dentification and characterization of Mesorhizobial communities associated with chickpea crop in Thal region of Pakistan

By Asad Ullah

dentification and characterization of Mesorhizobial communities associated with chickpea crop in Thal region of Pakistan


Chickpea (Cicer arietinum L.) being the 2nd most important short durational leguminous crop is grown throughout rain-fed areas of the world including Pakistan. In Pakistan, almost 1028.90 thousand hectares of land (4.3 % of the total cultivated area) is under chickpea cultivation with Punjab province contributing about 80% of the total produce. Under current scenario, output yield ratio from one hectare is less than 0.4 tons due to unavailability of nitrogenous sources and soil infertility. The only source of nitrogen in arid areas for chickpea is biological nitrogen fixation process through symbiotic relationship of Mesorhizobial communities. Hence in this study, to ensure the beneficial aspects of these communities towards nitrogen fixation through nodule formation, molecular characterization of the local Mesorhizobium  associated with chickpea roots was studied using 16s rDNA sequencing of the cultivable microflora. From 5 selected districts (Bhakkar, Miawali, Khushab, Jhang and Faisalabad) of Thal region, Twelve hundred and ten bacterial isolates were collected. 5 isolates were nominated for further molecular studies. The results depicted that out of these five selected isolates, four were assigned to genus Mesorhizobium based on 16S rDNA sequence analysis. The identity of isolates then were verified via BLAST method that depicted 80% to 90% similarities. Generally, this study has provided sustainable gateways for better understanding of Mesorhizobial association with chickpea and nitrogen fixation process at molecular level in Pakistan.


Chickpea (Cicer arietinum) is cultivated in 5 continents over 40 countries including India, China, Pakistan, Russia, Egypt, Greece, North Africa, Italy and Rumania. It is an oldest legume crop in Asia and Europe which preferably originated from Mediterranean or Himalayas region. Chickpea crop produced highly nutritive and protein rich (almost 21%) seeds that consumed in all over the World (Nawab et al., 2008) as green vegetable, fried and boiled seeds, and flour. The very popular chickpea dish in Pakistan is “Dal Chana”. The leading producers of chickpeas are developing countries which contribute 95% of overall production. Considering Pakistan, Chickpea alone contributes 80% of country’s pulse production with Thal region of Punjab as a leading producer. Being the 3rd largest producer, Pakistan is facing several challenges which includes international market policies, lack of high yielding cultivars, unsubstantiated extension facilities, soil barrenness, area reduction, environmental variation and pathological problems.

Considering the growth habit, it is a self-pollinated, small, herbaceous much branched crop of sixty centimeter height. The leaves of chickpea crop are pinnately compound, light green to dark green in color generally with one terminal leaflet and covered with granular hairs. Pinnate leaves have small 9-15 leaflet of various size, shape and color with serrated edges and red margins. Flowering process begins over early on the second day and anthesis starts from 9 AM- 10 AM and continues till 3 PM. The color of the flowers varies from white to shade of blue or pink. The pods usually are two cm long and contains two seeds per pod while a single plant have approximately 50-150 pods.

Chickpea plant has a developed nodule associated root system including central tap root, adventitious roots and extensive network of root hairs that spread out in all direction of soil’s upper layer. These nodules are filled up with Mesorhizobial bacteria that fix nitrogen form atmosphere on continual basis and transforms it into an energy rich source called ammonia.

On the basis of seed morphology and color, chickpea is divided in to two basic types, The Desi type: in which mostly the seeds are small, rough and angular in shape. The seed coat of desi type is thick and colorful while the common colors are black, green, yellow and brown. The flower of desi type are mostly of pink color due to the presence of anthocyanin pigmentation on the stem. In Pakistan, more than 70-80% of cultivated chickpea is of Desi type. Dal and flour are always made from this type. The Kabuli type chickpea are characterized by beige or white seed colored seed with ram’s head shape, white flower, thin seed coat, smooth seed surface and the absence of anthocyanin pigmentation. Kabuli type as compare to desi type has higher percentage of sucrose and lower percentage of fiber. The seed of Kabuli type generally large in size and due to this it gets higher market price as compare to desi type.

Usually chickpeas are grown as a rainfed cool weather crop or as dry climate crop in semi-arid regions. Desi type chickpeas are more tolerant to dry season than Kabuli type due to small gain size. Excessive rainfall, frost, drought, hailstorm and nutrient deficiency may led to the severe crop damage. Relative humidity for optimum seed setting must be between 21-41%.

Due to miraculous potential and drought resistant abilities, chickpea crop can be grown in wide agro-ecological zones and soil types. In Pakistan, chickpeas are generally grown on clay loam and sandy loam soils but the most suitable soil is the sandy loam soil of Punjab regions i.e. Thal and Cholistan. The soil excessive in soluble salts, neutral in reaction or having pH greather than 8.5 is not suitable for chickpea crop.

In leguminous crops nitrogen fixation mostly depends upon the symbiotic relationship of plants and Mesorhizobial communities. These communities are the group of gram negative bacteria that facilitate plant in nodule formation and atmospheric nitrogen fixation.(Babic, 2008).

The purpose of this study were to inspect the indigenous diversity of  Mesorhizobial species associated with chickpea crop in Thal region through PCR based 16S-rDNA sequence analysis, to determine the  relationship among these isolates, to understand the symbiotic relationship of rhizobia with host plant and finally to peruse scientist for development of proficient  bio-inoculant.

Before studying different biological, physical and molecular characteristics of rhizobia associated with chickpea, it is requires to isolate them first. The traditional methods for isolation of Rhizobia are much laborious and time consuming. Isolation actually involves treating each and every nodule separately while sterilizing and disrupting the nodule surface to release bacteria. Nodule sterilization is done by either washing nodules in distilled water or dipping in 70% of ethanol solution. While nodule disruption is accomplished through cutting, crushing or stabbing the nodules.

Rhizobia have symbiotic relationship with legumes and every specie has altered host range of plants with numerous metabolic proficiencies (Margaret et al., 2011). Under field conditions, various parameters for rhizobial symbiotic efficiencies are anticipated but the most important factors in chickpea symbiosis were: (i) the occupancy of nodules, that directly relates with endurance competitiveness among the bacterial community (ben Romdhane et al., 2007) and (ii) the nitrogen fixation affectivity, which strengthen the plant growth and yield (Ben Romdhane et at., 2008).

In recent years, 16S rDNA sequence analysis emerged as one of the divergent and important method for bacterial taxonomy and phylogenetic studies (Weisburg et at., 1991) but due to high sequence conservation

Sequence analysis of 16S ribosomal RNA (rRNA) has emerged as one of the most important methods in taxonomy and phylogenic analysis of bacteria (Weisburg et al. 1991).

However, due to the high sequence conservation, this techniques is insufficient to distinguish the strain of several species together.

In Pakistan farmers from “Barani tract/arid areas” in Thal and Cholistan desert areas (Bhakkar, Layyah, Muzafar garh, Jhang, Mianwali, Khoshab; scarce rainfall, hot and dry conditions with low nutrient availability through sandy soils) do not have the sufficient economical resources to buy farm inputs. In Pakistan, almost 479.5 thousand tone of chickpea (average yield of 466 kg/ha) is produced from 1028.90 thousand hectares (4.3 % of the total cultivated area), with Punjab province contributing about 80 percent of the total produce coming from Cholistan and Thal deseret areas. In these areas more than 90% agro-based income is generated from chickpea cultivation of Kabuli and Desi varieties. In these areas chickpea is sown on far separated sand dunes literally called “Tibbas” on residual soil moisture with no farm inputs through traditional agriculture systems. The total output yield is totally dependent upon ecological factors and farmers have very limited choices in terms of provision of better adapted germplasm, disease diagnostics, fertilizer and other farm inputs. Under such circumstances, for chickpea, the symbiotic nitrogen fixation by rhizobia species becomes crucial for supplying available nitrogen (Kaneko et al., 2000), salt and drought tolerance, general plant vigor and higher yields, and hence can be a target for developing low cost cropping solutions for sustainable chickpea production. Therefore, an immediate characterization of rhizobial strains associated with local chickpea germplasm is deemed necessary, for which the present research work is designed with following objective:

Review of Literature

Chickpea importance

Chickpea being the member of genus Cicer, family Fabaceae and subfamily papilionaceae is world’s 2nd largest leguminous crop which has been originated in southeastern Turkey (Ladizinsky, 1975). Genetically, chickpea is a self-pollinated, annual diploid crop having chromosome number 2n=16 (Rajput et al.). The name Cicer is derived from the Greek word “kirkus” which means the force. According to Duschak (1871), this word also have some roots in Hebrew word “kirkes” which means round in shape. While the 2nd part arietinum is derived from a Greek word “krios” which means ram, it is an insinuation to the shape of chickpea seed which actually resembles with the head of a ram (Van der Maesen 1987). The wild species of chickpea are found in all over the world but abundant in Pakistan, Turkey, Central Asia, Iran and Afghanistan (Duke, 1981).

            In central Asia, chcickpea is normally accredited as gram and is the most vital Rabi pulse crop of Pakistan. More than 10000 families in rainfed areas of Pakistan are dependent on chickpea crop for their survival.  In Pakistan, especially in Punjab, chickpea is progressively grown in Bhakkar, Mia wali, Khushab, Layyah, Jhang, Chakwal, Attock and Jhelum (Haqqani et al., 2000).

A lot of work has been done related to the genetic diversity of chickpea associated Mesorhizobia, effect of Mesorhizobial communities on nodulation or nitrogen fixation and production/yield variation of chickpea crop (Jeena et al., 2005) associated with above mentioned factors throughout the world. But the facts about genetic studies of Mesorhizobia and nodulation related to agro-climatic conditions in Pakistan are hardly accessible in literature. Being a main cash crop of arid areas, chickpea prefers to grow on sandy soil and its production completely depends upon judicious rainfall throughout growing season. Due to rainfall uncertainty, low attentive behavior towards research and development and abrupt changes in climatic conditions, it is difficult for the farmers to decide whether the input usability would be economical or not. Mesorhizobial community having symbiotic relationship with the roots of leguminous plant for root nodulation is collectively known as rhizobia. Nearly 13 genera of Proteobacteria are associated with nodule formation in chickpeas (Dudeja and Narula, 2008; Dudeja et al., 2012).

Phenotypic and genotypic characterization of chickpea Mesorhizobia

Phenotypic characterization of bacterial population includes morphological, biochemical, physiological and symbiotic compatibility of crop and its associated Mesorhizobia. For numerical taxonomic purposes, phenotypic based substrate tests are commonly used (Gao et al., 1994) which provides ecological information when organism use that substrate). 16S rDNA gene sequencing used for genotyping by various methods including polymerase chain reaction (PCR) (Boudewijns et al., 2006) and restriction fragment length polymorphism (RLFP) (Laguerre et al., 1994).

Genotypic characterization for nodulation and the impact of Mesorhizobia on nodulation

Significant nodulation variations and high heritability among different chickpea genotypes has been reported (Yadav et al., 2004; Gillani et al., 2005; Mensah and Olukoya, 2007). In 1992, it was reported that the frequency for Nod-plant fluctuated from 120 to 490 per million under field condition (Rupela, 1992). It was proved in 20th century that there is a significant correlation between nodulation and seed yield. (Corbin et al., 1977). It was studies that nitrogen requirement for plant or grains are mainly fulfilled by nodule fixated nitrogen rather than any other source i.e. nitrogen fertilizers/ supplements (Hungria and Neves, 1987). Highly specified symbiotic relationship among chickpea plant and Mesorhizobial communities is necessary for nodule formation and nitrogen fixation. Any ailment with Mesorhizobial communities such as poor growth, low population number, existence of inappropriate diversity or ineffectiveness of strains may led to nodulation and nitrogen fixation problems.

Different microsatellite markers have been used for the investigation of different chickpea genotypes and one of them is Simple Sequence Repeat (microsatellite) which is considered as efficient tool for assessment of genetic variability pattern (Winter et al., 1999) while for the Mesorhizobial species 16srDNA has been a successful technique.

Materials and methods

Survey and sample collection

Surveys was conducted during chickpea growing season in five districts of Thal region including Faisalabad (as model location), Jhang Miawali, Khushab and Bhakkar to collect samples of chickpea roots associated nodules.

Random sampling was performed in chickpea field and dunes of Thal region including Faisalabad. Twenty locations were selected from each of the five district and in each location, five sites were again selected and desired samples were collected. Major sampling sites from all districts was 7/1 Thal Janboobi, 7/2 Thal Janoobi, Athara Hazari, Nawan kot, Rudu Sultan, Jabboana, Chely Wala, Chenny wala, Burji Wala, Hedarabad Thal, Azri bhakkar, NIBGI Trail Fields, Mankera, Kalorkot, Darya khan, Khansar, Jhok Mehr Shah, Jhang Rd. Kaimar, Dera Dhona Wala, Pelo venus, Noor pur thal. Sha Hussan, Roda, Arsalpur, Chandni Chowk, Nawan Sago, Ali khel, Jhumtaan Wala, and hernoli   On the whole 100 samples were collected from each district and 500 samples from all districts.

Sample Harvesting Procedure

Roots of plants were harvested from field by digging up the area below the crown of plant using shovel. Soil was removed carefully from roots so that nodules remained fresh (washed nodules remain fresh up to 5 days) and then roots having nodule were placed in plastic bags.

Nodule separation and preservation

Nodules from all samples were separated, counted and preserved in sterilized 96 well plates containing 2-5 beads of silica gel. The filled plates were stored in refrigerator at -4◦C for further microbial studies such as for rhizobial isolation.

Isolation of Rhizobia from Nodules
Collected nodules associated with Mesorhizobial communities were isolated using different culture media including Nutrient Agar and Yeast Extract Mannitol (YEM) Agar with . Each nodule was surface sterilized with 70% ethanol for 30 seconds and rinsed twice with distilled water and then nodules were crushed into pieces. Crushed nodules were plated on the patriplates having rhizobia specific media (YEM). For crushing and plating, needles and scissors were used that were sterilized by dipping in methylated spirit and flaming several time. All the petriplates were incubated at 27◦C ± 1 for 4-7 days and daily observations were recorded.

Purification of Rhizobial Isolates

Rhizobial isolates were purified on YEMA (Yeast Extract Mannitol Agar) containing 0.0025% Congo red. YEMA is highly specific and routinely used media for rhizobia isolation and purification (Vincent, 1970).

YEMA was prepared by adding mannitol 10g; agar 15g; potassium hypochlorite 0.5g; Yeast extract 0.4g; Magnesium sulphate 0.2g;  NaCl 0.2g; Congo red 0.025g and 1 liter of distilled water in conical flask and then covered with cotton plug. Then the mixture was placed on stirrer for homogenization. After that the mixture was autoclaved at 121◦C temperature and 15 psi pressure for 15 minutes then prepared medium was placed at optimum conditions for cooling. Finally, medium was poured in Petri plates in laminar flow chamber. When the media was solidified, a single colony from each nodule extract was taken directly and streaked on congo red containing YEMA plates. Subsequent streaking (3 to 4) were done to obtain purified isolates.

Culture Preservation

Culture of isolated rhizobial isolates will be prepared in YEM broth medium through following steps.

Inoculum Preparation

5ml of sterile YEM broth medium was poured in each test tube then isolated subculture colonies was inoculated separately in each YEM tube via sterilized loop. Controlled was also maintained which contained only YEM liquid media for comparison. Finally, inoculated tubes were placed on shaker at 28◦C for 12-16 hrs for thorough mixing and better colonial growth.

Preparation of Glycerol stock

50% glycerol stock was prepared by mixing 50 g glycerol and add distilled water up to 100 ml then the mixture was autoclaved. After autoclaving, 0.5-0.75 ml culture was taken with equal volume of 50% glycerol in Ephondrof tubes.finally, Eppendrof tubes was placed at – 40◦C.

DNA extraction from isolates

Rhizobial isolates were grown in YEM broth and incubated at 27±1°C for overnight under shaking at 220 rpm. About 1.5 ml of prepared culture was taken in a micrcentrifule tube, spin for 7 minutes and supernatant was discarded. Now add 400 μL TE buffer to re-suspend the pallet, add 10 μL of 20% sodium dodecyl sulphate and vortex the mixture. Now add 100 μL of proteinase K, vortex the mixture complelety and incubate it at 37°C for 1hour. Now add 420μL phenol chloroform and vortex the mixture. Now centrifuge the suspension at 14ooo rpm for 15 minutes. Now carefully take the supernatant in to new eppondrof tube and add 15 μL of 3 molar sodium acetate and mix it well. Then add 570 μL of isopropanol and again mix it gently. Incubate the resultant at -80°C for 15 minutes and then again centrifuge at 14000 rmp for 15 minutes to get the pallet in the mixute. Now remove the supernatant by decanting the eppondrof tube and wash the pallet by using 200 μL of 70% ethanol. Again centrifuge at 14000 rpm for 1 minute and remove the liquid with the help of micropipette. In the last, air dry the pallet at 37°C for 15 minutes. Now add 25 microliter of TE buffer to re-suspend the pellet and store it at -20 °C. for PCR reactions

PCR based 16S rDNA sequencing

The rDNA of isolated rhizobial strains was amplified by using standard protocol described by Sambrook et al., 2001. PCR was performed in thermal cycler by using Universal Primers (Forward primer: fD15 CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3, Reverse primer: Rd15 CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3) (Wen et al., 1999). The Gel (1%) electrophoresis of the PCR product will be done to find out the band size.

Phylogenetic Relationship studies for Mesorhizobial communities

Phylogenetic studies for isolated Mesorhizobial strains will be conducted by using MEGA-6 phylogenetic software and the expected product fragment size will be 1500bp.

Phylogenetic relationship will be find out by BLASTING these 1 6S rDNA sequences of rhizobial bacteria on NCBI.

PCR amplification and Phylogenetic investigations: The standard protocols as discussed by Sambrook et al., 2001 will be followed for amplification of genomic DNA. PCR will be performed in thermal cycler. Specie specific forward primer and reverse primer will be used for PCR amplifications. PCR master mix will constitute of 18µLwater, buffer containing MgCl2 2.5µL, dNTP (2.5 mM) 0.5µL, forward Primer (1 µmol/µL) 1µL, reverse primer (1 µmol/µL) 1 µL, DNA polymerase (2U/µL) 1µL, template (25 ng/µL) 1µL. The final PCR products will be resolved in 1.0% agarose. The gel will be stained with ethidium bromide and photographed on UV trans-illuminator. Data analysis and interpretation of results will be done by comparison of banding pattern generated from each strain. The tree plot will be constructed by UPGMA (Un-weighed pair group arithmetic average) to analyze the chronological separation of isolates. Preliminary phylogenetic analyses were performed using the neighbour-joining method (Saitou and Nei, 1987) using the MUST package (Philippe, 1993) to facilitate the selection of representative sequences (Abram, 2014).

Total collected samples

District  Name

Total sample

Nodule Plant




















No. of nodules separated from all Districts

District  Name

Total sample

Nodule Plant

 Total nodules

























Total Isolates

District  Name

Total sample

 Total Isolates




















VIII- References:

Ali, M.A., N. N. Nawab, G. Rasool and M. Saleem. 2008. Estimates of variability and correlations for quantitative traits in Cicer arietinum. J. Agric. Sci. 4: 177-179.

Boudewijns, M., J. M. Bakkers, P. D. J. Sturm, and W. J. G. Melchers. 2006. 16S rRNA gene sequencing and the routine clinical microbiology laboratory: a perfect marriage? J. Clin. Microbiol. 44:3469-3470.

Corbin, E.J., J. Brockwell and R.R. Gault. 1977. Nodulation studies on chickpea. Aust. J. Exp. Agr.17: 16-134.

Dudeja, S.S., R. Giri, R. Saini, M.P. Suneja, and E. Kothe. 2012. Interaction of endophytic microbes with legumes. J. Basic Microbiol. 52: 248-60.

Gao, J.L., J.G. Sun, E.T. Wang and W.X. Chen. 1994. Numerical taxonomy and DNA relatedness of tropical rhizobia isolated from Hainan Province, China. Int. J. Syst. Bacteriol. 44: 151–158.

Gilani, G.S., K.A. Cockel, and E. Sepehr. 2005. ‘Effects of ant nutritional factors on protein digestibility and amino acid availability in food’, J. AOAC Int. Vol. 88, No. 3, pp.967–987.

Hungria, M. and M.C.P. Neves. 1987. Cultivar and strain effect on nitrogen fixation and transport in Phaseolus vulgaris L. Pl and S. 103: 111-121.

Jeena, A.S., P.P. Arora and O.P. Ojha. 2005. Variability and correlation studies for yield and its components in chickpea. Leg. Resc. 28:146-148.

Laguerre, G., M.R. Allard, F. Revoy and N. Amarger. 1994. Rapid identification of rhizobia by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 60: 56–63. PMID.

Kaneko, T., Y. Nakamura, S. Sato, E. Asamizu, T. Kato, S. Sasamoto, A. Watanabe, K. Idesawa, A. Ishikawa, K. Kawashima, T. Kimura, Y. Kishida, C. Kiyokawa, M. Kohara, M. Matsumoto, A. Matsuno, Y. Mochizuki, S. Nakayama, N. Nakazaki, S. Shimpo, M. Sugimoto, C. Takeuchi, M. Yamada, and S. Tabata. 2000. Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res. 7331-338.

Rupela, O.P. 1992. Natural Occurrence and Salient Characters of Non-nodulating Chickpea Plants. Crop Sci. 32: 349-52.

Vincent, J.M. 1970. A manual for the practical study of root-nodule bacteria, I.B.P. Handbook No. 15. Blackwell Scientific Publications, Oxford. p. 44.

Wen, A., M. Fegan, C. Hayward, S. Chakraborty, and L. I. Sly. 1999. Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al., 1987) gen. nov., comb. nov. Int. J. Syst. Bacteriol. 49:567-576.

Winter, P., T. Pfaff, S. M. Udupa, B. Hüttel, P. C. Sharma, S. Sahi, R. A. Espinoza, F. Weigand, F. J. Muehlbauer and G. Kahl. 1999. Characterization and mapping of sequence tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Mol Gen Genet. 262: 90-101.

Yadav, S.S., J. Kumar, S.K. Yadav, S. Singh, V.S. Yadav, N.C. Turner and R. Redden. 2006. Evaluation of Helicoverpa and drought resistance in desi and kabuli chickpea. Pl. Genet. Res. 4: 198-203.

El Hassan, G. A., Hernandez, B. S. and Focht, D. D. (1986) Comparison of Hup trait and intrinsic antibi- otic resistance for assessing rhizobial competitiveness axenically and in soil. Appl. Environ. Microbiol. 51, 546-551.

Brewin, N. J., Wood, E. A. and Young, J. P. W. (1983) Contribution of the symbiotic plasmid to the com- petitiveness of Rhizobium leguminosarurn. J. Gen. Microbiol. 129, 2973-2977.

Haqqani, A.M., Zahid, M.A. and Malik, M.R. 2000. Legumes in Pakistan. Pages 98-128. In: Legumes in rice and wheat cropping systems of the Indo-Gangetic Plainsconstraints and opportunities. (Johansen, C., Duxbury, J.M., Virmani, S.M., Gowda, C.L.L., Pandes, S. and Josh, P.K. eds.). Int. Crops Res. Institute for the Semi-arid Tropics and Cornel Univ. Ithaca New York, USA. pp.230.

Van der Maesen L.J.G. (1987): Origin, history and taxonomy of chickpea. In: Saxena M.C., Singh K.B. (eds.): The Chickpea. CAB Inter., Wallingford: 11–34.

Duke, J.A. 1981. Handbook of legumes of world economic importance. Plenum Press, New York. p. 52-57.

Babic K H, Schauss K, Hai B, Sikora S, Redžepović S, Radl  V & Schloter M, Influence of different Sinorhizobium  meliloti inocula on abundance of genes involved in nitrogen  transformations in the rhizosphere of alfalfa (Medicago  sativa L.), Environ Microbiol, 10 (2008) 2922.

Abram, F.2014. Systems-based approaches to unravel multi-species microbial community functioning. Computational and Structural Biotechnology Journal 13 (2015) 24–32.

Aslam M, Mahmood IA, Peoples MB, Schwenke GD, Herridge DF. 2003. Contribution of chickpea nitrogen fixation to increased wheat production and soil organic fertility in rain-fed cropping. Biol Fert Soils 38:59-64.

AKDAĞ, C. and O. DÜZDEMİR. 2001. The Effects of Bacterial (Rhizobium spp.) Inoculation on Some Plant Characteristics of Chikpea at Different Growth-Development Stages. Turkish Journal Of Field Crops. 6 (2):61-63.

Andersen, M.M., X. Landes, W. Xiang, A. Anyshchenko, J. Falhof, J.T. Østerberg, L.I. Olsen, A.K. Edenbrandt, S.E. Vedel and B.J. Thorsen. 2015. Feasibility of new breeding techniques for organic farming. Trends in Plant Science. Volume 20, Issue 7, July 2015, Pages 426–434.

Bergersen, F.J., G.L. Turner, R.R. Gault, D.L. Chase and J. Brockwell. 1985. The natural abundance of 15N in an irrigated Soybean crop and its use for the calculation of nitrogen fixation. Australian Journal of Agricultural Research, 36: 411-423.

Bezdicek DF, Kennedy AC. 1998. In: JM Lynch and JE Hobbie (eds). Microorganisms in Action. Blackwell Scientific Publications.

Bohlool BB, Ladha JK, Garrity DP, George T. 1992. Biological nitrogen fixation for sustainable agriculture: a perspective. Plant Soil 141:1-11.

Caldwell, B.A.2005.Enzyme activities as a component of soil biodiversity: A review. Pedobiologia 49 (2005) 637—644.

Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al.QIIME allows analysis of high-throughput community sequencing data. Nat Methods 2010;7(5):335–6.

Cha, C., Gao, P., Chen, Y.C., Shaw, P.D., and Farrand, S.K. (1998) Production of acyl-homoserine lactone quorum sensing signals by gram-negative plant-associated bacteria. Mol Plant Microbe Interact 11: 1119–1129.

Cuvelier, M.L., Allen, A.E., Monier, A., McCrow, J.P., Messié, M., Tringe, S.G., Woyke, T., Welsh, R.M., Ishoey, T., Lee, J.H. et al. (2010). Targeted metagenomics and ecology of globally important uncultured eukaryotic phytoplankton. Proc. Natl.Acad. Sci. U.S.A.107, 14679–14684.

D’Angelo-Picard, C., Faure, D., Carlier, A., Uroz, S., Raffoux, A., Fray, R., and Dessaux, Y. (2004) Dynamics of bacterial populations in the rhizosphere of tobacco plants producing or not the quorum sensing signals hexanoyl- and 3-oxohexanoyl-homoserine lactone. FEMS Microbiol Ecol 51: 19–29.

Da Rocha, U.N., van Overbeek, L., and Van Elsas, J.D. (2009). Exploration of hitherto-uncultured bacteria from the rhizosphere. FEMS Microbiol. Ecol. 69, 313–328.

Duan, Y., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H;, Lin, H., Liu, L;, Vahling, C.M., Gabriel, D.W.,. et al. (2009). Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus liberibacter asiaticus’ oBTained through metagenomics. Mol. Plant Microbe Interact. 22, 1011–1020.

Dixon R, Kahn D. 2004. Genetic Regulation of Biological Nitrogen Fixation. Nature Rev Microbiol 2:621-631.

Elasri, M., Delorme, S., Lemanceau, P., Stewart, G., Laue, B., Glickmann, E., et al. (2001) Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp. Appl Environ Microbiol 67: 1198–1209.

Fatima Z., Aslam M and Bano A.,2008.  Chickpea nitrogen fixation increases production of subsequent wheat in rain fed system. Pak. J. Bot., 40(1): 369-376, 2008.

FAO. 2008. FAOSTAT. Food and agriculture organization of the United Nations.

FAO. 2011. Current world fertilizer trends and outlook to 2015. Rome: Food and agriculture organization of the United Nations.

Fierer N, Ladau J, Clemente JC, Leff JW, Owens SM, Pollard KS, et al. Reconstructing the microbial diversity and function of pre-agricultural tallgrass prairie soils in the United States. Science 2013;342(6158):621–4.

Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A, Perret X. 1997. Molecular basis of symbiosis between Rhizobium and legumes. Nature. 387:394-401.

Friesen ML, von Wettberg EJB, Badri M, Moriuchi KS, Barhoumi F, Cuellar-Ortiz C, Chang PL, Cordeiro MA, Vu WT, Arraouadi S, Djebali N, Zribi K, Badri Y, Porter SS, Aouani MA, Cook DR, Strauss SY, Nuzhdin SV. 2014. The ecological and genomics basis of salinity adaptation in Tunisian Medicago truncatula. BMC Genomics, 15:1160.

Fuqua, W.C., Winans, S.C., and Greenberg, E.P. (1994) Quorum sensing in bacteria: the LuxR/LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176: 269–275.

Garg N, Geetanjali. 2007. Symbiotic nitrogen fixation in legume nodules: process and signaling. A review. Agron Sustain Dev 27:59-68.

González, J.E. and and Melanie M. Marketon. 2003. Quorum Sensing in Nitrogen-Fixing Rhizobia. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2003, p. 574–592 1092-2172/03/$08.00.

González, J.E. and Keshavan, N.D. 2006. Messing with bacterial Quorum sensing. Microbiology and Molecular biology reviews. 70: 859–875

GoP (Government of Pakistan). 2006. Agricultural Statistics of Pakistan (district-wise)”. Economic Wing, Ministry of Food, Agriculture and Livestock,Islamabad.

GoP (Government of the Punjab). 2005. Punjab Development Statistics, Government of the Punjab, Lahore.

GoP. (Government of the Punjab). 2014. : Prequalification Document: Prequalification of Consulting Firms for Selection of Consultants  for Feasibility  Study for Bringing Wastelands of Thal, Pothohar and Cholistan Areas under Cultivation  through a Comprehensive Strategy.  Directorate General Agriculture (water management) Punjab, Lahore .

Ginolhac, A., Jarrin, C., Gillet, B., Robe, B., Pujic, P., Tuphile, K., et al. (2004) Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol 70:5522–5527.

Grunz, et al., 2014. Genome-based analysis of the transcriptome from mature chickpea root nodules  doi: 10.3389/fpls.2014.00325. 5:325( 1). Waggoner PE. 1994. How much land can ten million people spare for nature? Council for Agricultural Science and Technology Task Force. Report 121. Ames, Iowa.

Haroon MF, Hu S, Shi Y, Imelfort M, Keller J, Hugenholtz P, et al. Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature. 2013;500:567–70.

ICRISAT. “Chickpea” International Crops Research Institute for the Semi-Arid Tropics 2005.

Jensen ES, Hauggaard-Nielsen H. 2003. How can increased use of biological nitrogen fixation in agriculture benefit the environment? Plant Soil 252:177- 186.

Kaneko, T., Y. Nakamura, S. Sato, E. Asamizu, T. Kato, S. Sasamoto, A. Watanabe, K. Idesawa, A. Ishikawa and K. Kawashima. 2000. Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA research. 7 (6):331-338.

Khan, C.M. Anwar. 1968. Sand dune rehabilitation in Thal. Pakistan. Journal Range Management. 21 :316-321. SSP. 1968. Reconnaissance Soil Survey of Thal. Soil Survey of Pakistan, Lahore.

Khattak,S.G., Khan, D.F., Shah,S.H.,  Madan, M,S and Khan T. 2006. Role of Rhizobial inoculation in the production of chickpea crop. Soil & Environ. 25(2): 143-145.

Kielak, A., Rodrigues, J.L., Kuramae, E.E., Chain, P.S., van  Veen J.A., and Kowalchuk, G.A. (2010). Phylogenetic and metagenomic analysis of Verrucomicrobia in  former agricultural grassland soil. FEMS Microbiol. Ecol. 71, 23–33.

Kwon, S.-W., J.-Y. Park, J.-S. Kim, J.-W. Kang, Y.-H. Cho, C.-K. Lim, M.A. Parker and G.-B. Lee. 2005. Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences. International Journal of Systematic and Evolutionary Microbiology. 55 (1):263-270.

Laranjo M, Rodrigues R, Alho L, Oliveira S. 2001. Rhizobia of chickpea from southern Portugal: symbiotic efficiency and genetic diversity. J Appl Microbiol 90:662-667.

Laranjo M, Machado J, Young JPW, Oliveira S. 2004. High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region. FEMS Microbiol Ecol 48:101-107.

Laranjo M, Alexandre A, Rivas R, Velázquez E, Young JPW, Oliveira S. 2008. Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species. FEMS Microbiol Ecol 66:391-400.

Laranjo M, Rodrigues R, Alho L, Oliveira S. 2001. Rhizobia of chickpea from southern Portugal: symbiotic efficiency and genetic diversity. J Appl Microbiol 90:662-667.

Laranjo, M., Alexandre, A and Oliveira, S. 2014. Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus. Microbiological Research169 (2014) 2–17.

Langridge, P. and M.P. Reynolds. 2015. Genomic tools to assist breeding for drought tolerance. Current opinion in biotechnology. 32:130-135.

Lauro FM, DeMaere MZ, Yau S, Brown MV, Ng C, Wilkins D, et al. 2011. An integrative study of a meromictic lake ecosystem in Antarctica. ISME J. 5:879–95.

Lindhal, V., and Bakken, L.R. (1995) Evaluation of methods for extraction of bacteria from soil. FEMS Microbiol Ecol.16: 135–142.

L'Taief B, Sifi B, Gtari M, Zaman-Allah M, Lachaal M. 2007. Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Tunisia. Can J Microbiol 53:427-434.

Mahmood, K., M. Munir, and S. Rafique. 1991. Rainfed Farming Systems and Socio-economic Aspects in Kalat Division (Highland Balochistan). Pak. J. Agri. Soc. Sci., 5: 15-20.

Maâtallah J, Berraho E, Sanjuan J, Lluch C. 2002b. Phenotypic characterization of rhizobia isolated from chickpea (Cicer arietinum) growing in Moroccan soils. Agronomie 22:321-329.

Maâtallah J, Berraho EB, Munoz S, Sanjuan J, Lluch C. 2002a. Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco. J Appl Microbiol 93:531-540.

McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra,S.R., Camara, M., et al. (1997) Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143: 3703–3711.

Nandwani R and Dudeja SS. 2009. Molecular diversity of a native mesorhizobial population of nodulating chickpea (Cicer arietinum L.) in Indian soils. J Basic Microbiol 49:463-470.

Nour SM, Cleyet-Marel J-C, Normand P, Fernandez MP. 1995. Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. Int J Syst Bacteriol 45:640-648.

Nour SM, Fernandez MP, Normand P, Cleyet-Marel J-C. 1994. Rhizobium ciceri sp. nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.). Int J Syst Bacteriol 44:511-522.

Peoples MB, Crasswell ET. 1992. Biological nitrogen fixation: investments, expectations and actual contributions to agriculture. Plant Soil 141:13-39.

Peix, A., A. Rivas-Boyero, P. Mateos, C. Rodriguez-Barrueco, E. Martınez-Molina and E. Velazquez. 2001. Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biology and Biochemistry. 33 (1):103-110.

Philippe, H. (1993) MUST, a computer package of Management Utilities for Sequences and Trees. Nucleic Acids Res 21: 5264–5272.

Peters JW, Fisher K, Dean DR. 1995. Nitrogenase structure and function: a biochemical-genetic perspective. Annu Rev Microbiol 49:335-366.

Preheim SP, Perrotta AR, Friedman J, Smilie C, Brito I, Smith MB, et al. Computational methods for high-throughput comparative analyses of natural microbial communities. Methods Enzymol 2013;531:353–70.

Riaz, K., Elmerich, C., Moreira, D., Raffoux, A., Dessaux, Y., and Faure, D. (2008). A metagenomic analysis of soil bacteria extends the diversity of quorum-quenching lactonases. Environ. Microbiol. 10, 560–570.

Rivas R, Laranjo M, Mateos PF, Oliveira S, Martínez-Molina E, Velázquez E. 2007. Strains of  Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum. Lett Appl Microbiol 44:412-418.

Qureshi, M. A. Shakir, M.A., Naveed. M and M. J. Ahmad. 2009. Growth and yield response of chickpea to co-inoculation with mesorhizobium ciceri and bacillus megaterium. The Journal of Animal & Plant Sciences 19(4): 2009, 205-211 ISSN: 1018-7081.

Riesenfeld, C.S., Schloss, P.D., and Handelsman, J. (2004). Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38, 525–552.

Romdhane S, Aouani ME, Mhamdi R. 2007. Inefficient nodulation of chickpea (Cicer arietinum L.) in the arid and Saharan climates in Tunisia by Sinorhizobium meliloti biovar medicaginis. Ann Microbiol 57:15-19.

Saitou, N., and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.

Schloss PD,Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 2009;75(23):7537–41.

Shah, N.A., K.M. Aujla, M. Abbas and K. Mahmood. 2007. Economics of chickpea production in the Thal Desert of Pakistan. Pakistan J. Life and Social Sci. 5 (1-2):6-12.

Siddique KHM, Loss SP, Regan KL, Jettner RL. 1999. Adaptation and seed yield of cool season grain legumes in Mediterranean environments of southwestern Australia. Aust J Agr Res 50:375-387.

Tyler, H.L., Roesch, L.F., et al., 2009). Confirmation of the sequence of ‘Candidatus Liberibacter asiaticus’ and assessment of microbial diversity in Huanglongbing-infected citrus phloem using a metagenomic approach. Mol. Plant Microbe Interact. 22, 1624–1634.

Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ, Richardson PM, et al. Community structure and  metabolism through reconstruction of microbial genomes from the environment. Nature 2004;428:37–43.

Uroz, S., Dessaux, Y., et al., (2009). Quorum sensing  and quorum quenching: the yin and yang of bacterial communication. Chembiochem. 10, 205–216.

ance, C. P. (2001). Symbiotic nitrogen fixation and phosphorus acquisition. Plant nutrition in a world of declining renewable sources. Plant Physiology 127: 390-397.

Varshney, R.K., R. Terauchi and S.R. McCouch. 2014. Harvesting the promising fruits of genomics: applying genome sequencing technologies to crop breeding.

VBrockwell, J. and P. J. Bottomley (1995). Recent advances in inoculant technology and prospects for the future. Soil Biology and Biochemistry 27: 683-697.

Ventura, M., Turroni, F., Canchaya, C., Vaughan, E.E., O’Toole, P.W., et al., (2009). Microbial diversity in the human intestine and novel insights from metagenomics. Front. Biosci. 14, 3214–3221.

Velthof GL, Oudendag D, Witzke HP, Asman WAH, Klimont Z, Oenema O. 2009. Integrated assessment of nitrogen emissions form agriculture in EU-27 using MITERRA EUROPE. J Environ Qual 38:402–417.

Warschefsky, E., R.V. Penmetsa, D.R. Cook and E.J. von Wettberg. 2014. Back to the wilds: Tapping evolutionary adaptations for resilient crops through systematic hybridization with crop wild relatives. American journal of botany. 101 (10):1791-1800.

Wang, F., Zhou, H., Meng, J., Peng, X., Jiang, L., Sun, P., Zhang, C., Van Nostrand, J.D., Deng, Y., He, Z. et al. (2009). GeoChip-based analysis of metabolic diversity  of microbial communities at the Juan de Fuca Ridge hydrothermal vent. Proc. Natl. Acad. Sci. U.S.A.2009  106, 4840–4805.

Young JPW. 1992. Phylogenetic classification of nitrogen-fixing organisms. In Biological Nitrogen Fixation (Stacey, G. et al., eds), pp. 43-86, Chapman & Hall.

Zhang et al., 2012.Mesorhizobium muleiense sp. nov., nodulating with Cicer arietinum L. International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2737–2742. DOI 10.1099/ijs.0.038265-0.

Zhang et al., 2014. Genotypic alteration and competitive nodulation ofMesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils. Syst Appl Microbiol.37(7):520-524. doi: 10.1016/j.syapm.2014.07.004.

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