Trypticase soy broth (tsb)

Microtitre plate method was performed as the gold standard quantitative method for characterizing biofilm-forming E. coli isolates. Briefly, overnight culture of E. coli isolates was inoculated into trypticase soy broth (3 mL, Merck, Germany), supplemented with 1% glucose, and incubated at 37 °C for 24 h. Afterward, the culture was diluted at 1:100 by adding sterile trypticase soy broth and 200 mL of dilution was added to each well of a sterile 96-well polystyrene microtiter plate. Three wells for each isolate were assessed in each microtiter plate. This pattern was repeated in three microtiter plates. The plates were covered and incubated aerobically for 24 h at 37 °C, and subsequently, washing (250 μL of sterile saline solution), fixing (200 μL of methanol), staining (200 μL 2% Hucker crystal violet per well), and drying were performed. Finally, a micro-enzyme-linked immunosorbent assay (ELISA) reader determined the absorbance of biofilm formation at 570 nm. Then, 200 mL of sterile TSB was inoculated in wells as negative control. Biofilm-forming isolates were categorized in four groups, including strongly adherent bacteria (4 × ODc < OD), moderately adherent bacteria (2 × ODc < OD ≤ 4 × ODc), weakly adherent bacteria (ODc < OD ≤ 2 × ODc), and non-adherent bacteria (OD ≤ ODc) [21 (link), 22 (link)].

Aerobic Endospore Forming Bacteria (AEFB) isolated from Musa sp. plants (Humboldt Institute Collection No. 191), B. subtilis NCIB 3610 (wild type, WT), B. subtilis SMY, B. subtilis PY79 and knockout strains of NCIB 3610 (Supplementary Tables S1 and S3) were used in the different inducible antagonism trials. These strains were stored in TSB (trypticase soy broth, Merck, Germany) with 20% v/v glycerol at −80 °C and activated in TSA (trypticase soy agar, Merck) or LB agar (Luria Bertani agar, Basingstoke, Oxoid, England) for 48 h at 30 °C before use. Strains R. solanacearum EAP-009 (GenBank accession N° KU603426), Serratia marcescens EAD-005 (GenBank accession N° KU603427)^{45 (link)} and R. solanacearum AW1^{46 (link)}, all were stored in BG medium⁴⁷ plus 20% v/v glycerol at −80 °C and activated at 30 °C for 72 h in BGA medium (BG plus 18 g/L agar (BD, Ontario, Canada)). BG medium was composed of 10 g/L special peptone (Oxoid), 1 g/L casamino acids (BD), 1 g/L yeast extract (Merck) and 5 g/L glucose (Merck). Pseudomonas putida UA-0095, Xanthomonas sp. UA-1539, B. cepacea UA-1541, S. marcescens UA-1538, Salmonella sp. ATCC 14028, Staphylococcus sp. G and E. coli DH5α (Supplementary Table S1) were stored in TSB (trypticase soy broth, Merck) with 20% v/v glycerol at −80 °C and activated in TSA or LB agar for 48 h at 30 °C before use.

Each sample was aseptically weighed in an analytical balance and 25 g were transferred into a sterile plastic bag. Then, 225 mL of buffered peptone water (Merck, Germany) was added and homogenized in a Stomacher Bagmixer 400 W (Interscience, Saint-Nom, France) for 2 min. Five milliliter aliquot of the enriched homogenate was transferred into 50 mL Trypticase Soy Broth (TSB, Merck, Germany) supplemented with 10% NaCl and 1% sodium pyruvate. After incubation at 35 °C for 18 h, a loopful of the culture was plated onto Baird-Parker agar supplemented with egg yolk tellurite emulsion (Merck, Germany) and incubated overnight at 37 °C. Black shiny colonies surrounded by 2 to 5-mm clear zones were further identified on the basis of Gram staining, hemolytic activity on sheep blood agar (Merck, Germany), catalase activity, coagulated test (rabbit plasma), oxidase test, glucose O/F test, resistance to bacitracin (0.04 U), mannitol fermentation on Mannitol salt agar (Merck, Germany), urease activity, nitrate reduction, phosphatase, deoxyribonuclease (DNase, Merck, Germany) test, voges-proskaver (Merck, Germany) test and carbohydrate (xylose, sucrose, trehalose and maltose, fructose, lactose, mannose) fermentation tests [11 (link)].