Selection of Effective Indigenous Rhizobium Strain for Seed Inoculation of Chickpea (Cicer aritenium L.) Production
Effective indigenous Rhizobium strains were selected and evaluated their ability for enhanced nodulation; grain yield and nutrient uptake of chickpea (Cicer aritenium L.) at eastern Uttar Pradesh region. Fifty Rhizobium strains were isolated from healthy chickpea root nodules. Eight Rhizobium strains were selected from out of fifty Rhizobium strains, on the basis of visual observations of effective nodules number and fast growth pattern on YEMA medium for field experiment. One reference strains USDA-3378 were selected for field experiment. In field experiment, the indigenous Rhizobium strain BHURC04 was found to be highly effective with significant increase in nodules number (73.53%) plant-1, dry weight of nodules (78.07%) plant-1 and grain (31.76%) and straw (24.37%) yields followed by BHURC05, USDA-3378 and BHURC03 over uninoculated control. Similarly uptake of N and P by grain (46.93 and 93.26%) and straw (47.01 and 79.56%), respectively maximum in seed inoculation of Rhizobium strain BHURC04 over uninoculated control. Indigenous Rhizobium strain BHURC04 was found to be highly effective symbiotic nitrogen fixer for uptake of nutrient content and grain yield of chickpea (Cicer arietinum L.).
Received: March 22, 2010;
Accepted: May 02, 2010;
Published: July 20, 2011
Chickpea (Cicer arietinum L.) is a leguminous crop. India is the largest
chickpea producing country accounting for 64% of the global chickpea production.
Pulses occupy a very important place in Indian diet because they constitute
the major source of protein to the predominantly vegetarian population. Many
legumes have the ability to form nitrogen (N2) fixing root nodules
with soil bacteria, collectively called rhizobia (Sprent,
2001) and thus contribute to the biological fixation of N2. Legume
and their rhizobia are often introduced to agricultural ecosystems to improve
soil fertility and farming systems flexibility (Brockwell
and Bottomley, 1995; Sessitsch et al., 2002).
Nitrogen is known to be an essential nutrient for plant growth and development.
Intensive farming practices that achieve high yield require chemical fertilizers,
which are not costly but may also create environmental problems. The extensive
use of chemical fertilizers in agriculture is currently under debate due to
environmental concern and fear for consumer health. Consequently, there has
recently been growing level of interest in environmentally friendly sustainable
agricultural practices and organic farming system (Rigby
and Caiceres, 2001; Lee and Song, 2007). Increasing
and extending the role of biofertilizers such as Rhizobium would reduce
the need for chemical fertilizers and decrease adverse environmental effects.
Therefore, in the development and implementations of sustainable agriculture
techniques, biofertilization is great importance in alleviating environmental
pollution and the deterioration of nature (Elkoca et
Rhizobium symbiosis with legumes species is of special importance, producing
50% of 175 million tons of total biological nitrogen fixation annually worldwide
(Sarioglu et al., 1993). Chickpea and Rhizobium
leguminosarum sub sp. ciceri association annually produce up to 176
kg N ha-1 depending on cultivar, bacterial strain and environmental
factors (Rupela and Saxena, 1987; Beck
et al., 1991). However, Rhizobium species producing nodules
in chickpea are specific only to this species and thus inoculation with effective
strains is advised in soils with no or weak bacterial presence (Rupela
and Saxena, 1987). The symbiosis between chickpea and its specific rhizoia
has been recently studied in several countries-Tunisia (Aouani
et al., 2001), Morocco (Maatallah et al.,
2002), Canada (Kyei-Boahen et al., 2002), Turkey
(Icgen et al., 2002) and Portugal (Laranjo
et al., 2002)-because of the promising agricultural usefulness of
this crop as a grain legume for human and animal nutrition and because of the
interesting extreme host specificity of its rhizobia. The most studied and longest
exploited PGPR are the rhizobia (including the Allorhizobium, Azorhizobium,
Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium)
for their ability to fix N2 in their legume hosts (Sawada
et al., 2003). The symbiotic association is highly specific in chickpea,
with a unique group of rhizobia necessary for formation of nodules and nitrogen
fixation. Nodulation problem in chickpea attributed to the rhizobial symbiont
may be due to absence of appropriate strains, low population, low ineffectiveness,
poor survival in soil, or competition amongst strains of rhizobia. Inoculation
trials in the eastern region of U.P. indicate yield response of cultivars to
application of appropriate rhizobial strains, but the importance of adequate
strain testing for their suitability and effectivity prior to an inoculation
effort in a given aria is emphasized. The objective of the current work was
selection of effective indigenous Rhizobium strain for seed inoculation
of chickpea (Cicer aritenium L.) production.
MATERIALS AND METHODS
Isolation of indigenous Rhizobium strains: Effective root nodules
were collected from chickpea rhizosphere soils of 50 villages from Varanasi,
Mirzapur, Jaunpur and Azamgarh districts of Uttar Pradesh during December, 2006.
The uprooted plants were washed in running water carefully and healthy nodules
were separated from root with the help of blade and forceps. We sterilized nodule
sample by soaking them in 0.1% HgCl2 for 3 min and in 70% ethyl alcohol
for 1 min, followed by rinsing in sterile distilled with sterilized water of
each step. The well surface sterilized nodules were kept in to sterilized glass
vial containing 90 μL sterile water and crushed with the help of sterilized
glass rod and made serial dilution up to 10-7 was made (Schmidt
and Coldwell, 1967) and spread on yeast extract mannitol agar (YEMA) medium
(Vincent, 1970). After spreading, Petri dishes were kept
in B.O.D. incubator for 2 to 3 days at 28±2°C. Further, sub-culturing
of milky and sticky colonies on other new YEMA medium was done by the streaking
method to find single and pure colonies of Rhizobium strain. The Rhizobium
strains were isolated and transferred on YEMA containing test tube and stored
at 4°C in the refrigerator. Eight Rhizobium strains were selected
from out of 50 Rhizobium strains, on the basis of visual observations
of effective nodules number and colour on the root and fast growth pattern on
YEMA medium for further testing in field conditions.
Culture, media and growth condition: Effective nitrogen fixing Rhizobium strain BHURC01, BHURC02, BHURC03 BHURC04, BHURC05, BHURC06, BHURC07, BHURC08 one reference strains USDA-3378 were grown in yeast extract mannitol (YEM) broth medium. Mesorhizobium ciceri strain USDA 3278 was obtained from Agricultural research services patent culture collection, United States Department of Agriculture, America. The culture were maintained by periodic transfer and stored in the refrigerator for further studies. All media component was purchased from Himedia Pvt Ltd., Mumbai.
Host seeds: Seeds of chickpea (Cicer arietinum L.) cv. Radhey were obtained from Indian Institute of Pulse Research (IIPR), Kalyanpur, Kanpur, Uttar Pradesh, India.
Seed bacterization: The Rhizobium strains were grown in YEM broth by incubation for 120 rpm at 28±2°C for 48 h. Healthy seeds weighed for each plot of 5 m2 (@ 100 kg ha-1) were separately inoculated as per treatments in plastic bags with 5 mL of 7 days old broth cultures grown in specific media of respective I noculants (mixed in 1:1 ratio for combined treatments) along with 1 mL of 1% (w/v) sticker solution of gum acacia to ensure bacterial population in the range of 107 to 108 CFU seed-1. After drying for one hour in shade, uninoculated seeds were sown first followed by inoculated seeds just to avoid contamination.
Field experiment for selection of indigenous Rhizobium strain:
Field experiments, one for selection of most appropriate indigenous Rhizobium
strain were conducted during winter season of October to March, 2006-2007
at Agricultural Research Farm, Banaras Hindu University, Varanasi. The first
experiment was conducted with 10 treatments and 3 replication of 8 Rhizobium
strain (BHURC01, BHURC02, BHURC03 BHURC04, BHURC05, BHURC06, BHURC07, BHURC08)
and an exotic strain (USDA-3378) and one uninoculated control on chickpea (Cicer
arietinum L.) cv. Radhey. The plot size was 12 m2 and spacing
25 cm between row and 10 cm between plants. The physico-chemical properties
of initial soil of filed experiment was sandy clay loam in texture with 40.83%
water holding capacity, neutral in reaction (pH 7.25) and electronic conductivity
0.155 dS m-1, organic carbon (0.778%) (Walkley
and Black, 1934); available N (213.24 kg ha-1) (Subbiah
and Asija, 1956); P2O5 (27.22 kg ha-1)
(Olsen, 1954) and K2O (254.76 kg ha-1) (Jackson,
1967); respectively in soil. The microbial population of total bacteria,
fungi and actinomycetes (4.5x10-8, 3.1x10-8 and 3.4x10-8
CFU g-1 soil) (Aneja, 2003), respectively.
Assessment of nodulation plant growth and yield: Study of nodulation and plant growth attributes was done 70 days after sowing (DAS). Five plants were randomly uprooted from each plot. Plant roots with the lump of the soil were washed in running water. Nodules were separated carefully. Different parameters like number of nodules and oven dried weight of nodules root and shoot plant-1 were recorded. Physiologically matured crop of both experiments was harvested during March.
Chemical analysis plant sample: The nodules, plants and grains samples
were digested in 4:1 of HNO3: HClO4 for total P (Vanadomolybdophosphoric
acid yellow colour methods) (Jackson, 1967) in diacid mixture
of 9:1 of H2SO4: HClO4 for the analysis of
total N (Nesslers reagent method) (Jackson, 1973)
and Fe in nodules (Atomic Absorption Spectrophotometer method) (Jackson,
Experimental design: The experiment was arranged in a randomized block design and was replicated three times. Statistical analysis was conducted using one-way Analysis of Variance (ANOVA) using SPSS 12.0 software. Comparisons of means were performed by the Fishers Protected LSD test at p≤0.05.
Effect of indigenous Rhizobium strain on nodulation, growth and yields: Results pertaining to performance of different indigenous rhizobial isolates used in field conditions had shown variability in nodulation and plant growth (70DAS) and yield of chickpea at harvesting stage (Table 1). The isolate BHURC04 was found to be most effective with maximum values of nodules number (73.53%) and dry weight (78.07%) of nodules dry weight of root (44.00%) and shoot (69.47%) per plant followed by reference strain USDA-3378 and BHURC05 significant increase over uninoculated control. The high nodulation and dry matter accumulation have shown competency of these three isolates in establishing symbiosis for nitrogen fixation which was very much conform from the iron content of 30.2, 26.95 and 24.69%, as the major constituent of nitrogenase enzyme in nodules caused by BHURC04, USDA-3378 and BHURC05, respectively. Therefore the stains BHURC04, USDA-3378 and BHURC05 showed significantly higher grain yield (31.76, 30.64 and 30.16%), respectively and straw yield BHURC04, USDA-3378 and BHURC05 (24.37, 26.02 and 19.17%), respectively as compare to control (Table 1). The other isolates which could not show better response in terms of nodulations growth and yield might be due to their competition for nutrition with native Rhizobium or their poor survivability and low infectivity in the soil.
Effect of indigenous Rhizobium strain on content and uptake of N
and P: The effect of indigenous Rhizobium strain on the fractions
of N and P (Table 2) was varying in the range of 3.50 to 4.06%
and 0.31 to 0.52% in grains and 0.44 to 0.56% and 0.11 to 0.21% in straw, respectively.
The maximum significant uptake of N and P in grain (46.93 and 93.26%) and straw
(79.56 and 47.01%), respectively over uninoculated control at harvesting stage
was recorded in Rhizobium strain BHURC04, followed by reference strain
USDA-3378 and BHURC05.
|| Effect of indigenous Rhizobium strains on nodulation,
growth and yield of chickpea
|HURC: Indigenous Rhizobium strain; USDA-3378, Exotic
strain of Rhizobium; wt, dry weight; g, gram; Fe, iron; Values are
the mean±SD, Mean values in each column with the same superscript
(s) do not differ significantly by LSD (p≤0.05)
|| Effect of indigenous Rhizobium strains on content
and uptake of N and P by chickpea
|Values are the mean±SD, Mean values in each column
with the same superscript (s) do not differ significantly by LSD (p≤0.05)
The significant accumulation of N-fraction and its uptake by grain and straw
relative to uninoculated control and some of the other inoculated treatment
were excellent indicator of symbiotic effectiveness with at par values of isolates
in order to BHURC04>USDA-3378>BHURC05>BHURC03>BHURC02. The relative
differences of N-accumulation in grains by these isolates were 46.69, 42.2,
41.26, 25.57 and 17.11% over uninoculated control. Considering all the above
facts, the Rhizobium strain BHURC04, USDA-3378 and BHURC05 were considered
to be effective and most synergistic seed inoculation of chickpea.
The plant rhizosphere is a major soil ecological environment for plant microbe interactions involving colonization of different microorganisms in and around the roots of the growing plant. This colonization may either result in associative, symbiotic, neutralist or parasitic interactions, depending upon the plant nutrient status in the soil environment.
Seed inoculation of chickpea with Rhizobium strains BHURC04 was found more significant nodulation, biomass production and grain yield over uninoculated control.
Rudresh et al. (2005) has been reported that
inoculation of seed with Rhizobium has been enhanced nodulation, growth
and yields response of legumes. The similar pattern of grain and straw yields
of chickpea was also observed. Increase in gain and straw yield of chickpea
due to effective Rhizobium inoculation has been also reported by Romdhane
et al. (2007) and Wani et al. (2007).
More significant nodule number and nodule dry weight were responsible for significant
increased in yield of grain and straw due to high rate of atmospheric nitrogen
fixation and its translocated in grain and straw. This finding agreed with the
finding of Kyei-Boahen et al. (2002) who have found
positive correlation between more number and weight of effective nodules and
yield of chickpea. Thibodeau and Jaworski (1975) have
reported that nitrate reductase activity peaks at early flowering and decreases
very rapidly at pod filling stage, while nitrogenase peaks at early pod filling
with a less rapid decline in activity. Rhizobium strains BHURC04 was
found more significant content of nitrogen and phosphorus in grain and straw,
respectively and also increase uptake of nitrogen and phosphorus in grain and
straw, respectively followed by USDA-3378 and BHURC05 as compare to uninoculated
control. Synergestic relationship was found between N and P uptake by grain
and straw of chickpea as also reported by Andrew (1977),
Arya et al. (2007) and Verma
et al. (2010) in legumes.
In this study, isolation and selection of indigenous Rhizobium strain BHURC04 was found to be highly effective symbiotic nitrogen fixer followed by USDA-3378 and BHURC05 for uptake of nutrient content and grain yield of chickpea (Cicer arietinum L.). Overall study of field experiment showed that Rhizobium strain BHURC04 may be effective symbiotic nitrogen fixing bacteria for chickpea production.
We thank my co-supervisor Dr. Janardan Yadav, Associate Professor in Department of Soil Science and Agricultural Chemistry for providing a laboratory and field carry out this research work at organic farm, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
1: Andrew, C.S., 1977. Nutritional restraints on legume symbiosis: Exploiting the legume-Rhizobium symbiotic in tropical agriculture. Coll. Trop. Agric. USA., 145: 253-274.
2: Aneja, K.R., 2003. Experiments in Microbiology, Plant pathology and Biotechnology. 4th Edn., New Age International (P) Ltd., India, ISBN-13: 9788122414943, Pages: 632
Direct Link |
3: Aouani, M.E., R. Mhamdi, M. Jebara and N. Amarger, 2001. Characterization of rhizobia nodulating chickpea in Tunisia. Agronomie, 21: 577-581.
4: Arya, R.L., J.G. Varshney and L. Kumar, 2007. Effect of integrated nutrient application in chickpea and Mustard intercropping system in semi-arid tropics of north India. Commun. Soil Sci. Plant Anal., 38: 229-240.
5: Beck, D.P., J. Wery, M.C. Saxena and A. Ayadi, 1991. Dinitrogen fixation and nitrogen balance in cool-season food legumes. Agron. J., 83: 334-341.
Direct Link |
6: Brockwell, J. and P.J. Bottomley, 1995. Recent advances in inoculant technology and prospects for the future. Soil Biol. Biochem., 27: 683-697.
7: Icgen, B., G. Ozcengiz and N.G. Alaeddinoglu, 2002. Evaluation of symbiotic effectiveness of various Rhizobium cicer strain. Res. Microbiol., 153: 369-372.
8: Elkoca, E., F. Kantar and F. Sahin, 2007. Influence of nitrogen fixing and phosphorus solubilizing bacteria on the nodulation, plant growth and yield of chickpea. J. Plant Nutr., 31: 157-171.
CrossRef | Direct Link |
9: Jackson, M.L., 1967. Soil Chemical Analysis. 1st Edn., Prentice Hall of India Pvt. Ltd., New Delhi, India
10: Jackson, M.L., 1973. Soil Chemical Analysis. 2nd Edn., Prentice Hall of India Pvt. Ltd., New Delhi, India
Direct Link |
11: Kyei-Boahen, S., A.E. Slinkard and F.L. Walley, 2002. Evaluation of rhizobial inoculation methods for chickpea. Agron. J., 94: 851-859.
CrossRef | Direct Link |
12: Laranjo, M., C. Branco, R. Soares, L Alho, M.D. Carvalho and S. Oliveira, 2002. Comparison of Chickpea rhizobia isolates from diverse portuguese natural populations based on symbiotic effectiveness and DNA fingerprint. J. Applied Microbiol., 92: 1043-1050.
13: Lee, J.Y. and S.H. Song, 2007. Evaluation of groundwater quality in coastal areas: Implications for sustainable agriculture. Environ. Geol., 52: 1231-1242.
14: Maatallah, J., E.B. Berraho, S. Munoz, J. Sanjuan and C. Lluch, 2002. Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco. J. Applied Microbiol., 93: 531-540.
15: Rigby, D. and D. Caceres, 2001. Organic farming and the sustainability of agricultural systems. Agric. Syst., 68: 21-40.
CrossRef | Direct Link |
16: Romdhane, S.B., F. Tajini, M. Trabelsi, M.E. Aouani and R. Mhamdi, 2007. Competition for nodule formation between introduced strains of Mesorhizobium ciceri and the native populations of rhizobia nodulating chickpea (Cicer arietinum) in Tunisia. World J. Microbiol. Biotechnol., 23: 1195-1201.
17: Rudresh, D.L., M.K. Shivaprakash and R.D. Prasad, 2005. Effect of combined application of Rhizobium, phosphate solubilizing bacterium and Trichoderma spp. on growth, nutrient uptake and yield of chickpea (Ciceraritenium L.). Applied Soil Ecol., 28: 139-146.
18: Rupela, O.P. and M.C. Saxena, 1987. Nodulation and Nitrogen Fixation in Chickpea. In: The Chickpea, Saxena, M.C. and K.B. Singh (Eds.). CAB International, Wallingford, Oxon, pp: 191-206
19: Sarioglu, G., S. Ozcelik and S. Kaymaz, 1993. Selection of effective nodosity bacteria (Rhizobium leguminosarum biovar. viceae) from lentil grown in Elazıg. Turk. J. Agric. For., 17: 569-573.
20: Sawada, H., L.D. Kuykendall and J.M. Young, 2003. Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts. J. Gen. Applied Microbiol., 49: 155-179.
CrossRef | Direct Link |
21: Schmidt, E.L. and A.C. Coldwell, 1967. A Practical Manual of Soil Microbiology, Laboratory Methods. FAO., Rome
22: Sessitsch, A., J.G. Howieson, X. Perret, H. Antoun and E. Martinez-Romero, 2002. Adavances in Rhizobium research. Crit. Rev. Plant Sci., 21: 323-378.
23: Sprent, J.I., 2001. Nodulation in legumes royal botanic gardens Kew, London. Ann. Botany, 89: 797-798.
24: Subbiah, B.V. and G.L. Asija, 1956. A rapid procedure for estimation of available N in soils. Curr. Sci., 25: 149-153.
25: Thibodeau, P.S. and E.G. Jaworski, 1975. Patterns of nitrogen utilization in the soybean. Planta, 127: 133-147.
26: Verma, J.P., J. Yadav and K.N. Tiwari, 2010. Application of Rhizobium sp. BHURC01 and plant growth promoting rhizobactria on nodulation, plant biomass and yields of Chickpea (Cicer arietinum L.). Int. J. Agric. Res., 5: 148-156.
CrossRef | Direct Link |
27: Vincent, J.M., 1970. A Manual for the Practical Study of the Root Nodule Bacteria. International Biological Programme. Blackwell Scientific Publications, Oxford
28: Walkley, A. and I.A. Black, 1934. An examination of the degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Sci., 37: 29-38.
CrossRef | Direct Link |
29: Wani, P.A., M.S. Khan and A. Zaidi, 2007. Synergistic effects of the inoculation with nitrogen-fixing and phosphate-solubilizing rhizobacteria on the performance of field-grown chickpea. J. Plant Nutr. Soil Sci., 170: 283-287.
CrossRef | Direct Link |