Ann. appl. Biol. (1980), 95,191-196 With 1 plate Printed in Great Britain

191

Properties of a carlavirus causing a latent infection of pepino (Solanurn muricatum)

BY WAYNE THOMAS, N. A. MOHAMED* AND MARGARET E. FRY Plant Diseases Division, D.S.I.R., Auckland, New Zealand

(Accepted 21 January 1980)

SUMMARY

Pepino (Solanum muricatum) cuttings imported from Chile contained a latent virus which was transmitted by inoculation of sap to Chenopodium quinoa but not to 21 other species. The virus was transmitted by the aphid, Myzus persicae. In C. quinoa sap, the virus lost infectivity when diluted between heated for 10 min between 65 and 70 OC, or stored at room temperature for 4 to 6 days.

The virus particles were straight or slightly flexuous filaments 660 to 680 nm long. Up to 15 mg virus per 100 g C. quinoa leaves was obtained by clarification with a mixture of chloroform and carbon tetrachloride. Purified preparations had A,,,/Amin = 1.1 1, A,,,/A,,, = 1-30, A&: = 2.8, and contained a single sedimenting component with a sedimentation coeficient of 149s and a buoyant density in CsCl of 1-3 18. The virus particles contained 5 .5% of single-stranded RNA of mol. wt 2.4 x 10, (estimated by gel electrophoresis of undenatured RNA) and sedimentation coefficient 38.5S, and a single polypeptide of mol. wt 33 000.

The virus is distantly serologically related to potato S and carnation latent viruses and is considered a new member of the carlavirus group. The name pepino latent virus is proposed. The cryptogram for this virus is R/ 1 : 2.415 - 5 : E/E: S/Ve/Ap.

and

I N T R O D U C T I O N

In 1976, vegetative cuttings of pepino (Solanurn muricatum Ait., cvs El Ceibo, Carrizo and Camino) were introduced into New Zealand for evaluation as a potential fruit crop. During the quarantine period no symptoms were observed on pepino leaves or fruit but when sap from the leaves of each cultivar was inoculated to Chenopodium quinoa Willd., the inoculated leaves developed chlorotic and necrotic local lesions followed by a systemic chlorotic mottle. This paper describes some of the properties of the pepino virus which appears to be a new member of the carlavirus group.

Virus isolate and test plants The virus isolate from pepino was maintained in C. quinoa by mechanical inoculation using

sap from systemically infected leaves as inoculum; C. quinoa was used throughout as a propagation and assay host and as a source of virus in host range studies and aphid transmission tests.

M A T E R I A L S A N D M E T H O D S

Virus purijication Batches of 80 to 100 g of frozen systemically infected C. quinoa leaves were homogenised in a

Warhg blender in 200 ml of a buffer containing 0.5 M borate, 0.5 M urea and 0.15% thioglycollic acid, pH 8.1. The extract was emulsified with a mixture of equal volumes of

Present address: c/o UNDP/FAO Coconut R & D Project, P.O. Box 7331, Manila International Airport, Philippines 3 120. CQ 1980 Association of Applied Biologists

192 W A Y N E T H O M A S , N . A . M O H A M E D A N D M A R G A R E T E . F R Y

chloroform and carbon tetrachloride (0.5 ml/g leaf tissue) and the emulsion was broken by centrifugation at 10 000 g for 15 min. Virus was precipitated from the aqueous phase by adding polyethylene glycol, mol. wt 6000, to 5% (w/v) and sodium chloride to 1.75% (w/v), stirring the suspension at 4 OC for 30 min, and then keeping it on ice for 1 h. The suspension was then centrifuged at 3000 g for 20 min and the resulting pellet resuspended in 40 ml of 0.5 M borate, 0.5 M urea, pH 8.1, by gentle agitation for 2 h at 4 OC. The suspension was clarified by centrifuging at 10 000 g for 10 min and the virus was concentrated by centrifugation at 78 000 g for 75 min. The virus pellet was resuspended in 0.05 M borate, 0.5 M urea, 0.005 M ethylene diamine tetra-acetate (EDTA), pH 8.1, and further purified by a cycle of differential centifugation followed by centrifugation in sucrose gradients prepared with the same buffer. For chemical studies and serology, the virus preparations were centrifuged twice through sucrose gradients.

Analytical Centrifugation The sedimentation coefficient of virus particles was determined in a Beckman Model E

analytical ultracentrifuge using Schlieren optics. For buoyant density determinations, 1-2 mg of purified virus in a CsCl solution of density 1.30 containing 5 mM borate, 0.05 M urea, pH 8.1, was centrifuged for 16 h at 39 000 rev./min in a Beckman 65 rotor. The gradient was displaced with a peristaltic pump and the ,4260 and refractive index of each 0.25 ml fraction measured. The densities of the fractions were calculated from the refractive indices.

Extinction coeficient Highly purified virus preparations were dialysed overnight against 5 mM borate, 0.5 M urea,

pH 8.1. Samples of known A2603 containing 0-5 to 1.0 mg of virus, were dried to constant weight at 110 OC using equal volumes of the dialysis buffer as a control.

Protein and nucleic acid composition Molecular weights of protein and nucleic acid and the phosphorus content of the virus were

determined as described by Mohamed (1978). Marker RNA species were: Escherichia coli ribosomal RNA (mol. wts 1.07 x lo6 and 0-55 x lo6; Stanley 8z Bock, 1965) and tobacco mosaic virus RNA (mol. wt 2-03 x lo6; Boedtker, 1960).

Antisera A rabbit was given a series of two intravenous and four intramuscular injections over a period

of 6 wk (1 mg of virus per injection). For the intramuscular injections, the virus was emulsified with an equal volume of Freund's incomplete adjuvant. The rabbit was bled 6 wk after the last injection.

Antisera to carnation latent virus, potato virus S and potato virus M were obtained from C. Wetter; antisera to pea streak virus and red clover vein mosaic virus from J. W. Ashby; and antiserum to cowpea mild mottle virus from M. Hollings.

Electron microscopy

JEOL lOOB electron microscope at 80 kv. Virus preparations were examined after negative staining on carbon coated collodion grids in a

RESULTS Symptoms and host range

two species were infected: The pepino virus was mechanically inoculated to a wide range of species but only the following

C. quinoa. Chlorotic local lesions, eventually forming necrotic centres. After 15-2 1 days systemic mottling developed in young uninoculated leaves (Plate, fig. 1).

A carlavirus from pepino 193

Solanum muricatum. Symptomless systemic infection. The virus could be detected by back inoculation to C. quinoa or by examination of leaf dips in the electron microscope (Plate, fig. 2). The following species did now show symptoms and no infection was detected by back

inoculation to C. quinoa or by examination of leaf extracts in the electron microscope: Brassica pekinensis (Lour.) Rupr., Capsicum anuum L. cv. California Wonder: Chenopodium amaranticolor Coste & Reyn.: Cucumis sativus L.; Cyphomandra betacea (Cav.) Sendt.; Datura stramonium L.; Dianthus barbatus L.; D. caryophyllus L.; D. chinensis L. cv. Heddewigii; Gomphrena globosa L.; Lycopersicon lycopersicum (L.) Karst. ex Farw. cv. Potentate; Nicotiana clevelandii Gray; N . glutinosa L.; N . tabacum L. cvs White Burley and Samsun; Petunia x hybrida Hort. Vilm. cv. Rose of Heaven; Phaseolus vulgaris L. cv. Top Crop; Pisum sativum L.; Spinacia oleracea L. cv. Royal Denmark; Vicia faba L.; Zea mays L.

Aphid transmission Virus-free Myzus persicae Sulz. were starved for 16 h and were then allowed to feed for 5-10

min on detached leaves of C. quinoa systemically infected with the pepino virus. The aphids were then transferred in groups of 5 to 10 to healthy C. quinoa test plants for 24 h. All the inoculated plants developed a characteristic systemic mottle after 28-35 days.

In vitro properties

65 O C but not at 70 'C, and after dilution to (18-20 "C) was infective after 4 days but not after 6 days.

Properties of purijied virus preparations Purified virus preparations in 0.05 M borate, 0.05 M urea and 5 mM EDTA, pH 8.1, showed a

single peak in sucrose gradients and in the analytical ultra-centrifuge. No host plant components were detected in purified preparations by electron microscopy, analysis of protein and nucleic acids by gel electrophoresis, or by reaction of antiserum to purified virus with host plant antigens. The purification procedure yielded 10-15 mg virus per 100 g leaf tissue.

Electron microscopy Examination of partially purified virus preparations in the electron microscope showed

numerous straight or slightly flexuous filamentous virus particles (Plate, fig. 3) with modal length

Sap from systemically infected C. quinoa leaves was infective after heating for 10 min at Sap stored at room temperature but not to

520 560 600 640 680 720 760 80C Particle length (nm)

Text-fig. 1. Histogram of the particle length distribution of the pepino virus. Particles were measured from negatively stained leaf dip preparations.

194 W A Y N E T H O M A S , N . A . M O H A M E D A N D M A R G A R E T E . F R Y

of 660-680 nm (Text-fig. 1). In some preparations the virus particules were aggregated (Plate, fig. 3) but the use of urea and EDTA in the resuspension buffer usually prevented aggregation.

Analytical centrifugation The sedimentation coefficient of the pepino virus in 0.05 M borate, 0.05 M urea, 5 mM EDTA,

pH 8.1 was 149s (not extrapolated to infinite dilution). In CsCl gradients, the virus formed a single band with a buoyant density of 1.3 18 g/cm3.

Spectrophotometry Purified virus preparations had an absorption spectrum typical of a filamentous virus with a

maximum at 258 nm and a minimum at 243 nm (Text-fig. 2). The Amax/Amin ratio was 1.11 f

1.2 x c v) .- 5

0.8 0 .- 4 2

0” 0.4

!25 250 215 300 325 Wavelength (nm)

Text-fig. 2. LJV absorption spectrum of a purified preparation of pepino virus in 0.05 M borate, 0.5 M urea, pH 8.1 .

0.02 and the A,,,/A,,, ratio was 1.30 & 0.03. The extinction coefficient at 260 nm (1 mg/ml, 1 cm path length) was 2.8 ? 0.2.

Molecular weight of coat protein The molecular weight of virus coat protein, estimated by electrophoresis in 7.5% and 10%

polyacrylamide/sodium dodecyl sulphate gels was 33 000 f 700 (average of seven deter- minations). Some virus preparations gave a faster migrating band corresponding to a molecular weight of 30000; this band constituted less than 5% of the total virus protein and may be a breakdown product of the main protein.

Properties of nucleic acid Samples of purified nucleic acid in 0.15 M NaC1, 0.015 M Na acetate, pH 7.0 (SSC) buffer,

were treated with 2 mg/ml of ribonuclease A or with 10 pglml of deoxyribonuclease plus 5 mM MgCl, for 10 min at 20 OC, and then electrophoresed on 2.2% polyacrylamide gels. The viral nucleic acid was digested by ribonuclease A in SSC buffer but not by deoxyribonuclease, showing that it was a single-stranded RNA. When the RNA was inoculated on to leaves of C. quinoa at a concentration of 100 pg/ml, the inoculated leaves developed numerous chlorotic local lesions. Phosphorus determinations on two virus preparations gave an average phosphorus content of 0.53% corresponding to a nucleic acid content of 5.5%.

A carlavirus from pepino 195

The molecular weight of the RNA, determined on 2% polyacrylamide gels under non-denaturing conditions, was 2-4 x lo6 (average of six determinations). The sedimentation coefficient of the RNA, determined in linear log sucrose gradients, was 38.58 which corresponds to a molecular weight of about 3.0 x lo6 (Hull, Rees & Short, 1969).

Serology Antiserum prepared to the pepino virus had a titre in microprecipitin tests of 11512 against

purified virus but did not react with normal plant extracts. In microprecipitin tests, the pepino virus reacted to a dilution of 11128 with antiserum to carnation latent virus (CLV) (homologous titre 118092) and to a dilution of 114 with antiserum to potato virus S (PVS) (homologous titre 1 /2048); neither of these antisera reacted with healthy plant extracts in microprecipitin or gel-diffusion tests. The pepino virus did not react with antisera to any of the following viruses in microprecipitin tests: potato virus M, cowpea mild mottle virus, pea streak virus and red clover vein mosaic virus.

D I S C U S S I O N

On the basis of particle morphology, host range, aphid transmission, physicochemical properties and serology, it is concluded that the pepino virus described here is a carlavirus. Members of this group have a narrow host range, induce few or no symptoms, are aphid-transmitted in a non-persistent manner, and are serologically inter-related; the particles are slightly flexuous filaments 620-690 nm long and contain about 6% single-stranded RNA (Fenner, 1976).

The physicochemical properties of the pepino virus are comparable to those of other carlaviruses (Veerisetty & Brakke, 1977). The protein molecular weight (33 000) is similar to that of pea streak virus; the molecular weight (2.4 x lo6) and sedimentation coefficient (38.5 S ) of the RNA are similar to those of alfalfa latent virus (Veerisetty & Brakke, 1977). The difference in RNA molecular weights determined by gel electrophoresis (2.4 x lo6) and sedimentation in sucrose gradients (3.0 x lo6) is probably due to the differential effect of the secondary structure of the RNA on migration in polyacrylamide gels and sedimentation in sucrose gradients.

The pepino virus is distantly serologically related to CLV and PVS, but the closer relationship is to CLV. However, in our tests the pepino virus did not infect three Dianthus spp. (which are hosts of CLV; Wetter, 1971b), or N . clevelandii or C. amaranticolor (which are hosts of PVS; Wetter, 1971~) ; this suggests that the pepino virus differs sufficiently from CLV and PVS to be considered a distinct member of the carlavirus group. We propose the name pepino latent virus; the cryptogram is RI1: 2.415.5: E/E: SIVelAp.

R E F E R E N C E S

BOEDTKER, H. (1960). Configurational properties of tobacco mosaic virus ribonucleic acid. Journal of

FENNER, F. (1976). Classification and nomenclature of viruses. Intervirology 7, 1-1 16. HULL, R., REES, M. w. & SHORT, M. N. (1969). Studies on alfalfa mosaic virus. I. The protein and nucleic

MOHAMED, N. A. (1978). Physical and chemical properties of cynosurus mottle virus. Journal of General

STANLEY, w. M. ~r BOCK, R. M. (1965). Isolation and physical properties of the ribosomal ribonucleic acid

Molecular Biology 2, 17 1- 188.

acid. Virology 37,404-4 15.

Virology 40, 379-389.

of Escherichia coli. Biochemistry 4, 1302-13 1 I.

196 W A Y N E T H O M A S , N . A . M O H A M E D A N D M A R G A R E T E . F R Y

VEERISETTY, v. & BRAKKE, M. K. (1977). Differentiation of legume carlaviruses based on their biochemical

WETTER, c. (19714. Potato Virus S . CMIIAAB Descriptions of Plant Viruses No. 60, 3 pp. WETTER, c. ( 197 Ib). Carnation latent virus. CMIIAAB Descriptions of Plant Viruses No. 6 1, 3 pp.

properties. Virology 83, 226-23 1 .

(Received I7 September 1979)

E X P L A N A T I O N O F P L A T E

Fig. 1. Symptoms of pepino virus in C. quinoa (a) healthy leaf; (b) inoculated leaf showing local lesions, and (c) systematically infected leaf showing chlorotic mottling. Fig. 2. Electron micrograph of a leaf dip from pepino leaves with characteristic virus particles in 2% phospho- tungstate, pH 7.0. Bar represents 250 nm. Fig. 3. Electron micrograph of a partially purified preparation of the pepino virus stained with 2% phosphotungstate, pH 7.0. Bar represents 250 nm.