@URIFICATEON, SEROLO’GY AND ELECTRON MiCROSCOPY 0F PEA ENATION MOSAIC VIRUS Thesis 50: the Degree of Ph. D. MkCHJG‘AN STATE flNlV'ERSEVTY Antonio Du .Bzustriiios 3.964 “1:515 LIBRARY Michigan State University This is to certify that the thesis entitled PURIFICATION, SEROLOGY AND ELECTRON MICROSCOPY OF PEA ENATION MOSAIC VIRUS presented by Antonio D. Bustrillos has been accepted towards fulfillment of the requirements for Ph. D. degree in Plant Pathology ){ajor professor Date / [26411.7 2” ’ // / f6 (,1 / / 0-169 ROOM USE Of’iLY, ROOM {ESE OE‘éLY. ABSTRACT PURIFICATION, SEROLOGY AND ELECTRON MIcmsmPY OF PEA ENATION MOSAIC VIRUS by Antonio D. Bustrillos The host range, physico-chemical properties, purifi- cation, serology and electron microscopy of pea enation mosaic virus (PEMV) were investigated. Isolates from Michigan (PEMV—Z), Washington (PEMV-W), Oregon (PW-0), and New York (PEMV—NY) were employed in this study. va infected only leguminous plant species. Mung bean (ghgseolus agrgus Beach.) was found to be a new host plant of PEMV. All pea varieties and 55 pea introductions were susceptible to infection. A pea introduction from Iran showed some tolerance to the virus. A number of species of plants of 6 different families were tested in an effort to find a local lesion host, but none were found. Dilution inactivation of PM occurred between 132000 to 133000. 'Ihe virus was still infective after 10 minutes at 65°C but not at 68°C: it resisted inactivation in 2.3252 for 4 days but lost its infectivity in air-dried diseased tissues after the third day. Infectivity of‘ the virus in sap extracts buffered at pH 7-7.8 was maintained for about 12 days storage in the refrigerator (3°C) . Antonio D. Bustrillos 2 The purification of PEMV was accomplished by low speed centrifugation of buffered extracts from frozen diseased tissues. The clarified extract was concentrated and further purified through 3 cycles of differential centrifugation of the appropriately buffered virus suspension. The purified virus preparation was infectious and electron micrographs revealed the presence of numerous apparently homogeneous nearly spherical particles. about 20 mp in diameter. Such particles were absent from a similar healthy control prepar- ation. The purified pEMV preparation was antigenic and re- acted serologically with its homologous antibody using Ouchterlony agar technique and micrOprecipitin tests. Sero- logical cross-reaction of the four PEMV isolates showed that they were closely related. Strains of cucumber mosaic virus, southern bean mosaic virus, bean yellow mosaic virus, Maryland bean virus, and tobacco :ringspot virus were not serologically related to PM. The presmce of an antigenic non-infectious host protein in the virus preparation was detected by the Ouchterlony agar double-diffusion technique. The specific PEMV antigen-antibody reaction formed curved precipitation bands while the non-specific host-antibody re- action formed a separate straight precipitation band, hence, its identification was possible. PM in clarified extracts could be detected and identified serologically with the anti- serum obtained. rusrncxrrou, ssaoroer AND memos Micnoscory ' er PEA manor: nosarc VIRUS BY Antonio D. Bustrillos A THESIS Submitted to Michigan State University in partial fulfillment of the requirmaents for the degree of DOCTOR OP PEILOSOPHY Deparusent of Botany and Plant Pathology 1964 A. ’ v C This thesis is sincerely dedicated to MY FAMILY AQ‘CNOWLBNMTS I sincerely appreciate the invaluable suggestions and criticisms of the members of my guidance committees Dr. H. H. Murakishi, Dr. J. L. Lodwood, Dr. A. H. Ellingboe, Dr. G. B. Wilson, Dr. W. N. Mack and Dr. S. '1‘. Bass. I am especially grateful to Dr. H. H. Murakishi, chairman of my committee, not only for his indispensable help in the development of the problem and preparation of the manuscript but also for his constant stimulation, intellectual and material aid throughout this thesis, and also to Dr. S. '1'. Bass for his help and guidance in the electron microscOpy part of this investigation. Furthermore, I wish to convey my sincere thanks and appreciation to the faculty and. staff, especially to Dr. W. S. Drew, Head, of the Department of Botany and Plant Pathology who have in one way or another helped me and made my graduate study at Michigan State University a pleasant one. I wish to acknowledge the financial support extended me through an assistantship." from the Michigan Agricultural Experiment Station and by an institutional grant from the American Cancer Society. Lastly, I wish to express my profound gratitude to the Rockefeller Foundation who provided travel funds and to the College of Agriculture, University of the Philippines for granting me a study leave at Michigan State University. ii TABLE OF CONTENTS Page mTROWCTION 00.0000...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 1 REVIEW or LITERATURE .................................. 3 MATERIALS AND METHODS .................................10 ’PMV isolates ..................................10 Source plant and biological assay ............ 10 Culture and method of inoculation ............ 10 Determination of physico-dhemical properties of PEMV ........................... ll Purification Of PM eeeeeeeeeeeeeeeeeeeeeeeee 13 Preparation of virus antigen and serum prOdQCtion geeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 1-4 Seroloqical tests ............................ 16 Electron microscopy .......................... 18 EXPERIMENTS AND RESULTS ........................o..... 20 Reaction of source plant to PEMV isolateea.... 20 Host range and search for local lesion host... 20 Reaction of pea introductions to PEMV 1'01at..OOOOOCOOOOOOOOOOOOOOOOO.0.0.0.0.000... 22 Physico-chemical prOperties of PEMV .......... 29 Effect of storage of virus-Juice extracts buffered at different pH levels on virus 1nfmt1V1ty OOOOOOOOOOOOOOOOOOOOOOO0.00.0.0... m Isolation, clarification and purification Of PEMV OOOOOOCOOOCCO0.0000COCOOOOCC0.0.0.0... 32 Serological properties of PEMV ............... 37 Electron microsc0py of PEMV .................. 48 iii Page DISCUSSION 0.00.00.00.000..0.0...OOOOOOOOOOOOOOOOOOOOOO 61 SWRY AND CONQIUSIONS .0.0.00.00.00.00...00.00.00.000 68 BIBLIOGWHY 00.0.00...OOOOOOOOOOOOOOOOOOOO00.0.0000... 71 iv 10. LIST OF TABLES Results of host range study of PEMV .......... A list of pea (Pisgm sativum L.) intro- ductions tested for their reactions to va iSOlates .eeeeeeeeeeeeeeeeeeeeoeeeeeeeeee Tolerance to dilution of the PEMV isolates.... Effect of pH of buffered virus-juice extracts on virus infectivity and its relation to inactivaflon';g vitro at 2" c eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Relative infectivities of PEMV fractions obtained from three purification trials ...... MicrOprecipitin test between the puri- fied PEMV-W preparation and its antiserum .... MicrOprecipitin test between PEMV-W anti- serum and purified healthy control pr‘Puation O...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO MicrOprecipitin test between normal rabbit serum and clarified extracts from PEMV-W infected and healthy control Pacific Freezer leaves ............................... Microprecipitin test between PEMV-W anti- serum and clarified preparations of PEMV- infected and healthy control Pacific Freezer lave: .geeeeeeeeeeeeeeeeeeeeeeeeeeeee MicrOprecipitin reaction between PEMV-W antiserum and clarified preparations of CMV, SBMV, msv, BYMV, and TRSV strains ....... Page 23 25 3O 31 38 41 41 43 43 45 LIST OF FIGURES Pa e FIGURE “2‘ 1. Purification rocedure of pea enation mosaic Vim! PEMV) eeeeeeeeeeeeeeeeeeeeeeeeeoe 39 2. Precipitin reaction band patterns obtained in the Ouchterlony agar double-diffusion test between the PEMV-W antiserum and the purified preparations of healthy control and PM 1.01atea .QQQQQQOOOOOOOO0.00.00.00.00. 49 3. Ouchterlony agar double-diffusion test between the PEMV-W antiserum and other plant viruses observed in ion agar (0.5%) prep- aration with sodium azide (135000) preserva— tive, 14 days inCUbation eeeeeeeeeeeeeeeeeeeeee 50 4. Ouchterlony agar double-diffusion test between the PEMV-W antiserum and other plant viruses observed in ion agar (0.5%) preparation with merthiolate (1310,000) solution preservative, 14 days incubation eeeeeeeeeeeeeeeeeeeeeeeeeeee 5]- 5. Electron micrograph of a purified PEMV-W from a distilled water suspension after the third Ultracentrifugation eeeeeeeeeeeeeeeeeeeee 54 6. Electron micrograph of a purified prep- aration from PEMV-W infected pea tissues ...... 55 7. Electron micrograph of a purified prep- aration from healthy pea tissues .............. 56 8. Electron micrograph of purified preparation from healthy pea tissues, 3 cycle of differ- ential centrifugation OOOOOOOOOOOQOOOOO00...... 57 9. Electron micrograph of partially purified PEMVSW preparation resuspended in phosphate buffer pH 7.5 obtained from the second ultracentrifugation ........................... 58 10. Electron micrograph of a purified prep- aration from PEMV-Z infected pea leaves, 3 cycles of differential centrifugation, in distilled wat.r aunpmsion eeeeeeeeeeeeeeoeeeee 59 11. Electron micrograph of purified PEMV-Z infected pod ggeparation showing the presence of t sad-like virus contaminant ..... 60 vi INTMWCT ION The application of serology and electron microscOpy to the detection, identification and characterization of plant viruses can give precise results only if purified in- fectious preparations are uployed. Such preparations permit a better study of the physical and cheaical nature of the viruses and also yield more meaningful interpretations of serological and electron microscopic findings. A knowledge of the morphology and size of the virus particles obtained from electron micrographs of purified preparations facili- tates their identification from ultra-thin sections of infected plant tissues. The usefulness of these preparations in qualitative and quantitative electron microscopy cannot be overeaphasized (48, 68, 78, 92, 93, 94). This investigation was initiated on the pea enation mosaic virus (PEMV), an unusual plant virus that causes tmnor- like growths similar to sarcoma in animals, with a view to gaining possible insight into the nature of virus-caused abnormal growths. Earlier work (39) reported the isolation of high molecular weight proteins from healthy and rm- infected broad bean plants by ultracentrifugation. Except for some infectivity in the latter, both proteins had similar sedimentation constants. Limited investigation on the puri- fication, serology and electron microscOpy of rmv is due 1 2 partly to the difficulty of obtaining infectious purified preparations, lack of adequate methods of assay and to some extent, on the instability of the PEMV. The present study attenpted to explore these specific areas3 tests were made on the host range in search for a local lesion assay plant and the physico-chmsical properties of the virus were studied to determine the conditions affecting its stability. REVIEW OF LITERATURE The pea enation mosaic virus (PM), also known as Enation Pea Mosaic Virus (50), Pea Virus 11(54), Pisum Virus 1 (72), and Mm Lie; (26), is a distinct legume virus which initiates enations on the infected pea plants, Piggy; m L.. The symptoms of PM disease are generally ex- pressed by the formation of hyaline spots or translucent areas on the foliage, the production of enations or out- growths of different sizes and develogaental forms (86) on the abaxial side of the stipules and leaves, on pods and rarely on stems. Occurrence of the di sease has been observed in most of the pea growing areas of the United States (24, 28, 29, 36, 37, 42, 4s, 55. 65, 73). The loss incurred on the crop from PEMV infection is manifested in (1) a general reduction of crop yield as a result of stunting and reduced productivi- ty of the infected plants and (2) the unmarketability of the deformed pods produced. Linford (36) first observed the occurrence of deform- ed pods on garden peas, the cause of which appeared similar to a pee virus causing enations and pod deformation reported by Snyder (74). Pm disease was first described by Osborn (so), then-by'Pierce (54); Stubbs (as), Ainsworth (1); and others. A detailed description of the macroscopic. symptoms and anatomical studies of PEN-diseased pea at different 3 4 stages of infection was reported by Tsao (86). Studies on the host range of PM were conducted by a number of investigators, Osborn (50) transmitted the virus to garden pea, sweet pea (Lathmg odogatgg L.) , field pea (g. m var. mg Poir.) and crimson clover (Trifglium ingarngtm L.) using the pea aphid which was allowed to feed on PEMV-infected broad bean (M! Lab; L.). Pierce (54) obtained infection on soybean (91m 9215 (L.) Sieb. SIZucc.) and on the following pea varieties3 Alderman, Alaska, Per- fection, Wisconsin Early Sweet, Surprise,'Progress, Green Admiral, Dwarf Telephone and floral. Stubbs (83) observed PFMV infection onyellow sweet clover (Mglilotus gfficigalis L. Lam.) and on ten more pea varieties, name1y3 Prenium Gan, Improved Gradus, Laxton's Progress, Gradus, Hereford, Bruce, Icer, Canada Field Pea, Black Eyed Marrowfat and Mammoth Luscious Sugar. Alfalfa (ESSA-£399. sgtiyg L.), alsike clover (Ti-gong; Mgidm L.) and perennial pea (m m L.) were also found susceptible to PEMV but it was not trans- mitted to yellow sweet cloVer and broad been (28). Ainsworth (1) observed PEMV infection on rough pea vine (W hirsutgs L.) and on other pea varieties in Great Britain. The susceptibility of crimson clover and sweet pea was con- firmed by Chaudhuri (14). He was unable to obtain infection on vetch (mg m L.), tobacco (31.92212: W L.) and trend: bean (My; SEQ-93.51.! L.). Bur clover V (W hispida Gaertn.) var. dulce and spotted medick 5 (£14,. m; All.) were susceptible to PEMV whereas sugar beet (Egg; vulgg; is L.), smooth leaf mustard (Bgssica juncig Coss.), cucumber (Cuggmig sativus L.) and celery (Aging gggveoleng L.) were not susceptible to PEMV (69). Hagedorn and Walker (24) observed PEMV infection on most of the leguminous hosts reported earlier but found nonsusceptibility to virus infection in the following 1egumes3 beans (Phggglus vulgEis L.), cowpea (Viggg mm), white lupine (My; 91;; L.), red clover (Trifolig 2. ragensg L.), white clover (3;. m L.), alsike clover (_T_. hfigidum L.), alfalfa (W m L.) and white sweet clover (Mglilotgg offigingis (L.) Lam.). Furthermore they obtained no infection on Nicotimg glutiposg L. and tomato (W W 1-.)- Mcfiveh and Schroeder (42) transmitted the virus to white clover including Ladino (Trifolig rgpgns L.) vetch, spring vetch (Vicig gillosa L.), white sweet clover (Melilotus Qba Dar.), and they confirmed PEMV infection on yellow sweet clover, rough pea vine, and on 6 alfalfa varieties3 Grimm, Hairy Peruvian, Kansas Common, Narragansett, Ranger and vernal. The same investiga- tors further confirmed (43) transmission of PEMV to alsike clover. In the meantime, a considerable number of leguminous plants were reported susceptible to the virus. Studies conducted by Hagedorn (23) showed that PEMV would infect milk vetch (Astggggus 53211.1. L.), chick pea (Cicg mm L.), flat pod pea vine (m cicgg'g L.) Tangier pea 6 (a. gnm L.), chester clover (Trigoligg W L.), Persian clover (g. mum L.),.subterranean clover (z. W L.), white lupine (M 93;; L.).blue lupine (L. W L.), wooly pod vetch (whm L.) and Hungarian vetch (Y.- W Crantz.). some physical properties Lg zit-£9 of PM have been reported. The virus was inactivated by a temperature of 56°C with no infection after 10 minutes exposure at 58°C (54). Johnson and Jones (28) reported a wide thermal inactivation ranged «to-80°C. Osborn (53) obtained infection at 64°C with no infection beyond 66°C. Ainsworth (1) indicated 55°C to: the thermal death point of em. a dilution and point of 133000 was reported by Stubbs (83). Osborn (53) found pmv infectivity at dilutions of 131000 but not at'l310,000. The virus has been reported to resist inactivation in 21292 at 20-22°C for 3 days (54). In a separate similar investi- gation (53, 83), PM resisted inactivation for 4 days. Early investigators reported that PEMV was difficult to tran-it by mechanical pans unless abrasive materials were mnployed. Stubbs (83) transmitted the virus by a rubb- ing method employing Norit' or carborundum powder. He also transferred PEMV by insertion of macerated diseased tissue into slits made in the stun of suscept plants, however, the percentage of infectlon was low. The use of carborundum in sap transmission of the virus was further desonstrated by Osborn (53) who further noted that repeated serial transfers 7 of PEMV through broad been by mechanical means resulted in the attenuation of the virus. Ainsworth (1) reported that the virus retained its virulence for more than two years through serial transfers mechanically in garden pea and sweet pea. The carborundum method was found superior to atomization of the virus suspension at a pressure of 28 lbs./ in.2 (23). Insect transmission of PEMV and the virus-vector relationship was first studied by Osborn (49) and reported in his subsequent papers (50, 52, 53). The pea aphid, W Lip; Kalt. and the potato aphid, 31. f 11 main.) I E0 g3; Koch, were observed vectors of PM. Furthermore, he reported that PEMV was a persistent virus requiring an incubation period of 12-18 hours in the vector before the aphid was able to tranmnit the virus. The green peach aphid, m m (Sulz) was first reported a vector of PEMV by Chaudhuri (l4) . He noted that the pea aphid was the most efficient I vector of the virus and that the aphid mains infective for a considerable period of time. Simone (69) showed that the ornate aphid, 22159.! ornatu. Laing, could tranmait PM and he suggested that the aphid vectors probe the phloem in order to accomplish in- fection. Infection could be accomplished also by probing the parenchymatous tissues by the vector (43). Two more aphid vectors. Escalate was (c1...) ‘ and Wishes * 8 m Harris (5. W, B.D.P.) have been reported to transmit PEMVTrom diseased broad baan to healthy clover plants (25). A search for resistance to PEMV in pea varieties was conducted by several investigators (64, 65, 73). The Geneva line No. 168 (PI No. 140295) was found to eldaibit tolerance to the virus. Schroeder and Barton (65) reported that resistance of pea to PM was conditioned by a single domi- nant gene. McWhorter and Cook (46) observed some strain differ- ences in PM. Cross-protection studies have been conducted by Hagedorn and Walker (24). They obtained no protection when PEMV was tested against Bean Virus 2, Wisconsin pea streak, Red clover vein mosaic and Cucumber mosaic virus. Reciprocal tests also showed negative protection. Some anatomical aspects of PEMV disease were reported by McWhorter (44) and McWhorter and Cook (46). They observed certain nuclear changes in the infected cells. The enations formed following PEMV infection arose from proliferated epi- demal cells on veins of the lower surface of leaflets and stipules. The primary effect of PM (California isolate) on the pea plant (Perfected Wales var.) was the stimulation of cell division (hyperplasia) and the enlargmsent of the phloem parenchpa cells (hypertrophy) of the vascular bundles resulting in the differentiation of the abnormal phloaa and necrosis of the mesOphyll areas (86, 87). furthermore, the 9 translucent areas were couposed of hyperplastic and hyper- trophic parenchyma cells containing degenerated chloroplasts arranged in canpact fashion. In a similar study, Ruppel and Hagedorn (62) found that the giant enations observed on Perfected‘Wales following infection with the California isolate of PEMV were composed of highly organized and differentiated tissues comparable to healthy or normal ones. However, this type of enation was not reproduced in the sub- sequmt inoculations from these. The spread of PM in the field was reported reduced by controlling insect vectors. Parathion, duneton, and diazinon insecticidal sprays have been employed (19, 43, 84). MATERIALS AN D MEEODS EEMV lflgtg! Four pea enation mosaic virus (PEMV) isolates were uployed in this study. The virus isolates from Washington (PEMV-W), Oregon (PEMV-O) and New York (PBMV-NY) were obtained from Dr. D. J. Hagedorn, Department of Plant Pathology, University of Wisconsin, Madison. The virus isolate from Michigan (PEMV-Z) was from infected dried pea leaf tissues stored frozen over cacl2 for about 3-1/2 years in the refrigerator. Unless otherwise stated, the va-W isolate was unpleyed in the purification, serology and the identification of the virus by electron microscopy. WW Garden pea variety Pacific Freezer was chosen as a suitable host plant for PW based on the uniformity of symptom emression, ease of infection and compactness of growth. Since no host plant responding to 9m infection with local lesions was known, Pacific Freezer variety was also used as a systunic assay plant for virus activity. Virus infectivity assays were made by rubbing the carborundum- dusted leaves and stipules with polyurethane pads which had been saturated with the virus preparation to be tested. WW Five - 8 pea seeds lightly dusted with Arasan 10 11 (tetramethylthiuramdisulfide) , to prevent pre-euergence damp- ing off, were planted in 4-inch pots filled with greenhouse soil. The plants were given small amounts of complete fertilizer in solution a week after germination and once in two weeks thereafter. The seedlings to be inoculated were dusted lightly with carborundum powder, 400 to 600 mesh, to aid in the inoculation (59). Either frozen dried or recent- ly .‘infiacted leaves were macerated with a mortar and pestle with small mounts of neutral phosphate buffer added to the macerate to make a light thin paste or slightly liquid suspension. Polyurethane pads, 1/2‘ x 1" x 1", soaked lightly with the inoculum were uployed. . The . inoculating pads caused no detectable injury on the inoculated plant tissues. Pea seedlings 10-14 days old were readily infected with PM. Infection of very young seedlings generally resulted in severe stunting due to retarded apical growth and leaf distortion. When older plarts were used a low per- cmtage of infection was observed and symptom expression was often del ayed. Dete inat on of h sico- ic rt es 0 PM The methods described by Bos :5 g, (9), slightly modified, were «uployed in the determination of some physico-chemical properties of PBMV. Leaves and stipules of Pacific Freezer peas, 12-14 days after inoculation, were used as source of PEMV. Where clarified extracts were 12 unployed, the fresh infected tissues were ground in a mortar and the macerate was squeezed manually through 4 layers of cheese cloth. (a) Dilution end-point. The following serial dilu- tions of the clarified juice with distilled water as diluent were preparedi'. 0,131000, 132000, 133000, 134000, and 135000. These dilutions were used to inoculate carborundun- dusted leaves and stipules of healthy 12-day old pea seedlings. The tolerance to dilution of PM was recorded 24 days after inoculation. (b) Inactivation in, m. A clarified Juice extract was stored in a steppered Erlenmeyer flask and held at 20-22°C. A sunple was drawn for infectivity test after 0, 6, 12 hrs., 1, 2, 3, 4, and 5 days storage. Disease symptoms were observed and recorded. (c) Longevity of PM in dried tissue. Leaves of PEN-infected plants were harvested and allowed to dry in loosely covered beaker (20-2200) protected from direct sun- light. A sample was taken every day for 5 days. An inoculum with small amount of phosphate buffer pH 7.5 was prepared for mechanical inoculation on healthy pea plants. Virus survival in the dried tissue was measured in terms of infect- ivity. (d) Thermal inactivation studies. Portions of 0.5 ml of the clarified extract were pipetted into micro-tubes and placed in a pre-set constant temperature water bath. After 13 10 minutes' eacposure to the desired temperature they were withdrawn immediately and cooled in flowing cold tap water. Three samples exposed to the same tanperature were pooled and tested for infectivity on healthy pea seedlings. The following temperatures were used3 50, 55, 58, 60, 63, 65, 68 and 70°C. The temperature at which loss of infectivity of PEMV occurred was recorded 25 days after inoculation. (e) Effect of storage of virus juice extract buffered at different pH levels on virus infectivity. Three ml of the clarified juice was pipetted into test tubes containing 3 m1 of 0.1 M phosphate buffer (K23P04-KHZ 904) at pH 5.0, 5.5, 6.0, 6.3, 6.5, 6.8, 7.0, 7.3, 7.5, 7.8 and 8.0. The inocula were stoppered and stored at 263°C. lnoculations were done at 4 days intervals for 12 days on healthy Pacific Freezer plants. Number of plants infected was recorded 3 weeks after inoculation. Pur c t f PM To the knowledge of the writer isolation and purifi- cation of PM has not been reported. Therefore known pro- cedures of virus purification successfully employed with other plant viruses (81, 82) were tried on PM. A milder method of virus purification employing techniques of alternate cycles of high and low speed centri- fugation was develOped for PEN. The purification procedure is described in Results. Unless otherwise stated, low speed l4 centrifugations were done in a Servall Superspeed angle head centrifuge Type SS-lA. For high speed centrifugation work a Spinco Model L preparative ultracentrifuge with rotor no. 50 was «played. Preparation of fins fitiggg and gm prodggign The methods described in the literature (41) for virus antigen preparation and serum production were slightly modified and adapted in this study. (a) Experimental animals and preparation of normal serum. Rabbits weighing about 3-4 pounds were kept singly in suitable wire cages. Normal serum was obtained a week before the virus antigen preparation was administered. (b) Preliminary work on antiserum production. The virus antigen preparations were obtained from clarified Pm- infected extracts or from resuspensions of a virus pellet following one high speed centrifugation. These were pre- pared as follows3 The filtered crude juice extracted from fresh leaf tissues of PEMV-infected peas was centrifuged at low speed (2590 g) for 15 minutes. The clarified virus suspension was mulsified with mineral oil adjuvant (63) containing 103931 of virus, Bayol F, and Arlacel A respect- ively. Four - 6 ml of the emulsion was injected intra- muscularly into the leg of the rabbit. Injections were administered once every 5-6 days for 4 weeks. After the third injection the rabbit was bled through the ear and an antiserum was obtained for antibody titre test. 15 A similar clarified virus suspension was subjected to one hour high speed centrifugation at 25,000 rpm (63,581 g) in a swinging budcet rotor of the ultracentrifuge. The resulting pellet was resuspended in 2-4 ml of 0.01 14 neutral phosphate buffer-physiological saline solution and clarified by low speed centrifugation for 15 minutes. An unulsion of the virus-mineral oil ad juvant was prepared and administered to the rabbit intramuscularly. Antiserum was obtained after the third injection and tested serologically with a similar antigen preparation. (c) Antiserum used in serological studies of PEN. The virus antigen was prepared from a resuspension of the pellet obtained from the second cycle of differential centri- fugation (Fig. l). The pellet was taken up in 1-2 ml of 0.01 M neutral phosphate buffer-physiological saline (0.85%) solution. The virus resuspension from the third cycle of ultra-centrifugation was used in two instances for intra- venous injection into the rabbit. An emulsified preparation of the resuspended virus and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan), 131 V/V, was injectedinto the thigh and calf muscles of the rabbit. Each leg received no more than 1 ml of the virus-adjuvant emulsion per injection. No attempt was made to determine the actual weight of the virus protein in any of the immunizing antigen preparations since virus yields were generally small. The injections were administered 16 4-6 days apart for about 6 weeks. When satisfactory titre was achieved the rabbit was sacrificed by bleeding from the heart completely with a 50 ml glass hypodermic syringe with a No. 18 needle. The blood collected in suitable glass con- tainers was allowed to clot at room tenperature for 2-3 hours. The serum layer was catrifuged once at low speed (1000-2000 rpm) for 10 minutes. The clear antisera contain- ing the antibodies were stored in sealed vials in the freezer until needed. 8 sts The technique anployed in the study of the sero- logical properties of PEMV were essentially a modification of the precipitin test. The methods for the microprecipitin and the Ouchterlony agar double-diffusion tests described by Ball (3) were adopted with some modifications. (a) Microprecipitin test. A titration reaction area was drawn in a checkerboard pattern with a wax pencil on a plastic petri dish. An apprOpriate dilution series of the antigen and of the antiserum was obtained in test tubes us- ing physiological saline solution as diluent. A microdrop of the desired dilution of the antisera was placed in the center of the reaction square and then layered with an equal volume of an apprOpriate dilution of the antigen. Physio- logical saline solution was added to the control series. To minimize the evaporation of the reaction drops during in- . cabation and observation, the petri dish was flooded l7 carefully with mineral oil such that all the reaction drops were submerged completely. After 2 hours incubation at room temperature the dish was observed for any reaction under a binocular dissecting microscope. Incubation was carried further in the refrigerator overnight and observed again. (b) Ouchterlony agar double-diffusion test. A 0.5% ion agar No. 2 (Consolidated Laboratories Inc., P.0. Box 234, Chicago Heights, I11.) in physiological saline solution was prepared in 3-500 ml Erlemneyer flasks and sterilized for 15 minutes in the autoclave. To insure sterile conditions aqueous stainless solution of merthiolate (1310.000 final dilution) or sodium azide solution (135000 final dilution) were added to the agar. Ten ml of agar was poured into each sterile glass petri dish and allowed to solidify. Ten mm lengths were cut from a 12 mm diameter cellulose centrifuge tube and sterilized for a minute in a solution of merthiolate (1310,000) or sodium azide (1315,000) to serve as molds for the wells. The molds were pressed lightly on the thin layer of solidified agar following a predetermined arranganent drawn on paper underneath the petri dish. A small amount of liquid agar was layered around each mold to secure it. Then 15 ml more of agar was added to the petri dish and allowed to harden. The molds were then carefully removed from the agar plate, and the wells were carefully sealed with small amount of liquid agar to prevent the liquid reactants from spreading between the glass and the agar layer. A suitable 18 dilution of the antigen and the antiserum was pipetted into each designated well. The reaction dish was incubated at room tanperature in a moist chamber. Formation of antigen- antisertrn reaction bands was observed for two weeks. The micrOprecipitin and Ouchterlony gel techniques were employed in the serological studies of the, four PEMV isolates. Similarly, preparations of cucumber mosaic virus (CMV) , southern bean mosaic virus (BBMV), Maryland bean virus (MBV) , tobacco ringspot virus (TRSV), and bean yellow mosaic virus (BYMV) strains were serologically tested against the PEMV antiserum for virus relationship determination. Electron microsggpy The homogeneity of the PEMV preparation was deter- mined by electron microscOpy (80, 82, 92) and the morphology and size of the virus particles were determined from the electron micrographs obtained. Following standard techniques in electron microscopy (21, 31) samples were obtained from the purification procedure and prepared for electron micro- scopy. Records of the examination were made on electron micrographs. (a) Preparation of film-coated grids. Copper or silver Lecktromesh grids No. 200 were used as the supporting material for the thin films prepared from a 0.25% Formvar (polyvinylformal) solution in ethylene dichloride. ‘ A clean glass slide was dipped into formvar solution, drained and dried at room temperature. The resulting thin 19 film layer was stripped from the glass slide over distilled water. The grids were placed on the floating film and tapped lightly to insure adhesion of the metal grids on the film. The film containing the grids was scooped out of the water, dried, and stored in petri dish until needed. (b) Size calibration. A 0.01% polystyrene latex (PSL) suspension (Dow Chemical’Company, Midland, Michigan) with an average particle size of 264 mu 1 6 mp was used as an internal standard. It was incorporated with the specimen prior to deposition on the film-coated grids. (c) Specimm deposition. A microdrOp of the pre- pared suspension of purified PM or healthy control prep- aration and P31. was placed on the filmed grid with a micro- pipette. The excess liquid was absorbed at the periphery of the drOp with a strip of filter paper and the preparation was air dried in a petri dish. Deposition was also accomplish- ed by spraying the specimen directly on the formvar-filmed grids (2). A commercial pyrex nebulizer, Vaponefrin No.166, was auployed. (d) Shadow-casting and electron microscopy examina- tion. Tungsten as the shadowing metal was evaporated on the specimen at an angle of about 30° in a Kenney vacuum evaporator unit. The shadowed specimens were examined and electron micrographs were taken with an RCA EMU-2C electron microscope. EXPERIMENTS AND RESULTS Reaction of ggurcg plapt go PEMV isglates The PEMV-2 isolate was regularly infective on Pacific Freezer pea. The infected host plants develOped typical symptoms of PEMV disease consisting of mottling, translucent or hyaline spots and enations in young and old leaves and stipules as well as enations on the pods. The symptoms ex- hibited by the virus isolates PEMV-O, va-W, and PEMV-NY were similar to those of PEMV-Z. The symptoms nonnally were observed about a week after inoculation. H ane dsear f l 1sioht A study on the host range in a search for a suitable local lesion assay plant for PM wasconducted in the green- house. Twenty-three species of plants belonging to six families3 Leguminoseae, Solanaceae, Chenepodiaceae, Amaranthaceae, Cucurbitaceae, and Compositae were used. The species tested and the results obtained are given in Table 1. Susceptibility to PEMV was confined to the leguminous plants. These included all varieties of garden pea (m m L.), two varieties of broad bean (£33.; £323 L.), crimson clover (Tgifolim W L.), subterranean clover (2. W L.), alfalfa “QM-£999. 9.92119. L.), cowpea (liens genesis 32:41.). mung bean (Ella-.219; 9.92232 Roxb-h and soybean (Glygine 3.0.12 (1..) Sieb. 3. mice). 1 20 21 The 15 susceptible pea varieties were the following3 Alderman, Alaska, Dwarf gray sugar, Dwarf telephone (Daisey), Dark skin, Early perfection, Green skin, Improved telephone, Little marvel, Mira green, Osaya endo (OSY 6212), Perfected Wales, Sugar pea (1842 and 2408), Thomas Laxton, and Wisconsin perfection. The infected host plants showed mottling and faint or conspicious chlorotic spots on the leaves. The production of hyaline spots and enations on the leaves and stipules were frequently observed on the garden pea varieties. The infected mung bean and cowpea showed vain clearing and faint mottling of the leaves with no enations produced. In addition, a slight leaf distortion was observed in the former host plant. Although the PEMV-infected alfalfa appeared symptomless except for some occasional vein clearing, recovery of the virus was demonstrated. The leguminous plants which were found not suscept- ible to va were the following3 white lupine (Mg £1133 L.), red clover (W W L.), white clover (3;. m L.), lima bean (W M Macf.) and bean (Phaseolgs m L.) var. Prince, Michelite, and Pinto. The non-susceptible solanaceous plants were jimson- weed (Dgtgrg W L. and 2. tgsulg L.), tomato (W W M111.). and two species of tobaccos W m L. var. Xanthi nc., Ky-57, Sansun, and Nigtigpa glut inosg L. 22 No infection was obtained on the two species of ChenOpodiaceae, W W L. and Q. amarantigglo; Costs and Reyn. In addition, three other plant species from different families were found not susceptible to PEMV. They were3 comon globe amaranth (W glgbgsa L.), cucumber (Cumis sativus L. var. National Pickling), and zinnia (Zinnia 22992.! Jacq.). Back inoculations of the non-susceptible plants to healthy Pacific Freezer plants showed negative results. None of the susceptible plant species were found to be a local lesion host of PEMV. R in it onst PEMVsats Fifty-five pea introductions, Table 2, were tested under greenhouse conditions for their reaction to inocula- tions with PEMV-o, PEMV-W, PENN-NY, and PEN-2 virus isolates. Three of the pea introductions, namely3 Iran (PI-140295), Turkey (PI-175878), and Netherlands (PI-197989) were suppos- edly PEMV-resistant ones. All of the pea introductions tested were to some ex- tent susceptible to PEMV. The pea introduction from Iran showed some tolerance to virus infection. The young leaves of some successfully infected peas with pmv-o and PEMV-w showed faint mottling but no enations were observed 1-2 weeks after inoculation. Mottling was also eXpressed in fully expanded leaves but became inconspicuous later with no discernible symptom of the disease even in young terminal 23 Table 1. Results of host range study of PM. W Host Plant Reaction to PEMV Alfalfa( (Mggigago sativa L.) + Bean (Phasglus vulgaris L.) var. Michelite - “ Pinto - 5 Prince - Broad bean (Vicig faba L.) Broad improved long pod + McWhorter's selection, 1957 Lot + Chen0podium (Chenopgdigm pgqugm,L.) - (2. amarantigglgg Coste and Reyn.) - Common globe amaranth (figmphgggg_glgbosglL.) - Cowpea (Viqpa sinensis Endl.) + Crimson clover (Trifolium _gg___g§gnfL. ) + Cucumber (Cucgmis sativgs L.) var. National Pickling - Garden pea (z;g§m_sativum L.) + Jimsonweed (Dgtgga W L.) - (Q. tatula L.) '- Lima bean (Phaseolus ligensis Macf.) - Mung bean (ghasgglus aureus Roxb.) + Red clover (Trifolium mg L.) - Soybean (Glycine 3913 (L.) Sieb. and Succ.) + subterranean clover (Tgifoligm ggyggggggigm,L. ) + Tobacco (W W L.) var. Xanthi nc - 3 agile I (N. glutiposg L.) - Tomato (Lycopersicum esculgptgg Mill.) - White clover (Trifoligm repgps L.) - White lupine (Lupinus £133 L.) - Zinnia (ginnig elm; Jacq.) - 24 leaves. All the infected plants grow to.mtturity, produced pods, and in most respects were comparable to the accompany- ing uninoculated plants. AA similar reaction was observed in most PEMV-Z inoculated peas except in one case where‘hyaline spots and blister-like enations appeared on the leaves and stipules much later, 3-4 weeks, after inoculation. Schroeder and Barton (65) observed a similar case which they described as the effect of an aberrant strain of PEMV. Although yellowing and drying out of inoculated leaves which remain attached to the plant were Observed in peas inoculated with PEMV-NY, no other recognizable symptoms of the virus disease were noticed throughout the whole period of observaflon. However, in a separate inoculation expernment of the virus isolate on the pea introduction from Iran, symptoms similar to those described in the earlier two virus isolates were obtained. The pea introduction PI Nos. 175878 and 197989, from Turkey and.Netherlands respectively eXhibited a rather similar and fairly uniform reaction to PEMV inoculations. While some peas inoculated with the virus isolates showed either no detectible sign of virus infection or only'mottling of leaves, other infected peas developed enation mosaic virus disease. Although the symptoms were generally'mild some plants exhibited an appreciable stunting effect to virus infection. In most cases, normal-looking and slightly dis- torted pods with mild enations were produced. 25 Table 2. A list of pea (Pisgm satizgm L.) introductions* tested for their reaction to PEMV isolates. J i J it Sample No. PI Number Origin 1 140295 Iran 2 175878 Turkey 3 197989 Netherlands 4 24409 2 Hell and, Anthonie 5 244093 ' Apollo 6 244094 * Artemes 7 244101 4 Bon Amie 8 244110 “ Ceroso 9 244121 ‘ Crescent 10 244122 5 Cupido 11 244124 “ De Grace 12 244129 5 Demi Main Debeve 13 244130 * Dutigdaagse 14 244137 * Engelsk Sabel 15 244144 5 Fortune 15 244145 5 Fuga 17 244150 Goldkonigin 18 244151 Holland, Gaudraatje 19 244153 ' Gryze Reuzen 20 244154 * Grote Slier 21 244158 * Halfstam 22 244151 Heraut Table 2. Continued ....... m 26 Sample No. PI Number Origin 23 244162 Holland, Hogs-Late Greene 24 244153 5 Insulende 25 244155 5 Juni Sabel 25 244170 5 Konig der Carouley 27 244174 5 Kungsart 23 244175 5 Large Smelt Peul 29 244175 5 Large 30 244175 5 Lar Sabel 31 244189 '3 Moerheimes Reuzen 32 244190 5 Monster 33 244191 5 Morgenster 34 244194 5 Namum 35 244195 5 Olympia 35 244199 5 Oscar 37 244201 5 Overload 38 244214 5 Rasperpeulen mixture 39 244215 5 Rembrandt 40 244217 5 Reuzenboter gekrenkt 41 244213 5 Reuzenboter rond 42 244225 5 Rubens 43 244235 5 Surpette Vert 44 244245 5 Superonaugetont 45 244246 5 Sylvester 27 Table 2. Continued ........ m Sample No. PI Number Origin 45 244248 Holland, Tilly 47 244251 5 Uhiversales 48 244252 5 Veense 49 244255 5 Vermeer 50 244255 5 Vesta 51 244257 5 Vincent 52 244251 5 Vroege Hendriks 53 244252 5 Vroege Sinkepeul 54 244254 5 Wilhelm Tell 55 244272 5 Zwitzerse Reuzen *Supplied by North Central Regional Plant Introduction Station, Ames, Iowa. The other pea introductions inoculated with the PEMV isolates generally developed typical symptoms of enation mosaic virus disease. The symptoms were quite uniform though they varied somewhat in their severity. In most cases they exhibited a rather inconsistent symptom develOpment to infection with the virus isolates. Nineteen of the pea introductions, sample nos. 5, 7, 13, 14, 25, 26, 27, 28, 30, 31, 32, 34, 35, 43, 44, 46, 47, 28 49, and 52, showed severe PFMV infection resulting in a con- siderable size reduction. The following symptoms were observed3 numerous hyaline spots and enations on leaves and stipules, numerous chlorotic spots or large blotches of chlorotic areas on the eXpanding leaves leading to severe distortion in apical leaves, sometimes tap necrosis and the subsequent production of side shoots with typical enation symptoms on them, and the development of enations varying in size from small blister-like to leaf-like outgrowths on older leaves and stipules. Large leaf-like outgrowths, also referred to as lamina-like or the true enations (86, 87) were frequently observed on matured stipules, occasionally on the older leaves of pea introduction sanple nos. 35, 38 and 44 infected with PEMV-O, PEN-W and PEMV-Z. The pods, if produced, were often distorted bearing a few crater-like irregular blisters or numerous about circular blisters or furrow-like enations. The former type of pod enation was observed in pea introduction sample nos. 26, 32 and 35 and the latter two were the common types. Pods of pea intro- duction sanple No. 4 exhibited small nipple-like projections of papillae when infected with PEMV-NY and PEMV-2. A some- what milder reaction to PEMV infection with no appreciable stunting effect was observed on pea introduction smnple Nos. 4, 8, 14, 16, 20, 33, 37, 39, 40, and 47. The rest of the pea introduttions showed an intermediate response to PM infection but as a rule they produced typical enation symptoms. 29 Physico-Chemicgl pggpgggies 2f PEMV (a) Tolerance to dilution. Variable results were obtained from two separate trials on the tolerance to dilu- tion studies of the PBMV isolates (Table 3). Furthermore, the values were generally lower than those reported in the literature. The PEMV-O was infective when diluted to 131000 but only slightly so at 132000. On the other hand PEMV-W was infective at 132000 dilution but not at 133000. A.muCh lower dilution tolerance was found with PEMV-NY. lAt 131000 dilution no infectivity was demonstrated in one trial whereas slight infectivity occurred at 132000 in another trial. PEMV-Z was infectious at dilutions of 132000 and slightly so at 133000. From these results it appeared that PEMV has a dilution end point between 132000 - 133000. 5(b) Inactivation $9,315gg. ‘Virus infectivity was completely lost after 4 days aging of sap from infected plants at 20°-22°C. (é) Longevity of PEMV in dried tissue. ZLeaves of Pacific at...“ pea infected with pmv when allowed to dry in loosely covered'beakers in the greenhouse (20°-22°C) lost its infectivity after the third day of drying. (d) Thermal inactivation point of PEMV. PEMV-2 was infective after 10 minutes exposure to 55°C but not to 60°C. In two separate experfments employing PEMV-W the thermal in- activation temperature was between 63°-65°C. No virus activity was observed after 10 minutes exposure to 68°C. The values obtained were found higher than those for PEMV-2 isolate. 30 Table 3. Tolerance to dilution of the PEMV isolates. M Rec‘gffcal pmv-o paw-w PEMV-NY va-z dilution 1 2 l 2 1 2 l 2 o 3/ 5* 4/ 5 6/6 5/7 4/5 3/6 6/7 5/6 1000 1/6 2/5 3/5 2/5 2/5 0/5 3/5 3/6 2000 0/5 1/5 2/6 1/5 1/6 0/5 2/5 1/3" 3000 0/5 0/6 0/5 0/6 0/6 0/5 1/6 0/5 4000 0/5 0/6 0/5 0/5 0/5 0/6 0/5 0/5 5000 0/5 - 0/5 - 0/5 - o/s - * No. of plants infected/no. of plans inoculated ** 2 inoculated plants died 5 days after inoculation, no PEMV infection detected. - not determined. ":01. __~‘.-.-. V1- W0- -. : t . 3- , s - v 1 s in ectfluit; The infectivity of buffered PEMV-w juice extracts was generally'higher at pH 7-8.0 than at lower levels in either freshly prepared inocula or when stored igflziggg for 12 days at 2°~3°C (Table 4). Symptoms of the virus generally appeared earlier, within 446 days after inoculation, in plants mechanically inoculated with fresh inocula at pH 6.5- pH 7.5. Also more plants showed infection within a week from date of inoculation. The symptoms were a faint mottle and/or translucent spots in developing leaflets, stunting and chlorosis. 31 A reduction in infectivity of the buffered virus ex- tracts occurred in all pH levels throughout the aging of 12 days. Plants inoculated with 4-day old inoculum stored at pH 5.0, 5.5. or 6.0 showed no symptoms to PEMV. This also occurred with 8-day old inoculum at pH 6.3 or 6.5 and with 12-day old inoculum at pH 6.8, 7.0 and 8.0. Additional tests with some preparations confirmed the complete loss of virus activity. The buffered inoculum at pH 7.3, 7.5, and 7.8 still retained some infectivity after 12 days. Table 4. Effect of pH of buffered-virus juice extracts on virus infectivity and its relation to inactivation __'—- *7... “ —_—._. ”7,7 Agig " o f -v M 1 (days) 5.0 5.5 6.0 6.3 6.5 6.8 7.0 7.3 7.5 7.8 8.0 0 4/6' 5/7 5/6 4/5 6/8 6/7 7/8 6/6 5/5 6/6 5/5 4 0/5 0/7 0/6 1/5 2/8 2/5 5/7 6/8 5/6 5/7 3/4 a - 0/5 0/5 0/5 0/9 1/8 2/6 3/7 4/7 3/5 3/9 12 - - 0/5 0/6 0/5 0/8 0/7 1/7 1/6 1/8 0/5 *number of plants infected / number of plants inoculated. -not determined. The addition of a buffer solution. pH 7-7.8, to crude virus extracts at the rate of 1:1 V/V increased the survival of PEMV more than any of the other pH levels. 32 I 0 cl tio and r £1 a o of EMV (a) Preliminary investigation. PEMV-infected leaf tissues of Pacifichreezer pea, 10-18 days after inoculation, were used in the preliminary experiments on virus extraction, clarification and.purification. Freshly harvested tissues were macerated with KZHPO4 powder, 3% by weight, in a mortar or in Waring blender. The crude juice extract was expressed.by squeezing the macerate through cheesecloth. The resulting extract was infectious on the assay plant. Various means of clarification of crude juice exp tracts were tried (82). To determine whether the treatments removed host components and other virus contaminants com- parable virus-free plant tissue extracts were subjected to similar clarification procedure. Low speed centrifugation of the buffered crude virus extracts eliminated.most of the heavy plant debris. The in- corporation of the Kzspo4 powder to the macerated tissues effected coagulation of the chloroplastic materials and other non-infectious fractions which upon centrifugation were thrown down in the green pellet. This was similarly observed in the clarification of other plant viruses (75, 76, 77). The addition of 0.1M phosphate buffer (K239043KHZTO4). pH 7.5, to the crude juice extracts, 1:2 V/V, prior to low’speed centrifugation gave similar results. The buffer facilitated the sedimentation of the host contaminants with no apparent loss of virus activity in the resulting clarified suspension. 33 The pH of the buffer used falls within the range of pH 7.0- 8.0 reported to yield infectious virus preparations (82). Addition of phosphate buffer at lower pH levels (below pH 6.8) to the crude juice gave much clearer supernatant liquids after centrifugation but loss of virus activity occurred. The pH of the crude juice extracts from PEMV- infected pea tissues ranges from 5.6-6.2 while these of comparable virus-free tissues ranges from 6.0-6.8. In an attempt to improve the clarification method, the buffered crude sap was subjected to high speed centri- fugation in the ultra-centrifuge (Swinging-bucket Type SW- 25-1 rotor) for about 5 minutes at 25,000 rpm (63,581 g). The clear slightly green supernatant was not infectious, and most, if not all, of the virus was lost to the heavy dark green pellet. This was similarly observed in the clarification of tomato bushy stunt virus (93). The green pigments which persisted in the low speed supernatant liquid were eliminated by adsorption with acti- vated charcoal, Merck NP-powder 18351 (l6, 17). It gave an almost colorless filtrate but no demonstrable virus activi- ty. These results were similarly obtained with crude sap extracts. The chloroformébutanol method of virus clarification (81) resulted in a clear light brown aqueous phase but the treatment reduced the infectivity of PEMV. 34 Buffered crude juice extracts from frozen infected tissues were as infectious as those obtained from recently harvested tissues (Table 5). Pre—treatment of the diseased tissues by freezing and the addition of phosphate powder or buffer facilitated the maceration and extraction of the crude virus juice. Based on the preceding results and observation, freezing the infected tissues, use of appropriate buffer and low speed centrifugation were satisfactory for the clarifica- tion of PEMV. The density gradient centrifugation method (10, 11) was tried for the purification of PEMV. Tubes layered with sucrose gradients and with the clarified virus suspension were centrifuged for l to 15 hours at 25,000 rpm in the ultracentrifuge. .A light brown upper band and a whitish lower band were obtained. The healthy control preparation runatthe same time yielded similar bands indistinguishable in appearance and position in the gradient from those of the PEMV-infected preparation. The lower band from the gradient layered with the virus suspension was usually infective but infectivity was quite low or sometimes lacking. The com- parable band in the healthy control preparation was never infectious. Electron microscopic examination of the in- fectious band revealed small masses of aggregated somewhat spherical virus-like particles. Microeomes were also present. The differential centrifugation technique produced more satisfactory results than the density’gradient centri- fugation method. The virus preparation obtained was invari- ably infectious. 35 (b) The purification of PM. Based on the results and observation in the preliminary war): pea enation mosaic virus was isolated and purified from the diseased tissue ex- tracts following the procedure outlined in Figure 1. Leaves and stipules from PEMV-infected peas, 12-14 days after inoculation, and diseased green pods were harvested and weighed. The tissues were wrapped in aluminum foil and stored in the freezer 24-36 hours. The tissues with the phosphate powder added were macerated while frozen. When completely thawed, the crude juice was expressed through 4 layers of cheesecloth. The crude juice filtrate contained masses of chloroplastic materials and other aggregated de- natured host components which tended to settle upon standing. The pulp residue was moistened with additional 0.1 M phos- phate buffer pH 7.5 and re-extracted. Recovery of more virus from the pulp by re-extraction with phosphate buffer (4. 57) was assumed but no infectivity test was conducted on the re-extracted juice. The two extracts were peeled and clarified by centrifugation for 15 minutes at 4,500 rpm (2, 590 g). A clear slightly green to light brown supernatant liquid and a heavy green pellet with a whitish layer mostly starch were observed in the centrifuge tube. The clarified juice retained most of the virus activity present in the original crude sap (Table 5). The clarified extract was frozen for 2 hours, allowed to thaw at room temperature, then cmtrifuged further at 36 6,500 rpm (5,400 g) for 15 minutes. Freezing the clarified suSpension, then thawing and subjecting to a higher centri- fugal speed removed some of the colored pigments and other denatured non-infectious host proteins with no apparent harmful effect on the infectivity of the virus. The virus in the clarified juice was sedimented in the ultracentrifuge (-5°C) at 40,000 rpm (96,592 g) for 1 hour. Infectious pellets, light brown and somewhat glassy in appearance, were obtained. The non-infectious super- natant liquids were discarded and the pellets were then covered with 2 ml of buffer (0.01 M.KCl, 0.005 M KiHP04, 0.0005 M KH 1’04: pH 7.5) and allowed to resuspend overnight 2 at 2-300. The resuspended pellets were pooled and then centrifuged for 15 minutes at 3,100 rpm (1,330 g). The clarified virus suspension was further subjected to two more cycles of differential centrifugation. Bash of these con- sisted of a l-hour high speed centrifugation (40,000 rpm), resuspension of the virus in buffer solution, then low speed centrifugation for 15 minutes at 3,100 rpm. A colorless gelatenous pellet was obtained from the final high speed centrifugation. The buffer resuspension of the virus pellet was infectious (Table 5). Extracts from comparable healthy control plant tissues subjected to the same purification procedure some- times gave variable results. Often no distinguishable pellets were observed from the second high speed centrifuga- tion. In some trials, however, small amounts persisted 37 throughout the preliminary purification. Nevertheless, no infectivity was associated with the preparation. The final ultracentrifugation of the clarified healthy suspension in- variably produced no pellet at all. Purified virus suspension in glass distilled water was inactivated when stored frozen overnight. Virus in- activation during storage was minimized by using buffer solution pH 7.5 as the suspending medium. Freezing of puri- fied virus aqueous suspension was reported deleterious to tobacco ringspot virus (75) and to bushy stunt virus (5). Differential centrifugation teChnique has been reported satisfactory in the purification of the following plant viruses: cucumber mosaic 3 and 4 (33, 66, 82): potato yellow dwarf virus (8); potato virus x (38): southern bean mosaic (57): squash mosaic (35, 60): tabacco mosaic and strains (82): tobacco necrosis (6, 58); tobacco ringspot (75): tomato bushy stunt (76, 82); turnip yellow mosaic (15): and wild cucumber mosaic virus (70). S o 1 es f PEMV (a) Microprecipitin reaction. In preliminary sero- logical studies the antiserum was obtained from rabbits immunized with clarified virus extracts or with preparations obtained after one cycle of differential centrifugation. Results indicated the presence of antigenic host components which interfered in the detection and identification of a specific virus-antiserum reaction. Therefore the virus 38 Table 5. Relative infectivities of PEMV fractions obtained from three purification trials. m Stage of Purification Fraction Infectivity 2 Buffered crude juice ex- 12/15* tract, fresh infected tissue - 2 Buffered crude juice exp 11/13 tract, frozen infected tissue 3 Clarified juice, first lowb 7/11 speed centrifugation 4 Clarified juice, second lowb 8/15 speed centrifugation ‘ 5 Supernatant of virus pellet, I 0/9 6 Resuspended virus pellet I in. 1/10 distilled water, clarified 6 Resuspended virus pellet I in 9/14 buffer, clarified 8 Resuspended virus pellet II 7/13 in buffer, clarified 10 Resuspended virus pellet III 6/15 in buffer, clarified *Total No. of plants infected / total no. of plants inoculated antigen used for the immunization of the experimental animals was further purified and concentrated. The purified PEMV-W preparation, which consisted of more or less homogeneous spherical particles in the e1ectron.microscope and containing viral activity, was satisfactory for PEMV antiserum produc- tion in rabbits. 39 Figure 1. Purification procedure of pea enation mosaic virus (PEMV) . 1. Infested tissue (pea, Pacific Freezer) freeze grind while frozen KZHP04, 3% by weight thaw extract juice through cheesecloth clarify - 4,500 rpm, 15 min. P04 buffer pH 7.5 __ _ _ * regrind 3. Clarified juice — pellet re-extract freeze, 2 hrs. thaw C de ice - pulp? clarify - 6500 rpm, 15 min. 4. Clarified juige _ ===pellet* centrifuge - 40,000 rpm, 1 hr. 5. ggllet ----§---- ------ supernatant liquid* resuspend in buffer pH 7.5 overnight in refrigerator shake tube gently clarify - 3100 rpm, 15 min. 6. Elagigied suspensiog -- ----- pellet* Centrifuge - 40,000 rpm, 1 hr. 7. P let -- —~ — -------ggpernatgnt liquid* resuspend in buffer pH 7.5, 2-3 hrs. shake tube gently clarify - 3100 rpm, 15 min. 8- WW ----- let" centrifuge - 40,000 rpm, 1 hr. 9. Pellet -------------ggpernatgnt liquid* resuspend in buffer pH 7.5 or in glass distilled water - 2 hrs. clarify - 3100 rpm, 15 min. 10. Eggified virus spgpgpsion - pellet* *discard P04 buffer (0.1M “3204432900 pH 7.5 Buff” PH 7.5 ‘0.01M KCl, 0.0054 KZHPO4,O.OOOSM KHZ P04 40 This antiserum gave a positive precipitin reaction when reacted with a similar preparation of PEMV. The characteristic precipitin reaction was indicated by a slight- ly fluffy white precipitate in the microdrOp of virus- antiserum mixture. Formation of the precipitate at lower dilutions of the antigen was observed within two hours of incubation, at room tenperature. When allowed to react further by incubating the titration dish in the refrigerator overnight, a virus titre of 1/1024 was observed (Table 6). The controls of the antigen and the antiserum, mixed with saline solution in neutral 0.01M phosphate buffer, showed no precipitation. In the micrcprecipitin test between the antiserum and a similar healthy preparation a non-specific reaction was observed after overnight incubation of the re- action dish. No characteristic precipitin reaction formed at any dilution of the healthy antigen and the PEMV antiserum (Table 7). These results denonstrated the specificity of the antiserum to PM and also the presence of some antigenic host components in the anti serms preparation, but the non- specific and virus-specific precipitation reaction with the antiserum can easily be distinguished. Tests were conducted with the normal serum of the rabbit and clarified extracts of PEMV-infected and non- infected leaves of Pacific Freezer pea. The crude juice 41 Table 6. Microprecipitin test between the purified PEMV-W preparation and its antiserum 1 Reciprocal °f Reciprocal of antigen dilution antiserum dilu- tion 4 s 16 32 64 123—6256 Efz 1024 AsC* 4 4+ 4+ 3+ 3+ 3+ 2+ 2+ + + :2 8 4+ 4+ 3+ 3+ 2+ 2+ + + + .— 16 3+ 3+ 2+ 2+ 2+ + + + + - 32 3+ 2+ 2+ + + + + .. .. .. Agcee .. .. .. .. .. .. .. - .. - * Antiserum control ** Antigen control - No reaction + Relative intensity precipitin reaction Table 7. Microprecipitin test between PEN-W antiserum and purified healthy control preparation. M miggoninudifu- Reciprocal of antigen glution 1 tion 4 8 16 _32 64 128 256 512 1024 AsC' 4 NS NS NS NS NS NS - - - .. 8 N8 NS NS NS NS - - - .. - 16 , NS NS NS - - - .. - - .. 32 NS NS - - - — - .. .. .. Agcee .. _ _. - .. .. .. .. .- .— * Antiserum control ** Antigen control - No reaction NS Non-specific precipitation 42 obtained from the frozen tissue was diluted to 1/4 with neutral phosphate buffer and clarified by cmtrifugation for 15 minutes at 4, 500 rpm. Neither clarified extracts from PEMV-infected nor healthy control leaves showed a specific precipitin reaction. A slightly milky greenish suspension was observed in first dilution but beyond which no precipi- tate was observed (Table 8). Crude extracts of PEMV-W and PEMV-O were centrifuged for 5 minutes at 8,000 g in a Colman Microcentrifuge (Cat. No. 6-810) to remove most of the cellular debris. A positive precipitin reaction was observed between the antiserun and the clarified QEMV-infected extracts. Preparations from healthy control plants gave no reaction (Table 9). This shows that the antiserum could be used to detect the presence of the virus in clarified extracts from diseased tissues. Clarified extracts of other plant viruses were allowed to react with the antiserum to determine their sero- logical relationship to PEI/N. The following viruses were obtained from leaves of infected source plants: two strains of cucumber mosaic virus (CMV-2 and CTN-9) from tobacco, Nicotiang tabgm L. mthi nc: a strain of southern bean mosaic virus (SBMV 15-2) from cowpea, 24.923 W In: four strains of bean yellow mosaic virus (BYMV 61-35), (BYMV 61-36) pod distortion strain, (BM-81928), and (MBV—Z) Maryland bean virus all of which were harvested from infected broad beans, m; gm 1'... var. minor, McWhorter strain: 43 Table 8. Microprecipitin test between normal rabbit serum and clarified extracts from PEMV-w infected and healthy control Pacific Freezer leaves. rw—q. _. cal of norm Reci serum dilution Antigen dilution 1/4 4 8 16 32 64 128 256 512 1024 .AgC* Healthy pre- PEMV-W preparation NS - - - - - - - - - ABC. * - - - '- - - - - .- - * Antigen control ** Antiserum control NS Non-specific reaction Table 9. Microprecipitin test'between PEMV-w antiserum and clarified extracts from PEMV-infected and healthy control Pacific Freezer leaves. Antigen dilution Reggprocal of antiserum dilggiog 1/4 4 8 16 32 64 128 256 512 102 AgC* Healthy preparation NS NS as - - - - - - - pmv-w 3+ 3+ 2+ 2+ 2+ + + + .. - PEMV-O 3+ 2+ 2+ 2+ + + s - .. .. A‘C** - _ - - - _ - - - _ * Antigen control ** Antiserwm control NS Non-specific reaction + Relative intensity of precipitin reaction 44 and three strains of tobacco ringspot virus (TRSV 60-21), (TRSV 60-22), and (TRSV-T) American type culture isolate AC-98 harvested from diseased tobacco, N. tabacum L. xanthi nc., leaves. The CMV strains were obtained from Dr. D. deZeeuw, the SBMV from Dr. H. H. Msrakishi, and the rest of the legume viruses from Dr. A. Andersen, Department of Botany and Plant Pathology,.Michigan State University. Crude virus extracts were obtained from the infected source plants and.clarified by centrifugation for 5 minutes at 8,000 g. Pre-chilled neutral phosphate buffer was used to form a serial twofold dilution of each clarified virus preparation. An antiserum dilution of 1/25 was made to re- act with the various virus dilutions. A similar preparation of PEMV-W and healthy control from P. Freezer pea was in- cluded in the test. After 2 hours incubation at room temperature specific precipitation reaction was observed in the PEMV-W antigen- antiserum drOps at lower dilutions. No reaction was noted at any dilution in the other viruses. Table 10 shows the results of the test after incubation overnight in the refrig- erator. Only the clarified PEMV-W preparation gave a posi- tive reaction to the antiserum. The CMV and TRSV strains showed no reaction to the antiserum while the SBMV, MBV, and BYMV strains exhibited trace amounts of non-specific precip- itation reaction whiCh resembled that of the'healthy control preparation. The presence of normal host proteins in the 45 clarified preparations of SBMV, MBV, and BYMV strains having similar antigenic sites as those in the healthy control may account for the trace reactions. Table 10. Microprecipitin reaction between the PEMV-W anti- serum* and the clarified preparations of CMV, SBMV, MBV, BYMV and TRSV strains. R 1 ant en dilution ““99" “up“ 4'“? 5.2;“ W32 6 {BET—F25 2W0 s . PEMV-W 4+ 3+ 3+ 2+ 2+ 4' + + - .. CMV-Z - - - - - - - - - - CMV-9 - - - - - - - - - - SBMV-lS-Z NS - — - _ - - - - - MBV-2 NS NS - - - - — - .. .- BYMV-6l-35 NS NS NS - - - — - - - BYMV-61-36 NS NS - - — — - - - - BYMV-81928 NS NS NS - - - - - - - TRSV-60-21 - - - - - — - - _ _ TRSV-60-22 - - - - - - - - - - TRSV-T - - - - — — - - - - Healthy extract NS NS NS NS - - .. - - .. P. Freezer * l/ 25 dilution ** Antiserum control NS Non-specific precipitation - No reaction + Intensity of precipitin reaction 46 The CMV and TRSV strains which were obtained from a taxonomically different host plant from the other viruses gave no precipitation reaction to the antiserum. Results of the microprecipitin tests demonstrated PEMV as an individual virus entity and serologically unrelated to CMV, MBV, SBMV, and TRSV. (b) Reaction of PEMV isolates and other plant viruses to the antiserum in agar gel diffusion test. Pre- cipitation reaction bands were Observed when the purified PEMV isolates and similar preparation of healthy control were allowed.to react with the homologeous antiserum in agar double-diffusion wells (Figure 2). These‘bands, formed in the region of optimal proportion, were noticed 8-10 days after the reaction dishes were incubated in the moist chamber. All of the PEMV isolates produced two types of precipitation bands, a more or less straight and a slightly curved one, in the reaction area. The straight reaction bands, fommed near the antiserum well, were usually observed earlier than the other. The reaction area between the‘healthy control and the antiserum showed only one precipitation band. It was similar in shape and position to those straight bands observed in the PBMV isolates. These bands were considered to represent a non-specific reaction between the normal host proteins and the antiserum. This type of reaction was also Observed in the microprecipitin test. 47 The succeeding bands, slightly curved towards the antigen wells and differing slightly in intensity, were believed to represent a specific precipitin reaction between each of the PEMV isolates and the homologous antiserum. This type of reaction band was absent in healthy control preparation. These results were observed in reaction dishes containing ion agar treated with either merthiolate or sodium azide solutions as preservative. In the control reaction dish with no preservative added, similar results were indi- cated although the Observation of the precipitation bands were at times obscured by bacterial contamination which appeared within a week of incubation in the moist chamber. Results of the Ouchterlony agar double-diffusion test showed close relationship of PEMV-W with the other PEMV isolates and also confirmed those observed from the microprecipitin test. The agar gel technique was found to be a more sensitive test for the specificity of the antiserum and the homogeneity of the virus antigen preparation (18). No precipitation bands were Observed between the reservoirs of PEMV antiserum and the clarified extracts of CMV, SBMV, BYMV, and TRSV strains when sodium azide was in the agar (Figure 3). ‘A clarified extract of PEMV-W showed two precipitation bands and one band in the healthy control. These results confinmed the microprecipitin test in showing no cross-reaction between the PEMV antiserum and the 10 other viruses tested. Extracts containing MBV and.BYMV 48 strains obtained from broad beans tended to darken during incubation at room temperature. A somewhat different result was obtained in the re- action dish treated with merthiolate solution. Clarified extracts of MBV, SBMV, CMV, and BYMV strains exhibited single reaction bands to PEMV antiserum similar to those of the healthy control (Figure 4). The CMV strains produced faint precipitation bands while those of MBV, SBMV, and BYMV strains were more distinct. The TRSV strains did not react with the PEMV antiserum. E ectron m sco of PEMV Attempts were made to examine PEMV-containing sap with the electron microsc0pe. The exudate method (27); dip method (12, 13): and the spray technique (22) were tried with no positive results in the identification of likely virus particles. Results of the electron microscOpy of shadowed puri- fied PEMV preparation showed that in gross appearance the PEMV particles were nearly spherical in shape as evidenced by approximately circular outline and their symmetrical shadows. A purified PEMV-W preparation in distilled water contained essentially'monodispersed spherical particles of uniform size and.morphology and generally free of microsomal contamination (Figure 5). A.similar preparation from a phosphate buffer resuspension showed some kind of aggregation (Figure 6). The electron micrograph also disclosed the 49 Figure 2. Precipitin reaction band patterns obtained in the Ouchterlony agar double-diffusion test between the pmv-w antiserum and the purified preparations of healthy control and PEMV isolates. No. (1) healthy control, (2) PEMV-O, (3) PEMV-NY, (4) PEMV-W, (5) PEMV-z, center well-PEMV-antiserum. Curved precipitin lines are caused by the virus antigens and the straight lines by the antigenic host proteins. This was observed in a preparation of 0.5% ion agar with sodium azide (135000) preservative incubated for 12 days in a moist chamber. Figure 3. Ouchterlony agar double-diffusion test between the PFMV-W antiserum and other plant viruses observed in ion agar (0.5%) preparation with sodium azide (185000) preservative, 14 days incubation. Center wells contained the antiserum and the side wells (1) PEMV-W, (2) CMV-2, (3) CMV-9, (4) SBMV 15-2, (5) MBV-2, (6) BYMV 61-35, (‘7) BYMV 61-36, (8) am 81928, (9) TRSV 60-21, (10) msv 60-22, (11) TRSV-T, and (12) Healthy control preparation from P. Freezer. Note that no precipita- tion bands are formed with the other viruses (Nos. 2-11). 51 Figure 4. Ouchterlony agar . -~ - e-diffusion test between the PEMV-W antiserum and other plant viruses observed in ion agar (0.5%) preparation with merthiolate (1310.000) solution pre- servative, 14 days incubation. Center wells contain the anti- serum and the side wells (1) PEMV-W, (2) CMV-2, (3) CMV-9, (4) SBMV 15-2, (5) MBV-Z, (6) BYMV 61-35, (7) BYMV 61-36, (8) BYMV 81928, (9) TRSV 60-21, (10) TRSV 60-22, (11) TRSV-T, and (12) Healthy control preparation from P. Freezer. No precipitation bands are formed with the TRSV strains and the antiserum. 52 presence of a few snaller particulates of less uniform morphology and size. In general, the purification procedure developed for PEMV eliminated most if not all of the host contaminants. No comparable virus-like particles were observed in similar preparations of the healthy control (Figure 7 and 8). Occasional bits of small particles and aggregates, few in number could be observed. Although some of these particles were spherical, their sizes were less than 12 mp. The existence of some host components of similar morphology but smaller in size corrOborated the results obtained from the serological studies. These contaminants were believed to be normal host proteins (32). Healthy samples were generally free of these particles. Numerous spherical particles were observed on a partially purified PEMV-W preparation (Figure 9). Aggrega- tions of the virus particles are evident in this preparation. Individual virus particles could be Observed along with some host contaminants in the preparation. Earlier microscopic studies of PEMV-2 isolate pre- pared by the same purification procedure revealed the presence of two types of characteristic particles, spherical and rod-shaped ones (Figure 10). The former were of the same size and morphology as those observed in the PEMV-W pre- parations and therefore are believed to be the PEMV-2 particles. Obviously, the rod-shaped virus particles were contaminations. This preparation was infective, giving rise S3 to disease symptoms of PEMV. In some instances, however, leaf spotting was observed to be masked with milder type of mottling or mosaic on the leaves. Another electron micrograph of PEMV-Z purified preparation shows long thread-like particles interspersed with the spherical PEMV particles (Figure ll). This puri- fied preparation was obtained from frozen extracts of in- fected green pods of pea plants showing marked mottling of the leaves and small enations on the stipules. None of the rod-shaped particles were observed on the healthy control preparations. PEMV particles, as compared with polystyrene spheres (264 mp I 6 mp), had an average diameter of about 20 mp. Figure 5. 54 Electron micrograph of a purified PREV-W from a distilled water suspension after the third ultra- centrifugation. The preparation was sprayed on formvar film coated grid and shadowed with tungsten (approximately x 57,000) . Figure 6. 55 Electron micrograph of purified preparation from psnv-w infected pea tissues. Air-dried from a drOplet of virus suspension in phosphate buffer pH 7.5 on formvar film coated grid, shadowed with tungsten and examined in the electron microscope (Res-EMU 2c). Large white sphere is a polysty ene latex particle which has a diameter of 264 mu- 6 mu. (x 56,844). Figure 7. 56 Electron micrograph of a purified preparation from healthy pea tissues, 3-cyc1es of differential centrifugation, obtained from the resuspended per- sistent pellet. Preparation in distilled water suspension, sprayed, air-dried and shadowed wit tungsten. Pol styrene latex particles, 264 my 6 mu (X 37,878 . Figure 8. 57 Electron micrograph of purified preparation from healthy pea tissues, 3—cyc1es of differential centrifugation. Preparation in phosphate buffer pH 7.5 suspension, sprayed, air-dried and shadowed with tungsten. Polystyrene latex particle, 264 mp t 6 up in diameter. (x 56,844) Figure 9. 58 Electron micrograph of partially purified PEMV-W preparation resuspended in phosphate buffer pH 7.5 obtained from the second ultracentrifugation. An air-dried preparation and shadowed with tungsten (x 56,844). L m Figure 10. 59 Electron micrograph of purified preparation from PEMV-Z infected pea leaves, 3 cycles of differ~ ential centrifugation, in distilled water suspension. Note the presence of some rod-shaped virus part- icles found a contaminant of the isolate, small spheres are PEMV €articles. Polystyrene latex particle, 264 mu 6 mu in diameter, shadowed with tungsten. (X 56,844) Figure 11. Electron micrograph of purified PEMV-2 infected pod preparation showing the presence of thread- like virus contaminant. Preparation was obtained after the third cycle of ultracentrifugation and resuspended in phosphate buffer pH 7.0, deposited as a microdrop on formvar film coated grid and shadowed with tungsten. Polystyrene latex part- icle has a diameter of 264 mu * 6 mu (1: 37.878). DISCUSSION The apparent limited range of PEMV infectivity to leguminous plant species (24, 42, 69) was further demon- strated in this study. Most alfalfa varieties were reported symptomless carriers of the virus (42) and this may be the reason why several investigators did not observe PEMV in- fection on the host plant. Results of the study on the susceptibility of alfalfa to virus inoculation agree with findings of other workers (42, 61). Although negative transmission of PEMV to broad bean has been reported (14, 69) most investigators agree on its susceptibility to the virus. This was also found true in the two varieties tested. Induced virus infection observed on cowpea corro- borated the observation made by'Hagedorn and Walker (24). This plant has been reported to be not susceptible to PEMV (83). The susceptibility of subterranean clover confirmed earlier observations (24, 61). Chaudhuri (14) was not able to transmit the virus to this host plant. PEMV failed to infect red clover, white clover, lima bean, and beans variety Prince,.Michelite and Pinto. Hagedorn and walker (24) transmitted the virus to red and white clover while McEwen and Schroeder (42) found.infection only on the latter. French bean variety Prince has been reported a nonAhost plant to the virus (14). Lima.bean, Pinto and.Midhelite were not tried in host range studies by earlier investigators. 61 62 They, however, reported other bean varieties susceptible to PEMV. The virus was not able to infect white lupine whiCh is in agreement with.the report by Stubbs (83). This was not found true by other investigators (23, 24). All solanaceous plant species employed in this study 'were not susceptible to PEMV. Earlier attempts to infect tomato and varieties of Nicotiana tabaggmlL. and also of E, glutinosa.L. were found unsuccessful (14, 24, 83). Cucumber has been reported not susceptible to PEMV infection (51, 69) and this was also true in this study. Plant spe- cies tested belonging to the family Compositae, Amaranthaceae, and Chenopodiaceae were observed non-host plants of PEMV. Except for sugar beets, Egg; vulgaris L., of family ChenOpodiaceae which has been reported not susceptible to PEMV (69), all the species of the three families:mentioned earlier have apparently not been investigated prior to this work. Discrepancies in the results obtained from host range studies of PEMV by several investigators may be attributed to several factors sudh as: low initial virus concentration in the inoculum, method of transmission employed, and age of the test plant or tissues inoculated. No distinct difference could be detected‘between the four PEMV isolates as far as varietal reaction or symptom expression on the inoculated peas. It seemed however, that 63 the infected pea varieties and introductions showed severe symptoms to PEMV-O infection similarly reported by Ruppel and.Hagedorn (61). In some cases, however, the infected plants were less adversely affected compared to those of the other virus isolates. The reaction of the pea introduction from Iran (PI-140295) to PEMV infection resembled that of the resistant “Geneva“ line, G-31 (86). Tolerance to dilution of the PEMV isolates seemed to be influenced by the initial concentraton of the virus in the infected sap extracts as shown by the variable dilution end points from the two trials of the same isolate. The samples used in the two separate trials were obtained from coulparable infected peas but each sanple came from a differ- ent batch of inoculated Pacific Freezer peas. A dilution between 132000 and 133000 was considered as the end point since most of the isolates lost its infectivity between this range. The value obtained agree with an earlier report (83). Inactivation of PEMV ip‘gisgg (ZOO-22°C) after 4 days was similarly observed by other investigators (53, 83). The longevity of PEMV in dried diseased leaf tissues stored at ZOO-22°C for three days was not so great compared with other legume viruses. No report has been made other than this study. PEMV can tolerate heat inactivation to 55°C (PEMV-z) and 63°-65°C (PEMV-W). The values obtained are within the range reported in earlier studies (1, 28, 53, 54). 64 The beneficial effect of phosphate buffers of pH 7.7.8 on PEMV activity in crude sap extracts stored 20-300 may have been to its neutralizing acton on the inactivating sub- stances present. Furthermore, at these pH levels the virus may be above its isoelectric point so that virus aggregation and precipitation in the sap was at minimum. Purified PEMV was more stable when resuspended in a weaker ionic concentra- tion of buffer at elevated pH levels. The non-specific reaction observed from similar preparations of PEMV-infected and healthy control duon- strated that an antigenic component of pea leaves cannot be separated completely from the virus by differential centri- fugation alone. The difficulty of Obtaining virus prep- arations free from an antigenic host component, identified as Fraction 1 protein, by ultracentrifugation technique has recently been reported (88). Non-specific reaction of SBMV, MBV, and BYMV strains with the PEMV antiserum in the micro- precipitin test may indicate the presence‘of a common anti- genic host protein (47, 67, 88, 90) with the Pacific Freezer pea. The CMV and TRSV strains which were extracted from tobacco host plants revealed no reaction to the specific PEMV antiserum. This observation may explain why antigenic host components are unlikely to be detected in serological cross-reaction with viruses prepared from unrelated hosts (40). 65 The Ouchterlony agar double-diffusion test disclosed two antigenic groups, indicated by a straight and a curved precipitation band, from the purified PEMV-infected prep- aration. The straight precipitation band, also formed in shmilar‘healthy control preparation and regarded as non- specific precipitin reaction, may represent residual smaller plant antigens (91). These host contaminants present as low as 1 percent in a purified virus preparation that appear homogenous in the electron microscOpe may still be revealed by the agar gel diffusion technique (47, 89). Results of the serological tests shows the possibility of ribosomal contamination possessing an antigenic prOperty of the host plant in the apparently purified PEMV preparation. The slightly curved precipitation band, interpreted as a specific virus antigen-antiserum reaction, was observed in all of the PEMV isolates. The precipitation band curves away from the antibody reservoir in the agar gel suggesting that the PEMV antigen has a heavieerolecular weight than its antibody (34). Precipitation bands were exhibited between the PEMV antiserum and (NV, MBV, SBMV, BYMV strains in the agar gel preserved in merthiolate. These bands were not of the characteristic type, i.e., curved band, so they were not the resultant serological reactions of each of the viruses with the prepared antiserum (89, 90). Furthermore, these pre- cipitation bands may suggest a false type of precipitation 66 (95) since they were not observed in the sodium azide treated agar gel preparation. Results of the serological studies show that the four PEMV isolates were closely related. The serological tests, however, reveal no information whether eath isolate can be regarded as an individual strain (41). The electron micrographs of purified PEMV preparation suggest the presence of characteristic virus particles with definite morphology and size. Although the majority of these particles appeared to be more like spheres than did the polyhedral-shaped tobacco ringspot virus (79, 81) slight irregularities in the outline of some particles could be observed. A pointed and flat-tapped shadow and several intermediate symmetrical forms are suggested in some of them in the micrograph. Shadows of these kinds are formed only by angular objects but the image of the PEMV particles in the micrographs are not clear enough to permit an un- equivocal inference. According to Kaesberg (30) the so called ”spherical” viruses invariably assume hexagonal contours in clean, lightlyeshadowed preparations. In comparison with the size of other spherical plant viruses, PEMV is similar to tobacco necrosis, 20 mp (72) and falls within the reported size range of tobacco ringspot virus, 13-26 mp (7, 20, 71, 75, 79, 81, 85). Riboscmes and other normal plant proteins were idemonstrated to have a similar morphology and were about the 67 size of small Spherical viruses (32, 56). The work reported herein showed that these components are smaller than PEMV. SUMMARY AND CONCLUSIONS The host range, physico-chemical prOperties, purifi- cation, serological properties and electron microscopy of pea enation mosaic virus (PEMV) were studied. PEMV was limited in infectivity to leguminous plants and attempts to infect plant species of five other families failed. No suitable local lesion host for the virus could be found. Mung bean (Phaseolus aureus RoXb.) was found to be a new'host of PEMV. A11 55 pea introductions were ex- perimentally infected but no distinct difference in their reaction to PEMV isolates were Observed. A pea introduc- tion from Iran (PI-140295) showed some tolerance to virus in- fection. The tolerance of PEMV to dilution was fairly low (182000-183000) as compared to most other spherical plant viruses. It was slightly infectious when exposed to thermal inactivation at 65°C for 10 minutes but not at 68°C. It resisted inactivation‘ig‘giggg for 4 days and was inactivated in airbdried infected tissues after the third day. Storage of infected crude sap extracts buffered at pH 7-7.8 in the refrigerator (20-300) maintained the infectivity of the virus to about 12 days. PEMV-W was concentrated and purified from buffered extracts of frozen infected tissues of garden pea (Pisum gativum L.) var. Pacific Freezer. Purification consisted 68 69 essentially of clarifying the extracts by low speed centri- fugation, and concentration by 3 cycles of alternate high and lowbspeed centrifugation of appropriately buffered virus suspension. The resulting purified virus preparation was infectious and stimulated antibody production in the rabbit. Electron microscOpic examination of shadowed air-dried PEMV suspension revealed monodispersed apparently homogenous and nearly spherical particles with an average diameter of about 20 mp. Such particles were not Observed in similar prep- arations from healthy control. Microprecipitin and Ouchterlony agar double-diffusion tests showed that the virus preparation was antigenic and gave a positive precipitin reaction to its homologous anti- body. Based on serological cross-reaction, PEMV-W isolate was demonstrated closely related to PEMV-O, PEMV-NY, and PEMV-2, but not to CMV nor to any of the following legume viruses: SBMV, MBV, BYMV, TRSV. Some antigenic non-infectious host protein, possibly ribosomes, were detected in the purified PEMV preparation by the Ouchterlony tedhnique. These contaminants are similar in morphology but smaller (12 mp) than the PEMV particles. 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Reported in Virology discussion session on serology, American PhytOpathological Society Meeting, Corvallis, Oregon, August 27, 1962 (Unpublished). MICIIWITIWINHITIAIW flfuimflfiil’m 1W WIN 3 1gl3 030121154