Materials and Methods

1
Relative importance of bacteriocin-like genes in
antagonism of T3 strains to T1 strains of
Xanthomonas campestris pv. vesicatoria.
A.P. HERT (1), S. Tudor
(2), P.D. Roberts (1), G.V. Minsavage (2), and
J.B. Jones (2). (1) University of Florida,
Immokalee, and (2) University of Florida,
Gainesville

Abstract Xanthomonas campestris pv. vesicatoria
tomato race 3 (Xcv T3) strain 91-118 produces at
least three different bacteriocin-like compounds
(BCN-A, BCN-B, BCN-C) antagonistic to Xcv
tomato race 1 strains. The genes were disrupted
in BCN activity by either transposon mutagenesis
or mutagenesis using a suicide vector method and
were marker exchanged into a T3 strain, 91-118.
All bacteriocin-like compounds were evaluated for
activity in antagonism to T1 strains. Prior
experiments demonstrated that the bacteriocins
conferred different levels of inhibition to a T1
strain. T1 populations were reduced when
inoculated in combination with the wild-type,
BCN-AC, and BCN-BC, but not with BCN-C alone.
BCN-AC and the wt T3 were significantly better
than the other mutants. In the field when all
strains were mixed and inoculated, BCN-AC had a
competitive advantage in the field over the wt T3
and other mutants.
T1 strain, we generated BCN- clones by transposon
and suicide assisted deletion mutagenesis and
used marker-exchange and suicide-assisted
mutagenesis to knock out individual
bacteriocin-related genes. For each mutant
designated, the letter corresponds to the
bacteriocin knocked out (eg. ME-A indicates
marker exchange A for a BCN-A- BCN-B BCN-C
phenotype).
Materials and Methods Six-week-old seedlings of
the tomato cultivar Florida 47 were used in field
experiments. Strains were grown in nutrient broth
for 24 hr, harvested by centrifugation, and
resuspended in a solution of 0.01M MgSO4 ? 7 H20.
The concentration of the bacterial suspension was
adjusted to 5x107 CFU/ml and contained 0.025
Silwet as a surfactant. Plants were dipped into
suspensions of the wild-type (wt) T1 strain 24 hr
prior to inoculation with the mutant strain.
Each treatment consisted of four replications.
Plants were transplanted to the field two days
after the second dip inoculation. Symptomatic
leaf samples were taken every two weeks beginning
45 days after transplanting to determine the
populations of each strain in the treated plots.
Thirty lesions were sampled from 10-20 leaflets
in each plot. Bacteria were isolated by
maceration of a lesion in 75?l of sterile DI
water and the suspension streaked on NA amended
with Pentachloronitrobenzene (PCNB) and/or
cycloheximide. Individual colonies were plated
onto two media, one amended with antibiotics for
the mutant strain and another amended with
antibiotics for the sensitive T1 strain.
Antibiotics were used to maintain selection for
resistance markers at the following
concentrations tetracycline (Tet) 10?g/ml
rifamycin (Rif) 100?g/ml spectinomycin (Spec)
50?g/ml kanamycin (Kan) 50?g/ml chloramphenicol
(Cm) 34?g/ml streptomycin (Strep) 200?g/ml and
naladixic acid (Nal) 20?g/ml.
Introduction Bacterial spot of tomatoes and
peppers is caused by the pathogen, Xanthomonas
campestris p.v. vesicatoria. It is a major
problem in tropical and subtropical locations
such as Florida. On tomato, three races,
designated T1, T2, and T3, have been identified
based on their reaction on three tomato
cultivarsHawaii 7998 (H7998), Hawaii 7981
(H7981), and Bonny Best (Table 1).
Results The wt T3 and ME-B (BCN-A, BCN-B- ,
BCN-C) mutant strains significantly reduced
incidence of the T1 strain than the other
strains. Furthermore, ME-B had significantly
higher percentage of recovery than the wt T3,
while ME-A, ME-C, and ME-ABC did not differ
significantly (Figure 1 2). In a separate
experiment in which all the mutant strains and
the wild-type T1 strains were co-inoculated, ME-B
averaged over 70 recovery from lesions, whereas
wt T3 91-118 accounted for slightly more than 8
of the recovered strains (Figure 3 4).
Discussion In the first field experiment in
which the T3 or mutant T3 strains were applied
individually in combination with the T1 strain,
the wt T3 and mutant T3 strains significantly
reduced recovery of T1 from lesions. There was
also evidence in this experiment to suggest the
BCN-B- (ME-B) strain most effectively inhibited
the T1 strain. In the second field experiment,
in which the wt and mutant T3 strains were
co-inoculated with the T1 strain, the BCN-B-
mutant clearly appeared to be more competitive
than all other strains, including the wt T3
strain. Two possible explanations for the high
percentage of ME-B (BCN-B-) recovered when
compared to the other mutants are (1)
Bacteriocin B is inhibitory toward the expression
of at least one other bacteriocin, most likely
BCN-A and/or (2) bacteriocin B has a negative
effect on virulence factors. The first
explanation is supported by previous in vitro
experiments in which ME-B consistently exhibited
the largest zone of inhibition, including
inhibition zones produced by the wt T3 strain.
The second explanation is supported by the second
field experiment in which wt and mutant T3
strains were co-inoculated with the T1 strain,
resulting with BCN-B- strains predominating over
all other strains in the field. No single
strategy has been successful for controlling this
disease. This evidence shows the possibility of
developing a biological control strategy
utilizing these compounds. One study already
demonstrated that a non-pathogenic T3 strain of
X. campestris pv. vesicatoria was able to reduce
the severity of bacterial spot disease in the
field. Further characterization of the
bacteriocins may lead to promising new control
strategies for bacterial spot of tomato.
Table 1. Tomato Genotype table for Tomato Race
Identification
Tomato races T1 and T2, are present worldwide.
In the early 1990s another race differing from
T1 and T2 was identified in Florida and
designated Tomato race 3. This new strain was
distinct from T1 and T2 strains based on various
phenotypic and genotypic tests, but was similar
to T1 stains based on carbon utilization
patterns. Based on DNA similarity values T3
strains are genetically most closely related to
T1 strains. Previous research has shown
that Xanthomonas campestris p.v. vesicatoria race
T3 strains are antagonistic toward T1 strains.
Multiple antagonistic compounds were identified
in T3 strains, that closely resemble
bacteriocins. These compounds were determined to
have a narrow inhibition spectrum restricted to
X. campestris pv. vesicatoria strains, and fit
the definition of a bacteriocin based on the
following three criteria (1) the presence of a
biological active protein moiety, (2) by
inducibility with mitomycin C, and (3) non-self
inhibition. A T3 genomic library was constructed
and ten clones were identified that had the
ability, when present in a T1 strain, to inhibit
a T1 strain in plate assays. Southern
hybridization analysis grouped the clones into
three subsets, BCN-A, BCN-B, and BCN-C. The
three were distinct from each other in activity
and specificity.
Citations Bouzar, H., Jones, J.B., Somodi, G.C.,
Stall, R.E., Daouzli, N., Lambe, R.C.,
Felix-Gastelum, R., and Trinidad-Correa, R. 1996.
Diversity of Xanthomonas campestris pv.
vesicatoria in tomato and pepper fields of
Mexico. Can. J. of Path. 18 75-77. Jones, J.B.,
Bouzar, H., Somodi, G.C., Stall, R.E., Pernezny,
K., El-Morsy, G., and Scott, J.W. 1998a. Evidence
for the preemptive nature of tomato race 3 of
Xanthomonas campestris pv. vesicatoria in
Florida. Phyopathology 88 33-38. Jones, J.B.,
Stall, R.E., and Bouzar, H.1998b. Diversity among
Xanthomonads pathogenic on pepper and tomato.
Annu. Rev. Phytopathol. 36 41-58. Jones, J.B.,
Stall, R.E., Scott, J.W., Somondi, G.C., Bouzar,
H., and Hodge, N.C. 1995. A third tomato race of
Xanthomonas campestris pv. vesicatoria. Plant.
Dis. 79 395-398. Lui, T. 1998. Biological
control of tomato bacterial spot wth a Hrp-
mutant of Xanthomonas campestris pv. vesicatoria.
Masters thesis. University of Florida,
Gainesville. Tudor, S.M. 1995. An analysis of
antagonism in tomato race three strains of
Xanthomonas campestris pv. vesicatoria. Masters
thesis. University of Florida, Gainesville. Tudor,
S.M. 1999. Molecular characterization of
bacteriocin-like activity in tomato race-three
strains of Xanthomonas campestris pv.
vesicatoria. PhD thesis. University of Florida,
Gainesville. Tudor, S.M., Jones, J.B., Stall,
R.E. 1998. Evidence for multiple bacteriocins
produced in tomato race 3 strains of Xanthomonas
campestris pv. vesicatoria and their role in
antagonism towards T1 strains. Pytopathol. 88S90.
Objective of the study In order to evaluate the
relative importance of the bacteriocin-like
activity under field conditions as they relate to
competition with a sensitive