科研之友
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摘要
Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been Underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA-extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purification of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1-5.8S-1TS2 rDNA-region were developed and tested for their specificity to DNA extracts from eleven R.-subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DIVA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specific primers in combination with the DNA extraction method for soil was high: as little as 100 fg mu l(-1) R.-subterranea-DNA was sufficient for a detection in soil samples and plant material. Given that specific primers are available, the presented method will also allow quick and large-scale testing for other root pathogens.
- 出版日期2009-4
