The Role of Pigmentation, Ultraviolet Radiation Tolerance, and Leaf Colonization Strategies in the Epiphytic Survival of Phyllosphere Bacteria

J.L. Jacobs1,2, T.L. Carroll1 and G.W. Sundin1,2,3

(1) Department of Plant Pathology and Microbiology, Texas A & M University, College Station, TX 77843, USA (2) Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA (3) Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, USA Received: 4 September 2003 / Accepted: 19 November 2003 / Online publication: 23 September 2004

Abstract

Introduction

Phenotypic mechanisms that enhance bacterial UVR survival typically include pigmentation and DNA repair mechanisms which provide protection from UVA and UVB wavelengths, respectively. In this study, we examined the contribution of pigmentation to eld survival in Clavibacter michiganensis and evaluated differences in population dynamics and leaf colonization strategies. Two C. michiganensis pigment-decient mutants were signicantly reduced in UVA radiation survival in vitro; one of these mutants also exhibited reduced eld populations on peanut when compared to the wild-type strain over the course of replicate 25-day experiments. The UVR-tolerant C. michiganensis strains G7.1 and G11.1 maintained larger epiphytic eld populations on peanut compared to the UVR-sensitive C. michiganensis T5.1. Epiphytic eld populations of C. michiganensis utilized the strategy of solar UVR avoidance during leaf colonization resulting in increased strain survival on leaves after UVC irradiation. These results further demonstrate the importance of UVR tolerance in the ability of bacterial strains to maintain population size in the phyllosphere. However, an examination of several bacterial species from the peanut phyllosphere and a collection of environmental Pseudomonas spp. revealed that sensitivity to UVA and UVC radiation was correlated in some but not all of these bacteria. These results underscore a need to further understand the biological effects of different solar wavelength groups on microbial ecology.

Correspondence to: G.W. Sundin; E-mail: sundin@msu.edu

The phyllosphere (plant leaf surface) presents a harsh environment for the growth and survival of microorganisms; nutrients are limited, and stress conditions, including uctuating water availability, osmotic stress, and exposure to solar UV radiation (UVR), are prevalent. Solar radiation is categorized by photobiologists into three wavelength classes (UVA, 320 to 400 nm; UVB, 290 to 320 nm; UVC, <290 nm) based on biological effects of photons of each wavelength class on organisms. Photons of shorter wavelength UVR are more energetic and potentially more damaging to all organisms dwelling in UVR-exposed habitats. However, UVC wavelengths do not penetrate to the earths surface, as these wavelengths are completely screened by ozone and oxygen. UVA wavelengths contribute more total energy (95%) than UVB (5%) in the suns UVR spectrum. Lethality due to UVA exposure is attributed to the broad-spectrum effects of the intracellular generation of reactive oxygen species [22], including hydrogen peroxide, superoxide anion, and singlet oxygen, and not due to direct DNA damage. For instance, the superoxide anion will release free iron from enzymes containing 4Fe-4S centers and oxidize SH proteins and lipids [15, 22]. Relatively little is known concerning the relevance of UVA radiation to the ecological tness of environmental bacteria, as most studies of UVR effects have focused on UVB/UVC [21]. The UVB component of solar UVR causes direct DNA damage by inciting the formation of lesions such as cyclobutane pyrimidine dimers and pyrimidine(64)pyrimidinone photoproducts in cellular DNA [23]. These lesions result in the blockage of DNA replication and RNA transcription; such blockages can be lethal in the absence of efcient cellular mechanisms for their removal. Although completely screened out of the earths
Volume 49, 104113 (2005) Springer Science+Business Media, Inc. 2004

104

DOI: 10.1007/s00248-003-1061-4

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

105

atmosphere by oxygen and ozone, high-energy UVC wavelengths have been substituted for UVB in many laboratory assessments of UVR survival involving DNA repair. For example, we have found that UVC-sensitivity assays are more effective at discriminating survival differences among closely related strains [30]; also, UVC irradiation provides sensitivity data for bacteria and fungi that are highly tolerant or insensitive to elevated doses of UVB [1, 25, 29]. Phyllosphere bacterial communities are diverse and are usually composed of organisms displaying a range of sensitivity to DNA-damaging UVR wavelengths and a high percentage of pigmented organisms [2, 9, 12, 29, 33]. Mechanisms to combat UVA-induced oxidative stress in bacteria are complex and interactive and include the production of carotenoid pigmentation and/or the expression of enzymes such as catalase and superoxide dismutase (SOD). Bacterial pigmentation also affords protection from UVA wavelengths or from visible wavelengths that can activate photosensitizing chemicals that are inhibitory to cells. Carotenoid pigments produced by Erwinia herbicola (Pantoea agglomerans) and Clavibacter michiganensis play an important role in UVA survival [3, 29]. The yellow pigment xanthomonadin produced by Xanthomonas campestris increased the survival of cells in the presence of visible light and the photosensitizing agent toluidine blue, and also protected lipids from peroxidation [24]. Similar to the phyllosphere, carotenoid pigmentation is prevalent in organisms inhabiting other solar UVR-exposed habitats such as freshwater phytoplankton assemblages [18], and in atmospheric bacterial isolates [34]. However, the contribution of pigmentation to environmental survival, especially in the phyllosphere, has not been adequately addressed. We are interested in the role of solar UVR in modulating phyllosphere bacterial communities and in the specic UVR survival strategies that contibute to increased tness of bacterial phyllosphere inhabitants. Our previous studies have focused on C. michiganensis as a model peanut epiphyte; most isolates of C. michiganensis recovered from peanut are pigmented and highly tolerant to all UVR wavelength classes, and this organism makes up the majority of the culturable bacterial community from peanut late in the growing season in Texas [12, 29]. In this study, we examined the contribution of pigmentation to eld survival and examined differences in the population dynamics of C. michiganensis strains using two UVR-tolerant and one UVR-sensitive strain. We also evaluated the ecological strategy of UVR avoidance in eld populations of C. michiganensis. Finally, we report the results of comparative examinations of the in vitro survival of phyllosphere bacteria and Pseudomonas spp. following irradiation with UVA or DNA-damaging UVC wavelengths.

Methods
Bacterial Strains and Growth Conditions. The bacterial strains used in this study are listed in Table 1. The peanut epiphytes C. michiganensis G7.1, G11.1, and T5.1 were chosen for comparative study because these strains differed in sensitivity to UVC radiation (G7.1, highly UVC tolerant; G11.1, medium UVC tolerant; T5.1, UVC sensitive) and produced different colored pigments in culture (Table 1). All bacterial strains were grown in LuriaBertani (LB) medium (Difco) or Kings medium B (KB) [16] at 28C. Pigment-decient mutants of C. michiganensis G7.1 and T5.1 were generated using ethyl methane sulfonate (EMS) as previously described [29]. The antibiotic rifampicin (75 lg mL)1) was added to media where necessary. For enumeration of inoculated strains in eld experiments, KB medium was amended with rifampicin (75 lg mL)1) for bacterial selection and nystatin (300 U mL)1) for fungal growth inhibition (KBrn). In Vitro UVA and UVC Sensitivity Analyses. UVA sensitivity was determined in vitro using an XX-15 lamp (UVP Products; San Gabriel, CA). Preparation of bacterial suspensions prior to irradiation was done as previously described [12, 30]. The cell suspensions were placed on a rocking shaker (ve revolutions per minute) under the UVA lamp source or agitated by hand under the UVC lamp source to ensure mixing to minimize cell survival due to shading. We examined cell survival (compared to a nonirradiated strain) following ve to seven incremental doses per strain; higher doses were achieved by increasing the time of exposure. The energy output of the UVA (4548 J m)2 s)1) and UVC (1.5 J m)2 s)1) lamps was monitored with a UVX radiometer (UVP Products) tted with the appropriate wavelength detector. Field ExperimentsBacterial Population Dynam-

Field experiments were conducted at a site adjacent to the Texas A & M campus in College Station, Texas, using replicated microplots of peanut (Arachis hypogeae cv. Florunner). In one set of experiments, we compared the population dynamics of C. michiganensis G7.1 and G7.1pig), and T5.1 and T5.1pig) inoculated separately onto plants. In a second set of experiments, we compared the dynamics of C. michiganensis G7.1, G11.1, and T5.1 inoculated separately or as a 1:1:1 mixture. The circular microplots (diameter = 0.7 m) contained ve peanut plants which were hand-sown at a depth of 2 to 3 cm. Each eld experiment consisted of three replicate plots per treatment arranged in a randomized block pattern, and the experiments were conducted twice. Temperature, relative humidity, and precipitation were monitored at a site 500 m from the eld plot. Data
ics.

106

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

Table 1. Bacterial strains used in the study and their relevant characteristics

Strain Brevibacterium sp. S9 Burkholderia cepacia DB01 Clavibacter michiganensis G7.1 G7.1pig) G11.1 T5.1 T5.1pig) Curtobacterium sp. G28 Pantoea sp. T18 Pseudomonas cichorii 302959 Pseudomonas uorescens Pf5 Pseudomonas syringae pv. syringae B728a B86-17 Pseudomonas syringae pv. tomato DC3000
a

Relevant characteristics Produces an orange pigment on KB Soil isolate RifR, produces an orange pigment on KB White, pigment-decient mutant of G7.1 RifR, produces a pink pigment on KB RifR, produces a cream-colored pigment on KB White, pigment-decient mutant of T5.1 Produces an orange pigment on KB Produces a yellow pigment on KB Plant isolate Soil isolate Bean pathogen Bean pathogen, encodes rulAB operon Tomato pathogen

UV MIDa c 50 NT 250 250 100 50 50 200 100 NT NT NT NT NT

Source or reference [29] C.F. Gonzalez [12] This study [12] [12] This study [29] [29] G.W. Sundin C.L. Bender G.A. Beattie [32] J. Murillo

The UV-MIDc is dened as the minimal dose of UVC radiation (J m)2) required to inhibit growth [29]. NT: not tested.

readings were taken every 15 s and logged using a CR-10 datalogger (Campbell Scientic, Logan, UT). Solar UVB irradiance during the hours 1100 to 1500 was monitored twice per week throughout the experiments using a UVB detector (SED240/UVB-1/W) attached to an IL-1700 research radiometer (International Light, Newburyport, MA). The detector was placed at 0.3 m above canopy height. UVB irradiance was measured every second, and the readings were integrated over the 4-h period yielding a quantitative output in J m)2. Cells of C. michiganensis strains were grown on KBrn for 48 h prior to use as inocula for plant experiments. The cells were resuspended in 0.1 M potassium phosphate buffer (pH 7.0), and suspensions were adjusted turbidimetrically to 1 108 cfu mL)1. Bacterial inoculum was applied to the peanut leaves (at approximately 0800) with a hand-held sprayer until the leaf surfaces were uniformly wet. Samples consisted of ve individual leaves taken from each replicate plot for a total of 15 leaves per strain per sampling date. Leaves of the same size and age were chosen randomly from the top of the plant canopy. Plant leaf samples were taken at 1200. Each leaf was placed in a sterile plastic bag and transported to the laboratory on ice for immediate processing. Leaves were weighed and placed in 10 mL prechilled buffer (0.1 M potassium phosphate, pH 7.0, 0.1% peptone), following which bacterial cells were removed by a 7-min sonication treatment in an ultrasonic bath (Model 250T, VWR Scientic; Houston, TX). Samples (0.1 mL) from

appropriate dilutions of the sonicate were plated on KBrn medium. Bacterial colonies were counted following 72 to 96 h incubation at 25C. In experiments examining the dynamics of mixed C. michiganensis populations, strains were simultaneously enumerated on the same plates as colonies of C. michiganensis G7.1 (orange), G11.1 (pink), and T5.1 (creamy light yellow) could be easily differentiated by color.
Enhanced UVR Survival of C. michiganensis During In these experiments, we evaluated Leaf Colonization.

the survival of C. michiganensis G7.1, G11.1, and T5.1 on UVC-irradiated peanut leaves by comparing populations on UVC-irradiated leaves with populations recovered from nonirradiated leaves. Inoculum was prepared as described above. Strains (G7.1, G11.1, T5.1) were either inoculated individually or as a 1:1:1 mixture. Immediately and at 1, 2, 3, 4, 6, 8, and 10 days after inoculation, four leaves per treatment were randomly excised, placed in sterile plastic trays, and brought to the laboratory on ice. The leaves were then irradiated on the abaxial and adaxial leaf surfaces with UVC radiation by using the XX15 lamp as described above. A UVC dose of 225 J m)2 was used for strains G7.1 and G11.1, and a dose of 100 J m)2 was used for strain T5.1; the UVC doses were chosen because they resulted in 10% cell survival in vitro. An additional four leaves were excised per treatment, and these leaves were not irradiated as a control. After irradiation, leaves were processed by sonication (to effec-

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

107

tively quantify cells located in external leaf sites) and bacterial populations were enumerated by dilution plating as described above. Percent survival values were determined by comparing counts of cells recovered from UVC-irradiated and nonirradiated leaves. A ratio was then derived by dividing the in planta percent survival values by the corresponding percent survival value determined in vitro. Independent experiments were conducted for each strain and for the mixture in both 2000 and 2001.

Results
Selection and Characterization of Pigment-Decient MuThe role of tants of C. michiganensis G7.1 and T5.1.

pigmentation in UVA radiation sensitivity and epiphytic survival on peanut leaves in the eld was studied using C. michiganensis strains G7.1 and T5.1. A chemical mutagenesis procedure involving EMS addition to stationaryphase cultures of C. michiganensis G7.1 and T5.1 [29] was utilized to generate pigment-decient mutants of these strains. White, nonpigmented colonies were selected on KBr plates, and their similarity to the parental strain was determined by comparison of proles generated using fatty acid methyl ester analysis (data not shown). Strain G7.1 produces an orange pigment in culture, and nonpigmented colonies were easily distinguishable on KBr. Nonpigmented colonies of strain T5.1 (originally creamy light yellow) were also selected. The in vitro sensitivity to UVA and UVC radiation was compared for both parental and pigment-decient mutants of G7.1 and T5.1. While UVC radiation sensitivity for the pigment-decient mutants was unchanged compared to the appropriate parental strain (data not shown), both mutants exhibited marked reductions in UVA survival following doses of 100 kJ m)2 or higher (Fig. 1). These experiments also revealed that strain T5.1 was reduced in UVA survival when compared to G7.1 (Fig. 1). We examined the population dynamics of C. michiganensis G7.1, G7.1pig), T5.1, and T5.1pig) in replicated eld plots of peanut. Prior to the initiation of eld studies, the results of growth chamber experiments (no UVR pressure) indicated that all strains consistently maintained equivalent populations on peanut leaves over 7- to 10-day periods (data not shown). Each strain was inoculated and evaluated individually, and the experiments were conducted simultaneously. Two separate experiments were conducted with each strain and corresponding pigmentdecient mutant. Maximum daily temperatures averaged 36C during the course of these experiments with no measurable rainfall occurring during any experiment. Four-hour (1100 to 1500) solar UVB irradiance averaged 12.4 kJ m)2 during these experiments. Although we did not monitor solar UVA irradiance during these experi-

T5.1 (r), and T5.1pig) ()) after UVA irradiation. Each datum point represents the mean ( the standard error of the mean) from three replicate experiments.

Figure 1. Survival of C. michiganensis G7.1 (h), G7.1pig) (m),

ments, the results of several other studies indicate that dose levels of solar UVA radiation are typically 2285 times greater than solar UVB levels [4, 7, 20]. If the solar UVA irradiance followed this range under the conditions of our studies, we would expect the average 4-h UVA doses to range from 272 to 1054 kJ m)2, levels that would be expected to signicantly affect the survival of the C. michiganensis strains that we examined. Following inoculation, populations of G7.1pig) exhibited small but consistent reductions of up to sevenfold when compared to G7.1 over the course of the 25day experiment (Fig. 2A). Differences were statistically signicant (P < 0.05) on ve occasions (indicated by asterisks in Fig. 2A). In contrast, populations of T5.1pig) and T5.1 on peanut leaves were nearly identical throughout the course of the experiment (Fig. 2B). Similar results were observed when the experiment was repeated (data not shown).
In Vitro UVC Survival and Population Dynamics on Field-Grown Peanut of C. michiganensis G7.1, G11.1, and The objective of these experiments was to T5.1.

108

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

Figure 2. Population dynamics of (A) C. michiganensis G7.1 (d), G7.1pig) (s), and (B) T5.1 (m), and T5.1pig) (n) recovered from the phyllosphere of eld-grown peanut from replicated eld plots. Each datum point represents the mean ( the standard error of the mean) from samples consisting of 15 individual leaves. Signicant differences in populations (P < 0.05) are indicated by asterisks (*).

compare the population dynamics of three strains from within a bacterial species that differed in UVR sensitivity. Although we would have preferred to use UVB radiation for the strain sensitivity assays, C. michiganensis strains G7.1 and G11.1 were insensitive to UVB doses as high as 12 kJ m)2 (data not shown); therefore, we used UVC radiation in these experiments. In vitro analyses of UVC sensitivity indicated that strain G7.1 was signicantly more tolerant than T5.1 with survival differences of >1000-fold following irradiation with a 250 J m)2 dose (Fig. 3). Strain G11.1 was only slightly more sensitive to UVC radiation than G7.1 (Fig. 3). Separate eld experiments were conducted to examine the population dynamics of C. michiganensis strains G7.1, G11.1, and T5.1 on peanut; strains were inoculated either individually or as a 1:1:1 mixture of all three strains. Comparisons of strains inoculated individually indicated that strain G7.1 consistently colonized peanut leaves at levels that were 10- to 100-fold higher than T5.1 (Fig. 4A). These results were observed in each replicated experiment performed (data not shown). Populations of strain G11.1 were similar to that of G7.1 throughout the 10-day experiments (Fig. 4A). The experiments involving mixed inoculum again showed that G7.1 and G11.1 maintained similar population levels on peanut while populations of T5.1 were reduced up to 25-fold compared to G7.1 in the three strain mixture (Fig. 4B).
Enhanced UVR Survival of C. michiganensis During The effect of Leaf Colonization in Field Experiments.

Figure 3. Survival of C. michiganensis G7.1 (h), G11.1 (s), and T5.1 (m) after UVC irradiation. Each datum point represents the mean ( the standard error of the mean) from three replicate experiments.

leaf colonization strategy on increasing strain survival on

leaves following UVC irradiation was evaluated in eld experiments. Populations of G7.1, G11.1, and T5.1 were established separately or as a 1:1:1 mixture on peanut leaves in replicated eld plots. We then enumerated populations from replicate sets of irradiated and nonirradiated leaves taken at eight time points over a 10-day period following inoculation. Mean populations were determined from four individual leaves, with an additional set of four leaves irradiated with UVC. An in planta UVC survival percentage was generated by dividing the mean values from the irradiated/nonirradiated populations, yielding a value which was then divided by the in vitro percent survival of the strain at the corresponding UVC dose to yield an in planta/in vitro survival (IPIVS) ratio. Ratios of >1 indicated that UVC survival was elevated for the in planta populations. The IPIVS ratio of strain G7.1 inoculated individually following a 225 J m)2 UVC dose was between two and three for the rst 3 days of the experiment, and then increased to greater than 10 on days 4, 6, and 8 (Fig. 5A).

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

109

A decrease to seven was observed on day 10; however, this value indicated that the in planta survival remained sevenfold elevated compared to the in vitro survival value (Fig. 5A). The IPIVS ratios for G11.1 remained less than 4 until day 10 (Fig. 5B) and uctuated considerably for T5.1 from a high of about 10 on day 2 to a value of 1 on day 4 (Fig. 5C). Although uctuations in the IPIVS ratio were observed in these experiments, we did not observe large uctuations in population numbers from nonirradiated leaves (data not shown). Experiments performed with the strains inoculated as a mixture revealed that the IPIVS ratios for G7.1 and G11.1 were similar to those observed when the strains were inoculated individually (Fig. 5A, B). However, the IPIVS ratio for T5.1 decreased considerably from days 6 to 10 in mixed inoculations compared to T5.1 inoculated alone (Fig. 5C).
Comparative UVA and UVC Sensitivity of PhylloLittle is sphere Isolates and Pseudomonas Strains.

Figure 4. Population dynamics of C. michiganensis G7.1 (h), G11.1 (s), and T5.1 (m) recovered from the phyllosphere of peanut in replicated eld plots. (A) Strains were inoculated and enumerated individually onto plants. (B) A mixture of the three strains was inoculated and populations of individual strains were enumerated based on pigmentation color. Each datum point represents the mean ( the standard error of the mean) from samples consisting of 15 individual leaves.

known about the interrelationship of UVB/UVC and UVA survival strategies within bacteria. We initiated studies to address this by rst determining the sensitivity of a group of peanut phyllosphere strains and Pseudomonas strains following UVC and UVA irradiation. We chose the peanut phyllosphere isolates Brevibacterium sp. S9, C. michiganensis G7.1, G11.1, and T5.1, Curtobacterium sp. G28, and Pantoea sp. T18; each of these isolates was previously characterized as UVC tolerant except for C. michiganensis T5.1 and Pantoea sp. T18, which were UVC sensitive [12]. We also included plant pathogen strains P. cichorii 302959, P. syringae pvs. syringae B728a and B86-17 and P. syringae pv. tomato DC3000, and soil isolates P. uorescens Pf5 and Burkholderia cepacia DB01. UVC and UVA irradiation experiments were done as described in the Methods section and were repeated at least three times for each strain. Each of the UVC-tolerant peanut phyllosphere strains produced a pink or orange pigment when cultured on KB medium, and C. michiganensis T5.1 and Pantoea sp. T18 produced a yellow pigment on KB. UVA irradiation experiments revealed that Brevibacterium sp. S9 and Pantoea sp. T18 were relatively insensitive exhibiting killing of <10% up to UVA doses of 350 kJ m)2 (Fig. 6). The UVA sensitivities of C. michiganensis G7.1 and G11.1 were similar, and C. michiganensis T5.1 and Curtobacterium sp. G28 were the most sensitive of the peanut isolates (Fig. 6). Evaluation of the Pseudomonas strains involved UVC and UVA sensitivity determinations which revealed that survival of P. syringae B728a, B86-17, and DC3000 was comparatively similar following UVC or UVA irradiation (Fig. 7A, B), and that P. syringae DC3000 was more sensitive to both UVC and UVA wavelengths than the other P. syringae strains. P. cichorii 302959 was relatively

110

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

Figure 6. Survival of Brevibacterium sp. S9 (n), Pantoea sp. T18 (h), C. michiganensis G7.1 (d), G11.1 (s), T5.1 (m), and Curtobacterium sp. G28 (n) after irradiation with UVA. Each datum point represents the mean from three replicate experiments.

pared to the P. syringae strains following UVA irradiation (Fig. 7A, B). P. uorescens Pf5 was the most sensitive organism examined (Fig. 7A, B).

Figure 5. In planta/in vitro survival ratio of C. michiganensis G7.1

(A), G11.1 (B), and T5.1 (C) on peanut leaves irradiated with UVC. Open symbols indicate strains inoculated alone; lled symbols indicate strains (G7.1, G11.1, and T5.1) inoculated in a 1:1:1 ratio. The in planta/in vitro survival ratio is determined by dividing the percent survival of strains from irradiated peanut leaves by the percent survival of strains irradiated in saline solution. The dashed lines denote a ratio of 1; a value >1 indicates enhanced survival in planta. UVC doses used were 225 J m)2 for G7.1 and G11.1 and 100 J m)2 for T5.1.
Figure 7. Survival of B. cepacia DB01 (m), P. cichorii 302959 (h), P. uorescens Pf5 (s), P. syringae pv. syringae B728a (r) and B8617 (n), P. syringae pv. tomato DC3000 (n) after irradiation with (A) UVC or (B) UVA. Each datum point represents the mean from three replicate experiments.

tolerant to UVC radiation and was more sensitive to UVA radiation; in contrast, B. cepacia DB01 was sensitive to UVC radiation, but showed increased survival com-

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

111

Discussion

The modulation of phyllosphere microbial populations by solar UVB radiation has been demonstrated in several recent studies [10, 12, 13, 28, 29], but the role of UVA radiation, bacterial pigmentation, and the effect of differential strain sensitivity to DNA-damaging UVR wavelengths are less understood. In this study, the eld survival on peanut leaves of C. michiganensis G7.1pig) (EMS-selected mutant that does not produce its orange pigment in culture) was reduced compared to the wildtype strain on all 11 sampling dates and signicantly reduced on ve of 11 sampling dates over a 25-day period. The size reductions were never greater than 10fold, but still implicate the importance of pigmentation as a UVA-survival mechanism in the environment and suggest that ambient solar UVA radiation can reduce phyllosphere bacterial populations. No difference was observed between populations of C. michiganensis T5.1 and T5.1pig) in eld experiments; however, the creamy yellow-colored pigment produced by T5.1 is not the color typical of most carotenoids [6]. Thus, although strain T501pig) was greatly reduced in UVA survival in vitro, it is possible that other UVAsurvival mechanisms compensated for the loss of pigmentation in this strain, enabling survival under the ambient UVA conditions existing in our experiments. An alternative explanation is that the UVR sensitivity of strain T5.1 is such that any additional mutations lowering in vitro UVR sensitivity do not affect (i.e., further reduce) epiphytic survival in nature. Since we used EMS mutagenesis to generate the G7.1pig) and T5.1pig) strains, it is possible that additonal mutations were also present in the mutants that also affected their epiphytic survival. However, because of the lack of tools available in Clavibacter genetics, we felt that the EMS method afforded the best opportunity to examine the effect of pigmentation on epiphytic survival using eld studies. The results of our studies, accompanied by recent increases in the genetic understanding of Clavibacter/Corynebacterium species [17], provides the justication for future examinations of additional phenotypes that affect UVA sensitivity and efforts to construct and examine the survival of dened genetic mutants in the eld. Assessment of the role of UVA radiation in phyllosphere ecology is difcult because of the lack of an effective screening lter that would eliminate UVA wavelengths above plant canopies while permitting UVB and visible wavelengths to pass. Recent experiments utilizing short-term sunlight exposures of the fungus Metarhizium anisopliae and the bacterium P. syringae pv. syringae comparing survival following nonscreened, UVB-screened, or UVB + UVAscreened exposures have highlighted the importance of both UVA and UVB

wavelengths in mediating damage to cellular components and cell death [5, 19]. Pigmentation has long been recognized as a potential mechanism for increased microbial survival in solar radiationimpacted ecosystems. In eukaryotic microbes, pigments such as melanin are effective sunscreens for solar UVB radiation and signicantly enhance survival [11, 36]. Bacterial cells are too small to effectively utilize UVR self-shading mechanisms [8], and this observation is borne out with the importance of active mechanisms such as DNA repair and photoreactivation in UVB radiation survival [14, 27, 28, 32]. The pigments produced by many phyllosphere bacteria resemble carotenoids, pigments whose function is to quench active oxygen species that are generated by exposure to UVA radiation [35]. Indeed, laboratory-derived nonpigmented mutants of C. michiganensis were more sensitive to UVA wavelengths, but their sensitivity to UVC wavelengths was unchanged [29]. Thus, although pigmented phyllosphere isolates are also typically more tolerant of UVC radiation than nonpigmented isolates [12], the pigmentation is most likely providing UVA protection only. In this study, we found that the UVC-tolerant C. michiganensis G7.1 and medium UVC-tolerant G11.1 maintained epiphytic populations on peanut that were 10- to 100-fold larger than the more sensitive strain T5.1 when these strains were either inoculated individually or inoculated as a mixture. The difference in population size may also be related to other aspects of epiphytic tness (e.g., desiccation tolerance, altered competitive ability) as strain T5.1 consistently exhibited reduced population size on peanut leaves. However, the increased tolerance of strains G7.1 and G11.1 to UVC and UVA radiation, relative to strain T5.1, could also indicate that UVR tolerance is an important phenotype for the maintenance of population size in the phyllosphere. Avoidance of solar UVR may be an important ecological strategy of a range of bacterial phyllosphere residents. We have previously demonstrated in growth chamber experiments that phyllosphere populations of C. michiganensis G7.1 and T5.1 on peanut survived UVC irradiation at increased levels compared to cells irradiated in vitro [10]. In this study, we observed that eld populations of C. michiganensis G7.1, G11.1, and T5.1 exhibited increased UVC survival when inoculated separately on peanut leaves. The reduction in IPIVS ratio of strain T5.1 on later sampling dates when inoculated as part of a mixture with G7.1 and G11.1 suggests that interstrain competitive interactions may also play a role in a strains ability to colonize leaves and/or access UVRshaded sites on leaves. It is unlikely that these nonpathogenic organisms are colonizing internal protected sites as pathogenic P. syringae strains inhabit on their cognate host plant [26, 37]. However, external physically

112

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

shaded leaf sites, such as those associated with leaf veination, or shading of cells within aggregates represent possibilities that might result in the survival increases observed in our studies. Our initial attempt to determine if correlations existed in bacterial survival following irradiation with DNA-damaging UVB/UVC or with UVA wavelengths showed that a relationship was not clear. For example, P. agglomerans exhibited the highest UVA survival of the peanut epiphytes tested, but also exhibited the lowest UVC survival. C. accumfaciens, an organism whose UVC survival level is similar to that of C. michiganensis G7.1, was increased in UVA sensitivity when compared to G7.1 at higher dose levels. UVA and UVC sensitivities were relatively similar for the P. uorescens and P. syringae strains; however, B. cepacia DB01 and P. cichorii 302959 differed in comparative sensitivity between the UVA and UVC wavelength groups. These results illustrate that a larger knowledge base on the effects of UVA and UVB/UVC wavelengths on microbial ecology is required before a generalized determination of UVA and UVB/UVC sensitivity of individual bacterial strains will provide revealing biological information. Bacterial survival in solar UVR-impacted habitats is mainly affected by UVR irradiance (UVA + UVB) and duration of exposure. UVR exposure affects ecological tness through necessitating a shift in energy resources from cellular growth to DNA repair or other processes to ameliorate UVR-mediated damage. Intuitively, higherenergy UVB wavelengths would seem to be of utmost importance because of their ability to damage DNA. Chromosomal DNA damage blocks replication and transcription and must be repaired to ensure cell survival. UVA wavelengths may have additional ecological effects; higher-energy, i.e., lower, UVA wavelengths are capable of killing cells, whereas UVA wavelengths >375 nm can be used as ecological cues for fungal sporulation [17]. An understanding of how bacterial cells simultaneously perceive and respond to the solar UVA and UVB radiation present within their environment, the coordination, if any, of cellular resources into DNA repair and UVAsurvival processes, and subsequent effects on tness represents an important unsolved problem in microbial ecology.
Acknowledgments

References
1. Ashtana, A, Tuveson, RW (1992) Effects of UV and phototoxins on selected fungal pathogens of citrus. Int J Plant Sci 153: 442452 2. Beattie, G, Lindow, SE (1995) The secret life of foliar bacterial pathogens on leaves. Ann Rev Phytopathol 33: 145172 3. Becker-Hapak, M, Troxtel, E, Hoerter, J, Eisenstark, A (1997) RpoS dependent overexpression of carotenoids from Erwinia herbicola in OxyR decient Escherichia coli. Biochem Biophys Res Commun 239: 305309 4. Bischof, K, Krabs, G, Wiencke, C, Hanelt, D (2002) Solar ultraviolet radiation affects the activity of ribulose-1,5-bisphosphate carboxylase-oxygenase and the composition of photosynthetic and xanthophyll cycle pigments in the intertidal green alga Ulva lactuca L. Planta 215: 502509 5. Braga, GUL, Flint, SD, Miller, CD, Anderson, AJ, Roberts, DW (2001) Both solar UVA and UVB radiation impair conidial culturability and delay germination in the entomopathogenic fungus Metarhizium anisophilae. Photochem Photobiol 74: 734739 6. Edge, R, Mcgarvey, DJ, Truscott, TG (1997) The carotenoids as anti-oxidantsa review. J Photochem Photobiol B 41: 189200 7. Flores-Moya, A, Gomez, I, Vinegla, B, Altamirano, A, PerezRodriguez, E, Maestre, C, Caballero, RM, Figueroa, FL (1998) Effects of solar radiation on the endemic Mediterranean red alga Rissoella verruculosa: photosynthetic performance, pigment content and the activities of enzymes related to nutrient uptake. New Phytol 139: 673683 8. Garcia-Pichel, F (1994) A model for internal self-shading in planktonic organisms and its implications for the usefulness of ultraviolet sunscreen. Limnol Oceanogr 39: 17041717 9. Goodfellow, M, Austin, B, Dickinson, CH (1976) Numerical taxonomy of some yellow-pigmented bacteria isolated from plants. J Gen Microbiol 97: 219233 10. Gunasekera, TS, Paul, ND, Ayres, PG (1997) The effects of ultraviolet-B (UV-B: 290320 nm) radiation on blister blight of tea (Camilla sinensis). Plant Pathol 46: 179185 11. Henson, JM, Butler, MJ, Daw, AW (1999) The dark side of the mycelium: melanins of phytopathogenic fungi. Ann Rev Phytopathol 37: 447471 12. Jacobs, JL, Sundin, GW (2001) Effect of solar UV-B radiation on a phyllosphere bacterial community. Appl Environ Microbiol 67: 54885496 13. Kadivar, H, Stapleton, AE (2003) Ultraviolet radiation alters maize phyllosphere bacterial diversity. Microb Ecol 45: 353361 14. Kim, J-J, Sundin, GW (2001) Construction and analysis of photolyase mutants of Pseudomonas aeruginosa and Pseudomonas syringae: contribution of photoreactivation, nucleotide excision repair, and mutagenic DNA repair to cell survival and mutability following exposure to UV-B radiation. Appl Environ Microbiol 67: 14051411 15. Kim, YC, Miller, CD, Anderson, AJ (1999) Transcriptional regulation by iron and role during plant pathogenesis of genes encoding iron-and manganese-superoxide dismutases of Pseudomonas syringae pv. syringae B728a. Physiol Mol Plant Pathol 55: 327339 16. King, EO, Ward, MK, Raney, DE (1954) Two simple media for the detection of pyocyanin and uorescein. J Lab Clin Med 44: 301 307 17. Kirchner, O, Tauch, A (2003) Tools for genetic engineering in the amino acidproducing bacterium Corynebacterium glutamicum. J Biotechnol 104: 287299 18. Laurion, I, Lami, A, Sommaruga, R (2002) Distribution of mycosporine-like amino acids and photoprotective carotenoids among freshwater phytoplankton assemblages. Aquat Microb Ecol 26: 283294

This work was supported by the Agricultural Experiment Stations of Texas and Michigan. We thank the researchers listed in Table 1 for bacterial strains, S. Sabaratnam and T. Gunasekera for critical reviews of the manuscript, and three anonymous reviewers whose comments strengthened the manuscript.

J.L. JACOBS

ET AL.:

ULTRAVIOLET RADIATION AND PHYLLOSPHERE BACTERIA

113

19. Miller, CD, Mortensen, WS, Braga, GUL, Anderson, AJ (2001) The rpoS gene in Pseudomonas syringae is important in surviving exposure to the near-UV in sunlight. Curr Microbiol 43: 374377 20. Parisi, AV, Kimlin, MG (1999) Horizontal and sun-normal spectral biologically effective ultraviolet irradiances. J Photochem Photobiol B 53: 7074 21. Paul, ND, Gwynn-Jones, D (2003) Ecological roles of solar UV radiation: towards an integrated approach. Trends Ecol Evol 18: 4855 22. Pourzand, C, Tyrell, RM (1999) Apoptosis, the role of oxidative stress and the example of solar UV radiation. Photochem Photobiol 70: 380390 23. Pfeifer, GP (1997) Formation and processing of UV photoproducts: effects of DNA sequence and chromatin environment. Photochem Photobiol 65: 270283 24. Rajogopal, L, Sundari, CS, Balasubramanian, D, Sonti, RV (1997) The bacterial pigment xanthomonadin offers protection against photodamage. FEBS Lett 415: 125128 25. Rotem, J, Aust, HJ (1991) The effect of ultraviolet and solar radiation and temperature on survival of fungal propagules. J Phytopathol 133: 7684 26. Sabaratnam, S, Beattie, GA (2003) Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic and endophytic colonization of leaves. Appl Environ Microbiol 69: 12201228 27. Sinha, RP, Hader, DP (2002) UV-induced DNA damage and repair: a review. Photochem Photobiol Sci 1: 225236 28. Sundin, GW (2002) Ultraviolet radiation on leaves: its inuence on microbial communities and their adaptations. In: Lindow, SE, Hecht-Poinar, EI, Elliott, VJ (Eds.) Microbiology of the Phyllosphere, APS Press, St. Paul, MN, pp 2741 29. Sundin, GW, Jacobs, JL (1999) Ultraviolet radiation (UVR) sensitivity analysis and UVR survival strategies of a bacterial com-

30.

31.

32.

33.

34.

35.

36.

37.

munity from the phyllosphere of eld-grown peanut (Arachis hypogeae L.). Microb Ecol 38: 2738 Sundin, GW, Jacobs, JL, Murillo, J (2000) Sequence diversity of rulA among natural isolates of Pseudomonas syringae and effect on function of rulAB-mediated UV radiation tolerance. Appl Environ Microbiol 66: 51675173 Sundin, GW, Kidambi, SP, Ullrich, M, Bender, CL (1996) Resistance to ultraviolet light in Pseudomonas syringae: sequence and functional analysis of the plasmid-encoded rulAB genes. Gene 177: 7781 Sundin, GW, Murillo, J (1999) Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to ultraviolet B (290320 nm) radiation and distribution of rulAB among P. syringae pathovars. Environ Microbiol 1: 7587 Thompson, IP, Bailey, MJ, Fenlon, JS, Fermor, TR, Lilley, AK, Lynch, JM, McCormack, PJ, McQuilken, MP, Purdy, KJ, Rainey, PB, Whipps, JM (1993) Quantitative and qualitative seasonal changes in the microbial community from the phyllosphere of sugar beet (Beta vulgaris). Plant Soil 150: 177191 Tong, Y, Lighthart, B (1997) Solar radiation is shown to select for pigmented bacteria in the ambient outdoor atmosphere. Photochem Photobiol 65: 103106 Tuveson, RW, Larson, RA, Kagan, J (1988) Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specic phototoxic molecules. J Bacteriol 170: 46754680 Wang, Y, Casadavell, A (1994) Decreased susceptibility of melanized Cryptococcus neoformans to UV light. Appl Environ Microbiol 60: 38643866 Wilson, M, Hirano, SS, Lindow, SE (1999) Location and survival of leaf-associated bacteria in relation to pathogenicity and potential for growth within the leaf. Appl Environ Microbiol 65: 1435 1443