2.2.84
PHYLOGENETIC PLACEMENT OF OPHIOSTOMA STENOCERAS-LIKE ISOLATES FROM HARDWOODS IN THE SOUTHERN HEMISPHERE

ZW DE BEER1, RC WITTHUHN2, H BRITZ 1, MJ WINGFIELD 1 and BD WINGFIELD 1

1 Tree Pathology Cooperative Programme, Forestry and Agricultural Biotechnology Institute (FABI), Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002, South Africa; 2Department of Microbiology and Biochemistry, University of the Orange Free State, PO Box 339, Bloemfontein, 9300, South Africa

Background and objectives
Ophiostoma stenoceras is a well-known cause of mild sapstain on softwood species in the Northern Hemisphere. Medical mycologists are familiar with this fungus, since it has previously been suggested that the human pathogen, Sporothrix schenckii is the anamorph species for O. stenoceras [1]. S. schenckii is phylogenetically related to the genus Ophiostoma [2], but it remains uncertain whether S. schenckii represents the anamorph of O. stenoceras. During a recent survey of ophiostomatoid fungi occurring in South Africa, a number of isolates resembling O. stenoceras were found on Eucalyptus spp. and Acacia mearnsii wood chips in South Africa's Eastern provinces, Mpumalanga and Kwazulu-Natal. In the Western Cape Province an almost identical isolate was found on Quercus. Similar isolates were also obtained from Eucalyptus and soil in Colombia, as well as from soil in Kenya. The aim of this study was to determine the phylogenetic relationships of these isolates to O. stenoceras and S. schenckii.

Materials and Methods
African and South American isolates were compared with the type strain and other authenticated isolates of O. stenoceras from the Northern Hemisphere and New Zealand, as well as with S. schenckii isolates. Colony morphology and growth rate were determined for all isolates. Cultures were incubated at temperatures ranging from 10 to 35C with 5C intervals and measurements were done after 8 ;days of incubation. The morphology of the anamorph states was investigated on slide cultures incubated for 24 ;h. Fifty conidia, as well as 50 conidiogenous cells, were measured for each isolate. All cultures were incubated on water agar, enriched with sterilised pieces of wood from the original hosts to induce teleomorph production. Fifty perithecia and 50 ascospores were measured and the thallism was determined. For the phylogenetic analysis DNA was extracted from all the isolates studied. A part of the ribosomal DNA operon was amplified using PCR. The internal transcribed spacer (ITS) regions 1 and 2 were sequenced using the ABI Prism 377 Automatic DNA sequencer (Perkin Elmer). The DNA sequence data were analysed using PAUP, with O. ulmi as the outgroup.

Results and conclusions
All the teleomorph-producing isolates were found to be morphologically similar to the type strain of O. stenoceras. The African isolates, however, frequently produced annuli on perithecial necks. These were absent in isolates from the Northern Hemisphere and South America. There were no obvious differences in the anamorph or colony morphology. The O. stenoceras isolates showed optimal growth at 25C, while those of S. schenckii grew optimally at 30C, Based on the DNA sequence data all the O. stenoceras isolates form a well-supported single, monophyletic group, sister to the clade formed by the S. schenckii isolates.

From these results we conclude that all the Southern Hemisphere isolates from woody substrates and soil are the same. They probably do not represent a species distinct from the type strain of O. stenoceras or the other Northern Hemisphere isolates. Our data do not support the view that S. schenckii might represent the anamorph of O. stenoceras.

References
1. De Hoog GS, 1974. Studies in Mycology 7, 84.
2. Berbee ML, Taylor JW, 1992. Experimental Mycology 16, 87-91.