ISOLATION AND CHARACTERIZATION OF ENDOPHYTIC ...

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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences

ISOLATION AND CHARACTERIZATION OF ENDOPHYTIC

BACTERIA IN SOYBEAN ROOT NODULES

*1Prof. Dr. Cao Ngoc Diep, Nguyen Thi Xuan My

2, Dr. Van Thi Phuong Nhu

3

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

Tho University, Vietnam.

2 MSc. Student of Biotechnotology, Biotechnology R&D Institute, Can Tho University,

Vietnam.

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

ABSTRACT

Sixty-eight entophytic bacterial isolates were isolated from 70 soybean

nodules of soybean plants which collected at Buonho town, DakLak

province (38), Cujut district, DakNong province (19) and Can Tho

city, Mekong Delta (11), Vietnam; they developed on two kinds of

medium (PDA and TSA) after 2 or 3 days incubation and they made

the pellicles on semi-solid media. The bacterial isolates were tested in-

vitro for plant growth promoting properties including nitrogen fixation,

phosphate solubilization and IAA production together with evaluating

effects on the growth of soybean plants on pots. All of them had the

ability of ammonium synthesis, phosphate solubilization and IAA

biosynthesis however the isolates originated from western highland

(DakLak and DakNong province(s) having the big potential of

phosphate solubilization in comparison to the isolates from Can Tho city (Mekong Delta).

The sequences from selected nitrogen-fixing and phosphate-solubilizing bacteria (16 isolates)

showed high degrees of similarity to those of the GenBank references strains (between 97%

and 99%). From 16 isolates, 9 belonged to Bacilli and 7 were Gamma-Proteobacteria. 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 gammaproteobacteria. From these

results showed that there are two strains as Paenibacillus lautus CJE17 and Bacillus

megaterium CJE10 revealed promising candidates with multiple beneficial characteristics and

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 6, 222-241 Research Article ISSN 2278 – 4357

*Corresponding Author

Prof. Dr. Cao Ngoc Diep

Lecturer in Department of

Microbiology

Biotechnology,

Biotechnology R&D

Institute, Can Tho

University, Vietnam.

Article Received on

05 April 2016,

Revised on 26 April 2016,

Accepted on 16 May 2016

DOI: 10.20959/wjpps20166-6971


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they have the potential for application as inoculants adapted to poor soils and local crops

because they are not only best strains but also combine with rhizobia strains for improvement

of good grain yield and quality seed of soybean cultivation on ferralsols in the future.

KEYWORDS: 16S rRNA Gene Sequence, Endophytes, Ferrasols, Nitrogen-Fixing Bacteria,

Phosphate-Solubilizing Bacteria, Root-nodule of soybean.

INTRODUCTION

In recent years, interest in endophytic micro-organisms has increased, as they play a key role

in agricultural environment and are promising because of their potential use in sustainable

agriculture (Dudeja and Giri, 2014). Endophytes have been found in almost every plant

studied (Ryan et al., 2008); endophytes are sheltered from environmental stresses and

microbial competition by the host plant, and they seem to be ibiquitous in plant tissues,

having been isolated from flowers, fruits, leaves, stems, roots, and seeds of various plant

species (Kobayashi and Palumbo, 2000). Endophyte-plant associations have been found to

improve plant health and may help host plant to rescue from various biotic and abiotic

stresses (Hasegawa et al., 2006; Sapak et al., 2008).

Soybean (Glycine max (L.) Merrill) is an Asiatic leguminous plant, occupying large acreages

of land worldwide for its oil and protein (Fayzalia et al., 2009). Rhizobia are perhaps the best

known beneficial plant-associated bacteria because of the importance of the nitrogen fixation

that occurs during the Rhizobium-legume symbiosis (Hung et al., 2007). Endophytic bacteria

have been isolated from legume plants such as alfalfa (Gagne et al., 1987), clover (Sturz et

al., 1997), pea (Elvira-Recuenco and van Vuurde, 2000) and soybean (Oehrle et al. 2000).

Besides that, Sturz et al. (1997) reported the isolation of 15 non-rhizobial species from clover

root nodules, eight of which were found only in root nodule tissues.

Root nodules also accommodate various non-nodulating bacteria having definite influence on

the survival, nodulation and grain yield of crop and their densities are reported to be very

high (Mishra et al. 2009; Tariq et al., 2012). These endophytic bacteria live inside the nodule

tissues without subtantially harming or gaining benefit other than shelter (Kobayashi and

Palumbo, 2000). Furthermore, these bacteria act synergistically with rhizobia to improve

nodulation and nitrogen fixation (Duangpaeng et al. 2012). Non-nodulating bacteria for the

first time isolated from nodules of legume plant were identified as Agrobacterium

radiobacter (Berjerinck and Delden, 1902).


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This study was aimed to isolate the non-nodulating endophytic bacteria from the root nodules

of soybean plants and characterize in vitro for plant growth promoting properties. Using 16S

rRNA gene sequence analysis, we also studied the taxonomic position of these non-

nodulating endophytic bacteria and compared endophytic bacterial isolates originated from

nodules of root soybean cultivating on ferralsols in western highland and alluvilal soil in the

Mekong Delta and their effects on growth of soybean plant (in-pot experiment).

MATERIALS AND METHODS

Plant sample and Isolation endophytic bacteria in soybean nodules

Soybean plants used in the experiment was a cultivar (cv. CuJut)(Glycine max L. Merr), was

cultivated at three sites (Cujut district, DakNong province; Buonho district, DakLak province,

highland of Vietnam and Can Tho city, Mekong Delta, Vietnam). Plant samples were

collected at the flowering-stage (35-40 days after sowing), five samples were carefully

removed, washed under tap water to remove soil, and separated into roots and nodules.

Nodules were put in beaker, soaked in distilled water, and drained. They were rinsed in 70%

ethanol for 30 s and then sterilized with 0.1% HgCl2 for 3 min (Hung et al., 2007). After that,

nodules were washed ten times with sterile water (Gagne et al., 1987). Surface-disinfected

tissue was aseptically macerated with homogenizers and tissues were diluted with 1 mL

sterile water. One hundred microliters from appropriate dilutions were palted on two different

media, viz potato dextrose agar (PDA) and tryptic soy agar (TSA)(Collins and Lyne, 1984).

Morphological characterization of the isolates was carried by Gram staining. For motility,

each isolate was spot-inoculated on the center of semi-soloid nutrient agar plates (0.2% agar)

and incubated at 30oC (Hung et al., 2007). Cell shape was observed under light microscope,

colony characterization as size, color, shape were recorded at 2 – 3 days after plating into

petri-dishes.

Characterization of endophytic bacteria for plant growth promoting attributes

Bacterial isolates were also studied in vitro for plant growth promoting properties including

indole acetic acid (IAA) production, plant growth in pots, nitrogen fixation, solubilization of

phosphate.

For indoleacetic acid production, 5 µl for log phase culture was inoculated in 5 ml of LB

(Luria-Bertani; Bacto-Tryptone 10.0 g/l, yeast extract 5.0 g/l, NaCl 5.0 g/l) broth with L-


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tryptophane and incubated on shaker for 24 h. Auxin quantification was carried out following

the method of Gordon and Weber (1951).

For nitrogen fixation ability and phosphate solubilization: the ability to fix N2 was tested on

Burk’N free liquid medium incubation 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

incubated at 30oC and the P2O5 concentration was measured by ammonium molypdate

method after 5, 10, 15 and 20 DAI (Tam and Diep, 2015).

16S rDNA gene amplification and sequencing

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

following primers were used for PCR amplification of 16S ribosomal DNA: p515FPL

(Relman et al., 1992) and p13B (Relman et al., 1990)(Zinniel et al., 2002). The 50 µL

reaction mixture consisted of 2.5 U Taq Polymerase (Fermentas), 0.1 mM of each

desoxynecleotide triphosphate, 1.5 mM magnesium chloride, 0.4 mM spermidine (Sigma),

10 pM of each primer (Fermentas) and 10 ng DNA, 10% (vol/vol) dimethyl disulfide

(Fermentas). The thermocycling profide was carried out with an initial denaturation at 94oC

(3 min) followed by 30 cycles of denaturation at 94oC (60 s), annealing at 57

oC (60 s),

extension at 72oC (120 s) and a final extension at 72

oC (4 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. Partial 16S rRNA gene of selectived

isolates in each site was 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 ability of nitrogen fixation, phosphate solubilization and IAA

synthesis) and 16 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 endophytic 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


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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).

Effect of soybean plants

According to the results, the best isolates together with rhizobia strains were selected for the

greenhouse assay. One hundred soybean seeds (Glycine max L. Merrill, cv. Cujut) were

sterilised with sodium hypochloride (25 g l-1

) followed six rinses in sterile distilled water. The

seeds were placed in a sterile dish and mixed with 4 ml of inoculant (mixture of each isolate

with rhizobia strain and population of each isolate over 108

cell/ml) in 3 hour incubation.

Four inoculated seeds were sown in each pot (220x180x250 cm), which had been previously

filled with 5 kg of soil and five replications (pots) per each treatment. The experiment was a

completely randomized design with eleven treatments as follows: control 1 (no fertilizer,

without bacteria), control 2 with 100 kg N/ha without P and bacteria, Endophytic bacteria

strain 1 + 20 kg N/ha, Endophytic bacteria strain 2 + 20 kg N/ha, Rhizobial strain 1 +

Endophytic bacteria strain 1 + 20 kg N/ha, Rhizobial strain 1 + Endophytic bacteria strain 2

+ 20 kg N/ha, Rhizobial strain 2 + Endophytic bacteria strain 1 + 20 kg N/ha, Rhizobial

strain 2 + Endophytic bacteria strain 2 + 20 kg N/ha, Endophytic bacteria strain 1 and strain 2

+ 20 kg N/ha, Rhizobial strain 1 + Endophytic bacteria strain 1 and strain 2 + 20 kg N/ha,

Rhizobial strain 2 + Endophytic bacteria strain 1 and strain 2 + 20 kg N/ha. Plants were

grown in the greenhouse for a 95-day period under natural temperature (avery day, 32-34oC

and average night, 26-28oC) and light condition (800 lux). One mililiter of each isolate was

added per seed by micropipette at 0 days after sowing (DAS). Plants were watered with

sterile water during 10 DAS after that they were irrigated by distilled water. At harvest, yield

component and grain yield were recorded and soil samples were analysed with parameters as


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pH, N total, Available P, and Organic matter.

Data analyses

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

completely randomized design with three replicates. Yield component and grain yield

together with pH and soil characteristics were analysed with five replications. Duncan test at

P=0.01 or P=0.05 were used to differentiate between statistically different means using SPSS

version 16.

RESULSTS AND DISCUSSION

Plant sample and Isolation endophytic bacteria in soybean nodules

From colonies were plated and develop on TSA and PDA media after incubation at 30oC; we

wished to isolate non-rhizobia from within soybean root nodules. Total of 68 endophytic

bacterila isolates (consisted of 38 isolates from soybean nodules at Buonho, DakLak, 19

isolates from Cujut, DakNong and 11 isolates from CanTho city). The entophytic bacteria

developed in the pelicles of semi solid (in two kinds of medium) after 36 h after incubation in

semi-solid (Figure 1) as the previous results of Thu Ha et al. (2009).

Figure: 1 Endophytic bacteria made pellicles in semi-solid (TSA and PDA media) after

36 h incubation at 30oC

Almost their colonies have round-shaped; milky, white clear (on PDA’s medium and TSA

medium); entire or loabate margin (Figure 2); diameter size of these colonies varied from 0.2

to 3.0 mm very large and all of them are Gram-positve and Gram-negative by Gram stain.

Pelicles appeared on surface of

semi- solid medium


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Figure: 2 Characteristics of colonies of bacterial isolates after grown on two kinds of

medium

From 70 soybean nodule samples of 3 sites, 68 endophytic bacterial isolates were isolated on

two kinds of medium (Table 1, Table 2 and Table 3).

Table: 1 Ammonium (NH4+) and Available P (P2O5)(mg/l) concentration of 38

endophytic bacterial isolates from soybean nodules at Buonho site

No Bacterial

name

Ammonium

(NH4+)*

concentration

Available P

(P2O5)**

concentration

No Bacterial

name

Ammonium

(NH4+)*

concentration

Available P

(P2O5)**

concentration

01 Control 0.00 o 0.00 n 21 BHE20 0.27 n 28.23 d

02 BHE01 0.78 m 15.19 i 22 BHE21 0.87 m 23.96 f

03 BHE02 0.31 n 9.20 lm 23 BHE22 4.90 e 35.27 b

04 BHE03 3.73 f 17.64 h 24 BHE23 0.33 n 27.69 d

05 BHE04 2.04 j 14.90 j 25 BHE24 5.14 e 28.58 d

06 BHE05 1.63 k 10.07 l 26 BHE25 0.31 n 18.49 gh

07 BHE06 3.43 g 0.31 n 27 BHE26 0.25 n 30.22 c

08 BHE07 3.90 f 19.43 g 28 BHE27 0.32 n 27.51 de

09 BHE08 4.07 f 1.74 n 29 BHE28 0.301 10.57 l

10 BHE09 1.21 l 10.66 l 30 BHE29 0.32 n 31.31 c

11 BHE10 0.25 n 14.27 i 31 BHE30 0.30 n 23.05 f

12 BHE11 0.19 no 15.19 i 32 BHE31 0.25 n 30.82 c

13 BHE12 8.51 c 10.22 l 33 BHE32 0.43 n 12.55 k

14 BHE13 10.14 b 13.81 j 34 BHE33 1.20 l 17.32 h

15 BHE14 0.30 n 13.99 j 35 BHE34 3.30 g 25.67 e

16 BHE15 3.39 g 8.82 m 36 BHE35 0.37 n 26.98 de

17 BHE16 7.60 d 17.68 h 37 BHE36 1.28 l 16.91 h

18 BHE17 13.12 a 23.26 f 38 BHE37 2.50 i 16.97 h

19 BHE18 7.63 d 10.74 l 39 BHE38 3.65 fg 42.69 a

20 BHE19 2.74 i 34.92 b C.V (%) 8.65 8.38


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*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

The result from Table 1 showed that there were two isolates having the highest ammonium

concentration (BHE17 and BHE13) however there were a lot of isolates having the high

ability of phosphate solubilization such as BHE38, BHE26, BHE26... while at Cujut site,

there was only one isolate having the highest ammonium concentration (CJ.E06)(Table 2),

and there was no isolate having the high ammonium concentration and several isolates having

the high phosphate solubization such as CTE05 and CTE01 (Table 3). In ferralsols, many

endophytic bacterial isolates from soybean nodules had the high ammonium and phosphate

solubilization in comparison to endophytic bacterial isolates from nodules in alluvial soil

(Can Tho site). Especially these isolates had the high ability of ammonium and phosphate

solubilization concentration in the first stage (2 and 5 days after incubation) while these

isolates isolated from nodules in Can Tho site had the ability of ammonium and phosphate

solubilization very slow, they only developed in the later stage (8 or 10 days after

incubation)(Table 4 and Table 5).


endophytic bacterial isolates from soybean nodules at Cujut site

No Bacterial

name

Ammonium

(NH4+)*

concentration

Available P

(P2O5)**

concentration

No Bacterial

name

Ammonium

(NH4+)*

concentration

Available P

(P2O5)**

concentration

01 Control 0.00 m 0.00 l 12 CJE11 0.32 e 0.29 i

02 CJE01 0.22 h 0.34 i 13 CJE12 0.25 g 2.12 d

03 CJE02 0.19 i 0.47 h 14 CJE13 0.18 i 0.29 i

04 CJE03 3.18 b 2.74 c 15 CJE14 0.17 j 0.30 i

05 CJE04 0.56 c 0.28 i 16 CJE15 0.12 l 0.38 i

06 CJE05 0.18 i 0.26 i 17 CJE16 0.22 h 0.69 g

07 CJE06 11.56 a 1.48 f 18 CJE17 0.37 d 1.94 e

08 CJE07 0.16 k 0.47 h 19 CJE18 0.28 f 3.44 b

09 CJE08 0.16 k 0.50 h 20 CJE19 0.16 k 0.30 i

10 CJE09 0.17 j 2.13 d CV(%) 9.50 9.57

11 CJE10 0.16 k 3.88 a





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endophytic bacterial isolates from soybean nodules at Can Tho site

No Bacterial

name

Ammonium

(NH4+)*

concentration

Available P

(P2O5)**

concentration

No Bacterial

name

Ammonium

(NH4+)*

concentration

Available P

(P2O5)**

concentration

01 Control 0.00 g 0.00 g 08 CTE07 0.68 a 0.51 f

02 CTE01 0.11 fg 3.96 b 09 CTE08 0.14 e 0.57 f

03 CTE02 0.06 g 0.81 e 10 CTE09 0.26 c 2.21 c

04 CTE03 0.22 d 0.64 ef 11 CTE10 0.31 b 1.28 d

05 CTE04 0.14 e 0.75 e 12 CTE11 0.68 a 0.48 f

06 CTE05 0.22 d 5.97 a C.V (%) 33.3 8.51

07 CTE06 0.27 c 0.49 f




On the contrary, the endophytic bacterial isolates from soybean nodues from Can Tho site

had the high biosynthetic IAA concentration with CTE01 isolate (79.88 mg/L) in comparition

to the isolates from Buonho (BHE29 with 11.28 mg/L) and Cujut sites (CJE19 with 36.92

mg/L)(Table 6).

Table: 4 Effects of endophytic bacterial isolates on Ammonium (NH4+)(mg/l)

concentration during 4 stages

Time Buonho site Cujut site Can Tho site

2 days after incubation 4.199 a 1.682 a 0.000 c

4 days after incubation 2.568 b 0.796 b 0.124 b

6 days after incubation 1.794 c 0.631 d 0.149 b

8 days after incubation 1.826 c 0.716 c 0.348 a

F calculated ** ** **

CV (%) 8.65 9.50 33.2


Table: 5 Effects of endophytic bacterial isolates on Available P (P2O5)(mg/l)

concentration during 4 stages

Time Buonho site Cujut site Can Tho site

5 days after incubation 8.29 d 0.081 c 0.053 d

10 days after incubation 17.05 c 1.492 a 1.511 a

15 days after incubation 27.84 a 1.515 a 1.047 b

20 days after incubation 22.39 b 1.407 b 0.921 c

F calculated ** ** **

CV (%) 8.38 9.57 8.51



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Table: 6 IAA (mg/l) concentration of 68 endophytic bacterial isolates from soybean

nodules at 3 sites (Buonho, Cujut and Can Tho site) at 2 days after incubation with

tryptophan

Buonho site Cujut site

No Bacterial

Name

IAA concentratrion

(ml/L) No

Bacterial

Name

IAA concentratrion

(ml/L)

01 Control 0.00 q 01 Control 0.00 l

02 BHE01 0.99 o 02 CJE01 3.14 j

03 BHE02 2.12 k 03 CJE02 6.57 h

04 BHE03 1.11 n 04 CJE03 4.32 i

05 BHE04 2.58 j 05 CJE04 2.12 k

06 BHE05 2.96 i 06 CJE05 8.16 g

07 BHE06 3.32 h 07 CJE06 4.63 i

08 BHE07 2.86 i 08 CJE07 7.46 h

09 BHE08 2.94 i 09 CJE08 3.19 j

10 BHE09 2.55 j 10 CJE09 6.77 h

11 BHE10 3.93 f 11 CJE10 2.43 k

12 BHE11 7.23 c 12 CJE11 7.03 h

13 BHE12 4.14 ef 13 CJE12 6.59 h

14 BHE13 4.37 de 14 CJE13 29.81 b

15 BHE14 4.41 d 15 CJE14 21.87 f

16 BHE15 1.52 m 16 CJE15 8.51 g

17 BHE16 0.62 p 17 CJE16 25.50 d

18 BHE17 1.58 m 18 CJE17 28.21 c

19 BHE18 2.45 j 19 CJE18 24.58 e

20 BHE19 1.88 ki 20 CJE19 36.92 a

21 BHE20 0.00 q CV(%) 0.96

22 BHE21 0.70 p

23 BHE22 0.11 p

24 BHE23 0.27 p

25 BHE24 2.11 k

26 BHE25 0.00 q

27 BHE26 0.18 q Can Tho site

28 BHE27 1.95 k 01 Control 0.00 h

29 BHE28 1.59 m 02 CTE01 79.88 a

30 BHE29 11.82 a 03 CTE02 12.16 e

31 BHE30 3.24 hi 04 CTE03 13.97 d

32 BHE31 3.78 fg 05 CTE04 5.27 f

33 BHE32 1.86 ki 06 CTE05 5.92 f

34 BHE33 3.34 h 07 CTE06 22.09 b

35 BHE34 2.50 j 08 CTE07 4.92 g

36 BHE35 1.75 l 09 CTE08 17.51 c

37 BHE36 7.71 b 10 CTE09 4.82 g

38 BHE37 3.41 h 11 CTE10 5.43 f

39 BHE38 3.70 g 12 CTE11 5.97 f

C.V (%) 9.03 C.V (%) 2.99



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16S rDNA gene amplification 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 (9/16 isolates),

7 isolates belonged to Gammaproteobacteria (Table 7).

Table: 7 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

BHE13 Bacillus pumilus strain RRLJ SMAD (DQ299945) 98

Bacillus safensis strain HKG214 (KJ741254) 98

BHE19 Bacillus megaterium, strain: PD22 (LC092937) 97

Bacillus flexus strain GS5 (KC608047) 97

CJE6 Bacillus flexus strain PHCDB20 (KF417548) 99

Bacillus megaterium strain p50_A06 (JQ833743) 99

CJE10 Bacillus megaterium strain DCU41 (HM594691) 99

Bacillus flexus strain p49_B07 (JQ8336620 98

CJE3 Bacillus flexus strain IK-MB14-518F (FJ906742) 99

Bacillus megaterium strain p56_D01 (JQ835316) 99

BHE12 Bacillus circulans strain HMF2507 (KT983982) 99

Bacillus nealsonii, strain A1 (LT547803) 99

CJE17 Paenibacillus sp., strain: SNHAPa-TKSA (LC054173) 99

Paenibacillus lautus strain RRT AY-2 (DQ299946) 99

CJE18 Lysinibacillus xylanilyticus (KF772240) 99

Lysinibacillus fusiformis strain LWJ2 (KT8618580 99

CTE5 Staphylococcus gallinarum strain W-61 (EU706285) 99

Staphylococcus xylosus, strain TU9, isolate TU9 (HF548353) 99

Gammaproteobacteria

BHE38 Acinetobacter calcoaceticus strain JO_1 (KF374680) 99

Acinetobacter pittii strain C_12 (KT748635) 99

CTE4 Acinetobacter pittii strain BR_12 (KT748634) 99

Acinetobacter calcoaceticus strain EH52 (GU339280) 99

CTE11 Acinetobacter junii strain NF110 (KP772089)

Acinetobacter calcoaceticus strain petra-09 (GQ141870)

CTE6 Enterobacter cloacae strain BCG11 (KT156816) 99

Enterobacter asburiae strain PW2a (KF673163) 99

CTE1 Klebsiella pneumoniae subsp. pneumoniae strain NF25 (KP772099) 99

Klebsiella pneumoniae strain NF89 (KP772068) 99

CTE7 Stenotrophomonas maltophilia strain RD_MAAMIA_22

(KU597502) 99

Xanthomonas retroflexus strain ZSB23 (KT825693) 99

BHE17 Proteus mirabilis strain IK-MB4-518F (FJ906732) 99

Proteus vulgaris strain CYPV1 (CP012675) 99


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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 A1 had 5 isolates as Bacillus

megaterium BHE19, Staphylococcus gallinarum CTE5 and Klebsiella pneumoniae CTE1 in

cluster A11 and Acinetobacter junni CTE11 and Bacillus pumilus BHE13 correlated very

closely in cluster A12. Cluster A2 composed of four strains: Enterobacter cloacae CTE5,

Lysinibacillus xylanilyticus CJE18, Bacillus flexus CJE6 and Bacillus flexus CJE3 had

relationship very closely. Cluster B had cluster B1 with two strains: Acinetobacter pittii

CTE4 and Bacillus megaterium CJE10 while cluster B2 composed of five strains:

Acinetobacter calcoaceticus BHE38, Paenibacillus lautus CJE17, Proteus miribalis BHE17,

Stenotrophomonas maltophilia CTE7 and Bacillus circulanas BHE12. This result showed

that relationship between endophytic bacterial isolates from nodules of soybean in ferralsols

of Buonho and Cujut (Western Highland) and endophytic bacterial isolates from nodules of

soybean cultivated on alluvial soil of the Mekong Delta closely.

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 Gammaproteobacteria

(Table 8).

Table: 8 Nucleotide diversity (Ɵ) values of two EST’s using the programme DNASp 4.0

(Watterson, 1975)

ESTs 15 isolates

Ncleotide diversity (Pi) 0.70300 ± 0.0009579

Theta (per sequence) from Eta 0.87421 ± 0.11179

Primer p515FPL 5’-GTGCCAGCAGCCGCGTAA-3’

Primer p13B 5’-AGGCCCGGGAACGTATTCAC-3’


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Figure: 3 Phylogenetic tree showing the relative position of endophytic bacterial isolates

by the neighbor-joining method of complete 16S rRNA sequence (p515FPL primer).

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

The endophytic bacterial strains have been studied and described as beneficial bacteria with

Gram-positive bacteria presented on both of media and its occupied over 50% among 9

strains in our result (Figure 4).

Figure: 4 The proportion of group and they distributed in three clusters


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Treatments of Paenibacillus lautus CJE17 mixed with Bradyrhizobium japonicum VNR71

and Paenibacillus lautus CJE17 mixed with Bacillus megaterium CJE10 had highest pod

number/plant and 100-seed weigh and grain yield (Table 9), this result showed that the high

effectiveness of endophytic bacterial strains on yield component and grain yield of soybean

cultivated on ferralsols.

Table: 9 Effects of soybean rhizobia and endophytic bacteria in soybean nodule on yield

component of soybean (cv. Cujut) cultivated on ferralsols (in pot experiment)

Treatment

Branche

number/

plant

Pod

number/

plant

Pod

number/

plant

100-seed

weigh

(g)

Seed weigh

/pot

(g)

No fertilizer,

without bacteria 1.33 g 11.17 bc 26.00 bcd 14.07 de 6.82 ghk

100 N 2.83 abcde 13.17 a 32.67 a 13.67 e 9.63 cd

CJ.E17 + 20 N 1.83 efg 12.00 bc 30.83 ab 14.26 de 7.03 fgh

CJ.E10 + 20 N 1.33 g 11.33 bc 23.50 d 15.05 abc 7.55 efg

VNR71* + CJ.E17 + 20 N 2.67 bcde 11.92 bc 32.17 a 15.24 ab 11.80 a

VNR71* + CJ.E10 + 20 N 1.83 efg 12.00 bc 33.00 a 14.53 cd 7.98 e

CJ02** + CJ.E17 + 20 N 1.50 fg 11.50 bc 29.33 abc 14.23 de 6.07 k

CJ02** + CJ.E10 + 20 N 2.67 bcde 12.17 ab 30.67 ab 14.70 bcd 6.47 hk

CJ.E17 + CJ.E10 + 20 N 2.50 cdef 11.42 bc 34.17 a 15.12 abc 10.70 b

VNR71 + CJ.E17+CJ.E10

+ 20 N 2.67 bcde 11.75 bc 24.83 cd 14.62 bcd 7.50 efg

CJ02 + CJ.E17+CJ.E10

+ 20 N 2.33 cdefg 11.08 c 29.67 abc 15.45 a 9.49 cd

C.V (%) 19.11 5.43 10.77 3.03 4.73

*, ** rhizobia from Biotechnology R&D Institute, Can Tho University


Besides endophytic bacterial strains improved soil fertility as available P and organic matter

in soil after harvesting (Table 10). Therefore endophytic bacterial strains or co-inoculation

between they and rhizobial strain not only supported yield component and grain yield but also

improved soil fertility of soybean cultivated on ferralsols.

Table: 10 Effects of soybean rhizobia and endophytic bacteria in soybean nodule on pH

and nutrient soil component of ferralsols (in pot experiment) after harvesting

Treatment pH N total

(%)

Available P

(mg/kg)

Organic matter

(%)

Initial 4.64 cde 0.135 c 8.177 a 3.575 c

No fertilizer, without bacteria 4.91 a 0.202 a 5.932 de 3.917 b

100 N 4.66 cd 0.139 b 6.159 bcd 3.909 b

CJ.E17 + 20 N 4.62 cde 0.139 b 5.991 cde 3.974 ab


236


CJ.E10 + 20 N 4.61 cde 0.137 bc 6.646 bc 3.870 b

VNR71* + CJ.E17 + 20 N 4.49 ef 0.139 b 6.734 b 4.203 a

VNR71* + CJ.E10 + 20 N 4.67 cd 0.137 bc 5.769 def 4.086 ab

CJ02** + CJ.E17 + 20 N 4.35 f 0.138 bc 4.910 g 3.909 b

CJ02** + CJ.E10 + 20 N 4.56 de 0.138 bc 5.178 fg 3.943 ab

CJ.E17 + CJ.E10 + 20 N 4.72 bc 0.140 b 5.115 fg 4.069 ab

VNR71 + CJ.E17+CJ.E10 + 20 N 4.84 ab 0.135 c 5.402 efg 3.978 ab

CJ02 + CJ.E17+CJ.E10 + 20 N 4.67 cd 0.138 bc 6.184 bcd 4.099 ab

C.V (%) 1.41 0.89 4.89 3.44


Plant-associated bacteria colonize the rhizosphere (rhizobacteria), the phyllosphere

(epiphytes) and the the inside of plant tissues (endophytes). Endophytes are sheltered from

environmental stresses and microbial competition by the host plant, and they seem to be

ibiquitous in plant tissues, having been isolated from flowers, fruits, leaves, stems, roots and

seeds of variuous plant species (McInroy and Kloepper, 1995; Kobayashi and Palumbo,

2000) and they have some beneficial effects on host plant.

Rhizobia are perhaps the best known beneficial plant-associated bacteria because of the

importance of the nitrogen fixation that occurs during the Rhizobium-legume symbiosis

(Hung et al., 2007). Soybean (Glycine max (L.) Merrill) is an Asiatic leguminous plant,

occupying large acreages of land worldwide for its oil and protein (Fayzala et al., 2009).

Besides endophytic bacteria from legume plants have been reported; endophytic bacteria

from roots and nodules of fieldpea and chickpea being grown in Northern India were isolated.

A total of 75 endophytic bacteria roots and nodules of fieldpea (Narula et al., 2013a) and 88

from roots and nodules of chickpea showed that 50% in roots and 93.4% in nodules were

Gram positive. Endophytic bacteria have been isolated from soybean (Oehrle et al., 2000) and

especially Sturz et al. (1997) reported the isolation of 15 non-rhizobial species from clover

root nodules; Bai et al (2002) reported fourteen strains of putative endophytic bacteria, not

including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized

soybean (Glycine max (L.) Merr.) root nodules; Hung et al. (2007) found that endophytic

population was highest in the nodules tissue with 31 nodule isolates, however, did not form

nodules on soybean (cv. Pusa-22) and they suggested that 31 endophytic bacterial isolates.

The studies have reported the identification of a range of endosymbiotic but non-nitrgen-

fixing bacteria (commonly including species of genus Pseudonomonas) from root nodules

(Zakhia et al., 2006; Li et al., 2008). Recent the report of Tariq et al. (2014) isolated ten

bacterial isolates from root nodules of cultivated pea plants and they were unable to nodulate


237


pea plants in nodulation assay but they had good plant-growth promoting characteristics such

as IAA production, nitrogen fixation and phosphate solubilization when testing in-vitro.

However, Bai et al, (2003) discovered that co-inoculating soybean plants with Bacillus

thuringiensis and Bradyrhizobium japonicum has produced consistent increases in yield.

Hung et al, (2007) reported that the isolation of Paenibacillus polymyxa HKA-15, a Gram-

positive bacterium from root nodules of soybean and this strain showed that potent biocontrol

activity towards soil borne fungal plant pathogens (Senthikumar et al., 2008). Our results

showed that over 50% endophytic bacterial strains were idenfified that are bacilli among

Paenibacillus lautus CJE17 which the best strain, it combined with the rhizobial strains as

VNR71 or it combined with another strain (CJE10) supported grain yield (Table 9). The

results of Bai et al. (2002) showed that three isolates were designated as non-Bradyrhizobium

endophytic bacteria (NEB4, NEB5 and NEB17) when soybean plants were co-inoculated

with one of three isolates and Bradyrhizobium japonicum which increased soybean weight

under nitrogen-free condition. Three isolates were belonged to genus Bacillus with NEB4 and

NEB5 are Bacillus subtilis strains and that NEB17 is a Bacillus thuringiensis strain. ‘Bacilli’

AEFB are a diverse group with wide distribution in agricultural soils that contribute both

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

Compared to Gram-negative bacteria, Gram positive bacteria strains have the advantages as

its ability to form endospores and produce different antibiotics. On the otherhand, Bacilli can

survive for a long time in carrier in comparison to other bacteria in inoculant production

commercially and especially endophytic bacterial Bacilli strains will be selected with

characteristic of biology safety.

CONCLUSION

Sixty-eight entophytic bacterial isolates were isolated from 70 soybean nodules of soybean

plants which collected at Buonho town, DakLak province (38), Cujut district, DakNong

province (19) and Can Tho city, Mekong Delta (11), they developed on two kinds of medium

(PDA and TSA) after 2 or 3 days incubation and they made the pellicles on semi-solid media.

They were identified as endophytic bacterila isolates and 16 isolates having good plant

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

Bacilli (more than 50%), and Gammaproteobacteria with seven strains. Among them, there

are two strains as Paenibacillus lautus CJE17 and Bacillus megaterium CJE10 supported

yield component, grain yield and improved soil fertility of soybean cultivated on ferralsols.


238


Bacilli are bacteria having endospore and this support their survival in drought condition of

ferralsols in dry season (from November to April). Together with rhizobial strains, they will

be suggested to produce as inoculant for soybean cultivation on ferralsols in the future.

ACKNOWLEDGEMENTS

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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