Abstract: Four different intergenic regions of mitochondrial DNA (mt-IGS), a fragment of the intergenic spacer (IGS) region of the rDNA (rDNA-IGS), and a fragment of the ras-related protein (Ypt1) gene were amplified and sequenced from a panel of 31 Phytophthora species representing the most significant forest pathogens and the breadth of diversity in the genus. Over 80 kbp of novel sequences were generated and alignments showed very variable (introns and non-coding regions) as well as conserved coding regions. The mitochondrial DNA regions had an AT/GC ratio ranging from 67.2 to 89.0% and were appropriate for diagnostic development and phylogeographic analysis. The IGS fragment was less variable but still appropriate to discriminate amongst some important forest pathogens. The introns of the Ypt1 gene were sufficiently polymorphic for the development of molecular markers for almost all Phytophthora species, with more conserved flanking coding regions appropriate for the design of Phytophthora genus-specific primers. In general, phylogenetic analysis of the sequence alignments grouped species in clades that matched those based on the ITS regions of the rDNA. In many cases the resolution was improved over ITS but in other cases sequences were too variable to align accurately and yielded phylograms inconsistent with other data. Key studies on the intraspecific variation and primer specificity remain. However the research has already yielded an enormous dataset for the identification, detection and study of the molecular evolution of Phytophthora species.
Abstract: In recent years, quantitative molecular diagnostic assays based on real-time PCR have been developed for many pests and pathogens of potato. In addition, simple sequence repeat markers have been developed and used to track isolates of Phytophthora infestans. These diagnostic assays are now being used as tools to study unresolved questions in the epidemiology of potato diseases including late blight, powdery scab and black dot. Examples of various investigations designed to examine the relative contribution of seed and soil-borne inoculum in causing black dot and powdery scab on progeny tubers, the effect of environmental factors on the incidence and severity of powdery scab and the survival of asexual and sexual inoculum of P. infestans in soil are described. Consideration is given to the development of appropriate diagnostic assays, their use in conjunction with relevant and robust sampling techniques, and the interpretation of results to inform disease risk assessment and control strategies through industry collaboration.
Abstract:Phytophthora nicotianae is an important soil-borne pathogen. The classical determination based on morphological or physiological characters is time and labour-consuming and is taxonomically unsure. Two specific primers designed from the spacer regions ITS1 and ITS2 were used to detect P. nicotianae by polymerase chain reaction (PCR). A PCR fragment of 737 bp was obtained from all P. nicotianae isolates assayed, but not from other Phytophthora spp. or other genera. Thus, the PCR characterization was species-specific. Polyclonal antisera against mycelium and zoospores were used for comparison and were only genus-specific.
Abstract:Phytophthora fragariae var. fragariae is pathogenic to strawberry only and is an EPPO A2 quarantine pest. Seventy-five isolates of P. fragariae from all over the world have been characterized by amplified fragment length polymorphism (AFLP) analysis. However, a sensitive, specific and fast method of detection is now needed, and polymerase chain reaction (PCR)-based methods have now been set up. Based upon sequences of the ITS (internal transcribed spacer) regions of rDNA of several Phytophthora species, PCR primers specific for P. fragariae have been developed together with SCRI (Dundee, GB). These primers were tested on DNA extracted from water, soil and plant material. A nested PCR procedure has been included in the currently used bait test to improve sensitivity and reliability. Also several methods have been studied to detect the amplicon of the PCR reaction: gel electrophoresis, DIAPOPS (detection of immobilized amplified products in a one-phase system), PCR-ELIS A, TaqMan and molecular beacon. With the last two methods, using the ABI 7700 detection system, quantification of target DNA is possible.
Abstract: Betelvine (Piper betle L.) is an important cash crop in Asia, with trade worth Rs 700 crores in India alone. One of the major limiting factors in yield is foot and leaf rot disease caused by Phytophthora parasitica var. piperina that sometimes causes 100% losses. The importance of molecular diagnostics in the management of this crop has been emphasized by Johri et al. because morphological identification is laborious and requires high level of expertise and could also lead to false determination. In this communication, we attempted a survey of betelvine conservatories for natural occurrence of the disease, isolation of pathogen, genomic DNA extraction, polymerase chain reaction (PCR), Southern hybridization and restriction fragment length polymorphism (RFLP) analysis of the PCR products to establish the identity at molecular level and the relationship/differentiation bet-ween Indian and other known isolates of Phytophthora.
Abstract:Ralstonia solanacearum, a soil-borne plant pathogen, causes lethal wilting disease of more than 200 plants worldwide. This very wide host range covers both monocots and dicots, extending from annual plants to trees and shrubs. Although generally it’s considered as a plant pathogen, R. solanacearum behaves primarily as a saprophytic bacterium able to survive for long periods of time in various natural habitats such as surface waters and different types of soils. Epidemiological and ecological studies on pathogen distribution in the environment are seriously hindered by the lack of efficient detection method especially when the concentration of the pathogen is either very low or is present in a latent, dormant or non-culturable state. With due attention to importance of R. solanacearum in Malaysia and several irreparable losses that every year caused by this bacterium, this is prompted current study to develop a sensitive PCR-Based method to improve the detection of R. solanacearum in natural sources. We selected the previously reported primers (OLI1;OLI2; Y2; JE2) for their sensitivity and specificity detection of the bacterium in water and soil by a modification of PCR assay.
Abstract: Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.
Abstract: The green fluorescent protein encoded by gfp gene and the luminescent protein encoded by luxAB genes were used as markers to detect p-nitrophenol (PNP)-degrading Moraxella sp. G21r and polychlorinated biphenyl (PCB)-degrading Ralstonia eutrophas H850Lr cells, respectively, in mixed liquid cultures and in soil samples using a most-probable-number (MPN) assay. Population estimates for both gfp-marked G21r and lux-marked H850Lr by using MPN assays were similar to viable colony counts. The MPN assay with microtiter plates permitted the simultaneous detection of fluorescent and luminescent bacteria in soil samples faster than conventional plate counting.
Abstract: Three primers from 16S rRNA were successfully assayed simultaneously in one reaction for sensitive detection of Ralstonia solanacearum in watercourses. The protocol is a modification of the Co-operational polymerase chain reaction (Co-PCR), which allows the simultaneous and co-operational action of the primers. It specifically amplified R. solanacearum strains belonging to biovars 1, 2 and 4. No products were obtained from any of the 162 unidentified isolates from river water. The sensitivity of the assay was <1 cfu/ml as determined by analysis of heat-treated water samples spiked with R. solanacearum, also containing indigenous microbiota up to 105 cfu/ml. The developed Co-PCR assay was more sensitive than other standard PCR assays in the analysis of 51 Spanish environmental water samples. Namely 31.3% of the samples were positive using the newly developed assay, whereas 13.7% or less positive samples were found with the other protocols. The Co-PCR improves the detection sensitivity of R. solanacearum and provides an important tool for its routine detection from environmental water samples and for epidemiological studies.
Abstract: A sensitive, selective, and rapid protocol for detecting Ralstonia solanacearum from soil and plant tissues was developed based on the integration of the rapid self-replicating ability of bacteriophages with quantitative PCR (q-PCR). Six bacteriophages were isolated and selected for their ability to specifically infect and lyse R. solanacearum. Sixty-three strains of R. solanacearum and 72 isolates of other bacterial species were tested for their susceptibility to the bacteriophages. Based on the large host range and observed replication speed and reproductive burst sizes in ginger infecting R. solanacearum strain GW-1, phage M_DS1 was selected for the development of the phage-based indirect assay. With primers based on the phage genome, the protocol was used to detect R. solanacearum from a number of substrates. In pure R. solanacearum cultures, the protocol consistently detected approximately 3.3 CFU/ml after an hour's incubation with 5.3 × 102 PFU/ml M_DS1. We used the protocol to confirm the presence of the pathogen in infected potted ginger plants, detecting levels near 102 CFU/g in 0.1 g of leaf tissue and levels near 103 CFU/ml in drainage water from the pots. In soils emended with the bacteria, we observed detection limits down to approximately 102 CFU/g.
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