dissertation submitted for the degree of m^ittx of $f)iios ... · i express my sincerest gratitude...
TRANSCRIPT
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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P A R E N T
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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