Novel application of methods to investigate epidemiology and management of botrytis bunch rot in wine grapes

Botrytis bunch rot (BBR) of grapes caused by the fungus Botrytis cinerea, can cause yield and quality implications. This study investigated the epidemiology and management of BBR in a cool climate, specifically in the Coal River Valley and Rokeby regions of Tasmania. Field trials were part of a larger project investigating spray timing and risk factors associated with BBR. Currently there is a shift to develop and use novel methods in the study of BBR epidemiology because they have the potential to provide assessment of total infection of the disease and not just visible disease. Symptoms may not be evident until fruit ripening, even though infection may have occurred weeks or months earlier. As part of this project, a duplex qPCR technique was developed based on a previously published qPCR technique targeting the intergenic region of the B. cinerea sequence. The assay was developed specifically to use on wine grapes with the internal control targeting Vitis vinifera DNA. The assay was modified and adapted to suit laboratory equipment available and then used to detect and quantify B. cinerea DNA from total nucleic acids extracted from grape berry samples. A study, using qPCR and visual assessment, was conducted during the 2008-09 season to track natural infections of B. cinerea in grape berries sampled from commercial vineyards in the period pre-bunch closure until harvest. Temporal progress curves of disease severity were generated using data from two V. vinifera cultivars: Riesling and Sauvignon Blanc from different vineyards. The qPCR results confirmed that infection and colonization of the fruit occurs during the early stages of berry development, followed by a latent period. Disease expression during fruit ripening increased with time. The latent infection pathway was determined to be more important than the necrotic tissue pathway, in a small plot trial conducted in 2007-08. Treatments included four different spray programs with and without removal of bunch trash (decaying floral parts which include calyptras and aborted berries). The trial was also used to investigate use of qPCR, an ELISA QuickStix™ test and mid-infrared spectroscopy to determine B. cinerea levels in juice samples. QPCR clearly showed that the fungicides reduced B. cinerea load while the ELISA tests were able to statistically separate the treatments. Spectroscopy and visual assessments were unable to statistically separate treatment effects, but there was a positive correlation between values measured using each method. A whole-of-block experimental procedure was conducted during the 2008-2009 growing season to investigate spatial variation of BBR within a vineyard. Vine vigour, measured as plant cell density, was found to positively correlate with BBR severity. Disease increase was attributed to berry-to-berry spread, not that of new infections. The trial also investigated the effect of early (flowering) versus midseason (PBC) spray application and both qPCR and visual assessments demonstrated that the PBC application was more effective than the flowering application. This project clearly demonstrated that qPCR methods can complement traditional visual assessments in the quantification of BBR, and showed the usefulness for assessing management practices such as fungicide application and vineyard variation, and for determining vineyard factors that contribute to increased disease.