reproduction, and crown gall incidence in combined infection with Agrobacterium and Meloidogyne spp. on tomato roots. Results suggest that tomato plants infected with pathogenic A. tumefaciens 2 days before the nematodes show enhanced plant defense against M. ethiopica resulting in lower egg and gall counts on roots 45 and 90 days postinoculation (dpi); no significantly enhanced defense was observed when the plant was inoculated with bacteria and nematodes at the same time. Split-root experiments also showed that the observed interaction was systemic. Reverse-transcription quantitative polymerase chain reaction analysis that targeted several genes under plant hormonal control suggests that the suppression was mediated via systemic acquired resistance by the pathogenesis-related protein 1 and that M. ethiopica did not enhance the defense reaction of tomato against Agrobacterium spp. Nematodes completely inhibited tumor growth in a 45-day experiment if inoculated onto the roots before the pathogenic bacteria. We conclude that the observed antagonism in the tested pathosystem was the result of initially strong plant defense that was later suppressed by the invading pathogen and pest. more...

Phytoalexins phenalenones (PNs) are phytochemicals biosynthesized inside the plant in responsive to exterior threat. PNs are excellent type-II photosensitizers, which efficiently produce singlet oxygen upon light irradiation. Based on the core functional structure of PNs, novel PN derivatives were synthesized here and their singlet oxygen generating abilities and their phototoxicity were evaluated. At the presence of light, these PNs have photoinduced toxicity towards Aedes albopictus larvae and nematode Meloidogyne incognita, while the activity lost in the dark. The obvious tissue damage was observed on the treated mosquito larvae and nematode due to the generation of singlet oxygen. Our results revealed the potential of phenalenones as photoactivated agents for mosquito and root-knot nematode management together with light., Competing Interests: The authors declare no competing financial interests. more...

Five viruses were previously discovered infecting soybean cyst nematodes (SCN; Heterodera glycines) from greenhouse cultures maintained in Illinois. In this study, the five viruses [ScNV, ScPV, ScRV, ScTV, and SbCNV-5] were detected within SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN from 43 greenhouse cultures and 25 field populations were analyzed using qRT-PCR. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Viruses were found at similar or lower levels within field populations of SCN compared with greenhouse cultures of North Carolina populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mixture of fewer viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Several of these SCN viruses were also detected in Heterodera trifolii (clover cyst) and Heterodera schachtii (beet cyst), but not the other cyst, root-knot, or reniform nematode species tested. Viruses were not detected within soybean host plant tissue. If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation., Competing Interests: The authors have declared that no competing interests exist. more...

Taxonomy: Superkingdom Eukaryota; Kingdom Metazoa; Phylum Nematoda; Class Chromadorea; Order Tylenchida; Suborder Tylenchina; Infraorder Tylenchomorpha; Superfamily Tylenchoidea; Family Meloidogynidae; Subfamily Meloidogyninae; Genus Meloidogyne., Biology: Microscopic non-segmented roundworm. Plant pathogen; obligate sedentary endoparasitic root-knot nematode. Reproduction: facultative meiotic parthenogenetic species in which amphimixis can occur at a low frequency (c. 0.5%); relatively fast life cycle completed in 19-27 days on rice depending on the temperature range., Host Range: Reported to infect over 100 plant species, including cereals and grass plants, as well as dicotyledonous plants. Main host: rice (Oryza sativa)., Symptoms: Characteristic hook-shaped galls (root swellings), mainly formed at the root tips of infected plants. Alteration of the root vascular system causes disruption of water and nutrient transport, stunting, chlorosis and loss of vigour, resulting in poor growth and reproduction of the plants with substantial yield losses in crops., Disease Control: Nematicides, chemical priming, constant immersion of rice in irrigated fields, crop rotation with resistant or non-host plants, use of nematode-free planting material. Some sources of resistance to Meloidogyne graminicola have been identified in African rice species (O. glaberrima and O. longistaminata), as well as in a few Asian rice cultivars., Agronomic Importance: Major threat to rice agriculture, particularly in Asia. Adapted to flooded conditions, Meloidogyne graminicola causes problems in all types of rice agrosystems., (© 2016 BSPP and John Wiley & Sons Ltd.) more...

Meloidogyne enterolobii is one of the most important plant-parasitic nematodes that can overcome the Mi-1 resistance gene and damage many economically important crops. Translationally controlled tumour protein (TCTP) is a multifunctional protein that exists in various eukaryotes and plays an important role in parasitism. In this study, a novel M. enterolobii TCTP effector, named MeTCTP, was identified and functionally characterized. MeTCTP was specifically expressed within the dorsal gland and was up-regulated during M. enterolobii parasitism. Transient expression of MeTCTP in protoplasts from tomato roots showed that MeTCTP was localized in the cytoplasm of the host cells. Transgenic Arabidopsis thaliana plants overexpressing MeTCTP were more susceptible to M. enterolobii infection than wild-type plants in a dose-dependent manner. By contrast, in planta RNA interference (RNAi) targeting MeTCTP suppressed the expression of MeTCTP in infecting nematodes and attenuated their parasitism. Furthermore, MeTCTP could suppress programmed cell death triggered by the pro-apoptotic protein BAX. These results demonstrate that MeTCTP is a novel plant-parasitic nematode effector that promotes parasitism, probably by suppressing programmed cell death in host plants., (© 2016 BSPP and John Wiley & Sons Ltd.) more...

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita . Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode., Competing Interests: The authors declare no conflict of interest. more...

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.) more...

Nematodes are important pests of soybean throughout the world and cause high yield losses. As a control strategy, the identification of resistance genes is an important aim of breeding studies. Plants possess resistance genes (R), which are responsible for the recognition of pathogens and activation of the defense system. R genes and resistance gene analogs (RGAs) possess conserved domains, from which nucleotide-binding site is the most common. Using degenerate primers originating from these domains, it is possible to identify and isolate sequences of R and RGA genes. In this study, soybean genotypes resistant to the nematodes Heterodera glycines, Meloidogyne incognita, M. javanica, and M. enterolobii were compared by the use of RGAs and simple sequence repeat (SSR) markers. Forty-six soybean genotypes were studied, including plant introductions (PIs), commercial crops, and source of resistance genotypes. Thirteen combinations of RGA primers and different SSRs linked to QTLs were used to confirm resistance to soybean cyst nematodes (SCN). Fragments associated with resistance to the studied nematodes were amplified in the source of resistance and PI genotypes. RGA markers were efficient at distinguishing groups of genotypes that were resistant and susceptible to Meloidogyne spp and SCN. Combinations of specific primers were identified through their ability to amplify nucleotide sequences from possible resistance candidate genes. SSR markers contributed to the analysis of SCN race specificity, showing that the QTLs identified by these markers are distinct from those identified by RGA markers., Competing Interests: The authors declare no conflict of interest. more...

The effect of carbon source on efficacy of anaerobic soil disinfestation (ASD) toward suppression of apple root infection by Rhizoctonia solani AG-5 and Pratylenchus penetrans was examined. Orchard grass (GR), rice bran (RB), ethanol (ET), composted steer manure (CM), and Brassica juncea seed meal (SM) were used as ASD carbon inputs, with plant assays conducted in natural and pasteurized orchard soils. Subsequent studies investigated the effect of GR application rate used in ASD on control of these pathogens. In general, apple root infection by R. solani AG-5 was significantly lower in ET, GR, RB, and SM ASD treatments compared with the control. Among different ASD treatments, apple seedling growth was significantly greater when GR or SM was used as the carbon input relative to all other ASD treatments. R. solani AG-5 DNA abundance was significantly reduced in all ASD treatments, regardless of amendment type, compared with the control. In independent experiments, ASD-GR was consistently superior to ASD-CM for limiting pathogen activity in soils. ASD treatment with a grass input rate of 20 t ha(-1) provided superior suppression of P. penetrans but grass application rate did not affect ASD efficacy in control of R. solani AG-5. The soil microbiome from ASD-GR-treated soils was clearly distinct from the control and ASD-CM-treated soils. In contrast, composition of the microbiome from control and ASD-CM-treated soils could not be differentiated. Comparative results from pasteurized and nonpasteurized soils suggest that there is potential for GR based ASD treatment to recruit microbial elements that persist over the anaerobic phase of soil incubation, which may functionally contribute to disease suppression. When ASD was conducted with GR, microbial diversity was markedly reduced relative to the control or ASD-CM soil suggesting that this parameter, typically associated with system resilience, was not instrumental to the function of ASD-induced soil suppressiveness. more...

Root-knot nematodes secrete proteinaceous effectors into plant tissues to facilitate infection by suppressing host defences and reprogramming the host metabolism to their benefit. Meloidogyne graminicola is a major pest of rice (Oryza sativa) in Asia and Latin America, causing important crop losses. The goal of this study was to identify M. graminicola pathogenicity genes expressed during the plant-nematode interaction. Using the dual RNA-sequencing (RNA-seq) strategy, we generated transcriptomic data of M. graminicola samples covering the pre-parasitic J2 stage and five parasitic stages in rice plants, from the parasitic J2 to the adult female. In the absence of a reference genome, a de novo M. graminicola transcriptome of 66 396 contigs was obtained from those reads that were not mapped on the rice genome. Gene expression profiling across the M. graminicola life cycle revealed key genes involved in nematode development and provided insights into the genes putatively associated with parasitism. The development of a 'secreted protein prediction' pipeline revealed a typical set of proteins secreted by nematodes, as well as a large number of cysteine-rich proteins and putative nuclear proteins. Combined with expression data, this pipeline enabled the identification of 15 putative effector genes, including two homologues of well-characterized effectors from cyst nematodes (CLE-like and VAP1) and a metallothionein. The localization of gene expression was assessed by in situ hybridization for a subset of candidates. All of these data represent important molecular resources for the elucidation of M. graminicola biology and for the selection of potential targets for the development of novel control strategies for this nematode species., (© 2015 BSPP AND JOHN WILEY & SONS LTD.) more...

Background: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes., Results: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking., Conclusions: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action. more...

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii. more...

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction. more...

Background: The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium., Results: The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure., Conclusion: This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process. more...

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism. more...

Within the phylum Nematoda, plant-parasitism is hypothesized to have arisen independently on at least four occasions. The most economically damaging plant-parasitic nematode species, and consequently the most widely studied, are those that feed as they migrate destructively through host roots causing necrotic lesions (migratory endoparasites) and those that modify host root tissue to create a nutrient sink from which they feed (sedentary endoparasites). The false root-knot nematode Nacobbus aberrans is the only known species to have both migratory endoparasitic and sedentary endoparasitic stages within its life cycle. Moreover, its sedentary stage appears to have characteristics of both the root-knot and the cyst nematodes. We present the first large-scale genetic resource of any false-root knot nematode species. We use RNAseq to describe relative abundance changes in all expressed genes across the life cycle to provide interesting insights into the biology of this nematode as it transitions between modes of parasitism. A multigene phylogenetic analysis of N. aberrans with respect to plant-parasitic nematodes of all groups confirms its proximity to both cyst and root-knot nematodes. We present a transcriptome-wide analysis of both lateral gene transfer events and the effector complement. Comparing parasitism genes of typical root-knot and cyst nematodes to those of N. aberrans has revealed interesting similarities. Importantly, genes that were believed to be either cyst nematode, or root-knot nematode, "specific" have both been identified in N. aberrans. Our results provide insights into the characteristics of a common ancestor and the evolution of sedentary endoparasitism of plants by nematodes., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.) more...

Heterodera glycines, the soybean cyst nematode (SCN), is a subterranean root pathogen that causes the most damaging disease of soybean in the USA. A novel nematode virus genome, soybean cyst nematode virus 5 (SbCNV-5), was identified in RNA sequencing data from SCN eggs and second-stage juveniles. The SbCNV-5 RNA-dependent RNA polymerase and RNA helicase domains had homology to pestiviruses in the family Flaviviridae, suggesting that SbCNV-5 is a positive-polarity ssRNA virus. SbCNV-5 RNA was present in all nematode developmental stages, indicating a transovarial mode of transmission, but is also potentially sexually transmitted via the male. SbCNV-5 was common in SCN laboratory cultures and in nematode populations isolated from the field. Transmission electron microscopy of sections from a female SCN showed virus particles budding from the endoplasmic reticulum and in endosomes. The size of the viral genome was 19 191 nt, which makes it much larger than other known pestiviruses. Additionally, the presence of a methyltransferase in the SbCNV-5 genome is atypical for a pestivirus. When cDNA sequences were mapped to the genome of SbCNV-5, a disproportionate number aligned to the 3' NTR, suggesting that SbCNV-5 produces a subgenomic RNA, which was confirmed by RNA blot analysis. As subgenomic RNAs and methyltransferases do not occur in pestiviruses, we conclude that SbCNV-5 is a new flavivirus infecting SCNs., (© 2014 The Authors.) more...