International Journal of Innovations in Engineering and Technology (IJIET) http://dx.doi.org/10.21172/ijiet.141.07 Isolation and identification of endophytic 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 [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. Pellet was collected and resuspended by adding 9 ml of STE buffer (0.1 mM NaCl, 10 mM Tris, 10 mM EDTA) 1 ml of SDS (10% Stock Solution). The suspension was incubated at 70˚C for 1 hour and centrifuged at 6000 rpm. for 10 min at room temperature. The supernatant was

Volume 14 Issue 1 August 2019 052 ISSN: 2319-1058 International Journal of Innovations in Engineering and Technology (IJIET) http://dx.doi.org/10.21172/ijiet.141.07 collected in fresh tube and add equal volume of Phenol : Chloroform : Isoamyl alcohol (PCI mix) (25:24:1) was added and mixed slowly. The suspension was centrifuged at 6000 rpm. for 10 min. The aqueous phase in fresh tube. Equal vol. of Chloroform: Isoamyl alcohol (24:1) and mix slowly and centrifuged at 6000 rpm for 10 min. The aqueous phase was collected and added double the vol. of absolute alcohol was added. The tube was subjected to overnight incubation in −20˚C. The solution was centrifuged at 6000 rpm. 4˚C for 10 min and the pellet was resuspended in 1/10 th ml of 3M sodium acetate and 10 ml of absolute alcohol and centrifuged at 6000 rpm. 4˚C for 10 min. The supernatant was discarded and the pellet was air dried. The pellet was dissolved in 1 ml sterile TE buffer. The DNA quality was checked using agarose gel electrophoresis and quantified using nanodrop.

2.4.2 PCR Amplification and Phylogenetic Analysis Amplification of 16S rDNA by PCR was carried out using the primers p515FPL and p13B [24]. The 50 µL reaction mixture consisted of 2.5 U Taq Polymerase (Fermentas), 50 µM of each desoxynecleotide triphosphate, 500 nM of each primer (fermentas) and 20 ng DNA. The thermocycling profide was carried out with an initial denaturation at 95oC (5 min) followed by 30 cycles of denaturation at 95oC (30s), annealing at 55oC (30s), extension at 72oC (90s) and a final extension at 72oC (10 min) in C1000 Thermal Cycler (Bio-Rad). Aliquots (10 µl) of PCR products were electrophoresed and visualized in 1% agarose gels using standard electrophoresis procedures. Aliquots (10 µl) of PCR products were electrophoresed and visualized in 1% agarose gels using standard electrophoresis procedures. Partial 16S rRNA gene of selectived isolates in each group were sequenced by MACROGEN, Republic of Korea (dna.macrogen.com). Finally, 16S rRNA sequence of the isolate was compared with that of other microorganisms by way BLAST (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi); In the best isolate(s) (high nitrogen fixation, phosphate solubilization ability, IAA biosynthesis and good salt tolerance) and 5 isolates of 3 sites were chosen to sequence and the results were compared to sequences of GenBank based on partial 16S rRNA sequences to show relationships between root-associated bacterial strains [25] and phylogenetic tree were constructed by the maximum-likelihood method using the MEGA software version 7.0 based on 1000 bootstraps [25].

2.4.3 Data Analyses Data from ammonium and orthophosphate concentrations in media were analysed in completely randomized design with three replicates and parameters of pot experiment also was arranged to completely randomized design with seven replications and Duncan test at P=0.01 or P=0.05 were used to differentiate between statistically different means using SPSS version 16.

III. RESULTS and DISCUSSION 3.1 Bacteria Isolation, Colony Characteristic and Microscopic Examination The endophytic bacteria developed to the pellicles of semisolid (in Burk’s N free and NBRIP media) after 24 h inoculation (Fig. 4a and Fig 4b) as the previous results of Weber et al. [26], and our previous result [27]. From 18 plant samples of 3 sites (villages of Vinh Chau and Tran De district), 68 isolates were isolated on two media included 38 and 30 isolates from NBRIP and Burk’N free media, respectively and 43 and 43 isolates from roots of Bruguiera sexagula and Ceriops decandra.

Figure 4a. Endophytic bacteric made a pellicle on the NBRIP (A) and Figure 4b. on Burk’s N free media (B)

Almost their colonies have round-shaped; milky, white clear (on Burk’s medium) and yellow, reddish yellow (on NBRIP medium); entire or loabate margin; diameter size of these colonies varied from 0.2 to 3.0 mm and all of them are Gram-positve and Gram-negative by Gram stain. Especially phosphate-solubilizing bacteria make a halo around colonies in NBRIP medium as described of Thanh and Diep [28], Nhu and Diep [29]; (Figure 5a and 5b). All isolates grew well on both of media (they have nitrogen fixation and phosphate solubilization ability) (Figure 5c and Figure 5d). The cells were observed by microscopic and appearded as short rods and most of them have motility.

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Figure 5c and 5d. The isolates from NBRIP grew on Burk’s N- Figure 5a. Colonies of the isolate Figure 5b. Colonies of the isolate free medium (5c) and the isolates from Burk’s N free grew on on Burk’s N free medium on NBRIP medium NBRIP medium (5d) very well

IV. SCREENING FOR BIOFERTILIZER ACTIVITIES Selection of good isolates for nitrogen fixation [from 2 media] were presented in Table 1a and 1b Table 1a. Nitrogen fixation of 20/68 isolates (mg NH4/l) on Burk’s N free medium Day 8 No Bacterial Isolate Day 2 Day 4 Day 6 Average 0 Control 0.00 xy 0.00 op 0.00 l 0.00 m 1 NVVC1a 0.16 h 0.00 gh 0.00 gh 0.10 h 0.07 2 NVVC2a 0.07 n 0.00 gh 0.20 a 0.07 q 0.09 3 NVVC2b 0.28 c 0.00 gh 0.00 gh 0.07 q 0.09 4 NVMO1b 0.32 b 0.00 gh 0.04 cdefgh 0.03 ac 0.10 5 NVMO1c 0.25 d 0.00 gh 0.01 gh 0.08 m 0.09 6 NVMO1e 0.28 c 0.00 gh 0.00 gh 0.07 r 0.09 7 NVMO2a 0.20 f 0.00 gh 0.10 bc 0.24 a 0.14 8 NVMO3a 0.00 xy 0.00 gh 0.23 a 0.10 i 0.08 9 NDMO1e 0.47 a 0.00 gh 0.00 gh 0.00 ah 0.12 CV 24.24%

Table 1b. Nitrogen fixation of 9/68 isolates (mg NH4/l) on NBRIP medium No Bacterial Isolate Day 2 Day 4 Day 6 Day 8 Average

Control 0 0.00 v 0.00 s 0.00 s 0.00 l

NVMO2b 11.90 de 10.00 cd 12.30 a 11.50 a 11 . 4 0 1 2 NVMO3a 13.20 a 10.40 b 11.60 bc 10.30 d 11.40 3 NVMO3d 9.18 n 9.74 e 11.20 c 10.60 c 10.20 4 NDMO1c 12.20 c 9.89 de 10.70 d 6.96 e 9.94 5 BVMO2e 11.90 cde 11.50 a 8.94 e 0.05 l 8.12 6 BVMO2h 11.20 g 10,2c 12,20 a 11,10 a 11.20 7 BVMO3a 9.74 kl 6.67 hi 6.25 f 4.55 f 6.80 8 BVMT1a 11.90 de 10.60 b 11.70 b 10.50 c 11.20 9 BVMT1e 10.60 ij 7.65 f 6.57 f 4.32 g 7.30 10 BVMO2f 11.00 gh 6.45 i 6.51 f 3.84 h 6.95

CV 3.27% The numbers followed by the same word not different at p<0.01 and good isolates for phosphate solubilization [from 2 media] were presented in Table 2a and 2b

Table 2a. Phosphate solubilization of 21/34 isolates (mg P2O5/l) on Burk’s N free medium No Bacterial Isolate Day 5 Day 10 Day 15 Day Average 20 0 control 000 q 000 q 000 q 000 q 1 NVVC2a 354 ef 439 hij 293 cde 234 hi 330

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2 NVVC2b 234 klm 243 no 255 fghijk 408 c 285 3 NVMO1a 338 fg 513 cd 249 ghijkl 265 f 341 4 NVMO1b 371 de 433 ijk 200 pqrstuvwxy 207 mnopq 303 5 NVMO1c 317 gh 489 e 180 wxyzaaabacad 235 ghijk 305 6 NVMO1d 515 a 535 c 210 nopqrstuvwx 235 ghij 374 7 NVMO1e 502 a 443 ghi 133 afag 142 t 305 8 NVMO1f 309 gh 491 de 223 jklmnopqrst 181 rs 301 9 NVMO2b 305 gh 277 m 292 cde 874 a 291 10 NVMO2c 352 ef 411 kl 243 ghijklmn 201 opqr 302 11 NVMO2e 302 h 500 de 294 cde 129 tuvwxy 306 12 NVMO3b 250 jkl 498 de 300 cd 139 tuv 297 13 NDMO1b 352 ef 418 jkl 198 rstuvwxy 223 jklmn 298 14 NDMO2a 192 pqrst 430 ijk 288 cdef 214 klmnopq 281 15 NDMO2b 372 de 450 fghi 219 klmnopqrstu 179 s 305 16 NVMT1a 394 cd 404 l 256 fghij 130 tuvwx 296 17 BVVC1a 425 b 747 a 625 a 453 b 563 18 BDMO1a 202 nopqrs 604 b 523 b 543 a 468 19 BVMT1a 301 h 462 fg 214 mnopqrstuvw 197 qrs 293 CV 6.81%

Table 2b. Phosphate solubilization of 39/52 isolates (mg P2O5/l) on NBRIP medium Day 20 No Bacterial Isolate Day 5 Day 10 Day 15 Average 0 Control 000 x 000 u 000 z 000 u 1 NVVC1a 165 qrst 386 hijk 352 opqrs 424 mn 332 2 NVVC1b 122 uvwx 355 jkl 384 mnopq 600 e 365 3 NVVC2a 191 opqr 464 cde 528 fghijk 694 bc 469 4 NVVC2b 385 b 360 ijkl 338 pqrst 515 ghi 399 5 NVVC2c 258 ghijk 328 lmno 296 qrstuv 410 n 323 6 NVVC2e 303 def 589 a 756 a 224 rs 468 7 NVVC2d 130 tuvw 313 mnop 318 pqrst 449 lmn 303 8 NVVC2f 381 b 623 a 652 bc 65 yzaa 430 9 NVMO1a 204 mnop 391 ghij 489 hijkl 351 o 359 10 NVMO1b 216 lmnop 268 qrstu 508 ghijk 732 ab 431 11 NVMO1c 242 ijkl 349 klm 401 lmnop 701 bc 423 12 NVMO1d 120 uvwx 388 ghijk 438 klmno 620 de 391 13 NVMO1f 290 efgh 300 opqr 186 wxyzaa 483 ijkl 315 14 NVMO2a 293 efg 281 pqrs 338 pqrst 600 e 378 15 NVMO2c 325 cde 254 stu 508 ghijk 501 ghijkl 397 16 NVMO2d 358 bc 449 def 542 efghij 544 fg 473 17 NVMO2e 204 mnop 398 ghi 483 hijkl 581 ef 417 18 NVMO3a 160 rst 389 ghij 364 nopqr 540 fgh 363 19 NVMO3b 230 jklmn 448 ef 523 ghijk 521 ghi 430 20 NVMO3c 434 a 425 efgh 641 bcd 676 c 544 21 NVMO3f 327 cde 488 bcd 654 bc 654 cd 531 22 NVMO3g 375 b 621 a 568 cdefgh 496 ghijkl 515 23 NDMO1b 192 opqr 359 ijkl 465 ijklm 608 de 406 24 NDMO1d 107 vwx 448 ef 336 pqrst 500 ghijkl 348 25 NDMO2a 271 fghi 240 tu 250 tuvwx 530 fghi 323 26 NVMT1a 336 cd 426 efg 516 ghijk 429 mn 427 27 NVMT1b 246 i jkl 507 b 515 ghijk 652 cd 480 28 NVMT1c 128 tuvw 341 lmn 498 ghijk 619 de 397 29 NVMT2a 159 rst 304 nopq 556 defghi 530 fghi 387 30 BVVC1a 192 opqr 193 w 447 klmn 758 a 398 31 BVVC1b 184 pqrs 294 opqr 548 efghi 676 c 426 32 BVMO1d 258 ghijk 497 bc 726 ab 503 ghijk 496

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33 BVMO1e 135 tuv 263 rstu 589 cdefg 510 ghij 374 34 BVMO2c 150 stu 303 nopq 630 cde 616 de 425 35 BVMO2d 184 pqrs 235 uv 525 ghijk 510 ghij 364 36 BVMO2f 255 hijk 312 mnop 525 ghijk 613 de 426 37 BVMO2g 223 klmno 234 uv 463 ijklm 350 o 318 38 BVMO3a 266 fghij 419 fgh 519 ghijk 523 ghi 432 39 BVMT1c 324 cde 303 nopq 618 cdef 606 de 463 40 BVMT1e 212 lmnop 173 w 403 lmnop 456 klmn 311 C.V 5.41% The numbers followed by the same word not different at p<0.01

From the results of Table 1a, 1b, 2a and 2b showed that amount of isolates (and nitrogen fixation concentration) having high biological nitrogen from Burk’s N free medium were lower than those of NBRIP medium. The same result of nitrogen fixation, quantity of isolates having high phosphate solubilization from NBRIP medium were much more than those of from Burk’s medium. Interestingly, there were 49/68 isolates isolated had IAA biosynthesis during 8 days inoculation in two media plus 2% NaCl without tryptophan; These isolates biosynthezed the highest IAA concentration at 2 day after inoculation and reduced to at 6 day after inoculation, after that they increased IAA bionsynthesis at 8 day after inoculation. The isolates had high IAA biosynthesis in Burk’s N free medium (Fig. 6a) and NBRIP medium (Fig. 6b) without tryptophan showed that number of good isolates (10) from NBRIP medium was two fold than number of good isolates (5) from Burk’s N free medium. There was no isolates producing siderophores.

Figure 6a. Some isolates had high IAA biosyntheis in Burk’s N free medium and Figure - 6b. in NBRIP medium

4.1 Salinity tolefance Almost rhizospheric bacteria from mangrove soil have ability of saline tolerant with4.0% NaCl however when increasing NaCl concentration from 2.5 to 4.0% NaCl in the media, amount of bacteria reduced when saline concentration in water increased (Table 3). There were 5 isolates developed well in water level 4.0% as NVVC2a, NVMO2b, BVMO2h, BVMO2f, BDMO2b (Figure 7) ; At 4.5%, there was no isolate developed. This explained that mangrove soil of Bruguiera sexagula and Ceriops decandra grew less saline concentration because they developed at higher than Rhirophora and Avicancia (Figure 1).

Table 3. Ratio (%) endophytic bacteria developed medium with the different NaCl concentration Ratio (%) NaCl concentration in medium (%) Bacteria 2.0 2.5 3.0 3.5 4.0 Developed 100 8/68 (96.51) 8/68 (95.35) 8/68 (86.05) 5/68 (60.45) No developed 100 60/68 (3.49) 60/68 (4.65) 60/68 (13.95) 63/68 (39.55)

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350/ 400/ 00 00

Figure 7. The isolates developed well on medium with salt concentration 3,5 đến 4%

Based on the characteristics as high nitrogen fixation, phosphate solublization, IAA, and well developed on medium high salt concentration, 6 good isolates together with 3 isolates having stable biofertilizer activities were chosen to identify with universal primers p515FPL and p13B and sequencing as NVVC2a, NVMO2b, NVMO3d, BVMO2e, BVMO2f, BVMO2h, BVMO3e, BDMO2a, BDMO2b DNA analysis and sequencing. The fragments of 900 bp 16S rRNA were obtained from PCR with p515FPL and p13B primers and sequencing. Homology searches of 16S rRNA gene sequence of selected strain in GenBank by BLAST revealved that they had similarity to sequences of Bacilli (3/5 isolates), 2 isolates belonged to Gamma- (Table 4) (Figure 8)

Table 4. Phylogenetic affiliation of isolates on the basis of 16S rRNA genes sequences by using BLAST program in the Gen Bank database based on sequences similarity Taxonomic Group Similarity nucleotide Closest species relative and Strain (%) Mangrovibacter plantisponsor strain UMTKB-3 99.4% NVVC2a 839 (KT025846) Mangrovibacter yixingensis strain TULL-A (NR_136780) 99.3% Acinetobacter radioresistens strain BMYN20_2 99.4% NVMO2b 841 (MG996812) Acinetobacter sp. strain JLT159 (KX989241) 99.4% Gallaecimonas sp. strain HK-28 (MF800856) 99.4% NVMO3d 843 Gallaecimonas xiamenensis strain 3-C-1 (NR_117541) 98.3% Martelella endophytica strain YC6887 (NR_109025) 99.6% BVMO2e 834 Martelella radicis strain BM5-7 (NR_134079) 99.4% Stenotrophomonas panacihumi strain MK06 (NR_117406) 99.1% BVMO2f 845 Stenotrophomonas sp. FSBSN2 (KJ185037) 98.9% Curtobacterium oceanosedimentum strain S52_MA1R 99.5% BVMO2h 840 (MK883133) Curtobacterium luteum strain IAE64 (MK415011) 99.5% Stenotrophomonas panacihumi strain MK06 (NR_117406) 99.3% BVMO3e 846 Stenotrophomonas sp. FSBSN2 (KJ185037) 99.2% Defluviimonas alba strain b45 (KC222647) 99.9% BDMO2a 817 Frigidibacter albus strain SLM-3 (KF944302) 99.5% Mangrovibacter plantisponsor strain UMTKB-3 99.6% BDMO2b 834 (KT025846) Mangrovibacter yixingensis strain TULL-A (NR_136780) 99.5%

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Figure 8. Phylogenetic tree showing the relative position of endophytic bacteria by the maximum-likelihood method of complete 16S rRNA sequence. Bootstrap values of 1000 replicates are shown at the nodes of the trees.

A maximum-likelihood analysis of phylogenetic tree in these isolates showed in the two clusters: cluster A composed of three strains with two species of genus Mangrovibacter as strains M. plantisponsor MVVC2a (Tran De district) and M. yixingensis BDMO2b (Vinh Chau district) related closely even though they were isolated at other sites in Soc Trang province and Gallaecimonas xiamenensis NVMO3d originated from Tran De district in cluster A1; in cluster A2 had three genera: Martelella endophytica BVMO2e, Stenotrophomonas panacihumi BVMO2f and Curtobacterium oceanosedimentum BVMO2h, all of them originated from Tran De district. Cluster B only 3 strains belong to Gamma-proteobacteria as Defluviimonas alba BDMO2a, Acinetobacter radioresistens NVMO2b and Stenotrophomonas panacihumi BVMO3e (Figure 8). Genus Mangrovibacter belonged to Enterobacteriaceae, Mangrovibacter plantisponsor found in roots of wild rice species (Porteresia coarctata Tateoka) in mangrove forest having nitrogen fixation Genus Mangrovibacter is micro- aerobic, rod and gram negative [30], Mangrovibacter yixingensis isolated agricultural soil in China and it is the novel species which nitrogen fixation ability and relationship with Mangrovibacter plantisponsor [31]. Mangrovibacter yixingensis developed from 0 to 60‰ salt concentration and Mangrovibacter plantisponsor to 70‰ in LB medium [31]. Gallaecimonas xiamenensis with similarity 98.3%, it related with Mangrovibacter yixingensis and Mangrovibacter plantisponsor (d=0,4) in comparison to others in [32]. Eight strains belonged to gram-negative (Proteobacteria) and one strain only was gram-positive (Actinobacteria) Curtobacterium oceanosedimentum BVMO2h, and it was classied at center of two clusters (Fig. 8) however it related with Mangrovibacter and Martelella & Stenotrophomonas than cluster B. Unless Acinetobacter and Stenotrophomonas, Defluviimonas alba was a novel species, belonged to Rhodobacteriaceae, isolated at oilfield, aerobic bacteria, gram-negative, rod and growth from 0 to 5% NaCl contration in LB medium [33](Pan et al., 2015) Mangrove forests develop at the interface between land and ocean in tropical and subtropical zones. Mangroves play a vital role in defending against the impact of tropical cyclones and protecting dyke systems Their total area was estimated at 167,387 km2 in 2012 [34]. Although Mangrove forests have contributed significantly to the socio- economic lives of coastal dwellers [35]. Rapid economic growth and population pressure have resulted in severe depletion of mangrove forest in the world. Exploitation of tree resources, conversion of mangrove areas to shrimp and mud crab ponds, agricultural use, salt pans and human settlement have contributed to the loss and degradation of mangrove resources [14, 35]. cupy only 2% of the world’s coastal ocean area [36]. In Vietnam, mangrove forests covered an area of about 400,000 ha in 1943, but only 252,500 ha remained in 1983 [14] and 270,000 ha in 2015 [37]. Most of these mangroves were planted in the late 70s and early 90s, after their destruction during the war. More than 80% of the total mangrove forest area in Vietnam develop in the south of the country, either in the Mekong Delta or in the Can Gio Estuary [38]. Microbes are major components of this biodiversity, with bacteria and fungi constituting 91% of the total biomass of mangrove ecosystems [39], Endophytes are microorganisms that colonize the intercellular space by establishing either a symbiotic or a mutualistic or a commensalistic or trophobiotic association with host plants.[40-42]. These microbes are often bacteria or fungi [found in various plant organs such as seeds, roots, stem, leaves, flowers, and

Volume 14 Issue 1 August 2019 058 ISSN: 2319-1058 International Journal of Innovations in Engineering and Technology (IJIET) http://dx.doi.org/10.21172/ijiet.141.07 fruits] that colonize host tissues similar to pathogen. Studies demonstrated that the endophytic association with the host contributes significantly in accelerated seedling emergence,[43] enhanced plant growth, [44-49] improved resistance against various phytopathogens, [50-52] and abiotic stresses.[53-55]. Some endophytes synthesize novel metabolites [56] known for its antibiotic and antimicrobial activities. Owing to its potential benefits, a number of endophytes have been isolated from wide variety of plant species and from diverse environmental conditions ranging from permafrost sediments to agricultural field. In a recent study, Khianngam et al. [57] isolated and screened endophytic bacteria from mangrove plants in Thailand for the presence of hydrolytic enzymes. Twenty isolates showed activities associated with proteases, lipases, amylases or cellulases. The Rhf-2 strain, which was isolated from the fruit of Rhizophora mucronata, produced all of these enzymes; the strain was later identified as Bacillus safensis. Castro et al., [58] isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and Avicennia nitida, that are found in streams in two mangrove systems in Bertioga and Cananéia, Brazil. Bacillus was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga. However, other common endophytic genera such as Pantoea, Curtobacterium and Enterobacter were also found. In our experiment, 8/9 strains were identified as Proteobacteria and one strain was actinobacteria; our result differed from with described results previously [59] when they determined Bacillus was the most abundant genus isolated from all samples. Occurrence of genus Mangrovibacter, a genus as a beneficial genus for rice biofertilizer production in China [31] and they can develop very well from 4 to 6% NaCl concentration in medium. In otherhand, two strains of Mangrovibacter had high nitrogen fixation, phosphate solubilization and IAA biosynthesis activity. They are not only good nitrogen fixation, high phosphate solubilization but also good IAA biosynthesis and salt tolerance, so that they become two promising strains to produce biofertilizer for the rice cultivation in saline soil.

V. CONCLUSION From 18 plant samples (Bruguiera sexagula and Ceriops decandra) on mangrove forest of Soc Trang province, Mekong delta, Vietnam, 68 isolates were isolated and identified as endophytes, 9 isolates having good plant growth promotion from 3 sites were chosen to analyse their relationship. The results showed that bacterial diversity was very high; 8/9 strains belonged to Proteobacteria and one strain was actinobacteria. Among them, two strains will be suggested to produce for crop cultivation on soil salinity in the future.

VI. ACKNOWLEDGEMENTS The authors thank the helpfulness of Microbiology BSc.Students and technicians in the Environment Microbiology

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