Pectate lyase genes from Radopholus similis and their application in pathotype identification.

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes. KEY POINTS: • Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed. • The expression of Rs-pels is different in two pathotypes of Radopholus similis. • A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Potential of different species of actinobacteria in the management of Meloidogyne javanica.

Root-knot nematodes (RKN) are one of the most harmful soil-borne plant pathogens in the world. Actinobacteria are known phytopathogen control agents. The aim of this study was to select soil actinobacteria with control potential against the RKN (Meloidogyne javanica) in tomato plants and to determine mechanisms of action. Ten isolates were tested and a significant reduction was observed in the number of M. javanica eggs, and galls 46 days after infestation with the nematode. The results could be explained by the combination of different mechanisms including parasitism and induction of plant defense response. The M. javanica eggs were parasited by all isolates tested. Some isolates reduced the penetration of juveniles into the roots. Other isolates using the split-root method were able to induce systemic defenses in tomato plants. The 4L isolate was selected for analysis of the expression of the plant defense genes TomLoxA, ACCO, PR1, and RBOH1. In plants treated with 4L isolate and M. javanica, there was a significant increase in the number of TomLoxA and ACCO gene transcripts. In plants treated only with M. javanica, only the expression of the RBOH1 and PR1 genes was induced in the first hours after infection. The isolates were identified using 16S rRNA gene sequencing as Streptomyces sp. (1A, 3F, 4L, 6O, 8S, 9T, and 10U), Kribbella sp. (5N), Kitasatospora sp. (2AE), and Lentzea sp. (7P). The efficacy of isolates from the Kitasatospora, Kribbella, and Lentzea genera was reported for the first time, and the efficacy of Streptomyces genus isolates for controlling M. javanica was confirmed. All the isolates tested in this study were efficient against RKN. This study provides the opportunity to investigate bacterial genera that have not yet been explored in the control of M. javanica in tomatoes and other crops.

Molecular Techniques for Root-Knot Nematode Identification.

Among plant-parasitic nematodes, root-knot nematodes (RKN), Meloidogyne spp., are the most important parasite infecting economically important crops globally and causing severe losses in crop production. The use of efficient nematode control methods against these parasites depends upon their correct detection in roots and soil samples. Currently, the use of integrated identification methods, including biochemical, molecular, and morphological-based characters, is preferred. But the techniques using morphology and phylogenetic analysis are time-consuming and not suitable for routine analysis. They have only been used for studies of cryptic species, which were identified using integrative taxonomy. Here we describe the enzymatic and molecular-based methods that have successfully been used in Brazil for more than 25 years in the Nematology Lab at Embrapa Genetic Resources and Biotechnology for routine analysis. This technique is a combination of isozyme esterase profiling and molecular markers, with the aim of having a rapid and correct diagnosis of Meloidogyne spp. populations from field and greenhouse.

Detection of Pathogenesis-Related (PR) Genes in Susceptible Tomato Plants Infected by Meloidogyne spp.

Meloidogyne species, as infective second-stage juveniles (J2s) larvae, are parasites able to attack host of relevant agronomic interest such as tomato plants. The identification of gene expression markers, useful to investigate the levels of root-knot nematode infection in the roots, is a fundamental tool in plant-pathogen interaction. The laboratory methods for analyzing the differential expression of pathogenesis-related (PR) genes constitute powerful tools for detecting the induced systemic acquired resistance defense response to M. incognita in infected plants and can be extended to all pathogen infection markers to obtain an early and sustainable control.

The completed mitochondrial genomes of Globodera vulgaris reveals new insights into the genus Globodera phylogeny.

Due to the highly conserved structure, animal mitochondrial genome (mtDNA) is widely used in classification, evolution, phylogeny, population genetic structure and other fields. We reported on the five circle multipartite mtDNAs of a newly described species of Globodera, Globodera vulgaris (Gv) from potatoes in China. The results showed that the mtDNA of Gv was obtained through second- and third-generation sequencing, with a total length of 42,995 bp. It contained 12 protein-coding genes, two rRNA genes and 17 tRNA genes, which were distributed in different subgenomic circles. Comparison of the differences in mtDNA among Gv, G. rostochiensis, G. pallida and G. ellingtonae showed that the size and arrangement of the genes in the mtDNA of the genus Globodera were variable and not conserved. The codon usage bias of the mitochondrial protein-coding gene of Gv showed that Gv might have originated from locally and more primitive group of existing Globodera. Based on the cytochrome c oxidase subunits I genes (COX1) and the nicotinamide adenine dinucleotide dehydrogenase subunits I genes (ND1), and the results showed that Gv was clustered with Globodera spp. according to the COX1 and ND1 in scmtDNA-V, while Gv was clustered with Meloidogyne spp. according to ND1 in scmtDNA-III. The results of this study provided a new basis for understanding the multipartite structure of mtDNA as a phylogenetic and taxonomic feature of the genus Globodera. The number of subgenomic circles is a diagnostic feature of species and the arrangement order and size of mitochondrial protein-coding genes also have important application value in species identification within the genus.

A Critical Appraisal of DNA Transfer from Plants to Parasitic Cyst Nematodes.

Plant-parasitic nematodes are one of the most economically important pests of crops. It is widely accepted that horizontal gene transfer-the natural acquisition of foreign genes in parasitic nematodes-contributes to parasitism. However, an apparent paradox has emerged from horizontal gene transfer analyses: On the one hand, distantly related organisms with very dissimilar genetic structures (i.e. bacteria), and only transient interactions with nematodes as far as we know, dominate the list of putative donors, while on the other hand, considerably more closely related organisms (i.e. the host plant), with similar genetic structure (i.e. introns) and documented long-term associations with nematodes, are rare among the list of putative donors. Given that these nematodes ingest cytoplasm from a living plant cell for several weeks, there seems to be a conspicuous absence of plant-derived cases. Here, we used comparative genomic approaches to evaluate possible plant-derived horizontal gene transfer events in plant parasitic nematodes. Our evidence supports a cautionary message for plant-derived horizontal gene transfer cases in the sugar beet cyst nematode, Heterodera schachtii. We propose a 4-step model for horizontal gene transfer from plant to parasite in order to evaluate why the absence of plant-derived horizontal gene transfer cases is observed. We find that the plant genome is mobilized by the nematode during infection, but that uptake of the said "mobilome" is the first major barrier to horizontal gene transfer from host to nematode. These results provide new insight into our understanding of the prevalence/role of nucleic acid exchange in the arms race between plants and plant parasites.

Unzipped genome assemblies of polyploid root-knot nematodes reveal unusual and clade-specific telomeric repeats.

Using long-read sequencing, we assembled and unzipped the polyploid genomes of Meloidogyne incognita, M. javanica and M. arenaria, three of the most devastating plant-parasitic nematodes. We found the canonical nematode telomeric repeat to be missing in these and other Meloidogyne genomes. In addition, we find no evidence for the enzyme telomerase or for orthologs of C. elegans telomere-associated proteins, suggesting alternative lengthening of telomeres. Instead, analyzing our assembled genomes, we identify species-specific composite repeats enriched mostly at one extremity of contigs. These repeats are G-rich, oriented, and transcribed, similarly to canonical telomeric repeats. We confirm them as telomeric using fluorescent in situ hybridization. These repeats are mostly found at one single end of chromosomes in these species. The discovery of unusual and specific complex telomeric repeats opens a plethora of perspectives and highlights the evolutionary diversity of telomeres despite their central roles in senescence, aging, and chromosome integrity.

Transcriptomic changes in soybean underlying growth promotion and defense against cyst nematode after Bacillus simplex Sneb545 treatment.

Bacillus simplex Sneb45 is a plant-growth-promoting rhizobacterium that promotes soybean growth and systemic resistance to cyst nematode. To investigate transcriptional changes in soybean roots in response to B. simplex Sneb45 treatment, transcriptome analysis and quantitative real-time PCR were conducted to detect and validate the differentially expressed genes (DEGs). In total, 19,109 DEGs were obtained. After B. simplex Sneb545 treatment, 970 and 1265 genes were up- and down-regulated at 5 days post-inoculation (dpi), respectively, and 142 and 47 genes were up- and down-regulated at 10 dpi, respectively, compared with untreated soybean roots. Functional annotation of DEGs indicated that B. simplex Sneb545 regulated soybean growth and defense against cyst nematode possibly through genes related to auxin, gibberellin, and NB-LRR protein. In addition, GO and KEGG enrichment analyses indicated that the DEGs were enriched in metabolism, signal transduction, and plant-pathogen interaction pathways. Moreover, the auxin and gibberellin contents were lower in B. simplex Sneb545-treated soybean roots than in untreated roots at 5 dpi. B. simplex Sneb545 possibly altered the expression of wound-induced protein and NAC transcription factor to regulate soybean growth and defense against cyst nematode. Our study provided deep insights into the alterations in soybean transcriptome after exposure to B. simplex Sneb45 and a theoretical basis for further exploring molecular functions underlying the biological control activity of B. simplex Sneb545.

Molecular and functional characterization of chemosensory genes from the root-knot nematode Meloidogyne graminicola.

BACKGROUND: Root-knot nematode Meloidogyne graminicola has emerged as a major threat in rice agroecosystems owing to climate change-induced changes in cultivation practices. Synthetic nematicides are continually being withdrawn from the nematode management toolbox because of their ill effects on the environment. A sustainable strategy would be to develop novel nematicides or resistant plants that would target nematode sensory perception, which is a key step in the host finding biology of plant-parasitic nematodes (PPNs). However, compared to the extensive literature on the free-living nematode Caenorhabditis elegans, negligible research has been performed on PPN chemosensory biology. RESULTS: The present study characterizes the five chemosensory genes (Mg-odr-7, Mg-tax-4, Mg-tax-4.1, Mg-osm-9, and Mg-ocr-2) from M. graminicola that are putatively associated with nematode host-finding biology. All the genes were highly transcribed in the early life stages, and RNA interference (RNAi)-induced downregulation of each candidate gene perturbed the normal behavioural phenotypes of M. graminicola, as determined by examining the tracking pattern of juveniles on Pluronic gel medium, attraction to and penetration in rice root tip, and developmental progression in rice root. In addition, a detrimental effect on nematode chemotaxis towards different volatile and nonvolatile organic compounds and host root exudates was documented. CONCLUSION: Our findings enrich the existing literature on PPN chemosensory biology and can supplement future research aimed at identifying a comprehensive chemosensory signal transduction pathway in PPNs.

Comparative Transcriptome Analysis Reveals the Molecular Mechanism of Bacillus velezensis GJ-7 Assisting Panax notoginseng against Meloidogyne hapla.

The rhizosphere bacteria Bacillus velezensis GJ-7, as a biological control agent (BCA), has significant biological control effects on Meloidogyne hapla, and has strong colonization ability in the root of Panax notoginseng. In this study, we conducted a comparative transcriptome analysis using P. notoginseng plant roots treated with B. velezensis GJ-7 or sterile water alone and in combination with M. hapla inoculation to explore the interactions involving the P. notoginseng plant, B. velezensis GJ-7, and M. hapla. Four treatments from P. notoginseng roots were sequenced, and twelve high-quality total clean bases were obtained, ranging from 3.57 to 4.74 Gb. The Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment showed that numerous DEGs are involved in the phenylpropane biosynthesis pathway and the MAPK signaling pathway in the roots of P. notoginseng with B. velezensis GJ-7 treatments. The analysis results of the two signaling pathways indicated that B. velezensis GJ-7 could enhance the expression of lignin- and camalexin-synthesis-related genes in plant roots to resist M. hapla. In addition, B. velezensis GJ-7 could enhance plant resistance to M. hapla by regulating the expression of resistance-related genes and transcription factors (TFs), including ETR, ERF, ChiB, WRKY22, and PR1. The expression of plant disease resistance genes in the roots of P. notoginseng with different treatments was validated by using real-time quantitative PCR (qRT-PCR), and the results were consistent with transcriptome sequencing. Taken together, this study indicated that B. velezensis GJ-7 can trigger a stronger defense response of P. notoginseng against M. hapla.

Integrative taxonomy and phylogenetic relationships between representatives of genera within Merliniinae (Nematoda: Merliniidae), with new data on fourteen known and one unknown species.

The application of integrative taxonomic approaches is useful to species delineation based on a combination of distinct types of characters, here morphological features and ribosomal DNA sequences. In this study, we surveyed ectoparasitic nematodes of the subfamily Merliniinae in cultivated and natural environments in Iran. Results of morphological and morphometrical studies, light and scanning electron microscopic observations, and molecular analyses allowed us the identification of fourteen known and one unknown species including representatives of the genera Amplimerlinius (five species), Geocenamus (one species), Merlinius (three species), Nagelus (two species), Paramerlinius (one species), Scutylenchus (two species), and Telomerlinius (one species). The unknown species, Scutylenchus sp., characterized by having 35-50 incisures at mid-body; lateral field with 6 longitudinal incisures; lip region slightly offset by a constriction, flattened at front end; bearing 5-7 annuli; cephalic framework not refractive; stylet robust, 18.3-27 µm long; post anal intestinal sac absent; tail elongate conical, dorsally convex, with 24 (19-28) annuli in ventral side, ending to a smooth terminus and males common; spicules 24.5-31 µm long. The phylogenetic analyses were carried out using molecular data from nuclear ribosomal DNA (rDNA) genes viz. D2-D3 expansion segments of the large ribosomal subunit (28S rRNA), partial small ribosomal subunit (18S rRNA), and internal transcribed spacer (ITS). The molecular variability of D2-D3 expansion segments of the 28S rRNA and partial 18S rRNA was low in this family in comparison to the ITS region, which could be a more helpful molecular marker in species and genus identification.

Pathogenicity of the root lesion nematode Pratylenchus neglectus depends on pre-culture conditions.

The ability of a plant parasitic nematode to infect and reproduce within a host plant depends on its genotype and the environmental conditions before and during infection. We studied the culturing conditions of the root lesion nematode Pratylenchus neglectus to produce inoculum for plant infection tests. Nematodes were either cultivated on carrot calli for different periods or directly isolated from the roots of the host plants. After infection of wheat and barley plants in the greenhouse, nematodes were quantified by RT-qPCR and by visual counting of the nematodes. We observed drastically reduced infection rates after long-term (> 96 weeks) cultivation on carrot callus. In contrast, fresh isolates from cereal roots displayed much higher pathogenicity. We recommend using root lesion nematodes cultivated on carrot calli no longer than 48 weeks to guarantee uniform infection rates.

Transcriptome-Based Comparative Expression Profiling of Sweet Potato during a Compatible Response with Root-Knot Nematode Meloidogyne incognita Infection.

M. incognita, a root-knot nematode (RKN), infects the roots of several important food crops, including sweet potato (Ipomoea batatas Lam.), and severely reduces yields. However, the molecular mechanisms underlying infection remain unclear. Previously, we investigated differential responses to RKN invasion in susceptible and resistant sweet potato cultivars through RNA-seq-based transcriptome analysis. In this study, gene expression similarities and differences were examined in RKN-susceptible sweet potato cultivars during the compatible response to RKN infection. Three susceptible cultivars investigated in previous research were used: Dahomi (DHM), Shinhwangmi (SHM), and Yulmi (YM). Of the three cultivars, YM had the highest number of genes with altered expression in response to infection. YM was also the cultivar with the highest susceptibility to RKN. Comparisons among cultivars identified genes that were regulated in more than one cultivar upon infection. Pairwise comparisons revealed that YM and DHM shared the most regulated genes, whereas YM and SHM shared the lowest number of regulated genes. Five genes were up-regulated, and two were down-regulated, in all three cultivars. Among these, four genes were highly up-regulated in all cultivars: germin-like protein, anthranilate synthase α subunit, isocitrate lyase, and uncharacterized protein. Genes were also identified that were uniquely regulated in each cultivar in response to infection, suggesting that susceptible cultivars respond to infection through shared and cultivar-specific pathways. Our findings expand the understanding of the compatible response to RKN invasion in sweet potato roots and provide useful information for further research on RKN defense mechanisms.

Species-specific microRNA discovery and target prediction in the soybean cyst nematode.

The soybean cyst nematode (SCN) is a devastating pathogen for economic and food security considerations. Although the SCN genome has recently been sequenced, the presence of any miRNA has not been systematically explored and reported. This paper describes the development of a species-specific SCN miRNA discovery pipeline and its application to the SCN genome. Experiments on well-documented model nematodes (Caenorhabditis elegans and Pristionchus pacificus) are used to tune the pipeline's hyperparameters and confirm its recall and precision. Application to the SCN genome identifies 3342 high-confidence putative SCN miRNA. Prediction specificity within SCN is confirmed by applying the pipeline to RNA hairpins from known exonic regions of the SCN genome (i.e., sequences known to not be miRNA). Prediction recall is confirmed by building a positive control set of SCN miRNA, based on a limited deep sequencing experiment. Interestingly, a number of novel miRNA are predicted to be encoded within the intronic regions of effector genes, known to be involved in SCN parasitism, suggesting that these miRNA may also be involved in the infection process or virulence. Beyond miRNA discovery, gene targets within SCN are predicted for all high-confidence novel miRNA using a miRNA:mRNA target prediction system. Lastly, cross-kingdom miRNA targeting is investigated, where putative soybean mRNA targets are identified for novel SCN miRNA. All predicted miRNA and gene targets are made available in appendix and through a Borealis DataVerse open repository ( https://borealisdata.ca/dataset.xhtml?persistentId=doi:10.5683/SP3/30DEXA ).

An Innovative Approach for Biocontrol of Meloidogyne incognita in Ginger Using Potential Bacteria Isolated from Indian Himalayas.

The prevalence of Meloidogyne incognita, a severe root-knot nematode, is alarmingly high in the production of ginger-a main cash crop of Himachal Pradesh, a Himalayan state of India. In order to control this through natural means, the nematicidal potential of plant growth-promoting rhizobacteria (PGPR) against M. incognita was analyzed. This is an effective alternative solution to manage nematode incidence as compared to hazardous chemicals under protected and field cultivation of ginger. In the present study an attempt has been made to isolate, characterize, and identify potential rhizobacteria associated with ginger rhizosphere and endosphere. In total, 169 bacterial isolates were isolated from ginger (Zingiber officinale) rhizosphere and endosphere of 4 different sites of Sirmaur district, screened out for multifarious PGP traits showing positive results. The combined cluster analysis and 16S rRNA genotypic analysis of selected bacterial isolates revealed that Serratia marcescens FS-23, Pseudochrobacter sp. GS-15, Stonotrophomonas pavanii HER-9, Pseudomonas brassicacearum HER-20 and Serratia marcescens IS-2 exhibited highest PGP traits. All tested bacterial isolates were capable of exerting a significant effect on mortality of juvenile M. incognita ranging upto 40-90% in laboratory experiments. Further a consortium of these screened isolates showed 86.67% reduction in gall formation by M. incognita in lab conditions. The remarkable increase to 93.24% with 138.73 q/ha with application of charcoal based bio-formulation of consortium without adding any chemical fertilizer was observed in field trials of Nohradhar of Sirmaur district. An alternative choice as a biocontrol agent as well as for PGP activities, the novel and most robust isolate Serratia marcescens IS-2 had revealed to have a variety of bioactive metabolic products with abilities against nematodes, bacteria, and fungi.

Host response of Nicotiana benthamiana to the parasitism of five populations of root-lesion nematode, Pratylenchus coffeae, from China.

In a recent survey of nematodes associated with tobacco in Shandong, China, the root-lesion nematode Pratylenchus coffeae was identified using a combination of morphology and molecular techniques. This nematode species is a serious parasite that damages a variety of plant species. The model plant benthi, Nicotiana benthamiana, is frequently used to study plant-disease interactions. However, it is not known whether this plant species is a host of P. coffeae. The objectives of this study were to evaluate the parasitism and pathogenicity of five populations of the root-lesion nematode P. coffeae on N. benthamiana.N. benthamiana seedlings with the same growth status were chosen and inoculated with 1,000 nematodes per pot. At 60 days after inoculation, the reproductive factors (Rf = final population densities (Pf)/initial population densities (Pi)) for P. coffeae in the rhizosphere of N. benthamiana were all more than 1, suggesting that N. benthamiana was a good host plant for P. coffeae.Nicotiana. benthamiana infected by P. coffeae showed weak growth, decreased tillering, high root reduction, and noticeable brown spots on the roots. Thus, we determined that the model plant N. benthamiana can be used to study plant-P. coffeae interactions.

Diversity of bioprotective microbial organisms in Upper Region of Assam and its efficacy against Meloidogyne graminicola.

Meloidogyne graminicola has a well-established negative impact on rice yield in transplanted and direct-seeded rice, resulting in yield losses of up to 20 to 90 percent. Studies were undertaken to isolate potential native strains of bio-control agents to manage the devastating Rice Root Knot Nematode (M. graminicola). Eighteen bacterial strains and eleven fungal strains were isolated from the rhizosphere of crops like rice, okra, ash gourd, chili, beans and cucumber, enveloping diverse soil types from the Upper Brahmaputra Valley region of Assam. Six bacterial strains were gram-positive according to morphological results, while twelve others stained negatively. Fifteen bacteria were rod-shaped, two were coccus and one was diplococcus, and all the bacterial isolates showed signs of movement. All the bacterial strains exhibited positivity for gelatin hydrolysis and catalase test. Seven bacteria showed positive, while eleven showed negative reactions to possess the ability to deduce carbon and energy from citrate. The study of the in vitro efficacy of the twenty-nine bacterial and fungal isolates tested against second-stage juveniles (J2) of Meloidogyne graminicola revealed that all the bacterial and fungal isolates potentially inhibited the test organism and caused significant mortality over sterile water treatment. The promising bacterial and fungal isolates that exhibited mortality above 50% were identified as BSH8, BTS4, BTS5, BJA15, FJB 11 and FSH5. The strain BSH8 exhibited the best result of mortality, with 80.79% mortality against J2 of M. graminicola. The strain BTS4 and BTS5 expressed mortality of 71.29% and 68.75% under in-vitro conditions and were significant. The effective and promising bioagents were identified using the 16 S rRNA sequencing as Bacillis subtilis (BSH8), Bacillus velezensis (BTS4), Alcaligenes faecalis (BTS5), Rhizobium pusense (BJA15), Talaromyces allahabadensis (FSH5) and Trichoderma asperellum (FJB11). These results indicated the microorganism's potential against M. graminicola and its potential for successful biological implementation. Further, the native strains could be tested against various nematode pests of rice in field conditions. Its compatibility with various pesticides and the implication of the potential strains in integrated pest management can be assessed.

DNA-based assessment of root lesion nematode infections in cereal roots.

Root lesion nematodes (RLN) of the genus Pratylenchus are causing significant damage in cereal production worldwide. Due to climate change and without efficient and environment-friendly treatments, the damages through RLNs are predicted to increase. Microscopic assessments of RLNs in the field and the greenhouses are time-consuming and laborious. As a result, cereal breeders have mostly ignored this pest. We present a method measuring RLN in infected cereal roots using a standardized PCR approach. Publicly available Pratylenchus neglectus primer combinations were evaluated. An optimal primer combination for RT-qPCR assay was identified to detect and quantify P. neglectus within infected cereal roots. Using the RT-qPCR detection assay, P. neglectus could be clearly distinguished from other plant parasitic nematodes. We could identify P. neglectus DNA in barley and wheat roots as low as 0.863 and 0.916 ng/µl of total DNA, respectively. A single P. neglectus individual was detected in water suspension and within barley and wheat roots. The RT-qPCR detection assay provides a robust and accurate alternative to microscopic nematode identification and quantification. It could be of interest for resistance breeding, where large populations must be screened to detect and quantify P. neglectus in farmer's fields.

Activity of root-knot nematodes associated with composition of a nematode-attached microbiome and the surrounding soil microbiota.

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

Morphologic, Morphometric, and Molecular Characterization of Vietnamese Populations of Meloidogyne incognita.

Meloidogyne incognita is considered the most damaging and common root-knot nematode to numerous host plants worldwide. During a survey of nematodes in Vietnam, 1,106 samples from 22 different plant species were collected. M. incognita was recorded on 13 of the 22 host plants. Four populations of M. incognita from four host plants were chosen for comparison and confirmation of their morphologic, morphometric, and molecular characteristics. Genetically based phylogenetic trees were constructed to show relationships among root-knot nematodes. Molecular barcodes of four gene regions, ITS, D2-D3 of 28S rRNA, COI, and Nad5 mtDNA, integrated with morphologic and morphometric data were used as reliable references for molecular identification of M. incognita. Our analyses indicated that tropical root-knot nematodes are very similar in characterization of ITS, D2-D3 of 28S rRNA, and COI regions. However, these gene regions can be used to separate the tropical root-knot nematode group from other groups. On the other hand, the analysis of Nad5 mtDNA and multiplex-PCR with specific primers can be used to distinguish tropical species.