IDENTIFICATION AND CHARACTERIZATION OF CUCUMBER MOSAIC AND BEAN YELLOW

IDENTIFICATION AND CHARACTERIZATION OF CUCUMBER MOSAIC AND BEAN YELLOW MOSAIC

VIRUSES AFFECTING CHICKPEA (CICER ARlETlNUM L.) IN INDIA

Thesis submitted to the Andhra Pradesh Agricultural University in part fulfilment of the requirements

for the award of the degree of

DOCTOR OF PHILOSOPHY

by

T. VENKATA CHALAM, M. Sc. (Ag.)

Department of Plant Pathology College of Agriculture Andhra Pradesh Agricultural University Rajendranagar, Hyderabad 600 030

Pulse Pathology Subprogram ICRISAT Patancheru P.O. 6 0 2 324 Andhra Pradesh India

November 1982

IDENTIFICATION AND CHARACTERIZATION OF

CUCUMBER MOSAIC AND BEAN YELLOW MOSAIC VIRUSES AFFECTING CHICKPEA (CICER ARlETlNUM L.) IN INDIA

Thesis submitted to the Andhra Pradesh Agricultural University in part fulfilment of the requirements

for the award of the degree of

DOCTOR OF PHILOSOPHY

by

T. VENKATA CHALAM, M . Sc. (Ag.)

Department of Plant Pathology College of Agriculture Andhra Pradesh Agricultural University Rajendranagar, Hyderabad 5 0 0 0 3 0

Pulse Pathology Subprogram ICRISAT Patancheru P.O. 5 0 2 324 Andhra Pradesh India

November 1982

CERTIF ICATE

Shr i T. Venkata Chalam has s a t i s f a c t o r i l y prosecuted t h e

course of research and t h a t t h e t h e s i s e n t i t l e d " I d e n t i f i c a t i o n and

cha rac t e r i za t i on o f cucumber mosaic and bean yellow mosaic v i r u s e s

a f f e c t i n g chickpea (Cicer , . ari,etinum L.) i n Indi,a," submitted i s t h e

r e s u l t o f o r i g i n a l research w o ~ k and d s of s u f f i c i e n t l y h igh s tandard

t o warrant i t s presentaf ion t o t h e examination. We,also c e r t i f y

t h a t t h e t h e s i s o r p a r t i t h e r eof has*?gt. peen previous ly submitted

by him f o r a degree of any Univers i ty .

Co- Chairman

Y.L. NENE Pr inc ipa l P l an t Pa thologis t and Leader, Pulses Improvement Program I CRISAT Patancheru P.O. 502 324 Andhra Pradesh India

Chairman

J . SUBBAYYA Professor and Head Department of P l an t Pathology S. V. Agr i cu l tu ra l College T i rupa t i 517 502 Andhra Pradesh India

CERTIFICATE

This i s t o c e r t i f y t h a t the t h e s i s e n t i t l e d " I d e n t i f i c a t i o n

and c h a r a c t e r i za t ion of cucumber mosaic and bean ye1 low mosaic

v i r u s e s a f f e c t i n g chickpea (cicer arietinum L .) i n India" submit ted

i n p a r t i a l f u l f i l m e n t of t h e requirements f o r t h e degree of Doctor

of Philosophy i n Agr icu l tu re , i n t h e major s u b j e c t o f P lan t Pathology

of t h e Andhra Pradesh A g r i c u l t u r a l Univers i ty , Hyderabad, i s a record

of the bond fide r e s e a r c h work c a r r i e d o u t by Mr. T. Venkata Chalam

under our guidance and ruperv i s ion . The s u b j e c t o f t h e t h e s i s has

been approved by t h e S t u d e n t ' s Advisory Committee.

No p a r t o f t h e t h e s i s has been submit ted f o r any o t h e r

degree o r diploma.

A l l a s s i s t a n c e and h e l p received dur ing t h e course o f t h e

i n v e s t i g a t i o n s have been duly acknowledged by him.

7 ' . -Y 1 7. \ +, .;..4 -.

- . _ - _. _. ... .- -- Y . L . NENE Co-Chairman Advisory Committee

- G -3

-:. 2

J . SUBBAYYA Chairman Advisory Committee

The t h e s i s approved by t h e Advisory Committee. - -'

Chairman (J. SUBBAYYA) 2

Co- Chairman (Y . L . NENE)

Member (S.P.S. BENIWAL)

Member (N. GOVINDA RAO)

Member (A. NARAYANAN)

Member (V. ARUNACHALAM)

CONTENTS

Page No,

List of i l l u s t r a t i o n s

L i s t of t a b l e s

Acknowl edgnent

Abstract

List o f symbols and abbrevia t ions

I . INTRODUCTION

11. REVIEW OF LITERATURE

111. MATERIALS AND METHODS

General

I d e n t i f i c a t i o n o f v i ruses by serology

Host range and symptoms

Physical p roper t i e s of v i ruses i n crude sap

Insect transmission

P u r i f i c a t i o n of v i ruses

Elect ron microscopy

Physico-chemical p roper t i e s of p u r i f i e d CMV

Production of antiserum t o CMV

b e t e r k n a t ~ o n o f t h e serological r e l a t i o n s h i p between t h e p resen t s t r a i n of 6n and o ther known st r a i n s

Screening f o r d i sease r e s i s t a n c e

I V . RESULTS

Symptomatology i n chickpea

Host range and symptoms

I n vi tro proper t i e s

Insec t transmission - P u r i f i c a t i o n o f v i r u s e s

Elect ron microscopy

I d e n t i f i c a t i o n of v i ruses by serology

Physico-chemical p roper t i e s o f p u r i f i e d CMV

Production o f antiserum t o CMV

v i i

v i i i

Serological r e l a t i o n s h i p between presen t s t r a i n . . of CMV and o ther known s t r a i n s

Screening f o r d i sease r e s i s t a n c e . . V. DISCUSSION AND CONCLUSION . .

V I . SUMMARY . . LITERATURE CITED D .

APPEND1 X . . VITA . .

Page No,

60

List of I l l u s t r a t i o n s

Fig.No. I l l u s t r a t i o n

1. Symptoms produced by CMV on chickpea . O

2. Symptoms produced by BYMV on chickpea 0 .

3 . Local l e s i o n s produced by CMV on Chenopodium . . m a n t i c o Z o r , broad bean, Phuseolus vulgaris v a r . Pinto , and P. vulgaris var. P o r r i l o

4 . Local l e s i o n s produced by CMV on P. vulgaris . . v a r . Lavica, Vigna mguiculata var . Pusa Dophasli and V. wtguiculata var. Barsa t i mutant

5 . Systemic symptoms produced by CMV on . . Nicotiana rus t i ca , cucumber and peas

6. Systemic symptoms produced by BYMV on . . P. vuZgaris

7 . Light s c a t t e r i n g zone o f CMV i n d e n s i t y . . grad ien t tube ( a ) , uv absorpt ion and i n f e c t i v i t y o f f r a c t i o n s of sucrose dens i ty g rad ien t tube @), and uv absorp- t i o n spectrum o f p u r i f i e d CMV (c)

8 . Elect ron photomicrograph of p u r i f i e d CMV . . 9. Electron photomicrograph of l e a f d i p . .

prepara t ion o f BYMV

10. Length d i s t r i b u t i o n histogram of BYMV . . 11. Elect ron photomicrograph o f p u r i f i e d BYMV . . 12. U l t r a t h i n s e c t i o n s of BYMV i n f e c t e d . .

chickpea l e a f

13. Agar double-di f fus ion t e s t f o r i d e n t i - . . f i c a t i o n o f CMV and BW

14. Enzyme- l i k e d immunosorbent assay f o r . . i d e n t i f i c a t i o n o f CMV

15. Schle i ren p a t t e r n o f p u r i f i e d CMV i n an . . a n a l y t i c a l u l t r a c e n t r i f u g e

16. UV absorpt ion and d e n s i t i e s o f C s C l gradient . . column (CMV)

17. Polyacrylamide gel e l ec t rophores i s of CMV . . coat p r o t e i n (a) and (;MY RNAs (b)

18. Photometric scanning o f CMV coat p r o t e i n ( a ) , . . marker p r o t e i n s (b), CMV coat p r o t e i n and markers (c) and re la t ic in between migra- t i o n d i s t ance and l o g mol .wt. o f markers (d)

Page No.

4 7b

4 7d

4 7f

Lis t of Tables

S.No. Table - - 1. Randomization o f t r ea tments t o determine

thermal i n a c t i v a t i o n p o i n t o f CMV

2 . Randomization o f t r ea tments t o determine d i l u t i o n end p o i n t of CMV

3. Host range and symptomatology of CMV and BY MV

4 . Inf luence o f hea t ing t h e s t andard e x t r a c t o f CMV a t d i f f e r e n t temperatures f o r 10 min on i n f e c t i v i t y

5. Influence of d i l u t i n g t h e s t andard e x t r a c t o f CMV on i n f e c t i v i t y

6. Inf luence o f s to rage o f t h e s t andard e x t r a c t of CMV a t room temperature on i n f e c t i v i t y

7 . Inf luence of hea t ing t h e s t andard e x t r a c t o f BYMV a t d i f f e r e n t temperatures f o r 10 min on i n f e c t i v i t y

8 . Inf luence o f d i l u t i n g t h e s t andard e x t r a c t o f BYMV on i n f e c t i v i t y

9. In f luence of s to rage o f t h e s tandard e x t r a c t of BYMV a t room temperature on i n f e c t i v i t y

10. Aphid t ransmiss ion o f CMV and BYMV

11. Sero log ica l a f f i n i t y o f p resen t CMV s t r a i n wi th o t h e r known s t r a i n s o f CMV

Page No,

34

ACKNOWLEDGMENT

1 de& hmende p t e a s w ~ e i n e ~ p h e b b i n g my h c a e 2hanh6 and deep senbe 0 6 g m f i d e la Dh. Y . 1 , N w e , P h i n c i p d P h n t Pa&o&ogAt and l e a d a , P u b e s lmpmvement P m g m , ICRlSAT, doh buggu. t ing t h e pmblem and doh kib enwwuzgement and guidance dwLing t h e c o w e 0 6 t k i d i n v e b t i g a t i a n , and in p / t e p W o n 0 6 .the manub+t.

1 am k i g M y h k 6 d ;to Dh. 3. Subbayya, Pkodedbofi and H a d , Dept. 0 6 PlanX P a t h o b g y , S . V. A g h i d W CoUege, T A W , doh kis v a l u a b l e buggebtLon6 m d doh b m v i n g M ChlLiRrnan 0 6 Xhe Advbotry Conanittee.

1 am g m t e d d RD Dh. M.V. Reddy, Chickpea PaZhotog-bt, lCARVA, S y ~ a , doh h d p i n g me ach ieve p a d e c f i o n in t h e 6iel .d 0 6 plant v h L o g y .

7 am aedo ; t hank id b Dh. S.P.S. Beniwd, S e n i o t P l a n t PadhoLogAist ( P u b e s I , ICRISAT, doh kis h d p dwLing A%Q h.W phA ad my wotrk, 60tr go ing t h o u g h & manuc&ipt, and 604 acting ad Membeh od isthe AdvAohy CormLittee.

T h a n k m e aedo due ;to Dh, D. V. R. Reddy, P ~ i c i p d V h t o g A t , ICRlSAT, doh kis v d u a b l e s u g g u k i o n b .

1 am thank6u.l RD D u . N , Govinda Rao, A. Nuhayanan and V. Atrunachdeam doh acaXng as membcm od t h e AdvAorry ComnuXee, a3 Dh. R. JambunaLhan, P a n c L p a t 12iochemist, 1CRlSAT; M u b ~ . Y. 8 b h , S.K. M a n a h , S.M. Raju, and b.tad6 0 6 P&e Pathology, ICRlSAT doh 2%eih b h c e h e h d p .

1 w h h ib t o k e f&.6 p /L i vdkge b exptredb y prto60Wld senbe 0 6 h e s p e d and Love b my s A & , Mu. T. V. S u b b m and my b m t h m Mk. T . Venkac teaodu, who bo tdey ltook up t h e b w d e n 0 6 my d u d o n eve4 b h c e t h e demide ad my dathm.

1 bpeciaeey Zbnh my w i d e S a v W , doh h m w o p , W n and LnbpjrLLttion d u g my Ph.D. prrogham,

The & U o u u k i p and 6acLlLiAa p w v i & d by ZCRISAT dwLing .the c o m e 0 6 a%& invesa%jat ian m e ghateduiQ acknavledged.

7 w h h a% euplle6b my appke-n Xu to. R. N a h s h g Rm, do t typ ing Xki.4 manttbchipt and to h. H.S. kis w U a g u ~ 604 pbhgrrapkic wohh.

. . T . VENKATA CHALAM

ABSTRACT

Author : T. Venkata Chalam

T i t l e of the t h e s i s : I d e n t i f i c a t i o n and charac te r i za t ion o f cucumber m s a i c and bean yellow m s a i c v i ruses a f f e c t i n g chickpea (Cicer m i e t i n t m L . ) i n India

Degree : Ph.D.

Facul ty : Plan t Pathology

Guides : Y.L. Nene (ICRISAT) J . Subbayya (APAU)

Universi ty : Andhra Pradesh Agricul tura l Universi ty

Year of submission : 1982

Two mechanically t r ansmiss ib le v i ruses ; v iz . , cucumber mosaic

v i r u s (CMV) and bean yellow mosaic v i r u s (BYMV) were found t o n a t u r a l l y

i n f e c t chickpeas. These were i d e n t i f i e d by studying t h e i r hos t range,

biophysi ca l p roper t i e s , i n s e c t transmission, e l ec t ron microscopy and

serology. Also e l i t e chickpea germplasm access ions were screened f o r

r e s i s t a n c e t o these v i r u s e s .

Both the v i ruses i n i t i a l l y produced twis t ing o f terminal bud

i n chickpea. Subsequently, CMV e i t h e r produced w i l t i n g and death of

p l a n t s o r p r o l i f e r a t i o n and bushiness of branches bearing very small

green leaves and very small pods, whereas BYMV produced p r o l i f e r a t i o n

of branches with narrow o r f i l i f o r m leaves, bearing very small pods.

The (51V could i n f e c t 13 p lan t species belonging t o f i v e

fami l i e s , whereas t h e hos t range o f BYMV was r e s t r i c t e d to Leguminosae

only. The r e s u l t s on i n vitro p r o p e r t i e s ind ica ted t h a t CMV was more

s t a b l e than BYMV. Both t h e v i ruses could be t ransmit ted by two aphid

species ; v i z . , Aphis maccivora and t$jzus perskae i n a non-pers is tent

manner.

An e f f i c i e n t p u r i f i c a t i o n procedure developed for CMV yielded

80-100 mg o f v i r u s per kg of p lan t t i s s u e . The CMV p a r t i c l e s were

s p h e r i c a l and measured about 30 nm i n d iameter . The p u r i f i e d CMV had

a buoyant d e n s i t y and sedimentat ion c o e f f i c i e n t o f 1.325 g/ml and 104S,

r e s p e c t i v e l y . Using t h e polyacrylamide ge l e l e c t r o p h o r e s i s (PAGE),

t h e molecular weight o f CMV coat p r o t e i n was determined t o be 25,000

da l tons . The PAGE o f CMV WAS revea l ed t h e presence o f f o u r RNA s p e c i e s ,

The p r e s e n t s t r a i n of CMV was r e l a t i v e l y more c l o s e l y r e l a t e d t o CMV-C

pool, fo l lowed by CMV-Ix and M-CMV. The ant i serum produced t o CMV had

a t i t r e o f 1/1024 a s determined by agar double-d i f fus ion t e s t .

A s a t i s f a c t o r y p u r i f i c a t i o n procedure was developed f o r BYMV.

The p a r t i c l e s of BYMV measured 750 nm i n l e n g t h and 15 nm i n width.

Pinwheels and l amina t e agg rega t e s t y p i c a l o f po tyv i ruses were observed

i n t h e cytoplasm of BYMV-infected ch ickpea c e l l s . S ince t h e v i r u s

p repa ra t ion f r e e o f hos t components cou ld n o t be obta ined , t he physico-

chemical p r o p e r t i e s o f BYMV were n o t determined.

E l i t e chickpea germplasm acces s ions were screened f o r r e s i s -

tance t o both CMV and BYMV, and sou rces o f r e s i s t a n c e were i d e n t i f i e d

f o r both t h e v i r u s e s .

List o f symbols and abbrevia t ions

A absorbance

absorbance a t 260 nm

absorbance a t 280 nm

A max absorbance maximum in a absorption spectrum

A min

APAU

BY MV

cm

am cv

DEP

absorbance minimum i n a absorption spectrum

Andhra Pradesh Agricul tura l Universi ty

bean yellow mosaic v i r u s

centimeter ( s )

cucumber m s a i c v i r u s

c u l t i v a r

d i l u t i o n end point

E an a l t e r n a t i v e symbol f o r A

s p e c i f i c ex t inc t ion coef f i c i en t = the absorbance value of a 0.1% so lu t ion i n a cuvet te wi th an o p t i c a l path length o f t h e l i g h t o f 10 mm

: idem, but a t a wave length of 280 nm

E DTA : ethyl enediaminete t raacet ic acid, sodium s a l t

EL I SA : enzyme-linked immunosorbent assay ( se ro log ica l 5

I . C q t e s t ) . .

g : gram(s) (a u n i t symbol of mass)

g ' : i n i t i a l acce le ra t ion i n f r e e f a l l . Values i n u l t r acen t r i fuga t ion a r e given in t h i s g r a v i t y u n i t

I CC

ICRISAT

: hydrochloric a c i d

: hour ( s )

: In te rna t iona l Center k r A g r i c u l t u r a 1 Research i n t h e Dry Areas

: ICRISAT chickpea

: In te rna t iona l Crops Research I n s t i t u t e f o r t h e Semi -Arid Tropics

: potassium chlor ide

: potassium phosphate, m n o b a s i c

mm

ml . w t .

N

NaCl

Na2CO3 NaHC03

NaH2POq

Na2HP04

NaOH

nm

OD PAGE

P B ~ '

PEG

P04 p s i

PSV

PVP

BNA

RNAs

RNase

rpm S

longevity i n vitm molar concentrat ion i n grams per l i t r e

m i l l i molar concentration = 0.001M

milligram = 0.001 g

m i l l i ampere

minute( s )

m i l l i l i t e r = 0.001 l i t r e

mil l imeter = 0.001 meter

molecular weight

normality

sodium ch lo r ide

sodium carbonate

sodium b i carbonate

sodium phosphate, monobasic

sodium phosphate, d ibas ic '

sodium hydroxide -9 nanometre = 10 m (formerly mp was used)

a l t e r n a t i v e symbol f o r A

polyacrylamide gel e lec t rophores i s

phosphate buffered s a l i n e

polyethylene glycol

hydrogen ion exponent ( a u n i t symbol f o r t he degree of a l k a l i n i t y o r a c i d i t y )

phosphate

per square inch

peanut s t u n t v i r u s

polyvinyl pyrrolidone

r ibonucle ic ac id

r ibonucle ic ac ids

r ibonuclease

revolu t ions per minute (cent r i fuga t ion)

svedberg un i t s , 10-l3 sec. Values o f sedime: t a t i o n c o e f f i c i e n t s

: sedimentation c o e f f i c i e n t a t 2 0 ' ~ i n water

: sodium dodecyl s u l f a t e

TAV

TEME D

TIP

T r i s

Tween-20

W

tomato aspermy v i r u s

N , N , N t , N ' t e t r amethy l e thy lene diamine

thermal i n a c t i v a t i o n p o i n t

t r i s (Hydroxymethyl) Aminomethane

polyoxyethylene s o r b i t a n mono1 a u r a t e

u l t r a v i o l e t

v a r i e t y

volume/volume

weight/volume

weight /weight

microgram = g

m i c r o l i t r e = l om6 l i t r e

d i f f e r e n c e i n t h e d i s t a n c e t r a v e l l e d between any two t ime i n t e r v a l s

: d i f f e r e n c e between any two fime i n t e r v a l s

: r o t o r ' s angu la r v e l o c i t y

: mean o f t h e d i s t a n c e s t r a v e l l e d

INTRODUCTION

Chickpea (Cicer a r i e t i m L . ) is an important g ra in legume crop of dry-

land a g r i c u l t u r e i n Asia, Africa and Central and South America. The

t o t a l c u l t i v a t e d a r e a o f chickpea i n the world i s about 9 . 3 mil l ion

hec ta res with annual production of about 4 .8 mi l l ion tonnes (FAO, 1980)

The average y i e l d s pe r hectare a r e es t imated t o be around 700 kg.

Chickpea i s known by o t h e r names such a s Bengal gram, gram, Egyptian

pea, Spanish pea, Chestnut bean ( a l l Engl ish) , Pois chiche (French),

chana, (Hindi) , homos (Arabic), grao-de-bico (Portuguese), garbanzo o r

garavance (Spanish), e t c .

About 50 pathogens have so f a r been repor ted on chickpea

from d i f f e r e n t p a r t s of t h e world (Nene, 1980). While many r e p o r t s

a r e mere records o f occurrence, severa l d i seases a r e widespread and a

few a r e devasta t ing. A survey o f t h e l i t e r a t u r e r e v e a l s t h a t on ly a

few d i seases have been inves t iga ted i n d e t a i l (Nene e t aZ . , 1978b) .

h r r g chickpea diseases, w i l t and r o o t r o t s a r e considered

t o be of ,major importance. Considerable confusion e x i s t e d i n t h e p a s t

i n diagnosing var ious chickpea d i so rders , p a r t i c u l a r l y w i l t s and r o o t

r o t s (Nene e t a t . , 1978a). A t ICRISAT ( In te rna t iona l Crops Research

I n s t i t u t e f o r t h e Semi-Arid Tropics), a p r o j e c t was i n i t i a t e d i n 1974

on ' w i l t complex1. During the course of i n v e s t i g a t i o n s i n t o t h e

e t i o l o g y o f so -ca l l ed w i l t c o q l e x , two v i ruses ; v i z . , a l f a l f a mosaic

v i r u s and pea l e a f r o l l v i r u s were i d e n t i f i e d and found t o be t h e compo-

n e n t s o f wilt complex (Nene e t a1 ., 1978a). Chickpea p l a n t s a f f e c t e d

by t h e s e two v i r u s e s o f t e n d ie prelpaturely and hence were wrongly

considered t o be a f f e c t e d by %wilt1'.

Present s t u d i e s were i n i t i a t e d t o know i f o t h e r v i ruses

a f f e c t e d chickpeas under natura l condit ions i n India, and t h e i r c o n t r i -

but ion, i f any, t o the " w i l t complexw.

With the exception of b r i e f r epor t s , very l i t t e has been

published on the i d e n t i t y o f v i ruses a f f e c t i n g chickpeas i n na tu re .

Viral d iseases of chickpea may have escaped detection i n many coun t r i e s

s ince the macroscopic symptoms produced i n such p l a n t s a r e o f t e n n o t

conspicuous. Because o f premature death of these p lan t s , symptoms a r e

f requent ly confused with those o f w i l t o r r o o t r o t s . I t i s therefore ,

necessary t o observe t h e suspected p l a n t s ca re fu l ly and the v i r a l

na tu re needs t o be confirmed by s p e c i f i c procedures (Kaiser and Danesh,

197 l a ) .

The present inves t igat ions were undertaken with t h e o b j e c t i v e

o f i s o l a t i n g v i ruses a f f e c t i n g chickpeas in nature . Wo mechanically

t ransmiss ible v i ruses ; v i z . , cucumber mosaic v i rus (CMV) and bean ye l low

mosaic v i r u s (BYMV) were i so la ted and t h e i r i d e n t i t y was confirmed by

procedures such a s host range, biophysical proper t ies , i n s e c t t r a n s - . . mis$ion, serolbgy, and e lec t ron, microscopy. I n addi t ion, production

of antiserum and screening of germplasm l i n e s f o r d i sease r e s i s t a n c e

were a l s o attempted.

REVIEW OF LITERATURE

I. MICKPEA VIRUSES

S i x v i ruses have been reported t o i n f e c t chickpea from

d i f f e r e n t p a r t s of the world (Nene, 1980). These were: a l f a l f a mosaic

v i r u s from Algeria, India , Iran, Morocco and U.S .A. ; bean yellow mosaic

v i r u s from I ran and U.S.A.; cucumber mosaic v i rus from Colombia, India ,

I ran and USSR; l e t t u c e n e c r o t i c yellows v i r u s from Austra l ia ; pea l e a f

r o l l v i r u s from Algeria, Bangladesh, Ethiopia, India, I ran , Lebanon,

b r o c c o , New Zealand, Pakistan, Sudan, Syria, Tunisia, and Turkey;

pea enat ion mosaic v i r u s from U.S.A.

I n India, four v i ruses have been reported t o n a t u r a l l y occur

on chickpea; v i z . , a l f a l f a m s a i c v i rus (Nene e t at., 1978a), pea l e a f

r o l l v i r u s (Nene and Reddy, 1976; Nene et at. , 1978a; ICRISAT, 1980),

cucumber mosaic v i r u s (Dhingra e t aZ., 1979) and an un iden t i f i ed v i r u s w

causing l e a f d i s t o r t i o n and k s a i c (Mali and Vyanjane, 1980).

11. CUCUMBER MOSAIC VIRUS (CMV)

A: F i r s t Description

The v i r u s was f i r s t described by Dool i t t l e (1916) and Jagger

(1916) i n cucumber. The natura l occurrence of W on chickpea was

f i r s t repor ted by Kaiser e t a2 . (1968). In India i ts na tu ra l occurrence

on chickpea was repor ted by Dhingra e t at. (1979). -

B, Symptoms on Chickpea

Kaiser and Danesh (1971a) repor ted t h a t W caused bushiness

and shortening of internodes i n chickpea. Dhingra e t aZ. (1979) a l s o

observed diseased p lan t s both infected natura l ly and experimentally,

developed ( i n 3-4 weeks a f t e r infect ion) excessive a x i l l a r y shoots

bearing very small, narrow, s l i g h t l y chlorot ic , and deeply denta te

leaves. Affected p lan t s remained stunted giving a bushy appearance.

Phloem of roo t s and c o l l a r region stem developed s l i g h t necros is i n

severe cases of infect ion. Diseased plants bore fewer flowers and

pods than t h a t of the healthy, and plants infected a t a very ea r ly

stage of growth died prematurely, Similar symptoms were observed i n

p lan t s a r t i f i c i a l l y inoculated with the causal v i rus .

C. Host Range

Host range of CMV a s reported by Pr ice (1940), Komuro (1958,

1973), Smith (1972), and Douine e t aZ. (1979) i s given below:

A 1 ZOACEAE

APOCYNACFAE . 9 '

ASCLEPIADACEAE

BALSAMINACEAE

BOUGINACEAE

BROMELIACEAE

CAPPARI DACEAE -

CARRY OPHY LLACEAE

CHENOPODIACEAE

: Mesmhyanthwn crystQZZinwn L . , TetragoniQ e;.cpansa Mum.

: W a n t h u s caudatus L., b a n t h u s r e t r o f l m L.

A. tricoZor L. , Celosia argent& L.

: Vinca minor L. , V. rosea L.

: ~ s c ~ e p i a s ' &iaca L . : Imptiens baZsamina L .

: Anchus sp. , CynogZosswn d i Z e Stapf and Drummond . , BeZiotropiun corynboswn Rui z and Pav . , H. perwiunun L , , Uyosotie syluatioa Hoffm.

: Amnas cornsus Merr.

: Lobetia cardiml is L.

: CZeome spinosa L . , Polanisia trcrchyspemna Torr and

Gray.

: Lychnis aZ& M i l l . , L. chakedonica L. , L. visoaria L.

: Beta vulgaris L . , Chempdiwn atbun L., C. mmZe L. ,

Spimcicr olemoea L.

: Cometinu commmis L., C. errsctu L . , C . nudiflora L . ,

lbadeeoanticr sp., Zebrinu p d u Z a Schni zl .

COMPOS I TAE : hnbrosia e ta t ior L . , CaZenduZa of f ic inaZis L . ,

C a l l i s k p h w chinensis Nees., Centaurea moschata

L . , Chrysanthemum sp. , Cichoriwn endivia L.,

&ilia sagi t t a ta (vahl) Dc., Helianthus annuus L . , H. debi l i s Nutt . , Helichrysm bmc teatwn Andr . , Lactwa satiuz L., Tagetes erecta L . , T. ptula L. , Zinnia ezegans Jacq.

COWOLVULACEAE : CuscutQ subinclusa Durr., C. cazifornica Hook 6 Am.,

Ipmoea batatus Lam., I. pwpurea Lam.

CRUCIFERAE : B m s s h a mpa L . , LobuZaria mritim Desv.,

Nasturtiwn of f ic inaze R. Br.

CUC URB I TAC EAE : Benincasa cerifera Cogn,, B. hispida Cogn., B ~ y o n k

aZba L., B. dwica L . , Bryonopsis lacimosa

Naudin., Chayota edulis Jacq., CitruZZus vulgaris Schrad., C w d s angwia L . , C. f i c i - f o l k Bouche., C. flellruosus Naudin., C. grossu-

h k e f o Z m i 6 Hort., C. melo L . , C. m t u z i f e r w -6

May., C. odoratissirnus Moensch., C. sativus L.,

C. uti l issima Roxbg. Cucmbita marima Duchesne.,

C . .mschata Duchesne., C. p e p L . , EcbQZZiwn' . .

e l a t e r i m A. Rich., Kedrostis foetidissima Cogn.,

Lugenaria leucantha Rusby., L. vulgaris Sch.,

Luffa acutanguk Roxb., L. cyZi&ica Roem.,

MeZothria scabra Naudin., ~ c n m r p e l i s l o b a h

(Mich.) Greene., Memrdica balsamina L . , M.

charantia L . , M. involwrata E. Meyer . , Physedm

barteri Cogn., Sicyos angutatus L., bichosanthes

- anguim L .

DIPSACEAE : Dipsacus sp,, Scabwsa atropurpurea L. , S. japonka

Miq . EUPHORB I ACEAE : hcphorbiu sptendens Bajer.

GERANI ACEAE : Cemniun cumlinianwn L., Pehgoniun hortorwn Bailey.

GRAMINEAE

HY DROPHY LL ACEAE

LAB I ATAE

LEGUMINOSAE

LILIACEAE

LOBELIACEAE

LOGAN I ACEAE

LY mRAC Ep . MALVACEAE

. t

MUSACEAE '

PASSIFLORACEAE

PHYTOLACCACEAE

POLEMONIACEAE

POLYGONACEAE -

P ORTUL AC AC EAE

RANUNCULACEAE

S CROP ULAR I AC EAE

: EuchZuena m e a m Schrad., HoZcus sorghwn L ., SeoaZe cereale L. , Ditiown aestivwn L , , Zea

m y s L. : PhaceZia tanacetifoziu Benth., P. whitlavia Gray,

: CoZeus blwneri Benth., Nepetu cataria L. , Salt&

spZendens Ker . : Cicer arietinun L . , CrotaZariu intermedia Kotschy.,

Lupinus angwtifoZius L. , L. leteus L . , Phaseolus

a m u s Roxb., P. lumtus L . , P. vulgarw L . ,

Piswn sativun L. , PifoZiwn incarmtmn L., V i c i a

faba I,. , Vigna sinensis End1 . : A Z Z i w n c e p L . , Hyacinthus orientalis L., LiZiwn

awrztwn Lindl ., L. candidun., L. formosanwn

Stapf., L. crocewn Chaix., L. Z0ngifLort.m Thunb.,

L. testacewn Lindl. , L. hrzrfisii Carr., L. tri-

grinwn Ker-Gawl . , filip gesneriana L .

: b b e l i a cardimzis L . , L. erinua L . , L. tenuitor

R . Br.

: Buddleia sp. : L y t h u n salicaria L.

: Hibiscus rianihot L . , Lcrtxrtem tmhestr is L .

: Musa cavendishii Lamb. M. paradisiuca Kuntze.,

M, sapbntun Cowan., M. t e x t i l i s Nees.

: &ssi f lom edulis Sims.

: Phytohca decandra L.

: Cilia capituta b u g l . , G. l ini f lora Benth ., PhZm d r m n d i i ~ o o k .

: Fagopyrun esculentta Gaertn., Rheun rhaponticm L.,

Rwnex crispus L.,

: B~tulaoa g m n d i f l m Hook.

: AqrciZegb camdensis L., Detphimwn consolida L.

: Antim?tinwn mjus L. , CymhaLark muralis Gaertn.,

Mey and Scherb, M - h . d ~ 8 mo8cham bug1 . ,

SOLANACEAE

Nemesk strwnosa Benth., Pentstenron sp. , Verbascwn phoenicewn L., Vemnica longifolia L., ZaZuzian-

skya vi l losa Schmlt.

: Atropo belladonna L., BromZZia specwsa Hook.,

Capsicwn annuwn L , , C. frutescens L , , Dztura mebtoides hnal. , D. metal L., D. stmmoniwn L., Hyo6cyamus niger L., Lycopersicon escuzenhm

M i l l . , Nicandra physaZoides (L.) Pers,, Nicotiunu g k w a Grah., N. glutinosa L. , N. b igelovi i Wats.,

N . rust ica L . , N, sanderae Sander, N. sy tves t r i s

Spegaz and Comes, N. tabacwn L. , Petunia hybrida V i l m . , P. v iohcea Lindl . , Physalis alkukengi

L . , P. anguIsrta L, , P. heterophylk Nees., P.

Isrgasoae R . and S. , P. peruviana L . , P. pubescens L., P. s u b g k h t a MacKenzie and Bush., Salpi- gZossis sinwzta Ruiz and Pav., S c h i m t h u a

wisetonemis Low., Solanwn aviculare Frost . ,

S. carolinense L. , S. melongem L. , S. nigrwn

L. , S. n o d i f l o r n Desv., S. tuberoswn L .

THYMELAEACF , : h p h rnezerewn . . L .

TROPAEO LACEAE : Tr.apaeoZun m jua L. , T. p e r e p i m L . ,

UMBELL IFERAE : Anethwn gmveolens L., Anthriscus cerefoziwn Hoffm.,

Apiwn p v e o l e n s L., Azucus camta L. , FoenicuZun

vulgare H i 1 1, Pas t i m a satiua L . , PetroseZinm hortense Hoffm.

V IOLACAEA : V i o k cornuta L.

D.- 1n v i m Properties

The The has thermal inact ivat ion point between 60' and ~o'c, the d i l u t i o n end point between 1:1000 and 1:10,000 and the longevity

i n v i t ro between 3 and 4 days (Gibbs and Harrison, 1970) . However,

d i f f e r e n t i s o l a t e s of t h e v i r u s d i f f e r i n t h e i r i n vitro proper t ies .

The reported i n v i t m propert ies fo r d i f f e r en t is01 a t e s / s t r a i n s a r e

given below:

TIP (OC) DEP L I V Virus source Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65-70 10" - 10 - 4 - P r unus Willison and

Weintraub (1957)

65-70 1 : 5000-1: 10000 - Chickpea Dhingra e t aZ. (1979)

60-65 1: 10000-1: 20000 3 days Sugarbeet Ragozzino (1973)

55-60 lom4 1 &Y Zinnia Sas t ry e t aZ. (1974)

80-90 1 0 ' ~ - 10" 35-48 hrs Ridge gourd Gael and Verma (1973)

68-70 lom4 - 10" 19-22 days Wild cucumber Horvath and Szirmai (1973)

7 0 3-5 days Cowpea Talens (1979)

65 l o m 4 5 days Gladiolus Kanikska (1977)

6 8 1 : 2000 10-12 days Cucumber Topchiiska and Topchiiski (1977)

50-55 1: 1000-1:5000 20-22 days Potato Matsunami e t aZ. (1972)

3

TIP = Therml inac t iva t ion point; DEP = Dilution end point; LI,V = I,ongevity i n vitro.

E. Transmission

1. Mechanical

- The UdV i s readi ly sap-transmissible inducing a var ie ty of

symptoms depending on v i rus s t r a i n and host cu l t i va r (Francki e t aZ.,

1979).

2. Graft - Graft transmission of UW has been documented by Schmidt and

The reported i n v i h w propert ies fo r d i f f e r en t i s o l a t e s / s t r a i n s a r e

given below:

TIP (OC) DEP LIV Virus source Reference

65-70 lom3 - 10 '~ - Prunus Willison and Weintraub (1957)

65-70 1: 5000-1: 10000 - Chickpea Dhingra e t aZ. (1979)

60-65 1: 10000-1: 20000 3 days Sugarbeet Ragozzino (1973)

55-60 10 '~ 1 day Zinnia Sas t ry e t aZ. (1974)

80-90 - 35-48 hrs Ridge gourd Goel and Verma (1973)

68-70 lo'4 - lo1' 19-22 days Wild cucumber Horvath and Szirmai (1973)

70 3-5 days Cowpea Tal ens (1979)

65 5 days Gladiolus Kanikska (1977)

6 8 1: 2000 10-12 days Cucumber Topchiiska and Topchiiski (1977)

50-55 1: 1000-1: 5000 20-22 days Potato Matsunami e t aZ. (1972)

- TIP = Thermal inac t iva t ion point; DEP = Dilution end point; LJV = Longevity i n uitro.

E . Transmission

1. Mechanical

- The i s readi ly sap-transmissible inducing a va r i e ty of

symptoms depending on v i rus s t r a i n and host cu l t i va r (Francki e t at . ,

1979) .

2 . Graf t - Graft transmission of C3.N has been docmented by Schmidt and

Karl (1970) i n case of sann hemp, Jones (1976) i n case o f wineberry,

and Dhingra e t a t . (1979) i n case o f chickpea.

3. Seed - The CMV has not been reported seed t ransmiss ible i n chickpea.

However, seed transmission occurs t o varying degrees i n 18 spec ies

(Neergaard, 1977), including some weed species. The hosts i n which

CMV is seed t ransmiss ible a r e given below with the transmission percent-

ages i n parentheses.

Benincasa hispi& ( I%) , Capsicwn a n n m ( I % ) , Cemstiwn holo-

s t e o 2 e s ( 2 % ) , Cucwnis me20 and C u d s sativus ( t r a c e ) , Ctdc~crbitu

moschata (0.7%), Cuctirbitz pepo and Echinocystis Zobata (9%), h i m

pmpirrewn (4%), Lupinus Zuteus (14%), ~ y c o ~ e r s i c o i escuzentwn (0.2%),

Phaseolus aureus ( 5 % ) , P. vuZgaris ( 7 % ) , SperguZa arwnsis (2%),

Stel laria media (21-40%), Vi'igna cyZindrica cv. k n a r c h , V . sesquipedalb,

V , tolguiculatu (4-28%).

4 . Dodder

Schmel zer (1957) obtained pos i t ive transmission o f CMV through

8 species of Cuscuta, v i z . , C. cqrnpestris, C. cali fomica, C. subinc'lusa,

C. gronovii, C. americuna, C. e m p e a , C. epithymwn, and C . ZupiZi- formis. In chickpea, p o s i t i v e transmission of the v i r u s through

C. r e f l a was obtained by Dhingra e t aZ. (1979).

5 . Insec t - CMV is t ransmiss ible i n a non-persistent manner by a number

o f aphid species . The aphid species involved i n t h e transmission a r e

1 is ted below :

Aphid species

Amphorophoro rhododendri

A. rubi A. sonchi Aphis cardui

A. craccivora

A. euoymi A. fabae A. gossypii

Brevicoryrre brassicae Rzc tyno tus hen& h i

Qsaphis era taegi

~ c h i m c y s t i s lobata

~is-ke+on&z*sktariae

k ros iphwn b m i M.' caZifornicwn M. duphnidis

M. euphorbiae

M. gei M. p i s i M , rosae M. sokmi fo l i i M. sonchifolii &aocaZZis asclepiadia

Virus source

Gladiolus

Raspberry

Gladiolus

Gladiolus

Capsicm amuwn

Chickpea

Pumpkin

Cucumber

Cherry

Cantaloups

Cucumber

Pumpkin

- Cucumber

Cantaloups

Cantaloups

Cherry

Cucumber

Cucumber

- Cantaloups

Gladiolus

Pumpkin

Gladiolus

Cucumber

Gladiolus

Reference

Swenson and Nelson (1959)

Harrison (1959)



Szalay-Marzso and Solymossy (1961)

Dhingra e t aZ. (1979)

Forghani e t aZ. (1966)

McClanahan and Guyer (1964)

S z a l a y - b r z s o and Solymossy (1961)

Swenson and Marsh (1967)

Goudriet (1962)

Heinze (1959)


Karl (1971)


Goudriet (1962)

Goudriet (1962)

Swenson and Marsh (1967)

Heinze (1959)


Vovk (1956)

Goudriet (1962)






Aphid species Virus source Reference

M. Zythri Cherry Swenson and Marsh (1967)

M, persicae Cherry Swenson and Marsh (1967)

Pen taZonia nigroner uosa Banana Medeiros (1963)

Phorodon cannubis Sann hemp Schmidt and Karl (1970)

RhophaZosiphwn m i d i s Cantaloups Goudriet (1962)

R. pisi Gladiolus Swenson and Nelson (1959)

F. P u r i f i c a t i o n

Several methods f o r p u r i f i c a t i o n of CMV have been used most

of which a r e modifications o f t h e one devised by S c o t t (1963). The

d i f f e r e n t methods used a r e summarized below:

1. The method of S c o t t (1963)

Infected leaves were homgenized i n 0.5 M sodium c i t r a t e ,

pH' 6.5, conta ining 5 mM EDTA and 0.5% th iog lyco l l i c a c i d . Af te r

s q u e e ~ i n p th,rqugh cheesecloth. t h e e x t r a c t was emulsified with ch lo ro l

form. The aqueous phase obtained a f t e r low speed c e n t r i f u g a t i o n was

d ia lysed aga ins t 0.005M borate buffer , pH 9.0 and subjected to t h r e e

cycles of d i f f e r e n t i a l centr i fugat ion.

Tremaine (1968) reported more s a t i s f a c t o r y preparat ion of Y

s t r a i n o f CMV by using S c o t t ' s method.

- Takanami and Tomaru (1969) improved S c o t t ' s method i n which

sephadex gel f i l t r a t i o n and EDTA were used t o avoid aggregation of the

v i r u s .

2 . The method of Lot e t aZ. (19721

They modified Sco t t ' s method i n which v i r u s was p r e c i p i t a t e d

by polyethylene glycol (10%) which was added to the aqueous phase,

obtained a f t e r chloroform emulsification. After incubat ion, it was

centrifuged a t 12,000 g f o r 10 min and the sediment was resuspended

i n 5mM sodium borate buffer , 0.5mM EDTA, pH 9.0, t o which Tri ton X-100

was added t o 2% and s t i r r ed . I t was then subjected t o three cycles

of d i f f e r en t i a l centrifugation. The v i rus was fur ther pur i f ied by

centr i fugat ion i n 5-25% sucrose density gradients (Francki, 1972).

3. The method of Tomlinson e t aZ. (1958)

Infected leaves were homogenized i n O.SM potassium phosphate

buffer , pH 7.5, containing 0.3% thioglycol l ic acid, Clar i f ica t ion of

the f i l t e r e d sap was done by 8.5% n-butanol. The v i rus was recovered

by one cycle of d i f f e r en t i a l centrifugation, resuspending the high

speed p e l l e t s i n 0,05M potassium phsophate buffer , pH 7.5.

Tomlinson e t aZ. (1959) used the same method a s above, but

fu r the r pur i f ied the virus by prec ip i ta t ion with 10% a c e t i c acid

followeQ by fur ther d i f f e r en t i a l centrifugation.

4. The method of Shohara and Osaki (1975)

They purif ied CMV by repeated prec ip i ta t ions with 8% PEG

and 0.2M NaC1, followed by PEG s o l u b i l i t gradient (0-6% PEG s t ab i l i zed

i n 35-5% sucrose) centrifugation.

5. Other methods

Other methods f o r CMV pur i f ica t ion (Morris e t aZ., 1978;

Mossop e t aZ., 1976; Watemorth and Povish, 1975) involved s l i g h t

modifications of one o r the other of the above methods.

Virus yields varied according to the virus s t r a i n and host

p l a n t used, but over 500 mg virus/kg o f l ea f material have been obtained

i n some ins tances (Francki e t at., 1979).

G . Proper t ies of Virw P a r t i c l e s

1. S t ruc tu re

The CMV p a r t i c l e s are isometric, about 28-30 nm i n diameter

(Francki e t ale , 1966, 1979; Debrot e t a l . , 1974; Betto e t aZ., 1964).

Waterworth and Povish (1975) reported t h a t a s t r a i n of CMV from Ixora

p lan t s (CMV-IX) d i f fe red i n having p a r t i c l e s of 24 nm i n diameter r a t h e r

than 28 nm reported f o r o the r CMV s t r a i n s . P a r t i c l e s of a s t r a i n of

CMV from Prunus measured 35 w (Willison and Weintraub, 1957). Matsunami

e t aZ, (1972) observed CMV p a r t i c l e s from potato to be 33 m i n d ia -

meter. The p a r t i c l e s of CMV a r e known t o d i s rup t when mounted unfixed

i n neutra l phosphotungstate fo r e l ec t ron m i c r o s c o ~ (Francki e t aZ.,

1979). However, p a r t i c l e s remain i n t a c t and contras t well i n uranyl

ace ta te , pH 4.5 (Francki e t aZ., 1966). .+-

2. G e d i d n t a f i ~ n coef f i c i en t ($~OW)

a The p a r t i c l e s of CMV sediment a s a s ing le component with

S20W (Swedbergs) of 98.6 (Francki e t aZ., 1966). A s l i g h t l y higher

sedimentation coef f i c i en t (101s) was reported by Tomlinson e t aZ.

(1959).

0 1% 3 . Extinction coef f i c i en t (E260

- I t has been determined as 5.0 (Francki e t aZ., 1966).

4. Az6o/Az80 r a t i o

I t has been determined a s about 1.7 (Francki e t aZ ., 1979).

5. Buoyant densi ty

I t has been determined a s 1.361 and 1.359 g/ml f o r CMV-S and

CMV-D, respec t ive ly (Lot and Kaper, 1976).

6 . Nucleic ac id

The CMV p a r t i c l e s contain single-stranded r ibonuc le ic ac id ,

about 18% of p a r t i c l e weight (Kaper and Re, 1974). The f a c t t h a t CW

contains a func t iona l ly divided genome has been revealed by Peden and

Symons (1973), who detected four species of RNA.

The exact molecular weights of t h e CMV RNAs i s a s u b j e c t o f

debate. Values, ranging between 1.01-1.35, 0.89-1.21, 0.68-0.93 and

0.33-0.38 x106 da l tons have been obtained f o r RNAs 1, 2, 3 and 4,

r espec t ive ly (Kaper and West, 1972; Peden and Symons, 1973; Marchoux

e t aZ., 1973). However, e lec t rophore t i c mobi l i t i e s o f RNA species

may d i f f e r s l i g h t l y between s t r a i n s (Hanada and Tochihara, 1980;

b s s o p , unpublished da ta c i t e d by Francki e t aZ., 1979).

. . A . sqal l , r ep l i ca t ing , single-stranded s a t e l l i t e RNA o f ab&t 5 1 x10 dal tons has been i s o l a t e d i n some s t r a i n s o f CMV (Mossop and

Francki, 1977; Could e t aZ., 1978). One of these, c a l l e d CARNA 5

(cucumber mosaic v i r u s associa ted RNA 5) appeares t o be responsible

f o r l e t h a l necros i s o f tomato (Kaper and Waterworth, 1977).

7 . Pro te in

- The CMV can be read i ly disrupted i n t h e presence of sodium

dodecyl sulphate (SDS) (Kaper, 1975). Analysis of SDS-treated v i r a l

p ro te in i n polyacrylamide gel e lec t rophores i s revealed t h a t t h e

p r o t e i n consis ted o f a s i n g l e polypeptide o f 24,500 da l tons (Habil i and

Francki, 1974) t o 25,200 da l tons (van Regenmortel e t a t . , 1972).

H. Serology

The immunogenicity o f t h e v i r u s is poor but , can be enhanced

by f i x a t i o n wi th formaldehyde (Francki and Habi l i , 1972; Habi l i and

Francki, 1975). The v i r u s p r e c i p i t a t e s on exposure t o physiological

sa l t so lu t ions and, therefore , se ro log ica l t e s t s a r e usua l ly done by

agarose double di f fus ion t e s t s i n buf fe r s o f low molar i ty o r i n water

(Francki e t aZ., 1966; Sco t t , 1968). Unstabil ized v i r u s may give one

o r two p r e c i p i t i n l i n e s , one near t h e ant igen wel l , caused by more o r

1 ess i n t a c t v i r u s and a second one near t h e antiserum well caused by

degraded v i r u s (Scott , 1968; Devergne and Cardin, 1970).

In i t s physical and chemical proper t ies , CMV has a f f i n i t i e s

with tomato aspenny v i r u s (TAV) and peanut s t u n t v i r u s (PSV) (Francki

e t aZ., 1979). More re l i ance has been placed on t h e r e s u l t s o f

se ro log ica l t e s t s than any o ther s ing le c r i t e r i o n t o t r a c e the degree

of r e l a t i o n s h i p s among the th ree v i ruses (Habil i and Francki, 1975) . Thorough serological ana lys i s o f CMV s t r a i n s indicated t h e presence of

a t l e a s t two serogroups both of which a r e se ro log ica l ly r e l a t e d t o

sohe' st;aina b f TAV and PSV (Devergne and Cardin, 1975). In c o n t r a i t ,

Hab i l i and Francki (1975) f a i l e d t o de tec t any serological r e l a t i o n s h i p

between t h e Q-CMV (Queensland s t r a i n of CMV) and V-TAV (Vic to r ia s t r a i n

of TAV). The lack of agreement on the serological r e l a t i o n s h i p between

TAV and W has been probably due t o the use o f d i f f e r e n t s t r a i n s o f

t h e v i ruses which appear t o be very var iable , and t o the l ack o f h igh

t i t r e d a n t i s e r a due t o the poor immunogenicity of t h e v i ruses (Rao,

1982). However, Rao (1982) showed by agar double-diffusion t e s t and

ELISA, a d i s t a n t serological r e l a t i o n s h i p between s i x s t r a i n s o f CMV

and two of TAV.

I . S t r a i n s

Many symptom var ian t s o f W a r e known t o occur making t h e

v i r u s o f ten d i f f i c u l t t o i d e n t i f y from symptoms alone. S t r a i n s well

character ized by t h e i r b iological proper t ies include t h e Y s t r a i n

which i n f e c t s cowpea systemically (Price, 1934), t h e spinach s t r a i n

(Bhargava, 1951) i s o l a t e d from ch loro t i c and deformed leaves o f

spinach, which produces l o c a l l e s ions i n several hosts , and M s t r a i n

o r i g i n a l l y i so la ted from Pr ice ' s (1934) yellow s t r a i n which induces

severe ch lo ros i s i n several p lan t species (Mossop e t aZ., 1976) . The

(5N s t r a i n s characterized physico-chemically include the Y s t r a i n

(Kaper e t aZ., 1965), the Q s t r a i n (Francki e t aZ., 1966; Habi l i and

Francki, 1974) and the S s t r a i n (van Regenmrtel e t aZ, , 1972).

However, o the r s t r a i n s reported a re :

r CMV-Ixora (Watemorth and Povish, 1975) which d i f fe red

from o ther s t r a i n s i n not inducing f e r n l e a f symptoms i n

Lycopersicon escuZentwn cv. Rutgers. I t a l s o produced a

few la rge ch lo ro t i c spots i n C u c k s s a t i v u s r a t h e r than

in fec t ing systemically.

r CMV-Comelina s t r a i n (Dool i t t le and Webb, 1960).

CMV-Cowpea s t r a i n (Anderson, 1955a) .

r CMV-Easter l i l y s t r a i n (Brierley and Travis, 1958).

CMV-Prunus s t r a i n (Willison and Weintraub, 1957)

a. Yield Losses

Kaiser e t aZ. (1968) i n a f i e l d inoculation t r i a l i n chickpea

wi th CMV reported a y i e l d l o s s of 52% due t o in fec t ion . 'Ihe y ie ld

losses depended on the s t age of p lan t in fec t ion . The v i rus caused

about 98% and 87% decrease i n chickpea y ie lds when p lan t s were inocu-

l a t e d a t 4 weeks (pre-bloom) and 9 weeks ( f u l l bloom) a f t e r p lant ing,

respect ively (Kaiser and Danesh, 1971b) .

K. Screening o f Germplasm

There is no repor t i n l i t e r a t u r e regarding screening o f

chickpea germplasm agains t CMV.

111. BE&' YELLOW MOSAIC VIRUS (BYMV)

A. F i r s t Description of the Virus

Pierce (1933), while working on the r e l a t i v e r e s i s t a n c e o f

bean v a r i e t i e s t o common bean mosaic (Bean v i rus I ) , i s o l a t e d another

v i r u s from a p lan t o f t h e Red Valentine va r ie ty . I t s s p e c i f i c i t y was

f i r s t suspected no t only from the f a c t t h a t it produced symptoms

d i s t i n c t from t h a t of the common bean mosaic v i rus , but a l s o because

it a f fec ted the v a r i e t i e s - Corbet Refugee, Great 'Northern U1 No.1

and Robust, which a re r e s i s t a n t t o common mosaic. The v i rus was

designated a s bean v i rus 2 (Bean yellow mosaic v i r u s ) . Occurrence o f

BYMV on chickpea which caused yellowing was f i r s t reported by Erwin

(1958), and Erwin and Snyder (1958) . However, they a t t r i b u t e d t h e

diskage t o othkr v i ruses a l s o ; . The f i r s t authent ic evidence on t h e

na tu ra l occurrence of t h i s v i rus on chickpea was provided by Kaiser

and Danesh (1971a). In India, the re i s no repor t on the na tu ra l

occurrence of BYMV on chickpea.

B . Symptoms on Chickpea

..- Kaiser and Danesh (1971a) repor ted two s t r a i n s of BYMV on

chickpea; a v i r u l e n t s t r a i n causing wi l t ing and yellowing, and a l e s s

v i r u l e n t s t r a i n inducing feathery and deformed leaves.

The sca t t e red incidence o f aphid-transmitted v i r u s yellowing

has been reported by Erwin and Snyder (1958) i n the causation o f which

the bean yellow mosaic virus is s t a ted t o be a dominant f ac to r , though

o the r aphid-transmitted viruses, including pea enation mosaic and a l f a l f a

mosaic v i ruses a l s o i n f e c t t h i s host i n Cal i fornia .

?he b r i g h t yellow color o f the leaves and discolourat ion of

the stem t i s s u e s i n the phloem o f p lan t s a f fec ted with a virus-induced

yellows disease was a t t r i b u t e d t o bean yellow m s a i c and poss ibly o the r

v i ruses (Erwin, 1958).

C. Host Range

BYMV, i n i t i a l l y thought t o be r e s t r i c t e d t o leguminous p lan t s ,

i s now known t o i n f e c t several non-leguminous p l a n t s (Bos, 1970a). Host

range o f the v i rus under d i f fe ren t famil ies i s given below,

A 1 ZOACEAE : Tetrugonia expansa Murr . AMARANTHACEAE : Gomphrena globma Murr.

CHENOPODIACEAE : Chenopodiwn m a n t i c o Z o r Coste e t Reyn ., C. quinoa Willd. . .

LEGUMINQSAE : AstragaZus s inkus Thunb . , Cassia t o m L. , Cicer crrietinwn L. , CrotoZaria spec tab i lb Roth., Glycine

nux Merr., Lathyrus odoratus L., Lars esculenta Moench., Lespedeza capitutu Michx., Lwinus aZbus L., L. Zuteus L . , L. pilosus Walt., MeliZotus alba Desr., M. dentatua Pers., M. o f f i c i m l i s Lam.,

PhaseoZus trulgaris L . , P. acutifoziuc Gray, P.

a m u s Roxb., P . coccineus L . , P . c a l w m t u s Roxb.,

Piswn satitrum L., Difo2it.m arvense L., T. compestre

Schreb., T . bbridwn L., T . incarmtwn L. , T.

micheZimrwn Gaud. ,T. prcrtense L. , T. repens L. ,

T. v e r s i c u l o m Savi. , V i C k faba L. , V. hirsutu

Gray, V. narbonenszk L., V . ViZZosa Roth.,

Vim sesquipedalis Wight.

PEDALIACEAE : SesumunindicwnL.

SOLANACEAE : Nicotiana benthamiam Domin.

D. In vitro Proper t i e s

In vi tro p r o p e r t i e s depend g r e a t l y on v i r u s source, t e s t

p l a n t and t e s t condi t ions , The thermal i n a c t i v a t i o n po in t i s u s u a l l y

between 55 and 6 0 ' ~ though i t may range between 50 and 70°c, t h e

d i l u t i o n end po in t usual ly - l om4 , and longevi ty i n v i tro normally

1-2 b u t some times over 7 days (Bos, 1970a).

I n vitro proper t i e s reported f o r some of t h e i s o l a t e s l s t r a i n s

o f t h e v i r u s a r e given below:

TIP(OC) DEP LIV Virus source Reference .-------------------------------------------.

60-70 - 2 days Red c lover Kme e t aZ. (1970)

55-60 lo-' - 2-3 days White beans Tu (1980)

60-62 , - l ow3 1 day Broad bean Shagrun (1973)

1 day Broad bean El-Attar e t a l . (1971)

50-55 1: 3000-1:5000 French bean Tosso and Vidal (19 76)

55-60 1:500-1:1000 1-2 days Broad bean Tho t tapp i l ly e t aZ. (1976)

TIP = Thermal i n a c t i v a t i o n point; DEP = Dilut ion end point ; L I V = Longevity in uitro.

E . Transmission

1. Mechanical

The v i r u s i s r e a d i l y mechanically t r ansmiss ib le (Bos, 1970a)

2 . Graft

No g r a f t transmission of t he v i rus has been reported i n t he

1 i t e r a t u r e .

3. Dodder

Schmelzer (1957) f a i l e d t o transmit BYMV through e i g h t

Cuscuta spp., which could transmit CMV.

4. Seed - The v i rus is not seed-borne i n French bean, but t ransmit ted

t o a small percentage i n Lqinus albus, L. luteus, MeZiZotus atba, Piswn sativwn and V i c k fQba (Bos, 1970a; Neergaard, 1977) .

5. Insect

More than 20 aphid spp. can transmit t he v i rus i n a non-

pe r s i s t en t manner (Kennedy e t a t . , 1962). Different aphid spp. \

re&rted t o 4)ansrnit the viru.s a r e given below:

Aphid species

Acyrthosiphon pis ton

A . sesbaniae

Amphorphom sp.

A . sonchi

Anuraphis t u Z i p e A: baker-i

Aph i s oardui

A . orac&wm

A . f a k e A . gossypi i

Virus source

Chickpea

Chickpea

Broad bean

Broad bean

Broad bean

White clover

Broad bean

Chickpea

Broad bean

Broad bean

Reference

Kaiser and Danesh (1971b)


Swenson (1957)

Swenson (1957)

Swenson (1957)

Manglitz and Kreitlow (1960)

Swenson (1957)


Kaiser e t aZ. (1971)


Aphid species

A. helianthi A. medimginis

Capi tophor us f iagaefotii HyaZopterus a tr ipz ic i s hhcrosiphwn barri

M. g r a m r i m M . rosae M. e,>ZanifoZii

Megowrz d c i u e Myzus c e m s i M. certus

M. persicae M . sotani

Virus source

Broad bean

Lupins

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Broad bean

Reference


Corbett (1958)

Swenson (1957)

Swenson (1957)

Swenson (1957)

Swenson C1957)

Swenson (1957)

Swenson (1957)

Tapio (1964)

Swenson (1957)

Swenson (1957)

Swenson (1954)

Swenson (1954)

&zus persicae was found t o be the most e f f i c i e n t vector

(Swenson, 1957; Aldez, 1959), followed by Mzcrosiphwn p is i and

~ n k z ~ h i s tuZipae (Swenson, 1957) . Anuraphis sonchi was found t o be

very i n e f f i c i e n t vector whereas Brev-icorp bmssicae f a i l e d t o

transmit , t h i s v i rus (Frandsen, 1952; Swenson, 1954; Swenson, 1957) .

F. Pur i f i ca t ion

Viruses of the potato Y group, to which BYMV belongs, a r e

c h a r a c t e r i s t i c a l l y d i f f i c u l t t o manipulate i n vi tro because o f t h e i r

tendency t o aggregate, both end-to-end and l a t e r a l l y (Shepherd and

Po-md, 1960). However, various methods have been followed s a t i s f a c t o r i l y

f o r pur i f i ca t ion of BYMV which a r e given below:

1. The method of Uyeda e t aZ. (1975)

They described pur i f i ca t ion of i s o l a t e no. 30 of BYMV

propagated i n broad bean. Homogenization of frozen leaves was done

i n 0.1M Tris-HC1 buffer, pH 7.0, containing 0.05M EDTA and 1% 2-Mercapto-

ethanol. After passing through cheesecloth, the e x t r a c t was emulsified

with 4 volume of carbon te t rachlor ide . 'Ihe v i rus was recovered from

aqueous phase by precipi ta t ion with PEG (4%), followed by d i f f e r e n t i a l

centr i fugat ion. The v i r u s pe l l e t s were suspended i n 0.01M phosphate

buffer . To avoid aggregation, 0.5M urea was added t o t h e suspending

medium. Further pur i f i ca t ion was achieved by 10-40% sucrose dens i ty

gradient centr i fugat ion.

2, The method of Huttinga (1973)

He pur i f ied BYMV by applying moderate centr i fugal fo rces .

Infected pea leaves were homogenized i n 0.1M t r i s - t h i o g l y c o l l i c ac id ,

pH 9.0, along with carbon te t rachlor ide and chloroform (40 m l each

f o r 150 m l of buf fe r ) . The virus was concentrated from aqueous phase

by applying 2 cycles of d i f f e r e n t i a l centr i fugat ion (1.5 h r a t 26,500 g

and 10 min a t 8000 g) . The virus was suspended i n 0.lM tris-HC1 buf fe r U'

pH 9.0. The low centr i fugal forces yielded unaggregated v i r u s with

neg l ig ib le breakage. On the contrary, higher centr i fugal fo rces

proguced %consi$erable amounts of broken p a r t i c l e s . * . .

3.- The method of Granett and Prowident i (1975)

They pur i f ied two s t ra ins , v i i . , BYMV' and BYMY' by using

a modification of Hut t ingats method. Each gram of c h i l l e d leaves was

homozenized i n 7.5 ml of 0.1M tris buffer , 2 ml each of carbon t e t r a -

chlor ide and chloroform. The pH of the e x t r a c t was adjus ted with

th iog lyco l l i c a c i d t o 6.5 f o r BYW' and 8 .5 fo r BYMV'. After breaking - the emulsion, the v i r u s was recovered from the aqueous phase by c e n t r i -

fugat ion a t 15,000 rpm f o r 1 hr. The p e l l e t was suspended i n ex t rac t ing

buffer and c l a r i f i e d a t 5000 r p m f o r 5 min. Higher cen t r i fuga l fo rces

during e i t h e r i n i t i a l c l a r i f i c a t i o n o r pe l l e t ing increased p a r t i c l e

aggregation and breakage, and reduced v i rus y ie ld .

4. The method of Uyemoto e t aZ. (1972)

They pur i f i ed BYMV and BCMV by the method followed by

Damirdagh and Shepherd (1970) f o r pur i f i ca t ion of tobacco e tch v i r u s .

One gram of infected t i s s u e was homogenized with 2 ml of 0.5M potassium

phosphate buffer , pH 7.0, containing 1% 2-Mercaptoethanol . After

s t r a i n i n g through cheesecloth the homogenate was s to red overnight a t

8 ' ~ i n butanol (8 m l per 100 ml). After c l a r i f i c a t i o n by c e n t r i -

fugation a t 8,500 rpm f o r 10 min, the v i rus was recovered from super-

natant with 4% PEG followed by d i f f e r e n t i a l centr i fugat ion (28,000 rpm

f o r 1 .S hr; 8,500 rpm f o r 10 min, suspending the v i r u s i n 0.025M

phosphate buffer , pH 7.0 containing O.5M urea and 0.1% 2-Mercaptoethanol)

Further pur i f i ca t ion was achieved by 10-40% sucrose dens i ty g rad ien t

centr i fugat ion.

G. Proper t ies of Virus P a r t i c l e s

1. Structure

P a r t i c l e s a r e elongated and flexuous, 750nm long and 1Snm \

wid; XBrandes 'and Quant z, 1955;. Brandes, 1964). P a r t i c l e s of g r e a t e r

length have a l s o been repor ted . Bos (1970b), using tobacco mosaic

v i rus a s an in te rna l s i z e standard, found p a r t i c l e s of 660 nm length .

Taylor and Smith (1968) and Bos (1970b) found t h a t p a r t i c l e s from

Chenopodiun amvanticolor were 50-60 nm longer than those from legumes.

Contrar i ly , no influence of host on p a r t i c l e morphology could be

noticed by Moghal and Francki (1981).

2 r Sedimentation coef f i c i en t (S*-jW)

The sedimentation c o e f f i c i e n t of 5 i s o l a t e s of BYMV ranged

between 140 and 145s (Huttinga, 1975).

0 1% 3. Ex t inc t ion c o e f f i c i e n t (E260 limm)

I t was determined as 2.4 (Uyeda e t at., 1975).

4 . A260/A280 r a t i o

The r a t i o f o r several preparat ions , corrected f o r l i g h t

s c a t t e r i n g var ied from 1.24 t o 1.31 wi th an average o f 1.28 (Uyeda

e t aZ., 1975).

5 . Nucleic acid

BYMV has a s i n g l e molecule o f s ingle-s t randed RNA (Matthews, 6 1979) wi th a presumed molecular weight o f about 3.0 x 10 a s f o r

t u r n i p mosaic, tobacco e tch and maize dwarf mosaic v i r u s e s ( H i l l and

Benner, 1976), and pea mosaic v i r u s (Abu-Samah and Randles, 1981) . Lisa e t aZ. (1981) es t imated t h e molecular weight of RNA of a poty-

v i r u s , which caused mosaic on ~Cucurbitu pepo and which showed s l i g h t

s e r o l o g i c a l r e l a t i o n wi th BYMV, t o be 2.93 x lo6 d a l t o n s .

6 . P r o t e i n

Huttinga (1975) determined t h e molecular weight o f c o a t

p r o t e i n of 5 i s o l a t e s of BYMV a s 34,000 daltons. Moghal and Francki

(1976) repor ted a molecular weight o f 33,000 da l tons f o r 3 i s o l a t e s

of BYMV. A s l i g h t l y higher molecular weight of 35,000 da l tons has

been repor ted f o r t h e BYMV i s o l a t e I of D r . D.J . Hagedorn, Univers i ty

o f Wisconsin (Uyemoto e t a t . , 1972). Y i l m z (1979) determined t h e

d e c u l a r weight of coat p r o t e i n of BYMV from broad bean a s 33,350 - 33,820 d a l t o n s .

H . Serology

Antisera t o BYMV with a t i t r e o f 112048 have been produced

(Bos, 1970a). These could be f u r t h e r increased by chemically concen-

t r a t i n g the globul in f r a c t i o n o f t h e se ra giving t i t r e s up t o 1/256,000

(Bercks, 1960; Bercks, 1961), but t h i s i s exceptional. Uyeda e t aZ.

(1975) obtained a n t i s e r a t o 3 s t r a i n s of BYMV with t i t r e s ranging from

1024-2048.

Microprecipit in and p r e c i p i t i n tube t e s t s a r e mostly used

because i n t a c t v i r u s p a r t i c l e s do no t d i f fuse i n agar g e l s . However,

t h e d i f fus ion r a t e of filamentous v i ruses through agar could be

increased by var ious detergents (Hamilton, 1964; Gooding and Bing,

1970; Purci ful and Batchelor, 1977) o r physical treatments (Tomlinson

and Wal key, 1967) which allowed degradation o f v izus p a r t i c l e s i n t o

r e a d i l y d i f f u s a b l e un i t s .

Granett and Provvidenti (1975) found t h a t 0.3% (w/v) o f SDS

i s ' good f o r degradation of BYMV i n t o d i f fusab le u n i t s than pyrrolidone

o r ethanblamiqe.

BYMV i s se ro log ica l ly d i s t a n t l y r e l a t e d t o bean common

mosaic v i r u s (Beemster and van der Want, 1951; Moghal and Francki,

1976), soyabean mosaic, sugar beet mosaic, watermelon mosaic v i ruses ,

and pota to v i r u s Y (Bercks, 1960) .

I . S t r a i n s - Various s t r a i n s d i f f e r i n g i n t h e symptoms they produce i n

c e r t a i n p l a n t species have been recognised. They a re :

Pea mosaic s t r a i n : Pea mosaic v i r u s ( D o o l i t t l e and

Jones, 1925) was f o r long considered a separa te v i r u s ,

d i f f e r i n g from BYMV by the b r i g h t yellow symptoms it causes

i n pea and broad bean, and by i t s i n a b i l i t y t o i n f e c t most

v a r i e t i e s of PhaseoZus vuZgaris (Schroeder and Provviden ti,

1966; Cousin, 1969). However, based on t h e i r c l o s e se ro-

l og i ca l r e l a t i onsh ip , i t was concluded t h a t pea mosaic

v i r u s was a s t r a i n of BYMV (Taylor and Smith, 1968).

Cowpea s t r a i n of Anderson (1955b) and Br i e r ly and Smith

(1962), i s o l a t e d from and produces systemic symptoms i n

V C g m sinemi4 which i s no t i n fec t ed by common s t r a i n o f

the v i r u s . The cowpea s t r a i n g ives severe systemic yellow

spo t t i ng i n Chenopodim amranticolor.

Red c lover necros i s s t r a i n of Zaumeyer and Goth (1963),

produced necros i s i n red c lover and pea, l oca l and systemic

necros i s i n C. amant-icoZor, death of DifoZiwn incarnatwn, T. subterranewn and Vicia faba-minor.

Pod d i s t o r t i n g s t r a i n of Grogan and Walker (1948), i s o l a t e d

from Idaho Refugee bean and o the r v a r i e t i e s r e s i s t a n t t~

common bean mosaic v i r u s .

Black r o o t o r X-disease s t r a i n o f McWhorter and Boyle

(1946) which produces purpl ing of t h e l e a f bases o f t h e

lower leaves . This purpl ing i s accompanied by dying of

the t i s s u e s within and o f t en leads t o prena ture dea th of

t he p l an t .

Necrotic l e s i o n s t r a i n : A l o c a l 1 esion-producing v i r u s

i s o l a t e d from in fec t ed bean pods by Zaumeyer and Fisher

(1953) . Top necros i s , produced by t h e pod d i s t o r t i n g and

X-disease s t r a i n s seldom develops with t h i s s t r a i n :

PhaseoZus Zunatus, V'igna sesquipedaZis and V. s inens i s

are suscept ib le t o the nec ro t i c l e s ion s t r a i n , but a r e

r e s i s t a n t t o o ther s t r a i n s of BYMV.

Severe yellow mosaic s t r a i n of Rex and Zaumeyer (1953)

which causes strong epinasty, ch lo ros i s and ve ina l

necros is of inoculated primary leaves of P. vulgaris .

These symptoms are followed by top necros is and death

of c e r t a i n bean v a r i e t i e s and severe yellow mosaic i n

o thers . No infect ion was obtained ou t s ide t h e Leguminosae,

Other s t r a i n s of BYMV reported are : BYMV-Q from Canna sp .

i n Queensland (Moghal and Francki, 1976), BYMV-G i s o l a t e d from

GZadwZw connntazis i n South Austral ia (Lee e t aZ., 1979) and BYMV-S

from V i c k faba i n South Austral ia (Randles e t aZ., 1980).

J. Yield Losses

Kaiser e t aZ., (1968), i n a f i e l d inocula t ion t r i a l s wi th

BYMV r e p ~ r t e d a y ie ld l o s s of 77-92% due to in fec t ion i n chickpeas. . . .

' Kaiser and Danesh (1971b) reported about 92-100% and 84-85%

decrease i n chickpea y ie lds when p lan t s were inoculated a t 4 weeks

(pre-bloom) and 9 weeks ( f u l l bloom) a f t e r planting, r e spec t ive ly .

K. Screening of Germplasm

There i s no r e p o r t i n the l i t e r a t u r e on the screening of

chickpea germplasm f o r r e s i s t ance t o BYMV.

MATERIALS A N D METHODS

I . GENERAL

A. Virus Source

Cucumber mosaic v i rus (CMV) was i so la ted from f i e l d s of

chickpea genetic resources uni t , ICRISAT, from the p lan ts showing t i p

necrosis . Bean yellow mosaic v i rus (BYMV) was i so la ted from f i e l d s

of the Agricultural College Farm, Parbhani, Maharashtra from p lan t s

showing the narrow leaf symptoms.

B. Maintenance of Viruses

Both the viruses were maintained i n the glasshouse on chick-

pea by periodic mechanical inoculations. A kabuli cu l t i va r obtained

from the loca l market and a kabuli germplasm accession, ICC-162, were

used f o r the maintenance of CMV and BYMV, respect ively. Besides chick-

pea, W was maintained on Nicoticzm rustica var . White pathar ,

obtained' f rop She Central Tobacco Research I n s t i t u t e , Rajahmundry,

Andhra Pradesh, India, and BYMV on PhaseoZus vuZgaris var . Dwarf

obtained from the local market.

C. Plants

A l l chickpea and other t e s t plants were raised i n a mixture

of Alf isol and farmyard manure (1:4, w/w) i n 15 cm pots i n the glass-

&e (temperature range 25-30'~).

D. Inoculum and Inoculations

The inoculum was prepared by grinding the infected chickpea

leaves w i t h a pre-chi l led pest le and mortar i n 0.05 M potassium

phosphate buffer , pH 7.5, containing 0.01M sodium s u l p h i t e and 0.01M

sodium d i e t h y l dithiocarbamate (4 m l per gram t i s s u e ) . For determining

physical p r o p e r t i e s of t h e v i ru ses , t he standard e x t r a c t was prepared

by grinding t h e i n f ec t ed chickpea leaves i n t h e same bu f f e r a s above

(1: 1, w/v) but without any add i t i ve s . C e l i t e ( I%, w/v) was added t o

t he e x t r a c t and rubbed onto t h e leaves of t e s t p l a n t s wi th t h e broad

end of t he p e s t l e , Inoculated p l a n t s were thoroughly r i n sed with t a p

water . All chickpea p l a n t s were inoculated when 10 days o ld , whereas

o t h e r p l a n t s were inocula ted when young.

11. IDENTIFICATION OF VIRUSES BY SEROLOGY

The v i r u s e s under s tudy were i d e n t i f i e d by serology employing

double- d i f f u s i o n t e s t i n agar (Ball , 1974) and enzyme-linked imrnuno-

sorbent a s say (ELISA) ( L i s t e r , 1978; Reddy e t a Z . , 1981).

A: lbublc+ Diffusion i n Agar

Agarose 0.5% (Sigma chemicals, U.S,A.) was prepared i n 0.01M

potassium phosphate bu f f e r , pH 7.0, t o which 0.15M sodium c h l o r i d e was

added. I t was then autoclaved a t 15 l b p s i f o r 15 minutes and 0.05%

Mer th io la te (Ethyl mercu r i t h io sa l i cy l i c ac id) was added t o a r r e s t

microbial contamination. Eight m l o f agar were poured i n t o p l a s t i c P e t r i

p l a t e s (5.3 cm diameter) . Af te r s o l i d i f i c a t i o n , w e l l s o f 7 mm

diameter were c u t with a cork borer . The p a t t e r n cons i s t ed of 4

pe r iphe ra l we l l s around a c e n t r a l well (5 nun d i s t ance from c e n t r a l w e l l ) .

Ant i sera t o W and BYMV were obtained from Dr. H.A, Sco t t , Univers i ty

o f Arkansas, U.S.A., and Dr. N. Conti, Laboratorio F i tov i ro log ia , Torino,

I t a l y , r e spec t ive ly . P u r i f i e d v i r u s and sap from in fec t ed chickpea

were used a s an t igens . Healthy sap and bu f f e r were kept a s c o n t r o l s .

The p l a t e s were incubated i n a mist chamber (des icca tor w i th moist

cot ton i n the bottom) a t room temperature (25-28'~) and observed d a i l y

f o r r eac t ion .

I n the case of BYMV, t he v i r u s was t r e a t e d wi th 0.3% sodium

dodecyl s u l f a t e before incorporating i n t o wel ls , t o break t h e v i r u s

i n t o d i f fusab le u n i t s (Purc i fu l l and Batchelor, 1977) .

B. Enzyme-Linked Immunosorbent Assay (ELISA)

1. Extract ion of y-globulins

One m l of antiserum was d i l u t e d with 2 ml o f phosphate

buffered s a l i n e (PBS) pH 7 . 4 (0.8% NaC1, 0.005M Na2HP04, 0.001M KH2P04,

0.02% K C 1 , 0.02% osdium azide) and mixed gent ly .

To t h e above, 3 m l of 36% sodium s u l f a t e were added, mixed

gen t ly and centr i fuged a t 5000 rpm f o r 15 min.

The p e l l e t was dissolved i n 4 m l of 18% sodium s u l f a t e , and

centr i fuged gt 5000 rpm f o r 15 .minutes.

The p e l l e t was resuspended i n 2 m l of PBS.

Dia lys i s was done agains t PBS i n r e f r i g e r a t o r a t about 4 ' ~

g iving two changes of 1 l i t r e each ( f i r s t d i a l y s i s was f o r 4 hours and

second overnight ) . The y-globulin so lu t ion was remove from t h e d i a l y s i s bag.

The concentrat ion of y-globulins was adjus ted t o 1 mg/ml, assuming an

Oo1' = 1.6). ex t inc t ion c o e f f i c i e n t of 1.6 OD (AZIO

2. Con jugat ion of y-globulins with a l k a l i n e phosphatase enzyme

The enzyme a l k a l i n e phosphatase (Sigma P-4502, type VII,

5 mg/ml) was p r e c i p i t a t e d from enzyme so lu t ion by cen t r i fug ing a t

5000 rpm f o r 15 minutes, and dissolved i n y-globulin s o l u t i o n (1.5 mg

o f enzyme per 1 nil of 1 mg/ml y-globulin so lu t ion) .

Dia lys is of conjugated y-globulins was done a g a i n s t PBS with

two changes of 1 l i t r e each ( f i r s t d i a l y s i s f o r 4 hours and second

overnight) .

Conjugated y-globulins were t r e a t e d with g lutara ldehyde by

d ia lys ing aga ins t PBS containing 0.06% glutaraldehyde f o r 1 hour a t

room temperature.

Dia lys is bags were t r ans fe r red t o PBS and dia lysed overnight ,

a t ~ O C , giving two changes of 1 l i t r e each.

Conjugated y-globul ins were remved and s to red i n g l a s s tubes

a t doc.

3. Preparation of p l a t e s \

a I ' . .

Micro t i t r e p l a t e s (Dynatech labs . , England) were demarcated

according t o the var ious d i l u t i o n s o f t e s t so lu t ions , l eav ing o u t

per iphera l we l l s .

y-globul in d i l u t i o n s were made i n coat ing buf fe r (0.02M

Na2C03, 0,02M NaHC03, 0.02% sodium azide , pH 9.6) and 0.2 m l was

added t o each well o f t h e p l a t e . Water was added t o the pe r iphera l

v e l l s and t h e p l a t e s were incubated a t 3 7 ' ~ f o r 2% hours.

y-globulins were poured o f f and t h e we l l s were washed t h r i c e

with PBS containing 0.05% Tween-20 (PBS-Tween) leaving the b u f f e r i n

w e l l s f o r 3 minutes f o r each washing.

Dilutions of t e s t samples (healthy and infected) were made by

grinding the t i s s u e i n antigen ext ract ion buffer (PBS-Tween containing

2% polyvinyl pyrrolidone (PVP) of mol. w t . 40,000) and s t r a i n i n g t h e

e x t r a c t through cheesecloth.

Test samples (0 .2 ml) were added t o each well and water t o

per iphera l wells . P la tes were incubated overnight a t 4 ' ~ .

Test samples were poured o f f and t h e wells were washed a s

e a r l i e r i n PBS-Tween.

Dilutions o f conjugated y-globulins were made i n conjugate

buf fe r (PBS-Tween containing 2% PVP; 0.2% ovalbumin).

Conjugated y-globulins (0.2 ml) were added t o each well and

water t o peripheral we l l s , P la tes were incubated a t 3 7 ' ~ f o r 3 hours.

y-globulins were poured o f f and t h e wel ls were washed i n

PBS- ween a s e a r l i e r .

The s u b s t r a t e (0.2 ml), p-nitrophenyl phosphate (1 mg/ml),

prepared i n subs t ra te buf fe r (9.7% aqueous diethanolamine, pH 9.8)

was added t o each well. P la tes were incubated f o r 30-45 minutes a t

room temperature.

One drop of 3M NaOH was added t o each well t o s t o p f u r t h e r

r eac t ion . A p o s i t i v e react ion was detected by the development of

yellow colour i n the subs t ra te .

111. HOST RANGE AND SYMPTOMS

Host range s tud ies were c a r r i e d out f o r CTW and BYMV, by

mechanical inocula t ion of d i f f e r e n t p lant species belonging t o the

families: Aizoaceae, Amaranthaceae, Chenopodiaceae, Compositae, Cucur-

b i taceae , Leguminosae and Solanaceae ra i sed under i n s e c t - f r e e glasshouse

condi t ions . Four p lan t s were inoculated with each v i r u s and an equal

number of p l a n t s were kept a s controls . P lan t s were observed d a i l y and

symptoms recorded a s and when they appeared.

IV. PHYSICAL PROPERTIES IN CRUDE SAP

A. Cucumber Mosaic Virus

Physical p roper t i e s of CMV were determined i n the standard

e x t r a c t following the procedure described by Bos e t aZ. (1960).

Chenopodiun m a n t i c o l o r was used a s an assay host .

1. Thermal inac t iva t ion point

Five m l samples o f the standard e x t r a c t i n t e s t tubes were

subjected separa te ly f o r 10 minutes t o heat treatments s t a r t i n g from

45' and 9 0 ' ~ with an i n t e r v a l of 5 ' ~ . Immediately a f t e r heat treatment,

the. sap Gas cooled by passing . t a p water over the outer surface o f t h e

tube. To t h i s was added c e l i t e (1%) and rubbed on the leaves o f the

assay host . Ten C. amrant icolor plants , each with 6 leaves were used

r e p l i c a t i n g each treatment 6 times (Table 1) . Observations were

recorded 3 days a f t e r inocula t ion, when the loca l l e s ions were conspi-

cuous i n control treatment.

2 . Dilution end m i n t

The standard e x t r a c t was d i lu ted t o ge t d i l u t i o n s varying

from 10-I through lom7. After adding c e l i t e to each, they were

separa te ly rubbed on the leaves of the assay host . Eight C. amrant i -

coZor plan t s , each w i t h 6 leaves were used and each treatment was

r e p l i c a t e d 6 times (Table 2 ) . Observations were recorded 3 days a f t e r

inocula t ion.

TABLE 1. Randomization of treatments t o determine thermal inac t iva t ion point (TIP)

Plant No.

1 2 3 4 5 6 7 8 9 1 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 l a g c i e j f b h d

& s 2 b h d j f a g c i e 0

N 0 ~ 3 c i e a g b h d j f +

j 8 4 d j f b h c i e a g c o

a = control ; b = 45'~; c = SOOC; d = 55'~; e = 60'~; f = 65'~; g = 70'~; h = 75'~; i = 80°c; j = 85'~.

TABLE 2 . Randomization of treatments t o determine d i l u t i o n end point , (DEP)

Plant No.

8 = control (undiluted); b = 1 0 c = d = 10'~; e =

f = 10-5; = 10-6; t, =

3 . Longevity i n tritro

The standard e x t r a c t was s tored i n a 50 m l beaker a t room

temperature (22'- 24'~) and was inoculated d a i l y on assay host , u n t i l

the sap was confirmed t o be non-infectious.

B . Bean Yellow Mosaic Virus

The preparation o f the standard e x t r a c t and the treatments

were s imi la r a s described f o r CMV. Since no loca l l e s ion host could

be found f o r t h i s v i rus , the assay was done on kabuli chickpea

(ICC-162), taking 10 seedlings f o r each treatment.

V. INSECT TRANSMISSION

Two aphid species, v i z . , Aphis crvrccivora and 4 jzus persicae,

were obtained from f i e l d grown pigeonpea and tobacco, r e spec t ive ly

and healthy cqlonies were ra i sed before using them i n transmission

s tud ies .

Aphids were fas ted i n P e t r i p l a t e s f o r an hour and then

allowed an aqu i s i t ion feeding period of one minute on vi rus- infected

chickpea leaves. Thereafter they were t r ans fe r red onto the healthy t e s t

p l a n t s f o r inocula t ion feeding period of 12-14 hours. Thereafter , the

insec t s were k i l l e d by spraying Rogor (0.02%). Five t o 10 aphids were

used per p lan t and 10-day-old healthy chickpea seedlings (10) were used

a s t e s t p lants .

V I . PURIFICATION OF VIRUSES

A. Cucumber Mosaic Virus

Chickpea was used a s a source p lan t s ince it supported a

high v i r u s concentrat ion. Plants were harvested 7-10 days a f t e r inocu-

l a t i o n , when they showed terminal drooping. The e n t i r e p lan t , except

roo t s , was used for pur i f i ca t ion . The p u r i f i c a t i o n procedure developed

by Tomlinson e t a'L. (1958) was used with modifications which i s given

below. A l l the s teps of pur i f i ca t ion procedure were c a r r i e d out a t

about 4 ' ~ by using pre-chi l led mater ia ls and keeping the prepara t ions

i n an i c e bucket.

Infected chickpea p lan t s were homogenized using a pre-chi l led

Waring blendor i n 0.5M potassium phosphate buffer , pH 7.5 containing

0.1% sodium th iog lyco l l i c ac id and ImM EDTA (4 ml/g t i s s u e ) . The sap

was s t ra ined through 2 l aye rs of cheesecloth. To the f i l t r a t e 8.5%

(v/v) of n-butanol was added dropwise with constant s t i r r i n g . S t i r -

r i n g was done f o r another 15 minutes. Then the mixture was c e n t r i -

fuged a t 5000 rpm f o r 10 minutes i n SS-34 r o t o r i n Sorval l centr i fuge.

The supernatant was saved t o which 8% polyethylene glycol (PEG) mol.

w t . 6000 (Carbowax, Fisher S c i e n t i f i c Company, U. S .A. ) and 0.2M sodium

chlor ide were added and dissolved f o r 30 minutes and incubated f o r 90

minutes a t 4 ' ~ . I t was then centrifuged a t 10,000 rpm f o r 10 minutes

and t h e ' r e s u l t i n g p e l l e t was resuspended i n hal f the o r ig ina l volume

of 0.025M potassium phosphate buffer pH 7.5, containing 1mM EDTA ( P E ) .

The p e l l e t was thoroughly dissolved using a vortex mixer and c e n t r i - t

fuged a t 5000 rpm fo r 10 m i n ~ e s . The supernatant was saved and c e n t r i -

fuged a t 30,000 rprn f o r 2 hours i n R-35 r o t o r i n a Beckman prepara t ive

u l t r acen t r i fuge (model L5-50) a t 4 ' ~ . The p e l l e t was resuspended i n

PE and centrifuged a t 5000 rpm f o r 5 minutes. The supernatant was

col lec ted and designated a s p a r t i a l l y p u r i f i e d v i rus .

Further pur i f i ca t ion of the v i r u s was achieved by r a t e - zonal

dens i ty gradient centr i fugat ion i n sucrose. Sucrose gradients were

prepared by layering 9, 9, 9 and 6 ml o f 40, 30, 20 and 10% sucrose

prepared i n PE, respect ively . me gradients were kept a t 4 ' ~ f o r

16 hours before use. Four m l of the p a r t i a l l y p u r i f i e d v i rus preparation

were layered over the gradients and centrifuged i n SW-27 ro to r f o r

90 minutes a t 24,000 rpm. A l i g h t s c a t t e r i n g zone was observed 2.4 cm

below the meniscus when a narrow beam o f l i g h t was passed through the

top of the tube i n a dark room. The zone was co l l ec ted by an ISCO

dens i ty gradient f r ac t iona to r (model 185) with UA-5 absorpt ion monitor.

I t was d i lu ted with an equal volume o f PE and centr i fuged

f o r 2 hours a t 30,000 rpm i n R-35 r o t o r o r 1 hour a t 45,000 rpm i n

R-50 r o t o r . The p e l l e t was dissolved i n 0.5 t o 1 m l of PE and des ig-

nated a s p u r i f i e d v i r u s . A t d i f f e r e n t s t eps of p u r i f i c a t i o n , t h e

i n f e c t i v i t y of the v i rus was assayed by mechanical inocula t ion on

C. amranticolor.

From another gradient , one-ml f r a c t i o n s were co l l ec ted by

the f r a c t i o n a t o r and each f r a c t i o n was separa te ly assayed on C. a m a n t i - color a f t e r d i l u t i n g 10 times, t o c o r r e l a t e t h e W a b s o r p t i o n component

wi th i n f e c t i v i t y . Three leaves were used f o r each f r a c t i o n and the

average number of loca l l e s ions was obtained.

For studying biochemical p roper t i e s of t h e v i rus , f u r t h e r

p u r i f i c a t i o n yas attempted by quasi-equil ibrium dens i ty gradient

cen t r i fuga t ion . The v i r u s zone co l l ec ted a f t e r the rate-zonal d e n s i t y

gradient cen t r i fuga t ion was d i l u t e d wi th PE ( 1 : l ) and layered over

60-30% sucrose gradients , prepared by layer ing 12, 7, 7, and 7 ml of

60, 50, 40, and 30% sucrose i n PE, r e spec t ive ly . After cen t r i fuga t ion

a t 24,000 rpm f o r 2 hours i n SW-27 r o t o r t h e l i g h t s c a t t e r i n g zone

was withdrawn by a bent needle a t tached t o a syr inge. I t was d i l u t e d ,

p e l l e t e d and dissolved i n PE a s described i n t h e rate-zonal dens i ty

g rad ien t cen t r i fuga t ion .

B. Bean Yellow b s a i c Virus

Several methods (Uyeda e t aZ. , 1975; Huttinga, 1973; and

Uyemoto e t aZ., 1972) were used f o r BYMV p u r i f i c a t i o n and the one

described below was found t o be s u i t a b l e . A l l t he s t eps of p u r i f i -

ca t ion procedure were carr ied ou t a t about 4 ' ~ a s i n the case of CMV.

BYMV infected chickpea t i s s u e was homogenized i n 0.1M Tris-PO4

buffer , pH 8.5 containing 0.1% th iog lyco l l i c ac id and 0.01M EDTA (1:3,

w/v). Carbon t e t r a chloride (4 volume of the buffer) was added during

homogenization i n a Waring blendor. After s t r a i n i n g through two-layered

cheesecloth, it was centrifuged a t 5000 rpm f o r 10 minutes. The aqueous

phase was recovered to which 4% PEG was added and incubated overnight

a t 4 ' ~ . Then it was centrifuged a t 10,000 rprn f o r 10 minutes. The

r e s u l t a n t p e l l e t was dissolved i n ha l f the o r i g i n a l volume of 0.01M

Tris-PO4 buffer , pH 8.5 containing 0.2M urea (PU) and centrifuged a t

5000 rprn f o r 5 min. Twenty-five m l of supernatant were layered over

13 m l of a sucrose pad (30% sucrose i n TPU containing 4% PEG) and

centrifuged a t 24,000 rprn f o r 2 hours. The pel le t 'was dissolved i n

TPU and centrifuged a t 5000 rprn f o r 5 minutes.

Four m l of the supernatant ( p a r t i a l l y p u r i f i e d v i rus ) were

layered over the preformed sucrose densi ty gradient column prepared

i n TPU a s in -case of W and centrifuged a t 24,000 rprn f o r 90 min.

An improminent, diffused v i rus zone formed j u s t above the midspoint

was co l l ec ted with a bent needle at tached to a syringe. I t was

d i lu ted with TPU and centrifuged a t 24,000 rprn f o r 90 min. The pel le '

was dissolved i n a small amount of 0.01M Tris-PO4 buffer (pur i f ied

v i rus ) .

/I1 . ELECTRON MICROSCOPY

Electron microscopy of p u r i f i e d preparations of (=MV and BYMV

was done. In addi t ion, e lec t ron microscopy of d i p preparations and

u l t r a t h i n sect ions of BYMV-infected chickpea leaves was a l s o done.

A. CMV-Purif i e d Preparat ion

A drop of p u r i f i e d v i r u s was kept on a paraf i lm on which a

formvar and carbon-coated copper g r i d (300 mesh) was f l o a t e d f o r 2 min,

The g r i d was removed with f i n e forceps , excess s o l u t i o n wiped o f f by

t h e pointed edge of f i l t e r paper and then f l o a t e d on a drop o f 1%

uranyl a c e t a t e f o r one minute. Thereaf ter , it was taken ou t , excess

s t a i n wiped o f f , d r i ed and observed under a P h i l i p s 201C e l e c t r o n

microscope. From negat ive e l e c t r o n photo micrographs, t h e average

diameter o f the v i r u s p a r t i c l e s was ca lcu la ted by measuring 100

p a r t i c l e s , using a Bousch and Lomb magnifier.

B . BYMV-Purified Preparat ion

The procedure was s i m i l a r t o t h a t described f o r CMV, except

t h a t n e u t r a l phosphotungstic ac id was used f o r negat ive s t a i n i n g .

C. Leaf Dip Preparation

The d i p preparat ion was done with BYMV-infected chickpea

leaves s ince i t i s necessary i n case of rod-shaped v i ruses t o measure

t h e p a r t i c l e s i n crude sap. A BYMV-infected chickpea leaf was

chopped i n a drop of water on a paraf i lm. The water drop was removed

with t h e h e l p o f a pas teur p i p e t t e and used f o r t h e p repara t ion o f a

g r i d f o r t h e e l e c t r o n microscopy. The length o f 100 p a r t i c l e s was

measured, a length d i s t r i b u t i o n histogram was p l o t t e d and a modal

l eng th ca lcu la ted .

D. U l t ra th in Sect ions o f BYMV-Infected Chickpea Leaves

U l t r a t h i n sec t ions of B W - i n f e c t e d chickpea leaves were

prepared t o observe inc lus ion bodies induced due t o in fec t ion .

Leaves were c u t i n t o small p i eces of 1 mm x 1.5 nun. They were

f i xed i n 3% glutaraldehyde f o r 2 hours, washed f o r 15 h r i n 0.1M phos-

phate buffer pH 7 . 3 , containing 0.5% sucrose. They were s to red over-

n i g h t i n r e f r i g e r a t o r i n the same bu f f e r . Next day, they were f i x e d i n

2% osmium t e t r o x i d e made i n 0.1M phosphate b u f f e r pH 7 . 3 containing 5%

sucrose f o r 2.5 h r s . They were washed i n d i s t i l l e d water f o r 20 minutes

and then dehydrated i n 50, 70 and 90% acetone (20 min each) . The f i n a l

dehydrat ion was done i n 100% acetone t h r i c e , each time f o r 20 minutes.

Acetone dehydrated t i s s u e s were t r a n s f e r r e d t o 1:l mixture o f 100%

acetone and epoxy embedding r e s i n (Ladd Inc . , U.S.A.) and shaken f o r

1 h r . The t i s s u e s were drained on a paper towel and soaked i n f r e s h

r e s i n fo r 4 h r s .w i th a change of r e s i n every hour. The t i s s u e s were

drained once aga in and t r a n s f e r r e d t o f r e s h r e s i n i n s i l i c o n rubber

blocks which were kept a t 6 5 ' ~ f o r 48 hours . Sec t ions were c u t (50-60

nm t h i ck ) wi th a ' Reichert-Jung Ul t r acu t ' , s ta ined ' i n s a t u r a t e d uranyl

a c e t a t e f o r 3 min and in 5% lead c i t r a t e f o r 3 min, washed, d r i ed

and observed under t he e l ec t ron microscope.

I11 I . PHYS I ~ O - QIEMICAL PROPERTIES OF PURIFIED CMV

A. W-Absorption

The W-absorpt ion spec t r a of t h e p u r i f i e d CMV ,were recorded

i n Beckman D. B . G . T. spectrophotometer a t wave l eng ths ranging from

200 t o 300 mm. Rat ios of 2601280, 280/260, and A max/A min were

ca l cu l a t ed . The y i e l d of t h e v i r u s was est imated using an ex t inc t ion

c o e f f i c i e n t of 5.0 OD = 5.0) (Gibbs and Harrison, 1970).

B . Sedimentation Coeff ic ien t

Sedimentation c o e f f i c i e n t of the p u r i f i e d CMV was determined

i n a Spinco model E a n a l y t i c a l u l t r a c e n t r i f u g e . The p u r i f i e d v i r u s

suspended i n 0.025M potassium phosphate bu f f e r was run a t 30,000 rpm.

After reaching the des i red speed, the Schleiren p a t t e r n was photo-

graphed a t 3 min in te rva l s . The sedimentation coef f i c i en t was deter .

mined by w i n g the following formula:

C. Buoyant Density

One m l of the pur i f i ed CMV was layered over 4 m l o f 40%

cesium chlor ide (density = 1.42 g/ml) i n 5 m l polyallmoer tubes, and

centrifuged i n SW-50 r o t o r a t 40,000 rpm f o r 18-20 h r s a t 4 ' ~ . The

gradient column was f rac t iona ted by puncturing the bottom of the tube

and c o l l e c t i n g 0 . 2 rnl f r ac t ions . These f r a c t i o n s were monitored i n t h e

Beckman Spectrophotometer a t 260 nm, and the d e n s i t i e s o f t h e f r a c -

t i o n s were determined by t h e i r r e f r a c t i v e indices measured with an

Abbe refractometer.

D. ~ o l ~ a c r ~ l a m ~ d e Gel Electrophoresis (PAGE) o f CMV Coat Prote in

The PAGE of CMV coat prote in was done following the method

of Laemmli (1970).

Stock so lu t ions

The stock so lu t ions were prepared a s follows:

Solution A (3M Tris-HC1 buffer , pH 8.91

Tri s : 36.6 g

1NHC1 : 48 .0ml

TEMED : 0 .23 m l

Made up t o 100 ml with d i s t i l l e d water and adjusted pH t o 8.9 with 1N HC1

Solution B (1M Tris-HC1 buffer , pH 6.8)

Tr is : 5.98 g

1 N HC1 : 48.0 m l

TEMED : 0.46ml

Made up t o 100 m l with d i s t i l l e d water and adjus ted pH t o 6.8 with 1N HC1

Solution C (Acrylamide : Bis)

Acrylamide : 30.0 g

B i s : 0.735 g

Made up t o 100 ml with d i s t i l l e d water.

Solution D (Acrylamide : Bis)

Acrylamide : 10.0 g

B i s : 2.5 g

Made up t o 100 ml with d i s t i l l e d water.

Preparation of the Gels

Resolving (rgnplraing) gel (10%)

Solution A : 2.0 ml

Solution C : 5 .2 ml

1% Sodium dodecyl s u l f a t e (SDS) : 1.6 m l

10% Urea : 4.8111.1

D i s t i l l e d water : 1.4 m l

1% Ammonium persu l fa te : 1.0 ml

Stacking (spacer) gel (3.6%)

Solution B : 0.5 m l

Solution D : 1.4 ml

1% SDS : 0.4 m l

10% Urea : 1 . 2 ml

Ammonium persu l fa te : 0.25 ml

With t h e he lp of a long-beaked Pas teur p i p e t t e , the reso lv ing

ge l was poured i n t o Bio-rod gel tubes up t o a l eng th o f 6 cms and

allowed t o polymerize f o r 15 minutes. Over t h i s s tacking gel was

poured up t o a length of 1 cm and allowed t o polymerize f o r 30 min.

After polymerization, t h e tubes were inse r t ed i n t h e e l e c t r o p h o r e t i c

apparatus and f i l l e d with e lec t rophores i s b u f f e r (0.05M Tr i s , 0.384M

glycine , 0.1% SDS, pH 8.3).

The v i r u s was d i s soc ia ted i n 0.0625M Tris-HC1 buf fe r , pH 6.8

conta ining 1% 2-Mercaptoethanol, 1% SDS and 6M urea . I t was then

incubated a t 1 0 0 ' ~ f o r 2 min ( i n b o i l i n g water) . Ten t o 100 p1 o f

t h e d i s soc ia ted v i r u s sample was layered on each ge l . A mixture o f

s i x marker p ro te ins (Bio-Rod Chemicals, U. S .A.) containing 20-100 pg

o f each polypeptide was a l s o layered separa te ly a s well a s mixed wi th

v i r u s p ro te ins ( a - e l e c t r o p h o r e s i s ) . Af ter e l e c t r o p h o r e s i s a t 30 v o l t s

f o r 6 hours a t room temperature, t h e g e l s were taken o u t o f t h e tubes

by l u b r i c a t i n g t h e inner wal ls o f t h e tubes wi th water, using a

syr inge with a b l u n t needle.

?h'e'gels were s t a ined f o r 4 hr i n 0.2% coomassie b r i l l i a n t

b lue prepared i n 20% methanol i n 7% a c e t i c a c i d . Destaining o f t h e

g e l s was done overnight with 20% methanol i n 7% a c e t i c a c i d . The g e l s

were than scanned a t 540 nm i n a Gi l l fo rd spectrophotometer .

The m l e c u l a r weight of CMV coat p r o t e i n was determined by

t h e l i n e a r r e l a t i o n s h i p between t h e migration d i s t a n c e o f marker

p r o t e i n s and t h e logari thm o f t h e i r known m l e c u l a r weights.

E. Polyacrylamide Gel Elect rophores is of CMV WAS

The PAGE of CMV RNAs was performed using t h e method a s

described by Adesnik (1970). A combination of 2.5% acrylamide and

0.5% agarose was used.

Stock solut ions

The stock solut ions were prepared a s follows:

Solution A (15% acrylamide)

Acrylamide : 7 .1 g

B i s : 0.4 g

Dissolved i n d i s t i l l e d water and made up t o 50 m l .

Solution B (15 x buffer , pH 7.5)

Tri s : 0.4M

NaH2P04 : 0.3M

E DTA : 0.01M

Solution C

10% ammonium persu l fa te (prepared j u s t before use)

Preparation of gels

. Mix

Solution A 5 .0 ml

15 x buffer 2 . 0 m l

D i s t i l l e d w a t e r 7 .7ml

TEMED 0.03 m l

1% agarose 15.0 ml

10% ammonium 0.3 m l (added a t t he end) per sul f a t e

The gels were immediately ca s t in to t he Bio-rod gel tubes

with the help of a long-beaked Pasteur p ipe t te . lbo drops of

d i s t i l l e d water were layered over the gel surface with the help of

a syringe and needle t o obtain a sharp meniscus. The gel tubes were

in se r t ed in to an electrophoret ic apparatus and f i l l e d with e lec t ro-

phoresis buffer (1 x buffer) t o which 0.05% die thyl pyrocarbonate was

added t o avoid poss ib le contamination with ribonudease (RNase) . Before

loading the samples, a pre-run was given a t 5 mA/gel f o r + h r . Virus

suspension was disrupted i n 1.5 x buf fe r conta ining 10M urea, 4 % SDS,

1% 2-Mercaptoethanol, 2mM EDTA, t o which 0.001% bromophenol b lue was

added a s a tracking dye.

The disrupted v i r u s containing about 50 t o 100 pg of nuc le ic

ac id was layered on the ge l s . Some ge l s were loaded with t h e d i s rup ted

v i r u s t h a t was t r e a t e d with RNase a t the r a t e of 1 pg/ml, and incubated

a t 2 5 ' ~ f o r 1 hour.

Electrophoresis was done a t 5mA/gel a t room temperature

u n t i l the t racking dye reached near the bottom of t h e g e l (about 3

hours) . The ge l s were removed from the tubes a s described i n p ro te in

gels . They were suspended i n 1 M a c e t i c ac id f o r 15-30*minutes, s t a i n e d

i n 0.2% to lu id ine blue, prepared i n 0.4M a c e t a t e b u f f e r , pH 4 . 7 f o r

15-30 min.

Destaining of the g e l s was done i n severa l changes of

d i s t i l l e d water i n order t o loca te RNA bands.

IX. PRODUCTION OF ANTISERUM TO CMV

An Albino rabb i t , weighing about 2 kg was used f o r immuni-

za t ion. One ml o f p u r i f i e d v i r u s (2-3 mg/ml) was emulsif ied with an

equal volume o f Fruend's incomplete adjuvant and i n j e c t e d i n t o the

rabb i t intramuscularly i n t h e th igh muscle of hind l e g . A t o t a l of

four i n j e c t i o n s were given a t 10-day-intervals. Ten days a f t e r t h e

l a s t in jec t ion , the r a b b i t a f t e r 16 h r s t a r v a t i o n was bled through a

marginal e a r vein by making an i n c i s i o n wi th a s t e r i l i z e d razor . The

blood was c o l l e c t e d i n a t e s t tube, allowed t o c l o t f o r one hour a t

room temperature and kept a t 4 ' ~ overnight. The serum was drawn with

a Pasteur p i p e t t e and centr i fuged a t 3000 rpm f o r 10 min i n a c l i n i c a l

cen t r i fuge t o separa te the serum from t h e blood plasma. Sodium azide (0.1%) was added a s a preservat ive t o the serum.

Ten-fold d i l u t i o n s of the antiserum were made i n normal

s a l i n e and t h e t i t r e was determined by employing t h e Ouchterlony agar

d o u b l e d i f fus ion t e s t .

X. DETERMINATION OF ?HE SEROLOGICAL RELATIONSHIP BETWEEN THE PRESENT S W I N OF CMV WITH OTHER KNOWN STRAINS

The se ro log ica l a f f i n i t y of t h e present s t r a i n of CMV with

o t h e r s t r a i n s was determined employing t h e Ouchterlony agar Quble-

d i f fus ion t e s t . Ant isera t o e igh t s t r a i n s of MV, obtained from

Dr. H . A . S c o t t (U.S.A.), Dr. H.E. Waterworth (U.S.A.), and Dr. R.I.B.

Francki (Aus t ra l i a ) , along with t h e homologous antiserum were made t o

form p r e c i p i t i n bands with pur i f ied vi rus . Thickness o f bands formed

was taken a s a method f o r determining se ro log ica l a f f i n i t y .

X I . SCREENING FOR DISEASE RESISTANCE

Chickpea germplasm l i n e s t h a t were repor ted t o be r e s i s t a n t

t o o t h e r d i seases ; v iz . , w i l t , r o o t r o t s , Ascochyta b l i g h t and s tun t

(pea l e a f r o l l v i rus ) were screened f o r r e s i s t a n c e t o CMV and BYMV.

For each gemplasm l i n e , 10 seeds were sown i n each 20 cm

p l a s t i c po t . Ten-day-old seedlings were separa te ly rub-inoculated

with t h e standard e x t r a c t of the two v i ruses . Observations on the

number of p l a n t s infected were recorded f o r each l i n e .

RESULTS

I. SYMPTOMATOLOGY IN CHICKPEA

A. Symptoms Produced by CMV on Chickpea

Two types of symptoms were produced by CMV i n chickpea. In

c e r t a i n v a r i e t i e s , t w i s t i n g o f terminal bud occu ed 4 - 5 days a f t e r ii i nocu la t i on (F ig . l a ) which was followed by n e c r o s i s o f te rmina l and

a x i l l a r y buds and w i l t i n g o f t h e p l a n t . P l a n t s d i ed 9-10 days a f t e r

i n o c u l a t i o n (Fig. lb ) . In o t h e r v a r i e t i e s , i nocu la t ed p l a n t s a f t e r

showing t w i s t i n g and n e c r o s i s o f terminal bud, d i d not w i l t , b u t showed

c h l o r o s i s followed by p r o l i f e r a t i o n of branches. The new branches

remained green which bore very small l eaves and very few, small pods.

Affec ted p l a n t s appeared b u h y (Fig. l c ) .

B. Symptoms Produced by BYMV on Chickpea

' h i s t i n g o f t h e te rmina l bud occurred 6-7 days a f t e r inocu-

l a t i o n which-was very s l i g h t a s compared wi th t h a t by CMV i n f e c t i o n .

This was followed by i n i t i a t i o n o f very narrow l eaves from new buds

and was more ev ident 15 days a f t e r i nocu la t i on (F ig . 2a). As t h e time

advanced, p r o l i f e r a t i o n of bo th terminal and a x i l l a r y branches occurred

r e s u l t i n g i n t o long and t h i n branches c o n s i s t i n g o f very narrow o r

f i l i form leaves . The leaves below t h e pro1 i f e r a t e d branches e x h i b i t e d

yellowing o r i n t e r v e i n a l c h l o r o s i s o r mosaic depending on t h e genotype

t e s t e d . The o v e r a l l he igh t of t h e p l a n t was reduced. Such p l a n t s

produced very few d i s t o r t e d f lowers t h a t were converted t o very small

pods (F ig . 2b) . The seeds from i n f e c t e d p l a n t s were b l ack , small and

very much s h r i v e l l e d (Fig. 2c) .

11. HOST RANGE

The r e a c t i o n s of d i f f e r e n t p l an t s p e c i e s t o CMV and BYMV a r e

p re sen ted i n Table 3 and Figures 3, 4 , 5 and 6 .

F i g . Symptoms produced by cucumber mosaic v i r u s on chickpea: ( a ) drooping of the terminal bud i n i a f e c t e d p l a n t ( l e f t ) , hea l thy p l a n t on r i g h t ; (b) w i l t i n g of t h e i n f e c t e d p l a n t ; (c) p r o l i - f e r a t i o n and bushiness of branches wi th small l e a v e s i n i n f e c t e d p l a n t ( l e f t ) ; hea l thy p l a n t on r i g h t

F i g . 2 . Symptoms produced by bean yellow mosaic v i r u s on chickpea: (a) an i n f e c t e d p l a n t wi th narrow o r f i l i f o r m leaves ; (b) showing p r o l i f e r a t i o n o f a branch with small pods ( r i g h t ) , h e a l t h y branch on l e f t ; (c) d iscoloured, deformed and s h r i v e l l e d chickpea seeds from i n f e c t e d p l a n t s ( l e f t ) , seeds from hea l thy p lan t on r i g h t .

Fig. 3 . Local l es ions produced by cucumber mosaic virus: (a) Chenopodiwn amaranticoZor, (b) Broad bean, (c) PhuseoZus ~uZgnris var. Pinto, (d) P. vuZgaris var. Porrilo

CMV - P a VULGAR IS

CMV - BAR SAT^ M U T A : ; ~

Fig. 4 . Local l e s i o n s produced by cucumber m s a i c v i r u s : (a) PRPBeoZw m 2 g a l . i ~ va r . Lavica; (b) V i g w u n g u i c u b t a v a r . Pusa Dophasli; (c ) V. urguiculata v a r . B a r s a t i mutant

CMV - CUCUMBER LONG F l g . 5 . Systemic symptoms produced by cucumber mosaic v irus on: (a) Nicotiana rustics; (b) cucumber

and (c ) peas

F i g . 6 . Systemic symptoms produced by bean yellow mosaic virus on P. vuZgaris

TABLE 3 . Host range and symptomatology of cucumber mosaic and bean yellow mosaic viruses

Symptoms Host CMV BYMV

A 1 ZOACEAE

Tetragoniu expansa Murr . AMARAN lMACEAE

Gomphrena globosa Murr . CHENOPODIACEAE

Chenopodiwn mant-LcoZor Coste e t NLL Ryne

COW 0s I TAE

Chry sun themwn sp . CUCURBI TACEAE

Systemic, inducing - large ch loro t ic patches

Cucurbitu pepo L . CLL - LEGUMINOSAE

Arachis hypogaea L. - - Detailed symptoms are described i n the t e x t

Cajanus cajan ( L . ) Millsp. NLL - CanavaZia ensi fomis D.C. - Mos, f r b t l .

&crotyZoma m i f l o r m (Lam.) Verdc. - - PhQseoZus vuLgaris L. 'Ababat NLL hb s.

P. vuZgaris ' Po r r i l o t NLL - P. vuZgaxis 'Red kidneyt N LL VN, M s . M t l .

P. vuZgaris Dwarf

P. vuZgaris 'Pinto

- a, m, b s , Motl, Ma

NLL VN, Mos, Motl, Ma.

P. vuZgaris 'Munroe1 NLL - P. vuZga- ' Lavica ' NLL -

contd.

TABLE 3 . Contd,

Host Symptoms

CMV BY MY

P i s m s a t i v m L. - Vicia faba L. 'Early long pod' NLL

V i g n a unguicuZatu (L.) Walp. NLL 'Early Ramshorn'

V. unguicu~ata ' Barsat i mutant ' NLL

V . unquiculatu 'Puss Ebphasli ' N L L

SOLAN ACEAE

Capsicm a n m m L. - - Datura stramoniwn L . CL L

Lycope~sicon esculentwn Mill. ' Pusa Rubyt - Nicotiam e a c w n L . ' Xanthi-ncl Mos, M t l . - N. rust ica L. 'White Pathar ' Mos, M o t l . - Petunia hybrida V i l m . - -

CLL = Chlorot ic l o c a l l e s ions ; Ma = Malformation of leaves; Mos = Mosaic; Motl = Mottle; NLL = Necrot ic l o c a l l e s ions ; TN = Top necros is ; VN = Veinal necros is ; - = No symptoms.

111. I N VITRO PROPERTIES

A . Cucumber Mosaic Virus

1. Thermal i n a c t i v a t i o n m i n t (TIPI

The assay p l a n t s inocula ted with t h e s tandard e x t r a c t t r e a t e d

a t 4S0, So0, 5S0, 60°, 6s0, and 7 0 ' ~ produced l o c a l l e s i o n s whereas no

loca l l e s i o n s developed with the e x t r a c t heated a t 7S0c o r more (Table 4)

The TIP o f t h e v i r u s was t h u s between 70' and 75'~.

TABLE 4. Influence of hea t ing t h e standard e x t r a c t of cucumber mosaic v i r u s a t d i f f e r e n t temperatures f o r 10 minutes on i n f e c t i v i t y

Exposure temperature (OC) Number o f loca l l e s ions*

4 5

5 0

5 5

6 0

6 5

7 0

75

80

8 5

9 0

Control

*Average o f s i x r e p l i c a t i o n s wi th range o f l e s i o n s i n parentheses

2 . Dilution end po in t (DEP)

Assay p l a n t s inocu la ted with 0, lo-', and lo - )

d i l u t i o n s of t h e s tandard e x t r a c t produced l o c a l l e s i o n s . No l o c a l

l e s i o n s were produced when t h e p l a n t s were inocu la ted wi th t h e

e x t r a c t d i l u t e d t o 1 0 ' ~ o r above (Table 5 ) . Thus t h e DEP o f t h e

v i r u s war between and ld4.

3 . Longevity in vitm

The s tandard e x t r a c t s t o r e d a t mom temperature (22' - 24'~)

l o s t i t s i n f e c t i v i t y on t h e f i f t h day (Table 6) .

TABLE*5. Influence o f d i l u t i n g t h e standard e x t r a c t o f cucumber mosaic v i r u s on i n f e c t i v i t y

Di lut ion Number of l o c a l l e s ions*

0

*Average of s i x r e p l i c a t i o n s wi th range o f l e s i o n s i n parentheses .

TABLE 6 . Infllrence of s to rage o f t h e standard e x t r a c t o f cucumber mqsaic v i r u s a t room temperature on i n f e c t i v i t y

Day on which p l a n t s were inocula ted Number of l o c a l l e s i o n s *

Immediately a f t e r e x t r a c t i o n of sap

F i r s t

Second

Third

Fourth

F i f t h

S i x t h

Seventh

*Average o f six r e p l i c a t i o n s wi th range o f l e s i o n s i n parentheses.

B. Bean Yellow Mosaic Virus *

1. Thermal inac t iva t ion point (TIP)

The chickpea p l a n t s inocula ted with t h e standard ex t rac t

heated a t 45', 50' and 5 5 ' ~ f o r 10 minutes produced disease symptoms

(Table 7). However, no symptoms were no t i ced when the p l a n t s were

inocula ted with the e x t r a c t heated a t 60°c o r more. The TIP o f t h e

v i r u s was thus between 55' and 60'~.

2. Dilution end po in t [DEP)

P lan t s inocula ted with t h e s tandard e x t r a c t d i l u t e d to 10"

and produced d i sease symptoms but no t with d i l u t i o n s t h e r e a f t e r

[Table 8) . The DEP o f t h e v i r u s was thus between and

3 . Longevity i n v i t r o

The v i r u s sap when s t o r e d a t room temperature (22-24'~) l o s t

i t s i n f e c t i v i t y on t h e 2nd day (Table 9 ) .

TABLE 7. Influence o f hea t ing the s tandard e x t r a c t of bean yellow m s a i c v i r u s a t d i f f e r e n t temperatures f o r 10 minutes on i n f e c t i v i t y

Expo sure temperature (OC)

4 5

50

5 5

60

65

7 0

75

80

85

9 0

Control

No. o f p l a n t s No. o f p l a n t s inocu la ted i n f e c t e d

10 7

10 5

10 3

10 0

10 0

10 0

10 0

10 0

10 0

10 0

10 10

TABLE 8. Inf luence o f d i l u t i n g t h e s t a n d a r d e x t r a c t o f bean yellow , mosaic v i r u s on i n f e c t i v i t y

Di lu t ion No. o f p l a n t s No. o f p l a n t s inocu la ted i n f e c t e d

TABLE 9 . In f luence o f s to rage o f t h e s t andard e x t r a c t o f bean yellow m s a i c v i r u s a t room temperature on i n f e c t i v i t y

Day on which p l a n t s were inocu la ted

Immediately a f t e r e x t r a c t i o n of p l a n t sap

F i r s t

Second

Third

Four th

No. o f p l a n t s No. o f p l a n t s ino cul a t ed i n f e c t e d

IV. INSECT TRANSMISSION #

The r e s u l t s on t h e transmission o f t h e two v i ruses by t h e

two aphid species a r e given i n Table 10. Both the vi ruses could be

t ransmit ted by Mzjzus persicae and Aphis cmccivora in a non-pers is tent

manner though t h e l a t t e r was found t o be m r e e f f i c i e n t than the

former.

V. PURIFICATION

A . Cucumber Mosaic Virus

The p u r i f i c a t i o n procedure adopted i n t h e present s tud ies

r e s u l t e d in highly p u r i f i e d and i n f e c t i o u s v i rus . .When butanol was

used f o r i n i t i a l c l a r i f i c a t i o n , m s t o f t h e chloroplas t mater ia l

could be e l iminated and thus a c l e a r amber-coloured supernatant was

obta ined a f t e r low speed cen t r i fuga t ion . m e v i r u s could e a s i l y be

concentrated by PEG p r e c i p i t a t i o n and d i f f e r e n t i a l cen t r i fuga t ion .

The high speed cen t r i fuga t ion r e s u l t e d i n a g lassy p e l l e t and by which

time almost a l l t h e green colour could be e l iminated. Further pur i -

f i c a t i o n by densi ty gradient cen t r i fuga t ion r e s u l t e d i n a s ing le ,

th ick, l i g h t s c a t t e r i n g zone in dens i ty gradient tube (Fig. 7a). When

t h e gradient tube was subjected t o f r a c t i o n a t i o n i n an ISCO f r a c t i o n -

a t o r , a s i n g l e absorption peak could be obta ined corresponding t o t h e

v i r u s zone (Fig. 7b). 'Ihe r e s u l t s of t h e i n f e c t i v i t y assays o f

gradient f r a c t i o n s ind ica ted t h a t t h e i n f e c t i v i t y was confined t o the

W absorbing aomponent (Fig. 7b). A c l e a r g lassy p e l l e t was obtained

i n high speed , cen t r i fuga t ion of t h e d i l u t e d v i r u s zone and could be

e a s i l y suspended i n buf fe r .

?he homogeneity of t h e p u r i f i e d v i r u s could b e f u r t h e r

assessed spectrophotometrically. The absorption spectrum was found

t o be typ ica l of nucleoprote in (Fig . 7c) , with absorpt ion maximum a t

TABLE 10. Aphid transmission o f cucumber mosaic and bean yellow mosaic v i r u s e 2

Aphid species

No. o f p lants Percent No. of aphids b No. o f p lants infected transmission used per p lant inoculatedC CMV BY MV CMV BYMV

Mjzw persicae

Aphis craccivora.

a. Data from two t r i a l s .

b. 'he aphids were given one hour f a s t i n g period and one minute acquisi t ion feed period.

c. Inoculation feeding period was 1 2 hours. Ten-day-old p lants were used f o r inoculation.

2d 260 240 W I W Length (nm)

Fig. 7 . a ) Sucrose dens i ty g rad ien t tube showing l i g h t s c a t t e r i n g zone of W; b) t h e W absorpt ion (-) and infectivity(-) of f r a c t i o n s from d e n s i t y g rad ien t column; c) t h e W absorpt ion spectrum of t h e p u r i f i e d CMV

260 nm and minirmun a t 240 nn. I t had A260/A280J A280/A260J and $

A max/A min r a t i o s o f 1.55, 0.64, and 1.27, r e spec t ive ly .

The v i r u s y i e l d ranged from 8 t o 10 mg/100 g of p lan t t i s s u e

when ca lcu la ted spectrophotometrically.

B. Bean Yellow Mosaic Virus

Since a s a t i s f a c t o r y loca l l e s i o n hos t could not be found,

the success o f t h e p u r i f i c a t i o n procedure adopted was assessed only

through t h e use o f t h e e l e c t r o n microscope. With t h e experience

gained from d i f f e r e n t p u r i f i c a t i o n procedures t r i e d , t h e following

conclusions could be drawn:

Carbon t e t r a c h l o r i d e was b e t t e r than chloroform o r butanol

f o r pre l iminary c l a r i f i c a t i o n .

r Addition o f carbon t e t r a c h l o r i d e i n t h e Waring blendor

during homgenizat ion was found t o be b e t t e r than adding

it a f t e r homogenization.

Use o f PEG alone was found t o be b e t t e r than PEG + NaC1.

Overnight incubation o f t h e PEG dissolved prepara t ion was

found t o be b e t t e r than incubat ion f o r 2 hours.

High speed cen t r i fuga l f o r c e s increased p a r t i c l e breakage.

Use of a sucrose pad was found use fu l i n e l imina t ing most

o f t h e green mate r i a l .

Out o f severa l methods used, t h e one adopted i n t h e present

i n v e s t i g a t i o n (descr ibed under Mater ia ls and Methods) was found t o be

s a t i g f a c t o r y . But i n densi ty gradient cen t r i fuga t ion , t h e v i r u s zone

was no t compact and c lea r . Rather, an inconspicuous, d i f fused zone

was obtained. However, t h i s zone, when p e l l e t e d consis ted o f high

concentrat ion o f v i r u s p a r t i c l e s ; though, the re was some degree of

breakage.

The o p t i c a l cha rac te r s and o t h e r physico-chemical p roper t i e s

could not be performed s i n c e t h e v i r u s p repara t ion , f r e e o f host

components could not be obtained.

VI . ELECTRON MICROSCOPY

A. CM! P u r i f i e d Prepara t ion

The negat ively s t a ined prepara t ion o f t h e p u r i f i e d CMV

contained a homogenous population o f spher ica l p a r t i c l e s which

measured about 30 nm i n diameter (Fig. 8 ) .


1. Leaf d ip prepara t ion

The negat ively s t a i n e d 1 eaf d ip prepara t ion revealed the

presence of f lexuous rods (Fig. 9 ) . From t h e l eng th d i s t r i b u t i o n

histogram (Fig. 10) i t i s c l e a r t h a t most o f t h e p a r t i c l e s measured

between 740 and 760 nm with a modal l e n g t h o f 750 nm. The width was

determined t o be about 15 nrn.

2 . Pur i f i ed prepara t ion

A negat ively s t a i n e d p repara t ion o f the p u r i f i e d BYMV

contained flexuous rods. Though the re was a breakage o f p a r t i c l e s

t o some ex ten t , aggregation was not observed (Fig . 11) .

m w o rd 4 h n O cdo .ri F 4 0 C, Q) 1- k k - rd a m 14 * a M.rl X c a -

* d

g % 2 5 2 :

P a r t i c l e s l e n g t h ( ~ 1 0 ) F i g . 10. An histogram showing the length d i s t r i bu t i on o f p a r t i c l e s

of bean yellow mosaic v i r u s f r o m l e a f d ip

3. Ul t ra th in sec t ions o f BYMV-infected chickpea leaves

Electron microscopy of u l t r a t h i n sec t ions o f BYMV-infected , chickpea leaves revealed the presence o f pinwheel inc lus ions with

curved p l a t e s , and laminate aggregates (Fig. 1 2 ) .

JII. IDENTIFICATION OF VIRUSES BY SEROLOGY

A. Double-Diffusion i n Agar

Clear p r e c i p i t i n bands could be observed when crude sap from

v i rus - infected chickpea p l a n t s and p u r i f i e d prepara t ions of CMV

(Fig. 13a) and BYMV (Fig. 13b) were made t o r e a c t wi th t h e i r respect ive

a n t i s e r a . The p u r i f i e d CMV sometimes produced two p r e c i p i t i n bands

with i t s antiserum, a s t r a i g h t one nea r t h e antiserum well and a

curved one near t h e ant igen well (Fig. 13c) . No reac t ion e i t h e r with

the heal thy sap o r b u f f e r control was observed wi th a n t i s e r a o f the

two vi ruses .

B. Enzyme-Linked Innnunosorbent Assay (ELISA)

For i d e n t i f i c a t i o n o f CMV by ELISA, t h e anti-CMV obtained

from D r . H.E. Waterworth, USDA, Maryland, U.S.A. was used (CMV-S) .

A p o s i t i v e reac t ion (development o f yellow colour) could be observed

both with i n f e c t i o u s crude sap and p u r i f i e d v i r u s a t y-globulin

concentra t ion o f 1:100 and conjugate concentra t ions o f 1: 100 and

1 :SO0 (Fig. 14) . No r e a c t i o n was observed e i t h e r with the heal thy

sap o r buf fe r .

The ELISA could not be performed w i t h BYMV due t o non-

a v a i l a b i l i t y o f good antiserum.

Fig. 12. Electron pbtomicrographs of uitratfuii sections cf BW- infected chickpea l ea f showing: (a) pinwheels (PW) and (b) laminate aggregates (LA)

Fig. 13. Agar gel double-diffusion t e s t o f p u r i f i e d CMV and i n f e c t - i v e crude sap. a ) Each o u t e r wel l c o n t a i n s : sap from CMV- i n f e c t e d chickpea ( I ) , p u r i f i e d CMV (2), b u f f e r (3 ) ) and saD from hea l thy chickpea (41. The c e n t r a l wel l c o n t a i n s - L . <

ant iserum t o CMV(A). Note a p o s i t i v e r e a c t i o n between p u r i f i e d CMV and i n f e c t i v e sap with ant iserum. b) Outer w e l l s con ta in : b u f f e r ( I ) , p u r i f i e d v i r u s ( 2 ) , sap from heal thy chickpea ( 3 ) , s a p from BYW-infected chickpea ( 4 ) . Note a p o s i t i v e r e a c t i o n o f p u r i f i e d v i r u s with antiserum. Reaction o f i n f e c t i v e sap with antiserum has n o t come i n t h e photograph. c) Both t h e o u t e r w e l l s c o n t a i n s p u r i f i e d CMV whereas t h e c e n t r a l well c o n t a i n s ant iserum t o CMV. Note t h e two types o f p r e c i p i t i n bands

Fig . 1 4 . I d e n t i f i c a t i o n o f cucumber mosaic v i r u s by ELISA. INF - s a p from i n f e c t e d chickpea; PV - p u r i f i e d virus; H - sap from h e a l t h y chickpea; CMV-H - homologous ant iserum, CMV-S - an t i se rum t o CMV s t r a i n S . Note a p o s i t i v e r e a c t i o n w i t h i n f e c t i v e s a p and p u r i f i e d v i r u s . Also n o t e a s l i g h t p o s i t i v e r e a c t i o n o f h e a l t h y s a p with CMV-H a t a conjugate c o n c e n t r a t i o n o f 1:100 .

V I 11. PHYSICO-CHEMICAL PROPERTIES OF ?HE PURIFIED CMV

A. Sedimentation Coeff ic ien t (SZOW)

The homogeneity o f t h e p u r i f i e d CMV was ind ica t ed by t h e

presence o f a s ing le Schle i ren peak (Fig. 15) i n t h e ana ly t i ca l u l t r a -

cent r i fuge . The SZ0W o f t he p u r i f i e d CMV was ca l cu la t ed t o be 104s.

B. Buoyant Density

In t h e Cesium c h l o r i d e equi l ibr ium dens i ty gradient c e n t r i -

fugat ion , t h e p u r i f i e d CMV banded a t a d e n s i t y l e v e l o f 1.325 g/ml.

The dens i ty l e v e l s and UV absorpt ion o f d i f f e r e n t f r a c t i o n s o f t he

g rad ien t a r e given i n Fig. 16.

C. The PAGE o f Coat Pro te in

The PAGE o f SDS-disrupted coat p r o t e i n o f CMV revea led t h e

presence o f a s i n g l e polypeptide. The banding p a t t e r n in t h e ge l s and

the scanning p a t t e r n o f t h e W - c o a t p r o t e i n and marker p r o t e i n s a r e

given i n Fig. 17a and 18. Based on t h e l i n e a r r e l a t i o n s h i p between

migrat ion o f marker p r o t e i n s and t h e logar i thm o f t h e i r known molecular

weights (Fig. l a d ) , t h e molecular weight o f CMV-coat p ro t e in was

est imated t o be 25,000 da l tons .

D. The PAGE o f Nucleic Acids

The PAGE o f SDS-disrupted CMV r evea led t h e presence o f fou r

RNA spec i e s (Fig. 1%). The e l ec t rophores i s o f t h e d i s rup ted v i r u s

t h a t was f i r s t t r e a t e d wi th RNase d id n o t produce any bands.

FRACTION No.

~ i g . 16, The UV absorpt ion ( - - - - - - - - ) and d e n s i t i e s (oeooee) of f r a c t i o n s from CsCl gradient column (CMV) . Note t h a t the W absorbing component had a buoyant dens i ty of 1.325 g/ml

Fig. 17. a ) The PAGE of (B) W - c o a t p r o t e i n , (C) marker p r o t e i n s and (A) coe l ec tmphores i s of B and C. Marker p r o t e i n s are: Phosphorylase B , ml . w t . 92,500(1); Bovine serum albumin, ml.wt. 66,200 ( 2 and 3 s ince t h i s m r k e r has s p l i t i n t o two); Ovalbumin, ml.wt. 45,000(4); Carbonic anhydrase, mol.wt. 31,000(5); Soybean t r y p s i n i n h i b i t o r y , ml . w t . 21.500(6) and Lysozyme, ml.wt. 14,400(7) . Note t h a t v i r u s p r o t e i n has not reso lved from carbonic anhydrase. Migration is from top t o bottom.

b) The PAGE o f CMV-RNAs, showing f o u r RNA s p e c i e s , Migration i s from t o p t o bottom.

IX. PRODUCTION OF ANTISERUM TO CMV

The immunization schedule followed produced a reasonably

high t i t r e d antiserum t o CMV. In Ouchterlony double-diffusion t e s t

the t i t r e was determined t o be 1/1024 when t e s t e d wi th the p u r i f i e d

v i rus . No reac t ion could be no t i ced with t h e heal thy sap i n t h i s

t e s t . However, when t e s t e d by ELISA, a very n e g l i g i b l e p o s i t i v e

react ion was no t i ced a t a conjugate concentrat ion of 1:100; no reac t ion

could be no t i ced a t a conjugate concentra t ion o f 1:500 pig. 14) .

X. SEROLOGICAL RELATIONSHIP BETWEEN 'IHE PRESENT STRAIN OF CM, WITH OTHER KNOWN STRAINS

The i n t e n s i t i e s of the p r e c i p i t i n bands formed i n Ouchter-

lony double-diffusion t e s t when t h e p u r i f i e d CMV was made t o r e a c t

with a n t i s e r a t o d i f f e r e n t known s t r a i n s o f CMV a r e given i n Table 11.

The r e s u l t s i n d i c a t e t h a t the p resen t CMV s t r a i n i s r e l a t i v e l y more

c losely r e l a t e d t o OW-C pool, followed by CMV-IX and M-CMV.

TABLE 11. Serological a f f i n i t y of p resen t CMV s t r a i n with o the r known s t r a i n s o f CMV

- - -

S t r a i n Source o f antiserum Reaction

CMV-C pool

am- I X

CMV- S

U-CMV

X- CMV

Q- m' M-w T- CMV

Homologous

H.A. S c o t t (U.S.A.)

H.E. Watemorth (U.S.A.)

H. E . Waterworth (U.S.A.)

R. I .B . Francki (Aus t ra l i a )

R. I.B. Francki (Aus t ra l i a )

R.I.B. Francki (Aus t ra l i a )

R . I . B . Francki (Aust ra l ia)

R.I.B. Francki (Aust ra l ia)

ICRISAT

XI. SCREENING FOR DISEASE RESISTANCE

A . Cucumber Mosaic Virus ,

The data on the react ions o f chickpea l i n e s to CMV a r e

presented i n Appendix I . I t i s evident t h a t only two l i ne s , ICC-1781

and -8203, remained f r e e from CMV infec t ion , one l i n e ICC-9006 showed

10% infec t ion and the remaining 140 l i n e s showed in fec t ion ranging

from 33 t o 100%.


'Ihe data on the reac t ions o f t h e 106 chickpea l i n e s to BYMV

a r e presented in Appendix 11. The r e s u l t s show t h a t 9 l i ne s ; ICC-607, -6999,

-1468, -2162, -2342, -3440, -3598, -4045, and -11550, remained f ree

from BYMV i n f ec t ion , four l i n e s ; ICC-1416, -3916, -4093 and -4192,

showed 10% infec t ion , 12 l i ne s ; ICC-187, -1121, -1754, -3634, -3918,

-3919, -3921, -3969, -4188, -6989, -7000 and -8207, showed infec t ion

between 10 and 20%. A l l the remaining 81 l i n e s showed infec t ion

ranging from 22 t o 100%.

D I S C U S S I O N A N D C O N C L U S I O N

Two v i ruses , v i z . , cucumber mosaic v i r u s (CMV) and bean

yellow mosaic v i r u s ( B W ) were found t o i n f e c t chickpeas under na tu ra l

condit ions. Their i d e n t i t y was confirmed by studying t h e i r character-

i s t i c s .

A d e t a i l e d study of t h e symptomatology by CMV and BYMV i n

chickpea ind ica ted t h a t both the v i ruses caused a s i m i l a r i n i t i a l

twis t ing of t h e terminal bud though the former caused a severe

twis t ing which terminated i n nec ros i s i n c o n t r a s t with a s l i g h t twist-

ing without nec ros i s caused by t h e l a t t e r . The CMV, a f t e r terminal

nec ros i s , caused two types of symptoms. In c e r t a i n v a r i e t i e s , i t

caused w i l t i n g and death of t h e p l a n t s whereas, i n o t h e r s p r o l i f e r a t i o n

and bushiness o f branches bearing small green l eaves . The l a t t e r

were commonly seen i n mechanically inocula ted chickpea and resembled

t h e symptoms produced by CMV under f i e l d condi t ions . The i n f e c t i o n

by BYMV a f t e r i n i t i a l twis t ing was followed by p r o l i f e r a t i o n o f both

the terminal and a x i l l a r y branches which a r e long and t h i n wi th

narrow leaves . The leaves below t h e p r o l i f e r a t e d branches showed

yellowing o r i n t e r v e i n a l ch lo ros i s o r mosaic depending upon t h e

genotypes used.

'Ihe present s t r a i n of CMV appears t o have a wide host range

as i t could i n f e c t 20 p lan t species belonging t o f i v e fami l i e s ;

A i zoaceae , Chenopodiaceae, Cucurbitaceae, Legminosae, and Sol anaceae,

Though i t produced l o c a l l e s ions on severa l p l a n t s t e s t e d , C. maranti- color was found t o be very good assay hos t and was, the re fo re , used

throughout t h e s t u d i e s . E a r l i e r , (=MV was repor ted t o produce systemic

mosaic on cucumber and f e r n leaf symptoms on tomato (Francki e t al . ,

1979). On t h e contrary , t h e p resen t s t r a i n of could n o t i n f e c t

tomato and produced l a r g e c h l o r o t i c patches on cucmber i n s t e a d o f a

systemic mosaic. Waterworth and Povish (1975) a l s o got s i m i l a r r e s u l t s

with CMV-IX. However, the cu l t iva r s of cucumber and tomato used i n the

present )s tudies were d i f fe ren t from those of Waterworth and Povish.

Though tobacco and pumpkin were reported t o be good hosts f o r v i rus

pur i f i ca t ion , i t was found t h a t chickpea supported b e t t e r v i rus multi-

p l i ca t ion , and therefore was used f o r v i rus pur i f i ca t ion i n the present

s tudies .

Though BYMV i s known to i n f e c t several non-legumes (Bos, 1970a),

the present s t r a i n appears to have a very r e s t r i c t e d host range as it

could not i n f e c t any p lan t species outs ide t h e Leguminosae. I t a l s o

produced veinal necros is of inoculated leaves and top necrosis o f some

french bean cu l t iva r s . The severe yellow mosaic s t r a i n of BYMV (Rex

and Zaumeyer, 1953) which a l so produced veinal necros is and top necros is

of french beans could a l s o not i n f e c t p l a n t s outs ide Leguminosae.

Based on the r e s u l t s of i n vitro proper t ies , the present

s t r a i n of CMV appears qu i t e s table . On the contrary, BYMV appears t o

be very unstable. However, it i s evident from t h e l i t e r a t u r e t h a t

in v i t r o proper t ies g rea t ly depend on the v i r u s source and t e s t p lan t

(Bos, 1970a).

Transmission of both the v i ruses by aphids d id not exceed

45%. I t i s well documented t h a t CMV t ransmission effici,ency va r ies

with p lant species (Simons, 1955; Simons, 1957). Similar ly , with CMV

and BYMV, Kaiser and Danesh (1971b) found t h a t transmission by

d i f f e r e n t aphid species was usually l e s s than 20% when chickpea was

used both as source and t e s t p lan t , whereas it was invar iably higher

when sweet clover and cucumber were used for CMV and BYMV, respect ively .

However, i n the present s tud ies t h e e f f e c t of d i f f e r e n t host species

on the transmission e f f i c i ency of aphids was no t studied.

The pur i f i ca t ion procedure adopted f o r CMV appeared e f f i c i e n t

a s judged by the W absorption i n ISCO f r a c t i o n a t o r and spectrophoto-

meter, i n f e c t i v i t y , and e lec t ron microscopy. A s ing le , sharp peak

obta ined i n the ISCO scanning p a t t e r n was an i n d i c a t i o n of i n t a c t

v i rus , without aggregation or degradation. In i n f e c t i v i t y t e s t s , the

f r a c t i o n s p resen t only i n the W absorbing component were found

i n f e c t i v e . Thus, the re was a c l o s e c o r r e l a t i o n between W absorbing

component and i n f e c t i v i t y which revea l s t h a t t h e zone i n t h e sucrose

gradient cons i s t ed of i n f e c t i v e v i r u s . The W absorbing p a t t e r n o f

f i n a l p u r i f i e d v i r u s i n spectrophotometer was dlso typ ica l o f nucleo-

p ro te in . Considerably higher y i e l d s (80-100 mg/kg o f t i s s u e ) were

obta ined by the p u r i f i c a t i o n procedure adopted. Yields o f 100-500

mg/kg t i s s u e have been repor ted i n the l i t e r a t u r e (Waterworth and

Povish, 1975; Francki e t aZ., 1979), though t h e y i e l d depended on

v i r u s s t r a i n and host p l a n t (Francki e t aZ., 1979).

The p a r t i c l e diameter (30 nm) obta ined f o r t h e present CMV

s t r a i n was i n agreement wi th o t h e r r e p o r t s (Francki e t aZ., 1966 and

Debmt e t at., 1974).

An e f f i c i e n t p u r i f i c a t i o n procedure could no t be evolved f o r

BYMV which belongs t o the pota to v i r u s Y (potyvirus) group. Viruses

of the potyvirus group a r e d i f f i c u l t t o p u r i f y because o f t h e i r

tendency t o aggregate, both end t o end and s i d e by s i d e (Shepherd and

Pound, 1960). Nevertheless, t h e r e have been severa l r e p o r t s on t h e

p u r i f i c a t i o n o f var ious v i ruses of t h i s group. The general conclusions

from these r e p o r t s a r e t h a t only small a m u n t s o f homogenous v i r u s . prepara t ions were obta ined wi th r a t h e r complicated procedures and t h a t

t h e methods success fu l ly used wi th a p a r t i c u l a r v i r u s could not

genera l ly be app l i ed f o r p u r i f i c a t i o n o f o t h e r e longate v i ruses . In

the present s t u d i e s , var ious methods used f o r BYMV p u r i f i c a t i o n d id

not y i e l d a high p u r i t y v i r u s . However, v i r u s p repara t ion o f s a t i s -

f a c t o r y p u r i t y could be obta ined by t h e procedure adopted. Urea t o

t h e concentra t ion ranging from 0.2 t o 1M has been used i n p u r i f i c a t i o n

o f d i f f e r e n t potyviruses t o avoid aggregation of t h e p a r t i c l e s

(Damirdagh and Shepherd, 1970; Uyeda e t aZ., 1975). In t h e present

s t u d i e s , 'the aggregat ion o f BYMV p a r t i c l e s could be avoided by using

0.2M urea i n a suspending medium. However, t h e breakage o f t h e v i r u s

p a r t i c l e s could no t be avoided completely d e s p i t e t h e use o f consi-

derably low c e n t r i f u g a l fo rces . The d i f f u s e d l i g h t s c a t t e r i n g zone

obta ined a f t e r a d e n s i t y g rad ien t c e n t r i f u g a t i o n might b e due t o t h e

breakage o f v i r u s p a r t i c l e s . Withdrawal o f t h e e n t i r e d i f fused zone

might have r e s u l t e d i n t h e contamination o f t h e v i r u s wi th p lan t

c o n s t i t u e n t s . With t h e r e s u l t , t h e v i r u s f r e e of host mater ia l could

no t be obta ined. For t h i s reason, t h e physico-chemical p r o p e r t i e s

c a r r i e d out f o r CMV could no t be performed f o r t h i s v i r u s . The mdal

length obta ined f o r this v i r u s was i n agreement wi th t h a t r epor ted f o r

o t h e r s t r a i n s of BYMV (Uyeda e t aZ., 1975; Kaiser and Danesh, 1971a).

Pinwheel inc lus ions observed i n u l t r a t h i n s e c t i o n s o f BYMV-infected

chickpea leaves were t y p i c a l o f po tyv i ruses (Mar te i l i and Russo, 1977)

t o which BYMV belongs.

In Ouchterlony double-di f fus ion t e s t , CMV sometimes produced

two p r e c i p i t i n bands. S c o t t (1968) and Devergne and Cardin (1970)

a l s o observed formation o f two p r e c i p i t i n l i n e s , a curved one n e a r

t h e ant igen we l l , caused by more o r l e s s i n t a c t v i r u s , and a s t r a i g h t

one near the antiserum wel l caused by degraded v i r u s . Sodium dodecyl

s u l f a t e - t r e a t e d BYMV, however, produced on ly a s i n g l e p r e c i p i t i n band.

The reason f o r a s l i g h t l y h igher sedimenta t ion value (104s)

obta ined f o r the p resen t (SiV s t r a i n than t h e repor ted values o f 98.6

(Francki e t aZ., 1966) and 101 (Tomlinson e t aZ., 1959) i s no t c l e a r .

The buoyant d e n s i t y of 1.325 g/ml obta ined f o r t h e p resen t

CMV s t r a i n was i n c l o s e agreement w i t h t h e va lues o f 1.361 and 1.359

f o r CMV-S and O W - D s t r a i n s , r e s p e c t i v e l y (Lot and Kaper, 1976).

E s s e n t i a l l y no W absorbing mate r i a l was p r e s e n t except i n t h e v i r u s

band, i n d i c a t i n g t h e high degree o f b m y a n t homgene i ty o f t h e

p u r i f i e d product .

The m l e c u l a r weight o f the CMV coat p ro te in obtained i n the , present s tud ies (25,000) was i n c l o s e agreement with the values o f

24,500 (Habil i and Francki, 1974) and 25,200 (van Regenmrtel e t aZ.,

1972).

The PAGE o f SDS-disrupted CMV revealed t h e presence of four

RNA species . The RNAs o f CMV have been va r iab ly described a s compris-

ing 4-6 d i f f e r e n t s i zed molecules. The presence o f f i t h and s i x t h

RNA components was claimed (Kaper and West, 1972; Peden and Symns,

1973) but was disputed when these components could not be cons i s t en t ly

ext racted from d i f f e r e n t s t r a i n s of CMV (Lot e t aZ., 1974) . However,

more recent work has demonstrated t h a t a t l e a s t one o f t h e s e components

(RNA 5) i s cons i s t en t ly present i n l a r g e proportions i n CMV-W (Wood and

b u t t s , 1975), and a l s o i n s t r a i n s D and R (Lot and Kaper, 1976).

Kaper and Waterworth (1977) discovered t h a t RNA 5 i s t h e causal agent

f o r a l e t h a l necros is d isease o f tomato p l a n t s . Though the presence o f

f i f t h and s i x t h WAS was doubtful , the presence o f t h e four l a r g e s t

components has never been i n doubt (Kaper and Tousignant, 1977). The

present s t r a i n o f Q.IV a l s o contained 4 RNAs. With CMV-bmmelina s t r a i n ,

Kaper and Tousignant (1977) demonstrated t h a t more than one passage i n

tobacco was required t o b u i l d up e l e c t r o p h o r e t i c a l l y de tec tab le

q u a n t i t i e s of RNA 5 . Thus, they a t t r i b u t e d t h a t c e r t a i n s t r a i n s of

CMV may contain undetectable q u a n t i t i e s o f RNA 5 whose syn thes i s can

be generated upon propagation i n c e r t a i n h o s t s . However, t h e e f f e c t

of propagation o f CMV i n d i f f e r e n t host p l a n t s on generation o f RNA 5

was not s tud ied i n t h e present s t u d i e s .

A good t i t r e d (1/1024) antiserum t o CMV produced i n t h e

p resen t s t u d i e s ind ica ted t h a t the i m u n i z a t i o n schedule followed was

good. The immunogenicity o f CMV was repor ted t o be poor (Francki

e t al., 1979), bu t could be enhanced by f i x a t i o n wi th formaldehyde

(Francki and Hab i l i , 1972) o r glutaraldehyde (Rao, 1982) . Using

t h e f i x e d v i r u s a s an ant igen, Rao (1982) produced antiserum to s i x

s t r a i n s o f CMV with a t i t r e ranging from 1/128 t o 1/512. Tomlinson

e t aZ. (1958) produced antiserum t o unfixed CMV which had a t i t r e of

1/1024. However, they used p a r t i a l l y p u r i f i e d v i r u s and thus the

r e s u l t a n t antiserum might have contained an t ibod ies t o p l a n t prote ins

a l s o which might have contr ibuted t o the high t i t r e of antiserum. In

t h e present s t u d i e s , no react ion wi th t h e hea l thy sap was detected i n

agar double-diffusion t e s t , and hence, the reac t ion was a t t r i b u t e d

t o the v i r u s only. I n ELISA, a s l i g h t r e a c t i o n could be not iced with

t h e heal thy sap a l so . However, when the reactions t o the i n f e c t i v e and

heal thy sap were compared, the colour development i n case o f heal thy

sap was very neg l ig ib le . Even t h i s r e a c t i o n however was not observed

when conjugate concentrat ion was increased from 1: 100 t o 1:500.

The na tu ra l occurrence o f these two v i ruses was found t o be

l e s s than 1%. Considering the cropping p a t t e r n in ' Ind ia which includes

severa l cucurbitaceous and leguminous p l a n t s , which no t only harbour

these v i ruses b u t a l s o a i d i n t h e vector population buildup, the re

e x i s t s a p o t e n t i a l p o s s i b i l i t y f o r these v i ruses t o become widespread

i n the f u t u r e . The n a t u r a l occurrence o f CMV has a l ready been repor ted

by Dhingra e t aZ. (1979). Though t h e n a t u r a l occurrence o f BYMV was

no t repor ted , the survey r e s u l t s ind ica ted t h a t i t was p resen t i n

severa l p laces i n India such a s Fai zabad and Pantnagar (Ut t a r Pradesh) , Junagarh (Guj a r a t ) , Parbhani (Maharashtra) , and Tapparwaripora (Jammu

and Kashmir) (Dr. S.P.S. Beniwal, personal communication) . Therefore,

keeping t h e t h r e a t posed by t h e s e v i r u s e s i n view, the e l i t e chickpea

germplasm access ions were screened f o r r e s i s t a n c e t o these two v i ruses ,

and a number o f access ions were found f r e e from i n f e c t i o n . These

access ions a f t e r confirming t h e i r r e s i s t a n c e , could be used in chickpea

breeding program t o incorporate r e s i s t a n c e i n t o commercially acceptable

chickpea cul t i v a r s t o thwart any p o t e n t i a l danger caused by t h e s e

v i ruses t o chickpea c u l t i v a t i o n i n f u t u r e .

S U M M A R Y

Wo mechanically t r ansmiss ib le v i r u s e s , v iz . , cucumber mosaic

v i r u s (CMV) and bean yellow mosaic virus (BYMV) were found t o

n a t u r a l l y i n f e c t chickpeas. Both t h e v i r u s e s were i d e n t i f i e d by

studying t h e i r hos t range, physical p roper t i e s , i n s e c t transmission,

e l ec t ron microscopy and serology. E l i t e chickpea germplasm accessions

t h a t were e a r l i e r found t o be r e s i s t a n t t o w i l t , r o o t r o t , Ascochyta

b l i g h t and promising t o s t u n t (pea l e a f r o l l v i rus ) were screened

f o r r e s i s t a n c e t o these v i r u s e s . The r e s u l t s a r e summarized below:

1. I n i t i a l 1 y, both the v i ruses produced t w i s t i n g o f terminal

bud i n chickpea. Subsequently, CMV e i t h e r produced w i l t i n g and death

of p l a n t s o r p r o l i f e r a t i o n and bushiness of branches bearing very

s m l l green leaves and very few small pods, whereis BYMV produced

pro1 i f e r a t i o n of branches with narrow leaves , followed by production

o f d i s t o r t e d f lowers t h a t were converted i n t o very small pods having

s h r i v e l l e d and black seeds.

2 . CMV appeared t o have a wide hos t range i n f e c t i n g t h e p l a n t s

o f Aizoceae, Chenopodiaceae, Cucurbitaceae, Leguminosae and Solanaceae,

whereas BYMV could n o t i n f e c t hos t s o u t s i d e Leguminosae.

3 . The CMV had thermal i n a c t i v a t i o n p o i n t between 70' and 7S0c,

the d i l u t i o n end po in t between and and longevi ty i n v i h o

between 4 and 5 days. The corresponding f i g u r e s f o r BYMV a r e between

55' and 60°c, lo-* and and 1 and 2 days.

4. Both v i ruses could be t r ansmi t t ed by two aphid species , v i z . ,

Aphis cmccivora and Myaus persicae i n a non-pers is tent manner.

5 . The p u r i f i c a t i o n procedure developed f o r CMV yie lded 8-10

mg virus/100 g o f i n f e c t e d t i s s u e . A s a t i s f a c t o r y p u r i f i c a t i o n

procedure was a l s o developed f o r BYMV.

6. m e CMV measured about 30 nm i n diameter, whereas the length

and width of BYMV were found t o be 750 and 15 nm, respec t ive ly . Ultra-

t h i n s e c t i o n s of BYMV-infected chickpea l eaves revealed the presence

of pinwheel inc lus ions and laminate aggregates.

7. The sedimentation c o e f f i c i e n t o f p u r i f i e d CMV was determined

a s 104S, and the buoyant dens i ty a s 1.325 g/cm5.

8. The (3.IV coat p r o t e i n i s made up o f s i n g l e polypeptide wi th

a m l e c u l a r weight o f 25,000 da l tons .

The W - R N A contains four RNA spec ies .

10. An antiserum t o W produced i n a r a b b i t had a t i t r e o f

1/1024 a s determined i n agar double-dif fus ion t e s t ;

11. The p resen t chickpea s t r a i n o f CMV was found to be c lose ly

r e l a t e d t o CMV-C pool, followed by CMV-lx and M-CMV.

1 2 . Out o f 143 chickpea germplasm access ions t e s t e d f o r r e s i s -

tance t o CMV, two (ICC-1781 and -8203) remained f r e e from in fec t ion ,

one (ICC-9006) showed 10% i n f e c t i o n , whereas a l l o t h e r s showed in fec -

t i o n ranging from 33 t o 100%. For BYMV, 106 access ions were t e s t e d

and nine remained free from i n f e c t i o n , four showed 10% i n f e c t i o n ,

1 2 showed i n f e c t i o n between 10 t o 20%, whereas t h e remaining 81 showed

i n f e c t i o n ranging from 22 t o 100%.

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A P P E N D I X

I . Reaction of chickpea l i n e s t o cucumber mosaic v i r u s

S1. Number Number Percent No. ICC No. i n o c u l a t e d i n f e c t e d i n f e c t i o n

9 9 0

contd

cont d .

contd.

1

II. Reaction o f chickpea germplasm l i n e s t o bean yellow mosaic virus

S l . Number Number Percent No. ICC No. inocu la ted i n f e c t e d i n f e c t i o n

contd.

contd.

contd.

I , T. Venkata Chalam, was born on 1 August 1949 t o

Smt. Kesamma and S r i Narasaiah, i n Cwnbum, Prakasam D i s t r i c t , Andhra

Pradesh, Ind i a . I marr ied Miss S a v i t h r i o f Gadwal on 1 May 1977 and

we have one son*. I ob ta ined my B.Sc. degree in Agr i cu l tu re from

Andhra Pradesh A g r i c u l t u r a l Un ive r s i t y , Hyderabad i n 1970 and was

awarded my M.Sc. degree i n Agr i cu l tu re (P l an t Pathology) by G . B. Pant

Univers i ty o f Agr i cu l tu re and Te,:hnology, Pantnagar i n 1973.

I am on r e g u l a r s e r v i c e (Ass i s t an t P ro fe s so r ) o f Andhra

Pradesh A g r i c u l t u r a l Univers i ty . I j o ined t h e Ph. D . program (on s tudy

leave) i n 1979 and worked i n p l a n t v i ro logy under t h e guidance of

D r . Y .La Nene of ICRISAT and D r . J. Subbayya of APAU. During t h e course

o f t h i s i n v e s t i g a t i o n , I r ece ived ICRISAT fe l l owsh ip .

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IDENTIFICATION AND CHARACTERIZATION OF CUCUMBER MOSAIC AND BEAN YELLOW