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.
Chapter: H.B. Singh, A. Jain, A. Saxena, A. Singh, C. Keswani, B.K. Sarma, S. Mishra (2014) Deciphering the Pathogenic Behaviour of Phyto-Pathogens Using Molecular Tools. In: Biological Controls for Preventing Food Deterioration: Strategies for Pre- and Postharvest Management, Sharma N. (Ed.), John Wiley & Sons, pp 377-408.Biological Controls for Preventing Food Deterioration: Strategies for Pre- and Postharvest Management, Edited by Sharma N., 07/2014: chapter Deciphering the Pathogenic Behaviour of Phyto-Pathogens Using Molecular Tools.: pages 377-408; John Wiley & Sons., ISBN: 9781118533062
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ABSTRACT: Anthracnose disease is an important fungal disease on Trichosanthes kirilowii Maxim. (T. kirilowii), which has recently become a major problem in production areas of T. kirilowii in Anhui Province, central China. A total of 78 putative Colletotrichum spp. isolates were obtained from infected leaves and fruits of T. kirilowii showing anthracnose symptoms during the years 2009-12. All the fungal isolates were consistently identified as Colletotrichum gloeosporioides by a combination of morphological characteristics, species-specific PCR assays and sequence analysis of the ITS region. The results of pathogenicity tests showed difference in virulence among 24 isolates of C. gloeosporioides representatively selected from different regions, when disease symptoms were evaluated after artificially inoculation on the detached T. kirilowii fruits for 2 weeks. The mycelial growth and germination rate of C. gloeosporioides isolates in vitro were determined under temperatures of 10, 15, 20, 25, 28, 30, 33 and 36A degrees C. The optimum growth temperatures of these isolates were observed at 25-30A degrees C, and the germination rates were rather high in the range of 20-33A degrees C. Host range study revealed that the pathogen, under experimental conditions, can infect nine cultivated and weed species belonging to six families (Cucurbitaceae, Rosaceae, Solanaceae, Convolvulaceae, Celastraceae and Cannabinaceae). Moreover, C. gloeosporioides can overwinter safely in infected tissues in field soil, suggesting the potential contribution of plant debris to disease epidemics in the region. The study presents useful information for epidemiological surveillance of T. kirilowii anthracnose caused by C. gloeosporioides in the central region of China.Phytoparasitica 09/2014; 42(4):549-558. DOI:10.1007/s12600-014-0393-6 · 0.68 Impact Factor
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ABSTRACT: Greeneria uvicola causes bitter rot on Vitis vinifera (bunch grapes) and Muscadinia rotundifolia (muscadine grapes) in warm moist temperate and subtropical regions. This study investigated the phylogenetic relationship of G. uvicola representatives from Australia (67 isolates), the USA (31 isolates), India (1 isolate) and Costa Rica (1 isolate) and compared their pathogenicity and fungicide sensitivity. Differences in cultural and conidial morphology were observed between the isolates from Australia and the USA. Phylogenetic relationships were determined based on three gene regions: the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1–58S–ITS2), 28S large subunit (LSU) nuclear rDNA and β‐tubulin‐2. Greeneria uvicola isolates were clearly differentiated into four groups: isolates from Australia and India; USA isolates from V. vinifera; USA isolates from M. rotundifolia; and the isolate from Costa Rica. All isolates were pathogenic on V. vinifera (cv. Chardonnay) berries although those originating from M. rotundifolia were not as aggressive as isolates from V. vinifera, irrespective of geographical origin. Sensitivity to pyraclostrobin and salicylhydroxamic acid (SHAM) was studied. Despite differences in fungicide applications, hyphal growth inhibition was not significantly different for geographical location, cultivar, tissue, year of collection or different spray regimes. For the Australian and USA isolates, fungal growth inhibition was significantly greater for pyraclostrobin than for SHAM, and was significantly greater for the combined treatment than for each of the fungicides applied singly. The aetiological and epidemiological knowledge of bitter rot collected through this study will aid better prediction and management strategies of this pathogen.Plant Pathology 08/2013; 62(4). DOI:10.1111/j.1365-3059.2012.02689.x · 2.97 Impact Factor