Purification, characterization and cDNA cloning of two filamentous viruses from Nerine : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

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PURIFICATION, CHARACTERIZATION AND eDNA CLONING OF TWO FILAMENTOUS VIRUSES FROM NERINE

A thes i s presented i n parti a l ful fi l ment of the requi rements for the degree of

Doc tor of Phi l osophy at

Massey Un i versi ty, Pal merston North ,

New Zeal and .

God w i n Bal asi ngam Jul y, 1989

( i i )

ABSTRACT

Vi r us-l i k e symptoms were evi d en t i n many of t h e c l on a l l y mass p r op ag at ed l i nes of t h e b u l b ous ornament a l Nerine , cul t i vated i n New Zea l and f or cut f l ower and bul b produc t i on . Al though f i l amentous v i r us p ar t i c l es were common i n el ectron mi c r oscop e i nvest i g at i on of Nerine t i ssue , cucumber mosai c vi rus was t h e on l y vi r us mec h an i cal l y t r an smi t t ed to herbaceous i nd i cator s .

Two o f t h e f i l amentous v i r uses f r om Nerine wh i c h wer e n ot amen ab l e t o mech an i c a l t r an smi ssi on were puri f i ed f rom f i el d­

i nf ec t ed Nerine t i ssue , c h ar ac t er i z ed and cDNA c l oned i n t h i s st ud y . An i so l ate of n er i n e vi rus X ( NeVX ) , a p ot ex vi r us , was pur i f i ed f rom Nerine fo therg illi � Major � l eaf t i ssue showi ng n o vi rus-l i ke sympt oms . An i so l ate of a h i therto unn amed and unc h ar ac t er i z ed potyv i r us was pur i f i ed f r om c l on a l l y mass p r op agated Nerine sarniensis h yb r i d l eaf t i ssue showi n g sever e yel l ow mosa i c sympt oms p r i or t o senesc en c e . Ner i ne vi rus V

( NeVY) i s t h e n ame p roposed f or t h i s potyvi rus .

T h e NeVX i sol ate was a sl i g h t l y f l ex uous f i l ament ous p ar t i c l e wi t h a normal l en g t h of 540n m . The mol ecul ar wei gh t of t h e si n g l e c oat p rotei n subun i t was 29 . 5kd and t h e s i z e o f t h e genom i c RNA was 6 . 3 k b . T h e NeVX RNA in vit ro t r ansl at i on p r of i l e r esemb l ed those of t h e potex v i ruses potato vi rus X and daphne v i rus X wi t h a maj or 180k d nonstructural p r ote i n b an d and a n umber of m i nor bands w i t hout a vi ral c oat p r ot e i n b an d .

Doub l e-stranded cDNA t o t h e 6 . 3 k b genomi c RNA was c l oned i nt o t h e P s t I s i t e of t h e p l asm i d vector pBR322 . N i c k-transl ated pBR322 DNA contai n i ng a 1 . 8 k b i nsert , represen t i n g 28 . 5% of t h e vi r a l genome , was f ound t o b e h i g h l y spec i f i c and sensi t i ve t o NeVX i sol ates i n dot-b l ot assays . The c l on ed cDNA probe d i d n ot hybr i d i z e t o seven ot her potex v i ruses , i nc l ud i ng a c a 540nm pot ex vi rus f r om Rgapanthus wh i ch , on the b asi s of ser o l og i c a l react i vi ty , was p r ev i ousl y d esc r i bed as an agap an t hus strai n of NeV X . A survey usi n g t h e c l oned c DNA i n dot-b l ot assays of Nerine spec i es i nd i c ated t h at NeVX was prevalent i n Nerine p l ants c u l t i vated i n d i f f erent p arts of t h e Nor t h I sl and of New Zea l an d .

Ner i n e vi rus V was f ound t o b e a t yp i c a l l y f l ex uous potyvi r us w i t h a n ormal l ength of 800nm , a si n g l e c oat p r ot e i n subun i t w i t h a mol ec u l ar wei gh t of 33.26kd and a g enomi c RNA of 1 0 . 0 k b . Ner i ne vi rus V-i n f ec t ed t i ssue had c haracter i st i c Typ e I potyv i rus cyl i nd r i cal i nc l us i on bod i es . S i z e f r ac t i onated vi r a l RNA w a s c l on ed i nto t h e l ambd a vec tor s g t l 0 and L47AB . Two cDNA c l ones of si z e 1 . 54kb and O . 56k b der i ved f rom l ambda g t l 0 and

(iii )

a 9 . 8 k b c DNA c l one der i ved f r om l ambda L47AB wer e sub c l oned i nto the p l asm i d vector pGEM3 . I n d ot-b l ot assays cDNA f rom al l c l ones hybr i d i z ed to t h e homol ogous v i rus but n ot t o n er i ne yel l ow st rip e vi rus, anot h er mor e c ommon p otyvi r us i n ner i nes . The 1 . 54 k b c DNA c l oned probe was f ound to b e hig h l y specif ic and sen si t i ve and did not hybr i d i z e i n dot-b l ot assays to f our other potyvi ruses . A sur vey usi ng t h e 1 . 54 k b c l oned c DNA p r ob e i n dot-b l ot assays i nd i c at ed t h at NeVV was n ot c ommon i n Herine sp ec i es c u l t i vated i n d i f f er e n t p arts of t h e Nor t h I sl and of New Z e a l and .

T h e use of r ec omb i nant tec h n o l ogy made i t possi b l e t o deve l op hig h l y sen s i t i ve and sp ec i f i c diagn ost i c tool s f or two f i lament ous v i r uses f r om Herine. These c l oned c DNA p r obes were f ound t o b e wel l suited f or c on d ucting f i el d sur veys to study t h e p r eva l ence of t h e vir us . The p r obes coul d be used i n subsequent studies i n scr ee n i ng f or v i r a l resi stance and i n v i r us e l i mi n at i on stud i es . Fur t h er , t h e c l oned c DNAs may i n t h e f ut ur e p r ove usef ul i n t h e c h ar acter i z at i on o f t h e g enomes of NeVX and NeVV .

I

^I

ACKNOWLEDGEMENTS

I w i sh to ex press my grat i tud e to the f o l l owi ng peopl e :

(i v)

My sup ervi sors Prof essor K S M i l n e ( Department of P l ant Hea l th , Massey Un i vers i ty , P a l merston North ) , Dr R L S Forster (Mt A l bert Research Centre , Department of Sc i enti f i c an d I ndustri a l Research ( DS I R ) , Auc k l and ) an d Dr D Cohen ( P l ant Phys i o l ogy D i v i s i on , Department of Sc i enti f i c and I n dustri a l Research , Pa l merston North ) f or gui d an ce , encouragement an d trai n i ng prov i d ed d uri n g the course of th i s proj ect .

To a l l the staf f of P l ant Phys i ol ogy Di v i s i on , DS I R , Pal merston North for ad v i c e an d ass i stanc e . I n part i cul ar , I wi sh to than k the D i rector , Dr J P Kerr , f or permi tt i ng me to comp l ete thi s research proj ect whi l e on staf f an d Ni cho l as E l l i son , f or assi st ance w i th c l on i ng i nto l ambda vectors .

R i chard G Chr i st i e and Stephen R Chri sti e of the Un i vers i ty of F l or i d a , Gai n esvi l l e , USA , f or trai n i ng provi ded i n the d etect i on of p l an t v i rus i nc l us i on bod i es by l i ght and e l ectron m i croscop y .

R i chard Benn ett a n d Dougl as Hopcrof t o f the E l ectron Mi croscopy Un i t, B i otechnol ogy Di v i s i on , DS I R , Pa l merston North , f or embedd i ng and sect i on i ng o f the p l ant mater i a l used f or tran sm i ss i on e l ectron m i c roscope stud i es and f or deve l opi ng the e l ectron m i crographs .

To my wi f e Suz anne my d eepest apprec i at i on f or her understan d i ng and support d ur i ng the course of thi s proj ect .

I a m greatl y i ndebted to my parents f or the person a l sacri f i ces they made i n send i ng me f rom Mal aysi a to c ompl ete my school i ng i n Austra l i a and supporti ng me through my undergraduate stud i es at the Austral i an Nat i on a l Un i versi ty .

God w i n B a l asi n gam

B Sc , Di p Ed ( Austral i a ) , D i p PI Sc , M Sc ( New Z e a l and )

CHAPTER 1 :

1 . 1 1 . 2 1 . 2. 1 1 .2 . 2 1 .3

CHAPTER 2:

2 . 1 2 . 1 . 1 2 . 1 . 2

2 . 1 . 3 2 . 1 . 4 2 . 1 .5

TABLE OF CONTENTS

T I TLE PAGE ABSTRACT

ACKNOWLEDGEMENTS TABLE OF CONTENTS L I ST OF F I GURES L I ST OF TABLES

V I RUSES I N THE GENUS NERINE

I nt r oduc t i on

V i r uses i n Neri ne - A l i ter ature r ev i ew C h r on o l og i cal pr esen t at i on of resear c h An a l ysi s of r esear ch

Object i ves of t h i s r esearch projec t

MATER I ALS AND METHODS MATER I ALS

Sou r c e of ner i n e v i r us i so l ates f or pr e l i mi nary stud i es and c DNA tests

Sour c e of other v i r u s i so l ates used i n t h i s study

I nd i c at or p l ants Source of an t i ser a B i och em i c a l s

( v )

Pace

( i ) ( i i ) ( i v ) ( v ) ( x ) ( x vi i )

1

1 2 2 6 1 0

1 1 1 1

1 1

1 1 1 3 1 3 1 3

2 . 2

2 . 2 . 1 2 . 2 . 2 2 . 2 . 3 2 . 2 . 4 2 . 2 . 4 . 1 2 . 2 . 4 . 1 ( a ) 2 . 2 . 4 . 1 ( b )

2 . 2 . 4 . 2 2. 2 . 4 . 3 2 . 2 . 4 . 4 2. 2 . 5 2 . 2 . 5 . 1 2. 2 . 5 . 2 2 . 2 . 5 . 3

2 . 2 . 5 . 3Ca ) 2 . 2 . 5 . 3 ( b ) 2 . 2 . 6

2. 2 . 6 . 1 2 . 2 . 6 . 2 2 . 2 . 7 2. 2 . 7 . 1 2 . 2 . 7 . 2 2 . 2 . 8 2.2 . 8 . 1 2 . 2 . 8 . 1 ( a ) 2 . 2 . 8 . 1 ( b )

METHODS

Ner i n es

P l ants f or e x p er i men t a l host r an g e t ests I noc u l at i on

E l ectr on m i c r osc opy

E l e c t r on m i c r oscope g r i d p r ep er at i on Crude sap

Par t i a l l y p ur i f i ed and p ur i f i ed vi r us p r ep ar at i on s

Par t i c l e s i z e deter m i nat i on

U l t r at h i n sect i on i n g and s t a i n i n g p r oced u r e L i ght m i c r oscopy

V i r u s p ur i f i cat i on

Pur i f i c at i on of n er i n e v i r us X Pur i f i c at i on of n er i n e v i r us Y C l ar i f i c at i on a n d concen t r at i on of v i r uses f r om smal l samp l es f or d ot-b l o t assays

P r o c e d u r e f or the c l ar i f i c at i on and

c on c en t r at i on of v i r uses f r om 'mi n i -samp l es' P r oc ed u r e f or the c l ar i f i c at i on and

c on c en t r at i on of v i r uses f r om 'm i c r o-samp l es' Mol e c u l ar w e i ght d etermi n at i on of v i r a l coat a n d i n c l us i on body p r ot e i n s

P r ot e i n d i ssoc i at i on

A n a l ys i s of prot e i n s on SDS-p o l yac r y l ami d e g e l s RNA e x t r ac t i on

RNA e x t r ac t i on f rom n er i n e v i r us X RNA e x t r ac t i on f r om ner i n e p otyvi r us

Agar ose g e l e l ec t r ophor es i s of nuc l ei c ac i d s Non-d e n a t u r i ng g e l s

Agar ose g e l e l ectr ophor es i s of DNA

N on - d en a t u r i ng g e l f or v i r a l RNA c on c ent r at i on d et er m i n at i on

2 . 2 . 8 . 2 Denatur i ng a g ar ose g e l e l ectr ophor e s i s 2 . 2 . 8 . 2 ( a ) A l k a l i ne a g arose g e l s f or DNA

2 . 2 . 8 . 2Cb ) Denatur i ng g e l s f or vi r a l RNA

2 . 2 . 8 . 2 ( b ) ( i ) Forma l d ehyde g e l s f or m o l ecul ar w e i ght d e t e r m i n at i on of vi r a l RNA

2 . 2 . 8 . 2 (b ) ( i i ) Methy l mercur i c hydr ox i d e g e l f or s i z e f r ac t i o n at i on of v i r a l RNAs

2. 2 . 9

2 . 2 . 9 . 1 2 . 2 . 9 . 2 2 . 2 . 9 . 3 2 . 2 . 1 0 2 . 2 . 1 0 . 1

T r a n s f e r o f n uc l ei c ac i d s f r om ag ar ose g e l s to s o l i d suppor t s

Souther n b l ots Northe r n b l ots

Dot-bl o t t i ng p r ocedure

Prep a r at i on of c DNA f or m o l ecu l ar c l on i ng

eDNA syn thesi s and molecu l ar c l on i n g of n er i n e v i r u s X RNA

( vi )

1 5 1 5 1 5 1 5 1 6 1 6 1 6 1 7 1 7 1 7 1 8 1 9 2 1 22

23 23 23

24 24 24 26 26 27 27 27 27

28 28 28 29 29 30

3 1 3 1 32 32 33 33

2.2. 10 . 2

2.2. 1 1 2 . 2 . 12 2.2 . 12 . 1 2 . 2. 12 . 2

CHAPTER 3 :

3. 1 3.2 3.3 3 . 4 3.5

CHAPTER 4 :

4. 1 4.2 4.3 4.4 4.5

CHAPTER 5 :

5. 1 5 . 2 5.3

CHAPTER 6 :

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

c DNA synthesis and molecular clonin g of th e potyvirus f r om n erines

In v i t ro t r an slation of n erin e virus Serology

Ouchterlony d ouble-dif f usion test I mmuno-dot blotting

SYMPTOMATOLOGY , ELECTRON M I CROSCOPY MECHAN I CAL TRANSM I SS I ON STUD I ES

I ntroduction Symptomatology

Electron mic roscopy Mechanical transmission Disc ussion

X RNA

AND

I DENT I F I CAT I ON OF CUCUMBER MOSA I C V I RUS I N HERIHE I N NEW Z EALAND

I ntroduction

Symptomatology in h erbac eous indicator s Ser ology

Partial purific ation Discussion

PREL I M I NARY PUR I F I CAT I ON STUD I ES OF F I LAMENTOUS V I RUSES F ROM SYSTEM I CALLY I NFECTED HERIHE

I ntrod uction

Purific ation studies Disc u ssion

PUR I F I CAT I ON , CHARACTER I ZAT I ON AND c DNA CLON I NG OF NER I NE V I RUS X

I ntroduction

Particle morph ology

Mech anic al tran smission ex periments Symptomat ology

Cytopath ology by light and electron mic rosc opy Purific ation

Molecular weight of c oat protein

Analysis of n erine virus X n ucleic acid In v i t ro translation pr oduc t s

( vi i)

37 46 46 46 46

47

47 47 5 1 55 55

59

59 59 62 62 62

64

64 64 66

70

70 72 72 74 74 76 77 80 82

6 . 1 0

6 . 1 1 6 . 1 2 6 . 1 3 6 . 1 4 6 . 1 5

CHAPTER 7:

7 . 1 7 . 2 7 . 3 7 . 4 7 . 5 7 . 5 . 1 7 . 5 . 2 7 . 5 . 3

7 . 6 7 . 7 7.8 7 . 9 7 . 1 0 7 . 1 1

7 . 1 2

(vi i i )

Ser o l og i c a l r e l at i on sh i p t o the �gapan t h us s t r a i n of n er i n e v i r us X

c DNA synthes i s a n d mol ecu l ar c l on i n g o f v i r a l RNA Restr i c t i on an a l ys i s of the c DNA c l on ed i n sert Deter m i n at i on o f hybr i d i z at i on spec i f i c i t y of

c DN A c l oned p r obe

Determ i n at i on of sensi t i v i t y o f the c DN A c l on ed p r obe f or d et ec t i on o f n er i n e v i r us X i n sap D i scus s i on

PUR I F I CAT I ON , CHARACTER I Z AT I ON AND c DN A CLON I NG OF NER I NE V I RUS

Y

In t r od uc t i on

Par t i c l e morpho l ogy

Mechan i c a l t r a n sm i ss i on expe r i ments Sympt omat o l ogy

An a l ysi s of i n c l us i on bod i es L i g ht m i c r osc o p y

E l ec t r on m i c r osc o p y

SDS-Page g e l e l e c t r opho r es i s o f c y l i n d r i c a l i nc l u s i ons

Pur i f i c at i on of n er i n e v i r us Y

Mol e c u l ar w e i ght of n er i ne v i r us Y c oat p r ot e i n Ext r ac t i on and an a l ys i s of n er i n e v i r us Y RNA S i z e f r act i on at i on , c DN A synthes i s and m o l e c u l ar c l on i n g of n er i ne v i r u s Y RNA

Estab l i shment of i de n t i ty and hybr i d i z at i on s p ec i f i c i ty of c DNA c l oned p r obes

Deter m i n at i on of sen s i t i vi ty of the 1 . 5 k b l ambd a g t l 0 d er i ved c DNA c l oned p r obe f or d et ec t i on o f n er i n e v i r u s Y i n sap

D i scussi on

83 84 86 88

9 0 9 3

96

96 96 1 00 1 00 1 03 1 03 1 06

1 1 2 1 1 5 1 1 7 1 1 9 1 22 1 27

1 32 1 34

CHAPTER 8 : TESTS FOR NERINE VIRUS X AND NERINE VIRUS Y USING cDNA PROBES

8 . 1 Int r od uc t i on

8 . 2 Test s f or n er i n e v i r us X usi ng c l oned c DNA p r ob e 8 . 3 Tests f or n er i n e v i r us Y us i ng t h e c l oned c DNA

probe

8 . 4 D i sc ussi on

CHAPTER 9 : GENERAL DISCUSSION AND CONCLUSIONS

APPENDICES

APPENDI X I :

APPENDIX II :

THE GENUS NERINE

SENSITIVE AND SPECIFIC DETECTION OF TWO FILAMENTOUS VIRUSES FROM NERINE USING CLONED c DN A PROBES

[ Paper pub l i shed i n �c t a Hortic ult urae� Numb er 234� 1 988�

pp 2 67-274] .

APPENDIX III : STRATEGIES AND TECHNOLOGIES TOWARDS BULB CROP IMPROVEMENT

[ P ap er pub l i shed i n Pl an t Breedi ng and Genet i c Eng i neeri ng�

Z a k r i � A . H . ( Ed i tor ) , Proc eed i ngs of t h e Inter n at i onal Symposium an d Wor kshop on Gene Man i pu l at i on f or P l ant Improvement i n Devel op i ng Coun t r i es h e l d i n Kua l a Lumpur, Ma l ays i a, 30 Novemb er - 3 December, 1 987, b y Th e Soc i et y f or t h e Advan cement of Breed i n g Researcher s i n Asi a and Ocean i a .

REFERENCES

( i x )

136

136 136

138 1 40

1 4 1

1 43

143

160

1 68

176

( x )

LIST OF FIGURES

Fi gur e

1 Some v i rus- l i k e symptoms on ner i n e f l ower s, 48 f l ower stems and l eaves

l ( A ) Di stor t i on of f l ower stem of a Nerine sarniensis

h yb r i d i n f ec ted w i t h 800nm potyvi rus part i c l es 48

l ( B ) Co l our-br ea k i n p eta l s of a Nerine sarniensis h yb r i d i nf ec ted w i t h a 540n m potex v i rus an d a

740n m potyv i rus 48

l ( C ) & ( D ) Mot t l i n g, str ea k i n g an d yel l ow i ng of Nerine bO Hdenii hyb r i d l eaves i nf ected w i t h a 540nm

p o t ex v i rus and a 740n m p ot yv i rus 49

l ( E ) Mar g i na l c h l or os i s and b l each i ng i n N erine bOHdenii l eaves i n f ec t ed w i t h 540nm p ot ex v i r us

part i c l es 50

2 Pot ex v i r us-l i k e p art i c l es, i n c rude sap h omogenat e of n er i ne l eaf t i ssue, w i t h a

moda l l en g t h of 540nm 53

3 Len g t h d i st r i but i on of f i l amen t ous v i r us par t i c l es f ound i n c r ude sap h omog ena t es of n er i ne l eaf t i ssue tested d ur i ng sur veys

usi n g t h e e l ec t r on mi c r oscope 54

4

4 ( A )

4 ( B )

Loc a l l esi ons i n t est p l ants i nocu l at ed w i t h v i r us p r eparat i on contai n i n g t h e

n er i ne i so l ate of cucumber mosai c v i r us (CMV )

Nec rot i c l oc a l l esi ons i n Cheno podiu. quino a 15 d ays af t er i nocul at i on w i th part i a l l y p ur i f i ed v i rus p r ep arat i on f r om

Nerine sarniensis l eaf t i ssue con tai n i ng CMV

Loc a l etc hed f l ec k s f or m i n g c oncentr i c r i ng s i n N ic o tiana tabac um 'Samsun� 2 3 days af t er i n ocu l at i on w i t h n er i n e crude sap p r e parat i on contai n i ng CMV

60

60

60

4 ( C )

4 ( D )

5 6

7

8

9

1 0

1 1

1 2 ( A )

1 2 ( B )

1 3

1 4

1 5

1 6

Loc a l l esi ons and system i c n ecrosi s i n

Cuc u. i s sa t i vus 'S l i c emaster' 22 d ays af ter i n oc u l at i on w i t h p art i a l l y puri f i ed n er i ne vi rus prep arat i on c o n t a i n i ng CMV

Fawn ' t arget -spot s ' i n N i c o t i ana g l u t i no sa 1 0 d ays af ter i nocu l at i on w i t h p art i a l l y

puri f i ed ner i ne v i rus preparat i on c on t ai n i ng CMV Ouc h t er l ony d oub l e-d i f f usi on t est i n agar

Part i a l l y puri f i ed prep arat i on of a Neri ne sarn i ensi s i sol at e of cucumber mosa i c v i rus f rom N i co t i ana t abac u. ' Wh i te Burl ey '

Fragment at i on of f i l ament ous v i rus p art i c l es af t er two c yc l es of d i f f erent i a l centri f ugat i on

A d i st i nct o p a l escent v i rus b an d a f t er i sopycn i c caesi um c h l or i d e c en tri f ugat i on

En d -t o-end aggregat i on of v i rus part i c l es f o l l ow i ng treatment w i t h pol yet h y l ene g l yc o l

C l ust er o f N eri ne fo t herg i l l i ' Maj or' b u l bs i n f ec t ed w i t h an i sol ate of n er i n e v i rus X

Lengt h d i str i but i on of an i so l ate of n eri ne vi rus X f ound i n Neri ne f o t herg i l l i 'Maj or'

Cl ose-up of Ner i ne fo t herg i l l i ' Maj or' l eaves systemi c a l l y i n f ec t ed w i th an i sol ate of n eri ne vi rus X but show i n g no v i rus- l i k e sympt oms

Fl o wers of Neri ne fo t herg i Il i ' Maj or ' i n f ec t ed w i t h an i sol ate of neri n e v i rus X show i ng n o

v i rus-l i k e symp t oms

Part i c l es of neri n e v i rus X i so l ate p ur i f i ed fro m Neri ne fo t herg i l l i ' Ma j or '

Sep arat i on o f SDS-d i ssoc i ated mark er protei n s and v i ral coat prot e i n o f n er i n e v i rus X i sol at e e l ectrop h oresed on a 1 2 . 5% p o l yacry l ami d e ge l

Ca l i brat i on c urve f or mol ecul ar wei ght

determi n at i on of n er i ne v i rus X coat prote i n

Agarose gel e l ectroph oresi s of v i ral R NAs under non-d enaturi ng c on d i t i ons

( x i )

6 1

6 1 63

63

68

68

69

7 1

73

75

75

76

77

79

80

17

1 8

1 9

20

2 1

22

23

24

25

26

27

28

Cal i br at i on c urve ob t a i ned by p l ot t i n g d i stan c e mi g r at ed b y RNA sp ec i es i n Beth esda Resear c h

( x i i )

Lab o r at or i es RNA l ad d er ver sus mo l ec u l ar we i g h t 8 1

Aut or ad i og ram of t r an s l at i on p r oduc t s made i n r ab b i t r et i cu l ocyte l ysat e of RNAs f r om p ur i f i ed vi r us par t i c l es of n er i n e v i rus X, d ap h n e v i rus X , p o t a t o v i rus X� n ar c i ssus mosa i c v i r us and wh i t e

c l over mosa i c v i r us ( i so l ate M ) 82

I mmuno d ot -b l ot assays usi n g ant i serum pr epar ed

t o t h e agapanthus st r a i n of n er i n e v i rus X ( NeV X-A ) ag a i n st: NeV X-A� h ea l t h y n er i n e sap and NeV X f r om

Ner i ne fo t hergi lli ' Major ' 83

Rest r i ct i on endonuc l ease map of pBR322 DNA�

show i n g Pst I c l on i n g s i t e 84

Agar ose g e l e l ec t r op h or es i s f or s i z e

d e t e r m i n at i on o f c DNA c l on ed i n ser t s 85

Rest r i c t i on endon uc l ease anal ysi s of c DNA

c l o n e of n er i ne v i r us X RNA 86

Locat i on of H i nd I I I r estr i ct i on endonuc l ease

s i t e i n n er i ne v i r us X c DNA c l on e 8 7

Hybr i d i z at i on of c l oned c DNA (32P ] - l ab e l l ed n i c k-t r an s l ated p r o b e w i t h homol ogous genomi c

v i r a l RNA 88

Aut or ad i og r ap h of a d ot -b l ot h yb r i d i z at i on of a c l oned c DNA (32P]- l abel l ed pr obe w i t h

c l a r i f i ed v i r a l c on c en t r ates o f seven ot h er p o t ex v i r uses

Aut or ad i og r aph of d ot -b l ot hyb r i d i z at i on of

p ur i f i ed n er i n e v i r us X at var i ous c o n c en t r at i ons

Aut orad i og r ap h of d o t -b l ot hybr i d i z at i on of sap ex t r ac t ed f r om f i e l d- i nf ect ed Ner i ne fo t hergi lli 'Major' l eaf t i ssue

Part i c l e l en g t h d i st r i but i on of ner i n e v i rus Y i n a f i el d- i n f ec t ed Ner i ne sarn i ens i s h yb r i d c u l t i var c l onal l y mass propag ated f or c ommer c i a l c u t -f l ower produc t i on

89

9 1

92

97

29

30

3 1

32

33

34

35

35 ( A )

35 ( 8 )

35 ( C )

36

37 ( A-E )

37 ( A )

37 ( 8 )

Puri f i ed v i rus preparat i on of neri n e v i rus Y i so l ate f rom Ner i ne sarni ensis hybri d

Storage-degraded n er i n e v i rus Y part i c l e s h ow i n g dar k l y s t a i n ed c ore o r ax i a l c anal when s t a i ned wi t h 2% PTA , pH7

St orage-d egraded n er i n e v i rus Y part i c l es s h ow i ng hel i c a l struc ture when st ai ned wi th 2% PTA , pH7

Severe yel l ow mosa i c symptoms i n senesc i n g l eaves of a c ommec i a l l y c u l t i vated Nerine sarniens i s hyb r i d c u l t i var system i c a l l y i nf ect ed w i t h ner i n e v i rus Y

Di s t orted f l ower st a l k of n er i ne vi rus Y i n f ec t ed Ner i ne sarniensis h y br i d

L i g h t mosa i c sympt oms i n young l eaves of n eri n e v i rus Y i n f ected Ner i ne sarni ensis hybr i d g rown i n a g l asshouse at an average temperat ure of 20C

Li g h t m i crograp h s of n eri n e v i rus Y i nc l us i on bod i es i n epi derma l c e l l s

An epi d ermal cel l c o n t a i n i n g a l oose mass of c y l i n d r i c a l i nc l usi o n s ( C I )

An epi d ermal cel l c o n t a i n i ng a c ompact mass of c y l i n d r i c a l i n c l us i ons ( C I ) and n uc l eus ( N )

A c ompac t mass of c y l i ndri c a l i n c l us i ons ( C I ) near t h e n uc l eus ( N ) of t h e epi dermal c e l l

El ec tron m i crograph o f a rec t angul ar stri ated port i on of a c y l i n d r i c a l i n c l usi on i nd uced i n Neri ne sarni ensi s l eaf t i ssue i nf ected b y ner i n e v i rus Y

Ul trath i n sect i on s of n eri ne vi rus Y c y l i n dr i c a l i nc l us i ons i nduced i n Neri ne sarniens i s h ybr i d

( x i i i )

98

99

99

1 0 1

1 0 1

1 02

1 04

1 04

1 05

1 05

1 07

l eaf c e l l s 1 08

Lon g i t ud i na l sec t i on of cyt op l asmi c c y l i n d r i c a l i n c l us i ons o r b un d l es ( B ) b et ween c h l orop l asts ( C ) an d m i t oc hondri a ( M ) i n mesophy l l c e l l s 1 08 Mass of b un d l es ( B ) i n the c ytopl asm of a l eaf

c e l l a d j acent to t h e c el l wal l ( CW ) 1 09

37 ( C )

37 ( 0 )

37 ( E )

38

39

40

4 1

42

43

44

45

46

47 48 49

( x i v )

Bun d l es ( B ) assoc i ated w i t h l on g i tud i na l

sec t i ons of f i l amen t ous v i rus p art i c l es ( V) 1 09

Cytop l asmi c c y l i n d r i c a l i nc l usi ons i n cr oss-sec t i on sh ow i n g sc r o l l s ( 5 ) and

p i n wh ee l s ( P ) 1 1 0

P i n w h ee l s sh owi n g cur ved l amel l a ( L )

rad i at i n g out of t h e c en t r a l c ore ( CC ) 1 1 0

Ul t r at h i n sec t i on of heal t h y Ner i ne bo wdeni i seed l i n g l eaf t i ssue show i n g c h l orop l asts ( C )

an d m i toch oandr i a ( M ) 1 1 1

Sep arat i on of SOS-d i ssoc i ated marker p r ot e i n s and ner i ne v i rus Y c y l i n d r i c al i n c l usi on p r ot e i n

el ect r op h or esed on a 1 2 . 5% p o l yacryl ami d e g e l 1 1 2

Cal i br at i on c urve f or mol ec ul ar we i g h t deter m i nat i on o f n er i n e v i rus Y i n d u c ed cyl i nd r i c a l i nc l usi ons

El ect r on m i c r og r ap h of c y l i n d r i cal i nc l us i on s assoc i ated w i th ner i n e v i r us Y par t i c l es

Cal i br at i on c ur ve f or mol ec u l ar we i g h t

det er m i n at i on o f ner i n e v i rus Y coat protei n

Sep ar at i on of 50S-d i ssoc i at ed marker p r ot e i n s an d n er i ne v i rus Y c oat p r ote i n

Ag arose g e l el ectrophores i s of ner i n e v i rus Y RNA under ( f ormal dehyd e / f ormam i d e ) d enatur i n g cond i t i on s

Cal i b r at i on c urve obtai ned by p l ot t i n g d i st ance mi g r a t ed b y RNA spec i es i n RNA l ad der (Bet h esda Researc h Laborator i es > ver sus mol ec u l ar wei g h t

Low mel t i ng p O i nt 1 . 21. agarose g e l

el e c t r o p h oresi s o f n er i n e v i r us Y RNA a n d RNA mar ker s d enatured b y met h y l mer c ur i c hydrox i d e Map of b a c t e r i ophag e l amb d a g t l 0

feo R I d i g ests of p GEM3 t o show i n ser t s i z e

Map o f 2 . 752kb p GEM3 vec t or s h ow i n g feo R I c l on i n g s i t e

1 1 4

1 1 6

1 1 8

1 1 9

1 20

1 2 1

1 22 1 23 1 24

1 25

50

5 1

52

53

54

55

56

57

58

59

60 ( A ) 60 ( B )

6 1

Mao o f p art of t h e 40 . 4 k b b ac ter i op h ag e l amb d a L47AB s h ow i n g l oc at i on of Eeo R I , Bam H I ,

( x v )

and H i nd I I I restri c t i on s i tes 1 26

Eeo R I d i g est of p l asmi d p GEM3 to show 9 . 8 k b

i n sert sub c l oned i n t o l amb d a L47AB 1 26

Autorad i ograph of unf ragme n t ed and d epur i n a t ed

[32P]- l ab e l l ed n i c k -tran s l ated c DNA c l oned probes 1 28

Autorad i ographs of PE l -c e l l u l ose c hromat ogram of

depur i nated [32P]-l abel l ed c DNA c l oned probes 1 29

Aut orad i ographs of d ot -b l ot h ybri d i z at i on of the 0 . 56kb, 1 .54 kb� and 9 . 8 k b [32P]- l a b e l l ed n i c k-tran s l ated c l on ed c DNA probe w i t h

ner i n e v i rus X, n er i n e v i rus Y , hea l t h y n eri n e

sap a n d a c a 750n m n er i n e potyv i rus 1 30

Autorad i ograph of n er i n e v i rus Y and f our o t h er potyv i ruses hybri d i z ed w i t h c l oned c DNA·

[ 32P]- l ab e l l ed probe 1 3 1

Aut orad i ograph of c l ar i f i ed v i ral c on cen trat es of n er i n e v i rus Y and an un c h arac ter i z ed ca 800nm n er i n e p ot yv i rus ob t a i ned f rom A A Brunt

hybri d i z ed w i th a c l oned c DNA [ 32P]- l ab e l l ed

probe 1 3 1

Aut orad i ograph of sap ex tract ed from f i e l d­

i n f ec t ed Neri ne sarn i ensi s hybr i d l eaf t i ssue hybri d i z ed w i th a c l on ed 1 . 54 k b neri n e v i rus Y

cDNA [ 32P]- l abe l l ed probe 1 33

Aut orad i ograph of c l ar i f i ed v i ral concen trat es (eVe) of n er i ne l eaf t i ssue hybrid i z ed w i t h

c l oned n er i n e v i rus X c DNA [32P]-l ab e l l ed probe 1 36

Aut orad i ograph of c l ar i f i ed v i ral c oncen trat es (eVe) of n er i ne l eaf t i ssue hybri d i z ed w i t h c l oned n er i n e v i rus Y c DNA [32P]-l ab e l l ed

probe 1 38

Lon g i t ud i n a l sec t i on of n eri n e bul b ( Ph otograph ) 1 46

Lon g i t ud i n al sec t i on of n er i n e bul b show i n g

ana t om i c a l and morp ho l og i c a l f eatures ( D i agram ) 1 47 Typ i c a l Neri ne sarn i ensi s hybri d p l an t i n f l o wer 1 49

62

63 64

65

66

Nerine sarniensis hybr i d 'mo t h er-bul b' w i t h b u l b l ets

Twi n-sc a l es of Nerine sarniensis hyb r i d b u l b

Nerine sarniensis hybr i d i n c u l t ure� der i ved f r om f l or a l stem e x p l ant s

Ran g e o f f l ower c o l our s o f mod ern Nerine sarniensis hybr i d s

Ner i ne Nurser i es Ltd , Pal merston Nor t h

( x vi )

1 49 1 50

1 50

1 5 1 1 58

Tab l e

1

2

3

4

L I ST OF TABLES

The re l at i ve preval en c e of v i ruses reported i n assoc i at i on w i th Ner i ne spp .

Source of pot ex v i rus and p ot y v i rus i sol ates t ested ag a i n st c l oned c DNA prob es

V i rus i nd i c at or spec i es tested i n mec h an i c a l transm i ssi on st ud i es

Part i c l e l en g t h ranges f or f i l ament ous v i ruses f rom t hree N e r i ne spec i es and h y b r i d s i nd ex ed by el ectron m i crosc opy

5 Resul t s of mec h an i c a l tran smi ssi on stud i es w i t h Neri ne c rude sap or part i a l l y pur i f i ed v i rus prepara t i on

6

7

8

9

10

Puri f i c at i on s t ud i es on potyvi rus-l i k e part i c l es f rom f i el d - i n f ec ted Ner i ne sarn i ens i s l eaf t i ssue

Cal i brat i on d a t a used f or mol ecu l ar wei g h t d eterm i n at i on o f n er i n e v i rus X c oat prot e i n

Cal i brat i on d at a f or mol ecul ar w e i g h t determ i n at i on of n er i n e v i rus X RNA

Cal i brat i on d a t a used f or mo l ec u l ar wei g h t determ i n at i on of n eri n e v i rus Y i nduced cyl i nd r i c a l i n c l us i ons

Cal i brat i on d at a used f or mol ec u l ar wei g h t determ i n at i on o f n eri n e v i rus Y c oat prot e i n

11 Cal i brat i on d a t a ob t a i ned b y g e l e l ectrophoresi s of an RNA l ad d er <Beth esda Research Laborat or i es >

( x v i i )

9

12

14

52

56

65

78

81

113

117

on a 1 1. ag arose g e l under den a t uri n g c on d i t i on s 121 12

13

Summary of resu l ts of t ests usi ng t h e c DNA c l oned n er i n e v i rus X prob e

Summary of resu l t s of t ests usi ng cDNA c l oned neri n e v i rus Y prob e

137

139

1 4

1 5

Approx i mate areas of n eri n es i n c u l t i vat i on i n several c ountri es

Maj or n er i n e growers i n New Z e a l and , t h e i r l oc at i on, s i z e of operat i on and spec i es grown

( x v i i i )

1 55

1 59