2.2.25
COMPARISON OF RIBOSOMAL DNA ITS REGION SEQUENCE BETWEEN CAUSAL FUNGUS OF APPLE VALSA CANKER AND VALSA CERATOSPERMA

K SUZAKI and K YOSHIDA

Apple Research Center, National Institute of Fruit Tree Science, 92 Nabeyashiki, Shimokuriyagawa, Morioka, Iwate, Japan

Background and objectives
Apple valsa canker is one of the severest disease for apple production in Japan. The causal fungus of apple valsa canker was once classified as Valsa mali, however, it was reclassified as V. ceratosperma from detailed study on Diaporthaceae in Japan. However, Kanehira et al. [1] reported, from the results of isozyme analysis, that isolates of V. ceratosperma from apple trees could be distinguished from those from some broadleaf trees, except apple. Suzaki et al. [2] investigated the relationship in V. ceratosperma between phloridzin-degrading ability and pathogenicity to apple branches and reported that while isolates of V. ceratosperma from some broadleaf trees had no phloridzin-degrading ability and no pathogenicity to apple branches, those from apple trees had both. It is doubtful whether V. ceratosperma as the causal agent of apple valsa canker in orchards and as the parasite of some other broadleaf trees are the same species. Recently, sequence analysis of ribosomal DNA ITS region of fungi has become a useful tool to investigate the relationship among closely related species. The purpose of this research is to analyse the sequence of the ITS region and to investigate the relationship between causal fungus of apple valsa canker and V. ceratosperma isolated from other broadleaf trees.

Results and conclusions
Four isolates from apple trees and four isolates from some other broadleaf trees were investigated in this experiment. The ITS region was amplified by PCR and the products of each isolate appeared as one fragment on agarose gel electrophoresis, with sizes were around 600 bp. These products were digested with restriction enzymes, AluI, HaeIII, HpaII, and SmaI. RFLP patterns of ITS region were same among apple isolates and among other broadleaf isolates but were different between the apple isolates and the broadleaf isolates. This indicates that apple isolates were genetically different from the other broadleaf isolates and this was confirmed by sequence analysis of the ITS region. While the ITS1 sequence was 207 bp in apple isolates, it was 210bp in broadleaf isolates; sequence homology between the isolates was about 85%. The ITS2 sequence was 235 bp in apple isolates and 229 bp in other broadleaf isolates and homology was about 91%. In all isolates investigated, the 5.8S ribosomal DNA gene was 154 bp and homology was more than 99%. Ribosomal DNA sequence analyses of other species in genus Valsa are now in progress. From morphological characteristics and these results, it was thought that the causal fungus of apple valsa canker belonged to genus Valsa but differed genetically from V. ceratosperma isolated from other broadleaf trees. Although apple valsa canker has been reported only in Japan, Korea, and China, it is still unknown whether the causal fungus of apple valsa canker occurs only in eastern Asia or in all of the apple-producing countries.

References
1. Kanehira T, Kameoka Y, Shinohara M, 1991. Transactions of the Mycological Society of Japan 32, 199-205.
2. Suzaki K, Yoshida K, Ito T, 1997. Annual Report of the Society of Plant Protection of North Japan 48, 145-147.