Minor, Scott M.

Nordeen, Russell O.

Paschall, Chad E.

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 50 Article 28 1996 Partial Characterization of Bacteriophages of Pseudomonas syringae pv.tomato Scott M. Minor University of Arkansas at Monticello Russell O. Nordeen University of Arkansas at Monticello Chad E. Paschall University of Arkansas for Medical Sciences Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Bacterial Infections and Mycoses Commons, and the Plant Pathology Commons Recommended Citation Minor, Scott M.; Nordeen, Russell O.; and Paschall, Chad E. (1996) "Partial Characterization of Bacteriophages of Pseudomonas syringae pv.tomato," Journal of the Arkansas Academy of Science: Vol. 50 , Article 28. Available at: https://scholarworks.uark.edu/jaas/vol50/iss1/28 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 General Note 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. 137Partial Characterization ofBacteriophages ofPseudomonas syringae pv. tomatoScott M.Minor and Russell O. NordeenUniversity ofArkansas at MonticelloDivision of Mathematics and SciencesMonticello, AR71656IntroductionSince the first major outbreak of bacterial speck in1978, this disease has been a major problem for tomatogrowers in certain regions of North America. This dis-ease, characterized by small brown to black lesions, iscaused by Pseudomonas syringae pv. tomato (Jones et. al.,1991). While bacterial speck is not a threat to the plantitself, it reduces the marketable value of the fruit bydecreasing its aesthetic appeal to the consumer. The taxo-nomic status ofP. tomato is uncertain at present. It is diffi-cult to distinguish morphologically and physiologicallyfrom other pathovars of the species, particularlyPseudomonas syringae pv. syringae. P. syringae pv. syringaehas also been characterized from necrotic lesions oninfected tomato fruit (Jones et. al., 1981). P. tomato is alsoof interest because it has been used as a model in plantpathogen studies with Arabidopsis thaliana, to gain a bet-ter understanding of the molecular basis of bacterial dis-ease resistance (Whalen and Staskawicz, 1990). A compre-hensive fine structure genetic analysis ofP. tomato wouldprovide useful information for these areas of interest.Transducing bacteriophages are useful genetic toolsfor analyzing closely linked bacterial genes.Bacteriophages are classified as either virulent, wherebyinfected cells lyse with liberation of new bacteriophageparticles, or temperate, in which case the infected bacte-ria either lyse with liberation of new bacteriophage proge-ny or become lysogenic (Hayes, 1968). In the lysogenicstate, the bacteriophage genome, now called a prophage,replicates in tandem with the bacterial chromosomeeither by establishing a plasmid form or by integratinginto the chromosome. Temperate bacteriophages capableof establishing lysogeny probably account for most trans-duction-mediated gene flow among bacteria in natureand provide systems that are most easily studied(Gerhardt et. al., 1981).The objectives of this study were to enrich for andcharacterize bacteriophages ofP. tomato suitable for trans-duction analysis. The characterization and analysis ofbac-teriophage isolates is useful for taxonomic purposesbecause they offer valuable methods to distinguish closelyrelated bacterial species (Cuppels, 1983). These methodsinclude bacteriophage sensitivity tests and fine structuremapping by transduction. Bacteriophage sensitivity pat-Chad E. PaschallUniversity ofArkansas for Medical Sciences4301 West MarkhamLittleRock, AR 72205terns have been used to show that the cherry and plumpathotypes of P. syringae pv. morsprunorum are two dis-tincthomogenous groups (Crosse and Garrett, 1963).Materials and MethodsIn preliminary tests, 44 strains of P. tomato wergrown in NBY media to determine the presence of ternperate bacteriophages and to determine which strainwould be suitable hosts for bacteriophage propagatioand enrichment (see Table 1). NBYmedia was used for abacterial strains (Vidaver, 1967). NBY soft agar (0.7 g/10ml) was used to create overlays for bacteriophage enrichment and titer (Adams, 1959). Media components werobtained from Fisher Scientific Co. (St. Louis, MO). Iithe initialsurvey for temperate bacteriophages, the bactcrialstrains tested were grown to stationary phase inNBYbroth at 28 °C and diluted in bacteriophage buffer to anoptical density of 0.15 at 600 nm. The bacteriophagbuffer consisted of 10 mM Tris-HCl and 10 mM MgSO,,pH 7.5. NBY thick agar plates containing 35-40 ml ofreshly prepared NBY agar were overlaid with 3 ml omolten NBY soft agar at 50 °C containing approximatelyx 107 cells per ml of the bacterial strain to be testedFollowing overnight incubation of these plates at 28 C(16-20 hours), 5 mlof bacteriophage buffer was added t(the surface of each overlay and the plates were incubatecat room temperature for 2 hours. The bacteriophagebuffer was drawn off each plate with a sterile Pasteupipet and placed into 30 ml Oak Ridge centrifuge tubeswhich were centrifuged at 10,000 rpm at 4°C for 10 to 3(minutes. The supernatants were removed and chloroformwas added to each of these preparations (5% v/v). Thesesupernatant solutions were tested immediately for bacteriophages or stored at 4"C for one to two days and theitested.To determine the presence ofbacteriophages, 5-10 (iof each supernatant was spotted onto overlays ofall 44 Ptomato strains in a 44 x 44 cross. Bacteriophages inplaques that formed were propagated by removing thesection of the soft agar containing the plaque by suctionusing a sterile Pasteur pipet and resuspending the agaiplug in 0.1 to 0.5 ml of bacteriophage buffer containingchloroform (5% v/v). This suspension was incubated atProceedings Arkansas Academy of Science, Vol.50, 1996137Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28Published by Arkansas Academy of Science, 1996138fable 1. Pseudomonas syringae pv. tomato strains tested forhage production.Source+Strains* Geographical OriginCanada10862AustraliaAustraliaNebraskaCaliforniaGeorgia3055531861AV80B19B88Bl17Bl18B120B121B122B125B181B191CNBP 1323JL1031LI053LI060LI075LI105LI120NCPPB880NCPPB2424PDDCC3357PT14iO /CNBP1318DC84-1(C92-10C3000PDDCC3647PST95SM78-1OKIDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsBenderGeorgiaFloridaDelawareNew JerseyCaliforniaCanadaGeorgiaGeorgiaFranceCaliforniaCaliforniaCaliforniaCaliforniaCaliforniaCaliforniaYugoslaviaSwitzerlandNew ZealandCaliforniaCaliforniaCaliforniaVenezuelaCanadaGreeceSwitzerlandCanadaCanadaEnglandAustraliaCanadaSouth AfricaCanadaGeorgiaCanadaOklahomaOklahomaOklahomaOklahomaBenderBenderBenderPT12PT17PT23.2*Strain numbers used are those devised by the originalsuppliers of the cultures (Denny, 1988). Abbreviations areCNBP, Collection National de BacteriesPhytopathogenes, Angers, France; NCPPB, NationalCollection of Plant Pathogenic Bacteria, Harpenden, UK;PDDCC, Plant Diseases Division Culture Collection,Auckland, NZ.t Sources of strains include the following: Bender, C.Bender, Oklahoma State University, USA; Cuppels, D. A.Cuppels, Agriculture Canada, Ontario, Canada; Denny, T.P. Denny, University ofGeorgia, USA.room temperature for 2 hours. Serial dilutions in bacte-riophage buffer were then spotted (5-10 fil)onto an over-lay of the host strains B19, PST6 or PDDCC3647. Isolatedplaques were picked and the entire procedure repeatedtwo more times to ensure purified bacteriophage prepa-rations. Purified bacteriophage isolates were titered by aspot titering method in which 10 \i\ aliquots of serial dilu-tions of bacteriophages in bacteriophage buffer werespotted onto soft agar overlays containing one of thethree bacterial hosts. A suitable dilution for enrichmentwas one that produced overlapping plaques (-1,000plaques per plate). The enrichment plates were incubatedovernight (16-20 hours) and overlaid with 5 ml of bacte-riophage buffer. The enriched bacteriophage prepara-tions were centrifuged and the supernatants were filteredinto 1.5 ml Eppendorf tubes through 0.2 urn SuporAcrodisc 32 filters (Gelman, Ann Arbor, MI). Tilers ofthe enriched bacteriophage isolates were determined bythe spot method or by the plate method in which 0.1 mlof appropriate bacteriophage dilutions in bacteriophagebuffer were added to 3 ml of molten soft agar at 50"Ccontaining the appropriate density of host cells (1 x 10"per ml). The tube was then mixed and poured over anNBY agar thick plate. Following overnight incubation at28°C, the plates were checked and the bacteriophage titerdetermined by counting plaques and multiplying by theplating factor and dilutions. High titer bacteriophagepreparations were stored at 4 C.Results and DiscussionPreliminary attempts to show bacteriophage produc-tion in broth cultures were unsuccessful. However, usingthe plate method described in the Materials and Methodssection, over 50% of strains gave evidence of plaque for-Proceedings Arkansas Academy of Science, Vol.50, 1996138Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28https://scholarworks.uark.edu/jaas/vol50/iss1/28139Table 2. Source, titer, and plaque morphology ofP. tomato bacteriophages.Host Strain Phage Isolate Source* Titert Plaque Morphology*PST6 7 AV80 1.5xlO10 <lmm,TPST6 9 PT30 1.2xl()l() l-2mm, TPST6 17 PT17 1.2x10"' l-2mm, TPST6 18 DC92-10 1..0xl() l() l-2mm, TB19 3 CNBP1323 2.3xlO10 2-3mm, C, HB19 21 PST95 4.7x10'° 2-3mm, T,HB19 27 T4B1 6.9x10'° 2-3mm, C, HB19 31 30555 3.0xl()10 2-3mm, C, HB19 33 B121 5.3x10'° 2-3mm, C, HB19 36 B125 6.1x10'° 2-3mm, C, HB19 40 10862 4.1x10'° 2-3mm, C, HB19 43 SM78-1 3.4x10'° 2-3mm, C, HPDDCC3647 5 JL1060 1.6x10'" l-2mm, TPDDCC3647 10 JL1053 1.6x10'° l-2mm, TPDDCC3647 14 PT23.2 1.5x10'° <lmm, TPDDCC3647 15 PT14 4.8x10'° l-2mm, TPDDCC3647 16 DC84-1 3.0x10'° l-2mm, TPDDCC3647 22 JL1075 1.0x10'° l-2mm, TPDDCC3647 23 PT21 4.2x10'° <lmm, TPDDCC3647 24 JL1031 2.3x10'° <lmm, T*Refer to Table 1t Titers (pfu/ml) are the highest achieved from plate enrichments as determined by either spot titers orplate titers asindicated in the Material and Methods. Titers were repeated at least twice for each bacteriophage isolate withduplicatesamples counted and an average determined in each instance.*C = clear, H= halo and T= turbidProceedings Arkansas Academy of Science, Vol.50, 1996139Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28Published by Arkansas Academy of Science, 1996140Partial Characterization ofBacteriophages ofPseudomonas syringae pv. tomatoobtained in this study have not been rigorously examined.Nonetheless, plaque forming ability in supernatants fromstrains of diverse geographical orgin was observed,bacteriophage isolates propagated and enriched on strain$19 from California came from such disparate locationsas Australia, France, South Africa and Venezuela. Incon-rast, strain PDDCC3647 from Australia showed goodDlaque formation using supernatants obtained only fromCalifornia and Canada strains. The basis of these differ-ences is not clear. It does indicate however that P. tomatotrains which harbor bacteriophage are ubiquitous andraises the possibility that bacteriophages may play a roleingene transfer on plant surfaces (Kidambi et. al., 1994).Demonstration of transducing activity for bacteriophagesisolated from supernatants of P. syringae pv. syringaestrains indicates that the bacteriophage isolates obtainedin this study also may be useful for this purpose (Nordeenand Currier, 1983). Further physical characterization ofthe bacteriophage isolates including electron microscopyof density gradient purified bacteriophage particles andrestriction enzymes digests of bacteriophage DNA is inprogress and should establish the identity of the bacterio-phage isolates.ACKNOWLEDGEMENTS.—Funding of this research wasprovided by a student undergraduate research fellowshipSURF) from the Arkansas Science Information LiaisonOffice (SILO) and an academic research grant from theJniversity ofArkansas at Monticello.Jones, J.B., J.P.Jones, R.E. Stall and T.A.Zitter. 1991.Compendium ofTomato Diseases. AmericanPhytopathological Society Press, pp. 26-30.Jones, J.B., S.M. McCarter and R.O. Gitaitis. 1981.Association ofPseudomonas syringae pv. syringae with aleaf spot disease oftomato transplants in southernGeorgia. Phytopathology 71:1281-1285.Kidambi, S.P., S. Ripp and R.V. Miller.1994. Evidencefor phage-mediated gene transfer among Pseudomonasaeruginosa strains on the phylloplane. Appl.Environ.Microbiol.60:496-500.Nordeen, R.O. and T.C. Currier. 1983. Generalizedtransduction in the phytopathogen Pseudomonassyringae. Appl. Environ. Microbiol.45:1884-1889.Nordeen, R.O., M.K.Morgan and T.C. Currier. 1983.Isolation and partial characterization ofbacterio-phages of the phytopathogen Pseudomonas syringae.Appl. Environ. Microbiol.45:1890-1898.Vidaver, A.K.1967. Synthetic and complex media for therapid detection of fluorescence ofphytopathogenicpseudomonads: effect of the carbon source. Appl.Microbiol.15:1523-1524.Whalen, M.C. and B.J. Staskawicz. 1990. Molecular basisof host range and specificity in two bacterialpathogens of tomato. Wiley-Liss, Inc. pp.285-290.Literature CitedAdams, M.K. 1959. Bacteriophages. IntersciencePublications Inc. New York. 592 pp.Baigent, N.L.,J.E. DeVay and M.P. Starr. 1963.Bacteriophages ofPseudomonas syringae. N.Z. J. Sci.6:75-100.rosse, J.E. and C.M.E. Garrett. 1963. Studies on thebacteriophagy ofPseudomonas mors-prunorum, Ps.syringae and related organisms. J. Appl.Bacteriol.26:159-177.fuppels, D.A. 1983. Isolation and characterization ofphages useful foridentifying Pseudomonas syringae pv.tomato. Phytopathology 73:1376-1381.Denny, T.P. 1988. Phenotype diversity inPseudomonassryingae pv. tomato]. Gen. Microbiol.134:1939-1948.Gerhardt, D.,R.G.E. Murray, R.N. Costilow, E.W.Nester, W.A. Wood, N.R. Kriegand G. BriggsPhillips. 1981. Manual ofMethods for GeneralBacteriology. American Society for Microbiology,pp. 222-265.Hayes, W. 1968. The genetics ofbacteria and their virus-es. Blackwell Scientific Publications, London. 925 pp.Proceedings Arkansas Academy of Science, Vol.50, 1996140Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28https://scholarworks.uark.edu/jaas/vol50/iss1/28

English

Partial Characterization of Bacteriophages of Pseudomonas syringae pv.tomato

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 50 Article 281996Partial Characterization of Bacteriophages ofPseudomonas syringae pv.tomatoScott M. MinorUniversity of Arkansas at MonticelloRussell O. NordeenUniversity of Arkansas at MonticelloChad E. PaschallUniversity of Arkansas for Medical SciencesFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Bacterial Infections and Mycoses Commons, and 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 General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the ArkansasAcademy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu.Recommended CitationMinor, Scott M.; Nordeen, Russell O.; and Paschall, Chad E. (1996) "Partial Characterization of Bacteriophages of Pseudomonassyringae pv.tomato," Journal of the Arkansas Academy of Science: Vol. 50 , Article 28.Available at: http://scholarworks.uark.edu/jaas/vol50/iss1/28137Partial Characterization ofBacteriophages ofPseudomonas syringae pv. tomatoScott M.Minor and Russell O. NordeenUniversity ofArkansas at MonticelloDivision of Mathematics and SciencesMonticello, AR71656IntroductionSince the first major outbreak of bacterial speck in1978, this disease has been a major problem for tomatogrowers in certain regions of North America. This dis-ease, characterized by small brown to black lesions, iscaused by Pseudomonas syringae pv. tomato (Jones et. al.,1991). While bacterial speck is not a threat to the plantitself, it reduces the marketable value of the fruit bydecreasing its aesthetic appeal to the consumer. The taxo-nomic status ofP. tomato is uncertain at present. It is diffi-cult to distinguish morphologically and physiologicallyfrom other pathovars of the species, particularlyPseudomonas syringae pv. syringae. P. syringae pv. syringaehas also been characterized from necrotic lesions oninfected tomato fruit (Jones et. al., 1981). P. tomato is alsoof interest because it has been used as a model in plantpathogen studies with Arabidopsis thaliana, to gain a bet-ter understanding of the molecular basis of bacterial dis-ease resistance (Whalen and Staskawicz, 1990). A compre-hensive fine structure genetic analysis ofP. tomato wouldprovide useful information for these areas of interest.Transducing bacteriophages are useful genetic toolsfor analyzing closely linked bacterial genes.Bacteriophages are classified as either virulent, wherebyinfected cells lyse with liberation of new bacteriophageparticles, or temperate, in which case the infected bacte-ria either lyse with liberation of new bacteriophage proge-ny or become lysogenic (Hayes, 1968). In the lysogenicstate, the bacteriophage genome, now called a prophage,replicates in tandem with the bacterial chromosomeeither by establishing a plasmid form or by integratinginto the chromosome. Temperate bacteriophages capableof establishing lysogeny probably account for most trans-duction-mediated gene flow among bacteria in natureand provide systems that are most easily studied(Gerhardt et. al., 1981).The objectives of this study were to enrich for andcharacterize bacteriophages ofP. tomato suitable for trans-duction analysis. The characterization and analysis ofbac-teriophage isolates is useful for taxonomic purposesbecause they offer valuable methods to distinguish closelyrelated bacterial species (Cuppels, 1983). These methodsinclude bacteriophage sensitivity tests and fine structuremapping by transduction. Bacteriophage sensitivity pat-Chad E. PaschallUniversity ofArkansas for Medical Sciences4301 West MarkhamLittleRock, AR 72205terns have been used to show that the cherry and plumpathotypes of P. syringae pv. morsprunorum are two dis-tincthomogenous groups (Crosse and Garrett, 1963).Materials and MethodsIn preliminary tests, 44 strains of P. tomato wergrown in NBY media to determine the presence of ternperate bacteriophages and to determine which strainwould be suitable hosts for bacteriophage propagatioand enrichment (see Table 1). NBYmedia was used for abacterial strains (Vidaver, 1967). NBY soft agar (0.7 g/10ml) was used to create overlays for bacteriophage enrichment and titer (Adams, 1959). Media components werobtained from Fisher Scientific Co. (St. Louis, MO). Iithe initialsurvey for temperate bacteriophages, the bactcrialstrains tested were grown to stationary phase inNBYbroth at 28 °C and diluted in bacteriophage buffer to anoptical density of 0.15 at 600 nm. The bacteriophagbuffer consisted of 10 mM Tris-HCl and 10 mM MgSO,,pH 7.5. NBY thick agar plates containing 35-40 ml ofreshly prepared NBY agar were overlaid with 3 ml omolten NBY soft agar at 50 °C containing approximatelyx 107 cells per ml of the bacterial strain to be testedFollowing overnight incubation of these plates at 28 C(16-20 hours), 5 mlof bacteriophage buffer was added t(the surface of each overlay and the plates were incubatecat room temperature for 2 hours. The bacteriophagebuffer was drawn off each plate with a sterile Pasteupipet and placed into 30 ml Oak Ridge centrifuge tubeswhich were centrifuged at 10,000 rpm at 4°C for 10 to 3(minutes. The supernatants were removed and chloroformwas added to each of these preparations (5% v/v). Thesesupernatant solutions were tested immediately for bacteriophages or stored at 4"C for one to two days and theitested.To determine the presence ofbacteriophages, 5-10 (iof each supernatant was spotted onto overlays ofall 44 Ptomato strains in a 44 x 44 cross. Bacteriophages inplaques that formed were propagated by removing thesection of the soft agar containing the plaque by suctionusing a sterile Pasteur pipet and resuspending the agaiplug in 0.1 to 0.5 ml of bacteriophage buffer containingchloroform (5% v/v). This suspension was incubated atProceedings Arkansas Academy of Science, Vol.50, 1996137Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28Published by Arkansas Academy of Science, 1996138fable 1. Pseudomonas syringae pv. tomato strains tested forhage production.Source+Strains* Geographical OriginCanada10862AustraliaAustraliaNebraskaCaliforniaGeorgia3055531861AV80B19B88Bl17Bl18B120B121B122B125B181B191CNBP 1323JL1031LI053LI060LI075LI105LI120NCPPB880NCPPB2424PDDCC3357PT14iO /CNBP1318DC84-1(C92-10C3000PDDCC3647PST95SM78-1OKIDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyDennyCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsCuppelsBenderGeorgiaFloridaDelawareNew JerseyCaliforniaCanadaGeorgiaGeorgiaFranceCaliforniaCaliforniaCaliforniaCaliforniaCaliforniaCaliforniaYugoslaviaSwitzerlandNew ZealandCaliforniaCaliforniaCaliforniaVenezuelaCanadaGreeceSwitzerlandCanadaCanadaEnglandAustraliaCanadaSouth AfricaCanadaGeorgiaCanadaOklahomaOklahomaOklahomaOklahomaBenderBenderBenderPT12PT17PT23.2* Strain numbers used are those devised by the originalsuppliers of the cultures (Denny, 1988). Abbreviations areCNBP, Collection National de BacteriesPhytopathogenes, Angers, France; NCPPB, NationalCollection of Plant Pathogenic Bacteria, Harpenden, UK;PDDCC, Plant Diseases Division Culture Collection,Auckland, NZ.t Sources of strains include the following: Bender, C.Bender, Oklahoma State University, USA; Cuppels, D. A.Cuppels, Agriculture Canada, Ontario, Canada; Denny, T.P. Denny, University ofGeorgia, USA.room temperature for 2 hours. Serial dilutions in bacte-riophage buffer were then spotted (5-10 fil)onto an over-lay of the host strains B19, PST6 or PDDCC3647. Isolatedplaques were picked and the entire procedure repeatedtwo more times to ensure purified bacteriophage prepa-rations. Purified bacteriophage isolates were titered by aspot titering method in which 10 \i\ aliquots of serial dilu-tions of bacteriophages in bacteriophage buffer werespotted onto soft agar overlays containing one of thethree bacterial hosts. A suitable dilution for enrichmentwas one that produced overlapping plaques (-1,000plaques per plate). The enrichment plates were incubatedovernight (16-20 hours) and overlaid with 5 ml of bacte-riophage buffer. The enriched bacteriophage prepara-tions were centrifuged and the supernatants were filteredinto 1.5 ml Eppendorf tubes through 0.2 urn SuporAcrodisc 32 filters (Gelman, Ann Arbor, MI). Tilers ofthe enriched bacteriophage isolates were determined bythe spot method or by the plate method in which 0.1 mlof appropriate bacteriophage dilutions in bacteriophagebuffer were added to 3 ml of molten soft agar at 50"Ccontaining the appropriate density of host cells (1 x 10"per ml). The tube was then mixed and poured over anNBY agar thick plate. Following overnight incubation at28°C, the plates were checked and the bacteriophage titerdetermined by counting plaques and multiplying by theplating factor and dilutions. High titer bacteriophagepreparations were stored at 4 C.Results and DiscussionPreliminary attempts to show bacteriophage produc-tion in broth cultures were unsuccessful. However, usingthe plate method described in the Materials and Methodssection, over 50% of strains gave evidence of plaque for-Proceedings Arkansas Academy of Science, Vol.50, 1996138Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28http://scholarworks.uark.edu/jaas/vol50/iss1/28139Table 2. Source, titer, and plaque morphology ofP. tomato bacteriophages.Host Strain Phage Isolate Source* Titert Plaque Morphology*PST6 7 AV80 1.5xlO10 <lmm,TPST6 9 PT30 1.2xl()l() l-2mm, TPST6 17 PT17 1.2x10"' l-2mm, TPST6 18 DC92-10 1..0xl() l() l-2mm, TB19 3 CNBP1323 2.3xlO10 2-3mm, C, HB19 21 PST95 4.7x10'° 2-3mm, T,HB19 27 T4B1 6.9x10'° 2-3mm, C, HB19 31 30555 3.0xl()10 2-3mm, C, HB19 33 B121 5.3x10'° 2-3mm, C, HB19 36 B125 6.1x10'° 2-3mm, C, HB19 40 10862 4.1x10'° 2-3mm, C, HB19 43 SM78-1 3.4x10'° 2-3mm, C, HPDDCC3647 5 JL1060 1.6x10'" l-2mm, TPDDCC3647 10 JL1053 1.6x10'° l-2mm, TPDDCC3647 14 PT23.2 1.5x10'° <lmm, TPDDCC3647 15 PT14 4.8x10'° l-2mm, TPDDCC3647 16 DC84-1 3.0x10'° l-2mm, TPDDCC3647 22 JL1075 1.0x10'° l-2mm, TPDDCC3647 23 PT21 4.2x10'° <lmm, TPDDCC3647 24 JL1031 2.3x10'° <lmm, T*Refer to Table 1t Titers (pfu/ml) are the highest achieved from plate enrichments as determined by either spot titers orplate titers asindicated in the Material and Methods. Titers were repeated at least twice for each bacteriophage isolate withduplicatesamples counted and an average determined in each instance.*C = clear, H= halo and T= turbidProceedings Arkansas Academy of Science, Vol.50, 1996139Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28Published by Arkansas Academy of Science, 1996140Partial Characterization ofBacteriophages ofPseudomonas syringae pv. tomatoobtained in this study have not been rigorously examined.Nonetheless, plaque forming ability in supernatants fromstrains of diverse geographical orgin was observed,bacteriophage isolates propagated and enriched on strain$19 from California came from such disparate locationsas Australia, France, South Africa and Venezuela. Incon-rast, strain PDDCC3647 from Australia showed goodDlaque formation using supernatants obtained only fromCalifornia and Canada strains. The basis of these differ-ences is not clear. It does indicate however that P. tomatotrains which harbor bacteriophage are ubiquitous andraises the possibility that bacteriophages may play a roleingene transfer on plant surfaces (Kidambi et. al., 1994).Demonstration of transducing activity for bacteriophagesisolated from supernatants of P. syringae pv. syringaestrains indicates that the bacteriophage isolates obtainedin this study also may be useful for this purpose (Nordeenand Currier, 1983). Further physical characterization ofthe bacteriophage isolates including electron microscopyof density gradient purified bacteriophage particles andrestriction enzymes digests of bacteriophage DNA is inprogress and should establish the identity of the bacterio-phage isolates.ACKNOWLEDGEMENTS.—Funding of this research wasprovided by a student undergraduate research fellowshipSURF) from the Arkansas Science Information LiaisonOffice (SILO) and an academic research grant from theJniversity ofArkansas at Monticello.Jones, J.B., J.P.Jones, R.E. Stall and T.A.Zitter. 1991.Compendium ofTomato Diseases. AmericanPhytopathological Society Press, pp. 26-30.Jones, J.B., S.M. McCarter and R.O. Gitaitis. 1981.Association ofPseudomonas syringae pv. syringae with aleaf spot disease oftomato transplants in southernGeorgia. Phytopathology 71:1281-1285.Kidambi, S.P., S. Ripp and R.V. Miller.1994. Evidencefor phage-mediated gene transfer among Pseudomonasaeruginosa strains on the phylloplane. Appl.Environ.Microbiol.60:496-500.Nordeen, R.O. and T.C. Currier. 1983. Generalizedtransduction in the phytopathogen Pseudomonassyringae. Appl. Environ. Microbiol.45:1884-1889.Nordeen, R.O., M.K.Morgan and T.C. Currier. 1983.Isolation and partial characterization ofbacterio-phages of the phytopathogen Pseudomonas syringae.Appl. Environ. Microbiol.45:1890-1898.Vidaver, A.K.1967. Synthetic and complex media for therapid detection of fluorescence ofphytopathogenicpseudomonads: effect of the carbon source. Appl.Microbiol.15:1523-1524.Whalen, M.C. and B.J. Staskawicz. 1990. Molecular basisof host range and specificity in two bacterialpathogens of tomato. Wiley-Liss, Inc. pp.285-290.Literature CitedAdams, M.K. 1959. Bacteriophages. IntersciencePublications Inc. New York. 592 pp.Baigent, N.L.,J.E. DeVay and M.P. Starr. 1963.Bacteriophages ofPseudomonas syringae. N.Z. J. Sci.6:75-100.rosse, J.E. and C.M.E. Garrett. 1963. Studies on thebacteriophagy ofPseudomonas mors-prunorum, Ps.syringae and related organisms. J. Appl.Bacteriol.26:159-177.fuppels, D.A. 1983. Isolation and characterization ofphages useful foridentifying Pseudomonas syringae pv.tomato. Phytopathology 73:1376-1381.Denny, T.P. 1988. Phenotype diversity inPseudomonassryingae pv. tomato]. Gen. Microbiol.134:1939-1948.Gerhardt, D.,R.G.E. Murray, R.N. Costilow, E.W.Nester, W.A. Wood, N.R. Kriegand G. BriggsPhillips. 1981. Manual ofMethods for GeneralBacteriology. American Society for Microbiology,pp. 222-265.Hayes, W. 1968. The genetics ofbacteria and their virus-es. Blackwell Scientific Publications, London. 925 pp.Proceedings Arkansas Academy of Science, Vol.50, 1996140Journal of the Arkansas Academy of Science, Vol. 50 [1996], Art. 28http://scholarworks.uark.edu/jaas/vol50/iss1/28

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Partial Characterization of Bacteriophages of Pseudomonas syringae pv.tomato

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ScholarWorks@UARK