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A study of five mechanically transmissable cherry virus isolates with herbacous hosts Hoes, Josephus Antonius Johannes Marie 1958

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A STUDY OF FIVE MECHANICALLY TRANSMISSABLE CHERRY VIRUS ISOLATES WITH HERBACOUS HOSTS by JOSEPHUS ANTONIUS JOHANNES MARIE HOES, B.S.A. A Thesis Submitted i n P a r t i a l F u l f i l l m e n t of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE We accept t h i s Thesis as conforming to the Standard required from candidates for the Degree of Master of Science. THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1958 A B S T R A C T Five virus i s o l a t e s RS 2, RS 25, RS 26, HS 28 and RS 29, were transmitted by juice-inoculation technique from sour and sweet cherry trees to cucumber. Four i s o l a t e s were obtained from trees growing i n the Kootenay cherry d i s t r i c t of B r i t i s h Columbia. Another one was i s o l a t e d from a tree growing i n the coastal area of t h i s province. Viruses known to occur i n the source trees are Necrotic Ring Spot Virus, Sour Cherry Yellows Virus, Twisted Leaf Virus and L i t t l e Cherry Virus. The relationship and the complexity of the virus i s o l a t e s was studied with herbaceous hosts, using a mechanical transmission technique. Pincherry (Prunus pennaylvanica L.) was inoculated by the same technique as a means for provisional i d e n t i f i c a t i o n of the virus i s o l a t e s . The cucumber syndrome of i s o l a t e RS 25 was very mild, that of i s o l a t e RS 2 mild, that of i s o l a t e RS 29 was of medium severity and those of i s o l a t e s RS 26 and RS 28 were very severe. Inoculates RS 2 and RS 29 varied greatly i n symptom expression on cucumber, whereas the symptom expression of the other i s o l a t e s was l e s s variable. Isolate RS 29 was characterized by symptomless systemic i n f e c -t i o n of Nemesia sp.. var. Triumph. Isolates RS 26 and RS 28 both infected i i Petunia hybr.. var. Blue Bee, without expressing symptoms, whereas the other i s o l a t e s did not i n f e c t t h i s species. Other host species too carried the i s o l a t e s without expressing symptoms, whereas symptoms were produced on cucurbit hosts. Isolates RS 2, RS 26, RS 28 and RS 29 appeared to consist of more than one v i r u s . Strains of a virus P occur i n a l l i s o l a t e s and i s o l a t e RS 25 i t s e l f i s also a s t r a i n of t h i s v i r u s . A l l f i v e strains of virus P express s i m i l a r very mild symptoms on cucumber, whereas a characteristic severe savoying type of symptom i s pro-duced on squash (var. Table Queen). Species susceptible to virus P are cucumber, pincherry, squash, sweet pea, tobacco (under conditions of long day) and other species. Lathyrus odoratua L. and Lens c u l i n a r i s Medic, are species useful i n separ-ating virus P from the other viruses occurring i n i s o l a t e s RS 2, RS 26, RS 28 and RS 29. I t i s possible that virus P i s related to cucumber-mosaic virus as suggested by symptoms on squash and tobacco. In previous work by other investigators a s t r a i n of cucumber-mosaic virus was also i s o l a t e d from Prunus hosts. On pincherry (P. pennsylvanica L.) i s o l a t e RS 28 caused acute symptoms of necrosis and shothole. The plants recovered but symptoms of mottling were systemic. Necrotic Ring Spot Virus caused s i m i l a r symptoms on Prunus hosts and t h i s virus and Sour Cherry Yellows Virus was present i n the o r i g i n a l source tree. i i i The other i s o l a t e s i n pincherry a l l caused s i m i l a r symptoms of mottling on the young leaves. A few necrotic lesions were produced also. On r e i s o l a t i o n from pincherry virus P was obtained i n case of i s o l a t e s RS 2, RS 26 and RS 29. No virus was rei s o l a t e d i n the case of i s o l a t e RS 25. The complete parent i s o l a t e was reisolated i n case of i s o l a t e RS 28. The r e s u l t s with pincherry suggest that virus P i s responsible f o r the mild symptoms whereas virus P i n conjunction with an additional v i r u s as i n i s o l a t e RS 28 i n c i t e s the severe shock symptoms. The i d e n t i f i -cation of the viruses present i n the i s o l a t e s can be carried out by scion inoculation of a set of suitable Prunus indicator hosts. In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of 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 permission f o r extensive copying of t h i s t h e 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 e p r e s e n t a t i v e . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. i v A C K N O W L E D G E M E N T I express my sincere thanks to Dr. N.A. MacLean, Dr. R.E. F i t z p a t r i c k , Dr. J.M. Wilks and Dr. J.J.R. Campbell, who as members of my Committee took an in t e r e s t i n my study. In p a r t i c u l a r do I thank Professor N.A. MacLean f o r his d i r e c -t i o n , encouragement and assistance throughout the course of t h i s inves-t i g a t i o n . In p a r t i c u l a r also do I thank Dr. R.E. F i t z p a t r i c k of the Science Service, Plant Pathology Laboratory, Canada Department of Agriculture, Vancouver, B.C., who allowed me the use of the greenhouse f a c i l i t i e s and who gave me valuable suggestions. I also thank Dr. R. Stace-Smith of the same Laboratory f o r photography work, (Figs. 11,12,13) and advice and who supplied me with virus i s o l a t e RS 25 used i n t h i s investigation. Acknowledgement i s due Dr. N.S. Wright of the same Laboratory f o r h i s valuable suggestions, I express my sincere thanks also to Dr, M.F, Welsh and Dr. J.M. Wilks of the Science Service, Plant Pathology Laboratory, Canada Department of Agriculture, Summerland, B.C., substation Creston, B.C., who suggested the problem and who allowed me time and use of the green V house and who gave me valuable advice and assistance. I also express my sincere thanks to Dr. T.M.C. Taylor and Dr. J.W. N e i l l , of the Botanical Gardens of the University of B r i t i s h Columbia, for provision of seed of many plant species used i n t h i s i nvestigation. I am grat e f u l too to the National Research Council i n Ottawa and the B.C. Sugar Refining Company at Vancouver for f i n a n c i a l a s s i s t -ance . F i n a l l y I express my sincere thanks to Mr. M i c h a i l Schaedle f o r much photography work, (Figs. 1 - 10). v i TABLE OF CONTENTS Page Introduction 1 Review of Literature 5 Relationship of Viruses of Stonefruits . . . . 6 Prunus Virus Isolates and Host Range Studies 11 Materials and Methods 19 The Isolates . . ' 19 Plant Growing and Environmental conditions 21 Inoculation Technique 22 General Course of Host Range Studies 22 I d e n t i f i c a t i o n of the Cherry Virus Isolates . . i 23 Preparation of Inoculum . 24 Cherry Inoculum . . 24 Cucumber Inoculum 25 Testplant.Inoculum . . 2 6 Photography 26 Observations and Results 27 Symptom Expression of the Isolates on Cucumber 27 Cucumber Syndrome of Isolate RS 2 . . . . 27 Cucumber Syndrome of Isolate RS 25 . . . 28 Cucumber Syndrome of Isolate RS 26 and RS 28 28 Cucumber Syndrome of Isolate RS 29 . . . 30 Comparison of the Cucumber Syndromes . . 31 Inoculation t r i a l s with Herbaceous Hosts . . . 31 Virus Reisolated from Host Species 33 Symptom Expression of the Isolates on Pincherry 39 Virus Reisolated from Pincherry 40 v i i Page Discussion and Conclusion 42 Literature Cited 50 Appendix: Figures 1-13 I - XIII I N T R O D U C T I O N General problems In research of virus diseases of plants. Identi f icat ion of the causal agent of a plant disease i s of primary importance i n plant pathological problems. Plant viruses are commonly ident i f ied by and their presence i s observed from the symptoms they produce on their hosts. Particular problems however arise when symptomology i s made the basis for ident i f i ca t ion . Different viruses may produce similar symptoms on a single host or a single virus may produce dissimilar symptoms on different hosts. Furthermore, a single host may be infected simultaneously by more than one virus and the symptoms expressed may be the resul t of the combined effects or of the effect of only one, the others being la tent . In addition there i s the complication of the existence of forms or s t ra ins . Different strains of the same virus may cause widely different symptoms i n one or more hosts. In virus diseases affecting plants the causal agent may there-fore be simple or complex, A single virus entity or two or more virus ent i t ies or virus strains or combinations thereof may be involved in the disease. Different virus diseases on the same host may therefore have a virus factor in common. Consequently these diseases w i l l be related 2 to each other. In the Agriculture Handbook 10 ( l ) which deals with virus diseases of stonefruits only and published i n 1951, forty-eight d i f f e r e n t diseases were described, another four are mentioned i n the same publica-t i o n and several more have been reported since. (6, 26, 27, 35). Transmission experiments by budding or grafting techniques were the means by which stonefruit virus diseases were studied, Symptomology of a descriptive and comparative nature, host range studies and cross-protection experiments yielded data by which stonefruit viruses were distinguished. The transmission experiments were confined to the Rosaceae. Pri m a r i l y Prunus species were used. Other species involved were apple (Malus s y l v e s t r i s M i l l . ) . Japanese K e r r i a (Kerrla japonica (L.) DC.) and Rosa sp. (33). Only when a virus i s sap-transmissable can i t s properties be investigated i n a convenient and thorough manner (2). Stonefruit viruses did not seem to be subject to |uice-inoculation and i n - v i t r o studies were v i r t u a l l y impossible. In 1948 however Moore, Boyle and K e i t t (28) transmitted a vir u s from sour cherry to cucumber using a mechanical transmission tech-nique. The same group of workers (5) showed that the i s o l a t e was d i s t i n c t from cucumber viruses. Other herbaceous hosts were thereafter reported, d i f f e r e n t i a t i n g between i s o l a t e s from Prunus hosts. The scope of the study of stonefruit viruses was widened by these discoveries. Cucumber proved to be useful as a host i n which a 3 number o f s t o n e f r u i t v i r u s e s c o u l d be m a i n t a i n e d . R e l a t i o n s h i p s b e -tween s t o n e f r u i t v i r u s e s c o u l d be i n v e s t i g a t e d on the b a s i s o f h e r b a c -eous h o s t r a n g e s . I n t h i s i n v e s t i g a t i o n r e l a t i o n s h i p s between f i v e i s o l a t e s f rom f i v e v i r u s i n f e c t e d c h e r r y t r e e s were s t u d i e s b y herbaceous h o s t range work. The i s o l a t e s were t r a n s f e r r e d t o and m a i n t a i n e d i n cucumber (Cucumis s a t i v u s L . ) . The t r e e s were s e l e c t e d because o f the range i n s e v e r i t y o f symptom e x p r e s s i o n o f r i n g s p o t v i r u s . The t r e e s were a l s o s e l e c t e d on the b a s i s o f r e g i o n a l o c c u r r e n c e . Four i s o l a t e s were o b t a i n e d from t r e e s growing i n t h e B r i t i s h Columbia Kootenay c h e r r y d i s t r i c t . Another one was i s o l a t e d from a t r e e growing i n the c o a s t a l a r e a o f B r i t i s h C o l u m b i a . Some v i r u s d i s e a s e s o c c u r i n t h i s p r o v i n c e which are o f economic i m p o r t a n c e . L i t t l e C h e r r y o f sweet c h e r r y i s o f importance because i t reduces the v a l u e o f t h e c r o p and i t s n a t u r a l spread i s e x t r a o r d i n a r i l y r a p i d (9). M o t t l e L e a f o f sweet c h e r r y i s Important because growth o f t r e e s i s s e r i o u s l y a f f e c t e d (22). Data on the economic importance o f N e c r o t i c R i n g s p o t o f sour c h e r r y are n o t c o n c l u s i v e b u t t h e t r e e may be s e v e r e l y d i s e a s e d i n the f i r s t y e a r when acute symptoms o c c u r (3). Sour C h e r r y Y e l l o w s i s e c o n o m i c a l l y the most i m p o r t a n t v i r u s d i s e a s e o f s o u r c h e r r y i n t h e U n i t e d S t a t e s and Canada (20). The t r e e s y i e l d i n g the i s o l a t e s were known t o be i n f e c t e d w i t h these v i r u s e s . P a r t o f the problem was concerned w i t h d e t e r m i n i n g i f the v i r u s e n t i t i e s t r a n s f e r r e d t o cucumber c o n s i s t e d o f o n l y one component o r whether a m i x t u r e o r a complex o f v i r u s e s was i n v o l v e d i n the t r a n s f e r . The identi ty of the cherry virus isolates was probed by inoculation of pincherry (Prunus pennsylvanica L . ) . The use of this species for such a purpose and the method of transmission was suggested by the work of Fulton (10). 5 R E V I E W OF L I T E R A T U R E The use of herbaceous species i n research on viru3 diseases of woody plants - and i n p a r t i c u l a r virus diseases of stone f r u i t s -has found wide application after the work of Kunkel and of Moore, Boyle & K e i t t . Kunkel (21) used dodder (Cuscuta campestris Yuncker) i n trans-mitting a virus from X-diseased peach trees to carrot (Daucus carota L.), parsley (Petroselinum crisoum Nym.). periwinkle (Vinca rosea L.) and to tomato (Lycopersicon esculentum M i l l . ) . Juice-inoculation technique was applied by Moore, Boyle & K e i t t (28) to i n f e c t cucumber (Cucumis sativus L. ) var. Ohio with a v i r u s from sour cherry (P. eerasus L.). The trees were infected with Necrotic Ring Spot Virus alone, or i n combination with Sour Cherry YellowsVirus. The v i r u s was e a s i l y transmissable between cucumber plants. The v a r i e t a l s u s c e p t i b i l i t y of cucumber was investigated by Boyle, Moore & K e i t t (5) and by Hobbs (19). The f i r s t mentioned workers reported that a l l twenty v a r i e t i e s tested were susceptible to v i r u s i s o l a t e d from f i f t e e n Prunus species. Hobbs reported that a l l except one out of forty-seven cucumber v a r i e t i e s tested could be infected with v i r u s from sour cherry. 6 Using mechanical transmission technique cucumber has been used i n many investigations to maintain virus isolated from"a variety of Prunus species. In some studies the physical properties of stonefruit v irus isolates were investigated. In other investigations the re la t ion-ships between virus isolates was elucidated when herbaceous hosts other than cucumber were found, which differentiated between the iso la tes . Some isolates also appeared to d i f fe r i n symptom expression on cucumber and other herbaceous hosts. The source of the differences between the isolates i n host range, symptomology and other properties can at least part ly be explained on the basis of differences i n virus contents of the source trees. In this thesis symptomology and host ranges are c r i t e r i a used i n ident i f icat ion and dif ferent iat ion of the isolates concerned. The relationships between stonefruit virus isolates w i l l re f l ec t the relationships i n virus contents of the source t ree. This review of l i terature w i l l deal f i r s t with those viruses which are presumably mechanically trahsmissable to cucumber. After that discussion symptomological - and host range studies with Prunus virus isolates w i l l be reviewed. Relationships of Viruses of Stonefruits. The study of stonefruit viruses was l imited i n scope pr ior to the discovery that they were mechanically transmissable to herbaceous hosts. 7 Valuable information however was obtained from transmission -and cross-protection experiments using scion-inoculation technique and from f i e l d observations. On the basis of symptomology on a variety of Prunus species c e r t a i n diseases could be grouped together and r e l a t i o n -ships between causal viruses were recognized. Generally recognized are the peach-yellows-little-peach group, the X-disease-little-cherry group, and the line-pattern group, the mottle-leaf-rugose-mosaic group and the cherry-yellows-necrotic r i n g -spot-group. (41). Viruses of the l a t t e r mentioned group and some other viruses are the subject matter i n t h i s t h e s i s . W i l l i s o n et a l . (41) consider that the following diseases -and by inference the causal viruses - balong to the cherry-yellows-necrotic-ring-spot group: a Sour Cherry Yellows (SCY) Green Ring Mottle of sour cherry (GRM) Necrotic Ring Spot of sour cherry (NRS) Tatter Leaf of sweet cherry (TL) Prune Dwarf (PD) was also included i n t h i s group by these workers because t h i s v i r u s gives shock symptoms on sour cherry (18). and because t h i s virus was-presumably - transmitted to cucumber (38). The groups however are defined by t h e i r symptomology on Prunus hosts only and not by t h e i r reaction on cucumber. Cameron & Moore (7) report, that PD should be considered a virus d i s t i n c t from the ones causing ringspot and (sour cherry) yellows. In many cases NRS alone or i n combination with SCY was r e -portedly present i n the source trees, y i e l d i n g i s o l a t e s mechanically 8 transmissable to cucumber. From budding and grafting experiments i t appears however that no tree with SCY has been found i n which NRS d i d not occur also. (7, 20 ) . On the other hand i t has been shown that NRS can occur i n trees without SCY being present ( 7 ) . Both NRS and SCY are appar-ently d i f f e r e n t v i r u s e n t i t i e s because they are able to i n c i t e d i f f e r e n t diseases i n the same host. I t was suggested, that Sour Cherry Yellows i s i n c i t e d by a complex that includes NRS (20) . MLlbrath (2$) suggested that SCY and Peach Ring Spot Virus are strains of each other. The l a t t e r v i r u s and NRS are considered to be i d e n t i c a l or clo s e l y related. ( 8 ) . Therefore i n cases where reference i s made to SCY, also i m p l i -cated i s NRS. The s i t u a t i o n i s more c l e a r l y expressed, when the term Sour Cherry Yellows Complex i s used instead of Sour Cherry Yellows Virus. Green Ring Mottle of sour cherry was also present i n source trees from which an i s o l a t e was mechanically transmitted to cucumber. GRM i s also a member of the SCY-NRS group because of i t s close r e l a t i o n -ship to SCY (30) . A s i m i l a r relationship exists between NRS and GRM as with NRS and SCY. A more appropriate term to be used i n cases where GRM i s concerned would to the Green Ring Mottle Complex. Another virus concerned i n t h i s discussion i s Recurrent Necrotic Ring Spot Virus of sour Cherry (R-NRS). This virus and NRS are thought to be strains of each other ( 3 ) . R^iJRS therefore also can be included i n the SCY-NRS group. Tatter Leaf of sweet cherry (TL) also may be considered to be a s t r a i n of NRS (8) and was present i n source trees y i e l d i n g mechanically 9 transmissable i s o l a t e s . Investigations where members of the SCY-NRS group were reported to be present i n the source trees are: NRS alone or i n combination with SCY (4, 5, 12, 15, 16, 19, 24-, 28, 34, 36, 37, 38, 39.) R-NRS alone or i n combination with SCY (5, 12, 15, 28) GRM (15, 34, 37, 38, 39) and TL(36, 37, 38, 39). In addition to members of the SCY-NRS group other viruses reportedly present i n Prunus hosts from which viruses were mechanically transmitted to cucumber are Prune Dwarf (15, 24, 36, 38). Peach Stunt (24), Rough Bark of plum (44) and Line Pattern of plum (15). A great v a r i e t y of Prunus sp. contain virus mechanically trans-missable to cucumber (5). Most of the studies however were concerned with virus i s o l a t e s obtained from sour cherry (P. cerasu3 L.). sweet cherry ( P. avium L.). peach (P. persica L.) and with plum (P. domestica L.). These four Prunus species are a l l susceptible to two or more of the viruses mentioned above and some of these viruses can occur i n a latent form. Prune Dwarf for instance can occur i n a masked form i n sour and sweet cherry and i n some P B domestica v a r i e t i e s (18). Whether Prune Dwarf i s present i n the source tree can be ascertained only by scion-inoculation of an indicator host such as I t a l i a n Prune or Lombard Plum (18). This same virus i s often found associated with Line Pattern Virus i n P. domestica L. var. I t a l i a n Prune. (18). Also Line Pattern can be latent i n sour cherry (29). Temperature conditions w i l l determine symptom expression of SCY and hence the presence of t h i s virus cannot 10 always be discerned. (20). The complexity of the s i t u a t i o n i s well i l l u s t r a t e d by symptom-less sour or sweet cherry, which may be carrying NRS, SCY, PD, Peach Stunt and possibly cither viruses not mentioned i n t h i s discussion. Hence, the known virus content of a Prunus host as reported by an investigator i s not necessarily i d e n t i c a l with the actual v i r u s "population" of the source tree. The reported virus contents can only be suggestive of the i d e n t i t y of the virus or viruses mechanically transmitted to cucumber. Ideall y studies concerned with mechanically transmissable i s o l a t e s from Prunus hosts should s t a r t with inoculation of a set of suitable Prunus indicator hosts with scions from the trees y i e l d i n g the i s o l a t e s . The r e a l v i r u s contents of the source tree would be demonstrated hereby. However, i t does not appear from the l i t e r a t u r e that such an index-i n g procedure has been followed. A l l the investigators concerned were therefore working with a subject virus which was defined only to the extent of the reported virus content. Summarizing the above i t i s stated that the SCY-NRS group i s comprised of NRS, R-NRS, SCY, GRM and TL. Cucumber appears susceptible to virus e n t i t i e s i s o l a t e d from trees known to be infected with Prune Dwarf, Peach Stunt, Rough Bark of Plum, Line Pattern of Plum and members of the SCY-NRS group. The reported virus contents of the Prunus host i s only suggestive of the i d e n t i t y of the i s o l a t e transmitted to cucumber because of the contamination with l a t e n t viruses. 11 Prunus Virus i s o l a t e s and Host Range Studies. Of in t e r e s t i n t h i s l i t e r a t u r e review are the vi r u s contents of the source tree, the symptom expression of the i s o l a t e i n cucumber, d i f f e r e n t i a l hosts and the i d e n t i t y of the Prunus virus i s o l a t e . In some eases the i d e n t i f i c a t i o n procedure was determinate i n nature as a r e s u l t of backtransfer of the i s o l a t e to Prunus sp. In other cases the conditions of the investigation i t s e l f were only suggestive of i d e n t i t y . In some publications evidence was presented suggesting that the i s o l a t e included more than one d i s t i n c t v i r u s . A s a l i e n t point as t h i s one w i l l be stressed also. .As stated before, Moore, Boyle & K e i t t (28) were the f i r s t to transmit a virus from a Prunus host to cucumber by juice-inoculation technique. The eight sour cherry trees used i n t h e i r investigation con-tained NRS alone or i n combination with SCY. The syndromes i n c i t e d i n cucumber were a l l s i m i l a r and were characterized by yellow r i n g s , coalescence of yellow blotches on the cotyledons and bud p r o l i f e r a t i o n a f t e r k i l l i n g of the ap i c a l growing point. Backtransfer to indicator sour cherry trees was carried out by placing small pieces of cucumber l e a f under the bark of the cherry trees. Symptoms i n d i c a t i n g NRS were observed. Boyle, Moore & K e i t t (;£) i s o l a t e d virus from a sour cherry, infected with NRS. A s i m i l a r syndrome as described above was obtained on cucumber. The i s o l a t e was studied i n comparison with several cucumber viruses. Characteristic differences between the cherry virus and the 12 cucumber viruses were noted. The same group of workers (5) transferred virus from s i x t y - s i x trees, comprising f i f t e e n Prunus species. The trees were known to be infected with NRS, NRS and SCY or R-NRS and SCY. The syndromes on cucumber were a l l quite s i m i l a r and are the same as described above. Afte r backtransfer to Prunus hosts by bark transmission, one out of s i x sour cherry trees developed symptoms of NRS. Only NRS was also observed on two out of twenty-three pincherries inoculated. Because of the low proportion of successful backtransfers, the true i d e n t i t y of the viruses transmissable to cucumber was not determined. The r e s u l t s on sour cherry and on pincherry do show, that NRS belongs to those viruses which are mechanically transmissable to cucumber. This i s also supported by the work of Moore et a l . (28) already described above. In an i n v e s t i g a t i o n by Heinis & Milbrath (17) twenty-three d i f f e r e n t stonefruit trees were indexed for ringspot virus on peach, Bing sweet cherry, and Kwanzan - and Shirofugen flowering cherry. A l l trees except one caused symptoms of ringspot i n a varying degree of severity, ranging from very mild to very severe. The syndromes i n c i t e d on cucumber also showed a range of severity. A close c o r r e l a t i o n was noted with severity of symptom expression on cucumber and on the index-ing hosts. This c o r r e l a t i o n also suggests that Ring Spot Virus or NRS -i s a virus mechanically transmissable to cucumber. Extensive host range studies were also carried out by Boyle et a l . ($). Fifty-seven species i n twenty families were tested. Only 13 cucumber and squash (Cucurblta maxima Duchesne, var . Giant Summer Grookneck) were found to be susceptible. Hobbs (19) made a comparative study of nine regional sour cherry iso la tes . A l l the trees showed symptoms of NRS. The isolates di f fered i n in fec t iv i ty and responded di f ferent ly to dif ferent temper-atures. A l l attempts to reinfect cherry with the isolates f a i l e d . Some herbaceous species were tested i n the same invest igat ion. O f v watermelon, none out of seven variet ies tested appeared to be susceptible. Of nine pumpkin var iet ies tested, four were found to be susceptible. None out of twelve squash variet ies proved to be susceptible, Milbrath (24.) however was able to transfer twenty-five stone-f r u i t virus isolates to a number of squash var ie t ies . The source trees were known to contain NRS, SCY, Peach Stunt and Prune Dwarf. Milbrath obtained his inoculum from peach trees which were bud-inoculated with scions from the source trees. On the variety Buttercup bright golden patterns developed when the inoculum was from source trees infected with SCY. Some strains developed loca l lesions on Hubbard squash. Other isolates a l l developed dissimilar symptoms. Gilmer (15) reported that an apparent latent virus i n cucumber developed d is t inct veinbanding symptoms i n squash Cocozelle. Such a syndrome was only observed when the isolate was derived from source trees containing Line Pattern V i rus . Gilmer suggested that th is or another virus was responsible for the veinbanding symptoms on Cocozelle. Apparently the part icular isolates comprised at least two d is t inct v i ruses. WilBson (39) & Weintraub were reportedly also dealing with isolates which were complex i n nature. An i s o l a t e designated G. 1 was obtained from a source tree infected with Green Ring Mottle. Another i s o l a t e T. 2 was obtained from a Prunus host infected with Tatter Leaf. Both these two i s o l a t e s could be separated i n t o two d i s t i n c t components, designated G. l.A and G. I.B.. and T.2.A and T.2.B. respectively. Components B infected cucumber, tobacco and other herbaceous hosts, whereas components A i n -fected cucurbit hosts only. From l a t e r work by the same investigators (4-0) i t appeared that G.l.B. and T.2.B. were strains of the same v i r u s . This v i r u s named CMVP infected bean, cowpea, cucumber, Datura  stramonium, petunia, tobacco, spinach, sugar beet, Swiss chard and zi n n i a . Because of host range, symptomology and physical properties of CMVP, i t was considered to be an a t y p i c a l s t r a i n of cucumber mosaic v i r u s . W i l l i s o n & Weintraub were of the opinion that t h i s virus was late n t i n Prunus hosts and was not implicated i n the etiology of cherry yellows and related stonefruit diseases. Besides source trees contain-ing Tatter Leaf and Green Ring Mottle, other Prunus hosts with d i f f e r e n t v i r u s contents were involved i n the studies of W i l l i s o n & Weintraub. Present i n these Prunus hosts were NRS, SCY, NRS and PD. Because of inoculation to tobacco i t was suggested that CMVP was also present i n these i s o l a t e s . Tobacco and zin n i a were d i f f e r e n t i a l hosts also reported by Varney & Moore (33). Virus was mechanically transmitted from leaves of some Prunus hosts to tobacco, z i n n i a and to cucumber. A l l the i s o l a t e s that transmitted to tobacco or zin n i a also transmitted to cucumber, but some sources were only transmittable to cucumber and did not i n f e c t 15 tobacco or zi n n i a . Also certain sources infected tobacco or zin n i a but not both. Isolates were obtained from sour cherry, mahaleb, peach and I t a l i a n Prune. Cowpea (Vigna sinensis Endl.) gave a l o c a l l e s i o n reaction to is o l a t e s of Thornberry (32) and of Milbrath (23). The source tree i n Thornberry*s study was a sour cherry. Thornberry suggested cherry r i n g -spot virus as the i n c i t a n t of the l o c a l l e s i o n reaction. Milbrath used two types of inoculum: one type was prepared from flower petals, the other type was made from leaves. The flower inoculum gave numerous l o c a l necrotic lesions on the primary leaves of cowpea. Cowpea did not react with the l e a f inoculum. Both the two inocula i n c i t e d symptoms on cucumber. The syndromes however d i f f e r e d from each other. The sour cherry trees contained latent viruses only, Milbrath suggested that d i f f e r e n t viruses or strains were transmitted. Cowpea i n investigation of W i l l i s o n & Weintraub (40) already discussed above did not react with l o c a l lesions to CMVP, In an investigation by Yarwood (42) Peach Yellow Bud Mosaic Virus was transmitted from peach to cowpea also. The plant reacted with l o c a l lesions on inoculated leaves and with systemic shock symptoms. Also susceptible were bean, cucumber, guar, sunflower and tobacco. Bean also reacted with a l o c a l l e s i o n symptom. The virus was backtransferred by mechanical inoculation to peach, aided by heat treatment. The same worker (43) reported that bean also reacted to a s t r a i n of peach r i n g spot obtained from apricot. I t appeared that 16 t h i s s t r a i n could be transmitted from bean to bean by contact rubbing. Other strains of peach ri n g spot could not be transmitted i n t h i s manner. Most extensive host range studies were ca r r i e d out by Fulton (12). Four i s o l a t e s were investigated by t h i s worker. Sour cherry was the source tree for a l l the i s o l a t e s used. In case of i s o l a t e A, the source tree was affected by R-NRS and by SCY. Isolate B was obtained from Varney & Moore (33), and had been maintained i n tobacco (Nicotiana tabacum L. ) . This i s o l a t e originated i n a tree, affected only by SCY. The source tree i n case of i s o l a t e E was also affected by SCY. Virus E was separated from another virus thought to be a s t r a i n of i s o l a t e B. The sour cherry tree y i e l d i n g i s o l a t e G did not show symptoms, but was known to be carry-ing necrotic ringspot v i r u s . Numerous differences i n hostrange between the i s o l a t e s were found. Investigations involving the same iso l a t e s and carrie d out by the same worker (13) at a l a t e r date showed, that the i s o -l a t e s also d i f f e r e d i n physical properties. I n c i d e n t a l l y , the four i s o l a t e s a l l caused s i m i l a r symptoms i n cucumber. Fulton however did not describe the symptoms produced i n t h i s species. When backtransferred to sour cherry, the symptoms produced by the four i s o l a t e s were also d i f f e r e n t and re f l e c t e d the differences i n vir u s content of the source trees. Fulton (14.) reported, that i s o l a t e A causes recurrent Necrotic Ringspot, i s o l a t e G causes ordinary Necrotic Ringspot, i s o l a t e E causes necrotic spotting s i m i l a r to ringspot i n sour cherry except that enations appear on the lower surface of the leaves. Isolate B produces a chl o r o t i c spotting i n sour cherry accompanied by some ring s . This i s o l a t e does not produce symptoms i n the second year. Isolate B, according to Fulton i s widely d i s t r i b u t e d i n infected stone-17 f r u i t s , including sweet cherries on the west coast. I n Fulton's opinion the symptoms produced by i s o l a t e B i n cherry are d i s t i n c t from previously described virus diseases i n stonefruits. Isolate B therefore would represent a new disease. Useful i n assay of virus i n f e c t i v i t y , Fulton (12) also reported hosts giving a l o c a l l e s i o n reaction. Local l e s i o n hosts were guar (r,ynmrrpfds tetragonoloba (L.) Taub.). Momordica balsamina L.. Segbania spp.. Grotalaria gpectabilis Roth, and C. capensis Jacq. Certain plant families seem to provide more species susceptible to Prunus i s o l a t e s than others. Computation of Eulton's data gives the following information. The number behind the family name gives the number of species susceptible to one or more of Fulton's i s o l a t e s . Apocynaceae 2 Compositae 9 Cucurbitaceae 24. Labiatae 2 Leguminosae 23 Scrophulariaceae 2 Solanaceae 26 Eleven other famili e s each 1 Of the twenty-six solanaceous species, twenty are of the genus Nicotiana. The importance of the Cucurbitaceae, Leguminosae, and Solanaceae i n host range work with stonefruit virus i s o l a t e s i s obvious. The v i r u s CMVP of W i l l i s o n & Weintraub (40) already discussed above infected a .o. some members of the Chaenopodiaceae (spinach, sugar beet, Swiss chard). This family might be useful also i n d i f f e r e n -t i a t i o n of is o l a t e s from Prunus hosts. 18 Some species found susceptible to Prunus isolates by workers other than Fulton (12) are bean, cowpea, spinach, sugar beet and Swiss chard, (23, 32, 4-0, 42, 4-3). Apparently these species were not suscep-tibile to any of Fulton's isolates. Fulton does not state however, whether they were tested or not. Other hosts reported by those workers are also susceptible to one or more of Fulton's isolates. 19 M A T E R I A L S AND M E T H O D S The Isolates Twelve cherry trees were selected i n the experimental orchard of the Science Service, Plant Pathology Laboratory, Summerland, B r i t i s h Columbia, substation Cireston, B.C. The range i n severity i n symptom expression of Necrotic Ring Spot Virus and related virus was the basis f o r t h e i r selection. Some of the trees were without v-irus symptoms. Others showed a severe c h l o r o t i c mottling, a shothole or a laceration e f f e c t . The f i r s t i s o l a t i o n was made on June 2, 1957, about three weeks after petal f a l l . After repeated attempts l a s t i n g throughout the months of June, July and August f i v e of the source trees yielded the iso l a t e s RS 2, RS 24, RS 26, RS 28 and RS. 29. No vir u s could be i s o l a t e d from the other seven cherry trees. The i s o l a t e RS 24 was l o s t because of a severe outbreak of powdery mildew on cucumber i n the greenhouse. The other four i s o l a t e s were used i n t h i s investigation. Also used was Isolate RS 25, which was received from the Science Service, Plant Pathol-ogy Laboratory, Vancouver, B.C. I t was i s o l a t e d from a cherry tree on the campus of the University of B r i t i s h Columbia at Vancouver, B.C. Nomenclature of these i s o l a t e s i s based on systems i n use with these laboratories. The i s o l a t e s were transferred to and maintained i n cucumber (Cucumis sativus L.). var. National P i c k l i n g . The same va r i e t y of 20 cucumber was also used i n making isolat ions from inoculated test plants. The identi ty of the cherry virus isolates was probed by inocula-t ion of pincherry (Prunus pennsylvanica L . ) . The use of th is species for such a purpose was suggested by the work of Fulton (10). Virus Contents of the Source Trees. No formal diagnostic studies were made to determine the virus contents of the source trees. Certain definite data are provided however from symptoms present on the trees and from scion-inoculation experiments of previous years i n the case of the Creston trees. The tree yielding isolate RS 25 did not show virus symptoms in the spring of 1958. Data are given i n Table 1. TABLE 1 Virus Contents of the Source Trees * Isolate Source Tree Virus Contents Remarks RS 2 RS 25 RS 26 RS 28 Sweet Cherry Oregon Lambert Sweet Cherry Mazzard Seedling Montmorency Sour Cherry RS 29 Mazzard Seedling NRS Tw. L f . LC NRS symptoms present only on uppermost leaves Symptomless Observed only in spring 1958 NRS Mottle Leaf Virus LC SCY NRS R-NRS Very strong chlorotic mottle. Symptoms confined to basal por-tion of the current season's growth and to the spurs. Also laceleaf appearance. Depending on weather conditions symptoms of NRS and SCY are apparent every year. No symp-toms i n 1957. Symptoms confined to the basal portions of the current season's growth. Laceleaf appearance. * LC - L i t t l e Cherry Virus NRS - Necrotic Ring Spot Virus R-NRS - Recurrent NRS SCY - Sour Cherry Yellow Virus Tw.Lf. - Twisted Leaf Virus 21 Plant Growing and Environmental conditions. Actual isolation work was carried out at Creston, B.C. during the summer of 1957. A l l other work was done at Vancouver, B.C. in the period October 1957 - June 1958. At Creston the cucumbers were grown in flats, incubated in a greenhouse where l i t t l e or no control of temperature could be exercised. Temperatures of 90 -100 F were common and often prevailed for the greater part of the day. Such high temperatures are thought to be a factor affecting the isolation of the virus. At Vancouver the cucumbers were grown from seed, sown directly into benches containing a 6 n deep layer of fertile greenhouse so i l . The distance between plants was three inches. Seedlings of pincherry were grown in A" flower pots. Other plant species were grown as transplants or they were seeded directly into the soil benches and thinned out when necessary. Artifici a l light was provided during the short day season. An area of approximately 300 square feet was used during the investigation and facilities of two greenhouses A and B were necessary. Environmental conditions in these greenhouses differed considerably. From October 1957 to April 1958 wben the greater part of the host range studies was carried out, the temperature in greenhouse A was at a constant 75 F, During the same period however, the temperature in greenhouse B fluctuated between 55 and 65 F. After April 1958 tempera-22 ture conditions i n the two greenhouses were comparable. Fluorescent l i g h t tubes i n greenhouse A gave 10 hours extra l i g h t of a high inten-s i t y . In greenhouse B l i g h t i n t e n s i t y was lower and l i g h t provisions were such, that often many plants had to be grown under conditions of natural l i g h t only. In general plants grew better i n greenhouse A and therefore stock cultures of the i s o l a t e s were maintained i n t h i s greenhouse. How-ever cucumbers used i n backtransfers had often to be grown i n green-house B. Pincherry was cultured i n greenhouse A. Inoculation Technique. A l l inoculations were made by mechanical transmission tech-nique. Expressed crude plant juice was rubbed onto the upper surface of leaves that had previously been dusted with carborandum 400 mesh. Using the forefinger three to f i v e strokes per l e a f were applied i n an inoculation. Previous work has indicated that such a technique i s sat i s f a c t o r y . (12, 16.) General Course of Host Range Studies. In general inoculations were made, when the plants were i n a , state of most rapid growth and presumably most susceptible to virus i n -f e c t i o n . Cucumber was inoculated i n the cotyledon stage, before the young bud had started to unfold. In t h i s stage i t i s most susceptible to i n f e c t i o n by cherry virus (5). I t seemed l o g i c a l to inoculate other members of the Cucurbitaceae i n the cotyledon stage as wel l and therefore 23 t h i s procedure was followed. For other plant species inoculations were made when 2-8 leaves had developed. In testing a species three to seven plants were used for each i s o l a t e . As a check upon the i n f e c t i v i t y of the inoculum three to f i v e cucumber plants were inoculated at the same time with the same inoculum. Three to f i v e plants of the species tested were kept as an additional control. Backtransfers to cucumber to determine whether i n f e c t i o n had taken place were made 12-18 days after inoculation. Three to f i v e cucumber plants were used f o r each backtransfer. Young leaves were taken from a l l plants inoculated with the same i s o l a t e and a representa-t i v e sample was used i n preparation of inoculum, regardless of whether the young growth showed symptoms or not. Inoculum prepared i n t h i s method would demonstrate systemic i n f e c t i o n . Local i n f e c t i o n was only investigated when inoculated leaves showed symptoms. In t h i s case inoculum was prepared from such symptom bearing leaves. A backtransfer sometimes yielded a syndrome c l e a r l y d i f f e r i n g from that connected with the parent i s o l a t e . Such a backtransfer was cultured i n cucumber f o r a better comparison with the parent i s o l a t e and with other s i m i l a r r e - i s o l a t e s . I d e n t i f i c a t i o n of the Cherry Virus Isolates. I t was thought that the i d e n t i t y of the cherry virus i s o l a t e s could be determined at lea s t p r o v i s i o n a l l y by inoculation of pincherry (P.' pennsylvanica L.). The use of t h i s species f o r such a purpose and the method of inoculation was suggested by the work of Fulton (10) 24 Ultimate i d e n t i f i c a t i o n however must take place by inoculation of a set of suitable Prunus indicator hosts, using scions of P. pennsylvanica. This l a t t e r phase of the i d e n t i f i c a t i o n procedure was not carr i e d out i n t h i s i n v e s t i g a t i o n . Pincherry seedlings i n the 6-leaf stage were inoculated on March 9, 1958. Three seedlings numbered 1, 2 and 3 were used f o r each i s o l a t e . Three young succulent leaves of a seedling were rubbed with cucumber-inoculum. Backtransfers to cucumber were carried out af t e r the period of incubation. The inoculum used i n r e - i s o l a t i o n was prepared from each seedling separately. For some seedlings, backtransfers were repeated several times. Preparation of Inoculum. Inoculum was prepared by macerating rubbed and mature leaves or leaves of the young growth i n a small amount of 0.03 M KgHPO^- KR"2P0^ buffer. The pH l e v e l used was eight. The p a r t i c u l a r pH l e v e l was suggested by the work of Fulton (11, 12, 13) and of Heinis (16). The work of Fulton also suggested the p a r t i c u l a r molar concentration of the buffer. In some cases tapwater was used as the diluent. Cherry Inoculum. Princ i p l e s of inoculation as outlined by Boyle, Moore & K e i t t (5) were applied here. Young succulent leaves, preferably those showing 25 i n i t i a l ringspot symptoms were selected as the source of inoculum. Tapwater was used i n making i s o l a t i o n s from the cherry trees, because no buffer was available at that time. In l a t e r work however when making r e - i s o l a t i o n s from pincherry the phosphate buffer formed the suspending medium. Cucumber Inoculum. Cucumber inoculum was used i n routine transfers to maintain the stock cultures and with inoculations of t e s t plants. Regular transfers of i s o l a t e s to maintain the stock cultures were made every 14 - 17 days. Inoculum prepared from the stock cultures showed a high degree of i n f e c t i v -i t y upon cucumber. In the case of i s o l a t e s RS 2, RS 25 and RS 29, the inoculum was prepared from systemically infected leaves. Plants inoculated with i s o l a t e s RS 26 and RS 28 showed l i t t l e or no growth beyond the cotyledon stage and l o c a l l y infected cotyledons had to be used. Phosphate buffer was used as a diluent with i s o l a t e s RS 25, RS 26, RS 28 and RS 29. The i n f e c t i v i t y of RS 2 dropped considerably with the beginning of the short day season when the phosphate buffer was used. However when tapwater was substituted, high i n f e c t i v i t y was restored and t h i s procedure was followed i n subsequent inoculations. Previous work has shown that cherry viruses i n cucumber extracts have only a short l i f e t i m e i n - v i t r o (11,38). I n t h i s investiga-t i o n cucumber inoculum applied to any plant was i n no case older than 26 f i v e minutes. Testplant inoculum. Testplant inoculum was used to demonstrate vi r u s i n f e c t i o n . I t was prepared as explained above, (p.23). Photography. Photopictures of figures 1-10 were taken with a 35 mm. Contra-f l e x camera (f =4.5) on Kodachrome f i l m . Those of figures 11-13 were taken with a 35 mm. Pr a c t i c a camera ( f =2.8) on Anscochrome f i l m . The photopictures presented are enlarged copies of the o r i g i n a l ones. 2 7 OBSERVATIONS AND RESULTS Symptom Expression of the Isolates on Cucumber. In general the symptom expression of the i s o l a t e s varied with length of daytime, temperature and season of the year. Some i s o l a t e s were more subject to v a r i a t i o n than others. Characteristic differences were noted i n degree of stunting, occurrence of necrosis, c h l o r o t i c lesions and bud p r o l i f e r a t i o n . Minor differences were noted i n color of the syndrome, and type and degree of mottling, ( F i g . 1 - 9 i n Appendix). Isolate RS 2 was characterized by a d i f f e r e n t requirement of pH of buffer as explained under Materials and Methods. Cucumber Syndrome of Isolate RS 2 (Fig. 2 ) . Bud p r o l i f e r a t i o n did not occur with t h i s i s o l a t e and c h l o r o t i c lesions on the cotyledons did not develop. Except during a prolonged hot s p e l l necrosis did not take place. The plants were only s l i g h t l y stunted and mottling was mild. The i n i t i a l symptom was a chlorosis of the f i r s t true l e a f , s t a r t i n g at the margin and becoming i n t e r v e i n a l l a t e r on. The l e a f then became mottled. As the plant aged the mottling of the leaves became less pronounced. In general symptom expression was more severe during the long day season. During the short day season symptoms pro-duced were very mild and the syndrome was very s i m i l a r to that of 28 i s o l a t e RS 25. Cucumber Syndrome of Isolate RS 25 (Fig. 3.4.5) The f i r s t symptom was observed on the f i r s t true l e a f . Pale green ch l o r o t i c areas appeared which followed the outline of the bigger veins. These chl o r o t i c areas were bordered by dark green bands and they enclosed islands of dark green tissue. When the second true l e a f had developed the dark green dissue of the f i r s t l e a f had become chlorotic also. By t h i s time the f i r s t l e a f often showed a chessboard effect where the l i g h t e r colored bigger veins formed the outline of the blocks. The youngest growth showed a few ch l o r o t i c areas of an i r r e g u l a r shape and often concentrated along the main vein. The chessboard ef f e c t on the f i r s t l e a f was persistent, whereas no symptoms remained on the other older leaves. Under short day conditions the syndrome was dark green, whereas under long day conditions i t was l i g h t green colored. Lesions on the cotyledons were observed only u n t i l the s t a r t of the short day season i n 1957. These lesions were pale yellow and had a rather d e f i n i t e outline. Development of symptoms was slow, stunting was not observed and the syndrome was very mild. Cucumber Syndrome of Isolates RS 26 and RS 28 (Fig. 6.7.8) Bud p r o l i f e r a t i o n , severe mottling, severe stunting and develop-ment of chlorotic lesions on the cotyledons were char a c t e r i s t i c s of both these i s o l a t e s . The lesions were of two types. 29 In the one case the lesions were diffuse pale green, c h l o r o t i c , c i r c u l a r 2-3 mm. i n diameter and appearing 3-6 days af t e r inoculation. These spots coalesced r e a d i l y r e s u l t i n g i n c h l o r o t i c areas, which often became yellow as the plant matured. In the other case the lesions were yellow, c i r c u l a r , measuring 1-2 mm. i n diameter and appeared during or a f t e r coalescence of the other l e s i o n s . This type however had a d i s t i n c t margin, remained mostly separate but sometimes two or seldom three adja-cent lesions coalesced also. These lesions were often suurounded by a dark green - complete or p a r t i a l - r i n g . One to ten lesions of t h i s type were present on a cotyledon. The greater part of the cotyledons was taken i n by the f i r s t mentioned ch l o r o t i c areas. Both the two i s o l a t e s showed necrosis of the f i r s t true l e a f . The cucumber plants were of a general c h l o r o t i c l i g h t green appearance, but with plant maturity the cotyledons became nearly completely yellow. The i s o l a t e s were distinguished by the degree of severity of the syndrome. K i l l i n g of the primary bud and the degree of bud p r o l i f e r a -t i o n were d i f f e r e n t i a t i n g features. The primary bud was k i l l e d mostly before the f i r s t true l e a f had f u l l y developed. The f i r s t l e a f became necrotic, and bud p r o l i f e r a -t i o n was immediate and conspicuous. The buds were severely mottled. Sometimes under conditions of hot weather the f i r s t true l e a f was not k i l l e d so soon and bud p r o l i f e r a t i o n was delayed. This was the more severe syndrome. Isolate RS 28 behaved i n t h i s manner during the summer and f a l l of 1957 u n t i l the s t a r t of the short day season. 3 0 When the primary bud was not k i l l e d , bud p r o l i f e r a t i o n was less pronounced. The internodes were very short measuring inch. The plants slowly increased i n length, though the leaves were severely stunted. Isolate RS 2 8 behaved i n t h i s manner from the s t a r t of the short day season i n 1 9 5 7 u n t i l J u l y 1 9 5 8 . During t h i s period i s o l a t e RS 2 6 was more severe than i s o l a t e RS 2 8 . Both the two i s o l a t e s , however, were very s i m i l a r i n symptom expression. Also, v a r i a b i l i t y i n expression of symptoms was minor when compared with i s o l a t e s RS 2 and RS 2 9 . Cucumber Syndrome of Isolate RS 2 9 (Fig. 9 ) Under long day conditions the syndrome was quite s i m i l a r to that of i s o l a t e RS 2 . The RS 2 9 syndrome however showed a more severe type of mottling, more stunting and chlorotic lesions were produced on the cotyledons. In the summer of 1 9 5 7 these lesions were 1 - 2 mm. i n diameter, were of a pale yellow color and had a d e f i n i t e margin. During the spring of 1 9 5 8 lesions were not produced. Under short day conditions the syndrome was e n t i r e l y d i f f e r e n t and was very si m i l a r to that of i s o l a t e RS 2 8 , The RS 2 9 syndrome however was dark green i n color i n contrast to that of i s o l a t e RS 2 8 which was l i g h t green i n appearance. Stunting was very pronounced, internodes were short and bud p r o l i f e r a t i o n occurred. On the cotyle-dons c i r c u l a r c hlorotic lesions developed 2 - 3 mm. i n diameter and had a d e f i n i t e margin. The true leaves were severely mottled, the cotyledons became dark green. 31 Comparison of the Cucumber Syndromes. Table 2 gives features distinguishing and d i f f e r e n t i a t i n g the syndromes of the respective i s o l a t e s . (p.31-A). Inoculation T r i a l s with Herbaceous Hosts. Seventy-five species i n twenty-three plant families were tested. On backtransfer virus was reisolated from twenty-three species and v a r i e t i e s belonging to nine families (Table 3, p.32 B-H). In general only the occurrence of systemic i n f e c t i o n was checked. The species which were not susceptible to systemic virus i n f e c -t i o n are l i s t e d on Table A (p. 32 I-K). I t i s possible that some of these species were l o c a l l y infected. Virus symptoms were shown by a few species. Most of the experi-ments were carried out during the short day season when growth of plants was slow. Symptoms usually occur on a c t i v e l y growing leaves (2) and one can expect that virus i n f e c t i o n i s also favored by an active state of growth. Symptoms on host plants were shown by members of the Cucurbitaceae, by Nicotiana tabacum L.. var. Haranova, by Lens c u l i n a r i s Medic, by Cro t a l a r i a s p e c t a b i l i s Roth and by Cassia marylandica L. The l a s t named species showed a l o c a l l e s i o n reaction on the terminal l e a f l e t s when inoculated with i s o l a t e RS 25. These lesions had a necrotic centre surrounded by a yellow chlorotic r i n g . Only a few lesions were produced on a plant and only a low proportion of inoculated 32 plants reacted with t h i s symptom. Hence only l i t t l e inoculum was a v a i l -able for backtransfer to cucumber and as a r e s u l t the causal virus could not be reisolated. The inoculation t r i a l s with Cassia were conducted three times and the same t y p i c a l symptom was reproduced on every occasion. For t h i s reason i t i s believed that i s o l a t e RS 25 i s the i n c i t a n t of the l o c a l l e s i o n reaction. The results with some species were inconsistent with time. On some occasions virus was recovered showing that systemic i n f e c t i o n had taken place whereas i n repeat t r i a l s v i r u s could not be r e - i s o l a t e d e Frequently only one out of four or f i v e cucumber plants used i n a backtransfer showed virus i n f e c t i o n . Results as these might be due to host materials which inactivate or i n h i b i t v i r u s . Another cause would be the low s u s c e p t i b i l i t y of the host to virus i n f e c t i o n . In some cases only a l i m i t e d quanitity of seed was available or only a low proportion of the seeds germinated. As a consequence repeat t r i a l s could not be carried out or not a l l i s o l a t e s could be tested i n an inoculation t r i a l . The experimental value w i l l be l i m i t e d of those inoculation t r i a l s which were performed only once and where only a low proportion of the cucumbers used i n r e - i s o l a t i o n became infected. The results with other experiments are however of more value and these w i l l be dealt with below. OBSERVATION AND RESULTS  TABLE 2 Comparison of the syndromes oft-cucumber of the cherry virus - i s o l a t e s . * Isolate Syndrome Bud Bud Pro- Necrosis Lesions Stunting Color Variation i n i n general K i l l i n g l i f e r a t i o n Diffuse Definite symptom Margin Margin expression RS 2 Mild No No Sometimes (H.T.) — Some LG(LD) DG(SD) Considerable RS 25 Very mild No No No ' - Yes (LD) No DG (SD) Negligible RS 26 Severe Yes (SD) Yes Yes Yes Yes Severe LG Some RS 28 Severe Yes (LD) Yes Yes Yes Yes Severe LG Some RS 29 Medium No Yes Yes (HT) No Yes Severe DG(SD) Considerable (SD) LG (LD) Medium (LD) * LG HT - Light greenj DG - Dark greenj LD - under high temperature conditions. - under day conditionsj SB - under short day conditionsj OBSERVATIONS AND RESULTS  TABLE 3 Host Range Studies of Five Cherry Virus Isolates with Herbaceous Species Family & Species Date of Inoculation Tested Symptom on Test Species Backtransfer to Cucumber Ratio Syndrome infected/ RS-0 Parent Isolate inoculated AMARANTHACEAE Gomphrena globosa L. Jan. 12"58 Mar. 8'58 n 9*58 BORAGINACEAE Brunnera macrophylla Johnston Ma. 25'58 CARYOPHYLLACEAE Silene armaria L. Mar. 9'58 CHAENOPODIACEAE . Chaenopodium ambrosi-doides L. Apr. 12«58 RS 2 A l l i s o l a t e s n n RS 2 -25 -26 -28 -29 -2 -25 -26 -28 -29 RS 2 -25 -26 -28 -29 2/4 0/4 0/4 0/4 0/4 i A 2/4 I A 0/4 0/4 0/4 1/4 0/4 0/4 I A 0/4 0/4 0/4 + + + TABLE 3 Cont'd. COMPOSITAE Dahlia sp., var. Ideal Bedding Helianthus annuus L. Apr. 4«58 Feb. 2? 58 RS 2 -25 -26 -28 -29 RS 2 -25 -26 -28 -29 l A oA oA 0/4 0/4 0/4 0/4 0/4 0/4 1/4 CUCURBITACEAE Cucurbita moschata Duchesne Nov, 26'57 var. Buttercup var. Cocozelle var. Table Queen Nov. 26'57 Nov, 26«57 May 22'58 RS 2 -25 -29 RS 2 -25 -26 -29 RS 2 -25 -26 -28 -29 RS 2 chlorosis mottle n n ? mottle n n n II savoying 0/4 5/5 0/4 3/4 5/5 4/5 3/4 3/4 3/4 4/4 4/4 3/4 5/5 + + + + + + + to TABLE 3 Cont'd var. Table Queen Cont'd. June 19'58 RS 2 -25 -26 -28 -29 Cyclanthera sp. Mar. 18'58 RS 2 -26 -28 -29 Momordica balsamina L. Feb. 22«58 RS 26 - _ 28 -29 Momordica 3P. Mar. 13'58 RS 2 -25 -26 LEGUMINOSAE Cassia marylandica L. Dec. 11'57 RS 25 -28 -29 Mar. 8'58 RS 2 -25 -26 -28 -29 savoying 4/4 M 4/4 " 2/4 " 4/4 II 5/5 mottle 2/4 " 3/4 0/4 0/4 mottle 2/4 0/4 mottle 0/4 » 2/4 mottle 2/4 " 3/4 n 0/4 necr.lesion 0/4 0/4 0/4 0/4 necr. l e s i o n 0/4 0/4 0/4 0/4 TABLE 3 Cont'd. LEGUMINOSAE Cont'd. Cassia marylandlca L. Lathyrua odoratus L. Mar. 25'58 RS 2 — oA -25 necr. l e s i o n oA -26 _ oA -28 — oA -29 - oA Jan. 26«58 A l l i s o l . - 0/5 Jan 26. .& Feb. 16'58 * • RS 2 ? oA -25 chlorosis 4/4 — -26 ? 0/4 -28 - 0/4 -29 - 1/4 + — Feb. 16 & Mar. 8'58* RS 2 chlorosis 3/4 + — & necrosis -25 chlorosis 5/5 — + -26 necr/chlor 0/4 -28 n II 1/4 + — -29 n II 1/4 + -Mar. 9'58 -26 chlorosis 2/4 + -Jan. 11'58 -26 3/3 + -28 - 1/3 + -Feb. 15'58 RS 2 0/4 -25 — 3/4 + -26 — 0/4 -28 — 0/4 -29 - 3/4 + - ro * Same plants reinoculated. TABLE 3 Cont'd. LEGUMINOSAE Cont'd. Feb. 22«58 RS 2 Lathyrua odoratus L. 25 28 -29 -May 22'58 RS 2 -25 -26 -28 -29 -Lens c u l i n a r i s Medic. Feb 2"58 RS 2 25 -26 -28 -29 -May 22«58 RS 2 c h l o r i s i s 25 " 26 " 28 " 29 Medicago sa t i v a L. Mar. 9«58 RS 2 var. Rhizome 25 26 28 29 SCROFHULARIACEAE Nemesia sp.. var. Triumph Dec.30'57 RS 2 25 26 28 29 it ro i TABLE 3 Cont'd. Nemesia sp. var. Triumph Mar.8'58 cont'd SOLANACEAE Nlcotiana tabacum L. yar. Havanna 38 and also var. STurkish Tobacco var. Turkish Tobacco var. Haranova Petunia Hybr.var. .. Blue Bee Mar.9'58 Jan. 11'58 Apr. 1«58 June 19'58 Dec. 30«57 RS 2 25 26 28 29 RS 2 25 26 28 29 RS 2 25 26 28 29 A l l i s o l a t e s RS 2 25 26 28 29 RS 2 25 26 28 29 Ringspot & mottle n n n it oA oA oA oA 3A oA oA oA oA 3A 2A oA oA oA oA oA 3/6 i A 3 A 3/6 oA 0/4 2/4 2/4 0/4 + + + + + TABLE 3 Cont'd. Petunia Hybr. var. Blue Bee Cont'd. Apr. 1'58 RS 2 - 0/4 25 0/4 26 - 4/4 - + 28 - 4/4 - + 29 - 0/4 ro 32-1 TABLE L LIST OF SPECIES HOT SUSCEPTIBLE TO SYSTEMIC  INFECTION BY THE ISOLATES Isolates not Tested.  APOCYNACEAE m Apocynum . .androsaemifolium L. ASCLEPIADACEAE Asclepias curassavica L. 28 BORAGINACEAE . _ Anchusa azurea M i l l , var. Dropmore 2, 26, 28 A. capensis Thumb. 2, 25, 29 Cynoglossum montanum L. 25 CAMPANULACEAE Adenophora f a r r e r i L.  Campanula medium L.. CARYOPHYLLACEAE Pianthus serotinus L. CHAENOPODIACEAE .Beta vulgaris L.i var Detroit Dark Red Chaenopodium album L. Ch. amaranticolor Coste & Rein. Spinacia oleracea L. COMPOSITAE Ageratum Houstoninum M i l l . , var. Blue Cap Aster sp.; var. Giant of C a l i f o r n i a Calendula o f f i c i n a l i s L. Centaurea moschata L. C. imperialis Hort. Cosmos bipinnatus Cav.: var. Early Sensation Lactuca s c a r i o l a L.  Tagetes erecta L.  Tharaxacum o f f i c i n a l e Weber  Zinnia elegans Jacq. CRUCIFERAE Cheiranthus cheiry L. var. Golden Wonder EUPHORBIACEfiE . . Euphorbia Lattsras L. TABLE L Cont'd. Isolates not Tested  LABIATAE Sa l v i a i i l i a e f o l i a e L. LEGUMINOSAE Baptisia a u s t r a l i s R. Br. Crotala r l a capensis Jacq. 25, 26, 28 Cyamopsls tetragonoloba L. Taub. Dolichos Lablab L. (D. soudanensis Hort). Glycine Max Merr. (G. hispida Maxim.) Lupinus polypfedlns L i n d l . Phaeseolus coccineus L. (P. multi f l o r a s Laa..) P. vulgaris. L.: var. Golden"Wax" P. vulgaris L. var Golden Wax Dwarf Pisum sativum L. var. L i t t l e Marvel P. - var. Onward P. - - var. Perfection V i c i a faba L. V. Sativa L. Vigna sinensis Savi. var. C a l i f o r n i a Black Eye MALVACEAE Althea rosea Cav. 26, 28 NYCTAGINACEAE M i r a b i l i s .jalapa L. 25 ONAGRACEAE Cla r k i a elegans Dougl. Bpilobium angustifolium L. Godetia amoena Don PAPAVERACEAE Papaver nudicaule L. PLANTAGINACEAE - „ Plantago lanceolata L. RANUNCULACEAE Delphinium sp. 25, 28, 29 SCROPHULARIACEAE . Antirrhinum majus L. var Ear l y Sensation D i g i t a l i s purpurea var. g l o x i n i a e f l o r a Vilm. TABLE A Cont'd Isolates not Tested SOLANACEAE Atropa belladonna L. Capsicum frutescens L. Datura Stramonium L. 2, 26, 28 D. innoxia L. 2, 26, 28 Sal p i g l o s s i s sinuata. Ruiz & Pav.« var. Emperor TROPAEOLAGEAE . Tropaeolum ma.ius L. VIOLACEAE V i o l a t r i c o l o r L. var. Red Giant 33 Virus Re-isolated from Host Species. In many cases virus obtained i n r e - i s o l a t i o n was maintained i n cucumber for a better comparison with the parent i s o l a t e s and for a better comparison of a l l re-isolates together. In some cases the cucumber syndrome of the re- i s o l a t e d virus was the same as that caused by the o r i g -i n a l parent i s o l a t e . In many cases however i t was d i f f e r e n t . Many re- i s o l a t e s originating from sources RS 2, RS 26, RS 28 and RS 29 a l l produced the same cucumber syndrome irrespec t i v e of the parent i s o l a t e . The parent i s o l a t e s however d i f f e r r e d i n greater or less e r d e t a i l as already described. For example, virus r e - i s o l a t e d from sweet pea, Lathyrus adoratus L.. inoculated with these d i s s i m i l a r i s o l a t e s a l l produced s i m i l a r symptoms on cucumber. This p a r t i c u l a r syndrome w i l l be referred to henceforth as SO. In a l l cases syndrome SO was reproduced constantly i n repeated transfers between cucumbers and i n no case did the syndrome of the o r i g i n a l parent i s o l a t e manifest i t s e l f . I t appeared that SO was indistinguishable from the cucumber syndrome of i s o l a t e RS 25. This does not mean however, that the causal viruses are i d e n t i c a l , because d i s s i m i l a r viruses can produce s i m i l a r syndromes i n the same plant species. The observations suggest only that the four i s o l a t e s concerned are not simple but complex i n virus compo-s i t i o n . For convenience however, the general symbol RS-0 w i l l be used to denote a virus factor found i n i s o l a t e s RS 2, RS 26, RS 28 and 34 RS 29, which produces the same syndrome SO i n cucumber. Some species notably Lathyrus odoratus L. and Lens c u l i n a r i s  Medic, appeared to be useful i n separation of RS-0 from the parent i s o l a t e s . Isolate RS 28 however did not y i e l d RS-0 i n i n f e c t i v i t y t r i a l s with Lens c u l i n a r i s . T r i a l s were repeated three times with sweet pea and two times with Lens c u l i n a r i s . Control plants of these species did not y i e l d RS-0. Symptoms were not observed on Lathyrus whereas i n one experiment with Lens conducted i n May 1958, the young l e a f l e t s showed a d i s t i n c t chlorosis. The other experiment with t h i s species was carried out i n February 1958. Other species useful i n separation of RS-0 from the parent i s o l a t e s are tobacco (var. Haranova) and squash (var. Table Queen). The re s u l t s with these species however are inconsistent and need explanation. In one experiment carried out i n greenhouse A i n January 1958 the tobacco v a r i e t i e s Havanna 38 and Turkish tobacco were r e a d i l y i n -fected with i s o l a t e RS 2. On r e i s o l a t i o n a l l f i v e cucumber plants used i n each backtransfer c l e a r l y showed the syndrome d i s t i n c t i v e of i s o l a t e RS 2. Symptoms on tobacco were not observed. No other i s o l a t e infected either of these two tofcacco v a r i e t i e s . Another experiment carried out i n the same greenhouse and taking place i n A p r i l 1958, using Turkish tobacco only was unsuccessful, however. Questionable re s u l t s were also obtained with squash (var. Table Queen). In an experiment conducted i n November 1957, taking place i n greenhouse A, a l l f i v e isolates proved to be i n f e c t i v e . Isolates RS 2 35 and RS 29 caused a s i m i l a r s l i g h t systemic mottle, i s o l a t e RS 25 caused a mottle accompanied by a l i g h t green ch l o r o s i s , i s o l a t e s RS 26 and RS 28 caused severe mottling and stunting. On r e i s o l a t i o n into cucumber i s o l a t e s RS 25, 26, 28 and 29 yielded a syndrome i d e n t i c a l to that of the parent i s o l a t e s . Isolate RS 2 yielded a mild syndrome, which might have been due to RS-O. In a l a t e r experiment conducted i n May 1958 and taking place i n greenhouse B where many of the seeds f a i l e d to germinate, i s o l a t e RS 2 only was used. After ten days the young growth showed a l i g h t green chlorosis. Nine days l a t e r the symptoms were very severe and conspicuous to the extreme. Stunting was very pronounced and the plant had a savoyed appearance. On the younger growth dark green b l i s t e r s with the opening pointing downwards were conspicuous amidst the pale green ch l o r o t i c areas. The b l i s t e r s were variable i n size and were often coalescing. The veins protruded beyond the l e a f edge giving i t a f r i n g e - l i k e appearance. Often the l e a f lamina was almost e n t i r e l y lacking and the l e a f was reduced to fringes of tissue only (F i g . 10). Spatula shaped leaves and other variations i n l e a f malformations were noted also. On the l e a f p e t i o l e s , e s p e c i a l l y of the older leaves, enations were produced appearing i n i t i a l l y as pin-point areas and l a t e r forming bands of tissue raised above the surface of the p e t i o l e . These bands were a l l running p a r a l l e l to each other. When backtransfered to cucumber, RS-O was produced. Smith (31) described a symptom on squash caused by cucumber mosaic virus which i s reminiscent of the squash symptom described above. The same writer related that cucumber mosaic vi r u s produces 3 7 c h a r a c t e r i s t i c symptoms on Nicotiana tabacum and on N. glutinosa. Another experiment, the t h i r d , with tobacco Haranova was therefore con-ducted. At the same time a more extensive t r i a l with squash (var. Table Queen) was carried out. Squash-inoculum of i s o l a t e RS 2 was used i n both these experiments, along with cucumber-inoculum of RS-0 as the sweet pea f i l t r a t e of i s o l a t e RS 28 and f i n a l l y a l l the regular stock i s o l a t e s . Symptoms on squash were the same for a l l v i rus sources. Also the symptom was s i m i l a r to the one already described above. In a l l backtransfers RS-0 was re i s o l a t e d . The results on tobacco however were i n contrast to those obtained i n e a r l i e r experiments. Ten days after inoculation ringspot symptoms were observed only i n the case of the stock i s o l a t e s . The ringspots were present on l o c a l l y infected leaves and the symptom did not become systemic. These spots were 3 - 4 mm. i n diameter and appeared as dark green water-soaked areas surrounded by a r i n g of l i g h t green tissue. On the day following t h i s observation and with careful examination no such ringspots could be discerned. The number of ringspots produced were counted: « w No. of Ringspots Plant 1 Plant 2 RS 2 2 0 - 25 6 0 - 26 1 4 0 (mottle) - 28 6 1 - 29 2 0 - 2 ( i n squash) 0 0 RS-0 Sweet Pea f i l -t r a t e of RS 28 0 -Control 0 0 38 On plant 2 of i s o l a t e RS 26 another systemic symptom was produced. I t consisted of an i n i t i a l dark green veinbanding e f f e c t , which l a t e r changed into a dark green mottle. I t was very reminiscent of the syndrome of a cucumber-mosaic v i r u s . Except for t h i s plant, a doubt-f u l inconspicuous mottle was noticeable on a l l the other plants. On backtransfer and irrespective of the vi r u s source, RS-O was obtained only. Plant 2 of i s o l a t e RS 26 from which separate inoculum was pre-pared also yielded t h i s syndrome. No virus was i s o l a t e d from the con-t r o l tobacco plants. A l l inoculum was prepared from systemically infected young leaves. No attempt was made to i s o l a t e virus from the l o c a l l y infected leaves. The experiments reported above were a l l concerned with rei s o l a t e d v i r u s which produced cucumber syndromes d i s s i m i l a r to those caused by i s o l a t e s RS 2, RS 26, RS 28 and RS 29. In other t r i a l s the rei s o l a t e d v i r u s consistently produced a cucumber syndrome which was always s i m i l a r to that caused by the parent i s o l a t e . This was the case with Nemesia sp.. var. Triumph which was susceptible only to i s o l a t e RS 29. I t was also apparent with Petunia  hybr.. var. Blue Bee, from which only i s o l a t e s RS 26 and RS 28 could be re i s o l a t e d . These experiments were repeated several times and the same res u l t s were reproduced on every occasion. When virus r e i s o l a t e d from Nemesia and Petunia was used to i n f e c t sweet pea, syndrome SO only was obtained i n backtransfer to cucumber. Evidently passage through Nemesia and Petunia had not deprived i s o l a t e s 39 RS 26, RS 28 and RS 29 from virus factor RS-0 responsible for syndrome SO. Symptom Expression of the Isolates on Pincherry. The f i r s t v irus symptoms were observed nine days a f t e r inocula-t i o n . Plants inoculated with i s o l a t e RS 28 were characterized by acute symptoms of necrosis and-curling of the young leaves. (Fig. 13). Another s i x days l a t e r these plants showed a severe shothole e f f e c t . Plants inoculated with the other i s o l a t e s a l l showed s i m i l a r symptoms of mottling on the young succulent leaves. (Fig. 12). In some cases a few necrotic lesions developed shortly after the i n i t i a l symptoms of mottling. Subsequent symptoms observed on inoculated plants are of doubt-f u l experimental value because of fungus contamination. The occurrence of the fungus was not noted u n t i l near the end of the investigation when a few necrotic lesions-pf a s i m i l a r type as found on vi r u s inoculated plants - were observed on one control plant. Some of these lesions on the control plant were accompanied by a white s u p e r f i c i a l mycelial growth occurring at the underside of the l e a f at the s i t e of the l e s i o n . Similar mycelial growth also occurred on leaves of inoculated plants. Microscopic examination on one occasion showed the presence of one c e l l e d hyaline globular catanulate conidia. The conidia of Coccomyces hiemalis Higglns and Coryneum b e i . j r i n c k i i Pud.. both i n c i t a n t s of l e a f spot on cherry, do not correspond to the description of the condia referred to above. No time was available to pursue a more 40 thorough investigation of the fungus contaminant and i t s association with the necrotic lesions. On a l l virus inoculated plants s i m i l a r symptoms of necrotic l e s i o n s , shothole e f f e c t , shredding and d e f o l i a t i o n were noted, whereas on one out of three control plants a few necrotic lesions developed. Conspicuous differences i n disease severity between control and virus inoculated plants are therefore present. A combined virus-fungus effect could explain the more severe symptoms on the inoculated plants. The inoculated plants however do r e t a i n t h e i r value as a stock of scions to be grafted or budded onto suitable Prunus indicator hosts. I d e n t i f i c a t i o n of the virus i s o l a t e s can be carried out by such a pro-cedure. Virus Reisolated from Pincherry. The cucumbers used i n backtransfers from pincherry inoculated with i s o l a t e s RS 2, RS 26 and RS 29 a l l yielded the syndrome SO. In other words only virus RS-O was reisolated i n these cases. No virus was reis o l a t e d from plants inoculated with i s o l a t e RS 25. Attempts to r e i s o l a t e virus were made on three d i f f e r e n t occas-ions. Plants inoculated with i s o l a t e RS 28 yielded on backtransfer a cucumber syndrome s i m i l a r to that of the parent i s o l a t e . Inoculation of sweet pea with t h i s reisolated virus and subsequent backtransfer to cucumber yielded syndrome SO. Evidently virus RS-O s t i l l formed part of the complex reis o l a t e d from pincherry inoculated with i s o l a t e RS 28. 41 The inoculum used i n backtransfer wasalways prepared from the seedlings with the most pronounced symptoms. In case of i s o l a t e RS 28 a l l three seedlings showed sim i l a r acute symptoms. Table 5 gives the resu l t s of backtransfer i n a tabulated form TABLE 5 Results of Backtransfer to Cucumber from Pincherry Inoculated  with the Virus Isolates Isolate No. of Seedling I n i t i a l Date of Symptom on Reisolation Seedling i n 1958 Ration infected/ inoculated plants Backtransfer to Cucumber Syndrome RS-O Parent i s o l a t e RS 2 RS 25 RS 26 RS 28 RS 29 1 2 3 3 2 3 mottle n acute necrosis shothole mottle July 6 Apr. 9 Hay 26 Apr. 9 " 28 May 5 May 26 Apr. 9 Apr. 9 Apr. 9 " 28 May 5 " 26 l A 1/4 3/3 3/3 O A 0/4 0/4 3/3 1/4 5/5 0/4 0/4 0/4 1/4 + + + + + + 42 D I S C U S I O N AND C O N C L U S I O N I t i s evident from the experiments that several species of herbaceous plants can be symptomless c a r r i e r s of viruses i s o l a t e d from cherry, whereas symptoms are produced i n cucurbit species. This confirms the work of Pulton (12) and of W i l l i s o n and Weintraub (40). Species as Lathyrus odoratus L.. Lens c u l i n a r i s Medic, and Nemesia sp. d i f f e r e n t i a t e between viruses i s o l a t e d from cherry and have not been reported by other workers. Another d i f f e r e n t i a t i n g host i s Petunia hybr. var. Blue Bee also reported by Fulton (12). From the inoculation experiments i t i s evident that i s o l a t e s RS 2, RS 26, RS 28 and RS 29 are complex i n nature, i . e . they consist of more than one v i r u s . Isolates complex i n nature were also involved i n work done by Gilmer (15), Milbrath (23) and W i l l i s o n and Weintraub (40). Lathyrus odoratus and Lens c u l i n a r i s are useful i n separating the virus RS-0 present i n i s o l a t e s RS 2, RS 26, RS 28 and RS 29. Virus RS-0 causes a mild syndrome SO i n cucumber which was very s i m i l a r to that of i s o l a t e RS 25. The same virus i n squash (var. Table Queen) causes a severe savoying type of symptom characterized by f r i n g e - l i k e outgrowths at the l e a f edge. Tobacco War. Haranova) was systemically infected by v i r u s RS-0 under conditions of long day. A3 On pincherry (P. pennsylvanica L.) i s o l a t e RS 28 causes acute symptoms of necrosis and shothole. The plants recovered but symptoms of mottling were systemic. Necrotic Ring Spot Virus caused s i m i l a r symptoms on Prunus hosts and t h i s virus and Sour Cherry Yellows Virus was present i n the o r i g i n a l source tree. The other i s o l a t e s i n pincherry a l l cause s i m i l a r symptoms of mottling on the young leaves. A few necrotic lesions were produced also. On r e i s o l a t i o n from pincherry virus RS-O was obtained i n the case of i s o l a t e s RS 2, RS 26 and RS 29. No virus was reisolated i n the case of i s o l a t e RS 25. The r e s u l t s with pincherry suggest that virus RS-O i s respon-s i b l e for the mild symptoms whereas virus RS-O i n conjunction with an additional virus as i n i s o l a t e RS 28 i n c i t e s the severe shock symptoms. The i d e n t i f i c a t i o n of the viruses present i n the i s o l a t e s can be carried out by scion inoculation of a set of suitable Prunus indicator hosts. Some species used i n extensive host range work by Fulton (12) proved to be useful i n d i f f e r e n t i a t i o n of Prunus virus i s o l a t e s . Cassia marylandica L. gave a systemic symptomless reaction to virus E of Fulton, whereas i n t h i s investigation i t reacted with a necrotic l o c a l l e s i o n symptom to i s o l a t e RS 25. C r o t a l a r i a s p e c t a b i l i s Roth reacted with symptoms of necrosis and chlorosis to a l l i s o l a t e s , whereas i n Fulton's work i t gave a l o c a l l e s i o n reaction to virus B. Cyamopsis  tetragonoloba (L.) Taub. reacted with symptoms to viruses A, E and G of Fulton, whereas t h i s species was not susceptible to any of the f i v e i s o l a t e s tested i n t h i s study. Gomphrena globosa L. became systemicaliy infected by virus A of Fulton. Isolate RS 2 infected t h i s species on one 44 occasion but the r e s u l t s could not be reproduced. Zinnia elegans Jacq. reacted with a mottle to virus B of Fulton whereas i t was not susceptible to any of the i s o l a t e s used i n t h i s investigation. Other cases s i m i l a r to Zinnia can be c i t e d . I t would appear that there i s l i t t l e r elationship between the v i r u s i s o l a t e s studied here and those studied by Fulton. Necrotic Ring Spot Virus and Sour Cherry Yellows Virus however occur i n the source trees concerned i n t h i s investigation as well as i n Fulton's study. Different viruses or d i f f e r e n t strains are evidently involved. Isolates RS 26 and RS 28 are distinguished from the other i s o l a t e s by systemic symptomless i n f e c t i o n of Petunia hybr.. var Blue Bee. Isolate RS 28 i n one experiment with Silene armeria L. infected t h i s species whereas i s o l a t e RS 26 f a i l e d to do so. The value of t h i s experi-ment i s not great because on r e i s o l a t i o n only one out of four cucumber plants became infected. Also on the basis of very s i m i l a r symptom expression on cucumber i s o l a t e s RS 26 and RS 28 appear to be closely related to each other. Isolate RS 29 i s characterized by symptomless systemic i n f e c -t i o n of Nemesia S P . . var. Triumph, Isolate RS" 25 d i f f e r s from the other i s o l a t e s by l o c a l i n f e c -t i o n r e s u l t i n g i n necrotic lesions on Cassia marylandica L. Under the conditions of the experiment i t does not appear that t h i s species i s useful f o r assaying the i n f e c t i v i t y of t h i s i s o l a t e because only a few lesions per plant were produced and because some plants escaped i n f e c t i o n . 45 No herbaceous host was found which gives consistent and reproduce-able results i n d i f f e r e n t i a t i n g i s o l a t e RS 2 from the other i s o l a t e s . The re s u l t s with Gomphrena globosa L, and with Nicotiana tabacum L. were obtained on one occasion only and could not be reproduced. I t appeared that i s o l a t e s RS 2, RS 26, RS 28 and RS 29 consist of more than one v i r u s . This was apparent by inoculation of Lathyrus  odoratus L. and of other species. The rei s o l a t e d v i r u s of these four i s o l a t e s a l l yielded a cucumber syndrome SO which was d i s s i m i l a r to that of the parent i s o l a t e s . At the same time syndrome SO could not be d i s t i n g -uished from the cucumber syndrome of i s o l a t e RS 25. The question arises whether syndrome SO i s caused by the same v i r u s , by dif f e r e n t viruses or by d i f f e r e n t strains of the same v i r u s , because s i m i l a r symptoms on the same host can be caused by viruses which are d i s s i m i l a r (I) or simi l a r ( I I ) . For convenience the symbol RS-0 i s replaced by P. In case I i s o l a t e RS 25 e.g. comprises virus P-25, i s o l a t e RS 2 comprises virus P-2, etc. In case I I the same virus e.g. P-25 i s present i n a l l i s o l a t e s . Virus P may consist of only one virus or may comprise more than one v i r u s . Because the parent is o l a t e s do d i f f e r i n symptom expression on cucumber and because i t i s assumed that v i r u s P alone always gives the same cucumber syndrome SO, an additional virus must be present i n a l l i s o l a t e s except RS 25. Virus P-25 alone distinguishes t h i s i s o l a t e already from is o l a t e s RS 2, RS 26, RS 28 and RS 29. The additional factor present i n the l a s t named is o l a t e s may be the same for a l l ( l ) or i t may be d i f f e r -ent (2). In case 1 the l e t t e r A w i l l denote the same factor, i n case 2, 46 l e t t e r s A, B, C and D w i l l denote the d i f f e r e n t factors. The s i t u a t i o n i s as follows: Isolate Virus Composition 1-1 1-2 I I - l II-2 RS 2 A P-2 A P-2 A P-25 A P-25 RS 25 P-25 P-25 P-25 P-25 RS 26 A F-26 B P-26 A P-25 B P-25 RS 28 A P-28 C P-28 A P-25 C P-25 RS 29 A P-29 D P-29 A P-25 D P-25 Case I I - l can be dismissed because then the parent i s o l a t e s RS 2, RS 26, RS 28 and RS 29 would give the same cucumber syndrome. This was not the case. In case II-2 one would expect that i n an experiment as with (e.g.) Brunnera macrophylla which appears susceptible to i s o l a t e s RS 26, RS 28 and RS 29, also i s o l a t e RS 25, would i n f e c t t h i s species. The con-centration of virus P-25 i n the respective cucumber inocula used f o r inocula-t i o n of Brunnera should be higher f o r i s o l a t e RS 25 than f o r the other i s o l a t e s where additional factors were present. Isolate RS 25 does not i n f e c t Brunnera. A si m i l a r way of reasoning applies i n the case of Dahlia (only i s o l a t e RS 2 inf e c t s ) and i n the case of Helianthus (only i s o l a t e RS 29 i n f e c t s ) . A7 One would also expect that i n case of Nemesia (susceptible to i s o l a t e RS 29) and of Petunia ( susceptible to is o l a t e s RS 26 and RS 28) i s o l a t e RS 25 also would i n f e c t t h i s species, because these i s o l a t e s RS 26 and RS 28 s t i l l contain the virus factor responsible f o r syndrome SO. Both these species could not be infected with i s o l a t e RS 25 i n spite of repeated attempts. Case II-2 therefore i s rejected and i t i s considered that Case 1-1 and 1-2 represent pictures closer to r e a l i t y . Case 1-1 seems possible only i f virus A alone i s l a t e n t i n cucumber. Gilmer (15) reported a Prunus virus latent i n cucumber which caused a veinbanding symptom i n the squash variety Cocoaelle. In the experiment with Nemesia the o r i g i n a l cucumber syndrome of i s o l a t e RS 29 was obtained i n backtransfer. One may assume that t h i s syndrome i s due to the interaction of both viruses A and P-29, because P-29 alone yields the d i s s i m i l a r cucumber syndrome SO. In t h i s case Nemesia i s apparently susceptible to virus A. Virus A present i n the other i s o l a t e s can only be discerned i f symptoms are produced. The cucum-ber used i n backtransfer for the other i s o l a t e s did not show symptoms how-ever. This can only be accounted f o r i f virus A i s latent i n cucumber. Case 1-2 accounts for the r e s u l t s obtained with Nemesia and with Petunia on the basis of the differences between viruses A, B, C and D. The res u l t s with Brunnera. Dahlia and Helianthus can be explained on the basis of the d i s s i m i l a r i t y between the s t r a i n s . Brunnera e.g. would be susceptible only to P-26, P-28 and P-29 and not to P-2 and P-25. 48 Similarly Dahlia would only be susceptible to strain P-2 and Helianthus only to strain P-29. The results with Nemesia and Petunia can be explained on the same basis. For example, Nemesia is susceptible to both viruses D and P-29 present in the parent isolate RS-29. The varying results with tobacco can be explained on the basis of differential susceptibility to virus P under long - and short day con-ditions. Virus P does not infect tobacco under short day conditions whereas conditions of long day are favorable to infection. Viruses P-2, P-25, P-26, P-28 and P-29 may be considered to be different strains of the same virus because of common host ranges. Cucumber, pincherry, squash, sweet pea and tobacco are a l l susceptible to each of the five strains of virus P. A l l strains also give the same or similar syndrome on cucumber, pincherry, squash and tobacco and a l l strains react without symptoms in sweet pea. The differences between the cucumber syndromes of the parent isolates can be explained on the basis of the differences in reaction of the strains of virus P with the same or different viruses. The results with Nemesia and Petunia and other species are suggestive also of a relationship between the strain of virus P and the other viruses present in the isolates. Obviously strain P-29 and virus E have common host ranges. Both viruses are isolated from cherry, and both infect cucumber and Nemesia. Strain P-26 and virus B and strain P-28 and virus C similarly have host ranges in common. 49 The identi ty of virus P has not been determined because of lack of time. It i s apparently widespread i n nature because i t occurs i n isolates of both the Kootenay d i s t r i c t (RS 2, RS 26, RS 28, RS 29) as well as i n isolate RS 25 originating from the coastal area. From experiments not described here i t appears that virus P i s resistant to aging in -v ivo . The symptoms on squash are reminiscent of a stra in of cucumber-mosaic virus (31). One of two tobacco plants infected with isolate RS 26 showed symptoms suggestive of a strain of cucumber-mosaic virus a lso . Species of a number of different famil ies, other than Cucurbitaceae are apparently susceptible, especial ly members of the Leguminosae. Wil l ison and Weintraub (4-0) isolated a virus CMVP from Prunus hosts. Its part ic le size and immunological reactions suggested that i t was a strain of cucumber-mosaic v i rus . A more complete discussion of the work of these investigators i s found in the review of l i te ra ture . Virus P and virus CMVP of Wil l ison and Weintraub may well be related to each other. 50 LITERATURE CITED 1. Agriculture Handbook 10, 1951. Virus Diseases and Other Disorders with Virus l i k e Symptoms of Stone Fruits i n North America, U.S.D.A., Washington D.C. pp.276. 2. Bawden, F.C. 1956. Plant Viruses and Virus Diseases, Ed. 3. Chronica Botanica Co. Waltham, Mass. pp. 335. 3. Berkeley, G.H., Donald Cation, E.M. Hildebrand, G.W. K e i t t and J . Duain Moore, 1951. Necrotic Ring Spot i n : Agriculture Handbook 10, 1951, U.S.D.A. Washington, D.C. pp. 164-170. A. Boyle, J.S., J . Duain Moore and G.W. K e i t t , 194-9. Comparative studies of a virus disease of cucumber transmitted mechanically from sour cherry and several cucumber virus diseases. (Abs.) Phytopathology 3^:3. 5. ' , 1954. Cucumber as a plant host i n stone f r u i t virus research. Phytopathology AA: 303-311. 6. Cameron, H. Ronald, 1954. A bark s p l i t t i n g virus disease of Montmorency sour cherry. (Abs.) Phytopathology 4^ *: 484. 7. , H.R. and J.D. Moore, 1956. Prune dwarf virus and the sour cherry viruses. (Abs.) Phytopathology 46:635. 8. Cochran, L.C., Lee M. Hutchins, J.A. Milbrath, G i l b e r t L. Stout and S.M. Z e l l e r , 1951. Ring Spot i n : Agriculture Handbook 10, 1951, U.S.D.A., Washington, D.C., pp. '71-80. 9. Foster, W.R., T.B. Lott and M.F. Welsh, 1951. L i t t l e Cherry i n : Agriculture Handbook 10, 1951, U.S.D.A., Washington, D.C., pp. 126 - 129. 10. Fulton, Robert, W., 1957. Mechanical transmission of Prunus viruses to cherry. (Abs.) Phytopathology 47:12. 11. . , 1957. Differences i n properties of mechanically transmitted Prunus viruses. (Abs.) Phytopathology Q± 12-13. 51 12. Fulton, Robert W. 1957. Comparative host ranges of certain mechanically transmitted viruses of Prunus. Phytopathology £7: 215-220. 13. , 1957. Properties of cer t a i n mechanically transmitted viruses of Prunus. Phytopathology L£h 683-687. 14. . , 1958. Private Correspondence. A l e t t e r dated A p r i l 28, 1958. 15. Gilmer, R.M. 1957. The behaviour of some stonefruit virus i s o l a t e s i n cucumber and a new d i f f e r e n t i a l cucurMt host for a stonefruit v i r u s . Plant Dis. Reptr. /yLi 11-16. 16. Heinis, J u l i u s L., 1956. Correlation of r i n g spot virus reactions on stonefruits and cucumber. Phytopathology 46: 163-167. 17. . ., J.L. and J.A. Milbrath, 1954. St r a i n relationships of the ringspot virus on cherry, peach and cucumber. (Abs.) Phytopathology 492. 18. Hildebrand, E.M., T.B. Lott and R.S. W i l l i s o n , 1951. Prune Dwarf i n : Agriculture Handbook 10, 1951, U.S.D.A. Washington, D.C. pp. 171-174. 19. Hobbs, G.A., 1951. Investigations on a cucumber virus mechanically transmitted from sour cherry. (Abs.) Phytopathology ^ l : 16-17. 20. K e i t t , G.W., G.H. Berkeley, Donald Cation, C.N. Clayton, E.M. Hildebrand, J . Duain Moore and E.J. Rasmussen, 1951. Sour Cherry Yellows i n : Agriculture Handbook 10, 1951. U.S.D.A. Washington, D.C. pp. 152-158. 21. Kunkel, L.O. 1944. Transmission of virus from X diseased peach trees to herbaceous plants. (Abs.) Phytopathology J3£: 1006. 22. McLarty, H.T., T.B. Lott, J.A. Milbrath, E.L. Reeves and S.M. Z e l l e r , 1951. Mottle Leaf i n : Agriculture Handbook 10, 1951, U.S.D.A. Washington, D.C. pp. 106-111.. 23. Milbrath, J.A. 1953 Transmission of components of the stonefruit l a t e n t v i r u s complex to cowpea and cucumber from cherry flower petals. (Abs.) Phytopathology l£: 479-480. 24. Milbrath, J.A. 1956. Squash as a d i f f e r e n t i a l host for s t r a i n s of stonefruit ringspot 5 2 viruses. (Abs.) Phytopathology 4 6 : 6 3 8 . 2 5 . Milbrath.J.A. 1 9 5 7 . The r e l a t i o n of peach ringspot v i r u s to sour cherry yellows, prune dwarf and peach stunt. (Abs.) Phytopathology 47: 5 2 9 . 2 6 . , 1 9 5 7 . Midleaf necrosis - a virus disease of sour cherry. Phytopathology A 7 : 637 -640 . 2 7 . and H.E. Williams, 1 9 5 6 , Bud abortion - a virus disease of cherry. (Abs.) Phytopathology 46 : 6 3 9 . 2 8 . Moore, J . Duain, J.S. Foyle and G.W. K e i t t . 1 9 4 8 . Mechanical transmission of a virus disease to cucumber from sour cherry. Science 1 0 8 : 6 2 3 - 6 2 4 . 2 9 . and H.R. Cameron. 1 9 5 6 . Separation of l i n e pattern virus from sour cherry yellows, necrotic ringspot, and prune dwarf viruses with Abundance plum. (Abs.) Phytopathology 46: 2 1 . 3 0 . Rasmussen, E.J., G.H. Berkeley, Donald Cation, E.M. Hildebrand, G.W. K e i t t and J . Duain Moore. 1 9 5 1 . Green Ring Mottle i n : Agriculture Handbook 1 0 , 1 9 5 1 U.S.D.A. Washington, D.C. pp. 1 5 9 - 1 6 1 . 3 1 . Smith, Kenneth M. 1 9 5 7 . A textbook of plant virus diseases. Ed. 2 . J . & A C h u r c h i l l Ltd. London, pp. 6 5 2 . 3 2 . Thornberry, H.H. 1 9 5 7 . A cowpea l o c a l - l e s i o n assay of presumably cherry ringspot virus (Abs.) Phytopathology £h 3 5 . 33. Varney, E.H. and J . Duain Moore, 1 9 5 4 . Tobacco and z i n n i a - two new herbaceous hosts f o r Prunus virus (Abs.) Phytopathology 5 0 9 . 3 4 . Weintraub, M and R.S. W i l l i s o n , 1 9 5 3 . Studies on stone-fruit viruses i n cucurbit hosts. I I I . The effects of cucurbit extracts on i n f e c t i v i t y . Phytopathology 4 3 1 3 2 8 - 3 3 3 . 3 5 . Williams, H.E. and J.A. Milbrath, 1 9 5 5 . Freckle f r u i t of sweet cherry (Abs.) Phytopathology 4 5 » 6 9 6 . 3 6 . W i l l i s o n , R.S. 1 9 5 1 . The effect of some stone-fruit viruses i n cucumber. Plant Dis. Reptr. 3 J ± 2 5 4 - 2 5 5 . 53 37. W i l l i s o n , R . S . and M . W e i n t r a u b . 1953. S t u d i e s on s t o n e - f r u i t v i r u s e s i n c u c u r b i t h o s t s . I . A method o f e v a l u a t i n g the i n f e c t i v i t y o f i n f e c t i o u s j u i c e . P h y t o p a t h o l o g y . £3.: 175-177. 38. , 1953. S t u d i e s on s t o n e - f r u i t v i r u s e s i n c u c u r b i t h o s t s I I . Some f a c t o r s a f f e c t i n g the a g i n g o f i n o c u l u m i n v i t r o . P h y t o p a t h o l o g y 324-328. 39. , 1954. S t u d i e s on s t o n e - f r u i t v i r u s e s i n c u c u r b i t h o s t s . I V . Some e f f e c t s o f hydrogen- ion c o n c e n t r a t i o n and d i l u t i o n on i n f e c t i v i t y . Phytopathology 533-537. 40. , 1957. P r o p e r t i e s o f a s t r a i n o f cucumber-mosaic v i r u s i s o l a t e d from Prunus h o s t s . Can. J. o f B o t . 2j>: 763-771. 41. and J . D . Ferguson, 1956. P u r i f i c a t i o n and e l e c t r o n m i c r o s c o p y o f v i r u s e s c a u s i n g c h e r r y y e l l o w s and r e l a t e d d i s e a s e s . Can. J . o f B o t . 34: 86-103. 42. Yarwood, C E . 1956. M e c h a n i c a l t r a n s m i s s i o n o f peach y e l l o w bud mosaic v i r u s . P l a n t D i s . R e p t r . ^0: 299. 43. : , 1957. C o n t a c t t r a n s m i s s i o n o f peach r i n g s p o t v i r u s . (Abs . ) P h y t o p a t h o l o g y £Z: 539. 44. _ and H . E . Thomas, 1954. M e c h a n i c a l t r a n s m i s s i o n o f 3 f r u i t t r e e v i r u s e s . ( A b s . ) Phytogathology hit'. 511. I I F i g . 1 Healthy cucumber plant, of same age as plants i n figures 3, 6, 7 , 8 . F i g . 2 Young cucumber l e a f showing t y p i c a l mottle of i s o l a t e RS 2 (right) Leaf of healthy plant at l e f t . F i g . 3. Isolate RS 25 i n cucumber, 11 days after inoculation. IV I—, F i g . U. F i r s t true l e a f of cucumber infected with i s o l a t e RS 25 showing the chessboard appearance ( r i g h t ) . Leaf of healthy plant at l e f t . V / F i g . 5. Cucumber leaves showing t y p i c a l mottle of i s o l a t e RS 25. VI F i g . 6 Isolate RS 26 i n cucumber, 11 days after inoculation. Apical bud has been k i l l e d . VII Fig. 7 Isolate RS 28 i n cucumber, 11 days after inoculation. P r o l i f e r a t i o n of buds has started, youngest l e a f shows necrosis at t i p . F i g . 8 Isolates RS 26 (left) and RS 28 (right) i n cucumber, 27 days after inoculation. Fig. 9 Isolate RS 29 in cucumber, 11 days after inoculation. F i g . 10 Typical syndrome of virus RS-O i n squash (var. Table Queen), showing dark green b l i s t e r s on chlorotic l e a f lamina and the f r i n g e - l i k e l e a f edges. F i g . 11 Healthy seedling of Pincherry. XII F i g . 12 Isolate RS 2 i n pincherry, 32 days after inoculation. The l e a f i n front of the picture i s mottled. XIII F i g . 13 Isolate RS 28 i n pincherry, 32 days after inoculation, l e a f with shock symptoms at ri g h t and mottled l e a f i n front of the picture. 

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