Potato dextrose agar (pda)

The B. cinerea LT13B_FRA_76 isolate used in this study was from the LAMMC IH Laboratory of Plant Protection isolate collection. B. cinerea from rotten strawberry fruit was morphologically identified and verified by a species-specific PCR, according to [4 (link)]. The single-spore isolates were cultivated on potato dextrose agar (PDA) (Liofilchem, Roseto degli Abruzzi, Italy).

Single spore C. acutatum F-05-I isolate was obtained from LAMMC IH Laboratory of plant protection isolate collection. The F-05-I origin was from the rotten strawberry fruit. The isolate was identified by PCR as C. acutatum by Xie et al. [40 (link)]. For experiments, isolates were cultivated in Petri plates containing potato dextrose agar (PDA) (Liofilchem, Roseto degli Abruzzi, Italy) at 25 ± 2 °C for 7 DAI. The isolates mycelial plugs (7 mm diameter) mycelium were placed side-down in the center of the new Petri with PDA. The morphological and phenotypic characteristics of C. acutatum were evaluated under different LEDs and PFD. The closed, controlled environment conditions were 23 ± 2 °C temperature and four hours (h) photoperiod. The control plates were incubated in complete darkness. There were four replicates per treatment.

All microorganisms used in this study are listed in Table 1. Cell stocks were kept at −80 °C and subcultured once before the experiments. Filamentous fungi strains were maintained in Sabouraud Dextrose Agar (SDA) (Liofilchem s.r.l., Roseto D.A., Italy) or Potato Dextrose Agar (PDA) (Liofilchem s.r.l., Roseto D.A., Italy) for approximately 7 days at room temperature. For each experiment, conidia were harvested by flooding the agar surface with sterilized saline solution containing NaCl 8.00 g·L⁻¹ (Sigma-Aldrich, Sintra, Portugal), KCl 0.2 g·L⁻¹, Na₂HPO₄·2H₂O 1.44 g·L⁻¹, and KH₂PO₄ 0.24g·L⁻¹ (all from José Manuel Vaz Pereira, Benavente, Portugal) (pH 7.4). Biomass was then suspended in the saline solution with a sterile loop and the final solution collected with a pipette tip to a sterile tube. The heavier fragments were allowed to deposit in the bottom for 5–10 min and subsequently the supernatant was transferred to a new sterile tube [26 (link),27 (link)]. Candida strains were maintained on Sabouraud dextrose agar for 48 h at 37 °C [28 (link)]. Bacterial strains were maintained on Tryptic soy agar (TSA) (Liofilchem s.r.l., Roseto D.A., Italy) for 24 h at 37 °C [29 (link)]. Strains were provided by Colección Española de Cultivos Tipo (CECT), Micoteca da Universidade do Minho (MUM), and the American Type Culture Collection (ATCC).

Microbiological control was carried out by seeding the antigen mixture undiluted and 1/10 diluted in different culture media, such as Tryptone Soy Agar (TSA) (Merck, Darmstadt, Germany) for aerobic bacteria; Thioglycolate Broth (TGB) (Merck, Darmstadt, Germany) for strictly anaerobic, facultative anaerobic, and microaerophilic bacteria; and Potato Dextrose Agar (PDA) (Liofilchem, Roseto degli Abruzzi, TE, Italy) for fungi. TSA plates and TGB tubes were incubated for 7 days at 37 °C, and PDA plates were incubated at 25 °C for the same time. All cultures were performed in duplicates. The manipulation was carried out in a biosafety cabinet (AirScience, Fort Myers, FL, USA).

The FB₁ standard solution (purity > 99%) was obtained from Sigma–Aldrich (St. Louis, MO, USA). The phenolic standards 1,2-dihydroxybenzene, 3,4-dihydroxicinnamic acid, benzoic acid, 3-phenylLactic acid, hydroxycinnamic acid, p-coumaric acid, protocatechuic, sinapic acid, vanillin, syringic acid, and ferulic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Lactic acid was obtained from Sigma–Aldrich (St. Louis, MO, USA).
Acetonitrile (ACN) (LC-MS/MS grade), ethyl acetate (EA), formic acid (FA), and methanol (HPLC-MS/MS grade) were obtained from VWR Chemicals (Randor, PA, USA). The deionised water used in chromatography analysis (<18 MΩ cm resistivity) was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA). The salts, magnesium sulphate (MgSO₄) and sodium chloride (NaCl), were provided from Sigma–Aldrich (St. Louis, MO, USA).
The Potato Dextrose Agar (PDA), Potato Dextrose Broth (PDB), and Buffered peptone water (BPW) were purchased from Liofilchem Bacteriology Products (Roseto, Italy). De man Rogosa Sharpe (MRS) Broth was obtained from Oxoid (Hampshire, UK).
The Yellow Mustard Flour (YM) (code #106) and Oriental Mustard Flour (OM) (code #107) were provided by G.S. Dunn Dry Mustard Millers (Hamilton, ON, Canada).