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.

Exploring the causes of small effective population sizes in cyst nematodes using artificial Globodera pallida populations.

The effective size of a population is the size of an ideal population which would undergo genetic drift at the same rate as the real population. The balance between selection and genetic drift depends on the effective population size ( Ne), rather than the real numbers of individuals in the population ( N). The objectives of the present study were to estimate Ne in the potato cyst nematode Globodera pallida and to explore the causes of a low Ne/ N ratio in cyst nematodes using artificial populations. Using a temporal analysis of 24 independent populations, the median Ne was 58 individuals (min Ne = 25 and max Ne = 228). Ne is commonly lower than N but in the case of cyst nematodes, the Ne/ N ratio was extremely low. Using artificial populations showed that this low ratio did not result from migration, selection and overlapping generations, but could be explain by the fact that G. pallida populations deviate in structure from the assumptions of the ideal population by having unequal sex ratios, high levels of inbreeding and a high variance in family sizes. The consequences of a low Ne, resulting in a strong intensity of genetic drift, could be important for their control because G. pallida populations will have a low capacity to adapt to changing environments.

The coexistence of generalist and specialist clonal lineages in natural populations of the Irish Famine pathogen Phytophthora infestans explains local adaptation to potato and tomato.

Phytophthora infestans, causing late blight on Solanaceae, is a serious threat to potato and tomato crops worldwide. P. infestans populations sampled on either potato or tomato differ in genotypes and pathogenicity, suggesting niche exclusion in the field. We hypothesized that such niche separation can reflect differential host exploitation by different P. infestans genotypes. We thus compared genotypes and phenotypes in 21 isolates sampled on potato (n = 11) or tomato (n = 10). Typing at 12 microsatellite loci assigned potato isolates to the 13_A2, 6_A1 and 1_A1 lineages, and tomato isolates to the 23_A1, 2_A1 and unclassified multilocus genotypes. Cross-inoculations on potato and tomato leaflets showed that all isolates were pathogenic on both hosts. However, tomato isolates performed much better on tomato than did potato isolates, which performed better on potato than did tomato isolates, thus revealing a clear pattern of local adaptation. Potato isolates were significantly fitter on potato than on tomato, and are best described as potato specialists; tomato isolates appear to be generalists, with similar pathogenicity on both hosts. Niche separation in the field may thus result mainly from the large fitness gap on tomato between generalists and unadapted potato specialists, while the small, but significant fitness difference on potato between both types of isolates may prevent population invasion by generalists. Extreme specialization to potato seems very costly relative to performance loss on the alternative host. This study therefore shows that local adaptation and niche separation, commonly expected to involve and generate specialists, can occur with generalists.

And the nasty ones lose in the end: foliar pathogenicity trades off with asexual transmission in the Irish famine pathogen Phytophthora infestans.

A trade-off between pathogenicity and transmission is often postulated to explain the persistence of pathogens over time. If demonstrated, it would help to predict the evolution of pathogenicity across cropping seasons, and to develop sustainable control strategies from this prediction. Unfortunately, experimental demonstration of such trade-offs in agricultural plant pathogens remains elusive. We measured asexual transmission of Phytophthora infestans isolates differing in pathogenicity in two sets of artificial infection experiments under controlled, semi-outdoor conditions. Higher foliar pathogenicity decreased mean daughter tuber weight, increased infection severity in daughter tubers, and increased stem mortality before emergence. The most pathogenic isolates thus suffer a double penalty for asexual transmission: a lower survival probability within small and severely infected tubers; and a lower infection probability of neighbouring healthy plants due to fewer infected stems produced by surviving tubers. Moderate tuber resistance favoured transmission of the least pathogenic isolates, while high levels of resistance almost abolished transmission of all isolates. These data demonstrate a trade-off between foliar pathogenicity and asexual transmission over seasons in P. infestans, which should stabilise pathogenicity over time in the potato late blight pathosystem and possibly favour clone replacement by less pathogenic lineages after demographic bottlenecks.

Heterozygote deficits in cyst plant-parasitic nematodes: possible causes and consequences.

Deviations of genotypic frequencies from Hardy-Weinberg equilibrium (HWE) expectations could reveal important aspects of the biology of populations. Deviations from HWE due to heterozygote deficits have been recorded for three plant-parasitic nematode species. However, it has never been determined whether the observed deficits were due (i) to the presence of null alleles, (ii) to a high level of consanguinity and/or (iii) to a Wahlund effect. The aim of the present work was, while taking into the possible confounding effect of null alleles, to disentangle consanguinity and Wahlund effect in natural populations of those three economically important cyst nematodes using microsatellite markers: Globodera pallida, G. tabacum and Heterodera schachtii, pests of potato, tobacco and sugar beet, respectively. The results show a consistent pattern of heterozygote deficiency in the three nematode species sampled at the spatial scale of the host plant. We demonstrate that the prevalence of null alleles is weak and that heterozygote deficits do not have a single origin. Our results suggested that it is restricted dispersal that leads to heterozygote deficits through both consanguinity and substructure, which effects can be linked to soil movement, cyst density, and the number of generations per year. We discuss potential implications for the durability of plant resistances that are used to protect crops against parasites in which mating between relatives occur. While consanguineous mating leads to homozygosity at all loci, including loci governing avirulence/virulence, which favours the expression of virulence when recessive, the Wahlund effect is expected to have no particular effect on the adaptation of nematodes to resistances.

Modelling the evolutionary dynamics of viruses within their hosts: a case study using high-throughput sequencing.

Uncovering how natural selection and genetic drift shape the evolutionary dynamics of virus populations within their hosts can pave the way to a better understanding of virus emergence. Mathematical models already play a leading role in these studies and are intended to predict future emergences. Here, using high-throughput sequencing, we analyzed the within-host population dynamics of four Potato virus Y (PVY) variants differing at most by two substitutions involved in pathogenicity properties. Model selection procedures were used to compare experimental results to six hypotheses regarding competitiveness and intensity of genetic drift experienced by viruses during host plant colonization. Results indicated that the frequencies of variants were well described using Lotka-Volterra models where the competition coefficients ß(ij) exerted by variant j on variant i are equal to their fitness ratio, r(j)/r(i). Statistical inference allowed the estimation of the effect of each mutation on fitness, revealing slight (s = -0.45%) and high (s = -13.2%) fitness costs and a negative epistasis between them. Results also indicated that only 1 to 4 infectious units initiated the population of one apical leaf. The between-host variances of the variant frequencies were described using Dirichlet-multinomial distributions whose scale parameters, closely related to the fixation index F(ST), were shown to vary with time. The genetic differentiation of virus populations among plants increased from 0 to 10 days post-inoculation and then decreased until 35 days. Overall, this study showed that mathematical models can accurately describe both selection and genetic drift processes shaping the evolutionary dynamics of viruses within their hosts.

Combining Masculinizing Resistance, Rotation, and Biocontrol to Achieve Durable Suppression of the Potato Pale Cyst Nematode: A Model.

The pale cyst nematode, Globodera pallida, is a pest that poses a significant threat to potato crops worldwide. The most effective chemical nematicides are toxic to nontarget organisms and are now banned. Alternative control methods are therefore required. Crop rotation and biological control methods have limitations for effectively managing nematodes. The use of genetically resistant cultivars is a promising alternative, but nematode populations evolve, and virulent mutants can break resistance after just a few years. Masculinizing resistances, preventing avirulent nematodes from producing females, might be more durable than blocking resistances, preventing infection. Our demo-genetic model, tracking both nematode population densities and virulence allele frequencies, shows that virulence against masculinizing resistance may not be fixed in the pest population under realistic agricultural conditions. Avirulence may persist despite the uniform use of resistance. This is because avirulent male nematodes may transmit avirulent alleles to their progeny by mating with virulent females. Additionally, because avirulent nematodes do not produce females themselves, they weaken the reproductive rate of the nematode population, leading to a reduction in its density by at least 20%. This avirulence load can even lead to the collapse of the nematode population in theory. Overall, our model showed that combining masculinizing resistance, rotation, and biocontrol may achieve durable suppression of G. pallida in a reasonable time frame. Our work is supported by an online interactive interface allowing users (i.e., growers, plant health authorities, researchers) to test their own control combinations.

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).

Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec, Canada.

The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world.

Virus adaptation to quantitative plant resistance: erosion or breakdown?

Adaptation of populations to new environments is frequently costly due to trade-offs between life history traits, and consequently, parasites are expected to be locally adapted to sympatric hosts. Also, during adaptation to the host, an increase in parasite fitness could have direct consequences on its aggressiveness (i.e. the quantity of damages caused to the host by the virus). These two phenomena have been observed in the context of pathogen adaptation to host's qualitative and monogenic resistances. However, the ability of pathogens to adapt to quantitative polygenic plant resistances and the consequences of these potential adaptations on other pathogen life history traits remain to be evaluated. Potato virus Y and two pepper genotypes (one susceptible and one with quantitative resistance) were used, and experimental evolutions showed that adaptation to a quantitative resistance was possible and resulted in resistance breakdown. This adaptation was associated to a fitness cost on the susceptible cultivar, but had no consequence either in terms of aggressiveness, which could be explained by a high tolerance level, or in terms of aphid transmission efficiency. We concluded that quantitative resistances are not necessarily durable but management strategies mixing susceptible and resistant cultivars in space and/or in time should be useful to preserve their efficiency.

Volatile Organic Compounds from a Lichen-Associated Bacterium, Paenibacillus etheri, Interact with Plant-Parasitic Cyst Nematodes.

Healthy food is one of the major challenges to develop in this century. Plant-parasitic nematodes cause significant damage to many crops worldwide and till now, the use of chemical nematicides is the main means to control their populations. These chemical products must be replaced by more environmental-friendly control methods. Biocontrol methods seem to be one promising option, and the number of biopesticides derived from living organisms has increased in the last decades. To develop new plant protection products, we have decided to combine our skills in natural products chemistry and nematology and to focus on the lichen microecosystem as underexploited ecological niches of microorganisms. We present herein the potential of lichen-associated bacterial suspensions from Paenibacillus etheri as nematicides against the beet cyst nematode Heterodera schachtii and the potato cyst nematode Globodera pallida, in particular the effects of volatile organic compounds (VOCs) produced by the bacteria. A solid phase micro-extraction method associated to gas chromatography-mass spectrometry analysis of 14 day cultures was used to analyze these VOCs in order to identify the main produced compounds (isoamyl acetate and 2-phenylethyl acetate) and to evaluate them on the nematodes.

Fitness costs associated with unnecessary virulence factors and life history traits: evolutionary insights from the potato late blight pathogen Phytophthora infestans.

BACKGROUND: In gene-for-gene models of plant-pathogen interactions, the existence of fitness costs associated with unnecessary virulence factors still represents an issue, both in evolutionary biology and agricultural sciences. Measuring such costs experimentally has proven difficult, especially in pathogens not readily amenable to genetic transformation, since the creation of isogenic lines differing only by the presence or absence of avirulence genes cannot be achieved in many organisms. Here, we circumvented this difficulty by comparing fitness traits in groups of Phytophthora infestans isolates sharing the same multilocus fingerprint, but differing by their virulence/avirulence spectrum. RESULTS: Fitness was assessed from calculations derived from the basic reproduction number, combining several life history traits (latent period, spore density and lesion growth rate) evaluated on leaflets of the potato cultivar Bintje, which is free of resistance genes. A statistically significant fitness cost was found in isolates virulent to the R10 resistance gene. That cost was due to a lower spore production in virulent isolates; however, the latent period was shorter in virulent isolates. Similar trends, although not statistically significant, were observed for the other genes tested. CONCLUSION: The data likely reflect the adaptive response of the pathogen to the cost associated with virulence. They suggest strong trade-offs between life history traits related to pathogenicity and adaptive biology of pathogens.

A point mutation in the polymerase of Potato virus Y confers virulence toward the Pvr4 resistance of pepper and a high competitiveness cost in susceptible cultivar.

To understand why the Pvr4 resistance of pepper against Potyvirus spp. remained durable in field conditions while virulent Potato virus Y (PVY) variants could be selected in the laboratory, we studied the molecular mechanisms which generated these variants and the consequences on viral fitness. Using a reverse genetics approach with an infectious cDNA clone of PVY, we found that the region coding for the NIb protein (RNA-dependent RNA polymerase) of PVY was the avirulence factor corresponding to Pvr4 and that a single nonsynonymous nucleotide substitution in that region, an adenosine to guanosine substitution at position 8,424 of the PVY genome (A(8424)G), was sufficient for virulence. This substitution imposed a high competitiveness cost to the virus against an avirulent PVY variant in plants devoid of Pvr4. In addition, during serial passages in susceptible pepper plants, the only observed possibility of the virulent mutant to increase its fitness was through the G(8424)A reversion, strengthening the high durability potential of the Pvr4 resistance. This is in accordance with the fact that the NIb protein is one of the most constrained proteins expressed by the PVY genome and, more generally, by Potyvirus spp., and with a previously developed model predicting the durability of virus resistances as a function of the evolutionary constraint applied on corresponding avirulence factors.

Microsatellite markers reveal two admixed genetic groups and an ongoing displacement within the French population of the invasive plant pathogen Phytophthora infestans.

Potato late blight is an example of a re-emerging disease of plants. Phytophthora infestans was first introduced into Europe during the 19th century, where it caused the Irish potato famine. During the 20th century several additional introduction events have been suspected, especially in the mid-70s due to the import of large quantities of potato needed after the shortage caused by drought in 1976. Here, we investigate the genetic population structure of Phytophthora infestans, at the first stages of a recent invasion process in France. A total of 220 isolates was collected from 20 commercial fields of the potato susceptible cultivar Bintje, during two consecutive years (2004 and 2005). Clustering analyses based on eight recently developed microsatellite markers reveal that French P. infestans populations are made of two differentiated genetic clusters of isolates (F(ST) = 0.19). This result suggests multiple introductions of P. infestans into France, either through the introduction of a composite population of isolates or through the successive introduction of isolates having differentiated genetic backgrounds. Both clusters identified have a strong clonal structure and are similar regarding genetic diversity and mating type composition. The maintenance of differentiation between the two genetic clusters should result from the low or non-existent contribution of sexual reproduction in French P. infestans populations.

The hidden diversity of the potato cyst nematode Globodera pallida in the south of Peru.

Our knowledge of the diversity of potato cyst nematodes in their native areas still remains patchy and should be improved. A previous study based on 42 Peruvian Globodera pallida populations revealed a clear south to north phylogeographic pattern, with five well-supported clades and maximum diversity observed in the south of Peru. In order to investigate this phylogeographic pattern more closely, we genotyped a larger collection of Peruvian populations using both cathepsin L gene sequence data and a new set of 13 microsatellite loci. Using different genetic analyses (STRUCTURE, DAPC), we consistently obtained the same results that led to similar conclusions: the presence of a larger genetic diversity than previously known suggesting the presence of cryptic species in the south of Peru. These investigations also allowed us to clarify the geographic borders of the previously described G. pallida genetic clades and to update our knowledge of the genetic structure of this species in its native area, with the presence of additional clades. A distance-based redundancy analysis (dbRDA) was also carried to understand whether there was a correlation between the population genetic differentiation and environmental conditions. This analysis showed that genetic distances observed between G. pallida populations are explained firstly by geographic distances, but also by climatic and soil conditions. This work could lead to a revision of the taxonomy that may have strong implications for risk assessment and management, especially on a quarantine species.

Plant-parasite coevolution: A weak signature of local adaptation between Peruvian Globodera pallida populations and wild potatoes.

Plant-parasite coevolution has generated much interest and studies to understand and manage diseases in agriculture. Such a reciprocal evolutionary process could lead to a pattern of local adaptation between plants and parasites. Based on the phylogeography of each partner, the present study tested the hypothesis of local adaptation between the potato cyst nematode Globodera pallida and wild potatoes in Peru. The measured fitness trait was the hatching of cysts which is induced by host root exudates. Using a cross-hatching assay between 13 populations of G. pallida and root exudates from 12 wild potatoes, our results did not show a strong pattern of local adaptation of the parasite but the sympatric combinations induced better hatching of cysts than allopatric combinations, and there was a negative relationship between the hatching percentage and the geographical distance between nematode populations and wild potatoes. Moreover, a strong effect of the geographic origin of root exudates was found, with root exudates from south of Peru inducing better hatching than root exudates from north of Peru. These results could be useful to develop new biocontrol products or potato cultivars to limit damages caused by G. pallida.

Hatching of Globodera pallida Induced by Root Exudates Is Not Influenced by Soil Microbiota Composition.

Plant-parasitic nematodes are among the most harmful pests of cultivated crops causing important economic losses. The ban of chemical nematicides requires the development of alternative agroecological approaches to protect crops against nematodes. For cyst nematodes, egg hatching is stimulated by host plant root exudates. Inducing "suicide hatching" of nematode second-stage juveniles (J2), using root exudates in the absence of the host plant, may constitute an effective and innovative biocontrol method to control cyst nematodes. However, before considering the development of this approach, understanding the effect of soil biotic component on cyst nematode hatching by root exudates is a major issue. The effectiveness of this approach could be modulated by other soil organisms consuming root exudates for growth as soil microbiota, and this must be evaluated. To do that, four different native agricultural soils were selected based on their physicochemical properties and their microbiota composition were characterized by rDNA metabarcoding. To disentangle the effect of microbiota from that of soil on hatching, four recolonized artificial soils were obtained by inoculating a common sterile soil matrix with the microbiota proceeding from each agricultural soil. Each soil was then inoculated with cysts of the potato cyst nematode, Globodera pallida, and low or high doses of potato root exudates (PREs) were applied. After 40 days, viable J2 remaining in cysts were counted to determine the efficiency of root exudates to stimulate hatching in different soils. Results showed that (i) when physicochemical and microbiota compositions varied among native soils, the hatching rates remained very high albeit small differences were measured and no dose effect was detected and (ii) when only microbiota composition varied among recolonized soils, the hatching rates were also high at the highest dose of PREs, but a strong dose effect was highlighted. This study shows that abiotic and biotic factors may not compromise the development of methods based on suicide hatching of cyst nematodes, using root exudates, molecules inducing J2 hatch, or trap crops.

Hatching Induction of Cyst Nematodes in Bare Soils Drenched With Root Exudates Under Controlled Conditions.

Cyst nematodes account for substantial annual yield losses in crop production worldwide. Concerns over environmental and health issues due to the use of chemical nematicides mean alternative sustainable and integrated solutions are urgently required. Hatch induction of encysted eggs in the absence of host plants, i.e., 'suicide-hatching,' could be a sustainable alternative in reducing population densities of cyst nematodes in infested soils. Here we examined in situ hatching of encysted eggs of Globodera pallida, Heterodera carotae, and Heterodera schachtii at varying soil depths, following exogenous applications of host root exudates in repeated glasshouse experiments. Cysts were retrieved 30 or 43 days post-incubation depending on the nematode species and assessed for hatching rates relative to the initial number of viable eggs per cyst. Hatching of the potato cyst nematode G. pallida depended on both soil moisture and effective exposure to root exudates, and to a lesser extent on exudate concentration. The carrot cyst nematode H. carotae had over 75% hatched induced by root exudate irrespective of the concentration, with better hatch induction at 20 cm as compared with 10 cm soil depth. Hatching of the beet cyst nematode H. schachtii largely depended on the soil moisture level at constant temperature, rather than the type or concentration of root exudates applied. As a conclusion, exogenously applied host root exudates may play a major role in inducing in situ hatch of encysted eggs of potato and carrot cyst nematodes in the absence of host plant under favorable soil temperature/moisture conditions. To improve such strategy, the characterization of chemical profiles of the root exudate composition and field validation are currently ongoing.

Microsatellite markers reveal two genetic groups in European populations of the carrot cyst nematode Heterodera carotae.

The cyst nematode Heterodera carotae, which parasitizes carrot roots, has been recorded in many countries in Europe (Italy, The Netherlands, Switzerland, France, Denmark, …), in South Africa and in North America (Canada, USA). To date, there is a lack of knowledge about the genetic structure of the populations of this economically important nematode. The aim of this work was to study the structuration of the genetic diversity of the carrot cyst nematode at the European scale. We have developed a set of thirteen polymorphic microsatellite markers and used it to genotype seventeen European populations of H. carotae coming from France, Switzerland, Italy, Denmark and one non-European population from Canada. As previously showed for other cyst nematode species, the H. carotae populations were characterised by a strong heterozygote deficit. A Bayesian clustering analysis revealed two distinct genetic clusters, with one group located in the north of Europe and a second one located in the south of Europe. Moreover, our results highlighted rather limited gene flow at small spatial scale and some events of long distance migration. This first investigation of the genetic diversity of H. carotae populations would be useful to develop sustainable control strategies.

Genetic structure and aggressiveness of Erysiphe necator populations during grapevine powdery mildew epidemics.

Isolates of the causal ascomycete of grapevine powdery mildew, Erysiphe necator, correspond to two genetically differentiated groups (A and B) that coexist on the same host. This coexistence was analyzed by investigating temporal changes in the genetic and phenotypic structures of E. necator populations during three epidemics. Group A was present only at the start of the growing season, whereas group B was present throughout all three epidemics. Group A was less aggressive in terms of germination and infection efficiency but was more aggressive than group B in terms of the latency period, lesion diameter, and spore production. Our results are consistent with a temporal differentiation of niches, preventing recombination, and suggest an association between the disease level and the frequencies of genetic groups.