Characterisation of three isometric viruses infecting daphne : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

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.

A thesis presented in partinl f'ulfilrnent of the req_uirements for -;;he degree o:f

ltaster of Science in 11.icrobioloG'{ at 1fo.ssey Uni versi "bJ

by

Bret Al ton llax Uorris-Krsinich

llarch

1976

ERRATA

p.16 Figure 7 : 'recrotic' should read necrotic

p. 23

&

26 'innoculation' mis-spelt should read inoculatio~

p. 27, 62 ^& 78 The names of families should begin with capital

po 29

P• 31

P• 32

P•

34

P•

44

P• 53

p. 62

P• 75

letters e.g. Solanaceae

mistake associated with the for~ula 'w rev/min 39,000' should read:

w ^{2 '11} rev/min rev/min 39,000 60

Table 2. 'HaOH' should read NaOH

line 15 anl 17 'distilled water' should read borate

line 3 'Lot et al., ' should read Lot et al., 1972;

Figure 13 in the heading the numbers ¹45,235, 16'5' should read 4s, 23s, 16s

Sentence begining 'nucleotide base ratios - -

shoul~ not be a new paragraph.

heading 2.6 'PHYSICAL PROTERTrnS' should read PHYSICAL PROPERTI~S - - -

'additional tests' should read additional test

AOOn V/LEDGE1lENTS

I run sinc;erely gr<!.teful to Dr. K.s. llilne for bis advice and encot<_·<i.c;ement throuchout the course of this study and. in the preparation of this·thesis.

I would also like to thank:

'l'he staff of the electron microscope laboratory, D.S.I.R.

for the preparation of sectioned material, grids and electron micro- graphs.

Dr. C.H. 1.:oore and Dr. J. ,'/. 'l\:eedie (Biochemistry Departnent, ::.iasscy Uni vorsi ty) for as::iistance and instruction in the following

techniques: analytical ul tracentrifu~u tion and polyacryl amide gel electrophoresis.

L'.r. H.F. Neilson for technical assistance and valuable discussion.

:.:r. J.R. Clouston (Photographic Unit, :.:assey University) for photOL,Taph:;r.

The s taf'f of the S::w.11 Animal .:. reduction Unit, l:assey University for assistance with antiseru productior:.

Duncan and Davie:..; Lini ted, Uu1·scrymen, i:cw Plymouth for daphne plants.

l.!rs. J. Llorric and K. I.~orris-Krsinich for typirl£'.

Special th~nlcs to Kathleen.

PREFACE

'l'he poor vigour of many cul ti vars bel on;:;:i.nc to the gerru.s Danhne (Thymelaeaceae) has been attributed to viral infection

iii

( Ch;:_ ... i<. .. ~:lain, 1954) and several viruses including alfalfa mosaic virus and cucw..Ler mosaic -~irus have been isolated (Chamberlain, 1954;

Liilbrath t°.:. Young, 1956; and Schmelzer, 1968).

Concern about daphne 'virus' problems expressed by nurserymen, has 1 ed to several progr2r;mies aimed at improving stock plants. He cent survey work on daphne viruses (Forster & Eilne, 1975; Sutton & Taylor, 19711; and Sweet & Campbell, 1973) has therefore been prompted by this req_uireu;cnt for high heal th and virus-free plants.

In a survey of viruses infecting Daphne species and cul ti vars in l:Tcw Zeclund., Forster and llilne, ( 1975) isolated four previously described viruses (alfclfa mosaic virus, arabis mosaic virus, cucwr1ber mosaic virus and tobacco rincspot virus) plus seven partially characte:::·- ised viruses (d.aphne isometric viruses 1,2 & 3, daphne-tobacco mosaic virus, dap}mo virus S, daphne virus X and daphne virus Y) o The lutte.c·

three anisomotric viruses (DVS, DVX, DVY) have been further characterL:- ed (R.L. l<'orster &

r: . s.

^Liilno, pers. corm!l., 197~), v1hile the reoairlirl,'.;

isometric viruses (DIV-1, DIV-2,& DIV-3) are tho subject of this studs.

Several isolates of each of tho three isometric viruses were obtained from their respective hosts and extensively characterised.

DIV-1, DIIJ-2 and DIV-3 could be readily differentiated from each other by host range and syr.1ptomatolo& in differential hosts and more detail- ed study led to their separate identification.

PREFACE iii

ABSTRACT ix

CHAP11ER 1. CHARACTERISATION OF CUCU!mER

IDSAIC VIRUS STRAIN D 1

1

.1 lla.terials and methods 2

1.2

Isolation from daphne

7

1.3

^{·Host range}

8

1.4

Vector transmission 17

1.5

^Physical properties in crudL sap

18

1.6

^Electron microscopy 18

1.7

Purification 26

1.8 Fhysical properties of purified virus 40

1.9

Chemical composition 40

1.10

ImmunoloQ'"

46

1

•

11

^Serology

46

1.12

Discussion

51

CHAP'l'.ER 2 • CHJt..RACrEmSA'l'IOH OF DA.PmlE

CAlli'IJ'ATIOlJ L~O'r'LLE VIRUS 53

2 .1 Materials and methods 54

2.2 ^{Proof of isolation fron daphne} 55

2.3 Transmission from' daphne 57

2.4 Host range 57

2.5 Vector transmission 62

2.6 Physical properties in crude sap 62

2.7 Electron microscopJ 63

2.8

Purification

67

2.9

Physical properties of purified virus

68

2.10

Chemical composition

68

2.11

Immunology

71

2.12

Serology 72

2.13

Discussion

73

Cl-L-\PI'llli 3. CW..RACTEHISATIOU OF DAPHNE LATEHT RIHG:JPOT VIRUS

3.1

l.b.teri~l and methods 3.2 Transmission from daphne 3. 3 Hp st rc:ri.ge

3.4

Vactor transmission

3.5

^Seed tr~nsmission

3.6 ^Physic~l properties in crude sap 3. 7 Electron microscopy

3.8

Purific2tion

3. 9

^Physic~l properties of purified vir~s

3 .10 In:iunology and serology 3.11 Discussion

Al'PElIDIX 1. .'i.ntiscr:: used in thi:; study BIJ3LIOGR.',Vi1Y

v

75 75 75

76 82 82

83 83 86 88 88 89 90

93

CUV-D infection in ,~aranthus caudc.tus.

2. Systemic chlorotic flecking and le:lf curling induced by cr.1V-D in Chenopodium quinoCJ..

3.

^{Systemic chlorotic flecking in} t:or.1ordica bnls2..r-'lin.J..

4. Systemic voinc;.l chlor·osis and flccl:inc in GucUT'.lis .. 1;.itivus 'CI"Jst~:l Apple' induceU. b,y CI.'.V- D.

5 .

Systor.,ic mos:lic u.nJ vein~l chlorosis i~ Cucurbi t;..;.

p.:;po 'S:n~ll Suc;..;.r' induced by vkV-D.

6. L·)cal necrotic line p;_i,tterns induced by G'.:.V-D in NicotL.n:... tccbi.i.cum 'Burley 21^1•

7. Irregul;..;.r - sh~1)ecl necrot ic lesions induced. on C~.:V-D

inocuL .. ted Ph;ys:.. .. lis frc;.nchetii.

8. Purified prep-.r<.!.ticns of CLV-D, resuspended in buffers co1~t:::.inin'-: ll.if'f.:orent :::;11rA concentrations, ncz:lti vely stained in cm1~1oniurn molybJ..:,te.

9. Purifi~.d pre1L::.·c;. tions of

c::..V- D ,

resu:..>}Jencled in

buffer~> cont::,inin___; clifi'ore ,t :i::DrA concentr~.tions, nec~-tively st:.incu in phosphotuncstic ~ .. cid.

10. Procod.ure !'or the rurific:..i.tion of cucunbcr r.:os:lic virus.

11 . ,·~nc.lytic,.tl Gedir.1ont: .. tion p: ttcrn, for ^::.i. puri.::'ied prep:..m:i.tion of O'..''J-D re::;uspcndcd in 1.Jor;_i,tc l.uffer

('.·,it hou t .I;D'rL) •

12. Analytical sedir:lcnt'-Lt ion p::;.ttcrn, for :i· purified preparat jon of CL:V- D resuspended in boro.te buffer containing EDTA.

13. Standard curve for the rnolecuh.r weight detGrminc.t- ion, by polyucrylrunide eel electrophoresis, of CJJV RNA species.

14. Densitogram showing polyacrylunide gel electrophoretic separation of CL!V-D RUA species.

15 .

Comparison of CllV-C ~nd ~~V-D in gel double diff\l.sion tests.

13

14 14

15

15

24

33

37

37

44

45

49

16. Compc..rison of C~~V-C :md CL:V-D in eel double diffusion tests using C:~V-CS (IIollcnd) antiserum.

17. Chcnopodium quinoa .:;y~;temically infected \'!i th D-cartlV.

18. Isor-.etric pc:.rticles of D-C::i.rl.:V in a squ<:lsh homoc;ennte fro.:i locally infectc..d Chenopodiu:.. r:uinoc le<...ves, ncc"-tively stc..ined ¹:.ith . .u.i-PI'.h oi..ture.

19. AL:,;1·e~:ltes of D-C:::.rLV pu.rticles .!.n a sq_u:..sh hor:logen::i.te fro;;i locally ir.1.'ectcd .. icoti;.rn:.i. clevelnndii le::i.ves, nE:~_ ti vely !:.lt.:.ined '.1ith Al.I-.1/I'.A r.:::.::.:ture.

20. D-0:~rl.'J pe.rticles in thin sectioned Cheno)odium

~~ le.:i.f tissue.

?1. V;_cuol _r vesicle::; contL-ininB D-C:_:::':.:v particles in t!:in scctioncu ~~.eno1)oc.liuo 'lUino:.. lc:.:.:f t:=-scue.

22. .tJ1·.lyticc.l secii.!.1en1,-_.tion p:...ttern :'or '1 purified

:::;c1 _.r:..tion of D-C:.r._, .:lL r~pecic::;.

24. Loc:.i.1 vein:::.l :·tro:::_in~; ~.nd irrc. 1-th:.r-sh:.i.pocl rinr:;spot leuion::; :=-nducGll C.'/ ... LC/ in Jypso ::il~. clo ·:ms.

25 .

Loc:.l veinal cl1lo:..~o.1iG induced l::· .DL:~\f in ~:_ .. vnc_ria

26. Syster:iic chlo:::'otic .':?.·on·~ :.mi :;,ub..,I) .ucnt rc.:;recnine imtu ceti by .i.iL., 'l in .;Leno )oC..iw:i r. ,, .:.nor..

27. Jy8temic chlorotic ,'lecLin[; u.nd r..osu.ic inJuced by DLHV in Chcno·.oditun :;u:i:.rcnticolor.

28. Uecrotic loc.:..l lesions <...nd veinu.1 necr9sis on ULRV- infected le<::.f of Ph~.seolus vuleci.rls 'Top Crop' .

29. DLRV purified prep:..r<~tion, negatively stu.incd vii th amr:1onium molybd::.te pH

5. 3.

30. DLRV purified prep:lr:...tion, nej~tivoly

.

st::i.ined with phosphotungstic ::i.cid pII

7. 0.

31. An J.gerecate of DLltV p~rticles in ~ squash hoooGenate from ChenopodiUI!). ouinoo., ri.e3di vely stained with Al!-Pl'A.

32 .

An::i.lyticol sediment~tion pattern for a p~rtially

purified prep~r~tion of DLRV.

vii

50

61

66

66

69

70

79

79

85

8 7

87

1. The effects on CJ.IV- D p:....rticle stability of sevornl neguti ve st3.ins ::i.ml different EDT.A concentrc.tions.

2. Compc.¹~lson of :cesus1_1<·nsion buffers used in CT,:'/-D purification.

3. SW;mary of UI/ ;,.cl uorr)tion spectroi~i1otometric

date:. for purified

a..:v .

4. lioloc.1.l::.r weichts o: Cl~V RUA s~e:;,;ies detemined

by ::ms -

polyucryl::i.1ide c;el electrophoresis.

20

31

39

43