Everything you need to know about bio-fertilizers. Some of the most frequently asked questions are as follows:-
Q.1. Define bio-fertilizers.
Ans: Bio-fertilizers or microbial inoculants can be defined as preparations containing live or latent cells of efficient strains of nitrogen fixing, phosphate solubilizing or cellulolytic microorganisms used for application with seeds, soil or composting areas with the objective of increasing the number of such cellulolytic microorganisms that accelerate certain microbial processes to increase the extent of the availability of nutrients in a form which can be easily assimilated by plants.
Q.2. Who were the first to propose biological nitrogen fixation?
Ans: J.B. Boussingault and Hellreigel were the first to open up the field of biological nitrogen fixation, in 1886.
Q.3. Who discovered the bacterium now known as Rhizobium and its role?
Ans: Beijerinck, a Ducth in 1888 discovered the bacterium now known as Rhizobium which is found in the root nodules of legumes and fix up atmospheric nitrogen.
Q.4. Who first cultured rhizobia in the laboratory?
Ans: Nobbe and Hiltner were the first to culture rhizobia in 1895 and called it “Nitragin”.
Q.5. Which are the bio-fertilizers in addition to Rhizobium?
Ans: The bio-fertilizers in addition to Rhizobium are: Azotobacter, Azospirillum, blue-green algae (BGA) also known as cyanobacteria, Azolla, an aquatic fern of freshwater inhabiting Anabaena azollae as a symbiont, phosphorus solubilizes Bacillus and Pseudomonas (bacteria) and Aspergillus (a fungus), phosphorus absorber, vesicular arbuscular mycorrhizal (VAM) fungi as Gigaspora and Glomus, lignocellulolytic organisms Cellulomonas and Arthrobacter (bacterial species) Agaricus and Trichoderma (fungal species), Frankia and Wilcoxina (Tricharina) both actinomycetous and liglinolytic orchid mycorrhizal fungi as Rhizoctonia.
Q.6. Which forest trees are benefited by actinomycetous N-fixing symbiosis of Frankia?
Ans: Casuarina and Alnus.
Q.7. What are nif genes?
Ans: These are genes whose expression is necessary for nitrogen fixation.
Q.8. How many nif genes are clustered in Klebsiella pneumoniae?
Ans: There are at least 17 nif genes in K. pneumoniae close to his operon in 8 contiguous transcriptional units (nif J, nif HDK, nif Y, nif ENX, nif USVM, nif F, nif LA and nif BQ) spanning Ca. 23 kb on the chromosome.
Q.9. What are the structural genes for nitrogenase?
Ans: nif HDK.
Q.10. Name the non-legume with which Rhizobia can fix up atmospheric nitrogen in symbiotic association.
Q.11. How can Rhizobium be isolated?
Ans: Rhizobium can be isolated on yeast extract manitol agar (YEMA) from the clean (surface sterilized by immersing in acidified 0.1% HgCl2) crushed nodules, incubating at 28-30°C for 3 to 10 days.
Q.12. What are the characteristics of the colonies of Rhizobium?
Ans: They usually appear as white, translucent, glistening and elevated along the entire margin, and shine against a pink background of Congo red dye mixed in the medium.
Q.13. Which are the groups of Rhizobia based on growth habit?
Ans: They comprise two groups based on their growth habits:
(1) Fast grower with generation time less than 4 hours (g = 4 hours) producing acid on the YEMA which includes R. phaseoli, R. trifoli, R. leguminosarum and R. melilot and
(2) Slow growing strains with generation time more than eight hours or equal to eight hours (g = 8 hours) producing alkali on YEMA which includes B. japonicum, R. lupine and rhizobia of cowpea miscellany groups.
Fast growing species form gummy raised colonies 2-4 mm in diameter with 3-4 days while slow growing species form granular colonies not exceeding 1 mm in diameter within 5-7 days.
Q.14. What are the structural parts of a mature nodule of a legume?
Ans: A mature nodule comprises a central bacteroid zone surrounded by many layers of cortical cells.
Q.15. What are bacteroids?
Ans: They are bacterium like or modified bacterial cells. The endosymbionts occur within the nodules as modified, membrane enclosed, often pleomorphic cells referred to as bacteroids.
Q.16. On which enzyme nitrogen fixation is dependent?
Ans: Enzyme nitrogenase.
Q.17. How will you differentiate Agrobacterium the most common contaminant of Rhizobium?
Ans: The Agrobacterium takes up the colour of Congo red and appears red in colour.
Q.18. Give the presumptive tests carried out to identify an isolate of Rhizobium.
Ans: (1) Rhizobium grows very poorly on glucose peptone agar medium.
(2) Doesn’t grow on Hofer’s alkaline broth at pH 11.0.
(3) Rhizobium does not utilize lactose.
(4) The best way to identify rhizobia is to conduct plant infectivity tests and check if the nodules are formed.
Q.19. How is a plant infectivity test carried out for rhizobia?
Ans: It is carried out under controlled and sterile conditions either in small tubes or pots. Small test tubes are used for small seeded legumes like clover, berseem, moong and urd while Leonard jar assembly or small pots are used for testing bold seeded legumes as chickpea, arhar and French beans. Agar-agar, vermiculite or sand can be used as the substrate for growing plants.
The surface sterilized seeds of the selected crop are sown in appropriate assembly and are inoculated with the bacterial isolate under study for test. The plants are watered at regular intervals with any nitrogen -free nutrient solution.
The plants are observed for nodulation, nitrogenase activity and dry biomass after 5 to 6 weeks. Hence an efficient strain needed for a specific crop can be selected. The strain so selected can be judged for its potential in the pots and fields.It is necessary to lyophilize the selected strain to maintain their efficiency.
Q.20. Why is a selected strain of Rhizobium lyophilized?
Ans: A selected strain is lyophilized because sub-culturing at monthly intervals on artificial medium may lead to deterioration of the activity of the organisms.
Q.21. What is the importance of carriers in preparation of Rhizobium inoculants?
Ans: The carrier works as a medium which allows the Rhizobium to multiply. The shelf life of the product mainly depends on the carrier.
Q.22. What are the suitable carriers used for Rhizobium inoculants?
Ans: The suitable carriers for the growth of Rhizobium are peat, lignite, Farm Yard Manure (FYM) and charcoal.
Q.23. Where is peat like material found in India?
Ans: The typical peat is not available in our country in large quantities but peat like material is available in Nilgiris (Ootacamund or Udagamandalam) has been found to be a very good carrier.
Q.24. Where is lignite available in India?
Ans: Lignite is available in Neyveli lignite mines of South India.
Q.25. What are the characteristics of a good carrier for Rhizobium?
Ans: (1) It should be cheap and easily available in the country,
(2) It should be nontoxic to the bacteria used for fertilizer,
(3) It should have near neutral pH,
(4) The organic matter should be 40%
(5) Above all it should have high water holding capacity and surface area.
Q.26. Give the method for inoculation of Rhizobium.
Ans: A 10 per cent sugar solution or 10 per cent solution of pharmaceutical grade of gum Arabic is prepared to help the Rhizobium cells to stick on the surface of the seeds. This solution is sprinkled on the seeds required for I hectare and are spread on polythene sheet and mixed uniformly. Then the contents of inoculants of Rhizobium are sprinkled on the sticky seeds and mixed thoroughly.
The seeds thereafter are spread on gunny bags or on the cement floor in shade to avoid direct sunlight and are sowed on drying. The pelleting of inoculated seeds is done by providing a uniform fine coat of calcium carbonate to protect the seeds from adverse effects of soil like acidity, alkalinity and pesticides, etc..