Influence of Bioinoculants on Growth and Yield of Cowpea (Vigna unguiculata L.) under Field Condition

Cowpea is an annual herbaceous legume from the genus Vigna and it is native to Africa (Carvalho et al., 2017). Cowpea grain offers key vitamins including thiamine, riboflavin, ascorbic acid, niacin and folic acid. It is low in fat  and represents a fair source of fiber of about 6% (Kushwaha and Kumar, 2013). It is relatively low in sulphur containing amino acids but high in lysine and other essential amino acids, making it a good complement to the main cereal diets (Ohler and Mitchell, 1992).
       
Bioinoculants naturally activate microorganisms found in the soil being cheaper, effective and eco-friendly agents that gaining importance for use in crop production, restoring the soil natural fertility and protecting it against drought, soil diseases and therefore stimulate plant growth (Suhag, 2016). Bioinoculants such as Rhizobium sp., Bacillus megaterium and Glomus mosseae were together known to give the best results in pulses.
       
Rhizobium is a Gram-negative, rod-shaped bacteria generally found as root nodule-forming bacteria in legumes which fix atmospheric nitrogen with the help of nitrogenase enzyme (Somasegaran and Hoben, 2012). Bacillus megaterium is a potential phosphate solubilizing bacteria (PSB) found in different rhizospheric soil. Glomus mosseae is now known as Funneliformis mosseae (Schubler and Walker, 2010). It has the ability to adapt to various agricultural management.
 
 

A field experiment of this study was conducted at Zonal Agricultural Research Station, GKVK, UAS Bangalore, during kharif 2020 and further microbiological and biochemical analysis were carried out in the Department of Agricultural Microbiology, UAS, GKVK, Bangalore. The experiment plot was laid out in the RCBD design with three replications. The cowpea variety selected for this study was KBC-2. The bioinoculants used for the study were Rhizobium sp., Bacillus megaterium and Glomus mosseae. Treatments are comprised of, T₁: Control, T₂: Recommended dose of fertilizer (N:P₂O₅:K₂O kg/ha: 10:20:10), T₃: 50% of N and P₂O₅+100% K₂O+Rhizobium sp. (R. sp.), T₄: 50% of N and P₂O₅+100% K₂O+B. megaterium (B. m.), T₅: 50% of N and P₂O₅+100% K₂O+Glomus  mosseae (G. m.),T₆: 50% of N and P₂O₅+100% K₂O+R.sp., B.m., G.m., T₇: 75% of N and P₂O₅+100% K₂O+R. sp., T₈: 75% of N and P₂O₅+100% K₂O+B.m., T₉: 75% of N and P₂O₅+100% K₂O+G.m., T₁₀: 75% of N and P₂O₅+100% K₂O+R. sp., B.m., G.m. Observations on traits viz., plant height, number of branches, number of nodules plant^-1,fresh weight of nodules, chlorophyll content (SPAD reading), plant N and P content, microbial biomass carbon, microbial biomass nitrogen, microbial biomass phosphorus, enumeration of N₂ fixers, phosphate solubilizers, spore load of AM fungi, enzymes such as dehydrogenase, urease, acid and alkaline phosphatase activity of soil were recorded.
 
Chlorophyll content (SPAD reading)
 
Observations were recorded from five randomly selected and tagged plants, during flowering stage of crop . The SPAD meter reading were taken from top, middle and bottom of completely opened green leaves and further average value was computed.
 
Nitrogen and phosphorus content in plant
 
Nitrogen and phosphorus content in plants were analysed at flowering and harvest stage of crop by micro kjeldhal distillation method (Piper, 1966) and vanodomolybdophosphoric  yellow colour method (Piper, 1966) using spectrophotometer, respectively.
 
100 Seed weight
 
One hundred seeds were counted from the produce of each treatment separately in each replication. These seeds were weighed on electronic balance mean 100 seed weight is expressed in grams.
 
Estimation of microbial activities in rhizospheric soil
 
Soil microbial biomass
 
Microbial biomass carbon, nitrogen and phosphorus were estimated during flowering and harvest stage of the crop. Microbial biomass carbon and nitrogen were estimated by using fumigation and extraction method proposed by Carter (1991). Microbial biomass P were estimated as per the procedure given by Brookes et al., (1982).
 
Enzyme activity
 
Soil enzymes like dehydrogenase, urease, acid phosphatase and alkaline phosphatase were estimated. These enzyme activities were estimated in the rhizospheric soil during flowering and harvest. Dehydrogenase activity in the samples was determined by following the procedure described by Casida et al., (1964). The urease activity and phosphatase activity of samples was determined by following the standard procedure of Eivazi and Tabatabai (1977).
 
Enumeration of functional groups of microorganisms
 
Enumeration of free-living nitrogen fixers and phosphate solubilizers in the rhizospheric soil was carried out using standard serial dilution and plating method. Using N free Jensen’s media and Pikovskaya’s medium Spore load of AM (Arbuscular Mycorrhiza) fungi was estimated using wet sieving and decanting method proposed by Gerdemann and Nicholson,1963.
 
Statistical analysis
 
The data obtained from field experiment was statistically analysed using WASP: 2.0 (Web Agri Stat Package 2) statistical tool (www.icargoa.res.in/wasp2/index.php) and means were separated by Duncan Multiple Range Test (DMRT).
 

Effect of bioinoculants on growth and yield attributing parameters of cowpea (Vigna unguiculata L.) under field condition
 
The growth and yield attributing parameters of cowpea during flowering and harvest were indicated in Table 1 and 2  respectively.




       
Growth parameters in cowpea revealed that, there is significant differences among the treatments at flowering and harvest. During flowering in treatment T₁₀ (75% of N₂ and P +100% K+R. sp., B.m., G.m.) recorded highest plant height, highest number of branches, number of nodules, fresh weight of nodules, chlorophyll content, plant nitrogen and phosphorus content i.e., 35.07, 10.44, 5.56, 1.74g, 35.82, 2.98% and 0.31% respectively, followed by T₆: 50% of N₂and P+100% K+R. sp., B.m., G.m. (33.80, 6.89, 4.44,1.07g, 35.48, 2.84% and 0.29% respectively). Yield attributes recorded in cowpea showed highest yield of the crop and seed index in T₁₀ (9.48q/ha and 24.89 g respectively) and followed by T₆ (9.20q/ha and 22.91g respectively).
       
Bioinoculants are known to increase the growth regulating hormones there by increase in the growth and yield attributing parameters. Similar results were obtained by Pramanik and Bera, (2012) in which they showed that the application of bioinoculants increased the yield due to production of growth regulators. Harireddy and Dawson, (2021) also mentioned that the application of inorganic fertilizer or compost along with the bioinoculants increase the growth and yield attributing parameters.
 
Effect of bioinoculants on functional group of microorganisms in the rhizosphere of cowpea (Vigna unguiculata L.) under field condition
 
Effect of bioinoculants on the microbial density in rhizospheric soil of different treatments during flowering and harvest in the rhizosphere of cowpea is depicted in the Table 3.


       
Changes in the population of the function group of microorganisms in the rhizospheric soil viz., nitrogen fixers, phosphate solubilizers and phosphorus mobilizers were significantly influenced during flowering and harvest by the application of bioinoculants. Highest population of N₂ fixers (5.9310³ and 5.82×10³ CFU g^-1 soil respectively), phosphate solubilizers (3.96×10³ and 3.87×10³ CF^{U g-1} soil respectively) and AMF spore load (169.67 and 167.53 spores/100 g of soil respectively) were noticed in the treatment T₁₀ (75% of N₂and P+100% K+R. sp., B.m., G.m.). The microbial load in the rhizospheric soil is gradually reduced from flowering to harvest.
       
Seed inoculation with bioinoculants increased the microbial population, could be due to modification of biochemical changes in soil. The microorganisms in rhizospheric soil is responsible for providing favourable physical properties, which help in the mineralization of soil nutrient leading to higher available phosphorus and potassium. The beneficial effect of microorganism on potassium availability includes minimization of the losses from leaching. Pradip, 2016 and Macik et al., 2020 conducted the experiment in cowpea in which application of bioinoculants improved the soil fertility and increased crop yields by nitrogen fixation, potassium and phosphorus solubilization, production of phytohormones, substances suppressing phytopathogens and guarding plants from abiotic and biotic stresses.
 
Effect of bioinoculants on microbial biomass carbon, nitrogen and phosphorous in the rhizosphere of cowpea (Vigna unguiculata L.) under field condition
 
Observations on the effect of bioinoculants on the soil microbial biomass carbon, nitrogen and phosphorus in the rhizosphere of cowpea during flowering and harvest are presented in Table 4.



Soil microbial biomass carbon, nitrogen and phosphorus have varied significantly among the different treatments as recorded in cowpea. Highest microbial biomass carbon, nitrogen and phosphorus was recorded in the treatment T₁₀: 75% of N₂ and P+100% K+R. sp., B.m., G. m. (246.49, 28.76 and 53.88 mg g^-1 soil respectively) during flowering and (238.46, 27.82 and 53.46 mg g^-1 soil respectively) during harvest, followed by the treatment T₆: 50% of N₂ and P+100% K+R. sp., B.m., G.m. (236.86, 27.63 and 52.80 mg g^-1 soil respectively) during flowering and (224.43, 26.18  and 49.72 mg g_-1 soil respectively) during harvest.
       
Treatment T₁₀ which received 75% RDF along with microbial consortia showed significantly higher soil microbial biomass carbon, nitrogen and phosphorous compared to the treatments that received inorganic fertilizers, single bioinoculant  and control. This could be due to higher amount of organic matter inhabiting microorganisms compared to the treatment received inorganic fertilizer to supply nutrients. The results obtained were in agreement with the work carried out by Balakrishna (2001) and Latkovic et al.,(2020).
 
Effect of bioinoculants on soil enzymes activityin the rhizosphere of cowpea (Vigna unguiculata L.) under field condition
 
Effect of bioinoculants on soil enzymes activity in the rhizosphere of cowpea under field condition is represented in the Table 5.


       
Significant difference was recorded with respect to the soil enzymes in the rhizosphere during flowering in cowpea. Highest dehydrogenase, urease, acid and alkaline phosphatase were recorded during flowering in the treatment T₁₀: 75% of N₂and P+100% K+R. sp., B.m., G.m. (6.39 mg TPF g^-1 of soil h^-1, 21.56 mg NH₄⁺-N g^-1 of soil h^-1, 27.60 mg PNP g^-1 of soil h^-1 and 21.97 mg PNP g^-1 of soil h^-1 respectively) followed by the treatment T6: 50% of N₂and P +100% K+R. sp., B.m., G.m. (5.19 mg TPF g^-1 of soil h^-1, 19.97 mg NH₄⁺-N g^-1 of soil h^-1, 25.20 mg PNP g^-1 of soil h^-1 and 18.04 mg PNP g^-1 of soil h^-1 respectively).
       
Higher enzyme activity in soil in above mentioned treatment might be due to the application of bioinoculants that resulted in higher biomass production and extensive root exudates in the rhizosphere and this might have promoted the build-up of microbial population in soil. Application of bioinoculants induces better proliferation of roots and is responsible for the higher soil enzymes activity. Similar results were reported by Abd elgwad. (2019) in cowpea with higher dehydrogenase due to microbial inoculation and Kaur et al.,(2017) in greenpea there was higher dehydrogenase, urease and alkaline phosphatase in soil due to addition of co-inoculation of bioinoculants. The lower enzymes activity in soil was observed in control with no bioinoculants seed inoculation might be due to restricted root growth and lesser exudates from roots which might have caused lower enzymes activity in soil.

Modern agriculture involves the use of hazardous chemical fertilizers, pesticides and high yielding varieties. These bioinoculants will help to overcome the usage of these  chemicals by  providing essential nutrients to plants and improve plant resistance against diseases.  Hence from the present study conclude that the use of bioinoculants will partly replace the  inorganic fertilizers.