APPLIED AND ENVIRONMENTAL MICROBlOIOOGY. JUlY 1986. p. 33-360099-2240/86/070033-04$02.00/0Copyright 17D 1986. American Society for Microbiology

Avirulent Isolates of Corynebacterium fascians That Are Unable toUtilize Agmatine and Proline

PAULA R. SABART1 DARLENE GAKOVICH, AND RICHARD S. HANSON'*Gray Fresh water Biological Iinstitiute, Unihersity of Mininesota, Nai arre, Minnesota 55392,1 aiid Departml enlt of

Ba-cteriology, University of Wisc onsin, Madison, Wisc onsin 537062

Received 26 December 1985/Accepted 20 March 1986

Growth of a highly virulent strain of the phytopathogen Corynebacterium fascians on rich media at 37Cresulted in a loss of virulence in a majority of the population within 10 generations. Strains retained virulenceduring cultivation at 30C on a minimal medium with ammonia as a nitrogen source. Populations of avirulentstrains on the surfaces of pea seedlings decreased, whereas the number of cells of the virulent strain increased1,000-fold during a 3-week period. All avirulent mutants isolated by growth on rich media at 37C were unableto grow on media containing agmatine or proline as sole sources of nitrogen. The ability of the mutants to growon pea seedlings and cause fasciation disease appeared to be related to their ability to utilize nitrogen sourcesavailable on plant surfaces.

Corvniebacterium Jscians causes fasciation disease indicotyledonous plants. The disease is characterized by a lossof apical dominance and outgrowth of lateral buds (2, 4, 5).The bacterium is epiphytic, living on the surface of the hosttissue, where it produces cytokinins (9, 11). The symptomsof the disease can be duplicated by treating seedlings withsynthetic cytokinins or cytokinins produced by virulentstrains of C. jcians (11). Strains of C. fiscians vary greatlyin their degree of pathogenicity. Virulent strains of C.J'asdianis were found to excrete more cytokinins into theculture medium than did weakly virulent and avirulentstrains (8). The major cytokinin found in culture media aftergrowth of C. fiscilass wasN6-(Ai-isopentenyl)adenine. Themost virulent strains also contained a large (Mr, ~108)plasmid, whereas plasmids were not detected in avirulentstrains (8).

It was shown that repeated cultivation of some plantpathogens on artificial media resulted in the loss of virulence(6). This report describes an attempt to find the physiologicalbasis for the loss of virulence when one C. fliscians strainwas grown at 37C on rich media. To determine whether theloss of virulence was progressive or due to a single event, wedeveloped a quantitative assay of the degree of pathogenicityof different strains.

MATERIALS AND METHODS

Bacterial isolates. Cornnebac'teriiintacn ias strain MW2was isolated by and obtained from M. A. Rahman (Univer-sity of Alexandria, Egypt). A hypervirulent strain of MW2,designated MW2V, was isolated in this laboratory from peaseedlings repeatedly infected with MW2. Strain MW2 is aweakly virulent parent of strain MW2V. Avirulent deriva-tives of strain MW2V (designated MW2VC) were isolated asdescribed in the text. Strain Cf-15 was provided by M. Starr(University of California, Davis, Calif.). Strain Cf-1 wasobtained from F. Skoog (University of Wisconsin, Madison,Wis.). Strains Cf-2 and MW11 were provided by J. Helgeson(University of Wisconsin, Madison, Wis.). Strain MW11V is

* Corresponding author.

a hypervirulent reisolate of MW11, obtained by passage onpea seedlings. Strain MW11VC is an avirulent mutant iso-lated as described in the text.

Media. Cultures were grown routinely at 30C on a mini-mal salts thiamine medium (MT) containing 3.0 g ofK,HPO4, 1.15 g of NaH2PO4- H.O, 0.3 g of MgSO4 7HO,0.15 g of KCI, 0.01 g of CaCl2 2H.0, 0.0025 g ofFeSO4- 7H.O, and 0.005 g of thiamine per liter of deionizedwater. The pH of the medium was adjusted to 6.8. Glucosewas autoclaved separately and added to the sterile MTmedium to give a final concentration of 0.5% (wt/vol). MTNmedium was MT medium supplemented with 1.0 g of NH4Clper liter. Cultures were also grown on CH medium, contain-ing 5.0 g of Bacto vitamin-free Casamino Acids (DifcoLaboratories, Detroit, Mich.; acid hydrolyzed) per liter ofMT medium. Nutrient broth (NB) containing 8.0 g of Bactonutrient broth and 0.5% glucose per liter of distilled waterwas used to isolate avirulent strains of C. Ja'scians MW2V.Bacto-Agar (Difco; 15 g/liter) was used to prepare a solidmedium.When the ability of C. fjascians to utilize hydrocarbon

fumes as a sole source of carbon was tested, cultures weregrown on glucose-free MTN medium. Sterile capillary tubeswere filled with the liquid hydrocarbon (n-tetradecane,undecane. or ni-pentadecane) and placed on the inner surfaceof the petri dish cover. Plates were then inverted and sealedwith adhesive tape before incubation.

Virulence tests. Seeds of Pisium sativumn L. var. AlaskaDwarf were surface sterilized in 0.1% mercuric chloride for10 min, followed by five washings with sterile distilled water.Washed seedlings were placed in petri dishes lined withsterile, moistened filter paper and allowed to germinate untilthe radicles were between 0.5 and 1.0 cm long (approxi-mately 5 days). Ten germinated seedlings were immersed ina 48-h culture of C. faiscians grown in the CH medium at30C (2.0 ml of bacterial culture per seedling) containingsterile 2% (wt/vol) Carbowax (polyethylene glycol 6000) tofacilitate adhesion of the bacteria to the seedlings. Theaddition of polyethylene glycol to the medium in which thebacteria were suspended gave more reproducible virulenceratings. Four to five times as many bacterial cells werebound to plants in the presence of polyethylene glycol than

33

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CD 10z

C):

z111

J 5

5;

0

/~~~~~~~~

/~~ ~~~ n*z *- a

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10 20

DAYS AFTER INOCULATIONFIG. 1. Time course of symptom development in pea seedlings

inoculated with three different strains of C. fliscians. The peaseedlings were inoculated with C. fiascians strains MW2V (U, O[two experiments]). Cf-2 (A), and Cf-15 (0) as described in Mate-rials and Methods. Symptom development was used to determinevirulence ratings as described in Materials and Methods.

in its absence. After incubation for 60 min at 30C on a rotaryshaker, the seedlings were removed and planted, one perpot, at a depth of 2.0 cm in sterile vermiculite. Another 1.0ml of the bacterial suspensions was added to the vermiculite.Five uninoculated germinated seedlings were planted pertrial as controls. All seedlings were incubated at 230C undernoncontinuous illumination (12 h on; 12 h off) from acool-white fluorescent plant light (General Electric Co.,Schenectady, N.Y.); the plants were watered with distilledwater. Plants were examined regularly for symptoms. Dif-ferences between highly virulent and weakly virulent strainswere obvious within 1 week after planting. The virulencerating was determined approximately 4 weeks after planting.A final virulence rating for a given strain was determined byaveraging virulence ratings from five separate trials.The following scheme was used to quantitatively define

virulence. Stunting was assigned a value of 1, each primaryshoot above one was given a value of 1, and each secondaryshoot was given a value of 0.5. The sum of these values forall seedlings inoculated with the same culture, divided by thenumber of seedlings used in the test, was the virulence ratingfor that culture. The first symptoms of C. ftisc ians infectionare multiple shoot development (fasciation), stunted growth,or both. In many separate trials, the virulence ratings ofdifferent strains and isolates maintained on the MTN me-dium at 30C agreed within 20%.

Tests for loss of virulence during growth of C. fascians at37C in a rich medium. C. fascians strain MW2V was grownin nutrient broth containing 0.5% (wt/vol) glucose at temper-atures of 30, 35, and 37C. After 3 and 10 generations ofgrowth, cultures were serially diluted. Dilutions were spreadonto nutrient agar containing 0.5% (wt/vol) glucose andincubated at 30C. Fifty or more colonies were selected,cultivated at 30C in the MTN medium supplemented with0.05% yeast extract, and tested for virulence.

Measurements of growth and survival of C. fascians strainson pea seedlings. Pea seedlings were surface sterilized andgerminated as described above. The bacterial strains used inthe experiments were grown in CH medium at 30C for 3days. Viable cell counts of each culture were determined byspreading dilutions onto CH agar immediately before theaddition of the germinated seedlings. Twenty germinatedseedlings were immersed in 40 ml of a bacterial suspensioncontaining sterile 2% (wt/vol) Carbowax (polyethylene gly-col 6000) for 1 h at 30C and transferred to petri dishes linedwith sterile, moistened filter paper. Two infected seedlingswere blotted on sterile filter paper to remove unabsorbedbacteria and transferred to separate sterile tubes with 3.0 mlof MT medium. The tubes were mixed on a vortex mixer andincubated on a rotary shaker at 30C for 1 h. This procedureremoved 95% of the cells that were bound. Less than 5%more bacteria were removed by additional washes or bygrinding the seedlings in a sterile mortar and pestle. Thecontents of each tube were serially diluted in sterile MTmedium, and the dilutions were plated on CH agar. Theplates were incubated at 30C, and the yellow colonies of C.fascians were counted. The initial counts were used todetermine the number of C. fascians cells bound to eachseedling. Two seedlings inoculated with each strain wereremoved at intervals and treated similarly to determine thesurvival and growth rate of each C. fascians strain on thehost plant.

Source of materials. All chemicals were purchased fromSigma Chemical Co., St. Louis, Mo., or Fisher ScientificCo., Fair Lawn, N.J.

RESULTS

The time course of symptom development in pea seedlingsinfected with different strains of C. fascians is shown in Fig.1. The virulence ratings of each strain are in agreement withthe highly virulent MW2V, moderately virulent Cf-2, andweakly virulent Cf-15 designations previously assigned tothese strains (8). Maximum virulence ratings were achievedafter 15 days of growth.As few as 2.4 x 104 MW2V cells per ml in the cell

suspension used to infect pea seedlings gave maximumvirulence when Carbowax was added to the suspendingmedium. Approximately i05 cells were required for maxi-mum virulence in the absence of Carbowax.

Isolates from cultures of C. fascians grown at 30 and 350Con the MTN medium supplemented with 0.05% yeast extractall had virulence ratings equal (+20%) to that of the parentstrain. In one experiment, colonies isolated from a culturegrown in nutrient broth containing 0.5% glucose at 37Crapidly lost virulence (Table 1). The loss of virulence in themajority of the isolates was complete. No detectable symp-toms were observed in seedlings infected by 43 of the 52isolates from a culture grown for 10 generations at 37C in

TABLE 1. Loss of virulence during growth of C. fascians strainMW2V on rich media at 37C

No. of Isolates with virulence rating afterVirulence generation:ratings

3 10

12.5-14.0 57 28.0-12.5 11 01.0-8.0 1 7

LOSS OF VIRULENCE IN C. FASCIANS 35

nutrient broth plus glucose. Strain MW2V retained virulencewhen cultivated at 37C on the MTN medium. The loss ofvirulence was found to be dependent both on the richmedium and growth at 37C.The number of virulent C. fascians MW2V cells on the

surface of pea seedlings increased exponentially from 3 daysto approximately 8 days (Fig. 2). When 2.1 x 105 cells werebound per seedling, the population reached 108 cells perseedling in 18 days. The generation time was approximately1 day. The mean virulence rating for these seedlings was 12.When 2.7 x 107 and 3 x 107 cells of MW2V and MW2VC

(an avirulent isolate) were bound per seedling, the popula-tion of MW2V increased to 8 x 108 cells per seedling in 24days (Fig. 3). In contrast, the population of MW2VC de-creased from 3 x 107 cells per seedling to approximately 2 x106 cells per seedling in 18 days. The virulence rating forstrain MW2V in this experiment was 14. Seedlings infectedwith strait MW2VC did not show symptoms of fasciationdisease.

In another experiment, MW2VC cells, concentrated bycentrifugation to 1010 cells per ml were used to inoculate peaseedlings. A total of 6 x 107 CFU per seedling wererecovered from newly infected seedlings. The same numberof cells were bound when the inoculum contained 1011 cellsper ml. The bacterial population decreased to less than 106per seedling in I days. None of the seedlings exhibitedsymptoms of fasciation disease. When peas were ihoculatedwith MW2V cells (2 x 106 cell per ml), symptom develop-ment was obvious within 7 days (Fig. 2). Reinoculation ofseedlings with a suspension of MW2VC cells (109 cells perml) at daily intervals failed to produce symptoms.

In five separate experiments, including the one describedin Table 1, 50 or more avirulent colonies (virulence ratings of

1o 8

az.1

a

-

1 0

106

0 5 10 15

DAYS AFTER INOCULATIONFIG. 2. Growth of C. fascians strain MW2V on pea seedlings.

The procedures used to infect seedlings and to determine the viablepopulation of C. fascians on pea seedlings were described inMaterials and Methods. Seedlings were immersed in a suspension ofC. fascians containing 2 x 106 viable cells per ml. Bars represent therange of viable cell counts per seedling in replicate samples.

Z 108_

0-~~~~

LL \00

Do '4

4 8 12 16 20 24

TIME (DAYS)FIG. 3. Growth and survival of C. fascians strains MW2V and

MW2C on pea seedlings. Pea seedlings were infected by immersionin suspensions of bacteria containing 2 x 109 viable cells per ml. *,MW2V; O, MW2C.

0) from a culture in which strain MW2V was grown in NB at37C for 10 generations were isolated. These isolates (ovet250 total) were all unable to grow on MT medium withagmatine or proline as sole nitrogen sources. The virulentparental strain, MW2V, grew well with these compoun'ds assole sou'rces of nitrogen. Nine of the isol'ates of MW2Vjobtai'ned after growth in the NB medium at 37C for thr'eegenerations, were weakly virulent (virulence ratings of 1.0 to3.6) and were found to be able to utilize agmatine or prolineas sole sotirces of nitrogen. All avirulent and virulent isolatesof MW2V grew on MT agar medium with gluiamate (0.25%[wt/vol]) as a sole source of nitrogen.A few avirullent isolates ofMW2V were found to be unable

to utilize n-tet'radecane, undecane, or n-pentadecane as thesole carbon and energy soulrce when grown on MTN me-dium. The parental MW2V strain and other virulent strainsof C. fascians were able to utilize all three hydrocarbonstested as the sole carbon and energy source. No growth wasobserved on MTN agar without a carbon source.Growth of MW2V and MW11V at 30C on nutrient agar'

containing ethidium bromide at concentrations that partiallyinhibited growth (1 ,ug/ml) resulted in a loss of virulence insome isolates. This treatment also resulted in a loss of theability of some isolates of MW2V and MW11V to grow onC10-C15 hydrocarbon fumnes as the sole carbon and energysource. Loss-of virulence, loss of the ability to grow on thesehydrocarbons, and the loss of the' ability to grow on agmatineand proline as nitrogen sources were found to be indepen-detit events in both strains when mutants induced byethidium bromide and by growth at 37C in rich media wereexamined (Table 2).

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TABLE 2. Relationship between hydrocarbon utilization andvirulence of C. fascians strains

Growth on glucose-free MTN agar with theStrain Virulence following carbon source:b

,designationa ratingUndecane n-Tetradecane n-Pentadecane

MW2V 14.0 + + +MWllV 20.0 + + +MW2VC 0 + + +MWllVC 0 + + +MW2V-1 0.6c - - -MWllV-l 0.9c - - -MW11V-2 0.6c + + +

a MW2VC and MW11VC are avirulent mutants isolated after growth ofMW2V on nutrient agar at 37C. MW2V-1, MW11V-1, and MW11V-2 aremutants isolated after growth of MW2V and MW11V on nutrient agarcontaining ethidium bromide at 30C.bHydrocarbons were supplied as fumes. +, Growth observed; -, no

growth observed.c These bacteria were able to grow on MTN medium with agmatine or

proline as the sole source of nitrogen.

DISCUSSION

A number of UV-induced auxotrophic mutants of C.fascians strain MW2V have been isolated (3). The mutantswere avirulent and had single requirements for glycine,arginine, methionine, aspartic acid, or adenine. We isolatedspontaneous mutants that became avirulent during growthon rich media at 37C. The loss of virulence in strain MW2Vwas correlated with the inability of the isolates to grow onpea seedlings and with the inability to use agmatine orproline as the sole source of nitrogen. None of these isolateswere auxotrophic.The ability of virulent strains to survive and grow on pea

seedlings appeared to be associated with their ability toutilize nitrogen sources provided by the host. Growth of C.fascians on pea seedlings is required for symptom develop-ment (10). Neither increasing the inoculum size of avirulentstrain MW2VC nor reinoculation of pea seedlings withMW2VC cells at daily intervals resulted in development offasciation disease symptoms.We cannot yet satisfactorily explain the consistent loss of

the ability of strain MW2V to utilize agmatine or proline asthe sole source of nitrogen. One possible explanation for thephenotype is that a single mutation occurred and that sib-lings of this mutant grew faster at 37C than did the virulentparental strain. However, this explanation is not consistentwith the following observations. (i) Avirulent mutants ofMW2V with the same phenotypes were obtained in fiveseparate experiments, (ii) differences in the growth rates ofMW2V (wild type) and several avirulent isolates of MW2V(including MW2VC) at 37C in NB with glucose were notobserved, and (iii) the large proportion of mutants to wild-type isolates obtained in the experiments described cannotbe accounted for by a single mutational event followed bygrowth of the mutant during 10 generations of growth.

It is not likely that proline and agmatine catabolism areencoded by plasmid DNA. The highly virulent C. fasciansstrain MW2V contains three plasmids (8). In numerousattempts, we failed to detect plasmid DNA in a weaklyvirulent strain, MW2, derived from the same parent. Thisstrain will grow on media containing agmatine or proline as

a sole source of nitrogen. Attempts to isolate mutants fromthis strain by growth at 37C in nutrient broth plus glucosehave consistently failed. The production of high levels ofcytokinins (8) and ability to undergo high-frequency muta-genesis under these conditions appears to be associated withone or more plasmids. We have not isolated plasmids fromstrains that have been cured by growth at 37C in rich media.This procedure appeared to eliminate the plasmids observedin the highly virulent strain.Attempts to produce mutants by growth of C. fascians in

the presence of ethidium bromide resulted in the isolation ofstrains unable to grow on hydrocarbons as well as avirulentisolates. Ethidium bromide is known to cure some bacteriaof plasmids (1). However, hydrocarbon utilization, viru-lence, and the utilization of agmatine and proline as nitrogensources were not correlated. It is not known whether hydro-carbon utilization is plasmid encoded in C. fascians.One possible explanation of the present data is that a

plasmid or part of a plasmid that is essential for cell survivalcannot replicate at 37C and integrates into the genome at aspecific site near or within genes essential for the catabolismof agmatine and proline. This hypothesis would explain whythe weakly virulent MW2 strain that is apparently devoid ofplasmids fails to produce avirulent mutants when grown onrich media at 37C.

ACKNOWLEDGMENTS

This research was supported by grant 5-T 32 GM 07094 from theDepartment of Genetics and Cell Biology, University of Minnesota,and by the University of Wisconsin College of Agricultural and LifeSciences.

LITERATURE CITED1. Bouanchand, D. H., M. R. Scavizzi, and Y. A. Chabbert. 1969.

Elimination by ethidium bromide of antibiotic resistance inenterobacteria and staphylococci. J. Gen. Microbiol. 54:417-425.

2. Dowson, W. J. 1957. Plant diseases due to bacteria, 2nd ed.Cambridge University Press, London.

3. Jacobs, S. E., and U. Mohanty. 1951. Factors influencing infec-tion by Corynebacterium fascians (Tilford) Dowson. Ann.Appl. Biol. 38:237-244.

4. Lacey, M. S. 1936. The isolation of a bacterium associated with"fasciation" of sweet peas, "cauliflower" strawberry plantsand "leafy gall" of various plants. Ann. Appl. Biol. 23:302-310.

5. Lacey, M. S. 1939. Studies on a bacterium associated with leafygalls, fasciations, and "cauliflower" disease of various plants.Part IIl. Ann. Appl. Biol. 26:262-279.

6. Lacey, M. S. 1948. Further observations on the pathological andphysiological reactions of Bacterium fascians. Ann. Appl. Biol.35:572-581.

7. Miller, H. J., et al. 1980. Recent observations on leafy gall inLiliaceae and some other families. Neth. J. Plant Pathol.86:55-68.

8. Murai, N., F. Skoog, M. E. Doyle, and R. S. Hanson. 1980.Relationships between cytokinin production, presence of plas-mids and fasciation caused by strains of Corynebacteriumfascians. Proc. Natl. Acad. Sci. USA 77:619-623.

9. Rathbone, M. P., and R. H. Hall. 1972. Concerning the presenceof the cytokinin N6-(A2-isopentenyl)adenine, in cultures of Co-rynebacterium fascians. Planta (Berlin) 108:93-102.

10. Rivain, J.-G., and J. Roussaux. 1982. Relationship betweengrowth and pathogenicity of Corynebacterium fascians (Tilford)Dowson. Agronomie 2:479-486.

11. Thimann, K. V., and T. Sachs. 1966. The role of cytokinins inthe "fasciation" disease caused by Corynebacterium fascians.Am. J. Bot. 53:731-739.

36 SABART ET AL.