Abstract: Genotypic variation among 32 single-zoospore isolates (SZI)of Phytophthora infestans, derived asexually from two hyphal-tip parental isolates (PI-105 and PI-1) of the US-8 genotype, was assessed with 80 random amplified polymorphic DNA (RAPD) primers and 18 amplified fragment length polymorphic DNA(AFLP) primer pairs. In previous investigations, the SZIs from parental isolate PI-105 showed high levels of virulence variability and were differentiated into 14 races, whereas the SZIs from PI-1 showed identical virulence to the parent. The purpose of this investigation was to determine if phenotypic variation observed among SZIs of P. infestans could be detected at the DNA level in these isolates.Polymorphism was detected with 51 RAPD primers and with all 18 primer pairs in PI-105 SZIs. In SZIs from
PI-1, polymorphism was also detected with 25 RAPD primers and 17 AFLP primer pairs. Cluster analysis using the unweighted pair-group method with arithmetic averages(UPGMA) separated the SZIs from parent PI-105 into six virulence groups, 11 RAPD groups and three AFLP groups. Cluster analysis of PI-1 SZIs, which all belong to the same virulence group, differentiated them into four RAPD groups and six >AFLP groups. No close correlation among RAPD,AFLP and virulence groups could be established within the two progenies of SZIs. Results of this study suggest that there is a considerable level of inherent genetic variability among SZIs derived asexually from the same parental isolate. The possible mechanisms and implications of this genetic variation are discussed.
Abstract: Cotton blight, caused by the oomycete Phytophthora boehmeriae, is a serious disease of cotton in China. In wet weather conditions, P. boehmeriae is usually the primary pathogen, followed by many saprophytic
fungi and pathogens such as Pythium spp., Fusarium spp., Rhizoctonia and others. As P. boehmeriae grows much slower than other pathogens, it is difficult to isolate and identify. A rapid and accurate method for its specific identification is necessary for the detection of blight in infected cotton tissue. The internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) from three isolates of P. boehmeriae were amplified using the polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. PCR products were cloned and sequenced. The sequences were aligned with those published of 50 other Phytophthora species, and a region specific to P. boehmeriae was used to construct the specific PCR primers PB1 and PB2. Over 106 isolates of 14 Phytophthora species and at least 20 other fungal species were used to check the specificity of the primers. PCR amplification with primers PB1 and PB2 resulted in the amplification of a product of approximately 750 bp only from isolates of P. boehmeriae. Using primers PB1 and PB2, detection sensitivity was approximately 10 fg DNA/ll. In inoculated plant material, P. boehmeriae could be detected in tissue 1 day after inoculation,prior to the appearance of symptoms. The PB primerbased PCR assay provides an accurate and sensitive method for detecting P. boehmeriae in cotton tissue.
Abstract: To identify markers for the Phytophthora resistance gene, Rps1-d,123 F2:3 families were produced from a cross between Glycine max(L.) Merr. Tanbakuro (a Japanese traditional black soybean) and PI103091 (Rps1-d) as an experimental population. The results of virulence tests produced 33 homozygous resistant, 61 segregating and 29 homozygous susceptible F2:3 families. The chi-squared test gave a goodness-of-fit for the expected ratio of 1:2: 1 for resistant,segregating and susceptible traits, suggesting that the inheritance of Rps1-d is controlled by a monogenic dominant gene. Simple sequence repeat(SSR) analyses of this trait were carried out using the cultivars Tanbakuro and PI103091. Sixteen SSR primers, which produced 19 polymorphic fragments between the two parents, were identified from 41 SSR primers in MLG N. Eight SSR markers were related to Rps1-d,based on 32 of the 123 F2:3 families, consisting of 16 homozygous resistant and 16 homozygous susceptible lines. The remaining 91 families were analysed for these eight markers, and a linkage map was constructed using all 123 F2:3 families. The length of this linkage group is 44.0 cM. The closest markers, Sat_186 and Satt152, are mapped at 5.7 cM and 11.5 cM, respectively, on either side of the Rps1-d gene. Three-way contingency table analysis indicates that dualmarker-assisted selection using these two flanking markers would be efficient.
Abstract: Aims:The primary objectives of this study were to determine if a single-strand conformation polymorphism(SSCP) analysis can be used for rapid identification of Phytophthora ramorum, an important quarantine plant
pathogen worldwide, and to further assess the potential of the SSCP technique as a taxonomic tool for the genus Phytophthora.
Methods and Results: SSCP of ribosomal DNA internal transcribed spacer 1 was characterized for 12 isolates of P. ramorum, using a recently reported protocol. The SSCP patterns of this species then were compared with those of 18 closely related Phytophthora species. Phytophthora ramorum had a unique pattern and was easily distinguished from genetically, morphologically and ecologically close relatives.
Conclusion: An immediate benefit of this study is provision of a highly effective and eficient identification tool for P. ramorum in the quarantine process.
Significance and Impact of the Study: This study also provides additional evidence demonstrating that the SSCP is an ideal DNA marker for species differentiation within the genus Phytophthora.
Abstract: Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses.We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans.The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex(Phytophthora extracellular protein) cDNAs.Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases.Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans.To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes,high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome.This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora.Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato.Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.
Abstract: In our researches we tried to convert the P.infestansRAPD markers into SCAR markers for a more operative diagnosis of potato late blight in the infected material. For this goal, several monomorphic and polymorphic RAPD sequences were recuperated from the agarose gel, and cloned into E.coli bacteria. The cloned monomorphic sequences were sequenced, establishing the nucleotide sequence. Relying on this several pair of primers was designed. One pair from them, with the following nucleotide sequences AGC CCA CAC TGA AAT TAC GC/GGC TAC CGG ATC AGT ACC AA), gave amplification product only with P.infestansDNA. Being specific, this pair pf primer could be used for a straightforward potato late blight diagnosis from the infected material, and our objective concerning the conversion of P.infestansRAPD markers into SCAR markers could be considered accomplished.
Abstract: Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phytophthora ramorum, although species such as P. nemorosa and P. pseudosyringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of
the coxI and II genes for detection of Phytophthora spp. in general, and
P. ramorum, P.nemorosa, and P.pseudosyringae in particular. The firstround multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P.ramorum, P.nemorosa, or P. pseudosyringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P.ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA,the limit of detection for P.ramorum using this marker system was ≈2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P.ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.
Abstract: A number of molecular diagnostic procedures for detection of Phytophthora ramorum have been reported in the literature. In an effort to evaluate the specificity of 10 of these techniques a standardized DNA library for 317 isolates was assembled that included 60 described species as well as 22 taxonomically unclassified isolates. These were sent blind at a concentration of ca. 10 ng/µl (a concentration greater than would be encountered in field samples, but was used in an effort to fully evaluate specificity) to collaborators to evaluate the various diagnostic procedures. In general the procedures worked well with varying levels of specificity observed among the different techniques. Low levels of nonspecific amplification were observed for the mitochondrial markers and most of the real-time assays based on nuclear markers. The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not standalone and is used in conjunction with two other assays for diagnostic purposes. The results from the APHIS lab and another lab indicated that using three assays improved the accuracy of the results compared to looking at a single assay alone. The SSCP procedure accurately identified P. ramorum and was helpful in classification of other isolates to a species level. Given that one of the objectives of the trials was to determine if there would be any false positives, the DNA concentrations that were tested in all but one of the assays (6 to 10 ng/amplification) was higher than would be expected when processing field samples. As a result false positives for some of the assays (in particular real-time assays that had high Ct values) may not be representative of what might be encountered with field samples. Additional evaluations for these samples with a dilution series of target DNA are needed to evaluate specificity at DNA concentrations more reflective of what would be encountered in field assays. Trials evaluating marker performance with samples recovered from the field are in progress.
Abstract: Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on-site techniques require expensive equipment, considerable expertise and are not suited for plant health inspectors. Therefore, an extensive evaluation of a commercially available lateral flow device (LFD) for Phytophthora species was performed involving four separate trials and 634 samples. The assay proved simple to use, provided results in a few minutes and on every occasion a control line reacted positively confirming the validity of the test. LFD results were compared with those from testing a parallel sample, using laboratory methods (isolation and real-time PCR). The diagnostic sensitivity of the LFD (87·6%) compared favourably with the standard laboratory methods although the diagnostic specificity was not as stringent (82·9%). There were a small number(n= 28) of false negatives, but for statutory purposes where all positive samples must be identified to species level by laboratory testing, overall efficiency was 95·6% as compared with visual assessment of symptoms of between 20-30% for P. ramorum and P. kernoviae. This work demonstrates the value of the LFD for diagnosing Phytophthora species at the time of inspection and as a useful primary screen for selecting samples for laboratory testing to determine the species identification.
Abstract: Sensitive and specific primer sets for polymerase chain reaction (PCR)
for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestansDNA,or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P.infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.
Abstract