Alison Wakeham

A new method to monitor airborne inoculum of the fungal plant pathogens Mycosphaerella brassicicola and Botrytis cinerea.

ABSTRACT We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores ofMycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores ofM. brassicicola correlation coefficients (r2) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants ofBrassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.

Serological profiling of the fungal genus Pythium

Two monoclonal antibodies and three polyclonal antisera were raised to cell wall/membrane fractions of Pythium violae and Pythium sulcatum . When screened with a collection of 40 isolates of the genus Pythium including 20 species and the H-S group there was extensive cross-reaction. However, when the binding of the antibodies was assessed in an enzyme-linked immunosorbent assay using cytoplasmic fraction antigens, the combined recognition patterns produced profiles unique to each species. Multivariate analysis methods were used to establish relationships between isolates on the basis of these profiles; isolates of the same species tended to group together. The affinity of these reagents for fungi other than members of the genus Phytophthora was low. The use of multi-antibody probes and appropriate statistical methods to differentiate species of fungi is discussed.

Risk assessment methods for the ringspot pathogen Mycosphaerella brassicicola in vegetable brassica crops

Mycosphaerella brassicicola causes ringspot on Brussels sprouts, which can result in substantial yield loss in commercial production. Brussels sprout buttons are downgraded if this pathogen occurs on them. In this study, the effect of temperature and wetness duration was investigated on infection of Brussels sprouts using controlled environments (CE). The effect of temperature and wetness duration on inoculum production and ascospore discharge was also investigated. Infection by M. brassicicola was described using a mathematical model and was compared to estimates of ascospore availability obtained via a volumetric air sampler and immunofluorescence (IF). Infection of M. brassicicola was correlated (r = 0.92) with temperature during leaf wetness periods. The relationship between temperature and time to discharge of 5 and 50% of the cumulative total number of ascospores from ringspot lesions was r = 0.99 and 0.98, respectively (P < 0.001). In field experiments, an optimal wind run (the product of the average wind speed and the period over which that average speed was measured) of 250 to 500 km day-1 was required for the dissemination of ringspot inoculum to field bait plants. Quantification of M. brassicicola inoculum in collected field aerosols was possible using a monoclonal antibody in a plate-trapped antigen enzyme-linked immunosorbent assay. Precoating of the air sampler wells with sodium azide prevented trapped spores from germinating. Ringspot inoculum could be detected and quantified in air samples from commercial crops of Brussels sprouts in the United Kingdom. Low levels of ringspot inoculum measured within crops did not lead to disease development.

Deleterious effect of soil‐applied metalaxyl and mancozeb on the mycoparasite Pythium oligandrum

Pythium oligandrum was recovered, identified and quantified from air‐dried soil plated on 1.5% water agar containing 0.1% glucose. Isolations of P. oligandrum over 2 years from soils treated with single applications of metalaxyl plus mancozeb were consistently lower than those from untreated soil from the same fields. In three fields in the first year P. oligandrum was reduced from a range of 43.3–115.0 to 17.0–43.2 isolates g‐1 soil. In the second year, results from 11 fields showed reductions from 27.8–141.8 to 2.8–44.5 isolates g‐1 soil. P. oligandrum was sensitive to both metalaxyl and mancozeb, with median effective dose (ED50) values of 0.13 ± 0.02 μg m‐1 and 3.33 ± 0.12 μg ml‐1, respectively. In a pot test with three soils treated with metalaxyl, mancozeb or the combination of fungicides, levels of P. oligandrum declined over 3 months, with effects first recorded 2 weeks after treatment. Levels of P. oligandrum were reducd by differing degrees in the three soils. Isolate counts from untreated soils...

Monitoring Infection Risk for Air and Soil Borne Fungal Plant Pathogens using Antibody and DNA Techniques and Mathematical Models describing Environmental Parameters

Economic losses resulting from disease development can be reduced by accurate and early detection of plant pathogens. Early detection can provide the grower with useful information on optimal crop rotation patterns, varietal selections, appropriate control measures, harvest date and post harvest handling. Classical methods for the isolation of pathogens are commonly used only after disease symptoms. This frequently results in a delay in application of control measures at potentially important periods in crop production. This paper describes the application of both antibody and DNA based systems to monitor infection risk of air and soil borne fungal pathogens and the use of this information with mathematical models describing risk of disease associated with environmental parameters.

Quality of the Governing Temperature Variables in WRF in relation to Simulation of Primary Biological Aerosols

We have evaluated three prognostic variables in Weather Research and Forecasting (WRF) model, mean daily temperature, daily maximum temperature, and daily minimum temperature using 9 months of model simulations at 36 and 12 km resolution, and compared the results with 1182 observational sites in north and central Europe. The quality of the results is then determined in the context of the governing variables used in crop science, forestry, and aerobiological models. We use the results to simulate the peak of the birch pollen season (aerobiology), growth of barley (crop science), and development of the invasive plant pathogen Hymenoscyphus pseudoalbidus (the cause of ash-dieback). The results show that the crop and aerobiological models are particularly sensitive to grid resolution and much higher quality is obtained from the 12 km simulations compared to 36 km. The results also show that the summer months have a bias, in particular for maximum and minimum temperatures, and that the low/high bias is clustered in two areas: continental and coastal influenced areas. It is suggested that the use of results from meteorological models as an input into biological models needs particular attention in the quality of the modelled surface data as well as the applied land surface modules.

Diagnostic Tests and their Application in the Management of Soil- and Water-borne Oomycete Pathogen Species

Oomycete diseases cause significant losses across a broad range of crop and aquaculture commodities worldwide. These losses can be greatly reduced by disease management practices steered by accurate and early diagnoses of pathogen presence. Determinations of disease potential can help guide optimal crop rotation regimes, varietal selections, targeted control measures, harvest timings and crop post-harvest handling. Pathogen detection prior to infection can also reduce the incidence of disease epidemics. Classical methods for the isolation of oomycete pathogens are normally deployed only after disease symptom appearance. These processes are often-time consuming, relying on culturing the putative pathogen(s) and the availability of expert taxonomic skills for accurate identification; a situation that frequently results in either delayed application, or routine ‘blanket’ over-application of control measures. Increasing concerns about pesticides in the environment and the food chain, removal or restriction of their usage combined with rising costs have focussed interest in the development and improvement of disease management systems. To be effective, these require timely, accurate and preferably quantitatve diagnoses. A wide range of rapid diagnostic tools, from point of care immunodiagnostic kits to next generation nucleotide sequencing have potential application in oomycete disease management. Here we review currently-available as well as promising new technologies in the context of commercial agricultural production systems, considering the impacts of specific biotic and abiotic and other important factors such as speed and ease of access to information and cost effectiveness

Field Evaluation of a Competitive Lateral-flow Assay forDetection of Alternaria brassicae in Vegetable Brassica Crops

On-site detection of inoculum of polycyclic plant pathogens could potentially contribute to management of disease outbreaks. A 6-min, in-field competitive immunochromatographic lateral flow device (CLFD) assay was developed for detection of Alternaria brassicae (the cause of dark leaf spot in brassica crops) in air sampled above the crop canopy. Visual recording of the test result by eye provides a detection threshold of approximately 50 dark leaf spot conidia. Assessment using a portable reader improved test sensitivity. In combination with a weather-driven infection model, CLFD assays were evaluated as part of an in-field risk assessment to identify periods when brassica crops were at risk from A. brassicae infection. The weather-driven model overpredicted A. brassicae infection. An automated 7-day multivial cyclone air sampler combined with a daily in-field CLFD assay detected A. brassicae conidia air samples from above the crops. Integration of information from an in-field detection system (CLFD) with weather-driven mathematical models predicting pathogen infection have the potential for use within disease management systems.