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

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Authors Lima, José Albersio de Araújo, 1940-

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Link to Item http://hdl.handle.net/10150/554643 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

1 9 7 2 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­ quirements 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 permission, 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 judg­ ment the proposed use of the material is in the interests of scholar­ ship. 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: mz. MERRITT R. NELSON Date Professor of Plant Pathology ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude and appre­ ciation for the counsel and helpful guidance of Dr„ Merritt R. Nelson,

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 manuscript.

The author also thanks Mr. Wilbur R. Hague for his helpful contributions in the greenhouse works.

Gratitude is extended to Dr. William G. Matlock, Campus Co­ ordinator, AID Brazil Program, The University of Arizona/University of

Ceara Contract, and to his secretary, Mrs. Evelyn Jorgensen, for their understanding and cooperation.

The author was supported by funds from the Agency for International Development (US AID), The University of Arizona/Univer­ sity of Ceara Contract, to whom sincere appreciation is expressed.

Special recognition is given to my wife, Diana, whose patience and support made this work possible. TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS...... v

LIST OF TABLES ...... vi

ABSTRACT...... vii

INTRODUCTION AND LITERATURE REVIEW ...... 1

MATERIAL AND METHODS ...... 12

Virus Strains and Hosts „ ...... 12 Virus Purification and Inoculation Methods ...... 13 Cross-protection Experiments between Strains of SMV in Pumpkin and Cantaloupe . . . . „ . „ „ . . . 14 Squash M osaic Virus Strain Dominance in Simultaneously Inoculated Plants .... „ „ . . . . . 16 Concentrations of Strains of in Pumpkin and Cantaloupe...... 17

RESULTS ...... 18

Virus Strains and Hosts ...... 18 Cross-protection between Strains of SMV in Pumpkin and Cantaloupe ...... 22 Squash M osaic Virus Strain Dominance in Simultaneously Inoculated Pumpkin and Cantaloupe Plants...... 24 Squash Mosaic Virus Strain Concentration in Pumpkin and Cantaloupe...... 26

DISCUSSION ...... 28

REFERENCES...... "...... „ . 32

iv LIST OF ILLUSTRATIONS

Figure Page

1 „ Systemic Symptoms in of pepo L. Inoculated with Strains of Squash M osaic Virus . . . . 19

2. Symptoms of Strains of Squash M osaic Virus in L „ „ ...... 20

3. Local Lesions on Cotyledons of Citrullus vulgaris Schrad. Caused by IH Strain of Squash Mosaic Virus ...... 21

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 ...... 21

v 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 ...... 25

2. The Relative Concentrations of IH and IIA Strains of Squash M osaic Virus in Pumpkin and Canta­ loupe Plants Grown in Greenhouse and Growth Chamber...... 27

v i ABSTRACT

Interactions between strains IH and IIA. of squash mosaic virus

(SMV) in pumpkin ( 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 multi­ plication to twice the extent of strain IIA. In pumpkin, the relative ex­ tent 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 range differ­ ences and the intragel absorption technique,

When opposite cotyledons of pumpkins were inoculated simul­ taneously with equal concentrations but different strains of purified virus, 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. INTRODUCTION AND LITERATURE REVIEW

Biological properties of can and have been used exten­

sively to determine the relationship between strains of plant viruses.

According to Matthews (1970), differences and similarities in the bio­

logical 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 viruses, cross-protection

studies have been widely and successfully used by several workers to

demonstrate identity and strain relationships of some plant viruses.

The phenomenon in which plant tissues 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 tissues has received wide attention.

Ainsworth (1934) compared viruses isolated from Cana­

dian material with standard English material. The fact that mild

strains conferred immunity in tomato against a virulent strain was con­

sidered as strong evidence of close relationship of the two isolates.

Kunkel (1934) demonstrated that leaves of Nicotiana 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. systematically invaded by

1 any one of four different mottling strains of 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 unre­ lated to CMV or TMV were susceptible to the necrotic-type strains of both.

The results obtained by Price (1936) evidenced the specificity of acquired immunity from tobacco ring spot diseases. 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 plants, Price (1937) concluded that the two viruses were closely related and that lily mosaic virus should be classified in the 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 inoculation and graft transmission, 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. 3 Strains of alfalfa mosaic virus (AMV) have been identified by- cross-protection tests. The results obtained by Berkeley (1947) in to­ bacco 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

Walker (1948) concluded that they were closely related viruses. Corbett

(1957) observed that when Crotalaria spectabilis Roth system atically in­ fected 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, vulgaris L. , was a strain of bean common mosaic virus.

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 virus.

Matthews (1949) studied the relationships between strains of potato virus X. He found a correlation between the degree of cross- protection 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 presence of a systemic 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"2 U caused no demonstrable symptoms . They observed that when strain Uj was in­ oculated 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 tobac­ co 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 similarities 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, sero­ logical and vector relationship studies indicated that the two viruses were distinct.

Cross-protection tests 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 ■ ' ■ 5 spot virus , These results were confirmed by serological tests reported in the same paper.

Jedlinski and Brown (1965) carried out cross-protection experi­ ments 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 virus.

Corbett and Price (1967) showed that citrus plants infected with psorosis virus were not protected against local and systemic infection by citrus variegation virus, thus suggesting that psorosis and infectious variegation were caused by distinct viruses.

Nelson, Mate jka, and McDonald (1965) reported that cross- protection tests of a strain of squash mosaic virus (SMV) with cucurbit viruses other than SMV failed to reveal any relationships.

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 be­ tween unrelated viruses. Silberschmidt (1957) observed a lack of pro­ tection in tomato plants between strains of potato virus X and pointed out certain limitations of the value of cross-protection tests for taxonomical purposes. Tu and Ford (1969) could not find any cross-protection between strains of 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 . Varney and Moore

(1952) and Fulton and Fulton (1970) observed unilateral cross-protection 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 unre­ lated to the former. Nevertheless, they pointed out that the phenomenon is quite different from the reciprocal protection, which is so success­ fully and widely used to test whether viruses.causing different symptoms are related strains.

Although the mechanism to account for the phenomenon of cross­ protection in plants remains undetermined, several theories have been put forward as to what underlies the protective process.

One of the earliest accounts of a protective effect between 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 one. 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 virus.

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 essential metabolites required for the second to multiply. Sadasivan (1940) sug­ gested that the degree of protection between plant virus strains is di­ rectly proportional to the number of active units of the protecting strain present in the tissue at the time of re inoculation. Critical analysis of reported cases in which one strain of virus protected plant tissues 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 ac- accounted for on the basis of competition for materials essential for virus multiplication. 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 water, 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 and bush after a mild strain of the same virus had become thor­ oughly 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 wilt 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 pro­ tection effects observed between tomato spotted wilt virus strains. The result obtained by Thomson (1961) indicated that from mixed infection of Nicotiana glutinosa L„ with two strains of potato virus X a new strain could be obtained.

Another possible explanation for cross-protection was sug­ gested 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 at­ traction forces and inactivated by the association.

The immunity phenomenon so characteristic of has not been observed in plants, and there is no evidence that antiviral anti­ bodies are produced in plants. 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 var. Pinto and suggested that multiplication of Uj was inhibited as a result of metabolic changes induced in cells sur­ rounding 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 sug­ gested that when a site in a susceptible cell was already occupied by one strain, a second strain of the same virus, requiring the same site, could not be able to attach itself and multiply. This idea has been sup­ ported 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 transmission, and serological studies have been used to identify squash mosaic virus

(SMV) (Takahashi and Rawlins, 1947; Freitag, 1952; and Lindberg, Hall, and Walker, 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 al., 1955). In the same study they also ob­ served that under the electron microscope some of the SMV particles appeared as polyhedrons.

Squash mosaic virus has been reported to be both bettle trans­ mitted and seedborn. Freitag (1941) first reported that the virus could be transmitted either by or by cucumber , but only ineffi­ ciently by aphids. In subsequent stu d ie s, Freitag (1952 and 1956) con­ cluded that SMV was but not transmitted. Bitterly (1960) demonstrated that balteata LeConte, the banded , was a new and efficient vector of SMV. . ■ : 10 Middleton (1944) observed that the virus was transmitted 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 transmitted by -commercial seed of cucurbit species, it was more prevalent in areas of where cucumber beetles were more common. The results obtained by Powell and Schlegel (19 70) indi­ cated that seed transmission 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 Matejka (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 (Nelson et a l., 1965). They observed that, al­ 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 Nelson, 1968).

Nelson and Knuhtsen (1969) observed a differential seed trans­ mission between both groups of SMV strains. Their data showed that group I was more efficiently transmitted by seed of several cucurbits, 11 which they considered important in the epidemiology of the diseases caused by these SMV strains.

In the present study, a series of experiments was designed to observe the relationship between strains of SMV in cucurbit plants. 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 be­ tween strains of plant viruses. 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 plants. 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 Wisconsin. They represent the two serological groups of SMV strains (Nelson afid Knuht- sen, 1972) „ Both isolates were obtained from Merritt R„ Nelson of the

Plant Pathology Department, 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 used, Cucurbita pepo L., Cucumis melo L., and

Citrullus vulgaris Schrad., 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. Most of the experiments were conducted during the summers . of 1971 and 1972 in a greenhouse at The University of Arizona, Tucson .

The greenhouse temperature ranged from 25°C (average night temperature) to 32°C (average day temperature). The greenhouse was fumigated at frequent intervals to control . The experiments carried out during the cold seasons were conducted in a growth chamber adjusted to a day­ time of 14 hours and night and day temperatures of 25°C and 30°C/ re­ spectively. During the daytime period, the growth chamber was kept under continuous illumination by incandescent and fluorescent lamps pro­ ducing approximately 2,000+ 200 foot candles at the level of the pots.

12 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 process.

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 result­ ing extract was strained through a double layer of cheesecloth and sub­ sequently clarified by centrifugation for 20 minutes in a VRA rotor at

10,000 rpm in a Lourdes Model A-2 Beta-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 minutes, 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 minutes, the precipitate was sedimented for 30 minutes at 10,000 rpm in a VRA rotor of the Lourdes centrifuge. The resulting pellet was resus­ 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 times, 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 „ 14 The viral concentrations were obtained in milligrams of virus per milliliters of purified preparations and converted to milligrams of virus nucleoprotein per gram of fresh weight of leaf tissue „

The method of inoculation used in the experiments was the same in all cases „ Carborundum was added to purified virus suspen­ sions 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 „

Cross-protection Experiments between Strains of SMV in Pumpkin and Cantaloupe

Cotyledons of 30 seedlings of pumpkin or cantaloupe were in­ oculated with a test 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 inocu­ lation, 15 uninoculated plants of the same age were also inoculated and

15 plants were.left uninoculated. All experiments were repeated at least twice. All inocula used in these experiments were constituted of puri­ fied virus suspensions in which the concentrations were adjusted to

0.1 or 0.5 mg/ml.

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 host). The IIA strain of SMV was inoculated to seedlings of_C. pepo plants. Ten days later, 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 plants, the . 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 seedlings. Three other batches of watermelon and cantaloupe seedlings, to serve as controls, were inoculated with IH, IIA, and a mixture of IH and IIA. At the same time, 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 as­ sayed in watermelon, 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. Nelson. Fifteen milli­ liters of 1.0 percent Ion Agar II (Consolidated laboratories, Chicago

Heights, Illinois) dissolved in glass-distilled 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.

Wells were punched into the agar with cork borers in a hexagonal ar­ rangement. The hexagonal arrangement consisted of a center well, 6 mm in diameter, with six peripheral wells,4 mm in diameter, spaced 10 mm from the center well as measured from the centers of the w e lls.

In the. first test, a high concentration (2.0 mg/ml) of IH antigen was placed in the center well, and 24 hours later the IIA antiserum was added in the same well. At the same time, IIA antigen and purified virus suspension obtained from double-inoculated pumpkin plants were alter­ nated in the six outer wells . 16 A similar test was carried out as follows: 24 hours after a high concentration of IIA antigen had been put in the center well, IH anti­ serum was added, and IH and the purified virus suspension from pump­ kin were alternated in the six outer wells.

After the wells were filled with the reactants, the plates were incubated for 1 to 5 days at room temperature. The development of pre­ cipitation patterns was observed by looking at the plates, 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 milliliter 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 in­ oculations were made simultaneously. In each experiment, the IH and

IIA strains were inoculated on opposite cotyledons of a,group of 100 seedlings. 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 domi­ nance , experiments were conducted with both cantaloupe and pumpkin in which concentrations of one strain was varied from 0.1 mg/ml to

0.0001 mg/ml while the concentration of the other strain was maintained at 0.1 mg/ml. 17 Concentrations of Strains of Squash Mosaic Virus in Pumpkin and Cantaloupe

This experiment was devised to try to find some degree of cor­ relation between the results obtained in cross-protection studies and concentrations of IH and IIA in pumpkin and cantaloupe plants. 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 plants, 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 milliliter of purified virus suspension, and an approximately equal amount of IIA was inoculated into the other two groups of plants. Ten days after inoculation, 50 gm

(wet weight) of systemically 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. 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 systemic 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 systemic chlorotic rings 5 to 7 days after inoculation

(Figs. 1-B and 1-C).

Both strains also produced systemic symptoms in cantaloupe,

_C. melo. 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 leaves. On the other hand, the strain IIA produced only a very mild mosaic 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 Figure 1. Systemic Symptoms in Leaves of Cucurbita pepo L. Inoculated with Strains of Squash M osaic Virus

A. Healthy leaf.

B. Leaf systemically infected with strain IH showing characteristic systemic chlorotic rings.

. C. Leaf inoculated with strain IH showing a mild generalized mottle.

D. Leaf systemically infected with IIA strain showing a severe mosaic.

E. Severe leaf distortion induced by strain IIA. Figure 1. Systemic Symptoms in Leaves of Cucurbita pepo L. Inoculated with Strains of Squash Mosaic Virus i—■ CD Figure 2. Symptoms of Strains of Squash Mosaic Virus in Cucumis melo L.

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. 20

Figure 2. Symptoms of Strains of Squash Mosaic Virus in Cucumis melo L. 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

Antisera to IH and IIA were those used by Nelson and Knutsen (1972); each had been shown to contain a significant proportion of strain- specific 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 plants. .

B„ Central well filled initially with IIA antigen, then 24 hours later with IH antiserum (as. I). Well II = IIA antigen; well X = purified SMV suspension obtained from double-inoculated pumpkin plants. Figure 3. Local Lesions on Cotyledons of Citrullus vulgaris Schrad. Caused by IH Strain of Squash Mosaic Virus

9 9 0

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 22

Demski (1969) pointed out. the value of these hypersensitive reactions in

_C. vulgaris for virus bioassay.

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 de­ veloped, although a like inoculation of previously uninoculated plants resulted in the production of typical severe symptoms of this strain.

Cross-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 lesions. 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 mosaic developed on all pumpkin plants inoculated with similar inoculum. 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 suspen­ sion 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 wells, cross-reacted with and.fully precipitated the cross-reacting antibodies at the region of optimal proportions close to the center w ells. The outer wells with the IH antigen did not show any reaction evidencing a complete cross-absorption. On the other hand, bands were observed close to the outer wells 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 wells showed any reaction (Fig. 4-B). These results evidenced a com­ plete cross-absorption and indicated that the strain IH was not present in the purified SMV suspension obtained from the double-inoculated pumpkin plants.

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. Nevertheless, in the double-inoculated plants, the appearance of IH symptoms was delayed

3 to 4 days in relation to the inoculated controls. 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 mosaic accompanied by leaf distortions, while 45 to 48 percent showed only a mild mottle.

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) de­ creased with decrease in IIA concentration (Table 1).

A similar experiment performed by Cohen et al. (1957) with and \J2 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 results. Plants simultaneously inoculated with the same concentrations of both strains on opposite cotyledons showed almost complete dominance 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%

10-3 . io -i 30% 70% 40% 60% CO i — 1 1 O r—i o I — i

1 40% . 60% 65% 35% 1 1 h-J — 10"1 I o 45% 55% 90% 10% r—1 I o 1 — 1 10" 2 55% 45% 90% 10%

10-1 10-3 80% 20% 95% 5% 1—1 o 1 r—i 10-4 95% 5% 100% ■ . 0%

a. Inoculum concentrations in mg of virus per ml of purified virus suspensions 26 of the IH over the IIA. strain. Ninety to 92 percent of these simultaneous­ ly 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 inocu­ lated 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 Mosaic Virus Strain Concentration in Pumpkin and Cantaloupe

In each experiment, the virus strain concentration in systemi- cally infected leaves was determined 10 days after inoculation. The IH strain of SMV multiplied to a greater extent than the IIA in cantaloupe plants, since the results shown in Table 2 revealed that the IH concen­ tration 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). 27

Table 2. The Relative Concentrations of IH and IIA Strains of Squash . Mosaic 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 0.25 0.26 0.23 0.24

Cantaloupe 0.45 0.18 0.52 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 pre­ viously described because of low nighttime temperatures in the green­ house „ 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 systemically infected tissue.

Cross-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 results--high or com­ plete 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 systemically infected with IIA strain of SMV. This is supported by Demski'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 others.

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 canta­ loupe even when used in lower concentration (Table 1). This exclusion

28 29 was confirmed when control plants were inoculated with a mixture of IH and IIA.

The use of intragel absorption tests to confirm this cross- protective result revealed the value of this serological technique in . demonstrating cross-protection between systemic 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 canta­ loupe 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 tissues fully infected by the first strain resist infection by the second. Sinclair and Walker (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 canta­ loupe plants than IIA strain. The suppression of IIA by IH in simulta­ neously 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 in­ dicates that part of the cells of these plants were protected against 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 tissues.

When the concentration study with cantaloupe was performed in a growth chamber, 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 temperature-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 factors.

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 cells. Bawden and Kassanis (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) 31 suggested that when two strains of TMV are simultaneously inoculated in N. sylvestris plants, 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 canta­ loupe 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 canta­ loupe plants (Table 2). REFERENCES

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