International Journal of Innovations in Engineering and Technology (IJIET) http://dx.doi.org/10.21172/ijiet.141.07 Isolation and identification of endophytic bacteria associated with Bruguiera sexagula and Ceriops decandra in mangrove forest of Soc Trang province, Vietnam Ho Thanh Tam1, Tran Vu Phuong2, Cao Ngoc Diep3 1Can Tho College, Can Tho City, Vietnam 2,3Biotechnology R&D Institute, Can Tho University, Can Tho City, Vietnam Abstract – Total of 68 bacterial isolates inside the roots of Bruguiera and Ceriops in mangrove forest of Soc Trang province, Vietnam. They were isolated from 16 root samples on the two kinds of Burk’ N-free and NBRIP semi-solid medium with the presence of pellice located below the surface of medium 1-2 mm as indicator. Almost their colonies have round-shaped; milky, white clear (on Burk’s medium) and yellow (on NBRIP medium); entire or lobate margin; diameter size of these colonies varied from 0.2 to 3.0 mm and all of them are Gram-negative by Gram stain, motility. 38/68 isolates grew on NBRIP medium and 30/68 isolates grew on Burk’s N-free medium and all isolates did not produce siderophore. From 68 isolates, screening of 6 isolates had high nitrogen fixation ability, 8 isolates insolved high phosphate and 9 isolates biosynthezed high IAA concentration in NBRIP tryptophan-free liquid medium; in Burk’s N-free without tryptophan medium, selecting of 11 isolates had high phosphate solubilization ability and 8 isolates biosynthezed high IAA concentration. Nine good isolates were selected to identify, sequencing and compared with bacterial 16s-rRNA genes in Genbank using BLAST N software. The results showed that 9 strains belonged to 7 genera as Mangrovibacter, Acinetobacter, Gallaecimonas, Martelella, Stenotrophomonas, Curtobacterium and Defluviimonas with 99% similarity. Keywords: 16S rRNA gene sequence, Bruguiera sexagula, Ceriops decandra, endophytic bacteria, nitrogen fixation, phosphate solubilization. I. INTRODUCTION Mangroves are a tropical coastal biome that is located in the transition zone between the land and the sea where the vegetation is dominated by a particular group of plant species [1]. This ecosystem is characterized by periodic tidal flooding, making environmental factors such as salinity and nutrient availability highly variable and resulting in unique and specific environmental characteristics [2]. Thus, the mangrove ecosystem provides a distinct environment harboring diverse groups of microorganisms [3-4]. Although the mangrove cosystem is rich in microbial diversity, less than 5% of the species present have been described; in many cases, neither their ecological role nor their technological potential is known [3]. Bacterial communities can be found living freely in mangrove sediments [5-7] or as endophytes associated with the native flora [8-11]. Endophytes are microorganisms that live inside of plants without causing any harm to their hosts [12]. Endophytic bacteria have been isolated from many different plant species but some endophytic communities remain unexplored in studies describing the bacterial communities from tropical native plants. Consequently, studies on the endophytic bacteria of plants from different ecosystems (mangroves, for example) offer a great opportunity to discover new compounds and resources with biotechnological potential that can be exploited [4]. Microorganisms from mangrove ecosystems contain useful enzymes, proteins, antibiotics and salt tolerant genes, all of which have biotechnological significance [3]. According to Ministry of Agriculture of Rural Development,Vietnam [13], had approx. over 209,741 ha mangrove forest, mainly mangrove located at esturies of the rives in the Mekong Delta to Ha Tien, Kien Giang with 128 537 ha (>53%). Phan Nguyen Hong and Hoang Thi San [14] reported that there were 78 plant species in mangrove in Vietnam and 69/78 species found in the Mekong Delta (from Vung Tau to Ha Tien) including 37 species, 20 genera, 14 families as Sonnertia caseolaris (bần chua), Avicennia alba (mắm trắng), Avicennia marina (mắm biển), Rhirophora apiculate (đước), Rhirophora mucronata (đưng), Bruguiera parviflora (vẹt tách), Bruguiera cylindrical (vẹt trụ), (dà), Lumnitzera racmose (cóc vàng), Xylocarpus granatum (xú ổi), Nypa frutican (dừa nước) and Threspecia populnea (tra) [15]. Depending on the salt content at estuary, Sonnertia, Avicennia, Rhirophora …appear in the pionees stage (Figure 1) at low tide level and high tide level and flooding water. After 4-5 years or high tide period, the salt content is also much lower than past period, suitable conditon for growth of Ceriop decandra and Bruguiera. [14]. Volume 14 Issue 1 August 2019 050 ISSN: 2319-1058 International Journal of Innovations in Engineering and Technology (IJIET) http://dx.doi.org/10.21172/ijiet.141.07 Figure 1. Growth of plants in mangrove depends on the salt content in sea water at estuary [14] The rationale of host plant selection largely relies on promotion of growth and development of the plant under adverse conditions by endophytes. In the present study, the mangrove tree (Bruguiera sexagula and Ceriops decandra) served as a promising source for examining endophytes as its ecosystem characterized by broad range of salinity, temperature, and moisture [16] is similar to lowland rice ecosystem. Further, mangrove trees have remarkable adaptation and grow abundantly in saline coastal sediment. It has been proved that the endophytic colonization has played a major role in the ecological adaptation of the host and increased their survival under adverse conditions.[17-18]. The aims of this study were (i) to isolate nitrogen-fixing bacteria and phosphate- solubilizing bacteria of endophytic bacteria associated with Bruguiera sexagula and Ceriops decandra (ii) in mangrove forest of Soc Trang province to obtain their characterization as salt-tolerance, colonies…and (iii) to identify by 16S rDNA techniques. II. MATERIALS AND METHODS 2.1 Collect of plant samples Plant samples were collected carefully from two species of mangroves viz. Bruguiera sexagula and Ceriops decandra from a 3 year old in plantation site, Trung Binh village, Tran Đe district (soil pH = 6.22, salinity 10‰; Vom Chua site, Vinh Hai village, Vinh Chau district (soil pH= 6.18, salinity 7‰; My Thanh site, Vinh Hai village, Vinh Chau district (soil pH= 4.16, salinity 8‰). (Lat. 09o 21’ 44” N; Long. 106o 00’ 11” E) (Figure 2). Figure 2. Sample collection sites in mangrove forests of Soc Trang province, Vietnam (the Mekong Delta, Vietnam) The samples were collected in December, 2018. For isolation of bacterial endophytic samples were collected during the low tide and brought to the laboratory immediately for analyses in day. 2.1.1 Bacterial isolation The tree samples (every species) were washed with water (Figure 3a and 3b) to remove adherent particles and were superficially disinfected according to Araújo et al. [19]. Then, the samples were cut into fragments, and roughly 1g was triturated in the presence of 5 mL of PBS (phosphate buffered saline) buffer, transferred to a 15 mL tube and shaken for 1 hour at 180 rpm. Isolation of nitrogen-fixing bacteria in Burk’N free media plus 2% NaCl [20] and Volume 14 Issue 1 August 2019 051 ISSN: 2319-1058 International Journal of Innovations in Engineering and Technology (IJIET) http://dx.doi.org/10.21172/ijiet.141.07 phosphate-solubilizing bacteria in NBRIP media plus 2% NaCl [21]; Cultures were streaked on media to obtain single colonies. To check for phosphate solubilization ability or nitrogen fixation ability, colonies from Burk’N free media were streaked to NBRIP media and colonies from NBRIP media were also cultivated to Burk’s N free media in order to select the colonies which developed on two media (or microbes having N2-fixing and phosphate- solubilizing ability). Endophytic isolates were purified and inoculated into liquid 5% Tryptic Soy Broth (TSB, Merck) medium supplemented with glycerol (15% final concentration) and stored at −80°C for future experiments. Fig.3a. Plant, flower and roots of Bruguiera sexagula Fig.3b. Plant, flower and roots of Ceriops decandra 2.2. Morphological Characterization The morphological characterization of the bacterial colonies were carried out according to on the basis of their shape, size, color, margin, elevation on the media and Gram staining were performed to decide the further determinative protocol. All isolates were tested on media (Burk’s or NBRIP) with higher NaCl concentration (.2.5 to 4.0% NaCl). 2.3. Screening for Biofertilizer Activities The ability to fix N2 was tested on Burk’N-free liquid medium incubating at 30oC and the ammonium concentration in medium was measured by Phenol Nitroprusside method after 2,4,6 and 8 days inoculated (DAI) and inorganic phosphate solubilizing ability was tested on NBRIP liquid medium and they were incubated at 30oC and the P2O5 concentration was measured by ammonium molypdate method. The qualitative detection of indole-3-acetic acid (IAA) production was carried out basing on the colorimetric method [22]. Precultures were grown in Burk’s N free (100 ml) without tryptophan in 250mL-flask at 30oC on a roller at 100 rpm and samples were taken from at 2, 4, 6, and 8 DAI, cell free supernatants were mixed 2:1 with Salkowki reagent (0.01 M FeCl3 in 35% perchloric acid) and incubated in the dark for 20 min at RT. IAA-containing solutions were indicated by reddish color with an absorption peak at 530 nm on Genesys 10uv Thermo Scientific spectrophotometer. Furthermore, siderophore production was assayed by the rhizopheric bacterial isolates according to Schwyn and Neilands [23] using NBRIP medium without tryptophan which was diluted fivefold. The isolates were spot inoculated onto Chrome azurol S agar plates divided into equal sectors, and the plates were incubated at 28oC for 48 h. Development of a yellow, orange or violet halo around the bacterial colony was considered to be positive for siderophore production. 2.4 Molecular Analysis 2.4.1 Genomic DNA Isolation Culture was centrifuged at 10,000 rpm for 5 min.