Potato dextrose agar medium

In both experiments, the plants or seeds were infected with IPO348–01 nivalenol chemotype mycelium of F. culmorum from the Plant Breeding Institute, Wageningen (Netherland). Mycelium box test was performed in in vitro conditions on seedlings grown on inoculated Potato Dextrose Agar (PDA) medium (Sigma–Aldrich, Poznań, Poland). The mycelium was grown in a microbiological thermostatic chamber (ST 5 Smart, Pol–Aura Aparatura, Wodzisław Śląski, Poland) at 21 °C, in darkness for seven days [32 (link)]. The mycelium production for the open tunnel experiment followed the method described by Wiśniewska et al. [81 ]. An Erlenmeyer glass flask (250 cm³) was filled with 50 g of spring wheat seeds, and 15 cm³ of water w added to obtain 40% humidity. After 24 h, the seeds were autoclaved at 101 325 Pa, at 121 °C for 30 min and then cooled. The infection was initiated by transferring three 1.5 cm discs of PDA medium. The glass flasks were placed in the microbiological thermostatic chamber (ST 5 Smart, Pol–Aura Aparatura, Wodzisław Śląski, Poland), and the mycelium was growing at 20 ± 1 °C for five to six weeks in darkness. The flasks were shaken thoroughly every day to prevent sticking the grain to the glass, and to also provide uniform inoculation of the grain.

Pathogenicity assays were performed to confirm the capability of the selected Colletotrichum strains to cause soybean anthracnose (Table 1). For sporulation, strains were cultured on Potato Dextrose Agar (PDA) medium (Sigma-Aldrich, St. Louis, MO, USA) for 15 days at 25 °C and conidia suspensions were prepared and adjusted to 1 × 10⁶ conidia/mL.
Soybean seeds of the IPRO7739 cultivar (Monsoy company, São Paulo, Brazil), were superficially disinfected for 1 min into a 1% NaClO solution, rinsed three times in sterile distilled water (SDW) and placed in Petri dishes containing 100 g of sterile sand, soaked with 10 mL of SDW. Seeds were incubated in the dark for 32 h at 25 °C.
Conidia suspensions of each Colletotrichum strain were placed on five germinated seeds as described previously [96 (link)]. SDW was used as a negative control. Inoculated seedlings were incubated for 4 h, transferred to pots filled with vermiculite and transferred to a greenhouse for 7 days. The virulence of Colletotrichum strains was evaluated using an adapted diagrammatic scale that ranges from 0 to 5 [15 (link)]. Severity data were analyzed with the post-hoc Tukey method at 0.05 significance level, using the ExpDes R package (v.1.2.0) (Alfenas, Brazil).

Fungal strains used are Fusarium culmorum Schltdl. and Cochliobolus sativus Drechsler ex Dastur. They are from the Belgian coordinated collections of microorganisms (BCCM-MUCL, Ottignies-Louvain-la-Neuve, Belgium) where they are recorded as MUCL 28166 and MUCL 46854, respectively.
Fungi were cultivated on Potato Dextrose Agar (PDA) medium (Merck KGaA, Darmstadt, Germany, 39 g.L⁻¹) at 23 °C and a 16:8 (L:D) photoperiod. Cultures were used after 14 days of growth to make spore suspensions.

The strain used in this study was L. theobromae LA-SOL3, isolated from Vitis vinifera in Peru. LA-SOL3 was one of the most aggressive strains in artificial inoculations trials of cv. Red Globe plants^{2 (link)}. The culture was routinely grown on Potato Dextrose Agar (PDA) medium (Merck, Germany) at 25 °C.
For DNA extraction, the mycelium harvested from a culture grown on Potato Dextrose Broth (PDB, Merck, Germany) at 25 °C for three days was ground in liquid nitrogen and DNA was extracted according to Möller et al.^{30 (link)}. For protein and RNA extraction, LA-SOL3 was inoculated into a 250 mL flask containing 50 mL of PDB and incubated at 25 °C or at 37 °C for 4 days, as described earlier^{31 (link)}. All assays were performed in triplicate.

Filamentous fungi were collected during a sugar beet harvesting campaign in June 2020. The contaminated sugar beet site was located 25 km from Beni Mellal city (32°29′34.2″ N 6°10′56.1″ W, center of Morocco). This infected site has a warm Mediterranean climate with a temperature ranging between 1.1 and 40 °C and a mean annual rainfall generally between 350 and 650 mm/year [46 (link)]. In sterile plastic bags, roots with symptoms of damage and fungi infection were harvested and isolated aseptically (Figure 1).
According to the modified protocol of Chenaoui et al. [47 ], the surface soil of contaminated sugar beet root was removed. To cultivate rhizoplane fungi that may be associated with the root symptoms and interact with rhizosphere Streptomycetes, the mycelium on the root surface was scraped off with a sterile platinum loop and plated on potato dextrose agar (PDA) medium (Merck, France). The PDA medium used contained potato infusion (4 g), dextrose (20 g) and agar (17 g) in 1L of deionized water at a final pH of 5.6 ± 0.2. Plates were incubated in the dark at 26 ± 2 °C for 7–8 days and checked every day. Hyphae were purified and preserved on PDA medium for further study.

In the present study the following polymers were used: poly(3-hydroxybutyrate) (Scheme 1a) (PHB, 330,000 g.mol⁻¹, Biomer, Schwalbach, Germany) and polyvinylpyrrolidone (Scheme 1b) (PVP K25; Fluka, Buchs, Switzerland) with Mr = 24,000 g.mol⁻¹. 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) (CQ) (Scheme 1c) was purchased from Sigma-Aldrich, Buchs, Switzerland. N,N-Dimethylformamide (DMF) (Merck, Darmstadt, Germany), chloroform (Merck, Darmstadt, Germany), acetone (Sigma-Aldrich, Buchs, Switzerland) and dimethyl sulfoxide (Merck, Darmstadt, Germany) were of an analytical grade of purity. The microbiological growth media was potato dextrose agar medium (Merck, Darmstadt, Germany).

Cellulose acetate (CA, Aldrich, St. Louis, MO, USA) with ${\overline{\mathrm{M}}}_{\mathrm{n}}$ at 30,000 g/mol and 39.8 wt% of degree of substitution in acetyl content, polyethylene glycol (PEG, Fluka, Buchs, Switzerland) with relative molecular mass (Mr) 1900–2000 g/mol, polyethylene oxide (PEO, Serva, Heidelberg, Germany) with Mr ca. 100,000 g/mol and 5-chloro-8-hydroxyquinolinol (5-Cl8Q, Sigma-Aldrich, Buchs, Switzerland) were used. Acetone (Sigma-Aldrich) of analytical grade of purity was used. Potato dextrose agar medium was purchased from Merck, Darmstadt, Germany. The disposable consumables were supplied by Orange Scientific, Braine-l’Alleud, Belgium.