Submitted to: European Journal of Plant Pathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/5/2007
Publication Date: 8/15/2007
Citation: Bowman, K.D., Albrecht, U., Graham, J.H., Bright, D.B. 2007. Detection of Phytophthora nicotianae and P. palmivora in citrus roots using PCR-RFLP in comparison with other methods. European Journal of Plant Pathology. 119:143-158. Interpretive Summary: The diseases caused by Phytophthora fungi can be highly destructive to orange, grapefruit, and other citrus crops. Much of the damage caused by these diseases is to the root system and is not readily visible to citrus growers or researchers. The impact of Phytophthora diseases is to increase mortality of trees and reduce productivity and fruit quality of trees that survive. Methods to study these diseases in citrus have relied heavily on excavation to assess root damage and plating on selective media along with characterization of fungal morphology to identify the species of Phytophthora involved. This paper describes the use of molecular markers to reliably and rapidly indentify Phytophthora species in citrus roots from small samples. These methods will be very beneficial in the development and testing of new cultivars that are resistant to this pathogen.
Technical 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 1000x 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. P. 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.