| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1986: ¥1,400,000 (Direct Cost: ¥1,400,000)
|
|---|
| Research Abstract |
Acculate and rapid method for the detection of plant pathogenic bacteria has been required in the fields of disease-forecasting and of plant quarantine. The applicability of DNA-DNA hybridization using the isolated genes of disease factors as the probes for the above-mentioned purpose was studied. An Eshcerichia coli clone which contains primarilly the structural gene of one of the pectate lyase isozymes of Erwinia carotovora subsp. carotovora, was first used as the probe. The photobiotin-labeled probe could detect upto 50 ng of total DNA preparations from various soft-rotting Erwinia strains by DNA-dott brotting on the nylon membrane. This result indicates that the isozyme may be common in this group of pathogen. DNA sequence of this isozyme gene was perfectly matched to the reported sequence of PL-E isozyme of E.chrysanthemi, EC16 strain. However, there was some nonspecific binding of the photobiotin probe to some unrelated colonies in the case of colony hybridization. The probe labeled with nick-translation by incorporating [^<32>P]-dCTP was more sensitive, and it could be used also for colony hybridization. The probe made from the gene of IAA-synthesizing enzyme, on the other hand, could detect specifically the pathogen of gall disease of Japanese whisteria. Thus, at least the pathogenes of these two types of plant diseases can be detected by DNA-DNA hybridization method. Especially, the advantages of the phtobiotin probe are no requirement of special facilities for handling radioactive materials and its stability (no difference in its sensitivity even after one year).
|
