Biofertilizer Production Technology AMB507 PDF

Biofertilizer production technology AMB507 (1+1) 1 Biofertilizer production technology of free living N2 fixers – Azotobacter Domain : Bacteria Phylum : Proteobacteria Class : Gammaproteobacteria Order : Pseudomonadales Family : Pseudomonadaceae Genus : Azotobacter Free living diazotrophs Diazotroph (Diazo = Dintrogen) :Organisms that can use atmospheric dinitrogen as sole source of nitrogen Diazotroph: prokaryotic microorganisms fixing atmospheric nitrogen Free living diazotrophs: Organisms living freely in the habitat and fixing N2. Isolation of efficient Azotobacter strain Culture /isolation Medium: Ashby’s , Jensen’s medium, Waksmann No 77 medium, Burks medium Method : Serial dilution plate method Selecting efficiency test based on 1. Test for N2 fixation in Pure culture by Kjeldahl Method 2. Determine the sucrose consumed Cyst formation : Cyst formation takes place in Burks medium containing n-butanol in place of carbon source after 4-5 days after incubation Table : Distinguishing features b/w Free living diazotrophs Diazotroph Flagallation slime pigment Azotobacter peritrichous moderate slime black –brown insoluble pigments chroococcum A vinelandii, A peritrichous Little to Green, fluorescent and soluble paspali, moderate slime A beijerinckii nil moderate slime Yellow light brown insoluble pigments A macrocytogenes Polar abundant Light rust brown insoluble pigments Derxia gummosa Polar abundant Yellow light brown insoluble pigments Cultural characteristics When grown at 30oC with sugar— usually sucrose—as the carbon source at 1–2% (w/v) on either Winogradsky’ s nitrogen-free agar medium or on Burk’s medium, colonies appear within 48 h at 30oC and reach a diameter of 2–6 mm in a week, depending on the numbers (crowding) per plate. Burk’s medium is now more commonly used than Winogradsky’ s medium for growth of laboratory strains of A. vinelandii and A. chroococcum 2 Colony characters :Generally smooth, glistening, opaque, low convex, and mucoid; however, variations may occur Azotobacter chroococcum : Colonies are opaque and convex, and, for most strains, are mucoid, glistening, and smooth. A brown-black nondiffusible pigment is produced in aging colonies Response of plants to Azotobacter inoculation Inoculation of soil or seed with Azotobacter is effective in increasing yields of crops in well – manured soil with high organic matter content. 1. Fixes N2 2. Synthesizes biologically active substances such as B-vitamins, IAA, GA 3. Produces fungistatic compounds : Preventing growth of pathogens such as Alternaria and Fusarium Results of Azotobacter inoculation : 1. N2 fixed in soil is 10-15kgN/ha/yr (Maximum) 2. Increase in yield in field crops is upto 12% over uninoculated control Azotobacter preparations : “Azotobacterin” in USSR and Europe is used for bacterization of seeds and increased the growth and yield. Why incoculation ? Population of Azotobacter in rhizosphere soil and uncultivated soil is generally low. used as a biofertilizer for all non-leguminous crops especially in cereals like rice, maize, wheat etc., and in crops like cotton, jute, tobacco, vegetables, ornamentals plants and plantation crops. They are known to secrete various growth promoting substances like indole acetic acid, gibberllic acid, cytokinins and vitamins which promote seed germination and induce vigour to the plant to get luxuriant growth. Azotobacter responds well to organic matter addition and exhibits better performance in presence of either compost or farm yard manure. This organism also has the ability to inhibit the growth of few soil – borne fungal pathogens. 3 Production of Azotobacter biofertilizer Selection of suitable Azotobacter species/strain which can fix N2 efficiently over wide suitable range of environmental conditions ↓ Lyophilize Azotobacter species/strain to maintain their efficiency since sub culturing at monthly interval on artificial medium may lead to deterioration of the activity of the N2 fixation ↓ Whenever it is required for use, the tube containing the lyophilized material is cut open and the lyophilized cells are reconditioned ↓ These are grown in sufficient numbers on agar slants to be used for mass multiplication during the season (Ashby’s , Jensen’s medium, Waksmann No 77 medium, Burks medium etc.,) ↓ There should be regular continued evaluation with the systematic long-stored, untouched culture in lyophilized conditions. This is necessary to ensure continued N2 fixation efficiency of the culture as seed inoculums Mass scale production of Azotobacter species/strain can be carried out using system of rotary shaker/ fermenter. About 100 ml aliquots of the the Jensens Broth/Burks broth are dispensed into 250 ml conical flasks, which are plugged with non-absorbent cotton and are sterilized at 15lbs pressure for 20 minutes and cooled after sterilization. ↓ 3-6 day old cultures of Azotobacter are examined for the purity and about 4-6 ml of sterile broth is transferred aseptically into the tubes. ↓ The bacterial growth is scrapped with the help of an inoculated needle and the resulting bacterial suspension is transferred to 250 ml conical flask containing Jensens Broth/Burks broth. ↓ The flasks are incubated at 300C on a rotary shaker for 2-7 days. Fermenters are used for industrial scale production of biofertilizers. Cultures vessels ranging from 5-1000 liters can be used depending upon the requirement. The amount of inoculum culture to be added into fermenter vessel depends on the size of the fermenters, but the ratio between the inoculum and medium in the vessel should be maintained at 1:20 (5% inoculums rate). ↓ When the number of rhizobia in the broth has attained the required standard (108-109 cells /ml) , the broth should be added to the carrier for preparation of carrier based inoculants. The broth is continuously aerated by forcing sterile air through porous stainless steel sparger. 4 Various fermentation requirements like aeration, agitation and fermentation time vary from strain to strain. Mass – scale production of Azotobacter species/strain can be carried out using system of rotary shaker/ fermenter. About 100 ml aliquots of the Jensens Broth/Burks broth are dispensed into 250 ml conical flasks, which are plugged with non-absorbent cotton and are sterilized at 15lbs pressure for 20 minutes and cooled after sterilization Preparations of carriers The carrier material is sun dried to 5 % moisture and pulverized to fineness of 72 - 100 mesh. Coarser materials form balls on mixing with broth and seeds are not properly coated with the inoculants. Lignite powder being acidic pH 5.5- usually 1 kg of lime (CaCO3) is mixed with 9 kg of lignite to bring the PH to about 7.00. Charcoal powder being neutral in PH dose not require lime. Finally the carrier is mixed with 10 % water before sterilization. The carrier material must be sterilized before blending with broth culture to suppress the proliferation of contaminants. Best method is ¥ gamma irradiation (5 megarods) for carrier sterilization. The carrier materials may also be packaged and sealed in autoclavable high density polypropylene bags which can withstand a temperature of 1210C and sterilized in an autoclave Packing and labeling The blended carrier is kept for 2-4 hours for curing. After curing, the inoculants is packed in low density polypropylene (LDPE) bags of 0.2 to 0.5 mm gauge size either manuaaly or by using automatic dispenser. The bags are sealed by an electric sealer leaving about two-third vacant space to give proper aeration to the inoculants. LDPE packets are used universally, as they can be readily sealed, allow high exchange with relative low moisture loss. The inoculants packet is labeled with relevant information regarding culture viz. name of the culture, crop for which intended, expiry date, directions for storage / use and precautions, certifications mark if any. Packets (Inoculants) must be incubated for a week in the room at 300C so that the bacteria multiplies and attains the required count. The packet may then be stored in a cold room (4-150C). The inoculants packets should have the following information: 1. Name of the product 2. Name and address of the manufacturer 3. Name of carrier 4. Batch number 5. Date of manufacture 5 6. Date of expiry 7. Net mass 8. Crops for which intended 9. Storage instructions The inoculants should not be subjected to excessive temperatures, ideally it should be held at 40C throughout transport and storage. Inoculum in bulk should be transported in strong cartoons on boxes. Distributions of cultures during summer and rainy seasons require suitable arrangements and strict quality control measures. The shelf life of Azotobacter inoculant thus prepared is about 6 months at room temperature and in a cold room it can be up to 4 years. During the end of this period each gram of the inoculants shall contain 107 cells /g of the carrier on dry weight basis Crops on which they are to be used Azotobacter biofertilizers can be used for cultivating all non-leguminous crops especially in cereals like rice, maize, wheat etc., and in crops like cotton, jute, tobacco, vegetables, ornamentals plants and plantation crops. They are not host specific. Dosage and seed treatment Usually 400-500 g lignite / charcoal based culture would be sufficient to the quantity of seed required per hectare. The method of seed inoculation includes preparation of 10% sugar/pharmaceutical grade gum Arabic /1% carboxy methyl cellulose (CMC) solution. This solution is sprinkled on the seeds and the seeds are thoroughly mixed so as to have a uniform coating. A count of 1000 viable cells per seed is to be attained at the time of treating the seed and quantity of culture used is accordingly adjusted. The seeds are spread uniformly for drying on a gunny bag/cement floor in shade avoiding direct sunlight. The seeds are sown immediately (preferably in the early morning or late evening). Dosage, method and time of application: Soil application: 10 kg/ha mixed in 20 kg FYM and can be applied by broadcasting or by row method. Slurry treatment/root dip method: Thick slurry of 1 kg inoculums in 5 liters water is made and roots of seedlings are dipped in slurry for a minimum period of 30 minutes and then planted in the main field. Inoculation to plantation crops: For plantation crops, the soil around the root zone is excavated in the form of ring. The Azotobacter biofertilizer is applied all along the root zone in the form of a basin or ring @ 25-50 g per plant after 6 mixing with well decomposed FYM / vermicompost. The excavated soil is then covered back around the trunk of the tree. Similarly, plantation crops, vines such as pepper can be inoculated. For crops more than 6 months duration Azotobacter biofertilizer can be applied to soil once in 6 months. Shelf life Biofertilizer packets normally have a shelf life of 6 months at room temperature. During the end of this period each gram of the inoculants shall contain 107 cells / g of the carrier (on dry weight basis). Certain precautions are to be observed in the use of Biofertilizers. The Azotobacter biofertilizer has to be used before the expiry date marked on the packet and the inoculated seeds are not allowed to come in direct contact with any pesticides/fertilizers. If fungicides, insecticides , herbicides and chemical fertilizers have to to be used, there should be a gap of at least 15 days between the use of these chemicals and biofertilizers. In order to protect Azotobacter from effects of fertilizers , pesticides, acid/saline and dry soils, pelleting of the seeds is the most commonly used practice. The most commonly used pelleting agents are calcium carbonate, rock phosphate, charcoal, talc, gypsum, bentonite etc. Co-inoculation with other biofertilizers like phosphate solubilizing microorganisms, AM fungi etc, have no deleterious effect on Azotobacter inoculants. Quality control specifications as per Bureau of Indian Standards (BIS) prescribed for Azotobacter 1 Base Carrier based* in form of moist/dry powder/granules/liquid based 2 Viable cell count Cfu minimum 5 X 107 cells / g of powder, granules or carrier material or 1 X 108 cells /ml of liquid 3 Contamination level No contamination at 105 dilutions 4 pH 6.5 – 7.5 5 Particle size in case of carrier based All material shall pass through 150 to 212 µ material (72 to 100 mesh) IS sieve 6 Moisture percent by weight, maximum in 30 - 40% case of carrier based 7 Efficiency character The strain should be capable of fixing at least 10 mg of nitrogen / g of sucrose consumed *Type of carrier: The carrier material such as peat, lignite, peat – soil, humus, wood charcoal or similar material favouring growth of the organism. NOTE- sources for supplying the mother culture of Azotobacter 7 National Biofertiliser Development Centre (NBDC), Ghaziabad and its six Regional Centres (RBDC) located at 1. Bangalore, 2. Bhubaneshwar, 3. Irnphal, 4. Hissar, 5. Jabalpur 6. Nagpur and 7. Indian Agricultural Research Institute (lARI), New Delhi 8. Tamil Nadu Agricultural University (TNAU), Coimbatore 9. University of Agricultural Science, Bangalore Packaging material : Low density poly ethylene /poly propylene bags with minimum thickness of 75 to 100 µ Quality control 1. Total plate count by SPC 2. Test for N2 fixation in Pure culture by Kjeldahl Method 3. Determine the sucrose consumed ********************************************************************************** 8 Biofertilizer production technology of – Azospirillum Domain : Bacteria Phylum : Proteobacteria Class : Alphaproteobacteria Order : Rhodospirillales Family : Rhodospirillaceae Genus : Azospirillum Azospirillum Azospirillum is a comma shaped bacterium containing large amount of lipid granules inside its cell. It enters cortical cells of the root and fixes atmospheric nitrogen. Since these bacteria enter the root cells and fix atmospheric nitrogen without forming root nodules, they are said to be in associative symbiosis with the host. They also produce plant growth promoting substances. Azospirillum Inoculant (ASI) - ASI is a product having large population of a strain or a combination of several strains of Azospirillum for a particular crop or a group of crops which shall develop efficient roots in the plant and enrich soil with nitrogen and thereby enhance nitrogen fixation, crop growth and productivity. Isolation of efficient Azospirillum strain Culture /isolation Medium: Nfb Method : Serial dilution plate method/from roots or soil Selecting efficiency strain based on 1. Test for N2 fixation in Pure culture by Kjeldahl Method 2. Determine the amount of carbon source consumed TEST FOR EFFECTIVE ROOT GROWTH 1. POT CULTURE TEST Inoculate the seeds and sow the seeds in pots 9 After three weeks of growth, thin down the number of plants in each pot to four uniform plants. At the end of three months, take one set of pot from control and inoculated series and separate the plants carefully from the soil under slow running water. Obtain the number, length and mass of the plant as a whole including branch, seeds if any and roots. At the end of three months, harvest the shoot system (depending on the crop). Dry at 60°C for 48 hours and determine the dry mass. For the above purpose maintain a minimum of four replications or a maximum of 16 replications. Better vigour of roots in inoculated seeds if established from the data when compared to the control, is the confirmation of effectiveness of ASI. If there is 10 percent increase in the plants over the dry mass of uninoculated control without nitrate, it may be concluded that the culture is of required quality. Isolation of A. brasilense and A. lipoferum Inoculate the semi-solid NFb medium with 0.1 mL of soil or root suspension and incubate at 30–34 °C. Observe for pellicles formation within 4–7 days, and Transfer the veil/ pellicles new semi-solid NFb medium and incubate again for 4–7 days. Streak the culture on solid NFb medium supplemented with 50 mg yeast extract and incubate for 3– 5 days and observe for colonies from both species are white, dry and small. Transfer individual colonies to semi-solid NFb medium and incubate again for 4–7 days and observe pellicle formation. After pellicle formation, streak cultures on solid potato medium. Observe for colonies formed in this medium which are initially yellowish- white, and eventually becoming pinkish. Transfer these colonies to new semi-solid NFb medium and observe for pellicle formation. Identify these bacteria through wet mounts under the microscope. A. brasilense : cells are medium sized (1 × 3–5 μm), very motile, curved rods with spirilloid movement. A.lipoferum : cells initially are indistinguishable from the former, but once the medium turns alkaline (blue colour), the cells change into large pleomorphic forms ,immootile (size may grow up to 10 μm in length and 3–5 μm thick). A. lipoferum is capable of metabolizing glucose as the sole carbon source A. brasilense is not capable of metabolizing glucose as the sole carbon source TABLE : Characteristics differentiating the species of the genus Azospirillum Characteristics A. lipoferum A. amazonense A. brasilense Cell width, µm 1.0–1.7 0.8–1.0 1.0–1.2 10 Enlarged, pleomorphic cells + - - develop in alkaline media Biotin requirement + - - Optimal growth temperature, oC 37 35 37 Carbon sources: Glucose + + - Mannitol + - - Glycerol + - + Sucrose - + - Colony type on BMS agar medium pink, raised, white, flat raised pink, raised, curled curled margin Congo red medium scarlet nd scarlet pH range for growth 5.7–6.8 5.7–6.5 6.0–7.8 Acidification of peptone-based + - - glucose broth: Acid from: Glucose + - - Fructose + - - Habitat: Soil and tissues + + + mainly of nonlegumes NFb medium (g/l): L-malic acid 5.0 K2HPO4 0.5 MgSO4 7H2O 0.2 NaCl 0.02 Trace element solution 2.0 ml Bromthymol blue 2.0 ml Fe EDTA (1.64% solution) 4.0 ml Vitamin solution 1.0 ml KOH 4.0 pH Adjusted to 6.8 with KOH Distilled water 1000 ml 11 For a semi-solid medium 1.75 g/l agar For a solid medium 15.0 g/l agar Bromthymol blue : 0.5% aqueous solution [dissolve in 0.2 N KOH Trace element solution Na2MoO4 2H2O 0.2 g MnSO4 H2O 0.235 g H3BO3 0.28 g CuSO4 5H2O 0.008 g ZnSO4 7H2O 0.024 g Distilled water 1000 ml Vitamin solution Biotin 0.01 g Pyridoxin 0.02 g Distilled water 1000 ml Group of Crops - Crops including cereals, millets, forage crops, vegetables, fruits, plantation crops and sugar crops which are supplied nitrogen efficiently by given strain of Azospirillum for a particular crop for a group of which shall develop efficient roots and the plant enrich soil with nitrogen and their by enhance nitrogen fixation crop growth and productivity. Selecting efficient strain based on 1. Amount of N2 fixation in Pure culture by Kjeldahl Method 2. Determine the malic acid/ carbon source consumed 3. Show effective root development on all cultivar/crops against which the inoculant is intented to be used. 4. Formation of white pellicle in semisolid nitrogen – free bromothymol blue media Production of Azospirillum biofertilizer Selection of suitable Azospirillum species/strain which can fix N2 efficiently over wide suitable range of environmental conditions ↓ Lyophilize Azospirillum species/strain to maintain their efficiency since sub culturing at monthly interval on artificial medium may lead to deterioration of the activity of the N2 fixation ↓ Whenever it is required for use, the tube containing the lyophilized material is cut open and the lyophilized cells are reconditioned ↓ 12 These are grown in sufficient numbers on agar slants to be used for mass multiplication during the season (Okon’s /Doberenier’s broth ,Nfb etc.,) ↓ There should be regular continued evaluation with the systematic long-stored, untouched culture in lyophilized conditions. This is necessary to ensure continued N2 fixation efficiency of the culture as seed inoculums Mass scale production of Azospirillum species/strain can be carried out using system of rotary shaker/ fermenter. About 100 ml aliquots of the the Okon’s /Doberenier’s broth ,Nfb are dispensed into 250 ml conical flasks, which are plugged with non-absorbent cotton and are sterilized at 15lbs pressure for 20 minutes and cooled after sterilization. ↓ 3-6 day old cultures of Azospirillum are examined for the purity and about 4-6 ml of sterile broth is transferred aseptically into the tubes. ↓ The bacterial growth is scrapped with the help of an inoculated needle and the resulting bacterial suspension is transferred to 250 ml conical flask containing Okon’s /Doberenier’s broth ,Nfb. ↓ The flasks are incubated at 300C on a rotary shaker for 2-7 days. Fermenters are used for industrial scale production of biofertilizers. Cultures vessels ranging from 5-1000 liters can be used depending upon the requirement. The amount of inoculum culture to be added into fermenter vessel depends on the size of the fermenters, but the ratio between the inoculum and medium in the vessel should be maintained at 1:20 (5% inoculums rate). ↓ Pellicle formation 1 to 2 mm below the upper surface of the medium is a clear indication of the growth of Azospirillum When the number of Azospirillum in the broth has attained the required standard (108-109 cells /ml) , the broth should be added to the carrier for preparation of carrier based inoculants. The broth is continuously aerated by forcing sterile air through porous stainless steel sparger. Various fermentation requirements like aeration, agitation and fermentation time vary from strain to strain. The process optimization for the some of the Azospirillum cultures using fermenter Preparations of carriers 13 The carrier material is sun dried to 5 % moisture and pulverized to fineness of 72 - 100 mesh. Coarser materials form balls on mixing with broth and seeds are not properly coated with the inoculants. Lignite powder being acidic pH 5.5- usually 1 kg of lime (CaCO3) is mixed with 9 kg of lignite to bring the PH to about 7.00. Charcoal powder being neutral in PH dose not require lime. Finally the carrier is mixed with 10 % water before sterilization. The carrier material must be sterilized before blending with broth culture to suppress the proliferation of contaminants. Best method is ¥ gamma irradiation (5 megarods) for carrier sterilization. The carrier materials may also be packaged and sealed in autoclavable high density polypropylene bags which can withstand a temperature of 1210C and sterilized in an autoclave Packing and labeling The blended carrier is kept for 2-4 hours for curing. After curing, the inoculants is packed in low density polypropylene (LDPE) bags of 0.2 to 0.5 mm gauge size either manuaaly or by using automatic dispenser. The bags are sealed by an electric sealer leaving about two-third vacant space to give proper aeration to the inoculants. LDPE packets are used universally, as they can be readily sealed, allow high exchange with relative low moisture loss. The inoculants packet is labeled with relevant information regarding culture viz. name of the culture, crop for which intended, expiry date, directions for storage / use and precautions, certifications mark if any. Packets (Inoculants) must be incubated for a week in the room at 300C so that the bacteria multiplies and attains the required count. The packet may then be stored in a cold room (4-150C). The inoculants should not be subjected to excessive temperatures, ideally it should be held at 40C throughout transport and storage. Inoculum in bulk should be transported in strong cartoons on boxes. Distributions of cultures during summer and rainy seasons require suitable arrangements and strict quality control measures. The shelf life of Azospirillum inoculant thus prepared is about 6 months at room temperature and in a cold room it can be up to 4 years. During the end of this period each gram of the inoculants shall contain 107 cells /g of the carrier on dry weight basis. Crops on which they are to be used It can be used for a variety of crops like cereals (paddy, ragi, maize, sorghum), fodder crops, tuber crops etc. 14 Crops including cereals, millets, forage crops, vegetables, fruits, plantation crops and sugar crops Dosage and seed treatment Usually 400-500 g lignite / charcoal based culture would be sufficient to the quantity of seed required per hectare. The method of seed inoculation includes preparation of 10% sugar/pharmaceutical grade gum Arabic /1% carboxy methyl cellulose (CMC) solution. This solution is sprinkled on the seeds and the seeds are thoroughly mixed so as to have a uniform coating. A count of 1000 viable cells per seed is to be attained at the time of treating the seed and quantity of culture used is accordingly adjusted. The seeds are spread uniformly for drying on a gunny bag/cement floor in shade avoiding direct sunlight. The seeds are sown immediately (preferably in the early morning or late evening). Dosage, method and time of application: Soil application: 10 kg/ha mixed in 20 kg FYM and can be applied by broadcasting or by row method. Slurry treatment/root dip method: Thick slurry of 1 kg inoculums in 5 liters water is made and roots of seedlings are dipped in slurry for a minimum period of 30 minutes and then planted in the main field. Inoculation to plantation crops: For plantation crops, the soil around the root zone is excavated in the form of ring. The Azospirillum biofertilizer is applied all along the root zone in the form of a basin or ring @ 25-50 g per plant after mixing with well decomposed FYM / vermicompost. The excavated soil is then covered back around the trunk of the tree. Similarly, plantation crops, vines such as pepper can be inoculated. For crops more than 6 months duration Azospirillum biofertilizer can be applied to soil once in 6 months. Shelf life Biofertilizer packets normally have a shelf life of 6 months at room temperature. During the end of this period each gram of the inoculants shall contain 107 cells / g of the carrier (on dry weight basis). Certain precautions are to be observed in the use of Biofertilizers. The Azospirillum biofertilizer has to be used before the expiry date marked on the packet and the inoculated seeds are not allowed to come in direct contact with any pesticides/fertilizers. If fungicides, insecticides , herbicides and chemical fertilizers have to to be used, there should be a gap of at least 15 days between the use of these chemicals and biofertilizers. In order to protect Azospirillum from effects of fertilizers , pesticides, acid/saline and dry soils, pelleting of the seeds is the most commonly used practice. 15 The most commonly used pelleting agents are calcium carbonate, rock phosphate, charcoal, talc, gypsum, bentonite etc. Co-inoculation with other biofertilizers like phosphate solubilizing microorganisms, AM fungi etc, have no deleterious effect on Azospirillum inoculants Quality control specifications prescribed for Azospirillum 1 Base Carrier based* in form of moist/dry powder/granules/liquid based 2 Viable cell count Cfu minimum 5 X 107 cells / g of powder, granules or carrier material or 1 X 108 cells /ml of liquid 3 Contamination level No contamination at 105 dilutions 4 pH 6.5 – 7.5 5 Particle size in case of carrier based All material shall pass through 150 to 212 µ material (72 to 100 mesh) IS sieve 6 Moisture percent by weight, maximum in 30 - 40% case of carrier based 7 Efficiency character Formation of white pellicle in semisolid nitrogen – free bromothymol blue media *Type of carrier: The carrier material such as peat, lignite, peat – soil, humus, wood charcoal or similar material favouring growth of the organism. Packaging material : Low density poly ethylene /poly propylene bags with minimum thickness of 75 to 100 µ Quality control 1. Total plate count by SPC/MPN technique 2. Test for N2 fixation in Pure culture by Kjeldahl Method DETERMINATION OF NUMBER OF AZOSPIRILLUM CELLS Visual counting and use of MPN table NFB Medium (Nitrogen free bromothymol blue medium) PREPARATION OF SERIAL DILUTION FOR COUNT BY MPN METHOD: Dispense 30 g of ASI in 270 ml of sterile water and shake for 10 minutes on a reciprocal shaker. Make serial dilutions upto 107 dilutions. Pipette out 0.2 ml aliquots of 10 5 to 107 dilution and deliver it to screw cap tubes or petridishes containing set medium as described in A - 2.1. Spread the aliquots over the plates. Invert the plates and place them in the incubator at 28 ± 2°C for 3 days. Use 5 replicates of 105. 106 and 107 level. COUNTING 16 Count the tubes or plates which have turned blue in colour after inoculation and ascertain the presence of pellicles in undisturbed medium. To determine usual contamination on the same examine doubtful objects carefully. Count all plates/tubes which have turned blue and consider them for the purpose of calculation. Count such type of tubes/plates and tally this count with MPN table to get the number of cells per gram of the carrier. Azospirillum count/g of carrier = MPN table value x Dilution level Dry mass of product METHOD FOR ESTIMATING MPN COUNT FOR AZOSPIRILLUM 1. Add 100 g ASI sample to 900 ml of sterile distilled water. 2. Shake for 10 minutes on a reciprocal shaker. 3. Make ten fold dilution series. 4. Pipette 1 rnl of each dilution (from 10-1 to 10-7) to each one of 5 replicates (5 tubes containing nitrogen free bromothymol blue semi-solid malate media for Azospirillum). 5. Begin by taking aliquot from the highest dilution and proceed down the series with same pipette 6. Collect the ubes from 105, 106 and 107 dilution level. 7. Incubate the inoculated series for 2 days at 30°C. 8. Check the characteristic sub-surface while particles (+ or -) in malate semi-solid medium, a change to dark blue colour of the medium. CALCULATION To calculate the most probable number of organisms in the original sample, select as P1 the number of positive tubes in the least concentrated dilution in which all tubes are positive or in which the greatest number of tubes is +ve, and let P2 and P3 represent the numbers of positive tubes in the next two higher dilution. Then find the row of numbers in Table 1 in which P1 and P2 correspond to the values observed experimentally. Follow that row of numbers across the table to the column headed by the observed value of P. The figure at the point of intersection is the most probable number of organisms in the quantity of original sample represented in the inoculum added in the second dilution. Multiply this figure by the appropriate dilution factor to obtain the MPN value 17 Biofertilizer production technology of Rhizobium Domain : Bacteria Phylum : Proteobacteria Class : Alphaproteobacteria Order : Rhizobiales Family : Rhizobiaceae Genus : Rhizobium, Allorhizobium, Sinorhizobium Family IV. Phyllobacteriaceae Genus VI. Mesorhizobium Family VII. Bradyrhizobiaceae Genus I. Bradyrhizobium Family VIII. Hyphomicrobiaceae Genus VI. Azorhizobium Mass culture production of rhizobia had its beginning in 1895 when Nobbe and Hiltner introduced a laboratory grown culture of Rhizobium sp. Under the name ‘Nitragin’. Prior to 1923 :cultures of rhizobia were mostly prepared in small bottles /cans 1927: Method for large scale production of rhizobia by submerged fermentation with the development of a device for artificial aeration was developed. There are several forms of Microbial Inoculant (MI) viz., to be used for inocultating seed /soil 18 1. Agar based 2. Broth cultures 3. Frozen concentrates 4. Carrier based granular soil inoculum 5. Porous gypsum granules 6. Natural peat granules 7. Freeze dried Rhizobium inoculants (RI) is a product having a large population of a strain or a combination of several strains of Rhizllbium for a particular leguminous plant or a group of leguminous plants which shall produce efficient root nodules on the relevant plants and thereby enhance nitrogen fixation, crop growth and yield Isolation of efficient Rhizobium strain 1. Collection of 20-25 fresh and firm nodules/plant (Avoid damaged and decaying nodules) – this large sampling is to ensure success in obtaining effective isolates since all the strains of rhizobia forming nodules on the host are not necessarily effective in N2 fixation. 2. Preserve, store and transport nodules in airtight container to the lab for isolation. 3. Isolation of Rhizobia from root nodule 4. Confirmation /Authentication of nodule forming ability of the isolated Rhizobium on a test host under bacteriologically controlled conditions (It is only the formation of nodules , which is taken as the only criterion required in this test). The test host should be that from which the original nodule was collected. 5. Characterization of Rhizobium – microscopically, physiologically and biochemically by following BMSB 6. Colony character :Rhizobium forms as white, translucent, glistening and elevated colonies Methods to test rhizobia for nodulation 1. Cotton Plugged tubes 2. Semienclosed Gibson’s seedling agar tubes 3. Leonard jar assemblies 4. Plastic pouches 5. Fahraeus seedling tube 6. Pot culture 7. Field trials MAINTENANCE OF PURE CULTURES Maintain pure cultures of rhizobia on yeast extract mannitol agar (YEMA ) slants 19 Transfer a loopful of the pure culture to each of the agar slants aseptically in an inoculation room and incubate at 28 + 2°C for 3 to 10 days depending upon the species of Rhizobium. Always keep pure cultures at 4°C Prepare 100 ml YEMB in 250 ml conical flask and plug with non-absorbent cotton. Autoclave the flasks at 120°C for 20 min, cool and inoculate with the required Rhizobium Examine a 4 to 6 days old pure culture visually and by Gram staining test for freedom from contamination and transfer approximately 2 to 3 ml of sterile broth aseptically into it. Scrape the bacterial growth with the help of an inoculation needle and transfer the resulting bacterial suspension into a 250 ml conical flask containing the broth. Incubate the flasks at 28 ± 2°C on a rotary shaker for 2 to 6 days depending upon the species of the Rhizobium. Preservation of the cultures Preserve cultures in their own isolation or potato agar media for many years at 1. −80 °C 2. in liquid N 2 3. after adding 50 % glycerol or dimethylsulfoxide (DMSO) to an exponentially growing culture. 4. lyophilization : The cells must then be collected by centrifugation and re-suspended to a dense cell suspension with 10 % sucrose solution containing 5 % peptone. Then, 0.1 ml portion is transferred into lyophilization ampoules, which are frozen and lyophilized according to the procedures recommended for Rhizobium spp. (Vincent 1970 ). 5. Another variation is to grow the bacteria in rich medium, collect the cells by scraping and mixing with glycerol 10 % and transfer to cryotubes. Efficiency character: Formation of root nodule on test plant Response of plants to Rhizobium inoculation Inoculation of seed with Rhizobium is effective in increasing yields of crops in well – manured soil with high organic matter content. 1. Fixes N2 in root nodules by living with symbiotic association with legume Results of Rhizobium inoculation : 1. Increase in yield in field crops is upto 15% over uninoculated control Why inoculation ? 1. Population of Rhizobium in rhizosphere soil and uncultivated soil is generally low. 2. Native rhizobia may not efficient 3. Native rhizobia may not competent 20 The first requirement for the mass production of a MI is that it should have certain desirable characteristic like 1. Ability to multiply under broth culture conditions 2. Survival in carrier material 3. Stability during storage 4. Ability to withstand adverse conditions (environmental stress, agrochemical inputs) and to perform desired action under natural /field conditions 5. Competitiveness in terms of enhanced/early colonization of soil/rhizosphere and internal plant organs as per the mode of action 6. Enhanced establishment in a wide range of host genotypes Hosts Botanical Name Common Name a) Fast – growing Rhizobia Rhizobium metiloti Medicago sativa Lucerne ( Medic-rhizobia) TrigoneIIa foenum-graecum Fenugreek Rhizobium trifolii Trifolium alexandrinum Egyptian clover ( Clover-rhizobia) Trifolium sp. Clovers Rhizobium leguminosarum Lathyrus sativus Grasspea, Khesari (Pea rhizobia) Lens culinaris Lentil Pisum sattivum Pea Ytcia saliva Common vetch Rhizobium phaseoli Phaseolus multiflorus Bean ( Bean-rhizobia) P vulgarts Kindney bean, French bean Rhizobium sp. Cicer arietinum Chickpea, Bengal Gram Hosts Botanical Name Common Name b) Slow-Growing Rhizobia Rhizobium lupini Lupinus alba White lupines ( Lupin-rhizobia) 21 ( Lupinus sp. ) Lupines Rhizobium japonicum Glycine max Soybean (Soybean group) Rhizobium sp. ( Cowpea Crotalaria juncea Sunnhemp Miscellany) Cyemopsis tetragonoloba Cluster bean ( guar) Arachis hypogaea Peanut Lespedeza sericea Lespedeza( Kudzu) Canavalia gladiata Jack bean or sword bean Lablab purpureus Lablab Macrotyloma unijlorum Horse gram( Kultht) Phaseolus aconitifolius Moth bean Vigna mungo Blackgram V radiata Greengram V sinensis Cowpea Cajanus cajan Redgram Calopogonium muconoides Calopogonium, Calopo Rhizobia is presently divided into 3 genera : 1. Rhizobium 2. Bradyrhizobium 3. Sinorhizobium PREPARATION OF MASS CULTURE Prepare conical flasks or suitable fermentors each containing required amounts of broth and proceed for sterilization. Into each one of these flasks, transfer requisite quantity of inoculum culture aseptically and incubate the flasks preferably on a rotary shaker at 28 ± 2°C for 3 to 10 days depending on the species of the Rhizobium. NOTE - If the medium (broth) is formulated in a fermentor the amount or inoculum culture to be transferred into the fermentor is variable depending on the size of the fermentor but the ratio between the inoculum culture and the medium formulated in the fermentor is recommended as 1 : 20 ( inoculum: quality of broth ) in the fermentor. TEST FOR NODULATION POT CULTURE TEST Plant Nutrient Solution Composition Concentration g/l 22 (a) Potassiumchloride 0.001 M 0.0745 (b) Potassium hydrogen phosphate ( K2HP0 4 ) 0.001 M 0.175 (c) Calciumsulphate ( CaSO42H20 ) O.002M 0.344 (d) Magnesium sulphate ( MgS04·7H2O) 0.001 M 0.246 (e) Trace elements solution: 0.50 ml (f) Iron solution 0.50 ml (e) Trace elements solution Composition Concentration g/l (1) Copper sulphate ( CuS04 5H20 ) 0.01 mg/kg 0.78 (2) Zinc sulphate ( ZnS047H20 ) 0.025mg/kg 2.22 (3) Manganese sulphate (MnS044H20 ) 0.25mg/kg 2.03 (4) Ammonium molybdate [(NH4)6M07024·4H2O] 0.0025 mg/kg 0.01 (5) Boric acid: (H3B04 ) 0.125 mg/kg 1.43 (f). Iron solution Composition g/100 ml (1) Ferrous sulphate 5 (2) Citric acid 5 Autoclave the nutrient solutionthus prepared at 120°C for 20min Surface sterilization of seeds ,sterilization of soil and sowing of seeds Immerse the seeds in 95 percent alcohol and followed by surface-sterilization in freshly prepared chlorine water ( for 15 to 20 min) or 0.1 percent mercuric chloride solution for 3 min in a suitable container such as a screw-capped bottle or a test-tube with a rubber hung. In case of seeds with tough seed coat, concentrated sulphuric acid may be used as a surface sterilant for 20 to 30 min. It is recommended that the seeds should be placed overnight in a desiccator containing calcium chloride before surface sterilization with sulphuric acid. Pour out the sterilant and wash the seeds in several changes of sterile water ( at least ten times) to get rid of the sterilant. Fill earthenware or glazed pots with soil (2 parts soil and 1 part washed coarse sand) ( pH 6 to 7 ) and autoclave for 2 h at 120°C. After two days incubation at room temperature, repeat autoclaving to ensure complete sterility of soil. Inoculate surface-sterilized seeds with a water slurry of the inoculant taken from a culture packet ( 15 to 100 g seeds per gram of inoculant depending on the size of the seed) and sow the seeds. Keep a set of pots with uninoculated seeds as control and also a set of pots with ammonium nitrate at the rate of 100 kgN/ha as control aid incubate them in a pot- culture house during appropriate seasons for appropriate plants, taking care to separate the inoculated pots from the control pots. If growth rooms or cabinets having facilities to adjust temperature and light are available, the pots may be incubated in such controlled environmental 23 conditions. Sterilize the nutrient solution at 120°Cfor 20 min and irrigate each pot once to the moisture holding capacity of soil. Subsequently, water the seedling periodically with sterilized water preferably through a plastic tube, taking care to prevent splashing of water from inoculated pots to uninoculated ones. Maintain required number of replicated pots ( 4 to 6 ) for each botanical species for statistical analysis. After two to three weeks of growth, thin down the number of plants in each pot to four uniform plants. At the end of 6 to 8 weeks, take one set of pots from both the control and inoculated series and, separate the plants carefully from the soil under slow- running water. Obtain data on the number, colour ( effective nodules are pink or red) and mass of nodules. At the end of 6 to 8 weeks, harvest the shoot system, dry at 60°C for 48 h and determine dry mass. For the above purpose, maintain adequate replications of pots ( 4 to 16). Record the nodulation data regarding formation of pink colour of nodules as revealed visually when nodules are cut open by a razor blade. After computing the data, based on the dry mass of plants and nodulation data decide the effectiveness of cultures. If good effective pink modulation is obtainable in inoculated plants together with local absence or sometimes presence of stray nodules in controls and if there is a 50 percent increase in the dry mass of plants over the uninoculated control without nitrate, it may be concluded that the culture is of the required quality. Production of Rhizobium biofertilizer Selection of suitable bacterial Rhizobium strain which can form effective nodules in association with selected legumes over suitable range of environmental conditions ↓ Lyophilize Rhizobium species/strain to maintain their efficiency since sub culturing at monthly interval on artificial medium may lead to deterioration of the activity of the N2 fixation ↓ Whenever it is required for use, the tube containing the lyophilized material is cut open and the lyophilized cells are reconditioned ↓ These are grown in sufficient numbers on agar slants to be used for mass multiplication during the season (YEMB) ↓ There should be regular continued evaluation with the systematic long-stored, untouched culture in lyophilized conditions. This is necessary to ensure continued N2 fixation efficiency of the culture as seed inoculums 24 Mass scale production of Rhizobium species/strain can be carried out using system of rotary shaker/ fermenter. About 100 ml aliquots of the YEMB are dispensed into 250 ml conical flasks, which are plugged with non-absorbent cotton and are sterilized at 15lbs pressure for 20 minutes and cooled after sterilization. ↓ 3-6 day old cultures of Rhizobium are examined for the purity and about 4-6 ml of sterile broth is transferred aseptically into the tubes. ↓ The bacterial growth is scrapped with the help of an inoculated needle and the resulting bacterial suspension is transferred to 250 ml conical flask containing YEMB. ↓ The flasks are incubated at 300C on a rotary shaker for 2-7 days. Fermenters are used for industrial scale production of biofertilizers. Cultures vessels ranging from 5-1000 liters can be used depending upon the requirement. The amount of inoculum culture to be added into fermenter vessel depends on the size of the fermenters, but the ratio between the inoculum and medium in the vessel should be maintained at 1:20 (5% inoculums rate). ↓ When the number of Rhizobium in the broth has attained the required standard (108-109 cells /ml) , the broth should be added to the carrier for preparation of carrier based inoculants.The broth is continuously aerated by forcing sterile air through porous stainless steel sparger. Various fermentation requirements like aeration, agitation and fermentation time vary from strain to strain. The process optimization for the some of the Rhizobium cultures using fermenter – based technology is presented in table given below Table : Optimum fermentation conditions for mass multiplication of Rhizobium strains Sl.No. Parameter Specification 01 Type of fermenter Stirred tank 02 Type of operation Batch 03 Carbon source Sucrose/molasses (3-5 g/l) 04 Nitrogen source Corn steep liquor/yeast extract 05 PH 7.8 (Controlled) 06 Temperature 280C 25 07 Inoculum rate 10% (v/v) 08 Inoculum count 109 cells/ml 09 Antifoam Silicones/ Polyethers/Sorbitan derivatives 10 Aeration 0.5 VVM (Volume of air/Vol. of liquid/min) 11 Agitation Depends on the fermentation size 12 Fermentation time Variable (18-40 hrs) 13 Cell processing Centrifugation/membrane filteration followed by lyophilization 14 Quality 108 / g carrier at the time of manufacture specification 15 Shelf life 6 months from date of manufacture A good carrier material should: 1. Have high water holding capacity 2. Be non-toxic to rhizobia 3. Be easy to sterilize by autoclaving / gamma radiation 4. Be readily and inexpensively available 5. Provide good adhesion to seed 6. Have PH buffering capacity 7. Have cations and/or anions exchange capacity Different carrier materials like peat, lignite, charcoal, rice husk, pressmud, flyash, vermiculite, soil and coir dust have been employed. Lignite and charcoal are more commonly used as carrier in India. Preparations of carriers The carrier material is sun dried to 5 % moisture and pulverized to fineness of 72 - 100 mesh. Coarser materials form balls on mixing with broth and seeds are not properly coated with the inoculants. Lignite powder being acidic pH 5.5- usually 1 kg of lime (CaCO3) is mixed with 9 kg of lignite to bring the PH to about 7.00. Charcoal powder being neutral in P H dose not require lime. Finally the carrier is mixed with 10 % water before sterilization. The carrier material must be sterilized before blending with broth culture to suppress the proliferation of contaminants. 26 Best method is ¥ gamma irradiation (5 megarods) for carrier sterilization. The carrier materials may also be packaged and sealed in autoclavable high density polypropylene bags which can withstand a temperature of 1210C and sterilized in an autoclave Packaging material : Low density poly ethylene /poly propylene bags with minimum thickness of 75 to 100 µ Packing and labeling The blended carrier is kept for 2-4 hours for curing. After curing, the inoculants is packed in low density polypropylene (LDPE) bags of 0.2 to 0.5 mm gauge size either manualy or by using automatic dispenser. The bags are sealed by an electric sealer leaving about two-third vacant space to give proper aeration to the inoculants. LDPE packets are used universally, as they can be readily sealed, allow high exchange with relative low moisture loss. The inoculants packet is labeled with relevant information regarding culture viz. name of the culture, crop for which intended, expiry date, directions for storage / use and precautions, certifications mark if any. Packets (Inoculants) must be incubated for a week in the room at 300C so that the bacteria multiplies and attains the required count. The packet may then be stored in a cold room (4-150C). The inoculants packets should have the following information: 1. Name of the product 2. Name and address of the manufacturer 3. Name of carrier 4. Batch number 5. Date of manufacture 6. Date of expiry 7. Net mass 8. Crops for which intended 9. Storage instructions The inoculants should not be subjected to excessive temperatures, ideally it should be held at 40C throughout transport and storage. Inoculum in bulk should be transported in strong cartoons on boxes. Distributions of cultures during summer and rainy seasons require suitable arrangements and strict quality control measures. The shelf life of Rhizobium inoculants thus prepared is about 6 months at room temperature and in a cold room it can be up to 4 years. During the end of this period each gram of the inoculants shall contain 107 cells /g of the carrier on dry weight basis. 27 Crops on which they are to be used Rhizobium biofertilizers can be used for cultivating legumes like redgram, Bengal gram, cowpea, greengram, blackgram, horsegram, soybean, beans, clusterbeans, groundnut etc. Care should be taken to use Rhizobium only to those crops for which they are meant as mentioned on the biofertilizer packet, as some of them are host specific. Dosage and seed treatment Usually 400-500 g lignite based culture would be sufficient to the quantity of seed required. per hectare (20g/kg seed). The method of seed inoculation includes preparation of 10% sugar/pharmaceutical grade gum Arabic /1% carboxy methyl cellulose (CMC) solution. This solution is sprinkled on the seeds and the seeds are thoroughly mixed so as to have a uniform coating. A count of 1000 viable cells per seed is to be attained at the time of treating the seed and quantity of culture used is accordingly adjusted. The seeds are spread uniformly for drying on a gunny bag/cement floor in shade avoiding direct sunlight. The seeds are sown immediately (preferably in the early morning or late evening). Shelf life Biofertilizer packets normally have a shelf life of 6 months at room temperature. During the end of this period each gram of the inoculants shall contain 107 cells / g of the carrier (on dry weight basis). Certain precautions are to be observed in the use of Biofertilizers. The Rhizobium biofertilizer has to be used before the expiry date marked on the packet and the inoculated seeds are not allowed to come in direct contact with any pesticides/fertilizers. If fungicides, insecticides , herbicides and chemical fertilizers have to to be used, there should be a gap of at least 15 days between the use of these chemicals and biofertilizers. In order to protect Rhizobium from effects of fertilizers , pesticides, acid/saline and dry soils, pelleting of the seeds is the most commonly used practice. The most commonly used pelleting agents are calcium carbonate, rock phosphate, charcoal, talc, gypsum, bentonite etc. Co-inoculation with other biofertilizers like phosphate solubilizing microorganisms, AM fungi etc, have no deleterious effect on Rhizobium inoculants. Quality control specifications prescribed for Rhizobium 1 Base Carrier based* in form of moist/dry powder/granules/liquid based 28 2 Viable cell count Cfu minimum 5 X 107 cells / g of powder, granules or carrier material or 1 X 108 cells /ml of liquid 3 Contamination level No contamination at 105 dilutions 4 pH 6.5 – 7.5 5 Particle size in case of carrier based All material shall pass through 150 to 212 µ material (72 to 100 mesh) IS sieve 6 Moisture percent by weight, maximum in 30 - 40% case of carrier based 7 Efficiency character Should show effective nodulation on all the species listed on the packet *Type of carrier: The carrier material such as peat, lignite, peat – soil , humus, wood charcoal or similar material favouring growth of the organism. Quality control 1. Total plate count by SPC 2. Test for N2 fixation in Pure culture by Kjeldahl Method 3. Root nodulation test QUALITY CONTROL TESTS RECOMMENDED AT BROTH STAGE Qualitative Tests 1. Check for freedom from visible contaminants 2. The pH of the bacterial broth shall normally be between 6.5 and 7.5 3. Smear and Gram Stain NOTE- A smear prepared from undiluted broth should be free from Gram positive cells. The presence of 8 few Gram positive cells in occasional fields which may be due to dead cells in the medium may be disregarded. 4. Absence of Growth on Glucose-Peptone Agar NOTE - When a loopful of the broth is streaked into this medium and incubated at 28 ± 20C for 24 h, the purple-violet colour of the medium ( due to the indicator bromocresol purple ) shall not change. If the colour changes to yellow ( acidic reaction) or blue ( alkaline reaction) the broth is grossly contaminated. Hence, the broth should be rejected. 5. Streak on Congo Red YEMA (CRYEMA) When a loop full of the broth is streaked into this medium and incubated at 28 ± 20C for 3 to 10 days, it shall show colonies of bacteria with growth characteristics same as that of the pure culture used in the preparation of the broth. Otherwise, the broth should be rejected. 29 When a loop full of the broth is streaked into this medium and incubated at 28 ± 20C for 3 to 10 days, it shall show colonies of bacteria with growth characteristics same as that of the pure culture used in the preparation of the broth. Otherwise, the broth should be rejected. Quantitative Test Viable or Plate Counts Serially dilute one millilitre of the broth to obtain dilutions of the order of 106 to 109 Plate 0.2 ml aliquots of the dilutions on YEMA plates and incubate at 28 + 2°C for 2 to 6 days, depending on the species of Rhizobium. The counts of viable Rhizobium in the final broth from shake culture or fermentors shall be not less than 108 to 109 cells/ml. Otherwise, the broth should be rejected INCUBATION OF PLATES Label the plates and incubate at 28 + 2°C for 3 to 5 days for fast growing rhizobia and 5 to 10 days for slow-growing ones. The common species of fast and slow-growing rhizobia are given below: COUNTING Count the colonies which are specific to Rhizobium. Count such type of tubes/plates and tally this count with MPN table to get the number of cells per gram of the carrier. Rhizobium count/g of carrier = Number of colonies x Dilution level Dry mass of product DETERMINATION OF NUMBER OF Rhizobium CELLS PREPARATION OF SERIAL DILUTION FOR SPC: Dispense 30 g of RI in 270 ml of sterile water and shake for 10 minutes on a reciprocal shaker. Make serial dilutions upto 107 dilutions. Pipette out 0.2 ml aliquots of 10 5 to 107 dilution and deliver it to screw cap tubes or petridishes containing set medium. Spread the aliquots over the plates. Invert the plates and place them in the incubator at 28 ± 2°C for 3 - 5 days. Use 5 replicates of 105. 106 and 107 level. COUNTING Count the colonies which specific to Rhizobium. Consider them for the purpose of calculation to get the number of cells per gram of the carrier. Number of Cfu of Rhizobium /g of carrier = Number of colonies x Dilution level Dry mass of product 30 Biofertilizer production technology of PSMs Domain : Bacteria Phylum : Proteobacteria Class : Gammaproteobacteria Order : Pseudomonadales Family : Pseudomonadaceae Genus : Pseudomonas Phosphorus Solubilizing Microorganisms (PSMs) 31 Plants generally take up phosphorus as primary orthophosphate ion (H 2PO4) and sometimes as secondary orthophosphate ion (HPO4-2). Phosphorus nutrition in the early stages benefits the plant by producing deeper and abundant roots. Tropical soil are mostly deficient in available Phosphorus, though the soil may contain P Phosphorus abundantly in unavailable form (it is a form roots cannot take). Several bacteria, fungi, actinomycetes have the capacity to solubilize insoluble mineral phosphates and have been reported to solubilize varying quantities of phosphorus depending on the efficiency of the strains. These groups of MOs are collectively called Phosphate Solubilizing Microorganisms (PSMs). The two important groups of SMOs that are used in commercial biofertilizer production. 1. Phosphate Solubilizing bacteria (PSB) : Pseudomonas striata, Bacillus polymixa, B cirulans, B megaterium var.phosphaticum, Burkholderia cepacia, etc., 2. Phosphate Solubilizing fungi (PSF) Aspergillus awamori, Penicillium digitatum, , etc. Their inoculation to different crops is found to be highly beneficial. Efficiency test of strain based on phosphate solubilising capacity in the range of minimum 30 percent in terms of zone formation minimum 10 mm solubilisation zone in a prescribed solid medium having at least 3 mm thickness Isolation of efficient Phosphorus Solubilizing Microorganisms 1. Isolation from rhizosphere soil 2. Name of culture medium : Pikov-skaya’s medium 3. Serial dilution technique 4. Pour plat / spread plate method 5. Characterization of Phosphorus Solubilizing Bacteria – microscopically, physiologically and biochemically by following BMSB 6. Characterization of Phosphorus Solubilizing Fungi – microscopically, physiologically and biochemically by following standard mycological methods Response of plants to PSMs inoculation Inoculation of soil or seed with PSMs is effective in increasing yields of crops in well – manured soil with high organic matter content. 1. Solubilizes P Results of PSMs inoculation : 1. Increase in yield in field crops is upto 12% over uninoculated control Why incoculation ? Population of PSMs in rhizosphere soil and uncultivated soil is generally low. Production of Pseudomonas striata biofertilizer Selection of suitable PSB - Pseudomonas striata strain which can form effective in solubilization of P over suitable range of environmental conditions 32 ↓ Lyophilize Pseudomonas striata strain to maintain their efficiency since sub culturing at monthly interval on artificial medium may lead to deterioration of the activity of Phosphate Solubilization ↓ Whenever it is required for use, the tube containing the lyophilized material is cut open and the lyophilized cells are reconditioned ↓ These are grown in sufficient numbers on agar slants to be used for mass multiplication during the season (Pikov-skaya’s medium ) ↓ There should be regular continued evaluation with the systematic long-stored, untouched culture in lyophilized conditions. This is necessary to ensure continued Phosphate Solubilization efficiency of the culture as seed inoculums Mass scale production of Pseudomonas striata strain can be carried out using system of rotary shaker/ fermenter. About 100 ml aliquots of the Pikov-skaya’s medium are dispensed into 250 ml conical flasks, which are plugged with non-absorbent cotton and are sterilized at 15lbs pressure for 20 minutes and cooled after sterilization. ↓ 3-6 day old cultures of Pseudomonas striata are examined for the purity and about 4-6 ml of sterile broth is transferred aseptically into the tubes. ↓ The bacterial growth is scrapped with the help of an inoculated needle and the resulting bacterial suspension is transferred to 250 ml conical flask containing Pikov-skaya’s medium. ↓ The flasks are incubated at 300C on a rotary shaker for 2-7 days. Fermenters are used for industrial scale production of biofertilizers. Cultures vessels ranging from 5-1000 liters can be used depending upon the requirement. The amount of inoculum culture to be added into fermenter vessel depends on the size of the fermenters, but the ratio between the inoculum and medium in the vessel should be maintained at 1:20 (5% inoculums rate). ↓ When the number of Pseudomonas striata in the broth has attained the required standard (108-109 cells /ml) , the broth should be added to the carrier for preparation of carrier based inoculants. The broth is continuously aerated by forcing sterile air through porous stainless steel sparger. Various fermentation requirements like aeration, agitation and fermentation time vary from strain to strain. The process optimization for the some of the Pseudomonas striata cultures using fermenter. Crops on which they are to be used 33 All cereals, pulses, oil seeds, plantations crops, vegetables, flower crops, fodder crops, commercial crops like sugarcane, tobacco, chilly, cotton etc. Dosage and seed treatment Usually 400-500 g lignite / charcoal based culture would be sufficient to the quantity of seed required per hectare. The method of seed inoculation includes preparation of 10% sugar/pharmaceutical grade gum Arabic /1% carboxy methyl cellulose (CMC) solution. This solution is sprinkled on the seeds and the seeds are thoroughly mixed so as to have a uniform coating. A count of 1000 viable cells per seed is to be attained at the time of treating the seed and quantity of culture used is accordingly adjusted. The seeds are spread uniformly for drying on a gunny bag/cement floor in shade avoiding direct sunlight. The seeds are sown immediately (preferably in the early morning or late evening). Dosage, method and time of application: Soil application: 10 kg/ha mixed in 20 kg FYM and can be applied by broadcasting or by row method. Slurry treatment/root dip method: Thick slurry of 1 kg inoculums in 5 liters water is made and roots of seedlings are dipped in slurry for a minimum period of 30 minutes and then planted in the main field. Inoculation to plantation crops: For plantation crops, the soil around the root zone is excavated in the form of ring. The Pseudomonas striata biofertilizer is applied all along the root zone in the form of a basin or ring @ 25-50 g per plant after mixing with well decomposed FYM / vermicompost. The excavated soil is then covered back around the trunk of the tree. Similarly, plantation crops, vines such as pepper can be inoculated. For crops more than 6 months duration Pseudomonas striata biofertilizer can be applied to soil once in 6 months. Shelf life Biofertilizer packets normally have a shelf life of 6 months at room temperature. During the end of this period each gram of the inoculants shall contain 107 cells / g of the carrier (on dry weight basis). Certain precautions are to be observed in the use of Biofertilizers. The Pseudomonas striata biofertilizer has to be used before the expiry date marked on the packet and the inoculated seeds are not allowed to come in direct contact with any pesticides/fertilizers. If fungicides, insecticides , herbicides and chemical fertilizers have to to be used, there should be a gap of at least 15 days between the use of these chemicals and biofertilizers. In order to protect Pseudomonas striata from effects of fertilizers , pesticides, acid/saline and dry soils, pelleting of the seeds is the most commonly used practice. 34 The most commonly used pelleting agents are calcium carbonate, rock phosphate, charcoal, talc, gypsum, bentonite etc. Co-inoculation with other biofertilizers like Rhizobium, AM fungi etc, have no deleterious effect on Pseudomonas striata inoculants Quality control specifications prescribed for PSMs 1 Base Carrier based* in form of moist/dry powder/granules/liquid based 2 Viable cell count Cfu minimum 5 X 107 cells / g of powder, granules or carrier material or 1 X 108 cells /ml of liquid 3 Contamination level No contamination at 105 dilutions 4 pH 6.5 – 7.5 for moist / dry powder, granulated or carrier based and 5.0-7.5 for liquid based 5 Particle size in case of carrier based All material shall pass through 0.15 – 0.212 material mm IS sieve 6 Moisture percent by weight, maximum in 30 - 40% case of carrier based 7 Efficiency character The strain should have phosphate solubilizing capacity in the range of minimum 30%, when tested spectrophotometrically. In terms of zone formation, minimum 5 mm solubilization zone in prescribed media having atleast 3 mm thickness. *Type of carrier: The carrier material such as peat, lignite, peat – soil, humus, wood charcoal or similar material favouring growth of the organism. Packaging material : Low density poly ethylene /poly propylene bags with minimum thickness of 75 to 100 µ Quality control 1. Total plate count by SPC 2. P Solubilization zone – 10 mm with 3 mm thickness Biofertilizer production technology of Mycorrhiza Arbuscular Mycorrhizal Fungi (AMF) The most common association occurring in majority of crop plants important in agriculture, horticulture, and tropical forest trees is the Arbuscular Mycorrhizal type. 35 Arbuscular Mycorrhizal Fungi are geographically ubiquitous and occur over broad ecological range from aquatic to desert environments. Majority of plants are colonized by Arbuscular Mycorrhizal Fungi (AMF) except those belonging to few families like Brassicaceae, Chenopodiaceae, Polygonaceae, etc. Arbuscular Mycorrhizal Fungi are formed by Glomeromycetous fungi, the common genera being Glomus, Gigaspora, Acaulospora, Entrophosphora and Scutellospora. These fungi are obligate symbionts and have not been cultured on nutrient media in the laboratory. Hence, they are maintained in the roots of living host plants as “pot cultures”. It is very well established that inoculation with efficient AMF improves the growth and yield of crop plants.This is particularly so in the soils deficient in nutrients, especially phosphorus. This is mainly because of increased surface area of absorption of nutrients by the hyphae. Other beneficial effects are better water uptake, control of root pathogens, production of plant growth regulators etc. AMF are not host specific. However, it has been shown to have host preference i.e., for every host there is always a best fungal partner. Thus after screening several AMF, efficient AMF for inoculating a particular crop has been developed. Because of difficulty in mass producing AMF, the best way to utilize AMF for crop production would be to concentrate on crops which are normally grown on nursery beds or root trainers or polybags, where they could easily be inoculated with desired AMF, and then transplanted to the field. Nearly 25-50% phosphatic fertilizer can be saved through inoculation with efficient AMF. Tissue cultured plantlets also respond very well to AMF inoculation. Co-inoculation of AMF with other beneficial SMOs like N fixers, P solubilizers is more useful in improving the growth and yield of crop plants. 36 Fig. 1: Schematic picture of arbusucular mycorrhizal fungi colonizing roots and their hyphal extension into soil Glomus , Diversispora, Paraglomus sporiferous saccule Acaulospora Entrophospora Gigaspora bulbous suspensor germination shield Scutellospora Fig. : Morphology of representative genera of arbuscular mycorrhizal fungi. 37 Recent classification of AM fungi Phylum Class Order Family Genera Glomeromycota GLomeromycetes Archaeosporales Ambisporaceae Ambispora Archaeosporaceae Archaeospora Intraspora Geosiphonaceae Geosiphon Diversisporales Acaulosporaceae Acaulospora Diversisporaceae Diversispora Otospora Entrophosporaceae Entrophospora Gigasporaceae Gigaspora Scutellosporaceae Scutellospora Racocetraceae Racocetra Cetrospora Dentiscutaceae Dentiscutata Fuscutata Pacisporaceae Pacispora Glomerales Glomeraceae Glomus Paraglomerales Paraglomeraceae Paraglomus A biofertilizer is a product containing living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of nutrients to the host plant Mycorrhiza is defined as a mutualistic symbiosis between plant and fungus localized in root or root like structures in which energy moves primarily from the plant to the fungus and inorganic nutrients and water move from fungus to plant. Mycorrhizal associations are formed between most plant roots and some specific fungi. Their association ensures increased efficiency of mineral uptake, especially of poorly mobile ions and plays an important role in enhancing plant growth and yield. Efficient strains of mycorrhizal fungus on application as biofertilizer to soil and roots colonize roots and form thick mycelial mat around the roots. This thick fungal colonization within and around roots, can also act as physical barrier against soil borne disease causing pathogens. Hyphal strands of fungi spread many folds in the adjoining soil far beyond the root zones for nutrient uptake developed around the non-mycorrhizal roots. These extending hyphae increase the root volume by 50 to 150% and ensure better uptake of water and nutrients from the deeper and inaccessible far away zone from plant roots. In brief, mycorrhizal hyphae ensure greater absorptive area and provide scope for exploration of larger soil volume. Mycrorrhizal association also ensures increased water use efficiency, resulting into less water requirement. Under rain fed marginal land conditions with low fertility indices conditions and those soils with mycorrhizal associations ensure better productivity. Plants inoculated with mycorrhiza have been found to be more resistant to drought (water stress conditions) and some root diseases. 38 Mycorrhizal plants can absorb and accumulate phosphate and other micronutrients from the soil or solution several times more than non-mycorrhizal plants. This advantage of mycorrhizal association helps in reducing the chemical fertilizer requirement of phosphorus and some micronutrients such as zinc. Under optimal conditions, mycorrhizal association can replace the requirement of chemical phosphorus up to 25 percent These mycorrhizal associations also ensure better utilization of applied phosphorus, which can be quickly fixed in insoluble form in certain soils. Researches conducted over last 30 years have proved beyond doubt that mycorrhizal fungi on application as biofertilizer can increase in crop growth and yield by 10 to 25% depending upon the type of fungal inoculum of crop selected and the method of inoculation. With the growing awareness for environment friendly technologies and the need for promoting renewable sources of nutrients, mycorrhiza is getting wider acceptance among the farming community. Keeping in view of the economical and environmental benefits of mycorrhizal fungi, large numbers of commercial manufacturers have set up production facilities. While some of them are producing pot culture based soil inoculum, some are producing large quantities through in-vitro culture technique. In soil based formulations mycorrhizal fungi is grown on the roots of host plant in pots or field soil of limited area and after sufficient growth has taken place, roots along with fungal spores and hyphae are harvested and formulated into products. The methodology is cumbersome and such products may have low spore counts. If soil culture is carried out under open conditions the product is prone to contamination by soil pathogens and weed seeds and hence enough care has been taken to ensure that soil for inoculum preparation is sterilized and preparation of soil culture is carried out under poly-houses. In in-vitro culture technique, the fungus is grown on fibrous root tissues under controlled laboratory conditions. Once adequate growth has taken place, the root biomass is harvested and formulated in to the product. The biofertilizer so prepared may have very high spore count. Also as the technology do not require open fields and the entire process can be carried out under sterile controlled conditions of the laboratory, it is easier to produce in bulk quantities under the industrial/commercial mode without the fear of being contaminated with soil pathogens and weed seeds. Commercialization of technology necessitates effective implementation and enforcement of quality control measures to protect the interests of consumers/farmers. Collection and Maintenance of AM fungal spore : 1. Method is wet sieving and decanting – 53µ , 105µ , 250µ, 710µ sieves. 2. Surface sterilization of AM fungal spore – 2% Chloramine T containing 200mg Streptomycin/l and trace of detergent for 15 minutes. 39 3. Maintenance of stock culture of AM fungi : Single spore through funnel technique to pot culture to get a single of AMF (Pure culture). This inoculum (pot) can be used to establish large number of stock plants that will supply inoculum for further use. Desirable qualities of AMF inocula 1. Infective inoculum : The infectivity of inoculum is measured by its ability to penetrate and spread in the roots of the crop under consideration. Benefits of AMF inoculation start realizing from early part of its growth. The benefits of AMF inoculation may not be realized if the inoculum is non-infective. It is therefore desirable to undertake preliminary testing before the inocula is passed on to the user. 2. Effective inoculum : The effectiveness of the inoculum is judged by the ability of AM fungi to induce stress tolerance to enhance growth under actual crop production conditions. Effective assay must involve indigenous AM fungi , fertility levels, pathogens and soil or appropriate growth media. Very often AMF isolated from low – P condition have been found to fail in colonising roots of plants growing at high concentrations of available P. Inoculant effectivity improves with concentration and enhanced infectivity. 3. Concentrated inoculum : The requirement for the concentrated inoculum is a practical desirable quality requirement. It is relatively cheaper to store, transport and apply an inoculum if it is concentrated one. It is desirable that the inoculum if it is concentrated it will not be bulky. It is possible to produce concentrated inocula containing 100s of spores/gram of substrate through pot culture. The inocula produced through aeroponic culture of roots may contain 1000s of spores / gram of root. Use of 100 g of aeroponically grown root inoculum may meet the requirement of a hectare of land area. 4. Freedom from pathogens : Sanitary and aseptic condition must be maintained during production of AMF inoculum 5. Shelf-life : AMF inoculum obtained from pot culture can be stored for about 4-5 weeks without refrigeration, its shelf-life could of course be extended to 25-50 weeks if stored moist with refrigeration (4oC). The viability of inoculum may be lost in less than a month if spore is dried without the host. Large scale inoculation with AM fungi is limited because of lack of production of high quality pathogen free inocula. The inocula now in use consists of 1. Fungal structures : Spores , hyphae 2. Infected roots 3. Whole rhizosphere : Infected roots fragments, Spores , hyphae, Procedure for mass production 40 Several methods like spores of AMF as inoculums, alginate entrapped inoculums, nutrient film technique, expanded clay as a carrier of AMF, aeroponic culture, root organ culture method etc., have all been tried to mass produce AMF but the best method, at present , is to produce these AMF is in association with host roots. This substrate method is described here. Substrate method of mass production of AMF : Substrate based inoculums is commonly used for mass production of AMF. The main objective of this production technology is to produce maximum number of infective propagules in a short time, with least contaminants. The inoculum production should be done in a glass house. Large plastic trough / cement cisterns can be filled with perlite : soilrite mix (1 : 1) by volume and inoculated with the starter culture of desired AMF and sown with Rhodes grass as the host. If the Rhodes grass is not available, Guinea grass / any other suitable grass can be used. Calcium ammonium nitrate (to give 80 ppm N) and rock phosphate (to give 10 ppm P) can be the N and P source. Ruakura nutrient solution can be added once in 8 days. Addition of fungicides Captan or Rilon, the acaricide Furadan and the insecticide, Sumicidin at half the recommended level can be mixed with the substrate to reduce the contaminants in the inoculums, with no effect on AMF. Plants can be harvested 75 days after sowing. Roots can be cut into small pieces, mixed with the substrate, air dried, packed in polybags and used as the inoculums. The inoculums must be free from root pathogenic organisms. Crops on which AMF inoculums can be used AM inoculums can be used on any crop important in agriculture, horticulture and tropical forestry that is raised in nursery bed or root trainer or ppolybag. Crops which donot respond to AMF inoculation are those belonging to Brassicaceae like mustard, cauliflower, cabbage, radish etc., Dosage, method and time of application Nursery application: About 1 kg of inoculums is sufficient for treating the nursery bed of 1 m2. The inoculums should be applied 2-3 cm below the soil. Sowing the seeds or planting the cuttings can be done after applying the inoculums. Polybag nurseries: About 10g of the inoculums can be applied around 6 cm below the soil surface and seeds can be sown 1-2 cm below the surface of the soil. Inoculum for out planting: About 10g of the inoculums can be applied to the planting hole before planting the seedling or hardened tissue cultured plantlets. Inoculation of established perennial crops : Already established fruit trees like papaya and crops like mulberry can respond well to mycorrhizal inoculation when AMF inoculums is applied in the region at the rate of 100 g of inoculums / tree or bush. 41 Mycorrhizal Inoculum: Mycorrhizal Inoculum is a product having a large population of one isolate or a combination of several isolates of mycorrhiza mixed with a carrier which can colonize plants and thereby enhance nutrient uptake, crop growth and yield of inoculant plants. REQUIREMENTS 1. The Mycorrhizal Inoculum shall be carrier based, the colour depending on the colour of the carrier. Soil/fly ash/charcoal/ lignite or any other neutral material shall be used as carrier. 2. Form/base of the Mycorrhizal Inoculum shall be fine powder/ tablets/ granules/ root biomass mixed with growing substrate. 3. Particle size for carrier based powder formulations shall be such that 90 percent shall pass through 250 micron IS sieve [see IS 460 (Part 1)]. 4. Mycorrhizal Inoculum shall have a minimum shelf life of one year from the date of manufacture. 5. The material shall also comply with the requirements given in Table 1. The Mycorrhizal Inoculum shall also comply with the requirements given in Table 1. Sl. No. Characteristic Requirement 1 Moisture content of Mycorrhizal Inoculum, percent by mass, 8% Maximum 2 pH 6.5 - 7.5 3 Total viable spores, number per gram of Mycorrhizal Inoculum, 100 Minimum 4 Infectivity potential, infection points in test roots/g of Mycorrhizal 120 Inoculum, Minimum Packing Mycorrhizal Inoculum shall be packed in plastic bags or any other container as agreed to between purchaser and the supplier. Quality control specifications prescribed for AMF 1 Form/Base Fine powder / tablets / granules/ root biomass mixed with growing substrate 2 Particle size for carrier based powder 90 % should pass through 250 micron IS sieve formulations (60 BSS) 3 Moisture content per cent maximum 8 4 pH 6.0 – 7.5 5 Total viable propagules/ g of product, 100 propagules / g of finished product minimum 6 Infectivity potential 120 infection points in test roots / g of mycorrhizal inoculums used 42 Each packet shall be marked legibly to give the following information: 1. Name of the product, specifically as Mycorrhizal inoculant; 2. Name and address of the manufacturer; 3. Type of the carrier; 4. Batch or Code number; 5. Date of manufacture; 6. Date of expiry (agreed between the manufacturer and the purchaser subject to minimum one year from the date of manufacture) 7. Net quantity and the area meant for; 8. Storage instructions worded as under : ‘STORE IN A COOL PLACE AWAY FROM DIRECT SUN AND HEAT’ 9. Any other information as required under FCO. Directions for the use of Mycorrhizal Inoculum shall be printed briefly on the packet. A separate pamphlet may preferably be given with it. BIS Certification Marking The product may also be marked with the Standard Mark. The use of the Standard Mark is governed by the provisions of the Bureau of Indian Standards Act, 1986 and the Rules and Regulations made there under. The details of conditions under which the License for the use of the Standard Mark may be granted to manufacturers or producers may be obtained from the Bureau of Indian Standards. Storage Mycorrhizal Inoculum shall be stored by the manufacturer in a cool and dry place away from direct heat preferably at a temperature of 15°C to 30°C. It shall also be the responsibility of the manufacturer to instruct the retailers and, in turn, the users about the precautions to be taken during storage. Shelf life AMF inoculum is made up of the substrate containing hyphal bits and thick walled chlamydospores, and root pieces containing mycelium, vesicles and arbscules. The inoculums can be stores at room temperature for one year. ESTIMATION OF VIABLE SPORES For estimation of viable spores, spores are harvested from the Mycorhizal Inoculum (finished product), stained and observed under stereomicroscope. Harvesting of Spores from Finished Product Two methods have been specified for harvesting spores – by 43 1. sieving 2. sucrose gradient The sucrose gradient method shall be taken as the referee method Harvesting of Spores from Finished Product : By sieving Apparatus 1. Stalking sieves with nylon or stainless steel mesh and a large range of pore sizes for isolating spores from the carrier or soil sample 2. 40-50 micron (0.04 mm) sieve for small sized spores 3. 100 micron (0.10 mm) sieve for medium sized spores 4. 250 micron (0.25 mm) sieve for very large spores and sporocarps 5. Wash bottles 6. Jars for collecting the sieving 7. Stereo zoom (stereomicroscope) 8. Petri dishes ( 11 cm ) for observing the sieving under stereomicroscope 9. Micropipettes for spore picking 10. Centrifuge Procedure 1. Mix 5-10 g inoculum in a substantial volume of water and decant through a series of sieves arranged in descending order of mesh size. 2. Roots and coarse debris are collected on a coarse (60-BSS) sieve, while spores are captured on one or more finer sieves. 3. Vigorous washing with water is necessary to free spores from aggregates of clay or organic materials. 4. Collect the sieving in jars. 5. Transfer the sieving onto the gridded petri dishes/plate and observe under stereomicroscope after staining Harvesting of Spores from Finished Product : By sucrose gradient Apparatus 1. 40-50 micron (0.04 mm) sieve for small sized spores 2. Wash bottles containing water 3. Jars for collecting the sieving 4. Stereo zoom (stereomicroscope) 5. Petri dishes ( 11 cm ) for observing the sieving under stereomicroscope 44 6. Micropipettes for spore picking 7. Centrifuge Reagents : 60 percent sucrose solution 1. Collect the sieving by the method described in sieving. 2. Transfer the sieving into centrifuge tubes and centrifuge for 5 minutes at 1750 rpm in a horizontal rotor. 3. Decant the supernatant liquid carefully and re-suspend pellet in 60 percent sucrose solution. 4. Again centrifuge for 2 –5 minutes. 5. Pour the supernatant (with spores) onto a 300 BSS sieve size and rinse with water to remove the sugar. 6. Transfer the sieving onto the gridded petri dishes/plate and observe under stereomicroscope after staining. Spore staining Apparatus 1. Centrifuge tubes of 1.5-2.0 ml size 2. Stereomicroscope 3. Petri-dishes Reagent 1. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) 2. Distilled water Procedure Prepare 0.25% solution of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). Avoid exposure of MTT solution to light, as the stain is light sensitive. Add freshly collected spores (approximately 100 in number) collected by any of the two methods previously described to the staining solution and incubate at 270C in sterile centrifuge tubes in dark. Observe the spores for different colour reactions using stereomicroscope under dark field after 24 hours, 48 hours and 72 hours of incubation. Spores, which stained red or pink, are treated as viable. Count the number of spores in plate/dish and express it as spores/g of the sample. PRINCIPLE The bioassay is used to determine the number of infective propagules present in the product. Once the infective propagules (spores, mycelia and vesicles in the root fragments) come in contact with the host roots they give out a turgid mycelial structure - the appressorium, which is the initial step in the penetration event. This appressorium facilitates the fungus to enter the root through an ‘entry point’. This entry point can be visualized by staining and enumerated as a measure of the infectivity 45 of the inoculum. Host plants are grown from pre germinated seeds and a known weight of the inoculum is applied to the test host plant in pots. These pots are maintained for 15 days if the ambient temperature is >25oC and for 30 days if the ambient temperature is 25oC and for 45 days if the ambient temperature is 90%, no seed rotting, seedling healthy, root and Highly Efficient shoot portions well developed (HE) 1-15 Germination 80-90%, infection on main as well as lateral roots, Efficient (E) seedlings are well developed 16-30 Germination 70-80%, development of roots restricted and Moderately growth is less compared to Score 1. Infection occurred on roots. Efficient (ME) Shoot portions developed but growth retarded compared to Score 1. 31-45 Germination 60-70%, length of roots and shoots short compared Moderately to Score 1. Germination of seeds inhibited. 50% of root area Inefficient (MI) infected. Shoot portions also showed infection 46-60 Seed germination 50-60%. Development of roots and shoots Efficient (I) greatly retarded. Shoot portions also showed infection. Above 60 Less than 50% germination and seed rotting Highly Inefficient (HI) For the root colonization assay, the rhizosphere region of the plants tested above have to be collected and the soil adhering to the root surface has to be removed by gently tapping the roots. The root bits have to be cut into 1 cm bits and randomly 25 bits should be selected for each treatment. They have to be plated on (TSM) and the percentage of root bits colonized has to be recorded. This has to be performed in the sterile soil and non sterile soil. One control treatment without the Biocontrol agent, being tested, should be kept for both the sterile and non-sterile soil to rule out of the possibility of interference of native micro flora in the bioefficacy assay. Annexure-1.1 UNDERTAKING BY MANUFACTURERS OF MICROBIAL PESTICIDES I,-------------------------------------,aged-------years, s/o-------------------------------------------, R/o------------------ -----------------------------and-----------------------------------------of M/s.------------------------------------------------ -------------------------------Registered Office at----------------------------------------------------------do hereby undertake as follows: That the product----------------------------------------------------based on--------------------, Strain------- ----------------------, manufactured by M/s.--------------------------------and /or imported by M/s……………………………………..does not contains any genetically modified organism (GMO). That I/We shall abide by the provisions contained in the Interna

Biofertilizer Production Technology AMB507 PDF

Document Details

Tags

Related

Summary

Full Transcript

Upgrade to continue