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


RESULTS
DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)-PCR. Four extraction methods (rapid alkaline lysis; microLYSIS-PLUS; DNeasy; FTA 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 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.


CONCLUSIONS
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.


CONCLUSIONS
This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi.
Manzanilla-López
R H
RH
Nematode Interactions Unit, Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, UK. rosa.manzanilla-lopez@bbsrc.ac.uk
Clark
I M
IM
Atkins
S D
SD
Hirsch
P R
PR
Kerry
B R
BR
D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D006999:Hypocreales / Q000235:genetics / Q000302:isolation & purification*; D018517:Plant Roots / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012988:Soil Microbiology
D017931:DNA Primers; D004271:DNA, Fungal