The tobacco cyst nematode, Globodera tabacum, is considered a serious and important pest of shade and broadleaf tobacco. In our study we provide phylogenetic analyses of 51 COI and 66 ITS rRNA gene sequences of G. tabacum populations from different geographical areas using statistical parsimony. These sequences included 17 new COI and 34 new ITS rRNA gene sequences obtained from 19 samples from the USA, France, Spain and Italy. Statistical parsimony COI gene sequence network confirmed that G. tabacum is a polytypic species consisting of four subspecies rather than three as previously reported. The four subspecies include the three known subspecies: G. tabacum tabacum, G. t. virginiae and G. t. solanacearum and a new subspecies, G. tabacum argentinensis subsp. n. Maximal intraspecific COI gene sequence diversity for G. tabacum was 14.1%. If COI gene sequences clearly differentiated subspecies showing large genetic distance between them, the statistical parsimony analysis of the ITS rRNA gene sequences differentiate four subspecies based on a few nucleotides only. Moreover, this discrimination could be compromised by the presence of recombinant ITS rRNA gene sequences due to subspecies hybridisation. The new subspecies, G. t. argentinensis subsp. n., presently found in Argentina and Italy, is proposed and morphologically described.

Polyandry is a common reproductive behaviour in nature, and it was observed in several plant parasitic cyst nematodes. For the potato cyst nematode Globodera pallida, polyandry is assumed but has never been demonstrated and quantified. Since the potato resistance used to control this nematode, i.e., the resistance conferred by GpaV from Solanum vernei, acts by masculinizing populations, and because polyandrous mating is more frequent in male-skewed populations, the level of polyandry can be expected to decrease during the nematode adaptation process to potato resistance. Aims of this study were thus to determine whether polyandry occurs in G. pallida and to explore the polyandry evolution during the adaptation process to the potato resistance. Using G. pallida lineages obtained from experimental evolution on susceptible and resistant potato cultivars, we explored and quantified the genetic evidences of multiple paternity within cysts by genotyping juveniles using microsatellite loci. Results clearly highlighted multiple paternity in G. pallida and showed that 100% of females were polyandrous, with an average of seven fathering males. Contrary to our expectations, the frequency of polyandrous females and the female mating rate with different males, estimated from the minimum number of fathers, appeared to remain stable throughout the adaptation process to a masculinizing resistance. The level of polyandry highlighted here may represent an important parameter to consider in demo-genetic models designed to compare nematode population control strategies.

The use of alternative methods to control cyst nematode populations has accelerated since the ban of chemical nematicides in Europe. The resistant QTL GpaVvrn, derived from the wild species Solanum vernei, is widely present in resistant European potato cultivars and provides strong protection against Globodera pallida populations although a risk of resistance breakdown has already been demonstrated in both experimental evolution studies and field populations. The wild relative S. sparsipilum, harbouring the resistant QTL GpaVspl, would be an interesting alternative source of resistance to control virulent G. pallida. The goal of the present study was to understand the genomics of adaptation of the nematode to these two colinear resistant QTLs. Starting with two natural populations, an experimental evolution approach allowed, after 10 generations on resistant potato genotypes, selecting independent nematode lineages adapted to each QTL. These virulent lineages were analysed through a combination of phenotyping and genome scans approaches. Phenotyping enabled the quantification of virulence levels and confirmed resistance breakdowns. Pool-Seq whole genome sequencing followed by genome scan analyses identified genomic regions under selection, potentially involved in the adaptive mechanisms to each resistance factor. Candidate genes within these regions provided insights into the genetic basis of adaptation, revealing effectors known to suppress plant immunity. As genome scans highlighted distinct genomic regions for the adaptation to both resistant factors, we were able to predict and phenotypically confirm the absence of cross-virulence between nematode lineages evolving on GpaVvrn and GpaVspl. These findings have significant implications for the design of effective and sustainable resistance management strategies.

BACKGROUND:Since the banning of chemical products used to control plant-parasitic nematode populations, the use of resistant plants has become the most effective management approach against the potato cyst nematode Globodera pallida. However, some populations, from experimental evolution setups and field samplings, are able to overcome these resistances. Herein, a transcriptomics approach was used to disentangle the mechanisms by which G. pallida adapts to the plant resistant factor GpaVvrn, and to elucidate the functions involved in this adaptation. RESULTS:Differential gene expression analysis between virulent and avirulent lineages originating from experimental evolution experiments identified candidate genes involved in the adaptation to GpaVvrn. GO enrichment analyses showed that virulent lineages up-regulated genes involved in cell wall destruction and stress response compared to avirulent lineages. In virulent lineages, a set of genes was up-regulated later in the parasitism stages and are thus potentially involved in adaptation. These genes encode effectors of the VAP and SPRYSEC families contributing to the suppression of plant immunity. CONCLUSION:These results will have a major impact on our understanding of the mechanisms by which nematodes adapt to resistant plants, and will contribute to identify effective and sustainable management strategies.

The use of alternative methods to control cyst nematode populations has accelerated since the ban of chemical nematicides in Europe. The resistant QTL GpaV vrn , derived from the wild species Solanum vernei , is widely present in resistant European potato cultivars and provides strong protection against Globodera pallida populations although a risk of resistance breakdown has already been demonstrated in both experimental evolution studies and field populations. The wild relative S. sparsipilum , harbouring the resistant QTL GpaV spl , would be an interesting alternative source of resistance to control virulent G. pallida . The goal of the present study was to understand the genomics of adaptation of the nematode to these two colinear resistant QTLs. Starting with two natural populations, an experimental evolution approach allowed, after 10 generations on resistant potato genotypes, selecting independent nematode lineages adapted to each QTL. These virulent lineages were analysed through a combination of phenotyping and genome scans approaches. Phenotyping enabled the quantification of virulence levels and confirmed resistance breakdowns. Pool‐Seq whole genome sequencing followed by genome scan analyses identified genomic regions under selection, potentially involved in the adaptive mechanisms to each resistance factor. Candidate genes within these regions provided insights into the genetic basis of adaptation, revealing effectors known to suppress plant immunity. As genome scans highlighted distinct genomic regions for the adaptation to both resistant factors, we were able to predict and phenotypically confirm the absence of cross‐virulence between nematode lineages evolving on GpaV vrn and GpaV spl . These findings have significant implications for the design of effective and sustainable resistance management strategies.

The use of resistant plants is an effective alternative to chemical products. But their sustainability is often compromised by the rapid adaptation of pathogen populations. For the cyst nematode Globodera pallida, a major parasite of potato, several quantitative trait loci (QTLs) conferring resistance have been identified, but their individual use could lead to resistance breakdown. Combining several resistance loci within a single potato genotype has been proposed as a strategy to improve both efficacy and durability. However, the evolutionary pathways leading to the circumvention of this pyramidal resistance remain unknown. The combination of experimental evolution, phenotyping and genome scan enabled us to study the genomic basis of G. pallida adaptation to individual (GpaV spl , GpaXI spl ) and pyramidal (GpaV + XI spl ) resistance QTLs. Experimental evolution over 10 generations revealed that adaptation to GpaV + XI spl pyramidal resistance was more difficult than to individual QTLs, but was nevertheless possible. Genomic analyses identified distinct regions under selection for each resistance, with a strong overlap between the adaptation to GpaV spl and to GpaV + XI spl , but a weaker overlap between the adaptation to GpaXI spl and to the pyramidal resistance. Known effector genes involved in immune suppression were systematically found in the selected regions, confirming their potential role in virulence. In addition, a two-generations experiment demonstrated that prior adaptation, particularly to GpaV spl , facilitated adaptation to pyramidal resistance. These results highlight the existence of preferential evolutionary trajectories favored by genomic proximity between nematode lineages adapted to different resistances. Our results show that pyramidal resistance can be compromised by the prior deployment of its individual components, and underline the importance of taking evolutionary pathways into account in resistance deployment.

Cyst nematodes of the genus Heterodera are obligatory sedentary endoparasites of great economic importance throughout the world. The Goettingiana group of this genus consisted of 17 species parasitising dicotyledons and are characterised by a lemon-shaped cyst having an ambifenestrate cone, long vulval slit, weak underbridge and presence or absence of bullae. In this study, we provided comprehensive phylogenetic analyses of 164 COI and 108 ITS rRNA gene sequences of several species of the Goettingiana group, including H. carotae, H. circeae, H. cruciferae, H. goettingiana, H. microulae, H. persica and H. urticae and several unidentified species using Bayesian inference, maximum likelihood, and maximum and statistical parsimony. 126 new COI and 46 new ITS rRNA gene sequences from 45 nematode populations representing six valid and two unidentified species collected in 13 countries were obtained in this study. Heterodera scutellariae syn. n. is considered as a synonym of H. circeae based on similar molecular and morphological characters. The carrot cyst nematode H. carotae is reported in France on a wild carrot species growing in natural ecosystems and in California from an agricultural field for the first time. The nettle cyst nematode in Spain and France is also reported for the first time. Our study showed that the ITS rRNA gene sequence can be used for discrimination of some species from the Goettingiana group; however, it did not allow differentiating H. carotae, H. cruciferae and H. urticae belonging to the H. cruciferae species complex from each other. The COI gene sequences clearly distinguished all studied species of the Goettingiana group from each other and can be recommended as a DNA barcoding marker for this group. It has been hypothesised that the majority of the Goettingiana group species originated and diversified in regions located in Western and Eastern Asia and Central and Western Europe during the Pleistocene and then dispersed from these regions across the world.

The present study reviews the knowledge to date on the carrot cyst nematode Heterodera carotae, which has become the primary threat to the carrot sector following the withdrawal of the last chemical soil fumigants in Europe. A keyword co-occurrence network was used to structure this review into five research areas: i ) biology and epidemiology of H. carotae; ii ) molecular identification and phylogeny; iii) population genetics and genomics; iv ) control methods and strategies; and v ) root exudates and hatching stimulation. Our findings indicate that H. carotae is an under-studied plant-parasitic nematode species, with several identified alternative control methods that require further investigation.

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).

The carrot cyst nematode, Heterodera carotae, is an important pest causing qualitative and quantitative production losses across several countries worldwide. In culture crops, Heterodera carotae and Heterodera cruciferae, can be found in mixture and are virtually unrecognizable from a morphological point of view. However, only H. carotae is able to develop on carrot. To monitor carrot production and set up suitable H. carotae control plans, simple and reliable diagnostic methods to detect and identify these two species are needed. In this study, we developed and successfully tested two real-time PCR protocols (SYBR Green singleplex and TaqMan multiplex real-time PCRs) against both species: H. carotae and H. cruciferae. Using two specific sets of primers, we managed, whatever the methods, to distinguish the two species. Moreover, we showed a higher specificity using the TaqMan assays but a better sensitivity with the SYBR Green assays. In this study, we are proposing two useful and reliable diagnostic tools to enable farmers and scientists to rapidly detect two morphologically and genetically closely related species and supervise agricultural management.

ABSTRACTThe carrot-parasitic nematode,Heterodera carotae, is an important pest causing qualitative and quantitative production losses across several countries worldwide and must be carefully identified and monitored. Indeed, in culture crops,Heterodera carotaeandHeterodera cruciferae, two genetically closely related species from the Goettingiana group, can be found in mixture and are virtually morphologically non-recognizable. However, onlyH. carotaeis able to develop on carrot. To monitor carrot production and set up suitableH. carotaecontrol plans, simple and reliable diagnostic methods to detect and identify these two species are needed. In this study, we developed and successfully tested two real-time PCR protocols (SYBR Green singleplex and TaqMan multiplex real-time PCRs) against both sister species:H. carotaeandH. cruciferae. Using two specific sets of primers targeting the two species, which were designed from sequences containing microsatellite loci, we managed, whatever the methods, to distinguish the two species. Moreover, we showed a higher specificity using the TaqMan assays (test on cysts) but a better sensitivity (tests on J2 juvenile larvae) with the SYBR Green assays. In this study, we thus proposed two useful and reliable diagnostic tools to enable farmers and scientists to rapidly detect two plant-parasitic nematodes and supervise agricultural management.

Plant-parasitic nematodes are a major threat to crop production in all agricultural systems. The scarcity of classical resistance genes highlights a pressing need to find new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality phased 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 parasitism stages. Analysis of the hologenome of the plant-nematode infection site identified 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 a highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is required for full pathogenicity. Knockout of either plant-encoded or now nematode-encoded steps in the pathway significantly reduces parasitic success. Our experiments establish a reference for cyst nematodes, further our understanding of the evolution of plant-parasitism by nematodes, and show that congruent differential expression of metabolic pathways in the infection hologenome represents a new way to find nematode susceptibility genes. The approach identifies genome-editing-amenable targets for future development of nematode-resistant crops.

Plant parasitic nematodes are highly abundant in all agrosystems and some species can have a major impact on crop yields. To avoid the use of chemical agents and to find alternative methods to manage these pests, research studies have mainly focused on plant resistance genes and biocontrol methods involving host plants or natural enemies. A specific alternative method may consist in supporting non-damaging indigenous species that could compete with damaging introduced species to decrease and keep their abundance at low level. For this purpose, knowledge about the biodiversity, structure and functioning of these indigenous communities is needed in order to carry out better risk assessments and to develop possible future management strategies. Here, we investigated 35 root crop fields in eight regions over two consecutive years. The aims were to describe plant parasitic nematode diversity and to assess the potential effects of cultivation practices and environmental variables on communities. Community biodiversity included 10 taxa of plant parasitic nematodes. Despite no significant abundance variations between the two sampling years, structures of communities varied among the different regions. Metadata collected for the past six years, characterizing the cultural practices and soils properties, made it possible to evaluate the impact of these variables both on the whole community and on each taxon separately. Our results suggest that, at a large scale, many variables drive the structuration of the communities. Soil variables, but also rainfall, explain the population density variations among the geographical areas. The effect of the variables differed among the taxa, but fields with few herbicide applications and being pH neutral with low heavy metal and nitrogen concentrations had the highest plant parasitic nematode densities. We discuss how these variables can affect nematode communities either directly or indirectly. These types of studies can help to better understand the variables driving the nematode communities structuration in order to support the abundance of indigenous non-damaging communities that could compete with the invasive species.

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.

Identification of plant parasitic nematode species is usually achieved following morphobiometric analysis, which requires a certain level of expertise and remains time consuming. Moreover, molecular and morphological discrimination of a number of emergent or cryptic species is sometimes difficult. Finding a way to achieve morphological characterisation quickly and accurately would greatly advance nematology science. Here, we developed a complete method in order to identify the two quarantine nematode species Globodera pallida and Globodera rostochiensis. First, we chose discriminative metrics on the stylet of nematodes that are able to be used by algorithms in order to build an automated process. Second, we used a custom computer vision algorithm (CCVA) and a convolutional neural network (CNN) to measure our metrics of interest. Third, we compared the CCVA and CNN predictions and their discriminative power to distinguish closely related species. Results show accurate identification of G. pallida and G. rostochiensis with the two methods, despite small-scale divergence (one to five mu m depending on the metric used). However, the error rate is higher for Globodera mexicana, suggesting that the algorithms are too specific. Nonetheless, these methods represent a promising novel approach to automated morphological identification of nematodes and Globodera species in particular.

Abstract Globodera ellingtonae was originally described from populations collected in the United States. In the original description, ribosomal DNA loci from Globodera sp. collected in Chile and Argentina were similar to G. ellingtonae, suggesting this nematode originated in this region of South America. In an effort to find additional populations of G. elllingtonae, collection trips were conducted in 2017 and 2020 in the Antofagasta and Arica y Parinacota Regions in Northern Chile, respectively. Globodera sp. were more prevalent in Antofagasta (17 samples collected, 53% positive for Globodera sp.) than in Arica y Parincota (16 samples collected, 13% positive for Globodera sp.). The genomes of single cysts (N ≥ 3) from four fields were sequenced. Additionally, the genomes of the G. ellingtonae population from Oregon and a Globodera sp. population originally collected in Antofagasta Region but maintained in culture in France were also sequenced. Based upon a HSP90 sequenced data mined from WSG data, all of the populations from the Antofagasta Region were G. ellingtonae and grouped in a monophyletic clade. A population collected from the Arica y Parincota Region was identified as G. rostochiensis based upon HSP90 data. Genome-wide SNP patterns of the G. ellingtonae populations showed strong clustering based on geographic location indicating that G. ellingtonae has high genetic diversity within Chile. A phylogenetic tree derived from 168,354 binary SNPs in the nuclear genome showed separate but distinct clustering of the Oregon population and the population from Antofagasta maintained in France. The Oregon G. ellingtonae population subtended the Chilean clades and placed on a long branch representing approximately twice the genetic variation observed among all Chilean G. ellingtonae populations. The possibility remains that G. ellingtonae from Chile may be sufficiently diverged to constitute a new species from G. ellingtonae originally described from a population collected in Oregon.

Global trading of plant materials, in combination with agricultural practices, may facilitate the spreading of cyst nematodes to so far non-infected areas. Recently Potato Cyst Nematode (PCN) was recognized to be present in Indonesia and both diversity and distribution require further study. Assessment of PCN populations was done by collecting soil samples, determination of morphological characteristics in combination with ITS rDNA and COI mtDNA sequencing. Thirty-seven soil samples were collected from potato fields in the Indonesia archipelago. The results showed the presence of Globodera rostochiensis in 22 out of 37 sampling fields, namely North Sumatra (6 fields), Central Java (12 fields), East Java (3 fields), and -for the first time- in Sulawesi (North Sulawesi) (1 field). The highest observed density was found in Banjarnegara (Central Java), i.e., 872 cysts 100 ml soil −1 . Globodera pallida was not recovered. Both ITS and COI characterisation of Indonesian PCN ( G. rostochiensis ) revealed the virtual absence of sequence variation as compared to most PCN from the rest of the world; the COI sequences were identical to the most common and mostly distributed haplotype around the world. Microsatellite genotyping indicated a higher genetic diversity for populations from East Java than for populations from North Sumatra, suggesting that cysts at the origin of populations in North Sumatra were coming from populations in East Java. These data on species identification, population density, genetic diversity, and distribution of potato cyst nematode over the Indonesian archipelago constitute the very basis for the design of environmentally-sound and effective PCN control strategies.

The recent ban of the most efficient chemical nematicides has left growers without methods for controlling the carrot cyst nematode Heterodera carotae . This phytoparasitic nematode species has a very narrow host range and causes severe crop losses in the main carrot‐growing regions worldwide. The development of alternative means of management of H. carotae is thus essential, and knowledge is required about the adaptive abilities of H. carotae , which mainly depend on gene flow among populations. The goal of this study was to describe the genetic structure of H. carotae populations at the spatial scale of the main infested French carrot‐producing region, i.e. Lower Normandy, and to disentangle the causes of the heterozygote deficit in this polyvoltine species. Microsatellite genotyping of populations collected at both the plant and field scales showed that: (i) the heterozygote deficit is mainly due to substructure; and (ii) there is strong gene flow among populations, leading to low F ST and to no clear genetic structure at the spatial scale explored here. Soil transport through both agricultural machinery and the transport of leek seedlings is probably responsible for the very strong H. carotae migration among fields and production areas. Measures should be considered to limit the passive spread of H. carotae .