J. M. Duncan

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.

Specific detection of Phytophthora nicotianae using the polymerase chain reaction and primers based on the DNA sequence of its elictin gene ParA1

Six primers based on the sequence of the flanking and coding regions of the elicitin gene ParA1 of Phytophthora nicotianae were tested for specific detection of the fungus by the polymerase chain reaction (PCR). One combination, IL7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others from a variety of hosts. The sequence of the amplification product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParA1 gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.

A Combination of Baiting and Different PCR Formats, Including Measurement of Real-Time Quantitative Fluorescence, For the Detection of Phytophthora fragariae in Strawberry Plants

Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined for export. Increasingly though, PCR is being incorporated into these testing procedures in an effort to increase sensitivity and speed. Various combinations of baiting and PCR assays were compared with existing testing procedures. Water and root samples from the bait test were screened by nested PCR and the PCR amplicon was detected by several methods, including fluorescent labelled probes (TaqMan™ and Molecular Beacon™). PCR amplification was monitored in real-time and semi-quantitative detection was possible. Because PCR reactions are sensitive to inhibitors present in extracted DNA samples, an internal control containing the primer sequences specific for P. fragariae was developed to avoid false negatives.

Identifying, diagnosing and detecting Phytophthora by molecular methods

The paper outlines the impact of molecular biology on the identification and detection of fungi and fungus-like micro-organisms. We have tried to use as little jargon as possible but avoiding it completely is very difficult. The article is centred around the ‘pseudofungus’ Phytophthora and its closest relatives, because that is the micro-organism with which we have had most experience. Nevertheless, most of the approaches and techniques mentioned are applicable to any fungus or fungus-like organism and, in that broader sense, the article should be of interest to those with no special interest in or knowledge of Phytophthora .

The Use of Nested PCR for the Detection of Phytophthora Fragariae in Strawberry

The fungus Phytophthora fragariae Hickman is a quarantine organism and is as well pathogenic on strawberry as on raspberry [1]. It means that all plantmaterial for export has to be tested on the presence of the fungus. A sensitive and specific detection method is therefore necessary. At IPO-DLO in the Department of Detection we work on the development of such a detection method. In the past work was done to solve this problem by serological methods [2,3,4,5]. Unfortunately with limited succes. In this study a detection method is developed using nested PCR.