Isolation of endophytic bacteria from E. colonum
The potential bacterial antagonists were isolated from the stems, roots, and leaves of healthy E. colonum collected from different locations of Binh Thuan province, Vietnam, during June 2019. The samples were immediately washed under tap water to remove dust and soil. The fragments were disinfected by successive treatment with ethanol 70% (1 min) and exhaustively rinsed several times with sterilized distilled water. The fragments were then submitted in mercuric chloride 0.01% (available chlorine) for 5 min and then rinsed again with sterilized distilled water for 3 times. Aliquots of 0.1 ml of the last wash water were inoculated onto the media plates of LB (yeast extract 5 g/l, tryptone 10 g/l, NaCl 5 g/l, agar 15 g/l, pH 7.0–7.5) to check the effectiveness of the disinfection process. After the disinfection, samples were cut into fragments (5 × 5 mm) and placed onto the LB plates for the isolation of bacteria. The sterile distilled water uninoculated on LB plates were done as respective controls. All the plates were incubated at 28 °C for 7 days and observed daily for the growth of a different type of bacterial colonies. The observed colonies were picked up and subcultured on new LB plates several times by repeated streaking (Quadrant streaking) till single type and isolated colonies were obtained. The obtained pure cultures were inoculated onto LB slants. After overnight incubation at 28 °C, they were stored at 4 °C till further use.
Antagonist activity of bacterial isolates in vitro
All isolated bacteria were tested for antifungal activity. On a sterile potato dextrose agar (PDA, potato infusion 200 g/l; dextrose 20 g/l; agar 15 g/l; pH = 7.0–7.3) plate, a cylinder of a 1-cm2 fungal plug (A. alternata) was placed in the center and each bacterial isolate was inoculated using a sterile stick at a distance of 2.5 cm from the fungus. Then, the plates were incubated at 28 °C for 72 h. The control plate was inoculated with only phytopathogenic fungi. The strains that showed an inhibition of the fungal growth were selected and preserved. The inhibition of mycelial growth was observed after 5 or 7 days. The antimicrobial activity was determined by measuring the diameter of the zone of inhibition that is the mean of triplicates ± SE of 3 replicates.
Biocontrol of combined strains on A. alternata
In addition, the inhibition of possible combinations of those bacteria against A. alternata was investigated. A separate experiment with the isolates’ strains was carried out to indicate the occurrence of competition on LB. The bacteria were cultured individually overnight. The cultures were mixed by a 1:1 ratio and inoculated as described in the standard antagonism test. After 2 weeks of incubation, the bacterial colony development was observed, and the antagonism ability of the combination was also determined.
Antifungal activity of extracellular filtrates
The overnight culture of the isolated bacterium with antifungal activity was used and filtrated to obtain extracellular filtrates. Then, biocontrol assays were carried out with the supernatant. After that, the plate incubation was done at 28 °C for 120 h and verified every 12 h. The indicator for the antifungal activity of filtrate was the fungal growth inhibition.
In vitro assay of antifungal isolates
Amylase, IAA, and biofilm production
Amylase detection
The bacteria were inoculated in the LB agar containing 1% starch (Biobasic, Canada). The starch agar plates were incubated at 37 °C for 2 days after which they were flooded with Lugol’s iodine solution (iodine 0.2 g, potassium iodide 0.4 g, distilled water 100 ml) for 2 min. The control experiment was also set up, using LB plates without the inducing substrate (starch). The plates were observed for a clear zone of hydrolyzed starch against a blue background of unhydrolyzed starch. The experiment was replicated thrice.
Detection of IAA
Indole acetic acid production was evaluated in LB agar media supplemented with 1% tryptophan (Shrivastava and Kumar 2011). The wells were made on the media plates by means of a sterile inverted 1-ml tip. Then, 200 μl of overnight grown culture was pipetted into each well and incubated at 30 °C overnight. After that, using tissue paper to remove the cultures gently from the wells was done and 200 μl of IAA reagent was added into the empty well. In the next 5–10 min, an appearance of the pink halo zone around wells means the IAA production and the halo zone was measured including the whole cavity.
Biofilm formation
A total of antifungal isolates was subjected to biofilm detection methods described by Christensen et al. (1995). A loopful of test organisms was inoculated in 10 ml of LB in test tubes. The tubes were incubated at 37 °C for 24 h. After incubation, tubes were decanted and washed with phosphate-buffered saline (pH 7.3) and dried. Tubes were then stained by crystal violet (0.1%). The excess stain was washed by deionized water. Tubes were dried in an inverted position. The scoring for the tube method was done according to the results of the control strains. Biofilm formation was considered positive when a visible film lined the wall and the bottom of the tube. The amount of biofilm formed was scored as 1—weak/none, 2—moderate, and 3—high/strong. The experiment was performed in triplicate and repeated three times.
Greenhouse experiment
Pitaya seeds were surface sterilized by 2% sodium hypochlorite for 2 min, rinsed thoroughly in sterile distilled water (SDW), and let dry on sterile discs of Whatman filter paper placed inside Petri dishes. Covering the pitaya seeds with the antagonistic bacterial suspensions (108 CFU ml−1) was carried out and then the bacterized seeds were incubated at 28 °C for 72 h before sowing. In parallel, a mixture that included equal parts of peat, sand, and field soil was prepared and autoclaved twice for 20 min at 120 °C with 24 h between autoclavings. Plastic pots (6 × 6 × 5.5 cm) were filled by a sterile substrate. The bacterized seeds were added 1 ml of bacterial suspensions before covered with soil. For negative control, seeds were treated with SDW instead of the bacterial suspension. Pots containing the treated seeds were placed in the greenhouse according to a completely randomized block experimental design. For each treatment, 15 pots each containing 3 seeds were used. All treatments were designed in completely randomized experiments and 15 replicates for each treatment. Plant growth parameters were recorded 45 days after sowing, using parameters as follows: percentage of seed germination, seedling height, and the fresh and dry weight of shoots. The experiment was repeated once.
Statistical analyses
Data analysis was done with significance (P < 0.05) of treatment effects using one-way ANOVA, followed by post hoc comparisons (Tukey’s HSD). Plant growth promoting activity of endophytic bacteria was determined by calculating the percentage increases in seed germination and plant growth parameters. The significance of the results was determined by Duncan’s tests.
