Molecular characterisation of Peronospora parasitica infecting Brassica species.
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Peronospora parasitica (Pers. ex. Fr.) Fr., pathotype, single spore isolate, Brassica spp., downy mildew, internally transcribed spacers, ITSI , 5.8S, ITS2 sequence, radial phylogram, phylogeny, peR, micro satellite markers, SSRs, DNA library, microsatellite primer sequences, allele, geographic and pathotype diversity, polyploidy The downy mildew disease caused by Peronospora parasitica (Pers. ex. Fr.) Fr. infects approximately lOO plant species in the family Cruciferae. It is primarily a foliage blight which causes 60-70% seedling losses to nurserymen and rural subsistence farmers in Kwazulu-Natal, South Africa who depend on this crop to maintain a healthy cash flow not provided by other products. The disease is also frequent in India, France, Portugal and in the UK. The pathogen is especially debilitating in winter when low temperatures and high humidity favour infection and spread of the disease. Control of the disease with the use of fungicides is inefficient since P. parasitica has developed resistance to metalaxyl-based fungicides. There is a need to establish patterns of genetic diversity in order to address the ambiguities surrounding species definition of P. parasitica. The present study focused on clarifying the relationships between different pathotypes of P. parasitica found on various Brassica species viz. B. oieracea, B. juncea, B. napus, B. rapa, as well as the Arabidopsis thaiiana pathotype, a wild host of P. parasitica. Co-evolution with plant hosts over long periods was suggested to have lead to the divergent forms of this pathogen adapted to different host taxa. Genetic analysis of host specificity was investigated, based on sequence analysis of intergenic spacer regions of ribosomal DNA and on micro satellite markers. The current study provides the first comprehensive ITS-based phylogeny of pathotypes of P. parasitica. Based on ITSl, 5.8S and ITS2 sequences all pathotypes of P. parasitica from Brassica species (viz. Brassica oieracea, Brassica napus, Brassica juncea and Brassica rapa) were monophyletic. Based on ITSl sequences, the pathotype of P. parasitica from the wild host, Arabidopsis thaliana, was found to be significantly different from the Brassica pathotypes (i.e. cultivated host pathotypes). Furthermore the genetic distance between the genus Peronospora and Phytophthora was closer compared to other taxa such as Pythium, supporting the observation that the downy mildews have derived from a Phytophthora ancestor. rDNA sequence analysis was unable to differentiate Brassica pathotypes of P. parasitica. The development of a powerful class of genetic markers known as microsatellites (SSRs) in the present study provided greater insight into the relationships between Brassica pathotypes of P. parasitica. A genomic DNA library highly enriched for various micro satellites was prepared and a large number of potential SSRs consisting primarily of dinuceotide repeats (CA)n and (CT)n were obtained. Sequence analysis of 351 clones yielded 120 clones containing SSR loci, and 29 (24%) potentially useful SSRs from which primers could be designed were identified. PCR amplification with radiolabelled probes at 8 loci yielded useful polymorphisms across 27 isolates representing four pathotypes of P. parasitica. The alleles showed pathotype specific diversity since isolates of the same pathotype could be grouped together. The value of micro satellites as a strong discriminatory tool for intraspecies variation was further demonstrated by the ability to significantly separate isolates of the Brassica oleracea pathotype by their geographic origin and even field population in some instances. Several monoconidial lines from the same field population were grouped together and in some instances were found to be alike. The large number of alleles observed per genotype in the present study suggests that P. parasitica is polyploid and more complex than previously known. This parallels similar observations in other downy mildews such as Phytophthora, the closest relative of Peronospora. Future work involving sequencing at microsatellite loci would provide more insight into the exact ploidy, as well as the mapping of characterised single nucleotide polymorphisms (SNPs) which may be useful in specific identification of pathotypes. The primers developed for P. parasitica were able to amplify genomic DNA of Bremia lactucae indicating the value of the library developed in this study in comparative diversity studies with other Oomycetes. ITS-PCR and micro satellites markers provide useful tools for improved classification and diagnosis of downy mildew diseases. Furthermore, knowledge on the genetic diversity in P. parasitica together with the wealth of information now available about RPP genes of P. parasitica in Arabidopsis thaliana provides many exciting avenues for a complete understanding of the genetics of host specificity.