1. Microbes play a central role in nitrogen availability and life on Earth. Certain bacteria can fix nitrogen from the atmosphere in forms usable by plants through free-living or symbiotic relationships. 2. The document discusses nitrogen fixation by bacteria, which make nitrogen available to support plant growth. It also describes symbiotic nitrogen fixation between rhizobia bacteria and legume plants in root nodules. 3. Organic farming aims to feed the soil by promoting nutrient cycling without synthetic pesticides or fertilizers. It uses techniques like crop rotation, cover crops, composting, and biological pest control.

1. ‫االولــــــى‬ ‫الوحدة‬
Discuss the role of microbes in nitrogen fixation
The growth of all organisms depends on the availability of mineral nutrients, and none is more
important than nitrogen, which is required in large amounts as an essential component of
proteins, nucleic acids and other cellular constituents. There is an abundant supply of nitrogen
in the earth's atmosphere - nearly 79% in the form of N2 gas. However, N2 is unavailable for
use by most organisms because there is a triple bond between the two nitrogen atoms, making
the molecule almost inert. In order for nitrogen to be used for growth it must be "fixed"
(combined) in the form of ammonium (NH4) or nitrate (NO3) ions. The weathering of rocks
releases these ions so slowly that it has a negligible effect on the availability of fixed nitrogen.
So, nitrogen is often the limiting factor for growth and biomass production in all environments
wherethere is suitable climate and availability of water to supportlife. ..Microorganisms havea
central role in almost all aspects of nitrogen availability and thus for life support on earth:..
some bacteria can convert N2 into ammonia by the process termed nitrogen fixation; these
bacteria are either free-living or form symbiotic associations with plants or other organisms
(e.g. termites, protozoa) other bacteria bring about transformations of ammonia to nitrate, and
of nitrate to N2 or other nitrogen gases any bacteria and fungi degrade organic matter,
releasing fixed nitrogen for reuse by other organism .. All the nitrogen-fixing organisms are
prokaryotes (bacteria). Some of them live independently of other organisms - the so-called
free-living nitrogen-fixing bacteria. Others live in intimate symbiotic associations with plants or
with other organisms (e.g. protozoa) .. microorganisms capable of transforming atmospheric
nitrogen into fixed nitrogen, inorganic compounds usableby plants. More than 90 percent of all
nitrogen fixation is effected by them... Two kinds of nitrogen fixers are recognized: free-living
(non-symbiotic) bacteria, including the cyanobacteria (or blue-green algae) Anabaena and
Nostoc and such genera as Azotobacter, Beijerinckii, and Clostridium; and mutualistic
(symbiotic) bacteria such as Rhizobium, associated with leguminous plants, and Spirillum
lipoferum, associated with cereal grasses..The symbiotic nitrogen-fixing bacteria invade the
root hairs of host plants, where they multiply and stimulate formation of root nodules,
enlargements of plant cells and bacteria in intimate association. Within the nodules the
bacteria convert free nitrogen to nitrates, which the host plant utilizes for its development. To
insuresufficient nodule formation and optimum growth of legumes (e.g., alfalfa, beans, clovers,
peas, soybeans), seeds are usually inoculated with commercial cultures of appropriate
Rhizobium species, especially in soils poor or lacking in the required bacterium…

2. Describe the method of organic farming
Organic farming methods combine scientific knowledgeof ecology and modern technology with
traditional farming practices based on naturally occurring biological processes. Organic farming
methods are studied in the field of agroecology. While conventional agriculture uses synthetic
pesticides and water-soluble synthetically purified fertilizers, organic farmers are restricted by
regulations to using natural pesticides and fertilizers. The principal methods of organic farming
include crop rotation, green manures and compost, biological pest control, and mechanical
cultivation. These measures use the natural environment to enhance agricultural productivity:
legumes are planted to fix nitrogen into the soil, natural insect predators are encouraged, crops
are rotated to confuse pests and renew soil, and natural materials such as potassium
bicarbonate[ and mulches are used to control disease and weeds.. Crop diversity Crop
diversity is a distinctive characteristic of organic farming. Conventional farming focuses on mass
production of one crop in one location, a practice called monoculture. The science of
agroecology has revealed the benefits of polyculture (multiple crops in the same space), which
is often employed in organic farming. Planting a variety of vegetable crops supports a wider
range of beneficial insects, soil microorganisms, and other factors that add up to overall farm
health. Crop diversity helps environments thrive and protect species from going extinct.. Soil
management Organic farming relies heavily on the natural breakdown of organic matter,
using techniques like green manure and composting, to replace nutrients taken from the soil by
previous crops. This biological process, driven by microorganisms such as mycorrhiza, allows
the natural production of nutrients in the soil throughout the growing season, and has been
referred to as feeding the soil to feed the plant. Organic farming uses a variety of methods to
improve soil fertility, including crop rotation, cover cropping, reduced tillage, and application of
compost. By reducing tillage, soil is not inverted and exposed to air. Weed management
Organic weed management promotes weed suppression, rather than weed elimination, by
enhancing crop competition and phytotoxic effects on weeds. Organic farmers integrate
cultural, biological, mechanical, physical and chemical tactics to manage weeds without
synthetic herbicides. Organic standards require rotation of annual crops, meaning that a single
crop cannot be grown in the same location without a different, intervening crop. Organic crop
rotations frequently include weed-suppressive cover crops and crops with dissimilar life cycles
to discourage weeds associated with a particular crop. Research is ongoing to develop organic
methods to promote the growth of natural microorganisms that suppress the growth or
germination of common weeds. Controlling other organisms See also: Biological pest control
and Integrated Pest Management Organisms aside from weeds that cause problems on organic
farms include arthropods (e.g., insects, mites), nematodes, fungi and bacteria. Organic practices
include,. Livestock Raising livestock and poultry, for meat, dairy and eggs, is another
traditional farming activity that complements growing. Organic farms attempt to provide
animals with natural living conditions and feed. While the USDA does not require any animal
welfare requirements be met for a product to be marked as organic, this is a variance from
older organic farming practices. Genetic modification Although GMOs are excluded from
organic farming, there is concern that the pollen from genetically modified crops is increasingly
penetrating organic and heirloom seed stocks, making it difficult, if not impossible, to keep
these genomes from entering the organic food supply. Differing regulations among countries
limits the availability of GMOs to certain countries, as described in the article on regulation of
the release of genetic modified organisms..

3. Symbiosis ))‫قصير‬ )) Root nodule symbiosis .. ((Legume family)).. Plants that contribute to
nitrogen fixation include the legume family – Fabaceae – with taxa such as kudzu, clovers,
soybeans, alfalfa, lupines, peanuts, and rooibos. They contain symbiotic bacteria called rhizobia
within nodules in their root systems, producing nitrogen compounds that help the plant to grow
and compete with other plants. When the plant dies, the fixed nitrogen is released, making it
available to other plants and this helps to fertilize the soil. The great majority of legumes have this
association, but a few genera (e.g., Styphnolobium) do not. In many traditional and organic farming
practices, fields are rotated through various types of crops, which usually includes one consisting
mainly or entirely of clover or buckwheat (non-legume family Polygonaceae), which are often
referred to as "green manure". Inga alley farming relies on the leguminous genus Inga, a small
tropical, tough-leaved, nitrogen-fixing tree.. ((Non-leguminous)) … Although by far the majority of
plants able to form nitrogen-fixing root nodules are in the legume family Fabaceae, there are a few
exceptions: Parasponia, a tropical genus in the Cannabaceae also able to interact with rhizobia and
form nitrogen-fixing nodules. Actinorhizal plants such as alder and bayberry can also form
nitrogen-fixing nodules, thanks to a symbiotic association with Frankia bacteria. These plants
belong to 25 genera distributed among 8 plant families .... The ability to fix nitrogen is far from
universally present in these families. For instance, of 122 genera in the Rosaceae, only 4 genera are
capable of fixing nitrogen. All these families belong to the orders Cucurbitales, Fagales, and
Rosales, which together with the Fabales form a clade of eurosids. In this clade, Fabales were the
first lineage to branch off; thus, the ability to fix nitrogen may be plesiomorphic and subsequently
lost in most descendants of the original nitrogen-fixing plant.. There are also several nitrogen-fixing
symbiotic associations that involve cyanobacteria (such as Nostoc): Some lichens such as Lobaria
and Peltigera , Mosquito fern (Azolla species) , Cycads ,Gunnera …
Composting ))‫قصير‬ )) is organic matter that has been decomposed and recycled as a fertilizer
and soil amendment. Compost is a key ingredient in organic farming. At the simplest level, the
process of composting simply requires making a heap of wetted organic matter known as green
waste (leaves, food waste) and waiting for the materials to break down into humus after a period
of weeks or months. Modern, methodical composting is a multi-step, closely monitored process
with measured inputs of water, air, and carbon- and nitrogen-rich materials. The decomposition
process is aided by shredding the plant matter, adding water and ensuring proper aeration by
regularly turning the mixture. Worms and fungi further break up the material. Aerobic bacteria and
fungi manage the chemical process by converting the inputs into heat, carbon dioxide and
ammonium. The nitrogen is the form of ammonium (NH4) used by plants. When available
ammonium is not used by plants it is further converted by bacteria into nitrates (NO3) through the
process of nitrification. Compost can be rich in nutrients. It is used in gardens, landscaping,
horticulture, and agriculture. The compost itself is beneficial for the land in many ways, including as
a soil conditioner, a fertilizer, addition of vital humus or humic acids, and as a natural pesticide for
soil. In ecosystems, compost is useful for erosion control, land and stream reclamation, wetland
construction, and as landfill cover . Organic ingredients intended for composting can alternatively
be used to generate biogas through anaerobic digestion. Anaerobic digestion is fast overtaking
composting in some parts of the world (especially central Europe) as a primary means of down
cycling waste organic matter. Composting organisms require four equally important ingredients to
work effectively: Carbon — for energy; the microbial oxidation of carbon produces the heat, if
included at suggested levels. High carbon materials tend to be brown and dry. Nitrogen — to grow
and reproduce more organisms to oxidize the carbon. High nitrogen materials tend to be green (or
colorful, such as fruits and vegetables) and wet. Oxygen — for oxidizing the carbon, the
decomposition process. Water — in the right amounts to maintain activity without causing
anaerobic conditions…

4. Organic manure ‫))قصير‬ )) is organic matter, mostly derived from animal feces except in the case
of green manure, which can be used as organic fertilizer in agriculture. Manures contribute to the
fertility of the soil by adding organic matter and nutrients, such as nitrogen, that are trapped by
bacteria in the soil. Higher organisms then feed on the fungi and bacteria in a chain of life that
comprises the soil food web. It is also a product obtained after decomposition of organic matter
like cow dung which replenishes the soil with essential elements and add humus to the soil.. There
are three main classes of manures used in soil management:1- Animal manure --Most animal
manure consist of feces. Common forms of animal manure include farmyard manure (FYM) or farm
slurry (liquid manure). FYM also contains plant material (often straw), which has been used as
bedding for animals and has absorbed the feces and urine. Agricultural manure in liquid form,
known as slurry, is produced by more intensive livestock rearing systems where concrete or slats
are used, instead of straw bedding. Manure from different animals has different qualities and
requires different application rates when used as fertilizer.
2- Human manure Some people refer to human excreta as human manure, and the word
"humanure" has also been used. Just like animal manure, it can be applied as a soil conditioner
(reuse of excreta in agriculture). Sewage sludge is a material that contains human excreta, as it is
generated after mixing excreta with water and treatment of the wastewater in a sewage treatment
plant. 3- Green manure Green manures are crops grown for the express purpose of plowing them
in, thus increasing fertility through the incorporation of nutrients and organic matter into the soil.
Leguminous plants such as clover are often used for this, as they fix nitrogen using Rhizobia
bacteria in specialized nodes in the root structure. Other types of plant matter used as manure
include the contents of the rumens of slaughtered ruminants, spent hops (left over from brewing
beer) and seaweed…

5. ‫الثانية‬ ‫الوحدة‬
Plant growth-promotingrhizobacteria (PGPR) Formulation and Applications
Free-living plant growth-promoting rhizobacteria (PGPR) can be used in a variety of ways when
plant growth enhancements are required. The most intensively researched use of PGPR has
been in agriculture and horticulture. Several PGPR formulations are currently available as
commercial products for agricultural production. Recently developing areas of PGPR usage
include forest regeneration and phytoremediation of contaminated soils. As the mechanisms of
plant growth promotion by these bacteria are unravelled, the possibility of more efficient plant-
bacteria pairings for novel and practical uses will follow. The progress to date in using PGPR in a
variety of applications with different plants is summarized and discussed here. PGPR with wide
scope for commercialization includes Pseudomonas fluorescens, P. putida, P. aeruginosa,
Bacillus subtilis and other Bacillus spp. The potential PGPR isolates are formulated using
different organic and inorganic carriers either through solid or liquid fermentation
technologies. They are delivered either through seed treatment, bio-priming, seedling dip, soil
application, foliar spray, fruit spray, hive insert, sucker treatment and sett treatment.
Application of PGPRformulations with strain mixtures perform better than individual strains for
the management of pest and diseases of crop plants, in addition to plant growth promotion.
Supplementation of chitin in the formulation increases the efficacy of antagonists. More than
33 products of PGPR have been registered for commercial use in greenhouse and field in North
America. Though PGPR has a potential scope in commercialization, the threat of certain PGPR
(P. aeruginosa, P. cepacia and B. cereus) to infect human beings as opportunistic pathogens has
to be clarified before large scale acceptance, registration and adoption of PGPR for pest and
disease management. PGPR are known to improve plant growth in many ways when compared
to synthetic fertilizers, insecticides and pesticides. They enhance crop growth and can help in
sustainability of safe environmentand crop productivity. The rhizospheric soil1 contains diverse
types of PGPR communities, which exhibit beneficial effects on crop productivity. Several
research investigations are conducted on the understanding of the diversity, dynamics and
importance of soil PGPR communities and their beneficial and cooperative roles in agricultural
productivity. Some common examples of PGPR genera exhibiting plant growth promoting
activity are: Pseudomonas, Azospirillum, Azotobacter, Bacillus, Burkholdaria, Enterobacter,
Rhizobium, Erwinia, Mycobacterium, Mesorhizobium, Flavobacterium, etc. This article presents
perspectives on the role of PGPR in agriculture sustainability.

6. Frankia )) ‫قصير‬ )) is a genus of nitrogen fixing, filamentous bacteria that live in symbiosis with
actinorhizal plants, similar to the Rhizobia bacteria that are found in the root nodules of legumes in
the Fabaceae family. Bacteria of this genus also form root nodules .. Frankia is the only named
species in this genus, but a great many strains are specific to different plant species. The bacteria
are filamentous and convert atmospheric nitrogen into ammonia via the enzyme nitrogenase, a
process known as nitrogen fixation. They do this while living in root nodules on actinorhizal plants.
The bacteria can supply most or all of the nitrogen requirements of the host plant. As a result,
actinorhizal plants colonise and often thrive in soils that are low in plant nutrients..Several Frankia
genomes are now available which may help clarify how the symbiosis between prokaryote and
plant evolved, how the environmental and geographical adaptations occurred, the metabolic
diversity, and the horizontal gene flow among the symbiotic prokaryotes. Frankia alni forms a
symbiotic relationship exclusively with trees in the genus Alnus. These are widely distributed in
temperate regions of the northern hemisphere. One species, Alnus glutinosa, is also found in Africa
and another, the Andean alder, Alnus acuminata, extends down the mountainous spine of Central
and South America as far as Argentina. Evidence suggests that this alder may have been exploited
by the Incas and used to increase soil fertility and stabilize terrace soils in their upland farming
systems. Alnus species grow in a wide range of habitats that include glacial till, sand hills, the banks
of water courses, bogs, dry volcanic lava flows and ash alluvium . The first symptom of infection by
Frankia alni is a branching and curling of the root hairs of the alder as the bacterium moves in. The
bacterium becomes encapsulated with a material derived from the plant cell wall and remains
outside the host's cell membrane.The encapsulation membrane contains pectin, cellulose and
hemicelluloses …
mass cultivation of azotobacter The effect of different aeration conditions during the culture of
Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake
flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in
conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by
the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as
compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in
the molecular mass distribution. In either cases, the mean molecular mass increased with culture
age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in
unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded,
the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in
baffled flasks decreased in size. Azotobacter Of the several species of Azotobacter, A.
chroococcum happens to be 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...
Azotobacter It is the important and 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 present on the rhizosplane but are abundant in the rhizosphere region.
The lack of organic matter in the soil is a limiting factor for the proliferation of Azotobaceter in the
soil. Field experiments were conducted in 1992, 1993 and 1994 during the pre-kharif wet seasons
to find out the influence on rice grain yield by the combined use of N- fixing organisms and
inorganic nitrogen fertilizer which recorded increase in was yield.

7. Rhizobium is a soil habitat bacterium, which can 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 1930s and the strange situation at that time has been
chronicled by Fred (1932). Initially, due to absence of efficient bradyrhizobial strains in soil,
soybean inoculation at that time resulted in bumper crops but incessant inoculation during the last
four decades by US farmers has resulted in the build up of a plethora of inefficient strains in soil
whose replacement by efficient strains of bradyrhizobia has become an insurmountable problem.
Rhizobium This belongs to bacterial group and the classical example is symbiotic nitrogen fixation.
The bacteria infect the legume root and form root nodules within which they reduce molecular
nitrogen to ammonia which is 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 Table-2 shows the N
fixation rates.
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. The bacteria of Genus 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.
Five species of Azospirillum have been described to date A. brasilense, A.lipoferum, A.amazonense,
A.halopraeferens and A.irakense. 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. Azospirllium It belongs to bacteria and is known to fix the considerable quantity of
nitrogen in the range of 20- 40 kg N/ha in the rhizosphere in non- non-leguminous plants such as
cereals, millets, Oilseeds, cotton etc. The efficiency of Azospirillium as a Bio-Fertilizer has
increased because of its ability of inducing abundant roots in several pants like rice, millets and
oilseeds even in upland conditions. Considerable quantity of nitrogen fertilizer up to 25-30 % can
be saved by the use of Azospirillum inoculant. The genus Azospirillum has three species viz., A.
lipoferum, A. brasilense and A. amazonense. These species have been commercially exploited for
the use as nitrogen supplying Bio-Fertilizers. One of the characteristics of Azospirillum is its ability
to reduce nitrate and denitrify. Both A. lipoferum,and A. brasilense may comprise of strains which
can actively or weakly denitrify or reduce nitrate to nitrite and therefore, for inoculation
preparation, it is necessary to select strains which do not possess these characteristics.Azospirllium
lipoferum present in the roots of some of tropical forage grasses uch as Digitaria, Panicum,
Brachiaria, Maize, Sorghum, Wheat and Rye.

8. ‫الثالثة‬ ‫الوحدة‬
Method of inoculation of BGA in rice field
Blue green algae may be applied as soil based inoculum to the rice field following the method
described below.
 Powder the soil based algal flakes very well.
 Mix it with 10 kg soil or sand (10kg powdered algal flakes with 10 kg soil / sand).
 BGA is to be inoculated on 7-10 days after rice transplanting.
 Water level at 3-4” is to be maintained at the time of BGA inoculation and then for a month so
as to have maximum BGA development… A week after BGA inoculation, algal growth can be
seen and algal mat will float on the water after 2-3 weeks. The algal mat colour will be green or
brown or yellowish green..
……………………………………………………………………………………………………………
Cyanobacteria (BGA) can be found in almost every conceivable environment. Blue green algae
are photosynthetic Cyanobacteria and promote the growth of lowland paddy by supplying fixed
nitrogen through exudation. Besides fixing nitrogen, these algae excrete vitamins and
hormones, which may also contribute to the growth of rice plants. Blue green algae found in
almost every environment, from oceans to fresh water to bare rock to soil. They occur as
plantonic cells or form phototrophic biofilm in marine environments, in damp soil, moistened
rocks ,deserts , Blue green algae are photosynthetic . Cyanobacteria ,promote the growth of
lowland paddy by supplying nitrogen. BGA strains used for biofertilizer are Anabaena variabilis
,Nostoc muscorum, Aulosira fertilissima and Tolypothrix tenuis.. Blue green algae excrete
vitamins and hormones, which enhance rice growth. BGA also enhances soil fertility ,,
Cyanobacteria include unicellular and colonial form. Colonies form filaments, sheets , hollow
balls. Filaments differentiate into vegetative cells, heterocysts and resistant spores.Heterocyst
is the site of nitrogen fixation.Heterocyst fix nitrogen into ammonia and nitrate Nitrate and
ammonia are absorbed by plants … Blue green algae are photosynthetic .They form
phototrophic biofilm in fresh water and marine environments, in damp soil. Promote the
growth of lowland paddy by supplying fixed nitrogen through exudation. Blue green algae also
excrete vitamins and hormones, which may also contribute to the growth of rice plants..
Method of inoculation of BGA in rice field Blue green algae may be applied as soil based
inoculum to the rice field following the method described .# Powder the soil based algal flakes
very well. ,, Mix it with 10 kg soil or sand (10kg powdered algal flakes with 10 kg soil / sand). ,,
BGA is to be inoculated on 7-10 days after rice transplanting. ,, Water level at 3-4” is to be
maintained at the time of BGA inoculation and then for a month so as to have maximum BGA
development. A week after BGA inoculation, algal growth can be seen and algal mat will float
on the water after 2-3 weeks. The algal mat colour will be green or brown or yellowish green

9. Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen in
association with nitrogen fixing blue green alga Anabaena azollae. Azolla fronds consist of
sporophyte with a floating rhizome and small overlapping bi-lobed leaves and roots. Rice growing
areas in South East Asia and other third World countries have recently been evincing increased
interest in the use of the symbiotic N2 fixing water fern Azolla either as an alternate nitrogen
sources or as a supplement to commercial nitrogen fertilizers. Azolla is used as biofertilizer for
wetland rice and it is known to contribute 40-60 kg N ha-1 per rice crop. The agronomic potential
ofAzolla is quite significant particularly for rice crop and it is widely used as biofertilizer for
increasing rice yields. Rice crop response studies with Azolla biofertilizer in the People’s Republic in
China and in Vietnam have provided good evidence that Azolla incorporation into the soil as a
green manure crop is one of the most effective ways of providing nitrogen source for rice. The
utilization of Azolla as dual crop with wetland rice is gaining importance in Philippines, Thailand,
Srilanka and India. The important factor in using Azolla as a biofertilizer for rice crop is its quick
decomposition in soil and efficient availability of its nitrogen to rice. In tropical rice soils the
applied Azolla mineralizes rapidly and its nitrogen is available to the rice crop in very short period.
The common species of Azolla are A. microphylla, A. filiculoides, A. pinnata, A. caroliniana, A.
nilotica, A. rubra and A. mexicana.
Method of inoculation of Azolla to rice crop … The Azolla biofertilizer may be applied in two ways
for the wetland paddy. In the first method, fresh Azolla biomass is inoculated in the paddy field
before transplanting and incorporated as green manure. This method requires huge quantity of
fresh Azolla. In the other method, Azolla may be inoculated after transplanting rice and grown as
dual culture with rice and incorporated subsequently. A. Azolla biomass incorporation as green
manure for rice crop (Collect the fresh Azolla biomass from the Azolla nursery plot. , Prepare the
wetland well and maintain water just enough for easy incorporation. . Apply fresh Azolla biomass
(15 t ha-1) to the main field and incorporate the Azolla by using implements or tractor.) B.
Azolla inoculation as dual crop for rice ( Select a transplanted rice field. , Collect fresh Azolla
inoculum from Azolla nursery. , Broadcast the fresh Azolla in the transplanted rice field on 7th day
after planting (500 kg / ha)., Maintain water level at 5-7.5cm. , Note the growth of Azolla mat four
weeks after transplanting and incorporate the Azolla biomass by using implements or tranctor or
during inter-cultivation practices. , A second bloom of Azolla will develop 8 weeks after
transplanting which may be incorporated again. , By the two incorporations, 20-25 tonnes
of Azolla can be incorporated in one hectare rice field.
Azolla is a free floating water fern that floats in water and fixes nitrogen in association with the
nitrogen fixing blue green algae, Anabaena azollae. Azolla is considered to be a potential
biofertilizer in terms of nitrogen contribution to rice. Long before its cultivation as a green manure,
Azolla has been used as a fodder for domesticated animals such as pigs and ducks. In recent days,
Azolla is very much used as a sustainable feed substitute for livestock especially dairy cattle,
poultry, piggery and fish. Azolla contains 25 – 35 per cent protein on dry weight basis and rich in
essential amino acids, minerals, vitamins and carotenoids including the antioxidant b carotene.
Cholorophyll a, chlorophyll b and carotenoids are also present in Azolla, while the
cyanobiont Anabaena azollae contains cholorophyll a, phycobiliproteins and carotenoids. The rare
combination of high nutritive value and rapid biomass production make Azolla a potential and
effective feed substitute for live stocks.

10. ‫الرابعة‬ ‫الوحدة‬
AM fungi
The transfer of nutrients mainly phosphorus and also zinc and sulphur from the soil milleu to
the cells of the root cortex is mediated by intracellular obligate fungal endosymbionts of the
genera Glomus, Gigaspora, Acaulospora, Sclerocysts and Endogone which possess vesicles for
storage of nutrients and arbuscles for funneling these nutrients into the root system. By far,
the commonest genus appears to be Glomus, which has several species distributed in
soil.Availability for pure cultures of AM (Arbuscular Mycorrhiza) fungi is an impediment in
large scale production despite the fact that beneficial effects of AM fungal inoculation to plants
have been repeatedly shown under experimental conditions in the laboratory especially in
conjunction with other nitrogen fixers. AM fungi Nursery application: 100 g bulk inoculum
is sufficient for one metre square. The inoculum should be applied at 2-3 cm below the soil at
the time of sowing. The seeds/cutting should be sown/planted above the VAM inoculum to
cause infection. For polythene bag raised crops: 5 to 10 g bulk inoculum is sufficient for each
packet. Mix 10 kg of inoculum with 1000 kg of sand potting mixture and pack the potting
mixture in polythene bag before sowing. For out –planting: Twenty grams of VAM inoculum is
required per seedling. Apply inoculum at the time of planting. For existing trees: Two hundred
gram of VAM inoculum is required for inoculating one tree. Apply inoculum near the root
surface at the time of fertilizer application.
Production of mycorrhizal fungi as fungal biofertilizers AM fungi are obligate symbiotic
microorganisms since they cannot be grown without the plant host on synthetic media . Therefore
AM fungal inocula must be produced in association with the host plant and therefore there are
many constraints to large scale commercial production. Mass production is by pot culture either in
the greenhouse or in growth chambers is the most commonly used production method . AM fungal
inocula have to be prepared by multiplication of the selected fungi in roots of susceptible host
plants growing in the sterilized soil or substrates for example perlite, vermiculite, peat, sand, or
mixture of them. The inocula of AM fungi can be applied as spores, or fragments of colonized roots.
The spores and hyphae can be isolated from the soil rhizosphere and mixed with carrier substrates
. Spore inocula are the most resistant and can survive unfavorable environmental conditions for a
long period, but they colonize new root systems more slowly than other preparations. Therefore
both types of inocula, e.g. spores and fragments of colonized roots should be combined in
commercial products

11. Inoculum preparation
 Prepare appropriate media for specific to the bacterial inoculant in 250 ml, 500 ml, 3 litre and 5
litre conical flasks and sterilize.
 The media in 250 ml flask is inoculated with efficient bacterial strain under aseptic condition
 Keep the flask under room temperature in rotary shaker (200 rpm) for 5- 7 days.
 Observe the flask for growth of the culture and estimate the population, which serves as the
starter culture.
 Using the starter culture (at log phase) inoculate the larger flasks (500 ml, 3 litre and 5 litre)
containing the media, after obtaining growth in each flask.
 The above media is prepared in large quantities in fermentor, sterilized well, cooled and kept it
ready.
 The media in the fermentor is inoculated with the log phase culture grown in 5 litre flask. Usually
1 -2 % inoculum is sufficient, however inoculation is done up to 5% depending on the growth of
the culture in the larger flasks.
 The cells are grown in fermentor by providing aeration (passing sterile air through compressor
and sterilizing agents like glass wool, cotton wool, acid etc.) and given continuous stirring.
 The broth is checked for the population of inoculated organism and contamination if any at the
growth period.
 The cells are harvested with the population load of 109 cells ml-1 after incubation period.
 There should not be any fungal or any other bacterial contamination at 10-6 dilution level
 It is not advisable to store the broth after fermentation for periods longer than 24 hours. Even at
4o C number of viable cells begins to decrease.
Glomus, A new arbuscular mycorrhizal fungal species of genus Glomus, G. perpusillum
(Glomeromycota), forming small, hyaline spores is described and illustrated. Spores of G.
perpusillum were formed in hypogeous aggregates and occasionally inside roots. They are globose
to subglobose, (10–)24(–30) μm diam, rarely egg-shaped, oblong to irregular, 18–25 × 25–63 μm.
The single spore wall of G. perpusillum consists of two permanent layers: a finely laminate,
semiflexible to rigid outer layer and a flexible to semiflexible inner layer. The inner layer becomes
plastic and frequently contracts in spores crushed in PVLG-based mountants and stains reddish
white to grayish red in Melzer’s reagent. Glomus perpusillum was associated with roots of
Ammophila arenaria colonizing sand dunes of the Mediterranean Sea adjacent to Calambrone, In
single-species cultures with Plantago lanceolata as host plant, G. perpusillum formed vesicular-
arbuscular mycorrhiza. Phylogenetic analyses of partial SSU sequences of nrDNA placed the species
in Glomus group A with no affinity to its subgroups. The sequences of G. perpusillum
unambiguously separated from the sequences of described Glomus species and formed a distinct
clade together with in planta arbuscular mycorrhizal fungal sequences found in alpine plants

12. Fungal biofertilizers
Biofertilizers comprise microbial inocula or assemblages of living microorganisms which exert
direct or indirect benefits on plant growth and crop yield through different mechanisms .
These microorganisms are able to fix atmospheric nitrogen or solubilize phosphorus,
decompose organic material, or oxidize sulfur in the soil properties that are beneficial to
agricultural production in terms of nutrient supply. One type of biofertilizer are the arbuscular
mycorrhizal fungi, which are probably the most abundant fungi in agricultural soil . The
inocula improve crop yield because of increased availability or uptake or absorption of
nutrients, stimulation of plant growth by hormone action or antibiosis and by decomposition
of organic residues . Selected fungal Mycorrhizal fungi used as biofertilizers Mycorrhizae form
mutualistic symbiotic relationships with plant roots of more than 80% of land plants including
many important crops and forest tree species . There are seven types of mycorrhiza: arbutoid
mycorrhiza, ectomycorrhiza, endomycorrhiza or arbuscular mycorrhiza, ect-endomycorrhiza,
ericoid mycorrhiza, monotropoid mycorrhiza, and orchidoid mycorrhiza . The two dominant
types of mycorrhizae are ectomycorrhizae (ECM) and arbuscular mycorrhizae (AM) which can
improve water and nutrient uptake and provide protection from pathogens but only a few
families of plants are able to form functional associations with both AM and ECM fungi .
However, AM fungi are most commonly found in the rhizosphere roots of a wide range of
herbaceous and woody plants . , we focus on ectomycorrhizal fungi and arbuscular
mycorrhizal fungi because they are the most widespread and economically important types of
mycorrhizal fungi. Endomycorrhizae from mutually symbiotic relationships between fungi
and plant roots . The plant roots provide substances for the fungi and the fungi transfer
nutrients and water to the plant roots . Endomycorrhizal fungi are intercellular and penetrate
the root cortical cells and form structures called arbuscular vesicles and known as vesicular
arbuscular mycorrhiza (VAM) but in some cases no vesicles are formed and they known as
arbuscular mycorrhiza (AM) The agriculturally produced crop plants that form
endomycorrhizae of the vesicular-arbuscular mycorrhiza type are now called arbuscular
mycorrhizal (AM) fungi .AM fungi belong to nine genera: Acaulospora, Archaeospora,
Enterophospora, Gerdemannia, Geosiphon, Gigaspora, Glomus, Paraglomus, and Scutellospora
. AM fungi are a widespread group and are found from the arctic to tropics and are present in
most agricultural and natural ecosystems. They play an important role in plant growth, health,
and productivity AM fungi help plants to absorb nutrients, especially the less available mineral
nutrients such as copper, molybdenum, phosphorus and zinc . They increase seedling
tolerance to drought, high temperatures, toxic heavy metals, high or low pH and even extreme
soil acidity .

13. Ectomycorrhizal (ECM) fungi form mutualistic symbioses with many tree species . Most ECM fungi
do not penetrate the living cells in the roots, but only surround them. ECM fungi occur naturally in
association with many forest trees, for example, pine, spruce, larch, hemlock, willow, poplar, oak
birch and eucalyptus . Most ECM fungi that are associated with forest trees are basidiomycetes,
such as Amanita sp., Lactarius , Pisolithus, and Rhizopogon sp. and many of these are edible Some
ascomycetes also form mycorrhizae such as, Cenococcum sp., Elaphomyces sp., and Tuber. The
importance of ECM fungi to trees is in their ability to increase the tree growth due to better
nutrient acquisition . ECM fungi help the growth and development of trees because the roots
colonized with ectomycorrhiza are able to absorb and accumulate nitrogen, phosphorus,
potassium, and calcium more rapidly and over a longer period than nonmycorrhizal roots. ECM
fungi help to break down the complex minerals and organic substances in the soil and transfer
nutrients to the tree. ECM fungi also appear to increase the tolerance of trees to drought, high soil
temperatures, soil toxins, and extremes of soil pH. ECM fungi can also protect roots of trees from
pathogens . The most commonly widespread ectomycorrhizal product is inoculum of Pisolithus
tinctorius . Pisolithus tinctorius has a wide host range and their inoculum can be produced and
applied as vegetative mycelium in a peat vermiculite carrier. These fungus inocula are applied to
nursery or forestry plantations. Piriformospora indica (Hymenomycetes, Basidiomycota) is another
ECM fungus used as a biofertilizer. This taxon can promote plant growth and biomass production
and help plant tolerance to herbivory, heat, salt, disease, drought, and increased below- and
above-ground biomass..
Pisolithus tinctorius
is a mycorrhizal fungus that is not at all picky about its plant and tree partnerships. For this reason
it is frequently used by foresters and gardeners to assist plant or tree growth (for impressive
photos of plants and trees grown with and without the mycorrhizal support of fungi... It also grows
well in poor soil, sandy areas, and so on, making it an even more valuable fungus for plant life.
Description: Ecology: Mycorrhizal with just about anything that makes roots; growing alone,
scattered, or in small groups; often found in gravel, sandy soil, in ditches, on lawns, and so on;
summer and fall; widely distributed in North America but more commonly found on the West
Coast and in the southeast. Studied was the effect of ecto mycorrhizal formed by the fungus
Pisolithus tinctorius on growth of loblolly and shortleaf pine seedlings in acid coal spoils. Seedling
production was tested on both Kentucky and Virginia coal spoils. Pisolithus ectomycorrhizal
improves the survival and growth of pine seedlings; seedlings treated with ectomycorrhizal from
Thelephora terrestris exhibited lower growth rates and accumulated higher levels of selenium,
iron, aluminum, and manganese. The use of Pisolithus ectomycorrhizae in land reclamation
projects should be studied further. In the present work the genetic transformation and the
expression of gene markers in transgenic Pisolithus tinctorius are reported. The
ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording
mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this
association (more than 80% of gymnosperms are associated with these fungi), its study
from both basic and applied viewpoints is relevant. We have transformed this fungus with
reporter genes and analyzed their expression in its saprophytic state. Genetic
transformation was performed by microprojectile bombardment and Agrobacterium-
mediated transformation. This last method proved to be the more efficient. Southern
analysis of biolistic-transformed fungi revealed the random integration of the transgene into
the genome. The accumulation of the transcript of the reporter gene was demonstrated by
RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia
confirmed the expression of the reporter gene…

14. Plant Growth Promoting Rhizobacteria (PGPR)
The group of bacteria that colonize roots or rhizosphere soil and beneficial to crops are referred to
as plant growth promoting rhizobacteria (PGPR). The PGPR inoculants currently commercialized
that seem to promote growth through at least one mechanism; suppression of plant disease
(termed Bioprotectants), improved nutrient acquisition (termed Biofertilizers), or phytohormone
production (termed Biostimulants). Species of Pseudomonas and Bacillus can produce as yet not
well characterized phytohormones or growth regulators that cause crops to have greater amounts
of fine roots which have the effect of increasing the absorptive surface of plant roots for uptake of
water and nutrients. These PGPR are referred to as Biostimulants and the phytohormones they
produce include indole-acetic acid, cytokinins, gibberellins and inhibitors of ethylene production.
Recent advances in molecular techniques also are encouraging in that tools are becoming available
to determine the mechanism by which crop performance is improved using PGPR and track survival
and activity of PGPR organisms in soil and roots. The science of PGPR is at the stage where
genetically modified PGPR can be produced. PGPR with antibiotic, phytohormone and siderophore
production can be made. Despite of promising results, biofertilizers has not got widespread
application in agriculture mainly because of the variable response of plant species or genotypes to
inoculation depending on the bacterial strain used. Differential rhizosphere effect of crops in
harbouring a target strain or even the modulation of the bacterial nitrogen fixing and phosphate
solubilizing capacity by specific root exudates may account for the observed differences. On the
other hand, good competitive ability and high saprophytic competence are the major factors
determining the success of a bacterial strain as an inoculant. Studies to know the synergistic
activities and persistence of specific microbial populations in complex environments, such as the
rhizosphere, should be addressed in order to obtain efficient inoculants. In this regards, research
efforts are made at Agricultural College and Research Institute, Madurai to obtain appropriate
formulations of microbial inoculants incorporating nitrogen fixing, phosphate- and silicate-
solubilizing bacteria and plant growth promoting rhizobacteria which will help in promoting the use
of such beneficial bacteria in sustainable agriculture..