STUDIES ON HISTOPATHOLOGICAL TRANSFORMATIONS INDUCED BY

Meloidogyne incognita IN SOME SELECTED CUCURBITS

DISSERTATION submitted in partial fulfilment of the requirements

for the award of the degree of

MASTER OF PHILOSOPHY IN

BOTANY

BY

MOHD. YAQUB BHAT

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 1994

DS2565

^^2.5"<^5"' > . "̂

It is Allah who sendeth down Rain from the skies:

With it We produce Vegetation of all kinds:

From some We produce Green (crops), out of which We produce grain, Heaped up (at harvest)

And (then there are) gardens Of grapes, and olives, And Pomegranates, Each similar (in kind) Yet different (in variety):

When they begin to bear fruit. Feast your eyes with the fruit And the ripeness thereof Behold ! in these things There arfe Signs for people Who believe. (SURA VI Vol. 99)

It is Allah Who causeth The seed-grain And the date-stone To split and sprout. (SURA VI Vol. 95

ZIAUDDIN A. SIDDIQUI M. Sc . Ph D. (Alig.)

Professor of Botany

Section of Plant Pathology and Nematolog

Department of Botan

Phone :0571-25676

ALIGARH MUSLIM UMIVERSITY, ALIGARH-202 002, INDIA

TO WHOM IT MAY CONCERN

Jh\s \s to certift̂ that the work embodied m this Dissertation entitled "Studies On Histopcrtfiologica/ Transformations \v\ducedByN\e\o'\doQyY\e\Y\coQmta In Sortie Selected Cucurbits" \s the bonafide work carried out by A/lr. /Wohd. Yaqub Bfiat \Ar\der rY\y s[Apery\s\or\ and is suitable for submission for the A/l.Phil. Degree of Aligarh l\Aus\\n\ University, Aligarh.

DATE: 24/6/1995 Prof. Ziauddin A. Siddiqui

ACKNOWLEDGEMENT

I w i t h i n s u f f i c i e n t and i n c o m p e t e n t words

e x p r e s s my s i n c e r e s t g r a t i t u d e t o my es t eemed

s u p e r v i s o r Dr. Ziauddin Ahmad Siddiqui Professor,

Department of Botany, A l i g a r h Muslim U n i v e r s i t y

Aligarh, who insp i red me to i n i t i a t e the research work

in the d i s c ip l i ne of p lant Nematology. I am highly

indebted to him for h i s s k i l l f u l guidance, constant

encouragement, sympathetic a t t i t u d e . Zealous i n t e r e s t

and a f f e c t i o n and o v e r a l l s u p e r v i s i o n d u r i n g t h e

p r e p a r a t i o n of t h i s manusc r ip t .

I extend my h e a r t f e l t sense of g r a t i t u d e to Dr.

Hisamuddin, L e c t u r e r , Department of Botany, A.M.U.

Aligarh, for h i s cons t ruc t ive c r i t i c i sm and valuable

suggestions and sparing h is precious time for going

through t h i s manuscript.

I am i n d e b t e d t o Professor Wazahat Husain,

Chairman, Department of Botany, A.M.U. Al igarh, for

providing me a l l the necessary f a c i l i t i e s poss ib le in

the department.

I t i s p leasurable render my s incere thanks and

a p p r e c i a t i o n t o Dr. Syed Munawar Fazal, Research

a s s o c i a t e , Department of Botany, who sac r i f i ced his

v a l u a b l e t ime d u r i n g t h e p r e p a r a t i o n of t h i s

manusc r i c t .

My humble thanks are also due to my parents,

brother and sisters and Mr. Nazir Ahmad Bhat, who have

kindled the flame of learning in me and whose

affection, encouragements and sacrificial devotions I

ascribe all my success.

Special thanks are extended to my friends and

colleagues M/s M. Imran Khan, M. Imran, Kunhalvi, M.

M. Mobin, M. Manzar, Imtiaz Ahmad, A. Hamid, Anvar

Javed, M. Ashiq, Ms Shreerali Patwari, Rajesh

Tripathi, Miss Regina, and Fahemida Hassan for their

whole hearted co-operation and encouragement.

I am highly thankful to Mr. Muneer Uddin for

the timely preparation of this Manuscript.

Finally, I am thankful and Bow in gratitude to

the Almighty Allah, who provided and guided all the

channels to work in cohesion and coordination and led

me to this path of success.

(MOHD. YAQUB BHAT)

CONTENTS

Page No.

Introduction 1-11

Plan of Work 11

Review of Literature 12-29

Materials and Methods 30-42

References 43-62

Introduction

IN THE NAME OF ALLAH THE MOST BENEFICENT THE MOST MERCIFUL

INTRODUCTION

The annual production of vegetables in India is

estimated to be around 45 million tonnes from a

cropped area of four million hectares, excluding

potato and tubers (Singh, 1991) .

The per capita availability of vegetable per day

in India is very low. The low per capita consumption

is mainly due to the low productivity levels in

vegetable crops. The vegetables are one of the

important items for export. The vegetables exported

are okra, bitter gourd, bottle gourd, round gourd,

chillies, green peas, cauli flowers, radish, pointed

gourd, and sponge gourd. It means that mostly

cucurbits are exported. The cucurbits are of trailing

habit and are cultivated during summer and rainy

seasons.

The family cucurbitaceae includes 90 genera and

700 species. Majority of the species are herbaceous

annual climbers. The cucurbit fruits are largest known

in the plant kingdom. Fruits of cucurbits are

extensively used as vegetables and consumed both when

young and ripe. They are used as fresh or processed

food. Fully matured fruits are also used for preparing

jams and sweets and fermented to give beverages. Some

varieties are excellent for culinary use during early

2

Stages of maturity and very poor for the same use at

later stages while the reverse may be true of other

varieties (Culpepper and Moon, 1945).

The roots of bitter gourd are astringent and are

used medicinally. They are applied in bleeding piles,

bowel affections and urinary complaints. The roots are

pasted and applied over the body as sedative in fevers

(Dymock et. al. , 1890-99; Kirt and Basu, 1935).

Fruits and leaves are used in external

application for lumbago, ulceration and fracture of

bones. In Honkong the plants are used as an

alternative bitter drug in place of strychnos (Burkill

II, 1486), Brown 1946; III, 399, Rama Rao 1914, Dymock

et al., 1890-99) .

The soil harbours a large number of plant

pathogenic organisms such as bacteria, fungi,

actinomycetes, insects, and nematodes. Cucurbit crops

are attacked by a number of diseases of which the

nematode diseases are of economic importance. The

plant parasitic nematodes are one of the most

important pathogens affecting cucurbits. The nematode

diseases not only cause yield loss but also reduce the

ability of infected roots to utilize available

nutrients.

The root-knot diseases are caused by Meloidogyne

spp. the most important pathogen of cucurbits. The

3

p a r a s i t e u s u a l l y p e n e t r a t e s u n d e r g r o u n d p a r t s of

p l a n t s where i t induces the development of abnormal

growth in r o o t s . Sometimes unusua l ly l a r g e g a l l s a r e

developed a t the base of stem ( S t e i n e r e t . al., 1934).

The s ize and charac ter of these enlargements vary in

d i f f e r e n t p l a n t s , as in Thunbergia laurifolia and

r h u b a r b enormous s t r u c t u r e s n e a r l y two f e e t i n

d i a m e t e r may be s e e n . Roo t -kno t nematodes a r e

s e d e n t a r y e n d o p a r a s i t e s . The female nematode remains

wholly embedded in the va scu l a r c y l i n d e r . I n f e c t i o n

with r o o t - k n o t nematode s t i m u l a t e s t h e format ion of

v a r i a b l e number of d i s c r e t e g i a n t c e l l s i n h o s t

t i s s u e s . H y p e r p l a s t i e d and h y p e r t r o p h i e d c e l l s o f t en

s u r r o u n d t h e r e g i o n of i n f e c t i o n c a u s i n g g a l l s

t e r m i n a l l y and s u b t e r m i n a l l y in the i n f e c t e d r o o t s .

Kostoff and Kendall (1930) be l i eved t h a t g i a n t c e l l s

were formed by w a l l d i s s o l u t i o n of a f f e c t e d c e l l s

fol lowed by coa l e scence of c e l l c o n t e n t s . Huang and

Maggenti (1969) in t h e i r d e t a i l e d s tudy on g i a n t c e l l s

development in Vicia faba roots found no evidence of

c e l l w a l l d i s s o l u t i o n in t he s t i m u l a t e d c e l l and

concluded t h a t g i a n t c e l l o r i g i n a t e d from s i n g l e c e l l .

They observed t h a t m u l t i n u c l e a t e c o n d i t i o n of g i a n t

c e l l s a rose from r epea t ed mi tcses wi thou t c y t o k i n e s i s

of a s i n g l e d i p l o i d c e l l . In a g ian t c e l l the n u c l e a r

changes may range from a nucleus having one s e v e r e l y

4

hypertrophied nucleolus to nuclei with various stages

of membrane deterioration and lobulated periphery. The

cytoplasm of the young giant cell becomes dense.

Christie (1936) found dense cytoplasm near the head

region of the nematode than the remainder of giant

cell cytoplasm. As the nematode matures and nutrient

demand from giant cell increases, the giant cell

cytoplasm shows regions of intense metabolic activity.

The giant cell cytoplasm disintegrates after egg

laying of the nematode. Formation of giant cell and

multiplication of pericycle and other parenchyma cells

around the nematode head cause the vascular tissue to

divert from its normal path. Xylem and phloem also

become abnormal with reference to structure and

orientation. Small as well as hypertrophied parenchyma

cells are transformed into vessel like elements and

sieve tube elements and constitute the abnormal xylem

and phloem. Since giant cells are highly metabolically

active cells, therefore, they must directly or

indirectly be connected with the vascular tissues.

Ediz and Dickerson (1976) found most of the

giant cells in phloem region. The root-knot nematode

readily penetrates the plant root near the apical

meristem (Nemec, 1910) within 24 hours after

inoculation (Siddiqui and Taylor, 1970). However other

regions of the root are not immune to attack, Christie

(1936) .

5

Once in side the root tze migration occurs both

inter and intracellularly, (Nr-aec, 1910; Krusberg and

Nielson, 1956; Christie, 192~)_ Nemec (1910) found

that the division of giant cell xiuclei takes place by-

mitoses, but early in the life of giant cell, nuclei

may coalesce. He also found tiiat the nuclei were

fusing in pairs or in the case older cells, all the

nuclei might be massed together mear the center of the

cell. The cytoplasm of young giant cells (within 48

hours) becomes dense and encloses a large vacuole. The

vacuole diminishes and cytoplasoi becomes more dense as

well as granular with the gian. cell development.

Christie (1936) fou-d more dense and

hyperchromatic cytoplasm nenr the head region of

nematode than remainder of th= giant cell cytoplasm.

Nuclei become highly lobed azi heterochromatic with

numerous prominent nuclear poxes. The cytoplasm

becomes vacuolated and vacuclation increases when

nematode reaches it's maturity. The giant cell

cytoplasm is disintegrated af.er egg laying of the

nematode. Nuclear changes ranged from a nucleus near

the stylet of nematode which shewed a hypertrophied

nucleolus with the apparent absence of nuclear

membrane to nuclei with varies stages of membrane

deterioration and a lobulated -eriphery. Irregularly

shaped, dumb-bell or sickle shaped nuclei were

6

o b s e r v e d on r o o t - k n o t i n f e c t i o n of sweet p o t a t o

(Krusberg and Nie l son , 1958) . Nuclear enlargement in

g i a n t c e l l s of r o o t - k n o t nematode i n f e c t e d tomato,

cucumber and hawk ' s - b e a r d Crepis capillaries

appeared to r e s u l t from swelling and in some cases

from nuclear fusion (Owens and specht, 1964).

Molliared (1900) observed g a l l s on the root of

melon Coleus and Begonia and reported tha t a f t e r

i n v a s i o n t h e r o o t t i p g rowth may be a r r e s t e d and

l a t e r a l r o o t s f r e q u e n t l y developed nea r the r eg ion of

i n v a s i o n . He a l s o r e p o r t e d i r r e g u l a r i t i e s in the form

and t h e number of g i a n t c e l l n u c l e i and t h e i r

s u b s e q u e n t c o a l e s c e n c e and d i s i n t e g r a t i o n . Dur ing

e a r l y s t a g e s of development the d i v i s i o n of g i a n t c e l l

n u c l e i i s by normal m i t o s e s . A f t e r c e r t a i n p e r i o d

a m i t o t i c d i v i s i o n and f r a g m e n t a t i o n a r e f o l l o w e d .

G a l l s are p a t h o l o g i c a l l y developed c e l l s , t i s s u e or

o r g a n s of p l a n t s t h a t have a r i s e n m o s t l y by

hyper t rophy and h y p e r p l a s i a under the i n f l uence of

p a r a s i t i c o rgan isms .

Schus te r and S u l l i v a n (1960) r e p o r t e d t h a t g a l l s

were induced in tomato by Meloidogyne incognita larvae

even when they did not enter the tomato r o o t s . They

c o n c l u d e d t h a t s t y l e t p e n e t r a t e d t h e r o o t s u r f a c e

c e l l s and s e c r e t e d m a t e r i a l s t h a t s t i m u l a t e d h o s t

t i s s u e t o form g a l l s . Dav i s and J e n k i n s (1960)

7

r e p o r t e d g a l l format ion in Gardenia spp. infected with

Meloidogyne incognita; M. incognita acrita and M.

hapla. Cor t ica l and s t e l a r p r o l i f e r a t i o n accompanied

a l l i n f ec t i ons .

Huang (1966) reported cork and l i g n i f i e d wall

thickening of endodermis and per icyc le a t in fec t ion

s i t e s in Zingiber officinale. Division of s t e l e in

root g a l l s of Lycopersicon pimpinellifolium infected

with M. incognita was observed.

J o n e s (1981) Pasha e t . al., (1987) reported

format ion of abnormal xylem in p l a n t s i n f e c t e d with

r o o t - k n o t nematode, Pasha e t . al., (1987) found tha t

abnormal xylem o c c u r r e d i n i r r e g u l a r p a t c h e s w i t h

s c a t t e r e d v e s s e l e l e m e n t s and r e s u l t e d i n

d i s c o n t i n u i t y of v a s c u l a r t i s s u e s and s i g n i f i c a n t

r e d u c t i o n in v e s s e l d imension.

P a t e l and P a t e l (1991) r e p o r t e d t h a t t h e

immature s t a g e s of nematode p e n e t r a t e d f r e e l y i n t o the

e p i d e r m i s and r e a c h e d t h e endodermal l a y e r

i n t e r c e l l u l a r l y through the c o r t i c a l c e l l s in p igeon

p e a . The c e l l s s u r r o u n d i n g t h e f e e d i n g s i t e were

d a r k e r and t h i c k e r t h a n normal c e l l s . G ian t c e l l

i n i t i a l s were observed near the head of the nematode

most ly in phloem parenchyma and r a r e l y in p e r i c y c l e

and xylem parenchyma in pea s e e d l i n g s , t h r e e days

a f t e r i n o c u l a t i o n of r o o t - k n o t nematode. The g i a n t

c e l l s bore appendages which grew i n t r u s i v e l y among the

ne ighbour ing c e l l s . Wall ingrowth or p ro tube rances

f o r m a t i o n in g i a n t c e l l s may i n d i c a t e a form of

t r a n s f e r c e l l . Sharma and Teobi (1989) and Da l la e t .

al . , (1991) reported the giant c e l l formation, 72

h o u r s a f t e r i n o c u l a t i o n i n t h e xylem and phloem

parenchyma i n Vigna radiata and Cyamopsis

tetragonaloba.

Hisamuddin and Siddiqui (1992) observed 5 to 6

g i a n t c e l l s i n r e g i o n of v a s c u l a r d i f f e r e n t i a t i o n

w i t h i n 24 hours of i n o c u l a t i o n in Luffa cylindrica.

Abnormal xylem formation progressed incessan t ly as the

g a l l grew older and annexed most of the parenchyma.

S u r r o u n d i n g t h e g i a n t c e l l s , howeve r , i s o l a t e d

abnormal v e s s e l s were wi tnessed even a f t e r 40 days of

i n o c u l a t i o n .

R e s i s t a n c e denotes t he g r e a t l y reduced r a t e of

r e p r o d u c t i o n of the p a r a s i t e on r e s i s t a n t c u l t i v a r .

In r e s i s t a n t r o o t s l a r v a e may e i t h e r f a i l to e n t e r or

e n t e r i n r e d u c e d numbers w i t h v a r y i n g d e g r e e s of

deve lopmen t ( B a r r e n s , 1939; C h r i s t i e , 194S) .

H y p e r s e n s i t i v i t y i s a common t y p e of r e s p o n s e of

r e s i s t a n t p l a n t s (Riggs e t . al., 1959; Rohde, 1965).

The larvae may enter the root of r e s i s t a n t p l an t s in

large numbers but hypersensi t ive c e l l s quickly die and

w a l l off t h e p a t h o g e n so t h a t i n j u r y t o h o s t i s

9

confined to a few cells (Riggs and Wintesel, 1959) .

This results in the subsequent death of nematode. The

hypersensitive reaction in some cases may be delayed

until after the parasitic relationship. Linford

(1939) reported penetration, development and migration

of cotton root-knot nematode, Meloidogyne incognita,

acrita in resistant and susceptible alfalfa varieties.

He found no development of nematode in resistant

cultivar but egg production began after 18 days of

penetration in susceptible cultivar. Attractiveness of

Meloidogyne species juveniles was observed towards the

root and excised shoot tissues of several host plants.

The juveniles of Meloidogyne hapla were equally

attracted towards the resistant and susceptible

alfalfa seedlings when an egg mass was placed mid way

between germinating seedlings (Griffin and Waite,

1971) . In Resistant varieties the juveniles of root-

knot nematode either did not enter or if entered, they

did not develop properly (Sasser and Taylor 1952).

Resistance in plants towards root-knot nematode may

develop with age (Griffin and Hemt 1972).

Three types of histological responses were

observed in infected resistant root. Some galls

contain fragments of nematode others contained no

detectable traces of developing larvae. Formation of

druses in galls but not in healthy tissues was ncted

10

in both cultivars, 20 days after inoculation. Massive

invasion of roots resulted in deep longitudinal

fissures of root cortex (Michael, et. al. , 1973).

Root penetration by second stage juveniles of

Meloidogyne incognita race I and M. Javanica in

resistant germplasm was significantly less than in

susceptible cultivars of vegetables like, cabbage,

cauliflower, cucumber, pepper and tomato (Khan and

Khan 1991) .

Pankaj and Swanand (1992) reported the efficacy

of chemicals as seed dresser against M. incognita in

bitter gourd and round gourd. It caused the inhibition

of larval penetration, female development, and gall

formation on bitter gourd and round gourd.

Fattah and Webster (1983) observed normal giant

cells and fungal infection in one week old inoculated

seedlings of tomato cultivars. After two weeks hyphae

were visible in xylem tissue of Fusarium susceptible

but not resistant plants. Dissolution of giant cell

wall occurred where giant cells were in direct contact

with hyphae. No such changes were seen in the giant

cell of control plant inoculated with nematode alone.

Cabanillas et. al., found no galling and giant cell

formation in tomato roots inoculated in with nematode

eggs infected with P. lilacinus. Few to no galls and

no giant cell formation were observed in roots dipped

11

in spore suspension of P. lilacinus and inoculated

with M. incognita.

The present work will be carried out:

i. To compare anatomical changes leading to the

formation of giant cells and galls, from earlier

findings.

ii. To investigate the origin of giant cells and their

fate after death of the nematode,

iii To find out any link of giant cells with phloem,

iv To observe abnormalities in xylem and phloem.

V. To examine response of giant cells after treatment

with some systemic nematicides in infected roots,

vi To study the response of vascular tissue after

nematicide treatment of the infected roots,

vii To study any resistance towards Meloidogyne

incognita infection in different varieties,

viii To study the difference in giant cells and

vascular tissues in resistant and susceptible

varieties.

ix To study the response of giant cells after

inoculation with Paecilomyces lilacinus in

infected roots.

X To study the response of vascular tissues after

inoculation with P. lilacinus in infected roots.

Review of Literature

REVIEW OF LITERATURE

Root-knot nematode, Meloidogyne spp., are most

important and best known nematodes have evolved very-

specialized and complex host parasite relationship.

Meloidogyne spp. parasitize a wide variety of host

plants belonging to monocotyledons and dicotyledons

including both herbaceous and woody plants, and cause

the formation of familiar knot or gall on roots of

susceptible host. Root-knot nematodes are known to

parasitize more than 2000 species, but host parasite

relationships have been investigated only in few

plants (Webster, 1969, 1975) .

Root galling induced by Meloidogyne spp. is a

well known host response which involves the production

of abnormally large multinucleate cells in the

vascular tissues of susceptible plants. Generally,

root damage caused by parasitic nematodes is reflected

on the above ground portion of the plant as poor shoot

growth, leaf chlorosis and even death of plants

resulting in low yield and poor quality produce.

The above ground symptoms are similar to those

associated with any root injury that results in

reduced amounts of water uptake by plants. Flowering

is scanty and fruits are either lacking or are of poor

quality (Jenkins and Taylor, 1967) . Meloidogyne also

13

interferes with the process of nitrogen fixation in

the nodules of leguminous plants.

Second stage juvenile is the first stage that

infects the plants and starts it's life cycle on

susceptible host. The juveniles penetrate the root

tips (Christie, 1936) and feed on root hairs and

epidermal cells. They accumulate either at the region

of the cell elongation just behind the root cap or at

the points where lateral roots emerge or at the site

of penetration of other juveniles and cut surfaces of

roots (Godfrey and Oliveira, 1932; Linford, 1939,

1942; Peacock, 1959; Bird, 1969; Green, 1971;

Siddiqui, 1971-a, 1971-b; and Prot, 1980). The

mechanism of penetration may involve mechanical action

by thrusting of the stylet or cellulytic and

pectolytic activity of enzymes (Linford, 1942; Bird

and Loveys, 1980).

Primary root penetration by second stage

juveniles occur at the tips of the young root in the

region of differentiation in sweet potato or any where

from root cap back to the region of root hair

formation and also through the loose ruptured cells of

enlarging tuberous roots (Krusberg and Nielson, 1958) .

Siddiqui and Taylor, 1970 found most of the juveniles

penetrating in the region of cell differentiation and

elongation. After penetration intercellular migration

14

of juveniles in the cortex was detected (Nemec, 1910,

Godfrey and Oliviera, 1932; Linford, 1937, 1942; Endo

and Wergin 1973; Jones and Payne, 1978).

During intercellular migration the cells were

separated along the middle lamella. Along the path of

migrating juveniles the cells were distended and

compressed and did not show any sign of rupture or

nematode feeding. Inter and intra-cellular migration

was reported by Christie (1936), Krusberg and Nielson

(1958), Roman (1961), and Bird (1959, 1960, 1962);

Siddiqui and Taylor (1970) and Siddiqui (1971-a,b).

Roman (1961) observed galleries and furrows of broken

and separated cells that indicated intracellular

penetration of juveniles.

Giant cell is a multinucleate transfer cell in

which the multi nucleate condition results from

multiple mitosis in absence of cytokinesis. Formation

of giant cell is not accompanied by wall dissolution

and are invariably formed by root-knot nematodes

(Endo, 1987) . Giant cells are highly specialized

cellular adaptations induced and maintained in

susceptible hosts by the feeding of Meloidogyne spp.

Primary phloem or adjacent parenchyma are the highly

preferred tissues for the development of giant cells

(Christie, 1936 Krusberg and Nielson, 1958; Byrne,

1977) .

15

Beille (1898) was the first who proposed that

giant cells are formed by disintegration of cell walls

and coalescence of cells in papaya roots. Observation

of cell wall fragments in giant cell cytoplasm

strengthened this view (Bird, 1961, 1972; Smith and

Mai 1965) .

Later studies supported this mode of giant cell

formation (Kostoff and Kendall, 1930; Christie, 1936;

Krusberg and Nielson, 1958; Dropkin and Nelson, 1960;

Bird, 1961; Roman, 1961; Krusberg, 1963; Owens and

Specht, 1964; Birchfield, 1965; Littrel, 1966;

Siddiqui and Taylor, 1970; Siddiqui, 1971, a,b; Bird

1972, 1973, 1974), Rohde and Mcclure (1975). Nemec

(1910) proposed a contrasting mode of giant cell

induction by root-knot nematode and reported that

around the nematode head the plerome enlarged, their

cytoplasmic contents increased and the number of

nuclei increased by mitosis without accompanying

cytokinesis. In this way multinucleate giant cells

were formed early in development of gall. Cell wall

break down and cell wall dissolution was not observed

at any stage in giant cell formation. This mode of

giant cell formation was supported by a number of

researchers (Myuge, 1956; Huang and Maggenti, 1969;

Paulson and Webster, 1970; Jones and Northcote, 1972;

Jones and Dropkin, 1976; Jones and Payne, 1978.

16

Huang and Maggenti (1969a,b) observed g i a n t c e l l

i n t h e r o o t s of Vicia faba infected with Meloidogyne

javanica formed by repeated mytoses of the o r ig ina l

d ip lo id c e l l with out subsequent cy tok ines i s . They

repor ted chromosome number as 4n, 8h, 16n, 32n and

64n. I n t e r p h a s e n u c l e i w i t h many chromosomes were

formed due t o fus ion of m i t o t i c appa ra tu s in g i a n t

c e l l e i t h e r a t me t aphase o r a n a p h a s e s t a g e s . Many

i n t e r p h a s e n u c l e i , however , were s i m p l y i n c l o s e

j u x t a p o s i t i o n i n s t e a d of a c t u a l l y b e i n g fu sed as

r e v e a l e d by e l e c t r o n microscopy. The i n t e r p h a s e n u c l e i

were i r r e g u l a r l y lobed and f r e q u e n t l y assumed amoeboid

a p p e a r a n c e . M i t o s e s i n g i a n t c e l l were g e n e r a l l y

s y n c h r o n i z e d . Some n u c l e i i n h y p e r p l a s t i c t i s s u e s

a r o u n d g i a n t c e l l s were a l s o l o b e d and d e f i n i t e l y

s m a l l e r than those of g i a n t c e l l s . The c e l l p l a t e

fo rmat ion was not observed from b i n u c l e a r s t a g e upto

the m u l t i n u c l e a r s t age in g i a n t c e l l s . They d id not

f i n d any e v i d e n c e of c e l l w a l l d i s s o l u t i o n .

Synchronous n u c l e a r d i v i s i o n s w i t h i n t he same g i a n t

c e l l wi thou t daughter c e l l wal l format ion have been

observed in many p l a n t s , (Krusberg and Nie l son , 1958;

Bi rd , 1961; Owens and Specht , 1964; Smith and Mai,

1965) .

J o n e s and Payne (1978). o b s e r v e d g i a n t c e l l

i n d u c t i o n i n r o o t s of Impatiens balsamina by

17

Meloidogyne spp. under light and electron microscope.

They reported that first sign of giant cell formation

was the division of cells around the larval head. Cell

plate alignment appeared to proceed normally but

cytokinesis was unsuccessful and binucleate cells

formed subsequently, no wall break down was evidenced.

Nuclei increased in number by repeated motoses without

cytokinesis. Nuclear divisions were detected 24 hours

after inoculation in the cells upto two cell layer

from head of nematode. After 48 hours 2, 4 and 8

nuclei were observed in the cells near the nematode

head. In early stages of giant cell formation, cell

wall break down and cell fusion were not evidenced

under electron microscope. "Wall gaps or holes in a

continuous cell wall were not detected. Cell wall of a

giant cell consisted of thick and thin areas. The area

of the cell wall where plasmodesmata occur is close to

the limit of resolution of light microscope. This

gives an impression of gaps in the wall. The irregular

outline of individual giant cell at many sites due to

cell wall ingrowth appeared as tylose like structure

which, according to Jones and Payne (1978), was

considered by certain workers as cell wall fragments.

In binucluate cells, 48 hours after inoculation, the

cell plate vesicles were found aligned normally but

dispersed without fusion and thus failed to form

18

daughter cell wall. The cell plates vesicles were also

thought to be the fragments of broken walls.

Krusberg and Nielson (1958) reported young root

tips lateral root ruptures and the surface of cracks

were three major infection courts in sweet potato

roots. After penetration the second stage larvae

migrated inter and intracellularly to the feeding

site. The feeding sites varied with the infection

court. Larvae came to rest primarily in the stele in

the region of cell elongation or in the cambial zone.

Nematode feeding stimulated the formation of several

atypical tissues i.e. giant cells, abnormal xylem,

hyperplastic parenchyma and cork. Giant cells were

initiated first. Cork formed around mature females and

their egg masses and was most abundant in root from

late harvests. Mcclure et. al., (1974) reported three

types of histological responses in infected resistant

roots and were co-related with the degree of nematode

development. Some galls were observed which contained

only fragments of nematodes, while others contained no

detectable traces of developing larvae. Formation of

druses in galls, but not in healthy tissue, was noted

in both resistant and susceptible plants, 20 days

after inoculation. Massive invasion of roots resulted

in deep longitudinal fissures of root cortex. In

sorghum roots, on infection with cotton root-knot

19

nematode, development of giant cells in the cortex or

stele of lateral roots was studied. Giant cells

developed either singly with few nuclei or in groups

with many nuclei. Interruption of pericycle and

endodermal tissue was absent upto the one third of

circumference of stele and extended 1.5 mm

longitudinally along the root. In the area where

pericycle and endodermis were absent, the pericycle

parenchyma of the cortex extended to the vascular

elements and abnormal xylem surrounding giant cells

extended into the region of cortex. Root galls

appeared on sorghum roots as elongated swellings,

discrete knots, or swellings with root proliferation.

(Orr and Morey, 1987) .

Pasha et. al., (1987) reported that feeding site

of nematode was mainly confined to steler region of

roots of Solanum melongena L., resulting from

infection with root-knot nematode Meloidogyne

incognita. Cells in the feeding site showed

hypertrophy, hyperplasia, thickening of cell wall,

granular cytoplasm and elarged nuclei and nucleoli.

Abnormal xylem formed in response to infection by

nematode, occurred in irregular patches with scattered

vessel elements resulting discontinutity of vascular

tissue. The findings supported the view that giant

cell originated from hypertrophy and repeated mitoses

20

without cytokinesis of single stimulated cell.

Penetration leading to intracellular migration

causing cortical hypertrophy, took place within 24

hours after inoculation. Development of giant cell

took place within 4 -5 days around the head of each

nematode in the stele. Initial pathological

alterations in giant cell formation consisted of

protophloem and protoxylem cells. Giant cells

contained 2-8 agglumerated multinucleolate nuclei,

synchrous mitotic divisions were first observed 9 days

after inoculation. After 21 days giant cells becames

highly vacuolated. Observations, 40 days after

inoculation revealed complete degeneration of cell

contents in many giant cells, but their thick walls

remained intact. Abnormal xylem completely surrounded

the degenerated or partially degenerated giant cell

(Siddiqui and Taylor 1969) . Siddiqui (1971) showed

that large number of Meloidogyne naasi larvae

penetrated root tips of Wintok oats within the first

24 hours and first sign of incipient giant cell

formation was visible, four days after inoculation.

Three types of responses were observed (a) necrosis of

inner cortical and endodermal cells in contact with

the nematode lip region and failure of larvae to enter

the stele, (b) necrosis of cells around the incipient

giant cells, resulting in degeneration of giant cells

21

and larvae (c) uninterrupced giant cell development

which is not distinguishable from that in susceptible

host.

Dropkin (1954) showed giant cell development and

maintenance in plants infected with Meloidogyne

javanica depends on continuous stimulus from the

nematode whose removal resulted in break down of the

giant cell. Normal giant cell development took place

under sterile conditions. A growth promoting substance

is present in galls but not in adjacent roots. Roots

of heavily infected plants are often so severely

damaged that individual galls coalesce into amorphous

masses containing large number of nematode. It was

noted that galls on tomato roots were all of about the

same size and number of larvae within a gall can be

predicted by measuring the size of the gall.

Fawali (1988) while observing naturally and

artifically infected white yam tuber with root-knot

nematode found intercellular movement of Meloidogyne

juveniles. Feeding sites were always in the ground

tissue layer where the vascular tissues were

distributed. Sharma and Tiabi (1989)observed giant

cell initials near the head of Meloidogyne incognita

larvae mostly in Phloem parenchyma and rarely in

pericycle and xylem parenchama. Kim and Ohh (1990)

suggested differentiation of tomato xylem vessels with

22

the formation of adjacent giant cells

Subbaton (1990) observed formation of giant

cells after 21 to 32 days in the root of Glycine max

infected with Meloidogyne javanica. Their activity

increased with progressive development of nematode.

The modified cells were enlarged and had larger

numbers of organelles which underwent structural

changes forming Lalyrenth of cell tissues. In the

Stelar region of wheat, root-knot nematode migrated

intracellularly and caused gall formation around its

head. Patel and Patel (1991). Severe damage to the

vascular tissues disrupted the continuity of xylem

tissue. Salawu (1991) observed extensive damage to

epidermis, cortex and vascular tissues due to giant

cell formation in the stele in roots of Celosia

argentia by Meloidogyne incognita. Datta et. al.,

(1991) reported the giant cell formation in xylem and

pholem parenchyma after 72 hours of inoculation by

Meloidogyne incognita in roots of Vigna radiata and

Cyamopsis tetragonolaha.

Khan and Khan (1991) reported siginificant

difference in the penetration of M. incognita race I

and M. javanica in resistant and susceptible germplasm

cultivars of vegetables. Abrantes et. al., (1992)

reported the induction of giant cells in roots of

Olive [Olea europia) by M. javanica. Hypertrophy and

23

hyperplasia were common phenomena in the cortical and

vascular parenchyma. Wyss etal (1992) observed the

invasion of Arabidopsis thaliana roots by Meloidogyne

incognita primarily in the region of elongation

close to the meristematic zone, by destroying

epidermal and subepidermal cells. Inside the root the

second-stage juveniles (J2) oriented them selves

always in the direction of root tip and migrated

towards it, between cortical cells. At the apex of the

root the juveniles turned round and migrated backwards

between cells towards and differentiating vascular

cylinder. Within the vascular cylinder, migration

eventually stopped and the giant cell induction was

intiated. Second-stage juveniles became surrounded by

multinucleate giant cells within about 24 hours. Bird

(1992) laid emphasis on stylet exudate proteins in

establishment and maintainence of giantcells.

Husain et. al., (1992) reported the abnormal

xylem development in response to Meloidogyne spp

infection. They observed siginificant reducation in

vessel element dimensions but no difference in fiber

elements in infected and uninfected plants. Hisamuddin

and Siddiqui (1992) reported the induction of 5 to 6

giantcells in the region of vascular tissues within 24

hours of inoculation at 23 to 33°C. Abnromal xylem

formation progressed incessently as the gall grew

24

o l d e r . Drought et. al. , (1992) reported tha t

Meloidogyne incognita. infected tomato roots showed

only s l i gh t f luorescence. The nematode induced giant

c e l l s accumulated f l u r e sc in from the apoplas t . Giant

c e l l s had increased net p ro te in extrusion a c t i v i t y

compared to other c e l l type a t the infec t ion s i t e This

s u g g e s t e d t h a t p r o t e i n e x t r u s i o n might be d r i v i n g

f o r c e fo r s o l u t e u p t a k e from a p o p l a s t . Vovlas and

S a s n e l l i (1993) r e p o r t e d t h a t i n f e c t i v e Meloidogyne

j u v e n i l e s p e n e t r a t e d r o o t s and stem of Helianthus

annuus s e e d l i n g d u r i n g g e r m i n a t i o n . Nematodes

e s t a b l i s h e d the feed ing s i t e s mainly in the cambium.

Stem s w e l l i n g s were caused by pronounced hyper t rophy

of cambial c e l l s a t t h e feed ing s i t e . The s i z e of

g a l l s and p r o d u c t i o n of g i a n t c e l l in t h e a e r i a l p a r t

i n d u c e d by s i n g l e n e m a t o d e s were s i m i l a r t o t h o s e

observed on r o o t s .

Gourd e t . al., (1993) studied the r a t e s of

p e n e t r a t i o n of f r e s h l y ha tched Second s t a g e j u v e n i l e s

(J2) of Meloidogyne arenaria, M. Hapla, M. incognita

and M. javanica and Heterodera glycines race I and V

in soybean r o o t s . H. glycines entered roots more

q u i c k l y t h a n Meloidogyne spp. Penetrat ion by most

nematodes was a c c o m p l i s h e d w i t h i n 48 h o u r s . Host

s u s c e p t i b i l i t y and r e s i s t a n c e , each r e s u l t from s e r i e s

of s e q u e n t i a l even ts t h a t a re e a s i l y d iv ided i n t o pre

25

and post infection stages. The sequential events may­

or may not lead to the establishment of disease.

Resistance is the absence or inhibition of disease

upon challenge by pathogenic agent. Thus any thing

that prevents, retards or restricts disease

development contributes to host resistance. Resistance

manifested at the time of infection or thereafter may

be constitutive or due to infection induced factors.

Root-knot nematode {Meloidogyne spp.) after

establishing a permanent feeding site in the host

roots induced the formation of enlarged symplastic

structures called giant cells (Jones and Northcote,

1972,a) The giant cells have the ability to transfer

metabolites from the host to developing larvae. There

is an ample evidence indicating that transfer, cells

are metabolically hyperactive and essential to the

development of nematode. ( Bird, 1961; Littrell, 1966;

Endo and Veech, 1969a,b; 1970; Veech and Endo, 1969;

Webester, 1969; Endo, 1971; Dropkin, 1972; Gommers and

Dropkin, 1977).

Failure of transfer cell to develop or function

will result in the death of nematode and is therefore

a mechanism of Resistance. There has been considerable

speculation in the role of nematode enzymes in the

transfer cell formation. Hussey and Sasser (1973)

observed peroxidase in the stylet exudate of

26

Meloidogyne incognita females and suggested tha t t h i s

enzyme i s i n v o l v e d in g i a n t c e l l i n d u c t i o n and

m a i n t e n a n c e . R e s i s t a n t p l a n t s i n f e c t e d w i t h

Meloidogyne incognita normally undergo a

h y p e r s e n s i t i v e response and the a f f e c t e d c e l l s t u r n

brown and d i e . However, i n r o o t s t r e a t e d w i t h

c y c l o h e x a m i d e , t h e t y p i c a l h y p e r s e n s i t i v e r e s p o n s e

does not occur or a t l e a s t the e f f e c t e d c e l l s donot

t u r n brown and t h s c e l l s remain a l i v e .

Sawheny and Webster (1975) observed browning of

a f f e c t e d c e l l s wi th c e l l dea th and lack of browning

w i t h l i v i n g c e l l s . Or ion and Minz (1971) t r e a t e d

t o m a t o s e e d l i n g s w i t h m o r p h a c t i n ( an a n t i b i o t i c

agent ) t h a t a p p a r e n t a l y does not a f f e c t nematode or

a t l e a s t does n o t a f f e c t Panagrellus redivives and

o b s e r v e d a marked d e l a y i n g i a n t c e l l f o r m a t i o n ,

reduced r a t e of nematode development and a tendency

t o w a r d s m a l e n e s s i n m a t u r i n g l a r v a e . Davide and

T r i a n t a p h y l l o u (1968) o b s e r v e d s i m i l a r e f f e c t s by

t r e a t i n g p l a n t s wi th ma l i chydraz ide ( m u l t i f u n c t i o n a l

i n h i b i t a r ) w i t h i n f o u r days a f t e r i n f e c t i o n by

M. incognita. Terpenoid aldehydes were h is tochimical ly

d e m o n s t r a t e d i n endode rmis and s t e l e n e a r s i t e of

n e m a t o d e . I n f e c t i o n i n d u c e d t e r p e n o i d a c c u m u l a t i o n

occured in both s u s c e t i b l e and r e s i s t a n t , but i t was

d e t e c t a b l e s o o n e r , r e a c h e d g r e a t e r i n t e n s i t y and

27

occur red more in host c e l l s in the r e s i s t a n t c u l t i v a r s

than in the s u s c e p t i b l e c u l t i v a r s . Thus the r a t e and

e x t e n t of accumulat ion seems to be impor tant f a c t o r s .

Wieser (1956) observed t h a t t h e exc i sed r o o t s of

soybean and egg p l a n t v a r i e d in t h e i r r esponse to

l a r v a e of M. hapla, some being r e p e l l a n t , some

a t t r a c t i v e and some n e u t r a l . He a t t r i b u t e d t h e s e

r e s u l t s an i n t e r p l a y be tween an a t t r a c t i v e a g e n t

p r e s e n t in the l i v i n g p l a n t and r e p e l l a n t a s s o c i a t e d

wi th t he chemical decay and break down of r o o t .

F a s s o l i t i s , (1970) o b s e r v e d no d i f f e r e n c e i n

l a r v a l p e n e t r a t i o n of Meloidogyne incognita acrita in

suscept ib le Cucumis melo and r e s i s t a n t C. ficifolius

and C. mituliferus. After 26 days of in fec to r there

was no observable difference in type of g i an t c e l l

development in regions of roo ts assoc ia ted with adult

females. However, in C. mituliferus immature nematodes

were a s s o c i a t e d w i t h s m a l l g i a n t c e l l s which were

l i m i t e d t o few c e l l s nea r t h e head of nematode, with

l i m i t e d amount of h y p e r t r o p h y and c e l l w a l l

d i s s o l u t i o n .

Reyno lds e t . al. , (1970) reported that

a p p r o x i m a t e l y some number of Meloidogyne incognita

acrita larvae penetrated the r e s i s t a n t and suscept ib le

roots of a l f a l f a p lan t . Number of larvae in r e s i s t a n t

roots decreased sharply a f t e r 3 - 4 days t i l l on 7th

28

days fewer than 5 larvae per seedling and no nematode

development could be detected. In susceptible roots

larvae became sedentary and developed nonnally.

Michael et. al. , (1974) reported that galls on

resistant roots, of cotton on infection by Meloidogyne

incognita, contained no nematodes penetration of the

larvae in the resistant cultivar was equal to that of

the susceptible cultivar and independent of the member

of nematodes.

Shepherd and Huck (1989) reported small cracks

caused by M. incognita in root epidermis and cortex.

Soon after the cotyledons expanded, the cracks became

longer and wider and extended through the cortex. Gall

formation on tap roots took place on fourth day, when

true leaf became visible. Root cracking, galling and

giant cell on twelvfth day led to predisposition of

cotton roots to pathogen resulting in synergistic

interactions and diseases. Windhen and Williams (1994)

reported similar penetration of Meloidogyne incognita

juvenile on 3rd day after inoculation (3DAI) in four

corn genotypes in both susceptible and resistant

genotypes.

Par.kaj and Siyanand (1992) reported that the

chemicals like carbofuran, carbcsulfan and phenamiphos

inhibit y.eloidogyne incognita larval penetration and

the development of females or. roots resulting in

29

reduced g a l l format ion and r e p r o d u c t i o n a f t e r 50 and

6 0 days , in case of bitter gourd and roiond melon.

Nanjegowda et. al. , (19S1) reported tha t the

a n t i b i o t i c KT199 m a r k e d l y d e c r e a s e d t h e number of

nematodes and number of g i a n t c e l l s and n u c l e i . Giant

c e l l s were a l s o found t o c o n t a i n l e s s i n s o l u b l e poly

s a c c h a r i d e , n u c l e i c a c i d and p r o t e i n s . A n t i b i o t i c

checked the damage caused by Meloidogyne incognita to

tomato p l a n t s .

C a b a n e l l a s , e t . al., (1988) s tudied the

h i s t o l o g i c a l i n t e r a c t i o n of Paecilomyces lilacinus

with Meloidogyne incognita on the excised roo ts of

tomato. They observed no g ian t c e l l in tomato roots

i n o c u l a t e d w i t h nematode eggs i n f e c t e d w i t h P.

lilacinus.

Few to no g a l l s and no giant c e l l formation were

found i n r o o t s d i p p e d i n s p o r e s u s p e n s i o n of P.

lilacinus and inoculated with M. incognita. Numerous

l a r g e g a l l and g i a n t c e l l s were p r e s e n t i n r o o t s

i n o c u l a t e d w i t h o n l y M. incogzilta.. P. lilacinus

colonized the surface of ep ide r r s l c e l l s as well as

the in t e rna l c e l l s of epidermis and cor t ex . (Dunn e t .

al., 1982; Fattah and Webster 1963;.

Materials and Methods

MATERIALS AND METHODS

The different materials to be used ar.d methods

to be employed during the course of proposed

experimental programme are generalized as follows :

Test Plant and Pathogens and Material:

In the proposed plan of work bitter gourd

Momordica charantia L ) will be selected as the test

plant, root-knot nematode as the test pathogen and

Paecilomyces lilacinus as the test fungus and aldicarb

as the test nematicide.

Collection of Nematode Inoculum {Meloidogyne

ineognita):

Roots of brinjal {Solanum melongena ) infected

with root-knot nematode will be collected fron brinjal

field. Root-knot nematode species and races will be

identified , on the basis of the characteristic perineal

patterns and differential host test.

Pure Culturing 2uid Maintenance of Inoculum :

A single egg mass of Meloidogyne incognita will

be surface sterilized in 1 : 500 solution of chlcrox

(calcium hypochlorite ) for five minutes ani washed

thrice in sterilized distilled water. The egg mass

will be allowed to hatch in distilled water at 27°C.

Egg-plant seedlings raised in 25 cm pots ccntaining

J±

autoclaved s o i l , wi l l be inoculated with the second-

stage juveni les(J2) thus obtained. After about f ive

months s o i l of inoculated pots wi l l be examined to

a s c e r t a i n t h e e s t a b l i s h m e n t of n e m a t o d e s . The

s u b c u l t u r i n g w i l l be done by i n o c u l a t i n g new egg

p l a n t s wi th a t l e a s t 15 egg masses ob ta ined from pure

c u l t u r e i n o r d e r t o m a i n t a i n s u f i c i e n t i nocu lum

throughout the course of i n v e s t i g a t i o n .

Isolat ion and Preparation of Nematode Inoculvun:

Egg masses wi l l be removed from the infected

p lan t s and allowed to hatch (Stemerding, 1963) a t 27°C

in an incubator . After every 24 hours the nematode

suspension wi l l be co l l ec t ed in beakers. The second-

s t a g e j u v e n i l e s of Me2oidog3/ne incognita w i l l be

c o u n t e d w i t h t h e h e l p of c o u n t i n g d i s h u n d e r t h e

s t e r e o s c o p i c microscope . An average of 3 to 5 counts

w i l l be made to de te rmine the d e n s i t y of nematode

j u v e n i l e s in the s u s p e n s i o n .

Raising of Test plant:

The s e e d s of t e s t p l a n t , b i t t e r gourd

{Momordica charentia ) w i l l be surface s t e r l i z e d with

0.5% sodium hypochlori te (NaOcl) for five minutes and

thoroughly washed five times with d i s t i l l e d water. The

seeds wi l l then be sown in autoclaved clay pots of

diameter 15 cm., containing steam s t e r l i z e d s o i l and

32

manure in fixed proportion of 2 : 1. One week old

seedlings will be used for inoculation.

Inoculation with Nematode :

After one week of seedling emergence, holes of

5- 7 cm depth around the plants within a radius of 2

cm. from the plant will be made in which a counted

number of nematode larvae { second stage infective

juveniles ) will be transferred with the help of

sterlized pipette. The holes will then be plugged with

sterilized soil. Regular watering will be done to

maintain the soil moisture till the termination of the

experiment.

Collection o£ Fungal Inoculiun:

The fungal plant pathogen for use in experimental

studies will be Paecilomyces lilacinus.The culture of

the P. lilacinus (Thorn) Samson, will be obtained from

International Potato Centre, Lima, Peru. It will be

maintained on Potato Dextrose Agar, (P D A) slant. The

Constituents of the P D A (Ricker and Ricker, 1936)

are given below:

Agar 17.00 g

Potato peeled and sliced 200.00 g

Dextrose 20.00 g

Distilled water 1000.00 ml

33

Richard's liquid medium (Ricker and Ricker,l935)

will be used for the mass production of P. lilacinus

The composition of Richard's liquid medium is as

follows.

Potassium nitrate{KN03) 10.00 g

Potassium dihydrogen phosphate(KH2PO4) 5.00 g

Magnesium sulphate (MgSo4.7H20) 2.50 g

Ferric chloride(Fecl2) 0.02 g

Sucrose (C-L2"22°11^ 50.00 g

Distilled water 1000.00 ml

The medium will be prepared, filtered through

muslim cloth and sterilized in autoculave at 15 lb.

for 15 minutes in 250 ml corning flasks, each

containing 100 ml of liquid medium. Liquid medium

taken in flask will be inoculated with small amount of

fungus maintained on P D A slants with the help of

sterilized inoculating needle under aseptic conditions

in aseptic chamber. These inoculated flasks will be

incubated at 28MC for about 15 days to allow rapid

fungal growth for experimental studies.

Preparation of Fxingal Inoculiun and its Inoculation:

After enmassing of Paecilowyces lilacinus on

Richard's medium, 100 g of mycelia well be blended in

1000 ml of distilled water, in warring blender so that

34

10 ml of suspension consist of one gm mycelial

suspension.

The fungus P. lilacinus will be incorporated

into the soil around the root zone of bitter gourd

simultaneously with nematode inoculum and following I,

II, III and IV week after nematode inoculation in the

form of suspension. The plants will be left as such

for two months and then harvested and processed.

Study the Effect of Systemic Nematicides :

To study the effect of systemic nematicides on

root-knot nematodes Meloidogyne incognita development

and gall formation. Aldicarb and Carbofuran will be

selected. Aqueous stock soluations conataining 1 mg

aldicarb per ml soluation will be added to the potted

plants after pre, post and simultaneous inoculations

of nematodes. The nematicide, aldicarp (2 - methyl- 2

-methylthio) Propionaldehyde o-(methyl carbomyle

(oxime) is a carbamate insecticide-nematicide. To

study the effect of pre inoculation treatment the

required dosages of nematicide will be applied to pots

containing autoclaved soil around the root zone.

Second treatment will be given simultaneously at the

time of inoculation of nematodes and after that the

third treatment will be followed after 2, 3 and 4

weeks respectively in different replicates and the

35

p l a n t s w i l l be l e f t a s such f o r 2 m o n t h s .

H i s t o p a t h o l o g i c a l s t u d i e s of the i n f e c t e d r o o t s w i l l

be c a r r i e d out a f t e r t e r m i n a t i o n of each expe r imen t s .

Histopathological Studies:

Inoculated seedlings wi l l be uprooted ca re fu l ly

of the s o i l from 24 hours u n t i l l the 10 th day. The

root w i l l be washed gent ly and thoroughly to remove

a l l s o i l p a r t i c a l adhering on them. Galled roots wi l l

be cut in to 1 cm long pieces and processed.

For h i s topa thologica l s tudies the infes ted root

a f t e r the t e r m i n a t i o n of every e x p e r i m e n t w i l l be

processed as fo l lowes :-

Fixation;

The roots collected from experimental pots will

be washed thoroughly and fixed in F. A. A. (Formalin

aceto alcohol) (Johanson, 1940) F. A. A. wil be

prepared as followes :

Ethanol 50 % - 90 ml

Formalin 37 % - 5 ml

Glacial acetic acid - 5 ml

The roots will be placed in the fixative for a

minimum period of 24 hours to several days, depending

on its thiknes. Material may also be fixed in the

fixative indefinitely.

36

Dehydration :

Dehydration is accomplished by moving the roots

step wise through increasingly higher concentrations

of alcohal. Tertiary butyl alcohal (TBA) dehydration

schedule (Table l) will be followed (Johnsen, 19-40)

TABLE 1

Quantity (ml) needed £or solution

Step

1

2

3

4

5

6

7

8

% Alcohol

50

70

85

95

100

100

100

100

Time

2h or more over­night

1-2 h

1-2 h

1-3 h

1-3 h

1-3 h

over­night

Distill Water

50

30 •

15

0

0

0

0

0

95% Ethanol

40

50

50

45

0

0

0

0

100% Ethanol

0

0

0

0

25

0

0

0

100% T.B.A.

10

20

35

55

75

100

100

100

It is important to keep the tertiary butyl

alcohol TBA changes in warm place as the chemcal

solidifies at 25.5]^C.

37

Infiltration:

In this step alcohols in the tissues will be

replaced by paraffin so that tissue is saturated with

pure solution of paraffin, when the TBA dehydration

schedule is followed , the 100% TBA solution in step 8

will be replaced with 1:1 mixture of 100% TBA and

paraffin oil. The tissues will be allowed to remain in

this solution for 1 h or more depending on its

thickness. Shortly before the next step, another

container will be filled 3/4th of its volume with

melted paraffin and allowed to solidify slightly. The

roots from TBA paraffin oil mixture will then be

placed on top of the solidified paraffin oil solution.'

This container will be placed uncovered in an oven set

at slightly above the melting point of paraffin. After

1 - 3 hours the TBA parafin oil mixture will be poured

off and replaced with pure melted paraffin wax and

kept in oven for about 3 hours. This step will be

repeated atleast oncemore.

Eimbedding:

The roots will be placed in metal base molds or

folded paper. Molds will first be coated with thin

layer of glycerine and then liquid paraffin will be

poured, roots will be placed into the mold with heated

forceps and additional melted paraffin will be added

38

to fill the mold. Once the paraffin begin to solidify

over the top of the mold, the mold be plunged into ice

water and left there untill solidification. After

hardening, it will be cut into smaller blocks and

trimmed. Then the blocks will be mounted on block

holders.

Sectioning :

Transverse and longitudinal sections of 8 - 12

urn thickness of the roots will be cut serially with

the help of rotary microtome. The paraffin ribbon thus

obtained will be mounted on a clean galss slide with

an amount of albumin and glycerine dissolved in water,

the slide will be kept overnight in an incubator at 40

degree C to allow water to evaporate. These slides

will then be kept at 60 degree C for one hour to melt

the paraffin.

Staining :

The process of staining will remove all paraffin

from the sections and increase the contrast in the

tissues. Staining will be done with saffranin and fast

green combination ( Table -2, Sass, 1951). Then slides

will be removed from the xylene, and laid on a flat

absorbent surface. The mounting madium will be applied

to surface of the slide before evaporation of xylene

and cover slip will be lowered gradually over the

slide.

Table 2

Sass Safranin and fast-green stain

39

step Solution Time

1.

2.

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Xylene

absolute ethanol

95% ethanol

70% ethanol

50 ethanol

30 ethanol

1% aqueous safranin

rense in tap water

30% ethanol

50% ethanol

70% ethanol

95% ethanol

0.1% fast green FCF in 95% ethanol

absolute ethanol

absolute ethanol

xylene-absolute ethanol i:i

xylene

xylene

5 min

5 min

5 min

5 min

5 min

5 min

1-12 h

3 min

3 min

3 min

3 min

5-30 sec

15 sec

3 min

3 min

5 min

5 min or larger

40

Finished slides will be left to dry for atleast 24

hours at room temperature. The medium will harden better if

the slides are held on 60^0 warming tray over night. The

Slides will be examined under laborlux-k-compound

microscope. Necessary photographs will be taken.

To Determine the Resistance of Different Cucurbit Varieties:

Seeds of several varieties of cucurbits will be

obtained from National Seeds Corporation (NSC), New Delhi

and different seed centres. The resistance will be

determined by taking different growth parameter and compared

over controlled plants. Each variety will be replicated six

times with 3 pots as experimental set and 3 pots as

controlled sets. Shoot length, leaf number and size of

leaf, number of flowers, number of fruits, number of galls,

shoot weight and root weight both fresh and dry weight will

be taken to compare. Host suitability designation to each

variety will be assigned according to Sasser et al. (1984)

scale.

41

Table III

Quantitative scheme for assignment of Sasser's host

suitability (resistance) designations

Plant damage (gall index)

Host efficiency (R factor)

Degree of resistance (DR) designation

< 2

<. 2

> 2

> 2

< 1

> 1

< 1

> 1

resistant

tolerant

hypersusceptible

susceptible

42

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