R2a broth

The evaluation of the secondary metabolites of bacterial isolates extracted with ethyl acetate was performed in three culture media—R2A broth (Himedia ref. 1687), ISP2 broth (Difco ref 277010), and nutrient broth (Termofisher Scientific ref CM0001)—as described in a previously published protocol [35 (link)]. The crude extracts of three representative strains were used for antimicrobial and antiproliferative activity tests. Simultaneously, the crude extracts were analyzed by ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) in a Thermo Scientific QExactive® Hybrid Quadrupole-Orbitrap Mass Spectrometer.

The microbial extract library preparation and screening platform for EPI activity is discussed in Supplementary Material. The 48 h old inoculum of soil isolate IMTB 2501 was grown in R-2A broth (Himedia^®, India), supplemented with 1% NaCl, and 0.2% calcium carbonate. After 96 h of incubation, cells were removed by centrifugation and extract was prepared using Diaion^® HP-20 resin. The crude extract was fractionated on Sephadex^TM LH-20 (GE Healthcare) column using a step gradient of methanol: water. The active fractions were pooled and processed further by reverse-phase high-pressure liquid chromatography (RP-HPLC) (Waters, XBridge^® BEH C18 OBD^TM Prep Column 130 Å, 5 μm, 10 mm × 250 mm). The mobile phase consisted of solvent A as 10 mM ammonium acetate (pH 5.5) and solvent B as 100% acetonitrile. The flow rate was kept constant at 3 mL/min. The following gradient was used; 5% solvent B for 5 min, 5 to 95% B in 45 min, hold at 95% B for 10 min. The peaks were collected and assayed for the bioactivity. The active peak (termed as RP2) was identified and subjected to lyophilization. The purified compound was subjected to gas chromatography (Varian 450-GC) coupled with mass spectrometry (Varian 20-MS). The spectrum was recorded in an electron-ionization mode. NMR spectra (¹H, ¹³C, DEPT, COSY, and HSQC) were recorded on a Bruker 400 MHz spectrometer in DMSO-d₆.

Subsequent to the in planta enrichment cycles, Ex-3 was selected for isolating bacteria present on the spike. With a similar methodology to the microbiome extract (Section 4.2), spikelets of Ex-3, cycle 3, were selected. The spike microbiome extract was used as the starting suspension for limiting dilution. In total, 94 isolates were selected and preserved in a glycerol stock. The spike microbiome extract was serially diluted in two different media, R2A broth (HiMedia, Mumbai, India) and a spike extract medium (SEM). SEM was prepared by boiling 50 g of spikes at anthesis in 1 L of RO water for 30 min followed by filter sterilization. The dilutions were plated in a 96-well plate by pipetting 100 µL diluted extraction per well. The plates were incubated for 14 days at 21 °C. The dilution factors were chosen in such a way that less than 50% of the wells showed visible bacterial growth after an incubation period of two weeks. The wells that showed growth in these plates were regarded as clonal cultures and subsequently handled as pure isolates. Glycerol was added to an end concentration of 25%, and isolates were stored at −80 °C.