1Tree Pathology Cooperative Programme (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002 South Africa; 2Medical Research Council (MRC), PO Box 19070, Tygerberg 7550, South Africa

Background and objectives

Fusarium subgiutinans f. ;sp. pini (FSP) is the causal agent of the pine disease, pitch canker. This fungus belongs to the Fusarium Section Liseoia, which includes F. subglutinans, F. moniliforme and F. proliferatum. The Section Liseoia is subdivided into seven separate mating populations, A-G. Each of these also include different host-specific groups. The B-mating population, for example, consists of FSP, as well as F. subgiutinans isolates pathogenic to sugarcane, mango and pineapple. The E-mating population is also morphologically identifiable as F. subglutinans, but these isolates are pathogenic to maize. FSP can be distinguished from isolates of F. subgiutinans from other hosts, by sexual compatibility and pathogenicity [1]. The aim of this study was to develop a technique to distinguish isolates of F. subglutinans pathogenic to different hosts. Part of the H3 histone gene was amplified by PCR and sequenced using a primer pair, previously described by Glass and Donaldson [2]. From sequence data potential restriction sites were identified, for use in PCR-RFLPS.

Materials and methods
Thirty-one Fusarium isolates were included in this study. In addition to the F. subgiutinans isolates from different hosts, the F. moniliforme and F. proliferatum mating tester strains were also included. An isolate of F. oxysporum was included as an outgroup. DNA was isolated from these fungi using standard techniques and PCR was performed as previously described by Glass and Donaldson [1]. The H3 primer pair was used in both the PCR amplification and sequencing of the H3 gene. The resulting DNA sequences were analysed with PAUP after the sequences were manually aligned by inserting gaps. Dendograms generated in this way were rooted to F. oxysporum as a monophyletic sister outgroup.

Results and conclusions
The H3 gene is a conserved gene that is known to contain at least one intron [2]. Nucleotide sequence analysis of this gene from the different Fusarium isolates revealed the presence of two introns. Although the position of these introns appeared to be conserved among the fungi studied, they were sufficiently variable for distinguishing among the different isolates. The sequence of both introns and the position of one of them were unique, since they shared no homology to any previously published H3 introns. The coding regions of the Fusarium H3 gene were highly conserved in both nucleic and amino acid sequences and shared a high degree of homology to those from other organisms. From the DNA sequence of both the introns and exons, three restriction enzymes, MspI, CfoI and DdeI, were identified for use in PCR-RFLPS. By using the enzyme DdeI, it was possible to distinguish between FSP and isolates of F. subgiutinans isolated from sugarcane and mango. The enzyme CfoI was used to differentiate between F. subglutinans isolates from maize and pineapple. By using MspI it was also possible to distinguish among the different F. oxysporum, F. moniliforme and F. proliferatum isolates.

Phylogenetic analyses of sequence data from the isolates, indicated that the different host-specific isolates of F. subglutinans, grouped according to host. Analyses of both the exon and intron regions further showed that the isolates included in this study form two separate clades. One clade included FSP and F. subgiutinans isolates from maize, pineapple and some from mango. F. subglutinans isolates from the B-mating population and others from mango resided in the second clade.

1. Viijoen A, Marasas WFO, Wingfield MJ, Viijoen CD, 1996. Mycological Research 101, 437-445.
1. Glass NL, Donaldson GC, 1995. Applied and Environmental Microbiology 61, 1323-1330.