Chapter 2: Review Literature

Chapter 2: Review of Literature

REVIEW OF LITERATURE 2.1 Rhizobium Legumes have been used in agriculture since ancient time and legume seeds or pulses were among the first source of human food and their domestication. Legumes plant posses a unique ability to establish symbiosis with nitrogen fixing bacteria of the family Rhizobiaceae. The bacteria belonging to the genera Rhizobium, Bradyrhizobium, Allorhizobium, Rinorhizobium and Mesorhizobium (Martinz Romero,2003; Willems, 2006) which are collectively referred to as rhizobia. Rhizobia are gram negative, motile rod shaped bacteria. Role of Rhizobia in nitrogen fixation were first identified in root nodule of legumes in 1888 (Hirsch et al., 2001). They are motile and able to move in damp soil through the water films surrounding the soil particles, but the movement by their own efforts is very slow (Hamdi, 1971). Rhizobia are bacteria that establish symbiosis with legumes, forming root or stem nodules and fixing atmospheric nitrogen (Quatrini et al., 2002). This symbiotic relationship reduces the requirements for nitrogenous fertilizers during the growth of leguminous crops and also enrich soil with nitrogen. Symbiotic nitrogen fixation by legumes is generally the dominant source of nitrogen input in soil for imparting fertility but also avoid soil stresses, such as temperature, acidity and salinity which pose a severe yield constraint in obtaining plant growth and development (Lawson et al., 1995). The bacterium’s enzyme system supplies a constant source of reduced nitrogen to the plant and this bacterial symbiosis with leguminous plant reduces the requirements for nitrogenous fertilizers during the growth of leguminous crops (Dilworth and Parker, 1969). Rhizobia show a typical translucent, viscid, slimy growth on Yeast Mannitol Agar media with individual colonies having domed shape, elevated feature with entire margins (Gupta et al., 2007). There is variation in specificity of interaction between rhizobia and legume species. Some rhizobia legume associations are very specific, where a host legume will form nodules with a range of rhizobia (Vance, 2000). Specificity involves the recognition of the bacterium by the host and of the host by the bacterium through the exchange of signal compounds, which induce differential gene expression in both partners (Broughton et al., 2000). Rhizobia have been utilized in agriculture to increase the yield of leguminous plants (Wadhwa et al., 2010) through their use as inoculants to seed or, less often, soil.

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2.1.1 Taxonomy of Rhizobium In 1888, Beijerinck reported isolation of the root nodule bacteria and established that they were responsible for this process of nitrogen fixation. He named these bacteria Bacillus radicicola (Beijerink, 1888). Later, Frank changed the name to Rhizobium with originally just one species, R. leguminosarum (Frank, 1889). The earliest classification of rhizobia was based on specificity of symbiotic plant range of bacterial species. Fred et al., 1932 they recognized six species in genus Rhizobium, viz. R. japonicum (Lathyrus, Lens, Pisum and Vicia), R. lupines, R meliloti (Melilotus, Medicago, Trigonella), R. phaseoli (Phaseolus) and R. trifolii (Trifolium) based on their host range, though they also described certain morphological and physiological properties of the identified species. Based on growth rate, bacteria were grouped as fast growers and slow growers, but were still placed in the genus Rhizobium till (Jordan, 1982) coined the new genus Bradyrhizbium. A single species, Bradyrhizobium japonicum, was described for isolates of Glycine max. (Norris, 1965) observed that fast growers and slow growers differed in their symbiotic affinity. Accordingly, alkali- producing slow growers were associated with the tropical legumes and acid producing fast growers with the temperate legumes; exceptions to this general observation, however, are known. Temperate legumes like Corollina and Lupinus (Allen et al., 1981) are infected by slow- growing rhizobia whereas tropical legumes, e.g. Acacia, Leucaena and Sesbania are infected by fast- growing rhizobia (de Lajudie et al., 1994; George et al., 1994) indeed, fast and slow growing rhizobia have been isolated from the same legume species, e.g. G. max (Scholla et al., 1984) or even from the same plant, e.g. Acacia, (Dreyfus et al., 1981), upin (Fulchieri et al., 1999) and Prosopis (Jenkins et al., 1987). Thus it is clear that classification of rhizobia on the basis of host range and physiological properties does not reflect the true phylogeny of the group. 2.1.2 CURRENT RHIZOBIUM TAXONOMY Rhizobium taxonomy primarily based on 16S ribosomal DNA sequences. Depending on this sequences, the symbionts of leguminous plants has main separate phylogenetic branches. Agrobacterium branch The first branch of Rhizobium is called Agrobacterium branch. It consist of several subbranches, each corresponding to four rhizobial genera, some plant- related bacteria (Agrobacterium, phyllobacterium ), soil bacteria (Mycoplana), and clinical bacteria (Brucella, Ochrobactrum, Bartonella). Page 8

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

The first subbranch corresponds to the s.s Rhizobium genus and includes R. leguminosarum (type species), R. tropici, (type species), R tropici, (Martinez- Romero et al., 1991) R. etli (Segovia et al., 1993) R. gallicum ( Amarger et al., 1997) R. mongolense (Van Berkum et al., 1998) and Agrobacterium biovar .



The second subbranch corresponds to the Sinorhizobium genus and includes the species S. fredii and S. xinjiangensis (Chen et al., 1988) S. meliloti, S. terangae and S.sahelens (de Lajudie et al., 1994; Truper et al., 1997) S. medicae, (Rome et al.,1996) kostiense and S. arboris (Nick et al.,1999).



The third subbranch corresponds to thegenus Mesorhizobium ( Jarvis et al.,1997), which contains M. loti (Jarvis et al., 1982), M. huakuii ( Chen et al., 1991), M. ciceri (Nour et al., 1994), M. mediterraeneum (Nour et al., 1995), M. tianshanense (Chen et al ., 1995), M. plurifarium ( de Lajudie et al., 1998) M. amorphae ( Wang et al., 1999).



The fourth subbranch includes Agrobacterium biovar, Agrobacterium rubi, Agrobacterium vitis, but also R. galegae (Lindstrom et al.,1989), R. giardinii ( Amarger et al.,1997), R. huautlense ( Wang et al., 1998) and Allorhizobium undicola ( de Lajudieet et al., 1998).

Very Recently Young et al. (2001) revisited taxonomic data from the literature and argued that discriminatory phenotypic features to distinguish between these genera were not convincing, and that phylogenetic relationships between genera inferred from comparative 16S rDNA sequence analysis differ depending on the chosen algorithm and most particularly on the selection of included sequences. As a consequence, the authors proposed to group the three genera Rhizobium, Agrobacterium and Allorhizobium in a single emended genus Rhizobium, including all species of Agrobacterium and Allorhizobium as new combinations: R. radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis (Young et al., 1989). 2. Azorhizobium branch The second branch, called Azorhizobium caulinodans, isolated from stem nodules of the tropical legume Sesbania rostrata (Dreyfus et al.,1988).These bacteria perform different functions, like in vitro nitrogen fixation and assimilation for growth under low O 2 partial tension (3%).A second genomic species has been documented in Azorhizobium, but naming was not done(Rinaudo et al.,1991).According to Phylogenetics, the species of the genera Azorhizobium, Xanthobacter and Aquabacter are so intermixed that their inclusion in a single Page 9

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genus Xanthobacter has been examined but not proposed because of their many divergent phenotypic features (Rainey et al.,1996). 3. Bradyrhizobium branch The Bradyrhizobium genus is comprised only one species, B. japonicum, including all soybean nodulating strains. According to (Hollis et al. 1981) this group was heterogeneous having DNA homology groups (I, Ia and II). For the group II, which differ from the species B. japonicum bymany features, (Kuykendall et al. 1992) created a new species, Bradyrhizobium elkanii. Other strains, with very slow growth (generation time between 16 and 24 hours), isolated from the nodules of Glycine max and Glycinesoya in China, were given the proposed name Bradyrhizobium liaoningense (Xu et al., 1995). Classification of nitrogen fixing bacteria forming symbioses with legume plants Species

Host plant

Reference

Rhizobium R.leguminosarum

Pisum Lathyrus, Vicia, Lens, Frank (1879) Phaseolus, Trifolium

R.lupiniib

Lupinus, Ornithopus

Lindstrom (1989)

R. etli

Phaseolus vulgaris

Segovia et al., (1993)

R. gallicum

Phaseolus vulgaris

Amarger et al., (1997)

R. giardinii

Phaseolus vulgaris

Amarger et al., (1997)

R. hainanense

Desmodium, Stylosanthes,

Chen et al., (1997)

Centrosema, Tephrosia, Acacia, Zornia, Macroptilium R. mongolense

Medicago ruthenica

van Berkum et al., (1998)

R. huautlense

Sesbania herbacea

Wang et al., (1998)

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R. yanglingense

Coronilla, Gueldenstaedtia, Tan et al., (2001) Amphicarpaea

R. sullae

Hedysarum coronarium

Squartini et al., (2002)

R. indigoferae

Indigofera

Wei et al., (2002)

R. loessense

Astragalus

Wei et al., (2003)

R. daejeonense 2005

Medicago

Quan et al., (2005)

Mesorhizobium Lotus,

Lupinus,

M. loti

Leucaena

M. huakuii

Astragalus(China)

M. ciceri,

Cicer

Anthyllis, Jarvis et al., (1982)

Chen et al., (1991)

arietinum

(Spain, Nour et al., (1994)

USA, India Russia, Turkey Morocco, Syria) M. tianshanense

Glycyrrhiza,

Sophora Chen et al., (1995)

Caragana, Halimodendron, Swainsonia, Glycine(China) M. mediterraneum

Cicer

arietinum

(Spain, Nour et al., (1995)

Syria, India, Lebanon, Syria, Tunesia) M. plurifarium

Acacia,

Prosopis, de Lajudie et al., (1998b)

Chamaecrista, Leucaena (Senegal, Sudan, Brazil) M. amorphae

Amorpha fruticosa (China)

Wang et al., (1999)

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M. chacoense

Prosopis (Argentina)

M. septentrionale

Astragalus

Vela zquez et al., (2001)

adsurgens Gao et al., (2004)

(China) M. temperatum

Astragalus

adsurgens Gao et al., (2004)

(China) M. thiogangeticum

Rhizosphere

of

Clitoria Gosh and Roy (2006)

ternatea (India) Senorhizobium S. meliloti

Melilotus,

Medicago, Dangeard (1926)

Trigonella S. fredii

Glycine, Vigna, Cajanus

Scholla and Elkan (1984)

S. xinjiangense

Glycine

Chen et al., (1988)

S. saheli

Sesbania, Acacia (Senegal)

de Lajudie et al., (1994)

S. terangae

Sesbania (Senegal)

de Lajudie et al., (1994)

S. medicae

Medicago (Syria, France)

Rome et al., (1996)

S. arboris

Acacia, Prosopis (Sudan,

Nick et al., (1999)

Kenya) S. kostiense

Acacia, Prosopis (Sudan)

Nick et al., (1999)

S. kummerowiae

Kummerowia stipulacea

Wei et al., (2002)

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S. morelense

Leucaena leucocephala

Wang et al., (2002)

(Mexico) S. americanum

Acacia spp. (Mexico)

Toledo et al., (2003) Dreyfus et.al., (1988)

Azorhizobium A. caulinodans

Sesbania rostrata

Dreyfus et.al., (1988)

Azorhizobium sp

Sesbania rostrata

Rinaudo et.al., (1991)

Bradyrhizobium B. japonicum,

Jordan (1982) Glycine max Glycine soja

Jordan et al.(1984) Kirchne.et.al.(1896)

B. elkanii

Glycine max

Kuykendall.et.al.(1992)

B. liaoningense

Glycine max, Glycine soja

Xu.et.al.(1995)

Bradyrhizobium sp.

Vigna, Lupinus, Mimosa

Jordan (1982)

Acaci Aeschynomene Allorhizobium A. undicola

de Lajudie.et.al(1998) Neptunia natans

de Lajudie.et.al(1998)

Table: 2.1 Classification of nitrogen fixing bacteria forming symbioses with legume plants. According to Willems et al., (2006) rhizobia classified on the basis of its growing speed in artificial culture medium, according to them Rhizobium and Sinorhizobium are fastest growing bacteria, Bradyrhizobia are slow growing bacteria and mesorhizobium shows intermediate growth. Dubey,(2011) also classified rhizobia into two categories, that is, rhizobia(Fast growers) and Bradyrhizobia (slow growers).Rhizobia (fast growers) are acid producers and Page 13

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Bradyrhizobia (slow growers) are alkali producers. Two dimensional gel electrophoresis should be used to identify and classify rhizobium strains (Leps et al., 1980). Kittiwongwattana et al.(2013 ) resolved uncertainties in the taxonomy and nomenclature within Rhizobiaceae family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the “R. galegae complex” (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera. Moukoumi et al.(2013) isolated rhizobium from Caragana arborescens and observed that Rhizobium isolates from C. arborescens root nodules are intermediate in 7 growth rate (g) (mean of 5 isolates g = 6.41) as compared to fast growers, Rhizobium 8 leguminosarum NRG457 (g: 4.44), R. tropici 899 (g: 3.19) and Sinorhizobium meloliti BALSAC (g: 3.45) but faster than the slow grower Bradyrhizobium japonicum USDA 10 110 (g: 13.86) and similar to Mesorhizobium amorpheae (g: 7.76). Strain identification was carried out by determining the sequences of three genes, 16S rRNA-encoding genes, cpn60, and recA. This analysis determined that the symbiotic partner of Canadian C. 15 arborescens belong to genus Mesorhizobium and seem more related to M. loti than to previously described Caragana symbionts like M. caraganae. This is the first report of Mesorhizobium sp nodulating C. arborescens in western Canada. Jial et al., 2015 worked on identification and classification of Rhizobia by MatrixAssisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry. Mass spectrometry (MS) has been widely used for specific, sensitive and rapid analysis of proteins and has shown a high potential for bacterial identification and characterization. Type strains of four species of rhizobia and Eischericha coli DH5α were employed as reference bacteria to optimize various parameters for identification and classification of species of rhizobia by (MALDI -TOF MS). The parameters optimized include culture medium states (liquid or

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solid), bacterial growth phases, colony storage temperature and duration, and protein data processing to enhance the bacterial identification resolution, accuracy and reliability. The medium state had little effects on the mass spectra of protein profiles. A suitable sampling time was between the exponential phase and the stationary phase. Consistent protein mass spectral profiles were observed for E.coli colonies pre- grown for 14 day and rhizobia for 21 days at 40C or 210C. A dendrogram of 75 rhizobial strains of 4 genera was constructed based on MALDI TOF mass spectra and the topological patterns agreed well with those in the 16S rDNA phylogenetic tree. 2.2 Rhizobium-legume symbiosis and Nodule Endosymbiotic interactions are characterized by the formation of specialized membrane compartments by the host in which the microbes are hosted, in an intracellular manner .For example arbuscular mycorrhizal symbiosis and the Rhizobium-legume symbiosis. In both symbiosis, the specialized host membrane that surrounds the microbes forms a symbiotic interface, which facilitates the exchange of nutrients and signals and therefore their formation is at the heart of endosymbiosis. In the Rhizobium-legume symbiosis, the rhizobium bacteria are hosted inside a novel organ, the root nodule (Ivanov et al., 2012). Amount of N gained through symbiosis is affected by three factors: genotype of host plant, genotype of Rhizobium micro-symbiont and the environmental factor (Heichel and Vance, 1979). In terms of symbiosis, flavonoids are most important of these compounds, as they trigger the induction of bacterial nodulation (nod) genes (Redmond et al.,1986).The legume-rhizobium symbiosis is the most important symbiotic association in terms of BNF, producing roughly 200 million tons of N annually (Graham and vance, 2003; Peoples et al., 2009).Symbiotic nitrogen fixation by rhizobia in legume root nodules injects approximately 40 million tones of nitrogen into agricultural systems each year (Herridge et al., 2008). Symbiotic host specificity between leguminous plant and rhizobia is determined by substituted and acylated glucosamine oligosaccharide signal (P.Lerouge; 1994). Legume plants are capable of developing an association with nitrogen- fixing bacteria generally known as rhizobia forming nodule. A nodule is unique organ commonly found on plant root where nitrogen fixation occurs. Nodule provides a suitable environment to nitrogenase complex of bacteria to convert nitrogen gas from atmosphere to ammonia which will be assimilated by the plant.

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The symbiosis process begins with change an interchange of molecular signal between both plant and rhizobia. The legume root secretes flavinoids to induce the synthesis of lipochitin – oligosachride signal called node factors by the rhizobia. Node factor activate the organogenesis on root plant through stimulation of cell division in root cortex, and pericycle. Induced mutant or natural variants of some legume are also available that nodulate in absence of rhizobia .Many stage of nodule development resembles with common plant development for example cell divide and differentiate, vascular tissue develops, nodule respond to external factor such as stress and nitrate. Legumes have two types of nodule: determinate and indeterminate. Determinate nodules are found on tropical (sub) legumes. The meristematic activity of determinate nodules are found on tropical (sub) legumes. The meristematic activity of determinate nodules gets finished after initiation of nodulation (transient meristem), thus growth of nodules is due to cell expansion which gives mature nodules that is spherical in shape. Indeterminate nodules are found on temperate legumes; it has meristematic activity after nodulation and produces new cell throughout the life of nodule (persistent meristem) (Fernandez-Lopez et al., 1998). Difference between the two nodule types are the site of first internal cell division, maintenance of a meristematic region, and the form of mature nodules (Newcomb et al., 1979; Rolfe and Gresshoff 1988). Ziegler et al.,(2015), investigated that ribosomal protein biomarkers provided root nodule bacterial identification by MALDI- TOF MS. Accurate identification of soil bacteria that from nitrogen- fixing association with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB).The labour- intensive and timeconsuming 16S ribosomal RNA (rRNA) sequencing and multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, they recently showed that matrix- assisted laser desorption ionization (MALDI) time-of- flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, they report on the development of a MALDITOF RNB- specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB- specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM)

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database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDITOF MS. Gomes et al., 2014 they worked on proteomic analysis of free- living Bradyrhizobium diazoefficiens; highlighting potential determinants of a successful symbiosis. They were recently reclassified Strain CPAC7 (=SEMIA 5080) into the new species Bradyrhizobium diazoefficiens; due to its outstanding efficiency in fixing nitrogen, it has been used in commercial inoculants for application to crops of soybean [Glycine max (L.)Merr.] in Brazil and other South American countries. Although the efficiency of B. diazoeefficiens inoculants strain is well recognized, few data on their protein expression are available. They provided a two- dimensional proteomic reference map of CPAC 7 obtained under free- living conditions, with the successful identification of 115 spots, representing 95 different proteins. The results highlighted the expression of molecular determinants potentially related to symbiosis establishment, fixation of atmospheric nitrogen (N2) (e.g. Nifh) defenses against stresses (e.g. chaperones). They concluded that proteomic map conditions and their approach combining bioinformatics and gene- expression assays resulted in new information about unknown genes that might play important roles in the establishment of the symbiosis with soybean. 2.2.1 Nodule organogenesis Nodule formation is initiated by the host plant roots secrete phenolic flavonoid compound into rhizosphere (Redmond et al., 1986) (Figure 2.1).The secrete partly determines the specificity of the symbiotic relationship as each rhizobia species responds to specific flavonoids. Most rhizobia species interact with only a select few legumes, some have been shown to have a broad host range (Pueppke and Broughton 1999). Flavonoid perception attracts the bacteria to the root activates nod(nodulation) gene expression, leading to the production and secretion of strain- specific lipo-chitooligosaccharides, also known as nod factors (Caetano- Anolles and Gresshoff 1991, Denarie et al., 1996, Spaink 2000).The exception are some recently identified photosynthetic Bradyrhizobium strains that can induce nodule development despite not having the critical nodABC genes required for NF biosynthesis (Giraud et al., 2007).NFs have an oligosaccharide backbone of N-acetyl-D-glucosamine units with a fatty acyl group attached to the non- reducing sugar. A major determinant of host-symbiont specificity is attributed to

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the different NF substituents attached to the oligosaccharide backbone (Lerouge et al 1990; Denarie et al., 1996). Rhizobia have two main ways of entering the plant root: via the root hair or through cracks in root epidermal tissue (Oldroyd and Downie 2008). Root hair infection is the most common and involves the formation of infection threads, which are tubular structure composed of plant cell wall components that act as a passage for the bacteria into the cortical cells of the plant (Gage 2004).The rhizobia enter through the deformed root hair tip, which encapsulates a small proportion of the dividing bacteria (Callaham and Torrey 1981; Turgeon and Bauer 1985).The enclosed microcolony presumably has an enriched NF concentration as well as cell wall degrading enzymes. Penetration of the cell wall, but not its plasma membrane is followed by resynthesis and re-digestion. It is possible that invading rhizobia, still capable of NF production as evidenced by NodC::LacZ fusion expression, stimulate ever-increasing NF levels that lead to mitotic activation of cortical cell in the root. This eventually result in the development of nodule primordium. The radial position of the cell divisions, and thus the primordium, is controlled by positional gradients for hormones such as ethylene (Heidstra et al., 1997; Lohar et al., 2009).Accordingly, most nodules develop close to the xylem radial cells, away from the phloem. The infection thread grows through the root hair into the root cortex and the newly induced dividing cells. Bacteria are released from near the growing tip of the infection thread into an infection droplet in the host cell cytoplasm. Through a process resembling endocytosis,the bacteria are surrounded by a plantderived membrane, called the peribacteroid membrane,which forms the symbiosome (Udvardi and Day 1997).the membrane-enveloped bacteria and start to fix nitrogen (Roth and Stacey 1989 a, b).

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Figure: 2.1 Nodule Development and autoregulation. Photo adapted from Journal of Integrative Plant Biology 2010 Topic – Molecular Analysis of Legume Development. 2.2.2 Nod Factor Different type of Nod factors produced by rhizobia, with the exception of one minor Nod factor produced by Sinorhizobium fredii USDA 191 Bec-Fert,1996.,it consist of an oligosaccharide backbone of β-1,4-linked N-acetyl-D-glucosamine. A fatty acyl group is always attached to the nitrogen of the non-reducing saccharide.Because of the resemblance of the oligosaccharide backbone to a fragment of chitin,the Nod factors are often called lipochitin oligosaccharide (LCOs).All rhizobia appear to produce complex mixtures of LCO species. Proteins encoded by the rhizobial nodulation gene (nod, nol and noe gene) are involved in the synthesis and secretion of Nod factors. The expression of these genes is Page 19

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activated when the bacteria perceive specific molecule, in general flavonoid that are secreted by plant root (Zuanazzi, et al., 1998). Flavonoid activates the bacterial transcriptional regulator NodD that in turn induce the transcription of the other bacterial nodulation gene involved in the synthesis of Nod factors. The basic structure of Nod factors produced by different rhizobial species is very similar. Generally, they consist of a β-1, 4-linked N- acetyl- d- glucosamine backbone with 4 or 5 residues of which the non reducing terminal residue substituted at the C2 position with an acyl chain. Depending on the rhizobial species, the structure of the acyl chain can vary, and substitution at the reducing and non-reducing terminal glucosamine residues can be present. The structure of Nod factors of different rhizobia and their function in nodulation has been reviewed recently (Mergaert et al., 1997; Cullimore et al., 2001).

Figure 2.2: The Major Nod Factor Produced by Sinorhizobium meliloti The major Nod factor produced by Sinorhizobium meliloti (fig: 2.2) contains four glucosamine units, an acyl chain of 16 C- atoms in length with two unsaturated bonds (determined by NodE and NodF), and an acetyl group at the non-reducing terminal sugar residue (determined by NodL), and a sulfate group at the reducing terminal sugar residue (determined by NodH, NodP and NodQ) (Lerouge et al., 1990). Nod factor binding Nod factor induce responses in their host at pico molar concentration. Therefore, it is often suggested that Nod factors are recognized by a high affinity receptor (Denarie and Cullimore,

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1993; Heidstra and Bisseling, 1996). Further, the amphiphylic nature of Nod factor, with their hydrophobic lipid tail and hydrophilic sugar backbone, suggests that Nod factor receptors are located in the plasma membrane. The latter is supported by in vitro studies showing that Nod factors rapidly insert into membranes (Goedhart et al., 1999). 2.3 REGULATION OF NOD FACTOR 2.3.1 Positive nod factor regulation Nod factors are produced in response to inducers that are secreted from the plant roots. The most potent of these inducers belong to the group of flavonoids(Schlaman et al.,1998).Other molecules, such as the betaines (e.g. erythronic acid and tetronic acid), are active as inducers2 in some rhizobial species at much higher concentration (Gagnon et al .,1998;Schlaman et al.,1998). The induction of Nod factor production is specific for the structure of the flavonoid. The specificity of this process has been shown to mediated by the protein NodD, which is a positive transcriptional regulator belonging to the LysR family and found in all rhizobial species (Horvath et al., 1987, Schlaman et al., 1991; Spaink et al., 1987). In several rhizobial strains, multiple isoforms of the nod genes are found which, for S. meliloti, have evolved so that the bacteria could adapt the structures of the Nod factors to their interactions with multiple hosts that secrete different flavonoids (Demont et al., 1994). The transcriptional regulation by NodD in several rhizobia is further complicated by the occurrence of one or more other LysR family members, called syrM gene, they coregulate the production of Nod factors apparently independently from flavonoids (Barnett et al., 1998; Hanin et al., 1998; Schlaman et al., 1998). The syrM gene also involved in the production of the extracellular polysaccharides (Dusha et al., 1999; Mulligan et al., 1989). In B.japonicum the NodV and NodW proteins are involved in the recognition of the iso- flavonoid genistein (Loh et al., 1997). Nod factor production in B. Japonicum is also regulated by the nolA gene (Loh et al., 1999). nolA, which encodes three functionally distinct proteins, is probably involved in mediating specificity toward different soybean genotypes via the regulation of nodD2(Garcia et al., 1996). 2.3.2 Negative regulation of Nod Factors production : A repressor nolR has been identified from the genera Sinorhizobium and Rhizobium and that repressor can bind to particular target sequences in the promoter regions of genes involved in Nod factor synthesis (Kiss et al., 1998). Nod factor production might also be regulated at the posttranscriptional level which was suggested by the results of a recent study showing that

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the presence of several modifications of the Nod factors is regulated by growth temperature (Olsthoorn et al., 2000). A likely mechanism for the finding reported by Olsthoorn et al is that the activity of the acetyltransferase NodX, which is involved in the substitution of an acetyl at position R5, is strongly temperature dependent. It has been shown that the nod genes that are essential for Nod factor synthesis are switched off at later stages of the symbiosis, there is no information on the regulatory mechanisms responsible for this down- regulation (Schlaman et al., 1991 and Sharma et al., 1990). Cerro et al., 2015 Observed that regulatory nodD1 and nodD2 genes of rhizobium tropic strain CIAT 899 play important role in early stage of molecular signaling and hostlegume nodulation. Phenotype, Nod factors and gene expression of nodD1 and nodD2 mutant of CIAT899 were compared with those of the wild type strain, both in the presence and in the absence of the nod- gene- inducing molecules apigenin and salt (NaCl). Difference between the wild type and mutant were observed in swimming motility and IAA (indole acetic acid) synthesis. They concluded that full nodulation of common bean by R. tropic requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1, nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis. 2.4 Nodule functioning Bacteria that have completed the bacteroid differentiation program express the enzymes of the nitrogenase complex and begin to fix nitrogen. The reduction by nitrogenase of 1 molecule of N2 to 2 molecules of NH4+ requires 16 molecules of ATP and 8 electrons (Jones et al., 2007). Thus, bacteroids require high rate flux of O2 to enable high rates of ATP synthesis, but this must be achieved whilst maintaining a very low concentration of free O 2 to avoid inactivation of O2 – labile nitrogenase. These conditions exist due to the presence of an O2 diffusion barrier and the synthesis of nodule- specific leghemoglobins, which accumulate to milli molar concentrations in the cytoplasm of infected cells prior to nitrogen fixation and buffer the free O2 concentration at around 7-11nM, while maintaining high O2 flux for respiration (Appleby, 1984; Downie, 2005; Ott et al.2005). The unique low- O2 environment provided for the bacteroid is a key signal in bacteroid metabolism, inducing a regulatory cascade controlling gene expression of the nitrogenase complex and the microaerobic respiratory enzymes of the bacteroid. The O2- sensing two- component regulatory system FixL- FixJ activates the transcription of the two intermediate regulators nifA and fixK genes, Page 22

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which induce the expression of nif and fix gene involved in nitrogen fixation and respiration (Reyrat et al.,1993). More generally, bacteroid differentiation is accompanied by global change in gene expression compared with free- living bacteria. There is down- regulation of many genes such as most housekeeping genes involved in synthesis of membrane proteins and peptidogycan in favour of symbiosis specific processes (Becker et al.,2004; Bobik et al.,2006; Capela et al.,2006; Karunakaran et al., 2009; Pessi et al.,2007). 2.5 BIOLOGICAL NITROGEN FIXATION (BNF) The demand of agriculture production is increases continuously resulting greater fertilizer application (Whitehead, 1995; Wood, 1996). Nitrogen(N) applied in fertilizer or manures which is not taken up by crops can be released into the atmosphere as nitrogenous greenhouse gases (Flechard et al., 2007) or leached into ground water (Stout et al., 2007;Trindade et al.,2001), with resulting environmental implications. Rather than relying purely on application of N fertilizer, alternative sources are needed to help develop more sustainable farming sustainable farming system. Legumes have potential to fulfill his requirement due to their unique ability to fix N biologically from the atmosphere, benefiting not only the legumes themselves but also the intercropped or subsequent crops. Biological nitrogen fixation is the conversion of atmospheric nitrogen into ammonia by symbiotic, associative and free- living bacteria. (Dixon &Khan, 2004). 2.5.1 Genes responsible for biological nitrogen fixation The bacterial genes which are responsible for nitrogen fixation are dividing into two broad categories. Those that can fix nitrogen in the free living state are known as nif while those that are unique to symbiotic nitrogen fixation are known as fix (Arnold et al., 1988; Long, 1989). The genetics of nitrogen fixation was initially elucidated in Klebsiella oxytoca strain M5a1 (first identified as pneumoniae). In that strain, nif genes necessary for Mo nitrogenase polypeptides are nifD and nifK for Mo protein subunit and nifH for Fe protein. Full assembly of nitrogenase requires products of other nif genes involved in synthesis of FeMoCo (nifB, nifQ, nifE, nifN, nifX, nifU, nifS, nifV, nifY also nifH) and in assembly of iron sulfer clusters (nifS and nifU) (Hu et al.,2007) and maturation of the nitrogenase components (nifW and nifZ) (Zheng et al.,1998; Dioxn and Khan 2004).It is now established that a core of nif genes (nifH, nifD, nifK, nifY, nifB, nifQ,nifE, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ) required for nitrogenase synthesis and catalysis conserved in all diazotrops. The gene nifW is involved in staility of dinitrogenase and protect the protein fromoxygen inactivation (Cheng,

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2008).Depending on the system, other gene are required for in vivo nitrogenase activity, such as those coding for components of physiological electron transport chains (flavodoxin, ferredoxin and the NADH- ubiquinone oxidoreductase (NQR) encoded by the mfABCDGEF cluster) to nitrogenase, molybdenum uptake and homeostasis, and oxygen protection and regulation, including respiratory chains adapted to oxygen conditions at which the nitrogen fixation process can operate. 2.5.2 Mechanism of biological nitrogen fixation Biological nitrogen fixation, a process utilized only by certain prokaryotes, is catalyzed by a two-component nitrogenase complex (Yan et al., 2010). Nitrogenase catalyzes the simultaneous reduction of one N2 and 2 H+ to ammonia and a molecule of hydrogen gas. N2 + 8H2 +16 ATP 2NH3+2NH3+2H2+16ADP+16Pi The immediate electron donor is the potent reducing agent ferredoxin and the reaction is driven by the hydrolysis of 2 ATP for each electron transferred (Wheelis, 2008).The best known Biological Nitrogen Fixation (BNF) system occurs between legumes and rhizobium bacteria (Carvalho et al., 2011). The symbiotic association between the roots of legumes and certain soil bacteria, generally known as rhizobia, accounts for the development of a specific organ, the symbiotic root-nodule, whose primary function is nitrogen fixation. Root nodules make a crucial contribution to the nitrogen content of the soil playing a key role in agricultural practices (Alla et al., 2010). Perception of legume root exudates triggers the production of rhizobial Nod factor signals which are recognized by compatible plant receptors leading to the formation of root nodules, in which differentiated bacteria (bacteroids) fix atmospheric nitrogen (Oldroyd and Downie, 2008). In the nodule, maintenance of nitrogenase activity is subject to a delicate equilibrium. Firstly, a high rate of oxygen respiration is necessary to supply the energy demands of the Nitrogen reduction process (Sanchez et al., 2011), but oxygen also irreversibly inactivates the nitrogenase complex. These conflicting demands are reconciled by control of oxygen flux through a diffusion barrier in the nodule cortex and by the plant oxygen carrier, leghemoglobin, which is present exclusively in the nodule (Minchin et al., 2008). In addition to fixing nitrogen, some rhizobia species are able to grow under low oxygen conditions using nitrate as electron acceptor to support respiration in a process known as denitrification by which bacteria reduce sequentially nitrate (NO3) or nitrite (NO2) to Nitrogen (N2). Nitrate is reduced to nitrite by either a membrane-bound or a periplasmic

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nitrate reductase, and nitrite reductases catalyse the reduction of nitrite to nitric oxide (NO). Nitric oxide is further reduced to nitrous oxide (N2O) by nitric oxide reductases and, finally, N2O is converted to N2 by the nitrous oxide reductase enzyme (Van Spanning et al., 2005, 2007). The significance of denitrification in rhizobia-legume symbiosis can be appreciated when oxygen concentration in soils decreases during environmental stress such as flooding of the roots, which causes hypoxia. Under these conditions, denitrifying activity could work as a mechanism to generate ATP for survival of rhizobia in the soil and also to maintain nodule functioning (Sanchez et al., 2011). 2.5.3 Nitrogenase – key enzyme in nitrogen fixation The fixation and reduction of atmospheric nitrogen is a complex phenomenon which requires a huge amount of energy (postgate, 1982).The nitrogen molecule consists of two nitrogen atoms joined by a triple covalent bond which renders the molecule almost chemically inert and nonreactive (Wagner,2012).It does not combine with other elements easily. The breaking of this bond is catalyzed by the microbial enzyme nitrogenase. Nitrogenase is the complex metalloenzyme that plays a crucial role in the reduction of biological nitrogen. The Modependent nitrogenases are the most important and best studied enzyme. These are most widely distributed (Burgess et al., 1996). Mo-dependent nitrogenase consists of two component proteins called as component I and component II. These proteins are soluble in nature. 2.5.4 Component I(Mo Fe protein) The component I is also known as dinitrogenase .The Mo Fe protein contains the active site for substrate reduction. It has a molecular weight of approximately 250kDa (Christiansen et al., 2001).The protein is associated with two Mo atoms, 30 non-heme Fe atoms and 32 acid labile sulfides (Rees et al., 2005). Polyacrylamide gel electrophoresis of Mo Fe protein revealed its structure which indicated that it is organized as an α2β2 heterotetramer (where α= Nif D and β=Nif K protein)(Raymond et al., 2003).The Mo, Fe and S protein of component I are organized into two unique mettalloclusters called P (or [8Fe-7S] cluster and ironmolybdenum cofactor (Fe-Mo cofactor)(Dos Santos et al.,2004).The actual reduction occurs in the Fe-Mo center (Rees et al., 2005). The specificity & efficiency of Fe-Mo nitrogenase in binding nitrrogenase is more than the alternative nitrogenase (Raymond et al., 2003).The auxillary p cluster mediates the transfer of electron from Fe protein to FeMo-co (Hoffmann et al., 2009).

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2.5.5 Component II (Fe protein) Component II has a molecular weight of 55000-65000Da (Christiansen et al., 2001). The Fe protein is a homodimer of consisting of γ subunit (Nif H protein). It contains four non-heme Fe atoms and four acid labile sulfides referred as [4Fe-4S] cluster which is bridged between the dimer (Howard et al., 1989).Two nucleotide binding sites (one on each subunit) are also present in the Fe protein. The Fe protein delivers electrons one at a time to component I (Hoffmann et al., 2009) (Figure 2.3).

8Fe- 7S P cluster

FeMo cofactor

cluster

Bouunded nucleotide

4Fe-4S cluster

Fe protein

MoFe protein

Fe protein

Figure: 2.3 Structure of the complex that is formed between the component Fe and MoFe proteins of Azotobacter vinelandii nitrogenase. Photo adapted from nature review 2004. Topic: Genetic regulation of biological nitrogen fixation.

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2.5.6 Significance of Biological Nitrogen Fixation Biological nitrogen fixation is an efficient source of nitrogen .The atmosphere is a nearly homogeneous mixture of gases, the most plentifully is nitrogen 78.1% (Garrison, 2006). About 96% of the N taken up by the crop has been measured as nitrogen derived from atmosphere (Lopez-Bellido et al., 2006). Biological nitrogen fixation involves conversion of atmospheric nitrogen (N) to ammonium, a form of N that can be utilized by plants (Vessey et al., 2005). The nature of biological nitrogen fixation is that the dinitrogenase catalyzes the reaction, splitting triple-bond inert atmospheric nitrogen (N2) into organic ammonia molecule (Cheng, 2008). Biological nitrogen fixation is regarded as a renewable resource for sustainable agriculture as it helps to reduce fertilizer N requirements and thus increases economic returns to producers (Walley et al., 2007). Furthermore, it plays a key role in assessment of rhizobial diversity, contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. The fixation of N2 by legumes has the potential to contribute greatly to more economically viable and environmentally friendly agriculture (Odair et al., 2006). It has been estimated that the 80–90% of the nitrogen available to plants in natural ecosystems originates from biological nitrogen fixation (Rascio and Rocca, 2008). Biological nitrogen fixation contributes to the replenishment of soil N, and reduces the need for industrial N fertilizers. It offers an economically attractive and ecologically sound means of reducing external N input. In recent years, agricultural systems have changed to improve environmental quality and avoid environmental degradation. One of the most promising techniques to avoid environmental degradation is the use of inoculants composed of diazotrophic bacteria as an alternative use of nitrogen fertilizers (Roesch et al., 2007). 2.6 Plant Pigeon pea (Cajanus cajan) also known as Congo pea, Red gram or Non-eye pea is perhaps one of most widely grown agriculture legumes in tropical and sub-tropical countries (Summerfeld & Robberts, 1985). Cajanus cajan (L.) Millsp. (Pigeon pea) is a perennial legume which is belongs to family Fabaceae. The drought tolerant legume is grown mainly in the semi-arid tropics though it is well adapted to several environments (Troedson et al., 1990). While most of the legume

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require inoculation to optimize their N-fixing ability but rarely pigeon pea needs inoculation because it can nodulate on rhizobium that is naturally present in most soil(Faris,1983). 

Pigeon pea provided most important benefit by improving soil quality and fertility when used as green manure(Onim et al.,1990).pigeon pea has been used successfully under coffee plantation as a cover crop to improve the soil fertility, reduce weed contamination and as well as act as a food source for predators(Venzon et al., 2006).



In cropping season the plant fix about 40gh-1 atmospheric nitrogen and add valuable organic matter to soil through fallen leaves (upto 3.1th-1 of leaf dry matter) (Rupela et al., 2004).



Its root help in releasing soil- bound phosphours to make it available for plant growth. Pigeon pea seed protein content (on average approximately 21%) compare well with that of other important grain legume .Owing to several unique characteristics and benefits, Pigeon pea has become an ideal crop for sustainable agriculture system in rain fed areas, because of large temporal variation (90-300days) for maturity, four major duration for pigeon pea varieties exist extra short (mature in < 100 days), short (100-120days), medium (140-180 days) and long duration (>200 days).

2.6.1 Origin The pigeon pea name was first reported from plants used in Barbados. Once seed of this crop were considered very important there as pigeon feed (Plukenet 1692). Based on range of genetic diversity of crop in India, Vavilov (1951) concluded that pigeon pea originated in India. Several authors considered eastern Africa to be the center of origin of pigeon pea, as it occurs there in the wild form (Zeven and Zhukovsky 1975). However based on the large diversity among the crop varieties, the presence of several related wild species, including the progenitor species, linguistic evidence and usage in daily cuisine, the most of the researchers have agreed on India as the original home of pigeon pea. India is now unequivocally accepted as the primary center of origin and Africa as the secondary centre of origin of pigeon pea (De, 1974, Royes Venon, 1976, Vander Maeson, 1980). Most probably in nineteenth century, immigrants from India introduced the crop into East Africa (Hillocks, Minja, Nahdy, & Subrahmanyam, 2000). Thereafter, pigeon pea moved into the Nile valley, then into west Aferica and eventually to the Americas (Odeny, 2007). It is now widely grown in the Caribbean region. Further, Reddy (1997)and De (1974)

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also postulated that the genus Cajanus probably originated from an advanced Alylosia(now classified as cajanus ), including cultivated species C.cajan occur in India. Geographical Distribution At present Pigeon pea is cultivated in the tropical and subtropical areas between 300N and 300S Latitude on 4.71 million hectares with an annual production of 3.69 million metric tons and productivity of 783kg/hq (FAOSTAT, 2010). Pigeon pea is widely grown in the Indian subcontinent which accounts for about 88% of the global pigeon pea production. The major pigeon pea growing countries in the region are India followed by Myanmar and Nepal. India alone represents about 75% of the area and about 67% of the global pigeon pea production, Africa, including major pigeon pea- growing countries, such as Malawi, Kenya, and Uganda, account for about 11% of the global production. The Americas and the Caribbean produce about 1% of the total Pigeon pea of the world. Pigeon pea is often cross- pollinated with an insect-aided natural out- crossing range from 20% to 70% (Saxena, Singh & Gupta 1990).With chromosome number 2n=2x=22 and genomic size 1C=858Mbp.

Figure 2.4: Pigeon pea plant. Adapted from: www. Google image.com.

2.6.2 Classification of pigeon pea.

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Kingdom: Plantae Division: Magnoliophyta Class:

Magnoliopsida

Order:

Fabales

Family: Fabaceae Genus:

Cajanus

Species: C.cajan 2.6.3 Nodules Pigeon pea is nodulated by the cowpea group of rhizobia, mainly on the upper 30cm of the root system. Nodulation starts approximately 15 days after sowing (DAS) and countinues up to 120 days. It declines towards pod filling (Kumar Rao; 1990). The nodule development is through the meristematic zone, arching around the apical end and medulla contains many bacteroid- filled cells. Sometimes the latter are highly vacuolated (Bisen and Sheldrake 1981). The nodules differ in size from 2-4mm. They may be spherical, oval, elongate, or branched. 2.6.4 Features of pigeon pea plant The pigeon pea plant is erect and branching. The stem is woody, leaves are trifoliate, and compound. It possesses a strong taproot system. The plant grow into woody shrubs, 1-2m tall when annually harvested. It may attain a height of 3-4m when grown as a perennial plant in fence rows or agroforestry plots (Figure 2.4). 2.6.4.1Temperature Pigeonpea is predominantly a crop of tropical areas mainly cultivated in semi- arid region of India and Kenya. It is also cultivated in subhumid regions of Uganda, the West Indies, Myanmar, and the Caribbean region. Reddy and Virmani (1981) concluded that Pigeonpea can be grown between 140N and 280N latitude, with temperature ranging from 260 to 300C in the rainy season and 170 to 220C in the post rainy season.

2.6.4.2 Soils Requirment

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In India, Pigeonpea is cultivated on Entisols, Alfisols, Enceptisols, and Vertisols. The Entisols found in the alluvial- soil belt of the Indo- Gangetic region are deep loam, slightly alkaline (pH 7.5 -8.5), with about 150-200mm available water storage capacity in 2m of soil. The Vertisols are characterized by 40%-60% clay in the surface soil horizons, pH around 8.0 with a water holding capacity between 150-300mm, and the available water in the top 1.5-2.0 m of soil. Alfisol are neutral in reaction (pH 6.5 -7.0) and relatively shallow with low- clay content. They are often sandy loam and can retain about 100mm available water in the root profile (Reddy and Virmani 1981). Pigeon pea, being sensitive to water logging, requires a well- drained soil. It does not grow well in saline soil, but can withstand drought reasonably well. Responses to lime indicated by increase in shoot growth and nodulation, have been reported in soils with pH below 5.0(Edwards; 1981). 2.6.5 Growth and Management Pigeon pea is normally sown directly into prepared ground. Seeding rates for pure stand are 12 to 25kg of seed/ha (Smartt 1976). Seeding depths of 2.5 to 5cm are recommended (Center for New Crops and Plant products 2002). No pregermination treatment of the seed is needed. Although some varieties mature seed is 5 to 6 months, longerlived, tall varieties including those that are more competitive in the wild take 10 to 12 month to mature seed. These plants live about 5 years (Smartt 1976). Experimental yield of 50 dry t/ha/year have been demonstrated; yield of 3 to 8 dry t/ha/year are obtained under normal management (Van Den Beldt 1988). Paliya et al., 2014 observed that the Efficacy of micronutrients influencing growth behaiour of Rhizobium of Pigeon pea. 2.6.6 Nutritive value of pigeon pea A wide variability exists in the chemical composition of pigeon pea seeds due to genotype, growth conditions of storage (Salunkhe et al., 1985; Amaefule and Onwudike, 2000). Pigeon pea is a rich source of protein, carbohydrate and certain minerals. The protein content of commonly grown pigeon pea cultivars ranges between 17.9 and 24.3g/100g for whole grain samples (Salunkhe et al., 1986). Wild species of pigeon pea have been found to be very promising source of high protein and several high protein genotype have been developed with a protein content as 32.5% (Singh et al., 1990). These high protein genotype contain protein content on average by nearly 20% higher than the normal genotypes (Reddy et al., 1979; Saxena et al., 1987). The high protein genotypes also contain siginificantly higher (about 25%), Sulpher containing amino acids, namely methionine and cystine (Singh et al., 1990).

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Pigeon pea seeds contain about 57.3-58.7% carbohydrate, 1.2-8.1 crude fiber and 063 -3.8% lipids (Sinha, 1977).The Supplementation of cereals with protein rich legumes is considered as one of the best solutions to protein-calorie malnutrition in the developing world (Chitra et al.,1996). Pigeon pea flour has been tested and found to be suitable as a protein source for supplementing backed products such as bread, cookies and chapattis due to its high level of protein, iron (Fe) and P (Harinder et al., 1999). Pigeon pea is a good source of dietary minerals such as calcium, phosphorus, magnesium, iron, sulpher and potassium. A study by singh et al. (1984b) showed that the mature pigeon pea contain calcium (120mg/100gm), magnesium (122mg/100gm), phosphorus (1290mg/100gm), and potassium (1302mg/100gm). The mature seed also contain trace minerals, iron (4mg/100gm), zinc (2mg/100gm). It is also a good soluble vitamins, especially thiamine, riboflavin, niacin and choline (Sinha, 1977). Copper (102mg/100gm) and magnesium (1.3mg/100gm). Anti- nutritional factors such as protease (trypsin and chymotrypsin) inhibitors, amylase inhibitors and polyphenols, which are a known problem in most legumes, are less problematic in Pigeon pea than soybean, peas and field beans (Singh and Eggum, 1984; Singh, 1988; Faris and Singh, 1990). Within pigeonpea cultivar, anti-nutritional factors are mainly found among dark seeded genotypes (Faris and Singh, 1990) that are typically grown in Asia. The native African Pigeon pea types are largely cream or white seeded with relatively less antinutritional factors. 2.7 Acidic Soils Soil acidity is term used to express the quantity of hydrogen (H+) and aluminum (Al3+) cations (Positively charged ions) in soils, and soil pH is an indicator of acidity. The pH is the negative logarithm of the hydrogen concentration, expressed on a scale from 1to 14. Approximately 30% of the world’s total land area consists of acid soils, and it has been estimated that over 50% of the world’s potential arable lands are acidic (Von Uexku II and Mutert 1995). Agriculture practices and climate changes increase the amount of land affected by acidity, and thus limit legume crop productivity. Worldwide, more than 1.5 Gha of acid soils limit agriculture production (Graham and Vance, 2000) and as much as 25% of Earth’s croplands are impacted by problems associated with soil acidity (Munns, 1986). 2.7.1 Causes of soil acidity Page 32

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Rainfall: Rainfall is most effective in causing soils to become acidic if a lot of water moves through the soil rapidly. Rainfall is most effective agent for removing basic cations over a long time period. Sandy soils are often the first to become acidic because water percolates rapidly, and soils contain only a small reservoir of bases (buffer capacity) due to low clay and organic matter contents. Since the effect of rainfall on acid soil development is very slow, it makes take hundreds of years for new parent material to become acidic under high rainfall. Weathering of minerals: Soil contains both primary and secondary minerals .As these minerals weather, some components such as Mg, Ca, and K, are taken up by plants, others such as Si are leached from the soils, but due to chemical properties, Fe and Al remain in the soil profile. Highly weathered soils are often characterized by having high concentrations of Fe and Al oxides. Use of nitrogenous fertilizers: The amount of acid added to the soil by nitrogenous fertilizers varies according to the type of fertilizer. The most acidifying are ammonium sulfate and diammonium phosphate (DAP). Less acidifying are urea, ammonium nitrate and anhydrous ammonia. Ammonium (NH +4) fertilizers react in the soil in a process called nitrification to form nitrate (NO3-), and in the process release H+ ions. Leaching of nitrogen: Leaching of nitrogen in the nitrate form is a very important factor in soil acidity. Nitrate is a major nutrient for plant growth. It is supplied either from nitrogenous fertilizers or atmospheric nitrogen fixed by legumes. When there is more nitrate than the plant can use, the nitrate is at risk of draining- leaching- below the plants roots and into the ground water system. This leaves the soil more acidic. Leaching of nitrate can happen through inappropriate use of nitrogen fertilizers and is more common in intensive production like horticulture- or because the plants are not at a suitable stage of growth to use the available nitrogen. Pastures based on annul species, the use of long fallow in crop rotations and heavy applications of nitrogen fertilizers are examples of practices that may increase the risk of nitrate leaching.

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Acid Rain: When atmospheric water reacts with sulpher and nitrogen compounds that result from industrial processes, the result can be the formation of sulfuric and nitric acid in rainwater. However the amount of acidity that is deposited in rainwater is much less, on average, than that created through agriculture activities Plant root activity: Plant take up nutrients in the form of ions (NO3-, NH4+, Ca2+, H2PO4-, etc.), and often, they take up more cations than anions. However plants must maintain a neutral charge in their roots. In order to compensate for the extra positive charge, they will release H+ ions from the root. Some plants will also exude organic acids into the soil to acidify the zone around their roots to help solubilize metal nutrients that are insoluble at neutral pH, such as iron (Fe) (Figure 2.5).

Figure 2.5: Cause of soil acidity. Adapted from www.google image.com

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2.7.2 Soil acidity effects Aluminum toxicity Aluminum is the third abundant element after oxygen and silicon present in the earth’s crust (Ma and Furukawa, 2003; Matsumoto and Motoda, 2012). It belongs to the non-essential category of metals, thus does not exert any known function in plant metabolism (Wang and Kao, 2004). However, the metal is considered to be a major growth- limiting factor particularly in acid soils (pH