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American Journal of Life Sciences

2014; 2(6): 361-368
Published online December 18, 2014 (http://www.sciencepublishinggroup.com/j/ajls)
doi: 10.11648/j.ajls.20140206.16
ISSN: 2328-5702 (Print); ISSN: 2328-5737 (Online)

Isolation, characterization and identification of endophytic
bacteria in sugarcane (Saccharum spp. L.) cultivated on
soils of the Dong Nai province, Southeast of Vietnam
Hoang Minh Tam1, *, Cao Ngoc Diep2
1
Dept. Natural Science Teacher Training, Sai Gon University, HCM City, Vietnam
2
Dept. Microbiology Biotechnology, Biotechnology R&D Institute, Can Tho University, Can Tho City, Vietnam

Email address:
hoangminhtam18@yahoo.com (H. M. Tam), cndiep@ctu.edu.vn (C. N. Diep)

To cite this article:
Hoang Minh Tam, Cao Ngoc Diep. Isolation, Characterization and Identification of Endophytic Bacteria in Sugarcane (Saccharum spp. L.)
Cultivated on Soils of the Dong Nai Province, Southeast of Vietnam. American Journal of Life Sciences. Vol. 2, No. 6, 2014, pp. 361-368.
doi: 10.11648/j.ajls.20140206.16

Abstract: Endophytic bacterial diversity in sugarcane plant cultivated on Latosols and Acrisols of the Eastern of South
Vietnam was studied. Sugarcane plant samples were taken in four sites (districts/towns) (Xuanloc, Dinhquan, Vinhc uu and
Trangbom) of Dong Nai province. Endophytic bacteria were isolated in LGI medium together with 16S rRNA gene fragments
amplified from DNA using eubacterial universal primers (p515FPL and p13B). A total of 27 isolates were isolated on LGI
medium and all of them have ability of nitrogen fixation and phosphate solubilization together with IAA biosynthesis but
there were 10 isolates having the best characteristics. The sequences from selected endophytic bacteria (10 isolates) showed
high degrees of similarity to those of the GenBank references strains (between 98% and 99%). All of them are Proteobacteria
(gram-negative bacteria) and 3 isolates belonged to Alpha-proteobacteria (30%), 2 isolates were Beta-proteobacteria (20%), 5
isolates were Gamma-proteobacteria (50%). Based on Pi value (nucleotide diversity), Gamma-proteobacteria group had the
highest theta values and Theta values (per sequence) from S of SNP for DNA polymorphism were calculated for each group
and Gamma-proteobacteria group had the highest values in comparison of two groups. From these results showed that
Enterobacter oryzae LT7, Achromobacter xylosoxidans T16, Achromobacter insolitus R15b and Pantoea agglomerans T12
revealed promising candidates with multiple beneficial characteristics and they have the potential for application as inoculants
or bio-fertilizer adapted to poor latosols and acrisols because they are not only famous strains but also are safety strains for
sustainable agriculture.
Keywords: Acrisols, 16S rRNA Gene Sequence, Latosols, the Eastern of the South Vietnam, Endophytic Bacteria,
Sugarcane

Gluconacetobacter diazotrophicus). Subsequently, several
1. Introduction endophytic N2 -fixing bacteria, such as Herbaspirillum sp.
Sugarcane (Saccharum spp.) is a tropical and sub-tropical [6], Pantoea sp. [7], Burkholderia tropica [8], B. unamae [9].
crop that can produce sugars and a large amount of chemical and B. silvatlantica [10], were isolated from sugarcane
fertilizers have been applied to sugarcane to promote early plants. Endophytes represent a subgroup of the
growth in many countries, especially in developing countries rhizobacterial communities that have the ability to enter the
[1]. In this sustainable sugarcane production, biological roots of their hosts after the rhizoplane is colonised [11].
nitrogen fixation (BNF) has replaced chemical fertilizers Endophytic bacteria colonise the intercellular spaces and the
[2][3]. There have been long-term search efforts to identify inside of xylem vessels and may promote plant growth
the N2-fixing bacteria involved in sugarcane production [4]. directly or indirectly [12][13].
Cavalcante and Dobereiner [5] first isolated endophytic N2 - The Southeast of Vietnam locates from 105o49’ to 107o35’
fixing bacteria from sugar juice and gave this bacterium its E and from 10 o20’ to 12 o17’ N, it is one of the two regions
species name, Acetobacter diazotrophicus (now of South Vietnam situated in the east of part of South
Vietnam, covering 2.34 millions ha, occupied approximately

Characterization and Identification of Endophytic Bacteria in Sugarcane (Saccharum spp. collected regions in the South Vietnam. Interactions with 70% ethanol for 30 s and finally washed five times and association between microorganism and their host plants with sterile distilled water. Vinhcuu (tenfold dilution) in sterile water.5 – 5.) Cultivated on Soils of the Dong Nai Province. of Vietnam. for biofertilizer activities were Burk’N free [19]. respectively were collected. Colony Characteristic and Microscopic Examination The characteristics of colony such as size. Recent interest has focused particularly upon PGPR bacterial growth after incubation at 28 oC for 3 days. The plates were examined for (PGPR).4.5 ton/ha and productivity was 2. The soils are mainly red Four sugarcane fields (Xuanloc. In the present work we report characterization of Samples were cut to 0.5% available CT) for 5 min. 2. Trangbom. Culture Media and Growth Condition Isolation media was LGI [5].4. 2.5-cm pieces and macerated with a endophytic bacteria isolates from sugarcane plants collected sterile mortar and pestle.3. the aliquots of the sterile distilled many of these bacteria are beneficial to their hosts. Trangbom latosols (from origin of volcanic mountain) and acrisols with and Dinhquan) in Dong Nai province were sampled. Fields a pH range of 4. After 48-72 h incubation. Materials and Methods cultures were streaked on media to obtain single colonies.395 ha.0. rinsed amounts of fertilizers (400-500 kg N ha-1 yr-1). Bacterial colonies were differentiated on the basis of colony 2. The geographic map and the locations of southeast of Vietnam examined in this study and sugarcane samples were collected at the these concentration in medium was measured by Phenol sites (Xuanloc. This isolation process carries out in shifts of the agar-based culture medium to the agar-based subculture medium until monocultures were obtained. their structure [14]. Vinhcuu.e.362 Hoang Minh Tam and Cao Ngoc Diep: Isolation.435 tons. the sugarcane area occupied the whole plant after that roots and stems (50-cm) of 12. and a very low available phosphorus. cell morphologies of the isolates were observed using an optical microscope and they were also observed on scanning electron microscope.3% of total of Vietnam area.19%. The Southeast of Vietnam and the Samples were obtained whole plant after that soil Mekong Delta (Southwest) are two big sugarcane cultivation rhizosphere was separated for further experiments.2. Sample Collection and Isolation of Endophytes morphology and pigmentation. Subsequently. cation 200-500 kg N ha-1 yr-1). Trangbom. Monocultures were culture on the agar-based culture medium slant in the test-tube (12 ml) and incubated at 30 oC for 4 days following by stored 10 oC in refrigerator. NBRIP [20].7% with 34. Vinhcuu and Dinhquan) of Dong Nai province Nitroprusside method after 2. tissue extracts were then serially from four agricultural soils (Xuanloc. are which have been Sugarcane stem and roots samples that were not reported to be associated with important crops such as rice. the production and productivity were sugarcane plant [hybrid variety](near 6 months old plant) 66. Southeast of Vietnam 20. shape…. Colonies were subculture on the agar-based subculture medium plates by striking technique and re-incubated at 30oC for 4 days. 2. To confirm that the sterilization may be inhibited by high levels of added fertilizer [17] and process was successful. L. bacteria growing in tubes as a white or yellow pellicle at a depth of 1 to 4 mm were streaked on LGI agar plates. the stems were immersed in yr-1 or more in most sugarcane cultivating countries [16] as 70% ethanol in 3 min. washed with fresh sodium in sugarcane fields in Spain farmers frequently use high hypochlorite solution (2. color. 200 µl-aliquot samples and Dinhquan district) in Dong Nai province which is one were used to inoculate in (in triplicate) Nitrogen-free in two important sugarcane cultivating regions of southeast semisolid LGI in 5 ml tubes. They are considered nutrient poor. Screening for Biofertilizer Activities The ability to fix N2 was tested on Burk’N-free liquid medium incubating at 30 oC and the ammonium Figure 1. contaminated as detected by culture-dependent sterility test wheat and sugarcane [18].14 – 0. exchangeable K and contain more sand in four sites of Dongnai province (Figure 1).6 and 8 day inoculation (DAI) .were presented in each group. stem were washed with tap water to remove [ 1 5] . and are water used in the final rinse were set on tryptic soy agar collectively termed plant growth-promoting rhizobacteria (TSA) medium plates. were used for further analysis. 2.1. PGPE).329. have been in monoculture for more than five years and with an average organic matter of 2%. It has been a general practice to apply 250 kg N ha-1 attached clay. Sugarcane plants were collected at exchange capacity. a total nitrogen range sugarcane plants were fertilized with different levels (from of 0. that are endophytic (i.

27 sequences of GenBank based on partial 16S rRNA sequences isolates were isolated on LGI medium (Table 1). It means a 1% chance basecall is spectrophotometer. (Fermentas) and 10 ng DNA. climy.macrogen. Precultures were grown in Burk’s The sequence data from 30 endophytic bacterial isolates N free (100 ml) without tryptophan in 250mL-flask at 30 oC were analysed with SeqScape@Software (Applied Biosystem.nlm. . From 14 sugarcane samples of 4 were chosen to sequence and the results were compared to sites (Xuanloc. 2(6): 361-368 363 and inorganic phosphate solubilization ability was tested phylogenetic tree were constructed by the neighbor-joining on NBRIP liquid medium and they were incubated at 30oC method using the MEGA software version 6. 16S rRNA sequence of the isolate was compared Microscopic Examination with that of other microorganisms by way BLAST (http://www. cell free supernatants were mixed 2:1 with tool for variant identification. incorrect. [29]. n is alleles and L is the total length of sequence (bp). and 8 DAI. The thermocycling profide was carried out with an initial denaturation at 94 oC (3 min) Data from ammonium. orthophosphate and IAA followed by 30 cycles of denaturation at 94 oC (60 s).com). Republic of Korea (dna. Thu Ha et al. yellow and some colonies appeared (Figure 3). by SEM and appeared as rod and most of them have motility colourless or milk-color.1.8. The 50 µL reaction mixture consisted of 2.7. electrophoresed and visualized in 1% agarose gels using standard electrophoresis procedures. Salkowki reagent (0.cgi). Foster City. 3. Colony Characteristic and Finally. Vinhcuu and Dinhquan). on a roller at 100 rpm and samples were taken from at 2.ncbi. Data Analyses disulfide (Fermentas).6.01 M FeCl3 in 35% perchloric acid) and It considers alignment depth. SNP discovery and validation. where K is the number of SNPs identified in an alignment 0.nih.gov/BLAST/Blast. amplification of 16S ribosomal DNA: p515FPL [23] and p13B [24] [25]. CA. their colonies had round-shape.01 were used to differentiate between statistically (Bio-Rad).4 mM spermidine (Sigma). Partial 16S rRNA gene 3. USA). Aliquots (10 µl) of PCR products were different means using SPSS version 16. Total of isolates were isolated from 4 sites (districts/towns) from roots and stems of sugarcane cultivated on Latosols and Acrisols of Dong Nai province (the eastern of South Vietnam) Site Sample number Sugarcane sample Number of isolated isolates Total Xuanloc 3 Stem 2 5 Root 3 Trangbom 4 Stem 5 9 Root 4 Dinhquan 4 Stem 4 9 Root 5 Vinhcuu 3 Stem 1 4 Root 3 They developed very well on the LGI medium from 36-48 to have much larger size (Figure 2).1 mM of each desoxynecleotide triphosphate. [27]. molypdate method. 4. the base calls in each of the incubated in the dark for 20 min at RT. 10% (vol/vol) dimethyl 2. American Journal of Life Sciences 2014. Putative solutions were indicated by reddish color with an absorption SNPs were accepted as true sequence variants if the quality peak at 530 nm on Genesys 10uv Thermo Scientific value exceeded 20.5 mM magnesium chloride. Trangbom.5. Results and Discussion of selectived isolates in each site was sequenced by MACROGEN. The following primers were used for PCR described by Halushka et al. The qualitative detection of indole-3- acetic acid (IAA) production was carried out based on the 2. 0.06 based on and the P 2O5 concentration was measured by ammonium 1000 bootstraps. 1. concentrations in media were analysed in completely annealing at 57 oC (60 s). 2. In the best The endophytic bacteria developed in the pellicles of semi isolate(s)(high ability of nitrogen fixation. smooth. The cells were observed h at 30oC. 10 pM of each primer length. SeqScape is a sequence comparison 6. Nucleotide Diversity (Ө) Bacterial DNA was isolated following published protocols Nucleotide diversity (Ө) was calculated by the method [22]. extension at 72 oC (120 s) and a randomized design with three replicates and LSD test at final extension at 72 oC (4 min) in C1000 Thermal Cycler P=0.5 Ө = K/aL a=∑ l/(i − l) U Taq Polymerase (Fermentas). solubilization and IAA synthesis) and 27 isolates of 4 sites [28]. Bacteria Isolation. to show relationships between endophytic strains [26] and Table 1. SNPs Discovery colorimetric method [21]. IAA-containing sequences and the associated base quality values. phosphate solid (in LGI medium) as the previous results of Weber et al. 16S rDNA Gene Amplification and Sequencing 2.

86 c 1. Beta- Figure 3. LR6 (Dinhquan). Endophytes increase plant growth Figure 2. indole acetic acid production [33].86 f 0. Dinhquan 0.801 ab Novosphingobium sp.259 hi 01.286 e isolated by the BLAST search program showed that they 27 MR4 Root.16 c 0.69 c 1.045 a 23.212 c LR6 98 (KJ9400520 04 LR2 Root.10 a 1.429 de grouped into two clusters (Figure 4).05 f 0. Xuanloc 0. Dinhquan 0.617 f 37.626 f 12. Trangbom 0. Dinhquan 0.) Cultivated on Soils of the Dong Nai Province. All 10 isolates have nitrogen fixation. Sequencing and Nif-H Gene All of them (10 isolates) were chosen to identify and the fragments of 900 bp 16S rRNA were obtained from PCR and sequencing (Table 3). L. Xuanloc 0. the means of 4 replications napthtlalenivorans LT3b. Phylogenetic affiliation of isolates on the basis of 16S rDNA Table 2.555 d Pantoea agglomerans strain TA22 19 R15b Root.99 f 0.00 f 0.627 d neighbor sequences for 16S rRNA gene sequences of the 10 26 MB9 Root.427 de 24 R10 Root. Dinhquan 0. LT7.714 ef 41.784 ab Novosphingobium naphthalenivorans 05 LR4 Root.833 cd LT7 99 (KC843381) 18 T12 Stem. Dinhquan 0. ZYY112 03 LT2 Stem.60 ab 1.227 hi 22. Dinhquan 0. Southeast of Vietnam Among 27 isolates. LR6 .331 c 23. 3. Dinhquan 0.47% Pantoea agglomerans T12.117 jk 00.31 c 1. Calculated F ** ** ** C.296 h 00.636 ab LT1 98 (KF758547) 14 MB4 Stem. LR6.2.429 g 02. phosphate solubilization ability) and all of them produced indole-3-acetic acid (IAA) in vitro.709 ab LR7 98 (KF747357) 16 MR1 Stem.21 c 0.96 f 1.312 gh 01. 10 isolates having good biofertilizer activity were chosen to study (Table 2).V 12. Trangbom 0.631 ab LR4 99 (KC456530) 22 RK4 Root.10.631 ab R15b 99 11 (KC633947) 11 LR8b Root.272 hi 00.374 de Enterobacter oryzae strain 20 17 T16 Stem.10 days after inoculation for Sphingomonas sp.000 f small cluters: cluster A11 with Klebsiella vaniicola LR7.50 b 1.859 a LT3b 99 strain VIT-DD2 (KJ716452) 06 LR7 Root.591 b Achromobacter insolitus strain zjsru- 10 LT4a Stem.24 f 1. Trangbom 1. PS5 (JX083381) 99 02 LT3a Stem. Trangbom 0.224 hi 22.593 d 23 MB6 Root.379 gh 01. Cluster A divided two 28 Control 0.000 k 00. Trangbom 0.364 Hoang Minh Tam and Cao Ngoc Diep: Isolation.671 f 35.18 d 1. LT3 (Xuanloc).3.768 ab Gammaproteobacteria 12 LR6 Root.728 ef 25.14% 15. LR2 and Novosphingobium ammonium and IAA and 5. Xuanloc 0.718 ab Betaproteobacteria 07 LT7 Stem. they are endophytic bacteria in sugarcane however all of them are gram-negative bacteria with three groups Alpha-proteobacteria (3 strains).20 e 1. T16 (Trangbom).826 b 26. Dinhquan 0. phosphate solubilization and IAA synthesis genes sequences by using BLAST programme in the GenBank database production of 27 isolates based on sequence similarity NH4 P2O5 IAA No Isolate Site Taxonomic group and strain Closest species relative (mg/L)* (mg/L)** (mg/L)* Alphaproteobacteria 01 LT3b Stem. Vinhcuu 0. Dinhquan 0. Screening for Biofertilizer Activities Table 3.97 f 0. LT7.8.364 de Raoultella planticola strain ALK314 21 MR5 Root. Vinhcuu 1.476 g 02.15 and 20 days inoculation for P2O5). phosphate and other nutrients [30].01 f 0. LR7.702 ab LR2 Sphingomonas sp.046 c T12 99 (KM269038) 20 R16 Root. Several isolates have good plant growth activities as LT1. Nitrogen fixation. Trangbom 0. Trangbom 0. 16S rDNA Gene Amplification.08 c 1. Klebsiella pneuminiae LT1 have relationship closely and cluster A12 composed of Data were recorded at 4 times (2.46 f 0. Endophytes also promote plant growth by a number of similar mechanisms as phosphate solubilization activity [31][32].074 k 01. Vinhcuu 2. Xuanloc 0. LR7.58 f 0. 3.183 ij 26.47 f 0. Characterization and Identification of Endophytic Bacteria in Sugarcane (Saccharum spp.32 b 1.104 jk 01. LR4.513 de With this level the determination of nearest phylogenetic 25 R13 Root.35 c 1.828 ab Achromobacter xylosoxidans strain 08 LR1a Root. Vinhcuu 0. Xuanloc 0. The colonies of several endophyte isolates from stems and roots through the improved cycling of nutrients and minerals such of sugarcane as nitrogen.233 hi 16. and T12.74 e 1.191 ij 10.401 de Klebsiella variicola strain CICR14 15 MB7 Stem.655 f 26. Trangbom 0.879 d 32.768 e 24. In cluster A2 with 2 strains: Numbers following the same word not difference at 1% level Enterobacter oryzae LT7 and Novosphingobium sp. Trangbom 0.27 c 1. R15b (Vinhcuu)(bold number in Table 2). Electron micrographs of cell proteobacteria (2 strains) and Gamma-proteobacteria (5 strains).57 f 0.4.334 gh 01.86% 11.844 ab T16 98 D32 (KM488475) 09 LT4b Stem.08 c 1. LR2.812 ab Klebsiella pneumoniae strain AG4 13 LT1 Stem.

based on 16S rRNA sequences. However. Many studies have reported the endophytic presence of Enterobacteriaceae members in various crop species [45].012 Achromobacter xylosoxidans T16 had a relationship closely Primer p515FPL 5’-GTGCCAGCAGCCGCGTAA-3’ because they were isolated on site. nucleotide wheat plant and our results also discovered Achromobacter diversity. [42] discovered 32 endophytic bacterial isolates in Brazilian sugarcane (stem and leaf tissues) and 14 strains were classified as the Enterobacteriaceae (Gamma-proteobacteria). most of the research on endophytic bacteria (Proteobacteria) [34][35][36] and three strains were classified has focused on diazotrophs. and Gamma-proteobacteria group had the highest values as comparison with Alpha and Beta. Herbaspirillum spp.73577 0. Genetic diversity of 10 strains Dinhquan and Trangbom. Achromobacter Polymorphism software version 4. are Gluconacetobacter diazotrophicus. Many studies have shown the potential of Pantoea spp. presence of diazotroph among the total of bacteria in sugarcane tissues seems to be low in Indian sugarcane [41]. the southeast of Vietnam belonged to representative strains along with related sequences retrieved from GenBank. Theta (Ɵ)(per group) etc… In this study. Raoultella planticola LR4 and 10 strains 0. Besides that. of which the main representatives to Alpha-proteobacteria (30%). p13B) showing relationships between Dong Nai province. Based on bio-safety and good characteristics. agglomerans has bases. Our results showed that 10 endophytic bacterial strains in sugarcane cultivating on Figure 4. Enterobacter oryzae on a Maximum-Likelihood analysis of 100 re-sampled datasets. Kluyvera (1 strain) and Klebsiella (1 strain).141 0. American Journal of Life Sciences 2014.0 [38]. Achromobacter insolitus R15b and Pantoea nucleotide diversity of sequences for each gene. rice seeds [48] and citrus leaves [49]. and its standard deviation (SƟ). Jha and Kumar [56] also identified a novel plant growth promoting Nucleotide polymorphism can be measured by many endophytic bacterium Achromobacter xylosoxidans from parameters. proteobacteria (20%) and Gamma-proteobacteria (50%) [39][5] and Azospirillum amazonense [40]. Magnani et al. The proportion of group and they distributed in three clusters sugarcane growth promotion successfully. the number fixation. Pi values explained xylosoxidans T16. the composed of five strains (Figure 5) in our result. For systemetic resistance induction [50] and protection against pests and plant-pathogenic microorganisms [51]. these bacteria may induce plant growth by increasing the nitrogen supply in nonsymbiotic associations [52]. This class includes Enterobacter cloacae and Pantoea agglomerans (formerly Erwinia herbicola)[44]. phosphate solubilization and IAA biosynthesis. proteobacteria group had the highest values. Gamma. xylosoxidans T16 having good characteristics as high nitrogen nucleotide diversity was estimated by Theta (Ɵ). isolates by using primers (p515FPL. solubilizing phosphorus [53] and stimulating phytohrmone production [54] and recent result of Quecine et al. 2(6): 361-368 365 were very closely but they orinated from sugar stems in Table 4. The scale bar indicates the phulogenetic distance sorresponding to 5 changes per 100 LT7 and Pantoea agglomerans T12. Theta values (per sequence) from S of SNP for DNA polymorphism were 4.1 as Figure 5. of segregating sites [37]. [55] applied Pantoea agglomerans 33. Phylogenetic tree for partial 16S rRNA gene sequences from 10 latosols and acrisols of two sites (Dinhquan and Trangbom). the more diversity among groups. such as halotypes (genes) diversity. Proteobacteria (gram-negative bacteria) with 50% strains are The numbers at the nods indicate the levels of bootstrap support (%) based Gammaproteobacteria among two strains. Primer p13B 5’-AGGCCCGGGAACGTATTCAC-3’ The bacterial endophytes has been studied and described as beneficial bacteria which are Gram-negative bacteria In sugarcane. Members of the Enterobactericeae family (Gamma-proteobacteria) are frequntly described as rhizosphere colonizers of sugarcane and other grasses [43]. Pantoea (3 strains). the higher agglomerans T12 to evaluate their effects on suganrcane values. among which were Enterobacter (9 strains). (Pi). Additionally. and P. Conclusion calculated for each group. acrisols in 4 districts of Dong nai province of the Eastern of . and it has also been isolated from potato stems [47]. From 14 field-grown sugarcane samples on latosol and proteobacteria group (Table 4).353±0.955±0. Nucleotide Theta (per site) Theta (per site) Cluster B composed of three strains: Achromobacter diversity from Eta from S (θ) insolitus R15b. this study These parameters were estimated by DNA Sequence selected 4 strains as Enterobacter oryzae LT7. been described to be an important corn and wheat endophyte [46]. two strains belonged to Beta. cultivated on acrisols in pot-experiment and the field trial.

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