STUDIES ON THE NATURE OP RESISTANCE TO VIRUS X IN POTATO (SPLANUM TUBEROSUM L.) by ALBERT P. BENSON AN ABSTRACT Submitted to the School fop Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1959 Approved by ProQuest Number: 10008552 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008552 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 Albert P. Benson ABSTRACT 1 Isolation of virus X from llimmune,, varieties of potato (Solanum tuberosum L.) This work was undertaken to determine the nature of the Immune type of resistance in potato (Solanum tuberosum L .) to virus X. Approach grafts were made to varieties of potato considered to be immune to virus X (USDA potato seedling clone S. 41956, Saco, ana Tawa). To these stocks, scions were grafted using Datura tatula (L.) Torr. and Erlaine selfed seedling clones free of virus X or Irish Cobbler infected with virus X. Inoculation to scions free of virus X were made li| days after grafting at which time grafts were cut to produce plants with a single scion carrying virus X on an immune stock. lation of virus X was attempted from: system, (2 ) 1+-6 Iso­ (1) the complete root mm. sections cut from below the graft union and at a distance of 1.5 to 2.5 mm. away from the graft, (3 ) a stem section at the soil line, (4 ) necrotic tubers when present. Symptom expression was erratic requiring incubation periods of 5 to 23 days before symptoms were expressed in the Datura tatula test plants. quency from all 4 locations. Virus X was Isolated in low fre­ Isolations were more frequent and consistent from the variety Tawa indicating possibly that this variety was more susceptible than others tested. 2 ABSTRACT Time of symptom response m Datura tatula L. (Torr.) to virus X as a function of virus concentration Under normal greenhouse conditions, D. tatula ordi­ narily requires 5 to 9 days for full symptom expression, after inoculation with virus X. Incubation periods in D. tatula were determined by serial dilutions of both clarified and highly purified preparations of virus X, strain X 5. Tests were also prepared using a constant concentration of virus and limiting factors of leaf area and inoculum volume. Plants inoculated with high concentrations of the virus preparations developed symptoms within 5 to 7 days. As the virus concentration was decreased, incubation periods progressively Increased. At the lowest concentration before reaching the dilution end point for the virus preparations, plants occasionally required up to pression. 25 days for symptom ex­ It was considered that Incubation periods were a function of initial virus concentration. Certain physiological and morphological reactions of potato seedling clone S. 1+1956 (Solanum tuberosum L. ) Studies were made of reactions of plants grafted either with virus X carrying scions, or scions free of the virus and later inoculated with virus X after the grafts had become established. Plants responded in the following waysr scions and stocks became completely necrotic. (2 ) (l) scions became necrotic, an apical bud proliferated producing a side ABSTRACT 3 branch either above or below the graft union. remained stunted and weak. (3 ) plants (I4 ) plants with bifurcated tops remained healthy and vigorous. Scions and stocks of grafted plants were cut longitudinally and tested for the presence of starch. Accumulation of starch in virus X carrying scions was observed when grafts were made to immune (S. i|.195b) and hypersensitive (Epicure) stocks. 3 Starch accumulated up to cm. below the graft as well as in susceptible scions when inoculation of the virus was delayed until after the graft had become established. Relative carbohydrate deficiencies in the presence of virus X were demonstrated in roots of immune and hypersensi­ tive stocks when grafted with virus X inoculated, susceptible scions• Evidence is presented suggesting that immunity to virus X in potato, was similar to the hypersensitive reaction to virus X. STUDIES ON THE NATURE OP RESISTANCE TO VIRUS X IN POTATO (SOIANUM TUBEROSUM I*.) toy ALBERT P. BENSON A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Botany and Plant pathology STUDIES ON THE NATURE OP RESISTANCE TO VIRUS X IN POTATO (SPLANUM TUBEROSUM L. ) by Albert P. Benson A Thesis Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of Doctor of Philosophy Major Subject: Plant Pathology Approved: In Chs&ge of Major Work Guidance Committee: Michigan Stat 1959 TABLE OF CONTENTS PAGE Section I, Isolation of virus X from "immune® varieties of potato (Solanum tuberosum L.) Introduction and review of literature 1 Materials and methods 2 Experimental results 5 A* Mechanical Isolations from growing plants 5 B. Isolations from tubers 9 Discussion If? Summary 19 Literature Cited 22 Section II. Time of symptom response In Datura tatula L. (Torr.) to virus X as a function of virus concentration 21| Introduction and review of literature 21+ Materials and methods 25 Experimental results 27 Discussion 36 Summary 38 Literature Cited I4O Section III. Certain physiological and morpho­ logical reactions of potato seedling clone S. 1i1956 (Solanum tuberosum L.) I4.I Introduction and review of literature Ij.1 Materials and methods 1+2 A. Reactions of grafted plants 1+2 B. Relative carbohydrate deficiency in the presence of virus X 1+3 Experimental results 1+7 A. Reactions of \+l grafted plants B. Relative carbohydrate deficiency In the presence of virus X Discussion A. Reactions of 51 58 grafted plants B. Relative carbohydrate deficiency in the presence of virus X 58 60 Summary 61 Literature Cited 63 ACKNOWLEDGMENT The author wishes to express his most sincere appreciation to Dr. W.J. Hooker under whose supervision this Investigation was undertaken. He is also greatly indebted for his suggestions, aid in preparation of the manuscript, and helpful encouragement when most needed. The other members of the guidance committee, Dr. R.L. Klesling, Dr. D.J. deZeeuw, Dr. N.R. Thompson, Dr. L.W. Mericle and Dr. R.S. Bandurski are tendered sincere thanks for their interest and assistance* To Barbara, his wife, for her patience, and encouragement, he is deeply grateful. Isolation of virus X from "immune" varieties of potato (Solanum tuberosum L.) Introduction and review of literature The identification of a very high type of resistance to virus X in potato (Solanum tuberosum L.) by Schultz and Raleigh (1933) was subsequently described as immunity in the seedling S.lj.1956 (Raleigh, 1936). Attempts to recover the virus from above ground portions of S. 1+1956 inoculated with virus X either by grafting or by mechanical means have in the past been unsuccessful (Salaman, 1937; Schultz et al, 1937; Schultz and Raleigh, 1933; Raleigh, 1936; Clinch, 191+2; Anonymous, 1952; Cockerham, 19i|3). These reports of attempts to isolate virus X from this seedling are often brief and lack detailed descriptions of methods, or listings of plant parts from which isolations were attempted. It is probable that isolations were attempted from either leaves or tubers rather than stem or root tissues. It has been shown in twice grafted plants with a susceptible stock and scion, and an intermediate scion of resistant S. lj.1956, that virus X was transmitted readily downward through the intermediate scion to infect the susceptible stock (Clinch, 19lj.4). The upward movement of the virus was extremely slow when the stock was carrying virus X. This condition suggested that possibly stems and roots of resistant stocks of plants grafted with a virus X carrying scion might possibly yield that virus upon 2 isolation. The purpose of this research was to determine if virus X could be isolated from immune types of potato which had been graft inoculated with virus X. Materials and methods Isolations were attempted from potato varieties highly resistant to virus X, USDA potato seedling clone S.i|1956 (Schultz and Raleigh, 1933) , Saco (Akely et al, 1955), and Tawa (Peterson and Hooker, 1959). These were graft inoculated using approach grafts to scions which were susceptible to virus X. At the time of grafting,scions were either free of virus X, or were infected with virus X depend­ ing upon the particular inoculation treatment. wrapped with Sealtex latex bandage. Grafts were The variety S.ij.1956 carries virus S in a latent condition. This virus, however, could not interfere with the isolation studies since the test plants used are considered to be immune from virus S. At intervals following virus X inoculation, attempts were made to isolate the virus from grafted stocks of highly resistant varieties. attempted were; Plant parts from which isolations were (1 ) the complete root system; (2 ) J4.-6 mm. stem sections cut from below the graft union and at least to 2.5 1.5 mm. away from the graft; (3 ) a stem section immediately at and below the soil line; and (I4.) necrotic tubers grown on stocks of grafted plants. Necrotic tubers were occasionally produced on plants grafted with virus X carrying scions. 3 luffhole tubers or excised necrotic tissues from such tubers were ground in a mortar and pestle and used for inoculating Datura tatula (L.) Torr. Plant parts to be tested were handled with sterile forceps and parts were excised with sterile razor blades, portions for isolation were ground using a steamed mortar and pestle. Only enough water was added to the triturated material to provide sufficient inoculum for transfer to an indicator plant. Carborundum,Lj.OO mesh was mixed with the inoculum. Tests for presence of virus X were made by mechanical inoculation of 2 plants of Datura tatula (L.) Torr. Leaves were sprayed with water, dusted heavily with lj.00 mesh car­ borundum, and inoculated with a glass spatula. After rubbing, inoculum was allowed to dry on the leaves and later thoroughly washed with water. In order to evaluate the possibility of chance contam­ ination or of visually unidentifiable incipient infection with virus X in the 2 Datura test plants, a third control plant of D. tatula was inoculated using one leaf from each of the 2 test plants. Thus, if either test plant were infected with virus X but not showing symptoms at the time of Inocula­ tion with potato juice, symptoms would have on the control plant. been expressed There was no evidence of uncontrolled infection with virus X in any of the Datura plants used in these tests. k .When virus X was recovered from tissues of highly resistant plants it was identified as such by each of the following tests: (1 ) cross protection in D. tatula pre­ viously inoculated with a mild strain of virus X, X 6 (Timian et al.1955); (2 ) serological chloroplast agglutination test with virus X antiserum (antiserum developed against strain X5); (3) synergistic reaction with virus Y in Nicotiana tabacum L.; and (I4.) inoculation to G-omphrena globosum L. Plants in the trials were grown in sand and watered twice a week with a production type nutrient solution^/ Greenhouse temperatures during the period of observation were approxi­ mately 20° C. Scions grafted to resistant stocks were of 3 types: (1) Irish Cobbler or Green Mountain naturally infected with virus X; (2) D. tatula plants free of virus X; and (3) sus­ ceptible potato plants (S3. tuberosum) free of virus X. It should be pointed out that all stocks of Cobbler and Green Mountain are naturally infected with virus X. Susceptible plants free of virus X were grown from true selfed seed of the variety Erlaine. Individual seedling plants were subse­ quently grown as clonal lines. Before being grafted these scions were demonstrated to be free from virus X using the serological chloroplast agglutination test. ^The following compounds were dissolved in 1 gallon of water; 1.39 gr. Ca (1103)2 *k 0.69 gr. NH^Cl 0.35 gr. KH^PO^ 0.69 gr. 0.67 gr. KNO 3 1.39 gr. MgSO^ . 7 H 2 O 1.73 gr. K Cl 5 Virus X free Erlaine selfed scions and D. stramonium scions were mechanically inoculated 12| days after grafting with a severe strain of virus X which had been subcultured from isolate X5 (Timian et al 1955)« inoculation procedures were followed. Standard trituration Several plants grafted as described were not inoculated and held as controls free of virus X. Approach grafted plants were cut llj. days after the initial graft producing a plant with a single scion and a single stock. Susceptible scions were inoculated at the time of cutting. Experimental results Mechanical Isolations from growing plants. In attempts to Isolate virus X from 20 grafted stocks of S.l|1956 (Table 1), the virus was successfully isolated from portions of 11 plants. Virus X was obtained from roots, from subterranean stems, and from above ground stems 1.5 to 2.5 mm. below the graft union. Isolations from plants grafted with clones of Erlaine selfed inoculated with virus X were slightly more successful than from plants grafted with D. tatula. Virus X was isolated from all 3 test portions of only one grafted plant of S.i+1956* It was isolated from both roots and stems below the graft union in only 2 instances, and from both roots and subterranean stems in 3 instances. Of the 11 plants from which virus X was isolated the virus was obtained 6 Table 1. Isolation of virus X from grafted stocks of USDA potato seedling clone S.Ij.1956 Source of tissue for isolation Virus X carrying scion Erlaine selfed 11 it if root t/ days 21 23 9 9 10 11 subterranean stem --------T T ~ days 12 Tf tt above ground stem below graft union ££^ 1 7 days 6 12 9 8 7 Ik 8 7 No virus X obtained from 3 plants Datura tatula n-- 11 22 7 8 11 - 8 7 11 - 8 - No virus X obtained from 6 plants 1/ Days required for development of virus X symptoms following rub inoculation of 2 D. tatula plants. 7 from only 1 of the test positions in 8 plants. Of the 16 successful isolations, only one of the 2 D. tatula test plants was- infected in 8 instances and both of the D. tatula test plants were infected in 8 instances. was isolated in greatest frequency Virus X from the area below the graft union, and least frequently from the subterranean stem. Furthermore, both test plants were infected in 5 the 7 successful isolations* This suggests that the virus concentration at the former location was considerably higher than in other portions tested. Tissues of S.i|1956 were examined macroscopically before being ground for inoculum. There were no differen­ tiating symptoms nor unusual conditions associated with tissues from which the virus was successfully isolated. At the time isolations were attempted roots of resistant stocks had become somewhat necrotic after grafting with virus X infected scions. Again the presence or absence of virus X could not be associated with macroscopic differences in root appearance. Isolation attempts were made with 27 grafted Saco plants (Table 2}. Virus X was obtained from only 1 of the 3 isolation locations in each of 10 plants. It was not obtained from 2 locations within a single plant. were not successful in 17 plants attempted. Isolations Both D. tatula test plants were infected in only 3 of the 10 successful 8 Table 2. Isolation of virus X from, grafted stocks of the Saco variety Source of tissue for Isolation Virus X carrying scion root T/ days' subterranean stem ---------- above ground stem below graft -----union days Erlaine selfed days 23 6 20 14 19 19 »t 13 17 No virus X obtained from 11 plants Datura tatula n— n 6 3 5 13 - No virus X obtained from 6 plants 1/ Days required for development of virus X symptoms following rub Inoculation of 2 D. tatula plants. 9 isolations. Roots of grafted stocks again tended to be slightly necrotic. Virus X was isolated in 10 of 19 attempts with grafted plants of the Tawa variety (Table 3)* Results of this series were in general agreement with the 2 previous trials except that the variety appeared to be somewhat more susceptible than S.I4 I9 5 6 and Saco as the frequency of successful isola­ tions was somewhat higher with Tawa. which may be of some importance Another difference is that virus X was recovered from roots in 7 of the 10 successful isolation attempts. In no instance was virus X recovered from the subterranean stem. It was isolated in only I4. instances from the stem portion below the graft union. in only one plant. The virus was isolated in 2 locations Furthermore there was considerably more uniformity of infection in both D. tatula test plants than was observed in isolations from either S. k 1956 or Saco. Isolations from tubers Unsuccessful attempts to isolate virus X from tubers of plants which had been grafted with scions carrying the virus have been reported (Clinch, 19i|2; Anonymous, 1952). Isolations were attempted from 22 tubers of S*!|.1956 harvested from plants with grafts described in the previous experiments. Of these, 6 tubers exhibited a diffuse, fleck type necrosis (Fig. 1A) which was generally distributed throughout the tuber. Usually, only a small percentage of tubers exhibited this 10 Table 3* Isolation of virus X from grafted stocks of the Tawa variety Source of tissue for inoculation Virus X carrying scion root 1/ days" subterranean stem y — days above ground stem below graft union Erlaine selfed it ------- days 9 7 it 9 7 No virus X obtained from 1 plant Datura tatula n - 10 10 21 10 22 11 18 19 21 10 17 13 19 7 7 8 9 No virus X obtained from 8 plants 1/ Days required for development of virus X symptoms following rub inoculation of 2 D. tatula plants. 11 limited necrosis. from only 1 of 6 Isolation of virus X was successful necrotic tubers. The presence of virus X in this instance was demonstrated by inoculation to D. tatula and subsequent tests which were previously described for the identity of the virus. Virus X could not be isolated from 16 tubers which did not exhibit necrosis. In tests determining resistance in potato to virus A virus X was present in scions grafted to a resistant stock. Some of the tubers from such plants developed severe necrosis (Fig. IB). This necrosis was of a more severe type than the diffuse necrosis described in earlier trials. An extreme necrosis of the cortex and vascular ring was obtained in one case when virus A and virus X infected Green Mountain scions were approach grafted to the Saco variety. Successful Isola­ tions of virus X from Saco tubers were made (Table i|) by the following method. Whole tubers were triturated in a mortar and pestle. A slight amount of water was added to the ground tissue to moisten the pulp and make it suitable for inoculum. Carbor­ undum I4OO mesh was dusted on the leaves of D. tatula and into the inoculum,and mechanical inoculations were made using a glass spatula. Inoculum was allowed to dry on the leaf surface after which the leaves were washed thoroughly with water. Temperatures during the experiment were 17° C. 12 FIGURE 1. A. Typical internal necrosis in freshly harvested tubers of S.Lj.1956 plants grafted with virus X infected Irish Cobbler. B. External and internal views of freshly harvested tubers of the Saco variety with advanced necrosis following graft inoculation with viruses X and A. C. Typical small, spindly plants grown from necrotic tubers produced on "immune" stocks grafted either with virus X carrying scions, or with scions doubly infected with viruses X and A. Healthy plants were grown from non-necrotic tubers pro­ duced under conditions similar to the necrotic tubers. 12 Table Lj.• Tuber Isolation of virus X from necrotic tubers produced on Saco stocks grafted with virus X infected scions, or grafted with scions doubly infected with viruses X and A. Scion___________ Variety Virus present 1 2 3 b Irish Gobbler tt it if « It tt i 2 3 b 5 6 7 Green Mountain X it it X it it X tt tt X 4* A ii 51 X + A 11 tt X 4 A tt If X 4 A X X X X Isolation of virus X from necrotic tubers 1/ 44+ 4* + + 4* 4 4- T ------------ +, virus X isolated; virus X not Isolated 1 ' U When a virus mottle was produced in the inoculated £• ifliula, the virus was successively transferred Ij. times through that host to eliminate virus A after the method of MacLachlan et. al. (1953). Specific tests for the identi­ fication of virus X were applied to all unknown virus cul­ tures following the fourth transfer. Virus X was identified in 9 of 12 isolation attempts (Table J+) indicating at least some survival of virus X in necrotic tubers of plants grafted with a virus X infected scion, or a scion doubly infected with viruses X and A. Necrotic and non-necrotic tubers from S.i|195& stocks grafted with Irish Cobbler were stored for a period of 3 months and planted in the greenhouse. Plants produced from necrotic tubers (Pig. 10) were always stunted and spindly while plants grown from non-necrotic tubers developed in a normal manner. Attempts to isolate virus X were made from locations from healthy appearing, and spindly plants. Locations included: 1) leaves, 2) stems, 3) roots, and JLj.) necrotic and non-necrotic seed pieces. not be isolated from ij. spindly plants. Virus X could Furthermore, I| healthy appearing plants or their seed pieces yielded no virus X. Successful isolations of virus X were made, how­ ever, from all lj necrotic seed pieces which produced the spindly plants. 15 Necrotic tubers produced by Tawa stocks which had been grafted with virus A carrying Green Mountain were tested under similar circumstances. was found to exist. A parallel situation No successful isolations were made from healthy tubers or spindly and healthy plant parts* Again, successful isolations were made from 3 of 12 necro­ tic seed pieces. In no instance was the virus re-isolated from tubers of mechanically inoculated plants of any immune variety. Discussion Three rather well defined types of resistance to virus X in potato have been reported from a number of sources. This literature has been recently reviewed by Hooker et. al. (195^)* Immune types have been described as those varieties in which the virus fails to become estab­ lished. Hypersensitive types are those in which top necrosis develops after virus inoculation. Certain tolerant varieties exhibit considerable resistance to field spread of the virus whereas many are completely susceptible. Investigations of the immune, and hypersensitive types of resistance have been carried out (Cadman, 191*2; Cockerham, 191*3; Hutton and Wark, 1952; Clinch, 191*2, and !9l*l*; Raleigh, 1936; Anonymous, 1952). Clinch (191*1*) using an intermediate scion of S.1*1956 grafted to an X-free susceptible stock with an X-infected scion found that the virus moved downward through the intermediate scion 16 in an unrestricted manner to infect the virus X-free stock but that upward movement was very slow. She failed to iso­ late the virus from the tissues of S.i|1956 and concluded that the inability of the virus to multiply in the cells of S.l|1956 was due to some substance or physical condition necessary for virus X synthesis. Hutton and Mark (1952) studied the immune reaction with a seedling similar to S.l|1956 in its resistance to virus X* Attempts were made to isolate virus X from mechanically inoculated leaves of plants with various types of resistance. They considered the "immunity" of S.41956 due to an inactivating system as evidenced by extremely restricted development of the virus in leaves of the X-immune type as compared to the susceptible types. Others (Anonymous, 1952) suggest that immunity and hypersensitivity are phases of the same reaction since necro­ tic spots developed on leaves of S.41956 following grafting to an X-infected stock. It is suggested by the isolations herein reported that the virus survives, at least to a limited extent, in the tissues of "immune” varieties. It seems probable that it is either restricted in its development or short lived in these "immune” varieties. Therefore, under these conditions, the term "immune" should be used advisedly since the virus is present in the tissues. to be clearly defined. The mechanism for resistance has yet Furthermore, the possibility exists 17 that the necrotic condition of the tubers could even be a phase of the hypersensitive reaction and that the assumptions of certain investigators (Hutton and Wark, 1952; Anonymous, 1952) who believe that the immune reaction is similar to the hypersensitive reaction, could be correct. The type of resistance found in S.ij.1956, Saco, and Tawa, to virus X is very useful in the breeding program. For purposes of potato breeding, these varieties should still be considered "immune11 but with some reservations since it has been demonstrated that the virus will not move from stored tubers into a growing plant. Spindly appear­ ances of plants produced from necrotic tubers were probably due to the necrotic condition of the tubers rather than virus X itself. When more refined techniques are available for virus assay, however, it is possible that the virus may then be isolated from plants one generation or more away from graft inoculation with virus X. Certain evidence suggests that virus X is confined to the vascular tissues and moves in the phloem of "immune" varieties. This evidence summarized is; (1) aerial tuber formation which suggests the impedance of translocation; (2 ) isolation of the virus from areas with abundant vascular tissues; (3 ) failure of the virus to move upward from necrotic tubers into growing portions of highly resistant varieties; and (1|) the impedance of upward transmission of the virus 18 through a highly resistant intermediate scion; and (5 ) early necrosis of the inner phloem in varieties hypersen­ sitive to virus X (Quanjer, 193D. That the virus was never isolated from leaf tissues in earlier trials or later experiments may be of some sig­ nificance. Possibly the succulence of tissues, and the comparative lack of vascular tissues as compared to stems and roots, lends itself to excess dilution of the virus,, and a smaller quantity of virus containing tissues. It is also probable, however, that the virus is precluded in its upward movement into the tissues of the leaves. In view of these assumptions, several reasons should be considered for success of virus isolation from tissues of highly resistant "immune,f varieties. (1) Tissues with abundant phloem were used in isolation rather than succulent leaves, and the triturated tissue was left as nearly undiluted as possible. (2) Refined inoculation techniques have been developed in recent years. (3) A necrotic strain of virus X was used which was easily distinguished upon Inoculation to D. tatula. (Lj.) The virus was Inoculated after the grafts had become established instead of using virus X infected scions at the time of grafting. (5) Since an extended period of time was sometimes necessary for symptom development in D. tatula test plants, earlier investigators may have dis­ carded their test plants prematurely. In general, length of incubation period in D. tatula was inconsistent. Incubation periods of up to 23 days were sometimes necessary before symptoms were observed. Delayed symptom development has been shown to be a function of low virus concentration (Hooker and Benson, 1958)- With lower concentrations of virus X the Incubation period was extended beyond the normal period required for expression of symptoms in D. tatula. 5 It is probable that an incubation period of days represented a considerably higher initial concentra­ tion of virus in the tissues than was the case with a 23 day incubation period. There is also some evidence that certain "immune" varieties may be more resistant than others. This is indi­ cated by increased frequency of successful isolations of virus X from the Tawa variety as compared to the number of successful isolations from other varieties. G-enerally, there was more uniformity of infection of the test plants, and greater numbers of successful isolations. Summary Varieties of potato considered to be immune from virus X were approach grafted with scions susceptible to the virus. These were either (1) free of virus X at the time of grafting and inoculated with the virus after the graft had become established, or (2 ) scions were infected with virus X at the time of grafting. All leaves were 20 trimmed from below the graft union on the highly resistant stocks. Isolations of virus X were attempted from such stocks at various time intervals following virus X inocula­ tion. These isolations were attempted from: (1) the com­ plete root system, (2 ) areas immediately below the graft union, (3 ) stem sections from below the soil line, and (I4 ) necrotic tubers grown on stocks of grafted plants. Tri­ turated tissues from the above mentioned plant portions were inoculated to Datura tatula (L.) Torr. which responded to infection with this strain of virus X by a strong systemic mottle. The presence of virus X in these varieties was con­ firmed by the following tests for the identification of virus X: (1) serological precipitin tests, (2) local lesions on G-omphrena globosa D., (3) cross protection with a mild strain of virus X on D. tatula, (4 ) synergistic reaction with virus Y on Nicotiana. tabacum L. Virus X was isolated at a very low frequency from all plant parts tested. Virus X was isolated successfully from 11 plants in 21 attempts from USDA seedling clone S.4l9jJ6, 10 in 27 attempts from the variety Saco, and 10 in 19 attempts from the Tawa variety. The evidence suggests the possibility of differences in degrees of resistance between "immune” varieties. Virus X was isolated more frequently and con­ sistently from Tawa than from either S.i|1956 or Saco. Incubation periods of the virus in D. tatula were inconsistent, varying from 5 to 23 days (before a definite virus mottle was distinguishable). It was later demon­ strated that the length of incubation period was a function of low initial virus concentration. In no instance was the virus isolated from leaves of highly resistant varieties grafted with virus X carrying scions. The virus was isolated from freshly harvested necrotic tubers with some consistency. The frequency of isolation was reduced when attempts were made to isolate the virus from germinated seed pieces after a period of storage. Even though the virus was isolated from the seed pieces, isola­ tions from plants produced from those seed pieces were not successful. Eor all practical purposes, highly resistant varieties of this type may still be considered "immune” since isola­ tions were not successful from plants one generation removed from the original inoculation. 22 Literature Cited Akeley, R.V., P. J. Stevenson, E. S. Schultz, R. Bonde, K. E. Nielson, and A. Hawkins. 1955. Saco: a new late-maturing variety of potato, immune from common race of late blight fungus, highly resis­ tant to, if not immune from net necrosis, and immune from mild and latent mosaic. Amer. Potato Jour. 32: I4.I—148. Anonymous. 1952. potatoes - Scotish Society for Research in plant breeding Ann. Rpt., Edinburgh, 22-27. Cad man, C. H# 19ij-2. Aut otetraploid inheritance in the potato: Some new evidence. Jour. Genetics 44: 33-52. * Clinch, Phyllis E. M. 1942. The identity of the topnecrosis virus in Up-to-Date potato. Roy. Dublin Soc. Proc. N.S. 23: 18-34. _______ . 1944. virus X. 273-299. Observations on a severe strain of potato Roy. Dublin Soc. Sci. Proc. N.S* 23: Cockerham, G. 1943* The reaction of potato varieties to viruses X, A, B, and C. Ann. Appl. Biol. 30: 338-344. Hooker, W.J., and A. P. Benson. 1958. Time of symptom response in Datura stramonium var. tatula to virus X as a function of virus concentration. Tabstract) Amer. Potato Jour. 35: 44^Hooker, ¥ . J., C.E. Peterson, and Roland G. Timian. 1954* Virus X resistance in potato. Amer. potato Jour. 31: 199-212. Hutton, E.M.,and D.C. Wark. 1952. A relationship between immunity and localized reaction to virus X in the potato (Solanum tuberosum). Aust. Jour. Sci. Res., Ser. B. 51 237-243. MacLachlan, D.S., R.H. Larson, and J.C. Walker. 1953. Strain interrelationships in potato virus A. Wis Agr. Exp. Sta. Res. Bui. 180. 1-36. 23 Peterson, C.E., and W. J. Hooker. Tawa: A new early potato variety resistant to late blight, scab, and immune to latent mosaic. Amer. Potato Jour. (in press,1959) Quanjer, H.M. 1931. The methods of classification of plant viruses, and an attempt to classify and name potato viruses. Phytopath. 21; 577-613* Raleigh, W.P. 1936. An abnormal graft reaction in potato resulting from virus infection of a scion on a resistant stock. Phytopath. 26: 7 9 5 -7 9 6 . Salaman, Redcliffe N. 1938. The potato virus "X”: its strains and reactions. Roy. Soc. Lond. Phil. Trans. Ser. B. 229: 137-217. Schultz, E.S., C.F. Clark, W.P. Raleigh, P.J. Stevenson, Reiner Bonae, and J.H. Beaumont. 1937- Recent developments in potato breeding for resistance to virus diseases. Phytopath. 27: 190-197. Schultz, E.S., and W.P. Raleigh. 1933* Resistance of potato to latent mosaic. (Abstract) Phytopath. 23: 32. Timian, Roland G., W.J. Hooker, and C.E. Peterson. 1955* Immunity to virus X in potato: Studies of clonal lines. Phytopath. Ij.5: 313-319. 2k Time of symptom response in Datura tatula L. (Torr.) to virus X as a function oi* virus concentration Introduction and review of literature In the re-isolation of virus X from inoculated plants of potato varieties considered to be immune to virus X, the time between mechanical inoculation and symptom expression in plants of Datura tatula (L.) Torr. was considerably longer than that normally required with this virus. Virus X in mechanically inoculated D. tatula ordinarily requires approx­ imately 5 to 9 days for symptom expression depending upon environmental conditions in the greenhouse. When isolations were made from graft inoculated immune varieties such as S.U1956, Saco, and Tawa (Benson and Hooker, 1958) the incuba­ tion period ranged from virus symptoms. 10 to 2L j. days for full expression of In all other characteristics, the virus iso­ lated under these conditions was identical to the virus used for original inoculation of the resistant potato varieties. It seemed probable that virus X was present at a very low concentration in the tissues of such inoculated plants, and that the delay in symptom expression was a function of initial virus concentration. Delay in symptom expression as a function of virus concentration has been described with a number of animal viruses which include: Shope papilloma virus in rabbits (Bryan and Beard, 1939); encephalomyetitis virus of mice 25 (G-ard, 191+0); meningopneumonitis virus of mice (Gogolah, 1953; Crocker, 195^); and the avian erythromyeloblastic leukosis virus (Eckert, Beard and Beard, 195U)« The research herein reported was undertaken to determine if a similar relationship exists in plant viruses. Materials and methods A virulent strain of virus X, isolate previously described by Timian et.al. (1955)* was cultured on Nicotiana glutinosa L. The virus was clarified by extraction from frozen leaves, grinding in a mortar and pestle, filtering through 1+ layers of cheesecloth, and centrifuging at 3000 x G. for 15 minutes. The supernatant was heated to 55° C for 10 minutes, cooled quickly and again centrifuged as before. This process of freezing and centrifuging was repeated and the virus preparation was transferred to 1 ml. ampoules which were sealed hermetically and frozen until used. Vigorously growing patura tatula (L.) Torr. plants of similar age were inoculated with a series of dilutions of the described virus preparation. The inoculation proce­ dure finally decided upon was as follows. One series of inoculations was made using a glass spatula in the conven­ tional manner. Prior to rubbing the virus suspension on the P. tatula leaf surfaces, the leaves were uniformly dusted with 1+00 mesh carborundum, and carborundum was also added to the inoculum. Approximately equal volumes of virus 26 suspension were used on each leaf, as the spatula was dipped into inoculum only once before rubbing each one of the sev­ eral leaves in an inoculation series* Each of 2 leaves of a single plant was supported with a paper towel and rubbed lightly by stroking II4. times with a glass spatula. There was practically no macroscopically visible mechanical injury with the inoculation method. The time required for expression of systemic symptoms in the uninoculated leaves was recorded for each plant after daily observations. In a second series of tests (Table 5), leaves were covered with the original virus suspension diluted uniformly dusted with 00 10^ mesh carborundum, and gently rubbed with a glass rod which had been drawn out to a flexible tip, and a small ball formed, by melting the end of the glass tip. An area approximately 1 mm. in diameter was gently rubbed at each inoculation site. Three leaves of each test plant were inoculated. In series 1, one site per leaf was inocula­ ted, in series three sites per leaf were inoculated and 2, in a third series, 15 sites per leaf were inoculated. Care was taken to inoculate the interveinal areas by avoiding as much as possible the larger veins of the leaves. This test approximated the situation obtained at the low levels of virus concentration in a dilution series by controlling the area of infection. Noninoculated controls were maintained, as well as controls in which undiluted virus was used with the conventional glass spatula inoculation procedure. 27 In other trials relative sensitivity of Gromphrena globosa L. as a local lesion assay plant for virus concen­ tration was compared with I). tatula. In a final confirmatory test on D. tatula at 17° C (Table 6 ), a highly purified stock preparation of isolate X 5 containing lation. 6 mg, of virus X per ml. was used for inocu­ Two drops of diluted virus preparation were placed on 3 leaves of each D. tatula plant by means of a thin glass tube of the type commonly used for melting point determina­ tions. Average weight of drops from such tubes was Virus X was therefore applied at rates of 9.18 x 9.18 x lO"^ mg., 9*18 x 1 0 "^ mg., 9*18 x 10“7 mg. for each plant In the test. x 1 0 “*^ mg. 10"3 mg., rag. and 9.18 A parallel test was prepared using half leaves of G. globosa to determine the sen­ sitivity of that host to virus X as compared to D. tatula. Experimental results A total of 5 dilution tests were made with clarified virus using the conventional mechanical inoculation technique with a glass spatula and carborundum. Initial tests were designed to determine the most reliable inoculation technique. Results were inconsistent and variable when limited amounts of carborundum were sprayed on leaf surfaces and into inoculum. The least variation in the experimental results (Table 1 ) were obtained with liberal amounts of carborundum dusted on the leaf surfaces and in the Inoculum. ment appeared to be the most reliable. The second treat­ Plants developed Comparison of 2 inoculation techniques, and time required for expression of systemic symptoms of virus X infection in Datura tatula following inoculation with virus at different coneentra1 1 ons. 28 © • hOCM ft © 40 P © ft P P ir \ C O • © O © © ft -=fr *-h P 0 © • p t>* 0 0 f t cU f j H 2 13 O' rd 1. 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The dilution end point of the clarified virus pre­ paration was determined in 2 experiments with limited num­ bers of plants (Table 2). In the first experiment, symptoms in non-inoculated leaves of D. tatula were not apparent until 11 days after inoculation. This was probably due to the fact that rather old plants were used for inoculation. The dilu­ tion end point of the virus preparation was 1/10& as shown in the second test. Results of these tests were similar to the following more critical experiments. In the fourth test (Table 3) ten plants were inoc­ ulated at each level of virus dilution. Average greenhouse temperatures were approximately 25° C during the month of August. Where a high concentration of virus X was used, systemic symptoms were expressed in non-inoculated leaves of all plants within 6 days. At lower concentration levels the expression of symptoms was progressively retarded. There was no infection at concentrations of 1/10^ and above. A fifth trial was prepared with average greenhouse temperatures approximating 20° C (Table I4). The average period for symptom development was slightly longer than that obtained at 2f?° C. Once again there was an increase in the Table 2. Dilution end point of clarified virus X as determined by 2 tests, and time required for expression of systemic symptoms of virus X infection in Datura tatula following inoculation with virus at different concentrations 30 £5*0 D— -cj- jzt 0 rP rP cd ra > t>» • ajovi) 'DCH >O•l A i—1 © J=ST cr\ c^\ _=}■ I I ra -P t>s • cd cd O ra £ O P CA VflH ra !>s • aJ O cr\ _X* c\i I I CM i— I i O - 0 iP CM | I & t >5 ra ra S i P •H £ • cd o cm

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•H P cd o o o o o o o o H H H H o O o CM o o o o o o o o o o o o CM CM CM CM 0 o ra a CD ra p CM 1T \ fA O O O O i—I i —1 i — I i—I _0- o rP 1A M3 O - CO o o o o i—I rP iP i—I CD rH 0 rP PU © t — 11 P=S P ra CD E-4 CM rP | Table 3* Number of local lesions on Gomphrena globosa and time required for expression of symptoms of virus X infection in Datura tatula follow­ ing inoculation with virus at different concentrations. 31 ra Sh © 09 C\J | f t !h *O cO S3 03 f t OJ * O H cd S3 XI O ft _zj* (A I I • o o ■LA OJ -P p 03 • P*s O cd S3 o co rO P S3 co • i>*o ft X> cd S ft TJ cd I I 01 O ft ft OJ f I I J>3 O cd £3 P cO P ‘ ~o cd |A u cd S3 'O •P cd P>s O CQ ft I I I I I I I I J h © P ft cd ra o ft o CQ • ft Cd £! fi A ro TJ © C\J £3 f t cd £S o CQ P P CO o ft o S3 I I f to I • o c5l o S3 ft o cd © ft OJ i —i S3 o OJ • r— OJ 1A • o o * o o• o o• o o• o o• o cd S3 o ra f t 2 P £< £5 ft ft f>ft x> ra S3 o ft ra © ft o CA -=f m o o o ft ft o ft oo u ft * > © © S h P 3 S3 ta O ft •H S3 o ft • P o cd o £i 1A 0 oj ra ft Pr> O ft © xi P o cd cd £ © •H X P o cd CO o ft ft 3 ft £ cd o 4^ © ft U £ S3 *>* P ra cd U W) © S3 ft ft £ Is 0 o P xi ra cd o o p I © ra ra £5 P o S3 x l cd S3 f t © ft 0 h ft Cl3 O ft O J| i —i OJ © u o 1-1 ftI 03 cd £3 CQ o o ft f t CQ o © <—I fciOl CQ oi S h cd OJ © ra !>> ft • 9. S3 © ft £i cd ft • p cd cd 03 ft (A 'ft r—I 3 o \ 32 ra "LA OJ Table 1|. Time required for expression of systemic symptoms of virus infection in Datura tatula following inoculation with virus at different concentrations. X 1 11 © > • O OJ C\] © g rO o C\J •H P cd i—1 G u © 03 -P vD f t rH cd OJ OJ cd T5 o CA iH P 03 5s CQ oif\h Cd 0 * 'O' £ OJ OJ ■ P H CQ _ P P 1 —1 £ © © > CQ © CO 1—I © G P ft G © ft G CQ =t i>» rH © C— C\J C\J C\J CQ 0 © ft CO P G P ^ © 3 hO t> CQ n Hi h _zf* OJ pi—i cd G cd T3 ft CQ CA rH A |H ft [x< © ra ft • 0 O C\J Cd |rH G P • l>*o cd G 1*P cd P Ift cd CQ 1 —1© • O _rd* «H co cd G G G p cd |o CQ • !>>O ft ft cd G ro © ra cd £ £ 0 •h 43 P 0 A OJ ft ft ft £ t>5 © cd faO CQ ca G * >a O © »H cd G © £ O G ft P ra cd G 0 rH ft -G O rH 1—I 1A O 1 —1 rH vO O rH ft Ci5 ft £ G G virus incubation period at low concentrations of virus X. Infection at dilutions of 1/10^ was obtained. In the test (Table 5) using known concentrations of purified virus, symptoms were produced in plants to the final concentration of 9 .1 d x 1 0 “ f, this being an actual dilution O of 1 / 1 0 ° of the original virus preparation, and being close to the reported dilution end point for virus X on Nlcotiana t abac urn L. (Bawden, 191+3) • produced only to the Local lesions on G-. globosa were x 10~$ concentration of the virus. 9.18 This was in general agreement with earlier trials although dilution end point in G. globosa using the clarified virus preparation (Table 3) was l/lt)3# In the second series of Inoculation trials small interveinal areas were rubbed with a small glass ball on the end of a flexible glass rod. The virus concentration in this instance was held constant at a dilution of l/lO^ of the original inoculum (Table 6 ). This concentration produced symptoms In the minimum 5 days period when rubbed uniformly over the leaf. In this trial, all plants ultimately developed symptoms even though leaf rubbing was confined to a very limited area on each leaf. The influence of temperature on the incubation period was apparent in each treatment. Where the complete leaf was Inoculated, the difference was not well defined. At 23° C one plant expressed systemic symptoms in 5 days and the remainder at '( days. in all plants after 7 days at 18° C. Symptoms were expressed In the other treatments 3k in cu Ed •a ft Table 5* Number of local lesions on Goirrphrena globosa and time required for expression of symptoms of virus X infection in Datura tatula following inoculation with virus at different concentrations. m m C O a o m cu I1 & OD m Pi •H ■a 3 *ft ft VO rH cu ^tr in m O C ft P< ft «h rH ,(Do £ • -m=1 2 (1 rH m O - 0 -=j- <6 m •H 0 rH m m o cr\ vo cu .rfcu =± to in cd +3 Pi © 09 in rH in r— IH ft © i—| 5H W VO ih §> •H ft ED o o ) CQ 40> +» Cfi in rH rH O in m in m in m in © m JH +P»* ft O w oj cD d C O •H • 5^ tfc •©*» +©* r° O & S'S K\ *3 O t mo in o © 3 * O O • p? 0 09 o pj o o P i o •H M 47 jH JCf > © O P f o o tiQ m 0 b rH M 00 1— 1 m o\ luD ^1- b rH K to rH • cr\ a in b rH H 00 rH • cn .3 a VO b rH W $ a r— b rH M 00 rH • 00 rH • cn a\ s •H CQ © © cd e qD f ©c © r© do rH rH rH ^3 1 Pi o ■*» o © O O O cd +JHf © rH| W| o & o CU| d ro H oa crv t* o cu rl flj rt rd m to >> o 35 cu rl to fl rH TrJ CQ CU rH ■3 d CQ V fl b O rH 3 •d CU d to LTk >> O 3 to VO 5 CU t ^ 00 ^ * CQ $ ft OS o o d ro iH VO CU iH LO CQ O to o LTV IO 00 j* IO IO to to i vo ro «h vo vo CU | rH rH I t 1 t I 1 1 io to to ro O LT\ to vo "5 ca 3 s JO r3 ^ 3 n CQ tO O cd d •d CQ r— J=5»d 2 VO CQ >> O * SJ I *d m CQ • H i l l >, o ' d Table 6. •a •d © CQ + » 4* 3 3 ■Od vo to ro vo o o ro cu o o IO cu o o IO cu o o to «H to ro io rH ft O § *rl Cl t <0 © { >§ 3 » Q > c d o CD w© +o lb dj cd 01 ft ro *d d 0 3 +©> o• rd o © -d © ,0 © CQ & d g Q O O B © U *©H •H © d rH rH © U <3w •h © d rH rH to rH l/ Figures represent cumulative numbers of plants showing symptoms at each observation interval. Time required for expression of systemic symptoms of virus X infection Datura tatula at different levels of inoculation severity. in r— »>> o 36 the incubation period was increased substantially and became more apparent with decreasing numbers of inoculation sites per leaf. The pattern of symptom development followed closely the trends exhibited in the first two dilution trials (Table 3 and I}.). The delay of symptom expression was evident with each decrease in inoculation severity. Discussion Although there have been plant virus research papers suggesting that length of incubation periods were influenced by virus concentration there are no clear cut discussions of the subject, Maromorsch (1950) demonstrated that the incubation period of the aster-yellows virus varies in its insect vector according to initial concentration. He also found (Maromorsch, 1953) that occasionally there was an abnormally long incubation period in the plant before devel­ opment of symptoms when inoculation was made by an insect on its first day of transmitting the virus. After the first day of transmission the incubation period was reduced in length. Jensen (1951) In 1° minute thermal inactivation tests, increased the incubation period of the mosaic virus of Cymbidium orchids with increases in temperature. periods ranged from ij.3 to 72 days. Latent This suggested reduced virus concentration following heat treatment. Apparently incubation periods of virus X in “ D * tatula are a function of initial concentration of the virus since 37 the average number of days for symptom development increased correspondingly with a reduction in virus concentration. Although there was overlapping of incubation periods in individual plants between treatments, the average number of days for symptom development of virus X in D. tatula was greater with lower virus concentrations. This phenomenon may have been caused in part by variations in the physiological conditions of the test plants in spite of efforts to select plants of uniform type. It is also reasonable to assume that differences in numbers of infection courts may have varied from plant to plant in spite of efforts to provide uniform inoculation. Possibly there is a threshold infection phenomenon giving rise to differences in incubation period. If the infection courts were relatively large in number the apparent incubation period might be in the normal 5 to 9 day range of symptom development. As the virus concentration decreased, little noticeable change in incubation periods might be observed until the number of infection courts were limited. Virus multiplication above this point apparently requires a certain minimum time for development of systemic symptoms. Below the threshold point, an abnormally long incubation period is necessary before virus X symptoms become apparent in D. tatula due to time required for spread from individual inoc­ ulation sites until the bulk of susceptible cells have been invaded. In this light it is interesting to speculate on the 38 possibility tbat plant viruses might display asynchrony of infection as well as some animal viruses (Cairns, 1957)* and that latent periods become constant when all initially susceptible cells become infected (Prince, 1958). The incubation period of virus X is increased at lower temperatures. This presumably is due to a slower rate of multiplication than at higher temperatures. The possibility exists that in this lower temperature range, the virus infects, spreads, and is synthesized at a slower rate than at higher temperatures. G. globosa is a commonly used local lesion test plant for indicating the presence of virus X, and for determining virus concentration. In these tests, D. tatula was approxi­ mately 100 times more sensitive than G. globosa in detecting the presence of the virus. Summary Serial dilutions of clarified virus X and a highly puri­ fied strain of the virus, X 5» were mechanically inoculated to Datura tatula (L.) Torr. Test plants which were inoculated with high concentrations of the virus developed symptoms within the expected minimum time limits. As the virus was decreased, time required for symptom development increased correspondingly and incubation periods in some cases were as long as 25 days. Delayed symptoms were obtained when leaf area and inoculum volume were the limiting factors. In this res­ pect results were similar to those obtained in the virus dilution studies. In 2 tests where known amounts of virus X were inocu­ lated to the Datura leaf surfaces and to G-omphrena globosa the latter host was considerably less sensitive to virus X D. tatula was approximately G. globosa. 100 times as sensitive as 40 Literature Cited Bawden, F.C. 19143* Plant viruses and virus diseases, 2nd ed. Boston. Chronica Botanica Co. Benson, A.P. and W. J. Hooker. 1958* Recovery of virus X from "immune" potato varieties (Solanum tuberosum L.) Amer. Potato.Jour. (abstract) 351 l-j.21. Bryan, W.R. and J.W. Beard. 1939. Estimation of purified papilloma virus protein by infectivity measurements. Jour. Infectious Diseases 65: 306-321. Cairns, J.H.F. 1957* influenza virus. The asynchrony of infection by Virology 3: I-II4 . Crocker, T.T. 1954* H 1® number of elementary bodies per 5 0 % lethal dose of meningo-pneumonitis virus as deter­ mined by electron microscopic counting. Jour. Immunol. 73: 1-7. Eckert, E.A., D. Beard,and J.W. Beard. 195U* Dose res­ ponse in experimental transmission of avian erythromyeloblastic leukosis. Ill Titration of the virus. Jour. National Cancer Inst. II4 : 1055-1066. Card, S. 19i|0. Encephalomyetitis of mice II. A method for the measurement of virus activity. Jour. Exptl. Med. 7 2 : 69-77. G-ogolak, F.M. 1953* Purification of murine pneumonitis virus from moose lung. Jour. Infectious Diseases V2: 214.8-2^3* Jensen, D. D. 1951* Mosaic or black streak disease of Cymbidium orchids. Phytopath, ill: I4OI-I4.H4 . Maromorsch, Karl. 1950. Effect of dosage on length of incubation period of aster-yellows virus in its vector. Soc. Exptl., Biol, and Med., Proc. 75: 7hk> p 1953. Do developmental stages occur in the reproductive cycle of aster-yellows virus? Cold Springs Harbor Symposia on Quantitative Biolo gy. h: 5l-5il* Prince, Alfred M. 1958* Quantitative studies on Rous sarcoma virus III Virus multiplication and cellular response following infection of the chorioallantoic membrane of the cbick embryo. Virology. 5: U35-i|57• Timian, Roland G., W. J. Hooker, and C.E.Peterson. 1955* Immunity to virus X in potato: studies of clonal lines. Phytopath. U5: 313-319* 41 Certain physiological and morphological reactions of potato seedling clone S. Ij.1956 (Solanum tuberosum L.) Introduction and review of literature Raleigh (1936) described the immunity of potato seed­ ling clone S. 1|1956. He demonstrated that aerial tubers were produced on a scion infected with virus X when grafted to a highly resistant stock. It was later shown (Timlan et al, 1955) that in certain instances such grafted scions failed to produce aerial tubers. Webb and Schultz (1957) later found that aerial tubers developed quite rapidly and with more uniformity under short day periods long days (12 or 16 hours). (8 hours) than under Their results were not completely uniform as one plant In 16 failed to produce aerial tubers, enlarged nodes or axillary buds under the most favorable c onditions. Rootstock deterioration of immune plants grafted with virus X carrying scions has been well known since Raleigh (193b) first described the immune reaction of S. 41956. It was speculated that root necrosis and lack of development of underground portions of the stock was caused by nutri­ tional deficiency of the grafted stock. This study was undertaken to determine; (1) graft- scion relationships of virus X - immune plants with various stock and scion graft types; and (2 ) whether the cause of necrosis in underground portions was either associated with k? roots, or more directly due to the necrotic action of virus x. Materials and methods Reactions of grafted plants. Graft experiments were made to determine the reaction of s. Ij.1956 when grafted with a number of virus X-infected and virus X-free scion types. Scions were approach grafted to S. 14.1956 and cut II4. days after grafting leaving a virus X susceptible scion on an immune stock. In other instances the S. i4.1956 top was left intact producing a side branch scion susceptible to virus X. In the first experiment the variety Irish Cobbler was grafted as the scion to S. 14-1956. The section of the scion below the graft union, and the stock above the graft union were cut leaving a single Irish Cobbler scion on a highly resistant stock. One series of plants was placed In a warm chamber at 25° C, and a second series was left on the greenhouse bench at 18° C under normal greenhouse winter conditions. A second trial was prepared in which grafted plants were placed in constant temperature tanks at temperatures of 16° C, 22° C, and 28° C. S. 1|1956, used as the stock In all cases, was approach grafted (1) to (healthy) virus X infected Irish Cobbler, (2) to Irish Cobbler doubly in­ fected with viruses X and X, and (3) to tomato plants of the Bonnie Best variety. Tomato scions were also grafted h3 to a virus Y infected S. J+1956 stock. Tomato plants were either inoculated 7 days after grafting with a severe strain of virus X, X 5 (Timian et al, 1955)f or left as uninoculated checks. Grafts were cut llj. days following grafting leaving half the plants with single tops and half the plants with bifurcated tops at each temperature range. Observations of plant reactions were recorded periodically. A third graft trial was conducted to determine patterns of starch accumulation in grafted plants using various stock and scion types (Table 1). single scion tops. All grafted plants were reduced to Grafts were harvested 12, 2\\ and 1|5 days after virus X inoculation of scions free from virus X. Upon harvest, grafts were cut longitudinally through both the scion and stock, and the presence of starch determined by iodine-potassium iodide applied to the exposed plant tissue. Similar trials were prepared using Datura tatula (L.) Torr. and tomato (Lycopersicum esculentum L.) as the virus Xinfected and X-free scions. Relative carbohydrate deficiency in the presence of virus X . To determine if root necrosis may have been caused by carbohydrate deficiency, roots of grafted plants were analyzed semi-quantitatively for relative deficiency of carbohydrate. Plants were grown in sand and watered twice a week with a production type nutrient solution. S. 1|1956 stocks were grafted with scions of potato S. 1+1956, potato seedling © •h o o o o o * o o o o o I * O pa I fd *W CD O 'H rd m EH © ■8 4o > ra s •H O CO nd © H Pt 5 X © ©© 0 ,P © © O p p* © •p Pi © £ .a © 3 •rl te EH © 7! p? © P Pi jH > nd © P O © © © EH P O *rl O © o r^S 0 0 o 4-> © P p O •H •rl O .p © o 0 U © 4» O © O +» © o o o Pi .a O <+-f ,p o 0 © S © 4* EH © Mo +o> CO «d © Mo +o> CO rd © cr> © © Pi +» © 0 rH © rO P O •H O © © X © P Pi *rl t> © © M d © © © ■+J P © H P © p •H P O © Pi O rH •H P Pi !> •H P3 l/ 0 negative starch text, f positive starch testf * occasional starch test, and - graft type not included in experiment. © Pi O *H_ O U •H © CD M Pi © 4* o 3 B M o +» © X Erlaine selfed, L. esculentum and D. tatula. Stocks of the Epicure variety, resistant to virus X by virtue of hyper­ sensitivity, were grafted with L. esculentum, and potato seedling clone Erlaine. Some plants of the varieties Epicure and S. !{.1956 were left ungrafted to serve as controls. Approach grafted plants were cut ll| days following grafting, leaving plants with both single and bifurcated tops. At the time grafts were cut, those plants intended for virus X inoculation were rubbed with a suspension of the X 5 strain of the virus. Grafted check plants were rubbed with a sus­ pension of carborundum and water. Plants were arranged in pairs according to size and general vigor. Roots were harvested for analysis 7, II4 , and 21 days after virus inoculation. Samples of roots were washed thor­ oughly in water to eliminate sand and debris, and constant weight in a 60° C oven. Dried samples were ground in a V/iley Mill over a. I4.0 mesh screen. samples were weighed to within 0.3 dried to Aliquots of paired mg. of one another. Somogyifs test for reducing sugars (somogyi, 1952) was used. Sugars were extracted from samples in 80$ ethanol, 2 ml. being added per 10 mg. of dried sample. placed at i|° C for washed with 0.5 36 hours and filtered. Samples were The residue was ml. of 80$ ethanol for each 10 mg. of sample and the washing portion collected with the original sample. 2 ml. of the prepared samples were placed in 25 ml. volumetric J|6 flasks, and 2 ml. of Somogyi*s reagent (Somogyi, 1952) were added. Checks consisted of 2 ml. of Q0% ethanol and 2 ml. Somogyit s solution. Flasks were placed in boiling water for 15 minutes, removed and allowed to reagent cool. (19l|l|) were added to bring out 2 ml. of Nelson*s a stable color. The final solution was diluted to 25 ml. with distilled water and read on a Klett-Summerson colorimeter. Readings of all solutions were converted to mg. equivalents of glucose which would produce the same color under the conditions of the reaction minus the check values. To substantiate the validity of the preceding test, a semi-quantitative glucose determination was run on certain samples. Commercial Glucostat, a specific glucose oxidase (Keilen and Hartree, 19l|8; Huggett and Nixon, 1958) wasused for the determination. possibility thatthe Because of the alcohol might interfere with the action of the enzyme, 3 ml. of each sample extracted in 3QJ& ethanol were evaporated in a water bath at 65° C. in 1 Residues of each were resuspended ml. of distilled water, and the complete sample was used in glucose determinations. 2.5 ml. of the reagent waa dispensed into a tube and water was added to make the final volume 5.0 ml. The 1 ml. sample was added to the enzyme preparation and after a reaction time of 10 of i| N HC1 was added to stop the reaction. minutes, 1 drop The resultant colored solutions were read in a Klett-Summerson colorimeter. In all cases, glucose standards were run with each set of determinations• k7 Experimental results Reactions or grafted plants» In the first experiment, Irish Cobbler plants, which are known to carry virus X in a latent condition, were approach grafted to S. ^1956. Grafts were cut leaving an Irish Cobbler scion on an immune stock and paired plants were placed at two temperatures, 18° C and 25° C. S. lj.1956 stocks following grafting reacted generally in the same manner at both temperatures. single tops, 10 Of 32 plants with stocks became completely necrotic 18 to 32 days after grafting, and died before completion of the test. Aerial tubers were produced on the remaining 22 plants with Irish Cobbler scions and growth continued to a limited extent. After 75 days, two differences were apparent'(Pig. la). half of the remaining 22 In plants, the scions at first wilted and later became completely necrotic. Necrosis proceeded down the stock 1 or 2 internodes below the graft union. At this point, apical buds proliferated and the stock began to grow vigorously. The balance of the plants in the test which did not become necrotic developed scions which were small and weak, with large aerial tubers in all cases. In these plants, the apical bud which was present above the graft union prolif­ erated greatly. The plant then grew in a normal manner. Roots of all plants which were harvested at the last observation were only slightly, if at all, necrotic. Thus approximately FIGURE 1 A. Scion-stock reaction in plants when virus X^-carrying scions were grafted to immune stocks. Upper left exhibits complete necrosis of scion and stock. Upper center shows necrosis of the scion, and necro­ sis of the stock 2 internodes below the graft. An axillary bud has produced a normal plant. Upper right shows a bifurcated graft in which scion and stock remain vigorous. A small aerial tuber may be seen at the graft. B. Starch accumulation In potato scions grafted to S. 1+1956 stocks. From left to right; 1. Virus X free scion on S. 1+1956. 2. Virus X-carrying Irish Gobbler scion on immune S. 1+1956 26 days after grafting. 3. Immune S. 1+1956 scion on an Erlaine selfed 12 days after the stock had been Inoculated with virus X. 1+. Erlaine selfed scion on S. 1+1956, 12 days after inoculation of the scion with virus X. 1+9 one third of the plants in the test became rapidly necrotic and died, another third became necrotic below the graft but the necrosis did not become general while a third of the group developed aerial tubers. With the possibility that viruses X and Y could react synergistic ally, and break down the immunity of S. i|.1956, a second experiment was carried out at 16° C, 22° C and 28° C soil temperatures. of temperature. Plant reactions were again independent Variations in graft reactions from the above trend differed in only 2 respects. When S. 1|1958 was grafted with virus Y infected Irish Cobbler 16 of 21}. plants developed complete necrosis. It is probable, if the remaining 8 plants had been left for a longer period, they also would have become necrotic. It is believed that this reaction was due in a large measure to the necrotic influence of virus Y. A sim­ ilar situation developed when tomato scions were grafted to a virus Y carrying S. llj.956 stock and later infected with virus X, strain X 5- Stocks of S. I}.1956 became almost com­ pletely necrotic before the tomato scions began to wilt and die. Virus X inoculated tomato scions on a healthy S. I}.195>6 stock occasionally displayed the same phenomenon with slightly less rapidity. Virus X was not isolated from any portions of the above grafted plants. Grafted plants in which only virus X was involved were cut leaving bifurcated tops. Plants of this type 50 continued to grow vigorously throughout the experiment. When virus X only was involved in the grafted plants reactions were usually similar to the previously described experiment. In previous trials it was determined that virus Xinfected scions grafted to an immune stock developed a strong positive test for starch immediately above the graft union and several cm. above when treated with an iodine solution, plants were grafted in a number of combinations to determine the starch reaction when virus X was, or was not present in the graft tissues. A strong positive test for starch was apparent 12 days (Fig. IB) following virus X inoculations of virus Xfree scions, and 76 infected scions. days after grafting with virus X- Positive tests for starch have been observed as early as 10 days following virus X inoculation. Accumulation of starch was not apparent in scions where virus X was absent from grafted plants (Table 1). There was similarity in starch accumulation patterns in the hyper­ sensitive (Epicure) and immune (S. ij.1956) types. When virus X-infected Irish Cobbler scions were grafted to either immune, or hypersensitive stocks, the scions. starch accumulated in The most striking similarity, however, was when virus X-free scions were grafted to both immune fc. Ij.1956) and hypersensitive (Epicure) types and later inoculated with the virus. S. Ij.1956 stocks reacted 31 consistently with iodine to give a positive test fop starch 3 cm. op more below the graft. sionally in the same manner. Epicure stocks reacted occa­ Apparently, delay in inoculation of virus X to the susceptible scions, shifted, the site of starch accumulation from the scion to an area somewhere in the stock. When either D. tatula or tomato was used as the scion and later inoculated with virus X> the same pattern of starch accumulation in stocks of S. J4 I9 S 6 below and above the graft was apparent. Starch accumulated occasionally when virus free scions were grafted to virus X-carrying, or virus X inoculated stocks. A phenomenon hitherto unreported was also noted in this graft test. When scions either carrying virus X or scions free of the virus and later inoculated were grafted to Epicure stocks, either enlarged nodes or enlarged axillary buds suggesting very small aerial tubers were sometimes pre­ sent on the scions. All aerial tubers reacted strongly with iodine indicating the presence of starch. Relative carbohydrate deficiency in the presence of virus X . This phase of the study was undertaken to obtain some information concerning the cause of necrotic roots on immune stocks which had been grafted with virus X free scions and later inoculated with the virus after the graft had become established. Semi-quantitative estimations were made 52 of reducing carbohydrates in roots of such grafted plants using Somogyi* s test (1952) for reducing sugars# For the purpose of this discussion, the phrase rela­ tive carbohydrate deficiency in the presence of virus X, has been used to indicate relative amounts of carbohydrates present in paired grafted plants. The scion of one of the paired plants was free of virus X, while the scion of the other plant was inoculated with virus X after graft estab­ lishment. Since carbohydrate levels were usually low when virus X was present in the scion, the term relative carbo­ hydrate deficiency is used to denote the difference. No relative carbohydrate deficiency in the presence of virus X was observed in roots of approach grafted S. 1|1956 7 days after inoculation with the virus (Table 2). At this time there was some relative carbohydrate deficiency in bifurcated plants. Relative carbohydrate deficiencies were apparent 11+ days after inoculation. When tomato and D. tatula were used as single scions, relative carbohydrate deficiency in the presence of virus X was more apparent than when Erlaine selfed potato clones were used as scions. Relative carbo­ hydrate deficiency with virus X, in roots of plants with bifurcated tops, was essentially similar at the reading as at the 7 1 day day reading. When roots were harvested 21 days after virus X inoculations, relative carbohydrate deficiency was not as •d p© o iri & CPi I to © rd • © • t> P llD •H to s 4 •H P Pi PI © *H Pf *H & (D r— 1 8 8PU o4 A © © o Pi © ^ ♦H Pi n © *a c[ SPd a* © •4 o p P © t Iso —• rH m o ir•^ t 0 s! • rH m in Ios• • o rH CM rH • CM CM O • rH in CM • rH i in n « CM © ?5) a p •H * t» 0 & © W).H CVJ o> 3<8 P £ p {PSi* H to 3w < P4 o & do cd o Pi Pi Pi c8 m rd © flj o u P p »rH ^=* d © & © «3 I © © U p (A Pi O o •rH H OOP o o3 o• cl B P i *H , 3 © P Pi *H © p cJ o • i —1 m 8 tS (C w O • CM CM ■8 O . TOHX P ■ **•3 -S i *d *d p O Pi 01 Vf • VO VO Pi Pi o •H O © in o> Or\ to cr\ e • • o o * © Pi d t o © >d © © p o cfl ■ 5* P O o o CT^ •H Pi o rH © O .g ttf r§ o 0 .7 5 it P rH S d i-h | 51+ great in roots of plants with single tops as in roots of similar plants harvested a week earlier. Relative carbo­ hydrate deficiencies in plants with bifurcated tops was greater at the 21 day period than at either of the earlier analyses. Relative carbohydrate deficiencies with virus X in roots of S. 1*1956 stocks grafted with potato scions were not as great from week to week as were the deficiencies in any comparable treatment with tomato or with D. tatula scions. Trends were similar when the hypersensitive variety Epicure was used as the stock (Table 3). Relative carbo­ hydrate deficiencies with virus X were greater in roots of Epicure than in roots of S. 1*1956 on the same harvest dates (Table 2). furthermore, relative carbohydrate deficiency remained relatively constant 7 to 21 days after inoculation with virus X (Table 3). The relative carbohydrate deficiency with virus X in roots of bifurcated Epicure plants remained fairly con­ stant throughout the test. In this respect the reaction was somewhat different from that of S. U 1956 in the 21 day analysis. Glucose analysis of certain samples from Tables 2 and 3 were made using a colorometric test with commercial glucose oxidase, Glucostat (Tables 1* and 5). Analyses were limited because of insufficient samples for analysis from & > V# * m cvi H • in Hi 9 $ 55 in in £ © O S ? * i1?o4 m • to ^J• CTV to CVJ f3 t o o m in CVJ in C V J to -8 in rH sv in CVJ vo m * © IV • © • t>> u tf l H -s* *H a 3 .1 Of1 CVJ © © © o < +H E ©r ^ iH u ^ © Q t +©> O § •H O • 03 fl S •rt O }*J m • • 1"r— -=f • m to tn € to m vo k\ -= i* OJ v=j o* E> i — cvi m -rH =!• in crv o' © to « © * f> 3* *• <1-4 a a* CVI © A ft EH VO CTV • CTV CVJ © © o © © o• -© • CVJ in CVI o* m vo rn CVI to • © • to m vo « to rH o• o r— ■Sb 0 © VO O <©w rH I — -=r to o ov to vo vo P{ © •rl ra +3 © 1 •O34 © © Fh « i> m to Cf< © © 4 Table 3* Carbohydrate reserves in roots of the variety Epicure grafted to scions either free of virus X or inoculated with the virus l/ © «HI H© F h^ Q J U 3 «i*©d © © o © oh 5 m CTv VO •©d +» fij o P4 & £u •H ^=> rH -a VO to CTV • o © o P* & « © "5 g •r ) m LT\ ■s© o u & 9 in to• CTV © © & o IP w © b *H h o -R T©i •R O • t•> © f*P«rj o* o EH rH t^s o m C vJ © P g ©i e 2 « Pi © % © «H 01 O o Q © P •> & ePTo1 J "wJsj i © rr} Pi Cil > n P\ rH -d* CD *d CD CD < W a CT1 •rH d 03•rH 0 rH 0> 0 t o o m d •rl 0 o u *>

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