Abstract
This project investigated techniques to differentiate three bunch rot pathogens, Botrytis cinerea, Aspergillus and Penicillium. Estimates of total fungal biomass examined chitin and ergosterol content of infected tissues. Laccase enzyme activity was variable, B. cinerea produced more laccase than either Aspergillus or Penicillium. Isolates of Aspergillus produced elevated levels of gluconic acid and had low glycerol:gluconic acid ratios. Chemical measures of off flavours and aromas using GC/MS provided an indicator of bunch rots; this technique offers an opportunity to more accurately establish bunch rot thresholds for contamination. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FT-IR) with appropriate modelling was able to discriminate between grapes infected with Aspergillus, Botrytis or Penicillium.
Summary
Bunch rot diseases of grapes are caused by fungal moulds, typically they occur close to harvest and are found worldwide. These fungal rots result in grape yield loses and a down grading of fruit as a result of the mouldy, earthy flavours they produce which are present in wine made from bunch rot affected grapes. Disease management in vineyards is not always successful, particularly in grape growing regions that experience rainfall close to harvest. Quantification and discrimination of the type of bunch rot found in the vineyard is problematic because many of the fungi involved look similar even to the trained eye. Often more than one type of rot may be present in the one vineyard and even on the one bunch. Three such bunch rots are grey mould, caused by Botrytis cinerea, Aspergillus rot caused by predominantly Aspergillus carbonarius and A. niger, and Pencillium rot caused by a number of Penicillium species.
This project was an initial scoping study to investigate methods of bunch rot quantification and discrimination. The study focused on techniques that had the potential to be utilised in an industry setting.
Results form laccase enzyme assays as an indicator of fungal contamination of grapes were variable. Isolates of B. cinerea produced laccase, but the amount of enzyme produced varied depending on the culture conditions. Isolates of A. niger and A. carbonarius were found not to produce laccase under the culture conditions used. More promising results were obtained with measures of glycerol and gluconic acid ratios. Species of Aspergillus and in particular A. carbonarius produced higher amounts of gluconic acid than either B. cinerea or Penicllium expansum and as a consequence had a low glycerol : gluconic acid ratio.
Methodology using GC/MS analysis was developed for the quantification of a number of secondary metabolites associated with off flavours in bunch rot affected fruit and inoculated berries. All three bunch rot pathogens investigated produced a similar spectrum of secondary metabolites associated with fungal taints, including geosmin, methyl isoborneol(MIB), 1-octen-3-ol and 1-octen-3-one. Measures of these unwanted secondary metabolites may be useful in setting thresholds for bunch rot contamination in the future.
Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FT-IR) with appropriate modelling (chemometric and machine learning algorithims) was able to successfully differentiate berries inoculated with A. carbonarius, A. niger or P. expansum from the other fungi. The differentiation of B. cinerea from the grapes with other pathogens; also shows promise as a tool for the rapid detection of the pathogen. The ATR-FT-IR methodology will require further validation with a greater number of isolates and samples from different cultivars.
The project examined two methods for estimating total fungal biomass in berries affected with bunch rot disease. The first method examined fungal chitin, a polysaccharide found in the cell walls of fungi. Techniques used to measure fungal chitin were found to have limited applicability for berry tissues. This was because the fungi examined degraded berry skins during the infection process, leading to interference with the fluorometeric measurements. The second method utilised a HPLC method to quantify the fungal sterol ergosterol from bunch rot affected grapes. Ergosterol was quantified from both pure fungal cultures and infected plant material and the technique may have some applicability in quantifying total fungal biomass present in grape bunches affected with fruit rotting fungi.
This pilot project has provided a base from which further research on determining thresholds for bunch rot tolerance can be set and for the further establishment of methods to objectively measure bunch rot contamination of grapes and the impact these rots have on grape and wine quality.