PEA ENATION MOSAIC mus ,r‘j * ’ ‘ ' CHARACTERISTICS. ,OF Puma 1, _ j 1 STRAINS DIFFERENTIALLY TRANSMITIED BY THEVECTOR; ;. ACYRTHOSIPHON .PISUM - (HARRIS); , ‘ :_ j_j;_ Thesés for the Regree of Ph. .D. RICHIGAR STATE UNIVERSITY J. VICTOR FRENCH 1973 ,, 4444 LIBRARY Michigan State University This is to certify that the thesis entitled Pea Enation Mosaic Virus: Characteristics of Purified Strains Differentially Transmitted by the Vector, Acxrthosiphon pisum (Harris) presented by J. Victor French has been accepted towards fulfillment of the requirements for PhD Entomology degree in Wage/e 0 Major professor Date W 3 I M 7 3 . 0-7639 g 1 BINDING BY V t . s . _. ‘ ' V \. it ‘1. . ,. I _: “I ‘ . . ¥ ‘ - v' ‘ Experime: membrane-feedi: missible FEW protein yields variants with The pea aphid, FEW pur 0f virus purit feeding assay. ABSTRACT PEA ENATION MOSAIC VIRUS: CHARACTERISTICS OF PURIFIED STRAINS DIFFERENTIALLY TRANSMITTED BY THE VECTOR, ACYRTHOSIPHON PISUM (HARRIS) By J. Victor French Experiments were conducted to identify some factors affecting membrane—feeding assay of purified suspensions of a highly trans- missible PEMV strain and to contrast UV absorption spectra and nucleo— protein yields of partially purified suspensions of PEMV strains and variants with widely different aphid transmission characteristics. The pea aphid, Acyrthosiphon piggm (Harris) was used throughout. PEMV purification procedure, plant source tissue age and degree of virus purity directly influenced aphid transmission via membrane— feeding assay. Transmissibility of a New York strain (NY-PEMV) 'varied with the purification procedure and was dependent on aphid— feeding behavior as well as concentration of virus in suspension. Greatly improved aphid feeding and subsequent virus transmission was achieved by removal of the solvent and/or chelating agent residues (used in certain of the purification procedures) from partially purified PEMV suspensions, either through dialysis or dilution. NY-PEMV partially purified from 6, 10, and 20-day infected pea tissue was transmitted by lst instar pea aphids with 80, 60 and 33% efficiency, respectively, but no transmission was obtained with virus c {- tron 30°clay inie aphid-transmitte sions, even tho = the latter. NucleopIO‘ were found to v and with age of tion. NY—PEMV bility could n ratios. Howex miss’ible (CI-1 on the basis Peas Used f0] mental Chan‘s Wheat and g CT‘FEMV. T‘: with abOut ' J. Victor French from 30-day infected tissues. Highly purified PEMV suspensions were aphid—transmitted with less efficiency than partially purified suspen— sions, even though virus concentrations were higher in the former than the latter. Nucleoprotein yields and sedimenting component ratios of PEMV were found to vary with season of the year when inoculations were made and with age of pea source tissues used for partial virus purifica- tion. NY—PEMV and CALIF-PEMV strains which vary in aphid-transmissi— bility could not be differentiated on the basis of yield or component ratios. However, nonaphid-transmissible (CNT—PEMV) and aphid—trans- missible (CT—PEMV) variants of the CALIF-PEMV strain were separable on the basis of nucleoprotein yield and component ratio when source peas used for partial purification were grown in controlled environ- mental chambers. CNT—PEMV produced higher concentrations of top com- ponent and greater nucleoprotein yields (often 10 X higher) than did CT—PEMV. The 2 sedimenting components of NY-PEMV were aphid—transmitted with about equal efficiency, 84% vs 89%. PEMV was successfully purified from pea aphids and in sufficiently high concentrations to be monitored by sucrose density gradient frac— tionation and UV-spectrophotometric analysis. Electron micrographs showed the particles to be indistinguishable from those isolated from infected plants. Furthermore, virus suspensions were highly trans— missible to pea plants by pea aphids or mechanical means. Virus purified from aphids was established in pea plants by mechanical transmission and compared as to aphid—transmissibility with purified virus from a plant source. The aphid-purified virus source J. Victor French line was transmitted by lst instar pea aphids with significantly (2 < 0.05) higher efficiency than the plant—source virus line after 1— and 4—hour acquisition—access periods and with characteristics superior to those recorded for any PEMV—pea aphid relationships. First instar pea aphids were 95.8% efficient in transmission of the aphid-purified virus source line after only a 4-hour acquisition— access period on infected pea plants and this virus line had a median latent period (LPSO) in the lst instar of only 5.7 hours at 25° C. Other comparable LP estimates on record are 25.0 hours at 20° C. 50 and 14.0 hours at 30° C. PEA ENATION MOSAIC VIRUS: CHARACTERISTICS OF PURIFIED STRAINS DIFFERENTIALLY TRANSMITTED BY THE VECTOR, ACYRTHOSIPHON PISUM (HARRIS) By J. Victor French A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1973 “Ibo ,) R» (:0 ACKNOWLEDGMENTS I wish to express my sincere thanks to Dr. James E. Bath for his encouragement and guidance throughout the course of this research. The enthusiastic giving of his time, ideas, and critical analysis are very gratefully acknowledged. Thanks also to the members of my graduate guidance committee, Drs. James E. Bath, Gordon E. Guyer, Harold D. Newson, Harry H. Murakishi and Gary R. Hooper. The opportunity to associate with and to learn from these knowledgeable scientists has been a most rewarding and invaluable experience. I express my appreciation to departmental chairman, Dr. Gordon E. Guyer, for granting not only financial assistance but also for pro- viding excellent laboratory and greenhouse facilities and equipment used in this research program. I extend thanks also to Dr. Paul R. Desjardins, University of California-Riverside, a very able and knowledgeable teacher, researcher and friend from whom I learned many of the virological techniques used in my research. Special thanks to my dear wife, Lee Ann, whose patience, under- standing and support made the purSuance of this doctoral degree possible. ii TABLE OF CONTENTS Page GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 4 PART I: APHID-TRANSMISSIBILITY OF PEA ENATION MOSAIC VIRUS PREPARED BY VARIOUS PURIFICATION REGIMES INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 ’MATERIALSANDMETHODS...................... 8 Virus purification . . . . . . . . . . . . . . . . . . 8 Analysis of purified preparations . . . . . . . . . . . . . . . 10 Bioassay of preparations . . . . . . . . . . . . . . . . . 10 RESULTS . . . . . . 11 Transmissibility of partially purified virus prepared by various differential centrifugation methods . . . . . . . ll Transmissibility of virus purified by rate zonal density gradient centrifugation . . . . . . . . 16 Transmissibility of partially purified virus obtained from source tissue of various ages . . . . . . . . . . . 19 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 25 PART II: CHARACTERIZATION OF NUCLEOPROTEIN COMPONENTS OF STRAINS 0F PEA ENATION MOSAIC VIRUS THAT DIFFER IN APHID-TRANSMISSIBILITY INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 28 Virus strains and variants . . . . . . . . . . . . . . 28 Aphid culturing and transmission bioassay . . . . . . . . . . . 29 Virus purification . . . . . . . . . . . . . . . . . . . . . . 29 iii Page RESULTS . . . . . . . . . . 30 Influence of tissue age on nucleoprotein yields and sedimenting components of NY- and CALIF-PEMV strains . . . . 30 Seasonal influence on nucleoprotein yield and sedimenting components of NY—PEMV . . . . . . . 34 Characterization of nucleoprotein yield and sedimenting component ratios of aphid- -transmissible and nonaphid- transmissible PEMV variants . . . . . . . . . . . 38 Aphid-transmissibility of separated sedimenting components of NY-PEMV . . . . . . . . . . . . . 42 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 54 PART III: PURIFICATION OF PEA ENATION MOSAIC VIRUS FROM ITS VECTOR, ACYRTHOSIPHON PISUM (HARRIS) AND APHID—TRANSMISSION CHARACTERISTICS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 57 Virus purification . . . . . . . . . . . . . . . . . . 58 Density gradient centrifugation . . . . . . . . . . . . . . . . 58 Electron microscopy . . . . . . . . . . . . . . . . . . . . . . 59 Infectivity assay . . . . . . . . . . . . . . . . . . . . . . . 59 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Preliminary tests . . . . . . . . 60 Infectivity of PEMV partially purified by chloroform— butanol technique . . . . . . . . . . 60 Frozen viruliferous aphids as a virus source . . . . . . . . . 62 Infectivity and UV analysis of density gradient- purified virus . . . . . . . . . . . . . . . . 63 Electron microscopy . . . . . . 64 Comparative transmissibility of virus after consecutive aphid- to-plant or plant- to-plant transfer . . . . . . . . . . 66 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 74 iv Table 10. LIST OF TABLES Legend to MEthods Used to Obtain Partially Purified Pea Enation Mbsaic Virus by Differential Centrifu- gation for Comparative Transmission Trials Pea Aphid (lst—Instar) Transmission of Pea Enation Mosaic Virus Partially Purified by Various Methods Pea Aphid (lst—Instar) Transmission of Pea Enation Mbsaic Virus Partially Purified by Modified Methods II, III and IV . . . . . . . Comparative Transmission of Pea Enation Mosaic Virus as Partially Purified and Density Gradient Purified Suspensions Aphid- and Mechanical-Transmissibility of Pea Enation Mosaic Virus Partially Purified from Source Plants of Varying Ages . . . . . . . . Influence of Tissue Age on Nucleoprotein Yield of Two Strains of Pea Enation Mosaic Virus . . . Influence of Seasonal Variation on Yield of NY—PEMV from P. sativum L. Grown Under Greenhouse Conditions Ratio of Top/Bottom Sedimenting Components of CNT— and CT— PEMV Variants Differentially Resuspended in Potassium Phosphate Buffer with and without 5% Sucrose . . . . . . . . . Comparative Yields of CNT—PEMV and CT—PEMV Purified at 3 Different Times from Infected Tissues of P. sativum L. grown in a Controlled Environmental Growth Chamber . . . . . . . Transmission of Sedimenting Components of PEMV Separated by Sucrose Density Gradient Centrifugation (DGC) and Acquired Through an Artificial Membrane by First-Stage Acyrthosiphon pisum . . . . . . Page l3 l6 18 21 33 36 41 42 . 46 Table Page 11. Infectivity of Successive Aqueous Fractions Obtained by Repeated Chloroform-Butanol Emulsification of Viruliferous Pea Aphids . . . . . . . . . . . . . . . . . 62 12. Aphid-Transmission of Partially Purified PEMV Prepared from Fresh or Frozen Viruliferous Pea Aphids . . . . . . . . . . . . . . . . . . . . . . . 64 13. Aphid-Transmission Characteristics of PEMV Lines after Consecutive Aphid-to-Plant or Plant—to- Plant Transfers . . . . . . . . . . . . . . . . . . . . . 69 vi LIST OF FIGURES Figure Page 1. UV Scanning Profiles at 254 nm of Sucrose Gradients (10— 40%) Layered with NY— or CALIF-PEMV Preparations Partially Purified from Infected Peas 10, 15, and 20 Days after Mechanical Inoculation . . . . . . . 32 2. UV Scanning at 254 nm of Sucrose Gradients (10—40%) Layered with NY- PEMV Partially Purified at Various Seasonal Periods . . . . . . . . . . . 35 3. UV Scanning Profiles at 254 nm of Sucrose Gradients (10-40%) Layered with CNT- or CT—PEMV . . . . . . . . . . 40 4. UV Scanning Profiles at 254 nm of Sucrose Gradients Layered with NY—PEMV . . . . . . . . . . . . . . . . . . 44 5. UV Scanning Profile at 254 nm of Preparations of Non-Viruliferous and Pea Enation Mosaic Virus- Infested Pea Aphids . . . . . . . . . . . . . . . . . . . 65 6. Electron Micrographs of Negatively-Stained Preparations of DGC-Purified Suspensions Obtained from PEMV- Carrying Pea Aphids . . . . . . . . . . 67 7. Scanning Pattern at 254 nm of Fractions from Preparations of Peas Infected with an Aphid Line and Peas Infected with a Plant Line . . . . . . . . 71 vii GENERAL INTRODUCTION Most plant viruses are dependent on arthropod or nematode vectors for dissemination and inoculation in nature. But in general only a relatively few viruses are transmitted by a given vector species and only a limited number of vector species transmit a given virus. While the degree of this specificity varies between viruses and vectors, it is most pronounced in the circulative aphid— and leafhopper-borne viruses. Not only is vector-virus specificity present between viruses and vector species but also between virus strains and vector biotypes or races. An understanding of the mechanism which controls vector-virus specificity should enable development of virus disease control procedures that are founded on breaking the compatibility between vector and virus rather than destroying the vector. The literature on vector-virus specificity has been reviewed (Rochow, 1963, 1969). The aphid-borne (circulative type) pea enation mosaic virus (PEMV) is well suited for studies of vector-virus specificity since its aphid-transmission characteristics are well defined (see review by Harris, 1971); it consists of at least 2 strains that are trans— mitted with widely different efficiencies by a common pea aphid bio- type (Bath and Tsai, 1969); a single strain can be transmitted with markedly different efficiencies by various pea aphid biotypes (Bath and Chapman, 1967; Tsai g£_al., 1972); and it can be perpetuated by l mechanical, as well as aphid means. The chemical and physical prop- erties of the virus have been extensively characterized (Bozarth and Chow, 1966; Gibbs g£.21., 1966; Izadpanah and Shepherd, 1966; Shepherd g£_§l., 1968; Musil £5 31., 1970; Gonsalves and Shepherd, 1972), and some information on the relationship of the virus to pea aphid vector and host plant tissues is available (Shikata, ££.§l-: 1966; Shikata and Maramorosch, 1966; de Zoeten, gt al., 1972; Harris and Bath, 1972). To date, research on vector—virus specificity within the pea enation mosaic virus and aphid-vector populations has centered on (a) definition of the transmission (plant-to—plant) characteristics of various strains and isolates by various vector species and biotypes (Bath and Chapman, 1966, 1967; Chapman and Bath, 1968; Bath and Tsai, 1969; Thottappilly E£.El'9 1972; Tsai gt al., 1972) and (b) elucidation of the relationship of a highly transmissible PEMV strain (NY) to the tissues of a most efficient pea aphid biotype (Harris and Bath, 1972). Future advances in this mission-oriented research program will un- doubtedly be achieved through studies of the fate of various PEMV strains and isolates in various vector biotypes and the relationship of viral and vector composition to transmission efficiencies. Success in both of these approaches is largely dependent on the use of purified virus preparations from both the infected plant and the infested (or infected?) aphid and on systems for assaying infectivity and trans- missibility of various experimentally manipulated virus preparations. Recently, the assay problem was alleviated by development of a technique whereby virus suspensions are injected into the vector's hemocoel and an artificial membrane-feeding technique through which virus suspensions are fed to aphids. Both techniques were shown to I‘llllllllll 3 be very suitable for PEMV—pea aphid studies (Thottappilly E£.El-: 1972; Clarke and Bath, 1973). My objectives were to: (a) characterize the nucleoprotein components and yields of PEMV strains and variants of widely different aphid-transmissibilities; (b) determine the suitability of several purification methods for the preparation of virus for membrane—feeding assay; and (c) develop a regime for purification of PEMV from infested (infected?) pea aphids and contrast aphid—transmissibility of virus purified from plant and vector sources. LITERATURE CITED Bath, J. E. and R. K. Chapman. 1966. Efficiency of three aphid species in the transmission of pea enation mosaic virus. J. Econ. Entomol. 59:631-634. Bath, J. E. and R. K. Chapman. 1967. Differential transmission of two pea enation mosaic virus isolates by the pea aphid, Acyrthosiphon pisum (Harris). Virology 33:503-506. Bath, J. E. and J. H. Tsai. 1969. The use of aphids to separate two strains of pea enation mosaic virus. Phytopathol. 59:1377—1380. Bozarth, R. F. and C. C. Chow. 1966. Pea enation mosaic virus: purification and properties. Contrib. Boyce Thompson Inst. 23:301-309. Chapman, R. K., and J. E. Bath. 1968. The latent period of pea enation mosaic virus in three of its aphid vectors with emphasis on adult versus nymph comparisons. Phytopathology 58:494-499. Clark, R. G., and J. E. Bath. 1973. Transmission of pea enation mosaic virus by the pea aphid Acyrthosiphon pisum following virus acquisition by injection. Ann. Entomol. Soc. Amer. 66: in press. de Zoeten, G. A., G. Gaard, and F. B. Diez. 1972. Nuclear vesicula- tion associated with pea enation mosaic virus-infected plant tissue. Virology 48:638-647. Gibbs, A. J., B. D. Harrison and R. D. Woods. 1966. Purification of pea enation mosaic virus. Virology 29:348-351. Gonsalves, D., and R. J. Shepherd. 1972. Biological and physical properties of the two nucleoprotein components of pea enation mosaic virus and their associated nucleic acids. Virology 48:709-723. Harris, K. F. 1971. The fate of pea enation mosaic virus in its pea aphid vector, Acyrthosiphon pisum (Harris). Ph.D. Thesis, Michigan State University, 139 p. Harris, K. F., and J. E. Bath. 1972. The fate of pea enation mosaic virus in its vector, Acyrthosiphon pisum (Harris). Virology 50:778-790. Izadpanah, K., and R. J. Shepherd. 1966. Purification and properties of the pea enation mosaic virus. Virology 28:463—476. Musil, M., K. Marcinka and F. Clampor. 1970. Some properties of pea enation mosaic virus. Acta. Virol. 14:285—294. Rochow, W. F. 1963. Variation within and among aphid vectors of plant viruses. Ann. N. Y. Acad. Sci. 105:713—729. Rochow, W. F. 1969. Specificity in aphid transmission of a circula- tive plant virus, p. 175-198. In Karl Maramorosch (ed.) Vectors, Viruses and Vegetation. Wiley (Interscience), New York. Shepherd, R. J., R. J. Wakeman and S. A. Ghabrial. 1968. Preparation and properties of protein and nucleic acid components of pea enation mosaic virus. Virology 35:255-267. Shikata, E., K. Maramorosch, and R. R. Granados. 1966. Electron microscopy of pea enation mosaic virus in plants and aphid vectors. Virology 29:426—436. Shikata, E., and K. Maramorosch. 1966. Electron microscopy of pea enation mosaic virus in plant cell nuclei. Virology 30:439—454. Thottappilly, G., J. E. Bath, and J. V. French. 1972. Aphid trans- mission characteristics of pea enation mosaic virus acquired from a membrane-feeding system. Virology 50:681—689. Thottappilly, G., J. E. Bath, and E. C. Igbokwe. 1972. Differential aphid transmission of two bean yellow mosaic virus strains and comparative transmission by biotype and stages of the pea aphid. Ann. Entomol. Soc. Amer. 65:912-915. Tsai, J. H., J. E. Bath, and E. C. Igbokwe. 1972. Biological and transmission characteristics of Acyrthosiphon pisum biotypes efficient and inefficient as vectors of pea enation mosaic virus. Ann. Entomol. Soc. Amer. 65:1114—1119. PART I: APHID—TRANSMISSIBILITY OF PEA ENATION MOSAIC VIRUS PREPARED BY VARIOUS PURIFICATION REGIMES INTRODUCTION Previously we showed that partially purified suspensions of the aphid—borne (circulative type) pea enation mosaic virus (PEMV) can be assayed for transmissibility by feeding aphids on virus suspensions across an artificial membrane (Thottappilly 35 21., 1972). Membrane- feeding of virus to test insects has much utility in the study of fundamental vector-virus relationships since the concentration of the virus in the source solution can be controlled and virus acquired during membrane—feeding presumably undergoes the same biological cycle in the vector as virus acquired from infected plants. However, the virus suspension used for membrane-feeding must be relatively free of chemicals which inhibit aphid feeding and thereby virus acquisition. During our initial study (Thottappilly E£.fll-’ 1972) in which we used a modification of the method reported by Bozarth and Chow (1966) to obtain partially purified virus that was efficiently trans- mitted by aphids after membrane—feeding, we occasionally and super- ficially tested the transmissibility, by membrane-feeding, of virus obtained through other purification procedures. Limited results indicated that virus suspensions yielded by various purification procedures varied considerably in suitability for use in membrane— feeding studies. In the study described herein, our objectives were to (a) com- pare directly the suitability of several procedures for preparing 7 8 partially purified PEMV suspensions that were both relatively free of nonviral contamination and transmissible by membrane-feeding assay; (b) test the transmissibility, by membrane-feeding, of virus purified by rate zonal density gradient centrifugation; and (c) determine the influence of source tissue age on assay of partially purified virus by membrane-feeding. MATERIALS AND METHODS Virus purification.——A highly aphid—transmissible isolate of NY PEMV (Bath and Tsai, 1969) was used exclusively; it was maintained in garden pea (Pigum sativum L. 'Midfreezer') and perpetuated by twice-monthly mechanical transfers of expressed sap from diseased plants. Virus source plants were obtained by mechanically inoculating young 2, sativum seedlings prior to time of 1st-leaf expansion. Unless otherwise noted, tissue for virus purification was harvested 10—12 days after inoculation; only tissue from plants with severe symptoms was used. Five methods were used to partially purify PEMV from its host tissues; these methods are contrasted in Table 1 and will hereafter be referred to by the designated Roman numeral. Methods I-IV were originally designed for PEMV purification and were followed precisely in our work. Method V was designed for purification of tobacco ring— spot virus and was modified as follows: 0.1 M, pH 7.0 potassium phosphate buffer with 52 sucrose was added (1:1 v/v) to the chloroform— butanol extraction solvent; the number of ultracentrifugations was .illllll 9 TABLE 1. Legend to methods used to obtain partially purified pea enation mosaic virus by differential centrifugation for compara— tive transmission trials Buffer used for virus extraction Reference for Method Clarification and Dialysis details no. ‘ solvent resuspension used? of method I Chloroform 0.05 g, pH 6.0 Yes Bozarth & Chow potassium (1966) phosphate II Chloroform 0.1 g, pH 6.0— Yes Thottappilly 7.0b potassium £2.21' (1972) phosphate with 5% sucrose III Chloroform a 0.03—0.08 if, pH No Gibbs, e_t a_1. +0.05 g EDTA 7.5 potassium (1966) phosphate IV None 0.1 g, pH 6.0 No Izadapanah & sodium acetate Shepherd (1966) V Chloroform & 0.1 g, pH 7.0 Yes Steere (1956)d Butanol (1:1) potassium phosphate with 52 sucrose aEDTA - ethylenediaminetetraaceticacid. pr 6.0 in extraction buffer and pH 7.0 in resuspension buffer. c0.08 ! used for extraction, 0.03 g used for resuspension. duodified from the original by (1) adding 0.1 5, pH 7.0 potassium phosphate buffer with 5% sucrose to the chloroform—butanol extraction solvent (v/v); (2) reducing the number of ultracentri- fugations to 2; (3) including a 48—hr dialysis of the lst lowspeed supernatant against 0.05 g, pH 7.0 potassium phosphate buffer; and (4) resuspending each pellet in 0.1 g, pH 7.0 potassium phosphate buffer with 52 sucrose. 10 reduced to 2; and a 48—hr dialysis of the lowspeed supernatant against 0.05 5, pH 7.0 potassium phosphate buffer was included. These methods varied mainly in the type, molarity, and pH of the buffers used in the virus extraction and resuspension phases of purification, and in the types of solvents and chelating agents used for virus extraction (Table 1). Analysis of pgrified preparations.——Rate—zonal density gradient analysis was performed by layering one-half to 1 ml of each virus preparation (either undiluted or adjusted to A, m = 2) on linear 10- 260 n 40% sucrose (in 0.02_§, pH 7.0 potassium phosphate buffer) columns, centrifuging the columns for 2 hr at 24,000 rpm in a SW25.2 rotor of the model L Beckman ultracentrifuge, and monitoring the columns for UV absorbance (at 254 nm) with an 1800 density gradient fractionator and UV analyzer. Virus concentrations, were determined through planimetry with viral absorbance peaks and conversion of areas to weight of virus per unit volume. Bioassay of preparations.—-The EL biotype (Tsai st 31., 1972) of the pea aphid, Acyrthosiphon piggm (Harris), was used to test aphid- transmissibility of the various virus preparations. Aphids were reared on El£1§.£2§£ L. First—stage nymphs were used exclusively and were obtained by transferring viviparous adults to X. fgba plants for a 12-18 hr nymph-deposition period. Aphids acquired virus by membrane— feeding for specific periods on 0.2 ml of the virus preparation which was adjusted with appropriate buffer to a specific 5260 nm concentration. The details of membrane-feeding were described earlier (Thottappilly £§‘§;., 1972). After completion of membrane—feeding, aphids were A r x 11 transferred singly to very young g. sativum 'Midfreezer' seedlings for inoculation-access periods of 3-4 days. Virus preparations also were mechanically inoculated to Mid- freezer pea (systemic host) and to Chenopodium amaranticolor Coste and Reyn. (local lesion host) with the aid of glass spatulas and carborundum. The former plants were in the pre-leaf stage and the latter in the 4—5 leaf stage. RESULTS Transmissibility of partially purified virus prepared by various differential centrifugation methods.—-In the first of 2 experiments, partially purified virus suspensions were obtained through each of 5 procedures (Table 1). Each purification procedure was initiated on the same day with 40—50 g of infected tissue and was completed within 3 days. Most of the virus pellets obtained by each method were re- suspended in the same buffer used for virus extraction except for minor changes in pH (Method II) and molarity (Method III); some pallets obtained by Methods 1, III and IV were resuspended in 0.1 M, pH 7.0 potassium phosphate buffer with 5% sucrose. Each suspension was held at 4 C until it was bioassayed. On the 4th day, 2 portions of each suspension were fed to aphids during a 4-hr acquisition-access period; one portion was adjusted with the appropriate buffer to £260 nm - 7.5, the other portion was not adjusted. Each portion was fed to 30 aphids in a single feeding—chamber (trial 1). Identical portions were mechanically inoculated to 10 pea seedlings and to the terminal 2 fully—expanded leaves on each of 2 g, amaranticolor plants. 12 The unused portions of each suspension were frozen. 0n the 5th day, a portion of each frozen virus suspension was thawed, and the aphid— transmission test was repeated (trial 2). On the 6th day, 0.5 m1 of each suspension was thawed, adjusted toA260 nm = 2 and subjected to rate—zonal density gradient analysis. The various virus suspensions varied considerably in absorbance at 260 nm, aphid-transmissibility, and weight of virus in suspension (Table 2). Variation in transmission rate between trials and between the adjusted and unadjusted suspensions was extraordinarily low, even though the latter suspensions usually contained much less virus than the former. Transmissibility appeared to be regulated by aphid-feeding behavior and the quantity and quality of the virus in suspension. Aphids hesitated to feed on the 0.03 M, pH 7.5 potassium phosphate suspension of virus prepared by Method III and on both suspensions obtained from Method IV; but aphids appeared to feed better on the £260 = 7.5 preparation of each suspension than on the unadjusted por- tion. Since virus from each of these 3 suspensions was transmitted with poorer efficiency (in relation to virus concentration in test suspension) than virus from all other suspensions, reduced ingestion rate was considered to be primarily responsible for poor transmission. Virus obtained by Method I and suspended in 0.05 M, pH 6.0 potassium phosphate buffer was transmitted with higher efficiency than all others tested, even though ané26 - 7.5 suspension contained only 0 18 ug of virus per m1——an amount exceeded by all other 5260 = 7.5 suspensions tested except that of Method I suspended in 0.1 M, pH 7.0 phosphate buffer with 5% sucrose (Table 2). Method II, while quite similar to Method I, yielded more virus per an £260 = 7.5 suspension, 13 .1- .msOHmcommam woumahwmss How wwsHvuou O0Nfi n mHmonusouoa aw .ozn .mzmu o>Hu=ummsoo so vouusvaoo mums N can H mHmHuu “mousom use use: whoa mVHnmm on "muemHm umou hauHuon ou szeHm wouuowmemuu use 0000 mmooom|=0HuHmH=vom sale a su>Hw onus mpHsnsm omouusm N0 nuHB oumnnmosm mm A00 00 n.0m m.m~ m.m~ n.0u asHmmouom o.n an «m H.o > omouosm N0 :uHa ouannmonm eHN AoHV men 0.0m 0.00 H.¢N 0.00 asHmmmuom 0.5 ma .m.H.o ouauoou mmm “HHV ohm 0.m o o o aaHvom 0.0 no .m.H.o >H omouusm N0 zuHs oumanmonm mm AmMHv n00 ¢.n0 5.0n o.m0 5.00 ssHmmmuom o.“ no .m.H.o oumanmona 2: God 82 53 new 9.3 T? 3:333 mg mm .m 86 SH owouosm um :uHa oumsnmonn m: 83 :3 TE 52 Ham 98 33333 or mm .m To Z omonosm N0 £uH3 oumnnmosn 0H Amwv «QH «.05 0.05 N.O0 0.05 ssHmmmuon o.n mm .m.H.o uuunmmonm wH ANOV mmH n.0m 5.0m H.mm m.ma ssHmmmuon o.0 ma .m.mo.o H m.n n 00mfl. Aboumsfivmsb N HmHuH H HmHuH N HmHuH H HmHHH uuHHom mauH> HmeHu .oe usunmsnou ou venue: msOHmaonmsm m.n u so o0mfl. woumsnwmub vows nomwsm voHMHowmm aH nHE\w:V m=HH> mo oOHumuusoonoo mmGOHmsonmsm usuH> vonHoomm no GOHmmHamnuuH N avenues msoHue> ha voHMHusd mHHaHuumA msuH> onmos GOHuucm use we eonmHamsuuu AumumeHlumHv annm mom .N mqn FIGURE 1. UV scanning profiles at 254 nm of sucrose gradients (lo-40%) layered with NY- or CALIF-PEMV preparations partially purified from infected peas 10, 15, and 20 days (1, 2, 3, respectively) after mechanical inoculation. Density gradient centrifugation was for 2 hrs at 24,000 rpm in the Spinco SW 25.2 rotor. 33 purified from lS-day-infected tissues was not discrete on the scanning profiles and appeared only as a shoulder on the ascending slope of the bottom component peak. The nucleoprotein yield of NY- and CALIF-PEMV decreased with increasing age of infected tissue used for partial purification (Table 6). Yield of NY-PEMV decreased approximately five-fold between tissues used for purification 10 and 15 days after inoculation, but yield was essentially the same from both 15- and ZO-day-infected tissues. Yields of CALIF-PEMV from 10- and 20—day-infected tissues were lower than the same age tissues infected with NY-PEMV; however, CALIF-PEMV yield was higher than that of NY—PEMV when both were partially purified from 15—day—infected tissues. Apparently the titre of NY-PEMV in plants declines with age of tissue at a faster rate than does the CALIF—PEMV strain. TABLE 6. Influence of tissue age on nucleoprotein yield of two strains of pea enation mosaic virus Yield (pg/g) of virus obtained from infected pea tissue at specified days after inoculation PEMV Strain 10 days 15 days 20 days NY 274.33 54.02 53.07 CALIF 183.11 87.22 12.29 aVirus was purified by the method of Thottappilly gp_§l, (1972). Peas used for purification were mechanically inoculated and grown under greenhouse conditions during November, 1970. 4.. .31.;33 a a. an..." 34 Seasonal influence on nucleoprotein yield and sedimenting components of NY—PEMV.--Beginning in September of 1970, lO-day NY- PEMV-infected pea tissues were harvested at about 2-month intervals for about 1 year. These tissues were sources for partial purifica- tion of virus and only tissues showing very severe symptoms were used. At each purification date nucleoprotein yield was computed, and UV scanning profiles were made of density gradient columns containing ultracentrifuged virus. Ultraviolet scanning profiles of centrifuged density gradient columns layered with partially purified NY—PEMV varied considerably with different seasons of the year (Figure 2). All profiles clearly exhibited the predominant 113Slbottom component, but the presence (in varying proportions) or complete absence of the 9481 top component was evidenced at certain seasonal periods of the year. No top com— ponent was recovered in purifications made in September of 1970, or in June and July of 1971, and only minimal but detectable amounts of top component were obtained in a purification made in August, 1971 (Figure 2). In those purifications of NY-PEMV made during the fall, winter and spring seasonal periods, the top component was readily apparent both as a visible zone in gradient columns and on UV scanning profiles. The highest level of top component was attained in virus preparations from tissues harvested and processed in March of 1971 (Figure 2), and occurred during a period when the total yield of extracted NY-PEMV 1Sedimentation coefficients for top and bottom nucleoprotein components of PEMV as derived by Bozarth and Chow (1966) and Musil 35 11. (1970). 35 9-2-70 11-30-70 L1 1-28-71 RELATIVE ABSORBANCE AT 254 nm 3-23-71 RELNHVE DEPTH FIGURE 2. 5‘13'71 Gw18471 7‘9‘71 8-2-71 9-23-71 IN TUBE -—-4> UV scanning at 254 nm of sucrose gradients (lo—40%) layered with NY-PEMV partially purified at various seasonal periods from lO-day infected peas. Density gradient centrifugation was for 2 hrs at 24,000 rpm in the Spinco SW 25.2 rotor. 36 was at or near its lowest level — 9.64 micrograms virus/gram of infected tissue (Table 7). TABLE 7. Influence of seasonal variation on yield of NY-PEMV from P. sativum L. grown under greenhouse conditions Purification Mean Yield (pg/g) of date temperature virus 9-2-70 82.6 36.83 11-30-70 39.7 274.33 1-28-71 28.5 56.60 3-23-71 41.3 9.61 5-13-71 68.9 8.19 6-18-71 81.3 36.28 7-9-71 82.4 39.78 8-2-71 79.5 22.85 9-23-71 71.4 160.06 aMean temperatures in East Lansing were computed for that period from time pea seedlings were inoculated and held in greenhouse to date of purification, or total of 10 days. Data obtained from U.S. Department of Commerce. bVirus was purified by the method of Thottappilly gp'al. (1972) from infected pea 10 days after mechanical inoculation. Virus symptoms on pea seedlings infected by NY—PEMV between June to September and December to March were not as severe as during other periods of the year. During those periods of mild symptoms, infected seedlings were not severely stunted and the active-growing portions of these plants often did not show PEMV symptoms; furthermore, very few enations (neoplasms) were evident. The most severe symptoms 37 were observed on seedlings inoculated and used as tissue sources for purification in the spring and fall. These seedlings, while showing the usual symptoms of PEMV infection were severely stunted and all leaves showed chlorotic flecking (windows). Symptom severity could not be used as a reliable visual means for estimating NY-PEMV yield, since the highest and lowest recorded yields were obtained at dif— :3] ferent periods of the year (Table 7), but the infected pea seedlings E used for partial purification in each case showed similar severity of symptoms. :4 Failure to recover NY-PEMV top component appeared to coincide with mild symptom expression on infected pea seedlings and occurred at periods in which higher light intensities and elevated temperatures were most likely to occur in the greenhouse. Although internal green- house temperatures were not monitored during plant incubation periods, outside temperatures (available for East Lansing from U.S. Dept. of Commerce) have a direct bearing on the internal greenhouse temperatures. Greenhouse coolers and shading compound on the glass are inadequate to hold temperatures much below 80° F when outside temperatures reach into the 80's on sunny days. Often greenhouse temperatures reach 90—100°, even though the outside temperature is at or near 80°. Through the use of climatological data, it was possible to compute the mean temperatures for the 10 days prior to each purifica- tion date (Table 7). It is of particular interest to note that in each of those purifications in which no top component was recovered (September 1970, and June, July, 1971, Figure 2), the mean temperature exceeded 80° F. This was opposed to an average temperature of 41.3° F recorded in March, 1971, when the highest level of top component was 38 recorded in any one purification. While an average temperature was computed for each lO-day period, temperatures often deviated 10 or more degrees from the mean. In each of those purification periods with a mean above 80° F, there were specific days in which temperatures approached or exceeded 90° F. Since greenhouse cooling systems and supplementary shading are often not adequate to control high light intensities and high temperature fluctuations, they could be particu- larly detrimental to pea seedling growth and, therefore, a limiting factor in virus production. Characterization of nucleoprotein yield and sedimenting component ratios of aphid-transmissible and nonaphid-transmissible PEMV variants.-- The aphid-transmissible (CT-PEMV) and nonaphid-transmissible (CNT- PEMV) variants of CALIF—PEMV were partially purified on several occasions from infected peas which were incubated in the greenhouse and compared by rate—zonal density gradient analysis. In each case source plants of CT-PEMV were obtained through aphid transfers of virus and CNT—PEMV by mechanical transfers of sap from infected tissue; each purification trial included both variants. Analyses were inconsistent between purification trials; however, top/bottom component ratio data tended to show ONT-PEMV to contain higher ratios of top component than did CT-PEMV. Three additional purification comparisons were made of CNT— and CT—PEMV, but to restrict inter-trial variability, source plants (inoculated as before) of each variant were incubated in a controlled environmental chamber at 23 i l C, ca 50% R.H., and ca lSOO-ft-candles, with a 12-hr photophase. Furthermore, the purification procedure was 39 modified to insure maximum nucleoprotein recovery by the addition of a reextraction of the sedimenting plant debris pellet from the first low-speed centrifugation. Since Bozarth and Chow (1968) showed that sucrose in buffer solutions, used for resuspension of the final high speed PEMV pellets, affected the yield and component ratios, I also tested its effect on TE? both variants by resuspending pellets in 0.1 M potassium phosphate buffer (pH 7.0) with and without 5% sucrose. Component ratios were computed from planimetry measurements of the UV-scanning profiles of top and bottom components of the 2 variants. Comparative UV-scanning profiles of gradient columns, layered with PEMV preparations from repeated purifications of the 2 variants consistently demonstrated ONT—PEMV to contain much greater proportions of top component than did CT—PEMV. When CNT-PEMV pelleted by ultra- centrifugation was resuspended in potassium phosphate buffer containing no sucrose the resultant preparation had a higher level of top than bottom component (Figure 3). The predominance of top component was also shown by a top/bottom ratio of less than 1 (Table 8). However, when the resuspending phosphate buffer contained 5% sucrose signifi- cantly higher proportions of bottom component were obtained and top/ bottom ratios varied from 2.8 to 4.4 for the 3 purification trials. If final high speed pellets of CT—PEMV were treated in a similar manner the level of top component was not appreciably changed by the presence or absence of sucrose in the resuspending buffer (Figure 3). Bottom component was always extracted in much higher proportions than top component; this was shown by top/bottom component ratios often exceeding 5.0 (Table 8). Like CNT-PEMV the amount of CT—PEMV bottom ONT-PEMV + sucrose I ONT-PEMV no sucrose E CT- PE MV LSUCIOSG CT- PEMV no sucrose RELATIVE ABSORBANCE AT 254 nm é RELATIVE DEPTH —-> FIGURE 3. UV scanning profiles at 254 nm of sucrose gradients (IO-40%) layered with CNT- or CT-PEMV partially purified and resuspended in potassium phosphate buffer either with or without 5% sucrose. Centrifugation was for 2 hrs at 24,000 rpm in the Spinco SW 27.1 rotor. 41 component could be further increased by resuspension of virus pellets in phosphate buffer containing 5% sucrose. TABLE 8. Ratio of top/bottom sedimenting components of CNT- and CT-PEMV variants differentially resuspended in potassium phosphate buffer with and without 5% sucrose3 Component ratios for purification trialsc Virus pellets Variant resuspended in l 2 3 CNT bufferb 0.60 —---d 0.59 CNT buffer + sucrose 2.81 4.40 3.00 CT buffer 5.83 ----° 5.00 CT buffer + sucrose 3.33 -—--d 5.33 aCNT—PEMV was mechanically inoculated to pea, and CT-PEMV was aphid inoculated to pea. Tissues used 10 days later for partial purification by method of Thottappilly 3; pl. (1972). b0.1 M potassium phosphate buffer, pH 7.0. cComponent ratios determined from planimetry measurements of UV-scanning profiles of sucrose gradients layered with partially purified CNT- or CT-PEMV preparations from 3 separate purification trials. dComponent ratios could not be determined because top component was unresolved from bottom component on UV-scanning profiles. Comparative nucleoprotein yield data from the 3 trials showed that consistently higher yields were obtained from plants infected with CNT—PEMV than from those infected with CT-PEMV (Table 9). In some cases the yield of CNT-PEMV was lO-fold higher than CT-PEMV. Nucleoprotein yields of both CT- and CNT-PEMV at the time of each purification was also substantially increased by resuspending final high speed virus pellets in potassium phosphate buffer containing 42 5% sucrose; in some instances the yield was 3 times greater than when the virus was resuspended in phosphate buffer having no sucrose (Table 9). TABLE 9. Comparative yields of CNT—PEMV and CT—PEMV purified at 3 different times from infected tissues of P, sativum L. grown in a controlled environmental growth chambera Yields (ug/g) when partially purified virus is resuspended inb Purification PEMV b b trial variant Buffer Buffer + 5% sucrose 1 CNT 84.4 244.4 CT 31.2 42.8 2 CNT 168.0 284.7 CT 14.4 44.8 3 CNT 122.2 180.6 CT 32.4 54.0 aCNT-PEMV was mechanically inoculated to pea, and CT—PEMV was aphid inoculated to pea. Tissues used 10 days later for partial purification by method of Thottappilly gp_§l. (1972). b0.1 M potassium phosphate buffer, pH 7.0. While the nucleoprotein yield from pea tissues infected with CNT-PEMV was substantially higher than from CT—PEMV—infected tissues, there was not a corresponding difference observed in symptom severity between the two variants. Independent of the variant used, symptoms on infected pea seedlings grown under the regulated environmental con- ditions of the growth chamber were always extremely severe. Aphid-transmissibilityyof separated sedimentinggcomppnents of NY—PEMV.-—Since there has been some controversy over the infectivity of PEMV top component (Bozarth and Chow, 1966; and Izadpanah and 43 Shepherd, 1966), it was decided to test both components (separate and mixed) for their aphid-transmissibility. In one experiment partially purified NY-PEMV was layered on each of 6 gradients and after centri- fugation 4 fractions were collected from each tube. These fractions as shown by the UV scanning profile (Figure 4A) correspond to top, top and bottom mixture, bottom and bottom aggregate component mixture. Care was taken to collect individual fractions from the gradient in the indi- cated areas of the scanning profile. Like PEMV fractions from each tube were combined, dialyzed 48 hours against 0.02 M potassium phosphate buffer pH 7.0 and virus pelleted from solution by ultra-centrifugation. Viral pellets were resuspended in the same dialysis buffer but with 5% sucrose added. The 4 PEMV suspensions were then adjusted to equal con- centrations of A_ 2 artificial membrane. After a 24-hr acquisition-access feeding period 60 - 2.0 and fed to first-stage pea aphids across an (AAP) aphids were transferred singly to healthy pea seedlings for a 3— day inoculation-access feeding period (IAP). Twenty-three to 30 aphids were used per treatment. The 4 suspensions also were each mechanically rubbed on 4-6 pea seedlings for systemic infectivity bioassay. In a second experiment NY-PEMV was again partially purified and subjected to sucrose density gradient centrifugation. However, this aphid feeding trial differed from the first in that fractions of 10 drops each were collected from the sucrose gradient corresponding to top, bottom and aggregated bottom components (Figure 4B). These fractions were fed to the aphids directly from the gradient without the recycling steps of dialysis, ultracentrifugation and concentration determination and adjustment. First-stage aphids were given only a 4-hr AAP on the component fractions followed by a 3—day IAP on healthy RELATIVE ABSORBANCE AT 254 nm RELATIVE DEPTH IN TUBE-——> FIGURE 4. UV scanning profiles at 254 nm of sucrose gradients layered with NY-PEMV. Arrows in (A) correspond to fractions containing virus components which were collected from the gradients, dialyzed and reconcentrated by ultra-centrifugation and membrane—fed to let- instar aphids. Arrows in (B) correspond to 3 virus component fractions collected from the gradient and without reconcentration membrane-fed Vdirectly to lst—instar aphids. 45 peas. Thirty-five to 45 aphids were used per treatment. Component fractions were each mechanically rubbed on 5 pea seedlings for in- fectivity bioassay. Sedimenting components of PEMV either separate or mixed were aphid-transmitted but with varying efficiency. When fractions from density gradient columns containing PEMV components were reconcentrated by ultracentrifugation and adjusted to equal A = 2.0 concentrations 260 before feeding, aphids transmitted the top and bottom components with approximately equal efficiency (Table 10). However, that fraction which contained a mixture of the 2 components was transmitted with a higher efficiency by aphids than were either the top or bottom com— ponents separately. While the ratio of top/bottom components in this mixture was not known it could be assumed that the bottom component predominated since collection of this fraction was restricted to that part of the gradient column between the 2 major component bands, and relative to the UV scanning profile collection included only the descending slope of the top component peak while including all of the ascending slope of the bottom component peak (Figure 4A). The fourth fraction contained an aggregate of the bottom component and was trans- mitted with the least efficiency — 17.4% (Table 10). All pea seedlings (100%) developed systemic PEMV symptoms that were mechanically inoculated with reconcentrated components from the 4 DGC fractions. Aphid transmission efficiency was vastly improved when PEMV components were fractioned from the gradient column and fed directly to first-stage pea aphids without any interceding dialysis and recon— centration steps. With only a 4-hr AAP (as opposed to a 24-hr AAP in Exp. 1) aphids transmitted top, bottom and aggregated components It... 1 I'J_ I .' 46 TABLE 10. Transmission of sedimenting components of PEMV separated by sucrose density gradient centrifugation (DGC) and acquired through an artificial membrane by first-stage Acyrthosiphon pisum DGC fraction PEMV Peas infected number component (percentage) Experiment 13 1 top 14/30 (46.7)b 2 top and bottom 17/24 (70.8) 3 bottom 12/29 (41.4) 4 bottom aggregated 4/23 (17.4) Experiment 2C 1 top 36/43 (83.7) 2 bottom 31/35 (88.6) 3 bottom aggregated 17/40 (42.5) aAfter separation PEMV component fractions were dialyzed 48 hours against 0.02 M potassium phosphate buffer, pH 7.0, reconcen- trated by ultracentrifugation and components resuspended in the dialysis buffer containing 5% sucrose. Component preparations were adjusted to equal 2260 a 2.0 concentrations and provided to aphids for a 24-hr acquisition-access feeding period (AAP) followed by a single aphid transfer to healthy pea seedlings for a 3-day inoculative- access feeding period (IAP). bEach component fraction was also mechanically inoculated on 4 to 5 healthy pea seedlings with a resultant 100% of the peas infected, except those inoculated with fraction 3 (of Exp. 2) where only 40% were infected. cAfter separation PEMV component fractions were fed directly to pea aphids (without dialysis and reconcentration) for a 4-hr AAP and single aphid transfer to pea seedlings for a 3—day IAP. 47 with about 2-fold greater efficiency than comparable component trans- mission in experiment 1 (Table 10). Top and bottom components were aphid-transmitted with approximately equal efficiency (84% vs 89%). In the mechanical infectivity tests of the top, bottom and aggregated components, -100, 100 and 40% respectively, of the inoculated pea seedlings developed systemic symptoms. In attempting to explain the difference in component trans- mission efficiency by aphids these data would appear to indicate that: either the component concentration in those fractions fed directly from the gradient column (Exp. 2) was much higher than the 5260 - 2.0 concentration used in the first experiment; or the lengthy procedure of dialysis and reconcentration of component fractions (Exp. 1) re— sulted in some loss of infectivity of the components (and mixtures) prior to aphid-membrane feeding. DISCUSSION Our investigations indicated that the nucleoprotein yield and sedimenting component ratios of PEMV varies with season and age of source tissue. Non—aphid-transmissible and aphid-transmissible variants of CALIF-PEMV were separable on bases of nucleoprotein yield and component ratio when source plants used for partial purification were grown in controlled environmental chambers. The CALIF- and NY-PEMB strains, which vary in transmissibility but to a lesser extent than the CALIF-PEMV variants (CT- and CNT-PEMV), could not be differentiated on basis of yield or component ratios. However, these strains were only tested under greenhouse growing conditions and 48 apparently unfavorable growing conditions, such as excessively high temperatures, may mask any differences between strains. Furthermore, the top nucleoprotein component of NY-PEMV was not recoverable from peas grown and used for purification during these periods of excessive temperature fluctuations. It is of interest to note that Lapido and de Zoeten (1972) recently published results on a study of host and seasonal variation on the sedimenting components of tobacco ringspot virus (TRSV). They found that with a single TRSV strain the type and amount of components were determined by the host that was used to increase the virus; moreover, with the same host the type and amount of components present were determined by the period of the year in which the host was inoculated. While it is likely that strains, and variants of those strains, differ in nucleic acid base sequence, it is also probable that coat protein differences are present. Both could account for the differ- ences in nucleoprotein yield which occur between strains and variants, but the coat protein is likely to play a prominent role in aphid transmission efficiency determination. Because of the circulative nature of viruses such as PEMV, protein compatibility (complementarity) is almost certainly involved at the membrane level of one or more tissues within the vector (Rochow, 1969). If protein—membrane comple- mentarity is essential or required before virus enters a cell, e.g., epithelial cells of the aphid mid-gut, it is reasonable to expect that 2 strains with identically infective nucleic acid could be transmis- sible or nontransmissible by vectors if the protein coat was comple- mentary or noncomplementary, respectively. Just as the coat protein is important in determining plant host range (due to differential 49 absorption of virus to membranes of some plant species and not others) (Atabekov, 1971; Novikev and Atabekov, 1969) it is likely to be similarly important in determining which if any aphid membranes the virus can attach to and penetrate, either intact or in the form of viral RNA. If CNT—PEMV and CT-PEMV do indeed differ in their coat protein composition, it would be reasonable that such an altered coat protein in either of the variants could determine vector—virus compatibility and subsequent aphid transmissibility. Thus, when there is a mixed infection with 2 or more variants (as in the case of CALIF-PEMV- infected peas) aphids would select and perpetuate that variant which is compatible with their internal system. Other variants of a mixed infection are undoubtedly acquired by the aphid but fail to be trans- mitted due to membrane incompatibility or variant susceptibility to degradative or inhibitory enzymes within the aphid. Differences in coat protein could again be the dominant factor in determining variant susceptibility or nonsusceptibility to these enzymes. Why aphids select a PEMV variant with low levels of top com- ponent while repeated mechanical inoculation selects a variant with high levels of top component remains unresolved and will require further investigation. However, Gonsalves and Shepherd (1972) utilizing a different PEMV strain than ours, provided experimental evidence to show that the top component is slightly more mechanically infective than the bottom component. Furthermore, they have found that when nucleic acid preparations from unfractionated PEMV are subjected to electrophoresis in polyacrylamide gels or to sucrose density gradient centrifugation, there occurred three separate RNA species of 50 34, 30 and 12S. Infectivity was associated only with the 308 viral RNA and was the only RNA species found in the top nucleoprotein com- ponent, whereas the bottom nucleoprotein component contained approxi- mately equal amounts of 303 and 128 RNA and larger amounts of 34S RNA. Thus, in light of their findings it would not be unreasonable to expect that continually repeated mechanical inoculation might gradually select a variant with more top component because of its slightly greater infectivity. The role of 128 and 34S RNA species are unknown but it could be theorized that they might have some function in aphid transmission. CT- and CNT—PEMV variants should be extremely useful tools in further studies on vector-virus relationships particularly since they are at opposite ends of the aphid transmission spectrum, i.e., CT-PEMV being highly aphid-transmissible and CNT-PEMV net transmissible even by our best transmitter-pea aphid biotype. Characterizational studies on coat proteins of the variants and PEMV strains are in progress to attempt to elucidate specific differences. Electrophoretic hetero- geneity between variants as resolved by polyacyrlamide gel electro- phoresis may provide preliminary evidence of protein dissimilarities. Furthermore, amino acid sequencing of coat protein will undoubtedly be required, and although detection of specific sequence differences would be an important contribution the ultimate objective will be to correlate these differences with aphid transmissibility or nontrans- missibility. de Zoeten and Rettig (1972) working with a single PEMV strain have already demonstrated differences in protein patterns be- tween infected and noninfected pea seedlings and between viruliferous and nonviruliferous pea aphids. 51 Possibly one of the best methods to establish such a correlation is based on the concept of "heterologous encapsidation" as proposed by Rochow (1972) this includes both phenotypic mixing and transcapsida— tion. Isolated capsids and nucleic acids of certain viruses and virus strains have been combined ipflyippo to form a wide spectrum of combina— tions with varying infectivities. Since PEMV has been successfully separated into its constituent protein coat and RNA (without the latter losing its infectivity), as shown by Shephard, op 31. (1968) and Gonsalves and Shephard (1972) it seems probable that under the right conditions ip_yip£p_reconstitution by transcapsidation or phenotypic mixing or both, could be successfully effected. This coupled with PEMV transmission bioassay by our aphid-membrane feeding system (Thottappilly, 35 gl., 1972), would provide a unique method in deter— mining the role of the protein capsid in vector-virus specificity. In other words, would successful transmission be effected with the coat protein of CT-PEMV on the RNA of CNT-PEMV or through phenotypic mixing would the addition of capsomeres of the coat protein of CT—PEMV in combination with capsomeres of the coat protein of CNT-PEMV result in degrees of aphid transmissibility and plant infectivity. Also, an intriguing question arises as to whether PEMV RNA free of the coat protein could be aphid transmitted. There is some support for this hypothesis since the salivary glands are the final steps in the postulated circulative route of the virus through the aphid vector and to date PEMV particles have not been observed through electron microscopy in these glands (Harris and Bath, 1972; Shikata gp_§l., 1965, 1966). It has further been postulated that PEMV may exist in the salivary glands as free RNA and may be introduced with the salivary 52 fluid into the plant host by the feeding aphid in this form (free RNA). This is a speculative issue and has a major drawback in the fact that RNA free of the protective coat protein is undoubtedly very sensitive to the enzymes of the aphid's digestive system. To what extent heterologous encapsidation takes place in plants with mixed virus infections is not known. But this phenomenon could F- be an explanation for the continuing emergence and isolation of new PEMV virus strains or variants (and for strains of other viruses as well), each with a differing capacity to be transmitted or non- transmitted by insects. Also, very little is known about the effect 8' strains of a given virus, or differing viruses, have upon each other in determining their transmissibility. There is some evidence, par- ticularly in the case of barley yellow dwarf virus (Rochow, 1969), that the capsids or nucleoprotein of a particular transmissible strain may serve as "helper viruses" in mixed infections to aid in the trans- missions of other normally non-transmissible strains. It is thought that the major barrier controlling transmission specificity is deter- mined by whether a particular strain can gain entrance into the salivary glands of the aphid vector. Certain strains, because of their comple- mentarity with the membranes of this structure, readily pass through (probably by pinocytosis) while others are unable to penetrate and are not transmitted. But through a mixed infection, the nontransmis- sible strain is carried through the membrane with the transmissible strain, or its capsid, and transmission is effected. These types of studies, in combination with radioactive labeling or florescent anti- body labeling should be able to show specific strain and aphid- membrane relationships. 53 If such a membrane barrier exists for certain PEMV strains and variants, it is likely at some site other than the salivary glands - perhaps the epithelial cells of the lining of the aphid midgut. Since as before noted, no PEMV particles have been found in the salivary glands of the pea aphid and furthermore, viral particles of NY—PEMV and CT-PEMV (but not CNT-PEMV particles) have been found in the cells of the mid-gut epithelium and in the hemocoel surrounding the gut. Whether this type of "helper phenomenon" can be used for strains and variants to effect aphid transmission, particularly of CNT-PEMV, has yet to be shown. Investigations on the intrinsic properties of PEMV strains and variants and their relationship to the pea aphid vector are just be- ginning to yield fundamental insights into the vector-virus specificity phenomenon. In the future the most profitable and beneficial research from PEMV and other circulative aphid-borne viruses will likely result from studies conducted to determine the association and fate of the virus at the histological and cytological levels within the vector. LITERATURE CITED Atabekov, J. G. 1971. Some properties and functions of the coat protein of plant viruses, including the function of host-range control. Acta Phytopathologica Academiae Scientiarum Hungaricae 6:57-60. Bath, J. E., and R. K. Chapman. 1966. Efficiency of three aphid species in the transmission of pea enation mosaic virus. J. Econ. Entomol. 59:631-634. Bath, J. E., and R. K. Chapman. 1967. Differential transmission of two pea enation mosaic virus isolates by the pea aphid, Acyrthosiphon pisum (Harris). Virology 33:503-506. Bath, J. E., and J. H. Tsai. 1969. The use of aphids to separate two strains of pea enation mosaic virus. Phytopathology 59:1377-1380. Bozarth, R. F., and C. C. Chow. 1966. Pea enation mosaic virus: purification and properties. Contrib. Boyce Thompson Inst. 23:301-309. Bozarth, R. F., and C. C. Chow. 1968. Pea enation mosaic virus: differential suspension of virus components in buffer and sucrose. Virology 36:506-508. de Zoeten, G. A., and N. Rettig. 1972. Plant and aphid protein patterns as influenced by pea enation mosaic virus. Phytopathology 62:1018-1023. French, J. V., J. E. Bath, J. H. Tsai, and G. Thottappilly. 1973. Purification of pea enation mosaic virus from its vector, Acyrthosiphon pisum (Harris), and aphid-transmission char— acteristics. Virology (In press). Gibbs, A. J., B. D. Harrison, and R. D. Woods. 1966. Purification of pea enation mosaic virus. Virology 29:348—351. Gonsalves, D., and R. J. Shepherd. 1972. Biological and physical properties of the two nucleoprotein components of pea enation mosaic virus and their associated nucleic acids. Virology 48:709-723. 54 55 Harris, K. F., and J. E. Bath. 1972. The fate of pea enation mosaic virus in its pea aphid vector, Acyrthosiphon pisum (Harris). Virology 50:778-790. Izadpanah, K., and R. J. Shepherd. 1966. Purification and properties of the pea enation mosaic virus. Virology 28:463-476. Ladipo, J. L., and G. A. de Zoeten. 1972. Influence of host and seasonal variation on the components of tobacco ringspot virus. Phytopathology 62:195-201. Fm Musil, M., K. Mercinka, and F. Ciampor. 1970. Some properties of pea enation mosaic virus. Acta Virol. 14:285-294. Novikov, V. K., and J. G. Atabekov. 1970. A study of the mechanisms controlling the host range of plant viruses. Virology 41: . 101-107 . B Rochow, W. F. 1969. Specificity in aphid transmission of a circula- tive plant virus, p. 175-198. lp_Karl Maramorosch (ed.) Vectors, Viruses and Vegetation. Wiley (Interscience), New York. Rochow, W. F. 1972. The role of mixed infections in the transmission of plant viruses by aphids. Ann. Rev. Phytopathol. 10:101-124. Shepherd, R. J., R. J. Wakeman, and S. A. Ghabrial. 1968. Prepara- tion and properties of the protein and nucleic acid components of pea enation mosaic virus. Virology 35:255-267. Shikata, E., and K. Maramorosch. 1965. Electron microscopy of pea enation mosaic virus in ultrathin sections of plants and pea aphids. Papers Conf. on Relationships between Arthropods and Plant-Pathogenic Viruses, Jap. Soc. Prom. Sic., Tokyo, 1965, p. 160. Shikata, E., K. Maramorosch, and R. R. Granados. 1966. Electron microscopy of pea enation mosaic virus in plants and aphid vectors. Virology 29:426-436. Thottappilly, G., J. E. Bath, and J. V. French. 1972. Aphid- transmission characteristics of pea enation mosaic virus acquired from an artificial feeding system. Virology 50: 681-689. Tsai, J. H., J. E. Bath, and E. O. Igbokwe. 1972. Biological and transmission characteristics of Acyrthosiphon pisum biotypes efficient and nonefficient as vectors of pea enation mosaic virus. Ann. Entomol. Soc. Amer. 65:1114-1119. ' 1“. PART III: PURIFICATION OF PEA ENATION MOSAIC VIRUS FROM ITS VECTOR, ACYRTHOSIPHON PISUM (HARRIS) AND APHID-TRANSMISSION CHARACTERISTICS 56 INTRODUCTION Among the persistent or circulative aphid-transmitted viruses only potato leafroll virus (Peters, 1967a,b; Peters and Van Loon, 1968) and barley yellow dwarf virus (Rochow and Brakke, 1964) have been successfully purified from their vectors. Another aphid-borne circulative virus, pea enation mosaic virus, has been purified on numerous occasions from plant tissue but it has not been purified from its pea aphid vector, Acyrthosiphon pisum (Harris). This paper describes the successful purification of PEMV from fresh or frozen aphid tissue. In addition the-virus purified from aphids was established in plants and compared as to aphid transmis- sibility with virus purified from infected plants. MATERIALS AND METHODS Stock aphid colonies were reared under controlled conditions of light (12-hr photoperiod) and temperature (25°) on broadbean (Vicia faba L.) or garden pea (Pisum sativum L. cv. Midfreezer). Pea aphid Biotype EL (Tsai g3 31., 1972) and the NY strain of PEMV (Bath and Tsai, 1969) were used exclusively. Non-viruliferous lst— to 3rd- instar aphids were transferred to PEMV-infected pea plants for acquisition-access periods of 2—7 days. PEMV was partially purified 57 . '-'U""—'2~la'.it.\2fi-- 1} 9 m1 -. g < V 58 from viruliferous aphids either freshly—collected or frozen. In any one purification trial 2 to 10 grams (g) of aphids were used. Virus purification.--Aphids were first homogenized in a Sorvall Omni-mixer together with 0.1 M potassium phosphate buffer (pH 6.0) and chloroform-butanol (1:1) at a ratio of: l g/lO ml/lO ml. The E5 homogenate was allowed to stand 15 to 90 min (time varied with experi- ments) in ice; phase separation was completed by centrifugation at 9,000 rpm for 10 min on a Sorvall SS-l centrifuge. The upper aqueous phase was decanted and saved, the bottom chloroform-butanol phase was a discarded and the interface of insect debris was saved for reextrac- tion. The aphid debris from the interface was homogenized with chloroform-butanol (l g/5 ml/S ml), allowed to stand for 15 to 90 min, centrifuged for 10 min at 9,000 rpm and decanted as before. This re- extraction of the interface residue was repeated 3-4 times to obtain maximum virus extraction. All aqueous phase collections were com- bined, unless otherwise specified, and dialyzed against several changes of 0.05 M_potassium phosphate buffer (pH 7.0) for 24 hr. Following dialysis the preparation was centrifuged in the No. 30 rotor with a Spinco model L ultracentrifuge at 29,000 rpm for 2 hr at 4°. Virus pellets were resuspended in 0.1 M potassium phosphate buffer (pH 7.0) with 5 or 30% sucrose with the intention of later use in density gradient centrifugation and aphid transmission experiments. Density gradient centrifugation.--Sucrose density gradient columns were prepared using 4, 7, 7, and 7 m1 of 10, 20, 30 and 40% sucrose, respectively, in 0.02 M, pH 7.0 potassium phosphate buffer. After standing over night at 4°, 0.5-1.0 m1 of partially purified 59 virus suspension was layered on each gradient. Centrifugation was done for 2 hr at 24,000 rpm in the SW 25.1 rotor of the Spinco Model L ultracentrifuge, refrigerated at 4°. Tubes were analyzed for sedi- menting components and fractionated with an 1800 model D density gradient fractionator and UA-2 ultraviolet analyzer monitoring at 254 nm. ”T Electron microscopy.--E1ectron microscopic investigations were . I made on partially and density gradient-purified virus from viruliferous I aphid tissues. In DGC material, fractions collected from above and i} below the sedimentation zone were examined without reconcentration; the fraction within the zone was recycled, pelleted by differential centrifugation and resuspended in deionized water before examination. Each preparation was negatively stained with 2% phosphotungstic acid (pH 7.0) and examined with a Philips 300 EM. Extracts from non- viruliferous aphids were subjected to identical purification procedures and examined under the electron microscope in the same manner as extracts from viruliferous aphids. Infectivity assgy.--Assays of virus infectivity were made on Midfreezer pea seedlings by mechanical inoculations or aphid- trensmissions of partially and density gradient-purified virus. In aphid—transmission assay, virus solutions were fed to lst-stage pea aphid nymphs across an artificial membrane in a manner identical to that described by Thottappilly gp_§l., (1972). While partially purified virus was fed in phosphate buffer containing 5 or 30% sucrose, virus purified by density gradient centrifugation was fractionated and fed to aphids just as it came from the sucrose gradient column. After 6O completing the virus acquisition period, aphids were placed singly on test plants for S-day inoculation periods. RESULTS Preliminary tests.--Peters' (1967a,b) chloroform extraction technique for partial purification of potato leafroll virus from Myzus pgrsicae (Sulzer) was initially employed for purification of PEMV from A. pispm. Three grams of young adult aphids were processed through 5 cycles of chloroform emulsification immediately after completing a 5-day feeding period on PEMV infected peas. The aqueous phase from each emulsification was saved and mechanically inoculated to 60 young pee seedlings. No infectivity was associated with aqueous phases from the lst, 3rd, 4th and 5th cycles and only 3 plants became infected when inoculated with the aqueous phase from cycle 2. This low level of infectivity prompted attempts to improve the purification procedure. Infectivity of PEMV partially purified by chloroform—butanol technique.--Because the chloroform-butanol method (Steere, 1956) has been used successfully to free virus from plant host material from which viruses are ordinarily difficult to purify, we substituted a chloroform-butanol mixture for the chloroform of Peters' (1967a,b) tech- nique. The technique was modified further by permitting the emulsion to stand for periods much longer than the 5 min used by Peters (see Materials and Methods). 61 In an initial trial, 2 g of young pea aphids served as sources of virus immediately after completing a 5-day period on PEMV-infected peas. Following the let solvent treatment in which the emulsion stood for 15 min, the interface material (aphid residue) was reclaimed and processed through 3 cycles of re-extraction; thus, 4 aqueous phase fractions were obtained. After high speed centrifugation and virus resuspension each fraction was mechanically inoculated to 30 pea seedlings. Aqueous fractions 1, 2, 3 and 4 infected 23.3, 80.0, 70.0 and 73.3%, respectively, of the plants inoculated. In an additional trial, 9.5 g of aphids that fed on virus source plants for 5 days immediately preceeding extraction and 10 g of aphids reared on healthy plants were used. The interface material of both non-viruliferous and viruliferous aphid preparations was recycled 4 times employing an emulsion standing time of 1 hr. Aqueous phases 2-4 were pooled and compared with phases 1 and 5 in transmissibility by aphids after the virus in the 3 fractions was reconcentrated and resuspended. These fractions were artificially fed in phosphate buffer containing either 5 or 30% sucrose to lst-instar aphids during a 12-hr acquisition-access period. Thirty-five to 63 aphids were tested per treatment. Aqueous phase 1 of the viruliferous aphid purification was more infectious than either pooled phases 2-4 or phase 5 as judged by resultant aphid-transmission efficiencies (Table 11). Transmission was not appreciably affected by the concentration of sucrose in the buffer medium. Transmission percentages indicated that most of the recoverable virus was extracted from the aphid homogenate during the 62 lst or 2nd cycle of solvent treatment. No transmissions of virus resulted from aphids fed on preparations from non-viruliferous aphids. TABLE 11. Infectivity of successive aqueous fractions obtained by repeated chloroform-butanol emulsification of viruliferous pea aphids % Transmission by pea aphidsb Aqueous fractiona Trial 1 Trial 2 1 77.1 80.0 2-4 11.9 36.5 5 7.1 16.7 8Fraction 1 was obtained from the initial chloroform-butanol emulsification of aphid extract. Aphid debris was reclaimed from the interface of the aqueous and chloroform-butanol phases and processed through 4 additional cycles of emulsification to produce aqueous fractions 2-5. blst-stage nymphs were given a 12-hr acquisition-access period on artificial membrane source containing concentrated virus in 0.2 m1 of 0.1 M, pH 6.0 phosphate buffer containing 5% sucrose (trial 1) and 30% sucrose (trial 2);.35-63 aphids were tested singly per treatment. Frozen viruliferous aphids as a virus source.--Because it was difficult to obtain more than 5 g of viruliferous aphids at one time, it was desirable to preserve aphids by freezing. We tested the efficacy of this procedure by purifying virus from two 5-grem batches of aphids; one was frozen at -10° for 2 weeks after a 5-day acquisition- access period and the other was used for purification immediately after an identical exposure to source plants. As a control, 10 g of aphids (freshly-collected) that were reared on healthy plants were processed concomitantly with the viruliferous samples. The emulsion stood for 63 1 hr in each case before phase separation was completed by centrifuga- tion. The interface material of each preparation was recycled twice and all 3 aqueous phase collections were pooled for high speed centri- fugation. Virus pellets were obtained from both viruliferous aphid preparations and when resuspended in phosphate buffer containing 30% sucrose were found to be identical in A. nm concentration. These I?! 260 suspensions were fed to lst-instar aphids during 4— and 17-hr acquisition-access periods to compare infectivity when transmitted by aphids. While no visible pellets were obtained from non-viruliferous b . aphid preparations, the preparation was treated identically to that of the viruliferous aphids and fed to test aphids. An average of 41 aphids were tested per treatment. The fresh preparation from viruliferous aphids was transmitted with greater efficiency after either acquisition period than was the frozen preparation (Table 12); however, the difference was small and both preparations were highly infective. No infectivity was associated with the preparation of non-viruliferous aphids when assayed by mechanical and aphid-transmission tests. Infectivigy and UV analysis of density gradient-purified virus.—— The virus preparation obtained from fresh aphids in the previous tests was further purified by density gradient centrifugation and monitored with UV light at wavelength 254 nm. A partially purified preparation from non-viruliferous aphids was treated identically. The preparation from viruliferous aphids had an absorbance pattern very similar to that obtained from PEMV preparations purified from plants and was char- acterized by a virus absorbance peak which consisted of a low intensity 64 top component and a high intensity bottom component (Figure 5). Both preparations contained a non-viral component near the meniscus of the tube; a virus peak was not detected in the healthy preparation. The virus absorbance peak of the viruliferous aphid preparation was col- lected by gradient column fractionation and artificially fed to lst- instar pea aphids during a 21—hr acquisition period. A fraction was collected from the density gradient column of the non-viruliferous aphid preparation at the same depth in the column as the UV-absorbing zone occurred in the column containing the viruliferous aphid prepara— tion and fed to aphids in a manner identical to that used for the viruliferous preparation. Twenty-two of 32 test aphids transmitted the density gradient-purified virus from viruliferous aphids. No transmission resulted from the non-viruliferous aphid preparation. TABLE 12. Aphid-transmission of partially purified PEMV prepared from fresh or frozen viruliferous pea aphidsa % Transmission after acquisition- access period of: Condition of aphids 4 hr 17 hr Fresh 85.7 95.1 Frozen 70.7 87.5 aArtificially fed to lst-instar pea aphids at a relative concen— tration 0f.5260 = 7.5 in phosphate buffer containing 30% sucrose; 40-42 aphids were tested singly per treatment. Electron microscopy.--Partially and density gradient-purified preparations of both non-viruliferous and viruliferous aphids were negatively-stained and examined with the electron microscope. Virus 65 I I. vuauursaous — I NON-VIRULIFEROUS nu : E I: .11 V I! C‘ p- < H E) z < II 8 c 05 ll < 0* \~----------------- RELATIVE DEPTH IN TUBE --) FIGURE 5. UV scanning profile at 254 nm of preparations of non-viruliferous and pea enation mosaic virus-infested pea aphids after centrifugation into sucrose density gradients (IO-40%) for 2 hrs at 24,000 rpm in the Spinco SW 25.1 rotor. r‘I 66 particles were abundant in both preparations from viruliferous aphids, but aphid tissue debris obscured virus particles in the partially purified preparation. The density gradient preparation contained very little cellular debris and virus was easily detected in samples from the sedimentation zone (Figure 6A and B). Virus particles were of uniform size and shape, and were ca. 27 nm in diameter. Although each grid opening (300) mesh contained numerous distinct virus particles, the real concentration might have been much higher as the distinct negatively-stained particles were apparently underlain with thousands of other particles which were only faintly stained (Figure 6A). Only a few scattered virus particles were found in samples from above and below the sedimentation zone. No virus-like particles were found in either preparation of non-viruliferous aphids. Comparative transmissibility of virus after consecutive aphid- to:p1ant or_plant-to-plant transfer.--Virus partially purified from aphids was mechanically inoculated to pee seedlings to establish an aphid-source PEMV line for subsequent aphid-transmission comparisons with a PEMV line that was established after purification from a plant source. Both lines originated from the same isolate of NY-PEMV. The plant-source line was initiated 2 months previous to the aphid-source line through use of the purification technique previously reported (Thottappilly g£_§l., 1972) and was maintained by thrice-monthly mechanical transfers of crude sap after the initial purification. Prior to the following experiment the aphid-source line had been mechanically transferred on 1 occasion and the plant-source line mechanically transferred on 6 occasions. FIGURE 6. Electron micrographs of negatively—stained preparations of DGC-purified suspensions obtained from PEMV—carrying pea aphids. A. Virus particles at magnification of 69,000 X. B. Particles at 198,000 X. Bar represents 100 nm. 68 To obtain source plants for comparative transmission tests, the plant- and aphid-source lines were inoculated to young pea seedlings by mechanical and aphid means, respectively. Ten days later plants of both lines were used as virus sources for aphid-transmission trials and for virus purification. First-stage_A.“pigpm_nymphs were starved 2 hr and given 1— and r“? 4-hr acquisition-access periods on source plants of both lines; source I plants of the 2 lines were indistinguishable and showed severe symptoms. Thirteen to 16 aphids were tested per acquisition period per each of ': 3 source plants (replicates) per virus line. The 4-hr treatment aphids L} were serially transferred at short intervals to healthy pea seedlings to enable detection of latent period differences. The l-hr treatment was allowed a 5-day inoculation period. The aphid-source line was transmitted with significantly (P < 0.05) greater efficiency after either the l- or 4-hr acquisition periods than was the plant-source line (Table 13). The median latent period (LP 1 of the aphid-source line was significantly shorter than 50) the plant-source line, and the former virus line completed latency in 50% of the test insects in less than 6 hr. Each virus line was partially purified from plants, assayed by UV scans (at 254 nm) of density gradient columns, and tested for 1The median latent period (LPSO) was calculated by transforming the time, in hours, at the midpoint of the transfer interval to logarithms and the cumulative percent of first transmissions to probits, calculating a least squares linear regression and solving for value of time when probit value of cumulative first transmission was equal to 5. Virus acquisition was assumed to have occurred at the start of an acquisition-access period and inoculation at the midpoint of a transfer interval. 69 rail-lid! . in...” n cos: .msOHussHaususo onuse unsusH you use: was cOHusuucsosoo 0.x < snu masseussuu use NstHm vsumsu sass sneaks oHsom use smsumnumuHm «NIHN muosaHusaxs sea 00 sowed ussuo sH musmHm sousom new mom: smocu mm uOH sass snu mo mussHm Boom vsHMHuse ms3 msuH> a .m<< onus see mo mvHsmo Scum oscHeususw sass vaHuse unsusH usOHumHsaom umsu mo Non sH ooHusm unsumH osust Iaoo msuH> cons sEHu vsmmmHs menu n ommH ”some assoc hus>s scum Amo.o v.mv unsusMMHv hHucmoHMstHm mH nose pOHHsn assumH was GOHmmHamssuu zoos mussaumsuu\souaoo\nsumsu muss menses pHses use swmum lumuHm 0HImH “AooHuse mmsoosICOHuHmHsvosv m<< use use: sass AmsusoHHmsuv muesHm sousom ssusam N.“ m.~s a.oo m.o~ a.s~ s.oa o.ma m.sm mouooo -ooosa a.NN o.HH oos o.om 0.0m a.m m.mm a.ms oooooo uoaso< 0; o v Anne m.a a.H as o v Anny be a an H ones we onusa ommH mo voHusa ommH msuH> unsusH nuHB as 00N¢.us oasumH nuHa ems: ”no m<< usums m0H£e< N GOHusHom msuH> so m<< were mpHsm< N GOHmmHamGsuu N sosz usumm GOHmmHamssuH N nmsOHusuoesum wusHusm hHHsHuuma Eouw psuHsvos hHHsHonHuus mauH> mmucsHe summon scum osuHsvom msuH> ucmHQIOuIanam s>Husosmsoo usums mssHH >2mm mo musmmssuu uasHmloulucsHe no moHumHusuosumno GOHmmHaossuulpH£e< .MH mHm FIGURE 7. Scanning pattern at 254 nm of fractions from prepara- tions of pea infected with an aphid line (initiated with partially purified virus from pea aphids) and a plant line (initiated with partially purified virus from pea plants) of pea enation mosaic virus. Sedimentation occurred in sucrose density gradients (lo-40%) centrifuged for l-1/2 hr at 24,000 rpm and 4° in the Spinco SW 25.2 rotor. 72 DISCUSSION The size and shape of the PEMV particles that we purified from viruliferous pea aphids were generally the same as particles purified from infected plants (Bozarth and Chow, 1966; Gibbs_g£_§l., 1966; ' Musil pp 51., 1970) and observed in ultra-thin sections of plants and aphids (Shikata_g£.§1., 1966; Harris and Bath, 1972), and the UV scanning pattern closely approximated those we have obtained with preparations from plant sources. However, after the purified aphid- source virus was established in plants it was transmitted by aphids with remarkably high efficiency. The median latent period (LPSO) of the aphid-source virus line (5.7 hr at 25°) was not only signifi- cantly shorter than that obtained for the comparable virus line from a plant source, but it was by far, the shortest LP50 recorded for any PEMV isolate in any pea aphid biotype. Even if inoculation was assumed to have occurred at the end of inoculation access periods, the LP50 was only 7.8 hr. Other comparable LP50 estimates on record are: 25.0 and 14.0 hr at 20° and 30°, respectively (Sylvester and Richardson, 1966), 20.6 hr at 24° (Chapman and Bath, 1968), and 19.5 hr at 22° (Bath and Tsai, 1969). In addition the efficiency with which lst instars transmitted the aphid-source virus line after 1- and 4-hr acquisition-access periods was approximately equal to the highest transmission efficiency (51.7 and 92.9% after 1- and 4-hr AAPs, respectively) known for a PEMV and pea aphid relationship (Bath and Chapman, 1968). Furthermore, after the aphid- and plant-source virus lines were purified from pea plants of the same lot as those used for virus source plants in the plant-to-plant transmission 73 trials, virus of the aphid line was much more efficiently transmitted by aphids after membrane feeding than that of the plant line. The enhanced aphid-transmissibility of the aphid-source line over the plant-source line apparently was not a function of virus concentration in the source plants or membrane-feeding system; for the source plants of the aphid line yielded less virus than those of the plant line and the UV scans of density gradient columns showed that less virus was present in the partially purified aphid-source preparation that was fed to test aphids than was present in the plant-source preparation. LITERATURE CITED Beth, J. E., and Chapman, R. K. 1968. Influence of aphid stage on acquisition and inoculation phases of pea enation mosaic virus transmission. Ann. Entomol. Soc. Amer. 61:906-909. Bath, J. E., and Tsai, J. H. 1969. The use of aphids to separate two strains of pea enation mosaic virus. Phytopathology 59: 1377-1380. Bozarth, R. F., and Chow, C. C. 1966. Pea enation mosaic virus: purification and properties. Contr. Boyce Thompson Inst. 23: 301-309. Chapman, R. K., and Bath, J. E. 1968. The latent period of pea enation mosaic virus in three of its aphid vectors with emphasis on adult versus nymph comparisons. Phytopathology 58:494-499. Gibbs, A. J., Harrison, B. D., and Woods, R. D. 1966. Purification of pea enation mosaic virus. Virology 29:348-351. Harris, K. F., and Bath, J. E. 1972. The fate of pea enation mosaic virus in its pea aphid vector, Acyrthosiphon_pisum (Harris). Virology. in press. Musil, M., Marcinka, K., and Ciampor, F. 1970. Some properties of pea enation mosaic virus. Acta Virology 14:285-294. Peters, D. 1967a. The purification of potato leafroll virus from its vector Myzus persicae. Virology 31:46-54. Peters, D. 1967b. Potato leafroll virus, its purification from its vector Myzus persicae. Meded. L.E.B. Fonds No. 45. H. Veenman and Zonen, Wageningen, The Netherlands. 108 p. Peters, D., and Van Loon, L. C. 1968. Transmission of potato leaf- roll virus by aphids after feeding on virus preparations from aphids and plants. Virology 35:597-600. Rochow, W. F., and Brakke, M. K. 1964. Purification of barley yellow dwarf virus. Virology 24:310-322. Shepherd, R. J., Wakeman, R. J., and Ghebriel, S. A. 1968. Preparation and properties of the protein and nucleic acid components of pea enation mosaic virus. Virology 35:255-267. 74 75 Shikata, E., Maramorosch, K., and Granados, R. R. 1966. Electron microsc0py of pea enation mosaic virus in plants and aphid vectors. Virology 29:426-436. Steere, R. L. 1956. Purification and properties of tobacco ringspot virus. Phytopathology 46:60-69. Sylvester, E. S., and Richardson, J. 1966. Some effects of temperature on the transmission of pea enation mosaic virus and on the biology of the pea aphid vector. J. Econ. Entomol. 59: 255-261. T' Thottappilly, G., Bath, J. E., and French, J. V. 1972. Aphid- transmission characteristics of pea enation mosaic virus acquired from an artificial feeding system. Virology (in press). Tsai, J. H., Bath, J. E., and Igbokwe, E. C. 1972. Biological and b! transmission characteristics of Acyrthosiphon pisum biotypes efficient and nonefficient as vectors of pea enation mosaic virus. Ann. Entomol. Soc. Amer. 65 (in press). ”murmumumn 93 03056 682 312