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Mixed infections with cucumber necrosis virus and tobacco necrosis virus Pekkala, David H. 1976

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MIXED INFECTIONS WITH CUCUMBER NECROSIS VIRUS AND TOBACCO NECROSIS VIRUS *y DAVID H. PEKKALA B..Sc..,-. Lakehead University, 1971 B.Ed., Lakehead University, 1972 H.B.Sc. Lakehead University, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS'FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Plant Science Department) We accept this thesis as conforming to the required standard THE UNIVERSITY: OF BRITISH COLUMBIA August, 1976. 0 David H. Pekkala, 1976 In p resent ing t h i s t he s i s in p a r t i a l fu 1 f i lmerit o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permiss ion fo r ex tens i ve copying of t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying o r p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l gain s h a l l not be al lowed without my w r i t t e n permis s ion. Department of ?Ll\Hl SCIENCE The Un i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 ABSTRACT Tobacco necrosis virus (TNV) interfered with cucumber necrosis virus (CNV) replication in mixed infections. TNV inhibited both the initiation of CNV infections and subsequent CNV multiplication. Evidence of interference was found in qualitative evaluations of symptoms on a wide host range and by quantitative evaluations (lesion counts and incidence of systemic symptoms) on cucumber, cowpea, and bean. Fresh weight measurements similarly indicated antagonism in mixed infections. Some evidence of CNV interfering with TNV replication was found, in the form of a reduction in the occurrence of systemic symptoms. Photometric scanning of sucrose density gradient columns after centrifugation revealed that in mixed infections CNV reached only half the concentration attained in single infections while TNV was unaffected or even very slightly increased in concentration. This interference continued even when high temperatures greatly limited TNV replication. Only at 3° C, where its replication was barely detectable, did TNV have no effect on the CNV concentration attained. CNV did not significantly aid TNV replication at any temperature. In serial passage of mixed infections TNV quickly gained in concentration relative to CNV. Although CNV and TNV symptoms could not readily be distinguished from one another on most hosts, Phaseolus vulgaris var. Topcrop kept at 23 C and Chenopodium capitaturn kept at 18 C were established as reliable indicators of CNV and TNV respectively. One preparation from doubly infected tissue showed evidence of possible phenotypic mixing based on polyacrylamide gel electrophoresis and on reactions with antisera, although this could not be confirmed by infectivity tests. i i i Through use of the antigen-antibody neutralization test, another virus preparation from doubly infected tissue gave evidence of possible genomic masking of TNV RNA in CNV coat protein. No evidence of possible genomic masking in the opposite direction was found. Supervisor's signature: iv. TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES ix ACKNOWLEDGEMENTS x i i INTRODUCTION 1 LITERATURE REVIEW 4 Effects of mixed infections oh the host 4 Effects of mixed infections on virus concentration and dist r i -bution 10 Genetic interactions between viruses in mixed infections.. 15 Structural interactions between viruses in mixed infections...... 17 METHODS AND MATERIALS 23 Viruses and inoculation procedure.. 23 Virus purification. 24 Estimation of the virus content of partially purified preparations 28 Antisera and serological tests 29 Sucrose density gradient centrifugation of virus preparations after reaction with antisera 38 Antigen-antibody neutralization 38 Polyacrylamide gel electrophoresis1 (PAGE) 40 (i) Whole virus PAGE 40 (ii) RNA PAGE 42 Electron microscopy. 43 V. RESULTS 44 Differential hosts for CNV and TNV 44 Virus propagation and purification 52 Effects of mixed infections on symptoms and systemic spread 62 Effects of mixed infections on fresh weight. 72 Effects of mixed infections on virus concentration. 75 Structural interaction studies... 92 Examination of mixed infection preparations for phenotypic mixing. 92 (i) Polyacrylamide gel electrophoresis (PAGE) of whole virus " (ii ) Antiserum treatments. 99 Examination of mixed infection preparations for genomic masking.. 100 Attempts to establish a method of polyacrylamide gel electro-phoresis to detect genomically masked viral RNA. 112 DISCUSSION 120 SUMMARY 132 LITERATURE CITED 133 v i LIST OF TABLES Figure Page I. A comparison of properties of cucumber necrosis virus and tobacco necrosis virus 3 II. Volumes of sucrose'solutions layered into each cellulose nitrate tube in the manual preparation of 10-40$ sucrose density gradients 27 III. Symptoms of CNV and TNV infections in five plants tested as possible differential hosts under cool greenhouse conditions..45 IV. Local lesion production on plants inoculated with CNV or TNV in host range trials..*. . . . . . . . . .46 V. The effect of temperature on the number of lesions induced by CNV and TNV inoculated singly to four hosts. 47 VI. The effect of temperature on the number of lesions induced by CNV and TNV inoculated singly to three Chenopodium species....51 VII. Response of Topcrop bean. The Prince bean, and Early Ramshorn cowpea to TNV inoculations at different temperatures..........53 VIII. Comparison of TNV yields from Straight Eight cucumber and Early Ramshorn cowpea.• .55 IX. Comparison of CNV yields from Straight Eight cucumber and Early Ramshorn cowpea .....56 v i i . X. Yields of CNV and TNV from singly inoculated cowpea grown at different temperatures 57 XI. Estimated amount of TNV recovered from cowpea primary leaves using two purification methods......... 53 XII. Estimated amount of CNV recovered from cucumber cotyledons using different purification methods.... 64 XIII. The effects of mixed infections on lesion numbers 66 XIV. The effect of CNV and TNV mixed infections on systemic symptoms in cowpea, bean and cucumber. 67 XV. Symptoms of virus on cowpea two weeks after inoculation of stems with a mixture of CNV and TNV 71 XVI. Fresh weight of buffer-inoculated and virus-inoculated primary leaves of cowpea 73 74 XVII. Fresh weight of buffer-inoculated and virus-inoculated cucumber. . XVIII. Comparison of cucumber necrosis virus yields from single and double infections. . gg XIX. Comparison of TNV yields from single and double infections go XX. Relative yields of CNV and TNV in each of three double infections created by serial inoculation to cucumber gj XXI. Preparations from tissues doubly infected with CNV and TNV used in tests for structural interactions. . . 9 3 v i i i . XXII. The effects of antisera treatments on CNV and TNV mixed infection preparations. 108 XXIII. Number of lesions produced on bean by in vitro virus mixtures and mixed infection preparations after antiserum treatment. 109 XXIV. Number of lesions produced on Chenopodium capitatum by in vitro virus mixtures and mixed infection preparations after antiserum treatment I l l XXV. RNA extraction buffers tested in conjunction with the use of polyacrylamide gel electrophoresis to monitor their effectiveness. 117 i s . LIST OF FIGURES Figure Page 1. Amount of cucumber necrosis virus (mg) versus area under virus peak of absorbance profiles........ 30 2. Amount of tobacco necrosis virus (mg) versus area under virus peak on absorbance profiles................... 32 3. Pattern of wells cut in agar for immunodiffusion tests 36 4. Symptoms of CNV and TNV infections on leaves of Chenopodium capitatum. 49 5. Examination of Vigna sinensis cv. Early Ramshorn root. stem. & and leaf tissue for evidence of TNV infection two weeks after inoculation of roots.... 60 6. Examination of Vigna sinensis cv. Early Ramshorn leaf and stem tissue for evidence of CNV and TNV two weeks after mixed inoculations of the roots and stems.... 69 7. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown under summer greenhouse condition&77 8. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown at 25 C. 79 9. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown at 30 C. 81 X 10. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown under autumn greenhouse conditions. • 83 11. Comparison of the relative amounts of CNV and TNV recovered from doubly infected cucumber cotyledons in each of three serial passages of infection...... 85 12. Amount of virus recovered from single and mixed infections of Phaaeolus vulgaris cv. Topcrop primary leaves grown under autumn greenhouse conditions.. 87 13. Comparison of staining and photometric scanning for the detection of CNV and TNV following electrophoresis in 2.6$ polyacrylamide gels * 95 14. Relative positions of CNV and TNV after electrophoresis in 2,6% polyacrylamide gels.. 101 15. The effects of antiserum treatments on virus mixtures formed in vitro as detected in standard sucrose gradients. 103 16. The effects of antiserum treatments on mixed infection preparation CNV/TNV #6 as detected in standard sucrose gradients, 105 17. The effects of antiserum treatments on mixed infection preparation CNV/TNV #2 as detected in standard sucrose gradients.................. IQ6 x i . 18, The effects of antiserum treatments on mixed infection preparation CNV/TNV #2 as detected in linear log gradients.... 107 19, A comparison of the effects of BNA extraction buffer 2 (Table XXV), as detected by polyacrylamide gel electro-phoresis, on CNV and TNV suspended in sodium phosphate and potassium phosphate buffers. ilk 20, The effects of an SDS-EDTA BNA extraction buffer on CNV and TNV as detected in standard sucrose density gradients , . , 118 ACKNOWLEDGEMENTS I would like to thank my supervisor. Dr. R. I. Hamilton, for suggesting this study and for his understanding and helpful guidance throughout. I also thank Dr. J. A. Dodds for his many suggestions and Dr. M. Weintraub, Director, for making available the facilities of the Agriculture Canada Research Station. Vancouver. Thanks are extended as well to the members of my supervising committee. Dr. R. I. Hamilton. Dr. J. H. Tremaine. Dr. R. J. Copeman, and Dr. V. C. Runeckles, for their assistance, and to Anabel Cole for typing the final copy. The financial support provided me through a National Research Council of Canada operating grant to Dr. Hamilton and. later, through a National Research Council of Canada Postgraduate Scholarship to me is gratefully acknowledged. 1. INTRODUCTION The study of mixed infections with plant viruses generally involves examination of the effects of the mixed infections on one or more of four broad aspects: the host (and disease), virus concentration and distribution, genetic interactions between the viruses, and structural interactions between the viruses. In recent years, more interest has been centered on structural interactions, largely due to the potential they may have in altering host range and vector specificity. While the literature is replete with reports of structural interactions between animal viruses and between bacteriophages, l i t t l e information exists on the situation with plant viruses. Studying the effects of structural interactions involves careful selection of the virus-host system. The system usually must offer some possibilityJof interactions and certainly no obvious barriers to their occurrence. A review of the properties of cucumber necrosis virus (CNV) and tobacco necrosis virus (TNV) reveals the features that made this system apparently suitable to such studies. The two viruses have similar physical properties (Table i ) , yet are serologically unrelated. They are also similar in biological behaviour. TNV has a wide host range and mechanical inoculation of leaves results in infections localized in necrotic lesions. TNV has a cosmopolitan distribution and natural infections are commonly confined to the roots of infected plants. Systemic spread is rare, but certain TNV isolates become systemic in Phaseolus vulgaris var. The Prince and occasionally in Vigna sinensis. CNV has been reported only from greenhouse infections in Southern 2 Ontario (McKeen 1959), but this limited distribution may be more apparent than real due to the ease with which CNV and TNV symptoms may be confused (Tremaine 1972; Dias and McKeen 1972). The host range of CNV is similar to that of TNV and the viruses induce indistinguishable local lesions on inoculated leaves of most hosts. CNV frequently becomes systemic in Cncumis sativns and occasionally in Vigna sinensis and Zinnia elegans. The similarity in size and structure and in host range indicated no obvious barriers to either mixed infections or structural interactions. At the same time, the fact that CNV and TNV are unrelated serologically and are transmitted by distinct fungal vectors provided a means by which CNV and TNV might be distinguished and separated and a means by which the occurrence of structural interactions in mixed infections might be detected. The CNV/TNV mixed infection system is similar to that of barley yellow dwarf virus isolates, where serology and changes in vector trans-mission (breakdown of vector specificity) in mixed infections were used to indicate the occurrence of structural interactions (Rochov 1970). In this investigation, the effects of viral interactions on symptoms, host fresh weight, and virus concentration were examined. The possibility of structural interactions was also explored. 3 Table I. Ascoraparison of the properties of CNV and TNV Property CNV TNV Particle shape Particle diameter Components Sedimentation coefficient Particle molecular weight Composition - protein - RNA 260/280 ratio Isoelectric point Thermal inactivation point Serological relatedness Classification Infection Vectors icosahedral * 31nmb* (35 nm)g* one plus non-infectious accessory^' 133 s b* (136 s ) g > fia. 8.6 x 10>g. 9.3 x 10° I6gc#(l5-18.5$0G* 1.7 pH 3.9* 75-80 C (10 min) f # unrelated to tested virus ungroup ed b. icosahedral^* 26 nmb* (30 nm)a# one a. 118 s*' (112-130 s a*; 120 s 1*; 116 s) e. 7.0 x 10° 80#d# 18.7$e* 1.7d* ( l . 5 ) a # pH 4.5 85-90C d. f. unrelated to^any tested virus * ungrouped local (occasionally local (occasionally systemic in cucumber, ^ systemic i^French bean copea, Zinnia elegans) * and cowpea) * * Olpidium cucurbita- Olpidium brassicae cearum D.J.S. Barr and (Wor.) Dang. Dias sp. n. °» d. References: a. Babos and Eassanis 1963 b. Dias and Doane 1968 c. Dias and McEeen 1972 d. Kassanis 1970 e. Lesnaw and Reichmann 1969 f. McKeen 1959 g. Tremaine 1972 4. LITERATURE REVIEW Mixed virus infections of plants occur commonly in the field. Two recently noted examples are double infections of cabbage black ring and cucumber mosaic viruses in radish seed crops (Shukla 1972) and mixed infections of barley yellow dwarf virus isolates in winter grains (Rochow and Muller 1974). The replication of two or more viruses together in the same host may modify symptom expression and virus concentration and d i s t r i -bution in that host. Simultaneous replication in the same cell allows the possibility of genetic recombination and structural interactions between the viruses. Of these four aspects of mixed infections, a change in symptom expression is the most readily detectable and the most studied. Effects of mixed infections on the host The symptoms of some naturally occurring plant diseases are characteristic of a virus mixture and are not induced by any of the viruses alone. This was f i r s t noted with the finding that tomato streak disease in Quebec was caused by double infections of tobacco mosaic virus and potato virus X (Vanterpool 1926). Other diseases attributable to specific mixed virus infections include rugose mosaic disease of potato caused by potato virus X and potato virus Y (Smith 193l)» potato crinkle disease caused by potato virus X and potato virus A (Murphy and McKay 1932), l i l y fleck disease caused by l i l y symptomless virus and cucumber mosaic virus (Brierley and Smith 1944), blossom necrosis of Cattleva orchids caused by Cymbidium mosaic virus and Odontoglossum ringspot virus (Thornberry et a l . 1968), cotton mosaic disease caused by Brazilian tobacco streak virus and cotton 5 anthocyanosis virus (Costa 1969), and raspberry mosaic disease caused by Rubus yellow net virus and heat labile mosaic virus components (Freeman and Stace-Smith 1970). Symptoms have been used to indicate when i n the infection process virus interactions occur. Interactions may take place at the i n i t i a t i o n of infection, usually i n the form of competition for available infection sites, or they may occur later i n the replication cycle. Interactions affecting the establishment of infection have been demonstrated by comparing the number of lesions induced by a lesion -forming virus inoculated alone and together with another virus. Com-petition i s most common between related viruses. For example, a mottle strain of tobacco mosaic virus (TMV) reduced the number of lesions induced by a local lesion strain of TMV i n tobacco (Sadasivan 1940). However, reductions i n lesion numbers are not necessarily restricted te interactions between related viruses. A reduction i n lesion number was also reported for the unrelated viruses potato virus X (PVX) and potato virus Y ( F V Y ) i n potato seedling USDA 41956 (Ross 1950). In the latter case, PVX i n the inoculum reduced the number of FVY lesions even though the host was not susceptible to PVX infection, as determined by the absence of PVX microlesions and the absence of any detectable PVX multiplication (Ross 1974). The importance of the host i n virus interaction i s em-phasized by the fact that i n Physalis floridana. PW lesion number was unaltered i n mixed infection with PVX, although the plant i s a nest for both viruses. Other examples include the reduction i n the number of cabbafee black ringspot virus lesions on tobacco i n mixed infection with tobacco mosaic virus, potato virus Y, or severe etch virus (Thomson I960). Interference between tobacco necrosis v i r u s (TNV) and i t s serologically unrelated s a t e l l i t e v i r u s no t e n l y reduced the number »f TNV l e s i o n s 6. bat also decreased their average size (Eassanis 1962). Similarly, simultaneous inoculation of bean yellow mosaic virus and alfalfa mosaic virus (AMV) to beam-reduced both the number and size of AMV lesions (Ford 1967). Interferenee is often enhanced when the inhibiting virus is allowed to become fully systemic before introduction of the lesion-forming virus. Nitzany and Sola (1962) demonstrated that ene strain of cucumber mosaic virus reduced the number ef tobacco mosaic virus lesions by 50% while another cucumber mosaic strain caused ne inhibition at a l l . When tobacco mosaic or tobacco necrosis viruses were inoculated to tobacco systemically infected with potato virus Y, the resulting lesions were reduced in size and number (Davis and Ross 1968). In some cases, interactions increase the number of lesions pro-duced by the lesion-forming virus. The number of potato virus X lesions on White Burley tobacco was greater in mixed infection with tobacco mosaic virus or potato virus Y than in single infection (Thomson 1961). Under certain conditions, satellite virus doubled the number of tobacco necrosis virus lesions elicited on tobacco, although their size was smaller than usual.(Eassanis 1962). Tobacco necrosis virus had no effect on the number of tobacco mosaic virus lesions produced on Nicotians glutinesa. a local lesion host for both viruses (Wu and Hudson 1963)* Virus interactions occurring after the initiation of infection have also been detected by their effect on symptom expression* Antagonism, resulting in reduced symptom severity from that observed in single in-fections, is encountered most frequently between strains. This observation was put to use as the "cross protection test" in determining relationships between viruses, but the numberous exceptions to the rule now make i t of limited value. Although interference is more common among virus 7 strains and synergism more common among unrelated viruses, there is no cor-relation between the type of interaction and serological relatedness. Many mechanisms have been suggested to explain virus interactions but the current view is that interactions between strains and between unrelated viruses are not qualitatively different (Kassanis 1963). although this has been'chal-lenged (Ross 1974). Ross bases this view partly on the observation that in sequential inoculations maximum interference between unrelated viruses occurs earlier than the maximum interference between strains. Antagonism between viruses repliating in the same host is often evi-denced by milder symptoms in mixed infections or by a delay in the appearance of symptoms. When potato virus Y was challenge inoculated to tobacco system-ically infected with potato virus C, symptom inducation by the challenge virus was completely inhibited (Bawden and Sheffield 1944). However, when another closely related virus, tobacco veinal necrosis virus, was challenge inoculated, i t evoked its typical symptom response in the host (Bawden and Kassanis 1951). With the more distantly related viruses tobacco severe etch-infected tobacco plants exhibited no symptoms of PVY infection when subsequently inoculated with PVY (Bawden and Kassanis 1945). The presence of sugarcane mosaic virus in a particular sorghum variety reduced the symptoms induced by the related maize dwarf mosaic virus (Gillaspie and Koike 1973). The pattern of suppression of apple leaf pucker disease and apple blotch disease symptoms following bud-graft inoculation to certain Mcintosh and Spartan apple clones was suggestive of viral interaction, although infectivity tests failed to isolate any of the common apple viruses (Welsh and May 1973a, b). Mixed infections may have a synergistic effect, resulting in symptoms more severe than the sum of the effects of both viruses in single infections. This occurs in tobacco where potato virus X and potato virus Y or tobacco mosaic virus induce a synergistic increase 8 « in vein-clearing, necrosis, and mottling (Rochov and Ross 1953). Other examples of synergistic symptom response in mixed infections of plant viruses include the effects of barley stripe mosaic virus, brome mosaic virus, and vheat streak mosaic virus on stunting, necrosis, and death of wheat plants (Lai and S i l l 1959); the effects of citrus yellow-vein and citrus vein-enation viruses on dwarfing of lemon (Weathers I960); the effects of soybean mosaic virus and bean pod mottle virus on dwarfing and foliage distortion,of soybean (Ross 1968); the effects of alfalfa mosaic virus and pea streak virus on stunting and necrosis of peas (Hampton and Sylvester 1969); the effects of cowpea chlor-otic mottle virus and southern bean mosaic virus on the height of cowpea (Euhn and Dawson 1973); and the synergistic effect of barley stripe mosaic virus and brome mosaic virus on stunting in barley (McKinney 1956). At least two examples of related viruses inducing a synergistic symptom response in mixed infections are known. Both of these involve the appearance of new symptoms not characteristic of either virus alone. When the type strain of tobacco mosaic virus, which produced a systemic green mosaic, and the aucuba strain of tobacco mosaic virus, which produced necrotic local lesions with rare systemic involvement, were inoculated together to Nicotians syIvestris. the host responded with typical type strain symptoms, plus white spots on the f i r s t systemically infected leaves. The aucuba strain was isolated from the white spots and the ordinary strain from the green portions of the same leaves (Benda 1957). Similarly, localized necrotic strains of tomato spotted wilt yirus spread systemically when in mixed infections with the ringspot strain (Finlay 1952; Norris 1951). A new symptom was evident on certain soybean cultivars, doubly infected with soybean mosaic virus and bean pod mottle virus, in the form of filiform enations produced 9. on the midribs of trifoliate leaves (Quiniones and Dunleavy 1971). , Synergism may also be evident at the cellular level* In single infections, only 5% of leaf cells shoved cucumber mosaic virus-induced chloroplast abnormalities while in double infections with TMV, 7°$ of leaf cells showed such abnormalities (Honda and Matsui 1968). In contrast to the examples so far cited, peanut mottle virus replication had no effect on the degree of stunting induced in peanut by peanut stunt virus (Kuhn 1969)* and tobacco mosaic virus replication did not alter the symptoms of barley stripe mosaic virus in barley (Dodds and Hamilton 1972). Cucumber mosaic virus and tomato aspermy virus produced a nearly additive symptom response in tomato (Holmes 1956), as did alfalfa mosaic virus and bean yellow mosaic virus in pea (Ford 1967), and cowpea chlorotic mottle virus and soybean mosaic virus in soybean (Demski and Jellum 1975), the effects in mixed infections being the sum of those in both single infections. Host yields, expressed as fresh or dry weight of the plant or as the weight of fruit or seed produced may also be affected by mixed virus infections. A synergistic effect is often seen, with the weight loss due to mixed infections being greater than the losses due to both single infections combined. Examples include the reduction in dry weight of wheat leaves infected with barley stripe mosaic, brome mosaic, and wheat streak mosaic viruses (Lai and S i l l 1959); the reduced seed yield, by weight, of soybean infected with soybean mosaic virus and bean pod mottle.virus (Boss 1963, 1968, 1969); the reduced yield (indicator not stated) of soybean infected with soybean mosaic virus and tobacco ringspot virus (Schmitthenner and Gordon 1969); and the reduction in the number of seeds and seed pods, and the reduction in dry weight of individual seeds and plant parts in cowpea infected with cowpea chlorotic mottle and 10. southern bean mosaic viruses (Kuhn and Dawson 1973)* Occasionally, the yield loss is only the additive effect of that in both single infections, as with the effect of barley stripe mosaic virus and brome mosaic virus on-the fresh weight of barley (Morris 1970),3-and the effect of bean pod mottle virus and bean yellow mosaic or tobacco ringspot viruses on the yield of soybean (Schmitthenner and Gordon 1969)* Some virus combinations do not alter the host yield from that in either single infection. The yield of soybean doubly infected with soybean mosaic virus and bean yellow mosaic virus was approximately the same as that from either single infection (Schmitthenner and Gordon 1969)* The fresh weight of barley infected with both tobacco mosaic virus and barley stripe mosaic virus was no different from that of barley singly in-fected with barley stripe mosaic (Dodds and Hamilton 1972). When bean yellow mosaic virus was in mixed infections with tobacco ringspot virus in soybean, yield loss was actually less than in plant singly infected with tobacco ringspot virus (Schmitthenner and Gordon 1969), implying a possible interference with tobacco ringspot virus replication. Effects of mixed infections on virus concentration and distribution The concentration of one or both components in mixed infections may differ from that of each in single infections. Virus concentration has been estimated most frequently in mixed infection studies by the severity of symptoms elicited. However, while symptom expression • generally parallels virus concentration, such is not always the case. For this reason, concentration estimations made solely on the basis of symptom severity have not been cited here. Virus concentration may be l i t t l e changed although symptoms are more severe, as with alfalfa mosaic virus and potato aucuba mosaic virus 11. in tobacco (Oswald et a l . 1955)* In mixed infections with cowpea chlorotic mottle virus (CCMV"), southern bean mosaic virus concentration was reduced by 50$ and G6MV concentration was unaltered, while the host exhibited a synergistic symptom response (Kuhn and Dawson 1973)* Bochow and Boss (1955) were able to correlate increased PVX concentration, as determined by dilution end point infectivity assay, with the number of virus particles seen by electron microscopy. Lee and Boss (1972), however, found a substantial discrepancy between the two methods applied to the determination of soybean mosaic virus concentration in mixed infections with bean pod mottle virus. To obtain accurate estimates of virus concentration, several workers have employed physical means such as serology (Close 1964), optical density (Kuhn and Dawson 1973), sucrose density gradient centrif ugation and photometric scanning (Dodds and Hamilton 1972; Peterson and Brakke 1973), or electron micro-scopy (Allen and Lyons 1969; Lee and Boss 1972). Mixed injections with some virus strains do not occur due to complete antagonistic exclusion of one of the viruses; especially when inoculation with the inhibiting virus occurs several hours or days before a challenge inoculation with a second virus. This occurred in Nicotiana sylvestris infected with mottle-inducing aucuba strains of tobacco mosaic virus and challenged with a lesion-forming aucuba strain. Subsequent infectivity tests revealed that no multiplication of the challenge strain occurred in the doubly-inoculated plants (Bennett 1953)* More frequently, especially with simultaneous inocu-lations, antagonism takes the form of a reduction in concentration of one or both viruses, rather than complete elimination. This occurred with mild strains of tobacco etch virus in tobacco, which reduced potato virus Y concentration from that attained in single infections, 12 although in mixed infection with severe etch, no PVY multiplication was detectable (Bawden and Eassanis 1945). Sugarcane mosaic virus reduced the concentration of the serologically related maize dwarf mosaic virus in corn (Tu and Ford 1969)* Unrelated viruses exhibiting antagonism resulting in reductions in virus concentration include potato virus Y ('PVY) and Datura necrosis virus, where PVY concentration was reduced in doubly infected tobacco (Badami and Eassanis 1959). In mixed infections of tobacco mosaic and cucumber mosaic viruses, tobacco mosaic virus concentration was reduced (Garces-Orejuela and Pound 1957)* and cowpea chlorotic mottle virus reduced the concentration of southern bean mosaic in mixed infections (Kuhn and Dawson 1973)* The concentration of both barley stripe mosaic and brome mosaic viruses was reported to be reduced in doubly-infected barley (Petersonaand Brakke 1973)* although other work with the same system (Morris 1970) found no difference in the concentration of each virus in single or mixed infections. Increases in the concentration of one or both viruses may result from mixed infections. Under certain conditions, two to four times more potato aucuba mosaic virus was produced in mixed infections with potato virus Y or A than in single infections, although there was no increase in symptom severity (Eassanis 1961). Other examples include the increase of dodder latent virus when infected plants were challenged with tobacco etch virus or tobacco mosaic virus (Bennett 1949); the increase of tobacco mosaic virus in mixed infections with tobacco ringspot virus (Garces-Orejuela and Pound 1957) or with potato virus X (Boss 1969); the increase of l i l y symptomless virus in mixed infections with cucumber mosaic virus (Allen and Lyons 1969); the increase of potato virus X by up to ten times in mixed infections with potato virus Y in tobacco (Rochow and Boss 1955)* 13 or up to five times in mixed infections with tobacco mosaic virus in tomato (Eassanis 1963); and the marked increase in tobacco mosaic virus concentration in barley also infected with barley stripe mosaic virus (Dodds and Hamilton 1972). The number of soybean mosaic virus particles increased by two to three times in mixed .infections of soybean with bean pod mottle virus (Lee and Boss 1972). By contrast. PVX concen-tration in double infections with alfalfa mosaic virus in tobacco was no different than that in PVX single infections (Bochow and Boss 1934). Mixed infections may alter virus distribution. A normally localized virus may be induced to spread systemically when in mixed infections with another virus, as occurs with tomato spotted wilt virus strains (Finley 1952; Norris 1951), and with certain tobacco mosaic virus strains (Benda 1957)* It has been suggested that the synergism in these two cases might be the result of strain interference suppressing virus concentration such that the hypersensitive response in the host fails to be expressed sufficiently to restrict spread of the normally localized strain (Boss 1974). Examples of an unrelated virus inducing systemic distribution include the spread of the normally localized Brazilian tobacco streak virus when in mixed infections with cotton anthocyanosis virus in cotton (Costa 1969) and the spread of potato virus X in tobacco infected with potato virus Y at temperatures restrictive to potato;virus X multiplication in single infections (Close 1964). Mixed infections may also alter the intracellular distribution of a virus. Honda and Matsui (1969, 1971) commonly found TMV particles in nuclei and proplastids of tobacco doubly infected with CMV, but rarely in cells singly infected with TMV, There are at least two examples of mixed inoculation apparently allowing a virus to multiply in a host normally resistant to infection by i t . When sugarcane mosaic virus 14. was inoculated together with the related maize dwarf virus, i t was able to multiply in Sorghum halepense. a plant resistant to sugarcane mosaic virus alone (Gillaspie and Koike 1973)* Citrus yellow-vein virus inoculated alone to Ponicims trifoliata did not reach detectable levels of multiplication, but following mixed inoculations with citrus vein-enation virus, both viruses multiplied (Boss 1974). The changes in virus concentration or distribution due to mixed infections may alter the pattern of seed or vector transmission. For example, seed transmission of soybean mosaic virus in double in* fections with bean pod mottle virus was reduced by 40$ from that in single infections (Boss 1968). Seed transmission of southern bean mosaic virus was increased by 50$ in double infections with cowpea chlorotic mottle virus despite the fact that SBMV concentration was reduced by 50$ (Kuhn and Dawson 1973). The frequency of transmission of pea streak virus by Acyrtho- siphon pi sum dropped from 72$ in single to 46$ in mixed infections with alfalfa mosaic virus, while the frequency of alfalfa mosaic virus transmission rose from 8$ in single to 22$ in mixed infections (Hampton and Sylvester 1969). That individual plant cells can host mixed infection has been demonstrated in several ways. Light microscopy (McWhorter and Price 1949) and electron microscopy (Fujisawa et a l . 1967) were used to reveal the characteristic crystalline inclusions of tobacco mosaic virus and tobacco etch virus occurring together in doubly infected cells. The latter authors noted that challenge inoculation, by contrast to simul-taneous inoculation, produced few doubly Infected cells. Benda (1956) inoculated individual leaf hair cells of Nicotiana glutinosa with type tobacco mosaic virus and aucuba tobacco mosaic virus, assayed the 15. lesions produced, and. found that at least one-quarter of them supported a double infection. It is possible, however, that each strain multiplied in separate cells adjacent to the inoculated leaf hair. Electron microscopy detected the characteristic aggregation bodies of two strains of alfalfa mosaic virus in the same cell (Hull and Plaskitt 1970), and the occurrence of particles and inclusions of the BNA-containing turnip mosaic virus and the DNA-containing cauliflower mosaic virus together in the same cell (Kernel et a l . 1969)* Inclusion bodies of soybean mosaic virus and particles of bean pod mottle virus were found in the same cells of soybean plants•(Lee and Ross 1972) and Allen and Lyons (1969) detected aggregates of the rod-shaped l i l y symptomless virus particles in the same cell with cucumber mosaic virus particles. These studies clearly reveal that two plant viruses can infect the same ce l l . Genetic interactions between viruses in mixed infections A possible consequence of mixed infections of the same cell is the production of new virus strains resulting from genetic recombina-tion between the parental viruses. This is well documented with animal viruses and bacteriophages} for example, genetic recombination has been reported to occur between poliovirus strains (Hirst 1962) and between P22 and P221 phages (Yamamoto and Anderson 196l). Genetic recombination and the production of new virus strains in plant cells having mixed virus infections has,not yet been satisfactorily demonstrated. Genetic recombination between strains of tomato spotted wilt virus (Best 1954, 1961; Best and Gallus 1955)* between potato viruses C and Y (Watson i960), between two strains of tobacco mosaic virus (Sukhov 1956), and between potato virus X strains (Thomson 1961) has been reported or suggested. The first is the best studied. When a mild and a virulent strain of 16 tomato spotted wilt virus were inoculated to tomato, three new strains eould he detected through single lesion isolations. The possibility exists, however, that these represented mutations (Kassanis 1963). The evidence was also insufficient to rule out mutations in the ex-periments with tobacco mosaic virus strains and potato virus X strains. The observation that aphid transmission of FVC was dependent on the presence of PVY led Watson (i960) te suggest the possibility of genetic recombination between FVC and PVY. However, subsequent work (Kassanis and Govier 1971) determined that actual mixed infection of these two viruses was not necessary for aphid transmission of FVC to occur. Acquisition of PVY prior to FVC in feedings from singly infected plants also resulted in FVC transmission and i t was suggested that PVY-induced modifications of the adsorptive properties oftthe aphid stylet might be responsible. Interference between virus strains, the use of whole plants as hosts, and the possible topographical separation of viruses during replication within the comparatively large plant cell may be factors hindering the demonstration of genetic recombination between plant viruses (Kassanis 1963). While true genetic recombination between plant viruses has not been demonstrated, new virus strains have been created in vitro by selectively mixing the components of multi-partite virus systems. A multi-partite virus has the genetic information necessary for fully competent replication divided among several nucleoprotein components. For example, replication of alfalfa mosaic virus requires three different classes of nucleoproteins which differ in size and can be separated by zonal density gradient centrifugation. Heterologous mixing of component classes from different strains results in a hybrid, or new strain, having the protein coat of one of the original viruses 17. and inducing symptoms that are a combination of those of both original viruses (Franck and Hirth 1976; van Vloten-Doting et al . 1968. 1970). Such new hybrids have been created using other multi-partite viruses (van Eammen 1972). Structural interactions between viruses in mixed infections When two or more viruses infect the same cell , the potential for viral structural interactions arises. One of the possible outcomes is the production of progeny virions with the genome of one parent but with a protein coat consisting of subunits from both parental types. This has been called phenotypic mixing (Hershey et al . 1951). Such pheno-typic mixing has been demonstrated between the related bacteriophages T2 and T4 (Novick and Szilard 1951), between strains of 0X174 bacterio-phages (Hutchison et al . 1967), between BNA phages SP and Fl (Miyake and Shiba 1971)» and between two T4 mutants (Carlson and Eozinski 1974). It also occurs with some animal viruses, for example between polio virus types 1 and 2 (liedinko and Hirst 1961), between group A arboviruses (Burge and Pfefferkorn 1966), between members of the avian tumour virus group (Vogt 1967)* and between human and simian adenoviruses (Alstein and Oodoneva 1968). To date, only one example of in vivo phenotypic mixing between plant viruses has been reported. Atabekova et a l . (1975) detected phenotypically mixed particles from plants doubly infected with the aucuba and T (thermotolerant) strains of TMV. In vitro re-assembly experiments with the related brome mosaic, cowpea chlorotic mottle, and broad bean mottle viruses have produced particles with mixed coat proteins (Wagner and Bancroft 1968, 1971)» as have in vitro experiments with various TMV strains (Atabekova et al. 1975)* Phenotypic mixing of capsid proteins has occurred only between 18 viruses related either serologically or structurally or both, although with bacteriophages this may be partly a reflection of the mutual exclusion of unrelated phages (Adams 1959)* With enveloped viruses, phenotypic mixing of structurally dissimilar and serologically unrelated viruses has been reported; for example, mixed infections of parainfluenza virus SV5 and vesicular stomatitis virus produced phenotypic mixing of envelope proteins (Choppin and Compans 1970). Phenotypic mixing apparently requires that production and assembly of proteins of the different replicating viruses occur simultaneously (Miyake and Shiba 1971) in a common pool within the cell (Streisinger 1956). As well, the different protein subunits probably have to be similar in configuration and chemical composition to assemble into stable particles (Caspar and King 1962). Genomic masking (Tamamoto and Anderson 1961) refers to the production, in mixed infections, of progeny virions with the nucleic acid of one parental type enclosed in a protein coat completely of different parental origin. Phenotypic mixing and genomic masking may occur together (Miyake and Shiba 1971; Lagwinska et a l . 1975) or i t may be difficult to determine which phenomenon is being exhibited (Burge and Pfefferkorn 1966). While some degree of relatedness, either structural or serological, appears to be necessary for phenotypic mixing to occur, the requirements are not as stringent for genomic masking. Between unrelated but structurally similar viruses, genomic masking was reported with the BNA bacteriophages f2 and f24 (Valentine and Zinder 1964), with the BNA phages P22 and P221 (Yamamoto and Anderson 1961), and with phages lambda and 080 (ikokuchi and Ozeki 1970). Similarly with animal viruses, poliovirus RNA was encapsulated in coxsackie Bl protein (Cords and Holland 1964), foot and mouth disease 19 virus nucleic, acid was encapsulated in bovine enterovirus protein (Trautman and Sutmoller 1971)» and poliovirus has been genomically masked in a heterologous poliovirus capsid (Wecker and Lederhilger 1964). However, genomic masking has also been reported for viruses unrelated in both structure and serology. Simian virus 40 genome was masked in adenovirus protein (Easton and Hiatt 1965) and 0X174 DNA was masked in phage fd coat protein (Snippers and Hoffraann-Berling 1966). Genomic masking has been reported to occur between several plant viruses. In mixed infections with a thermostable strain of tobacco mosaic virus and a temperature sensitive strain under conditions preventing the latter virus from producing a functional coat protein, the tem-perature sensitive strain RNA was genomically masked in thermostable. strain protein (Atabekova et al . 1975; Sarkar 1969). Similarly, in mixed infections of tobacco ringspot virus (TRSV) and its defective satellite, TRSV produced the capsid subunits for both viruses (Schneider 1971)* The hybrid virus produced by mixing nucleoprotein component classes from different strains of multi-partitie viruses can be considered to be genomically masked (Dodds and Hamilton 1976) as only one parent strain determines the protein coat for the progeny RNA of both. Genomic masking also occurs between structurally similar but serologically unrelated isolates of barley yellow dwarf virus in mixed infections of barley (Rochow 1965, 1970, 1975)* RNA of the rod-shaped barley stripe mosaic virus was genomically masked in the protein coat of the unrelated spherical brome mosaic virus (Peterson and Brakke 1973), and tobacco mosaic virus RNA was encapsulated by barley stripe mosaic virus protein (Dodds and Hamilton 1974). Barley stripe and TMV are unrelated rod-shaped viruses of different dimensions. The factors determining whether genomic masking occurs, as between 20 certain TMV strains (Kassanis and Bastow 1971a, b), or not. as between other TMV strains (Tal'Yanskii et a l . 1974), and the factors deter-mining whether one or both viral nucleic acids in a double infection become genomically masked are s t i l l l i t t l e understood (Dodds and Hamilton 1976). The specificity of protein-RNA and protein-protein interactions during virion assembly is high (Atabekova et a l . 1975). Thus, the assembly of certain heterologous protein subunits around' a nucleic acid molecule may be prohibited by incompatibility at binding sites due to configurational or chemical differences (Breck and Gordon 1970). The occurrence of genomic masking between phages QB and MS2 in vitro but not in vivo (Ling et a l . 1970) however, suggests that other factors may also be involved. Because the replication and assembly of different viruses may occur at different sites within the cell (Schlegel et a l . 1967; Hamilton 1974), two viruses may be essentially sequestered even though in mixed infection. Replication may be separated in time rather than (or as well as) spatially. Structural interactions might be pre-vented by different rates of virus replication. However, in one case, a difference in maturation rates is a direct cause of genomic masking. Phage lambda matures earlier than phage 080, and because of this i t : supplies the protein to coat both viral genomes. Cell lysis occurs before much 080 protein production has occurred (ikokuchi and Ozeki 1970). Differences in the total amount of each virus produced may also be important. Synthesis of large amounts of brome mosaic virus protein relative to barley stripe mosaic virus is cited as a possible factor in the genomic masking of BSMV RNA in BMV protein (Peterson and Brakke 1973). Genomic masking may occur but be transitory due to instability of the particle. It has been suggested, on the basis of in vitro studies, that the efficiency of reconstitution is greater with 21. homologous than with heterologous protein (Atabekov et a l . 1970b) and i t was observed that reconstituted particles of potato virus X RNA and TMV protein were susceptible to ribonuclease (Breck and Gordon 1970). Reconstituted spherical particles appear more stable, as with broad bean mottle, cowpea chlorotic mottle, and brome mosaic viruses (Hiebert et a l . 1968). and with TMV-BNA encapsulated i n vitro i n cowpea chlorotic mottle virus protein (Grouse et a l . 1970). These particles were not only resistant to ribonuclease but infectious as well. Phenotypic mixing and genomic masking alter the host range of bacteriophages (Hutchison et a l . 1967) and of animal viruses (Alstein and Dodonova 1968) but the situation with plant viruses i s not clear. Both with hybrid plant viruses reconstituted i n vitro (Breck and Gordon 1970) and with genomically masked plant viruses formed i n vivo (Peterson and Brakke 1973) host range of the masked BNA was apparently unaltered. However, reconstituted virions of brome mosaic virus (BMV) BNA i n TMV protein were unable to infect plants susceptible to BMV but resistant to TMV, although infections of plants susceptible to both viruses did occur (Atabekov et a l . 1970a). This i s an important consideration i n the use of i n f e c t i v i t y tests to detect genomic masking. Host range has been altered by mixed infections without any probable v i r a l structural interaction, as with the conditioning of sorghum by maize dwarf mosaic virus.allowing the replication of sugarcane mosaic virus i n this normally resistant host (Gillespie and Koike 1973)* - Certain plant viruses can be transmitted by vectors only from mixed infections and there i s substantial evidence to indicate that their transmission i s dependent on structural interactions between the viruses. With barley yellow dwarf virus (BYDV) isolates, MAV transmission by the aphid Bhopalosiphum padi i s dependent on mixed infections of the plant 22. host with the helper isolate SPY. Treatment with MAY antiserum of mixtures derived from single infections of RPV and MAY completely re-moved MAY infec t i v i t y . However, the same treatment applied to preparations derived from actual mixed infections of RPV and MAY, although i t apparently removed a l l MAY virions, did not remove a l l MAY infec t i v i t y . These results are consistent with the occurrence of genomic masking of MAY RNA i n RPV protein (Rochow 1970). Dependent transmission of the MAY isolate by another aphid, Rj_ maidis. with the RMV isolate as helper, i s also the apparent result of genomic masking (Rochow 1975). As stated i n the introduction to this review, many mixed infections occur naturally i n the f i e l d . Some, such as cabbage black ring and cucumber mosaic viruses i n mixed infections of radish (Shukla et a l . 1972) are of direct importance i n the crop losses they cause. Others, such as mixed infections with barley yellow dwarf virus isolates of up to 28$ of winter barley plants (Rochow 1974), are of potential importance. RMV was isolated from every natural BYDV multiple infection and was the'predominating isolate i n Manitoba i n most seasons. Thus, a virus known to function i n the laboratory as a helper for the dependent transmission of other BYDV isolates i s common i n mixed infections i n the f i e l d , where i t may similarly be involved i n virus structural interactions that increase the potential for disease spread by vectors. 23. METHODS AND MATERIALS Viruses and inoculation procedure CNV and TNV isolates were supplied by Dr. H. F. Dias, Agriculture Canada. Vineland, Ontario, in the form, of freeze-dried infected tissue. CNV was propagated in Cncumis sativus L. cv. Straight Eight or, occasionally, cv. National Pickling (cucumber) and TNV was propagated in Vigna sinensis (L.) Engl, cv. Early Ramshorn (cowpea) whenever possible. Soil quality and unfavourable winter lighting sometimes contributed to unsatisfactory rates of cowpea germination and growth and at such times TNV was propagated in cucumber. Plants were grown under prevailing greenhouse conditions, with « supplemental lighting in the winter, or in a controlled environment chamber with 60$ relative humidity and a photoperiod of 18 hours. Temperature in the chamber was varied depending on the purposes of the experiment and on which virus was present. For the purpose of virus propagation temperature was maintained at a constant 18 C for TNV and a constant 21 C for CNV. Cowpea primary leaves or cucumber cotyledons were inoculated with a sterilized cheesecloth pad soaked in virus preparation. When the preparation volume was small, inoculations were accomplished using cotton swabs. Celite (Johns-Manvilie's trademeark for diatomaceous silica) was included either as a suspension in the virus preparation or sprinkled on leaves prior to inoculation to increase abrasion. For root inoculations, coarse sand was compared with perlite as a substrate, either with or without a water-retaining gel. Perlite in well-drained flats without the water-retaining gel produced cowpea seedlings with the best root 24 branching development and with root systems easily cleaned of debris for inoculation and purification. As primary leaves were unfolding, cowpea seedlings germinated in perlite were carefully removed and the roots inoculated in the manner described above. Seedlings were re-planted and watered with a nutrient solution every four days until harvest at two weeks post-inoculation. Inocula were either crude (unclarified)extracts or partially purified preparations from single infections. For crude extracts, infected tissue was homogenized in 0.01 M potassium phosphate buffer, pH 7*1 (2 ml buffer/l gm tissue) and placed in 4 ml-capacity glass test tubes for frozen storage. CNV and TNV inocula were used at approximately the same relative concentrations except where otherwise indicated. The relative concentrations of crude inocula were estimated by infectivity assay on cowpea while the virus content of partially purified preparations was estimated from ultraviolet absorption profiles. Mixed inocula were prepared by mixing samples of singly infected tissue prior to virus extraction or by mixing crude extracts or partially purified preparations. Mixed inocula contained the same concentration of each virus, and there-fore twi ce the total virus of single inocula. Control plants were inoculated with 0.01 M potassium phosphate buffer, pH 7.1, with Celite added. Virus purification Both CNV and TNV were purified by the same method, regardless of host. The method was adapted from that of Dias and Doane (1968) and that of Tremaine (unpublished). (i) Infected tissue was mixed with extraction buffer on a 1:1 (weight to volume) basis and homogenized by grinding for three 25 minutes in a Waring blendor. The extraction buffer was 0.2 M sodium acetate. pH 5.0* containing 0.02 M diethyldithio-carbamate (DIECA) and 0.2% NSgSO^. After being expressed through cheesecloth, the crude extract was adjusted to pH 5*0 and stored overnight at approximately 4 C. The precipitated plant materials were removed by low speed centrif ugation at approximately 10,000 g in a Sorvall SS-34 or GSA rotor, depending on the volume of extract. The pH was again adjusted to 3.0 with acetic acid and the preparation left for another 24 hours at 4 C. Another low speed centrif ugation left the supernatant clear but deep yellow-brown in colour. This supernatant was centrifuged in a Beckman Type 30 rotor for 150 minutes at 28,000 rpm (approximately 80,000 g). Such a cycle of alternate low speed and high speed centri-fugation (differential centrif ugation) proved more effective in virus recovery than did precipitation with polyethylene glycol (PEG). The pellet from high speed centrifugation was resuspended at 4 C overnight in a small volume (usually 0.5 ml but this was varied depending on the size of the pellet) of 0.01 M potassium phosphate buffer, pH 7*0. Besuspension was completed by taking up the pellet several times in a pasteur pipette and by agitating the suspension on a high speed mixer. The remaining insoluble material was removed by centrifugation at 9,000 rpm for 20 minutes in an SS-34 rotor (approximately 10,000 g) and the partially purified virus preparation was stored at 4 C with chlorobutanol added as a preservative. Further purification, where desired, was by preparative sucrose 26 density gradient centrif ugation in Beckman SW-25 or SW-27 rotors with virus bands being detected on anISCO UA-5 absorbance monitor. By determining the time delay between the passage of virus in front of the absorbance monitor window and its emergence from the drainage tube, virus could be easily collected manually. Sucrose density gradient centrifugation was conducted in an SW-41 rotor for analytical work and in SW-25 or SW-27 rotors for pre-parative purposes. Gradient columns were prepared from density gradient grade (ribonuclease free) sucrose (Schwarz/Mann) dissolved in 0.01 M potassium phosphate buffer pH 7*0 or in some cases (as indicated) in 0.1 M sodium acetate buffer pH 5.0. Gradients were formed in cellulose nitrate tubes by manually layering with a pipette volumes of 40, 30, 20, and 10$ sucrose solutions and allowing the layers to diffuse overnight at 4 C. The volumes used in the different sized tubes are indicated in Table II. Alternatively, gradient columns were prepared using a peristaltic pump drawing from a reservoir containing a 40$ sucrose solution with the withdrawn liquid being replaced by siphon action from a reservoir of 10$ sucrose. The resulting gradients were ready for immediate use but could be stored for at least 24 hours without affecting the quality of virus separation. Gradients of 10 to 40$ sucrose at pH 7*0 will be referred to as standard gradients. For analytical work a sample of 0.2 ml was applied to each gradient column which was then centrifuged at 39,000 rpm for 60 minutes in an SW-41 rotor. Gradients were analyzed for virus content on an ISCO UA-5 absorbance monitor connected to a chart recorder and fractions were collected manually or by an ISCO density gradient fractionator Model 640. For preparative work SW-25 or SW-27 rotors were used. A 2.0 ml sample 27. Table II. Volumes of sucrose solutions layered into each cellulose nitrate tube i n the manual preparation of 10 - k0% sucrose density gradients Rotor """45]? sucrose Volume (ml) ! o £ sucrose 20$ sucrose 10% sucrose SW-25 SW-27 S¥-41 7 9 2.8 7 9 2.8 7 9 2.8 4 5 2.8 28. was applied to each gradient column and the columns were then centrifuged at 23,000 (SW-25) or 25*000 (SW-27) for 150 minutes. To examine TNV-infected tissue for the presence of satellite virus a purification method based on that of Fridborg et al . (1965) and Eassanis (1970) was employed. Tissue was extracted by grinding in a Waring blendor for 3 minutes with 1:1 (weight to volume) 0.1 M sodium acetate buffer pH 5.0 containing 0.02 M DIECA and 0.2$ NagSO^. After being expressed through cheesecloth the extract was left at room temper-ature for 24 hours and then centrifuged at 7500 rpm for 15 minutes in a Sorvall GSA rotor (approximately 9200 g). Ammonium sulfate was added to the supernatant at the rate of 0.25 gm/ml of preparation and left to stir for 2-3 hours at 4 C. After a low speed centrifugation, pellets were resuspended in 0.1 M sodium acetate buffer pH 5.0 at 4 C for at least three hours or overnight, and then subjected to another low speed centri-fugation to remove insoluble materials. The ammonium sulfate precipitation cycle was repeated twice and the final supernatant dialysed against 0.1 M sodium acetate buffer pH 5*0 to remove a l l ammonium sulfate. Samples were analysed by photometric scanning after sucrose density gradient centrifugation in gradients made with 0.1 M sodium acetate pH 5*0. Estimation of the virus content of partially purified preparations The virus content of partially purified (one cycle of differential centrifugation) preparations was measured in one of two ways. Early in this work a Beckman DU spectrophotometer was used to measure the absorbance at 260 nm and from this virus concentration was calculated. An extinction coefficient of E 0 , ^ = 5.6 was used for both TNV (Lesnaw and Beichmann 1969) and CNV (Tremaine, unpublished) The 260/280 ratio, however, was consistently low (see Results) 29. indicating the presence of contaminating proteins that reduced the accuracy of concentration estimation. Subsequently, the: method of Brakke (1967) was used. The concentrations of dilutions of gradient purified CNV and TNV preparations vere determined on the Beckman DU spectrophotometer. Then, for each dilution, the concentration was correlated with the area under the virus peak in ultraviolet absorption profiles of SW-41 sucrose density gradient columns after centrifugation. The area was determined by mechanical planimetry or by a computer plotter. A standard curve was thus prepared which could be used to establish the concentration of virus in partially purified preparations from the area under the absorption profile. The absorbance area was approximately proportional to the concentration of the virus (Figures 1 and 2). This method was particularly useful in estimating the concentrations of CNV and TNV in preparations from mixed infections. Antisera and serological tests Virus identity was confirmed and checked periodically through this work by serological tests using CNV antisera supplied by Dr. H. F. Dias and by Dr. J. Tremaine and TNV antisera supplied by Dr. H. F. Dias and by Dr. R. Stace-Smith. Later, antisera produced in this work were routinely used to check the identity of virus preparations, especially for contamination by TNV. Prior to antiserum production, each rabbit was bled of 20-30 ml blood from an ear vein for the production of normal sera. Antisera to CNV and TNV were prepared by intramuscular injection of rabbits with gradient purified virus at a concentration of 2 mg/ml thoroughly mixed 1:1 with Freund's complete Bacto adjuvant (Difco Laboratories). One mi H i litre of the mixture was injected into each hind thigh muscle and 30 Figure 1. Amount of cucumber necrosis virus (mg) versus area under virus peak on absorbance profiles. Milligrams of virus applied to gradient column (dilutions calculated from optical density readings at 260 nm of gradient-purified virus) plotted against the area under virus peak on ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. 31 ' • • i i i i i i i i i Area under virus^eak in ul^raviole?*absorbance%rofiles (sensitivity 0.5 scale; chart speed 2.0 ml/min) (square inches) 32 Figure 2. Amount of tobacco necrosis virus (mg) versus area under virus peak on absorbance profiles. Milligrams of virus applied to gradient column (dilutions calculated from optical density readings at 260 nm of gradient-purified virus) plotted against the area under virus peak on ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. 33 i i i • * • • « i i i i 0 7 2 O O 0.8 I 7 o 1 . 2 Area tinder vir?-.s peak in ultraviolet absorbance profiles (sensitivity 0.5 scale; chart speed 2.0 ml/min) (square inches) 34. the inoculation procedure repeated three times at approximately ten day intervals. Three weeks after the third inoculation the rabbits were bled. Seven months later, in order to replenish antisera stocks, the rabbits were given a "booster" inoculation to increase antiserum titre and held two weeks after inoculation. Serum was prepared by incubating blood at 37 C for 30 minutes, then at 4 C overnight, and collecting the liquid portion by draining i t away from the clot. Centrif ugation at 4000 rpm for 20 minutes in a Sorvall SS-34 rotor removed red blood cells leaving a clear serum which was tinged pink due to hemolysis. Serum was preserved by adding glycerin (l t l ) and storing in a freezer or by adding sodium azide (0.01$) (Noordam 1973) and storing in a refrigerator (approximately 4 C). Antisera were tested for specificity and titre using the Ouchterlony (1962) agar double immunodiffusion method. One per cent Noble agar was prepared using 0,01 M, phosphate buffered saline (0.01 M potassium phosphate buffer with 0.8$ sodium chloride). Sodium azide was added to 0.02$ as a preservative and 10 ml of agar was poured into each plastic petri plate. The use of plastic obviated the need for a water-repellent coating such as Formvar. When the agar had set, 0.6 cm diameter wells were cut with a No. 2 cork borer in the pattern illustrated in Figure 3(a) such that the distance between the centres of opposite wells in adjacent rows was 0.8 cm and well edges were 0.2 cm apart. Wells in the central row were f i l l e d with serial dilutions of the antiserum being tested and the wells of each outer row were f i l l e d with either CNV or TNV at a constant concentration. The plates were kept in plastic bags with moist paper towelling to maintain humidity and incubated at room temperature for 3 days. Observations of precipitin lines were made at 48 and 72 hours. Normal sera were similarly 35. tested and a l l antisera vere tested against sap from uninfected plants. Later, agar immunodiffusion on glass, slides vas used. The advantages of this method vere economy of time and materials and greater sensitivity. Clean microscope slides vere rinsed in 100$ ethanol. then dipped into a solution of 4$ collodion in 1:1 ether and ethanol and allowed to dry. Two millllitres of 1$ Noble Agar (as above) vere applied to each slide. When the agar had set, a template vas used to punch veils in the pattern shown in Figure 3(b). The centres of opposite wells vere 0.7 cm apart but the outer edges of the veils vere separated by only 0.1 cm of agar. Antigen at constant concentration vas placed in the central veils and serial dilutions of antiserum vere placed in the outer veils. Incubation vas at room temperature for 48 hours and precautions vere taken as above to maintain humidity. Observations vere taken at 24 and 48 hours. The microprecipitin test (van Slogteren 1955; Ball 1974) vas routinely used to check single infection preparations for contamination by the other virus. This test has the advantage of using small volumes of antigen and antiserum and of providing results quickly. A grid of four by six 1 cm squares vas drawn in wax pencil on the inside surface of a plastic petri dish. A dilution series of antigen vas prepared and a drop of each dilution transferred to a separate square. A drop of antiserum at a constant dilution vas added to each drop of antigen. The dish vas flooded with paraffin o i l . covering the droplets in order to prevent evaporation, and incubated at 37 C for one hour at which time observations of precipitation vere made. The dish was incubated in a refrigerator overnight and the reaction read again. Precipitation vas viewed against a dark field with incident light under low power (25X) magnification. 36. Figure 3 (a) Pattern of wells cut in agar for immunodiffusion tests in petri plates. (b) Pattern of wells cut in agar for immunodiffusion tests on glass slides. 37. (a) (b) ° o ° o 0 c r o o 38. Sucrose density gradient centrif ugation of virus preparations after  reaction with antisera If treatment of a mixed infection preparation with an antiserum previously shown to remove only homologous virus from an in vitro virus mixture results in complete or partial removal of heterologous as well as homologous virus, then the presence of virions with capsids composed of protein subunits from both viruses in the mixed infection would be indicated. Visualizing the results of antigen-antibody reactions by photometric scanning of centrifuged sucrose density gradient columns (Whitcomb and Spendlove 1966; Ball and Brakke 1969) was used here as a sensitive assay for the occurrence of phenotypic mixing. Samples of an in vitro mixture of CNV and TNV were treated with different dilutions of antisera to determine the optimum concentration for complete removal of homologous virus. It was found that a l/lOth dilution of either antiserum completely (within the range of detection of photometric scanning) removed a l l homologous virus at virus con-centrations less than 0.75 mg/ml when mixed on a 1:1 basis. Mixed infection preparations and in vitro mixtures of CNV and TNV were treated as follows and compared. Each preparation was divided into four parts, mixed with an equal volume of buffer, normal serum, antiserum to CNV, or antiserum to TNV, and incubated at 37 C for one hour with frequent agitation. From each sample 0.2 ml of supernatant was carefully removed without disturbing the precipitate i f one was present and floated on a standard sucrose density gradient in an SW-41 tube. After centrifugation at 39,000 rpm for 60 minutes the gradients were scanned and the areas under the virus peaks measured. Antigen-antibody neutralization The antigen-antibody neutralization method, adapted from 39. Dodds and Hamilton (1974). vas used to examine mixed infection pre-parations for the occurrence of genomic masking. If a particular antiserum treatment removed a l l homologous viral infectivity from control (in vitro) mixtures hut failed to do so from mixed infection preparations of similar composition, the possibility of genomic masking would be indicated. every group of three mixed infection preparations tested and, consequently, the virus concentrations in some preparations vere sometimes higher or lover than in the controls (see Results). The in vitro mixture consisted of CNV and TNV in an approximate 1:1 ratio with the concentration of each virus being similar to that in the majority of mixed infection preparations. Each test or control preparation was divided into four aliquots of 0.25 ml each and mixed with an equal volume of buffer (0.01 M potassium phosphate, pH 7.l)» normal serum, antiserum to CNV, or antiserum to TNV. Sera vere diluted 1 in 10 with potassium phosphate buffer prior to use. Three treatment cycles vere used: l) 0.25 ml preparation (i) incubated at 37 C for 1 hour + 0.25 ml buffer or serum ( i i ) incubated at 4 C for 1 hour Because several mixed infection preparations from different sources vere tested, an equivalent in vitro mixture (control) was not prepared for each preparation. Instead, a single control vas used for ( i i i ) centrifuged at 9500 g for 20 minutes to pellet precipitate. 2) supernatant from step (l) + 0.25 ml buffer or appropriate serum (i) incubated at 37 C for 1 hour (ii ) incubated at 4 C overnight ( i i i ) centrifuged at 9500 g for 20 minutes to pellet precipitate. 40. 3) supernatant from step (2) + 0.25 ml buffer or appropriate serum (i) incubated at 37 C for 1 hour ( i i ) incubated at 4 C for 3 hours ( i i i ) centrifuged at 9500 g for 20 minutes to pellet precipitate. The final supernatants vere inoculated to indicator plants to assay infectivity remaining after treatment. Chenopodium capitatum (L.) Asch. vas used to assay for TNV infectivity and Phaseolus vulgaris L. cv. Topcrop vas used to detect CNV infectivity. Polyacrylamide gel electrophoresis (PAGE)  (i) Whole virus PAGE Electrophoresis of vhole virus samples from in vitro mixtures and from double infection mixtures vas conducted in 2 .6$ polyacrylamide gels in an attempt to detect phenotypic mixing. Gels vere prepared by mixing the folloving: 5.2 .ml of a 20.0 $ acrvlamide and 0 .6$ N,N•-methy1ene-bis-acrylamide (Bis) solution 3.0 ml of gel buffer stock 1.0 ml of N, N, N', N'-tetramethyl-ethylenediamine (TEMED) 27.8 ml of distilled vater 0.15 ml of a 10$ ammonium persulfate solution (prepared fresh every two weeks). Gel buffer stock vas prepared by dissolving 36*3 gm of 2-amino-2(hydroxy-methyl)-l,5-propanediol (Tris) in 48.0 ml of 1.0 N HC1 and bringing the volume to 100 ml with distilled vater. The gel buffer stock was at pH 8 .9 . Tray buffer stock was prepared by dissolving 4.5 gm of Tris and 21.6 gm of glycine in a total volume of 750 ml distilled water. The pH was adjusted to 8.3 (if necessary) with NH^ OH. One hundred millilitres of this tray buffer stock vas diluted to 2 litres with distilled vater 41 before use. This system will be referred to as the Tris-HGl/Tris-glycine system. Another system was tried and will be referred to as the Tris-HCl/Tris-diethylbarbituric acid system. Gel buffer stock here was prepared by dissolving 6.85 gm Tris in 48.0 ml of 1 N HCI and bringing the volume to 100 ml with distilled water (Maurer 1971). This Tris-HCl gel buffer stock was at pH 7*5 and was substituted for the pre-viously described stock in the preparation of gels for this second system. The tray buffer was prepared by dissolving 1.0 gm of Tris and 5*52 gm of diethylbarbituric acid in 1000 ml of distilled water (Maurer 1971). The final pH was 7*0. Gels were cast in plastic tubes or. where ultraviolet absorbance profiles were desired, in quartz tubes having an internal diameter of 0.6 cm and a length of 10 cm. The gels themselves were 8.5 cm in length. A layer of distilled water was applied immediately after pouring of the gels to allow them to set with a flat surface. This allowed uniform penetration of virus into the gels during electrophoresis. When the gels had set (approximately 30 minutes) the tubes were inserted into the upper tray of the electrophoresis apparatus where they were held in place by water-tight seals. One litre of tray buffer was placed in each of the upper and lower trays so that both ends of the gels were immersed in buffer. Tray buffer was pipetted down onto the gel surface to remove any air pockets. Virus preparations were diluted to a concentration of 1 mg/ml in a diluent of 5% sucrose in tray buffer containing a few drops of bromo-phenol blue as a front marker. The sucrose increased the density of the sample and prevented i t from floating when layered on the gels, as samples were applied with a micropipet under the surface of the tray buffer. A current of 2-3 ma per gel was applied until the front marker reached 42 the bottom of the gel, approximately 3 to 4 hours. Gels were removed from the plastic tubes by blowing them out and they were stained in a solution of 0.01$ Coomassie blue in 20$ trichloroacetic acid (TCA) overnight. They were destained in approximately 7$ acetic acid overnight and then placed in distilled water with a drop of glacial acetic acid for temporary storage. Gels were observed on a light table and the positions of the stained virus bands recorded. (ii) RNA PAGE Electrophoresis of viral RNA samples was attempted on 2.4$ polyacrylamide gels prepared by mixing the following: 8.3 ml of 3E gel buffer (30.0 ml of 10E buffer and 70.0 ml of distilled water) 2.0 ml of a 1.0$ TEMED solution 0.2 ml of a 10.0$ ammonium persulfate solution 4.0 ml of a 15.0$ acrylamide and 0.75$ bis-acrylamide solution 10.3 ml of distilled water. These volumes were sufficient to cast six gels. The 10E stock buffer was prepared from 43.62 gm Tris, 24.51 gm sodium acetate (C2R^02Na*3H20), 3*36 gm disodium ethylenediaminetetraacetate (EDTA), and 18.0 gm sodium dodecyl (lauryl) sulfate (SDS) made up to 1000 ml with distilled water; the pH was adjusted to 7*2 with acetic acid. Gels 8.5 cm in length were cast in 10 cm long quartz tubes having an internal diameter of 0.6 cm and after the gels had set, the tubes were inserted into the electrophoresis apparatus and tray buffer was poured into the upper and lower chambers. The tray buffer (IE) was prepared by mixing 200 ml of 10E buffer with 1800 ml of distilled water. Virus samples were diluted 1:1 in RNA extraction buffer and after incubation a 50 ul sample was applied to 43. each gel with a micropipet. The diluted samples were sufficiently dense to allow their direct application to the gels. A current of 8 ma/gel was applied for 2-2.5 hours. Gels in their quartz tubes were scanned at 254 nm and 280 nm in an ISCO gel scanner coupled to a UA-5 absorbance monitor and a chart recorder, and then removed by injecting water from a syringe between the gel and the tube. Gels were stained in 0.03$ Toluidine blue in 40$ ethylene glycol overnight and then de-stained for two weeks in daily changes of distilled water. Electron microscopy A few partially purified virus preparations were examined by electron microscopy. Virus particles were stained with 2$ aqueous uranyl acetate. Photographs were taken at a magnification of 19*237 times and final print magnification was 134.659 times. 17 44. RESULTS Differential hosts for CNV and TNV Cowpea. bean, cucumber, and Gomphrena globosa were tested for suitability as differential hosts of CNV and TNV. Although differences between CNV- and TNV-induced symptoms were noted in these hosts (Table III), they were not distinctive. Consequently, a wide range of plants was inoculated in an attempt to find suitable specific or differential hosts for CNV and TNV (Table IV). Each species tested reacted to both viruses in a similar manner. While lesion type varied from species to species, CNV and TNV induced the same or similar lesion type in any given host. Plants were kept two to three weeks at which time no evidence of systemic infection was visible in any case. However, three of the hosts were notable for their differential response with respect to numbers of CNV and TNV lesions in single infections. Datura stramonium and Plantago major produced large numbers of CNV lesions but a highly infectious (as determined on cowpea) TNV inoculum induced only a few scattered lesions. In contrast, Chenopodium capitatum developed large numbers of lesions after inoculation with the same TNV preparation while producing very few lesions to CNV, In an attempt to enhance this differential response to the point of exclusion of the less-favoured virus, plants of the test species along with cucumber as a control, were singly or doubly inoculated and placed under three temperature regimess constant 1 8 C, ambient green-house temperature, and constant 30 G. Dj_ stramonium and Pj_ ma.ior were found unsuitable as specific indicators of CNV under these conditions (Table V). C. capitatum. when kept at 17 C after inoculation, produced 45 Table III. Symptoms of CNV and TNV infections in five plants tested as possible differential hosts under cool greenhouse conditions Host CNV-induced symptoms TNV-induced symptoms Gomphrena globosa Vigna sinensis cv. Early Bamshorn Cucumis sativus cv. Straight Eight Phaseolus vulgaris cv. Bountiful Phaseolus vulgaris cv. The Prince tan-coloured necrotic lesions generally less than 2 mm diameter red lesions, 2 mm or less in diameter; heavily infected leaves f a l l at approximately 7 days tan-coloured necrotic lesions approximately 2 mm in diameter but up to 4 -5 mm; seedlings frequently killed by systemic necrosis of stem; less severe infections completely localized red lesions 3 -5 mm in diameter red!lesions up to 2 mm in diameter tan-coloured necrotic lesions of same appear-ance as CNV lesions, but larger ( 3 - 4 mm) red lesions like those produced by CNV except smaller; heavily infected leaves drop at 7 days tan-coloured necrotic lesions 2 mm in diameter with lighter coloured pinpoint centres; seed-lings occasionally killed by systemic necrosis of stem, but infections more commonly localized red lesions as produced by CNV but ranging from pinpoint to 3-4 mm in diameter lesions darker red than CNV lesions, almost black; pinpoint to 1 mm in diameter 46 Table IV, Local lesion production on plants inoculated with CNV or TNV in host range trials Plant inoculated Local lesion reaction to CNV TNV Nicotiana clevelandii Gray + Nicotiana tabacum cv, Haranova + cv, Havana 425 + cv. Xanthi-nc + N. sylvestris " + Pelargonium sp. Zinnia sp. + Brassica nigra (L.) Eoch. cv. Tender Green Capsicum a n n u u m L. cv. California Wonder + Trifolium pratense L. _ Trifolium hybridum L. Medicago sativa L. -Lycopersicon esculentum Mill, cv. Subarctic -Triticum aestivum L. -Hordeum vulgare L. cv. Black Hulless -Cucumis B a t i v u s L. cv. National Pickling + cv. Straight Eight + + Gomphrena globoaa L. + Vigna sinensis (L.) Engl. cv. Early Bamshorn + Datura stramonium L. + Plantago major L. + Plantago lanceolata L. Phaseolus vulgaris L. cv. Bountiful + cv. Pinto + cv. The Prince + cv. Topcrop + Chenopodium amaranticolor Coste and Beyn. + C. amaranticolor cv. Greenleaf + C. quinoa Willd. + C. capitatum (L.) Asch. + + + + + + Table V. The effect of temperature on the number * of lesions induced by CNV and TNV inoculated singly to four hosts*** Number of CNV lesions at Number of TNV lesions at Host 18 C greenhouse temperature 30 C 18 C greenhouse temperature t Chenopodium capitaturn 0 3 4 100 100 1 Datura stramonium 4 50 0 14 12 0 Plantago ma.1or 2 11 0 5 2 0 Cucumis sativus 17 100 7 C' 18 100 0 a. lesion numbers are averages of 4-leaf samples b. counts vere made at 3 or 5 days post-inoculation (depending on environment) although plants vere maintained up to two weeks for observation of significant changes c. very faint ohlorotic spots 48. large numbers of TNV lesions by the third day post-inoculation while lesions on GNV-inoculated leaves did not appear until the f i f t h or sixth day and were few in number (Figure 4). In addition, lesion appearance was different. TNV lesions were 3*4 mm in diameter with pinpoint necrotic centres surrounded by a wide band of tissue having a "water-soaked" appearance. CNV lesions were pinpoint, tan coloured necrotic spots. Mixed inoculations did not alter the time of lesion appearance or any lesion characteristics; however. CNV lesions were not easily distinguished from the pinpoint centres of earlier formed, coalescing TNV lesions when on the same leaf. Subsequent tests esta-blished winter greenhouse conditions to be generally as effective as a controlled 17 C temperature for use of C, capitatum as a differential host. The rate of TNV lesion development, however, was slower in the greenhouse, with lesion appearance being delayed by a day. C. capitatum took several months from seeding to reach the desirable size for use as a TNV indicator. Because of this, i t was in limited supply and plants of two more readily available Chenopodium species were singly inoculated with CNV or TNV under winter greenhouse conditions and at 17 C to determine i f they might be substituted for C. capitatum. On both C^ qninoa and C. amaranticolor TNV symptoms developed slightly faster than did CNV symptoms, although both viruses elicited approximately the same number of lesions (Table VI). However, the difference in time of appearance was not definitive, making these hosts unsuitable for use as TNV indicator plants. The host range tests of CNV and TNV failed to reveal an indicator plant for CNV. Among the plants tested were four varieties of Phaseolua vulgaris: Bountiful, Pinto, The Prince, and Topcrop. Under the autumn greenhouse conditions of the test, a l l four responded to CNV 49. Figure 4. Symptoms of CNV and TNV infections on leaves of Chenopodium capitatum. Plants vere kept under cool (approximately 18 C) greenhouse conditions. (a) Symptoms at 3 days after CNV inoculation (b) Symptoms at 3 days after TNV inoculation (c) Symptoms at 3 days after mixed CNV and TNV inoculation (d) Symptoms at 6 days after CNV inoculation (e) Symptoms at 6 days after TNV inoculation (f) Symptoms at 6 days after mixed CNV and TNV inoculation Table VI. The effect of temperature on the number * of lesions induced by CNV and TNV inoculated singly to three Chenopodium species * (with Vigna  sinensis cr. Early Bamshorn included as control) Number of CNV lesions at Number of TNV lesions at Host 18 C winter greenhouse 18 C winter greenhouse temperatures temperatures Chenopodium capitatum 0 C. quinoa 100C' C. amaranticolor 100°* Vigna sinensis 71 cv. Early Bamshorn 'lesion numbers are averages of 3-leaf samples 'counts were made three days after inoculation c*chlorotic spots d*severe, spreading necrosis 0 100d' 100 100 100d* 100 1009 100d' 100d* 90 48 100 52. and TNV with the production of red lesions which were of similar size on any given variety. The inclusion of varieties Topcrop and The Prince in similar experiments conducted under summer greenhouse conditions, however, revealed the failure of a highly infectious TNV preparation to induce lesions on Topcrop bean. Different trials (summarized in Table Vii) under summer and f a l l greenhouse conditions revealed erratic lesion production, with apparently only slight environmental changes determining the appearance or absence of TNV lesions. When primary leaves of Topcrop bean were TNV-inoculated while s t i l l unfolding, some lesions were produced, while comparison plants under the same conditions but inoculated on fully opened primary leaves developed no symptoms. CNV inoculated to Topcrop bean produced necrotic lesions ringed with red. which often extended into adjacent minor veins. When inoculated Topcrop plants were kept in a growth chamber at a constant temperature of 23 C (18 hour photoperiod; 60$ humidity). TNV consistently failed to induce lesions even after 10 days. CNV. on the other hand, produced numerous large red lesions at 3-4 days post-inoculation. Under such controlled conditions. Topcrop bean was useful as an indicator of CNV. Virus propagation and purification The results of the virus host range trials revealed that cowpea and cucumber responded to inoculations with the production of numerous lesions, with a similar degree of symptom severity induced by either CNV or TNV. Cucumber and cowpea were thus selected as the best potential propagation hosts and virus yields were measured to obtain an indication of which of the two was preferrable. Two experiments were done to determine the more suitable host for Table VII, Response of Phaseolus vulgaris cv. Topcrop and cv. The Prince (bean) and Vigna sinensis cv. Early Ramshorn (cowpea) to TNV inoculations at different temperatures Plant inoculated Temperature Symptoms Topcrop bean with primary leaves winter greenhouse fully opened Topcrop bean with primary leaves fully opened Topcrop bean with primary leaves fully opened Topcrop bean with primary leaves fully opened Topcrop bean with primary leaves only partially opened The Prince bean with primary leaves fully opened Cowpea with primary leaves fully opened 18 C 23 C summer greenhouse summer greenhouse summer greenhouse summer greenhouse numerous red pinpoint lesions often difficult to detect unless viewed with backlighting scattered red pinpoint lesions none none scattered red pinpoint lesions with only one-third ( 2 of 6 ) inoculated leaves affected numerous pinpoint lesions, very dark red to almost black (difficult to detect) numerous red lesions, up to 2 mm in diameter 54. TNV propagation. Virus concentration (mg/lOO gm fresh weight) was determined hy measuring the optical density of partially purified preparations at 254 nm. Two to five times more TNV was recovered from cowpea than from cucumber (Table VIII). Cowpea and cucumber were similarly examined for their relative yields of CNV in single infections (Table IX). Cowpea yielded only three-quarters the amount of virus produced in cucumber in this experiment. No replicate experiments were conducted. A preliminary comparison of virus yields from cowpeas singly infected with CNV and TNV under warm (26 or 30 C) and cool (spring greenhouse) temperatures was used to establish suitable temperature ranges for virus propagation. Virus concentration (mg/100 gm fresh weight of infected tissue) was estimated by measuring the optical density of partially purified preparations at 254 nm. The results are summarized in Table X. The ratio of the amount of CNV recovered from infections at 26 C to that recovered from infections in the cool greenhouse (7.8/6.3 • 1.24) indicated the warmer temperature was more suitable for CNV replication, with 24$ more virus being recovered. The ratio of the amount of TNV recovered from infection in the cool greenhouse to that recovered from infection at 30 C (l7»7/5.0 * 3.54) indicated the cooler temperature vas more suitable for TNV replication, with three and one-half times more virus being recovered. Several attempts to induce systemic infections in bean, cowpea. and cucumber met with only limited success. Plants of Phaseolus vulgaris varieties Bountiful. Pinto, The Prince, and Topcrop, inoculated with TNV and kept under mid-summer greenhouse conditions, failed to develop local lesions or any other sign of infection. Purification followed by observation of ultraviolet absorption profiles after sucrose density gradient centrifugation failed to detect any TNV replication. The Table V I I I . Comparison of TNV yields from Cncmnis sativus cv. Straight Eight (cucumber) and Vigna sinensis cv. Early Ramshorn (cowpea) Host Dilution of virus preparation Ultraviolet absorbance 260/280 at 260 nm ratio 280 nm Estimated ' TNV recovered TNV yield from (mg/100 gm fresh weight cowpea/TNV yield of tissue) from cucumber at each dilution average cucumber 1/50 0.106 0.09 1.2 0.8 cucumber 1/100 0.047 0.04 1.2 0.7 0.75 cowpea 1/50 0.48 0.325 1.5 3.4 cowpea 1/100 0.255 0.176 1.4 3.7 3.55 cucumber 1/50 0.204 0.17 1.2 1.4 cucumber 1/100 0.11 0.08 1.4 1.6 1.5 cowpea 1/50 0.382 0.27 1.4 2.7 cowpea 1/100 0.249 0.19 1.3 3.5 3.1 3.55/0.75 = 4.7 (trial l) 3.1/1.5 « 2 i l (trial 2) 'sample size 250 gm; resuspension buffer volume 2.0 ml; extinction coefficient E 0 , ^ « 5.6 (Lesnaw and Beichmann 1969) Table IX. Comparison of CNV yields from Cncnmis sativus cv* Straight Eight (cucumber) and Vigna sinensis cv. Early Ramshorn (cowpea) Estimated *GNV recovered Host Dilution of Ultraviolet 260/280 (mg/100 gm fresh weight CNV yield from virus preparation absorbance at ratio of tissue) cowpea/CNV'yield 260 nm 280 nm at each dilution average from cucumber Cucumber 1/50 0.215 0.14 1.5 3.8 4.0 Cucumber 1/100 0.12 0.09 1.3 4.2 3.0 = 0.75 1/50 4.0 Cowpea 0.16 0.11 1.5 2.8 3.0 Cowpea 1/100 0.09 0.065 1.4 3.2 a*sample size 250 gm; resuspension buffer volume 2.0 ml; extinction coefficient E g ^ « 5.6 (T remaine unpublished) Table X. Yields of CNV and TNV from singly inoculated Vigna sinensis C T . Early Ramshorn (cowpea) grown at different temperatures Virus and Dilution of Ultraviolet 260/280 Calculated virus Preparation Weight of Estimated virus temperature virus absorbance at ratio concentration** volume (ml) tissue recovered (mg/ preparation 2 6 o m ^ ^ (me/ml) prepared 100 gm fresh at each average (gm) weight of tissue) dilution CNV, 26 C CNV, 26 C 1/50 1/100 0.126 0.066 0.098 0.053 1.3 1.2 1.1 1.2 1.2 13 200 7.8 CNV, spring greenhouse CNV, spring greenhouse 1/50 1/100 0.125 0.071 0.096 0.056 1.3 1.3 1.1 1.3 1.2 19 360 6.3 TNV, 30 C TNV, 30 C 1/50 1/100 0.103 0.052 0.075 0.037 1.4 1.4 0.92 0.93 0.93 14 260 5.0 TNV, spring greenhouse TNV, spring greenhouse 1/50 1/100 0.393 0.288 0.290 0.210 1.4 1.4 3.5 5.1 4.3 7 170 17.7 a ,extinction coefficient E 0 , ^ = 5.6 for both CNV (Tremaine unpublished) and TNV (Lesnaw and Reichmann 1969), 58. inoculations of TNV were repeated on varieties Topcrop and The Prince under late f a l l or winter greenhouse conditions conducive to TNV replication. This time small dark red lesions appeared on the inoculated leaves of both hosts while trifoliate leaves formed after inoculation remained symptomless. The latter tissues were nevertheless harvested, purified, and examined by sucrose density gradient centri-fugation for the presence of TNV that would indicate systemic infections. None could be detected. The host range trials produced no indication of systemic infections by CNV and TNV, whether inoculated singly or combined, in any host. Over a period of time, however, during which numerous cowpea and cucumber plants were inoculated for virus propagation at various times of the year i t was noted that occasionally CNV elicited streaking of petioles and stems in cowpea and. more rarely, in Topcrop bean. The red streaks often became necrotic and were distinct from the normal red streaks seen on stems and petioles of older, healthy cowpea plants. In no case did systemic infections spread upwards into uninoculated leaf tissue. A l l necrosis and reddening occurred in veins of inoculated leaves, in leaf petioles, and less frequently downwards into the stem. Both viruses sometimes caused a greyish necrosis descending down stems of cucumber seedlings inoculated on the cotyledons with highly infectious preparations. This was especially pronounced in CNV infections, where necrosis quickly involved the whole stem and killed up to three-quarters of inoculated seedlings. More restricted TNV systemic infections occurred in approximately one-quarter of the plants; necrosis was re-stricted to streaks and sunken areas on the stems and seedlings were seldom killed. Attempts to induce systemic infections in mature cucumber were 59. unsuccessful. Eight cucumber plants approximately fifteen inches in length were inoculated on apical leaves with CNV alone. The tests were done under summer and f a l l greenhouse conditions. No symptoms of systemic infections or even of localized infections became evident, although plants were kept four to eight weeks after inoculations. The inoculum was shown to be infective on cucumber cotyledons. Attempts were made to initiate systemic infections by root inoculations of cowpea with TNV. Two weeks after root inoculations, cowpea seedlings were harvested and root. stem, and leaf tissues prepared separately for examination of virus content. The only symptoms were a few brown spots on the roots and occasional necrotic streaks at the bases of stems. Electron microscopy of partially purified preparations revealed no virus from leaf tissue but considerable virus from the stems. The root preparation was yellowish-white and opaque and despite additional treat-ments of incubation at 37 C for one hour, or stirring with either chloroform added to 10% or ether added to 7%, i t could not be sufficiently purified to detect the presence of virus by electron microscopy. Sucrose density gradient centrifugation and photometric scanning similarly revealed virus from stems but none from leaves, but in addition, revealed evidence of TNV in preparations from roots (Figure 5). During i n i t i a l virus purification trials, the results of modifying extraction buffers and clarification methods were compared. For the purification of TNV from cowpea primary leaves, extraction with 0.1 M sodium acetate buffer at pH 5.0 and clarification by a 24 hour pH 5.3 treatment (method 2) proved superior to extraction with 0.07 M sodium phosphate - 0.1 M ascorbic acid buffer at pH 7.0 and clarification by a ten minute treatment with 10% chloroform and a one hour pH 5.3 treatment (method l ) . The ratio of TNV recovered by method 2 to TNV 60. Figure 5. Examination of Vigna sinensis cv. Early Bamshorn root, stem, and leaf tissue for evidence of TNV infection two weeks after inoculation of roots. Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples. Centrifugation at 39000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedi-mentation is to the left, as indicated by horizontal arrow. Vertical arrows indicate the TNV peak or i t s expected position. (a) Preparation from stems (b) Preparation from roots clarified by incubation at pH 5.0 (c) Preparation from roots clarified by incubation at pH 5*0 and stirring with 10$ chloroform (d) Preparation from roots clarified by incubation at pH 5*0 and stirring with 7$ ether (e) Preparation from roots clarified by incubation at pH 5*0 and heat treatment (57 C for 1 hour) (f) Preparation from leaves 61 62. recovered by method 1 (3*6/2.1 = 1*7; Table Xl) indicated that method 2 allowed greater TNV recovery. For purification of CNV from cucumber cotyledons, method 2 was again superior to method 1 and also to method 3* where extraction was in 0.1 M sodium acetate buffer at pH 5.0 but clarification was by stirring for one hour with 10$ chloroform. The ratios of the amount of CNV recovered by method 2 to the amounts recovered by methods 1 and 3 (7.2/2.8 = 2.6, 7.2/1.24 « 5*8 respectively; Table Xii) indicated that method 2 allowed greater CNV recovery. A comparison of TNV yield from fresh and frozen cowpea tissue revealed that freezing caused no loss in the amount of virus recovered. Effects of mixed infections on symptoms and systemic spread When plants of each species tested in the host range trials were inoculated with a mixture of CNV and TNV and observed for up to three weeks, no new symptom type uncharacteristic of either virus in single infections could be detected. The virus combination did not induce systemic symptoms in any host at that time. A qualitative assessment of symptoms on these hosts indicated slight interference in Pinto bean and a slight increase in symptom severity in Chenopodium amaranticolor. C. quinoa. 6. globosa. and cowpea. This increase was apparently less than additive, symptoms in mixed infections being only slightly more severe than i n either of the single infections. No synergism with respect to symptoms was evident. Doubly infected cucumber were no more seriously affected than singly inoculated plants. A quantitative assessment was made of the effects of mixed infections on lesion numbers on cowpea, Topcrop bean, and cucumber Table XI. Estimated amount of TNV recovered from Vigna sinensis cv. Early Ramshorn primary leaves using two purification methods Purification method Dilution of virus preparation Ultraviolet absorbance at 260 nm 280 nm 260/280 Estimated0* virus ratio recovered (mg/100 gm fresh weight of tissue) at each dilution average 1. 1/50 1/100 0.308 0.135 0.209 0.096 1.5 1.4 2.2 2.0 2.1 2. 1/50 1/100 0.48 0.255 0.325 0.176 1.5 1.4 3.5 3.7 3.6 0 \% 'sample size 250 gm; resuspension buffer volume 2.0 ml; E_*-0^  = 5.6 (Lesnaw and Reichmann 1969; b. method 1: extraction with sodium phosphate - ascorbic acid buffer. pH 7.0; clarification with 10$ chloroform and pH 5.3 treatment method 2: extraction with sodium acetate buffer. pH 5.0; clarification by pH 5.5 treatment Table XII. Estimated amount of CNV recovered from Cucumis sativus cv. Straight Eight cotyledons using different purification methods Purification Dilution of Ultraviolet Calculated virus Preparation Weight of Estimated method * virus absorbance at concentration volume (ml) tissue virus preparation 260 nm (mg/ml) * prepared recovered (gm) (mg/lOO gm fresh weight of tissue) 1. 1/50 0.161 1.4 2.0 100 2.8 2. 1/50 0.204 1.8 2.0 50 7.2 3. 1/50 0.069 0.62 1.0 50 1.24 a*E260 * 5 , 6 C1160"11116 unpublished) "'method 1: extraction with sodium phosphate - ascorbic acid buffer, pH 7.0; clarification with 10$ chloroform and pH 5.3 treatment method 2: extraction with sodium acetate buffer. pH 5.0; clarification by pH 5*3 treatment method 3* extraction with sodium acetate buffer, pH 5.0; clarification with 10$ chloroform (Table XIII). Particularly notable was the marked reduction in the number of CNV lesions on doubly infected Topcrop bean from that on Topcrop singly infected with CNV. despite the presence of almost no lesions on TNV singly inoculated plants. Work subsequent to the host range trials revealed that CNV and TNV sometimes exhibited limited systemic spread in single infections. The percent occurrence of systemic symptoms and the symptom severity after leaf inoculations were compared in single and mixed infections with CNV and TNV on cowpea. Topcrop bean, and cucumber (Table XIV). In cowpea. CNV was almost completely localized in lesions, of which there were approximately the same number as induced by TNV. By ten days after inoculation. 95$ of TNV-infected cowpea leaves exhibited some systemic symptoms and many were senesced; CNV-inoculated leaves, however, were s t i l l intact and only 11$ of the cowpea plants showed signs of systemic infections. In mixed infections, the presence of CNV resulted in a slight reduction in the occurrence of systemic symptoms from that observed in TNV single infections. In Topcrop bean, TNV induced no systemic symptoms, although on the 40 plants (80 leaves) inoculated, four lesions were formed. Yet in mixed infections, TNV reduced the occurrence of petiole streaking to two-thirds of that observed in single CNV infections. As usual, systemic infections did not spread upwards into uninoculated leaf tissue. In cucumber, no signs of systemic infections were visible at five days after inoculation. However, by ten days, CNV had killed three-quarters of the inoculated seedlings, a l l of which exhibited desiccated cotyledons and necrosis extending downwards in the stem. Only 3$ of TNV-inoculated seedlings died, although TNV lesion numbers were similar to those of CNV. The number of plants killed in mixed 66. Table XIII. The effects of mixed infections on lesion numbers Average number 'of lesions induced by Host CNV TNV CNV/TNV (mixed infections) Vigna sinensis 3 5 4 cv. Early Bamshorn Phaaeolus vulgaris 73 1 23 cv. Topcrop Cncumis sativna 55 47 51 cv. Straight Eight Cowpea: average number of lesions/half leaf; sample size 20 leaves. Topcrop bean: average number of lesions/leaf; sample size 5 leaves. except for TNV infection where 4 lesions were produced on a total of 80 inoculated leaves. Cucumber: average number of lesions/cotyledon; sample size 4 cotyledons. 67. Table XIV. The effect of CNV and TNV mixed infections on systemic symptoms in Vigna sinensis cv. Early Bamshorn. Phaseolns  vulgaris cv. Topcrop, and Cucumis sativus cv. Straight Eight Plants with symptoms of Host Virus Number of plants inoculated Bed necrosis of veins and inoculated leaves  Number % Red necrotic streaks of petioles and/or stem Number % Vigna sinensis CNV cv. Early Bamshorn TNV (5 days post- CNV/TNV inoculation) Vigna sinensis CNV cv. Early Bamshorn TNV (10 days post- CNV/TNV inoculation) Phaseolus vulgaris CNV cv. Topcrop TNV (10 days post- CNV/TNV inoculation) 55 0 55 40 73 14 26 55 39 71 7 13 55 4 7 6 11 55 52 95 21 38 55 49 89 16 29 40 33 83 27 68 40 0 0 0 0 40 29 73 19 48 Host Virus Number of plants inoculated Plants killed by infection  Number % Plants alive and bearing systemic symptoms  Number $> Cucumis sativus CNV cv. Straight Eight TNV CNV/TNV 104 79 76 6 6 101 3 3 24 24 103 44 43 18 17 68 infections vas only half the number killed in CNV single infections. Systemic symptoms included, besides grayish streaking in the stems. a mild chlorotic mottling of the f i r s t true leaves on some of the plants The effects of mixed inoculations of the roots of cowpea with CNV and TNV on the systemic spread of these viruses was also examined. As with single TNV inoculations, there vas some streaking in the lover stem but no foliar symptoms. Subsequent analysis of a purified virus preparation from leaf and stem tissues detected the presence of only TNV in small amounts. No indication of CNV replication vas found (Figure 6 (a)). As TNV had been readily recovered from stem tissues, an experiment vas conducted to determine i f mixed inoculations of stems would result in systemic infections and high yields of both CNV and TNV. By two weeks after inoculation of the stems, symptoms were visible on the primary leaves of some plants. They ranged from a reddening restricted to major veins at their junction in the petiole, to involvement of virtually a l l leaf veins with inter-veinal necrosis and some chlorosis. A "water-soaked" appearance about the major veins vas seen in a fev plants. Some plants, although having fev or no stem symptoms, exhibited red necrotic streaking of tha petioles of primary leaves which resulted in the affected leaves being twisted such that some were completely inverted. The occurrence of symptoms is summarized in Table XV. Stem tissues and symptom-bearing leaf tissues were prepared for virus purification. The final stem tissue preparation was highly opalescent and ultraviolet absorption profiles after sucrose density gradient centrifugation confirmed a high virus concentration. However, a l l the virus was apparently TNV with no evidence of CNV being present. Leaf tissues similarly yielded only TNV, although in a much smaller amount 69 Figure 6. Examination of Vigna sinensis cv. Early Ramshorn leaf and stem tissue for evidence of CNV and TNV two weeks after mixed inoculations of the roots or stems* Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples* Centrifugation at 39000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by horizontal arrow. Vertical arrows indicate the expected positions of CNV and TNV peaks. (a) Leaf and stem tissue preparations (pooled) from root-inoculated plants. (b) Leaf tissue preparation from stem-inoculated plants (c) Stem tissue preparation from stem-inoculated plants (diluted 1:9 Table XV. Symptoms of virus infection on Vigna sinensis cv. Early Bamshorn two weeks after inoculation of stems with a mixture of CNV and TNV Number of plants inoculated Number of symp tomless plants Number of plants with symptoms of Necrosis of both stems Leaf necrosis and primary leaves Stem necrosis (with or without (dead) (alive) (total) (dead) (alive) (total) stem necrosis) Leaf necrosis ONLY (with no stem necrosis) 338 132 (39.1$) 13 21 34 95 102 197 43 (10.1$) (58.3$) (12.7$) 9 (2.7$) 72. than stem tissues (Figure 6(b)). Three groups of cowpea plants were stem-inoculated with CNV, TNV, or mixed CNV-TNV. The latter two inocula induced symptoms as already described; the CNV stem-inoculated cowpea, however, showed no indication of virus infections, no reddish streaking on stems or petioles, and no leaf symptoms. The CNV inoculum did induce lesions when inoculated to cowpea leaves. Effects of mixed infections on fresh weight Four experiments were conducted to determine the effect of mixed infections as compared to single infections on the fresh weight of virus-inoculated cowpea primary leaves. In experiments 1 and 2 the viruses were at equal concentrations, while in experiments 3 and 4, infectivity of the CNV inocula was substantially less than that of TNV. The results (Table XVI) were variable, making i t difficult to establish whether the effect of mixed infections on fresh weight was antagonistic, synergistic, or additive. In experiments 2, 3, and 4, the weight loss in mixed infections was generally greater than the loss in either of the single infections but less than the additive effect of both single infections. Only in experiment 1 was an antagonistic trend evident, leaves singly infected with TNV generally showing a greater weight loss than leaves doubly infected. Five similar experiments were conducted using cucumber cotyledons as the host tissue (Table XVII). Experiments 1-4 indicated a fresh weight loss in mixed infections greater than that in either of the single infections but less than additive. However, when cotyledon size and conformation at the time of inoculation and tissue sample size (extraction of tissue plugs with a cork borer) were a l l made constant the effect of mixed infections appeared additive. Table XVI. Fresh weight of buffer-inoculated (control) and virus-inoculated primary leaves of Vigna sinensis cv. Early Bamshorn Experiment Hours post-inoculation Fresh weight of tissue Difference in fresh weight of harvest (gm) inoculate with: (per cent of control) buffer CNV TNV CNV/TNV CNV TNV CNV/TNV 24 36.7 34.5 35.1 31.8 6.0 4.4 13.4 36 32.8 30.2 28.0 29.8 7.9 14.6 9.1 48 31.2 30.3 29.4 30.4 2.9 5.8 2.6 72 31.0 31.2 16.8 23.0 - 45.8 25.8 84 34.0 27.4 10.1 23.6 19.4 70.3 30.6 36 21.9 20.4 24.3 20.7 6.8 - 5.5 48 29.8 29.8 28.8 27.5 0.0 3.4 7.7 60 24.3 26.3 21.7 17.1 - 10.7 29.6 72 31.7 26.8 20.9 20.2 15.5 34.1 36.6 84 27.0 26.2 9.6 11.6 2.2 64.4 57.0 24 50.3 48.7 50.2 50.6 3.2 0.2 — 48 58.7 58.0 57.1 50.4 1.4 2.7 14.1 72 57.2 48.0 34.0 29.1 16.1 40.6 49.1 96 61.5 57.4 26.5 15.0 6.7 57.0 75.6 24 38.4 38.4 37.7 38.0 0.0 1.8 1.0 36 48.4 41.9 44.7 39.8 13.4 7.6 17.8 60 56.9 44.3 39.0 34.4 22.1 31.5 39.5 72 48.9 41.1 24.6 17.1 16.0 49,7 65.0 84 51.4 46.8 31.0 15.6 8.9 39.7 70.0 Table XVII. Fresh weight of buffer inoculated (control) and virus inoculated Cucumis sativus cv. Straight Eight Experiment Hours post-inoculation Fresh weight of tissue (gm) Difference in fresh weight of harvest inoculated with: (per cent of control) buffer CNV TNV CNV/TNV CNV TNV CNV/TNV 1. 48 (2 days) 4.4 3.3 2.8 2.5 26 35 43 96 (4 days) 6.4 3.5 2.5 1.9 45 61 70 144 (6 days) 6.8 2.9 2.8 2.3 58 59 66 192 (8 days) 8.0 3.6 2.5 2.1 55 69 74 240 (10 days) 10.2 2.8 2.7 1.3 73 73 87 288 (12 days) 18.3 2.6 3.3 0.7 86 82 96 2. 24 60.4 50.2 50.2 45.6 17 14 25 48 58.8 18.8 18.9 15.4 68 68 74 72 58.4 10.7 10.3 9.0 82 82 85 3. 36 52.4 49.0 46.7 44.6 6 11 15 60 49.7 43.7 36.0 34.7 12 28 30 72 52.0 31.9 24.2 28.8 39 53 45 4. 24 36.5 32.6 29.2 28.7 11 20 21 36 34.6 41.2 33.4 32.4 — 4 6 48 39.6 35.8 17.6 13.2 10 56 67 60 36.1 32.3 12.7 10.3 11 65 72 72 39.9 35.4 10.0 8.6 11 75 78 5. 24 2.71 2.83 2.81 2.67 _ _ 1.5 36 2.82 2.75 2.55 2.48 2.5 9.6 12.1 48 2.91 2.77 2.42 2.21 4.8 16.8 24.1 60 2.94 2.52 1.52 1.05 14.3 48.3 64.3 72 2.96 2.29 1.21 0.65 22.6 59.1 78.0 75 Effects of mixed infections on virus concentration Ihe concentrations reached by CNV and TNV in single and mixed infections of cucumber cotyledons at different temperatures vere compared. Ultraviolet absorption profiles after sucrose density gradient centri-fugation of partially purified preparations revealed that CNV yield from cucumber under summer greenhouse conditions vas substantially higher than that of TNV in single infections. In mixed infections. CNV yield vas significantly reduced while TNV yield vas slightly increased (Figure 7.) At 25 C constant temperature. TNV replication vas severely inhibited (Figure 8). CNV replication, although reduced from that in the greenhouse, vas s t i l l high. The antagonistic effect of TNV on CNV was s t i l l evident despite the extremely low level of TNV replication. It was not until at 30 C constant temperature that TNV no longer exerted a detectable antagonistic effect on CNV replication. CNV attained the same concentration in mixed infections as in single infections (Figure 9). A comparison of virus concentrations vas also made from cucumber grown under f a l l greenhouse conditions, where temperatures could be expected to be conducive to the replication of both viruses, with warm days and cool nights. The results (Figure 10) indicated CNV and TNV reached similar concentrations in single infections. In mixed infections, the antagonistic effect of TNV on CNV vas marked and a slight increase in TNV concentrations from that attained in single infections vas evident. The antagonistic effect of TNV on CNV replication vas further evidenced in serial passages of mixed infections. The f i r s t mixed infections vere those from summer greenhouse conditions vhere CNV vas at a high concentration relative to TNV. A sample of this mixed preparation vas inoculated to a second flat of 100 cucumber seedlings. 76 The virus yield recovered from each of the three infected tissue lots vas examined. Although comparison cannot he made amongst the three passages because resuepension volumes were not standard, the relative yields of CNV and TNV within each of the mixed infections can be compared (Figure l l ) . The yield of TNV relative to CNV increased markedly from l/20th to about 2/3 the concentration of CNV. This occurred under greenhouse conditions established to be favourable for both CNV and TNV replication in single infections and despite the high i n i t i a l CNV concentration relative to that of TNV. In addition to the work with cucumber, plants of Phaseolus  vulgaris cv. Topcrop were singly or doubly inoculated and kept under autumn greenhouse conditions. No detectable TNV was recovered from singly or doubly inoculated tissue. The concentration of CNV appeared l i t t l e altered in mixed infections with TNV from what i t was in single infections (Figure 12). The amounts of virus recovered, as estimated by comparisons of areas under virus peaks on ultraviolet absorption profiles to standard curves, are tabulated in Tables XVIII (CNV yield in single and mixed infections). XIX (TNV yield in single and mixed infections), and XX (serial passage of mixed infections). 77. Figure 7. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown under summer greenhouse conditions. Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by the horizontal arrow. Vertical arrows indicate the position of CNV and TNV peaks. (a) Preparation from CNV-inoculated cotyledons (diluted 1:4) (b) Preparation from TNV-inoculated cotyledons (c) Preparation from CNV and TNV doubly-inoculated cotyledons (diluted 1:4) 78. © O 7 9 Figure 8. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown at 25 C (18 bour photoperiod; 60$ relative humidity). Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by the horizontal arrow. Vertical arrows indicate the position of CNV and TNV peaks. (a) Preparation from CNV-inoculated cotyledons (b) Preparation from TNV-inoculated cotyledons (c) Preparation from CNV and TNV doubly-inoculated cotyledons 81 Figure 9* Amount of virus recovered from single and mixed infections of cucumber cotyledons grown at 30 C (18 hour photoperiod; 60$ relative humidity). Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by the horizontal arrow. Vertical arrows indicate the positions of CNV and TNV peaks. (a) (b) (c) Preparation from CNV-inoculated cotyledons Preparation from TNV-inoculated cotyledons Preparation from CNV and TNV doubly-inoculated cotyledons nm ijQz V s aotreqjosqv 83. Figure 10. Amount of virus recovered from single and mixed infections of cucumber cotyledons grown under autumn greenhouse conditions. Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by the horizontal arrow. Vertical arrows indicate the positions of CNV-and TNV peaks. (a) Preparation from CNV-inoculated cotyledons (b) Preparation from TNV-inoculated cotyledons (c) Preparation from CNV and TNV doubly-inoculated cotyledons 85 Figure 11. Comparison of the relative amounts of CNV and TNV recovered from doubly infected cucumber cotyledons i n each of three ser i a l passages of infection. Plants were grown under autumn greenhouse conditions. Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of p a r t i a l l y purified samples. Centrifugation was at 39,000 rpm for 60 minutes i n standard sucrose gradients i n an SW-41 rotor. Direction of sedi-mentation i s to the l e f t , as indicated by the horizontal arrow. Vertical arrows indicate the positions of CNV and TNV peaks. (a) Preparation from f i r s t mixed infection (diluted 1:4) (b) Preparation from second mixed infection (diluted 1:9) (c) Preparation from third mixed infection (diluted 1:9) (Note: Comparison of absolute amounts of virus among infections i s not v a l i d as re suspension buffer volumes were not constant. 86 . ran ijQz 1* aotreqaosqy 87 Figure 12. Amount of virus recovered from single and mixed infections of Phaseolus vulgaris cv. Topcrop primary leaves grown under autumn greenhouse conditions. Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples. Centrifugation was at 39,000 rpm for 60 minutes in standard sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by the horizontal arrow. Vertical arrows indicate the positions of CNV and TNV peaks. (a) Preparation from CNV-inoculated leaves (b) Preparation from TNV-inoculated leaves (c) Preparation from CNV and TNV doubly-inoculated leaves Table XVIII. Comparison of cucumber necrosis virus yields from single and double infections Host and growth conditions Yield a*of CNV (mg/100 seedlings1* *) in single infection double infection Yield in double infection as a % of yield in single infection Cncumis sativns cv. Straight Eight in 11.25 summer greenhouse (Figure 7) C. sativns cv. Straight 2.7 Eight at 25 C (Figure 8) C. sativus cv. Straight 2.55 Eight at 30 C (Figure 9) C. sativns cv. Straight 2.25 Eight in autumn greenhouse (Figure 10) Phaseolus vulgaris cv. Topcrop in autumn greenhouse 0*39 (Figure 12) 7.05 2.19 2.55 1.01 63 81 100 45 0.32 82 a , Y i e l d estimated by comparison of area under virus peaks in Figures cited to standard curves relating area to amount of virus. Adjustments made where necessary for dilution factors and changes in absorbance monitor scale. ^'Except for Phaseolus vulgaris cv. Topcrop where 50 plants were taken per sample. Table XIX. Comparison of tobacco necrosis virus yields from single and double infections Host and growth Yield 'of TNV (mg/lOO seedlings ) in Yield in double infection conditions single infection double infection as a % of yield in single infection Cucumis sativus 0.36 0.42 117 cv. Straight Eight in summer greenhouse (Figure 7) C. sativus cv. Straight 0 0 Eight at 25 C (Figure 8) C. sativus cv. Straight 0 0 — Eight at 30 C (Figure 9) C. sativus cv. Straight 1.28 1.35 106 Eight in autumn greenhouse (Figure 10) Phaseolus vulgaris 0 0 — cv. Topcrop in autumn greenhouse (Figure 12) 'Yield estimated by comparison of area under virus peaks in Figures cited to standard curves relating area to amount of virus. Adjustments made where necessary for dilution factors and changes in absorbance monitor scale used. 'Except for Phaseolus vulgaris cv. Topcrop where 50 plants were taken per sample. Table XX. Relative yields of CNV and TNV in each of three double infections created by serial inoculations to Cucumis sativus cv. Straight Eight (from Figure l l ) Double infection Yield a*of virus (mg/lOO cucumber seedlings) Ratio of TNV to CNV CNV TNV expressed as a % 1. 7.05 0.42 6 2. 8.2 2.2 27 3. 2.2 1.4 64 'Yield estimated by comparison of area under virus peaks in Figure 14 to standard curves relating area to amount of virus. Adjustment made for dilution of sample. 92 Structural interaction studies For the structural interaction studies of both phenotypic mixing and genomic masking, tissues from different hosts doubly infected with CNV and TNV were prepared and examined separately. A l l the preparations studied are numbered and described (as to host tissue) in Table XXI. The purpose of this table is to supply a simple means of identifying each preparation in order that, where the same preparation was tested by different methods for phenotypic mixing or genomic masking, the results may be easily compared.. Examination of mixed infection preparations for phenotypic mixing (i) Polyacrylamide gel electrophoresis (PAGE) of whole virus If an in vitro mixture of CNV and TNV produced two distinct bands after polyacrylamide gel electrophoresis, a mixed infection preparation containing phenotypically mixed virions might be expected to produce one to several bands or perhaps a diffuse area intermediate to the CNV and TNV band positions due to the mixing of coat protein subunits. In the Tris-HCl pH 8.9/Tris-glycine pH 8.3 buffer system. CNV migrated as a single heavily staining band with the occasional appearance of a slower migrating, fainter band. TNV was clearly dis-tinguished in whole virus gels from CNV and its occasional companion, migrating to a point between the two but closer to the main CNV band. However, equivalent CNV and TNV concentrations did not appear as equally intense staining bands after electrophoresis, relatively high TNV concentrations eliciting only faint bands. Although the TNV band could be enhanced by doubling or tripling the volume of sample applied to the gel, in mixed infection preparations the resulting excessive 'amount of CNV masked the zone intermediate to the CNV and TNV bands, obscuring 93. Table XXI. Preparations from tissues doubly infected with CNV and TNV used in tests for structural interactions Designation of double Description of source of double infection infection^preparation CNV/TNV #1 Inoculated cowpea * primary leaves CNV/TNV #2 Inoculated cowpea primary leaves Inoculated cucumber1*" cotyledons CNV/TNV #3 CNV/TNV #4 Inoculated cucumber cotyledons CNV/TNV #5 Inoculated cucumber cotyledons CNV/TNV #6 Inoculated cucumber cotyledons CNV/TNV #7 Inoculated cowpea stems CNV/TNV #8 Leaves from cowpea inoculated via stems CNV/TNV #9 Leaves and stems from cowpea inoculated via stems CNV/TNV #10 c Inoculated bean 'primary leaves a*Vigna sinensis cv. Early Ramshdrn k'Cucumis sativns cv. Straight Eight c'Phaseolus vulgaris cv. Topcrop 94. the area in which evidence of phenotypic mixing, i f any. was expected. The results obtained by photometric scanning of gels concurred with those obtained by staining (Figure 13). The comparisons were direct, with each gel being examined fi r s t by photometric scanning and then by staining. The comparisons indicated that the difficulty in detecting TNV was not due to a low affinity for the stain used but rather to a failure of a significant portion of the TNV virion population to migrate into the gel to form a distinct band. To determine i f the gel buffer and/or chamber buffer were causing degradation of TNV. comparisons were made of both CNV and TNV subjected to sucrose density gradient centrifugation in sucrose columns formed with three buffers: (i) 0.01 M potassium phosphate buffer. pH 7.0 (control) (i i ) 0.24 M Tris-HCl buffer. pH 8.9 (gel buffer) ( i i i ) 0.0025 M Tris-0.19 M glycine buffer. pH 8.3 (chamber buffer). No appreciable,.differences could be detected in the concentration of either virus in any of the three gradient systems, indicating that degradation of TNV was not likely the cause of its failure to form a band in PAGE. The effects of electrophoresis in a Tris-HCl pH 7.5/Tris-diethylbarbituric acid pH 7*0 system were detected by photometric scanning. High absorbance along the whole length of the gel scanned at 254 nm masked virus peaks and consequently only absorbance profiles at 280 nm were useful. This system resulted in CNV migrating as a single sharp peak while TNV appeared as a squat double peak. When the two viruses were co-run on the same gel the CNV peak migrated to the same position as the faster-migrating component of the TNV preparation leaving only a small, somewhat indistinct peak as evidence of TNV. Although 95. Figure 13. Comparison of staining and photometric scanning (at 254 nm and 280 nm) for the detection of CNV and TNV following electrophoresis in 2.6$ polyacrylamide gels. Gel buffer vas Tris-HCl (0.24 M Tris at final dilution in gel) at pH 8.9 and chamber buffer vas Tris-glycine (0.0025 M Tris, 0.19 M glycine) at pH 8.3. Sample size vas 25 u l . A current of 2 ma/gel vas applied for 4 hours. Direction of migration is to the left, as indicated by horizontal arrow. (a) CNV (i) stained gel (i i ) ultraviolet absorbance profile at 254 nm ( i i i ) ultraviolet absorbance profile at 280 nm (b) TNV (i) stained gel (i i ) ultraviolet absorbance profile at 254 nm ( i i i ) ultraviolet absorbance profile at 280 nm (c) CNV + TNV in vitro mixture (i) stained gel (i i ) ultraviolet absorbance profile at 254 nm ( i i i ) ultraviolet absorbance profile at 280 nm Note: C & refers to the top a accessory component of CNV. 96. ct v© JJ« <N » • • o o o ma T,c,z ya eotreqaosqv Absorbance at 254 nm •H •H VO o CVJ o ma ijQz Vs ootreqaosqv 99 changing the pH of this system resulted in substantial changes in the appearance of the virus profiles, none of the results were of sufficient quality to make the method useful in testing for phenotypic mixing. Several actual mixed infection preparations, along with controls (single infections preparations and in vitro mixtures), were subjected to PAGE using the Tris-HCl/Tris-glycine system. Virus bands were detected by staining (Figure lk). The presence of both CNV and TNV vas evident from only three of the mixed infection preparations and there vere no detectable intermediate bands to indicate a phenotypically mixed virion population. (i i ) Antiserum treatments Incubation in normal serum slightly reduced the concentration of both viruses in in vitro mixtures from that observed after incubation in phosphate buffer. The normal serum treated mixture vas used as a control standard against which the effects of CNV and TNV antisera treatments were compared. Antiserum to CNV specifically removed CNV from an a r t i f i c i a l mixture but caused no significant alteration in the cm centration of TNV. Similarly. TNV antiserum specifically removed TNV but did not affect CNV concentration in an in vitro mixture. Nine different mixed infection preparations were similarly treated and compared to the standard in vitro mixture treatments. Ultraviolet absorbance profiles of a control and a typical mixed infection preparation are shown in Figures 15 and 16. The effects of antisera treatments on virus concentrations were quantified by measuring the areas under the virus peaks (Table XXII). With the possible exception of one preparation, no evidence of phenotypic mixing was found. The particular preparation (CNV/TNV #2J Figures 17 and 1 8 ) 100. was from inoculated primary leaves of cowpea and in sucrose density gradients appeared as a single peak with a shoulder or as a close "doublet" co-sedimenting with TNV. Treatment with TNV antiserum removed a l l trace of the virus peak while antiserum to CNV reduced the area under the curve by l/4. Attempts to identify the RNA(s) present by assaying for TNV on Chenopodium capitatum and for CNV on 'Topcrop' bean failed, as the preparation was not infectious. No symptoms were produced on cowpea either. This same preparation was shown to contain both CNV and TNV in whole virus PAGE (Figure 14). The small volume of the preparation precluded further tests. Examination of mixed infection preparations for genomic masking (l) Antiserum treatments The "antigen-antibody precipitation" method was used to examine mixed infection preparations for the occurrence of genomic masking. Where a particular antiserum treatment removed a l l homologous viral infectivity from control (in vitro) mixtures but failed to do so from mixed infection preparations of similar concentration, genomic masking would be indicated as a possible explanation. Inoculations of treated preparations to Topcrop bean revealed (Table XXIII) that in only one case—CNV/TNV #4—was CNV infectivity not eliminated by treatment of the preparation with CNV antiserum. Only one lesion (average over sample of 0 lesions/leaf) was evident and examination of the virus preparation ultraviolet absorbance profile at 254 nm revealed a very high CNV concentration in this preparation relative to that in the control. This indicated that the single lesion probably resulted from incomplete removal of CNV from the mixture by the CNV antiserum, rather than from genomic masking of CNV RNA in TNV protein. 101. Figure 14. Relative positions of CNV and TNV after electrophoresis in 2.6$ polyacrylamide gels. Gel buffer vas Tris-HCl (o.24 M Tris at final dilution in gel) at pH 8.9 and chamber buffer vas Tris-glycine (0.0025 M Tris. 0.19 M glycine) at pH 8.3. Sample size vas 25 ul. A current of 2 ma/gel vas applied for 3.5-4 hours. Direction of migration is to the left, as indicated by horizontal arrow. (a) CNV; concentration 1.0 mg/ml (b) TNV; concentration 1.0 mg/ml (c) CNV -i- TNV; concentration 1.0 mg/ 'ml each (in vitro mixture) (d) CNV; 0.5 mg/ml (e) TNV; 0.5 mg/ml (*) CNV/TNV #1 (g) CNV/TNV #2 (b) CNV/TNV #3 (i) CNV/TNV #4 (j) CNV/TNV #5 (k) CNV/TNV #9 Note: C refers to the top a accessory component of CNV* a* 102 I 1 I i n 103 Figures 15 to 18. Pig. 15. Fig. 16. Pig. 17. Fig. 18. Ihe effects of antiserum treatments on virus mixtures formed in vitro or derived from doubly infected tissue. Ultraviolet absorbance profiles at 254 nm following sucrose density gradient centrifugation of partially purified samples reacted with specific antisera. Centrifugation was at 39*000 rpm for 60 minutes in sucrose gradients in an SW-41 rotor. Direction of sedimentation is to the left, as indicated by horizontal arrow. Vertical arrows indicate CNV and TNV peaks. In vitro mixture (control) treated with: !a) normal serum b) antiserum to CNV c) antiserum to TNV CNV/TNV #6 treated with: !a) normal serum b) antiserum to CNV c) antiserum to TNV CNV/TNV #2 treated with: (a) buffer (b) normal serum (c) antiserum to CNV (d) antiserum to TNV CNV/TNV #2 in linear log gradients, treated with: (a) buffer normal serum antiserum to CNV antiserum to TNV 106 0.10 107 108. Table XXII. The effects of antisera treatments on CNV and TNV mixed infection preparations. Virus preparations were treated with antisera and centrifuges in sucrose density gradients. The relative amounts of virus remaining after antisera treatments were compared by measuring the areas under virus peaks in ultraviolet absorbance profiles at 254 nm. Area * under virus peak in ultraviolet absorbance Preparation profiles after treatment with  normal serum antiserum to CNV antiserum to TNV CNV + TNV in vitro mixture 0 . 5 2 0 . 2 1 0 . 3 0 CNV/TNV #1 1.42 0.04 1 . 3 8 CNV/TNV #2 0 . 2 5 0 . 1 9 0 CNV/TNV #2 (in linear log gradients) 0 . 2 6 0 . 2 0 0 CNV/TNV #3 0 . 5 1 0.08 0.42 CNV/TNV #4 0 . 7 0 0.14 0 . 5 1 CNV/TNV #5 0.82 0 . 5 2 0 . 2 6 CNV/TNV #7 0 . 2 5 0 . 2 3 0 "average area from at least 5 planimetric measurements Table XXIII. Number of lesions produced on Phaseolus vulgaris cv. Topcrop by in vitro virus mixtures and mixed infection preparations after antiserum treatment Preparation Propagation host Number of lesions after treatment with buffer normal serum CNV antiserum TNV antiserum in vitro mixture #1 in vitro mixture #2 in vitro mixture #3 CNV/TNV #1 CNV/TNV #2 CNV/TNV #3 CNV/TNV #4 CNV/TNV #4 repeated CNV/TNV #4 with additional antiserum treatment CNV/TNV #7 CNV/TNV #10 cowpea leaves cowpea leaves cucumber cotyledons cucumber cotyledons cucumber cotyledons cucumber cotyledons cowpea stems Topcrop bean leaves 46 32 2 29 0 9 76 28 0 57 30 8 3 2 0 110 56 60 13 26 0 0 0 0 0 0 0< 0 0 0 46 7 13 10 0 56 24 105 17 0 28 + one lesion was produced in the sample of 3 leaves. 110. Inoculation of Chenopodium capitatum with antibody-treated preparations indicated the presence of TNV infectivity after treatment in two trials (Table XXIV). One was again CNV/TNV #4, the other a single lesion from the standard #3. The latter could be explained as incomplete removal of TNV from the mixture by the TNV antiserum treatment. The mixed infection preparation CNV/TNV #4, however, produced an average of 13 TNV lesions per inoculated leaf while its control (standard #l) produced no TNV lesions after treatment with homologous antiserum. When preparation CNV/TNV #4 was included in a second set of tests (standard #2), an average of 3 lesions per leaf resulted, the standard again producing none. The TNV concentration in this preparation was closely comparable to that in the standards, and inadequate removal of TNV virions due to excessive concentration was considered unlikely. An additional TNV antiserum treatment was given the already-treated preparation run with standard #2 and the preparation inoculated to C^ capitatum. The antiserum was diluted l/lOth to avoid antibody excess. This time, despite the additional TNV antiserum treatment, an average of 3 lesions per leaf was elicited. Although C_j_ capitatum was considered a reliable specific indicator for TNV under these conditions, additional observations were made to corroborate the identity of the lesions as TNV. Individual, isolated lesions were harvested at 3 days post-inoculation and inoculated to C. capitatum leaves. Lesions were characteristic of TNV in their time of formation and in appearance. Similarly, lesion isolates were ino-culated to Gomphrena globosa. a host useful in distinguishing the two viruses when single infections are compared. The resulting lesions were characteristic of TNV in size and distinct from the smaller CNV lesions. Table XXIV. Number of lesions produced on Chenopodium capitatum by in vitro virus mixtures and mixed .infection preparations after antiserum treatment Preparation Propagation Number of lesions after treatment with host buffer normal serum CNV antiserum TNV antiserum in vitro mixture #1 - 51 26 46 0 in vitro mixture #2 - 32 11 3 0 in vitro mixture #5 - 22 31 100 0* CNV/TNV #1 cowpea leaves 83 47 51 0 CNV/TNV #2 cowpea leaves 0 0 0 0 CNV/TNV #3 cucumber cotyledons 57 18 27 0 CNV/TNV #4 cucumber cotyledons 46 100 100 13 CNV/TNV #4 repeated cucumber cotyledons 92 62 70 3 CNV/TNV #4 with additional antiserum treatment cucumber cotyledons ammm 5 CNV/TNV #7 cowpea stems 12 32 22 0 CNV/TNV #10 Topcrop bean leaves 14 15 62 0 one lesion was produced in the sample of 3 leaves. 112. Attempts to establish a method of polyacrylamide gel electrophoresis  to detect genomically masked viral RNA Sodium dodecyl sulfate (SDS), present in the electrophoresis buffers and in most of the RNA extraction buffers tested, precipitates potassium ions. Although most of the virus preparations being examined were suspended in 0.01 M potassium phosphate buffer, a comparison in-dicated they were as suitable for use as were suspensions in sodium phosphate buffer (Figure 19). The need need for dialysis to remove potassium ions was therefore considered obviated. Dialysis was to be avoided, as a substantial portion of virus was lost in dialysis of low concentrations of virus. CNV and TNV samples were separately treated in several RNA extraction buffers (Table XXV). The best results.Lin terms of a large distinguishable peak in ultraviolet absorbance profiles, are shown in Figure 19, and were obtained using extraction buffer #2 from Table XXV. Because TNV or TNV RNA did not migrate during electro-phoresis to form a detectable peak and because the peaks that were evident were not distinguishable as whole virus or RNA, the effects of an RNA extraction buffer were monitored by sucrose density gradient centrifugation and subsequent photometric analysis of the columns. CNV incubated in extraction buffer consisting of 0.05$ SDS and 0.01 M EDTA at pH 7.0 (Tremaine, unpublished) for one hour at 37 C showed substantial breakdown compared to the control with the apparent release of RNA (Figure 20). A substantial proportion of TNV similarly treated was degraded, but only partially (Figure 20). The position of the peak formed after treatment in extraction buffer was intermediate to that expected for whole virus and pure RNA. In vitro mixtures of CNV and TNV were similarly treated with 113. the same extraction buffer. The results (Figure 20) indicated that TNV was partially degraded, as before, but that CNV was l i t t l e affected. This was in direct contrast to the degradation of CNV and the apparent release of RNA in the absence of TNV. Increasing the percent SDS and the molarity EDTA had l i t t l e effect, indicating that these were not in some way limiting factors to virus degradation. Work was terminated at this stage, and as satisfactory BNA extraction could not be accomplished, use of polyacrylamide gel electro-phoresis to detect genomically masked RNA after antiserum treatment could not be attempted. 114 Figure 19. A comparison of the effects of BNA extraction buffer 2 (Table XXV), as monitored by polyacrylamide gel electro-phoresis, on CNV and TNV suspended in sodium phosphate and potassium phosphate buffers. Virus samples were incubated in extraction buffer 2 at 50 C for 15 minutes and 50 ul samples were applied to standard BNA gels. Electrophoresis was conducted with a current of 8 ma/gel applied for 2 hours. Ultraviolet absorbance profiles of gels at 254 nm were recorded. Direction of migration is to the left. Vertical arrows indicate possible RNA peaks. (a) CNV in 0.01 M potassium phosphate buffer (b) CNV in 0.01 M sodium phosphate buffer (c) TNV in 0.01 M potassium phosphate buffer (d) TNV in 0.01 M sodium phosphate buffer Table XXV. RNA extraction buffers tested in conjunction vith the use of polyacrylamide gel electrophoresis to monitor their effectiveness Identification a number Description of buffer * 1. 0.2 M ammonium carbonate. 0.002 M EDTA. 2$ SDS at pH 9.0 2. 0.04 M Tris. 0.002 M EDTA, , 2$ SDS. 2.0 M urea at pH 9.0 3. 0.5 M MgCl2 4. 0,2 M ammonium carbonate. 0.01 M EDTA. 2% SDS 3. 0.04 M Tris. 0.01 M EDTA. 2$ SDS. 2.0 M urea 6. 0.04 M Tris. 0.05 M EDTA. 2% SDS. 2.0 M urea a*virus samples were incubated in extraction buffer ( l x l , volume to volume) in a 50 C water bath for 15 minutes. 118. Figure 20. The effect of an SDS-EDTA RNA extraction buffer on CNV and TNV as monitored by sucrose density gradient centri-fugation. Virus samples vere incubated in 0.05$ SDS and 0.01 M EDTA. or in 0.01 M potassium phosphate (control), for 1 hour at 37 C and then applied to standard gradient columns and centrifuged at 39,000 rpm for 60 minutes in an SW-41 rotor. Ultraviolet absorbance profiles at 254 nm vere recorded. Sedimentation is to the left. Suspected virus and RNA peaks are indicated by vertical arrows. (a) CNV incubated at 37 C for 1 hour (control) (b) CNV incubated with extraction buffer at 37 C for 1 hour (c) TNV incubated at 37 C for 1 hour (control) (d) TNV incubated with extraction buffer at 37 C for 1 hour (e) CNV and TNV in vitro mixture incubated at 37 C for 1 hour (control) (f) CNV and TNV in vitro mixture incubated with extraction buffer at 37 C for 1 hour 119 120 DISCUSSION AND CONCLUSIONS The fi r s t part of this work centered on determining conditions that might improve virus replication and recovery and that might enhance mixed infection; that i s , conditions that might increase the proportion of doubly infected cells in the host. CNV yields have been reported to be the highest in cucumber (Tremaine 1972), although McKeen (1959) reported greater CNV recovery from inoculated cowpea and Samsun tobacco than from cucumber. TNV isolates vary widely in their abilities to replicate in certain hosts and, depending on the isolate, have been propagated in French beans (The Prince), tobacco, or Nicotiana clevelandii (Kassanis and Phillips 1970), cucumber (Dias and Doane 1968), and cowpea (Stace-Smith personal communication). These literature reports and the severity of CNV and TNV symptoms in the host range trials were the factors determining the selection of cucumber and cowpea as propagation hosts. None of three tobacco varieties tested, nor Nicotiana clevelandii. produced more than a few scattered local lesions when inoculated with either CNV or TNV. Four bean varieties tested, including The Prince, produced few or no lesions in response to TNV, although they produced numerous lesions in response to CNV inoculations. At cool temperatures a few scattered TNV lesions were produced, while in a warm greenhouse no symptoms at a l l were evident and no virus could be recovered. The identity of the TNV isolate was not known and its behaviour was perhaps unusual for TNV, although Kassanis and Phillips (1970) reported a TNV isolate that, when inoculated to tobacco or French bean, produced few and small lesions containing very l i t t l e virus. 121 TNV reached substantially higher concentrations (2 to 5 times) more virus in cowpea than in cucumber. The finding that CNV yield was greater from cucumber than from cowpea is in agreement with the findings of Tremaine (1972) but in contrast with those of McKeen (1959). Temperature was important in determining the extent of virus replication. In the warm summer months TNV yields were low. while CNV yields were lowest in the cooler winter months. These observations were supported by direct comparisons of virus yields under warm and cool temperature regimes. The findings are in general agreement with those of Babos and Eassanis (1963) and Tremaine (1972). Some changes in the purification procedure were examined for their effect on virus recovery. An extraction buffer of sodium acetate pH 5*0 (Tremaine personal communication) resulted in greater virus recovery than one of sodium phosphate pfi 7.0 and ascorbic acid (Bias and Doane 1968). Although Dias and Doane (1968) reported good CNV recovery when sap was clarified by treatment with 10$ chloroform and pH 5.3, i t was found here that better virus yields were obtained by omitting the organic solvent. A comparison revealed that freezing TNV-infected tissue for 24 hours prior to purification had no effect on the amount of virus re-covered. TNV-infected tissue was also purified for the extraction of satellite virus which, i f present, could have been responsible f o r the apparent low rates of TNV replication (Eassanis 1962); however, no satellite tobacco necrosis virus was detected. In the work so far discussed, concentration of partially puri-fied virus preparations was estimated by optical density readings at 260 nm. However, in a l l cases, the 260/280 ratio was low, indicating the presence of contaminating protein in excess to that contained in 122 virions. This placed in doubt the accuracy of the concentration estimates, especially where no replicate trials were performed or where differences in virus yield vere minor. To obtain greater accuracy, the concentrations of highly purified (gradient purified) virus preparations were determined by optical density readings. The 260/280 ratio approached the expected value of 1.7* Samples of the same preparations were then subjected to sucrose density gradient centrifugation and the areas under virus peaks on ultraviolet absorbance profiles were plotted against concentration. Through use of the standard curve so derived, virus content of partially purified preparations could be accurately measured. This eliminated the need for further purification, a process in which much virus would have been lost along with contaminants. Attempts to induce systemic infections with CNV and TNV were considered important for two reasons. First, i t was hoped virus titre would be higher in systemically infected plants than in plants with localized infections, although McKeen (1959) reported CNV yields were higher from locally infected tissue. Second, i t was hoped the incidence of double infections would be increased i f both viruses were moving systemically through the host. CNV systemically infects cucumber and, occasionally, cowpea and Zinnia elegans (McKeen 1959). Some TNV isolates become systemic in French bean (Kassanis and Phillips 1970) and infre-quently in cucumber and cowpea (McKeen 1959). Thomas (1973) and Thomas and Fry (1973) reported a TNV isolate with a high incidence of systemic infection in cucumber. In this investigation, however, very l i t t l e evidence of systemic infections could be found in any host, and where i t did occur systemic spread was very limited in extent. Infections never moved upwards into uninoculated new growth. Under ideal conditions, CNV systemically infected up to 75$ of inoculated cucumber seedlings, 123 but killed them a l l . Few i f any systemically infected plants survived. CNV inoculations to apical leaves of flowering cucumber plants—a pro-cedure which has induced systemic infections in up to 100$ of inoculated plants (McKeen 1959)—failed to induce any systemic infections in this work. In view of the observed tendency of CNV and TNV to move downwards from the site of inoculation and of their natural occurrence as root-infecting viruses, attempts were made to initiate systemic infections by root and stem inoculations to cowpea. CNV failed to reach detectable levels in roots or stems and, although root tissue could not be adequately purified, TNV appeared present only in small quantities in the roots. In the stems, however, TNV reached the highest conentrations observed in this work. The failure of CNV replication in cowpea stems and the absence of extensive systemic infections by CNV or TNV in any other host prevented the establishment of a high-yielding mixed infection system. The remainder of this study dealt with the effects of CNV and TNV in mixed infections both on the hosts and on the viruses. A qualitative assessment on a wide host range revealed that the type and severity of symptoms in mixed infections of CNV and TNV were l i t t l e different from those in either single infection. This could be interpreted as evidence of competition between the viruses in replication, with the total virus concentration in mixed infections being l i t t l e different than that in either single infection. No evidence of synergy was seen and in the few hosts where symptoms were slightly more severe in mixed infections than in either of the single infections, the increase in severity was less than additive. CNV and TNV infections were indis-tinguishable from single infections. This was confirmed quantitatively by lesion counts in single and mixed infections of cucumber and cowpea. 124. Because CNV and TNV lesions vere identical in appearance and in time of formation, no determination of the virus against which inter-ference was directed could be made simply by symptom observations on most hosts. However, the marked reduction in the number of CNV lesions on Topcrop bean in mixed infections with TNV, despite the near absence of lesions on plants singly inoculated with TNV, indicated that TNV inter-ference with CNV replication may have been the major source of the antagonism. When doubly inoculated Topcrop bean from a separate experiment vas purified no evidence of TNV replication vas found and the yield of CNV vas essentially the same as that from singly inoculated plants. These results indicated that TNV interfered with CNV in the early stages of the infection process, perhaps competing for binding sites (Kassanis 1963). The situation appears closely analogous to that of PVX and PVY in potato (Ross 1930), where PVX reduced the number of PVY lesions although apparently not replicating itse l f . As with CNV and TNV, PVX and PVY are unrelated viruses. Antagonism was evident in the occurrence of systemic symptoms. In Topcrop bean, TNV reduced the spread of CNV while inducing no systemic symptoms itself, and in doubly infected cucumber TNV reduced the number of plants killed to almost half the number killed by CNV single infections. This could have been the result of TNV reducing the titre of CNV in doubly infected plants. CNV interfered slightly with TNV replication in cowpea, as indicated by a reduction in the incidence of systemic symptoms in mixed infections from that observed in TNV single infections. The results of fresh weight measurements of singly and doubly infected tissues generally supported the findings based on symptom observations. In cowpea, the weight loss in mixed infections vas greater than in either of the single infections, but less than additive, indicating 125. an interference between the viruses. In one case, the interference was particularly pronounced, with the weight loss in doubly infected tissue being less than in tissue infected with TNV alone. This was another indication of the ability of CNV to interfere with TNV re-plication in cowpea. Interference was also indicated by fresh weight measurements of virus-infected cucumber cotyledons, although one experiment indicated an additive weight loss in mixed infections. This could be interpreted as reflecting independent CNV and TNV replication but might also represent a situation where replication of one virus was decreased and replication of the other correspondingly increased. A comparison of the amounts of virus recovered from singly and doubly infected cucumber seedlings revealed more of the nature of the interference between TNV and CNV. In a l l cases. CNV yield was reduced substantially (up to 50$) in the presence of TNV and in most cases a slight increase in TNV yield was evident. The situation is similar to that reported for cowpea chlorotic mottle virus (CCMV) and southern bean mosaic virus (SBNV) by Kuhn and Dawson (1973)» where CCMV reduced SBMV concentra-tion by 50$ while itself being unaltered in concentration. The host, however, exhibited a synergistic symptom response. In this investigation, the 50$ reduction in CNV yield from mixed infections could be correlated with an observed 50$ reduction in the incidence of systemic symptoms. However, the severity of the local lesion response was the same in single and mixed infections, emphasizing the uncertainty of using symptom responses to gauge the effect of mixed infections on virus concentrations. Even at temperatures unfavourable for TNV replication, TNV clearly inhibited CNV replication. Only when TNV replication was almost non-existent did CNV reach the same concentrations in mixed infections as in single infections. When mixed infections were serially inoculated the proportion 126. of TNV relative to CNV increased dramatically. This confirmed ob-servations based on symptoms (McKeen 1959) that TNV displaced CNV in repeated transfer of mixed infections. There was no evidence that CNV actually aided TNV replication at temperatures permissive to CNV but restrictive to TNV, and TNV yields were similar in mixed and single infections under such conditions. Only limited evidence was found to indicate the occurrence of structural interactions between CNV and TNV in double infections. Structural interactions between viruses require mixed infections of cells and close association of the viral replicative processes in such cells. The interference between CNV and TNV may have reduced the number of cells in which both viruses became successfully established or may have reduced the incidence of structural interactions by severely reducing the replication of one of the potential interacting partners, as discussed by Dodds and Hamilton (1976). Conversely, the interference may indicate a close spatial and temporal association between the replicative processes during mixed infections that might, although reducing virus concentration, increase the proportion of virions exhibiting structural interactions. While no phenotypic mixing was evident in eight of the nine mixed infection preparations tested, the one exception is worthy of note. Polyacrylamide gel electrophoresis distinctly revealed three bands in this preparation. Two corresponded in position to those in CNV preparations and one corresponded in position to TNV. Analytical sucrose gradient centrifugation of this preparation had revealed only a single species— apparently TNV. When a sample of this preparation was mixed with a standard in vitro CNV/TNV mixture,: i t was clearly seen to co-sediment with the TNV component of the mixture after gradient centrifugation. Ultraviolet absorbance profiles of this preparation centrifuged alone in sucrose 127 gradients revealed that i t could he resolved into a "doublet" or a main peak with a shoulder. Treatment of the preparation with TNV antiserum prior to sucrose density gradient centrifugation removed a l l trace of the peak while treatment with CNV antiserum reduced the area under the peak by one-quarter. A virion population composed of normal TNV and phenotypically mixed particles, perhaps containing either CNV or TNV nucleic acid but with no capsids composed solely of CNV protein, might produce such results. Although this evidence might indicate a phenotypically mixed particle population, i t must be interpreted cautiously, especially in the absence of further evidence. Phenotypic mixing of plant viruses in vivo has been recently reported to occur between two strains of TMV (Atabekova et a l . 1975) but no reports of unrelated viruses doing so could be found. If a high degree of specificity exists in the replication and assembly of CNV and TNV, the frequency of genomic masking could be expected to be low and thus difficult to detect. Infectivity is often considered the most sensitive assay for the presence of virus (Matthews 1970) and was adopted here to detect genomic masking. It was necessary, before using this method, to locate a specific host for each virus or, alter-natively, a host in which the viruses could be accurately distinguished on the basis of distinct symptoms. While cowpea, French bean, cucumber, and Cromphrena globosa have a l l been used to distinguish CNV and TNV (McKeen 1959; Dias and Doane 1968) the differences noted in these hosts in this work were insufficient to allow accurate detection of a low concentration of one virus in a relatively high concentration of the other. Symptoms are not reliable in distinguishing CNV from TNV and other methods, such as serology, must be employed (Dias and McKeen 1972; Tremaine 1972). As earlier noted, temperature plays a significant role in CNV and TNV replication. By using a host-temperature combination 128. preferential to one virus but restrictive to the other, i t vas possible here to specifically detect one virus in the presence of the other. Phaseolus vulgaris cv. Topcrop at 23 C vas used to indicate the presence of CNV and Chenopodium capitatum at 18 C vas used to detect TNV. The sensitivity of the systems vas not established, as might have been done by counting lesions produced by dilutions of a single-virus preparation inoculated to the indicator host. In addition, since both indicator hosts vere hosts to both viruses, interference for binding sites following a mixed inoculation might interfere with the number of lesions produced by one virus even in the absence of symptoms or replication of the other. This was seen to occur with Topcrop bean, where the presence of TNV in the inoculum caused a significant reduction in the number of CNV lesions produced. Such a situation may have occurred with C^ capitatum too, although the possibility was not investigated. Interference may even have been greater in Cj_ capitatum because both viruses replicated and produced symptoms in this host, while only CNV produced symptoms on 'Topcrop' at 23 C. Such interference would reduce the effectiveness of the infectivity assay in detecting genomic masking. The results of antiserum treatments of mixed infection preparations and subsequent analysis of infectious BNA by recovery of specific indicator hosts revealed no evidence of CNV BNA being genomically masked by TNV coat protein. However, one mixed infection preparation produced TNV lesions on Cj_ capitatum after being subjected.to the same treatments that completely removed a l l TNV infectivity from in vitro mixtures of comparable composition. Subjecting the preparation to an additional cycle of TNV antiserum treatment caused no reduction in the number of TNV lesions produced on the indicator. This could be considered evidence indicating the genomic masking of TNV BNA in CNV coat protein during mixed infections. However, i t is possible that 129 some other unknown factors in this particular virus preparation may have been responsible for incomplete removal of TNV virions or of free TNV RNA. hence giving the appearance of genomic masking. In the absence of con-firmatory tests, genomic masking of TNV RNA in CNV protein is only indicated but not proved. The absence of reciprocity in the apparent genomic masking i s analogous to the unidirectional genomic masking of MAY RNA in RPV protein (Rochow 1970). The barley yellow dwarf virus system, like the CNV/TNV mixed infections system, i s composed of unrelated, structurally similar viruses. No other structural interaction studies using unrelated but structurally similar plant viruses have been reported. Such systems present certain problems to study. In the case of the CNV/TNV system, similarities in virus size, shape, and behaviour that made this system a likely one for structural interactions, also presented difficulties in separation and identification of the infection partners. These problems necessitate approaches employing serological and vector studies. The CNV/TNV system was considered ideal for this because the viruses, as in the barley yellow dwarf system, are serologically distinct and have specific vectors. Further testing to determine the incidence of genomic masking between CNV and TNV should make use of the specific fungal vectors, Olpidium cucnrbitacearum for CNV and Olpidium brassicae for TNV. If, for example, from a mixed infection preparation treated with TNV anti-serum, 0^ brassicae transmitted no TNV to test plants but 0j_ cucnrbita- cearum transmitted not only CNV but also TNV, genomic masking of TNV EKA in CNV protein would be indicated. A comparison of the frequency of apparent TNV transmission by 0^ cucurbitacearum from treated in vitro mixtures and double infection preparations would be required. Test hosts would be plants the roots of which the Olpidium spp. would readily infect 130. and which CNV and TNV could infect. Infected root tissue could he assayed for virus on the indicator hosts C_j_ capitatum and Topcrop bean. A potential problem with such work is that the very small amounts of TNV BNA transmitted with relatively large amounts of CNV BNA might not be sufficient for TNV replication to reach detectable levels in the roots of test plants. While suitable for detecting TNV genomically masked in CNV protein, the method would not likely be useful in detecting genomic masking in the reverse direction due to the tendency of TNV to overwhelm CNV in mixed infection. The use of sucrose density gradient centrifugation to separate the components of a mixed infection (Peterson and Brakke 1973) would probably be less effective than antiserum treatment for the CNV/TNV system due to the similarity in size and molecular weight of the viruses and the consequent incomplete separation in gradients. Preliminary trials indicated several gradient centrifugation cycles would be required and the amount of virus lost could seriously affect the detection of genomic masking. The different thermal inactivation points of the viruses (CNV 75-80 C for 10 minutes; TNV 85-95 C) might possibly be put to use in conjunction with antiserum treatments in detecting genomic masking, at least invone direction. With the assumption that heating destroys the BNA rather than protein in small isometric viruses, heating at 83 C for 10 minutes would destroy a l l CNV infectivity while allowing TNV, including that TNV BNA genomically masked in CNV protein, to survive. Subsequent treatment with TNV antiserum would leave only TNV BNA genomically masked in CNV protein, which could be detected on the indicator host. Careful control treatments of in vitro mixtures would be required. The sensitivity of detection of genomic masking would likely be improved using this method, 131. as a l l CNV would be destroyed and the possibility of CNV interference with TNV lesion on Chenopodium capitatum would not exist. Different Physalis species have been noted to react differently to TNV inoculations (Horvath 1974. 1973) and investigation of their reaction to CNV should be conducted to determine their suitability as indicator plants. For future work on this system i t would be desirable to use systemically infecting isolates of CNV and TNV. Especially useful might be a TNV isolate which has a high incidence of systemic spread in cucumber (Thomas 1973; Thomas and Fry 1973) and which might be used to establish systemic mixed infections with CNV in cucumber. 132. SUMMARY Symptoms i n mixed infections vere similar to those i n single infections. Where an increase i n symptom severity vas evident, i t vas less than additive. No synergy vas found. The reduction i n host fresh veight i n mixed infections vas also generally less than additive. TNV interfered with the replication of CNV i n mixed infections, substantially reducing CNV yields. Virus concentration measurements indicated TNV yi e l d vas l i t t l e affected by mixed infections, although some evidence of CNV interfering v i t h TNV replication i n cowpea was apparent i n symptom expression. The symptoms of mixed infections i n cucumber did not reflect the concommitant reductions i n virus concentration. The occurrence of phenotypic mixing of CNV and TNV coat proteins i n mixed infections of cowpea and the occurrence of genomic masking of TNV RNA i n CNV coat protein i n mixed infections of cucumber were indicated but not proved. 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