Abstract: Field-based experiments were conducted to evaluate the combined application of Rhizobium sp. BHURC01 and plant growth promoting rhizobacteria on chickpea production in two consecutive years. A positive influence of plant growth promoting rhizobactria and Rhizobium sp. BHURC01 on nodulation, plant biomass, nitrogen and phosphorus in nodule, grain and straw and yield related parameter were recorded in two year of field experiments. The maximum significant increase in nodule number, dry weight of nodule, root and shoot were recorded in co-inoculation of Rhizobium sp. BHURC01 and Pseudomonas fluorescens followed by co-inoculation of Rhizobium sp. BHURC01, Azotobacter chroococcum and Bacillus megaterium over uninoculated control in two year of field study while, nitrogen and phosphorus content increase in nodules, grain and straw. The Rhizobium sp. BHURC01 and P. fluorescens showed significant increase in all parameter due to more available nitrogen by Rhizobium sp. BHURC01 and more available of phosphorus, iron and plant hormones like indole-3-acetic acid (IAA) and antifungal activity by P. fluorescens in comparison of B. megaterium and A. chroococcum. Therefore, co-inoculation of Rhizobium sp. BHURC01 and P. fluorescens could be effective biofertilizer for chickpea (Cicer arietinum L.) production.
INTRODUCTION
The plant rhizosphere is an important soil ecological environment for plant-microbe interactions. It involves colonization by a variety of micro-organisms in and around the roots which may result in symbiotic, associative, neutralistic or parasitic relations within the plant, depending on the type of microorganism, soil nutrient status and plant defense system and soil environment. Due to excessive use of the chemical fertilizers and plant protection chemicals, the rhizosphere microflora has been greatly affected and in place of the beneficial associative bacteria, harmful types now predominate in the rhizosphere. Therefore, more attention being paid to the search for early root colonizers which directly or indirectly influence plant growth and productivity. Rhizobacteria from the rhizosphere and rhizoplane of healthy chickpea (Cicer ariectinum L.) plants with growth-promoting and pathogenic-suppressive abilities were isolated and identified as species of Pseudomonas and Bacillus (Parmar and Dadarwal, 1999).
The use of mixed biofertilizers is advocated to get the maximum benefits due to additive and synergistic effect. The role of symbiotic nitrogen fixing bacteria, Plant Growth Promoting Rhizobacteria (PGPR) and phosphate solubilizing microorganisms in crop productivity is well documented (Kennedy et al., 2004; Hariprasad and Niranjana, 2009). The PGPR play a crucial role in soil health and plant growth and have been used for biocontrol of plant pathogens. But the studies regarding coinoculation of Mesorhizobium sp. with phosphate solubilizer or/and PGPR in the presence/absence of inorganic fertilizers under field conditions are scarce. Co-inoculation studies with PGPR and Rhizobium, Bradyrhizobium sp. have shown to increase root and shoot weight, plant vigor, nitrogen fixation and grain yield in various legumes (Valverde et al., 2006; Yadegari et al., 2008). Phosphate-solubilizing Bacillus sp. stimulates plant growth through enhanced P nutrition and increasing the uptake of N, P, K and Fe (Biswas et al., 2000). Combined inoculation of Rhizobium with Pseudomonas striata or Bacillus polymyxa and with Bacillus megaterium have shown increased dry matter, grain yield and phosphorus uptake significantly over the uninoculated control in legumes (Elkoca et al., 2008).
Chickpea (Cicer arietinum L.) is a major grain legume crop important pulse crop. It is contributes to 38% of national pulse production in India. Chickpea can obtain a significant portion of its N requirement through symbiotic N2-fixation to give high grain yield when grown in association with effective and competitive Rhizobium strain (Stephen et al., 2002). Interactions between these PGPR with Rhizobium may be antagonistic or synergistic and the beneficial effects of such interactions could be exploited for economic grain (Dubey, 1996). The objective of the present study was evaluation of chickpea (Cicer aritenium L.) seed inoculation with indigenous Rhizobium strain and plant growth promoting rhizobacteria on nodulation, plant growth and yield.
MATERIALS AND METHODS
Culture, Media and Growth Condition
The pure cultures of Azotobacter chroococcum strain MTCC-446 was
obtained from MTCC (microbial technology culture collection), Institute of Microbial
Technology, Chandigarh, Punjab, India. Pseudomonas fluorescens strain
BHUPSB06 and Bacillus megaterium strain BHUPSB14 was isolated from rhizosphere
soil of eastern Uttar Pradesh region at September, 2006. Rhizobium sp.
BHURC01 was isolated from root nodules of chickpea plant from Eastern Uttar
Pradesh. Pseudomonas fluorescens strain BHUPSB06, B. megaterium strain
BHUPSB14 and Rhizobium sp. strain BHURC01 were characterized by biochemical
and molecular methods. 16S rDNA gene sequencing had been done and sequence deposited
in NCBI with different accession number GU124814 (P. fluorescens strain
BHUPSB06), GU124821 (B. megaterium strain BHUPSB14) and GU124816 (Rhizobium
sp. strain BHURC01). The bacterial strains of A. chroococcum, P.
fluorescens and B. megaterium were maintained on nutrient agar medium.
Rhizobium sp. strain BHURC01 was maintained on Yeast Extract Mannitol
Agar (YEMA) medium. The pure fungal culture of Fusarium oxysporum and
Rhizoctonia solani were obtained from Department of Plant Pathology and
Mycology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi.
The culture were maintained by periodic transfer and stored in the refrigerator
for further studies.
In vitro Screening of Bacterial Strain for their Plant Growth Promoting
Activities
The IAA (indole-3-acetic acid) production was estimated by the modified
method as described by Brick et al. (1991). Phosphate
solubilization was screened on Pikovskaya's agar plates as described by Gaur
(1990). NH3 production was tested in peptone water by the method
of Cappuccino and Sherman (1992). Siderophore production
was analyzed on the Chrome azurol S agar medium (Sigma, Pvt. Ltd.) as described
by Schwyn and Neilands (1987). The HCN production was
determined on nutrient agar plates supplemented with glycine (4.4 g L-1)
as described by Jha et al. (2009). Antifungal
assay was determined by the method of Rajendran et al.
(2008).
Host Seeds
Seeds of chickpea (Cicer arietinum L.) cultivar (C-235) were obtained
from Indian Institute of Pulse Research (IIPR), Kalyanpur, Kanpur, Uttar Pradesh,
India.
Seed Bacterization
The Rhizobium sp. BHURC01 was grown in YEM broth and A. chroococcum,
B. megaterium and P. fluorescens were grown in Nutrient broth
by incubation for 120 rpm at 28±2°C for 48 h. Healthy seeds weighed
for each plot of 5 m2 (at 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 inoculants (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 colony
forming unit (cfu) seed-1. After drying for 1 h in shade, uninoculated
seeds were sown first followed by inoculated seeds just to avoid contamination.
Field Experiments
The field experiments were set up in the first week of October 2006 to March,
2007 (first-year experiment) and October 2007 to March, 2008 (second-year experiment)
at Agricultural Research Farm, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi Uttar Pradesh, India. The field experiments were conducted
with 5 treatments and 3 replication of indigenous Rhizobium sp. strain
BHURC01 and appropriate synergistic combination of Plant Growth Promoting Rhizobacteria
(PGPR) as (A. chroococcum P. fluorescens and B. megaterium) and
one uninoculated control. The plot size was 10x2 m and spacing 25x25 cm between
row and 10x10 cm between plants. The physico-chemical properties of the initial
soil of experimental field was sandy clay loam in texture with 40.83% water
holding capacity, neutral in reaction (pH 7.25) and electronic conductivity
(dS m-1) 0.155. The content of 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 et al., 1954) and K2O (254.76
kg ha-1) (Jackson, 1973), 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), respectively.
Assessment of Nodules Number, Dry Weight of Root and Shoot, Yield
For assessment of root shoot dry weight, ten plants were randomly uprooted
at flowering time (80 days after sowing) from inoculated and control plots and
washed several time with running water. First roots were cut off and root and
shoots were dried at 70°C for 72 h. separately, for each plant. For nodules
number, another ten plants were uprooted from control and inoculated plots.
The nodules were separated and counted from each plant and dry weight was recorded
after drying the nodules at 70°C for 72 h. The ends of the growth period,
another ten plants were randomly harvested from control and inoculated plots
to determine yield parameters. The plants were dried at 70°C for 72 h and
weighted for calculating the biological yield. Grain weight was also recorded
at the time of harvesting.
Determination of Nitrogen and Phosphorus in Nodules, Grain and Straw
For determination of nitrogen and phosphorus in nodules, ten representative
plants from control and inoculated plots at flowering time were taken. The dry
nodules (0.2 g), grains (0.2 g) and straw (0.5 g) samples were digested in 10
mL of 4.1 ratio of HNO3: HClO4 for total P (Vanadomolybdophosphoric
acid yellow color methods) (Jackson, 1967) in 10 mL diacid
mixture of 9:1 ratio of H2SO4: HClO4 for the
analysis of total N (Nessler’s reagent method) (Jackson,
1973). Protein content in grain was calculated according to the formula:
nitrogen (%) x6.25 (conversion factor).
Experimental Design
The experiment was arranged in a randomized block design and was replicated
four times. Statistical analysis was conducted using One-Way Analysis of Variance
(ANOVA). Comparisons of mean were performed by the Least Significant Deferent
(LSD) test at p≤0.05 by using SPSS software version 12.0.
RESULTS AND DISCUSSION
In vitro Screening Test of Bacterial Strain for their Plant Growth
Promoting Activity
Rhizobium sp. strain BHURC01, P. fluorescens BHUPSB06,
A. chroococcum and B. megaterium BHUPSB14 were found to be positive
for ammonia, IAA production (Table 1). Only P. fluorescens
BHUPSB06 was found to be positive for HCN, siderophore production and positive
for inhibition of growth of soil born phytophatogen (Fusarium oxysporum
and Rhizoctonia solani). Tryptophan is a precursor of IAA biosynthesis.
Pseudomonas fluorescens showed significant increase concentration of
IAA as compared to other PGPR strains when tryptophan was added as the precursor.
In broth, IAA production in bacterial culture varies from 35.11 to 114.19% at
100 μg mL-1 tryptophan after 72 h incubation. IAA production
showed more significant in P. fluorescens (114.19%), Rhizobium sp.
(70.37%) and A. chroococcum (35.11%) as compare with B. megaterium
at 100 μg mL-1 tryptophan after 72 h incubation. Phosphate solubilization
was most frequently encountered by P. fluorescens followed by B. megaterium
least by Rhizobium sp. and A. chroococcum. Pseudomonas fluorescens
produces largest halos, 20 mm (approx.) around their colonies within 5 days
of incubation than others isolates. Bacillus megaterium produces 15 mm
(approx.) halo zone around colonies on Pikovaskaya medium. However, production
of ammonia was a common trait in all isolates of bacteria. Experimental results
revealed that P. fluorescens is capable of siderophore and HCN production;
where as other strains were not.
| Table 1: | In vitro screening test of bacterial strain for their plant growth promoting activity |
+: Positive, -: Negative test, PSB: Phosphate solubilizing Bacteria; NH3: Ammonia production; HCN: Hydrogen cyanide, IAA-Indole Acetic Acid (μg mL-1±SD). Data are average values of three replicates±SD. Mean with different letter(s) in the same column differ significantly at p≤0.05 according to Fisher’s Protected LSD | |
| Table 2: | Effect of Plant Growth Promoting Rhizobacteria (PGPR) and Rhizobium sp. BHURC01 inoculation on nodule related parameters of chickpea in field experiments of two years |
Values are the Mean±SD. Mean values in each column with the same letter(s) do not differ significantly by LSD (p=0.05) | |
Pseudomonas fluorescens also showed mycelial growth inhibition against Fusarium oxysporum and Rhizoctonia solani.
Effect of Plant Growth Promoting Rhizobacteria (PGPR) and Rhizobium sp.
BHURC01 Inoculation on Growth of Nodule
The three PGPR isolates (A. chroococcum, P. fluorescens and
B. megaterium) did not antagonize Rhizobium sp. BHURC01 when grown
together on plates. The Rhizobium sp. BHURC01 interacted differentially
with PGPR isolates and showed significant variation in nodulation, dry weight
of nodules (Table 2). Dual inoculation of seed with Rhizobium
sp. BHURC01 and P. fluorescens, B. megaterium and A. chroococcum
were produced 30.77, 19.23 and 11.54%, respectively, more nodule number in first
year experiment and 36.21, 24.14 and 6.90%, respectively, more nodule in second
year experiment than Rhizobium strain inoculation alone. The maximum
significant increased nodule dry weight 44.53 and 37.6%, nitrogen 39.37 and
47.84% and phosphorus in nodules 13.91 and 19.15% in the first and second year
study, respectively in combination of Rhizobium sp. BHURC01 and P.
fluorescens, followed by combination of Rhizobium sp. BHURC01, B.
megaterium and A. chroococcum over uninoculated control.
Effect of PGPR and Rhizobium sp. BHURC01 Inoculation on Biomass Production
and Yield
Table 3 shows that co-inoculation of seed with Rhizobium
sp. BHURC01 and P. fluorescens showed significant increase dry weight
of root 23.66 and 30.23% in first and second year of field study, respectively
followed by combination of Rhizobium sp. BHURC01, B. megaterium and
A. chroococcum as compare with uninoculated control. Similarly, shoot
dry weight was more increased significant, 46.56 and 79.46% in the first year
and second year of field experiment, respectively. The higher grain yield 37.26
and 23.64% and straw yield 27.95 and 21.70% in first and second year of field
study, respectively was increased significantly in co-inoculation of Rhizobium
sp. BHURC01 with P. fluorescens followed by B. megaterium and
A. chroococcum over uninoculated control (Table 3).
Effect of PGPR and Rhizobium sp. BHURC01 Inoculation on Nitrogen
and Phosphorus Content in Grain and Straw and Grain Protein
The maximum nitrogen in grain 31.75 and 39.67% and in straw 55.17 and 48.38%
in first and second year of study, respectively, while maximum significant phosphorus
in grain 52.78 and 34.21% and in straw 36.04 and 33.04% in inoculation of Rhizobium
sp. BHURC01 and P. fluorescens over uninoculated control (Table
4).
| Table 3: | Effect of PGPR and Rhizobium sp. BHURC01 inoculation on biomass production and yield-related parameters of chickpea in field experiments of two years |
Values are the Mean±SD. Mean values in each column with the same letter(s) do not differ significantly by LSD (p=0.05) | |
| Table 4: | Effect of PGPR and Rhizobium sp. BHURC01 inoculation on nitrogen and phosphorus content in grain and straw of chickpea in field experiments of two consecutive years |
Values are the Mean±SD. Mean values in each column with the same letter(s) do not differ significantly by LSD (p=0.05) | |
The protein content in grain was also increased significant 20.16 and 25.45% at first and second year of field study, respectively in co-inoculation of Rhizobium sp. BHURC01 with P. fluorescens followed by B. megaterium and A. chroococcum over uninoculated control.
DISCUSSION
The IAA production showed significant increase in P. fluorescens, Rhizobium sp. and A. chroococcum as compare with B. megaterium. Similar high level of IAA production was recorded in Pseudomonas by Ahmad et al. (2008). Wani et al. (2007) have been reported Rhizobium sp. increased IAA production by 79, 98 and 152% over Bacillus PSB1, Bacillus PSB10 and A. chroococcum A4, respectively. Generally, the IAA production increased with increasing tryptophan. Moreover, it has been reported that IAA production by Plant Growth Promoting Rhizobacteria (PGPR) can vary among different species and strains and that it is also influenced by culture condition, growth stage and substrate availability (Beneduzi et al., 2008). According to De Freitas et al. (1997) good phosphate-solubilizers were able to produce halos of more than 15 mm diameters around their colonies. Several species of fluorescent pseudomonas such as P. fluorescens NJ101 (Bano and Musarrat, 2004), P. aeruginosa (Jha et al., 2009) and Bacillus sp. (Ahmad et al., 2008) were reported as good phosphate solubilizers. However, production of ammonia was a common trait in all isolates of bacteria. Pseudomonas fluorescens was detected positive for siderophore and HCN production than other strains of plant growth promoting bacteria. Pseudomonas fluorescens prevents the wilting and root rot disease in chickpea plant by inhibiting the growth of soil pathogenic fungi Fusarium oxysporum and Rhizoctonia solani than other strains under culture condition. In this study, P. fluorescens was found to produce HCN. The HCN affects the respiratory system of pathogenic fungi and results in their growth inhibition (Kirimura et al., 1987). Several studies have demonstrated that production of siderophores, other secondary metabolites and lytic enzymes by pseudomonas strains was most effective in controlling the plant root pathogens including F. oxysporum and R. solani (Nagrajkumar et al., 2004; Ahmad et al., 2008).
Co-inoculation studies with PGPR and Rhizobium, Bradyrhizobium sp. have been shown to increase root and shoot biomass, nodule dry matter, N2-fixation and grain yield in chickpea (Gull et al., 2004) and various legume such as common bean green gram (Sindhu et al., 1999) and pigeonpea (Tilak et al., 2006). Furthermore, combined inoculations with N2-fixing and P-solubilizing bacteria were more effective than single inoculation possibly by providing a more balanced nutrition for plants (Belimov et al., 1995). Over all maximum significant result have been shown in combination of Rhizobium sp. BHURC01 with P. fluorescens followed by other combination as Rhizobium sp. strain with B. megaterium and A. chroococcus over uninoculated control in two year of field experiment. Co-inoculation of Rhizobium sp. and P. fluorescens has been found more significant for chickpea production because Rhizobium sp. strain is an effective nitrogen fixer and P. fluorescens is an effective PGPR than other bacterial strains. Direct mechanism of PGPR has stimulated plant growth by the production of phytohormone (IAA), improved nutrient acquisition increasing crop nutrient uptake of N from nitrogen fixing bacteria (Rhizobium sp.) and uptake of P from phosphate mineral solubilizing bacteria (P. fluorescens and B. megaterium) and uptake of iron from siderophore producing bacteria (P. fluorescens). Indirect mechanism of PGPR has suppressed plant diseases like wilt disease (Fusarium oxysporum) and root rot (Rhizoctonia solani) by Pseudomonas fluorecsens. Thus, the present finding supports that a composite application of symbiotic N2-fixing organisms and plant growth promoting rhizobacteria could improved plant growth and nutrient uptake, leading to significant increases in the grain yield and protein content of field-grown chickpea. Wani et al. (2007) have been reported the synergistic effect of nitrogen fixing and phosphate-solubilizing rhizobacteria on plant growth, yield, grain protein and nutrient uptake of chickpea plants. That all of the bacterial inoculations resulted in considerable yield increases compared with control and co-inoculations, especially microbial fertilization could be an alternative to NP fertilization in chickpea (Cicer arietinum L.) (Elkoca et al., 2008; Rudresh et al., 2005).
CONCLUSION
In over all study of two year field experiment data, we have been found synergistic combination of Rhizobium sp. BHURC01 and P. fluorescens for better growth and yield of chickpea production. The potential of these rhizobacteria to inhibit the growth of the phytopathogenic fungi leading to suppression of the plant disease, along with their nodule promoting and plant growth promoting effects on co-inoculation with Rhizobium sp. BHURC01 suggested that these bacteria could be exploited to improve chickpea (Cicer arietinum L.) production.
ACKNOWLEDGMENTS
We acknowledge Co-supervisor Dr. Janardan Yadav (Associate Prof.) in Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India for providing necessary facilities to carry out this research work.