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Legume Inoculation
The practice of applying artificially prepared cultures of rhizobia to leguminous seed before sowing can be referred to as legume inoculation. This practice is known since the beginning of this century. Agar based cultures were used for a long time which was later replaced by soil based ones. Finely ground and neutralized peat is now being generally used in Australia and U.S.A as a carrier in the preparation of legume inoculants. Peat as a carrier has decided advantages over agar or soil.
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Besides possessing high moisture holding capacity and organic matter content, so essential for better shelf life of bacterial cultures, peat improves the survival of rhizobial cells on the seed coat, especially under dry soil conditions.
In Australia, peat is harvested, dried in the field and ground to pass a 200 mesh sieve. Peat is generally acidic in nature and hence is neutralized by adding sufficient CaCO3. The neutralized peat is packed in low density, 0.05 mm gauge polythene bags and sterilized by gamma rays at a dose of 5.0 x 106 rads. Australians have found that sterilization by gamma radiation is generally superior to autoc1aving for 4 hr at 121°C in promoting the growth of rhizobia.
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Rhizobia are grown on yeast extract mannitol broth in suitable fermentor vessels until the numbers of rhizboia in the broth have reached the minimum standard or even more than the standard figure. The acceptable minimum Australian standard for rhizobial count in the broth culture is 500 x 106 viable rhizobia/ml, although in practice the numbers usually reach the range of 1000-4000 x 106 viable cells/ml.
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If the broth is added to unsterilized peat, mechanical mixers are used to mix peat with the broth so as to provide a moisture content of 45-50% on a wet weight basis. The peat is sieved through a coarse sieve to remove lumps and then matured for 4 days at 26°C in trays covered with polythene and packaged according to convenience in polythene bags. The polythene bags are made from polythene sheeting of 0.0015 inch or 0.0375 mm. If the broth is added to sterilized peat, the peat is initially packaged in polythene bags and the required quantity of broth is added through a syringe to provide a moisture content of 60%. The puncture is sealed by adhesive tape, the contents mixed by rolling in hand, incubated at 26"C for two weeks and then stored at 4"C. The minimum Australian standard for rhizobia in peat are 108 -109 viable cells/g of peat at manufacture and 107 -108 cells/g of peat during the full shelf life of the culture.
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The Australian method of using cultures grown on agar is to make a suspension of the bacterial cells in water. This suspension may be directly applied to seed or improved by the use of 10% sugar or .40% neutral gum arabic in the suspending fluid. If peat based cultures are used, 25 g of culture is added to 100 ml of water or a solution of sugar and gum arabic. The resultant slurry is used for application on seed.
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Pelleting of inoculated seed with lime (finely divided CaCO3) or rock phosphate improves survival of rhizobia on seed and hence secures better root nodulation under adverse soil conditions. For pelleting, a sticker is generally used. The recommended stickers are pharmaceutical fine grade gum arabic at approximately 40% level, 5% methyl ethyl cellulose (cellofas A) and methyl hydroxypropyl cellulose (methofas) or carboxy methyl cellulose.
It is necessary that finely ground calcium carbonate must pass through 300 mesh Small quantities of seeds may be handled in dishes for pelleting. For larger quantity of seeds, a concrete mixer may be used. Initially, the seeds are mixed thoroughly in the peat-adhesive slurry. Finely ground CaCO3 is then added to the inoculated seed while it is still wet and rolled evenly so as to obtain uniform pelleting of lime over the seed. The pelleted seed may be sown immediately and if absolutely necessary may be stored up to 2-3 weeks at temperatures not exceeding 18°C.
In U.S.A., large quantities of legume inoculants are produced for internal use as well as for exporting to other countries. The usual method followed in manufacture of inoculants is as follows: Inocula are transferred from agar slants into starter flasks containing yeast extract mannitol broth. After 4 days growth, the culture from starter flasks is transferred into a small seed-tank fermentor. At this stage, yeast extract mannitol broth is formulated in a battery of large production fermentors, pH adjusted to 7.0, sterilized, cooled and kept ready for use.
The contents of see tank fermentors are transferred to production fermentors. The temperature range for fermentation is 30-35°C, depending on the species of rhizobia. Aeration is done by forcing sterile air through porous carborundum or stainless steel spargers at the bottom of the fermentors. A cell population of 5 x 109 can be attained in 96 hours which is again dependent on the amount of initial inoculum added.
A minimum of 109 cells per ml is needed in the broth for being used in the preparation of peat cultures. In U.S.A., the broth is sprayed to powdered, neutralized and flash-dried peat (partial sterilization) while the mixture is being agitated in a ribbon or pa dle-type batch mixer. After mixing, the inoculant is spread in thin layers on floor for 48 to 72 hours at 22 to 24°C. The product is then milled to break up aggregates, finely pulverized and packed in polythene bags. The general standards advocated are to provide massive inoculum and sound recommendations for the use of the inoculant since various factors affect the number of viable cells in peat.
In India, peat-like material is available in the Nilgiri valley to the extent of 5.5 million tonnes as estimated by the soil survey department. An unknown quantity is also known to occur in the Kashmir Valley. Lignite is another carrier which is widely used and it is estimated that about 3 million tonnes of mined lignite is available annually from Neyveli lignite mines in Tamil Nadu. Powdered peat or lignite is neutralized with CaCO3 (passing through 200 mesh sieve) and is autoclaved at 15 lb pressure for 4 hours before use. Upon cooling, the broth from shake cultures (or from a fermentor) is poured into powdered peat and mixed by hand or a mixer in such a way that the finished product retains 40% moisture. After curing for few hours at room temperature the product is packed in polythene packets. Inoculant slurry is prepared in 5% aqueous solution of jaggery or sugar before being applied to seed.
In practice, the following bacterial counts have been achieved while making peat based inoculants at the Indian Agricultural Research Institute: (1) the minimum count for broth is 10 x 107/ml and maximum count being 80 x 109, (2) the minimum peat count at manufacture is 100 x 106 and the maximum being 90 x 108, (3) the minimum peat count at peak shelf life (4 weeks) is 10 x 108 and the maximum being 10 x 1010 at 12 weeks, and (4) the minimum count on seed at sowing is l03 per seed and the maximum being 105 per seed.
A schematic presentation of processes involved in the mass production of rhizobia
| 1. Culture broth in flask |
2. Peat mixed with bacteria |
| 3. Bacteria in culture packet |
4. Culture |
| 5. Air Compressor |
6. Dehumdfer |
| 7. Air |
8. Primary Air Filter |
| 9. Glass Wool |
10. Secondary Air Filter |
| 11. Stoorm |
12. Bacteria Broth |
| 13. Fermentor |
14. Broth |
| 15. Agitator |
16. Boiler |
| 17. Stoorm |
18. Shaker Culture (Primary inoculum) |
| 19. Flask |
20. Test Tube |
| 21. Nodules Petrosh |
22. Legume Plant |
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