Genetic diversity of the potato cyst nematode in the Netherlands

The potato cyst nematodes Globodera rostochiensis (Woll.) Skarbilovich and G. pallida (Stone) originate from the Andes region in South America and have been introduced into Western Europe since 1850. Both species are successful colonizers. Once primary founders have established vital populations, an area is rapidly colonized by secondary founding events. The mode of spread results in patchy distribution patterns. Analyses of the processes that influence the spatial variations in virulence are of major importance for the control by means of host plant resistance. The ability to unravel the mosaic distribution patterns of the two species and their pathotypes enables breeders and growers to anticipate on the dynamics of virulent populations. The aim of this thesis was to analyse the intra- and interspecific variation of G. rostochiensis and G. pallida in the Netherlands and to obtain insight in the processes that determine the spatial variations in protein, DNA and (a)virulence polymorphisms.interspecific variationThe molecular variation between the sibling species G.rostochiensis and G.pallida is remarkably large. The RAPD technique revealed a total of 250 DNA fragments, of which only nine DNA fragments were common to both species (Chapter 3). Similar results were obtained with the AFLP assay. A total of 1000 AFLP fragments was amplified, of which only 64 fragments could be identified in both species (Chapter 4). These results agreed with previous investigations using 2-DGE and show that morphologically nearly indistinguishable organisms can be quite distinct the molecular level.The extensive genetic differentiation of G.rostochiensis and G . pallida offers perspectives for the development of a diagnostic assay. In addition the PCR technique enables the amplification of species specific fragments from small numbers of cysts extracted from soil samples. For example, G. rostochiensis specific DNA fragments can be amplified from single juveniles (Chapter 1).intraspecific variationThe intraspecific variation of G.rostochiensis as revealed by RAPDs and AFLPs is relatively low (Chapter 3 and 4). The proportion of polymorphic DNA fragments among nine G. rostochiensis populations was 19% and 15.8%, respectively. Three clusters of populations were identified and each cluster could be described by one or more specific DNA fragments.The intraspecific variation of G.pallida, as revealed with RAPDs or AFLPs, is larger in comparison with G.rostochiensis (Chapter 3 and 4). The proportion of polymorphic RAPD fragments among 17 populations was 46%. For the AFLP assay this figure was 23% polymorphic AFLP fragments among 15 populations. The majority of the populations displayed a continuous range of variations. Only a few clearly diverged clusters could be discriminated on the basis of specific DNA fragments.Various groups have applied the RAPD technique to study the genetic divergence among conspecific populations of plant parasitic nematodes. The validity of this technique was evaluated in chapter 4 by comparison of the clustering of 36 G.pallida populations based on RAPD and 2-DGE data. Both data sets demonstrated that the majority of the G.pallida populations were not clearly differentiated from each other. The overall correlation between the distance matrices derived from both data sets was low. Careful examination of the separate dendrograms showed similarity in clustering only for clearly diverged populations or groups of populations.Although the AFLP and RAPD technique are simple, fast and require only minute amounts of biological material, they are not suitable to resolve the subtle differences among potato cyst nematode populations. Quantitative variation in allele frequencies is often not resolved with those techniques, which is, among others, due to the virtual inability to recognize co-dominant alleles and the non-linear amplification of DNA fragments. In chapter 6 and 7 the genetic variation was studied by analysing pools of individuals with 2-DGE. The ratios between the protein quantities produced by the codominant alleles are appropriate measures for the allele frequencies. The correctness of this method has been confirmed by 2-DGE of single individuals (De Boer etal ., 1992)bottleneck effects on the secondary foundersThe intraspecific variation among potato cyst nematode populations in Europe is predominantly determined by the genetic constitution of the primary founders, directly or indirectly introduced from South America, and the effect of random genetic drift on the secondary founders. To obtain insight in these processes, 226 G.pallida populations from the Netherlands were analyzed with 2- DGE. The results strongly suggest that these populations originate from one source, or in case of multiple introductions, from a number of sources with a similar genetic makeup.The genetic differentiation of the 226 G.pallida populations indicate that the colonization of the Netherlands has been accompanied by extensive random genetic drift. Only a limited proportion of the populations appeared to be identical. It is also shown, that the bottleneck effects differ between regions. Significant variation in population structure was observed between the three investigated areas. The genetic variation within G.pallida populations from north Friesland and the IJsselmeerpolders is in general smaller than within populations from the northeast Netherlands. A plausible explanation for this phenomena is the low multiplication rate of potato cyst nematodes due to wider crop rotation schemes in the IJsselmeerpolders and north Friesland. These factors result in a slow expansion of newly founded populations, which enhances the effect of drift.gene-pool similarity conceptThe gene-pool similarity concept rests on the hypothesis that in the absence of selection pressure by host plant resistance, degrees of similarity between populations revealed by molecular techniques are also reflected at virulence loci, including those not yet resolved. To test this concept, the genetic variation revealed by 2-DGE among 102 G.pallida populations was compared with the variation in virulence towards two resistant cultivars. This analysis showed that a decrease in genetic distance among populations is accompanied with a decrease in variation in virulence. In addition it is demonstrated that the gene pool similarity concept is also applicable to loci determining the variation in fitness among populations. The variation in Pf/Pi values among the 102 populations on Désirée is in general smaller between closely related populations than between distantly related populations.breeding for resistanceBreeding for resistance has been dominated by trial-and-error approaches, which has stimulated the view that control by means of host plant resistance is unavoidably a short-term approach due to the 'appearance' of virulent populations. The pervasive myth that breeding for resistance against potato cyst nematodes is a lost arms race is challenged by the results of this thesis. Until recently it was assumed that the genetic variation of G.pallida in the Netherlands was too large to produce potato cultivars with broad-spectrum resistance. In this thesis it is shown that the genetic diversity introduced from the Andes region has been limited and that the variation among the Dutch G.pallida populations is mainly the result of random genetic drift. The elaborate analysis of 226 Dutch G.pallida populations offers perspectives to obtain potato cultivars with broad and durable resistance. The gene pool similarities revealed by 2-DGE can be used as guidance in testing the effectiveness of new sources of resistance.