ISOLATION AND CHARACTERIZATION OF RHIZOSPHERIC BACTERIA …

www.wjpps.com Vol 5, Issue 2, 2016.

34

Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences

ISOLATION AND CHARACTERIZATION OF RHIZOSPHERIC

BACTERIA IN SOYBEAN (Glycine max (L.) Merrill) CULTIVATED ON

FERRALSOLS OF BUONHO TOWN, DAKLAK PROVINCE, VIETNAM

*1Prof. Dr. Cao Ngoc Diep, Nguyen Ba Trung

2 and Dr. Van Thi Phuong Nhu

3

1Lecturer in Department of Microbiology Biotechnology, Biotechnology R&D Institute, Can

Tho University, Vietnam.

2Agronomy Engineer, Vinasoy Factory, Quang Ngai Sugar Company, Vietnam.

3Lecturer in Biology Department, Phu Yen University, Vietnam.

ABSTRACT

Rhizobacterial diversity and population dynamics in the ferralsols

rhizosphere of soybean grown in BuonHo town, DakLak province,

highland of Vietnam was studied. Soil rhizosphere samples were taken

in 24 sites of this region. Physical and chemical characteristics of soil

samples and total nitrogen-fixing and phosphate-solubilizing bacteria

counts were determined by drop plate count method together with 16S

rRNA gene fragments amplified from DNA using eubacterial universal

primers (8F and 1492R). A total of 43 isolates were isolated on two

media (Burk’s N-free and NBRIP) and all of them have ability of

nitrogen fixation and phosphate solubilization together with IAA

biosynthesis. Population of rhizobacteria correlated with soil pH and

organic matter content in soil closely (P<0.05) and there was

relationship between nitrogen-fixing and phosphate-solubilizing bacteria significantly. The

sequences from selected rhizobacteria (17 isolates) showed high degrees of similarity to those

of the GenBank references strains (between 98% and 99%). From 17 isolates, 10 belonged to

Bacilli while Proteobacteria having 1, 1 and 4 were Alpha, Beta and Gamma-Proteobacteria,

respectively while there was one was Bacteroides. Based on Pi value (nucleotide diversity),

Bacilli group had highest Theta value and Thete values (per sequence) from S of SNP for

DNA polymorphism were calculated from each group and Bacilli group had the highest

values in comparison to four groups. From these results showed that three strains (Bacillus

methylotrophicus BHN5, Bacillus amyloliquefaciens BH8 and Bacillus subtilis BHN16)

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 5.210

Volume 5, Issue 2, 34-50 Research Article ISSN 2278 – 4357

Article Received on

23 Nov 2015,

Revised on 14 Dec 2015,

Accepted on 03 Jan 2016

*Correspondence for

Author

Prof. Dr. Cao Ngoc Diep

Lecturer in Department of

Microbiology

Biotechnology,

Biotechnology R&D

Institute, Can Tho

University, Vietnam.


35


revealed promising candidates with multiple beneficial characteristics and they have the

potential for application as inoculants adapted to poor soils and local crops because they are

not only famous strains but also are safety strains for agricultural sustainable.

KEYWORDS: 16S rRNA, Ferrasols, Gene Sequence, Rhizobacteria, Rhizosphere, Soybean.

INTRODUCTION

Plant-microbial interactions have been a premier area of research interest; as plants present an

excellent ecosystem for microorganisms where different niches are exploited by a wide

variety of bacteria (Kuklinsky-Sobral et al., 2004). The rhizosphere is an environmental that

the plant itself helps create and where pathogenics and beneficial microorganisms constitute a

major influential force on plant growth and health (Baldani et al., 2002). Most plants depend

on soil, but plants and their associated microorganisms also play a crucial role in the

formation or modification of soil (Teraja et al., 2005) and microorganisms present in the

rhizosphere play important roles in the growth and in the ecological fitness of their plant host

(Ahemed and Kilbert, 2013). Microbial interactions with roots may involve either endophytic

or free living microorganisms and can be symbiotic, associative or casual in nature; beneficial

microorganisms include N2-fixing bacteria in association with legumes and interaction of

roots with mycorrhizal fungi and phosphate-solubilizing microorganisms in relation to plant

P uptake, enhancement of root growth (i.e. through plant growth promoting rhizobacteria.

Daklak province is situated in the highland of Vietnam, it locates from 107o20’03” to

108o59’43” E and from 12

o10’00” to 13

o24’59” N and Buon Ho is a district of Daklak

province, it locates the north of province (Figure 1). The soils are mainly red latosols (from

origin of volcanic mountain) or ferralsols (FAO classification) with a pH range of 5.28 - 5.45.

They are considered a good nutrient, with an average organic matter of 3%, a total nitrogen

range of 0.18 – 0.20%, but it has concentrations of low available phosphorus, cation

exchange capacity, exchangeable K (Trinh, 2012). Many kinds of crop such as rubber, coffee,

pepper, upland-rice, corn and soybean have been cultivated on ferralsols permenantly.

Soybean [Glycine max (L.) Merr.], is an important protein and oil source and is one of the

most important grain legumes in the world (Li et al., 2011); soybean is also planted on

ferralsols during wet season (April to November in year) but low grain yield (1 – 2 ton(s)/ha).

The application of native, adapted microorganisms might improve yields by direct plant

growth promotion and increasing grain yield, decreasing cost in soybean cultivation in order

to enhance income for the farmers. The aims of this study were (i) isolation and


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characterization of rhizopheric bacteria, (ii) analysis of relationship of beneficial bacterial

population with characteristics of ferralsols (iii) selection of best bacterial isolates and (iv)

identification of these bacterial isolates. These bacteria can be considered promising

candidates for application in sustainable agricultural management for this region.

Figure: 1 The location was examined in this study and BuonHo town (Daklak

province)[with dark blue] and ferralsols were presented soils with reddish brown

latosols and red & brown latosols

MATERIALS AND METHODS

Soil sample and isolation of bacteria

The soybean plants were sampled at the stage of flowering during the rainy season (July

2013) from the fields of BuonHo town, DakLak province which is the biggest soybean

cultivation area in highland of Vietnam. Samples were collected on 8 sites (3 samples in each

site) of this town because soybean has been planted on ferralsols. Samples were taken whole

plant with stem, root (10-20 cm depth) together with soil which around soybean plants;

samples were kept in 18oC plastic box before tranferred to laboratory in Can Tho University.

Rhizosphere soil around soybean plants were collected to moving the soil that adherered to

the roots (stem and root of soybean plant will be used in further experiment) and they were

kept to refrigerator for counting by viable drop plate count (Hoben and Somasegaran, 1982)

and isolation of nitrogen-fixing bacteria in Burk’N free media (Park et al., 2005) and

phosphate-solubilizing bacteria in NBRIP media (Nautiyal, 1999); 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


37


colonies which developed on two media (or microbes having N2-fixing and phosphate-

solubilizing ability).

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 day inoculation (DAI) and inorganic phosphate solubilization 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 based on the colorimetric method (Gordon and Weber

1951). 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.

Besides that, the pH of rhizosphere soil was measured in a 1:5 soil to water (w/v) mixture in

20 min and read on a Jenway 3510 pH meter, N total were measured using the micro-

Kjeldahl digestion method, the colorimetric P determination was based the method of

ammonium molypdate method (Murphy and Riley, 1962), organic carbon measured by

Walkley Black method (Andersen and Ingram, 1993).

16S rDNA gene amplification and sequencing

Bacterial DNA was isolated following published protocols (Neumann et al., 1992);

Amplification of 16S rDNA by PCR was carried out using the universal primers 8F and

1492R (Turner et al., 1999). The 50 µL reactionmixture 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 95

oC (30 s), annealing

at 55oC (30 s), extension at 72

oC (90 s) and a final extension at 72

oC (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


38


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

and phosphate solubilization ability) and 17/43 isolates of 24 sites were chosen to sequence

and the results were compared to sequences of GenBank based on partial 16S rRNA

sequences to show relationships between PGPR strains (Tamura et al., 2011) and

phylogenetic tree were constructed by the neighbor-joining method using the MEGA

software version 6.06 based on 1000 bootstraps.

SNPs Discovery

The sequence date from 17 root-associated bacterial isolates were analysed with

SeqScape@Software (Applied Biosystem, Foster City, CA, USA). SeqScape is a sequence

comparison tool for variant identification, SNP discovery and validation. It considers

alignment depth, the base calls in each of the sequnces and the associated base quality values.

Putative SNPs were accepted as true sequence variants if the quality value exceeded 20. It

means a 1% chance basecall is incorrect.

Nucleotide Diversity (Ө)

Nucleotide diversity (Ө) was calculated by the method described by Halushka et al. (1999)

n

Ө = K/aL a = ∑ l/(i - l)

i=2

where K is the number of SNPs identified in an alignment length, n is alleles and L is the

total length of sequence (bp).

Data analyses

Data from ammonium, orthophosphate and IAA concentrations in media were analysed in

completely randomized design with three replicates and Duncan test at P=0.01 or P=0.05

were used to differentiate between statistically different means using SPSS version 16.

RESULTS AND DISCUSSIONS

Soil Characteristics

DakLak province in highland of Vietnam has large cultivated soybean area and soybean has

been cultivated on ferralsols mainly with coffee, corn, pepper, rubber and the results from 24

soil samples (8 villages of BuonHo town) showed that characteristic of ferralsols is low soil

http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi


39


pH together with low available P concentration, and N total in ferralsols also are low (Table

1) except organic matter content in soil is high (4-5%). However nitrogen – fixing and

phosphate-solubilizing bacterial population in ferralsols were low (approx. from one hundred

to ten thousand cells per dry soil gram). According to Boku (www.bodenkunde-projekte-

berlin.de/tropics), ferralsols are the classical deeply weathered red or yellow soils of the

humid tropics. They are dominated by low activity clays (LAC), manly kaolinite and

sesquioxides, ferralsols (FAO) and their international names are latosol (Brasil), Oxisols (Soil

Taxonomy, USA), Sols ferraltiques (France) and Lateric soils (Russia); their chateristics are

low soil-pH, low concentrations of dissolved weathering products but contains relatively

much Fe and Al. From the unavailable factors affect to survival of nitrogen-fixing and

phosphate-solubilizing bacteria in soil.

Table: 1 Soil characteristics and N2-fixing and phosphate-solubilizing bacterial

population in ferralsols rhizosphere

Soil

sample

site

Soil

pH

N

total

(%)

Available P

(mg P2O5/kg

soil)

Organic

matter

content

(%)

N2-fixing

bacteria

population

P-solubilizing

bacteria

population

CFU log10/g soil

DT26CS1.1 5.42 0.112 6.669 3.705 2.924 4.548

DT26CS1.2 5.39 0.124 6.617 3.665 2.911 4.448

DT26CS1.3 5.28 0.131 6.718 3.689 2.891 4.514

DT26CS2.1 5.32 0.121 5.927 3.978 2.832 4.643

DT26CS2.2 5.22 0.134 5.901 3.914 2.811 4.612

DT26CS2.3 5.28 0.137 5.814 3.885 2.745 4.558

DP1.1 5.47 0.122 6.369 3.783 2.777 3.865

DP1.2 5.44 0.131 6.314 3.714 2.717 3.815

DP1.3 5.31 0.125 6.225 3.681 2.512 3.844

DP2.1 5.38 0.111 6.322 3.622 2.919 3.888

DP2.2 5.22 0.104 6.125 3.278 2.612 3.018

DP2.3 5.27 0.118 6.231 3.655 2.845 4.012

CJCS.1 5.42 0.101 6.659 3.705 3.045 3.881

CJCS.2 5.32 0.112 6.607 3.978 3.025 3.815

CJCS.3 5.47 0.124 6.632 3.811 3.121 3.912

CJCM.1 5.38 0.112 6.322 3.822 2.716 4.806

CJCM.2 5.24 0.107 6.258 3.759 2.701 4.709

CJCM.3 5.31 0.122 6.268 3.771 2.691 4.759

DT26CM.1 5.12 0.112 6.658 3.001 2.442 5.112

DT26CM.2 5.27 0.124 6.625 3.907 3.314 5.147

DT26CM.3 5.33 0.133 6.558 3.814 3.369 5.089

SI DT26CM.1 5.22 0.102 6.325 3.771 2.653 4.514

SI DT26CM.2 5.29 0.115 6.258 3.901 2.615 4.125

SI DT26CM.3 5.11 0.121 6.159 3.812 2.581 5.211


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The results from Table 2 showed that there were a significant linear relationship between

population of N2-fixing and phosphate-solubilizing bacteria and soil pH at P<0.05 and

P<0.01 (y=1.0201x - 2.5988, r=0.501*; y= - 2.681x + 18.654, r=0.515**, respectively) and

both of microbes with organic matter content were a linear relationship significantly at

P<0.05 (y=0.421x + 1.2459, r=0.450*; y=1.8546x - 2.6569, r=0.523*, respectively) and there

was a significant linear relationship between N2-fixing bacteria population with available P

concentration in soil at P<0.05 (y=0.3504x – 0.592, r=0.461*) but there were no difference

between population of phosphate-solubilizing bacteria with available phosphorus

concentration in soil significantly; N total concentration in soil did not relate with N2-fixing

and phosphate-solubilizing bacteria population. Besides, there was relationship between

phosphate-solubilizing bacteria and N2-fixing bacteria population in soil significantly

(y=0.9412x + 1.63, r=0.451*). These results showed that soil pH and organic matter content

in soil are two important factors affecting to populations of nitrogen-fixing bacteria and

phosphate-solubilizing bacteria in soil while N total in soil did not correlate with nitrogen-

fixing and phosphate-solubilizing bacteria population in ferralsols. Soil pH decreased but

phoshate-solubilizing bacterial population increased (P>0.01) perhaps these P-solubilizing

bacteria strains released organic acids to dissolve non-soluble P in soils to soluble P, however

organic matter is an important factor which affects to survival of both of bacterial strains in

ferralsols.

Table: 2 The relationship between population of N2-fixing and phosphate-solubilizing

bacteria with pH, N total, and available phosphorus and organic matter content in

ferralsols.

Characteristics Population (cfu/dry soil gramme)

N2-fixing bacteria Phosphate-solubilizing bacteria

Soil pH r = 0.401* r = 0.515**

Y = 1.0201X – 2.5988 Y = - 2.6817X + 18.654

N total concentration (%) r = 0.191 (ns) r = 0.268 (ns)

Y = 3.6969X + 2.3798 Y = 13.572X + 2.7123

Available P (mg/kg soil) r = 0.461* r = 0.125 (ns)

Y = 0.3504X – 0.592 Y = 0.249X + 2.7444

Organic matter (%) r = 0.450* r = 0.523*

Y = 0.4214X + 1.2459 Y = 1.8546X – 2.6569

Forty-three bacterial isolates were isolated from 24 soil samples in two media (Burk’N free

and NBRIP medium)(Table 3) and all isolates grew well on both of media (they have nitrogen

fixation and phosphate solubilization ability)(Figure 2a and Figure 2b).


41


The results showed that these bacterial isolates synthesized high ammonium concentration

but they solubilized big quantity of phosphorus. Especially BHN5 and BHN8 isolates

synthesized the highest ammonium comcentration and BHN8 isolate solubilized high amount

of phosphorus (Table 4).

Figure: 2b The colonies of four isolates in NBRIP medium with the halos around the

colonies and changed the color of medium because of organic acids

Table: 4 Ammonium (NH4+) and available P (P2O5)(mg/L) of 43 bacterial isolates

No Bacterial

name

Ammonium

(NH4+)

conc.

(ml/L)*

Available P

(P2O5)

conc.

(ml/L)**

No Bacterial

name

Ammonium

(NH4+)

conc.

(ml/L)*

Available P

(P2O5)

conc.

(ml/L)**

01 control 0.000 l 000.00 l 23 BHN22 0.286 k 138.15 i

02 BHN1 0.731 d 108.98 j 24 BH1 0.294 k 324.91 d

03 BHN2 0.425 i 135.71 i 25 BH2 0.340 j 228.57 fg

04 BHN3 0.882 b 140.34 i 26 BH3 0.690 d 219.81 g

05 BHN4 0.297 k 86.60 k 27 BH4 0.477 i 215.81 g

06 BHN5 0.961 a 305.68 de 28 BH5 0.452 i 344.34 c

07 BHN6 0.413 i 276.94 f 29 BH6 0.567 fg 209.91 g

08 BHN7 0.691 de 263.07 f 30 BH7 0.441 i 194.61 gh

09 BHN8 0.957 a 460.51 a 31 BH8 0.514 g 221.03 fg

10 BHN9 0.875 b 353.15 c 32 BH9 0.494 hi 144.61 h

11 BHN10 0.588 f 201.98 h 33 BH10 0.762 c 178.55 h

12 BHN11 0.395 j 212.27 gh 34 BH11 0.456 i 166.81 h

13 BHN12 0.539 g 382.55 b 35 BH12 0.442 i 241.41 f

14 BHN13 0.455 i 302.58 de 36 BH13 0.471 i 171.21 h

15 BHN14 0.608 ef 234.74 g 37 BH14 0.611 e 119.41 i

16 BHN15 0.446 i 149.46 i 38 BH15 0.678 d 165.91 h

17 BHN16 0.915 b 136.04 i 39 BH16 0.611 e 161.59

18 BHN17 0.872 c 144.96 i 40 BH17 0.738 cd 154.41 h

Figure: 2a The colonies of several isolates

on Burk’s N free


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19 BHN18 0.892 b 235.87 g 41 BH18 0.666 d 127.71 ij

20 BHN19 0.641 de 225.66 gh 42 BH19 0.609 e 150.72

h

21 BHN20 0.440 i 324.89 d 43 BH20 0.709 cd 170.83 h

22 BHN21 0.717 d 288.26 ef 44 BH21 0.667 d 136.65 i

CV (%) 10.2 8.97 CV (%) 10.2 8.97

*means of 4 times (2,4,6 and 8 days after incubation in Burk’s free N medium)

**means of 4 times (5,10,15 and 20 days after incubation in NBRIP medium)

Means within a column followed by the same letter/s are not significantly different at p<0.01

However, all isolates were chosen to test IAA concentration in vitro in NBRIP medium

adding with 100 mg tryptophan/L. After 4 days incubation, there were twenty isolates

synthesed high IAA concentration (>1 mg/L)(Table 5).

Table: 5 IAA concentration (ml/L) of 20 isolates

No Bacterial

name

IAA

(mg/L) No

Bacterial

name

IAA

(mg/L) No

Bacterial

name

IAA

(mg/L)

01 BHN1 2.523 08 BHN18 3.884 15 BH5 1.608

02 BHN3 2.693 09 BHN21 3.439 16 BH8 1.652

03 BHN5 3.041 10 BHN13 3.339 17 BH10 1.237

04 BHN8 2.692 11 BH1 1.569 18 BH12 1.570

05 BHN9 3.813 12 BH2 1.689 19 BH17 1.149

06 BHN16 3.071 13 BH3 1.437 20 BH20 1.742

07 BHN17 2.684 14 BH4 1.189

Almost their colonies have round-shaped; milky, white clear (on Burk’s medium) and yellow,

reddish yellow (on NBRIP medium); entire or loabate margin (Figure 2a and 2b); 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 (2014), Tam and Diep

(2014)(Figure 2b).

The cells were observed by SEM and appearded as short rods and most of them have motility

(Figure 3).


43


Figure: 3 Electron micrograph of cells

The fragments of 1495 bp 16S rRNA were obtained from PCR with 8F and 1492R 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 (10/17

isolates), 4 isolates belonged to Gammaproteobacteria, 1 was Alpha, 1 was

Betaproteobacteria and 1 was Bacteroides (Table 5).

Table: 5 Phylogenetic affiliation of isolates on the basis of 16S rRNA genes sequences by

using BLAST programmes in the GenBank database based on sequences similarity

Taxonomic Group and

Strain Closest species relative

Similarity

(%)

Bacilli

BH5 Bacillus subtilis A2 (KC433738) 99

BH8 Bacillus sonorensis BGAS 39I (KT895841) 99

BHN5 Bacillus methylotrophicus SH1 (KM096464.1) 99

BHN8 Bacillus amyloliquefaciens AB-525 (KJ879953.1) 99

BHN9 Bacillus flexus KP031r (KT200442.1) 99

BHN12 Bacillus megaterium BLZ01 (KP343685.1) 99

BHN16 Bacillus subtilis strain D29 (KC441767.1) 99

BHN17 Bacillus aryabhattai PS15 (KR063195.1) 98

BHN18 Bacillus megaterium SH6-1 (FJ461752.1) 98

BHN20 Bacillus megaterium strain HB22 (KM659224.1) 98

Alphaproteobacteria

BH20 Brevundimonas vesicularis KK6 (KF975414) 99

Betaproteobacteria

BH12 Burkholderia vietnamiensis AU4i (KF114029) 99

Gammaproteobacteria

BH1 Acinetobacter schindleri BL AcIso69 (FJ860880) 99


44


BH2 Acinetobacter schindleri YNB103 (JQ039984) 98

BH3 Acinetobacter schindleri URT27(JX315564) 99

BH17 Acinetobacter schindleri BGSLP29 (KP192007) 99

Bacteroides

BH10 Sphingomonas hankookensis S5-278 (JQ660181) 99

A neighbor-joining tree phylogenetic tree in these isolates showing the two clusters: cluster A

divided into two cluster A1 and A2. Cluster A1with cluster A111 had 5 isolates as Bacillus

arybhattai BHN17, B. megaterium BHN20 and Burkholderia vietnamiensis BH15 correlated

very closely and B. flexus BHN09 and B. sonorensis BH13 had the high relationship while

cluster A112 had Bacillus methylotrophicus BHN05, B. megaterium BHN12 and

Acinetobacter schindleri BH06, especially cluster A12 only had one strain Acinetobacter

schindleri BH11. Cluster A2 composed of three strains: Bacillus megaterium BHN18, B.

amyloliquefaciens BHN08 and Bacillus subtilis BH12. Cluster B had cluster B1 with two

strains: Acinetobacter johnsonii BH16 and Brevudimonas vesicularis BH17 while cluster B2

composed of three strains: Bacillus subtilis BHN16, Acinetobacter schindleri BH09 and

Sphingomonas hankookensis BH14.

Figure: 4 Phylogenetic tree showing the relative position of rhizobia (PGPR) by the

neighbor-joining method of complete 16S rRNA sequence.


45


Bootstrap values of 1000 replicates are shown at the nodes of the trees.

Theta values (per sequence) from S of SNP for DNA polymorphism were calculated for each

group and Bacilli group had the highest values as comparison with Proteobacteria and

Bacteroides (Table 6) and Pi and Theta of Alpha, Betaproteobacteria and Bacteroides groups

were very low.

Table: 6 Nucleotide diversity (Ɵ) values of two EST’s using the programme DNASp 4.0

(Watterson, 1975)

ESTs Bacilli Gammaproteobacteria

Ncleotide diversity (Pi) 0.0337 ± 0.0053 0.0109 ± 0.0033

Theta (per sequence) from Eta 0.0288 ± 0.0122 0.01134 ± 0.0064

The rhizospheric bacteria has been studied and described as beneficial bacteria with Gram-

positive bacteria presented on both of media and its occuoied over 50% among 17 strains in

our result (Figure 5).

Figure: 5 The proportion of group and they distributed in four clusters

Nucleotide polymorphism can be measured by many parameters, such as halotype (genes)

diversity, nucleotide diversity, Pi, Theta (θ)(per gourp) etc...In this study, nucleotide diversity

was estimated by Theta, the number of segregating site (Watterson, 1975), and its standard

deviation (Sθ). These parameters were estimated by DNA Sequences Polymorphism sofware

version 4.0 (Rozas and Rozas, 2005). Pi values explained nucleotide diversity of sequences

for each gene, the higher values, the more diversity among group, Bacilli group had the

highest.


46


In 10 bacilli strains had variation of nucleotide from position 200 to position 500 (Figure 6).

Figure: 6 Variation of nucleotide from position 200 to position 500

Chen et al. (2006) showed that phosphorus solubilization activity of PSB is associated with

the release of organic acid and a drop in the pH of the medium. Especialy, N2-fixing and

phosphate-solubilizing bacterial population in ferralsols were lower than in acrisols (Thanh

and Diep, 2014; Tam and Diep, 2014, 2015), even through organic matter content and soil pH

of ferralsols are higher than acrisols, perhaps Fe and Al concentrations in ferralsols are high

and this affects to survival of both of them. The plant-beneficial rhizobacteria may decrease

the global dependence on hazardous agricultural chemicals which destabilize the agro-eco-

systems (Ahemad and Kilbert, 2013). The rhizobacteria are the dominant deriving forces in

recycling the soil nutrients and consequently, they are crucial for soil fertility (Glick, 2012).

The plant growth promoting rhizobacteria (PGPR), are characterized by the following

inherent distinctiveness: (i) they must be proficient to colonize the root surface (ii) they must

survive, multiply and compete with other microbiota, at least for the time needed to express

their plant growth promotion/protection activities, and (iii) they must promote plant growth

(Kloepper, 1994). Many plant-associated bacteria are well known for their capacity to

promote (Compant et al., 2010).

‘Bacilli’ AEFB are a diverse group with wide distribution in agricultural soils that contribute

both directly and indirectly to plant development (McSadden, 2004). Numerous Bacillus and

related genera with plant growth promoting (PGP) activities have been isolated from

soybean, corn, sorghum and wheat rhizospheres (Cohelo et al., 2007; Beneduzi et al, 2008a;

Beneduzi et al., 2008b), Bacilli are bacteria having endospore and this support their survival

in drought condition of ferralsols in dry season (from November to April). Genus

Sphingomonas discovered and presented the physiology and ecology by White et al. (1996)

and Ali et al. (2010) reported genus Sphingomonas has a ability of nitrogen fixation,


47


phosphate and potassium solubilization and Sphingomonas together with Burkholderia,

Bacillus, Pseudomonas have been used biofertilizing microbes. Recently Bumunang and

Babalota (2014) examined the rhizobacteria from field grown GM maize in South Africa as

follows species of Pseudomonas, Aeromonas, Sphingomonas, Burkholderia, Bacillus,

Stenotrophomonas, Achromobacter, Ewingella, they have catalase activity, ammonia

production, IAA production, phosphate solubilisation and antifungal activity. Our results also

found the rhizospheric bacteria in ferralsols with species of bacilli, alphaproteobacteria as

Brevundimonas vesicularis, betaproteobacteria as Burkholderia, gammaproteobacteria as

Acinetobacter and Bacteroides as Sphingomonas.

CONCLUSION

From 24 ferralsols samples of soybean regions in BuonHo town, DakLak province, the

highland of Vietnam, 43 isolates were isolated on two media (Burk’s N free and NBRIP) and

they were identified as rhizospheric bacteria and 17 isolates having good plant growth

promotion were chosen to analyse their relationship. These isolates were identified as Bacilli

(more than 50%), Brevundimonas, Acinetobacter, Burkholderia, Sphingomonas on ferralsols.

Among them, there are three strains will be suggested to produce for soybean cultivation on

ferralsols in the future.

ACKNOWLEDGEMENTS

This work was supported by Vinasoy Factory, Quang Ngai Sugar Company, Quang Ngai

province, Vietnam. The authors thank the helpness of Microbiology BSc. Students and

technicians in the Environment Microbiology Laboratory, Biotechnology R&D Institute, Can

Tho University, Vietnam; especially Associate Professor Dr. TRUONG TRONG NGON,

Head of Molecular Biotechnology Department, Biotechnology R&D Institute, Can Tho

University, Vietnam for analysing molecular data.

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ISOLATION AND CHARACTERIZATION OF RHIZOSPHERIC BACTERIA …