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

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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 1 fg 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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Acknowledgements

This research was supported in part by the Florida Citrus Production Research Advisory Council, Project No. 025-02I. Mention of a trademark, warranty, proprietary product, or vendor does not imply an approval to the exclusion of other products or vendors that also may be suitable.

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Authors and Affiliations

  1. US Horticultural Research Laboratory, US Department of Agriculture, Agricultural Research Service, 2001 South Rock Road, Fort Pierce, FL, 34945, USA

    Kim D. Bowman & Ute Albrecht

  2. Citrus Research and Education Center, University of Florida, Institute of Food and Agricultural Sciences, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

    James H. Graham & Diane B. Bright

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  1. Kim D. Bowman
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  2. Ute Albrecht
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  3. James H. Graham
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  4. Diane B. Bright
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Correspondence to Kim D. Bowman.

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Bowman, K.D., Albrecht, U., Graham, J.H. et al. Detection of Phytophthora nicotianae and P. palmivora in citrus roots using PCR-RFLP in comparison with other methods. Eur J Plant Pathol 119, 143–158 (2007). https://doi.org/10.1007/s10658-007-9135-7

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  • DOI: https://doi.org/10.1007/s10658-007-9135-7

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