G.S Deora and K.

Singhal

Nutrient enrichment of soil by nitrogen fixing symbiotic bacteria present in legumes has

been known for centuries. Scientific demonstration of this symbiosis was stared in 19th

century and it established the fact that bacteria present in nodules on legume roots are

responsible for fixing atmospheric nitrogen (Zsbrau, 1999). Rhizobium spp. are well known

group of bacteria that act as the primary symbiotic fixer of nitrogen. These bacteria infect

the root nodules of leguminous plants, leading to the formation of lumps and nodules where

the nitrogen fixation takes place

The bacterium's enzyme system supplies a constant source reduced nitrogen to the host

plant and the plant furnishes nutrients and energy for the activities of the bacterium. This

symbiosis reduces the requirement for

nitrogenous fertilizer during the growth of leguminous crops (Dilworth and Parker, 1969;

Olevera et al., 2007;

Booling et al., 2007; Hunter et al., 2007). In the present study, strains of Rhizobium were

isolated from the root nodules. Further characterization was done by performing various

biochemical tests and also determine if the rhizobial cells be efficiently immobilized on

matrices to produce industrially important biofertilizer.

Shefali Poonia

Nitrogen is one of the essential elements required for the synthesis of amino acids which, in

turn, are used by the plant to form protein. Plants primarily take nitrogen in the ionic form as
+ -
either ammonium (NH4 ) or nitrate (NO3 ). Leguminous plants are also able to utilize

nitrogen derived from the symbiotic relationship they form with root nodule bacteria.[1].

Legumes play an important role in sustainable management of dry arid regions. Rhizobia are

the gram negative bacteria which have been widely used in agricultural systems for
enhancing the ability of legumes to fix atmospheric nitrogen [2]. These inhabit the root

nodules of most legumes which can provide enough nitrogen for their physiological needs

[1]. Each major legume crop is nodulated by different species of Rhizobium. Intensive

farming practices that accomplish high yields need chemical fertilizers, which are not only

costly but also create environmental problems [3]. The extensive use of chemical fertilizers in

agriculture is currently under debate due to environmental concern and fear for consumers’

health. Consequently, there has recently been a growing level of interest in environment

friendly sustainable agricultural practices and organic farming systems which include the use

of biofertilizers as a substitute of chemical fertilizers[4]. Thus, in the development and

implementation of sustainable agriculture techniques, biofertilization is of major importance

in decreasing environmental pollution and the conservation of nature [5].

Zeenat Wadhwa

With increased concern about the production of adequate amount of food to feed the

constantly increasing human population which is going to touch a mark of 9 billion by 2050

has forced us to reinforce the importance of sustainable increase in crop productivity. One of

the methods for sustainable agricultural includes the use of beneficial microorganisms for

plants as they are able to promote plant growth by growing endophytically on plants, in

symbiotic association with plants or as free-living cells in soil. The requirement for good

agricultural practices is revitalizing the interest in biological nitrogen fixation and Rhizobia-

legumes symbiosis, particularly those involving economically important legume crops in

terms of food and forage.

(Datta et al., 2015)

Legumes are agronomically and ecologically important symbionts that lead to the development of

new plant organ (legume nodule) in response to nitrogen fixing bacteria. In developing countries
like India and other South Asian countries, chickpea is an important source of protein for millions

of people. Other than having high protein content (20-22%), chickpea is rich in fiber, minerals

(phosphorus, calcium, magnesium, iron and zinc) β-carotene and large amount of unsaturated

fatty acids (Gaur, 2010). Besides playing an important role in human diet it also improves soil

fertility by fixing the atmospheric nitrogen (Siddiqi and Mahmood, 2001; Kantar et al., 2007).

Maximum nitrogen requirement (4-85%) of chickpea as a legume is obtained through symbiotic

Nitrogen fixation in association with compatible Rhizobium strain (Chemining and Vessey,

2006). The genus Mesorhizobium includes species with high geographical dispersion and able to

nodulate a wide variety of legumes, including important crop species, like chickpea. It has been

estimated that 1g of soil may contain a community of 10 9 microorganisms with Rhizobia

representing around 0.1% of soil microbes or 106g-1 soil.

Rhizobia are one of the most efficient bacterial symbionts of legumes that fix atmospheric

nitrogen by the process of biological nitrogen fixation (BNF).

Rhizobia are able to metabolize atmospheric nitrogen and convert it into plant usable form in

specialized structures called nodules where aerobic condition are maintained by leghaemoglobin.

In return, Rhizobia utilize the carbon substrates derived from the plant photosynthesis. In

agriculture, perhaps 80% of the biologically fixed nitrogen comes from symbiosis involving

leguminous plants and bacteria of family Rhizobiaceae.

The biological nitrogen fixation because of Rhizobia-legume symbiosis benefits not only the host

crop but also the subsequent crops in that field. Besides this, it may also act as a non-symbiotic

PGPB as in the case of certain non-legume crops such as rice or wheat, which are the best-studied

examples that benefit from Rhizobia as endophytes (Biswas et al., 2000). For all these reasons,

the Rhizobium-legume symbiosis has been widely studied as a model of mutualistic associations

and as a beneficial association for sustainable agriculture. With increasing use of Rhizobium and

other beneficial microbes as bio fertilizers, reduction in the need for chemical fertilizers can be

observed. Therefore, bio fertilization has great importance in decreasing environmental pollution
and deterioration of nature (Vessey, 2006; Erman et al., 2011). The inoculation of seeds with

Rhizobium is known to increase nodulation, N uptake, growth and yield parameters of legume

crops (Erman et al., 2011). Keeping in view the importance of Rhizobia in legume plants, the

present study was undertaken to shed some light on different morpho-physiological and

biochemical properties of Rhizobial strain isolated from chickpea plant.

Mitchell Andrews

The Leguminosae (Fabaceae, the legume family) is comprised of ca. 19,300 species within

750 genera that occur as herbs, shrubs, vines or trees in mainly terrestrial habitats and are

components of most of the world’s vegetation types. Currently, the legume family is divided

intothree sub-families, the Caesalpinioideae, Mimosoideae and Papilionoideae. Members of

the Caesalpinioideae are grouped into four tribes, the Caesalpinieae, Cassieae, Cercideae and

Detarieae comprising ca. 170 genera and 2250 species. The Mimosoideae are grouped into

two tribes, the Ingeae and Mimoseae with ca. 80 genera and 3270 species, while the

Papilionoideae consists of 28 tribes with ca. 480 genera and 13,800 species. However, a new

classification of the legumes has been proposed with six sub-families based on the plastid

matK gene sequences from ca. 20% of all legume species across ca. 90% of all currently

recognized genera. The six sub-families proposed are a re-circumscribed Caesalpinioideae,

Cercidoideae, Detarioideae, Dialioideae, Duparquetioideae and Papilionoideae. In this

system, the currently recognized Mimosoideae is a distinct clade nested within the re-

circumscribed Caesalpinioideae. Species within the Cercidoideae, Detarioideae, Dialioideae

and Duparquetioideae do not nodulate.

Most legume species can fix atmospheric nitrogen (N2) via symbiotic bacteria (general term

“rhizobia”) in root nodules, and this can give them an advantage under low soil nitrogen (N)

conditions if other factors are favourable for growth. Furthermore, N2 fixation by legumes

can be a major input of N into natural and agricultural ecosystems.. Generally, legume
nodules can be classified as indeterminate or determinate in growth. Indeterminate nodules

maintain meristematic tissue, while determinate nodules have a transient meristem. Nodule

type is dependent on host plant, and legume species that can produce both determinate and

indeterminate nodules are rare. All genera examined in the Caesalpinioideae and

Mimosoideae had indeterminate nodules. Within the Papilionoideae, most tribes had

indeterminate nodules, but the Desmodieae, Phaseoleae, Psoraleae and some members of the

Loteae show “desmodioid” determinate nodules and the Dalbergieae “aeschynomenoid”

determinate nodules. Desmodioid nodules have lenticels, and rhizobia “infected” tissue

within them also contains uninfected cells. Aeschynomenoid nodules do not have lenticels,

have uniform infected tissue and are always associated with lateral or adventitious roots.

Lewis et al., 2005

The general classification of the Leguminosae follows Lewis et al., 2005 [1], with updates.

The sub-families Caesalpinioideae, Mimosoideae and Papilionoideae are considered

separately. The Papilionoideae is split into those that show indeterminate nodules and those

that show determinate nodules. Those that show indeterminate nodules are further split into

the IRLC and all other clades. Nodulating bacteria were classified at the genus level, on the

basis of sequences of the 16S rRNA gene (almost all cases), and/or the 16S–23S DNA

intergenic spacer region, and/or common house-keeping genes, and/or DNA-DNA

hybridisations, and these results are presented in the tables. Sequences for nif and nod genes

are considered in the text. Rhizobial genus and species names validated in the International

Journal of Systematic and Evolutionary Microbiology were used with one exception:

Burkholderia was retained as opposed to using Paraburkholderia, as a case to reinstate

Burkholderia is being prepared by workers in the field. The term symbiovar (sv.) is used

when describing rhizobial strains within the same species that differ with respect to the

legume species they effectively nodulate.

A comprehensive collation of published legume-rhizobia symbioses up until 30 September

2016 was carried out. Articles were collected by searching the Institute for Scientic

Information (ISI) Web of Science using each legume genus partnered with each of the

rhizobia, Bradyrhizobium, Burkholderia, Cupriavidus, Ensifer, Mesorhizobium, Rhizobium

and Sinorhizobium as the keywords. Further searches were carried out on the literature

quoted in the selected papers and those listed as quoting the selected papers in the ISI Web of

Science. Only data for plants sampled under field conditions, or for plants grown in soils

taken from the field, or supplied field soil extracts were used. Bacteria isolated from legume

nodules were accepted as rhizobia if they were shown to produce functional (N2 fixing)

nodules on inoculation of their original legume host or a species within the original host

legume genus under axenic conditions. The range of measurements and visual assessments

used as evidence of the occurrence of N2 fixation were accepted. These were acetylene

reduction activity, red/pink nodules (evidence of leghaemoglobin and, hence, nodules

assumed to be active), increased total plant or shoot dry matter or N content, visually greener

(increased chlorophyll) and increased plant vigour. However, it is acknowledged that in some

cases, greater growth, vigour and/or greenness could have been caused by plant hormone

production by the bacterium. All data obtained for all species are presented with three

exceptions. Representative data are presented for Glycine max, Phaseolus vulgaris and Vigna

unguiculata due to the large number of publications on these three species.

(Bromfield et al., 1986) and (Singleton and Tavares, 1986)

Inoculated Rhzobium species often fails to compete with indigenous soil rhizobia and

not enough to increase nodulation as these strains have to complete with native

rhizosphere community for nutrients (Bromfield et al., 1986) and (Singleton and

Tavares, 1986). Therefore, effective inoculants strains have been selected which are

able to compete with the native Rhizobium and thus from a high percentage of

nodulation (Hafeez et al., 1991), (Hafeez et al., 2000) and (Hafeez et al., 2001). There

are different type of legumes from various size and shapes of nodules on their roots.

Although many more differences have been even found within the same species of

legumes. Hence, nodules are collected when plants are in flowering stage and one can

make out an effective nodule which is large in size and red in colour. Such nodules

are used for separation of Rhizobium in the laboratory. An isolation of Rhizobium is

made by following usual techniques. Yeast Extract Manitol Agar (YEMA) is the

special medium used to get the Rhizobium growth. Following tests are performed in

the laboratory.

i. Complete Plant cover test

ii. Hennaed Jar test

iii. . Test in earthen pots.

iv. Separation of root and their testing.

v. Tissue culture test.

vi. Field experiment.

This cross inoculation groups as a new concept that is separate Rhizobium species

only nodulate specific related legumes. For example Braddyrhizobium Japonicum

nodulating soya bean cannot infect groundnut plants and vice versa. The cross

inoculation groups have naterbigul compartment and no exceptions are found from
the published information so far it is not known what are the colonies grown on a

medium may not be only of Rhizobium they can be even of Agrobacterium for

definite conclusion one has to inoculates seeds of particular sequence and wait for

formation of nodules on such plants isolated colonies are of Rhizobium for this

purpose one has to see that inoculated plants form effective nodules on roots. For

further combination following tested are performed in the laboratory.

i. Growth on yeast mannitol agar.

ii. Examination under microscope

iii. Congored test

iv. Alkaline mixture test of Hoffer

v. Lactose agar test

Besides, there are several tests of nodule formation on roots of legume conducted, of

which following are most important reasons for existence of cross inoculation groups

in the nature.

Sharma (2007)

He carried out an experiment at Gwalior and found that higher consumption of

nutrients from soil which rescue the lower amount of nutrients in soil. On the other

hand lower consumption of nutrients from soil which rescue the higher amount of

nutrients in soil.

Jain (2008)

He found that application of Rhizobium and phosphate solubilizing bacteria (PSB) as

co-inoculants with and without different sources of organic increased the available

nutrients and balance sheet of nutrients in soil and economic viability of chickpea.

Dalal and Nandkar (2010)

They observed that Rhizobium sp. and Azotobacter chroococcum. Phosphorus can be

made soluble in boil by phosphate solubalizing bacteria (Pseudomonas striate). They

possess ability to bring insoluble phosphate in to soluble from by secreting organic

acids. NPK is most important elements in controlling the normal growth and yield of

crops including Pigeon pea. Generally the crops need recommended doses of 25kg P:

50 kg/ha and to get the 1.2 a tonnes/ha yield, the crop plant required 8.5, 8.0 and

16kg NPK/ha respectively

Singh et al. (2013)

He observed that Rhizobium spp. are gram-negative soil bacteria that have a

profound scientific and agronomic significance due to their ability to establish

nitrogen-fixing symbiosis with leguminous plants, which is of major importance to

the maintenance of soil fertility. For this reason and taking into consideration the

importance of legumes in animal and human consumption, some attention has been

given to the effects that heavy metals exert on Rhizobium isolates as free-living

organisms or symbiotically associated with legumes.

REFERENCES

[1] J.C. Cleyet-Marel, R. Di Boniot and D.P. Beck. Chickpea and its root-nodule bacteria:

implications of their relationships for legume inoculation and biological nitrogen fixation. In:

Saxena, M.C. (ed.), present status and future prospects of chickpea crop production and

improvement in the Mediterranean countries. Zaragoza (Spain). CIHEAM-IAMZ, 6: 101-

106.1990 [2] N. Teaumroong and N. Boonkerd. Detection of Bradyrhizobium spp. and B.

japonicum in Thailand by primer-based technology and direct DNA extraction. Plant and

Soil, 204:127-134. 1998 [3] D. Rigby and D. Caceres. Organic farming and the sustainability

of agricultural systems. Agricultural Systems, 68:21-40.2001 [4] J.Y. Lee and S.H. Song

2007. Evaluation of groundwater quality in coastal areas: implications for sustainable

agriculture. Environmental Geology, 52:1231-1242.2007 [5] H. Ogutcu, O.F. Algur, E.

Elkoca and Kantar, F. The determination of symbiotic effectiveness of Rhizobium strains

isolated from wild chickpea collected from high altitudes in Erzurum. Turkish Journal of

Agriculture and Forestry,32: 241-248.2008.

Dilworth MJ, Parker CA (1969). Devolopment of the nitrogen fixing system in legumes. J.

Theor. Biol. 25: 208-218.

Hunter WJ, Kuykendall LD, Manter DK (2007). Rhizobium selenireducens sp. nov.: A

Selenite-Reducing _-Proteobacteria Isolated From a Bioreactor. Curr. Microbiol. 55: 455-

460.

Zsbrau HH (1999). Rhizobium-Legume symbiosis and nitrogen fixation under sever

conditions and in arid climate. Microbiol. Mol. Biol. Rev. 63: 968-989.

G.S Deora and K. Singhal, Department of Biotechnology, B. N. (P.G.) College, Udaipur,

India, Isolation, biochemical characterization and preparation of biofertilizers using

Rhizobium strains for commercial use, Vol. (3) No. (2) December, 2010(132-136)
Shefali Poonia, Department of Botany, D.N. College, Meerut, U.P, INDIA, International Journal of

Engineering and Management Research Vol. 1, Issue-1, November-2011 ISSN No.: 2250-0758

Zeenat Wadhwa1, Vivek Srivastava2, Raj Rani1, Tanvi1, Kanchan Makkar1 and Sumit

Jangra3*, 1Department of Biotechnology, Dayanand College, Hisar-125001, Haryana, India

2Department of Botany, Dayanand College, Hisar-125001, Haryana, India 3Department of

Molecular Biology, Biotechnology and Bioinformatics, CCS Haryana Agricultural

University, Hisar-125004, Haryana, India, Isolation and Characterization of Rhizobium from

Chickpea (Cicer arietinum), International Journal of Current Microbiology and Applied

Sciences. ISSN: 2319-7706 Volume 6 Number 11 (2017) pp. 2880-2893