Molecular evolution, identification and epidemiology of North American species of a root-infecting fungus, Armillaria

Piercey-Normore, Michele D. (1997) Molecular evolution, identification and epidemiology of North American species of a root-infecting fungus, Armillaria. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Armillaria (Fr.:Fr.) Staude is a genus of root-infecting fungal pathogens which cause disease in forests and orchard plantations. Biological species of Armillaria are identified by sexual incompatibility mating interactions. Epidemiological studies of North American species of Armillaria indicated that some are saprotrophic, some mycotrophic, and others are necrotrophic pathogens often producing death of the host. Stress of the host tree is often considered a critical factor in disease development. -- Three objectives to this study examined different aspects of the pathogenicity of Armillaria. The first was to estimate phylogenetic relationships among North American biological species (NABS) of Armillaria. This allowed inference of inheritance of pathogenic traits among species. The second was to develop species-specific molecular markers for NABS Armillaria. This would provide a method of identification for pathogenic species. The third was to examine disease development of Armillaria inoculum using different types and degrees of stress inflicted on the host tree. Molecular techniques were employed to examine the first two objectives. The third objective involved a field inoculation trial with two Newfoundland isolates of A. ostoyae. -- This study is consistent with previous phylogenetic hypotheses concerning relationships among species of Armillaria. Phylogenetic analysis of randomly amplified DNA regions of unknown function provided strong support for intraspecies clustering. Most NABS Armillaria were resolved using four anonymous nucleotide sequences combined within a single data set. There was stong support for the clustering of NABS I and II, as well as NABS III and VII. Isolates of NABS V showed sequence polymorphism. -- Species-specific molecular markers were developed for most NABS Armillaria. PCR amplification using a combination of different random primer sets in each of the reaction mixtures, yielded species size-specific bands on an agarose gel for each of NABS I, II and VI. A common band was found specific for NABS III and VII. Although NABS V, IX and X contained less variation, 10 nucleotide primers could be used to confirm their identity. Results from inverse PCR suggested that secondary DNA structure and primer/template competition played a significant role in determining species-specificity with SWAPP 10 nucleotide primers. -- Results from the field inoculation trials suggested that two years was sufficient time to allow forest managers to make informed decisions regarding stand management. Host stress appeared to influence Armillaria root disease development. The black spruce plantation had more infection than the naturally regenerated stand. There was more disease in the balsam fir thinned stand than the un-thinned stand. Significant correlation between infection and above ground tree symptoms occurred in a very severely defoliated balsam fir sawfly plot only. Well-drained sandy soil seemed to increase the aggressiveness of the Armillaria isolate used as inoculum. -- The utilization of molecular techniques, combined with knowledge of ecological processes, would greatly enhance the efficiency of forest management.

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