Botrytis cinerea

Laccase activity

Botrytis cinerea is a weather-driven fungus which causes the grapevine diseases botrytis bunch rot and grey mould, as well as the ‘noble rot’ used for sweet wines. Botrytis produces the enzyme laccase, which in the presence of oxygen can cause serious oxidative spoilage. Early detection of laccase in grapes or juice before fermentation allows winemakers to employ strategic winemaking techniques during grape processing or during and after fermentation. The AWRI Managing Botrytis-infected fruit fact sheet details strategies to help minimise any potential damage before it occurs.

Qualitative test for laccase activity

A simple bench test can be used to obtain a qualitative result for laccase activity. Sulfur dioxide is added to the sample in question to give a total SO₂ concentration of about 60 mg/L. The sample is then poured into two wine glasses (approximately 50 mL of sample in each glass) and each glass is covered with a watch glass or petri dish lid. One sample is placed in a refrigerator, while the other sample is left ‘on the bench’. The samples are examined after 24 hours and compared for any change in colour or quality. If there is laccase activity, the sample left on the bench should be browner that the sample left in the fridge and may have an oily film on the surface of the wine.

Quantitative test for laccase activity

Quantitative determination of laccase activity can be achieved using a number of commercially available test kits. These kits assess laccase activity with a spectrometer, measuring syringaldazine as a purple-coloured oxidation product.

Affinity Labs performs simultaneous quantitative and qualitative analysis for laccase activity in grapes, juice and wine. Laccase results are reported semi-quantitatively in units of laccase activity/mL (U/mL) and as positive/negative for the 24-hour qualitative test.

Laccase activity ranges from 0 in sound fruit, up to 140 u/mL in fully Botrytis-infected fruit. For dry white and red table wines, values greater than 3 indicate that it is likely that there will be some influence of laccase activity. Botrytis also produces glycerol and gluconic acid as major products.

Sampling and limitations

Grape or juice samples can be submitted fresh or frozen, but juice must be submitted in a plastic container. The presence of free sulfur dioxide and/or ascorbic acid in samples will slow the reactions in the tests and give a false negative or lower apparent laccase activity.

Gluconic acid analysis

Botrytis can oxidise glucose in grapes to produce gluconic acid. As gluconic acid is not utilised by yeast or bacteria it is sometimes used as an indicator of Botrytis infection. Gluconic acid is present at low levels in musts and in wines derived from a usual harvest at levels not exceeding 200 – 300 mg/L. At elevated levels it can contribute to a sour taste in musts or wine. Early detection of elevated levels > ~300 mg/L of gluconic acid associated with Botrytis cinerea infection allows for effective screening of grapes pre-harvest, and gives the winemaker the opportunity to determine the most appropriate winemaking strategy.

This method involves determining D-gluconic acid using a commercial enzymatic kit and a Discrete Analyser. The lower limit of reporting for gluconic acid with this method is 50 mg/L and the upper limit is 5,000 mg/L. Results outside these limits will be reported as < 50 mg/L or > 5,000 mg/L. Grape or juice samples can be submitted fresh or frozen, and juice must be submitted in a plastic container.