Abstract: RAPD polymerase chain reaction analysis was used to study the genetic diversity among a wild potato variety Solanum demissum (very resistant to late blight) and six potato cultivars (Hanna, Lady-Olympia, Lady-Rosetta, Spunta, Diamant and Cara) varied in their resistance to Phytophthora infestans. Cluster analysis of six potato genotypes showed that, all tested genotypes were separated into two clusters (1 and 2). Cluster 1, included only the wild potato variety (S. demissum), whereas cluster 2 divided into two groups (G1 and G2). Late blight high resistant cultivars
Hanna and Cara were grouped in G1. Group 2 included the moderate resistant cultivar Spunta and the susceptible cultivars Diamant, Lady-Rosetta and Lady-Olympia. The potato cultivars that showed highest genetic similarity to the wild potato variety were the resistant cultivars Hanna and Cara. Lowest genetic similarity was obtained with the susceptible cultivars Lady-Rosetta, Diamant and Lady-Olympia. RAPD primer K17 yielded a band with molecular weight of 936 bp found in all susceptible potato cultivars (Lady-Rosetta, Lady-Olympia and Diamant). On the other hand, band with molecular weight of 765 bp were detected in the wild potato and the resistant cultivars Hanna and Cara. Results of this study suggested that, the RAPD marker technique could be beneficial for revealing the genetic variability of different genotypes of potato varied in their resistibility to late blight.
Abstract: Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HHO6-23 and EST-SSR primer pair PiO8N. The results showed that the optimal annealing temperature was 63°C, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol.L-1 dNTPs, 2 µL 10 × Buffer (Mg2+ free), 1.75 mmol.L-1 MgCl2, 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20µL reaction system. The PCR program was initial denaturation at 94°C for 2 mm, followed by 35 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s, then a final extension step was 72°C for 7 min, and held at 4°C. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity of P. infestans population.
Abstract: A collection of 432 single-lesion isolates of Phytophthora infestans collected from blighted potato foliage during 2004-2007 in Estonia, were analyzed for virulence (all isolates), mating type (424 isolates) and response to metalaxyl (412 isolates). The samples came from 25 fields comprising conventional production in central, northern, southern, south-eastern and south-western regions and from untreated experimental field trials at Jõgeva Plant Breeding Institute in eastern Estonia. Of the isolates 33% were A2 mating type. Both mating types were present in all fields; the frequency of A2 mating type varied from 3% to 71%. In the context of specific virulence, the Estonian population had a very low frequency of virulence against R5 (17%) and R9 (3%). The most common pathotype was 1.3.4.7.10.11. A subgroup of 57 isolates was assessed for mtDNA haplotype and RG57 fingerprints. Three mitochondrial DNA haplotypes, i.e. Ia (51%), IIa (42%) and IIb (7%), were found. Twenty-one RG57 fingerprints were detected. The four most common fingerprints represented more than half of the isolates (67%). On the basis of combined markers, thirty-three multilocus genotypes were identified, of which 81% were detected only once. Genotypic diversity measured by the normalized Shannon diversity index was 0.79. The data indicate that the Estonian population of P. infestans is diverse, having a large number of multilocus genotypes and both mating types within fields, with potential for sexual recombination and spread of fungicide resistance.
Abstract: A total of 104 isolates including two Korean isolates and three Japanese isolates of Phytophthora infestans collected from Heilongjiang and Jilin Provinces from 2006 to 2008 were used to determine their mating types, metalaxyl resistance, and RAPD genotypes. All the isolates of P. infestans collected from Heilongjiang and Jilin Provinces belonged to A1 mating type, and no A2 mating type was detected. Frequencies of metalaxyl resistant isolates were 94.4%, 47.8% and 75.0% in 2006, 2007 and 2008, respectively. According to RAPD analysis by using OPD-20 primer, 104 isolates including two Korean isolates and three Japanese isolates were separated into 10 RAPD groups at 90.8% similarity level. A total of 10 RAPD groups were not correlated to groups defined by mating type, locations, and metalaxyl resistance.
Abstract: The present study was undertaken to evaluate the diversity of Phytophthora infestans isolates collected between 1996 and 2006 from potato-growing regions of India. Thirty-two genotypes were distinguished among 70 isolates characterized using mating type, metalaxyl resistance, mitochondrial DNA (mtDNA) haplotypes and microsatellite markers. The mating type analysis revealed that the A1 mating type prevailed (90%) in the subtropical Indo-Gangetic plains, whereas the A2 mating type was dominant (93%) in temperate highland regions. All 11 isolates collected before 2000 were metalaxyl sensitive, whereas all 28 isolates from after 2004 were metalaxyl tolerant. Before 2002, the Ib mtDNA haplotype was seen in all 26 isolates, but a shift was observed from 2004 to 2006 after which the Ia mtDNA haplotype was found to have replaced the Ib haplotype everywhere except in the north eastern hills of India. Microsatellite analysis revealed that isolates from the north Himalayan hills after 2000 were distinct from the all the others. Little divergence was observed among earlier isolates from the same region along with earlier isolates from the north Indian plains and the 2005 isolates from the north-eastern hills. The highest variability was observed in regions with high pathogen build-up and where frequency of exchange of planting materials was relatively high. The results of this study indicate a drift in the pathogen population in the first half of the present decade throughout India.
Abstract: Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates. The genotypic diversities, quantified by a normalized Shannon's diversity index (Hs), were 0.95 for the four Nordic countries. The low FST value of 0.04 indicates that the majority of variation is found within the four Nordic countries. The large number of genotypes and the frequency distribution of mating types (60% A1) support the hypothesis that sexual reproduction is contributing notably to the genetic variation of P. infestans in the Nordic countries.
Abstract: Current molecular detection methods for the genus Phytophthora are specific to a few key species rather than the whole genus and this is a recognized weakness of protocols for ecological studies and international plant health legislation. In the present study a molecular approach was developed to detect Phytophthora species in soil and water samples using novel sets of genus-specific primers designed against the internal transcribed spacer (ITS) regions. Two different rDNA primer sets were tested: one assay amplified a long product including the ITS1, 5.8S and ITS2 regions (LP) and the other a shorter product including the ITS1 only (SP). Both assays specifically amplified products from Phytophthora species without cross-reaction with the related Pythium s. lato, however the SP assay proved the more sensitive and reliable. The method was validated using woodland soil and stream water from Invergowrie, Scotland. On-site use of a knapsack sprayer and in-line water filters proved more rapid and effective than centrifugation at sampling Phytophthora propagules. A total of 15 different Phytophthora phylotypes were identified which clustered within the reported ITS-clades 1, 2, 3, 6, 7 and 8. The range and type of the sequences detected varied from sample to sample and up to three and five different Phytophthora phylotypes were detected within a single sample of soil or water, respectively. The most frequently detected sequences were related to members of ITS-clade 6 (i.e. P. gonapodyides-like). The new method proved very effective at discriminating multiple species in a given sample and can also detect as yet unknown species. The reported primers and methods will prove valuable for ecological studies, biosecurity and commercial plant, soil or water (e.g. irrigation water) testing as well as the wider metagenomic sampling of this fascinating component of microbial pathogen diversity.
Abstract: Phytophthora foot rot of black pepper caused by Phytophthora capsici is a major disease of black pepper (Piper nigrum) throughout Vietnam. To understand the population structure of P. capsici, a large collection of P. capsici isolates from black pepper was studied on the basis of mating type, random amplified microsatellites (RAMS) and repetitive extragenic palindromic (REP) fingerprinting. Two mating types A1 and A2 were detected in four provinces in two climatic regions, with A1:A2 ratios ranging from 1:3 to 1:5. In several instances A1 and A2 mating types were found to co-exist in the same farm or black pepper pole, suggesting the potential for sexual reproduction of P. capsici in the field in Vietnam although its contribution to disease epidemics is uncertain. RAMS and REP DNA fingerprinting analysis of 118 isolates of P. capsici from black pepper showed that the population was genetically more diverse where two mating types were found, although the overall genetic diversity was low with most of the isolates belonging to one clonal group. The implication of these findings is discussed. The low diversity among isolates suggests that the P. capsici population may have originated from a single source. There was no genetic differentiation of isolates from different climatic regions. In addition to the large clonal group, several isolates with unique RAMS/REP phenotypes were also detected. Most of these unique phenotypes belonged to the minority A1 mating type. This may have significant implications for a gradual increase in overall genetic diversity.
Abstract: Eight strains belonging to the Oomycete genus Phytophthora were isolated from Zostera marina (seagrass) in The Netherlands over the past 25 y. Based on morphology, isozymes, temperature-growth relationships and ITS sequences, these strains were found to belong to two different Phytophthora species. Five strains, four of them isolated from rotting seeds and one isolated from decaying plants, could not be assigned to a known species and hence belong to a new species for which we propose the name Phytophthora gemini sp. nov. Three strains were isolated from decaying plants and were identified as Phytophthora inundata, thereby expanding the known habitat range of this species from fresh to brackish-saline areas. The possible role of both Phytophthora species in the decline of Z. marina in The Netherlands and the evolutionary significance of the presence of Phytophthora species in marine environments are discussed.
Abstract: A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trnY and rns and trnW and cox2 were identified by comparing the whole mitochondrial genomes of Phytophthora infestans, Phytophthora ramorum, and Phytophthora sojae and amplified using primers designed from the flanking conserved genes. These regions were sequenced from 51 isolates of P. nicotianae of both A1 and A2 mating type recovered from different hosts and geographic regions. Amplicon length varied from 429 bp to 443 bp (trnY/rns) and 322 bp to 373 bp (trnW/cox2) with intraspecific variation due to single nucleotide polymorphisms and indels. Seventeen, seven and 20 different haplotypes were detected by individually analyzing regions trnY-rns, trnW-cox2 and the combined data set of sequences from both regions, respectively. Phylogenetic analysis inferred with three different methods enabled the grouping of isolates in five clades, each containing different mitochondrial haplotypes and revealed diversity in the mitochondrial genome of P. nicotianae. The majority of isolates from citrus grouped in a single clade indicating either movement of isolates on planting stock or an association of particular isolates with this host. Phylogenetic groups were not correlated with the radial growth rate of the isolates or the rapidity of apple flesh colonization. The method developed in the present study represents an innovative molecular tool for the characterization of natural populations of P. nicotianae and should be easily expanded to other species of Phytophthora as well as other plant pathogens. It can be used to track specific haplotypes and, thanks to its high genetic resolution, it could be standardized and applied in a DNA barcoding like strategy for the precise identification of sub-specific taxa. Compared to alternative molecular methods, a major advantage is that results are unbiased (a list of nucleotides) and highly reproducible, thus enabling the comparison of data from different laboratories and time periods. Furthermore, the method could be further enhanced by the identification of additional variable mitochondrial and/or nuclear genomic regions.
Abstract