Sample collection and cultivation
Seeds of S. chirata and D. stramonium were collected from Gossaigaon, Kokrajhar (26.4371° N, 89.9767° E), and Bijoynagar, Jorhat (26.7509° N, 94.2037° E) of Assam, respectively. Subsequently, they were grown in the experimental field for further study.
Isolation of endophytes
Bacterial endophytes were isolated at flowering stages of both the plant species. After proper surface sterilization as described by Devi et al. (2017), the shoots (5 g) and roots (1 g) of the both the plant samples were weighed and grinded using mortar and pestle, separately. The extracts were then centrifuged at 10,000 rpm for 10–20 min. The aliquots were then spread over nutrient agar plate amended with fungicide, cycloheximide. Nutrient agar plates prepared were incubated at 37 °C for 24–48 h. Colonies with different morpho-types were selected and purified for further study.
Mass multiplication of endophytic isolates
Pure cultures of endophytic bacteria were fermented at shake-flask level in 1 L Erlenmeyer flasks that contained 600 ml nutrient broth. Bacterial culture for each bacterial isolate was grown at 28 °C and 200 rpm in a mechanical shaker incubator, and growth curve was drawn to know the best time of harvest for secondary metabolites. The point just after entering the stationary phase was considered the best harvest time.
Molecular analysis of endophytic bacterial isolates
Genomic DNA isolation was done by using the modified method of Ligozzi and Fontana (2003). Amplification of 16S rRNA gene was performed using the 27 F-AGAGTTTGATCCTGGCTCAG forward and 1492 R-GGTTACCTTGTTACGACTT reverse primers. The PCR was carried out with an initial denaturation at 94 °C for 2 min, 30 cycles of 94 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min, and final extension at 72 °C for 10 min. The amplified products of the isolates were sequenced using 454 automated Pyrosequencer (Agrigenome, Kerala, India). The sequenced 16S rRNA genes from both the ends were further pre-processed and assembled to a consensus sequence using Codon Code Aligner version 4.1(Codon Code Corporation, USA).
Phylogenetic analysis of 16S rRNA
Phylogenetic analysis of 16S rRNA gene sequences for each bacterial strain along with their closest relatives was performed using the Kimura 2-parameters method incorporated in MEGA version 5.1.0 by NJ method to assign each bacterial isolate in their respective taxonomic position (Tamura et al. 2007). The 16S rRNA gene sequences of the 9 different strains were submitted to GenBank of NCBI through sequence submission tool, Bank. It was where they were assigned GenBank accession numbers. In addition, the strains identified were taken together for construction of phylogenetic tree using NJ method incorporated in MEGA.
Culture of plant pathogenic bacteria and fungi
Two bacterial plant pathogenic species, Xanthomonas oryzae and Ralstonia solanacearum, and two plant pathogenic fungal species, Fusarium oxysporum and F. solani, were collected from the Plant Pathology Department of Assam Agricultural University, Jorhat, Assam.
Screening of bacterial endophytes for their antimicrobial activities
The isolated bacterial endophytes were screened for their antimicrobial activity using agar well diffusion method with few modifications (Schillinger and Lücke, 1989) against plant pathogenic bacteria and with modified dual culture method (Gkarmiri et al. 2015) against fungal plant pathogens. For antimicrobial assay, 3 replications were taken and mean value was reported. The bacterial phytopathogens used were X. oryzae and R. solanacearum, and the fungal phytopathogens were F. oxysporum and F. solani. The bacterial endophyte isolates were grown in NA broth for 48 h, at 28 °C and 200 rpm in a mechanical shaker incubator, and then centrifuged at 10,000 rpm for 10 min. The supernatant obtained was filtered through micro-filter (0.22 μm pore size). The filtrate obtained was used for the determination of antimicrobial activity. To assay antibacterial activity, 500 μl of active broth cultures (overnight grown) of phytopathogenic bacteria, X. oryzae, and R. solanacearum were transferred to the NA agar Petri plates, separately and spread uniformly with a glass spreader. Four wells with 7-mm diameter were made in each of these plates using sterile gel puncher. Out of the 4, 2 wells were used for test samples, one was used as a positive control, and the other was used as a negative control. An aliquot of 200 μl of the sample was loaded into the wells of the plates. In the case of positive control, instead of the sample, 200 μl streptomycin was used (50 μg/ml), and in the negative control, the same amount of sterile water was used. All the plates were then kept in incubation at 30 ± 2 °C. After 24 h of incubation, plates were observed for formation of clear zone of inhibition around the well and measured. To evaluate antifungal activity, a loop-full of active fungal culture was placed on one edge of the sterile plates containing PDA media. One well with 7-mm diameter was made in the center of the plate using sterile gel puncher. An aliquot of 200 μl of the sample was loaded into the well of the plate. In place of test sample, 200 μl cycloheximide (50 μg/ml) was used in the positive control and the same amount of sterile water was used in the negative control. Plates were then incubated at 30 ± 2 °C and observed for formation of zones of inhibition every 24 h for 6 days.