Bioinsecticide /Biofertilizers isolation production purification and assay

BACKGROUND

Farmers pay billions of dollars in costs due to insects and other pests, which increase production costs by reducing product quality and quantity. Chemical pesticides are frequently used in underdeveloped nations due to their efficiency and affordability.

Chemical pesticides are frequently used in India, especially to manage lepidopteron pests of the Noctuidae family, including Spodoptera litura (cutworm), Heliothis armigera (pod borer), and Plutellidae, including Plutella xylostella. Chemical pesticides typically poison non-target species, including humans. Additionally, frequent usage may result in the development of resistance traits, resulting in an insect variety.

Crop rotation, which avoids the target host and encourages the development of adventitious root systems, is one cultivation technique that can help with the resistance issue. Biopesticides (both living organisms and chemical compounds of organic origin) are target specific, easily biodegradable, better having less self-life, and user pleasant for sustainable agriculture, making them a better alternative to synthetic chemical pesticides.

A variety of soil microorganisms engage in metabolic activity in the rhizosphere, the area of soil around the roots. These microorganisms may influence plant development and productivity in a positive, neutral, or adverse way. Plant growth-promoting rhizobacteria (PGPR), a group of microorganisms that are helpful to plant growth, are of interest to the agricultural industry as possible biofertilizers and (or) biopesticides.

The selection of the most promising microbes for further development as biofertilizers or biopesticides can be aided by the creation of a database of isolates classified for physiological features that promote plant growth.

Rhizosphere bacteria are one type of biofertilizer that helps plants grow by producing phytohormones like indole-3-acetic acid (IAA) and cytokinins, increasing the availability of nutrients like phosphorus and iron, and aminocyclopropane-1-carboxylic acid (ACC) deaminase, which reduces the production of ethylene, a plant growth inhibitor.

Rhizosphere bacteria are a type of biopesticide that inhibit the activity of phytopathogens by producing antagonistic compounds like antibiotics, competing with the plant for vital nutrients, and inducing both systemic and localized defence mechanisms.

REQUIREMENTS

Healthy plant (Fenugreek or Pea)
Sterile distilled water
0.1% mercuric chloride
70% ethanol
Glass rod
BOD incubator
Plant seed
Soft agar
Congo red yeast extract mannitol agar (CRYMA media):	Composition (g/L): Yeast extract-1, Mannitol-10, Dipotassium phosphate-0.5, Magnesium sulphate-0.2, Sodium chloride-0.1, Congo red-0.25, Agar-20, pH-7.0
Yeast extract mannitol agar (YEMA):	Composition (g/L): Yeast extract-1, Mannitol-10, Dipotassium phosphate-0.5, Magnesium sulphate-0.2, Sodium chloride-0.1, Agar-20, pH-7.0
Jensen’s medium:	Composition (g/L): Sucrose-20.0, Dipotassium phosphate-1.0, Sodium chloride-0.5, Magnesium sulphate-0.5, Ferrous sulphate-0.10, Sodium molybdate-0.005, Calcium carbonate-2.0, Agar-20.0
Pikovskaya medium:	Composition (g/L): Glucose-10.0, Ammonium sulphate-0.5, Potassium chloride-0.2, Magnesium sulphate-0.1, Yeast extract-0.5, Tricalcium phosphate-0.5, Sodium chloride-0.2, Ferrous sulphate- 0.0001, manganese sulphate- 0.0001, Agar- 20, pH 7.2

PROCEDURE

1. Take healthy plant (Fenugreek or Pea) with root nodules and its surrounding soil. ^[2]
2. Separate the heathy root nodules and its surrounding soil, wash root nodules with distilled water.
3. For Rhizobium isolation immersed root nodules into the 0.1% mercuric chloride (HgCl₂) and 70% ethanol for 2-3 min. ^[1]
4. Transfer the root nodules to the beaker containing sterile distilled water and crushed with the help of glass rod.
5. The root nodule suspension was plated on the CRYMA media, incubate in the BOD incubator at 28˚C for 24hr.
6. Observe for the small round, colourless or white with central red dot colonies.
7. Selected colonies transfer to yeast extract mannitol agar medium for pure culture maintain till further use.
8. Prepare 300 ml yeast extract mannitol broth, inoculate with the isolated culture.
9. Incubate at 28±2˚C in shaking condition for 3-5 days depending on the culture growth.
10. For Azotobacter isolation, 1 gm root nodule surrounding soil add to sterile distilled water and prepare dilution of it upto the 10^-5.
11. 0.1 ml of 10^-3 or 10^-4 suspension is spread on the Jensen’s agar plate, incubate at 30˚C in BOD incubator.
12. Observe for the light brown to black pigmented colonies, select those and prepare pure culture till further use.
13. Prepare 300 ml Jensen’s broth, inoculate with the isolated culture.
14. Incubate at 28±2˚C in shaking condition for 3-5 days depending on the culture growth.
15. For bioassay of the isolates, ^[1][2]

1. Seed germination assay:
   • Surface sterilize the seeds with 0.1% HgCl₂ for 2-3 min, followed by washing with sterile distilled water and decant the water.
   • Add the seeds to cultures grown in respective medium containing 10⁶ cells/ml for 48 hrs.
   • Transfer the seed to soft agar plate and incubate at 30˚C for 3-5 days.
   • Observe for the root and shoot length.
2. Phosphate solubilization:
   • Prepare the Pikovskaya medium, autoclave it and prepare the plates of it.
   • Add 10 µl isolated microbial suspension, incubate at 30˚C for 48 hr in BOD incubator.
   • Observe for clearing zones around the colonies for phosphate solubilization.

CONCLUSION

Plant growth Rhizobium isolated from root nodules and nitrogen-fixing Azotobacter isolates from soil are among the bacteria. Their production is done by using the selective production medium, and their conformation is done by the bioassay methods.

REFERENCES

1. Dr. Ajar Nath Yadav, “Production Technology for Bioagents and Biofertilizers”, A Laboratory Manual, 2021.
2. Russell K. Hynes, Grant C.Y. Leung, Danielle L.M. Hirkala, and Louise M. Nelson, “Isolation, selection, and characterization of beneficial rhizobacteria from pea, lentil, and chickpea grown in western Canada”, Can. J. Microbiol, 2008.
3. Irda Safni, Lisnawita, Khairunnisa Lubis, Ahmad R. Tantawi, Surya Murthi, “ISOLATION AND CHARACTERIZATION OF RHIZOBACTERIA FOR BIOLOGICAL CONTROL OF ROOT-KNOT NEMATODES IN INDONESIA”, Journal ISSAAS, Vol. 24 (1), 2018.
4. Pritam Chattopadhyay, Sandipan Chatterjee, Shrikanth Gorthi and Sukanta K. Sen, “Exploring Agricultural Potentiality of Serratia entomophila AB2: Dual Property of Biopesticide and Biofertilizer”, British Biotechnology Journal, Vol. 2 (1), 2012.

Also read:

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• Strain improvement of the industrially important isolate A. niger using EtBr for higher yield of the product
• Alcohol Fermentation using different substrates and its downstream processing
• Fermentation process of alcohol production
• Preparation of Baker’s Yeast from molasses

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