ISOLATION AND CHARACTERIZATION OF RHIZOBIA FROM ROOT

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117

Diep et al. World Journal of Pharmacy and Pharmaceutical Sciences

ISOLATION AND CHARACTERIZATION OF RHIZOBIA FROM

ROOT NODULES OF SOYBEAN (GLYCINE MAX L. MERR.) GROWN

IN FERRALSOLS OF DAKNONG AND DAKLAK PROVINCE,

VIETNAM

Prof. Dr. Cao Ngoc Diep1*

and Duong Ba So2

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

Tho University, Vietnam.

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

ABSTRACT

Total of 44 isolates were isolated from nodules of soybean grown on

ferralsols of CuJut district (24 isolates) and Buonho town (20 isolates).

The microsymbionts causing nodules on the roots of soybean (Glycine

max L. Merr.) and 34/44 rhizobial isolates were analysed with 16S-23S

intergenic spacer and PCR-RFLP for repeated sequence RSα a 1195-bp

DNA fragment, indole acetic acid production and nitrogen fixing

activity. Almost isolates produced IAA but they had the different

nitrogen fixation capacity. All the strains were fast-growing soybean

rhizobia (Sinorhizobium [Ensifer) fredii except two isolates (CJ02 and

CJ09) were slow-growing soybean rhizobia (they were identified with

sequence RSα as molecular marker for Bradyrhizobium japonicum).

The study also showed the low presence of soybean nodulating slow

growers in ferralsols of western highland of Vietnam.

KEYWORDS: Bradyrhizobium, ferralsols, Sinorhizobium, soybean, western highland of

Vietnam.

INTRODUCTION

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

most important grain legumes in the world. Soybean, like other legumes, fixes atmospheric

nitrogen in association with gram-negative soil bacteria of the genera Bradyrhizobium and

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

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Volume 5, Issue 03, 117-134. Research Article ISSN 2278 – 4357

*Correspondence for

Author

Prof. Dr. Cao Ngoc Diep

Lecturer in Department of

Microbiology

Biotechnology,

Biotechnology R&D

Institute, Can Tho

University, Vietnam.

Article Received on

03 Jan 2015,

Revised on 27 Jan 2015,

Accepted on 15 Feb 2016

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Sinorhizobium (Jordan, 1982; Scholla and Elkan, 1984). In 1982, the fast-growing rhizobial

strains were also isolated from soybean nodules and from soil of People‟s Republic of China,

within the center of origin and diversity of this legume (Keyser et al., 1982). Later, fast-

growing strains were isolated from other primary nd secondary centers of soybean origin

(e.g., Xu and Ge, 1984; Dowdle and Bohlool, 1985; Young et al. 1988; Rodriguez-Navarro et

al., 1996). The fast growers were classified as the new species Rhizobium fredii (Scholla and

Elkan, 1984), later reclassified as Sinorhizobium fredii and Sinorhizobium xinjiangensis

(Chen et al., 1988), and recently proposed to change to genus Ensifer (Young, 2003).

Although it was originally thought that Sinorhizobium (Ensifer) fredii was specific for Asian

soybean lines (Keyser et al., 1982; Devine, 1985).

Soybean was introduced in Vietnam for a long time, and it has become a crop which to

produce many kinds of food for human everyday. According to Shurleff and Aoyaki (2010),

Soybean products (soy sauce) are first ordered for use in Hanoi, Vietnam by Dutch East India

Company officers in 1652; soybean cultivation is first reported in the Van-dai Loai-ngu

(Encyclopedia of Vietnam), by Le Quy Don (discovered by Tran Van Lai 1993, p. 143) and

soybean cultivation reported again (Jao de Loureiro 1790, in Latin). He calls it Dolichos Soja,

Dau nanh, Hoam te1u (yellow bean) and he also mentions soy sauce and tofu (Tau hu).

Vietnam also has small local production, but the country‟s booming feed industry imported

600,000 tonnes of soybean meal in 2002. Vietnam is the only country in the region

experiencing double-digit growth in grain demand. Soybean milk requirement of Vietnamese

is more and more with 613 million litres (2014)[3rd country in the world drink soy-milk] and

Vietnam imported over 1.2 million tonnes soybean seed. Soybean cultivation also introducted

on red yellow latosol (ferralsols) in the begining of 1965-1970 with new cultivars from

UDSA at EakMat experimental station at Ban Me Thuot (Know, 1969). An area of soybean

cultivation of two provinces (DakNong and DakLak) of western highland of Vietnam reduced

from 20.000 ha (2000) to 8.000 ha (2014) with 15.000 tonnes soybean production (Trac,

2015)(http://daunanhdinhduonglanh.vn/).

The aim of this study were (i) isolation and selection good brady(rhizobia) isolates, (ii)

analysis of characterization and identification of soybean brady(rhizobia)(slow-growing or

fast-growing soybean rhizobia) from nodules of soybean grown on ferralsols of western

highland of Vietnam by PCR 16S-23S IGS and PCR of RSα methods.


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MATERIALS AND METHODS

Sampling of nodules and isolation of brady(rhizobia) strains

In the present study, root nodules were collected from soybean plants (at the flowering stage)

grown in 25 sites in two provinces (DakNong and DakLak) mainly at CuJut district

(DakNong) and BuonHo town (DakLak). DakNong and DakLak provinces are situated in the

western highland of Vietnam, CuJut district is a district of DakNong which locates the north

of DakNong (107o44‟44” E and 12

o40‟56” N) and Buon Ho is a town of Daklak province, it

locates the north of province (108o16‟13” E and 12

o51‟16” N)(Figure 1).

Figure:1 The location was examined in this study and CuJut district [DakNong

province] and BuonHo town [DakLak province] and ferralsols were presented soils with

reddish drak brown

Isolation of brady(rhizobium)

The rhizobia were isolated from nodules and purified with the standard protocol using yeast

extract-mannitol agar (YMA) supplied with 0.02% congo red (Vincent, 1970). The nodules

were surface-sterized in 95% ethanol for 1 min, and then in 1% sodium hypochlorite for 50

min. After the nodules were washed in sterile water, each nodule was pricked by a sterile

needle, streaked onto a YMA agar plate containing Congo red and incubated in the dark at

30oC until the appearance of single colonies, which were selected after 24 h of showing

different morpholotypes and subcultured until the purity of cultures was confirmed (Marsudi

et al. 1999). Pure cultures were stored in 20% glycerol at – 80oC. Cell shape and size were



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observed under light microscope by taking a drop of bacterial culture suspension in saline.

Gram‟s reaction was performed according to Vincent (1970).

Nodulation test

The nodulation capacity of the strains on soybean was confirmed by inoculation tests as

described previously (Vincent, 1970) with sterile black rice-hull ash (substitutive

vermiculite). Leonard jar assemblies consist of plastic bottle [drinking water bottle 500mL],

bottom of bottle was cut and up-down in another bootle which was cut at neck of bottle;

above bottle was filled by streile sand and mouth bottle was put with sterile cotton

(connecting from above bottle to jar or under bottle) to irrigate mineral solution N-free

(Winarno and Lie, 1979)(Figure 2).

Figure:2 Leonard jar assemblies with sterile black rice-hull ash and sand

Briefly, the Glycine max (cv. CuJut) seeds were surface sterilized with ethanol 70% in 5 min,

washed with sterile water 5 times and then germinated on 0.8% agar-water plates in the dark

at 30oC for 24 to 36 h. Two germinated seeds were placed in the drinking bottle 500-ml filled

with sterile black rice-hull ask (Leonard jar model) and moistened with nutrient solution free-

N (Winarno and Lie, 1979). Two days later, the seedlings were inoculated with each of the

isolates (approx. 106 CFU per bottle). The negative controls were inoculated with sterile

water. All the plants were grown in a greenhouse with temperature and light control. Nodules

were checked and harvested after 28 days. Effectiveness of the nodules was identified by

observing the pink color within nodules and dark green color of leaves, especially nodules

presented near tap-roots as good isolates together with their size and dry weight.



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Nitrogen fixation capacity

The good isolates were chosen from “nodulation test” experiment, they were evaluated their

effectiveness in Leonard jar assembly model (Krisnan and Puepphe, 1991). Leonard jar

assemblies consisted of plastic bottle [drinking water bottle 500mL], bottom of bottle was cut

and up-down in another bootle which was cut at neck of bottle; above bottle was filled by

sterile sand and mouth bottle was put with sterile cotton (connecting from above bottle to jar

or under bottle) to irrigate mineral solution N-free (Winarno and Lie, 1979). These systems

were heat steriled (70oC) in 48 hours before use. In order to select the isolates having high

effectiveness, The experiment was designed with positive control (with N fertilizer) and

negative control with four replications. Nodules were checked and harvested after 40 days.

Effectiveness of the nodules was identified by observing the pink color within nodules and

dark green color of leaves, especially nodules presented near tap-roots as good isolates

together with their size and dry weight. Shoot and Root dry weight of each treatment were

recorded at the havesting stage.

IAA production

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 10 uv Thermo Scientific spectrophotometer.

PCR 16S-23S IGS rDNA sequence (Massol-Deya et al., 1995)

The isolates were grown on TY medium (Beringer, 1974) on agar slants in 2 days, a loopful

from each tube (each isolate) was asceptically suspended in a Eppendorf vial 1.5ml

containing one sterile nano-pure water by vortexing, this suspension was washed with 1 ml

NaCl 1M two times and 1 ml nano-pure water one time, adding 30-50 µl lysis buffere (NaOH

50 mM + 2.5% SDS)(Rodriguez-Navarro et al., 1996) and inoculated at 70oC in 2 hours,

centifuge 12000 rpm in 2 minutes, adding 50 µl sterile nano-pure water into suspension and it

was used as a template for PCR.

Reactions were performed in a Perkin-Elmer 9700 thermocycler, USA; A 50 µl reaction

volume contained 25 pmol primer pHr and 25 pmol primer p23SRO1, 5 µl 10X PCR buffer



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(0.5 M KCl, 0.1 Tris-HCl, pH=8.3), 1.5 µl 2.5 mM MgCl2, 1.25 µl W1-detergent, 2.5 µl 5

mM dNTP, 32 µl sterile nano-pure water, 2.5 U of Taq DNA polymerase and 2.5 µl cell

suspension as template. The following temperature profile was used for PCR amplication

94oC 7 minutes for an initial denaturation, 35 cycles of denuturation at 94

oC in 20 sec,

annealing at 52oC for 20 sec and extension for 40 sec at 68

oC and a final extension at 68

oC

for 7 minutes. Amplified 16S-23S IGS rDNA was examined by electrophoresis in 1.5%

agarose NA gel with 5 µl aliquots of PCR produces, gels were stained with ethidium bromide

and photographed under UV illimination. Elecphoresis was carried at 80 volt for 60 minutes

with standard (6 by 8 cm).

Amplication of RSα (Annapura et al., 2007)

Total genomic DNA was isolated as described above. Amplication of RSα was carried out

by PCR using a Perkin-Elmer 9700 thermocycler. The expected size of the amplification

product was 1195 bp. Primers RSAL 1 (23 mers, 5‟ AGC GGG CGC GGA TAG TTC TGT

TG 3‟) and RSAR 1 (23 mers, 5‟ GGC TCG GCT CTG TCG TTG TAT GC 3‟) were

synthesized by Phu Sa Co (Vietnam). Amplification was carried out in a 50-µl reaction

mixture containing: 150 ng genomic DNA as template, 5 µl of PCR buffer (with 15 mM

MgCl2), 2.5 µl of dNTP mix (2 mM), 50 ng of each primer, and 0.4 µl of Taq polymerase

(3U l-1

). The cycling parameters used were initial denaturation at 95oC for 5 min, 20 cycles of

denaturation for 40 s at 94oC, annealing at 60

oC for 90 s, and extension at 74

oC for 3 min, and

10 cycles of denaturation for 40 s at 94oC, annealing at 60

oC for 90 s, and extension at 74

oC

for 6 min, and a final extension at 74oC for 15 min. Elecphoresis was carried at 80 volt for 60

minutes and gels were visualized under UV and photographed with Gel documentation

system.

Data analyses

Data from nodule number/plant, dry weight of nodule, dry weight of shoot 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 DISCUSSION

Isolation of brady(rhizobium) and nodulation test

Total of 44 isolates were isolated from nodules of soybean grown on ferralsols of CuJut

district (24 isolates) and Buonho town (20 isolates). The isolates grew on YMA with Congo



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red and their colonies were stained with red color clearly, and this result recognized rhizobia

in comparison to other bacteria (Figure 3). Furthermore, two isolates (CJ02 and CJ09) grew

very slow onto YMA and single colonies only appeared after 4 or 5 days incubation;

especially their colonies had red color very dark (Figure 3a).

Besides that, these isolates were determined capacity of nodulation on soybean plants

(Leonard jar assemblies) and they were also selected through nodule number/plant and dry

weight (DW) of one nodule (Table 1).

A B

Figure:3 The colonies of several isolates on YMA with congo red, the rhizobial isolates

were identified through red colonies

CJ02 (CuJut) and BH18 isolates (Buonho) nodulated the highest nodule numbers on soybean

plants (cv. CuJut) but the highest DW of nodule/plant of CJ10 (CuJut) and BH07 isolate

(Buonho) and especially the BH07 isolate also had bigger nodules than others while CJ14

isolate had the biggest nodules in comparison to 23 isolates (Cujut)(Table 1). At Cujut, many

isolates nodulated many nodules/plant and high DW of nodules as isolate: CJ02, CJ06, CJ08,

CJ10 but the highest DW of one nodule was CJ14 isolate (the lowest nodule number/plant)

however many isolates had high DW of one nodule as CJ03, CJ13 and CJ28, this results

showed that these rhizobial isolates nodulated many nodules but small nodule size. At

Buonho, 11/20 rhizobial isolates nodulated many nodules/plant but there were 4 isolates had

high DW of nodules, especially BH07 isolate had the highest DW of one nodule but DW of

one nodule of this isolate was nearly half (47.8%) of DW of nodule of CJ14 isolate.



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Furthermore, two isolates (CJ02 and CJ09), originated from nodules of soybean grown on

ferralsols of CuJut district, nodulated nodules early and near tap-root of soybean plants this

showed that they had high effectiveness of biological nitrogen fixation because these rhizobia

isolates supported the early nodulation and host soybean plant symbiosis while rhizobia

originated from nodules of soybean grown on ferralsols of Buonho town which nodulated

nodules slowly, nodules did not present near tap-root of soybean plant, they nodulated

nodules on rootlets (far from tap-root)(Figure 4).

Table:1 Nodule number/plant, dry weight (DW) of nodule/plant and DW of one nodule

of rhizobia isolates were isolated from nodules of soybean cultivated on ferralsols of Cu-

jut district (DakNong province) and Buonho town (DakLak province) in Leonard jar

assemblies with sterile black rice-hull ash

Cu-Jut district Buonho town

Rhizobia

isolate

Number

nodule

/plant

DW of

nodule

/plant (mg)

DW of one

nodule (mg)

Rhizobia

isolate

Number

nodule

/plant

DW of

nodule

/plant (mg)

DW of one

nodule

(mg)

Control 3.33 f 4.91 f 0.624 h Control 4.00 f 8.66 g 0.258 g

CJ01 20.01 cd 42.27 c 2.138 e BH03 35.67 bc 27.33 cd 0.784 e

CJ02 39.67 a 64.27 b 1.774 f BH05 28.33 cde 31.67 c 1.106 bc

CJ03 20.33 c 62.97 b 3.138 b BH06 23.67 e 21.01 def 0.895 d

CJ04 22.67 c 38.71 cd 1.741 f BH07 26.00 de 49.33 a 1.895 a

CJ05 18.67 d 29.57 d 1.586 fg BH08 19.01 e 12.01 fg 0.642 ef

CJ06 34.01 ab 46.37 c 1.415 g BH09 34.33 bcd 19.67 def 0.576 f

CJ07 22.33 c 48.11 c 2.166 e BH10 39.00 bc 42.67 ab 1.100 bc

CJ08 33.01 ab 51.17 bc 1.556 fg BH11 24.33 de 28.33 cd 1.255 b

CJ09 25.33 c 64.01 b 2.551 d BH12 20.00 e 14.33 def 0.720 e

CJ10 34.67 ab 82.57 a 2.445 d BH13 35.67 bc 32.33 c 0.906 d

CJ11 12.67 e 21.87 d 1.702 f BH14a 30.00 bcd 30.67 c 1.060 c

CJ12 10.67 e 14.73 e 1.381 g BH14b 23.33 e 20.01 def 0.907 d

CJ13 11.33 e 31.47 cd 2.800 bc BH15a 34.33 bcd 35.01 bc 1.028 c

CJ14 7.01 ef 21.17 d 3.963 a BH15b 33.33 bcd 23.33 de 0.698 e

CJ15 13.01 e 20.51 e 1.569 fg BH16 25.33 de 27.33 cd 1.095 bc

CJ16 23.67 c 34.41 cd 1.458 g BH17a 25.33 de 15.33 efg 0.619 f

CJ18 27.67 bc 37.97 cd 1.440 g BH17b 32.67 bcd 20.67 def 0.641 ef

CJ19 22.67 c 41.83 c 1.849 fg BH18 49.33 a 23.33 de 0.515 f

CJ20 21.01 cd 35.43 cd 1.664 f BH19 41.33 ab 36.01 bc 0.960 cd

CJ26 25.67 c 44.87 c 1.794 f BH20 30.33 bcd 18.67 def 0.617 f

CJ27 11.01 e 23.27 de 2.079 e

CJ28 10.01 e 27.17 de 2.880 bc

CJ29 19.67 cd 52.91 bc 2.699 cd

C.V (%) 16.47 15.93 7.72 C.V (%) 18.04 18.50 8.87

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



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From each site, 10 isolates were chosen to evaluate nitrogen fixation capacity through

nodulation, DW of nodules/plant and DW of one nodule.

Figure: 4 Early nodulation of two isolates (CJ02 and CJ09) and nodules presented near

tap-root and on main root (left), nodules presented on rootlets (far from tap-root) of

soybean roots by BH06 isolate (right)

Nitrogen fixation capacity

Table: 2 Effects of 24 rhizobial isolates on nodule number/plant, DW of nodule/plant

and DW of one nodule (mg) on soybean plant (cv. Cujut) cultivated on Leonard jar with

sand in 35 days

Cujut site Buonho site

Rhizobia

isolate

Number

nodule

/plant

DW of

nodule

/plant (mg)

DW of one

nodule

(mg)

Rhizobia

isolate

Number

nodule

/plant

DW of

nodule

/plant (mg)

DW of one

nodule

(mg)

Control * 3.33 e 4.49 f 1.467 c Control * 1.67 gh 2.17 gh 1.533 de

NH4NO3 * 9.33 d 21.57 e 2.311 ab NH4NO3 * 3.00 fg 2.37 gh 0.758 e

CJ01 21.67 b 47.11 bc 2.183 ab BH03 2.00 g 10.17 ef 6.222 b

CJ02 28.67 a 64.43 a 2.254 ab BH05 16.67 c 39.83 a 2.392 d

CJ03 31.01 a 52.57 b 1.706 c BH07 19.01 b 9.03 fg 0.472 e

CJ04 18.67 bc 36.53 c 1.973 bc BH10 1.33 gh 12.43 ef 10.083 a

CJ06 18.33 bc 47.31 bc 2.586 a BH13 3.00 fg 18.43 d 6.144 b

CJ07 15.33 c 28.77 d 1.928 b BH14 10.33 e 31.87 abc 3.088 c

CJ08 18.33 bc 31.50 cd 1.752 bc BH15a 23.33 a 38.13 ab 1.648 de

CJ09 21.01 b 42.20 bc 2.037 ab BH17a 12.00 d 25.47 cd 2.156 d

CJ10 28.33 a 50.63 b 1.789 bc BH17b 15.33 c 12.11 ef 0.793 e

CJ19 18.00 bc 37.77 c 2.114 ab BH18 13.33 d 30.13 bc 2.244 d

CJ26 19.67 bc 41.23 bc 2.135 ab BH19 3.01 fg 3.12 gh 1.667 de

CJ29 19.33 bc 41.27 bc 2.143 ab BH20 4.67 f 16.93 d 3.723 c

C.V (%) 13.15 6.68 12.92 C.V (%) 9.72 25.51 16.22



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The result from Table 2 showed that there were three isolates (CJ02, CJ03, CJ10) had the

highest nodule number/plant, DW of nodules/plant and DW of one nodule from CuJut site,

and at Buonho site, BH15a isolate had the highest nodule number/plant and DW of

nodules/plant and two isolates (BH05 and BH14) had the highest DW of nodules/plant and

they did not differ with BH15a isolate significantly.

Effect of biological nitrogen fixation was evaluated through DW of shoot, the result from

Figure 5 showed that CJ09 isolate supported the highest DW of soybean shoot and CJ01 and

CJ10 isolates also had the high DW of shoot and they also did not differ from CJ09 isolate

and soybean plant applying N inorganic fertilizer (1 mM NH4NO3) in rhizobial isolates which

were isolated from soybean nodules cultivating in Cujut site. Furthermore, many isolates

(CJ02, CJ04, CJ06, CJ26 and CJ29) also supported DW of shoot equivalent as DW of shoot

from treatment applying NH4NO3 and all rhizobial isolates had DW of shoot higher than

control treatment. This results showed that all rhizobial isolates had different effectiveness on

soybean plant and the rhizobial isolates had the highest effectiveness as CJ01, CJ02, CJ04,

CJ06, CJ09, CJ10, CJ26 and CJ29 and their effectiveness did not differ from with N

inorganic fertilizer treatment without inoculation.


Figure 5. Effect of rhizobial isolates (from soybean nodule of Cujut site) and N

inorganic fertilizer on DW of soybean shoot cultivated on Leonard jar assemblies

C.V = 7.7%



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Note: control: no rhizobial isolate inoculation, no fertilizer, NH4NO3: no rhizobial isolate

inoculation and applied 1 mM NH4NO3

From Figure 6 showed that DW of shoot of N inorganic fertilizer (1 mM NH4NO3) was the

highest but it did not differ from DW of shoot of BH03, BH14, BH15b, BH18 significantly

however there were shoot DW of rhizobial isolates lower than control treatment as BH07 and

BH10 isolates. This result showed that rhizobial isolates from soybean nodules in Buonho

site had effectiveness lower than rhizobial isolates from Cujut site.


Figure:6 Effect of rhizobial isolates (from soybean nodule of Buonho site) and N

inorganic fertilizer on DW of soybean shoot cultivated on Leonard jar assemblies

Note: control: no rhizobial isolate inoculation, no fertilizer, NH4NO3: no rhizobial isolate

inoculation and applied 1 mM NH4NO3

The relationship between nodule number/plant with DW of nodule/plant closely very

significantly (P<0.01) and DW of nodule/plant with DW of shoot also was closely (P<0.01)

while nodule number/plant with DW of shoot correlated significantly (<0.05) with rhizobial

isolates isolated from nodules of CuJut site (Table 3). While nodule number/plant did not

relate with Shoot DW but it had a relationship closely with nodule DW (P<0.01) and the

relationship between nodule DW with shoot DW was closely very significantly (P<0.01)

from rhizobial isolates isolated from soybean nodules at Buonho site (Table 3). These results

showed that nodule DW is an important factor which affect to shoot DW of soybean in

comparison to nodule number/plant.

C.V = 11.57%



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Table 3. The relationship between Nodule number/plant and Nodule DW with Shoot DW.

Characteristics Cujut site

Nodule number/plant Nodule dry weight

Shoot dry weight r = 0.396* r = 0.466**

Y = 11.517 X + 687.92 Y = 7.424 + 613.33

Nodule dry weight r = 0.786**

Y = 1.4359 X + 12.529

Buonho site

Nodule number/plant Nodule dry weight

Shoot dry weight r = 0.037 ns r = 0.467**

Y = 2.598 X + 723.65 Y = 6.137 + 623.84

Nodule dry weight r = 0.542**

Y = 0.917 X + 11.157

Almost rhizobial isolates showed IAA production in-vitro (with tryptophan) except three

isolates (CJ05, CJ11 and CJ28) synthesized IAA very low.

PCR 16S-23S IGS

16S-23S rDNA IGS region of 16/20 rhizobial representative isolates (Buonho site) was

amplified with two suitable primers and the results showed that there was a big variation in

16S-23S IGS (Figure 7) with band size of approx from 1250 to 1600 bp and 18 rhizobial

isolates (Cujut site) (Figure 8) with band size from 1150 to 1500 bp.

Figure:7 Gel electrophoresis showing variability of PCR amplified 16S-23S rDNA IGS

region from 16/20 soybean rhizobial isolates (Buonho site)

Note: M: Ladder 100 bp plus

1: BH9, 2: BH15b, 3: BH20, 4: BH3, 5: BH6, 6: BH7, 7: BH10,

8: BH14, 9: BH15a, 10: BH11, 11: BH13, 12: BH16, 13: BH17,

14: BH18, 15: BH17b, 16: BH19

1500 bp

1000 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16



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Figure:8 Gel electrophoresis showing variability of PCR amplified 16S-23S rDNA IGS

region from 8/24 soybean rhizobial isolates (Cujut site)

Note: M = ladder

1 = CJ28 (1500 bp), 2 = CJ14 (1500 bp), 3 = CJ15 (1450 bp), 4 = CJ26 (1250 bp),

5 = CJ09 (1250 bp), 6 = CJ01 (1250 bp), 7 = CJ03 (1250 bp), 8 = CJ04 (1250 bp)

With results from gel electrophoresis of PCR amplified 16S-23S rDNA IGS showed that the

isolates had different size (table 4)

Table:4 The isolates had different size on gel electrophoresis 16S-23S

Buonho site Cujut site

No Bacterial name Size (bp) No Bacterial name Size (bp)

01 BH3 1300 01 CJ01 1250

02 BH6 1250 02 CJ03 1250

03 BH7 1400 03 CJ04 1250

04 BH9 1300 04 CJ05 1150

05 BH10 1400 05 CJ06 1200

06 BH11 1600 06 CJ07 1200

07 BH13 1400 07 CJ08 1200

08 BH14 1400 08 CJ09 1250

09 BH15a 1300 09 CJ10 1200

10 BH15b 1350 10 CJ13 1500

11 BH16 1400 11 CJ14 1500

12 BH17a 1400 12 CJ15 1450

13 BH17b 1500 13 CJ18 1200

14 BH18 1400 14 CJ20 1250

15 BH19 1350 15 CJ26 1250

16 BH20 1450 16 CJ27 1250

17 CJ28 1500

18 CJ29 1250

Amplication of RSα

There were two isolates made a band at 1195 bp in 20 rhizobial isolates (Figure 9) when all

of them were tested with primers RSAL 1 and RSAR 1, this results demonstrated that two

isolates are Bradyrhizobium japonicum.

M 1 2 3 M 4 5 6 7 8

1600 bp

1000 bp



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1600bp

1195bp

1000 bp

Figure:9 PCR amplification of RSα in the two rhizobia isolates (CJ02 and CJ04)

nodulating soybean at 1195 bp

Lane M: 100-bp ladder, lane 1: CJ02 and lane 2: CJ09

Hungaria et al. (2008) studied on 30 fast-growing rhizobial strains isolated from nodules of

Asian and modern soybean genotypes by rep-PCR and RADP methods, the result showed

that none of the strains was related to Sinorhizobium (Ensifer) fredii, whereas most were

related to Rhizobium tropici (although they were unable to nodulate Phaseolus vulgaris) and

to Rhizobium genomic species Q. One strain was related to Rhizobium sp. OR 191, while two

others were closely related to Agrobacterium (Rhizobium) spp.; furthermore, symbiotic

effectiveness with soybean was maintained in those strains; especially 5 strains were related

to Bradyrhizobium japonicum and Bradyrhizobium elkanii. However when studied on

rhizobia associated with soybean grown in the subtropical and tropical regions of Chiana,

Man et al. (2008) reported that 252 rhizobial strains isolated from 5 eco-regions of China

with four genomic groups including of Bradyrhizobium japonicum complex (including B.

liaoningense, B. japonicum and a B. japonicum realted genomic species) and Bradyrhizobium

elkanii as major groups. Bradyrhizobium yuanmingense and Sinorhizobium fredii as the

minor groups, were identified by ribosomal/housekeeping genes analyses. Analysis of

diversity of 215 rhizobia strains isolated from root nodules of Glycine max grown in Hebei

province, China; All the strains were classified into nine genospecies in the genera of

Bradyrhizobium and Sinorhizobium (Ensifer) except one (Mesorhizobium). While Adhikari et

al. (2012) recognized the soybean rhizobial isolates isolated from root nodule in Nepal were

bolonged to Bradyrhizobium (slow-growing rhizobia).

Several reports have appeared that demonstrate the presence of repeated sequences (RSα) in

the genome of Rhizobium species (Kuykendal et al., 1988; Kuykendall et al., 1992;

Wheatcroft and Laberge, 1991). Kaluza et al. (1985) discovered two different RSα in the

Bradyrhizobium genome that process the structural characteristic on prokaryotic insertion

M 1 2



131


sequence elements. Hartmann et al. (1996) demostrated the usefulness of RSα for

Bradyrhizobium strain identification. Annapurma et al. (2007) demonstrated that RSα can be

a useful molecular marker for slow-growing soybean rhizobia.

When Keyser et al. (1982) isolated a new group of fast-growing rhizobia, from nodules of

domesticated soybeans and from the wild type Glycine soja, in the east-central provinces of

China; These rhizobia, called Sinorhizobium fredii (Scholla and Elkan, 1984). In Vietnam,

Saeki et al. (2005) isolated 120 isolates from soybean nodules which collected from the fields

of Hanoi Agricultural University (HAU), Hanoi and Can Tho University (CTU), Can Tho,

Vietnam and based on 16S rDNA and 16S-23S rDNA ITS region techniques to analyse

phylogenetic tree of these isolates, the results showed that most of the isolates on

Bradyrhizobium japonicum were extra-slow-growing and their ITS were similiar to that of

Bradyrhizobium japonicum USDA 135. Using PCR-ARDRA IGS 16S-23S, we isolated 223

isolates from nodules of cultivated soybean and wild legumes cultivars in the Mekong Delta,

Vietnam and the results showed that most of they were fast-growing soyben rhizobia

(Sinorhizobium fredii) and several rhizobial strains had high nodule number/plant (>100

nodules/plant) and high DW of shoot in comparison to control and reference strain (USDA

110)(Diep et al., 2004). Their results showed that most of the isolates were Sinorhizobium

fredii except two isolates (CJ02 and CJ04) were Bradyrhizobium japonicum as Annapurna et

al. (2007) previous described.

CONCLUSION

Total of 44 root nodule isolates of soybean rhizobia were isolated from two field sites (Cujut,

DakNong province and Buonho town, DakLak province). They were characterized using

PCR-16S-23S rDNA IGS and PCR for repeated sequence RSα a 1195 bp DNA fragment. The

results showed that two isolates (CJ02 and CJ09) were Bradyrhizobium japonicum and others

were Sinorhizobium fredii. Three strains Sinorhizobium fredii CJ01 and CJ10 and

Bradyrhizobium japonicum CJ09 revealved promising candidates to produce a inoculant for

soybean cultivation on ferralsols.

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.



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REFERENCES

1. Adhikari, D., M. Kaneto, K. Itoh, K. Suyama, B.B. Pokharei, and Y.K. Gaihre, Genetic

diversity of soybean-nodulating rhizobia in Nepal in relation to climate and soil

properties. Plant Soil., 2012; 237: 131-145.

2. Annapurma, K., Balakrihman and Vital., L.,Verification and Rapid Identification of

Soybean Rhizobia in India Soils. Current Microbiology., 2007; 54: 287-291.

3. Beringer, J.E., R-factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol., 1974;

84: 188-196.

4. Chen, W.X., Yan, G.H and Li, J.L.,Numerical taxonomic study of fast-growing soybean

rhizobia and a proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int.

J. Syst. Bacteriol., 1988; 38: 393-397.

5. Devine, T.E., Nodulation of soybean plant introduction lines with the fast-growing

rhizobial strain USDA 205. Crop Sci., 1985; 25: 354-356.

6. Diep, C.N., N.N. Dang, N.T. Phong and T.P. Duong., Biodiversity of soybean rhizobia

isolated from nodules of soybeans and wild legumes growing in the Mekong Delta,

Vietnam using Amplified 16S-23S Intergenic Spacer Ribosomal DNA Gene Sequences

and Restriction Endunuclease Analysis (PCR-ARDRA IGS). Journal of Agricultural

Science and Technoly, Nong Lam University, Vietnam, 2004; 4: 86-91.

7. Dowdle, S.F. and Bohlool, B.B., Predominance of fast-growing Rhizobium japonicum in

a soybean field in the People‟s Republic of China. Appl. Environ. Microbiol,, 1985; 50:

1171-1176.

8. Gordon, S.A. and Weber, R.P.,Colometric estimation of indolacetic acid. Plant Physiol.,

1951; 26: 192-195.

9. Jordan, D., NOTES: transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium

gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int. J.

Syst. Bacteriol., 1982; 32: 136-139.

10. Kaluza, K., M. Hahni, and H. Hennecke., Repeated sequences similar to insertion

elements clustered around the nif region of the Rhizobium japonicum genome. J.

Bacteriol., 1985; 162: 535-542.

11. Keyser, H.H. Bohlool, B.B., Hu, T.S., and Weber, D.F., Fast-growing rhizobia isolated

from root nodules of soybeans. Science, 1982; 215: 1361-1632.

12. Krishnan, H.B. and Pueppke, S.G., Sequence and analysis of the nod ABC region of

Rhizobium fredii USDA 257, a nitrogen-fixing symbiont of soybean and other legumes.

Mol. Plant-Microbe Interact., 1991; 4: 512-520.



133


13. Kuykendall, L.D., Roy, M.A., O‟Neill, J.I., and Devine, T.E., Fatty acids, antibiotic

resistance and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum.

Intnt. J. Syst. Bacteriol., 1988; 38: 358-361.

14. Kuykendall, L.D., Saxena, B., Devine, T.E., Udell, S.E, Short interpersed repetitive DNA

sequence in prokaryote genomes. J. Bacteriol., 1992; 174: 4525-4529.

15. Kwon, S.H., Soybeans and soybean products in Vietnam., Agricultural Research

Institute, Ministry of Land reform and development of Agriculture and Fisheries, Saigon,

Vietnam., 1969.

16. Hartmann, A., M. Gomez, I.J. Giraud, and C. Revllin., Repeated sequence RS α is

diagnostic for Bradyrhizobium japonicum and Bradyrhizobium elkanii. Biol. Fert., Soils.,

1996; 23: 15-19.

17. Hungria, M., Chueire, L.M.O., Megias, M, Lamrabet, Y, Probanza, A, Guttierrez-

Mafiero, F.J., and Campo, R.J., Genetic diversity of indigenous tropical fast-growing

rhizobia isolated from soybean nodules. Plant Soil., 2008; 288: 343-356.

18. Man, C.X., Wang, H., Chen, W.F., Sui, X.H., Wang, E.T., and Chen, W.X., Diverse

rhizobia associated with soybean grown in the subtropical and tropical regions of China.

Plant Soil., 2008; 310: 77-87.

19. Massol-Deya, A.A., Odelson, D.A., Hickey, R.F., and Tiedje, J.M., Bacterial community

fingerfrinting of amplified 16S and 16S-23S ribosomal DNA gene sequences and

restriction endonucleases analysis (ARDRA). In; Molecular Microbial Ecology Manual,

A,D.L. Akkermans, J.D., van Elsas, F.L., de Bruin, F.J. (eds.). Kluwers Academic

Publishers, Dordrect, The Netherlands., 1995; 2.3: 1-8.

20. Rodriguez-Navarro, D.N., Ruiz-Sainz, J.E., Buendia-Claveria, A., Santamaria, C., Balatti,

P.A., Krisman, H.B., Pueppke, S.G., Characterization of fast-growing rhizobia from

nodulated soybean [Glycime max (l.) Merr] in Vietnam. Syst. Appl. Microbiol., 1996; 9:

240-248.

21. Saeki, Y., Kaneko, A., Hara, T., Suzuki, K., Yamakawa, T., Nguyen, M.T., Nagatomo, Y,

Akao, S., Phylogenetic analysis of soybean-nodulating rhizobia isolated from alkaline

soils in Vietnam. Soil Sci. Plant Nutri., 2005; 51: 1043-1052.

22. Scholla, M.H. and Elkan, G.H., Rhizobium fredii sp. nov., a fast-growing species that

effectively nodulates soybeans. Inst. J. Syst. Bacteriol., 1984; 34: 484-486.

23. Shurtleff, W. and Aoyagi, A., History of Soybeans and Soyfoods in Southeast Asia (13th

Century to 2010): Extensively Annotated Bibliography and Soucebook, Soyinfo Center.

2010.



134


24. Vincent, J.M., A manual for the practical study of root-nodule bacteria. Blackwell

Scientific, Oxford, UK, 1970.

25. Wheatcroft, R and Laberge, S., Identification and nucleotide sequence of Rhizobium

meliloti insertion sequence ISRm3: similarly between the putative transposase encoded by

ISRm3 and those encoded by Staphylococcus aureus IS256 and Thiobacillus ferroxidans

IST2. J. Bacteriol., 1991; 173: 2530-2538.

26. Winarno, R and Lie, T.A., Competition between Rhizobium strains in nodules formation:

interaction between nodulating and non-nodulating strains. Plant and Soil, 1979; 51:

135-142.

27. Xu, L.M., and Ge, C., Physiological-biochemical characteristics and symbiotic responses

of the fast-growing Rhizobium japonicum. Soybean Sci., 1984; 3: 102-109.

28. Young, C.C., Chang, J.Y. and Chao, C.C., Physiological and symbiotic characristics of

Rhizobium fredii isolated from subtropical-tropical soils. Biol. Fert. Soils., 1988; 5: 350-

354.

29. Young, J.M., The genus name Ensifer Casids (1982) takes priority over Sinorhizobium

Chen et al. 1988. and Sinorhizobium morelense Wang et al. (2002) is a later synonym of

Ensifer adhaerens Casida 1982. Is the combination „Sinorhizobium adhaerens’ (Casids

1982) Willems et al. (2003) ligitimate? Request for an Opinion. Int. J. Syst. Evol.

Microbiol., 2003; 53: 2107-2110.


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