Enumeration of endophytic bacterial population in leaves and pneumatophores of mangrove plant Avicennia officinalis L.
The endophytic bacterial population in leaves (3.5 × 105
cfu/g) and pneumatophores (4.2 × 105
cfu/g) of mangrove plant Avicennia officinalis
were isolated. Three isolates from leaves and eight isolates from pneumotaphores of Avicennia officinalis
were obtained and purified by Streak plate technique.
Among 11 isolates, which belongs to the genus Bacillus, Exiguobacterium, Salinicola, Pseudomonas, Enterobacter and Vibrio, Strain AOL2 (Aveicenia officinalis
Leaf strain-2) is of particular interest due to its salt tolerance at 10-15% NaCl and also showed distinct morphology and phenotypic characters and was selected for further studies (Fig 2 to 5) (Table 1).
Colony and cell morphology
Table 1: Coding of endophytic bacterial isolates.
The colonies of AOL2 strain on agar medium were medium, round in shape, yellow in colour, convex in elevation. The cells when viewed microscopically are single rods and motile. Gram’s staining was carried out and the cells were gram negative in appearance. When grow in nutrient broth cells showed aerobic in nature and are non-endospore forming.
Growth at saline conditions
AOL2 grows well at temperature 25-30°C with 3% NaCl nutrient agar, pH ranges 05-10 and salt tolerance up to 15% NaCl nutrient agar. Similar findings were presented by Ali et al. (2017)
in their study with Bacillus pumilus
AM11 and Exiguobacterium
AM25 showed significantly higher growth in saline media.
Biochemical characteristic of endophytic bacterial isolates
Biochemical studies were carried out by the isolate AOL2 and presented in Table 2. The results showed that the AOL2 isolate were KOH positive with stringy appearance. Catalase positive when 3% H2
was added and oxidase negative when para-aminodimethyl aniline oxalate solution were added. IMViC test showed Indole positive with red colour when Kovac’s reagent was added, Methyl red positive with the production of red colour, Voges-Proskeaur test negative with no colour and Citrate utilization positive with production of blue colour due to pH change in the medium. AOL2 showed positive result with Gelatin (0.5 cm) and Starch (0.8 cm) hydrolysis (Table 2).
Indole-3-acetic acid (IAA) production
Table 2: Phenotypic studies of endophytic bacteria isolates strain AOL2.
IAA production was screened for the presence of Indole compounds by colorimetric assay using Salkowski reagent. Strain AOL2 was cultured and inoculated in 3% Nutrient broth with 0.5% Tryptophan. After 48 hrs incubation the culture were subjected to centrifugation and the supernatant was mixed with 1.5 ml of Salkowski’s reagent. The colour intensities were measured by using UV Spectrophotometer at 536 nm. The OD values were compared with standard graph of IAA and 7.5 μg/ml of IAA production was recorded with strain AOL2 (Table 2). Similar results were obtained by Hardoim et al., (2015).
Phosphate solubilizing ability
AOL2 culture were inoculated in 4 ml of 3% pikovskaya’s broth in ria vials and kept for 48 hrs incubation as mentioned in material and methods. After incubation 750 µl of phosphate reagent to the 4ml of the sample and blue colour intensities were measured by using UV Spectrophotometer at 680 nm wavelength. 3.5 ppm was recorded with strain AOL2. Similar results were also reported Rylo et al. (2011)
in their study isolated Endophytic bacteria from the surface sterilized pneumatophores of Avicennia marina.
They identified isolates as GU930357 (Bacillus
sp.), GU930358 (Enterobacter
sp.) and GU930359 (S
sp., fixed nitrogen, S
produced siderophore and all the three strains solubilized phosphate molecule. Same findings were also obtained by El-Tarabily and Youssef, (2010)
conducted a study on mangrove Avicennia
marina rhizosphere identified 129 bacterial strains with the ability to solubilize rock phosphate, with Oceanobacillus picturae
able to mobilize 97% of this mineral. Richardson (2009)
in their study stated that phosphate solubilization by microorganisms has an important function in supplying phosphorus (P) to plants with the potential to be used as inoculants. Kim et al., (1997)
also reported that the ability of microorganisms to solubilize phosphate correlates with the ability to produce organic acids and/or extracellular polysaccharide.
Microbial identification using 16S rRNA gene based molecular method
Isolated DNA from the culture was evaluated on 1.0% agarose gel and single band of high molecular weight DNA was observed. Fragment of 16S rRNA gene was amplified by 16SrRNA-F-5'-TCTTCGGACTTCGCGCTATC-3' and 16SrRNA-F-5'-CAGACCAGCTACGGATCGTC-3' primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. A comparison using 16S rRNA gene sequences from the databases revealed that the 16S rRNA gene sequence of the type strain of AOL2 displayed high levels of similarity to those of Salinicola salarius.
The percentage of 16SrRNA sequence similarity between strain AOL2 and Salinicola salarius
was 99.19%. The phylogenetic tree reconstructed using the neighbour-joining algorithm showed that the strain AOL2 and Salinicola salarius
clustered together and constituted a separate group from the other closely related species (Fig 6).
Fig 6: Phylogenetic tree based on nucleotide sequence of the 16S rRNA sequence of AOL2 and other related type strains.