Differentiating individuals of Armillaria species in New Zealand forests

Background: Armillaria novae-zelandiae and A. limonea occur naturally as wood decay fungi in native forests in New Zealand. As pathogens they are responsible for significant root disease in trees and shrubs in plantations, crops and urban parks and gardens. A thorough understanding of their population dynamics entails knowledge of the spatial arrangement of their individual mycelia or genets. In previous work the distributions of vegetative compatibility groups (VCGs) of these fungi were mapped in an area of native forest prior to and after replacement by a young Pinus radiata plantation. With the advent of molecular technology, it has become possible to test species identities made earlier using culture techniques and to verify whether or not their VCGs, determined by incompatibility reactions between paired cultures, represent distinct individual genets. Methods: Stock subcultures of isolates representing each VCG were recovered from storage in order to obtain DNA. Extracted DNA was subjected to a polymerase chain reaction procedure (UP-PCR) using 11 universal primers to assess genetic variation between subcultures. Bands were scored as either present or absent for each primer-subculture combination and cluster analysis was undertaken by generating dendrogram trees to reveal genetic groupings among subcultures.   Results: DNA cluster analysis divided subcultures of isolates into two species groups, A. novae-zelandiae and A. limonea, corresponding to identities determined through culture morphology. Within species, subcultures grouped into clusters that matched VCGs determined by earlier culture pairing. There was little indication of genetic variation within VCGs, except for one of A. limonea, which comprised two sub-clusters. Conclusions: The Armillaria species and VCGs identified by culture techniques in the laboratory were verified by independent molecular methodology. In general, the VCGs represent discrete individual genets or colonies in the field. Techniques that differentiate isolates based on differences in their DNA sequence provide a quick alternative to time-consuming laboratory culture methods for resolving population spatial structure. However, some complementary isolate pairing may be necessary when rationalising the significance of groupings in dendrogram trees.