STUDIES ON HISTOPATHDLOGY OF SOME ROOT-KNOT NEMATODE

INFECTED CUCURBITS

DISSERTATION SUBMITTED FOR THE DEGREE OF

M^ittx of $f)iIos(opI)p

BOTANY

BY

HISAMIUDDIN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

19 8 7

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READER

DEPARTMENT OF BOTANY

ALIGARH MUSLIM UNIVERSITY

ALIGARH-202001 ( INDIA )

Date. 1 2 . 1 0 . 1 9 8 7

C E i < T I F I C A E

C e r t i f i e d t h a t t h e work embodied i n t h i s d i s s e r ­

t a t i o n e n t i t l e d "STUDIES CN HI ST O PATHOLOGY OF SOME

ROOT-KNOT rEMATODL IKFECTED CUajRBITS" i s t h e b o n a -

f i d e work c a r r i e d o u t by Mr. Hisamuddin u n d e r my

s u p e r v i s i o n and i s s u i t a b l e f o r s u b m i s s i o n f o r t h e

M . P h i l Degree of A l i g a r h Muslim U n i v e r s i t y , A l i g a r h .

( DR. ZIAUDDIN A. SIDDIQUI )

/^ • /o. s-y

DEPARTMENT OF BOTANY

ALIGARH MUSLIM UNIVERSITY

M. Sc. (Alig.) ALIGARH-202001 ( INDIA )

Date 1Z...X.Q...X981

ACKNOIVLEDGEMEN T

I 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 D r . Z i a u d d i n Ahmad

j i a a i q u i , Ph .U ( A l i g . ) , D e p a r t m e n t of B o t a n y , A l i g a r h M u s ­

l i m u n i v e r s i t y / A l i g a r h / who i n s p i r e d lue -co i n i t i a t e t h e

r e s e a r c h w o r k i n t h e f i e l d o f p l a n t n e m a t o l o g y / e s p e c i a l l y

h i s t o p a t h o l o g y . I am h i g h l y i n d e b t e d t o h i m f o r h i s k e e n

i n t e r e s t , c o n s t a n t e n c o u r a g e m e n t 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 m a n u s c r i p t .

T h a n k s a r e a l s o d u e t o D r . M. w a j i d Khan f o r h i s c o n s ­

t r u c t i v e c r i t i c i s m and v a l u a b l e s u g g e s t i o n s and f o r s r ^ a r i n g

h i s p r e c i o u s t i m e f o r g o i n g t h r o u g h t h i s m a n u s c r i n t c r i t i ­

c a l l y . F u r t h e r , I am g r a t e f u l t o P r o f . K h a l i d Mahmood,

C h a i r m a n , D e p a r t m e n t o f B o t a n y , f o r p r o v i d i n g l i b r a r y and

l a b o r a t o r y f a c i l i t i e s arid t o t h e U . G . C f o r t h e a w a r d of

J . R . F .

And l a s t l y , I s h a l l n o t f o r g e t t o t h a n k my c o l l e a g u e s

a n d f r i e n d s y ^ s M . J . P a s h a , K a l i m u l l a h , Mu j a h i d A. Khan

a n d A t h a r A a i l h a s h m i who h e l p e d me, e v e r y now and t h e n ,

i n o n e way o r t h e o t n e r d u r i n g t h i s p e r i o d .

H13AMUDDIN

C O K T E K T S

1 . I n t r o d u c t i o n 1

2 . H o s t - P a r a s i t e u e l a t i o n s h i p 5

3 . H i s t o p a t h o l o g i c a l Changes 7

4 . P l a n of work 62

5 . i M a t e r i a l s and Methods 65

6 . R e f e r e n c e s 79

/ , /n/roc/uc/'/o/?

1

INTRODUCTION

I n p l a n t s , nematode a c t i v i t i e s r e s u l t i n m o r p b o l o g i c a l and

p h y s i o l o g i c a l changes of t h e a f f e c t e d t i s s u e s . These c h a n g e s may

b e d e s t r u c t i v e , a d a p t i v e o r n e o p l a s t i c . I n d e s t r u c t i v e c h a n g e s ,

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

by r e m o v i n g t h e i r c o n t e n t s r a p i d l y o r s l o w l y b u t c o m p l e t e l y . I n

a d a p t i v e c h a n g e s t h e h o s t c e l l s a d a p t t o nema todes by e n l a r g i n g and 4

by increasing the i r metabolic a c t i v i t i e s . In neop las t i c changes the

c e l l s undergo growth and mu l t i p l i ca t i on , various types of g a l l s

associa ted with ce r ta in nematodes are examples of neoplasm

(Dropkin, 1980),

All phytoparas i t ic nematodes remove c e l l contents through

t h e i r s t y l e t s , but each kind of nematode has i t s own way of feeding,

and each induces i t s own type of t i s s u e damage. All plant - para­

s i t i c nematodes are c l a s s i f i e d in to two broad groups ec toparas i t es

and endoparasites depending upon t h e i r feeding sit&sand mode of

feeding. Ectoparasi tes are fu r the r of two types surface feeders

and subsurface feeders , Itie former feed on c e l l s of the root

epidermis and on root h a i r s , e ,g . , Trichodorus, Par at r ich odor us,

Tylanchorhynchus e t c , of which Trichodorus i s the most damaging

nematode of t h i s group. The l a t t e r feed on c e l l s in the cor tex

or c lose to s t e l e by pene t ra t ing t h e i r s t y l e t s and sometimes with

a small portion of the anter ior body. This group includes

Belonolaimus/ Hoplolaimus, Longidorus, Xiphinema e t c . Three

genera of th i s group (Hemicycliophora, Longidorus and Xiphinema)

induce ga l l s with enlarged and p ro l i f e ra t ed c e l l s . Endoparasites

are also of two types, migratory endoparasites and sedentary endo­

p a r a s i t e s . c e r t a i n migratory endoperasi t ic nematodes feed on roots

of herbaceous plants l i k e Radopholus, Hiirschmaniella, Pratylenchus.

Radopholus s i m i l i s makes extensive tunnels through the cor tex of

banana roots and rhizomes. Certain other feed on ae r i a l par t s of

herbaceous p lants l i k e i^helenchoides D. dipsaci e t c . Aphelen-

choides damage, severely, buds and leaves of s t rawber r ies , chrysan­

themums, begonias. APhelenchoides infec t ing chrysanthemum leaves

crawl through stomata in to mesophyll t i s sues where they move about

ac t ive ly in the in te rve ina l a reas , Ditylenchiis d ipsac i induces

even more extensive damage in non-vascular t i s sues of stems,

leaves , and cotyledons. Some of the migratory endoparas i t ic nema­

todes feed on woody t rees e .g . , Rhadinaphelenchus invades paren­

chyma t i s s u e of trunk and roots of coconut and other palms,

s e s s i l e endoparasites or sedentary endoparasites may e i t h e r be

p a r t l y embedded in the t i s s u e ana ca l l ed as semi endoparasi tes comp­

r i s e Tylenchulus semipenetras and Rotylenchulus reni formis , or be

e n t i r e l y embedded l i k e N acobbus, Heteroder a Globodera, Meloidogyne,

Tyler>chulus senipenetrans and Rotylenchulus reniformis penet­

r a t e t h e i r an te r io r portion of the body in to the roots and feed on

3

a se t of normal s ized c e l l s . Macobbus invades .the s t e l e and st imu­

l a t e s both ce l l d iv is ion and the fusion of c e l l p ro top la s t a f t e r

p a r t i a l d issolut ion of c e l l wa l l s , A spindle-shaped mass of c e l l s

with p a r t i a l l y dissolved walls r e su l t s and the nonatode feeds at

one end of th i s group of interconnected c e l l s , Ihe feeding s i t e

i s at the cen t re of a conspicuous ga l l formed by the c e l l d iv i s ion

in cor tex and vascular t i s s u e s , Heterodera induces c o r t i c a l c e l l s

to enlarge and the walls between then to break down in p a r t . The

r e s u l t i n g syncytium invades the s t e l e . Syncytium walls next to

xylem vesse ls develop finger l i k e ingrowths, and the c e n t r a l

vacuoles disappear. Nuclei of incorporated c e l l s enlarge but do

not d iv ide (Dropkin, 1980). Meloidogyne species usua l ly cause g a l l ­

ing of the host t i s sues in the roots to and occasional ly in steins

and l eaves . Galling i s due p a r t l y to hyperplas ia and hypertrophy

of the roo t cor tex and p a r t l y to the developnent of g ian t c e l l s on

which the nematodes feed. The g iant c e l l s develop from undi f fe ren­

t i a t e d c e l l s , usual ly of per icyc le , in response to feeding by the

nematodes, TSie giant c e l l s are multinucleate and hav« i r r e g u l a r l y

thickened walls (Jones and Northcote, 1972) . Usually four to s i x

c e l l s are assocj ated with each nematode and t h e i r functioning

appears to depend on continuous s t imulat ion by the nematode ( Bird,

1962), Heavy ga l l ing of p lan ts r e s u l t s in s tun t ing of top growth

and r e l a t i v e l y greater root to shoot r a t i o . Infes ted p lan t s show

increased s u s c e p t i b i l i t y to water s t r e s s and there i s incx^eased

s y s t h e s i s of prote ins in the g a l l s with dis turbance of th« noxroal

t r anspor t of substances between root and shoot.

Meloldogyne in t e r f e r e s with the physiological funct ions of

the p l a n t s . Rate of r e sp i r a t i on i s a l t e red depending upon the hos t

specis and physiological age of the hos t . Due to d i s rup t ion of

vascular t i s sue , flow of nu t r i en t s and water i s af fected. Heavy

i n f e s t a t i on of Meloidogyne in the root decreases r a t e of photo­

synthesis in the leaves, considerably. All these anatomical and

physiological abnormalities lead to s tun t ing , ch loros i s and u l t i ­

mately death of the p l a n t s .

z; nosrraros//-e /^e/a//b/?j/i/}?

H 0 : J T - P A < A 3 I T £ RELATIOSl 3HIP

The p h y t o p a r a s i t i c nematodes are of two broad types , e c t o ­

p a r a s i t e s and e n d o p a r a s i t e s . E c t o p a r a s i t e s feed a t or near p l a n t

s u r f a c e s and complete t h e i r l i f e c y c l e in t h e s o i l wh i l e endopara-^-

s i t e s embed t h e i r e n t i r e bod ies w i th in p l a n t t i issues and feed

w i t h i n . Endoparas i t e s pass t h e i r e n t i r e l i f e c y c l e i n r o o t t i s s u e

except fo r a b r i e f pe r iod when p a r a s i t i c s t a g e Ictrvae may remain

i n t h e s o i l a f t e r h a t c h i n g . some of t h e nematodes have been

observed wi th up to o n e - t h i r d of t h e body embedded in the r o o t and

t h e remainder of t h e body i n t h e s o i l , a r e l a t i o n s h i p sometimes

termed semiendoparas i t i s ra . However, a d i s t b t c t d i v i s i o n between

e c t o p a r a s i t i s m and semiendoparas i t i sm does no t always e x i s t , as

shown, f o r i n s t a n c e by t h e feed ing of jtfnplimerlinus s p p . (Bridge

and Hague, 1974) . Development of modes of p a r a s i t i s m wi th in t h e

Tylenchida has been proposed in a sequence from e c t o p a r a s i t e s t o

s ^ i e n d o p a r a s i t e s t o e n d o p a r a s i t e s by Paramonov (1967); Maggenti

(1971) .

Depending upon t h e i r m o b i l i t y du r ing p a r a s i t i s m , t h e nematod»f

axe f u r t h e r d iv ided i n t o mig ra to ry and s eden t a ry g r o u p s . Migra to ry

nematodes, whether e c t o p a r a s i t e s o r e n d o - p a r a s i t e s , move from p l a c e

t o p l a c e throughout t h e i r l i f e c y c l e . Some nematodes, such as

females of He te rode ra and Meloidogyne e n t e r t he r o o t s as m i g r a t o r y

JLarvae, Their body s w e l l s t o n e a r l y s p h e r i c a l and t h e m i g r a t i o n i s

6

no longer poss ib le . iTiey pass t h e i r remainder l i f e cycle in a

s ing le feeding s i t e . "These nematodes are termed sedentary

endo p a r a s i t e s .

J, Hfs/o/7a//?o/o anorm/oa/ca/ C/iames

HISTOPATHOLOGICAL CHANGES

P h y t o p a r a s i t i c nema todes f e e d i n g on be low g r o u n d p l a n t

p a r t s c a u s e i n j u r i e s of v a r i o u s l e v e l s t o t h e r o o t s y s t e m s .

The t i s s u e o r t h e p l a n t p a r t on wh ich t h e y f eed , i n d u c e v a r i o u s

a n a t o m i c a l and p h y s i o l o g i c a l c h a n g e s l e a d i n g t o t h e d e v e l o p m e n t

of s p e c i f i c and c h a r a c t e r i s t i c symptoms,

some m i g r a t o r y e c t o p a r a s i t e s t h o s e f e e d on r o o t h a i r s

e p i d e r m a l c e l l s i n a r a t h e r c u r s o r y manner do n o t m a r k e d l y

a f f e c t t h e r o o t m o r p h o l o g y . A n o t h e r g r o u p of m i g r a t o r y e c t o ­

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

l a y e r s of c o r t i c a l c « l l s . D e a t h of c e l l s i s p r o b a b l y d u e t o

c e r t a i n enzymes which a r e s e c r e t e d i n t o t h e h o s t b y t h e p a r a s i t e ,

some of t h e m i g r a t o r y e n d o p a r a s i t e s c a u s e s u r f a c e l e s i o n s . When

l e s i o n s a r e formed, t h e y f r e q u e n t l y c o a l e s c e t o g i v e r o o t s y s t e m s

a g e n e r a l l y d i s c o l o r e d a p p e a r a n c e . F o r m a t i o n of l e s i o n s r e s u l t

f rom t h e p r o d u c t i o n of p h e n o l i c compounds by enzymes s e c r e t e d

b y t h e n e m a t o d e . Much more complex , however , a r e t h e h o s t -

p a r a s i t e r e l a t i o n s h i p s of t h e s e d e n t a r y e n d o p a r a s i t e s w h i c h

c a u s e h y p e r t r o p h y and h y p e r p l a s i a o f t h e s t e l a r pa renchyma

(Dropk in , 1955 ; Mounta in , 1 9 6 0 ) ,

3 . 1 . ECT0PARA3ITKO

All e c t o p a r a s i t i c nematodes remove c e l l c o r t e n t s of t h e

s u r f a c e t i s s u e through t h e i r s t y l e t s , bu t each kirsd c^- nematode

has i t s own way of feeding , and each induces i t s own type of t i s s u e

damage. C h r i s t i e and Perry (1951) were t h e f i r s t t o e s t a b l i s h an

exper imenta l evidence fo r d i r e c t damage t o p l a n t s by an e c t o p a r a ­

s i t i c nematode. The e c t o p a r a s i t e s i nc luded in t h i s t e x t a r e :

T r i chodorus , Tylenchorhynchus, Belonol aimus, Longidorus, Xiphingna,

Hemicycl iophora , cr iconerooides, Hoplolaimus, He l i co ty l enchus and

Roty lenchus .

3 . 1 . 1 . Tr ichodorus Cobb, 1913.

Rohde and Jenk ins t l 957) found t h a t "s tubby root" nematode,

Tr ichodorus C h r i s t i e a f f e c t e d tomato r o o t s l ack a d e f i n i t e r o o t

c a p , a reduced m e r i s t s n a t i c reg ion and t h e p re sence of protoxylem

n e a r t h e roo t spex. I t caused m a t u r a t i o n of ap i ca l merisi:«n in

g r a p e - f r u i t ( c i t r u s p a r a d l s i ) r oo t s (S tand i f e r , 1960). Root t i p s

a r e , i n many cases , t he f a v o u r i t e feed ing s i t e s of Tr ichodorus spp ,

(Zuckerman, 1961; Russel and Pe r ry , 1966; Alhassan and H o l l i s , 1966;

Wyss, 1971 a, b , c ; HOgger 1971; S c h i l t and Cohn, 1975; S t i r l i n g ,

1976) . There was a d i s t i n c t s u p e r f i c i a l d i s c o l o r a t i o n of e p i - ,

hypo - and a few ou t e r c e i l s of c o r t i c a l parenchyma changiiug from

ye l low through orange t o brown in apple r o o t s a t t acked by

T. Virul i ferus ana the aggregation of nematodes was in the zone

between the apical meristeni and the beginning of the root h a i r

zone (Pitcher, 1967).

3 . 1 . 2 . Tylenchorhynchus cobb, 1913.

Ear l ie r s tudies of ' s tun t nematodes' Tyler^chorhynchus did

not show h i s to log ica l changes as a r e s u l t of t h e i r feeding. Southey

(1982) s t a t e s tha t a l l s tages of T. dubius feed ac t ive ly on epider­

mal c e l l s of roots and root h a i r s . Cell function i s affected and

roo ts of some crops can be discoloured l i g h t to dark brown a f t e r

feeding. According to Sutherland and j^ams (1964) the only apparent

damage to the roots of red pine pa r a s i t i z ed by T. c lay ton i was a

disarrangement of the organisat ion of the roo t cap c e l l s r e s u l t i n g

from the feeding a c t i v i t y . Nematodes which fed on non-apical por­

t i ons of the root caused no v i s i b l e damage. Wyss (1973) found t h a t

a f t e r feeding of T. dubius on epidermal c e l l s and root h a i r s of

Brass ica rapa var, s i l v e s t r i s , the cyc los i s eventually stopped and

then the cytoplasm coagulated. Root h a i r s were k i l l e d but the

growth of the root was not g rea t ly affected even when severa l

nematodes fed close together on epidermal c e l l s in the roo t h a i r

r eg ion .

10

3 . I . 3 . Belonolainms Steiner, 1949.

Belonolaimus is commonly known as s t i ng nematode, Owens

(1951) found minute necrot ic les ions along the tap root of peanuts

infec ted with B. graci l i s^ as a r e s u l t of feeding of the nematode,

Standifer and Perry (1960) observed les ions on grapef ru i t ( c i t rus

pa rad i s i ) roots cons is t ing of a cavi ty surrounded by affected c e l l s

which were character ized by ruptured c e l l walls and coagulated

protoplasm. Xylon and phloom were found c lose to the root t i p , the

roo t cap was reduced to a few c e l l s in depth, and branch roots

arose very close to the apex, All of these observations ind ica ted

a general maturation of the e n t i r e root t i p .

3 .1,4 Longidorus Micoletzky, 1922

Feeding by 'needle nemat-.ode Lcmgidorus spp. was inva r i ab ly

a t or j u s t behind, root t i p s and caused a c h a r a c t e r i s t i c roo t t i p

swel l ing or ga l l ing associated with a general reduction of the root

system. The meristematic a c t i v i t y was found to be disturbed? root

elongation was ha l ted but not c e l l maturation so tha t vascxilar t i s a i e

extended c lose t o the apex of the r o o t . Root t i p swell ing were

caused by L. africanus, r e s u l t i n g from hyperplas ia of the c o r t i c a l

parenchyma (cohn & Orion, 1970; wyss 1970). Root t i p g a l l s of

chicory, caused by L. apulus, showed hyperplas ia in the cor tex and

11

hypertrophy in cambial c e l l s , but root t i p ga l l s of ce lery only

contained hypertrophied c e l l s (Bleve-zachet) et a l . , 1977). The

syncyt ia in these ga l l s resu l ted from the break down of c e l l w a l l s ,

Itiey were l a t e r ca l led "lysogenous cav i t i e s" (Bleve-Zacheo et a l . ,

1979). The cytoplasm of these cav i t i e s consisted of an amorphous

mass in which s t ruc tu res or organelles were no longer de tec tab le ,

and therefore , the "syncytia" could not be compared, at l e a s t in

t h e i r function, to those induced by sedentary endoparasi t ic

ty lenchids (Bleve-Zacheo £ t a l . , 1979). Gr i f f i ths and Robertson

(1984) s tudied the histochemical changes occuring during the l i f e

span of root t i p g a l l s on Lolium perenne induced by L. elongatus

in d i f f e ren t s t ages . In the i n t i a l s tage hypertrophy occurred and

the c e l l s contained enlarged nucle i and nucleol i , a g rea te r propor­

t i on of cytoplasm, and increased concentration of p ro te in . I'his

was followed by hyperplasia; c e l l s divided to give two or four

daughter c e l l s , accompanied by a proport ionate reduction in volumes

of cytoplasm, nucle i , and nuc leo l i and reduced concentrat ion of

RNA and p ro te in . The th i rd s tage was secondary hypertrophy with

enlarged, amoeboid nuclei and nucleol i and a s ign i f i can t increase

in concentrat ions of RKA and p ro t e in , Xn the f ina l two s tages ,

as feeding by L, elongatus progress ively removed c e l l contents ,

most c e l l s were devoid of inclus ions and g a l l s col lapsed and were

invaded by s o i l b a c t e r i a .

12

3 , 1 , 5 . Xiphinema Cobb, 1913,

Schindler (1957) denonstrated severe s tun t ing of top growth

as well as ga l l ing of root t i p s in r o s e s . Davis and Jenkins (i960)

while inves t iga t ing the his tology of the g a l l s caused by

^« dlversicaudatujT) on rose roots found tha t ga l l s were forined by

a hyperp las t i c response of c o r t i c a l t i s s u e . Some hypertrophy

occurred, with c o r t i c a l c e l l s two to three times l a rge r than normal

and with a granular-appearing cytoplasm, TSiese c e l l s were some­

what s imi l a r to giant c e l l s except tha t they were smaller and even

had mul t ip le (two to th ree ) , hypertrophied n u c l e i , Meristematic

a c t i v i t y of the root t i p s was re tarded to varying degrees . In

some roo t s vascular d i f f e r en t i a t i on ranged from the usual lack of

development to fu l l development.

While studying host range and pathogenic i ty of X, index,

Radewald and Raski (1962), observed c e l l u l a r hypertrophy, necros i s

and n u l t i n u c l e a t e condition of und i f fe ren t i a t ed c o r t i c a l c e l l s

near the nematode feeding s i t e s . The cortex of infected roots of

p lan t s by Xiphinema spp., d i s i n t eg ra t ed in severa l a reas ,

P.i tcher and Ponsette (1963) found s t y l e t of X, diversica.)datum

apparent ly passing through the vascular t i s s u e and coming to r e s t

with i t s t i p near the outer wall of a s ieve tube in Petunia

faybr^da, in jury to c o r t i c a l parenchyma was obvious on both s ides

of the s t e l e and one of the protoxylem elements appeared s l i g h t l y

13

X. index attacked roots of grapevine turned brown and swelled

at t i p s . Epidermal and outer c o r t i c a l c e l l s collapsed at feeding

s i t e s and showed necros i s . In growing roots mult inucleate c e l l s ,

which were considerably enlarged and contained dense cytoplasm,

were formed beneath the layer of necro t ic c e l l s (Weischer & Wyss,

1976) . Roots of grapevine, heal thy and attacked by X, index wete

s t u d i e d . F i r s t v i s i b l e symptoms of nematode feeding were hyper­

trophy of c e l l s and a d r a s t i c reduction of c e l l d iv i s i ons . At

feeding s i t e s mult inucleate g ian t c e l l s and mononucleate hyper-

t rophied c e l l s developed. The walls of these c e l l s showed enlarged

p i t f i e l d . incomplete c e l l separa t ion and i r r e g u l a r wall formation

were found in giant c e l l s (Rump«:ihorst and Weischer, 1981) .Gr i f f i t h s

and Robertson (1984) observed tha t feeding of X. diversicauTaatxim

on strawberry caused a progress ive increase in ENA content and s i z e

of n u c l e i . Multinucleate c e l l s were present a f t e r 2 and 4 days

feeding.

3 . 1 . 6 . Hemicycliophora de Man, 1921.

Gall ing by sheath nematodes, Hjgnicycliophor a i s caused

due to the i n i t i a t i o n of c e l l d iv i s ion in the infec t ion s i t e . TSie

s t y l e t may be inser ted from s i x t o e ight c e l l s in to the cortex,

hyperp las ia occuring in t h i s area^ Hypertrophied nuclei occur in

c e l l s about the inser ted s t y l e t , but no necros is has been observed,

van Gundy and Rackham (1961) observed in rough lemon tha t the

14

areas in the immediate v i c i n i t y of the nematode s ty l e t appeared

t o be undergoing ce l l d iv i s ion , givfing a dense pat tern of small

c e l l s , as compared with the l a rge r c e l l s surrounding the area .

The nucle i were more deeply s t a ined and la rger than those in adj a -

cent a r ea s . Feeding of Honicycliophora on tomato roots on water

agar in Pe t r i dishes exhibi ted inh ib i t i on of c e l l elongation

i n i t i a l l y , evidently due t o lack of c e l l d i f f e r en t i a t i on and

matura t ion , "Snen hyperplas ia of the cor tex in the feeding s i t e s

formed the root swel l ings . When the nematodes were removed the

swel l ing stopped and one or more branch roots or iginated from t h e

swe l l i ng .

In cranberry, feeding zone of H. s i m i l i s was probably l imi t ed

by the secondary growth of the roots and the formation of cortesc

t i s s u e . These changes prevented the s t y l e t frcro penet ra t ing the

vascu la r t i s s u e (Zuckerman, 1961) .

H, s imi l i s fed on eleven of the th i r t een plants near the

roo t t i p , mostly between the meristematic region and the region of

d i f f e r e n t i a t i o n , occasional ly in the region of elongation (Khera

and Zuckerman, 1963), Mc Elroy and van Gundy (1968) observed

accumulation of protein and enlargement of nucleol i in c e l l s

adjacent and bas ipe ta l to t he fed c e l l s during the f i r s t s i x hrs

feeding of H, ar en a r i a on toroa.to. In next 6 - 1 2 h r s . the

cytoplasmic contents were removed, and the nucleus d i s t o r t e d and/or

reduced in s i z e . The nucleoias was enlarged and the feed c e l l s

15

appeared to double or t r i p l e in volume. The feed c e l l s were l a t e r

crushed and pushed to the root surface by the new meristem formed

by the p e r i c y c l e .

Most feeding by H, s imi l i s on cranberry was at the root t i p

with the s t y l e t penet ra t ing to the meristematic zone. However,

a few nematodes fed fur ther back from the t i p and, where t i s s u e

d i f f e r o i t i a t i o n had occurred, the s t y l e t penetrated the xylem which

were d i f f e r e n t i a t e d by the safranin s ta in ing of the l i g n i f i e d c e l l

wal ls (Kies ie l , M, et a l . , 1971) .

3 . 1 . 7 . Criconemoides Taylor, 193 6.

Ring nematodes' criconemoides, feeding caused extensive

l e s i o n s End p i t s on r o o t s . Nematodes were observed with t h e i r

a n t e r i o r aids enbedded several c e l l layers deep (Hung and Jenkins ,

1969; Ratanaworabhan and smart, 1970). C. curvatom and C. xenoplax

were observed feeding near root t i p s . Single worms had e i t h e r

t h e i r s trylet or pa r t of t h e i r anter ior end embedded in the root

t i s s u e . In cranberry root system, some root t i p s became d i s ­

colored arid ceased growing, i n many cas-es a brown " f rass l i k e "

ma te r i a l was seen near these discolored areas . * s imilar ma te r i a l s

were not seen in areas where nematodes had not been feeding, suggest­

ing t h a t i t was a r e s u l t of nematftde parasi t ism (Bird and Jenkins,

1964).

3 . 1 . 8 . Hoplolaimus Dayday, 1905.

The "lance neitiatOGe" Hoplolaimus feeds on outer root t i s s u e s

of many plants sometimes as ec toparas i t e and sometimes as endo-

pa ra s i t e . Krusberg and Sasser (1956) found tha t H, coronatus caused

considerable damage in cortex of cotton roots and formed c a v i t i e s .

Many undamaged c e l l s were devoid of c e l l con ten t s . Cell walls of

damaged c e l l s and deformed c o r t i c a l c e l l s appeared th icker and

s t a ined deeper with fas t green than did undamaged c e l l s . I t penet­

r a t ed the endodermis to feed eas i ly on the vascular t i s s u e .

H. coronatus fed on phloem parenchyma and phloem elements, causing

a death of these c e l l s . The xylem elements those in c lose p rox i ­

mity of pa ras i t i zed phloem were damaged even though no xylem

elsnents had been attacked. Feeding and migration of H^ coronatus

caused extensive co r t i c a l damage in pine roots (Ruehle, 1962) . Singh

and Misra (1976) while studying sugarcane infected with H. indicus

found t h a t i t caused damage to vascular parenchyma espec ia l ly the

endodexmis and parenchyma af ter 10 days of inocu la t ion . After 20

days the cav i ty enlarged and vascular elements became d i s to r t ed^

The roots became f lacc id with completely d i s in t eg ra t ed cor tex and

a few s t rands of xylem elements leading to wi l t i ng and yellowing

of f o l i a g e . H. indicus remained confined to the cor tex of sugar

cane, cotton, soybean r o o t s . I t r a r e l y crossed the endodermis.

Much of t he cytoplasm of invaded c e l l s and some adjoining c e l l s ,

disappeared or shrank together with the nucleus against the c e l l

17

wall (Singh and Misra, 1976; Lewis e t a l . , 1976). Kourcr< and

Osman (1980) observed that H. aegypti and H. columbus caused

extensive damage to the c o r t i c a l parenchyma and p rec ip i t a t i on of

t y loses in the s t e l e region of soybean r o o t s . Ng and Chen (1985)

while studying histopathology of a l f a l f a roots infected by

H, gal eat us found hypertrophy and ear ly c e l l d iv is ion of host c e l l s

in the per icyc le soon af ter nematode feeding began, As the

pe r i cyc l e c e l l s increased in s i z e and number^ the oidodermis :> '•

f l a t t ened and collapsed, vascular damage included feeding c a v i t i e s ,

lysed phloem t i s sue , and the associa t ion of electron-dense mate­

r i a l with the xylem elements,

3 , 1 . 9 , Helicotyl enchus Stein^r , 1945.

The ' s p i r a l nonatode* Helicotylenchus invades the cor tex ,

both as ecto-and endoparasite but does not move aboat ac t ive ly in

t h e t i s s u e . Blake (1966) when inoculated banana with H. mu l t i -

c lna tu s , observed the nematxxiss t e^J ing d i r e c t l y on c o r t i c a l

parenchyma evacuating t h e i r contents p a r t i a l l y within th ree days

of inocu la t ion . After 4 days the nematodes emtened the cor tex

wholly sometimes upto a depth of 4 - 6 c e l l s . In c e l l s in the

v i c i n i t y of a nematodes' head, the cytoplasm had contracted, some

c e l l wal ls were d i s to r t ed or ruptured and the nucleus had increased

in s i z e , ihe c e l l s were often dis<x)lored and some n e c r o t i c . The

wal l s of c e l l s invaded by the noa^tode cffid 6 - 1 0 adjoining c e l l s

were thickened (Orbin, 1973), jscotmd the point of pene t ra t ion of

18

nematodes small brown les iorswere observed. In case of severe

i n f e s t a t i on sloughing off of epidermal c e l l s and pa r t of t he co r t ex

was also observed. The walls of c e l l s invaded by the nematode

took deep saf - ran ine st--ain indica t ing the presence of l i g n i n

(Rao and swarup, 1975). The nonatodes • s t y l e t penetrated the

cytoplasm of food c e l l s each of which was surrounded by 4 or 5

c e l l s with an enlarged cytoplasm (Jones, 1978).

3.1.10 Rotylenchus F i l ip jev , 193 6

Goodey (1935) recorded c e l l u l a r des t ruc t ion due to the

formation of c a v i t i e s , p a r t i c u l a r l y in the area adjacent to t h e

periderm, t h a t were usual ly f i l l e d with nematodes, Rotylenchus

bradys and the i r eggs in yams. He found ne i the r hyperp las ia nor

hypertrophy. Golden (1956) found les ions formed by R. buxophilus

extended i n t o the cor tex to varying depths . Ttie les ions contained

c e l l u l a r d ^ r i s , p a r t i c u l a r l y broken c e l l walls and the c e l l s

adjacent to the lesicxis w^re idggoid of protoplasm. Such damage

was due to mechanical des t ruc t ion of c e l l s as a r e s u l t of nonatode

movement, chemical a t t r ac t i on of c e l l u l a r contents , and t h e ronoval

of c e l l contents (iiring feeding, Vovlas e t , a l . , (1980) r epo r t ed

^» LQU-J^entinus feeding semi-endoparas i t ica l ly on small l a t e r a l

c a r r o t r o o t s . Nematodes were p a r t i a l l y embedded in the r o o t s , "ttie

a n t e r i o r ends of nematodes were penet ra ted in the wall of epidermal

c e l l s and some layers of the cxartex. The vascular t i s sues were

not damaged.

19

3 , 2 . SEMI-END0PARA3ITES

The s e d e n t a r y s emi -endopa ra s i t e s l i k e Tylenchulus and

R o t y l e n c h u l u s , b r i n g about many c y t o l o g i c a l , anatomical and

p h y s i o l o g i c a l changes in t h e h o s t .

3 . 2 . 1 Tylenchulus Cobb, 1913.

C i t r u s nematode, as i t i s commonly known as, e n t e r s t h e h o s t

c o r t e x and makes c a v i t i e s i n i t . van Gundy and K i r k p a t r i c k (1964) ,

i n o c u l a t e d C i t r u s s i n e n s i s and C. j a m b h i r i wi th t h e l a r v a e of

T. semi p e n e t r a n s , and found l a r v a e s i n g l y or in groups in t h e e p i ­

dermal and hypodermal c e l l s , young females p e n e t r a t e d c o r t i c a l c e l l i

by r u p t u r i n g t h e c e l l wal l and then e s t c b l i s h e d at a d e p t h of

s e v e r a l c e l l s i n t h e c o r t i c a l parenchyma. At t h e eand of 4 t h week

t h e females formed a permanent " f e e d i n g s i t e " which c o n s i s t e d of

6 - 1 0 c o r t i c a l c e l l s *nu r s^ c e l l s " , su r round ing a c a v i t y where

n a n a t o d e head was found, c e l l s su r round ing t h e c a v i t y were a l t e r e d

by nematode feed ing as evidenced by dark s t a i n i n g r e a c t i o n s and

c y t o p l a s m i c and n u c l e a r c±ianges. No i n c r e a s e i n c e l l s i z e o r

number of c e l l s was observed due t o nematode fee<iing. At l a t e r

s t a g e s nematode feed ing caused s w e l l i n g of t h e cy top lasm. Tliis

p roceeded u n t i l t h e c e l l was comple te ly f i l l e d , i t i e r e was an

en la rgement of t h e nuc leus and nuc l eo lu s and a movement of n u c l e u s

t o t h e c e n t r a l a r e a of t b e c<»11. Advanced s t a g e s of c e l l change

20

were a thickened ce l l wall/ very dense cytoplasm, and an amoeboid -

shaped nucleus, ihere was no apparent effect on c e l l s tha t

impinged on the "nurse c e l l . "

Schneider and Baines (1964) found t h a t young females of

X, semipenetraas penetrated midway or deeper i n to the cor tex of

C. aurantivun and C, Sinensis by breaking i n to one c o r t i c a l c e l l

a f t e r another and then became seden ta ry . The c e l l s fed upon by

the female nematodes did not d ie , bu t became g rea t l y a l t e r e d , the

cytoplasm thickaued and f i l l e d the c e l l s , and the nucleus and

nucleolus enlarged g rea t ly . After successful host p a r a s i t e r e l a t i o n

sh ip f a i l e d to become es tabl i shed, the c o r t i c a l t i s s u e surrounding

the nematode died, and the host i s o l a t e d the area by wound r e s ­

ponses, Cohn (1955) observed d i f f e r e n t s tages of t i s s u e d e t e r i o ­

r a t i o n in the feeding zone around the head of embedded feriale of

T. semipenetrans in the c o r t i c a l parenchyma of sweet li'oae, C,

a u r a i t i f o l i a . Both the nucleus and nucleolus of host c e l l s were

enlarged, but t±ie c^l l i t s e l f d id not appear to increase in s i z e .

3 , 2 , 2 . Rotyl enchulus Linford Sc Ol iv ie r a, 1945

Reniform nematode, Roty 1 enchu 1 us reniformis ch ie f ly a phloen

feeder causes vas t damage to the c rops . While studying host para?-

s i - t e - r e l a t ionsh ip of Rotylenchulus reniformis on Gogsyp-iuro

hlxjsuturo, Birchf ie ld (1962) observed t h a t young females i n i t i a t e d

21

in fec t ion by destroying epidermal c e l l s and causing a s l i g h t

browning and necrosis of the surrounding c o r t i c a l c e l l s as they

col lapsed. Phloem c e l l s near the head of the nematode s t a ined

darker than normal t i s s u e s ; t h i s apparent damage to the phloan

extended several c e l l s along the root ax i s . Necrosis in the

phloon and parenchyma apparently r e su l t ed in severe root prunning

of seedl ing roots and consequent dv/arfing of co t ton , R, ra^ifomnis

on G, barbadense caused des t ruc t ion of the epidermis and c o l l a p s e

of f i r s t 2 or 3 layers of the c o r t i c a l parenchyma. The phloem

c e l l s became compact. Cel ls of the per icyc le exhibi ted hypertrophy,

along the axis of the root , and developed in to g i an t c e l l s

(Oteiffa and Salem, 1972), Siveikumar and Seshadri (1972) found

R, reniformis to be a phloem feeder in cas tor , papaya and tomato.

The infec ted t i s s u e at the feeding s i t e showed hypertrophy, hyper­

p l a s i a , thickening of c e l l wal ls , granular cytoplasm and enlarged

n u c l e i . No changes were observed in the xylem, Cohn (1973)

observed R, reniformis feeding in the s t e l a r region of cot ton ,

tomato and mint . The c e l l s of the per^icycle were hypertrophied

and c e l l wal ls in endoderrais were thickened. Some of the hyper­

t rophied per icyc le c e l l s were mul t inucleate in feeding zone of

^« ^g>^ifQ^"'is in mint and there was break-down of c e l l wal ls in

t h i s zone in mint as well as upland cotton (Cohn, 1976). R. macro-

do ra t a s in oak and soybean induced a s ingle , l a rge and well defined

22

syncytium, extending deep i n t o the s t e l e , and bordered by a

thickened wall which c l ea r l y i so l a t ed i t from normal t i s s u e

(Cohn, 1976) . R. reniformis in sweet potato roots fed in endo-

dermis. The g iant c e l l s were observed in xylan parenchyma opposi te

to the feeding s i t e , I sod iane t r i ca l c e l l s were associated with

the g iant ce l l s^ (Brathwaite e t a l . , 1974). In r e s i s t a n t and

suscep t ib l e soybean roots , Rebois e t ^ . (1974, 1975) reported

some s t r u c t u r a l changes induced by R, reni formis . The fenale nema­

tode penetra ted i n t r a c e l l u l a r l y through the cor tex to the endo-

dermis where they inser ted t h e i r s t y l e t s , secreted, and i n i t i a t e d

syncy t ia l formation and c e l l hypertrophy. Syncytia pr imar i ly

involved per icyc le t i s sues and to a l e s s e r extent , xylan paren­

chyma and Qfidodermis, They observed i n i t i a l c e l l of syncytium (ICS)

usua l ly in endodermis, suscep t ib le t i s sues esdiibited two bas i c

developmental phases the period of infect ion : an i n i t i a l p a r t i a l

c e l l wall l y s i s and separat ion phase; and an anabolic phase, charac-

t a r i z e d by organized p ro l i f e r a t ion and development accompanied by

th ick secondary wall deposi ts , which provided n u t r i t i o n for s e s s i l e

female development, Hie r e s i s t a n t or hypersens i t ive react ion (HR)

lacked the anabolic phase and was charac ter ized by a continued, ,

u sua l ly accelerated, and uncontrol led l y s i s phase. R. reni f ormis

feeding, in cantaloup, s t imulated the per icyc le to e i the r s i d e of

the endodermal feeding ce l l and caused hypertrophy with enlarganent

of the nuc leo l i and granular thickening of the cytoplasm (Heald, 1975)

fo«

The feeding area of R. ren i for ro is on cowpea r o o t s comprised of

6 - 1 2 c e l l s on e i t h e r s i d e of the nematode head was observed by

Razak e t al_. (1976). A group of 4 - 6 c e l l s c l o s e s t to nematode

l i p s formed t h e " feeding zone" . The " feed ing c e l l " (or p o r o s y n c y t e

o r i n i t i a l c e l l of syncyt iumr was u s u a l l y l o c a t e d near a p r o t o -

xylem p o l e . A " feed ing peg" surrounded the nematode s t y l e t where

i t p e n e t r a t e d t h e th ickened c e l l wal l of t h e i n i t i a l c e l l . From

t h e feed ing pegs o p p o s i t e t h e s t y l e t a p e r t u r e , emerged a c o i l e d

" f e e d i n g tube" (Razak and Evans, 1976). Robinson and Orr (1980)

found t h a t t h e feeding c e l l was of p e r i c y c l e o r i g i n a t two f e e d i n g

s i t e s , c o r t i c a l o r i g i n a t t h r e e feeding s i t e s and of «idodermal

o r i g i n a t 23 feeding s i t e s i n sunflower i n f e c t e d with R. r a a i f o r m i s .

3 . 3 . ENDOPARA3ITE3

The endopa ras i t e s comple t ing t h e i r e n t i r e l i f e c y c i e almost

i n s i d e t h e h o s t t i s s u e s , b r i n g about most comprehensive anatcoaical^

c y t o l o g i c a l and p h y s i o l o g i c a l changes i n s i d e t h e h o s t c aus ing grerat:

damages t o p l a n t s . The e n d o p a r a s i t e s a re of t h r e e types , m i g r a t o r y

e n d o p a r a s i t e s comprising P r a t y 1 enchus, Radopholus and H i r s c h m a n i e l l a ;

p a r a s i t e s of a e r i a l organs l i k e D i ty l enchus , Anguina, Aphelenchoides ,

Rh ad in aph e l enchus and Burs aphel enchus; and s e d e n t a r y a n d o p a r a s i t e s

c o n t a i n i n g Heterod^ra , Glcdaodrera, Meloidogyne, Nacdbbus, Melo idodera .

24

3 , 3 . 1 . MIGRATORY ENDOPARASITES

M i g r a t o r y e n d o p a r a s i t i c nematodes e n t e r t h e p l a n t t i s s u e s

and move abou t a c t i v e l y . They f e e d on a c e l l , k i l l i t , and

move t o an a d j a c e n t c e l l . iSne damages a r e i n t h e form o f n e c r o ­

t i c a r e a s , l e s i o n s and even t u n n e l s .

3 . 3 . 1 . 1 . P r a t y l e n c h u s F i l i p j e v , 193 6.

P r a t y l e n c h u s s p p . a r e mos t commonly r e f e r r e d t o as ' l e s i o n *

o r ' r o o t l e s i o n ' nematodes b e c a u s e of t h e c o n s p i c u o u s n e c r o t i c

s p o t s which t h e y p r o d u c e on t h e r o o t s of i n f e c t e d p l a n t s . P i t c h e r

e t al^., (1960) s t u d i e d t h e e f f e c t s of P . p e n e t r a n s on a p p l e s e e d ­

l i n g s u n d e r a s c e p t i c c o n d i t i o n s , Th-ere was a r a p i d d i s c o l o r a t i o n

of t h e o u t e r m o s t ( i . e . e p i d e r m i s and h y p o d e r m i s ) and i n n e r m o s t

( i . e . i n n e r c o r t e x and e n d o d e r m i s ) c o r t i c a l t i s s u e s , b u t l i t t l e

o r no r e a c t i o n i n t h e i n t e r v e n i n g c o r t i c a l p a r e n c h y m a . I n p e a c h

r o o t s , a l l t h e c o r t i c a l t i s s u e s were x e a d i l y d i s c o l o r e d . H i s t o -

c h e m i c a l t e s t s u g g e s t e d t h a t s e n s i t i v i t y t o P . p e n e t r a n s i s

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

t a n c e s i n t h e v a r i o u s t i s s u e s of t h e s e two h o s t s . P e p p e r r o o t s

p a r a s i t i z e d by P . p e n e t r a n s were damaged due t o t h e d e s t r u c t i o n

o f pa renchyma c e l l s of t h e r o o t c o r t e x ( s h a f i e e and J e n k i n s 1963)

P , c o f f e a e f ed p r i m a r i l y i n t h e pa renchyma of r o o t c o n ; e x o f

25

of Citrus jambhlri, when invaded a root t i p , i t often destroyed

meristine ana l a t e r a l root i n i t i a t i o n usua l ly occured near the

destroyed root t i p (Radewald, 1971). Oyekan et al_., (1972)

observed tha t within 24 h r . of inoculat ion of pea roots by

P. penetrans the nematodes were mostly in the mid cor tex which

showed orange d i sco lo ra t ion ; t h e i r feeding and reproduct ive a c t i ­

v i t i e s r e su l t ed in extensive breakdown of the cor tex , No nema­

todes were observed within the s t e l e , co rbe t t (1972) observed

t h a t P. fa l lax after penetra t ion in root t i p s r e su l t ed rap id

necros i s in the region of root h a i r development and the junct ion

of main and l a t e r a l roots of wheat, ba r ley and sugarbeefe.

The axenic penetra t ion and colonizat ion of tu rn ip (Brassica

rapa) and corn (Zea mays) root t i s sues by P, penetrans produced

brownish necro t ic l e s i o n s . "The nematode fed on both h o s t s ,

e c t o p a r a s i t i c a l l y for a while, on a l l zones of root t i p except

t be roo t cap. After penet ra t ion i t migrated around the S t e l a r

regions in the cortex to produce l a rge c a v i t i e s . Although the

paa^ t ra t ion appeared to be mechanical, t he re was often a d i s ­

co lo ra t ion of the c e l l s as the nematode advanced, suggest ive for

an erizymatic action (Ogiga and Ester , 1975) . P. s c r i b n e r i i n f e c ­

t i on showed l i t t l e c e l l u l a r necros is in snap bean', whereas c e l l s

surrounding the nematode in lima bean were extensively nec ro t i c

(Thomason et a l . , 1976) . 01 owe and Corbett (1976) found both

28

P. brachyurus and P, zeae in all par ts of the maize roots inc lud­

ing the s t e l e . Cavit ies were founa in the cortex with l i t t l e

accompanying necrosis and in the s t e l e with much, including the

deposi t ion of a dense s ta in ing substance tha t occluded xylem and

phloem t i s s u e s , P. zeae caused more mechanical damage but l e s s

necros i s than P. brachyurus.

Huang and Chiang (1976) s tudied the effects of P. coffeae

on sunki orange (Citrus sunki) in the green-house and found

nematodes feeding primari ly in the cortex, both in te r -and i n t r a -

c e l l u l a r l y , r e su l t ing in extensive c e l l u l a r des t ruc t ion . Ttie

nematode did not penetra te the endodermis and did not cause hyper­

p l a s i a , hypertrophy or any s t imulat ion in c e l l u l a r growth at the

i n f ec t ion s i t e s . Cells at infect ion s i t e s showed r e t r a c t i o n and

disappearance of cytoplasm, thickening of c e l l walls and necros i s

of c e l l s around feeding s i t e s ( inse r ra e t £ 1 . / 1980; A::osta and

Malek, 1981; Onapitan and Anosu, 1982).

3 .3 ,1 .2 Radopholus Thome, 1949.

Burrowing nematodes, Radoptiolus spp. cause in jury e s sen t i a l ly

l i k e t h a t a t t r ibu ted to Pratylenchus spp. Females and l a rvae a l l

en t e r the root and destroy c o r t i c a l c e l l s , thus producing small

n e c r o t i c les ions which enlarge with ixivasion by b a c t e r i a and

fungi . Du Charme (1959) found tha t R. s i m i l i s entered ac t ive ly

27

growing root t i p s in the region of ce l l elongation and root

h a i r production in c i t r u s r o o t s . As the nematode progressed

through the root, tunnels and c a v i t i e s were formed in the cor tex

and s t e l e . All types of parenchyma t i s sues were a t tacked.

Ins ide the root the nematodes entered the s t e l e through the

passage ce l l s of the endodermis. 'the phloem cambium r ing , where

accumulated in la rge numbers, seemed oreferred. Cells exposed

to the nematode metaboli tes sometimes became hypertrophied.

Hyperplasia and tumour formation from the per icyc le occurred

when the nematodes passed through the endodermis and became s i tua ­

ted next to the pe r i cyc le . These per icycle tumours were a lso

a t tacked by the nematodes. Blake (1961) found R. s i m i l i s in

l e s ions in the root cor tex of banana, containing females, l a rvae

and eggs, originated as a puncture in the epidermis of the roo t .

Beneath the epidermis was a zone of necro t ic c e l l s . iSie c e l l s

forming the boundary of the les ion showed mild hypertrophy.

Within 12 hr of entering the cor tex, the s^ize of the nucleus and

nucleolus in the few c e l l s immediately surrounding the nematode

had increased s i g n i f i c a n t l y . This suggested t h a t the nematodes

f-ed d i r e c t l y on the cytoplasm of the c e l l (Blake, 1961, 1966).

O' Bannon et _al_., (1967) made h i s t o log i ca l comparisons in four

c i t r u s rootstocks infected with R. s i m i l i s . In a l l four

v a r i e t i e s ; i . e . 'Ridge pineapple ' and 'Milam' r e s i s t a n t ; ' E s t e s '

t o l e r a n t and 'Duncan' highly suscep t ib le to R. s i m i l i s , the

28

ner(,atodes penetrated the epidermis, cortex and endodermis,

causing ce l l wall d i s in teg ra t ion , hypertrophy, hyperplasia , and

wound gum formation. Ent i re vascular cylinder was invaded in

'Duncan' and 'Milam' but not in 'Ridge pineapple ' and ' E s t e s ' .

There was l i t t l e or no nuclear change in ' E s t e s ' and 'Duncan*.

However, in 'Milam' and 'Ridge pineapple roots the nucle i and

nuc leo l i in c e l l s adjacent to the infected area became enlarged.

R. s imi l i s penetrated the t i s sues in rhizomes of ginger (Zingiber

of f ic ina le) i n t r a c e l l u l a r l y forming channels or g a l l e r i e s

(Vilsoni et a l . , 1975).

3 , 3 . 1 . 3 . Hirschmaniella Luc & Goodey, 1963.

Hirschmajiiella g r a c i l i s causes a disease of r i c e c a l l e d

•Mentek' beginning as roo t r o t with small discolored areas which

gi>adually enlarge involving the e n t i r e cor tex . In severely

in fec ted plants only the epidermis and cen t ra l cyl ind«r remains,

the parenchyma being t o t a l l y destroyed. Secandaxy roots and

root h a i r s become dead (Jenkins and Taylor, 19 67) .

Babatola and Bridge (1980) s tudied the feeding beh^aviour

and histopathology of H. oryzae, H. imamuri and H. sp in icaudata

on r i c e . They found les ions extending along the burrowed channels

of the nan atode for some dis tance from the entry point , u n t i l ,

eventual ly the nematodes were surrounded by broken non-necrot ic

29

c e l l s . Mechanical damage caused l a r g e c a v i t i e s and c o l l a p s e of

c e l l w a l l s , mainly wi th in t h e c o r t e x . The s t e l e was dan.aged only

in r o o t s i n f e c t e d by H. s p i n i c a u d a t a . The burrowing a c t i v i t i e s

of H. imamuri almost severed or imord ia of l a t e r a l r o o t s , c o r t i c a l

and l a c u n a l c e l l s were c o l l a p s e d , and c e l l w a l l s were broken by

nematode f eec ing .

3 . 3 . 2 . SEDENTARY END0PARA3ITE3

Nematodes of s e v e r a l genera a re adopted t o a s e s s i l e l i f e

w i t h i n h o s t p l a n t s . A i u l t swollen females remain in one p o s i t i o n

and induce formation of s p e c i a l i z e d c e l l s in t h e h o s t . These

c e l l s s u r v i v e r epea t ed removal of p o r t i o n s of t h e i r c o n t e n t s .

3 . 3 . 2 . 1 . He te rodera Schmidt, 1871.

Ttie c y s t forming nematode H e t e r o d e r a i s p r i m a r i l y a r o o t

p a r a s i t e though i t may a l so occur on underground s t e n s , e . g . ,

s t o l o n s and tube r s of p o t a t o (Oostenbrink, 1950) and, excep t ion ally^

on l e a v e s of wh i t e c love r Ross , (1960) . Syncy t i a l formatrion

appears t o be an e s s e n t i a l phenomenon of s u c c e s s f u l p a r a s i t i s m in

H e t e r o d e r a . Syncyt ia induced by cys t -nematodes o r i g i n a t e i n t h e

p e r i c y c l e , endodermis, or ad jacen t c o r t e x , f r e q u e n t l y o p p o s i t e

protoxylem poles (Endo, 1978) . H. o r y z i c o l a i n f e s t a t i o n on r i c e

30

v a r . CRM - 13 - 3241 roots caused ce l l wall d i s in t eg ra t ion of

a group of phloeni parenchyma c e l l s of s t e l e and formed a syncyt ia

near the head region were highly thickened and the degree of

thickness gradually declined in c e l l s away from the nematode. The

cys t nematode broken open tl-je cor tex and epidermis to protrude

outs ide the root (jayapraXash and Rao, 1982) .

PeTietrarion i

Mankaa end Linford (i960) observed t h a t H. t r i f o l i i penet­

r a t ed clover roots within 14 min af ter coming in to contac t with

the root surface. The la rvae general ly entered the young p r o l i ­

ferous zone of the roots , but oenetrat ion was also common along

the length of the root up-to the mature t i s s u e .

Formation of Syncytium :

The juveni le i n s e r t s i t s s t y l e t in to the i n i t i a l c e l l of

syncytium (ICS), and aroumd t h i s a plug develops (Endo, 1978).

The wall near the s t y l e t does not dissolve, but becomes evenly

thickened as the syncytium develops. Walls i n t e r n a l to the

s t y l e t are degraded, however, so tha t p ro top las t s of neighbouring

c e l l s fuse . Wall d i sso lu t ion occurs at p i t f i e l d s , where t he

wall i s t h innes t . Affected c e l l s also expand, enlarging wall

gaps . syncytia i n i t i a t e d in the cortex extend towards the s t e l e ,

with r e l a t i v e l y l i t t l e c e l l expansion. Cel ls incorporated within

31

the s t e l e exoand considerably, especia l ly per icycle , vascular

parenchyma, and cambial c e l l s . Within the s t e l e , extension of

the syncytium occurs long i tud ina l ly on both sides of the i n i t i a l

region to reach a length of upto 2 - 3 mm. c e l l wall d i s so lu t ion ,

in Heterodera infect ion, in the process of syncyt ia l formation

has been reported on a large number of plant species (Nemec, 1932;

Mankau and Linford, 1960; Endo, 1964, 1965; <Ambrogioni and

Porc inai , 1972). syncytial walls of heterodera infect ion were

espec ia l ly thickened near the l i p region of the nematode and along

the syncytial walls adjacent to the xylan (Tr i f f i t , 1931; Kanec,

1932; Mankau and Linford, I960; Endo, 1964) . Cell wall thickening

•was minimal where syncytia appear to merge with normal t i s s u e s .

In an electron microscopic study of the ear ly stages of H. g lyc ine

infec t ion of soybean, Gipson et a l . , (1969) s t a t ed that c e l l wall

d i s in t eg ra t ion appear to be the i n i t i a l phenomenon leading to

syncyt ia l development. Within 42 hr of inoculat ion, abnormal

per fora t ions of c e l l walls were v i s i b l e . Progressive d e t e r i o r a ­

t i o n of ce l l walls occurred such tha t by 196 hr after inocula t ion

por t ion of the ce l l walls were no longer presen t .

Nuclei and Nucleoli in Syncytia :

Syncytia induced by nematodes have been reported to a t t a i n

t he polynuclear s t a t e by (1) mitoses in the absence of cy tok ines i s ,

(2) amitosis or nuclear fragmentation, and (3) the merging of

32

nucle i frcxn several c e l l s t ha t coalesce as a r e s u l t of c e l l wall

d i s s o l u t i o n . Within the polynuclear syncytium, nuclear hyper­

trophy has been a t t r ibu ted to swelling, nuclear coalescence, and

fusion or incorporation of chromosomal s e t s . Tischler (1902)

repor ted that an increase in the number of nuclei in young g ian t

c e l l s i n i t i a l l y occurs through normal mi to s i s . Later, fur ther

mult inucleat ion involved amitosis or nuclear fragmentation through

budding, in which nuclei gave r i s e to a whole se r ies of new n u c l e i .

In a repor t of giant c e l l s in gal led glorybower (clerodendron)

roots , Nemec (1910) observed tha t when the head region of the

nematode reached the plerome, the host c e l l s surrounding the oral

opening of the larvae enlarged. The plasma content of these c e l l s

increased, and the i r nucle i divided without the formation of c e l l

wal l s ; thus, mult inucleation of c e l l s occurred. Owens and Specht

(1964) found tha t mult inucleat ion in the ear ly stages of syncy­

t i a l development was due to the d i s so lu t ion of host c e l l wal ls

and the coalescence of t h e i r protoplasm, in the study of H. g ly­

c ines infec t ion of soybean root (Endo, 1954), mitosis was not

evident xn syncytia, Nuclear morphology of syncytia of c lover

roots infected by H. t r i f o l i i was described by Mankau and Lin ford

(i960) . upon st imulat ion by the nematode, the c e l l nucleus

enlarged and the cytoplasm increased in dens i ty and became hyper-

chromatic, c e l l s adjacent to syncyt ia showed nuclear enlargement

and c e l l u l a r hypertrophy, which was followed by a d i s so lu t ion of

33

the c e l l wall and a merging of cytoplasm.

syncyt ia l Cytoplasm :

The granular t ex ture of cytoplasm of giant c e l l s in

Heterodera spp. infect ions has been reported for numerous h o s t -

p a r a s i t e i n t e r a c t i o n s . Bergman (1958) noted the fine granular

t e x t u r e of syncytial cytoplasm in sugar beets during ear ly s tages

of H, schacht i i i n f ec t ion . At l a t e r s tages, the cytoplasm became

tu rb id and s ta ined heavi ly with fuchsin. The pers i s tance and

t e x t u r e of syncyt ia l cytoplasm in soybean roots infected by H.

g lyc ines varied with the feeding habi t s of the male and female

nematode (Endo, 1964). I n i t i a l l y both sexes st imulated a r e t i ­

c u l a t e cytoplasmic network which l a t e r became granular . Syncytia

induced by males were ephemeral, and t h e i r de t e r io ra t ion began

wi thin a few days af ter inocu la t ion . This i s i nd i ca t i ve of the

r e l a t i v e l y shor t feeding time of developing male l a rvae . The

ex tens ive syncyt ia l development and dense granular cytoplasm,

c h a r a c t e r i s t i c of syncyt ia induced by female nematodes, can be

a t t r i b u t e d to the prolonged feeding time required for t h e i r growth

and development. Gipson et a l . (1969) reported on the u l t r a -

s t r u c t u r e of syncyt ia induced by H. g lyc ines . They found t h a t

i n the f i r s t 6 days of development, the cen t ra l vacuoles of the

34

c e l l s being incorporated in to the syncytium appeared to be r e ­

placed with cytoplasm containing l a rge numbers of microtubules

and resembling smooth endoplasmic reticulum. The remaining vacuo­

l e s progressively decreased in s i z e as the syncytium developed.

Mankan and Linford (1960) reported the presence of hyaline, tubu­

l a r s t ruc tu res in the syncyt ia l cytoplasm of host ce l l s stimu­

l a t e d by H. t r i f o l i i . s imilar elongated bodies were observed

(Endo, 1964) in syncyt ia near the la rva l l i p region of the soy­

bean cyst nematodes feeding on soybean roo t s .

3 . 3 , 2 . 2 . Globodera Skarbilovich, 1959,

Potato cyst neir.atode (potato root eelworm, golden nematode,

formerly Heterodera ros toch iens i s ) Globoderra ros tochiens is

l a rvae af ter entering a potato r o o t l e t usual ly s e t t l e to feed

with t h e i r head end in the vascular t i s s u e . Certain c e l l s in the

s t e l e around the head of the nematode enlarge and coalesce to

form syncytia, which i s e s sen t i a l for normal female development

(Webster^ 1972) .

Feldmesser (1952, 1953) reported for the f i r s t time root

g a l l i n g on tomato roots infected by G. ros toch iens i s . Large

syncyt ia (giant ce l l s ) were developed during th i rd developmental

s tage of the nematodes, in the cor tex and in the s t e l e causing

d i scon t inu i ty of some vascular elements and the re ta rda t ion of

35

other vascular elements. During the fourth ana f i f th s tages

g i an t c e l l areas became more extensive, and individual g ian t

c e l l s became larger ana more i r r e g u l a r in shape, cu t t ing off

g rea te r areas of vascular t i s s u e . The processes involved in

the formation of giant c e l l s are fragmentation and d i s so lu t ion

of c e l l walls, coalescence of the cytoplasmic masses of the

previously i n t ac t c e l l s the aggregation of the nucle i from these

c e l l s ,

in potato roots infected with G, ros toch iens i s , g i an t c e l l s

were confined to per icycle and parenchyma c e l l s of the vascular

str.and between the primary xylam and phloem groups. Giant c e l l

formation was much less frequent in the cor tex (Cole Howard, 1958)

Hyperplast ic react ion in the c e l l s of tomato roots infecrted with

G. ros tochiens i s were d i f f e r en t i a t ed i n to three types by Serribdner

(1953) : (1) Extensive i r r egu l a r c e l l d iv is ion proceeding mainly

from the cent ra l cy l inder . (2) ^iul t iple subdivision of s i n g l e

c e l l s in the endodermis, in the neighbouring layer of cor tex

parenchyma and/or in the endodermis. (3) Hyperplast ic r eac t i ons

in a wound-periderm-like manner. Kaczmarek and Gieixel (1980)

reported mitot ic disturbances in presence of G. ro s toch iens i s in

the c e l l s of l e n t i l (Lens culin a r i s ) . lAA and CHA (chloragenic

acid) stimulated most miotic d is turbances , e .g . , mi tos is i n h i b i ­

t ion , various chromosomal aberra t ions , lack of cy tokines is , and

the formation of 2 - or 3 - nuc lea te c e l l s .

33

3 . 3 . 2 . 3 . Meloidogyne Goeldi, 1887.

infect ion with root knot nematodes (Meloidogyne spp.)

s t imulates the formation of e var iab le number of d i s c r e t e g ian t

c e l l s (usually about 6, but reported to vary from 2 to 12) in

host t i s s u e s . Hyperplasia and hypertrophy often surrounded the

region of infection and usual ly cause iga l l s to be formed t e rmi ­

n a l l y or sub-terminally on the infected r o o t s .

Penetrat ion ;

Root-knot nematode la rvae read i ly pene t ra te p lant roots

near the apical meristam (Nonec, 1910), within 24 hrs a f t e r

inoculat ion (Siddiqui & Taylor 1970) ; however, other regions of

the root are not immune to at tack (chr is lde , 1936). Once in the

root , migraticxi occurs both i n t e r - and i n t r a c e l l u l a r l y (Nemec,

1910 ; Chr i s t i e , 1936; Krusberg and Kielson, 1958).

Formation of Syncytiujan :

The formation of g iant c e l l s or syncyt ia through the d i s s o ­

lu t ion of ce l l waHs and the coalescing of t h e i r contents was

reported for root-knot infec t ion of Nicot iana hybrids (Kostoff and

Kendall, 1930). Ch r i s t i e (1936) gave fur ther support to t h i s

process of syncytial fTDrmation in the study of root-knot in fec t ions

of tomato. He observed t h a t undi f fe ren t ia ted c e l l s near the head

37

of the neri.atode enlarged s l i g h t l y and, following a s l i gh t swell ing

of nuc le i , the ce l l walls adjacent to the i n i t i a l l y s t imula ted

c e l l d i s i n t e g r a t e d . There was progressive d i sso lu t ion of c e l l

wal ls followed by the coalescence of protoplasm during the expan­

sion cf the giant c e l l . s imilar evidence of ce l l wall d i s s o l u ­

t ion has been reported for a number of hosts infected by species

of Meloidogyne (Krusberg and Nielsen, 1958; Dropkin and ^'elsen,

1960; Owens and Specht, 1964; L i t t r e l l , 1966). In c o n t r a s t ,

Huang and Maggenti 1969; Paulson and Webster, 1970; Jones and

Morthcote, 1972; Jones and Dropkin, 1976; Jones and Payne, 1978

found no evidence of c e l l wall d i sso lu t ion or breakdown in t he

formation of syncytia in root-knot infec t ions of Vicia faba when

syncy t i a l boundaries were observed through the electron micro­

scope. They concluded that c e l l wall breakdown played no pa r t in

g i an t c e l l formation and the mult inucleate condition arose p r i ­

marily from mitosis without cy tokines is .

Nuclei and Niicleoli in Syncytia :

The multinuclear s t a t e in syncytia induced by root-knot

nematodes have been reported to a r i se as described in case of

Heterodera infect ions (Tischler, 1902; Nemec, 1910; Krusberg

and Nielsen, 1958; Dropkin and Nelson, I960; Owens and specht,

1964; L i t t r e l l , 1965). Owens and specht (1964) found t h a t number

of nuc le i within the developing syncytium could be cor re la ted with

3^

the number of host c e l l s tha t would normally occupy the volume

of the syncytium. Thus multir.ucleation in the ear ly s tages of

syncyt ia l development was thought to r e s u l t so l e ly from the

d i sso lu t ion of host walls and the coalescence of t h e i r p ro to ­

plasm. Nuclear changes ranged from a nucleus near the s t y l e t of

the nonatode which showed a hypertrophied nucleolus with the

apparent absence of a nuclear manbraine, to nucle i with var ious

s tages of membrane de te r io ra t ion and a lobulated per iphery.

Other nuclei aberrat ions included nucleolar fragn.entation so t h a t

small granules, which s ta ined l i k e nuc leo l i , were s ca t t e r ed

throughout the nucleus. I r r e g u l a r l y shaped dumbbell^-or s i c k l e -

shaped nucle i were observed. s imilar observat ions of s i c k l e

shaped nuclei were reported on root-knot in fec t ions of sweet pota^

to (Krusberg and Nielsen, 1958), Nuclear enlargement in syncyt ia

of root-knot infected tomato, cucumber, and hawk's - beard

(crepis c a p i l l a r i s ) (Owens and Specht, 1964) appeared to r e s u l t

from swelling and in some cases from nuclear fusion. Such nuc­

l e a r fusion in syncytia of tomato account for the extreme en la rge­

ment of some nuclei which had diameters of 3 ^ as compared t o

normal oe l l nuclei of 6/u. Other da ta (Rubinstein and Owens, 1964)

i n d i c a t e tha t a 10 - 12 - fold increase in nuclear volume i s more

usual in ton.ato r o o t s . Hairy vetch plants inoculated with M.

i n c o g n i t a had syncyt ia l nuc le i with over 100 chromosomes, whereas

normal c e l l s have a 2N number of 14 (Dropkin 1965) ,

33

Syncytial cytoplasm :

The granular t ex tu re of syncyt ia l cytoplasm of g iant c e l l s

in root-knot infect ion has been reported for a number of hos t -

p a r a s i t e in te rac t ion (Chr is t ie , 193 6; Owens and 3pecht, 1964;

Heald, 1969; Rif f le , 1973). There was a tendency for the syncy­

t i a l cytoplasm near the head region of the nenatode to be more

dense aiid hyper-chromatic than the remainder of the syncy t ia l

contents (Christ ie , 1936). AS the nematode matures and n u t r i e n t

demands from giant c e l l s increase with egg laying, the giant c e l l

cytoplasm shows signs of in tense metabolic a c t i v i t y . Nuclei

become highly lobed and heterochromatic with prominent and

numerous nuclear pores, ind ica t ing rapid nucleus - cytoplasm

exchange. Starch gra ins are l o s t , and the secondary vacuoles

become more numerous and smal ler . Final ly , the cytoplasm becomes

ext rac ted as the giant c e l l s senesce, leaving some organel les and

the ingrowths, but l i t t l e ground cytoplasm.

3 , 3 . 2 . 4 . Gall Formation j

Mani (1964) descr ibes p lant ga l l s as "pa thologica l ly

developed c e l l s , t i s s u e s or organs of plants t ha t have ar isen

mostly by hypertrophy (over-growth) and hyperplasia (cel l p r o l i ­

f e ra t ion) under the inf luence of p a r a s i t i c organisms). Gall ing

i s the one of the e a r l i e s t hos t responses of root-knot nematode

40

in the roots of the p l a n t s . Schuster and Sullivan (I960) reported

t h a t g a l l s were induced in tomato roots by M, incogni ta l a rvae

even when they did not enter the tomato roots , thus concluding

t h a t the s t y l e t penetrated the root surface and secreted mate r ia l s

t h a t s t imulated host t i s sues to form g a l l s , Davis and Jenkins

(1960)/ reported gal l formation in Gardenia sp . infec ted with

^ » incogni ta , M, incognita a c r i t a and M. hapla. Cor t i ca l and

s t e l a r p ro l i f e ra t ion accompanied a l l i n fec t ions . In p l an t infec ted

with M, hapla although c o r t i c a l and s t e l a r parenchyma p r o l i f e ­

ra t ed extensively, c o r t i c a l c e l l s exhibited l i t t l e hypertrophy.

This lack of increase of c e l l s i z e probably accounts for the

smaller ga l l s i z e c h a r a c t e r i s t i c of M. hapla infected r o o t s .

Eversraeyer and Dickerson (1966) observed two types of g a l l s on

peony roots infected with M. incogni ta and M. hapla . 'Regular '

type of swellings involved the e n t i r e surface of the root ,

whereas the ' s ide* type involved only a l imi ted por t ion . Huang

(1966) reported gal l formation on the fibrous and f leshy roo t s

of ginger (Zingiber o f f i c i n a l e ) . Abnormal xylem and hype rp la s t i c

parenchyma were also present several small abnormal g a l l s on

newly formed adventi t ious roots as well as on r o o t l e t s of t ap

roo t of solanum melongena infected with M. incogni ta a c r i t a

were found by Vdrghese and Kamlesh (1970). Gall formation by

M, naas i in wheat and oat roots was studied by s iddiqui and

Taylor (1970); Siddiqui (1971a, 1971b,). S l igh t root g a l l i n g and

41

thickening of roots was found in conifers ghuja o r i e n t a l i s and

juniperus hor izon ta l i s infected with M, incogni ta . The ga l l i ng

in conifers was deviated from typ ica l root g a l l - i n g in other

p lan t s (Nemec and Morrison, 1972). Gall formation in d i f f e r en t

p l an t s infected with M. incogni ta has been reported by Tala t

Farooq (1973); Ibrahim et a l , , (1973); Mc d u r e e t a l . , (1974);

Ngundo and Taylor (1975); Jones and Payne (1978); Arens e t a l . ,

(1981); Glazer e t a l . , (1983).

Mechanism of Gall Formation s

B e i l l e , as early as 1898, suggested tha t g ian t c e l l forma­

t i o n involved ce l l wall d i s so lu t ion and subsequent cytoplasmic

fusicai between the neighbouriTng c e l l s ; t h i s view was suppoxted

by C h r i s t i e (1936); Krusberg (1963); Bi rchf ie ld (1965); Bird

(1973, 1974) ; Rohde and Mc d u r e (1975) . Nemec in 1910 gave an

a l t e r n a t i v e explanation for the mechanism of formation of g ian t

c e l l s . He Qbs-erved tha t nuclei in g ian t c e l l s induced by a root

knot nematode divided without forming new c e l l wa l l s . The same

was observed by Krusberg a©d Nielsen (1959); Bird (1961); Owens

and specht (1964); Smith and Mai (1965); L i t t r e l l (19€€) . Sw-SEiy

and Krishnamurthy (1971) and Prakaso Rao e t a l . , (1973) repor ted

t h a t the ga l l formation was a t t r i b u t e d t o the hypertrophy and

hyperp las ia of parenchyma c e l l s and p a r t l y of vascular c e l l s in

51* JQV^Q J-ca infected p l a n t s . Ibrahim and Massoud (1974) repor ted

ttea^ M. j avanica was foind feeding in cortex, endodennis, p^^icycle

42

and s t e l e of soybean roots r e su l t i ng hypertrophy, hyperolasia

and g iant ce l l formation in the t i s sues immediately surrounding

the nematode head and consequently developing g a l l s .

Ga l l s on 4?t>ove-ground Plant Parts :

Sometimes root-knot nonatodes cause ga l l s on above-ground

p l a n t p a r t s . Linford (1941) inoculated stems, petioles and leaves

of tomato and cowpea and obtained g a l l s . M. incogni ta caused

leaf g a l l s in s ide ras i s fuse at a (^4iller and Di Edwardo, 1962) .

Wong et al_,t (1969) observed ga l l formation in ae r i a l pa r t s of

tomato inoculated with M, javanica , s l i gh t swellings appeared

around the point of infac t ion a few days af te r inoculat ion due

t o hypertrojAiy sr.d hyperplasia of the c e l l s around the neRistocte

head. Gall formation on in ae r i a l par ts of p lants l i k e stCTa, leaf,

p e t i o l e , inflorescence vas also observed by Steiner (1940) y

F a s s u l i o t i s and Deakin (1973); Taylor (1976); Mac Gowaan e t a l . ,

(1979); Lehman arrcl Mac Gowan (1933); Lehman (1985); L^raan and

Mac Gowan (1986) .

induct ion of Gall Formation :

Root-knot nornatode, once within the root, secre tes a

ma te r i a l which causes hypertrophy and to sora e extent hypeirplasia.

The comprehensive study of root-knot nematodes on tcmmto rcKrts

by C h r i s t i e (193 6) anxi the study of ga l l ing respcaises xepoTLi ad

43

by the ear ly •workers Treub (1887) and Meroec (1910, 1911), bave

l a i d foundation for recent s tud ies on the agents of ga l l forma­

t i o n . The process of ga l l ing by nematodes which i s brought

about la rge ly by hyperplasia and syncyt ia l formation, appear to

be two qu i t e d i f fe ren t responses . The former s t a r t s r e l a t i v e l y

rap id ly , i . e . , within a few hours of infect ion, and may be l a r v a l

feeding at the root surface without actual ly enter ing the root

(LO 'wenberg et a l . , 1960; Schuster and Sull ivan, I960), The

d i f f e r e n t shapes and s t r u c t u r e s of the syncytia induced by the

four Meloidogyne species in tobacco provide some ins igh t in to

the or ig in and development of syncyt ia . The compact rounded

syncy t ia associated with M. incogni ta and M. hapla appear to be

the r e s u l t of increased nuclear a c t i v i t y without subsequent cy to­

k i n e s i s , m con t ras t the fonnation of very extensive, h ighly

lobed g iant c e l l s associated with M. aren a r ia and M, javanica

on tobacco may involve c e l l d i sso lu t ion and coalescence of

cytoplasm (sosa - Moss et a l , , 1983). The r o l e of growth pro­

moting substances are propos ed to be involved in the production

of galXs by Ustinov (1951); Owens and Specht (1964)? Yu ^id

V ig l i e r ch io (1964); Dropkin (1972).

Muge (1956a, l956b) suggested tha t amino acids which were in

much g rea te r concentration in g a l l s than in heal thy t i s sues were

probably responsible for g a l l i n g and tha t possibly l i b e r a t i o n of

amino acids by nematodes caused the growth of the g a l l , Good«y

44

(1948), for the f i r s t time suggested that ga l l s were i n i t i a t e d

by excretion, similar to auxins, AiXins have been i d e n t i f i e d

in g a l l s caused by root-knot nematodes, and frequently g rea te r

auxin concentrat ions occur in nematode infected t i s s u e than in

non-gal led t i s s u e (Balasubramanian and Rangaswamy, 1962; Set ty

and Wheeler, 1968), Certain auxins, are unique to ga l led t i s s u e

and t h e i r concentrations axe influenced by the Meloidogyne and

h o s t species involved (YU and v i g l i e r c h i o , 1964; Vig l ie rch io and

Yu, 1968). Cytokinin l eve l s may also increase in Meloidogyne-

i n f e c t e d plants (Krupasagar and Barker, 1966)# and have been

repor ted to be higher in noninfected root-knot suscep t ib l e p lan t s

than in r e s i s t a n t plants (V3t\ staden and Dimalla 1977), Auxins

alone (Kochba and samish, 1971), cytokinins alone (Dropkin e t a l . ,

1969), and ccxnbinations of these two (Kochba and samish, 1971;

Sawhney and Webster, 1975), when exogenously supplied to i t r o o t -

knot r e s i s t a n t pjlants, reversed the r e s i s t ance response and made

p l a n t s suscept ib le to infect ion by Meloidogyne juveni les^ Aixins

and cytokinins have also been iden t i f i ed in d i f f e ren t srtages of

Meloidogyne (v ig l ierchio and Yu, 1968; Dimalla and van Stadec,

1977) , If growth regula tors are involved in g ian t c e l l formation,

y e t i t i s not c lea r whether they are of nematode or hos t o r i g i n .

Ethylene, another p lant growth regula tor , has been assoc ia ted with

g a l l formation. Ethylene production was highly correlatred with

i n c r e a s e s in ga l l weight on M, javanica infec ted , excised tcanato

43

roo ts , suggesting tha t i t may be involved in the hypertrophy of

c o r t i c a l parenchyma t i s s u e during ga l l formation (Glazer e t a l .^

1985).

Effect of Galls on vascular Elonents :

The hos t -pa ra s i t e r e l a t i onsh ip of the root-knot nen\atode

in fec t ion causes disrupt ion of xylem and phloon t i s s u e s r e s u l t i n g

in hindrance in the t ranspor ta t ion of water and mineral n u t r i e n t s

and t rans loca t ion of food mater ia ls in the host plan, Davis and

Jenkins (1960) observed the d is rupt ion of s t e l e in Gardenia s p .

in fec ted with Meloidogyne sp, by g ian t c e l l s . P ro l i f e ra t ed

par«ichyma c e l l s caused the conducting t i s sues to s c a t t e r so tha t

xylem and phloeni occurred in i r r e g u l a r patches ra the r than in

one coluiBC, They also observed a periderm l i k e formation devel ­

oping from the outer layers of cor tex on the infec ted s i t e of the

root , e v ^ though the nematode was embedded severa l c e l l s beneath

t he sur face . In ginger (zingiber o f f i c ina le ) infec ted with M.

incoT^ni ta» wcmnd cork and l i g n i f i e d wall thickenings of endoder-

mis cffid por icycle are reported at infec t ion s i t e s (Huang, 1966).

CXiihir±n and Jenkins (1965) a lso repor ted d is rupt ion of vascu la r

cy l i nde r so that the cont inu i ty of xylem and phloonn i s broken,

Ta la t Farooq (1973) found the d iv i s ion of s t e l e in root g a l l s of

Licopersicon piropinel 1 ifolium infec ted with M, incogni ta , vftien

the giant; ce l l complex was in the vascular cyl inder , the abnormal

43

vesse l s connected with the phloem f ib res ; when the g iant c e l l

coniplex was in the cortex, vessels were independent of surround­

ing c e l l s (Ediz and Dickerson, 1976) . In infected roots the

an t e r io r ends of the larvae were invar iably embedded in the

phlo«n and the giant c e l l s were frequently bordered by xylem

v e s s e l s . This resu l ted in a morphological aberration of the

vascular cylinder (Finley, 1981). Hhe formation of abnormal xylesm

has been reported by d i f fe ren t workers in d i f fe ren t p lan ts i n f e c ­

t ed with root-knot nesmatodes (L i t t e r e l , 1966; Siddiqui and Taylor,

1970; Siddiqui e t a l , , 1974; Ngundo and Taylor, 1975; Jones

e t a l , , 1976; Byrne et al_., 1977; Meon et a l , , 1978; Jones 1981;

pasha e t , a^.# 1987). Eversmeyer (1966) reported vascular t i s s u e s

in Peony roots in which xylem and phloom were mostly parenchyma­

tous having much s t a r ch . Ri f f le (1973) observed tha t xylen

t r ach i eds in immediate v i c i n i t y of g iant c e l l s were d i s t o r t e d ,

crushed and even sca t t e r ed in i r r e g u l a r i so l a t ed pa tches . The

complete suppression of xylem and phloor t i s sues has also been

reported (Wong and f i l l e t s 1969; swamy and Kirishnamurthy, 1971).

Siddiqui and Ghous^ (197S) found t h a t af ter the des t ruc t ion of

primary phloem a t tiie s i t e of infect ion abnormal elements with

unusual o r ien ta t ions are formed in the roots of Lagenaria

l eacan tha infected with M. incogni ta . Pasha et a l . , (1987) found

t h a t abnormal xylem occurred in i r r e g u l a r patches with s c a t t e r e d

ve s se l elonents resu l ted in d i scon t inu i ty of vascular t i s s u e s .

47

Effect of Galls on Physiology of Plants :

The root-knot nematode not only induces cyto logica l and

anatomical changes in the host plant but they also affect the

host physiology in one way or the o ther .

i ) Respiration s

Muge (1956) found tha t r e sp i r a t i on of Meloidogyne induced

g a l l s of cucumber was three times grea ter than that of non-

infec ted root t i s s u e . Bird (1962) found tha t r e sp i r a t i on of g a l l

t i s s u e was no grea te r than tha t in adjacent t i s sues or in un­

infec ted roots in tomato. The e f fec t of Meloidogyne in fec t ion on

r e sp i r a t i on r a t e var ies with nematode-host combinations and p l an t

age^ increased enzyme a c t i v i t y (Veech cmd Endo, 1969) and chaanges

in biochemical components of g ian t c e l l s Owens and specht (1966)

show t h a t giant ce l l metabolism i s affected and regulated by the

nematode.

i i ) Translocation of water and Nut r ien t s j

His to logica l s tudies (Byrne e t a l . , 1977; Meon et a l , , 1973)

of Meloidogyne ga l l s revealed a d i s rup t ion in th-e contrinuity of

vesse l elements and the presence of abnormal vessel elements. Such

a l t e r a t i o n d is rupt nu t r i en t and water flow in the p l an t . Hunter

(1958) s tudied nu t r ien t absorption in tomato infected by M.

incogn i t a . He found suppressed shoot growth and ch lo ro t l c

4S

fo l i age on diseased plants with no accompanying a l t e r a t i on of

N, P/ K, Ca, Mg and Fe content in the fo l iage . Roots of infected

p l an t s , however, contained la rger quan t i t i e s of N, P, K and Mg

than did uninfected p l a n t s . Translocation of other substances

i s impaired in Meloidogyne-infected p l an t s . Nicotine, which

i s synthesized in tobacco root t i p s , increased in root t i s s u e of

suscep t ib l e and r e s i s t e n t plants and in leaves of only r e s i s t a n t

p lan t s following M, incogni ta infect ion (Hanowanik and Osborne,

1975), However, leaf content of n icot ine decreased by ha l f in

the suscept ib le c u l t i v a r where the root damac(e was the g r e a t e s t .

This occurrence ind ica tes tha t t ranslocat ion may have been

affected,

O' Bainon and Reynolds (1965) reported tha t cotton p lan ts

in fec ted by M, incogni ta consumed s l i g h t l y more water than

noninfected plants when water was supplied to the p lants c o n t i ­

nuously, Mecan et el^,, (1978) found tha t water flow in infec ted

tomato roots decreased with increased inoculum dens i t i^^ &nd

with decreasing so i l moisture content . They concluded tha t

d i s rup t ion and development: of vascular elements resu l ted in r e s ­

t r i c t e d water flow.

'\\i^ Photosynthesis :

Meloidogyne infect ion of roots decreases the r a t e of

photosynthesis in leasves. Loveys and Bird (1973) reported, t h a t

43

high inoculum leve ls of M. j avanica on tomato caused a dec l ine

in net photosynthetic r a t e within two days after inocu la t ion .

This lower r a t e was maintained throughout subsequent growth of

t he infected p lan t . Wallace (1974) reported that decrease in

• the photosynthetic r a t e of young tomato plants var ied with

inoculum leve ls and was not g rea t ly influenced by plant age.

W) Biochemical Consti tuents :

The protein content of tomato syncytia has been shown to

be about 4 - fold in 22 days old syncytia (Bird, 1961) and also

to range from 3-to 20-fold depending on age (Ovens and specht,

1966) and to about 3-fold in soybean and 10-fold in balsam

(Gommers and Dropkin, 1977). Balasubramanian and Purushothanicffi

(1972) s tudied the phenolic contents of root-knot affected t i s s u e s

and observed not much difference in t o t a l phenolic contents in

g a l l e d and healthy r o o t s . However, orthodihydroxy phenolic

content was s l i g h t l y higher in ga l led t i s s u e than in normal t i s s u e s ,

Veech and Endo (1969) reported increased a c t i v i t y of enzymes

l i k e a lka l ine phosphatase, acid phosphatase, peroxidase, adeno­

s ine t r iphosphatase , e s te rase and cytochrome oxidase at the s i t e

of infec t ion than in adjacent non-infected t i s s u e . Owens and

Specht (1966) found that in ga l led t i s sues carbohydrates and

pec t in s decreased 2&A and c e l l u l o s e and l ignin decreased 31%.

Other components in the ga l l increased as follows : hemicel lulos=^

so

3 6%, o rgan ic acids eTAt f r e e amino ac ids 304%, p r o t e i n s 80%,

n u c l e o t i d e s 2^/o, KNA 87%, nsiA 70% l i p i d s 154%, and mine ra l s 4%.

s e v e r a l workers Endo and Veech, (1969) Veech and Endo, (1969),

(1970) Orion e t a l . , (1973); Wang and Bergeson, (1974); Lewis

and Mc d u r e , (1975). Jones and Payne, (1978). Kasr , e t a l .

(1980); Rashid, e t a l . (1981); Haseeb e t a l . (1982); Basu, e t a l , *

(1983). Ganguly and Dasgupta, (1983), have ca r r i ed out many

experiments to es tabl i sh a r e l a t i onsh ip between an increase or

a decrease of biochemical cons t i tuen ts and root-knot i n f ec t i on .

3 , 3 . 2 . 5 . Nacobbus Thome & Alle i , 1944.

False root-knot nematode, Nacobbus causes ga l l ing through

both hypertrophy and hyperplas ia . The c e l l s resemble Meloidogyne

induced g ian t c e l l s occurring in the cortex adjacent to the

s t e l e . These c e l l s , however, are described as spindle-shaped

area of amorphous t i s s u e with atypical c e l l walls which co l l apse

and coalesce (Jenkins and Taylor 19-67).

Clark (1967) observed damage to the host roots caused by

N_, se rend ip i t i cus la rva l feeding, followed by necrosis of c o r t i ­

ca l c e l l s . Galls were associated with adult females conta in ing

a sp indle shaped mass of small c e l l s having s ta rch g r a i n s , Jones

and Payne (19?7) found a c l e a r cut bouirdary between the g a l l

c e l l s surrounding the syncytium eand the transformed c e l l s in

51

tomato roots ^infected with bj_. aberrans. syncytia c e l l s were

interconnected by perforat ions through the i r wa l l s .

According to Inser ra et ^ . » (1983), a l l the juveni les

of N. aberrans invaded c o r t i c a l parenchyma of sugarbeet roots

and formed cav i t i e s in the c o r t i c a l c e l l s . Cells with dense

cytoplasm and hypertrophic nuclei were seen in cor tex and endo-

dermic c e l l s as a r e s u l t of nsnatode feeding. In some cases

c a v i t i e s extended from the c o r t i c a l parenchyma to the per iphery

of the cen t ra l cy l inder .

3 . 3 . 2 , 6 . Meloidodera Chitwood, 1956.

Pine cystoid nanatode, Meloidodera does not produce roo t

g a l l s . Giant c e l l s adjacent to the head of the female of Meloi

dodera , developed in the region of cortex, in protoxylem and

pxotophloera in both s lash and l o b l o l l y pine r o o t s . iSie pa r t of

t b e g ian t c e l l wall i n contact with the head of the female nema­

tode became thicJcer than the remaining wall area . Hyperplasia

of the c o r t i c a l and vascular parenchyma c e l l s surrounding the

giaant c e l l s was very common, Trachieds around giant c e l l s were

somewhat pushed out of pos i t ion , but t h e i r con t inu i ty remained

unbroken. 1*he c e l l s adjacent to the swollen female were col lapsed

and compressed (Ruehle, 1962 and Cohn e t a l . , 1984),

52

Mundo e t a l . , (1983) s t u d i e d g i a n t c e l l formation in

l o b l o l l y pine , peony and sage i n f e c t e d V7ith M. f l o r i d e n s i S / M.

c h a r i s and M, b e l l i r e s p e c t i v e l y . M. f l o r i d e n s i s i n i t i a t e d t h e

g i a n t c e l l in the p e r i c y c l e of p i n e , and i t was r e s t r i c t e d t o

t h e i n n e r per iphery of t h e v a s c u l a r c y l i n d e r . The g i a n t c e l l

remained surrounded by v a s c u l a r parenchyma c e l l s , which were

con t inuous with those of t h e p e r i c y c l e . Hyperp las i a commonly

o c c u r r e d in c e l l s adjacent t o t h e g i a n t c e l l s , however, no h y p e r ­

t r o p h y or n e c r o s i s was obse rved . M, c h a r i s and M, b e l l i a l s o

i n i t i a t e d g i a n t c e l l s in t h e p e r i c y c l e , b u t as t h e c e l l s en la rged

they extended i n t o t h e v a s c u l a r c y l i n d e r which i n some ca se s

became d i s t o r t e d . The mature g i a n t c e l l d i r e c t l y c o n t a c t e d

xylean and phloem which were major components of t he v a s c u l a r

c y l i n d e r . Both h y p e r p l a s i a and hyper t rophy of t h e ad jacen t t i s s u e s

o c c u r r e d .

3 . 3 . 3 . PJ^RASLITES QF JSERIJtti ORGj^S

A few of t h e p l a n t - p a r a s i t i c nematodes are a s s o c i a t e d wi th

t h e d i s e a s e s of f lowers , l e a w ^ ancL s te ins . They cause e x t e n s i v e

damages in the form of l e s i o n s and t u n n e l s in nonvascu la r t i s s u e s ,

3 . 3 . 3 , 1 Di ty lenchus F i l i p j e v , 1936.

Swollen t i s s u e s a s s o c i a t e d wi th stem nematodes D i t y l e n c h u s

s p p . have t h e same g e n e r a l h i s t o l c ^ i c a l appearance r e g a r d l e s s of

53

t h e hos t . Cells of the c o r t i c a l parenchyma are enlarged, more

spher i ca l , and more numerous in the swollen areas, with l a rge

i n t e r c e l l u l a r spaces. As ear ly as 1927 Quanjer suggested tha t

nematodes r e l ease a pect inase during feeding r e s u l t i n g in a

d i s so lu t ion of the middle lamella; t h i s allowed c e l l s to become

more spherical and produced la rger i n t e r c e l l u l a r spaces. Potato

tubers infected with D* dipsaci and D. des t ructor usua l ly have

a cracked epidermis with sunken les ions and a dry r o t condi-

t i o n . Lesions caused by D, des t ruc to r increase in s i z e as d i s ­

ease progresses, giving r i s e to the production of a r e t i c u l a t e

system of cav i t i e s and tunne ls . Cytoplasm and nuclei become

or ien ted toward the s ide of the s ide c loses t to the nematodes.

Coxtical cav i t i e s are found within roots pa ras i t i zed by D. des ­

t r u c t o r . In addit ion, hypertrophy and hyperplas ia occur in

both the s t e l e and the cor tex (Jenkins and Taylor, 1967).

Feder and Feldmesser (1953) pointed out tha t D. d i p s a c i

in fec t ion of Narcissus pseudonarcissus r e su l t ed in "Spikkeled*

areas on leaves, sickle-shaped leaves, s tunted unsaleable flowers

and eventual des t ruct ion of the bu lbs . The external Leaf moxpho-

logy and the r a i s i ng of the "spikkeled" area above the leaf s u r ­

face were the r e su l t s of hypertrophy and hyperplasia in areas

adjacent to nematode l o c i . Groups of affected epidermal c e l l s

enlarged and buckled away from underlying t i s s u e forming l o c u l a t e

54

"spikkels" containing the nematodes. Giant c e l l s were often

found in areas containing D, d ipsac i , and were charac te r ized by

heavi ly s ta ined cytoplasm, smaller vacuoles and abnormally la rge ,

often pycnotic c e l l s . Mitotic a c t i v i t y was not observed.

The effects of two populations of nematodes, waynessvi l le .

North Carol ina (WNC) and Raleigh, North Carolina (RNC) on Alaska

pea and Wando pea were s tudied by Hussey and Krusberg (1963, 1970).

WNC gave r e s i s t a n t response (necrosis) while (RNC) gave suscep­

t i b l e response (ga l l ing) . In WNC - infected seedl ings, seven

days a f te r inoculat ion, necrosis in the older par ts was more

extensive , although s t i l l r e s t r i c t e d to the epidermis and adja­

cent t i s s u e . After 14 days, nec ro t i c c e l l s were s t i l l mostly

continued to the epidermii:, but in scsne sec t ions , adjacent c o r t i ­

ca l c e l l s were also affected. These co r t i c a l c e l l s had under­

gone mi to t i c d iv is ions giving the appearance of cork formation.

Ce l l s in hyperplas t ic areas were much smaller than adjacent un­

affected c o r t i c a l c e l l s , and the hyperplasia was d i rec ted toward

the nec ro t i c c e l l s . In RNC-nematodes inoculation seedl ings , t i s s u e

d i s rup t ion , a suscept ib le response, was observed. Large c a v i t i e s

bordered by g rea t ly misshapen c e l l s occurred in the cor tex and

parenchymatous mesophyll t i s s u e of infected seedlings 7-days a f te r

i nocu la t ion . The large c a v i t i e s r e s u l t i n g from des t ruc t ion of

parenchymatous c e l l s , extended acropeta l ly in to the embryonic

55

leaves and bas ipe t a l l y in to the stem. Epidermis adjacent to

infec ted t i s s u e was hyperp las t ic and convoluted. No necrosis

was observed in t i s sues ga l led by RNC nematodes. After 14-days

of inoculat ion, the damage was moDe extensive causing general

des t ruc t ion of the cor tex . Portions of the epidermis were des ­

t royed . Outer layers of the cor tex were necro t ic and p a r t i a l l y

sloughed, with the adjacent c o r t i c a l c e l l s dividing and giving »

t he appearance of corX formation.

Reed e t a l . , (1979) inoculated Buffalo (susceptible) and

Washoe ( r e s i s t an t ) v a r i e t i e s with D. d i p s a c i . Sections of g a l l s

of Buffalo showed la rge c o r t i c a l c a v i t i ^ with darkly s t a ined and

granula r c e l l s on the p e r i p h e r i e s . Some hypertrophied c e l l s

surrounding cav i t i e s were evident . Nematodes were found in a l l

p a r t s of the cor tex from j u s t in s ide the epidermis to near the

vascular cy l inder , s imi lar g a l l s developed in Washoe, but c e l l u ­

l a r des t ruc t ion and cavi ty formation were l e s s extensive tha?^ in

Buffalo, Washoe had fewer enlarged c o r t i c a l c e l l s with granular

cytoplasm than Buffalo. Stem g a l l s on Ci'trus arv^nse by D. d i p -

s a c i were character ized by extensive hypertrophy and hyperplasia*

d i f f e r e n t i a t i o n of n u t r i t i v e t i s s u e , nuclear modification and a

c e n t r a l cavi ty containing nematodes (Watson and Shorthouse, 1979) .

5o

3 . 3 . 3 . 2 . Anguina scopoli , 1777.

Seed and Leaf ga l l nematode, Anguina feeds e c t o p a r a s i t i c a l l y

on young leaves near the growing point (Southey, 1982). Anguina

t r i t i c i produces g a l l s from undi f ferent ia ted flower buds. Stami-

na te t i s sues are f i r s t involved than c a r p e l l a t e t i s s u e s . F i n a l l y

t issu.es between carpels and stamens are involved. Larvae of

fescue leaf ga l l nematodes (A. graminis) enter the t i s sues of

young leaves and form g a l l s resembling knots or swoller nodes..

All por t ions of leaf t i s s u e become enlarged and hypertrophied,

the c e l l s being much l a rge r than normal, A. graminophila form

g a l l s on the leaves of f ine b e n t - g r a s s . Transverse sec t ions

revea l tha t c e l l hypertrophy and mul t ip l ica t ion involve ep ider -

mal» inesoi^iyll and vascular t i s s u e . Cells over the vascular

bundles form r idges , while those between are depressed in to

fuxrows (Thorne, 1961).

Goodey (1932) worked out on plants infected with d i f f e r en t

Anjguina spp. A, agroat is was, located in the p i t h and in the

ou^eo: pa r t s of the cor tex in red fusiform swell ings of the hos t

and. i n terminal r o s e t t e s of thickened leaves of Thymus v u l g a r i s .

A. p ra t ens i s made i t s way through root t i s s u e s , broken down

c e l l walls , and fed on c e l l contents , brought oibout d i s t o r t a t i o n

of c o r t i c a l t i s s u e of r o o t s . A. agropyronif lor is invaded only

57

f e r t i l e f lo re t s of wheat grass forming elongated g a l l s as a

consequence of celj. elongation ra ther than by c e l l d i v i s i o n .

A. Agrostis induced g a l l s in Lolium rigidum. Galls t y p i c a l l y

developed in place of ovules, l e ss commonly in place of stamens,

and r a r e ly on glume or r a c h i s . The g a l l s cons is ted of a wall

several c e l l layers deep surrounding a cen t r a l c av i ty , c e l l s

adjacent to the cavity were modified, presumably to provide meta­

b o l i t e s for development and reproduction of the nematodes, c e l l s

at the surface remained unchanged, thus maintaining the

s t r u c t u r e of g a l l s ,

Norton and sass (1965) showed tha t the only pa r t of the

inf lorescence, of wheat-grass (Agropyron smi th i i ) invaded by

A. agropyronif loris was the undeveloped f e r t i l e ovary, p r i o r

to stamen enlargement. Invasion of the ovary occurred down to ,

but not including the procscnbium. Cel ls in the d i s t a l half of

the undeveloped ovary became enlarged, vacuolate and loose ly

arranged. Stynes and Bird (1932) s tudied the develonment of

g a l l s induced by A. agros t i s in ryegrass (Lolium rigidum) , The

g a l l s which grew rapidly as nematodes developed, cons is ted of

a wall several c e l l l ayers deep surrounding a cen t ra l c a v i t y .

Ce l l s adjacent to the cavi ty were modified, presumably to provide

metabol i tes for development and reproduction of the nametodes,

which completed a s ingle generation before p lant senescence

occurred and the ga l l s dr ied ou t . c e l l s at the surface were un­

changed, thus maintaining the s t r u c t u r e of g a l l s .

58

3 , 3 . 3 . 3 . Aphelenchoides Fischer, 1894.

The fo l ia r leaf nematode, ^he lenchoides enters through

stoinata in to leaf mesophyll and destroys t i s sues during feeding.

Lesion become brown; usua l ly appearing as angular sec tors between

l a r g e leaf veins , itiey also reach flower buds. C h r i s t i e and Arndt,

(1936) frequently found A. par ie tans associated with l e s ions on

the underground par ts of p lan t s , espec ia l ly on the hypocotyle

of seed l ings , sometimes these nemas migrated beyond the diseased

area penet ra t ing apparently heal thy c o r t i c a l t i s s u e , and were

found coiled within c e l l s or i n t e r c e l l u l a r spaces,

Todd and Atkins (1958), washed root , culm, leaf and o a r i -

c l e s of various p lants , then s ta ined and examined. No nematodes

were observed within s ta ined t i s s u e s , however, nematodes were

recovered from leaf sheath washings confirming the e c t o p a r a s i t i c

n a t u r e of t h i s f o l i a r nematode, A r i t z^na -bos i fed almost e n t i r e l y

e c t o p a r a s i t i c a l l y on the epidermal c e l l s of anbryonic leaves aid

tiae shoot apex, outer 2 - 3 layers of c e l l s were k i l l e d and a

th i ck red s ta ined layer was formed. The red s ta ined layer i n h i b i t e d

growth and divis ion of the underlying i n t a c t c e l l ^ which b^ccOBe

enlarged, vacuolated, contained l i t t l e cytoplasm, and whose nuc le i

were l a rge , reddish and granular with apparently normal n u c l e i .

They SOTietimes penetrated through stomata i n t o mesophyll t i s s u e .

N^Tiatodes were o^t«i seen in substomatal c a v i t i e s and the

59

accompanying guard c e l l s were swollen causing the apertures

to enlarge. Cavit ies in which many eggs were seen were most

freqaent in the spongy mesophyll especia l ly around vascular

bundles (Krusberg, 1961).

3 . 3 , 3 , 4 . Rhadinaphelenchus Goodey, 1960

The coconut nematodes Rhadinaphelenchus spp. mul t iply rapid­

l y within palms and onormous numbers invade a l l par t s of t he

p l a n t , A band of nec ro t i c t i s s u e appears in the lower por t ions

of t he steras. This band may be 3 cm wide enci rc l ing the sten»

2,5 cm boieath the su r face . Local areas of necrosis not forming

a complete r ing develop in t he upper po r t ions . The reddish

co lor r e s u l t s from the death of c e l l s upcaa -^ ich the nematodes

have fed. The infec t ion causes occlusion of xylem vessels

(Dropkin, 1980),

Blai r and Darling (l968) found R. cocophilus in ground

parenchi«n-a c e l l s in stems, pe t io l e s sand cortex of roots in the

coconut palm. Nematodes were not confined to the discolored zone,

bu t a lso occurred in smaller numbers i n t e r c e l l u l a r l y in the

white paroichyma for a d i s tance of 1,5 cm on the outs ide and

4 cm on the ins ide of the discolored t i s s u e as well as in t he

meristematic region above the area of d i s c r e t e l e s i o n s . In the

region of d i s c r e t e l e s ions , the nematode occurred i n t e r c e l l u l a r l y

in the ground parenchyma of the stem. In c ross - sec t ion ,

they sometimes apoeared as heavi ly

60

s ta ined rounded dots, or they followed the shapes of i n t e r ­

c e l l u l a r spaces they occupied. In longi tudinal sec t ion , they

were intertwined between c e l l s . Occasionally, they were seen

nea t ly coi led in some c e l l s and in large i n t e r c e l l u l a r spaces .

They also occurred i n t e r c e l l u l a r l y in the bundle sheath ly ing

p a r a l l e l to immature f ib res , but not in the xylem nor phloem

t i s s u e s .

3 . 3 , 3 , 5 , Burs aphel enchus

Pine wood nematode i s t ransmit ted by a wood boring b e e t l e

from dead to uninfected t r ees of pines in Japan.

Y^uhara and Kiyohara (1972) found in Japanese red pine

(Pinus densif lora) and black pine (Pinus thanbergi i ) i n fec ted

with B. l ign ico lus tha t the nematode was present in both axial

and r ad i a l res in canals . The res in csaial p^ranchyma c e l l s ,

e p i t h e l i a l c e l l s , of nematode infes ted TSfocai were conspicuously

damaged anct in many cases, they were ctmpietelY dest royed. The

r e s i n canals were supposed to be the sd te of i n f e s t a t i on and

t h e e p i t h e l i a l c e l l s the most l i k e l y feeding s i t e . Myres (1982)

found pine wood nematode in the axial r e s in canals of Japanese

black, scot, and southern white p ine . The nematodes k i l l e d

parenchyma rays, cambial layers and rays , Myres (1986) inocu­

l a t e d ten d i f fe ren t species of pine with B, xylophilus and

61

observed that the nematodes migrated from the cor tex through

the rad ia l rays or d i r e c t l y across the sieve c e l l s of the phloem

to the cambium. Ttie nematodes moved v e r t i c a l l y as well as

c i rcumferent ia l ly destroying the fusiform and ray i n i t i a l s and

t h e i r de r iva t ives . Nematodes also occurred between xylem and

phloen.

i P/c an l^or^

6<^

P L ^ OF WOKK

s y n c y t i a o r g i a n t c e l l s f o r m - a t i o n i s must f o r t h e

s u c c e s s f u l e s t a b l i s h m e n t of h o s t - p a r a s i t e r e l a t i o n s i n r o o t - k n o t

n e m a t o d e i n f e c t i o n . R e g a r d i n g t h e f o r m a t i o n and d e v e l o o m e n t of

s y n c y t i a v a r i o u s h y p o t h e s e s and e x p l a n a t i o n s h a v e b e e n p u t f o r ­

w a r d e d by d i f f e r e n t v?o rke r s . Many p rob l ems s t i l l r emain u n ­

s o l v e d and i t i s n o t p r e c i s e l y knovnn w h e t h e r g i a n t c e l l f o r m a t i o n

and d e v e l o p m e n t a r e d e p e n d e n t on a c o n t i n u o u s s t i m u l u s from t h e

n e m a t o d e o r w h e t h e r o n l y an i n i t i a l s t i m u l u s i s r e q u i r e d t o

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

c e l l w i t h o u t f u r t h e r s t i m u l u s from t h e n o m a t o d e . Mankau and

L i n f o r d ( i960) s u p p o r t e d t h e f o r m e r h y p o t h e s i s b u t f u r t h e r

e x p e r i m e n t a l e v i d e n c e i s r e q u i r e d t o e s t a b l i s h i t .

To e s t a b l i s h t h e c o r r e l a t i o n s be tween t h e s t r u c t u r ^ a l cbanxres

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

t h e e f f e c t s on h o s t p l a n t s d e p r i v i n g them of n u t r i e n t s b y d i s ­

t u r b i n g t h e i r n o r m ^ p h y s i o l o g i c a l f u n c t i o n i s a l s o e s s e n t i a l .

sponge gouxd (Ltrff-a c y l i n d r i c a L . ) s a i d t o b e i n d i g e n o u s

t o I n d i a , i s a l a r g e c l i m b e r , grown t h r o u g h o u t I n d i a , smooth,

c y l i n d r i c a l , t e n d e r f r u i t s of s p o n g e g o u r d a r e u s e d as v e g e t c b l e , ^

I t i s i m p o r t a n t n o t o n l y as a v e g e t a b l e b u t a l s o as a m e d i c i n e

»

A clear l iquid , extracted from the stem i s useful in r e s p i r a t o r y

complaints. Ripe f r u i t , af ter burning and pulverizing, i s

used as carminative and anthelmint ic . Mature seeds are b i t t e r ,

emetic and c a t h a r t i c , seed o i l i s used for skin a f fec t ions .

Apart from fungal and v i r a l d i seases , i t i s also attacked by

root-knot nematodes. The g a l l s formed, due to root-knot nematodes

in the root are well developed and prominent. As far as h i s t o -

pathological studies of spong gourd roots , infected with r o o t -

knot nematodes, are concerned, the l i t e r a t u r e , as the review

ind ica t e s , i s s i l e n t . Ko work t i l l now has been car r ied out on

anatomical ananolies, Attenpts , also have not been iisaade to

e s t a b l i s h the effects of root -knot nematodes on anatomical, cy to -

log i ca l and physiological changes of the phloem t i s s o e . There­

fore , i t i s proposed to i n v e s t i g a t e the h is topa thologica l changes,

in complex t i ssues ; , with spec ia l emphasis on phloem, due t o r o o t -

knot in fec t ion . Following aspects wi l l be included ±n t he

proposed plan of work j

1. To study th-e e f f ec t of d i f fe ren t inoculum l e v e l s

of M. incogpxta on the development of g iant c e l l s

in Luffa cy l i nd r i ca r o o t s .

2, TO t race the d i f f e r en t cytologic el changes leading

t o the formation of syncyt ia .

61

3 , To study the damage caused by root-knot nematode

infect ion to vascular t i s sues in L, cy l indr i ca

roots as a consequence of root-knot nematode

i n f ec t i on .

4 . TO t race the h is topa thologica l changes, in roo ts

leading t o g a l l formation,

5 . To study the co r r e l a t ion between extent of damage

caused to vascular elements and reduction in growth

of p lants and physiological symptoms r e s u l t i n g from

dis turbed physiology of infected p l an t .

6. To study the di f ference in h is topa thologica l r e s ­

ponse of some v a r i e t i e s r e s i s t a n t and suscept±ble

to root-knot nematode.

7 . TO study the e f fec t of d i f fe ren t ecological s-tresses

on the formation of g iant ce l l and development of

g a l l .

^ /l/Jakrb/s one/ /l^e//?oc/j v

65

MATERIja^S J^D METHODS

T^e d i f f e r e n t m a t e r i a l s t o b e u s e d and methods t o b e

employed d u r i n g t h e c o u r s e of p r o p o s e d e x p e r i m e n t a l programme

a r e g e n e r a l i z e d as f o l l o w s j

5.1 Test Plants and Pathogens :

In the proposed plan of work sponge gourd (Luffa c y l i n d r i c a

L. ) w i l l be se lec ted as t e s t p lan t and root-knot nematode

(Heloidogyne incognita) as t e s t pathogen.

5.2 Collect ion of InocvaTum o± M. incogni ta :

Root-knot nematode infec ted roots wi l l be co l l ec t ed from

vegetable crop f i e l d s . Species of root-knot nonatodes and races

w i l l be i den t i f i ed on the b a s i s of the c h a r a c t e r i s t i c of pe r in ­

e a l p.atterns and d i f f e r e n t i a l host t e s t .

5.3 Pure Culturing and Maintenance of Inoculum :

A s ing le eggmass of M. incogni ta wi l l be surface s t e r i l i z e d

in 1 J 500 solut ion of Chlorox (calcium hypochlori te) fa r f i v e

minutes and washed t h r i c e in s t e r i l i z e d d i s t i l l e d water . iSne

egpg-raass w i l l then be allowed to hatch in d i s t i l l e d water at

68

27°C. Egg-plant seedlings ra i sed in 25 cm pots containing

autoclaved s o i l wi l l be inoculated with the la rvae thus obtained.

After about two months the so i l of inoculated pots w i l l be exa­

mined to ascertain the establishment of nematodes. Then sub-

cu l tu r ing wi l l be done by inocula t ing new egg-plants with at

l e a s t 15 egg masses obtained from pure cu l t u r e in order to main­

t a in su f f i c ien t inoculum throughout the course of i n v e s t i g a t i o n .

5 .4 . InoculatioD of Nematodes j

Pr ior t o inoculat ion the egg masses wi l l be removed from

the infected roots of egg plant and allowed to hatch (St«nerding,

1963). The counting of second s tage juveni les of M. incogni ta

w i l l be done with the help of countLng dish under the s t e r e o ­

scopic microscope. Three days a f te r seedl ing emergence holes of

5 - 7 cm depth around the p lants within a radius of 2 cm from

the p lan t w i l l be made, in which a counted number of nematode

lairvae w i l l be t ransfer red with the help of s t e r i l i z e d p i p e t t e .

The ho les \«ill then be plugged with s t e r i l i z e d s o i l . Regular

watering wi l l be done to maintain the so i l moisture t i l l t he t e r ­

mination of the experiment,

5 . 5 . Hjgtopathological Studies i

Inoculated seedlings wi l l be uprooted ca re fu l ly of the s o i l

frcan t h e f i r s t uir t i l the s i x t h day, then every th ree days u n t i l

67

t h e 30th day and f i n a l l y on t h e 40th day . The r o o t s w i l l be

washed gen t ly , but thoroughly t o remove a l l s o i l p a r t i c l e s

adher ing to them. Gal led r o o t s w i l l be c u t i n t o one cm long

p i e c e s and processed as fo l lows t

5 . 5 . 1 . F i x a t i o n :

The r o o t s c o l l e c t e d from exper imenta l po t s w i l l be f i x e d

i n FAA (Johansen* 1940) . FAA (Fo rma l in - ace to - a l coho l ) w i l l be

p repa red as follows j

Ethanol 5CPA 90 ml

Formaline 37% 5 ml

G l a c i a l a c e t i c a c i d 5 ml

The r o o t s w i l l be p laced i n t h e f i x a t i v e fo r a minimum p e r i o d of

24 h r t o s e v e r a l days , depending on i t s t h i c k n e s s . M a t e r i a l may

a l s o be s t o r e d in the f i x a t i v e i n d e f i n i t e l y .

5 . 5 « 2 . P ^ y d r a t i o n :

Dehydra t ion i s accomp-l ished by moving t h e r o o t s s t e p w i s e

th rough i n c r e a s i n g l y h i g h e r c o n c e n t r a t i o n s of a l c o h o l s . T e r t i a r y

b u t y l - a l c o h o l (TBA) dehydra t ion schedu le (Table 1) w i l l b e

fo l lowed ( johansen, 1940) .

TABLE - I

6b

S t e p % Alcohol Time D i s t i l l e d 99»/o 100% lOO/o w a t e r E t h a n o l E t h a n o l TBA (ml) (ml) (ml) (ml)

1 .

2 .

3 .

4 .

5 .

Ir

6. •

7 .

8 .

50 2 hr or more 50

70 ove rn igh t

85 1 - 2 h r

95 1 - 2 h r

100 1 - 3 h r

100 1 - 3 h r

lOO 1 - 3 h r

100 overn igh t

30

15

0

0

0

40

50

50

45

0 25

0

0

10

20

35

35

75

100

100

100

* TBA chapgeg~mast be c a r r i e d out i n a warm p l a c e as i t

s o l i d i f i - ^ ! a t 25 .5 C.

69

5 . 5 . 3 . I n f i l t r a t i o r :

in th i s step» alcohols in the t i s sues wi l l be replaced

by paraffin so that the t i s s u e i s saturated with a pure so lu t ion

of paraf f in , when the TBA dehydration schedule i s followed, the

10C% TBA solution in s tep 8 wi l l be replaced with a 1 : 1 mixture

of 100J4 TBA and paraffin o i l . The t i s s u e wi l l be allowed t o

remain in t h i s solut ion for 1 hr or more, depending on i t s t h i c k ­

n e s s , short ly before the next s t ep , another container wi l l be

f i l l e d ^ th of i t s volume with melted paraffin and allowed t o

s o l i d i f y s l i g h t l y . "Uie roots from the TBA-paraffin o i l mixture

w i l l then be placed on top of the s o l i d i f i e d paraffin o i l so lu ­

t i o n , •ftiis container wi l l be placed uncovered in an oven s e t a t

s l i g h t l y above the melting point of the para f f in . After 1-3 hr ,

t he TBA - Paraffin o i l mixture wi l l be poured off and replaced

with pure melted paraffin wax, and kept in oven for aibout 3 h r .

This s t e p wi l l be repeated at l e a s t once more.

5 . 5 . 4 . ESnbedding :

The roots wi l l be placed in metal base iiKJlds or folded

paper . Molds wi l l f i r s t be coated with a thin layer of g lyce r ine

and then, l iqu id paraffin w i l l be poured. Roots wi l l be placed

i n to t he mold with heated foceps and addi-tiorral melted paraff in

-will be added to f i l l the mold. Once the paraffin begins to

70

s o l i d i f y over the top of the mold, the mold wi l l be plunged

i n t o ice water and l e f t the re u n t i l s o l i d i f i c a t i o n . After harden­

ing, i t wi l l be cut i n to smaller blocks and trimmed. The blocks

w i l l be mounted on block ho lde r s .

5 . 5 . 5 . Sectioning :

Transverse and longi tud ina l sec t ions of 8 - 12 Aim th ickness

of the roots wi l l be cut s e r i a l l y with the help of r o t a r y micro­

tome. The paraffin ribbon thus obtained wi l l be mounted on a

clean g lass s l i d e with an amount of albumin and g lycer ine

d issolved in water. The s l i des w i l l be l e f t overnight in an o

incubator at 40 C to allow water to evaporate. These s l i d e s wi l l

then be kept at 6D C for on« hr to melt t h e pa ra f f in ,

5 . 5 .6 , Staining :

Th« process of s t a i n i n g wi l l remove the paraffin from the

sec t ions sBd increase the con t ra s t in the t i s s u e s . Staining w i l l

be (&a3€ with sa^rsoin and Fast Green combination (Table 2)

(•Sass, 1951). ThF«i, s l ides wi l l be r^noved from the xyleiae, t h e

f i n a l s tep in a s ta in ing procedures and l a i d on a f l a t , absorb ant

su r f ace . The mounting medium wi l l be applied to the surface of

t h e s l i d e before evaporation of the xylene, and cove r - s l i p w i l l

be lowered gradually over the s l i d e .

TABLE - 2

71

Step So lu t ion Time

1 .

2 .

3 .

4 .

5 .

6 .

7 .

8 .

9.

10 .

1 1 .

1 2 .

1 3 .

14 .

1 5 .

1 6 .

1 7 .

1 8 .

xylene

a b s o l u t e e thano l

95% e thano l

70% e thano l

50% e thano l

3 0% e thano l

1% aqueous safranim 0

r i n s e i n t a p water

30% e thano l

5C(% e thano l

70% e thano l

95% e thano l

5 min

5 tnin

5 min

5 min

5 min

5 min

1-12 h r

3 min

3 min

3 min

3 min

0..!% f a s t green FCF i n 95% e thanol 5-3 0 sec

abs^^olute e thano l 15 sec

a issolute e thano l 3 min

x y l e n e - a b s o l u t e e thano l 5 rain

xy lene 5 min

s y l e n e 5 min or l o n g e r

72

Finished s l i des wi l l be l e f t to dry for at l e a s t 24 hr at

room temperature. The medium wi l l harden b e t t e r i f the s l i d e s

are held on a 60°C warming t r ay overnight. The s l i d e s w i l l be

examined under Laborlux - K compound microscope. Necessary

photographs wil l be taken. To study the morphology of ind iv idua l

vascular elements nanely xylem of the infected roo t s , they wi l l

be macerated in hot HNO,* following the methods prescr ibed by

Ghouse and Yunus (1972).

5.6, Physiological s tudies :

To c o r r e l a t e . the extent of damage to the vascular elements

due to M, incogni ta in fec t ion and deficiency symptoms of n i t rogen ,

phosphorus aoid potassium exhibited by the fol iage, the seed l ing

of spo-nge gourd wi l l be grown in glazed pots having acid leached

sand and fed with 25 ml of complete Long Ashton solut ion (Hewitt,

1966), the ccanpositricm given in t ab l e 3,g, 4^

iimhE - 3

Macronutrients ppm

KNO3 K 156 N 57

Ca(N03)2 Anhyd. Ca 160 K 113

MgSD^. 7H2O Kg 36 S 48

NSHjPO^ Na 31 P 41

TABLE - 4

73

Micronutri ents ppm

Fe . C i t r a t e . 3H2O

M n s o ^ .

CUSO4.

Z n s O ^ .

N a c l

H3BO3

4H^0 s

SHgO

7H2O

(NH^)^ M V 2 4 4H2O

F e

Mn

Cu

Z r

CI

B

MD

5 . 6

0 . 5 5

0 . 0 6 4

0 . 0 6 5

3 . 5 Na 2 . 3

0 . 5 4

0 . 0 4 8

Plants w i n Ise ixtocul ated with d i f f e ren t inoculum l e v e l s in

log scale^ The his-topathology of infected roots wi l l be done.

Nitrogen*^ phosphorus antd potassium pres-ent in the leaves of p lan t s

inocula ted with d i f fe ren t inoculum leve l s wi l l be es t imated and

compared with the amounts in the leaves of uninoculated p l a n t s .

For the esrtLmetion of ni t rogen and phosphorus 'Spec t ronic

2-0 • (Bausch aui L^Ar) wtU be used. 'Sys t ronix ' flame-photometer

74

wil l be used for the estimation of potassium. Nitrogen wi l l be

est imated by Linder (1944) and phosphorus by !Fiske and subba Row

(1925) method respec t ive ly . Potassium wi l l be estimated d i r e c t l y

from al iquot by flame-photometer.

5 . 6 . 1 . Leaf Analysis for N, P and K. j

Leaf analysis i s an es tabl ished p r » : t i c e for assessing

the n u t r i t i o n a l s t a tus of the p lan ts (Lundegardh, 1951). Fu l ly

mature and healthy leaves of dr ied samples wi l l be powdered f ine ly ,

and passed through a 72 mesh screen, "Hie leaf powder wi l l be

kept a t 70°C overnight before acid digest ion according to the

mfitbiod. o± Linder (1944) t h a t i s b r i e f l y described below.

prom each sample, 100 mg of the dr ied leaf powder w i l l be

ca re fu l ly t ransfer red to a 50.0 ml K j e l d ^ l f lask and 2,0 ml of

chemically pure sulphuric acid w i l l be added. The f lask wi l l be

kept fo r digest ion for about two b r tx) allDw complete reduction

x>± n i t r a t e s present in the p l an t material^ giving off dens-e white

furoes u n t i l the contents turn b l^ck .

Flask wi l l then be allowed to cool down for about 15 min,

a f t e r which 0,5 ml of 30% hydrogen peroxide wi l l be added dropwise

and the solut ion wil l be heated again t i l l i t s colour changes

from black to l i gh t yellow. Heating wi l l b e continued for about

75

30 min. The flask wi l l be cooled for 10 min and an addi t ional

amount of 3-4 drops of 30% hydrogen peroxide wil l be added^

followed by gentle heat ing for another 15 min, to get a c l e a r

and co lor less ex t rac t . At t h i s s tage, care w i l l be taken in the

addit ion of hyd-rogen peroxide because f i t i s added in excess

t he r e i s a po s s i b i l i t y t h a t i t would oxid ise the ammonia in the

absence of organic mat ter . The sulphuric a: id-peroxide d iges ted

ma te r i a l wi l l be di luted with double d i s t i l l e d water and wi l l be

t r ans fe r red with 3 or 4 washings to a lOO ml volumetric f l ask

and f i n a l l y the volume wi l l be made upto the mark. For the ana­

l y s i s of ni trogen, phosphorus and potassium, a l iquots w i l l be

tcGcen from these digested sanples, the methods employed for t h i s

ana lys is are b r i e f ly summarized below :

Nitrogen :

Nitrogen content of the saa^ le ysAl be estimated according

to the method of Linder (1944), A l<X,-0 lal a l iquot of ttie diges^ted

raat:erial wil l be taken in a 50 ml voluroetr.ic f l a sk . The excess of

ac id w i l l be p a r t i a l l y neu t r a l i zed with 2,0 ml of 2,5 N sodium

hydnaxide and 1.0 ml of 1C?4 sodltwi s±l±xrste w i l l be added to

prevent t u r b i d i t y . Final ly , the volume wi l l be made upto t he

mark. A 5.0 ml aliquot of t h i s so lu t ion wi l l be taken in a 10 ml

-graduated t e s t tube and 0»5 ml of Ness le r ' s reagent wil l be added

drop by drop, mixing thoroughly af ter ejach ins ta lment . The volume

wi l l again be made upto the mark with -aie he lp of d i s t i l l e d water

76

and the contents wi l l be allowed to stand for 5 min for itiaximuin

color development. The solut ion wi l l be t ransfer red to a c o l o r i -

metr ic tube and the op t i ca l densi ty wi l l be measured at 525 nm on

a colorimeter , A blank wi l l be run with each s e t . The amount

of nitrogen in the al iquot wil l be read from a c a l i b r a t i o n curve,

obtained by using known d i lu t ion of a standard ammonium su lpha te

s o l u t i o n .

Phosphorus s

Total phosphorus in the sulphuric acid - peroxide d iges ted

so lu t ion wi l l be determined by following the method of P iske and

Subba ROW (1925), A 5,0 ml of a l iquot wi l l be taken in a 10.0 ml

graduated tube and 1,0 ml molybdic acid (2^9% oEunonixim molybdate

in 1.0N H SO ) wi l l be careful ly added, followed by 0.4 ml of

1, 2, 4 - aminonaphthol - sulphonic acid, which wi l l turn t he

contents b lue . D i s t i l l e d water wi l l be added -to make up the

volume up-to 10,0 ml and the solut ion wi l l be allowed to stand for

about 5 min aEter mixing thoroughly. I t wi l l be then t r ans f e r r ed

t o a co lor imet r ic tube and the op t i ca l densiiry wi l l be read at

620 nm on a colorimeter , A blank wi l l be run s ide by s i d e , A

c a l i b r a t i o n curve wi l l be prepared by using known d i lu t ion of a

s tandard monobasic potassium phosphate so lu t ion .

77

Potassium :

Potassium wil l be estimated using a flame photometer. A

1,0 ml al iquot wi l l be taken and read at 768 nm with the help of

potassium f i l t e r . A blank wi l l be run for each determinat ion.

The readings wi l l be compared with a ca l ib ra t ion curve p lo t t ed

for d i f fe ren t di lut ions of a s tandard potassium sulphate so lu t i on .

5 .7 , s tudies on Effects of Soil Moisture and Soil Temperature :

To study the effect of d i f f e ren t s o i l moisture and s o i l

temperature l eve l s on the h is topathologica l changes in Lxrffa

seed l ings , p lants wil l be grown at d i f fe ren t s o i l moisture l eve l s

as described below and wi l l be inoculated with M. incogrtita and

in fec ted roots wi l l be processed for h is topathologica l s t u d i e s .

5 ,7 .1 so i l Moisture j

In ordea: tx) study the effect of d i f fe roa t so i l roal^-tuxe

l e v e l s on the h i s to log i ca l changes, ICP/o, 15%, 20%, 23% ^sd 3C%

moisture l^eveCLs wi l l be usfid. F i r s t of a l l th=e wat€sr hnl iiTrg^

capac i ty and moisture content of the s o i l to be used wi l l be deter­

mined. Then the desired moisture leve ls wi l l be maintained by

adding r e q u i s i t e amount of water to the s o i l con trained in p o t s .

The sui;fa:e of the pots w i l l be covered with cellof^gane sheets

in order -to ch-eck the loss of water.

78

5 . 7 . 2 . s o i l Temperature t

E f f ec t of s o i l t e m p e r a t u r e w i l l be s t u d i e d in t h e Wisconsin

t y p e t empera tu re t a n k s . Seed l ings of t e s t p l a n t s w i l l be t r a n s ­

p l a n t e d in 15 cm c l a y po ts hav ing au toc laved s o i l and i n o c u l a t e d

wi th nematodes. Ihe po t s w i l l then be t r a n s f e r r e d t o t e m p e r a t u r e

t anks running a t 10°, 15°, 20°, 25°, and 30°C.

The d a t a w i l l b e ana lysed s t a t i s t i c a l l y and w i l l b e i n c o r ­

p o r a t e d i n t h e form of a t h e s i s fo r t he award of Fh.D degree of

t h e JttilGARH MUSLIM UNIVERSITiT, MJIGWH.

6. ^e/ere/7ces

7J

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