Abstract:Phytophthora clandestina, a serious pathogen of subterranean clover (Trifolium subterraneum), has only been recorded in Australia. A rapid method was used to characterise genetic variation among 61 isolates of P. clandestina belonging to eight pathogenic races based on DNA sequencing and single-strand conformation polymorphism (SSCP) analyses of the β-tubulin gene of the pathogen. The β-tubulin gene among those tested displayed a high degree of variability among isolates. Cluster analysis of the SSCP profiles grouped the 61 isolates into two main clusters (A and B). Cluster A with 3 subclusters viz; I (race 177), II (race 000) and III (races 001, 121, 101, 143 and 157) and B with race 173 alone. In addition, SSCP of β-tubulin also successfully differentiated between races 173 and 177, the two most prevalent and most virulent of the races studied. This is the first time that the β-tubulin gene has been used to study intra-species variation in this pathogen. In addition to showing relationship among the strains, it also provides a practical means for rapid monitoring of current and future differences in the distribution of P. clandestina strains, giving subterranean clover breeders and farmers a sound basis for the selection/breeding and deployment of appropriate cultivars to counter the predominant strain populations in specific localities.
Abstract: Pink rot of potatoes caused by Phytophthora erythroseptica is known to infect all underground potato (Solanum tuberosum) tissues such as roots, stolons, tubers and basal stems. Potato leaves and stems do not normally exhibit symptoms except when extensive infection of underground plant parts results in plant wilt, chlorosis and necrosis. This is the first study to investigate the spread of P. erythroseptica in above-ground potato tissues using both traditional isolation and PCR methods originally developed to detect the pathogen in tubers. P. erythroseptica was detected in 66% of leaf and stem tissue samples originated from artificially-inoculated and naturally-infected tubers of Yukon Gold and Shepody by PCR methods. However, it was only recovered in pure cultures from 38% of stem and leaf tissue samples. The pathogen was also detected in leaf and stem tissues and aerial tubers of plantlets grown in potting mixtures infested with P. erythroseptica. Pure cultures of P. erythroseptica obtained from stem and leaf tissues, together with the 95% of the detection with the real-time PCR assay and 45% with the conventional PCR assay confirmed the viability of the pathogen in above-ground potato tissues. Furthermore, the pathogen was detected in progeny tubers and stolons produced by the infected potato plants. P. erythroseptica was also detected in a few samples of debris taken from naturally senesced above-ground potato tissue after harvest.
Abstract: As part of a study to examine the phylogenetic history of the taxonomically challenging species Phytophthora cryptogea and P. drechsleri, a distinct monophyletic group of isolates, previously described as P. drechsleri or P. cryptogea, were characterised. Analysis of their rDNA ITS sequences indicated that these isolates were distinct from P. drechsleri, P. cryptogea, and all members of Phytophthora ITS clades 1–8, clustering instead alongside basal groups previously described as clades 9 and 10. This group comprised six isolates all of which were isolated from woody plants, such as pistachio (Pistacia vera, Iran and USA), fig (Ficus carica, Iran), and almond (Prunus dulcis, Greece). Analysis of sequence data from nuclear (b-tubulin and translation elongation factor 1α) and mitochondrial (cytochrome c oxidase subunit I) genes confirmed the ITS-based analysis as these isolates formed a distinct monophyletic group in all NJ trees. The isolates were fast growing with a relatively high optimum growth temperature of 30 °C and, in most cases, rapid colony growth even at 37 °C. The isolates produced complex colony patterns on almost all media, especially corn meal agar (CMA). Phylogenetic analysis and examination of all the other morphological and physiological data lead us to infer that this taxon has not been described previously. As this taxon was first isolated and described from Iran we propose that this taxon be formally designated as Phytophthora parsiana.
Abstract: In California and Oregon, two recently described oomycete forest pathogens, Phytophthora nemorosa and P. pseudosyringae, overlap in their host and geographic ranges with the virulent P. ramorum, causal agent of sudden oak death. Epidemiological observations, namely broader geographic distribution and lack of landscape-level mortality, led to the hypothesis they are native to this region, whereas multiple lines of evidence indicate P. ramorum is exotic to North America. We used AFLP analysis to measure genetic variability in the homothallic P. nemorosa and P. pseudosyringae and to evaluate the hypothesis of endemism. We analysed 39 P. nemorosa and 48 P. pseudosyringae isolates (29 American and 19 European) from throughout their geographic and host ranges. In the US, both P. nemorosa and P. pseudosyringae have a dominant AFLP clone with several closely related variants. There is no evidence that genetic diversity is partitioned by host or location in P. nemorosa, but the US P. pseudosyringae clonal lineage is largely nested within a more genetically variable European group. Though the absence of highly variable sampled source populations does not allow us to determine whether each species is native or introduced in the western US with certainty, the results are most consistent with the hypothesis that both are introduced — P. pseudosyringae perhaps from Europe. Invasive Phytophthora species are increasingly being implicated in emergent forest diseases, highlighting the need to identify and characterize both native and previously unknown introduced forest Phytophthoras.
Abstract: The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.
Abstract: Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species of Phytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianae specific primers a delayed and lower fluorescence increase was also obtained from P. cactorum DNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg ll-1 for P. nicotianae and 100 pg ll-1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg ll-1 was detected for both pathogens. Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianae and P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.
Abstract: An unnamed ITS clade 6 Phytophthora was frequently isolated from rhizosphere soil of reed (Phragmites australis) growing on the littoral zone of Lake Constance. The isolates closely resembled P. gonapodyides, having internally proliferating, non-papillate sporangia, a rather high temperature optimum for growth (300C), and being sexually sterile. ITS sequence analysis revealed that they were identical to the as yet unnamed Phytophthora taxon Salixsoil, originally isolated from Salix roots in the UK and Alnus debris in Denmark. The taxon was readily isolated from permanently as well as occasionally flooded reed sites using
standard baiting procedures, indicating a wide distribution in the Lake Constance littoral zone. In an in vitro leaf inoculation assay P. taxon Salixsoil proved to be more aggressive towards Salix alba than
P. gonapodyides. The new taxon may be of significance as a root pathogen of woody plants in moist or flooded situations occurring in alluvial forest/plant communities. We propose that due to its close resemblance to P. gonapodyides the taxon might have passed unnoticed in the past, and possibly is much more widely distributed than previously recognised.
Abstract: Five isolates collected from different peppercropping regions in Guangdong Province, China were determined. Based on their morphological characteristics and symptoms after being re-inoculated to pepper, these isolates were identified as Phytophythora capsici Leonian. The sporangia induced on carrot medium (CA) were morphologically similar and most of their shapes were ovate or elliptic, and obviously papillate. The mean size of the sporangium was 40.8−45.9 (l) µmx23.2–30.9 (b) µm, with l/b ratio 1.4 1.8. There were evident differences in mycelial growth rate, productivity of sporangia and pathogenicity to pepper among the isolates. A test of physiological characteristics showed that one isolate was determined as Race 1, and the other four isolates belonged to Race 3. It is concluded that Race 3 is most likely to be the predominent race in Guangdong Province, China.
Abstract: The purpose of this study was to develop a new technique to evaluate the number of spores incorporated in splash droplets by the use of an engineered fluorescent pathogen strain and image analysis hardware and software. The inoculum source consisted of a tomato leaflet infected with a Phytophthora infestans strain transformed with the gene encoding the green fluorescent protein (GFP). Splash droplets formed after impact of incident drops on sporulating lesions were collected on microscope slides located at different distances from the source. Each slide was examined using a fluorescent microscope to visualize the GFP expressing sporangia. Photographs were taken and assessed using image processing to count the sporangia incorporated in each droplet. Data analysis confirmed characteristics of splash dispersal shown using other methods. The use of fluorescent sporangia also facilitates the selective detection and counting of viable (living) sporangia, and is a tool that can be use in the study of splash dispersed diseases.
Abstract: Introns are generally highly polymorphic regions within genes and were proven to be of great interest for discriminating among phylogenetically-close Phytophthora species. Phytophthora ramorum and P. fragariae are considered as quarantine pathogens by the European Union and accurate detection tools are therefore necessary for their monitoring. From introns located in different single copy genes (GPA1, RAS-like, and TRP1), we developed a series of PCR primers specific to P. ramorum and P. fragariae. The specificity of these primers was successfully checked with a wide collection of Phytophthora isolates and a protocol was developed to detect both pathogens directly in infected plant tissues. These genes should be of particular interest for the development of additional species-specific detection tools within the Phytophthora genus.
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