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 the permission of the Author.

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,

Pa l merston North ,

New Zea l and .

God w i n B a l 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 d­i 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 p o t ex 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 en g t h 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 e n c e 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 ow i ng peopl e :

(i v)

My sup erv i 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 , Dep artmen t of Sc i enti f i c an d I ndust r i a l Research ( DS I R ) , Auc k l an d ) an d Dr D Cohen ( P l ant Phys i o l ogy D i v i s i on , Dep artmen t 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 o l ogy D i v i s i on , DS I R , Pal merston North f or ad v i c e an d ass i stan c e . I n part i c u l ar , I w i sh to than k the D i rector , Dr J P Kerr , f or perm i tt i ng me to comp l ete thi s research proj ect whi l e on sta f f an d Ni cho l a s E l l i son , f or assi st ance w i th c l on i ng i nto l am b d a vectors .

R i chard G Chr i st i e an d Stephen R Chri sti e of the Un i vers i ty of F l or i d a , Ga i n es v i l l e , USA , f or trai n i ng prov i d ed i n the d etect i on of p l an t v i ru s i n c l us i on b od i es by l i ght and e l ectron m i croscop y .

R i chard Benn ett a n d Doug l as Hopcrof t o f the E l ectron M i croscopy Un i t, B i otechnol ogy D i v i s i on , DS I R , Pa l merston North , f or embed d i ng a n d 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 d e ve l op i ng the e l ectron m i crographs .

To my w i f e Suz a n n e my d eepest apprec i at i on f or her und erstan d i ng and support d ur i ng the course of thi s proj ect .

I a m greatl y i n d ebted to my parents f or the person a l sacri f i c es they mad e i n send i ng me f ro m Mal aysi a to c ompl ete my school i ng i n Austra l i a and s upporti ng me through my und ergraduate stud i es at the Austra l i an N at i on a l Un i vers i ty .

God w i n B a l a s i 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 n t 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 r esear c h An a l ysi s of r esear c h 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 m i nary stud i es and c DNA tests Sour c e o f other v i r u s i so l ates used i n t h i s study I n d i c at or p l ants Sou r c e o f an t i ser a B i oc h 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 a n t s f or e x p er i men t a l host r an g e t ests I n oc u l a t i on E l e c t r 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 C r u d e sap Par t i a l l y p ur i f i ed and p ur i f i ed v i r us p r ep ar at i on s Par t i c l e s i z e d e t er m i n at 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 c at 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 c o n c en t r at i on of v i r uses f r o m 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 a r 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' M o l e c u l ar w e i ght d et e r m i 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 p r o t 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 r om n er i n e v i r us X RNA e x t r ac t i on f r om n er i n e p otyv i r us Agar ose g e l e l ec t r ophor es i s of nuc l e i c ac i d s Non-d e n a t u r i n g 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 n g 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 n g a g ar ose g e l e l e c t r ophor e s i s 2 . 2 . 8 . 2 ( a ) A l k a l i n e a g arose g e l s f or DNA 2 . 2 . 8 . 2Cb ) Denatur i ng g e l s f or v i r a l RNA 2 . 2 . 8 . 2 ( b ) ( i ) Forma l d ehyd e 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 v i r a l RNA 2 . 2 . 8 . 2 (b ) ( i i ) Methy l mercur i c hyd r 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 e i 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 n g p r oc e d u r e Prep a r at i on of c DNA f or m o l ec u l ar c l on i n g eDNA syn thesi s and molec u l ar c l on i n g of n er i n e v i r u s X RNA

( v i )

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 synth esis and molec ular clonin g of th e potyvirus f r om n erines In v i t ro t r an slation of n erin e vir us Ser olo g y Ouchterlony d ouble-dif f usion test I mmun o-d ot blotting

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

I n troduction Symptomatology Elec tron mic r oscopy Mech anic al tran smission 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 ntrod uction Symptomatolog y in h erbac eous indic ator s Ser olo g y Partial purific ation Disc ussion

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 nt r od uction Purific ation studies Disc u ssion

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

I nt r o duction Partic le morph ology Mec h anic al tr an smission ex periments Symptomat olog y Cytopath ology by lig ht and electron mic r osc opy Purific ation Molecular weight of c oat pr otein An alysis of n erine vir us X n ucleic acid In v i t ro tran slation 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

(v i i i )

S er o l o g 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 syn thes i s a n d m o l 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 D e t er 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 D e t e rm i n at i on of s e n s i 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 s c us 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 exp e r i me n t s Symp t omat o l ogy An a l y s i 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 n c 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 n e v i r us Y c oat p r ot e i n Ext r ac t i on a n d 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 an d m o l e c u l a r c l on i n g of n er i n e v i r u s Y RNA Estab l i shment of i d e n t i t y an d hybr i d i z at i on s p ec i f i c i t y of c DNA c l oned p r obes D e t er m i n at i on of sen s i t i v i t y 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

8 3 8 4 8 6

8 8

9 0 9 3

9 6

9 6 9 6

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 c DNA PROBES

8 . 1 In t 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 o n ed c DNA p r ob e 8 . 3 Tests f or n er i n e v i r us Y us i n g 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 G ENUS 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 t or ) , P r o c eed i ngs of t h e Inter n at i on a l Symposium an d Wor k shop o n Gene Man i p u l at i on f or P l ant Improv ement i n Devel op i ng Coun t r i es h e l d i n Kua l a Lump ur, 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 A d van cement of Breed i n g Resear c h er 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 sym p t oms on ner i n e f l ower s, 48 f l ower stems an d l eaves

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

h yb r i d i n f ec t ed w i t h 800nm p o t yvi 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 n f ec t ed w i t h a 540n m p o t ex v i r u s an d a 740n m pot yv i rus 48

l ( C ) & ( D ) Mot t l i n g, st r ea k i n g an d yel l ow i ng of Nerine bO Hdenii h yb r i d l eaves i n f e c t ed w i t h a 540n m 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 eac h i n g i n N erine bOHdenii l eaves i n f ec t ed w i t h 540n m 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 ar t i c l es, i n c r ude sap h omog enat e of n er i n e l eaf t i ssue, w i t h a moda l l en g t h of 540n m 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 n e l eaf t i ssue t ested d ur i n g sur veys usi n g t h e e l ec t r on m i c r oscope 54

4

4 ( A )

4 ( B )

Loc a l l es i ons i n t est p l an t s i n ocu l at ed w i t h v i r us p r eparat i on c on t a i n i n g t h e n er i n e i so l ate of cuc umber mosai c v i r us (CMV )

Nec rot i c l oc a l l es i o n s i n Cheno podiu. quino a 15 d ays af t er i n o c u l at i on w i t h par t 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 c on tai n i n g CMV

Loc a l etc hed f l ec k s f or m i n g c on c en t r i c r i n g s i n N ic o tiana tabac um 'Samsun� 2 3 days af t er i n o c u l at i on w i t h n er i n e c r u d e sap p r e p arat i on c o n t a i n i n g 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 es i o n s and system i c n ecros i s i n C uc u. i s sa t i vus 'S l i c emaster' 22 d ays af t er 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 t er i n ocu 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 a i 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 so l at e of cucumber mosa i c v i rus f rom N i c o t i ana t abac u. ' Wh i te Burl ey '

Fragmen t at i o n of f i l ament ous v i rus p art i c l es af t er t w o c yc l es of d i f f eren t i a l centri f ugat i on

A d i st i n c t o p a l escent v i rus b an d a f t er i sopycn i c c aes i 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 p o l 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 so l ate of n er i n e v i rus X

Lengt h d i str i b ut i on of an i so l ate of n eri ne vi rus X f ou n d 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 e aves systemi c a l l y i n f ec t ed w i t h an i so l ate of n eri ne vi rus X but show i n g n o v i rus- l i k e sym p t 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 so l 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 at e 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 so l at e e l ectrop h oresed o n a 1 2 . 5% p o l yacry l a m i 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 c oat prote i n

Agarose gel e l ectrop h oresi s of v i ra l 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 b r at i on c ur v e ob t a i ned by p l ot t i n g d i s t an c e mi g r at ed b y RNA sp ec i es i n Bet h 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 w e i g h t 8 1

Aut or ad i og r a m 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 c u l ocyte l ysat e of RNAs f r om p ur i f i ed v i 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 r u s X , p o t a t o v i r u s X� n ar c i ssus mosa i c v i r us a n d wh i t e c l over mosa i c v i r us ( i so l a t e M ) 82

I mmun o d ot -b l ot as says usi n g ant i serum pr e p ar ed t o t h e agapanthus st r a i n of n er i n e v i r u s 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 c t i on endonuc l ease map of pBR322 DNA� show i n g Ps t 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 e n d on uc l ease a n a l ysi s of c DNA c l o n e of n er i n e v i r us X RNA 86

Locat i on o f H i nd I I I r estr i c t i on endonuc l ease s i t e i n n er i n e v i r us X c DNA c l on e 8 7

Hybr i d i z at i on o f c l o n ed 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 og ous g e n o m i c v i r a l R NA 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 o f a c l o n e d c DNA (32P]- l abel l ed p r 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 ap h of d ot -b l ot h yb 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

Par t i c l e l en g t h d i st r i b ut i on of ner i n e v i r u s Y i n a f i e l 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 o n a l l y mass p r o p ag ated f or c ommer c i a l c u t -f l ower p r o d uc 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 w i 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 n g h e l i c a l struc ture when st a i ned w i 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 n f ec t ed w i t h n er i n e v i rus Y

D i s t orted f l ower st a l k of n er i ne v i 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 er i n e v i rus Y i n f ec t e d Ner i ne sarni ensis h y br i d g rown i n a g l assh ouse at a n average temperat ure of 20C

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

An epi d erma l c e l 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 n c l us i o n s ( C I )

An epi d ermal c e l l c o n t a i n i n g a c ompa c t 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 n d r i c a l i n c l us i o n s ( C I ) near t h e n uc l eus ( N ) of t h e epi dermal c e l l

E l e c tron m i crograph o f a rec t angul ar stri a t ed port i on of a c y l i n d r i c a l i n c l us i on i n d uc e d i n Neri ne sarni ens i s l eaf t i ssue i n f ected b y ner i n e v i rus Y

Ul trath i n sect i on s of n er i ne v i rus Y c y l i n dr i c a l i n c l us i ons i n duced 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 ea f c e l l s 1 08

Lon g i t ud i n a l sec t i on of c yt 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 h ondri a ( M ) i n mesophy l l c e l l s 1 08

Mas s of b un d l es ( B ) i n t h e c ytopl asm of a l eaf c e l l a d j acent to t h e c e l 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 t ud i n a l sec t i o n s o f f i l amen t ous v i rus p art i c l es ( V) 1 09

Cyt op l asmi c c y l i n d r i c a l i n c l u s i ons i n cr oss-sec t i on sh ow i n g sc r o l l s ( 5 ) an d 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 or e ( CC ) 1 1 0

Ul t r at h i n sec t i on of h e a l 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 or op l asts ( C ) an d m i t o c h oandr i a ( M ) 1 1 1

Sep arat i on of SOS-d i s soc i ated marker p r ot e i n s and n er i n e v i r us Y c y l i n d r i c al i n c l usi on p r ot e i n e l e c t 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 b r at i on c urve f or mol ec u l ar we i g h t d e t er m i nat i on o f n er i n e v i r us Y i n d u c ed c y l i n d r i c a l i n c l u s i ons

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

Cal i b r at i on c ur ve f or mo l ec u l ar we i g h t det er m i n at i on o f n er i n e v i rus Y coat p r o t e i n

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

Ag arose g e l e l e c t r o p h o r es i s of n er i n e v i r u s Y RNA u n d e r ( 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 o b t a i n e d b y p l ot t i n g d i st a n c e mi g r a t ed b y R N A spec i es i n RNA l ad d er (Bet h esda Resear c h Laborator i es > ver sus mo l ec u l ar wei g h t

Low m e l t i n g p O i nt 1 . 21. agar ose 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 h ydrox 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 t er 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 t e s 1 26

Eeo R I d i g est of p l asmi d p GEM3 to sh ow 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 u n f 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

Aut orad i ographs of PE l -c e l l u l ose c hromat ogram o f depur i n a t e d [32P]- l a b e l l ed c DNA c l o n ed 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 k b � 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 er i n e sap a n d a c a 750n m n er i n e p o t yv 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 hybr i 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 ra l c on c en trat e s of n er i n e v i rus Y a n d an un c h arac t er i z ed ca 800 n m n er i n e p ot yv i rus ob t a i n e d f rom A A Brunt hybr i d i z ed w i th a c l o n ed 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 d­i 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 ner i n e v i rus Y cDNA [ 32P]- l ab e l l ed probe 1 33

Aut orad i ograph of c l ar i f i ed v i ral c o n c e n trat e s (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 pro b e 1 36

Aut orad i ograph of c l ar i f i ed v i ral c on c e n trat e s (eVe) of n er i ne l eaf t i ssue hybri d i z ed w i t h

c l o n e d 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 er i 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 s h ow i n g an a t om i c a l and morp h o 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-b u l 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 h y b r 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 an t 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 n e Nurser i es Ltd , P a l 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 preva l en c e of v i ruses reported i n assoc i at i on w i t h Ner i ne spp .

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

V i rus i n d i c at or spec i es t ested i n mec h an i c a l transm i s s i on st ud i es

Part i c l e l en g t h ran ges 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 n d ex ed by el e c tron 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 Ner i ne c rud e 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 e l 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 ec u l ar wei g h t d et erm i n at i on o f n er i n e v i rus X c oat prot e i n

Cal i br at i on d at a f or mol ecul ar w e i g h t determ i n at i on o f 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 w e i g h t d et erm i n at i on o f n er i n e v i rus Y i n duced c y l i n d r i c a l i n c l us i ons

Cal i brat i on d at a used f or mo l ec u l ar wei g h t determ i n at i on o f n er i 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 ec troph ores i s of an RNA l ad d er <Bet h esda Researc h Lab orat 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 d en a t ur i n g c on d i t i on s 121

12

13

Summary of resu l t s 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

Ap prox i ma t e 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 a n d , 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