# Biofertilizers - Structure and characteristic features of bacterial biofertilizers

Ajeet Kumar

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Ajeet Kumar. Biofertilizers - Structure and characteristic features of bacterial biofertilizers. Master. Biopesticide and Biofertilizer (Elective course), Dr. Rajendra Prasad Central Agricultural University, Samastipur, India. 2025, pp.1-16. hal-05267034

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Submitted on 18 Sep 2025

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Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur) - 848125, Bihar

A Lecture notes

On

Biofertilizers - Structure and characteristic
features of bacterial biofertilizers

<figure><img src="image_2.png" /></figure>

COURSE TITLE: Biopesticide and Biofertilizer

*(Elective course)

COURSE NO.: ASS-307

CREDIT HOUR: 3 (2+1)

Dr. AJEET KUMAR
Assistant Professor-cum-Scientist (Soil Science)
Department of Microbiology
Collage of Basic Sciences and Humanities, Dr. RPCAU, Pusa

2025
# BIO-FERTILIZERS

Dr. AJEET KUMAR<fnref n="1" />

## Bio-Fertilizers

Bio-fertilizers are defined as preparations containing active (living cells) or latent cells of efficient strains of certain microorganisms that help crop plants to utilize the atmospheric nitrogen to increase the nitrogen content of soil, and can dissolve the insoluble phosphate of the soil to release the phosphorus in the rhizosphere when applied through seed or soil. They accelerate certain microbial processes in the soil which augment the extent of availability of nutrients in a form easily assimilated by plants.

## Processing of carrier material

The use of ideal carrier material is necessary in the production of good quality biofertilizers. Peat soil, lignite, vermiculite, charcoal, press mud, farmyard manure and soil mixture can be used as carrier materials. The following points are to be considered in the selection of carrier material; Cheaper in cost, Should be locally available, High organic matter content, No toxic chemicals, Water holding capacity of more than 50%, Easy to process, friability and vulnerability.

## Preparation of carrier material

The carrier material (peat or lignite) is powdered to a fine powder so as to pass through 212 micron sieve. The pH of the carrier material is neutralized with the help of calcium carbonate (1:10 ratio), since the peat soil / lignite are acidic in nature (pH of 4 - 5). The neutralized carrier material is sterilized in an autoclave to eliminate the contaminants

Use of biofertilizers is one of the important components of integrated nutrient management, as they are cost effective and renewable source of plant nutrients to supplement the chemical fertilizers for sustainable agriculture. Several microorganisms and their association with crop plants are being exploited in the production of biofertilizers. They can be grouped in different ways based on their nature and function.

<footnote n="1">¹Assistant Professor-cum-Scientist (Soil Science-Soil Microbiology, Soil fertility, Soil Chemistry), Department of Microbiology, College of Basic Sciences & Humanities (CBS&H), RPCAU, Pusa-848125, Bihar. E-mail: ajeet.sri@rpcau.ac.in</footnote>
<table><thead><tr><th>S. No.</th><th>Groups</th><th>Examples</th></tr></thead><tbody><tr><td colspan="3">N<sub>2</sub> fixing Biofertilizers</td></tr><tr><td>1.</td><td>Free-living</td><td>Azotobacter, Beijerinkia, Clostridium, Klebsiella, Acetobacter, Anabaena, Nostoc,</td></tr><tr><td>2.</td><td>Symbiotic</td><td>Rhizobium, Frankia, Anabaena azollae</td></tr><tr><td>3.</td><td>Associative Symbiotic</td><td>Azospirillum</td></tr><tr><td colspan="3">P Solubilizing Biofertilizers</td></tr><tr><td>1.</td><td>Bacteria</td><td>Bacillus megaterium var. phosphaticum, Bacillus subtilis Bacillus circulans, Pseudomonas striata</td></tr><tr><td>2.</td><td>Fungi</td><td>Penicillium sp, Aspergillus awamori</td></tr><tr><td colspan="3">P Mobilizing Biofertilizers</td></tr><tr><td>1.</td><td>Arbuscular mycorrhiza</td><td>Glomus sp., Gigaspora sp., Acaulospora sp., Scutellospora sp. & Sclerocystis sp.</td></tr><tr><td>2.</td><td>Ectomycorrhiza</td><td>Laccaria sp., Pisolithus sp., Boletus sp., Amanita sp.</td></tr><tr><td>3.</td><td>Ericoid mycorrhizae</td><td>Pezizella ericae</td></tr><tr><td>4.</td><td>Orchid mycorrhiza</td><td>Rhizoctonia solani</td></tr><tr><td colspan="3">Biofertilizers for Micro nutrients</td></tr><tr><td>1.</td><td>Silicate and Zinc solubilizers</td><td>Bacillus sp.</td></tr><tr><td colspan="3">Plant Growth Promoting Rhizobacteria</td></tr><tr><td>1.</td><td>Pseudomonas</td><td>Pseudomonas fluorescens</td></tr></tbody></table>

## Types of Bio-fertilizers:

There are following types of biofertilizers: Bacterial, fungal, algal, aquatic fern and earthworms

### Bacterial Bio-Fertilizers

Here, live cells of bacteria are used as fertilizers. They are the principal **nitrogen fixers** in the soil and may be either free-living or symbiotic (within the nodules of legume roots, leaf nodules of certain plants and stem nodules of *Sesbania grandiflora*). Some bacterial genera can **solubilize phosphorus** from the bound form (*Bacillus*, *Pseudomonas* sp.).

Bacteria like *Agrobacterium* sp. and *Pseudomonas fluorescence* supply growth hormones like indole acetic acid to the plants. Certain groups of bacteria like the *Pseudomonas fluorescence* living in association with the rhizosphere of most of crop plants (rhizobacteria promoting plant growth) supply all the essential nutrients required for the growth of the crop and in addition, protects the plant roots from the
attack by soil-borne pathogens (saprophytic suppression). The nitrogen fixing ability of the bacteria can be studied under the following heads.

**Free-living nitrogen fixers:** Among the large number of free-living nitrogen fixers, the *Azotobacter* requires oxygen to flourish and *Klebsiella* and *Rhodospirillum* can survive both in the presence and absence of oxygen. Apart from fixing nitrogen, *Azotobacter chroococcum* has the ability to synthesize and secrete B-vitamins, growth hormones and antifungal antibiotics into its environment. *Azotobacter* has one drawback, its nitrogen fixing ability is regulated by the presence of nitrogenous compounds in its environment. Thus it cannot be used along with a nitrogenous fertilizer. *Klebsiella* is another promising free-living nitrogen fixer, and it has been possible to isolate its 'nif' genes (17 of them) and to introduce them into E.coli.

## Benefits of Liquid Bio-fertilizer

The advantages of Liquid Bio-fertilizer over conventional carrier based Bio-fertilizers are listed below:

* Longer shelf life -12-24 months.

* No contamination.

* No loss of properties due to storage upto 45° c.

* Greater potentials to fight with native population.

* High populations can be maintained more than 10⁹ cells/ml upto 12 months to 24 months.

* Easy identification by typical fermented smell.

* Cost saving on carrier material, pulverization, neutralization, sterilization, packing and transport.

* Quality control protocols are easy and quick.

* Better survival on seeds and soil.

* No need of running Bio-fertilizer production units through out the year.

* Very much easy to use by the farmer.

* Dosages is 10 time less than carrier based powder Bio-fertilizers.

* High commercial revenues.

* High export potential.

* Very high enzymatic activity since contamination is nil.
# Structure and characteristic features of bacterial biofertilizers

(Azospirillum, Azotobacter, Bacillus, Pseudomonas, Rhizobium and Frankia)

## 1. Rhizobium

Rhizobium is a soil habitat bacterium, which is able to colonize the legume roots and fixes the atmospheric nitrogen symbiotically. The morphology and physiology of Rhizobium will vary from free-living condition to the bacteroid of nodules. They are the most efficient biofertilizer as per the quantity of nitrogen fixed concerned. They have seven genera and highly specific to form nodule in legumes, referred as cross inoculation group. Rhizobium inoculant was first made in USA and commercialized by private enterprise in 1930. The bacteria infect the legume root and form root nodules within which they reduce molecular nitrogen to ammonia which is in reality utilized by the plant to produce valuable proteins, vitamins and other nitrogen containing compounds. The site of symbiosis is within the root nodules. It has been estimated that 40-250 kg N / ha / year is fixed by different legume crops by the microbial activities of Rhizobium. The percentage of nodules occupied, nodules dry weight, plant dry weight and the grain yield per plant the multistrain inoculant was highly promising.

**Structure:** Rhizobia are rod shaped bacteria, 0.8 µm in diameter and 2 µm in length, often with flagellae. They assume a different shape when inside their host, being irregularly shaped or often 'Y' -shaped

<figure><img src="image_3.png" /></figure>

### Physical features of liquid Rhizobium

* Dull white in colour

* No bad smell

* No foam formation,

* pH 6.8-7.5

## 2. Azotobacter

Of the several species of Azotobacter, A. chroococcum is the dominant inhabitant in arable soils capable of fixing N₂ (2-15 mg N₂ fixed /g of carbon source) in culture media. The bacterium produces abundant slime which helps in soil aggregation. The numbers of A. chroococcum in Indian soils rarely exceeds 10⁵/g soil due to lack of organic matter and the presence of antagonistic microorganisms in soil. It is well known free living nitrogen fixing aerobic bacterium. It is used as a Bio-Fertilizer for all non leguminous plants especially rice, cotton, vegetables etc. Azotobacter cells are not

<figure><img src="image_4.png" /></figure>
present on the rhizosplane but are abundant in the rhizosphere region.

## Physical features of liquid Azotobacter

The pigmentation that is produced by *Azotobacter* in aged culture is melanin which is due to oxidation of tyrosine by tyrosinase enzyme. Some of the pigmentation are described below-

* A. chroococcum : Produces brown-black pigmentation in liquid inoculum.

* A. beijerinchii : Produces yellow-light brown pigmentation in liquid inoculum.

* A. vinelandii : Produces green fluorescent pigmentation in liquid inoculum.

* A. paspali : Produces green fluorescent pigmentation in liquid inoculum.

* A. macrocytogenes: Produces, pink pigmentation in liquid inoculum.

* A. insignis : Produces grayish-blue pigmentation in liquid inoculum.

* A. agilities : Produces green-fluorescent pigmentation in liquid inoculum.

## Role of liquid Azotobacter as a Bio-control agent

Azotobacter produces antifungal substance which inhibits the growth of soil fungi like Aspergillus, Fusarium, Curvularia, Alternaria, Helminthosporium, Fusarium etc.

### 3. Acetobacter

This is a sacharophillic bacterium and is associated with sugarcane, sweet potato and sweet sorghum plants and fixes 30 kg N/ha/year. Mainly this bacterium is commercialized for sugarcane crop. It is known to increase yield by 10-20 t/ acre and sugar content by 10-15 %.

<figure><img src="image_5.png" /><figcaption>Acetobacter colony</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Acetobacter colony</figcaption></figure>
## 4. Azospirillum

Azospirillum lipoferum and A. brasilense are primary inhabitants of soil, the rhizosphere, and intercellular spaces of root cortex of graminaceous plants. They perform the associative symbiotic relation with the graminaceous plants. Azospirillum are N₂ fixing organisms isolated from the root and above ground parts of a variety of crop plants. They are Gram negative, Vibrio or Spirillum having abundant accumulation of polybetahydroxybutyrate (70 %) in cytoplasm.

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Five species of Azospirillum have been described to date

* A. brasilense

* A. amazonense

* A. irakense

* A. lipoferum

* A. halopraeferens

The organism proliferates under both anaerobic and aerobic conditions but it is preferentially micro-aerophilic in the presence or absence of combined nitrogen in the medium. Apart from nitrogen fixation, growth promoting substance production (IAA), disease resistance and drought tolerance are some of the additional benefits due to Azospirillum inoculation.
Phosphatic Bio-fertilizers: Phosphate solubilizing microorganisms (PSM)

Several soil bacteria and fungi, notably species of *Pseudomonas*, *Bacillus*, *Penicillium*, *Aspergillus* etc. secrete organic acids and lower the pH in their vicinity to bring about dissolution of bound phosphates in soil. Increased yields of wheat and potato were demonstrated due to inoculation of peat based cultures of *Bacillus polymyxa* and *Pseudomonas striata*. They act in different ways. Some bacteria secrete organic acids such as lactic acid, acetic acid and citric acid which solubilize the bound phosphate to forms which are available to plants. Others produce sulphuric acid by oxidation of sulphur which acts like the organic acids in making phosphate available. Carbonic acid formed by the action of carbon dioxide released by bacteria during respiration and water acts in a similar way. Some organisms give off hydrogen sulphide which reacts with the iron salt, ferrous phosphate, to form ferrous sulphide and thus releases the phosphate. Microbes are also responsible for decay of dead animal and plant litter in the soil during which humic and fulvic acids are formed which bind the metal ions such as Fe, Al, Mn and Ca and release the phosphate ions for the plants. They are used as seed inoculants.

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
## Cynobacterial biofertilizers: (Algae as Bio-fertilizers)

Another group of free-living nitrogen fixers are the **cyanobacteria** commonly called the **blue-green algae (BGA)**. More than a hundred species of BGA can fix nitrogen.

Nitrogen fixation takes place in specialized cells called the **heterocysts** (large, thick walled and metabolically inactive cells) which depend on vegetative cells for energy to fix nitrogen while the fixed nitrogen is utilized by the vegetative cells for growth and development. BGA are very common in the rice fields (the micro-aerophilic condition and alkalinity are conducive to the algal population).

If no chemical fertilizers are added, inoculation of the algae can result in 10-14 % increase in crop yields. Unlike *Azotobacter*, the BGA are not inhibited by the presence of chemical fertilizers. On the other hand, presence of inorganic minerals (superphosphate) accelerates their growth. They are easy to produce and usually they are mass produced in cement tanks filled with fresh-water. Since they do not require any processing, they are quite cheap. Cost of 10 kg may be only Rs.30-40. Algal effect can be seen in subsequent years without the use of fresh inoculants.

Algal bio-fertilizers or cyanobacterial biofertilizers are Anabaena, Nostoc and Oscillatoria. Algae have proved beneficial in the case of certain crops like vegetables, cotton and sugarcane when added along with chemical fertilizers.

### Aquatic Fern (Azolla) as a Bio-fertilizer:

Azolla is a tiny water fern common in ponds, ditches and rice fields. It has been used as a bio-fertilizer for rice. The nitrogen accumulated in the Azalla is made available to the rice crop when the fern decomposes. The nitrogen fixing work is accomplished by the symbiotic relationship between the fern and a BGA, *Anabaena azollae*. The alga inhabits some of the cells on the underside of the Azolla frond and fixes atmospheric nitrogen. It is dependent on the fern for photosynthesis which supply the energy for nitrogen fixation. In addition to nitrogen, the decomposed Azolla also provides K, P, Zn and Fe to the crop. It also controls aquatic weeds which would otherwise compete with the crop for nutrients.

### AZOLLA (Botanical name is Azolla pinnata)

It belongs to the family Azollaceae. Azolla is a water fern. It is considered as aquatic weed commonly found in floating in idle ponds, tanks shallow ditches and canals. Azolla is associated with rice field. A blue green algae (BGA) *Anabaena azollae* living in the epidermal cavity of lower side of the leaf of Azolla. The symbiotic association of *Azolla pinnata* and *Anabaena azollae* is termed as Azolla Anabaena complex. This algae fixes atmospheric nitrogen for Azolla and in exchange, the plant provides shelter and food to the algae. As this complex fixes atmospheric nitrogen, it has great potentiality for use, in agricultural field as biofertilizer and can be used as an alternative to nitrogen fertilizers. It is recommended as green manure
because of its large biomass and high N content ( 4-6%N) for sub merged rice fields, with in maximum temperature of 38°C. The yield increase is 15-20% Fresh Azolla contains 90-95 water. Decomposed Azolla contains the following plant foods.

<table><thead><tr><td>S.No.</td><td>Nutrient</td><td>Per cent (%)</td></tr></thead><tbody><tr><td>1</td><td>Nitrogen</td><td>4 to 6</td></tr><tr><td>2</td><td>Phosphorus</td><td>0.5 to 0.9</td></tr><tr><td>3</td><td>Potassium</td><td>2 to 6</td></tr><tr><td>4</td><td>Calcium</td><td>0.4 to 1</td></tr><tr><td>5</td><td>Magnesium</td><td>0.5</td></tr><tr><td>6</td><td>Manganese</td><td>0.11 to 0.16</td></tr><tr><td>7</td><td>Iron</td><td>0.06 to 0.16</td></tr><tr><td>8</td><td>Total ash</td><td>9 to 10</td></tr></tbody></table>

## Ideal conditions for growth and multiplication of Azolla

1. Depth of water should be maintained at 8-10 cm in the field

2. Temperature in between 14 to 35°C. Optimum for quick growth and multiplication is 20 to 28°C.

3. Suitable soil pH is 6.0 to 8.0

4. Single Super Phosphate should be applied at the rate of 20-25 kg ha⁻¹, should be applied

5. Furadon granules @2-3 kg /week should be applied to control leaf eating insects and snails.

## Methods of growing Azolla

1. Green manuring

2. Dual cropping

3. Composting

4. Just growing

### 1. Green manuring

When there is standing water in the field, Azolla is grown by spreading 200-500 kg /ha fresh Azolla. After 25 days of the whole field will be covered by Azolla, if condition is ideal. Green Azolla is incorporated by ploughing after dewatering in the field. This will add 30-40 kg of N fertilizer /ha.

### 2. Dual cropping

1. Azolla nursery is prepared simultaneously with the paddy nursery

2. The area of Azolla nursery should be half that of paddy nursery (seed bed).

3. The Azolla nursery should be kept free from other aquatic weeds and there should be 8-10 cm standing water in the Azolla nursery.

4. Where the paddy seeds are sown in the paddy nursery fresh Azolla is spread in the Azolla nursery @40-80 grams per square meter.
5. After 25 days the Azolla will increase an extent of 60 times and super phosphate is applied @ 25 grams/Sq. Meter/week.

6. Within 25 days the paddy seedlings will be ready for transplanting. Before transplantation of paddy seedlings, there will be standing water in the main field. Azolla is taken out from the Azolla nursery and spread in the transplanted field @ 200-500 kg/ha.

7. Single super phosphate @25-50 kg/ha/week is applied for 3 weeks and water level is maintained 5-10 cm.

8. After another 25 days, the whole field may be covered by Azolla. Azolla is incorporated after dewatering the field. This will add 30-40 kg N/ha besides another nutrients.

3. **Composting:** Azolla is grown in permanent Azolla nursery. The Azolla is collected every week from the nursery and kept in layers on the side of nursery. Within 10 days, the Azolla will decompose and can be used in any crop.

4. **Just growing :**

Azolla can be grown along with paddy. Nitrogen will be added where it is not incorporated. Azolla when grown in one hectare of land through out the year and ideal conditions are maintained it can fix 800 kg of nitrogen per hectare zolla is suitable for only flooded rice and fixes up to 40-80 kg N/ha. Symbiotically with *Anabaena azollae*. The yield increase is 15-20%.

**Advantages of azolla:**

1. It contains higher Nitrogen content

2. Release of nitrogen is easy

3. There is no leaching loss

4. Releases nutrient slowly

5. Weed interference is less where grown as dual crop

V. Blue green algae or Cyanobacteria:

BGA is phototrophic in nature. These are photosynthetic, prokaryotic microorganisms and capable of fixing molecular nitrogen. BGA are found to grown in a paddy field and marshland. It is used as biofertilizer for irrigated rice crop. The rice field ecosystem provides an environment favorable for the growth of BGA with respect to their requirements viz., light, water and temperature. Nitrogen fixation by BGA have been estimated to be 25-30 kg N/ha. Yield increases by 10-15%. BGA has some species viz., Nostoc, Anabaena, Olocia, Calothrix, Tolipothix etc., They fix nitrogen in the presence of sunlight. They also contain growth promoting substances like auxin, Indole acetic acid and Gibberellic acid.

Recommendation: application of 10 kg/ha flakes of BGA. It is applied 10 days after the transplanting of rice crop.
Algal production technology:

Mainly three methods are followed.

1) Trough method

2) Pit method

3) Field scale

1. Trough method:

<figure><img src="image_10.png" /></figure>

Take 3 m x 2 m x 20 cm size (L x B x h) galvanized iron sheet trough or to the convenient size. Add about 8 to 10 kg of soil and well mixed with 200 grams of SSP.

<figure><img src="image_11.png" /></figure>

The soil pH should be about neutral. If acidic correct by lime. Add water to the trough 5-15 cm (height) to protect from insects, and add Carbofuran 3.0 G at the rate of 25 grams / tray.

<figure><img src="image_12.png" /></figure>

Sprinkle the algal culture on the surface of the standing water and keeping it in air (open) and exposed to sunlight.

<figure><img src="image_13.png" /></figure>

In hot summer months, the growth of algae will be rapid in about 7 to 10 days and form a thick mat, where algal growth becomes sufficiently thick stop watering and allow the water to evaporate completely, the dry algae cracks in to flakes.

<figure><img src="image_14.png" /></figure>

Collect the dry algal flakes from the trough and store in bags for the use in the field. One trough gives 1.5 to kg mats.

2. Pit-method:

<figure><img src="image_15.png" /></figure>

Adopted by number of farmers in Bihar and India

<figure><img src="image_16.png" /></figure>

Demark the area in the field for algal production

<figure><img src="image_17.png" /></figure>

Flood the area with a depth of about 12.5 cm

<figure><img src="image_18.png" /></figure>

Apply SSP @12.0 kg /40 m² area

<figure><img src="image_19.png" /></figure>

Apply the algal culture

<figure><img src="image_20.png" /></figure>

In clayey soils good growth of algae takes place in about two weeks in clear sunny weather while in loamy soils it takes about 3-4 weeks

<figure><img src="image_21.png" /></figure>

Once algal growing mats are formed, they are allowed to dry up in the field and deed algal flakes are thin collected and stored in bags.

<figure><img src="image_22.png" /></figure>

BGA benefit the rice crop up to 25-30 kg N/ha

<figure><img src="image_23.png" /></figure>

Dose: Apply BGA @10-15 kg/ha after transplanting.
## Actinomycetes as Bio-fertiliser

Frankia is an actinomycete and forms nitrogen fixing nodules in trees and shrubs. The organism invades the cells of a developed lateral root and causes it to fuse into a nodule. Entry into the host changes the structure of the microbe.

## Fungi as Bio-fertiliser

Some nonpathogenic fungi help in plant growth by forming associations with the host plant roots called mycorrhizae (myca- fungi, rhiza -root). examples are Trichoderma, Gigaspora, Glomus, etc. One group of mycorrhizae forms a sheath around the fine lateral roots and replaces the root hairs by dichotomous branching of the fungal hyphae. They are called ectomycorrhizae because they do not traverse intracellularly. The ectomycorrhizae help the plant by solubilizing nutrients near the plant roots and making it easy for the plants to feed. They also prevent the roots from being attacked by nematodes (by entangling them). Another group called the endomycorrhizae penetrate the roots and establish symbiotic relation with the plants.

The fungi helps the roots in obtaining inorganic nutrients while obtaining essential organic nutrients from the host. There is yet another group called ect-endomycorrhiza or vesicular-arbuscular mycorrhiza (VAM fungi) wherein they are partly outside the host roots and partly intracellular. The mycorrhizae are of particular significance as they assist plants to absorb phosphates from the soil. Phosphate ions are not mobile in the soil, thus when a plant uses up all the available phosphate near its roots, it starves for want of phosphates. The mycorrhizal mycelia spread far and wide, away from the depleted zone and translocate the phosphates directly to the host. Plants with restricted root system, having short stubby roots and few root hairs are particularly benefited by association with mycorrhiza. Absorption of elements which are required in minute quantities by plants is also facilitated by mycorrhizae. Mycorrhizae are valuable particularly in times of drought. They enable plants to survive and grow in the driest and poorest of soils, even in leached soils that have been mined by deforestation and overcropping.

## Cultivation of mycorrhizal fungi

It is easy to mass cultivate ectomycorrhizal fungi since they can grow in an artificial fungal medium. They are mass produced in liquid media, dried and pressed into cakes or cultivated in the form of spores and packaged. The seed inoculation is performed just as that of Rhizobium. Cultivation of endo mycorrhizal fungi is quite difficult since they need a live host for survival. Hence the host plants harbouring such mycorrhizal fungi are specially grown in laboratory conditions, their roots excised, powdered and the mixture used as a fertilizer.
## MYCORRHIZA

The term 'mycorrhiza' (literally, fungus root) was first used by A. B. Frank to characterise the association between higher plants and fungi. The symbiotic association between the roots of some plants and some fungi is called mycorrhizal association. The fungus is highly habitat limited and is usually found in the immediate vicinity of or within the roots.

### Occurrence of Mycorrhiza

They occur on almost all terrestrial plants though not as specific as the nitrogen fixing symbiosis. Thus a plant may have several mycorrhizae that can form symbiosis with its roots. Extent of symbiosis depends on fertility. High soil fertility leads to low mycorrhizal infection and poor symbiosis and vice versa. Roots supply carbohydrates to the fungi which absorb nutrients from the soil and supply them to the crop.

### Types of mycorrhizae

Mycorrhizae are of two kinds:

**Ectomycorrhiza** Ectomycorrhizal symbiosis is a mutually beneficial union between fungi and the roots of vascular and non-vascular plants. The host of an ecomycorrhizal fungus is usually a gymnosperm (pine). The typical ectomycorrhizal fungi are basidiomycetes (Agaricus), ascomycetes or phycomycetes members. Usually more common in the temperate regions, they are capable of growing apart from the host on media containing simple sugars and vitamins. Ectomycorrhizae have poor competitive saprophytic ability hence they have a tough time competing with other microbes in the soil.

<figure><img src="image_24.png" /></figure>

During the infection process, ectomycorrhizal fungi in soil are stimulated by the root metabolites to grow toward the root. The hyphae aggregate around the root
and penetrate between the root epidermis and the cortex. A structure called Hartig net is formed which is a fungal sheath surrounding the root in which fungal hyphae penetrate between the root cells.

Eventually, the root gets surrounded by a fungal mantle. The fungal hyphae replace the fine lateral root hairs of the host root system thus modifying it structurally. Thus the host root system that is infected with ectomycorrhiza looks stunted and dichotomously branched. The fungal hyphae on the exterior of the roots usually serve as an extension of roots and store large amounts carbohydrates

**Endomycorrhiza** In this case, the fungal hyphae penetrate the host root cells. They are quite common among the Ericaceae and Orchidaceae members of higher plants as well as fruit trees like citrus, coffee, rubber, etc.

## Vam Fungi

Vesicular-arbuscular mycorrhiza (VAM) fungi shown in are geographically ubiquitous. They are commonly found in association with agricultural crops, shrubs, tropical tree species and some temperate trees. Their nutritional requirements are not specific. VAM associations are formed by non septate Zygomycetes and Phycomycetes. Some examples are *Glomus*, *Gigaspora*, *Acaulospora*, *Entrophospora* and *Scutellospora* of which *Glomus* is the most common fungus.

The fungi are obligate biotrophs and do not grow on synthetic media and hence are classified according to the morphological characteristics of the spores formed in the soil. VAM fungi produce large resting spores (0.2 mm). The spores can survive adverse conditions. The germ tube dies if it is unable to encounter and successfully penetrate the host root. The fungal hyphae traverses and ramifies in the root cortex. Branches from the intercellular hyphae enter cortical cells where further branching results in highly branched hyphal structures called arbuscles. They are short-lived and serve as the nutrient transfer

<figure><img src="image_25.png" /></figure>
Mechanism between the fungus and the host. Phosphate transfer possibly occurs across living membranes of the host and the fungus via arbuscles. When the association is well established, hyphal swellings called vesicles form on the mycelium inside and outside the root. The vesicles are sac-like terminal swellings at the tip of hyphae and contain many lipid droplets and function as storage organs. External mycelium form very thick-walled chlamydospores. These fungi are disseminated both actively and passively. Active dissemination is form one root to another by mycelial growth through soil and passive dissemination is through biotic agents like rodents, worms, insects and birds or abiotic agents like wind and water. VAM fungi interact with other soil microbes like the free-living and symbiotic nitrogen fixers and phosphate solubilisers to improve their efficiency for the biochemical cycling of elements to the host plants.

## Constraints in usage of biofertilizers

1. Farmers acceptance has been for away from satisfaction

2. Difficult to demonstrate striking increase in yield due to biofertilizers unlike chemical fertilizers

3. Strains fail to establish if the soil moisture in the surface in the surface zone

4. Fungicide treatment is a problem in crops like groundnut

5. Inoculants manufacturing is for below the requirement

6. Availability of quality of inoculants is another problem

7. Seed inoculation is no more suitable when seeds are sown on dry soil under

8. When seeds are coated with toxic chemicals and pesticides

9. Soil containing higher number of ineffective native Rhizobia