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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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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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).
Table: 2 Ammonium (NH4+) and Available P (P2O5)(mg/l) concentration of 19
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
*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
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Table: 3 Ammonium (NH4+) and Available P (P2O5)(mg/l) concentration of 11
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
*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
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
Means within a column followed by the same letter/s are not significantly different at p<0.01
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
Means within a column followed by the same letter/s are not significantly different at p<0.01
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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
Means within a column followed by the same letter/s are not significantly different at p<0.01
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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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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
Means within a column followed by the same letter/s are not significantly different at p<0.01
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
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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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
Means within a column followed by the same letter/s are not significantly different at p<0.01
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
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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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.
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Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences
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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