Evaluation of the Global Access Diagnostics Botrytis rapid test method

Abstract

Grapes are typically assessed for grey mould by visual inspection, which is subjective and prone to error. This project investigated Botrytis Alert, a commercially available rapid antigen detection kit as an alternative to current industry practices for grey mould (Botrytis cinerea) detection. Six wine industry participants analysed 376 grape samples and a further 346 samples were analysed by Charles Sturt University. The genus-specific nature of the Botrytis Alert kit provided a definitive and rapid quantitative determination of the presence of grey mould in grape samples that was superior to visual assessments and other assessment measures currently used in the wine industry.

Summary

Botrytis cinerea, responsible for grey mould, is a fungus that frequently attacks grape bunches, particularly if climatic conditions are conducive to infection. Aside from a loss of yield, wine made from grey mould-affected grapes suffers from various faults, such as a loss of red wine colour, browning in white wine, formation of mouldy and earthy characters and diminished desirable aromas and flavours. As with any plant disease, early detection and ideally quantification of grey mould in grapes is essential for management decisions, including timing of harvest and grape processing procedures. Unfortunately, in the case of grey mould, the fungus responsible for the disease is often hidden from view within the interior of the bunch. Furthermore, the disease is harder to identify on red-skinned grape varieties and can sometimes be confused with other physiological or pathological disorders of the vine. Current wine industry practices largely rely on visual assessment of vineyards and grapes at harvest however this subjective method is fraught with difficulty, given that the fungus is often hidden within the interior of the bunch. A number of alternative methods for grey mould assessment are carried out by some grape growers and wine makers. These include measurement of gluconic acid, activity of the fungal enzyme laccase (which is responsible for wine oxidation), and in some cases measurement of grape and / or wine colour. Other methods of assessment, such as molecular methods (e.g. qPCR) and chemical analysis for fungal metabolites (e.g. ergosterol) are only suitable for the research laboratory and are not readily applied to a field situation where a rapid determination of the amount and severity of grey mould is required, (e.g. immediately prior to harvest). A further technique involving immunodetection, where monoclonal antibodies are employed to detect fungal antigens, does have potential to be packaged into a rapid field-based kit and a number of such devices have already been commercialised.

This project investigated the use of a commercially available Botrytis antigen detection kit, Botrytis Alert, which employs a hand-held reader. This kit is specific for Botrytis and will not detect other fungi, thereby removing the uncertainty that is often associated with subjective methods of detection.

The Botrytis Alert kit was evaluated alongside visual assessments of grey mould contamination and measurements of ergosterol, a sterol unique to fungal cells that can be used to quantify the amount of fungal biomass present in a grape sample. In laboratory studies, using hand harvested grapes from a number of vineyard sites and grape varieties, the Botrytis Alert kit was found to be more reliable than visual assessments of grey mould. The linear relationship between the amount of fungus present in a grape sample and the signal intensity of the kit was confirmed by adding known amounts of freeze-dried B. cinerea mycelium to clean grape sample homogenates.

Aside from the studies conducted in the research laboratory, the Botrytis Alert kit was also evaluated by six wine industry participants. The industry participants used the kit alongside whatever method they routinely used for grey mould evaluation. Analysis of the data gathered by the wine industry participants further confirmed that visual assessment of grey mould contamination is prone to error.

Some samples originally assessed (by visual inspection) as contaminated with grey mould by the wine industry participants were found to be uninfected when assessed with the Botrytis Alert kit. Conversely some samples that were visually assessed as disease free were subsequently found to contain B. cinerea, and in some cases this was at relatively high levels of contamination. For other winery operators who were maybe more skilled in visually assessing grapes for grey mould, there was stronger correlation between their assessment of the amount of grey mould present and the signal intensity of the Botrytis Alert kit.

The genus specificity of the Botrytis Alert kit makes it significantly more reliable and accurate method for Botrytis grey mould detection and quantification, than visual assessments and other methods currently used by the wine industry for Botrytis monitoring. Furthermore, the kit is suitable for field use, requires minimal training, and a result is obtained in a matter of minutes.