The application of species-specific assays based on the polymerase chain reaction to analyse Fusarium crown rot of durum wheat
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Crown rot of wheat in Australia is caused by species of Fusarium, particularly F pseudograminearum, formerly known as F. graminearum Group 1. Rapid assays are required to identify the species responsible for disease symptoms, especially those with similar morphology. Previously developed assays based on the polymerase chain reaction (PCR) were able to identify F. pseudograminearum, F. graminearum, F. culmorum and F. crookwellense isolates, but not F. acuminatum. To design novel F. acuminatum and F. pseudograminearum species-specific primer sets, randomly amplified polymorphic DNA profiles were amplified that differentiated F. acuminatum and F. pseudograminearum from the other species and polymorphic bands were cloned and sequenced. The specificity of the PCR assays was verified on 79 isolates from 12 different Fusarium species. For two isolates an apparent misidentification occurred using the F. pseudograminearum PCR assay. These isolates were fingerprinted using Amplified Fragment Length Polymorphism analysis, which showed that they had genotypes more similar to F. graminearum than F. pseudograminearum. The PCR-based assays were validated using seedlings infected with single or multiple isolates. A method was also devised to rapidly identify Fusarium species associated with crown rot symptoms on mature wheat stems by culturing the fungi and extracting DNA directly from infected tissue. This assay can be used for routine diagnosis and for epidemiological studies of this disease.
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