Malt extract agar mea

For mycological analysis, 20 g of the sample was mixed with180 ml of 0.1% peptone solution (achieving a 10–1 dilution) and further diluted until 10^–4. Dilution plating was carried out according to Samson et al. (2019 ), utilizing selective mycological media, namely, Malt Extract Agar (MEA; Merck, Darmstadt, Germany) supplemented with 100 μg/ml of chloramphenicol (Roth, Karlsruhe, Germany) and Dichloran Rose Bengal chloramphenicol Agar (DRBC; Roth, Karlsruhe, Germany). These media have been used in studies of mycology of silages (O'Brien et al. 2005 (link); O'Brien et al. 2007 (link); Manfield and Kuldau 2007 (link)). For inoculation of the plates, 0.1 mL aliquots representing 10^–2, 10^–3 and 10^–4 dilutions were used, in triplicates. Plates were incubated at 25 °C for 5–7 days in the dark. Additional cultivation at 37 °C for 5 days was used for the isolation of opportunistic fungal pathogens. Each fungal colony isolated from a sample was considered as an individual isolate. Morphological identification of dominant fungal genera/species was performed by evaluation of macro- and microscopic morphological traits according to Samson et al. (2019 ) and de Hoog et al. (2020 ).

Yeast strains isolated from grape must were obtained from the culture collection of the South African Grape and Wine Research Institute (SAGWRI), Stellenbosch University. Thirty-one strains (Supplementary Table S1) were routinely grown and maintained on Wallerstein Nutrient (WLN) agar (Merck Millipore, South Africa). For long-term storage, the strains were stored at −80°C in 25% (v/v) glycerol in cryogenic tubes. Three strains of B. cinerea, laboratory strain (B05. 10), grape strains (IWBT FF1 and IWBT FF2) isolated from Cabernet Sauvignon grapes obtained from Thelema Mountain vineyard, South Africa (33°54′46.1′S 18°56′30.7′E), one strain of Aspergillus niger and Alternaria alternata isolated from soil collected from Stellenbosch University’s Welgevallen experimental farm (33°57′03.0′S 18°52′05.6′E), were used in this study. Filamentous fungal cultures were revived on Malt Extract agar (MEA; Merck Millipore, South Africa) containing 2% (w/v) bacteriological agar. Yeast inoculums were prepared from overnight cultures grown in 5 ml YPD broth containing per litre (10 g yeast extract, 20 g peptone and 20 g glucose). Fresh yeast cultures were collected by centrifugation at 10,625 g for 5 min and washed twice with sterile 0.9% (w/v) NaCl solution. The yeast suspensions were adjusted to OD₆₀₀ 0.1 (≈ 10⁶ CFU/ml) using 0.9% (w/v) NaCl.

The antifungal activity of EOs of basil, savoury, thyme, oregano, lemon, and fennel at concentrations of 1.0%, 0.5%, and 0.1% was evaluated in vitro, using the sandwich plates method, to evaluate the mycelial growth inhibition of two strains (PEN2 and PEN3) of P. expansum, taken from the collection of the University of Turin and isolated from apples. For the preparation of the EOs, Petri dishes with a diameter of 90 mm (VWR, Milan, Italy) were used, where Potato Dextrose Agar (PDA, Merck, Darmstadt, Germany) medium was poured, after autoclaving, with the selected EO. The EO was added at 1.0%, 0.5%, or 0.1% (v/v) to sterile deionised water (98.0, 98.5, or 98.9% v/v) and Tween 20 (1.0% v/v). For the pathogen growth, Malt Extract Agar (MEA, Merck) plates were inoculated with mycelium plugs (0.4 cm) taken from cultures grown on MEA for 21 days. Plates with the EOs were placed on top of plates with the mycelium plug to build a sandwich and then closed with parafilm. The plates were incubated at 25 ± 1 °C and the mycelium diameter of the pathogen was measured after 24 h, 48 h, 72 h, 96 h, and 11 days of incubation. Control plates were set up in the same manner as described above but using a plate containing only PDA. The test was performed twice, each time with 5 biological replicates.