Despite public’s growing concerns for human health and environmental pollution, synthetic fungicides are still preferred and massively used for the protection of agricultural crop plants against fungal diseases. However, more and more research is focused on finding potential alternatives in the form of effective biological control agents. Although there are reports of yeast’s biocontrol activity, they are up to date poorly commercialized for such purposes. As the yeasts represent an important part of the grape microflora, competing with other microorganisms (including pathogens) for nutrients and space, we decided to examine the potential of autochthonous yeasts as "green" alternatives in fighting against phytopathogens such as Botrytis cinerea in viticulture. With this aim we tested biocontrol activity of various strains / species of indigenous wine yeasts that were isolated from the vineyard environment in Slovenia and cross-border Italy. The yeasts were tested for the presence of siderophores, hydrolytic enzymes (chitinase, β-glucosidase and β-glucanase) and sensitivity to fungicides (copper, iprodion, cyprodinil + fludioxonil combination). The tests of phytopathogenic fungi growth inhibition in the presence of antagonistic yeast were also performed. The double Petri dish test was used to verify the inhibition of conidia germination by the formation of antifungal volatile organic compounds (VOC), synthetized by the antagonistic yeasts. The presence of hydrolytic enzymes was confirmed in many wine yeasts, particularly in the strains of Hanseniaspora uvarum and Metschnikowia pulcherrima species and in some strains of the Pichia genus. Using a medium, based on natural diluted grape juice in a double Petri dish test, we found that yeasts of the Debaryomyces hansenii, Lachancea thermotolerans, P. kudriavzevii, Saccharomyces kudriavzevii, S. cerevisiae and Torulaspora delbrueckii species were able to inhibit the germination of fungal conidia at the expense of the VOC formation. However, none of the up to date tested yeasts formed siderophores. On the other hand, the yeasts were generally resistant to the tested fungicide concentrations, thus they could be used as potential biofungicides in the combination with chemical fungicides.
B.06 Other
COBISS.SI-ID: 4924155At the COST conference we presented first preliminary results, based on testing of 26 autochthonous yeasts for siderophore production, efficacy of antifungal VOCs production by biocontrol yeast on the pathogen conidia germination, and chitinase and laminarinase activity. Yeasts included in testing were selected based on phenotypic characterization of the WRC yeast collection (Dashko, 2015). The purpose of this preliminary study was also to set-up and modify methods for testing biocontrol yeasts. Thus, the most promising results we obtained in the double petri dish assay, where we introduced naturally diluted grape juice agar for yeast growing, and on this agar D. hansenii, L. thermotolerans, P. kudriavzevii, S. kudriavzevii, S. cerevisiae and T. delbrueckii were able to inhibit conidia germination, while generally used YPD media did not indicate any clear antifungal pattern. Probably this was also due to release of some VOCs (like glycosidically-bound aroma) from grape juice because of the presence of yeast enzymes. Therefore, based on this we decided to perform in future screening for other glucoside hydrolase activity. With the DNS method we were not able to detect laminarinase and chitinase activity, however with a simple medium based on chitin as carbon source and pH indicator bromocresol purple we were able to detect positive-chitinase yeast.
B.06 Other
COBISS.SI-ID: 4709371In a study, involving 80 different strains that belong to 25 different grape / wine-related yeasts, the yeast selection was tested for the presence of siderophores, hydrolytic enzymes (chitinase, β-glucosidase and β-glucanase) and sensitivity to fungicides (copper, iprodion, cyprodinil + fludioxonil combination). The tests of phytopathogenic fungi growth inhibition in the presence of antagonistic yeast were also performed. The double Petri dish test was used to verify the inhibition of conidia germination by the formation of antifungal volatile organic compounds (VOC), synthesized by the antagonistic yeasts. Based on these screening tests we selected the biocontrol yeast P. guilliermondii ZIM 624 for the in vitro experiment on vine plantlets (V. vinifera L. c. Schioppettino and Sauvignon). In this experiment we observed after 2 weeks of incubation the development of symptoms on plantlets, which were treated with a selected biocontrol yeast P. guilliermondii ZIM 624 and infected with phytopathogenic fungus B. cinerea F61. In plantlets that were treated with biocontrol yeast and infected with phytopathogenic fungus, no symptoms of infection were observed, or they were less pronounced compared to treatment where tissue culture was infected with phytopathogenic fungi. In addition, during the season, the biocontrol yeast P. guilliermondii ZIM624 was applied in a vineyard field experiment at a concentration of 5 x 107 CFU / ml of fresh cells. The presence of the applied biocontrol yeasts on the grapes was monitored until harvested on a solid WL (Biolife) medium with added chloramphenicol. Biocontrol yeasts were present on the grapes even during the harvest. On the day following the treatment of grapes, bio-control yeasts were on average 2.9 log10 CFU / g and 2.3 log10 CFU / g at harvest time.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 4950267Grapevine canopy microclimate may be of crucial importance for predisposing the grapes to smaller or greater incidences of microbial infections but can also significantly alter plant biosynthetic behaviour. Leaf removal is one of the viticulture techniques that can affect fruit-zone microclimate and consequently the yielding of many important metabolites. Phenolics are grapevine secondary metabolites known to have a significant technological value in winemaking but also an important role in plant natural self-protection. A field trial with a completely randomized experimental design was set up in 'Pinot gris' (Vitis vinifera L.) vineyard (Potoče, Slovenia). The aim was to evaluate the effect of canopy microclimate manipulation trough leaf removal applied at different phenological stages (pre-flowering, at berry set, untreated control) on a comprehensive number of phenolics, including some rarely studied to date. A modern analytical approach of metabolic profiling using UPLC/QqQ-MS/MS was applied to analyse the grape berry samples of all the treatments, collected at harvest time. Cluster compactness, critical factor related with grape microbial diseases was also observed. The results revealed that out of 140-targeted phenolic compounds in the observation, 49 were present in our samples. Of these, significant differences in concentrations were observed in case of 16 individual phenolic representatives depending on the observed microclimate scenario. Comparing to untreated control, leaf removal at berry set significantly altered (decreased) the concentrations of p-hydroxybenzoic acid, phlorizin, apigenin-7-glucoside, catechin, gallocatechin and procyanidin B1, whereas pre-flowering treatment caused an increase in concentrations of vanilic acid, catechin and flavonols isorhamnetin-3-glucoside and quercetin-3,4-rutinoside. In case of pre-flowering leaf removal additional promising results were obtained for grape compactness. Focusing on only a few targeted phenolics in the past, new advanced analytical approaches are opening up a much wider range of possibilities to fill in the gaps of missing knowledge about plant reactions to purposely-induced abiotic stresses, including microclimate shifts.
B.06 Other
COBISS.SI-ID: 4918523Grapevine canopy microclimate, particularly temperature and relative humidity conditions around the clusters may be of crucial importance for predisposing the grapes to smaller or greater incidences of microbial infections. Different leaf removal timings, leading to different microclimate scenarios, were therefore introduced to Pinot noir (Vitis vinifera L.) vineyard in order to compare the leaf removal effectiveness with the effectiveness of fungicide application for Botrytis cinerea suppression. Prospective for cost and energy savings was also calculated based on all the observations. The results confirmed that early, pre-flowering treatment is more effective in controlling microbial infection then late (veraison) leaf removal. In case of pre-flowering leaf removal additional promising results were obtained for cluster compactness, another critical factor related with microbial diseases. Furthermore, a lower yield obtained by pre-flowering approach can reduce the need for cluster thinning and consequently total energy consumption thus this novel technique can be considered as a good option toward more sustainable and cost effective Pinot noir vineyard management in Vipava valley geo-climatic conditions. Similarly promising are also the results of its further investigation on ‘Pinot Gris’ (Vitis vinifera L.) variety. Beside variety and geo-climatic conditions also viticulture practices and winemaker’s choices of technology approaches play a significant role in the final wine style and quality. To reveal the effects of different technology approaches on wine chemical and sensory characteristics, the experimental wines, produced from grapes of some locally important varieties, were subjected to the modern analytical approaches of metabolic profiling. The wines were also sensory evaluated. The results contributed important data regarding the technological effects on phenolic and aromatic profiles of the observed wines as well as their related sensory characteristics.
B.04 Guest lecture
COBISS.SI-ID: 4833019