Detection of Phytophthora nicotianae and P. palmivora in citrus roots using PCR-RFLP in comparison with other methods

European Journal of Plant Pathology (Impact Factor: 1.71). 09/2007; 119(2):143-158. DOI: 10.1007/s10658-007-9135-7

ABSTRACT Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly
and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal
transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the
method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested
PCR with universal primers ITS4 and ITS6, the detection limit was 1fg of fungal DNA, which made it 1000× more sensitive than
a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific
patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification
of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to
the interpretation of data.

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    Plant Disease 08/2012; 96(8):1080-1103. · 2.74 Impact Factor
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    ABSTRACT: The goal of this study was to develop a procedure that could be used to evaluate the potential susceptibility of aquatic plants used in con-structed wetlands to species of Phytophthora commonly found in nurseries. V8 agar plugs from actively growing cultures of three or four isolates of Phytophthora cinnamomi, P. citrophthora, P. cryptogea, P. nicotianae, and P. palmivora were used to produce inocula. In a labora-tory experiment, plugs were placed in plastic cups and covered with 1.5% nonsterile soil extract solution (SES) for 29 days, and zoospore presence and activity in the solution were monitored at 2-or 3-day intervals with a rhododendron leaf disk baiting bioassay. In a green-house experiment, plugs of each species of Phytophthora were placed in plastic pots and covered with either SES or Milli-Q water for 13 days during both summer and winter months, and zoospore presence in the solutions were monitored at 3-day intervals with the baiting bioas-say and by filtration. Zoospores were present in solutions throughout the 29-day and 13-day experimental periods but consistency of zoo-spore release varied by species. In the laboratory experiment, coloniza-tion of leaf baits decreased over time for some species and often varied among isolates within a species. In the greenhouse experiment, bait colonization decreased over time in both summer and winter, varied among species of Phytophthora in the winter, and was better in Milli-Q water. Zoospore densities in solutions were greater in the summer than in the winter. Decreased zoospore activities for some species of Phy-tophthora were associated with prolonged temperatures below 13 or above 30°C in the greenhouse. Zoospores from plugs were released consistently in aqueous solutions for at least 13 days. This procedure can be used to provide in situ inocula for the five species of Phy-tophthora used in this study so that aquatic plant species can be evalu-ated for potential susceptibility.
    Plant Disease 11/2013; 98(4):551-558. · 2.74 Impact Factor
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    ABSTRACT: With the increased attention given to the genus Phytophthora in the last decade in response to the ecological and economic impact of several invasive species (such as P. ramorum, P. kernoviae, and P. alni), there has been a significant increase in the number of de-scribed species. In part, this is due to the extensive surveys in historically underexplored ecosystems (e.g., forest and stream eco-systems) undertaken to determine the spread of invasive species and the involvement of Phytophthora species in forest decline worldwide (e.g., oak decline). Brasier (41) cited the number of described species in 1999 at approximately 55, and there has been an increase of an additional 50 species or distinct taxonomic enti-ties described between 2000 and 2007. This represents a near dou-bling in eight years! Érsek and Ribeiro (80) recently updated the list of described species to 100; since then, additional species have been named (some provisional), raising the total to 117 (Table 1) with a number of other distinct taxonomic entities in the process of formal description. The number of species will likely continue to increase as more surveys are completed and greater attention is devoted to clarifying phylogenetic relationships and delineating boundaries in species complexes. The development of molecular resources, including the recent comprehensive multigene phyloge-netic analysis of the genus (32), the availability of credible se-quence databases to simplify identification of new species (,,, and, and the sequencing of several genomes (107,278) have provided a solid framework to move for-ward. Gaining a better understanding of the biology, diversity, and taxonomic relationships within the genus will be important for the improvement of identification and diagnostic protocols. This infor-mation is much needed considering the impact invasive or exotic Phytophthora species have had on natural ecosystems and the regulatory issues associated with their management. Reviews by Cooke et al. (61) and O'Brien et al. (217) are noteworthy for providing additional information on molecular identification and detection. Taxonomy of the Genus Phytophthora Numerous studies on the taxonomy of Phytophthora have been published since the initial description of the type species of the genus, Phytophthora infestans (Mont.) de Bary, in 1876 (reviewed in Erwin and Ribeiro, 82). Most of the publications contain dichotomous keys for species identification with the exception of Stamps et al. (253), which includes a tabular format based on keys developed by Waterhouse (286,287). Waterhouse (286) introduced the concept of morphological groupings (I-VI) "with the intent to solely serve as an aid to species identification, and not meant to imply a natural classification". Newhook et al. (213) and Stamps et al. (253) also included these groupings in their taxonomic evalua-tion of the genus. More recent attempts to simplify identification of species include a manual for identification of 60 species of Phy-tophthora by integration of a dichotomous key with a DNA finger-printing technique based on polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) (88) and a Lucid Key for identification of 55 common Phytophthora spp. based on a series of interactive computer matrices (230). Cline et al. (55) have published an online list of Phytophthora spp. occur-ring in the United States as well as species occurring elsewhere with a hyperlink for each species to the USDA SMML database that includes host range, distribution, and supporting literature. Recently, Kroon et al. (166) provided an update on the taxonomy of the genus.
    Plant Disease 01/2012; 96(8):1080-1103. · 2.74 Impact Factor


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