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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: 1.61). 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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    ABSTRACT: A PCR-based system based on the sequence and restriction digests of the ITS region of the genomic ribosomal RNA gene repeat (rDNA) was used as a tool to characterize fourteen Phytophthora spp. isolates collected from the citrus orchards of Vidarbha region of Maharashtra. The results of RFLP analysis of internal transcribed spacer (ITS) region of ribosomal DNA of all the isolates revealed a difference in their restriction pattern for MspI and AluI digestions. Comparative data of RFLP analysis suggest 12 isolates as Phytophthora nicotianae and the rest two as P. palmivora. These two species were further detected with species-specific primers and sequence analysis of rDNA. The study conclusively revealed that P. nicotianae and P. palmivora are the most prevalent species in citrus orchards of Vidarbha region causing the widespread problem of citrus decline.
    Indian Phytopathology. 01/2011; Vol. 64.