Changes in fungal associate abundance over mountain pine beetle lifecycle using target-specific primers and quantitative PCR

• Main Author: Khadempour, Lily
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/38068

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, in particular, lodgepole pine. It is closely associated several ophiostomatoid fungi, with which it has a mutually beneficial relationship. This thesis is comprised of two sections. The first objective was to develop target-specific PCR primers that could identify the major fungal species associated with MPB: the pathogenic Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an un-described Ceratocystiopsis species (Cop. sp.1). Growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, and these associates can be difficult to differentiate morphologically. I designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp.1. and the pine Leptographium clade (i.e. G. clavigera and L. longiclavatum). I also designed two primer sets, from another gene, that can differentiate G. clavigera and L. longiclavatum. The primers reliably identify their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The second objective was to use these target-specific primers in conjunction with qPCR to compare the relative abundance of MPB fungal associates during the beetle life cycle. To determine the changes in relative abundance of the fungal species, MPB galleries were sampled at four phases in the beetle life cycle: eggs, larvae, pupae and teneral adults. Multivariate analysis of covariance indicated that changes in the relative abundance of the fungi over the lifecycle of the MPB were statistically significant. Univariate analysis of covariance showed a statistically significant difference in the abundance of Cop. sp.1 through the lifecycle, and pair- wise analysis showed that the difference occurs after the larval phase. The staining fungi O. montium and the Leptographium species did not change significantly through the MPB lifecycle. The work described in this thesis contributes to our understanding of the interactions between the MPB and its fungal associates and provides a tool for further studies that require rapid detection, identification and quantification of MPB fungal associates.