Molecular characterisation of Peronospora parasitica infecting Brassica species.
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Date
2002
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Abstract
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.
Description
Thesis (Ph.D.)--University of Durban-Westville, 2002.
Keywords
Peronospora., Peronospora -- Parasites -- Control -- Environmental aspects., Brassica.