3.3.2
A DNA PROBE FOR DIFFERENTIATING THREE STRAINS OF PERONOSPORA PARASITICA

M SATOU1, F TERAMI2, F FUKUMOTO3 and S HORIUCHI1

1National Research Institute of Vegetables, Ornamental Plants and Tea, Shimokuriyagawa, Morioka 020-0123, Japan; 2Hokkaido National Agricultural Experiment Station, Hitsujigaoka, Sapporo 062-0045, Japan; 3Hokuriku National Agricultural Experiment Station, Inada, Joetsu 943-0154, Japan

Background and objectives
Downy mildew of crucifers is caused by Peronospora parasitica. A large number of studies have been carried out on host range and host reaction. We have found three host-specific strains of P. parasitica [1, 2] , i.e. oleracea strain (on broccoli), campestris strain (on turnip) and raphanus strain (on Japanese radish). However, the genetic relationship between the strains is still unclear. Establishment of molecular probes is therefore essential for phylogenetical analysis on the three strains.

Materials and methods
Total DNA was prepared from conidia of the strains. DNAs of P. parasitica on broccoli were used for cloning with Escherichia coli DH5a and pUC19. Cloned DNAs were constructed into probes with digoxigenin. Total DNAs were extracted also from ten other pathogenic fungi affecting cruciferous crops and 34 cruciferous cultivars by the same procedure as P. parasitica. Probes were applied for hybridization tests with all the DNAs extracted. Furthermore, DNAs of the three P. parasitica strains digested with restriction enzymes were electrophoresed and transferred to a nylon membrane. DNA polymorphisms were examined by the hybridization tests with the probe.

Results and conclusions
One probe was capable of hybridizing with DNA of the three strains of P. parasitica. The probe hybridized neither with DNAs of other crucifer-pathogenic fungi nor with DNAs of cruciferous plants. Thus the DNA probe proved to be specific to P. parasitica. Polymorphisms were shown among DNAs from the three P. parasitica strains. The DNA probe obtained in the present study may be useful for determination of latent infection, and possibly for practical identification of the three strains of P. parasitica.

References
1. Satou M, Fukumoto F, 1996. Annals of the Phytopathological Society of Japan 62, 393-396.
2. Satou M, Fukumoto F, 1996. Annals of the Phytopathological Society of Japan 62, 402-407.