Rapid assessment for prediction and quantification of Botrytis cinerea off-flavours in grapes using mass spectrometry

Infection of grapes (Vitis vinifera) by Botrytis cinerea is a frequent occurrence in vineyards during prolonged wet and humid conditions, and it can lead to a significant detrimental impact on grape yield and overall quality. Growth of B. cinerea causes the oxidisation of phenolic compounds resulting in a loss of colour and the formation of a suite of off-flavours and odours in wine made from excessively infected fruit. Such contamination may therefore lead to huge economic losses. To eliminate such loss, a method to identify B cinerea infection in grapes at an early stage prior to harvest is critical. Ideally, a rapid quality assessment of grapes must be done to establish an objective measurement of the phytosanitary aspects of the crops. However, with the techniques currently available, including visual inspection of grapes, ergosterol measurement, detection of B. cinerea antigens, or molecular diagnostic techniques such as qPCR, rapid identification and quantification of B. cinerea infection in grape berries remains a challenge due to the time frames for analysis, low accuracy, and lack of fungal species specificity. This work investigated an innovative approach for rapid detection and prediction of B. cinerea infection in grapes with high sensitivity and reproducibility, that could potentially be suitable for non-destructive in-field detection prior to harvest.

In the first experiment, volatile organic compounds (VOCs) were detected and quantified from naturally infected or lab-inoculated wine grapes of different B. cinerea infection severities using headspace solid phase microextraction (SPME) gas chromatography (GC) mass spectrometry (MS). The correlation between the detection of volatile compounds and the degree of B. cinerea infection as assessed by ergosterol and B. cinerea antigen analysis was investigated. Partial Least Squares (PLS) predictive models were established with detected VOCs such as trans-2-hexen-1-ol, 1-octen-3-ol, 1-octen-3-one, 3-octanone, 3-octanol, and trans-2-octen-1-ol, for B. cinerea infection severities from different wine grape cultivars including Chardonnay, Cabernet Sauvignon, Semillon, and Shiraz. Potential volatile markers for B. cinerea infection in grapes were assessed and selected from the predictive models by variable importance in projection (VIP) and selectivity ratios. A time course experiment was conducted to confirm and monitor the presence of key markers such as 1,5-dimethylnaphthalene, 1,5-dimethyltetralin, 3-octanol, and phenylethyl alcohol, which were accumulated in the inoculated grapes and were detectable around six days post B. cinerea infection. Potential early markers for B. cinerea infection such as trans-2-octen-1-ol were detected as early as two days post inoculation.

Having successfully identified the key markers for the detection and prediction of B. cinerea infection in grapes, a rapid in-field sampling method for volatile extraction was investigated. In the second experiment, a new metal-organic solid phase material compromising zeolitic imidazolate framework-8 (ZIF-8) was utilised as the absorbent material for volatile collection coupled with thermal desorption GC-MS. The method validation was conducted with whole bunches of grapes with different B. cinerea infection severities sampled in a sealed glass container to control the temperature, sampling flow rate and the air movement. The infection severities were assessed in laboratory conditions by quantifying B. cinerea antigens using a lateral flow device and the volatile compounds associated with infections were quantified using SPME GC-MS. In the field sampling experiment, ZIF-8 was proved to be more sensitive and efficient for the absorption and detection of B. cinerea-related volatile compounds with increased peak areas of 8.29, 4.65, 13.13 and 18.52 times higher than a commercially available material, Tenax^®-TA, for 3-octanone, 1-octen-3-one, 3-octanol, and 1-octen-3-ol, respectively.

Owing to time-consuming chromatographic separation process, a rapid, high-throughput detection method is important for the application to vineyard samples on an industrial scale. In the third experiment, direct electrospray ionisation (ESI) MS was applied for the metabolomic analysis of grapes with B. cinerea infection. Healthy or infected grape samples from different cultivars, vintages, regions, and maturity stages were prepared with a simple acetonitrile extraction and automatically analysed by direct MS, achieving in the full detection cycle of approximately two minute per sample, from sample injection, needle wash and preparation for the next injection. A robust predictive model was then established for the discrimination of healthy and B. cinerea infected grapes by a two-class PLS discriminant analysis, with an overall predictive error of less than 10%, for an independent test samples, in both negative and positive ion modes. The variable of importance was assessed using a statistics methods with combined VIP and selective ratios. The important molecular features were annotated with a separate experiment using a high-resolution quadrupole time-of-flight (qTOF) ultra-high performance liquid chromatography (UHPLC)-MS. Grape derived metabolites such as linoleic acid, oleic acid, and succinic acid were identified with high relevance for B. cinerea infection.

Such analytical methods, including in-field sampling, rapid detection and data processing methods can be applied for grape quality assessment and the detection of fungal disease and related contamination. It would also be beneficial for monitoring post-harvest infection levels of B. cinerea in horticultural fruit crops and other biological and chemical analysis.