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.

A Lab-on-a-Chip Approach for the Detection of the Quarantine Potato Cyst Nematode Globodera pallida.

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor's electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor's potential as a bioanalytical device for field applications and border phytosanitary inspections.

Characterization of a Root-Knot Nematode Infecting Aconitum carmichaelii in Southwest China.

Growth of the Chinese herbal medicine industry has resulted in several new pests and diseases. China is one of the world largest producers of monkshood (Aconitum carmichaelii Debx.), but an unidentified root-knot nematode has become a significant pest in the southwestern provinces of Yunnan and Sichuan. Morphological characteristics and the ribosomal DNA-internal transcribed spacer and D2-D3 region of the 28S ribosomal RNA gene sequences were used to identify the nematode as Meloidogyne hapla. Through investigation, this is the first report of M. hapla infecting monkshood in Yunnan and Sichuan Provinces.

A Novel Rhabdovirus Associated with the Idaho Population of Potato Cyst Nematode Globodera pallida.

Globodera pallida, a potato cyst nematode (PCN), is a quarantine endoparasitic pest of potato (Solanum tuberosum) in the US due to its effects on yield and quality of potato tubers. A new rhabdovirus, named potato cyst nematode rhabdovirus (PcRV), was revealed and characterized in the G. pallida populations collected in Idaho through use of high-throughput sequencing (HTS) and RT-PCR and found to be most closely related to soybean cyst nematode rhabdovirus (ScRV). PcRV has a 13,604 bp long, single-stranded RNA genome encoding five open reading frames, including four rhabdovirus-specific genes, N, P, G, and L, and one unknown gene. PcRV was found present in eggs, invasive second-stage juveniles, and parasitic females of G. pallida, implying a vertical transmission mode. RT-PCR and partial sequencing of PcRV in laboratory-reared G. pallida populations maintained over five years suggested that the virus is highly persistent and genetically stable. Two other Globodera spp. reproducing on potato and reported in the US, G. rostochiensis and G. ellingtonae, tested negative for PcRV presence. To the best of our knowledge, PcRV is the first virus experimentally found infecting G. pallida. Based on their similar genome organizations, the phylogeny of their RNA-dependent RNA polymerase domains (L gene), and relatively high identity levels in their protein products, PcRV and ScRV are proposed to form a new genus, provisionally named "Gammanemrhavirus", within the family Rhabdoviridae.

A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase.

Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.

Comprehensive analysis of codon usage pattern in Withania somnifera and its associated pathogens: Meloidogyne incognita and Alternaria alternata.

Meloidogyne incognita (Root-knot nematode) and Alternaria alternata (fungus) were among the dominant parasites of the medicinal plant Withania somnifera. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation is lacking. The present study elucidates evolutionary and codon usage bias analysis of W. somnifera (host plant), M. incognita (root-knot nematode) and A. alternata (fungal parasite). The results of the present study revealed a weak codon usage bias prevalent in all the three organisms. Based on the nucleotide analysis, genome of W. somnifera and M. incognita was found to be A-T biased while A. alternata had GC biased genome. We found high similarity of CUB pattern between host and its nematode pathogen as compared to the fungal pathogen. Inclusively, both the evolutionary forces influenced the CUB in host and its associated pathogens. However, neutrality plot indicated the pervasiveness of natural selection on CUB of the host and its pathogens. Correspondence analysis revealed the dominant effect of mutation on CUB of W. somnifera and M. incognita while natural selection was the main force affecting CUB of A. alternata. Taken together the present study would provide some prolific insight into the role of codon usage bias in the adaptability of pathogens to the host's environment for establishing parasitic relationship.

Opposite Beet Cyst Nematode Infection Phenotypes of Transgenic Arabidopsis Between Overexpressing GmSNAP18 and AtSNAP2 and Between Overexpressing GmSHMT08 and AtSHMT4.

The rhg1-a GmSNAP18 (an α-SNAP) and Rhg4 GmSHMT08 are two major cloned genes conferring soybean cyst nematode resistance in Peking-type soybeans, but the application of α-SNAPs and SHMTs in cyst nematode management remains elusive. In this study, GmSNAP18 and GmSHMT08, together with their orthologs in Arabidopsis, AtSNAP2 (an α-SNAP) and AtSHMT4, were individually transformed into Arabidopsis Col-0 to generate the transgenic lines, and the growth of transgenic plants, beet cyst nematode (BCN) infection phenotypes, and AtSNAP2, AtSHMT4, and AtPR1 expression patterns were analyzed using Arabidopsis-BCN compatible interaction system, in addition with protein-protein interaction assay. Pulldown and BiFC assays revealed that GmSNAP18 and GmSHMT08 interacted with AtSHMT4 and AtSNAP2, respectively. Plant root growth was not impacted by overexpression of GmSNAP18 and AtSNAP2. However, overexpression of GmSHMT08 and AtSHMT4 both increased plant height, additionally, overexpression of GmSHMT08 decreased rosette leaf size. Overexpression of GmSNAP18 and GmSHMT08 both suppressed AtPR1 expression and significantly enhanced BCN susceptibility, while overexpression of AtSNAP2 and AtSHMT4 both substantially boosted AtPR1 expression and remarkably enhanced BCN resistance, in transgenic Arabidopsis. Overexpression of GmSNAP18 reduced, while overexpression of AtSNAP2 unaltered AtSHMT4 expression. Overexpression of GmSHMT08 and AtSHMT4 both suppressed AtSNAP2 expression in transgenic Arabidopsis. Thus, different expression patterns of AtPR1 and AtSHMT4 are likely associated with opposite BCN infection phenotypes of Arabidopsis between overexpressing GmSNAP18 and AtSNAP2, and between overexpressing GmSHMT08 and AtSHMT4; and boosted AtPR1 expression are required for enhanced BCN resistance in Arabidopsis. All these results establish a basis for extension of α-SNAPs and SHMTs in cyst nematode management.

Early Detection and Temporal Dynamics of Pratylenchus scribneri Infection in Potato Roots Determined Using Quantitative PCR and Root Staining.

The root-lesion nematode, Pratylenchus scribneri, is a migratory endo-parasitic nematode that impacts potato production on a large scale. Effective management of this nematode requires an understanding of its population dynamics alongside early detection. Typically, the nematode population estimates are made from infested soil; however, considering the endo-migratory lifestyle of this nematode, it also is crucial to determine the nematode population residing inside the host roots. In this study, a SYBR green-based quantitative real-time PCR (qPCR) assay was developed for detection and quantification of P. scribneri in potato roots. The assay used a previously reported primer pair (ITS-2F/ITS-2R), and it proved to be specific and sensitive, detecting as low as 1/128th equivalents of a P. scribneri individual per 0.2 g of potato roots. The robustness of the assay was reflected in high correlation observed between the P. scribneri densities determined microscopically and the densities detected by qPCR in artificially inoculated (R2 = 0.93) and naturally infected (R2 = 0.73) root samples. A time-course experiment conducted in the greenhouse using qPCR detected P. scribneri in potato roots as early as 5 days after planting. The results correlated well with the microscopic observations (R2 = 0.80) and were complemented further with root staining. Additionally, three P. scribneri reproduction peaks were observed during one 3-month growth cycle of potato. Overall, the assay developed in this study is specific to P. scribneri in DNA extracts of root tissue and allows early detection and understandings of reproduction timings of this important nematode of potato.

Genomic Reconstruction of the Introduction and Diversification of Golden Potato Cyst Nematode Populations in Indonesia.

Potato cyst nematodes (PCNs), the umbrella term for Globodera rostochiensis and G. pallida, coevolved with their Solanaceous hosts in the Andean Mountain region. From there, PCN proliferated worldwide to virtually all potato production areas. PCN is a major factor limiting the potato production in Indonesia. In our survey, only G. rostochiensis was found. Fourteen field populations were collected on Java and Sumatra, and unique variants were called by mapping resequencing data on a G. rostochiensis reference genome. A phylogenetic tree based on 1.4 million unique variants showed a genotypic separation between the outgroup, a Scottish Ro1 population, and all Indonesian populations. This separation was comparable in size with the genotypic distinction between the Javanese and the Sumatran PCN populations. Next, variants within PCN effector gene families SPRYSEC, 1106, 4D06, and venom allergen-like protein (VAL) that all interfere with the host innate immune system were compared. Distinct selective pressures acted on these effector families; while SPRYSECs (4,341 single-nucleotide polymorphisms [SNPs]/insertions or deletions of bases [indels]) behaved like neutral genes, the phylogenetic trees of 1106, 4D06, and VAL proteins (235, 790, and 150 SNPs/indels, respectively) showed deviating topologies. Our data suggest that PCN was introduced on Java not too long after the introduction of potato in the middle of the eighteenth century. Soon thereafter, the pathogen established on Sumatra and started to diversify independently. This scenario was corroborated by diversification patterns of the effector families 1106, 4D06, and VAL. Our data demonstrate how genome resequencing data from a nonindigenous pathogen can be used to reconstruct the introduction and diversification process.

Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology.

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10-4 single cysts and single females, 4-3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.

Comparative genomics of two inbred lines of the potato cyst nematode Globodera rostochiensis reveals disparate effector family-specific diversification patterns.

BACKGROUND: Potato cyst nematodes belong to the most harmful pathogens in potato, and durable management of these parasites largely depends on host-plant resistances. These resistances are pathotype specific. The current Globodera rostochiensis pathotype scheme that defines five pathotypes (Ro1 - Ro5) is both fundamentally and practically of limited value. Hence, resistant potato varieties are used worldwide in a poorly informed manner. RESULTS: We generated two novel reference genomes of G. rostochiensis inbred lines derived from a Ro1 and a Ro5 population. These genome sequences comprise 173 and 189 scaffolds respectively, marking a ≈ 24-fold reduction in fragmentation as compared to the current reference genome. We provide copy number variations for 19 effector families. Four dorsal gland effector families were investigated in more detail. SPRYSECs, known to be implicated in plant defence suppression, constitute by far the most diversified family studied herein with 60 and 99 variants in Ro1 and Ro5 distributed over 18 and 26 scaffolds. In contrast, CLEs, effectors involved in feeding site induction, show strong physical clustering. The 10 and 16 variants cluster on respectively 2 and 1 scaffolds. Given that pathotypes are defined by their effectoromes, we pinpoint the disparate nature of the contributing effector families in terms of sequence diversification and loss and gain of variants. CONCLUSIONS: Two novel reference genomes allow for nearly complete inventories of effector diversification and physical organisation within and between pathotypes. Combined with insights we provide on effector family-specific diversification patterns, this constitutes a basis for an effectorome-based virulence scheme for this notorious pathogen.

Developing a Real-Time PCR Assay for Direct Identification and Quantification of Soybean Cyst Nematode, Heterodera glycines, in Soil and Its Discrimination from Sugar Beet Cyst Nematode, Heterodera schachtii.

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the Heterodera schachtii sensu stricto group is not only difficult and time-consuming but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes and to quantify SCN directly from DNA extracts of field soils. SCN- and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated on the basis of target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and they may serve as useful diagnostic tools in research, diagnostic laboratories, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.

Potato cyst nematodes Globodera rostochiensis and G. pallida.

TAXONOMY: Phylum Nematoda; class Chromadorea; order Rhabditida; suborder Tylenchina; infraorder Tylenchomorpha; superfamily Tylenchoidea; family Heteroderidae; subfamily Heteroderinae; Genus Globodera. BIOLOGY: Potato cyst nematodes (PCN) are biotrophic, sedentary endoparasitic nematodes. Invasive (second) stage juveniles (J2) hatch from eggs in response to the presence of host root exudates and subsequently locate and invade the host. The nematodes induce the formation of a large, multinucleate syncytium in host roots, formed by fusion of up to 300 root cell protoplasts. The nematodes rely on this single syncytium for the nutrients required to develop through a further three moults to the adult male or female stage. This extended period of biotrophy-between 4 and 6 weeks in total-is almost unparalleled in plant-pathogen interactions. Females remain at the root while adult males revert to the vermiform body plan of the J2 and leave the root to locate and fertilize the female nematodes. The female body forms a cyst that contains the next generation of eggs. HOST RANGE: The host range of PCN is limited to plants of the Solanaceae family. While the most economically important hosts are potato (Solanum tuberosum), tomato (Solanum lycopersicum), and aubergine (Solanum melongena), over 170 species of Solanaceae are thought to be potential hosts for PCN (Sullivan et al., 2007). DISEASE SYMPTOMS: Symptoms are similar to those associated with nutrient deficiency, such as stunted growth, yellowing of leaves and reduced yields. This absence of specific symptoms reduces awareness of the disease among growers. DISEASE CONTROL: Resistance genes (where available in suitable cultivars), application of nematicides, crop rotation. Great effort is put into reducing the spread of PCN through quarantine measures and use of certified seed stocks. USEFUL WEBSITES: Genomic information for PCN is accessible through WormBase ParaSite.

Nematode Genome Announcement: Draft Genome of Meloidogyne chitwoodi, an Economically Important Pest of Potato in the Pacific Northwest.

Meloidogyne chitwoodi is one of the most devastating pests of potato in the U.S. Pacific Northwest (PNW). Nematode-infected tubers develop external as well as internal defects, making the potato tubers unmarketable, and resulting in economic losses. Draft genome assemblies of three M. chitwoodi genotypes-race 1, race 2 and race 1 pathotype Roza-were generated using Illumina and PacBio Sequel RS II sequencing. The final assemblies consist of 30, 39, and 38 polished contigs for race 1, race 2 and race 1 pathotype Roza, respectively, with average N50 of 2.37 Mb and average assembled genome size of approximately 47.41 Mb. On average, 10,508 genes were annotated for each genome. Benchmarking universal single-copy ortholog (BUSCO) analysis indicated that 69.80% of the BUSCOs were complete whereas 68.80, 0.93, and 12.67% were single copy, duplicated, and fragmented, respectively. These highly contiguous genomes will enrich resources to study potato-nematode interactions and enhance breeding efforts to develop nematode-resistant potato varieties for the PNW.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Resources of Two Pathotypes of the Potato Cyst Nematode Globodera rostochiensis from New York.

The potato cyst nematode Globodera rostochiensis is a regulated pest posing a serious threat to potato production worldwide. Although the endemic pathotype (Ro1) of G. rostochiensis has been confined to New York State for several decades as a result of quarantine regulations and management with resistant potato cultivars, a virulent pathotype, Ro2, has emerged, for which control measures are scarce. The ability to detect Ro2 early in fields is necessary to sustain the success of G. rostochiensis quarantine in the United States. Here, we report the comparative analysis of whole-genome sequences of multiple single-cyst-derived Ro1 and Ro2 lines, propagated from original field populations. The identified discriminant variants are good targets for developing molecular diagnostic tools for differentiating G. rostochiensis pathotypes in New York.

Initial responses of the trap-crop, Solanum sisymbriifolium, to Globodera pallida invasions.

Many researchers today are looking for mechanisms underlying plant defenses against nematodes by identifying differentially expressed genes in domesticated hosts. In order to provide a different perspective, we analyzed the root transcriptome of an undomesticated non-host species, Solanum sisymbriifolium Lamark (SSI) before and after Globodera pallida infection. Utilizing RNAseq analyses, we identified changes in the expression of 277 transcripts. Many of these genes were not annotated; however, the annotated set included peroxidases, reactive oxygen species-producing proteins, and regulators of cell death. Importantly, 60% of the nematode-responsive genes did not respond to physical damage to root tissues, or to exogenous treatments with either salicylic acid or methyl jasmonate. Based on this, we speculate that the majority of changes in SSI gene expression were promoted by either nematode effectors, pathogen-associated molecular patterns (PAMPs), or by exposure to untested endogenous signaling molecules such as ethylene, or by exposure to multiple stimuli. This study incorporates our findings into a model that accounts for part of this plant's unusual resistance to nematodes.

Evolutionarily conserved plant genes responsive to root-knot nematodes identified by comparative genomics.

Root-knot nematodes (RKNs, genus Meloidogyne) affect a large number of crops causing severe yield losses worldwide, more specifically in tropical and sub-tropical regions. Several plant species display high resistance levels to Meloidogyne, but a general view of the plant immune molecular responses underlying resistance to RKNs is still lacking. Combining comparative genomics with differential gene expression analysis may allow the identification of widely conserved plant genes involved in RKN resistance. To identify genes that are evolutionary conserved across plant species, we used OrthoFinder to compared the predicted proteome of 22 plant species, including important crops, spanning 214 Myr of plant evolution. Overall, we identified 35,238 protein orthogroups, of which 6,132 were evolutionarily conserved and universal to all the 22 plant species (PLAnts Common Orthogroups-PLACO). To identify host genes responsive to RKN infection, we analyzed the RNA-seq transcriptome data from RKN-resistant genotypes of a peanut wild relative (Arachis stenosperma), coffee (Coffea arabica L.), soybean (Glycine max L.), and African rice (Oryza glaberrima Steud.) challenged by Meloidogyne spp. using EdgeR and DESeq tools, and we found 2,597 (O. glaberrima), 743 (C. arabica), 665 (A. stenosperma), and 653 (G. max) differentially expressed genes (DEGs) during the resistance response to the nematode. DEGs' classification into the previously characterized 35,238 protein orthogroups allowed identifying 17 orthogroups containing at least one DEG of each resistant Arachis, coffee, soybean, and rice genotype analyzed. Orthogroups contain 364 DEGs related to signaling, secondary metabolite production, cell wall-related functions, peptide transport, transcription regulation, and plant defense, thus revealing evolutionarily conserved RKN-responsive genes. Interestingly, the 17 DEGs-containing orthogroups (belonging to the PLACO) were also universal to the 22 plant species studied, suggesting that these core genes may be involved in ancestrally conserved immune responses triggered by RKN infection. The comparative genomic approach that we used here represents a promising predictive tool for the identification of other core plant defense-related genes of broad interest that are involved in different plant-pathogen interactions.

Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium.

A transcriptome analysis of G. pallida juveniles collected from S. tuberosum or S. sisymbriifolium 24 h post infestation was performed to provide insights into the parasitic process of this nematode. A total of 41 G. pallida genes were found to be significantly differentially expressed when parasitizing the two plant species. Among this set, 12 were overexpressed when G. pallida was parasitizing S. tuberosum and 29 were overexpressed when parasitizing S. sisymbriifolium. Out of the 12 genes, three code for secretory proteins; one is homologous to effector gene Rbp-4, the second is an uncharacterized protein with a signal peptide sequence, and the third is an ortholog of a Globodera rostochiensis effector belonging to the 1106 effector family. Other overexpressed genes from G. pallida when parasitizing S. tuberosum were either unknown, associated with a stress or defense response, or associated with sex differentiation. Effector genes namely Eng-1, Cathepsin S-like cysteine protease, cellulase, and two unknown genes with secretory characteristics were over expressed when G. pallida was parasitizing S. sisymbriifolium relative to expression from S. tuberosum. Our findings provide insight into gene regulation of G. pallida while infecting either the trap crop S. sisymbriifolium or the susceptible host, S. tuberosum.

The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum.

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

What determines host specificity in hyperspecialized plant parasitic nematodes?

BACKGROUND: In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host. RESULTS: RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species. CONCLUSIONS: Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes.