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Feb 3, 2016 - eyed Juncos (Junco hyemalis) after mist-net capture in south-central Kansas, USA. Bacterial loads were measured by standard plate counts ...
Volume 133, 2016, pp. 155–167 DOI: 10.1642/AUK-15-126.1

RESEARCH ARTICLE

Isolation and characterization of bacteria from the feathers of wild Darkeyed Juncos (Junco hyemalis) John W. Dille, Christopher M. Rogers, and Mark A. Schneegurt* Department of Biological Sciences, Wichita State University, Wichita, Kansas, USA * Corresponding author: [email protected] Submitted July 8, 2015; Accepted November 22, 2015; Published February 3, 2016

ABSTRACT We dislodged microbes from samples of composites of ventral feathers from different birds of overwintering Darkeyed Juncos (Junco hyemalis) after mist-net capture in south-central Kansas, USA. Bacterial loads were measured by standard plate counts and .300 isolates were purified by repetitive streak-plating on R2A medium (þ cycloheximide). Biochemical and physiological characterization included identification by 16S rRNA gene phylogeny. Nearly half of the isolates grew on keratin and 80% exhibited lipase activity, suggesting that these isolates can degrade feathers and thus may affect survival and reproduction. Individual bacterial loads from 8 juncos varied within a 3-fold range, 105– 106 colony-forming units g1 feather. At 97% DNA sequence identity (species-level), 63 operational taxonomic units were detected among 202 sequences; the Chao1 estimate was 123. The Shannon diversity index (H; 97% identity) was 3.75, Simpson’s diversity index (1/D) was 16.1, and Good’s coverage was 82.4. Gram-positive bacteria dominated the culture collection, balanced between low and high GþC clades. Bacillus spp. were abundant, including B. asahii, B. cereus, B. megaterium, and B. pumilus. Lysinibacillus, Paenibacillus, and Staphylococcus also were isolated. Remarkably, substantial numbers of Actinomycetes were isolated, including representatives of Clavibacter, Curtobacterium, Microbacterium, and Rathayibacter, genera recognized as being populated by xylem-filling crop plant pathogens. Apposed to these were feather isolates implicated as beneficial to host plants, Frigoribacterium and Kitasatospora, being antagonists to plant pathogens or acting as plant growth promoters. High GþC Gram-positive bacterial isolates included Blastococcus, Cellulomonas, Humicoccus, Nocardioides, Promicromonospora, and Rhodococcus. Proteobacteria dominated the Gram-negative bacteria, with Alphaproteobacteria most abundant, including the potential plant pathogens Agrobacterium and Sphingomonas, and the oligotrophs Aurantimonas, Brevundimonas, Methylobacterium, Rhizobium, and Rhodobacter. Gammaproteobacteria included Pantoea, Pseudomonas, and Stenotrophomonas. Ours is the first report of abundant helpful and harmful phyllosphere bacteria on wild bird feathers. The clear implication is that free-living migratory birds may carry bacteria throughout their geographic ranges and may transmit pathogens and beneficial bacteria to plants. Keywords: feather bacteria, beneficial bacteria, plant pathogens, Dark-eyed Junco, cladistics, Junco hyemalis, migration ´ de Bacterias provenientes de Plumas Silvestres de Junco hyemalis Aislamiento y Caracterizacion Silvestres RESUMEN Obtuvimos microbios provenientes muestras compuestas de plumas ventrales de Junco hyemalis, luego de capturarlos con redes de niebla en el sur-centro de Kansas, EE.UU. Las cargas bacterianas fueron medidas recuentos en placas esta´ndares, y .300 aislamientos fueron purificados mediante el sembrado repetido en placas en un medio de R2A ´ bioqu´ımica y fisiologica ´ ´ por secuenciacion ´ filogen´etica del (þcicloheximida). La caracterizacion incluyo´ la identificacion gene 16S ARNr. Cerca de la mitad de los aislamientos crecieron en queratina y 80% mostro´ actividad lipasa, sugiriendo ´ La carga bacteriana que estos aislamientos pueden degradar las plumas, afectando la supervivencia y la reproduccion. proveniente de ocho individuos de J. hyemalis presento´ un rango de tres veces de 105–106 unidades de colonias en ´ de la secuencia de ADN (nivel de especie), se detectaron 63 ´ g1 pluma. Con un 97% de identificacion formacion ´ unidades taxonomicas operativas entre 202 secuencias; el estimado de Chao1 fue 123. El ´ındice de diversidad de ´ fue 3.75, la diversidad de Simpson (1/D) fue 16.1 y la cobertura de Good fue 82.4. Shannon (H; 97% de identificacion) ´ de cultivos, con un balance entre clados GþC bajos y altos. Las Las bacterias Gram-positiva dominaron la coleccion ´ se aislaron especies de Bacillus fueron abundantes, incluyendo B. asahii, B. cereus, B. megaterium y B. pumilus. Tambien ´ Lysinibacillus, Paenibacillus y Staphylococcus. Llamativamente, se aislaron numeros sustanciales de Actinomicetos, incluyendo representantes de Clavibacter, Curtobacterium, Microbacterium y Rathayibacter, g´eneros reconocidos por ´ verse poblados de patogenos del xilema de las plantas de cultivo. En sentido contrario, Frigoribacterium y

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´ Kitasatospora fueron aislados de las plumas, que son reconocidos como beneficos para las plantas hospederas, siendo ´ antagonistas de los patogenos de las plantas o actuando como promotores del crecimiento de las plantas. Aisalmiento bacterias Gram-positiva de GþC alto incluyeron a Blastococcus, Cellulomonas, Humicoccus, Nocardioides, Promicromonospora y Rhodococcus. Proteobacteria domino´ las bacterias Gram-negativa, siendo Alphaproteobacteria la ma´s ´ ´ abundante, incluyendo los patogenos potenciales de las plantas Agrobacterium y Sphingomonas, y los oligotroficos Aurentimonas, Brevundimonas, Methylobacterium, Rhizobium y Rhodobacter. Gammaproteobacteria incluyo´ a Pantoea, ´ ´ Pseudomonas y Stenotrophomonas. Esta es la primera cita de abundantes bacterias filosferas utiles y perjudiciales en plumas de aves silvestres. Una clara implicancia es que las aves migratorias silvestres pueden transportar bacterias a ´ ´ trave´ s de sus rangos geogra´ficos y transmitir bacterias patogenas y beneficas a las plantas.

´ Palabras clave: bacterias de plumas, bacterias beneficiosas, patogenos de plantas, cladistica, Junco hyemalis, ´ migracion

INTRODUCTION Plumage is a key point of interaction between birds and the microbial world. Feathers filter air, capturing particles and bacteria, and frequently contact soil and plants. Our limited knowledge of the diversity of avian microbial communities diminishes our ability to include microbial community interactions in discussions of avian survival and intersexual selection. Previous studies on the bacterial community of feathers have focused mainly on isolates that degrade keratin, rather than specifically on describing diversity (Shawkey et al. 2003, 2009, Lucas et al. 2005, Whitaker et al. 2005, Peele et al. 2009, Bach et al. 2011, Saag et al. 2011, Verea et al. 2014). Measurements of bacterial loads on feathers also have been focused mainly on keratinolytic bacteria (Burtt and Ichida 1999a, 2004, Møller et al. 2009, Giraudeau et al. 2010, Czirja´k et al. 2013). We counted and isolated bacteria from feathers of the Dark-eyed Junco (Junco hyemalis) and captured a more diverse microbial community than has been described previously. Feathers are critical not only to flight and protection, they also play a central role in sexual displays, sexual selection, and reproductive success (Freeland 1976, Hamilton and Zuk 1982, Hamilton 1990, Hill 1991, Møller 1991, Swaddle et al. 1996, Leclaire et al. 2014). Bacteria can influence avian ecology by degrading feathers, thereby altering coloration and physical properties, or featherassociated bacteria may be pathogenic to birds (Burtt and Ichida 2004, Peele et al. 2009). Bird–microbe interactions have broad implications for the conservation of birds and for the protection of domesticated birds and the food supplied through aviculture. Preen oils, sanitation behaviors, social interactions, and environmental conditions likely play central roles in shaping microbial community structure on plumage (Pugh 1971, 1972, Jacob and Ziswiler 1982, Altizer et al. 2004, Kulkarni and Heeb 2007, Archie and Theis 2011). Feathers may be a challenging environment for microbes, presenting low-quality nutrients, high UV irradiation, and relatively low moisture (Mathison 1964, Korniłłowicz-

Kowalska 1999, Saranathan and Burtt 2007). Feather microbial communities are under substantial selection pressures, and feathers may have microbial assemblages distinct from the microbial communities of the soil and the phyllosphere with which birds regularly interact. Our work characterized the bacterial community on the feathers of wild Dark-eyed Juncos, migratory songbirds (18–28 g) that are common and widespread across North America (Nolan et al. 2002). Juncos spend considerable time during their annual cycle in diverse habitats, ranging from boreal forest in the breeding season to forest edges, old fields, and suburban landscapes during migration and winter. In addition, this species is easily captured at winter feeding stations in Kansas, USA, and responds well to capture and banding. Junco ecology and behavior are well studied, providing a useful background for the interpretation of new findings. Bacterial isolates from juncos were identified by 16S rRNA gene sequencing and characterized through biochemical and physiological assays that address the potential of these isolates to degrade feathers and hence influence reproductive success and sexual selection. Our surveys of bacterial abundance and the large bacterial isolate collection that we generated provide the best analysis of a feather microbial community to date. METHODS Feather Sampling and Processing Adult Dark-eyed Juncos were haphazardly captured using mist nets at winter feeding stations in the Ninnescah Reserve of the Wichita State University Biological Field Station (37831 0 N, 97842 0 W) in Kansas. Sterile gloves were used to grip birds from the dorsal side. Ventral contour feathers (9) were collected from each bird and placed in sterile Whirl-Pak bags (Nasco, Fort Atkinson, Wisconsin, USA) held at ambient temperature. Ventral feathers are easy to collect, relatively consistent in size, and removal has little effect on the bird. The area near the cloaca was avoided, since fecal bacteria could then dominate the microbial collection. Samples were transported and processed within 90 min. Microbes were dislodged from

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feathers in 10 mL of 0.1% Na pyrophosphate (a chaotropic agent), with shaking on a rotary platform at 150 rpm for 35 min before plating (Caton et al. 2004). Three distinct sets of feather samples were taken for different experiments in this study. Feathers from 8 individual birds were used for measuring bacterial loads. Separate composite feather samples from different groups of individual birds were used for measuring growth tolerances and for microbial isolation. A third set of feather samples was used for determining average feather weight in order to quantitate bacterial abundance per gram of feather. This third set of feathers was collected because it was important for cultivation experiments to control contamination introduced through manipulation of feather samples and sufficient numbers of feathers (at least 10; more than could be safely taken from an individual bird) were needed to obtain a reliable average weight. We did not record the age or sex of the birds sampled for any part of this study; however, wintering juncos are mainly female and are of similar size. Of the juncos (n ¼ 221) captured during recent winters (2010–2013), 60% were female, 28% were older than 1 yr, and only 5 of 141 birds were outside the range of 17.5 to 22.0 g (C. M. Rogers personal observation). Since feather size scales with body size, the feathers were expected to be within 25% of each other in weight. Bacterial Load and Growth Tolerances Microbes were separately dislodged from 9 feathers collected from each of 8 individual juncos, and 100-lL aliquots were spread onto triplicate plates of Reasoner’s 2A agar (R2A) supplemented with 0.2% cycloheximide. This medium is commonly used for soil microbiology and suppresses fast-growing organisms that can dominate mixed cultures. Cycloheximide was used to inhibit the growth of fungi. Colonies were counted after incubations of 1, 3, 7, and 14 days. Blank controls with no feathers were not performed, however all materials used were sterile. Surveys of thermotolerance and halotolerance used composite samples of 3 feathers from each of 3 birds. Triplicate R2A plates (with cycloheximide) were maintained at 4, 25, and 378C to measure temperature tolerance. R2A medium (with cycloheximide) was supplemented with 1, 5, 10, 15, and 20% NaCl for survey counts of halotolerant bacteria on triplicate plates. Individual feather weights were very low, so batches of 10 feathers each were carefully weighed using a closed scale (on a marble block) to determine an average feather weight of 5.6 3 104 6 3.7 3 105 g per feather (n ¼ 20 batches of 10 feathers each) to use for calculations. Counts are expressed as colony-forming units (CFU) g1 feather 6 SD. Microbial Isolation A composite sample of 3 ventral feathers from each of 3 birds (9 feathers total) was processed to dislodge microbes

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as above and aliquots (100 lL) were spread-plated on R2A medium (with cycloheximide) at 25 or 378C. Over a 2week period, isolated colonies were collected sequentially as they arose, without discrimination by colony morphology. Isolated colonies were purified by 6 rounds of repetitive streak-plating on R2A medium. All isolates were maintained on R2A plates (with cycloheximide) and archived as 20% glycerol stocks at 808C. Identification and Characterization of Bacterial Isolates Bacterial isolates were identified by 16S rRNA gene sequence analysis. Pure cultures grown in R2A broth were harvested by centrifugation and lysed by 6 freeze–thaw cycles in liquid N2 and 908C water as previously described (Caton et al. 2004). Crude DNA extracts were subjected to PCR amplification with TaKaRa Ex Taq polymerase (Takara Bio, Kusatsu, Shiga, Japan) using universal bacterial primers that give nearly full-length 16S rRNA gene sequences: pA 5 0 -AGAGTTTGATCCTGGCTCAG-3 0 and pH 0 5 0 -AAGGAGGTGATCCAGCCGCA-3 0 (Edwards et al. 1989). PCR was performed as described previously (Kilmer et al. 2014), with an annealing temperature of 538C. Amplicons were confirmed by 2% agarose gel electrophoresis, with visualization after ethidium bromide staining. PCR products were cleaned with the Promega Wizard SV Gel and PCR Cleanup System (Promega Corporation, Madison, Wisconsin, USA) before Sanger sequencing of 202 isolates at the University of Kansas Biodiversity Institute (Lawrence, Kansas, USA) using the pA primer. DNA sequences were aligned using CLUSTAL W (Thompson et al. 1994), including contextual sequences selected with BLAST from the GenBank database (Altschul et al. 1990). Aligned sequences were trimmed to equal length in MacClade 4.08 (Sinauer Associates, Sunderland, Massachusetts, USA) and realigned in CLUSTAL W. Phylogenetic analysis was performed with PAUP* 4.0 b10 (Swofford 1998) through distance analysis by neighborjoining using Jukes-Cantor rules, giving ~350 positions suitable for analysis after removing missing and ambiguous bases. Jackknife resampling was used to assess the relative support for each branch, with a total of 100 bootstrap replicates conducted heuristically using the distance-based neighbor-joining algorithm and the nearest-neighborinterchange algorithm in PAUP*. No putative chimeras were identified using Pinwheel within Sequin (http://www. ncbi.nlm.nih.gov/Sequin/). Sequences appear in GenBank with accession numbers KM513853–KM514055. Diversity indices, Chao estimators, and rarefaction curves were generated using the mothur statistical package (Schloss et al. 2009). Individual isolates were assayed for halotolerance on R2A medium supplemented with 1, 5, 10, 15, and 20%

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FIGURE 1. Bacterial loads of ventral feathers from 8 individual Dark-eyed Juncos. Bacteria were dislodged from feather samples and serially diluted onto triplicate R2A plates. Values are colonyforming units (CFU) g1 feather 6 SD. The SD includes variance due to errors in microbial counting and batch feather weight measurement.

NaCl. The water activity of each medium was measured using an AquaLab Series 3 water activity meter (Decagon Devices, Pullman, Washington, USA) calibrated with standard NaCl solutions and run at room temperature. Lipase was assayed using agar plates infused with 2% tributyrin oil, noting zones of clearing. Starch hydrolysis was tested on agar plates infused with 0.4% soluble starch. Two days after inoculation, plates were flooded and incubated with Gram’s iodine for 5 min. The stain solution was removed and colonies were examined for zones of clearing. Gelatin stab tubes were inoculated with isolates and incubated for 48 hr. A liquefied mixture indicated that the isolate was positive for protein hydrolysis. Keratin utilization was determined by growth on BSM plates (33.3 mM KH2PO4; 33.3 mM K2HPO4; 15.1 mM (NH4)2SO4; 0.79 mM MgCl26 H2O; 0.18 mM CaCl2; 9.67 lM NaMoO42 H2O; 7.98 lM MnCl24 H2O; 6.58 lM FeSO4) supplemented with 1% (w/v) feather meal (Down to Earth organic feather meal 12-0-0 fertilizer; Everwood Farm, Brooks, Oregon, USA), as compared with growth on BSM plates without an exogenous carbon source. RESULTS Survey of Microbial Abundance The abundance of aerobic bacteria dislodged from the feathers of overwintering Dark-eyed Juncos, as determined

J. W. Dille, C. M. Rogers, and M. A. Schneegurt

FIGURE 2. Abundance of feather bacteria after cultivation at different temperatures. Bacteria dislodged from composite Darkeyed Junco feather samples were serially diluted and plated in triplicate onto R2A medium incubated at 4, 25, or 378C. Values are colony-forming units (CFU) g1 feather 6 SD. The SD includes variance due to errors in microbial counting and batch feather weight measurement.

by standard plate counts on R2A medium (with cycloheximide), was in the range of 105 to 106 CFU g1 feather (Figure 1). Values for the 8 individual birds varied within a 3-fold range (compare bird B with bird H; Figure 1). Variation in feather batch weights was too small to explain the differences observed in bacterial loads. Salinity testing found that microbial growth (1.2 3 106 CFU g1 feather with no added salt) was inhibited as salinity increased. However, 17% of the cultivable bacteria grew at 5% NaCl, twice the salt concentration of seawater. Approximately 2% of the bacteria grew at 10% NaCl, a medium with relatively low water activity (aw ¼ 0.92). Only 6.0 3 103 CFU g1 feather grew at 15% NaCl (aw ¼ 0.88), and no growth was observed at 20% NaCl. The cultivable bacteria were predominantly mesophilic. Growth was best at 258C, with lower counts observed at 378C (Figure 2). Colonies developed most slowly at 48C, but reached nearly 40% of the counts observed at 258C. Identification of Bacterial Isolates All separated colonies were collected from plates of microbes dislodged from composite samples of junco feathers, so that community analysis would not be biased by colony morphology. More than 300 bacterial isolates were obtained by repetitive streak-plating of clonal colonies, although some expired before the completion

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J. W. Dille, C. M. Rogers, and M. A. Schneegurt

FIGURE 3. Phylogenetic tree for Gram-positive bacteria from Dark-eyed Junco feathers, based on 16S rRNA gene sequences. Bootstrap values greater than 50% are shown. A full tree with GenBank accession numbers can be found in Appendix Figure 4.

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of the study and were not identified by 16S rRNA gene sequencing. Results from the 202 isolate sequences accepted by GenBank are given here. Isolates from genera within the Gram-positive bacteria were more abundant in the collection than those of other clades and were nearly balanced between high GþC Actinomycetes and low GþC Firmicutes (Figure 3 and Appendix Figure 4). In the low GþC Gram-positive clade, isolates were predominantly from the genus Bacillus, mainly clustering with B. pumilus. Bacillus species isolated from juncos included B. asahii, B. cereus, B. drentensis, B. megaterium, B. pumilus, and B. subtilis. Closely related Lysinibacillus, Paenibacillus, and Staphylococcus were recovered as well. Isolates in the high GþC Gram-positive cluster were predominantly Actinomycetes. Microbacteriaceae were abundant, including Clavibacter, Curtobacterium, Microbacterium, and Rathayibacter. These genera are known to be predominantly or entirely populated by plant pathogens, affecting crop plants such as wheat and ryegrass (Bergey’s Manual Trust 2011). Other high GþC Grampositive isolates were from genera (e.g., Frigoribacterium and Kitasatospora) implicated as being beneficial to plant hosts, acting as antagonists to fungal and bacterial plant pathogens or as plant growth promoters (Sessitsch et al. 2004, Haesler et al. 2008). Isolates of Cellulomonas, Promicromonospora, and Rhodococcus were identified, along with representatives of Blastococcus, Humicoccus, and Nocardioides. Isolates within the Proteobacteria dominated the collection of Gram-negative bacteria (Figure 5 and Appendix Figure 6). Alphaproteobacteria were most abundant and included representatives of the genera Agrobacterium, Aurantimonas, Brevundimonas, Methylobacterium, Rhizobium, Rhodobacter, and Sphingomonas, oligotrophic organisms observed in the phyllosphere and soils (Bergey’s Manual Trust 2011). The Gammaproteobacteria included Pantoea, Stenotrophomonas, and Pseudomonas, the latter clustering with Pseudomonas syringae, considered a plant pathogen (Bergey’s Manual Trust 2011). Variovorax was the only Betaproteobacteria genus observed. The 2 isolates from the Bacteroidetes group were related to Pedobacter and Spirosoma. Diversity indices determined from the 202 rRNA gene sequences are given in Table 1. At the species level (97% identity), 63 operational taxonomic units (OTUs) were observed. Chao1 estimates suggest that this represents approximately half of the species present in the sample. Good’s coverage suggests somewhat higher recovery (82%). At the 88% sequence identity level, it appears that nearly all of the bacterial diversity on feathers was observed within the collection. This is supported by the rarefaction curves for different levels of identity (Appendix Figure 7). Only the 88% sequence identity curve appears to be

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TABLE 1. Diversity analyses of the bacterial assemblage isolated from Dark-eyed Junco feathers. Level of sequence identity Parameter

99%

97%

94%

88%

Operational taxonomic units 78 63 52 29 Chao1 Average 143 123 90 33 95% CI 108–217 88–207 66–155 30–47 Good’s coverage 76.9 82.4 87.4 96.0 Shannon diversity (H) 4.10 3.75 3.49 2.59 Shannon evenness (E) 0.94 0.91 0.88 0.77 Simpson’s diversity (1/D) 19.2 16.1 14.6 6.60

isolates were more balanced between bacilli and cocci, with some coccobacilli (Figure 8B). The only filamentous organism was F64, related to Kitasatospora, a Streptomycete. Neither catalase nor oxidase was detected in the majority of the isolates (Figure 8A). Catalase was somewhat more prevalent in the Gram-negative isolates than in the Gram-positive isolates. Approximately a third of all isolates were positive for amylase or gelatinase (Figure 8C). More than 80% of isolates were lipase-positive (Figure 8C). Nearly half of the isolates could use keratin as a sole carbon and energy source (Figure 8C). Isolates in the collections had time to acclimate to the laboratory environment during streak-plate isolation and showed more robust halotolerance than microbes from the direct survey. Most of the isolates (77%) grew at 5% NaCl, and 20% of the isolates grew at 10% NaCl. Nine isolates (F33, F107, F116, F126, F154, F168, F233, F303, and F314) grew at 15% NaCl, and 1 isolate (F107) grew at 20% NaCl. DISCUSSION

FIGURE 5. Phylogenetic tree for Gram-negative bacteria from Dark-eyed Junco feathers, based on 16S rRNA gene sequences. Bootstrap values greater than 50% are shown. A full tree with GenBank accession numbers can be found in Appendix Figure 6.

flattening. Shannon indices suggest a diverse community, giving a value near 4 at the species level. Community diversity appears to be relatively even, especially at higher sequence identity levels. Characterization of Bacterial Isolates The isolate collection from junco feathers was dominated by Gram-positive bacteria. In all cases, Gram staining results agreed with phylogenetic placement based on 16S rRNA gene sequences (Figure 8A). Most of the Gramnegative bacteria were cocci, while the Gram-positive

Estimating the bacterial load of feathers is challenging because cells may be firmly attached to or inside feather structures, potentially leading to underestimates of microbial abundance. It is difficult to compare estimates published previously with our current results as different methods were used, e.g., using feather presses on agar or standardizing values on a per-feather basis (Lucas et al. 2005, Shawkey et al. 2009, Saag et al. 2011, Leclaire et al. 2014). A study of captive male House Sparrows (Passer domesticus) found 2–4 3 106 CFU g1 feather (Czirja´k et al. 2013). On Mallard (Anas platyrhynchos) feathers, 105 CFU g1 feather were reported (Giraudeau et al. 2010). Bacterial loads on junco feathers, 105–106 CFU g1 feather, were in a similar range. However, it should be noted that R2A medium is not suitable for all bacteria and our conditions selected for aerobic heterotrophs. We also examined only feather washes, thus our results likely underestimated bacterial loads.

The Auk: Ornithological Advances 133:155–167, Q 2016 American Ornithologists’ Union

J. W. Dille, C. M. Rogers, and M. A. Schneegurt

FIGURE 8. Characterization of bacterial isolates from Dark-eyed Junco feathers by (A) Gram stain and catalase and oxidase assays (solid ¼ positive; open ¼ negative); (B) cell morphology (solid ¼ Gram-positive isolates; open ¼ Gram-negative isolates); and (C) catabolic enzyme activities (solid ¼ positive; open ¼ negative).

Most previous studies of bird feather microbial communities were not focused on diversity and did not detect the diversity of bacteria that we recorded from juncos (Muza et al. 2000, Shawkey et al. 2003, 2005, 2009, Lucas et al. 2005, Whitaker et al. 2005, Bisson et al. 2007, 2009, Peele et al. 2009, Bach et al. 2011, Saag et al. 2011, Kilgas et al. 2012a, 2012b, Verea et al. 2014). These earlier studies

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detected low diversity for a variety of methodological reasons, including selective growth conditions and quantification schemes (Lopez-Velasco et al. 2011, Schneegurt 2013). For instance, a study of American Redstarts (Setophaga ruticilla) reported a Shannon diversity index of ,1, with nearly 80% of the isolates within the Pseudomonads, after storage of feathers at 48C before cultivation (Bisson et al. 2007, 2009). Our study gave a Shannon diversity index near 4 for the bacterial assemblage on fresh junco feathers. Cultivation inherently selects for certain populations of microbes, which in our case were aerobic, heterotrophic bacteria. The feather microbial community is surely more diverse than what was observed here using a single culture medium and growth conditions. R2A is an oligotrophic medium commonly used for soil microbiology, as it slows rapidly proliferating microbes that overgrow on plates. Cycloheximide was needed to suppress the copious growth of the fungi that are common on feathers (Pugh 1971, 1972). While cultivation yields microbial isolates that can be characterized biochemically and physiologically, culture-independent metagenomic techniques can reveal greater diversity through nextgeneration sequencing of rRNA genes (Epstein 2013). Ours is the first report of abundant culturable recognized plant pathogens and beneficial bacteria on the feathers of wild migratory birds. Although juncos are ground-feeding birds, bacteria found in the phyllosphere were abundant on their feathers. Common in both the soil and the phyllosphere, bacteria related to Bacillus were abundant, as expected from previous work (Burtt and Ichida 1999a, 2004, Whitaker et al. 2005, Hasegawa et al. 2006, Izhaki et al. 2013, Singh et al. 2014). Most surprising was the abundance of Actinomycetes, specifically Microbacteriaceae, which are predominantly populated by recognized plant pathogens of crops that are widely cultivated in Kansas and across North America. Note that pathogenicity and transmission were not directly assayed in our study. While previous work suggests some degree of taxon specificity of keratinolytic fungi on wild bird feathers (Huba´lek 1976), it seems unlikely that juncos are the only common birds carrying putative plant pathogens and beneficial bacteria on their feathers. Sample contamination is possible and difficult to detect; however, our isolate collection did not include human-associated bacteria such as certain Staphylococcus species. It is possible that minute particles of plant debris or soil were caught in feathers and not casually observed. We did not fractionate feathers to determine the location of microbes dislodged for isolation. The Actinomycetes recovered from juncos clustered with recognized xylem-filling plant pathogens that cause gumming disease in wheat and annual ryegrass. Winter wheat is a primary agricultural crop in Kansas. All reported Clavibacter isolates are plant pathogens, and transmission is believed to be by contaminated seeds, asymptomatic

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seedlings, and plant debris (Bergey’s Manual Trust 2011). Curtobacterium, Okibacterium, and Plantibacter have been isolated primarily from the phyllosphere, with the latter reported to be transmitted by nematodes (Jurkevitch and Shapira 2000, Behrendt et al. 2002, Bergey’s Manual Trust 2011). Rathayibacter transmission also appears to be by nematodes. Rathayibacter toxicus is a U.S. Department of Agriculture (USDA) select agent, serious enough to be reportable as a severe threat to agriculture. Agrobacterium and Sphingomonas include members that are recognized plant pathogens (Bergey’s Manual Trust 2011). Frank bird or animal pathogens were not found in the junco bacterial collection, with the exception of Lysinibacillus, a reported insect pathogen (Berry 2012, Yang et al. 2012). However, only ventral feathers were sampled, while enteric pathogens have been associated with cloaca (Scullion 1989, Lombardo et al. 1996). Migratory birds previously have been found to carry pathogenic microbes on their feathers, including avian pathogens (Lombardo et al. 1996, Corry and Atabay 2001, Bengis et al. 2004, Abulreesh et al. 2007, Tsiodras et al. 2008). Interest in avian hosts as potential vectors and reservoirs for the transmission of human disease has arisen from serious concerns about emerging infections and the spread of pathogens. It is not surprising that birds similarly may carry plant pathogens and potentially act as vectors. Bashan (1986) included birds as carriers of the plant pathogens Pseudomonas syringae and Xanthomonas campestris, but concluded that their low abundance on feathers may preclude transmission. We found a variety of abundant culturable bacteria on feathers that are implicated as plant pathogens. A number of junco feather isolates clustered with bacterial groups known to be beneficial to plants. Lysinibacillus, Lysobacter, Stenotrophomonas, and several Bacillus spp. include members shown to be antagonistic to fungal pathogens (e.g., Fusarium) in the rhizosphere and phyllosphere (Hayward et al. 2010, Kavroulakis et al. 2010, Izhaki et al. 2013, Singh et al. 2014). Frigoribacterium and Labedella have few cultured representatives, are associated with the phyllosphere, and are suggested to be beneficial bacteria (Sessitsch et al. 2004, Lee 2007, Yashiro et al. 2011). Kitasatospora is antagonistic to Phytophthora (Haesler et al. 2008). Cellulomonas degrades cellulose, but also has been shown to promote growth in the wheat rhizosphere (Egamberdiyeva and H¨oflich 2002). While Bacillus licheniformis has been isolated from feathers previously (Burtt and Ichida 1999b), it was not observed here; however, in earlier studies samples were cultured at 508C specifically to select for B. licheniformis strains. Various Bacillus species were recovered from juncos, but these species are common in soils and the phyllosphere (Bergey’s Manual Trust 2011).

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A majority of the bacterial isolates grew on keratin as a sole carbon and energy source, including many of the putative plant pathogens (Dille et al. 2011, 2012). This could have significant consequences for bird survival, should the integrity of feathers be compromised, and for fecundity, should the bird appear less attractive due to duller or damaged feathers (Møller 1991, Swaddle et al. 1996, Shawkey et al. 2009). Similarly, the high proportion of isolates expressing lipase suggests that feather oils may be a nutritional source for bacteria, potentially leading to feather degradation by the removal of water-proofing (Al Musallam and Radwan 1990). There is likely interplay between the microbial community and antimicrobial compounds found on feathers and in preen oils (Pugh 1971, Jacob and Ziswiler 1982, Møller et al. 2009, Giraudeau et al. 2013). Enhanced halotolerance would be consistent with organisms adapted to dry, oily environments, such as feathers, and suggests that the microbial community on feathers may have some specificity for feathers, since the halotolerance of bacteria from common soils (Porazka et al. 2011) is substantially less pronounced than the halotolerance of feather bacteria. The clear implication of our study is that migratory birds carry potential plant pathogens and beneficial bacteria on their feathers and may transmit these organisms to plants after dispersing long distances. Microbial pathogens on crop plants lead to broad societal costs, being detrimental to the production of commodities from foods to biofuels. Demonstration of a novel vector for phyllosphere bacteria, both helpful and harmful, especially one with such great potential for widespread dissemination, would abrogate our current understanding of the epidemiology of plant pathogens and beneficial bacteria. It is an intriguing system. Birds interact with phyllosphere and soil communities that include pathogenic and beneficial bacteria. They use preen oils and sanitation behaviors that presumably select for microbial communities favorable to feather health and reproductive success, with no regard to plant pathogenicity or benefit. Then birds potentially disseminate and transmit the resultant microbial community over broad spatial scales. There are major implications for avian biology and ecology suggested by our findings. The high bacterial species diversity on junco contour feathers suggests that bacterial feather communities are much richer than known from previous studies. Keratinolytic isolates that can degrade feathers may have real impacts on feather quality, and hence survival, mating success, and intersexual selection. Examining bacterial assemblages on feathers adds a dimension to microbe–host interactions, enhancing our understanding of avian ecology and conservation. Studying a new vertebrate host–microbe system also can enhance our understanding of human and animal systems. Finally, juncos may be utilizing agricultural habitats to an

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appreciable extent, reflecting suboptimal habitat selection caused by loss and/or degradation of historically important winter habitats, such as forest edges, old fields, and riparian zones. Factors reducing winter habitat quality include decreased food supply resulting from lower plant species diversity and elevated predator density due to increased abundance of Cooper’s Hawks (Accipiter cooperii), forcing juncos to use a broader selection of habitat types (Ratajczak et al. 2012, Sauer et al. 2014). Ecological habitat loss and alteration in this case may lead to greater exposure of birds to bacterial plant pathogens and potentially to broader spread of these pathogens to agricultural plants. ACKNOWLEDGMENTS This is publication #28 in the series of reports from the Wichita State University Biological Field Station. We are grateful for the preliminary laboratory work performed by Sarah Jack, Shanna Kelzenberg, and Brian Kilmer. Water activity was kindly measured by Fadi Aramouni. Funding statement: This work was supported by funding from Kansas INBRE NIH NIGMS IDeA (P20 GM103418), Kansas NSF EPSCoR (EPS-0903806), and NSF GK-12 (DGE 0537844). None of the funding agencies had any input into the content nor required approval of the manuscript prior to submission or publication. Ethics statement: Ethical animal handling protocols were approved by the Wichita State University Institutional Animal Care and Use Committee (WSU IACUC permit #196). Author contributions: C.M.R. and M.A.S. conceived the idea, design, and experiment (supervised research, formulated question or hypothesis); J.W.D., C.M.R., and M.A.S. performed the experiments (collected data, conducted the research); C.M.R. and M.A.S. wrote the paper (or substantially edited the paper); J.W.D., C.M.R., and M.A.S. developed or designed the methods; J.W.D., C.M.R., and M.A.S. analyzed the data; and M.A.S. contributed substantial materials, resources, or funding.

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APPENDIX FIGURE 4. Phylogenetic tree for Gram-positive bacteria from Dark-eyed Junco feathers, based on 16S rRNA gene sequences. Bootstrap values greater than 50% are shown.

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APPENDIX FIGURE 7. Rarefaction curves based on 16S rRNA gene sequences of bacteria collected from Dark-eyed Junco feathers. The curves represent different levels of DNA sequence identity.

APPENDIX FIGURE 6. Phylogenetic tree for Gram-negative bacteria from Dark-eyed Junco feathers, based on 16S rRNA gene sequences. Bootstrap values greater than 50% are shown.

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