Isolation of Cellulolytic Bacillus sp . from the Viviparous Seedling Hypocotyls of Red Mangrove – Rhizophora mucronata Lam.

Mangroves are one of the world’s most unique tropical coastal ecosystems. They are a rich repository of biological wealth, including specially adapted flora and fauna. The microbiome component of this ecosystem is a fascinating world that is yet to be fully explored for its functional and ecological inter-relationships with its hosts. The mangrove ecosystem is a hidden treasure of microbial diversity, without which mangrove biology is incomplete. In the present study, the isolation of a cellulase-producing, endophytic Bacillus sp. from the hypocotyl region of viviparous seedlings is described. This study urges us to look into the microbial diversity of mangrove propagules, by presenting a glimpse of a member of the endospheric microbiome of viviparous hypocotyls.


INTRODUCTION
][9][10][11][12][13][14] Microbiome component, especially endophytes of 'mangrove holobiont', 15,16 is one such area that needs further investigations to unveil the functional relationship between endophytes and their host. 17- 224][25][26] In the case of mangroves, the interactions between the microbes and its mangrove host, forming a single dynamic entity can be apprehended as 'mangrove holobiont'.][34][35][36][37] Rhizophora mucronata Lam. is a mangrove tree species, belonging to the family Rhizophoraceae.9][40][41] Vivipary is an important developmental phenomenon seen associated with the mangrove tree species. 39,40ere, the seeds germinate while still attached to the mother plant and later, seedlings detach and drop into the muddy environment where it grows further into an adult plant, overcoming extreme pH and salinity. 40,42 xploration of the microbiome of these seedlings will help us to identify and understand the role of microorganisms in the establishment and growth of mangroves, and further, it will shed light on mangrove biology and its survival.In the present study, attached viviparous seedling hypocotyls of Rhizophora mucronata are explored for the presence of culturable endophytes.

Isolation of pure culture
The samples were cleaned thoroughly using 5% teepol solution, followed by surface sterilisation using 70% ethanol for 1 min and treatment with 5% sodium hypochlorite for 3 min. 43,44After surface sterilisation, the samples were washed three times using sterile distilled water and air-dried.Afterwards, the seedlings were cut into small pieces of 1-2 cm in length.The samples were split open longitudinally and the outer green-coloured epidermal portion was removed.Inner 'core' parts were placed on Luria-Bertani agar medium (Titan Biotech, Kerala, India) and incubated for 1 to 2 days at room temperature along with a control plate with sterile water.

Biochemical characterisation of endophytes
The following tests were carried out to characterise the biochemical properties of the bacterial isolates.

a) Test for Indole acetic acid (IAA) production
The bacterial isolates were cultured in Luria-Bertani broth containing L-tryptophan (1 µg/ ml) for 24 h. 45,46Culture supernatant was collected by centrifugation (8000 rpm; 10 min) and 1 ml of it was taken and mixed with freshly prepared Salkowsky reagent (2 ml).The reaction mixture was incubated at 28°C for 25-30 min., and colour change noticed.

b) Cellulase activity
Fresh cultures of the bacterial isolates were spot inoculated on nutrient agar containing carboxy methyl cellulose (0.2%) and incubated at 30°C for 3-4 days. 47After the incubation, the culture plates were flooded with Congo-red Freshly grown bacteria was inoculated on nutrient gelatin medium and incubated for 3 days at 30°C along with control tubes.These tubes were then placed in ice to test the gelatinase activity. 49,50

g) Phosphate solubilisation
To detect phosphate solubilisation activity, freshly grown bacterial cultures were spot inoculated on Pikovskaya agar medium, and incubated at 30°C for 1-8 days.The amplified products were analysed on 0.8% EtBr-Agarose gel.

b) Sequencing of PCR product
The PCR product were purified and subjected to direct sequencing in an automated sequencing machine (ABI prism, Applied biosystems, CA,USA).Forward primer given above was used for the sequencing purpose.

c) BLAST analysis of sequences
Good quality sequences were subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov).The sequences were searched against nucleotide database of NCBI to identify the bacterial strain.

d) Phylogenetic analysis
Phylogenetic analysis of the current bacterial isolate were carried out based on the 16SrRNA sequences using the software MEGA version 11. 52 Neighbour-Joining Method was used to generate the phylogenetic tree (cladogram).

Isolation and characterization of endophytes from plant tissues
Out of the several bacterial colonies obtained, the bacterial colony which is found fast growing and emerging directly out of the tissues was selected and named as VpR.It was found to have rod-shaped morphology when observed under a microscope.The culture was positive for Gram's stain reaction and also for biochemical tests like cellulase, catalase and ammonia production.The culture did not show any prominent hydrolytic activity for starch or gelatin.Tests for IAA production and Phosphate solubilisation were also negative.A summary of morphological and biochemical characterization is shown in Table .Figure 1 shows the results of the biochemical test for cellulase activity.

16S rRNA amplicon sequence analysis of bacterial isolates
Molecular analysis of bacterial isolates was carried out based on the 16S rRNA region.16S rRNA region of the bacterial culture samples was amplified and separated on 0.8% EtBr-Agarose gel.Sequencing of the purified PCR products was carried out in ABI prism automated sequencer.The 16S rRNA sequence showed maximum homology to that of Bacillus subtilis.The sequence obtained was deposited in GenBank: MT968034 (VpR).Result of the phylogenetic analysis is shown as a bootstrap consensus tree in Figure 2. The evolutionary relatedness of the present bacterial isolate with other bacterial groups can be inferred from this cladogram.

DISCUSSION
The microbiome of mangroves is an underexplored ecological as well as functional component of 'mangrove holobiont'. 15Several studies have highlighted the importance of microbial associates of mangrove plants which includes both plant protection and plant growth promotion. 53,54Isolation of salt-tolerant microbes from the mangroves is an example to apprehend the role microbes in mangrove ecosystem which is a unique ecosystem characterized by salinity. 8They can facilitate the uptake of nutrients like Phosphorus, Nitrogen, etc. which is very much crucial for the plant growth.Many of the resident microbes are capable of producing growth promoting phytohormones like IAA (indole acetic acid) besides the production of hydrolytic enzymes like cellulases.They can also 'prime' the plant defense mechanism. 55][58][59][60] In the present study, isolation and identification of Bacillus subtilis from the core tissues of viviparous seedling hypocotyls of Rhizophora mucronata is described.The occurrence of Bacillus subtilis in the endosphere of the undetached viviparous seedling hypocotyl of R. mucronata points to the various possibilities that this endophyte could have in the lifecycle of its host.This particular bacterial culture showed cellulase, catalase and ammonia-producing activities.Ammonia production by endophytic bacteria is a feature that is generally considered to be plant growth promotion in nature, 61 but, contrary views and opinions are also presented by researchers. 62,63From an ecological point of view, the feature of ammonia production by bacteria and its relevance to mangrove biology will be an interesting study to follow.Microbial cellulase activity is considered to contribute significantly to the carbon cycle at the ecosystem level. 64,65atalase activity is generally considered as a cellular mechanism to mitigate abiotic stress, but the endophytic catalase activity observed here could be an attribute of the microbe to adapt itself to the endophytic lifestyle. 66 In most cases, endophytes are known to impart plant-beneficial attributes.They are found to reduce the severity of stress encountered by the host plant. 30-32, 61,67,680][71] Endophyte-mediated plant resistance has emerged as a successful alternative agriculture strategy in many instances.][25][26] Endophytes of these salt-tolerant trees are important partners of the 'holobiont' without which the mangrove biology will be incomplete.Besides, the microbiome component of the mangrove ecosystem is a promising resource for novel microbes with hitherto unknown attributes or features with biotechnological and agricultural application potential.

51
Molecular analysis of 16S rRNA region of bacterial isolates a) Amplification of 16S rRNA F o r w a r d p r i m e r 5 ' -C C G A AT T C G TCGACAACAGAGTTTGACCCTGGTTCAG-3'; andReverse primer 5'-CCCGGGATCCAAGCTTAC GGCTACCTTGTTACGACTT-3' was used to amplify the 16S rRNA gene from the microbe under the following PCR conditions: 98°C -2 min; 30 cycles of 98°C -30 sec; 55°C -30 sec; 72°C -1 min and Final extension, 72°C -10 min.

Figure 1 .
Figure 1.Cellulase activity by endophytic isolates from Rhizophora mucronata Lam.a) Control plate (E.coli), b) VpR.The 'clear zone' seen around the colony is due to the cellulolytic activity of bacteria

Figure 2 .
Figure 2. Phylogenetic analysis of Bacterial isolate VpR -Bacillus subtilis (MT968034.1).Bootstrap consensus phylogenetic tree is shown here.The sequences were aligned and analysed using MEGA Software (version 5.0.5).The branching pattern of the tree is derived by Neighbour-Joining method