Genetic and phenotypic diversity in the graminaceous cyst nematode complex, inferred from PCR-RFLP of ribosomal DNA and morphometric analysis

Graminaceous cyst nematodes form a group of eleven valid species including Heterodera avenae, Heterodera filipjevi and Heterodera latipons and constitute major pests to cereals. They are widely spread in circum-mediterranean areas where they are presumed to cause yield losses on bread and durum wheat. The objective was to document the diversity of these cereal cyst nematodes, in particular samples from Mediterranean regions, in comparison to species which develop on cultivated or wild grasses (H. arenaria, H. hordecalis, H. mani) and on rice or sugarcane (H. sacchari). Studies involved PCR-RFLP of ITS and morphometrics of the juvenile and cyst characters. UPGMA analysis of molecular data showed that the isolates segregated in two main clusters which seem to represent different evolutionary lineages. The H. avenae sensu lato cluster (I) contained H. arenaria, H. avenae, H. filipjevi and H. mani. The second cluster (II) contained isolates of H. hordecalis and H. latipons. Within H. avenae sensu lato, H. filipjevi was separated from the other related species with significant bootstrap value. The differentiation of H. arenaria, recognized for the first time based on molecular data, and H. mani with few restriction enzymes were the least significant. Intraspecific polymorphism allowed differentiation of isolates originating from Australia within H. avenae sensu stricto. The group H. hordecalis–H. latipons showed the greatest genetic variability between and within isolates. Two representatives of Heterodera sacchari, taxonomically included in the ‘schachtii’ group, were genetically as distant to this group as to the other graminaceous species belonging to either H. avenae sensu lato or H. hordecalis–H. latipons group. Results inferred from multivariate analysis applied on morphometrics of the cysts and juveniles showed agreement between genetic and phenotypic classifications. This study demonstrates the utility of combined molecular and classical methods to enhance our knowledge about the diversity within the complex of graminaceous cyst nematodes and to establish robust techniques to identify a wider set of nematode species.