Detecting airborne plant pathogenic fungal spores by polymerase chain reaction (PCR) assays

Conventional methods for identifying and enumerating airborne plant pathogenic fungal spores rely on microscopic or cultural techniques, and as a consequence are time consuming and laborious. Additionally, microscopy is unreliable for detection of the small, nondescript spores produced by many fungi, whilst cultural techniques are unsuitable for detection of spores that are slow growing or non-germinable in vitro. For these reasons, routine air sampling is rarely done in the study of plant diseases. The lack of reliable, fast, accurate and simple to use methods for assessing airborne inoculum has limited both epidemiological studies of plant disease and the use of information on airborne inoculum in the management of disease. This paper describes experiments to assess the potential for using polymerase chain reaction (PCR) methods to detect specific plant pathogenic fungal spores in air samples. Experiments were done using Sclerotinia sclerotiorum, Pyrenopeziza brassicae and Leptosphaeria maculans, pathogens of canola (Brassica napus) and the ubiquitous fungus Penicillium roqueforti. These fungi have different spore types.

Three different treatments of spore samples were tested: (1) adding spores direct to a PCR assay; (2) disrupting spores before adding to a PCR assay; (3) disrupting spores followed by DNA purification and adding the purified DNA to a PCR assay. Spores were disrupted by milling them with Ballotini beads.

Three PCR assays were also tested: (1) a single step PCR using primers specific for the target fungus; (2) a PCR using consensus fungal primers followed by a PCR using specific primers (nested PCR); (3) PCR using consensus fungal primers followed by Southern blotting and probing.

The relative consistency of the different spore sample treatments varied according to the species being assayed. Generally spore disruption followed by DNA extraction gave the most sensitive results, allowing DNA from less than 10 spores to be detected in most cases. Tests using target and non-target spores, and with target spores in air samples, suggest that the sensitivity of PCR-assays may be reduced when large numbers of non-target spores are present. In such circumstances a DNA extraction step may be needed to yield acceptable detection limits.

Experiments were also done to extract DNA from samples collected by conventional spore traps such as a Hirst-type trap and rotating arm traps. Spore samples were placed on cellophane (Sellotape) and melinex tape with and without wax coatings. PCR assays could successfully detect spores if the samples were milled and the DNA extracted. The PCR assays detected DNA from P. roqueforti spores in wind tunnel air samples collected using conventional rotating-arm samplers.

The following conclusions could be drawn from the results of the experiments. PCR technology has great potential for detecting spores of a specific species in air samples. Spores may need to be disrupted to release DNA before being processed by PCR and a DNA extraction step may be needed before PCR processing depending on the cleanliness of the air sample.