Purification of pea enation mosaic virus

345

DISCUSSION AND PRELIMINARY REFERENCES L., GIBBS; A. J., and WOODS, II. I>.,

REPORTS

The sediments were resuspended in 0.03 M pH 7..i phosphate buffer (1 ml/40 g tissue), and the suspension was clarified by cm21, 390-395 (1963). 2. BAILEY, L., GIBBS, A. J., and WOODS, 12. T)., trifuging it for 3 minutes at 8000 g. The whole procedure was done at 2-Z’. Virology 23, 425-429 (1964). 3. LEE, P. E., and FURGALA, B., Virology 25, 38iElectron microscopy showed that the 392 (1965). resulting preparations contained many iso4. BAILEY, L., J. Invert. Pathol. 7, 132-140 (1965). metric particles about 30 mp in diameter, 5. LEE, P. E., and FURGALA, B., J. Inve~l. Pathol. which tended to aggregate when mountBed 7, 170-174 (1965). in pH 7.U sodium phosphotungstate. 6. BAILEY, L., and GIBBS, A. J., J. Insect. I’athol. Schlicren diagrams obt,aincd when the 6, 395-407 (1964). virus preparat#ions were examined in the B. FCI~GAIA~ analytical ultracentrifuge showed two spePETER E. 1,~s~ -. cific components with sedimentation coeffcients at infinite dilution of 95 S and 115 S, 1Entomology Research Institute the faster occurring in slight’ly larger Canada Department of Agriculture Ottuwa, Canada amount. To study the differences between &se components, the virus preparations 2 Biology Department were fract,ionated by centrifuging them in Carleton University sucrose density gradients at about, 5”; l-2 Ottawa, Canada ml of the preparation was floated on each Accepted February 10, 1966 gradient made from 4, 7, 7, and 7 ml of solutions containing 100, 200, 300, and 400 g per litre of sucrose, respectively, prcparcd Purification of Pea Enation Mosaic Virus in G.03 111 phosphate buffer (pH T..‘,), and t.hen cent’rifuged for 3 hours at 70,000 g. Only a few plant viruses are known that l’rcparwtions from infected plants gave t’wo are transmitted both by manual inoculation light-scattering layers at8 about 19 mm and of sap and by aphids that have mou!ted 23 mm from the meniscus, arid samples since last feeding on infected plants. Lettuce from these layers, together with samplrs necrotic yellows, one such virus, has been from above, between, and below them were purified and found to have large complex dialyzed at 5” against 0.01 M phosphat,e bacilliform or bullet-shaped particles (1, 2). buffer (pH 7.5) to remove the sucrose. Pea enation mosaic virus is another, and Isometric particles were abundant (I3g. our experiments, summarized here, show it 1 a-c) in the samples from in and between has particles quite different from those of the light-scat#t,ering layers, fewer iI1 samples lettuce necrotic yellows virus, and that, it, is from below, and were not, found in samples best purified in a different way. from above the layers. The inftlctivity of An isolate of pea enation mosaic virus these samples, estimated by inoculating (PEMV), obtained from a field bean plant t#hem to lc:~vcs of C’htvwpodium amarantion Rothamsted Farm, was transmitkd to color Coste wnd Reyn, (Fig. 1 d), W;IScrudely batches of field bean (Kick Jaba I,. “Herz proportional to the numb(Jr of particles they E’reya”) by the pea aphid (Rc~~*thosiph~~ contained. pisum Harris). Syst8emically infected bean Table 1 summarizes the properties of shoots were triturated mechanically in a samples taken from the two light-scattering mixture of pH 7.5 buffer (1.5 ml/g plant layers. Particles from the upper layer aptissue) containing 0.08 M phosphate and peared irregular and distorted, and tm0.05 X ethylenediamine tetraacetatc, and trastcd poorly with the phosphotungstat,e chloroform (1 ml/g tissue). The resulting emulsion was centrifuged for 13 minutes at (Wig. la). They appeared more regular and cont8rasted better when treated for 1 hollr 2000 y, and the aqueous supernatant layer v&h 1 5%fornlaldehyde before being mixrcl was poured off and centrifuged for 2 hours and mountjctl fot at 75,000 g to sediment the virus part,icles. with phosphotungstate 1. BAILEY, Virology

Frc. 1. (a) Electron micrograph of particles from upper light-scattering layer in sucrose density gradients, mounted in neutral phosphotungstate. (b) As in (a), but treated with 1% formaldehyde before mixing with phosphotungstate. (c) Particles from lower light-scattering layer in sucrose density gladienls, mounted in neutral phosphot,ungstate. Note two kinds of pa.rticle. All micrographs at. the s:rme magnification. (d) Local lesions in leaves of Chenopotli~m amarnn~icolo~ inoculated with PEMV. .31!)

350

DISCUSSION

AND 1’1IELIMINAI1Y TABLE

PROPERTIES

1

OF SAAXPLES OF PEG ENATION MOSAIC V~xas FROM THE UPPN~ LIGHT-SCATTERING LAE-ERS IN SVCROSE DENSITY-GRADIENTS

Property Morphology

REPORTS

of particles

Sedimentation CoefficientsU (s~O,,~) Number of components detected serologically Infectivit,yb at 1:100 1:1ooo

Sample from upper layer Isometric, c. 30 rnp diameter, irregular outline 95 R 1

675 96

AXIJ

LOLVER

Sample from lower layer Isometric, c. 30 rnr diameter, outlines regular or irregular 116 s (f 95 S) 2

1310 207

a At infinite dilution. 1,Total number of lesions on 6 leaves of C. amaran~icoZor.

electron microscopy (Fig. lb). The lower layer contained some particles indistinguishable from those in the upper layer, and also many others, which had a similar diameter but a more discrete and regular outline than the particles from the upper layer, and coiltrasted bet’ter mit’h the phosphotungstate (Fig. lc). Analytical ultracent,rifugation showed that samples from t,he upper layer contained a single component8 with a sedimentation coefficient of 95 S, whereas those from the lower layer contained, usually, about a quarter as much of the 9<5S conlponent together with a 115 S main component in an amount similar t,o that’ of the 95 S component in the upper layer. Samples from each of the two layers produced symptoms typical of PEMV in manually inoculated Y. f&z plants, and aphids, which had moulted since last feeding on these plants, infected other bean plants. Double-diffusion serological tests were done in 0.8 % agar gel using an antiserum (kindly provided by Dr. R. J. Shepherd, University of California, Davis) to an American isolate of PEMV. Samples from the upper light-scatt’ering layer gave one faint line of precipitate, and samples from the lower layer gave two lines, one of which was confluent with the line given by the samples from the upper layer; both lines were co11fluent wit#h the two lines of precipitate given by t,he unfractionated virus preparations. The unfract,ionated preparat,ions did

not react specifically with antisera prepared against cherry necrotic ringspot,, cowpea mosaic, prune dwarf, red clover mottle, and t)rue broad bean mosaic \firusc>s (cherry necrotic ringspot virus, an antiserum prcpared against it, and another prepared against. prune dwarf virus were kindly provided by Dr. I<. R. Bock, East’ Mailing R,esearch S tat#ion). The PEMV preparations thus contain two kinds of particles of similar shape and size but different appearance, sedimentation coefficient’, and antigenic constitut,ion. The serological results suggest that part,icles similar to these two kinds also occur in plants infected wit’h American isolates of PEMV, prcparat’ions of which contain particles with two slightly differing sedimentat,ion coefficients (Dr. R. *J. Shepherd, personal communication). Our infcct,ivity tests indicate t’hat the 95 S particle is infect’ivc, but as the 115 S particle has not been obtained free from 95 S particles, whethel it too can cause lesions in C. amwanticolor is not known. Although t’here is no evidence that PE-\IV is closely related t,o any other virus, its two kinds of particle resemble in infectivit’y, appearance and sedimentation behaviour those of cherry riccbrotic ringspot virus (3; and K. R. Bock and R. D. Woods, unpublishcd rcsultsj,

which is pollen-borne

but not,

reported to be t’ransmissiblc by aphids. The 115 S particles of PTL\IV also rcsmlble

DISCUSSION

AND

PRELIMINARY

those of barley yellow dwarf (41, another virus that is transmissible by aphids that have moulted since feeding on infect’ed plants, but further work is needed to examine the extent of the similarity. Note added in proof. Our results confirm and extend some of those of Izadpanah and Shepherd l’irology 28, 463476 (1966). REFERENCES 1. CRO\I.LEY, N. C., HARRISON, B. D., and FRANCKI, R. I. B., ViroZogy26,290-296 (1965). ,$. HARRISON, B. D., and CROWLEY, N. C., Virology 26, 297-310(1965). 8. TOLIN, S. Phytopatho2ogy 55, 1080 (1965). 4, Rocaow, W. F., and BRAKKE, M. K., Virology 24, 310-322 (1964). A. J. GIBBS B. D. HARRISON R. D. WOODS R~&amsted

Experimental

Station

H’wpenden Htvffordshire, England ilccepted March 21, 1966

Magnesium

Trisilicate

Increases

Virus

Transmission

The successful use of bentonite in some plant virus transmissions (1, 2) and the knowledge that magnesium, aluminum, and silicon are major components of bentonite (::) suggest’ed the use of magnesium trisilic:cte (Mg,SisOs~5Hz0, here abbreviated R IgSi) and other magr esium, aluminum, atld silicon compounds. Of those tested, T\IgSi has been the most effective and is also tile most effective chemical supplement to virus inoculum, with the possible exception of K2HPOh, we have tested. MgSi has a pH oi 10.5 in water and 9.4 in 1% I&HI’04 while bcntonite has a pH of 9.5 in water and 5.8 irl 1% I&HP04 . MgSi is easily suspended in water in contrast to bentonite, which has objectionable gelatinous properties. Tnfcbcted leaves and either MgSi (technical grade) or bentonite (crude material) were ground in a mortar with water or 1% &HP04 in such proportions as to give a final concentration of 0.1-I % each of leaf

REPORTS

351

tissue and of MgSi or bentonite, and inoculated on indicator plants. Mostly, the inoculum had been mixed with Celite, but sometimes the indicator plants were dusted with Corundum prior to inoculation. Controls were inoculated with virus suspended in water. Lesions were counted 3-10 days after inoculation. Tobacco mosaic virus (TMV) was assayed on Phaseolus vulgaris var. Pinto, tobacco necrosis virus (TNV) on Vigna sinensis var. Blackeyc, and cucumber mosaic virus (CMV) on 1’. sin~ensis. Representative results from more than 100 trials are given in Table 1. Other viruses tested were tomato ringspot virus (TRSV) on P. vulgaris or V. sinensis, tomato spotted wilt virus (TSWV) 011 I/‘. sinensis, sugar beet mosaic virus (SBMV) on Nicotiana cleve2andii, pear ringspot virus (PRSV) OII Cherlopodium quinoa, and citrus tatter leaf virus (TLV) OII 17. sine&s. MgSi increased t,ransmission of all these viruses except PRSV, although for optimum results the concentration of PtIgSi and the age of the inoculum appear to differ for each virushost combination. The addition of K2HI’04 to MgSi-virus mixtures usually greatly increased their infectdvity, even when AlgSi or K2HPOI alone had little effect. The combination of K2HP04 and MgSi usually resulted in greater infectivity than Ohat of K~HPOJ and bentonite (see Table 1). The donor host. tissue seemed to be decisive. MgSi increased Oransmission of TlUV from systemically infected leaves of sugar beet and from local lesions on Pinto bean; of TNV from local lesions on sugar beet and Chenopodium amaranticolor; of CI\IV from sugar beet’, C. amaranticolor and cucumber; of TRSV from Erigeron glnuaus; of TSWV from cowpea; of TLV from lemon; of SBMV from sugar beet, but not of I’RSV from C. quinoa. The number of lesions resulting from inoculations with JIgSitreated inoculum was usually 5-500 times that of the cont’rol. i\IgSi did not increase infectivity of, or had only a slight effect, with purified T&IV or TMV from tobacco or TNV from cowpea. With CMV, the position and/or the age of the cucumber and sugar beet’ leaves appeared to have a bearing on the result,. Addition of MgSi had little