Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres

A - Papers appearing in refereed journals

Manzanilla-Lopez, R. H., Clark, I. M., Atkins, S. D., Hirsch, P. R. and Kerry, B. R. 2009. Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres. Letters in Applied Microbiology. 48 (1), pp. 71-76.

AuthorsManzanilla-Lopez, R. H., Clark, I. M., Atkins, S. D., Hirsch, P. R. and Kerry, B. R.
Abstract

To develop a simple, rapid, reliable protocol producing consistent polymerase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chlamydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes. DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS((R))-PLUS; DNeasy((R)); FTA((R)) cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA((R)) cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs. ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study. This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi.

KeywordsBiotechnology & Applied Microbiology; Microbiology
Year of Publication2009
JournalLetters in Applied Microbiology
Journal citation48 (1), pp. 71-76
Digital Object Identifier (DOI)doi:10.1111/j.1472-765X.2008.02489.x
PubMed ID19018961
Open accessPublished as non-open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeSEF
ISSN02668254
PublisherWiley
Grant IDD17565

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