3.5.9
DNA FINGERPRINTING OF WEED PATHOGENS FOR BIOLOGICAL CONTROL


DNA FINGERPRINTING OF WEED PATHOGENS FOR BIOLOGICAL CONTROL

DG LUSTER and WL BRUCKART

USDA, ARS, Foreign Disease-Weed Science Research, 1301 Ditto Ave., Ft. Detrick, MD 21702-5023, USA

Background and Objectives
Government regulatory agencies require some form(s) of identification of microbes proposed for release into the environment. We are developing molecular identification techniques in addition to morphological characters to identify and fingerprint fungal weed pathogens collected from outside the US and tested for release as biological control agents against domestic weeds.

Results and conclusions
Obligate rust pathogens collected for control of thistle spp. have yielded only enough DNA for procedures utilizing the polymerase chain reaction (PCR), so we have amplified ribosomal RNA gene internal transcribed spacer (ITS) regions for characterization of this group of organisms. A previous restriction analysis [1] of PCR-amplified ITS regions from 15 isolates of Puccinia carduorum, a pathogen of Carduus thistles, revealed sufficient differences among the P. carduorum isolates to warrant further investigation. Sequence analyses of PCR-amplified ITS regions from the P. carduorum isolates as well as 13 isolates of Puccinia jacea, a pathogen of Centaurea thistles, have indicated minor intraspecies differences, which correlate to the respective pathogens' host ranges on groups of thistle species. Automated DNA sequencing of PCR-amplified ITS2 regions has been performed on 9 urediniospore field samples of P. carduorum that were collected across the U.S. from infected musk thistle (Carduus thoermeri), which was the target of a controlled release of the pathogen in Virginia in 1989 [2]. The ITS2 nucleotide sequences from all of the isolates were identical. The evaluation of classical urediniospore morphological characters and ITS2 DNA sequences from the field samples confirmed the identity of the spreading musk thistle pathogen as P. carduorum. We intend to utilize the differences observed in ITS2 sequences between pathogen host range groups to develop PCR-based diagnostic assays for risk assessment following field releases.
References
1. Berthier YT, Bruckart WL, Chaboudez P, Luster DG, 1996. Applied Environmental Microbiology 62, 3037-3041.
2. Baudouin ABA, Abad RG, Kok LT, Bruckart WL, 1993. Biological Control 3, 53-60.