Isolation and Characterisation of Endophytic Bacteria from Holostemma ada-kodien Schult

Plants with medical properties are often enriched with endophytes that have the potential to produce important bioactive compounds. Endophytes after entering the plant tissue may either colonize a particular tissue or may spread throughout the host plant without causing damage. The possession of pharmaceutical and biological properties has made the Holostemma ada-kodien Schult as one of the widely used plants of medicinal importance in India. Following the direct cut method three endophytic bacterial strains (UC H1, UC H4 and UC H7) were isolated, identified and characterized from the healthy looking rhizome of H. ada-kodien. Among these isolates, UC H1 and UC H4 were found to have many properties like antibacterial compounds, hydrolytic enzymes and plant growth promoting traits. The isolate UC H4 have ability for Indole-3-Acetic Acid (IAA) production of 513.54 U/ml and very good protease and pectinase activities of 20.65 U/ml and 16.09 U/ml respectively. So far no reports are available on the endophytic microflora of H. ada-kodien.

dipped in diluted ethanol (70%) for a period of 1 min, which was then subjected to 5 minutes treatment with 1% of sodium hypochlorite. This followed by subsequent 1 minute treatments and again treated with 70% ethanol. Then the samples were washed several times and the last treated wash was spread on the nutrient agar (NA) plates as the control for confirming that the isolates are surely endophytes Sterilized filter paper assisted the drying of samples which was cut into small pieces of about 1 cm 3 . The surface of each segments were removed (Islam et al. 2010) and then placed on NA plate and were incubated for 48 hrs at 30°C. The morphologically distinct bacterial colonies were isolated, then sub-cultured for further purification and used for the further studies. The lack of bacterial colonies on the control plates confirmed that the isolated bacteria were endophytes and the surface sterilization programme was effective in removing the contaminants.

Identification of the isolates by morphological characterization and 16S rrnA gene sequencing
The macro-morphological evaluation of bacterial colonies and micro-morphological characterization of bacterial cell was done as per standard protocols in bacteriology.
The identification of isolates was further clarified by using the genetic information of 16S rRNA gene. The forward and reverse primers used were 16S-RS-F (5'CAGGCCTAACACATGCAAGTC3') and 16S-RS-R (5'GGGCGGWGTGTACAAGGC3') respectively. The amplification studies were carried out using Applied Biosystems PCR-GeneAmp System 9700, The amplified DNA was checked by electrophoresis using agarose which was then subjected to purification and for sequencing. The sequence quality was checked using Sequence Scanner Software v1 then checked by BLAST analysis to construct phylogenetic tree and viewed in software Phylodraw.

Study for anti-bacterial activity
The anti-bacterial activity was determined using the paper disc susceptibility test. Isolated endophytic bacterial strains were grown in 100ml nutrient broth (peptone: 0.5g; yeast extract: 0.5g and NaCl: 0.5g in 100ml distilled water) and incubated for five days in rotary shaker at 120rpm. The culture media were centrifuged at 10,000rpm for 15 minutes. The supernatant of probiotic isolates were monitored for antibacterial activity against human pathogenic bacteria inoculated on nutrient agar. A total of 20μl of cellfree supernatant was applied on 6mm diameter cellulosic disc on to the lawn of 200µl of indicator bacteria swabbed on nutrient agar along with Amoxicillin as the standard antibiotic and control as nutrient broth. Six bacteria such as Vibrio cholera, Salmonella typhi, Serratia marcescens, Escherichia coli, Bacillus subtilis and Arthrobacter nicotianae were used as indicator organisms. They are cultured in 10ml nutrient broth and incubated for 18hrs at 37°C. The diameter of the inhibition clear zone was measured after 48 hrs of incubation at 30°C.

Study for enzyme production Primary screening
Primary screening was effected with detection of metabolites like enzymes that have been diffused throughout the agar. The bacteria were screened for cellulase, xylanase, pectinase, protease, lipase, and amylase. The bacterial isolates were cultured on different indicator media for each enzyme study. After the inoculation, the culture was kept in incubator at 30°C for 48 hrs.

Enzyme assay
The enzyme activities were carried out by initially growing the isolates in specific medium for 24 hrs at 120 rpm. After that, 5ml pre-inoculum of each bacteria was transferred to 95ml culture medium with similar composition to pre-inoculum and incubated at 120 rpm for 96 hrs. Samples were collected at 24, 48, 72, 96 hrs and centrifuged at 10000 rpm at 4°C for 20 mins. The cell free supernatant served as crude enzyme. In each enzyme assay, specific substrates were used such as such as casein for protease activity; starch for amylase; CMC for cellulase; pectin for pectinase; p-nitrophenol palmitate (p-NPP) for lipase and xylan for xylanase. Twenty four hour old cultures were transferred to centrifuge tubes and centrifuged at 10000 rpm for 20 min. Cells were discarded and resultant supernatant was used as the crude enzyme for various enzyme assays.

Protease Assay
Protease assay was conducted using casein as the substrate. The tyrosine liberated by the action of enzyme was estimated using Folins reagent at 660 nm. One unit enzyme activity is defined as the amount of enzyme that releases 1μg of tyrosine per ml per minute under the assay conditions.

Amylase Assay
Amylase assay was conducted using starch as the substrate and the reducing sugar liberated was estimated using DNS reagent. The absorbance was measured at 575 nm. Enzyme activity was expressed in units (1 unit/ml = amount of enzyme which releases 1 μ mole glucose under the assay condition.

Cellulase Assay (CMCase assay)
CMCase assay was conducted by using CMC as substrate. Enzyme was added to the substrate and the reaction was proceeded for 10 minutes. Dinitro salicylic acid reagent was used for quantifying the reducing sugar at 540nm.

Pectinase assay
Pectinase assay was conducted using pectin, the substrate pectin was prepared by dissolving 1gm pectin in 100 ml 0.2M phosphate buffer. After the reaction for specified time the end products were measured at 540 nm. Enzyme activity expressed as μmol glucose released per min-1ml-1 of culture filtrate as enzyme solution.

Lipase Assay
Lipase assay was conducted using p-NPP. The absorbance of the products was measured at 410nm. One unit of enzyme activity is defined as the amount of enzyme that released 1μmol p-nitrophenol per minute from the substrate. p-nitrophenol was used as the standard.

Xylanase Assay
Xylanase assay was conducted using oat spelts xylan. The concentration of reducing sugars released was estimated against xylose standard by noting the absorbance at 540nm. 1 unit of xylanase activity was defined as moles of xylose liberated per minute per ml of enzyme preparation.

Study for the screening of plant growth augmenting traits Screening for Indole 3-acetic acid
Nutrient broth (20 ml) containing L-tryptophan was used for screening bacterial isolates. The bacteria were allowed to grow for period of 10 days and inoculated cultures were subjected to centrifugation at 3000 rpm for 20 mins (Rahman et al. 2010). Supernatant was collected and used for the further analysis. A mixture of 1 ml culture supernatant and 2 ml of Salkowski solution was incubated for 20 minutes in the absence of light. After incubation, the presence of pale red-dark red colour indicated IAA production. The un-inoculated medium served as the control. The mixture was read in spectrophotometer at absorbance of 530 nm. Indole acetic acid was estimated using pure IAA as standard and the values were denoted as mg/ml. In order to confirm the production of IAA positively answered microbes were again subjected to growth in NB supplemented with L-tryptophan. The pH value of this NB preparation was then adjusted to 2.5 using dilute HCl (1N) which was then extracted with ethyl acetate. The extract was vacuum dried and the powder mixed with one ml methanol and subjected to RP-HPLC. The elution was monitored at 280 nm.

Screening for phosphate solubilization
The inorganic phosphate solubilization studies were conducted using the method developed by Jasim et al. (2013). The bacterial cultures were streaked onto modified Pikovaskaya's nutrient medium consisting of inorganic phosphate, which is then kept at 30°C for 96-168 hours. A yellow halo like zone of clearance around the bacterial colony indicated mobilisation of mineral phosphate. The solubilization index for phosphate mobilization was calculated by Solubilization index (SI) = (Diameter of Colony + Diameter of halo zone) / Diameter of Colony Screening for ACC deaminase production Method of Jasim et al. (2013) was followed for screening the production of ACC deaminase. The microbial cultures were streaked onto Dworkin and Foster salt medium supplemented with 0.2% w/v ammonium sulphate and the petriplates were incubated for a period of 2-3 days at 30°C. The growth bacterial colony indicated production of ACC deaminase.

rESULTS And dISCUSSIon
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.
T h e m a c ro a n d m i c ro m o r p h o l o g i c a l characterization was used for the identification of isolates (

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 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.

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).
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

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

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  +  +  -2  Amylase  ---3  Cellulase  +  +  +  4  Pectinase  +  +  +  5  Lipase  ---6 Xylanase + + + 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 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

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

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).

ConCLUSIon
H. ada-kodien is a very important medicinal plant highly exploited in Indian system of medicine. The rhizome is enriched with terpenoid, lupeol, amyrin, sitosterol, alanine, aspartic acid, glycine, threonine, serine and valine. This is the first report regarding the isolation of endophytic bacteria from Holostemma ada-kodien. Three distinct endophytic bacteria that were isolated from the rhizome of H. ada-kodien Schult, designated as UC H1, UC H4 and UC H7. After the morphological, biochemical and molecular characterization, UC H1 was identified as Bacillus pumilus, UC H4 as Micrococcus luteus and UC H7 as B. pseudomycoides. M. luteus (UC H4) have potential to produce antibacterial compounds, hydrolytic enzymes such as protease, cellulase, pectinase and xylanase as well as IAA. B. pumilus (UC H1) have the ability to produce antibacterial compounds, protease, pectinase, xylanase, ability for phosphate solubilization and ACC deaminase production. B. pseudomycoides (UC H7) have potential to produce antibacterial compounds. From the results it can be concluded that M. luteus (UC H4) and B. pumilus (UC H1) are the