Interactions between strains of squash mosaic virus in pumpkin and cantaloupe plants.

Transcription

1 Interactions between strains of squash mosaic virus in pumpkin and cantaloupe plants. Item Type text; Thesis-Reproduction (electronic) Authors Lima, José Albersio de Araújo, Publisher The University of Arizona. Rights Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 19/07/ :50:54 Link to Item

2 INTERACTIONS BETWEEN STRAINS OF SQUASH MOSAIC VIRUS IN PUMPKIN AND CANTALOUPE PLANTS by Jos6 Albersio de Aratijo^Lima A Thesis Submitted to the Faculty of the DEPARTMENT OF PLANT PATHOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA

3 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special perm ission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: MERRITT R. NELSON Professor of Plant Pathology Date mz.

4 ACKNOWLEDGMENTS The author w ishes to express his sincere gratitude and appreciation for the counsel and helpful guidance of Dr Merritt R. N elson, Professor of Plant Pathology, The University of Arizona, under whose leadership this investigation was conducted. Appreciation is extended to Dr. Milton Zaitlin and Dr. Richard B. Hine for their useful advice and efforts in criticizing the m anuscript. The author also thanks Mr. Wilbur R. Hague for his helpful contributions in the greenhouse w orks. Gratitude is extended to Dr. William G. M atlock, Campus Coordinator, AID Brazil Program, The University of Arizona/University of Ceara Contract, and to his secretary, M rs. Evelyn Jorgensen, for their understanding and cooperation. The author was supported by funds from the United States Agency for International Development (US AID), The University of Arizona/University of Ceara C ontract, to whom sincere appreciation is expressed. Special recognition is given to my wife, D iana, whose patience and support made this work possible.

5 TABLE OF CONTENTS LIST OF ILLUSTRATIONS v LIST OF TABLES... vi ABSTRACT vii INTRODUCTION AND LITERATURE REVIEW MATERIAL AND METHODS Page Virus Strains and Hosts Virus Purification and Inoculation Methods C ross-protection Experiments between Strains of SMV in Pumpkin and Cantaloupe Squash M osaic Virus Strain Dominance in Simultaneously Inoculated Plants Concentrations of Strains of Squash M osaic Virus in Pumpkin and Cantaloupe RESULTS Virus Strains and Hosts Cross-protection between Strains of SMV in Pumpkin and Cantaloupe Squash M osaic Virus Strain Dominance in Simultaneously Inoculated Pumpkin and Cantaloupe Plants Squash M osaic Virus Strain Concentration in Pumpkin and Cantaloupe DISCUSSION REFERENCES " iv

6 LIST OF ILLUSTRATIONS Figure Page 1 Systemic Symptoms in Leaves of Cucurbita pepo L. Inoculated with Strains of Squash M osaic Virus Symptoms of Strains of Squash M osaic Virus in CuCumis melo L Local Lesions on Cotyledons of Citrullus vulgaris Schrad. C aused by IH Strain of Squash M osaic Virus Results of Intragel Cross-absorption Tests with Purified SMV Suspension Obtained from Pumpkin Plants Inoculated First with IIA and 10 Days Later with IH Strains v

7 LIST OF TABLES Table Page 1 Dominance of One SMV Strain over the Other When Different Concentrations of IH and IIA Strains Are Simultaneously Inoculated into Pumpkin and Cantaloupe Plants The Relative Concentrations of IH and IIA Strains of Squash M osaic Virus in Pumpkin and C antaloupe Plants Grown in Greenhouse and Growth Chamber v i

8 ABSTRACT Interactions between strains IH and IIA. of squash mosaic virus (SMV) in pumpkin (Cucurbita pepo L ) and cantaloupe (Cucumis melo L.) plants were studied. Complete reciprocal cross-protection was observed in pumpkin. In cantaloupe, strain IH was able to overcome any initially suppressive effect of IIAi This phenomenon in cantaloupe is believed related to the fact that strain IH was determined to be capable of multiplication to twice the extent of strain IIA. In pumpkin, the relative extent of multiplication of the two strains is roughly equal. The detection of cross-protection, when the challenge strain is the milder of the two, was accomplished by utilizing host range differences and the intragel absorption technique, When opposite cotyledons of pumpkins were inoculated simultaneously with equal concentrations but different strains of purified v iru s, each strain dominated in roughly 50 percent of the plants to the exclusion of the other. When the concentration of one strain was reduced in relation to the other, the number of double-inoculated plants in which it dominated was reduced proportionately. Identical experiments with cantaloupe resulted in almost complete dominance of strain IH even when one-tenth of the concentration of strain HA was used.

9 INTRODUCTION AND LITERATURE REVIEW Biological properties of viruses can and have been used extensively to determine the relationship between strains of plant viruses. According to Matthews (1970), differences and sim ilarities in the biological properties of virus isolates are probably related to functions of the viral genome not involved with viral coat protein synthesis. Among the biological properties of v iru ses, cross-protection studies have been widely and successfully used by several workers to demonstrate identity and strain relationships of some plant v iru ses. The phenomenon in which plant tissu es infected with one strain of a virus are protected against related strains of the same virus was first observed by McKinney (1929). Since then the interaction between virus strains in plant tissu es has received wide attention. Ainsworth (1934) compared potato viruses isolated from Canadian tomato material with standard English m aterial. The fact that mild strains conferred immunity in tomato against a virulent strain was considered as strong evidence of close relationship of the two iso lates. Kunkel (1934) demonstrated that leaves of N icotiana sylvestris Spegaz. and Comes mottled by tobacco mosaic virus (TMV) strains were protected against the aucuba mosaic strain of TMV. He also showed that viruses unrelated to TMV gave no protection against the aucuba. mosaic strain of TMV, suggesting a certain degree of specificity in the protection process. Similar results were obtained by Price (1935). He observed that leaves of Zinnia elegans Jacq. system atically invaded by 1

10 any one of four different mottling strains of cucumber mosaic virus (CMV) were protected against a strain of CMV which produced necrotic lesions in healthy zinnia plants but not against a necrotic-type TMV strain. He also found that leaves of plants mottled by TMV strains became immune from the necrotic-type TMV but not from a necrotic-type CMV. He further demonstrated the specificity of the protection action in zinnia for both CMV and TMV by demonstrating that plants infected with viruses unrelated to CMV or TMV were susceptible to the necrotic-type strains of b oth. The results obtained by Price (1936) evidenced the specificity of acquired immunity from tobacco ring spot d isea ses. Strains of tobacco ring spot virus protected tobacco plants against each other but did not confer protection against unrelated viruses. From cross-protection studies between lily mosaic virus and CMV in zinnia p la n ts, Price (1937) concluded that the two viruses were closely related and that lily mosaic virus should be classified in the cucumber mosaic virus group. Again, Price (1941) observed that zinnia plants infected with Hawaiian commelian mosaic virus were protected against the indicator strain of CMV, thus demonstrating that the virus was a strain of CMV. Using insect inoculation and graft transm ission, Crowdy and Posnette (1947) carried out experiments on cross-immunity reactions between viruses attacking Theobroma cacao L. The results revealed some degree of protection afforded by one virus against infection with the other.

11 3 Strains of alfalfa mosaic virus (AMV) have been identified bycross-protection te s ts. The results obtained by Berkeley (1947) in tobacco plants indicated that a pepper virus was closely related to the AMV. Oswald (1950) used cross-protection studies to identify a strain of AMV which was causing vine and tuber necrosis in potato. Because common bean mosaic and yellow mosaic viruses were very similar in dilution end point, thermal inactivation, longevity in vitro, and protected against one another in bean varieties, Grogan and W alker (1948) concluded that they were closely related viruses. Corbett (1957) observed that when Crotalaria spectabilis Roth system atically infected with a strain of bean yellow mosaic virus was inoculated with a necrosis-producing strain good protection was evidenced. On the other hand, lack of protection between a strain of bean yellow mosaic virus and tobacco ring spot virus indicated some specificity on the protection action. The results of cross-protection studies obtained by Silbernagel (1969) provided evidence that a Mexican virus isolated from the bean line, Phaseolus vulgaris L., was a strain of bean common mosaic v iru s. Ross (1948) reported that, although mild strains of potato virus X produced no marked symptoms on Physalis floridana Rydb., they could protect the plants against severe strains of the v iru s. Matthews (1949) studied the relationships between strains of potato virus X. He found a correlation between the degree of crossprotection among strains in tobacco and Datura tatula L. and serological relationships. Siegel (1959) observed that the number of local lesions produced by a necrotic strain of TMV (Hg) on 14. sylvestris was inhibited by the

12 presence of a system ic strain (U^) in the inoculum. These two strains of T,MV were used by Wu and Rappaport (1961) to study competition in vulgaris var. (pinto bean). The strain produced local lesions on the primary leaves of bean plants whereas the related TMV strain U"2 caused no demonstrable symptoms. They observed that when strain Uj was inoculated mixed with Ug strain, the number of Uj lesions was inhibited. Their data were similar to those reported by Siegel (1959) and suggested that the inhibitory effect was the result of an active competition between the two strains of TMV for the same infection site or loci within cells. Wu and Hudson (1963) studied the interference between TMV and tobacco necrosis virus (TNV) in the initiation of infection. They observed that the presence of an excess of TNV in a TMV inoculum did not interfere with TMV lesion formation. The lack of interference between unrelated plant viruses suggests that unlike related viruses, they do not utilize the same site to institute infection. Varney and Moore (1952) suggested that an elm mosaic virus was closely related to tomato ring spot virus (TmRSV). In addition to sim ilarities in host range and physical properties, these investigators demonstrated unilateral protection between the two viruses. Although Fulton and Fulton (1970) confirmed these cross-protection results, serological and vector relationship studies indicated that the two viruses were distinct. Cross-protection te sts in Petunia hybrida Vilm. showed close relationship between yellow bud mosaic and TmRSV (Cadman and Lister, 1961). In contrast, they observed that neither peach yellow bud mosaic nor TmRSV protected. hybrida plants from infection with tobacco ring

13 ' 5 spot virus, These results were confirmed by serological tests reported in the same paper. Jedlinski and Brown (1965) carried out cross-protection experiments with three strains of barley yellow dwarf virus (BYDV) in Avena sativa L. under field conditions. They observed that plants infected with a mild strain of BYDV were protected against subsequent invasion of severe strains of the same v iru s. Corbett and Price (1967) showed that citrus plants infected with psorosis virus were not protected against local and system ic infection by citrus variegation v iru s, thus suggesting that psorosis and infectious variegation were caused by distinct viruses. N elson, Mate jka, and McDonald (1965) reported that cro ss- protection tests of a strain of squash mosaic virus (SMV) with cucurbit viruses other than SMV failed to reveal any relatio n sh ip s. Although most of the cross-protection studies between strains of viruses performed by different investigators have shown cross- protection, some have failed to reveal any degree of protection. On the other hand, a certain degree of interference has been demonstrated between unrelated viruses. Silberschmidt (1957) observed a lack of protection in tomato plants between strains of potato virus X and pointed out certain limitations of the value of cross-protection te sts for taxonomical purposes. Tu and Ford (1969) could not find any cross-protection between strains of maize dwarf mosaic virus in corn, Zea mays L. Paulsen and and Sill (1969) also reported negative cross-protection results between strains of maize dwarf mosaic virus in grain sorghum. Varney and Moore (1952) and Fulton and Fulton (1970) observed unilateral cross-protection

14 between serologically distinct viruses Bawden and Kassanis (1945) found that plants infected with severe etch virus were protected against subsequent infection with potato virus X, which was serologically unrelated to the former. N evertheless, they pointed out that the phenomenon is quite different from the reciprocal protection, which is so su ccessfully and widely used to test whether viruses.causing different symptoms are related stra in s. Although the mechanism to account for the phenomenon of cro ssprotection in plants remains undetermined, several theories have been put forward as to what underlies the protective p ro c ess. One of the earliest accounts of a protective effect between plant virus strains was put forward by Salaman (1933), who observed a certain degree of protection between two strains of potato virus X when a nonvirulent strain was inoculated before a virulent o n e. He observed that the secondarily inoculated virulent strain was completely excluded, and he suggested that once the plant cell had formed a symbiotic union with the nonvirulent strain, it had no capacity to enter into relations with any other strain of the same v iru s. Some investigators have suggested a certain degree of compe-. tition between plant virus strains for limited amount of material from which viruses reproduce. The first strain should use the same essen tial metabolites required for the second to m ultiply. Sadasivan (1940) suggested that the degree of protection between plant virus strains is directly proportional to the number of active units of the protecting strain present in the leaf tissue at the time of re inoculation. Critical analysis of reported cases in which one strain of virus protected plant tissues

15 against infection by related strains led Valleau (1941) to the conclusion that the cross-protection effects could be satisfactorily explained on the basis of competition between virus strains for the same building blocks essential for strain replications. Giddings (1950) suggested that the apparent resistance observed in susceptible beets already infected with one strain of curly top virus to infection by another strain could be acaccounted for on the basis of competition for materials essential for virus m ultiplication. Hageman (1964) studied the interaction between strains of TMV in tobacco leaves and suggested a precursor exhaustion theory to explain the degree of protection observed between U 2 and green aucuba strains He observed that when discs from fully protected U2 -infected leaves were floated on nutrient solution, the protection decreased as the precursor exhaustion replenished, whereas if these discs were floated on tap w ater, they remained fully protected. Best (1954), working with tomato, observed that a severe strain of tomato spotted wilt virus produced a significant reduction on the yield of fruit and bush after a mild strain of the same virus had become thoroughly established throughout the plant. On the other hand, he observed that these double-inoculated tomato plants yielded significantly more fruit and bush than the controls inoculated with the severe strain alone. These results led him to the conclusion that a new strain was formed by genetic recombination with severe and mild strains of tomato spotted w ilt virus in the double-infected tomato plants. He observed that the new strain was intermediate in severity between the mild and severe strains and pointed out that this mechanism could account for the protection effects observed between tomato spotted wilt virus strain s. The result obtained by Thomson (1961) indicated that from mixed infection

16 of Nicotiana glutinosa L with two strains of potato virus X a new strain could be obtained. Another possible explanation for cross-protection was suggested by Kavanau (1949), who put forward the idea that plant protection could be conferred by the persistence of the first inoculated virus strain in relative inactive aggregates which have specific adsorptive properties. These aggregates had the ability to protect the plant against a second related strain. Additional virus strains introduced into the protected plant were adsorbed to the aggregates under the action of specific a t traction forces and inactivated by the association. The immunity phenomenon so characteristic of animals has not been observed in p la n ts, and there is no evidence that antiviral antibodies are produced in p la n ts. However, the possibility that protection between strains of plant viruses might be explained with the mechanism that the earlier replication of the first inoculated strain could cause metabolic changes releasing inhibiting substance has been suggested by Helms (1965). The author worked with Uj and Hg strains of TMV in leaves of vulgaris v ar. Pinto and suggested that multiplication of Uj was inhibited as a result of metabolic changes induced in cells surrounding U"2 infections. Finally, the theory that related virus strains combine with and multiply at the same site whereas different viruses combine, with and multiply at different Sites was advanced by Bawden and Kassanis (1945). To explain the cross-protection effects observed with severe and mild etch virus strains, potato virus Y, andhyoscyamus virus 3, they suggested that when a site in a susceptible cell was already occupied by

17 one strain, a second strain of the same virus, requiring the same site, could not be able to attach itself and m ultiply. This idea has been supported by other investigators. Siegel (1959) and Wu and Rappaport (1961) suggested that the cross-protection phenomenon with plant viruses was the result of competition between strains for the same site within cells However, no clear idea has yet been put forward to explain what sites in the cells might be specific for each virus replication and what virus part would be involved, or for that matter what the site itself consists of. Physical properties, host range, symptoms, insect transm ission, and serological studies have been used to identify squash mosaic virus (SMV) (Takahashi and Rawlins, 1947; Freitag, 1952; and Lindberg, H all, and W alker, 1956). Nelson et al. (1965) and Knuhtsen and Nelson (1968) differentiated two groups of SMV strains, one which infected watermellon (group I) and one that did riot. Purified preparations of SMV showed three distinct schlieren peaks in the ultracentrifuge, but only one peak upon electrophoresis at different ph values (Rice et a l., 1955). In the same study they also observed that under the electron microscope some of the SMV particles appeared as polyhedrons. Squash mosaic virus has been reported to be both bettle transmitted and seedborn. Freitag (1941) first reported that the virus could be transmitted either by aphids or by cucumber b eetles, but only inefficiently by aphids. In subsequent stu d ie s, Freitag (1952 and 1956) concluded that SMV was beetle but not aphid transm itted. Bitterly (1960) demonstrated that Diabrotica balteata LeConte, the banded cucumber beetle, was a new and efficient vector of SMV.

18 . : 10 Middleton (1944) observed that the virus was transm itted in squash seed and demonstrated that poor-quality seed carried a higher percentage of SMV than did good-quality seed taken from the same seed population. Grogan, H all, and Kimble (1959) observed that, although SMV was commonly transm itted by -commercial seed of cucurbit sp ecies, it was more prevalent in areas of California where cucumber beetles were more common. The results obtained by Powell and Schlegel (19 70) indicated that seed transm ission of SMV was influenced not only by virus invasion of the seed but also by host-pathogen relationship during seed formation, storage, seed germination, and seedling development. Nelson and M atejka (1963) isolated an apparently unidentified virus from cucurbits in Arizona. Subsequent studies indicated that the Arizona isolate was a strain of SMV which caused a severe systemic infection in watermelon (Nelson et a l., 1965). They observed that, a l though the Arizona isolate produced symptoms on several cucurbits quite different from those of common SMV (group II), its physical properties were very similar to those previously described for SMV (Lindberg et a l., 1956; Freitag, 1956). Serological studies showed a close relationship between the common (group II) and the watermelon (group I) strains of SMV and also indicated that there is a certain degree of antigenic difference between these two groups of SMV strains (Nelson et al., 1965; and Knuhtsen and N elson, 1968). Nelson and Knuhtsen (1969) observed a differential seed transmission between both groups of SMV stra in s. Their data showed that group I was more efficiently transm itted by seed of several cucurbits,

19 11 which they considered important in the epidemiology of the diseases caused by these SMV stra in s. In the present study, a series of experiments was designed to observe the relationship between strains of SMV in cucurbit p lan ts. The serological intragel cross-absorption technique used by Nelson and Knuhtsen (1972) to reveal the presence of two serotypes of SMV was used in this study to demonstrate cross-protection between strains of SMV where symptoms were inadequate. No previous report indicates the utilization of this serological technique to show cross-protection between strains of plant v iru se s. An attempt was made to correlate the results obtained in cross-protection tests with the relative concentra-. tions of SMV strains in pumpkin and cantaloupe p la n ts.

20 MATERIAL AND METHODS Virus Strains and Hosts Two squash mosaic virus isolates were used in this study: strain I isolate H originated from infected Colorado cucurbit seeds, and strain II isolate A originated from a wild cucurbit in W isconsin. They represent the two serological groups of SMV strains (Nelson afid Knuhtsen, 1972) Both isolates were obtained from Merritt R Nelson of the Plant Pathology Departm ent, The University of Arizona These two strains of SMV Were propagated in a greenhouse either in plants of small sugar pumpkin, Cucurbita pepo L., or cantaloupe, Cucumis melo L. The plants u se d, Cucurbita pepo L., Cucumis melo L., and Citrullus vulgaris S ch rad., were grown from seeds in 4-inch plastic pots with a soil mixture of sand and peat in a proportion of 2:1 plus fertilizer. M ost of the experiments were conducted during the summers. of 1971 and 1972 in a greenhouse at The University of A rizona, Tucson. The greenhouse temperature ranged from 25 C (average night temperature) to 32 C (average day tem perature). The greenhouse was fumigated at frequent intervals to control in se c ts. The experiments carried out during the cold seasons were conducted in a growth chamber adjusted to a daytime of 14 hours and night and day temperatures of 25 C and 30 C/ respectively. During the daytime period, the growth chamber was kept under continuous illumination by incandescent and fluorescent lamps producing approximately 2, foot candles at the level of the pots. 12

21 13 Virus Purification and Inoculation Methods The precipitation of plant protein by butyl alcohol method and the precipitation of virus by polyethylene glycol 6,000 (PEG) method (Hebert, 1963) were used in the virus purification p ro c e ss. Fifty to 100 gm of infected leaves were homogenized in a blender with two volumes of 0.1 M phosphate buffer ph 7.0. The resu lting extract was strained through a double layer of cheesecloth and subsequently clarified by centrifugation for 20 minutes in a VRA rotor at 10,000 rpm in a Lourdes Model A-2 B eta-fuge. The pellet was discarded and enough butyl alcohol was added to the supernatant to make a final concentration of 8 percent. This mixture was stirred for 20 to 30 m inutes, and coagulated green debris obtained was removed by centrifugation for 20 minutes at 10,000 rpm in a VRA rotor. For precipitation of virus, while stirring, PEG 6,000 and NaCl were added to a final concentration of 8 percent and 4 percent (w/v), respectively. After stirring for 30 to 40 m inutes, the precipitate was sedimented for 30 minutes at 10,000 rpm in a VRA rotor of the Lourdes centrifuge. The resulting pellet was re su s pended in 0.1 M phosphate buffer ph 7.0 and clarified by centrifugation for 10 minutes at 10,000 rpm in the same rotor. The PEG precipitation was repeated two more tim es, using a 9RA rotor, to concentrate the virus. The concentration of purified viral suspensions was determined with the optical density obtained at 260 hm wavelength in a Beckman DB-G Grating Spectrophotometer. Using an extinction coefficient of 7, the virus concentration of each strain was calculated by this formula: ^..,.. Optical Density at 260 nm. Concentration (mg/ml) Extinction Coefficient x Dilution Factor

22 14 The viral concentrations were obtained in milligrams of virus per m illiliters of purified preparations and converted to milligrams of virus nucleoprotein per gram of fresh weight of leaf tissu e The method of inoculation used in the experiments was the same in all cases Carborundum was added to purified virus suspensions or crude sap, and inoculations were performed by rubbing the SMV inoculum over the adaxial surface of cotyledons or true leaves of the plants with a brush C ross-protection Experiments between Strains of SMV in Pumpkin and Cantaloupe Cotyledons of 30 seedlings of pumpkin or cantaloupe were inoculated with a te st strain (IH or IIA) and ten days later, when these plants had developed symptoms, the youngest leaves of half of them were inoculated with the challenge strain. At the time of second inoculation, 15 uninoculated plants of the same age were also inoculated and 15 plants w ere.left uninoculated. All experiments were repeated at least tw ice. All inocula used in these experiments were constituted of purified virus suspensions in which the concentrations were adjusted to 0.1 or 0.5 m g/m l. A cross-protection experiment was carried out with pumpkin plants to determine if HA (severe, strain in pumpkin) could protect the plants against IH (mild strain in this h o st). The IIA strain of SMV was inoculated to seedlings of_c. pepo plants. Ten days la te r, when the inoculated plants were exhibiting severe symptoms characteristic of IIA in this host, they were superinoculated with IH. In order to determine whether IH had infected and multiplied in these IIA-infected p la n ts, the.

23 15 youngest leaves were ground in a sterile mortar using one volume of 0.1 M phosphate buffer ph 7.0 for each gram of fresh leaf, 10 and 20 days after the challenge inoculation. The extract was strained through two layers of cheesecloth and inoculated on the cotyledons of groups of watermelon, pumpkin, and cantaloupe seed lin g s. Three other batches of watermelon and cantaloupe seed lin g s, to serve as controls, were inoculated with IH, IIA, and a mixture of IH and IIA. At the same tim e, purified viral suspensions were also obtained from the youngest leaves of these double-inoculated pumpkin plants. To determine the presence or absence of IH antigen in these purified preparations, they were a s sayed in w aterm elon, pumpkin, and cantaloupe plants and were tested in serological reactions. Two intragel absorption tests were carried out, using IH and IIA antisera obtained from Merritt R. N elson. Fifteen m illiliters of 1.0 percent Ion Agar II (Consolidated laboratories, Chicago H eights, Illinois) dissolved in g lass-d istilled water with 0.85% NaCl and 1:5000 w /v sodium azide added as a weak preservative were poured into each 85x15 mm plastic petri dish and allowed to harden overnight. W ells were punched into the agar with cork borers in a hexagonal arrangement. The hexagonal arrangement consisted of a center w ell, 6 mm in diameter, w ith six peripheral w ells, 4 mm in diameter, spaced 10 mm from the center well as measured from the centers of the w e lls. In the. first te st, a high concentration (2.0 mg/ml) of IH antigen was placed in the center w ell, and 24 hours later the IIA antiserum was added in the same w ell. At the same time, IIA antigen and purified virus suspension obtained from double-inoculated pumpkin plants were alternated in the six outer wells.

24 16 A similar te st was carried out as follows: 24 hours after a high concentration of IIA antigen had been put in the center w ell, IH antiserum was added, and IH and the purified virus suspension from pumpkin were alternated in the six outer w e lls. After the wells were filled with the re ac tan ts, the plates were incubated for 1 to 5 days at room tem perature. The development of precipitation patterns was observed by looking at the p la te s, which were illuminated from the bottom, and photographs taken. All antigens used in these tests consisted of purified virus suspensions in which the concentrations were adjusted to 1 or 2 mg of virus per m illiliter of purified suspension. Squash M osaic Virus Strain Dominance in Simultaneously Inoculated Plants Two further experiments were designed to observe, interaction between the strains of SMV in pumpkin and cantaloupe plants when inoculations were made sim ultaneously. In each experiment, the IH and IIA strains were inoculated on opposite cotyledons of a,group of 100 seed lin g s. Purified virus suspensions were used as inocula, and final virus concentrations were adjusted to 0.1 mg, of virus per milliliter of purified preparation. \ " To observe the effect of inoculum concentration on strain dominance, experiments were conducted with both cantaloupe and pumpkin in which concentrations of one strain was varied from 0.1 mg/ml to mg/ml while the concentration of the other strain was maintained at 0.1 mg/ml.

25 17 Concentrations of Strains of Squash M osaic Virus in Pumpkin and Cantaloupe This experiment was devised to try to find some degree of correlation between the results obtained in cross-protection studies and concentrations of IH and IIA in pumpkin and cantaloupe p la n ts. The experiment with cantaloupe was first conducted in the greenhouse and repeated in the growth chamber previously described. Both replications with pumpkin were carried out in the greenhouse. ' Four groups of 20 p lan ts, two of pumpkin and two of cantaloupe, were used. One group of each cucurbit was inoculated with an inoculum containing 0.5 mg of IH strain of SMV per m illiliter of purified virus suspension, and an approximately equal amount of IIA was inoculated into the other two groups of p la n ts. Ten days after inoculation, 50 gm (wet weight) of system ically infected leaves of each group of plants were separately harvested, and virus strains were purified as described previously. Great care was taken to duplicate the purification procedure in each group. Each final viral precipitate was resuspended in 20 ml of 0.1 M phosphate buffer ph. 7.0, and after final clarification the optical density was determined using a Beckman DB-G Grating Spectrophotometer and calculations made as previously described.

26 RESULTS ) Virus Strains and Hosts The virus strains were readily differentiated on the basis of symptoms produced on pumpkin, cantaloupe, and watermelon. The symptoms induced by IH and IIA strains of SMV in these hosts were observed to be as described for I and II groups of SMV strains by Nelson and Knuhtsen (1972). The IIA strain produced a severe system ic mosaic accompanied by severe leaf distortion in pumpkin (Figs, 1-D and 1-E). The symptoms appeared 5 to 7 days after inoculation as small chlorotic pinpoints. In the same host the IH strain induced a mild mosaic often beginning with characteristic system ic chlorotic rings 5 to 7 days after inoculation (Figs. 1-B and 1-C). Both strains also produced system ic symptoms in cantaloupe, _C. m elo. The IH strain induced an initial characteristic green vein banding (Fig. 2-C) with subsequent chlorotic mottle. Leaf distortions, as shown in Figure 2-D, could also occur in IH -infected leav es. On the other hand, the strain IIA produced only a very mild m osaic pattern on this host (Fig. 2-B) Watermelon was not susceptible to the IIA strain of SMV, but local necrotic lesions developed on the cotyledons or true leaves when they were rubbed with inoculum containing strain IH (Fig. 3). This symptom in watermelon was first reported by Grogan et al. (1959). 18

27 Figure 1. Systemic Symptoms in Leaves of Cucurbita pepo L. Inoculated with Strains of Squash M osaic Virus A. Healthy leaf. B. Leaf system ically infected with strain IH showing characteristic systemic chlorotic rings.. C. Leaf inoculated with strain IH showing a mild generalized m ottle. D. Leaf system ically infected with IIA strain showing a severe m osaic. E. Severe leaf distortion induced by strain IIA.

28 Figure 1. Systemic Symptoms in Leaves of Cucurbita pepo L. Inoculated with Strains of Squash Mosaic Virus i CD

29 Figure 2. Cucumis melo L. Symptoms of Strains of Squash M osaic Virus in A. Healthy leaf. B. Leaf infected with strain HA showing a mild mottle pattern. C. Green vein banding induced by IH strain. D. Leaf distortion induced by strain IH.

30 Figure 2. Symptoms of Strains of Squash M osaic Virus in Cucumis melo L. 20

31 Figure 4. Results of Intragel C ross-absorption Tests with Purified SMV Suspension Obtained from Pumpkin Plants Inoculated First with IIA and 10 Days Later with IH Strains Antisera to IH and IIA were those used by Nelson and Knutsen (1972); each had been shown to contain a significant proportion of strainspecific antibodies. A. Central well filled initially with IH antigen, then 24 hours later with IIA antiserum (as. II). Well I = IH antigen; well X = purified SMV suspension obtained from double-inoculated pumpkin p la n ts.. B Central well filled initially with IIA antigen, then 24 hours later with IH antiserum (as. I). W ell II = IIA antigen; well X = purified SMV suspension obtained from double-inoculated pumpkin p la n ts.

32 Figure 3. Local Lesions on Cotyledons of Citrullus vulgaris Schrad. Caused by IH Strain of Squash Mosaic Virus Figure 4. Results of Intragel Cross-absorption Tests with Purified SMV Suspension Obtained from Pumpkin Plants Inoculated First with IIA and 10 Days Later with IH Strains

33 Demski (1969) pointed out. the value of these hypersensitive reactions in _C. vulgaris for virus bioassay. 22 Cross-protection between Strains of SMV in Pumpkin and Cantaloupe The results of cross-protection studies with strains of SMV in : pumpkin and cantaloupe plants were variable. In pumpkin experiments, a complete reciprocal protection was observed, while the cross- protective effect in cantaloupe plants was unilateral. Leaves of pumpkin plants inoculated with strain IH showed a mild mottle 10 days after inoculation. When such leaves were rubbed with inoculum containing the IIA strain of SMV, no severe mosaic developed, although a like inoculation of previously uninoculated plants resulted in the production of typical severe symptoms of this strain. C ross-protection was also observed when pumkin plants were first inoculated with IIA strain and IH was the challenge virus strain. No evidence of IH in the double-inoculated pumpkin plants was observed when extract from these plants was bioassayed in watermelon, pumpkin, and cantaloupe seedlings. Cotyledons of watermelon inoculated with crude sap or purified virus preparation from these double-inoculated pumpkin plants did not develop any le sio n s. However, when similar Cotyledons were inoculated either with strain IH alone or a.mixture of IH and IIA strains, necrotic lesions were produced. Similarly, cantaloupe plants inoculated with extracts from these double - inoculate d pumpkin plants did not show a green vein banding but only a mild mottle, while a severe m osaic developed on all pumpkin plants inoculated with similar inoculum.

34 23 These results were confirmed by serological tests carried out with purified virus suspensions obtained from these double - inoculate d pumpkin plants. The absence of IH antigen in the purified virus suspension was demonstrated in intragel absorption tests shown in Figure 4. A complete cross-absorption was observed in both serological tests. When the IIA antiserum was used, the heterologous IH antigen, which was first placed into the antiserum w ells, cross-reacted with an d.fully precipitated the cross-reacting antibodies at the region of optimal proportions close to the center w e lls. The outer wells with the IH antigen did not show any reaction evidencing a complete cross-absorption. On the other h an d, bands were observed close to the outer w ells with the purified virus preparation, indicating the presence of IIA strain in this SMV suspension (Fig. 4-A). When the center wells were initially filled with IIA antigen and 24 hours later with IH antiserum, different results were observed. Neither IIA antigen nor the purified preparation placed into the outer w ells showed any reaction (Fig. 4-B). These results evidenced a complete cross-absorption and indicated that the strain IH was not present in the purified SMV suspension obtained from the double-inoculated pumpkin p la n ts. Mildly mottled cantaloupe leaves infected with the systemic IIA strain of SMV were not protected against infection by IH since a green vein banding developed on IIA-infected plants as well as on the control plants inoculated with IH strain alone. N evertheless, in the double-inoculated plants, the appearance of IH symptoms was delayed 3 to 4 days in relation to the inoculated controls.

35 24 This raised the question of why strain IIA protected pumpkin but not cantaloupe plants against infection by IH. Other experiments were set up to observe the relationship between these protective effects, or lack of them, and the concentrations of the virus strains in pumpkin and cantaloupe plants at the time of challenge inoculations. Squash M osaic Virus Strain Dominance in Simultaneously Inoculated Pumpkin and Cantaloupe Plants When pumpkin plants were simultaneously inoculated with equal concentrations of IH and IIA strains of SMV on opposite cotyledons, 52 to 55 percent of these plants came down with severe m osaic accompanied by leaf distortions, while 45 to 48 percent showed only a mild m ottle. These results showed no consistent dominance of either strain when used at the same concentration. As the concentration of the IH strain was decreased while the IIA concentration was maintained constant, the number of plants showing the IH symptom decreased with decrease in IH concentration (Table 1). Similarly, when the IH concentration was kept. constant, the number of plants with severe mosaic (IIA symptom) decreased with decrease in IIA concentration (Table 1). A similar experiment performed by Cohen et al. (1957) with and \J 2 strains of TMV in Nicotiana tabacum L, indicated that the Uj dominance in simultaneously mixed inoculated plants increased with H^-Hg inoculum.ratio. The experiments performed with cantaloupe produced different re s u lts. Plants simultaneously inoculated with the same concentrations of both strains on opposite cotyledons showed almost complete dominance

36 Table 1 Dominance of One SMV Strain over the Other When Different Concentrations of IH and IIA Strains Are Simultaneously Inoculated into Pumpkin and Cantaloupe Plants (Both IH and IIA strains of SMV were simultaneously inoculated onto different cotyledons of each pumpkin and cantaloupe seedling.) Percentage of Plants Showing SMV-symptoms Inoculum Concentrations (mg/ml) a Pumpkin Plants Cantaloupe Plants IH Strain IIA Strain ih-symptom IIA-symptom IH-symptom IIA-symptom 10-4 io -i 10% 90% 5% 95% io -i 30% 70% 40% 60% CO 1 o I 1 i i 1 O r i 40%. 60% 65% 35% 10"1 I 1 o 1 h-j 45% 55% 90% 10% r 1 I o " 2 55% 45% 90% 10% % 20% 95% 5% o r i % 5% 100%. 0% a. Inoculum concentrations in mg of virus per ml of purified virus suspensions

37 26 of the IH over the IIA. strain. Ninety to 92 percent of these sim ultaneously double-inoculated cantaloupe plants showed a green vein banding and severe mottle (IH symptoms), while only 8 to 10 percent came down with a mild mottle. The results obtained with plants simultaneously inoculated with different concentrations of SMV strains are shown in Table 1. The strain IH suppressed the IIA even when IH concentration was ten times lower than the concentration of IIA inoculum. Squash M osaic Virus Strain Concentration in Pumpkin and Cantaloupe In each experiment, the virus strain concentration in system i- cally infected leaves was determined 10 days after inoculation. The IH strain of SMV multiplied to a greater extent than the IIA in cantaloupe p la n ts, since the results shown in Table 2 revealed that the IH concentration was about twice the concentration of IIA. On the other hand, the concentrations obtained in pumpkin plants suggested that both strains of SMV multiply equally in this host (Table 2).

38 27 Table 2. The Relative Concentrations of IH and IIA Strains of Squash. M osaic Virus in Pumpkin and Cantaloupe Plants Grown in Greenhouse and Growth Chamber Virus Concentrations (mg/gm wet weight) a First Experiment Second Experiment Host. IH Strain IIA Strain IH Strain IIA Strain Pumpkin Cantaloupe b 0.27 b a. Virus concentrations determined spectrophotometrically and converted to mg of virus per gm of fresh infected leaves b This experiment was conducted in the growth chamber previously described because of low nighttime temperatures in the greenhouse

39 DISCUSSION The combination of butyl alcohol and PEG 6,000 methods used to purify squash mosaic virus resulted in high yields of purified virus suspensions with high infectivity. Virus concentrations determined spectrophotometrically ranged from 0.10 to 0.52 mg of virus per gram (wetweight) of system ically infected tissu e. C ross-protective results obtained during the course of this study confirmed that a close relationship exists between the two groups of SMV strains. The absence of cross-protection observed in.cantaloupe plants does not invalidate this relationship, since Loebenstein (1972, p. 46) stated that "the absence, or low order of cross-protection should not be regarded as conclusive, whereas positive resu lts--h ig h or complete protection can be regarded as good evidence for a close relation between two viruses." The absence of local lesions in watermelon plants inoculated with extracts from double-inoculated pumpkin plants indicates that IH might not have infected and multiplied in pumpkin plants system ically infected with IIA strain of SMV. This is supported by D em ski's (1969) work which indicated the use of Citrullus /vulgaris Schrad. to detect the presence of local lesion strain of SMV in a mixture with o th e rs. No cantaloupe plants showed IH symptoms when inoculated with crude sap or purified viral suspension from pumpkin. This evidenced the cross-protective effect in pumpkin since IH excluded IIA. in cantaloupe even when used in lower concentration (Table 1). This exclusion 28

40 29 was confirmed when control plants were inoculated with a mixture of IH and IIA. The use of intragel absorption tests to confirm this crossprotective result revealed the value of this serological technique in. demonstrating cross-protection between system ic strains of plant viruses where symptom differences are inadequate A degree of association was found between the cross-protective results and relative concentrations of IH and IIA in pumpkin and cantaloupe plants (Table 2). The absence of cross-protection in cantaloupe could be explained by the fact that strain IIA does not invade this host uniformly, leaving certain areas of the leaves or cells unprotected. It has been shown by Salaman (1933), Kunkel (1934), and Sinclair and Walker (1956) that only those tissu es fully infected by the first strain resist infection by the second. Sinclair and W alker (1956) attributed the lack of cross-protection between strains of CMV in field-grown cucumber to a failure of the mild strain to become thoroughly established in the host plant. The results shown in Table 1 and Table 2 suggest that IH is more invasive and would quickly reach a greater concentration in cantaloupe plants than IIA strain. The suppression of IIA by IH in simultaneously inoculated cantaloupe plants suggests a certain degree of association with their ability to multiply in cantaloupe plants. The delay of appearance of green vein banding and severe mottle in IIA-infected cantaloupe plants after challenge inoculation with IH indicates that part of the cells of these plants were protected against

41 30 infection with IH during the time of second inoculation, thus at least delaying build-up of IH. The complete reciprocal protection observed in pumpkin plants suggests that the plants were fully infected with either strain of SMV at the time of challenge inoculation Crowdy and Posnette (1947) stated that the protection acquired by a plant against one virus strain because of previous infection with a related strain depends upon the complete invasion and continued presence of the first strain in the plant tissu e s. When the concentration study with cantaloupe was performed in a growth cham ber, the concentrations of both strains were higher than those obtained under greenhouse conditions (Table 2). The reason for this could be the fact that the environmental conditions are more stable in the growth chamber than in the greenhouse. In the growth chamber, day and night temperatures were 25 C and 30 C, respectively, while the temperature in the greenhouse reached a minimum of 20 C during the night and a maximum of 35 C during the day. Bancroft (1958) studied the effects of temperature and tem perature-light on the concentration of SMV.in leaves of growing cucurbits and observed a relationship between the virus concentration and the combined effect of these environmental facto rs. The results obtained with pumpkin plants during the course of this study suggest that the two strains of SMV compete for the same sites of infection in these plant c e lls. Bawden and K assanis (1945), Bawden (1950), Siegel (1959), and Wu and Rappaport (1961) suggested that plant virus strains actively compete for the same sites within cells of double-inoculated plants. The data obtained by Siegel (1959)

42 31 suggested that when two strains of TMV are simultaneously inoculated in N. sylvestris p la n ts, either one or the other, but not both, could initiate infection. On the other hand, the suppressive effect and cross-protection results observed in cantaloupe plants suggest that strain IIA is incapable of occupying all the available sites in this host necessary to prevent multiplication Of strain IH introduced later. It is presumed that in can taloupe strain IH multiplies at " sites" that are unavailable to strain IIA while still utilizing all sites that IIA can utilize. This could also be the explanation for the greater concentration of IH observed in cantaloupe plants (Table 2).

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48 M