Get Complete Information on Biofertilizers

Lgumes are the rich source of proteins in our diet. In India there are different legumes which are sown in different agro-climatic conditions. Even then most of the people are still sufferring from protein dificiency. It shows different types of legume seeds used as dal which are the rich source of protein.

Nitrogen is a major element required by plants. The atmosphere comprises of 78% nitrogen but unlike oxygen or carbon dioxide, this cannot be accessed directly by plants or animals. It has to be acquired by microbes before it becomes available to plants and then to animals.

Such microorganisms have been explited commercially and now sold in market in the form of microbial products called bioinoculants. Bioinoculants are the microbial preparations that enhance the uptake of nutrients by plants from the soil, increase the avaliability of the nutrients and stimulate plant growth.

There are two types of organisms which are used as inoculants: symbiotic organisms (such as Rhizobium, Synorhizobium, Bradyrhizobium, Nostoc, Anabaena, etc.) and non-symbiotic organisms (such as Azotobacter, Azospirilum, Beijerinckia, etc.) Much work has been done on Rhizobium and Anabaena spp.

Methods have also been developed for their commercial production. Therefore, these inoculants have become popular as biofertilizers. Some termed them as biostimulants because they stimulate plant growth by poorly understood mechanisms.

I. Rhizobium Biofertilizer

Rhizobium (Gr. riza – root; bios = life) develops a mutualistic symbiotic association with the roots of legume plants such as pea, beans, alfalfa, cowpea, etc. The association of species of Rhizobium with distinct legume species is highly specific.

In this association bacterium is attracted towards roots and infects plant root hair causing the hair to curl. The bacterium moves through the root hair cell to adjacent root cells, to which it causes to enlarge greatly to form a nodule. Thus the root system bears a large number of root nodules of varying size and shape.

The process of invading the root hair and induction of nodule formation is controlled by a series of genes in the bacterium which are termed as nod (nodulation) genes. Within the enlarged cells of the nodule the bacterium changes its form to a different structure which is called bacteroid.

It shows cross section though a soybean (Glycine max) root nodule. The bacterium, Bradyrhizobium japonicum infects the roots and establishes a nitrogen fixing symbiosis.

Inside the bacteroids, atmospheric N” is converted into NH₄ ⁺ by a specific enzyme called nitro- genase. Then nitrogen becomes available to plants.

This process involves at least 20 nif genes (nitrogen fixation genes) that act in concert to make sure that the right protein (usually an enzyme) is produced at the right time.

Thus, the process of nitrogen fixation in legumes involves many genes operating in a coordinated manner. It is often asked if non-legume crops such as wheat or maize could be genetically modified to fix nitrogen thus reducing the need for artificial fertilizers.

Currently this would not be possible because of the complexity of the mechanism but research is actively being undertaken on this subject.

Cultures of Rhizobium are commercially available for use as biofertilizer but there are various problems. There is considerable specificity between Rhizobium strains and legume species and so the correct strain has to be identified and applied.

The process is relatively inefficient and there is an energy penalty on the plant in supporting the symbiotic bacterium.

(a) Mass Production of Rhizobial Inoculant:

Suitable species of Rhizobium from a specific host is isolated and characterized. It is multiplied in large scale in a fermentor. Then the suspension is filtered and cells are isolated. It is used as Rhizobium inoculant.

(b) Methods of Application:

There are three ways of using these N₂-fixing bacteria: seed treatment, root dipping and soil applications.

(i) Seed treatment:

It is the most common method adopted for all type of inoculant. The seed treatment is effective and economical also. This method is most suitable for small quantity of seeds i.e. up to 5 kg.

Coating can be done in a plastic bag having 21″ x 10″ or big size. 10% sugar solution, 40% gum Arabic, synthetic glue, 10% molasses or rice starch, etc. is collected. This solution acts as sticker. It is added @ 15-25 ml/kg of seeds (depending on size of seeds).

The sticker increases the amount of inoculant that adheres to seeds so that number of rhizobia on the each seed must retain higher population i.e. 10³ to 10⁶ (depending upon seed size). The bag is shut in such a way so as to trap the air as much as possible.

The bags have to be twisted for 2 minutes or more until all the seed were uniformly wetted with the sticker solution. The bags have to be opened, inflated and shaken gently. Shaking is to be stopped when each seed got a uniform layer of culture coating.

The bag is opened and seeds are spread under the shade for 20-30 minutes to dry. For large amount of seeds, coating can be done in a bucket or mixer of sticker and inoculant can be mixed directly with the hand.

i. Use of lime:

The problem of acidic soil is about 80% in Orissa where the pH is less than 6.0. In acidic soils where pH is 5.5 Rhizobium fails to nodulate in higher mass and greater number. Therefore, application of lime is beneficial to nodulation in acidic soil.

ii. Pelleting materials:

The quantity of lime for effective and economical use is required for small seeds (40-50 g lime/kg seeds) and for medium and big size seeds (30-40 g lime/kg seeds). The materials used for pelleting include: calcium carbonate, various grades of dolomite, bentonite, talc, activated charcoal, rock phosphate, etc.

The pelleting materials should be finely grounded to 200-250 mesh size. Pelleting has to be done after seeds have been treated with inoculant. When the seeds are wet the pelleting materials have to be dusted in such a way so that each seed must get a layer of pelleting material.

iii. Seed treatment with other bacteria- the concept of microbial consortium:

Seed treatment can be done with any of two or more bacteria. There is no antagonistic (inhibitory) effect. The important things to be kept in mind are that the seeds must be coated first with Rhizobium or Azotobacter or Azospirillum.

When each seed gets a layer of above bacteria, the phosphate solubilizing microorganisms (e.g. Pseudomonas aeruginosa, P. fluorescens, etc.) have to be treated on outer layer of the seeds. This method will provide the maximum number of population of each bacterium required for better results.

(ii) Root dipping:

This method is applied before transplantation of paddy and vegetable plants. Depending on legume or non-legume plant, Rhizobium or Azospirillum inoculant is mixed with sticker suspension.

The required quantity of Azospirillum has to be mixed with 5-10 litre of water at one corner of the field. Roots of such plants are dipped in the suspension and all the plants have to be kept for minimum V2 an hour before transplantation.

(iii) Soil application:

This method is most suitable for phosphate solubilizing microorganisms (PSM) e.g. Pseudomonas spp. The PSM is used @ 2 kg per acre as soil application. PSM is mixed with 400-600 kg of cow dung farmyard manure (FYM) along with V2 bag of rock phosphate, if available.

The mixture of PSM, cow dung and rock phosphate should be kept under the shade of a tree for overnight maintaining 50% moisture. The mixture is used as soil application in rows or during leveling of soil.

(c) Precautions:

The following precautions must be taken in account:

(i) Store biofertilizer packets in cool and dry place away from direct sunlight and heat.

(ii) Use right combination of biofertilizers.

(iii) Rhizobium is crop specific, so use in specified legume crops.

(iv) Do not mix them with chemicals.

(v) While purchasing the biofertilizers, ensure that each packet is provided with necessary information like name of the product, name of the crop for which intended, name and address of the manufacturer, date of manufacture, expiry date, batch No. and instructions for use.

(vi) Use the packet before expiry date, only on the specified crop, by the recommended method.

(d) Genetic Modification in Rhizobium:

Two approaches are being used with genetic modification of Rhizobium. Firstly, strains of the related Sinorhizobium melilotii have been modified to increase nitrogen fixation; however, this modification did not give any yield benefits in the field.

The second application of GM technology is to insert marker genes into Rhizobium so that bacteria released into the environment can be monitored. In a long-term experiment, it was found that the marked bacteria could persist in the microbial population but they did not dominate the non-transgenic bacteria.

II. Blue-Green Algal (Cyanobacterial) Biofertilizer :

Blue-green algae are recently called as cyanobacteria. They are oxygenic photo-autotrophic prokaryotes. They are the members of Cyanophyta. The cyanobacteria bear many properties of bacteria.

They occur in all types of soil and water and have the capabilities of growing at any extreme environments. This property makes them capable to grow at adverse conditions. Presence of heterocysts has increased their significance to be used as biofertilizer.

The most common representative members of the BGA are the strains of Anabaena variabilis, Nostoc muscorum, Aulosira fertilissima and Tolypothrix tenuis. It shows the contorted filaments of Nostoc sp. The filaments bear heterocysts which are present terminally or at intercalary position. Due to the presence of heterocysts, they fix N₂ like bacteria (Fig. 11.5).

In recent years the Government of India and State Governments have started establishing BGA production technologies on the basis of agro-climatic conditions. During 1960s Banaras Hindu University and later the Indian Agricultural Research Institute (IARI), New Delhi have contributed.

1. Mass Production of BGA

(a) Construction of Ponds/tanks:

BGA are photosynthetic in nature and require sunlight as any other higher green plants. Development of closed units should have enough provision of light by using transparent material.

To overcome this problem, multiplication units (ponds) are constructed in a polyhouse or greenhouse. BGA are grown in polythene lined shallow pits containing formulated medium in a polyhouse/greenhouse.

Refinement in polyhouse technology can be done by making a permanent infrastructure facility for BGA mass multiplication. A two tier system with cemented tanks is developed inside the glass/ green house. For the lower tier of tanks, light source is provided.

Luxuriant growth of BGA can be observed within 5-6 days. The height of liquid medium is kept at 5 cm. A thick mat of BGA could be observed within 5 days of incubation. Now it is ready to mix with a suitable carrier.

(b) Formulation of Medium:

The important environmental parameters that affect the growth of BGA are: pH, light and temperature. These need to be optimized by growing the BGA growth media buffered with pH ranging from 7.0 to 10.0. A pH of 7.3±0.2 was found to be the most suitable.

Although the cultures can tolerate a temperature of 40°C, yet sustained biomass production and nitrogen fixation can be observed at 30±1°C. The BGA inoculants are transferred in cemented tanks having sufficient amount of water, phosphate, lime, etc.

(c) Inoculum Carrier:

A large variety of carriers including thermocol, riverbed sand, rice straw, wheat straw and saw dust have been tested. Wheat straw performed the best result. The individual strain can be grown in the formulated medium in polyhouse/greenhouse independently in a multiplication unit.

After 4-5 days of growth it is mixed with wheat straw (400 g/pond of 1.5 m²). BGA immobilized in wheat straw is sun dried and can be packed in polythene packets for storage. Such dried cultures can be stored for 2 years without loosing viability.

(d) Packing and Storage of Cyanobacterial Biofertilizer:

The individual strain of cyanobacteria is mixed in carrier, sun dried and packed at 400 g per pack. The packets are finally sealed and stored for a long time (more than 2 years) in a dry state at normal room temperature in shade without any loss in quality of the inoculum. Shelf life of BGA is about 2 years.

(e) Field Application:

BGA biofertilizer produced in a formulated medium contains 100,000 to 1,000,000 units (propagules) per gram of carrier. Hence 1 kg/ha (400g/acre) straw- based BGA inoculum is enough for field application. Application should be done immediately after the transplantation of rice seedlings is completed.

Application of excess BGA inoculum is not harmful but it accelerates the fast growth in fields. The users must ensure that there is enough water at least 1 week after application of cyanobacterial biofertilizer. Recommended pest control measurers and the other management practices do not normally interfere with the establishment and activity of the BGA in the field.

2. National Centre for Conservation and Utilization of Blue-Green Algae:

A national centre has been established for conservation and utilization of blue green algae under the leadership of Dr. RK. Singh. Main activities of the Division in brief are given below:

(i) To act as a National Germplasm Centre for blue-green algae (BGA).

(ii) The Centre functions as a service as well as depository centre in the country for algal isolates.

(iii) To conduct basic and applied research on blue-green algae.

(iv) To act as a resource centre for training subject matter specialists, students, extension workers and farmers.

(v) Protocol development and mass production of BGA biofertilizer.

III. AZOLLA spp.

Azolla is an aquatic fem that belongs to the Family Salviniaceae, although some authorities now place it in the monotypic family, Azollaceae. There are seven species of Azolla distributed worldwide.

Any sample of Azolla examined under a microscope will have filaments of a cyanobacterium called Anabaena living within ovoid cavities inside the leaves. Like nitrogen-fixing bacteria living inside the root nodules of legumes, the relationship appears to be mutually beneficial. Azolla is easy to maintain in aquarium cultures.

It is an excellent source of prokaryotic cells and heterocysts for laboratory exercises of general biology on cell structure and function. In addition, this little fern and its algal partner provide an important contribution toward the production of rice for a hungry world.

Individual Azolla plants have slender, branched stems with minute and overlapping scale like leaves only one millimeter long. Each plant resembles a little floating moss with slender roots on its underside.

The plants often form compact mats on the water surface. Azolla may produce reddish anthocyanin in the leaves, in contrast with the bright green carpets and filamentous green algae.

Filaments of Anabaena are found in the ovoid central cavity of Azolla leaves. These filaments become visible with larger and oval heterocysts around the crushed fern leaf.

The thick-walled heterocysts often appear more transparent and have distinctive ‘polar nodules’ at each end of the cell. Polar nodules are visible in some of the heterocysts.

The heterocysts are the site of nitrogen-fixation where atmospheric nitrogen (N₂) is converted into ammonia (NH₃). The water fern benefits from its bacterial partner by an ‘in house’ supply of usable nitrogen.

This provides at least 100,000 tons of nitrogen fertilizer per year worth more than $ 50 million annually. Extensive propagation research is being conducted in China to produce new varieties of Azolla that will flourish under different climatic and seasonal conditions.

Excellent research work on production of Azolla biofertilizer and its application method have been developed at Central Rice Research Institute (CRRI) (Cuttack). According to some report, Azolla can increase form mycorrhiza with all groups of plants such as bryophyta, pteridophyta, gymnosperms and angiosperms.

The members of Zygomycotima (especially Glomales) form vesicular-arbuscular mycorrhiza (VAM); hence they are called VAM fungi.

There are six members of VAM fungi such as the species of Glomus, Sclerocystis, Gigaspora, Aculospora, Scutellospora and Entrophospora. Currently two terms are used: VA-mycorrhiza and AM-mycorrhiza. The term AM refers to arbuscular mycorrhiza i.e. such fungi that it form only arbuscules.

Vesicles formed by VAM fungi that differ from each other. Since the fungal mycelia are present inside the cortex of plant roots, it is also called endomycorrhiza. It shows a vesicle of Glomus with a substending hypha.

The other type of mycorrhiza is called ectomycorrhiza. It is formed by the members of Basidiomycotina and Ascomycotina. There are over 5,000 fungi of Basidiomycotina and Ascomycotina which are involved in forming ectomycorrhiza on about 2000 woody plant species.

Ectomycorrhiza is characterized by the presence of mantle (a sheath formed by fungal mycelia), and Hartig net (intercellular hyphae present on outer cortex). The fungi that form ectomycorrhiza are the species of Amanita, Boletus, Cantharellus, Cenococcum, Geastrum, Scleroderma, Lactarius, Russula, Rhizopogon, etc.

1. Mass Inoculum Production:

The application of mycorrhiza on a large scale is limited by the nature of its proliferation which is strictly biotrophic. Tata Energy Research Institute (TERI), New Delhi has developed technology for mass production of mycorrhizal fungi by exploiting genetically modified host.

A vesicle of Glomus roots using a bacterium Agrobacterium rhizogenes that carries the Ri (root inducing) T-DNA plasmid. This method allows a huge recovery of inoculum in very short time and using very little space compared to conventional modes of multiplication in pots.

2. Transfer of Technology and Launching of Product(s):

These industries have already started manufacturing the mycorrhiza- based biofertilizer and marketed them with the brand name Ecorrhiza-VAM and Nurserrhiza-VAM.

KCP Sugar and Industries (Pvt.) Ltd has been awarded by the All India Biotechnology Associa­tion. Similarly, Cadila Pharma-ceuticals (Ahmedabad) has also launched a product with the name Josh – a root booster and it is being used by the Gujrat State Forest Department.