synergism of vam and rhizobium on production and metabolism of iaa in roots and root nodules of...

Synergism of VAM and Rhizobium on Production and Metabolismof IAA in Roots and Root Nodules of Vigna Mungo

Jayanta Chakrabarti • Sabyasachi Chatterjee •

Sisir Ghosh • Narayan Chandra Chatterjee •

Sikha Dutta

Received: 10 October 2009 / Accepted: 15 January 2010 / Published online: 21 March 2010

� Springer Science+Business Media, LLC 2010

Abstract Mature and healthy root nodules of Vigna

mungo appeared to contain higher amount of indole-acetic

acid (IAA) than non-nodulated roots. Dual effect of VAM

fungus, Glomus fasciculatum and the nitrogen-fixing bac-

teria, Rhizobium sp. on the nodulation of roots of V. mungo

was studied. It was recorded that the roots which were

inoculated simultaneously with both the symbionts i.e., G.

fasciculatum and Rhizobium exhibited greater amount of

IAA production than the non-inoculated roots. A trypto-

phan pool present in the mature nodules and young leaves

might serve as a precursor for IAA production in the roots

and in the nodules. Activity of IAA-metabolizing enzymes,

such as IAA oxidase, peroxidase, and polyphenol oxidase

was investigated which indicates the active metabolism of

IAA in roots and nodules. The Rhizobium symbiont iso-

lated from fresh nodules of V. mungo produced significant

amount of IAA under in vitro condition when tryptophan

was added to the medium as precursor. Present study rep-

resents some beneficial effects of Rhizobium and G. fas-

ciculatum on the production and metabolism of IAA in

roots and nodules of V. mungo. The important physiolog-

ical implication of the study on IAA production and its

metabolism in Rhizobium–Legume–VAM tripartite sym-

biosis is certainly representing a new approach to satisfy

the hormonal balance in the host plant.

Introduction

The legume, Vigna mungo is an important pulse having

good amount of protein and phosphoric acid and as such

adorable to our daily diet. The plant produces numerous

root nodules from very beginning of its early develop-

mental stage. The appreciable amount of different phyto-

hormones present in the roots of many leguminous plants

play important roles in formation [25] and development

[33, 41] of root nodules.

The term ‘‘symbiosis’’ was first used by de Bary [17] to

refer to ‘‘the living together of differently named organ-

isms’’. Mycorrhizal association between fungi and more

than 80% of land plants [31] and the nitrogen-fixing

interaction between bacterial members of Rhizobiaceae

and legumes are the two most commonly studied symbio-

ses. The association between Rhizobium and legumes and

that between vesicular arbuscular mycorrhizal fungi

(VAM) and most of the land plants display a remarkable

degree of similarity. Both the events of symbiosis involve

the recognition of the host for entry and coexistence within

the plant roots, with the development of a specialized

interface that always separates the two partners and at

which nutrient exchange occurs. Development of both the

fungal and bacterial symbioses can be explained as an

analogous progression of events like attraction, recogni-

tion, contact, entry, growth, development, and differentia-

tion. The wide spread occurrence of VAM fungi in

nodulated legumes and the mycorrhizal role in improving

nodulation and Rhizobium activity within the nodules are

universally recognized [3, 6, 12–14, 22, 37].

Plants colonized by VAM fungi have altered levels of

auxin and cytokinin [7]. The purpose of present investi-

gation is to throw some light on the interactive effects

of vesicular arbuscular mycorrhizal fungus Glomus

J. Chakrabarti � S. Chatterjee � S. Ghosh �N. C. Chatterjee � S. Dutta (&)

Mycology and Plant Pathology Laboratory, UGC Centre of

Advanced Study, Department of Botany, The University of

Burdwan, Burdwan, West Bengal 713104, India

e-mail: [email protected]; [email protected]

S. Chatterjee

e-mail: [email protected]

123

Curr Microbiol (2010) 61:203–209

DOI 10.1007/s00284-010-9597-2

fasciculatum and Rhizobium on the production of indole

acetic acid (IAA) and its metabolism in the roots and root

nodules of V. mungo.

Materials and Methods

Conduction of Pot Culture

A pot culture experiment was conducted in the net house of

Botany Department, Burdwan University during the crop-

ping season of 2004 and 2005.

The certified seeds of V. mungo (native variety) were

purchased from the Crop Research Farm (CRF) of Burdwan

University. The plants were grown in pots. Mature and fresh

nodules and young roots were selected for the experiment.

Collection and Maintenance of VAM and Rhizobium

Culture

VAM

The vesicular arbuscular mycorrhizal fungus, G. fascicul-

atum was collected from the Bidhan Chandra Krishi Vis-

vavidyalaya, West Bengal, India, and was maintained in

roots of Zea mays in the net house of Botany Department,

Burdwan University, West Bengal, India.

Rhizobium Culture

Mature and fresh root nodules of the plant V. mungo were

selected for isolation of the symbiont. The isolated sym-

biont was grown in pure culture on basal yeast extract

mineral medium of Skerman [36] with 1% mannitol and

0.1% CaCl2, 2H2O (instead of NaCl and CaCO3) at pH 7.0

and at 37�C temperature for 30 h through repeated dilution

plating and colony selection. The bacteria were also rou-

tinely checked under microscopic observations for studying

the cell morphology of the growing bacteria. The medium

was supplemented with isomers of tryptophan (D-, DL-, and

L-tryptophan). Bacteria were incubated in 30 ml medium in

conical flasks (100 ml) in three replicates at 30 ± 2�C on a

rotary shaker. Growth of the bacteria was measured tur-

bidometrically by a spectrophotometer at 540 nm. After

bacterial growth, the culture medium was centrifuged and

the cell-free supernatant was used for extraction of IAA,

and the extracted IAA was estimated by the method of

Gordon and Weber [20].

Incubation of V. mungo Roots with Mycorrhizal Inocula

Mycorrhizal inocula of G. fasciculatum consisting of

spores and mycelia isolated from soil and mycorrhiza

infected root segments of Zea mays were introduced in the

soil of pot culture at a depth of 2 inches below the seeds

sown in 9 inches pots having 2 kg of soil per pot.

For the study of production and metabolism of IAA as a

result of VAM Rhizobium interaction, the IAA levels in

young roots, nodules, and VAM ? Rhizobium infected

roots of V. mungo were analyzed. As tryptophan is the

precursor of IAA, so in our study tryptophan level was also

estimated.

Extraction and Estimation of IAA

IAA was extracted from fresh tissues of both the VAM

infected and non-VAM infected roots and nodules of V.

mungo following the method of Sinha and Basu [35]. The

extracted IAA was purified by repeated thin layer Chro-

matography (TLC) following Sinha and Basu [35] and was

estimated colorimetrically by Salkowski reagent following

the method of Gordon and Weber [20] using a standard

curve prepared from authentic IAA. This purified IAA

extract was fully devoid of tryptophan, ascertained by

spectrophotometric test for tryptophan following Hassan

[21].

Estimation of Tryptophan

Tryptophan was extracted according to Nitsh [32] and

estimated colorimetrically at 360 nm following the method

of Hassan [21]. Total phenol content of the tissue was

estimated after Bray and Thorpe [9].

Enzyme Assay

Peroxidase and polyphenol oxidase were extracted and

partially purified by (NH4)2SO4 saturation up to 95% fol-

lowed by dialysis and was estimated according to Kar and

Mishra [24]. Oxidation of IAA by Peroxidase activity of

the partially purified extract was estimated following

Kokkinakis and Brooks [26] with slight modification fol-

lowing Datta and Basu [15, 16]. The protein content in the

enzyme extract was estimated following Lowry et al. [28].

Results and Discussion

The herbaceous legume V. mungo produced a plenty of root

nodules, which were pink in color and oval to circular in

shape. Mature root nodules of the plant contained higher

amount of IAA than the non-nodulated roots. Dutta and

Basu [15, 16] and Ghosh and Basu [18] reported that the

IAA content in the roots of leguminous plants apart from

nodules was below detection level. However, it is evident

204 J. Chakrabarti et al.: Synergism of VAM and Rhizobium

123

from present investigation (Table 1) that the amount of

IAA in VAM-inoculated roots of V. mungo was 12.13 lg/g

of fresh tissue and that to was much higher (38.25 lg/g of

fresh tissue) in the nodules which were inoculated with

VAM (VAM ? Rhizobium infected root nodules).

Among the IAA metabolizing enzymes, the activity of

IAA oxidase, peroxidase, and polyphenol oxidase was

higher in the roots than in nodules (Table 2). From

Table 1, it is observed that a high tryptophan pool exists in

the nodule–Rhizobium–VAM symbiosis. Higher levels of

total phenol in the nodules (Table 1) may well be due to

the lower peroxidase activity in the nodules [38] than in the

roots (Table 2). The probable explanation of the difference

in phenol levels in different parts of the plant would also be

due to the variation in the metabolism and synthesis of

phenols in different tissues by phenylalanine-ammonia

lyase and tyrosine-ammonia lyase [38, 40]. Higher amount

of total phenols in the VAM-inoculated roots and nodules

of V. mungo than non-VAM inoculated roots and nodules

may well be attributed with increased resistance of the host

due to VAM infection.

An enhancement in IAA production in VAM ? Rhizo-

bium-infected roots may well be due to the synergistic

interaction between the endosymbionts which is reflected

by enhanced rhizobial activity in terms of increased nodule

number, size of the nodules and their biomass, nodule

protein, leghaemoglobin content, nitrogenase activity, etc.

[34]. IAA production by the nodule bacteria might have

important physiological implications. IAA alone or in

conjugation with other plant hormones, might be involved

in different stages of the symbiosis. Genes induced by IAA

are probably involved in execution of vital cellular func-

tions and developmental processes [41].

Plant hormone levels are altered in legume–Rhizobium–

VAM tripartite symbiosis. Plant colonized by AM fungi

has altered levels of auxins and cytokinin [7]. AM fungi are

known to synthesize some plant hormones like IAA and

cytokinins [4, 5]. Van Rhijn et al. [39] found that alfalfa

roots colonized by Glomus intraradices contain higher

levels of trans-zeatin riboside than non-mycorrhizal roots

and such trend was enhanced by Nod factor application.

The reported increase in the production of IAA and cyto-

kinins in VA mycorrhizal plants [1] raises the question of

whether the growth regulators are produced by the plant

itself in response to fungal infection. Barea and Azcon-

Aguilar [4, 5] have showed that at least a part of the total

Table 1 IAA and other indole compounds present in the root nodules of V. mungo

Indole compound Rf of authentic sample Rf of root nodule extracts of

VAM Rhizobium VAM ? Rhizobium

Indole-3-acetic acid (IAA) 0.84 0.82 0.82 0.84

Indole-3-pyruvic acid 0.76 0.74 0.76 0.76

Indole-3-propionic acid 0.85 – 0.85 0.85

Indole-3-butyric acid 0.87 0.86 – 0.87

Indole-3-glyoxylic acid 0.81 0.81 0.81 0.81

Indole-3-carbinol 0.92 – – –

Tryptamine 0.93 0.93 0.91 0.93

Tryptophol 0.94 0.94 – 0.95

Indole-3-acetal-dehyde 0.96 0.96 0.96 0.96

Others (unknown) NR ? ? ?

The solvent system was isopropanol:Ammonia:Water (10:14:0.6)

‘?’ = present, (–) = absent and NR not recorded. The hormones were identified by comparing the Rf (Relative frequency) values of the

extracted indole compounds with those of authentic samples

Results presented are the mean of 3 individual experimental set up

Table 2 Content of tryptophan,

IAA and total phenol (lg/g

fresh tissue) in nodules and

roots of VAM infected and non-

VAM infected plants of V.mungo

Plant parts IAA (lg/g

fresh tissue)

Tryptophan (lg/g

fresh tissue)

Total Phenols

(lg/g fresh tissue)

Roots 4.27 ± 0.01 535 ± 2.00 1108 ± 1.15

Nodule (Rhizobium) only 20.33 ± 0.01 1875 ± 1.73 1568 ± 1.73

VAM ? Nodule (= VAM ? Rhizobium) 38.25 ± 0.02 3528 ± 1.15 2150 ± 1.15

VAM ? Roots (= VAM only) 12.13 ± 0.01 1119 ± 3.06 1215 ± 0.58

CD at 5% 11.10 988.80 358.06

SEM ±3.803 ±338.630 ±122.623

J. Chakrabarti et al.: Synergism of VAM and Rhizobium 205

123

contribution is from the endosymbiotic mycorrhizal fungi

itself. Assuming that hormones like IAA play a role in the

infection mechanism of legume roots by Rhizobium [33],

the formation of these substances by endomycorrhizal

fungi could help to explain some interactions between

these microorganisms in establishing dual symbiosis with

legumes [2].

The beneficial effect of VAM in symbiotic activity of

Rhizobium and in nodulation and N2 fixation is due to the

relatively high phosphorus demand during N2 fixation

process by Rhizobium which is mitigated with Phosphorous

supplied by the mycorrhizal fungus. However, nutrients

other than P, such as Zn, Cu, Mo, and Ca can affect the

infectivity and symbiotic efficacy of Rhizobium. Enhanced

uptake of different nutrients like P, K, N, Ca [11, 27], Zn

[8], and S [10] by the VAM fungi has been demonstrated

using radioactive isotopes. A tripartite symbiosis of Cy-

amopsis tetragonoloba, VAM fungi, and Rhizobium

resulted in higher accumulation of Ca, Mg, and Mn in leaf

tissue [30]. Concentrations of Fe, Cu, Al, Zn, Co, and Ni

were considerably greater in the shoots of plants coinocu-

lated with the PGPR (Pseudomonas putida) and VAM

fungi than in plants inoculated with the PGPR or VAM

fungi alone [29]. Therefore, enhanced uptake of these

elements could also be involved in the synergestic inter-

action of VAM fungus and Rhizobium conversely. Supply

of Nitrogen by N2 fixation, as carried out by rhizobial ac-

tivivity, could be critical to maintain a balanced physio-

logical status in the host plant, which is important for

mycorrhiza formation and functioning [23].

In order to have an idea about the source of IAA in the

nodules as well as in the roots, the symbiont (Rhizobium

sp.) from the plant was isolated and checked to study its

ability for IAA production under in vitro condition. When

the bacteria were grown in tryptophan supplemented yeast

extract mannitol medium, production of IAA appeared to

be the highest (Table 4). The bacteria preferred L-trypto-

phan for growth and IAA production than D-tryptophan and

DL-tryptophan (Table 3). Although an increase in the con-

centration of L-tryptophan enhanced growth and IAA pro-

duction by the Rhizobium symbiont (Fig. 1), but it was

appended from the curve that concentration of L-tryptophan

higher than 2.0 mg/ml in the growth medium happened to

be the inhibitory for growth and IAA production by the

Table 3 IAA oxidase peroxidase and polyphenol oxidase activity in nodules and roots of V. mungo

Plant parts IAA oxidase (lg/g IAA

oxidase/mg protein/hour)

Polyphenol

oxidase

Peroxidase (lg/g IAA

oxidase/mg protein/hour)

Roots 44.12 ± 0.012 13.00 ± 0.058 15.01 ± 0.006

Nodule (Rhizobium) only 14.11 ± 0.006 9.10 ± 0.006 9.86 ± 0.012

VAM ? Nodule (= VAM ? Rhizobium) 12.96 ± 0.012 8.85 ± 0.000 9.81 ± 0.012

VAM ? Roots (= VAM only) 11.25 ± 0.12 9.14 ± 0.006 10.72 ± 0.012

CD at 5% 11.984 1.886 1.518

SEM ±4.104 ±0.646 ±0.520

Fig. 1 Effect of different

concentrations L-tryptophan on

IAA production and growth by

Rhizobium sp. in culture


123

bacteria. Both the growth and IAA production by the

bacteria started simultaneously and reached to the sta-

tionary phase after 18 h of growth period (Fig. 2). Level of

IAA production in the medium declined during late sta-

tionary phase of growth. Decrease in the level of IAA

might be due to the release of some IAA degrading

enzymes [42].

The authenticity of the extracted IAA was checked by

TLC and UV-spectrophotometry (Tables 4, 5). A com-

parison of the UV-spectra of extracted IAA with that of

authentic sample also proved the authenticity of the

extracted IAA. It is evident from the results that the levels

of IAA in nodules, in VAM-infected roots and in

VAM ? Rhizobium-infected roots were always higher in

colorimetric assay (Table 1) than in the UV-spectropho-

tometric assay (Table 5). The estimated differences in the

levels of IAA in UV-spectrophotometry and colorimetry

(Tables 4, 5) were probably due to the presence of some

unknown chemicals of similar physicochemical nature in

the extracts.

From the present study, it has been established that

legume root nodules contain higher amount of IAA and a

part of it is transported to the host. If nitrogen fixation in

the root nodules and supply to the host be taken as first line

of symbiosis then IAA content and its supply to the host

may be taken as a second line of symbiosis between

legume and Rhizobium [19]. Moreover, it should also be

mentioned here that there is a molecular and chemical

dialog that occurs between VAM fungi and Rhizobia with

their hosts and it appears to be crucial for recognition of the

hosts by the symbionts and initiation of the symbioses. In

case of nitrogen-fixing symbiotic association, plant exu-

dates induce the production of bacterial ‘‘Nod factor’’,

the molecular signal which dictates the host-symbiont

Fig. 2 Growth of the

Rhizobium sp. and its IAA

production in culture

Table 4 Effect of different

tryptophan isomers on growth

and IAA production by

Rhizobium sp. in culture

Isomers

of tryptophan

Growth at

540 nm (O.D.)

IAA production

(lg/ml)

Specific productivity

(IAA production/growth)

D-tryptophan 1.12 ± 0.006 21.30 ± 0.015 19.01 ± 0.000

L-tryptophan 1.36 ± 0.012 114.25 ± 0.017 84.00 ± 0.305

DL-tryptophan 1.28 ± 0.012 89.18 ± 0.012 69.67 ± 0.017

CD at 5% 0.105 40.535 28.686

SEM ± 0.036 ± 13.882 ± 9.824

Table 5 Content of IAA (lg/g fresh tissue) in the nodules and young

roots infected either alone with VAM or both with VAM and Rhi-zobium as depicted by UV absorbance (UV-spectrophotometry) after

purification by TLC

Plant parts IAA (lg/g

fresh tissue)

Nodule (= Rhizobium infected) 14.39 ± 0.023

Young roots (VAM ? Rhizobium infected) 27.08 ± 0.012

Young roots (only VAM infected) 8.59 ± 0.023

Young roots (without VAM and Rhizobium) 4.27 ± 0.023

CD at 5% 7.554

SEM ± 2.587

Data presented are the mean of three individual experimental sets

After purification through TLC plates, IAA was checked for its UV

absorption pattern and compared with that of the authentic sample.

The UV absorption pattern of IAA (k max at 280 nm) from the

nodules and young roots of the plant with that of authentic IAA


123

specificity and progression of the symbiosis. Interestingly,

there appears to be some overlap in the signals involved in

such synergistic associations. For example, ‘‘Nod factor’’

application enhances mycorrhizal colonization [43]. Thus,

from present discussion, it is logical to conclude that in

addition to nitrogen fixation, hormone production is a

beneficial aspect of the legume–Rhizobium–VAM tripartite

symbiosis.

Acknowledgments The authors are thankful to UGC (SAP-II,

Phase-III), Govt. of India for financial assistance.

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