Project reference: 2008-03
Herbarium specimens: opening the treasure trove
Project description:
Objective: Improved identification capabilities for fungal needle pathogens:
Species–specific DNA diagnostic assay to detect D. septosporum (previously called D. pini) in infected needles.
Mating–type specific assay to monitor any appearance of MAT1 D. septosporum in NZ.
In the longer term diagnostic assays could be developed for other needle pathogens and quantitative as well as qualitative assays could be performed.
Being PCR–based, these assays will be sensitive and capable of detecting very low levels of infection.
Background: This project follows on from FHRC project 2007–04 (DNA–based identification of needle pathogens). As well as developing identification tools for Dothistroma spp., there were two interesting findings from this work:
Firstly, when targeting dothistromin biosynthesis genes to develop a species–specific PCR–based diagnostic method, an unexpectedly high level of genetic variability was found between different strains of D. septosporum. This will enable the development of DNA identification tools to distinguish New Zealand D. septosporum from overseas isolates of this species. The intention is to complete development of these tools as part of the Better Border Security programme in collaboration with Tod Ramsfield (Ensis).
Secondly, although extraction of DNA from dried herbarium needles was achieved, it was inefficient and required sacrificing several lesion samples in order to obtain suffcient DNA for PCR analysis. We trialed a “Whole Genome Amplification (WGA)” method (Foster & Monahan, 2005) that increases the amount of all the DNA in the sample by several orders of magnitude. The rationale for using this method is that only very small amounts of starting material are required in order to get sufficient DNA for identification or further analysis. In a preliminary trial, PCR amplification of “WGA” DNA was not successful but we have identified several technical modifications that can be done to improve the success rate (eg. Gadkar & Rillig, 2005). It is this avenue of investigation that will be pursued in this project.
This project will be carried out by Massey University in collaboration with Scion.
The aims of this project are:
1. To optimise the recovery of PCR–amplifiable DNA from small samples (e.g. single Dothistroma lesions) and herbarium specimens by:
Making technical improvements to the “Whole Genome Amplification” & subsequent PCR.
Exploring different options for grinding pine needles thoroughly to improve the efficiency of DNA extraction.
2. To use the increased–sensitivity assay developed above, to:
Test surface–sterilised asymptomatic needles for Dothistroma to determine if it is present as an endophyte, using PCR primers developed in FHRC 2007–04.
Commence comparisions of Dothistroma DNA extracted from NZFRI herbarium samples with modern–day isolates, by targeting the highly variable dothistromin genes.
Although Dothistroma is the target organism in this study, the methods will be generally applicable to all pathogen and endophyte species from forest samples.
Research Provider:
Other
