AbstractA specific and sensitive PCR assay for the detection of Phytophthora infestans, the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of internal transcribed spacer regions (ITS) of a range of Phytophthora species. Following PCR amplification with primers INF FW2 and INF REV, a 613 bp product was generated from DNA of an isolate of P. infestans. No product was amplified when DNA from isolates of various other Phytophthora species and potato blemish pathogens was tested, showing that the primers were species-specific. These primers can detect as little as 0.5 pg of pure P. infestans DNA. In a nested assay, sensitivity was increased by two fold, and as little as 5 fg P. infestans DNA was detected. The primer pair can also detect as few as two oospores, two sporangia and four zoospores of P. infestans. Using primer pair INF FW2 and INF REV, ten oospores of P. infestans could be detected in 0.5 g soil, besides detecting the pathogen in symptomatic and symptomless leaves, stems and tubers. The assay was validated on field soil samples and commercial potato seed stocks.
Studies on the long term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans were undertaken under natural field conditions. PCR detection of oospores was possible up to 24 months (total length of the study) after burial in soil. The sporangia, although detectable up to 12 months, were less efficiently detected at the nine and twelve months sampling dates, presumably representing a degradation of inoculum not adapted to long term survival. In a baiting assay, sporangial inoculum proved non-viable whereas leaf material containing oospores remained viable up to 24 months after burial.
Population studies are dependant on the development of co-dominant markers. Eight single nucleotide polymorphisms (SNPs) were identified in the P. infestans genes 2- phosphoglycerate dehydratase, transaldolase, glutamine synthetase, ubiquitin conjugating enzyme and ADP/ATP translocase which represents a rate of ~ 2 SNP per kb. The majority of the SNPs were synonymous (i.e. did not code for a different amino acid). Screening of randomly selected fragments of non-coding DNA from a BAC (Bacterial Artificial Chromosome) library yielded an additional 28 SNPs which represents a rate of 2 SNP per kb. Allele specific PCR was used to develop SNPs into useful markers. Use of fluorescently labelled primers markedly increased the assay throughput. Segregation analysis revealed that with the exception of 56E14R and glutamine synthetase (linked in the coupling phase), the markers segregated independently. Seventeen genotypes were detected amongst 42 Scottish P. infestans isolates. Cluster analysis based on SNP markers grouped isolates into two clades and broadly supported previous studies in which AFLP markers were used.
In the present studies, there was some evidence of cultivar-specific adaptation of P. infestans isolates. The isolates caused larger lesions on the cultivars on which they were maintained for seven successive weeks. The effect of cultivar adaptation was however lost upon removal of the selection pressure. The results were validated in an inter-isolate competition experiment. Generally, more sporangia were produced on a cultivar by the isolate adapted to that cultivar than by its non-adapted competitor isolate. The use of the above developed SNP markers allowed tracking of isolates in the inter-isolate competition within a single lesion. The availability of SNP markers will provide a powerful tool for epidemiological studies of P. infestans on a field scale.
|Date of Award||Feb 2003|