New Zealand Journal of Crop and Horticultural Science abstracts
Short communication
Amplification and cloning of a þ-tubulin gene fragment from strains of
Botrytis cinerea resistant and sensitive to benzimidazole fungicides
JOANNE E. LUCK
MICHAEL R. GILLINGS
CHRISTOPHER C. STEEL
Biological and Chemical Research Institute
NSW Agriculture
PMB 10, Rydalmere
NSW 2116, Australia
Abstract A series of oligonucleotide primers was used to
amplify portions of the
Botrytis cinerea þ-tubulin gene, in
particular, the region where mutations conferring resistance to benzimidazoles
occur in other fungal species. The optimum primer pair produced three distinct
fragments from
B. cinerea DNA: a strong band of 440 base pairs (bp); a
faint band of 480 bp; and an additional band of 700 bp. There was no apparent
difference between isolates that were resistant or sensitive to benzimidazoles.
Amplifications from other fungal species produced a 440 bp band (
Monilinia
fructicola and
Phomopsis viticola), a 480 bp band (
Aspergillus
nidulans), or both (
Penicillium digitatum), but did not produce a
700 bp band. Raising the primer annealing temperature from 55 to 58deg.C
eliminated the 700 bp product from the
B. cinerea amplifications. To
test the identity of the amplified fragments, DNA transfers were probed with a
biotin-labelled þ-tubulin clone from
Leishmania major. Both 440
and 480 bp products responded to the probe but the 700 bp product did not. The
hybridisation and annealing data suggest
B. cinerea has two distinct
þ-tubulin genes (represented by the 440 and 480 bp products), and that
the 700 bp fragment may be a distantly related tubulin gene or the result of
non-specific amplification. All products from the
B. cinerea isolates
were purified by electroelution and inserted into plasmid vectors. Insertion of
the þ-tubulin gene fragments was confirmed using restriction digestion
and hybridisation with the
L. major þ-tubulin clone. The identity
and relationship of the various amplified fragments will be tested using DNA
sequencing, which will also determine if sequence changes to this portion of
the þ-tubulin gene are associated with benzimidazole resistance in
B.
cinerea.
Keywords benomyl; fungicide resistance; polymerase chain
reaction; grey mould; Botrytis cinerea
PDF file of entire paper: medium quality (1706K); (scanned from paper original: notes about this process)
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