Influence of psychrotolerant plant growth-promoting rhizobacteria (PGPR) as coinoculants with Rhizobium on growth parameters and yield of lentil (Lens culinaris Medikus)

Cold-tolerant plant growth-promoting rhizobacteria (PGPR) are of immense agronomic importance due to the fact that the winter season crop growing cycle is subject to varying spells of low temperatures and such PGPRs are metabolically functional at cold conditions. Thirty-five (35) rhizobacterial isolates were isolated from lentil rhizospheric soil, six isolates showing maximum exponential growth at both 10 and 15°C after 24 h were selected and characterized belonging to genera Bacillus and Pseudomonas. Production of indole acetic acid (IAA) ranged from 12.7-17.5 μgml at 10°C to 18.4-20.4 μg/ml at 15°C. At 15°C, an isolate J-17 was found to be strong HCN producer whereas at 10°C, only two isolates: J-26 and J-30 were moderate producers. P-solubilizion at 15°C, ranged from 62.5 77.2 μgml. However, at 10°C none of the isolates solubilized P. At 15°C, catechol type siderophore production ranged from 523.4 606.1 μgml. On the basis of the PGP traits, four isolates J-3, J-17, J-18 and J-30 were selected for evaluation under field conditions of lentil at research farm of PAU, Ludhiana, Punjab, India. Coinoculation exhibited a significant increase in nodule number, nodule dry weight, plant and root dry weight, chlorophyll and leghaemoglobin content over Rhizobium alone. Application of J-3, J-17, J-18 and J-30 along with Rhizobium further enhanced the grain yield (1.8, 4.4, 3 and 1.4%).


INTRODUCTION
Lentil is the world's fifth largest pulse crop with annual production of 3-4 Mt (Sharpe et al., 2013). The important lentil-growing countries of the world are India, Canada, Turkey, Bangladesh, Iran, China, Nepal and Syria (Ahlawat, 2012). In Punjab, lentil occupied an area of 1.0 thousand hectares with a production of 0.73 thousand tones during the year 2011-2012. The average yield was 725 kg/ha (Anonymous, 2013). Lentil seeds contain 1-2% fat, 24-32% proteins and minerals (iron, cobalt and iodine) and vitamins (lysine and arginine). Being a legume crop, it can fix its own nitrogen (N) from the atmosphere and help in restoring the soil fertility. Presently, agricultural research has shifted to sustainable agriculture by supplementing chemical fertilizers with organic amendments. Lentil is constrained by extremities of temperature which are detrimental to the survival and functioning of most introduced mesophilic microorganisms. Such climatic conditions warrant the use of highly adapted microbial strains that retain their biological functions (Mishra et al., 2008).
The seed inoculation with the appropriate rhizobia at sowing is a recommended practice, but in recent years, the potential of combined inoculation of N 2 -fixers and plant growth-promoting rhizobacteria (PGPR) is more effective than single organism (Khanna and Sharma, 2011). Sindhu et al. (2002) reported that coinoculation of some Bacillus strains with effective Bradyrhizobium resulted in enhanced nodulation and plant growth of green gram (Vigna radiata L.).
The present investigation deals with the isolation, characterization, determination of the growth promotion of cold tolerant PGPR and their coinoculation effects with Rhizobium leguminosarum in lentil (Lens culinaris L. Medikus) under field conditions.

Isolation of rhizobacteria
Rhizobacteria were isolated from ten different soil samples collected from different lentil growing fields in Punjab. Pour plating was done on nutrient agar (NA) for Bacillus and on King's B for Pseudomonas. The isolates were grown at 10 and 15°C in respective media. Growth in terms of optical density was recorded at 10 and 15°C. The cultures showing maximum growth were selected for further analysis.

Biochemical characterization of rhizobacteria
Biochemical characterization of bacterial isolates was done on the basis of Gram reaction, catalase production, nitrate reduction, starch hydrolysis and methyl red test were conducted as per the standard methods (Cappuccino and Sherman, 1992;Holt et al., 1994).

Indole acetic acid production
Characterization of isolates for the production of IAA was done by method of Gordon and Weber (1951).

HCN production
HCN production was inferred by the qualitative method of Bakker Kaur et al. 259 and Schipper (1987).

Phosphate solubilization
Phosphate solubilizing isolates were screened by spotting on Pikovskaya's medium (Pikovskaya, 1948). Inoculated plates were incubated at 10 and 15°C and the diameter of clear zone (halo) surrounding the bacterial growth as well as the diameter of colony was measured after four weeks and P-solubilization index was calculated.
Phosphate solubilization index = colony of Diameter zone) halo (colony diameter Total  The colonies forming clear halos were considered as phosphate solubilizers. Microbial solubilization of insoluble phosphates in liquid media was detected by the method of Jackson (1973). The total phosphorus was estimated with the help of standard curve using different concentrations of phosphorus.

Production of siderophore
Siderophore production by the isolates was estimated qualitatively at two different incubation temperatures, that is, 10 and 15°C. Siderophore production was detected by the Chrome Azurol-S (CAS) assay given by Schwyn and Neilands (1987) in 100 mm Petri dishes. Catechol-type siderophores were estimated using the method given by Arnow (1937).

Field study
Field experiments were conducted during Rabi season of 2013-2014 on lentil at the research farms of Punjab Agricultural University, Ludhiana, Punjab, India ( 30°54'5"N 75°47'53"E). Lentil seeds of variety LL 931 were inoculated with recommended Rhizobium strain and PGPR, as per treatments. In single inoculation of Rhizobium (1x10 8 cell/g of carrier), inoculant was applied to seeds. In dual inoculation (Rhizobium + PGPR), 500 mg each of the inoculant was used. In uninoculated treatment (absolute control), seeds were treated with sterilized charcoal only. Before inoculation, equal volume of different organisms were mixed and allowed to stand for 30 min at room temperature. Inoculated seeds were dried at room temperature under shade before sowing. Crop was sown on 14 th November, 2013 following the recommended agronomic practice and harvested on 11 th April, 2014. The experimental design used was randomised block design and five treatments were used having three replications each. Each plot size was 12 m 2 . The experiment was performed as per the recommended practices.
Symbiotic and plant growth parameters such as nodule number and nodule dry weight, root dry weight, shoot dry weight, chlorophyll and leghaemoglobin contents were recorded both at vegetative and flowering stages, and grain yield was recorded at harvest. Five randomly selected plants were carefully uprooted from each plot at 60 and 90 days after sowing (DAS) with root system intact. The roots were washed in running tap water and nodules were detached carefully with forceps, and number of nodules per plant was counted as average. The detached nodules were dried in oven at 60°C for two days and their dry weight per plant was recorded in mg.
Dry weights of shoots and roots of five randomly uprooted plants from each plot were taken after drying at 60°C for 2 days. (1971) and leghaemoglobin content by the method of Wilson and Reisenauer (1963). Grain yield from each plot (g/plot) was recorded at final harvest and was expressed in kg/ha. Data was analysed using an analysis of variance appropriate for completely randomized block design (RBD). Statistical analysis was done using CPCS1 software developed by the Department of Mathematics, Statistics and Physics, PAU, Ludhiana.

Isolation and characterization
Thirty-five rhizobacterial isolates were isolated from lentil rhizospheric soil sample collected from 10 different locations of Punjab and six isolates showing maximum growth in terms of optical density at both 10 and 15°C when incubated for 24 h were selected (Figures 1 and 2). Two isolates from Kings B medium showed the characteristic yellowish-green pigmentation, whereas the other 4 isolates from NA showed off-white to creamish colour. The isolates were evaluated for cultural, morphological and biochemical characteristics as per Bergey's manual of systematic bacteriology. On the basis of these tests, the isolates were tentatively assigned to genera Bacillus and Pseudomonas (Table 1).

Indole acetic acid production
The auxin IAA is an important phytohormone produced by PGPR, and reports have shown that treatment with IAA producing rhizobacteria increase the plant growth (Hayat et al., 2010). Indole acetic acid production ranged from 12.7 -17.5 µgml -1 in the presence of tryptophan after 5 days of incubation at 10°C, whereas at 15°C, IAA production ranged from 18.4 -20.4 µgml -1 in the presence of tryptophan after 5 days of incubation (Table 2). Isolate J-17 produced IAA at the same rate at 10 and 15°C. Similar results were reported by Selvakumar et al. (2008) in a cold tolerant Serratia marcescens strain SRM (MTCC 8708) which produced 11.1 µg ml -1 IAA at 15°C.

HCN production
At 15°C, isolate J-17 was found to be strong producer of HCN (colour changed from yellow to reddish-brown), whereas J-18 was moderate producer (orange brown colour) as compared to yellow coloured control (Table 2). However, at 10°C, isolates J-26 and J-30 were found to be moderate producers. Pseudomonas sp. strain PGERs17 was reported to exhibit the HCN (cyanogenic compound) production at 15 and 4°C (Mishra et al., 2008).

Phosphate solubilization
At 15°C, P-solubilisation on solid media started on the 5th day and increased till the 15th day. P-solubilizing index ranged from 2.9 to 5.4, highest being recorded with isolate J-17 followed by J-18. The quantitative estimation showed that P-solubilization ranged from 62.5 -77.2 µgml -1 (Table 3). The bacterial isolate J-17 was found to be the most potent P-solubilizer. Selvakumar et al. (2008) reported that at 15°C, the isolate S. marcescens strain

Siderophore production
The six isolates produced distinct halo (golden yellow colour) after three days of incubation, reaching a maximum after seven days. Diameter of halo varied from 1.5 -3.0 cm on CAS agar plates, maximum being produced by fluoresecent Pseudomonas isolate J-18 (3 cm) (Table 3). Arnow's test gave positive reaction, indicating the presence of the catecholate group of siderophores. At 15°C, isolate J-17 was the highest catechol producer. However, at 10°C no zone was formed. The isolates J-3, J-17, J-18 and J-30 were selected for coinoculation with Rhizobium in lentil under field conditions.

Effect of co-inoculation of Rhizobium and PGPR on symbiotic parameters and grain yield of lentil
Isolate J-17 showed highest nodule number (27.1 and 52.8 nn/plant) followed by J-18 (26.8 and 52.3 nn/plant) as compared to Rhizobium (23.2 and 30 nn/plant) at 60 and 90 DAS, respectively (Table 4). This may be credited to the abilities of PGPRs to produce IAA. An increase of 50 to 98% was reported in nodulation through dual inoculation of Pseudomonas and Serratia with Rhizobium leguminosarum, when compared with control in lentil (Zahir et al., 2011).  Table 3. P-solubilisation and siderophore production by rhizobacterial isolates at 15°C.
Application of J-3, J-17, J-18 and J-30 alongwith Rhizobium further increased nodule dry weight (28.7, 34.6, 34.5 and 26.6 mg/plant) as compared to Rhizobium alone. Seed inoculation of lentil with effective strain of rhizobia ensures enhanced nodulation and N 2 fixation and combined use of Rhizobium with PGPR being more beneficial is well documented (Khanna et al., 2006;Siddiqui et al., 2007). Coinoculation of R. leguminosarum-PR1 with PGERs17 culture has also been reported to improve the symbiotic effectiveness in terms of nodule number (15.2%) and nodule dry weight (4.1%) in comparison with inoculation with R. leguminosarum-PR1 alone (Mishra et al., 2012).
At 60 DAS, maximum shoot dry weight was recorded (Table 4) in Rhizobium along with J-17(1.2 g/plant) followed by Rhizobium along with J-18 (1.03 g/plant) as compared to Rhizobium (0.71 g/plant). The findings are in agreement with the studies of Ogutcu et al. (2008) and Elkoca et al. (2008).
Coinoculation of non rhizobial Bacillus thuringiensis-KR1 with Bradyrhizobium japonicum-SB1 was reported to increase nodule number, root and shoot biomass, root length of soybean in comparison with B. japonicum-SB1 alone (Mishra et al., 2009). At 90 DAS, analogous results  (Table 4). Highest chlorophyll content was recorded by Rhizobium+J-17 (1.07 and 2.10 mg/g) and Rhizobium+J-18 (0.919 and 2.02 mg/g) which was at par with Rhizobium (0.649 and 1.64 mg/g), respectively at 60 and 90 DAS (Table 5). A study showed that treatment with Pseudomonas sp. strain PGERs17 supernatant was also effective in reducing chlorosis and increased total chlorophyll (6.9%) over non-inoculated control plants in field pea as reported by Mishra et al. (2012). Rhizobium inoculation along with PGPR application showed significantly higher leghaemoglobin content, that is, 4.5, 4.9, 4.6 and 4.4 mg/g of nodule with J-3, J-17, J-18 and J-30, respectively, in comparison with Rhizobium (3.2 mg/g) alone (Table 5). Coinoculation of Pseudomonas sp. strain PGERs17 with R. leguminosarum-PR1 resulted in 17.4 and 4.76-fold increase in leghaemoglobin content over control and R. leguminosarum-PR1 treated plants, respectively (Mishra et al., 2012).
Grain yield is the most important economic trait. Improvement in grain yield due to Rhizobium inoculation has been reported in legume crops Togay et al., 2008). Application of PGPRs J-3, J-17, J-18 and J-30 along with Rhizobium enhanced the grain yield (1.8, 4.4, 3.1 and 1.4%) over Rhizobium inoculation alone. The results are in corroboration with Saini and Khanna (2012) who reported that dual inoculation enhanced grain yield of lentil by about 13-15% over control. Kumar and Chandra (2008) reported that dual inoculation of Rhizobium sp. + PGPR showed significant increases in grain yields (20.8 and 23.5%) over Rhizobium sp. and PSB alone inoculation, respectively. The increase in yield can be attributed to complementation of the functionality traits of the co-inoculants, resulting in improved symbiosis and nutrient uptake.

Conclusion
The present study reveals that a vast diversity of PGPRs possessing an array of functionality traits is available for exploiting as bio-inoculants for sustainable crop production. In the present study, native PGPR isolate Pseudomonas species (J-17) alongwith recommended Rhizobium inoculant improved plant growth, symbiotic parameters and enhanced grain yield by 4.43% over Rhizobium alone.