This chapter focuses on the general principles concerning resistance to cyst nematodes together with detailed information that relates specifically to soyabean, cereal, potato or sugarbeet cyst nematodes. The results of extensive breeding programmes on improving the resistance of some of the most economically important crops against plant parasitic nematodes are also presented.

Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study interpopulation variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast andcompared to sequencing approaches-inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.

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Summary Characterising the non-neutral genetic variation within and among populations of plant-parasitic nematodes is essential to determine factors shaping the population genetic structure. This study describes the genetic variation of the parasitism gene vap1 within and among geographic populations of the beet cyst nematode Heterodera schachtii. Forty populations of H. schachtii were sampled at four spatial scales: 695 km, 49 km, 3.1 km and 0.24 km. DGGE fingerprinting showed significant differences in vap1 patterns among populations. High similarity of vap1 patterns appeared between geographically close populations, and occasionally among distant populations. Analysis of spatially sampled populations within fields revealed an effect of tillage direction on the vap1 similarity for two of four studied fields. Overall, geographic distance and similarity of vap1 patterns of H. schachtii populations were negatively correlated. In conclusion, the population genetic structure was shaped by the interplay between the genetic adaptation and the passive transport of this nematode.

Summary The stem nematode, Ditylenchus dipsaci, causes severe damage in sugar beet. To date, nematode inoculation through the leaf axil has been used as the standard method to investigate D. dipsaci interaction with sugar beet under in vivo conditions. To get as close as possible to field conditions, we established a new screening mechanism to perform soil inoculation. The most suitable inoculation time point, inoculum level and positioning on sugar beet, as well as rearing process on carrots, were determined. At a 15:8°C day:night temperature regime, penetration rates of D. dipsaci were at maximum following soil inoculation at plant emergence. Up to 115 nematodes penetrated sugar beet seedlings 22 days post-planting with an inoculum level of 1000 nematodes into the soil at plant emergence. Ditylenchus dipsaci penetration rate was higher in plants with soil inoculation than with inoculation on to the leaf axil. High soil moisture increased nematode migration into seedlings when D. dipsaci inoculation was carried out in four holes 1 cm from the plant base. Rearing the nematodes for 35 days at 20°C on carrot discs resulted in an infective inoculum containing up to 50% eggs. We recommend a soil inoculation of 1000 freshly extracted nematodes per pot at plant emergence. The nematode suspension has to be previously reared for 35 days on carrot discs to obtain active D. dipsaci inoculum. This system will allow for the selection of suitable sugar beet genotypes that suppress nematode penetration, in support of breeding for resistance against D. dipsaci.

The stem nematode, Ditylenchus dipsaci, is a severe pest in European sugar beet production. In France, Germany, and Switzerland, D. dipsaci damage in sugar beet varies among specific geographic areas. In this study, the reproduction potential of four geographically distinct D. dipsaci populations was determined using sterile carrot disc cultures. In addition, virulence and pathogenicity were investigated in-vivo using sugar beet. No difference was found in the reproduction potential on carrot discs, as well as penetration rate in sugar beet seedlings. The reproduction rate in sugar beet tissue was significantly affected by the D. dipsaci population used. The population from Seeland (CH) showed the highest number of nematodes per plant at 60 dpi (21,071.8 ± 5340.0), compared to the three other populations contained 3588.6 ± 3858.3, 5136.9 ± 4950.8, and 3579.7 ± 5174.2, respectively. Furthermore, the reproduction rate of D. dipsaci was negatively correlated with fresh biomass of sugar beets at 60 dpi. Based on these results, the D. dipsaci population “Seeland” is suitable for breeding programs to detect resistance in sugar beet. After selecting candidate genotypes/varieties, these should be further evaluated for their field resistance in their targeted growing regions.

In this study fungal strains were investigated, which had been isolated from eggs of the cereal cyst nematode Heterodera filipjevi, and roots of Microthlaspi perfoliatum (Brassicaceae). The morphology, the interaction with nematodes and plants and the phylogenetic relationships of these strains originating from a broad geographic range covering Western Europe to Asia Minor were studied. Phylogenetic analyses using five genomic loci including ITSrDNA, LSUrDNA, SSUrDNA, rpb2 and tef1-α were carried out. The strains were found to represent a distinct phylogenetic lineage most closely related to Equiseticola and Ophiosphaerella, and Polydomus karssenii (Phaeosphaeriaceae, Pleosporales) is introduced here as a new species representing a monotypic genus. The pathogenicity tests against nematode eggs fulfilled Koch’s postulates using in vitro nematode bioassays and showed that the fungus could parasitise its original nematode host H. filipjevi as well as the sugar beet cyst nematode H. schachtii, and colonise cysts and eggs of its hosts by forming highly melanised moniliform hyphae. Light microscopic observations on fungus-root interactions in an axenic system revealed the capacity of the same fungal strain to colonise the roots of wheat and produce melanised hyphae and microsclerotia-like structure typical for dark septate endophytes. Confocal laser scanning microscopy further demonstrated that the fungus colonised the root cells by predominant intercellular growth of hyphae, and frequent formation of appressorium-like as well as penetration peg-like structures through internal cell walls surrounded by callosic papilla-like structures. Different strains of the new fungus produced a nearly identical set of secondary metabolites with various biological activities including nematicidal effects irrespective of their origin from plants or nematodes.