Abstract:Ralstonia solanacerum and Clavibacter michiganensis subsp. sepedonicus are the two most relevant bacterial pathogens of potato for which a large number of molecular diagnostic methods using specific DNA sequences have been developed. About one hundred oligonucleotides have been described and thoroughly tested experimentally. After having compiled and evaluated all these primers and probes insilico to check their specificity, many discrepancies were found. A detailed analysis permitted the recognition of different possible reasons for such discrepancies: sequencing errors in public sequences, wrong supposed specificity (sometimes due to more recent sequences than the oligonucleotides being evaluated) or even typing errors in the oligonucleotides. Although this study is an exercise about insilico evaluation using two potato bacterial pathogens as a model, the conclusions reflect not only information useful for phytopathologists but, in a broader scope, draw the main situations that can be found during an evaluation of probes, which can be surely found in other scenarios.
Abstract: We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.
Abstract: Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.
Abstract: Eighty single-oospore offspring of Phytophthora infestans from a mating of isolates, which had previously been analyzed for segregation of avirulence/virulence, were assessed for the inheritance of 20 RFLP markers. Three offspring were triploid; they inherited three alleles at all loci where this could be detected and when heterozygous, showed unequal intensities of hybridization with most probes. Twenty-four offspring were trisomic, as each had three doses of one or a few markers, evident from their inheritance of three alleles or from unequal hybridization to one probe. Coinheritance of the extra allele(s) and mitochondrial haplotype in the majority of trisomic offspring suggested that meiosis in oogonia was more aberrant than in antheridia. Linkage analysis was performed on 50 offspring, which were assumed to be euploid; six small linkage groups were detected and several avirulence loci were found to be linked. The origins of aberrant offspring are discussed.
Abstract:Phytophthora infestans is the cause of late blight disease in potato and is an economically important pathogen worldwide. Early disease detection is important to implement disease control measures. In this study a surface plasmon resonance (SPR) immunosensor for detection of P. infestans sporangia is presented. The specificity of an existing mouse monoclonal antibody (phyt/G1470 mAb) against P. infestans was investigated in plate-trapped antigen ELISA and in subtractive inhibition ELISA. No or only limited cross-reactivity was observed against representatives having air-borne spores from Ascomycetes, Deuteromycetes as well as Basidiomycetes. phyt/G1470 mAb was incorporated in a subtractive inhibition SPR assay, consisting of a pre-incubation of mAb and sporangia, a centrifugation step to remove sporangia-bound phyt/G1470 mAb and quantification of remaining phyt/G1470 mAb by SPR. Good intra- and interday assay variability was observed and the assay had a detection limit of 2.2 × 106 sporangia/ml. Analysis time was 75 min, which is superior to existing P. infestans detection methods.
Abstract:Phytophthora ramorum is a recently described species responsible for sudden oak death syndrome and also causes symptoms such as twig wilt and dieback, stem lesions, necrosis of leafmidrib from the petiole and leaftip necrosis on a range of ornamental plant species. In the USA, a reported epidemic of P. ramorum infections on trees belonging to several families including Fagaceae, Lauraceae and Ericaceae seems to be increasing and there are ears of similar epidemics occurring in woodlands in the UK and mainland Europe. This paper reviews the current state of knowledge and the research efforts being made to understand the biology, manage the disease and prevent widespread outbreaks of P. ramorum infections across Europe and the USA.
Abstract: A previously unknown Phytophthora was recovered more than 60 times from evergreen hybrid azalea leaves collected during surveys for the sudden oak death pathogen Phytophthora ramorum in California and Tennessee. The novel Phytophthora was discovered when genomic DNA from this species cross-reacted with the ITS-based diagnostic PCR primers used to screen plants for the presence of P. ramorum. This species had caducous, semi-papillate sporangia, was homothallic with both paragynous and amphigynous antheridia, and was pathogenic on both wounded and intact azalea leaves. Nuclear and mitochondrial sequence data indicate that this species is related to, but distinct from, P. ramorum. AFLP analysis indicates that the isolates of this species have limited genotypic diversity and share no markers with P. ramorum. This paper presents the formal description of P. foliorum as a new species and underscores the need for caution when relying solely on DNA-based diagnostic tools.
Abstract: A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol–chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri ) with a sensitivity of 102 –103 cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies.
Abstract: We investigate themobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.
Abstract: In June 2008, Phytophthora syringae was baited from soil at the base of a dying pear tree in an orchard in the outer eastern suburbs of Melbourne, Australia. Phytophthora syringae has been rarely reported in Australia. The identification was made using rDNA ITS sequence data. State checklists record P. syringae on Prunus in South Australia and on Pyrus in Victoria. Neither of these records is linked to cultures or herbarium specimens. The only preserved specimen of P. syringae in Australia is from Cymbidium collected in New South Wales in 1991, but this specimen has been re-identified as P. multivesiculata, a species not previously recorded in Australia. These two cultures appear to be the only verifiable records of P. syringae and P. multivesiculata in Australia.
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