Czapek dox agar

Fifty-two mould colonies grown on MA were isolated from the spoiled goose sausages, purified and transferred onto three different agar media: Czapek Dox Agar (Oxoid, Milan, Italy), MA, and salt-malt agar (5% malt extract, 5% NaCl, distilled water 1 mL, pH 6.2; Oxoid, Milan, Italy). According to Samson et al. [39 ], the moulds were identified by morphological characters by macroscopic and microscopic examination (hyphae, spores and reproduction, colour of colony, and type of mycelium). The identification was confirmed by PCR-DGGE and sequencing according to the method reported by Iacumin et al. [41 (link)]. Briefly, the DNA of each colony was amplified by nested PCR (2-step amplification). Each amplicon was run in an acrylamide gel (DGGE), excised by gel cutting tips, and subjected to reamplification with the same primers without a GC clamp. The product was cloned into the pGEM-T easy vector (Promega, Milan, Italy) following the instructions of the manufacturer. The insert of the appropriate clone was sequenced by a commercial facility (Eurofins MWG GmbH, Martinsried, Germany). Sequence comparisons were performed using the Blast program [42 (link)].

Complete ldpA and ldpB CDSs were amplified from pCR2.1-LdpA and pCR2.1-LdpB. The PCR products were inserted into pHAN02-GFP (Fig. S3) between the HindIII and SmaI sites by using the In-Fusion HD Cloning Kit (Takara Bio). The resulting plasmids were transformed into chemically competent E. coli strain HST08. The plasmids were extracted using the GenElute^TM Plasmid Miniprep kit (Sigma-Aldrich) and linearised using the restriction enzymes BamHI or EcoRI. The A. fumigatus niaD⁻ mutant AfS35-niaD⁻ was isolated by positive selection using chlorate according to the methods described by Unkles et al.^{35 (link)} and Ishi et al.^{36 (link)}. The resulting plasmids were used for the transformation of AfS35-niaD⁻ by PEG-mediated protoplast transformation methods. The niaD⁺ revertants were selected for growth on Czapek–Dox agar (Oxoid), which contains 1.2 M sorbitol and sodium nitrate as the sole source of nitrogen. Gene integration was confirmed by PCR.

After incubation, incidence of infected with black aspergilli berries and distribution of most dominant grape mycoflora at genus level, were recorded. A representative number from the black aspergilli from every vineyard were isolated from DRBC and sub-cultured on Malt Extract Agar (MEA, LabM) and Czapek Dox Agar (CD, Oxoid) for further identification at species level. The initial identification of the different strains of Aspergillus section Nigri was performed using macroscopic and microscopic morphological criteria in accordance with appropriate keys [29] (link), [43] , [44] . The reference strains of A. carbonarius, A. niger, A. tubingensis, A. ochraceus and A. westerdijkiae were kindly provided by Prof. N. Magan from the Mycology Group of Cranfield University. The isolates were preserved at −80°C in the culture collection of the Department of Food Science and Human Nutrition of the Agricultural University of Athens, Greece.