Newly isolated endophytic bacteria from the healthy looking rhizome of H. ada-kodien were designated as UC H1, UC H4 and UC H7. There was no growth of microorganisms on the control plate. So, it clearly confirmed that all the isolates are endophytes and also all the epiphytic microorganisms were removed at the surface sterilization steps. The endophytes obtained from the isolation were considered for further analyses.

The macro and micro morphological characterization was used for the identification of isolates (Table-1, 2). The isolates UC H1 and H7 and showed positive results for Indole Production test, Starch hydrolysis, Catalase, Voges-Proskauer’s test, Methyl Red test, Nitrate Reduction and Citrate utilisation and negative for Urease test. UC H4 answered positive for Catalase and negative for oxidase test, TSI test (Tripple sugar Iron agar) and Simmons citrate agar test.

Table (1):
Colony characteristics of endophytes isolated from H. ada-kodien.

Table (2):
Micromorphological characteristics of endophytes from H. ada-kodien.

Molecular characterizations of three isolates
The identification and characterisation of selected isolates were done at the molecular level by amplifying the 16S rRNA gene from each isolate using suitable primers which was then subjected to partial sequencing. The gene sequence of UC H1, UC H4 and UC H7 were 1249 bp, 947 bp and 1243 bp long respectively. The DNA sequence analysis of 16S rRNA gene sequences of UC H1, UC H4 and UC H7 in BLAST showed its maximum similarity of 100% to Bacillus pumilus, Micrococcus luteus and Bacillus pseudomycoides respectively. The tree output was viewed in the tree viewing software Phylodraw (Fig. 1, 2, 3). These bacterial isolates were considered as the endophytes from the rhizome of H. ada-kodien.

Bacillus pumilus INR7, previously isolated as an endophyte from heavily infested cucumber plant has been commercialized as a biological control product against both soil borne pathogens and foliar pathogens. They act by the means of direct antagonism along with the triggering of systemic resistance (Jeong et al. 2014). Symbiotic influence of endophytic B. pumilus in Ocimum sanctum was noted by Murugappan et al. (2013). The survival of B. pumilus as an endophyte in alcohol treated in vitro cultured grapes has been observed (Thomas 2004). The endophytic bacterium Micrococcus luteus has been isolated from the leaves of Phaseolus vulgaris by Costa et al. (2012). M. luteus strain K39 occurring as endophytes in the roots of Cyperus conglomeratus was obtained from the Red Sea shore of the Kingdom of Saudi Arabia. This strain is of common occurrence as an endophytic flora of rice plant (Mano and Moriskai 2008). Micrococcus luteus (NR_075062) was recently isolated from the leaf and stem of Panax ginseng by Hong et al. (2018). B. pseudomycoides was isolated from yam plants (Souza et al. 2017).

Antibacterial activity
Preliminary antibacterial activity of three endophytic bacteria estimated based on the clear zone production reveals that supernatants obtained from all the isolates against the tested bacteria was found to be negative except in the case of Serratia marcescens and Arthrobacter nicotianae. The presence of antibacterial activity of probiotic bacteria is shown in Table-3. The presence of zone of inhibition clearly revealed that Micrococcus luteus and Bacillus pseudomycoides exhibited maximum inhibition against gram positive bacteria A. nicotianae (12mm).

Table (3):
Antibacterial activity of three endophytic bacterial isolates.

Information on the antibacterial activity of these two strains against the tested organisms is not reported. So the far related works are used for the specification. Sadrati et al. (2013) reported that all of the selected fungal isolates have potential to produce antimicrobial compounds against Serratia marcescens with inhibition zone (IZ) value in each isolates ranging from 10.7 mm to 14.3 mm. Another report regarding the isolation of endophytic fungi with antibacterial activity from Calotropis procera showed maximum IZ (17.3 mm) with respect to Aspergillus nomius, A. oryzae, Fusarium solani and Curvularia hawaiiensis against S. marcescens (Rani et al. 2017). Reports on endophytic bacteria showing negative results in the antibacterial compound production are many. The bacterial endophyte, B. pumilus and M. luteus isolated from the leaf of Plectranthus tenuiflorus have no potential for producing antibacterial compounds against Salmonellae typhi, Staphylococcus aureus, E. coli, Klebsiella pneumonia and Streptococcus agalactiae (El-Deeb et al. 2013).

Enzyme production
Primary screening
The newly isolated endophytic bacteria were screened for the ability to produce of enzymes like, protease, cellulase, xylanase, pectinase, lipase and amylase, (Table-4). In protease screening, only B. pumilus and M. luteus showed the clear zone of paracasein formation around the spotted isolates. All the three endophytic bacterial strains showed positive result in the screening test for the synthesis of cellulase, xylanase and pectinase and negative result for amylase and lipase. Genera like Bacillus, Paenibacillus and Pseudomonas are highly recognized as the members of protease producers. The endophytic bacteria B. pumilus from Plectranthus tenuiflorus have potential to produce protease, pectinase, xylanase, cellulase and M. luteus from the same plant could produce protease, xylanase and cellulase (El-Deeb et al. 2013). Previous studies suggested that Bacillus sps. are the most important genera for the production of xylanase (Irfan et al. 2016).

Table (4):
Primary screening of three endophytic bacterial isolates.

Enzyme assay
In the present study two strains showed protease production that is B. pumilus and M. luteus (Fig. 4). M. luteus showed the highest activity of 20.65 U/ml after 48 hrs that declined to 1.35 U/ml in the next 24 hrs. B. pumilus also showed the maximum activity of 6.52 U/ml after 48 hrs. Endophytic Bacillus strain from Mangifera indica showed a mild protease activity after 24 hrs i.e. 0.621±0.025; strain from Hibiscus rosa-sinensis had activity that increased with time (0.223±0.014 at 24 hrs; 0.474±0.057 at 48 hrs and 0.576±0.003 at 72 hrs) and an endophytic bacterial strain from Calotropis gigantea with increased activity at 48 hrs that declined at 72 hrs (0.093±0.006 at 24 hrs; 0.577±0.010 at 48 hrs and 0.315±0.013 at 72hrs) was reported by Vijayalakshmi et al. (2016). The growth associated protease production was studied by Chaudhuri et al. (2017) in Bacillus amyloliquefaciens UEF01, an endophyte in the carnivorous plant Utricularia exoleta. Regarding cellulase production (Fig. 5), M. luteus showed the highest activity at 24 hrs and 48 hrs than all other strains i.e. 13.02 and 13.33 U/ml respectively. Ray et al. (2017) observed maximum cellulase yield when the growth of the microorganisms was tested at 40°C, and minimum yield was obtained at 45° C by B. subtilis and B. circulans. The optimum medium conditions for cellulase production by Bacillus subtilis AS3 resulted in comparatively low level of activity with a value of 0.43 U/ml (Deka et al. 2011).

The isolate, UC H7 that was positive in the plate assay for pectinase production did not show considerable activity during the quantitative assay. Pectinase activity of the isolated bacterial strains was comparably negligible. Only B. pumilus and M. luteus showed activity at 72 hrs and 48 hrs respectively (Fig. 6). Paudel et al. (2015) reported that Bacillus sp. HD2 showed maximal PGase activity at 72 hrs. According to Kavuthodi et al. (2015), the required period of incubation time for highest pectinase activity of the isolated B. subtilis was in 72 hrs. These reports are supporting the present work. The enzyme production increased in 60 hrs (1.278 U/ml) and reached the maximum at 72 hrs (1.288 U/ml). El-Deeb et al. (2011) reported the production of pectinase activity by Micrococcus sp., Acinetobacter sp., Planococcus sp., Acetobacter sp. (TUB6) and Methylcoccus sp. (TUB4) isolated from the rose plant. The endophytes in Lonicera japonica such as Bacillus and Paenibacillus, exhibited detectable levels of cellulase and pectinase activities (Zhao et al. 2015). The maximum xylanase activity was observed in B. pumilus at 24 hrs (33.22 U/ml). At 48 hrs the activity became reduced which again got enhanced at 96 hrs (Fig. 7). Archana and Satyanarayana (1997) reported that several species of Bacillus and fungi secrete high amount of extracellular xylanase.

Plant growth promotion traits
Production of Indole 3-acetic acid (IAA)
During the primary screening study, IAA production was observed in M. luteus due to the development of red colour in the screening medium on the addition of Salkowski reagent (Fig. 8). The concentration of IAA was calculated by noticing the absorbance at 530nm. It was noticed that maximum IAA production was exhibited by M. luteus (513.54 U/ml). B. subtilis LK14 resulted in considerable level of IAA production with a value of 165.53 µM in 50 ml culture supernatant at the same time B. subtilis LK15 was less efficient with a production value of 32.01 µM in 50 ml culture supernatant (Khan et al. 2016). According to Raddadi (2008) 1.53-9.71 µg/ml IAA was produced by B. thuringiensis. In the present study, confirmation by RP-HPLC analysis ensured the potential of UC H4 to produce IAA into the medium (Fig. 9). El-Deeb et al. 2011 reported that six endophytic bacterial isolates from Rosa damascena trigintipeta had the capacity to produce IAA when the culture medium was supplemented with L-tryptophan, strains TUB3 and TUB5 showed a higher production of IAA (18.6±1.6 and 38.8±3.6 mg IAA/mL, respectively) than TUB1, TUB2, TUB4, and TUB6 (10.2±2.2, 16.6±1.8, 7.8±0.8 mg IAA/mL, respectively). Ribeiro et al. (2018) reported that the bacterial strains isolated from sap, roots and leaves of maize plant effected IAA production with an yield ranging from 3.2 mg/l (Bacillus subtilis) to 61.6 mg/l (Bacillus pumilus ). Similar results were observed in bacteria like Bacillus and Lactobacillus with an IAA production range of 30 to 60 gm/l (Mohite 2013) and 0.81 and 86.82 gm/l (Wahyudi et al. 2011).

Phosphate solubilization
B. pumilus was positive for phosphate solubilization (PS) assay in solid medium (Pikovaskaya’s medium) where in tricalcium phosphate was the phosphate source. Phosphate solubilization capacity was evidenced by the presence of yellow hallow zone of 25mm around the colony after the incubation period (Fig.10). The solubilization index (SI) of B. pumilus was 3.78. Matos et al. 2017 isolated forty endophytic bacteria from banana tree root, out of them Bacillus pumilus EB.102, B. pumilus EB.12, Bacillus sp. EB. 42, Bacillus sp. EB.17, Bacillus sp. EB.81 and B. pumilus EB.05 resulted in the higher levels of solubilized phosphate. B. megaterium and Pseudomonas fluorescens strains showed high phosphorous solubilizing ability (El-komy 2005). The phosphate solubilizing bacteria (PSB) KUPSB12 resulted in phosphate solubilizing halo zone around its colony of 13.0 mm with SI of 2.85 (Paul and Sinha 2016).

Production of ACC deaminase
Among the endophytic bacteria under investigation Bacillus pumilus was positive for ACC deaminase production test in solid medium (Dworkin and Foster salt medium) as shown in Fig.11. Previous reports revealed that various taxa of endophytes especially Bacillus (Xu et al. 2013), Burkholderia (Ali et al. 2014) and Pseudomonas (Qin et al. 2014) have the potential to produce ACC deaminase. Xu et al. (2013) reported that 6% of bacterial isolates from Lycopercium esculentum exhibited an ACC deaminase activity with prominent production by B. subtilis. Thus Bacillus found to be one of the most significant ACC deaminase producing bacteria (Khan et al. 2016).