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.

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 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.

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.

Metabolic Analysis of the Development of the Plant-Parasitic Cyst Nematodes Heterodera schachtii and Heterodera trifolii by Capillary Electrophoresis Time-of-Flight Mass Spectrometry

The cyst nematodes Heterodera schachtii and Heterodera trifolii, whose major hosts are sugar beet and clover, respectively, damage a broad range of plants, resulting in significant economic losses. Nematodes synthesize metabolites for organismal development and social communication. We performed metabolic profiling of H. schachtii and H. trifolii in the egg, juvenile 2 (J2), and female stages. In all, 392 peaks were analyzed by capillary electrophoresis time-of-flight mass spectrometry, which revealed a lot of similarities among metabolomes. Aromatic amino acid metabolism, carbohydrate metabolism, choline metabolism, methionine salvage pathway, glutamate metabolism, urea cycle, glycolysis, gluconeogenesis, coenzyme metabolism, purine metabolism, pyrimidine metabolism, and tricarboxylic acid (TCA) cycle for energy conversion (β-oxidation and branched-chain amino acid metabolism) energy storage were involved in all stages studied. The egg and female stages synthesized higher levels of metabolites compared to the J2 stage. The key metabolites detected were glycerol, guanosine, hydroxyproline, citric acid, phosphorylcholine, and the essential amino acids Phe, Leu, Ser, and Val. Metabolites, such as hydroxyproline, acetylcholine, serotonin, glutathione, and glutathione disulfide, which are associated with growth and reproduction, mobility, and neurotransmission, predominated in the J2 stage. Other metabolites, such as SAM, 3PSer, 3-ureidopropionic acid, CTP, UDP, UTP, 3-hydroxy-3-methylglutaric acid, 2-amino-2-(hydroxymethyl-1,3-propanediol, 2-hydroxy-4-methylvaleric acid, Gly Asp, glucuronic acid-3 + galacturonic acid-3 Ser-Glu, citrulline, and γ-Glu-Asn, were highly detected in the egg stage. Meanwhile, nicotinamide, 3-PG, F6P, Cys, ADP-Ribose, Ru5P, S7P, IMP, DAP, diethanolamine, p-Hydroxybenzoic acid, and γ-Glu-Arg_divalent were unique to the J2 stage. Formiminoglutamic acid, nicotinaminde riboside + XC0089, putrescine, thiamine 2,3-dihydroxybenzoic acid, 3-methyladenine, caffeic acid, ferulic acid, m-hydrobenzoic acid, o- and p-coumaric acid, and shikimic acid were specific to the female stage. Overall, highly similar identities and quantities of metabolites between the corresponding stages of the two species of nematode were observed. Our results will be a valuable resource for further studies of physiological changes related to the development of nematodes and nematode–plant interactions.

Glutathione contributes to plant defense against parasitic cyst nematodes

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) in Arabidopsis thaliana upon infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate–cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All GSH-deficient mutants exhibited impaired activation of defense marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant susceptibility to CN infection and the production of camalexin upon nematode infection. These results suggest that GSH levels affects plant susceptibility to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defense against infection.

Marasmius oreades agglutinin enhances resistance of Arabidopsis against plant-parasitic nematodes and a herbivorous insect

Background Plant-parasitic nematodes and herbivorous insects have a significant negative impact on global crop production. A successful approach to protect crops from these pests is the in planta expression of nematotoxic or entomotoxic proteins such as crystal proteins from Bacillus thuringiensis (Bt) or plant lectins. However, the efficacy of this approach is threatened by emergence of resistance in nematode and insect populations to these proteins. To solve this problem, novel nematotoxic and entomotoxic proteins are needed. During the last two decades, several cytoplasmic lectins from mushrooms with nematicidal and insecticidal activity have been characterized. In this study, we tested the potential of Marasmius oreades agglutinin (MOA) to furnish Arabidopsis plants with resistance towards three economically important crop pests: the two plant-parasitic nematodes Heterodera schachtii and Meloidogyne incognita and the herbivorous diamondback moth Plutella xylostella. Results The expression of MOA does not affect plant growth under axenic conditions which is an essential parameter in the engineering of genetically modified crops. The transgenic Arabidopsis lines showed nearly complete resistance to H. schachtii, in that the number of female and male nematodes per cm root was reduced by 86–91 % and 43–93 % compared to WT, respectively. M. incognita proved to be less susceptible to the MOA protein in that 18–25 % and 26–35 % less galls and nematode egg masses, respectively, were observed in the transgenic lines. Larvae of the herbivorous P. xylostella foraging on MOA-expression lines showed a lower relative mass gain (22–38 %) and survival rate (15–24 %) than those feeding on WT plants. Conclusions The results of our in planta experiments reveal a robust nematicidal and insecticidal activity of the fungal lectin MOA against important agricultural pests which may be exploited for crop protection.

Bacillus firmus I-1582 promotes plant growth and impairs infection and development of the cyst nematode Heterodera schachtii over two generations

Plant-parasitic nematodes wreak havoc on crops by root parasitism worldwide. An approach to combat nematode root parasitism is the application of antagonistic microbes like the rhizobacterium Bacillus firmus I-1582 which is promoted as biological control agent. Although B. firmus is a known nematode antagonist in general, the underlying mechanisms about its interaction with nematodes and plants have not yet been elucidated. Therefore, we explored the influence of B. firmus I-1582 as well as its extracellular and secreted molecules on plant–nematode interaction utilizing the plant–pathogen system Arabidopsis thaliana–Heterodera schachtii. We demonstrated that B. firmus I-1582 is attracted by A. thaliana root exudates, particularly by those of young plants. The bacterium colonized the root and showed a strictly pH-dependent development and plant growth promotion effect. Our results revealed that root colonization by B. firmus I-1582 significantly protected A. thaliana from infestation by the beet cyst nematode whereas dead bacterial cells or the culture supernatant were not effective. The bacterium also negatively affected nematode reproduction as well as pathogenicity and development of next generation nematodes. The obtained results highlight B. firmus I-1582 as a promising biocontrol agent that is well suited as an element of integrated control management strategies in sustainable agriculture.

Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.