Difcotm

The yeast Hanseniaspora uvarum (strain: LB-NB-2.2, accession number GenBank NCBI: MK567898) was isolated from feeding tunnels of SWD larvae in infested grapes in South Tyrol, Italy [18 (link)]. Yeast cultures were grown under sterile conditions in 220 mL autoclaved potato dextrose broth (PDB; 4 g/L potato starch, 20 g/L dextrose, Difco^TM, Becton Dickinson, Le Pont de Claix, France) at 25 °C, 120 rpm for 30 h under light in a 250-mL Erlenmeyer flask closed with cotton and aluminum foil. The inoculum (0.5 mL) was prepared with a loop full of yeast cells cultivated on potato dextrose agar (4 g/L potato starch, 20 g/L dextrose, 15 g/L agar, Difco^TM, Becton Dickinson), which were transferred in a 2-mL Eppendorf tube filled with 1 mL PDB and vortexed for 10 s at 1800 rpm. After 30 h of growth, the yeasts reached the stationary phase. The number of cells per mL (Fuchs Rosenthal counting chamber, Assistent^®, Sondheim vor der Rhön, Germany) was 6.4 × 10⁷, optical density (OD) at 600 nm (Cary 60 UV-Vis, Agilent, Santa Clara, CA, United States) was 1.8, and the pH (pH meter, Crison GLP 21, Hach, Düsseldorf, Germany) of the fermentation broth was 4.13. The yeast fermentation broths and autoclaved PDB were stored at −80 °C and thawed at room temperature before use.

This study used two sets of isolates. The first set included the interfertile fungal isolates F. circinatum (CMWF 350; FSP34) and F. temperatum (CMWF 389; Netza9) and their 94 F₁ hybrid progeny (Supplementary File S4, Table S1) that were previously used for GLM construction [26 (link)]. The second set included 19 genetically diverse F. circinatum isolates that were collected from various locations in South Africa, Mexico, California, and Florida during previous studies (Table 1).
Isolates were grown on one of two types of growth media, potato dextrose agar (PDA; 15 g/L; Biolab) and pine-tissue-derived agar medium. The latter was prepared by chopping the above-ground parts of 6-month-old Pinus patula seedlings into small pieces, of which 40 g was added to 1 L of H₂O and autoclaved at 120 °C for 30 min. The extract was filtered through Whatman Grade 1 filter paper (GE Healthcare Life Sciences) to remove the plant debris. The filtrate was mixed with malt extract (20 g/L; BD Difco^TM) and agar (10 g/L; BD Difco^TM), autoclaved, and poured into plates. The medium is referred to as PMEA (for pine-tissue extract containing malt extract agar), and Supplementary File S1 provides an overview of the metabolites it contains, as determined according to Hammerbacher et al. [52 (link)] and Gonzalez-Cabanelas et al. [53 (link)].

The antimicrobial activities of the compound were assessed and the minimum inhibitory concentrations (MIC) were determined against a panel of human pathogens, some of them multi-resistant to clinically-used antibiotics (Table 2). Some of them were isolated and identified in clinical microbiology laboratories from samples obtained in patients with clinical infections. Mueller-Hinton agar (Becton, Dickinson and Company, Le Pont de Cloix, France) was the culture medium in bioassays against E. coli, S. aureus, E. faecalis, E. faecium, M. luteus, H. influenzae, being supplemented according to the CLSI conditions for S. pneumoniae, S. pyogenes and N. meningitidis. Trypticasein soy agar (5% w/v) sheep blood DIFCO^TM (Becton, Dickinson and Company) was used for C. urealyticum. Brucella Broth (SIGMA-ALDRICH, St. Louis, MO, USA) supplemented with hemin (5 µg/mL), vitamin K1 (1 µg/mL) and lysed horse blood (5% v/v) was used for B. fragilis and C. perfringens.
For most Gram-positive and Gram-negative bacteria, the antimicrobial assays were performed according to CLSI performance standards [29 ]. For M. tuberculosis susceptibility testing was done in Middlebrook 7H10 agar medium (Becton, Dickinson and Company) supplemented with 10% OADC and 0.5% glycerol according to the agar proportion method for slowly growing mycobacteria [30 ].