2. REVIEW OF LITERATURE

2.1. Rhizobia

Rhizobia are a genetically diverse and physiologically

heterogenous group of bacteria (Somasegaran and Hoben, 1994) and they

are able to elicit nodule formation on legumes are called rhizobia (Denarie

et al., 1996). Rhizobia comprises of the genera Rhizobium,

Bradyrhizobium and AzoRhizobium (Denarie et al., 1992) and

Sinorhizobium (Denarie et al., 1996). Rhizobia are a ubiquitous part of the

soil micro-flora in a free-living state in the rhizosphere of legumes (Allen

and Allen, 1981 and Somasegaran and Hoben, 1994) until the point where

nodulation becomes possible (Rendig and Taylor, 1989). The nature and

properties of soil allows billions of organisms of coexist (Pepper and

Upchurch, 1991).

The ability of form symbiotic relationships with members of the

plant family Fabaceae is a unique feature associated with bacteria

belonging to the family Rhizobiaceae (Pepper and Upchurch, 1991). The

number of symbiotic relationships that can form between rhizobia and

hosts is restricted and vice versa (Denarie et al., 1992).Rhizobia elicit on

their host and the formation of nodules in which they fix nitrogen

(Denarie et al., 1992; Denarie et al., 1996 and Prescott et al., 1996) and

provide the plant with ammonia for growth (Rendig and Taylor, 1989).

Despite the widespread distribution of leguminous crops, many

soils remain void of rhizobial strains (Brockwell et al., 1995). Grobbelaar

and Clarke (1974) concluded that under local conditions nodulation may

8 not occur with introduced plants due to the lack of suitable Rhizobium

strains.

2.1.1. Rhizobial characteristics

Rhizobia are bacteria that selectively infect the roots of some

legumes and have the following characteristics; gram negative, motile

rod-shaped (approximately 0.5-0.9 m in width and 1.2-3.0 m in length)

and heterotropic (Pepper and Upchurch, 1991; Somasegaran and Hoben,

1994; Prescott et al., 1996).

Root nodule bacteria generally grow under the following

conditions 25-30°C (optimum) in the pH range of 6-7 (Vincent, 1970;

Somasegaran and Hoben, 1994). Rhizobium growth normally occurs under

aerobic conditions. However, when fixing nitrogen, low levels of oxygen

are required to protect the enzyme nitrogenase (Rending and Taylor,

1989) and hence, Rhizobium are able to grow in microaerophilic

conditions (Somasegaran and Hoben, 1994).

Fujihara (2000) described that the rhizobia are of great importance

for nitrogen acquisition through symbiotic nitrogen fixation in a wide

variety of leguminous plants. These bacteria differ from most of other soil

microorganisms by taking dual forms, i.e., a free-living form in soils and a

symbiotic form inside of host legumes. Therefore, they should have a

versatile strategy for survival, whether inhabiting soils or root nodules

formed through rhizobia-legume interactions. Rhizobia generally contain

large amounts of the biogenic amine homospermidine, an analog of

spermidine which is an essential cellular component in most living

systems. The external pH, salinity and a rapid change in osmolarity are

9 thought to be significant environmental factors affecting the persistence of

rhizobia. The regulation of homospermidine biosynthesis in response to

environmental stress and its possible functional role in rhizobia. Legume

root nodules, an alternative habitat of rhizobia, usually contain a variety of

biogenic amines besides homospermidine and the occurrence of these

amines is closely associated with rhizobial infections. In the second half

of this review, novel biogenic amines found in certain legume root

nodules and the mechanism of their synthesis involving co-operation

between the rhizobia and host legume cells are also described.

A large amount of Extra Cellular Polysaccharides (EPS) was

produced in yeast extract mannital medium by Rhizobium sp. isolated

from the root nodules of a monocotyledonous tree, Roystonea regia.

Maximum extra cellular polysaccharide was produced when the medium

was supplemented with mannital (2%) Triton X 100 (0.005% v/v),

MnCl2.4H2O (10 g/ml) and L-glutamic acid (0.3%). The extra cellular

polysaccharides contained xylose, rhamnose and arabinose. Possible role

of rhizobial EPS production in the symbiosis has been discussed (Basu

and Ghosh, 1999).

The Rhizobium sp. isolated from the root nodules of the

leguminous shrub Derris scandens produced a large amount of

extracelluar polysaccharide in a yeast extract-mannitol medium in the

stationary phase of growth. The production was maximum when the

medium was supplemented with mannitol (3%), (+) – biotin (3 mg/L) and

KNO3 (0.3%). The extracellular polysaccharide contained glucose,

galactose and mannose. The possible role of the rhizobial extracellular

polysaccharide is discussed (De and Basu, 1996).

10

2.2. Nodule formation

The presence of appropriate rhizobia the soil is the first critical

factor for nodule formation (Pepper, 1991; Pepper and Upchurch, 1991),

with the relationship between certain bacteria and legumes being highly

selective (Rending and Taylor, 1989). In some cases, only one Rhizobium

spp. is effective on a particular species of legume (Salisbury and Ross,

1992).

2.2.1. Signalling between host and root nodule bacteria

Rhizobial nod genes are important in the determination of host

specificity, infection and nodulation and are involved in the exchange of

signals between the plant and bacteria (Denarie et al., 1992). Legumes

release several flavonoids, some of which may be specific to a particular

Rhizobium. Both Bradyrhizobium and Rhizobium spp. are attracted by

flavonoids (Denarie et al., 1992).

2.2.2. Linkage of host and root nodule bacteria

The attachment of the bacteria to the hairs is the start of the

infection process (Rending and Taylor, 1989; Prescott et al., 1996). The

infection process involves a complex series of interactions, before the

rhizobia enter the root hairs of the host (Somasegaran and Hoben, 1994).

Rhizobium spp. produce nod genes (nodulation genes) that promote

the binding between bacteria and root hairs (Pepper and Upchurch, 1991;

Prescott et al., 1996). Attraction between the rhizobia and root surface

occurs due to van der Walls forces and leads to the linking of free

carboxyl groups in the peptidoglycan of the rhizobial cell to organic acids

11 found on the root surface (Howieson, 1995). Specialised proteins, known

as lectins, found on the surface of the roots are believed to act as

recognition sites (Allen and Allen, 1981; Raven et al., 1992). Rhizobial

multiplication occurs in the rhizosphere and on the root surface (Denarie

et al., 1992).

2.2.3. Rhizobial entry into the root

The method by which root infection occurs includes simple ‘crack

entry’ or through the root hairs. ‘Crack entry’ involves the entry of

rhizobia through intercellular spaces in the epidermis or in the middle

lamella, as seen in Arachis hypogaea L. (Denarie et al., 1992 and Denarie

et al., 1996) and Sesbania rostrate Bremek and Oberm (Webster et al.,

1997). Alfalfa and vetch both display infection of the roots by rhizobia

through the root hairs. Infection by rhizobia through the root hairs is

described in greater detail below.

2.2.4. Rhizobial entry through root hairs

The nodulation gene nod D is a regulatory proteins that is activated

by plant flavonoids, which in turn control the transcription of the nod

operons. Structural nod genes are involved in the synthesis of specific

lipo-oligosaccharides, which signal back to the plant to elicit root hair

deformations, cortical cell divisions and nodule-meristem formation

(Denarie et al., 1992 and Denarie et al., 1996).

Once the rhizobia are attached to the root hairs they are entrapped

as the root hairs they are entrapped as the root hairs curl tightly during

extension (Rending and Taylor, 1989; Raven et al., 1992; Salisbury and

Ross, 1992; Somasegaran and Hoben, 1994). The cell wall of the root

12 hairs is then degraded with the release of specialized enzymes by the

rhizobia, which allows for entry of the rhizobia into the cell itself

(Salisbury and Ross, 1992).

2.2.5. Infection thread formation

Tubular structures known as infection threads allow the invasion of

the root hairs and the underlying cortical cells by the rhizobia. The

infection thread (Figures 1 and 2) is important to the rhizobia as it

provides a means of avoiding the plant defence mechanisms (Pepper and

Upchurch, 1991). Somasegaran and Hoben (1994) reported that the

rhizobia are released from the infection thread into the cytoplasm of the

host cell, where bacterial cell multiplication takes place.

2.2.6. Bacteriod development

The movement of rhizobia from the infection thread into the host

cell results in the rhizobia being surrounded by a membrane known as the

peribacteroid membrane (Prescott et al., 1996). This is the formation of

nodules and leads to the proliferation of the enlarged rhizobia and cortical

cells. The enlarged rhizobia are often referred to as bacteroids in which

the fixation of nitrogen by legumes occurs (Denarie et al., 1992; Raven

et al., 1992; Salisbury and Ross, 1992; Somasegaran and Hoben, 1994).

The further differentiation of the bacteriod results in the nitrogen fixing

structure known as a symbiosome (Prescott et al., 1996).

13

Fig.1. Infection thread formations.

The cytosol of bacteroids is the site of synthesis of nitrogenase, the

enzyme responsible for the reduction of atmospheric nitrogen to

ammonium (Somasegaran and Hoben, 1994). Rhizobium spp. are unable

to reproduce once a functioning nodule is formed (Prescott et al., 1996).

Root nodules are genuine organs, not mere tumors (Denarie et al., 1992

and Denarie et al., 1996). The formation of nodules allows for the control

of oxygen concentration and the conditions for nitrogen gas to be fixed

into ammonia. The rate of nitrogen fixation is dependent on the size of the

nodules and the activity of nitrogen fixation (Rendig and Taylor, 1989).

According to Rendig and Taylor (1989), the time taken for the

host-rhizobia relationship to be formed and be fully functional is

dependent on four main factors: presence of Rhizobium, inoculants that

are competitive with background populations, provision of nutrients by

the host to the rhizobia and the influence of the environment and soil

factors. Nitrogen fixed from the atmosphere is eventually stored within

the legume or used for the assimilation of protein (Allen and Allen, 1981).

14

Fig.2. Rhizobial infections and nodule formation.

2.2.7. Root nodule formation in Sesbania rostrata

Rhizobia colonize their legume hosts by different modes of entry

while initiating symbiotic nitrogen fixation. Most legumes are invaded via

growing root hairs by the root hair-curl mechanism, which involves

epidermal cell responses. However, invasion of a number of tropical

legumes happens through tissues at lateral root bases by cortical,

intercellular crack entry. In the semiaquatic Sesbania rostrata, the bacteria

entered via root hair curls under nonflooding conditions. Upon flooding,

root hair growth was prevented, invasion on accessible root hairs was

inhibited and intercellular invasion was recruited. The plant hormone

ethylene was involved in these processes. The occurrence of both invasion

pathways on the same host plant enabled a comparison to be made of the

structural requirements for the perception of nodulation factors, which

were more stringent for the epidermal root hair invasion than for the

cortical intracellular invasion at lateral root bases (Goormachtig et al.,

2004).

15 2.2.8. Determination of nodules

Khan et al. (1999) suggested that the dot immunoblot assay was

used for determination of nodules produced on soybean, French bean,

pigeon pea and Urdbean by Bradyrhizobium japonicum USDA-110;

R. leguminosarum bv. phaseoli FB-77 and N-3; Rhizobium sp. A-3 and

U-1, respectively. Nodule occupancy by inoculated strains as determined

by the test ranged between 73 to 93 per cent. Replica immunoblot assay

reduced the time required for enumeration of rhizobia and was suitable for

strain specific enumeration of Rhizobium strains in nodules. Correlation

between immunoblot and traditional plate counts was r = 0.96 for

5 rhizobial strains tested.

2.3. Nitrogen fixation

Nitrogen fixation is the biological process by which atmospheric

N2 gas (nitrogen diatom) is reduced to 4NH (Raven et al., 1992; Salisbury

and Ross, 1992). Nitrogen can thus be added to ecosystems (Boddey et

al., 2000). Biological nitrogen fixation (BNF) is of great importance in a

number of environments, such as, terrestrial, freshwater, marine and arctic

(Salisbury and Ross, 1992).

Raven et al. (1992) reported that the fixation of nitrogen is a

process upon which all living organisms are dependent. Biological

nitrogen fixation is estimated to be approximately 150 to 200 million

tonnes annually on the earth’s surface. The symbiotic relationships

between specific soil micro-organisms and plants are the most significant

contributor of BNF in most terrestrial ecosystems (Boddey et al., 2000).

16 2.3.1. Symbiotic nitrogen fixation (SNF)

Symbiotic nitrogen fixation is a process carried out by root nodule

bacteria in association with legumes (Raven et al., 1992). The fixation of

nitrogen provides the plant with available ammonium, whilst the plant

provides the rhizobia with simple sugars (Pepper, 1991; Pepper and

Upchurch, 1991). The increase of rhizobia numbers in the rhizosphere is a

response to the release of nutrients by the host legume (Somasegaran and

Hoben, 1994).

Pathak et al. (2008) reported that the nitrogen use efficiency (NUE)

in plants is a complex phenomenon that depends on a number of internal

and external factors, which include soil nitrogen availability, its uptake

and assimilation, photosynthetic carbon and reductant supply, carbon-

nitrogen flux, nitrate signaling and regulation by light and hormones, and

physiological, biochemical and molecular aspects of NUE improved the

crop plant, N flux for Indian crop cultivars.

Urzua (2005) reported that advantages of symbiotic nitrogen

fixation in plants and improved the efficiency of SNF by some methods

included strain characterization and laboratory selection, green house

studies, N-accumulated, nodulation, C2H2 education. SNF facilitated their

management, achieving higher productions and improving the quality of

the crops, saving N-fertilizer and at the same time reducing the

environmental impacts associated with nitrogen fertilization (Urzua,

2005).

Azam (2001) suggested that the nitrogen (N) is the key nutrient

element, limiting crop production under most situations. A major reason

for insufficient N supplies being its presence in soil in organic forms

17 which must be mineralized before being used by the plants. Leguminous

plants are equipped with the facility to acquire a major portion of N

directly from atmospheric N2 through bacterial fixation. The bacteria

(Rhizobium spp.) reside inside the special structures on plant roots i.e.,

nodules and reduce atmospheric N at the expense of C supplied by the

plant.

2.4. Importance of host-rhizobia interactions to agriculture

The symbiotic fixation of nitrogen is crucial for the provision of

nitrogen in the plant world (Allen and Allen, 1981). Nitrogen fixing

associations are of significant ecological and agricultural importance

(Denarie et al., 1996). Nutrients are lost through natural processes, such

as, leaching or volatilization and therefore, in sustainable ecosystems

these nutrients must be replaced either by fertilizers or through natural

processes (Boddey et al., 2000). The long-term effect of poor nodulation

and nitrogen fixation is a decrease in soil nitrogen reserves and a

reduction in production potential (Peoples and Herridge, 1990). Brockwell

et al. (1995) stated that legumes account for approximately 40 per cent of

total nitrogen fixation. According to Pepper and Upchurch (1991) the

ability of legumes to fix nitrogen makes them an obvious choice for use in

agricultural areas considered to be semiarid or arid. The commercial use

of rhizobial strains within Australian agriculture is largely based around

the ability to fix nitrogen optimally across a broad range of species. This

is driven by the economics of commercial inoculant production.

Australian farmers have a greater need for optimal performance of their

legumes due to the return they receive on their goods and the relative cost

of inputs (Howieson et al., 2000).

18 2.5. Effect of Rhizobium inoculants

Bambara and Patrick (2010) reported that the Rhizobium

application in Phaseolus vulgaris L. significantly improved the number of

pods per plant, number of seeds per plant, 100-seed weight and seed yield.

Molybdenum supply significantly increased the number of pods per plant,

number of seeds per plant, 100-seed weight and seed yield. Lime supply

significantly increased in number of seeds per pod and seed yield.

Delic et al. (2009) investigated that the Vigna mungo (L.) hepper is

an important annual leguminous species in tropical and sub-tropical

regions. Vigna mungo seeds inoculated with highly effective rhizobial

strains in the form of microbial nitrogen (N) fertilizer. Inoculated plants

produced significantly higher shoot dry weight (SDW) yield, total N

content as well as protein yield in respect to untreated control. According

to plant shoot yield and yield attributes strain 542 was highly effective

without significant differences in comparison to its treatment in

combination with mineral N as well as uninoculated control with full rate

of mineral N, 80 kg ha-1.

Pot culture experiment was conducted by Mishra and Mishra

(2008) to examined the effect of Rhizobia, cowpea miscellany (P) and

native strains isolated from the nodules of two plant species namely

Phaseolus aconitifolla and Alysicarpus monilifer. Number of leaves, dry

weight, leghaemoglobin content of nodules, rhizobial cell count/g soil,

AM root colonization and N and P content in soil were recorded during

the growth period of plants. The Rhizobia cowpea miscellany were found

to be better than native strains in increasing plant growth and biomass

19 yield of both the species. Also the number of rhizosphere microflora in

soil were maximized by CP strains in both the legume plants.

Neeraj et al. (2008) found that the rhizobial inoculants to enhance

legume production and simultaneously reduce the use of inorganic

fertilizers. Selected strains of Vigna radiata on the basis of polysaccharide

that rhizobial strains and its mutant nodulate promote growth differently

under stressed environments. R0132 (1106): Tn5 inoculant of

Sinorhizobium fredii is a prime candidate as a commercial inoculant. It

benefited growth of V. radiata and it was more easily cultured on solid

and liquid media than any of the other strains and exhibited superior

growth promoting ability under extreme environmental condition;

therefore it was potential to be used in India.

The field experiment was carried out by Singh et al. (2008) in

randomized block design with six treatment including control and three

replications. T-9 was used as test variety of black gram. Application of S,

Mo and Rhizobium alone or in combination significantly increased the

vegetative growth, nodule number, grain and straw yield as compared to

control. Maximum growth and yield were recorded when S was applied at

20 kg ha-1 with Rhizobium inoculation plus Mo at 2 kg ha-1. S and Mo

content and uptake on straw was significantly affected by the T3,T4 and T5

protein content was also increased due to application of S, Mo and

Rhizobium.

A field experiment was conducted by Gayatridevi et al. (2007) to

evaluate the effect of method of Rhizobium and PSB application with

different rates of chemical fertilizers and FYM on groundnut crop.

20 Application of Rhizobium, PSB, chemical fertilizers and FYM resulted in

significant dry matter production. 100-pod weight, 100-kernel weight

shelling percentage, pod yield and haulm yield than application of

Rhizobium and PSB with either chemical fertilizers or with FYM, alone.

PSB performed equally well as seed treatment, soil applications or

application along with FYM, in respect of influencing yield and yield

parameters.

Jones et al. (2007) suggested that the nitrogen-fixing rhizobial

bacteria and leguminous plants have evolved complex signal exchange

mechanisms that allow a specific bacterial species to induce its host plant

to form invasion structures through which the bacteria can enter the plant

root. Once the bacteria have been endocytosed within a host-membrane-

bound compartment by root cells, the bacteria differentiate into a new

form that can convert atmospheric nitrogen into ammonia. Bacterial

differentiation and nitrogen fixation are dependent on the microaerobic

environment and other support factors provided by the plant. In return, the

plant receives nitrogen from the bacteria, which allows it to grow in the

absence of an external nitrogen sources. The mutual recognition process

that allows the model rhizobial symbiont Sinorhizobium meliloti to invade

and differentiate inside its host plant Medicago sativa and Medicago

truncatula.

Rondon et al. (2007) examined the potential, magnitude and causes

of enhanced biological N2 fixation (BNF) by common beans (Phaseolus

vulgaris L.) through bio-char additions. Bio-char was added at 0, 30, 60

and 90 g kg-1 soil and BNF was determined using the isotope dilution

method after adding 15N- enriched ammonium sulfate to a Typic Haplus-

21 tox cropped to a potentially nodulating been variety in comparison to its

non-nodulating isoline, both inoculated with effective Rhizobium strains.

The proportion of fixed N increased from 50 to 72 per cent. Rhizobium

and bio-char applications to improve N input into agroecosystems.

Ahmed et al. (2006) observed that the effect of Rhizobium

inoculation and nitrogen on performance of Vigna radiata L.

Investigations were conducted at University of Arid Agriculture,

Rawalpindi, during spring 2004. The research material consisted of

mungbean variety (NM-98) with treatments of seed and soil inoculation

and nitrogen levels at 15, 30 and 45 Kg ha-1. Rhizobium inoculation

significantly increased the number of nodules per plant, root length, plant

height at maturity and biological yield per plant. Soil and seed inoculation

in combination with N fertilizer positively affected the growth and nodule

formation of green gram.

Pryor and Crush (2006) reported that the Rhizobium populations

were counted in random soil samples from a ryegrass, in soil around white

clover plants and also in the rhizoplane of the white clover roots. The

random soil samples contained 3 105 cells per g dry soil of Rhizobium

leguminosarum bv. trifolii effective on its host and the same number of

Mesorhizobium loti. Soil around white clover plants had 106 cells per g

dry soil of each Rhizobium. In the rhizoplane of white clover there were

106 effective R. leguminosarum bv. trifolii poer mg root dry weight and

only 103 M. loti per mg root dry weight. The increase in the ratio of

effective white clover rhizobia to M. loti in the rhizoplane demonstrates

species-specific stimulation of effective rhizobia on the root surface.

22 A greenhouse experiment was conducted by Srivastava et al.

(2006) to examined the effect of N (20, 40 and 60 mg N kg-1 soil) and Zn

(0, 2.5 and 5.0 mg Zn kg-1 soil) and their interaction in French bean

inoculated with R. leguminosarum bv. phaseoli on nodulation at 40 days

after sowing (DAS) and shoot and root dry weights, seed and straw yields

at maturity, nutrient concentration and uptake at both 40 DAS and

maturity. At 40 DAS, application of N (or) Zn and their interaction effect

significantly increased both root and shoot dry weight. Seed was

significantly affected by NZn interaction and highest seed yield was

recorded with 40 mg N+5 mg Zn Kg-1 soil treatment combination. Zinc

application increased the concentration and uptake of N in shoots at 40

DAS and also in seed and straw at maturity. N application, especially at

medium and higher rates decreased Zn concentration and uptake in shoots

at 40 DAS and also Zn concentration in seed at maturity.

Field experiments were conducted by Swaroop (2006) during 2001

and 2002 at Research farm of CARI, Port Blair using Arka Garima variety

of vegetable cowpea to observed the pod yield, nutrient uptake and

residual available soil NPK by various levels of phosphorous, Potash and

Rhizobium inoculation with and without nitrogen. Maximum average

yield of green pods was obtained with the application of 80 kg, P, 120 kg

K, 20 kg N/Na+ Rhizobium inoculation. The length of root, uptake of

phosphorus, available phosphorus and potash in soil was recorded

maximum with the application of 120 kg P, 120 kg K and 20 kg N+

Rhizobium inoculation.

A comparative study of the response of French bean to local

Rhizobium isolate AAURh and an isolate FB-9-2 obtained from IARI

23 New Delhi was conducted using two French bean varieties. In both the

varieties the plant height and the number of leaves per plant were reduced

when inoculated with either of the strain. However nodules were formed

by both the strain but the nodule number and the weight of the nodules per

plant were comparatively more in the plants inoculated with the local

isolate (Johan and Talukdar, 2005).

Thies et al. (1991) studied that the effect of Rhizobium in Glycine

max, Phaseolus lunatus, Vigna unguiculata, Phaseolus vulgaris, Arachis

hypogaea, Leucaena leucocephala, Lathyras tingeatus, Medicago sativa

and Trifotium repens. Each legume was (i) inoculated with an equal

mixture of 3 effective strains of homologous rhizobia (ii) fertilized at high

rates with urea, or (iii) left uninoculated. Inoculation increased economic

yield for 22 of the 29 (76%) legume species-site combinations and yield

also increased greater than 100 kg ha-1 in all cases, Yield was significantly

increased 6 per cent when numbers of indigenous rhizobia were greater

than 10 cells g of soil-1. The symbiotic yield of crop was, on average, only

88 per cent of the maximum yield potential, as defined by the fertilizer N

treatment. The difference between the yield of N-fertilized crops and that

of N-fixing crops indicates a potential for improving inoculation

technology, the N2 fixation capacity of rhizobial strains, and the efficiency

of symbiosis.

2.5.1. Effect of Bradyrhizobium

Elevan strains of Rhizobiuam and five strains of Bradyrhizobium

were examined for their viability as well nodulation and nitrogen fixation

ability after storage under different conditions for two years. All the slow

growing strains showed better viability than the fast growing strains in

24 any of these conditions. The survival strains maintained their nodulation

ability about 50-60 per cent after one year and 40-50 per cent after two

years of preservation as compared to control, but the nodulation ability in

sterile distilled water was very poor. Acetylene reduction activity in the

nodules was found to be 70-90 and 50-70 per cent. The strains retained

their phenotypic characters like antibiotic resistance and salt tolerance up

to their highest survivability in respective nutritional condition (Bakshi

et al., 2006).

Bogino et al. (2006) investigated that the Bradyrhizobium form

symbiotic relationships with peanut root cells and fix atmospheric

nitrogen by converting it to nitrogenous compounds. Inoculation of peanut

with rhizobia can enhance the plants ability to fix nitrogen from the air

and thereby reduce the requirement for nitrogen fertilizers.

Bradyrhizobium increased nodule number, nodulation and nitrogen

fixation.

Provorov (1998) reported that the inoculation of Phaseolus aureus

Roxb. with Bradyrhizobium sp. increased the herbage mass, seed mass,

starch content in seeds and number of nodules.

2.5.2. Effect of Rhizobium and vermicompost

An investigation was carried out by Singh and Prasad (2008)

during the winter seasons of 2004-05 and 2005-06 at Kanpur to evaluate

the effect of vermicompost, Rhizobium and diammonium phosphate

(DAP) on Chickpea. Application of Rhizobium increased in higher dry

matter, dry weight of nodules/plant, number of pods/plant, seed

weight/plant and grain and straw yields of Chickpea. Seed inoculation

25 with Rhizobium also markedly enhanced growth and yield attributes, grain

and straw yield of Chickpea.

Yadav and Malik (2005) found that the application of organic/

inorganic sources of nitrogen at 20 Kg N ha-1 + seed inoculation with

Rhizobium significantly increased plant height, dry matter accumulation,

yield attributes viz., branches per plant, seeds per pod. pods per plant,

yield per ha over control. Combined application of organic/inorganic

sources of N+ seed inoculation further induced significant increase in all

these above mentioned parameters of plant over their individual

application. Vermicompost inoculation produced significantly highest

whereas, urea inoculation produced lowest number and dry weight of

nodules per plant. Application of vermicompost at 20 Kg N ha-1 was

found superior in respect of most of the growth and yield attributes than

FYM.

2.6. Factors affecting the growth of Rhizobium and host

Keneni et al. (2010) studied that the density of Rhizobium

population in Vicia faba L. The native rhizobial strains tolerated a higher

salt concentration (5% NaCl) than the exotic rhzobial strains. Both native

and exotic strains failed to grow at pH 4 and 4.5 levels in the laboratory

conditions. In the soil adjusted to pH 4-7, all the native rhizobial strains

persisted while those of the exotic strain failed to survive at pHs below

5.5. The native strains were more versatile than the exotic ones in utilizing

different carbohydrates as a sole carbon source and were found to be more

resistant to many antibiotics (Streptomycine, chloramphenicol,

rimfampenicillin, oxytetracycline, penicillin and tetracycline) than exotic

strains which are found resistant to chloramphenicol only percentage of

26 nitrogen fixation is also higher for native rhizobial strains these isolates

being found to be superior to the exotic strains in stimulating growth, dry

matter yield, nodulation and nodule wet weight of faba bean in pouch

culture.

972 rhizobia isolated from nodules of different varieties of Vigna

radiata, 67 isolates were found to be potential ones on the basis of plant

growth promotion/increased plant biomass and only six potential isolates,

showed higher plant biomass, maximum nodule numbers and higher

nitrogenase activity. Maximum range of fresh weights of nodule per plant

was shown again by isolate RO132 and noticed to be 261.37 mg

nodule/plant. Nitrogenase activity of plant nodule inoculated with

different rhizobial isolates varied from 2.56-3.21 g/mg/nodule. Isolate

RO132 was found to be the best candidate as it utilized a wide range of

nitrogen and carbon sources and was able to grow at slightly higher

temperature i.e., 32°C and slightly acidic (pH 6.0) and alkaline (pH 8.0)

conditions, ability to grow in presence 50 mM concentration of NaCl

(Neeraj et al., 2009).

Rajasekar et al. (2002) observed that the rhizobial isolates of

Pterocarpus santalinus L. were fast growers and acid producers. The

growth characteristics were determined on different media and they

produced effective nodules on P. santalinus. Isolates showed good growth

of on different carbon sources including mono, di and polysaccharides,

but moderate growth on sucrose, maltose and least on citrate. The isolates

grew well with glutamic acid, aspergine, yeast extract and potassium

nitrate as a source of nitrogen, but glycine was least preferred. Isolates

grew well at a pH range of 5.0-8.0 but not below 4.0 and above 9.0 and

27 they showed good growth at 0.1, 0.5 and 1.0 per cent of NaCl in the

medium. All the rhizobial isolates of red sanders nodulated Vigna radiata,

Dalbergia sissoo and produced ineffective nodules on Leucaena

leucocephala. The preliminary identification of the rhizobia of red sanders

showed it to be similar to Rhizobium phaseoli.

2.7. Molecular studies of Rhizobium sp.

Rhizobial isolates were identified and characterized on the basis of

colony morphology and biochemical traits viz gram staining, catalase and

oxidase tests and carbon and nitrogen source utilization pattern. The

survival efficiency of isolates was measured in culture. The genetic

diversity among the isolates assessed by RAPD-DNA finger printing and

PCR was done for the presence of 16S-rRNA gene. On the basis of carbon

/ nitrogen source utilization patterns, Rhizobium isolates placed in five

different groups and were designated as Rkh1, Rkh2, Rkh3, Rkh4 and

Rak5 but RAPD tests categorized the isolates into two clusters. The

RAPD results were further analyzed by MVSP software; similarity matrix

was measured and converted into dendrogram using UPGMA clustering

method (Naz et al., 2009).

EL-Fiki (2006) reported that Rhizobia are soil bacteria which

specifically nodulate legume roots thus forming a nitrogen fixing root

nodule symbiosis, which has a great importance to agriculture in nitrogen

deficient environments. RAPD fingerprinting was used for strain

identification and the assessment of genetic diversity within a field

population of Rhizobium (Bradyrhizobium archus, Bradyrhizobium

japonicum and Rhizobium leguminosarum bv. Trifolii).Total genomic

DNAs from different field isolates were amplified using two different

28 arbitrary primers. Different band patterns were obtained for all strains.

Cluster analysis showed the ralationship of R. leguminosarum bv. Trifolii

with B. archus (69%) and B. japonicum (63%)

Kucuk et al. (2006) studied that the physiological and biochemical

characteristics of Rhizobial isolates from Phaseolus vulgaris L. Most

isolates produced abundant extracellular polysaccharides, tolerated high

salt concentration, grew at a temperature of 400 C, and synthesized

melanin. They were able to grow at pH ranging from 3.5 to 9.0. The

majority of the isolates showed an intrinsic resistance to the antibiotics

chloramphenicol, erythromycin, kanamycin, and streptomycin. Plasmid

DNA profiles of the isolates were identified and it was determined that 2

isolates contained plasmid DNA.

Based on phenothypic characteristics and intrinsic antibiotic

resistance pattern, 110 out of 350 cultures of rhizobia, isolated from high

nodulating (HN) and low nodulating (LN) selection of two Cicer

arietinum L. cultivars viz., ICC4948 and ICC5003, were selected and

examined for their protein pattern by SDS-PAGE. To assess the

heterogeneity of rhizobia, two criteria were used. One based on the

presence of proteins of different molecular weight and Rm value, while

second was variation in the presence of major protein bands in an isolated.

In majority of the isolated, both form HN and LN variants. Proteins of

more than 45kD were presents and slight variation in the presence of

proteins of lower mol wt in different isolated were observed. Based on the

presence of major and minor bands, large heterogeneity in rhizobial

isolated was observed. In isolates from HN selections, all the 13 major

bands were present; while in case of isolates form LN selection, 2 major

bands were absent in all the isolates. All the isolates from HN and LN

29 selections were able to from nodules of their respective host (Chaudhary

et al., 2002).

2.8. Stress conditions

Hamida and Shaddad (2010) suggested that several environmental

factors adversely affect plant growth and development and final yield

performance of a crop. Drought, salinity, nutrient in balances (including

mineral toxicities and deficiencies) and extremes of temperature are

among the major environmental constraints to crop productivity

worldwide. Development of crop plants with stress tolerance, however,

requires, among others, knowledge of the physiological mechanisms and

genetic controls of the contributing traits at different plant developmental

stages. In the past 2 decades, biotechnology research has provided

considerable insights into the mechanism of biotic stress tolerance in

plants at the molecular level. Furthermore different abiotic stress factors

may provoke osmotic stress, oxidative stress and protein denaturation in

plants, which lead to similar cellular adaptive responses such as

accumulation of compatible solutes, induction of stress protein and

acceleration of reactive oxygen species scavenging systems. To improved

plant tolerance to salinity injury through either chemical treatments or

biofertiliers treatments (Asymbiotic nitrogen-fixing bacteria, symbiotic

nitrogen-fixing bacteria and Mycorrhiza) or enhanced a process used

naturally by plants to minimize the movement of Na+ to the shoot, using

genetic modification to amplify the process.

Ali et al. (2009) evaluated that the effect of salt, pH and

temperature on the growth of rhizobia isolated from Leucaena

leucocephala, Tephrosia purpurea and Crotalaria medicaginea grown in

arid and semiarid regions of Rajasthan with a view to screen out stress

30 tolerant isolates. A total of 27 isolates have been used for screening their

stress tolerating ability with contrast to environmental abiotic soil

conditions commonly prevailing in arid and semi-arid regions of

Rajasthan. All the isolates were phenotypically and biochemically

characterized followed by their plant assay test in growth pouches and pot

experiment under controlled environmental conditions. Growth of pure

rhizobial isolates on Yeast Extract Mannitol (YEM) medium having

variable range of pH (4-10) and different concentrations of NaCl

(0.01-4.5%) were recorded at 540 nm using UV-Vis spectrophotometer

after incubation at 28 2°C for 2 days. The stress tolerant traits of these

rhizobia are of potential value from the point of view of biofertilization of

legume seedlings during of forestation of degraded areas in arid and semi-

arid tropics of Rajasthan (Ali et al., 2009).

Hung et al. (2005) reported that the root-nodulating bacteria were

isolated and characterized from 7 native shrubby legumes and measured

growth rates in various media, colony morphology and tolerances to

extremes of temperature, salt and pH. Among the 83 isolates that were

screened, the majority were fast-growing rhizobia, 28 strains tolerated

high concentration of salt (45% NaCl) and grew well between

temperatures of 37 and 45°C. The majority of the strains also tolerated

extreme pH in their medium from 3.5 to 12. All strains formed nitrogen

fixing nodules and the highest activity was detected in the legume

Hedysarum crinita L. PCR restriction fragment length polymorphism

(PCR-RFLP) and sequencing of the small subunit ribosomal RNAs

revealed that the majority of the isolates belonged to the genera

Rhizobium, Bradyrhizobium and Agrobacteirum.

31 Serraj and Gyamfi (2004) suggested that the inclusion of a legume

in a cropping system does not always ensure the attainment of optimal

levels of symbiotic nitrogen fixation (SNF) in the field. Several

environmental factors including drought, temperature and soil nutrient

status are known to dramatically affected process at molecular/functional

level and thus play a part in determining the actual amount of nitrogen

fixed by a given legume in the field.

Natural occurrence of Sinorhizobium meliloti nodulate Medicogo

sativa and Rhizobium leguminosarum bv. trifolii nodulate and Trifolium

alexandrinum L. were examined in soils of 125 locations. S. meliloti

occurred in almost all the soils, while R. leguminosarum bv. trifolii

occurred in a very few soils. The reasons of the difference in their

occurrence were attributed to the variations in the occurrence or

cultivation of their respective host legumes and also the variation in the

ability of these nodule bacteria to survive under extremes of the

environment. Inoculation of these two legumes with their respective

nodule bacteria improved the forage yield; and the response was more on

Trifolium alexandrinum than on Medicago sativa (Gaur et al., 2002).

Wild legumes (herb or tree) are widely distributed in arid regions

and actively contribute to soil fertility in these environments. The

N2-fixing activity and tolerance to drastic conditions may be higher in

wild legumes than in crop legumes. The wild legumes in arid zones harbor

diverse and promiscuous rhizobia in their root-nodules. Specificity existed

only in few rhizobia from wild legumes, however, the majority of then are

with wide host range. Based on phenotypic characteristics and molecular

techniques, the root-nodule bacteria that was isolated from wild legumes

32 and been classified in to 4 genera (Rhizobium, Bradyrhizobium,

Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid

zones, exhibit higher tolerance to the prevailing adverse conditions, e.g.

salt stress, temperatures and desiccation. These rhizobia may be used to

inoculate wild, as well as, crop legumes, cultivated in reclaimed desert

lands. Recent reports indicated that the wild-legume rhizobia formed

successful symbioses with some grain legumes. Rhizobia have specific

traits that can be transferred to other rhizobia through genetic engineering

tools or used to produce industrially important compounds. Therefore,

these bacteria are very important from both economic and environmental

points of view (Zahran, 2001).

2.9. Salt stress on the leguminous plants

Predeepa and Ravindran (2010) observed that the Rhizobium-

legume symbiosis is one of the most well-established symbiotic nitrogen

fixing system for agronomic studies. Salt-tolerant rhizobial strains or

salt-tolerant cultivar does not necessarily promise a salt-tolerant symbiotic

system, as the symbiotic system is more sensitive to salt stress than the

bacterium and/or the plant. Salt tolerance of the symbiotic system

decreased by 1 ds/m, and also that there is a gradual shift in the spatial

distribution of the nodules from the primary roots to the secondary roots

under increased salt levels and is time-dependent.

Arachis hypogaea L. is considered to be one of the most important

crop have high nutritive value and a source of edible oil and tested the

effects of different salt levels on mineral nutrient partitioning and

morphological characteristics of plant. Three concentrations of salt

solution including 50, 100 and 200 mM NaCl and the control were used in

irrigation. The leaf relative water content (LRWC) rovoked by the salinity

33 in nutrient solution decreased from 85.08 to 79.70 per cent. Salt stress

reduced significantly the plant height, the number of leaves, dry weight of

roots, the dry weight of stems, plant and dry weight of leaves and also K+,

Mg2+, Ca2+, P, N, K+/Na+ and Ca+/Na+ uptake of peanut plant organs were

significantly reduced with increasing salinity (Desire et al., 2010)

Dardanelli et al. (2009) studied that the effects of saline and

osmotic stress on four peanut rhizobia, plant growth and symbiotic

N2-fixation in Arachis hypogaea were studied. Abiotic stress was applied

by adding 100 mM NaCl. At the rhizobial level, Bradyrhizobium ATCC

10317 and TAL 1000 showed stronger tolerance to stress than TAL 1371

and SEMIA 6144. The effect of salinity on the bacterium – plant

association was studied by using the variety Blanco manfredi M68. In the

absence of stresses, all the strains induced a significantly higher number

of nodules on the roots, although TAL 1371 and SEMIA 6144 were more

effective. Both stresses affected the interaction process, while TAL1371

was the best partner.

Taffouo et al. (2009) found that the effects of NaCl concentrations

on physiological behaviour of organs of five leguminous plants. Plants

were submitted to 5 levels of salt stress at the roots (0, 50, 100, 150 and

200 mM of NaCl). NaCl had an understanding effect on growth of stems

and seed germination of species. The reduction of stems growth rate were

not significant in P. adenanthus whereas in M. poggei and V. unguiculata

this inhibition was observed just when nutritive solutions were enriched

with 200 mM. The lipid contents were reduced in all the species under salt

stress, whereas proteins and proline contents in the leaves were

substantially increased in tolerant species of M. poggei, P. adenanthus and

34 V. unguiculata. Proteins and leaf proline contents were negatively affected

by salt concentrations to G. max and P. vulgaris. Seed germination,

proteins, proline contents could be used as physiological criteria of early

selection for salt tolerant leguminous plants.

Effect of low temperature, salinity, nutrient level and photoperiod

has been studied on 3 varieties of Pisum sativam var., P-48, meteor and

AM-inoculated with Rhizobium strain PS-1. The plants were grown at

5, 10, 15, 20 and 25°C. The number of viable cells was counted at each

temperature by most probable number technique. The number of viable

cells were constant at 5 and 10°C but increased between 15-30°C. Nodule

formation was not observed at 5, 10 and 25°C at 15°C the nodules were

less in number, whereas at 20°C best nodulation was observed. The pea

plants were also subjected to different salinity levels i.e., 0, 1, 2, 3, 4, 5,

10 and 15 dsm-1. High number of nodules was formed at 0 and 1 dsm-1

whereas at 2 and 3 dsm-1 the nodule number was reduced and yet at higher

salinity levels no nodules were formed. Pea plants nodulated best at 12

hours photoperiod and 1/6th concentration of Hoagland’s nutrient

solutions. The mature nodules developed at different temperatures and

salinity levels (Naeem et al., 2008).

Gaballah and Gomaa (2005) investigated that the impact of

Rhizobium inoculation and/or sodium benzoate application on

performance of two constrasting fababean varieties i.e., Giza Blanka (salt

tolerant) and Giza 634 (salt sensitive) grown in sandy soil under two

levels of salinity (3000 and 6000 ppm). High salinity (6000 ppm) greatly

reduced nodules formation in both varieties. Rhizobium inoculation

reduced the inhibitory effect of salinity and plants were able to survive

35 better. Rhizobium inoculation increased plant leaf area, reduced MDA

content in plant leaves and did not show a significant effect on SOD

activity in plant roots, but the interaction between Rhizobium inoculation

and sodium benzoate resulted in a significant increase in SOD enzyme

activity plant roots.

Bouhmouch et al. (2005) studied that the effect of salt stress on the

Rhizobium-common bean symbiosis. The comparison of the behaviour of

five cultivars of Phaseolus vulgaris differing in seed colour, growing on

nitrates and different concentrations of NaCl, showed genotypic variation

with respect to salt tolerance. R. tropici strain RP163 and R. giardinii

strain RP161. Their relative growth was moderately decreased at 250 mM

NaCl, but they were able to grow at a low rate in the presence of 342 mM

NaCl. Their viability at the minimal inhibitory concentration was slightly

affected. In the absence of salinity, the strains induced a significantly

higher number of nodules on the roots of the cultivar, SMV 29-21

compared to those of Coco Blanc. In the presence of salinity, Coco Blanc

was more severely affected when associated with RP163 than with

RP161. Salinity affected the nodulation development more than it affected

the infection steps. Neither of the 2 strains was able to nodulate SMV

29-21 under saline conditions, in spite of the fact that this was considered

the most salt-tolerant variety.

Common bean plants inoculated with salt-toleant Rhizobium tropici

wild-type strain CIAT899 formed a more active symbiosis than did its

decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12) or

almost ineffective (HB8, HB13) modules (Fixd) under non-saline

conditions. The DST mutant formed nodules that accumulated more

36 proline than did the wild-type nodules, while soluble sugars were

accumulated mainly in ineffective nodules. The salt stress affected the

plant growth, nitrogen fixation and the activities of the antioxidant

defense enzymes of nodules. Mutant nodules showed lower antioxidant

enzymes activities than wild-type nodules (Tejera et al., 2004).

Anthraper and DuBois (2003) studied that the effect of varying

NaCl concentrations on growth, N2 fixation and percentage of total tissue

nitrogen in different organs in L. leucocephala. Seeds were germinated

and grown for either 0, 7, 14, 21 or 28 week with either deionized water

(control), 0.000625 mol/L, 0.0125mol/L, 0.025 mol/L, 0.05 mol/L or 0.1

mol/L NaCl in addition to the fertilizer every 2 week. Growth was

measured as plant height, nodule number and mass, dry tissue mass. N2

fixation was measured by the acetylene reduction assay. Percentage of

tissue nitrogen was determined using Kjeldahl analysis. In younger plants

(7 weeks treatment), major fluctuations in NaCl tolerance were observed

in the different plant organs. As plant matured (14 and 21 week treatment)

NaCl concentrations of 0.025 mol/L and higher caused the greatest

reduction in growth and tissue nitrogen. The NaCl concentrations of 0.025

mol/L and greater caused a major decrease growth, N2 fixation and

percentage of tissue nitrogen in L.leucocephala plants.

Dash and Panda (2001) presented that the NaCl salt stress induced

changes in growth and enzyme activities in black gram seeds during

germination. A decrease in germination percentage, root length, shoot

length and fresh mass was noticed with an increase in NaCl concentration.

With the increase in NaCl concentration and duration of stress proline

content increased and catalase, peroxidase and polyphenol oxidase

activities decreased.

37 Strain Ch-191 of M. Ciceri was grown with different NaCl

concentrations. Protein and lipopolysaccharide patterns were determined

by electrophoresis. The strain Ch-191 tolerated up to 200mm0l1-1 NaCl,

although highest salt dosages limited its growth and induced changes in

the slowest band and appearance of an intermediate mobility band. The

accumulation of proline in response to salt stress surpassed that of

glutamate. The protein profile showed major alterations at salinity levels

which inhibited growth. The alterations in the LPS profile and

accumulation of compatible solutes were evident from the lowest levels,

suggesting that these changes may constitute adaptive responses to salt,

allowing normal growth. The selection and characterization of salt-

tolerant strains, which also show efficient symbiotic (Soussi et al., 2001).

The rhizobia from the alkaline soil showed significantly higher salt

tolerance than those isolated form neutral soil. Rhizobium sp. NBRI0102

Sesbania and Rhizobium sp. NBRI2502 Sesbania tolerated yeast extract

mannitol broth (YEB) containing 10 and 28 per cent salt (NaCl, wt/vol)

for up to 18h of incubation at 30°C. Growth of Rhizobium sp. NBRI0102

sesbania and Rhizobium sp. NBRI2505 Sesbania at pH 7.11, and 12 was

identical, except for a lag period of about 10 h in the growth of Rhizobium

sp. NBRI0102 Sesbania at pH 11 and 12, as compared with pH 7.

Rhizobium sp. NBRI0102 sesbania and Rhizobium so. NBRI2505

sesbania survived at 50°C and 65°C, in YEB at pH 7 (Kulkarni et al.,

2000).

A commercial cultivar Vicia faba L.var.minor was inoculated with

salt-tolerant Rhizobium leguminosarum biovar, Viciae strain GRA 19 in

solution culture with different salt concentrations (0,50,75 and 100 m

38 moles1-1 NaCl) added immediately at the time of inoculation. Rhizobium

leguminosarum strain GRA 19 formed an infective and effective

symbiosis with faba bean under saline and nonsaline conditions. Salinity

significantly decreased shoot and root dry weight, nodule weight and

mean nodule weight. Roots were more sensitive to salinity than was plant

growth. Analyses of ammonium assimilating enzymes in the nodule

showed that glutamine synthase, and that it limits ammonium assimilation

under saline stress (Cordovilla et al., 1999).

Singly and doubly – labelled antibiotic resistant mutants of 250

mg/ml of streptomycin and spectinomycin were isolated from chickpea

Rhizobium 2-ICAR-UNK-Ch-191 (Ch191). All mutants exhibited similar

characteristics to the wild-parent type in their response to nodulation and

tolerance of salinity and temperature. Salinity (1.0ds/m) decreased root

and shoot dry weight, total nodule number and nodule weight. Inoculated

plants accumulated more N compared to N fertilized plants. The

Rhizobium is more salt-tolerant than the cultivar and Rhizobium strain

Ch191 is effectively fixing N in saline and non-saline conditions

(Elsheikh, 1992).

Zahran (1991) suggested that the Rhizobium-legume symbiosis in

arid ecosystem is particularly important for locations where the area of

saline soils is increasing and becoming a threat to plant productivity.

Legumes, which are usually present in arid ecosystems, may be adapted to

fix more N2 under saline conditions than legumes grown in other habitats.

Legumes are known to be either sensitive or moderately resistant to

salinity. The salt sensitivity can be attributed to toxic ion accumulations in

different plant tissues, which disturb some enzyme activities. Among the

39 basic selection criteria for salt-tolerant legumes and rhizobia are genetic

variability within species with respect to salt-tolerant legumes and

rhizobia are genetic variability within species with respect to salt

tolerance, correlation between accumulations of organic solutes and salt

tolerance and good relationships between ion distribution and

compartmentation and structural adaptations in the legumes. Salt stress

reduces the nodulation of legumes by inhibiting the very early symbiotic

events. Levels of salinity that inhibit the symbiosis between legumes and

rhizobia are different from those that inhibit the growth of the individual

symbionts. The poor symbiotic performance of some legumes under saline

environments include rhizobial colonization and invasion of the

rhizosphere, root-hair infection and the formation of effective salt-tolerant

nodules.

2.10. Effects of salt concentrations on growth of Rhizobium

Rhizobacteria, being soil microorganisms are confronted with

fluctuating osmotic pressures of the rhizosphere. Rhizobium is an

important microbe because of its impact and interaction with the host

plant. Changes in salt concentration affect the growth and functioning of

Rhizobium. The effects of varying salt concentrations from 0.2 M to

0.00625 M are reported. Rhizobium is capable of osmoadaptation; it can

tolerate high salt concentrations, the growth tends to be inversely

proportional to salt concentration. In other worlds, growth decreases with

increasing salt concentration Rhizobial growth is more abundant at lower

salt concentrations ranging from 0.00625 M to 0.0125 M (Rafiq, 2007).

Mensah et al. (2006) found that the effects of salt concentrations

verifying from 0.005 M to 0.0200 M NaCl and a pH ranging from pH 3-9

40 on growth of Rhizobium as well as the cowpea associated with the

Rhizobium. The Rhizobium species from cowpea were capable of

osmoadaptation and were found to tolerate a relatively high salt

concentration of up to 0.200 M NaCl. The population count was inversely

proportional to the salt concentration with high growth

(30-31.6 104 cfu/ml) at lower concentration of 0.005-0.010 M and low

growth (7.1-19.2 104 cfu/mL) at higher salt concentrations of 0.050-

0.300 M. The optimal pH range for the growth of the Rhizobium sp. was

pH 6-7 while lower or higher pH values recorded lower population counts.

Low yield observed for the cowpea at higher salinity and low pH. To

improve the yield of cowpea in a saline soil with low pH, it is essential to

reduce the soil pH to a range of 6-8 and desalinate to enhance the growth

of the cowpea as well as the Rhizobium sp. associated with it.

The halotolerant strain Rhizobium meliloti EFBI modifies the

production of extracellular polysaciharides response to salt EFBI colonies

grown in the presence of 0.3M NaCl show a decrease in mucoidy and in

salt-supplemented liquid medium this organism produces 40 per cent less

exopolysaccharides. Transposon-induced mutant that, when grown in the

absence of salt, had a colony morphology similar to the colony

morphology of the wild type grown in the presence of salt. Calcoflour

fluorescence, proton nuclear magnetic resonance spectroscopy and genetic

analysis of the mutant indicated that galactoglucan, which is not produced

under normal conditions by other R. meliloti strains, is produced by strain

EFBI and that production of this compound decreases when the organism

is grown in the presence of salt (Lloret et al., 1998).

41 A halotolerant strain of Rhizobium meliloti was isolated from

nodule of a Melilotus plant growin in a salt marsh. The strain, EFB1, is

able to grow at NaCl concentrations of up to 500mM and no effect on

growth is produced by 300 mM NaCl. EFB1 showed alterations on its

lipopoly-saccharide (LPS) structure and modifications on LPS form part

of the adaptive mechanism of this bacterium for saline environment

(Lloret et al., 1995).

2.11. Water stress on the leguminous plants

Sinclair and Ludlow (2010) studied that the water balance of

Glycine max, Vigna unguiculata, Vigna mungo and Caganus cajan grown

in pots. The response of the plants was analysed for three distinct stages of

dehydration. In stage I, the rate of transpiration remained constant and

equal to that of well watered plants even though soil water status fell by

more than 50 per cent. Stage II began when the rate of soil water supply to

the plant was less than potential transpiration and stomates closed

resulting in the maintainance of plant water balance. Stage III occurred

once stomates had reached minimum conductance and water loss was then

a function of the epidermal conductance and the environment around the

leaf.

Pimratch et al. (2008) determined the effect of drought on biomass

production and N2 fixation by evaluating the relative values of these two

traits under well watered and water-stress conditions. Twelve peanut

genotypes were tested under three water regimes [field capacity (FC), 2/3

available soil water (AW) and 1/3 AW]. Genotypic variations in biomass

production and N2 fixation were found at all water regimes. Biomass

production and N2 fixation decreased with increasing levels of drought

42 stress. High N2 fixation under drought stress also was due largely to high

N2 fixation under well-watered conditions with significant but lower

contributions from the ability to maintain high nitrogen fixation under

drought stress. N2 fixation at FC was not correlated with the reduction in

N2 fixation at 2/3 AW and 1/3 AW. Positive relationships between N2

fixed and biomass production of the tested peanut genotypes were found

at both levels of drought stress and the relationship was stronger the more

severe the drought stress.

Pimratch et al. (2008) determined the effects of drought stress on

nitrogenase activity, nodule numbr and nodule dry weight of 11 peanut

(Arachis hypogaea L.) genotypes with different degrees of drought

resistance. The relative values of these nitrogen fixation traits were

evaluated under well watered and water-stressed conditions. Severe

drought stress reduced nitrogenase activity, nodule number and nodule dry

weight. Nodule dry weight was closely related with nitrogenase activity

under drought conditions. High nitrogenase activity under mild drought

conditions was related to nitrogenase activity under well watered

conditions and to a low rate of reduction in nitrogenase activity in

response to stress. The contribution of the potential was lower under more

severe drought conditions.

Legume-Rhizobium nitrogen fixation is dramatically affected under

drought and other environmental constraints. Nodulated pea plants were

grown in a split-root system, which allowed for half of the root system to

be irrigated at field capacity, while other half was water deprived, thus

provoking changes in the nodule water potential. Nitrogen fixation only

decline in the water-deprived, half-root system and this result was

43 correlated with modifications in the activities of key nodules enzymes

such as sucrose synthase and isocitrate dehydrogenase and in nodular

malate content. The use of partially droughted split-root system provides

evidence that nitrogen fixation activity under drought stress is mainly

controlled at the local level than by a systemic nitrogen signal (Marino

et al., 2007).

An experiment was conducted by Prathap et al. (2006) found that

the effect of PEG induced water stress at different cultivars. Water stress

was imposed by treating the seeds of those cultivars at different water

potentials. Across the cultivars, there was significant reduction in

germination, seedling growth and seedling vigour index with the

decreasing water potential from -0.3 to -0.1 mPa. Among the cultivars,

ICGV -86031 showed higher resistance to water stress. It has shown

germination and seedling growth even at higher water stress situation

(-1.0 mpa) where all other cultivars completely failed. Next to ICGV-

86031, TMV-2 TLGS-41 were found better in terms of drought resistance.

Field study was carried out by Ramesh et al. (2006) at M.S.

Swaminathan Research foundation, Ariyamuthupatti Pudukottai district of

Tamilnadu to evaluate the effect of different in situ soil moisture

conservation and nutrient management practices on root growth and

nodulation characteristics of cowpea under rainfed condition. The root

growth (length, volume and dry weight) nodulation characteristics

(numbers and dry weight) and grain yield of cowpea were increased

significantly due to in situ soil moisture conservation.

Two indigenous bradyrhizobia strains displaying different natural

behaviours towards water regime (strain ORS 3257, nodulating more

44 frequently in favourable-water conditions and strain ORS 3260, in

limited-water conditions were studied for their competitivity for

nodulation of cowpea under favourable and limited water conditions in

non-sterile soil. The nodule occupancy was studied by PCR-RELP

analysis. Both strains showed good competition with other indigenous

rhizobia populations under favourable – and limited-water conditions.

Competition between the inoculated strains in the mixture varied between

water regimes. In non-limited-water conditions, strain ORS 3257 was the

best competitor, whereas in limited-water conditions, strain ORS 3260

was the best competitor (Wade et al., 2006).

Groundnut (Arachis hypogea L.), is an important legume cash crop

for the tropical farmers and its seeds contain high amount of edible oil

(43-55%) and protein (25-28%). Drought resistance characteristics of

groundnut with a view toward developing appropriate genetic

enhancement strategies for water-limited environments. Increasing soil

moisture storage by soil profile management and nutrient management for

quick recovery from drought are some of the areas that need to be

explored further (Reddy et al., 2003).

Wahab et al. (2002) reported that the Lablab purpureus is a

drought-tolerant legume widely grown as a high-protein grain food and

forage legume within a wide range of neotropical regions with extensive

production in India. L. purpureus inoculated with Rhizobium sp. strain I4

tolerant to mild levels of salinity, but the nodule number was reduced to

about 35 per cent of the control plants when subjected to a high salt level

(120 mM NaCl). Lablab plants were similarly affected by different rates

of water deficits. The legume was tolerant to moderate levels of drought.

45 The nodule number and weight at 50 percent of field capacity was about

70 per cent of the control. These values were reduced to 45-55 per cent at

a fc of 16.5 per cent. Absolute nitrogenase activity, leghaemoglobin

content of nodules and protein content of bacteroids and cytosol were

moderately affected by mild levels of NaCl and drought but significantly

reduced to about 25-35 per cent of the control treatments.

John et al. (2001) observed that the significant differences in total

plant dry weight by the different treatments in pure stands, 21 days after

emergence with higher values under mixed cropping system in common

beans. At 42 days after emergence, plant dry weights in uninoculated

common bean pure stands with N application were significantly higher

than under other treatments. The inoculated common bean and N

application treatment recorded the largest seed dry weights and

subsequently yields per hectare. Inoculation of common bean with the

commercially available Rhizobium strain 446 on the other hand was

effective and improve yields.

Daniel et al. (2001) reported that the response of Vigna mungo L.

and Vigna radiata (L.) wilczek to cowpea-Rhizobium inoculation and

supplementary UV-B radiation on growth, nodulation and nitrogen

fixation. Supplmeentary UV-B radiation caused a reduction in plant height

(1-55%), dry matter yield (29-56%), total nitrogen (23-80%) and soluble

sugars (28-51%) in both the plant species. The enhanced UV-B radiation

reduced nodule number (10-74%), nodule leghaemoglobin (25-82%) and

nodule nitrogenase activity (27-72%) in V. mungo and V. radiata. There

was a reduction in total chlorophyll and carotenoids in the UV-B treated

plants, in contrast to an increase in anthocyanin and flavonoids in the

46 leaves of V. mungo and V. radiata. Rhizobium inoculation enhanced the

growth (6-133%), biomass yield (30-109%), total nitrogen (32-115%) and

total soluble sugars (71-342%) in both the plant species. V. radiata

responded more to Rhizboium inoculated conditions, V. radiata was more

sensitive to UV-B radiation stress, which is evidenced by reduced root

growth (up to 28%), total nitrogen content (80%), nodule leghaemoglobin

content (82%) and nodule nitrogenase activity (47%).

Symbiotic nitrogen fixation is highly sensitive to drought and

decreased N accumulation and yield of legume crops. The effects of

drought stress on N2 fixation usually have been perceived as a

consequence of straight forward physiological responses acting on

nitrogenase activity and involving exclusively one of three mechanisms:

carbon shortage, oxygen limitation, or feedback regulation by nitrogen

accumulation N feedback especially important in explaining the response

mechanism in nodules. Nitrogenous signal, associated with N

accumulation in the shoot and nodule, exists in legume plants so that N2

fixation is inhibited early in soil drying. The existence of genetic variation

in N2 fixation response to water deficits among legume cultivars opens the

possibility for enhancing N2 fixation tolerance to drought through

selection and breeding (Serraj et al., 1999).