CHAPTER II

REVIEW OF LITERATURE

2.1.0. AM Fungi

2.1.1. Taxonomy of AM Fungi

2.1.2. Morphology of AM Fungi

2.1.3. Distribution of AM Fungi

2.1.4. Isolation techniques of AM Fungi

2.1.5. Multiplication of AM Fungi

2.1.6. Physiology of AM Fungi

2.1.7. Functions of AM Fungi

2.1.8. Response Study of AM Fungi

2.1.9. Uptake of Nutrients

2.1.10. Benefits of AM Fungi

2.1.11. Interaction of AM with Rhizobium

2.2.0. Rhizobium

2.3.0 Cowpea

2.4.0. Maize

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REVIEW OF RELATED LITERATURE

2.1.0 : AM (ARBUSCULAR MYCORRHIZAL FUNGI)

2.1.1 : TAXONOMY OF AM

Taxonomically AM come under the class Zygomycetes in the

subdivision Zygomycotina, family–Endogonaceae, order-Endogonales.

Seven genera, Endogone, Glomus, sclerocystis, Entrophospora

Acaulospora, Scutellospora, and Gigaspora constitute Endogonaceae.

Of these only Endogone forms zygospores, while others probably lack

sexual reproduction. Species of Endogone do not form arbuscular

mycorrhizal association, Bagyaraj (1991,a), in contrast of report of

Potty (1978), that it has been stated that Endogone species are

involved in VA mycorrhizal type of association with Cassava, Sweet

potato and Coleus roots. The other six genera form VA mycorrhizal

association. According to Bagyaraj (1987), there are one hundred and

twenty species of VAM fungi. Schenck and Perez (1988), reported

that genus Endogone do not form VAM .According to Bagyaraj

(1991,a) VAM mycorrhizal fungi being obligate biotrophs, do not grow

on synthetic media and hence are classified according to

morphological characteristics of the spores formed in the soil.

Sieverding (1991,a) reported that Endogone form

ectomycorrhiza with certain tree species. Cavalier-Smith (1998) has

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recognized the fungal Phylum Glomeromycota with a single class

Glomeromycetes containing 150 described species all over the world.

Gupta and Ali (1993) reported that few members of the

cyperaceae family were found to be mycorrhizal. Plants like Cyperus

iria,C.pilosus, C. triceps, were found to be colonized with VAM

fungi. Morton and Redecker (2001) based on concordant molecular

morphological characters, discovered two new families

Archeosporaceae and Paraglomaceae with two new genera

Archeospora and Paraglomus respectively.

2.1.2 : MORPHOLOGY OF AM FUNGI

According to Schenck and Perez (1988), out of the six genera of

the fungi forming VA mycorrhizae with plants, two of the genera

Glomus and Sclerocystis produce chlamydospores only. Four genera

forming spores that are similar to azygospores are Gigaspora,

Scutellospora. Acaulospora, and Entrophospora. The genera are

distinguished by their spore characteristics and relationship of the

spore to the associated hyphal attachments. The existant genera are

established to reflect the manner in which the spores are produced

except for the genus Scutellospora. Species in this genus are

distinguished from species in the closely allied genus Gigaspora by

wall characteristics, method of germ tube formation and auxillary cell

characteristics.

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Bagyaraj (1991,a) reported that Glomus, the most common

genus of AM fungi has over 50 species which form globose, ellipsoid

irregularly shaped spores, that range from 20-400m. These spores

are thick walled (up to 30m) and hyaline, yellow, red-brown, brown

or black in colour. Spores are attached to a single subtending hypha.

Sclerocystis species produce chlamydospores that are very similar to

those of Glomus, except that they tend to be clavate rather than

globose. All Sclerocystis species form spores only in sporocarps up to

700 m in diameter.

Spores of the genus Acaulospora appear to form laterally on the

neck of a small thin walled saccule called differently as a mother

spore, a vesicle, a hyphal terminus, a sporiferous saccule, or a

sporogenous saccule. The spores are globose or ellipsoid from less

than 100 to greater than 400 m in diameter and hyaline yellow or

reddish brown in colour. The surface of the spore wall may be

ornamented with pits, projections of various shapes, folds spines or

reticulations, and the wall is up to 12um thick. Entrophospora is

more or less similar to Acaulospora except that spore is formed inside

the parent hypha just below the vesicle. Scutellospora spores are

greater than 600 m in diameter and the smallest ones are just

under 200 m in diameter. Gigaspora produce spores which are

superficially similar to those of Scutellospora spores. The striking

difference is the germination of spores. Schwarzott et al., (2001)

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reported that recently two genera viz: Archeospora, and Paraglomus,

have been separated from genus Glomus.

Characterization and Identification

There are eight AM fungal genera which are valid and known to

be distributed throughout the globe. The genera are: Acaulospora,

Archaeospora, Entrophospora, Glomus, Paraglomus, Gigaspora,

Scutellospora, and Sclerocystis. The following are the

morphotaxonomic criteria employed in the identification of AM fungi

(Manoharachary, 2004).

1. Hyphal characters (Hyphal dimorphism, Hyphal connections,

Swollen hyphae, knobe like projections, coloration, branching).

2. Vesicles (Shape, size, colour, pleomorphism).

3. Auxillary cells (ornamentation, single or clusters.

4. Subtending hypha (simple, straight, recurved, swollen,

bulbous).

5. Spore (terminal, formation of spore in soporiferous saccule

spore remain in the neck of saccule, spore terminal with

bulbous stalk).

6. Spore germination (Direct germination, multiple germ tubes,

indirect germination).

7. Germinating shields (shields form below a semi rigid unit wall

layer, germ slits located in the wall layer, membranous and

coriaceous wall layers).

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8. Sporocarp (loose sporocarps, compact sporocarps, peridium

present or absent).

9. Spore wall (laminated layer with permanent unit layer,

evanescent layer).

10. Ornamentation (Scrobiculate, granular, excrescences, pores,

warts, blunt).

Besides conventional methods of identification charaters like

isozyme analysis, ELISA technique and immunological, serological

techniques are also employed for authentic taxonomic identification.

Molicular studies include nudeotide sequence or RFLP of DNA

regions, analysis of glycoproteins and fatty acid profile.

Glomous mosseae (Schenck and Perez, 1988)

Sporocarp 1-10 spored, globose to ellipsoid upto 1mm diameter

peridium of loosely interwoven irregularly branched, hyaline, septate

hyphae 2-12 diameter, the walls upto 0.5 thick, irregularly

anastomising to form a thin network enclosing the chlamydospores

entirely in completely, or with somespores unenclosed. Endocarpic

and ectocarpic spores similar. Chlamydospores yellow to brown,

globose to ovoid, obovoid, or some what irregular, 105-310110-305

with one or occasionally two funnel shaped bases 20-30 (-50)

diameter divided from subtending hypahe by a curved septum; walls

2-7 thick, with a thin often barely perceptible hyaline outer

membrane, and a thick brownish-yellow inner layer.

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Glomus microcarpum

Clamydospores borne free in soil in loose aggregations in small

compact clusters unenclosed in a peridium or in sporocarps with a

peridium. Sporocarps upto 5mm broad; irregularly globose, light

brown. Peridium a thin layer of interwoven hyphae similar to those in

gleba, spores interspersed but less abundantly than in gleba. In

sporocarps, chlamydospores 35-49 diam. Globose to subglobose,

chlamydospores free in soil 25 25-55 32, globose, subglobose

ellipsoid obovoid or irregular. Spore wall upto 7 thick; laminate,

hyaline to light yellow, smooth or appearing roughened from adherent

debris. Opening into subtending hypha nearly occluded in mature

spores by wall thickening.

Glomus fasciculatum

Chlamydospores borne free in soil, in dead rootlets, in loose

aggregations, in small compact clusters and in spore carps.

Sporocarps upto 8x5x5mm irregularly globose or flattened,

tuberculate, grayish brown peridium absent – chlamydospores 35-

105 diam when globose, 75-150 35-100 when subglobose to

obovate, ellipsoid, sublenticular, cylindrical or irregular; smooth or

seeming roughened from adherent debris. Spore walls highly variable

in thickness (3-17), hyaline to light yellow or yellow brown, the

thicker walls often minutely perforate with thickened inward

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projections. Hyphal attachments 4-15 diam; occluded at maturity.

Walls of attached hyphae often thickened to 1-4 near the spore.

Acaulospora (Sieverding et al.,1989)

Acaulospora denticulata

Sporocarps unknown. Spores yellow brown to dark brown,

globose to subglobose 130-170m diameter. Spores formed laterally

on the soporiferous saccule. Soporiferous saccule is 80-160, diam

globose to subglobose with a tapering neck of 4-28 diameter; 1-2m

soporiferous saccule, at point of spore formation the saccule neck is

14-25m diam. Soporiferous saccule and neck breaking off after

spore formation.

Composite spore wall consists of four walls in two separable

groups (groups A & B), wall group A is composed of one wall (wall 1)

yellow brown to red.

Description of spores

(1) Gigaspora calospora

Spores are globose to subglobose measuring 200-500m at

maturity with smooth surface having multiple wall layers with thick

outer layer over two or more thin separable inner layers. Subtending

bulbous hyphae are orange brown with a diameter of 30-50m.

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(2) Sclerocystis sp.

Chlamydospores are formed in sporocarps with spores arranged

in a single layer around a central plexus of global hyphae. Individual

spores measuring 90-120m are light yellow in colour.

(3) Glomus monosporum

Spores are globose or subglobose 125-300m. Spore surface is

dull roughened with minute spines or fractures in hyaline matrix.

Double wall layers with outer thinner than inner is present. Outer

layer is hyaline and the inner layer is yellow to brown. Wall thickness

is 9-12m. Subtending hyphae is hyaline.

(4) Glomus macrocarpum

Spores are subglobose to ovate measuring 100-200m with

smooth surface and single wall layer having a thickness of 5-20m.

Spores are yellowish brown. Subtending hyphae is single and

cylindrical at the point of attachment. Wall colour is yellowish brown.

Closure at spore wall is by wall thickening.

(5) Glomus fasciculatum

Spores are globose mixed with subglobose and ellipsoid ones

measuring 75-125m. They are smooth with single, yellowish brown

wall layers. The wall thickness is 5-8m. The subtending hyphae is

single, cylindrical and yellow to brown in colour. The spore is

separated by spore wall thickening. Sporocarp is formed of irregular

aggregations of spores.

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(6) Acaulospora scrobiculata

Spores are globose, measuring 100-300m with pitted surface

wall. They are having multiple wall layers with thick outer layer over

one or more thinner layers. The wall thickness vary from 2-8m.

Outer wall layer is yellow and the inner layers are hyaline.

(7) Acaulospora laevis

Single globose spore developed laterally on hyphae below apical

swelling. Hyaphal apex is inflated and with maturity of the spore the

stalk cell collapses. Spores are with reticulate wall, mature ones

measure 100-300m. Multiple wall layers are present with outer thick

layer over one or more thin layers. The wall colour is yellowish brown.

2.1.3: DISTRIBUTION OF AM FUNGI

Law (1985) estimated that about 85-95% of 2,31,000 species of

angiosperms are mycorrhizal despite the fact that only 120 species of

AM fungi are described so far. However it is absent in certain families

such as Pinaceae, fagaceae and Betulaceae. According to Bagyaraj

(1991,b), Arbuscular mycorrhiza are geographically ubiquitous and

occur in plants growing in arctic, temperate and tropical regions. In

general AM population is more in cultivated soil of up to15 –30cm

from the surface and decrease the number markedly below the top

15cm and in virgin soil. The distribution of species of AM fungi vary

with climatic and edaphic environment. Acaulospora laevis is

common in Western Australia and New Zealand, but less common in

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Eastern Australia. Glomus spp. appear to have widest distribution.

Gigaspora and Sclerocystis spp. are more common in tropical soils.

Acaulospora spp.seems to be better adapted to soils with pH less

than 5.0; Glomus mosseae with fine textured, fertile high pH soils;

Acaulospora laevis with coarse-textured, acidic soils and Gigaspora

spp. with sand dune soils.

Sieverding (1991,a), reported that the tropical crop plants such

as cassava, sweet potato, cowpea, soybean, maize, sorghum, barley,

upland rice, sugarcane, tobacco, cotton, cacao, rubber, tea, oil palms,

tropical pasture grasses and legumes are often heavily colonized by

AM fungi. Other crops like wheat, beans, coffee and tomato may only

be infected to a moderate extent. He reported that the environmental

conditions, and high level of NPK fertilization inhibit mycorrhiza. AM

fungi are an important group of soil fungi colonizing the roots of 90%

of plant families (Rajan, et al.,2000).

According to Schwarzott (2001), the largest accepted genus is

the Glomus, in the family of Glomaceae, even after the very recent

separation of Archeospora and Paraglomus from Glomus. Around 70-

80%of plants ranging from bryophytes to flowering plants including

aquatic plants have obligate symbiotic association of AM fungi

(Manoharachary, 2004) Among different AM fungi, Glomus and

Acaulospora spp. were found association with almost all the grasses,

(Deepak and Anuradha, 2004).

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2.1.4: ISOLATION TECHNIQUES OF AM FUNGI

There are a lot of techniques are available for the isolation of

AM from the soil. However some of the important techniques that are

accepted universally are summed up in this chapter.

2.1.4.1: Isolation techniques of AM Fungi

Isolation of AM spores from soil by wet sieving and decanting

technique developed by Gerdemann and Nicolson (1963) is as given

below: About 50 g soil is suspended in 200 ml of luke warm water

in a 500 ml beaker and stirred thoroughly. The contents are

decanted through 1mm,450 m, 250m,105m and 45m sieves

placed one below the other. The residue is resuspended in fresh water

and the process is repeated until 1 litre suspension is passed

through above series of sieves. The spores can be collected by

observing under a stereo microscope. Quantitative estimation by

counting the number of spores isolated under the same Microscope.

The separated spores are maintained in sterile distilled water in a

refrigerator after surface sterilization with 2% w/v Chloramine T or

200 ppm Streptomycin for 15 minutes, (Mosse and Phillips, 1971).

2.1.4.2: Method by Phillips and Hayman

The percentage of mycorrhizal colonization determined by a

method suggested by Phillips and Hayman (1970). In this method,

the roots are washed gently in tap water and are cut into one cm, size

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and the bits are immersed in 10% KOH solution to clear the host

cytoplasm and nuclei for stain penetration and autoclaved at 15

lb/sq inch pressure for 20 minutes. The KOH solution is then poured

and roots are rinsed in tap water until no brown colour appeared in

the rinsed water. Then the roots are acidified for three to four

minutes with 2% HCl for proper staining. The acid is then poured out

and root bits are stained with 0.05% Trypan blue in lactophenol and

boiled for ten minutes and examine under binocular microscope :

Percentage of AM colonization = 010examined bitsroot ofnumber Total

infection with bitsroot ofNumber

2.1.4.3 Flotation and adhesion technique

Developed by Sutton and Barron (1972) based on ability of AM

spores to float on water surface and adhere to glass surface is as

follows. Ten gram soil is initially transferred to a 50 ml measuring

cylinder and mixed with water to separate out spores along with the

scum. It is decanted into a separating funnel of 250 ml capacity.

More water is added and the process is repeated three or four times

so that all the spores from the cylinder will get completely transferred

to the separating funnel. Allow the liquid to settle for two to three

minutes and then slowly drain out water from the funnel into another

separating funnel placed vertically below the first one. Wait for four

to five minutes before draining out water from the second funnel.

Wash both the funnels with the help of a mild jet of water to transfer

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the spores adhering to glass sides to a Petridish or filter paper. The

spores can be separated by observing under a stereomicroscope.

2.1.4.4 Colorimetric method

A colorimetric method for estimating AM infection in roots

developed by Christine (1977): Roots are cut into three to five

centimeter lengths mixed by dispensing in water collected and blotted

between filter papers. Blotted root samples are weighed between 30-

400 mg. Fresh weight is found to be suitable according to the amount

of infection. Similarly blotted root samples are reweighed after drying

at 800c. Each sample is ground thoroughly in mortar with 0.2 –0.4 ml

water and the suspension is transferred quantitatively to a centrifuge

tube and sedimented at 650g for 15 minutes. The supernatant is

removed and the residue is heated at 1300c for 45 minutes in a

sealed tube with three ml KOH solution. The chitosan that formed is

precipitated with aqueous ethanol with an addition of water and any

trace of ethanol remaining at this stage may give an erroneous result.

The residue is made up to 1.5ml with water deaminated and reacted

with 3-methyl 2-benzothiazolone hydrazone hydrochloride and FeCl3.

Optical density at 650 nm is read in5 mm path length cells after 45

minutes.

2.1.4.5 Quantitative method by plate method

Developed by Smith and Skipper (1979) is as follows: One gram

soil is initially suspended in nine ml of distilled water .After stirring

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vigorously, one ml is pipetted in parallel lines on to a filter paper disc

kept in a Petridish. The number of spores present can be counted

directly under a stereomicroscope.

2.1.4.6 Spore Isolation Procedure

Quantitative estimation method of AM spores by Daniel and

Skipper (1982) is as given below :- One ml of sample containing the

extracted spores in water is pipetted in to a nematode counting slide.

Spores per ml can be calculated by counting the number of spores

present in a portion of the slide and multiplying it with the dilution

factor.

2.1.4.7 Mycelium extraction procedure

Vilarino et al., (1993) developed a method for the extraction of

Arbuscular mycorrhizal mycelium from sand samples. Substrate for

this method is sand with a particle size of 100-1000 m. Here an

external mycelium extractor (EME) is used for extracting mycelium.

The substrate is centrifuged with 50% saccharose, using 25m sieve

and collection with an external mycelium extractor.

The EME is evaluated using three 60gm samples of the

experimental substrate divided into 10gm samples. Each sub-sample

is suspended in 75 ml of ringer solution and 0.1 gm of CaCl2 in

1000ml of distilled water (pH 7.4) stirred by hand for 30 seconds and

centrifuged for 5 minutes at 2200g. The sediment is sieved (<100m)

25

to remove sand particles and root fragments, and washed in to a

200ml beaker with 150 to 170 ml of ringer solution. Mycelium is

extracted with 5-4 minutes application of the EME. Mycelia become

entangled in the wire frame work, and then washed of with ringer

solution on to a 0.45 m millipore filter after each application. When

the mycelium of six sub samples of each 60gm sample has been

pooled, its fresh weight, total length and viability are determined.

Marianne (1993) developed a method for quantification of

mycorrhizal infection in the roots of Calluna vulgaris hull is as given

below: root samples of C. vulgaris are oven dried at 600c for 48 hrs.

Sub samples between 10 and 50 mg dry weight are found to be

suitable according to the amount of infection present .For the chitin

assay, each sample is hydrolysed. The resulting chitosan is

precipitated with aqueous ethanol and washed three times with

distilled water to remove any trace of ethanol. The final residue is

deaminated and assayed colorimetrically. The mycorrhizal infection in

the sub samples of each root sample is done by using line intersect

method after staining in 0.2% cotton blue in lactoglycerol.

2.1.5:- MULTIPLICATION OF AM FUNGI

Since Arbuscular mycorrhizae are obligate symbiont, they

cannot be cultured on the artificial media. However they can be

multiplied by cultivating them on the living roots of host plants like

maize, onion, cowpea, millets, sorghum, clover, strawberry, soybean,

26

several grasses etc. since they possess fibrous root system and are

highly susceptible to AM colonization. AM fungi could be conveniently

multiplied by cultivating them on the living roots of these host plants

in pots.

AM fungi are ecologically obligate biotrophs that have not been

cultivated in defined media in the absence of living roots (Siqueria et

al., 1985). Chlamydospores of Glomus mosseae and Glomus

fasciculatum were multiplied on sorghum in green house pot cultures.

(Safir et al., 1990). At plant senescence, soil containing spores were

separated from the roots, thoroughly mixed, air dried and divided in to

sub-samples of 25g each. Some sub samples were immediately used

as “fresh” spores. While all others were transferred to petridishes,

sealed with para film and stored at- 100C or at room temperature.

Studies of Janardhanan et al., (1990) demonstrated of the first

time that Glomus aggregatum could be grown in axenic culture. They

described methods of isolation and culturing of Glomus aggregatum on

a synthetic medium. A monoculture of Glomus aggregatum was

established by inoculating Palmarosa plants in steam sterilized pots;

with spores. Infected roots collected from these plants were used for

the isolation of the fungus on White’s medium supplemented with 1g.

1-1 yeast extract. Isolation was carried out by two steps using 1-1.5cm

root segments from six-month-old plants. Root segments surface

sterilized with sodium hypochlorite (1-2% available chlorine) were

transferred aseptically to petirplates containing 20 ml medium. The

27

petriplates were incubated in the dark at 25 ± 10C. After 10 days, the

root segments were again transferred to fresh medium. This process

was repeated twice in order to keep the root pieces moist. Each root

piece was lifted from the petriplates, transferred to sterile distilled

water, cut in to two pieces, and aseptically placed approximately 1 cm

away from the roots of axenic plants raised from surface sterilized

palmarosa seeds on white’s medium in 150 ml conical flasks. The

isolate that emerged from the root pieces was transferred, free from the

parent root segments to white’s medium in 10 cm petriplates after 15

days of growth.

A monoxenic cultivation was developed to study the

morphological, biochemical and molecular characters of Glomus

proliferum by Declerck et al., (2000). In this method the fungal isolate

was propagated for several months in pot cultures with Allium porrum.

The leek plants were grown under green house conditions set at

24/200c, day/night with natural light intensity. Plants were fertilized

at regular intervals; and rain water was delivered every 2-3 days. Root

pieces were extracted from six months old plants and those containing

numerous vesicles were disinfected and that root pieces were

incubated in the dark at 270c on the Modified Strullu-Romand (MSR)

medium but solidified with 4g l-1 Gelgro instead of 8 g l-1 Bacto agar.

Fracchia et. al., (2001) described simple technique for obtaining

monosporic culture of AM fungi, that is penetration of plant roots by

the mycelia produced by one single spore. Plant seedlings were grown

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in 5cm diameter petridishes with 10 ml of autoclaved vermiculite-

perlite mixture (1/1., V/V). The vermiculite and perlite were previously

sieved through 500 µm mesh. Clover known to be good host for AM

fungi, was used as a test plant. Seeds were surface sterilized with 10%

sodium hypochlorite for 2 min and 30 seeds were sown on each

vermiculite perlite petridish. Plants were grown in a chamber with

Sylvania incandescent and cool white lamp 400- 700 nm with a 16/8

h day/dark cycle at 25/190C and 50% relative humidity. Plants were

regularly watered and fed with 1ml of a diluted (1/2) Long-Ashton

nutrient solution. Spores were isolated by wet sieving and decanting

technique. All spores were surface sterilized with 50 ml sterile water, 1

gm chloramine- T together with 20 µg of streptomycin and a trace of

surfactant. The spores were rinsed with sterile water; these were

transferred to a 5cm petridish with a sterile capillary pipette with 10

ml of 10mM ethane sulphonic acid buffer plus 0.04 g of Gel Gro.

petridishes were incubated at 250C for eight days and spore

germination observed under binocular microscope. Ten petridishes

with hyphal length about 5mm for Glomus or 2cm for Gigaspora strins

were selected. The contents of vermiculite perlite dish with 2 weeks old

clover seedlings were transferred on to the Gel-Gro medium with the

germinated spore. The hyphal development was observed every two

days through the bottom of the petridish under the binocular

microscope. After three weeks of growth on the chamber, the content

of one dish was put in to one pot.

29

A modified glass bead compartment cultivation system for AM

fungi was developed by Chen et al., (2001). The containers were acrylic

boxes separated in to five compartments using screens with pores.

Compartments contained host plants, roots and extra matrical

hyphae, or hyphae. Plant growth compartments were filled with 1:1

(W/W) mixtures of sandy soil and sand. Root and hyphal growth

compartments were filled with coarse river sand or glass beads and the

central hyphal compartments contained glass beads, water was added

every few days. After growth period of eight weeks, spores and hyphae

that had developed between the glass beads were removed with a

spoon, transferred to a beaker and stirred in distilled water. The glass

beads sank rapidly and the fungal materials were decanted on to a

70µm sieve. This procedure was repeated twice to obtain full recovery.

There were two host plant species, two fungal symbionts, two different

substrates in the hyphal compartment and four replicates giving a

total of 32 containers. Mycorrhiza inoculum (30 g for maize and 15 g

for red clover) was mixed in to the soil/sand mixture in host plant

containing compartments before sowing. Five seeds of maize or about

50 seeds of clover were sown in each of the two plant growth

compartments. The plants grew under a 250C/220C temperature

regime with a 14h photoperiod at a light intensity of 250µmol M-2 per

second provided by supplementary illumination.

AM fungi could be conveniently multiplied by cultivating them

on the living roots of these host plants in pots. (Laxmilal, 2002). The

30

pot culture involved a complex system comprising of host plants,

mycorrhizal fungi, and soil microflora, the pH and other physical and

chemical properties of the soil. The initial inoculum in the form of the

fungal spores or the other propagules is collected from the soil by the

sieving. Any of the above mentioned plant could be used as host. The

surface sterilized spores are placed 4-5cm below the sterilized sand

soil mixture in pots. Surface sterilized seeds of the host plants are

simultaneously sown. The inoculated plants are kept in glass house

and are maintained well. The host plants are regularly examined for

the development of AM fungi. The new crops of fungal propagules in

the form of resting spores, juvenile hyphae and vesicles are collected

after 3-4 months. The root system with soil is macerated to constitute

bulk inoculum.

Soils less media used by different workers for cultivation of AM

are bark, expanded clay aggregates, peat, perlite, vermiculite and

sand. Other methods of multiplication of AM include Tissue culture,

Hydroponic culture, Aeroponic culture etc, (Lakshmilal, 2002). Whole

plant root-organ and transformed root cultures have been used for

producing AM inoculum through tissue culture. Since no plant growth

regulators are required for the sustained growth, use of transformed

root culture is considered to be the most effective method of producing

colonised roots. Here symbiosis is initiated by bringing roots and fungi

together on agar. Vermiculite or vermiculite with peat could be used

after autoclaving for mass production of AM inoculum by adding

31

nutrient solution to the substrate. The autoclaved and sterilized

material is inoculated from agar cultures. Adequate nutrient

concentration form, and quantity of nitrogen, sucrose, content,

optimum pH and ‘P’ concentration are required for successful

production of AM.

Hosts are grown for 30-40 days in sand or vermiculite before

being placed in a hydroponic system. Inoculum is produced by

growing colonized plants in defined nutrient solution allowed to flow

over the host roots in a thin film which facilitates adequate aeration as

compared to deep solution hydroponics. Inorganic expanded clay

pellets as a substrate that ensures large surface area for flooding with

nutrient solution. Many AM fungi sporulate vigorously in the cavities

of these pellets. Aeroponic culture of AM allows both efficient

production of AM inoculum and soil free investigation of mycorrhiza.

This technique provides highly aerated environment than hydroponics.

In this method, application of fine mist of defined nutrient solution to

the roots of the host plants are required. It results in greater

concentration of spores as compared to soil based pot cultures, and

spores are more infective. Aeroponic culture requires a claim and

unshaded green house, disinfected spores, demineralized water,

inorganic nutrient salts and supply of electricity. Periodic rot pruning

can minimize interplant root contact: Aeroponic culture has been

reported to be cost effective technology.

32

A technique for the mass production of AM fungi include the

following steps, (Clarson, 2002,a). A trench of one sq.m and depth

30cm is to be prepared, and lined with a black polythene sheet to

make a tube for plant growth. Vemiculite and sterilized soil (10:1) were

mixed and packed in the trench up to 20 cm height. Spread two kg

selected pure AM inoculum (mother culture) approximately 2-5 cm,

below the surface of vermiculite. Sow maize seeds that were surface

sterilized with 5% sodium hypocholrite for two minutes. Applied

sufficient nutrients, at the time of sowing of maize seeds and there

after 30th and 40th days of sowing. Host plants were grown for 60 days.

The inoculum is obtained by cutting all the roots of host plant along

with the medium. It consists of spores, pieces of hyphae, vermiculite,

plant roots etc.

A culture technique for the establishment and maintenance of

vesicular arbuscular mycorrhiza on rhode grass was developed by

Gosal et al., (2003). Here a pot experiment was conducted using p

deficient disinfected loamy sand and soilrite mix soil to study the

establishment and maintenance of Glomus mosseae and G.

fasciculatum on rhode grass as a host. The pots, each of which

contained sandy loam soil or soil soilrite mix soil, were irrigated weekly

with nutrient medium. Mycorrhizal inoculum containing spore

suspension was added, 100 spores pot-1 at the time of sowing. Spore

density in soil and percent mycorrhizal colonization in roots were

studied microscopically at different intervals.

33

The sand based AMF (Glomus fasciculatum) consisting of

mycelial fragments, spores and root bits were multiplied on sorghum

roots can be used for inoculum in chilli nursery. Application of AMF in

nursery furrows were at the rate of 2 kg (m2)-1 observed after 20,30, 40

days of inoculation. AMF in nursery furrows showed maximum

colonization of the roots. Inoculation of chilli plant with 850 g (m2)-1

was most economical (Kavitha et al., 2004).

Mass multiplication of AMF Glomus mosseae was influenced by

plant growth promoting rhizobacteria namely, Azospirillium sp.,

Azotobacter chroococcum, Pseudomonas fluorscens, P. striata and yeast,

Saccharomyces cerevisiae. A pot culture experiment was conducted by

Bhowmik and Singh, (2004) showed the effect. Rhode grass

(Chloris sp.) was the host plant. The grass seeds were surface sterilized

with 0.01% HgCl2 for four minutes washed ten times with sterile

deionised water and dried on blotting paper. AM root colonization

studies were carried out in plastic pots containing sterile soil and sand

(3:1) at the rate of 2kg pot-1. The potting soil at 1 cm depth was

removed from each pot and mixed thoroughly with microbial

preparations (50ml) of different organisms separately and left for light

drying. Twenty holes were made on the surface soil in each pot and 50

sterile Glomus mosseae spores were pipetted into each hole followed by

sowing of one sterile rhode grass seed. Open seeded holes were then

covered evenly with microbial preparation mixed soils to give rise to

individual treatments. After 90 days of sowing, observed. All the

34

treatments increased, percent of mycorrhizal infection and number of

spores. Of the different treatments of Azospirillum enhancing it to the

maximum followed by Pseudomonas fluorescens, Azotobacter

chroococcum, pseudomonas striata.

2.1.6: PHYSIOLOGY OF AM FUNGI

The symbiosis between plants and AM fungi is mutualistic and

necessary for the survival of the fungi. Successful mycorrhizal

formation depends on the presence of appropriate host, fungus and

environmental conditions. There are several physical chemical and

biological factors affecting the development of mycorrhizae.

Physical factors:- Light can strongly affect the development of

mycorrhizae. Shading not only reduces the root colonization and

spore production but also plant responses to VA mycorrhizae

(Gerdemann, 1968). The stimulating effect of light on development of

the VA mycorrhizae has been shown by Furlan and Fortin.(1977).

Daft and El-Giahmi(1978) postulated that day length play an

important role in VA mycorrhiza development.

Higher temperature generally result in greater root colonization

and increased sporulation (Furlan and Fortin,1973). Schenck and

Schroder (1974) observed that the maximum arbuscular development

occurred near 300C, but mycelial colonization of the root surface was

greatest between 28 and 340C. Schenck et al., (1975) reported that

two isolates of Glomus from Florida germinated best at 340C where as

35

one from Washington had an optimum of 200C. According to Black

and Tinker (1979) mycorrhizal infection in agricultural crops in

temperate soil is slow compared to tropical soil, is related to the low

soil temperature. Daniels and Trappe(1980) observed that the

optimum temperature for the germination of Glomus and Acaulospora

spores is around 20-250C where as Gigaspora had a much higher

optima. Bagyaraj (1991,b) reported that temperature has been shown

to have significant influence on colonization and sporulation by

VAM fungi under green house conditions. The Glomus genus was

reported to be dominated in arid and semi arid climates due to its

resistance to high soil temperatures, so Glomus species are in higher

frequency in different samples from North Jordan (Jamil Mohammad,

et al.,2003).

Heinemeyer and Fitter (2004) observed that the growth of

Plantago lanceolata increased with temperature. Specific leaf area

(SLA) and specific root length (SRL) increased independently of plant

size. Percentage of root colonization were also positively correlated.

But the growth of Holcus lanatus was little affected by temperature.

AM occur over a wide range of soil water regimes. Khan (1974)

reported that colonization of VAM in arid regions was noticed in

xerophytes. Bagyaraj and Manjunath (1979) observed in free floating

plants while Clayton and Bagyaraj (1984) in submerged aquatic

plants. According to Redhead (1977), the soil moisture exerts

36

influence on mycorrhizal association. Bowen (1984) reported that

more spore germination occured at soil moisture levels at which roots

did not normally grow. Oxygen concentration can inhibit VAM spore

germination and root colonization, (Saif, 1981).

Chemical factors:- According to Baas Becking et al., (1960) soil pH

of the world cover the range of 2.8 to 10 and soil pH may affect the

distribution of mycorrhizae in a subtle way. Daft et al., (1975)

observed considerable AM colonization in plants growing in a mine

soil of pH 2.7. Sparling and Tinker (1978) found no obvious effect

of pH on infection in three grassland sites at pH 4.9, 5.9 and

6.2. Lambert and Cole (1980) reported six isolates of Glomus tenue

differed in their ability to form mycorrhizae at low pH. According to

Daniels and Trappe (1980), more than 40% germination of Glomus

epigeum spores were found in soil over the pH range between 4.8-

8.0, the optimum being 7.0. The histochemical experiments of Martin

and Guttenberger (2000) revealed that the staining of arbuscules was

based on the ion –trap mechanism, which indicates acidic membrane

bound compartments that is arbuscules of vesicular arbuscular

mycorrhizal fungi inhabit an acidic compartment within plant roots.

Bowen (1980) found typical AM colonization and spores in a

soil with more than 5000 ppm of chloride. But Gildon and Tinker

(1983) reported that sodium and chloride ions inhibit spore

37

germination of AM fungi. Sengupta and Chaudhuri (1990) observed

AM fungi occur naturally in saline soils.

Jasper et al., (1979) reported that in virgin as well as cultivated

soil, addition of phosphorus was associated with decrease in

mycorrhizal development. The endophyte population of each soil differ

in their sensitivity to phosphorus supply. The proportion of root

volume infected decreased with high rates of application of

phosphorus to the soil. Soil without phosphorus application showed

55% colonization, where as two gm phosphorus application showed

only 18% infection . It is assumed that phosphorus influences AM

colonization by affecting concentration of root carbohydrates. Graham

et al., (1981) reported that phosphorus influences AM colonization

by affecting the amount of root exudates.

According to Bolan et al., (1984) adding phosphorus increased

both root growth and percentage of root length infected by

mycorrhizal fungi. At higher rates of phosphorus addition, there was

a decrease in percentage of root length infected. Sreenivasa and

Bagyaraj (1989) observed that rock phosphate applied at 100 ppm

phosphorus level resulted in more of infective propagules of Glomus

fasciculatum. Nagahashi et al., (1996) reported that addition of

phosphorus as K2HPO4 affected growth of hyphae from pre

germinated spores of percentage of AMF infection of the roots was

Gigaspora margarita. Phosphorus at one mM significantly decrease

38

the number of branches rising from the primary germ tube without

affecting the germ tube length, total hyphal growth or number of

auxillary cells. Phophorus at 10 mM inhibited both hyphal growth

and branching .

Jarstfer et al., (1998) observed that sporulation was depressed

by addition of magnesium to the nutrient solution. With the higher

level of MgSo4, the average number of spores per plant was four fold

lower than the base line level. Mean root colonization was very low.

Mg application depressed tissue calcium levels with lower calcium in

the tissues, colonization was reduced from 30% of root length to

10% and sporulation from 1200- 200 spores per plant.

Eason et al., (1999) stated that root colonization by AMF and

AMF spore density were significantly lower in grass land and soils

with a history of high input, conventional management, than in soils

with a low input or organic management. The percentage of AMF

infection of the roots was approximately one–third greater than AMF.

Spore density in the soil was approximately three times higher

under organic than conventional.

According to Brundrett et al., (1999) changes in soil nutrient

levels especially phosphorus had a large impact on plant growth

,substantially increasing root growth for both sorghum and clover.

For clover mycorrhizal formation expressed as a proportion of root

length reached at a peak at intermediate nutrient levels; but the

39

total root length was substantially greater at the highest fertility

levels .This resulted in increase in the overall length of mycorrhizal

roots at these nutrient levels. But spores were not numerous at any

nutrient levels.

Ruiz-Lozano and Azcon (2000) observed the effect of salinity on

mycorrhizal growth. Mycorrhizal colonization positively affected plant

growth at all the salt levels. Under low salinity, mycorrhizal effect on

growth was 52% in Giomus deserticola colonized plants, while it was

insignificant for plants colonized by other Glomus species. At highest

salt level, the mycorrhizal effect on shoot biomass production was

100% in Glomus deserticola colonized plants and 82% in other

Glomus sp. colonized plants. In contrast, increasing the salinity did

not affect the growth of control plants. Increasing salinity reduced

the root weight by 47% in control plants and by 21% in G. deserticola

colonized plants .In contrast 26% in Glomus species colonized plants.

The increase in salinity of soil decreased the hyphal growth and or

viability of Glomus species to a higher extend than those of Glomus

deserticola.

Hayman (1975) showed that nitrogen fertilizers had a large

negative effect on mycorrhizal population. Alexander and Fairly

(1983) reported reduction in mycorrhizal colonization following the

application of 300 kg N/ ha as (NH4)2SO4. Menge (1984) noted that

daily fertilization of citrus with more than 100 ppm N as a mixture

40

of NO3- and NH4+ retarded mycorrhizal development. Davis and

Young (1985) reported nitrate salts have more inhibitory effect to AM

development than ammonium salts.

Bagyaraj (1991,b) reported that Nitrogen levels with greater

than 80 ppm decreased the number of infective propagules. The

demand of AM fungi for elemental nutrients such as NPK, Ca and

Mg is low, (Sieverding,1991,b).

Jackson et al., (2002) reported that in lettuce percentage

of mycorrhizal root colonization decreased with increasing P

concentrations ie: under no addition of P, 79.8 % colonization where

as under 0.25mMol P Kg-1 colonisation become decreased to 60.6%.

number of arbuscules and vesicles also decreased. In wild lettuce,

the results are more or less similar. Plant responses to two levels of

N also showed decreased levels of mycorrhizal colonization at higher

levels of nitrogen. But plant dry weight was found to be higher with

higher N supply.

Micronutrients such as manganese and zinc inhibit spore

germination of mycorrhizal fungi, (Hepper,1979). Gildon and Tinker

(1983) reported that Zn and Cu were shown to inhibit mycorrhizal

colonization in clover, onion, maize and soybean.

Sieverding (1991,c) estimated that AM fungi require 1-17% of

the carbohydrate which the plants submit for root biomass

production for the development and functional activity. AM fungi

41

need carbohydrate for spore production, spore dry matter in the field

can vary from 10-1000 Kg/ha. (Sieverding et al.,1989).

Organic matter influences soil structure, pH, nutrient profile

and water holding capacity; all of which may directly or indirectly

influence AM development and efficiency (Bagyaraj,1991,b). Redhead

(1977) suggested that the seasonal die back of Sudan Sahel Savanna

grasses could stimulate endogenous spore production, thus

increasing spore populations .as observed when arable crops such as

maize, barley and wheat are harvested. Rives et al., (1980) also

suggested that in areas with low annual rain fall, contact between

colonised root debris and roots of uninfected plants may constitute

the most efficient mode of mycorrhizal spread.

Bio-accumulation of phenanthrene, one of most abundant

polycyclic aromatic hydrocarbon in the environment significantly

decreased the colonization of maize plant in an experiment conducted

by Gaspar et al.,(2002).

Addition of chitin improved the colonization of roots and growth

of AMF and spore production were also significantly stimulated and

percentage of alkaline phosphatase also increased (Gryndler et al.,

2003).

42

Biological factors

Fester et al., (1999) reported that three essential stages of AM

fungal life cycle could be followed in different parts of the wheat roots.

Stage one was charecterised by appressoria formation and by

predominance of arbuscules. This could be localised in all of two

week old root systems. This stage was mainly observed in young

parts of secondary roots. Stage two was characterised by a

predominance of vesicles, observed in older parts of the root branches

and the main roots. In stage three, spore formation was predominant.

The fungus produced massive amounts of spores, enclosing the

vascular cylinder of root parts older than five weeks.

The presence or absence of host plant obviously play an

important role in colonization and sporulation of AM fungi. Non host

plants such as Chenopodiaceae and Brassicaceae can become

minimally colonized by AM fungi (Kruckelmann,1975). The influence

of non host plants on the colonisation of host plants has been studied

with contradictory results. The presence of non mycorrhizal plants

has resulted in reduced colonization of mycorrhizal host plants

possibly because of toxic non host exudates, (Iqbal and Qureshi,1976

and Glenn,1982). Incontrast, Ocampo et al., (1980) detected no

reduced colonization of mycorrhizal plants cropped together with

nonmycorrhizal hosts .

43

Schenck and Kinloch (1980) reported that crop species itself

can exert a selective effect on which AM species in mixed indigenous

population become predominant. They found marked differences in

population of AM fungi between different crops grown for seven years

in monoculture. Three species of Gigaspora were most prevalent in

association with soybean roots, in contrast to cotton and pea nut,

where two species of Glomus were most numerous. The largest

number of AM species were found with sorghum.

AM fungi have been observed colonizing the root surface of

non hosts, there by procuring an ecological niche in which it can

survive in the absence of host root (Bevege and Bowen,1975; Ocampo

et al.,1980).

St. John (1980) reported that one possibility of variation in

mycorrhizal dependency of different host plants in that, plants with

coarse and relatively fewer hairs are more dependent on mycorrhiza

compared to these plants with fine roots and long root hairs. Krishna

et al., (1985) stated that mycorrhizal colonization could be a genetic

trait .Like resistance or susceptibility of crop plants to infection by

fungal pathogens, the extend of root colonization is a plant heritable

trait.

Reduction in plant size by pruning and defoliation of plants can

decrease mycorrhizal root colonization and sporulation (Sreenivasa

and Bagyaraj,1988).

44

Although AM fungi have extremely wide host ranges the

existence of host preference has been suggested by Mosse (1975) and

Bagyaraj et al., (1988). Powell et al., (1980) reported that species and

strains of AM fungi have been shown to differ in the extend to which

they increase nutrient uptake and plant growth. They vary in their

physiological interactions with different plants and hence in their

effects on plant growth. Generally those fungi that infect and colonise

the root system more rapidly are considered to be ‘efficient’ strains or

‘effective’ strains, (Munns and Mosse,1980; Abbott and Robson,

1981).

The ability to form extensive external hyphae and the ability to

absorb phosphorus from soil solution and to improve plant growth

are also important in determining the effectiveness of AM fungi

(Abbott and Robson,1982; Bagyaraj et al., 1988).

Spore dormancy in AM fungi was first reported by Tommerup

(1983). Acaulospora laevis spores are dormant for about six months.

Gigaspora calospora for six to twelve weeks; and Gigaspora

decipiens,Glomus caledonicum and Glomus monosporum for a few

days. Dormancy periods were much shorter in dry soils than in moist

soils. Short dormancy periods could prevent spores from germinating

immediately after formation around the root. Long dormancy may

protect spores against early false breaks in a season. Dormancy can

be broken by dessication or cold treatment, (Gemma,1987).

45

Of the various microorganisms colonizing the rhizosphere,

AM fungi occupy a unique ecological position, as they are partly

inside and partly outside the host. The part of the fungus within

the root does not encounter competition with other soil

microorganisms.Bagyaraj (1984) and Lindermann (1988) reported

about the different aspects of biological interactions of AM fungi. AM

markedly improve nodulation and nitrogen fixation by legume

bacteria mainly by providing the high phosphorus requirement for

fixation process. Mycorrhizal colonization allows introduced

populations of soil organisms like Azotobacter and phosphate

solubilising bacteria to maintain higher numbers than around

nonmycorrhizal plants and to exert synergistic effects on plant

growth. Bagyaraj (1984) reviewed various mechanisms of suppression

of pathogens by AM. Secila and Bagyaraj (1987) suggested another

possible mechanism of suppression of root pathogens by AM fungi, by

stimulation of antagonistic actinomycetes in the rhizosphere.

The influence of mixed AM fungal species on root colonization

and sporulation, i.e. their synergistic or competitive effects on each

other were also studied. Studies of competitive ability of AM fungal

species came from Ross and Ruttencutter (1977) who found that less

colonization occurred in Glomus macrocarpum inoculated with

peanuts and soybeans than when these hosts were inoculated with

Gigaspora gigantea and Glomus macrocarpum together. The

46

percentage of Glomus macrocarpum colonization as measured by

vesicle formation was reduced when combined with Gigaspora

gigantea, suggesting the better competitive ability of as Gigaspora

gigantea

The competitive ability of Glomus tenue (Powel and

Daniel,1978) use of techniques like fluorescent antibody technique

(Kough et al., 1983), enzyme linked immunosorbent assay or ELISA

(Aldwell and Hall, 1986) and gel electrophoresis (Hepper, 1987)

helped to understand the competition between different AM fungi in

colonising a root.

According to Praveen kumar and Bagyaraj (1998), the

mycorrhizal parameters were enhanced when Glomus mosseae was

inoculated together with Bacillus coagulans and Pseudomonas

flourescens. But the fungus Trichoderma harzianum did not seem to

enhance mycorrhizal colonization and sporulation. Co-inoculation of

Glomus mosseae with both these plant growth promoting

Rhizobacteria can be used for the mass production of Glomus

mosseae inorder to get more infective propagules of AM fungus. That

is a synergistic interaction was observed when Glomus mosseae was

co-inoculated with these two Rhizobacteria.

Relationship between soil aggregation and mycorrhizae after

inoculating Bradyrhizobium and fertilizing with various nitrogen

sources were studied by Bethlenfalvay et al., (1999). The water stable

47

soil aggregates was correlated positively with root and AM soil

mycelium development. Soil arthropod numbers were negatively

correlated with AM hyphal length. The numbers of soil invertebrates

(nematodes) and protozoans did not correlate with bacterial count

or AM soil hyphal length. AM fungi and roots interacted as the

factors that affect soil aggregation, regardless of nitrogen nutrition.

Agrobacterium rhizogenes, Pseudomonas flourescens and

Rhizobium leguminosarum, when co-inoculated with Glomus

intraradices enhanced the fungal colonization upto 60%, 40%, and

50% respectively, but Glomus intraradices alone was only about 20%

in Barley roots. ie these three species of potential MHB tested on

barley roots, increased mycorrhiza levels two to three fold

(Fester et al., 1999). Angela (2000) reported that some of the

mycorrhizal interactions such as grazing of the external mycelium are

detrimental, while others including interactions with plant growth

promoting rhizobacteria promotes mycorrhizal functioning. Following

mycorrhizal colonization the functions of the root become modified.

Studies of Vierheilig et al., (2003) showed that the inhibitory

effect of root exudates of Brassicaceae (mustard) and chenopodiaceae

(sugarbeet) on AMF is at the level of root colonization. Mustard and

sugar beet apparently reduced root colonization by AMF in cucumber

at least partially through inhibitory compounds in their exudates,

48

root exudates of lupin had no effect on cucumber root colonization

2.1.7: FUNCTIONS OF AM

Under natural field conditions, almost all crop plants are

infected by AM fungi, but it is difficult to demonstrate the function of

AM fungi in the field. Most results on the function of AM are from

experiments conducted under artificial and controlled conditions in

the laboratory or greenhouse.

Plants with profusely branched root system having fine rootlets

of less than 0.1mm in diameter and long root hairs ie graminoid

roots are less dependent on AM than those with coarse roots. ie

magnoloid roots, having rootlets of more than0.5 mm in diameter

(Baylis,1975).

Several field, laboratory and greenhouse experiments

have demonstrated that VA mycorrhizal inoculation can

greatly improve growth and nutrition of host plants, (Mosse,1973;

Sanders et al., 1977).

The phosphate absorbed by AM fungi from soil is mainly

accumulated in the form of polyphosphate granules. This represents

nearly 16-40% of total P in AM fungi and are propelled through

hyphae by cytoplasmic streaming to arbuscules and then broken

down to inorganic phosphate by specific enzymatic activities, (Callow

et al.,1978). This will results in the ultimate release of phosphate ions

into the plant cytoplasm. Bagyaraj and Manjunath (1980) reported

49

that inoculation of cowpea with Glomus fasciculatum increased their

shoot and root dry weight besides increasing the phosphorus and

zinc content of inoculated plants.

Inoculation of barley with a mixture of Glomus mosseae,

Glomus fasciculatum and Gigaspora margarita stimulated seed yield

by 27% and seed phophorus content by 37% (Powell,1981).

Plants with arbuscular mycorrhizal association are capable of

mobilizing more of the available nutrients from the soil. This is

because AM can significantly modify the overall nutrient uptake

properties of a plant root system through its external mycelium

extending to several centimeters from root surface often beyond the

rapidly developing nutrient depletion zone around an active root

system, (Hayman , 1982).

The specific activity of phosphorus is significantly higher in the

mycorrhizal plants than in the non mycorrhizal plants. Formation of

mycorrhiza increased total P uptake, but did not enable seedlings to

use relatively more non labile P. On the contrary relatively more P

was found in non mycorrhizal seedlings (Thomas et al., 1982).

Cooper (1984), reported that AM fungal hyphae were not able to

extract other elemental nutrients from the soil solution than those

which could be taken up by the non mycorrhizal root. Hence the

principal function of mycorrhiza is to increase the soil volume

50

explored for nutrient uptake and to enhance the efficiency of nutrient

absorption from the soil solution .

Bolan et al., (1984) reported that when inoculated with a

mycorrhizal fungus, the roots of subterranean clover plants were

heavily infected. Control plants were uninfected. Mycorrhizal plants

took up more ‘P’ than nonmycorrhizal plants, at all levels of addition

of phosphate and iron oxide. For mycorrhizal plants, addition of iron

oxide had little effect on the amount of ‘P’ in the shoots. In contrast,

for non mycorrhizal plants adding iron oxide decreased the amount

of ‘P’ in the shoots .For these plants there was no increase in the

‘P’ content in the shoots until large amounts of phosphate had been

added .

According to Peterson et al., (1984), mycorrhizal fungi

associated with plant roots increased the absorption of nutrients,

particularly phosphorus and thus enhanced the growth of crop plants

and trees. These symbiotic associations are important in crop and

biomass production.

Mycelial linkage between plants may be an important

mechanism of nutrient transfer between plants. The absence of

nutrient transfer from a mature host to a young non host indicates

the inability of the atypical infection of non host plants to take up

nutrients. The direction of nutrient transfer seemed to be depend on

the nutrient status of the plant, ( Ocampo,1986).

51

According to Maria and Justo (1988), plant growth increased

and AM infection decreased significantly with increasing ‘P’ doses in

acid soils. Plants mainly utilized inorganic ‘P’ of the soil fraction but

no absorption of ‘P’ from the organic ‘P’ fraction. AM root infection

was greatly diminished by the lowest ‘P’ dose. Most AM root infection

was found when no ‘P’ was added. The highest ‘P’ dose did not result

in a significantly increased yield .

Bagyaraj and Manjunath(1980) observed that when crops like

cowpea, cotton and ragi were inoculated with Glomus fasciculatum,

it enhanced the uptake of Phosphorus and Zinc. Raj and Bagyaraj

(1981) studied the phosphorus uptake from 32p labelled tricalcium

phosphate and superphosphate and concluded that mycorrhizal

plants produced more dry matter and removed more 32p from the soil

than non mycorrhizal plants .Johnson and Hummel (1985) reported

an increased leaf phosphorus content upon AM inoculation in citrus

seedlings. The beneficial effect of AM on plant growth has mostly

been attributed to an increase in the uptake of nutrients especially

phosphorus, (Hetrick,1989).

Seedlings of Acacia nilotica and Calliandra calothyrsus

inoculated with AM fungi had significantly higher phosphorus

content when compared to uninoculated control (Reena and

Bagyaraj,1990).

52

Shashi et al., (1990) reported that the responses of various

parameters in mycorrhizal plants were dependent on plant age and

phosphorus level. Higher rates of growth have been reported in

mycorrhizal plants than in non mycorrhizal plants on the soils having

low in available phosphorus. Root and shoot fresh weight responded

positively to control mycorrhizal plants and to low level P applicated

mycorrhizal plants at 38 DAS but negatively at 50 DAS with high

level P.

According to Morton et al., (1990), root dry weight of

mycorrhizal plants increased as P increased in soil. Colonisation by

Glomus diaphanum stimulated plant growth and nitrogenase activity

above that of non mycorrhizal controls. Mycorrhiza induced

enhancement of nodulation and nitrogenase activity with increase in

dry weight and N and, P uptake.

Sivaprsad et al., (1990) reported that percentage of infection of

AM varied with their crops and cultivars. Maximum colonization was

in sweet potato cultivars which ranged from 28 – 54%, followed by

Cassava with 22 – 42% and in dioscorea it was 17 – 22%. The

population of spores in the rhizosphere varied with crops as well as

cultivars. Rhizoshere spore count was higher in cassava and sweet

potato. The highest spore count being 25–26/g soil in Cassava

cultivar sreevisakh and lowest was eight in Dioscorea alata cultivar

53

Kaduvakkaiyan spore count was relatively less in cultivars of

Dioscorea and Coleus.

Bolan (1991) reviewed the role of mycorrhizal fungi in the

phosphate physiology of host plant and summarized the mechanisms

for increased uptake. That include exploration of larger soil volume,

faster movement of phosphorus into mycorrhizal hyphae and

solubilisation of soil phosphorus.

Sieverding (1991,a) reported that the P concentration in the soil

solution is very low in tropical soils and the soil around the growing

root is rapidly depleted of P ions within a distance of a few millimeter.

Due to the extremely slow diffusion rate of P, this zone can not be

adequately replenished with it. VAM mycelium grows far beyond this

zone and increases the soil volume which is exploited for P uptake.

The uptake must be brought about due to the lower P concentration

in the soil solution than in the fungal hyphae. P is absorbed in the

form of orthophosphate and transported actively in the hyphae as

polyphosphate.

AM fungi are not always equally infective to any one plant

species and they certainly vary in their physiological interactions

with different plants and hence in their effect on plant growth. Those

fungi that infect and colonise the root system more rapidly are

considered to be efficient strains. ( Peter and John,1991)

54

Habte and Aziz (1993) reported that Leucaena leucocephala

inoculated with AM exhibited increased uptake of phosphorus. AM

fungi help in the selective absorption of immobile P, Zn, and C and

mobile S, Ca, K, Fe, Mn, Cl, Br, and N elements to plants. It reduces

the soil stresses. They increase the resistance in plants and with

their presence reduce the effect of pathogens and pests on plant

health. In general, plant health, growth, survival and yield are

increased due to AM colonization, (Dubey, 1993).

Potassium and Magnesium are often found in higher

concentrations in mycorrhizal than non mycorrhizal plants.These

elements are more mobile in soil solution than P, (Sieverding,1991,b).

Micronutrients such as Zn, Cu, S, B and Mo are proven to be actively

taken up by fungal hyphae and transported to the host plant. Some

heavy and often toxic metals Cd, Ni, Sr, Cs, and some nutritional

anions, Br and I are known to be taken up and transported to the

host by AM fungal hyphae.

Joner and Jacobson (1994) recovered 5.5 and 8.6% of 32p from

cucumber plants colonized by Glomus spp and Glomus caledonium

respectively but only 0.6% for the non mycorrhizal control.

Azcon and Barea (1992) reported that there was a significant

increase in N and P uptake of alfalfa plants inoculated with

Rhizobium meliloti and AM fungi. Improved plant growth, nodulation

and nitrogen uptake were observed in AM inoculated plants of Acacia

55

and Eucalyptus compared to uninoculated control, (Michelsen and

Sprent,1994). AM fungus increased the Potassium content in neem

seedlings, (Karthikeyan,1994).

Rough lemon and trifoliate orange inoculated with AM showed

higher iron concentration in comparison with non mycorrhizal plants,

suggesting the role of mycorrhiza in host iron nutrition,

(Treeby,1992).

Warburton et al., (1993) observed that Eucalyptus plants

inoculated with AM fungi had increased levels of Ca and Mg in roots

and shoots when compared to control. Michelsen (1993) reported that

Glomus mosseae increased Mg uptake in Acacia abyssinica, Glomus

fasciculatum increased the uptake of Zn, Fe, Cu, and Mn in neem

seedlings, (Karthikeyan, 1994). There was a significant increase in Zn

uptake and utilization in neem seedlings which are inoculated with

Glomus constrictum, (Anandan,1996).

Miroslav (1995) observed positive growth response in onion by

most of AM inocula and concentrations. The best endophyte was

Glomus etunicatum, which in all concentrations significantly

increased the growth of plants. The plants inoculated by Glomus

etunicatum and Glomus intraradices showed increased bulb biomass

23-43 % over the control. The bulb fresh weight of non inoculated

plants was the lowest. The average enhancement of bulb biomass was

61% for Glomus intraradices and 47% for Glomus vesiculiferum.

56

Glomus intraradices inoculation increased the bulb yield 45% higher

than control.

Mycorrhizae offer resistance to host plant against heavy

metals. Senthil kumar and Arockiasamy (1995) studied the

distribution of Zn in the leaves and roots of sorghum bicolor in the

presence and absence of AM fungi. In the absence of AM, about 18%

to 35% of the Zn content was present in the root system. Thus in

normal conditions most of the heavy metals that are absorbed are

transported to the leaves, (82% and 65% respectively). In the

presence of AM, in the root, there was 32 % to 60% of Zn. The

differences observed in the heavy metal accumulation in the roots

due to the presence of AM fungi was found to be significant. At higher

concentrations of Zn treatment, Zn was relatively higher in roots than

in leaves .Root protein was significantly more in the presence of AM,

in plants treated with heavy metal. Thus synthesis of newer proteins

that could bind the heavy metal and retain in the root itself giving

heavy metal resistance to plants due to the presence of AM fungi.

Acid lime is a slow grower and it needs VA mycorrhizal fungi for

better growth. Plants inoculated with Glomus macrocarpum, Glomus

mosseae and Glomus caledonicum had significantly greater plant

girth compared to uninoculated plants. Plants inoculated with

Glomus macrocarpum produced maximum shoot biomass followed by

Glomus mosseae and Glomus fasciculatum. Maximum root biomass

57

was produced in plants inoculated with Glomus macrocarpum

followed by Glomus mosseae. In general plants inoculated with AM

fungi had higher plant height, number of leaves, leaf area, plant girth

,shoot and root biomass and P content, (Reddy et al.,1996).

Bhardwaj and Dudeja (1998) reported that soil samples from

rhizosphere of pigeon pea when used as AM inoculam showed more

AM colonization but were not efficient in P and N uptake than the

standard AM inoculants of Glomus mosseae and Glomus

fasciculatum.

According to Sharma et al., (1999) P uptake capacity of

mycorrhizal plants was 1.83 and 1.61 fold higher than non

mycorrhizal plants under low and high P supply respectively. During

the early duration P accumulation by root and its translocation to

stem was greater and more metabolically dependent in mycorrhizal

than non mycorrhizal plants under low P supply. At a later phase, P

accumulation in roots of mycorrhizal plants remained highly

metabolic but the translocation to stem and leaves became much less

dependent on cell metabolism.

Rajan et.al., (2000) studied that mycorrhizal inoculation

resulted in a significant increase in plant height, stem girth, plant

biomass and plant phosphorus content of teak seedlings. Seedling

raised in the presence of Glomus leptotichum showed an increase of

130%,113.5% and 149% in tissue phosphorus, Zinc and Copper

58

contents respectively, as compared to seedlings raised in the absence

of inoculation. These nutrients significantly higher in seedlings

inoculated with Glomus leptotichum followed by Glomus macrocarpum.

Khaliq and Sanders (2000) studied the effect of inoculation of

Glomus mosseae and phosphrus fertilizer, on the mycorrhizal

colonization of roots, crop yield and P uptake, of barley in a natural

and fumigated soil. Inoculated treatments had significantly higher

mycorrhizal infection than un inoculated treatments in nonfumigated

as well as fumigated soil. The plots given phosphate had lower

amounts of infection than their respective control plots not supplied

with phosphate.

Elizabeth and Alan (2000) studied the effect of three

commercial arbuscular mycorrhizal fungi, Vaminoc, Endorize IV and

Glomus intraradices on potato microplants. The Vaminoc and

Endorize treatments promoted flowering 80% and 76% respectively at

two months after planting where as control plants only 60%.

Mycorrhizal inoculation influenced the yield quality of potato

microplants. The percentage of seed size tubers, were 27% of

Endorize tubers, 24% of Vaminoc tubers, 21% of Glomus intraradices

and 20% of control microplants .

The efficiency of eight arbuscular mycorrhizal species collected

from rhizosphere soil of Moringa concanensis was tested, (Jitendra

and Anil,2002). According to their observations, the overall growth

59

and nutrient content of mycorrhizal plants were higher as compared

to control plants. Different AM fungi varied in their influence on test

plant Gigaspora margarita resulted in more than 50% increase in

shoot and root dry weight and more than two fold increase in uptake

of P and N. Mycorrhizal colonization resulted in increased

accumulation of nutrients, chlorophyll, carotenoids, sugar and

proteins. Among the eight AM fungi used, Gigaspora margarita proved

to be the most efficient.

Studies of Karasawa et al., (2002) on crop rotation have

suggested that cultivation of mycorrhizal host crops increases the

arbuscular mycorrhizal colonization of succeeding crops. Growth

and P uptake of succeeding maize were significantly affected by the

preceding cropping. Shoot weight and P uptake of maize after sun

flower cropping were significantly greater than those after cultivation

of mustard plants. The AMF inoculum from the soil after sun flower

cropping improved the growth and AM colonization of maize and

shoot weight was increased from 17–49 % of that in the soil after

sunflower cropping without inoculum. Anthoniraj (2002) reported

that mycorrhizal hyphae extend far away from the roots, absorbing

water, mobilizes phosphorus and micronutrients. VAM mobilizes P by

absorbing it far away from the roots and transport to the roots. Along

with P uptake Zn, Cu, S, Mg, Fe etc. are also taken up by mycorrhizal

plants.

60

Martin et al., (2002) investigated the influence of olive mill dry

residue on the growth of soybean and lettuce, colonized with the

arbuscular fungi, Glomus mosseae and Glomus deserticola. As the

residue contained soluble phenolic compounds, it decreased the

growth of plants colonized with AM fungi. These results showed that

AM fungi increased the phytotoxicity of olive mill dry residue in

lettuce and soybean. The application of residue deceased the

percentage of colonization of AM in plants except those were

inoculated with Glomus mosseae or Glomus deserticola four weeks

before the application of the residue.

Choudhury et al., (2002) reported that AM fungi inoculated in

the seedlings of assam lemon showed better growth response than

the uninoculated seedlings. Phosphorus uptake was also more in the

inoculated seedlings compared to the un inoculated seedlings. The

increase in weight of shoot and root, and nutrient content of

mycorrhizal plant over control was a result of absorption of higher

amount of P from the low P containing soil. Higher amount of P

uptake may be the result of higher activity of acid phosphatase, as

the total and the specific activity of this enzyme in root extract from

the inoculated seedlings was found to be significantly higher than

uninoculated control. The total and specific activity of enzyme alpha

mannosidase was also significantly more in AM inoculated seedlings

than in the un inoculated seedlings. This glycosidic enzyme was

61

found to help in the break down of complex polysaccharides into oligo

saccharides.

Aboidun and Oluwole (2002) discovered that when AM

agrotechnology was introduced, the crop yield was increased with

every year of cultivation. Anil kumar and Muraleedhara kurup (2003)

reported that the association of AM enhanced the leguminous plants

to withstand the various stresses to some extent. The nutrient uptake

level under drought and different levels of salinity were studied.

Among the three leguminous plants studied, the mycorrhizal Cicer

arietinum showed more growth rate. It was found that the growth rate

of mycorrhizal plants increased according to the increase in the level

of salinity and percentage of mycorrhizal infection. Under drought

stress conditions, the Vigna sinensis showed more nitrogen content

than the other two legumes. Phaseolus aureus showed more nitrogen

uptake in low and normal salinity stresses. It was noted that the

percentage of mycorrhization was directly proportional to the growth

and nutrient uptake levels under stress conditions.

Rubio et al., (2003) reported that the inoculation of Glomus

etunicatum, increased significantly the extent of P plant acquisition,

spore number, length of extra radical mycelium and phosphatase

activity, when compared with indigenous AM fungi fertilized with

partially acidulated rock phosphate.

62

According to Azcon et al., (2003), the availability of N and P in

the soil reduced the content of macro and micro nutrients in AM

plants. At the lowest P application the AM colonization increased

nutrient acquisition at different N levels. The highest application of N

and P to the soil reduced the uptake of N,P,K, Mn, and Zn in AM

compared to non AM lettuce plants. AM colonization increased

specific absorption rate values at the lowest N and P levels for nearly

all nutrients.

Reiko and Katsuya (2003) reported that pea nuts and pigeon

pea utilized non labile P sources more efficiently than other plants

due to their mycorrhizal association. Without mycorrhizal inoculation

both peanuts and pigeon peas showed poor P uptake. P uptake

responses were significant and similar for different P sources like

Al-P, Fe-P and Ca-phytate in pigeon peas. But in pea nuts the

response was significant for both Al-P and Fe-P but not for

Ca-phytate.

Feng et al., (2003) observed about the contribution of AM fungi

to utilization of organic sources of phosphorus. Inoculation of

Glomus versiformae increased shoot and root P concentrations and

this mycorrhizal effect become more pronounced as plant growth

proceeded. Contribution of AM fungi to plant P nutrition by absorbing

from the organic P pool indicated that AM fungi may play an

important role in cycling of organic P in soil. The P in organic

63

compounds can become available to plants after hydrolysis by

phosphatase enzymes. It has been suggested by Joner and Johansen

(2000) that AM fungi are able to produce acid phosphatase that is

released into the soil.

VAM colonization patterns are very much related to soil

pH, phosphatase activity, and phosphorus content of soil

(Lakshmipathy et al., 2003). AM colonization was more in acidic soil

compared to neutral and alkaline soils. Soils with a total P content of

six to seven mg/kg soil supported higher AM colonization. Acid

phosphatase activity was more in the root zone soil where the AM

colonization was more and alkaline phosphatase activity was less.

Mycorrhizal colonization increased the phosphatase activity in root

zone soil and altered the Phosphorus supply to plants.

Tarafdar and Marschner (1994) reported that inoculation of

AM increased the acid and alkaline phosphatase activity. That is the

rhizosphere soil of wheat plants inoculated with Glomus mosseae

showed increased alkaline and acid phosphatase activity. Compared

to the control prior to planting, in the rhizosphere soil acid

phosphatase activity was increased 14–18 fold and alkaline

phophatase activity1.6–2.1 fold.

Peter et al., (2004) reported that AM fungi can exert protective

effects on host plants under conditions of soil metal contamination.

Mycorrhizal infection enhanced metal uptake by roots. In highly

64

contaminated soils metal concentration were enhanced in the roots

but decreased in the shoots compared with non mycorrhizal plants. It

was reported that AM fungi decreased the metal phytotoxicity

particularly Zn toxicity by both direct and indirect mechanisms.

Binding of metals in mycorrhizal structures and immobilization of

metals in the mycorrhizosphere may contribute to the direct effects.

Indirect effects may include the balanced plant mineral nutrition

especially P nutrition due to mycorrhizal colonization, leading to

increased plant growth and enhanced metal tolerance.

Hosamani et al., (2004) demonstrated the response of Withania

somnifera plants to different AM fungal inoculation. Inoculated plants

with Glomus fasciculatum, Scutellospora reticulata, Glomus mosseae

and Glomus macrocarpum generally showed greater plant height,

biomass production and nutrient uptake compared to un inoculated

plants. There was a significant increase of phosphorus in

mycorrhizal inoculated plants with Glomus fasciculatum and Glomus

mosseae But there was no positive correlation between plant growth

parameters and mycorrhizal colonization.

According to Geeta, (2004) agricultural importance of vesicular

arbuscular mycorrhizal fungi is mainly due to their ability to increase

the phosphate uptake and their major and minor nutrient

requirement of crop plant. Inoculating crop plant with vesicular

arbuscular mycorrhizal fungi alone or in combination of

65

phosphate solubilising microorganisms can lead to more economical

use of costly inorganic fertilizers and better exploitation of

inexpensive biofertilizers.

The effect of Glomus fasciculatum on foxtail millet was studied

by Laxman et al., (2004). The response of foxtail millet to indigenous

Glomus fasciculatum was moderately influenced. The introduced

Glomus fasciculatum influenced very significantly the growth. The

plants have greater height, biomass production, root shoot ratio and

P content in shoots compared to the un inoculated controls. The

phosphorus contents of the shoots were significantly higher than un

inoculated plants. Higher root colonization allows host fungus

contact and exchange of nutrients, which helps in better growth of

the plants with introduced Glomus fasciculatum, more effective than

the indigenous Glomus fasciculatum in the soil.

According to Chandra et al., (2004) AM fungi enhanced the

uptake of macronutrients such as K and S and micronutrients such

as Cu and Zn. Mycorrhizal colonization affected hormone

accumulation in host tissues with changes in the levels of cytokinin,

abscissic acid and Gibberellin like substances. These changes altered

the biomass. AM infection reduced the susceptibility or increased the

tolerance of the host to certain soil born pathogens.Mycorrhizal

colonization also enhanced the nodulation and nitrogen fixation in

legumes.

66

Rohyadi et al., (2004) reported that the effects of AM

inoculation on growth of cowpea differed with AM fungal species and

pH of the growth medium. Gigaspora margarita increased both shoot

and root weights of plants compared to non mycorrhizal plants and

plants colonized by Glomus etunicatum irrespective of pH, but

increased root/ shoot ratios. No significant effect of Glomus

etunicatum in increasing plant growth was observed at any pH level

tested. But colonization of AM exceeds 30% in all treatments.

Increased root colonization by Gigaspora margarita was positively

correlated with increased growth response of the plants, based on

significant differences in dry weight between inoculated and non

inoculated plants. Gigaspora margarita treatments had higher P

concentrations in shoots at pH 5.2. Total uptake of P, Fe, Mn and

Zn was consistently increased in plants colonized by Gigaspora

margarita at all soil pH values.

Sorensen et al., (2005) reported that a cover crop of black

medic established the previous autumn increased the colonization of

leek roots by mycorrhizal fungi. But cover cropping did not

significantly increase nutrient concentration or growth.

2.1.8: RESPONSE STUDIES OF AM FUNGI

Experiments have demonstrated that mycorrhizal colonization

can greatly influence growth and nutrition of plants. Kuo and Huang

(1982) reported that inoculation of Glomus fasciculatum increased

67

seed yields and other agronomic traits of soybean plants. Inoculation

of soil with AM fungi has resulted in greater growth and dry matter

yield of winter wheat and upland rice, (Hetrick and Bloom,1983;

Ammani et al.,1985).

Inoculation of Glomus fasciculatum to maize crop improved

their growth and phosphorus nutrition (Mohan et al.,1984). Bala and

Singh (1985) reported that growth, dry matter accumulation,

nodulation, nitrogen fixation in lentil were improved in AM inoculated

plants over uninoculated control at low and medium levels of plant

available soil phosphorus.

Manjunath and Bagyaraj reported that inoculation of Glomus

fasciculatum with or without added phosphorus recorded significant

increase in dry weight, phosphorus content of shoot and root in black

gram, chick pea, and mung bean. Chick pea inoculated with Glomus

versiformae increased shoot dry weight and grain yield by 12% &

25% respectively, (Singh and Tilak,1989).

Raju et al., (1990) reported that Glomus macrocarpum colonised

sorghum roots, enhanced plant growth and mineral uptake than

other AM fungal species especially at 300C. Glomus intraradices

enhanced yield of sorghum plants under drought conditions (Ibrahim,

et al.,1990). Hamel and Smith (1991) reported that the growth of corn

and soybean plants were greatly enhanced when inoculated with

Glomus intraradices. The corn :soybean dry mass ratio was almost

68

doubled in the mycorrhizal plots as compared to the non fertilised

controls. Mycorrhizal plants also had the highest tissue P content,

mycorrhizal roots appeared more efficient at transferring nitrogen

than the non mycorrhizal roots.

Wellings et al., (1991) reported that pigeon pea inoculated with

mycorrhiza yielded on an average 3.3 times the dry weight of non

mycorrhizal plant. Sankaran et al., (1992) observed that Acacia

auriculiformis inoculated with Glomus and Acaulospora cultures have

increased the plant height, shoot dry weight and root dry weight

significantly over control. Inoculation of Glomus mosseae improved

the growth of Leucaena leucocephala under mine soils, (Thatoi,

et al.,1993). Rao and Tarafdar (1993) reported that inoculation with

AM fungi improved the dry matter production and grain yield of

cluster bean.

Geetha kumari et al., (1994) reported that VA mycorrhizal fungi

exhibited a favourable effect in promoting the yield of cowpea grown

in pots.Inoculation of Glomus mosseae and Gigaspora margarita to

Leucaena leucocephala resulted in increased VAM colonization

percentage, plant dry weight, and nodule dry weight, (Byra Reddy,

et al., 1994). Inoculation with Glomus mosseae increased the root

length, root and shoot dry weight, phosphatase activity in the

rhizosphere, and shoot concentration of phosphorus in wheat,

(Tarafdar and Marschner,1995).

69

Dutra et al., (1996) reported that Glomus intraradices increased

the shoot and root growth of citrus. Balakrishna Reddy et al., (1996)

reported that Glomus mosseae significantly improved the growth and

nutrition of papaya resulting in greater plant biomass production,

plant height, plant girth, leaf area and phosphorus and Zn content of

plants.

Munyanziza et al., (1997) reported that AM occur in most of the

agricultural and horticultural crops and several tropical tree species,

provided a greater absorptive surface than root hairs and thus helped

in the absorption of immobile ions in soil such as phosphate, copper

and zinc. Purakayastha et al., (1998) reported that the VAM fungi,

Glomus macrocarpum and Glomus fasciculatum had influenced the

mobilization of iron in Brassica oleracea, and there by enhanced the

Fe2+ content in leaf tissue and total uptake of iron, and resulted in

increased curd and straw yields of Brassica compared to this

observed with N,P,K alone.

Osundina (1998) observed that mycorrhizal inoculation

contributed flood tolerance of Casuarina equisetifolia by enhancing

root nodulation in the upper zone of flooded soil. Inoculation with

mycorrhizae caused an increase in P and N uptake in flooded plants

up to four weeks into the flooding period.

Boswell et al., (1998) suggested that the management of

mycorrhizal fungi by cover cropping might be a useful practice in

sustainable agriculture. They found out that an autumn-sown winter

70

wheat cover crop increased VAM fungal inoculam potential of field

soil. Infective extra radial hyphal densities were significantly

increased by cover cropping. The degree of mycorrhizal infection of

maize was correlated with maize growth and yield. Mycorrhizal

infection of maize plants was significantly correlated with the height

of mature plants.Height was significantly correlated with yield. Thus

fractional mycorrhizal infection was significantly correlated with

yield.

Hemla Naik et al., (1995) reported that the shoot dry weight

and flower yield of china aster plants were significantly higher in

mycorrhizal plants as compared with un inoculated control plant.

Inoculation with Gigaspora margarita resulted in a significantly

higher shoot dry weight and flower yield at 75% of recommended dose

of P. Plant P uptake was increased significantly with increase in level

of P up to 75% of the recommended P level.

AM fungi, Glomus etunicatum produced positive effects on

Tagetes erecta and Zinnia elegans compared with the control (Aboul-

Nasr,1996). The root length of infected plants increased to 46.67%.

The number of flowers per plant, shoot and root dry weight etc

increased. The uninoculated plants had no flowers. VAM significantly

increased shoot height compared with the un inoculated plants. Root

analysis showed that inoculated Tagetes had a decrease in

phosphorus of 40%. Inoculated Zinnia showed a decrease in

71

phosphorus, potassium and sodium root contents compared with the

un inoculated plants.

Response of arbuscular mycorrhizal fungi in eucalyptus species

were studied by Adjoud et al., (1996). Twenty weeks after,

mycorrhizal inoculation, most species tested showed a significant

growth stimulation compared to control plants. The increase in plant

height reached 44% (Eucalyptus bosistoana/ Glomus mosseae) while

stem dry weight was increased by up to 49% (Eucalyptus dives

Glomus caledonium). Following mycorrhizal inoculation, leaf

phosphorus concentration was significantly increased by up to 41%

in most of the Eucalyptus species.

Sequeira et al., (1998) reported that AM inoculation and

superphosphate application enhanced the plant development and

yield of coffee in Brazil. Plant height and stem diameter were greatly

enhanced in inoculated plants than in controls, up to 19 months

after transplantation. Inoculation effects on tree canopy diameter

were significant up to 26 months after transplantation. The bean

yield was highly enhanced by all pre colonization treatments upto

38% over control. Three isolates of Glomus etunicatum showed yield

enhancements above 50%. Plants raised from pre colonized seedlings

exhibited lower external P requirements achieving maximal yield,

than those without precolonisation.

72

Puthur et al., (1998) observed cent percent transplantation

success of micro propagated, Leucaena leucocephala, plantlets using

two vesicular arbuscular mycorrhizal fungi, Glomus fasciculatum and

Glomus macrocarpum Only 20% of plantlets transferred to sterilized

garden soil without mycorrhizal fungi survived four weeks after their

transfer. Transplanted mycorrhizal plantlets showed higher degree of

nodulation. Roots of mycorrhizal plants were found to be healthier

and stronger than that of nonmycorrhizal plants and thereby

enhanced the survival rates. Twelve weeks after transplantation to

soil the shoot length of mycorrhizal plants was about 70% higher

than that of non mycorrhizal plants.

According to Clark et al., (1998) AMF can enhance plant ability

to grow and withstand acidic soil induced stresses.Inoculation of

Panicum virgatum with eight isolates of AMF showed significant

increase in shoot, root and total plant drymatter, shoot to root dry

matter ratio, total and specific root length and root colonization.

Drymatter of AMF plants were 52 fold greater than non AMF plants.

Total drymatter for AMF plants followed the sequence. Glomus clarum

> Glomus diamphanum > Acaulospora morrowiae >Gigaspora

margarita >Glomus etunicatum >Gigaspora albida > Gigaspora rosea =

Glomus intraradices = non AMF. Specific root length was higher for

AMF plants.

The growth of Castanospermum australe and production of

castanospermine was increased in plants inoculated with AM fungi,

73

(Abu- Zeyad et al.,1999). The total dry weights were significantly

higher for inoculated seedlings than for the controls. The dry weights

of seedlings inoculated with Glomus intraradices were significantly

higher of leaves from mycorrhizal plants inoculated with Gigaspora

margarita or Glomus intraradices were significantly higher than those

of control seedlings. Mycorrhizal plants contained higher amounts of

castanospermine in their leaves than non mycorrhizal plants.

Mean yield response to inoculation by AMF spore populations

isolated from fields under organic management was significantly

greater than the yield response to inoculation with spores isolated

from conventional high input systems for allium and trifolium (Eason

et al.,1999). Allium was highly responsive to all the AMF inoculum

applied, increasing the dry shoot weight by up to a factor of 40 over

that of un inoculated control. Root weight also tended to be greater in

the organic compared with the conventional AMF treatment.

Rajan et al., (2000) reported that mycorrhizal inoculation

showed an increase in plant growth and plant nutrition. It was found

that Glomus leptotichum was the best AM symbiont for teak compared

to others used under the experiment.

Effect of AM fungi on growth of lemon was studied by

Fidelibus et al., (2000). Plants treated with AM fungal population had

positive effects on plant growth. Plants treated with dry soil had lower

total root length and weight than other inoculum treatments. The

74

dry soil was distinct in that greater than 80% of the total number of

AM fungal spores were from a single species, Glomus occultum.

Khaliq and Sanders (2000), reported that the application of

phosphorus increased the dry matter yield significantly but

suppressed mycorrhizal infection. Phosphorus concentration in grain

and straw were increased in plants both by P application and by

mycorrhizal infection.

Experiments on cereal/legume rotation showed that roots of

rotation cereals had higher early AM infection rates compared to

continuous cereals can be explained by higher N and AM infection

levels early in the season, (Bagayoko et al.,2000).

Sequeira and Saggin-Junior (2001) reported that there were

very large differences between plant species in AM colonisation,

responsiveness to inoculation, mycorrhizal dependency and efficiency

of phosphorus uptake. Thirty four percent of the total woody species

of plants studied were found to be mycorrhiza independent or non

mycotrophic; where as the rest were highly dependent. Mycorrhizal

responsiveness and dependency were not related and some species

were responsive to increased P in the soil solution only when

mycorrhizal. Plant growth measured as shoot dry matter was highly

affected by P and AM in most of the species.

The majority of species grew very poorly in the soil with the

original P level (0.002mg) even when inoculated with the AM fungus.

75

In ten species, no significant effect of AM or AM by P interaction was

found. The remaining species responded differently to both factors

and exhibited significantly AM by P interactions.

Vander Heijden (2001) found that inoculation of AMF, Glomus

mosseae on Salix repens, a mycorrhizal plant showed large short

term effects on shoot growth and root length. Glomus mosseae

colonization resulted in higher shoot P uptake, shoot growth and root

length.

According to Jackson et al.,(2002) remarkable difference in the

growth response of Lactuca sativa and Lactuca serriola occurred to

either nutrient supply or to mycorrhizal colonization at four to six

weeks of growth. The treatment that caused the highest biomass

production for both species was the Glomus treatment with no added

P. Non mycorrhizal plants showed a positive response to increased P

supply, but for mycorrhizal plant, dry weight was decreased at

added P.

AM fungi differing in their response to soil pH may influence

the relative abundance of mycorrhizal fungi inside roots

(Sano et al., 2002). Addition of CaCo3 reduced the growth rate of

Acaulospora laevis. Mycorrhiza of Glomus invermaium predominated

in soil containing Acaulospora laevis and CaCo3. Shoot and root

weights of plants inoculated with Glomus invernaium were not

affected by application of CaCo3. But the addition of CaCo3 decreased

76

dry weights of both roots and shoots of plants inoculated with

Acaulospora laevis alone by more than 60% shoot dry weight

gradually decreased with increasing application of lime irrespective of

fungal species.

According to Rao and Richa Tak (2002) soil inoculation with

Glomus mosseae had significantly enhanced plant growth and

biomass production in different tree species of lime stone mine spoils

but enhancement varied from one species to the other. Activities of

various enzymes like dehydrogenases, phosphatases and

nitrogenases and population of nitrifying bacteria and Azotobacter

were also improved. Improvement occurs in the uptake of many

nutrients except Na and K. Ability of AM fungus in improving the

water uptake and transport in plants enabling the plants to

withstand high temperatures.

Gaur and Adholeya (2002) demonstrated the effectiveness of

indigenous mycorrhizal endophytes in increasing crop yield of five

fodder crops. Inoculation of AM fungi in non sterile P deficient sandy

loam soil showed significant increase in shoot and root dry weights

and total uptake of P and N of all the test plants. Mycorrhizal

inoculation increased yield in terms of shoot dry weight by 257% in

Trifolium alexandrinum, 50% in Avena sativa, 28% in Zea mays, 20%

in Medicago sativa and 6% in Sorghum vulgare. Mycorrhizal

dependency was maximum in Trifolium alexandrinum.

77

Inorganic N influenced total AM colonization, percentage of root

length, and photosynthesis of cucumber (Valentine et al., 2002).

Despite differences in mycorrhizal colonization, there was no

significant effects on total dry mass, leaf area or the allocation of dry

mass with any of the N treatments. There were significantly higher

stem, leaf, and fruit P concentrations in AM plants. Significant

variations observed in maximum photosynthetic rate.

Bi et al., (2003) found out the comparative effects of two AM

fungi, Glomus mosseae and Glomus versiformae on the growth and

nutrient uptake of maize grown in different depths of soil layer

overlying coal fly ash. Non mycorrhizal plants had the lowest shoot

and root yields. Highest root and shoot yields were displayed by

plants colonized with Glomus mosseae and growing in ten centimeter

of soil overlying five centimeter of fly ash. There was a decreasing

trend in yield with decreasing soil depth, and plants inoculated with

Glomus mosseae had significantly lower yields in five centimeter of

soil overlying ten centimeter of fly ash compared with the deepest soil

treatment. Inoculation of Glomus versiformae gave lower yields than

with Glomus mosseae at the two soil/fly ash combinations in which

the two fungi are compared. Mycorrhizal plants absorbed more

nutrients than non mycorrhizal controls and translocated less

sodium to the shoots, protecting the plants from excessive sodium

accumulation.

78

Effect of four different VAM fungi on medicinal ginger plant

was studied by Selvaraj and Selvaraj (2003). Plants inoculated with

Glomus geosporum spores showed higher levels of colonization

percentage followed by Glomus fasciculatum and Glomus mosseae and

least in Sclerocystis sinuosa. The dry matter content of plant was also

more in Glomus geosporum inoculated plants followed by Glomus

mosseae, Glomus fasciculatum and Sclerocystis sinuosa. In control,

the dry matter content was least. The P uptake in plants were more

in both root and leaves of Glomus geosporum inoculated plants; and

was least in the control.

According to Persson et al.,(2003) plants differ in their N uptake

from distinct N sources. Three forest plants irrespective of their

mycorrhizal association rapidly aquired N from the entire range of

added N sources. It was suggested that a wide range of N compounds

having importance in N nutrition and the type of mycorrhiza may be

of great importance for N scavenging but less important to the N

uptake capacity of plants.

Glomus fasciculatum inoculated plants showed significant plant

height, dry weight of shoot and root percentage of colonization,

spore number and P uptake than the control plants, (Hosamani et al.,

2004). Introduced Glomus fasciculatum significantly influenced the

growth more than indigenous Glomus fasciculatum in fox tail millet,

(Laxman et al.,2004).

79

According to Geeta (2004) application of arbuscular

mycorrhizae as bio-inoculant for cereals, pulses, fruits and

vegetables had been found to be beneficial both under field trials and

demonstration trials. Mycorrhizal inoculation in citrus had improved

root growth, nutrient uptake and plant growth. The applications of

Glomus mosseae and Glomus fasciculatum in papaya were found to

save P and to increase in yield up to 20%. In banana, the crop

responded excellently to VAM fungal inoculation. Chilly cultivars

treated with Glomus mosseae and G. fasciculatum also showed better

performance when compared with inoculated counterparts. The

inoculation of soil with Glomus fasciculatum, Glomus mosseae and

Gigaspora margarita in conjunction with soil inoculation with

Pseudomonas striata produced significantly higher yield in soybean

than VAM alone. In tomato combined inoculation of VAM Glomus

fasciculatum and Pseudomonas striata increased the yield and

improved the quality attributes of tomato.

Giri and Mukerji (2004) reported that mycorrhizal seedlings

had significantly higher root and shoot dry biomass production than

non mycorrhizal seedlings grown in saline soil. The content of

chlorophyll was greater in the leaves of mycorrhiza inoculated plants

as compared to uninoculated. The number of nodules was

significantly higher in mycorrhizal than non mycorrhizal plants.

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According to Lakshmipathy et al., (2004) inoculation of VAM

fungi improved the growth of cashew root stock. Inoculation of

Acaulospora laevis resulted in maximum plant height, number of

leaves, stem girth, total biomass and P uptake.

2.1.9: UPTAKE OF NUTRIENTS

2.1.9.1. Nitrogen uptake:-

Increased nitrogen concentration have been reported in VA

mycorrhizal plants. Ross (1971) observed higher nitrogen

concentration in mycorrhizal soybean plants. Isolated spores of

Glomus mosseae and Glomus macrocarpum reduced nitrate to

nitrite, (Ho and Trappe, 1973).

A number of mechanisms were suggested and investigated to

explain the VAM effect on nitrogen uptake. They include direct

uptake of nitrogen from soils (Ames et al.,1983; Johansen et al.,1992;

Frey and Schuepp, 1993,a) improvement of biological nitrogen

fixation (Barea and Azcon–Aguilar,1983) and N transfer between host

plants, (Harnel et al., 1991). Good development of nodules and

nitrogen fixation in Leucaena leucocephala was reported by Habte

and Aziz (1991).

2.1.9.2. Phosphorus uptake

Most of the research on plant nutrition by VAM fungi has been

concerned with phosphorus because it is one of the limiting as well

81

as major plant nutrient. The uptake of phosphorus and growth are

consistently improved in mycorrhizal plants compared to non

mycorrhizal plants has been reported by several workers, (Ross,1971;

Powell,1980;Huaay et al., 1983; Krishna and Bagyaraj, 1984;

Alexander et al., 1984; Thompson et al.,1986).

Mycorrhizal roots on a unit weight basis absorbed much higher

amounts of phosphorus than did nonmycorrhizal plants both in

solution cultures (Cress et al.,1979) and in soils (Bolan et al.,1987).

This suggest that mycorrhizal fungus hyphae have higher affinity for

phosphate ions and lower threshold concentration for absorption

than do plant roots.

In soils low in available phosphorus, mycorrhizal plants had

higher rates of growth than nonmycorrhizal plants and increased the

concentrations of total phosphorus in tissues in early stages of plant

development. The flow of phosphorus from soils in to the roots of

mycorrhizal plants is faster than into nonmycorrhizal plants, (Smith,

1980; Hale and Sanders, 1982;Sanders and Sheik, 1983).

Bagyaraj and Sreeramulu (1982) reported that VAM fungi

significantly enhanced the phosphorus and zinc nutrition in chillies

under field conditions. Habte and Fox (1993) reported that Leucaena

leucocephala inoculated with VAM exhibited increased

uptake of phosphorus. VA mycorrhiza increased the uptake of

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phosphorus in Azadirachta indica seedlings, (Kalavathi and

Santhana-Krishnan, 1995).

2.1.9.3. Ptassium uptake

Mosse (1957) observed a higher concentration of potassium

in mycorrhizal plants. Higher concentration of potassium in shoots

of mycorrhizal Trifolium subterranean was reported by

Smith et al., (1979). Mosse (1973) indicated an increase in potassium

uptake by mycorrhizal plants in infertile soils.Huang et al., (1983)

reported greater potassium concentrations in mycorrhizal Leucaena

leucocaphala.

Srinivas (1987) conducted experiments to study the effect of

VAM inoculation on potassium uptake of Acacia nilotica and found

that inoculated seedlings showed increased uptake of nutrients as

against control.

2.1.10: BENEFITS OF AM FUNGI

The mycorrhizal endophytes as well as root pathogens in the

soil environment are part of a complex interacting system. The

dynamics of such microbial systems in the rhizosphere excert

measurable effects on plants by altering the morphology and

physiology of root system. Most important effect of microbial

interaction is suppression of soil born pathogens. A variety of

pathogenic fungi, bacteria, viruses and nematodes derive their energy

from plant roots. Several reports have indicated that a host plant

83

previously inoculated with an arbuscular mycorrhizal fungal

symbiont exhibited increased resistance to several root diseases.

Host plants previously inoculated with fungal symbionts have

been shown to exhibit an increased resistance to fungal root rot and

wilting, (Jalali and Thareja,1981; Rosendahl,1985). Growth of

mycorrhizal seedlings infected by Phytophthora parasitica was greater

than that of infected nonmycorrhizal citrus seedlings (Davis and

Menge,1981). Zambolium and Schenck (1983) while studying the

interactions between Glomus mosseae and Macrophomina phaseolina

in soybean, observed that the colonization of the host root by the

mycorrhizal symbiont compensated for the effect of pathogen without

changing incidence of disease in the host.

In a series of investigation by Caron et al., (1986) root

colonization by Glomus was not affected by the presence of pathogen

Fusarium oxysporum. The number of propagules of this pathogen

was consistently lower when the plants inoculated with the

mycorrhizal endophyte. The presence of fungal symbiont Glomus

intraradices significantly inhibited root necrosis caused by the

pathogen.

Since very few pathogen–mycorrhiza studies have investigated

the role of phosphate nutrition, it may not be possible to conclude

whether responses of mycorrhizal plants to pathogen would differ

from nonmycorrhizal plants at a comparable nutritional status.

84

Smith (1988) reviewed this dimension and examined those studies

that compared the influence of phosphate nutrition with VA

mycorrhizal fungi on the host- pathogen relationship.

Kaye et al., (1984) reported that mycorrhizal root colonization

was greater in plants grown in soils inoculated with Pythium ultimum.

Enhanced phosphate uptake in mycorrhizal plants and reduction in

the density of Pythium ultimum in the rhizosphere of mycorrhizal

plants, it has been suggested that mycorrhizal plant’s tolerance to

Pythium ultimum could be attributed to factors other than improved

plant nutrition alone.

Bath and Hayman (1983) carried out studies with mycorrhizae

and verticillium sp. on tomato plants. Irrespective of mycorrhizal

status of the host plants, inoculation with verticillium sp. decreased

plant growth compared to noninoculated controls. Addition of extra

phosphorus to the soil enhanced plant growth, but this enhancement

was nullified by Verticillium sp. These studies indicate that

mycorrhiza might not always be helping a plant to tolerate a root

pathogen.

Prevalence and severity of sweet potato wilt at 32οC was the

highest when host plants were inoculated with Fusarium oxysporum

alone but at 21οC more wilt development when plants were dually

inoculated with the pathogen and the mycorrhizal fungi, (Ngeve and

Roncadori, 1984).

85

Garcia-Garrido and Ocampo (1989) reported that the growth of

nonmycorrhizal infected tomato was decreased by Pseudomonas

syringae. However in mycorrhizal plants neither growth nor

percentage of VA infection was negatively affected by the pathogenic

bacteria. The production of tomato by Glomus mosseae against the

pathogen and the reduction in the number of colony forming units

of Pseudomonas syringae in the rhizosphere of mycorrhizal plants

was independent of P plant concentration and timing of inoculation

with the two microorganisms. Inoculation of nonmycorrhizal plants

with Pseudomonas syringae depressed shoot and root growth.

Mycorrhizal inoculation over came any growth depression caused by

the bacteria in dually inoculated plants. Shoot weight of plants

inoculated with both microorganisms at the same time was similar to

plants inoculated only with Glomus mosseae. But when Pseudomonas

syringae was inoculated three weeks after Glomus mosseae dually

infected plants were heavier than mycorrhiza only plants. Shoot

phosphorus content was increased by the VA mycorrhizal fungus but

was unaffected by the pathogenic bacteria.

A giass house experiment with linseed conducted by Thompson

(1994) showed that naturally occurring propagules of VAMF extracted

from a cropped field soil and added in to a long fallow soil would

overcome the disorder. Supplying additional VAMF propagules to the

long fallow soil had a similar effect on linseed as supplying zinc

86

fertilizer increasing growth in the presence of P fertilizer. Soil

irradiation killed all VAMF inoculum in the long fallow soil, resulting

in uncolonised linseed plants that grew very poorly. Inoculation of

irradiated soil with VAMF spores from the cropped soil resulted in

early VAMF colonization of the linseed roots and good growth of the

linseed tops.

The arbuscular mycorrhizal fungus Glomus intraradices

increased P uptake and the P concentration in the plant, but reduced

root rot disease of peas caused by Aphanomyces euteiches (Bodker

et al.,1998). In mycorrhizal plants the pathogen developed fewer

oospores than in nonmycorrhizal plants. This effect was statistically

significant. Mycorrhizal plants had a significantly lower disease index

in at all harvests.

Arbuscular mycorrhizal barley plants were not susceptible to

the obligate biotrophic shoot pathogen Erysiphe graminis, (Gernns

et al.,2001). Erysiphe graminis produced nearly three fold more

conidia on leaves of mycorrhizal than non mycorrhizal plants,

whereas there was no differences in infected leaf area. This higher

pathogen activity on AM plants was independent of the growth

conditions. However the mildew fungus infected a greater leaf area

and produced more conidia irrespective of mycorrhizal infection.

Powdery mildew significantly reduced the grain number per ear. But

pathogen produced no yield loss in mycorrhizal plants. There was no

87

AM mediated increase in yield parameters in non mildewed plants.

AM induced late tillering during grain formation.

Two micropropagated potato cultivars were inoculated with

either Glomus etunicatum or Glomus intraradices, and seven days

later with Rhizoctonia solani At different times after R. solani

infection, disease severity, mortality rate, root colonization levels,

various growth parameters and shoot mineral content were

evaluated. Inoculation with Glomus etunicatum led to a significant

reduction in disease severity ranging between 60% and 71% on both

shoot and crown. Glomus etunicatum significantly increased shoot

fresh weight, root dry weight and number of tubers produced per

plant; whereas Glomus intraradices only significantly increased the

number of tubers, (Yao et al.,2002).

Mutualistic microbial associations with aerial plant part such

as those in grasses involving fungal endophytes, they are relatively

rare among plant families and studies have revealed that shoots and

leaves are well adapted to initiate rapid and effective defence

responses against microbial invasion, (Heath,2002).

Mycorrhizal casuarina seedlings adapted to flooding better than

non inoculated seedlings, (Osundina,1998). This was achieved partly

by the greater development of adventitious roots and hypertrophied

lenticels, which increased oxygen availability. The inability of non

mycorrhizal, flooded Casuarina equisetifolia plants to nodulate

88

suggest that mycorrhizal infection is essential for the nodulation of

this species under flooded soil conditions.

Mycorrhizal symbiosis is a key component in helping plants

cope with adverse environmental conditions, (Ruiz-Lozano and

Azcon, 2000). Two AM fungi, Glomus species isolated from saline soil

and Glomus deserticola from non saline soils were inoculated on

lettuce plants.Both AMF assayed, efficiently protected the host plant

against the detrimental effects of salt. Mycorrhizal plants were not

negatively affected by increasing salinity in growth or in nutrient

acquisition. Both the AMF were differed in their symbiotic efficiency.

These differences were more evident at the two highest salt levels.

Glomus sp. colonized plants grew less and accumulated slightly less

N and P, but they formed higher amount of mycorrhizae. But

mycorrhizal responses increased in Glomus deserticola colonized

plants.

Effects of drought stress and arbuscular mycorrhiza on the

growth of two tropical legume trees under simulated eroded soil

conditions were studied by Fagbola et al., (2001). Biomass production

of both species Gliricida sepium and Leucaena leucocephala was

reduced in the sub soil compared to the topsoil. In the top soil,

drought stressed plants had a significantly lower biomass compared

to their adequately watered counter parts for both the tree species.

Inoculation of Glomus sepium with Glomus deserticola in the sub soil

89

significantly increased plant height, stem girth, leaf and stem dry

weights and root length under both watering regimes, but not leaf

dry weight under drought conditions. After ten drought cycles, the

growth of Gliricida sepium in the sub soil was enhanced by

mycorrhizal inoculation, but there was no significant contribution of

mycorrhizal inoculation to the growth of Leucaena leucocephala.

Drought stress significantly reduced most growth parameters for the

two tree species in both soils with or without fungal inoculation.

Cuenca et al.,(2001) reported that in the absence of AMF the

seedlings of Clusia multiflora did not grow indicating that this species

was highly dependent on AMF. When these plants were exposed to a

very acidic solution plants inoculated with AMF from acidic soils were

taller than those inoculated with AMF from neutral soils. All plants

accumulated high quantities of aluminium in the roots, but plants

inoculated with AMF from acidic soils accumulated less aluminium in

roots, than plants from other treatments. In mycorrhizal plants

aluminium was bound to the cell walls in the mycelium of the

fungus. It was evaluated that AM fungi could be responsible partially

for the tolerance to acidity and to aluminium, so that an inoculum of

AM fungi coming from acid soils contribute more to the tolerance of

acidity of Clusia multiflora than one coming from neutral soils.

It is known that number of physiochemical factors are

associated with drought tolerance in plants. Proline accumulation

correlates with drought resistance in various plant species,

90

(Smith, 1962; Aspinall and Paleg,1981). Ramakrishna et al., (1988)

found that mycorrhizal maize plants yielded considerably greater

amounts of proline than the non mycorrhizal plants at different water

deficit conditions. Thus mycorrhizal infection can increase drought

resistance in host plants. At the specific water stress treatment,

there was more increase in membrane permeability in drought

sensitive plant tissues, (Gupta, 1977,b;1981;1983). Drought sensitive

plant tissues revealed greater amounts of phospholipids,

(Gupta, 1977,a;1983;1984).

Sumithra and Reddy (2004) reported that there was a

significant increase in proline content in cowpea seedlings with

progressive increase in water stress. Approximately a five fold

increase in proline content at the end of sixth day of water deficit

treatment. Withholding of water in cowpea showed increase in the

activities of proline biosynthetic enzymes. Among various solutes

proline serves to be a typical osmo protectant during stressful

conditions. The main reason for the accumulation of proline in water

stressed cowpea might be due to the enhanced activities of proline

biosynthetic enzymes .

Anil Kumar and Muraleedhara kurup (2003) reported that

under drought stress condition, the Vigna sinensis showed more

nitrogen content than other legumes. At different levels of salinity,

the Phaseolus aureus showed more nitrogen uptake in low and

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normal salinity stresses. Vigna sinensis showed more nitrogen

content in high salinity. In this case, the percentage of

mycorrhization affected the nitrogen uptake.

Selvaraj and Selvaraj (2003) reported that Glomus geosporum

was the most efficient effluent tolerant VAM strain not only in

colonization but also in enhancing P nutrition and growth.

Eventhough treated with toxic industrial effluent, the VAM inoculated

plants of Zingiber officinale proved their efficiency to tolerate the toxic

industrial effluents.

Under salinity stress conditions Sesbania spp. showed a high

degree of dependence on mycorrhizae increasing with the age of

plants, (Giri and Mukerji, 2004). The reduction in Na uptake together

with the concomitant increase in P, N and Mg absorption and high

chlorophyll content in mycorrhizal plants may be important salt

alleviating mechanisms for plants growing in saline soil.

Various mechanisms allow VAM to increase plant stress

tolerance. These include increased uptake of water, storage and

movement back into plants, enhanced tolerance to drought stress.

Mycorrhizal fungi produce specific antibiotics, which immobilize and

kill soil pathogens. In addition net work of filaments act as a physical

barrier against the invasion of root diseases, (Nachiappan, 2004).

Mycorrhizal fungi and plant parasitic nematodes are commonly

found inhabiting the rhizosphere and colonizing roots of their host

92

plants. These two groups of microorganisms exert a characteristic but

opposite effect on plant health, (Schenck, 1983). Generally, severity of

nematode diseases is found to reduce in mycorrhizal plants, (Cooper

and Grandison, 1986; Olivera and Zambolim, 1986). Establishment

of VA mycorrhiza in plants usually confers resistance to nematode

parasitism, (Smith et al.,1986) or adversely affects nematode

reproduction, (Hussey and Roncadori, 1978).

The expression of interaction response depends upon, the

species of mycorrhizal fungus host plant, nematode and nematode

inoculum densities, the susceptibility of the host cultivar to

nematode and soil fertility, (Strobel et al., 1982; Smith et al., 1986).

Green house testing to evaluate the influence of Glomus

mosseae on Rotylenchulus reniformis penetration and development on

bushbean, cucumber and musk melon revealed that reduction in the

number of nematodes that penetrated mycorrhizal bushbean was 35

and 41% lower than the control, (Sitaramaiah and Sikora, 1981). A

similar reduction in larval penetration was also observed on

mycorrhizal cucumber and musk melon eight days after nematode

introduction. These studies have shown that VA mycorrhizal fungus

Glomus mosseae increased plant resistance to nematode infection. In

further studies, inoculation of tomato transplants or seed beds with

the fungal endophyte appreciably reduced juvenile penetration and

93

development on mycorrhizal plants, compared with control plants,

(Sitaramaiah and Sikora, 1982).

Cooper and Grandison (1986) reported that mycorrhizal plants

exhibited more resistance to root rot nematode (Meloidogyne hapla)

at all phosphate levels, and growth benefits were generally more

significant in plants pre infected with mycorrhizal fungi. In

mycorrhizal root systems, nematode numbers increased in the lower

phosphate soils. However at higher phosphate levels nematode

numbers were either unaffected or reduced in mycorrhizal

treatments; mycorrhizal root length remained unaffected by

nematode inoculation.

Although the role of phophorus in mycorrhiza nematode

interactions remains incompletely understood phosphorus status of

soil was known to influence plant growth and nematode

reproduction favourably ( Roncadori and Hussey, 1977) or adversely,

(Oliveira and Zambolim, 1986) or may exert no effect, (Thomson

et al.,1983).

Bagayoko et al., (2000) studied cereal/legume rotation effects

on cereal growth. Roots of rotation cereals had higher early AM

infection rates compared to continuous cereals. Nematodes extracted

from soil and root samples in millet/cowpea rotations generally

belonged to the groups of Helicotylenchus sp. Rotylenchus sp. and

pratylenchus sp. In sorghum/groundnut cropping systems nematode

94

densities were consistently lower in rotation sorghum compared to

continuous sorghum. Continuous groundnut had the lowest

nematode densities indicating that groundnut was a poor host for the

three nematode groups. In millet/ cowpea cropping systems with

inherently high nematode densities crop rotations barley affected

nematode densities indicating that both crops were good hosts. Here

the total dry matter also increased by rotation cereals compared with

continuous cereals. These results related to the efficiency of the

legume species to suppress nematode populations and increase

plant available nitrogen through N2 fixation.

Among various kinds of organisms engaged in the biological

control of nematodes, VAM suppresses the root pathogenic

nematodes through morphological, physiological and biochemical

alterations in the host plant. In a pot culture experiment of cotton,

among different VAM treatments with nematodes, Glomus mosseae

recorded maximum height of 109.1cm which was on par with Glomus

mosseae alone treatment. The minimum plant height of 81.3cm was

recorded in Rotylenchulus reniformis alone treatment control. The

similar trend was maintained for all the growth parameters. The

maximum yield of 67.9 g/ plant was obtained in Glomus mosseae

and it was on par with Glomus mosseae + Rotylenchulus reniformis

treatment. The next best treatment was Glomus fasciculatum, with a

yield of 55.5g/plant and it was also on par with Glomus fasciculatum

95

+ Rotylenchulus reniformis treatment. The Rotylenchulus reniformis

alone recorded least of 35.5g/plant. All the VAM alone and VAM+

nematode treatments were significantly superior than nematode

alone, (Sreenivasan et al., 2004) .

Yu et al., (2005) reported that AMF inoculation sharply

increased both shoot and root cadmium concentrations in rye grass

at different dosage of cadmium, when compared with control.

Although the shoot biomass decreased, the total cadmium uptake by

mycorrhizal rye grass was enhanced significantly except under the

highest dosage of cadmium. It was found that mycorrhizal interaction

have a potential role in elevating phytoextraction efficiency in low to

medium level metal contaminated soil.

Experiments of Adholeya and Cheema (1990) showed positive

responses in micro propagated Populus deltoids to VAM inoculation.

The beneficial effect evident here showed a marked reduction in

survival by the end of 60 weeks. The colonization of root by VAM

fungi in inoculated micro propagated plants was observed to be 30-

40% after two weeks of in vitro inoculation prior to transplantation.

The positive responses of VAM association were manifest in terms of

active root growth. This seems to have supported shoot bud break

and its growth into leafy shoots after one to two weeks following

transplantation. The control however showed delayed bud break

ranging from 8–22 weeks. Inoculation also markedly affected survival,

96

establishment and growth of transplants in field soil. Comparisons

over an extended period of time showed that control plants could not

withstand out door ambient summer temperature ranging between

40-450C, while the VAM inoculated plants successfully survived such

harsh conditions.

Field experiments of china aster with Gigaspora margarita

showed positive responses, (Hemla Naik,1995). Inoculation of

Gigaspora margarita improved flower yield when compared with

uninoculated control plants. Higher yield was obtained with

Gigaspora margarita at a P rate of 90 Kg, P2O5 ha-1 that is 75% of the

recommended rate; suggesting that the application of phosphate

fertilizer could be reduced through inoculation with mycorrhizal

fungi.

Adjoud et al., (1996) reported that there was a significant

growth stimulation in AM inoculated Eucalyptus plants, when

compared to noninoculated control plants. Faster flowering and

increased number of flowers were observe in Tagetes erecta and

Zinnia elegans when inoculated with Glomus etunicatum,, compared

with control plants, Aboul-Nasr,1996). The uninoculated plants had

no flowers .

Degree of mycorrhizal infection of maize was correlated with

maize growth and yield, (Boswell et al.,1998). Cover cropping of maize

significantly increased the number of ears per plant ie, about 20%

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and the grain dry weight per ear 66% and thus significantly increased

the grain dry weight per plant, 104%. That is the fractional infection

of maize which was significantly higher in cover crop plots than in

non cover crop plots.

Coffee bean yield was significantly greater in all

AM precolonised plants than those without precolonization,

(Siqueira et al.,1998). Maximum yield was obtained in the range of

800 Kg ha-1 as compared with 580 Kg ha-1 for noninoculated controls.

This corresponds to a yield increase of 38%. In addition precolonized

seedlings required only lower amounts of phosphorus.

No significant increase of nutrient concentrations or above

ground biomass due to the presence of mycorrhiza was observed in

Calluna vulgaris plants. Phosphorus concentration was 16% lower in

the shoots from the plants inoculated with mycorrhiza. Increased

growth rate in the plants with mycorrhiza diluted the nutrient

concentrations in the shoots from these plants. The mean nitrogen

content in shoots of these plants was 10% higher in mycorrhizal

plants than non mycorrhizal plants, (Strandberg and Johansson,

1999) .

Effect of VAM inoculation on the yield and phosphorus

uptake on field grown barley was studied by Khaliq and

Sanders (2000). An overall small increase of 3% in total P uptake and

a decrease upto 2% in grain and straw yield were observed as a result

98

of inoculation which were statistically non significant. But the

application of P fertilizer significantly increased grain, straw and

grain plus straw weight.

Inoculation of potato micro plants with AM fungi affected the

flowering time, percentage of flowering tuberisation etc., (Elizabeth

and Alan, 2000). The Glomus intraradices treatment showed late

flowering. Other AM treatments showed early flowering of about 80%

and 76% after two months. In control plants 60% at two months. In

Glomus intraradices 14% at two months. Tuberisation was also

delayed in Glomus intraradices. The yield of the tuber grown crop

was significantly higher than any of the treatments and was

approximately 34% greater than that of microplant control. Average

tuber yield for a seed tuber derived control was 1.2 Kg/ plant. In

control, it was 0.9 Kg. Here the mycorrhizal inoculation can

influence the yield of microplants of potato.

The overall growth and nutrient content of mycorrhizal plants

were higher as compared to control plants in Moringa concanensis,

(Jitendra and Anil, 2002). Gigaspora margarita resulted in more than

50% increase in shoot and root dry weight and more than two fold

increase uptake of phosphorus and nitrogen. Physiological

parameters were also changed due to Gigaspora margarita infection.

There was more than 25% increase in total chlorophyll and protein

content and more than 60% increase in carotenoid and total sugars.

99

Inoculation of Glomus mosseae significantly enhanced uptake

of nitrogen and phosphorus and this enhancement was attributed to

the higher biomass production in different tree species grown on

limestone mine spoils, (Rao and Richa Tak,2002).

According to Geeta (2004), inoculation of Glomus mosseae and

Glomus fasciculatum in papaya increased the yield upto 20% and in

banana, up to 20-30%. In chilly cultivars also the yield increased and

that led to 50% saving of super phosphate.

Uptake of zinc from solution by endomycorrhizas of Araucaria

cunninghamii was studied by Bowen et al., (1974). It was 2.6 times

greater than uninfected roots that was significant at one per cent

level.

VA mycorrhizal plants contained significantly greater cytokinin

concentrations in both the roots and leaves than did non mycorrhizal

controls, (Allen et al.,1980).

VAM is known to enhance the uptake of heavy metals in the

host. The effect of mycorrhizal infection on two grasses grown in

zinc polluted soils were studied. Both zinc and VAM influenced the

amount of biomass produced by both grasses. While zinc inhibited

the yield, VAM infection stimulated the growth of roots and shoots.

Apparently the negative effects of zinc on growth of root is alleviated

or even absent in VAM + zinc treatment. The zinc concentration in

the roots and shoots of both species were significantly increased by

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the zinc treatment but were not mitigated by VAM infection. The two

grass species reacted differently to zinc and VAM in the uptake and

translocation of minerals. Mycorrhizal Festuca rubra accumulated

higher amounts of iron and manganese in the root than non

mycorrhizal plants. The zinc treatment significantly increased the

concentration of phosphorus in the roots of both grass species and

the translocation of potassium in Festuca rubra, ( Dueck et al.,1986).

The inoculation of mycorrhizal fungi increased shoot iron

concentrations alone in comparison to inoculation with non

mycorrhizal microorganisms in citrus plants, (Treeby,1992). But this

effect was only in acidic conditions. Inoculation of mycorrhizal fungi

in alkaline conditions had no effect on shoot Fe concentrations.

Experiments of Frey and Schuepp (1993,a) studied the transfer

of nitrogen from legume to non legume plants via the hyphae of VA

mycorrhizal fungi. In the first experiment Trifolium alexandrinum as

legume and maize were selected as non legume plant. In the second

experiment apple was selected as the non legume. 4.7% of nitrogen

content of Trifolium was transferred to apple. In contrast the amount

of nitrogen transfered to mycorrhizal maize were smaller with 0.1%.

The uptake of zinc by the hyphae of three vesicular

arbuscular mycorrhizal fungi were studied by Barbara and

Alan (1994). At all the harvests zinc activity in shoots of plants

colonized by Acaulospora laevis was greater than non mycorrhizal

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plants and plants colonized by either Glomus spp. or Scutellospora

calospora, Acaulospora spp. inoculated plants absorbed zinc placed

40mm from the roots where as no zinc uptake from this distance

occurred with other treatments. The ability of plants colonized by

Acaulospora laevis to access zinc from such a distance was

associated with a much greater hyphal length.

Distribution of zinc in various parts of sorghum bicolor was

studied by Senthil Kumar and Arockiasamy (1995). In the absence of

VAM about 18-35% of zinc content was present in the root system.

Thus in normal conditions most of the heavy metals that are

absorbed are transported to the leaves. In the presence of VAM ,in

the root there was 32% to 60% of zinc. The differences observed in

the heavy metal accumulation in the roots are to the presence of

VAM fungi was found to be statistically significant. At higher

concentrations heavy metal content in the root is higher than that of

leaves. In the non infected plants a steady decrease in the root

protein was observed from 0.6-0.09 mg/g fresh weight. But the root

protein of infected plants showed a remarkable increase from 0.8-

1.54 mg/g fresh weight. The present study indicated that in sorghum

bicolor synthesis of newer proteins in response to heavy metal could

be the mode of resistance offered by VAM fungi. VAM fungi play an

important role in synthesis of newer proteins and thus help the host

to tolerate heavy metal toxicity.

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Inoculation of Glomus constrictum caused an increase of 50%

in P content in wheat plants over control plants, (Omar, 1998). Dual

or mixed inoculation of plants with Glomus constrictum and rock

phosphate solubilising fungi considerably increased P uptake and

enhanced plant growth. These results could be attributed to the

ability of these microorganisms to solubilise organic and inorganic

phosphorus already present in soil .

Sanginga et al., (2000) demonstrated that there were highly

significant differences in shoot dry weight among cowpea lines at

high (60 Kg P ha-1) and low P (0 Kg P ha-1) levels. Phosphorus

application increased nodule weight. Total N accumulation in the

shoots ranged from 15.7 to 34 K g N ha-1. The average total N values

were significant. No response to P was observed for total N in seed.

Differences in seed total N between cowpea lines ranged between

16.3 and 29.0 K g N/ ha. All cowpea lines nodulated profusely, the

number and weight of nodules increasing with the addition of P.

Liu et al., (2000) demonstrated that contribution of

mycorrhizae to uptake of copper, zinc, manganese and iron by maize

as influenced by soil P and micro nutrient level. There was an

interaction between P and micronutrient levels for the length of extra

radical hyphae. The highest amount of extra radical hyphae was

produced in the soil at low P without micronutrient addition, while

the lowest amount was at high P with high micro nutrient addition.

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Shoot biomass production was increased by mycorrhizal colonization

and when plants were grown at the high P level or with micro

nutrient application. At low application rates of both P and

micronutrients or when no micronutrients were added, shoot Zn

content of mycorrhizal plants was higher than that of non

mycorrhizal plants Shoots of mycorrhizal plants had enhanced total

Cu contents only when plants were grown without micronutrient

application. At the highest dose of micronutrients, mycorrhizal plants

had lower Mn contents in shoots than non mycorrhizal plants. Total

shoot iron content was higher in mycorrhizal than in non mycorrhizal

plants grown with no added micronutrients.

Zhu et al., (2001) reported that mycorrhizal infection and zinc

application had little effect on plant growth. Increasing zn application

rate led to increased uptake of zn in roots and shoots but the

increase was significantly greater in non mycorrhizal controls than

in mycorrhizal treatments. Zn uptake were similar in shoot and root

when no zn was applied but as zn application rate increased, shoot

zn uptake increased much less than root zn uptake and decrease in

zn uptake by mycorrhizal roots and shoots compared with controls.

Fester et al., (2002) reported that AM fungal roots stimulated

carotenoid metabolism. AM stimulated accumulation of various

isoprenoids, like mycorradicin and cyclohexanone derivatives.

Carotenoides were not detected in nonmycorrhizal roots.

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2.1.11: INTERACTION OF AM WITH RHIZOBIUM

Most of the leguminous plants are simultaneously symbiotic

with nodule forming Rhizobium and AM fungi. Nitrogen fixing

Rhizobium benefits plant growth and AM fungi benefit the host by

increasing the efficiency of uptake of mineral elements and water from

the soil, altering other physiological parameters of the host. This

tripartite relationship of host-Rhizobium- AM fungi have been

extensively studied by various scientists. When legumes are symbiotic

with both Rhizobium and AM fungi plant growth is generally much

greater than control plants. Many reports indicate that the AM

increased ‘P’ up take (Bethlen Falvay and Yoder, 1981; Manjunath and

Bagyaraj, 1984; Purcino et al., 1986) which under P stress conditions

would itself increase plant growth. Improved ‘P’ nutrition in turn would

favour the nitrogen fixation process by Rhizobium. The combination of

effects resulted in further growth enhancement (Manjunath et al.,

1984, Subba Rao et al., 1986; Kawai and Yamamoto, 1986).

Different strains of Rhizobium or strains or species of VA

mycorrhizal fungi do not affect their host plant in the same manner to

the same degree. Different edaphic factors influence the behaviour an

effectiveness of the symbionts and also would affect the combination

(Hicks and Loynanchan, 1987; Bethlenfalvay et al., 1987).

Azcon-Aguilar and Barea (1981) reported that the dual

inoculation of Rhizobium with Glomous mosseae is an efficient

biological fertilizer. Field inoculation of Medicago sativa with its

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symbiotic partners, Rhizobium meliloti and the AM fungi, Glomus

mosseae resulted in more than doubled yield compared to an

uninoculated control. During the first harvest all inoculation

treatments significantly increased plant growth. The best treatment

was Rhizobium with Glomus but there was no significant differences

between Rhizobium with Glomus and Rhizobium. The plants inoculated

with Glomus (Glomus, and Rhizobium with Glomus) possessed higher

N, P and K content than uninocualted control or Rhizobium

treatments. At the second harvest (after 10 weeks of spring growth) the

dual inoculum Rhizobium with Glomus was significantly more effective

than any other treatment.

Increased number of nodules on mycorrhizal plants than non

mycorrhizal plants has been demonstrated in numerous studies. But

one study demonstrated that advanced formation of AM inhibited

nodulation. (Bethlenfalvay et. al., 1985). Even when nodule number

has not been significantly increased by AM, the size and nitrogen

fixing activity of the nodules has been shown to be increased.

(Pacovsky et al., 1986). Establishment of AM increased total number of

bacteria isolated from the rhizoplane of sweet corn and clover, but did

not affect number of actinomycetes, but the total number of bacteria

and actinomycetes in rhizosphere soil were not affected by AM, (Meyer

and Linderman, 1986).

AM plants absorb more micronutrients from the soil which plays

a role in the enhanced nitrogen fixation process, (Pacovsky, 1986;

O’ Hara et al., 1988).

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Formation of AM generally results in increased P levels in the

plant tissues and thus changes in membrane permeability. The

resultant changes in the quality and quantity of root exudates can

induce changes in the rhizosphere population which could influence

the competition between Rhizobium and other Rhizobacteria. If

bacteria selectively favoured in the rhizoplane enhanced Rhizobium

competitiveness, then nodulation would be favoured, (Paulitz and

Linderman, 1991).

Increasing available fertilizer frequently has increased the rate of

nitrogen fixation has led many to conclude that the role of AM is to

increase P nutrition of the plant. Ames and Bethlenfalvay (1987) used

as split root system to demonstrate a localized nonsystemic, non P

mediated influence of AM on cowpea root growth and nodule activity.

Nearly all leguminous plants with nodules formed by Rhizobium

grow better if they are also colonized by AM fungi. The growth increase

was explained by increased photosynthesis and increased carbon flow

to the nodule resulting in more and larger nodules that fix more

nitrogen for the plant (Paulitz and Linderman, 1991).

Field studies using inoculants of Bradyrhizobium, AM fungi, and

phosphate solubilising microbes on soybean conducted by Singh

(1994) vesicular arbuscular mycorrhizae and phosphate solubiliisng

microorganisms individually stimulated the activity of native rhizobial

strains resulting significant increase in nodulation, plant growth and

grain yield in soybeans; over the uninoculated controls. Increase in

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grain yield in soybean due to individual inoculant of Bradyrhizobium,

AM and PSM ranged from 8.66 to 13.82%, 0.0 to 46.55% 0.0 to

41.38% respectively over their respective controls. But none of the

inoculants significantly increased the grain yield of soybean. Dual

inoculation with Bradyrhizobium and AM of PSM did not cause

significant increase in any of the parameters studied.

Stamford et al., (1977) studied about the comparative effect of

phosphorus fertilization, inoculation of Bradyrhizobium and

mycorrhizal fungi on the growth of Mimosa caesalpiniaefolia.

Statistical analysis indicated that P sources affected mycorrhizal

infection only when plants were not inoculated with Badyrhizobium

spp. Plant height and dry matter in uninoculated Bradyrhizobium

plants were increased significantly only when ‘P’ was added as triple

super phosphate. In unfertilized soil certain strains produced a clear

increase in plant height (63%) and in plant dry matter (148%)

Mycorrhizal inoculation did not contribute to increased yield in any

test. Addition of AM had little effect on N accumulation in plants

inoculated with Bradyrhizobium spp.

Bradyrhizobium sp (vigna) strain S 24 interacted differently with

eight AM fungi and caused significant variations in nodulation and

growth parameters of green gram. (Saxena et al., 1997) Co-inoculation

with Scutellospora calospora resulted in the highest nitorgenase

activity and dry biomass. Maximum nodule weight was obtained for

treatment inoculated with Glomus mosseae. In presence of Glomus

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mosseae, Glomus fasciculatum, and scutellospora calospora the nodule

occupancy of strain was significantly higher than (63%) single

inoculation (51%). However co-inoculation of Glomus versiforme,

Glomus macrocarpum, Gigaspora margarita and Endogone duseii

exhibited a decreasing trend on the nodule occupancy (26-43%) with

Bradyrhizobium. Root colonization by AM varied from 18-40% in green

plants inoculated with different AM fungi in the presence of

Bradyrhizobium. But AM infection was negligible in roots inoculated

with Bradyrhizobium alone and in the uninoculated control.

Pigeon pea- rhizobial strain interaction studies were performed

to study the symbiotic parameters such as nodule number, nodule

fresh weight, nitrogenase activity and dry matter accumulation,

(Anand and Dogra, 1977). With four pigeon pea cultivars, the

symbiotic effectiveness of three of the four isolates of Bradyrhizobium

was superior to that to all the four strains of Rhizobium species.

Among the fast growing strains, two strains did not perform well. The

nodule number as well as nodule fresh weight showed no relationship

with total nitrogenase activity and dry matter with different strains of

Bradyrhizobium spp. From the results Bradyrhizobium were fond to be

superior to be Rhizobium strains and the superiority was ascertained

to be due to the higher enzyme activity of he tricarboxylic acid cycle in

comparison to Rhizobium spp strains. The symbiotic performance of

isolates varied with the host cultivar.

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Miller et al., (1977) studied the effects of infection with

Rhizobium leguminosarum biovar trifoli and Gigaspora margarita on

response of subterranean clover to ozone. Biomass was usually

greatest for plants infected by Rhizobium leguminosarum only; and

least for plants infected by Gigaspora margarita only. When both

symbionts were present, the effects on plant growth by the mycorrhizal

fungus and Rhizobium effectively cancelled, and the plants grew

similarly to the noninoculated controls. Shoot/root ratio were greater

for all inoculated plants compared to noninoculated controls. Under

low ozone stress plants inoculated with both Rhizobium leguminosarum

and Gigaspora margarita transported a greater proportion of

photosynthate to roots than did noninoculated plants. At the highest

level of ozone stress this did not occur probably because little

photosynthate was available and this shoots retained most of it for

repair of injury.

The effect of drought on nutrient control and leaf water status in

aflafla plants inoculated with a mycorrhizal fungus and or Rhizobium

was studied by Goicoechea et al., (1997). The four treatments include

plants inoculated with Glomus fasciculatum and Rhizobium meliloti,

plants inoculated with Rhizobium meliloti only, plants inoculated with

Glomus fasciculatum only and non inoculated plants. Plants exhibited

similar plant dry weight in all treatments under well watered

conditions; but drought stressed noninoculated plants were

significantly lower in plant dry weight. When compared with other

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treatments. Nodule dry weight was higher in Rhizobium inoculated

plants than mycorrhiza with Rhizobium inoculated plants. Drought

stress decreased the nutrient content of leaves and roots of

noninoculated plants. Rhizobium inoculated plants showed a decrease

in nutrient content of leaves but maintained some micronutrients in

roots. Drought stress reduced leaf and root ‘N’ content in

noninoculated plants; whereas Rhizobium, mycorrhiza and Rhizobium

with mycorrhiza inoculated plants had higher leaf ‘N’ content.

The effectiveness of dual inoculation of Bradyrhizobium and

Glomus microcarpum in Yam bean, Pachyrhyzus was observed by

Rajith Kumar and Potty (1998). There was a favourable increase in the

overall development of the inoculated plants especially dual

inoculation. The plants which were treated with AM and

Bradyrhizobium produced the biomass more than three times the

weight of the control plants. In addition to the biomass, the production

of nodules, soil spores and percentage of AM colonization etc also had

much increase. AM showed its efficiency in absorption of phosphate

from the soil. Rhizobium absorbed nitrogen and supplied to the plants.

Both these processes enhanced the total production of the plant body.

Control plants have shown less amount of nodules, spore production

and root colonization.

Bhatia et al., (1988) assessed the growth of Prosopis juliflora and

its contribution to soil enrichment following inoculation with three

vesicular arbuscular mycorrhizae and an indigenous strain, and two

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Rhizobium isolates. There was a significant increase in the biomass of

closely spaced Prosopis juliflora inoculated with Glomus caledonius

alone. The girth at 50 cm above the ground values of plants inoculated

with Glomus caledonius and one of the Rhizobium isolates together and

those inoculated with Gigaspora calospora with Rhizobium isolate were

similar to those of the uninocualated controls and significantly lower

than the other treatments. The measurement of plant height during

the early years of growth, recorded at intervals of six months revealed

that the height of the plant was at par and significantly higher in

plants inoculated with Glomus caledonius and Rhizobium isolate alone.

Plant height increased significantly when a single inoculation with

Glomus caledonius, and dual inoculation with Gigaspora calospora and

Rhizobium together, in comparison to the other treatments. Biomass of

plants inoculated with Glomus caledonius alone suppressed the

biomass produced in the other treatments, but when plants were

inoculated with this AM in association with either of the Rhizobium

strains there was no statistical difference between the biomass

produced in these treatments and those uninoculated controls.

The dual inoculation of Rhizobium leguminosarum and AM

resulted in disease resistance in vicia faba and associated growth

improvements (Rabie, 1998). Infection of vicia faba with Botrytis fabae

caused significant decrease in growth viogur, total nitrogen content,

number of nodules and nutrient accumulation. Co-inoculation of

Glomus mosseae and Rhizobium leguminosarum improved plant growth

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of both diseased and non diseased faba bean plants, indicated that

mycorrhizae and Rhizobia were still functioning in the presence of the

pathogen. The root: shoot fresh and dry weight ratios were smaller for

all healthy or diseased mycorrhizal plants than for the corresponding

non mycorrhizal plants. But Mycorrhizal plants exhibit significantly

higher stem dry weights than those of corresponding non-mycorrhizal

plants. The morphological symptoms of infection were much lower in

mycorrhizal than in non mycorrhizal plants. Root colonization by AM

improved nodulation of healthy and diseased bean plants grown on

soil treated with Rhizobium leguminosarum.

Total and free phenol contents were significantly higher in

mycorrhizal plants. Thus is untreated plants the percentage of

infection of Botrytis was 83%, and if infected plants received

Rhizobium leguminosarum alone, Glomus mosseae alone or both of the

two symbionts the percentage of Botrytis infection decreased to 69.8%,

32.3% and 19.2% respectively.

Effect of mycorrhizae helperbaceteria on AM fungal colonization

and accumulation of secondary compounds, in barely and wheat roots

were studied by Fester et al., (1999). The three species of potential

MHB tested in the present study for their effects on barely root

mycorrhiza increased mycorrhiza levels two to three fold. The

frequency of colonization achieved with Glomus intraradices alone was

about 20%. Co-inoculation with Pseudomonas fluorescens, Rhizobium

leguminosarum or Agrobacterium rhizogenes enhanced fungal

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colonization up to, 40, 50 and 60% respectively. Level of one

prominent isoprenoid cyclohexanone derivative Bluemenin showed a

significant increase in this co-inoculation.

Goicoechea et al., (2000) observed the co-inoculation effects of

Rhizobium and/or Glomus on medicago. In his study the growth of

plants inoculated with both mycorrhiza and Rhizobium was compared

with that of four groups of Rhizobium inoculated, but not associated

with mycorrhizal fungus; plants, which received ‘P’ in the nutrient

solution shoot height, leaf, stem and root dry matter and number of

plant leaves and stems were similar in mycorrhizae with Rhizobium

plants. Rhizobium with 0.1mM P and R with 0.2mM P treatment, and

significantly higher than those measured in R with 0.02mM P and R

with 0.05m MP plants. The best nodulation was observed in

mycorrhizal with Rhizobium plants and in non mycorrhizal ones which

received the highest supply of P.

Dual inoculation of woody legume with Rhizobium and

mycorrhizal fungi were studied by Marques et al., (2001). Complete

fertilization was compared to treatments of inoculation with selected

Rhizobia strains, associated or not to AM fungi. The dual inoculation

increased the height and growth in relation to the plants treated with

Rhizobia alone. Plants undergo dual inoculation were taller and had

significantly greater dry weight- as compared to the control plants. The

combined inoculation increased the biomass production by 56.4% as

compared to the N-fertilized plants. The N content was also greater in

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dual inoculated plants, when compared with the uninocualted

fertilized plants. Survival index was also greater in co-inoculated

plants. AM seemed to favour the nodule occupation by Rhizobia

strains as compared to the nonmycorrhizal plants.

Plant drymatter weight and phosphorus content of shoots

showed a strong relationship between the type of microbial inoculants

introduced into the soil plant system and plant responses, (Vassilev

et al., 2001). Interactions of AM fungi with free or encapsulated cells of

Rhizobium trifoli and Yarowia lipolytica into soil-plant system was

studied. Single inoculation of Rhizobium trifoli resulted in the lowest

plant growth and ‘P’ content. Dual inoculation with Glomus deserticola

and Rhizobium trifoli considerably enhanced the responses. Whereas

the inclusion of Yarowia lipolytica provided the most effective

treatment combination. In general inoculation with the AM fungus

alone or in combination with a type of yeast by name Yarrowia

lipolytica positively affected the development of Rhizobium trifoli.

Mycorrhizal plants possessed higher nodule numbers than did the

nonmycorrhizal treatments. It seems that triple inoculation with an

AM, nitrogen fixing bacterium and phosphate solubilising yeast

culture resulted in highly effective relationship that provoke benefits

for all participants including the plant.

The rate of mycorrhizal colonization by the two AM was not

affected by the ‘N’ level in the medium (Vazquez, 2001). The effect of

nitrogen in the medium affecting the interaction between

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Sinorhizobium and AM fungi was studied, in Medicago sativa. Growth

response, nutrient acquisition, protein content, and nitrate reductase

activity were measured in plant shoots and roots. Results showed that

Nitrogen level in soil did not affect mycorrhizal colonization but they

strongly influenced nodulation, in mycorrhizal plants Glomus

intraradices was the most effective AM fungus at increasing alfalfa

growth, over the nonmycorrhizal controls. N addition increased shoot

nitrate reductase activity. This effect was more evident in mycorrhizal

plants ie, Glomus intraradices colonized plants had the highest nitrate

reductase activity in roots. Protein content was higher in Glomus

intraradices inoculated plants.

Mycorrhizal and Rhizobium inoculation resulted in a significant

increase in the phenolic acid content of groundnut plants as compared

to uninoculated plants Maximum total phenol content was recorded in

AM fungus and Rhizobium inoculated roots and shoots. Ortho dihydric

phenolic acid content was maximum in dual-inoculated plants

compared to individual inoculations, both in roots and shoots. No

qualitative changes as observed in the uninoculated plants while

inoculation resulted in an overall increase in the existing phenolic

acids and also in the appearance of some new compounds in both

roots and shoots, (Devi and Reddy, 2002). The phenolic compounds in

plants are known to play an important role in the host defense

mechanism against disease.

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Co-inoculation responses with AM Fungi and Rhizobium in lentil

were studied by Xavier and Germida, (2002). The growth and yield

responses of lentil to co-inoculation with AMF and Rhizobium strains

dependent on the particular AMF-Rhizobium strain combination. In

some cases productivity of lentil inoculated with an effective Rhizobium

strain was significantly reduced by an apparently incompatible AMF

species compared to the Rhizobium treatment. In contrast to the yield

of lentil inoculated with some ineffective Rhizobium strain was

significantly enhanced by an apparently compatible AMF species

compared to the Rhizobium treatment. The effect of Glomus clarum or

Glomus mosseae inoculation on the shoot growth of plant was not

significantly different from each other. But the dry weight of shoots

inoculated with Glomus clarum and Glomus mosseae was higher than

that of the uninoculated control plants, regardless of the Rhizobium

strains. Shoot biomass was significantly increased in co-inoculation

plants compared to the uninoculated control. Glomus clarum

significantly enhanced total root dry weight including nodules, of

plants compared with Glomus mosseae inoculated, or control plants,

irrespective of the Rhizobium strain in combination. Analysis of main

AM treatment effect on lentil yield revealed that the native AM

community was more effective at enhancing lentil yield compared with

Glomus clarum or G. mosseae; regardless of the Rhizobium strain.

Plants inoculated with Glomus clarum contained the highest amount of

total shoot N compared to uninoculated plants or those co-inoculated

117

with Glomus mosseae; regardless of the Rhizobium treatment. The

total shoot ‘P’ content of lentil inoculated with Glomus clarum was

significantly higher than Glomus mosseae among the AM treatments,

regardless of the Rhizobium treatments.

The studies of Rao et al., (2003) revealed that dual inoculated

Dalbergia sissoo plants performed better showing enhanced values of

all the parameters in comparison to single inoculated plants. Of

several dual combinations of rhizobial isolates and AM fungi tested,

the seedlings inoculated with Rhizobial isolates and Glomus

fasciculatum recorded maximum enhancement of growth, in terms of

shoot length, shoot dry weight, root length, root dry weight, and in

values of total plant protein, chlorophyll, total sugar total nitrogen and

total phosphorus content as compared to the seedlings inoculated with

Rhizobium isolate alone. Nodulation and total leghaemoglobin content

were greatly influenced by dual inoculation in test plants. Maximum

enhancement of nodulation in terms of nodule number, nodule dry

weight, maximum nodule size and total leghaemoglobin content was

found in seedlings inoculated with isolate Rhizobium with Glomus

fasciculatum as compared to other dual inoculated plants. While

seedlings with isolate Rhizobium alone recorded maximum nodule

number, nodule dry weight, nodule size and total leghaemoglobin

content. Plants inoculated with AM and uninocualted control did not

bear nodule. Maximum notrogenase activity of nodulated roots was

recorded in seedlings inoculated with Rhizobium with Glomus

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fasciculatum in comparison to seedlings inoculated with Rhizobium,

and thereby showing 32% enhancement.

According to Xavier and Germida (2003) dual inoculation of AM

fungi and Rhizobium leguminosarum enhanced the pea yield and

nutrition in Pisum sativum. Plants were inoculated with the AM species

Glomus clarum or Glomus mosseae and ten Rhizobium strains. The

shoot dry weight of pea was not affected by Glomus clarum compared

to the control and was significantly lower than that of Glomus mosseae

inoculated plants irrespective of the Rhizobium stain. Irrespective of

Rhizobium strain used, the total root weight of plans inoculated with

Glomus clarum and Glomus mosseae was not significantly different

from each other. The effect of the Glomus clarum inoculants on the

seed yield of pea was inferior to the native AM and Glomus mosseae.

Inoculation of pea with Rhizobium strains significantly altered the yield

of pea plants, and regardless of AM species, most inoculants increased

grain yield relative to the uninoculated control; Glomus clarum with

Rhizobium combination resulted in higher yields than Glomus mosseae

with Rhizobium combination. This yield increase was around 116%

higher than the control plants, 34% higher than plants inoculated with

Rhizobium alone and 48% higher than Glomus mosseae with

Rhizobium, combination. In general, treatments with effective

Rhizobium strains or co-inoculation treatments with effective

Rhizobium strains and a compatible AMF species produced the best

results.

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Tian et al., (2003) studied the co inoculation responses of ecto

and arbuscular mycorrhizae and Rhizobium on the growth and

nitrogen fixation by black locust (Robinia pseudoacacia). In pure

culture, the growth and nitrogen fixing ability of the seedlings in all

the inoculated treatments improved significantly and the treatment

with dual inoculation was better than single inoculation treatment.

The growth of seedlings inoculated with ecto and arbuscular

mycorrhizae and Rhizobium was significantly better than that with

only one or two microbes. The treatment with best effect was the one

inoculated with ecto and arbuscular mycorrhizae and Rhizobium in

which the seedlings had the fastest growth rate, the strongest nitrogen

fixing ability and the best mycorrhizal development.

The dual inoculation effect with Rhizobium and mycorrhizae on

two native forage legumes were studied by Shockely et al., (2004).

Seeds of legumes were planted in a green house and inoculated with

one of the two species on AM and or one of two strains of Rhizobium.

In Illinois bundle flower (one of the leguminous plants), the number of

nodules per plant was statistically similar for both Rhizobium strains.

Both species of AM Glomus intraradies and Glomus etunicatum

successfully colonized the Illinois bundle flower roots, and percentage

of colonization was similar for the two species. Plants colonized by

Glomus intraradices had grater leaf dry weight, stem dry weight and

root fresh weight compared to control plants. Glomus intraradices and

Glomus etunicatum both successfully colonized the roots of panicled

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tick clover. Plant colonized by Glomus intraradices had increased stem

height and leaf dry weight compared to the control; but plants

colonized by Glomus etunicatum did not. Both species of AM increased

P and K concentrations 41% and 55% respectively in panicled tick

clover. These results suggest that growth of these can be improved by

the use of proper AM species and/or Rhizobium strains. Both legumes

exhibited enhanced growth implants inoculated with Glomus

intraradices compared to the controls Glomus etunicatum also tended

to enhance plant growth but the differences were not significant.

2.2.0 RHIZOBIUM

Rhizobium is a chemoheterotrophic organism, coming under the

family Rhizobiaceae. This family include five distinct genera,

Azorhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and

Rhizobium (Lum and Hirsch, 2003). Genus Rhizobia has been

considered as fast growing Rhizobia and Bradyrhizobia has been

considered as slow growing Rhizobia, (Giller and Wilson 1991). Fast

growers grow much faster in culture than the slow growers. Six genera

coming under this single genus Rhizobium are Rhizobium

leguminosarum, R. trifoli, R. Meliloti, R. Lupini, R.phaseoli and R.

Japonicum, (Nutman, 1975).

It is an aerobic heterotroph, it is easily grown on a range of

bacteriological media that contain yeast extract as a source of

accessory growth factors. It survives at low temperature and can be

freeze dried but is soon killed at temperature above 500c for more than

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a few hours or by drying, (Diatloff, 1970). Growth is usually best at

neutral or slightly acidic pH. Most strains produce acid in culture but

this is not related to the acidity of the soil of origin, (Jones and

Burrows, 1969). Rhizobium produces plant growth substances such as

indole derivatives, cytokinins and trace of Gibberellic acid, although

more is produced in the nodule than in culture, and some strains

excrete B vitamins, plant Wilting factors, bacteriocins and porphyrins,

(Hamdi, 1971).

The species and strains of Rhizobium are differently affected

by acidic or alkaline soil conditions, (Van Schreven, 1972). Rhizobium

was shown to be intolerant of salinity. The growth of Rhizobium

japonicum is strongly retarded in media containing 0.008M Nacl

and does not grow with 0.16M Nacl. (Wilson and Norris 1970)

Subbarao et al., (1972) found Rhizobium meliloti more tolerant of salt

than its host plant. In sterile soil cultures allowed to dry slowly,

Rhizobia can survive for decades.

Because Rhizobium is similar culturally and biochemically to

many other soil organisms, it can not easily be distinguished on soil

plates. More reliable counts can be made by using the host plant in a

biological assay in which dilutions made from a weighed quantity of

soil are added to sterile grown plants. Any rhizobia present multiply

rapidly in the root surroundings, so that all dilutions containing at

least only one organism will modulate the test plant, (Wilson, 1930).

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Rhizobium inoculation is a promising fertilizer, because it is

cheap, easy to handle and improves plant growth and seed quality.

The efficiency of inoculation could be improved with the addition of

biological, chemical or organic fertilizers. Inoculation of Rhizobium

strain significantly increased yield and 100-seed weight of the faba

bean, and also increased the total nitrogen of faba bean, (Elsheikh and

Elzidany, 1997). Nitrogen fixation was improved in Acacia mangium

through inoculation with Rhizobium, (Galiana et al., 1998).

Among 5 strains of Rhizobium tested, the Bradyrhizobium

strains were found to be most efficient. Positive and significant effect

on plant height, and basal area was observed. Acacia mangium

inoculated with Rhizobium fixed more nitrogen and had a better

growth than uninoculated ones. Rhizobium etli strains increased

nodulation in phaseolus vulgaris than Rhizobium tropici strains and

there by increasing the capacity to fix nitrogen, (Martinez-Romero et

al., 1998). ie, the nodule, numbers were dependent out on the strain

and the cultivar used. But differences in nodule number were not

reflected in plant biomass.

The nitrogen fixing activity of inoculated plants was measured

by means of acetylene reduction assay. Diazotrophic activity in all

uninoculated plants was very low and significantly differed from that

of inoculated plants. Plant inoculation with the Rhizobium strain

induced a significant increase of net photo synthetic rate in both

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strains of Rhizobium. Total biomass of inoculated plants was always

higher than that of uninoculated owns (Lippi et al., 1999).

Effect of Rhizobium inoculation in two varieties of Pisum

sativum on nodulation and nitrogen fixation has been studied by

Bajaj et al., (1999). Inoculated plants showed increase in number of

nodules, nodule fresh weight, shoot dry weight nodule leghaemoglobin

content and nitrogenase activity over uninoculated control plants.

Mixed inoculation of vicia faba with four different

Rhizobium/Azospirillum and Rhizobium/Azotobacter combinations led

to changes in total content, concentration and/or distribution of the

mineral macro micrountrients K, P, Ca, Mg, Fe, B, Mn, Zn and Ca

when compared with plants inoculated with Rhizobium only, (Rodelas

et al., 1999).

Rai and Singh (1999) were screened Rhizobium strains in order

to select for suitable lentil (Lens culinaris) genotypes, that produced

high yields in normal and salt affected soils. Interaction between salt

tolerant lentil genotypes and Rhizobium strains were found to be

significant and resulted in greater nodulation, N2 fixation, total

nitrogen, plant height root length and grain yield in sodic soils under

field conditions compared to the uninoculated controls.

Rhizobium inoculation significantly increased the total nodule

number per plant, 100 seed weight and protein content of seeds of

chickpea cultivars (EI Hadi and Elsheikh, 1999). The results indicated

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that the three Rhizobium strains are ineffective in Nitrogen fixation,

and increased yield by 72%.

Nodulation at the late flowering stage was significantly

influenced by Rhizobium leguminosarum inoculation in faba bean and

the N2 fixation was highly positively correlated with the dry matter

production and total N yield of faba bean, (Amanuel et al., 2000).

Inoculation of soybean cultivars with several rhizobial strains

showed good nodulation and fixed as much as N as four

Bradyrhizobium japonicum/B. elkanii strains. Cutivar Davis was more

compatible with the fast growers and nodule occupancy was about

43%, (Hungria et al., 2001).

Combined of Rhizobium strains tested enhanced the growth and

total nitrogen accumulation of the lupin cultivars. Rhizobium

inoculation increased seed and straw yield compared with the non

inoculated control. It was suggested that the improvement through

development of combined inoculation strains could be possible and

would offer security for nodulation, (Raza et al., 2001).

Co-inoculation responses of Rhizobium meliloti strains, AM fungi

and plant growth promoting bacteria on Medicago arborea were

studied by Valdenegro et al., 2001). PGPR increased growth of Glomus

mosseae colonized plants associated Rhizobium WT strain by 36% and

those infected by Glomus deserticola associated with Rhizobial GM

strain by 40%.

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Pea root zones differing in their susceptibility to the Rhizobium

leguminosarum infection are characterized by different directions of

changes in peroxidase activity. Peroxidase is an indicator of plant

stress as a response to the biotic factor. Peroxidase in the case of

symbiosis, is likely to determine the nodulation self-regulation

mechanism, controlling its degree via bacteria penetration in the

susceptible root zone and preventing it in other root zones,

(Akimova et al., 2002).

Leucaena leucocephela plants inoculated with alginate beads of

Rhizobium were significantly more developed and more nodulated than

plants inoculated with other methods, (Forestier et al., 2001). Plants

here showed very significant difference. It corresponds to an increase

of 35% in the biomass of the nodules, and proximately 50% of shoot

dry weight compared to the inoculated plants with the other four

classical methods.

Strain-cultivar interrelationship and specificity of Rhizobium-pea

symbiosis was studied by Labidi et al., (2003). Different classes were

identified on the basis of efficiency parameters and identified to

isolates as efficient strains in fixing nitrogen with peas.

Effect of inoculation of mot effective strains of Rhizobium tropici

on Phaseolus vulgaris showed that nodule occupancy increased from

an average of 28% in the first experiment to 56% after four inoculation

procedures. The establishment of selected strains increased,

nodulation, N2 fixation rates and yield. A synergistic effect between

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low levels of N fertilizer and inoculation with superior strains was also

observed; resulting in yield increase, (Hungria et al., 2003).

Rhizobium etli which normally forms nitrogen fixing nodules on

Phaseolus vulgaris is a natural maize endophyte. Some strains of R.

etli were more competitive maize root colonizers than other strains

from the rhizosphere or from bean nodules, (Rosenblueth and

Martinez-Romero, 2004).

The nodule occupancies by an individual strain Bradyrhizobium

japonicum were directly correlated with the proportions of that

strain in the inoculum mixtures. Strain USDA 110 showed higher

nodulation competitiveness than other strains in soybean cultivars,

(Payakapong et al., 2004).

High molecular weight lectins from seeds of the legumes

temporarily stimulated the respiration of Rhizobium tropici and R. etli.

These stimuli induced by the lectins increased the significance of the

interaction lectin Rhizobium, (Martinez et al., 2004).

The division of Rhizobium into species is based on interaction

with plants; those bacteria that nodulate clovers are put in R. trifoli

and those that nodulate peas and vetches put in R. leguminosarum. In

the slow growing Rhizobia there is a group has wide specificity

that it has not been assigned to any species but is called as

cowpea miscellany group. Some strains of Rhizobium can form stem

nodules, eg: Sesbania rostrata and Aeschynomene afraspera,

(Mishra et al., 2005).

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The interaction between VA mycorrhizae Glomus mosseae, root

nodulating symbiont Rhizobium leguminosarum and root rot pathogen

Fusarium solani on common bean was studied by Hassan Dar et al.,

(1999). Mycorohizal root colonizing ability was reduced by 27% in the

presence of Fusarium solani, where Rhizobium enhanced the ability by

37%. In presence of Rhizobium, Glomus mosseae inoculated plants

were more tolerant of the fungal root pathogen. The Glomus with

Rhizobium inoculated plants had maximum plant biomass and root

nodulation, higher alkaline phosphatase activity and available

phosphorous in the rhizosphere.

2.3.0: COWPEA

Pulses which form an important grain all over India as dry seed,

as green pod, as edible oil, or as a solvent in pharmaceutical aid etc.

Taxonomically pulse crops coming under Kingdom - Plantae,

subkingdom - Phanerogamia, division - Angiospermia, class -

Dicotyledons, subclass - Calyciflorae; order – Leguminoase and sub

order- Papilonaceae, (Bose, 1987). The pulses production in the

country is about 13.78 million tonnes from an area of 22.6 million ha

India has largest area under. Chickpea, pigeon pea, lentil, dry beans

and total pulses in the world. Except chickpea and pigeaopea the

average productivity of other pulses in the country is significantly

lower than the average yield in the world, (Masood Ali and

Shivakumar, 2005). Pulses were taken up as the option of providing

protein component of the Indian diet and research on pulses was

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initiated at IARI during the 1940s, beginning with the collection of

germ plasm from local as well as exotic sources, (Sharma and Tickoo,

2005).

Cowpea is an old and revered crop apparently native to central

Africa. These are of ancient cultivation in Asia, Africa and Greece. It is

one of the principal pulses of common use in India. Cowpea is adapted

to a great variety of climatic and soil conditions. But it requires

considerable heat and extremely sensitive to cold. It withstands shade

and drought, (Ahlgren, 1956).

Morphologically it is twining rarely sub erect herbs or under

shrubs; stem scabrid, hairy at the nodes; leaves pinnately trifoliate,

membranous ovate, rhomboidal, entire or slightly lobed, stipellate,

stipels subulate; stipules large, basifixed or rarely peltate.

Inflorescence usually few flowered, axillary, racemes or fascicles,

penduncle long, flowers often in alternate pairs. Pods, lines, slender

often very long, rounded or compressed. Seeds, small but varied

usually subreniform, (Sambamurthy and Subrahmanyam, 1989,b).

It is rarely grown as a pure crop. Commonly it is grown as one of

the subsidiary corps of either jowar, bajra or ragi. It can be grown

through out the year under Kerala conditions; as a floor crop in

coconut garden and as an intercrop in tapioca during May-Sep. It can

be grown in homstead garden through out the year and in Kole lands

of Thrissur during summer. But as a rainfed crop sowing is done in

the month of June. During the second crop season ie Sep-Dec, it can

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be grown as a fringe crop along the rice field bunds. During summer it

can be grown as a pure crop in rice fallows after the harvest of paddy,

(Jose, 2002). Cowpea seeds should be inoculated with Rhizobium and

pelleted with lime. Mix the inoculant uniformly with the seeds using

minimum quantity of water, or 2.5%, starch solution or Kanjivellam of

the previous day, to make better stickiness of the inoculant with the

seeds. Dry these seeds under shade over a clean paper or gunny bag.

Add finely powered calcium carbonate to moist fresh Rhizobium treated

seeds and mix for 1-3 minutes until each seed is uniformly pellted.

Spread them on a clean paper to harden (Lime coating is required only

for acid soils) sow the seeds immediately.

Lime may be applied at the time of first ploughing. Half the

quantity of nitrogen, whole of phosphorus and potash may be applied

at the time of final ploughing. The remaining nitrogen may be applied

15-20 days after sowing. Giving two irrigations is highly beneficial ie at

15 days after sowing and at the time of flowering. Irrigation at the

flowering stage induces better flowering and pod set.

The crop comes flowering in about one and half months after

sowing and is ready for harvest in about three months. All pods do not

mature at the same time. After maturing they are likely to shatter, at

least in some varieties. There fore these are hand picked now- and

then and stored. The average yield of grain for a rainfed crop is

dependent on the seed rate giving 113-360 kg/ha as a pure crop,

(Sambamurthy and Subrahmanyam, 1989). The pods are ready for

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picking when they turn yellowish brown and seeds are firm, (Desai, et

al., 1997).

It is an important source of nutrient. Its protein content is 3-4%

in green leaf 4-5% in immature pods and 25-30% in immature seeds.

Aminoacids like lysine, leucine, phenyl alanine etc. are relatively high.

It contains approximately water 11%, fat 1.3%, carbohydrate 56.8%,

fibre 3.9%, ash 3.6%, (Som and Hazra, 1993).

2.4.0: MAIZE

Maize is one of the important cereal crops of the world. In terms

of area and production it ranks only next to wheat and rice, while in

yield it surpasses all cereal crops. On an average the yield of maize in

the world is 3082 kg/ha. The most important maize growing countries

of the world are: USA, Mexico, Brazil, Argentina, Yugoslavia, Rumania

etc, (Sambamurty and Subrahmanyam, 1989). In India it is grown in

6.5 million ha and its 45% of the total production is consumed as

food. It is used as food, forage and in industry. Maize occupies an

important position in Indian agriculture. Among cereals it ranks fifth

in area and third in production. Out of various speciality corns, sweet

corn (Zea mays L. saccharata) is one of the most popular vegetables in

the USA and Canada. It is becoming increasingly popular in India and

other Asian countries (Rai et al., 2005).

Maize may have originated in Mexico or Central America

because this area is considered to be the home of teosinte grass

(Euchlaena) a near relative of maize (Bland, 1971).

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Maize (zea mays) belongs to the family poaceae (Graminae),

Zea mays L. is a tall annual grass having broad leaves arranged in two

vertical ranks. The inflorescence are monoecious ie: the tassel is

staminate and sheds pollen while the ear shoot is pistillate, producing

silks or styles. The flowers of the tassel are born in numerous spike

like racemes which together form large spreading panicles which

terminated the stems. A pistillate inflorescence is born in one or more

axils of the leaves. The spikelets are arranged in 8 to as high as 30

rows on a thickened almost woody axis known as the cob., the whole

being enclosed in foliaceous bracts or husks. The long style or silks

protrude from the top of the bracts. The spikelets are unisexual.

The staminate spikelets occur in pairs in the tassel and are two

flowered. The pistillate spikelets are sessile and occur in pairs,

consisting of one fertile and one sterile floret (Sambamurty and

Subrahmanyam, 1989,a).

Maize is becoming increasingly popular in India, it may be grown

in all types of soil and is moderately salt tolerant, it can successfully

be grown in well drained soils with pH of 5.5-7.0 requires through

ploughing followed by land levelling. Ridges are to be laid out with

rows 75cm apart. Planting June-July in Kharif and September-October

in rabi both white and yellow grain type are grown, 10-11 kg of corn

seeds/ha are required. Two seeds/hill are dibbled manually or

mechanically. Depending upon the soil, number of irrigations varies

from 4-5 or 7-8. Fertilizers dose of 100-120 kg nitrogen, 50-60 kg

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phosphate and 4-60 kg potash/ha. Sweet corn matures early and can

be harvested 75-80 days after sowing. Sweet corn yields about 66,000

ears/ha. High density planting can give higher number of harvested

ears. (Rai et al., 2005).

Maize plant is extensively used as a cattle feed both as fresh

green fodder, and about 85% of maize production is used as food. It is

used in industry to produce a large number of industrial products,

in fermentation industry in the manufacture of ethyl and butyl

alcohol, acetone etc. Total sugar content in sweet corn ranges from

25-30% (Sambamurty and Subrahmanyam, 1989). Maize is used

as a host plant for large inoculum production of VAM fungi

(Sharma, et al., 1999).

Gryndler and Vosatka (1996) reported that Pseudomonas putida

has an effect on growth and mycorrhiza formation of maize.

Mycorrhizal infection of the roots was significantly higher when plants

were inoculated with Glomus fistulosum together with the living cells of

Pseudomonas putida.

Sahoo and Mahapatra (2004) reported that increase in levels of

nitrogen increased the number if cob, length and weight of green cob,

yield of fresh grain and ear of maize. Increase in nitrogen levels

increased the net profit. Thus the application of 120 kg N/ha to a

population of 66,666 plants/ha is recommended for sweet corn

cultivation (Growth regulators like Gibberellic acid, Indole acetic acid

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and benzyladenine were found to influence the growth and yield, and

quality of sweet corn (Vani et al., 2004).

Under salt stress, Glomus mosseae inoculated maize plants

showed higher soluble sugars and electrolyte concentrations. The

above findings suggested a higher osmoregulating capacity of these

plants and showed higher root and shoot dry weights. The

concentration of chlorophyll and P were higher in mycorrhizal maize

plants (Feng et al., 2002).