Influence of Plant Growth Promoting Rhizobium on Growth of Pigeon Pea [Cajanus cajan (L.) Millsp.] 

Pigeon pea [Cajanus cajan (L.) Millsp.], is the second most important Kharif pulse crop grown in India after chickpea. In India, it is predominantly grown in rainfed conditions. It is used as food, fodder and fuel wood. It also maintains soil fertility through nitrogen fixation by its microsymbiont Rhizobium, as well as from the leaf fall and recycling of the nutrients. One of the major problems that limit economically successful agricultural production in yield of pigeon pea worldwide is poor soil fertility. Way to correct this problem is the addition of biofertilizers, which improves soil fertility by supplying nutrients needed for optimum crop growth by fixing an atmospheric nitrogen, mineral solubilisation etc., (Osman et al., 2011).
       
Biological nitrogen fixation plays a major role in the production of million tons of nitrogen. The significance of these nitrogen fixing microbes is an evident from the fact that although a total of 100 million metric tons of synthetic nitrogen is produced per year, nitrogen fixing microorganisms yearly converts about 200 million tons of nitrogen to ammonia (NH₄) (Glazer and Nikaido, 2007).
       
All bacteria nodulating legumes described so far belongs to the Proteobacteria class. The majority of them belong to the genera of the α- Proteobacteria class, in the genera of Rhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium and Ensifer (formerly Sinorhizobium) (Berrada and Benbramhim, 2014). Rhizobia  are  gram-negative, rod shaped, motile, non sporulating bacteria that fix nitrogen from atmosphere after becoming established inside the root nodules of leguminous crops. Pigeon peas are often described as non-specific in their Rhizobium requirements and can be effectively inoculated by the indigenous soil populations. Inoculation of pigeon pea with Bradyrhizobium increased the number of primary branches, reduced the time to flowering and maturity. It also increased the nodulation and grain yield. Finally it was concluded that biological nitrogen fixation is one alternative to the inorganic N fertilizers (Ahmed et al., 2014).  BNF values estimated for legume crops fall in the range of 200 to 300 kg of N ha^-1year^-1. Yield increases of crops planted after harvesting of legumes are often equivalent to those expected from application of 30 to 80 kg of fertilizers/N/ha. Apart from N₂ fixation, Rhizobium also known to produce plant growth promoting substances thus stimulates plant growth.
               
Hence, the present study was carried out to characterize the Rhizobium isolates for their functional properties and plant growth promotional activities in pigeon pea.

The present investigation was carried out in the Department of Agricultural Microbiology, College of Agriculture, Vijayapur, UAS, Dharwad. Five of the isolates obtained from Northern district of Karnataka were subjected for functional characterization viz., IAA (Gordon and Paleg, 1957), GA production (Paleg, 1965), ACC deaminase activity (Penrose and Glick, 2003), P (Vazquez et al., 2000), Zn (Di Simine et al., 1998), Si solubilization (Bunt and Rovira, 1955),  HCN (Wei et al., 1991), siderophore production (Schwyn and Neilands, 1987) and antagonistic activity (Sakthivel and Gnanamanickam, 1987).
 
In vitro screening of Rhizobia isolates against fungal pathogen (Fusarium oxysporum f. sp. udum) of pigeon pea
 
The fungal pathogen was grown on potato dextrose agar plates until they completely cover the agar surface. With the help of a sterile cork borer (10 mm diameter), discs of fungal growth from the plates was taken and placed at the center of the fresh PDA plates. Each test isolate was then streaked parallel on either sides of the fungal disc leaving 1.5 cm distance from the edge of the plate. The PDA plates inoculated with only fungal pathogens were considered respective controls. The plates were incubated at 30oC for 96 h. The colony diameter of the fungus in control plate and the plates streaked with Rhizobium were recorded (Sakthivel and Gnanamanickam, 1987). The zone of inhibition (ZOI) of each fungal pathogen by different isolates were calculated by using the following formula:
 
ZOI = Colony diameter (control plate) - Colony diameter (in dual inoculated plates)
 
The per cent inhibition of pathogen was assessed by using the formula given below: Where,
I = Per cent inhibition.
C = Radial growth of the pathogen in control.
T = Radial growth of the pathogen in treatment.
 
Evaluation of Rhizobium isolates for their growth promotional activities on pigeon pea under pot culture conditions
 
Rhizobium isolates were evaluated for their growth promotional activities under pot culture condition using pigeon pea as a test crop. The experiment was conducted during Kharif season 2018-19 at Department of Agricultural Microbiology, College of Agriculture, Vijayapur, University of Agricultural Sciences, Dharwad, Karnataka.
 
Inoculum preparation
 
The individual flask containing 20 ml of sterilized yeast extract mannitol broth was inoculated with overnight grown culture of each Rhizobium isolate separately and incubated on rotary shaker for 96 h at a temperature of 28±2^oC to get a population of 108cfu/ml and mixed with sterilized talc powder in 1:3 proportions to prepare carrier based formulations. These formulations were used for seed treatment. Plastic pot of 35cm top diameter was filled with 8 kg sterilized soil and 30 grams of vermicompost. Pigeon pea seeds of variety TS-3R were surface sterilized in 0.1 per cent mercuric chloride for 3 minutes followed by repeated washing in sterile distilled water for 6 times to remove traces of mercuric chloride and seeds were treated with Rhizobium formulations. Treated pigeon pea seeds were sown in each pot by dibbling method at five equidistant spots of 4-5 cm depth where one seed was placed in each hole and immediately covered with soil. In control pots, untreated seeds were sown. After germination, thinning was done to retain only one plant in each pot. The pots were watered regularly to maintain optimum moisture.
       
Observations on plant growth parameters viz., number of nodules, nodule dry weight (g/plant) and chlorophyll content (SPAD value) were taken after 60 DAS and the observation on shoot dry weight, root dry weight and total dry matter (g/plant) were recorded after 90 days of sowing. Plant nitrogen and phosphorous were estimated at 90 DAS.
 
Molecular characterization of selected isolates
 
The isolate AMVPR98 found to be positive for all functional traits and performed better under pot culture condition was subjected for molecular characterization through 16S rDNA analysis.
 
Statistical analysis of the data
 
The data obtained from the experiment were subjected to statistical analysis by Completely Randomized Design. Interpretation of the data was carried out in accordance with Pansey and Sukhatme (1985). The levels of significance used in the ‘f’ and ‘t’ test was P=0.01. The critical difference values were calculated wherever the ‘f’ test values were significant.

Functional characterization of Rhizobium isolates
 
All the isolates were positive for IAA and GA production. The IAA production was ranged from 20.50 to 22.85 mg IAA/ ml of broth (Table 1) and GA production from 13.40 to 14.51mg/25ml of broth. Highest IAA production was observed in the isolate AMVPR 98 (22.85 mg/ml) followed by the reference strain PPM35B (22.45 mg/ml). With respect to GA production, the maximum of 14.51 mg/25ml of broth was observed in the reference strain which was on par with the isolates AMVPR98 (14.43 mg/25ml broth). ACC deamination activity in all the isolates was ranged from 57.50 to 75.5 nmoles of α-ketobutyrate/mg/h of broth (Table 1). The reference strain PPM35B reported 75.5 nmoles of α-ketobutyrate/mg/h ACC deamination activity which was followed by the isolates AMVPR98 and AMVPR79 with ACC deamination activity of 73.5 and 68.5 nmoles of α-ketobutyrate/mg/h respectively. All the isolates exhibited their ability to solubilize P and Zn (Table 2). The dissolution and mobilization of soil minerals by microorganisms is well known (Calvaruso et al., 2006; Sheng and He, 2006; Uroz et al., 2007). The isolate AMVPR 98 showed highest diameter of zone of P-solubilization (10.3 mm) which was followed by reference strain (PPM35B), with zone of solubilization of 9.8 mm. The diameter of zone of zinc oxide solubilization was ranged from 9.5 to 20.5 mm. It was highest in reference strain PPM35B (20.5mm) followed by AMVPR98 (16.5 mm).Whereas only one isolate (AMVPR-98) and reference strain PPM35B exhibited their ability to solubilize silica.The results are in conformity with the work of Chandrakala et al., (2019) who reported that the Rhizobium isolate obtained from rice rhizosphere, besides possessing mineral (P, K, Si and Zn) solubilising capacities, also exhibited phytostimulatory behaviour by producing IAA (0.95 +0.14 μg IAA /μg bacterial cell protein) and ACC deaminase activity (2.52+0.73 μg α ketobutyrate /μg bacterial cell protein/24 h).
 

 

       
All the isolates were found to be positive for siderophore and HCN production (Table 2). With respect to siderophore production, the diameter of zone of clearance on CAS agar in different isolates ranged from 6.5 to 10.5 mm. Maximum diameter of zone of clearance was observed in the isolate AMVPR98 (10.5 mm). Among six isolates, 2 isolates viz., AMVPR98 and AMVPR79 were strong (+++) HCN producers. The reference strain has also exhibited strong (+++) HCN production ability. The results of this study are in line with the findings of Manasa et al., (2017) they studied HCN production in fifteen Rhizobium isolates, among those 53% isolates showed positive for HCN and siderophore production. Siderophore production and utilization in rhizobia is of particular interest due to the dominant role of iron in the N₂ fixation and assimilation process (Schwyn and Neiland, 1987). Bacteria with the ability to produce siderophore can enhance plant growth by increasing the availability of iron near the roots for plant uptake (Alexander and Zuberer, 1991).
 
In vitro screening of Rhizobium isolates against fungal pathogen (Fusarium oxysporum f.sp. udum) of pigeon pea
 
With respect to in vitro screening of Rhizobium isolates against Fusarium oxysporum f.sp. udum, out of ten isolates, four isolates exhibited potential to inhibit mycelial growth of Fusarium oxysporum f. sp. udum (Table 3). Per cent inhibition was ranged from 48.75 to 73.75. The maximum per cent inhibition of 73.75 was observed in AMVPR-98. Several studies on the mode of action of Rhizobium spp. have shown that the growth inhibition of plant pathogens is ensured by the production of toxic compounds. Early work has allowed the characterization of antimicrobial activities related to extracellular compounds of Rhizobium spp. such as trifolitoxin (Malajczuk et al., 1984) that antibiosis may be part of their reported biocontrol efficacy.
 

 
Evaluation of Rhizobium isolates for their growth promotional activities on pigeon pea under pot culture conditions
 
Plant height
 
All the treatments with Rhizobium isolates differed significantly over the absolute control at 90 DAS with respect to plant height of pigeon pea (Table 4). The highest plant height of 114.58 cm was recorded in T₆ (PPM35B), which was on par with the treatment T₄ (AMVPR98) (114.36 cm)). Lowest plant height (93.72 cm) was recorded in control (T₇). Increase in plant height might be due to the production of phytohormones by the Rhizobium and increased mobilization of nutrients (Ahmad et al., 2014).
 

 
Nodule number
 
All the treatments with Rhizobium isolates differed significantly over control with respect to nodule number to at 60 DAS (Table 4). The treatments T₆ (PPM35B) and T₄ (AMVPR98) were on par with each other recording 12.80 and 12.60 number of nodules/plant. The next highest nodule number was recorded in T₃ (AMVPR79) (11.60) followed by T₂ (AMVPR53) (10.8). Lowest number of nodules (4.40/plant) was recorded in control T₇ which was not inoculated with Rhizobium.
 
Nodule dry weight
 
Significant differences were observed between the treatments with respect to nodule dry weight at 60 DAS (Table 4). At 60 DAS, maximum nodule dry weight (76.40 mg) was recorded in treatment T₆ (PPM35B) which was significantly superior over all other treatments. The next highest was observed in the treatment T₄ (AMVPR98) (70.25 mg) followed by T₃ (AMVPR79) (62.32 mg). Lowest nodule dry weight per plant (23.10 mg) was recorded in treatment T₇.  The results are in agreement with the work of Thakare and Rasal (2000) who reported significant increase in nodule number and dry weight of ground nut when inoculated with native isolates of Rhizobium. 
       
Growth promotional ability of these isolates could be attributed to production of higher amounts of IAA, GA and also greater ability to solubilize P, Zn and Si. Nascimento et al., (2012) reported that selection and use of rhizobial strains with high ACC deaminase activity is a promising strategy to improve the performance of rhizobia-legumes symbiosis by enhancing the nodulation. Similarly, in the present study the isolate AMVPR98 and reference strain exhibited highest ACC deaminase activity resulted in more number of nodules. Increase in dry weight of nodules may be due to enhanced nodulation, higher nitrogen fixation rate and general improvement of root development (Dakora, 2003).
 
Chlorophyll content (SPAD value)
 
All the treatments with Rhizobium isolates differed significantly over the treatment with absolute control. At 60 DAS, treatment T₆ (PPM35B) has recorded significantly highest chlorophyll content of 62.56 SPAD value, which was followed by treatments T₄ (AMVPR98) (60.62 SPAD value) and T₃ (AMVPR79) (58.42 SPAD value). Lowest chlorophyll content of 42.58 SPAD values was recorded in T₇ (control). Results are well in accordance with Samavat et al., (2012) and Bejandi et al., (2012) who reported that Rhizobium treatment had significantly high chlorophyll content as compared to control.  Iron uptake by siderophore production might also increase chlorophyll content of legumes (Kamble et al., 2006). In the present study, Rhizobium isolates were found to be siderophore producers.
 
Shoot dry weight, root dry weight and total dry matter production
 
All the treatments with Rhizobium isolates differed significantly over the treatment with absolute control (Table 5). At 90 DAS, highest shoot dry weight was recorded in the treatment T₆ (PPM35B) (8.38 g/plant) which was on par with the treatments T₄ (AMVPR98) (8.37 g/plant), T₃ (AMVPR79) (8.34 g/plant) and T₂ (AMVPR53) (8.29 g/plant). Lowest shoot dry weight was recorded in T₇ (3.43 g/plant). With respect to root dry weight at 90 DAS, highest root dry weight of 1.86 g/plant was recorded in the treatment T₆ (PPM35B) which was on par with the treatments T₄ (AMVPR98) (1.85 g/plant), T₃ (AMVPR79) (1.84 g/plant) and T₂ (AMVPR53) (1.81 g/plant). Lowest root dry weight was recorded in T₇ (1.30 g/plant). At 90 DAS, highest total dry matter content was recorded in the treatment T₆ (PPM35B) (10.23 g/plant) which was on par with the treatments T₄ (AMVPR98) (10.22 g/plant) and T₃ (AMVPR79) (10.19 g/plant).  Lowest total dry matter production was recorded in T₇ (4.73 g/plant). The results are in line with the findings of Rufini et al., (2016) observed the symbiosis of Bradyrhizobium sp. with pigeon pea Cv. Fava-larga increased the shoot dry weight and N accumulation in the plant under field conditions. Samudin and Kuswantoro (2018) also observed significant increase in the number of nodules, nodule dry weight, root length and root dry weight of soybean when inoculated with Rhizobium.
 

       
There are evidences that Rhizobium can enhance plant growth through the changes in root physiology and morphology along with nitrogen fixation (Biswas et al., 2000), which increases root dry weight. Root development is influenced by the phytohormone auxin which is known to affect a number of plant functions including promotion of cell division, elongation, root initiation and ethylene biosynthesis (Chasan, 1993).
 
Nitrogen and Phosphorous uptake
 
All the treatments with different Rhizobium isolates differed significantly over the treatment with absolute control at 90 DAS. The highest nitrogen uptake was recorded in the treatment T₆ (PPM35B) (0.85g/plant) which was on par with the treatments T₄ (AMVPR98) (0.83g/plant) (Table 5). Lowest nitrogen uptake was recorded in T₇ (0.48g/plant). With respect to P uptake, highest phosphorous uptake was recorded in the treatment T₄ (AMVPR98) (0.156g/plant) which was on par with the treatment T₆ (PPM35B) (0.152g/plant). Lowest phosphorous uptake was recorded in T₇ (0.084g/plant). It has been confirmed that IAA producing bacteria are reported to produce high levels of ACC, which inhibit ethylene levels reported to promote plant growth, enhanced rhizobial nodulation and mineral uptake (Glick, 2012).
 
Molecular characterization and phylogenetic analysis of efficient Rhizobium isolate
The isolate AMVPR98 was subjected for molecular identification using 16S rDNA sequence analysis. The alignment and assessment of the gene sequences data were performed by comparing with the sequences available in Gene Bank database of NCBI, using the algorithm BLAST program. The phylogenetic tree was constituted using BLAST-Webpage (NCBI). The isolate AMVPR98 showed 99% similarity with Rhizobium pusense strain AB3 (gene bank accession number KY392993.1). Similarly, Girija et al., (2020) identified the isolates obtained from cowpea root nodules as Rhizobium sp. based on 16S rRNA gene sequence analysis.

PGPR are beneficial bacteria, which are able to establish a symbiotic or nonsymbiotic association with plants in the rhizosphere. Rhizobium is the most important PGPR, which is able to develop a symbiotic association with its specific host plant and increase its growth and yield by biologically fixing atmospheric N2. In addition, they support plant growth via synthesis of phytohormones or other growth-promoting or protecting substances like siderophores and antibiotics. The use of PGPR strains in agricultural practices is strongly encouraged as they may constitute a sustainable solution that can improve the efficiency of chemical fertilizers.