Genomic Profiling of Virulence in the Soybean Cyst Nematode Using Single-Nematode Sequencing

Soybean cyst nematode (SCN) is one of the most important diseases in soybean. Currently, the main management strategy relies on planting resistant cultivars. However, the overuse of a single resistance source has led to the selection of virulent SCN populations, although the mechanisms by which the nematode overcomes the resistance genes remain unknown. In this study, we used a nematode-adapted single-cell RNA-seq approach to identify SCN genes potentially involved in resistance breakdown in Peking and PI 88788 parental soybean lines. We established for the first time the full transcriptome of single SCN individuals allowing us to identify a list of putative virulence genes against both major SCN resistance sources. Our analysis identified 48 differentially expressed putative effectors (secreted proteins required for infection) alongside 40 effectors showing evidence of novel structural variants, and 11 effector genes containing phenotype-specific sequence polymorphisms. Additionally, a differential expression analysis revealed an interesting phenomenon of coexpressed gene regions with some containing putative effectors. The selection of virulent SCN individuals on Peking resulted in a profoundly altered transcriptome, especially for genes known to be involved in parasitism. Several sequence polymorphisms were also specific to these virulent nematodes and could potentially play a role in the acquisition of nematode virulence. On the other hand, the transcriptome of virulent individuals on PI 88788 was very similar to avirulent ones with the exception of a few genes, which suggest a distinct virulence strategy to Peking.

The Two Faces of Nematode Infection: Virulence and Immunomodulatory Molecules From Nematode Parasites of Mammals, Insects and Plants

Helminths stage a powerful infection that allows the parasite to damage host tissue through migration and feeding while simultaneously evading the host immune system. This feat is accomplished in part through the release of a diverse set of molecules that contribute to pathogenicity and immune suppression. Many of these molecules have been characterized in terms of their ability to influence the infectious capabilities of helminths across the tree of life. These include nematodes that infect insects, known as entomopathogenic nematodes (EPN) and plants with applications in agriculture and medicine. In this review we will first discuss the nematode virulence factors, which aid parasite colonization or tissue invasion, and cause many of the negative symptoms associated with infection. These include enzymes involved in detoxification, factors essential for parasite development and growth, and highly immunogenic ES proteins. We also explore how these parasites use several classes of molecules (proteins, carbohydrates, and nucleic acids) to evade the host’s immune defenses. For example, helminths release immunomodulatory molecules in extracellular vesicles that may be protective in allergy and inflammatory disease. Collectively, these nematode-derived molecules allow parasites to persist for months or even years in a host, avoiding being killed or expelled by the immune system. Here, we evaluate these molecules, for their individual and combined potential as vaccine candidates, targets for anthelminthic drugs, and therapeutics for allergy and inflammatory disease. Last, we evaluate shared virulence and immunomodulatory mechanisms between mammalian and non-mammalian plant parasitic nematodes and EPNs, and discuss the utility of EPNs as a cost-effective model for studying nematode-derived molecules. Better knowledge of the virulence and immunomodulatory molecules from both entomopathogenic nematodes and soil-based helminths will allow for their use as beneficial agents in fighting disease and pests, divorced from their pathogenic consequences.

Molecular variation among virulent and avirulent strains of the quarantine nematode Bursaphelenchus xylophilus

Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD.

Whole Genome Sequencing and Comparative Genomics of Two Nematicidal Bacillus Strains Reveals a Wide Range of Possible Virulence Factors

Bacillus firmus nematicidal bacterial strains are used to control plant parasitic nematode infestation of crops in agricultural production. Proteases are presumed to be the primary nematode virulence factors in nematicidal B. firmus degrading the nematode cuticle and other organs. We determined and compared the whole genome sequences of two nematicidal strains. Comparative genomics with a particular focus on possible virulence determinants revealed a wider range of possible virulence factors in a B. firmus isolate from a commercial bionematicide and a wild type Bacillus sp. isolate with nematicidal activity. The resulting 4.6 Mb B. firmus I-1582 and 5.3 Mb Bacillus sp. ZZV12-4809 genome assemblies contain respectively 18 and 19 homologs to nematode-virulent proteases, two nematode-virulent chitinase homologs in ZZV12-4809 and 28 and 36 secondary metabolite biosynthetic clusters, projected to encode antibiotics, small peptides, toxins and siderophores. The results of this study point to the genetic capability of B. firmus and related species for nematode virulence through a range of direct and indirect mechanisms.

Effects of Cover Cropping on Microbial Communities Associated with Heterodera schachtii and Nematode Virulence

Nematode-resistant cover crops can suppress populations of plant-parasitic nematodes. Samples of a loamy and a sandy loam soil were collected from two sugar beet fields in Lower Saxonia, northern Germany (“Jeinsen” and “Gross Munzel”) to measure the effects of cover cropping on the population genetic structure and infectivity of Heterodera schachtii as well as the composition of soil microbial communities. These fields allowed for a comparison of cover cropping with Brassica species resistant to Heterodera schachtii to fallow. In a series of radish bioassays with H. schachtii populations from Jeinsen and Gross Munzel, ratios of second-stage juveniles in roots per eggs in soil were higher in soil from under Brassica cropping than from under fallow. In denaturing gradient gelelectrophoresis, profiles of the parasitism gene vap1 differed between Brassica and fallow treatments in both populations. At Gross Munzel, microbes of soils and within nematode cysts differed between Brassica and fallow areas. Specifically, the frequency and occurrence of isolates of Pochonia chlamydosporia and Exophiala salmonis were lower within the cysts from Brassica than from fallow treatments. Overall, cover cropping with resistant Brassica species affected the bacteria and fungi infecting the cysts and subsequently, the infectivity of the H. schachtii population. Cover crop effects on nematode virulence (vap1 gene) and microbial colonization of the cysts could affect long-term nematode population dynamics.

Durable resistance against Meloidogyne chitwoodi and M. fallax

The EU-funded Project QLRT-1999-1462 DREAM (Durable Resistance Against Meloidogyne) aims to contribute to sustainable production systems by developing a strategy for durable resistance management for two polyphagous quarantine root-knot nematodes Meloidogyne chitwoodi and M. fallax. The objective will be achieved by integrating expertise in breeding, nematology, botany and molecular biology. The project combines three areas of research: 1. Identification and incorporation of resistance in important arable crops: potato, pepper, ryegrass and fodder radish, 2. Study of variation in nematode virulence and of durability of the resistance, and 3. Optimising of production systems by rotation schemes. The main results expected are: resistant germplasm, characterised pathogen collections, breeding methods, knowledge of the stability of resistance, molecular markers linked to resistance and (a)virulence, resistance mechanisms and genes, genetic maps, and improved rotation schemes. The strategy and some first results will be discussed.

Soybean Resistance to the Soybean Cyst Nematode Heterodera glycines: An Update

The soybean cyst nematode (SCN), Heterodera glycines, remains a serious threat to soybean production throughout the world. A lack of genetic diversity in resistant soybean cultivars has led to a widespread shift toward virulence in SCN populations, leaving farmers with few proven options other than nonhost rotation to manage this nematode. Recent advances in our understanding of the genes controlling resistance to the nematode have led to improved molecular markers, which are, in turn, increasing the efficiency and precision of the breeding pipeline. A better understanding of the molecular and biochemical basis of SCN resistance and nematode virulence will provide information useful for the development of a long-term strategic plan for diversification and the deployment of cultivars that protect current sources of natural resistance while identifying new targets for engineering novel resistance.

Pathogenicity of selected isolates of the quarantine pinewood nematode Bursaphelenchus xylophilus to Scots pine (Pinus sylvestris L.)

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD). This nematode is considered to be an indigenous to North America and was introduced to Japan in the late 19th century. Subsequently, it has spread throughout Japan and in many other countries, China, Taiwan, and South Korea. In 1999, B. xylophilus was discovered in Portugal, and in 2008 in Spain. So far the studies have revealed that the pathogenicity of B. xylophilus varies between different isolates. The conducted study compared the pathogenicity of five isolates of B. xylophilus, originating from different parts of Japan, to 3-year-old Pinus sylvestris, and their ability to reproduce in the seedlings. The results revealed diverse virulence of B. xylophilus resulting in plant mortality. Three isolates S10, Ka4, and T4 caused 100% mortality of plants within three months while at the same time, the other two isolates, C14-5 and OKD-1 did not cause any disease symptoms on plants. After seven months, some dieback occurred on two seedlings, but similar symptoms were also found on the control plant. Moreover, a significant positive correlation was found between nematode virulence and the number of nematodes reproducing on pine seedlings.