The effect of dilution on necrotic ringspot virus infectivity and the enhancement of infectivity by noninfective virus

VIROLOGY

The

18,

Effect

477-485

of

(1962)

Dilution

Enhancement

on

Necrotic of Infectivity ROBERT

Department

Ringspot

of Plant

Pathology, Accepted

Virus

Infectivity

by Noninfective

Virus

and

the

W. FULTON UGversity July

of Wisconsin,

Madison

7, 1962

The infectivity dilution curves of sour cherry necrotic ringspot (NRSV) and prune dwarf (PDV) viruses on local-lesion hosts were significantly steeper than a theoretical one-hit curve. The slope of the curve varied with the host; on Dolichos biflorus each twofold dilution of NRSV resulted in a threeto fourfold decrease in infectivity. On Momordica balsamina twofold dilution resulted in eightto tenfold decreases in infectivity. Dilution curves of other viruses on these hosts resembled typical one-hit curves. Supplementing dilute NRSV extracts with additional serologically related virus caused disproportionate increases in infectivity, but the serologically unrelated PDV had no effect. A strain of NRSV noninfective to D. biflorus markedly increased infectivity when added to dilute extracts of a strain causing lesions on this host. UV- or heatinactivated virus also increased infectivity of dilute extracts of active virus, but virus inactivated by formalin or freezing did not. These results suggest that a dose of two or more particles of NRSV at an infection site is necessary to initiate infection and that the dose requirement may be satisfied partly by virus that is unable to infect.

The dilution curves of many plant viruses tend to have the same shape. This would be the case if variations in host susceptibility were due to differences in numbers of susceptible sites. Dilution curves wouId be expected to differ, however, if differences in susceptibility were due to differences in numbers of particles necessary to initiate a lesion at a susceptible site. This is cited by Lauffer and Price (1945) as evidence favoring the single-hit curve hypothesis for plant virus dilution curves. Price and Spencer (1943) have pointed out, however, that the slopes of the dilution curves of tobacco necrosis, alfalfa mosaic, and t.obacco ringspot viruses were steeper than the dilution curve of tobacco mosaic virus. Best (1937) found that tomato spotted wilt virus at high dilutions produced somewhat fewer lesions than expected theoretically, and related this deviation to leaf age and external environment. Bawden and Pirie (1959) described an abnormally steep dilution curve for infectious nucleic acid of tobacco mosaic vi-

INTRODUCTION

Local-lesion hosts for prune dwarf and sour cherry necrotic ringspot viruses have greatly facilitated development of methods for stabilizing and purifying the viruses. Wide and unexplained deviations, however, sometimes occurred between actual and expected numbers of lesions. Preliminary trials showed that the dilution curves of these viruses were different from those described for many other plant viruses. Lauffer and Price (1945) and Bald (1950) have discussed the theoretical interpretations of plant virus dilution curves. They point out that experimental curves best fit the theoretical curve based on Poisson distribution, assuming that a single particle is sufficient to initiate a lesion. The multiplehit curves that would result if two or more virus particles were necessary for infection have significantly steeper slopes than the single-hit curve when the number of lesions is much smaller than the number of susceptible sites.

I-US.

477

478

PULTON

A characteristic of the single-hit dilution curve is that for a large part of the curve, where only a small portion of susceptible sites actually become infected, the number of lesions is nearly directly proportional to the amount of infective virus in the inoculum. Exceptions occur with high dilutions, which usually induce more lesions than would be expected theoretically. Bald (1950) suggested that this was due to dissociation of aggregated virus on dilution. Kleczkowski (1950) pointed out that there was no evidence for a degree of dissociation at these dilutions sufficient to explain the dilution curves. Differences in the slopes of dilution curves of different viruses suggest that, in some cases, the supposition that infection results from chance encounters of single infectious particles with discrete, uniformly susceptible sites may be an oversimplification of events leading to infection. Kleczkowski (1950) proposed an explanation for plant virus dilution curves based on the assumption that susceptible regions may vary in the number of infectious particles necessary to initiate a lesion. Many of the data fitting theoretical single-hit curves were obtained with viruses much more stable than prune dwarf and necrotic ringspot viruses. As Kleczkowski (1950) pointed out, there is no reason to assume that all viruses cause infection in the same way. The dilution curves of the two Prunus viruses were investigated because they resembled multiple-hit curves, characteristically different from dilution curves of more stable viruses. This response to dilution may be involved in the difficulties sometimes encountered in transmitting these, or other unstable plant viruses. MATERIALS

AND

cage Pickling) inoculated 3-5 days previously and kept at day lengths of 18-20 hours. Disks were ground in 0.03 M pH 8.0 phosphate buffer containing 0.01 M sodium diethyldithiocarbamate. When PDV was extracted (from squash leaves, Cucurbita maxima L. var. Buttercup) the solution contained, in addition, 0.02 M sodium t’hioglycolate. Furt.her dilutions of the original extract were made volumetrically in the same diluent. Usually, five 4-mm disks ground in 1 ml of diluent (approximately a 1:50 dilution) induced between 25 and 75 lesions per halfleaf. Because assay leaves were small, more than about 75 lesions per half-leaf could not be counted accurately. Seasonal variations in extract infectivity or susceptibility of the assay hosts necessitated increasing or decreasing the amount of tissue per milliliter to prevent too few or too many lesions. Dilution series are expressed, therefore, in terms relative to the most concentrated extract. The series X, 0.5 X, 0.25 X, etc. (or X, X/2, X/4) represent a twofold dilution series, where the least dilute preparation (X) varied, in different trials, between about 1:30 and 1: 100. Momordica balsamina L. was used for local-lesion assays of both strains of NRSV. NRSV-H also was assayed on Dolichos biflorus L., a plant not susceptible to NRSVG. Usually four treatments or extracts were paired on opposite halves of six primary leaves on three plants. Each experiment was repeated three to ten times. PDV was assayed by counting lesions on cotyledons of Sesbania ezaltata (Raf.) Cory. Treatments were paired on opposite cotyledons so that six cotyledons were inoculated with each extract in four-dilution trials.

METHODS RESULTS

Two strains of sour cherry necrotic ringspot virus (NRSV) were used. These, designated NRSV-G and NRSV-H, differed somewhat in host range, but not in other properties. Prune dwarf virus (PDV) was used in some experiments. Most inoculum was prepared by punching a number of 4-mm disks from cucumber cotyledons (Cucumis sativus L. var. Chi-

The Slope of the Dilution

Curve

NRSV and PDV are highly unstable in extracts of infected tissue. Virus inactivation during only a few minutes between tissue homogenization and inoculation can result in abnormally low lesion counts. Thus counts inconsistent with dilution might be due to differing rates of virus inactivation

EFFECT

OF DILUTION

ON NRSV INFECTIVITY

479

in extracts at different dilutions. Stabilization of NRSV infectivity by sodium diethyldithiocarbamat,e (Hampton and Fulton, 1961)) however, did not eliminate lesion counts inconsistent with dilution. When uniformity tests were run by comparing lesion counts of a series of four threefold dilutions, markedly fewer lesions than expected were induced by dilutions of moderately infective extracts. In several series of trials (Fig. 1) each threefold dilution of NRSV-G caused more than a tenfold decreasein infectivity when assayed on 111. balsamina. The presence of virus-inhibiting substances in crude ext.racts may result in atypical dilution curves. Usually such inhibitors are effective at low dilution and relatively ineffective at higher dilutions, conditioning a curve representing maximum infectivity at some intermediate dilution. Presumably, however, an inhibitor effective over a wide range of concentrations might be responsible for the abnormally large decreases in infectivity with dilution. To test this possibility, dilution curves were obtained with extracts of NRSV-infected Petunia hybrida Vilm. and Prunus pennsylvanica L. and with purified virus (Fulton, 1959). The slopes of these curves (Fig. 1) were nearly identical to those obX tained wit’h cucumber extracts. Purified vi‘/27 v3 X/9 rus was diluted in buffer without sodium DILUTION diethyldithiocarbamate. These results indiFIG. 1. Dilution curves of necrotic ringspot cated that an inhibitor probably was not (NRSV) and prune dwarf (PDV) viruses from involved, since its presence in similar amounts would be unlikely in extracts of various sources.Data were derived from lesion counts on Momordica balsamina (NRSV) and different species and in purified virus. Effect of Extract Constituents on the Slope of the Dilution Curve Each diluted crude extract differed from the others in concentration of extraneous material as well as in virus content. If extraneous material stimulated infectivity at low dilution, this might result in the observed deviation from typical dilution curves. Accordingly, dilution series were prepared in which the concentration of host material was kept constant by additions of appropriate amounts of healthy plant extracts. The same marked decrease in in-

Sesbania ezaltata (PDV) and are averages of 3-5 trials with each source of virus. The solid line represents the slope of a theoretical one-hit curve.

fectivity with moderate dilution occurred with these as with other extracts (Fig. 2). When NRSV-G was diluted in supernatants of infected cucumber extracts from which the virus had been removed by high speed centrifugation, the slope of the dilution curve was similar to those obtained with other diluents. It was shown previously (Fulton, 1957) that the infectivity of NRSV was markedly affected by the concentration and pH of the

FULTON NRS” o......*)

IN: pii

7 B”FFER

tions. Other curves, therefore, are based on twofold dilutions. Eject

of Assay Host on Dilution Curves

Serologically related isolates of NRSV produced, on M. balsamina, dilution curves similar to those of NRSV-G. One of these isolates, NRSV-H, also induced discrete lesions on Dolichos biflorus. On this host, twofold dilutions caused three- to fourfold decreases in infectivity of NRSV-H extracts, whereas on M. balsamina eight- to tenfold decreases resulted from twofold dilution (Fig. 3). This suggested that variation of assay host susceptibility was not due simply to differences in numbers of susceptible sites. Only trials in which three or more dilutions produced lesions were included in Fig. 3. Of a number of more stable viruses tested, one strain of cucumber mosaic virus (CMVN) (Troutman and Fulton, 1958) induced .. countable lesions on M. balsamina. Dilu:. tion curves of this virus did not resemble . i those of NRSV; at the dilutions tested, in: fectivity was proportional to dilution within b the limits of accuracy of the tests (Fig. 3). I I I I Similarly, on D. biflorus, numbers of lesions X induced by dilutions of tobacco streak virus x/3 x/9 were approximately proportional to diluDILUTION tion. The atypical dilution curves of NRSV evidently were not due entirely to peculiFIG. 2. Dilution curves of necrotic ringspot viarities of the assay host. rus (NRSV) in 0.03 M phosphate buffer at different pH levels or with other additives altering inIn Fig. 3 the solid lines represent theofectivity. Each curve is an average of 3-5 trials. retical curves, assuming a Poisson distribuThe solid line represents the slope of a theoretical tion of one, two, or three particles necesone-hit curve. sary at an infection site for the induction of a lesion. The slope of the dilution curve buffer used to prepare extracts. These fac- of NRSV-G on M. balsamina differed sigtors appeared not to affect the slope of the nificantly (1% level) from slopes of theodilution curve (Fig. 2). Of a number of sur- retical one- or two-hit curves, but did not factants increasing the infectivity of ex- differ significantly from the slope of a threetracts containing NRSV, Tween 85 (polyhit curve. The slope of the dilution curve of oxyethylene sorbitan trioleate) was the NRSV-H on D. biflorus differed signifimost stimulatory. Its presence (0.03%) in cantly (1% level) from theoretical one- and extracts, however, did not affect the slope three-hit curves, but was not significantly of the dilution curve. The slope of the dilu- different from a two-hit curve. The slope of tion curve of PDV on Xesbania exaltata was the CMV-N dilution curve was not signifisimilar to that of NRSV on M. balsamina. cantly different from that of a theoretical It was evident in these trials that four one-hit curve, but did differ significantly threefold dilutions usually resulted in no (5% and 1% levels) from slopesof two- and lesions from the two most dilute prepara- three-hit curves.

EFFECT

OF DILUTION

NRSV-H preparations producing 50-75 lesions per half-leaf of M. balsamina usually produced only slightly fewer on the somewhat smaller leaves of D. biflorus. Thus the difference in dilution curves obtained on these two hosts was apparently due to a qualitative rather than a quantitative difference in susceptibility. The typical one-hit curve obtained with CMV-N on M. balsamina is evidence that both the virus and the host are involved in the interaction resulting in multiple-hit dilution curves. Effect of Treatment of Assay Host on Dilution Curves Although some property of NRSV and PDV apparently conditioned dilution curves peculiar to these viruses, the assay host species modified the slope of the curve. Trials were run, therefore, to determine whether treatments of assay plants that altered their susceptibility would alter the slope of the dilution curve. Darkening M. balsamina and D. biflorus before or after inoculation, exposing plants to 20 hours of light per day before inoculation, and the t,ime of day of inoculation did not affect the slope of the dilution curves, although these treatments did alter total numbers of lesions obtained. The differing slopes of NRSV-H dilution curves on M. balsamina and D. biflorus are evidently conditioned by factors not readily altered by changes in environment. Increasing Infectivity Virus Con tent

by Supplementing

Previous experiments did not differentiate between a multiple hit curve and an actual loss of infective virus during dilution procedures. Conceivably, since small volumes were used, adsorption of virus to glass surfaces could have been an important factor. Experiments were devised to determine whether there was potent,ially infective virus in diluted extracts or whether virus had been lost or inactivated. Dilutions of 1:50 and 1: 100 were prepared from a 1: 10 extract of NRSV-infect,ed cucumber had shown that

cotyledons. Previous the 1: 100 dilution

trials would

ON NRSV INFECTIVITY

481

300. 200

-

I 00

-

cn 5

50.

2 -I IL 0

iii

IO-

mw 2 z

I -

FIG. 3. A comparison of dilution curves of cucumber mosaic virus strain N (CMV-N) and necrotic ringspot virus strains (NRSV-G, NRSVH) resulting from lesion counts on Momordica balsamina or Dolichos bifE0ru.s. Curves are averages of 4-10 trials. The solid lines represent the slopes of theoretical one-, two-, and three-hit curves. The experimental curves were positioned along the X-axis to bring each curve adjacent to a theoretical curve of similar slope.

induce 5-10 lesions on three half-leaves of M. balsamina, and a 1:50 dilution, about 50-75. Then, to one portion of the 1:lOO dilution, 0.1 volume of the 1: 10 stock extract was added, making the final dilution (1:55) approximately equal to the portion diluted 1:50. To another portion of the 1: 100 dilution, 0.1 volume of diluent without virus was added. If the virus in the 1: 100 dilution was largely inactive, then the addition of an equal amount of virus should result in lesion counts about double

482

FULTON TABLE

1

THE EFFECT ON INFECTIVITY OF SUPPLEMENTING DILUTE, WEAKLY INFECTIOUS EXTRACTS OF APPROXIMATELY NRSV-G WITH EQUAL AMOUSTS OF ANOTHER STRAIN (NRSV-H) OR AS UNRELATED VIRUS (PDV). INFECTIVITY WAS ASSAYED ON Momordica balsamina Relative concentration

Virus

NRSV-G NRSV-G NRSV-G NRSV-H NRSV-H __-~__~ NRSV-G NRSV-G NRSV-G PDV PDV

x 0.5 x 0.5 x + 0.5 x

+

I 0.5

+

a Averages

0.5 x

I of 5 and

X 0.5 x x + 0.5 x

x

Total lesions three half leaves”

on

63 9 70 9 177 41 40 0

3 trials.

those of the 1: 100 dilution. If all virus in the 1: 100 dilution were potentially infective, then doubling the concentration should result in lesion counts similar to those obtained with the 1:50 dilution. The results of several trials were consistent; infectivity of 1:lOO extracts was low but increased about fivefold when the virus content was doubled. Similar results were obtained with mixtures of NRSV-G and NRSV-H. Mixtures containing 1: 100 NRSV-G and 1: 100 NRSV-H were five to seven times as infective as 1: 100 dilutions of either strain alone, and were as infective as 1:50 dilutions of NRSV-G (Table 1). When the experiment was repeated with mixtures of NRSV-G and the serologically unrelated PDV there was no increase in infectivity of dilute NRSV-G in the presence of PDV (Table 1). In this experiment PDV also infected the assay host (M. balsamina) , but without inducing recognizable primary lesions. In a few trials, purified southern bean mosaic virus, which did not infect M. balsamina, did not alter infectivity when added to diluted NRSV extracts. These results indicated that virus had not been lost or permanently inactivated during dilution procedures.

The Supplementing Efiect of NRSV-G on an Insusceptible Host An opportunity to determine whether the infection-stimulating property of NRSV-G was related to its ability to infect was provided by D. bijlorus. This host is susceptible to NRSV-H, but inoculations with NRSV-G induced no symptoms, and no virus could be recovered from the inoculated leaves. Dilute extracts of NRSV-H supplemented with approximately equal amounts of NRSV-G gave markedly higher lesion counts than unsupplemented extracts. Average lesion counts for four trials were: NRSV-H (X cont.) , 92 ; NRSV-H (0.5 X cont.) , 26; NRSV-H plus NRSV-G (0.5 X + 0.5 X cont.), 88; and NRSV-G (X cont.), 0. Thus, although NRSV-G apparently did not infect D. biflorus, when used to supplement dilute NRSV-H preparations the infectivity was nearly as great as though all virus were NRSV-H. The assumption that NRSV-G did not infect D. biflorus was based on failure to induce symptoms and failure to recover virus. Neither criterion may be conclusive. With most other viruses, however, inoculating mixtures of two strains to a host reacting with lesions to one strain resuhs in fewer lesions than inoculation with an equal amount of the unmixed lesion-inducing strain (Beale, 1947; Sadisivan, 1940; Wu and Rappaport, 1961). Stimulation of NRSV-H infectivity by NRSV-G, instead of interference, is additional evidence that infection by NRSV-G sufficient to block infection by NRSV-H does not occur. Supplementing rus

Effect

of Noninfectious

Vi-

The stimulatory effect of one strain on infection by another, on a host insusceptible to one strain, indicated that some property of the virus other than its infectivity was involved. To check this, trials were run to determine whether virus inactivated in different ways would affect infectivity of active virus. Tissue extracts containing inactive NRSV-G were prepared by freezing disks of tissue for 30-60 minutes at -6”, and by heating extracts at 56” for 10 minutes. Vi-

EFFECT

OF

DILUTIOiK

OX

rus in purified preparations was inactivated by ultraviolet radiation by exposing a 2mm layer of virus in buffer for 4 minutes, 28 cm from a Westinghouse Sterilamp WL 78221 (2537 A). Preparations exposed for 2 minutes retained about 5% of the original infectivity; virus was apparently completely inactivated in 4 minut,es. Purified virus also was inactivated by exposing a concentrated preparation to 0.25% formalin for 1 hour, then diluting 1:lO in buffer and dialyzing 24 hours against buffer. Absence of infectivity was demonstrated as part of the assay t,rial or by separate inoculations.

KRSV

The effect of inactive NRSV-G on the infectivity of dilut,e crude ext’racts of NRSVH was compared with the effect of active NRSV-G and with unsupplemented extracts (Table 2). Virus inactivated by freezing or formalin did not increase the infectivity of dilute NRSV-H. UV-inactivated SRSV-G, however, increased infectivity of NRSV-H, although the effect was less than that of an equal amount of untreated virus. Heat-inactivated NRSV-G increased infectivity of NRSV-H somewhat less than UV-inactivated virus. These increases occurred in each of twelve trials

TABLE THE

EFFECT

Assay

Dolichos

OF ADDING

VIRUS

2

INACTIVATED IX DIFFERENT PREPARATIOKS OF ACTIVE

host

Strain

or mixture

WAYS VIRUS

Relative

H H formalin-inactivated active G

G

H H + heated sap H + heat-inactivated H + active G

concentration

x 0.5 x 0.5 x + 0.5 0.5 x + 0.5 0.5 0.5 0.5 x + 0.5 x +

G

H H H f H +

UV-inactivated active G

G

OF

DILUTE

i\vg.” number of lesions on 3 half-leaves

0.5 0.5

x x 0.5 0.5

x 0.5 x x + 0.5 x + 0.5

x x

154 72 80 149

x x

40 45 68 111

x x

90 36 77 99

balsamina G G G + form&n-inactivated Formalin-inactivated

G G

G G G f frozen-inactivated Frozen G

G

G G G f UV-inactivated UV-inactivated G 0 Figures

ON THE INFECTIVITY

bijlorus

H + H +

Momordica

483

IKFECTIVITY

are

averages

of 3-12

trials.

G

x 0.5 x 0.5 x + 0.5 X 9 0.5 x 0.5 x + 0.5 x X 0.5 x 0.5 x + 0.5 x

x

x

x

112 55 29 0 114 39 21 0 180 55 79 0

484

FIJLTON

with UV-inactivated virus and in each of six trials with heat-inactivated virus. Numbers of lesions were significantly different from the unsupplemented controls at the 1% level by the Duncan multiple range test. Apparently the ability of NRSV-G to supplement infectivity of dilute NRSV-H is not necessarily related to its ability to multiply. Other experiments showed that heat- or UV-inactivated NRSV-H did not infect D. biflorus when supplemented with active NRSV-G.

might require a dose of several particles rat,her than one. The stimulatory effect of NRSV-G on the serologically related NRSV-H when inoculated to a host insusceptible to NRSV-G suggests that this effect is related to antigenic similarity. Although NRSV-G is unable to multiply, it evidently does take part in some process preliminary to replication. The stimulation of infectivity of active virus by the addition of virus inactivated by ultraviolet radiation or heat, but not by freezing or formalin, suggests that a specific portion of the virus particle is involved DISCUSSION in the stimulation. None of the treatments, On t,he assay hosts used, prune dwarf and however, is sufficiently selective to provide evidence of the nature of the portion capasour cherry necrotic ringspot virus infection seemedto result from some sort of coopera- ble of st,imulating infectivity of active virus. tion among two to three virus particles at The dilution curves of PDV and NRSV an infection site. The sensitivity of these vi- obviously resemble multiple-hit curves ruses to inactivators in plant extracts sug- more than single-hit curves and differ in gests that inactivators may play a part, in this respect from many other plant viruses. vivo, in conditioning a multiple-hit curve. It seems possible that plant viruses may If, at an infection site, a limited amount of vary from those able to initiate infection inactivator were distributed among the vi- by single particles to those requiring doses rus particles present,, the chances of one of infective particles not present in conparticle not receiving an inactivating dose ventional extracts. might increase greatly if two or more parACKNOWLEDGMENTS ticles were present. The available evidence does not support This work was supported in part by a grant from this hypothesis. Supplementing dilute ex- the National Science Foundation (G-10897) and tracts of NRSV-G with PDV did not in- is a contribution from the Wisconsin Agricultural crease numbers of NRSV-G lesions as did Experiment Station as a collaborator under North supplements of serologically related virus, Central Regional cooperative research project although multiple-hit dilution curves are NC-14 entitled “Stone and pome fruit virus diseases and their control.” characteristic of both viruses. Thus, if the dilution curves are conditioned by an inREFERENCES activator in vivo, the inactivator must be BALD, J. G. (1950). Measurement of concentration virus specific. This seems unlikely. of plant virus suspensions. In ‘Viruses 1950” (M. A multiple-hit dilution curve might reDelbriick, ed.), pp. 17-29. California Institute of sult from genetic insufficiency of many or Technology, Pasadena. most virus particles, as described by Luria BAWDEN, F. C., and PIRIE, N. W. (1959). The inand Dulbecco (1949) for UV-inactivated fectivity and inactivation of nucleic acid prepbacteriophage. Two or more virus particles arations from tobacco mosaic virus. J. Gen. Microhid.21,438-456. at one site might provide a complete complement of genetic units, one or more of BEALE, H. P. (1947). The interference phenomenon between the rib-grass and tobacco-mosaic viruses which were lacking in single particles. The in bean. (Abstract.) Phytopathology 37, 847. lack of suitable genetic markers and diffiBEST, R. J. (1937). The quantitative estimation of culty in reisolating these viruses from single relative concentrations of the viruses of ordinecrotic lesions have discouraged direct atnary and yellow tobacco mosaics and of tomato tempts to investigate this hypothesis. spotted wilt by the primary lesion method. AusA third possibility is that stimulation of tralian .I. Exptl. Biol. Med. Sci. 15, 65-79. host responses leading to virus synthesis FULTON, R. W. (1957). Properties of certain me-

EFFECT chanically

transmitted

pathology

47,683-687.

OF DILUTION

viruses of Prunus. Phyto-

necrotic

R. W. (1959). Purification of sour cherry ringspot and prune dwarf viruses. Vi-

rology

9,522-535.

FULTON,

R. E., and FULTON, R. W. (1961). The of polyphenol oxidase to instability in vitro of prune dwarf and sour cherry necrotic ringspot viruses. Virology 13, 44-52. KLECZKOWSKI, A. (1950). Interpreting relationships between t.he concentrations of plant viruses and numbers of local lesions, J. Germ. Microbial. 4, 53-69. LAUFFER, M. A., and PRICE, W. C. (1945). Infection by viruses. Arch. Biochem. 8, 449-468. LCRIA, S. E., and DULBECCO, R. (1949). Genetic recombinations leading to the production of active HAMPTON,

relation

ON NRSV INFECTIVITY

485

bacteriophage from ultraviolet inactivated bacteriophage particles. Genetics 34,93-125. PRICE, W. C., and SPENCER, E. L. (1943). Accuracy of the local lesion method for measuring virus activity. II. Tobacco-necrosis, alfalfa-mosaic, and tobacco-ringspot viruses. Am. J. Botan?~ 30, 340346.

T. S. (1940). A quantitative study of the interaction of viruses in plants. Ann. Appl.

SADISIVAN,

Biol.

27,359-367.

J. L., and FULTON, R. W. (1958). Resistance in tobacco to cucumber mosaic virus. Virology 6,30%316. Wu, J.-H., and RAPPAPORT, I. (1961). An analysis of the interference between two strains of tobacco mosaic virus on Phaseolus vulgaris L.

TROuTmN,

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