Abstract: A PCR-based ‘molecular tool box’, based on a region of the ras-related protein gene Ypt1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum,
P. cambivora,P. cinnamomi,P. citricola,P. europaea,P. inundata,P. lateralis,
P. megasperma,P. nemorosa,P. kernoviae,P. pseudosyringae,P. psychrophila,
P. quercina,P. ramorum and P. ilicis. Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium. Exceptions were primers designed for P. cactorum and P. ilicis, which cross-reacted with P. idaei and P. nemorosa, respectively. Amplification with Phytophthora-genus-specific primers before amplification with the various species-specific primers(nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µL–1 in the latter, compared with 100 fg µL–1 in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales,whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.
Abstract:Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and
herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.
Abstract:Phytophthora diseases cause major losses to agricultural and horticultural production in Australia and worldwide. Most Phytophthora diseases are soilborne and difficult to control, making disease prevention an important component of many disease management strategies. Detection and identification of the causal agent, therefore, is an essential part of effective disease management. This paper describes the development and validation of a DNA-based diagnostic assay that can detect and identify 27 different Phytophthora species.We have designed PCR primers that are specific to the genus Phytophthora. The resulting amplicon after PCR is subjected to digestion by restriction enzymes to yield a specific restriction pattern or fingerprint unique to each species. The restriction patterns are compared with a key comprising restriction patterns of type specimens or representative isolates of 27 different Phytophthora species. A number of fundamental issues, such as genetic diversity within and among species which underpin the development and validation of DNA-based diagnostic assays,are addressed in this paper.
Abstract:Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree,shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min
using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.
Abstract: The oomycete genus Phytophthora includes many of the world’s most destructive plant pathogens, which are generally disseminated by asexual sporangia. To identify factors relevant to the biology of these propagules,
genes induced in sporangia of the potato late blight pathogen Phytophthora infestans were isolated using cDNA macroarrays. Of ~1,900 genes known to be expressed in sporangia, 61 were up-regulated >5-fold in sporangia versus hyphae based on the arrays, including 17 that were induced >100-fold. A subset were also activated by starvation and in a nonsporulating mutant. mRNAs of some genes declined in abundance after germination,while others persisted through the germinated zoospore cyst stage. Functions were predicted for about
three-quarters of the genes, including potential regulators (protein kinases and phosphatases, transcription factors, and G-protein subunits), transporters, and metabolic enzymes. Predominant among the last were several dehydrogenases, especially a highly expressed sorbitol dehydrogenase that accounted for 3% of the mRNA. Sorbitol dehydrogenase activity also rose during sporulation and several stress treatments, paralleling the expression of the gene. Another interesting metabolic enzyme resembled creatine kinases, which previously
were reported only in animals and trypanosomes. These results provide insight into the transcriptional and cellular processes occurring in sporangia and identify potential targets for crop protection strategies.
Abstract: A description is given of the use of a combination of polymerase chain reaction (PCR) and baiting techniques for the specific detection of Phytophthora quercina and Phytophthora citricola from soil around declining oak trees. The soil was fooded with water and subjected to a specific baiting procedure using Quercus robur leaflets as baits. Single round or nested PCR, respectively, with species-specific primers allowed the detection of P. quercina and P. citricola in infected oak leafets used as baits and in the water from the same bait tests. PCR detection of both fungi was also possible after soil samples had been thoroughly mixed with water and the floating organic debris had been collected. Phytophthora quercina and P. citricola could be readily detected in almost every case in the water from these tests by PCR but less frequently in the organic debris. The identities of P. quercina and P. citricola were confirmed by restriction digests of the corresponding PCR amplicons. The presence of both fungi was also confirmed in parallel in soil samples tested by baiting with oak leaflets. Nested PCR with the primers used allowed the detection of as few as five zoospores of P. citricola and 300 zoospores of P. quercina in a volume of 100 ll. The methods presented here allow detection and identification of species of Phytophthora in soil without the need for direct extraction of soil samples, and without specific knowledge of the morphological characteristics of the genus.
Abstract: This paper describes the identification of PCR primers for the specific detection of Phytophthora cinnamomi. An internal standard DNA fragment amplified by the same PCR primers but giving an amplicon of a different size is added to the PCR reactions to detect false negative reactions caused by inhibition of amplification.
Abstract: Background: Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex,quantitative detection methods available suffer from compromises between the level of multiplexing,
throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput,ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides
with target complementary regions at their 5 and 3 ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system.
Results: The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar
ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays.
Conclusion: This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.
Abstract: Co-dominant microsatellite molecular markers for Phytophthora infestans were developed and their potential for monitoring the genetic variation in populations was demonstrated in the UK, across Europe and worldwide. Markers were developed according to two strategies. First, several thousand P. infestans expressed sequence tag (EST) and bacterial artificial chromosome (BAC) sequences were screened for the presence of simple sequence repeat (SSR) motifs, and, of these,100 candidate loci were selected for further investigation. Primer pairs developed to these loci were tested against a panel of 10 P. infestans isolates and approximately 10% were shown to be polymorphic and therefore appropriate for further testing. Secondly, the construction and screening of a partial genomic library resulted in the development of one additional polymorphic marker. The resulting 12 SSR markers were converted to higher-throughput fluorescence-based assays and used in combination with two previously published markers to characterize a wider collection of 90 P. infestans isolates from the UK and six other countries. Several isolates from the closely related species P. mirabilis, P. ipomoea and P. phaseoli collected from around the world were also genotyped using these markers. Amongst the 90 isolates of P. infestans examined, considerable SSR diversity was observed, with 68 different genotypes and an average of 3·9 (range 2–9) alleles per locus. When other Phytophthora species were genotyped, all loci were successfully amplified and the majority were polymorphic, indicating their transferability for the potential study of other closely related taxa.
Abstract:Phytophthora cinnamomi is an ecologically and economically important pathogen. In this study, PCR assays were developed with primer pair LPV2 or LPV3 for rapid detection and identification of this organism. Both primer pairs were selected from putative storage protein genes. The specificity of these primer pairs was evaluated against 49 isolates of P. cinnamomi, 102 isolates from 30 other Phytophthora spp., 17 isolates from nine
Pythium spp. and 43 isolates of other water moulds, bacteria and true fungi. PCR with both primer pairs amplified the DNA from all isolates of P. cinnamomi regardless of origin. The LPV3 primers showed adequate specificity among all other species tested. The LPV2 primers cross-reacted with some species of Pythium and true fungi, but not with any other Phytophthora species. PCR with the LPV3 primers detected the pathogen at levels of a single chlamydospore or 10 zoospores in repeated tests. The PCR assay was at least 10 times more sensitive than the plating method for detection of the pathogen from artificially infested soilless medium, and, to a lesser extent, from naturally infected plants. PCR with LPV3 primers can be a useful tool for detecting P. cinnamomi from soilless media and plant tissues at ornamental nurseries, whereas the LPV2 primers can be an effective alternative for identification of this species from pure culture. Applications of these assays for detection of P. cinnamomi in other environments were also discussed.
Abstract