STUDIES ON HISTOPATHOLOGICAL TRANSFORMATIONS INDUCED … · STUDIES ON HISTOPATHOLOGICAL...
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STUDIES ON HISTOPATHOLOGICAL TRANSFORMATIONS INDUCED BY
Meloidogyne incognita IN SOME SELECTED CUCURBITS
DISSERTATION submitted in partial fulfilment of the requirements
for the award of the degree of
MASTER OF PHILOSOPHY IN
BOTANY
BY
MOHD. YAQUB BHAT
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA) 1994
DS2565
^^2.5"<^5"' > . "̂
It is Allah who sendeth down Rain from the skies:
With it We produce Vegetation of all kinds:
From some We produce Green (crops), out of which We produce grain, Heaped up (at harvest)
And (then there are) gardens Of grapes, and olives, And Pomegranates, Each similar (in kind) Yet different (in variety):
When they begin to bear fruit. Feast your eyes with the fruit And the ripeness thereof Behold ! in these things There arfe Signs for people Who believe. (SURA VI Vol. 99)
It is Allah Who causeth The seed-grain And the date-stone To split and sprout. (SURA VI Vol. 95
ZIAUDDIN A. SIDDIQUI M. Sc . Ph D. (Alig.)
Professor of Botany
Section of Plant Pathology and Nematolog
Department of Botan
Phone :0571-25676
ALIGARH MUSLIM UMIVERSITY, ALIGARH-202 002, INDIA
TO WHOM IT MAY CONCERN
Jh\s \s to certift̂ that the work embodied m this Dissertation entitled "Studies On Histopcrtfiologica/ Transformations \v\ducedByN\e\o'\doQyY\e\Y\coQmta In Sortie Selected Cucurbits" \s the bonafide work carried out by A/lr. /Wohd. Yaqub Bfiat \Ar\der rY\y s[Apery\s\or\ and is suitable for submission for the A/l.Phil. Degree of Aligarh l\Aus\\n\ University, Aligarh.
DATE: 24/6/1995 Prof. Ziauddin A. Siddiqui
ACKNOWLEDGEMENT
I w i t h i n s u f f i c i e n t and i n c o m p e t e n t words
e x p r e s s my s i n c e r e s t g r a t i t u d e t o my es t eemed
s u p e r v i s o r Dr. Ziauddin Ahmad Siddiqui Professor,
Department of Botany, A l i g a r h Muslim U n i v e r s i t y
Aligarh, who insp i red me to i n i t i a t e the research work
in the d i s c ip l i ne of p lant Nematology. I am highly
indebted to him for h i s s k i l l f u l guidance, constant
encouragement, sympathetic a t t i t u d e . Zealous i n t e r e s t
and a f f e c t i o n and o v e r a l l s u p e r v i s i o n d u r i n g t h e
p r e p a r a t i o n of t h i s manusc r ip t .
I extend my h e a r t f e l t sense of g r a t i t u d e to Dr.
Hisamuddin, L e c t u r e r , Department of Botany, A.M.U.
Aligarh, for h i s cons t ruc t ive c r i t i c i sm and valuable
suggestions and sparing h is precious time for going
through t h i s manuscript.
I am i n d e b t e d t o Professor Wazahat Husain,
Chairman, Department of Botany, A.M.U. Al igarh, for
providing me a l l the necessary f a c i l i t i e s poss ib le in
the department.
I t i s p leasurable render my s incere thanks and
a p p r e c i a t i o n t o Dr. Syed Munawar Fazal, Research
a s s o c i a t e , Department of Botany, who sac r i f i ced his
v a l u a b l e t ime d u r i n g t h e p r e p a r a t i o n of t h i s
manusc r i c t .
My humble thanks are also due to my parents,
brother and sisters and Mr. Nazir Ahmad Bhat, who have
kindled the flame of learning in me and whose
affection, encouragements and sacrificial devotions I
ascribe all my success.
Special thanks are extended to my friends and
colleagues M/s M. Imran Khan, M. Imran, Kunhalvi, M.
M. Mobin, M. Manzar, Imtiaz Ahmad, A. Hamid, Anvar
Javed, M. Ashiq, Ms Shreerali Patwari, Rajesh
Tripathi, Miss Regina, and Fahemida Hassan for their
whole hearted co-operation and encouragement.
I am highly thankful to Mr. Muneer Uddin for
the timely preparation of this Manuscript.
Finally, I am thankful and Bow in gratitude to
the Almighty Allah, who provided and guided all the
channels to work in cohesion and coordination and led
me to this path of success.
(MOHD. YAQUB BHAT)
CONTENTS
Page No.
Introduction 1-11
Plan of Work 11
Review of Literature 12-29
Materials and Methods 30-42
References 43-62
Introduction
IN THE NAME OF ALLAH THE MOST BENEFICENT THE MOST MERCIFUL
INTRODUCTION
The annual production of vegetables in India is
estimated to be around 45 million tonnes from a
cropped area of four million hectares, excluding
potato and tubers (Singh, 1991) .
The per capita availability of vegetable per day
in India is very low. The low per capita consumption
is mainly due to the low productivity levels in
vegetable crops. The vegetables are one of the
important items for export. The vegetables exported
are okra, bitter gourd, bottle gourd, round gourd,
chillies, green peas, cauli flowers, radish, pointed
gourd, and sponge gourd. It means that mostly
cucurbits are exported. The cucurbits are of trailing
habit and are cultivated during summer and rainy
seasons.
The family cucurbitaceae includes 90 genera and
700 species. Majority of the species are herbaceous
annual climbers. The cucurbit fruits are largest known
in the plant kingdom. Fruits of cucurbits are
extensively used as vegetables and consumed both when
young and ripe. They are used as fresh or processed
food. Fully matured fruits are also used for preparing
jams and sweets and fermented to give beverages. Some
varieties are excellent for culinary use during early
2
Stages of maturity and very poor for the same use at
later stages while the reverse may be true of other
varieties (Culpepper and Moon, 1945).
The roots of bitter gourd are astringent and are
used medicinally. They are applied in bleeding piles,
bowel affections and urinary complaints. The roots are
pasted and applied over the body as sedative in fevers
(Dymock et. al. , 1890-99; Kirt and Basu, 1935).
Fruits and leaves are used in external
application for lumbago, ulceration and fracture of
bones. In Honkong the plants are used as an
alternative bitter drug in place of strychnos (Burkill
II, 1486), Brown 1946; III, 399, Rama Rao 1914, Dymock
et al., 1890-99) .
The soil harbours a large number of plant
pathogenic organisms such as bacteria, fungi,
actinomycetes, insects, and nematodes. Cucurbit crops
are attacked by a number of diseases of which the
nematode diseases are of economic importance. The
plant parasitic nematodes are one of the most
important pathogens affecting cucurbits. The nematode
diseases not only cause yield loss but also reduce the
ability of infected roots to utilize available
nutrients.
The root-knot diseases are caused by Meloidogyne
spp. the most important pathogen of cucurbits. The
3
p a r a s i t e u s u a l l y p e n e t r a t e s u n d e r g r o u n d p a r t s of
p l a n t s where i t induces the development of abnormal
growth in r o o t s . Sometimes unusua l ly l a r g e g a l l s a r e
developed a t the base of stem ( S t e i n e r e t . al., 1934).
The s ize and charac ter of these enlargements vary in
d i f f e r e n t p l a n t s , as in Thunbergia laurifolia and
r h u b a r b enormous s t r u c t u r e s n e a r l y two f e e t i n
d i a m e t e r may be s e e n . Roo t -kno t nematodes a r e
s e d e n t a r y e n d o p a r a s i t e s . The female nematode remains
wholly embedded in the va scu l a r c y l i n d e r . I n f e c t i o n
with r o o t - k n o t nematode s t i m u l a t e s t h e format ion of
v a r i a b l e number of d i s c r e t e g i a n t c e l l s i n h o s t
t i s s u e s . H y p e r p l a s t i e d and h y p e r t r o p h i e d c e l l s o f t en
s u r r o u n d t h e r e g i o n of i n f e c t i o n c a u s i n g g a l l s
t e r m i n a l l y and s u b t e r m i n a l l y in the i n f e c t e d r o o t s .
Kostoff and Kendall (1930) be l i eved t h a t g i a n t c e l l s
were formed by w a l l d i s s o l u t i o n of a f f e c t e d c e l l s
fol lowed by coa l e scence of c e l l c o n t e n t s . Huang and
Maggenti (1969) in t h e i r d e t a i l e d s tudy on g i a n t c e l l s
development in Vicia faba roots found no evidence of
c e l l w a l l d i s s o l u t i o n in t he s t i m u l a t e d c e l l and
concluded t h a t g i a n t c e l l o r i g i n a t e d from s i n g l e c e l l .
They observed t h a t m u l t i n u c l e a t e c o n d i t i o n of g i a n t
c e l l s a rose from r epea t ed mi tcses wi thou t c y t o k i n e s i s
of a s i n g l e d i p l o i d c e l l . In a g ian t c e l l the n u c l e a r
changes may range from a nucleus having one s e v e r e l y
4
hypertrophied nucleolus to nuclei with various stages
of membrane deterioration and lobulated periphery. The
cytoplasm of the young giant cell becomes dense.
Christie (1936) found dense cytoplasm near the head
region of the nematode than the remainder of giant
cell cytoplasm. As the nematode matures and nutrient
demand from giant cell increases, the giant cell
cytoplasm shows regions of intense metabolic activity.
The giant cell cytoplasm disintegrates after egg
laying of the nematode. Formation of giant cell and
multiplication of pericycle and other parenchyma cells
around the nematode head cause the vascular tissue to
divert from its normal path. Xylem and phloem also
become abnormal with reference to structure and
orientation. Small as well as hypertrophied parenchyma
cells are transformed into vessel like elements and
sieve tube elements and constitute the abnormal xylem
and phloem. Since giant cells are highly metabolically
active cells, therefore, they must directly or
indirectly be connected with the vascular tissues.
Ediz and Dickerson (1976) found most of the
giant cells in phloem region. The root-knot nematode
readily penetrates the plant root near the apical
meristem (Nemec, 1910) within 24 hours after
inoculation (Siddiqui and Taylor, 1970). However other
regions of the root are not immune to attack, Christie
(1936) .
5
Once in side the root tze migration occurs both
inter and intracellularly, (Nr-aec, 1910; Krusberg and
Nielson, 1956; Christie, 192~)_ Nemec (1910) found
that the division of giant cell xiuclei takes place by-
mitoses, but early in the life of giant cell, nuclei
may coalesce. He also found tiiat the nuclei were
fusing in pairs or in the case older cells, all the
nuclei might be massed together mear the center of the
cell. The cytoplasm of young giant cells (within 48
hours) becomes dense and encloses a large vacuole. The
vacuole diminishes and cytoplasoi becomes more dense as
well as granular with the gian. cell development.
Christie (1936) fou-d more dense and
hyperchromatic cytoplasm nenr the head region of
nematode than remainder of th= giant cell cytoplasm.
Nuclei become highly lobed azi heterochromatic with
numerous prominent nuclear poxes. The cytoplasm
becomes vacuolated and vacuclation increases when
nematode reaches it's maturity. The giant cell
cytoplasm is disintegrated af.er egg laying of the
nematode. Nuclear changes ranged from a nucleus near
the stylet of nematode which shewed a hypertrophied
nucleolus with the apparent absence of nuclear
membrane to nuclei with varies stages of membrane
deterioration and a lobulated -eriphery. Irregularly
shaped, dumb-bell or sickle shaped nuclei were
6
o b s e r v e d on r o o t - k n o t i n f e c t i o n of sweet p o t a t o
(Krusberg and Nie l son , 1958) . Nuclear enlargement in
g i a n t c e l l s of r o o t - k n o t nematode i n f e c t e d tomato,
cucumber and hawk ' s - b e a r d Crepis capillaries
appeared to r e s u l t from swelling and in some cases
from nuclear fusion (Owens and specht, 1964).
Molliared (1900) observed g a l l s on the root of
melon Coleus and Begonia and reported tha t a f t e r
i n v a s i o n t h e r o o t t i p g rowth may be a r r e s t e d and
l a t e r a l r o o t s f r e q u e n t l y developed nea r the r eg ion of
i n v a s i o n . He a l s o r e p o r t e d i r r e g u l a r i t i e s in the form
and t h e number of g i a n t c e l l n u c l e i and t h e i r
s u b s e q u e n t c o a l e s c e n c e and d i s i n t e g r a t i o n . Dur ing
e a r l y s t a g e s of development the d i v i s i o n of g i a n t c e l l
n u c l e i i s by normal m i t o s e s . A f t e r c e r t a i n p e r i o d
a m i t o t i c d i v i s i o n and f r a g m e n t a t i o n a r e f o l l o w e d .
G a l l s are p a t h o l o g i c a l l y developed c e l l s , t i s s u e or
o r g a n s of p l a n t s t h a t have a r i s e n m o s t l y by
hyper t rophy and h y p e r p l a s i a under the i n f l uence of
p a r a s i t i c o rgan isms .
Schus te r and S u l l i v a n (1960) r e p o r t e d t h a t g a l l s
were induced in tomato by Meloidogyne incognita larvae
even when they did not enter the tomato r o o t s . They
c o n c l u d e d t h a t s t y l e t p e n e t r a t e d t h e r o o t s u r f a c e
c e l l s and s e c r e t e d m a t e r i a l s t h a t s t i m u l a t e d h o s t
t i s s u e t o form g a l l s . Dav i s and J e n k i n s (1960)
7
r e p o r t e d g a l l format ion in Gardenia spp. infected with
Meloidogyne incognita; M. incognita acrita and M.
hapla. Cor t ica l and s t e l a r p r o l i f e r a t i o n accompanied
a l l i n f ec t i ons .
Huang (1966) reported cork and l i g n i f i e d wall
thickening of endodermis and per icyc le a t in fec t ion
s i t e s in Zingiber officinale. Division of s t e l e in
root g a l l s of Lycopersicon pimpinellifolium infected
with M. incognita was observed.
J o n e s (1981) Pasha e t . al., (1987) reported
format ion of abnormal xylem in p l a n t s i n f e c t e d with
r o o t - k n o t nematode, Pasha e t . al., (1987) found tha t
abnormal xylem o c c u r r e d i n i r r e g u l a r p a t c h e s w i t h
s c a t t e r e d v e s s e l e l e m e n t s and r e s u l t e d i n
d i s c o n t i n u i t y of v a s c u l a r t i s s u e s and s i g n i f i c a n t
r e d u c t i o n in v e s s e l d imension.
P a t e l and P a t e l (1991) r e p o r t e d t h a t t h e
immature s t a g e s of nematode p e n e t r a t e d f r e e l y i n t o the
e p i d e r m i s and r e a c h e d t h e endodermal l a y e r
i n t e r c e l l u l a r l y through the c o r t i c a l c e l l s in p igeon
p e a . The c e l l s s u r r o u n d i n g t h e f e e d i n g s i t e were
d a r k e r and t h i c k e r t h a n normal c e l l s . G ian t c e l l
i n i t i a l s were observed near the head of the nematode
most ly in phloem parenchyma and r a r e l y in p e r i c y c l e
and xylem parenchyma in pea s e e d l i n g s , t h r e e days
a f t e r i n o c u l a t i o n of r o o t - k n o t nematode. The g i a n t
c e l l s bore appendages which grew i n t r u s i v e l y among the
ne ighbour ing c e l l s . Wall ingrowth or p ro tube rances
f o r m a t i o n in g i a n t c e l l s may i n d i c a t e a form of
t r a n s f e r c e l l . Sharma and Teobi (1989) and Da l la e t .
al . , (1991) reported the giant c e l l formation, 72
h o u r s a f t e r i n o c u l a t i o n i n t h e xylem and phloem
parenchyma i n Vigna radiata and Cyamopsis
tetragonaloba.
Hisamuddin and Siddiqui (1992) observed 5 to 6
g i a n t c e l l s i n r e g i o n of v a s c u l a r d i f f e r e n t i a t i o n
w i t h i n 24 hours of i n o c u l a t i o n in Luffa cylindrica.
Abnormal xylem formation progressed incessan t ly as the
g a l l grew older and annexed most of the parenchyma.
S u r r o u n d i n g t h e g i a n t c e l l s , howeve r , i s o l a t e d
abnormal v e s s e l s were wi tnessed even a f t e r 40 days of
i n o c u l a t i o n .
R e s i s t a n c e denotes t he g r e a t l y reduced r a t e of
r e p r o d u c t i o n of the p a r a s i t e on r e s i s t a n t c u l t i v a r .
In r e s i s t a n t r o o t s l a r v a e may e i t h e r f a i l to e n t e r or
e n t e r i n r e d u c e d numbers w i t h v a r y i n g d e g r e e s of
deve lopmen t ( B a r r e n s , 1939; C h r i s t i e , 194S) .
H y p e r s e n s i t i v i t y i s a common t y p e of r e s p o n s e of
r e s i s t a n t p l a n t s (Riggs e t . al., 1959; Rohde, 1965).
The larvae may enter the root of r e s i s t a n t p l an t s in
large numbers but hypersensi t ive c e l l s quickly die and
w a l l off t h e p a t h o g e n so t h a t i n j u r y t o h o s t i s
9
confined to a few cells (Riggs and Wintesel, 1959) .
This results in the subsequent death of nematode. The
hypersensitive reaction in some cases may be delayed
until after the parasitic relationship. Linford
(1939) reported penetration, development and migration
of cotton root-knot nematode, Meloidogyne incognita,
acrita in resistant and susceptible alfalfa varieties.
He found no development of nematode in resistant
cultivar but egg production began after 18 days of
penetration in susceptible cultivar. Attractiveness of
Meloidogyne species juveniles was observed towards the
root and excised shoot tissues of several host plants.
The juveniles of Meloidogyne hapla were equally
attracted towards the resistant and susceptible
alfalfa seedlings when an egg mass was placed mid way
between germinating seedlings (Griffin and Waite,
1971) . In Resistant varieties the juveniles of root-
knot nematode either did not enter or if entered, they
did not develop properly (Sasser and Taylor 1952).
Resistance in plants towards root-knot nematode may
develop with age (Griffin and Hemt 1972).
Three types of histological responses were
observed in infected resistant root. Some galls
contain fragments of nematode others contained no
detectable traces of developing larvae. Formation of
druses in galls but not in healthy tissues was ncted
10
in both cultivars, 20 days after inoculation. Massive
invasion of roots resulted in deep longitudinal
fissures of root cortex (Michael, et. al. , 1973).
Root penetration by second stage juveniles of
Meloidogyne incognita race I and M. Javanica in
resistant germplasm was significantly less than in
susceptible cultivars of vegetables like, cabbage,
cauliflower, cucumber, pepper and tomato (Khan and
Khan 1991) .
Pankaj and Swanand (1992) reported the efficacy
of chemicals as seed dresser against M. incognita in
bitter gourd and round gourd. It caused the inhibition
of larval penetration, female development, and gall
formation on bitter gourd and round gourd.
Fattah and Webster (1983) observed normal giant
cells and fungal infection in one week old inoculated
seedlings of tomato cultivars. After two weeks hyphae
were visible in xylem tissue of Fusarium susceptible
but not resistant plants. Dissolution of giant cell
wall occurred where giant cells were in direct contact
with hyphae. No such changes were seen in the giant
cell of control plant inoculated with nematode alone.
Cabanillas et. al., found no galling and giant cell
formation in tomato roots inoculated in with nematode
eggs infected with P. lilacinus. Few to no galls and
no giant cell formation were observed in roots dipped
11
in spore suspension of P. lilacinus and inoculated
with M. incognita.
The present work will be carried out:
i. To compare anatomical changes leading to the
formation of giant cells and galls, from earlier
findings.
ii. To investigate the origin of giant cells and their
fate after death of the nematode,
iii To find out any link of giant cells with phloem,
iv To observe abnormalities in xylem and phloem.
V. To examine response of giant cells after treatment
with some systemic nematicides in infected roots,
vi To study the response of vascular tissue after
nematicide treatment of the infected roots,
vii To study any resistance towards Meloidogyne
incognita infection in different varieties,
viii To study the difference in giant cells and
vascular tissues in resistant and susceptible
varieties.
ix To study the response of giant cells after
inoculation with Paecilomyces lilacinus in
infected roots.
X To study the response of vascular tissues after
inoculation with P. lilacinus in infected roots.
Review of Literature
REVIEW OF LITERATURE
Root-knot nematode, Meloidogyne spp., are most
important and best known nematodes have evolved very-
specialized and complex host parasite relationship.
Meloidogyne spp. parasitize a wide variety of host
plants belonging to monocotyledons and dicotyledons
including both herbaceous and woody plants, and cause
the formation of familiar knot or gall on roots of
susceptible host. Root-knot nematodes are known to
parasitize more than 2000 species, but host parasite
relationships have been investigated only in few
plants (Webster, 1969, 1975) .
Root galling induced by Meloidogyne spp. is a
well known host response which involves the production
of abnormally large multinucleate cells in the
vascular tissues of susceptible plants. Generally,
root damage caused by parasitic nematodes is reflected
on the above ground portion of the plant as poor shoot
growth, leaf chlorosis and even death of plants
resulting in low yield and poor quality produce.
The above ground symptoms are similar to those
associated with any root injury that results in
reduced amounts of water uptake by plants. Flowering
is scanty and fruits are either lacking or are of poor
quality (Jenkins and Taylor, 1967) . Meloidogyne also
13
interferes with the process of nitrogen fixation in
the nodules of leguminous plants.
Second stage juvenile is the first stage that
infects the plants and starts it's life cycle on
susceptible host. The juveniles penetrate the root
tips (Christie, 1936) and feed on root hairs and
epidermal cells. They accumulate either at the region
of the cell elongation just behind the root cap or at
the points where lateral roots emerge or at the site
of penetration of other juveniles and cut surfaces of
roots (Godfrey and Oliveira, 1932; Linford, 1939,
1942; Peacock, 1959; Bird, 1969; Green, 1971;
Siddiqui, 1971-a, 1971-b; and Prot, 1980). The
mechanism of penetration may involve mechanical action
by thrusting of the stylet or cellulytic and
pectolytic activity of enzymes (Linford, 1942; Bird
and Loveys, 1980).
Primary root penetration by second stage
juveniles occur at the tips of the young root in the
region of differentiation in sweet potato or any where
from root cap back to the region of root hair
formation and also through the loose ruptured cells of
enlarging tuberous roots (Krusberg and Nielson, 1958) .
Siddiqui and Taylor, 1970 found most of the juveniles
penetrating in the region of cell differentiation and
elongation. After penetration intercellular migration
14
of juveniles in the cortex was detected (Nemec, 1910,
Godfrey and Oliviera, 1932; Linford, 1937, 1942; Endo
and Wergin 1973; Jones and Payne, 1978).
During intercellular migration the cells were
separated along the middle lamella. Along the path of
migrating juveniles the cells were distended and
compressed and did not show any sign of rupture or
nematode feeding. Inter and intra-cellular migration
was reported by Christie (1936), Krusberg and Nielson
(1958), Roman (1961), and Bird (1959, 1960, 1962);
Siddiqui and Taylor (1970) and Siddiqui (1971-a,b).
Roman (1961) observed galleries and furrows of broken
and separated cells that indicated intracellular
penetration of juveniles.
Giant cell is a multinucleate transfer cell in
which the multi nucleate condition results from
multiple mitosis in absence of cytokinesis. Formation
of giant cell is not accompanied by wall dissolution
and are invariably formed by root-knot nematodes
(Endo, 1987) . Giant cells are highly specialized
cellular adaptations induced and maintained in
susceptible hosts by the feeding of Meloidogyne spp.
Primary phloem or adjacent parenchyma are the highly
preferred tissues for the development of giant cells
(Christie, 1936 Krusberg and Nielson, 1958; Byrne,
1977) .
15
Beille (1898) was the first who proposed that
giant cells are formed by disintegration of cell walls
and coalescence of cells in papaya roots. Observation
of cell wall fragments in giant cell cytoplasm
strengthened this view (Bird, 1961, 1972; Smith and
Mai 1965) .
Later studies supported this mode of giant cell
formation (Kostoff and Kendall, 1930; Christie, 1936;
Krusberg and Nielson, 1958; Dropkin and Nelson, 1960;
Bird, 1961; Roman, 1961; Krusberg, 1963; Owens and
Specht, 1964; Birchfield, 1965; Littrel, 1966;
Siddiqui and Taylor, 1970; Siddiqui, 1971, a,b; Bird
1972, 1973, 1974), Rohde and Mcclure (1975). Nemec
(1910) proposed a contrasting mode of giant cell
induction by root-knot nematode and reported that
around the nematode head the plerome enlarged, their
cytoplasmic contents increased and the number of
nuclei increased by mitosis without accompanying
cytokinesis. In this way multinucleate giant cells
were formed early in development of gall. Cell wall
break down and cell wall dissolution was not observed
at any stage in giant cell formation. This mode of
giant cell formation was supported by a number of
researchers (Myuge, 1956; Huang and Maggenti, 1969;
Paulson and Webster, 1970; Jones and Northcote, 1972;
Jones and Dropkin, 1976; Jones and Payne, 1978.
16
Huang and Maggenti (1969a,b) observed g i a n t c e l l
i n t h e r o o t s of Vicia faba infected with Meloidogyne
javanica formed by repeated mytoses of the o r ig ina l
d ip lo id c e l l with out subsequent cy tok ines i s . They
repor ted chromosome number as 4n, 8h, 16n, 32n and
64n. I n t e r p h a s e n u c l e i w i t h many chromosomes were
formed due t o fus ion of m i t o t i c appa ra tu s in g i a n t
c e l l e i t h e r a t me t aphase o r a n a p h a s e s t a g e s . Many
i n t e r p h a s e n u c l e i , however , were s i m p l y i n c l o s e
j u x t a p o s i t i o n i n s t e a d of a c t u a l l y b e i n g fu sed as
r e v e a l e d by e l e c t r o n microscopy. The i n t e r p h a s e n u c l e i
were i r r e g u l a r l y lobed and f r e q u e n t l y assumed amoeboid
a p p e a r a n c e . M i t o s e s i n g i a n t c e l l were g e n e r a l l y
s y n c h r o n i z e d . Some n u c l e i i n h y p e r p l a s t i c t i s s u e s
a r o u n d g i a n t c e l l s were a l s o l o b e d and d e f i n i t e l y
s m a l l e r than those of g i a n t c e l l s . The c e l l p l a t e
fo rmat ion was not observed from b i n u c l e a r s t a g e upto
the m u l t i n u c l e a r s t age in g i a n t c e l l s . They d id not
f i n d any e v i d e n c e of c e l l w a l l d i s s o l u t i o n .
Synchronous n u c l e a r d i v i s i o n s w i t h i n t he same g i a n t
c e l l wi thou t daughter c e l l wal l format ion have been
observed in many p l a n t s , (Krusberg and Nie l son , 1958;
Bi rd , 1961; Owens and Specht , 1964; Smith and Mai,
1965) .
J o n e s and Payne (1978). o b s e r v e d g i a n t c e l l
i n d u c t i o n i n r o o t s of Impatiens balsamina by
17
Meloidogyne spp. under light and electron microscope.
They reported that first sign of giant cell formation
was the division of cells around the larval head. Cell
plate alignment appeared to proceed normally but
cytokinesis was unsuccessful and binucleate cells
formed subsequently, no wall break down was evidenced.
Nuclei increased in number by repeated motoses without
cytokinesis. Nuclear divisions were detected 24 hours
after inoculation in the cells upto two cell layer
from head of nematode. After 48 hours 2, 4 and 8
nuclei were observed in the cells near the nematode
head. In early stages of giant cell formation, cell
wall break down and cell fusion were not evidenced
under electron microscope. "Wall gaps or holes in a
continuous cell wall were not detected. Cell wall of a
giant cell consisted of thick and thin areas. The area
of the cell wall where plasmodesmata occur is close to
the limit of resolution of light microscope. This
gives an impression of gaps in the wall. The irregular
outline of individual giant cell at many sites due to
cell wall ingrowth appeared as tylose like structure
which, according to Jones and Payne (1978), was
considered by certain workers as cell wall fragments.
In binucluate cells, 48 hours after inoculation, the
cell plate vesicles were found aligned normally but
dispersed without fusion and thus failed to form
18
daughter cell wall. The cell plates vesicles were also
thought to be the fragments of broken walls.
Krusberg and Nielson (1958) reported young root
tips lateral root ruptures and the surface of cracks
were three major infection courts in sweet potato
roots. After penetration the second stage larvae
migrated inter and intracellularly to the feeding
site. The feeding sites varied with the infection
court. Larvae came to rest primarily in the stele in
the region of cell elongation or in the cambial zone.
Nematode feeding stimulated the formation of several
atypical tissues i.e. giant cells, abnormal xylem,
hyperplastic parenchyma and cork. Giant cells were
initiated first. Cork formed around mature females and
their egg masses and was most abundant in root from
late harvests. Mcclure et. al., (1974) reported three
types of histological responses in infected resistant
roots and were co-related with the degree of nematode
development. Some galls were observed which contained
only fragments of nematodes, while others contained no
detectable traces of developing larvae. Formation of
druses in galls, but not in healthy tissue, was noted
in both resistant and susceptible plants, 20 days
after inoculation. Massive invasion of roots resulted
in deep longitudinal fissures of root cortex. In
sorghum roots, on infection with cotton root-knot
19
nematode, development of giant cells in the cortex or
stele of lateral roots was studied. Giant cells
developed either singly with few nuclei or in groups
with many nuclei. Interruption of pericycle and
endodermal tissue was absent upto the one third of
circumference of stele and extended 1.5 mm
longitudinally along the root. In the area where
pericycle and endodermis were absent, the pericycle
parenchyma of the cortex extended to the vascular
elements and abnormal xylem surrounding giant cells
extended into the region of cortex. Root galls
appeared on sorghum roots as elongated swellings,
discrete knots, or swellings with root proliferation.
(Orr and Morey, 1987) .
Pasha et. al., (1987) reported that feeding site
of nematode was mainly confined to steler region of
roots of Solanum melongena L., resulting from
infection with root-knot nematode Meloidogyne
incognita. Cells in the feeding site showed
hypertrophy, hyperplasia, thickening of cell wall,
granular cytoplasm and elarged nuclei and nucleoli.
Abnormal xylem formed in response to infection by
nematode, occurred in irregular patches with scattered
vessel elements resulting discontinutity of vascular
tissue. The findings supported the view that giant
cell originated from hypertrophy and repeated mitoses
20
without cytokinesis of single stimulated cell.
Penetration leading to intracellular migration
causing cortical hypertrophy, took place within 24
hours after inoculation. Development of giant cell
took place within 4 -5 days around the head of each
nematode in the stele. Initial pathological
alterations in giant cell formation consisted of
protophloem and protoxylem cells. Giant cells
contained 2-8 agglumerated multinucleolate nuclei,
synchrous mitotic divisions were first observed 9 days
after inoculation. After 21 days giant cells becames
highly vacuolated. Observations, 40 days after
inoculation revealed complete degeneration of cell
contents in many giant cells, but their thick walls
remained intact. Abnormal xylem completely surrounded
the degenerated or partially degenerated giant cell
(Siddiqui and Taylor 1969) . Siddiqui (1971) showed
that large number of Meloidogyne naasi larvae
penetrated root tips of Wintok oats within the first
24 hours and first sign of incipient giant cell
formation was visible, four days after inoculation.
Three types of responses were observed (a) necrosis of
inner cortical and endodermal cells in contact with
the nematode lip region and failure of larvae to enter
the stele, (b) necrosis of cells around the incipient
giant cells, resulting in degeneration of giant cells
21
and larvae (c) uninterrupced giant cell development
which is not distinguishable from that in susceptible
host.
Dropkin (1954) showed giant cell development and
maintenance in plants infected with Meloidogyne
javanica depends on continuous stimulus from the
nematode whose removal resulted in break down of the
giant cell. Normal giant cell development took place
under sterile conditions. A growth promoting substance
is present in galls but not in adjacent roots. Roots
of heavily infected plants are often so severely
damaged that individual galls coalesce into amorphous
masses containing large number of nematode. It was
noted that galls on tomato roots were all of about the
same size and number of larvae within a gall can be
predicted by measuring the size of the gall.
Fawali (1988) while observing naturally and
artifically infected white yam tuber with root-knot
nematode found intercellular movement of Meloidogyne
juveniles. Feeding sites were always in the ground
tissue layer where the vascular tissues were
distributed. Sharma and Tiabi (1989)observed giant
cell initials near the head of Meloidogyne incognita
larvae mostly in Phloem parenchyma and rarely in
pericycle and xylem parenchama. Kim and Ohh (1990)
suggested differentiation of tomato xylem vessels with
22
the formation of adjacent giant cells
Subbaton (1990) observed formation of giant
cells after 21 to 32 days in the root of Glycine max
infected with Meloidogyne javanica. Their activity
increased with progressive development of nematode.
The modified cells were enlarged and had larger
numbers of organelles which underwent structural
changes forming Lalyrenth of cell tissues. In the
Stelar region of wheat, root-knot nematode migrated
intracellularly and caused gall formation around its
head. Patel and Patel (1991). Severe damage to the
vascular tissues disrupted the continuity of xylem
tissue. Salawu (1991) observed extensive damage to
epidermis, cortex and vascular tissues due to giant
cell formation in the stele in roots of Celosia
argentia by Meloidogyne incognita. Datta et. al.,
(1991) reported the giant cell formation in xylem and
pholem parenchyma after 72 hours of inoculation by
Meloidogyne incognita in roots of Vigna radiata and
Cyamopsis tetragonolaha.
Khan and Khan (1991) reported siginificant
difference in the penetration of M. incognita race I
and M. javanica in resistant and susceptible germplasm
cultivars of vegetables. Abrantes et. al., (1992)
reported the induction of giant cells in roots of
Olive [Olea europia) by M. javanica. Hypertrophy and
23
hyperplasia were common phenomena in the cortical and
vascular parenchyma. Wyss etal (1992) observed the
invasion of Arabidopsis thaliana roots by Meloidogyne
incognita primarily in the region of elongation
close to the meristematic zone, by destroying
epidermal and subepidermal cells. Inside the root the
second-stage juveniles (J2) oriented them selves
always in the direction of root tip and migrated
towards it, between cortical cells. At the apex of the
root the juveniles turned round and migrated backwards
between cells towards and differentiating vascular
cylinder. Within the vascular cylinder, migration
eventually stopped and the giant cell induction was
intiated. Second-stage juveniles became surrounded by
multinucleate giant cells within about 24 hours. Bird
(1992) laid emphasis on stylet exudate proteins in
establishment and maintainence of giantcells.
Husain et. al., (1992) reported the abnormal
xylem development in response to Meloidogyne spp
infection. They observed siginificant reducation in
vessel element dimensions but no difference in fiber
elements in infected and uninfected plants. Hisamuddin
and Siddiqui (1992) reported the induction of 5 to 6
giantcells in the region of vascular tissues within 24
hours of inoculation at 23 to 33°C. Abnromal xylem
formation progressed incessently as the gall grew
24
o l d e r . Drought et. al. , (1992) reported tha t
Meloidogyne incognita. infected tomato roots showed
only s l i gh t f luorescence. The nematode induced giant
c e l l s accumulated f l u r e sc in from the apoplas t . Giant
c e l l s had increased net p ro te in extrusion a c t i v i t y
compared to other c e l l type a t the infec t ion s i t e This
s u g g e s t e d t h a t p r o t e i n e x t r u s i o n might be d r i v i n g
f o r c e fo r s o l u t e u p t a k e from a p o p l a s t . Vovlas and
S a s n e l l i (1993) r e p o r t e d t h a t i n f e c t i v e Meloidogyne
j u v e n i l e s p e n e t r a t e d r o o t s and stem of Helianthus
annuus s e e d l i n g d u r i n g g e r m i n a t i o n . Nematodes
e s t a b l i s h e d the feed ing s i t e s mainly in the cambium.
Stem s w e l l i n g s were caused by pronounced hyper t rophy
of cambial c e l l s a t t h e feed ing s i t e . The s i z e of
g a l l s and p r o d u c t i o n of g i a n t c e l l in t h e a e r i a l p a r t
i n d u c e d by s i n g l e n e m a t o d e s were s i m i l a r t o t h o s e
observed on r o o t s .
Gourd e t . al., (1993) studied the r a t e s of
p e n e t r a t i o n of f r e s h l y ha tched Second s t a g e j u v e n i l e s
(J2) of Meloidogyne arenaria, M. Hapla, M. incognita
and M. javanica and Heterodera glycines race I and V
in soybean r o o t s . H. glycines entered roots more
q u i c k l y t h a n Meloidogyne spp. Penetrat ion by most
nematodes was a c c o m p l i s h e d w i t h i n 48 h o u r s . Host
s u s c e p t i b i l i t y and r e s i s t a n c e , each r e s u l t from s e r i e s
of s e q u e n t i a l even ts t h a t a re e a s i l y d iv ided i n t o pre
25
and post infection stages. The sequential events may
or may not lead to the establishment of disease.
Resistance is the absence or inhibition of disease
upon challenge by pathogenic agent. Thus any thing
that prevents, retards or restricts disease
development contributes to host resistance. Resistance
manifested at the time of infection or thereafter may
be constitutive or due to infection induced factors.
Root-knot nematode {Meloidogyne spp.) after
establishing a permanent feeding site in the host
roots induced the formation of enlarged symplastic
structures called giant cells (Jones and Northcote,
1972,a) The giant cells have the ability to transfer
metabolites from the host to developing larvae. There
is an ample evidence indicating that transfer, cells
are metabolically hyperactive and essential to the
development of nematode. ( Bird, 1961; Littrell, 1966;
Endo and Veech, 1969a,b; 1970; Veech and Endo, 1969;
Webester, 1969; Endo, 1971; Dropkin, 1972; Gommers and
Dropkin, 1977).
Failure of transfer cell to develop or function
will result in the death of nematode and is therefore
a mechanism of Resistance. There has been considerable
speculation in the role of nematode enzymes in the
transfer cell formation. Hussey and Sasser (1973)
observed peroxidase in the stylet exudate of
26
Meloidogyne incognita females and suggested tha t t h i s
enzyme i s i n v o l v e d in g i a n t c e l l i n d u c t i o n and
m a i n t e n a n c e . R e s i s t a n t p l a n t s i n f e c t e d w i t h
Meloidogyne incognita normally undergo a
h y p e r s e n s i t i v e response and the a f f e c t e d c e l l s t u r n
brown and d i e . However, i n r o o t s t r e a t e d w i t h
c y c l o h e x a m i d e , t h e t y p i c a l h y p e r s e n s i t i v e r e s p o n s e
does not occur or a t l e a s t the e f f e c t e d c e l l s donot
t u r n brown and t h s c e l l s remain a l i v e .
Sawheny and Webster (1975) observed browning of
a f f e c t e d c e l l s wi th c e l l dea th and lack of browning
w i t h l i v i n g c e l l s . Or ion and Minz (1971) t r e a t e d
t o m a t o s e e d l i n g s w i t h m o r p h a c t i n ( an a n t i b i o t i c
agent ) t h a t a p p a r e n t a l y does not a f f e c t nematode or
a t l e a s t does n o t a f f e c t Panagrellus redivives and
o b s e r v e d a marked d e l a y i n g i a n t c e l l f o r m a t i o n ,
reduced r a t e of nematode development and a tendency
t o w a r d s m a l e n e s s i n m a t u r i n g l a r v a e . Davide and
T r i a n t a p h y l l o u (1968) o b s e r v e d s i m i l a r e f f e c t s by
t r e a t i n g p l a n t s wi th ma l i chydraz ide ( m u l t i f u n c t i o n a l
i n h i b i t a r ) w i t h i n f o u r days a f t e r i n f e c t i o n by
M. incognita. Terpenoid aldehydes were h is tochimical ly
d e m o n s t r a t e d i n endode rmis and s t e l e n e a r s i t e of
n e m a t o d e . I n f e c t i o n i n d u c e d t e r p e n o i d a c c u m u l a t i o n
occured in both s u s c e t i b l e and r e s i s t a n t , but i t was
d e t e c t a b l e s o o n e r , r e a c h e d g r e a t e r i n t e n s i t y and
27
occur red more in host c e l l s in the r e s i s t a n t c u l t i v a r s
than in the s u s c e p t i b l e c u l t i v a r s . Thus the r a t e and
e x t e n t of accumulat ion seems to be impor tant f a c t o r s .
Wieser (1956) observed t h a t t h e exc i sed r o o t s of
soybean and egg p l a n t v a r i e d in t h e i r r esponse to
l a r v a e of M. hapla, some being r e p e l l a n t , some
a t t r a c t i v e and some n e u t r a l . He a t t r i b u t e d t h e s e
r e s u l t s an i n t e r p l a y be tween an a t t r a c t i v e a g e n t
p r e s e n t in the l i v i n g p l a n t and r e p e l l a n t a s s o c i a t e d
wi th t he chemical decay and break down of r o o t .
F a s s o l i t i s , (1970) o b s e r v e d no d i f f e r e n c e i n
l a r v a l p e n e t r a t i o n of Meloidogyne incognita acrita in
suscept ib le Cucumis melo and r e s i s t a n t C. ficifolius
and C. mituliferus. After 26 days of in fec to r there
was no observable difference in type of g i an t c e l l
development in regions of roo ts assoc ia ted with adult
females. However, in C. mituliferus immature nematodes
were a s s o c i a t e d w i t h s m a l l g i a n t c e l l s which were
l i m i t e d t o few c e l l s nea r t h e head of nematode, with
l i m i t e d amount of h y p e r t r o p h y and c e l l w a l l
d i s s o l u t i o n .
Reyno lds e t . al. , (1970) reported that
a p p r o x i m a t e l y some number of Meloidogyne incognita
acrita larvae penetrated the r e s i s t a n t and suscept ib le
roots of a l f a l f a p lan t . Number of larvae in r e s i s t a n t
roots decreased sharply a f t e r 3 - 4 days t i l l on 7th
28
days fewer than 5 larvae per seedling and no nematode
development could be detected. In susceptible roots
larvae became sedentary and developed nonnally.
Michael et. al. , (1974) reported that galls on
resistant roots, of cotton on infection by Meloidogyne
incognita, contained no nematodes penetration of the
larvae in the resistant cultivar was equal to that of
the susceptible cultivar and independent of the member
of nematodes.
Shepherd and Huck (1989) reported small cracks
caused by M. incognita in root epidermis and cortex.
Soon after the cotyledons expanded, the cracks became
longer and wider and extended through the cortex. Gall
formation on tap roots took place on fourth day, when
true leaf became visible. Root cracking, galling and
giant cell on twelvfth day led to predisposition of
cotton roots to pathogen resulting in synergistic
interactions and diseases. Windhen and Williams (1994)
reported similar penetration of Meloidogyne incognita
juvenile on 3rd day after inoculation (3DAI) in four
corn genotypes in both susceptible and resistant
genotypes.
Par.kaj and Siyanand (1992) reported that the
chemicals like carbofuran, carbcsulfan and phenamiphos
inhibit y.eloidogyne incognita larval penetration and
the development of females or. roots resulting in
29
reduced g a l l format ion and r e p r o d u c t i o n a f t e r 50 and
6 0 days , in case of bitter gourd and roiond melon.
Nanjegowda et. al. , (19S1) reported tha t the
a n t i b i o t i c KT199 m a r k e d l y d e c r e a s e d t h e number of
nematodes and number of g i a n t c e l l s and n u c l e i . Giant
c e l l s were a l s o found t o c o n t a i n l e s s i n s o l u b l e poly
s a c c h a r i d e , n u c l e i c a c i d and p r o t e i n s . A n t i b i o t i c
checked the damage caused by Meloidogyne incognita to
tomato p l a n t s .
C a b a n e l l a s , e t . al., (1988) s tudied the
h i s t o l o g i c a l i n t e r a c t i o n of Paecilomyces lilacinus
with Meloidogyne incognita on the excised roo ts of
tomato. They observed no g ian t c e l l in tomato roots
i n o c u l a t e d w i t h nematode eggs i n f e c t e d w i t h P.
lilacinus.
Few to no g a l l s and no giant c e l l formation were
found i n r o o t s d i p p e d i n s p o r e s u s p e n s i o n of P.
lilacinus and inoculated with M. incognita. Numerous
l a r g e g a l l and g i a n t c e l l s were p r e s e n t i n r o o t s
i n o c u l a t e d w i t h o n l y M. incogzilta.. P. lilacinus
colonized the surface of ep ide r r s l c e l l s as well as
the in t e rna l c e l l s of epidermis and cor t ex . (Dunn e t .
al., 1982; Fattah and Webster 1963;.
Materials and Methods
MATERIALS AND METHODS
The different materials to be used ar.d methods
to be employed during the course of proposed
experimental programme are generalized as follows :
Test Plant and Pathogens and Material:
In the proposed plan of work bitter gourd
Momordica charantia L ) will be selected as the test
plant, root-knot nematode as the test pathogen and
Paecilomyces lilacinus as the test fungus and aldicarb
as the test nematicide.
Collection of Nematode Inoculum {Meloidogyne
ineognita):
Roots of brinjal {Solanum melongena ) infected
with root-knot nematode will be collected fron brinjal
field. Root-knot nematode species and races will be
identified , on the basis of the characteristic perineal
patterns and differential host test.
Pure Culturing 2uid Maintenance of Inoculum :
A single egg mass of Meloidogyne incognita will
be surface sterilized in 1 : 500 solution of chlcrox
(calcium hypochlorite ) for five minutes ani washed
thrice in sterilized distilled water. The egg mass
will be allowed to hatch in distilled water at 27°C.
Egg-plant seedlings raised in 25 cm pots ccntaining
J±
autoclaved s o i l , wi l l be inoculated with the second-
stage juveni les(J2) thus obtained. After about f ive
months s o i l of inoculated pots wi l l be examined to
a s c e r t a i n t h e e s t a b l i s h m e n t of n e m a t o d e s . The
s u b c u l t u r i n g w i l l be done by i n o c u l a t i n g new egg
p l a n t s wi th a t l e a s t 15 egg masses ob ta ined from pure
c u l t u r e i n o r d e r t o m a i n t a i n s u f i c i e n t i nocu lum
throughout the course of i n v e s t i g a t i o n .
Isolat ion and Preparation of Nematode Inoculvun:
Egg masses wi l l be removed from the infected
p lan t s and allowed to hatch (Stemerding, 1963) a t 27°C
in an incubator . After every 24 hours the nematode
suspension wi l l be co l l ec t ed in beakers. The second-
s t a g e j u v e n i l e s of Me2oidog3/ne incognita w i l l be
c o u n t e d w i t h t h e h e l p of c o u n t i n g d i s h u n d e r t h e
s t e r e o s c o p i c microscope . An average of 3 to 5 counts
w i l l be made to de te rmine the d e n s i t y of nematode
j u v e n i l e s in the s u s p e n s i o n .
Raising of Test plant:
The s e e d s of t e s t p l a n t , b i t t e r gourd
{Momordica charentia ) w i l l be surface s t e r l i z e d with
0.5% sodium hypochlori te (NaOcl) for five minutes and
thoroughly washed five times with d i s t i l l e d water. The
seeds wi l l then be sown in autoclaved clay pots of
diameter 15 cm., containing steam s t e r l i z e d s o i l and
32
manure in fixed proportion of 2 : 1. One week old
seedlings will be used for inoculation.
Inoculation with Nematode :
After one week of seedling emergence, holes of
5- 7 cm depth around the plants within a radius of 2
cm. from the plant will be made in which a counted
number of nematode larvae { second stage infective
juveniles ) will be transferred with the help of
sterlized pipette. The holes will then be plugged with
sterilized soil. Regular watering will be done to
maintain the soil moisture till the termination of the
experiment.
Collection o£ Fungal Inoculiun:
The fungal plant pathogen for use in experimental
studies will be Paecilomyces lilacinus.The culture of
the P. lilacinus (Thorn) Samson, will be obtained from
International Potato Centre, Lima, Peru. It will be
maintained on Potato Dextrose Agar, (P D A) slant. The
Constituents of the P D A (Ricker and Ricker, 1936)
are given below:
Agar 17.00 g
Potato peeled and sliced 200.00 g
Dextrose 20.00 g
Distilled water 1000.00 ml
33
Richard's liquid medium (Ricker and Ricker,l935)
will be used for the mass production of P. lilacinus
The composition of Richard's liquid medium is as
follows.
Potassium nitrate{KN03) 10.00 g
Potassium dihydrogen phosphate(KH2PO4) 5.00 g
Magnesium sulphate (MgSo4.7H20) 2.50 g
Ferric chloride(Fecl2) 0.02 g
Sucrose (C-L2"22°11^ 50.00 g
Distilled water 1000.00 ml
The medium will be prepared, filtered through
muslim cloth and sterilized in autoculave at 15 lb.
for 15 minutes in 250 ml corning flasks, each
containing 100 ml of liquid medium. Liquid medium
taken in flask will be inoculated with small amount of
fungus maintained on P D A slants with the help of
sterilized inoculating needle under aseptic conditions
in aseptic chamber. These inoculated flasks will be
incubated at 28MC for about 15 days to allow rapid
fungal growth for experimental studies.
Preparation of Fxingal Inoculiun and its Inoculation:
After enmassing of Paecilowyces lilacinus on
Richard's medium, 100 g of mycelia well be blended in
1000 ml of distilled water, in warring blender so that
34
10 ml of suspension consist of one gm mycelial
suspension.
The fungus P. lilacinus will be incorporated
into the soil around the root zone of bitter gourd
simultaneously with nematode inoculum and following I,
II, III and IV week after nematode inoculation in the
form of suspension. The plants will be left as such
for two months and then harvested and processed.
Study the Effect of Systemic Nematicides :
To study the effect of systemic nematicides on
root-knot nematodes Meloidogyne incognita development
and gall formation. Aldicarb and Carbofuran will be
selected. Aqueous stock soluations conataining 1 mg
aldicarb per ml soluation will be added to the potted
plants after pre, post and simultaneous inoculations
of nematodes. The nematicide, aldicarp (2 - methyl- 2
-methylthio) Propionaldehyde o-(methyl carbomyle
(oxime) is a carbamate insecticide-nematicide. To
study the effect of pre inoculation treatment the
required dosages of nematicide will be applied to pots
containing autoclaved soil around the root zone.
Second treatment will be given simultaneously at the
time of inoculation of nematodes and after that the
third treatment will be followed after 2, 3 and 4
weeks respectively in different replicates and the
35
p l a n t s w i l l be l e f t a s such f o r 2 m o n t h s .
H i s t o p a t h o l o g i c a l s t u d i e s of the i n f e c t e d r o o t s w i l l
be c a r r i e d out a f t e r t e r m i n a t i o n of each expe r imen t s .
Histopathological Studies:
Inoculated seedlings wi l l be uprooted ca re fu l ly
of the s o i l from 24 hours u n t i l l the 10 th day. The
root w i l l be washed gent ly and thoroughly to remove
a l l s o i l p a r t i c a l adhering on them. Galled roots wi l l
be cut in to 1 cm long pieces and processed.
For h i s topa thologica l s tudies the infes ted root
a f t e r the t e r m i n a t i o n of every e x p e r i m e n t w i l l be
processed as fo l lowes :-
Fixation;
The roots collected from experimental pots will
be washed thoroughly and fixed in F. A. A. (Formalin
aceto alcohol) (Johanson, 1940) F. A. A. wil be
prepared as followes :
Ethanol 50 % - 90 ml
Formalin 37 % - 5 ml
Glacial acetic acid - 5 ml
The roots will be placed in the fixative for a
minimum period of 24 hours to several days, depending
on its thiknes. Material may also be fixed in the
fixative indefinitely.
36
Dehydration :
Dehydration is accomplished by moving the roots
step wise through increasingly higher concentrations
of alcohal. Tertiary butyl alcohal (TBA) dehydration
schedule (Table l) will be followed (Johnsen, 19-40)
TABLE 1
Quantity (ml) needed £or solution
Step
1
2
3
4
5
6
7
8
% Alcohol
50
70
85
95
100
100
100
100
Time
2h or more overnight
1-2 h
1-2 h
1-3 h
1-3 h
1-3 h
overnight
Distill Water
50
30 •
15
0
0
0
0
0
95% Ethanol
40
50
50
45
0
0
0
0
100% Ethanol
0
0
0
0
25
0
0
0
100% T.B.A.
10
20
35
55
75
100
100
100
It is important to keep the tertiary butyl
alcohol TBA changes in warm place as the chemcal
solidifies at 25.5]^C.
37
Infiltration:
In this step alcohols in the tissues will be
replaced by paraffin so that tissue is saturated with
pure solution of paraffin, when the TBA dehydration
schedule is followed , the 100% TBA solution in step 8
will be replaced with 1:1 mixture of 100% TBA and
paraffin oil. The tissues will be allowed to remain in
this solution for 1 h or more depending on its
thickness. Shortly before the next step, another
container will be filled 3/4th of its volume with
melted paraffin and allowed to solidify slightly. The
roots from TBA paraffin oil mixture will then be
placed on top of the solidified paraffin oil solution.'
This container will be placed uncovered in an oven set
at slightly above the melting point of paraffin. After
1 - 3 hours the TBA parafin oil mixture will be poured
off and replaced with pure melted paraffin wax and
kept in oven for about 3 hours. This step will be
repeated atleast oncemore.
Eimbedding:
The roots will be placed in metal base molds or
folded paper. Molds will first be coated with thin
layer of glycerine and then liquid paraffin will be
poured, roots will be placed into the mold with heated
forceps and additional melted paraffin will be added
38
to fill the mold. Once the paraffin begin to solidify
over the top of the mold, the mold be plunged into ice
water and left there untill solidification. After
hardening, it will be cut into smaller blocks and
trimmed. Then the blocks will be mounted on block
holders.
Sectioning :
Transverse and longitudinal sections of 8 - 12
urn thickness of the roots will be cut serially with
the help of rotary microtome. The paraffin ribbon thus
obtained will be mounted on a clean galss slide with
an amount of albumin and glycerine dissolved in water,
the slide will be kept overnight in an incubator at 40
degree C to allow water to evaporate. These slides
will then be kept at 60 degree C for one hour to melt
the paraffin.
Staining :
The process of staining will remove all paraffin
from the sections and increase the contrast in the
tissues. Staining will be done with saffranin and fast
green combination ( Table -2, Sass, 1951). Then slides
will be removed from the xylene, and laid on a flat
absorbent surface. The mounting madium will be applied
to surface of the slide before evaporation of xylene
and cover slip will be lowered gradually over the
slide.
Table 2
Sass Safranin and fast-green stain
39
step Solution Time
1.
2.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Xylene
absolute ethanol
95% ethanol
70% ethanol
50 ethanol
30 ethanol
1% aqueous safranin
rense in tap water
30% ethanol
50% ethanol
70% ethanol
95% ethanol
0.1% fast green FCF in 95% ethanol
absolute ethanol
absolute ethanol
xylene-absolute ethanol i:i
xylene
xylene
5 min
5 min
5 min
5 min
5 min
5 min
1-12 h
3 min
3 min
3 min
3 min
5-30 sec
15 sec
3 min
3 min
5 min
5 min or larger
40
Finished slides will be left to dry for atleast 24
hours at room temperature. The medium will harden better if
the slides are held on 60^0 warming tray over night. The
Slides will be examined under laborlux-k-compound
microscope. Necessary photographs will be taken.
To Determine the Resistance of Different Cucurbit Varieties:
Seeds of several varieties of cucurbits will be
obtained from National Seeds Corporation (NSC), New Delhi
and different seed centres. The resistance will be
determined by taking different growth parameter and compared
over controlled plants. Each variety will be replicated six
times with 3 pots as experimental set and 3 pots as
controlled sets. Shoot length, leaf number and size of
leaf, number of flowers, number of fruits, number of galls,
shoot weight and root weight both fresh and dry weight will
be taken to compare. Host suitability designation to each
variety will be assigned according to Sasser et al. (1984)
scale.
41
Table III
Quantitative scheme for assignment of Sasser's host
suitability (resistance) designations
Plant damage (gall index)
Host efficiency (R factor)
Degree of resistance (DR) designation
< 2
<. 2
> 2
> 2
< 1
> 1
< 1
> 1
resistant
tolerant
hypersusceptible
susceptible
42
References
REFERENCES
Abrantes, I.M. DeO., Vovlas, N., Santos, M.S.N. DeA 1992 . Host p a r a s i t e r e l a t i o n s h i p of Melodogyne javanica and M. Cusitanica with Olea europea. Nematologica 38: 320-327.
Araya M. and Caswell-Chen, E.P. 1994. P e n e t r a t i o n of Crotalaria juncea, Dolichos labla^ and Sesamum indicum roots by Meloedogyne Javanica, Nematol. 26: 238-240.
A r e n s , M.L, ; R i c h , J . R . and Dickson , D.W, 1 9 8 1 . Comparative s t u d i e s on roo t i nvas ion , roo t g a l l i n g , and f e c u n d i t y of t h r e e Meloidogyne spp. on a s u s c e p t i b l e tobacco c u l t i v a r . J . Nematol. 13: 201-205.
Barker , K.R. 1985. Nematode e x t r a c t i o n and b i o a s s a y s . pp . 19 -35 . In 'An Advanced T r e a t i s e on Meloidogyne, Vol .1 : Methodology' Eds. K.R. Barker, C.C. Carter and J . N . S a s s e r . A c o o p e r a t i v e p u b l i c a t i o n of t h e Depa r tmen t of P l a n t P a t h o l o g y and t h e U . S . A . I . D . , N.C.S.U. Graph ics , 223, pp .
B e i l l e , L. 1988. Sur l e s a l t e r n a t i o n s p r o d u i t s p a r I ' H e t e r o d e r a r a d i c i c o l i s u r l e s r a c i n e s du Papaya gracilis. Comp. Rend. Assoc. France. Avanc. Sc i . 27: 413-416.
Bergeson, G.B. 1966. Mobilization of minerals to the in fec t ion s i t e of root-knot nematodes. Phytopathology 56: 1287-1289.
44
Birchfield, W. 1965. Host-parasite relations and host
range of a new Meloidogyne species in southern U.S.A.
Phytopathology 55: 1359-1361.
Bird, A.F. 1959. The attractiveness of roots to the
plant parasitic nematodes, Meloidogyne javanica and M.
hapla. Nematologica. 4: 322-335.
Bird, A.F. 1960. Additional notes on attractiveness of
roots to plant parasitic nematodes. Nematologica 5:
217 (Abstr.) .
Bird, A.F. 1961. The ultrastructure and histochemistry
of a nematode induced giant cell, J. Biophys. Biochem.
Cytol. 11: 701-715.
Bird, A.F. 1962. The inducement of giant cells by
Meloidogyne javanica Nematologica 8: 1-10.
Bird, A.F. 1969. Changes associated with parasitism in
nematodes. Ultrastructure of the stylet exudation and
and dorsal esophageal gland contents of female
Meloidogyne javanica J. Parastol. 55:337-345.
Bird, A.F. 1972. Cell wall break-down during the
formation of syncytia induced in plants by root-knot
nematodes. Int. J. Parasitol. 2: 431-432.
Bird, A.F. 1973. Observation on chromosomes and nuclei
in syncytia induced by Meloidogyne javanica Physiol.
Plant Pathol. 3: 387-391.
Bird, A.F. 1974. Plant response to root-knot nematode.
Annu. Rev. Phytopathol. 12: 69-85.
45
Bird, A.F. and B.R. Loveys 198 0. The involvement of
cytokinin in a host-parasite relationship between the
tomato {Lycopersicon esculentum) and a nerr3.tode
{Meloidogyne javanica) parasitology 80: 497-505.
Bird, D.M.K. 1992. Mechanism of the Meloidogyne-host
interactions pp.51-59. 'In Nematology from a molecule
to ecosystem. Ed. of Gommers, F.J.; Mass P.W. Th. and
Dundell, U.K; European Society of Nematologists.
Brom, W.H. 1946. Useful plants of the Philippines, by
W.H. Brom (Department of Agriculture and Comnerce,
Manila); Vol.1, 1941. (reprinted 1951); vol.2, 1941
(reprinted 1954); and vol.3. 1964.
Burkil, I.H., 1909. A working list of flowering
plants of Baluchistan, by I.H. Burkill
(Superintendent, Govt. Printing, Calcutta.
Byrd, D.W., Ferris Jr. H. and Nusbaum C.J. 1972. A
method for estimating numbers of eggs of Meloidogyne
sp. in Soil. J. Nematol. 3: 378-385.
Byrne, J.M., Pesacreta, T.C. and Fox, J.A. 1977.
Vascular pattern change by a nematode, Meloidogyne
incognita, in the lateral roots of Glycine max (L.)
Merr. Am. J. Bot. 64: 960-965.
Cabanillas. E, Barker, K.R. and Daykin, M.B. 1988.
Histology of the interactions of Paecilo-yces
lilacinus with Meloidogyne incognita on tomato. J.
Nematol. 20 362-365.
46
C h i r , B . ; H o r r i g u e M.M. and Raouan i , N. 1 9 9 1 . V a r i a b i l i t y of h i s t o p a t h o l o g i c a l r e a c t i o n s induced by Meloidogyne spp . Ri jks U n i v e r s i t i t Gent, 56: 31-55 .
C h r i s t i e , J . R . 1936. The deve lopmen t of r o o t - k n o t nematode g a l l s . Phytopathology 26: 1-22.
C h r i s t i e , J .R. 1946. 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 the r o o t - k n o t nematodes, Heterodera warioni I I . The effect of the host on the p a r a s i t e . Phytopathology 36: 340-352.
Chr i s t i e , J.R. 1949. Hos t -paras i te r e l a t i onsh ip s of t h e r o o t - k n o t n e m a t o d e , Meloidogyne spp. I I I . The n a t u r e of r e s i s t a n c e in p l a n t s to r o o t - k n o t . Proc . Helm. Soc. Wash. 16: 104-108.
C r i t t e n d e n , H.W. 1954. F a c t o r s a s s o c i a t e d with r o o t -knot namatode r e s i s t a n t soybeans i n f e c t e d w i t h Meloidogyne incognita acrita. Phytopathology 44: 388 (Abs t r . ) .
C r i t t e n d e n , H.W. 1958 . H i s t o l o g y and c y t o l o g y of s u s c e p t i b l e and r e s i s t a n t soybeans i n f e c t e d w i t h Meloidogyne incognita Phytopathology 48: 461 (Abs t r . ) .
C.M.I. (1983). Plant Pathologis t Pccketbook. 2nd Ed. CAB Publ ica t ion . 43 9 p .
Datta, S; Trivedi P.G. and Tiagi , B. 1991. Development of the root-knot nematode Meloidogyne incognita in Vigna radiata and Cyamopsis tetragonaloba. Indian P h y t o p a t h . 4 3 : 4 9 6 - 4 9 9 .
47
Davide, R.G. and T r i a n t a p h y l l o u , 1968. Inf luence of
the environment on development and sex d i f f e r e n t i a t i o n
of r o o t - k n o t n e m a t o d e s ( 1 ) . E f f e c t of f o l i a r
o p p l i c a t i o n of m a l e i c h y d r a z i d e . Nema to log i ca 14; 37 -
46 .
Davis E.L. Kaplan D.T. Deckson D.W. and M e l c h e l l . D . J . 1 9 8 9 . Root t i s s u e r e s p o n s e of two r e l a t e d s o y b e a n c u l t i v a r s t o i n f e c t i o n by l e c t i n t r e a t e d Meloidogyne spp. J. Nematol. 21: 219-228.
Davis, R.A. and Jenkins, W.R. 1960. Histopathology of Gardenia jasminoides veitchi, i n f ec t ed with three s p e c i e s of Meloidogyne. Nematologica 5: 228-230.
D a y k i n , M.F. and H u s s e y R . S . 1 9 8 5 . S t a i n i n g and h i s t o p a t h o l o g i c a l t e c h n i q u e s i n nematology , p p . 3 9 - 4 8 in K.R. Barker, C.C. Carter and J .N . S a s s e r , e d s . An advanced t r e a t i s e on Meloidogyne, v o l . 2 . Methodology, Rele igh: North Carolima S ta te Univers i ty Graphics.
Dean, J.L. and Strubble, F.B. 1953. Resistance and s u s c e p t i b i l i t y to root-knot nematodes in tomato and sweet pota to . Phytopathology 43: 290 (Abstr.)
D r o p k i n , V.H. 1 9 5 4 . I n f e c t i v i t y and g a l l s i z e of t o m a t o and cucumber s e e d l i n g s i n f e c t e d w i t h Meloidogyne incognita var . acrita (root-knot nematode) . Phytopatho logy 44 : 4 3 - 4 9 ,
Dropkin, V.H. 1972. P a t h o l o g y of Meloidogyne g a l l i n g , g iant c e l l formation, e f f e c t s on host physio logy. Hur. Mediterr. Plant. Prot. Organ. (OEPP/EPPO) Bul l . 6: 23-32.
D r o p k i n , V.H. 1980. In ' I n t r o d u c t i o n t o P l a n t Nematology ' , John Wiley and Sons, New York, 293 pp.
D r o p k i n , V.H. and N e l s o n , P .E . 1960. The h i s t o p a t h o l o g y of r o o t - k n o t nematode i n f e c t i o n s i n soybeans . Phytopathology 50: 442-447.
D r o u g h t , R. K a l l o f f e l , C. Gommers, F . J . 1992 . A l t e r a t i o n of d i s t r i b u t i o n of r eg ions with high ne t p r o t o n e x t r u s i o n i n t oma to r o o t s i n f e c t e d w i t h Meloidogyne incognita Physiol , and Mol. Plant pa tho l . 40: 153-162.
Dunn, M.T., R.M. Sayre. A Car re l l , and W R. Wergin, 1982. Colonization of nematode eggs ''' ^aecilomyces lilacinus (Thom) Samson as o b s e r v w i t h s c a n n i n g e l e c t r o n microscopy, Scanning E lec t ron Microscopy 3 : 1351-1357.
Dymock W. Warden C . J . H . and Hooper D. In pharmacographia I n d i c a , (Trubner and Co. , London, 3 v o l s . 1890-99.
Ediz , S.A. and Dickerson, O.J . 1976. L i f e cyc l e and p a t h o g e n i c i t y , h i t o p a t h o l o g y and host range of race 5 of the b a r l e y r o o t - k n o t nematode. J . Nematol. 8: 228-232 .
E i s e n b a c k , J . D . 1 9 9 1 . Method f o r c o l l e c t i o n and p r e p a r a t i o n of nematodes P r e p a r a t i o n of nematodes for s c a n n i n g e l e c t r o n m i c r o s c o p e . In A Manual of A g r i c u l t u r a l Nematology. ed. Nickle W.R. New York, USA; Marcel Dekker, I nc . pp .87 -96 .
49
Endo, B.Y. 1 9 7 1 . Nematode i nduced s y n c y t i a ( g i a n t c e l l s ) Host p a r a s i t e r e l a t i o n s h i p of H e t e r o d e r i d a e , pp .91 -117 . In p l a n t p a r a s i t i c nematodes, v o l . 1 1 , ed. B.M. Zukerman, W.F. Mai and R.A. Rhode. Academic p r e s s . New York/London, 347 pp.
Endo, B.Y. 1987. H i s t o p a t h o l o g y and u l t r a s t r u c t u r e of c r o p s i n v a d e d by c e r t a i n s e d e n t a r y e n d o p a r a s i t i c nematodes, pp .196-210 . In ' V i s t a s on Nematology: A Commemoration of the Twen ty - f i f t h Anniversary of the S o c i e t y of Nema to log i s t s , e d s . , J .A. Veech and D.W. Dickson, Maryland, 509 pp .
Endo, B.Y. and Veech, J .A. 1969. The h i s t ochemica l l o c a l i z a t i o n of o x i d o r e d u c t i v e enzymes of s o y b e a n s i n f e c t e d w i t h t h e r o o t - k n o t nematode Meloidogyne incognita, acrita, Phytopathology. 59: 418-425.
Endo, B.Y. and Wergin , W.P. 1973 . U l t r a s t r u c t u r a l i n v e s t i g a t i o n of c l o v e r r o o t s dur ing e a r l y s t a g e s of i n f e c t i o n by the r o o t - k n o t nematode, M. incognita. Protoplasma 78: 365-379.
Eve rysmeyer , H.E. and D i c k e r s o n O . J . 1966. H i s topa tho logy of r o o t - k n o t nematode i n f e c t e d r o o t s . Phytopathology 56: 816-820.
Fa rooq , T. 1973 . The anatomy of a r o o t g a l l of Lycopersicon pimpinel li folium by Meloidogyne incognita. Nematologica 19: 118-119.
Fas suo l i t i s , G. 1970. Resistance of Cucumis to the root-knot r.ematode, Meloidogyne incognita acrita, J. Xemato l . 2 : 1 7 4 - 1 7 7 .
50
F a t t a h , F. and Webs te r J .M. 1 9 8 3 . U l t r a s t r u e t u r a l changes caused by Fusarium oxysporum-f-sp. Lycopersici i n Meloidogyne javanica induced giant c e l l s in Fusarium R e s i s t a n t and s u s c e p t i b l e tomato c u l t i v a r s . J . of Nematol. 15: 128-135.
Fawole, B. 1988. H i s topa tho logy of Root-not nematode [Meloidogyne incognita i n fec t ion on white Yam [Dioscorea rotundata) t ube r s . J . Nematol. 20: 23-28.
F i n l e y , A.M. 1 9 8 1 . H i s t o p a t h o l o g y of Meloidogyne chitwoodi (Golden et a l . , ) on Russet Burbank Potato. J . Nematol. 13: 486-491.
Godfrey, G.H. and Ol ivei ra , J . 1932. The development of root-knot nematode in r e l a t i o n to root t i s sue of pineapple and cowpea. Phytopathology 22: 325-348.
Golden, A.M. and Shafer, T. 1958. Unusual responses of Hesperis watronalis L. to root-knot nematodes (Melodigyne spp.) Plant Dis. Rep, 42: 1163-1166.
Gommers, F . J . and Dropkin, V.H. 1977. Quant i ta t ive h i s t o c h e m i s t r y of nematode - i n d u c e d t r a n s f e r c e l l s Phytopa tho logy , 67: 869-873.
Gourd T.R. Schmit, D .P . , and Baraker , K.R. 1993. P e n e t r a t i o n r a t e s by s e c o n d - s t a g e J u v e n i l e s of Meloidogyne spp. and Heterodera glycine in to soybean r o o t s . J . Nematol. 25: 38-41.
Green, CD. 1971. Mating and host finding behaviour of p l a n t n e m a t o d e s , p p . 2 4 7 - 2 6 6 . In p l a n t p a r a s i t i c nematodes, v o l . 1 1 , ed. B.M. Zukerman. W.F. Mai, and R.A. Rohde Academic P r e s s , New York, 347 pp.
51
G r i f f i n , G.D. and Hunt, O . J . J . 1972. Ef fec t of p l a n t age on r e s i s t a n c e of a l f a l f a to Meloidogyne hapla, J. Nematol. 4: 87-90.
Heald, CM. 1969. Pathologenici ty and histopathology of Meloidogyne graminis in fec t ing 'Tifdwarf Bermuda grass roo t . J . Nematol. 1: 31-34.
Hisamuddin and S i d d i q u i , Z.A. 1992a. P a t h o g e n e t i c i n d u c t i o n of xylem in Luffa root g a l l s induced by Meloidogyne incognita. Indian J . Applied and Pure B i o l . 7: 15-17.
Hisamuddin and S i d d i q u i , Z.A. 1992b. The e f f e c t of d i f f e r e n t inocu lum l e v e l s of r o o t - k n o t n e m a t o d e , Meloidogyne incognita on growth of Luffa cylindrica var Ghiya. Geobios 19.
Hisamuddin Siddiqui , Z.A. 1992. Pathogenetic Induction of xylem in luffa cylindrica root g a l l s induced by Meloidogyne incognita (Kofoid and white) chitwood Ind ian J . of Applied and Pure B i o l . 17: 15-17.
Hooper D.J . 1985. E x t r a c t i o n of nematodes from p l a n t m a t e r i a l , p p . 5 1 - 5 8 . In 'Labo ra to ry Methods for Work With P lan t And So i l Nematodes ' , ed. J . F . Southey, Her Ma je s ty ' s S t a t i o n e r y Of f i ce , London, 191 pp .
Huang, C .S . and M a g g e n t i , A.R. 1969a . M i t o t i c a b e r r a t i o n s and n u c l e a r changes of deve loping g i a n t c e l l s i n Vicia faba caused by root-knot nematode, Meloidogyne javanica. Phytopathology 59: 447-445.
52
H u a n g , C . S . and M a g g e n t i , A .R . 1 9 6 9 b . W a l l m o d i f i c a t i o n s i n d e v e l o p i n g g i a n t c e l l of Vicia faba and Cucumis sativus induced by r o o t - k n o t namatode, Meloidogyne javanica. Phytopathology 59: 931-937.
Husain , S . I . , Pasha M.J. S idd iqu i Z.A. 1992. S t u d i e s
on a b n o r m a l x y l e m i n t o m a t o and b r i n j a l r o o t s
d e v e l o p e d due t o i n f e c t i o n of r o o t - k n o t n a m a t o d e .
Meloidogyne s p e c i e s Current Nematol. 351-521 .
Hussey, R .S . and S a s s e r , J .N . 1973. Pe rox idase from
Meloidogyne incognita P h y s i o l . p l a n t p a t h o l . 3 : 223-
229.
Ibrah im, I .K.A. and Lewis, S.A. 1985. 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 Meloidogyne arenaria and M. incognita
on s u s c e p t i b l e soybean. J . Nematol. 17: 381-385 .
Ibrahim, I .K.A. and Massoud, S . I . 1974. Developent of
p a t h o g e n e s i s of. a r o o t - k n o t n e m a t o d e s , Meloidogyne
javanica. P roc . Helm. Soc. Wash. 4 1 : 68-72 .
J e n k i n s , W.R. and T a y l o r , D . P . 1 9 6 7 . In ' P l a n t
Nema to logy ' R e i n h o l d P u b l i s h i n g C o r p o r a t i o n , New York,
523 p p .
J o h a n s e n , D.A. 1940. In ' P l a n t M i c r o t e c h n i q u e ' . McGraw
H i l l Book C o . , New York, 523 p p .
J o n e s , M.G.K. 1 9 8 1 . The deve lopmen t and f o r m a t i o n of
p l a n t c e l l s m o d i f i e d by e n d o p a r a s i t i c n e m a t o d e s . In
' P l a n t P a r a s i t i c N e m a t o d e s V o l . I I I ' . E d s . B.M.
Zuckerman and R.A. Rohde. Academic P r e s s , New York,
508 p p .
53
Jones, M.G.K. and Dropkin, V.H. 1976.Scanning electron
microscopy of nematode induced giant transfer cells.
Cytobios. 15: 149-161.
Jones, M.G., K. and Gunning, B.E.S, 1976. Transfer
cells and nematode induced giant cells in Helianthernum
Protoplasma 87: 273-279.
Jones, M.G.K. and Northcote, D.H. 1972. Multinucleate
transfer cells induced in Coleus roots by the root-
knot nematode, Meloidogyne arenaria. Protoplasma 75:
381-391.
Jones, M.G.K. and Payne, H.L. 1978. Early stages of
nematode induced giant cell formation in roots of
Impatiens balsamina. J. Nematol. 10: 70-84.
Kaplan, D.T. and Davis E.L. 1991. A bioassay to
estimate root penetration by nematodes, J. Nematol.
23: 446-450.
Khan, A.A. and Khan, M.W. 1991. Penetration and
development of Meloidogyne incognita race I and
Meloidogyne j avanica in susceptible and resistant
vegetables. Nematropica 21: 77-77.
Khan, A.M. 1969. In 'Technical Report. Studies on
plant parasitic nematodes associated with vegetable
crops in Uttar Pradesh'. Botany Department, Aligarh
Muslim University, Aligarh, India, 238 pp.
Khan, F.A. and Thomas, Y.T. 1993. Studies on host
parasite relationship between selected onion cultivars
and Meloidogyne incognita race I in ncrthern Nigeria.
Afro Asian J. Nematol. 3: 1-4.
54
Kim, Y. and OHH., S.H. 199 0. Anatomical evidence on the d i f f e r e n t i a t i o n of xylem vesse ls around the g ian t c e l l s induced by the root-knot nematode. Korean. J . of Plant pa tho l . 6: 417-420.
K r i t i k a r , K.R., Basu , B.D. and I .C.S . (retd) ; 1935. Indian Medicinal p l a n t s ; r i v i s e d by E. Bla t l e r J . F . Caius and K.S. Mhaskar (Lal i t Mohan Basu, Allahabad), 4 v o l s . 2nd ed. 1935.
Koenn ing , S.R. and B a r k e r , K.R. 1 9 8 5 . G n o t o b i o t i c t e c h n i q u e s for p l a n t - p a r a s i t i c nematodes, pp .49 -66 . In 'An Advanced T r e a t i s e on Meloidogyne. Vol .11: Methodology' e d s . K.R. Barker , C.C. C a r t e r and J .N. S a s s e r . A Coopera t ive P u b l i c a t i o n of the Department of P l a n t Pathology and the U . S . A . I . D . , N.C.S.U. Graph ics , 22 3 pp .
Kostoff , D. and Kendal l , J . 1930. Cytology of nematode g a l l s on Nicotiana r o o t s . Cent ra lb l , Bakt. , I I Abt. 81: 86-91.
K r u s b e r g , L.R. 1963 . Host r e s p o n s e s t o nematode i n f e c t i o n s . Annu. Rev. P h y t o p a t h o l . 1: 219-240.
Krusberg , L.R. and Nie l sen , L.W. 1958. Pa thogenes i s of r o o t - k n o t nematodes to Por to Rico v a r i e t y of sweet p o t a t o . Phytopathology 48: 30-39 .
L iao , S.C. and Dunlap, A.A. 1970. A r r e s t e d invas ion of Lycopers icon peruvianum roo t s by the root-knot namatode. Phytopathology 60: 216-218.
55
Linford , M.B. 1937. The feed ing of r o o t - k n o t namatode in roo t t i s s u e and n u t r i e n t s o l u t i o n . Phytopathology 27: 824-835.
L i n f o r d , M.B. 1939 . A t t r a c t i v e n e s s of r o o t s and e x c i s e d r o o t t i s s u e t o c e r t a i n n e m a t o d e s . Proc . Helminthol . Soc. Wash. 6: 11-18 .
L inford , M.B. 1942. The t r a n s i e n t feeding of r o o t - k n o t nematode l a r v a e . Phytopathology 32: 580-589.
L i t t r e l l , R.H. 1966. C e l l u l a r r e sponses of Hibiscus esculentus t o Meloidogyne incognita acrita. Phytopathology 56: 540-544.
Loewenberg, J . R . , S u l l i v a n , R.T. and S c h u s t e r , M.L. 1960. G a l l i n d u c t i o n by Meloidogyne incognita by su r face feed ing and f a c t o r s a f f e c t i n g the behaviour p a t t e r n of t he second s t a g e l a r v a e . Phytopathology 50: 322-323 .
McClure , M.A., E l l i s , K.C. and Nigh, E .L . 1974. R e s i s t a n c e of c o t t o n t o t h e r o o t - k n o t nematode Meloidogyne incognita. J . Nematol. 6: 17-20.
McClure, M.A. and Vig l ie rch io , D.R. 1966. Penetrat ion of Meloidogyne incognita in r e l a t i o n to growth and n u t r i t i o n of s t e r i l e , e x c i s e d cucumber r o o t s . Nematclogica 12: 237-247.
Meon, S . , F i s h e r , J.M. and Wal lace , H.R. 1978. Water r e a c t i o n s of tomato {Lycopersicon eszulentum Mil l . cv. Early dwarf Red) with Meloidogyne javanica (Treub.) Chitwccd, Physiol . Plant Pathol . 13: 275-281.
Michael, A. McClure, K.C, E l l i s , and Edward, L. Nigh, 1974. Post- infect ion development and histopathology of Meloidogyne incognita in r e s i s t a n t co t ton . J . Nematol. 6: 21-26.
Minton, N.A. 1962. Factors influencing res i s tance of c o t t o n t o r o o t - k n o t nematode [Meloidogyne spp. ) . Phytopathology 52: 272-279.
Myuge, S.G. 1956. The n u t r i t i o n a l phys io logy of the g a l l nematodes {Meloidogyne incognita) Dokl Akad. Nauk SSSR 108: 164-165.
Nanjegowda, D. S e t t y K.G.H. and Syamasunder J o s h i 1991 . Effect of A n t i b i o t i c K-T-199 on h i s t o l o g i c a l changes in Meloidogyne incognita - induced g a l l s on tomato. Indian J . Nematol. 21 : 58-60.
Nemec, B. 1910. Das problem der Befruchtun-gsvorgauge und a n d e r e z u t o g i s c h e F r a g e n . G e b r u d e r B o r t a e g e r , B e r l i n . P a r t IV. V i e l k e r n i g e R i e s e n z e l l e n i n Heterodera ga l len . 155-173.
Odihir in , R.A. and Jenkins , W.R. 1965. Host -paras i te r e l a t i o n s h i p of Impatiens balsamina and ce r t a in nematodes. Phytopathology 55: 165-176 .
O r i o n , D. and Minz , G. 1 9 7 1 . The i n f l u e n c e of m o r p h a c t i n on t h e r o o t - k n o t nema tode , Meloidogyne javanica and i t s g a l l s . Nematologica. 17: 107-112.
Orr, C.C. and Morey, E.D. 1978. Anatomical response of gra in sorghur. roots to Meloidogyne incogniza acrita. J . Nemato l . i : : 4 8 - 5 3 .
Owens, R.G. and Specht, H.N. 1964. Root-knot
h is togenes is . Contrib. Boyce Thomp. Ins t . 22: 471-489.
Pankaj and Siyanand - 1992. Efficacy of chemicals as
seed dresser against Meloidogyne incognita on b i t t e r
gourd and round melon. Indian J. Nematol. 22: 110-116.
Pasha, M.J., Siddiqui, Z.A., Khan, M.W. and Qureshi,
S.I. 1987. Histopathology of eggplant roots infected
with root-knot nematode, Meloidogyne incognita. Pak.
J. Nematol. 5: 27-34.
Patel, Y.C.; Patel, D.J. 1991. Studies on root viral
nematode on wheat Pak. J, Nematol. 9: 119-126.
Paulson, R.E. and Webster, J.M. 1970. Giant c e l l
formation in tomato roo t s caused by Meloidogyne
incognita and M. hapla (Nematode) infection. A light
and electron microscope study. Can. J. Bot. 48: 271-
276.
Peacock, F.C. 1959. The development of a technique for
studying the host-parasite relationship of the root-
knot nematode, Meloidogyne incognita under controlled
conditions. Nematologica 4: 43-55.
Powell, N.T. 1962. Histological basis of resistance to
roo t -kno t nematodes in flue cured tobacco.
Phytopathology 52: 25 (Abstr . ) .
Prakaso Rao, C.G. a n d A r u n e e , K . K . 1 9 7 3 .
H i s t o p a t h o l o g i c a l s t u d i e s o f °^\LliiiiAMA\m, ^^='"'"'"''^'^^^='
c a u s e d by Meloidogyne javani^rftf^i»Gh.--<-->48§BalRath. 2 4 :
5 5 8 - 5 7 2 .
58
P r o t , J . C . 1980. M i g r a t i o n of p l a n t p a r a s i t i c nematodes towards p l a n t r o o t s , Rev. Nematol. 3 : 305-318 .
Rama Rao; M. 1914. In f lower ing p l a n t s of t r a v a n c o r e , by M. Rama Rao (Govt. P r e s s , Tr ivandrum).
Reynolds , H.W., C a r t e r , W.W. and O'Bannon, J .H. 1970. Symptomless r e s i s t a n c e of a l f a l f a t o Meloidogyne incognita acrita. J . Nematol. 2: 131-134.
Ricker, A.J. and Ricker, R.S . , 1935. Introduct ion to research on plant d i seases . St. Louis and New York, John ' s Swift Co., 117 p .
Ricker, A.J. and Ricker, R.S . , 1936. Introduct ion to research on plant d i seases . St . Louis and New York, John ' s Swift Co., 117 p .
Rohde, R.A. and McClure, M.A. 1975. Autoradiography of d e v e l o p i n g s y n c y t i a i n c o t t o n r o o t s i n f e c t e d w i t h Meloidogyne incognita. J . Nematol. 7: 64-69.
Roman, J . 1 9 6 1 . P a t h o g e n e c i t y of f i v e i s o l a t e s of r o o t - k n o t nematodes {Meloidogyne spp.) to sugarcane r o o t s . J . Agric. Universi ty of Puerto Rico, 45: 55-84.
Samson, R.A. 19 75 . Paecilomyces and some a l l i e d hyphomycetes. S tud ie s in Mycology 6: 1-119.
Saluwu, E.G., 1991. Anatomical changes induced in r c o t of celosia argentia by Meloidogyne incognita Pak. J . N e m a t o l . 9: 9 1 - 9 4 .
59
S a s s , J . E . , 1 9 5 1 . In ' B o t a n i c a l M i c r o t e c r m i q u e ' . Iowa
S t a t e C o l l e g e P r e s s , Ames, Iowa, 228 p p .
S a s s e r , J . N . and T a y l o r , A.L. 1952. S u i a d i e s on t h e
e n t r y of l a r v a e of r o o t - k n o t ner .a todes i n t o r o o t s of
s u s c e p t i b l e and r e s i s t a n t p l a n t s . P h y t o p a t h o l o g y 4 2 :
4 7 4 ( A b s t r . ) .
S a s s e r , J . N . , C a r t e r , C . C . H a r t m a n , K.M. 1 9 8 4 .
S t a n d a r d i z a t i o n o f h o s t s u i t a b i l i t y s t u d i e s a n d
r e p o r t i n g o f r e s i s t a n c e t o r o o t - k n o t n e m a . t o d e R a l e i g h ,
N o r t h C a r o l i n a , N o r t h C a r o l i n a S t a t e U n i v e r s i t y
G r a p h i c s , 7 p p .
S a w h n e y , R. a n d J . M . W e b s t e r , 1 9 7 5 . The x o l e o f p l a n t
g r o w t h h a r m o n e s i n d e t e r m i n i n g t h e r e s i s t a n c e o f
t o m a t o p l a n t s t o t h e r o o t - k n o t n e m a t o d e , Meloidogyne
incognita N e m a t o l o g i c a . 2 1 : 9 5 - 1 0 3 .
Sharma K .S . and T i a g i , B. 1989 . G ian t c e l l f o r m a t i o n
i n p e a r o o t s i n c i t e d b y Meloidogyne incognita
i n f e c t i o n . J . P h y t o l . R e s e a r c h 2 : 1 8 5 - 1 9 1 .
S h e p h e r e d , R.L. and Huck, M.G. 1989. P r r o g r e s s i o n of
r o o t - k n o t n e m a t o d e , s y m p t o m s a n d i n f e c t i o n o n
r e s i s t a n t a n d s u s c e p t i b l e c o t t o n s . J . o f N e m a t o l . 2 1 :
2 3 5 - 2 4 1 .
S i d d i q u i , I . A . 1 9 7 1 a . H i s t o p a t h o g e n e s i s o f g a l l s
i n d u c e d b y Meloidogyne naasi in o a t r o o t s ,
N e m a t o l o g i c a . 1 7 : 2 3 7 - 2 4 2 .
S i d d i q u i , I . A . 1 9 7 1 b . C o m p a r a t i v e p e n e t r a t i o n a n d
d e v e l o p m e n t o f Meloidogyne naasi i n w h e a t and o a t
r o o t s . N e m a t o l o g i c a 1 7 : 5 6 6 - 5 7 4 .
60
S i d d i q u i , I . A . and T a y l o r , D.P. 1970 . Hi s t o p a t h o g e n e s i s of g a l l s i n d u c e d by Meloidogyne nassi in wheat roo t s . J . Ner.atol. 3: 239-247.
Siddiqui , Z.A. and Ghouse, A.K.M. 1975. Formation of phloem in the roots of Laaenaria leucantha infected with Meloidogyne incognita, Ind. J . Nematol. 5: 102-104. .
S i d d i q u i , Z . A . , R a s h i d , A . , Yunus, M. and Ghouse, A.K.M. 1974. S t u d i e s on r e a c t i o n xylem developed due to Meloidogyne incognita in the roots of Lagenaria leucantha. Ind. J . Nematol., 4: 46-52.
S i n g h , B. and Choudhury , 1 9 7 3 . The c h e m i c a l c h a r a c t e r i s t i c of tomato c u l t i v a r s r e s i s t a n t t o r o o t -kno t nematode {Meloidogyne spp.) Nematologica. 19: 443-448.
S i n g h , K i r t i , 1 9 9 1 . V e g e t a b l e p r o d u c t i o n *Tota l Sys t em ' a p p r o a c h w a n t e d . The Hindu Survey I n d i a n Agri.**f92_ 195
S m i t h , J . J . and Mai, W.F. 1965 . 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 Allium cepa and Meloidogyne hapla. Phytopathology, 55: 693-697.
Soomro, M.H., Hague N.G.M. 1992. Effect of Meloidogyne graminicola on root growth of gra-inaceons p l a n t s . Nematol. Med. 120: 143-147.
Subbotin, S.A. 1990. U l t r a s r r u c t u r a l changes in root c e l l s of Glycin max (Fabaceae infected with cyst , and root-knot nematode. Botan ick isk i i Zhurnae, 75: 315-324.
61
Swamy, B.G.L. and K r i s h n a m u r t h y , K.V. 1 9 7 1 . O n t o g e n e t i c s t u d i e s on p l a n t g a l l s . I I . The h i s t o p a t h o l o g y of r o o t s of Basella alba with M. javanica. Phytomarphology, 21: 36-46.
T a y l o r , A.L. and S a s s e r , J . N . 1978 . In ' B i o l o g y , i d e n t i f i c a t i o n and c o n t r o l of r o o t - k n o t nema todes {Meoidogyne s p e c i e s ) ' . Coop. Publ. Dep. Plant Pa tho l . ,
N.C.S.U., and U.S.A.I.D. Raleigh, N.C. I l l pp.
Veech, J.A. and Endo, B.Y., 1969. The histochemical l o c a l i z a t i o n of several enzymes of soybean infected with root-knot nematode Meloidogyne incognita acrita J . Nematol. 1: 265-276.
Veech, J . A . and Endo B . Y . , 1970. C o m p a r a t i v e l y Morphology and enzyme h i s t o c h e m i s t r y i n r o o t - k n o t r e s i s t a n t and s u s c e p t i b l e soybeans phy topa tho logy , 60: 896-902.
Vovlas , N, S a s n e l l i , N. 1993. Anatomy of sunf lower stem g a l l s induced by r o o t knot-nematode M. Javanica. Afro Asian J . of Nematol. 3 : 99-102.
Webster, J.M. 1969. The hos t -pa r a s i t e r e l a t i o n s h i p s of p lant p a r a s i t e nematodes. Adv. p a r a s i t o l . 7: 1-40.
W e b s t e r , J .M. 1975 . A s p e c t s of 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 p l a n t - p a r a s i t i c n e m a t o d e s . Adv. P a r a s i t o l . 13: 225-250.
Wieser , 1956. The a t t r a c t i v e n e s s of p l a n t s to l a r v a e of r o o t - k n o t n e m a t o d e . 1. The e f f e c t of t o m a t o s e e d l i n g and e x c i s e d r o o t s on Meloidogyne hapla chitwood 22: 106-112.
62
W i e s e r , W. 1956. The a t t r a c t i v e n e s s of p l a n t s t o l a r v a e of r o o t - k n o t n e m a t o d e s . 2 . The e f f e c t of e x c i s e d b e a n , e g g p l a n t and soybean r o o t s on Meloidogyne hapla, Chitwood. Proc. Helm. Soc. Wash. 23: 59-64.
Windham G.L. and William, W.P. 1994. Penetrat ion and d e v e l o p e m e n t of Meloidogyne incognita in roots of r e s i s t a n t and s u s c e p t i b l e corn genotypes J . Nematol. 26: 80-85 .
Wyss. U, Grund le r ; F.M.W, and Munch, A. 1992. The P a r a s i t i c b e h a v i o u r of s e c o n d - s t a g e J u v e n i l e s of Meloidogyne incognita in roots of Arabidopsis thaliana, Nematologica 38, 98-102.