Determination of thresholds for bunch rot contamination of grapes and techniques to ameliorate associated fungal taints

Abstract

This project investigated measures of bunch rot contamination of wine grapes. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR FTIR) successfully discriminated individual grape berries inoculated with the different types of fungi (Aspergillus spp., Botrytis cinerea and Penicillium expansum) responsible for bunch rots. Each of the fungi investigated produced a different suite of secondary compounds, some of which were implicated in deleterious effects on wine quality. Based on the ergosterol content of fungal cultures, thresholds for Botrytis contamination of grapes was estimated to be between 0.3 and 1.0 g dry weight of fungus per kg wet weight of grapes.

Summary

Bunch rot of grapes causes economic losses to grape and wine industries worldwide. The causative agents are phytopathogenic filamentous fungi. Botrytis cinerea (responsible for grey mould) is the principal fungal species associated with rotting grapes close to harvest. Other fungal organisms include species of Aspergillus, Cladosporium, Penicillium and Rhizopus. These other fungi are often opportunistic pathogens, infecting plant tissues when the host is compromised (e.g. when the berry skin is damaged). The impact these other fungi have on grape and wine quality is different from the impacts of B. cinerea.

This work focused on four fungal species associated with bunch rots, B. cinerea, Aspergillus carbonarius, A. niger and Penicillium expansum. The studies involved detached inoculated individual grape berries, detached inoculated whole bunches and naturally infected commercially-grown bunch samples. The work examined a single wine grape cultivar, Chardonnay. Using a combination of techniques in plant pathology, analytical chemistry and sensory science, the aims of this work were to more fully characterise the impacts bunch rotting fungi have on grape and wine quality. 

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR FTIR) in conjunction with chemometric modelling and machine learning algorithms, was successfully applied to objectively differentiate Aspergillus carbonarius, A. niger, Botrytis cinerea or Pencillium expansum fungal mycelium and mature wine-grape berries (Vitis vinifera, cultivar Chardonnay) infected with either of these bunch rot pathogens. The differentiation of B. cinerea infected grape berries from those infected with either Aspergillus or Penicillium species shows promise as a tool for the rapid detection of the pathogens when grapes are received at the winery for processing.

An untargeted metabolomics approach was used to classify volatile compounds produced in grapes infected with either Aspergillus carbonarius, A. niger, Botrytis cinerea or Pencillium expansum. Juice obtained from these grapes was subjected to GC/MS analysis. PLS-DA of the normalised summed mass ions across the chromatograms indicated sample classification according to pathogen. Compounds identified from those mass ion matrices that were responsible for classification included 1,5-dimethylnaphthalene and several unidentified sesquiterpenes that were relatively higher in B. cinerea infected samples. A. niger and A. carbonarius samples were higher in 2-carboxymethyl-3-hexylmaleic acid anhydride, while P. expansum samples were higher in γ-nonalactone and m-cresol. The differentiation of wines made from the different bunch rotting fungi was not as clear as for grape juice, suggesting that the compounds responsible for discriminating grape juices were lost during fermentation.

Grapes were sourced from a commercial vineyard and based on visual observations were sorted into one of five levels of Botrytis cinerea (grey mould) severity. Measurement of the fungal sterol, ergosterol, allowed more accurate quantification of the fungal biomass present in the batches of grapes. Based on this technique the amount of fungal biomass was determined and ranged from 0.07 to 5.16 g dry weight of fungus per kg wet weight of grapes for the five levels of grey mould severity. The five batches of grapes with the five different levels of grey mould severity were made into wine, the grape juice and finished wine were analysed by GC/MS and 1-octen-3-ol, 1-octen-3-one and 3-octanone, key volatile organic compounds associated with fungal contamination, were quantified. The finished wines were also subjected to sensory discrimination testing to determine the infection rate required to produce a significant difference in comparison to the control.. Volatile organic compounds associated with fungal taints in wine diminished during the wine making process, but were still above the sensory perception thresholds in wine made from grapes with the higher levels of grey mould contamination. The findings suggest that the

threshold for Botrytis contamination in Chardonnay grapes is between 0.35 and 1.00 g dry weight of fungus per kg wet weight of grapes, equating to a range from 1-2 berries in the bunch to 10% of the bunch affected with Botrytis.

During the project a collaborative working relationship was established with researchers at the University of Cádiz, Spain. This facilitated the identification of some of the hitherto unknown compounds found in grape berries in response to fungal infection. It is anticipated that this collaboration will be on-going.

Findings from the work have been communicated to the wine industry and the broader scientific community via presentations at grape grower and wine maker meetings, international scientific conferences, on-line and printed extension publications and peer-reviewed scientific manuscripts.

The project has provided grape growers and wine makers with a better understanding of how fungal rots affect wine quality. While thresholds for bunch rot contamination still require some fine tuning, and studies have to be conducted on additional grape varieties, these improvements in objective measures of wine quality will allow decisions to be made around harvesting fruit that is affected with fungal bunch rots.