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Jurnal Mikrobiologi Sabart Et Al., 1986 Isolasi Corynebacterium Fasciens
Jurnal Mikrobiologi Sabart Et Al., 1986 Isolasi Corynebacterium Fasciens
to
arre,
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.
pea
isolates.
was
Corresponding author.
33
SABART ET AL.
34
/~~~~~~~~
CD
10
/~~ ~~~
*z
*-
C):
111
5;
*
...
10
20
RESULTS
ratings
12.5-14.0
8.0-12.5
1.0-8.0
<1.0
10
57
11
1
2
2
0
7
43
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.
1o 8
35
10
.1
a
-
106
10
15
36
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
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
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
of the cytokinin N6-(A2-isopentenyl)adenine, in cultures of Corynebacterium fascians. Planta (Berlin) 108:93-102.
10. Rivain, J.-G., and J. Roussaux. 1982. Relationship between
growth and pathogenicity of Corynebacterium fascians (Tilford)
Dowson. Agronomie 2:479-486.
11. Thimann, K. V., and T. Sachs. 1966. The role of cytokinins in
the "fasciation" disease caused by Corynebacterium fascians.
Am. J. Bot. 53:731-739.