Bacterial strains.

Isolates and type strains used in this study are listed inTable 1. In the time course experiments, mixtures of fiveMethylobacterium and fiveSphingomonas strains were used to inoculate plants. These strains reflect part of the phylogenetic diversity within the respective genus and were isolated previously from the phyllosphere of different plants. As an exception, the two genome-sequenced type strainsSphingomonas wittichii andSphingopyxis alaskensis were included in theSphingomonas inoculation mixture. Because the classification and nomenclature of the sphingomonads still is under debate (55), here the termSphingomonas is used in sensu lato also for the generaSphingopyxis,Sphingobium, andNovosphingobium. In the screening experiment, additionalSphingomonas strains were included. These were previously isolated from plant surfaces (phyllosphere and rhizosphere), or their 16S rRNA genes show an identity of more than 97% to the majority (75%) of the clone sequences recovered from leaf-colonizing sphingomonads described in Delmotte et al. (9). Type strains were obtained from the DSMZ culture collection in Braunschweig, Germany, and the CIP culture collection of the Institute Pasteur in Paris, France.Pseudomonas syringae pv. tomato DC3000 andXanthomonas campestris pv. campestris LMG 568 were kindly provided by Thomas Kroj (UMR BGPI, Montpellier, France) and Emmanuelle Lauber (LIPM, Castanet-Tolosan, France), respectively.

Growth conditions ofArabidopsis thaliana plants.

A. thaliana Col-0 plants were cultivated on standard MS nutrient medium (36) including vitamins (Duchefa, Haarlem, Netherlands) and supplemented with plant agar (Duchefa, Haarlem, Netherlands) and 3% sucrose. The autoclaved medium was poured into full-gas microboxes (Combiness, Nazareth, Belgium), and a sterile Biofoil 25 (IVSS, Göttingen, Germany) with eight holes (diameter, 4 mm) was placed on the solidified agar surface to avoid the contact of growing plant leaves with the medium.A. thaliana seeds were surface sterilized by treating them with 70% ethanol for 2 min and then sodium hypochlorite solution (7% available chlorine) containing 0.2% Triton X-100 for 8 min. Seeds were washed seven times with sterile double-distilled H₂O and placed on the agar surface at the positions of the eight preformed holes. Plants were grown for 1 week under long-day conditions (16-h photoperiod) followed by short-day conditions (9-h photoperiod) in a standard growth chamber at a constant temperature of 22°C. Upon plant infection with the pathogen, 30 small holes (diameter, 1.2 mm) were pierced into the microbox plastic walls to reduce relative humidity to 80%.

Inoculation and infection of plants.

Methylobacterium strains were grown in liquid mineral salt medium (38) with 0.5% succinate as the carbon source.Sphingomonas strains were cultivated in nutrient broth without additional NaCl (NB). All cultures were incubated at 28°C and grown until early stationary phase. Just before inoculation, cells were washed once and resuspended in 10 mM MgCl₂ solution, the optical density at 600 nm (OD₆₀₀) was adjusted, and bacterial suspensions were added to the plants using seed or leaf inoculation as follows: for seed inoculation, 5 μl of cell suspension (OD₆₀₀ = 0.5) was pipetted onto each seed at the time of sowing; for leaf inoculation, 5 to 10 μl (depending on plant size and leaf number) of cell suspension (OD₆₀₀ = 0.02) was pipetted onto the leaves when plants were 2 weeks old (i.e., 1 week beforeP. syringae pv. tomato DC3000 infection). The cell density ofSphingomonas in the leaf inoculation suspension was adjusted to correspond roughly to the bacterial carrying capacity of seed-inoculatedA. thaliana plants of the same age. Axenic plants were mock treated by applying sterile 10 mM MgCl₂ solution to plant seeds or leaves. The plant pathogenP. syringae pv. tomato DC3000 was cultivated on King's B (26) agar plates at 28°C. Three-week-old plants were infected by spraying ca. 100 μl of infection suspension (or 10 mM MgCl₂ when mock treated) per box over the eight plants. The infection suspension was prepared from freshly grownP. syringae pv. tomato DC3000 cultures as described previously (45) but without Silwet L-77, and the OD₆₀₀ was adjusted to 0.0001, corresponding to approximately 5 × 10⁴ cells per ml. Plants were kept in darkness until infection.

In an additional experiment,X. campestris pv. campestris LMG 568 was used as a pathogen to infectA. thaliana plants.X. campestris pv. campestris LMG 568 cultures were grown on NB agar plates, and the infection suspension was prepared as described above. In this paper, the term inoculation refers to the application of commensal bacteria, i.e.,Methylobacterium andSphingomonas, and the term infection applies to the application of pathogenic bacteria, i.e.,Pseudomonas andXanthomonas.

Enumeration of phyllosphere bacteria.

At different time points postinfection (and the day of infection), commensal and pathogen cell numbers in the phyllosphere were determined on randomly selected plants (out of replicate microboxes). For this purpose, plants were taken out of the microboxes, the roots carefully removed, and the remaining aboveground plant parts placed individually into 2-ml tubes containing 1.3 ml of 100 mM phosphate buffer (pH 7) with 0.2% Silwet L-77 (GE Bayer Silicones, Leverkusen, Germany), a protocol that results in the recovery of bacteria from leaf surfaces and the apoplast, and this is as efficient as grinding the leaf tissue (45). The fresh weight of each plant was recorded. To dislodge bacterial cells from plant material, the tubes were shaken horizontally in a Retsch tissue lyser (Retsch, Haan, Germany) for 15 min at 25 Hz (plants were not destroyed) and sonicated in a water bath for 5 min. Tenfold serial dilutions were spotted onto selective agar plates, and CFU were counted after the incubation of the plates at 28°C. To select forMethylobacterium, mineral salt medium with 0.5% methanol as the carbon source was used; to select forSphingomonas, NB medium supplemented with streptomycin (20 μg/ml) was used. Pathogen CFU were counted on plates with rifampin (50 μg/ml) added to the respective medium. In the few cases early after infection where the number of CFU was lower than the detection limit of the method, values just below the detection limit were included to calculate the mean. In this way,P. syringae pv. tomato DC3000 cell numbers may have been slightly overestimated, but they represent a better estimate than if these values were omitted. The program Prism 5 (GraphPad Software, La Jolla, CA) was used to check for significantly different pathogen cell numbers on axenic and inoculated plants. The values were log transformed, and an unpairedt test was performed for each time point independently.

Plant disease severity index.

Disease phenotypes ofA. thaliana plants were scored according to their visual appearance at 21 days postinfection (dpi). Disease levels were rated from 1 (completely healthy plants) to 5 (dead plants) as described in Whalen et al. (50). When the average disease index was below 2.5, the testedSphingomonas strain was considered fully protective (+), between 2.5 and 3.5 was intermediately protective (±), and above 3.5 was not effective (−) againstP. syringae pv. tomato DC3000. In addition, the automated ground cover tool implemented in the image analysis software Assess 2.0 of the American Phytopathological Society (St. Paul, MN) was used to quantify plant disease.

Carbon utilization profiles.

GN2 MicroPlates (Biolog, Hayward, CA) were used to compare the substrate utilization patterns of representative methylobacteria, sphingomonads, andP. syringae pv. tomato DC3000. Plates were inoculated according the manufacturer's instructions with slight modifications:Methylobacterium cells were precultured at 28°C on Difco R2A agar plates (BD, Sparks, MD) with an additional 0.5% methanol and resuspended in the provided inoculating fluid to result in an OD₆₀₀ of 0.35.Sphingomonas strains andP. syringae pv. tomato DC3000 were grown at 28°C in liquid NB, washed twice, and resuspended in the inoculating fluid (OD₆₀₀ of 0.2). Aliquots of the bacterial suspensions were pipetted into each well, and plates were sealed with parafilm and incubated at 28°C on a rotary shaker moving at 80 rpm. The OD₆₀₀ was measured every 2 to 3 days with a Victor3 plate reader (Perkin Elmer, Waltham), and final values were recorded after 7 days. They were blank corrected and normalized by dividing each value by the highest measured absorption value per plate, resulting in final values between 0 and 1. The utilization of a carbon compound was considered positive at a value of ≥0.4, weakly positive from 0.4 to 0.2, and negative at <0.2. The nutritional similarity of the commensal bacteria and the pathogen was estimated by calculating the niche overlap index (NOI) as described by Ji and Wilson (22). Substrates for which a positive or weakly positive signal was obtained were included in the calculation.

Nucleotide sequence accession numbers.

Partial 16S rRNA gene sequences ofSphingomonas strains isolated in this study have been deposited in the GenBank/EMBL/DDBJ databases under accession numbersFR696367 toFR696370.

Development of a gnotobioticin planta assay.

To study the antagonistic effects of common phyllosphere colonizers against plant-pathogenic bacteria, the tested strains were analyzed under gnotobiotic conditions, i.e., on plants that do not harbor other common colonizers that may mask antagonistic effects. To prevent undesired colonization, the model plantA. thaliana was cultivated in microboxes. A special feature of these microboxes is a filter that allows gas exchange, holds back moisture, and keeps the plants free of airborne microbial contaminants. This allowed the cultivation of axenic plants as well as plants with a defined bacterial community. Several growth regimens, infection methods, and pathogen titers were tested to find a procedure that gives reproducible results under conditions that mimic natural conditions as much as possible. In place of dipping or infiltration, a more moderate infection procedure was applied, which consisted of spraying a relatively small number of cells (ca. 10 to 20 per plant) onto the leaf surface ofA. thaliana plants.

Disease symptom development and pathogen proliferation in absence and presence of commensal bacteria.

The potential plant-protective effects of methylobacteria and sphingomonads were investigated separately. To reflect the situation that plants are naturally colonized by several different strains of these genera, a mixture of fiveMethylobacterium isolates and fiveSphingomonas isolates, respectively, was used for the inoculation ofA. thaliana seeds in time course experiments. The presence of commensal bacteria alone did not affect the growth and development ofA. thaliana plants: compared to axenic plants grown under the same conditions, no difference was evident with respect to visual appearance (Fig. 1 and2, 0 dpi) or plant fresh weight (data not shown). When plants were 3 weeks old, they were infected withP. syringae pv. tomato DC3000. Appearance and bacterial cell numbers were monitored over time. Phenotypic differences between axenic plants and plants seed inoculated withMethylobacterium spp. were not observed after infection withP. syringae pv. tomato DC3000: severe disease symptoms were visible after 22 days for both treatments (Fig. 1A). With the exception of results at 1 dpi, pathogen population size was not significantly different between axenic andMethylobacterium preinoculated plants (P < 0.05) (Fig. 1B). On the contrary,Sphingomonas spp. revealed a conspicuous plant-protective effect when seed-inoculated plants were challenged withP. syringae pv. tomato DC3000: plants appeared to be very similar to the noninfected mock-treated control plants (Fig. 2A). Consistently with the healthy plant phenotype, the multiplication ofP. syringae pv. tomato DC3000 onSphingomonas preinoculated plants was significantly reduced compared to that of axenic plants (P < 0.001). The differences in CFU ranged from 340-fold at 1 dpi to 10-fold at 26 dpi (Fig. 2B). The average population sizes of methylobacteria and sphingomonads were in the range of 10⁸ to 10⁹ CFU/g leaf fresh weight. Also after the application ofP. syringae pv. tomato DC3000, cell numbers remained stable throughout the monitoring period (see Fig. S1 in the supplemental material).

In a second step, plants were analyzed that were leaf inoculated withSphingomonas spp. 1 week beforeP. syringae pv. tomato DC3000 infection. When we applied this preinoculation scheme, again a pronounced plant-protective effect ofSphingomonas spp. was observed in terms of disease suppression and reduced pathogen population size (Fig. 3). We also tested whetherSphingomonas spp. had the capability to suppress disease caused by another bacterial pathogen known to occur in the phyllosphere:Xanthomonas campestris. Preinoculation withSphingomonas spp. reduced efficientlyX. campestris pv. campestris LMG 568 population size (50-fold at 32 dpi;P < 0.001) and kept plants free of disease symptoms (Fig. 4).

Screening of individualSphingomonas isolates.

To investigate whether the observed plant-protective effect is a wide-spread feature within the genusSphingomonas in sensu lato (55), isolates from various environments, including phyllosphere, rhizosphere, air, dust, and water, were tested individually onA. thaliana plants (Table 1; for phylogenetic placement based on 16S rRNA gene sequence analysis, see Fig. S2 in the supplemental material). Moreover, the fiveSphingomonas isolates that were used in the mixture of the time course experiment were included. The cell numbers of bothSphingomonas andP. syringae pv. tomato DC3000 were determined at 21 dpi (Fig. 5). For theSphingomonas spp., population sizes ranged between 10⁷ and 10¹⁰ CFU/g leaf fresh weight;S. alaskensis, an isolate from the Atlantic Ocean, reached a population density as low as 10⁴ CFU/g leaf fresh weight.P. syringae pv. tomato DC3000 cell numbers were in the range of 10⁵ to 10¹⁰ CFU/g leaf fresh weight depending on theSphingomonas strain used for inoculation. The disease severity index as described by Whalen et al. (50) was determined to score the 18 testedSphingomonas isolates according to their plant-protective effect (Fig. 5). The isolates were grouped into fully protective, intermediately protective, and nonprotective phenotypes. To confirm the results of the disease severity analysis, a software-based tool for disease quantification was used. A high positive correlation was found between the data derived from the two different methods (R = 0.95,P < 0.001). To evaluate correlations between the three parameters commensal population size, pathogen cell number, and plant disease index, a regression analysis was performed. TheSphingomonas cell numbers correlated with neither theP. syringae pv. tomato DC3000 population sizes (R = 0.16,P = 0.52) nor with the plant disease index (R = 0.38,P = 0.12). However, a correlation between the pathogen population size and the disease index was evident (R = 0.87,P < 0.001).

Concerning the origin ofSphingomonas strains, it was evident that all plant-derived isolates with one exception (S. asaccharolytica) showed full or intermediate plant protection. In particular, all tested phyllosphere isolates were effective againstP. syringae pv. tomato DC3000, although the degree of protection varied. On the other hand, four out of five air- and waterborne isolates were not beneficial forA. thaliana plants.

Carbon utilization profiles ofP. syringae pv. tomato DC3000 and representativeSphingomonas andMethylobacterium strains.

Carbon utilization is a crucial factor among subpopulations in the phyllosphere (51) and might explain differences betweenSphingomonas andMethylobacterium strains with respect to pathogen suppression. To verify whether this aspect is important for the observed differences in plant protection, the carbon utilization of representative strains was analyzed in Biolog assays. The results for the selected strains, which included twoSphingomonas strains with a plant-protective effect in the screening experiment, two nonprotectiveSphingomonas strains, and twoMethylobacterium strains, are displayed inFig. 6. Overall, the carbon substrate utilization profile ofP. syringae pv. tomato DC3000 was more similar to that of the protectiveSphingomonas strains than to the nonprotective commensals. The most striking difference was seen in carbohydrate metabolism: not a single sugar (out of 28) was used by one of the nonprotective strains. Accordingly, the niche overlap indices (NOI; where 0 indicates a completely different and 1 an identical substrate utilization pattern), calculated for the commensal bacteria relative to the pathogen and based on all 95 tested substrates, were higher for the protective antagonists (Sphingomonas sp. Fr1, 0.43;S. phyllosphaerae, 0.41) as for the nonprotective strains (S. aerolata, 0.26;S. wittichii, 0.30;M. extorquens PA1, 0.17;Methylobacterium sp. 32, 0.20).

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