Journal of the Arkansas Academy of Science

Volume 61 Article 14

2007 Characterization of Bacteriophages of syringae pv. tomato Sara E. Prior University of Arkansas at Monticello

Amanda J. Andrews University of Arkansas at Monticello

Russell O. Nordeen University of Arkansas at Monticello, [email protected]

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Recommended Citation Prior, Sara E.; Andrews, Amanda J.; and Nordeen, Russell O. (2007) "Characterization of Bacteriophages of pv. tomato," Journal of the Arkansas Academy of Science: Vol. 61 , Article 14. Available at: http://scholarworks.uark.edu/jaas/vol61/iss1/14

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 [email protected], [email protected]. Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Characterization ofBacteriophages ofPseudomonas syringae pv. tomato

I 1 SARA E. PRIORI, AMANDA 1. ANDREWS , AND RUSSELL O. NORDEEN ,2 an WI 'School ofMathematical and Natural Sciences, University ofArkansas at Monticel1o, 397 University Drive, Monticel1o, AR 71656 11 ac 2Correspondence: [email protected] S(I to Abstract.-Bacteriophages from supernatants ofthe plant pathogenic Pseudomonas syringae pv. tomato (P. tomato) were isolated, enriched, and purified by density block centrifugation in cesium chloride (CsCl) step gradients. The DNA from purified O. phage was isolated and digested with the restriction endonucleases EcoRl or HindIII. Three different DNA fingerprint patterns were iJJ determined indicating 3 unique phage isolates. Genome sizes ofthe phage ranged from 40 to 52 kilobases (kB). Buoyant densities of N phage particles in CsCI varied from 1.36 to 1.51 glml. Electron microscopy revealed a single morphological type with an elongated polyhedral head and a long tail indicating the family Siphoviridae.

Key words:- Bacteriophages, Pseudomonas syringae pv. tomato, DNA, endonucleases, EeoR1, HindlII, Siphoviridae. CI e: Introduction the .suitability of phage in controlling bacterial plant diseases o (Flaherty et al. 2000, Flaherty et aJ. 2001, Obradovic et al. a Bacterial speck, first described and identified in Taiwan and 2004). Further genetic knowledge ofphage used inthese studies F the United States in 1933, caused a major outbreak in 1978, and would be beneficial and might lead to a more efficient use ofthis v the disease has become a major problem for tomato growers in disease-control strategy. certain regions ofNorth America. Characterized by small brown The objectives of this study were to enrich for, then to black lesions on various tomato tissues, this disease is caused characterize bacteriophages ofP. tomato by electron microscopy by the bacterial pathogen Pseudomonas tomato (Jones et al. and restriction endonuclease digestion. This approach should 1991). Bacterial speck is not significantly harmful to the plant reveal unique phage isolates for further transduction and itself, but severe spotting on the fruit can reduce marketable possible DNA sequencing studies. The infonnation from these yield. studies should clarify the role these phage play in host-pathogen The taxonomic status ofP. tomato is unclear. It is difficult relationships between P. tomato strains and appropriate host to distinguish morphologically and physiologically from other . pathovars ofthe species, particularly Pseudomonas syringae pv. s}Tingae (P. syringae). Pathovars ofP. syringae cause disease on different plant hosts, but P. syringae has also been characterized Materials and Methods from necrotic lesions on infected tomato fruit (Jones et al. 1981). Bacterial Strains and Media.-Three P. tomato strains, Pseudomonas tomato is also of interest as it has been used B19, Pst6, and PDDCC3647, which had proven to be suitable as a model pathogen in studies with Arabidopsis tha/iana, a hosts for phage propagation and enrichment (Minor et al. 1996), common lawn weed ofearly spring that has proven amenable to were used for the isolation and characterization ofphages in this molecular genetic manipulations. Researchers are studying P. study. NBY media was used for all bacterial strains (Vidaver tomato infections ofA. thaliana to gain a better understanding 1967). NBYsoft agar (0.7g1100 rnl) was used to create overlays ofthe molecular basis of bacterial disease and bacterial-disease for phage enrichment andtiter(Adams 1959). Media components resistance (Whalen and Staskawicz 1990). The genomes ofA. as well as all reagents for phage enrichment and DNA isolation thaliana and P. tomato strain De3000 have been sequenced; were obtained from Fisher (St. Louis, MO). however, there is limited knowledge of P. tomato temperate Propagation and Enrichment ofBacteriophages.-NBY phage (Cuppels 1983, Minor et at. 1996). thick agar plates containing 35 to 40 ml offreshly prepared NBY Temperate phages capable of lysogeny probably account agar were overlaid with 3 ml ofmolten NBY soft agar at 50° C for most transduction-mediated gene flow among bacteria in containing approximately 5 x 107 cells per ml ofthe appropriate nature and provide genetic systems that are most easily studied propagating strain. These strains were grown to stationary phase (Gerhardt et at. 1984, Kidambi et al. 1993). Genomic sequencing in NBY broth at 28° C and diluted in phage buffer to an optical of a number of plant pathogenic bacteria has revealed that a denslty of 0.15 at 600 nm. The phage buffer consisted of 10 • substantial portion of these genomes contain mobile genetic roM Tris HCI. pH 7.5, and IO roM MgS04 Following overnight elements, including up to 7% prophage sequences (Buell et al. incubation at 28° C (I5 to 18 hours), 5 ml ofphage buffer was 2003, Simpson et aI. 2000). The role of phage in the disease added to the surface ofthe overlay, and the plates were incubated process caused by these bacterial pathogens is unclear at at room temperature for 30 minutes to 2 hours or 4° C overnight. present. Studies have recently been performed to investigate The phage buffer was then drawn offwith a sterile Pasteur pipet

Published by Arkansas Academy of Science,Journal 2007 oftheArkansas Academy ofScience, VoL 61, 2007 84 Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Characterization ofBacteriophages ofPseudomonas s)'ringae pv. tomato

and placed into 30-ml Oak Ridge centrifuge tubes. The tubes gels in TAE buffer at a constant voltage of 5 to 6 volts per cm. 56 were centrifuged at 10,000 rpm at 4° C for at least 10 minutes. EcoRI and HindlII endonucleases were purchased from Promega The supernatants were filtered through a 0.2-mm cellulose (Milwaukee, WI). Size standards for gel electrophoresisconsisted acetate filter into sterile 1.5-ml Eppendorf tubes. These phage ofbacteriophage lambda DNA digested with HindU!. Following solutions were stored at 4° C until they were used in experiments staining of gels with ethidium bromide (4 mg/mI), gels were to determine phage titer. destained in distilled water and documented with Polaroid film were Phage enrichments for DNA isolation were prepared using or an Alpha Innotech imaging system. Sizes of DNA fragments ilied 0.35% (w/v) of the synthetic gelling agent gelrite to avoid were determined by plotting the log of the molecular weight of were inhibition of restriction endonucleases (Bouche 198I ). The DNA fmgments observed against the distance traveled in mOl. :s of NBY thick-plate underlay contained 0.74% (w/v) gelrite. The ated enrichment plates were incubated overnight (15-I 8 hours) at 28° Cand overlaid with 5 ml ofphage buffer for 30 min at room Results temperature. The enriched bacteriophage preparations were centrifuged and filtered as previously described. Titers of the Three strains of P. tomato (819, Pst6, and PDDCC3647) enriched bacteriophage isolates were determined by the plate­ were used to enrich bacteriophages. These strains were selected overlay method (Adams 1959). Following overnight incubation on the basis ofa preliminary survey for bacteriophage production lSe5 al. at 28° C, the plates were checked and the phage titer determined. by 44 P. tomato stmins (Minor et al. 1996). Plate enrichments 9 10 lies High-titer phage isolates, >10 pfu (plaque forming units) perml, using these 3 hosts produced titers of>10 pfu (plaque forming 10 this were further concentrated to >10 pfu per ml by polyethylene units) per ml after concentmtion with PEG6000. (Table I). glycol 6000 precipitation (Yamamoto et al. 1970). High-titer Initial attempts to purify phage using CsCI block gradients len phage stocks were purified by CsCI block density gradients containing three 2.4-ml layers of 1.7 glml, 1.5 g/ml, and 1.45 lpy (Davis et al. 1980). glml caused the phage to band in the upper third of the 12-ml tJ1d Electron Microscopy.-Phages purified from CsC! gradients tubes used. Centrifugations were performed in a SW41 rotor Uld were dialyzed in two changes ofphage buffer overnight at 4° C at 23,000 rpm for 2 hours at room tempemture. Subsequent :se to remove CsCI. The phage titers were determined and samples purifications used five 2-ml layers of 1.7 glml, 1.5 glml, 1.45 with a titer of >I0 10 pfu per ml were spread onto grids with glml, 1.3 glml, and 1.2 glml and 1.0 ml of enriched phage in :en )st carbon-eoated Fonnvar support films, negatively stained with phage buffer. Using this protocol, phage banded near the middle 1% uranyl acetate, and examined with a Joel 1200EX electron of the centrifugation tubes; although both upper diffuse and microscope. Phage DNA Extraction and Digestion with Restriction Endonucleases.-EDTA was added to purified phage at a Table 1. Source ofP. tomalo bacteriophages. 20-mM final concentration. The phage were treated with Phage Source proteinase K at 20 mglml and 0.5% Sodium Dodecyl Sulphate Geographicat Propagation IS, Isolate Strain" Origin (5DS) at 56° C for 1 to disrupt phage heads and digest phage Strain" Ie hr proteins. An equal volume of phenol was added to extmct i), Pra7 AV80 Nebraska PST6 proteins followed by an equal volume of chloroform:isoamyl is Pra9 PI30 California PST6 alcohol (24:1 v/v) to remove remaining proteins and the phenol. Pro 17 PIl7 Oklahoma PST6 is The supernatant containing phage DNA was precipitated by Pral8 DCn-1O Canada PST6 adding a one-tenth volume of3-M sodium acetate (pH 5.2) and 2 Pra3 CN BP1323 France ts BI9 volumes ofabsolute alcohol followed by overnight incubation at Pra21 PST95 South Africa n BI9 -20" C. DNA was collected by centrifugation at 14,000 rpm in a Pra27 14BI Canada BI9 Pra31 30555 microcentrifuge for 10 minutes and washed in 70% ethanol and Australia 819 '/. Pra33 BI21 centrifuged again at 14,000 rpm for 2 minutes. New JerS<.')' HI9 ( Pro36 BI25 Canada BI9 After pellets had dried at room tempemture, they were Pro4O 10862 Canada 819 resuspended in 50 microliters ofTE buffer (IO mM Tris 1 tllivt Pr043 SM78-1 Georgia BI9 EDTA pH 8.0). Ten-microliter samples were run on 0.7% Pro5 JLlO6O California PDDCC3647 ProlO agarose gels to determine purity and estimate the volume JLl053 California PDDCC3647 Pro I4 needed for restriction endonuclease digestion. Approximately PT23.2 Oklahoma PDOCC3647 Prol5 PII4 one microgmm of phage DNA was digested in appropriate California PDOCC3647 Pro16 OC84-1 bUffers containing RNase at a final concentmtion of 10 mglml to Canada PDOCC3647 Pra22 JLl075 eliminate RNA, which was abundant in most samples. California PDOCC3647 hol3 PI21 Restriction endonuclease digests were incubated with 2 to California PDOCC3647 Pro24 JLI031 California 6 units of EcoRI or HindIlI per microgmm of DNA at 37° C PDOCC3641 for 3 hours. DNA digests were electrophoresed in 0.7% agarose "Minor et al. 1996

http://scholarworks.uark.edu/jaas/vol61/iss1/14 85 Journal oftheArkansasAcademy ofSciencey VoL 61,2007 Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Sara E. Prior, Amanda J. Andrews, and Russell O. Nordeen

c

Fig. I. Electron micrographs ofnegatively-stained Pseudomonas tomato phages A, PtaI6, B, Pto21,C, Pto33. Bar in A and B is 100 nm. Bar in C is 200nm.

lower intense bands were noted for most phage. Multiple bands, especially oflower density, may contain phage ghosts devoid of DNA. Phage titers oflower-density bands had 2-10 times fewer pfulml than higher-density bands (data not shO\\n). Phage were removed from the sides of the centrifuge tubes using a sterile 18-gauge needle. They were dialyzed twice overnight against 1.0 liter ofphage buffer at 4° C in dialysis tubing with an 8,000 dalton molecular weight cutoff. Phage densities were determined using a refractometer. Phage densities ranged from 1.36 to 1.51 glml as shown in Table 2. Purified phage with titers of >10 10 pfu/ml were used for EM analysis and DNA extraction. Only one morphological group was observed in EM studies of the enriched and purified phage (Fig. I). This morphology has been previously reported Fig. 2. Electrophoretic patterns ofEeoRI digests ofbacteriophage for phage from P. tomato strains (Cuppels 1983). The phage DNA (O. 7% gel). A. bacteriophage lambda digested \\ithHindlU. examined by EM in this study each had a long flexible tail and a Size oflambda standards are indicated to the left in kB. polyhedral head. Dimensions ofphage heads and tails are given in Table 2. sequence since these are known to be common in the P. tomato EeoRI and HindUI restriction endonuclease patterns genome (Buell et al. 2003). Pto21 appears to be unique based indicated 3 unique phage isolates as sho\\n in Fig. 2. Phage from on restriction fragment patterns with EeoRI and HindIlI. group I has a genome size of52 kB, phage from group II has a genome size of40 kB, and phage from group III has a genome size of48 kB (Table 2). All phage propagated on P. tomato strain Discussion 3647 (group I) gave similar restriction fragment patterns with EeoRI and HindIII (data not sho\\n). All phage except Pto21 Our results confirm earlier investigations of P. tomato (group II) propagated on P. tomato strain B19 and strain Pst6 also phage that indicated that approximately 50% ofP. tomato strains gave similar EeoRI and HindIU DNA fingerprints (group Ill) contain what appear to be temperate phage (Cuppels 1983). ~vitb the exception ofPta18. Pto18 has an extra DNA fragment Induction studies using UV and mitomycin C failed to yield of approximately 5 kB for a total genome size of 53 kB. We phage from the propagating strains (data not shown). Group I suspect that this additional DNA fragment may be an insertion phage failed to plaque onP. tomato strains PST6 and B19. Group

Published by Arkansas Academy of Science, 2007 86 Journal oftheArkansas Academy ofScience, Vol. 61, 2007 Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Cbaracterization ofBacteriophages ofPseudomonas syringae pv. tomato

Table 2. Phage characteristics.

Phage Size (nm) Phage Plaque Morphology* Genome Size (kB) Head Tail Density (glml) Type

Pto7

*C '" clear, H == halo, T = turbid NlD=not detennined

II and TIl phage failed to plaque on P. tomato strain 3647. This host strains that produced these phage were isolated in North ~ay indicate a host range difference ofthese phage or possibly America, Europe, and even Australia (strain 30555), and yet ~unity due to prophage in the propagating strains that can not DNA fingerprinting indicates the phage isolates are identical. be mduced. Phage Pto2I appears to produce plaques with clear Phage Ptoll from a South African strain appears to be distinct e edges indicating that this phage may be capable ofrapid lysis. from the other phage based on DNA fingerprinting. Although !he phage in this study appear similar in morphology to those this is not surprising given the geographical origin ofthis strain, ISolated by Cuppels (1983) and Nordeen (Nordeen et al. 1983); the significance ofthis finding is not clear at present. All ofthe aIth~Ugh the heads of group I and II phage were elongated and phage isolated in this study appear to be suitable for transduction not ISometric as those isolated by Cuppels. analyses based on their genome size. These studies would be The long, flexible, tail and polyhedral heads place these useful in determining lateral transmission of virulence genes Phage in the family Siphoviridae (Ackennan 1973). The phage on the phylloplane surface (Kidambi et al. 1993). Sequencing propagated on P. tomato strain 3647 appear to be identical of these phage should elucidate the role they play in disease based on DNA fingerprinting with EeoRI and HindIII. This development and may reveal additional novel genes of interest result is not surprising since the P. tomato strains from which as demonstrated in the recent sequencing of 18 P. aeruginosa ~ese phage were isolated all came from California initially phage (Kwan et aI. 2006) in which over 50% of open reading ~mclUding strain PT23.2) except Ptol6, which was isolated frames were novel. . om the Canadian strain DC84-1. The history ofthese strains IS unknown, but given their limited geographic origin, it was not ACK..,",oWLEIXiME1'ITS.-This study could not have been tln:xpected that the phage isolated from them might be similar completed without the assistance ofa number of people, some ~r '~tical. Surprisingly, the type III phage isolates came from of whom deserve special mention. Thanks to Drs. Ed Bacon aetenal host strains with a diverse geographical origin. The and Marvin Fawley of UAM for use ofequipment and supplies

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R7 - Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Sara E. Prior, Amanda J. Andrews, and Russell O. Nordeen

located in their labs. Thanks to Dr. John Iandolo ofthe University Pseudomonas syringae pv. syringae with a leafspot disease \1 ofOklahoma Medical School Department ofMicrobiology and oftomato transplants in southern Georgia. Phytopathology Immunology for the use of his lab and department equipment 71:1281-1285. for most of this study. I also am indebted to Dr. landolo for Kidambi S, PS Ripp, and RV Miller. 1993. Evidence for Phage- Y his expert advice and encouragement of this research. Thanks Mediated Gene Transfer among Pseudomonas aernginosa to Ben Fowler of the Oklahoma Medical Research Foundation Strains on the Phylloplane. Applied and Environmental for the excellent EM of phage in this study. Finally I thank the Microbiology 60:496-500. UAM Academic Research granting agency for funds used in this Kwan T, J Liu, M DuBow, P Gros and J Pelletier. 2006. investigation. Comparative Genomic Analysis of 18 Pseudomonas aeruginosa Bacteriophages. Journal of Bacteriology 188:1184-1187. Literature Cited Minor SM, RO Nordeen and CE Paschall. 1996. Patrial Characterization ofBacteriophages ofPseudomonas syringae Ackerman HW 1973. Tailed bacteriophages: Listing by pv. tomato. Proceedings oftheArkansas Academy ofScience morphological group. In: A.I. Laskin and H. A. Lechevalier, 50:137-140. editors. CRC handbook ofMicrobiology. Vol. 1. Cleveland, Nordeen R, K Morgan, and T Currier. 1983. Isolation OH : Chemical Rubber Company Press. p 579-607. and Partial Characterization of Bacteriophages of the Adams M K 1959. Bacteriophages. New York: Interscience Phytopathogen Pseudomonas syringae. Applied and Publications Inc. 592 p. Enviromental Microbiology. 45:1890-1898. Buell CR, VJordan, M Lindeberg, J Selengut, ITPaulsen, M L ObradovicA, JBJones, MT Momol, B Balogh, and SM Olson. Gwinn,RJ Dodson, RT Deboy, AAS Durkin,JFKolonay, 2004. 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Journal of the Arkansas Academy of Science, Vol. 61 [2007], Art. 14 Characterization ofBacteriophages ofPseudomonas syringae pv. tomato

sease Whalen Me and BJ Staskawicz. 1990. Molecular Basis of )logy Host Range and Specificity in Two Bacterial pathogens of Tomato. Wiley-Liss, Inc. p. 285-290. hage- Yamamoto KR, B.l\'lAlberts, R Benzinger, L Lawhorne, and ;nosa G Treiber. 1970. Rapid Bacteriophage Sedimentation in ental the Presence ofPolyethylene Glycol and Its Application to Large-Scale Virus Purification. Viriology 40:734-744. :006. mas logy trial ngae ence

tion 'the and son. dby :rs.

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