The infection of leafhoppers by Western X-disease virus

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

The Infection

12, 192-201

of Leafhoppers V. Properties

R. F. D&&ion

(1968)

by Western

of the Infectious

WHITCOMB,~

D. D.

of Entomology,

University

JENSEN,

of Ca&fornia,

Received Februanj The

AND

X-Disease

Virus

Agent

JEAN &&e&y,2

RICHARDSON

California

%i’.%

9, 1968

concentration

of extractable Western X-disease virus (WXV) in a vector, (the mountain leafhopper), was much higher than in any of several herbaceous plants tested. Leafhoppers were therefore used as virus source material for a survey of viral properties in crude extracts. A new method of bioassay was employed, based on the concentration dependence of the incubation period, in the insect, between injection of virus and transmission of WXV to plants. This bioassay permitted more accurate study of viral properties than has heretofore been possible with leafhopper-borne viruses. WXV activity was not recovered after saturation of saline extracts from leafhoppers with butanol. However, WXV survived brief ultrasonic treatments, brief emulsification with Genetron 113, and in limited trials, emulsification with chloroform, Freezing of whole viruliferous insects or saline extracts from such insects did not destroy the virus. Infectivity was present in centrifugal supernatants after 10 min at 8,000 g, but was almost completely sedimented after 10 min at 25,000 g. After rate zonal densit! gradient centrifugation for 20 or 25 min at 20,000 or 25,060 rpm, infectivity was found throughout the gradient column, but was most concentrated in the bottom one-third of the column. Viral infectivity was greatly reduced by overnight dialysis against saline, but not by dialysis against a buffer containing glycine, divalent cations, and cysteine, or by buffer transfer through Sephadex G-75 into a protective buffer containing Mg?’ and glycine. Infectious WXV was recovered after brief exposure to various buffers at pH values from 5.1 to 9.7. Brief exposure to 0.01 M glycine or 0.01 M sodium borate buffers at pH 8.2 and 8.9, respectively, increased viral yield, but longer exposures to these buffers decreased the virus yield. Stability seemed best between pH 6.0 and 7.1. Recovery of virus from resuspended pellets after mntrifugation at 80,000 g for 30 min WFLS much better when 0.3 M glycine was used than any lower concentration of glycine whether the ~.IH was 6.1, 7.5, or 8.5.

colladonus

montanus

mitted to plants by leafhoppers of the tribe Deltocephalini, was found (Jensen, 1959; Jensen et al., 1967) to be lethal to one of its vectors, the mountain leafhopper (Colkzdonus montunus), to reduce fecu&ty (Jensen, 19&Z), and to produce cytopathological and, probably, proliferative symptoms in certain tissues of that leafhopper (Whitcomb et al., 1967, 1968a; Whitcomb and Jensen, 1968). In view of the biological interest in this virus, its iso-

INTF~ODUCTI~N

Western X-disease virus ( WXV), one of a number of “yellowing’‘-type viruses trans1 Present address: U. S. Department of Agriculture, Agricultural Research Service, Entomology Research Division, West Building, Plant Industry Station, Beltsville, Maryland 20705. 2 This study was aided by U. S. Public Health Service research grant No. AI-03490 from the National Institute of Allergy and Infectious Diseases to the University of California. 192

WESTERN

X-DISEASE

VIRUS

lation would be highly desirable; however, purification of a virus transmitted persistently by its insect vector has proved, historically, to be a complex problem. When this was accomplished, it was usually possible to use infected plant tissue as a virus source. As yet, no plant hosts of WXV have been found to contain high concentrations of virus. As a result of our discovery (Whitcomb et al., 1966a) that diseased insects contain much higher virus concentrations than diseased plants, a survey of virus properties and preliminary steps of virus purification was made using insects as virus source material. MATERIALS

AND

METHODS

Biological materials and methods have been previously described (Jensen, 1959; Whitcomb et al., 1966a,b). Two types of bioassay were employed in this study. One was based on the percentage of insects becoming infected, a statistic which has been shown to be related to WXV concentration (Whitcomb et al., 1966a). This method was useful when relatively few samples were to be assayed, or, especially, when the w 44. z = z z E =1 5 2 I

IN

LEAFHOPPERS.

193

v.

effect of a treatment was dramatic and obvious. Another type of bioassay was based on the concentration dependence of the incubation period, in the insect, between injection of virus and transmission of WXV to plants. This method allowed many samples to be assayed, inasmuch as each insect which eventually transmitted WXV yielded a statistic (the estimated latent period), which was an estimate of the injected virus concentration. Although it would be best for assays of this sort to include a “dilution series” control in each experiment, we found that an occasional dilution series sufficed to indicate the relationship between latent period and virus concentration. In Fig. 1 we have plotted the latent periods for all experiments as a function of virus concentration; this curve was used as a reference curve for estimating the relative virus concentrations given in our tables. Aimost all work reported herein was performed in a cold room or refrigerated ultracentrifuge at 4°C. Virus preparations were held in a refrigerator at 4°C or in an ice bath prior to injection. “Saline,” whereever used, was 0.85% NaCl. “Protective buffer” was 0.1 hf glycine, 0.03 M MgCL, 0.01 M Na2S03 at pH 7.3.

40.

RESULTS

36.

Sources

32. 28. 24

20’

1

3 2 LO&, VIRUS CONCENTRATION

A 4

5

FIG. 1. Relationship between mean latent period in the leafhopper and relative virus concentration (based on tenfold dilution series). Tests intervals were 3, 4, 5, or 6 days in duration. The median day of the test interval in which an insect first transmitted was used as an estimate of the WXV latent period. Points in the graph are means of latent periods of groups of insects, sample size varying from 4 to 54. The line was eye-fitted by inspection.

of

virus

Attempts were made to extract WXV by grinding plant tissues in 2 to 3 ml of 0.08 M KZHPO, and 0.01 M Na$03 per g of fresh tissue weight. The pH of the resulting extracts was about 7. The extracts were filtered through cheesecloth and centrifuged for 10 min at 1,800 g. Extracts diluted with 0.01 M glycine, 0.03 M MgC12, or 0.01 M Na,SO, (pH 7.3) were never infectious at a dilution of BY3 (based on original fresh weight of tissue) and often were not infectious at lO-*. The following tissues were extracted: roots of turnip and radish 30-50

194

WHITCOMB,

JENSEN,

days after inoculation by viruliferous insects; turnip shoots, which at the time of harvest, 30 days after inoculation, showed recently apparent and conspicuous vein clearing; and celery leaves and stems from plants with recently apparent symptoms. These trials exhausted the known herbaceous range for WXV with the exception of Vincu roseu. It was concluded that the available plant hosts contained virus concentrations several orders of magnitude below that required for purification. Because extracts from the leafhopper, C. montunus, were still infectious at dilutions from 1O-3 to 16”, insects were normally used as a virus source. Subsequent experiments, however, indicated that solvents of different molarity or ionic species might have proved superior to the buffer actually chosen for attempted extraction of WXV from plants. Furthermore, during some period of virus multiplication in plant hosts there may be a peak of WXV concentration at which useful amounts of virus could be extracted. This is true of WXV infection of the vector (Whitcomb et al., 1966b), and was also found in tomato spotted wilt virus infection of Nicotiuna rustica (Black, 1955). It is TABLE STABILITY

Percent

Time

powerQ

(&I

None

-

20% 20% 30% 50% 70%

2 5 1 1 1

OF

WXV

2” 5” 12” 20” 15” 18”

RICHARDSON

possible, and perhaps more likely, that inefficient acquisition of WXV by leafhoppers from celery and peach (Jensen, 1957) reflects a low concentration of virus in those plant hosts. Brakke et al. (1954) found that wound tumor virus occurred in lower concentrations in sweet clover than in crimson clover, paralleling the availability of virus to the vector Agullia constrictu from the two plants.

Ultrusonicuti4m Two preliminary experiments indicated that ultrasonic treatment might be used to disperse WXV aggregates. A third experiment measured the effects of various

dosages of ultrasonic energy upon yield of virus. Extraction of 1.7 g of viruliferous adults in 7 ml of saline was followed by emulsification with Genetron. The emulsion was centrifuged at 1,800 g for 10 min, and then passed through a Sephadex G-75 column, 20 x 1 cm, into protective buffer. The effluent fraction, which was visibly opalescent, comprising about 25 ml, was collected. Six-ml portions were placed in plastic centrifuge tubes and with a micro1

TO ULTRASONIC

Temperature( “C) during sonication Start Finish 2” 2” 2” 2” 2” 2”

AND

TREATMENT

Fraction infected

Average latent period

EstimatedWXV concentration

15/18 b 13/20 7/17 11/17 18/18 9/18

28.9 31.8 36.5 36.1 31.9 32.4

3.2 0 2.6 1.0 1.1 2.7 2.1

aBiosonik,Be-l, Bronwill Corporation. bFractions of insectstransmittingWXV to healthy celery test plants. 0Cakulated from Fig. 1. Virus, after transfer into protective buffer by Sephadexgel filtration, was given’ ultrasonic treatment as indicated. The resultswere erratic, but indicated loss of infectivity due not only to sonicenergy (especiallyNone+ 20% 2 min+ 20% 5 min), but also to elevation of the genera1 temperatureof the solution (30% 1 min, comparedwith 50% 1 min).

WESTERN

X-DISEASE

VIRUS

probe, given ultrasonic treatments 3 of different intensities (Table 1). The results indicate that not only increasing temperature of the entire solution but also sonication itself was responsible for loss of viral infectivity. Clarification Butanol. Addition of butanol, in sufficient quantity to saturate saline extracts from infected leafhoppers, destroyed all WXV infectivity. Lower concentrations of butanol were not tested. Chloroform. In one experiment, emulsification of a saline extract from infected leafhoppers with an equal volume of chloroform destroyed all infectivity. In a second experiment, however, infectivity was recovered after chloroform treatment. The temperature during all operations was maintained at about 2°C. Viruliferous leafhoppers were extracted in saline, and the extract centrifuged for 10 min at 1,800 g. One ml of this extract was emulsified briefly with 1 ml of chloroform. The emulsion was broken by centrifugation at 1,866 g, and the aqueous supernatant was removed and clarified by centrifuging for 10 min at 8,000 g. Fifteen of 28 leafhoppers injected with this supematant transmitted WXV, whereas only 7 of 20 leafhoppers injected with the untreated control suspension transmitted virus. Because an unsatisfactory level of infection was obtained in the control, the yield of WXV after the chloroform treatment cannot be assessed. Fluorocarbons. Several preliminary experiments indicated that Genetron 113 could be used for clarification of extracts. This treatment was therefore incorporated into routine preparation of crude inocula. Leafhoppers were extracted in the selected solvent, usually saline. An equal volume of Genetron was added to the extract. The 3 Biosonik BP-I, Bronwin Corporation.

IN

LEAFHOPPERS.

V.

195

mixture was thoroughly emulsified, using a 5-ml syringe without a needle. A short (5-10 min) centrifugation at 1,800 g was The sufficient to break the emulsion. aqueous (top) phase was removed, and, in some experiments, again emulsified with Genetron. In one experiment a third emulsification with Genetron appeared to destroy infectivity; ( 11/14 leafhoppers injected with the first supernatant were infective; 15/19 infective with second supernatant; O/60 with third supematant ). Another experiment suggested that Genetron could improve an otherwise poor exIt was estimated that approxitraction. mately 10 times as much infectivity was recovered after a single emulsification with Genetron, as compared with an untreated control. Such results could occur if cellassociated virus were extracted by destrucof memtion of the lipid components branes. Stability of wxv Ionic Environment. Dialysis preparations against solvents not containing divalent cations and amino acids destroyed infectivity (Table 2). It is recognized that results indicating apparent instability could be interpreted as loss of virus due to aggregation. However, the necessity of divalent cations and amino acids for stability of virions of potato yellow dwarf virus (Brakke, 1956) and wound tumor virus (Black and Markham, 1963), suggests that true stability (and not solubility) is involved. Freezing. Infectious WXV could be recovered 3 weeks after freezing of either whole viruliferous insects or saline extracts of such insects. Centrifugation Diflerential Centrifugation. A number of differential centrifugation experiments were performed in the SW-39L rotor of the

196

WHITCOMB,

JENSEN,

TABLE OVERNIGHT

Experiment

1

2

3

STABILITY

b

0.3

M glycine 0.1 hl glycine 0.03 h4 glycine 0.01 M glycine Not dialyzed

INFECTIOUS VARIOUS

Rotor

speed a

-

6.1 6.1

-

8.5 8.5 8.5 8.5 6.1 viruliferous

0.03 M CaCl,, transmitting

(rpm) 10,000 18,000 31,600 39,000

10 10 10 20

Dilution

injected lo-’

10-l lo/12 l/14 O/18 O/18

6

9/12 3/20 O/18 o/12

a SW-39 rotor, Spinco Model L preparative ultracentrifuge, 5-ml tubes. Viruliferous leafhoppers were extracted in 0.85% NaCI, and the extracts clarified for 10 min at 1,800 g prior to treatment. b Fraction of insects transmitting WXV to healthy celery test plants.

lo-1 8/24 16/24 13/21

2

o/13 19/25

0.1 M cysteine, WXV to healthy

3

Injected

0 0

in

DIALYZED

~_

0 0 0 0 0

leafhoppers

VIRUS IN SUPERNATANTS AFTER CENTRIFUGAL TREATMENTS

Time (min)

Buffer changes

6.1 7.0 6.1

Spinco preparative ultracentrifuge. After centrifugation of extracts at 10,000 rpm (S,OOO g) for 10 min, supernatants were always highly infectious. Further clarification, at 18,000 rpm ( 22,000 g), for 10 min, sedimented most of the viral particles, while 10 min at 31,600 rpm (65,000 g) completely sedimented the infectious particles (Table 3). In a later experiment, TABLE

2

PH

0.85% NaCl Not dialyzed

a Extracts prepared by grinding various buffers. b 0.1 M glycine, 0.03 M MgC12, ~Fractions of injected insects

RICHABDSON

OF CRUDE WXV PIWPARATIONS AGAINST VARIOUS BUFFERS

Dialyzed a vs. 2000 ml of0.85% NaCl Complex buffer Not dialyzed

AND

c

4138 l/38 o/35 2/36 7/30 saline

were

dialyzed

dilution lo-?

lo-3

O/24 5/24 7124

O/24 O/24 3124

-

-

-

overnight

against

pII 7.0. celery test plants.

one of 40 leafhoppers transmitted WXV after injection with the supernatant from 15 min centrifugation at 30,000 rpm. Rate Density Gradient Centrifugation. Rate density gradient zonal centrifugation (RZDGC; Brakke, 1953) was used in a number of experiments as an analytical tool to assess the thoroughness of dispersion of WXV in extracts. It was hoped that the information obtained could be used in the development of a fractionation technique. Gradient columns, prepared by layering 3, 6, 6, and 3 ml, respectively, of 100, 200, 300, and 400 g sucrose/liter of protective buffer in tubes for the SW 25.1 Spinco rotor, were allowed to stand overnight. Extracts from vitiliferous leafhoppers were clarified for 10 min at 8,000 g. One- or 2-ml aliquots from the supematant liquid were layered upon the gradient columns and centrifuged for 20 or 25 min at 20,000 rpm or 25,000 rpm. Virus yield from source material was variable, but eight RZDGC experiments gave consistent scanning patterns. The best

WESTERN

X-DISEASE

VIRUS

IN

LEAFHOPPERS.

197

V.

4

‘\

12

4

I

\

i

5

D2

I

6

LO;,, UNITS VIRUS

FIG. 2. Estimate of virus concentration and zonation in a density gradient column after centrifugation for 20 min at 25,000 rpm in the SW 39 L rotor of the Spinco ultracentrifuge. The column was prepared by layering 1 ml of 400, 300, 200, and 100 g sucrose/liter, and allowed to stand overnight before use. A sample of I/c, ml, prepared by clarification of an extract of viruliferous leafhoppers emulsified with Genetron 113, was layered on the column. Fifty droplets comprising the contents of the column, were collected by puncturing the bottom of the tube. Ten insects were injected with each drop, and their latent periods used (with help of Fig. 1) to estimate virus concentration. The concentrations were used to construct the scanning pattern. Zonation in the column: L, residual supematant, containing pigment; and R, the top of the gradient were somewhat opalescent. A heavy zone T apparently contained components reaching equilibrium. The region M contained very little opalescent material. The zone B was less intensely opalescent than T, but conspicuous. This zone graded off into regions D, and D,, which contained relatively few components, but which occasionally were somewhat more opalescent than healthy controls.

scanning pattern is shown in Fig. 2. Whereas more infectious particles were present in the top third of the gradient column than the middle third, the largest number of infectious particles was always in the bottom third of the column. Infectivity was

never associated with a distinctly visible zone, but occasionally the lower portions of columns where infectivity was highest were somewhat more opalescent than control columns in which corresponding healthy components were centrifuged.

3.7 5.0 6.1 7.0 7.8 8.5 9.0 10.0

Sodium citrate Sodium citrate Glycine Potassium phosphate Glycine Glycine Sodium borate Sodium bicarbonate

3.8 5.1 6.5 7.1 7.6 8.2 8.9 9.7

3 3 3 3 3 3 3 3

Final

4 AGAINST

0.01

2 2 1?4 1 1% 1% 2 2

Hours of dialysis vs. protective buffer

TABLE Acrr~vrrv AFTER DIALYSIS

PH

WXV

Hours of dialysis

OF M

OF VARIOUS

O/28 19/24 22/24 23/24 22/28 23/23 22/28 Q/18

Fraction of infected insects b

BUFFERS

aExposure to the dialysis treatment resulted in a pH-time course, so that actual exposure time at the nominal pH was short. Before injection, pH was brought back to 7.3 by dialysis vs. protective buffer. 0 Fraction of insects transmitting WXV to healthy test plants. 0 Calculated from Fig. 1.

PH

Nominal

Ionic species a (0.01 M buffers )

RECOVERY

35.7 28.9 29.3 31.3 24.9 27.3 32.3

Average latent period

pH a

1.0 3.2 2.8 2.2 4.3 3.5 2.0

concentration

VhS

Estimated C

Q

B

5 E

2 B 3

Y1

i3

W’ESTERN

Ionic

X-DISEASE

VIRUS

Environment

pil-i Effects. In three experiments extraction of plant material or insect material was not improved by using the buffer 0.08 M &HPOI, 0.01 M Na2S03 at pH 7.68.2 instead of pH 7.0. Two other experiments were designed to test pH stability. In one experiment, 306 infected adult leafhoppers were extracted in 6 ml of saline, emulsified with Genetron, and then clarified at 8,000 g for 10 min. One-ml portions were then dialyzed against 0.01 M buffers of various ionic species (Table 4), chosen for their buffering capacities at the test pH. For brief periods, at least, WXV was stable over the pH range 5.1-9.7. Higher viral titers were obtained after dialysis at pH 8.0 and 8.9; virus may have been eluted from aggregates or complexes under these conditions. However, in a subsequent experiment, when a density gradient zone carrying many infectious particles (28/28 of injected insects proved to be infective) was dialyzed for 3 hr against 0.01 M H3B03 adjusted to pH 8.6 with TABLE RECOVERY

OF

WXV

ACTIVITY

AITER

STORAGE

Minutes

PH=

IN

LEAFHOPPERS.

199

V.

NaOH, most of the virus became noninfectious, (l/25 injected insects were infective), perhaps as a result of the removal of dialyzable protective factors. In a second experiment, when the pH of extracts, prepared by grinding viruliferous leafhoppers in distilled HZO, was adjusted by adding 0.1 N NaOH dropwise, there was a marked downward drift in the pH of solutions during standing for 4 hr. Nevertheless it was indicated that brief exposures to pH values of 8.9 did not destroy WXV (Table 5). At pH 8.0 and 8.9 there was a significant loss of activity-estimated to be less than a log unit. Probably more virus was destroyed than is apparent, because any inactivation occuring at higher pH may well have been partially offset by increased solubility. A third experiment (Table S), discussed at length in the section on molarity, indicated that in the pH range 6.1-8.5, molarity was much more important than pH in resuspension of pellets. 5 FOR VARIOUS

TIMES AT 2°C AT VARIOUS

pH

after adjustment

Nominal

Final

10-30

58-74

110-126

180-200

8.9 8.5 8.0 7.1 6.6

7.8 7.2 6.8 6.4 6.0

33.6~ 29.5 26.9 24.3 21.5

29.6 25.7 28.3 23.8 29.6

32.7 34.8 28.5 29.9 23.8

37.9 28.2 29.2 26.6 30.3

Lumped data: (Average latent periods ) 33.2 d 29.8 28.0 25.9 27.9

Estimated virus concentration

b

1.0

2.1 2.5 3.2 2.5

a The adjustment of pH of extract of viruliferous leafhoppers in saline by addition of 0.1 N NaOH was followed by a drift toward acidity upon standing. b Calculated from Fig. 1. 0 Average latent period in insects infected by injection of viral inoculum stored for the indicated time (in this case, 10-30 min.). dAverage latent period of all insects injected with extract originally adjusted to indicated pH (in this case 8.9 ) .

200

WHITCOMB,

JENSEN,

AND

TABLE Recovmtv oE‘ WXV

AtWxa ATTEXWTED MOLARITIES

Molarity of glycine 0.32 M

0.16 M 0.08

M

0.04

M

0.01

M

Nominal PH of buffer

pH after resuspension

6.1 7.8 8.5 6.1 7.8 8.5 6.1 7.8 8.5 6.1 7.8 8.5 6.1 7.8 8.5

6.1 7.8 8.5 6.1 7.2 8.5 6.1 6.8 8.0 6.1 6.1 7.1 6.1 6.1 6.1

RICHARDSON

6

HESUSPENSI~N AND pH VALUES'~

IN C:~.VCINE

or

Fraction insects infected

Average latent periods

11/H u/11 9/10 lO/lO 617 14/14 lO/lO 12/12

23.63 24.86 25.22 26.40 27.58 31.61 28.75 26.08 28.42 30.25 27.09 27.45 27.62 27.95 30.75

w3 819 13/13 11/12 8/g 10/13 10/12

VARIOUS

Estimated virus concentration

b

3.2 2.9 2.8 2.4 2.1 1.0 1.8 2.5 1.9 1.3 2.3 2.1 2.2 2.0 1.2

‘J Pellets after ultracentrifugation at 80,000 g for 30 min were resuspended in glycine solutions of various pH and molarity, then clarified 10 min at 8,000 g, and the supernatants injected into leafhoppers. * Calculated from Fig. 1.

The significance of tolerance for the pI-1 range 5.040 was not thoroughly explored. Possibly a useful differential solubility might be obtained in this range, if buffers of suitable molarity are used.

pellets resuspended in glycine solutions of various pH’s and molarity. For this operation, a 2-ml syringe fitted with a blunttipped needle was used to homogenize the extract until no visible lumps remained. This caused a certain amount of foaming Mohity. Molarity may be a key factor of the extract. The extracts were then clariin viral solubility (Black et al., 1963) or fied for 10 min at 8,006 g, and the superstability (Brakke, 1956). Unfortunately, natants were injected into leafhoppers. solubility of virus in crude extracts is difExtracts resuspended in buffers whose ficult to measure, since many factors may nominal pH was higher than 6.1 tended to influence the total apparent virus conbecome more acidic upon standing, unless centration. We chose resuspension of centhe molarity was 0.16 hi or higher. In this trifugation pehets as a convenient method and a subsequent confirming experiment, of testing viral solubility. WXV suspenpH seemed to be of little importance, but sions were prepared by extracting virulia molarity of 0.3 was required for solubility ferous C. montanus in saline, and clarifyof the virus (Table 6). ing with Genetron. WXV was then pelleted by centrifugation at 80,000 g for 30 min. DISCUSSION This operation was performed in Spinco 0.8-ml tubes in adapters previously deBioassay of WXV requires 50-80 days. scribed (Whitcomb and Black, 1961). This simple fact limits, at present, the rate Supernatant liquids were decanted, and the of experimental progress. We are forced to

WESTERN

X-DISEASE

VIRUS

recall the cytopathic action and eventual lethality of WXV in an insect vector, its probable ability to induce proliferative symptomatology in a vector (Whitcomb and Jensen, 1968), and to speculate upon the many similarities [including virus properties as determined in extracts (Lee and Chiykowski, 1963)] between WXV and the large group of “yellows viruses,” whose morphology and chemical composition are at present unknown. If it were not for these matters of uncommon biological interest, the problem of WXV isolation would hardly seem worth the laborious measures required at present. Even so, perhaps a tissue culture assay, such as that of Chiu et al. (1966), will have to be devised before final purification becomes feasible. Recent reports that hlycoplasmalike bodies have been found in plants infected with several yellows-type diseases raise the possibility that the Western X-disease agent might even be cultured on an artificial medium. This possibility is more fully discussed in the following paper. NOTE:

REFERENCES

L. M. 1955. Concepts and problems concerning purification of labile insect-transmitted plant viruses. Phytopathology, 45, 208-216. BLACK, L. M., BRAKKE, M. K., AND VATTER, A. E. 1963. Purification and electron microscopy of tomato spotted-wilt virus. Virology 20, 120130. BLACK, L. M., AND MARKHAM, R. 1963. Basepairing in the ribonucleic acid of woundtumor virus. Neth. J. Plant Pathol., 69, 215. BRAKKE, M. K. 1953. Zonal separations by density-gradient centrifugation. Arch. Biochem. Biophys., 45, 275-290. BRAKKE, M. K. 1956. Stability of potato yellowdwarf virus. Virology, 2, 463476. BRAKKE, M. K. 1960. Density gradient centrifugation. Advan. Virus Res., 7, 193-224. BRAKKE, M. K., BLACK, L. M., AND VATTER, A. E. 1954. Size and shape of wound-tumor virus. Brookhauen Symp. Biol., 6, 137-156. CHIU, R. 1966. Inoculation and infection of leafhopper tissue cultures with a plant virus. Virology, 30, 562-566. BLACK,

IN

LEAFHOPPERS. D.

JENSEN,

v.

201

D. 1957. Differential transmission of yellow leaf roll virus to peach and by the leafhopper, Colladonus monPhytopathology, 47, 575-578.

peach celery tanus.

D. D. 1959. A plant virus lethal sect vector. Virology, 8, 164-175.

JENSEN,

to its in-

D. D. 1962. Pathogenicity of Western X-disease virus of stone fruits to its leafhopper vector, Colludonus montanus (Van Duzee) . Proc. Intern. Congr. Entomol. Ilth, Vienna, 1960, pp. 790-791.

JENSEN,

JENSEN,

D.

SON,

J.

Western vector. I,EE,

D., WHITCOMB, R. F., AND RICHARD1967. Lethality of injected peach X-disease virus to its leafhopper Virology, 31, 532-538.

P. F., AND CHIYKOWSKI, L. N. 1963. Infectivity of aster-yellows virus preparations after differential centrifugation of extracts from viruhferous leafhoppers. Virology, 21, 667-669. R. 1965. Clarification taining potato yellow dwarf puthology, 55, 752-756.

WHITCOMB,

of extracts convirus. Phyto-

R., AND BLACK, L. M. 1961. Synthesis and assay of wound-tumor soluble antigen in an insect vector. Virology, 15, 136-145.

WHITCOMB,

R., AND JENSEN, D. D. 1968. tive symptomatology in leafhoppers Western X-disease virus. Virology, 177.

WHITCOMB,

Prohferacarrying 35, 174-

R., JENSEN, D. D., AND RICHARDSON, J. The infection of leafhoppers by 1966a. Western X-disease virus. I. Frequency of transmission after injection or acquisition feeding. Virobgy, 28, 448453.

WHITCOME.,

R., JENSEN, D. D., AND RICHARDSON, 1966b. The infection of leafhoppers Western X-disease virus. II. Fluctuation virus concentration in the hemolymph injection. Virology, 28, 454458.

WHITCOMB,

J.

by of after

R., JENSEN, D. D., AND RICHARDSON, J. 1967. The infection of leafhoppers by Western X-disease virus. III. Salivary, neural, and adipose histopathology. Virology, 31, 539549.

WHITCOMB,

R., JENSEN, D. D., AND RICHARDSON, The infection of leafhoppers virus. IV. Cytopathology in tract. Virology, 34, 69-78.

J. by the

R., JENSEN, D. D., AND RICHARDSON, 196813. The infection of leafhoppers Western X-disease virus. VI. Cytopathological interrelationships. J. fnoertcbrate Pathol., 202-221.

J. by

WHITCOMB,

1968a. X-disease alimentary \VHITCOMB,

12,