3.3.3
RAPID DETECTION OF BOTRYTIS CINEREA AND B. ELLIPTICA USING SPECIES-SPECIFIC DNA PROBES

LC CHEN1, TZ CHEN1 and E YEH2

1Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, ROC; 2Council of Agriculture, Executive Yuan, Taipei, Taiwan, ROC

Background and objectives
Botrytis cinerea (telomorph Botryotinia fuckeliana) is a widespread pathogen producing infections on more than 200 hosts. Diseases caused by the fungus result in considerable losses to crops grown in the field, in greenhouses and during storage. Most of these crops are economically important, such as vegetables, fruits, flowers, ornamentals, bulbs and forest-tree seedlings. Host attack can occur before harvest or later during transport or storage. The fungus forms both conidia and microconidia as well as sclerotia. Infection by means of sclerotia is rarely observed. Mycelium from infected tissues and conidia produced on dead or dying plants are the most important sources of infection.

B. cinerea has become one of the most important threats to the production and export of cut flowers and to some important crops. Conidia produced in large amounts in greenhouses are easily spread through the air. After landing on the flowers the conidia remain dormant until water, for instance through condensation, is available for the spores to germinate, and in this way flowers become infected within a few hours. Infection frequently takes place at blossom time through the flower, but the fungus remains latent or quiescent until the fruit ripens. Detection of the fungus at the latent stage before packing for market is difficult because it is too time-consuming and tedious by classical methods. We describe here the development of a species-specific DNA probe.

Materials and methods
Mitochodrial DNA from an isolate of B. cinerea, WFFr103, and from an isolate of B. elliptica, EPL803, was treated with restriction endonuclease EcoRI, and each DNA fragment was ligated with vector pBluescript SK+ and transformed into E. coli DNA. Three transformants were selected randomly from genomic libraries. The mtDNA fragments from transformant clones were prepared for DNA probes. These DNA probes were tested via dot-blot hybridization with total DNA of the other isolates.

Results and conclusions
The sensitivity of DNA probe Be 83-8 (0.9 kb) is highest up to 10-5 mg among all DNA probes. But the DNA probe of Be 83-4B (1.2 kb) hybridized with all isolates of B. cinerea and B. elliptica. According to RAPD band pattern analyses with primer OPB-17, DNA of B. cinerea and B. elliptica has 600- and 800-bp fragments in amplified DNA after gel electrophoresis. The 600-bp fragments from transformant clones were prepared for DNA probes. The highly specific DNA probe was proved with Southern hybridzation with all isolates of B. cinerea and B. elliptica. Using this highly specific and reliable detection could prove valuable for regulatory, epidemiological and ecological studies.