Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.Conventional methods for identifying and enumerating airborne fungi and other microorganisms 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, while cultural techniques are unsuitable for detection of spores that are slow growing, or nonculturable in vitro and the choice of medium may influence which species can grow. These difficulties have restricted the use of routine air sampling in the study of plant, animal, and human diseases.Recently, however, molecular methods have been used in the development of diagnostic tests for a variety of fungi involved in plant diseases (8,25,26,28,30). While the potential of these techniques for detection of airborne spores has been recognized for some time (12,14), there have been few reports on progress in this area. The use of immunoassay in the detection of airborne plant pathogenic fungi has been investigated (10,20,21). However, the application of these methods is restricted by the difficulties of developing antibodies showing the required specificity. DNA-based detection methods offer greater potential for sensitive and specific detection, and some progress has been made in the detection of airborne bacteria using these techniques (1, 2, 9, 15, 17). Progress with fungi has been slower, although dete...

Stem canker or blackleg of brassicas, caused by Leptosphaeria maculans, is one of the most damaging diseases of winter oilseed rape in the UK. Airborne ascospores, released in autumn and winter, initiate leaf infections which may lead to colonization of the petiole and, later in the season, formation of stem lesions and cankers. Although isolates of the pathogen differ in ability to cause damaging stem cankers, this is not readily apparent from leaf spotting or stem lesion symptoms. However, several cultural, biochemical and genetic characteristics appear to be associated with the ability to form damaging stem cankers and isolates can be assigned to one of two groups, termed A and B, on the basis of differences in these characteristics. To investigate the relationship between leaf spotting symptoms and subsequent stem canker formation, and to improve understanding of the epidemiology of this pathogen, it is desirable to differentiate between the stem canker forming A group and the less damaging B group of L. maculans. Characterization of isolate type is also important in seed testing and crop breeding programs, particularly in countries such as Canada and Poland where the A type is not ubiquitous. This article reviews methods, including plant assays, assessments of growth characteristics in vitro, isozyme analyses, secondary metabolite profiling, serology, and nucleic acid analyses, that can be used to differentiate the A and B groups

The mobility in phloem of several substituted phosphonic acids and a sulfonic acid was studied in the castor bean plant, Ricinus communis L. For a series of14C‐labelled phosphonate mono‐esters applied to the petioles of mature leaves, phloem transport was modest, becoming poor over longer distances in the plant. Substituted phenylphosphonic acids were more efficiently moved in phloem; uptake from the petiole and subsequent redistribution were slow, but these dibasic compounds were very stable in plants and substantial amounts reached the roots after 72 to 120 h. Glyphosate was very efficiently transported to phloem sinks even within 24 h, with high concentrations in phloem sap. Toluene‐4‐sulfonic acid moved predominantly in the xylem to the mature leaves and its phloem transport was poor. Transport patterns are considered in relation to the physico‐chemical properties of the compounds. Ion trapping appears to play little part in the phloem transport of these strong acids, though the good accumulation and transport in phloem of the complex molecule glyphosate cannot at present be explained.

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Summary Coniothyrium minitans, a mycoparasite with biocontrol activity against Sclerotinia sclerotiorum, was found to disperse during glasshouse trials where overhead irrigation was used. Consequently, the role of water splash in dispersal of C. minitans was investigated using soil‐incorporated inoculum and a range of irrigation regimes found to occur in the glasshouse. The resulting inoculum deposition over horizontal distances up to 2 m was measured. Using drops < 6 mm diameter at 680 mm h‐1, C. minitans was splash‐dispersed at least 2.0 m, whereas with drops > 6 mm diameter at 30 mm h‐1 it was dispersed to only 1.75 m. Irrigation with droplets < 1mm diameter at 49 mm h‐1 failed to disperse inoculum beyond 0.5 m. The dispersal gradient produced by drops < 6 mm diameter at 680 mm h‐1 was best described mathematically by the power function, whereas irrigation with drops > 6 mm diameter at 30 mm h‐ resulted in a gradient described well by power or exponential functions. The latter regime produced a significantly steeper gradient than irrigation with drops < 6 mm diameter at 680 mm h‐1. C. minitans was isolated using an Andersen air sampler at concentrations of 2839 cfu m‐3 or 22 cfu m‐3 during irrigation with drops < 6 mm diameter at 680 mm h‐1 or > 6 mm diameter at 30 mm h‐1, respectively. After irrigation, deposition of C. minitans‐canying aerosol particles declined exponentially and distance from source had no effect on the amount of inoculum isolated. Conidia of C. minitans, splash‐dispersed by irrigation with drops < 6 mm diameter at 680 mm h‐1 were able to infect sclerotia of S. sclerotiorum such that almost all sclerotia at 0.5 m from the inoculum source, and c. 50% of those at 2.0 m, became infected with the mycoparasite.