Biofertilizers pk mani


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Biofertilizers, merits, usefulness, methodology of preparation and application methods etc.

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  • Figure: 19-71
    Schematic diagram of major metabolic reactions and nutrient exchanges occurring in the bacteroid. The symbiosome is a collection of bacteroids surrounded by a single membrane originating from the plant.
  • Finally, for a partner choice model to adequately explain the evolutionary persistence of symbiotic nitrogen fixation,
    Prior to infection - signalling mechanisms
    e.g. Nod genes
    However, signalling vulnerable to dishonesty
    More than 50 symbiotic genes have been discovered in rhizobia
    Mutations that inhibit symbiont quality (e.g. nif genes) will not necessarily disrupt symbiont recognition (e.g. nod genes)
    That is, plants may be infected by strains that signal reciprocity but have lost the ability to fix N effectively
  • Biofertilizers pk mani

    1. 1. Bio-fertilizers Dr. P. K. Mani Bidhan Chandra Krishi Viswavidyalaya E-mail: Website:
    2. 2. Bio-fertilizers or microbial inoculants are the carrier-based preparations containing sufficient number of microorganisms in a viable state inoculated to soil or seed to augment the nutrient availability to plant by enhancing the growth and proliferation of microorganisms. A biofertilzer is an organic product containing a specific microorganism (microbial inoculant) in concentrated form (107 to 109 g-1), which is derived either from the nodules of plant roots or from the soil of root zone (Rhizosphere). Biofertilizers may be referred to as inoculants after the name of microorganisms they contain, viz. Rhizobium inoculant or Azospirillum inoculant
    3. 3. Energetics Industrial fixation N≡N Haber-Bosch (100-200 atm, 400-500°C, 8,000 kcal kg-1 N) 3CH4 + 6H2O --> 3CO2 + 12H2 4N2+12H2 --> 8NH3 Biological Nitrogen Fixation Nitrogenase (4,000 kcal kg-1 N)
    4. 4. Fritz Haber (1868-1934) Carl Bosch (1874-1940) Haber and Bosch: Most influential persons of the 20th Century ( Nature, July 29,1999)
    5. 5. In 1828, the German chemist Friedrich Wöhler obtained urea artificially by treating silver cyanate with ammonium chloride.[5][6][7] AgNCO + NH4Cl → (NH2)2CO + AgCl This was the first time an organic compound was artificially synthesized from inorganic starting materials, without the involvement of living organisms. The results of this experiment implicitly discredited vitalism: the theory that the chemicals of living organisms are fundamentally different from inanimate matter.
    6. 6. The relationship between activation energy ( ) and enthalpy of formation (ΔH) with and without a catalyst, plotted against the reaction coordinate. The highest energy position (peak position) represents the transition state. With the catalyst, the energy required to enter
    7. 7. In chemistry, activation energy is a term introduced in 1889 by the Swedish scientist Svante Arrhenius that is defined as the minimum energy that must be input to a chemical system, containing potential reactants, in order for a chemical reaction to occur. Activation energy may also be defined as the minimum energy required to start a chemical reaction. The activation energy of a reaction is usually denoted by Ea and given in units of kilojoules per mole. Activation energy can be thought of as the height of the potential barrier (sometimes called the energy barrier) separating two minima of potential energy (of the reactants and products of a reaction). For a chemical reaction to proceed at a reasonable rate, there should exist an appreciable number of molecules with energy equal to or greater than the activation energy.
    8. 8. Chemical fertilizers FEATURES Vs CHEMICAL FERTILIZER Biofertilizers BIOFERTILIZER Raw material Non-renewable Renewable Energy Reductant Fossil Fuel H2 Solar Organic Catalyst Al, Fe, Mo oxides Nitrogenase enzy Temp. & Pr. 750oF, 200-600atm Ambient T, P Energy reqt. 680 kJ.mol-1.NH4+ 355 kJ.mol-1.NH4+ Efficiency 40-45% Exists due to indiscriminate use 90% Pollution free Cost High cost input @ Rs. 6/kgN Rs. 14/kg P2O2 Low cost input @Rs. 0.20/kg Soil Health Deteriorates Improves Pollution effect
    9. 9. Classification of Bio fertilizers Nitrogen fixer i) Symbiotic ii) Associative iii) Free living (Nonsymbiotic) Rhizobium (legume) Frankia (non-legume) BGA: Anabaena (Azolla) Azospirillum Beijerinckia, Azotobacter , Clostridium, Acetobacter Phosphate fixers Phosphate solubilizers: Organic matter decomposer Phosphate a) mobilizers: Cellulolytic: Bacillus VAM Trichoderma Pseudomonas Glomus, Aspergillus Gigaspora Penicillium b) Lignolytic: Agaricus, Polyporus Frankia-filamentous gram positive actinomycetes (vesicles for N fixation site). Actinorhizal plants are Alnus, Myrica, Casuarina
    10. 10. Symbiotic Nitrogen Fixation Microorganisms Host plants Location Isolated Plant group Tissue Inside or outside plant cell Rhizobium, Bacteria Bradyrhizobium (α-Proteobacteria) Azorhizobium Legumes and Parasponia Nodule (induced) Inside Yes Actinomycetes Frankia Betulaceae and 8 family(trees) Nodule (induced) Inside Yes Nostoc Bryophytes (Antheros etc.) Leaf cavity Outside Yes Nostoc (Anabaena) Pteridophyte (Azolla) Leaf cavity Outside No Nostoc Cycadophyta (Cycas,Macro zamia etc.) Collaroid root Outside Yes Nostoc Angiosperm (Gunnera) Gland tissue Inside Yes Large group Cyanobacteria (BGA) Genera
    11. 11. Types of Biological Nitrogen Fixation Cyanobacteria Azospirillum Rod shaped rhizobia in the nodule of cowpea (Vigna unguiculata).
    12. 12. Frankia and Actinorhizal Plants Actinomycetes (Gram +, filamentous); septate hyphae; spores in sporangia; thick-walled vesicles Azolla
    13. 13. Ecology of nitrogen-fixing bacteria
    14. 14. Significance of Biofertilizer: Biological N-fixation (BNF): 69% of global N-fixation. Legume-Rhizobium symbiosis is the most significant as it supplies 80-90% of the total N reqt. of legumes, increases grain yield by 10-15% (Verma and Bhattacharyya, 1990). Rhizobium bacteria can fix 50-100 kg N/ha/yr. Azotobacter and Azospirillum inoculation on several non-legumes crops experienced 5-15% yield increase and N contribution about 25 kg/ha. Use of Azospirillum as seed inoculant can save 20-30 kg N/ha in crops like Barley, Sorghum and Millets (Subba Rao et al., 1980) Use of Blue Green (submerged condition) Algae provides 25-50 kg N/ha to rice crop The use of Phosphobacterin has been found to increase the efficiency of ground rock phosphates and superphosphates applied in in neutral to alkaline soils Vesicular-Arbuscular Mycorrhizae (VAM) has prominent role in P availability.
    15. 15. Merits/ advantages/ usefulness of Biofertilizer use Reqd. in smaller quantities. 1g carrier of a BF contain 10 million cells of a specific strain (500g/ha material may be sufficient) Tandon (1991) reported estimates of Nutrient equivalent potential as follows: Rhizobium: 19-22 kg N/ha Azotobacter and Azspirillum: 20 kg N/ha BGA: may fix 20-30 kg N/ha Azolla : may fix 3-4 kg N/ton of Azolla BF increases the yield : 10-30 % by supplying N in the soil, adding organic matter to the soil. Bio fertilizers provides residual effect on soil fertility BF like Azospirillum and phosphobacterin produce growth promoting substances like hormone, vitamin etc favouring root growth. The fixed P become available by the application of phosphobacterin and the demand of P to the plants meets accordingly.
    16. 16. Class-8
    17. 17. legume Fixed nitrogen (ammonia) Fixed carbon (malate, sucrose) rhizobia
    18. 18. Rhizobium: A bacteria having the capacity to form morphologically well defined nodules on the roots of leguminous plants Gram negative Short rod 1.2-3.0 μm (L) x 0.5-0.9 μm (B) Have flagella (single polar or peritrichous Do not form endospore Aerobic, mesophilic , Chemoorganotroph 2 types of growth habit : Fast and slow (Bradyrhizobium) 2 distinct type of colony colour: Pink and creamy white Rhizobium has been placed in Bergey’s Manual of Systematic Bacteriology (1984) , belongs to the family Rhizobiaceae Rhizobium, Bradyrhizobium and Azorhizobium Azorhizobium ( stem nodule of Sesbania rostrata) A. caulonodans
    19. 19. Buchanon (1926) Shape: spherical, elongated, palmate Nodule Size: max. 6 cm Rhizobium Colour of nodule: white, green , black, pink Pink colour is effective strain: leghaemoglobin. 15-17kd, 1 peptide bond epiderm cortex Meristem Central zone having bacteriods Vascular bundle Cross section of nodule:
    20. 20. Nodulation process: (i) Multiplication in rhizosphere 1. Pre-infection: (ii) Attachment of root surface (iii) Branching of root hair (iv) Root hair curling 2. Infection and nodule formation 3. Nodule function (v) Formation of infection thread VI. Nodule development VII.Releasing Rhizobia from Infection thread VIII. Bacteriod formation IX. Reduction of N2 to NH3 X. Complementary functions XI. Nodule persistence
    21. 21. Model developed by Dazzo and Hubbell, 1975 Root Lectin R. trifolii hair Saccharide receptor Lectin = glycoprotien Root exudates like flavonoids and iso-flavonoids stimulate ‘Nod-d’ whose product binds with ‘nod-box’ which activate other nodulating genes
    22. 22. For nodulation to take place there has to be a molecular dialogue between the plant and the bacterial partner. •Root hair deformation •Membrane depolarization •Induction of early nodulin expression Flavonoids •Formation of nodule primordia nod genes •etc Nod factors
    23. 23. The Colonization Process Signaling • Rhizobia sense flavonoid compounds release by roots • specific species sense particular flavonoids specific to a plant • Rhizobia move by use of flagella propelling cell through soil water • Rhizobia produce lipo-oligosaccharides or nod factors • these initiate root hair deformation and curling and the division of cortical cells in the root at very low concentrations (< 10-9 M soil solution).
    24. 24. Flavonoids secreted by the root of their host plant help Rhizobia in the infection stage of their symbiotic relationship with legumes like peas, beans, clover, and soy. Rhizobia living in soil are able to sense the flavonoids and this triggers the secretion of Nod factors, which in turn are recognized by the host plant and can lead to root hair deformation and several cellular responses such as ion fluxes and the formation of a root nodule.
    25. 25. Sequence of molecular communication a) Plant excrete certain flavonoid compounds (differ between plants) b) Rhizobia recognize certain flavonoids (gene nod D product is a sensor) c) If Nodulin protein (product of nodD genes) recognizes right flavonoids, switch of other nod genes on, and products of nod genes coded proteins are formed--Nod factors (oligochitin compounds) d) Plant, in return, recognize right Nod factors. Early processes of nodulation is triggered by Nod factors. e) In addition to Nod factors, extracellular polysaccharides of bacteria may function in recognition of bacteria at later process of nodulation (bacteria spreading inside plant cells) nod genes: nodulation, nif genes: common with free-living nitrogen fixation, fix genes; Unique to symbiotic nitrogen fixation
    26. 26. Nodule development process 1. Bacteria encounter root; they are chemotactically attracted toward specific plant chemicals (flavonoids) exuding from root tissue, especially in response to nitrogen limitation naringenin (a flavanone) daidzein (an isoflavone)
    27. 27. 2. Bacteria attracted to the root attach themselves to the root hair surface and secrete specific oligosaccharide signal molecules (nod factors). Nod factors structurally are lipochitooligosaccharides (LCOs) that consist of an acylated chitin oligomeric backbone with various functional group nodsubstitutions at the terminal or non-terminal factor residues. N-acetylglucosamine
    28. 28. Nod gene expression is induced by the presence of certain flavonoids in the soil, which are secreted by the plant to attract the bacteria.[1]  These chemicals induce the formation of NodD, which in turn activates other genes involved in the expression of nod factors and their secretion into the soil. Nod factors induce root-hair curling such that it envelops the bacterium. This is followed by the localized breakdown of the cell wall and the invagination of the plant cell membrane, allowing the bacterium to form an infection thread and enter the root hair. The end result is the nodule, the structure in which nitrogen is fixed. Nod factors act by inducing changes in gene expression in the legume, most notable the nodulin genes, which are needed for nodule organogenesis.[
    29. 29. Rhizobium Attachment and infection Nod factor (specificity) Flavonoids (specificity) Invasion through infection tube Bacteroid differentiation Formation of nodule primordia Nitrogen fixation From Hirsch, 1992. New Phyto. 122, 211-237
    30. 30. 3. In response to oligosaccharide signals, the root hair becomes deformed and curls at the tip; bacteria become enclosed in small pocket. Cortical cell division is induced within the root.
    31. 31. 4. Bacteria then invade the root hair cell and move along an internal, plant-derived “infection thread”, multiplying, and secreting polysaccharides that fill the channel.
    32. 32. Rhizobium cells expressing GFP (green fluorescent protein) invade a host root hair infection thread
    33. 33. 5. Infection thread penetrates through several layers of cortical cells and then ramifies within the cortex. Cells in advance of the thread divide and organize themselves into a nodule primordium. 6. The branched infection thread enters the nodule primordium zone and penetrates individual primordium cells. 7. Bacteria are released from the infection thread into the cytoplasm of the host cells, but remain surrounded by the peribacteroid membrane (PBM). Failure to form the PBM results in the activation of host defenses and/or the formation of ineffective nodules.
    34. 34. transporters bacteroid peribacteroid membrane
    35. 35. The Colonization Process • Infection Thread – Protein called recadhesin and polysaccharides from Rhizobia and lectins from plants interact to adhere the bacterium to the root hair – curling of the root hair and hydrolysis of root epidermis – Rhizobia move down centre of the root hair toward the root cortex – plant produces tube called an infection thread – in the cortex Rhizobia enter enclosed area within a plant-derived peribacteroid membrane. – membrane protect the rhizobia from plant defense responses.
    36. 36. 8. Infected root cells swell and cease dividing. Bacteria within the swollen cells change form to become endosymbiotic bacteroids, which begin to fix nitrogen. The nodule provides an oxygen-controlled environment (leghemoglobin = pink nodule interior) structured to facilitate transport of reduced nitrogen metabolites from the bacteroids to the plant vascular system, and of photosynthate from the host plant to the bacteroids.
    37. 37. Shepherd crook Infection thread Nodulation Proliferation of IT Bacteriod formation
    38. 38. Rhizobium Root Nodules
    39. 39. Bio-chemical considerations
    40. 40. Enzymology of N fixation • • • • Only occurs in certain prokaryotes Rhizobia fix nitrogen in symbiotic association with leguminous plants Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates All nitrogen fixing systems appear to be identical They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits and NH4+ inhibits expression of nif genes
    41. 41. Nitrogenase Complex • • • • • Two protein components: nitrogenase reductase and nitrogenase Nitrogenase reductase is a 60 kD homodimer with a single 4Fe-4S cluster Very oxygen-sensitive Binds MgATP 4ATP required per pair of electrons transferred Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2
    42. 42. Nitrogenase A 220 kD heterotetramer • Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc) • Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor • Nitrogenase is slow - 12 e- pairs per second, i.e., only three molecules of N2 per second
    43. 43. Why should nitrogenase need ATP ?? • N2 reduction to ammonia is thermodynamically favorable • However, the activation barrier for breaking the N-N triple bond is enormous • 16 ATP provide the needed activation energy
    44. 44. Stereochemistry of Nitrogenase reaction + 8e- +8H+ + 16 Mg ATP N≡N 2NH3+ H2 + 16 Mg ADP+ 16Pi Nitrogenase enzyme e - e- α β α Amino acid β α α ATP N2 NH4+ produced reacts with glutamate to Nitrogenase form glutamine by (Azofermo) 260kd combined activities of GS and GOGAT ADP + Pi Nitrogenase reductase (Azofer) 60kd H2 NH4+ 2H+ +2e- Hydrogenase Glutamate + NH4+ +ATP ETC GS Mg2+ Glutamine + ADP + Pi (H3PO4) GOGAT Glutamine + 2-Oxoglutarate NADPH + H+ Amino acids are 2 Glutamate transpoted by Xylem NADP+
    45. 45. O2 Carbon Photo synth ate 1 2 l obi n haemog leg O2 TCA cycle e- H2O Ubiquinone → cyt b → cyt c → cyta/a3 ADP+ Pi Cyt 559-H2 e- ATP Ferredoxin 3 N≡N Hydrogen uptake H+ H2 Nitrogenase complex Bacteroid Nodule cytosol 2NH3 Amino acid Nodule amino acid pool 4
    46. 46. (i) Glutamate + NH4+ + ATP GS Mg 2+ Glutamine + 2-Oxoglutarate Glutamine + ADP + Pi (H3PO4) GOGAT NADPH + H+ 2 Glutamate NADP+ Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4+ GS= Glutamine synthatse, GOGAT= glutamine Oxo-glutarate amino-transferase GDH (ii) 2-Oxoglutarate + NH4+ NADPH + H+ Glutamate + NADP+ + H2 O GDH= glutamate dehydrogenase, km of GDH for NH4+ is very high, hence it has low affinity for NH4+
    47. 47. Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).
    48. 48. Bacteroid Contains less amount of ribosomes, mesosomes than free living rhizobial cell Presence of large quantities of PHB (Poly β-hydroxy butyrate) in conspicous amount, nearly 50% of bacteroid consists of PHB Absence of cytochrome-a in N-fixing bacteroid (about 20-30% dry wt of nodule is due to bacteroid) Soybean nodule: A nodule has 3.5 X 104 plant cells 1 plant cell has about 105 bacteroids So, Number of Bacteroid/ nodule =3.5 x 104 x105 = 3.5 x 109 Again, 1 g root has about 106 cells, So, 1 kg root has about 109 cells 1 kg root-mass may contain bacterial population equivalent to 1 medium sized nodule of Soybean
    49. 49. Class-9
    50. 50. Examples of using the cross-inoculation groups for selecting the proper rhizobial inoculant for the legume host. The proper combination of rhizobia and legume will result in the best nodulation and most nitrogen fixation. We see that using soybean rhizobia with soybean forms an effective symbiosis, while soybean rhizobia on leucaena does not. Using information from Table shows that cowpea rhizobia nodulates both mungbean and peanut.
    51. 51. Cross-inoculation group: Rhizobium sp. Host Legumes 1. Rhizobium leguminosarum biovar. trifolii Rhizobium leguminosarum biovar. phaseoli Rhizobium leguminosarum biovar. viceae Trifolium Phaseolus Vicia 2. Rhizobium meliloti Medicago 3. Rhizobium loti Lotus 4. Rhizobium friedii Glycine 5. Rhizobium galegae Galega orientalis 6. Rhizobium huakii Astragalus sinicus 7. Rhizobium tropici Phaseolus vulgaris Bradyrhizobium japonicum Nodulating soybean Bradyrhizobium sp. Vigna sinensis A cross inoculation group refers to a collection of leguminous species that are capable of developing nodules when exposed to bacteria obtained from the nodules of any member of that particular plant group
    52. 52. Azotobacte r Azotobacter is a free living, aerobic, chemoheterotrophic N fixing bacteria Important spp: Azotobacter beijerinckii. , A. chroococcum, A.vinelandii Azotobacter paspalum is closely associated with Paspalum notatum cv batatis. Producing 107 cfu / g of root (colony forming unit)), by ARA, it has been found that it can fix 15-93 kg N/ha/Yr Azotabacter has protective mechanism to safeguard the nitrogenase enzyme from oxygen.  (i) Respiratory protection in Azotobacter (specialised respiratory system  (ii) conformational protection in Azotobacter (a phenomena whereby nitrogenase activity becomes reversibly “switched on” or “off” in response to decreased or increased pO2 is known as conformational protection) Azotobacter inoculants commercially known as Azotobacterin Slurry of the carrier-based inoculant is made with minimum amount of water and seeds are mixed with the slurry, dried in shade and sown. Seedling dip (10-13 min) in slurry is done for transplanted crops and planted immediately.
    53. 53. Azotobacter have a full range of enzymes needed to perform the nitrogen fixation: ferredoxin, hydrogenase and an important enzyme nitrogenase. The process of nitrogen fixation requires an influx of energy in the form of adenosine triphosphate (ATP). Nitrogen fixation is highly sensitive to the presence of oxygen, and therefore Azotobacter developed a special defensive mechanism against oxygen, namely a significant intensification of metabolism that reduces the concentration of oxygen in the cells.[40] There is also a special nitrogenase-protective protein called Shethna, which protects nitrogenase and is involved in protecting the cells from oxygen. Mutants not producing this protein, are killed by oxygen during nitrogen fixation in the absence of a nitrogen source in the medium.[41] Homocitrate ions play a certain role in the processes of nitrogen fixation by Azotobacter
    54. 54. Azospirillu  m Azospirillum could be isolated from the root of tropical grass Digitaria decumbens (Dobereiner and Day, 1976) Ubiquitious in nature, capable of forming colony in roots and stems Gram negative, motile, vibroid in shape, contain Poly β-hydroxy butyrate granules, mesophilic but can tolerate 30-400C  Azospirillum lipeoferum (C4- Maize, sorghum, tropical forages D d are host plant) can fix 30 kg N / ha / yr  Azospirillum brasilense (C3- Rice/ wheat ) Azospirillum culture known to increase root biomass of rice and wheat (Dewan and Subba Rao, 1979) Produce growth hormones in pure culture (Tien et al, 1982). Seed inoculation with VAM together with Azospirillum brasilense increase the yield and P content of barley and pearl millet (Subba Rao et al., 1985) Carrier: FYM, FYM + soil, FYM + Charcoal
    55. 55. Blue Green Algae: Ubiquitious in distribution Singled cell or consists of branched/ unbranched filaments  possesses specialised type of cell- Heterocyst (where N fixation occurs)  Important species are Anabaena, Aulosira , Nostoc In submerged rice fields bnf is essentially an algal process, contributing 30 kg N/ha Symbiotic association between floating aquatic fern, Azolla and its partner Anabaena azollae (BGA) forms Azolla-Anabaena complex Anabaena is contained endophytically in ellipsoidal cavities of aerial dorsal lobes of the fern. Mature heterocysts have an almost normal content of Chlorophyll a, but are devoid of phycobiliprotein, the principal antenna pigment of Photosystem II Due to lack of PS-II and ribulose-bis phosphate Carboxylase; they can neither fix CO2 nor produce O2 in light The lack of photosynthetic O2 generation coupled with H dependent respiration, pO2 becomes very low
    56. 56. Electron donor Oxygenic photosynthesis Amino acids Keto acids Oxidised α pdt. gln KG gln N2 Fd red Oxidised FdOxid pdt. Electron donor PS1 ATP ADP glu glu glu NH3 Heterocyst Vegetative cell Microplasmadesmata channel Biochemical pathway of N fixation in BGA gln = glutamine; glu = glutamate
    57. 57. A profile of different biofertilizers Biofertilizer Function/Contribution Limitation Target Crops Rhizobium Fixation of 50-100 kg N/ha 10-35% increase in yield, leaves residual N Fixation only with legumes, Visible effect not reflected in traditional area, Needs optimum P , Mo Pulse Legumes Oilseed Legumes Forage legumes Tree legumes Azotobacter Fixation of 20-25 kg N/ha 10-15% increase in yield, Production of growth Promoting substances Demands high organic matter Wheat, maize, cotton, mustard, vegetable crops Azospirillum Same Poor performance in winter crops Sorghum. Pearl millet minor millets, maize, rice sugarcane Blue Green Algae and Azolla Fixation of 20-30 kg N/ha (BGA):30-100kg N/ha F (Azolla) Effective only in submerged rice Demands bright sunlight Survival difficult at high temp. Flooded rice Phosphate 5-50% increase in yield - All crops 10-15% increase in yield
    58. 58. Bio-super It is a granular material containing raw rock phosphate and finely ground elemental sulfur. The product is inoculated with sulfur-oxidizing bacteria Thiobacillus thiooxidans to ensure the conversion of the sulfur to sulfuric acid. The acid in turn reacts with the phosphate rock and making the contained phosphorus more available to plants. IARI-Microphos Culture Gaur and Gaind (1984) developed improved techniques for isolation of rock phosphate solubilizing microorganisms and by systematic investigation new efficient bacteria such as Pseudomonas striata, Bacillus polymixa and fungi like Aspergillus awamori have been selected for preparation of carrier based inoculation known as IARI-microphos culture (Tilak, 1991).
    59. 59. Phosphate Solubilising Micro-organisms : Bacillus, Pseuduomonas, Brevibacterium, Corynebacterium, Flavobacterium, Micrococcus, Sarcina, Achromobacter, Streptomyces, Schwanniomyces, Aspergillus, Penicillum etc. Phosphobacterin: Bacillus megatherium var phosphaticum Release about 10-25 kg P2O5/ha/season  Around 1350 t/a currently used in India
    60. 60. Biofertilizers General Dosages :  For Paddy - 3 kg/ha  For pulses, oilseeds, vegetables etc. - 2 kg/ha  For fruit and plantation crops - 8-10 kg/ha
    61. 61. Biofertilizers(BF) Applications Methods :  Seed Treatment (200 g(BF) in 400 ml water, make slurry, mix with seed, dry in shade and sown to the field)  Seedling Root Dipping (200 g(BF) in 1-1.5 L water, dipping root seedling for 30 min to 1 hr.)  Veg. Propagule treatment (200 g(BF) in 4-5 L water , spray it) (Potato, sugarcane,ginger-100 no. propagule)  Soil Treatment (2 kg Bf are mixed in 100 kg Compost, keep it overnight mixture is incorporated in the soil at the time of sowing or planting)
    62. 62.  Wheat, Maize, Cotton, Mustard etc. Azotobacter + PSM at 200 g each per 10 kg of seed as seed treatment For transplanted rice, the recommendation is to dip the roots of seedlings for 8 to 10 hours(whole night) in a soln of Azospirillum + PSM at 1kg each in 40L water  Jute, Azospirillum+PSM 200g each as seed treatment  Vegetables like Tomato, Brinjal, Chilli, Cabbage, Cauliflower etc., Mustard, Sunflower, Cotton use Azotobacter/ Azospirillum + Phosphobacterin 1 kg each as seedling root dip.
    63. 63. For Potato, Ginger, Colocasia, Turmeric, Paddy -use Azospiillum/ Azotobacter +PSM @ 4 kg each/acre mixed with compost and applied as soil treatment. Sugarcane use Acetobacter + Phosphobacterin 4 kg each/ acre as seed sett dipping. Plantation crops use Azotobacter+phosphobacterin 4 kg each/ acre with compost & applied in soil in two splits per year.
    64. 64. Chickpea seeds before (left) and after (right) treatment with biofertilizer
    65. 65. Mother culture Carrier Powder Flask culture Neutralization (lime) Bottle culture Sterilisation (γ-irradiation) Fermentor broth Prepared carrier Mixing @3:7 , moisture 40-50% (in trays) Curing (2-7 sprays at 28-30°C) Mass production of biofertilizer Packing Product for despatch (storing at 15-30°C)
    66. 66. What precautions one should take before using biofertilizers? •Biofertilizer packets need to be stored in cool and dry place away from direct sunlight and heat. •Right combinations of biofertilizers have to be used. •Other chemicals(Fertilizers and pesticides) should not be mixed with the biofertilizers. • Seed treatent chemicals like Bavistine etc. should mix 3 days prior to mix with biofertilizer treatment. •Sow the treated seeds(with Bio fertilizer) immediately preferably in the morning or afternoon avoiding scorching sunlight •The packet has to be used before its expiry, only for the specified crop and by the recommended method of application.
    67. 67. Biofertilizer – BCKV PSB Trichoderma
    68. 68. Phosphate Solubilizing Microorganisms (PSM) Several soil bacteria & fungi secrete organic acids & lower the pH in their vicinity to bring about dissolution of bound phosphate in soil. e.g.- Bacillus polymyxa Pseudomonas striata Aspergillus awamori
    69. 69. PGPR (Plant Growth promoting Rhizobacteria) Genera : Bacillus, Pseudomonas, Actinoplanes, Alcaligenes, Arthrobacter, Enterobacter, Azotobacter, Azosprillum, Clostridium etc. Activities :  Enhanced nutrient uptake  Hormone production  Vitamin production  Enzyme production  Biocontrol
    70. 70. Why biofertilizers are not so popular? Inspite of several long lasting benefits the bio-inoculants are not very popular among the farming community. There may be several reasons for this, however some of the important ones are as given below: • • • • • • • • • • Nutrient contribution is dependent on survival of organisms. Soil with high nutrient status do not show instant visible benefits. Low carbon content of soils- low proliferation. Water scarcity -possibility of desiccation. Fluctuating soil pH- variable microflora. Extreme temperature -in summer months. Shelf life of organisms. Poor storage and transportation Lack of awareness. Eagerness to look for instance effects.
    71. 71. The Dream….. If a way could be found to mimic nitrogenase catalysis (a reaction conducted at 0.78 atmospheres N2 pressure and ambient temperatures), huge amounts of energy (and money) could be saved in industrial ammonia production. If a way could be found to transfer the capacity to form N-fixing symbioses from a typical legume host to an important non-host crop species such as corn or wheat, far less fertilizer would be needed to be produced and applied in order to sustain crop yields
    72. 72. Azolla - the untold story
    73. 73. • Signals early in infection – Complex handshaking between legume root and rhizobium Correct signal Incorrect signal
    74. 74. Genetics of Nitrogenase Gene Properties and function nifH nifDK nifA nifB nifEN nifS fixABCX fixK fixLJ fixNOQP fixGHIS Dinitrogenase reductase Dinitrogenase Regulatory, activator of most nif and fix genes FeMo cofactor biosynthesis FeMo cofactor biosynthesis Unknown Electron transfer Regulatory Regulatory, two-component sensor/effector Electron transfer Transmembrane complex
    75. 75. Fe Protein Fe-Mo Protein a b a b Regulation
    76. 76. Fe Protein Fe-Mo Protein a b Regulation Activation of NifA a b NtrC-RNA polymerase
    77. 77. Electron Transport Fe Protein Fe-Mo Protein a ATP Ferredoxin b a b Regulation
    78. 78. Electron Transport Reduced Fe Protein Fe-Mo Protein a ATP Ferredoxin b a b Regulation
    79. 79. Electron Transport Reduced Fe Protein Reduction of Fe-Mo Protein a ATP Ferredoxin b a b Regulation
    80. 80. Electrons Donated to N2 Electron Transport Reduced Fe Protein Reduction of Fe-Mo Protein a ATP Ferredoxin b a b Regulation
    81. 81. Electrons Donated to N2 Formation of NH3 Electron Transport Reduced Fe Protein Reduction of Fe-Mo Protein a ATP Ferredoxin b a b Regulation
    82. 82. Genetics of Nitrogenase Summary NITROGENASE acetyl-CoA + CO ATP 2 N≡N NifH NifJ NifF NifH pyruvate + CoA ADP r educing equivalents NifD NifK NifK NifD 2NH 3 +H2
    83. 83. Bio-technological considerations
    84. 84. Nitrogenase enzyme complex Nitrogenase Electron transport MoFe protein Fe protein Assembling β α γγ α β Fe-Mo-Cofactor Regulator J H D K T Y E NX U SVWZM F L A BQ Physical association of nif genes in Klebsiella pneumoniae Redrawn from
    85. 85. Rhizobia and the cross-inoculation groups of legumes they nodulate.
    86. 86. Soybean nodules Stem nodules of Aeschynomene afraspera
    87. 87. Short Test on 30th July, 2012 at 1.30 p.m. Total Marks: 20 Time: 30 No. of questions: 40
    88. 88. VAM (Endomycorrhiza): The hyphae often form swellings (vesicle) and minute branches (arbuscles) within the cell of the host Genera :Glomus, Gigaspora, Acaulospora, Sclerocystis Activities : (Hacskaylo, 1972)  P - Uptake (15-30 kg/ha/season)  Availability of K, Mg, S, Fe, Mn, Zn, Cu, etc.  Tolerance to adverse env. Stresses (draught resistance)  Tolerance to disease and Nematodes  Produce plantVAM is mixed with Rhizobium to inoculate legume Dual inoculation: when growth hormones plant, it is known as Dual inoculation------better (i) nodulation, (ii) N-fixation and (iii) P-uptake
    89. 89. Vesicular Arbuscular Mycorrhiza Inside root • Intercellular mycelium • Intracellular arbuscule • tree-like haustorium • Vesicle with reserves Outside root • Spores (multinucleate) • Hyphae •thick runners •filamentous hyphae Form extensive network of hyphae even connecting different plants
    90. 90. GS + (i) Glutamate + NH4 + ATP Glutamine + ADP + Pi (H3PO4) Mg2+ GOGAT Glutamine + 2-Oxoglutarate NADPH + H+ 2 Glutamate NADP+ Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4+ GS= Glutamine synthatse, GOGAT= glutamine Oxo-glutarate amino-transferase (ii) 2-Oxoglutarate + NH4 + NADPH + H GDH + Glutamate + NADP+ + H 2O GDH= glutamate dehydrogenase, km of GDH for NH4+ is very high, hence it has low affinity for NH4+
    91. 91. GS + (i) Glutamate + NH4 + ATP Glutamine + ADP + Pi (H3PO4) Mg2+ GOGAT Glutamine + 2-Oxoglutarate NADPH + H+ 2 Glutamate NADP+ Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4+ GS= Glutamine synthatse, GOGAT= glutamine Oxo-glutarate amino-transferase (ii) 2-Oxoglutarate + NH4 + NADPH + H GDH + Glutamate + NADP+ + H 2O GDH= glutamate dehydrogenase, km of GDH for NH4+ is very high, hence it has low affinity for NH4+
    92. 92. Sinorhizobium meliloti Bacteroids
    93. 93. Glutamine and glutamate are usually at much higher concentrations than other amino acids in cells. This is due to their role as nitrogen carriers.
    94. 94. Glutamine can combine with a-ketoglutarate to yield a pair of glutamates. This is a reduction, catalyzed by glutamate synthase. Glutamate synthase (bacteria and plants)