MECHANISM OF ROOT GROWTH ANDPROMOTION OF NODULATION IN

VEGETABLE SOYBEAN BY AZOSPIRILLUMBRASILENSE

Abul Hossain Molla,1,* Zulkifli H. Shamsuddin,2 and

Halimi Mohd. Saud2

1Department of Crop Botany, Bangabandhu Sheikh Mujibur

Rahman Agril. University, Gazipur 1703, Bangladesh2Department of Land Management, Faculty of Agriculture,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,

D.E., Malaysia

ABSTRACT

Stimulation of root growth and promotion of nodulation in

vegetable soybean (Glycine max ) by Azospirillum brasilense was

studied under lightroom conditions to understand the possible

mechanism of root growth as well as the enhancement of nodule

initiation when co-inoculated with Bradyrhizobium. Root growth

stimulation of vegetable soybean was positively influenced by the

cell-free supernatant of Azospirillum brasilense Sp7 as well as Sp7

itself, and by indole acetic acid (IAA) application. The cell-free

supernatant of Sp7 treated plants produced the highest number of

roots and root length plant21 followed by bacterial cells of Sp7

2177

Copyright q 2001 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author. Current address: Biochemical Engineering Lab., Dept. of

Chemical and Environmental Engineering, Universiti Putra Malaysia, 43400 UPM

Serdang, Selangor DE, Malaysia. E-mail: jcd4086@hotmail.com

COMMUN. SOIL SCI. PLANT ANAL., 32(13&14), 2177–2187 (2001)

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and IAA log109M) application. Control plants were the lowest.

The cell-free supernatant of Sp7 may contain growth stimulants

similar to IAA, which was responsible for enhanced root growth.

Nitrite and nitrate did not show positive role in increasing root

growth in vegetable soybean. Co-inoculation of Azospirillum with

Bradyrhizobium significantly ðP , 0:05Þ promoted nodulation in

vegetable soybean. Bradyrhizobium strain UPMR48 performed

superior role on promotion of nodule growth by co-inoculation to

the strain TAL102. Azospirillum has the potential as a co-

inoculant with Bradyrhizobium in vegetable soybean cultivation.

INTRODUCTION

Vegetable soybean is a nutritious leguminous vegetable, not only rich in

protein, cholesterol-free fat, and vitamins (1), but also sugar, iron, phosphorus

and globulin (2). Traditionally, soybean is cultivated with inoculation of

Bradyrhizobium. In recent years, there have been studied on the possible

enhancement of root growth and nodulation of legumes when inoculated with a

mixture of inoculants. One of the possible inoculant is the free-living diazotroph,

such as Azospirillum sp.

Azospirillum is a free-living atmospheric N2 fixer and potential PGPR

(plant growth promoting rhizobacteria), which positively influences plant growth,

and yield of several leguminous and non-leguminous crops (3,4,5,6,7). A positive

influence on root growth, resulting in higher nutrient and water uptake, is the key

benefit to plants of Azospirillum application. It has many modes of stimulating

root growth but most studies cited phytohormones synthesis as one of the

principal mechanisms (8). Phytohormones, mostly IAA, are synthesized by

Azospirillum and plays a key role in enhancing root growth (9,10,11). Auxin

(IAA) has no effect on lateral root growth formation, nitrite, either added directly

or excreted by Azospirillum in nitrate respiration, causes a drastic change in

lateral root formation in wheat (12), although the negative effect of nitrite to root

growth is well known. In addition to increase root growth in non-legumes (4,5,13)

and legumes (14) by Azospirillum application, it also enhances biological

nitrogen fixation activity (15,16). Encouraging but inconsistent results on

increased nodulation, acetylene reduction activity, nutrient uptake, and yield have

been reported in different legumes (6,7,17). Besides inhibition of nodulation,

decreased infection thread developments were also reported in white clover (18)

and Medicago (19) when co-inoculated with Azospirillum. The issues of

increased root growth and enhanced nodulation in co-inoculation by Azospirillum

are still contradictory as well as inconsistent. The cited reports were, to some

extent, crop specific.

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In the present study, attempts were made (a) to evaluate the stimulation of

root initiation by Azospirillum brasilense, in comparison to combined N, in the

form of nitrate and nitrite with IAA application, and (b) to investigate the effect of

co-inoculation on the promotion of nodulation in vegetable soybean.

MATERIALS AND METHODS

Root Growth Stimulation

The experiment was conducted in test tubes (2.6 cm� 20 cm) with seven

treatments; (i) control (2N, without N), (ii) Sp7 (Azospirillum brasilense )

inoculation, (iii) cell-free supernatant of Sp7, (iv) 0.4 mM nitrite, (v) 0.4 mM

nitrate, (vi) IAA (log106M) and (vii) IAA (log10

9M). Strain Sp7 (A. brasilense )

was used from lab oratory stock obtained from Dr. Johanna Dobereiner, Embrapa

National Centre for Agrobiol. Res., Seropedica 23851-970, Brazil. A 30-mL

amount of nutrient (N-free) solution (20) was added to each test tube and a single,

uniform, healthy, surface sterilized, sprouted seed of vegetable soybean

(AGS190) of AVRDC (Asian Vegetable Research and Development Centre,

Taiwan) was placed in mouth of each test tube and was sealed by aluminum foil.

The seedlings were allowed to grow inside the test tube on the surface of nutrient

solution and kept upright with a suitable piece of sponge. The whole set of test

tubes was prepared in advance and sterilized before sowing the sprouted seeds.

For cell-free supernatant of Sp7, the inoculant was centrifuged (17310 g) 10 min

followed by sieving through 0.20-mm membrane Millipore to remove the

bacterial cells. Forty-eight-hour old bacterial cells (Sp7) and its cell-free

supernatant were used with a concentration of 1.83� 109 cfu mL21 were grown in

Okon broth (21). Half (0.5) mL of cell-free supernatant was applied to the

respective test tubes before placement of the sprouted seeds. For Sp7 treatment

the same amount (0.5 mL) of bacterial cell’s suspension was applied in each test

tube similar to cell-free supernatant. All of these tasks were ensured at sterile

environment. The plants were allowed to grow for 10 days at room temperature

with 12 h light (37.4mmol m22 s21) provided by cool-white fluorescent lamps in

an incubation room.

The experiment was conducted in completely randomized design (CRD)

and replicated seven times. Analyses of variance and comparison means were

done separately by Statistical Analysis System (SAS) package (22) and the least

significant difference (LSD) test was used to compare among the treatments.

Root numbers, root length, root fresh and dry weight were recorded and root

length was measured by grid line intersections method proposed by Newman (23)

and Tennant (24). For dry weight measurement, the samples were kept at 808C to

constant weight.

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Promotion of Nodulation

Promotion of nodulation was studied in glasshouses in Leonard jars by co-

inoculation of Azospirillum (Sp7) and Bradyrhizobium japonicum (TAL102 and

UPMR48) in vegetable soybean (AGS190), with six treatments; (i) control with N

(+N), (ii) control without N (2N), (iii) TAL102, (iv) UPMR48, (v) Sp7 + TAL102

and (vi) Sp7 + UPMR48. Bradyrhizobium strain TAL102 was obtained from

University of Hawaii, College of Tropical Agriculture & Human Resources,

Department of Agronomy & Soil Science (NifTAL), Hawaii 96779, USA and

UPMR48 was locally isolated from soybean nodules. Surface-sterilized uniform

sized sprouted seedlings were sown in sterile jars containing vermiculite, and

nutrient solution (20). The inocula were twenty-four-hour old Azospirillum Sp7

in Okon broth (21) with a concentration of 1.01 � 109 cfu ml21 and

Bradyrhizobium TAL102 & UPMR48 were 72 h old in YM broth (25) with

concentrations of 4.9� 106 and 4.6� 106 cfu mL21 respectively. Sprouted seeds

were inoculated by immersing it in respective Bradyrhizobia bacterial suspension

for 1 h for single inoculation. For co-inoculation, the seeds were first soaked for

30 min in Azospirillum suspension followed by 30 min in Bradyrhizobia

suspension. Non-absorbent cotton wool was used on the surface of vermiculite to

avoid contamination. The plants were harvested 30 days after sowing.

The average temperatures 34.2 (max.) and 22.5 (min.), available sun shine

6.5 h and 77.3% humidity were recorded in the glasshouse. Completely

randomized design was laid out to conduct this experiment along with six

replications. ANOVA and means comparison were done in SAS package and to

compare differences among the treatments it was tested by Duncan’s Multiple

Range Test.

RESULTS AND DISCUSSION

Root Growth Stimulation

Root growth of vegetable soybean was influenced by the cell-free

supernatant of Sp7 and Sp7 inoculation, compared to combined N and control

(uninoculated) plants. Enhanced root initiation was also observed in IAA

(log109M) treated plants (Table 1). Secondary and tertiary root numbers increased

significantly ðP , 0:05Þ, especially by the cell-free supernatant of Sp7 followed

by Sp7 inoculation and IAA (log109M) application. Primary and total root length

also increased significantly ðP , 0:05Þ by Sp7 inoculation, cell-free supernatant

of Sp7 and IAA (log109M) application. Nitrite and nitrate did not have any effect

on root growth except for tertiary roots and total root length for nitrate.

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Table 1. Root Growth of Vegetable Soybean (AGS190) Studied Under IAA, Combine N and Azospirillum Application. In Each Column,

Common Letters Are Not Significantly Different ðP , 0:05Þ by LSD

Treatments

Secondary Root

(Number Plant21)

Tertiary Root

(Number Plant21)

Primary Root

Length (cm/Plant21)

Total Root Length

(cm/Plant21)

Root Fresh Weight

(mg/Plant21)

Root Dry Weight

(mg/Plant21)

Control (2N) 07.20 e 04.80 c 2.90 b 31.90 d 206.00 ab 12.80 c

Sp7 17.20 b 10.60 b 6.36 a 70.00 a 172.00 b 13.60 bc

Supernatant (Sp7) 25.00 a 14.20 a 6.38 a 73.20 a 222.60 ab 17.00 abc

0.4 mM NaNO2 06.00 e 05.40 c 2.80 b 30.80 d 195.60 ab 18.80 abc

0.4 mM NaNO3 09.80 de 08.60 b 3.64 b 45.20 c 224.00 ab 21.40 ab

IAA (log106M) 12.20 cd 07.60 bc 2.92 b 32.20 d 228.00 ab 18.20 abc

IAA (log109M) 14.80 bc 09.20 b 5.30 a 58.30 b 236.00 a 24.80 a

LSD (5%) 04.90 03.03 1.09 11.32 63.61 07.93

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The root fresh weights were similar in all treatments, but the highest value

of fresh and root dry weight were observed with IAA (log109M) application. The

lowest values of all root parameters except root fresh weight were observed in

control treated plants. The lowest root fresh weight was observed in Sp7

inoculation. Increased root dry weight was observed in all treatments compared

to control plants, but values were not statistically significant.

Azospirillum brasilense Sp7 stimulated root growth of vegetable soybean,

similar trend of observations in other crops were reported by several authors

mainly in non-legumes (4,5,13). The cell-free supernatant of Sp7 showed

superior results in root growth stimulation compared to Sp7 bacterial cells. This

might be a consequence of environmental factors of test tubes on bacterial cells

growth. The limited space, environmental factors and solution pH of the test tubes

may not allow the bacterial cells to grow and function optimally, whilst the cell-

free supernatant would not have this problem. Bacterial cells multiplication

induced the pH of the test tubes solution into more acidic than the initial (data

were not presented). The stimulation of root growth of vegetable soybean by the

cell-free supernatant of Sp7 might be due to the production of phytohormone(s).

Azospirillum brasilense synthesis of IAA, Indole Lactic Acid (ILA) and

Gibbrellic Acid (GA) in broth culture which increased root development in pearl

millet has been reported by Tien et al. (9). Bashan and Levanony (26), and Patten

and Glick (11) also reported about production of IAA, GA and cytokinins by

Azospirillum and which is liable for enhancement of plant cell growth and

multiplication. Nitrate and nitrite did not increase root growth in vegetable

soybean, but the opposite results were reported by Bothe et al. (12) in wheat

seedlings. It might be that the concentration of nitrite and nitrate, which was used

in this study, was not favorable for soybean root growth. For the two

concentration of IAA, the higher concentration (log106M) produced poor root

growth compared to the lower concentration (log109M).

Promotion of Nodulation

Promotion of crown, lateral and total nodules number, nodule dry weight

and specific nodule weight were recorded in co-inoculation rather than single

inoculation (Table 2). Higher values of all nodule parameters were observed by

co-inoculation than the respective single inoculation of Bradyrhizobium except

for specific nodule dry weight of Sp7 with TAL102. Total nodule number, nodule

dry weight, and specific nodule dry weight increased significantly ðP , 0:05Þ

with co-inoculation of Sp7 and UPMR48 compared to the single inoculation of

UPMR48. The crown and lateral nodule numbers did not increased significantly

for co-inoculation treatment than the respective single inoculation of

Bradyrhizobium, but the total nodules plant21 was significantly higher in number.

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Table 2. Co-inoculation Effect of Azospirillum and Bradyrhizobium Strains on Nodulation and Plant Growth of Vegetable Soybean. In

Each Column, Common Letters Are Not Significantly Different ðP , 0:05Þ by DMRT

No. of Nodules Plant21 Dry wt. (g Plant21)

Treatments Crown Lateral Total

Nodule Dry wt.

(mg Plant21)

Specific Nodule dry

wt (mg) Root Shoot

Cont (+N) 00.00 b 00.00 c 00.00 e 00.00 c 00.00 c 0.58 a 2.53 a

Cont (2N) 00.00 b 00.00 c 00.00 e 00.00 c 00.00 c 0.28 b 1.05 b

TAL102 15.00 a 19.67 ab 34.67 c 60.00 b 1.73 b 0.29 b 1.48 b

UPMR48 14.33 a 15.00 b 29.33 d 49.00 b 1.68 b 0.23 b 1.12 b

Sp7 & TAL102 15.67 a 28.67 a 44.33 a 65.00 b 1.41 b 0.26 b 1.36 b

Sp7 & UPMR48 20.33 a 19.67 ab 40.00 b 101.33 a 2.51 a 0.27 b 1.39 b

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Promotion percent of all nodule parameters in co-inoculation of Sp7 with

UPMR48 were higher compared to co-inoculation of Sp7 with TAL102 except

for lateral nodule number. In Sp7 with TAL102, the value of specific nodule dry

weight declined compared to its single inoculation (TAL102), i.e. this parameter

was not influenced in co-inoculation (Sp7 with TAL102). Nodule dry weight was

highly promoted (.100%) with Sp7 and UPMR48 (Fig. 1) compared to its single

inoculation (UPMR48). Shoot characters were not influenced (Table 2).

Significantly increased nodule numbers with co-inoculation of Azospir-

illum in soybean (grain) has been reported, but nodule weight and C2H2 reduction

were not affected (17). In the present study co-inoculation of Sp7 with TAL102

increased lateral and total nodules number significantly ðP , 0:05Þ, but specific

nodule weight decreased (Table 2). The observed results implied that the smaller

sized but higher in number nodules plant21 produced in co-inoculation of Sp7

with TAL102 compared to Sp7 and UPMR48 co-inoculation. It was observed

higher nodules number in co-inoculation but not statistically significant in

common bean was reported by Burdman et al. (7) at 41 days after sowing. Similar

trend of results was also reported in chickpea at 52 and 62 days of growth when

co-inoculated with Azospirillum (6). In co-inoculation of Azospirillum with

UPMR48, all nodule parameters were significantly greater ðP , 0:05Þ than the

Figure 1. Promotion percent of nodule growth in vegetable soybean by co-inoculation of

Azospirillum (Sp7) with Bradyrhizobium strains (TAL102 & UPMR48) over its

(Bradyrhizobium ) respective single inoculation. (C.COD = Crown nodule, NDW =

Nodule dry weight, L.NOD = Lateral nodule, T.NOD = Total nodule SNDW = Specific

nodule dry weight).

HOSSAIN MOLLA, SHAMSUDDIN, AND SAUD2184

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single inoculation of UPMR48, except crown and lateral nodule numbers. The

promotion in percent of nodule growth was highly significant in Sp7 with

UPMR48 compared to Sp7 with TAL102. The specific nodule dry weight did not

increased, but rather it decreased (around 20%) in co-inoculation of Sp7 with

TAL102 compared to its single inoculation. Although the total nodule number

was the highest, the specific nodule weight clearly implied that the nodule sizes

was smaller than that of Sp7 with UPMR48. This may be due to immature growth

of nodules caused by insufficient food supply, delayed initiation of nodules in Sp7

with TAL102 or strain specificity.

CONCLUSIONS

Azospirillum (Sp7) has the potential to synthesize plant hormone, which

can replace IAA to stimulate root growth in vegetable soybean. Nitrate and nitrite

did not have positive effect on root growth of vegetable soybean. In co-

inoculation with Bradyrhizobium, Azospirillum (Sp7) could play an encouraging

role to enhance nodulation in vegetable soybean. UPMR48 is superior to TAL102

for vegetable soybean cultivation.

ACKNOWLEDGMENTS

The authors express their gratitude to the Ministry of Environment and

Technology, Malaysia for providing funds for this study.

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