Vol. 52. No.

APPLIED AND ENVIRONMENTAL MICROBlOIOOGY. JUlY 1986. p. 33-36

0099-2240/86/070033-04$02.00/0
Copyright 17D 1986. American Society for Microbiology

Avirulent Isolates of Corynebacterium fascians That Are Unable

Utilize Agmatine and Proline

to

PAULA R. SABART1 DARLENE GAKOVICH AND RICHARD S. HANSON'*

Minnesota 55392,1 aiid Departml enlt of
Gray Fresh water Biological Iinstitiute, Unihersity of Mininesota, Nai
,

arre,

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 37C
resulted in a loss of virulence in a majority of the population within 10 generations. Strains retained virulence
during cultivation at 30C on a minimal medium with ammonia as a nitrogen source. Populations of avirulent
strains on the surfaces of pea seedlings decreased, whereas the number of cells of the virulent strain increased
1,000-fold during a 3-week period. All avirulent mutants isolated by growth on rich media at 37C were unable
to grow on media containing agmatine or proline as sole sources of nitrogen. The ability of the mutants to grow
on pea seedlings and cause fasciation disease appeared to be related to their ability to utilize nitrogen sources
available on plant surfaces.

hypervirulent reisolate of MW11, obtained by passage on

seedlings. Strain MW11VC is an avirulent mutant isolated as described in the text.
Media. Cultures were grown routinely at 30C on a minimal salts thiamine medium (MT) containing 3.0 g of
K,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 of
FeSO4- 7H.O, and 0.005 g of thiamine per liter of deionized
water. The pH of the medium was adjusted to 6.8. Glucose
was autoclaved separately and added to the sterile MT
medium to give a final concentration of 0.5% (wt/vol). MTN
medium was MT medium supplemented with 1.0 g of NH4Cl
per liter. Cultures were also grown on CH medium, containing 5.0 g of Bacto vitamin-free Casamino Acids (Difco
Laboratories, Detroit, Mich.; acid hydrolyzed) per liter of
MT medium. Nutrient broth (NB) containing 8.0 g of Bacto
nutrient broth and 0.5% glucose per liter of distilled water
was used to isolate avirulent strains of C. Ja'scians MW2V.
Bacto-Agar (Difco; 15 g/liter) was used to prepare a solid
medium.
When the ability of C. fjascians to utilize hydrocarbon
fumes as a sole source of carbon was tested, cultures were
grown on glucose-free MTN medium. Sterile capillary tubes
were filled with the liquid hydrocarbon (n-tetradecane,
undecane. or ni-pentadecane) and placed on the inner surface
of the petri dish cover. Plates were then inverted and sealed
with adhesive tape before incubation.
Virulence tests. Seeds of Pisium sativumn L. var. Alaska
Dwarf were surface sterilized in 0.1% mercuric chloride for
10 min, followed by five washings with sterile distilled water.
Washed seedlings were placed in petri dishes lined with
sterile, moistened filter paper and allowed to germinate until
the radicles were between 0.5 and 1.0 cm long (approximately 5 days). Ten germinated seedlings were immersed in
a 48-h culture of C. faiscians grown in the CH medium at
30C (2.0 ml of bacterial culture per seedling) containing
sterile 2% (wt/vol) Carbowax (polyethylene glycol 6000) to
facilitate adhesion of the bacteria to the seedlings. The
addition of polyethylene glycol to the medium in which the
bacteria were suspended gave more reproducible virulence
ratings. Four to five times as many bacterial cells were
bound to plants in the presence of polyethylene glycol than

Corvniebacterium Jscians causes fasciation disease in

dicotyledonous plants. The disease is characterized by a loss
of apical dominance and outgrowth of lateral buds (2, 4, 5).
The bacterium is epiphytic, living on the surface of the host
tissue, where it produces cytokinins (9, 11). The symptoms
of the disease can be duplicated by treating seedlings with
synthetic cytokinins or cytokinins produced by virulent
strains of C. jcians (11). Strains of C. fiscians vary greatly
in their degree of pathogenicity. Virulent strains of C.
J'asdianis were found to excrete more cytokinins into the
culture medium than did weakly virulent and avirulent
strains (8). The major cytokinin found in culture media after
growth of C. fiscilass
was
The
N6-(Ai-isopentenyl)adenine.
most virulent strains also contained a large (Mr, ~108)
plasmid, whereas plasmids were not detected in avirulent
strains (8).
It was shown that repeated cultivation of some plant
pathogens on artificial media resulted in the loss of virulence
(6). This report describes an attempt to find the physiological
basis for the loss of virulence when one C. fliscians strain
was grown at 37C on rich media. To determine whether the
loss of virulence was progressive or due to a single event, we
developed a quantitative assay of the degree of pathogenicity
of different strains.

pea

MATERIALS AND METHODS

Cornnebac'teriiintacn ias strain MW2

isolated by and obtained from M. A. Rahman (University of Alexandria, Egypt). A hypervirulent strain of MW2,
designated MW2V, was isolated in this laboratory from pea
seedlings repeatedly infected with MW2. Strain MW2 is a
weakly virulent parent of strain MW2V. Avirulent derivatives of strain MW2V (designated MW2VC) were isolated as
described in the text. Strain Cf-15 was provided by M. Starr
(University of California, Davis, Calif.). Strain Cf-1 was
obtained 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
Bacterial

isolates.

was

Corresponding author.
33

SABART ET AL.

34

APPL. ENVIRON. MICROBIOL.

0

/~~~~~~~~
CD

10

/~~ ~~~
*z

*-

C):

111

5;
*

...

10

20

DAYS AFTER INOCULATION

FIG. 1. Time course of symptom development in pea seedlings
inoculated with three different strains of C. fliscians. The pea
seedlings were inoculated with C. fiascians strains MW2V (U, O
[two experiments]). Cf-2 (A), and Cf-15 (0) as described in Materials and Methods. Symptom development was used to determine
virulence ratings as described in Materials and Methods.

in its absence. After incubation for 60 min at 30C on a rotary

shaker, the seedlings were removed and planted, one per
pot, at a depth of 2.0 cm in sterile vermiculite. Another 1.0
ml of the bacterial suspensions was added to the vermiculite.
Five uninoculated germinated seedlings were planted per
trial as controls. All seedlings were incubated at 230C under
noncontinuous illumination (12 h on; 12 h off) from a
cool-white fluorescent plant light (General Electric Co.,
Schenectady, N.Y.); the plants were watered with distilled
water. Plants were examined regularly for symptoms. Differences between highly virulent and weakly virulent strains
were obvious within 1 week after planting. The virulence
rating was determined approximately 4 weeks after planting.
A final virulence rating for a given strain was determined by
averaging virulence ratings from five separate trials.
The following scheme was used to quantitatively define
virulence. Stunting was assigned a value of 1, each primary
shoot above one was given a value of 1, and each secondary
shoot was given a value of 0.5. The sum of these values for
all seedlings inoculated with the same culture, divided by the
number of seedlings used in the test, was the virulence rating
for that culture. The first symptoms of C. ftisc ians infection
are multiple shoot development (fasciation), stunted growth,
or both. In many separate trials, the virulence ratings of
different strains and isolates maintained on the MTN medium at 30C agreed within 20%.
Tests for loss of virulence during growth of C. fascians at
37C in a rich medium. C. fascians strain MW2V was grown
in nutrient broth containing 0.5% (wt/vol) glucose at temperatures of 30, 35, and 37C. After 3 and 10 generations of
growth, cultures were serially diluted. Dilutions were spread
onto nutrient agar containing 0.5% (wt/vol) glucose and
incubated at 30C. Fifty or more colonies were selected,
cultivated at 30C in the MTN medium supplemented with
0.05% yeast extract, and tested for virulence.

Measurements of growth and survival of C. fascians strains

on pea seedlings. Pea seedlings were surface sterilized and
germinated as described above. The bacterial strains used in
the experiments were grown in CH medium at 30C for 3
days. Viable cell counts of each culture were determined by
spreading dilutions onto CH agar immediately before the
addition of the germinated seedlings. Twenty germinated
seedlings were immersed in 40 ml of a bacterial suspension
containing sterile 2% (wt/vol) Carbowax (polyethylene glycol 6000) for 1 h at 30C and transferred to petri dishes lined
with sterile, moistened filter paper. Two infected seedlings
were blotted on sterile filter paper to remove unabsorbed
bacteria and transferred to separate sterile tubes with 3.0 ml
of MT medium. The tubes were mixed on a vortex mixer and
incubated on a rotary shaker at 30C for 1 h. This procedure
removed 95% of the cells that were bound. Less than 5%
more bacteria were removed by additional washes or by
grinding the seedlings in a sterile mortar and pestle. The
contents of each tube were serially diluted in sterile MT
medium, and the dilutions were plated on CH agar. The
plates were incubated at 30C, and the yellow colonies of C.
fascians were counted. The initial counts were used to
determine the number of C. fascians cells bound to each
seedling. Two seedlings inoculated with each strain were
removed at intervals and treated similarly to determine the
survival and growth rate of each C. fascians strain on the
host plant.
Source of materials. All chemicals were purchased from
Sigma Chemical Co., St. Louis, Mo., or Fisher Scientific
Co., Fair Lawn, N.J.

RESULTS

The time course of symptom development in pea seedlings

infected with different strains of C. fascians is shown in Fig.
1. The virulence ratings of each strain are in agreement with
the highly virulent MW2V, moderately virulent Cf-2, and
weakly virulent Cf-15 designations previously assigned to
these strains (8). Maximum virulence ratings were achieved
after 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 maximum
virulence when Carbowax was added to the suspending
medium. Approximately i05 cells were required for maximum virulence in the absence of Carbowax.
Isolates from cultures of C. fascians grown at 30 and 350C
on the MTN medium supplemented with 0.05% yeast extract
all had virulence ratings equal (+20%) to that of the parent
strain. In one experiment, colonies isolated from a culture
grown in nutrient broth containing 0.5% glucose at 37C
rapidly lost virulence (Table 1). The loss of virulence in the
majority of the isolates was complete. No detectable symptoms were observed in seedlings infected by 43 of the 52
isolates from a culture grown for 10 generations at 37C in
TABLE 1. Loss of virulence during growth of C. fascians strain
MW2V on rich media at 37C
Virulence

ratings

12.5-14.0
8.0-12.5
1.0-8.0
<1.0

No. of Isolates with virulence rating after

generation:
3

10

57
11
1
2

2
0
7
43

LOSS OF VIRULENCE IN C. FASCIANS

VOL. 52, 1986

nutrient broth plus glucose. Strain MW2V retained virulence

when cultivated at 37C on the MTN medium. The loss of
virulence was found to be dependent both on the rich
medium and growth at 37C.
The number of virulent C. fascians MW2V cells on the
surface of pea seedlings increased exponentially from 3 days
to approximately 8 days (Fig. 2). When 2.1 x 105 cells were
bound per seedling, the population reached 108 cells per
seedling in 18 days. The generation time was approximately
1 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 population of MW2V increased to 8 x 108 cells per seedling in 24
days (Fig. 3). In contrast, the population of MW2VC decreased from 3 x 107 cells per seedling to approximately 2 x
106 cells per seedling in 18 days. The virulence rating for
strain MW2V in this experiment was 14. Seedlings infected
with strait MW2VC did not show symptoms of fasciation
disease.
In another experiment, MW2VC cells, concentrated by
centrifugation to 1010 cells per ml were used to inoculate pea
seedlings. A total of 6 x 107 CFU per seedling were
recovered from newly infected seedlings. The same number
of cells were bound when the inoculum contained 1011 cells
per ml. The bacterial population decreased to less than 106
per seedling in I days. None of the seedlings exhibited
symptoms of fasciation disease. When peas were ihoculated
with MW2V cells (2 x 106 cell per ml), symptom development was obvious within 7 days (Fig. 2). Reinoculation of
seedlings with a suspension of MW2VC cells (109 cells per
ml) at daily intervals failed to produce symptoms.
In five separate experiments, including the one described
in Table 1, 50 or more avirulent colonies (virulence ratings of

Z 108_

0-~~~~

Do
LL

00

'4

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 immersion
in 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 at

37C for 10 generations were isolated. These isolates (ovet
250 total) were all unable to grow on MT medium with

1o 8

agmatine or proline as sole nitrogen sources. The virulent

parental strain, MW2V, grew well with these compoun'ds as
sole sou'rces of nitrogen. Nine of the isol'ates of MW2Vj
obtai'ned after growth in the NB medium at 37C for thr'ee
generations, were weakly virulent (virulence ratings of 1.0 to
3.6) and were found to be able to utilize agmatine or proline
as sole sotirces of nitrogen. All avirulent and virulent isolates
of MW2V grew on MT agar medium with gluiamate (0.25%
[wt/vol]) as a sole source of nitrogen.

35

10

.1

a
-

A few avirullent isolates of MW2V were found to be unable

n-tet'radecane, undecane, or n-pentadecane as the
sole carbon and energy soulrce when grown on MTN medium. The parental MW2V strain and other virulent strains
of C. fascians were able to utilize all three hydrocarbons
tested as the sole carbon and energy source. No growth was
observed on MTN agar without a carbon source.
Growth of MW2V and MW11V at 30C on nutrient agar'
containing ethidium bromide at concentrations that partially
inhibited growth (1 ,ug/ml) resulted in a loss of virulence in
some isolates. This treatment also resulted in a loss of the
ability of some isolates of MW2V and MW11V to grow on
C10-C15 hydrocarbon fumnes as the sole carbon and energy
source. Loss-of virulence, loss of the ability to grow on these
hydrocarbons, and the loss of the' ability to grow on agmatine
and proline as nitrogen sources were found to be independetit events in both strains when mutants induced by
ethidium bromide and by growth at 37C in rich media were
examined (Table 2).
to utilize

106

10

15

DAYS AFTER INOCULATION

FIG. 2. Growth of C. fascians strain MW2V on pea seedlings.
The procedures used to infect seedlings and to determine the viable
population of C. fascians on pea seedlings were described in
Materials and Methods. Seedlings were immersed in a suspension of
C. fascians containing 2 x 106 viable cells per ml. Bars represent the
range of viable cell counts per seedling in replicate samples.

36

APPL. ENVIRON. MICROBIOL.

SABART ET AL.
TABLE 2. Relationship between hydrocarbon utilization and
virulence of C. fascians strains
Strain

Virulence

,designationa

rating

MW2V
MWllV
MW2VC
MWllVC
MW2V-1
MWllV-l
MW11V-2

14.0
20.0
0
0
0.6c
0.9c
0.6c

Growth on glucose-free MTN agar with the

following carbon source:b

Undecane

n-Tetradecane

n-Pentadecane

+
+

+
+

+
+

+
+
+
+

+
+
+

a
MW2VC and MW11VC are avirulent mutants isolated after growth of
MW2V on nutrient agar at 37C. MW2V-1, MW11V-1, and MW11V-2 are
mutants isolated after growth of MW2V and MW11V on nutrient agar
containing 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 mutants
were avirulent and had single requirements for glycine,
arginine, methionine, aspartic acid, or adenine. We isolated
spontaneous mutants that became avirulent during growth
on rich media at 37C. The loss of virulence in strain MW2V
was correlated with the inability of the isolates to grow on
pea seedlings and with the inability to use agmatine or
proline as the sole source of nitrogen. None of these isolates
were auxotrophic.
The ability of virulent strains to survive and grow on pea
seedlings appeared to be associated with their ability to
utilize nitrogen sources provided by the host. Growth of C.
fascians on pea seedlings is required for symptom development (10). Neither increasing the inoculum size of avirulent
strain MW2VC nor reinoculation of pea seedlings with
MW2VC cells at daily intervals resulted in development of
fasciation disease symptoms.
We cannot yet satisfactorily explain the consistent loss of
the ability of strain MW2V to utilize agmatine or proline as
the sole source of nitrogen. One possible explanation for the
phenotype is that a single mutation occurred and that siblings of this mutant grew faster at 37C than did the virulent
parental strain. However, this explanation is not consistent
with the following observations. (i) Avirulent mutants of
MW2V with the same phenotypes were obtained in five
separate experiments, (ii) differences in the growth rates of
MW2V (wild type) and several avirulent isolates of MW2V
(including MW2VC) at 37C in NB with glucose were not
observed, and (iii) the large proportion of mutants to wildtype isolates obtained in the experiments described cannot
be accounted for by a single mutational event followed by
growth of the mutant during 10 generations of growth.
It is not likely that proline and agmatine catabolism are
encoded by plasmid DNA. The highly virulent C. fascians
strain MW2V contains three plasmids (8). In numerous
attempts, we failed to detect plasmid DNA in a weakly
virulent strain, MW2, derived from the same parent. This
strain will grow on media containing agmatine or proline as

a sole source of nitrogen. Attempts to isolate mutants from

this strain by growth at 37C in nutrient broth plus glucose
have consistently failed. The production of high levels of
cytokinins (8) and ability to undergo high-frequency mutagenesis under these conditions appears to be associated with
one or more plasmids. We have not isolated plasmids from
strains that have been cured by growth at 37C in rich media.
This procedure appeared to eliminate the plasmids observed
in the highly virulent strain.
Attempts to produce mutants by growth of C. fascians in
the presence of ethidium bromide resulted in the isolation of
strains unable to grow on hydrocarbons as well as avirulent
isolates. Ethidium bromide is known to cure some bacteria
of plasmids (1). However, hydrocarbon utilization, virulence, and the utilization of agmatine and proline as nitrogen
sources were not correlated. It is not known whether hydrocarbon 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 survival
cannot replicate at 37C and integrates into the genome at a
specific site near or within genes essential for the catabolism
of agmatine and proline. This hypothesis would explain why
the weakly virulent MW2 strain that is apparently devoid of
plasmids fails to produce avirulent mutants when grown on
rich media at 37C.
ACKNOWLEDGMENTS
This research was supported by grant 5-T 32 GM 07094 from the
Department of Genetics and Cell Biology, University of Minnesota,
and by the University of Wisconsin College of Agricultural and Life
Sciences.
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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 infection 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 plants
and "leafy gall" of various plants. Ann. Appl. Biol. 23:302-310.
5. Lacey, M. S. 1939. Studies on a bacterium associated with leafy
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86:55-68.
8. Murai, N., F. Skoog, M. E. Doyle, and R. S. Hanson. 1980.
Relationships between cytokinin production, presence of plasmids and fasciation caused by strains of Corynebacterium
fascians. Proc. Natl. Acad. Sci. USA 77:619-623.
9. Rathbone, M. P., and R. H. Hall. 1972. Concerning the presence
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