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Research Article

Legume Research, volume 44 issue 5 (may 2021) : 527-538

Effect of Liquid and Charcoal Based Consortium Biofertilizers Amended with Additives on Growth and Yield in Chickpea (Cicer arietinum L.)

Anjali1,*, Poonam Sharma1, Sharon Nagpal1
1Punjab Agricultural University, Ludhiana-141 004, Punjab, India.

  • Submitted04-03-2019|

  • Accepted21-10-2019|

  • First Online 03-12-2019|

  • doi 10.18805/LR-4131

Cite article:- Anjali, Sharma Poonam, Nagpal Sharon (2019). Effect of Liquid and Charcoal Based Consortium Biofertilizers Amended with Additives on Growth and Yield in Chickpea (Cicer arietinum L.) . Legume Research. 44(5): 527-538. doi: 10.18805/LR-4131.

ABSTRACT

The present study was carried out to compare the shelf life and bio-efficacy of liquid and charcoal based biofertilizers of LGR33+RB1 amended with different additives viz.0.1% CMC, 2% PVP and 10mM trehalose stored at 4oC and 28oC upto 300 days in chickpea. Significantly high viable cell count of bacterial population as well as plant growth promoting (PGP) traits viz. IAA production and P solubilization at an interval of 30 days were recorded in liquid based biofertilizer treatments with additives during the entire study period (300 days) at 4oC and 28oC as compared to charcoal based biofertilizer treatments. However, among different additives amendment with 0.1% CMC showed maximum viable population and PGP traits in both liquid as well as charcoal based biofertilizers. In vivo studies, all liquid as well as charcoal based biofertilizer treatments with additives improved growth parameters in chickpea as compared to recommended consortium treatment without additives. Liquid biofertilizer treatment LGR33+RB1+0.1% CMC recorded maximum plant height, chlorophyll content, number of nodules, dry weight of nodules as well as  leghaemoglobin content, soil enzyme activity, total N and P content of shoot and soil along with improvement in grain yield over uninoculated control. 

INTRODUCTION

Chickpea (Cicer arietinum L.) is one of the largest pulse crop grown and consumed all over the world. Seeds of chickpea are supreme source of carbohydrates and proteins with high nutritive value of essential amino acids and vitamins like riboflavin, niacin and thiamin. India is the major chickpea producing country of the world contributing for 64% of the global chickpea production (Shukla et al., 2013). In India area under chickpea production is 9.93 million hectare with productivity of 9.53 million tones and average yield of 960 kg/ha (Dixit 2015). In the past 50 years, the chemical fertilizers have played a fortified role in boosting the agricultural production, by enhancing the nitrogen (N) as well as phosphorus (P) levels in the soil. Intensive farming practices that bring off high yields make use of chemical fertilizers, which are expensive as well as are noxious to environment (Rigby and Caceres 2001). Indiscriminate and excessive use of chemical fertilizers has greatly influenced soil health due to toxic residual effect. Thus, minimizing the doses of agrochemicals applied to soil without dropping the crop yield is the need of time. To revive and renew, the soil health and productivity, the focus is shifting towards the biofertilizers, which are an alternate source for chemical fertilizers.
 
The term “biofertilizer” is synonymous with microbial inoculants or bioinoculants. It is generally defined as the preparations containing live formulations of proficient strains of nitrogen fixing, phosphorus solubilising phototrophic or cellulolytic microorganisms used for application to seed or soil. Conventionally, the use of carrier materials such as peat, lignite, charcoal and mineral soils for biofertilizer production was in trend. Poor survival rate or death of the organism after seed inoculation is one of the major factors resulting in the failure of field inoculation response (Mugilan et al., 2011). The use of carrier based biofertilizers pose constraints due to low shelf life, poor survival rate, high degree of contamination and low water activity of inoculum. To cater, the problem faced with use of conventional carrier based biofertilizers the advanced technology of liquid formulations came forward stepping into new era of biofertilizer technology. Liquid biofertilizers have greater potential to fight against native population, by maintaining large cell number per ml. The required dosage of liquid biofertilizer is 10 times less as compared to carrier based biofertilizers. Certain additives are supplemented to the broth for improving quality of liquid biofertilizer. Additives have ability of protecting the cells during dessication and high temperature with better seed adhesion, product stabilization and soluble seed toxin inactivation. Various polymers such as carboxy methyl cellulose (CMC), gum arabic, lignite, polyvinyl pyrrolidone (PVP), trehalose etc. are used for their capacity to limit heat transfer and better seed adhesion (Buntic et al., 2019). Polymers soluble in the liquid inoculant formulation are also more convenient for batch processing of microbial inoculants. Different organic polymers for inoculant production have been tested, including chitin, chitosan, gellan gum and polyvinyl alcohol (Zommere and Nikolajeva 2017, Namasivayam et al., 2014). PVP with the high water binding capacity maintains the seed vigour by providing water as the seed dries. The sticky consistency may serve enhanced adhesion to seeds. Trehalose promotes cell tolerance to desiccation along with temperature and osmotic fluctuations. Glucose and mannitol serve as additional carbon sources for cell proliferation in liquid formulations Optimum concentration of polymers, adjuvants and surfactants for liquid inoculant formulations can sustain the bacterial shelf life (Navi 2004). On comparing the growth conditions, shelf life and bio efficacy of liquid formulations of Azotobacterchrococcum, Bacillus megaterium var. phosphaticum and Azospirillum brasilense, Alamraj et al. (2013) observed that liquid formulations amended with 2% PVP, 0.1% CMC and 0.025% polysorbate 20 maintained prolonged survival of Bacillus megaterium var. phosphaticum (5.6×107cfu/ml), Azospirillum (1.9×108cfu/ml) and Azotobacter (3.5×107cfu/ml) respectively after 480 days of formulation when stored at 30°C. Trivedi et al. (2016) revealed that rhizobial formulation having PVP (2.5%) + glycerol (2%) showed maximum survival rate upto 720 days of storage.In vivo appliance of liquid inoculants is required in an appropriate formulation and their viability for a definite extended time interval is main focus area for technology commercialization (Bashan et al., 2014).

Still the rate of consumption of agrochemicals is higher as compared to biofertilizers. The state and central government is making serious efforts to popularize biofertilizers and making them available to the farmers. Presently in Punjab, Mesorhizobium (LGR33) and rhizobacteria (RB1) have been recommended as charcoal based consortium biofertilizer in separate packets for chickpea. Due to non-availability of single window delivery system of liquid and charcoal based consortium biofertilizer (Mesorhizobium+RB1) supplemented with additives to increase the shelf life is the need of the hour to improve chickpea productivity.

MATERIALS AND METHODS

In vitro experiment was conducted at Pulses Microbiology Laboratory, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana to study the effect of storage period on bacterial population in consortium of charcoal and liquid based biofertilizers of LGR 33+RB1 supplemented with additives viz. 0.1% CMC, 2% PVP and 10 mM trehalose stored at 4°C and 28°C separately with following treatments:
 
1) Mesorhizobium sp. (LGR33) + Rhizobacteria (RB1) + 0.1% CMC +0.025 ml/L tween 20.
2) Mesorhizobium sp. (LGR33) + Rhizobacteria (RB1) + 2% PVP +0.025 ml/L tween 20.
3) Mesorhizobium sp. (LGR33) + Rhizobacteria (RB1) + 10mM trehalose + 0.025 ml/L tween 20.
4)  Recommended consortium (LGR33+RB1) without any additive.
 
Cultures used for bioformulations
 
LGR 33 (Mesorhizobium sp.) and RB-1 (Pseudomonas argentinensis, JX239745.1). Liquid formulations were made in sterilized Luria Bertanni broth amended with the above mentioned additives.
 
Preparation of liquid and charcoal biofertilizers with different additives
 
Eighty autoclavable washed plastic bottles were used for preparation of liquid biofertilizers. Twenty five ml of autoclaved Luria broth having additives was dispensed into these bottles. Plastic bottles were autoclaved at 121°C for 15 minutes. Inoculation was done in the sterilized bottles from the previously inoculated Luria broth containing consortium of LGR33 and RB1 (108cells/ml) and incubated at room temperature for 3-5 days in order to get a uniform growth of inoculated bacteria. Similarly, finely powdered charcoal was used as the carrier material, which was autoclaved at 121°C for 15 minutes. Eighty zipper plastic packets were sterilized with ethanol inside the laminar air flow chamber and 20 gm of sterilized charcoal was added to each packet. The previously inoculated Luria broth (108 cells/ml) amended with different additives at the rate of 0.1% (w/v) was added to charcoal packets.
 
Determination of shelf life
 
The prepared liquid and charcoal based biofertilizers were assessed for viable cell count at an interval of 30 days upto 300 days. Each treatment of consortium biofertilizer placed at 4°C and 28°C was taken out and opened inside the laminar air flow chamber every time. Shelf life was determined by evaluating viable cell count using serial dilution method and calculating the CFU/ml.
 
Screening of plant growth promoting traits: IAA production
 
Estimation of IAA production from liquid and charcoal based consortium biofertilizer was done at an interval of 30 days upto 300 days by following the methodology suggested by Gordon and Weber (1951). Overnight grown cultures from different treatments were inoculated in fresh sterile 10 ml Luria Bertanni (LB) broth supplemented with tryptophan at 0.01% followed by incubation at 28±2°C for 3-6 days. At 3rd day, broth was centrifuged at 10,000 rpm for 20 min at 4°C and supernatant was collected. Two ml of Salkowski reagent was added to 1ml of culture supernatant and absorbance of pink color was measured at 535 nm after keeping samples in dark for 20 min.
 
Quantitative estimation of phosphate solubilisation
 
At the end of storage period both liquid and charcoal based biofertilizers kept at 4°C and 28°C were tested for their potential to solubilise phosphorus. Hundred ml of Pikovaskaya’s broth was dispensed in conical flasks of 250 ml capacity and 0.1g P2O5 (tri-calcium phosphate) was added separately to each flask as an inorganic phosphate source. Contents were autoclaved at 121°C for 15 min, allowed to cool down followed by inoculation with 1 ml of overnight grown culture (108 cells/ml) from charcoal and liquid biofertilizers. Inoculated flasks were incubated at 28±2°C for 15 days. Phosphorus solubilization was recorded at 3rd, 6th, 9th, 12th and 15th day by centrifuging the cultures at 10,000 rpm for 15 minutes. One ml of supernatant was taken in test tube along with 1ml each of ammonium molybdate and ammonium vandate were added with 2 ml of HNO3 and 5 ml of distilled water. Yellow colour intensity was measured at 420 nm after 25 min of incubation.
 
Evaluation of bio-efficacy of charcoal and liquid based consortium biofertilizers after the end of storage period for growth, symbiosis and yield of chickpea under field conditions
 
The experiment was conducted in rabi season (2017-18) using randomized block design (RBD) at Pulse Research Farm, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana.
 
Variety: desi chickpea variety PBG7.
 
Bioinoculants
 
Liquid formulations (in LB broth)
 
1.     LGR33+ RB1+0.1% CMC
2.     LGR33+RB1+2% PVP
3.     LGR33+RB1+10mM trehalose
4.     LGR33+RB1(Recommended consortium)
 
Charcoal formulations
 
1.     LGR33+ RB1+ 0.1% CMC
2.     LGR33+RB1+2% PVP
3.     LGR33+RB1+10mM trehalose
4.     LGR33+RB1(Recommended consortium)
 
The experiment was conducted in 24 plots using randomized block design. Size of each plot was 9 m2.
 
Observations
 
Observations on seed emergence, plant height (cm), chlorophyll content, nodule number, nodule dry weight (g/plant), leghaemoglobin content, total nitrogen (N), phosphorus (P), potassium (K) content of soil and shoot and grain yield (kg/ha) were recorded.
 
Chlorophyll content of leaves (Witham et al., 1971).
 
One gram of fresh leaf was mashed properly using pestle mortar in 20 ml of 80% acetone and magnesium carbonate in order to aid fine grinding followed by centrifugation at 4000 rpm for 5 minutes. The supernatant was added to volumetric flask and pellet was again crushed in 15 ml of 80% acetone and was repeated for 3-4 minutes. The final volume made up to 50 ml with 80% acetone. Absorbance of this extract was recorded at 645 and 663 nm by taking 80% acetone as reference.
 
Leghaemoglobin content (Wilson and Reisenauer 1963)
 
One gram fresh nodules were crushed well adding 3 ml of Drabkin’s solution and the mixture was centrifuged at 1500 rpm for 15 min. Supernatant was collected in volumetric flask (10 ml) and final volume was made to 10 ml with Drabkin’s solution and again centrifuged for 30 min at 20,000 rpm. Absorbance was recorded at 540 nm with reference to Drabkin’s solution.
 
Soil Dehydrogenase (Tabatabai 1982)
 
One gram of soil from each treatment was weighed into screw-cap vials (15 ml capacity), added 0.2 ml of 3.0% (w/v) solution of 2,3,5-triphenyltetrazolium chloride (TTC), 0.5 ml of 1% glucose solution and incubated at 28°C for 24 hours at 30°C. Once the process of incubation was over, it was followed by addition of 10 ml of methanol to it. After mixing the whole material for 1 min, the vials were placed in refrigerator for 3 hours. The intensity of triphenyl formazan produced was read at 485 nm using Elico VIS spectrophoto- -meter directly after centrifugation.
 
Total N and P content of soil
 
Determination of total N content of soil
 
Total N content of soil (Kg/ha) was estimated at flowering stage. 5g of soil sample was mixed with 25 ml of 0.32% KMnO4 and 25 ml of 2.5% NaOH, further the distillation was done  and the distillate so obtained was collected in flask containing 5 ml of 4% boric acid and titrated with N/70 H2SO4.
 
Determination of total P content of soil
 
One g of soil was mixed with activated Dargo G and 20 ml of 0.5 NaHCO3 and shaken for 1.5 hour and filtered. 5 ml of extract was mixed with 0.5 ml of 5N H2SO4. Four ml of reagent B was added and final volume was made to 25 ml with distilled water and OD was recorded at 760 nm.
 
Total N and P content of shoot
 
Determination of total N content of shoot
 
Plant shoots were allowed to dry and nitrogen estimation was carried out by Kjeldahl’s technique with slight modification of McKenzie and Wallace (1954). Half gram of straw was taken in digestion flask, mixed with five millilitre of conc. H2SO4 and 1-2 g of digestion mixture (CuSO4: K2SO4). The digestion flask was heated on digestion heater to get the clear solution. Once the clear solution is obtained, the digested contents were allowed to cool down and final volume was made 25 ml in volumetric flask using distilled water. Five ml of this solution was distilled in Microkjeldahl distillation apparatus by adding 5-10 ml of 40% NaOH. The liberated ammonia was trapped in 5 ml boric acid containing 2-3 drops of mixed indicator.
 
Determination of total P content of shoot
 
Plant material (0.5g) was digested with 20 ml of triacid mixture (HNO3: HClO4: H2SO4) (v/v) (9:3:1). The volume was made up to 50 ml with distilled water; specific aliquots were used to estimate the phosphorus by reacting with 5 ml of ammonium molybdate reagent in nitric acid. The volume was made up to 50 ml and the intensity of yellow colour was estimated at 470 nm using Elico VIS spectrophotometer. The amount of P was calculated from the standard graph.
 
Statistical analysis
 
Data was statistically analyzed using an analysis of variance (ANOVA) appropriate for randomized block design. Further, mean separation of treatment effect was accomplished by Fisher’s protected least significant difference test. Data analysis was carried out by using SAS-Software.

RESULTS AND DISCUSSION

Shelf life study
 
The data in Table 1 shows the comparative viable cell count of liquid and charcoal based consortium biofertilizer of LGR33 and RB1 stored at 4°C and 28°C temperature upto storage period of 300 days. Non-significant difference for viable population in all liquid vs charcoal treatments was observed upto 30 days of biofertilizers storage at 4°C and 28°C temperatures. Highest number of viable cells (8.48 log CFU/ml) was recorded with consortium of liquid as well as charcoal based biofertilizer each amended separately with 0.1% CMC at 4°C and 28°C as compared to control treatments (without additives). At 60 days of storage period with liquid based biofertilizer, all the treatments were at par for viable population with additives over recommended consortium broth (non-additive) as control. After 90 days of storage period consortium biofertilizer (both liquid and charcoal) treatments with additives maintained the viable population significantly high over recommended consortium broth as well as recommended consortium charcoal (non-additive) at 4°C. At 28°C, liquid formulation of consortium biofertilizer treatments with additives supported growth of inoculants significantly high as compared to charcoal based consortium biofertilizer treatments. A sharp decline in viable population was noticed in recommended consortium charcoal (without additive) treatments (7.43 and 7.42 log CFU/g at 4°C and 28°C, respectively). However, the interaction between I×T×A was significant. At 120 days of storage period, all liquid biofertilizer treatments with additives were at par with each other and registered significantly high viable cell count over recommended consortium broth (non-additive) at 4oC. All charcoal based biofertilizer treatments with additives were at par with each other at 4°C. On the overall basis, it was concluded that all liquid and charcoal biofertilizer treatments with additives maintained significantly high viable cell count as compared to recommended consortium treatment at 4°C. At 28°C liquid biofertilizer treatments with additives maintained significantly high viable cell count over non additive control treatments.
 

Table 1: Viable cell count of liquid vs charcoal based consortium biofertilizer amended with additives during storage period of 300 days at different temperatures.


 
Viable cell count after 150 days of storage period revealed that all liquid biofertilizers with additives registered significantly high count over charcoal based biofertilizer treatments with and without additives at 4°C and 28°C. However, liquid biofertilizer treatment supplemented with 0.1% CMC maintained significantly high viable population (8.46log CFU/ml) as compared to recommended consortium broth (non-additive) as well as recommended consortium at 4oC. Liquid biofertilizer treatments supplemented with 0.1% CMC and 2% PVP were at par with each other with significant viable cell count over other treatments with and without additives at 28°C. A sharp decline in viable cell count in charcoal based biofertilizer treatments with additives was observed after 150 days. Interaction between I×T, I×A and I×A×T were significant.
 
Observations recorded at 210, 240, 270 and 300 days of storage period revealed that the effect of liquid biofertilizer treatments on viable population was significantly high over charcoal based biofertilizer treatments. Liquid formulation supplemented with 0.1% CMC stored at 4°C retained maximum number of viable bacterial cells. Viable cell count was improved with all additives (CMC, PVP and trehalose) in both liquid and charcoal inoculants stored at 4°C and 28°C. However, 0.1% CMC and 2% PVP were found to be most effective in maintaining viable population density during the entire storage period (300 days) in charcoal as well as liquid formulations.
 
The results are in concurrence with Trivedi et al. (2016) who observed that amendments made with additives and emulsifiers in nutrient broth (containing 2% glycerol and 2.5% PVP) showed higher survival of bacterial cells till 720 days. Amendment of YEM broth with 2% of gum arabic for Rhizobium gave better shelf life upto 180 days (Sehrawat et al., 2015). Kumaresan and Reetha (2011) studied the effect of six different polymeric additives viz. PVP, glycerol, gum arabica, trehalose, PEG and PVA with different concentrations on the survival rate of Azospirillum brasilense in liquid medium during the storage period of 11 months under 28°C ± 4°C temperature. Of all the treatments studied, 2% PVP recorded better survival rate in liquid formulations as compared to carrier based formulations.
 
Quantitative estimation of IAA production
 
Liquid as well as charcoal based consortium biofertilizers stored at 4°C and 28°C were assessed for their potential to produce IAA during the storage period at an interval of 30 days upto 300 days (Table 2). A decreasing trend was observed in both liquid as well as charcoal based biofertilizers stored at 4°C and 28°C. After 30 days of storage period, at 4°C liquid biofertilizer treatments viz. LGR33+RB1+0.1% CMC and LGR33+RB1+ 2% PVP were at par for IAA production and recorded significantly high IAA production (35.15 and 34.64 µg/ml respectively) over LGR33+RB1+10mM trehalose and recommended consortium broth (non-additive). However, among charcoal based biofertilizer treatments LGR33+ RB1+0.1% CMC (31.64 µg/ml) recorded significantly high IAA production over different additive treatments and recommended consortium charcoal (non-additive). Similarly at 28°C, liquid biofertilizer treatment LGR33+RB1+0.1% CMC (37.51µg/ml) registered high IAA production over other additives as well recommended consortium broth (non-additive) treatments. Charcoal biofertilizer treatment viz. LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP (33.20 and 32.59 µg/ml) were at par for IAA production and supported significantly high IAA production over recommended consortium charcoal (non-additive). However all the liquid biofertilizers (with and without additives) were significantly superior over charcoal based biofertilizers at both the temperatures. At 120 days of storage period, at 4°C liquid biofertilizer treatment LGR33+RB1+0.1% CMC recorded significantly high IAA production (30.71µg/ml) over recommended consortium broth (non-additive). LGR33+RB1+2% PVP and LGR33 + RB1+10 mM trehalosetreatment varied considerably for IAA production and showed values viz. 28.81 and 22.75 µg/ml respectively. Recommended consortium broth (non-additive) reported low IAA production (17.8 µg/ml) over additive treatments. With charcoal biofertilizer treatments significantly high IAA production was shown by LGR33+ RB1+0.1% CMC (25.77µg/ml). Charcoal based biofertilizer treatments with additives showed low IAA production in comparison to liquid biofertilizer treatments with additives. At 28°C LGR33+RB1+0.1% CMC in liquid formulation registered significantly high IAA production (31.77µg/ml) as compared to other liquid as well as charcoal biofertilizer treatments. Interactions between I×T, T×A and I×T×A were non-significant. IAA production recorded at 240 days of storage period revealed that liquid biofertilizer LGR33+RB1 with additives viz. 0.1% CMC, 2% PVP and 10mM trehalose showed significantly high values (23.36, 21.53 and 8.62 µg/ml respectively) as compared to recommended consortium broth (non-additive) at 4°C. Charcoal biofertilizer treatments with additives also showed significantly high IAA production as compared to recommended consortium charcoal (non-additive). All liquid biofertilizer treatments at 28°C also recorded significantly high IAA production as compared to charcoal biofertilizer treatments.
 

Table 2: IAA production of consortium liquid vs charcoal based biofertilizers amended with additives during storage period of 300 days at different temperatures.


 
At 270 days of storage period, all liquid biofertilizer treatments at 4°C and 28°C registered significantly high IAA production as compared to charcoal biofertilizer treatments. Significantly high IAA production was recorded with liquid biofertilizer treatment LGR33+RB1+0.1% CMC (22.68 µg/ml) as compared to other treatments. Charcoal biofertilizer treatments with additives viz. 0.1% CMC, 2% PVP and 10mM trehalosedocumented high IAA production over that of recommended consortium charcoal (non-additive). At the end of the storage period (300 days), liquid biofertilizer treatment LGR33+RB1+0.1% CMC recorded significantly high IAA production at both temperatures.
 
The IAA secreted by rhizobacteria adds to the auxin pool of plant and regulates the developmental processes of plants. Biosynthesis of IAA in Pseudomonas fluorescence isolates boosted with an increase in levels of tryptophan concentration from 1 to 5 mg/ml (Verma et al., 2010). IAA production by Mesorhizobium sp. and Pseudomonas aeruginosa positively stimulated K and P uptake by chickpea inoculated with these microorganisms (Verma et al., 2013). Two potential indole producing bacteria Pseudomonas and Trichoderma were also able to solubilise large quantities of P and showed antagonistic activities against Fusarium oxysporumandRhizoctonia solanias compared to other strains (Saharan and Nehra 2011). The IAA producing bacteria may be efficient biofertilizer inoculants to promote plant growth and promoting medicinal plants for future generation (Pant and Agrawal 2014).
 
Quantitative estimation of P solubilization
 
P-solubilization activity of different treatments of dual inoculants of Mesorhizobium (LGR33) and Pseudomonas sp. (RB1) with different additives at varying concentrations was measured at different time intervals (3, 6, 9, 12 and 15 days) in Pikovaskaya’s broth amended with 0.1% TCP as an inorganic phosphate substrate after storage of 300 days (Table 3). It was observed that amount of P released by different treatments increased with increase in period of incubation up to 9 days.
 

Table 3: Assessment of P solubilization in consortium liquid vs charcoal based biofertilizers amended with additives stored at different temperatures after 300 days.


 
After 3 days of incubation, liquid biofertilizer treatment LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP were at par for P solubilization and recorded high P solubilization (8.29 and 8.24 mg/100ml, respectively) as compared to recommended consortium broth (non-additive). Charcoal based biofertilizer treatments of LGR33+RB1+ 0.1% CMC and LGR33+RB1+2% PVP were at par and registered significantly high P solubilization (5.40 and 5.23 mg/100 ml, respectively) over recommended consortium charcoal (non-additive). At 28°C, all liquid biofertilizer treatments showed significantly high P solubilization as compared to charcoal based biofertilizer treatments. Among liquid biofertilizer treatments, LGR33+RB1+0.1% CMC registered significantly high P solubilization over recommended consortium broth (non-additive). Similarly for charcoal based biofertilizer treatments, maximum P solubilization was recorded with LGR33+ RB1+0.1% CMC and LGR33+RB1+2% PVP (5.61 and 5.32 mg/100ml, respectively) over recommended consortium charcoal (non-additive).
 
After 6th day of incubation observations revealed that liquid biofertilizer treatments at 4°C recorded significantly high P solubilization as compared to charcoal biofertilizer treatments. However liquid biofertilizer treatment LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP (9.75 and 9.09 mg/100 ml, respectively) were at par and showed almost equivalent P solubilization in Pikovaskaya’s broth. Similarly charcoal biofertilizer treatments LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP were at par and recorded high P solubilization over recommended consortium charcoal (non-additive). Similar trends were shown by liquid biofertilizer treatment LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP at 28°C, recording maximum P solubilization (10.11 and 10.01mg/100 ml, respectively) over recommended consortium broth (non-additive). Interactions between I×T, I×A, T×A and I×T×A were non-significant.
 
Decline in P-solubilization was observed after 9 days of incubation. P-solubilization ranged between 3.55-7.01 mg/100 ml with all treatments at 12th day of incubation. Liquid biofertilizer treatment LGR33+RB1+0.1% CMC (7.01mg/100ml) registered significantly high P solubilization over recommended consortium broth (non-additive) at 4°C, whereas charcoal biofertilizer treatments LGR33+RB1+ 0.1% CMC and LGR33+RB1+2% PVP were at par and observed significantly high P solubilization over recommended consortium. P solubilization recorded with liquid and charcoal based biofertilizers at 28°C revealed that liquid biofertilizer treatment LGR33+RB1+0.1% CMC showed significantly high value (7.00mg/100ml) in comparison to the recommended consortium (3.67mg/100ml). At 15th day of incubation observations suggested that liquid biofertilizer treatment LGR33+ RB1+0.1% CMC recorded maximum P solubilization at 4°C as well as 28°C (5.24 and 5.01mg/100 ml, respectively).
 
Pandey et al., (2007) studied P solubilization in Cajanuscajan by the consortium of Burkholderia sp. MSSP and Sinorhizobiummeliloti PP3, where maximum solubilization of P was achieved on 8th day, level of solublization gradually increased up to 7 days with a maximum value of 10.95 mg/100 ml. Similar findings by Fankem et al., (2006) using strain EDJ6 documented solubilization of tricalcium phosphate up to 308 mg/L P in broth medium even though this strain showed no clear zone on tricalcium phosphate containing agar plates. Another strain DR5 was able to solubilize 191 mg/L P from tricalcium phosphate form in liquid broth condition in oil palm tree.
 
Plant growth parameters
 
Inoculation with consortium liquid biofertilizer as well as charcoal biofertilizers and LGR33+RB1 (with additives) showed numeric increase in emergence count, plant height and chlorophyll content (Table 3) as compared to uninoculated control treatment. Liquid biofertilizers supplemented with 0.1% CMC and 2% PVP showed higher plant height as well as chlorophyll content as compared to other treatments at vegetative as well as flowering stage. Increased emergence count, plant height and chlorophyll content could be attributed to supplementation and better adhesive nature of polymeric additives in liquid and charcoal based biofertilizers which allow better adhesion of bacterial inoculants on the seed after sowing in the field, thereby establishing a good population of inoculated bacteria over control treatment. Besides, bacterial cells might have been protected by cell protectants against adverse conditions prevailing in rhizosphere. Prakash (2010) documented liquid rhizobial inoculants with PVP and gum arabic improved plant growth and grain yield in winter legumes. Oad et al., (2002) evaluated the effect of different doses of liquid Rhizobium japonicum culture in soybean and recorded significantly different plant height of the soybean crop under different culture doses.
 
Symbiotic parameters
 
Significantly high number of nodules were recorded with liquid biofertilizer treatment LGR33+RB1+0.1% CMC (32 nodules) followed by LGR33+RB1+ 2% PVP (30 nodules) over uninoculated control (23 nodules). However in charcoal based biofertilizer with additives all the treatments varied non-significantly over uninoculated control and nodule number ranged from 24-27 nodules/plant. Treatment LGR33+RB1+ 0.1% CMC and LGR33+RB1+2% PVP recorded equal NN/plant with numeric increase of 17.4% over uninoculated control (Table 4). At flowering stage, a significant increase in nodulation was observed with both liquid and charcoal based biofertilizers with additives except charcoal based biofertilizer with trehalose over control treatments. Seed inoculation with consortium liquid biofertilizer treatment with additives viz. LGR33+RB1+ 0.1% CMC and LGR33+RB1+ 2% PVP recorded 79 and 77 NN/plant respectively registering a significant increase in nodulation over uninoculated control treatment. Sahai and Chandra (2011) concluded that liquid inoculants exhibited better nodulation than carrier based inoculants registering significant increase of 35 % and 30.9% in nodule number with Mesorhizobium ciceri and Pseudomonas diminuta in chickpea.
 

Table 4: Effect of liquid and charcoal based consortium biofertilizers amended with additives on plant height, total chlorophyll content, number of nodules and leghaemoglobin content in chickpea.


 
Maximum leghaemoglobin content was recorded with the liquid biofertilizer treatment LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP (3.89 and 3.84 mg/g of fresh weight of nodules respectively) as compared to uninoculated control. Seed inoculation with charcoal biofertilizer treatment LGR33+RB1+0.1% CMC, LGR33+RB1+2% PVP and LGR33+RB1+ 10mM trehalose registered an increment of 10.5%, 9.3% and 8.16% respectively over uninoculated control. Recommended consortium (without additive) showed 8.16% increase over uninoculated control.
 
Tagore et al., (2013) recorded higher leghaemoglobin content in the nodular tissue of chickpea seeds with Rhizobium and PSB. Increase in leghaemoglobin content may be probably because of better nodulation with inoculated cultures. Better response of liquid having additives might be due to high population density of effective Rhizobium sp. which could out-compete native rhizospheric microflora, causing better nodulation and higher leghaemoglobin content in chickpea. Sharma et al., (2006) evaluated the efficacy of liquid and carrier based Rhizobium inoculants with respect to nodulation, leghaemoglobin content and grain yield in mungbean, urdbean and pigeonpea and reported similar trend in symbiotic parameters.
 
Total nitrogen (N) and phosphorus (P) content of soil and shoot
 
All the treatments significantly improved the N content of soil over uninoculated control (Table 5).  Liquid biofertilizer treatments LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP were at par with each other and recorded significantly high total N content of soil (120.32 and 119.65kg/ha) followed by LGR33+RB1+ 10mM trehalose (118.60kg/ha) over uninoculated control. Similarly, liquid biofertilizer treatment LGR33+RB1+0.1% CMC and LGR33+RB1+2% PVP recorded maximum P content of soil (29.9 and 28 ppm respectively) over uninoculated control treatment (20.4 ppm). Rhizobium and PSB inoculation recorded better plant height, number of nodules, and rhizospheric environment which ultimately resulted in more nutrient acquisition (NPKS) in chickpea soil (Meena et al., 2006). Significant increase in total N content of shoot was observed with both liquid and charcoal based biofertilizer treatments with additives over uninoculated control treatment. All liquid biofertilizer treatments viz. LGR33+RB1+ 0.1% CMC, LGR33+RB1+ 2% PVP and LGR33+RB1+10mM trehalose marked  significantly high total N content of shoot (1.48, 1.46 and 1.38% respectively) over uninoculated control. However, The effect of all liquid and charcoal based biofertilizer treatments with additives on total P content of shoot was non-significant. Raja and Takankhar (2018) revealed that the seed inoculation of soybean with liquid formulation of Bradyrhizobiumsp. significantly increased plant nitrogen content at maturity by 7.93% and at harvest by 3.62% over uninoculated control. Ullah et al. (2016) reported that dual inoculation of Mesorhizobium ciceri with endophytic bacteria increased nitrogen content in chickpea by 7.06% over uninoculated control. Navi (2004) documented that total P of shoot was superior over other treatments and uninoculated control with inoculation of Bradyrhizobium grown in broth having PVP.
 

Table 5: Effect of liquid and charcoal based consortium biofertilizers amended with additives on total shoot and soil nitrogen, phosphorus, dehydrogenase activity and grain yield in chickpea.


 
Dehydrogenase activity
 
At vegetative stage, significantly high dehydrogenase activity was recorded with liquid biofertilizer treatment LGR33+RB1+ 0.1% CMC (24.78 µg TPF/g soil/hr), followed by LGR33+RB1+2% PVP and LGR33+RB1+10mM trehalose (22.54 and 21.35 µg TPF/g soil/hr) over uninoculated control (Table 5). At flowering stage both liquid and charcoal based biofertilizer treatments with additives except charcoal biofertilizer with trehalose significantly improved dehydrogenase activity as compared to uninoculated control. Liquid biofertilizer treatments viz. LGR33+RB1+0.1% CMC, LGR33+RB1+2% PVP and LGR33+RB1+10mM trehalose were at par with each other and showed significantly high dehydrogenase activity with values 47.60, 46.28 and 45.33 µg TPF/g soil/hr over uninoculated control. The effect of liquid and charcoal based biofertilizers in chickpea studied by Sahai and Chandra (2009) revealed that dual liquid inoculants of Mesorhizobium+ Pseudomanas gave high soil dehydrogenase activity (150 µg TPF g-1 24hr-1) as compared to single inoculants in liquid and charcoal biofertilizers. Increase in dehydrogenase might be due to inoculation of Mesorhizobium + rhizobacteria with additives like CMC and PVP resulting in overall enhanced microbial activity under field conditions. The adhesive properties of these polymeric additives allow the better survival on inoculated seeds.

Grain yield
 
The effect of liquid and charcoal based consortium biofertilizer on grain yield was non-significant among different treatments (Table 5). Highest enhancement in yield was recorded with liquid biofertilizer treatment LGR33+RB1+ 0.1% CMC and LGR33+RB1+ 2% PVP (1782 kg/ha and 1765 kg/ha respectively) over uninoculated control (1625 kg/ha).Charcoal based biofertilizer treatments LGR 33+RB1+0.1% CMC and LGR33+RB1+2% PVP registered a numeric increase of 6.3 and 6.1%, respectively over uninoculated control. Recommended consortium (without additive) showed enhancement of 5.85% over uninoculated control. Increase in grain yield could be a result of better plant growth and better nutrient uptake due to seed inoculation with liquid and charcoal based consortium biofertilizers. Superior grain yield due to liquid biofertilizers amended with different additives could be due to higher survival rate of consortium inoculants prior to seed application. Chandra and Pareek (2007) observed an increase of 5.2 to 22.5% in grain yield of chickpea upon dual inoculation with Mesorhizobium sp. with various PGPR over uninoculated control. Bramprakash et al., (2007) evaluated the performance of liquid and carrier-based formulations of Rhizobium in leguminous crops and found that liquid Rhizobium inoculants exhibited better yield than carrier-based inoculants. Gupta et al., (2005) recorded an increase in grain yield with liquid biofertilizers in comparison with charcoal based biofertilizers of Rhizobium in chickpea.

CONCLUSION

Liquid biofertilizers are an emerging and modern technology in Indian agriculture with unique production methods. Supplementation of biofertilizers with various polymeric additives like sodium alginate, gum arabic, PVP, PVA and PEG is in trend. The adhering nature of these polymers let on the better sticking of the inoculants to the seeds. The present study reveals that liquid inoculant formulations added with polymeric additives improved shelf life and PGP traits (In vitro) as well as growth, symbiotic parameters, soil health and yield (In vivo) in chickpea.

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