Abstract: Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum-specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.
Abstract: The present work studies the seasonal variation of Phytophthora infestans concentrations in the atmosphere of a potato crop grown in A Limia. Different models have also been tested to predict the attack of this pathogen in order to establish the necessary treatments. Sampling has been carried out during crop cycles in 2004, 2005 and 2007 by using a volumetric spore trap, located in the centre of the plot at a height of 1.5 m. The collection of meteorological data was done with an automatic gauge. The highest concentrations of Phytophthora infestans were registered during June and July, with maximum daily levels ranging from 82 to 145 spores/m3, as a result of the maximum temperature average, around 16–23°C. Three prediction models for Phytophthora infestans have been adjusted to our area of study. The Smith Periods model provides better results in years with low and medium levels of the oomycete inoculum. The NegFRY model is useful to adjust the day of first treatment application, when used together with the Negative Prognosis model. To observe the influence of meteorological parameters on Phytophthora infestans spore concentration, besides applying these models, a Spearman correlation analysis was carried out. This test allowed the establishment of a correlation between the temperature parameter and the oomycete, obtaining positive and significant correlations (p < 0.01).
Abstract: This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae, the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein (Ypt1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae. The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt1F/Ypt1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.
Abstract:Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora, the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pgu1 in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/ul and in TaqMan PCR 1.2 fg/ul, and real-time PCR was 104–105 times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/ul. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method.
Abstract: Pink rot caused by Phytophthora erythroseptica is found in most major potato-growing regions of the world. The pathogen can survive for many years in soil by means of oospores which are disseminated from diseased potato tissues. The ability to detect the pathogen in soil could be a valuable management tool that enables growers to plan control strategies depending on the presence of pathogen propagules in a particular field. However, soils are one of the most challenging environmental matrices to obtain microbial DNA that will support PCR. A method was developed that combined traditional baiting technique with PCR methods to detect P. erythroseptica in infested soil samples. Hairy nightshade (Solanum sarrachoides Sendt.) and bitter nightshade (Solanum dulcamara L.) two leaf stage (TLS) seedlings and cotyledon leaves successfully baited P. erythroseptica from zoospore suspensions, artificially inoculated soils and naturally infested soils. The pathogen was detected in the bait tissue with PCR methods. PCR increased the precision of the bait test. However, time was still required for the pathogen to infect and develop on the bait tissues. Although P. erythroseptica was detected from some bait plants only after 2 days of incubation, 10 days of incubation produced consistent results across the replicates with hairy and bitter nightshade cotyledon leaves and TLS seedlings.
Abstract: A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5×103 P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.
Abstract: The plant trade is unwittingly accelerating the worldwide spread of well-known and new or undescribed Phytophthora species and creating novel niches for emerging pathogens. The results of a survey carried out from 2001 to 2006 in garden centres and nurseries of the Balearic Islands and eastern Spain combined with the analysis of samples received from ornamental nurseries from northern Spain reflected the extent of this global issue at the local scale. A total of 125 Phytophthora isolates were obtained from 37 different host species and 17 putative species identified on morphological features and direct sequencing of the internal transcribed spacer and four mitochondrial and nuclear genes. Five species, P. ramorum, P. hedraiandra, P. niederhauserii, P. kelmania and P. taxon Pgchlamydo were formally unknown to science prior to 2001. In addition, 37 new host/pathogen combinations were first records for Spain, highlighting the risk of non-coevolved organisms from different biogeographic origins coming into contact under managed environments. The problem generated by new or rare taxa of Phytophthora found in nurseries for which no prior information on natural habitat and ecology is available for pest risk analysis is discussed.
Abstract:Phytophthora ramorum, the cause of sudden oak death and ramorum blight, has three major clonal lineages and two mating types. Molecular tests currently available for detecting P. ramorum do not distinguish between clonal lineages and mating type is determined by cultural methods on a limited number of samples. In some molecular diagnostic tests, cross-reaction with other closely related species such as P. hibernalis, P. foliorum or P. lateralis can occur. Regions in the mitochondrial gene Cox1 are different among P. ramorum lineages and mitochondrial genotyping of the North American and European populations seems to be sufficient to differentiate between mating types, because the EU1 lineage is mostly A1 and both NA1 and NA2 lineages are A2. In our study, we were able to identify P. ramorum isolates according to lineage using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) of the Cox1 gene, first by using ApoI to separate P. ramorum from other species and EU1 from North American populations, and then AvaI to distinguish between NA1 and NA2 genotypes. However, P. foliorum had the same restriction profile as P. ramorum NA1 isolates.
Abstract: Experiments were conducted in irrigation runoff containment basins to assess the effects of bait species (Camellia japonica, Ilex crenata or Rhododendron catawbiense), bait type (whole leaf vs. leaf disc), baiting duration (1, 2, 7 or 14 days), baiting depth and growth media (modified PARP-V8 or PARPH-V8) on the recovery of Phytophthora species. A two-rope, flexible bait-deployment system was compared with a one-rope fixed system for bait stability at designated locations and depths. A total of 907 Phytophthora isolates were subjected to PCR-based single-strand conformation polymorphism (PCR-SSCP) analysis to identify to species level. Seven distinct SSCP patterns representing six morphospecies: P. citricola (Cil I), P. citrophthora (Cip I), P. hydropathica (Hyd), P. insolita (Ins), P. megasperma (Meg I & II) and an unidentified Phytophthora species were identified. Irrespective of culture medium, 7 days of baiting with rhododendron leaves consistently resulted in the recovery of the greatest diversity and populations of Phytophthora species with minimum interference from Pythium species. The flexible bait-deployment system was superior to the fixed system, minimizing the risk of bait loss and dislocation of baiting units and allowing baits to remain at designated depths from the surface under inclement weather.
Abstract: Species of Phytophthora are ubiquitous in ornamental production resulting in significant crop losses. In Tennessee, national surveys for the sudden oak death pathogen Phytophthora ramorum in 2004 and 2005 led to the isolation of Phytophthora species causing disease in nursery-grown or handled woody ornamentals or both. Isolates recovered were identified to species using direct sequencing of the internal transcribed spacer and examination of morphological characters. Six known species (P. cactorum, P. citricola, P. citrophthora, P. nicotianae, P. palmivora, P. tropicalis) and one newly described species (P. foliorum) were recovered from ericaceous hosts. The most common species recovered were P. citricola and P. citrophthora. Genetic analysis using amplified fragment length polymorphism (AFLP) markers revealed intraspecific genotypic diversity as well as isolates with identical AFLP genotypes from multiple locations across multiple years. This work provides evidence for species and genotypic diversity of Phytophthora recovered in Tennessee as well as insight into the movement of individual genotypes in woody ornamental production.
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