Bio-fertilizers

Dr. P. K. ManiBidhan Chandra Krishi Viswavidyalaya

E-mail: pabitramani@gmail.com Website: www.bckv.edu.in

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

Energetics

Industrial fixation NNHaber-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)

Carl Bosch(1874-1940)

Fritz Haber(1868-1934)

Haber and Bosch: Most influential persons of the 20th Century ( Nature, July 29,1999)

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. 

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 transition state decreases, thereby decreasing the energy required to initiate the reaction.

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.

Chemical fertilizers Vs Biofertilizers

FEATURES CHEMICAL FERTILIZER BIOFERTILIZER

Raw material Non-renewable Renewable

Energy Fossil Fuel Solar

Reductant H2 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% 90%

Pollution effect Exists due to indiscriminate use Pollution free

Cost High cost input @ Rs. 6/kgN Rs. 14/kg P2O2

Low cost input @Rs. 0.20/kg

Soil Health Deteriorates Improves

Classification of Bio fertilizers Nitrogen fixer Phosphate fixers Organic

matter decomposer

i) Symbiotic Rhizobium (legume)

Frankia (non-legume)

Phosphate solubilizers: Bacillus Pseudomonas Aspergillus Penicillium

Phosphate mobilizers:

VAMGlomus, Gigaspora

a)Cellulolytic:

Trichoderma

b)Lignolytic:

Agaricus, Polyporus

ii)Associative

BGA: Anabaena (Azolla)

AzospirillumBeijerinckia,

iii)Free living(Non-symbiotic)

Azotobacter , Clostridium,Acetobacter

Frankia-filamentous gram positive actinomycetes (vesicles for N fixation site). Actinorhizal plants are Alnus, Myrica, Casuarina

Symbiotic Nitrogen Fixation

Microorganisms Host plants Location Isolated

Large group Genera Plant group TissueInside or outside plant cell

 

Bacteria (-Proteobacteria)

Rhizobium, Bradyrhizobium Azorhizobium

Legumes and Parasponia

Nodule (induced)

Inside Yes

Actinomycetes FrankiaBetulaceae and8 family(trees)

Nodule (induced)

Inside Yes

Cyanobacteria(BGA)

Nostoc Bryophytes (Antheros etc.)

Leaf cavity

Outside Yes

Nostoc (Anabaena)

Pteridophyte(Azolla)

Leaf cavity

Outside No

Nostoc Cycadophyta (Cycas,Macrozamia etc.)

Collaroid root

Outside Yes

Nostoc Angiosperm(Gunnera)

Gland tissue

Inside Yes

Types of Biological Nitrogen FixationCyanobacteria

Azospirillum

Rod shaped rhizobia in the nodule of cowpea (Vigna unguiculata).

Azolla

Frankia and Actinorhizal Plants

Actinomycetes (Gram +, filamentous); septate hyphae; spores in sporangia; thick-walled vesicles

Ecology of nitrogen-fixing bacteria

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 Algae provides 25-50 kg N/ha to rice crop (submerged condition)

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.

Merits/ advantages/ usefulness of Biofertilizer useReqd. 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.

Class-8

legume

rhizobia

Fixed nitrogen(ammonia)

Fixed carbon(malate, sucrose)

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

Buchanon (1926) Rhizobium

Nodule Size: max. 6 cm

Shape: spherical, elongated, palmate

Colour of nodule: white, green , black, pink

Pink colour is effective strain: leghaemoglobin. 15-17kd, 1 peptide bond

Cross section of nodule:

epiderm

cortex

Meristem

Central zone having bacteriods

Vascular bundle

Nodulation process:

1. Pre-infection:(i) Multiplication in rhizosphere

(ii) Attachment of root surface

(iii) Branching of root hair

(iv) Root hair curling

2. Infection and

nodule formation

(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

3. Nodule function

Lectin

Saccharide receptor

R. trifoliiRoot

hair

Model developed by Dazzo and Hubbell, 1975

Root exudates like flavonoids and iso-flavonoids stimulate ‘Nod-d’ whose product binds with ‘nod-box’ which activate other nodulating genes

Lectin = glycoprotien

For nodulation to take place there has to be a molecular dialogue between the plant and the bacterial partner.

Flavonoids

nod genes

Nod factors

•Root hair deformation

•Membrane depolarization

•Induction of early nodulin expression

•Formation of nodule primordia

•etc

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).

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.

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)

Sequence of molecular communication

nod genes: nodulation, nif genes: common with free-living nitrogen fixation, fix genes; Unique to symbiotic nitrogen fixation

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

daidzein (an isoflavone)

naringenin(a flavanone)

2. Bacteria attracted to the root attach themselves to the root hair surface and secrete specific oligosaccharide signal molecules (nod factors).

nod factorN-acetylglucosamine

Nod factors structurally arelipochitooligosaccharides (LCOs) that consist of an acylated chitin oligomeric backbone with various functional group substitutions at the terminal or non-terminal residues.

Nod gene expression is induced by the presence of certain flavonoids in the soil, which are secreted by the plant toattract 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.[

From Hirsch, 1992.New Phyto. 122, 211-237

Rhizobium

Formation ofnodule primordia

Bacteroiddifferentiation

Nitrogenfixation

Nod factor(specificity)

Invasion through infection tube

Attachment and infection

Flavonoids(specificity)

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.

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.

Rhizobium cells expressing GFP (green fluorescentprotein) invade a host root hair

infection thread

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.

peribacteroidmembrane

bacteroid

transporters

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.

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.

Nodulation

Bacteriod formation

Shepherd crook

Infection thread

Proliferation of IT

Rhizobium Root Nodules

Bio-chemical considerations

Enzymology of N fixationOnly 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

Nitrogenase ComplexTwo 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

NitrogenaseA 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

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

+ 8e- +8H+ + 16 Mg ATP 2NH3+ H2 + 16 Mg ADP+ 16PiN ≡ N

α

α

α

α

β

β

ATP ADP + Pi

e- e-

N2

NH4+

Nitrogenase reductase (Azofer) 60kd

Nitrogenase (Azofermo) 260kd

Nitrogenase enzyme

H2 2H+ +2e-

Hydrogenase ETC

NH4+ produced reacts

with glutamate to form glutamine by combined activities of GS and GOGAT

Stereochemistry of Nitrogenase reaction

Glutamate + +ATP Glutamine + ADP + Pi (H3PO4)

Glutamine + 2-Oxoglutarate 2 Glutamate

NH4+ GS

Mg2+

GOGAT

NADPH + H+ NADP+

Amino acid

Amino acids are transpoted by Xylem

Ubiquinone → cyt b → cyt c → cyta/a3

ADP+ Pi ATP

Nitrogenase

complex

Hydrogen

uptake

H+

N ≡ N

H2

2NH3

Amino acid

e-

Cyt 559-H2

Ferredoxin

Bacteroid

Nodule cytosol

Nodule amino acid pool

Carbon

Photo synthate

e-

TCA cycle

O2

leghaemoglobin

O2

H2O

1

2

3

4

(i) Glutamate + + ATP Glutamine + ADP + Pi (H3PO4)

Glutamine + 2-Oxoglutarate 2 Glutamate

NH4+ GS

Mg2+

GOGAT

NADPH + H+ NADP+

GS= Glutamine synthatse, GOGAT= glutamine Oxo-glutarate amino-transferase

Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4

+

(ii) 2-Oxoglutarate + NH4+ NADPH + H+ Glutamate + NADP+

+ H2O

GDH

GDH= glutamate dehydrogenase, km of GDH for NH4

+ is very high, hence it has low affinity for NH4+

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).

BacteroidContains 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

Class-9

Examples of using the cross-inoculation groups for selecting theproper rhizobial inoculant for the legume host. The proper combination of rhizobia and legume willresult in the best nodulationand most nitrogen fixation. We see that using soybeanrhizobia with soybean formsan effective symbiosis, while soybean rhizobia on leucaena does not. Using information from Table shows that cowpea rhizobia nodulates both mungbean and peanut.

Rhizobium sp. Host Legumes

1. Rhizobium leguminosarum biovar. trifolii Rhizobium leguminosarum biovar. phaseoli Rhizobium leguminosarum biovar. viceae

TrifoliumPhaseolusVicia

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

Cross-inoculation group:

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

AzotobacterAzotobacter 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.

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

AzospirillumAzospirillum 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

Blue Green Algae:

Ubiquitious in distributionSingled cell or consists of branched/ unbranched filaments

possesses specialised type of cell- Heterocyst (where N fixation occurs)

Important species are Anabaena, Aulosira , NostocIn 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

Keto acids

Amino acids

glu

α KG

glu

gln gln

glu

N2

NH3FdOxid

Fd red

Electron donor

Oxidised pdt.

ATPADP

Oxygenic

photosynthesis

Vegetative cell Heterocyst

Electron donor

Oxidised pdt.

Microplasmadesmata channel

PS1

Biochemical pathway of N fixation in BGA gln = glutamine; glu = glutamate

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)

10-15% increase in yield

Effective only in submerged rice Demands bright sunlight Survival difficult at high temp.

Flooded rice

Phosphate Slubilisers

5-50% increase in yield - All crops

A profile of different biofertilizers

Bio-superIt 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).

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

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

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)

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.

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.

Chickpea seeds before (left) and after (right)

treatment with biofertilizer

Mother culture

Flask culture

Bottle culture

Fermentor broth

Carrier Powder

Neutralization (lime)

Sterilisation (γ-irradiation)

Prepared carrier

Mixing @3:7 , moisture 40-50% (in trays)

Curing (2-7 sprays at 28-30°C)

Packing

Product for despatch (storing at 15-30°C)

Mass production of biofertilizer

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.

Biofertilizer – BCKV PSB Trichoderma

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

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

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.

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

The Dream…..

Azolla - the untold story

• Signals early in infection– Complex handshaking between legume

root and rhizobium

Incorrect signal

Correctsignal

Genetics of NitrogenaseGene 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

ba

ab

Fe Protein Fe-Mo Protein Regulation

ba

ab

Fe Protein Fe-Mo Protein Regulation

NtrC-RNA polymerase

Activation of NifA

ba

ab

Fe Protein Fe-Mo Protein Regulation

Electron Transport

Ferredoxin

ATP

ba

ab

Reduced Fe Protein

Fe-Mo Protein Regulation

Electron Transport

Ferredoxin

ATP

ba

ab

Reduced Fe Protein

Fe-Mo Protein Regulation

Electron Transport

Ferredoxin

ATP

Reduction of

ba

ab

Reduced Fe Protein

Fe-Mo Protein Regulation

Electron Transport

Ferredoxin

ATP

Reduction of

Electrons Donated to N2

ba

ab

Reduced Fe Protein

Fe-Mo Protein Regulation

Electron Transport

Ferredoxin

ATP

Reduction of

Electrons Donated to N2

Formation of NH3

NN

2NH3pyruvate + CoA

acetyl-CoA + CO 2

NifJ NifF

NifH

NifD

NifKNifD

NifK

NITROGENASE

NifH

+H2

reducing equivalents

ATP

ADP

Genetics of Nitrogenase Summary

Bio-technological considerations

Redrawn from www.asahi-net.or.jp/~it6i-wtnb/BNF.html

Nitrogenase enzyme complex

Physical association of nif genes in Klebsiella pneumoniae

NitrogenaseMoFe protein

Fe proteinElectron transport

AssemblingFe-Mo-Cofactor

Regulator

H D K T Y E NX U SVWZM F L A B QJ

Rhizobia and the cross-inoculation groups of legumes they nodulate.

Soybean nodules Stem nodules of Aeschynomene afraspera

                                        

               

                                          

              

Short Test on 30th July, 2012at 1.30 p.m.

Total Marks: 20Time: 30

No. of questions: 40

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 plant growth hormonesDual inoculation: when VAM is mixed with Rhizobium to inoculate legume plant, it is known as Dual inoculation------better (i) nodulation, (ii) N-fixation

and (iii) P-uptake

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 hyphaeeven connecting different plants

(i) Glutamate + + ATP Glutamine + ADP + Pi (H3PO4)

Glutamine + 2-Oxoglutarate 2 Glutamate

NH4+ GS

Mg2+

GOGAT

NADPH + H+NADP+

GS= Glutamine synthatse, GOGAT= glutamine Oxo-glutarate amino-transferase

Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4

+

(ii) 2-Oxoglutarate + NH4+ NADPH + H+ Glutamate + NADP+

+ H2O

GDH

GDH= glutamate dehydrogenase, km of GDH for NH4

+ is very high, hence it has low affinity for NH4+

(i) Glutamate + + ATP Glutamine + ADP + Pi (H3PO4)

Glutamine + 2-Oxoglutarate 2 Glutamate

NH4+ GS

Mg2+

GOGAT

NADPH + H+NADP+

GS= Glutamine synthatse, GOGAT= glutamine Oxo-glutarate amino-transferase

Low km of GS for NH4+ (0.02 mM), high affinity to bind NH4

+

(ii) 2-Oxoglutarate + NH4+ NADPH + H+ Glutamate + NADP+

+ H2O

GDH

GDH= glutamate dehydrogenase, km of GDH for NH4

+ is very high, hence it has low affinity for NH4+

Sinorhizobium meliloti Bacteroids

Glutamine and glutamate are usually at much higher concentrations than other amino acids in cells.

This is due to their role as nitrogen carriers.

Glutamate synthase (bacteria and plants)

Glutamine can combine with a-ketoglutarate to yield a pair of glutamates. This is a reduction, catalyzed by glutamate synthase.