ANISHA SHRIVASTAVA, Ph.D. Scholar 
Dept. of Agricultural Microbiology COA, 
IGKV, Raipur Chhattisgarh
DR. SUBUHI NISHAD, Programme officer (NSS Girls unit), 
Indira Gandhi Krishi Vishwavidyalaya Raipur Chhattisgarh
DR. DIPTIMAYEE DASH, Department of Agricultural Microbiology, 
College of Agriculture,
Indira Gandhi Krishi VishwaVidyalaya, Raipur Chhattisgarh


INTRODUCTION
There is always a challenge to intensify agriculture, agriculture being the backbones of many economies worldwide makes it essential to ensure the production and productivity to fulfill entire population. Many hurdles comes in the way to make it sustainable like the environmental factors which have different biotic and abiotic stresses which includes disease and insect incidence and to overcome these; farmers have use synthetic fertilizers to improve soil fertility. Especially nitrogenous fertilizers are used as almost all soils are deficient in nitrogen. Further Nitrogen is one of the important nutrients required by plants being components of nucleotides, nucleic acids, amino acids, chlorophyll, hormones, and enzymes. Synthetic fertilizer is costly as well as rarely available to many local farmers these are also not safe for our environment many of them cause various detrimental effects on soil health like soil acidification, reduce in the efficiency of soil also. The impact of continuous applying fertilization in agricultural practices can lead to a detrimental effect on the environment. Microorganisms establish associations with plants and promote plant growth using several beneficial characteristics. For inoculation, endophytes are suitable, which reflects the ability of these organisms for plant colonization.

Nitrogen is important and one of the major nutrients which is essential for plant growth. Nitrogen can be easily obtained by leguminous plants from air by working symbiotically with specific bacteria (Bradyrhizobium or Rhizobium species) in root nodules of plant. There are several reasons which limit growth and N2 fixing activity of the organisms in environmental. Zahran (1999) reported that the behavior of some nitrogen fixing systems under severe environmental conditions such as drought stress, salt stress, alkalinity, acidity, fertilizer, nutrient deficiency, pesticides and heavy metals. Throughout the world Bradyrhizobium is popularly used as a bio-inoculants which is using is slow growing root nodule symbiont, in soybean fields.

Several approaches appreciate the mechanisms and deliver tools to intensify plant tolerance under environmental stresses. Plant growth-promoting rhizobacteria (PGPR) is one of the best option and emerging technologies used to resolve this problem naturally. Effectiveness of PGPR is shown by various reports increasing plant growth and development (Glick, 2012; Paul and Nair, 2008; Zahir et al., 2004).

Isolation of Bradyrhizobium and Pseudomanas from Soybean rhizosphere soil samples by serial dilution method

The plant growth promoting rhizobacteria were isolated from rhizosphere soil samples collected from soybean crop grown in research field of (Glycine max (L.) Merr.) by using serial dilution method. For isolation of Bradyrhizobium japonicum and Pseudomonas fluorescens, YEMA and KING’S B medium was used respectively (Shrivastava, A., & Dash, D. 2020)

3.3.2.1 Serial dilution method
Collected soil sample were weighed 1gram of each separately and labeled the sterilizes water blank test tubes as 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8.
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Prepare initial dilution by adding 1 gram soil sample in 9 ml sterilized distilled water labeled 10-1. Thus, diluting original sample 10 times.
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Soil sample were shaken vigorously on a vortex mixture.
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Test tube were left 5min for settlement.
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Transfer 1ml of soil suspension and mixed with 9ml sterilized distilled water in tube to 10-2 dilution.
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Soil sample were shaken vigorously on a vortex mixture.
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Again one ml of suspension from 10-2 to 10-3 was taken and mixed with a fresh sterile pipette.
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Repeat the procedure till it reaches to 10-8 dilution.
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Approximately 20ml melted medium added to each sterilized petriplate after it get solidify 1ml aliquot from 10-5, 10-7 dilutions were pore plated on YEMA media and KING’S B media.
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Plates were incubated at 300c ± 20c for 48 hrs.
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Discrete bacterial colonies were picked on the basis of their morphological appearance and subculture for purification in case of F. pseudomonas colonies were detected by viewing under U.V. light then purified.


Mass production of Bradyrhizobium and Pseudomanas
Mass production of Bradyrhizobium and P. fluorescence involves the large-scale cultivation of these bacteria to produce a sufficient quantity of biomass for use in agriculture as a bio-fertilizer or bio-pesticide. The process of mass production of Bradyrhizobium and P. fluorescence typically involves several steps, including isolation and selection of the best-performing strains, inoculums preparation, large-scale fermentation, harvesting and processing, formulation and packaging, and quality control. Each of these steps requires careful optimization to ensure consistent production of high quality biomass that is effective in promoting plant growth and controlling plant diseases. Fluorescent Pseudomonas has gained popularity as an alternative to chemical fertilizers and pesticides, which can have negative impacts on the environment and human health. Mass production of Bradyrhizobium and P. fluorescence can help to reduce the dependence on synthetic chemicals in agriculture and promote sustainable agricultural practices. Overall, mass production of Bradyrhizobium and P. fluorescence represents a promising approach to enhance plant growth and protect plants from diseases in an environmentally friendly and sustainable manner. With continued research and development, the use of fluorescent Pseudomonas in agriculture is likely to become increasingly widespread in the coming years.

STEPS INVOLVED IN MASS PRODUCTION OF Bradyrhizobium and Pseudomanas

Isolation and Selection
Isolation of Bradyrhizobium and Pseudomonas fluorescence strains from soil or plant roots (based on their ability to promote plant growth and control plant diseases.)
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Inoculums Preparation | Preparing a starter culture (mother culture)
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Large-Scale Fermentation
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Formulation and Packaging
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Formulating the final product (e.g., liquid, powder, granules)
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Quality Control
(May involve testing the product for bacterial viability, purity, and potency, as well as conducting field trials to assess its efficacy under different conditions.)

Application methods of Bradyrhizobium and Pseudomanas
Bradyrhizobium and Pseudomanas can be applied in various ways to enhance plant growth and protect plants from diseases. Here are some common methods of application:

Seed Treatment: Bradyrhizobium and P. fluorescence can be applied to seeds before planting to promote seed germination, seedling growth, and protect seedlings from soil-borne pathogens. The bacterial cells can be mixed with a seed treatment solution and applied to seeds using seed coating or soaking methods.

Soil Application: Bradyrhizobium and P. fluorescence can be applied to soil directly or through irrigation systems to establish beneficial populations of bacteria in the rhizosphere, the soil around the roots of plants. The bacterial cells can be applied in the form of liquid suspensions or granules that are mixed into the COMPOST or applied to the soil surface.

Foliar Application: Bradyrhizobium and P. fluorescence can be applied to plant leaves and stems to promote plant growth and protect plants from foliar diseases. The bacterial cells can be applied in the form of a spray solution that is applied to the plant foliage.

Root Drench: Bradyrhizobium and P. fluorescence can be applied as a root drench, where a liquid suspension of the bacterial cells is poured directly onto the soil around the base of the plant. This method allows the bacterial cells to establish a beneficial population in the rhizosphere and protect the plant roots from soil-borne pathogens.

Compost Tea: Bradyrhizobium and P. fluorescence can be added to compost tea, a liquid fertilizer that is made by steeping compost in water. The bacterial cells help to break down organic matter and release nutrients that are available to plants.

The choice of application method depends on the type of crop, the target disease, and the desired outcome. To achieve optimal results, it is important to follow recommended application rates and timing and to use high-quality, viable bacterial cells.

Benefits of Bradyrhizobium and Pseudomanas on plant growth promotion and disease management.

Biological nitrogen fixation: In a study conducted by Arora and Singh (2017), Bradyrhizobium and P. fluorescence strains were found to fix atmospheric nitrogen and enhance plant growth in chickpea.

Production of plant growth-promoting hormones: In a study by Shukla et al; (2016), Bradyrhizobium and P. fluorescence strains were found to produce plant growth-promoting hormones and enhance plant growth in wheat.

Solubilization of phosphate: In a study by Vyas and Gulati (2009), Bradyrhizobium and P. fluorescence strains were found to solubilize phosphate and increase the availability of phosphorus for plant uptake in rice.

Production of antibiotics: In a study by Kloepper et al; (1980), Bradyrhizobium and P. fluorescence strains were found to produce antibiotics that protect plants from soil-borne pathogens.

Induction of systemic resistance: In a study by De Vleesschauwer et al. (2008), Bradyrhizobium and P. fluorescence strains were found to induce systemic resistance in rice plants against bacterial blight disease.

Biocontrol of plant pathogens: In a study by Höfte & Altier (2010), Bradyrhizobium and P. fluorescence strains were found to act as bio-control agents against soil-borne pathogens in tomato plants. Overall, these studies and others support the potential of Bradyrhizobium and P. fluorescence bacteria as a sustainable and eco-friendly solution for improving plant growth and protecting plants from diseases.

Conclusion
Bradyrhizobium and P. fluorescence can have a significant positive impact on sustainable agriculture. By promoting the use of these beneficial microbes, farmers can reduce their reliance on chemical fertilizers and pesticides, which can have negative environmental and health impacts.

Educating farmers about the advantages of Bradyrhizobium and P. fluorescence and demonstrating its effectiveness in promoting plant growth and controlling plant diseases can encourage widespread adoption of these eco-friendly agricultural practices. Empowering farmers with knowledge and resources to utilize Bradyrhizobium and P. fluorescence can not only benefit their crops, but also contribute to a healthier and more sustainable food system.

Reference

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