Ff microplate

Biolog FF MicroPlate™ (Biolog Company, USA) were used to obtain the metabolic profile of FocTR4 in comparison to isolates of A. fumigatus, F. solani, three other species of Fusarium spp. that occurred at high abundances during fungal isolation in the treatment containing banana residues (Table S4), and three other species of Aspergillus spp. known to have no antagonistic effect on FocTR4. Each fungal strain was cultured on PDA medium to obtain spores. A 0.1 ml of spore suspension of each strain (adjusted to 75% ± 2% with a turbidity meter in FF inoculation fluid, FF-IF) was inoculated into each well in a Biolog FF MicroPlate™. Triplicated MicroPlates per treatment were placed in an aerobic Omnilog incubator reader (Biolog Inc., USA) at 20 °C (i.e., the optimal growth temperature as recommended by the Westerdijk Fungal Biodiversity Institute culture collection). Colorimetric values at 590 nm were measured at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 96 h, 120 h, 144 h, and 168 h, and blanked against the control wells. Results were considered positive only when differences between the first and last days were observed across all replicates. The percentage of common carbon source utilization between each strain and FocTR4 was obtained by (A∩B)/B*100%, where A is the available carbon source of antagonistic taxa and B is the available carbon source of FocTR4.

The biochemical profile of Aspergillus niger environmental isolates was obtained using the Biolog MicroStation test system (MicroLog3 ver. 4.20.05, Biolog Inc., United States). By using a 96-well Biolog FF MicroPlate, unique phenotypic fingerprinting can be obtained for individual strains; this is a manifestation of their catabolic potential. Metabolic reactions, dependent on the production of suitable enzymes for the oxidation of the tested substrates, are quantified by the formation of tetrazolium dye (Bochner, 2009 (link)). These results, as fingerprint reaction patterns, provide a lot of information about the individual differences in the metabolic properties of each fungus tested.
All A. niger environmental isolates were cultured in tubes on 2% slants of MEA (Malt Extract Agar HiMedia Laboratories, Mumbai, India) at 26°C for 5–7 days. Conidia were then inoculated into FF-IF inoculating fluid (72106 FF-IF inoculating fluid, Biolog, United States) according to the Biolog protocol and the density was adjusted to the recommended value of 65%, which was measured using a turbidimeter. Subsequently, we inoculated 100 μL of conidia suspension onto plates containing a pre-set of test substrates. We incubated the microplates for 72–168 h and then evaluated them using the Biolog MicroStation system.

Metabolic abilities of selected phytopathogens were performed on the FF MicroPlate (Biolog^®, Hayward, CA, USA) containing 95 different carbon sources in the wells. Inoculation was performed according to the manufacturer’s protocol with modifications described by Oszust et al. [17 (link)]. After the homogenization of the mycelium suspension in inoculating fluid (FF-IF, Biolog^®, Hayward, CA, USA), the transmittance was adjusted to 75% using a turbidimeter (Biolog^®, Hayward, CA, USA). A volume of 100 μL of the mycelium suspension was added to each well. The inoculated microplates were incubated in darkness at 25 °C for 10 days. Absorbances were measured daily at the wavelengths of 490 nm and 750 nm, then the ratio of those values was calculated.

Growth rates on different carbon sources, except on apple pectin, were analyzed in biological duplicates using a phenotype microarray system for filamentous fungi (Biolog Inc., Hayward, CA), as described by Atanasova and Druzhinina [70 (link)]. Briefly, strains were cultivated on PDA for 7 days. Conidial inocula were prepared by rolling a sterile, wetted cotton swab over sporulating areas of the plates. The conidia were then suspended in sterile Biolog FF inoculating fluid (0.25% Phytagel, 0.03% Tween 40), gently mixed, and adjusted to a transmission of 75% at 590 nm (using a Biolog standard turbidimeter calibrated to the Biolog standard for filamentous fungi). A total of 90 μL of the conidial suspension was dispensed into each of the wells of the Biolog FF microplates (Biolog Inc.), which were incubated at 28 °C in darkness. The optical density (OD) at 750 nm (for detection of mycelial growth) was measured after 24, 48, 72, 96 and 120 h using a microplate reader (Biolog Inc.). Statistical analyses were performed using the Statistica software package (version 6.1; StatSoft Inc., Tulsa, OK). Growth rates on pectin were determined by cultivation of the fungi on solid Mandels-Anderotti minimal medium supplemented with 2% agarose and 1% apple pectin. The growth of mycelia was measured every 24 h.

The growth rates and carbon utilization profiles of the strains were monitored using a phenotype microarray system with Biolog FF microplates for filamentous fungi (BIOLOG, Hayward, USA) as described previously [81 (link)], with the following modifications. Briefly, Trichoderma spores were collected and suspended in sterile Milli-Q water in disposable borosilicate tubes. Then, 90 μl of the adjusted spore suspension (with a transmission of 75% ± 2% at 590 nm, ca. 10⁷ cells ml^-1) was dispensed into each well. Microplates were sealed in the original bags and incubated at 25°C in darkness, and the OD₇₅₀ and OD₄₉₀ were measured at 12, 18, 24, 36, 48, 60, 72, 96, 120, 144 and 168 h. OD₇₅₀ values were adopted for the biomass measurements [81 (link)], while respiratory activity (activity of the succinate dehydrogenase proportional to the formation of red color of the formazan dye integrated into the FF Biolog microplate) was calculated by subtracting the OD₇₅₀ from the OD₄₉₀ [82 (link)].