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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.
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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.
80
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
82
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.
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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
91
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%
97
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
112
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
113
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
128
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).
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