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Analysis of the distribution of Phytophthora cinnamomi in soil at a disease site in Western Australia using nested PCR
Author(s) -
Williams N.,
Hardy G. E. St. J.,
O’Brien P. A.
Publication year - 2009
Publication title -
forest pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.535
H-Index - 49
eISSN - 1439-0329
pISSN - 1437-4781
DOI - 10.1111/j.1439-0329.2008.00567.x
Subject(s) - phytophthora cinnamomi , biology , nested polymerase chain reaction , polymerase chain reaction , environmental dna , soil water , soil test , pathogen , botany , agronomy , veterinary medicine , phytophthora , microbiology and biotechnology , ecology , biodiversity , genetics , gene , medicine
Summary The oomycete plant pathogen Phytophthora cinnamomi has infected a very large area of native vegetation in the south western corner of Australia. An important aspect of effective disease management depends on being able to accurately map areas of infestation. For this purpose, we have developed a nested polymerase chain reaction (PCR) protocol for the detection of P. cinnamomi in soil. The test uses two sets of primers developed from the rRNA ITS sequences of P. cinnamomi and can detect as little as 1 pg DNA. The degree of sensitivity was reduced with DNA extracted from soil although this depended on the type of soil. Soils with a high organic content, such as eucalypt forest soil and potting mix were more inhibitory than sandy soils. Inhibition by soil DNA could be reduced by the addition of bovine serum albumin and formamide to the reaction. Taq DNA polymerase was very sensitive to inhibitors compared with Tth + or Taq F1*. In comparison with baiting (0–10% positive samples), nested PCR proved to be a very much more efficient (90–100% positive samples) method for the detection of P. cinnamomi in soil.