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Canadian populations and melanin biosynthetic genes of Ceratocystis resinifera Loppnau, Peter Arne

Abstract

This thesis extends our knowledge concerning the sapstain fungus Ceratocystis resinifera. The presented work covers two subject areas of research. The first reports the collection and identification of Canadian populations of C. resinifera, while the second reports the isolation and characterization of three genes encoding melanin biosynthetic enzymes. C. resinifera was identified as a deep stain fungus present in softwood logs across Canada. It was a generalist, not specific to any one-host tree since both Pinus and Picea species were colonized. Molecular methods (PCR-RFLP) were used to positively identify the collected isolates. DNA sequence information indicated that there was low genetic variability within these Canadian populations of C. resinifera, and this conclusion was confirmed by our collaborators using RAPD analysis of genomic DNA markers (Morin 2002). Three genes, PKS1, 4HNR, and SD1, encoding for melanin biosynthetic enzymes were isolated. PKS1 encoded for a typical type I fungal pentaketide synthase responsible for 1,3,6,8,- tetrahydroxynaphthalene synthesis. 4HNR encoded for a 1,3,6,8,-tetrahydroxynaphthalene reductase and the gene product was also found to have 1,3,8,-tetrahydroxynaphthalene reductase activity in Magneporthe grisea. SD1 encoded for a scytalone dehydratase. The pigment responsible for wood stain in C. resinifera was confirmed to be DHNmelanin. Targeted disruption of PKS1 resulted in strains with an albino phenotype. The addition of scytalone to disrupted strains and the recovery of pigmentation confirmed that the genetic lesion occurred in an enzyme at the beginning of a DHN-melanin pathway. Other than the loss of pigmentation, no secondary effects on the growth of albino strains were observed. The presented work should stimulate further research into the molecular genetics of melanin production in sapstain fungi. Also, it should contribute to our knowledge concerning the pigmentation and population structure of C. resinifera, which we propose as a candidate organism for biological control to prevent sapstain in Canadian softwood logs.

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