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Previous Article | Next Article 
Applied and Environmental Microbiology, November 2007, p. 7210-7217, Vol. 73, No. 22
0099-2240/07/$08.00+0 doi:10.1128/AEM.00960-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada-Náquera, km 4.5, Moncada 46113, Valencia, Spain,1 Departamento de Microbiología y Ecología, Universidad de Valencia, Av. Dr. Moliner, 50, Burjasot 46100, Valencia, Spain2
Received 29 April 2007/ Accepted 29 August 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fate of R. solanacearum in environmental waters may also depend on the presence of native microorganisms reported to be involved in predatory, competitive, or parasitic events in other bacterial models (17, 19, 20, 33, 35, 43). Within the range of water microbiota, bacteriophages and protozoa represent relatively constant sources of mortality for bacterial populations in water systems (5, 21, 22, 33, 48, 49). For instance, in river water, the disappearance of Escherichia coli has been related to the presence of bacteriophages and bacteria (17) as well as predatory protozoa (4, 19). In the same way, a strong effect of indigenous microbiota on the survival of R. solanacearum in agricultural drainage water microcosms has been reported (45) but the nature and influence of the main groups of the aquatic biota were not investigated.
Our research was motivated by the scarce information available on the fate of R. solanacearum in natural water, together with the fact that this pathogen had been detected in some watercourses in Spain (9, 39). The objectives were to establish whether R. solanacearum ph II can survive in microcosms of natural water from a Spanish river and to ascertain the influence of aquatic microorganisms on its survival, which is related to the persistence of the bacterium in the environment. River water microcosms inoculated with relatively large populations of this pathogen were selected as a model that would mimic what would happen in environmental water systems when occasionally polluted by R. solanacearum.
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MATERIALS AND METHODS |
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Water samples.
Water samples were collected from the river Tormes (Northwestern Spain) at three sampling sites contaminated with R. solanacearum (9, 39), as described by Caruso et al. (9).
Protozoa were counted in each water sample after 2% (vol/vol) formaldehyde fixation. Samples were filtered through a 100-µm-pore-size sieve, and the particles between 30 and 100 µm were counted by a Coulter Z series device (Coulter Corporation). After staining by acridine orange (37), microscopic observation of the samples was also performed by epifluorescence to confirm the presence of protozoa and to discard protophyta. A Nikon Eclipse E800 microscope was used at magnifications of x400 and x600.
Culturable counts of native bacteria and R. solanacearum were performed with YPGA and a modified semiselective medium South Africa (SMSA) agar (14), respectively, after incubation for 72 h at 29°C. Semiselectivity for R. solanacearum is mainly based on the actions of four antibiotics (penicillin, polymyxin, chloramphenicol, and bacitracin), triphenyl-tetrazolium chloride, and crystal violet (1, 3, 14). Identification of putative R. solanacearum water isolates and biovar characterization were performed as described above (1, 3).
Bacteriophage enrichment detection assays for R. solanacearum lytic phages were performed in triplicate by adding 1-ml aliquots of each 0.22-µm filtered river water sample to log-phase cultures of R. solanacearum cells (about 109 CFU/ml) in 10 ml and 5 ml of a modified Wilbrink broth (8), according to Hendrick and Sequeira (24). Bacterial suspensions without filtered water were used as negative controls. Incubations were done in flasks of 50 ml with shaking (165 rpm) at 29°C and 35°C, because both temperatures had previously been used to improve the recovery of R. solanacearum in water samples from this river (9). The cleared suspensions (lysates) were tested for phage enumeration on YPGA plates according to a standard surface plating method (10). Based on this kind of enrichment assays, the initial concentrations of lytic phages of R. solanacearum in the river water were estimated according to the most-probable-number technique (49), by determining the number of tubes in each group that became cleared and testing for the presence of plaques.
Survival experiments in river water microcosms.
Natural river water microcosms were prepared with water samples as described for other models (17, 33). Briefly, four types of microcosms were prepared in duplicate for each of the three water samples used in each of the survival experiments: (i) untreated river water, with the whole microbiota; (ii) 0.8-µm-filtered water, to keep most protozoa apart; (iii) 0.2-µm-filtered water, to remove most of bacteria; and (iv) control microcosms, with 0.2-µm-filtered and autoclaved water to inactivate virus. R. solanacearum strain IVIA-1602.1 was inoculated at a final concentration of 0.5% (5 x 106 CFU/ml) in a volume of 100 ml for each microcosm. Samples were incubated in the dark in the static state at either 24 ± 1°C or 14 ± 1°C for 1 month. Both temperatures were selected because they were within the range in which R. solanacearum had been detected in this river (9, 39).
For some experiments, supplementary microcosms prepared as described above were also inoculated with either the Dutch strain IPO-1609 or a variant of the Spanish strain resistant to river water phages isolated during the experiments (from 0.2-µm-filtered water microcosms), as described below.
Total, viable, and culturable R. solanacearum cell counts.
Sampling from the microcosms was initially performed at inoculation time (day 0) and 3, 6, 9, 15, 21, and 33 days postinoculation (dpi) in preliminary assays and at 0, 1, 2, 4, 8, 15, 22, and 28 dpi in subsequent experiments. Plate counts were done in duplicate, after incubation for 72 h at 29°C on YPGA and SMSA media, with a detection limit of about 10 CFU/ml. Total and viable cells were microscopically counted by a modified direct-viable-count method (29, 45). According to this method, yeast extract and nalidixic acid were added to aliquots to reveal the presence of viable cells, which elongated without dividing. Subsequently, cells were stained by acridine orange as described previously (37). For microcosms with indigenous bacteria, R. solanacearum cell staining was performed by indirect immunofluorescence (IF) (1, 3) with a polyclonal antiserum, 1546-H IVIA (9). The detection limit of this technique is around 103 cells/ml (1, 3), but it was 10-fold increased for some samples by processing higher volumes. Cells were visualized with a Leika epifluorescence microscope at an amplification of x1,250.
Coculture of R. solanacearum strain IVIA-1602.1 and a purified river water phage.
To confirm the influence of indigenous river water phages on R. solanacearum populations, sterile river water was coinoculated with the strain IVIA-1602.1 at about 106 CFU/ml (similar to that inoculated in river water microcosms) and a selected phage at 2 x 102, 2 x 103, 7 x 103, or 2 x 104 PFU/ml (within the range for river water estimated by the most-probable-number method) at 24°C. Similar bacterial concentrations and phage at 2 x 103 and 7 x 103 PFU/ml were coinoculated and monitored at 14°C. Sterile river water with either R. solanacearum or the phage was used as a control. Aliquots for bacterial and phage counts were taken regularly over 24 h at 24°C or 48 h at 14°C, serially 10-fold diluted in phosphate-buffered saline and plated on YPGA. For phage enumeration, aliquots were previously filtered through 0.22-µm-pore-size membranes and plated as described above (10). Total bacterial counts were performed as described above, in some of the assays at both temperatures. All the assays were done in duplicate, in separate experiments.
Statistical analysis.
Each survival experiment was done at least in duplicate in independent experiments, and data for total, viable, and culturable R. solanacearum cell counts were analyzed by using mean values of log-transformed data from duplicate samples. The null data from culturable counts below the detection limit were not included for the statistical analysis. Significant differences were assessed by variance analysis. Factors considered for the analysis were day, media, experiment, water treatment, and incubation temperature. Differences were recorded as significant at P values below 0.05.
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RESULTS |
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Culturable counts of river water bacteria on YPGA (from samples taken at water temperatures above 14°C) were similar in all water samples analyzed (103 CFU/ml), while counts for R. solanacearum populations on SMSA agar ranged from 15 to 45 CFU/ml. When water temperature dropped below 14°C, river water bacterial counts decreased to 102 CFU/ml and the pathogen was not detected by direct isolation. For survival experiments, only water samples taken at temperatures of 
14°C were used.
Lytic phages of R. solanacearum were detected and isolated from water samples, but only from those where the pathogen was recovered on SMSA agar (at 14°C). Initial concentrations of these lytic phages in the river water samples were estimated between 102 and >103 lytic viral particles/ml. In the assays for phage detection at 29°C, suspensions were observed to clear after overnight incubation for a 10:1 (vol/vol) bacterium/phage ratio and earlier for a ratio of 5:1 (vol/vol) in all water samples, while control suspensions became more turbid. Phage enumeration on YPGA plates spread inoculated with fresh cultures of R. solanacearum yielded plaques which were visible after 36 to 48 h of incubation at 29°C and continued expanding on plates up to 72 h. Plaque counts were similar in all cases (108 to 109 PFU/ml). Plaques were usually round with irregular edges and transparent, and some variation in size was observed. In contrast, at 35°C, phage detection assay results were negative for the same water samples that had proved positive at 29°C. Characterization of lytic activity by selected river water phages against R. solanacearum strains IVIA-1602.1 and IPO-1609 in modified Wilbrink broth confirmed such activity at 29°C but not at 35°C.
Survival of R. solanacearum strain IVIA-1602.1 in river water microcosms.
Preliminary survival assays monitoring R. solanacearum culturability in river water microcosms at 24°C were done for three water samples, with similar results. In sterile river water, the culturability of the strain was maintained at 107 CFU/ml, while in all microcosms with indigenous microbiota, the strain was not detected on plates from the second sampling day, at 3 dpi. From that time, in 0.2-µm-filtered water microcosms, colonies which were slightly different from those of the inoculated strain appeared on YPGA and SMSA media and were identified as R. solanacearum (1, 3). These colonies were comparably smaller, with less fluid and irregular edges, and were further proved to be phage resistant. Regarding 0.8-µm-filtered and untreated water microcosms, only indigenous bacteria were observed from 3 dpi on YPGA (105 CFU/ml) and SMSA (102 to 103 CFU/ml) agar and no colonies of phage-resistant variants were detected. The declines in the inoculated R. solanacearum populations in all the nonsterile microcosms suggested the involvement of river water microorganisms. Similar results were obtained for the other two water samples.
To confirm these initial results, another three water samples taken from the same river sites were used to prepare new microcosms, which were monitored in more detail at 24°C and 14°C. Figure 1 shows survival curves for strain IVIA-1602.1 in the different microcosms for one representative water sample. Trends in the dynamics of total, viable, and culturable R. solanacearum cells were similar for the other two water samples, showing significant differences (P < 0.05) between the two incubation temperatures.
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Survival at 14°C.
Cell counts of R. solanacearum in sterile control microcosms at 14°C (Fig. 1E) were similar to those at 24°C (Fig. 1A), whereas the declines in total, viable, and culturable cell numbers for nonsterile water microcosms showed comparably less pronounced slopes (Fig. 1B and F, C and G, and D and H). Likewise, significant differences were observed between sterile and nonsterile microcosms (P < 0.05). Microscopic counts in all nonsterile river water microcosms (Fig. 1F, G, and H) decreased similarly to culturability within the first 2 days. Afterwards, for 0.2-µm-filtered water (Fig. 1F), there were declines in total and viable cell numbers to 105 or 106 cells/ml and subsequent increases, followed by stabilization at around 106 to 107cells/ml. As at 24°C, a decrease in the culturability of the inoculated strain was observed, as well as the appearance of phage-resistant colonies, although 3 days later (at 4 dpi), and their counts increased progressively until stabilization at 104 to 105 CFU/ml. Unlike at 24°C, at 14°C colonies of the phage-sensitive strain were present simultaneously with those of the resistant strain, but at slightly lower levels (Fig. 1F). For 0.8-µm-filtered and untreated water microcosms, microscopic R. solanacearum cell counts decreased progressively to about 105 and 104 cells/ml, respectively, up to 8 dpi (Fig. 1G and H); culturability remained at around 103 to 102 CFU/ml on SMSA agar to the end of the experiment, while it dropped below detection on YPGA plates (Fig. 1G and H). As at 24°C, in both types of microcosms only culturable indigenous bacteria were observed on YPGA (data not shown) after some days, at similar levels (104 to 105 and 103 to 104 CFU/ml, respectively).
Survival of R. solanacearum strain IPO-1609 in river water microcosms.
To discard the possibility that the observed biotic effects concerned only the Spanish strain IVIA-1602.1, the Dutch strain IPO-1609 was also used for survival experiments in the four types of river water microcosms. Trends in the population dynamics of the Dutch strain in the microcosms were very similar to those of the Spanish strain, with no significant differences for up to 1 month, including the appearance of phage-resistant variants (data not shown).
Survival of a phage-resistant variant of strain IVIA-1602.1 in river water microcosms.
To assess whether the influence of phages on R. solanacearum ph II survival in river water microcosms may mask to some extent other biotic effects, the four types of microcosms were inoculated with one randomly selected phage-resistant variant and its survival was monitored at 14°C. In control river water microcosms (Fig. 2A), total, viable, and culturable counts of this variant remained slightly higher than the initial values of 107 cells/ml and 106 CFU/ml, showing a behavior similar to that of the wild type in sterile water (Fig. 1E). In the nonsterile microcosms, different trends were observed (Fig. 2B, C, and D). In 0.2-µm-filtered water (Fig. 2B), microscopic and CFU counts confirmed the resistance of this variant to the phages present in this water. However, in 0.8-µm-filtered water (Fig. 2C) there were decreases in total, viable, and culturable numbers, mainly from 4 to 8 dpi, to about 104 cells/ml and 103 CFU/ml, respectively, and then stabilization around 1 log unit lower for the following 3 weeks. In untreated river water (Fig. 2D), reductions to around 105 to 104 cells and CFU/ml from 2 to 4 dpi were observed; these reductions slowly continued, reaching about 104 cells/ml and 103 CFU/ml by 8 dpi, followed by stabilization until the end of the experiment. Culturable indigenous bacterial counts in 0.8-µm-filtered and untreated river water microcosms stabilized at about 104 CFU/ml in the first days of the experiment.
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DISCUSSION |
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The numbers of indigenous protozoa and culturable bacteria in the river water samples were similar to others reported for freshwater systems (9, 42, 45). Likewise, population levels of culturable R. solanacearum ph II in these waters were in accordance with previous reports at water temperatures above 14°C (9). Estimations of the relative abundance of R. solanacearum lytic phages in the river water were always higher than those for host cells, similar to other studies of environmental waters (49). The fact that lytic phages were isolated from the river water samples from which the bacterium was recovered suggests an association between them. Interestingly, positive phage detection at 29°C always became negative with testing at 35°C using the same water samples. This would explain previous results showing a more efficient recovery of R. solanacearum from samples from the same river by enrichment at 35°C than at 29°C (9), not only because the pathogen is able to grow at 35°C over most of the native water bacteria but also due to phage inactivation at this temperature.
With respect to the survival of R. solanacearum in control microcosms of sterilized river water, total, viable, and culturable populations of the inoculated Spanish strain remained largely constant at inoculation levels at 24°C and 14°C throughout the experiments. The survival of this pathogen under the natural nutrient limitation conditions of aquatic ecosystems was thus confirmed since, according to previous studies (28, 45, 47), both temperatures favor its persistence. In contrast, R. solanacearum counts in the microcosms with water biota declined in total, viable, and culturable cells within the first days, showing that a great proportion of the inoculated population was affected by native microbiota with lytic and/or predatory activity. A negative effect of biotic factors on R. solanacearum was previously reported for Dutch water microcosms, but after studying only the culturability, the authors did not analyze the nature of the agents involved (45).
At 24°C, decreases in microscopic counts observed within the first days were progressively more noticeable as more biotic fractions were present in the nonsterile microcosms, and for culturable counts, declines were comparably more pronounced. Lytic activity by phages was likely to be involved, since all the nonsterile microcosms contained the river water viral fraction. Predation by other water microorganisms also contributed to the decline when present in the nonsterile microcosms. The differences between microscopic and CFU counts, especially in those microcosms with 0.2-µm-filtered water, could be due to the physiological state of the host cells: when they are under nutrient-depleted conditions, as in river water microcosms, phages may be in an intracellular nonreplicative state for an extended period and/or have a reduced replication rate (36, 49). On the contrary, for CFU counts, lysis occurred for the 72 h of incubation, with host cells actively growing on nutritive media. In the microcosms keeping only the viral fraction, the decline in culturability was followed a few days later by a slight increase due to the appearance of small and less fluid colonies with irregular edges. Such colonies were identified as phage-resistant R. solanacearum variants and were also observed in the experiments performed with the Dutch strain. Their existence might be hindering the observation of higher decreases in the populations of the inoculated strain induced by phage activity in 0.2-µm-filtered water, compared to what occurs with 0.8-µm-filtered and untreated water microcosms, from which these variants were not recovered.
The resistant variants would have appeared in the microcosms at 24°C within the first 48 h, but they would have remained undetected because of their low population levels. Afterwards, the growth of the variants would have taken place at the expense of leakage and cellular debris from phage-sensitive lysed cells. This phenomenon, called cryptic growth (40), might be considered a survival strategy that bacteria would employ to cope with adverse conditions (41). The levels of these phage-resistant bacterial populations remained about 2 log units lower than those for the inoculated strain. This could be explained by the metabolic cost often involved in the acquisition of bacterial resistance to phage infection (18), resulting in a population that is not as competitive as the nonresistant population (6).
At 14°C, in nonsterile microcosms there were slighter declines in R. solanacearum populations than at 24°C, which implied that biotic interactions were slower at a lower temperature. Similarly to what happened at 24°C, in 0.2-µm-filtered water, colonies of the phage-resistant variant appeared on plates, but simultaneously with remaining small populations of the inoculated strain. Such phage-sensitive populations were also observed on the selective media from microcosms with 0.8-µm-filtered water and with untreated river water. They were not affected by viral lysis, probably because of their low numbers, which may be below a threshold for phage infection, as suggested in other models (49, 50). This would result in an ecological balance between prey (strain IVIA-1602.1) and predator (bacteriophage) achieved within time in the river water microcosms, as observed in other freshwater systems (49, 50). In the nonsterile river water microcosms, the temperature of 14°C was more favorable for the survival of the pathogen than 24°C.
Trends in the dynamics of R. solanacearum populations in the different river water microcosms were similar for the Dutch strain, suggesting that the biotic effects influencing the pathogen persistence were not related only to one strain.
The influence of nonspecific protozoan grazing and/or bacterial activity on R. solanacearum survival could be observed more precisely in the experiment in which the river water microcosms were inoculated with a selected phage-resistant variant that exhibited a cellular size similar to that of the wild type. In untreated water, the declines in the pathogen populations after 2 to 4 days pointed out the protozoa grazing R. solanacearum cells, released into the microcosms in larger numbers than native bacteria. A similar period for protozoa to reach sufficient density to effect a detectable removal of E. coli in natural water has been reported for a similar temperature (21). Afterwards, a slight decrease in population was observed in untreated water; this decrease was more noticeable in 0.8-µm-filtered water and was due to predatory activity by small, flexible remaining protozoa able to pass through the filters. No increase of the R. solanacearum populations occurred during the following 3 weeks of the experiment, with such populations remaining at densities lower than those of native competitor bacteria that grew faster than R. solanacearum on plates. Bacterial competition for the scarce water nutrients was also observed in the experiments with the phage-sensitive strain, where the phage-resistant variant populations could increase only in the nonsterile microcosms without native bacteria. This is in agreement with other studies reporting the influence of indigenous protozoa and bacteria on other bacterial species introduced in freshwater (4, 19, 20, 22).
The time course of the interaction between R. solanacearum and a selected river water phage in sterile river water confirmed the lysis of bacterial cells at different phage concentrations and at 24°C and 14°C. In spite of the fact that bacterial lysis by phages is usually proved by culturable data, in this work it has been further demonstrated by microscopic counts of total bacterial cells. Thus, at both temperatures, in the absence of native water protozoa and bacteria, lysis occurred as in nonsterile river water microcosms, as evidenced by declines in both total and culturable cell numbers, from millions to several thousands or hundreds of R. solanacearum cells. Differences between total and culturable counts at 24°C might be due to starvation conditions, as already discussed. At 14°C, smaller differences were recorded, probably because of a lessened effect of starvation occurring when the host has lower metabolic activity, and the lysis was delayed in time.
In summary, this study has revealed that R. solanacearum survival was less favored in nonsterile than in sterile river water microcosms and that biotic factors influenced this persistence in freshwater. After an increase in R. solanacearum populations in environmental watercourses (due to occasional spilling or dumping of polluted waste, multiplying, and leaching from infected roots of Solanum dulcamara or other plants), lytic phages would reduce the densities of the bacterium, and protozoa and bacteria may also have an influence on R. solanacearum abundance. All these complex interactions would be affected by water temperature. Evidence presented in this work may broaden our knowledge on the epidemiology of the bacterial wilt pathogen in environmental waters, which might improve the strategies for the management of the disease.
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ACKNOWLEDGMENTS |
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This work has been funded by projects FAIR 5-CT97-3632 and QLK 3-CT-2000-01598 of the European Union and FD 1997-2279 of the Ministerio de Educación y Ciencia of Spain.
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FOOTNOTES |
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Published ahead of print on 14 September 2007.
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), viable (
), and culturable cell counts on YPGA (
) and SMSA (
) plates at 24°C (left) and 14°C (right) in river water microcosms. Results are shown for sterile control water (A, E), 0.2-µm-filtered water (B, F), 0.8-µm-filtered water (C, G), and untreated water (D, H). For 0.2-µm-filtered water microcosms, dashed lines refer to a phage-resistant variant of the inoculated strain of R. solanacearum. The detection thresholds of the techniques were 
102 cells/ml for IF and 
) were both on YPGA. The bacterium (CFU/ml)-to-phage (PFU/ml) ratios were 106 to 2 x 102 (A), 106 to 2 x 103 (B, E), 106 to 7 x 103 (C, F), and 106 to 2 x 104 (D). Dashed lines refer to a phage-resistant variant of the inoculated strain of R. solanacearum. Points are the means for two separate assays performed in duplicate, and error bars indicate variation as the standard deviation for each point.