Plant Physiol. (1967) 42, 1433-1441

Purification and Properties of a Phytotoxic PolysaccharideProduced by Corynebacterium sepedonicum'

Gary A. StrobelDepartment of Botany and Microbiology, Montana State University, Bozeman, Montana 59715

Received June 12, 1967.

Summary. A polysaccharide possessing a capacity to wilt plant cuttings has beenisolated and purified from cultures of Corynebacterium sepedonicum. The molecularweight, based on the average of molecular weights determined by 3 physical methods, is21,450. The empirical formula of the polysaccharide is C48Hg9648N. It is antigenicand the borate complex migrates in an electric field. It has an initrinsic viscosity of0.125 and an S20W of 0.76. A hydrolysate of the compound yields glucose, mnannose,2 unidentiified reducing compounds and 1 unidentified non-reducing compound. The nitro-gen in the toxin can be accounted for in 6 amino acids. Although the toxin is primarilypolysaccharide it might more aptly be termed a glycopeptide.

Some information regarding the physiological activity of this compound as well as adiscussion of how it acts to bring about wilting is included.

Corynebacterium sepedonicum causes ring rot ofpotato. This disease is characterized by chlorosis,flagging of leaflets as well as stunting and wilting insome cases. The disease is found in all potato grow-ing areas of the world and is a constant menace tothe potato industry.

Hodgson et al. (3) showed that polysaccharidesproduced by a bacterial pathogen may be responsiblefor wilting symptomatology. Paquin, Lachance, andCoulombe (6) showed C. sepedonicum did not producedetectable quantities of pectic enzymes reported to beresponsible for disease symptoms in other situationsand suggested that a bacterial secretion might beresponsible for the symptoms. Spencer and Gorin(9), following this hypothesis, reported the occurrenceof physiologically active gumns in cultures of C. insi-diosumvZ and C. sepedonicum and showed that crudepolysaccharide preparations possessed the ability towilt plant cuttings. There was circumstantial evi-dence of the presence of bacterial! polysaccharides innaturally infected iplants. The present report de-scribes the purification and properties of a toxicpolysaccharide from C. sepedonicum. Information re-garding the physiological activity of this compoundis also presented.

Methods

A pure culture of C. sepedonicum was obtainedfrom a potato tuber showing typical ring rot symp-

1 Supported in part by NSF grant GB-5557. Mon-tana Agricultural Experiment Station Paper Number825, Journal Series.

toms. The culture was grown on the media describedby Spencer and Gorin (9). Polysaccharide was pro-duced by growing the bacterium in 0.5 liter of theliquid medium, incuibated 1 week aerdbically at 230in 1 liter filasks.

The crude toxic polysaccharide was prepared bycentrifugation of the medium at 20,000 X g; passageof the supernatant liquid through a 20.0 cm X 2.5 cmcolumn of iDowex 1 200 to 400 mesh (formate form)

5 10 15 24TUBE NUMBER

FIG. 1. The separation of labeled polysaccharidesby Sephadex column chromatography. Ten mg of acrude preparation with a specific activity of 8000 dpm/mg was placed on a Sephadex G-200 column and elutedwith distilled H,O. Fractions (1.5 ml) were collectedand 0.1 ml aliquots counted by liquid scintillation spec-troscopy. The total dpm in each tube is plotted againstthe tube number. The shaded area represents the tubesthat contained the toxin polysaccharide as manifestedby the tomato cutting assay test.

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PLANT PHYSIOLOGY

and then a 2.0 cm X 2.5 cm column of Dowex 50200 to 400 mesh (H+). The effluent was flashevaporated to 20 to 30 ml and the polysaccharidesprecipitated 'by the addition of 3 volumes of 95 %ethanol. The precipitate was centrifuged and storediln a vacuum desiccator over P205. Further purifi-cation was carried out by placing 10 to 100 mg ofcrude polysaccharide 'preparation on a 1.4 X 25 cmcolumn of Sephadex 'G-200 and eluting with H20.Approximately one-third of the pollysaccharide prep-aration, by weight, was recovered as toxic polvsac-charide. A typical elution curve of the separationof '-C labeled ,polysaccharides is shown in figure 1.All studies in this report were done with the poly-saccharide illustrated as peak No. 2 in figure 1. PeakNo. 1 was a polysaccharide, but did not possess bio-logical activity. Polysaccharides were labeled bygrowing C. sepedonicum in 25 ml of the regularmedium in the presence of 25 Mxc of nmannose 14C-ULwith a specific activity of 4.9 mc/mmole ( NuclearChicago Corp.).

RadIoactivity on all samples was determined witha model 6804 Nuclear Chicago liquid scintillationspectrometer. The channels ratio method was usedto correct for quenching. The sample, up to 0.1 mlaqueouis solution, was 'placed in a vial along with3.0 ml of absolute methanol. Added to this was12.0 ml of scintillation solut on containing 4.0 g2,5-diphenyloxazole and 100 mg- of p-bis-2(5-phenvl-oxazolyl)-benzene per Iliter of toluene.

The biological activity of the polvsaccharide wasassayed bv the time required for a 7.5) cm youngtomato cutting to begin wilt!ng at the margin of theprimary lea!f. There was a linear relationsh!ip of thetime required to wilt aiid the toxin concentration(Ifig 2).

The polysaccharide was acid hydrolysed by re-fluxing (0.2 %) in 0.5 N H1S04 for varying periodsof time after which the acid was neutralized with anexcess of BaCO3. The precipitate was removed bycentrifugation and the suipernatant liquid taken todryness.

200

150

w100 1

z50-

0.12 0.5 101 2.0(JJG/ML) X 100

FIG. 2. Time, in minutes, of 7.5 cm tomato leavesprior to wilting as a function of purified polysaccharideconcentration. Polysaccharide concentration is expressedas the reciprocal of ,ug/ml X 100. The values oni thegraph (left to right) are equal to 0.8, 0.4, 0.2, 0.1 and0.05 % solutionis, rcspectivelv.

Separation of the sugar residues was done onWhatmani No. 1 paper using ethyl acetate: pvridine:H.,0 8:2:1. The sugars were detected with the re-agent of Trevelyan et al. (10). After elution fromchronmatograms, reducing sugars were determined bythe method of Nelson (4). Amino acids were deter-mined qualitatively and quantitativelx with a Tech-nicon Aniino Acid Analvzer.

Nuclear magnetic resonance studies were done ona Varian NMIR A60. The samiples w\vere preparedby dissolv:ng 100 mg of polysaccharide in D.,O, re-m1oVing the D,0 by flash evaporation antI repeatin,gthe process 2 to 3 times. Finall1 1.(0) l of D20was added to the polysaccharicle for analysis. Aniniportant resonance line of D.,0 remlained about4.75 ppm down field from tetramethylsilane.

The antiserum u,sed in this sttudv was (preparedbv Mr. P. V. Rai of this departmiienit. It \xas obtainedfroimi the serum of rabbits injectedI intermiiuscularlytwice at weekly interval's w,ith 1() mg of crude toxinwith adjuvant. A titer of 1:4 was obtained in thisantigen-antibody sylsteim.

Carbon, hydrogen and oxygen were determined bySchwarzkopf Microanalytical Lalboratory, Woodside,New York. Nitrogen was determined b\ the imiicro-kjeldahl technique. Number average molecularwveig,ht \vas done bv Dr. George Bogart. HewlettPackard Corrporation, using the 500 series membraneosmometer.

Ultracentrifugation analyses wx ere done on aSpinco model E analytical ultracentrifuge using1.0 ml of a 1 % aqueotus solution of pol'vsaccharilefor t,he sedimentation constant (letermination. Theshort column method of Vaan Holde and Baldwin (12)for equilibrium sedimentation was emp'loyed formolectular we&ght determination using 0.2 iil of a0.02 % solution. Sedimentation calculations werebased on those of Schachmuan (8). The partialspecific volume of the toxic polvsaccharide wasas6su'med to be 0.62.

A Cannon-Ubbel'ohde semi-micro viscomiieter, model75. (Cannon Instrument Co.) wvas used to (leterminethe s,pecific viscosity of the *polvsaccharide at concen-trations of 5.0, 3.3 and 2.5 mlg/nil at 25°. Theintercept of the plot of specific viscosity/concenitrationas a function of concentration \as the intrinsicvi scositv.

Results

Elemental analysis of the poly-saccharide yieldedthe empirical foriiiula of C,,H1904\N. The infraredspectrum o,f the polysaccharide wvas consistent withthose generally observed for polysaccharldes; absorp-tion bands at 3300 cni-i and 1100 cim -1 for OHgroups and a sharp band at 290X) ciii-I for -CH-.No assignment, however, has beeii given to a bandat 1615 cm-.

Acid hydrolysis Nxas doiie by refluxing for 8 hourson 2 mg of polysaccharide containiiig 132,000 dpm.The solution was neutralized alnd( pa.sSed through a

1 434

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STl'ROBItI.PII'ROI'I-RTI .OF'SAF PHXTONIC' PO1'YSAti (IHARID 143

3 5 7 9 11 13 15C \ inches

G M

I 1(;I . 3. A radl(whro atoc,-()l.)Si f thct nctiltral,mlionulfr itcti it t)tt til ptlv s cct.tir de aftci acit l ols dis.-111C i11Sll-lillll(11t .st.lli1ed at I c'111/111ii1 with1 a1 coCllil11iiator\itith of 5.s 111111 iti(l titilt. c t)i lttlit otf 30. T Ile tiark

ttrt.as i1 tilt' c'hr mitatog-ravo r epixseilt rcd(tiilg Ctt0111

p)t()nI itds. (,,i. (il Ni, i tl icateti the l(t.' ttittii (if (g uc tse ittidillmllllmso,its thct 1lt1h- redtcclill-) *( )llll)(f)llll(IS arle 1111dli(ltified(,ail(1 Owl imnl-r-(dw:.1110, lllxil( is 1-cprlesliltc(I hy! tiltskshcd1.( arc-t. wl tile chrllomiato>-ra.li

sOlltl cotltiniti of Doxxecx ( IT). 'I'lic eltiatc conl-

talnedlC( tile llt'Lltl-1-1 anId anIionl fra-ctionls a111C tlzt Catioll

fraction e'tited( xxith 2.0 (if I[C l. A fterchromatil-trtarpt,lix, .luttiioii frotii tile chroIllmtto-ramil,s. ailCi

aialvs-. the ratio ot gluco'e to maimiinos xa<; 1.5 :1.0.

T o otlhr Ireducillg compounds ailtid 1111-i reducillo

comiptiltid re at1so preseilt htt the quantities xxere

not tletertiiiii-d. A radiocllroill'ttoscail aild reproduc-tiCoIl of tue clirtolimatogram of tue letitrtl-anillil frac-tioil ohtaiined frtoiii lhydrol-siis of tile labeled polysac-charide aII-c llutrate(i ill figire 3. 'I'Tle catioll

fraction \t-eltledl tllreotliile, serile, glyi lle,:tlatlile,lxvsle, i1l-ti Iiietii,onilne ill a ratio otf 1 :1 :1 :1 :1 :2.

Part al ac)d livxdrolysis of the polisaccharite Ie-siulteti inl thet dra-astic redtiction of hiolog cl activitvillustrated ii figuire 4. Virtutally all of the activitvxas lost after 3 iltinltites of refluxin- ill (0.5 HN 1SOf

''eln otf a labeled crudle toxill plireiltrattionl xitiha specific actixity of 9230 dpm/ililg xas appited to tileSephiatdex (;-20)() coluitmini and(I eltit.0n Wa begni. 'Thlettibcs contlttln:n-- radioactix itv crc ploole(l andi takenlto (Idrv ncss ith ft5lsh eVaporaitt:l0. Tlit, toniltto Clitt:ilgassay xas- tiset! fOr 0.2 % siollitonl of polystcchlaridefroiii easli 11eak. No xilttug as Oibserxed in thetomiatIo Itcaf ae'( dinill -t stlut on iptrepared fi-ct teak 1

xxihcreas tle !t.af xxwilttd tlhiii 3)) iiiiititts in thetit lli)tlpre l t1ed front pt.'atk 2. T1 iti, tlit nolpt asa.,

charilde 1s tiated fio1I)efetk 2 xxwas cotsdertd.tl its tilt.tox:in. .\ftcr litturs ini the sttltititmls the avx- xxwcrc

entox t.I ,iitd n1aced tl otd tk- itt 'reti Xi-ra - fillll

toi 2 wxt.t.k .tt ii) .\ ilusti-ation if a le,f atdltli1t)OtiStl\ e pratt of tile N-rax filiti of the sa te lt.itt

si it fig s. \ stliut1l tllittotn t otf e iosedt ftiltllCtI c-pt'' ' _i tot ttt. xt.t1. 1d e'nlets xxais thiir i-bo tthit. ict t Ii - tltt s t!tltit1i frltul pteak 1. I loxxc xcri, f(r

tilea toxi"ll tr <att< l lcaf. r1-td:IocitCst1v (ZcIC,_,1111111ted( at tilei l1ll(t:()l o)f tlC e(ttt aZll" the jwtil(, (.sce arrmv'00\'iii tiO,- D ). I 's leaf xxw as (t.xLt ttt (l \xx tli 2.)) nil of

stItt. ti 1 (1 ) oitrifti-ed. ctit.l tile stiltp ilnttilit liqu idtak;- t(-i) xrvi -e> 'I'lit t.xt: tct waits d ti. to () .2 ol

nti(l (Lis' t.1iv its c:ltpacitx tit xxilt a tol a-Ito leltf.

After 2Sf) minuEltes the primary leaflet began to wilt.The renain:ng, soluition was tested by the Ouchterlony(5) double di f'fusion-gel method against the antiserumto tile crucle toxiin. An antigen-antibody precipitationband was ol)serve(d betwxeen the 2 wells. No re-actioIn w as oiserx'ed wNitlh an extract fronm a controlplanlt treate(Id wxith li ).

.lIolc'llar 11 'ig/lt J)cterminations. ColtuimnI chro-iatographl with Seplhadex has been used effectivelyfor inolecult r weigh-lt estimations of polysaccharides27. U sin Sel)hadex G-200, the equation for molec-

ular xxeight estillmation is Ve = 3.20 to 0.58 logVt

titolecular x eiglht. WNlhere Ve equals the voltulmie atwxlhichl tile sanlide is eluited from the columln ImliUs thev oi(l volutiiie of tile columni an(i V, equals the total'oluille of tile colunlni xilicilh xas 35.5 nl. 'File void

voluimie xas (leter-illed Ihy the use of blue dextranalid wxas 1 0.5 nil xhereas the sailiple volunle xxas 36 ml.Tius- Ve equaled 0.718. Tile molecular xeig-lt from

Vtthlis eqluationl is 18,850.

The silort coluillil illetliod of equilibriuln cen-trifu-gationl vielded a xx eight average imlolecular weEight of21,900. 'I'le nlulllier av-erage molecular xxeighlt was23,60) as (letertitiied lbv menibrane osnlonletrv. Theaxerage itiolectilar -weight froin these 3 values is21,450.

O ther' Physical Properties. TI'he polvsaccliaride(0.5 mg) xith a specific activity of 7200 (Iprn/mg

nowilt

200

u100-

EO 1 2 3 4minutes

F it. 4. itl ntiles of exposure of .t 0.2 % solutionof polys'tcchtaritie to r-fluxin ii, 0.5 N 1 ,SOC (N axis)iipittt(d ao-ailtst niinttss rc quired to cauise xxilt in thletomato assa\- test (Y axis). After each exposure tille,ain ti-Itnot 1f tll i at i(I st titioll wx its remioved alln etratlz(ld xitit in exess of BaCf).-. After reniovatl oftile pltCt.ciptttc tile sointn)il itas tatk 11 to tdrvit-s a(1ndrediss ixveti ill an 11 (aimnt of xwater resulting ill a 0.2 %sol0 titi( ttof tilet I)I -tI,LIIiix i(t 0drCoi (I 1I So lvsa Ce -i:trIe.

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PLANT PHYSIOLOGY

ISO,

75

B:

U9175 1 2.5 7.5 2.5F~

__fFIG. 6. Electrophoretic mobility of the purified

polysaccharide in 0.1 M borate buffer pH 8.7. Afterelectrophoresis, a portion of the strip exposed x-rayfilm for 2 to 3 weeks after which 1 spot at the loca-tion of the slashed area on the paper was noticed likewisethis was the only area possessing weak reducing propertyas represented by the shaded area on the paper. Bio-logical activity was detected between the 7.5 and 10.0cm mark. A radiochromatoscan was done and is locatedabove the reproduction of the paper. Conditions ofscanning are given in the legend to figure 3. Migra-tion was from right to left and details of the procedureare given in the text.

was applied in a 5 cm streak to a 14" X 2" strip ofWhatman No. 1 fil.ter paper and gently wetted with0.1 iborate buffer of pH 8.7. Electrophoresis was

carried out at 40 volts/2.5 cm for 1 hour with theends of the ipaper strip immersed in borate buffercontaining the electrodes. After drying, the paperwas ipassed through a Packard radiochromatogramscanner at a rate of 1 cm/min. The paper was

also placed on x-ray film for 2 weeks after which itwas cut in half, longitudinally. One half was treatedwith the reagent of Trevelyan (10) for reducinggroups and the other half was cut at 2.5 cm intervalsand the papers eluted with H20. The tomato leafassay was used to determine where biological activitywas located. It was noted that the spot on the x-rayfilm coincided with the weakly reducing spot on thepaper strip which corresponded to the area containingbiological activity as depicted in figure 6. The poly-saccharide borate complex had a mobility of 1.5 X10-4 cm volt-1 seC-1.

The intrinsic viscosity of the polysaccharide was

0.125 deciliters/g which favorable compares with a

value of 0.148 for a dextran with a molecular weightof 22,500 obtained from Pharmacia Inc.

The sedimentation constant of a 1 % aqueoussolution the polysaccharide was 0.76 at 200. TheSchlerien diagrams of the migration of the polysac-charide in the centrifugal field are illustrated infigure 7.

Small droplets of a 1% solution of the polysac-charide were placed on the surface of a collodionmembrane on a copper grid, dried and then observedin a Zeiss EM-9 electron microscope. Figure 8shows a typical field of ipolysaccharide crystalis (C);amorphous globules (G) are also seen. The globulesburst into crystals during the viewing process whichwas probajbly due to the high vacuum of the columnof the electron microscope or iheating due to exposureto the beam. The crystals were 20 m,/ X 40 muin size.

That the polysaccharide was obtained in a purifiedstate is satisfied by several physical interia of purity,namely, 1 peak when subjected to electrophoresis, 1peak by ultracentrifugation, 1 evenly shaped peakafter elution from a column of Sephadex G-00, andthe appearance of homogenity in preparationis viewedunder the electron microscope.

Natufre of Bonding and Degree of Branching.Proton nuclear magnetic resonance was done on 1 mlof a 10 % D,0 solution of the polysaccharide. Thespectrum is 'shown in figure 9. Peak 1 is attributedto the C5 and C6 protons and -peak to the 'CkC3 andC4 protons according to Pasika and 'Cragg (7). Peak4 may be attributed to the proton on the C1 carbonat the acetal linkage. The ratio of the area of peaks1+2 to the area under peak 4 is 8.3:1 indicating thatthe polysaccharide is a branched structure. If it werea linear chain of residues a ratio of 6:1 would beexpected. The glycosidic anomeric proton absorptionoccurs at 5.1 ppm (peak 4) and has a coupling con-stant of 4.0 count/sec. A coupling constant of3.2 ± 0.6 count/sec is reported by van der Veen (11)to occur in oligosaccharides with an a linkage and acoupling constant of 7 + 0.7 count/sec to occur inoligosaccharides with a /8 linikage. Therefore, a pre-dominance of a linkages is suggested to be presentin this polysaccharide. Peak 3 in, figure 8 is due tothe resonance line of DO.

Discussion

Acid hydrolysis followed by chromatography ofthe purified "-C lalbeled polysaccharide revealed 2unidentified reducing compounds and 1 unidentifiednon-reducing compound. Glucose and mannose werein a ratio of 1.5 :1.0 and 6 amino acids were identiified.The mro!ecular weight, emperical formula, and aminoacid analysis would allow for 2 residues of each aminoacid, except 4 for methionine, per molecule of toxin.Since it has a peptide mioety, this toxin might morecorrectly be termed a glycopeptide. This is in contrastto the work of Spencer and Gorin whose data indicatedthat galactose and 'fucose were also present in the poly-saccharide, but it should be pointed out that theirwork was done on the noni-purified toxin preparation.Unpubl.ished data from this laboratory suggested thatthe chemical and physical environment of the bacterialculture affected the sugar ratio in the toxin. Thatmore than 1 polysaccharide was present in crudetoxin ipreparations was pointed out by Rai (unpub-lished, Ph.D. thesis) who followed the incorporationof mannose-14C into the components of the crudetoxin preparation of C. michiganense. He alsoshowed that mannose, rather than glucose, served asthe better precursor to labeled toxin'. That more than1 polysaccharide was present in the crude prepara-tions was verified as shown in 'figure 1. Peak 1contained a polysaccharide that had a molecular weightgreater than 2 X 105. It was water soluible andwas taken up by the plant, but did not possess physio-logical activity. This suggested that there is some

.1*

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STROBEL-PROPERTIES OF A PHYTOXIC POLYSACCHARIDE

FIG. 5. A reproduction of a plant cutting (left) which was placed in a 0.2 % solution of purified polysac-charide (sp. act. of 27,000 dpm/mg) for 2 hours and then exposed to a Kodak No-Screen X-ray film for 2 weeks(right). Initial wilting was observed after 30 minutes figure 4. The location of the toxin in the planit is repre-sented by the exposed spot oni the x-ray film which is the juncture of the tomato leaflet to the petiole. Reisolationof the toxini from this leaf showcd that antigenicity and toxicity were retained.

Insert

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STROBEL-PROPERTIES OF A PHYTOXIC POLYSACCHARIDE 1439

FIG. 7. Ultracentrifugal pattern of a 1 % aqueous solution of the purified polysaccharide. Analysis was per-formed at 60,000 rpm with a bar angle of 600. The pictures were taken 35, 67, and 99 minutes after 60,000 rpmhad been reached. The sedimentation is toward the right.

5.

V;~~~~). ,,. t a S

FIG. 8. Electrui micrograph of crystalline andamorphous polysaccharide toxin. During viewinig theamorphous electron transparenit particles were seeni toburst into crystals approximately 20 mgt X 40 mAog insize. The field is ma-Lgnified X 60,000. C, crystal; Gglobule.

Insert

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STROBEL-PROPERTIES OF A PHYTOXIC POLYSACCHARIDE

4.0parts / mill ion

FIG. 9. Proton nuclear magnetic resonanice of a 10 %solutioin of the polysaccharide. Peaks 1 and 2 are

attributed to the C. and C. protons and the C2, C3 andC4 protons respectively. Peak 4 is the proton on theCl involved in the acetal linkage. The D,O peak isNo. 3.

intrinsic property, other than mere size of a molecule,that may be involved in causing a plant to wilt. Boththe work of Rai and the present work have shownthat only 50 jug of the toxin, taken up by a 7.5 cm to-mato leaf, is required for wilting to occur. The mostconmmonly accepted theory for wilting caused bby poly-saccharides involves plugging of the vessels in thestem (1). It is difficult, however, to imagine how50 jug of polysaccharide could block water iflow in a

tomato leaf with a diameter of 1 to 2 mm. Eventhough water movement in the stem is curtailed, an

explanation other than plugging seems to be required.It may be suggested that the toxin affects the waterrelations of the cells in the area in, which it becomeslocated, figure 5. At the present time electronmicroscope studies as well as 3H2O studies are inprogress to determine the nature of the wiltingphenomenon.

Approximately one-half of the biological activityof the polysaccharide was lost by refluxing for 1minute in 0.5 N H2S04 (fig 4). It is not likely thatcomplete hydrolysis oif the polysaccharide occurredin th-is time. The data may be interpreted as the lossof critical sugar or amino acid residues from the {parentmolecule resulting in loss of biological activity.

The serological reaction of the polysaccharide withan antiserum prepared to the crude toxin with a titerof 1:4 implies that high titers could be expected inantisera prepared to the purified polysaccharide.Since the serological technique was useful in detectingthe ,polysaccharide in artificially treated leaves, itmay eventually prove useful as a technique for thedetection of C. sepedonticwtnii in naturalily infectedplants in the field.

Acknowledgments

The author acknowledges the assistance of Dr. J.Robbins and G. Baker of the Department of Chemistry,Montana State University in the ultracentrifugation andNMR studies presented in this report. The assistanceof Dr. T. Carroll of this department in the E.M. study-is also appreciated.

Literature Cited

1. BUDDENHAGEN, I. AND A. KELMAN. 1964. Bio-logical and physiological aspects of bacterial wiltcaused by Pseudomjiontas solanacearumn. Anni. Rev.Phytopathology. 2: 203-30.

2. GRANATH, K. A. 1965. Gel filtrationi, fractiona-tion of dextran. In: Methods in CarbohydrateChemistry. Vol. V. R. Whistler, ed. AcademicPress, Inc., New York. 20-28.

3. HODGSON, R., A. J. RICHER, AND W. H. PETERSON.1947. The toxicity of polysaccharides and otherlarge molecules to tomato cuttings. Phytopath-ology. 37: 301-18.

4. NELSON, N. 1944. A photometeric adaptation ofthe Somogyi method for the determination ofglucose. J. Biol. Chem. 153: 37540.

5. OUCHTERLONY, 0. 1958. Diffusion in gels miieth-ods for immunological analysis. Vol. V. In:Progress in Allergy. D. Kallos, ed. S. Karger,Basel and New York. p 1-78.

6. PAQuIN, R., R. A. LACHANCE, AND L. J. COULOMBE.1960. La production des enzymes pectiques parCorynebactcrium sepedon icutin. Can. J. Microbiok6: 435-38.

7. PASIKA, WV. AI4. AND L. H. CRAGG. 1963. Thedetection and estimation of branching in dextrarnby proton magnetic resonance spectroscopy. Can.J. Chem. 41: 293-99.

8. SCHACHMAN, H. K. 1957. Ultracentrifugation,diffusion, and viscometry. Methods in Enzym-ology. Vol. IV. S. P. Colowich and Nr. 0.Kaplan, eds. Academic Press Inc., Nexv Yorlkp 32-103.

9. SPENCER, J. F. T. AND P. A. J. GORIN. 1961. Theoccurrence in the host plant of physiologicallyactive gums produced by Corynebacterium ittsidiJosuinit and Corynebacteriunin sepedonicumii. Canl. J_Microbiol. 7: 185-88.

10. TREVELYAN, W. E., D. P. PROCTOR, AND J. S. HAR-RISON. 1950. Detection of sugars on paper chro-matograms. Nature 166: 444-45.

11. VAN DER VEEN, J. M. 1963. An N.M.R. study ofthe glycoside link in glycosides of glucose andgalactose. J. Org. Chem. 28: 564-66.

12. VAN HOLDE, K. E. AND R. L. BALDWIN. 1958Rapid attainment of sedimentation equilibriun-J. Phys. Chem. 62: 734-40.

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