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Effect of Microbial Inoculants on Plant Attributes and Nutrients Uptake by Soybean in Vertisols

The present study was carried out during kharif season 2019-20 at the Research Farm, Department of Soil Science & Agricultural Chemistry, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh (INDIA), to assess the effect of microbial inoculants on plant attributes and nutrients uptake by soybean in Vertisols. The experiment was laid out under randomized block design (RBD) with three replications. The 15 treatments comprised of different beneficial microbial consortia in possible combinations applied as seed treatments. The crop was supplemented with recommended dose of fertilizers 20 N : 80 P2O5 : 20 K2O kg ha -1. Besides these, two control plots were maintained as fertilized un-inoculated control (FUI) and unfertilized un-inoculated control (UFUI). The findings revealed that the significant improvement were noticed by the application of consortia NPK+EM+PGPR in plant growth attributes of nodulation at 25, 45 & 65 DAS (71, 70 & 59% respectively), over control (9.5, 33.4 & 34.7 nodule plant-1) and its biomass, (62, 69 & 74% respectively),over the control (0.58, 1.16 & 0.99 g plant-1), plant height at 25, 45 & 65DAS were increased 61, 40, 41% respectively, over the control (16.20, 34.90 and 44.30 cm) and plant biomass, (48, 62 & 53%), over the control 1.67, 4.73 and 6.1 g plant. Similarly, nutrient uptake (seed & stover) Original Research Article Kumar et al.; IJPSS, 33(18): 102-109, 2021; Article no.IJPSS.72709 103 were also increased at 25, 45 and 65 stages of crop growth, with 36.6, 34.8 & 51.3% in seed and 66.7, 98.2 & 67.2% in straw respectively over the control (98.5, 63.8, 5.2, and 7.4, 24.9 and 44.4 kg ha-1 respectively). Thus, it may be concluded that the consortium of NPK + EM + PGPR was superior for sustainable crop production and soil health.


INTRODUCTION
Soybean [Glycine max (L.) Merri.] is an important leguminous oil seed crop and due to nutritional value, it has been considered as "Protein hope of future". Generally, soybean contains 40-45% protein and 18-20% oil [1]. The top two soybean growing states in India are Madhya Pradesh and Maharashtra with 45% and 40% shares of this crop, respectively. In Madhya Pradesh the soybean cultivation spreads over 5.2 M ha with total annual production of 6.7 M tones and productivity of 1285 kg ha -1 [2]. Soybean rhizosphere harbors vast proportions of soil microorganisms, whose activities largely determine the biological condition of the soil and influence the plant growth right from seed germination to maturity [3].
Different microbial consortia i.e. Pseudomonas as PGPR is the most efficient and effective strain with significant remarks on isolates of P. fluorescens and P. putida increasing growth and yield of different crops, especially legumes. Rhizobium (diazotroph) is a Gram-negative bacterium and symbiotic N 2 -fixer with roots of legumes. It colonizes the roots of specific legumes to form tumor like growths called root nodules, which acts as the factories of ammonia production [4]. Bacillus subtilis a soil Grampositive catalase-positive bacterium is known also as the hay bacillus or grass bacillus. The Bacillus sp. produces soluble exudates which is composed of five organic acids; gluconic acids, succinic acids, lactic acetic and propionic acids. The action of organic acids is recognized as a major mechanism responsible for the release of phosphates from the hydroxyl apatites [5]. Rhodopseudomonas palustris is a photrophic, rod-shaped, Gram-negative purple non-sulfur bacterium, having ability to switch between four different modes of metabolism [6]. Its role in degrading aromatic compounds has been in evidence extensively.
R. palustris utilizes a reductive coenzyme to convert via reduction and dehalogentation of 3chlorobenzoate into acetyl-CoA and CO 2 . The purple non-sulfuric bacteria can also perform N 2fixation due to having unique genome encodes for three different nitrogen fixing enzymes. Saccharomyces cerevisiae is saprophytic, single celled egg-shaped yeast-fungi generally larger than most bacteria that grow on sugary solution, grapes etc. and known to contribute pleasant smell of bread. Soil yeast and yeast-like fungi produce a variety of biologically active compounds viz. phytohormones, vitamins, amino acids, enzymes etc. [7]. It has active stimulating effect on the plant growth, development and productivity. In addition, yeasts produce antimicrobial substances helping to reduce phytopathogenic infection.
Isolates of constitutional microorganisms of EM culture (Effective Microbial Culture) individually have already been evidenced beneficial but their consortium could be more valuable to augment the supply of nutrients through solubilization, anti-phytopathogenicity, induced phytoresistance and phytostimulator. In view of the above, the present investigation was carried out to find the effect of different microbial inoculants on plant attributes and nutrient uptake by soybean in a Vertisols.

MATERIALS AND METHODS
The present investigation was focused on effect of different microbial inoculants on nodule attributes, plant growth and nutrient uptake of soybean in Vertisols. The study was carried out during kharif season 2019-20 at the Research Farm, Department of Soil Science & Agricultural Chemistry, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh (INDIA). It is situated at 23°10'N latitude and 79°57" E longitude at 393 meters above the mean sea level.
The soil of the experimental site is Vertisol belonging to Kheri series of fine montmorillonitic hyperthermic family of Typic Haplusterts with pH of 7.15, electrical conductivity 0.24 dS m -1 (1 : 2.5 soil : water ratio) and organic carbon 5.2 g kg -1 . The soil available N, P and K were 226, 15.8 and 282 kg ha -1 , respectively. The soil available N was estimated by alkaline permanganate method [8], available P by Olsen's method [9] and available K by ammonium acetate extraction method [10]. At the start of the experiment, microbial population counts ware also analyzed by Serial Dilution Technique [11], which is presented in Table 1. (N : P2O5 : K2O kg ha -1 ). The sources of N, P and K used were urea, single super phosphate and muriate of potash. Besides these, two types of control plots were maintained as fertilized uninoculated control (FUI) and unfertilized uninoculated control (UFUI) to measure the comparative effects of different microbial inoculants. The treatment combinations are presented in Table 2.

Seed Treatment and Inoculation
Soybean seed was weighed 40 g separately for each plot in clean polythene bags. One ml of liquid formulation of each microorganism was transferred aseptically to previously sterilized appendage tubes (micro centrifuge tubes). Sterilized gum acacia (2%) was used as sticker solution and one ml of this solution was also transferred aseptically to micro centrifuge tubes separately. Seed treatment was done in shade and seeds were sown manually as early as possible. After germination, thinning was done to maintain the required plant population. Necessary plant protection measures were taken as per recommended package of practices [12]. 1.51 X 10 3 Lactobacillus sp.

Nutrients Uptake
The seed and stover of soybean was taken to determine nutritional consistence. For the determination of nitrogen content in soybean crop micro kjeldahl method was used [13], for phosphorus content of the plant extract was determined using Vanado Molybdo phosphoric yellow colour method [14] and potassium was determined with the help of flame photometer [14]. On the basis on nutrient content the nutrient uptake of soybean was calculated in kg ha -1 in relation to (dry matter production) yield ha -1 . All observations recorded on nodule attributes, crop growth and nutrients uptake were analyzed statistically [15].

Nodulation of soybean at different growth stages
Studies on nodulation (nodule enumeration and its biomass) and the data on number of nodules and its oven dried weight per plant at 25, 45 and 65 DAS of crop growth stages have been presented in Table 3, which clearly indicated that the microbial consortium of NPK+EM+PGPR recorded significantly higher nodule number . The increase in nodulation might be due to synergistic effect of these microorganisms for biological nitrogen fixation as against their individuals [12]. Similar findings were also reported by [16] which clearly revealed that the number of nodules increased 30% by inoculation over un-inoculated.  [17] also agreed with the findings that the consortia of bradyrhizobia and phosphate solubilizing bacteria (Pseudomonas sp.) as liquid inoculants on soybean enhanced the nodule number, fresh weight and dry weight of nodules. Similar findings have also been recorded by [12].

Plant height and its biomass at different Growth Stages of Soybean
Among all the treatments at 25 DAS, the consortium of NPK+EM+PGPR responded best for plant height of 26.1 cm and 61% increase over that of FUI (16.2 cm), followed by PK+EM+PGPR, PK+EM, NPK+EM and Rhizo+PGPR, with response of 50.1, 40.0, 33.9, and 33.1%, respectively (Table 4). Similar findings ware also reported at 45 and 65 DAS. It was reported that salicylic acid increases the cell division of apical meristem of seedlings and thereby improve plant growth. Effective microbial culture (Ems) enhance plant growth (plant height) and productivity by fixing atmospheric nitrogen and supplementing the plants with the fixed nitrogen as ammonia. Additionally, the release of trace elements, secreted antioxidant, exo-polysaccharides, bioactive compounds (vitamins, hormones and enzymes) by the EMs stimulate plant growth and productivity [18].
The plant biomass (  73 g plant -1 ). Further, at 65 DAS the treatment NPK+EM+PGPR performed best with maximum plant biomass of 9.35 g plant -1 and 53.30 % increase over that of FUI (6.10 g plant -1 ). The higher plant biomass under this treatment combination was mainly due to more availability of N, P, K and S in soil for soybean plants. Moreover, growth promoting substances (phytohormones) are produced by these organisms which further promote plant biomass [19].

Nutrients Uptake by Soybean
The data pertaining to nutrient uptake by soybean was presented in Table 5 revealed that the nutrient uptake differs significantly with different treatment combinations. In seed, maximum uptake of N, P and K recorded under treatment receiving NPK+EM+PGPR was 138, 15.5 and 44.4 kg ha -1 , respectively. Applied NPK and EM with PGPR helped to increase the N uptake by soybean seed as compared to the application of PGPR alone (104, 12.3 and 33.8 NPK kg ha -1 , respectively). This might be attributed to potential of the endophytic actinomycetes that produces plant growth promoters used as co-inoculants with Bradyrhizobium japonicum to enhance the growth of soybean crop. These endophytes exhibited the potential to enhance plant growth, nitrogenase activity of root nodules and plant nutrients uptake as well as the co-inoculation significantly increased the nutrient levels of nitrogen within a soybean plant [20]. Similar findings were also reported by [ 21& 22]. Further, NPK consortium gave the 127, 15.5 and 38.8 kg ha -1 NPK uptake, respectively. In stover the highest NPK uptake (100, 31.5 & 96.8 kg ha -1 , respectively) was recorded with NPK+EM+PGPR treatment combinations, followed by PK+EM+PGPR treatment combination gave 28.8 kg ha -1 P uptake and 91.6 kg ha -1 K uptake, while Rhizobium alone treatment gave 98 kg ha -1 N uptake.

CONCLUSION
It was concluded that maximum number of nodules and its biomass was recorded with the consortium of NPK + EM + PGPR. This consortium also exhibited the best in term of plant height and its biomass and nutrients uptake (NPK) by seed and stover of soybean crop. The consortium of NPK + EM + PGPR was superior for N uptake by soybean crop and 36% response over FUI, while for P and K uptake by soybean crop, response of the consortium NPK + EM + PGPR over Fertilizer Un Inoculated (FUI) control were 44.6% and 51.3%, respectively.