Horn, Wayne G.

Walters, H. J.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 22 Article 15 1968 Purification of Soybean Mosaic Virus Wayne G. Horn University of Arkansas, Fayetteville H. J. Walters University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Plant Pathology Commons Recommended Citation Horn, Wayne G. and Walters, H. J. (1968) "Purification of Soybean Mosaic Virus," Journal of the Arkansas Academy of Science: Vol. 22 , Article 15. Available at: https://scholarworks.uark.edu/jaas/vol22/iss1/15 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 72iArkansas Academy of Science Proceedings, Vol. 22, 1968PURIFICATION OF SOYBEAN MOSAIC VIRUSWayne G. Horn1 and H. J. WaltersDepartment of Plant PathologyUniversity of ArkansasSymptoms of soybean mosaic virus (SMV) in soybean includemosaic, rolling and puckering of foliage, and chlorosis of older leaves.The virus may significantly reduce yields of soybeans and cause amottling of the seed (1). The characterstic particles associated withSMV are long flexuous rods about 740 mm in length. Galvez (2)reported aggregation of the particles during purification whichprevented isolation of the virus sufficiently pure to produce antiserumfree of antibodies to normal host antigens. Ross (4) reported purifica-ton of SMV and production of antisera free from host antibodiesby using 0.5 M citrate buffer during low speed centrifugation andsuspending the pelleted virus after high speed centrifugation in aweak borate buffer. However, this procedure did not eliminatevirus loss due to aggregation of particles during the high speedcentrifugation.Several viruses, including those with particles that are longflexuous rods, have been purified by filtration through granulatedagar columns (3, 5, 6, 7, 8). The object of this investigation was topurify SMV without aggregation by using agar gel filtration com-bined with alternate high and low speed centrifugation.MATERIAL AND METHODSThe virus used was a strain of SMV used in past host-rangestudies (9). Soybean (Glycine max var. Hill) was used for in-creasing the virus and as a systemic indicator host. Inoculationswere made by rubbing carborundum-dusted leaves with gauze padseither soaked in sap produced by grinding infected soybean leaveson 0.5 M potassium phosphate buffer (pH6.5) or soaked in viruspreparations obtained during the course of purification.Low-speed centrifugations were performed in a Lourdes LCA-1centrifuge at 4°C. The virus was pelleted at 30,000 rpm for 60 min.in the No. 30 rotor of a Spinco model L ultracentrifuge.. In this study a column similar to that described by VanRegenmortel (7) was used. Four percent lonagar No. 2 was pre-pared and ground in a Waring Blender until the granules couldpass through a 40-mesh sieve but not a 60-mesh sieve. The granules'Participant—Undergraduate Research Participation Program inPlant Pathology—NSF Grant No. GY2609.72Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15Published by Arkansas Academy of Science, 196873iPurification of Mosaic Viruswere packed in a glass column 60 cm by 2 cm, covered with a thinpad of glass wool, and washed with a liter of 0.01 M sodium boratebuffer (pH8.3). Ths buffer was used throughout purification unlessotherwise noted. Column experiments were conducted at about 22°C.Dialysis was performed in 4 liters of 50% polyethylene glycolwith an Oxoid multiple dialyzer at 4°C.Ultraviolet absorption spectra were taken with a BeckmanDB-G spectrophotometer with virus diluted sufficiently to placepeaks within an absorbance range of 0.2-0.8.Virus antiserum was produced by injecting a rabbit intra-muscularly with 1mg of purified virus in 1ml of buffer emulsifiedwith 1 ml of Freund's incomplete adjuvant (Difco), with 3 in-jections given at 6-day intervals. Blood samples of 50 ml were col-lected on each of 2 days at a 7-day interval starting 7 days afterthe last injection. Antiserum dilution end points were determinedby microprecipitin tests. Results were recorded after the tests hadbeen incubated 1hour at 37°C and left overnight at 4°C.Partial purification of the virus followed closely the tech-nique used by Ross (4). Harvested SMV-infected soybean leaves(100 g) were homogenized in 200 ml of 0.5 M sodium citrate with1% 2-mercaptoethanol. The pulp was extracted by straining theextract through 2 layers of cheese cloth. While the extract was stirred,16 ml of n-butanol was slowly added. After curdling was evident,the preparation was placed in a cold room overnight at 4"C. Theextract was then clarified at 10,000 rpm for 10 minutes in the 9RArotor of the Lourdes centrifuge. The supernatant was decantedthrough glass wool and an amber liquid was obtained. The volumeof the liquid was reduced to 25 ml by dialysis against 50% polyethy-lene glycol. To reduce loss of virus adhering to the tubing duringdialysis, 50 ml of the liquid in each of 4 tubes was reduced to 25 ml,the contents of the 4 tubes combined into 2 tubes, and process re-peated until 25 ml remained in one tube. Following concentrationsufficient sucrose was added to increase density of the liquid 10%by weight and the liquid placed on the column under 70 ml of buffer.The liquid was filtered through the column at th^e rate of 20 ml/hour.The effluent passed through an ISCO ultraviolet analyzer with wavelength set at 254 mu and was collected in 6 ml volumes by a fractioncollector.RESULTS AND DISCUSSIONInfectivity was associated with the initial peak on the UVanalyzer graph indicating that the virus was eluted between 50 and120 ml and followed by plant components. Considerable overlappingof the two fractions was evident.73Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15https://scholarworks.uark.edu/jaas/vol22/iss1/1574Arkansas Academy of Science ProceedingsFractions containing the 50 to 100 ml portion of the elutionvolume were combined and the virus pelleted by high speed centri-fugation. The pellets were resuspended in 2 ml borate buffer. Ab-sorption spectra of fractions taken in the 50 to 70 ml range of theelution volume showed the maxium peak at 257 mu and a minimumat 234 mu.Virus antiserum tested against the purified virus preparationand against clarified sap from SMV-infected plants gave the floc-culent type of precipitate characteristic of rod shaped viruses. Someprecipitation was observed with clarified healthy plant sap. Theantiserum dilution-end-point against the purified virus was 1:16.The purification process was carried out several times. Dry weightdeterminations indicated that approximately 2.1 mg vrus were ob-tained per 100 g plant tissue.Separation of SMV and host protein using the agar columnwas difficult due to overlap of the two components during filtration.High speed centrifugation was found to be the most efficientmethod of concentrating the purified virus, although this procedureresulted in partial loss of infectivity. A sharp peak was obtained atboth the maximum of 257 mu and minimum of 234 mu on the spec-trophotometer. Ross (4) reported a broad maximum peak at 260-265mu and a sharp minimum peak at 245 mu, along with a tryptophanshoulder at 290 mu, which was not evident in the present studies.The overlapping of the virus and plant protein during filtra-tion through the column explains the presence of precipitate whenclarified healthy plant sap was reacted with the virus antisera.LITERATURE CITED1. Dunleavy, J. M., D. W. Chamberlain, and J. P. Ross. 1966. Soy-bean diseases. U. S. Dept. Agric, A. R. S., Agriculture Hand-book No. 302.2. Galvez, G. E. 1963. Host range, purification, and electronmicroscopy of soybean mosaic virus. Phytopathology 53:388-393.3. Poison, A. 1961. Fractionation of protein mixtures on columnsof granulated agar. Biochem. Biophys. Actra. 50:565-567.4. Ross, J. P. 1967. Purification of soybean mosaic virus for antiseraproduction. Phytopathology 57: 465-467.5. Steere, R. L., and G. K. Ackers. 1962. Purification and separa-tion of tobacco mosaic virus and southern bean mosaic virus byagar-gel filtration. Nature. 194:114-116.74Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15Published by Arkansas Academy of Science, 1968Ii 75! Purification of Mosaic Virusi 6. Van Regenmortel, M. H. V. 1962. Purification of a plant virusj by filtration through granulated agar. Virology 17:601-602.I 7. 1964. Separation of an antigenic plant proteinI from preparations of plant viruses. Phytopathology 54:282-289.f 8. J> Brandes, and R. Bercks. 1962. InvestigationsI on the properties of watermelon mosaic virus. Phytopathology2. 45:205-216.j 9. Walters, H. J. 1963. Leguminous hosts of soybean mosaic virus.Plant Disease Reptr. 47:726-728.75Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15https://scholarworks.uark.edu/jaas/vol22/iss1/15

Purification of Soybean Mosaic Virus

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 22 Article 151968Purification of Soybean Mosaic VirusWayne G. HornUniversity of Arkansas, FayettevilleH. J. WaltersUniversity of Arkansas, FayettevilleFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Plant Pathology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationHorn, Wayne G. and Walters, H. J. (1968) "Purification of Soybean Mosaic Virus," Journal of the Arkansas Academy of Science: Vol. 22 ,Article 15.Available at: http://scholarworks.uark.edu/jaas/vol22/iss1/1572iArkansas Academy of Science Proceedings, Vol. 22, 1968PURIFICATION OF SOYBEAN MOSAIC VIRUSWayne G. Horn1 and H. J. WaltersDepartment of Plant PathologyUniversity of ArkansasSymptoms of soybean mosaic virus (SMV) in soybean includemosaic, rolling and puckering of foliage, and chlorosis of older leaves.The virus may significantly reduce yields of soybeans and cause amottling of the seed (1). The characterstic particles associated withSMV are long flexuous rods about 740 mm in length. Galvez (2)reported aggregation of the particles during purification whichprevented isolation of the virus sufficiently pure to produce antiserumfree of antibodies to normal host antigens. Ross (4) reported purifica-ton of SMV and production of antisera free from host antibodiesby using 0.5 M citrate buffer during low speed centrifugation andsuspending the pelleted virus after high speed centrifugation in aweak borate buffer. However, this procedure did not eliminatevirus loss due to aggregation of particles during the high speedcentrifugation.Several viruses, including those with particles that are longflexuous rods, have been purified by filtration through granulatedagar columns (3, 5, 6, 7, 8). The object of this investigation was topurify SMV without aggregation by using agar gel filtration com-bined with alternate high and low speed centrifugation.MATERIAL AND METHODSThe virus used was a strain of SMV used in past host-rangestudies (9). Soybean (Glycine max var. Hill) was used for in-creasing the virus and as a systemic indicator host. Inoculationswere made by rubbing carborundum-dusted leaves with gauze padseither soaked in sap produced by grinding infected soybean leaveson 0.5 M potassium phosphate buffer (pH6.5) or soaked in viruspreparations obtained during the course of purification.Low-speed centrifugations were performed in a Lourdes LCA-1centrifuge at 4°C. The virus was pelleted at 30,000 rpm for 60 min.in the No. 30 rotor of a Spinco model L ultracentrifuge.. In this study a column similar to that described by VanRegenmortel (7) was used. Four percent lonagar No. 2 was pre-pared and ground in a Waring Blender until the granules couldpass through a 40-mesh sieve but not a 60-mesh sieve. The granules'Participant—Undergraduate Research Participation Program inPlant Pathology—NSF Grant No. GY2609.72Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15Published by Arkansas Academy of Science, 196873iPurification of Mosaic Viruswere packed in a glass column 60 cm by 2 cm, covered with a thinpad of glass wool, and washed with a liter of 0.01 M sodium boratebuffer (pH8.3). Ths buffer was used throughout purification unlessotherwise noted. Column experiments were conducted at about 22°C.Dialysis was performed in 4 liters of 50% polyethylene glycolwith an Oxoid multiple dialyzer at 4°C.Ultraviolet absorption spectra were taken with a BeckmanDB-G spectrophotometer with virus diluted sufficiently to placepeaks within an absorbance range of 0.2-0.8.Virus antiserum was produced by injecting a rabbit intra-muscularly with 1mg of purified virus in 1ml of buffer emulsifiedwith 1 ml of Freund's incomplete adjuvant (Difco), with 3 in-jections given at 6-day intervals. Blood samples of 50 ml were col-lected on each of 2 days at a 7-day interval starting 7 days afterthe last injection. Antiserum dilution end points were determinedby microprecipitin tests. Results were recorded after the tests hadbeen incubated 1hour at 37°C and left overnight at 4°C.Partial purification of the virus followed closely the tech-nique used by Ross (4). Harvested SMV-infected soybean leaves(100 g) were homogenized in 200 ml of 0.5 M sodium citrate with1% 2-mercaptoethanol. The pulp was extracted by straining theextract through 2 layers of cheese cloth. While the extract was stirred,16 ml of n-butanol was slowly added. After curdling was evident,the preparation was placed in a cold room overnight at 4"C. Theextract was then clarified at 10,000 rpm for 10 minutes in the 9RArotor of the Lourdes centrifuge. The supernatant was decantedthrough glass wool and an amber liquid was obtained. The volumeof the liquid was reduced to 25 ml by dialysis against 50% polyethy-lene glycol. To reduce loss of virus adhering to the tubing duringdialysis, 50 ml of the liquid in each of 4 tubes was reduced to 25 ml,the contents of the 4 tubes combined into 2 tubes, and process re-peated until 25 ml remained in one tube. Following concentrationsufficient sucrose was added to increase density of the liquid 10%by weight and the liquid placed on the column under 70 ml of buffer.The liquid was filtered through the column at th^e rate of 20 ml/hour.The effluent passed through an ISCO ultraviolet analyzer with wavelength set at 254 mu and was collected in 6 ml volumes by a fractioncollector.RESULTS AND DISCUSSIONInfectivity was associated with the initial peak on the UVanalyzer graph indicating that the virus was eluted between 50 and120 ml and followed by plant components. Considerable overlappingof the two fractions was evident.73Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15http://scholarworks.uark.edu/jaas/vol22/iss1/1574Arkansas Academy of Science ProceedingsFractions containing the 50 to 100 ml portion of the elutionvolume were combined and the virus pelleted by high speed centri-fugation. The pellets were resuspended in 2 ml borate buffer. Ab-sorption spectra of fractions taken in the 50 to 70 ml range of theelution volume showed the maxium peak at 257 mu and a minimumat 234 mu.Virus antiserum tested against the purified virus preparationand against clarified sap from SMV-infected plants gave the floc-culent type of precipitate characteristic of rod shaped viruses. Someprecipitation was observed with clarified healthy plant sap. Theantiserum dilution-end-point against the purified virus was 1:16.The purification process was carried out several times. Dry weightdeterminations indicated that approximately 2.1 mg vrus were ob-tained per 100 g plant tissue.Separation of SMV and host protein using the agar columnwas difficult due to overlap of the two components during filtration.High speed centrifugation was found to be the most efficientmethod of concentrating the purified virus, although this procedureresulted in partial loss of infectivity. A sharp peak was obtained atboth the maximum of 257 mu and minimum of 234 mu on the spec-trophotometer. Ross (4) reported a broad maximum peak at 260-265mu and a sharp minimum peak at 245 mu, along with a tryptophanshoulder at 290 mu, which was not evident in the present studies.The overlapping of the virus and plant protein during filtra-tion through the column explains the presence of precipitate whenclarified healthy plant sap was reacted with the virus antisera.LITERATURE CITED1. Dunleavy, J. M., D. W. Chamberlain, and J. P. Ross. 1966. Soy-bean diseases. U. S. Dept. Agric, A. R. S., Agriculture Hand-book No. 302.2. Galvez, G. E. 1963. Host range, purification, and electronmicroscopy of soybean mosaic virus. Phytopathology 53:388-393.3. Poison, A. 1961. Fractionation of protein mixtures on columnsof granulated agar. Biochem. Biophys. Actra. 50:565-567.4. Ross, J. P. 1967. Purification of soybean mosaic virus for antiseraproduction. Phytopathology 57: 465-467.5. Steere, R. L., and G. K. Ackers. 1962. Purification and separa-tion of tobacco mosaic virus and southern bean mosaic virus byagar-gel filtration. Nature. 194:114-116.74Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15Published by Arkansas Academy of Science, 1968Ii 75! Purification of Mosaic Virusi 6. Van Regenmortel, M. H. V. 1962. Purification of a plant virusj by filtration through granulated agar. Virology 17:601-602.I 7. 1964. Separation of an antigenic plant proteinI from preparations of plant viruses. Phytopathology 54:282-289.f 8. J> Brandes, and R. Bercks. 1962. InvestigationsI on the properties of watermelon mosaic virus. Phytopathology2. 45:205-216.j 9. Walters, H. J. 1963. Leguminous hosts of soybean mosaic virus.Plant Disease Reptr. 47:726-728.75Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 15http://scholarworks.uark.edu/jaas/vol22/iss1/15

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Purification of Soybean Mosaic Virus

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