In planta, experiments have been published recently showing that Arabidopsis, over-producing C6 aldehydes (e.g. hexanal and hexenal) were more resistant to Botrytis infection, mainly through a direct effect of the aldehydes on the fungus growth rather than through an elicitor role that the aldehydes might have had [17]. This study also leads to think that the spraying of such aldehydes on grapevines may reduce the development of various fungi, however the cost and the hazard of manipulating such molecules have to be considered (Fig. 3.3).

Utama et al. [4] tested in vitro the bactericidal and fungicidal properties of several aldehydes and benzaldehyde. Benzaldehyde was shown to be slightly more efficient than acetaldehyde.

3.5 Ascorbic Acid

It is a natural compound which accumulates in many fruit, but in grapes it does not reach high levels as in citrus, as it is a precursor of tartaric and oxalic acids [18] (Fig. 3.4). It accumulates at low levels in berries, some ^moles per gram of fresh weight [19]. Whether it shows some antifungal activity at this level, is unknown. Authors have observed antimicrobial effects of ascorbic acid at higher concentration such as 2.5% [20], but no test has been recorded on grapevines. However the role of ascorbic acid on phytopathogens is probably complex, indeed Barth et al. [21] have shown that Arabidopsis mutants deficient in ascorbic acid are more resistant to bacteria and fungi, may be through an increase in salicylic acid accumulation, thus leading to activation of plant natural defences.

This compound is also naturally present in plant and fruit tissues, and can be found under normal aerobic conditions when the inside of the cells become too acidic or under hypoxic conditions [22, and refs therein] (Fig. 3.5).

3.6 Ethanol

Our work regarding applications of ethanol has been initiated by reading an article by Beaulieu and Saltveit [23]. They observed that exogenous ethanol was stimulating ethylene production and tomato fruit ripening, so we tested it on grapes in order to modulate ripening and anthocyanin accumulation [24] and wine colour [25] using hand-held sprayers directed towards the bunches. Later we found that ethanol sprays with commercial sprayers increases mostly the berry diameter [26].

Subsequently, we tested ethanol for its efficacy to limit fungus growth. The first application was post-harvest, we will present pre-harvest applications later. The idea came from a paper by Lichter et al. [27] showing that dipping grapes at harvest in ethanol solutions was decreasing the Botrytis cinerea growth. The ethanol dip has two drawbacks. Firstly, there is a need to promptly dry the grapes after treatment to prevent berry cracking [28], and secondly, there is possibility of cross-contamination with fungus spores from a previously infected grape crate, when working with low ethanol concentrations. We adapted these ethanol treatments to commercial practices using ethanol in the vapour phase [29]. Indeed the industry is already using fumigation with SO2 in adapted chambers, or in crates with SO2 pads releasing the SO2 in contact with air humidity, so if ethanol was going to be efficient and accepted by the industry, a simple change of the active ingredient was possible. The application of ethanol vapours was optimised over two seasons for 'Chasselas' table grapes. At a dose rate of 2 ml kg-1 of grapes, the ethanol vapour was as effective as sulphur dioxide pads to prevent rot development, caused by Botrytis cinerea, and stem browning. Further tests with consumer panels showed no significant difference in sensory perception between controls and treated grapes. The application of evenly distributed ethanol vapours is critical, as higher concentrations of ethanol may enhance stem browning. Materials releasing ethanol are already on the market, such as the "ethanol powder" [30]. Post harvest applications of ethanol may also present potentials to reduce berry shatter [31] but these need further development.

Then we tested pre-harvest applications of exogenous ethanol in the vineyard, to prevent fungus development ahead; the results that are detailed below have been reported recently [32]. The idea came from the reading of an article by Karabulut et al. [33]. These authors found that spraying 1 l per 5 vines of a solution at 50% ethanol, 24 h prior harvest, was effective in reducing the rots over the postharvest period. We then adapted this to commercial practices, reducing the amount of solution to 150 l/ha (using a mist blower) and no treatment in the last 2 weeks prior to harvest. The treatments were performed every 2 weeks from veraison, we tested a late harvest, scheduled 2 months and a half after veraison, was chosen for these trials, so the conditions were optimised for high Botrytis development. The fungus development was assessed at harvest and after 4-6 weeks cold storage. We always found a higher impact of the treatments after cold storage when Botrytis development had occurred at higher rates.

In preliminary trials, we found that even very low concentration of ethanol i.e., 2% was reducing the Botrytis growth. It is not likely that ethanol would have had a direct effect on fungus growth at this low percentage. This can also be inferred by the studies of Lichter et al. [34]. They showed that at least 30% ethanol is necessary to prevent Botrytis cinerea spore germination. Thus, the 2% ethanol dose is more likely to induce plant defence.

The optimal dose of ethanol to reduce Botrytis growth by pre-harvest spraying was found to be around 16%. However, to match the industry demand, we had to combine it with calcium chloride in order to further reduce the gray mold growth. This was done after reading an article by Nigro et al. [1] in which the authors reported the efficacy of various salts to reduce the Botrytis development. This will be detailed in a paragraph below in this chapter. Thus, we reported that preharvest applications of a 16% ethanol solution, containing 1% CaCl2, reduced gray mold development. At harvest the losses due to rotten clusters dropped from 15% in controls to 5% in grapes treated with ethanol and CaCl2. Over 6 weeks of cold storage, the losses due to gray mold were reduced by 50% in bunches treated with ethanol and CaCl2, compared to untreated controls. These treatments did not induce significant changes in fruit quality assessed by sensory analysis of healthy berries.

The ethanol has a much higher boiling point than acetaldehyde and is not oxidative, therefore it is safe to use. Furthermore, it is already used by industry as a wetting agent or for its solvent properties. As it is flammable, precautions are necessary. Ethanol is rather cheap to produce, and its worldwide production is increasing, mainly due to its use as ethanol fuel, so its cost will decrease.

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