Download GROWTH INHIBITION OF CLAVIBACTER MICHIGANENSIS

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Journal of Plant Pathology (2009), 91 (1), 221-224
Edizioni ETS Pisa, 2009
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SHORT COMMUNICATION
GROWTH INHIBITION OF CLAVIBACTER MICHIGANENSIS subsp.
MICHIGANENSIS AND PSEUDOMONAS SYRINGAE pv. TOMATO
BY OLIVE MILL WASTEWATERS AND CITRIC ACID
Z. Özdemir
Department of Plant Protection, Faculty of Agriculture, Adnan Menderes University, 09100 Aydın, Turkey
SUMMARY
Effects of citric acid and olive mill wastewaters on
the growth of seed-borne bacterial pathogens of tomato,
Clavibacter michiganensis subsp. michiganensis and
Pseudomonas syringae pv. tomato were investigated.
Bacteria were exposed to citric acid (0.1 mol l-1) and 10
fold-diluted filter-sterilized olive mill wastewaters and
their growth was determined by the plate count
method. Citric acid inhibited the growth of both bacteria. The minimum inhibitory concentration (MICs) of
citric acid, determined by the broth dilution method
ranged between 1.56 to 3.13 mmol l-1 for C. m. michiganensis, and was 1.56 mmol l-1 for P.s. tomato. Olive mill
wastewaters inhibited the growth of both bacteria in
most experiments. In some, however, a slight growth or
reduction in size of the colonies of C. m. michiganensis
was observed. Neither treatment showed negative effects on seed germination. Results of this study suggest
that citric acid at 0.1 mol l-1 concentration can prove
useful for the elimination of both pathogens from tomato seeds.
Key words: Plant pathogenic bacteria, organic acids,
biophenols, antimicrobial agent, growth inhibitors
INTRODUCTION
Olive mill wastewaters (OMW) cause environmental
concerns due to their richness in toxic compounds such
as low-degradable biophenols (De Marco et al., 2007).
OMW can reduce the population of the soil bacterium
Bacillus megaterium (Paredes et al., 1986; González et
al., 1990), are phytotoxic (Capasso et al., 1992), can
seep through the soil reaching underground water, and
can increase soil salinity (Obied et al., 2007). On the
other hand, OMW can be used as fertilizer (Paredes et
al., 1999), weed suppressor (Boz et al., 2003) and show
Corresponding author: Z. Özdemir
Fax: +90.256.7727233
E-mail: [email protected]
antifungal and antibacterial properties due to the high
content of phenolics (Capasso et al., 1995; Aziz et al.,
1998) and other compounds (González et al., 1990; Capasso et al., 1992). Capasso et al. (1995) studied the effects of raw OMW on Pseudomonas savastanoi pv. savastanoi and Clavibacter michiganensis subsp. michiganensis showing their bactericidal effects. Ciafardini and
Zullo (2003) proved that OMW phenols, caffeic acid in
particular, had no negative effect on cauliflower seed
germination when used to control Xanthomonas
campestris pv. campestris.
Citric acid is effective in controlling food-borne
yeasts and bacterial pathogens (Ananou et al., 2007;
Nielsen and Arneborg, 2007), its antimicrobial properties resting primarily in the chelation of divalent cations
(Imai et al., 1970; Graham and Lund, 1986). However,
citric acid has not been tested against many plant pathogenic bacteria. A study was therefore conducted for
evaluating the effects of OMW and citric acid on the
growth of the tomato seed-borne pathogens Clavibacter
michiganensis subsp. michiganesis and Pseudomonas syringae pv. tomato.
C. m. michiganensis ICMP 2550 (International Collection of Micro-organisms from Plants, Auckland, New
Zealand) and P. s. tomato ICMP 2844 were used in the
experiments. Tested inhibitors were 10-fold diluted
OMW at pH 4.6, and citric acid (Carlo Erba, Italy) at
0.1 mol l-1 concentration and pH 2.0. OMW was obtained from a mill near Çakırbeyli, (Aydın, Turkey) and
was filter-sterilized before use because in preliminary
experiments some unidentified bacteria and fungi were
observed when it was plated on agar medium.
To test bacterial growth inhibition by the plate count
method, C. m. michiganensis and P. s. tomato cultures
were grown on nutrient broth yeast extract agar (NBY,
Schaad et al., 2001) at 24 ºC for 96 h and 48 h, respectively. Four colonies from each culture were transferred
to 9 ml of 1% nutrient broth glucose medium (NBG) in
glass tubes. Liquid cultures were grown overnight by
shaking at 55 rpm with a twist shaker (Isolab, Germany)
at 26ºC. Aliquots of 0.1 ml from these liquid cultures
(108-109 cfu ml-1) were transferred to tubes containing
0.1 mol l-1 citric acid and 10-fold diluted OMW in 10
ml NBG, which were incubated under shaking at 26ºC
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Growth inhibitors of bacteria by OMW and citric acid
Journal of Plant Pathology (2009), 91 (1), 221-224
Fig. 1. Growth inhibition of citric acid (0.1 mol l-1) and OMW (diluted 1:10) on Clavibacter michiganesis subsp. michiganensis
ICMP 2550. A. Experiment 1; B. Experiment 2. Results of the third experiment are not shown here since growth was inhibited
completely by citric acid and OMW treatments as in Fig. 1B.
for 1.5 and 8 h. Untreated tubes were used as control
and 0.1 ml aliquots from treated and untreated tubes
were plated onto tryptic soya agar (TSA, Difco, Becton
Dickinson, England) plates in triplicate. Plates were incubated for 24 to 48 h at 26ºC for allowing the development of P. s. tomato and C. m. michiganensis colonies,
respectively. Growth inhibition of P. s. tomato by OMW
was also assessed after exposure to treatment for 1 h
and 45 min.
To determine the minimum inhibitory concentrations
(MICs) of citric acid against both bacteria, the broth dilution method (EUCAST, 2003) was used. Liquid cultures of C. m. michiganensis (1-2 x 109 cfu ml-1) and P. s.
tomato (1-2 x 108 cfu ml-1) grown overnight (17 h) in
NBG were serially diluted to adjust bacterial concentrations to ca. 1-1.5 x 105 cfu ml-1 . To obtain descending
half concentrations, citric acid at the initial concentration of 0.1 mol l-1 was prepared and diluted in 4 ml final
volume of NBG in glass tubes. Serial dilutions followed
so as to reach a final citric acid concentration of 0.024
mmol l-1. Tubes with each citric acid concentration (500.024 mmol l-1) were additioned with 0.1 ml of liquid
bacterial cultures (1-1.5 x 105 cfu ml-1) and incubated at
26ºC for 24 or 48 h for allowing growth of P. s. tomato
and C. m. michiganensis, respectively. A duplicate set of
citric acid concentrations with no bacterial cultures
added was prepared in NBG as control. Bacterial
growth was assessed visually. MICs were determined in
duplicate experiments as the lowest concentration
where no visible bacterial growth (turbidity) was observed.
Effects of citric acid and OMW on seed germination
were tested on tomato cv. H-2274. Citric acid at 0.1 mol
l-1 concentration was dissolved in 10 ml sterile distilled
water (SDW) in sterile glass tubes. SDW was used as
control. Tomato seeds were added to each tube, mixed
briefly and incubated at 26ºC for 1 h. Seeds were then
removed by filtration and rinsed three times with 10 ml
SDW. Rinsed seeds were dried on sterile filter paper,
then placed onto sterile double-layered wet filter paper
in sterile plastic 90 mm Petri dishes and incubated at
26ºC. Each treatment had five replicates. Twenty three
seeds were used per replicate Seed germination was
recorded at initial germination (4 days after incubation)
and again at 6 to 7 days, when most of the seeds had
germinated.
The effect of OMW on seed germination was determined as in citric acid-treated seed assays. Seeds treated
with OMW for 1 h at 26ºC, were rinsed three times in
25 ml sterile distilled water, dried and incubated as
above.
Citric acid and OMW assays were repeated twice.
Statistical analysis of the data from the first and second
readings on seed germination was done using the SPSS
Program (USA). Since no statistical difference was ob-
Fig. 2. Growth inhibition of Pseudomonas syringae pv. tomato
ICMP 2844 by citric acid (0.1 mol l-1) This figure is representative of three independent experiments.
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Journal of Plant Pathology (2009), 91 (1), 221-224
served between the two readings, results of first reading
only are presented.
Whereas citric acid completely inhibited the growth
of C. m. michiganensis and P. s. tomato (Fig. 1 and 2).
OMW was not equally effective in all experiments, but
Özdemir
223
strongly reduced the growth of both pathogens (Fig 1
and 3). In particular, growth of P. s. tomato was completely inhibited in two experiments, while in two others bacterial colonies developed on TSA plates after 8 h
incubation. OMW treatment caused approximately 7
log/ml reduction in the growth of colonies as compared
with the control (Fig. 3). C. m. michiganensis showed
very slight growth after 1 and 5 h incubation with
OMW in one out of three experiments. In both cases,
growth reduction was ca. 5 log/ml in comparison with
the control (Fig. 1).
MICs of citric acid were 1.56 mmol l-1 for P. s. tomato
and ranged between 3.13 and 1.56 mmol l-1 for C. m.
michiganensis,
Citric acid and OMW had no negative effect on the
germination of tomato cv. H-2274 seeds (Tables 1 and
2), as shown by the lack of statistical significance between the means of control (SDW), citric acid and
OMW treatment data. In the first experiment, citric
acid-treated and control seeds showed the same germination level (73%) and in the second experiment, germination of citric acid-treated seeds was 78% compared
to 81% of the control (Table 1). OMW-treated seeds
had 79% germination, slightly higher than that of the
control (74%) in the first experiment and slightly lower
in the second (69% versus 80%) (Table 2). The slight
reduction in germination detected in the second experiment, may have been caused by residual OMW on the
seed surface.
Plant pathogenic bacteria such as Pseudomonas savastanoi pv. savastanoi and C. m. michiganensis (Capasso et
al., 1995) were previously shown to be negatively affected
by OMW (up to 100% growth inhibition at 1:1 and 1:2
dilution). A positive although lower effect (55% growth
reduction) was also reported for Pseudomonas tolaasii
Table 1. Effect of citric acid on tomato seed germination.
Treatments
Experiment 1a
(% germination)
Experiment 2
(% germination)
Citric acid (0.1 M)
73.40+12.05
77.80+9.39
Control (SDW)
73.80+8.76
81.20+9.39
a
Means ± standard deviation.
Table 2. Effect of OMW on tomato seed germination.
Fig. 3. Growth inhibition of Pseudomonas syringae pv. tomato
ICMP 2844 by olive mill waste waters (OMW) (diluted 1:10).
A-C. Experiments 1-3 respectively. Results of the fourth experiment are not shown since complete inhibition of bacterial
growth was obtained as in Fig. 3B.
Treatment
Experiment 1a
(% germination)
Experiment 2
(% germination)
OMW (1:10 diluted)
78.60+11.74
68.60+8.26
Control (SDW)
74.40+7.64
79.80+8.53
a
Means ±standard deviation
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Growth inhibitors of bacteria by OMW and citric acid
(Soler-Rivas et al., 2006). This is in line with the results of
the present study showing that two seed-borne bacterial
pathogens of tomato, i.e. the Gram negative P. s. pv.
tomato and the Gram positive C. m. michiganensis are
strongly inhibited by 10 fold-diluted OMW.
Some of the OMW-treated C. m. michiganensis
colonies showed very slow growth (data not shown).
This type of colonies was recognized 18 to 24 h after
reading OMW-treated TSA plates. Interestingly, an adverse effect was reported by Della Greca et al. (2001)
four days after exposure of algae to OMW, consisting of
modification of the shape and colour of the cells.
Examples of the use of citric acid as seed treatment
for controlling plant pathogens are few (Haggag and
Abd El-Khair, 2007). The present results now show that
citric acid can be a potential control agent against some
plant pathogenic bacteria. Citric acid is inexpensive,
easily applied to and removed from seeds, and does not
cause irritation to the operators. Thus, it is a good candidate product to use in organic agriculture (Davies and
Maw, 1972).
Although confirmation by further and more extensive experiments would be desirable, it seems plausible
to conclude that citric acid and OMW can be safely employed as a seed treatment with no concern for phytotoxicity or negative impact on germination.
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