Effect of Rhizobium and Rhizobacterial Inoculation on Nitrogen Content and Uptake in Green Gram

Introduction

Green gram (Vigna radiata L.) is a nutritionally rich pulse crop valued for its high protein content, essential amino acids, vitamins, and minerals. It plays a vital role in vegetarian diets and sustainable agriculture due to its ability to fix atmospheric nitrogen and improve soil health. In India, green gram is cultivated over a wide range of agro-ecological regions, with major production concentrated in Rajasthan, Maharashtra, Andhra Pradesh, Telangana, Karnataka, Madhya Pradesh, Tamil Nadu, and Odisha. Its short duration, low water requirement, and compatibility with multiple cropping systems make it an important component of rainfed and irrigated agriculture. However, green gram productivity is often limited by physiological constraints arising from nutrient stress, moisture stress, poor photosynthetic efficiency, and suboptimal nitrogen metabolism. Physiological traits such as chlorophyll content, nodulation efficiency, nitrogen uptake and assimilation, leghemoglobin concentration, and overall metabolic activity directly influence plant growth, yield formation, and grain quality. Under field conditions, these physiological processes are highly sensitive to environmental fluctuations and soil nutrient availability, leading to inconsistent crop performance. Microbial inoculation has gained increasing attention as a sustainable approach to improve plant physiological efficiency without reliance on excessive chemical inputs. Symbiotic Rhizobium and associated rhizobacteria enhance physiological functioning by improving nodulation, biological nitrogen fixation, chlorophyll synthesis, enzymatic activity, and nutrient translocation within the plant. Increased chlorophyll content enhances photosynthetic capacity, while effective nodulation and higher leghemoglobin levels ensure efficient nitrogen fixation and transport, ultimately improving nitrogen use efficiency and plant metabolism. The identification and utilization of efficient microbial strains capable of exerting consistent physiological benefits is a critical requirement in modern pulse production. Single strains often show variable performance due to soil and climatic factors, whereas microbial consortia offer synergistic effects by combining multiple functional traits such as nitrogen fixation, nutrient mobilization, and stress tolerance. The present agricultural scenario demands such biologically driven solutions to enhance crop productivity while maintaining soil health and environmental sustainability. In this context, the present investigation emphasizes the physiological influence of microbial inoculants on green gram, focusing on parameters such as relative chlorophyll content, number and effectiveness of nodules, nodule biomass, shoot and root nitrogen content, and overall physiological performance across different growth stages. A clear understanding of microbial-mediated physiological responses will support the development of efficient bio-inoculant technologies aimed at improving nitrogen economy, photosynthetic efficiency, and sustainable productivity of green gram.

Materials and Methods

Current study was conducted under greenhouse conductions at university of agricultural Sciences, Dharwad with treatments designed with best performing plant growth promoting rhizobia and rhizobacteria after series of invitro characterization. This study was conducted to study the effect of rhizobia alone and with in combination with rhizobacteria for their effect on root nodulation and leghemoglobin content along with their effect on plant physiology which was studied as effect on chlorophyll content. The chlorophyll content in leaves was determined using SPAD (Soil Plant Analysis Development) instrument at 30, 45 and 60 DAS. Parallelly number of nodules and biomass of nodules was estimated. Pink and firmly attached nodules were carefully detached from uprooted plants and were counted. Further, fresh weight of nodules was recorded. The mean values were expressed in gram per plant. For dry weight the samples were dried at 75°C until there was no reduction in weight and the mean values were expressed in gram per plant. Further, the nodules ranging in weight from 50-100 mg were crushed in 9 volumes of Drabkin's solution in a microfuge tube with a glass rod and then they were centrifuged at 12000 rpm for 15 min. The supernatant was filtered through 0.2 µm syringe filter. The filtrate was taken in a micro cuvette and its absorbance was noted using spectrophotometer at wavelength of 540 nm. The nitrogen content in shoot and root Micro-Kjeldhal method was followed (Jackson, 1967). Dried and powdered samples (0.5 g) of shoot and root samples at 30, 45 and 60 DAS were separately digested with 5 ml of concentrated H 2 SO 4 and 200 mg digestion catalyst (K 2 SO 4: Cu 2 SO 4 , Selenium) (100:10:1 ratio) until the contents became clear. After cooling, the volume was made up to 25 ml with distilled water. Then 5 ml of aliquot was transferred to micro-Kjeldhal distillation unit. An aliquot of 10 ml of 40 per cent sodium hydroxide was added and steam distilled. Ammonia evolved was collected into 2% boric acid (20 ml) containing 2 drops of double indicator (83.3 mg bromocresol green and 16.6 mg methyl red indicator dissolved in 10 ml of 95 per cent ethanol) and back titrated against 0.005 N H 2 SO 4 , until the colour changed from green to red. The percentage of nitrogen was calculated by recording the volume of acid run down. The plant nitrogen was calculated by using the formula,

Results

Relative chlorophyll content

The relative chlorophyll content was measured using SPAD meter at 30, 45 and 60 DAS and the results are presented (Table 1). Inoculation of consortium for green gram varieties, increased the relative chlorophyll content of green gram when compared to controls. Among all observation recorded at different DAS i.e., 30, 45 and 60 the treatment M7 inoculum (44.

Number of effective nodules

The number of effective nodules were counted in triplicates at 30, 45 and 60 DAS and the results are documented and represented in Table 2 andplate

Effective nodule fresh weight

The observations pertaining nodules fresh weight were recorded at 30, 45 and 60 DAS and the results are presented in Table 3. Inoculation of consortium for green gram varieties, increased the nodules fresh weight of green gram at 30, 45 and 60 DAS when compared to controls. Among all observation recorded at different DAS i.e., 30, 45 and 60 the treatment M7 inoculum (215.83, 284.17

Root nitrogen content

The nitrogen content of root was analyzed at 30, 45 and 60 DAS and the results are presented. (Table 6) Inoculation of consortium for green gram varieties, increased the root nitrogen content of green gram at 30, 45 and 60 DAS when compared to controls. Among all observation recorded at different DAS i.e., 30, 45 and 60 the treatment M7 inoculum (1.00, 1.44, 0.85) shown highest percentage of root nitrogen content when compared to other treatments followed by M5 inoculum (0.85, 1.33, 0.82), M8 inoculum (0.79, 1.18, 0.67) and least percentage of root nitrogen content among all treatments was shown by M9 inoculum (0.05, 0.20, 0.00) whereas, M1 inoculum with NC-92 showed root nitrogen of 0.32, 0.82, 0.17%. Among the varieties, DGGV-2 with an average of 0.60, 1.00, 0.47 shown higher percentage root nitrogen content when compared to DGG-96 (0.52, 0.94, 0.39) at 30, 45 and 60 DAS respectively. The interaction effect of microbial inoculum with varieties DGGV-2 and DGG-96 was found significant in root nitrogen content at 30, 45 and 60 DAS with root nitrogen content range of 1.07-0.04, 1.47-0.03, 0.97-0.09. The treatment M7 with DGGV-2 recorded significantly highest percentage of root nitrogen content with values of 1.07, 1.47, 0.97 at 30, 45 and 60 DAS respectively.

Leghemoglobin content

The leghemoglobin content in treatments was analyzed at 45 DAS and the results are presented. (Table 8) Inoculation of consortium for green gram varieties, increased the Leghemoglobin content of green gram significantly as compared to controls. Among all observation recorded across all different treatments, microbial consortium with M7 inoculum (0.270) shown highest Leghemoglobin content when compared to other treatments followed by M5 inoculum (0.245), M6 inoculum (0.218) and least Leghemoglobin content among all treatments was shown by M9 inoculum (0.173) whereas, M1 inoculum with NC-92 showed Leghemoglobin content of 0.180. Among the varieties, DGGV-2 with an average of 0.196 shown Leghemoglobin content higher Leghemoglobin content when compared to DGG-96 (0.188) at 45 DAS. The interaction effect of microbial inoculum with varieties DGGV-2 and DGG-96 was found significant in Leghemoglobin at 45 DAS with a range of 0.287-0.137. The treatment M7 with DGGV-2 recorded significantly highest Leghemoglobin of 0.287 at 45DAS. [2] reported inoculation of Rhizobium + VAM+ PSB increased effective root nodule plant -1 and nitrogen use efficiency after conducting an experiment at West Bengal. Justin et al. (2022) [4] conducted an experiment at Cameron and reported that rhizobial isolates significantly increased the plant growth and nodule weight as compared to uninoculated control. They revealed that identified Rhizobium belonging to the same cross inoculation group were useful in symbiotic N₂ fixation, plant growth and yield and enhancing nodule weight in the studied legume.

The results revealed that two varieties were on par with each other and DGGV-2 being the best host for inoculums which was indicated by nodule number, fresh and dry weight of nodules. The efficiency of rhizobial strains was improved with combination of rhizobacteria which is depicted in leghemoglobin content where higher values were observed for microbial inoculum with rhizobacteria. Improved efficiency of rhizobial strains leads to higher nitrogen content in plants, which was proved in present study with higher values of shoot and root nitrogen content for seeds treated with M7(HR-73+ BR-29+ AUDP 203 + AUDP 279) microbial inoculum.

From the above observations, the higher values of shoot nitrogen content, root nitrogen content, number of effective nodules, nodule fresh weight, nodule dry weight, leghemoglobin where observed in plants with consortium treatment followed at 45 DAS when plants are at peak flowering and pod filling stage highest content of nitrogen in plants and number of nodules along with weight of nodules was observed then at 30 DAS indicating the need of plant nutrients for meeting plant nutritional requirement in metabolic activities. Decline of nitrogen content and number of nodules along with weight of nodules at 60 DAS was observed with symptoms of crop harvest and beginning of senescence (Sharma et al., 2024) [5] .

Conclusion

The study conclusively demonstrated that inoculation with Rhizobium and rhizobacteria significantly improves nitrogen content and uptake in green gram. Consortium treatments were more effective than individual inoculants, reflecting enhanced biological nitrogen fixation and better nutrient translocation within the plant system. Improved nitrogen nutrition under microbial treatments highlights the importance of selecting efficient and compatible microbial strains for sustainable nutrient management. These findings support the use of microbial consortia as an eco-friendly strategy to enhance nitrogen availability and productivity in pulse-based cropping systems. Further field-level studies are recommended to validate the consistency of these results under diverse agroclimatic conditions.

Acknowledgement

AcknowledgmentsI am extremely grateful to AICRP on EAAI (Bioconversion Technology) for timely issuing materials, chemical and assistance whenever required.

References

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