13

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

14

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

15

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.

16

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-

17

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

18

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.

19

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

20

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).

21

Fig. 1: Location of study area map.

22

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

23

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.

25

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

26

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

27

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

28

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.

29

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

30

(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.