ECO-FRIENDLY MANAGEMENT OF RENIFORM NEMATODE, ROTYLENCHULUS RENIFORMIS

INFECTING COWPEA

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

ifSlasiter of $t)tIo£(opIip IN

BOTANY

(PLANT PATHOLOGY)

BY

BHAVNA SHARMA

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2009

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DS3942

11

No words could adequately express all that my parents have

done for me throughout my life. They have always been a source of

inspiration and whose constant admonitions help me in my academic

pursuits. Their sacrifices, prayers, affection and love made me to reach

this stage. I am immensely thankful to my respected in-laws for their

amenity encouragement and moral support.

My appreciations also due to Mr. H.K. Sharma, Hind Typing

Centre for his excellent typing work.

Mrs. Bhavna Sharma

Dedicated to

Almiglity t i od

Prof, Mohd, Farooq Azam M.Sc, Ph.D. (Alig.), FNSI

Professor of Botany (Plant Nematology)

Ex-Vice-President, Nematological Society of India.

91-0571-2700920 Extn-3303

Ph: 91-0571-2702016(0) 91-0571-2403503 (R)

09358256574(M) E-mail: azam03@rediff.coni

mfazam05(5),vahoo.com farooqazam07@gmail.com

Department of Botany Aligarh Muslim University

Aligarh-202002 (U.P.) India

Date:..i2.£;f2.

Certificate

This is to certify that the dissertation entitled "Eco-Friendly Management of

Reniform Nematode, Rotylenchulus reniformis Infecting Cowpea" submitted to the

Department of Botany, Aligaiii Muslim University, Aligarh in partial fulfillment for

the degree of Master of Philosophy in Botany (Plant Pathology), is a bonafide WCMIC

carried out by Mrs. Bhavna Sharma under my supervision.

U%. (Prof. Mohd Farooq Azam)

Residence: 4/35, "AL-FAROOQ", Bargad House Compound, Dodhpur, Civil Lines, Aligarh-202002 (U.P.) INDIA.

ACKNOWLEDGEMENT

Behind every success there is, certainly an xinseen power of "Almighty

God", the most adorable, deity and paramoimt to whom I am debted. It

is his Die gratia and Deo volent that guided all the channels to the work

in cohesion and coordination to make this study possible.

I wish to express my sincere thanks and deep sense of requital

gratitude to my reverend and esteemed supervisor, Mohd. Farooq

Azam, Professor, Department of Botany, Aligarh Muslim University,

Aligarh for his savvy navigation, excellent supervision, able guidance,

constructive criticism and keen interest during the preparation of this

dissertation.

I owe an immense debt of gratitude to Prof. Arif Inam,

Chairman, Department of Botany, Aligarh Muslim University, Aligarh

for providing me all necessary facilities to carry this work.

Good people are rare and I express my debetedness to Dr.

Tabreiz Ahmad Khan, Rader, Department of Botany, Aligarh Muslim

University, Aligarh for his unstinted help, moral support, worthwhile

suggestions and cooperation in completing this work.

I extend my deep sense of indebtedness to my lab colleagues Mr.

Nasiruddin, Mr. Suraj, Neelu and Athar for their generous help,

worthwhile suggestions and encouragement during the course of this

work.

I am immensely thankful to Mrs. Shweta, Ms. Farha, Zehra and

Nikhat for their unstinted help to enhance my dedication and also for

their valuable cooperation.

11

No words could adequately express all that my parents have

done for me throughout my life. They have always been a source of

inspiration and whose constant admonitions help me in my academic

pursuits. Their sacrifices, prayers, affection and love made me to reach

this stage. I am immensely thankful to my respected in-laws for their

amenity encouragement and moral support.

My appreciations also due to Mr. H.K. Sharma, Hind Typing

Centre for his excellent typing work.

Mrs. Bhavna Sharma

CONTENTS

Pages

1. Introduction 1 - 6

2. Review of Literature 7-32

3. Materials and Methods 33 - 42

4. Results 43 - 51

5. Discussion 52-61

6. Summary and Conclusion 62 - 64

6. References 65 - 80

^ntrodi tu uction

CHAPTER-1

INTRODUCTION

Cowpea (Vigna unguiculata L. Walp.), an annual legume is also

commonly referred to as southern pea, black eye pea, crowder pea, Lubia,

niebe, coupe and Frijole. Cowpea originated in Africa and is widely grown in

Africa, Latin America, South east Asia and in the southern United States. It is

chiefly used as a grain crop for animal fodder or as a vegetable. The history of

cowpea dates to ancient West Africa cereal farming, 5 to 6 thousand years

ago, where it was closely associated with the cultivation of sorghum and pearl

millet.

Cowpea is propagated by seeds. Sowing commences in March when

the legume is grown as a hot weather crop under irrigation. As a rainfed crop,

particularly for seed, it is sown during June-July after the commencement of

rains. Though mainly a Kharif crop, it is also grown to some extent as a rabi

crop, sown in September.

World wide cowpea production has increased dramatically in the last

25 years. United states production of dry cowpea has declined from V* Million

across to a few thousand over the same period.

Cowpea seed is a nutritious component in the human diet, as well as a

nutritious livestock feed. Nutrient content of cowpea seed is as follows :

Protein

Fat

Fiber

Carbohydrate

24.8%

1.9%

6.3%

63.6%

Thiamine

Riboflavin

Niacin

0.00074%

0.00042%

0.00281%

The soil constitutes a complex ecosystem that harbours a wide variety

of plants and pathogenic organisms such as bacteria, fungi, actinomycetes,

insects and nematodes etc, which make its biological system. Out of these

organisms which inhibit the soil ecosystem, the plant parasitic nematodes are

the most important ones which constitute about 12% of the soil flora and

fauna. These are found around the roots of plants and are said to play a vital

role in their growth and production. On our globe rarely is any crop free from

their attack, yet we are unaware of their appearance because of their

microscopic size and protected position in the soil. These active slender worm

like creature are found not only inhabiting the soil but also in fresh and salt

water wherever organic matter exists.

Plant parasitic nematodes constituting a substantial portion of soil biota

are cosmopolitan and attack a large number of plants and reducing their

productivity substantially over a period of time. They are considered worst

enemies of mankind because of devastations they cause to the crops.

Plant parasitic nematodes continue to threaten agricultural crops,

throughout the world. Estimated over all average annual yield loss of the

major crops due to damage caused by phytoparasitic nematodes is 12.3%. The

national loss due to plant parasitic nematodes in 24 different crops in

monetary terms has been worked out to the tune of 21068.73 million rupees

(Jain era/., 2007).

Rotylenchulus reniformis Linford and Oliveira (1940) is one of the major

nematode problems of pulse crops in India. Siddiqui and Basir reported this

nematode for the first time in India on coffee in 1959. It is widely distributed

throughout the world and is known to be a serious problem of tropics, semi

tropics and warmer areas of temperate zone.

It feeds on almost all leguminous crops especially Pigeon pea,

chickpea, black gram, cowpea and beans. A large number of plants have been

recorded as hosts of Rotylenchulus reniformis from various countries.

To control the nematode, use of nematicides is better but it is now

being discouraged because of its impact on human health and enviroimiental

risks associated in their manufacturing and application. Due to problems

caused by chemical control, development of alternative control measures are

of great concern. Biological control of plant parasitic nematodes offer an

alternative tool to chemical control. Biological control mainly deals with the

application of biological agents or by stimulation of natural enemies of the

nematodes like fungi and bacteria in the soil enviroimient. Many natural

enemies attack phytonematodes in soil and reduce their populations.

Biological control has been defmed as the reduction of inoculum density or

disease producing activities of a pathogen or parasite in its active or dormant

stage, by one or more organisms, accomplished naturally or through the

manipulation of the enviroiunent, host or antagonist or by mass introduction

of one or more antagonists.

Biological controlling agents and organic amendments are the only

solution to maintain plant health. That's why investigators are concentrating

their efforts on integrating biological control agents in plant disease

management strategies (Jatala, 1986). The use of micro-organisms that can

grow in the rhizophere are ideal for use as biological control agents

Rhizosphere provides the initial barrier against pathogen attack to the root

(Weller, 1988). However, despite this wide interest, only a few biocontrol

products are commercially available at present (Upadhyay and Rai, 1988). Of

the various microorganisms present in the rhizosphere, PGPR (Plant growth

promoting rhizobacteria) viz; Bacillus spp., Pseudomones jluorescens,

antagonistic fungus, Trichoderma harzianum, Paecilomyces lilacinus and oil­

cakes with high nitrogen contents, biofertilizers, organic amendments are

noteworthy as biocontrol agents. Trichoderma is a cosmopolitan saprophytic

fungus widely used as a biological control agent in the fight against plant

diseases caused by economically important plant pathogens. Trichoderma are

common in soil, dung and decaying plant materials. Trichoderma spp. are

strongly antagonistic to nematodes and other fungi. Trichoderma spp indeed

have the ability to control plant diseases.

Paecilomyces lilacinus is primarily a saprophyte, being able to

compete for and used in wide range of common substrates in soil (Domsch et

al., 1980). In the laboratory test this fungus infects eggs of M incognita and

destroys the embryos within 5 days (Jatala, 1986). The infection process

begins with the growth of fungal hyphae in the gelatinous matrix and

eventually the eggs of the nematodes are engulfed by the mycelial network.

The colonization of eggs appears to occur by sunple penetration of the egg

cuticle by individual hyphae aided by mechanical and/or enzymatic activities.

The serine protease produced by P. lilacinus might play a role in penetration

of the fungus through the egg shell of the nematodes (Sonants et al., 1995).

Plant growth promoting rhizobacteria {Pseudomonas jluorescens and

Bacillus subtilis) control the damage to plants from phytopathogens by a

number of different mechanisms including out competing the phytopathogen

by physical displacement, secretion of siderophores to prevent pathogens in

the immediate vicinity from proliferating, synthesis of antibiotics, synthesis of

variety of small molecules, production of enzymes that inhibit phytopathogen

and stimulation of the systemic resistance in plants.

Vesicular arbuscular mycorrhizal (VAM) fungi are found to occur on

roots of most of the food and horticultural crops and tropical trees. The

presence of vesicles and arbuscules is the diagnostic criterion for identifying

VAM fungi in a root. VAM fungi improve plant growth through improved

uptake of nutrients, especially phosphorus. They also improve uptake of

minor elements like zinc and copper and of water. They also produce plant

hormones and increase the activity of nitrogen fixing organisms. VAM fungi

have been observed in 1000 genera of plants, representing some 200 families

(Bagyaraj, 1991).

Use of plant residues and organic amendments has been recognized as

an effective way of achieving substantial population reduction of plant

pathogenic life form like nematodes, fungi etc. Organic amendments such as

oil cakes and biocontrol agents have been reported by several workers for the

management of plant diseases (Anver, 2003, Ahmad et ai, 2004). For a

healthy environment there is a need to develop safe and effective methods for

biocontrol of nematode diseases.

The aim of the present study is to focus attention on microbes which

can be used for the management of reniform nematode (Rotylenchulns

reniformis) on cowpea (Vigna unguiculata). The present work is having

following aspects -

1. Studies on the management of reniform nematode, Rotylenchulus

reniformis through fungal bioagents on cowpea.

2. Studies on the management of reniform nematode, Rotylenchulus

reniformis through biofertiiizer (VAM) and bacterial bioagents on

cowpea.

3. Studies on the management of reniform nematode, Rotylenchulus

reniformis through organic amendments on cowpea.

f^euiew of cJLiterat ravure

CHAPTER-2

REVIEW OF LITERATURE

Phytoparasitic nematodes regarded as serious pests constituting an

important major limiting factor in the production of agricultural and

horticultural crops, thus attracting the attention of most nematologists and

pathologists who are seriously concerned to manage them. Various chemical,

cultural, physical and biological measures are adopted for the management of

plant-parasitic nematodes. Biological control at present seems to be practically

demanding and have the most relevant approach due to greater awareness of

the essentiality of pollution free environment (Khan, 1990).

Biological control implies total or partial destruction of a pathogen by

bio-organism except man. The term was mtroduced in scientific literature by

G.F. Van Tabuef in 1914 (Baker, 1987). Interest in biological control first arose

in 1920s and 1930s, when some plant pathogens were suppressed by

introducing some antibiotics producing microbes. A turning point for research

on biological control of plant pathogens came after a gap of more than 30 years

when in 1963 an International Symposium on 'Ecology of soil-borne plant

pathogens prelude to biological control' was held in Berkeley, USA.

The possibility of biological control of nematodes was first suggested by

Lodhe (1874). His study generated interest in many early workers organized

research on this aspect of nematode management, however, started in 1960s in

recent past, biocontrol of plant parasitic nematodes has been subject of several

reviews (Sayre, 1980; Jatala, 1986; Khan, 1990; Siddiqui and Mahmood,

1998).

The biocontrol agents provide protection against plant diseases either by

direct action against the pathogen i.e. antagonism which includes parasitism,

antibiosis, competition or indirectly by reducing host susceptibility i.e. host

mediated interaction and includes exudation, altered rhizosphere, induced

resistance, hypovirulence etc. (Chaube et al., 2003).

Microorganisms have been extensively used for the biological control of

soil-borne plant diseases as well as for promoting plant growth. The important

genera of fungi studied as biocontrol agents are Trichoderma, Paecilomyces,

Verticillium, Glomus, Gliocladium, Aspergillus etc., out of these genera

Paecilomyces, Trichoderma, Gliocladium are being extensively studied for

their use in biocontrol.

Exploitation of microbes (i.e. fungi, bacteria and viruses) in crop

protection appears to be one of the safer and lasting methods of crop disease

management. Agriculture is one of the major sectors of the Indian economy

and 70% of the population is dependent upon it for their livelihood and

contributes over 40% of the gross national production.

A number of nematode problems of national importance have emerged.

Root-knot nematodes are prevalent in 90% of agricultural crops and are

considered to be the major cause of number of problems. Cyst forming

nematodes are restricted to wheat, barley rice, maize, sorghum, potato and

pulses. Reniform and lesion nematodes are associated with wide range of

agricultural crops. Burrowing, citrus, wheat gall, rice and mushroom

nematodes are major constraints onregional basis (Sasser, 1980).

Mahapatra and Mohanty (2002) estimated the avoidable yield loss in

blackgram due to reniform nematode, Rotylenchulus reniformis under field

conditions in Orissa, India. The treatments comprised 1 kg carbofuran/ha and

the untreated control soil application of carbofuran increased the yield of

blackgram by 19.3% with a reduction of reniform index by 36.3% as compared

to the control. A yield loss of 16.2% due to Rotylenchulus reniformis

infestation was incurred.

Patel et al. (2003) reported potential damage of Rotylenchulus

reniformis on cowpea cv. Pusa Falguni and indicated that an inoculum level of

100 and above of young female/plant significantly decreased plant height and

fresh shoot and root weight. The nematode reproduction rate decreased with an

increase in inoculum level.

Prasad (2008) studied on assessment of avoidable yield loss of

sunflower and groundnut due to Rotylenchulus reniformis. Significant

reduction in yield due to nematode was calculated to be 48.7% and 40.8% in

2002-03 and 2003-04 respectively.

Roy et al. (2008) examined yield loss due to Rotylenchulus reniformis,

Linford and Oliveira, 1940 in cowpea {Vigna unguiculata) L. Walp. The

application of carbofuran 3G at 23.0 kg a.i./ha increased the green pod yield of

cowpea by 10.5-14.5% and significantly reduced the eggmass index and

nematode population.

MANAGEMENT BY BIOAGENTS

Paecilomyces lilacinus is a chitin degrader (Okafar, 1967). Chitin

constitutes the largest portion of the nematode egg shell, while the larval

10

cuticle lacks it. This explains the effectiveness of the fungus as an egg

destroyer, which, however, become most effective. Once the nematode larvae

are formed, Paecilomyces lilacintis exhibits chitinase activity when grown on

chitinase agar plates (Gintis et ai, 1983).

Paecilomyces lilacinus was isolated in early 1978 from egg masses of

Meloidogyne incognita acrita infecting potato roots. It frequently infects the

eggs and occasionally the females of M incognita acrita (Jatala et ai, 1979).

Paecilomyces lilacinus was also effective as a biocontrol agent of Globodera

pallida. Eggs of Globodera pallida on potato roots were infected and hatching

declined due to increased infection of eggs by P. lilacinus (Jatala et ai, 1981).

Paecilomycs lilacinus parasitized the eggs and egg masses of nematode

(Silva et ai, 1992, Lm et al, 1993), destroyed 80 to 90% of Meloidogyne eggs

and attacked both eggs and developing females (Strathier, 1979; Jimenez and

Gallo, 1988; Reddy and Khan, 1989; Croshier et al., 1985; Morgan-Jones and

Rodriguez-Kabana, 1984).

P. lilacinus also inhibited the hatching of juveniles (Khan et al, 1988;

Oduor and Wando, 1995) reduced population density, root galling mature

females and nematodes juvenile count in Meloidogyne spp. (Rohana et al.,

1987; Mousa and Mausa, 1995; Noe and Sasser, 1995) and root gall formation

in M.javanica (Zaki and Maqbool, 1996).

Khan and Esfahani (1990) studied the efficiency of P. lilacinus for

controlling M javanica on tomato in green house. P. lilacinus also reduced

damage to cowpea caused by M incognita and R. reniformis (Khan and

Hussain, 1990).

11

Khan and Saxena (1996) found that presence of P. lilacinm along with

either M. arenaria, M. incognita, M. javanica or R. reniformis significantly

reduced the root galling and multiplication, and their egg masses, eggs and

females were more vulnerable to fungal infection.

Reddy and Khan (1989) studied the effect of Paecilomyces lilacinm

(Thorn.) Samson, alone and in combination with carbofiiran @ 0.5 and 1.0 kg

a.i./ha for the management of reniform nematode, Rotylenchulus reniformis,

Linford and Oliveria infecting brinjal, Solanum melongena L. Inoculation of

plant with P. lilacinus grown on 4 g sterilized rice was effective in increasing

plant height and root weight and in reducing the nematode population both in

soil and roots. The fungus gave least reproduction factor and increased the

percentage of males. Paecilomyces lilacinus infected both eggs and mature

females of the reniform nematode.

Khan and Husain (1991) tested the application of Paecilomyces lilacinus

at 2.0 gm mycelium/pot, reduced damage to Vigna unguiculata caused by M

incogntia, R. reniformis and Rhizoctonia solani.

Mahmood, and Siddiqui (1993) studied on integrated management of

Rotylenchulus reniformis by green manuring and Paecilomyces lilacinus. P.

lilacinus and green manuring of maize and Sesbania aculeate were used for

management of R. reniformis on pigeonpea. Application of P. lilacinus found

to be the best followed by amendment of soil by S. aculeata and maize.

Walters and Barker (1994) tested efficacy of Paecilomyces lilacinus in

suppressing R. reniformis on tomato. Effect of rice cultured Paecilomyces

12

lilacinus on R. reniformis was studied in both greenhouse and field microplot

tests with 'Rutgers' tomato. P. lilacinus reduced the population ofR. reniformis

and increased the growth of tomato under both green house and field microplot

condition.

JChan and Saxena (1996) examined comparative efficacy of P. lilacinus

in the control of Meloidogyne sp. and R. reniformis on tomato. Presence of P.

lilacinus with either M arenaria, M. incognita, M. javanica or R. reniformis

significantly reduced the root galling and multiplication of the nematodes

which resulted in improvement of plant growth over nematode infected plants

in control. Use of P. lilacinus showed significant control of Meloidogyne

incognita followed by M javanica, M. arenaria and R. reniformis on tomato

plants.

Anjum Ahmad and Alam (1998) reported that a combined treatment of

organic amendments and P. lilacinus suppressed R. reniformis on tomato,

castor, beans and basil, which subsequently increased the yield of test crops.

Jaya Kumar et al. (2002) conducted an experiment to evaluate the

efficacy of Paecilomyces lilacinus as seed treatment, soil application in single

dose and soil application in split doses as individually and in different

combinations against the reniform nematode on cotton revealed that the

combination of seed treatment with P. lilacinus @ 10 g/kg seed (2.4x10^

spores/g) + soil application in split doses @ 100 and 50 kg/ha (2.4x10^

spores/g) at the time of sowing, 30 and 60 DAS respectively, was the most

effective method of application in checking the reniform nematode,

Rotylenchulus reniformis in cotton. The same treatment also promoted the plant

growth characters and increased the yield.

13

P. lilacinus parasitised the eggs and mature females of/?, renifromis and

reduced its multiplication on cowpea (Khan and Hussain, 1988), tomato and

eggplant (Reddy and Khan, 1989).

Jaya Kumar et al. (2002) studied bioefficacy of Paecilomyces lilacinus

against R. reniformis infesting cotton. The experiment was conducted to

evaluate the efficacy of seed treatment (smgle dose) and soil application (split

doses) of P. lilacinus against the reniform nematode on cotton cv. MCU5. P.

lilacinus as seed treatment at 10 g/kg seed (2.4x10^ spores/g)+ soil application

in split doses of 100 and 50 kg/ha (2.4x10^ spores/g) at the time of sowing and

at 30 or 60 days thereafter was the most effective treatments in controlling the

reniform nematode.

Sanyal and Prasad (2005) studied the effect of Paecilomyces lilacinus

against root-knot and reniform nematodes. Secondary metablites from dried

mycelia of Paecilomyces lilacinus were isolated using various extraction

solvents and nematicidal tests of the crude extracts were conducted in vitro

against freshly hatched second stage juveniles of root-knot nematode,

Meloidogyne incognita and Rotylenchulus reniformis. Methanol extract was

found to be more active against both Meloidogyne incognita and R. reniformis.

The LC50 value against M. incognita was 84.2 ng ml"' at 24 hours, 122.4 \ig

ml'' at 48 hours and 68.1 ng ml"' at 72 hours. The LC50 value (activity) of the

methanol extract against R. reniformis was found to be 91.4, 127.5 and 54.4 (ig

ml"' at 24,48 and 72 hours respectively.

Anver and Alam (1999) studied the control of Meloidogyne incognita

and Rotylenchulus reniformis singly and concomitantly on chickpea and

14

pigeonpea. Meloidogyne incognita caused greater reduction in plant growth,

chlorophyll content, water absorption capacity of roots and root nodulation of

chickpea and pigeonpea and bulk density of pigeonpea stem than Rotylenchulns

reniformis. These nematodes inhibited each other in concomitant infections.

However, both the nematodes together caused more damage to the test plants

than was caused by either of them singly but it was less than the sum total of

the damage caused by them individually. Oil-seed cakes of neem/margosa,

castor, mustard, rocket salad were found to be highly effective in reducing the

multiplication of nematodes. Consequently, plant growth, the water absorption

capacity of the roots, root nodules and bulk density of woody stem of

pigeonpea increased significantly. The test nematodes were found to be less

damaging in the presence of Paecilomyces lilacinus. The multiplication rate of

nematodes was less in the presence of Paecilomyces lilacinus as compared to

the absence of Paecilomyces lilacinus. Damage caused by the nematodes was

further reduced when P. lilacinus was added along with oil-cake. The most

effective combination of P. lilacinus was with neem-cake.

Anitha and Subramanian (1998), studied the management of reniform

nematode on tomato by application of carbofuran 3G at 0.3 g a.i/m ,

Paecilomyces lilacinus at 2 g/m^ and neem cake at 100 g/m^ were evaluated

either individually or in combination in the nursery followed by application of

carbofuran 3G at 1 kg a.i/ha in the main field 15 days after transplanting.

Application of neem cake or carbofuran 3G in the nursery significantly

increased tomato yield by 59.4% and 58.48% respectively and suppressed the

reniform nematode population in the roots by 44.5 and 46.6%, respectively.

15

Application of neem cake in the nursery followed by carbofuran @ 1 kg a.i./ha

in the main field. Significantly reduced the rate of multiplication of the

nematode and increased the crop yield.

Tiyagi and Ajaz (2004) studied the biological control of reniform

nematode associated with chickpea usmg oil-cakes and Paecilomyces lilacinm.

The addition of organic matter to the soil was explored as an alternative means

of nematode control. Oil-seed cakes of neem, castor bean, groundnut, linseed,

sunflower and soybean were highly effective in reducing the multiplication of

soil nematodes (Hoplolaimus indicus, Rotylenchuliis reniformis,

Tylenchorhynchus brassicae, Meloidogyne incognita, Longidorous,

Pratylenchus coffeae, Dorylaimus) resulting in significant plant growth

increase. The multiplication rate of nematode was less in the presence of

Paecilomyces lilacinus compared to the absence of P. lilacinm. The damage

caused by the nematodes was further reduced when P. lilacinus was added

along with the oil-seed cakes. The most effective combination oi Paecilomyces

lilacinus was with neem cake under field conditions.

Ashraf et al. (2005) investigated that the management of reniform

nematode, Rotylenchulus reniformis infecting okra {Abelmoschus esculentus)

by integrating eco-friendly components such as oil-cake viz. neem

(Azadirachta indica), castor (Ricinus communis), black mustard (Brassica

nigra), sunflower {Helianthus annuus) and linseed (Linum usitatissimum) @ 15

g/kg soil and a biocontrol agent Paecilomyces lilacinus @ 1 g mycelium +

spores/kg soil. The treatments were evaluated individually and in integration

16

against Rotylenchulus reniformis under glass house conditions. It was noted

that all the treatments quite effectively suppressed the nematode population and

kept the infection at significantly low level. Application of P. lilacinus showed

better results in improving plant growth and reducing the nematode population

build up as compared to oil-cake treated plants whereas neem cake gave better

results than other oil-cakes. However, integration of neem cake with P.

lilacinus gave best result causing increased plant growth and reduced

population build-up of reniform nematode.

Interest in arbuscular mycorrhizal fungi propagation for agriculture is

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

1969; Tinker, 1975; Linxian and Hao, 1988). The agricultural importance of

AMF (arbuscular mycorrhizal fungi) 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 products of

quality and efficiency is not possible.

Lingaraju and Goswami (1993) studied interactive relationship of a

vesicular arbuscular mycorrhizal fungus. Glomus fasciculatum and reniform

nematode, Rotylenchulus reniformis on cowpea in greenhouse. Four levels each

of these organisms were used either singly or in combinations. Mycorrhizal

fungus enhanced total biomass as well as fresh root weight while the nematode

reduced them and the interaction effects were significant. The final numbers of

17

the nematode were more in the presence of fungus whereas the nematode did

not influence the spore numbers though it interfered with mycorrhizal

colonization. The VAM induced tolerance in cowpea to the nematode even in

the presence of damaging levels of the nematode under phosphorus deficient

conditions.

Sikora and Sitaramaiah (1995) studied pre-inoculation and simultaneous

inoculation of cotton (Gossypium hirsutum cv. "Coker Carolina Queen").

Plants with Glomus fasciculatum reduced Rotylenchulus reniformis juvenile

penetration, eggmass production and population build up on mycorrhizal

inoculated plants when compared to non-mycorrhizal controls. Very few

nematodes were located in and around the root tissues extensively colonized by

the fungus. The decreased number of nematodes and egg masses per root

system was also reflected in reduced soil population. Simultaneous inoculation

of G. fasciculatum and R. reniformis at planting resulted in reduced nematode

penetration conversely the number of chlamydospores recovered from the soil

of mycorrhizal roots.

Lingaraju and Goswami (1995) studied the effect of two organic

amendments mustard and neem oil cakes in an interaction involving a vesicular

arbuscular mycorrhizal fungus, Glomus fasciculatum (GF) and a plant parasitic

nematode, Rotylenchulus reniformis on cowpea in a greenhouse study with a

view to know the fate of Glomus fasciculatum m oil-cake amended soils.

Mustard oilcake + G. fasciculatum amendment resulted in high growth

responses in presence of the nematode. Mycorrhizal plants grown in oil-cake

18

amended and inoculated with R. reniformis produced as many spore counts as

those of G. fasciculatum alone plants. Also, nematode egg numbers/g of root

were least on such plants. More actinomycetes were estimated to be present in

oil cake amended + R. reniformis inoculated mycorrhizal soils.

Sitaramaiah and Sikora (1996) evaluated the influence of different

spore levels of Glomus fasciculatum on growth of cotton, Gossypium hirsutum

cv. "Coker Carolina Queen" and on the population dynamics of Rotylenchulus

reniformis. Lower numbers of aduU females with eggmasses were recorded on

mycorrhizal roots inoculated with a spore concentration of 300 and above when

compared to controls. Increasing the spore concentration of the fungus from

300 to 1800/plant reduced soil population ranging from 40-80% at low

nematode inoculum level (100/plant). In general, the nematode did not have

any effect on the number of spores produced or root colonization of the fimgus.

There was a positive vegetative growth response of cotton plants to increase

spore concentration of G. fasciculatum. The mycelium and chlamydospores of

G. fasciculatum were consistently observed in eggmasses ofR. reniformis.

Jothi and R. Sundarababu (1998) studied the interaction of Glomus

fasciculatum and R. reniformis on ragi. It was observed that the yield was

highest in VAM (100 g/m ) inoculated plots and the confrol of nematodes was

effective in phorate lOG (1 kg a.i./ha) followed by carbofiiran 3G 1 kg a.i./ha

treated plots.

Sreenivasan et al. (2003) reported that four vesicular arbuscular

mycorrhizal (VAM) fimgi viz., Glomus mosseae, G. fasciculatum, G.

19

intraradices and G. fulvum were screened to select efficient VAM firngi for

management of Rotylenchulus reniformis in cotton Gossypium barbadense cv.

TCB 209. Plants inoculated with G. fasciculatum were significantly better in

all respects of plant growth, mycorrhizal colonization and yield of cotton, G.

fasciculatum and G. mosseae were equally effective in reducing R. reniformis

population in root and soil.

Sikora and Hofinann (1993) examined that the biological control of

nematodes is based on the introduction of bacteria colonizing the rhizosphere

of the host plant. These plant health-promoting rhizobcteria adversely influence

the intimate relationship between the plant-parasitic nematode and its host by

regulation of nematode behaviour during the early root penetration phase of

parasitism. Significant levels of control have been observed in penetration of

sugarbeet and potato cyst nematodes. Two mechanisms of action are thought to

be responsible for reduction in nematode infection (i) production of metabolites

which reduce hatch and (ii) degradation of specific root exudates which control

nematode behaviour.

Ali (1996) tested five bacterial antagonists (Arthrobacterium sp.,

Bacillus sp., Corynebacterium sp., Serratia sp. and Streptomyces sp.) for

controlling Meloidogyne javanica and R. reniformis on sunflower in pot

experiment. Results indicated that populations of both nematode species were

affected by application of the tested bacterial isolates. Inoculum levels of any

of these bacterial isolates, relative to nematode population, also had significant

effect on their suppressive potential on either nematode species. In this regard,

20

application of bacteria one week prior to nematode inoculation surpassed that

of simultaneous, or one week post nematode inoculation treatments. In all

cases, the suppressive effect relative to check ranged between 46-96% for

Meloidogyne javanica and 36-100% for Rotylenchulus reniformis. The carry

over effect of the five bacterial isolates continued, upto 15 months at which a

reduction in nematode population was 91% for Rotylenchulus reniformis and

5-88% for Meloidogyne javanica.

Vats and Dalai (1998) studied the interrelations between Rotylenchulus

reniformis and Rhizobium leguminosarum on pea (Pisum sativum). Results

revealed that inoculations with the nematode reduced number and weight of

nodules, and dry shoot weight of pea plants as compared to the uninoculated

check. The nematode infection also interfered with the symbiotic nitrogen-

fixation and reduced the N2 content of shoot. Inoculation of Rhizobium,

resuhed increased nodule number and weight. At both the stages of

observation, N2 content and dry shoot weight increased significantly in

simultaneous inoculation of nematode and Rhizobium over nematode alone.

Reniform nematode infection and multiplication was reduced significantly in

the presence of Rhizobium leguminosarum.

Jaya Kumar et al. (2003) examined various Pseudomonas fluorescens

isolates collected from cotton rhizosphere and TNAU commercial formulation

strain of PFI were evaluated for management of reniform nematode in cotton.

Maximum colonization of rhizobacterium on cotton roots was observed in

plants treated with PFI strain @ 2.5 kg/ha. Significant reduction in soil and root

21

population of R. reniformis and subsequent increase in cotton plant growth

characteristics were observed in P. fluoresces treated plants. Strain PFI

effected maximum population reduction of nematode to the extent of 70.4% in

soil and 44.8% in roots. It was followed by isolates PFSPI, PFTH and PFC03

which were at par with each other in reducing reniform nematode population in

soil and roots of cotton.

Niknam and Dhawan (2003) showed effect of seed bacterization and

methods of application of Pseudomonas fluorescens on the control of R.

reniformis infecting tomato. Seed bacterization resulted in 90% germination

compared to 81% in non-bacterized seeds seedling weight and shoot length

were improved by 54 and 18%, respectively, 21 days after sowing. Root

colonization by the rhizobacterium in term of cfii/g and cm., fresh root of the

seedlings raised from bacterized seeds increased with increasing observation

time. Furthermore, strain Pf, when applied as seed bacterization, soil drench

and bare root dip either singly or in combination, caused a significant reduction

in the penetration of Rotylenchulus reniformis as compared to the untreated

control. The maximum reduction of 55% of nematode penetration occurred

when the bacterium was applied as seed bacterization followed by a soil

drench. Application of the bacterium alone or in combmation with the seed

bacterization and soil drenching/root dip improved growth characters of the

tomato plants, except root length. The multiplication rate (Rf) of the nematode

was also significantly reduced in all of the treatments receiving bacteria with

the maximum (45%) in seed bacterization followed by a soil drench.

22

Jayakumar et al (2003) evaluated the effect of P. fluorescens strain on R.

reniformis penetration in cotton. The biological control agent, P. fluorescens

was reported to be effective in management of nematode in cotton. The results

of study indicated that, under growth chamber conditions set at 27 ± 1**C 15000

lux for 9 h and 70% relative humidity, seed treatment with strain Pf 1 at 10*

cfu/ml reduced the root penetration by the reniform nematode in sand medium

by up to 64%.

Jayakumar et al. (2004) studied biological control of cotton reniform

nematode, R. reniformis in cotton cv. MCU-5. The treatments comprised: (TO

soil application (SA) with Pseudomonas fluorescens at 2.5 kg/ha before

sowing; (T2) seed treatment (ST) with P. fluorescens at 10 g/kg seed (6x10*

cfu/g); (T3) ST + SA; (T4) split application with P. fluorescens at 1.0, 0.75 and

0.75 kg/ha at 0, 30 and 60 days after sowing (DAS), respectively; (T5) ST +

split application, and (Te) untreated control. Observation on plant growth

parameters and nematode population, both in the soil and roots, and cotton

yield were recorded at 180 DAS. Among the various application methods of P.

fluorescens, T5 showed maximum increase m plant height (19.6%) over the

control, further, T5 registered the maximum number of bolls per plant (75.3%).

The growth and yield of cotton were significantly improved due to P.

fluorescens, irrespective of the application method. P. fluorescens significantly

reduced the soil and root populations of/?, reniformis. T5 significantly reduced

the soil and root nematode population by 74.2 and 53.9%, respectively over the

control. The efficient reduction in nematode population due to split application

23

in different intervals might be due to the maximum building up of bacterial

population or root colonization by the bacterial inoculum.

Niknam and Dhawan (2003) revealed induction of systemic resistance

by Bacillus subtilis isolate BSt against R. reniformis in tomato. The results

showed that the nematode penetration was reduced by 44.5% in the split root in

which one half received a bacterial cell suspension (10*° cells/ma) as a soil

drench and a week after the other half was inoculated with the nematode. All

growth characters measured were increased to some extent by the bacterium,

but only fresh shoot length and fresh shoot weight significantly differed from

the control. The nematode multiplication rate was reduced significantly when

the bacterial soil drench was applied a week before nematode inoculation,

indicated that BSt could induce systemic resistance to tomato against R.

reniformis.

Niknam and Dhawan (2003) studied the effect of three application

methods of Bacillus subtilis isolates Bst on the penetration and multiplication

of R. reniformis infecting tomato. The study evaluated the efficacy of Bacillus

subtilis as seed treatment (ST), soil drench and bare root dip alone or seed

treatment followed by soil drench or bare root dip on tomato (cv. Pusa Ruby).

The results revealed that seed treatment resulted in 88% germination (81.3% in

non-treated seeds), seed treatment also increased seelding fresh weight (39.3%)

and shoot length (15.3%) at 21 days after sowing. Root colonization by the

rhizobacterium showed that cfu/g and fresh roots of seedlings raised from

treated seeds increased with the increase in observation time. Bacillus subtilis

24

applied as seed treatment, soil drench and bare root dip either singly or in

combination resulted in a significant reduction in nematode penetration

compared to the control. The maximum reduction (50.5%) in nematode

penetration was obtained when the bacterium was applied as seed treatment

followed by soil drench. The application of the bacterium either alone or in

combination improved all the evaluated growth characters of tomato except

root length. The multiplication rate of the nematode was also significantly

reduced in all the treatments receiving the bacterium, but the least

multiplication rate (5.9 times) or the highest reduction (48.7%) in terms of

PfiTi was obtained with seed treatment followed by soil drench.

Senthamizh and Rajendran, G. (2002) conducted an experiment on

cotton (cv. MCU5) infested with Rotylenchulm reniformis to evaluated

potential biological control agents that could be profitably used in minimizing

nematode damage in cotton. The treatments comprised 4g Trichoderma

viridefkg seed (ST); 2 g Pseudomonas fluorescenslkg seed (ST); 10x10^ spores

Paecilomyces lilacinus/kg seed (SA); 10x10^ spores Verticillium

chlamydosporium/kg soil (SA); 10 g P. fluorescemlkg soil SA); 10 g T.

viride/kg soil (SA), combined application of T. viride (ST) and T. viride (SA);

and P. fluorescens (SA) and P. fluorescens (SA). The highest plant height (44.2

cm), root length (13.9 cm), shoot weight (21.5 g) and root weight (2.2 g) were

recorded with the combined application of P. fluorescens (ST + SA). The same

treatments recorded the highest reduction in the nematode population by 61%

compared with the control.

25

Visalakshi et al. (2002) examined the biocontrol potential of

Pseudomonas fluorescens, Paecilomyces lilacinus, Verticillium lecani, V.

chlamydosporium, Trichoderma viride, Arthrobotrys spp., Bacillus subtilis, B.

cereus and VAM for the management of reniform nematode in Papaya (var.

C0.3) under glasshouse conditions. The bioagents were applied through

nursery soil mixture before sowing. The experimental results revealed that

nursery soil application of VAM was effective in reducing reniform nematode

population by 80% significantly and improved the growth of the seedlings in

terms of length and weight of shoot and root.

Jayakumar et al. (2003) studied on the compatibility of avermectine with

carbofuran 3G, Pseudomonas fluorescens, T. viride^ for the management of/?.

reniformis in bhendi and revealed that avermectin 75% as seed treatment in

combination with soil application of carbofuran 3G at 1 kg/ha recorded a

maximum shoot length (47.8 cm), fresh shoot weight (32.47 g), dry shoot

weight (14.76 g) and fruit yield (53.21 g/plant). It was followed by Avermectin

+ P. fluorescens and avermectin + T. viride. The combined treatment of

avermectin 75% as seed treatment + carbofuran 3G recorded the maximum root

length (29.37 g), fresh weight (6.23 g) and dry weight (4.97 g). It was followed

by (Avermectine + P. fluorescens) and {Avermectine + T. viride), the

maximum reduction in R. reniformis soil population (by 71.01%) root-knot

nematode population (by 66.89%) over the untreated control was affected by

the combined application of Avermectine + carbofuran 30. It was followed by

the combined application of avermectine + P. fluorescens + T. viride.

26

On the other hand, the application of avermectin 75% as seed treatment

effected reduction in soil and root population by 35.6 and 21.7% respectively

compared to the untreated control.

Sharma et al. (2003) studied the effect of Azotobacter chrococcum,

Azospirillum brasilense and VAM fungi {Glomus mossae) alone and in

combmatiorv with carbofuran on the reniform nematode, Rotylenchulus

reniformis infecting okra. Azotobacter showed an increase in shoot length and

shoot weight compared to inoculated check. Shoot length of Azospirillum and

VAM treated plant were at par with each other though it improved to the extent

of 80% over inoculated check. However, root weight was better in VAM

treated plants in comparison of Azospirillum treated plants. The number of

eggmass produced with Azotoacter application was similar to the inoculated

check and thus there was little effect of this treatment on nematode

multiplication. The reproduction factor in Azotobacter treated soil was highest

at 12 followed by Azospirillum and VAM as 8.5 and 5.7 respectively compared

to inoculated check with 5.8. Low doses of carbofuran (0.5 kg/ha) interacted

positively with Azotobacter and Azospirillum in bringing down the nematode

population in contrary to higher dose comparison to inoculated check and

carboftiran alone treated plants.

Subramanian and Senthamizh (2006) investigated the effect of culture

filtrates of Pseudomonas fluorescens and Trichoderma viride as well as root

exudates of G. mosseae each at 0, 25, 50, 75 and 100% on egg hatching of/?.

reniformis in vitro. The culture filtrate of T. viride and P. fluorescens caused

27

marked suppression in egg hatching of reniform nematode as compared to the

control. The root exudates of G. mosseae treated plants significantly decreased

the hatching of juveniles over the control. The reduction in hatching was the

highest at 100% (3.5 J2/egg mass) followed by 75, 50 and 25% concentration

(5.5, 7.5 and 8.5 J2) eggmasses, respectively.

Subramaaiaa and Senthamizh (2006) examined effect of caUure filtrates

of Psendomonas fluorescens and Trichoderma viride and root exudates of

Glomus mosseae on hatching of R. reniformis. All the three biocontrol agents

screened for egg hatching ofR. reniformis, significantly reduced egg hatching

of juveniles over control.

Subramanian and Senthamizh (2006) also reported effect of culture

filtrate of P. fluorescens and T. viride and root exudates of G. mosseae on

mortality ofR. renifromis and found that there was an increase in the mortality

of young females with increase in the exposure period of all biocontroUing

agents.

Sreenivasan and Sundarababu (2007) examined the efficacy of

commercial formulations of vesicular arbuscular mycorrhiza. Glomus mosseae,

plant gowth promoting rhizobacterium, Pseudomonas fluorescens and the

antagonistic fungus, Trichoderma viride against reniform nematode, R.

reniformis in cotton under green house conditions. Among the three biocontrol

agents tried, G. mosseae as soil application and Pseudomonas fluorescens as

seed treatment performed well in improving the plant gcowth and yield of

cotton. G. mosseae as soil application recorded maximum reduction of

28

nematode population in roots (72.1%) and soil (81.1%). Trichoderma viride

both as seed treatment and soil application was less effective in the

management of/?, reniformis.

MANAGEMENT BY ORGANIC AMENDMENTS

Padhi and Mishra (1987) applied four organic materials viz., cakes of

neem and karanj, prawn exoskeleton and organic manure at 1 and 2 tonnes/ha

against reniform nematode, R. reniformis infesting frenchbean var. Premier.

Among different treatments, neem and karanj cake at 2 ton/h were more

effective in reducing nematode population and producing better plant growth.

Akhtar and Alam (1989) reported that the incorporation of chopped

leaves of Azadirachta indica (neem), Lantana camara, Calotropis procera,

Eucalyptus citriodera at 50 or 100 g/pots significantly suppressed build up of

Hoplolaimus indicus, Helicotylenchus indicus, Tylenchorhynchus brassicae,

Rotylenchulus reniformis and Tylenchus filiformis on Capsicum annum cv. NP-

46A. Higher doses gave better results and chopped leaves of Calotropis

procera produced the greatest reduction in nematode population.

El-Nagar et al. (1994) tested some biocides on the reproduction of R.

reniformis. All the combination of biocides against R. reniformis on sunflower

were effective in reducing the number of nematodes in soil and the number of

females and egg masses on the plant roots. The combination of margosa (neem

extract) and gurdar gave the best results followed by the combination of Gurdar

and Sincocin. The latter gave the best improvement in plant growth.

29

Yassin and Ismail (1994) studied the effect of some oil seed cakes as

soil amendments and aldicarb on cowpea plants infected with R. reniformis.

Cotton, linseed, soyabean and sunflower oil seed cakes as well as aldicarb were

used to study their effect on the reproduction of Rotylenchulus reniformis and

growth of cowpea plants under greenhouse conditions. All treatments of oil

seed cakes and aldicarb reduced final population rate of build-up and

reproduction of nematode than those of untreated soil in all soil types. The

reduction in such values varies greatly according to the oil seed cake and soil

type.

Patel et al. (1995) studied the effect of periwinkle {Catharanthus

roseus) on reniform nematodes. Pink and white flowered periwinkle plants

significantly reduced the final population of Rotylenchulus reniformis 45 days

after inoculation in pot experiments. The reduction was 99% with a

reproduction rate of 0.008. Different inoculum levels of nematodes had no

adverse effect on plant growth, however, root length and weight were

significantly reduced at the initial inoculum levels of 10 and 10,000

nematodes/plant respectively. There was 100, 98, 96 and 97% reduction in

nematode development at the initial inoculation levels of 10, 100, 1000 and

1,000 nematodes/plant, respectively.

Anjum and Alam (1996) showed the efficacy of some organic

amendments against R. reniformis on black gram. Oil seed cakes and chopped

leaves of neem {Azadirachta indica) and castor and rice were found to be

effective against Rotylenchulus reniformis resulting in improved plant growth

30

of blackgram. Neem cake was the most effective (41.9%) reduction over

untreated control followed by castor cake (39.0%), rice polish (34.8%), neem

leaf (30.3%) and castor leaf (23.9%).

Youssef and Amin (1997) evaluated effect of soil amendment in the

control of M.javanica and R. reniformis infection on cowpea. The effect of soil

amendment with ricebran, castor cake, donkey dung, leaf extract powders of

basil (Ocimum kilmemdescharicum)^ castor, filaya {Mentha sylvestris), maize

mulberry, olive, water hyacinth or willow and fenamiphos lOG, on control of

M javanica and R. reniformis on cowpea cv Baladi was tested under

greenhouse conditions. Fenamiphos was more effective in reducing nematode

infection and reproduction then the organic amendments, with improvement

also in plant growth as compared to control. A greater reduction in the

nematode populations was found in soil treated with the amendments one week

prior to infestation with nematodes than in soil treated at the time of

inoculation.

Dalai and Vats (1998) studied the use of phytotherapeutic substances for

the management ofR. reniformis in pea. Chopped leaves oiAzadirachta indica,

Eucalyptus tereticornis, Lantana camera and Parthenium hysterophorus each

at 3 doses viz. 5, 10 and 20 gm/kg of soil were evaluated for the management

of R. reniformis in pea. Maximum increase in plant growth and yield was

obtamed with the higher dose (20 g/pot) of A. indica followed by the 10 g dose.

However, the 10 and 20 g doses of . indica, L. camara and P. hysterophorus

did not differ significantly. Phytotoxicity was noticed when chopped leaves of

31

Eucalyptus tereticornis were used at 10 and 20 g/pot. The maximum reduction

in nematode fecundity and population build up was recorded with the higher

dose of A. indica chopped leaves followed by the higher dose of P.

hysterophorus chopped leaves.

Anvar and Alam (2000) studied on the organic management of

concomitant M incognita and R. reniformis on chickpea (cv. K-85). M

incognita caused greater reduction in plant growth, chlorophyll content, water

absorption capacity of roots and root nodules than R. reniformis. These

nematodes inhibited each other in concomitant infections. Both the nematodes

together cause more damage to the test plants than was caused by either of

them singly, but it was less than the cumulative damage caused by them

individually. Oil-seed cake of neem/margosa, castor, mustard, rocked salad

(Eruca sativa) and nematicides significantly reduced the multiplication of

nematodes and thus improved plant growth, water absorption capacity of roots

and root nodules.

Patel et al. (2003) reported that application of neem, castor, mahua,

mustard, piludi and karanj cakes, farmyard and poultry manures, dry and fresh,

AzoUa, pressmud and urea significantly increased the plant growth and

decreased the host infestation by Rotylenchulus reniformis over control.

Among them, pressmud followed by fresh AzoUa and farm yard manure were

proved to be the best. Application of piludi cake and urea were least effective.

Budhram and Baheti (2003) studied the management ofR. reniformis on

cowpea through seed treatment with botanicals. Seed soaking with aqueous

32

extracts of neem and karanj seed kamel at 20% proved to be most effective

among various plant products tested in improving plant growth of cowpea and

minimizing infection of/?, reniformis.

Budhram and Baheti (2004) examined that the plant products were

effective in improving plant growth and reducing nematode reproduction over

the untreated control. However, neem seed kernel was the most effective

among all the plant products.

Umamaheshwari et al. (2005) conducted two glass house experiments to

study the impact of neem {Azadirachta indica), leaf extract and leaf powder on

Rotylenchulus reniformis infecting cowpea. Aqueous leaf extract of neem was

applied as foliar and soil application at two concentrations (10 and 15%). Soil

application with 15% extract recorded maximum growth parameters and yield

(25.5 g/plant) and minimum nematode population in soil (109.5/250 cc) and

root (10/g). Foliar application also showed significantly higher growth

parameters and lesser nematode population as compared to control. In another

experiment neem leaf powder as seed treatment at 10% w/w recorded the

highest growth parameters and yield (36.5/plant) and the lowest nematode

population in soil (43.5/250 cc) and roots (3.5/g).

rui

and

irlethodd

CHAPTER-3

MATERIALS AND METHODS

The different materials to be used and the methods to be employed

during the course of proposed experimental programme.

Selection of test pathogen and test plant:

In the present study, the most widely occurring and economically

important pathogen i.e. reniform nematode, Rotylenchulus reniformis (Linford

and Oliveira, 1940) was selected as a test pathogen. Cowpea {Vigna

unguiculata L. Walp var. Pusa Komal) was used as a test plant.

Preparation and sterilization of soil mixture :

Sandy loam soil collected from a fallow field of Aligarh Muslim

University Campus, was sieved through 16 mesh sieve and mixed with sieved

river sand and organic manure in the ratio of 3:1:1 respectively. Throughout the

course of studies, unless stated otherwise, 6" pots were filled with this soil

mixture @ 1 kg/pot. A little amount of water was poured in each pot to just

wet the soil before transferring to an autoclave for sterilization at 20 lb pressure

for 20 minutes. Sterilized pots were allowed to cool down at room temperature

before use for experiments.

Raising and maintenance of test plants ;

Seeds of test plant were surface sterilized with 0.1% mercuric chloride

for 2 minutes and washed thrice in sterilized water. Four seeds of cowpea were

34

then sown in 6" earthen pots for raising the seedlings and after their

germination thinning was done so as to maintain only one plant per pot. One

week old, well established and healthy seedlmgs were used for experimental

purposes throughout the course of investigations.

Raising and maintenance of pure cultures of nematodes :

Pure culture of R. reniformis was raised on castor plants using a single

eggmass collected from infected castor roots. The egg mass was surface

sterilized by treating it with 1:500 aqueous solution of chlorox (Calcium

hypochlorite) for 5 minutes as described by den Ouden (1958). Treated egg

mass was washed thrice in distilled water. The eggs, in the eggmass were

allowed to hatch out at 27°C under aseptic conditions on a coarse sieve leaving

tissue paper and kept in a pefridish containing sufficient amount of sterilized

distilled water touching the lower surface of coarse sieve. Castor seedlings

grown in 12" clay pots containing autoclaved soil were inoculated with the

immature females so obtained. Nematodes were extracted from the pot soil

after a month through graded sieves of 16, 60 and 400 mesh according to

modified Cobb's shifting and gravity method followed by Baermann fimnel

technique (Southey, 1970). Nematodes so obtained were used for inoculating

fresh castor seedlings growing in several 12" clay pots containing sterilized

soil. Immature females of reniform nematode infested the roots and multiplied

there on in respective pots. After 6-8 weeks, a little of soil from near the root

zone and roots of inoculated plants were examined separately to confirm the

establishment and multiplication of nematodes. Fresh castor seedlings planted

35

in already nematode infested soil from time to time in order to maintain a

regular supply of the respective inoculum. Culture of R. reniformis multiplied

and maintained in this way and thereafter used for obtaining required inoculum.

Preparation of nematode inoculum :

For extraction of reniform nematode, soil was collected from the root

zone of heavily infected castor plants in which pure culture of this nematode

was raised. This soil was processed for extraction of immature females of

reniform nematode using the technique described earlier. Separate water

suspension of above mentioned nematodes were thoroughly stirred for making

homogenous distribution of nematodes before taking 10 ml suspension in the

counting dish (Southey, 170) for counting the number of nematodes from each

sample under the stereoscopic microscope. An average of five counts were

taken to determine the density of nematodes in the suspension. Volume of

water in the nematode suspension was so adjusted that each ml contain about

100 nematodes. Each 20 ml of this suspension used for inoculation of plants to

provide required inoculum level (i.e. 2000 nematodes/pot).

Isolation of fungi from Rhizosphere and infected cowpea roots :

Cowpea plants showing distinct galls and exhibiting root-rot and wilt

symptoms were collected in polythene bags from around the University

Agricultural Farm. Serial washing techniques were employed to isolate ftmgus

from infected roots. Roots were transferred to a sterilized dish containing

sterile distilled water and gently freed of soil particles. The process was

repeated till all the soil particles were removed. The roots were cut into small

36

pieces of about approximately 5 mm and transferred to petridishes containing

0.1% mercuric chloride. Root pieces were then washed at least thrice in

distilled water and dried on filter paper. In aseptic chamber, five of these root

pieces were then placed in each of 10 sterilized petridishes containing potato

dextrose agar (PDA) having following composition.

Peeled potato 200 g

Dextrose 20 g

Agar-Agar 20 g

Distilled water 1000 ml

Petridishes were then incubated at 28+2°C. The fungi which developed

on root segments were examined and identified.

Raising and maintenance of fungus cultures:

The isolated fungi viz. Paecilomyces lilacinus, Trichoderma harzianum,

Trichoderma virens (GUocladium virens), Trichoderma viride were separately

raised on semi-solid substrate. Pre-soaked semidried sorghum seeds were

autoclaved at 15 lb pressure for 1 hr. Flasks filled with these substrates were

inoculated with spore suspension (10** ml' ) of 7 days old fungal cultures and

incubated at 25±2°C for 20 days in BOD incubator.

Raising and maintenance of bacterial culture:

Soil samples around the cowpea roots were collected fi-om fields of

University Agricuitural Farm Aligarh. These samples were selected for the

37

isolation of Pseudomonas fluorescens and Bacillus spp. using following

procedure :

• For stock solution, 10 gm soil was dissolved in 200 ml normal saline

solution.

• Seven test tubes each with 9 ml normal saline solution were prepared and

marked as 1 to 7.

• One ml from stock solution was aseptically transferred to first test tube

making the dilution 10"\

• Solution in the test tube was mixed and 1 ml from this tube (10'') was

transferred aseptically to next making it as 10".

• Similar transfers were made till 10 dilution was achieved.

• From 10"' dilution, 0.1 ml was transferred to sterile nutrient agar plate and

spread properly. Above step was repeated with all other dilutions.

• All 7 plates were kept for incubation at 32+l°C for 24-48 hrs.

Plates were observed for different colonies of bacteria. Required

bacterial colonies were picked, properly streaked on separate nutrient agar

plates to get pure discreate colonies. Slants cultures of these strains were

maintained in duplicate for various biochemical tests for fiirther confirmation

of Pseudomonas and Bacillus spp.

1. Confirmatory test

1.1 Staining

Gram staining was done by method given by Christian Gram (1884).

38

• Thin smears were prepared on a clean glass slides.

• Smears were stained with crystal violet for 30 seconds and rinsed with

sterile distilled water.

•

•

Films were flooded with gram iodine for 30 seconds and again rinsed with

sterile distilled water.

Decolorization was done with 95% alcohol and rinsed again with sterile

distilled water.

Counter stainmg was done with safranin for 20-30 seconds, finally rinsed

with sterile distilled water and blot dried.

• Slides were observed under microscope.

1.2 Biochemical tests:

1.2.1 Casein acid glucose (CAG) medium

Composition

Casein acid hydrolysate 10.0 gm

Yeast extract 5.0 gm

Glucose 5.0 gm

K2HPO4 4.0 gm

Agar 20.0 gm

CAG medium plates were prepared and incubated at 30+l°C overnight to

check sterility and to remove excess moisture. Each plate was divided into

eight equal sections. Each section was streaked fi-om freshly prepared nutrient

39

broth cultures. Plates were incubated at 32±1°C for 24-48 hour, growth in the

streaked region confirms Bacillus spp.

1.2.2 Fluorescence on King's B medium:

Composition

Proteose peptone (Difco) 20 gm

K2HPO4 1.5 gm

MgS04.7H20 0.5 gm

Agar 15.0 gm

Glycerol 15.0 gm

King's B medium plates were prepared and incubated at 30+l°C overnight to

check sterility and to remove excess moisture. Each plate was divided into

eight sections and each section was streaked from freshly prepared

Pseudomonas culture plates were left in incubator at 32+l°C for 72 hour.

Production of fluorescence pigment were checked under UV light to confirm

Pseudomonas fluorescens.

Preparation of bacterial inoculum:

Nutrient agar plates were prepared by pouring sterile nutrient agar in

petriplates and incubated overnight at SO C to check sterility and remove

excess moisture Single colony of isolates from freshly cultured sub-plate was

inoculated into each nutrient broth flask and incubated at 32+l°C for 72 hrs.

One ml contain about 10 cfti/ml bacterial inoculum.

40

Inoculation Techniques:

Feeder roots of cowpea seedlings, just before inoculation were exposed by

carefully removing the top layer of soil and the required quantity of nematode

suspension, fungus and bacterial inoculum was poured uniformly all around the

exposed roots using sterilized pipette. Exposed roots were immediately covered

by soil properly. Untreated and uninoculated plant served as control. Each

treatment was replicated thrice and suitably randomized on a glasshouse bench.

Plants were watered as and when required and terminated after 60 days of

inoculation.

Efficacy of organic amendments for the management of reniform

nematode on cowpea:

Chopped leaves of Datura stromonium (Linn.), fresh AzoUa {Azolla

pinnata R.Br.Syn.), Catheranthus rosea (Linn.), Calotropis procera (Ait.),

Tagetes erecta (Linn.), Lantana camara (Liim.), Euphorbia hirta (Linn.) and

neem cake (Azadirchta indica (A.Juss.)) were grounded in a grinder to make

into a fine powder. These organic materials separately mixed with soil @ 20

g/kg soil. The pots were watered after treatment to ensure proper

decomposition of organic matter then seeds were sown in these pots and at

three leaf stage 2000 freshly hatched immature females of Rotylenhulus

reniformis were inoculatd on it. Untreated and uninoculated plants served as

control. Three replicates for each treatment was taken.

41

Management of Rotylenchulus reniformis by using fungal bioagent,

bacterial bioagent and biofertilizer (VAM) *

Bioagents (Paecilomyces lilacinus, Trichoderma harzianum, Trichoderma

virens {Gliocladium virens), Trichoderma viride, Pseudomonas fluorescens and

Bacillus subtilis (isolated from the soil) and AM fungi {Glomus fasciculatum)

obtained from division of Microbiology lARI, New Delhi) for the management

oi Rotylenchulus reniformis on cowpea. These bioagents and biofertilizer were

thoroughly mixed with the soil separately @ 1 g (2.4x10^ spores/g), 10 ml

(6x10*' CFU/ml) and 10 g (VAM) per kg soil, respectively (100 spores/g of

inoculum).

RECORDING OF DATA

Plant growth determination:

Plants were uprooted after 60 days of inoculation and roots were washed

thoroughly in slow running tap water. Utmost care was taken to avoid loss and

injury of root system during the entire operation. For measuring length and

weight, the plants were cut with a sharp knife just above the base of root

emergence zone. The length of shoots and roots were recorded in centimeters

from the cut end to the tip of first leaf and the longest root respectively. The

excess water was removed by putting the plant parts between the folds of the

blotting sheets, for sometime before taking their fresh weight. For measuring

dry weight, the shoot and root were kept in envelops separately for drying in an

oven running at 80°C for 24 hours and the weight was recorded in grams. For

42

interpretation of results, the reduction in plant growth was calculated in terms

of percentage dry weight reduction.

Nematode population estimation:

For extraction of nematodes, the soil from the pot of each treatment was

mixed thoroughly and a sub-sample of 200 gm soil was processed through

sieves according to Cobb's decanting and sieving method followed by

Baermann funnel technique. The nematode suspension was collected in a

beaker and volume made up to 100 ml. For proper distribution of nematodes,

the suspension was bubbled with the help of pipette and 2 ml suspension from

each sample was drawn and transferred to a counting dish. The number of

nematodes were counted in three replicates for each sample. Mean of three

such countings was calculated and the final population of nematodes per kg soil

was determined.

To estimate the nematode population in roots, 1.0 g root stained in

tryphanblue from each replicate and placed under the stereoscopic microscope.

The roots were teased with the help of needles and the number of females were

counted.

Reproduction factor (Rf) : of reniform nematode was calculated by the

formula Rf = pfi pi where 'pf represented the final and 'pi' initial population of

the nematode.

Statistical Analysis

The data obtained were analyzed statistically and significance of variance

was calculated at P = 0.05 and P = 0.01 levels of probability.

f\e6uu6

CHAPTER-4

RESULTS

The data presented in table 1.1 reveals that the inoculation of cowpea

seedlings with Rotylenchulus reniformis caused significant reduction in plant

growth characters viz., plant length, plant fresh weight, plant dry weight,

nodules per plant and pods per plant as compared to untreated uninoculated

plants. The individual application of fungal biocontrol agents viz., Trichoderma

harzianum, Trichoderma viride, Trichoderma virens and 2 isolates of

Paecilomyces lilacinus viz. (PU and P12) showed significant increase in plant

growth parameters as compared to control, however, the plants inoculated with

both isolates of Paecilomyces lilacinus did not significantly affect the plant

growth parameters as against control. The maximum increase in the length and

dry weight of plant was observed in the soil treated with Trichoderma

harzianum followed by T. viride and T virens. Similarly, the highest number

of nodules and pods per plants was also recorded in T. harzianum followed by

T. viride, T. virens, Paecilomyces lilacinus (PU and P12) respectively.

The application of fungal biocontrol agents in presence of Rotylenchulus

reniformis resulted the control of nematodes to a varying degree. The highest

improvement in growth parameters viz., length, fresh and dry weight and yield

was recorded in the plants treated with T harzianum followed by T. viride, T.

virens. Paecilomyces lilacinus (Pll) and P. lilacinus (P12) as compared to

untreated and inoculated plants.

44

In the corresponding treatments, the shoot length of plant was recorded

by 32.90, 31.60, 30.10, 24.50, 24.10 cm as against 22.40 cm, root length was

21.40,18.30,17.80,16.90,16.60 cm as against 14.20 cm, total plant length was

54.30, 49.90, 47.90, 41.40, 40.70 as against 36.60, fresh shoot weight was

66.20, 62.60, 60.90, 56.30, 56.00 as against 44.60 gm; fresh root weight was

16.80, 13.80, 13.20, 12.10, 11.80 gm as against 11.10 gm; total plant fresh

weight was 83.00, 76.40, 74.10, 68.40, 67.80 as against 55.70 gm; dry shoot

weight as 36.60, 33.80, 33.20, 31.30, 31.00 as against 26.30 gm; dry root

weight was 11.50, 10.30, 10.10, 7.7, 7.3 as against 5.6 gm; and total plant dry

weight was 48.10, 44.10, 43.30, 39.00, 38.30 as against 31.90 gm and number

of nodules was 39, 36, 33, 26, 25 as agamst 31 and number of pods was

observed as 28, 24, 213, 20, 19 as against 17 (uninoculated control), m the test

plants inoculated with only biocontroUing agents viz., Trichoderma harzianum,

T. viride, T. virens, Paecilomyces lilacinus (PI 1) and (P12) as against control

(uninoculated) plants respectively.

When test plants were inoculated with above biocontrol agents and with

2000 immature females of Rotylenchulus reniformis simultaneously, the shoot

length of plant was observed as 19.00, 17.50, 16.90, 15.20, 15.00 as against

13.10 cm, root length was 13.10, 11.70, 11.00, 10.50, 10.20 as against 7.20 cm

and total plant length was 31.10, 29.20, 27.90, 25.70, 25.20 as against 20.30

cm; shoot fresh weight was 39.60, 35.20, 33.60, 29.60, 29.20 as against 25.30,

fresh root weight was 8.30, 7.0, 6.60, 6.10, 5.90 as against 4.80 and total plant

fresh weight was 47.90, 42.30, 40.20, 35.70, 35.10 as against 30.10 gm; plant

shoot dry weight was 21.30, 17.30, 16.60, 14.50, 14.10 as against 12.60 gm,

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root dry weight was 4.70, 3.80, 3.60, 3.30, 3.10 as against 2.5 gm and total

plant dry weight was 26.00, 21.10, 20.20, 17.80, 17.20 as against 15.10 gm;

number of nodules per plant was 25, 19, 16 and 13 as against 12 and number of

pod per plant was 13, 11, 10, 9, 9 as against 7 in inoculated control

respectively.

The percentage reduction in plant length was recorded as 15.03, 20.22,

23.77, 29.78 and 31.15, plant fresh weight was as 14.0, 24.06, 27.83, 35.91 and

36.98 and dry weight was 18.50, 33.86, 36.68, 44.20 and 46.08 and in number

of pods was 23.53, 35.29, 41.18, 47.06 and 47.06 over control respectively.

However, in the absence of biocontrol agents reduction in plant length, fresh

weight, dry weight and number of pods was 44.54, 45.96, 52.66 and 58.82%

over confrol (untreated, uninoculated) respectively.

The number of eggmasses/root system and multiplication of

Rotylenchulus reniformis was also significantly reduced by the application of

fungal biocontrol agents {Trichoderma harzianum, Paecilomyces lilacinus, T.

viride and T. virens) as compared to untreated and inoculated plants. The

highest reduction in reproduction factor was observed in the plants treated with

T. harzianum followed by Paecilomyces lilacinus (Pll and P12), T. viride and

T. virens.

In the corresponding treatments the reproduction factor of nematodes

was recorded as 1.58, 1.94, 2.03, 2.99 and 3.04 as against 4.37 and average

number of egg masses per root system were observed as 68, 70, 73, 84 and 86

46

as against 106 and average number of eggs per mass were 42,48, 52, 53 and 54

as against 64 over inoculated control respectively (Table 1.2).

The data presented in table 2.1 clearly reveals that inoculation of

cowpea seedlings with Rotylenchulus reniformis caused significant reduction in

plant growth parameters as compared to untreated uninoculated plants

(control). The application of biofertilizer (VAM) and bacterial bioagents viz.,

two isolates of Pseudomonas fluorescens (Pfl and Pf2) and two isolates of

Bacillus subtilis (Bstl and Bst2) showed significant increase in plant growth

parameters as compared to control. The highest improvement in plant growth

parameters viz. length, fi-esh weight, dry weight and number of nodules and

pods per plant was observed in plants treated with VAM {Glomus

fasciculatum) followed by Pseudomonas fluorescens 1 (Pfl), P. fluorescens 2

(Pf2), Bacillus subtilis (Bstl) and (Bst2) significantly reduced the damage in

terms of plant growth parameters viz. length, fi-esh weight, dry weight and

number of nodules and pods per plant caused by Rotylenchulus reniformis as

compared to those plants inoculated with Rotylenchulus reniformis only. The

highest improvement in plant growth parameters was recorded in the plants

treated with biofertilizer (Glomus fasciculatum) followed by bacterial bio-agent

(Pfl, Pf2, Bstl and Bst2) respectively.

In the corresponding treatments, the shoot length of plant was recorded

by 33.40, 32.60, 21.70, 26.70, 26.50 as against 22.40 cm; root length was

22.50, 21.60, 19.60, 17.50, 17.40 as against 14.20 and total plant length was

55.90, 54.20, 51.30, 44.20, 43.90 as against 36.90 cm the shoot fi-esh weight

was 67.80, 66.10, 64.30, 58.50, 56.40 as against 44.60 gm, fresh root weight

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was 17.60, 16.70, 14.20,12.80, 11.70 as against 11.10 gm and total plant fresh

weight was 85.40, 82.80, 78.50, 71.30, 68.10 as against 55.70 gm; shoot dry

weight was 35.20, 35.10, 34.70, 31.10, 30.70 as against 26.30 gm; root dry

weight 14.30, 14.20,12.60, 10.50, 9.80 a against 5.60 gm, and total dry weight

was 49.50, 49.30, 47.30, 41.60, 40.50 as against 31.90 gm in the test plant

inoculated with only biocontroUing agents viz. VAM {Glomus fasciculatum),

two isolates of Pseudomonas fluorescens i.e. (Pfl and Pf2), and two isolates of

Bacillus subtilis i.e. (Bstl and Bst2) respectively and number of nodules as 44,

34, 42, 29, 28 as against 31 and number of pods was 32, 27, 27, 22, 21 as

against 17 (untreated and uninocualted control).

When test plant inoculated with 2000 immature females Rotylenchulus

reniformis with bioagents simultaneously, the shoot length of plant was

observed as 19.90, 18.90,18.10,16.10,15.80 as against 13.10; root length was

13.30, 11.10, 12.30, 10.80, 10.30 as against 7.2 cm and total plant length was

33.20, 30.40, 30.00, 26.90, 26.10 as against 20.30 cm, shoot fresh weight was

41.80, 39.10, 38.50, 31.50, 31.60 as against 28.80 gm, root fresh weight was

8.5, 8.3, 7.6, 6.3, 6.0 as against 5.6 gm and total plant fresh weight was 50.30,

47.30,46.10,37.80 and 37.60 as against 30.10 gm, shoot dry weight was 22.80,

19.70,19.30,15.10,14.90 as against 12.60, root dry weight was 4.90,4.50,4.0,

3.6, 3.4 as against 2.5 and total plant dry weight was 27.70, 24.20, 23.30,

18.70,18.30 as against 15.10 gm (inoculated confrol).

The number of nodules per plant was 28, 23, 21, 17, 16 as against 12

and number of pod per plant was 14. 12, 11, 9, 9 as against 7 (inoculated

control), the percentage reduction in length was recorded as 9.28,16.93, 18.03,

48

26.50 and 28.68, fresh weight as 9.69, 15.08, 17.24, 32.14 and 32.50 and in

number of pods 17.65, 28.82, 35.29, 47.06 and 47.06. In the absence of VAM

(Glomus fasciculatum) and bacterial bioagents reduction in length, fresh

weight, dry weight and number of pods was found to be as 44.54, 45.96, 52.66

and 58.82%, respectively in the plants inoculated with R. reniformis.

The number of eggmasses per root system and multiplication of R.

reniformis was also significantly reduced by the application of biofertilizer

(VAM) and bacterial bioagents viz. {Pseudomonas fluorescens (Pfl) and P.

fluorescens (Pf2), and Bacillus subtilis (Bstl and Bst2) as compared to

untreated and uninoculated plants. The highest reduction in reproduction factor

was observed in the plants treated Glomus fasciculatum followed by P.

fluorescens 1, P. fluorescens 2, Bacillus subtilis 1 and B. substilis 2

respectively. In the corresponding treatments the reproduction factor of

nematodes was recorded as 1.62, 1.71, 1.72, 2.08 and 2.16 as against 4.37 and

number of egg masses per root system observed as 62, 66, 67, 84 and 86 as

against 106, and number of eggs per egg mass observed as 40, 42, 42, 52 and

55 as against 64 over inoculated control (Table 2.2).

The data presented in table 3.1 clearly reveals that the inoculation of

cowpea seedlings with R. reniformis caused significant reduction in plant

growth characters viz. plant length, fresh weight, dry weight, number of

nodules and pods as compared to untreated uninoculated plants. The individual

application of neem cake (Azadirachta indica) and leaves of some plants viz.

fresh Azolla {Azolla pinnata), datura {Datura stramonium), vmca rosea

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49

iCatharanthus roseus), Mudar (Calotropis procera), marigold (Tagetes erecta),

lantana (L camara) and asthma weed {Euphorbia hitra) significantly improved

the plant growth parameters as compared to control.

The maximum increase in plant length, fl-esh weight dry weight, number

of nodules and pods was observed in plants treated with firesh Azola (Azolla

pinnata), followed by neem cake {A. indica). Datura stramonium,

Catharanthus roseus, Calotropis procera, Tagetes erecta, Lantana camara and

Euphorbia hirta. The significant improvement in plant growth parameters was

observed in the plant growing in the soil amended with leaves of Azolla

pinnata, neem cake, D. stramonium, C. roseus, C. procera, T. erecta, L

camara and E. hirta and inoculated with R. reniformis as compared to

unamended and inoculated plants.

The highest improvement in plant growth parameters viz. plant length,

fi-esh weight, dry weight, number of nodules and pods was recorded in the

plants treated with leaves of fi-esh Azolla followed by neem cake {A. indica),

Datura stramonium, Catheranthus roseus, Calotropis procera, Tagetes erecta,

L. camara and E. hirta. In the corresponding treatments, the shoot length of

plant was recorded by 30.80, 3060, 29.90, 28.60, 2850, 28.30, 27.90, 26.0 as

against 1.10 cm, root length of plant was 20.8, 20.7, 20.1, 19.5, 19.5, 19.4,

18.7,17.8 as against 15.5 and total plant length was 51.6, 51.3, 50.0,48.1,48.0,

47.7, 46.6, 43.8 as against 34.6 cm, shoot fi-esh weight was 65.8, 65.6, 64.5,

63.8, 62.0, 61.6, 61.2, 60.3 as against 44.0; root fi-esh weight was 16.7, 16.4,

15.8, 15.1, 14.8, 14.6, 13.9, 11.8 as against 11.1 and total plant fresh weight

50

was 82.5, 82.0, 80.3, 78.9, 76.8, 76.2, 75.1, 72.1 as against 55.1 gm and shoot

dry weight was 38.1, 37.9, 37.3, 36.8, 36.5, 36.4, 36.1, 34.6 as against 26.1 gm,

root dry weight was 9.1, 9.0, 8.8, 5.7, 5.6, 5.4, 5.1, 4.6 as against 5.3 and total

plant dry weight was 47.2, 46.9, 46.1, 42.5, 321, 41.8, 41.2, 39.2 as against

31.4 gm and number of nodules per plant was 41, 38, 36, 32, 29, 28, 28, 27 as

against 30 and number of pods was 32, 30, 28, 27, 25, 23, 22, 21 as against 18

(uninoculated control) in the test plant inoculated with only biocontroUing

agents viz. fresh AzoUa, neem cake, datura, Vinca rosea, aak, marigold, lantana

and Asthma weed (Euphorbia hirta) respectively.

When tested plant inoculated with 2000 immature females of

Rotylenchulus reniformis and bioagents simultaneously, the shoot length of

plant was observed 19.20, 18.50, 18.10, 17.80, 17.20, 16.80, 15.10, 15.10 as

against 19.10 cm, root length was 12.7, 11.0, 9.8, 9.3, 8.0, 7.8, 7.2, 7.0 as

against 15.5 cm, and total plant length was 29.9, 29.5, 27.9, 27.1, 25.2, 24.6,

22.3, 22.1 as against 34.6 cm, shoot fresh weight was 38.4, 38.3, 37.5, 37.0,

36.6, 36.3, 34.9, 33.3 as against 44.0 gm root fresh weight was 7.4, 7.2, 7.0,

6.9, 6.6, 6.3, 6.2 and 5.9 as against 11.1 and total plant fresh weight was 45.8,

45.5,44.5,43.9,43.2,42.6,41.1, 39.2 as against 55.1 gm shoot dry weight was

20.5, 20.3, 20.1,19.8, 19.6, 19.1,18.7,18.2 as against 26.1 gm, root dry weight

was 4.8, 4.8, 4.6, 4.4, 4.3, 3.1, 3.0, 2.9 as against 5.3 gm and total plant dry

weight was 25.3, 25.1, 24.7, 24.2, 23.9, 22.2, 21.7, 21.1 as against 31.4 gm and

number of nodules per plant was 25,23,21,19,18, 18,16, 15 as against 12 and

number of pods was observed as 15, 14, 13, 13, 12, 11, 11, 10 as against 8

(inoculated control).

51

The percentage reduction in length was recorded as 13.58, 14.74, 19.36,

21.68, 27.17, 28.90 and 35.55, in fresh weight as 16.88, 17.42, 19.24, 20.33,

21.60, 22.69 and 25.41, in dry weight as 19.43, 20.06, 21.34, 22.93, 23.89,

29.30, 30.89 and number of pods per plant observed as 16.67, 22.22, 27.78,

27.78, 33.33, 38.89 and 38.89. However, in the absence of botanicals, reduction

in length, fresh weight, dry weight and number of pods was recorded as 43.64,

46.10, 52.87 and 55.56%, respectively in the plants infected with Rotylenchulus

reniformis.

Similarly, the multiplication of reniform nematode, Rotylenchulus

reniformis was reduced by the application of leaves of Azolla pinnata, neem

cake {A. indica), Datura stramonium, Calotropis procera, Tagetets erecta,

Lantana camara and Euphorbia hirta. The greatest reduction in reproduction

factor was observed in A. pinnata followed by neem cake, D. stramonium,

Calotropis procera, T. erecta, L camara and E. hirta. In the corresponding

treatments the reproduction factor was recorded as 1.93, 1.96, 2.09, 2.35, 2.46,

2.60, 3.04 and 3.10 and number of egg masses per root system recorded as 58,

60, 69, 72, 76, 78, 82 and 88 and number of eggs per egg mass observed as 54,

56, 58, 65, 65, 66, 70, 71, 79 (Table 3.2).

2> Ldcuddvon

CHAPTER- 5

DISCUSSION

According to Stirling (1991) biological control is "A reduction of

nematode populations which is accomplished through the action of living

organisms other than the nematode resistant host plant, which occurs naturally

or through the manipulation of the environment or the introduction of

antagonists". The main aim of biological control is to increase the natural

enemies of nematodes in the soil so as to reduce nematode density.

Microorganisms that can grow in the rhizosphere provide the frontline defence

for roots against pathogens attack and are ideal for use as biological controlling

agents (Weller, 1988). According to Kim and Riggs (1992), the characteristics

of good biological control agents are: (i) highly parasitic to nematodes, (ii) host

range primarily nematode spp. but not pathogenic to crop plants or higher

animals, (iii) grow at suitable pH and temperature ranges, (iv) grow on artificial

media, (v) indentifiable and with mode of action known, (vi) can be formulated

in usable form, competition with other soil microorganisms should be

considered.

Successfiil biological control is achieved with little effort. Bioagents in

addition to being cheap and effective are the safest in terms of environmental

consideration and want relatively less technical skill for their application. Fungi

possess a number of characteristics that make them potentially ideal biocontrol

agent i.e. saprophytic species antagonize all pest organisms, plant pathogens,

weeds and insects, grown readily in cultures so that large quantities can be

53

economically produced. Besides fiingi many bacteria and actinomycetes have

also been identified and are in use, but fungal antagonists take a major share.

In our studies application of fungal biocontrol agents viz. Trichoderma

harzianum, T. viride, T. virens and two isolates of Paecilomyces lilacinus

significantly reduced the reproduction factor, number of eggs and females

which consequently improved the plant growth parameters viz., plant length,

dry weight, fresh weight, number of nodules and pods in the plants inoculated

with Rotylenchulus reniformis to a varying degree. It is clear from the results

given in Table (1.1) that the antagonistic fungi {Trichoderma harzianum) was

best among all the biocontroUing agents and T. virens was less effective on

nematode management.

It has been reported that Trichoderma is a biocontrol agent, antagonistic

to several soil inhibiting fungi, bacteria, nematodes etc. (Pathak et al, 2007).

The mycelium of Trichoderma quickly grew towards the pathogen, entangles it

or curls around it and penetrates inside the pathogen by its haustoria/appresoria

and cause lysis. The lysis resulted by extracellular secretion of lytic enzymes

(B-1, 3 glucanase, chitinase, protease, lipase etc.) by Trichoderma spp. Once

Trichoderma is introduced in the soil either with treated seeds or mixed with

farmyard manure it grows so rapidly consuming essential nutrients from soil

that the other soil inhibiting microorganisms fail to compete with the antagonist

and finally die due to scarcity of food and space. The antagonist is also known

to release alkaloids like alkylpyrones, isonitriles, polyketides, peptiabols,

deketopiperzones, sesquiterpenes, steroids etc. which are harmful to pathogen

54

and therefore inhibits the growth of pathogen (Elad et al, 1982; Lorito et al.,

1993), such mechanism is called antibiosis. Some antibiotics produced by

Trichoderma spp. are identified as Trichodermin, Gliovirin, viridol and

Gliotoxins. Trichoderma spp. are also known to solubilise rock phosphate,

metallic zinc, manganese, iron and copper and also enhance nitrogen efficiency

in plants. Because of these properties, Trichoderma spp. not only become a

bioagent for disease control but also an agent which enhance the growth of

plants by providing soil nutrition in soluble forms for absorption by the roots

resulting in better health of plants (Pathak et al, 2007). These results are also

in agreement with those of Mahmood and Siddiqui (1993), Senthamizh and

Rajendran (2002), Visalakshi et al. (2000), Jayakumar et al. (2003), who

reported the use of fungal biocontrol agent viz., Trichoderma spp. and

Paecilomyces lilacinus to control the reniform nematode, Rotylenchulus

reniformis on different plants. These results are also supported by Shukla and

Haseeb (2004) who reviewed the management of reniform nematode by

Trichoderma harzianum. The nematicidal effect of Trichoderma viride against

reniform nematode have been reported by Senthamizh and Rajendran (2002),

Visalakshi et al. (2002), Subramanian and Senthamizh (2006), Sreenivasan and

Sundarababu (2007).

The efficacy of Paecilomyces lilacinus against R. reniformis has been

reported by various workers, Reddy and IChan (1989), Khan and Husain

(1990), Walters and Barker (1994), Khan and Saxena (1996), and Jayakumar et

al. (2002) in brinjal, cowpea, tomato and cotton respectively. Earlier reports

showed that the hatched out juveniles become incapacitated in the presence of

'fVi' ?7 Y^ .• , 55

% . ^ ^ ^ . - / ^

fungal hyphae, indicate a primarily ditfeig::!]^^'effect rendering them

subsequently vulnerable to colonization. It is possible that partial disintegration

of vitelline layer may be due to exoenzyme production by the fungal hyphae,

possibly involving the presence of translocable, physiologically disorganizing

factor, such as diffusible toxic metabolites. This disruption not only

predisposes the egg to fungal infection by physical weakening of the shell, but

also increases permeability thus facilitating inward passage of fungal

metabolites both toxic and enzymatic. This exopathic effect might be enough to

abort the reproductive process. Once a fungal hypha enters an egg, enzymatic

dissolution of the chitic layer takes place (Okafor, 1967). It has been reported

that mycelial proliferation on the nematode body results in probable

biosynthesis of destructive metabolites endogenously. Thereafter, endogenous

mycelial proliferation might support the lysis of egg-shell material. Later,

hyphae penetrate the larval cuticle. This endopathic activity of the fungus

causes total degeneration of the egg contents and leads to the ultimate demise

of the larvae. Paecilomyces lilacinus isolates from silkworm yielded oxalic,

dipicolic and succinic acid and some unidentified aminoacids, as well as large

amounts of D-mannitol (Domsch et al., 1980). These chemicals might also be

responsible for the killing of nematodes and inhibitions of fungal growth.

The application of biofertilizer {Glomus fasciculatum) significantly

increased the plant growth parameters viz. length, fresh weight, dry weight and

number of pods and nodules as compared to untreated uninoculated plants.

These results are also in agreement with those of (Lingaraju and Goswami,

1993, 1995; Sikora and Sitaramaiah, 1995; Jothi and Rajeshwari, 1998) who

56

reported that the application of bioferitlizer (G. fasciculatum) increased plant

growth parameters of several crops as compared to control. The reasons for

enhanced growth is attributed to the growth promoting substances, viz. Indole-

acetic acid, gibberellins and cytokinins produced by G. fasciculatum (Martinez-

Toledo et al., 1988) which enhance root growth and thereby help the plants to

absorb nutrients better which have been reported to elongate stem, enlarge cells

and leaves, expand root system, induce cell division and cause early flowering

and fruiting. The improvement in plant growth in the presence of G.

fasciculatum might be due to improve uptake of nutrients, especially

phosphorus. In addition to phosphorus, G. fasciculatum are known to enhance

uptake of Ca, Cu, S and Zn (Gerdemann, 1968). It also produce plant hormones

and increase the activity of nitrogen fixing organisms.

Sikora and Sitaramaiah (1995) observed that the mycelium of

endomycorrhizal fungus (G. fasciculatum) grew irrespectively through the egg

matrix when the fimgal colonization was extremely heavy. However, the

individual eggs were not colonized by the fungus. The adverse effects on

nematode penetration, development restricted reproduction rate and

multiplication in mycorrhizal roots, could be the net resuh of complex

physiological changes associated with the mycorrhizal colonization of roots.

The individual application of Rhizobacteria (Bacillus subtilis,

Pseudomonas fluorescens) significantly increased the plant growth parameters

viz. length, fresh and dry weight, number of nodules and pods as compared to

untreated-uninoculated control. It is clear from the results given in Table (2.1)

57

that the biofertilizer Glomus fasciculatum was best among all the biocontroUing

agents viz. Pseudomonas fluorescens and Bacilus subtilis. Bacillus subtilis was

less effective on nematode management in plants inoculated with

Rotylenchulus reniformis. These results are in agreement with those of (Ali,

1996; Dalai and Vats, 1998; Jayakumar et al., 2003; Niknam and Dhawan,

2003) who reported that the application of PGPR (plant growth promoting

rhizobacteria) increased plant growth parameters of several crops as compared

to inoculated control. It has been reported that the rhizobacteria are known to

induce reduction in hatching, interference with normal host recognition and

reduce nematode penetration because bacteria envelop or bind the root surface

with carbohydrate lectin (Oostendorp and Sikora, 1989). Although the exact

mechanisms involved in growth promotion are still unclear but various

mechanisms have been suggested to explain the phenomenon of plant growth

promotion, when agronomic crop are inoculated with rhizobacteria. These

include increase in the nitrogen fixation, production of auxins, gibberellins,

cytokinins, ethylene, solubilization of phosphorus, oxidation of sulfur, increase

in nitrate availability, extra cellular producton of antibiotics, lytic enzymes,

hydrocyanic acid, increase in root permeability, strict competition of the

available nutrients at root sites, suppression of deleterious rhizobcteria and

enhancement in the uptake of essential plant nutrients, could be the best

possible explanations (Subba Rao, 1982; Pal et al., 1999; Enebak and Carey,

2000).

Sikora and Hoffinan-Hergarten (1993) suggested two possible

mechanisms of action, responsible for reduction of nematode infection of roots

58

(1) production of metabolites which reduce hatch and attraction and/or (2)

degradation of specific root exudates which control nematode behaviour.

Pseudomonas fluorescens generally acts through direct antagonism to

pathogens through antibiotic production, through competition with the

pathogens for essential nutrients such as iron or more directly through plant

growth promotion (Gamliel and Katan, 1993). Recently, systemic resistance

induced by plant-growth promoting bacteria has also been considered as a

mechanism for biocontrol of pathogens (Wei et al., 1996). Mechanisms for

induced systemic resistance may be due to increase in activity of chitinases,

p-l,3-gluconases, peroxidases and other pathogenesis related proteins (Lawton

and Lamb, 1987), accumulation of phytoalexin (Kuc and Rush, 1985) and

formation of protective biopolymers eg. Lignin, callose and hydroxyproline

rich glycol-proteins (Hammerschmidt et al., 1984).

The individual application of neem cake (Azadirachta indica) and

chopped leaves of Azolla pinnata, Datura stramonium, Catharanthus rosetis,

Calotropis procera, Tagetes erecta, Lantana camara and Euphorbia hirta

significantly increased the plant growth parameters as compared to untreated

and uninoculated plants. It is clear from the results given in Table (3.1) that

fresh Azolla was the best among all the organic amendments and Euphorbia

hirta was less effective on nematode multiplication. These results are also in

agreement with those of Patel et al, 1995; Dalai and Vats, 1998; Anvar and

Alam, 2000; who reported the manurial effect of botanicals which lead to

increase in plant growth parameters in amended soil. These organic additives

improved soil structures and water holding capacity of the soil. These

59

materials also release some nutrients after their decomposition which accelerate

rapid root development and overall plant growth.

The application of neem cake and chopped leaves of A. pinnata, D.

stramonium, C. roseus, Calotropis procera, T. erecta, L. camara and E. hirta

significantly reduced the reproduction factor of reniform nematode which

consequently improved the plant growth parameters in plants inoculated with

Rotylenchulus reniformis. These results are inconformity with those of Mishra

and Padhi, 1985; Patel et ai, 2003; Tiyagi and Ajaz, 2004; Budhram and

Baheti, 2004; Umamaheswari et ai, 2005; who reported that application of

plant parts caused significant reduction in reniform nematode infestation which

consequently lead to increase the growth of different plants. Metabolites of

microbes which are activated by organic amendments, are toxic to nematodes

(Pandey and Singh, 1990). As a result of the application of organic

amendments, plant nutrients are released which accelerate root development

and overall plant growth and thus helping the plants to escape nematode attack.

Plants have an almost limitless ability to synthesize aromatic substances

as secondary metabolites such as phenols, alkaloids, flavonoids, terpenoids and

phytosterol which are produced from carbohydrate, amino acids and

chlorophyll i.e. primary metabolites. These secondary metabolites do not play

any role in growth and development of the plant but they provide protection to

the plants against predators and pathogens (Harbome, 1984; Wink, 1999).

The common occurring chemical in neem e.g. Azadirachtin,

Kaempferol, nimbin, nimbidin, nimbidic acid, querctin and thionemone are

60

thought to be responsible for the management of reniform nematode. Neem-

cake organic manure is the bye-product obtained in the process of cold pressing

of neem fruit and kernels and the solvent extraction process for neem oilcake.

Neemcake organic manure has more nitrogen, phosphorus, potassium, calcium

and magnesium than the farmyard manure. It is rich in sulphur compounds as

well as bitter limonoids. According to research calculations, neem cake seems

to make soil more fertile due to an ingredient that blocks soil bacteria from

converting nifrogenous compounds into nifrogen gas. It is a nitrification

inhibitor and prolongs the availability of nitrogen to both short duration and

long duration crops.

Thiophenes, one of the several compound classes foimd in marigold

show significant antiviral capabilities (Soule, 1993). Infact, 7 of the 10 most

effective antiviral thiophenes are found in Tagetes (Atkinson et al, 1964;

Hudson, 1990). A chemical released by marigold roots called a-terthienyl has

drawn much attention for its nematicidal characteristics. The presence of a-

terthienyl inhibit the hatching of nematode eggs (Siddique and Alam, 1988). In

addition, penefration of and development of Rotylenchulus reniformis was

reduced within T. patula, a poor host (Caswell et al., 1991) and Meloidogyne

spp. juveniles were unable to fully develop in roots of trap crop T. erecta

(Ploeg and Maris, 1999). The suppression of nematodes in amended soil may

be because of the effect of several combined factors. Produciton of volatile

fatty acids, phenols, ammonia, amino acids, HCN etc. during decomposition of

plant products, which may cause inhibitory effect to the nematodes. The

decomposed products may be directly toxic to nematodes or the microbial

61

metabolites produced during decomposition may be toxic to nematodes or

enhance activity of predators and parasites which feed on the nematodes.

The use of botanical possessing the antifeedent properties offers a

harmonious approach to manage the reniform nematode on cowpea. In the

present study while using these botanicals did not observe any phytotoxic

effect on plants.

Antagonist plant also offer basis for finding and developing natural

nematicides and fungicides which will help to maintain a clean environment.

However, it will be desirable to isolate the active principle fi-om the extract of

these plants for testing against some plant disease under field conditions.

^ummaru and C^oncu V onciudion

SUMMARY AND CONCLUSION

The efficacy of fungal biocontrol agents {Trichoderma harzianum,

Trichoderma viride and Trichoderma virens and two isolates of Paecilomyces

lilacinus i.e. PU and P12) were tested for the management of reniform

nematode, Rotylenchulus reniformis on cowpea. The individual application of

biocontrol age.nte {T. harzianum, T. viride wA T. virens) in the absence of

nematode caused significant increase in plant growth parameters as compared

to control. However, the plant inoculated with P. lilacinus did not

significantly affect the plant growth parameter as compared to control.

The simultaneous inoculation of plant with Rotylenchulus reniformis

and either of the biocontrol agents {T. harzianum, T. viride, T. virens and P.

lilacinus (PU), P. lilacinus (P12)) significantly reduced the reproduction

factor, number of eggmasses per root system and number of eggs per egg

mass which consequently improved the plant growth parameters viz. length,

fresh weight, dry weight, number of nodules and pods of cowpea as compared

to those plants inoculated with R. reniformis alone. Among these biocontrol

agents T. harzianum was found to be the most effective in the management of

reniform nematode followed by P. lilacinus (Pll and P12), T. virens and T.

viride.

The individual application of biofertilizer {Glomus fasciculatum) and

bacterial bio-agents (2 isolates of Pseudomonas fluorescens i.e. Pfl and Pf2,

and 2 isolates of Bacillus subtilis i.e. Bstl and Bst2) significantly improved

the plant growth parameters as compared to control.

63

The simultaneous inoculation of plant with Rotylenchulus reniformis

and either of the biocontrol agents (G. fasciculatwn, P. fluorescem 1 and P.

fluorescens 2, Bacillus subtilis 1 and B. subtilis 2) significantly reduced the

reproduction factor, number of egg masses per root system and number of

eggs per egg mass which consequently improved the plant growth parameters

as compared to untreated and inoculated plants with Rotylenculus reniformis.

Among the biofertilizer (G. fasciculatvm) and all the bacterial bioagents, G.

fasciculatum was found to be the most effective in the management of

reniform nematode, Rotylenchulus reniformis.

The individual application of neem cake and leaves of some plants

(fresh Azolla, Datura stramonium, Catheranthus roseus, Calotropis procera,

Tagetes erecta, Lantana camara and Euphorbia hirta) significantly improved

the plant growth parameters as compared to control. The simultaneous

inoculation of plants with R. reniformis and amendment of either of the leaves

of some plants viz. fresh Azolla, Datura stramonium, Catheranthus roseus,

Calotropis procera, Tagetes erecta, Lantana camara and Euphorbia hirta

significantly improved the plant growth parameters (length, fresh and dry

weight and number of nodules and pods) and reduced the reproduction factor,

number of eggmass per root system, number of eggs per egg mass as

compared to untreated inoculated plant. Among these amendments the leaves

of Azolla pinnata was found to be the most effective in the management of/?.

reniformis followed by neem cake {A. indica), Datura stramonium,

Calotropis procera, Tagetes erecta, Lantana camara and Euphorbia hirta.

64

The present investigations showed that fungal biocontrol agents (T.

harzianum, T. viride, T. virens, Paecilomyces lilacintds), biofertilizer (G.

fasciculatum), bacterial bioagents {Pseudomonas fliiorescens, Bacillus

subtilis) and certain botanicals viz., fresh Azolla, neem cake, D. stramonium,

C. procera, T. erecta, L camara and E. hirta significantly reduced the

reproduction factor, number of egg masses and eggs which consequently

increased the plant growth parameters of cowpea inoculated with

Rotylenchulus reniformis and among all the treatments, VAM {Glomus

fasciulatum) was most effective.

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