STUDIES ON A VIRUS CAUSING NOSAIC DISEASE ON

VEGETABLES

DISSERTATION SUBMITTED FOR THE DEGREE OF

Muittx of ^^iloiop^ IN

BOTANY (Plant Virology)

BY

MALIK M. ASHAQ RAZA

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 1993

IIIMIII DS2263

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T^GZJ.^?>IJ •t" \ -

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B>omt am tofio niebe it poisiihlt

Qamar A. Naqvi M.Sc.Ph.D. ,FPSI., CIES (Paris) Reader

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 Phone : 401259 Dated...P1...03,19.94

CERTIFICATE

This is to certify that this d i sse r ta ­

tion entitled "Studies on a virus causing mosaic

disease of vegetables" submitted by Mr.Mohd.

Ashaq to the Aligarh Muslim University, Aligarh

for the award of the degree of Master of

Philosophy, is a faithful record of the bonafide

research work carried out by him under my

supervision. No part of this dissertation has

been published or submitted for any other

degree or diploma.

QVI )

A C K N O W L E D G E M E N T

I bow in gratitude to the Almighty Allah who

enabled me to achieve this target.

I consider it a golden chapter of my life to

have worked under the kind supervision of my benevolent

teacher Dr. Qamar A. Naqvi/ Reader Department of Botany,

A.M.U., Aligarh to whome I find myself at = loss of

words to express my sincere gratitude. During the

entire period of persuit of my studies, I had a good

furtune to enjoy his constant and healthy criticism,

sympathetic attitude and valuable advice without which,

indeed present work would not now has been what it is.

I am indebted to Prof- Khalid Mahmood/ Deptt.

of Botany, A.M.U., Aligarh who has provided me unstinted

advice and always been a source of inspiration to me.

I am also thankful to Prof. Wazahat Hussain,

Chairman, Department of Botany, A.M.U., Aligarh, for

providing all the necessary facilities during the entire

course of this work.

I wish to express my sincere thanks to my Lab.

colleagues Dr. Miss Zarina B- Zaidi, Mr. Abdul Rashid

Malik and Mr. Mustaq R. Mir, for their selfless

assistance and co-operation.

My sincere thanks are also due to my friends

Messers. M. Farooq Malik, Tahseen H- Shah, M. Shafait

Azad and Mohd. Younis Malik for their encourageous

help and co-operation during the manuscripts.

I present gift of thanks to Messers. Anjum

Parwez Ansari, Munawar Fazal and Naseer H. Shah for

their keen interest in taking various problems

encountered by me during the course of my research work.

I would miss my duty if I do not make a mention

of my parents and my elder brother Mr. Kafait Dllah

Malik whose sacrifices and blessings gave me impetus

to achieve this goal.

Malik M.^Ashaq Raza

C O N T E N T S

Page No.

INTRODUCTION „, „ Ul-Uo

REVIEW OF LITERATURE 06-45

1 . Cucumber a n d o t h e r c u c u r b i t s 05-22

2 . C h i l l i e s 22-27

3 . B r i n j a l 27-

4 . P o t a t o 28-

5 . R a d i s h 29-

6 . S p i n a c h 30

7 . Tomato 31-38 8. Turnip 38 9 . L i t e r a t u r e on c o n t r o l of CMV 39

PLAN OF WORK AND METHODS 46-69

1 . 0 M a i n t e n a n c e o f v i r u s i n o c u l u m 46

1 . 1 R a i s i n g o f t e s t p l a n t s 46

1 .2 V i r u s c u l t u r e 46

1 .3 S o u r c e of I n o c u l u m 47

2 . 0 T r a n s m i s s i o n 47-55

2.1 Mechanical ^^

2.2 Biological

2.2.1 Insect transmission

48

48

(a) Transmission by aphids

(b) Raising of virus free aphids 48

(c) Mode of transmission 49

( i ) N o n - p e r s i s t a n t 49

( i i ) P e r s i s t a n t 49

2.2.2 Transmission by white flies 50

(a) Source of virus free white flies 50

(b) Handling of white flies 50

(c) Transmission 50

2.2.3 Graft 51

2.2.4 Dodder 51

2.2.5 Soil Transmission 52

(a) Search for fungal vectors 52

(b) Search for nematode vectors 53

2.2.5 Seed Transmission 54

2.2.7 Transmission through Pollen 55

3.0 Host range studies 55

4.0 Virus vector relationship 55

5.0 Effect of different buffers on 56

the infectivity

6.0 Virus concentration in different parts

of the host. 56

7.0 Selection of suitable propagation host 57

and an assay host.

8.0 Biophysical properties 57-59

8.1 Dilution end point (DEP) 57

8.2 Longivity in vitro (LIV) 58

(a) In sap 58

(b) In dried leaves 58

8.3 Thermal inactivation point 59

9.0 Effect of various additives 59

on virus infectivity.

10.0 Purification 59

10.1 Clarification of sap. 59

(a) Celite and charcoal 60

(b) Organic solvent 60

(c) Calcium phosphate gel 61

(d) Silver nitrate 61

10.2 Concentration of virus 62

(a) Differntial centrifugation 62

(b) Precipitation 62

(i) Polythylene glucol(PEG) 62

(ii) Ammonium phosphate 63

11.o Further purification by density 63

gradient centrifugation

12.0 UV-Spectrophotometry 64

13.0 Electron Microscopy 64

13.1 Leaf dip-method 65

13.2 Procedure with purified virus 55

preparation.

a7 14.0 Serology 65

14.1 Raising of antisera 55

14.2 Ouchterlony double-diffusion test. 67

14.3 Precipitation test in tubes. 57

15.0 Isolation of Nucleic Acid 68-69

15.1 Infectivity of viral nucleic Acid 59

15.2 Type of Nucleic Acid 59

16.0 Studies on Proteins of varions 59

References 66-94

INTRODUCTION

INTRODUCTION

Vegetables are most important article of human diet.

They are important sources of mineral elements specially calcium

and iron, valuable sources of vitamins, proteins and carbohydrates.

They play important role in neutrilizing the acids, produced

during the course of digestion of meat, cheese, and other fatty

foods. Their importance and essentiality as a balanced human diet

has been ooserved throughout the world. In India, a majority of

population is vegetarian, but their per capita consumption is

somewhat low. The uptake of vegetables needs to be augmented.

Most, if not all vegetables may become infected with viruses.

Virus diseases besides, affecting the yield directly, cause losses

by reducing the quality. They have not received considerable attention

due to the difficulties involved in obtaining a monetary evaluation

of crop loss.

Many viruses have been reported to cause diseases on

vegetables but the infection caused by cucumber mosaic virus is

considerably high and wide. All the important vegetables like

cucumber^ spinach, brinjal, tomato, potato, chillies, radish,

carrot, various beans, and other cucurbits and crucifers have

been reported to be infected by this (CMV) virus. It has also been

isolated from many ornamentals and weeds. According to a survey of

its host range made oy Komuro (1973), more than 191 sps. of 40

families have been found to be infected by cucumber mosaic virus.

Cucumoer is its native host, is an important vegetable crop

of world wide distribution, used as salad, pickle, and also as

cooked vegetable. Being perishable, cucumber is consumed shortly

after harvast. Its seeds are reported to De cooling, tonic and

diuretic. A large number of varieties of cucumber are grown

throughout India which vary from small pickling to very large and

thick varieties grown in Rajasthan and Gujarat. Most important are

Japanese long green,'Balam Khira'and 'Khira Poona' varieties. A

large number of viruses have been reported to cause mosaic disease

in cucumDer, Cucumis sativus L. The most common and important has

been reported to be caused by cucumber mosaic virus i.e. cucumis

virus 1, cucumis virus 2 and 3 etc.

Tomatoes are the next important vegetables, found to be

infected by cucumber mosaic virus. However, symptoms caused here

by this virus are different from those caused on cucumber; i.e.

tomato necrosis, fern leaf appearance etc.

Similarly, chillies, spinach, potato, brinjal and turnip

are infected by this virus, show mosaic symptoms in addition to

necrosis, stunting, vein clearing or flower breeking.

Like tobacco mosaic virus, this virus exsists in a number

of allied strains, some of which produce symptoms very different

from those characteristic of the type virus.

The detailed information about its host range, transmission

modes, biophysical properties, serology, electron microscopy and

relationships is being given just for an introduction.

Discription formula R/1 : 1 ^ + ^ + Q-^^Q-^:S/S:S/c

Ve/Ap

Discovery, Author-Described, Doolittle(1916) and Jagger(1916),

U.S.A.

Synonyms : Cucumber virus 1 (Rev. appl. Mycol. 6 : 501)

Cucumis virus 1 (Rev. appl. Mycol. 17; 52)

Marmor cucumeris (Rev. appl. Mycol. 28: 514)

Spinach blight virus (f. agric. Res. 14 : 1)

Tomato fern leaf virus (Rev. appl. Mycol. 9 : 417)

Geographical distribution ; World wide especially in temperate

regions.

Virus group ; Cucumovirus group.

Enveloped or non-enveloped : Non - enveloped

Shape of Particle : Isometric

Size : 28 - 30 nm

Type of nuclic acid : Single stranded RNA (ssRNA), linear

genome has 4 parts, MW of Largest; 2nd;

3rd ; 4th = 1.3x10^ : 1.1x10^ : 0.8x10^

0.35x10^

Nucliec acid composition : G = 24^, A = 23%, C = 23? , U = 30%

Composition of virion: Nuclic acid - ^8%, Proteins - 8256,

Lipids and compounds - C^.

Strains : Y (Cowpea) strain (Price, 1934), Spinach strain

(Bhargava, 1951), M strain (Mossop £t al ., 1976).

Two groups of strains 1, ToRS (Q and S strain) and

2, DLT group

Dilution end point (DEP) : 10'" to 10*^

Thermal inactivation point : SS' to 70* C.

Longivity in vitro : 1 to 10 days.

Sedimentation co-e f f i c i en t : 1 compound, S2o» W = 98.65

(-1.04xvirus cone.)

Density ( in caesium chloride) 1,367/cm

I s o - e l e c t r i c point : pH 5.5 (Q s t r a i n )

Replication : Independent, sub-genomic raRNA found in infected ce l l s

Serology : Poor immunogen, low s a l t in ge l diffusion and

non-specif ic pp t . in sa l ine (best c o n d i t i o n s ) , ISEM.

Transmission : Mechanical, Seed (19 s p e c i e s ) . Vectors (non-

p e r s i s t e n t l y by more than 60 spec ie s ) . Dodder

(by a t l e a s t 10 species of cuscuta ) .

Host range ; Wide, 19'' species in 40 fami l ies (Komuro, 1973)

Assay host : Viqna unquiculata (Crowley, 1954), Chenopodiurn

amaranticolar and C. quinoa.

Diagnostic species : Chenopodium amaran t ico la r . C. quinoa

(Chlorot ic and nec ro t i c l o c a l l e s i o n s ) , Gucumls

sa t ivus (Cucumber), Systemic mosaic and s tun t i ng ,

and Nicotiana g lu t inosa . Viqna unquiculata.Lycopersicon

esculentum and Cucurbita moschata are su i t ab l e

h o s t s .

Propagation species : N. glutinosa and N. tabacum cv. Xanthi

(convenent for maintaining c u l t u r e s ) , N. tabacum

and Cucurbita pepo ( s u i t a b l e for virus source) ,

but ^ . c levandi i (bes t for some s t r a in s )

Main d i seases : Mosaic of cucumber and c u c u r b i t s , b l igh t of

spinach, fern leaf of tomato, mosaic of ce le ry ,

and mosaics of many o ther species of dicotyledonous

and monocotyledonous crops, ornamentals and weeds. Dwarfing

and flower breaking is also reported (Smith, 19'72).

Relationships : Tomato aspermy, peanut stunt, cowpea ringspot

(virus particles serologically related)

Remarks : The physical, antigenic and biological properties

overlap the properties of some other viruses, Inclusion

bodies-present are, crystals and tonoplasts particles found

in cytoplasm.

Reviews : VIDE (Virus identification data exchange) DPV-13, 201-215,

1979 (CMI/AAB Description of plant viruses, July, 1979).

In the present study an attempt has been made to review the

literature on certain properties of cucumber mosaic virus. However,

it may not be a complete record of the properties of this virus.

A wider coverage of literature would add many more citations.

Moreover, it has been proposed to investigate a virus

causing mosaic disease on any of the vegetables reviewed here for

virus disease in this dissertation.

REVIEW OF LITERATURE

REVIEW OF LITERATURE

Almost all the vegetables have been reported to be infected

by cucumoer mosaic virus. Among most important are : cucumber,

tomato, spinach, chilli, brinjal,potato and turnip. The chapter

deals with only a limited number of vegetables infected by this

virus.

CUCUMBER AND OTHER CUCURBITS

Doolittie (1921), Doolittle and Walker (1925) and Walker

(1925) reported a mosaic disease of cucumber from U.S.A. where

they showed the role of weeds (Milkweed-Ascleoias s/rica. Martyma

Louisiana and pokeweed-Phytolacca decandra and wild cucumber

Micrampelis looata) and Capsicum annuum, and the insect vectors

(aphid-.Myzus persicae and beetle-D. vittata) in the spread of

this disease. However, they later reported Physalis heterophylla

and P. subqlabrata as important source of inoculum for cucumber

mosaic virus.

Ainsworth (1934, 1935) reported mild mosaic or ordinary

mosaic (named it as green mottle mosaic of cucumber), and yellow

mosaic (named as yellow-mottle mosaic) caused by cucumber virus

3 and cucumber virus 1, respectively. A third aucuba mosaic re-named

yellow mosaic of cucinnber was reported to be caused by cucumber

virus 4, occuring naturally only on cucumber. Cucumoer virus 4

remained infective after a storage period of more than 9 months,

tolerated a temperature of QO°C. The virus was mechanically

transmissible to cucumber and otner cucurbits.

The occurrance of a mosaic disease of vegetable marrow and

rock melon (Cucumis melo Var. cantalupensis) was reported by

Chamberlin (1939). He showed that the virus was transmitted

mechanically as well as by aphids, Aphis qossypii. Myzus persicae

and Macrosiphum solani, retained its infectivity upto a thermal

inactivation point of 62-66°C, remained infective upto 4 days in

vitro and had a dilution end point 10 . Raino (1943) described

same type of virus in Finland. The symptom included protuberances

on the aerial organs accompanied by arching and crinkling of the

leaves and in severe infection, sterility. The aphids and centri-

pedes were shown to be the vector of virus.

Occurrance of cucumis 2 (a strain of cucumber green mottle

mosaic virus) on cucumber was reported by Valentin (1958), from

a nursery of Berlin (Germany). Affected plants showed stunted

growth and occasional shrivelling of upper leaves. Vein clearing

and pale yellow mosaic with asteroid spots, changing into silvery

mosaic was ooserved on the affected leaves. The virus was sap

transmissible, persisted in dried material and remained infective

in sap after a storage period of 100 days. Thermal inactivation

point ranged between 80-90°C (10 minute exposure).

Liera (1959) reported cucumis virus 2 (cucumber green mottle

virus) on cucumber in the Netherlands.

A virus causing vein-clearing, chlorosis and a green necrosis

of cucumber in Jordan Valley, Israel, was reported by Cohen and

Nitzany (1960). They claimed it to be the first virus on cucurbits

and the only plant virus known to be transmitted by white fly

8

Bemisia tabaci as well as mechanically. Harpaz and Cohen (1965)

named this virus as cucumber vein yellowing virus (CVYM) to avoid

confusion with viruses given the name "bottle gourd mosaic virus"

in India.

Moskovets and Glushak (1968) reported cucumoer mosaic virus

as the causal agent of a virus disease of cucumber in Ukrane.

Protsenko (1969) gave a description of cucumber virus I

(cucumber mosaic virus) .& cucumber virus 2 (cucumber green mottle

virus). The former usually appears one month after planting, over

winters in the roots of perennial weeds and is transmitted by aphids

while the later is seed transmitted. The air temperature in glass

house affected the symptoms of mosaic i.e. at over 25 C, the mosaic

observed was white and at lower temperature it was green.

Silberschmidt and Herbas (1969) observed rings on the leaves

of Allamanda cathartica L. sap inoculation induced local lesions

on Chenopodium quinoa and systemic symptoms on the leaves of

Nicotiana qlutinosa. Petunia hybrida and cucumber. The virus

retained its infectivity upto 65 C and concluded it to be a new

strain of cucumber mosaic virus.

Demeski (1969) reported that cucumber mosaic virus is

mechanically transmissible to Summer squash in the glass house

conditions.

Havramek (1970) in a survey of cucumber diseases, found

tobacco necrosis and five strains of cucumber mosaic virus as the

only viruses attacking cucumbers in different localities of

Czechoslovakia.

Verma et al.. (1970) reported a mosaic disease of vegetaole

marrow similar to cucumis virus 1 (cucumber mosaic virus).

Karl (1971) reported the Dysaphis crataeqi. a new non-

persistant vector of cucumber mosaic virus.

Palet and Hani (1971) described various methods of concen­

trating CMV, campared, and assayed 39 preparations. The methods

gave heterogenous results except gel filtration on Sepharose which

gave highly infections solutions showing a low level of contami­

nation but a poor yield.

Twardowicz-Takusz Anna (1971) observed that the infection

of cucumber in the Poznan area varied from 0.04 to 93;- . The virus

was dectected from 10 of 27 vegetables, ornamentals and weeds

examined from the vicinity of infected cucumber.

Peden et aJL. (1972) reported that the difference in the size

of the soluble cucumber mosaic virus and tobacco ringspot virus-

induced RNA polymerases and in the solubilization of these enzymes

from the particular fractions, may provide evidence for the virus-

coded differences in the life cycles of the 2 viruses.

Boatman et al_. (1973) compared the peanut stunt virus (PSV) nut

with cucumber mosaic virus and concluded, pea/stunt virus a strain

of cucumber mosaic virus as the PSV had same properties as of CJ.W.

Devergne and Cardin (1973) used the technique like double

diffusion Agar to study the antigenic properties of 11 isolates

of cucumber mosaic virus in France, Belguim, Italy and U.S.A.

They found 12 catogories of antibodies with a fairly high specific

10

corresponding to 12 possible epitopes of the intact capsid of the

virus, four serological types, To, R,S, and DTL were distinguished

each with highly specific epitopes and shov.'ed that all isolates

inducing ringspot lesions on Xanthi taoacco belongs to the group

To, R, S.

Sharma and Chohan (1973) reported the transmission of

cucumis virus 1 and 3 through seeds of cucurbits. In seed trans­

mission tests cucumis virus 1 (cucumber mosaic virus) was found

to be seed borne in vegetable marrow, ash gourd (Benincasa hispida)

and pumpkin and cucumis virus 3 (cucumis green mottle virus) in

bottle gourd (Laqenaria siceraria),

Shohara and Osaki (1974) purified cucumber mosaic virus by

repeated precipitation with 8% PEG and 0,Z*A Nacl followed PEG

solubilit gradient (0.6? PEG stabilized in 35-5^ sucrose) centri-

fugation. Virus yields were 500-800 mg/kg fresh tissue. Purified

preparations were highly infectious and contained uniform virus

particles. The sedimentation pattern showed a single peak,

indicating that no aggregation occurred.

Chung et £l. (1975) infected 145 plants (24, previously

unreported) from 45 families, with cucumber mosaic virus, but

negative results were obtained with 23 spp. reported to be

susceptible by previous workers.

Devergne and Cardin (1975) analysed the antibody composition

of 37 antisera prepared against 16 viruses isolates and found that

4 serological groups can be distinguished, CMV (To R,S), CW-DTL,

^^

TAL and PSV-V, each possessing specifiv antigenic patterns. The

TAV (Tomato aspermy virus) group, which included all aspermy

viruses is related to PSV-V and, much more remotely to CtAV (To,

RS).

Singh ^ . (1977) reported the significant reduction in

chlorophyll contents and increased respiratory loss in the leaves

of cucumber infected by cucumber mosaic virus.

Bhargava and Bhargava (1977) reported pumpkin yellow-vein

mosaic virus, pumpkin virus and cucumis virus 3 (cucumber green

mottle virus) from seven cultivated and 2 wild cucurbits from

Gorakhpur (Uttar Pradesh). They identified 3 str. of cucumber

mosaic virus, and 7 of watermelon mosaic virus and concluded their

close serological relationship.

Amemiya and Misawa (1977) studied the induction of resistance

of cucumber by cucumber mosaic virus. They shoved that virus multip­

lication in the cotyledens disks of susceptible and resistant

cultivars was similar during the initial stages of infection but

the virus contents of later fell after 36 h whereas that of the

former remained constant. Treatment with actinoraycin D,e^-amonitin

or heat increased virus multiplication in the resistant cultivars.

Loent'eva Kosheleva and Konovalova (1978) reported the

occurrence of cucumber mosaic virus in the field in Kuybyshev region

in Mid-August. Losses were reported to be highest in the late

crops protected against wind.

12

Lee et_ a_l. (1978) multiplied the cucumber mosaic virus

(GAV) in the tobacco cv. Ky-57 and purified it to give 24.25 mg/ml.

The epidemiological survey of viruses affecting cucurbits

crops in Massachusetls of Komm et aj. (1978) showed that 17%

infection was caused by cucumber mosaic virus.

Rahiman and Izadpanah (1978) identified the cucumber mosaic

virus on the basis of transmission, host range, electron micro­

scopy and serology. Twelve isolates of cucumoer mosaic virus from

cucurbits, zinnia, petunia and viola hybrids were divided into

4 groups, according to the type of symptoms produced and into

2 serological catogories.

Shanrayganathan (1980) in a survey of the viral diseases of

three islands reported the occurrance of cucumber mosaic virus on

cucumber and watermelon mosaic virus on pumpkin.

Omar £t £1. (1982) isolated 3 strains of cucumber mosaic

virus, from naturally infected cucumber, bean and squash plants.

Their studies revealed that cucumber mosaic virus need at least

48 hours to multiply in inoculated leaves, after which the/ could

oe detected in all leaves of the plant, showing the virus particles

more with assimilates. No virus was detected in uninoculated leaves

24 hours after inoculation. All components of cucumber and tomato

fruits possessed the virus particles but at maturity the virus

concentration decreased.

Kyriakopolou and Bern (1982) reported the occurrence of

cucumber mosaic virus on squash from Greece. They isolated the

virus and identified as CMV on the basis of host range, aphid

13

transmission, particle size and morphology, physical properties

and serology. The virus was widely distributed through out Greece

on many cultivated and wild plants. The plants showed severe

malformation and natural infection of Globe artichoke was noted.

Yamamoto and Ishii (1983) while studying aphid transmission

in cucumoer cultivars infected with watermelon mosaic virus and

cucumber mosaic virus reported that the virus in leaves inoculated

with either virus showed a highly positive correlation with the

rate of transmission of Aphis qossypii. It was noted that doubly

infected leaves showed greater severity as compared to singly

infected leaves with CMV, However, reverse was the case with W.N

i.e. the leaves infected only with WiMV showed higher severity as

compared to the leaves infected with both the viruses. These

results presented a elude to observe the difference between the

concentration of the two viruses in leaves infected with both

simultaneously.

Glasshouse trails of Erdiller and Ozyanar (1983) with

cucumber seedlings grown in perlite and inoculated with cucumber

mosaic virus (CMV) on cotyledons, revealed that on 1st leaf the

protein content was increased, but respiration, starch and sugars

were reduced, compared with the healthy ones.

In an investigation, made by Dikova (1983) of cucumber

cultivars to CMV, it was found that out of 89 cultivars inoculated

with race B and C, 44 cultivars with race C of virus in glass house,

13 were resistant.

14

Noga/ and Vorganci (1984) reported that of 269 cucurbit

samples with virus-like symptoms collected in 1979 and 1980, 142

contained cucumoer mosaic virus, 118 watermelon mosaic virus, 2

and 9, GMV + WivIV-2. Physical properties and particle-dimensions

of the viruses were determined.

A new structure designated CMV-K obtained from melon in

Ahvaz was characterized by Elahina and Habili (1984).

In the year 1985, Nogay and Yorganci investigated that

cucumoer mosaic virus and watermelon mosaic virus 2 were not

transmitted by seed in the cucumber, pumpkin, melon and water­

melon cultivars. The G W induced local and systemic infection on

cucumber, pumpkin and melon, without inducing local lesions on

inoculated leaves.

Atiri (1985) isolated a virus from fluted pumpkin (Telfairia

occidentalis) in Nigeria, which produced symptoms in some members

of so:lanaceae and Legurainosae and was transmitted by Aphis

spiracola. These properties distinguished it from Telfairia

mosaic virus, which neither caused symptoms in members of these

families, nor transmitted by insects. The virus in crude sap or

purifified preparations resulted with antiserum to G W but not

with antisera to several common viruses in Nigeria. Electron

microscopy examination revealed isometric particles of 29+1nm

diameter. These properties confirmed that the virus is an isolate

of CMV.

Bedlan (1985) studied the variation in cucumber mosaic virus

symptoms with temperature on cucumoer, melon, pepper (Capsicum ,Spa.

15

tomato, spinach, celery and lettuce. He pointed out that latent

infection and weed hosts e.g. Stellaria and Metha spp. pla/

important role in the aphid transmission of this non-persistant

virus.

Rosemeyer et a_l. (1986) isolated cucumber mosaic and four

other viruses from field grown buffalo gourd (Cucurbita foetidissima

near Tucson AZ. Both single and mixed viral infections were

associated with symptomatic plants grown in field. Viruses were

distinguished from one another by mechanical or insect transmission,

particle morphology, experimental host range and serology. Four

were mechanically transmissible (CMV, watermelon mosaic virus 1,

squash mosaic virus 2 and watermelon curly mottle virus) while the

5th, lettuce infectious yellows virus is exclusively whitefly-

transmissible. Although these plant viruses are known to infect

cultivated cucurbits, an investigation of naturally occuring

viruses of buffalo gourd in Arizona had not been undertaken.

Haack (1986) studied the ecology of cucumber mosaic virus.

He found that samples from several thousands cultivated and wild

plants in Aschersleben and Dresden, Yield 609 isolates of N group

of CMV. During the suamer the preportion of the U group of GMV

increases in certain crops to j> 50%. It is suggested that the

relative frequencies of the virus groups may be related to their

thermosensitivity. No structure appeared to be specially acapted

to particular plants or weeds nor was there evidence of particular

isolates among epidemics.

16

Garcia-arenal et £l. (1987) analysed the nucleotide sequence

of six satellite RN'As of cucumber mosaic virus differing in their

pathological properties. They concluded that primary sequence and

secondary str, alterations do not correlated with differences in

pathogenicity.

Gu £t_ a_l. (1987), produced and characterized the monoclonal

antibodies against QAV, belonging to the 1g M sub group, were

obtained by fusion of sp 20 myeloma cells with spleen cells with

spleen cells from BALB/C mice immunised with QA\f, They were

specific to QAV and cross-reacted with aW-Q, GW-? and CW-B,

but not with arlV-6 and TAV.

Katul and Makkouk (1987) detected Zucchini yellow fleek virus,

Zucchini yellow mosaic virus papaya ringspot (type H) virus, water­

melon mosaic II virus and cucumber mosaic virus in cucurbits from

Lebanon and Syria. More than one virus was found in 66.4% of the

samples. Detection by ELISA, using 0.1M phosphate + 0.1M EDTA

buffer at pH 7.4, was the most efficient for all the viruses.

Simultaneous incubation of the leaf sap and enzyme-conjugate reduced

the assay period by at least 4 h. Cross reactivity was detected

between PRS (type W) V antigen WM IIV and ZYMV antisera and between

ZYFV antigen and ZYMV antiserum.

Hseu et al. (1987) reported that in 1985, 583 leaf samples

were collected from cucurbits in Taichung, Yuchia-Nan and Kao-?ing,

Taiwan. These included cucumber, Luffa spp. bitter gourd, was gourd,

pumpkin and bottle gourd (Laqenaria leucantha). Direct ELISA was

used to detect zucchini yellow mosaic poty virus, watermelon

17

mosaic-I poty virus and cucumber green mottle mosaic tobacco

virus. ZYMV was rare in cucumber, Luffa spp., 3. hispida and

pumpkin. In 1986, 908 samples from the same species were tested

for the aoove viruses plus watermelon mosaic II poty virus and

cucumber mosaic cucumov.irus in an extended area covering Hwa-

Tung and Penghu islands, ZYMV was most prevalent, then WMIV

followed by GMV, but WM 11V was only predominent in L. siceraria,

L. lacantha W411V was only detected in Hwa-Tung samples and no

ZYMV was detected on Penghu Islands. ZYMV thought to be the most

i.Qportant virus in cucurbit cultivation in Taiwan.

Yang et al . (1987) reported that isolates causing small

necrotic lesions (SNL-Isolate) and large chlorotic lesions (LCL-

isolates) were oDtained from cucumber leaves showing vein-banding

symptoms. On the basis of host range, inclusions characters,aphids

transmission, serological relationships and particle morphology,

the SNL isolate was identified as cucumber mosaic virus and the CL

isolate as zucchini mosaic virus. Both the viruses were transmitted

by the cotton aphid in a non-persistant manner.

Yoshida and lizuka (1987) isolated cucumoer mosaic virus from

cucurbiteous plants like squash, bottle gourd (1,. siceraria) and

cucumber.

Erdiller and Ertung (1988) reported the occurrence of

cucumber mosaic virus on muskmelon in Ankra Province. Structures

5 and 6 of cucumber mosaic cucumovirus and watermelon mosaic poty

virus 1 and 2 were isolated from melon in 1981-84 and identified

by host range, serology, physical properties and ElA.

18

Owen and Palukaitis (1988) while characterizing the cucumber

mosaic virus, divided RNAs into two groups, on the basis of their

ability to hybridize cDNA of either Fny CMV RNA or YIL-QAV RNA from

its 13 strs. The extent of cross-hybridization within one of these

groups was analysed by an RNA protection assay. The patterns of

RNA fragments protected from digestion were specific for each C.W

str. and revealed the extent and location of heterogenicity among

the viruses as well as within the Fny-Cf4V natural population.

Nitta et al. (1988) determined the complete sequence of

CvIV RNA^ of str. Y and compared with the reported sequence of

RNA^ of structure Q.

Rizzo and Palukaits (1988) determined and compared the

nucleotide sequence of RNA- of the Fny-str. (sub-gp I) of C\V at

both the nucleic acid and proteins levels with the previously

determined corresponding sequence of RNA^ of the Q str. (sub gp. II)

of CMV. They found that Fny-OAV RNA II-2 consisted of 3050 nucleo­

tides and contained a single open reading frame (ORF) of 2571

nucleotides whereas Q-CMV RNA2 consisted of 3035 nucletide level,

there was 71% sequence homology while at the protein level 73%

sequence homology was noted between the two RNAs.

Maeda et £1,. (1988) produced monoclonal antibodies and

discussed their use in enzyme-linked immunosorbent assay (ELISA).

They reported that efficiency and sensitivity of virus detection

was superior with MAb conjugate than with PAb and also noted that

DAS ELISA with htJKb conjugate could efficiently detect heterologous

serotypes with higher ELISA values than with PAb.

19

Saric and Stefanac (1988) reported the indicence and

variation of G W in four vegetables (Squash, Capsicum annuum.

£. vulgaris and Lettuce) frona Crotia. The/ found that all isoletes

of QAW caused similar s/mptoms typical on cucumber. Datura stromo-

nium, Nicotiana qlutinosa. N. meqlosiphan white burley tabacco

and Chenopodium amaranticolar. Reaction of other test plants and

serological tests showed that there were at least 3 str. including

5 variants of GAV on the 4 vegetables host examined.

Romero £jt a_l. (1988) isolated double stranded RNA by using

a modification of Moris and Dodd's method, double stranded RNA

was ootained virtually free from contamination with other nucleic

acids. They concluded that this method is especially useful for

analysis of large number of mixed virus infections and studies of

cryptic viruses, satellite viruses and viroids. Other possible

applications would be separation of mixed infections and the use of

ds RNAs as probes in hybridization experiments.

Hayakawa ejt a_l. (1989) determined the complete nucleotide

sequence (3369 nt) of RNA I of cucumber mosaic virus strain 0 that

(GAV-O). A comparative study of the 3 viruses revealed/CfW-0 is

more homologous to CMV-Fny (Sub gp. II) than to C4V-Q (Suo-gp. I).

Hsu e_t aj.. (1989) while investigating host reaction, serology

and RNA pattern of C\W isolates found that out of 19 cucumoer

mosaic cucumovirus (GIV) isolates tested, 14 produced differential

reactions, but were serologically indistinguishaole in gel diffusion

tests and western blot analysis. Agrose gel electrophorosis revealed

that all isolates had similar MW for their FJ As 1-4. RMA 5 was

20

m found/15 isolates, but was undectable in isolates NT 9, 904, GLI

and 1003 propagated in tobacco at the second transfer.

In the same year, Haase £t a^. (19S9) developed two variants

of direct antibody sandwich ELISA; as ELISA with PAbs and . Mbs for

detection of cucumber mosaic cucumo virus and a IMb-HLISA with

2 tAAbs directed against different epitopes of CMV for specific

detection of N-serotype. Mixed ELISA was more sensitive than ELISA

with Polyclonal antisera in detecting C W in crude sap of infected

plants. The 1st step procedure took less tine and appeared to give

a lower detection thresh hold.

Lee e_t ad. (1990) detected plant viruses by protein A-enzyme

linked immunosorbent assay. They found that the effective concen­

tration of protein A was 0.1-3 Jlig/ml in coating buffer solution

and 0.001 tig/ml in enzyme conjugate buffer. Protein A (PA)

prevented specific reactions in different virus strains among

cucumber mosaic cucunovirus strain O, C and Y. Viruses in different are

gps. detected by this method/turnip mosaic, ppotyvirus, cucumber

mosaic cucumo virus etc,

Richter (1990) used blocking ELISA as a confirmatory test

in the detection of cucumber mosaic virus and found that this

technique is suitable for confirmatory identification of C'V.

3ansal et al . (1990) reported the occurrance of C W on

summar squash: Transmission tests, host range, physical properties

and serology, the pathogen causing this major disease in North

iVestern India was identified as cucumber mosaic cucuraovirus.

21

Shoji et. a_l. (1991) investigated the infectivity of 18

cucumber mosaic cucumovirus isolates from cucumber plants in

Japan, Severe s/mptoms suggested that the isolate could be used

in investigations of cross protection, confirmed by attenuated

virus.

Wahyuni (1991) characterized strains of C W from Australia

b^ their host range symptomatology. They were classified as sub

group I and II strains by a dot-blot molecular hybridization assay

oetween their total viral RNA and selected cDNA. A range of sero­

logical tests was used to compare these isolates. Both molecular

bybridization with total RNA and specific MAbs may be useful for

separating isolates of CMV into sub group I and II.

Stoimenova (1991) reviewed the structure and function of

cucumber mosaic cucumovirus.. (CMV) genome and satellite RNA/cDNA.

The possibility of protecting cuJtLvated plants using vaccine strains

of G W (with or without cRNA) or transgenic plants expressing R>tA

or capsid protein was discussed,

Matsuo et al. (1991) reported the occurrence of mosaic

disease of melon caused by Laqenaria strain of G/iV from Nagasaki

perfecture, ,

Daniels and Cambell (1992) characterized G^V isolates of

California, A total of 30 cucumber mosaic cucumovirus (C<\V)

isolates from California, U.S.A. were characterized biologically

(host reaction and thermosensitivity), Serologically (protein A

sandwich ELISA) and physically (viral capsid protein migration

in PAGE, peptide mapping and migration of viral ds RNA in PAGE.

22

All methods supported the classification of CMV isolates into 2 main

sub group. QMV-I and CW-II, Peanut stunt cucumovirus., tomato

aspermy cucumo virus and CMV were distinguished from each other

by PAS-ELISA, peptides mapping and host reaction.

Paras et £l. (1992) differentiated the biologically distinct

cucumber mosaic virus isolates oy PAGE of double-stranded RMA.

Ertunc (1992) reported that out of 40 cucurbitaceae seed

samples tested, cucumber mosaic cucumo virus (CfAV) was identified

by DAS-ELISA. Although non-precoated indirect ELISA gave higher

aoserbance values, both assays were suitable for detection of C'.V

in cucurbits.

Polak e_t al , (1992) isolated cucumber mosaic virus (C/iV) and

tooamovirus from water of an irrigation ditch in the vegetables

growing area north of Prague, The achieved results give the 1st

proof of the presence of C W in surface waters in Czechoslovakia

and pointed out possible danger of virus contamination by means of

irrigation water.

Chillies

Marrou et £1 . 1971 studied the rhythm of periodic variations

in concentration of cucumber mosaic virus and influence of various

factors on the course of infection. They found that concentration

of virus, estimated by symptoms on cowpea, showed periodic varia­

tions in infected leaves of tobacco, melon and pepper (C. annuum).

Maximum concentration in an 8 hr. cycle,Temp, and light had no

effect on the rhythm.

23

Herbas 1971 reported mosaic of pepper (C. frutescens var,

grossum) in Bolivia and some physical properties of the causal

agent. The symptoms of a serious virus disease of capsicum were

descrioed. Mechanical transmission was successful and the virus

is possibly a str. of cucumber mosaic virus.

Barrios £t a^. (1971) reported the inheritance of resistance

to Tobacco etch and cucumber mosaic viruses in Capsicum frutescens.

Several hundred capsicum introduction and the C. frutescens var.

LP'l was resistance followed a simple Mendalian Inheritance, TEV

resistance being dominent and CMV recessive.

Conti et £^. (1974) reported cucumber mosaic virus, nasturtium

ringspot virus, potato virus y and tobacco mosaic virus from

piemonte and Umoria. Cucumber mosaic virus alone caused mild mosaic

of leaves and ringspots on fruits.

Dubey and Joshi (1974) conducted transmission studied on

cnillies mosaic by its vector Aphis qossypii Glove, They ooserved

that the relation between the virus on Capsicum annuum and its

vector is non-persistent, and the higher incidence of the virus

may be due to the presence of this efficient vector.

Wongkgew and Sutabutra (1974) isolated cucumber mosaic virus,

the causal agent of pepper mosaic in Thialand from the virus

complex.

Bock et £l. (1975) reported the occurrance of cucumber mosaic

virus 9 in Eastern Africa. The str. of C.W were isolated from

capsicu3i and Notonia abyssinica in the field in kenya and identified

24

on the oasis of particles morphology, serology, host range and

reaction. 3oth the strg.were apparently closely related to the

str. of OViV occur in field in U.K.

The occurrence of cucumber mosaic along with other viruses

was reported from 4 major capsicum growing districts of Srilanka

by Sugiura and Sandaranayake (1975).

Awasthi and Singh (1976) observed that GMV infection caused

a decrease in the ascorbic acid and capsicum content of fresh

capsicum fruits samples; this was more pronounced in the susceptible

var. then in the toirentjOne, ^ e reduction of ascorbic acid content

might be associated with the increase in ascorbic acid oxidation

activity and thus pungency may be correlated with resistance.

Shukla and Ram (1978) reported the natural occurrence of

cucumber mosaic virus from Rajasthan. They collected eleven

isolates from capsicum growing in Udaipur and Mathania, cucumber

mosaic virus was detected in both C. frutescens and C. annuum.

Histological and histochemical changes in the authers of

Capsicum annuum inoculated with C W were reported by Chouhan j_t al.

0L981).

In the same year Chouhan and Srivastava reported that the

extent of pollen sterility, dehiscent and non-dehiscent anthers

and tapetal behaviour in plants (Capsicum annuum L.)-,inoculated

with GMV at different growth stages.

Horvath (19B6) reviewed the literature, and reported that

at present 13 spp. and 9 varieties of capsicum are known to be

25

susceptible to some 43 viruses of C 18 viruses occurring

sponteneously are tobacco mosaic, tomato mosaic, cucumber

mosaic, potato virus y and alfalfa mosaic are most important

in Hungary. Apparantl/ 15 capsicum spp. and varieties are

resistant to 45 viruses.

Salamon and Kobler (1986) identified the viruses infecting

capsicum annuum by ELISA. The most important were cucumber mosaic

and alfalfa mosaic viruses.

Khalil (1987) used ELISA technique as a rapid and quantative

detection method for cucumber mosaic virus in pepper.

Pares and Gunn (1989) studied the role of non-vectored

soil transmission (NVST) as a primary source of infection by

pipper mild mottle tobamovirus and cucumber mosaic virus in glass

house-grown capsicum for the 1st time in New South Wales.

Wei and Fang (1989) identified a pepper isolate of cucumber

mosaic cucumovirus from capsicum plants with yellowing and fern

leaves was transmitted by aphids and induced systemic mosaic

on Datura stromonum and N. qlutinosa and red and brown narotic

spots on Vicia faba.

Gowda and Reddy (1989) reported that cucumber mosaic

cucumovirus was transmitted by Myzus persicae (50^). Aphis

qossypii {40%) and A. cracivora (20%) to chillies.

Abdel Salam e_t £1. (1989) purified a severe isolate of

GViV from nurseries growing pepper from Egypt. Extraction was 0.3V.

phosphate buffer, pH 7.5, containing 1m M EDTA and ^'i Thioglycolic

acid. Chloroform was used in clarification, followed by 1 cycle of

26

differential centrifugation, and acid precipitation, at the

virus isoelectric point, then 1 cycle of differential centri-

fugation and acid precipitation centrifugation. Purified virus

was highly infectious to Chenopodium amaranticolar.

Subasic et al^. (1990) reported the occurrence of QAV on

pepper (Capsicum annuum L.) from Bosnia and Herzegovina. Based

on host range and sap tests, the virus isolated from C. annuum

plants in Yugoslavia was identified as cucumber mosaic cucumber

virus. Double radial immunodiffusion showed its affinity with

unstable GMV isolates.

Singh and Shukla (1990) described the properties of a virus

causing necrosis and mosaic in capsicum around Kanpur, Uttar

Pradesh, India. The virus was transmitted mechanically by seeds

and by 2 aphids. Aphis qossypii and Myzus persicae and could

infect 44 plant species, inducing a range of symptoms. EM of the

Purified virus showed spherical particles of C. 30 nm diameter

and serological tests produced a positive reaction to CVIV antiserum.

It was concluded that virus is a new strain of CM cucumovirus.

Kim et £l. (1990) reported the incidence of virus diseases

of Capsicum annuum in the Euiseng area, Korea, averaged 5.3% in

30-d-old seedling and 21.2% in 50-d-old seedling in the green

house and ranged from 23.3 to 83.3'^ 20 d after transplanting in

the field. The main symptoms included mosaic, mild mosaic, mild

necrosis and rugose, varying according to the season. The major

virus was TMV with an incidence of 89%. Cucumber mosaic cucumo­

virus (12.5%) and undetermined virus 26.9' also occuring single

or in mixed infections.

27

Kim and Lee (1991) reported virus disease incidence and

symptom expression on pepper cultivated in Vinyl-house and

serological relationship of Ci'JIV isolates from pepper. Viruses

were isolated from sample material and identified by EM and by

reaction in 11 indicator host plants. Tobacco mosaic virus was

isolated from 75; of plants from Namgie and CMV from 12.5% plants.

Both viruses were isolated from 12.5^ of samples.

Nono-womdium et al. (1993) studied the multiplication of

CMV in susceptible and resistant Capsicum annuum lines. The rate

of CMV multiplication as estimated by DAS-ELISA was lower in

Perennial (the tolerent line) then in vania or yolo wonder

(resistant and susceptible CVS. respectively). The specific

infectivity of the virus extracted from perennial was less then

that from Vania or Yolo wonder, suggesting that perennial is

resistant to CMV multiplication, while restriction of the virus

in inoculated leaves of vania is not due to the inhibition of the

virus replication. However, PAGE revealed that the RNA profiles

of CiA\f purified from the 3 pepper lines were similar.

Brinjal

Sharma (1969) made a comparative study of five sap-trans-^

missible viruses isolated from mosaic infected Brinjal (Solanum

melonqena L.). The five viruses isolated were identified by

difference in physical characteristics, transmission, host range

and cross protection. Isolate LL. (egg plant mild mosaic) is a

distinct str. of tobacco mosaic virus and isolate 4 (common egg

plant mosaic) a str. of cucumber mosaic virus (CW), isolates

28

SS (egg plant severe mosaic), 6 (egg plant ring mosaic) and 17

(egg plant crinkle mosaic) differ from one another and are not

related to TiW and concluded as new records for this host.

Spontaneous occurrence of cucumber mosaic virus on

Solanum melonqena and on some other hosts in Yogoslavia has been

reported by Mamula £t_ al . (1977). The isolates were similar in

symptoms on test plants to Bhargav5?s spinach str. of C?»1V.

Potato

Occurrence of cucumber mosaic virus on wild spp. of potato

was reported by Oertel et al_. (1978) Alafalfa mosaic, tabacco

mosaic and tobacco rettle viruses were also found on this spp.

Although these viruses (including QAV) are relatively infrequant

on cultivated hybridization with wild spp. may be dangerous.

Occurrence of cucumber mosaic virus on potato in India was

reported by Sangar and Agarwal (1986). A str. of QvlV causing

chlorosis and blistering on potato plants at the Central Research

Institute, Kufri was identified from transmission host range

serology and properties in sap.

Cohen et al . (1988) reported that GMV infects sweet potato

severely in Israel, causing stunting, chlorosis and yellowing of

plants was identified by host range, serology and ElA. All G W

infected field plants carried sweet potato feathery mottle virus

(SPFMV) transmission of CMV by mechanical grafting and aphid

inoculation to healthy sweet potato plants failed, but it was

achieved easily if the accepter plants carried SPFMV. Appearantly

29

a required factor for CMV replication in sweet potato is provided

by the presence of SPFMV. This seems to be the 1st report that

G.W has a helper virus requirement.

Richter £t a_l. (1989) while using various ELISA tests,

identified a previously described virus structure in the Estonian

SSR as cucumber mosaic cvixus. -. .,

Radish

Kono and Sakai (1974) observed the infection of turnip

mosaic virus alone {75%) or in combination with cucumber mosaic

virus in May-June and Sept.-Nov. Inoculation tests with aphid vector

showed that with aphid vector infection occurred readily at av.

temps. 10°C. Symptoms were masked in winter but not always in

summer.

Sano and Kojima (1989) reported that dual infection by

turnip mosaic Potyvrirus (TuTW) and cucumber mosaic cucumov^ifus,

(CIV) causes a severe mosaic disease in Japanese radish plants.

TuW caused mild mosaic symptoms when present alone, but GMV did

not. Plants inoculated with both viruses displayed more severe

mosaic symptoms then plants inoculated only with'TuMV. Thus

suggesting that systemic transport and spread of G W in radish

plants were enhanced in the presence of TuMV.

Ishiraoto _e_t al.. (1990) also reported that the concentration

of OA cucumovirus was higher in doubly infected plants (G.lV+Tui'W)

then alone,Observations suggested that this increase was mainly

due to an increase in the number of GMV-infected cells co-infection

30

with turnip mosaic potyvirus had little effect on cell to cell

transport of GMV, but enhanced long distance transport of the

virus.

Spinach

Joseph P. Fulton (1950) reported eight virus diseases from

field grown spinach (S.. oleracea L.) from United States. These

were cucumber mosaic (cucumber virus I) aster yellows, sugar-

beet curly top, beet mosaic spotted wilt, spinach yellow dwarf,

western cucumber mosaic and celery calico. Cucumber virus 1

originally called spinach blight on this host, has been considered

the predominent virus disease of spinach.

Eenink (1974) reported that in spinach vars. and crosses

tested, there appeared to be a weak linkage in the repulsion

phase between the gene for resistance to P. spinaciae and the

one for tolerance of cucumber virus I (GViV).

Bednareic and Slusarek (1976) reported occurrence of C'V

in spinach and found that the contents of oxalates was lower

in spinach infected Dy the virus than in healthy plants. In cv.

koda the content of insoluble oxalates increased considerably

with age infected plants, this being particularly marked 28 days

after inoculation.

Stefanac (1978) while investigating some viruses

on spinach cv. Matador near zegreb, in Crotia isolated cucuniber

mosaic virus, turnip mosaic virus and tomato bushy stunt virus.

Each was re-transmitted to spinach, causing severe infection.

31

Chod (1983) reported susceptibility of some spinach

cultivars and hybrids to cucumoer mosaic virus and oeet mosaic

virus, plants of 11 varieties and 1 new selection were sap-

inoculated with mv and CMV, while 3YV was transmitted by

M. persicae. In most cultivars except caramble total sacchirides

were substantially higher on inoculation with CA1V (Chod and Chodova,

1985).

Wilson and Halliwell (1985) characterized the C'.V isolate

from spinach in winter Garden area of Taxas. The isolate was

very closely related serologically higher on inoculation with

GV.V (Chod and Chodova 1985).

Wilson and Halliwell (1985) characterized the CW isolate

from spinach in winter Garden area of Taxas. The isolate was

very closely related serologically to str., of CMV and is designated

the Texas spinach isolate of G/iV-6. The virus infected 39 s?p. of

13 families tested. M. persicae efficiently transmitted the virus

experimentally. The isolate had a sedimentation co-efficient of

91.8+0.1 S as determined by analytical Ultracentrifugation analysis.

Virus with a mean diameter of 28.9+0.3 nm were found in purified

preparations with electron microscopy. A single protein unit (mean

mol. wt. 25300+2550 daltons), and four separate RNA species were

found. The mean mol. wt. distribution of the yiral genome was

1.22, 1.09, 0.77 and 0.36 x 10 daltons. The isolate reduced

yield of 3 spinach cultivars by 23.8-47.4;) .

Tomato

Mintc (1969) reported the serological relationships a-ong

CMV, tomato aspermy type viruses and peanut stunt virus. He

32

determined .. that antisera against an eastern str. of groundnut

stunt virus (GSV), reacted with a low mol. wt. antigen associated

with the cowpea str. of CMV. Some CMV antisera reacted with one

or both GSV strs. (Eastern and V/estern) although cross reactions

in gel diffusion plates suggested non-identity between the viruses,

G W and GSV-W both showed a distinct serological relationship with

tomato aspermy virus.

In 1971, Tomaru and Udagowa, reported that phenol extracts

from leaf tissues systemically infected with 6 C.V strs. and 2 TAV

isolates, showed higher infectivity than that of control phosphate

buffer-homogenata, regardless of the assay host. This increased

infectivity was used as a criterion for CAW diagnosis, and the

similar property in TAV and chrysenthemum mild mottle virus was

regarded as further evidence of the close relationship between

tomato aspermy virus and cucumber mosaic virus.

Zimmerman let al . (1974) reported for the 1st time viral

diseases of tomato in the Jericho area. They isolated tomato

yellow leaf curl, alfalfa mosaic,cucumber mosaic,tobacco mosaic

and potato y viruses infecting tomatoes,

Putz et a^, (1974) reported variation in pathogenicity of

isolates of cucumber mosaic virus associated with tomato necrosis.

A comparative study of isolates of G W from necrotic plants showed

variations in symptoms expression, including latency, true fern

leaf and lethal necrosis, on inoculated plants. The isolates were

attenuated after passage through cucumber. It was suggested that

a mixture of a new CMV str. with common strs. may be responsible

for the recent development of the disease in Alsace.

33

In the same year, Habili and Francki compared physical and

chemical properties of tomato aspermy and cucumber mosaic virus I.

They showed that the size, morphology, sedimentation rate, RNA

base ratio and buoyant density of the TAV str. V and GMV str. Q

are indistinguishable. The amino acid compositions of protein from

the 2 viruses, although similar, were distinguishable and the

calculated mol. wts. of proteins sub units were 26100 and 26300

daltons for TAV and QAV respectively. The two viruses were sero­

logically distinct and the data presented, suggests that in

preparations of both viruses, 3 distinct particles are present with

identical protein shells but different RNA cores.

Beczner and Horvath (1974) while studying the etiology of

fern leaf diseases of tomato, reported that appearance of fern

leaf symptoms depend on the presence of virus complexes (tobacco

mosaic and cucumoer mosaic viruses) or CMV that could produce the

symptoms in single infections. It was observed that fern leaf

symptoms were produced with CW-V1 + aW-T, TMV-U, TMV-T + aW-T

and G.W-T. The other single and complex inoculations caused only

4&rK green mild mosaic symptoms and leaf distortion.

Effect on disease development of inoculum concentration

and seedling age in the assessment of CMV resistance in tomatoes

was reported by Kuniyasu et a_l. (1975). They observed that of

11 tomato CVS. resistant to tomato mosaic (str. of tooacco mosaic)

virus tested, iMR 9, MR 12, Ohio 690712 and ^ 253 had the fewest

symptoms following inoculation with GMV.

Mossop £t £l_. (1976) described a str. (M) of C W inducing

brilliant yellow mosaic symptoms in Nicotiana spp. purified by

34

a new method. The physical and chemical properties of MCMV were

typical of a cucumovirus and an antigenic relationship with str.

of DAV, was demonstrated.

'Kaper and Waterworth (1977) reported RNAs of cucumber mosaic

virus as causal agent of tomato necrosis, was probably the same

as that which in 1972 destroyed most of the field tomato crop in

large regions of the French Alsace.

Ignash (1977) investigated biological and physical proper­

ties of CivIV str. 1, isolated from cucumber and tulip, str. 2 from

tomato and str. 3 of tomato aspermy virus. Apart from some common

features, the 3 isolates, 1 and 2 showed similar symptoms and

serology.

Villemson and Agur (1979) while comparing Host range,

morphological, physical and chemical features of tomato aspermy,

potato N and CA viruses observed that all are related, PVN being

nearer to G W than TAV.

Tobias and Andrasfalvy (1984) in an investigation of

Necrotic and other virus diseases of tomatoes, using ELISA and

indicator plants detected LM, GM and potato viruses from Hungary.

loannou (1985) isolated five mechanically transmissible

viruses, identified on bases of symptamatology, indicator host

reactions and serology were yellow leaf curl, tobacco mosaic

virus, potato viruses x and y and GW. These viruses especially

TMV were commonly associated with mosaic and occasionally with

other leaf or fruit disorders.

35

Feng and Cai (1987) reported that in the preparatory-

stages of a breeding programme for resistance, TMV, GMV and to

a lesser extent, potato virus x and potato virus y were identified

as the main agents of viral diseases of tomato in China.

Xuan £jt a^. (1987) isolated two structures (ordinary and

yellow) of cucumber mosaic cucumovirus (C?AV) from tomatoes in

the phillipines were distinguished by the symptoms induced on

Vinca rosea. Cucurbita maxima, cucumber, Nicotiana qlutinosa and

Datura metal. Both were mechanically as well as aphid transmissible

(in non-persistant manner). Aphis qossypii and Myzus persicae. Both

—4 had a DEP of 10 , However differed in longivity, 2 and 3 days m

yellow and ordinary strs. respectively. The thermal inactivation

point was 60 and 75 C. The yellow str. was serologically related

to C/iV-Y and ordinary str. to aAV-0.

The occurrence of cucumber mosaic virus in tomatoes was

reported by Lima e;t £1. (1963) from Brazil.

Goto et a^. (1988) and Kosaka et al.. (1989) isolated of

cucumber mosaic viruses causing necrosisytomatoes from Oshima

perf«cture of Hokkaido in 1982, and from Kyoto, Japan in 1984

respectively. Both found that severe necrotic disease of tomato

was caused by CIA cucumovixas^. In the former case, the symptom

appearrance depended upon the time of inoculation, at the young

seedling stage symptom appeared within 12-13 days whereas at the

flower and cluster stage no symptom developed. In case of Kyoto

isolate of 14 commercial tomato cultivars all produced necrotic

symptoms and died 7-10 d after inoculation.

35

From the host range symptomatology and serology the virus

(kosakaet al. . 1988) containing RNAs was found to belong to a

peper str. of CMV. While Hokkaido Strain, be a new SATR to

Japan.

Occurrence of CMV in tomatoes, causing necrosis of fruits

was reported by Benetti (1989) from Italy. The virus was trans­

mitted by aphids.

Badr (1989) reported prevalenceof GAV and T?.iV in tomatoes

grown in commercial green house and field. This was the 1st

report of natural infection of tomatoes by these viruses in

Algeria. The TMV was more common in green house tomatoes, whereas

CMV infected more field grown plants.

Zheng et aj . (1990) conducted studies on strain types of

tomato in the Tianjin Suburbs (China), It was explicit that TMV

and G W are the main virus diseases of tomato in China.

Giri and Mishra (1991) reported from India that loses in

number and weight of tomato fruits from inoculated Pusa Ruby

seedlings were 34 and 59.8,^ respectively, due to tobacco mosaic

tooamovlruso (TMV) and 40.8 and 49.^ respectively, due to cucumber

mosaic cucumovirus-' {OAV) infection. The percentage of non-viable

pollen was 12.2 and 5.95^ in tomato plants affected by CMV and

TMV respectively, compared with 7.23% in healthy plants.

Kvriakopoulou et_ al_. (1991) reported that tomato fruit

toughness, two new diseases in Greece probably related to CMV.

Jorda et^ a_l. (1992) while studying the epidemics of CAV

plus satallite RNA in tomato in eastern spain found that CMV,

37

accompanied by a satallite RNA of necrogenic phenotype was the

causative agent of tomato necrosisi A sub 6pidernial necrosis of

the fruits which also occured at epidemic levels was induced by

isolates of CMV without satRNA.

The nucleotide sequence of a cucumber mosaic cucuraovixus

satellite RNA (Tfn-CARNA 5), isolated from tomato plants in

Southern Italy was given by Crescenzi et al.. (1992).

Liu ejt a_l. (1992) conducted studies on strains of GViV on

tomato and reported that during 1986-1989, 111 isolates of

cucumber mosaic virus were obtained from tomato fields in Shandong

strains of G W which were identified as tomato fern leaf strain

(aiV-ToF), tomato mosaic strain (Q^V-Tc^O and Tomato latent (light

mosaic) strain (GW-ToL) due to the difference not only in oio-

characteristics, but also in electrophoretic mobility and aorpho-

logy or purified virus particle, mol. wt. of capsid polypeptide

components of viral RNA and in serological relationships.

Pilotti and Barba (1992) reported that CMV and potato virus

play important role in morphological changes in tomato. The

symptoms observed in the field were reproduced on tomato cultivars

Rezano, TM VF, San Marzano and Hypeal 244, inoculated with cucumber

mosaic cucumd^ixus^ and potato y potyvirus. singly and in combina­

tion. They concluded that in mixed infections, G W infection is

predomenent, while PYV intensifies the symptoms produced by GVW

on tomato plants.

Moriones et a .. (1992) reported the differential interactions

among strains of tomato aspermy virus and satellite R JA of cucumber

mosaic virus.

38

Gusui ejt a_l.. (1993) reported the tomato necrosis and 359

nucleotide y satellite of G W , and factor affecting satellite

biological expression. The results showed that in a standardized

tomato bioassay at 24°C, the /-satellite, when supported by either

QylV-l or CMV-y, did not induce: tomato necrosis in the Rutgers

variety by elicited a slower necrotic response in the best of all

varieties that was variable lethal, as compared to the faster

inheritably lethal response induced by a prototype necrogenic D

satellite variant in both tomato varieties. At higher temperature

(26.5 to 32°C) an extremely fast-killing necrosis caused by CMV-y,

itself was observed.

Turnip

Mohamed and Quacquarelli (1976) while studying virus

diseases of market garden, isolated cucumber mosaic virus from

turnip plants showing syndrome.

Mixed infection of turnip mosaic and cucumber mosaic viruses

on turnip (Brassica rapa) var. §ilvestris Perko PV^ (Turnip) was

reported in 2 localities in Czechoslovakia by Novak et a^. (1984)

plant growth was retarded, young leaves showed vein clearing and

chlorosis. Old leaf veins were dark green or lemon coloured also

with interveinal chlorosis serological tests and reactions in

indicator confirmed mixed infection of TMV and CtAV.

Fujisawa (1985) reported that cucumber mosaic virus 2 could

be transmitted by Aphids M. persicae and Aphis qossypii with its

interaction with turnip mosaic virus. However, Aphis qossypii

transmitted G W acre effectively in alone infections.

39

Davino and Areddia (1987) reported an isolate of G'.W on

wild crucifer (Brassica fruticolosa) from Silicity. The virus

isolated from Brassica fruticolosa showing leaf discolouration,

mosaic and stunting was identified as OAV on the basis of host

range, serology and physico-chemical properties.

LITERATURE ON CONTROL OF CMV

Unlike mycologists and bacteriotogists, virologists have

no array of chemicals with which to attack and control virus

diseases. However, considerable efforts and time have been spent

to find out chemicals that will directly eliminate or restrict

the virus multiplication in crop plants. Cucumber mosaic virus,

in last few decades has been restricted rather controlled by the

following measures.

Sharma and Cohan (1973) reported that cucumber mosaic virus

was completely inhibited by leaf and seed extracts and phenolics

from the leaves and seeds of Syzyqium cummini as indicated by

the lack of local lesions on Chenopodium amaranticolar. The

virus was inhioited 77 and 63?o by extracts from the seeds of

mango and Callistemon citrinus, respectively cucumber mosaic

virus was also inhibited by floral glucinol and gallic, salicylic

and tannic acids.

Parshin (1974) reported that in Uzbekistan (cucumoer mosaic)

virus attack, cucumber (9.5-4.5?^, tomato (8.7-44?^), egg plant

(8.5-21; ) and red pepper (Capsicum spp.) (18.8-44.2;^. It was

also isolated from lucerne, carrot, lettuce, melon and pumpkin.

40

Control measures should include treatment of tomato seeds with

20,0 hydrochloric acid for 30 min and of seedlings 15 days after

transplanting with 0.1% boric acid, destruction of aphid vectors

and infected plants and isolation of crops.

Schmelzer and Wolf (1975), reported old and new methods of

controlling cucumber mosaic virus and green mottle mosaic viruses

on cucumber and tooacco mosaic, tomato aspermy and cucumber mosaic

viruses on tomato. For both crops in the glass house seed dis­

infection by heat treatment is of special importance. Cross

immunization of tomatoes with str. against normal strs. of tobacco

mosaic virus is also successful.

Almaniyazov (1975) reported the spread and control of virus

(G\W1 etc.) diseases of cucurbits from Kzylorda district.

Dubey and Joshi (1976) studied the effect of Pyriraidine

analogues and 2, 4-D. Both structures tested were inhibited by

3-azugucinine^ , 5-nitrouracil and 2-thiouracil. 2, 4-D was

effective against the severe str. and more so against the mild

str.

Devaux (1977) reported that low temperature in June-July

led to increased incidence. Transmission was mainly by insect

vectors. Seed transmission seemed not to occur. Good control was

obtained by growing cucumber on plastic film or oetween maize

wind breaks, as long as very susceptible cvs.were excluded. Oil

sprays were not very efficient. Most of the 42 table cultivars

were susceptible, while most of the 40 pickling cvs.were

resistant.

41

Leont'eva jet £l.. (1978) reported that in the tcuybyshev

region the virus normally attacks field cucumber in mid-August.

Seed treatment and 2 sprayings with 0.2% saiphos reduced the

number of aphids and infection (from 65 to 46%). Early planting

of seedlings is also recommended.

Yasui and Yamakawa (1978) conducted studies on resistance

of CMV and tomato mosaic virus in tomato I. The relationship

between the doze of the virus and the proportion of plants

remaining healthy after inoculation was examined for tomato fcvs.

and breeding resistant lines, using a basic model, described,

All evs»except I were resistant.

Deol and Rataul (1984) carried out the studies in Punjab

India, in 1976 to determine the effect of various row spacing on

the yield of chilli (C. annuum) and the incidence of the aphid-

transmitted cucumber mosaic virus in the crop. The row spacing

tested were 40x45, 50x45, 60x45 and 80x45. In the spring crop,

there was no difference in disease incidence between the various

row-spacing, but in the main-season crop plots with close row

spacing (40, 50 and 60 cm) had a much lower incidence of disease

than the wider row-spaced plots (70x80 cm), in both seasons the

plots with the closer row spacing gave significantly higher yield

then the wider row spaced plots. The higher yields were thought

to be due to the higher number of plants per plots as compared

with the wider spaced ones.

Lei £t a_l. (1984) reported that the mixture of fatty acids

present in the seed oil of Brassica compestris var. oleifera was

almost as inhibitary to TMV in tomato plants as pure docosyl acid

42

alone. The preparation NS-83 was processed from the seed oil and

was more effective and cheaper than any other compound used in in

the practical control the virus diseases in tomato. Some evidence

was obtained that tolerance of both TMV and CMV infections could

be induced in tomato by WS-83.

Eisbein and Haack (1985) reported the changes in the

resistance behaviour of spinach towards a strain of cucumber

mosaic virus under the influence of higher temperature. He found

that at 30°C temperature the infection was reduced considerably

(20%). In spinach vars. Metador and A-498.

Following crosses and backcrosses between the G W tolerant

Capsicum annuum accession MRCH, which has fruit weighing 10.04 g

and susceptible Bell-pepper cultivars with fruits weighing 150 g

selection for CMV resistance gave resistant BC^F^ with a maximum

fruit weight of 5.58g and a mean fruit weight of 4.03 g (Shifriss

and Cohen, 1986). It was suggested that the many genes for small

fruits are linked with those for resistance.

Nasser and Basky (1987) reported the inhibition of aphid

transmission of plant viruses by light summer oils in seed

cucumber field from Hungary. The effects of 3 mineral oil sprays

on the spread of CMV and watermelon mosaic virus in seed cucumber

were investigated. Sprays were applied weekly as 15! emulsions

from 19 June to 20 July. The cucumber mosaic and watermelon

mosaic viruses were isolated from untreated plots in the ratio

11.89?^, but watermelon mosaic virus was isolated from treated

plots hense suggesting^the G W is inhibited by these sprays.

43

Peshney and Moghe (1987) reported that out of 14 fungicides

tested, pre and post inoculation sprays of Agrimycin, Aureofungin,

fosetyl-AL, thiabendazole, triforine and chlorothalonil at 0.1%^

suppressed multiplication of TMV and OM isolates from capsicum.

The fungicides were more effective on Capsicum annuum Cv. Jwala,

than on Nicotiana tabacum cv. Burley-21 and more effective when

applied before inoculation than at the same time.

Tien £t £^. (1987) reported that satellite RNA could control

the plant diseases caused by QW. Results from 14 localities in

China indicated that protecting strs. (551 and 552) decreased the

disease index by 21.6-82.8^ and increased fruit yields by 10.S-

55. S%,

Pink (1987) reported the genetic control of resistance to

cucumber mosaic virus in Cucurbita pepo.

Masuta £t _al. (1988) reported that satellite RNA (Y str.)

SR-Y, induced a lethal necrosis on tomato in combination with

CVIV str. y but not with Q W str. However, SR-Y induced symptoms

attenuated on several plants infected by C W str. 0. It was

concluded that bright yellow symptoms on tobacco and capsicum

and the intensification of CMV str. Y on tomato could be over­

come, application of satellite RNA may be suitable for field

control of some CMV strs,

Kroll (1988) carried out the comparative studies of the

effect of alkane monosulfonate (emulsifier E 30) and saponin on

virus infected plants. Reduction in the virus concentration

depended more on the virus than on the host, great reductions

44

usually occuring with EXV, However, CMV also affected by the

activity of these chemicals.

Reiss et_ aj.. (1988) reported the application of bacterial

culture filtrate from an unpigmented isolate of jE. herbicola to

cucumber and tobacco plants 12 h before inoculation inhibited the

development of symptoms due to CMV and TMV, respectively and

resulted in better growth than in untreated inoculated plants.

Tien (1990) reported that satellite RNA (sat-RNA) is

considered as a molecular parasite of viruses. There are

possibilities of using sat-RNA for control of virus diseases.

One way is by adding sat-RNA to virus genomic RNAs to

constract a control agent. Two such agents, 551 and 552, were

obtained for the control of cucumber mosaic cucumovirus (CMV).

Both were found to protect capsicum pepper, tomato and tobacco

against virulent OW. Eight years of results on about 8000

hectares indicated that the agent decreased the disease index

by about 50^ and increased yield by about 30^.

Another way is transformation of plants by sat-RNA. cDNA

A full length cDNA of OK\f sat-RNA-I was synthesized and cloned.

The plasmid with cDNA monomer was introduced into A. tumefaciens

containing T-,-plasmid by triparantal mating. The tire of C W

in transgenic plants was only 8-23, is compared to non-transgenic

ones. Progeny of transgenic plants resisted, wild C/4V infections

in the field.

Smith e;t a ,. (1993) reported that multiplication and

movement of tomato virus and cucumber mosaic virus is restricted

by somaclones (resistant tomato somaclone).

45

Partial resistance of bellpeper to cucumber mosaic virus

movement within plants and its efficiency in Southern France was

determined by Nono-Womdium (1993). During the three test years,

CMV spread was delayed 4 to 6 weeks in var. Milard and in var.

Vania as compared to var. Yolo Wonder. Final CMV incidence and

the apparent infection rate, the latter calculated from vander

plank logistic analyses were lower in var. Milord and in var.

Vania then in var. Yolo wonder. The resistance of var. Milard

and var, Vania expressed as a delay of spread and a reduced

GMV incidence over the past three years, provides practical

disease control.

PLAN OF WORK AND METHODS

PLAN OF WORK AND METHODS

1. Maintenance of virus inoculum :

1.1 Raising of test plants :

All the plants will be grown in x;lay pots of 4 and 6"

diameter, filled with a mixture of soil, sand and compost in a

ratio of 2:1:1. The soil mixture will be sterilized by autoclaving

for one hour at a pressure of 20 Lb per square inch. The clay pots

will be sterilized by rinsing 4per cent formalin solution and

prepared by filling with sterilized soil mixture autoclaved 24 hrs.

earlier and seived before use.

Cucumber (Cucumis sativus L.) plants and other plants

belonging to families cucurbitaceae and leguminosae will be raised

singly by direct sowing in clay pots. However, formalin rinsed

wooden trays (IS^xIS^xS") containing sterilized soil mixture will

be used in raising seedlings of various other plants. Young seedlings

of uniform size will be transplanted singly to 6" and 4" pots

containing the autoclaved soil, sand and compost mixture.

For inoculations the plants will be used two weeks after

transplantation. The plants belonging to family cucurbitaceae will

be inoculated at 2 leaf stage (seedling). All the plants will be

raised and kept in an insect proof glass house (20-30°C, normal

day length) and given a uniform care with respect to fertilizer,

water and other requirements.

1.2 Virus culture :

The virus culture will be obtained from naturally infected

plants showing symptoms of virus infection and maintained on

47

suitable propagation host b/ mechanical (sap) inoculations.

Attempts of single lesion inoculations will be made to maintain

a pure virus culture. Virus/es not transmitted mechanically will

be knowingly omitted and only mechanically transmitted virus/es

will be taken for investigations. Once the culture of the virus/es

has been maintained on suitable propagation host, it will be kept

in active state by fresh inoculations at regular intervals on

young propagation hosts.

1.3 Source of inoculum :

Young leaves from infected propagation host plants will be

used as source of inoculum. Inoculum will be prepared by macerating

them in a mortar with pestle in 0.1M phosphate buffer pH 7.0. For

each gram of leaf material 1 ml of buffer will be used and the

macerate will be filtered through two layered cheese cloth. The

sap thus obtained will be used as standard inoculum.

2. Transmission ;

2.1 Mechanical :

The fully expanded leaves of the plants to be inoculated will

be dusted uniformly with carborandum 500 mesh as an abrasive and

the standard inoculum will be applied gently but firmly on the

upper surface of leaves with the help of forefinger by keeping the

other hand beneath the leaf to be inoculated. The inoculated leaves

will be rinsed with gentle stream of water before the inoculum on

the surface of leaves dries up. If the rate of transmission is not

promising, some chemicals will be mixed with the inoculum so as

48

to enhance the rate of transmission. Additive in the inoculum will

include sodium sulfite, 2-mercapto-ethanol, ethylene diamine-

tetracatic acid, sodium diethyl-dithio carbomate and thio-glycolic

acid either alone or in various possible combinations if needed.

2.2 Biological :

Attempts will be made to find out the vector/s of virus in

the field, transmission by insects, soil, dodder (Cuscuta spp.),

seeds, graft nematode and pollen will be studied.

2.2.1 Insect transmission :

2.2.1(a) Transmission by aphids :

Adult aphids found transmitting the disease during preliminary

investigations will be used to study aphid-virus relationship (non-

persistant, semi-persistant or persistant).

2.2.1(b) Raising of virus free aphids :

Viviparous adults will be starved for 2,4,6 & 8 hours at

room temperature in a petridish and then placed upon a detached

leaf of an appropriate healthy host in a petridish. The atmosphere

inside the petridish will be made humid by covering the inner

surface of the petridish with wet filter paper. Newly born nymphs

will be transferred to a fresh and healthy plant immune to the virus

under investigation. The aphid colonies thus developed will be used

as healthy colonies of the virus free aphids. The aphids from one

plant to other will oe transferred with the help of moistened tip

of camel's hair brush type A, No. 1.

49

2.2,1 (c) Mode of transmission :

To establish the mode of transmission following procedure will

be adopted.

i) Non-persistant :

Virus free aphids will be first starved for 4-3 hours in a

glass vial before an acquisition access feeding of 1-2 min on the

detached leaf of the diseased plant placed on moist filter paper

in a petridish. After acquisition feeding, 10-20 aphids will be

transferred to each healthy seedlings of test plants for an

inoculation feeding period of 24 h. The plants will be covered

with lantern chimney having its top covered with muslin cloth to

avoid aphids from escaping. The aphids, after the end of the

inoculation feeding will be killed by spraying an insecticide. The

test plants will be kept in insect proof glass house to observe

the development of symptoms.

Summary

1. Pre-acquisition starvation period 4-8 hours.

2. Acquisition access period 1-2 minutes.

3. Inoculation access period 24 hours

4. Number of aphids per plant 10-20.

ii) Persistant :

The virus-free aphids, without subjecting them to starvation

will be allowed 24 h acquisition feeding time on diseased leaves

placed on a moist filter paper in a petridish. After the completion

of acquisition feeding, 10-20 aphids will be transferred to each

50

test plant where the/ will be given an inoculation feeding period,

aphids will be killed by spraying an insecticide. The test plants

will be kept in an insect proof glass house to ooserve the develop­

ment of symptoms. Back inoculations from the plants on which aphid*

were given inoculation feedings will be made on an appropriate

diagnostic host.

Summary

1. Acquisition access period 24 hours

2. Inoculation access period 48 hours

3. Number of aphids per plant 10-20.

2,2.2 Transmission by white flies :

a) Source of virus free white flies :

White flies (Bemisia tabaci Genn.) collected from Clitoria

tumatea will be caged on a healthy plant of C. turnatea for egg

laying. After 10 days the adults will be removed from the cage.

New white fly adults developing after 7-8 days would oe further

multiplied. Insect colonies so raised will be virus free and would

be further multiplied. Insect colonies so raised will be virus

free and would be used for transmission studies,

b) Handling of white flies :

Same method as used by Srivastava jet al.. (1977) will be

carried out for handling of whiteflies.

c) Transmission :

Non-viruliferous white flies would be allowed for acquisitior

51

and inoculation access periods of 24 h each on diseased and healthy

plants, respectively. Rogor (0.1%) will be sprayed to kill the

whiteflies after the end of inoculation feeding. The test plants

will be kept for a month for observation of symptoms.

2.2.3 Graft ;

Attemps will be made for side wedge grafting. Infected

scions will be grafted on healthy stock and kept under appropriate

light and humidity conditions to allow successful union which is

necessary for transmission.

2.2.4 Dodder :

Seeds of dodder (Cuscuta spp.) will be germinated on moist

filter paper placed in petriplates and then transferred in 12" clay

pots, sterilized with formalin (4%) and containing sterilized soil

mixture when the plants are about 6" long, they will be trained on

a suitable host plant susceptible to the virus being studied, and

the host plant (on which the dodder is being trained) will be

inoculated after one week. When the dodder has been established on

inoculated plant, a healthy test plant in another pot will be

placed near the pot (having inoculated plant with dodder established

on it) and the tips of the branches of dodder will be placed on

the healthy test plant or, the branches of the dodder will be

detached, placed in the axil of the healthy test plant and allowed

to establish there. The plants thus inoculated will be ooserved for

the development of symptoms, if any, for about 6 weeks. Back

52

inoculation will be made on local lesion host to confirm the

presence of virus (transmitted by dodder).

2.2.5 Soil transmission :

Soil around the naturally infected plants will be collected

from the field and sieved to remove roots and debris etc. such soil

will be divided into two parts. One part will be filled in a gunny 2

oag and will be autoclaved at 15 Lbs/m for 30 minutes and the other

part of soil will be left unautoclaved and will be filled in pots

as such. Seeds will be sown in pots containing sterilized and

unsterilized soil. Plants of both the sets will be kept for

observation in an insect proof glass house for a period of two

months. Back, inoculation tests will be carried out to ascertain

the presence of virus on them.

Any appearance of the symptoms in the seedlings grown in

unautoclaved soil will indicate the presence of soil borne vectors

or the seed transmissible nature of virus. Soil borne vectors may

be either nematodes or fungi.

2.2.5(a) Search for fungal vectors :

Roots of naturally infected plants not watered since 2-3 days

will be immersed in double distilled water for 15-30 min. The

suspension will be tested in two ways :

I. Suspension (roots water) will be poured around the roots

of healthy test seedlings. Development of symptoms on such test

plants will indicate that the zoospores carried the virus

internally.

53

II. Fungal suspension obtained as described above will be

mixed with the standard virus inoculum. The mixture will be allowec

to incubate for 15-20 minutes and thereafter poured around the

roots of healthy test seedlings. Symptom development if any will

indicate the association of fungal vector carrying the virus

externally on zoospores.

2.2.5(b) Search for nematode vector :

Nematodes from soil samples usually Lonqidorus . Trichodorus

and Xiphinema known as the vector of some of plant viruses will be

isolated from the soil samples using Cobb's method (Cobb. 1918).

To assure whether the nematode isolated from the soil samples

carry virus or not, the following tests will be performed.

i) A drop of concentrated nematode suspension on a glass

slide will be macerated with a glass spatula and inoculated on the

leaves of local lesion host.

ii) Nematode suspension will be poured around the roots of

test plants. The plants will be kept for the observation of the

symptoms for about one to two months.

Similarly varying number of nematodes isolated will be poured

around the roots of virus infected plants in a insect proof glass

house inoculated priorly. After an acquisition feeding of 15 days,

infected plants will be uprooted and the healthy ones will be

planted in the same pots. Plants will be kept for observation of

symptoms. Back inoculations from all nematode inoculated plants

be made on a local lesion host.

54

In case of positive observations the studies will extended

to identify the specific nematode species acting as vectors and

its relationship with the virus under investigation.

2,2.6 Seed transmission :

A few inoculated plants will be kept till flowering and

fruiting. After the seed maturation, they will be collected and

dried. A considerable number of seeds will be sown in the wooden

tra/s containing sterilized soil mixture. The germinating seeds

will be countedi The number of healthy and diseased plant, if any,

in the trays will be counted. To compare their percentage geraina-

tion, seeds from the healthy plants will also be treated in tne

same way.

Seeds transmissible nature of the virus under study will be

tested by the following method :

a) 3y macerating the seeds from diseased plants in 0.1M

phosphate pH 7.0, giving macerate a low speed centrifugation and

inoculating the sap thus obtained on the local lesion host.

b) 3y keeping the plants, developed from the seeds collected

from diseased plants under insect proof glass house for about one

month to observe the development of symptoms,

c) By inoculating sap ootained from young seedlings developed

from seeds collected from diseased plants on local lesion host.

55

2.2.7 Transmission through pollen :

Attemps will be made to determine the transmission of virus

through pollen by the same methods described by Frosheiser .(1974)

3. Host range studies :

Several species of plants, belonging to different families

will be screened for the susceptibility to the cucumber mosaic

virus. Standard inoculum will be used for inoculation of all plants.

At a time at least five plants of each species will be inoculated

and the same number will be kept as a control. Plants at 5-6 leaf

stage (cucurbits at seedling stage) will be used and all the fully

expanded leaves will be inoculated. The inoculated plants will be

observed upto two months for the development of symptoms. The time,

sequence and severity of the symptoms will be noted. Inoculated

plants exhibiting no symptoms will be kept for about 8 weeks for

observation. Back inoculations *'ill be made to a test plants from

all the inoculated plants.

4. Virus vector relationship :

In order to determine the relationship between the virus and

the vector, the method would depend on the type of the vector

56

group involved in transmission. However, in general, the varia­

bilities including number of insect per plant, different pre-

acquisition starvation periods, varying acquisition and inoculation

access periods will be worked out along with effect of moulting of

insect on various retention and latent period in the vector.

5. Effect of different buffers on the infectivity :

Various buffers (Phosphate, borate, citrate, acetate, glycine-

Na OH and tris-Hcl) at different pH and molarities will be used and

tested to find out the most suitable one in which virus infectivity

is retained most.

Young infected leaves will be macerated in a mortar with

pestle using a Duffer (any of the above mentioned) as extraction

medium. The sap obtained after filtrating it through double layered

cheese cloth will be inoculated on the leaves of local lesion host

following the usual method of inoculation. All buffers will be

tested in the same way, and a buffer at a pH and molarity in which

virus infectivity is higher will be selected and used regularly as

an extraction medium for the virus being used.

6. Virus concentration in different parts of the host :

To determine the virus concentration in different parts of

the host plant, 10-15 days earlier inoculated plants will oe uprooted

carefully and washed. The plants will be blotter dried. Root, stem

and leaf tissues will be cut separately into pieces. Equal amount

of root, stem and leaf tissue will be macerated separately in mortar

and pestle using a suitable buffer. Sap obtained from each sample

57

will be inoculated separetely on a local lesion host using usual

method of inoculation.

7. Selection of suitable propagation host and an assay host :

To find out a suitable propagation host several plants,

susceptible to the virus will be inoculated and showing most

prominent symptoms will be selected. A plant exhibiting following

characters will be selected.

i) Rapid seed germination and fast growth;

ii) Short incubation period of the virus;

iii) Peak concentration of the virus within a short period

after inoculation;

iv) Absence of virus inhibitors and

v) More yield of infected tissue with good virus

concentration.

Assay of virus will be carried on a local lesion host. To

search out a local lesions.^veral commonly used plants will be tested!

However, in case of non-availability of a local lesion host,

assay tests of the virus will be carried out on a systemic host.

8. Biophysical properties :

To determine the dilution end-point, thermal inactivation

point and Longivity in vitro, methods described by Noordam (1973)

will be employed.

8.1 Dilution end-point (PEP) :

By adding suitable buffer, ten fold dilutions (10*'', 10'^,

"•O 10' , 10" ) will be made of the sap obtained

58

from infected leaves of the propagation host after homogenizing

them in a mortar with pestle. Each sample will be inoculated on

to the leaves of the local lesion host following the usual method

of sap inoculation. In this way the dilution at which virus loses

its infectivity will be determined.

8.2 Lonqivity in vitro :

a) In sap :

The infected leaves of the propagation host will be homogeni­

sed in a mortar with pestle, while using a suitable buffer and the

homogenate will be filtered through two layers of cheese cloth and

the sap thus obtained, will be kept at room temperature (20-25°C).

After every 6 h interval, a small amount of the sap will be taken

and inoculated on the leaves of local lesion host. The process will

be counted on the inoculated leaves for each interval and the time

after which the virus loses its infectivity will be recorded.

b) In dried leaves :

The young infected leaves of the propagation host will be

cut into small pieces and dried over anhydrous calcium chloride

in a desicator. After 24 h interval, such pieces will be homo­

genised, by using a suitable buffer, in a mortar with pestle.

The sap thus obtained after passing through two layered cheese

cloth will be inoculated onto the leaves of local lesion host.

This will be continued upto the time, until the virus loses its

infectivity in the tissue dried over anhydrous calcium chloride.

59

8.3 Thermal inactivation point :

The sap obtained by the same method mentioned above, will be

divided into 12 aliquots of 5 ml each and kept in glass vials. The

glass vials will be held in a water bath in such a way that the

sap^level in the vial is below the water in the bath. The different

aliquots will be heated at 40, 45, 50 85, 90°C, for ten minutes

and cooled under running tap water, immediately after heating. Each

heated aliquot will be inoculated on the leaves of a local lesion

host. One aliquot left at room temperature will also be inoculated

and will be served as control.

9. Effect of various additives on virus infectivity :

To .work out whether the stability and infectivity of the virus

will get increased, several additives (sodium sulfite, DIECA, EDTA,

sodium thioglycollate, mercaptoethanol)will be used. In case, the

infectivity get enhanced, the most suitable additive will be

selected and routinely added to the medium for virus extraction.

10. Purification :

After selecting a suitable buffer, a propagation host(s),

an assay host(s) and oiophysical properties, atteraps will be made

to purify the virus under consideration.

10.1 Clarification of sap :

leaves The infected/of the propagation host will macerated by usual

and suitaole method and the macerate will be passed through a double

cheese cloth. The sap thus obtained will be given a low speed

60

centrifugation at 5000 g for 10 minutes. The supernatent (sap)

will be subjected to various clarification procedures.

10.1(a) Celite and charcoal :

Celite and activated charcoal will be mixed with sap at the

rate of 5 g per 100 ml, either separately or, in combination. When

both are to be used 5g of activated charcoal will be mixed with

100 ml of sap and after 1/2 minute stirring 5g of celite will be

added. Shaking will be continued for another half minute. The

absorbent will be removed by the following methods.

i) Centrifugation at 2,000 rpm for 5 minutes;

ii) Filteration through Buchner funnel supported by a 2-3 mm

thick celite pad and filter paper (Whatman No. 1) or,

iii) Filteration through a filter paper (Watman No. 1) only

in a Buchner funnel,

10.2 Organic solvent ;

Organic solvents (butanol, ethyl alcohal, chloroform, carbon

tetrachloride and di-ethylether) either seperately or in combination

such as (chloroform-butanol) will be used in two ways for the

removal of the extraneous plant material from the infected tissue.

a) by macerating the infected tissue by using a mixture of

suitable buffer and organic solvents, or

b) by adding requisite amount of solvent in crude sap obtained

after macerating the infected tissue in buffer and filtering

through two layers of cheese cloth.

61

The mixture will be incubated for 30 minutes and then

centrifuged at 5000 g for 15 minutes. The aqueous la/er will be

separated. The effect of solvent on the virus infectivity will be

tested by assaying the aquous layer for active virus content on

a local lesion host,

c) Calcium phosphate qel :

The gel will be prepared by mixing 0.1M sodium dibasic

hydrogen phosphate (Na2 H PO^. 2 H2O) and 0.1M calcium chloride (Gel)

in equal volume. The mixture after continuous stirring for 15 minutes

will be allowed to settle. Then the supernatent will be decanted.

To the remaining precipitate double distilled water will be added

and the resuspended precipitate will again be allowed to settle

down. In this way precipitate (gel) will be washed 15-20 times to

assure the removal of chloride ions (CL"). Ultimately it will be

equilibrated with phosphate buffer (O.IM pH 7.0). Such freshly

prepared gel will be mixed with sap obtained after low speed (5000 g

for 10 minutes) centrifugation of the crude sap, stirred vigorously

and centrifuged for 5 minutes at 5000 g. The clear supernatant will

be assayed for virus activity on local lesion host.

d) Silver nitrate :

Different volumes of 1 per cent silver nitrate solution will

be added drop by drop to the standard inoculum (1/5) and stirred

simultaneously. The mixture will be left at room temperature for

30 minutes and thereafter, centrifuged at 5000 g for 15 minutes.

The supernatent thus ootained will be bioassayed on local lesion

host for virus infectivity.

62

Out of the clarification methods described above, one will

be standardized and used as clarification method in the purifi­

cation of the virus being studied.

10.2 Concentration of virus :

The sap obtained after clarification treatment as described

above will be used for concentration of virus by any of the

following methods.

a) Differential centrifuqation :

The ultracentifugation will be worked out in model L3-50

Backman preparative ultracentrifuge using roter type 50. Normally,

high speed centrifugation will be done at 97,000 g unless otherwise

stated. The pellet, thus ootained will be dissolved in a suitable

buffer. Low speed centrifugation will be performed at 10,000 g in

a Remi T-24 centrifuge or anyother same type of centrifuge. The

numoer of cycles and the time of centrifugation at different rpm

will be carried out keeping in view the stability of the virus and

its sedimentation. Activity of different samples in supernatent

and the pellet will be assayed on local lesion assay host.

b) Precipitation :

i)Pdlv ethylene glycol (PEG)

Poly ethylene glycol 6,000 will be used for precipitating

the virus in clarified sap. Precipitation of the virus will be

tried with 2,4,6,8,10 and 12 percent PEG separately. In every

case, the variation in salt (NaCl) concentration and its impact

63

on precipitation of the virus will be standardized. After the

addition of requisite quantity of PEG and NaCl to the clarified

sap, the mixture will be stirred on a magnetic strirrer till both

(PEG and NaCl) are dissolved completely and kept in a refrigerator

at 4 to 8* 0 for 6h to allow complete precipitation,

ii) Ammonium sulphate;

Different quantities (10-40?$) of ammonium sulphate (NH^)2

SO. (w/v) will be added to clarified sap (1/1). The mixture will

be stirred at 8+2* C in an ice Ducket till the (NH4)2 SO^ crystals

are dissolved completely. The mixture will then be incubated at

4+1°C for 2 hours and centrifuged at 5,000 g for 15 min. to collect

the precipitate.

The pellet thus obtained by PEG and (NH4)2 ^^4 Precipitation

will oe dissolved seperately in a suitable buffer and recentrifuged

at 5,000 g for 5 minutes. Supernatent thus ootained will be assayed

on local lesion host.

11. Further purification by density gradient centrifuqation :

Concentrated virus samples obtained by the methods described

above will be suojected to further purification using density

gradient centrifugation (Brakke, 1951, 1960),

Linear sucrose gradient columns will be prepared by layering

7,7,7 and 4 ml of O.IM phosphate buffer pH 7.0 having 400,300,200

and 100 mg sucrose per ml, respectively, in a IxS" tube. The

sucrose solutions of different concentrations will be layered

using a pipette with a broad orifice.

64

The heaviest solution will be layered 1st and the solutions

of decreasing concentration will be layered on the top of each

other. The column will be used after standing for 24 h in a

refrigerator usually 2 ml of the virus preparation will be

floated on the top of the column and the column will be centrifuged

immediately after floating the virus preparation to avoid droplet

sedimentation. The column will be centrifuged in SW-25.1 rotor in

L3-50 preparative ultracentrifuge. The acceleration upto a few

hundred rpm will be made gradually. The tubes will be centrifuged

for 2V2 - 4 hours. After centrifugation the tubes will be examined

in a dark room by projecting a beam of light down the tube from the

top. The virus zone scattering the light will be removed from the

tube by 20 guaze 10 cm long needle bent twice at right angles and

attached to a hypodermic syringe.

12. UV-»spectrophotometry :

The virus preparation will be examined in Beckraan DU-2 model

ultraviolet absorption spectrophotometer to evaluate the different

methods of purification and to ascertain the purity virus isolated.

Ultraviolet radiations are absorbed in a characteristic manner

by the virus (nucleo-protein) containing solutions. Absorbance of

samples will be studied in UV-range (230-320 nm) and graphs will be

plotted. Values of A-max/min, A-280/260 and A-260/280 will be

calculated to know the approximate percentage of nucleic acid.

13, Electron microscopy :

Shape and size of virus particles will be studied in electron

microscope ISEWr) ,

65

13.1 Leaf dip method :

Method described by Brandes (1964) will be followed for leaf

dip preparations. One drop each of 2% potassium phosphotungstic

acid (PTA) and uranyl acetate will be placed separately on several

fonovar coated copper grids having carbon backing. The freshy cut

ends of infected leaves will be dipped in the drop of 2-4 seconds.

Such grids will be allowed to dry for sometime and thereafter

examined under electron microscope at various magnifications.

13.2 Procedure with purified virus preparation :

A small droplet of purified virus preparation will oe placed

on formvar coated copper grids having caroon oacking, then a small

drop of suitable stain (either PTA or uranyl acetate) will be

added to the virus suspension. The excess fluid will be absorbed

with a small piece of filter paper leaving a very thin film of

fluid on the grids, which will be dried at room temperature. Such

grids will be examined under SEM.

14. Serology :

Specific antigen and antibody reaction is one of the useful

techniques either for assigning the virus to a particular group

or to differentiate it at the strain level. Antisera to the virus

under consideration will be prepared for the identification of the

virus as well as for testing the latent infection in certain hosts.

Besides, it would also be useful for ascertaining the seed trans­

missible nature of the virus through routine serological methods

or by enzyme-linked immunosoroent assay (ELISA).

66

14.1 Raising of antisera :

Young healthy rabbits, approximetely 3 Lbs. in weitht will

be used for production of antisera. The purified or partially

purified virus preparation will be used as antigen.

To work out the effect of injection on the formation of

antibodies as well as titre of the antiserum, antigen (virus

preparation) will be injected intravaneously or intramuscularly

or in both ways.

The antigens will be administered intravenously through the f

marginal ear vein of the rabbit using a clinical syringe with a

fine needle. Five to seven weekly injections of virus preparation

of 2ml each will be administered intravenously through the marginal

vein of the ear. For intramuscular injections, antigens will be

emulsified with an equal volume of Freund's incomplete adjuvant.

Two injections of the virus-adjuvant mixture of 3 ml each at an

interval of 2 weeks will be administered intramuscularly in thigh

of the same rabbit which has been given intravenous injections of

antigens, test bleedings will be made several times from the ear

of the raboit at different intervals after the administration of

last intramuscular injection to check the antibody titre in serum.

After the titre has reached its maximum, the immunized rabbits

will be finally oled by giving a sharp incision on the marginal

vein of the ear, which has not been used for injecting the antigen.

About 10-15 ml of the blood will be collected and allowed to clot

at room temperature (20+5°C) for 2 hours and kept overnight in a

refrigerator. Serum containing antibodies (antiserum) will there-

67

after be separated and centrifuged at 1,000 rpm for 5 minutes to

remove fibron, blood cells etc. The straw yellow coloured antiserum

will be collected and stored for serological studies.

To identify the virus under investigation upto group or

strain level, the Ouchterlony.'s double diffusion test (Ouchterlony,

1962) will be performed.

14.2 Ouchterlony double-diffusion test :

One per cent agar will be prepared in 0,85? saline containing

0.1-0.2^ sodium azide. Suitable amount of agar will be poured into

sterilized petridishes so as to get a 2-3 (ran thick agar bed. Using

a cork borer of 5 mm diameter well will be made into the agar and

the cut portions of agar will be removed by aspiration. The distance

between two wells will be kept 5 mm apart. The central well will

be filled with antiserum. The remaining wells will be loaded with

various dilutions of antigens made in physiological saline (0.85^

NaCl solution) and the clarified sap from healthy plant. Such

treated petridishes will be incubated at room temperature and

observed for formation of precipitation lines.

14.3 Precipitin test in tubes :

Equal proportions of antisera and antigen after making 2 fold

dilutions in 0.857o saline will be mixed together in serological

tubes (6x1 cm) and incubated at 37°C in a water bath. The formation

of precipitate and its intensity will be observed using a magni­

fying lens.

68

15. Isolation of nucleic acid :

Phenol detergent method will be used to isolate the nucleic

acid of viruses. To a 2.5 ml of purified virus preparation will be

added 0.05 ml of 6? sodium dodecyl sulphate and 2.6 ml of water

saturated phenol. The phenol used will be redistilled and stored

at 10^C after adding distilled water. The mixture will be stirred

in a glass tube on a magnetic stirrer for 10 minutes and then

centrifuged for 5 minutes at 3,000 rpm in a clinical centrifuge.

The mixture will separate into two layers, the upper aquous layer

and the lower phenol layer containing sodium dodecyl sulphate.

The top aqueous layer will be dra/m with a pippette. To the lower

phase 2.5 ml of O.lM phosphate buffer pH 7.0 will be added and

stirred for 10 minutes and then centrifuged for 5 minutes at

3,000 rpm. The aqueous phase will be drawn off and pooled together

with the aqueous phase obtained at previous step and stirred for

10 minutes with an equal volume of phenol followed by centrifugation,

The aqueous phase will be extracted once more with half the volume

of phenol. Traces of phenol will be removed from the aqueous phase

by extraction with ether. The nucleic acid will be precipitated by

the addition of 2 ml of ice-cold ethanol to the solution. The

precipitate will be pelleted out by centrifugation for 15 minutes

at 7,500 rpm. The pellet will be suspended in 0.1M phosphate buffer

pH 7.0 and centrifuged for 15 minutes at 10,000 rpm to remove any

insoluble material present in the precipitation, and the super­

natant thus obtained will be tested for infectivity and type

{RNA or DNA) of the nucleic acid.

69

15.1 Infectivitv of viral nucleic acid :

The infectivity of viral nucleic acid will be assessed by

inoculating the nucleic acid preparation on local lesion host.

Several dilutions of nucleic acid preparation will be made and

inoculated on the local lesion host and the number of local lesions

developed will be compared with the corresponding dilutions of the

virus preparation.

15.2 Type of nucleic acid :

It is well known that RNA and DNA differ in their chefnical

composition with respect to the base and sugar involved in their

composition. RNAs are known to contain ribose sugar and uracil

base (other three bases being adenine, cytosine & guanine) while

DNAs contain thymine - (other three oases are same as in case of

RNA) and deoxyribose sugar.

Thus, test will be performed to studv the type of sugar.

Diphenylamine test for deoxyribose or orcinol test for ribose

sugar will be used for ascertaining the type of nucleic acid in

virus under investination.

16. Studies on proteins of the virions :

Attemps will be made, while using standard methods to

deter.-Tiine the approximate molecular weight of proteins associated

with the virions.

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