ROLE OF VAM FUNGI IN THE MANAGEMENT OF PHYTONEMATODES ON TOMATO

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

IN

BOTANY

" I • :•• t ' • • ' " ^ '

• • ; - - ^^ " B y • •

ZEHRAKHAN

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DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2007

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DS3808

dedicated To

My Loving (Parents

Irshacf MaRmoocf M.Sc. Phi).

Reader

Department of Botany Aligarh Muslim University Aligarh 202 002 India Phone: 0571-2702016(0) Mob: 09412535631

Date: August 10,2007

Certifitate

This is to certify that the dissertation entitled "Role of VAM fungi in the

management of phytonematodes on tomato" embodies original and bonafide

work carried out under my supervision by MINS Zchra Khan. It may be

submitted to the Aligarh Muslim University, Aligarh towards the fulfillment of

requirements for the Degree of Master of Philosophy in Botany.

(Dr. Irshad Mahmood) Research Supervisor

ACKNOWLEDGEMENT

/ bow to the Almighty for all that He has blessed me with and I have never, ever-felt abondoned by His Grace. I pray to Him to keep it the same way for me in this life and the life thereafter

I acknowledge, with all respect and regard to my supervisor Dr. Irshad Mahmood for the help and guidance. His invaluable help, support and teaching shall remain the guiding force for the rest of my life.

I am also thankful to Professor Ainul Haque Khan, Chairman, Department of Botany, A.M.U. Aligarh for providing necessary laboratory facilities.

I would fail in my duty if I do not acknowledge my heartfelt thanks to Prof. K.G. Mukherji and Prof. Bhatnagar, Department of Botany, Delhi University for the help. My gratitude particularly to Prof Mukherji who taught me the identification technique ofVAMis beyond the reach of my expression.

It is my privilege to mention my deep sense of reverence to Mr. Sonal Bhatnagar and Mr. Sayeed Ahmad, for his advice and valuable suggestion.

I feel pride and privilege for my loving father Mr. Mohd. Wall Khan and beloved mother Mrs. Nasreen Begum who lit the flame of learning in me. Their prayer and sacrifices helped me to gain this success.

A special note of thanks to my seniors and friends, Farha, Bushra, Humera, Sheeba, Zeba, Shweta, Sangeeta, Swam, Qaisar and Uzma for their constant help, encouragement throughout the writing of this dissertation.

I have no words to express my special feelings for my brothers and sisters Hammad, Nadeem, Wajahat, Nasir, Fatima and Shagufta who always provided me warm shoulder with affection and regards. It is their love, care and inspiration that has been instrumental in overcoming the complexities at every step of this work.

I am sincerely behold to Mr. H.K. Sharma who very painstakingly typed the manuscript despite his personal engagements.

Zehra Khan

CONTENTS

Page No.

INTRODUCTION 1

REVIEW OF LITERATURE 6

SECTION I The occurrence of arbuscular mycorrhizal fungi (AMF) Characterization of soil, spore population of AMF in soil And root colonization of some commonly cultivated crops In Pilibhit District - A survey

Introduction 27

Materials and Metliods 27

Results 29

Discussion 39

SECTION II

Comparative efficacy of different Arbuscular-mycorrhizal Fungal spp. (AMF) on tomato (Lycopersicon esculentum Mill.)

Introduction 42

Materials and Methods 43

Results 47

Discussion 55

SECTION III Effect of AM fungus, Glomus fasciculatum and root-knot nematode, Meloidogyne incognita on growth and development of tomato

Introduction 57

Materials and Methods 58

Results 70

Discussion 82

LITERATURE CITED 87

LIST OF TABLES

Table Page No.

number

Section I

1. Soil characteristics of four sites of Pilibhit district 30

2. Total spore counts at different sites in different crops 32

3. Spore counts of G. fasciculatum at different sites 32 under different crops

4. Spore counts of G/oww^ wo^^eae at different sites 32 under different crops

5. Spore counts of Glomus etunicatum at different sites 35 under different crops

6. Spore counts of G/owwi'co«6'?nc/wm at different sites 35 under different crops

7 Spore counts of ^caw/o^pora/aevw at different sites 35 under different crops

8 Spore counts of Gigaspora gigantea at different sites 38 under different crops

9 Mycorrhization at different sites under different crop 38

Section II

10 Effect of different arbuscular mycorrhizal (AM) 48 flingi on shoot, root and total lengths of tomato var. Pusa Ruby at different stages of growth

11 Effect of different arbuscular mycorrhizal (AM) 50 fungi on shoot, root and total fresh weights (g/plant)

of tomato var, Pusa Kuby at different stages of growth

Effect of different arbuscular mycorrhizal (AM) 12

13

fungi on shoot, root and total dry eights (g/plant) of tomato var. Pusa Ruby at different stages of growth

Effect of different arbuscular mycorrhizal (AM) fungi inoculation on mycorrhizal infection of tomato var. Pusa Ruby at different stages of growth

52

54

Section III

14 North Carolina differential host test reaction chart 61

15 Inoculation Schedule (Section III) 63

16 Interaction effect of spore density of mycorrhizal 71 fungus G. fasciculatum and population of nematode Meloidogyne incognita on the length of tomato plants

17 Interaction effect of spore density of mycorrhizal 73 fungus G. fasciculatum and population of nematode Meloidogyne incognita on the fresh weight of tomato plants

18 Interaction effect of spore density of mycorrhizal 74 fungus G fasciculatum and population of nematode Meloidogyne incognita on the dry weight of tomato plants

19 Interaction effect ofspore density of mycorrhizal 75 fungus G. fasciculatum and population of nematode Meloidogyne incognita on the N, P and K content of tomato plants

20 Interactioneffectof spore density of mycorrhizal 77 fungus G. fasciculatum and population of nematode Meloidogyne incognita on mycorrhization G. fasciculatum in tomato plant

21 Interaction effect of spore density of mycorrhizal 80 fungus G. fasciculatum and population of nematode Meloidogyne incogntia on the root-knot disease parameters in tomato plant

LIST OF FIGURES

Figure No.

Section I

a Showing the sHde of Glomus fasciculatum

b. Showing the sUde of Glomus mosseae

c Showing the sHde of Glomus etunicatum

d Showing the shde of Glomus constrictum

e Showing the shde of Acaulospora laevis

f Showing the shde of Gigaspora gigantea

INTRODUCTION

INTRODUCTION

Agriculture is one of the major sector of the Indian economy. 70 per

cent of the population is dependent upon it for their livelihood and contributes

over 40 per cent of the gross national production. Food production has always

remained a matter of great concern for the people all over the world. Any effort

to improve food production necessarily involve step to protect the crop against

pests and diseases. Among the pest, the plant parasitic nematodes are

recognized as potentially serious constraint to agricultural production

throughout the world.

During the last few decades plant parasitic nematodes have definitely

made their presence felt as important pest of economic crops besides insects,

bacteria, fungi, viruses, weeds etc. The prevalent pest complexity has

accentuated due to induction of new production input such as high yielding

varieties, increased irrigation, water fertilizers, pesticides etc.

Use of biocontrol agents for the management of plant parasitic nematode

or to integrate management studies are the current emphasis area. For obvious

reason many natural enemies attract nematodes in soil and reduce their

populations. Exploitation of these enemies for practical use in management of

plant parasitic nematodes at present seems to be practically demanding and the

relevant approach in the greater awareness of pollution free environment (Khan

andHussain, 1990).

In India, the importance of the nematodes as a constraint to successful

crop production was first realized with the discovery of the cyst nematodes on

wheat (Vasudeva, 1958) and potato (Jones, 1961). Since then number of

nematode problems of national importance have emerged.

Plant parasitic nematodes, cause diverse kinds of damages to crop

plants. They are of global importance as parasites of agricultural crops. Among

all the plant parasite nematode, root-knot nematodes Meloidogyne spp. being a

polyphagus in nature is a major threat in the production of various crops, all

over the world and cause enormous crop damage (Sasser, 1990).

Tomato, Lycopersicon esculentum an important vegetable crop and its

cultivation is worldwide. Fresh ripe fruits are refreshing and appetizing tomato

are consumed in the form of juice, puree, paste, ketchup, sauce, soup and

powder. Ripe tomatoes contain glucose and fructose as principal sugars.

Tomato contains a gluco-alkaloid tomatine which is used as a precipitating

agent for cholesterol. There are several constraints for the successful

cultivation of tomato, out of which root-knot nematode alone causes about

20.60% world wide yield loss (Sasser, 1989). In India several nematologists

have assessed the loss in tomato crop caused by root-knot nematode, 26.5-

73.30% by Seshadri, (1970); Bhatti and Jain (1977), observed a loss of 46% ,

whereas Reddy (1985) in his crop loss assessment study reported 39.11% yield

loss of tomato due to Meloidogyne incognita. Jain et al. (1994) reported 47.3-

71 % yield loss due to M javanica and M incognita.

Limited importance has been placed on the management of nematodes

by non chemical approaches to plant disease control. Due to excessive use of

pesticides large number of once effective chemicals have become uneffective

due to development of resistance by pathogen (Delp, 1980). Several

nematicides have been withdrawn from the market because of health and

environmental problems associated with their production and use (Thomson,

1987). As a result of increasing public concern over the use of pesticides on

food production, some pests management researches have focused efforts on

developing an alternative approach to synthetic chemicals by introducing the

use of biological agents. Weller (1988) reported that the Microorganism

present in the rhizosphere provides a front line defense for pathogens attacking

roots. In recent years much attention has been generated in minimizing the

excessive use of chemicals against the pest by using an alternative measures

such as biological methods (Baker and Cook, 1974; Cook and Baker, 1983,

Blakeman and Fokkema, 1982; Vidauer, 1982; Kerry, 1992; Dalpe and

Monreal, 2004; Pal and Gardner, 2006).

Among the natural biological processes that contribute to soil nutrients

management are symbiosis between plant and bacteria (as in nitrogen

fixation)or fungi (as in mycorrhiza). Arbuscular mycorrhiza (AM)increase the

plant ability to absorb phosphorus, minor elements, translocate certain mineral

elements and water (Hayman, 1982; Ratnnayake et al, 1978). They are also

known to reduce damage caused by pathogen (Dehne and Schoenbeck,1975;

Bagyaraj et al, 1979; Hussey and Roncadori, 1982; Sitaramaiah and Sikora,

1982 and Smith 1987). Increased flow of nutrients was observed in mycorrhizal

plants which impart mechanical strength to the host .AM fungi improved plant

growth, control root pathogens, increased nutrient uptake and hormone

production withstand water stress and are potential means of management of

waste lands.

The AMF belongs to the Zygomycetes and have recently regrouped in a

single order, the Glomales (Morton and Benny, 1990), the majority of known

3

species belong to the family Glomaceae (Pirozynski and Dalpe, 1989). VAM

are characterized by the presence of two specialized structures, vesicles and

dichotomously branched haustoria with the host cell called arbuscules.

Arbuscules are formed intracellular^ as internal mycellial structure which

penetrate mechanically and enzymatically into cortical cells in the form of

highly ramified aborescences within a few days of infection. Bifurcated

arbuscules hyphae are enveloped by host derived encasement layer and

continuously invaginate host plasmalemma and form a wide contact between

the two symbionts but are short lived and can be digested with in few days

after formation of host vesicles .The so called vesicles have now been termed

chlamydospores (Mehrotra.,1993; Mehrotra and Baijal,1994; Will et al., 1995).

A second type of structure which have swollen thick walled appearance are

also produce by internal mycelium intercellularly. They can act as infective

propagules and remain viable for long periods (Biermann and Linderman,

1983; Diop et al., 1994) and are also known to absorb and release phosphorus

in the process of transfer between the symbionts. Plants provides the ftingus

upto 80 per cent or more of the net energy fixed as sunlight to below ground

processes. Some of this energy goes into root growth; but a high proportion

may be used to feed mycorrhizal fungi. On the contrary the AM fungi living in

the hot zone greatly influence the ability of plants to establish through effects

on plants in number of ways.

1. increased nutrient uptake

2. increased disease resistance

3. enhanced water relation

4. increased soil aggregation

Use of microbial inoculants seem to be better alternative to achieve the

objective of management of root knot nematode disease and improvement of

plant growth which at present seems to be practically demanding in view of the

greater awareness of health and safe environment. Keeping in view of the

above, the present study was undertaken to explore the possibility of achieving

an effective biocontrol of nematode disease by AM fungal exploitation on

tomato plant. That resulted to decreased input cost, increased yields and

environmental benefits.

The present work consist of following aspects

1. A survey of some cultivated crops of Pilibhit district, to determine the

frequency of occurrence and distribution of AM fungi.

2. Collection of AM fungi from agricultural fields and their multiplication

by single spore inoculation technique and maintenance of pure culture

under pot condition.

3. Screening of comparative efficacy of different species of AM fungi to

determine the efficient AM inoculant in tomato in sterilized soil under

greenhouse condifion.

4. Efficient strain of AM fungus Glomus was tested alone in various

combination with Meloidogyne incognita on tomato (var. Pusa Ruby).

REVIEW OF LITERATURE

REVIEW OF LITERATURE

While various epiphytes and endophytes may contribute to biological

control. The importance of mycorrhizae deserved special consideration.

Mycorrhizae are formed as the result of mutalistic symbiosis between fungi

and plants and occur on most plant species. Because they are formed early in

the development of the plants, they nearly represent ubiquitous to root colonists

that assist plants with the uptake of nutrients and also prevent root infections by

reducing the access sites and stimulating root defense. VAM fungi have been

found to reduce the incidence of root nematode (Linderman, 1990). Numerous

reviews over the past 15 years on the efficacy of AM fungi as biological

control agent have been done (Schenck and Kellam, 1978; Schoenbeck, 1979;

Dehne, 1982; Schenck, 1987; Caron, 1989; Jalali and Jalali, 1991; Siddiqui and

Mahmood, 1995). The main objective of this review is to analyse the role of

AMF on biological control of plant diseases. However the information

regarding the interaction of these organisms with nematodes are meagre and is

completely lacking in Indian context. It is therefore a need to give a short

review on this aspect.

VAM as biofertilizers :

Much interest has been shown in the recent past on the root fungus

association and the possible beneficial role of fungal mycelia which often

mantle plant roots. This was brought about for first time in 1842 and was given

name as Mycorrhiza by Frank (1885). The importance of AMF as a tool, for

improving the growth and productivity in diverse growth of plants was

recognized only after the work of Gerdemann (1968) and Mosse (1972).

However, during last two decades a lot of information has been gathered about

the taxonomy, ecology, physiology and anatomy of AM fungi and their relation

with host especially in relation to uptake of phosphorus and also other mineral

nutrients, hormone production and root diseases (Gianinazzi et al, 1982;

Harley and Smith, 1983; Gianinazzi and Gianinazzi-Pearson, 1986; Grahm,

1988). Attempts have been made to cultivate them in artificial culture media

and use them in different plant production system (Gianinazzi et al., 1984;

Hall, 1988; Gianinazzi etal., 1990).

Wong et al. (2005) studied the effect of biofertilizer containing N, P and

K solubilizers and AM fungi on maize growth. They identified biofertilizer as

an alternative to chemical fertilizers to increase soil fertility and crop

production in sustainable farming. They concluded that the use of biofertilizer

resulted in the highest biomass and seedling height. This study also indicated

that half the amount of biofertilizer application had similar effects when

compared with organic fertilizer or chemical fertilizer treatments.

AM fungi are not host specific but can exhibit certain preferences if

screened against different plants and therefore, have a very wide host range.

They were also occurring over a wide range of agroclimatic condition (Mosse,

1972). AMF produce three types of spores, zygospores, azygospores and

chlamydospores (Trappe and Schenck, 1982; Koske et al., 1983).

The vesicular arbuscular mycorrhizal fungi (VAM) also known as

arbuscular mycorrhizal or endomycorrhizal fungi) are all members of the

Zygomycota. The order glomales of class zygomycetes which encompasses

representing 6, the genera Glomus, Sderocystis, Acaulospora, Entrophosphora,

Gigaspora, Scutellospora with around 170 described species (Verma and

Hock, 1995; Morton and Benny, 1990; Smith et ai, 1997).

AM fungi are ubiquitous and have been recovered from the soils of a

variety of habitats. In India, VAM fungi are very well distributed in cultivated

than in non-cultivated soil. Mosse and Bowen (1968), Sparling and Tinker

(1978), Schenck and Kinloch (1980). Mukerji and Kapoor (1990) reported that

more number of spores in cultivated lands than non-cultivated land.

Janardhanan et al. (1994) reported presence of VAM fungi from alkaline soil.

AM fungi develop association with nearly all cultivated plants whether

they are agricultural, horticultural or fruit crops. There are about 300,000

receptive hosts and 170 AM fungal species reported (Anonymous, 1974;

Mukerji, 1996). Barea et al. (1993) pointed out that indigenous or introduced

VAM fungi are involved in the development of different crop production

system. Important crops associated with VAM include wheat, maize, all

millets, potato, beans, soybean, tomato, oranges, grapes, sugarcane (Bagyaraj,

1991).

Seasonal variation have a remarkable influence on the spore number

(Khan, 1975; G.P. and Tinker, 1978;). The spore populations of VAM fungi

also vary at different sites in the same geographical area. Powell and

Sithamparanathan (1977) noted that spore number and type also influenced by

soil factor. Bayles (1967) observed that spores is also influenced by the soil

pH. Khan (1975, 1978) reported that Glomus mosseae is very common in near

neutral soil and very rare in acidic soils.

Schmidt and Snow (1986) found that AM fungi were present to varying

degree in the roots of at least some members of all plant communities sampled.

Medina et al. (1988) observed that legume species differed in per cent root

colonization and total spore density among locations. Mohankumar et al.

(1988) revealed that most plant harboured AM fungi were influenced by

temperature and moisture status. Kim et al. (1989) noted that out of 103 plant

species (41 families) collected from limestone sites in Korea republic, 98

species were associated with AM fungi. Hussain et al. (1995) have been

observed in the roots of 14 hydrophytes and suggested that high AM.

Colonization in plant root is due to the availability of oxygen. Venkataramanan

et al. (1990) had given qualitative and quantitative distribution of AM spores in

soil samples from Indian habitats viz., Assam, Arunachal Pradesh, Manipur,

Mizorum and Nagaland. The highest counts were made in the planes of Assam.

The most abundant species were found to be Scutellospom nigra, Sclerocystis

rubriformis and Glomus macrocarpum. They suggested that the inoculation

trials in hilly areas with AM fungi proved beneficial to crops such as chilli,

soybean and maize.

Peng and Shen (1990) observed that spore density and frequency of

occurrence of AM fungi are greater in autumn than on the spring. Payal et al.

(1994) reported that AM colonization happens to be the lowest during winter

and highest during late summer and autumn. Kuhn et al. (1991) reported that

out of 43 species of flowering plants examined, 40 are nearly associated with

VAM fungi. Root colonization by vesicular arbuscular mycorrhizae ranged

from 6 to 28 per cent and total spore density ranged from 23-256 per 100 g soil.

Level of AM colonization varies between the two crops from site to site and

not related to soil properties (Talui<;clar and Germida, 1993) and species to

species (Muthukumar et al. 1996).

VAM as growth promoter :

Interest in arbuscular mycorrhizal fungi propagation for agriculture is

increasing due to their role in the promotion of plant growth and health

(Gerdemann, 1968; Harley, 1969; Tinker, 1975; Howeler et al, 1987; Linxian

and Hao, 1988; Raju et al, 1989). The use of AMF for crop productivity

requires a selection of an efficient and appropriate fungus and it has been

assessed by many workers (Jensen, 1982; Krishna et al, 1984). There are

several reports of some species being more efficient than others (Kuo and

Huang, 1982; Mosse, 1977). Glomus tenius was more efficient than the

indigenous fungi at a particular location. The agricultural importance of AMF

is mainly due to their ability to increase phosphate uptake and other major and

minor nutrients of crop plants (Mosse et al, 1973). The main constraints in

exploiting beneficial effects of AM fungus in improving agricultural

productivity is the obligate nature of symbiont large scale production of

commercial inoculum that provide users with product of quality and efficiency

is not possible. Presently, use of AM fungi is confined to green house and pot

culture studies and to a limited extent in plant propagation nurseries

(Rangaswami, 1990). Effect of VAM fungi on various kinds of plants has been

studied by various workers (Asif er al, 1995; Muhammad and Hussain, 1995;

Kehri and Chandra, 1990; Menge et al, 1978; Plenchette et al, 1981;

Ragupathy and Mahadevan, 1995; Adjoud etal, 1996).

10

Growth yield and dry matter increase by AM inoculation had been

reported in many crops such as barley, onion, soybean and rice (Owersu and

Mosse, 1979; Bagyaraj et ai, 1979; Sanni, 1976). Koch et al. (1997) observed

that VAM inoculated garlic plants had larger and more green leaves and

increased photosynthetic rate, especially at low light intensities, and higher

fresh and dry weights than plants in uninoculated plots. Root colonization of

AM fungus Glomus species is known to increase phosphorus content in the

mycorrizal root tissue of maize plants to 35 percent by G. mosseae and 98% by

G. fasciculatum.

Almomany (1991) studied the response of beans, broadbean and

chickpea plant to inoculation with Glomus species. Eight different locally

obtained Glomus species including G. pallidum, G. mosseae, G. fasciculatum,

G. etunicatum and G. monosporum were inoculated into bean, broad bean and

chickpea plant to examine their effect on plant growth. He concluded that G.

fasciculatum and G. mosseae were the most effective in colonizing the roots of

beans, broad bean and chickpea plants, G. mosseae increased the shoot growth

of beans, broad beans and chickpea by 52, 117 and 190% respectively. Effect

of G. fasciculatum, G. mosseae, Gigaspora calospora and Acaulospora laevis

on sorghum variety C026 was seen by Prabhakaran et al. (1995) who found

increased biogrowth of both the plants by G. fasciculatum.

Edathil et al. (1996), studied the interaction of multiple VAM fungal

species on root colonization plant growth and nutrient status of tomato

seedlings. Tomato seedlings were grown in sterile, phosphorus deficient soil

and inoculated with 4 species of Glomus i.e. Glomus aggregatum, G.

11

fasciculatum, G. geosporum and G. sinuosum that resulted in a significantly

higher shoot length and biomass, than non-mycorrhizal plants. The VAM

combination, containing all endophytes promoted markedly better shoot length

and biomass than other combination.

Iqbal and Mahmood (1998) studied the effect of single and multiple

VAM inoculants on the growth parameters of tomato. G. mosseae resulted

maximum growth of plants followed by G. constrictum.

Utility of mycorrhizal application in horticultural crop production were

under evaluation in many fruit crops like citrus (Menge et ai, 1978; Onkarayya

and Sukhada, 1993), apple (Plenchette et ai, 1981; Sharma and Bhutani, 1995)

and strawberry (Hughes et ai, 1978). In all cases, beneficial effect of

inoculation has been recorded. Sivaprasad et al. (1995) reported a maximum

plant heights (12 cm) and fresh weight (4.38 kg) in jack fruit (Artocarpus

heterophylla) inoculated with G. mosseae. Different VAM fungi have been

found to have different efficiencies in increasing fruit yield and nutrient uptake.

In tomato plant (Raverkar et al, 1994; Raverkar and Bhandari, 1995).

Mamatha and Bagyaraj (2002) studied the effect of different levels of

VAM inoculum application on growth and nutrition of tomato under

greenhouse conditions. Different levels of G. fasciculatum inoculum tried (0,

0.5, 1.0, 1.5, 2.0 and 2.5 kg/m" of nursery bed). 2.0 kg inoculum per m of the

nursery bed improved the plant growth, biomass, P content, mycorrhizal root

colonization. Future increase in inoculum level did not significantly improved

seedling growth and nutrition. Again Mamatha and Bagyaraj (2003) conducted

a green house study to determine the effect of different method of VAM

12

inoculum application on growth and nutrient uptake of tomato seedlings grown

in raised nursery beds. G. mosseae application resulted an increase of plant

height, number of leaves, leaf areas, plant biomass, phosphorus content and per

cent colonization.

Increased growth responses and nutrient uptake has been reported

because of AM symbiosis in several field grown forage and seed legumes. In

case of pigeon pea, the inoculated plants had higher mycorrhizal root

colonization, shoot and root dry weight and phosphate content (Manjunath and

Bagyaraj, 1984; Ramraj and Shanmugam, 1990). Glomus leptotrichum, G.

macrocarpum, Acaulospora laevis, Gigaspora margarita and G. fasciculatum

were equally good in promoting shoot and root dry weight (Reddy and

Bagyaraj, 1990). In black gram dry weight were found to be significantly

higher on inoculation with G. fasciculatum, G. constrictum, G. versiforms and

Acaulospora species (Umadevi and Sitaramaiah, 1990). Sylvia et al. (2001)

evaluated the influence of arbuscular mycorrhzial (AM) fiangi on the

competitive pressure of bahiagrass on the growth of tomato. Tomato grown

alone was very responsive to mycorrhizal colonization, shoot dry weight of

inoculated plants was upto 243 per cent greater than that of non-inoculated

plants. Tomato grown with bahiagrass had reduced root and shoot growth

across all treatments compared with tomato grown alone but there was an

increase in shoot weight following AM fungal inoculation. They concluded that

the role of mycorrhizae in plant competition for nutrients is markedly impacted

by soil nutrient status and reduced P application.

13

Mycorrhizal fungi enhance the absorption of nutrients by increasing the

total surface area of the roots. Absorbed phosphorus is probably converted into

polyphosphate granules in the external hyphae (Callow et al, 1978) and passed

to the arbuscules for transfer to the host (White and Brown, 1979). This flow of

phosphorus occurs in the presence of acid phosphatase (Gianinazzi et al,

1979). The mycelial network in mycorrhizal plants enables them to extract

phosphorus from places beyond the zone of low concentration around the roots

and depend on the distribution and phosphate uptake of external hyphae

(Jakobson et al, 1992). AM fungi also stimulates the plant uptake of zinc,

copper, sulphur, potassium and calcium although not as markedly as

phosphorus (Copper and Tinker, 1978).

Anion (1982) reported the production of phytohormones by G. mosseae

and considered them as important for evaluation of isolates for inoculum

production.

Effect of fertilizers on host benefit by mycorrhizal fungi

Phosphorus deficiency is one of the major limiting factor for crop

growth and yield in the tropics, mycorrhiza help to conserve and use

phosphorus efficiently. Improved phosphorus nutrition when colonized with

AMF has been demonstrated for hundred of cultivated plants. By extending

past the P-depletion zone formed around the root system, the flingal soil

network is able to maintain P transports to plant for longer periods (Hodge,

2000; Lange et al, 2000; Miller et al, 2001).

Many researches have shown that under high phosphorus soil

conditions, AMF are almost of no use to the plant and symbiosis is temporarily

14

inhibited. However, very low phospiiate fertilizer addition to soil, can increase

per cent colonization of root system, possibly through direct effect on AM

fungus symbiosis efficiency (Bolan et al., 1984).

Hao et al. (1991), estimated the development of mycorrhizal infections

by assessing the percentage of VAM infection of mungbean in pot culture with

greyed alluvial soil and red soil. Most of which were deficient in available

phosphorus. Results showed that infections of indigenous VAM fungi in most

soil were low and there was usually a lag phase. But after introducing

mycorrhizal fungi into the soil lag phases of infections were decreased or

disappeared and plant growth was greatly stimulated. Phosphorus encouraged

mycorrhizal infection in most soils deficient in available phosphorus, but the

optimum amount of P fertilization was different between alluvial soil and red

soil.

The P status of the plant strongly affects AM colonization and spore

production and sporulation. This intum determines cell membrane permeability

and root exudation of carbohydrates and amino acids available metabolites to

the fungus (Grahm et al, 1981). In field addifion of P fertilizer to soil reduces

either AM fungal colonization or sporulation in a variety of crops, including

maize, corn, soybean, clover, small grains and teak stumps (Lu and Miller,

1989; Durga et al, 1995). Tawarya and Saito (1996) also found that addition of

P to host plant influenced the composition of root exudates and thereby

increase hyphal growth of AM fungi.

Susai (1991) studied the effect of phosphate applicafion on infection of

vesicular arbuscular mycorrhizal fungi in some horticultural crops. In yield

15

• tests on maize, soybean, tomato, carrot and Artium lappa, the application of

phosphorus fertihzers increased shoot dry weight, increased shoot phosphorus

content after second cropping (86 day after sowing) and decreased mycorrhizal

infection rate to varying degree. Mycorrhizal spore number on rhizosphere soil

(soybean, tomato and maize) was much higher in soil without added

phosphorus. It is concluded that VAM fungi promote phosphate uptake in low

phosphate soils during the early stages of plant growth.

Elwan (1993) observed that maize shoot and root dry weight, root and

shoot length, root surface and transpiration rate were increased with P fertilizer

inoculated with AM fungi. In another experiment Elwan (1993) determined

that the uptake of P, K, Ca, Mg, Fe, Mn, Cu and Zn were highest when plant

received P + AM in combination followed by P fertilizer alone.

Araujo et al. (1996) studied the effect of phosphate fertilizer on tomato

inoculated with Glomus etunicatum. They observed that plants received 60 mg

P/kg soil resulted in enhancing the growth and P accumulation rate while at

120 mg P/ka soil resulted a sharp decline in growth, P accumulation and P

utilization rate at the early growth stages and increased at the later stage.

Stimulatory effect on plant growth by fertilizer application were evident

from several studies (Fabig et al., 1989; Zaghloul et al., 1996; Singh, 1996).

Dhinakaran and Savithri (1997) studied the effect of different P treatments and

VAM fungal inoculation in tomato cv C03 grown in pots filled with calcareous

soil. Phosphorus was applied as single phosphate at 75 or 100 kg ^lOslha.

Phosphorus was applied as single superphosphate at 100 kg PjOs/ha that

resulted a significant increase on the yield of tomato, phosphorus contents and

16

P uptake of tomato fruits. These pattern increased more with the increase in

applied P and VAM fungal inoculation increase fertilizers.

Khaliq et al. (1997) found that VAM inoculation at three phosphorus

application levels (3, 10 and 30 mg/kg) significantly suppressed maize seedling

growth and phosphorus inhibition of mycorrhizal infection increased with

increasing rates of application.

Posta et al. (1997) reported that the positive effect of mycorrhizal

inoculation on shoot growth decreased as P rate increased. For the various soil

volumes, the effect of mycorrhizal inoculation on the shoot mass increased as

soil volume increased. In all cases mycorrhizal plants had higher P contents. P

application reduce the degree of root colonization and also the quantity of

external hyphae.

Olsen et al. (1996) studied the effect of VAM fungal inoculum, Glomus

mosseae on capsicum {Capsicum annum L.), sweet com {Zea mays L.) and

tomato {Lycopersicon esculentum) grown in a low P sandy loam and along with

different levels P (0, 10.3, 30.9, 9 to 278 mg P/kg oven dry soil; P,, P2, P3,, P4 or

P5 respectively) and 2 levels of N (50 or 200 mg N/L). At low P rates, dry

weight of sweet corn and tomato, plants colonized with VAM did not differ

from uninoculated plants, despite inoculated plants having higher P

concentrations in index tissues. For all three crops, lack of VAM response at

high P was related to a lower VAM colonization.

Asmah (2004) studied the effect of two-phosphorus sources (triple

superphosphate and Ghafsa phosphate rock), applied at rates equivalents to 44

kg ha' and 22 kg ha'', on VAM fungal infection in roots, dry matter yield and

17

nutrient content of maize grown in an oxisol and an alfisol. The application of

44 kg P ha"' resulted in root infection by VAM fungi was not significantly

different (p < 0.01) from when no P was applied. Root infection was

significantly greater when P was applied as triple, superphosphate at the rate of

22 kg ha"' phosphate rock treatment at both rates of application that resulted in

significantly greater root infection than in control or with triple superphosphate

was applied at 44 kg ha'V Plant P uptake increased in all soils with different P

treatments compared with the control.

VAM fungal in nematode control:

Plant parasitic nematodes and mycorrhizal fungi are commonly found

inhabiting the same soil and colonizing roots of their host plants. These two

groups of micro-organism exert a characteristic, but opposite effect on plant

health. The potential role of mycorrhizal fungi for the control of nematode

diseases has recently received considerable attention (Osman et al., 1990;

Ahmad and Aslayed, 1991; Sankaranaryanan and Sundarbabu, 1994; Santhi

and Sundarbabu, 1995; Price et ai, 1995; Kerry, 1996).

Arbuscular mycorrhizal fungi (AMF) variously influence a number of

plant nematode interactions (Atilano et ai, 1981; Grandison and Cooper, 1980;

Hussey and Roncadori, 1982). High populations of endoparasitic nematodes

and spores of endomycorrhizal fungi were found in a survey made by Hasan

and Jain (1987) indicating that these nematodes do not affect the VAM flingi

and vice versa. However, in some crops like gram, cowpea and pigeonpea, low

incidence of root-knot nematode in the roots having high level of VAM fungi

was observed (Hasan and Jain, 1987). Jain and Hasan (1986) in a different

18

investigation indicated that the presence of nematodes did not adversely affect

VAM sporulation and reported that, where there was only 50 per cent root

colonization, nematode number was lower and they rarely infect VAM

colonized region of roots, but on the other side, the roots infected by

endoparasitic nematode were not colonized by VAM fiingi while the damage

done by plant parasitic nematodes is compensated by mycorrhizae unless close

to the nematode feeding site. Stimulation of root growth by VAM provides

greater habitat and sometimes resulted in increase of nematode population.

Increased spore production and higher root colonization by VAM fungi in the

presence of nematodes have also been observed (Ingham, 1988). Jain and Sethi

(1989) showed that the occurrence of Heterodera cajani and VAM fungi.

Glomus fasciculatum and G. epigaeus in Vigna unguiculata were largely

independent of each other and the organism modify the effect of each other to

some extent. However, an increase in nematode inoculum invariably resulted in

reduced root infection and spore production by mycorrhizal fungi. The

presence of G. fasciculatum showed a profound adverse effect on cyst

production and multiplication of nematode, while G. epigaeus exhibited a

reverse trend.

The antagonistic effects of AM fungi on nematodes may be either

physical or physiological in nature. The nematode control may be through

improved plant vigour, physiological alteration of root exudates or through

direct role of mycorrhizae in retarding the development and reproduction of

nematodes within root tissues. Several studies report an antagonistic effect of

mycorrhizal fungi on plant parasitic nematodes (Fox and Spasoff, 1972;

Grandison and Copper, 1986; Macguidwin et al, 1985; Osman et ai, 2005).

19

Sitaramaiah and Sikora (1980) observed that Glomus mosseae increased the

resistance of tomato plants to Rotylenchulus reniformis infection. AM fungi

can alter the physiology of the root, including root exudates which were

responsible for chemotactic attraction of nematode. Sikora (1978) suggested

that attractiveness of the root system to M. incognita larvae was altered by the

presence of G. mosseae. Sitaramaiah and Sikora (1982) expressed the other

version of increasing resistance in tomato plants colonized by Glomus

fasciculatum against Rotylenchulus reniformis by delaying the nematode attack

in roots and less formation of egg/eggsac. Glomus fasciculatum adversely

affected R. reniformis during several phases of its life cycle.

Several investigations Atilano et al. (1981), Cason et al. (1983),

Roncadori and Hussey (1977), Healed et al. (1989) have suggested that

increased nutrient uptake of mycorrhizal fungi enhances plant tolerance relative

to detrimental effects on nematode. Similarly it had been found that the

presence of mycorrhizae increase the tolerance of plants to diseases (Chandra

and Kehri, 1996). The increase of giant cells formed by mycorrhizal plants

was significantly low in tomato plants, infected with root-knot nematode M.

incognita and mycorrhizal root did not prevent penetration by the nematode

larvae (Suresh et al, 1985).

Baghel et al (1990) observed that Glomus mosseae stimulated the

growth of Citrus jambhiri seedlings. The nematode Tylenchulus semipenetrans

decreased plant growth and simultaneous inoculation with AMF and nematode

partly neutralized the adverse effect of the nematode, as the fungus was

limiting the development of the nematode. Sivaprasad et al. (1990) showed that

20

the pre-inoculation of Piper nigrum cv. Panniyur cuttings with Glomus

fasciculatum or G. etunicatum reduced the root knot (Meloidogyne incognita)

index by 32.4 and 36.0 per cent respectively, reduced nematode population in

roots and surrounding soil, and significantly increased growth even in the

presence of nematode. The tolerance of kiwi plants to M javanica was

increased in presence of G. etunicatum (Verdejo et al., 1990).

Srivastava et al. (1990) found that acid phosphatase activity in leaf was

greater in Meloidogyne incognita inoculated tomato plants than Glomus

fasciculatum inoculated plants while phosphorus concentration was generally

higher in Glomus mosseae inoculated plants. Singh et al. (1990) noted that pre­

occupation of tomato (Pusa Ruby) roots with Glomus fasciculatum resulted in

an increase in lignin and phenols and this might increase the resistance in

tomato plants against root knot nematode, Meloidogyne incognita. Mittal et al.

(1991) showed that tomato tissues forming galls following inoculation with

Meloidogyne incognita had no AM fungi while roots lacking nematode galls

had vesicles and arbuscules of Glomus fasciculatum, which inhibits the

formation of nematode galls. Glomus fasciculatum was found to be the better

and most effective mycorrhizal fungus when compared to G. mosseae in its

overall performance (Sharma and Trivedi, 1994).

Glomus fasciculatum application in nursery beds helped the mycorrhiza

to colour the tomato roots before transplantation to the main field, thereby

preventing the penetration and development of nematode in the infected plants

(Sundarbabu and Sankaranarayanan, 1995).

21

Mishra (1996) studied the inter-relationship oi Meloidogyne incognita,

G. fasciculatum and the three commonly used herbicides. Higher level of VAM

after 60 days of inoculation improved growth of tomato plant, whereas higher

levels of M incognita caused progressive reduction in plant growth.

Simultaneous incorporation of VAM and nematode resulted in maximum

multiplication of VAM. Pre-establishment of G. fasciculatum increased plant

growth, decreased the size and number of galls and improved NPK uptake over

plants inoculated with the nematode alone or pre-inoculated with the nematode

to VAM. Herbicidal combination with VAM and nematode showed overall

decrease in growth of tomato plants, multiplication of Glomus and uptake of

NPK.

Sundarbabu et al. (1996) observed that when Glomus fasciculatum was

inoculated 15 days prior to nematode inoculation, that resulted an enhancing

the growth of tomato cv. COS and suppress M incognita multiplication.

Simultaneous inoculation followed the similar pattern. G. fasciculatum was

unable to suppress nematode growth when the nematode was inoculated 15

days prior to fungus.

Mishra and Shukla (1997) reported that simultaneous inoculation of G.

.fasciculatum with M incognita caused greater reduction in the number and size

of the nematode induced root galls. Application of G. fasciculatum 15 days

prior to the nematode significantly decreased the number of size of the galls

and increased growth of tomato var. Pusa Ruby as compared to plant

inoculated with nematode alone or inoculation with nematode 15 days prior to

22

VAM. NPK contents of tomato plants were significantly higher with prior an

simultaneous application of the VAM and nematode.

Cofcewicz et al. (2001) under greenhouse conditions studied the

interaction of arbuscular mycorrhizal fungi Glomus etunicatum and Gigaspora

margarita and root knot nematode, Meloidogyne javanica and their effects on

the growth and mineral nutrition of tomato. The result pointed out that the

shoot dry matter yield was reduced by nematode infection and this reduction

was less pronounced in plants colonized with G. etunicatum than plant

colonized with G. margarita and non-mycorrhizal plants. The higher tolerance

of plants colonized with G. etunicatum to M. javanica appeared to be

associated with P nutrition.

Labeena et al. (2002) evaluated the ability of five arbuscular mycorrhiza

viz., Glomus fasciculatum, Glomus macrocarpum, Gigaspora margarita,

Acaulospora laevis and Sclerocystic dussi to mitigate damage caused by M.

incognita on tomato cv. Pusa Ruby. They found that Glomus fasciculatum was

the most efficient in promoting plant growth in the presence of nematode. The

developmental stages of nematodes in the roots and nematode density in soil

were suppressed by the AM fungi and the most pronounced effect was

exhibited by Glomus fasciculatum.

Kantharajan et al. (2003) carried out a survey to determine the

biodiversity of vasicular arbuscular mycorrhiza (VAM) Glomus fasciculatum in

the rhizosphere of different crops in seven district of southern Kamataka, India.

Among 108 soil collected, only 8 isolates of G. fasciculatum were selected on

the basis of spore morphology and spore load and were evaluated against root

23

knot nematode. Meloidogyne incognita race-1, on tomato. Amongst the isolates

tested, the Shimoga, areca isolate was the most effective in increasing the plant

growth parameters, the most number of chlamydospores and highest per cent

colonization. Nematode population in soil, number of galls and egg masses per

root system were lowest in Shimoga banana isolate followed by Shimoga areca

isolate.

Carling et al. (1995) studied the individual and combined effects of two

arbuscular mycorrhizal fungi (AMF). Meloidogyne arenaria and phosphorus

(P) fertilization (0, 25, 75 and 125.7 g/g soil) on peanut plant growth and yields

in green house. Best growth and yields usually occurred at 75 and 125 g P

regardless of inoculation treatment. In challenge inoculations, VAM increased

peanut plant tolerance to the nematode and offset the growth reduction caused

by M. arenaria at the two lower of P levels.

Osman et al. (2005) studied the interaction of root-knot nematode and

VAM fungi on common bean plants {Phaseolus vulgaris L.) in the greenhouse.

They concluded that the inoculation with VAM fungus caused a significant

increase in plant height and fresh weight compared with non-treated plants.

Meloidogyne infection significantly decreased plant height and dry weight.

When fungus was inoculated at 15 and 30 days prior to M incognita infection,

a significant increased in fresh weight was observed. The inoculation with

VAM fungus caused a significant increase phosphorus content. However, it

was significantly decreased in plants inoculated with nematode alone and in

plants inoculated with VAM fungus and M incognita at the same time. Gall

index and final nematode population were significantly increased when

nematodes were inoculated at the same time with fungus, although there were

24

significant decrease in nematode final population and gall index when plants

were inoculated with nematodes at 15 and 30 day after mycorrhzial infection.

A decreased in percentage of fungal colonization was observed when

nematodes were inoculated with fungus simultaneously.

Castillo et al. (2006) studied the effect of single and combined

inoculation of olive planting stocks cvs. Arbequina and Picual with the

arbuscular mycorrhizal ftmgi (AMF) Glomus intraradices, Glomus mosseae or

Glomus viscosum and the root knot nematodes M. incognita and M. javanica on

plant performance and nematode infection. They concluded that prior

inoculation of olive plants with AMF may contribute to improving the health

status and vigour of cvs Arbequina and Picual planting stocks during nursery

propagation.

In several studies AM fungi had shown an antagonistic influence on the

population of plant parasitic nematode (Bagyaraj et al, 1979; Saleh and Sikora,

1984; Cooper and Grandison, 1986; Sitaramaiah and Sikora, 1982; Carling et

al, 1989; Sivprasad et al, 1990; Rao et al, 1992; Sankaranarayanan and

Sundarbabu, 1994; Kerry, 1996). Nematode susceptible plant colonized by AM

fungi were better able to tolerate plant pathogenic nematode (Kellam and

Schenck, 1980; Sitaramaiah and Sikora, 1982; Grandison and Cooper, 1986;

Diederichs, 1987; Jain and Sethi, 1989; Osman et al, 1990; Sankaranarayanan

and Sundarbabu, 1994). As a result of interaction in general, the severity of

nematode diseases was reduced in mycorrhizal plants. Biological control is

more inconsistent although improvements in performance might be expected

from more research on individual agents and their successful application.

25

SECTION I

SECTION -1

THE OCCURRENCE OF ARBUSCULAR MYCORRHIZAL FUNGI (AMF), CHARACTERIATION OF SOIL, SPORE

POPULATION OF AMF IN SOIL AND ROOT COLONIZATION OF SOME COMMONLY CROPS IN PILIBHIT DISTRICT -

A SURVEY

INTRODUCTION

An extensive survey of agricultural fields of Pilibhit district of some

important cultivated crops was conducted for quantitative and qualitative

assessment of AM fungal spores population and per cent root colonization by

AMF at four sites under field conditions. The four sites selected were Puranpur

(Ai), Bisalpur (A2), Nyorea (A3), and Mainakote (A4), having some commonly

cultivated crops like tomato {Lycopersicon esculentum Mill.), chickpea {Cicer

arietinum Linn), turmeric {Curcuma longa Linn.), chilli {Capsicum annum

Linn.), pigeonpea {Cajanus cajan (Linn.) Millsp.), sugarcane {Saccharum

officinarum Linn.), wheat {Triticum aestivum Linn.).

The soils of the experimental sites were sandy loam and the

characteristics of the soils are given in (table 1). The temperature of the sites

ranges 21.3 to 33.4°C while relative humidity from 65.5 to 78.20% in a

calendar year. All the sites are moderately rainfed and have satisfactory

irrigational facilities with a soil moisture 5 to 9.80%.

MATERIALS AND METHODS

Soil sampling and root sample collection :

Sampling was done for each crop separately from the fields of cultivated

crops at site Ai, A2, A3 and A4 in different months i.e. tomato December, 2006;

chickpea - January, 2006; turmeric - June, 2006; chilli - May, 2006;

pigeonpea - September, 2006; sugarcane - April 2006, wheat - March, 2006.

Soil samples (soil cones of 5 cm diam.) were collected at random from

each soil with the help of soil auger upto a depth of 15 cm near the plant base.

Forty such samples were collected from each plant species and were

thoroughly mixed to make a composite sample, seven samples of lOOg soils

were used for recovery of spores.

Seven root samples for each plant species were collected at random in

order to assess root colonization of AM fungi. A representative sample of the

entire root system was obtained from five different portions of the root system

after washing by tap water.

Soil characteristics :

The soil samples were brought to laboratory, marked and packed in

polythene bags and their electric conductivity (EC) and pH were measured with

EC meter and pH meter respectively in the extract collected from 1:1 soil/water

suspension. The texture of soil in relation to particle size was determined by

hydrometer method (Allen et al, 1974); total organic carbon by the method

given by Walkley and Black (1934); total nitrogen by microkjeldahl method

(Nelson and Sommers, 1972) and total phosphorus by molybdenum blue

method (Allen et al, 1974).

Quantitative estimation of spores from the soil:

Spores of AM fungi were isolated by wet sieving and decanting method

(Gerdeman and Nicolson, 1963). For this a sample of 100 g dry soil was mixed

27

in water (1000 ml) and the heavier particles were allowed to settle for few

seconds. The liquid was poured through a coarse soil sieve to remove large

piece of organic matter the liquid passed through this sieve was collected and

again passed through a sieves of 80, 100, 150, 250 and 400 mesh. Spores

obtained on sieves were collected with water in separate beakers. The spores

were counted in 1 ml of the suspension in nematode counting dish under the

stereoscopic microscope. The final number of spores/100 g of soil was

calculated accordingly for each crop.

Assessment of colonization by AM Fungi:

Clearing and staining (Phillips and Hayman, 1970)

Roots were washed with tap water and cut it into 1 cm long segments

and then boiled in 10% KOH solution at 90°C for 45 minutes. KOH solution

was then poured off and roots were rinsed well in a beaker until no brown

colour appeared in the rinsed water. Alkaline H2O2 which was used to bleach

the roots was made by adding 3m of NH4OH to 30 ml of 10% H2O2 and 567 ml

of tap water. The roots were rinsed thoroughly at least three times using tap

water to remove the H2O2. Roots were then treated with 0.05% trypan blue (in

lactophenol) and kept for overnight for destaining in a solution, prepared with

acetic acid (laboratory grade) 875 ml, glycerine 63 ml and distilled water

63 ml.

The cellular contents were removed by this method and the AM fungal

structures get stained dark blue. This stained root segments were used for

determining the root colonization by AM flingi.

28

Percentage of root colonization and per cent arbuscules were determined

by slide method (Giovanretti and Mosse, 1980). The root segments were

selected at random from the stained sample and mounted on microscopic slides

in groups of ten. One hundred to one hundred and fifty root segments from

each samples were used for the assessment. The presence or absence of

colonization in each root segment was recorded and result was expressed as

percentage of root colonized. The root colonization (mycorrhizal infection in

the roots) was calculated as follows :

Number of mycorrhizal segments % AM association =

Total number of segments served

Isolated spores were identified with the help of keys provided by

different workers (Trappe, 1982; Hall and Fish, 1979; Bakshi, 1974; Rani and

Mukerji, 1988; Srinivas et al., 1988). The data collected during this study were

statistically analyzed in simple randomized design by the method of Panse and

Sukhatmi (1985).

RESULTS

Soil collected from the four sites were alkaline with pH ranging from 7.1

at site Ai to 8.4 at site A4 and sandy loam in texture. The electric conductivity

was low at site A3 and high at site A4 in comparison to other two sites. The

organic carbon content of the soil at site A4, is highest, followed by A2, Aj and

A3. As far as the soil nutrient is concerned, nitrogen content was highest at site

A4 while P ranged between 7.0 to 8.1 kg/ha, highest being at site Ai. The

percentage of soil samples having a particular species of AM fungi idenfified

are given in table 1.

29

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The data provided in table 2 indicate that the total spore number per

lOOg of soil varied with the crop as well as with the site. The sugarcane crop

showed the highest number of spores, whereas chickpea show the minimum at

almost all the four sites investigated.

At site I, wheat showed the second highest number of spore followed by

pigeon pea, tomato and turmeric. The statistical analysis of the data clearly

shows that the spore number of chickpea, chilli and pigeon pea were varied

significantly from one another.

At site II, the highest and lowest spore numbers were recorded in wheat

and chickpea, respectively. The number of spore in turmeric was close to

pigeon pea. Similar trend were shown by chickpea, turmeric and chilli. The

second highest number of spores were recorded in sugarcane followed by

tomato and pigeon pea. The spore number in chickpea, chilli and sugarcane

were statistically significant than other crops while pigeon pea was at par with

sugarcane.

At site III, although the general trend was the same as in others the

highest number of spores were recorded in sugarcane and lowest in chickpea.

The spore numbers obtained from turmeric, chilli and pigeonpea were

significantly higher than other crops.

At site IV, the spore number in general was found to be poor as

compared to other sites. The highest and lowest spore number were recorded in

wheat and chilli. Turmeric show the second highest followed by crops like

tomato, chickpea, sugarcane and pigeon pea.

31

Table - 2 : Total spore counts at different sites in different crops

Crop

Tomato

Chickpea

Turmeric

Chilli

Pigeon pea

Sugarcane

Wheat

CD.

Site I

346

320

341

326

361

440

380

12

Site II

360

310

322

332

356

370

400

14

Site III

360

280

281

336

354

420

367

9

Site IV

220

270

280

187

216

260

314

7

Table -3 : Spore counts of Glomus fasciculatum at different sites under different crops.

Crop Site I Site II Site III Site IV Tomato Chickpea Turmeric Chilli Pigeon pea Sugarcane Wheat CD.

109

125

132

80

164

186

162

8

64

72

177

180

108

126

128

10

121

88

158

64

76

168

177

7

120

56

62

105

74

96

77

6

Table -4 : Spore counts of Glomus mosseae at different sites under different crops.

Crop Tomato Chickpea Turmeric Chilli Pigeon pea Sugarcane Wheat CD.

Site I Site II Site III Site IV 160

^9 95

75

55

141

136

4

107

112

36

74

80

148

194

5

167

100

64

104

130

148

130

7

48

59

73

47

36

85

130

6

32

Fig. a. Showing the slide of Glomus Fasciculatum

\ \ 4.^' * •>

fi

\

n

Fig.b. Showing the Slide of Glomus Mosseae

1:

Fig. e. Showing the Slide of Acaulospora laevis

Fig. f. Showing the Slide of Gigaspora Gigantea

Table -5 : Spore counts of Glomus etunicatum different sites under different crops.

Crop Site I Site II Site III Site IV

Tomato Chickpea

, Turmeric Chilli Pigeon pea Sugarcane Wheat CD.

31

39

50

74

40

14

37

5

0

66

35

31

104

13

%9 6

34 41

29

55

25

68

28

5

43

38

40

13

25

35

37

4

Table -6 ; Spore counts of Glomus constrictum at different sites under different crops.

Crop Site I Site II Site III Site IV

Tomato • Chickpea Turmeric Chilli Pigeon pea Sugarcane Wheat CD.

37

67

50

9

6

50

20

4

80

15

32

7

16

14

57

6

20

48

18

46

38

8

16

5

6

25

35

12

56

26

28

5

Table -7 : Spore counts of Aculospora laevis at different sites under different crops.

Crop Site I Site II Site III Site IV

Tomato

Chickpea

Turmeric

Chilli

Pigeon pea

Sugarcane

Wheat

CD.

9

0

8

44

50

37

19

3

41

0

24

24

48

66

26

5

12

0

12

65

47

28

12

3

3

48

40

10

25

0

6

2

35

Fig. c. Showing the Sh'de of Glomus etunicatum

• ^ • - ,

a>.

Jt -a* •

Fig. d. Showing the Slide of Glomus constrictum

At site IV, the highest number of spores was recorded under Tomato

cultivation which was closely followed by Turmeric, Chickpea, wheat and

sugarcane with minor variations. The lowest number of spores were recorded

from chilli field.

It was observed that the number of spores of a G. etunicatum of all the

four sites were very low as compared to G. fasciculatum and G. mosseae.

Table 6 gives the data of spore population of Glomus constrictum (Fig.

• d) in different sites and different crops.

At site I, the highest number of spores were obtained under chickpea

cultivation while turmeric and sugarcane resulted the same number of spores.

The lowest number of spores were recovered from pigeonpea followed by chilli

and are insignificant to each other.

At site II, highest number of spores were recorded under Tomato

cultivation followed by wheat and lowest under chilli cultivation. Chickpea,

chilli and sugarcane were more significant than others.

At site III, the spore number happened to be the highest under chickpea

cultivafion. The least number of spores were obtained under sugarcane while

chilli and chickpea remained almost at the same level.

At site IV the highest number of spores were recorded under Pigeon Pea

cultivation which is followed by Turmeric cultivation. The least number of

spores were obtained under tomato cultivation. Chickpea and chilli were more

significant than others.

Table 7 show spore population ofAculospora laevis (Fig. e) at different

sites and crops. At site I, the highest number spores were recorded under

36

Pigeon pea cultivation followed by chilli and sugarcane while the lowest was

obtained from Turmeric and Tomato where spore number remained at the same

level. No spores were obtained under chickpea cultivation.

At site II, the highest number of spores were recorded under sugarcane

cultivation, followed by pigeonpea and tomato and were statistically significant

to each other. No spore was recovered from chickpea field.

At site III, chilli yielded the highest number of spores while tomato,

turmeric and wheat remained at the same level and were statistically are

insignificant.

At site IV, the highest number of spores were obtained under chickpea

and lowest under Tomato cultivation. At this site tomato and chilli were

significant than others. No spore was recovered from sugarcane field.

The table 8 show, the number of spores of Gigaspora gigantea (Fig. f)

under different sites and different crops. At site I highest spores were recorded

under Pigeon pea followed by chilli and lowest were obtained under turmeric

cultivation. No. spores were obtained under, Tomato, chickpea and wheat

cuhivation.

At site II, highest number of spores was recorded under tomato

cultivation followed by chickpea, sugarcane and wheat crops resulted a very

small number of spores while no spore was recovered from pigeonpea field.

At site III, the highest spores were recorded under pigeonpea cultivation

while chilli, chickpea and wheat show a close proximity and were insignificant

to each other.

37

Table - 8 : Spore counts of Gigaspora gigantean at different sites under different crops.

Crop

Tomato

Chickpea

Turmeric

Chilli

Pigeon pea

Sugarcane

Wheat

CD.

Site I

0

0

6

44

46

12

0

3

Site II

68

45

18

16

0

3

6

3

Site III

6

3

0

2

38

0

4

2

Site IV

0

44

30

0

0

18

36

4

Table -9 : Mycorrhization at different sites under different crops.

Crop

Tomato

Chickpea

Turmeric

Chilli

Pigeon pea

Sugarcane

Wheat

CD.

Site I

87

65

76

68

89

94

93

6

Site II

85

63

65

71

83

90

91

6

Site III

78

60

61

68

70

81

77

4

Site IV

76

58

60

56

62

74

71

5

38

At site IV, the highest spores were recorded under chickpea followed by

wheat and turmeric. No spores were recorded under tomato, chilH, and

pigeonpea cultivation. The spore number were poor as compared to other

species.

Table 9 shows, the percent colonization of AM fungi in different sites

under different crops. A glance at the data clearly indicates that the percentage

colonization occurred to the maximum extent under sugarcane cultivation in all

the sites yielding an average of 84.75%. In wheat, the colonization percentage

recorded at second highest level in all the four sites with an average of 83.0%

followed by tomato with an average of 81.50%. Almost similar percentage of

colonization were recorded from chilli, turmeric and chickpea. In pigeonpea

percent colonization happened to be 76.0%. The per cent colonization in

, different crops followed the following descending order viz. sugarcane, wheat,

tomato, pigeonpea, chilli, turmeric and chickpea. It was also evident from the

data given in table 9, that the percent colonization differ widely at different

sites under all the crops surveyed depending on the soil conditions and

environmental factors.

DISCUSSION

The results of this investigation clearly indicate that most of the plants in

all the sites are mycorrhizal. The degree of AM formation varied in all crop

species. AM colonization in roots was below 84.75% at all four sites. The

average AM colonization in all crops at four site ranged between 61.50% to

84.75%. The colonization increased gradually with an increase in the spore

39

number. Due to difference in pH and soil characteristics, the number of spores

. as well as root percentage colonized varied the highest being at site Ai.

All the crops were colonized by the AM fungi extensively and there was

no definite relationship between crop and AM fungi as the spores number in

soils collected during different crops season varied at different sites. But AM

fungi preferred sugarcane and wheat crops than the others. In general the

leguminous as well as the other crop species are mycorrhizal in nature (Mosse,

1975). Multiple infection was observed in all the cultivated crops at all the four

sites in this survey and the diversity in AM fungal spore types was seemingly

very high and AM fungal chlamydospores were quite common in all the

samples. Similar results were obtained by (Khalil et ai, 1992, Mosse 1973,

Hayman and Strold '1979. Padmavathi et ai, (1991) also observed that spores

belonging to more than one species of AM fiingi are often found in agricultural

soils. Glomus was the dominant genus followed by Acaulospora and

Gigaspora. The spores of only three genera were found in the sample collected

from all the four sites in the present study, but the level of colonization varied

from site to site. Similar results were also obtained by (Sulochana et ai, 1990;

Peng and Shen 1990 and Blaszkowski 1993). Muthukumar et al, (1996)

pointed out that AM fungal colonization varied considerably between the

species., Difference in the per cent colonization and sporulation of the

symbiont in association with different crops receiving similar environment may

be attributed to the specificity of the symbiont to the crop (Mosse, 1973;

Kruckteman, 1975).

40

It has been reported that availability of the substrate in root and the

various soil factors affected the production of external hyphae. The complex

environmental factors, seasonal fluctuations, agricultural practices and there

soil microflora may also influence the development of mycorrhizal fungi

(Schwab et al, 1983; Abott and Robson, 1985).

AM fungi are wide spread in occurrence and due to their potential for

crop improvement they have been investigated extensively (Mukerji, 1995;

Mukerji and Dixon, 1992; Powell and Bagyaraj, 1984). Natural occurrence and

predominance Glomus, Acaulospora and Gigaspora in the cultivated soils at all

the four sites indicates that there is a need of maintaining them in cultivated

soils through proper management. They may be utilized as biofertilizer and

also as biocontrol agents. The bi-directional transport enhanced the plant

growth and lead to completion of fungal life cycle. For this reason mycorrhizal

symbiosis are attractive systems in agriculture.

41

SECTION II

SECTION II

COMPARATIVE EFFICACY OF DIFFERENT ARBUSCULAR-MYCORRHIZAL FUNGAL SPP. (AMF) ON TOMATO

(LYCOPERSICONESCULENTUM MILL.)

INTRODUCTION

Arbuscular mycorrhizal (AM) association is known to improve plant

grow1;h through better uptake of nutrients and increased resistance or tolerance

to drought and root pathogens in many species of leguminous and other crop

plants (Singh, 1994; Mosse, 1973). AM fungi vary in their physiological

interaction with different hosts and hence, in their effect on plant growth.

Species and strains of AM fungi have been shown to differ in the extent to

which they increase nutrient uptake and plant growth (Powell et al, 1980).

These observations have led to introduction of the term "efficient" or effective

strains (Abbot and Robson, 1981). Generally those fungi that infest and

colonize the root system more rapidly are considered to be "efficient" strain

(Mums and Mosse, 1980). The usefulness of mycorrhizae is especially

appropriate in the development of sustainable system of agriculture (Mosse,

1986), so as to produce desirable effect of improving plant growth and

inducing resistance to pathogen in given environmental conditions (Bali et al.

1987). Tomato is one of the important vegetable plants, used in different forms

viz ., juice, paste, ketchup, soup and powder. The information to select efficient

AM fungi for inoculating tomato (var. Pusa Ruby) to achieve better growth and

drought resistance is still meager. Hence, there is need to identify specific host

-endomycorrhizal association and to define conditions under which these

association function efficiently. The present study is a step in this direction to

identify the efficient AM fungi for tomato crop.

MATERIALS AND METHODS

Starter culture of AM fungus (inoculum production)

Collection of soil sample:

Morphologically different types of spores recovered from the

rhizosphere soils were collected separately. In order to collect spores of AM

fungi from each sites, fifty soil samples were collected from the crop fields in

with the help of soil auger upto a depth of 15 cm from the rhizosphere of the

plants.

Isolation of spores

Spores of AM fungi present in the soil samples were isolated by wet

sieving and decanting method described by Gerdemann and Nicolson (1963).

Samples of 100 g dry soil was taken in 1000 ml of water thoroughly shaked

and left for a minute to settle down the heavier particles. The soil solution was

passed through coarse sieve first and then decanted on to a series of sieves of

varied size i.e. 80, 150, 250 and 300 mesh. The spores obtained on sieves were

collected with water in separate beakers. The spore suspensions were

repeatedly washed by Ringers' solution (NaCl 6g 1"', KCl 0.1 g 1'' and CaCli

0.1 g r ' in disfilled water of pH 7.4) in order to remove the adhered soil

particles from the spores. The following species were found to be of common

occurrence in the agricultural fields.

Glomus fasciculatum

Glomus mosseae

Glomus etunicatum

Glomus constrlctum

Acaulospora laevis

Gigaspora glgantea

43

All the above mentioned species were evaluated for their potential as

effective AM inoculant for tomato (var. Pusa Ruby). The spores of AM fungi

were identified under a dissecting microscope with the help of the synoptic

keys suggested by Trappe (1982). The spores of AM fungal species were

separated by picking and used for pot culture. Spores were separated with a

microspatula and picked up by a Pasteur pipette fitted with a rubber bulb.

These tools were surface sterilized for 2 minutes in a solution containing

chloroamine T 20 g/1, streptomycin 300 mg/1 and Tween 80 in trace amount/1

of distilled water.

Maintenance of AM fungi culture :

Pure cultures of six AM fungi viz., Glomus fasciculatum, Glomus

mosseae, Glomus etunicatum, Glomus constrictum, Acaulospora laevis,

Gigaspora gigantea collected during the survey (Section-1) were raised on

Rhode's grass {Chloris gay ana Kunth) grown in pots under glasshouse

conditions. To raise Rhode's grass, seeds were surface sterilized with 0.1 per

cent solution of HgCl2 and sown (5 seeds per pot) in 9 cm clay pots,

containing sterilized soil (66% sand, 24% silt, 8% clay, OM 2%, pH 7.5).

Fifty spores of each AM fiingal species per pot were layered at 6 and 2 cm

depth in 50 clay pots. After emergence, seedlings were thinned and one

seedling was maintained in each pot. After 125 days, the plants were

uprooted and the spores were isolated by wet sieving and decanting method

from the pot soil and the roots were stained and examined for the AM

colonization. The spores, hyphal fragments and small plant root segments

were then used for further experiments. The population of different AM

44

fungi in tiie inoculum was assessed by the most probable number method

(Porter, 1979).

Inoculation of AM fungus :

In order to select efficient AM inoculant for tomato, the AM fungi

recovered from agricultural fields were evaluated for their efficiency in

improving the performance of the crop (tomato: var. Pusa Ruby). The

following AM fungi were used in this experiment Glomus fasciculatum, G.

mosseae, G. etunicatum, G. constrictum, Acaulospora laevis and Gigaspora

gigantea.

Seedlings of tomato (Lycopersicon esculentum Mill.) cv. Pusa Ruby

were raised in clay pots (25 cm diam.) from seeds surface sterilized with -

0.01% mercuric chloride. The surface sterilized seeds were sown in the pots

filled with autoclaved sandy loam soil (66% sand, 24% silt, 8% clay, 2%0M,

pH 7.7) and one-week - old seedling were transplanted in 15 cm diam. pots. In

each pot filled with 920 g sterilized soil; 80g soil with AM inoculum was

added later to make the amount of soil 1 kg/pot. Before transplantation of

seedlings, the mycorrhizal inoculum of different AM fungi was separately

placed below the seedling by the layering method (Menge et al, 1977). The

inoculum was spread as a layer at a depth of 3-5 cm in the pot at the time of

planting. The seedlings were recovered with a layer of soil to ensure the

development of an efficient host fungus association. The inoculum consisted of

a mixture of infected root segments and soil with extramatrical hyphae and

spores (1000 spores/pot) from cultures of different AM fiingi maintained on

Rhode's grass, as described earlier. For each treatment 15 replicates were

45

maintained. A control series was also maintained where no inoculum of AM

fungi was added to the soil. Pots were watered appropriately and maintained in

a glass house bench with air temperature ranging from 30±2°C.

The plants were examined 20, 40 and 60 days after the transplantation

for determining the plant growth and mycorrhization of the plants. The

samples of the roots and soil were processed as described earlier in Section I.

Mycorrhization was recorded in terms of mycorrhizal intensity in roots i.e.,

external colonization percentage, internal colonization percentage, per cent

arbuscules, average number of chlamydospores in 1 cm root segment and

number of spores recovered from 100 g dry rhizosphere soil. All the data

related to growth of shoots and roots, root infection, spore population were

analyzed statistically by the method of Pause and Sukhatme (1985). Minimum

difference, required for significance (CD) at 5% level was calculated by the

ANOVA model.

The performance of the crop raised with added inoculum of selected

AM fungal species was compared with that of control and the AM fungus

causing maximum improvement in the performance over control was selected

as efficient AM inoculants for the tomato var. Pusa Ruby.

Parameters studied :

During experimentation the following parameters were determined for

each treatment of the experiments at different growth periods.

Shoot and root lengths

Fresh and dry weights of shoot and root

Af>

Mycorrhization in term of:

External and internal colonization percentage, per cent arbuscules,

number of spores recovered from 100 g dry rhizosphere soil.

Plant growth parameters :

Length, fresh weight as well as dry weight of shoots and roots at

different stages of growth were recorded for each treatment. Plants of each

treatment were taken out from the pots and soil particles adhering to roots

were removed by washing in tap water and properly labelled. In the

laboratory, lengths of shoot and root were measured by measuring tape and

fresh weights of shoot and root were determined with the help of a physical

balance. For determining dry weights of root and shoot, plants from each

treatment were wrapped in blotting paper sheets, labelled and dried in a hot air

oven running at 60°C for 24-48 h till a constant weight is obtained.

Root colonization and the spore estimation :

At the termination of the experiments root colonization in term of

percentage external and internal colonization, per cent arbuscules, estimation

of spores (in 100 g rhizosphere soil) in the same soil samples were used for

assessment of the root colonization, as described in the section I.

RESULTS

Plant length :

The effect of different AM fungi at the different intervals on plant length

was examined in terms of shoot, root and total length of the tomato plants

(Table 10). Shoot length increased as the time intervals after inoculation

47

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48

increased from 20 to 60 days. Glomus fasciculatum treated plants, showed

increase in shoot at each interval, compared to the control as well as to those

inoculated with Acaulospora laevis and Gigaspora gigantea. Shoot lengths of

plants inoculated with either A. laevis or G. gigantea did not differ from

control. Inoculation of tomato plants with G. mosseae or G. constrictum

showed a better performance at 40 days interval. The root length was also

promoted by G. fasciculatum at all the growth intervals. Treatment of G.

fasciculatum improved the root length to the same extent as that of G.

constrictum at 40 and 60 days intervals but to a lesser extent at 20 days

seedling compared to the plants inoculated with G. constrictum or G. mosseae.

The growth effects due to treatment with AM fungi on total plant length

are given in Table 10. Total plant length in control, A. laevis or G. gigantea

were similar and lower compared to the ones treated with G. fasciculatum. No

significant difference in plant length observed in plants inoculated with A.

laevis or G. gigantea compared to control.

The highest (37%) growth promotion was recorded in case of G.

fasciculatum and it was closely followed by G. mosseae (36%) at 60 days

interval. However, on 40 days old plants, G. constrictum promoted the highest

level of growth (26.60%) followed by G. mosseae (24.41%) and G

fasciculatum (23.13%).

Plant fresh weight:

At all the growth intervals G. fasciculatum promoted the highest value

of shoot fresh weight, compared to those treated with the other five AM fungi

and control, but it was apparently at par with G. constrictum inoculated plants.

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50

Inoculation of G. etunicatum, G. gigantea and A. laevis however, resulted in no

significant increase in the shoot fresh weight over the control at 20 days

interval. The fresh weight of plants inoculated with G. gigantea did not differ

from control at 40 days interval but slight increase in shoot fresh weight

occurred at 60 days interval compared to control. Inoculation of plants with G.

consthctum resulted in higher increase in shoot fresh weight than those

inoculated with G. etunicatum at 20, 40 and 60 days intervals.

At 40 days intervals the plants treated with G. fasciculatum showed

higher root fresh weight than all the other AM fungi but it was almost

equivalent to those plants inoculated with G. constrictum and G. etunciatum. At

60 days interval, the root fresh weight was the highest in the plant treated with

G. fasciculatum.

Total fresh weight of tomato plants was improved by inoculation with

G. fasciculatum, plants inoculated with G. etunicatum and G. constrictum at 40

days interval were at par. At the final stage of growth A. laevis and G. gigantea

treatments resulted significantly higher plant fresh weight than control but

lower than that of G. fasciculatum (Table 11). Inoculation of G. fasciculatum

resulted in significant increase in total fresh weight percentage of the plant over

the control as well as the other five AM fungi.

Plant dry weight:

At 60 days interval, G. fasciculatum inoculation resulted in significantly

superior shoot dry weight than all others. At 40 days interval G. mosseae and

G. constrictum resulted almost the same amount of dry weight equal value of

dry weight was supported in G. fasciculatum inoculated plants at 20 days

51

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52

interval. No significant difference in siioot dry weight was recorded in plants

inoculated with A.laevis or G. gigantea compared to control at 40 days

intervals.

As in all the other cases, G. fasciculatum supported significantly high

root dry weight at 60 days interval. The root dry weight supported by G.

fasciculatum and G. mosseae did not vary to any significant level at 40 days

intei-val. Root dry weight in the control plants was found to be at par with those

inoculated with G. constrictum at 20 days interval.

The total dry weight of tomato plants inoculated with G. fasciculatum

proved to be significantly high at 60 days growth interval compared to other

treatments (Table 12). Plants inoculated with G. constrictum resulted in

significantly high dry weight over that of G. mosseae at 60 days interval. Equal

values of plant dr>' weight was recorded in plants treated with G. constrictum

and G. etunicatum at 20 days interval. The highest total plant dry weight was

supported by G. fasciculatum at all the three intervals, which was closely

followed by G. mosseae and G. constrictum at 40 days interval.

MYCORRHIZATION

The mycorrhization of AM fungi was estimated by using four

parameters as given in (Table 13).

The value for all the parameters in plants inoculated with G.

fasciculatum was higher than all the other treatments. The values of

mycorrhization in case of A. laevis and G. gigantea were lesser than the other

AM fungi. The highest percentage of outer colonization (78.2%), internal

colonization (66.2%), arbuscules (53.20%) was recorded in plants treated with

53

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(N C^ (N

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00

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54

G. fasciculatum which was followed by G. constrictum (68%), (55%) and

(46%) at 40 days interval. G. fasciculatum inoculated plants also resulted in the

highest spore population in 100 g soil/pot (300, 326,an4^820yc6iftBared to all

other AM fungi at all the three intervals. / " f ' ^ *7, p \ G

DISCUSSION ' V

In the present study, it has been found that the mycorrliigat^-^tus and

growth responses are higher in all treatments compared to the control and the

different strains of AM fungi differ in their capacity to promote plant growth.

Better plant growth in AMF infected plants could be due to enhanced nutrient

contents of the plant. Enhanced absorption and accumulation of several

nutrients such as N, P, K, Zn, Mn, Fe, Ca and S infected plants have been

reported (Bowen et al., 1975; Selvaraj et al, 1986 and Dhillion, 1992). AM

fungi also enhance the concentration of different organic compounds in roots

and can improve the productivity of the host plant (Selvaraj et al., 1995). Out

of the six different AM fungi tested, G. fasciculatum caused highest increase in

dry weight over the control. The results of the present study are due to

increased P uptake and other nutrients, resulting in better growth, yield and dry

matter. This has also been reported for many crops such as barley, onion,

soybean, rice, blackgram (Sanni, 1976; Bagyaraj et al., 1979; Owusu and

Mosse, 1979; Kuo and Haung, 1982; Luis Brown, 1986; Jeffries, 1987;

Umadevi and Sitaramiah, 1990). Sundaram and Arangarasan (1995) reported

that out of four cultures of AM fungi G. fasciculatum gives the highest fruit

yield in tomato plants. Bagyaraj et al. (1989) reported that different strains of

AM fungi have different capability to increase the nutrients uptake and plant

55

growth, and therefore there is a need for selecting the efficient ones. It has been

found in the present study that the extent of mycorrhization in soil varies with

different AM fungal species. They also vary in their preference to the host.

Root colonization has been found to facilitate more host fungal contact and

exchange of nutrients resulting in better plant growth. Similar observation has

been made by Abbott and Robson (1982) out of the six AM fungi investigated

G. fasciculatum promoted better plant growth and nutrient content of the plants

than others, G. fasciculatum inoculated plants also showed the highest

percentage of outer colonization (78.2%), and internal colonization (66.2%),

arbuscles (53.2%) and spore production (820) compared to others. A similar

kind of result has been reported by Abbott and Robson (1985). These

mycorrhizal mycelia coupled with increased nutrient uptake results in the better

performance of the mycorrhizal plants (Rhodes and Gerdemann, 1975). In the

present study it has been found that G. fasciculatum support the highest plant

growth in terms of length and biomass of the plants at maturity. Biomass

production, mycorrhizal colonization. Sporulation have been found to be

significantly high in the G. fasciculatum inoculated plants as compared to all

the other five AM fungal species. It could be, therefore, designated as the

potential AMF inoculant in sandy clay loam soil for tomato var. Pusa Ruby for

the successful plant growth and yield.

56

SECTION III

SECTION III

EFFECT OF AM FUNGUS, GLOMUS FASCICULATUM AND ROOT-KNOT NEMATODE, MELOIDOGYNE INCOGNITA ON

GROWTH AND DEVELOPMENT OF TOMATO.

INTRODUCTION

The arbuscular mycorrhizal fungi (AMF) are associated with all the

cultivated crop plants and can significantly increase the nutrient uptake

(Hayman, 1982, Mosse, 1975). AMF and plant parasitic nematodes occur

together in the rhizosphere of the plants in association with the same root tissue

for their growth and development and while doing so they exert a characteristic

but opposite effect on plants. The interaction between AM fungi and plant

parasitic nematodes has been studied by several workers (Fox and Spasoff,

1972; Sikora and Schoenbeck, 1975., Schenck and Kellam, 1978., Bagyaraj et

al, 1979., Sikora, 1981., Hussey and Roncadori, 1982; Mishra and Shukla,

1995; Edathil et al, 1996; Mamatha and Bagyaraj, 2002).

Concomitant colonization and infection of roots by mycorrhizal fungi,

pathogens and other microbes inevitably interfere with each other's activities

(Linderman, 1985). Several studies on interaction have shown that AM fungi

associations generally induce tolerance to nematode susceptible plants (Heald

et al., 1989; Cooper and Grandison, 1987; Lingaraju and Goswamia, 1993) and

mitigate the adverse effects of plant parasitic nematodes on plant growth

(Sikora, 1979; Jain and Sethi, 1987). Fox and Spasoff (1972) demonstrated that

as little as 10 spores/pot of Gigaspora gigantea suppressed the reproduction of

Heterodera solanacearum on tobacco. Pratylenchus brachyrus levels on cotton

were reduced with 250 spores/pot (Hussey and Ronchdori, 1982). Schenck and

Kellam (1978) obtained reduction in the number of galls of Meloidogyne

incognita on soybean with 25 spores of Glomus macrncarpiis var.

macrocarpus. (Similarly population of Rotylenchulus reniformis reduced by

150 chlamydospores and egg masses and the adult females of M incognita

with 300 chlamydospores of Glomus fasciculatum on tomato and cotton

respectively (Sitaramaiah and Sikora, 1982, 1996). In the present study,

glasshouse experiment was designed to find out the individual and concomitant

effects of Glomus mosseae and Meloidogyne incognita on the growth of tomato

plants and their population dynamics.

MATERIALS AND METHODS

Plant culture :

Seedlings of tomato cv. Pusa Ruby were raised in clay pots (30cm

diam.) from seeds surface sterilized with 0.01% mercuric chloride. The surface

sterilized seeds were sown in the pots filled with autoclaved sandy loam soil

(66% sand, 24% silt, 8% clay and 2% OM) of pH 7.7.

Root-knot nematode culture :

In the study root-knot nematode, Meloidogyne incognita (Kofoid and

White) Chitwood, one of the commonest root-knot nematode species in the

area, was used. Field populations of M. incognita were collected from tomato

and egg plant, (Solanum melogena L.), species of root-knot nematode, infecting

roots of tomato or egg plant in fields were tentatively identified on the basis of

the characteristics of perineal patterns of the females. Roots infected with M.

incognita were chopped and added to the pots containing sterilized field soil

and the tomato plants raised earlier as mentioned above (3 weeks-old) were

58

transplanted. After 50 days, single egg mass culture of the nematode was

established. Seedlings of tomato plants were transplanted in clay pots (30cm

diam.) containing autoclaved soil. Single egg mass of the nematode M

incognita obtained from the roots of the plants was added in each pot near the

roots of the seedlings. The pots were kept in glasshouse at 27°C (+2°C).

Subculturing was done approximately at every 3 months by inoculating new

tomato seedlings with at least 15 egg masses, each obtained from a single egg

mass culture in order to maintain sufficient inoculum for further experimental

studies.

Identification of the root-knot nematode :

The identity of the root-knot nematode was confirmed by studying the

characteristics of perineal patterns of females and conducting North Carolina

differential host test (Eisenback et al., 1981., Taylor and Sasser, 1978.,

Hartman and Sasser, 1985). Fifteen perineal patterns of females of each single

egg mass population were prepared and their characteristics were examined in

order to identify the species (Eisenback et al., 1981).

For North Carolina differential host test (Taylor and Sasser, 1978,

Hartman and Sasser, 1985) seedlings of tomato cv. Rutgers, tobacco cv. NC95,

pepper cv. California Wonder, peanut cv. Florunner, watermelon cv.

Charleston Grey and cotton cv. Deltapine-61 were grown in clay pots (one

seedling/pot) having sterilized soil in triplicate. Two additional replicates of

tomato were included to determine the time of termination of the test.

After determining the number of second stage juveniles (J2) per ml,

plants in each pot were inoculated with 5000 J2. Juveniles were added to a

<Q

depression made in the soil at the time of transplanting. Inoculated plants were

kept at glasshouse at 27-30°C). Fifty to sixty days after inoculation, roots were

harvested and thoroughly washed with tap water and examined for the presence

of galls. Roots with very high infection were stained with Phloxin B to

determine the number of egg masses. Number of galls and egg masses were

counted and GI and EMI were rated on 0-5 scale;

0 = 0

1 = 1-2

2 = 3-10

3 = 11-30

4 = 31-100 and

5 = more than 100 galls or egg masses per root system (Taylor and Sasser,

1978).

After the rating of root system, results were compared with the

differential host test reaction chart (Table 14). This confirmed the identity of

the species determined by the perineal pattern method.

Preparation of inoculum and inoculation :

Second stage juveniles (J2) of the nematode were used as inoculum in

the study. Second stage juveniles were obtained by incubating egg masses

collected from the roots of tomato plants maintaining single egg mass culture

of M incognita. Egg masses were incubated in coarse sieve fitted with double

layered tissue paper and placed on Baerman funnel containing water. The

sieves were then placed in an incubator (temp 25°C). After 72h, number of

hatched juveniles (J2) were collected in a beaker and number of juveniles per

60

V2

T5 O

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61

ml was standardised by counting the juveniles from ten, 1 ml samples.

Average number of juveniles was then calculated to represent the number of

juveniles per ml of the suspension.

For inoculation, the suspension containing J2 was taken in micropipette

controller and added near the roots of the seedlings. The holes were covered

with soil after inoculation. Nematode inoculation density consisted of uniform

quantity of suspension containing either 0, 150, 300, 600, 1200, 2400 and

4800 freshly hatched second stage juveniles (J2)/pot.

Preparation of inoculum of the AM fungus:

Glomus fasciculatum (Nicol & Gerd) Gerdemann & Trappe, inoculum

was maintained and multiplied on Rhodes grass (Chloris gayana) as in

section II.

Inoculation

For inoculation, inoculum of G. fasciculatum for each pot consisted of

uniform quantity of suspension containing 0, 50, 100, 200, 400 and 800

chlamydspores which were retrieved from the soil using wet-sieving and

decanting technique (Gerdemann and Nicolson, 1963) and were placed just

3 cm below the soil surface.

Six levels of G. fasciculatum and seven levels of Meloidogyne

incognita were used in combination for their effect on plant growth and on

each other (Table 15). The chlamydospores number as given constituted the

levels of G. fasciculatum (designed as GFQ, GFi, GF2, GF3, GF4 and

GF5). Similarly freshly hatched second stage juveniles numbering as above

62

> o a

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+ + + + +

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53

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63

constituted M. incognita levels (designated as MIQ, MIi, MI2, MI3, MI4, MI5

and MIe). The treatments were replicated five times, arranged in a

completely randomized block design and maintained in a glasshouse at 28-

35°C. The plants were harvested at 60 days after inoculation and all the

parameters studied in section II were studied along with the following

additional parameters viz., Nematode population (both in soil and root),

number of galls/root system, number of egg masses/root system, number of

eggs/egg mass (Fecundity). Data were analysed statistically using analysis of

variance in factorial design as mentioned by Fisher (1950) and CD was

calculated at P-0 .05 .

Plant growth :

Plant growth parameters and chemical parameters were studied by the

methods mentioned in section II.

Galls and egg masses :

At termination of the experiment, roots of harvested plants were

washed under the tap and examined for the presence of galls. Number of galls

per root system was counted. Roots were immersed in a aqueous solution of

Phloxin B (0.15 g/lit tap water) for 15 minutes to stain the egg masses. Egg

masses per root system were counted (Taylor and Sasser, 1978).

Fecundity :

The number of eggs per egg mass is known as fecundity. It was

measured by shaking vigorously 10 egg masses in 5.25 % NaOCl solution.

The eggs were separated from egg mass and collected over 500 mesh sieve.

64

From the sieve, eggs were transferred into a beaker and 0.35% acid fuchsin (in

25% lactic acid) was added into 20 to 25 ml of suspension with boiling for 1

minute for staining the eggs. After cooling, the eggs were counted and the

eggs per egg mass were calculated to find out the fecundity.

Nematode population :

For estimating root population of nematodes (J2 + J3 + J4, mature

females), root from each replicate was weighed and cut into 1 cm length. One

gram of root pieces was stained with acid fuchsin and lactophenol (Byrd et al.,

1983). The root pieces were placed between two slides, examined under

stereoscopic microscope and number of J2 + J3 + J4 counted. Then total number

of J2 + J3 + J4 for the whole root system of the replicate was calculated. For

counting the number of females, 1 g of root pieces were transferred in 5%

HNO3) and incubated at 25°C. After 72h root pieces were gently teased to

release the females. The number of females/g of root was counted and total

number of females for the whole root system was calculated (Hussey and

Barker, 1973). The means of replicates were then calculated.

The eggs were extracted from the roots of each treatment separately by

Chlorox method of Hussey and Barker, (1973). Roots from each treatment

were cut into l-2cm pieces. One gram of the root pieces were shaken

vigorously in 200 ml of 1.0% NaOCl solution for 1 to 4 minutes. Then NaOCl

solution was passed through a 200 mesh sieve, rested over a 500 mesh sieve to

collect free eggs. After this 500 mesh sieve with eggs was placed under a

stream of cold water to remove residual NaOCl (rinsed for several minutes).

The rinsed roots were put under water to remove additional eggs and were

65

collected by sieving. The number of eggs was then counted in counting dish

under stereoscopic microscope. The total number of eggs was calculated by

multiplying the number with the fresh weight of the root in the treatment. Soil

population (J2 + male) of the nematode was estimated by modified Cobb's

sieving and decanting technique (Southey, 1986).

Plant analysis :

Plant samples of each treatment from the glasshouse experiment were

processed for estimating phosphorus (P), nitrogen (N) and potassium (K)

contents of shoots and roots.

Estimation of nitrogen, phosphorus and potassium

For estimation of nitrogen, phosphorus and potassium in plant materials

(shoot + root) from each treatment, samples were digested in the following

way. Estimations were done from the mixed powder of plants of various

treatments, in a given experiment. Hundred mg of oven dried powder of the

shoots and roots from each treatment was transferred separately in 50 ml

Kjeldahl flask, then 2 ml of chemically pure H2SO4 was added and flask was

heated on Kjeldahl assembly for about 2 h till dense fumes were given off and

the contents turned black. Then 0.5ml of pure 30% H2O2 was added after 15

min. of cooling. Heating was continued till the colour changed to light yellow.

Extract was heated again for half an hour and cooled for 10 min. for getting it

clear and then 3-4 drops of 30% H2O2 was added drop wise followed by

heating for 15 minutes. After that, the digested material was transferred in 100

ml volumetric flasks with 3-4 washings and volume was made upto capacity.

This digested material was used for estimating N, P and K, (Linder. 1944;

Lundegardh, 1951)

66

Nitrogen

Prior to estimating nitrogen present in the digested plant material,

standard curve was prepared by the following method.

Nitrogen standard curve

Different dilutions in 10 test tubes (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8,

0.9 and 1.0 ml) were made from the solution prepared by dissolving 0.236g of

ammonium sulphate in 100 ml of distilled water. The volume was then made

upto 5 ml in each test tube by adding distilled water. A control was also run

side by side. After that 0.5ml Nesseler's reagent was added followed by 5 ml of

distilled water. Per cent transmittance was read at 525nm from spectrophoto­

meter on developing yellow orange colour after half an hour. Then a curve was

drawn between concentration in X axis and O.D obtained in Y axis.

Estimation ofN in plant material

An aliquot of 10 ml was transferred to 100 ml volumetric flask to

which 2ml of 2.5N NaOH was added so as to neutralize the excess of acid.

10% sodium silicate was added to prevent turbidity. The volume was made

upto capacity. 5ml of aliquot was taken in three test tubes followed by the

addition of 0.5 ml of Nesseler's reagent with shaking. Then 10 ml volume was

made by adding distilled water. After waiting for 5 min, the per cent

transmittance was read at 525 nm. The concentration was read with the help

of 0.0. from standard curve (Linder,1944).

Phosphorus standard curve

Different dilutions of potassium dihydrogen orthophosphate (KH2PO4)

viz., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml concentrations were

67

taken into separate test tubes and final volume was made upto 5 ml by adding

required amount of distilled water. One ml ammonium molybdic acid and 0.4

ml. of I -amino 2-nepthol 4-sulphonic acid were added in each test tube and

the volume was made upto 10 ml with distilled water. After half an hour, the

percent transmittance was read at 625nm. Standard curve was drawn between

concentration and O.D. in X and Y axis respectively.

Estimation ofP in plant materials

Five ml of aliquot of digested plant material was taken in three test

tubes, to which 5 ml of distilled water was added. After that I ml of ammonium

molybdic acid was added, with shaking, followed by addition of 0.4 ml of 1-

amino 2-nepthol 4-sulphonic acid and the volume was made up to 10 ml. The

control was run side by side and per cent transmittance was read at 625 nm.

after half an hour. The P concentrations in plant materials was calculated from

the standard graph using O.D. (Fiske and Row, 1925)

Potassium standard curve

Potassium was estimated using flame photometer. Different dilutions of

potassium chloride viz., 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ppm were prepared by

dissolving 91 mg of oven dried KCl in 100 ml of distilled water and diluting 0,

1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml of the stock solution to 100 ml. The flame

photometer was calibrated to zero using distilled water and to 100 using 10

ppm KCl solution and readings for other standard solutions were taken. A

standard graph was drawn taking concentration in X axis and readings obtained

from the flame photometer in Y axis.

68

Estimation ofK in plant materials

The digested plant materials were diluted with distilled water so as to

maintain the K concentration in the final solution ranged between 2 to 8 ppm

and the reading was taken in the flame photometer and with the help of the

standard graph, the concentration of K in plant materials was calculated.

The data collected for morphometries were statistically analysed as

under to determine the degree of authenticity of results.

Standard deviation (S.D)

Standard deviation is a measure of fluctuation if. a population produced

as a result of chance factors of sampling from the same population. S.D. for

each parameter of morphometries were calculated by the following formula

(X-X!)^ + ( X - X 2 ) ^ + . . . + (X-XN)^

S.D. = ± N-1

where,

X = Meanof observations i.e. arithmetic mean

X], X2, ... XN = observations

N = Number of observations

Coefficient of variation (C.V)

This measures the relative magnitude of variation present In

observations relative to the magnitude of their arithmetic mean. It IS defined

as the ratio of S.D. to arithmetic mean expressed as percentage

SD C.V.- xlOO

X

69

Data presented in the tables of the morphometries are in the form of Mean ±

S.D. and C.V. Like other parameters, CD was calculated for morphometries

also using the mean values obtained for each replicates.

RESULTS

Plant length:

The combined effect of Meloidogyne incognita and Glomus

fasciculatum was studied at different levels of inoculum of the nematode and

the mycorrhizal fungal spores. The nematode caused adverse effect on the

growth of tomato plants, while the AM fungus G. fasciculatum promoted their

shoot and root growth. When both were applied together, the adverse effect of

the nematode was mitigated to some extent by the AM fungus (Table 16). The

addition of the AM fungal spores had reduce the adverse effect of the nematode

at all population levels studied i.e. from 150 to 4800 J2/plant which was

proportional to the AM fungal spores/plant. For instance at 150 J2 level the

shoot length was reduced by 1.64 per cent while the reduction was 37.82 at

4800 J2/plant. At the same level of nematode, the addition of spore (400/plant)

shoot length increased by 41.44 per cent over the control. Further addition of

spores 800/pot did not bring any significant increase in shoot length at 2400 J2

level of nematode population.

Similar trend was observed for root length and total length, the

maximum effect of nematode was exhibited by a population of 4800 J2/plant

resulting in a loss of 31.60 per cent in root length. This loss was completely

eliminated when the culture medium was supplied with 400 spores/plant,

resulting in a gain of 11.49 per cent instead a loss of 31.62 per cent.

70

Table 16: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the length of tomato plants.

Spore density No/Pot Cont. 50 100 200 400 800 Mean CD

Spore density No/Pot

Cont. 50 100 200

Cont 30.4 34.1 36.8 41.3 41.9 44.8 38.2

150 29.9 33.3 37.4 40.9 43.0 43.8 38.0

Glomus fasciculatum

Cont 17.4 17.7 17.7 19.1

150 16.8 17.5 17.8 18.8

{ Shoot Length (cm) Nematode inoculum density (J2)

300 26.8 31.0 36.8 41.1 43.3 42.8 36.9

= 1.53

600 22.9 28.8 34.5 39.8 42.4 43.4 35.3

1200 21.0 25.9 30.7 37.8 44.1 41.9 33.5

2400 19.8 24.8 28.3 35.9 43.0 41.8 32.2

4800 18.9 24.4 28.1 33.3 39.7 40.4 30.8

M incognita ~ 1.65 Interaction =

Root Length (cm) Nematode inoculum density (J2)

300 15.4 15.0 17.4 18.0

600 14.5 15.3 16.8 18.4

1200 14.0 13.8 14.9 17.7

2400 13.8 13.2 13.8 16.6

4800 11.9 12.5 14.2 15.4

M 24.2 28.9 33.2 38.5 42.4 42.7

-4.1

M 14.8 15.0 16.2 17.7

400 19.8 20.0 20.4 19.4 18.6 19.4 17.4 19.2 800 20.4 19.4 19.9 18.8 20.5 18.5 18.6 19.4 Mean 18.6 18.3 17.6 17.2 16.5 15.9 14.9 CD

Spore density No/Pot Cont. 50 100 200 400 800 Mean

Glomus fasciculatum

Cont 47.8 51.8 54.5 60.4

61.7

64.5 56.8

150 46.7 50.8 55.2 59.1

63.0

63.2 56.3

= 0.947 M. incognita =

Total length (cm)

1.02

Nematode inoculum density (J2) 300 42.2 46.0 54.2 59.1

63.7

62.7 54.6

600 1200 38.2 35.0 44.1 39.7

51.3 45.6 58.2 55.5

61.8 62.7

62.2 62.4 52.6 50.15

2400 33.7 38.0 42.1 52.5 62.4

60.3

48.1

Interaction

4800 31.0 36.9 42.3 48.7 57.1

59.0

45.8

= 2.4

M 39.0 43.9 49.4 56.2

61.6

62.1

CD Glomus fasciculatum = 2.1 M. incognita = 2.0 Interaction = 6.2

71

The nematode caused a loss of 35.14 per cent in the total length, the loss

was completely overcome when the fungal spores where applied at the rate of

400/.plant and gain of 30.54 per cent in total length resulted.

Plant fresh weight:

The interaction effects of M. incognita and G. fasciculatum on the fresh

weight of shoots and roots are given in Table 17. Shoot fresh weight gradually

declined with the increase in the number of nematode (J2) per plant. No

significant reduction in fresh weight of the plant occurred at 150and 3OOJ2

inoculum levels compared to control. Effect of G. fasciculatum on other hand

was found to be increasingly positive with the growing number of spores,

although it was significant at 50 levels.

In case of shoot fresh weight the maximum interaction effect (55.14 per

cent) was found at 2400 J2 level with 400 spore application per plant. Higher

level of spores i.e.800/plant did not bring out any change in shoot fresh weight,

root fresh weight as well as the total fresh weight. The root fresh weight was

reduced at marginal reduction at 150 J2 levels of M incognita and it increased

at higher nematode levels bringing about significant reduction with the

maximum (44.87), occurring at 2400 J2 level (Table 17).

Plant dry weight:

The data on shoot, root and total dry weight are given in Table 18.

Inoculation of M incognita caused decreased in the dry weight of the plant.

Significant reduction occurred when nematode number was increased above

300 J2 level. In all the three cases of weight analysis, 2400 and 4800

J2 produced the same degree of reduction. G. fasciculatum produced beneficial

72

Table 17: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the fresh weight of tomato plants.

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 21.4 23.1 27.8 31.3 33.8 36.4 28.9

150 20.9 22.6 26.3 31.2 34.7 35.4 28.4

Shoot fresh weight (g/plant) Nematod(

300 20.0 21.0 25.7 31.7 35.3 34.9 28.1

; inoculum density (J2) 600 17.2 18.9 23.8 29.2 33.7 32.8 25.9

1200 15.9 18.0 20.7 28.1 32.4 33.5 24.7

2400 14.4 16.6 18.5 26.8 33.2 32.4 23.6

4800 14.6 16.0 18.3 27.3 32.2 31.9 23.3

M 17.7 19.4 23.0 29.3 33.7 33.9

CD Glomus fasciculatum = \ .Q M. incognita = \ .2 Interaction = 2.8

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 7.8 8.7 9.8 12.0 13.3 14.0 10.9

150 7.6 8.6 10.0 11.6 13.2 13.7 10.7

Root fresh weight (g/plant) Nematode inoculum density (J2)

300 6.6 8.0 9.4 11.3 12.1 13.3

10.11

600 6.0 6.9 8.7 10.3 12.7 12.8

9.5

1200 5.3 6.1 7.9 9.2 12.0 13.4 8.9

2400 4.3 4.5 6.6 8.7 11.3 12.7 8.0

4800 4.5 4.7 6.0 8.1 11.7 12.3 7.8

M 6.01 6.7 8.3 10.2 12.3 13.1

CD Glomus fasciculatum "= 0.691 M. incognita-Q Ml Interaction = 1.3

Spore density No/Pot Cont. 50 100 200 400 800 Mean

CD

Cont 29.2 31.8 37.6

43.3 47.1

50.1 39.8

150 28.5 31.2

36.3 42.8 47.9

48.9 39.2

Glomus fasciculatum

Total fresh weight (g/plant) Nematode inoculum density (J2)

300 26.6 29.0 35.1 43.0 47.4 48.2 38.2

= 1.5

600 23.2 25.8

32.5

39.5 46.4 45.4 35.4

1200 21.2 24.1

21.6 37.3 44.4

46.7 32.5

M. incognita = 0.\1

2400 18.7 21.1 25.1 35.5

44.5 45.1

4800 19.1 20.7

24.3 35.4

43.9 44.2

31.6 31.2

Interaction =

M 20.7 26.2

30.3 39.5 45.9

46.9

3.0

73

Table 18: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the dry weight of tomato plants.

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 5.10 5.27 6.33 7.27 8.36 8.64 6.82

150 5.03 5.36 6.28 7.41 8.40 8.54 6.83

Shoot dry we ight (g/plant) Nematode inoculum density (J2)

300 4.75 4.89 6.11 7.63 8.51 8.39 6.71

600 4.10 4.88 5.79 6.91 7.62 7.54 5.99

1200 3.78 4.25 4.98 6.71 7.86 7.77 5.89

2400 3.35 3.84 4.53 6.29 7.98 7.84 5.63

4800 3.20 3.74 4.13 6.48 7.59 7.86 5.50

M 4.18 4.54 5.45 6.95 8.04 8.08

CD Glomus fasciculatum = {).Ill M. incognita-025 Interaction = 0.61

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 2.13 2.28 2.61 3.12 3.31 3.50 2.82

150 2.08 2.33 2.70 3.03 3.40 3.48 2.83

Root dry wei ght (g/plant) Nematode inoculum density (J2)

300 1.80 2.13 2.48 2.95 3.21 3.52 2.68

600 1.64 1.77 2.10 2.48 3.13 3.10 2.37

1200 1.35 1.61

1.98 2.45 3.22 3.21

2.30

2400 1.15 1.28 1.81 2.25 2.81 3.28 2.09

4800 1.12 1.41 1.65 2.13 2.85 3.13 2.04

M 1.61 1.83 2.19 2.63 3.13 3.31

CD Glomus fasciculatum = 0.07 M. incognita = 0.08 Interaction = 0.15

Spore density No/Pot Cont. 50 100 200 400 800 Mean

CD

Cont 7.23

7.55 8.94

10.39

11.67 12.14 9.65

150 7.11

7.69 8.98 10.44

11.8

12.0 9.67

Glomus fasciculatum

Total dry wei ght (g/plant) Nematode inoculum density (Jj)

300 600 6.55 5.74 7.02 6.21 8.59 7.89

10.58 9.39

11.72 10.75 11.91 10.64 9.39 8.43

1200 5.13

5.86 6.96 9.16

11.08

10.98 8.31

2400 4.50 5.12 6.34 8.54

10.76 11.12 7.56

4800 4.32

5.15 5.78

8.61 10.44

10.99 7.54

= 0.27 M. incognita = 0.30 Interaction =

M 5.79 6.37 6.51

9.58

11.17

11.39

= 0.72

74

Table 19: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on the N, P and K content of tomato plants.

Spore density No/Pot Cont. 50 100 200 400 800 Mean

Cont 1.75 2.0 2.15 2.11 2.23 2.18 2.07

150 1.84 2.03 2.13 2.17 2.06 2.30 2.08

Nitrogen contents Nematode inoculum density (J2)

300 1.71 1.90 1.98 1.96 2.19 2.21 1.99

600 1.78 1.94 2.16 2.08 2.02 1.98 1.99

1200 1.64 1.83 2.01 2.16 2.07 2.22 1.98

2400 1.58 1.73 2.14 1.92 2.15 2.09 1.93

4800 1.69 1.80 1.95 2.06 2.11 2.18 1.96

M 1.71 1.89 2.07 2.06 2.11 2.16

CD Glomus fasciculatum-Q.\6 M. incognita = ]^S Interaction = NS

Spore density No/Pot "

Cont. 50 100 200 400 800 Mean

Cont 0.215 0.277 0.301 0.311 0.339 0.334 0.296

150 0.201 0.271 0.294 0.297 0.336 0.323 0.287

Phosphorus contents Nematode inoculum density (J2)

300 600 0.221 0.199 0.269 0.281 0.286 0.267 0.296 0.313 0.331 0.329 0.347 0.316 0.291 0.270

1200 0.204 0.257 0.281 0.285 0.317 0.338 0.280

2400 0.191 0.260 0.271 0.282 0.344 0.336 0.280

4800 0.205 0.269 0.256 0.273 0.327 0.331 0.276

M 0.205 0.269 0.279 0.293 0.331 0.384

CD Glomus fasciculatum = 0.02 M. incognita = NS Interaction = NS

Spore density No/Pot Cont. 50 100 200 400 800 Mean

CD (

Cont 1.45 1.54

1.75 1.85 2.01

2.06 1.77

jlomusfas

150 1.32 1.58

1.65 1.82 2.16

2.01 1.75

ciculatun

Potassium contents Nematode inoculum density (J2)

300 600 1.46 1.42 1.48 1.38

1.55 1.47 1.87 1.74 1.91 2.11

2.08 2.06 1.72 1.69

I = 0.033 M. inci

1200 1.32 1.39

1.40 1.75 1.95 1.94 1.62

2400 1.22

1.35 1.41

1.76 1.87

2.09 1.61

ognita = NS

4800 1.31 1.41

1.32

1.65 1.96

2.06 1.61

Interaction =

M 1.35 1.44

1.50

1.77 1.99 2.04

= NS

75

effect by promoting the plant dry weight. Shoot, root and total dry weight were

enhanced significantly as the spore number was increased upto 800. Significant

increase in dry weight was obtained in the plants inoculated with 800 spores

than that of 400 spores/plant. The maximum loss of dry weight caused by M

incognita amounted to 37.25, 47.41 and 40.24 per cent respectively in shoot,

root and total dry weight at the population level of 4800 J2 per plant. This loss

in all three cases was totally overcome by the addition of 800 spores of G.

fasciculatum per plant which resulted in a gain of 54.II, 46.94 and 52.00 per

cent, in case of shoot, root and total dry weight respectively.

Nutrient status :

In general M. incognita reduced the N, P and K contents of the plants at

different levels of inoculum. While Glomus fasciculatum treatments improved

the nutrient status significant (Table 19).

MYCORRHIZATION

The effect of M. incognita on the growth and development of G.

fasciculatum was studied. G. fasciculatum extensively colonized the tomato

plants when inoculated with spores. The external colonization percentage

(Table 20) increased significantly with the increasing number of spores per

plant. The colonization declined to 22.6 per cent from 27.1 per cent at the spore

level of 50 per plant when the nematode level was raised to 4800 J2 from 150

J2. With the increasing spore concentration, from 150 to 800/plant the per cent

colonization increased to 64.5 per cent at the level of 150 J2.

76

Table 20: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on mycorrhization G. fasciculatum in tomato plants.

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 0.0 26.7 35.4 48.9 60.5 62.3 38.9

150 0.0

27.1 34.8 50.1 59.5 64.5 39.3

External colonization (%)

Nematode inoculum density (J2) 300 0.0 25.1 36.8 45.5 57.6 63.9 38.15

600 0.0

21.3 33.7 39.7 54.5 66.2 35.9

1200 0.0 23.7 27.1 33.1 56.3 64.7 34.1

2400 0.0 17.3 20.3 26.4 46.5 53.2 27.2

4800 0.0 17.0 18.8 24.9 43.3 46.9 25.1

M 0.0 22.6 29.5 38.3 54.2 60.2

CD Glomus fasciculatum = 2.\ M. incognita = 2.3 In tera^rf^A6 ^^''^•-)S

1 •^ il 80%

Spore density No/Pot Cont 150

Internal colonization (%) Nematode inoculum density (J2)

300 600 1200 2400

\ \ X V

4800

^^ ---' '

. -:

M Cont. 50 100 200 400 800 Mean

CD

0.0 34.4 43.3 38.8 50.5 49.8 36.1

0.0 33.4 45.4 37.8 50.0 51.1 36.3

Glomus fasciculatum '-

0.0 30.4 31.8 30.5 40.3 41.8 29.1

-2.6

0.0 28.4 43.5 46.0 52.1 55.0 37.5

0.0 27.4 32.2 35.0 41.4 39.6 29.2

M incognita = 31

0.0 25.1 32.4 27.8 35.4 34.0 25.7

0.0 19.4 28.9 31.0 38.6 37.8 25.9

Interaction = 6.0

0.0 28.3 36.7 35.2

44.04 44.1

Spore density No/Pot Cont 150

Percent Arbuscules Nematode inoculum density (J2)

300 600 1200 2400 4800 M Cont. 50

100 200 400 800 Mean

0.0 28.8 37.3 36.0 41.3 39.8

30.5

0.0 30.0 38.7 34.1 39.5 42.0 30.7

0.0 23.0 36.9 37.8 35.8 38.0 28.7

0.0 18.8 34.6 38.4 41.3 40.8 28.9

0.0 20.8 39.1 37.4 34.4 38.4 28.3

0.0 22.0 32.4

37.3 38.8 36.4 27.8

0.0 18.4

31.1 37.1 40.3 38.4

27.5

0.0 23.1 35.7 36.8 38.7 39.1

CD Glomus fasciculatum = 2.^ M. incognita ^'HS Interaction = NS

77

Spore density No/Pot

Cont. 50 100 200 400 800 Mean CD

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 0.0 19.8 21.2 23.4 25.3 27.2 19.4

Number ol

150 0.0 20.5 21.0 22.6 24.4 26.4 19.1

Glomus fasciculatun

Cont 0.0 515 514 528 550 625 455

• chlamydospores / ( cm root Nematode inoculum density (J2)

300 0.0 18.2 20.2 21.4 24.1 25.8 18.2

2 = 2.0

Number of

150 0.0 511 504 541 551 521 545

600 0.0 16.8 18.4 20.3 19.1 21.8 16.0

1200 0.0 18.6 21.4 17.1 18.6 21.4 16.1

M incognita = NS

2400 0.0 17.1 18.6 21.8 23.1 24.4 17.5

4800 0.0 14.4 17.1 19.6 21.8 23.2 16.0

Interaction = NS

chlamydospores /100 g soil Nematode inoculum density (J2)

300 0.0 520 477 596 541 600 455

600 0.0 507 457 566 570 560 443

1200 0.0 405 411 541 550 610 419

2400 0.0 401 388 420 339 350 316

4800 0.0 200 437 255 268 275 239

M 0.0 17.9 19.7 20.8 22.3 24.3

M 0.0 437 418 492 481 519

CD Glomus fasciculatum = 50 M. incognita - 56 Interaction = 58

78

The internal colonization increased with the increasing level of spore

concentration, from 28.3 to 44.04 per cent, when the spores increased from 50-

800/plant.

The development of arbuscules followed the same trend as that of

external and internal colonization.

The number of chlamydospores per centimeter root length also depicted

a similar trend as that of external and internal colonization. The number of

chlamydospores per lOOg of soil show the maximum at 800 spore level. At

1200 and 2400 Ji levels of inoculum there was a significant decrease in the

chlamydospores.

Root-knot disease :

The effect of G. fasciculatum on the growth and development of M.

incognita has been studied in terms of root-knot disease. Population of the

nematode in soil increased significantly as the number of inoculated juveniles

increased. Nematode population showed a significant decrease compared to

control, irrespective of spore concentration (Table 21).

Root population of the nematode was suppressed by G. fasciculatum.

Significant reduction in the root population occurred. The reduction was

proportionate to the spores concentration upto 400 spore level but at 800 level,

there was no appreciable difference in the nematode population.

The number of egg masses per root system increased with increasing

level of M incognita. The number of egg masses per root system was reduced

with increasing spore concentration oiG. fasciculatum. It was observed that the

200, 400 and 800 spore levels caused the same degree of reduction.

79

Table 21: Interaction effect of spore density of Mycorrhizal fungus Glomus fasciculatum and population of nematode Meloidogyne incognita on root-knot disease parameters in tomato plants.

Spore density No/Pot

Cont.

50 100 200 400 800 Mean

Cont

0 0 0 0 0 0 0

150 1046

1170

908 1006

974 1028

1022

Nematode population

Nematode inoculum density (J2)

300 4114

3967

3817

3966

4395

4496

4125

600 6723

6826

6266

6194

6683

6469

6526

1200

8266

9744

8667

8844

9746

9193

9076

2400

13481

12516

11216

11767

12479

11762

12203

4800

16074

14002

15744

15001

14147

14084

14842

M 7100

6889

6659

6882

6917

6718

CD Glomus fasciculatum = 400 M. incognita = 475 Interaction ==936

Spore

density

No/Pot

Cont.

50 100 200 400 800 Mean

Cont

0 0 0 0 0 0 0

150 25 18 12 6 4

11

Nematode population/root

Nematode inoculum density (J2)

300 48 38 25 10 3 5 21

^n 89 67 32 26 11 12 39

1200

146 108 55 38 8 13 61

2400

168 143 68 58 21 11 78

4800

180 156 84 60 15 13 84

M 93 75 39 28 9 8

CD Glomus fasciculatum = 7 M incognita = 9 Interaction = 15

Spore

density

No/Pot

Cont.

50 100 200 400 800 Mean

Cont

0 0 0 0 0 0 0

150 25 18 12 6 4 3 11

Nematode population/root

Nematode inoculum density (J2)

300 48 38 25 10 3 5 21

600 89 67 32 26 11 12

39

1200

146 108 55 38 8 13 61

2400

168 143 68 58. 21 11 78

4800

180 156 84 60 15 13 84

M 93 75 39 28 9 8

CD Glomus fasciculatum = 2 M. incognita - 3 Interaction = 5

80

Spore density No/Pot Cont. 50 100 200 400 800 Mean

Cont 0 0 0 0 0 0 0

150 52 44 53 35 41 44 44

Nematode of c !ggs/egg masses Nematode inoculum density (J2)

300 64 51 50 43 40 43 48

600 69 62 52 51 40 38 52

1200 81 84 80 90 78 70 80

2400 73 82 69 84 74 70 75

4800 96 94 90 79 68 65 82

M 62 59 56 54 48 47

CD Glomus fasciculatum = 5 M. incognita = 6 Interaction = 10

Spore density No/Pot

Cont. 50 100 200 400 800 Mean

Cont 0 0 0 0 0 0 0

150 85 65 47 18 12 8 39

Nematode of egg; j/egg masses Nematode inoculum density (J2)

300 76 75 42 30 14 7

40

600 116 87 48 33 16 11 51

1200 150 107 56 40 12 14 63

2400 182 124 65 44

17 12 74

4800 194 144 72 52 15 13 81

M 114 86 47 31 13 9

CD Glomus fasciculatum = 8 M. incognita - 10 Interaction = 18

81

Fecundity of the nematode increased at different inoculum levels of M.

incognita. Treatment with 1200,2400and 4800 J2/plant level had more eggs/egg

mass than at the other inoculum levels. Inoculation of G. fasciculatum at the

rate oflOO/plant and above caused significant reduction in the fecundity.

The number of galls per root system was reduced to a significant level

by different treatment of G. fasciculatum. The reduction in gall number

occurred proportional to the spore level with the maximum (90.58% per cent)

occurring with the highest level of spores (800 level).

DISCUSSION

The present study showed that Glomus fasciculatum is beneficial to the

tomato plants as it helps in plant growth by promoting shoot and root

elongation and enriching nutrient status. The improvement in plant growth

characteristics in mycorrhizal plants compared to control indicates the

existence of complex physiological and biochemical relationship between

Glomus fasciculatum and tomato. AM fungi have been reported to alter the

physiology of root and reduce the root exudation (Grahm, 1981) causing

changes in phytohormone levels (Allen et al., 1980) and photosynthetic rates

(Aliened a/., 1981).

The isolates of G. fasciculatum has been found to improve the nutrient

status of tomato plants, as the phosphorus, nitrogen and potassium contents

were enhanced greatly in the plants improved nutrient status in the mycorrhizal

plants resulted in increased biomass production and growth potential. This

supports the earlier findings that mycorrhizal infection can cause a beneficial

physiological effect on host plants by increasing uptake of soil phosphorus

82

(Gerdemann, 1968, 1975). Better growth of cotton plants with Gigaspora

calospora and G. margarita has been observed earlier by Roncadori and

Hussey (1977). Similar results have been obtained for sorghum, upland rice

and other crop plants by Singh and Tilak (1990; Asif e/ ai, 1995 and Mosse et

ai, 1973). It has been suggested by a number of workers that mycorrhizae can

influence plant water relationship by reducing plant resistance to water

transport (Safir et ai, 1972; Hardic and Layton, 1981; Allen et al., 1982) and

thus may also improve drought tolerance.

In general root-knot nematode caused adverse effect on tomato piant,

while AM fungus produced beneficial effects by promoting the growth when

both were applied together, the adverse effect of the nematode was nullified to

a great extent by the endomycorrhizal fungus. The addition of G. fasciculatum

spores reduced the adverse effect of the nematode M incognita. The nematode

caused a maximum loss amounting to 37.25, 47.41, 40.24 in shoot, root and

total dn/' weights of the host plant at 4800 J2 level and this loss was overcome

by the addition of 800 spores/plant and there was also an improvement in the

shoot, root and total dry weight by 54.11, 46.94 and 52.0 respectively,

compared to control. A vegetative correlation has been found between

mycorrhizal development and the populations of plant parasitic nematode by

Schenck and Kinloch (1974); Sitaramaiah and Sikora (1996) on cotton with G.

fasciculatum and Rotylenchulus reniformis.

Nematode population in the soil generally decreased in concomitant

inoculation. Nematode population in root, egg mass production, fecundity, root

galling and also root-knot indices and egg mass indices were found to be

83

affected in the present study in the presence of endomycorrhizal fungus.

Reproduction and development of M. incognita have been found to be affected

markably by G. fasciculotum at the rate of 400 and 800 spores/plant at all the

inoculum levels of nematode. Saleh and Sikora (1984) also recorded that

symbiont caused reduction by 59 per cent in neamtode population at a spore

level of 750/plant. In present study it has been found that M. incognita reduced

the mycorrhizal colonization percentage, as well as arbuscules and

chlamydospores number, although the values improved with the increasing

inoculum levels of G. fasciculatum.

Mycorrhizal fungi which form the symbiotic association with plant

roots, promote plant health and growth have also been suggested by others

(Gerdemann, 1968; Harley, 1969; Howeler et al, 1987; Lin-Xian and Hao,

1988; Raju et al, 1990 and Tinker, 1975). The present observations clearly

show that increasing levels of G. fasciculatum improve plant growth and

health, while M. incognita suppresses the same, and the adverse effect of the

nematode become lesser and lesser as the number of G. fasciculatum spores

increase. Mycorrhizal plants with low newmatode inoculum showed maximum

growth potential than the mycorrhizal plants with higher inoculum levels of

nematode. The number of nematodes in the roots, the number of egg

masses/root system have also been noted to be lower in the mycorrhizal plants

compared to non-mycorrhizal ones. The reduction in the development and

severity of root-knot disease may be due to complex physiological and

biochemical changes in the mycorrhizal roots and the resistance of the host

induced by the AM fungus. This is in agreement with earlier investigations

which reveal that nematode susceptible plants colonized by endomycorrhizal

84

fungus are better able to tolerate plant pathogenic nematodes by showing better

growth (Verdejo et al, 1990; Palacino and Leguizaman, 1991; Lingaraju and

Goswami, 1993 and Sikora and Sitaramaiah, 1995). In a previous study Singh

et al. (1990) found that pre-infection of tomato roots with G. Jasciculatum

made Pusa Ruby resistant to root-knot nematode by bringing about biochemical

changes in the levels of ligneous and phenolic compounds.

Mycorrhizae help to withstand the antagonistic effects of the nematode

and increase plant growth. Application of G. fasciculatum at the higher

inoculum level benefits the plant most and promotes the plant health by

significantly reducing the nematode population in root, egg mass production,

root galling and fecundity, the above results also indicate that very few

nematodes are present in severely colonized roots. It is also evident that G.

fasciculatum colonized roots are less penetrated by the nematode. Similar

results have also been obtained by Kellam and Schenck (1980) and Grandison

and Cooper (1986). The damage due to nematode disease is generally reduced

in roycorrhizal plants (Carling et al, 1989; Osman et al., 1990; Price et ah,

1995).

Improved phosphorus nutrition by VAM fungi results in root exudation

(Graham et al, 1981) and has been correlated with reduction in soil borne

diseases (Graham and Menge, 1982). Thus, it may be the reason for the

reduced damage by the neamtode in the mycorrhizal plants and may also be

nematode parasitism by AM fungi changes in root morphology,

histopathological changes, physiological and biochemical changes, changes in

the host nutrition (Siddiqui and Mahmood, 1995a,b and Siddiqui and

85

Mahmood, 1998) and competition for food and space (O'Bannon and Nemec,

1979; Saleh and Sikora, 1984) and may also be due to the production of

antifungal and antibacterial metabolities.

Application of M. incognita decreased by the mycorrhizal infection and

sporulation of G. fasciculatum through the mycorrhization increases with

increase in G. fasciculatum inoculum levels. Schenck and Kinloch (1974) and

Linderman (1985) also obtained similar results in the field observations relating

the incidence of mycorrhizal fungi to the incidence of endoparasitic nematodes.

Nematode influenced with mycorrhizal colonization G. fasciculatum on

cowpea (Lingaraju and Goswami, 1993).

86

LITERATURE CITED

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