Europe as a secondary distribution hub in the worldwide invasion of the potato cyst nematode Globodera rostochiensis.

The potato cyst nematode Globodera rostochiensis originates from the Andean Mountain region in South America and has unintentionally been introduced to all inhabited continents. Several studies have examined the population genetic structure of this pest in various countries by using microsatellite markers. However, merging microsatellite data produced from different laboratories is challenging and can introduce uncertainty when interpreting the results. To overcome this challenge and to explore invasion routes of this pest, we have genotyped 22 G. rostochiensis populations from all continents. Within populations, the highest genetic diversity was observed in the South American populations, the European populations showed an intermediate level of genetic diversity and the remaining populations were the less diverse. This confirmed pre-existing knowledge such as a first introduction event from South America to Europe, but the less diverse populations could originate either from South America or from Europe. At the continental scale, STRUCTURE genetic clustering output indicated that North America and Asia have experienced at least two introduction events. Comparing different evolutionary scenarios, the Approximate Bayesian Computation analysis showed that Europe served as a secondary distribution centre for the invasion of G. rostochiensis into all other continents (North America, Africa, Asia and Oceania).

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 ).

Characterization of microRNAs in the cyst nematode Heterodera glycines identifies possible candidates involved in cross-kingdom interactions with its host Glycine max.

The soybean cyst nematode (SCN - Heterodera glycines) is one of the most damaging pests to the cultivated soybean worldwide. Using a wide array of stylet-secreted effector proteins, this nematode can restructure its host cells into a complex and highly active feeding structure called the syncytium. Tight regulation of these proteins is thought to be essential to the successful formation of this syncytium. To date, multiple mechanisms have been proposed to regulate the expression of these proteins including through post-transcriptional regulation. MicroRNAs (miRNAs) are a class of small, roughly 22-nucleotide-long, non-coding RNA shown to regulate gene expression through its interaction with the 3' untranslated region of genes. These same small RNAs have also been hypothesized to be able to cross over kingdom barriers and regulate genes in other species in a process called cross-kingdom interactions. In this study, we characterized the miRNome of the SCN via sequencing of small-RNAs isolated from whole nematodes and exosomes representing all developmental stages. We identified 121 miRNA loci encoding 96 distinct miRNA families including multiple lineage- and species-specific candidates. Using a combination of plant- and animal-specific miRNA target predictors, we generated a unique repertoire of miRNA:mRNA interacting partners in the nematode and its host plant leading to the identification of a set of nine probable cross-kingdom miRNA candidates.

Large-scale data mining pipeline for identifying novel soybean genes involved in resistance against the soybean cyst nematode.

The soybean cyst nematode (SCN) [Heterodera glycines Ichinohe] is a devastating pathogen of soybean [Glycine max (L.) Merr.] that is rapidly becoming a global economic issue. Two loci conferring SCN resistance have been identified in soybean, Rhg1 and Rhg4; however, they offer declining protection. Therefore, it is imperative that we identify additional mechanisms for SCN resistance. In this paper, we develop a bioinformatics pipeline to identify protein-protein interactions related to SCN resistance by data mining massive-scale datasets. The pipeline combines two leading sequence-based protein-protein interaction predictors, the Protein-protein Interaction Prediction Engine (PIPE), PIPE4, and Scoring PRotein INTeractions (SPRINT) to predict high-confidence interactomes. First, we predicted the top soy interacting protein partners of the Rhg1 and Rhg4 proteins. Both PIPE4 and SPRINT overlap in their predictions with 58 soybean interacting partners, 19 of which had GO terms related to defense. Beginning with the top predicted interactors of Rhg1 and Rhg4, we implement a "guilt by association" in silico proteome-wide approach to identify novel soybean genes that may be involved in SCN resistance. This pipeline identified 1,082 candidate genes whose local interactomes overlap significantly with the Rhg1 and Rhg4 interactomes. Using GO enrichment tools, we highlighted many important genes including five genes with GO terms related to response to the nematode (GO:0009624), namely, Glyma.18G029000, Glyma.11G228300, Glyma.08G120500, Glyma.17G152300, and Glyma.08G265700. This study is the first of its kind to predict interacting partners of known resistance proteins Rhg1 and Rhg4, forming an analysis pipeline that enables researchers to focus their search on high-confidence targets to identify novel SCN resistance genes in soybean.

Single Nematode Transcriptomic Analysis, Using Long-Read Technology, Reveals Two Novel Virulence Gene Candidates in the Soybean Cyst Nematode, Heterodera glycines.

The soybean cyst nematode (Heterodera glycines, SCN), is the most damaging disease of soybean in North America. While management of this pest using resistant soybean is generally still effective, prolonged exposure to cultivars derived from the same source of resistance (PI 88788) has led to the emergence of virulence. Currently, the underlying mechanisms responsible for resistance breakdown remain unknown. In this study, we combined a single nematode transcriptomic profiling approach with long-read sequencing to reannotate the SCN genome. This resulted in the annotation of 1932 novel transcripts and 281 novel gene features. Using a transcript-level quantification approach, we identified eight novel effector candidates overexpressed in PI 88788 virulent nematodes in the late infection stage. Among these were the novel gene Hg-CPZ-1 and a pioneer effector transcript generated through the alternative splicing of the non-effector gene Hetgly21698. While our results demonstrate that alternative splicing in effectors does occur, we found limited evidence of direct involvement in the breakdown of resistance. However, our analysis highlighted a distinct pattern of effector upregulation in response to PI 88788 resistance indicative of a possible adaptation process by SCN to host resistance.

A Broad Review of Soybean Research on the Ongoing Race to Overcome Soybean Cyst Nematode.

Plant pathogens greatly impact food security of the ever-growing human population. Breeding resistant crops is one of the most sustainable strategies to overcome the negative effects of these biotic stressors. In order to efficiently breed for resistant plants, the specific plant-pathogen interactions should be understood. Soybean is a short-day legume that is a staple in human food and animal feed due to its high nutritional content. Soybean cyst nematode (SCN) is a major soybean stressor infecting soybean worldwide including in China, Brazil, Argentina, USA and Canada. There are many Quantitative Trait Loci (QTLs) conferring resistance to SCN that have been identified; however, only two are widely used: rhg1 and Rhg4. Overuse of cultivars containing these QTLs/genes can lead to SCN resistance breakdown, necessitating the use of additional strategies. In this manuscript, a literature review is conducted on research related to soybean resistance to SCN. The main goal is to provide a current understanding of the mechanisms of SCN resistance and list the areas of research that could be further explored.

Recent Advances in Population Genomics of Plant-Parasitic Nematodes.

Plant-parasitic nematodes are a costly burden of crop production. Ubiquitous in nature, phytoparasitic nematodes are associated with nearly every important agricultural crop and represent a significant constraint on global food security. Population genetics is a key discipline in plant nematology to understand aspects of the life strategies of these parasites, in particular their modes of reproduction, geographic origins, evolutionary histories, and dispersion abilities. Advances in high-throughput sequencing technologies have enabled a recent but active effort in genomic analyses of plant-parasitic nematodes. Such genomic approaches applied to multiple populations are providing new insights into the molecular and evolutionary processes that underpin the establishment of these nematodes and into a better understanding of the genetic and mechanistic basis of their pathogenicity and adaptation to their host plants. In this review, we attempt to update information about genome resources and genotyping techniques useful for nematologists who are thinking about initiating population genomics or genome sequencing projects. This review is intended also to foster the development of population genomics in plant-parasitic nematodes through highlighting recent publications that illustrate the potential for this approach to identify novel molecular markers or genes of interest and improve our knowledge of the genome variability, pathogenicity, and evolutionary potential of plant-parasitic nematodes.

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.

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.

The Genomic Impact of Selection for Virulence against Resistance in the Potato Cyst Nematode, Globodera pallida.

Although the use of natural resistance is the most effective management approach against the potato cyst nematode (PCN) Globodera pallida, the existence of pathotypes with different virulence characteristics constitutes a constraint towards this goal. Two resistance sources, GpaV (from Solanum vernei) and H3 from S. tuberosum ssp. andigena CPC2802 (from the Commonwealth Potato Collection) are widely used in potato breeding programmes in European potato industry. However, the use of resistant cultivars may drive strong selection towards virulence, which allows the increase in frequency of virulent alleles in the population and therefore, the emergence of highly virulent nematode lineages. This study aimed to identify Avirulence (Avr) genes in G. pallida populations selected for virulence on the above resistance sources, and the genomic impact of selection processes on the nematode. The selection drive in the populations was found to be specific to their genetic background. At the genomic level, 11 genes were found that represent candidate Avr genes. Most of the variant calls determining selection were associated with H3-selected populations, while many of them seem to be organised in genomic islands facilitating selection evolution. These phenotypic and genomic findings combined with histological studies performed revealed potential mechanisms underlying selection in G. pallida.

PIPE4: Fast PPI Predictor for Comprehensive Inter- and Cross-Species Interactomes.

The need for larger-scale and increasingly complex protein-protein interaction (PPI) prediction tasks demands that state-of-the-art predictors be highly efficient and adapted to inter- and cross-species predictions. Furthermore, the ability to generate comprehensive interactomes has enabled the appraisal of each PPI in the context of all predictions leading to further improvements in classification performance in the face of extreme class imbalance using the Reciprocal Perspective (RP) framework. We here describe the PIPE4 algorithm. Adaptation of the PIPE3/MP-PIPE sequence preprocessing step led to upwards of 50x speedup and the new Similarity Weighted Score appropriately normalizes for window frequency when applied to any inter- and cross-species prediction schemas. Comprehensive interactomes for three prediction schemas are generated: (1) cross-species predictions, where Arabidopsis thaliana is used as a proxy to predict the comprehensive Glycine max interactome, (2) inter-species predictions between Homo sapiens-HIV1, and (3) a combined schema involving both cross- and inter-species predictions, where both Arabidopsis thaliana and Caenorhabditis elegans are used as proxy species to predict the interactome between Glycine max (the soybean legume) and Heterodera glycines (the soybean cyst nematode). Comparing PIPE4 with the state-of-the-art resulted in improved performance, indicative that it should be the method of choice for complex PPI prediction schemas.

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.

Preharvest UV-C treatment affected postharvest senescence and phytochemicals alternation of strawberry fruit with the possible involvement of abscisic acid regulation.

As an environmentally friendly approach for fruit quality improvement, the effect of preharvest UV-C on the physiology of strawberry fruit during postharvest storage remains to be assessed. Strawberry fruit developed with supplementary UV-C were stored at room temperature for 2 weeks. Preharvest UV-C attenuated fruit postharvest senescence and altered phytochemicals composition. Higher ester titer was found in the treated fruit at harvest, whereas higher terpene and furanone contents were detected after 72 h of storage. At harvest, polyphenolics accumulated to a higher level in UV-C group, but the difference disappeared after 24 h of storage. Meanwhile, the intrinsic level of abscisic acid and the expressions of FaPYR1, SnRK2, and FaASR in the UV-C-treated fruit was enhanced at harvest but returned to a lower level as storage proceeded. This study highlights the time-dependent effect of preharvest UV-C on strawberry fruit postharvest biochemical indexes and the possible involvement of abscisic acid signaling factors.

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.

"Piler Dirt" Survey for the Sampling and Detection of Potato Cyst Nematodes.

Potato cyst nematodes are a significant threat to potato production worldwide and have important economic impacts due to yield losses but also because of the expenses associated with regulation procedures. In order to reduce the sampling labor, an alternative strategy named the "Piler Dirt" that collects the soil carried with potato tubers during their transfer to storage was proposed. The method showed a better sensitivity than the reference method to detect fields infested with G. rostochiensis. The quantification of the number of cysts per kilogram of soil was proportional between the two methods at low and moderate population densities (R2 = 0.885) but no correlations were found at high density. However, the quantity of soil generated by the method was exceedingly large to be treated by diagnostic labs. It was shown that subsampling six aliquots, each equivalent to 5,000 cm3/ha, from the total quantity of soil generated by the Piler Dirt method, resulted in a probability of 97% to detect infested fields, 95% of the time in our dataset. Overall, Piler Dirt appears as a good compromise to reduce labor time and cost without significantly affecting sensitivity. However, it will be challenging to implement because it needs to be done simultaneously with harvest and will require the participation of farmers during a busy period.

Current Status of Potato Cyst Nematodes in North America.

The potato cyst nematodes (PCNs) Globodera rostochiensis and Globodera pallida are internationally recognized quarantine pests. Although not widely distributed in either the United States or Canada, both are present and are regulated by the national plant protection organizations (NPPOs) of each country. G. rostochiensis was first discovered in New York in the 1940s, and G. pallida was first detected in a limited area of Idaho in 2006. In Canada, G. rostochiensis and G. pallida were first detected in Newfoundland in 1962 and 1977, respectively, and further detections of G. rostochiensis occurred in British Columbia and Québec, most recently in 2006. Adherence to a stringent NPPO-agreed-upon phytosanitary program has prevented the spread of PCNs to other potato-growing areas in both countries. The successful research and regulatory PCN programs in both countries rely on a network of state, federal, university, and private industry cooperatorspursuing a common goal of containment, management/eradication, and regulation. The regulatory and research efforts of these collaborative groups spanning from the 1940s to the present are highlighted in this review.

The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes.

BACKGROUND: Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock. RESULTS: Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123 Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7 Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons. CONCLUSIONS: This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines.

Host Range and Genetic Characterization of Ditylenchus dipsaci Populations from Eastern Canada.

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode affecting over 500 plant species worldwide. Since 2012, garlic producers from Ontario and Quebec have been particularly affected with economic losses caused by this pest. Reproduction of D. dipsaci on a particular host depends on its biological race, and races are unknown for these populations from eastern Canada. As a polyphagous pest, D. dipsaci can possibly be a threat and have negative impact on many crops grown in Quebec, such as field and vegetable crops (e.g., onion). In this study, the host range of four populations of D. dipsaci from Quebec and Ontario was determined in a greenhouse experiment using 11 crops. Garlic, onion, and green onion showed high susceptibility to the nematode, whereas reproduction on potato was poor. No reproduction was observed on corn, soybean, barley, alfalfa, mustard, carrot, and lettuce. These crops could therefore be used as rotational crops in a control program. Thirty-two populations of D. dipsaci were also genetically characterized using genotyping-by-sequencing. The comparison of allele frequencies at 481 loci showed that most of the populations had a genotype similar to a reference population from northern Ontario. However, a sample from eastern Quebec exhibited a distinct genotype and will require further phenotyping in a greenhouse to preclude the possibility of a different race.

Preharvest Ultraviolet C Treatment Affected Senescence of Stored Strawberry Fruit with a Potential Role of MicroRNAs in the Activation of the Antioxidant System.

Recent studies presented preharvest ultraviolet C (UV-C) as an environmentally friendly approach for the management of horticultural crop diseases. The effect of this approach on quality preservation during postharvest storage has not yet been investigated. Strawberry fruit harvested from plants grown with supplemental UV-C were stored at room temperature for 72 h, and their postharvest shelf-life biochemical indicators were evaluated. The involvement of microRNAs (miRNAs) in the activation of UV-C-induced antioxidant systems was investigated. Preharvest UV-C contributed to the preservation of sugar and organic acid and reduced overall lipid peroxidation in strawberry fruit during storage. We found that miR159 and miR398 were downregulated by preharvest UV-C and that their respective targets were upregulated at the early stage of storage with enhancement of the activity of antioxidant enzymes. The initial burst of H2O2 and O2• - suggested that preharvest UV-C primed the fruit in an antioxidative activated state via reactive-oxygen-species-mediated feedback control with post-transcriptional involvement of miRNAs.