iMastter of ^fiilogopl)? JSotanp · growth of bottle gourd cv. 'Kanchan' in pots Table 12 a&b...

studies on the Integrated Management of Meloidogynejavanica (Treub) Chitwood

infecting Bottle gourd, Lagenaria siceraria (Molina) Standi.

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

iMastter of ^fiilogopl)?

in JSotanp

BY

OCavita TarlKar

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2010

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ff if if if ff if if if If if if if if if If

Dedicated to

My

Loving Grand Parents

i - ^ fjOfc i-^l* f « h »•<* I-*!* »••• !•«» f ^ f j * ^<*• ^«l^ f«fc- Mfc ^5I^ •••fc 1.4111.1.4I1. ,.4tt^ ^4lt ^4«|. |.4iii. ^.m^. ^4«t. •.4«^

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if ff if if

(Dr, MansoorA* Sidifiqui M.Sc, M.Phil., Ph.D , D.Sc, FPSI, FNSI, FBS, FBRS

Professor

Plant Pathology and Nematology Laboratories DEPARTMENT OF BOTANY Aligarh Muslim University Aligarh - 202 002 (U.P.). India Off.. 0571-2702016. Res: 0571-2409561 Mob:+91-9411414895 E-mail: [email protected]

mansoor_t)[email protected]. in

Daied-MJl^.Xpl^

^BvixiltntB

This is to certify that the dissertation "Studies on the Integrated

Management of Melokhgyne javanica (Treub) Chitwood infecting

Bottle gourd, Lagenaria siceraria (Molina) Standi." embodied a record

of bonafied research work carried out by Miss. Kavita Parihar at the

Department of Botany, Aligarh Muslim University, Aligarh under my

guidance and research supervision and that no part of it has been

submitted for the award of any other degree or diploma. She is allowed to

submit her dissertation to the Aligarh Muslim University, Aligarh for the

consideration of the award of the degree of Master of Philosophy in

Botany.

Prof. MansoorA. Stddiqui (Research Supervisor)

Resi.: H. No. 4/905 Gl, Ek Miner Colony, Dodhpur, Aligarh - 202 002 (U.P.V INniA

mailto:[email protected]

mailto:[email protected]

ACKNOWLEDGEMENT

Thanks to Almighty "God" the most beneficent and merciful.

I ejq)ress my immense gratitude and sincere thanfis to- my respected

supervisor (Prof. Mansoor Jlfimad Siddiqui, (Department of (Botany, A^'O,

JiCigarfi. Out of his Susy scheduk fie gave me RSerty to encroacd upon fiis

vaCuaSle time to complete my worli. I wish to express my heartiest feeCings

and deepest sense of gratitude for his f{een interest, enthusiastic support,

vaCuaSk guidance, suggestions and constant supermsion.

I feet immense pleasure to express my gratitude to (Prof Jirif Inam,

Chairman, (Department of (Botany, JUMl), Jifigarh for providing aCC the

necessaryfaciCities.

With ad regards, I acf{nowledge the co-operation and heCp rendered 6y the

teaching and non-teaching staff of the department.

I express my sincere thanf{s to my seniors Or. 9^ohd. JLzhar (Bother, (Dr

(paheem Ahmad, (Dr. Moin, Miss, geetesh (Baghet andMr (Rgyees Mtnad

who were aCways hefpfufto me during the course of my wor^

I am thanfifuCto my Aunt Mrs. (Rsnu Sharma without her hefp and support

I couCdnot initiate my experiment.

(Diction is not enough to express my thanfis to my friends fNefia, SHafieen,

Zarafshan, <bushra, Vijay <Pratap, !Nitin, ^ijay <Hgj, Sarah, ^Farah, !Nazia,

Anju^h Saifudcfin, Jifshan, Saima, Asim, Vsman A- and all my nears and

dears for their hefp, constant encouragement and moraf support.

I wish to egress my deep gratitude, Cove and respect to my parents, my sister

Shaitesh and 6rothers 1(aushaf and(Prattl{^-who have afways Seen a constant

source of inspiration to me and whose prayers, affection and Cove made me to

reach this stage.

LastCy, I -wish to place in records my thanl{s to Mr. SaCman.Hamidfor his

ey^ellent typing and formatting the dissertation with meticulous care and

devotion.

> ^ - ^ ^ -

Kavita Parihar

CONTENT

Page No.

Chapter 1: Introduction 1-12

Chapter 2: Review of Literature 13-22

Chapters: Materials and Methods 23-32

Chapter 4: Results 33-55

Chapter 5: Discussion 56-58

Chapter 6: Summary , 59-60

References 61-87

LIST OF TABLES

Table 1 Effect of water extracts of leaves of different plant species on the

hatching oiMeloidogyne javanica in vitro

Table 2 Effect of water extracts of flowers and fruits of different plant

species on the hatching of Meloidogyne javanica in vitro

Table 3 Effect of water extracts of leaves of different latex bearing plants

species on the hatching of Meloidogyne javanica in vitro

Table 4 Effect of water extracts of leaf,flower and root of Catharanthus

roseus and Zinnia elegans on the hatching of Meloidogyne

javanica in vitro

Table 5 Effect of water extracts of leaves of different plant species on

mortality of the root-knot nematode, Meloidogyne javanica in

vitro

Table 6 Effect of water extracts of flower and fruit of different plant

species on mortality of the root-knot nematode, Meloidogyne

javanica in vitro

Table 7 Effect of water extracts of leaves of different latex bearing plants

species on mortality of the root-knot nematode, Meloidogyne

javanica in vitro

Table 8 Effect of water extracts of leaf, flower and root of Catharanthus

roseus and Zinnia elegans on mortality of the root-knot nematode,

Meloidogyne javanica in vitro

Table 9a & b Effect of seed dressing with latex of different plant species on the

root-knot development caused by Meloidogyne javanica and plant

growth of bottle gourd cv. 'Kanchan' in pots

Table 10 a & b Effect of chopped leaves of latex bearing plants on the root-knot

development caused by Meloidogyne javanica and plant growth

of bottle gourd cv. 'Kanchan' in pots

Table 11 a & b Effect of Chopped leaves of different plant species on the root-

knot development caused by Meloidogyne javanica and plant

growth of bottle gourd cv. 'Kanchan' in pots

Table 12 a & b Effect of soil amendment with urea coated with different

commercial Neem products on the root-knot development caused

by Meloidogyne javanica and plant growth of bottle gourd cv.

'Kanchan' in pots

Table 13 a & b Effect of oilcakes (undecomposed), nematicide and biocontrol

agents on the root-knot development caused by Meloidogyne

javanica and plant growth of bottle gourd cv. 'Kanchan' in pots

Table 14 a & b Effect of oilcakes (decomposed), nematicide and biocontrol agents

on the root-knot development caused by Meloidogyne javanica

and plant growth of bottle gourd cv. 'Kanchan' in pots

IT 1

Chapter 1 Introduction

_j

CHAPTER!

INTRODUCTION

The root-knot nematode, Meloidogyne spp. are major agricultural pests of a

wide range of crops more than 3000 hosts species including monocots, dicots and

several weed species (Abad et a/.,2003). The root-knot nematode is sedentary

endo-parasite. Numerous females of the root-knot nematodes are present in root

galls. These survive in the soil and leaf debris. The primary infection is initiated

by eggmass that persists in the soil and from which second stage juveniles (J2)

hatch. The genus comprises of more than 90 described species with some species

having several races of Meloidogyne but the four most common occurring species

are M.incognita (Kofoid and White, 1919) Chitwood 1949, M.javanica (Treub,

1885) Chitwood 1949, M.hapla (Chitwood, 1949) and M arenaria (Neal, 1889)

Chitwood 1949 (Karsen, 2000; Hunt et al., 2005).These species are distributed

worldwide (Karsen and Van Hoenselaar, 1998).

The root-knot nematode larvae infected plant roots, drain the plant's

photosynthate and nutrients (Ashworth, 1991).Infection caused by root-knot

nematode in young plants may be lethal, while infection in mature plants cause

decreased yield, but high level of root-knot nematode damage can lead to crop-

loss. Symptoms of nematode damaged plants generally non-specific and are

characterized by poor-growth, plant stunting, crop patchiness, wilting and

chlorosis (yellowing and discoloration of leaves).Root-knot infected plants are not

weakened, but their root systems are more susceptible to secondary mfection

caused by fungi and bacteria.

Newly hatch juveniles have a short free-living stage in the soil in the

rhizosphere of the host plant. They may reinvade the host plant of their parent or

migrate through the soil to find a new host root. Second stage juveniles (J2) do not

feed during the free living stage, but use lipids stored in the gut (Eisenback and

Triantaphyllou, 1991). They invade in the root elongation region and migrate in

the root until they became sedentary. Signals from the second stage juveniles

promote parenchyma cells near the head of the juvenile to became multinucleate

(Hussey and Grundler, 1998) to form feeding cells, generally known as "giant

cells", from which the second stage juveniles (J2) and later they adults feed

(Sijmons et al., 1994).Concomitant with giant cell formation, the surrounding root

tissue give rise to a gall in which the developing juveniles are embedded. Juveniles

first feed from the giant cells about 24 hours after they become sedentary. After

ftirther feeding the J2 undergo morphological changes and become saccate

Without further feeding they moult three times and eventually become adults. In

females, which are close to spherical, feeding become stopped and the

reproductive system develops (Eisenback and Triantaphyllou, 1991).The life span

of an adult female may extend to three months and many hundreds of eggs can be

produced. M. javanica develops in a temperature range of 13-34°C with optimal

temperature is about 29°C for the development of adult nematode (Dropkin, 1980).

The root-knot nematode M. javanica females lay eggs into a gelatinous

matrix (GM) which is produced by six rectal glands and secreted before and

during egg laying (Maggenti and Allen, 1960). The matrix, providing a barrier to

water loss by maintaining a high moisture level around the eggs (Wallace,

1968). As the gelatinous matrix ages, it becomes tanned and shrinked turning from

a sticky colorless jelly to an orange brown substance which appears layered.

The root-knot nematode Meloidogyne species cause most extensive damage

on Solanaceous and Cucurbitaceous plants. Among the cucurbits, cucumber and

bottle gourd were found severely infected crops. As parasites, nematodes affect

plant growth and consequently productivity by disrupting physiological processes

ofthe host plant.

Host Description.

Lagenaha siceraria (Molina) Standi, cv 'Kanchan' Family Cucurbitaceae, is

popular vegetable grown almost all the year around, particularly in frost-free

areas, it can be cultivated in all kinds of soil, but thrives best in heavily manured

loams. It requires a warm humid climate or plenty of watering and sunshine when

grown during dry weather. Seeds may be sown directly, 4-5 seeds together, m

manured beds or kits 5-6 feet apart. They may be also sown in nursery beds and

seedlings transplanted when they are in 2-3 leaves stage. The stem of bottle gourd

is stout 5 angled and bifid tendrils found either wild or cultivated. Leaves are long

petioled, 5 lobed, flowers large, white solitary, monoecious or dioecious vines are

allowed to trail on the ground or trained over walls, tree or other support. Young

and tender finits are eaten as vegetables, large up to 1.8m long usually bottle or

dumb-bell shaped , almost woody when ripes . The flesh is soft, spongy and

insipid when old seeds are numerous, long white, smooth 1.6-2.0 cm long

horizontally compressed with marginal groove. Analysis of the edible portion of

the fruit gave following value:

• Moisture = 96.3 %

• Protein = 0.2 %

• Fat = 0 .1%

• Carbohydrates = 2.9 %

• Mineral matters = 0.5 %

• Calcium = 0.02 %

• Phosphorus < 0.01 %.

The fruit is a good source of B vitamins and a fair source of Ascorbic acid. Edible

portion of fruit contains:

• Thiamine =44 |ig/100g

• Riboflavin = 23 [ig /I OOg

• Niacin = 0.33 mg /I OOg

• Ascorbic acid = 13 mg /100 g

The pulp of the fruit is considered emetic, purgative, antioxidant,

immunomodulatory and cardiotonic agent. It works as coolant, diuretic, antibilious

(The Wealth of India, Raw Materials Vol. VI: L-M).

Crop losses

The need for food is increasing continuously due to constantly expanding

population; with currently more than 6.4 billion people worldwide (Food and

Agriculture Statistics Global Out look, Statistics division, June 2006). Based on

the Food and Agriculture Organization (FAO) estimates, the world population will

increase to 7.8 biUion by 2025 and around 84 % of these people will live in the

developing countries.

A loss of yield and quality can occur without specific above ground

symptoms. The crop losses are defined as the differences between the attainable

yield and the actual yield (Chiarappa 1971). However, it is difficult to determine

the attainable yield because it depends on many cultural, environment and pest

factors. Actual yield is more precisely measured as that obtained without

protection from any harm caused by various plant pathogens. Reducing yield

losses caused by pathogens of tropical agricultural crops is one measure that can

contribute to increased food production. Damage and yield caused by the

pathogens are on average greater in tropical than in temperate regions because of

greater pathogen diversity, more favorable environmental conditions for pathogen

colonization, development, reproduction and dispersal, technical and financial

resources.

It has been estimated that the global losses due to the root-knot nematode,

M incognita amounts to $78 billion (US dollar) (Chen ei al, 2004). The

production (tonnes) of pumpkins, squash and gourds is 35,00000 in India (FAO

2008). United States Department of Agriculture (USDA) estimated worth US

$372,335,000 loss per annum in 16 crops (Taylor, 1967). Earlier, Feldmesser et

al, (1971) Hutchinson et ai, (1960) and Cairns (1955) reported $ 500 million

annual loss respectively. The annual loss on agriculture has been estimated as US

$100 billion worldwide (Oka et al, 2000a) and recently Chitwood (2003) reported

that plant parasitic nematodes are serious pathogens on most food and fiber crops,

M. incognita alone accounts for 21.91% yield loss and M.javanica up to 40% yield

loss (Jain ? a/., 1986).

The use of chemicals are effective in the management of nematodes but

their hazardous effect on environment, ground water contamination and ill effects

on human health create a necessity to search newer and harmless methods of

nematode control (Chitwood, 2003).

Management of Root-Knot Nematode

Various methods are adopted for the control of nematodes but a system

advocated nowadays i.e. integrated pest management (IPM). The Integrated Pest

Management is a broad ecological approach which aims at keeping the pest

population below economical threshold level by employing all available methods

of pest control such as use of resistant /tolerant varieties of crops, cultural,

mechanical, biological and chemical methods in a compatible manner. The IPM

approach with a main goal of significantly reducing or eliminating the use of

nematicides / pesticides while at the same time managing pest populations at an

acceptable level.

Today integrated pest management is a combination of biological control

with host plant resistant, appropriate farming practices and a minimal use of

nematicides/ pesticides is an important part of agriculture and horticulture world

wide and provide a basis for pest management in future (Hillocks, 2002; Phipps

and Park, 2002; Feder et al, 2004).

The basis of sustainable approach to nematode management will integrate several

tools including the use of cover crops, crop rotations, soil solarization, least toxic

pesticides / nematicides, organic amendments and plant varieties resistant to

nematode damage. When the soil environment is rich in diverse population of

microorganisms then integrated management of nematodes works best. Some

organisms that are associated with well managed crop soil e.g. rhizobacteria and

mycorrhizae may induce systemic host resistance to nematode and some foliar

diseases (Barker and Koenning, 1998).

To maintain healthy environment of soil, the routine application of organic matter

is required .The addition of organic matter in the form of compost or manure will

decrease nematode pest population and associated damage to crops (Akhtar and

Alam, 1993 a,b; McSorely and Gallaher, 1995a,b). This could be a result of

improved soil structure and fertility, alteration of the level of plant resistance,

release of nemato-toxic or increased population of fungal and bacterial parasites

and other nematode antagonistic agents (Bongers and Ferris 1999; Akhtar and

Malik, 2000; Riegal and Noe, 2000).

Now many methods are applied for the management of nematode. That can

be divided into five broad categories viz., chemical, regulatory, physical, cultural

and biological methods.

Chemical Method

Nematicides are usually categorized as fumigants and non-fumigants, two basic

chemicals which can be incorporated into soil during the chemical means of

nematode control. Soil fumigation began to be used extensively since the

discovery of DD mixture (Dichloro-propane-dichloropropene) in 1943 and EDB

(Ethyl-di-bromide) and DBCP (Dibromo-chloro-propane) were formulated as

efficient soil fumigant for nematode management in 1945 and 1954 respectively.

Fumigants required water to activate toxicity in soil, which directly penetrate the

cuticle and reacts with amino acids, oxidizes and nucleophilic sites on protein. As

a result of this many enzymes or metabolic sites are inhibited simultaneously, thus

stopping several vital processes and leading to a rapid death.

Non-fumigants are systemic in nature and grouped into organophosphates

and carbamates. These nematicides directly penetrate nematodes and inhibit

neuromuscular activity of nematodes which reduces their movement, inversion,

feeding and rate of reproduction.

Nematicidal chemicals besides being expensive, non-available when it

needed and photo-toxic (Jesse and Jada, 2004) , also found to be unsafe , i.e.,

highly toxic aldicarb and methyl bromide used to control nematodes have been

detected in ground water (Xaki et al, 1982; Thomas , 1996) having good

systemic activity in plants against nematode (Goswami et ai, 2006). The

incorporation of chemicals in soil lead to the elimination of soil microflora.

Now more emphasis put onto those chemicals which are bio-degradable,

eco-friendly and easily available.

Regulatory Methods

Several attempts have been made to prevent introduction of nematodes into

countries and provinces by means of plant quarantine. Plant quarantine is an

effective and possible method to control plant parasitic nematodes. It is the

process to prevent the introduction or spread of nematodes or disease. This is

achieved by legal restrictions on the movement of commodities for the purpose of

preventives or inhibiting the establishment of nematodes and diseases in areas

where they are not known to occur. There are some other measures which can

prove beneficial in preventing human assisted spread of nematode to uninfested

areas like using certified planting material , cleaning soil from equipments /

working tools before moving between fields , reducing animal movement from

infested to uninfested fields and eliminating important weed host such as crabgrass

and ragweed (Kodira and Wasterdahl, 1995).

Physical methods

In nematode management various methods adopted for eradicating or

reducing the nematode population include heat treatment, steam sterilization,

electrical soil heating, hot water treatment, radiation treatment, ultrasonics,

osmotic pressure process and seed cleaning etc.(Alam and Jairajpuri, 1990).Soil

solarization is a method which entails laying clear plastic sheets over moist soil for

approximately 6-8 weeks, can effectively suppress nematode population. The

combination of various organic amendments (soybean oil cake, groundnut oil

cake, cotton oil cake, Brassica residues), different plant parts with soil solarization

were more effective than the amendment or soil solarization alone in reducing

nematode population and gall indices of M.incognita and M.javanica in pot and

greenhouse (Oka et al, 2007; GamUel and Stapleton, 1993; Siddiqui et al, 1998,

Siddiqui,2005).Steam sterilization of soil is for killing nematode, in which

autoclaved soil used. The suspension of nematode in steam sterilization is similar

to soil solarization.

Though all these practices are effective for infected material like

propagating tuber, shoot or root, but temperature regulation is quite difficult.

Besides that there are some problems for large scale infestations.

Cultural methods

The various cultural measures applied for the management of root-knot

nematode, include means like crop rotation, soil sanitation and flooding which had

been considered as potential and cultural practices. However, they are adaptable in

certain regions. Cultural practices implementation include prevention of spread,

fallowing, selection of appropriate propagating material, field sanitation at the

time of planting, harvesting, deep ploughing, nutrition general care of host crops

(proper irrigation and timely weeding), cropping and manuring etc.

Crop rotation

10

Crop rotation involves growing a crop that is not a host for the nematode

present in the field, before growing a crop i.e. susceptible. The non-host is a

immune crop will cause nematode population to decline, giving the subsequent

crop a chance to establish a good root system. The rotation must be selected

carefully because some nematodes have a very wide host range. Also, crop

rotation is a difficult to use with most perennial crops.

Flooding of the soil for 7-9 months kills nematode by reducing the amount

of oxygen (O2) available for respiration and increasing concentration of naturally

occurring substances like organic acids, methane (CH4) and hydrogen sulfide

(H2S) that are toxic to nematode (Mc.Guidwin, 1993). Flooding work best if soil

and air temperature remain warm.

These cultural control practices have been found to be economically

feasible in reducing disease losses. The growers should properly identify the

disease that limits the production and then use a variety of most appropriate

controls in combination.

Biological methods

According to Garrett (1965) biological control is "Any condition under

which or practice whereby survival or activity of a pathogen is reduced through

the agency of any other living organism (except by man himself), with the result

that there is reduction in incidence of the disease caused by the pathogen".

Biological control of nematode thus aims at increasing the natural enemies

of nematode (parasite and predators of nematode) in the soil to increase the

11

mortality of nematode. Now a days biological method are most acceptable and

widely used to control the nematode population.

Addition of organic amendments contribute to the biological activity in the

soil and stimulate the activity of microbial population of actinomycetes, bacteria

and fungi, elements of which might be antagonistic to nematode (Badra et a I.,

1979; Godoy et al., 1983, Sikora, 1992). These organic amendments also possess

some nematicidal properties (Pandey, 2002; Saxena and Gangopadhyay, 2005;

Jesse et. al., 2006).Some alternative methods are being sought to control the

population of root-knot neihatode by using non chemical methods which are safer

and more compatible among which natural products are of first importance.

Therefore, present studies were under taken in in vitro and glass-house

conditions to evaluate the potential of organic soil amendments and biocontrol

agents, alone and in combination with least use of nematicides, to combat the

harmful impact of chemicals in the effective and rational management of root-knot

nematode M.Javanica infesting bottle gourd cv. 'Kanchan'.

12

r '"I

Chapter 2 Review of Literature

^ }

CHAPTER-2

REVIEW OF LITERATURE

The main goal of the present research work is to observe the nematicidal

and nematostatic properties of agricultural wastes and biocontrol agents which can

be successfully used for the control of crop losses caused by the root-knot

nematodes. The agricultural wastes, antagonistic plants and phytochemicals are

easily available, cheap and biodegradable. These naturally occurring chemicals

offer alternative strategy to the prevalent use of synthetic chemicals. By the

addition of these organic amendments in the soil, ultimately increase microbial

activity in the soil. Application of plant products and biocontrol agents are

ecofriendly and possess no threat to plants and human beings. Therefore a brief

review is summarized here of various agricultural practices being employed for

the management or root-knot nematodes.

Plants are an important source of naturally occurring pesticides. Many

nematicidal compounds viz., alkaloids, fatty acids, diterpenes, glucosinolates,

isothiocynates, phenols, polyacetylenes, sesquiterpenes and thienyls are found in

plants (Gommers, 1981; Chitwood, 2002).

Bello et a I., (2006) reported the inhibitory effect of water extract of seed,

leaf and bark of five plants viz.,Tamarindm indica, Cassia siamea, C. sieberiana,

Isoberlinia doka and Delonix regia against larval hatching of Meloidogyne

incognita.

13

The seed extract of Areca catechu recorded the highest inhibition rate at

0.1% concentration and latex of Carica papaya caused 98.22% inhibition of

hatching at 0.1 and 10% concentration respectively against root-knot nematode

(Saravanpriya et al, 2004; Singh and Dabur, 2004, 2008; Ahmed et al.,2007;

Siddiqui and Alam, 1990, 1991) observed Ihat among other Neem parts viz.,

leaves, bark, Neem kernels proved most effective which did not allow hatching of

Meloidogyne incognita even upto 11 days of exposure period.

Crude extract of ten plants viz., Lycopersicon esculentum,Petroselinum

sativum, Artimesia campestris, Zea mays, Apium graveolens, Melia azedarach,

Asterales chrysanthemum, Adahtoda visca, Diplolaxis herra and eight essential

oils against root-knot nematode M.incognita at different concentrations. Maximum

nematicidal activity was found in oils of Eucalyptus citriodora, E.hybrida and

Osimum basilicum followed by Pelargonium graveolens, Cymbopogon martini,

Mentha arvensis, M.piperata and M.spicata oil respectively were found toxic to

eggmasses of M. javanica root-knot nematode. The maximum mortality (100%)

were found at 500 ppm (Stephan et al., 2001).

In vitro aqueous and chloroform methanol extracts of plants viz., Tagetes

erecta, Parkin javanica, Clerodendrun indicum, C. serratum, Tectona grandis,

Mussenda glabra, Melia azedarach, Xylosoma longifolia inhibited the egg

hatching of M.incognita and M.Javanica. Fresh plant parts viz., leaves, stem and

seeds of Lantana camara and Jatropa spp have strong nematicidal properties

(Jyomati Devi, 2008; Javed et al, 2007).

14

Oka et al, (2000b) reported that essential oils extracted from spices and

aromatic plants immobilized more than 80% juveniles of M.javanica at

concentration of 1 OOO il/litre and also inhibited nematode hatching. The essential

oils of Foeniculum vulgare, M.rotundifolia and M. spicata showed highest

nematicidal activity in vitro and those from Origanum vulgare, O.syriacum and

Coridothymus capitatus reduced root-galling of cucumber seedlings when mixed

with sandy soil.

Extract of Aloe, Cannabis sativa, Vinca rosea were toxic to larvae of

M.incognita and mortality was correlated with concentration of extracts. Spot

application of groundnut cake, sawdust, coal ash and sunflower cake significantly

reduced the root-knot nematode population on okra (Singh and Singh, 2002;

Bhosle et al., 2006).

The soil amending with diffrent parts of Neem/Margosa (Azadirachta

indica A. Juss.^ were found highly effective in reducing the population of different

plant parasitic nematodes attacking variety of plants (Hellap and Dreyer, 1995;

Rao et a/., 1996; Zaki, 1998; Siddiqui and Alam, 2001; Oka and Pivonia, 2002,

Yasmin et al, 2003; Shah et al, 2004; Raman and Venkateshwarlu,2006;

Siddiqui, 2006a; Rather and Siddiqui, 2007a,b,c,d; Akhtar, 1998, 2000; Tariq and

Siddiqui, 2005).

The solvent extract of the plant species viz., Allophylus cobbe, Lepisanthes

tetraphylla, Sarcococca zeylanica and Hedyotis lawsoniae showed significant

nematicidal activity against M. incognita affecting tomato plants (Jayasinghe et al,

15

2003). Neem cake is best non -chemical alternative for the management of

nematode in tomato and other vegetable crops. The effect of mustard cake,

groundnut cake and Neem cake were equally effective in reducing the damage

caused by nematode and improved the growth of tomato (Khan and Rathi, 2001;

Bhattacharya and Goswami, 1987; Siddiqui and Alam, 1997b; Siddiqui et ai,

1997; Alam, 1989; Byomkesh and Pathi. 1998; Ram et ai, 2009; Alam et ai, 1982).

In in vitro studies inhibitory effect of Neem cake and rakshak gold (neem

based product) was observed. Plant growth of host increased along with significant

reduction in root-knot infestation in pot experiments when seed was treated with

botanical extracts (Randhavan et al, 2002).Dried powder of turmeric rhizome,

Neem cake, Neem leaf, Neem seed kernel and root powder of some plants viz.,

Tagetes erecta, O. sanctum. Calendula officinalis, Carica papaya and Azadirachta

indica were used which suppressed the nematode effectively and consequently the

plant growth improved (Chakraborti, 2001; Sharma and Trivedi, 1992; Rajendran

and Saritha, 2005).

In addition to this a large number of indigenous plants have been reported

to possess nematicidal/nematostatic properties capable of managing the population

of root-knot nematode. Some of these plants were tested for their antinemic

properties by different workers viz., Ipomea camea, Sargassum spp. fAra et

ai,\991). Allium sativum, Tagetes spp. ^ a l i a and Gupta, 1997), Linum

usitatissimum, Brassica campestris (Butool et al, 1998), Salvia spp. fIdowa,1999),

Parkia biglobosa (Umar and Jada, 2000), Murraya koengii (Pandey,2000),

Calotropis procera, Datura fastuosa, A.indica (Zarina et al,2003), Catharanthus

roseus, I.fistulosa (Hassen et a/.,2003), Ricinus communis, B.juncea, Enica saliva

(Khan et or/.,2004), Thevetia peruviana, Pedilanthus tithymaloides. Euphorbia

timcalli, E.neriifolia (Siddiqui,2006b), Parkia biglobosa, Hyptis spicigera (Jesse

et al.,2006), Blechum piramidatum, Stenandrium nanum, Ageratum gaumeri,

Bidens alba, Croton chinensis, Trichilia arborea, T.minutiJJora, Randia longiloba

(Cristobal-Alejo et al,2006; Ajaz and Tyagi, 2003), Ficus benghalensis, F.virens

(Ahmad et a/,2007), Avicennia marine (Tariq et al., 2007), B.rapa, B. napus

(Randhawa and Sharma, 200^), Argemone mexicana (Shaukat et al.,2002)

The application of rapeseed and Sudangrass green manure were more

effective in reducing root-galling caused by M.arenaria and M.hapla in squash

roots (Crow et al., 1996; Widmer and Abawi, 2000). The addition of Brassica

green manures stimulate soil microbial activities and increases accumulation of

plant decomposition products and natural plant bio-chemicals that can be

deleterious to nematodes (Stirling and Stirling, 2003).

The oil cakes are rich source of Nitrogen, Phosphorus and Potassium

(NPK).Various oil cakes take about 1-2 weeks for decomposition and then it is

more effective in soil and lead to increase in soil microflora and fauna, resulting in

suppression of population of root-knot nematode. The oil cake when amended

with moist soils are more effective then amending with dry soil. The oil cakes of

Neem/Margosa {A. indica A.Juss.), Castor (Ricinus communis L), Cotton seed

(Gossypium herbaceum L), Groundnut (Arachis hypogea L), Linseed {Linum

17

usitatissimum L), Mustard {Brassicajuncea L), Soyabean {Glycine max Merr),

Mahua (Madhuca indica Gmel), Duan/Rocket salad {Eruca sativa Mill), Sesame

(Sesamum indicum L.), Bakain/Persian lilac (Melia azedarach L.) and Karanj

{Pongamia pinnata L.) have been extensively used for the control of a wide range

of root-knot nematode.

The residual effect of various oil cakes against plant parasitic nematodes

has been reported in the succeeding crops (Singh and Sitaramaiah, 1966; Akhtar

and Alam, 1991; Goswami and Meshram,! 991; Akhtar and Malik, 2000),

Large number of workers carried out the study that various oil cakes when

amended in combination with different nematicides are more effective in reducing

the root-knot infestation caused by M.incognita and increased the plant growth and

yield than either of them alone (Alam, 1989; Verma and Anwar, 1997;

Sankranarayana and Sundarababu, 1997; Goswami et ai, 2006; Rather et al,

2007; Ahmad et al, 2008). The combined application of different oil cakes and

biocontrol agents have been reported to be an effective approach to minimize the

losses caused by various plant parasitic nematodes (Rao et a/., 1995; Tiyagi et

a/.,2002; Borah and Phukan,2004; Zareena and Kumar,2005, Kerry e/«/., 1993,

Kerry, 2000).

The biological control of nematode by using rhizosphere micro-organisms

were potential effort, contributed by several workers including plant growth

promoting rhizobacteria (Siddiqui and Ehteshamul -Haque,2001; Bharadwaj and

18

Sharma,2006; Siddiqui and Shaukat, 2003),bacterial parasites (Singh and

Dhawan,1994),obligate fungal parasite (Kok and Papert, 2002).

Therefore pre-sowing apphcation of amendments was more effective than

post sowing (Ramakrishnan et al., 1999). Akhtar (1993) pointed out that pressmud

vegetables and fruit processing waste and tobacco waste were most effective in

reducing root-knot nematode. Soil amended with spent tea, sugarcane bagasse,

paddy husk, wheat straw, domestic garbage, saw dust, flyash, cellulosic waste,

waste water and sewage sludge etc also decreased the root-knot population (Eyal

etal., 2006).

Some plant parts viz., roots, shoots, leaves, flowers etc. are left intentionally

in the field after harvesting and ploughed deep into the soil, after some time these

organic additives are decomposed and release different kind of biochemical that

allow to reduce the population of root-knot nematode and other plant parasitic

nematodes were found. (Siddiqui and Alam, 1995,1997a, 1999; Wani, 2008).

Crushed shells of crustaceans (shrimp, crab etc) are nitrogenous amendments,

these are made up of chitin or mucopolysacchride Clandosam''"**, a nematicide

made up of crab shells and agricultural grade urea can be effectively used (Fiola

and Lalancettle, 2000).

Kalaiarasan et al., f2006) and Jayakumar et al., f2004) that the soil

application of chitin (applied @l%w/w) and chitinolytic biocontrol agents

{Pseudomonas flourescens and Trichoderma viride @ 2.5 kg/ha each) promoted

19

the plant growth of groundnut cv.'Co.3' to the tune of 39.7% and also increased

the yield of crop upto 27.8% compared to control.

Ram and Maheshwari (1999) found the seeds of bottle gourd treated with

fungal cultural filterates were effective to enhancing the germination and plant

growth. Papaya seeds were treated with P.Jluorescens (10* cfu/g) and application

of P.Jluorescens and T.harzianum (10 cfu/g),each at 5g/kg soil reduced

significantly the eggs/eggmasses oi M. incognita and 4ml of fungal suspension

(8x10^ spore/3kg) was found to be optimum dose for effective reduction of

M. incognita population (Rao, 2007; Priya and Kumar, 2006).

Khan et al.,(2009) reported that, integrated management of root-knot

nematode in pointedgourd (Trichosanthes dioica) by T.viride was found to be

most effective and reduced root-gall and enhanced plant growth. When T.viride

and P.lilacinus in combination with mustard oil cake reduced maximum root

galling caused hyM.incognita on tomato (Goswami et al.,2006).

P.Jluorescens, Asprgillus niger, T.harzianum and P.lilacinus @ 50kg/ha

(2x10* cfu /g) each and organic amendments viz., fresh chopped and murraya

leaves, farmyard manure, mint manure and Neem seed powder @ Iq/ha,

carbofuran 3G and TopsinM [email protected] kg a.i./ha. T.harzianum was found

superior among all the treatments followed by P.Jluorescens, carbofiiran (Haseeb

et al, 2006; Saikia and Borah 2008; Tripathi and Singh, 2006).

The potential of soil solarization to control (M.Javanica and M.incognita)

root-knot nematode and soil amended with cow dung 40T/ha alone or in

20

integration were studied in cucumber field. Soil solarization alone could reduce

the incidence of root-knot nematodes to 52.56% and in amended soil to 56,00%

and integration of soil solarization and amendment could effectively reduce the

nematode incidence by 83% (Esfahani, 2008; Siddiqui et al, 2009). Mixed

cropping practice, sometimes referred to as intercropping. Several workers

reported that different cropping sequences reduce the population of plant parasitic

nematodes (Haider et al., 2001; Haider and Pathak, 2001; Siddiqui and Alam,

1987a,b; Siddiqui and Saxena,1987; Tiyagi et al., 1986),

The intercropping of two rows of yellow sarson {Brassica campestris var.

Toria / B. campestris var. Sarson) with sugarcane highly reduced (23.7%)

nematode population followed by sugarcane+yellow mustard and exhibited the

highest cane yield. Similar results of inclusion of mustard, a poor host for several

nematode in different cropping sequences for reducing nematode population have

been reported by many other workers (Singh and Sitaramaiah, 1993; Haider et al,

2004; Kumar et al., 2006).

Prasad et al. f2004) found the highest linseed yield when linseed was

incorporated with mustard followed by gram. The presence of 2-propenyl

isothiocynate in mustard, when intercropped supposed to suppress the nematode

population and having nematicidal activity. The crop rotation may provide a short

term suppression of nematode population densities (Starr et al., 2002).

The mix culture of marigold (Tagetes erecta) inhibited the root-knot

development caused by M. incognita and other plant parasitic nematode population

21

densities and exhibiting a better plant growth (Siyanand et ai, 1994; Dhanger et

al., 2002; Yen et al, 1998; Moussa etal, 1997; Wani, 2005).

22

If -A

Chapter 3 Materials and Methods

^ - - ^

CHAPTER-3

MATERIALS AND METHODS

3.1. Test plant and pathogen

The root-knot nematode Meloidogyne javanica (Treub, 1885) Chitwood,

1949 was selected as test pathogen and bottle gourd Lagenaria siceraria (Molina)

Standi, cv 'Kanchan' was used as test plant.

3.2. Preparation and sterilization of soil mixture

A mixture of soil and organic manure was prepared in the ratio of 3:1. The

clay pots (15 cm in diameter) were filled with this mixture at the rate of 1 kg/pot.

Later pots with soil were sterilized in an autoclave at 15 lbs pressure for 20

minutes.

33 . Raising and maintenance of bottle gourd seedlings

The seeds of Bottle gourd, Lagenaria siceraria (Molina) Standi, cv.

'Kanchan', Family - Cucurbitaceae, were surface sterilized in 0.01% HgCl2 for

two minutes and then rinsed three times with Double Distilled Water (DDW).

Three seeds were sown in autoclaved soil in clay pots (15 cm in diameter). One

week after germination, two seedlings were removed from each clay pot

containing 1 kg soil treated with different doses of decomposed and

undecomposed oil cakes, fresh leaves of different plant species, commercial Neem

products, a nematicide and different fungal biocontrol agents. Pots containing soil

without amendments served as control. After seven days when seedlings got

23

established, each of them were inoculated with 1000 freshly hatched second stage

juveniles (J2) ofMeloidogyneJavanica. The experiment was replicated four times.

3.4. Maintenance of nematode culture

The healthy eggmass of root-knot nematode, M javanica was collected

from the infected roots of brinjal. Solatium melongena L. Family Solanaceae

(collected from a heavily infected brinjal field near Sasni, Agra Road, Hathras)

and placed on a small coarse sieve (1mm pore size) lined with tissue paper and

placed in 10cm diameter Petridish containing double distilled water. The second

stage juveniles, which hatched out, were collected along with water from the

Petridish. The infective second stage juveniles of M Javanica in water suspension

was adjusted so that 1000 freshly hatched juveniles could be added in 10 ml of

water suspension, used for in vitro test and served as initial inoculum level for

glasshouse experiments The nematode species was ascertained by close

examination of perineal pattern of the female from the first eggmass collected. It

was confirmed by similar examination of the randomly collected females from the

inoculum raised as above.

3.5. Preparation of plant extracts

Different parts of plant species viz., Lantana (Lantana camara Linn.

Family-Verbenaceae), Asgandh/Ashwagandha {Withania somnifera Dunal,

Family-Solanaceae), Pili katheri/Mexican Poppy (Argemone mexicana Linn.

Family-Papaveraceae), Datura (Datura stramonium Linn. Family-Solanaceae),

Congress grass (Parthenium hysterophorus Linn. Family-Asteraceae), Aak/Madar

24

{Calotropis procera (Ait.) Ait.f. Family-Asclepiadaceae), Kathel/Jackfruit

{Artocarpus heterophyllus Lam. Family-Moraceae), Euphorbia {Euphorbia

cotonifolia Linn. Family-Euphorbiaceae), Bargad {Ficus benghalensis

Linn.Family-Moraceae), Rubber Plant {Ficus elastica Roxb. Family- Moraceae),

Sadabahar/Red I'Qnwmk\e{Catharanthus roseus Linn. Family-Apocynaceae) and

Zinnia {Zinnia elegans Jacq. Family-Asteraceae) were thoroughly washed,

chopped and 50 g of each macerated in grinder and then mixed in 150 ml double

distilled water and extract were centrifUged at 2000 rpm for 15 minutes and after

it, filtered through Whatman No.l filter paper (Whatman International Ltd,

Maidstone, UK) and extracts arbitrarily termed as standard solution (S), were

stored at 4°C. Other different dilutions S/2, S/10 and S/10 were prepared from

standard solution (S) with adding the requisite amount of double distilled water at

the time of experiment.

3.6. Mortality test

Ten ml of water suspension containing 1000 second stage juveniles of M

javanica were poured in different concentrations (S, S/2, S/10 and S/100) of plant

part extracts of different plant species separately. Petridish containing distilled

water served as control. There were four replicates of each treatment. The

Petridishes were kept at 28°C temperature. Number of mobile and immobile

nematodes were counted after 12, 24, 48 hours of exposure period. The death of

nematodes was ascertained by transferring the immobilized juvenile into water for

6 hour and percent mortality was calculated.

25

3.7. Hatching test

The eggmasses were taken from thoroughly washed roots of eggplant

infected with root-knot nematode M javanica. Five eggmasses of average size

were kept in 40 mm Petridishes containing lOmi solution of different

concentration of extracts separately. Petridishes containing distilled water served

as control. These were 4 replicates of each treatment. Petridishes were left for 5

days at 28°C temperature. Then the total number of hatched juveniles were

counted with the help of counting dish under stereoscopic microscope.

3.8. Nematode control with latex used as seed dresser

Latices were collected from Artocarpus heterophyllus, Calotropis procera.

Euphorbia cotonifolia, Ficus benghalensis and F. elastica arbitrarily termed as

Standard (S). Seeds of bottle gourd were thoroughly mixed with different plant

latices so as to give a uniform and smooth coating over the seeds. The treated

seeds were then spread in an enamel tray and allowed to dry in shade before

sowing. The seeds were directly sown after treating them with the plant latices as

3 seeds/pot. One week after germination, two seedlings were removed from each

clay pot containing 1 kg autoclaved soil. Each treatment is replicated four times.

3.9. Nematode control with chopped plant leaves

For evaluating the efficacy and nematostatic potential of organic soil

amendments, chopped leaves of Argemone mexicana, Datura stramonium,

Lantana camara, Parthenium hysterophorus and Withania somnifera, were

applied in two doses (50 g and 100 g) to the pots. Each treatment including

26

untreated uninoculated and untreated inoculated control were replicated 4 times.

The pots were watered immediately for ensuring proper decomposition of the

organic additives. After 2 weeks of waiting period the seeds of bottle gourd cv

Kanchan were sown directly into the pots. The acclimatized plants were inoculated

with 1000 freshly hatched second stage juveniles (J2) of the root-knot nematode,

M.javanica (Treub) Chitwood.

3.10. Nematode control with different commercial Neem products

For evaluating the efficacy of different commercial Neem products viz.,

Nimin (a triterpene rich Neem product of Godrej Cooperation Limited, India),

Neem Kernel oil (a Neem product of Khadkeshwar oil Mills Ltd., Aurangabad,

Maharashtra, India), Achook (a Neem product of Godrej Co-operation Ltd., India),

Neem Raj (a Neem product of Khadkeshwar Oil Mills Pvt. Ltd., Aurangabad,

Maharashtra) as urea coating agents (1, 2 or 3 ml of above Neem products mixed

with 100 g urea). One ml/lOOg urea considered as single strength (SS), while 2

ml/lOOg urea as double strength (DS) and 3 ml/lOOg urea as triple strength (TS)

against root-knot nematode M. javanica and their effect on the plant growth of

bottle gourd, pots were filled with soil and incorporated with urea coated with

different amounts of Nimin, Neem Kernel oil, Achook and Neem Raj. The healthy

seeds of bottle gourd were sown singly to pots containing autoclaved soil. The

pots were inoculated with 1000 second stage juveniles oiM. javanica I pot. There

were 4 replicates for each treatment including control (uncoated urea).

27

3.11. Nematode control with chopped leaves of latex bearing plants and

biocontrol agents.

For evaluating the efficacy and nematostatic potential of organic soil

amendments, chopped leaves of latex bearing plants viz., Calotropis procera.

Euphorbia cotonifolia, Ficus benghalensis, F. elastica and Artocarpus

heterophyllus (@ 50 g and lOOg/pot) seperately to the pots and thoroughly mixed

with soil. Each treatment including untreated uninoculated and untreated

inoculated control were replicated four times. The pots were watered immediately

for ensuring proper decomposition of the organic additives. After 2 weeks of

waiting period the seeds of bottle gourd cv. 'Kanchan' were sown directly into the

pots. The acclimatized plants were inoculated with 1000 freshly hatched second

stage juveniles (J2) of root-knot nematode Myavan/co.

3.12. Nematode control with oil cakes, nematicide and biocontrol agents

For evaluating the efficacy of oil cakes of various plants viz., Mustard,

Cotton and Neem cake alone and in combination with nematicide, carbofuran/

Furadan 3-G (2, 3-dihydro -2, 2-dimethyl 7-benzo-furanyl methyl carbamate) and

two fungal biocontrol agents viz., Trichoderma harzianum and Paecilomyces

lilacinus against the root-knot development caused by root-knot nematode, M.

javanica and their potential in enhancing the plant growth characters of test plant.

The pots treated with different oil cakes applied @ 50g/pot alone and in

combination with carbofuran @ Ig/pot and T.harzianum and P. lilacinus @

Ig/pot. The pots were watered soon afler the application of different treatments for

28

facilitating the proper decomposition of organic additives. It was left for 3 weeks

termed as decomposed oil cake while for 1 week termed as undecomposed oil

cake. There were four replicates for each treatment including the untreated

uninoculated and untreated inoculated control. Each pot was inoculated with an

initial inoculum level of freshly hatched second stage juveniles of root-knot

nematode, M. Javanica @ 1000 Jz/pot.

3.13 Chlorophyll content

The chlorophyll content in the fresh leaf was estimated following method

worked out by Mackinney (1941).

One g of finely cut fresh leaves was groimd to a fine pulp using a mortar

and pestle after pouring 20 cm^ of 80% acetone The mixture was centrifiiged at

5,000 rpm for 5 minutes. The supernatant was collected in 100 cm^ volumetric

flask. The residue was washed three times using 80% acetone. Each washing was

collected in the same volumetric flask and volume was made upto the mark, using

80% acetone. The absorbance was read at wavelength of 645 and 663nm against

the (80% acetone) blank on spectrophotometer (UV.1700, Shimadzu, Japan), The

chlorophyll content present in extract (mg g"' tissue) was calculated using the

following equations:

1 V

mg chlorophyll 'a' g' tissue = 12.7 (Agss) - 2.69 (A645) x lOOOxW

-1,- .. . . . . . . . V mg chlorophyll 'b ' g'' tissue = 22.9 (A645) - 4.68 (Ages) x lOOOxW

29

mg total chlorophyll g"' tissue = 20.2 (A645) + 8.02 (A663) x ^ lOOOxW

A = Absorbance at specific wave length (X)

V = Final volume of chlorophyll extract in 80% acetone

W == Fresh weight oftissue used for extract

3.14. Preparation of fungal inoculums and inoculation

The culture of Trichoderma harzianum (strain no.6276) and Paecilomyces

lilacinus (srtrain no.4899) were obtained individually from microbiology division

of lARI, New Delhi. It was maintained on Potato Dextrose Agar (PDA) slant. The

constituent of PDA (Riker and Riker, 1936) are given below:

Agar 17.00 g

Potato peeled and sliced 200.00 g

Dextrose 20.00 g

Double Distilled water 1000.00 ml

Richards mediirai (Riker and Riker, 1936) was used for the mass production T.

harzianum and P. lilacinus The composition of Richard's medium is as follows:

Potassium nitrate (KNO3) 10.00 g

Potass ium Dihydrogen phosphate 5.00 g

Magnesium Sulphate (MgS04) 2.5 g

Ferric Chloride (FeCls) 0.02 g

Sucrose (C12H22O11) 50.00 g

Double distilled water 1000.00 ml

30

The medium was prepared, filtered through muslin cloth and then sterilized in an

autoclave at 15 lbs, for 15 minutes in 250 ml flasks, the liquid medium was then

inoculated with small amount of fungus maintained on PDA slants with the help of

inoculation needle in an aseptic chamber. These inoculated flask then kept in an

incubator at 25-28°C for about 15 days to allow rapid growth of fungus for

experimental studies. Afler enmassing of T. harzianum and P. lilacinus on

Richard's medium, 100 gm of mycelia was blended in 1000 ml of double distilled

water in Waring blender so that 10 ml of suspension contained of Ig mycelia. The

fungal suspension of T. harzianum and P. lilacinus were then inoculated into the

soil around the bottle gourd, by making holes, 5-7 cm deep within the radius of

20m. After inoculation of the fungus, these holes were plugged with the sterilized

soil. Regular watering was done to maintain soil moisture.

3.15. Observations

Plants were uprooted after 60 days from the date of inoculation. Roots were

gently washed with tap water taking utmost care to avoid loss or injury to the roots

during the complete operation, the excess water of plants was removed by putting

them between the folds of blotting sheets. The plants were cut with a sharp knife

just above the base of the root emergence zone. Fresh and dry weight of the root

and shoot of all replicates was taken in grams. The length of root and shoot (in

cm) and chlorophyll content were also recorded.

The nematode population in the soil from every replicate was determined

by processing 250g of soil using Cobb's sieving and decanting method,

31

followed by the modified Baermann funnel technique (Southey, 1986).

Nematode suspensions were collected after 24 hours, and the number of

nematode were counted with the help of a counting dish (Doncaster, 1962) in

three, 1 ml aliquots of the suspension from each sample. The means of three

counts were used to calculate the population of nematodes per Kilogram soil.

Number of adult females within the roots examined by obtaining 1 g fresh root

sub sample per replicate and stained using lactic acid-fuchsine technique (Byrd

et al., 1983). The number of adult female present in root was calculated by

multiplying the number of adult females present in Ig of root by the total

weight of root. The degree of root-knot nematode infection was recorded

according to rating degree given by Taylor and Sasser, 1978 as under:

Root knot index (RKI) Number of galls/root system

0 0

1 1-2

2 3-10

3 11-30

4 31-100

5 >-100

3.16. Statistical Analysis

The data were analyzed by one-way analysis of variance (ANOVA) using

SPSS 12.00 software (SPSS Inc., Chicago, IL, USA) critical difference (CD.)

were calculated atp = 0.05 and at/? = 0.01 to test for significant differences.

32

CHAPTER-4

RESULTS

4.1. Effect of water extracts of leaves of different plant species on the hatching

of Meloidogynejavanica in vitro

The experiment was conducted under in vitro conditions to assess the

antinemic properties of the water extracts of leaves of different plant species viz.

Argemone mexicana. Datura stramonium, Lantana camara, Parthenium

hysterophorus and Withania somnifera, against the hatching of second stage

juveniles (J2) of root-knot nematode, Meloidogynejavanica. Leaf extract of all the

test plants were foimd inhibitory to juvenile hatching. However, the inhibitory

effect increased with the increase in concentration of the extracts (Table-1).

The results presented in Table-1, Figure-1, first clearly indicate that average

number of juveniles hatched in S, S/2, S/10, and S/100 concentration of leaf

extracts of ^. mexicana were 0, 24, 46, and 68 respectively. The corresponding

figures for different concentration of leaf extracts of D. stramonium were 0, 10,

24, and 48 , for leaf extracts of Z. camara were 0,40, 67, and 96, for leaf extracts

of P. hysterophorus 0, 56, 86, and 118 and for leaf extracts of Withania somnifera

the figures were 3, 72, 105, and 134 respectively. The distilled water control

showed maximum juvenile hatching where average number of juvenile hatched

were 460 (Table-1).

In case of leaf extracts of A.mexicana, D.stramonium, L.camara and

P.hysterophorus 100% inhibition in juvenile hatching were observed while

33

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99.34% inhibition was observed in S concentration of W.somnifera It was

followed by S/2 concentration of A.mexicana 94.78%, D.stramonium 97.82%,

Lcamara 91.30%, P.hysterophorus 87.82% and W.somnifera 84.34%.

The range of inhibition in the juvenile hatching in S/10 concentration were

observed in leaf extracts of D.stramonium 94.78% followed by 90.00%, 85.43%,

81.30% and 77.17% in leaf extracts of A.mexicana, Lcamara, P.hysterophorus

and W.somnifera respectively. The highest inhibition in juvenile hatching in

S/100 concentration in above mentioned leaf extracts were 89.56%, 85.21%,

79.13%, 74.34% and 70.86% respectively (Table-1, Figure-1).

4.2. Effect of water extracts of flower and fruit of different plant species on

the hatching of Meloidogynejavanica in vitro


antinemic properties of the water extracts of flower of different plant species viz.,

Argemone mexicana. Datura stramonium, Lantana camara, Part hen ium

hysterophorus and fruit extracts of Withania somnifera against the hatching of

second stage juveniles of root knot nematode, Meloidogyne jax'anica. Flower and

fruit extracts of all the test plants were found inhibitory to juvenile hatching.

However, the inhibitory effect increased with increase in concentration of the

extract (Table 2).

The results presented in Table-2, Figure-2 clearly indicate that average

number of juveniles hatched in S, S/2, S/10, and S/100 concentration of the flower

extracts of 4. mexicana were 0, 32, 59 and 82 respectively. The corresponding

34

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a -•-» c o o __ >-< its

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at

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figures for different concentrations of flower extracts D. stramonium were 0, 15,

38 and 56 , for flower extracts of L camara were 0, 48, 78 and 107, for flower

extracts of P. hysterophorus the figures were 0, 64, 92, and 126 and for fruit

extracts of W. somnifera were 5, 80, 115 and 147 respectively. The distilled water

(DW) control showed maximum juvenile hatching when average number of

juvenile hatched were 460 (Table-2).

In case of the flowers of the D. stramonium, A. mexicana, L. camara,

P.hysterophorus 100% and in water extracts of fiiiits of W. somnifera 98.91%

inhibition in the juvenile hatching was observed in S concentration. It was

followed by S/2 concentration of flower extracts of D. stramonium 96.73%, A.

mexicana 93.04%, L. camara 89.56% and fhiit extracts of W. somnifera 82.60%.

The range of inhibition in the juvenile hatching in S/10 concentration was

observed in flower extracts of D. stramonium 91.73% followed by 87.17%,

83.04%, 80.00%) mA. mexicana, L camara, P. hysterophorus and fruit extracts of

W. somnifera 75.00%. The highest inhibition in juvenile hatching in S/100

concentration in above mentioned extracts were 87.82%,82.17%,76.73%,72.60%

and 68.04% respectively (Table-2 , Figure-2 ).

43. Effect of water extracts of leaves of different latex bearing plant species

on the hatching of Meloidogynejavanica in vitro.


antinemic properties of the water extracts of leaves of latex bearing plant species

viz., Artocarpus heterophyllus, Calotropis procera. Euphorbia cotonifolia, Ficus

35

benghalensis and F. elastica against the hatching of second stage juvenile of root-

knot nematode, Meloidogyne javanica. Leaf extracts of all the test plants were

found inhibitory to juvenile hatching. However, the inhibitory effect increased

with increase in concentration of the extracts (Table-3).

The results presented in Table-3, Figure-3, clearly indicate that average

number of juveniles hatched in S, S/2, S/10 and S/100 concentration of leaf extract

of C. procera were 0, 28, 72, and 94 respectively. The corresponding figures for

different concentrations of £". cotonifolia 0, 44, 87, and 112, for F. benghalensis

were 0, 52, 99, and 128, A. heterophyllus were 0, 65, 117, and 136 and F. elastica

were 7, 73, 123 and 154 respectively. The Distilled Water (DW) control showed

maximum 460 average number of juvenile hatching (Table-3).

In case of leaf extract of C. procera, E. cotonifolia, F. benghalensis, A.

heterophyllus 100% and in F. elastica, 98.47% inhibition in juvenile hatchmg

were observed in S concentration. It was followed by S/2 concentration of leaf

extracts of C procera 93.91%, E. cotonifolia 90.43%, F. bengalensis 88.69%, A.

heterophyllus 85.86% mdFelastica was 84.13% (Table-3).

The range of inhibition in the juvenile hatching in S/10 concentration were

observed in leaf extracts of C. procera 84.78% followed by E. cotonifolia

81.08%, F. benghalensis 77.26%, A. heterophyllus 74.56% and F. elastica

73.26%. the highest inhibition in juvenile hatching in S/100 concentration were

observed in C procera 86.08% followed by 75.65%, 72.17%, 70.43% and 66.52%

36

I o e

es

t i

V

a

IS

>

o

I

. . OS

/""s

^ « T3 •« h 3i C -5 a o •o

2 # 63

S fl o u 1 «s S a

< S ( H

o U 00,

o o

o VO • « *

<s t ^

00 n

e

U g •a

1

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£ N ^

s? r~-

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^ «5 fO OS w -

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r~-00

• > * • *

e

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S

1

/^^ tr> ^ •« t -• ^

0? o

•^ o 9 \ ^ ^

O o

o >« "»

00 1-H

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o\

«s l/>

e

•a A

a

^•s t -

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fS

^ NO

00 96, N ^ '

o" e

O «« • ^

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f * l t ^

r~-

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e

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t

c o o

u

u

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60

IE u x:

v^ V-o J3 trt tu

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c

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cn U

i CQ o.

_c c (l> >

m u 3 « >

I I I I r •I I

n

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o o

C/5

o • oe S ^ •-^ s « «s 5 "5, a u) •- a .2 « S •* S **

U en b «

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S «M '5 O

(Z)

DJtUDAtff'J^ JO

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S 08 tm UD O

• * *

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u 9 gjD

in E.cotonifolia, F. benghalensis, A. heterophyllus and F. elastica respectively

(Table-3 , Figure-3 ).

4.4. Effect of water extracts of leaf, flower and root of Catharanthus roseus

and Zinnia elegans on the hatching of Meloidogynejavanica in vitro


antinemic properties of the water extracts of plant parts i.e. leaf, flower and root of

Catharanthus roseus and Zinnia elegans against the hatching of second stage

juveniles of root-knot nematode, Meloidogyne javanica. The water extracts of all

parts of the test plants were found inhibitory to juvenile hatching. However the

inhibitory effect increased with in increase in concentration of the extract.

The results presented in Table-4, Figure-4, clearly indicate that average

number of juveniles hatched in S, S/2, S/10 and S/100 concentration of leaf

extracts oiC.roseus were 0, 15, 44 and 68 respectively. The corresponding figures

for different concentrations of flower extracts oiC.roseus were 0,22,56 and 82,for

root extracts of C.roseus were 11,34,67 and 93,for leaf extracts of Z.elegans

0,27,59 and 74,for flower extracts of Z elegans. were 5,31,65 and 88 and for root

extracts of Z.elegans were 16,37,76 and 99 respectively. The Distilled Water

(DW) control showed maximum juvenile hatching where average number of

juvenile hatched were 460 (Table 4).

In case of leaf and flower extracts of C. roseus and leaf extracts of Z.elegans

100% inhibition and in root extracts of C.roseus 97.60%,flower and root extracts

of Z.elegans 98.91% and 96.52% inhibition in juvenile hatching were observed in

37

en a

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S « e 5 ^ <£ a •

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•

15

1 Q §> <*i

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^

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2 -^ X

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js 'Si

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1/^ r t CI (N .-^

VJtUVAVf W J® 3uii(3|Bi| 3|!U9Anf ui uoi4iqii|ui luaajaj

S concentration respectively. It was followed by S/2 concentration of leaf extracts

of C.roseus 96.73%, flower extracts of C.roseus 95.21%, root extracts of

Z.elegans 94.13%,flower extracts of Z.elegam ,93.26% and root extracts of

Z.elegans, 91.95% respectively (Table-4).

The range of inhibition in juvenile hatching in S/10 concentration were

observed in leaf extracts of C.roseus, 90.43% followed by 87.82%, 85.43%,

87.17%, 85.86%) and 83.47% in flower extracts of C.roseus, root extracts of

C.roseus, leaf extracts of Z elegans, flower extracts oiZ.elegans and root extracts

of Z.elegans respectively. The highest inhibition in juvenile hatching in S/100

concentration in above mentioned extracts were 85.21%, 82.17%, 79.78%,

83.91%, 80.86% and 78.47% respectively (Table-4, Figure-4).

4.5. Effect of water exracts of leaves of different plant species on mortality of

the root-knot nematode, Meloidogyne javanica in vitro

The data presented in Table-5, Figure-5(a-e) clearly indicate that the

mortality of root-knot nematode, Meloidogyne javanica increased with increase in

concentrations and the duration period. The leaf extracts of Datura stramonium

proved highly toxic to nematode followed by Argemone mexicana, Lantana

camara, Parthenium hysterophorus and Withania somnifera.

In leaf extract of D. stramonium after 12, 24 and 48 hours of exposure

period, the percent mortality of nematode was 84, 95 and 100% in S; 75, 80 and

95% in S/2; 40, 55 and 74% m S/10 and 34, 50 and 65% in S/100 concentration

respectively (Table-5, figure-5a).

38

I §

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a o on 4)

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es

• •

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H

d o ••c es S

a o •pit «

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g ii 13 W c« >

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a»

g 1 — ° ft ««

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s 5 <"

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i| 0 js

^ S 1 1 2 t ^

iJ S o

( M 9> «? 0 ^ t: s 0 ,2 0 - - r e 2 = ^ Z 0 V

1-1 ^

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After 12, 24 and 48 hours of exposure period the percentage of nematode mortality

was 65, 76 and 88% in S concentration of A. mexicana leaves. The corresponding

figures were 55, 63 and 87% in S/2; 32, 43 and 60% in S/10 and 26, 40 and 54%

in S/100 concentration (Table-5, Figure-5b).

In the leaf extract of L. camara after 12, 24 and 48 hours of exposure

period the corresponding figures for nematode mortality were 41, 61 and 75% in

S; 36, 50 and 65% in S/2; 22, 35 and 47% in S/10 and 19, 30 and 42% m S/100

concentration respectively (Table-5, Figure-5c).

After 12, 24 and 48 hours of exposure period the percentage of nematode

mortality was 32, 43 and 72% in S concentration off. hysterophorus leaves. The

corresponding figures were 20, 33 and 52% in S/2; 15, 22 and 38 m S/10 and 11,

19 and 30% m S/100 concentration (Table-5, Figure-5d).

In the leaf extracts of W. somnifera after 12, 24 and 48 hrs of exposure

period the percentage of nematode mortality was 22, 35 and 55% in S; 15, 22 and

38% in S/2; 11, 15 and 26% in S/10 and 0, 12, 19% in S/100 concentration

respectively (Table-5, Figure-5e).

In distilled water control no nematode was killed even after 48 hours of

exposure period in all concentrations of the test plants (Table-5).

4.6. Effect of water extracts of flower and fruit of different plant species on

mortality of root-knot nematode, Meloidogyne javanica in vitro

The data presented in Table-6, Figure-6(a-e), clearly indicate that the


39

concentrations and the duration period. The flower extracts of Datura stramonium

proved highly toxic to nematode followed by Argemone mexicana, Lantana

camara, Parthenium hysterophorus and fruit extracts of Withania somnifera.

In flower extracts of D. stramonium, after 12, 24 and 48 hours of exposure

period, the percent mortality of nematode was 77, 87 and 100% in S; 62, 77 and

92% m S/2; 35, 48 and 65% m S/10 and 29, 44 and 60% in S/100 concentration



mortality was 51, 67 and 75% in S concentration of 4. mexicana flower. The

corresponding figures were 45, 54 and 72% in S/2; 28, 40 and 55% in S/10 and 23,

36 and 49% in S/100 concentration (Table-6, Figure-6b).

In the flower extract of I. camara after 12, 24 and 48 hours of exposure


S; 29, 43 and 60% m S/2; 18, 32 and 41% in S/10 and 16, 25 and 35% m S/100



mortality was 30, 40 and 65% in S concentration of P. hysterophorus flower. The

correspondmg figures were 18, 28 and 44% in S/2; 13, 18 and 32 in S/10 and 9, 15

and 24% in S/100 concentration (Table-6, Figure-6d).

In the fruit extracts of W. somnifera after 12, 24 and 48 hrs of exposure

period the percentage of nematode mortality was 18, 28 and 45% in S; 11, 18 and

40

4>

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32% m S/2; 10, 13 and 21% in S/10 and 0, 10, 14% in S/100 concentration




4.7. Effect of water extracts of leaves of latex bearing plants species on

mortality of root-knot nematode, Meloidogynejavanica in vitro

The data presented in Table-7, Figure-7(a-e), clearly indicate that the


concentrations and the duration period. Among all latex bearing test plants, the

leaf extracts of Calotropis procera proved highly toxic to nematode followed by

Euphorbia cotonifolia, Ficus benghalensis, Artocarpus heterophyllus and F.

elastica.

In leaf extract of C procera, after 12, 24 and 48 hours of exposure period,

the percent mortality of nematode was 78, 92 and 100% in S; 69, 85 and 95% in

S/2; 45, 60 and 79% in S/10 and 39, 55 and 70% in S/100 concentration



mortality was 67, 86 and 100% in S concentration of £". cotonifolia leaves. The

corresponding figures were 53, 75 and 88% in S/2; 41, 54 and 72%o in S/10 and 35,


In the leaf extract of F. benghalensis after 12, 24 and 48 hours of exposure


41

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S; 47, 68 and 80% in S/2; 39, 48 and 62% in S/10 and 31, 43 and 54% m S/100



mortality was 52, 76 and 85% in S concentration of 4. heterophyllus leaves The

corresponding figures were 41, 63 and 72% in S/2; 35, 42 and 56 in S/10 and 28,

36 and 48% in S/100 concentration (Table-7, Figure-7d).

In the leaf extracts of F. elastica after 12, 24 and 48 hrs of exposure period

the percentage of nematode mortality was 48, 70 and 80% in S; 36, 58 and 62% in

S/2; 29, 37 and 49% in S/10 and 22, 31, 40% in S/100 concentration respectively

(Table-7, Figure-7e).



4.8. Effect of water extracts of leaf, flower and root of Catharanthus roseus

and Zinnia elegans on mortality of root-knot nematode, Meloidogyne javanica

in vitro

The data presented in TabIe-8, Figure-8(a-f) clearly indicate that the


concentrations and the duration period. The leaf extracts of Catharanthus roseus

proved highly toxic to nematode followed by leaf extracts of Zinnia elegans,

flower extracts of C.roseus, flower extracts of Z.elegans, root extracts of C. rose us

and root extracts of Z.elegans.

42

In leaf extract of C. roseus, after 12, 24 and 48 hours of exposure period,

the percent mortality of nematode was 80, 92 and 100% in S; 72, 84 and 90% in

S/2; 60, 70 and 82% m S/10 and 52, 63 and 75% in S/100 concentration



mortality was 68, 80 and 100% in S concentration of C. roseus flower. The

corresponding figures were 56, 74 and 85% in S/2; 50, 62 and 70% in S/10 and 42,


In the root extracts of C. roseus after 12, 24 and 48 hours of exposure


S; 51, 62 and 78% m S/2; 40, 53 and 60% m S/10 and 30, 45 and 54% m S/100



mortality was 75, 88 and 100% in S concentration of Z. elegans leaves. The


55 and 65% in S/100 concentration (Table-8, Figure-8d).

In the flower extracts of Z. elegans after 12, 24 and 48 hours of exposure

period the percentage of nematode mortality was 62, 70 and 85% m S; 54, 60 and

72% in S/2; 46, 55 and 62% in S/10 and 40, 48, 57% m S/100 concentration



mortality was 50, 65 and 78% in S concentration of Z. elegans root. The

43

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• 5 «50

C i

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c

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i g

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fs

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2

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tc

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u e o U

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ri

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s =

B Q,

a, e

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a s <^ on ^

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S I .S 2 — « s >« S ^ « «*-I- O OS

f

cs

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8 g S 3 8

-V3tUVAVf 3U^OpiOJ3J^ JO X)i|RMOID ) a 3 3 J 3 J -


40 and 45% in S/100 concentration (Table 8, Figure 8f).


exposure period in all concentrations of the test plants (Table 8).

4.9. Effect of seed dressing with latex of different plant species on the root-

knot development caused by Meloidogyne javanica and plant growth of bottle

gourd cv. 'Kanchan' in pots

The results presented in Table-9 a & b clearly indicate that all treatments

brought about significant reduction in the root-knot development caused by M

javanica and also improved the growth parameters of bottle gourd. Highest

reduction in the root-knot development was observed in soil amended with

Calotropis procera (1.91), it was followed by Euphorbia cotonifolia (2.42), Ficiis

benghalensis (2.70); Artocarpus heterophyllus (2.90) and Ficus elastica (3.14). In

untreated inoculated pots the root-knot development was highest (4.90). The

reduction in the root-knot development by the application of various latex

treatments resulted in a better growth of the plant.

The total length of plant was increased by 170.40, 157.96, 145.62, 125.48

and 116.74 cm in C. procera, E. cotonifolia, F. benghalensis, A. heterophyllus and

F. elastica respectively as against 65.72 cm in untreated inoculated and 193.58 cm

in untreated uninoculated control plants (Table-9a, Figure-9).

The total fresh weight of plant was mcreased by 90.00, 79.88, 72.26, 66.90

and 62.71 g in C. procera, E. cotonifolia, F. benghalensis, A. heterophyllus and F.

44

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untreated uninoculated plants( Table 9 a,Figure 9).

The total dry weight of plant was 23.84, 20,42, 18.42, 16.38 and 15.36 g in

C. procera, E. cotonifolia, F. benghalensis, A. heterophyllus and F. elastica

respectively as against 11.30 g in untreated uninoculated and 30.56 g m untreated

uninoculated control plants ( Table-9 a, Figure-9).

The total chlorophyll content (leaf) was increased by 1.564, 1.426, 1.380,

1.261 and 1.227 mg/g in C. procera, E. cotonifolia, E. benghalensis, A.

heterophyllus and F. elastica respectively as against 0.903 mg/g in untreated

uninoculated and 1.891 mg/g in untreated uninoculated control plants ( Table-9 b).

The adult female of M javanica per root system was 50.00, 64.34, 72.76,

87.00 and 102.70 in C. procera, E. cotonifolia, F. benghalensis, A. heterophyllus

and F. elastica respectively as against 143.00 in untreated inoculated control

plants. The nematode population per kg soil was 1678.70, 1936.34, 2398.54,

3062.76 and 3518.16 in C. procera, E. cotonifolia, F. benghalensis, A.

heterophyllus and F. elastica respectively as against 8012.00 in untreated

inoculated control plants (Table 9b).

4.10. Effect of chopped leaves of latex bearing plants on the root-knot

development caused by Meloidogyne javanica and plant growth of bottle

gourd cv.' Kanchan' in pots



45

javanica and also improved the growth parameters of bottle gourd. The highest

reduction in the root-knot development was observed in soil amended with

chopped leaves of Calotropis procera (100 g), 1.65. This was followed by 1.80,

2.00, 2.20, 2.40, 2.70 and 3.00 in Euphorbia cotonifolia, Ficus benghalemis (100

g), C procera (50 g), E. cotonifolia (50 g), Artocarpus heterophyUus (100 g) and

F. elastica (50 g) respectively as against 4.90 in untreated inoculated control.

The total length was observed highest 185.65 cm when plants treated with

chopped leaves of C procera @ 100 g/pot. This was followed by 182.44, 175.80,

174.64, 168.00, 155.44 and 115.23 cm in C. cotonifolia (100 g), F. benghalensis

(100 g), C. procera (50 g), E. cotonifolia (50 g), A. heterophyUus (100 g) and F.

elastica (50 g) respectively as against 65.72 cm in untreated inoculated and 193.58

cm in untreated uninoculated control plants (Table-10 a. Figure-10).

The total fresh weight and dry weight was observed highest 105.23 and

26.47 g when plant was treated with chopped leaves of C. procera @ 100 g/pot

This was followed by 93.71 and 24.19 g in £". cotonifolia applied @ 100 g/pot,

86.91 and 22.79 g in F. benghalensis @ 100 g/pot; 84.93 and 19.55 m C. procera

(50 g) and the lowest fresh and dry weight was observed 59.40 and 14.70 g in F.

elastica (50 g) respectively as against 43.80 and 11.30 g in untreated .inoculated

and 107.65 and 30.56 g respectively in untreated uninoculated control plants

(Table-10a,Figure-10).


1.361, 1.342 and 1.034 mg/g in C. procera (100 g), E. cotonifolia (100 g), F.

46

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benghalensis (100 g), C. procera (50 g), E. cotonifolia (50 g) and F. elastica (50

g) respectively as against 0.903 mg/g in untreated inoculated and 1.891 mg/g in

untreated uninoculated control (Table-10 b).

The adult female of M javanica per root system was 42.73, 56.12, 59.70,

62.67 and 90.00 in C. procera (100 g), E. cotonifolia (100 g), F. benghalensis

(100 g), C. procera (50 g) and F. elastica (50 g) respectively as against 143.00 in

untreated inoculated control (Table-10 b).

The nematode population per kg soil was lowest 1380.30 when plants

treated with chopped leaves of C procera @ 100 g/pot and highest was 3062.00 in

F. elastica leaves applied @ 50 g/pot as against untreated inoculated (8012.00)

(Table-1 Ob).

4.11. Effect of chopped leaves of different plant species on the root-knot

development caused by Meloidogyne javanica and plant growth of bottle

gourd cv. 'Kanchan' in pots



javanica and also improved the growth parameters of bottle gourd. The highest

reduction in the root-knot development was observed in soil amended with Datura

stramonium (50 g), 1.60; it was followed by Argemone mexicana (100 g), 1.70; D.

stramonium (50 g), 1.75; Lantana camara (100 g), 1.87; A. mexicana (50 g), 1.94,

Parthenium hysterophorus (100 g), 2.00; L. camara (50 g), 2.10; P. hysterophorus

(50 g), 2.24; Withania somnifera (100 g), 2.60; W. somnifera (50 g), 2.75. In

47

untreated inoculated pots the root-knot development was highest (4.90). The

reduction in the root-knot development by the application of chopped leaves

resulted in a better growth of the plant.

The total length was observed highest when the plants were treated with

chopped leaves of D. stramonium (100 g), 189.48 cm. It was followed by plants

treated withv4. mexicana (100 g), 180.44 cm; D. stramonium (50 g), 175.56cm; L

camara (100 g), 171.94 cm; A. mexicana (50 g), 171.19 cm; P. hysterophorus

(100 g), 165.40 cm; L camara (50 g), 162.65 cm; P. hysterophorus (50 g), 153.81,

W. somnifera (100 g), 153.34 cm; W. somnifera (50 g), 142.66 cm respectively as

against 65.72 cm in untreated inoculated control . In untreated uninoculated

control the plant length was highest 193.58 cm (Table 11 a,Figure 11).

The total fresh weight was observed highest when the plants were treated

with D. stramonium (100 g), 104.96 g. It was followed by A. mexicana (100 g),

101.34 g; D. stramonium (50 g), 93.84 g; L camara (100 g), 85.57 g; A. mexicana

(50 g), 82.64 g; P. hysterophorus (100 g), 74.41 g and lowest fresh weight was

observed in W. somnifera (50 g), 60.64 g as against 43.80 g in untreated inoculated

control plants (Table-11 a. Figure-11).

The total dry weight was observed highest when plants were treated with D.

stramonium (100 g), 27.47 g. It was followed by A. mexicana (100 g), 25.36 g; D.

stramonium (50 g), 24.24 g; L camara (100 g), 19.81 g; A. mexicana (50 g), 18.31

g; P. hysterophorus (100 g), 17.16 g and lowest dry weight was observed m W.

somnifera (50 g), 11.59 g as against 11.30 g in untreated inoculated control plants

48

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In untreated uninoculated pots the dry weight was highest 30.56 g (Table-11a,

Figure-11).


1.647, 1.643, 1.572 and 1.490 mg/g in D. stramonium (100 g), A. mexicana (50 g),

P. hysterophorus (100 g) and W. somnifera (50 g) respectively as against 0.903

mg/g in untreated inoculated and 1.891 mg/g in untreated uninoculated control

plants(Table-llb).

The adult female of M. Javanica per root system was 48.30, 54.00, 56.00,

61.00, 62.73, 68.05 and 84.12 in Z). stramonium (100 g), A. mexicana (100 g), D.

stramonium (50 g), L camara (100 g), A. mexicana (50 g), P. hysterophorus (100

g) and W. somnifera (50 g) respectively as against 143.00 in untreated inoculated

control (Table-lib).

The nematode population per kg soil was 1329.47, 1724.47, 1823.12,

2278.00, 2440.00, 2815.20 and 3879.00 in D. stramonium (100 g), A. mexicana

(100 g), D. stramonium (50 g), L camara (100 g), A. mexicana (50 g), P.

hysterophorus (100 g) and W. somnifera (50 g) respectively as against 8012.00 in

untreated inoculated control plants (Table-11 b).

4.12. Effect of soil amendment with urea coated with different commercial

Neem products on the root-knot development caused by Meloidogyne javanica

and plant growth of bottle gourd cv.'Kanchan' in pots

The results presented in Table-12 a & b clearly indicate that the treatments


49

javanica and also improved the growth parameters of bottle gourd. In untreated

inoculated control, the root-knot index was maximum (4.90). The highest

reduction in root-knot development was observed in plants treated with higher

dose (triple strength) of Nimin (1.64). This was followed by Achook (1.76), Neem

kernel oil (1.92) and Neem Raj (2.00). All these treatment when applied at their

lower doses also inhibited the root-knot development, however to varying extent

The total length was observed highest when plants treated with higher dose

(Triple strength) of Nimin (184.52 cm). This was followed by Achook (180,62

cm), Neem kernel oil (175.50 cm) and Neem Raj (170.90 cm) respectively. When

the plants treated with double strength of dose, then the length was m Nimm

(166.00 cm), Achook (159.82 cm), Neem kernel oil (155.94 cm) and Neem Raj

(150.35 cm) respectively. The lowest plant length was observed in Neem Raj

(135.67 cm) when applied at their single strength as against 65.72 cm in untreated

inoculated control (Table-12 a, Figure-12).

The total fresh and dry weight was observed highest when plants treated

with higher dose (triple strength) of Nimin (103.64 and 28.20 g). This was

followed by Achook (94.50 and 27.23 g), Neem kernel oil (89.55 and 25.42 g) and

Neem Raj (87.26 and 24.27 g) as compared to untreated inoculated 43.80 and

11.30 g (Table-12 a, Figure-12).

The total chlorophyll content (leaf) was observed highest when plants

treated with higher dose (triple strength) of Nimm (1.792 mg/g). This was

followed by Achook (1.758 mg/g), Neem kernel oil (1.702 mg/g) and Neem Raj

50

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(1.660 mg/g) as compared to untreated inoculated plants (0.903 mg/g) (Table-

12b).

The adult female of M. javanica per root system was observed lowest when

plants treated with higher dose (Triple strength) of Nimin (41.00). This was

followed by Achook (45.34), Neem kernel oil (50.00) and Neem Raj (56.34) as

compared to untreated inoculated control plants (143.00) (Table-12 b).

The nematode population per kg soil was observed lowest when plants

treated with higher dose (triple strength) of Nimin (1318.67), followed by Achook

(1669.67), Neem kernel oil (1894.67) and Neem Raj (2089.00) as compared to

untreated inoculated control (8012.00) plants (Table-12 b).

4.13. Effect of oilcakes (undecomposed), nematicide and biocontrol agents on

the root-knot development caused by Meloidogyne javanica and plant growth

of bottle gourd cv. 'Kanchan' in pots

The results presented m Table-13 a & b clearly indicate that all the

treatments brought about significant reduction in the root-knot development

caused by M. javanica and also improved the growth parameters of bottle gourd

The highest reduction in the root-knot development was observed in soil amended

with Neemcake (50 g) + Trichoderma harzianum (1.62). This was followed by

Neem cake (50 g) + Paecilomyces lilacinus (1.90); Neem cake (50 g) + carbofliran

(2.10); Neem cake alone (2.43); mustard cake (50 g) + T.harzianum (2.50), cotton

cake (50) + T.harzianum (2.65) and the lowest reduction was observed m cotton

51

cake alone (3.30) respectively as against 4.90 in untreated inoculated control

plants.


Neem cake (50 g) + T.harziamim (180.27 cm). It was followed by Neem cake (50

g) + P.lilacinus (175.95 cm); Neem cake (50 g) + carbofuran (174.96 cm); Neem

cake alone (169.51); cotton cake (50 g) + T.harziamim (153.90 cm); mustard cake

(50 g) + P.lilacinus (147.58 cm) and cotton cake alone (102.50 cm) respectively as

against 65.72 cm m untreated inoculated and 193.58 cm m untreated uninoculated

control plants (Table-13a, Figure-13).

The total fresh and dry weight of plant was observed highest when plants

were treated with Neem cake + T.harzianum (97.64 and 26.86 g). It was followed

by Neem cake + P.lilacinus (92.68 and 25.15 g); Neem cake + carbofriran (89.43

and 24.00 g); Neem cake alone (84.41 and 21.96 g); mustard cake + T.harzianum

(86.89 and 23.97 g) and lowest was observed in cotton cake alone (54.87 and

12.91 g) respectively as against 43.80 and 11.30 g in untreated inoculated plants.

In untreated uninoculated the fresh and dry weight was 107.65 and 30.36 g

respectively (Table-13 a, Figure-13).

The chlorophyll content (leaf) was observed highest when plants were

treated with Neem cake + T.harzianum (1.664 mg/g), followed by Neem cake +

P.lilacinus (1.601 mg/g) Neem cake + carbofuran (1.572); Neem cake alone

(1,460 mg/g) and lowest observed in cotton cake alone (1.083 mg/g) respectively

52

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as against 0.903 mg/g in untreated inoculated and 1.891 mg/g in untreated

uninoculated control plants (Table-13 b).

The adult female of M javanica per root system was observed lowest when

plants treated with Neem cake + T.harzianum (50.00) and highest in cotton cake

alone (125.34) respectively as against 143.00 in untreated inoculated control plants

(Table-13 b).

The nematode population per kg soil was lowest in Neem cake +

T.harzianum (1311.37) and highest in cotton cake alone (5164.67) respectively as

against 8012.00 in untreated inoculated control (Table-13 b).

4.14. Effect of oilcakes (decomposed), nematicide and biocontrol agents on the

roOt-knot development caused by Meloidogyne javanica and plant growth of

bottle gourd cv. 'Kanchan' in pots

The results presented in Table-14 a & b clearly indicate that all the

treatments brought about significant reduction in the root-knot development

caused by M. javanica and also improved the growth parameters of bottle gourd.

The highest reduction in the root-knot development was observed in soil amended

with Neem cake (50 g) + Trichoderma harzianiim (1.50). This was followed by

Neem cake (50 g) + Paecilomyces lilacimts (1.65); Neem cake (50 g) + carbofuran

(2.00); Neem cake alone (2.20); mustard cake (50 g) + T.harzianum (2.40), cotton

cake (50 g) + T.harzianum (2.52) and the lowest reduction was observed in cotton

cake alone (3.20) respectively as against 4.90 in untreated inoculated control

plants.

53


Neem cake (50 g) + T.harzianum (188.35 cm). It was followed by Neem cake (50

g) + P.lilacinus (183.24 cm); Neem cake (50 g) + carbofuran (180,67 cm); Neem

cake alone (177.02 cm); cotton cake (50 g) + T.harzianum (164.53 cm); mustard

cake (50 g) + P.lilacinus (154.32 cm) and cotton cake alone (109.85 cm)

respectively as against 65.72 cm m untreated inoculated and 193.58 cm m

untreated uninoculated control plants (Table-14a, Figure-14).

The total fresh and dry weight of plant was observed highest when plants

were treated with Neem cake + T.harzianum (105.42 and 28.47 g). It was followed

by Neem cake + P.lilacinus (102.35 and 26.16 g); Neem cake + carbofuran (98.12

and 25.14 g); Neem cake alone (95.08 and 23.62 g); mustard cake + T.harzianum

(93.67 and 26.39 g) and lowest was observed in cotton cake alone (61.66 and

13.99 g) respectively as against 43.80 and 11.30 g in untreated inoculated. In

untreated unmoculated the fresh and dry weight was 107.65 and 30.36 g

respectively (Table-14 a, Figure-14).

The chlorophyll content (leaf) was observed highest when plants were

treated with Neem cake + T.harzianum (1.720 mg/g) followed by Neem cake +

P.lilacinus (1.684 mg/g); Neem cake + carbofuran (1.625); Neem cake alone

(1.536 mg/g) and lowest observed in cotton cake alone (1.127 mg/g) respectively

as agamst 0.903 mg/g in untreated inoculated and 1.891 mg/g m untreated

unmoculated control plants (Table-14 b).

54

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plants treated with Neem cake + T.harzianum (44.34) and highest in cotton cake

alone (121.00) respectively as against 143.00 in untreated inoculated control plants

(Table 14 b).

The nematode population per kg soil was lowest in Neem cake +

T.harzianum (1302.87) and highest in cotton cake alone (5015.00) respectively as

against 8012.00 in untreated inoculated control (Table-14 b).

55

*i

Chapter 5 Discussion

^

CHAPTER-5

DISCUSSION

The root-knot nematode {Meloidogyne spp.) occupy a paramount position

as they have wide host range, worldwide distribution and interaction (disease

complex) (Sikora and Fernandez, 2005) with bacteria, fungi and viruses make

them potentially serious constraints and limiting factors to the crop produce. They

reduce yield and quality of susceptible vegetable and other crops (Jonathan and

Hedwig, 1991). The plant parasitic nematodes severely caused damage to the

cultivated crops (Sasser and Frackman, 1987).

Majority of plant species (member of Family- Solanaceae and

Cucurbitaceae) that account for the major world's food supply are susceptible to

attack from root-knot nematode which are capable of causing sustamable

economic losses in the quantity and quality of the crops.

The root-knot nematodes (Meloidogyne spp.) cause an annual monetary

loss to the tune of Rs. 547.50 million in cucurbits (Jain et ai, 2007). The use of

chemicals is not sustainable, it affects the agro-ecosystem adversely and have

toxic effect to human health (Noling and Becker, 1994; Jesse and Jada, 2004).

Attentions have been diverted to standardize methods for eco-friendly

management of nematodes. All these factors led to a search for safer and more

compatible alternatives among which the nematostatic compounds from the plants

or plant products. These naturally occurring compounds contain polythienyls,

isothiocyanates and glucosinolates from Brassicaecae (Brown and Morra, 1997),

56

polyacetylenes from Asteraceae (Kogiso et al, 1976), Alkaloids (Matsuda et al.

1989), phenolics (Evans et al, 1984) and pentacyclic triterpenoids from Laniana

camara (Qamar et al, 2005) show nematicidal activity products are of first

importance, for the management of plant parasitic nematodes (Chitwood, 2002),

Hence the present studies were undertaken to evaluate the nematicidal

potential of various plant parts (leaf, flower, fruit and root), Neem products,

nemticide (carbofuran), oil cakes viz.. Mustard cake, cotton cake and Neem cake

and frmgal biocontrol agents viz., Trichoderma harzianum and Paecilomyce.s

lilacinus against the root-knot nematode.

One of the cheapest and effective methods of altering the soil environment

is its amendment with decomposable organic matter. The materials used for soil

amendment include dry or green crop residues, oil cakes and other organic wastes.

The materials are allowed to decompose in the field. Decomposing products are

also helpful in increasing the predacious or parasitic activity of soil biota, change

in the physical and chemical properties of the soil .However, the efficacy of an

organic amendment against plant parasitic nematode depend on many different

factors including the nematode species present (McSorley and Gallaher, 1996).

In in vitro studies it was observed that leaf and flower extract of all the test

plants were found highly deleterious to root-knot nematode. It proves that they

have strong nematicidal properties.

57

. „ r o « m found effective as the

latex coated seeds

. « , . o t a e v e . P ™ e n t ( N a * . . . . ^ j ^ ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ , , , , „ . . e . .

rnc^c^er^a haniann. were found mos ^ ^ ^ ^ ^ ^^^^^^^ ^^ ^,^^

The other plants spec.es were ^ _ ^ ^ ^ ^

thenlantgro«th«henamendmgtnso,l. ,a,Ung and enhance the plants. ^^„„„ , ^ e ) b.ocontrol agents and

Trichoderma harziamm and Pa c/Zomj „ „ s h o w e d h e t t e r r e s . t , n c o — „ w . * N e e » c a . e . c o . p a r e d t o P .

, „ an. reduce the adult female per root s,ste. and nen^tode popu.at.on .

the soil.

Thus .t can be concluded after *e .horou^ study of above mentioned

observations *at extracts of plant spec.es. 0.1 cakes and biocontrol agents alone

» d m comb.nat.on w.th nematicde could be of great use m solvmg some of the

nematode problems. These above test plants can be used for mtegrated and

biological control strategies.

58

http://spec.es

http://popu.at.on

http://spec.es

^ t

Chapter 6 Summary

J

CHAPTER-6

SUMMARY

Water extracts of leaves and flowers of Datura stramonium and Calotropis

procera, were found highly deleterious to the root-knot nematode, Meloidogyne

javanica, however other test plants were also effective to varying extent. All the

water extracts tested, significantly inhibited the juvenile hatching of root-knot

nematode. There was a direct relationship between mortality of root-knot

nematode and the exposure period. The mortality increased with an increase in the

concentration of the extract and exposure period.

Soil amended with chopped leaves of Datura stramonium (lOOg);

Calotropis procera; Nimin (Triple strength) and Neem cake (50g) with

Trichoderma harzianum were found highly satisfactory in reducing the root-knot

development caused by Meloidogyne javanica on bottle gourd cv. 'Kanchan'

Highest reduction in root-knot development was observed in decomposed Neem

cake (50g) in combination with Trichoderma harzianum (1.50) and lowest in

Ficus elastica (50g) alone (3.40). As a consequence in reduction in root-knot

development the plant growth in terms of length (cm), fresh and dry weight (g),

chlorophyll content (mg/g) also increased. The highest reduction in nematode

population per Kg soil was observed in decomposed Neem cake (50) in

combination with T.harzianum (1302.87), followed by undecomposed Neem cake

in combination with T.harzianum (1311.37).Other treatments were also showed

nemato-toxic effect against the root-knot nematode.

59

When the latices of different plant species, among all used, the latex of

Calotropis procera found to reduce the root-knot infestation (1.91) and increased

the plant length (170.40cm) as against untreated inoculated control (65.72 cm)

Among various commercial Neem products, the higher dose of Nimm

(triple strength) found to be more effective (root-knot index-^1.64, nematode

population /kg soil= 1318.00) in controlling nematode damage followed by triple

strengths of Achook (1.79 and 1669.67), Neem kernel oil (1.92 and 1894.67) and

Neem raj (2.00 and 2089.00) respectively.

60

r 1

References

tS

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