Environmental factors affect the activity of biocontrol agents against ochratoxigenic Aspergillus carbonarius on wine grape.

The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents-the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2-against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5 days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30 °C). The three biocontrol agents were able to prevent infections at 60% RH and 20 °C. At 60% RH and 25 °C only strain AU34-2 achieved some protection on day 5, whereas at 30 °C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20 °C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25 °C, and only strain LS16 at 30 °C. After 5 days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30 °C and 100% of RH for all the three strains, and at 25 °C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25 °C than at 30 °C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25 °C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius.

Strains of Aureobasidium pullulans can lower ochratoxin A contamination in wine grapes.

Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin alpha and the aminoacid L-beta-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin alpha in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.