SOME PROPERTIES OF A VIRUS FROM GALINSOGA PARVIFLORA*

By G. M. BEHNCKENt

During an investigation of stipple streak disease of French beans near Nambour in south-eastern Queensland (Behncken 1968), the roots of a number of weed species were indexed for tobacco necrosis virus (TNV). A virus was regularly isolated from the roots of Galinsoga parvijlora Cav., an annual commonly called potato weed. It was distinguished from TNV on the basis of differences in host reactions, absence of any serological reaction with TNV antisera, and its failure to be transmitted to the roots of seedlings of mung bean (Phaseolu8 aureU8 Roxb.) by zoospores of the fungus Olpidium bra88icae (Wor.) Dang. The only viruses reported to cause natural infections of G. parvijlora are cucumber mosaic virus (Hein 1957) and an unidentified aphid-transmitted virus (Herbert 1939). As this virus appears to be one which has not previously been reported either from this or any other host, it will be referred to as Galin80ga mosaic virus (GMV).

H08t Range, Symptom8, and Transmi88ion No obvious symptoms of virus infection were noted in the leaves of infected G. parvijlora plants in the field but the possibility of symptomless infection was not checked at the time of isolation of the virus from the roots. However, when the virus was inoculated onto the leaves of G. parvijlora plants, severe systemic symptoms were produced on the new leaves. For host range studies, inoculations were made to at least eight plants of each species, previously dusted with carborundum, with both sap from leaves ground in neutral 0 ·lM K 2HP04 buffer containing 0·1 % Na2S0a and partially purified preparations of the virus. Chenopodium amaranticolor Coste & Reyn. was used as an assay host for infectivity tests. All tests were carried out in an insect-screened glasshouse at temperatures of 16-27°C. Although GMV infected 41 species in eight families (Table 1), the virus usually remained restricted to the inoculated leaves. Only in G. parvijlora did GMV react with severe systemic symptoms. These began with chlorotic and necrotic lesions on the inoculated leaves and vein clearing and chlorotic spots on the new leaves (Fig. 1). This was followed by the development of a severe chlorotic mosaic and areas of leaf necrosis with most of the affected leaves showing marked distortion (Fig. 2). Leaf size was reduced and plant growth was inhibited but flowers and seeds were still produced. A number of other members of the Compositae developed local lesions on inoculated leaves or were susceptible to local infection without symptoms but only Chinese aster [Calli8tephU8 chinen8i8 (L.) N ees], saffiower (CarthamU8 tinctoriU8 L.), and cosmos (COsm08 bipinnatu8 Cav.) were systemically infected, all without symptoms, as was one member of the Amaranthaceae, Celo8ia cri8tata L. * Manuscript received September 15, 1969. t Department of Primary Industries, Indooroopilly, Qld.; present address: Department of Entomology, University of California, Berkeley, California 94720.

AUBt. J. biol. Sci., 1970, 23, 497-501 498 SHORT COMMUNICATIONS

The French bean varieties Brown Beauty, Redlands Autumncrop, and Redlands Pioneer also became systemically infected but the only symptoms were occasional very fine flecks of vein necrosis. Assays to O. amaranticolor suggested that the con­ centration of infective virus in these leaves was very low. In spinach (Spinacia oleracea L.) systemic infection was accompanied by chlorotic spots and faint vein necrosis but later growth was virtually symptomless. A similar systemic reaction was

TABLE 1 HOST RANGE OF GALINSOGA MOSAIC VIRUS +, species infected by sap inoculation; -, no infection; S, symptomless infection

Local Systemic Local Systemic Host Species Host Species Infection Infection Infection Infection

AMARANTHACEAE LEGUMINOSAE Celo8ia cri8tata L. + +S Dolicho8 uniflorus Lam. + Gomphrena globo8a L. + Glycine max (L.) Merr. + APOCYNACEAE M elilotus alba Desr. + Catharanthus r08eU8 (L.) Phaseolu8 aureus Roxb. + G.Don. +S P. lathyroides L. + CHENOPODIACEAE P. vulgari8 L. Chenopodium album L. + cv. Bountiful + C. amaranticolor Coste av. Brown Beauty + + & Reyn. + ± cv. Pinto + C. quinoa Willd. + cv. Redlands Autumn- Spinacia oleracea L. + + crop + + COMPOSITAE cv. Redlands Pioneer + + Ageratum hou8tonianum Trifolium incarnatum L. +S Mill. + Vigna 8inen8is (L.) Bidem pilo8a L. + Endl. ex Hassk. Calendula officinali8 L. +S cv. Blackeye +S Calli8tephus chinensis av. Caloona + (L.) Nees + +S cv. Poona + Carthamu8 tinctoriu8 L. + +S SOLANACEAE Cichorium endivia L. + N icotiana clevelandii COsm08 bipinnatus Cav. + +S A. Gray + Eclipta alba Hassk. + N. glutino8a L. + Emilia 8onchifolia (L.) N. tabacum L. DC. +S av. Turkish + Galin80ga parviflora Cav. + + Petunia hybrida Vilm. + Helianthu8 annUU8 L. + UMBELLIFERAE Hypochoeris radicata L. + Daucus carota L. +S Lactuca 8ativa L. + Sonchus oleraceU8 L. + Zinnia elegan8 Jacq. + noted in a single O. amaranticolor plant but reinoculation to a second plant resulted in local infection only. The local lesions developed by most hosts were small and usually necrotic. Those produced on O. amaranticolor (Fig. 3) had a necrotic centre surrounded by a chlorotic, translucent border while those on zinnia (Zinnia elegans Jacq.) were indistinct chlorotic ringspots. SHORT COMMUNICATIONS 499

The following species were found to be insusceptible to mechanical infection: Aster subulatus Michx., Capsella bursa-pastoris (L.) Medic, Cucumis sativus L., Datura stramonium L., Lycopersicon esculentum Mill., Stellaria media (L.) Vill., Tagetes minuta L., Trifolium repens L., Vicia faba L., and Vigna sinensis (L.) Endl. ex Hassk. cv. Black.

Fig. I.-Vein clearing in systemically infected leaves of Galinsoga parviflora. Fig. 2.-Severe systemic mosaic on G. parviflora. Healthy leaf below. Fig. 3.-Locallesions on Chenopodium amaranticolor. Fig. 4.-Isometric particles of GMV after partial purification. TMV included as a size reference. X c.72,000. 500 SHORT COMMUNICATIONS

The aphids Aphis (Jossypii Glover and Myzus persicae (Sulzer) were tested for ability to transmit the virus from systemically infected leaves of G. parvifiora . . The former species was occasionally found breeding on this plant. Thirty aphids of each species were starved for 1 hr, allowed to make a single probe, and then transferred to young G. parvifiora seedlings in groups of five. A further 30 of each species were allowed access for a minimum of 20 hr on the source plants before being transferred. Although no transmission was demonstrated by these tests, the possibility of aphid transmission is not ruled out especially if the virus is one requiring a long latent period in the vector.

Stability and Purification Some infectivity was retained after heating unbuffered sap from the inoculated leaves of C. amaranticolor for 10 min at 75°C but not at 80°C. Sap stored at 25°C was still infective after 6 weeks and samples of partially purified GMV were still highly infective after 4 months at approximately 4°C. The virus had a dilution end­ point in distilled water of 10-6. GMV also appeared to be stable in a concentrated salt solution as assays of partially purified virus to C. amaranticolor after incubation for 24 hr in an equal volume of 1M CaCh indicated little loss of infectivity. The virus was readily purified from small quantities (20-50 g) of inoculated leaves of C. amaranticolor harvested 5-7 days after inoculation. Sap was extracted by homogenizing leaf tissue in 2 volumes (w/v) of K 2HP04 buffer (0 'IM, pH 7 ·0) containing 0·1 % N a2S03 and then clarified by homogenizing with a further volume of an equal part chloroform-butanol mixture. After standing for 30 min at 4°C, the sap was centrifuged at 5000 (J for 10 min in a Servall centrifuge and the aqueous phase collected and given two cycles of differential centrifugation (100,000 (J for 2 hr in the 30 rotor of a Beckman model L ultracentrifuge followed by 8500 (J for 10 min in a Servall centrifuge). Pellets were resuspended in phosphate buffer (0'02M, pH 7·5). 1 ml of this partially purified preparation was layered on gradients of 10--40% sucrose (25 ml total) in O' OlM neutral phosphate buffer and centrifuged for 2·5 hr at 60,000 (J in a Beckman SW25·1 rotor. After centrifugation the tubes were examined in a narrow, vertical beam of light and samples were withdrawn by puncturing the tubes with a hypodermic needle, diluted with phosphate buffer (0 'OlM, pH 7 '0), and given a further cycle of differential centrifugation. A dense light-scattering zone at a depth of 24-28 mm below the meniscus was formed after density-gradient centrifugation through sucrose. This band appeared to contain the bulk of the infective virus. Two faint narrow bands at 9-10 mm and 14-15 mm below the meniscus produced only a few lesions on assay plants. Dr. A. J. Gibbs (personal communication) confirmed that GMV sedimented as a single entity with a sedimentation coefficient of approximately 120S. This procedure resulted in preparations that were highly infectious and contained high concentrations of virus-like particles. The particles, when negatively stained with neutral 2% potassium phosphotungstate and viewed in an electron microscope, were mainly spherical in appearance but some had a slight polyhedral shape (Fig. 4). Particle diameter was between 26 and 28 nm, based on the width of tobacco mosaic virus rods (taken as 15 nm) that were included on each grid. Many of the particles had been penetrated by the stain. SHORT COMMUNICATIONS 501

Serology An antiserum to GMV was obtained by injecting a rabbit with a partially purified virus preparation. Two intramuscular injections of a 1 : 1 emulsion of virus in Freund's adjuvant were given 10 days apart and these were followed by two intravenous injections given 14 and 19 days respectively after the second intra­ muscular injection. Antiserum collected 1 week after the final injection was stored as a 1 : 1 mixture with glycerol. The titre of the antiserum was 1 : 1024 in both microprecipitin and gel-diffusion tests. When purified GMV was used as antigen only a single line of precipitation was formed in agar gel (0'75% agar dissolved in neutral 0 'OIM borate buffer). Serological relationships of GMV with a number of other isometric viruses were tested by gel diffusion using various antisera. Purified GMV did not react with anti­ sera prepared against any of the following viruses: arabis mosaic, bean pod mottle, belladonna mottle, carnation mottle, cowpea chlorotic mottle, cowpea mosaic, cucumber mosaic (Q strain), lucerne mosaic, raspberry ringspot, southern bean mosaic, sowbane mosaic, squash mosaic, tobacco necrosis (A and D serotypes), tobacco ringspot, tomato blackring, tomato bushy stunt, tomato ringspot, turnip yellow mosaic, and an unidentified isometric virus isolated from white clover. Although GMV is very similar to sowbane mosaic virus, southern bean mosaic virus, and tomato bushy stunt virus, which have been grouped together by Gibbs (1969), in that it is highly infectious, similar in size and appearance, and has similar stability in salt solutions, it differs from them in having a lower thermal inactivation point. It is very similar in a number of properties, including its thermal inactivation point, to a virus isolated in Victoria from white clover by R. H. Taylor (personal communication) but it was not serologically related to this virus. A more detailed study of the chemical and physical properties of GMV will be necessary before it can be allocated to any particular group of isometric viruses.

Acknowledgments I wish to thank Mr. J. Hardy, Electron Microscope Unit, University of Queens­ land, for taking the electron micrographs; Dr. A. J. Gibbs, Department of Micro­ biology, John Curtin School of Medical Research, Australian National University, Canberra, for the analytical ultracentrifugation; and also Messrs. J. B. Bancroft, R. S. Greber, B. D. Harrison, B. Kassanis, K. Kimble, R. E. F. Matthews, H. L. Paul, R. H. Taylor, D. S. Teakle, and A. van Kammen for donations of antisera. I should also like to thank Mr. R. H. Taylor, Victorian Plant Research Institute, Burnley, Vic., for his most helpful criticism of this work.

References BEHNCKEN, G. M. (l968}.-Stipple streak disease of French bean caused by a tobacco necrosis virus in Queensland. Aust. J. agric. Res. 19, 731-8. GIBBS, A. J. (1969}.-Plant virus classification. Adv. Virus Res. 14, 263-327. HEIN, A. (1957}.-Beitrage zur Kenntnis der Viruskrankheiten an Unkrautern. III. Das Gurken­ mosaikvirus. Phytopath. Z. 29, 204-29. HERBERT, D. A. (1939}.-Plant viruses in Queensland. I. Pap. Dep. Bot. Univ. Qd 1 (Il), 1-4.