PHYLOGENY OF CRYPHONECTRIA CUBENSIS AND ALLIED SPECIES INFERRED FROM DNA SEQUENCE (ITS1-ITS2) DATA
BD WINGFIELD, H MYBURG and MJ WINGFIELD
Tree Pathology Cooperative Programme, Forestry and Agricultural Biotechnology Institute (FABI), Department of Genetics, University of Pretoria, Pretoria, 0002, Republic of South Africa
Background and objectives
Cryphonectria cubensis causes a serious canker disease of Eucalyptus in many tropical and subtropical areas of the world. The importance of C. cubensis increased with the recent and extensive planting of Eucalyptus species in many countries, Very little is known about the origin or phylogeny of C. cubensis. isozyme studies have, however, suggested that the fungus is the same as the clove pathogen Endothia eugeniae . This would imply that C. cubensis might have originated in Indonesia where clove is native. A report of C. cubensis on the roots of Eucalyptus marginata in Western Australia is also intriguing . The niche and climate in this case is atypical of that usually associated with Cryphonectria canker. The aim of this study was to determine the phylogenetic relationships among strains of C. cubensis from various parts of the world. C. ;parasitica, the causal agent of chestnut blight, Endothia gyrosa from eucalypts and an isolate of Endothia eugeniae from dove were included, in order to determine the relatedness of these fungi to C. cubensis. A second objective was to develop a rapid diagnostic method to distinguish between these taxa.
Materials and methods
A wide range of isolates of C. cubensis as well as other fungi were obtained from culture collections and colleagues in various parts of the world. Isolates were grown in malt extract broth on rotary shakers for 7 ;days. The mycelium was harvested, lyophilized and DNA was isolated. The variable ITS1 and ITS2 regions as well as the conserved 5.8S gene of the RRNA operon were amplified and sequenced. The amplified PCR products were also used in AluI and CfoI restriction digests.
Results and conclusions
Analysis of the restriction profiles indicate that it is possible to distinguish between C. cubensis, C. parasitica,and E. gyrosa using RFLPs. This will facilitate the identification of isolates thought to represent C. cubensis that sporulate poorly in culture. Analysis of the sequence data showed that C. cubensis isolates form a distinct clade that includes strains from clove that otherwise might have been assigned the name Endothia eugeniae. Data also confirmed the fact that isolates from Western Australia are typical of C. cubensis, despite the atypical niche in which they occur. C. parasitica isolates formed a separate clade distinct but not distantly related to C. cubensis. E. ;gyrosa grouped separately from both C. cubensis and C. parasitica.
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