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ISOLATION, IDENTIFICATION AND CLASSIFICATION OF AM FUNGAL SPORES FROM RHIZOSPHERIC SOIL OF FOUR
EXPERIMENTAL PLANTS
INTRODUCTION
Various types of microorganisms are present in soil, play vital roles in numerous
physiological activities. These dynamic activities are mediated by association of
microorganisms participating in saprophytic, pathogenic and symbiotic association with root.
Mycorrhizal fungi are ubiquitous, occurring in all natural ecosystems in most climatic zones
throughout the world. The mycorrhizal habitat probably evolved as a survival mechanism for
both partners in the association, allowing each to survive in environment of low fertility,
drought, disease and temperature extremes where, alone they could not.
Early morphological classifications separated mycorrhizas into endomycorrhizal,
ectomycorrhizal and ectendomycorrhizal associations based on the relative location of fungi
in roots (Peyronel et al., 1969). These three types were not enough to describe the diversity of
mycorrhizal associations. Harley and Smith (1983) had given the generally accepted
classification. These include Ectomycorrhizae, Endomycorrhizae, and Ectendomycorrhizae,
Arbutoid mycorrhizae, Monotropoid mycorrhizae and Orchid mycorrhizae.
Arbuscular Mycorrhizal (AM) fungi are a type of endomycorrhizae. The diagnostic
feature of arbuscular mycorrhizae (AM) is the development of a highly branched arbuscule
within root cortical cells. The fungus initially grows between cortical cells, but soon
penetrates the host cell wall and grows within the cell. As the fungus grows, the host cell
membrane invaginates and envelops the fungus, creating a new compartment where material
of high molecular complexity is deposited.
The AM fungi are the most intensively studied types of mycorrhizae because they are
present in most agricultural and natural ecosystems and play an important role in plant
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growth, health and productivity (Harley and Smith, 1983; Gianinazzi et al., 1990; Hosamani,
2005; Manimegalai, et al., 2011, Kanchana and Gupta 2012.). There are only a few genera
belonging to Brassicaceae; Chenopodiaceae and Cyperaceae where they are not found due to
the presence of glucosinolates and their hydrolysis products isothiocyanates in and around the
roots (Glenn et al., 1988; Hui, 2011), which are toxic to the growth of fungi.
Mycorrhizal colonization begins with the hyphae that arise from soil-borne propagules,
large resting spores of the AM fungi or mycorrhizal root fragments. The fungal hyphae
penetrate the root between the epidermal cells and form an appressoria in the first cell layers.
This stage marks the autotrophic growth of the fungus. The colonizing hyphae pass through
the intercellular spaces and then enter the root tissues spreading between and through cells of
the cortical root layers. Once the hyphae have reached the inner cortex they grow into the
cells and form tree-like structures called 'arbuscules'. These branched hyphae are closely
surrounded by the intact host plasmalemma and represent a large surface of cellular contact
between both symbionts. These facilitate the exchange of metabolites between host and
fungus. The arbuscules are probably the main transfer site of mineral nutrients, mainly
phosphorus, from the fungus to the plant and of carbon compounds to the fungus (Smith and
Gianinazzi-Pearson, 1990; Bonfante and Bianciotto, 1995). As internal colonization spreads,
the extraradical hyphae ramify, and grow along the root surface forming more penetration
points. They also grow outwards into the surrounding soil, thus developing an extensive tri-
dimensional network of mycelium which interfaces with soil particles. Smith and Gianinazzi-
Pearson (1990) had demonstrated that the length of the external hyphae growing in soil
associated with mycorrhizal roots reaches an average of 1m cm-1 root, but values of up to 10-
14 m cm-1 root have also been recorded. These mycelial network can extend several
centimeters outwards from the root surface, bridging over the zone of nutrient depletion
around roots to absorb low-mobile ions from the bulk soil (mineral nutrients). In return the
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plant provides the fungus with sugars, amino acids and vitamins essential for its growth
(Harley and Smith, 1983).
Arbuscular mycorrhizal (AM) fungi found in rhizosphere of several vascular plants and
have important roles on sustainable agriculture as well as agricultural ecosystems
management. The beneficial effect of indigenous AM fungi on the nutrition of agricultural
plants depends on both the abundance and type of fungi present in the soil (Abbott and
Robson, 1982). However, the potential for employing AM fungi on a wide scale in
agriculture is dependent on the development of crop-growth-promoting strains of AM, which
are superior to native soil population of AM fungi (Menge, 1983). Therefore, field study is
necessary to understand the abundance and type of indigenous AM fungi present in the
rhizosphere of the crop. In this view the present study was undertaken to isolate, identify and
classify the indigenous AM fungi associated with four experimental plants grown at different
localities of Dharwad taluk, in Dharwad district of Karnataka, INDIA.
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REVIEW OF LITERATURE
The occurrence of arbuscular mycorrhizal fungi in South Africa (old name:
Endogonaceae) was first reported by Hattingh (1972) when he discovered a honey coloured
sessile spore attached to the stalk of an empty mother spore. This was found in large numbers
in the rhizosphere soil of maize from the Outeniqua Farm, George, Cape Province and was
later designated as Acaulospora laevis (Coetzee, 1982; Hattingh, 1972). Arbuscular
mycorrhizal (AM) fungi are obligate symbiotic fungi and endosymbionts of a variety of
plants within the Angiosperms, Gymnosperms and Pteridophytes (Steinberg and Rillig, 2003;
Smith and Read, 1997). AM fungi have three major components: the root itself which
provides carbon in the form of sugars to the fungus, fungal structures within cortical cells of
plant root that provide contact between fungus and the plant cytoplasm and the extraradical
hyphae that aid uptake of nutrients and water (Smith and Read, 1997).
The evolution of AM fungi can be dated back 460 million years ago from fossil
records of the Ordovician age. These records suggest that AM fungi may have played a
crucial role in colonisation of most terrestrial plants (Brundrett, 2002; Redecker et al., 2000;
Smith and Read; 1997). The taxonomy of AM fungi has been based on morphological and
anatomical characteristics of their spores. Other modern techniques such as serology,
isozyme variation revealed by electrophoresis (Hepper et al., 1988), fatty acid variation
(Bentivenga and Morton, 1994) and DNA based methods (Helgason et al., 1999, Schupler et
al., 2001, Morton and Redecker, 2001)
Muthukumar et al., (2000) have recorded the distribution of VAM spores in semi-arid
zone of Western Ghats of India. Beena et al., (2000) studied the diversity of AM fungi on
coastal sand dunes of the West Coast of India. Lakshman et al., (2000) studied seasonal
fluctuation of VAM fungi in cultivated crops of Dharwad district of Karnataka. Gupta et al.,
(2002) was studied the arbuscular mycorrhizal association of mangroves in saline and non-
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saline soil. Since then similar species and others like Glomus fasciculatum, Gl. intraradices,
Gl. etunicatum and Gigaspora sp. have been found present in Fouriesberg in the Free State
Province, Nylsvley Nature Reserve as well as in association with indigenous plants such as
Vangueria infausta, Acacia saligna and Acacia cyclops (Dames, 1991; Hoffman and
Mitchell, 1986; Coetzee, 1982). Previously, there was lack of detailed information on the
influence of abiotic factors on indigenous AM fungal species. However, Uhlmann et al.,
(2004) carried out a comparative study on species diversity of AM fungi in different seasonal
areas of South Africa and Namibia. Results revealed that geographical distance and rainfall to
a lesser extent, influenced species diversity. A consideration of seasonal changes was also
suggested by Dames (1991) when AM fungal species responded differently to soil fertility
factors such as pH, moisture, percentage carbon, phosphorus and cations.
Morton and Redecker (2001) recognized two other families, the Archaeosporaceae
and Paraglomaceae, with two new genera, Archaeospora and Paraglomus. The recent
classification proposed by Schwarzott et al., (2001) has been adapted in the present
investigation. So far there are 164 species of AM fungi reported from all over the world.
Following is the dichotomous key for the segregation of AM fungal genera.
Phylum: Glomeromycota
Class: Glomeromycetes
Order I: Glomerales
Family: Glomeraceae
Genus: Glomus (Group-A and B)
Order II: Diversisporales
Family: Gigasporaceae
Genus: 1. Gigaspora
2. Scutellospora
Family: Acaulosporaceae
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Genus: 1. Acaulospora
2. Entrophospora
Family: Pacisporaceae
Genus: 1. Pacispora
Family: Diversisporacae
Genus: 1. Diversispora
2. Glomus (Group-C)
Order III: Paraglomales
Family: Paraglomaceae
Genus: 1. Paraglomus
Order IV: Archaesporales
Family: Geosiphonaceae
Genus: 1. Geosiphon
Family: Archaesporaceae
Genus: 1. Archaespora
According to Morten et al., (2001) the number of species of this group of fungi may
reach up to 2700. Tiehang Wu et al., (2002) studied screening of AM fungi for the re-
vegetation of eroded red soils in subtropical China. Harinikumar and Potty (2002) recorded
VAM spores from cultivated soils, uncultivated soils, moist forests, open woodlands, scrub
jungles, grass lands, sand dunes and semi deserts. Khade et al., (2003) has studied the
incidence of arbuscular mycorrhizal colonization in tubers of Gloriosa superba L. Hasan et
al., (2005) has reported the status of arbuscular mycorrhiza in mango in six district of Uttar
Pradesh. Bhat et al., (2005) was studied on AM fungi associated with tree species planted in
arboretum at Mangalore University, Mangalore, Karnataka. Khade et al., (2008) has studied
the arbuscular mycorrhizal association in Banana from the state of Goa. Parkash et al., (2009)
was studied the endomycorrhizal association of Acacia catechu Willd., in Himachal Pradesh.
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MATERIALS AND METHODS
Study site and Sample collection
Occurrence of Arbuscular Mycorrhizal (AM) fungal association was investigated on
the four experimental plants such as Corchorus capsularis L., Crotalaria juncea L.,
Gossypium hirsutum L. and Hibiscus cannabinus L. The soil and root samples were collected
from the selected seven different locations of Dharwad taluk in Dharwad district of
Karnataka, India, namely, University of Agricultural Science Campus Dharwad, Ettinagudda,
Kyarakoppa, Kelageri, Amminabhavi, Dasanakoppa and Beluru (Fig 1). Geographically
Dharwad is located between 140 15' and 150 5’ North longitude and 740 49’ and 760 21’ East
latitude. There is a marked diurnal temperature differences: The temperature can be as below
as 20.20C in June and high as 34.420C in March. The annual rain fall is 600-850mm. The
climatic regions are semi humid or humid. Soil is covered with a hard, compact crust having
dark brown colour. Then the samples were brought to the laboratory and the fine roots in
each sample were removed, rinsed with tap water and fixed in formalin acetic alcohol (FAA),
for the determination of root colonization. The soil samples were then air dried in the shade at
laboratory temperature for spore counting.
Isolation and Quantification of AM fungal spores
The AM fungal spores were separated from the soil by wet sieving and decanting
technique (Gerdman and Nicolson, 1963). Fifty gram of rhizospheric soil sample was mixed
in 200 ml of distilled water in a large beaker. After 1 hrs the contents of the beaker were
decanted through the sieves which were arranged in a descending order from 400 µm to 25
µm size. The process was repeated for thrice. The procedure was repeated until the upper
layer of soil suspension is transparent. The retained material on the sieve was decanted into a
beaker with a stream of water and estimation of spores was carried out by modified method
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of Gaur and Adholeya (1994). A circular filter paper was taken and folded into four equal
quadrants. The paper was reopened; two lines were drawn along the two folds to divide the
filter paper into four equal quadrants. Vertical lines were drawn on one half of the filter paper
so as to divide it into approximately 20 columns about 0.5cm apart. Each column was then
numbered and the direction of counting was marked by an arrow. The filter paper was then
folded in such a way that the marked portion becomes the receiving surface for the sample
during filtration. This filter paper along with sample spores was spread in a bigger Petri dish.
The Petri dish was observed under stereo binocular microscope. Two lines were focused in
the field and moving the Petri plate, the spores were counted in every space between the two
lines and since the lines were numbered and the direction was set, it was easy to keep track of
each spore on the filter paper.
For the identification of AM fungal spore, single spore or sporocarps were easily
picked up from the filter paper with the help of syringe or fine point camel brush and
mounted on a glass slide with a drop of polyvinyl lactophenol (PVL) and a cover slip was
placed. Subsequently, recovered spores were identified with the help of manual (Schenck and
Perez, 1990) and different taxonomic keys proposed by different workers (Frank and Mortan,
1994; Mehrotra, 1997; Schwarzott et al., 2001). The following characters are considered for
identification sporocarps, spore morphology, size, shape and peridium of spore, sporocarps
colour, wall ornamentation, subtending hyphae and mode of attachment. Some of the
important and selected spores were recorded and documented in the form of photographs
(Plate III-VIII).
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Fig. 1: Location of study area map.
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Evaluation of AM Fungal colonization
Arbuscular mycorrhizal fungal structure in roots is usually not observed without
appropriate staining. Freshly collected root samples should be washed gently and be free
from soil particles. Ultrasonic treatment is effective to disperse soil particles closely adhered
to roots.
Roots were treated with 10 % KOH solution for 30 min to 1-2 hours in a hot bath,
depending on thickness of root structure. Treated roots were washed with water and treated
with 2 % HCl solution. Acidified root samples are stained with 0.05 % trypan blue (or acid
fuchsin) in lactic acid for 10-15 min in a hot bath or for a few hours without heating. The
roots are destained with lactic acid or lacto-glycerol and are now ready for microscopic
observation. The stained roots may be observed first under a dissecting microscope with
transmitted illumination and then observed under a compound microscope. Fungal structures
are stained and can be easily recognized (Phillips and Hayman, 1970).
Root colonization:
Per cent of AMF colonization was estimated by microscopically examination at 10 X
magnification, after clearing of roots in 10% KOH and staining with 0.05% trypan blue in
lactophenol according to method described in Phillips and Hyman (1970). The mycorrhizal
colonization was determined by using following formula.
Per cent of mycorrhizal colonization = Number of root segments colonized X 100
Total number of root segments examined
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Table. 1.1: The physico-chemical characteristics of soil samples collected form seven different localities of Dharwad taluk.
Parameters
University of
Agricultural
Science Campus
Dharwad (UAS)
Ettinagudda Kyarakoppa Kelageri Amminabhavi Dasanakoppa Beluru
Soil Black soil Black soil Red soil Sandy loam Red soil Black soil Red soil
pH (1:2.5) 7.230d 7.333de 7.067d 7.267d 7.133d 6.95d 7.095d
Moisture (%) 315.333a 325.000a 313.333a 329.333a 330.667a 314.333a 326.310a
Conductivity (Fc)us/cm 3.543de 3.873f 3.613e 4.210e 4.447e 4.267e 3.516e
Total organic carbon (%) 0.813e 0.813g 0.807f 0.623f 0.567f 0.629f 0.816g
Nitrogen (%) 0.057e 0.060g 0.043f 0.080f 0.077f 0.070f 0.065g
Potassium (%) 6.533d 7.793d 7.400d 6.697d 7.620d 6.985d 7.796d
Phosphorus (%) 4.313de 4.163ef 4.433e 4.323e 4.493e 4.323e 4.268e
Magnesium (%) 0.140e 0.132g 0.133f 0.130f 0.133f 0.150e 0.136g
Calcium (%) 0.453e 0.461g 0.511f 0.610f 0.417f 0.459e 0.416g
Zinc (ppm) 3.650de 3.500f 3.410e 3.710e 3.653e 3.659e 3.569f
Copper (ppm) 0.017e 0.027g 0.013f 0.017f 0.027f 0.018e 0.022f
Manganese (ppm) 0.780e 0.770g 0.797f 0.867f 0.740f 0.845f 0.774g
Iron (ppm) 8.620d 7.847d 8.317d 9.001d 8.410d 8.798d 7.857d
Mean values followed by the same letter within a column do not differ significantly at P<0.05 according to DMRT.
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Table: 1.2: Population of AM fungal spores and per cent of root colonization in four fiber yielding Plants (Corchorus capsularis L.,
Crotalaria juncea L., Gossypium hirsutum L. and Hibiscus cannabinus L.) at various places of Dharwad taluk.
Parameters
University of Agricultural Science Campus Dharwad
(UAS)
Ettinagudda Kyarakoppa Kelageri Amminabhavi Dasanakoppa Beluru
Corchorus capsularis L.
AMF spore/50g of soil 95.6d 110.5b -- -- -- -- --
Percent colonization (%) 32.52f 39.62e -- -- -- -- --
Crotalaria juncea L.
AMF spore/50g of soil 129.6a 136.9a 98.8d 109.5c 112.3c 142.9a 138.6a
Percent colonization (%) 39.6e 42.6d 29.3g 32.6f 34.8e 45.6e 43.6f
Gossypium hirsutum L.
AMF spore/50g of soil 96.8c 99.9c 106.8c 125.6b 129.7a 130.0b 134.5c
Percent colonization (%) 25.6g 26.8g 29.6g 31.3g 33.5f 34.6f 35.8g
Hibiscus cannabinus L.
AMF spore/50g of soil 99.8b 110.6b 121.6a 132.6a 126.5b 129.5b 136.8b
Percent colonization (%) 29.8h 33.9f 40.6e 45.8d 42.5d 44.5e 48.6e
Mean values followed by the same letter within a column do not differ significantly at P<0.05 according to DMRT.
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Table: 1.3: AM fungal spores recovered from Dharwad district in seven places with respect to the rhizosperic soil of four fiber
yielding Plants (Corchorus capsularis L., Crotalaria juncea L., Gossypium hirsutum L. and Hibiscus cannabinus L.)
Locality Spore type Shape Spore diameter (µm)
Colour of wall layers
Number of wall layers
A1,A2,A3, A4, A5, A6, A7
Glomus faciculatum (Thaxter) Gerdemann & Trappe emend. Walker and Koske.
Globose-Subglobose
75-150x35-100 Light brown Single layered
A1, A2, A4 Glomus albidum Walker & Rhodes. Globose 143-330(-350) Yellow-golden yellow
Single layered
A1,A2,A3, A4, A5, A6, A7
Glomus macrocarpum Tulasne & Tulasne.
Globose-Subglobose
(90-)120(-140)x(70-)110(-130)
Yellowish brown Single layered
A2, A3, A7 Glomus caledonium (Nicolson & Gerdemann) Trappe & Gerdemann.
Globose-Ellipsoid
124-394 Yellowish brown Double layered
A1, A5, A6, A7
Glomus pallidum Hall. Globose-Subglobose
32-78x28-68 Pale yellow Single layered
A3, A4, A5 Glomus tenebrosum (Thaxter) Berch. Globose or Subglobose
(200-)240(-270) x(205-)230(-270)
Dark brown Single layered
A1, A2, A3, A4
Glomus multicauli Gerdemann & Bakshi.
Elliptical 149-249x124-162 Dark brown Single layered
A4, A5, A6 Glomus australe (Berkeley) Berch. Globose-Subglobose
(120-)160(-180) Yellowish brown Double layered
A2, A3, A7 Glomus fuegianum (Spegazzini) Trappe & Gerdemann.
Globose-Subglobose
65-80 Radish brown Single layered
A2, A3, A5 Glomus clarum Nicolson & Schenck. Globose-Subglobose
68-290 Yellowish brown Single layered
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A1,A2,A3, A4, A5, A6, A7
Glomus mossae (Nicolson & Gerdemann) Gerdemann & Trappe.
Globose-ellipsoid
105-310x110-305 Brownish yellow Double layered
A1, A2, A3, A6
Glomus fistulosum Skou & Jakobsen. Globose-Subglobose
(78-)120-160(-200) Yellowish brown Double layered
A1,A2,A3, A4, A5, A6, A7
Glomus microagreegatum Koske, Gemma & Olexia.
Globose 30(-50)x(15-)30(-40)
Brownish-yellow 1-2 layered
A4, A5, A6, Glomus occultum Walker. Globose-Subglobose
15-100x20-120 Hyaline white 1-2 layered
A2, A3, A4, A5, A6
Glomus taiwanensis Wu & Chen. Chlamydospores 40-85 Yellowish brown Double layered
A4, A6, A7 Glomus gerdemannii Rose, Daniels & Trappe.
Globose-Subglobose
140-198x149-230 Yellowish brown Five layered
A2, A3, A4, A6
Glomus globiferum Koske & Walker. Globose-Subglobose
150-260x150-270 Red brown 1-4 layered
A1, A3, A4, A5
Glomus flavisporum (M. Lange & Lund) Trappe & Gerdemann.
Globose 149-202x95-152 Yellowish brown Double layered
A5, A6, A7 Glomus fragilistratum Skou & Jakobsen.
Globose 108-191 Yellow Double layered
A1, A3, Glomus geosporum (Nicolson & Gerdemann) Walker.
Globose-Subglobose
110-290x100-290 Yellowish brown Single layered
A1,A2,A3, A4, A5, A6, A7
Acaulospora laevis Gerdemann & Trappe.
Globose-Subglobose
119-300x119-520 Red brown Three layered
A1, A3, A4, A5, A7
Acaulospora taiwania Hu. Globose-Subglobose
425-475 Dull yellowish brown
Double layered
A1, A4, A6, A7
Acaulospora scrobiculata Trappe. Globose 100-240x100-220 Greenish yellow Four layered
A1, A2, A3, A4, A7
Acaulospora thomii Hu. Globose-Subglobose
425-475 Yellowish brown Single layered
A1, A2, A4, Acaulospora denticulata Sieverding & Globose- (112-)130-170(- Red brown 1-4 layered
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A5, A6 Toro. Subglobose 175) A1, A2 Acaulospora foveata Trappe & Janos. Globose 250-300x185-250 Yellowish brown Three layered A4, A5, A6 Acaulospora delicata Walker, Pfeiffer
& Bloss. Globose-Subglobose
80-125(-150) x80-110(-140)
Yellowish cream Double layered
A1,A2,A3, A4, A5, A6, A7
Gigaspora margarita Becker & Hall. Globose 260-480 Hyaline-white Four layered
A1, A2, A6, A7
Gigaspora albida Schenck & Smith. Globose 232-252x234-250 Greenish yellow 1-6 layered
A2, A4, A5, A6
Sclerocystis pachycaulis Wu & Chen. Chlamydospores 170-230x175-270 Yellowish brown Double layered
A2, A3, A4, A6
Sclerocystis pakistanica Iqbal & Bushra.
Chlamydospores 65-205x32.5-55 Dark brown Single layered
A3, A4, A6, A7
Sclerocystis dussii (Patouillard) von Hohnel.
Chlamydospores 50-80x32-54 Brown Single layered
A1, A2, A3, A4
Scutellispora erythropa (Koske & Walker) Walker & Sanders.
Globose-Subglobose
170-551x205-660 Pale yellow 4 or 5 layered
A1, A3, A5, A7
Scutellispora scutata Walker & Diederichs.
Globose-Subglobose
350-667x350-713 Hyaline Six layered
A1, A3, A4, A6
Entrophospora schenckii Sieverding & Toro.
Globose-Subglobose
(37-)50-60(-77) Hyaline Three layered
A1= University of Agricultural Science Campus Dharwad (UAS) A2= Ettinagudda, A3=Kyarakoppa, A4= Kelageri A5= Amminabhavi A6= Dasanakoppa A7=Beluru
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RESULTS
Rhizosperic soil sample from seven different locations were subjected to the recovery of
AM fungal spores. The soil samples of different location showed different types of spores. All
the recovered spores represent six genera. Namely Acaulospora, Gigaspora, Sclerocystis,
Scutellispora, Glomus and Entrophospora. The entire collected rhizospere sample exhibited the
presence of varied range of spore population in the soil profile. Highest spore number was
observed in the rhizosphere soil of Crotalaria juncea L. collected form Dasanakoppa. Lowest
spore number was noticed in the rhizosperic soil of Gossypium hirsutum L. collected from
University of Agricultural Sciences Dharwad. Per cent root colonization was observed in
experimental plants grown with soil samples taken from various places. Highest per cent root
colonization was noticed in Hibiscus cannabinus L. grown at Beluru and Kelageri whereas,
lowest per cent root colonization was obsereved in the Gossypium hirsutum L. grown at
University of Agricultural Sciences, Dharwad.
The pH of the soils varied from 6.95 to 7.33 showing slight acidic to moderately alkaline
in nature at most of the localities.Total organic carbon content varied 0.567 to 0.816 at various
localities (Table 1.1). The total AM fungal spore number at different localities varied from 96.8
to 138.6 per 50 g of soil and per cent root colonization was 37. 9 to 58.6 (Table 1.2). In the
present investigation highest spore density was observed in the soil with rich organic matter and
slight acidic soil compare to neutral and alkaline soils. There was a wide variation in spore
number especially in Glomus species followed by Acaulospora species (Plate-II to V). However,
the distribution of Sclerocystis, Gigaspora, Scutellispora and Entrophospora (Plate VI to VIII)
was very less at all the localities.
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Maceration and anatomical studies followed by tryphan blue staining revealed different
stages with distinct components of AM fungi. Microscopic measurements provided an
assessment of the relative abundance of mycelium in root, the density of hyphae within root, the
number of entry points, wall thickness and pattern of outer epidermal cells. The coarse aseptate
hyphal coils were often seen from initial penetration points (Plate-IX). Most remarkable
morphological feature was the variation in the diameter range (3-25µm) among the hyphal
filaments. The thick walled hyphae were almost filled with dense cytoplasmic matrix with oil
globules and most of the thick walled hyphae were smooth with a few irregularities in outline.
The thin walled hyphae measuring 2-5µm in diameter arises laterally from the main hyphae. It
was noted that the lateral walled hyphae arise directly from the hyphae of the cytoplasmic
connection. Later content of hyphae disappeared with formation of appressoria.
It was observed that the fungus grows throughout the cortex, but it does not invade the
endodermis or stele. The fungus penetrated from one cell to another forming a new coil.
Intracellular hyphae were usually found in the intermediate layers of the cortical parenchyma
with a diameter of about 3-6µm. The hyphae run parallel in between the parenchyma to a
considerable distance. In some areas, the hyphae exhibited intermittent projections and were at
times swollen. Longitudinal hyphal branches in the form of H shape (Plate-IX 3).
Arbuscules of various stages of the growth were observed and some were in the state of
disintegration. Morphologically arbuscular branches were short, deteriorating and collapsing.
Senescent arbuscules were observed in older portion of the mycorrhizal roots as compared to the
young mycorrhizal roots. In present study vesicles were less in numbers and were seen
intercalary and intracellularly within the infected roots (Plate-IX 2). Vesicles size and shape
diffused depending on the anatomy of the root, varying in size (42-48µm), subglobose and large
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(89-106µm) in diameter. The intercellular vesicles and host walls have distinct contact, whereas
the intracellular vesicles usually enclosed in a layer of cytoplasm. The dense granular cytoplasm
with full of fat globules were observed in matured vesicles (Plate- IX 2). The outer walls of the
vesicles appeared smooth without ornamentation.
PLATE II
A. Aculospora appendicula Spain, Sieverding and Schenk. (400X)
B. Aculospora denticulate Sieverding and Tora. (400X)
C. Aculospora foveata Trappe and Janos. (400X)
D. Aculospora Spinosa. Walker and Trappe (400X)
E. Aculospora nicolsonii Walker , Reed and Sanders (400X)
F. Aculospora thomii Blaszkowski. (400X)
G. Aculospora denticulate Sieverding and Tora. (400X)
H. Aculospora denticulate Sieverding and Tora. (400X)
PLATE III
A. Aculospora sporocarpa Berch. (400X)
B. Aculospora dilatata Morton. (400X)
C. Aculospora tuberculata Janos and Trappe. (400X)
D. Aculospora undulate Sieverding. (400X)
E. Aculospora sp. (400X)
F. Aculospora sp. (400X)
G. Glomus hoi Berch and Trappe. (400X)
H. Glomus hoi Berch and Trappe. (400X)
PLATE IV
A. Entrophosphra schenckii Sieverding and Toro. (400X)
B. Entrophospora colombinana Spain and Schenck. (400X)
C. Glomus microagreegatum Koske, Gemma and olexia. (400X)
D. Glomus macrocarpum Tul and Tul. (400X)
E. Glomus macrocarpum Tul and Tul. (400X)
F. Glomus dimorphicum Boye tchko and tewari. (400X)
G. Glomus fugianum (Spegazzini) Trappe and Gerdemann. (400X)
H. Glomus multicauli Gerdemann and Bakshi. (400X)
PLATE V
A. Glomus flavisporum (M. Lange and Lund) Trappe and Gerdermann. (400X)
B. Glomus gerdemanni Rose, Daniels and Trappe. (400X)
C. Glomus pallidum Hall. (400X)
D. Glomus occultum Walker. (400X)
E. Glomus fuegianum (Spegazzini) Trappe and Gerdemann. (400X)
F. Glomus sp. (400X)
G. Glomus halonatum Rose and Trappe. (400X)
H. Glomus clarum Nicolson and Schenck. (400X)
PLATE VI
A. Glomus fasciculatum (Thaxtex) Gerd and Trappe emend walker. (400X)
B. Glomus sp. (400X)
C. Glomus sp. (400X)
D. Glomus formosanum Wu and Chen. (400X)
E. Glomus sp. (400X)
F. Glomus maculosum Miller and Walker. (400X)
G. Glomus geosporum Nicolson and Gerderman (400X)
H. Glomus warcuppi Mc Gee. (400X)
PLATE VII
A. Glomus sp. (400X)
B. Glomus sp. (400X)
C. Scutellospora species showing spores inside the spore. (400X)
D. Scutellospora persica (Koske and walker) Walker and Sanders. (400X)
E. Scutellospora arenicola Koske and Halvorson. (400X)
F. Scutellospora dipurpusescrns Mortan and Koske. (400X)
G. Glomus fasciculatum (Thaxtex) Gerd and Trappe emend walker. (400X)
H. Scutellospora calospora (Nicon and Gerd) Walker and Sanders. (400X)
PLATE VIII
A. Gigaspora albida Schenck and Smith (400X)
B. Gigaspora albida Schenck and Smith (400X)
C. Gigaspora rosea Nicolson and Shcenck. (400X)
D. Gigaspora rosea Nicolson and Shcenck. (400X)
E. Sclerocystis sinuosa Gerdemann and Bakshi. (400X)
F. Sclerocystis taiwanensis Wu and Chen. (400X)
G. Scutellispora calospora (Nicolson and Gerdemann) Walker and Sanders. (400X)
31
DISCUSSION
The present survey was conducted to study spore distribution and population of AM
fungal spores in rhizosphere of fiber yielding plants at different localities in Dharwad taluk,
Karnataka. All the plants growing under natural conditions had possessed AM fungal spores as a
regular component of the soil microflora. Among the recovered AM fungal spore population
Glomus species was dominant in most of localities. This might due to the high sporolation
capacity and high viability of the Glomus species, while, others were scanty /intermediate due to
the adverse edaphic conditions, longer reproductive times and short viability. Similar observation
was made by Sieverding (1991) and Jamuluddin et al., (2002). Another important factor in the
AMF distribution is related to the edaphic and climatic conditions against which every species
struggles for existence and the best suited species multiples quickly and gets established in soil.
Mohan kumar and Mahadevan, (1987), have reported that high organic carbon reduced the
mycorrhizal spore abundance in the soil. Soil acidity is an important factor regulating spore
germination and also may influence the distribution of AM fungi that is in accordance with
Green et al., (1976); Dickman et al., (1984); Porter et al., (1987); and Lakshmipathy et al.,
(2003) Lakshman (1992) and Mulla (2003) Sundar, and Meera (2011). Selecting the suitable
mycorrhizal fungi for medicinal plants is a high priority. Effectiveness of AM fungi is
determined by their ability to colonize the root, produced external hyphae and absorb and
transport P effectively (Abbott and Robson, 1991).
There were abundant AMF spores and the largest number was noticed in the rhizosphere
soil of Crotalaria juncea L., while the least in Gossypium hirsutum L., other plant species
carried over but could not express their efficiency for root colonization accordingly. Similar
results were observed by Scheitema et al., (1987) under field conditions.
32
When the AM fungal spore comes in content with root surface, the extradical hyphae
swells apically and increases in size forming a more or less pronounced appressorium-like
structure. Fan like appressoria have been observed by Gianinazzi-Pearson et al., (1980) on the
root surface of wild raspberry colonized by the fungal endophyte, identified as Glomus species.
According to Hall, (1979) the outer cortical layers of the root are after colonized by intracellular
hyphae, characterized by a linear or more often a looped arrangement, without any signs of
branching. The infected and similar coils subsequently spread to the neighboring cells.
The population of AMF spores in seven different areas of Dharwad taluk was determined
in terms of resting spores and sporocarps in the rhizospheric soils of selected experimental
plants. The AMF spore the widespread in all the soils investigated but varied in both number and
type of spore and sporocarps. No relationship was found between pH and spore numbers. Reddy
(1993) and Prabhakar (1995), also may similar observations. It is well know fact that
nutritionally deficient soils, phosphorus soils in particular, harbour more AMF. The present
research findings also support this. This is corroborated by the findings of Janaki Rani and
Manoharachary (1994), and Manoharachary et al., (1996).
In the present study it was observed that in many cases more than one appressorium is
located at an entry point. In most cases, adjacent appressoria probably results from the branching
of single external hyphae before or after contact with the root, which is accordance with
Brundrett et al., (1985) described characteristic branching of the patterns of the internal hyphae
of Glomus species. Occasionally numerous branches were produced by intercellular hyphae.
However, a convoluted of “comb” like structure was observed Furlan et al., (1973). Frequent
branching of the host root. In the present study the formation of vesicles was observed at later
stage of growth, containing storage lipid droplets, which are similar in structure and possible
33
function as soil borne spores of the fungus (Biermann and Linderman, 1983). Wide diversity
exists within the group of fungi responsible for the formation of AM fungi by most plants in the
majority of terrestrial ecosystems. The AM fungi at different levels of their organization (intra-
interspecific population), conservation and efficient utilization of their biodiversity are of crucial
importance for sustainable fiber plant production.