Detection and Monitoring of Greeneria uvicola and Colletotrichum acutatum Development on Grapevines by Real-Time PCR
ABSTRACT Bitter rot (Greeneria uvicola) and ripe rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in subtropical grape-growing regions of Australia, where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute toward their effective management and control, a rapid and reliable species-specific real-time polymerase chain reaction (PCR) method was developed based on the polymorphic portion of the internal transcribed spacer region of the two fungi. It was found that, within 6 to 8 h postinoculation, the assay could detect as little as 20 fg of genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid, and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.
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ABSTRACT: Abstract Anthracnose is an economically important disease of grapes in India. Apart from Elsinoe ampelina, Colletotrichum gloeosporioides and C. acutatum are also implicated in this disease in India. To resolve the etiology of the disease, cultural, morphological and molecular methods of identification were employed. Three hundred and eighty two isolates representing different geographical areas, cultivars and plant parts were isolated and purified. Sixteen morphological groups were formed based on colony characteristics and conidial morphology. Three hundred and sixty eight isolates belonging to morphological groups 1–13 produced straight cylindrical shaped conidia of 10.1×4.2μ to 20.7×6.8μ size and exhibited 6.1±0.4 mm rate of growth per day. These isolates resembled C. gloeosporioides. The amplification of a single specific fragment of approximately 450 base pairs with CgINT-ITS4 primer pair from the 363 isolates of morphological group 1–11 confirmed that these isolates belonged to C. gloeosporioides sensu lato. The five isolates belonging to morphological groups 12 and 13 did not amplify with CgINT-ITS4 primer pair. Fourteen isolates of morphological groups 14–16 produced falcate conidia resembling C. capsici and also gave expected band of approximately 460 base pairs with CcINT-ITS4 primer pair thus confirming their identity. None of the 382 isolates produced conidia resembling C. acutatum and also none of the isolates resembled E. ampelina in their cultural or morphological characteristics. Single selected isolate from each of the 16 morphological groups also did not amplify with CaINT2-ITS4 primer pair, except group 13 isolate which gave non-specific polymorphic bands, thus none of the isolates belonged to C. acutatum. These isolates also did not give expected single 500 base pairs amplicon when amplified with E. ampelina specific primers, thus none belonged to E. ampelina. Setae were found in the acervuli of isolates of morphological group 11–16 only. The sequences of the ITS region of two isolates of C. gloeosporioides showed 92–100 % homology with other sequences from C. gloeosporioides species complex and had only 52 % homology with the sequence of ITS region of E. ampelina available in genebank.Australasian Plant Pathology 01/2012; 41:493-504. · 1.02 Impact Factor