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

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 of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients (r(2)) 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 of Brassica 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.

Detection and Quantification of Botrytis cinerea by ELISA in Pear Stems During Cold Storage.

Botrytis cinerea was detected and quantified in pear stems from six orchards in the Pacific Northwest, and changes in fungal biomass in the stems after 6 and 8 months of cold storage in regular (air) atmosphere were studied. The fungus was detected by plating stem halves on selective medium and by enzyme-linked immunosorbent assay (ELISA) using the Botrytis-specific monoclonal antibody BC-12.CA4. Both methods demonstrated that the incidence of B. cinerea increased from 6 to 8 months, but ELISA was more sensitive than standard isolation. Quantitative ELISAs on stems indicated that over 200 µg of B. cinerea biomass per gram of stem tissue was present in the stems of visibly rotted fruits, but usually less than 35 µg/g was present in stems from fruits without visible gray mold. Aureobasidium pullulans, Penicillium spp., Alternaria spp., and Cladosporium spp. were commonly isolated from stem tissue. A. pullulans was present in 86% of the stems from which B. cinerea was detected. Use of the monoclonal antibody BC-12.CA4 could help in determining the infection path of B. cinerea in pear stems and detection of latent infections, enabling the timing and method of control of stem end rot to be optimized.