Isolation of endophytic bacteria from weed
Healthy E. indica plants were collected from different pitaya farmland of Nam Dinh province, Vietnam. The samples (stems, roots, and leaves) were immediately washed with clean water to remove dust and soil. The samples were then disinfected by successive treatment procedures including soaking in 75% alcohol for 30 s, rinsing once in sterile water, soaking for 5 min in 1% mercury chloride, and rinsing 3 times with sterile water. Aliquots of 0.1 ml of the last wash water were inoculated onto the plates containing Luria Broth (LB) media (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. The disinfected samples were cut into fragments (5 × 5 mm) and placed onto the LB plates to isolate bacteria living in the samples. The sterile distilled water was also inoculated on LB plates as controls. All the plates were incubated at 28 °C for 7 days and observed daily for the appearance of different types of bacterial colonies. The observed colonies were picked up and transferred to new LB plates several times by repeated streaking (quadrant streaking) till single type and isolated colonies were obtained. Obtained pure cultures were inoculated onto LB slants. After overnight incubation at 28 °C, they were stored at 4 °C till further use.
Identification of endophyte isolates
The modified cetyltrimethylammonium bromide (CTAB) method was used to extract the DNA of the endophytic bacteria isolated (Liu et al. 2019). Then, the extracted DNA was used as a template to amplify the target fragments by using polymerase chain reaction (PCR) amplification with the 16S rDNA universal primers (27F: 5′-CAGAGTTTGATCCTGGCT-3′, 1492R: 5′-AGGAGGTGATCCAGCCGCA-3′). The amplified PCR products were sequenced by Apical Scientific (1st BASE, Singapore). Obtained sequences were BLAST on the NCBI website and subsequently determining the possible species of the isolates. Closed BLAST results were downloaded to construct the phylogenetic tree using MEGA7.0 (Kumar et al. 2016).
Dual culture assays for antagonistic tests
Dual culture assays were used to test the antagonistic activity of isolated bacteria on A. alternata (Luu et al. 2021). For each repetition, a cylinder of a 1-cm2 fungal plug (A. alternata) was placed in the center of 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 and the bacterial isolates were inoculated between the fungal plug and the plate edge. The plates that grew only A. alternata were used as controls. Then, inoculated and control plates were incubated at 28 °C in the dark for 72 h. The antimicrobial activity was determined by measuring the diameter of the inhibition zone and subsequently was statistically analyzed. The diameter of the inhibition zone was measured by the longest line that was drawn from the plate center to the edge and also overlapping the endophyte growth area. Each endophyte/pathogen combination was done with triplicated and 2 independent experiments were repeated for each strain.
To identify the bacterial spectrum, the same method was used to detect the inhibitory effects of antagonistic endophyte on other common plant pathogens, including Fusarium moniliforme (a cotton pathogen), Sclerotium rolfsii (a rice pathogen), Alternaria alternata (a pear fruit pathogen), Curvularia lunata and Cladosporium perangustum (two pitaya pathogens), and Colletotrichum gloeosporioides (an apple fruit pathogen). Each treatment included 3 repetitions.
Antifungal activity of extracellular filtrates
The overnight culture of the isolated bacterium with antifungal activity was used and filtrated to obtain culture filtrate (CF) (Shafique et al. 2019). Then, biocontrol assays (0, 15, 30, 45, 60, and 75% dilutions of stock) were made. After that, a 5-mm plug of PDA with actively growing A. alternata was inoculated in a potato dextrose medium amended with CF at different concentrations. The inoculated and control media were incubated at 28 °C for 7 days and observed every 12 h. After that, the biomass of A. alternata was collected by filtrating it through filter paper and oven-dried at 40 °C overnight. The reduction of fungal biomass was calculated by using the following formula (Luu et al. 2021): Growth inhibition (%) = [(Growth in control – Growth in treatment)/Growth in control] × 100. The fungal growth inhibition was used as an indicator for the antifungal activity of filtrate.
Disease suppression on the twigs under laboratory conditions
Antagonistic bacteria were assessed for their efficacy in suppressing the A. alternata YZU under laboratory conditions (modified from Mohd et al. 2013). The bacterial antagonist was cultured in LB at 28 °C for 48 h. Two milliliters of this bacterial suspension (at 108 cfu ml−1) was pipetted onto a pitaya twig, wounded with a sterile needle, and placed in a closed plastic container with a layer of moistened filter paper at the bottom. The treatments were designed randomly with 3 replications including (1) the bacterial suspension was sprayed onto the wounded twig immediately before the PDA plug of A. alternata YZU was placed onto the wounded tissue, (2) the bacterial suspension was sprayed onto the wounded twig for 24 h before inoculation with a PDA plug of A. alternata YZU, (3) the PDA plug of A. alternata YZU was placed onto the wounded twig for 24 h before spraying the bacterial suspension, (4) only the bacterial suspension was sprayed onto the wounded twig, and (5) only the PDA plug of A. alternata YZU was placed onto the wounded twig. Symptom of stem end rot disease was observed after 15 days inoculating the wounded pitaya with A. alternata YZU and accessed by measuring the length of lesions produced.
Disease suppression on the twigs in the greenhouse
The efficacy of antagonistic bacteria against A. alternata YZU was also assessed in a greenhouse (Mohd et al. 2013). A similar procedure described above was applied, except some modifications including 10 replications were used under the greenhouse conditions.
Data analysis was done with significance (p < 0.05) of treatment effects using one-way ANOVA, followed by posthoc comparisons (Tukey’s HSD). The significance of the results was determined by Duncan’s tests.