1.10.8
PHYLOGENETIC STUDY OF FUSARIUM OXYSPORUM STRAINS IN RELATION TO THEIR MATING TYPES

T ARIE1, T YOSHIDA2, S GOUTHU1, T SHIMIZU1, K IIZUKA1, K YONEYAMA3, OC YODER4, BG TURGEON4 and I YAMAGUCHI1

1Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan; 2Tohoku National Agricultural Experiment Station, MAFF, Arai, Fukushima 960-2156, Japan; 3Meiji University, Tama, Kanagawa 214-0033, Japan; 4Cornell University, Ithaca, NY 14853, USA

Background and objectives
Fusarium oxysporum is one of the main phytopathogenic fungi causing soilborne diseases, and endopolygalacturonase (PG), a hydrolytic enzyme secreted by the fungus, has been reported as a pathogenesis-related factor. Recently, we obtained the DNA sequence of 1318 bp of a PG-encoding gene in F. oxysporum f. sp. lycopersici and also confirmed that the genome has a single copy of the gene [1]. In this study, through PCR amplification, a part (ca 970 bp) of the PG-encoding gene was amplified from F. oxysporum strains, and the DNA sequence analysed to determine a phylogenetic tree of the strains. We have also cloned the putative mating type genes of the asexual ascomycete [2], which made it possible to determine the mating type of each strain simply by PCR without crossing. In the present study, the phylogeny based on the PG-encoding gene sequence is discussed in relation to the mating types of the strains.

Materials and methods
Pathogenic strains of F. oxysporum belonging to f. sp. lycopersici, f. sp. radicis-lycopersici, other formae speciales and non-pathogenic F. oxysporum strains were used. Primers PG1 (5' GAYAAYGAYTTYRAYCCBATYG) and PG2 (5' CANGTRTTNGTVGGRTARTTRC) were used to amplify the 970-bp fragment of the PG-encoding gene [1]. The amplified fragment was cloned to pCR2.1 vector (Invitrogen) and was sequenced. The sequence data of the isolates were analysed for similarity by GeneWorks (IntelliGenetics) program. Primers FHMG11+ FHMG12 and Falpha1+ Falpha2 were used to amplify the HMG-box on the putative MAT-2 and alpha-box on the putative MAT-1 genes, respectively [2], and the mating type of each strain was determined.

Results and conclusions
All the strains of F. oxysporum used in the study, including pathogenic and non-pathogenic ones, possessed a PG-encoding gene on their genome. This suggests that pathogenicity of F. oxysporum does not depend directly on the possession of the PG-encoding-gene. The phylogenetic tree was drawn based on the sequence data of the DNA fragment amplified with primers PG1+ PG2.

All the strains of f. sp. lycopersici, from widely separated areas of the world, came out in a cluster in the tree, suggesting the close relatedness of these strains. Mating type of the strains was also determined, and all the f. sp. lycopersici strains were identical with MAT-1.

From the branching trend of f. sp. lycopersici, it is possible that a strain obtained pathogenicity toward tomato and has been distributed worldwide while reproducing asexually. This hypothesis is in line with that of Leslie and Klein [3], who proposed that fungal species are originally heterothallic and hermaphroditic, and that asexual (female sterile) strains emerge only under certain conditions.

References
1. Arie T, Gouthu S, Shimazaki S et al., 1998. Annals of the Phytopathological Society of Japan 64, in press.
2. Yoshida T, Arie T, Yoder OC et al., 1998. Abstracts of the 7th ICPP.
3. Leslie JF, Klein KK, 1996. Genetics 144, 557-567.