Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples

Sugar beet cyst nematode (SBCN, Heterodera schachtii) is an important nematode that causes significant yield losses of 25–50% or more in most areas of sugar beet production worldwide. Rapid and accurate identification of this species is essential to support decisions on pest management. However, the difference between H. schachtii and other Heterodera spp. based on morphology is a challenging task. In the present study, a SCAR-PCR assay was developed to identify and differentiate H. schachtii in infected root and soil samples. H. schachtii-species-specific SCAR-PCR primers OPA06-HsF and OPA06-HsR were designed from the randomly amplified polymorphic DNA (RAPD) marker amplified with random primer OPA06. The developed primers specifically amplify a 922-bp fragment from the target populations but did not amplify DNA from non-target cyst nematodes including Heterodera, Globodera, Cactodera, and other related species tested in this study. The sensitivity detection indicated that 5 × 10−4 of a single cyst, 1/320 of a single second-stage juvenile (J2), or 10 pg of genomic DNA could be detected. The assay accurately identifies the different stages of H. schachtii in sugar beet and oilseed rape roots as well as a single J2 in 10 g of soil. Finally, the SCAR-PCR assay detected H. schachtii in seven samples out of the fifteen field samples. The assay will not only be useful for differentiating H. schachtii from mixed populations of Heterodera spp. but also for effective detection of the species directly from infested samples. The assay also requires no expertise in the taxonomy and morphology of the species but serves to improve the diagnosis of H. schachtii in infested fields.

Phenotypic and DNA Marker-Assisted Characterization of Russian Potato Cultivars for Resistance to Potato Cyst Nematodes

Potato is one of the most important food crops in the world and also in the Russian Federation. Among harmful organisms reducing potato yield potential, the potato cyst nematodes (PCN) are considered to be ones of the most damaging pests. Information on PCN resistant cultivars is important for potato breeding and production. Russian potato cultivars are characterized in the state-bio-test program for resistance to only one PCN species Globodera rostochiensis and one pathotype Ro1 which is reported to be present in the country. This study aimed to find domestic cultivars with multiple resistances to different PCN species and different pathotypes using phenotyping coupled with molecular marker analysis due to the risk of the occasional introduction of new pests. The phenotypic response was determined by the inoculation of plants with pathotypes Ro5 of G. rostochiensis and Pa3 of G. pallida. The obtained results were supplemented by the state-bio-test data on resistance to Ro1 of G. rostochiensis. Nine of 26 Russian cultivars were resistant both to Ro5 and Ro1 pathotypes and two cultivars possess multiple resistances to both PCN species. Most tested molecular markers associated with the Gpa2, GpaVvrn, GpaVsspl, Grp1 loci showed discrepancies with phenotyping. However, a predictive haplotype and epistatic effect were detected.

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.

A new strain of Volutella citrinella with nematode predation and nematicidal activity, isolated from the cysts of potato cyst nematodes in China

Background Plant parasitic nematodes (PPNs) are responsible for causing many plant diseases and are extremely difficult to control at present. Currently, due to the negative effects of chemical agents on the environment and human health, the development of new biological pesticides has become an important part of plant nematode control. Nematophagous fungi refers to a class of fungi that kill plant nematodes. Notably, a large number of nematophagous fungi resources remain to be studied. The objective of our study was to use in vitro screening to identify nematophagous fungi and select strains that were highly active against nematodes, providing a primary research for the development and utilization of new nematophagous fungi. Results A new nematophagous fungal strain (GUCC2219) was isolated from cysts of possibly Globodera spp. and Heterodera spp., identified as Volutella citrinella. The hyphae of V. citrinella produced ring structures of variable size and exhibited predatory and nematicidal activity. The hyphal predation rates (in vitro) against three species of nematodes, Aphelenchoides besseyi, Bursaphelenchus xylophilus, and Ditylenchus destructor, averaged 59.45, 33.35, and 50.95%, respectively, while the fermentation broth produced by the fungus exhibited mortality rates of 100, 100, and 55.63%, respectively, after 72 h. Conclusion V. citrinella is a new strain with nematophagous properties, which are a novel discovery. At the same time, this is the first report of nematicidal and nematode predation activity in the genus Volutella.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100–200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Comparative genomics among three cyst nematode species reveals distinct evolutionary histories among effector families and an irregular distribution of effector-associated promoter motifs

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are an essential handle for PCN control. However, the poor delineation of G. pallida pathotypes hampers the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCN after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (DOG boxes) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Re-sequencing of PCN populations with deviant virulence characteristics will allow for the linking of these characteristics with the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

Assessment of resistance of potato hybrids from the Russian Potato Research Center’s collection to Globodera rostochiensis

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are the most common pests feeding on potato roots. These pests have an aggressive distribution dynamic and bring a lot of damage to yield. The study presents results of comprehensively test new potato genotypes for resistance to the golden potato nematode (Globodera rostochiensis), for its further use in breeding and the creation of economically valuable resistant varieties. Almost half of the samples showed laboratory resistance to golden potato nematode, the rest were susceptible. 26 samples have shown the presence of two molecular markers (N146, N19), indicating the presence of H1 gene. Almost all samples showed a correspondence between laboratory resistance and the presence of H1 gene markers.

The genome and lifestage-specific transcriptomes of a plant-parasitic nematode and its host reveal susceptibility genes involved in trans-kingdom synthesis of vitamin B5

Plant-parasitic nematodes are a major, and in some cases a dominant, threat to crop production in all agricultural systems. The relative scarcity of classical resistance genes highlights a pressing need to identify new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major stages of the interaction. This novel approach enabled the analysis of the hologenome of the infection site, to identify metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that the highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is critically important for parasitism. Knockout of either the plant-encoded or the now nematode-encoded steps in the pathway blocks parasitism. Our experiments establish a reference for cyst nematodes, use this platform to further our fundamental understanding of the evolution of plant-parasitism by nematodes, and show that understanding congruent differential expression of metabolic pathways represents a new way to find nematode susceptibility genes, and thereby, targets for future genome editing-mediated generation of nematode-resistant crops.

Применение биологических препаратов в защите картофеля против возбудителя рака Synchytrium endobioticum (Schilb.) Perc. и золотистой картофельной цистообразующей нематоды Globodera rosthochiensis (Woll.)

The researches results of the following biological preparations Planrise, Paurin, Micosan, Actofit, Phitodoctor usage were presented. The biological preparations Planrise and Paurin (32,6%) against causa-tive agent of wart potato Synchytrium endobioticum (Schilb.) Perc. were shown the highest efficiency among the biological preparations by the research results. The efficiency of Phitodoctor consisted 25,8 %, Micosan was 23,1, respectively. The preparations efficiency were Micosan (42,8%), Phitodoctor and Paurin (30,2%); Actofit (34,3%); Bitobacsicillin (28,5%) against potato yellow cyst nematodes Globod-era rostochiensis (Woll.).