Plant-associated bacterial populations on nativeand invasive plant species: comparisons between2 freshwater environments

Ola A. Olapade and Kayleigh Pung

Abstract: Plant–microbial interactions have been well studied because of the ecological importance of such relationships inaquatic systems. However, general knowledge regarding the composition of these biofilm communities is still evolving,partly as a result of several confounding factors that are attributable to plant host properties and to hydrodynamic conditionsin aquatic environments. In this study, the occurrences of various bacterial phylogenetic taxa on 2 native plants, i.e., may-apple (Podophyllum peltatum L.) and cow parsnip (Heracleum maximum Bartram), and on an invasive species, i.e., garlicmustard (Alliaria petiolata (M. Bieb.) Cavara & Grande), were quantitatively examined using nucleic acid staining and fluo-rescence in situ hybridization. The plants were incubated in triplicates for about a week within the Kalamazoo River andPierce Cedar Creek as well as in microcosms. The bacterial groups targeted for enumeration are known to globally occur inrelatively high abundance and are also ubiquitously distributed in freshwater environments. Fluorescence in situ hybridiza-tion analyses of the bacterioplankton assemblages revealed that the majority of bacterial cells that hybridized with the differ-ent probes were similar between the 2 sites. In contrast, the plant-associated populations while similar on the 3 plantsincubated in Kalamazoo River, their representations were highest on the 2 native plants relative to the invasive species inPierce Cedar Creek. Overall, our results further suggested that epiphytic bacterial assemblages are probably under the influ-ences of and probably subsequently respond to multiple variables and conditions in aquatic milieus.

Key words: epiphytes, fluorescence in situ hybridization, hydrodynamics, rivers.

Résumé : Les interactions plante–microbe ont été bien étudiées à cause de l’importance écologique de telles relations dansles systèmes aquatiques. Cependant, les connaissances générales en ce qui concerne la composition de ces communautés debiofilms évoluent toujours, dû en partie à différents facteurs confondants attribuables aux propriétés des plantes hôtes ainsique des conditions hydrodynamiques des environnements aquatiques. Dans cette étude, la fréquence de différents taxonsphylogéniques bactériens sur deux plantes indigènes, le podophylle pelté (ou pomme de mai, Podophyllum peltatum L.) etla berce laineuse (Heracleum maximum Bartram) et une espèce invasive, l’alliaire officinale (Alliaria petiolata (M. Bieb.)Cavara & Grande), a été examinée de manière quantitative par coloration des acides nucléiques et par hybridation in situ enfluorescence (FISH). Les plantes ont été incubées en triplicats pendent environ une semaine dans l’eau de la Kalamazoo Ri-ver et de la Pierce Cedar Creek, ainsi qu’en microcosme. Les groupes bactériens ciblés pour la numération étaient connuspour survenir globalement en relativement forte abondance et être aussi distribués de manière ubiquiste dans les environne-ments d’eau douce. Les analyses en FISH des assemblages de bactérioplanctons ont révélé que la majorité des cellules bac-tériennes qui s’hybridaient aux différentes sondes étaient similaires sur les deux sites. Cependant, les populations associéesaux 3 types de plantes incubées dans la Kalamazoo River étaient similaires, alors qu’elles étaient davantage représentées surles deux plantes indigènes comparativement à l’espèce invasive au site de la Pierce Cedar Creek. Dans leur ensemble, nosrésultats suggèrent que les assemblages de bactéries épiphytes sont probablement influencés et répondent probablement àdifférentes variables et conditions des milieux aquatiques.

Mots‐clés : épiphytes, hybridation in situ en fluorescence, hydrodynamique, rivières.

[Traduit par la Rédaction]

Introduction

There is currently ample information regarding the influ-ence of various plant species on the occurrence and possiblythe distribution of different bacterial taxa in aquatic environ-ments (e.g., King 1994; Weidner et al. 2000; Lindow and

Brandl 2003; Whitman et al. 2003, 2005; McNamara andLeff 2004; Olapade et al. 2006). Generally, copious amountsof microorganisms found associated with plant surfaces (es-pecially on leaves) are mostly in response to various endoge-nous nutrient sources, including varieties of organic andinorganic compounds released by the plant host (e.g., Mercier

Received 4 January 2012. Revision received 5 April 2012. Accepted 5 April 2012. Published at www.nrcresearchpress.com/cjm on 24 May2012.

O.A. Olapade and K. Pung. Department of Biology and the Center for Sustainability and the Environment, Albion College, 611 EastPorter Street, Albion, MI 49224, USA.

Corresponding author: Ola A. Olapade (e-mail: oolapade@albion.edu).

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Can. J. Microbiol. 58: 767–775 (2012) doi:10.1139/W2012-053 Published by NRC Research Press

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and Lindow 2000). While the availability and quality of nu-trients on the hosts, including various sugars, salts, aminoacids, methane, and phenolic compounds (Fiala et al. 1990;King 1994; Mercier and Lindow 2000), are mostly dependenton the plant’s surface properties (e.g., Fiala et al. 1990),some of these properties, especially leaf age, vary as growthconditions change between several plant species, and there-fore potentially influence the colonization and developmentof associated microbial assemblages (Knoll and Schreiber2000). Additionally, variations in several site-specific hydro-dynamic conditions could also significantly influence therate of release of these required nutrients from resident planthosts in aquatic systems.Therefore, despite the well-documented contributions of

various plant-specific properties as well as several environ-mental variables to plant–microbial associations, currentknowledge regarding bacterial population dynamics in plant–microbial relationships in aquatic milieus is still very muchunresolved, especially, given that past research efforts havebeen solely based on examining bacterial presence on eithersingle plant species (Whitman et al. 2003; McNamara andLeff 2004; Olapade et al. 2006) or several plant specieswithin the same aquatic environment (e.g., Crump and Koch2008; Olapade et al. 2011). For instance, an earlier study byOlapade et al. (2011) found close similarities in the occur-rences of most of the bacterial taxa examined within plant-associated assemblages on 2 native plants, i.e., Podophyllumpeltatum L. (mayapple (MA)) and Heracleum maximum Bar-tram (cow parsnip (CP)) after being anchored in a freshwatercreek. Basically, the study indicated that these 2 nativeplants, probably with comparable nutritional compositions,equally stimulated the occurrences of the leaf-associated bac-terial assemblages under comparable environmental condi-tions within the same freshwater system.Therefore, the main goal of this present study was to better

assess and further understand the influences of various planthosts on associated bacterial assemblages in aquatic environ-ments, given the diverse nutritional and physiological re-quirements that have been well documented within surface-associated bacterial assemblages in lotic systems (e.g.,McNamara and Leff 2004; Olapade and Leff 2004; Olapadeet al. 2006). Based on previously acquired information, wedecided to numerically examine bacterial populations onboth native, i.e., MA and CP, and invasive species, i.e., Allia-ria petiolata (M. Bieb.) Cavara & Grande (garlic mustard(GM)) of Michigan, USA. These plants known to have con-trasting nutritional compositions (e.g., Berenbaum and Feeny1981; Moraes et al. 2000; Bedir et al. 2006) were subjectedin our study to different environmental conditions within 2separate freshwater systems. Additionally, to be able to ac-count for potential impacts of several confounding factorsthat could be associated with several site-specific hydro-dynamic variables at the 2 study sites, we also examined trip-licate leaf packs of the plants in microcosms, undercontrolled laboratory conditions.The presence and abundance of targeted bacterial taxa

within the biofilm assemblages were enumerated with combi-nations of nucleic acid staining (i.e., 4′,6-diamidino-2-phenylindole (DAPI) for total direct counts) and fluores-censce in situ hybridization (FISH). Even though most of thebacterial taxa targeted in this study are known to occur in rel-

atively high abundance and are ubiquitously distributed, es-pecially on surfaces in aquatic systems (Brümmer et al.2000; Olapade and Leff 2004), we hypothesized that theirpresence on the plant hosts will be variably influenced bythe nutritional and physical characteristics of the individualplant hosts. Also, the occurrences of the bacterial taxa enum-erated could also be potentially dictated by various site-specific environmental and hydrodynamic factors within the2 study sites.

Materials and methods

Selected plant speciesIn this study, MA and CP were both selected as the 2

plants native to Michigan (USDA 2009), while GM, consid-ered an aggressive invader in deciduous forests throughoutNorth America (e.g., Nuzzo 2000), was selected as the inva-sive species. The potential phytotoxic influences attributed tovarious defensive compounds produced by these plants arewell documented (Camm et al. 1976; Berenbaum and Feeny1981; Moraes et al. 2000; Bedir et al. 2006). MA is a spring-time perennial wildflower found in deciduous forests ofNorth America (USDA 2009) with leaves containing podo-phyllotoxin, a precursor to several anti-cancer drugs (Moraeset al. 2000; Bedir et al. 2006). CP is a plant known to betoxic to humans and several other organisms (USDA 2009)and contains furanocoumarins that are generally used asstrong defensive compounds (Berenbaum and Feeny 1981).Upon direct contact with skin, furanocoumarins cause theskin to blister and discolor in the presence of light (McCloudet al. 1992), and this phytotoxicity is relatively commonwithin the genus Heracleum (Camm et al. 1976). In contrast,GM contains various allelochemicals, including flavonoids,glycosides, and glucosinolate, used in suppressing growth ofcompetitors in the environment (Cipollini 2002). Generally,these plants, while widely distributed, mostly occur in moist,shaded soils of river floodplains, edges of woods and trails,as well as forest openings (USDA 2009), from where theycan potentially influence the organic carbon dynamics in ad-jacent aquatic environments.

Study sitesThe study was conducted at separate and contrasting sites

within 2 freshwater environments in Michigan, USA (Ola-pade and Weage 2010; Olapade et al. 2011). The first studysite was at Albion (42.242550N, 84.735417W), within theKalamazoo River, with an extensive watershed along thesouthwestern portions of the Lower Peninsula of Michiganthat connects with Lake Michigan. The second study sitewas within the river reaches of Pierce Cedar Creek, a smalllowland stream surrounded by black muck soil types in Hast-ings, central Michigan (42.536166N, 85.301111W). Whilethe study site located along the north branch of the Kalama-zoo River is open and fast flowing, with an average dischargerate of 833 ft3/s (1 ft3/s = 28.316 85 dm3/s), in contrast, thePierce Cedar Creek site, is canopied and slow moving atabout 167 ft3/s (USGS 2010).

Field studyTriplicate water and leaf pack samples were aseptically

collected at the study sites as previously described (Olapade

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et al. 2011). Briefly, plant leaves were aseptically collected 4separate times at the White House Nature Center within thepremises of Albion College starting from mid-April throughmid-June in 2009. The leaves were then prepared as leafpacks (about 2 g wet mass) and bound together in triplicatesand immediately anchored within the riffle of the 2 riverreaches with plastic tent stakes as previously described byMcNamara and Leff (2004). The leaf packs were retrievedfrom the sites after 1 week of incubation and returned to thelaboratory on ice for preservation and subsequent analyses.Triplicate leaf discs (1 cm in diameter) and water sampleswere removed and preserved at each study site during the 4sampling dates, and then preserved in 8% (m/v) paraformal-dehyde and 1× phosphate-buffered saline solution for bacte-rial enumeration. Sodium pyrophosphate (0.1%) was lateradded to each preserved sample to aid in the effective detach-ment of attached bacteria, followed by sonication (Branson2210 Sonicator; Danbury, Connecticut) for approximately5 min (McNamara and Leff 2004). The YSI model 556 MPSmultiparameter-probe systems (YSI Incorporated, USA) wasused to measure various environmental variables at each lo-cation during the study period. Also, we applied an ion chro-matography method at the Dow Analytical Laboratory,Albion, USA, to quantify the presence of various anions (seeTable 1) in the samples using conductivity detectors thatwere standardized according to the manufacturer (Accustan-dard, Inc., New Haven, Connecticut).

Microcosm experimentLeaf packs of the plants were also employed for micro-

cosm study under controlled laboratory conditions to limitseveral confounding factors that are associated with variablehydrodynamic conditions at these 2 study sites. Briefly,

freshly collected leaves were incubated in triplicates for1 week in artificial stream water (containing (per litre)12 mg NaHCO3, 7.5 mg CaSO4·2H2O, 7.7 mg MgSO4,0.5 mg KCl, 10 mg CaCO3, pH 6.4) (APHA 1998) at roomtemperature and in the dark in sterile jars on a 6-paddlemodel PB-700 Jar tester (Phipps and Bird, Richmond Vir-ginia, USA). Subsequently, triplicate subsamples of leaf discsand water were removed and preserved as already describedabove every 24 h for the next 7 days for enumeration ofboth epiphytic and planktonic bacterial populations. We re-peated the microcosm experiment 3 separate times withfreshly collected leaf samples to ensure consistency and re-producibility of obtained results.

Bacterial enumerationTotal bacterial numbers in the preserved samples were de-

termined according to Porter and Feig (1980), while FISHwas used to determine the abundances of selected bacterialphylogenetic taxa (see Table 2) that are commonly found infreshwater environments as previously described in Manz etal. (1992) and Olapade et al. (2006). We concentrated bacte-rial cells in the preserved samples onto 0.2 µm pore size ano-disc or polycarbonate filters (Whatman, Maidstone, UK),rinsed with deionized water, and then treated with 1 mL of0.1% Nonidet P-40 (Sigma Aldrich, St. Louis, Missouri). A40 µL volume of Texas red-labeled probe (Sigma Genosys,The Woodlands, Texas; 5 ng/µL final concentration) dis-solved in hybridization buffer (6× standard saline citrate,0.02 mol/L TRIZMA base at pH 7.0, 0.1% sodium dodecylsulfate, 0.01% polyadenylic acid, and 30% formamide) wasadded to the filters before incubating for 4 h at the appropri-ate temperature (Table 2). After the incubation, filters werewashed twice with 400 µL of wash buffer (0.9 mol/L NaCl,

Table 2. Oligonucleotide sequences, target and hybridization conditions for probes used in this study.

Probe Target Taxon Sequence (5′–3′)Hybridizingtemp. (°C) Reference

EUB338 16S rRNA gene Domain Bacteria GCTGCCTCCCGTAGGAGT 48 Amann et al. 1990BET42a 23S rRNA gene Betaproteobacteria GCCTTCCACTTCGTTT 50 Manz et al. 1992GAM42a 16S rRNA gene Gammaproteobacteria GCCTTCCCACATCGTTT 57 Manz et al. 1992CF319a 16S rRNA gene Cytophaga–Flavobacterium TGGTCCGTGTCTCAGTAC 52 Amann et al. 1995HGC69a 23S rRNA gene Gram-positive high-GC bacteria TATAGTTACCACCGCCGT 48 Roller et al. 1994PLA886 16S rRNA gene Planctomycetes GCCTTGCGACCATACTCC 56 Neef et al. 1998ECO1482 16S rRNA gene Escherichia coli TACGACTTCACCCCAGTC 46 Fuchs et al. 2001ENF343 23S rRNA gene Enterococcus faecalis GGTGTTGTTAGCATTTCG 48 Beimfohr et al. 1993

Table 1. Environmental variables measured at the 2 river sites during the study period.

Date SiteTemp.(°C) pH

Cond.(mS/cm) ORP

Iron(mg/L)

Chlorine(mg/L)

NO3(mg/L)

PO4(mg/L)

SO4(mg/L)

15 April PCC 15.70 8.64 0.66 –34.20 0.03 0.04 1.37 0.33 40.30KR 14.30 8.01 0.48 –7.70 0.07 0.03 0.08 0.25 9.33

28 May PCC 17.40 8.27 0.73 –12.70 0.05 0.02 1.37 0.23 36.70KR 17.70 7.67 0.50 –7.10 0.10 0.06 1.07 0.13 9.67

5 June PCC 15.30 7.89 0.80 1.80 0.08 0.02 0.93 0.26 37.70KR 16.70 8.44 0.51 10.70 0.34 0.02 0.80 0.18 8.67

12 June PCC 14.80 8.01 0.68 15.70 0.24 0.01 1.17 0.72 32.30KR 15.80 8.22 0.52 1.67 0.17 0.03 1.17 0.31 8.30

Note: PCC, Pierce Cedar Creek; KR, Kalamazoo River; Temp., temperature; Cond., conductivity; ORP, oxidation–reduction potential; NO3,nitrate; PO4, phosphate; SO4, sulfate. Values are means of 3 replicates.

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0.02 mol/L Tris, pH 7, 0.1% sodium dodecyl sulfate), incu-bated with 80 µL of wash buffer for 10 min at the hybridiza-tion temperature, rinsed twice with 400 µL of steriledeionized water, and mounted on glass slides with immersionoil; hybridized cells were then enumerated using the epifluor-escence microscope by counting between 300 and 500 fieldson triplicate slides.

Statistical analysisSPSS for Windows (version 15, SPSS Inc., Chicago, Illi-

nois) was used in this study to perform all statistical analyseson samples that were collected in triplicates for each date and

site. Specifically, we utilized the Student’s t tests, ANOVA,and multivariate analyses to determine differences in boththe planktonic and epiphytic bacterial populations followedby the multiple range (i.e., Student–Newman–Keuls) test.Statistical significance was set at p ≤ 0.05.

Results

Environmental variablesOn average, most of the environmental variables measured

were similar between the 2 freshwater sites, except for con-centrations of phosphate (p < 0.05), sulfate (p < 0.0001),

Fig. 1. Box-plot representation of major bacterial phylogenetic taxa (as enumerated by fluorescence in situ hybridization) in the bacterio-plankton assemblages in Pierce Cedar Creek (A) and Kalamazoo River (B) during the study period. The bar in the middle of each box repre-sents the median, while the lower and upper hinges represent the 25th and 75th percentiles, respectively. EUB338, domain Bacteria; BET42a,Betaproteobacteria; GAM42a, Gammaproteobacteria; CF319a, Cytophaga–Flavobacterium cluster; HGC69a, high-GC Gram-positive bac-teria; PLA886, Planctomycetes; ENF343, Enterococcus faecalis; ECO1482, Escherichia coli. (N = 12 per site (i.e., 3 replicates × 4 samplingdate)).

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and conductivity (p < 0.05). Although, significant temporalvariations were recorded for all the parameters except foroxidation–reduction potential (i.e., ORP) values (p > 0.05)at each site (Table 1).

Bacterial abundanceThe numerical occurrences of the various bacterial taxa ex-

amined within the bacterioplankton communities in bothfreshwater sites varied during the study period (Fig. 1). Spe-cifically, while the numbers of cells that hybridized with theEUB338, CF319a, ENF343, and ECO1482 probes were sim-ilar (p > 0.05), in contrast, the abundance of cells that weredetected by BET42a, HGC69a, GAM42a, and PLA886probes differed significantly between the 2 sites (p < 0.05).Total bacterial counts as determined by DAPI staining dif-

fered significantly (Table 3) within the epiphytic assemblageson the 3 plant species within each site examined (F = 7.45,df = 2, p = 0.008) as well as between the 2 study sites (F =6.40, df = 1, p = 0.026). Specifically, total bacterial num-bers, while similar between CP and MA (p = 0.753), weresignificantly higher on GM than on both CP (p = 0.009) andMA (p = 0.032) plants. When individual bacterial taxa wereexamined, the numbers of cells that hybridized with most ofthe probes were generally similar on the 3 plants between the2 sites, with the exception of bacterial members belonging tothe Betaproteobacteria and Gammaproteobacteria.The abundance of Betaproteobacteria, while similar on the

plants incubated within the same river reaches (F = 0.32,df = 2, p = 0.734), differed significantly between the 2 sites(F = 30.80, df = 1, p < 0.001). Conversely, the numbers ofGammaproteobacteria, while significantly different (F =4.81, df = 2, p = 0.030) between the plant assemblages incu-bated at the same site, were similar on plants between thesites (F = 0.03, df = 1, p = 0.861). Comparatively, althoughGammaproteobacteria numbers were similar between MAand CP (p = 0.873) and between MA and GM (p = 0.079),the cell numbers recorded on GM were found to be signifi-cantly higher than those on CP (p = 0.033).The percent representation of total bacterial numbers (as

determined by DAPI staining) of each of the bacterial taxa

examined within the plant-associated assemblages are as pre-sented in Fig. 2. The percentage of total bacterial numbers byall the bacterial taxa examined on PCC were, in general,highest and similar (p ≥ 0.05) on both cow parsnip andmayapple and lowest on garlic mustard. For instance, the per-centage of total bacterial counts of Betaproteobacteria werehighest on both MA (15.4%) and CP (12.4%) compared with6.5% on GM (p < 0.05) at this river site. Conversely, wefound the percentage of total bacterial numbers recorded formost of the taxa examined to be relatively similar within theassemblages on the 3 plants developed at the KR site, exceptfor the occurrence of members the Gram-positive bacteriawith high-GC contents, which was lower on MA (5.7%) andGM (8.8%) compared with the 12.4% recorded on CP. Col-lectively, the 5 bacterial taxa enumerated in this study ac-counted for about 50%, 55%, and 62% of total bacterialnumbers on the epiphytic assemblages developed within theKR reaches on MA, GM and CP, respectively.When the occurrences of the 2 bacterial species (i.e., Es-

cherichia coli and Enterococcus faecalis) considered to be ofsignificant public health importance were examined withinthe developed epiphytic assemblages using the ECO1482and ENF343 probes, respectively, their percentage of totalbacterial counts were found to be similar on the 3 plants be-tween the 2 freshwater sites. Although in general, the occur-rences of E. coli cells were much higher on the plants thanthose recorded for E. faecalis at both study sites (Figs. 3Aand 3B).The numbers of DAPI-stained cells in the bacterioplankton

populations followed a similar trend in the 3 microcosm con-ditions examined on the 3 plants (p > 0.05, Fig. 4A). Gener-ally, the numbers of DAPI-stained cells increasedprogressively from the beginning of the experiment throughday 6 within the bacterioplankton communities associatedwith the 3 plants (CP: F = 8.87, p < 0.01; MA: F = 9.38,p = 0.01; and GM: F = 11.51, p < 0.006). Similarly, totalbacterial numbers within the epiphytic assemblages followedthe same trend at least through day 3 on the plants (p >0.05), although there was a sudden surge in the numbers onCP by the 4th day compared with the other 2 plants. Overall,

Table 3. Statistical values (based on multivariate analysis) on the abundance of majorbacterial taxa on cow parsnip, mayapple, and garlic mustard incubated at the 2 freshwatersites (i.e., Pierce Cedar Creek and Kalamazoo River).

Plants (df = 2) Sites (df = 1) Plant × site (df = 2)

Bacterialtaxon F p F p F pDAPI 7.45 0.008* 6.40 0.026* 4.13 0.043*EUB338 0.02 0.978 0.03 0.873 0.08 0.927BET42a 0.32 0.734 30.80 <0.001* 0.47 0.639HGC69a 1.78 0.210 2.14 0.169 12.99 0.001*CF319a 2.33 0.140 0.00 0.996 1.24 0.323GAM42a 4.81 0.030* 0.03 0.861 0.06 0.941PLA886 0.66 0.534 0.29 0.598 0.66 0.534ECO1482 1.24 0.324 4.32 0.060 0.03 0.974ENF343 0.10 0.907 0.00 0.974 1.88 0.195

Note: F, F statistic; df, degrees of freedom; DAPI, total bacteria; EUB338, domain Bacteria;BET42a, Betaproteobacteria; HGC69a, high-GC Gram-positive bacteria; CF319a, Cytophaga–Flavobacterium cluster; GAM42a, Gammaproteobacteria; PLA886, Planctomycetes; ECO1482, Es-cherichia coli; ENF343, Enterococcus faecalis. Within a column, values with an asterisk indicate asignificant difference at p ≤ 0.05.

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Fig. 2. Percent representation of total bacteria numbers (as determined by DAPI staining) by major bacterial taxa examined within the epi-phytic bacterial assemblages on leaves of cow parsnip (CP), mayapple (MA), and garlic mustard (GM) in Pierce Cedar Creek (A) and Kala-mazoo River (B) during the study period. PLA, Planctomycetes; Gamma, Gammaproteobacteria; CF, Cytophaga–Flavobacterium; HighGC,high-GC Gram-positive bacteria; Beta, Betaproteobacteria.

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while changes in total numbers between the days were simi-lar in the assemblages on CP (F = 4.06, p = 0.07) and MA(F = 5.15, p > 0.05), the counts differed significantly on GM(F = 7.25, p = 0.02) through the 6th day of the microcosmexperiment (Fig. 4B).

Discussion

In general, the results from this study revealed differencesin the occurrences of the various plant-associated bacterialpopulations examined between the 2 freshwater environ-ments. Although the 3 plants examined appeared to havemostly supported as well as stimulated bacterial colonizationand development within the 2 freshwater environments, thetotal bacterial numbers were more numerically dominant onthe plants incubated at Pierce Cedar Creek than those withinthe reaches of the Kalamazoo River, with the exception of to-tal numbers recorded on the invasive (GM) plant. These nu-merical differences in bacterial colonization on the plantscould be attributable to combinations of several site-specificenvironmental and hydrodynamic conditions between thesites. Additionally, variations in several plant propertiescoupled with differences in the nutritional and physiologicalcharacteristics of the individual bacterial taxa enumeratedmay have also collectively dictated the structural and compo-sitional profiles within the plant-associated assemblages. Forinstance, the 2 freshwater sites were primarily selected be-cause of differences in most of their environmental and

watershed characteristics, including discharge rates, canopycoverage and size (e.g., Olapade and Weage 2010; Olapadeet al. 2011). While PCC is a relatively small lowland creek,surrounded by black muck soil types, and is slow flowing,KR is fast flowing and located within an extensive volumi-nous watershed. Therefore, differences in several in-streamand watershed characteristics including water flow velocityand erosional pressure that differed between these 2 fresh-water sites could have probably influenced bacterial coloniza-tion and development on the plants during the study period(e.g., McNamara and Leff 2004).Additionally, differences in leaf properties could have also

influenced associated bacterial community structure and com-position. The very large and deeply lobed leaves of both CPand MA, compared with the stalked, triangular to heart-shaped, coarsely-toothed-margined leaves of GM, probablycreated variations in surface area required for microbial colo-nization and may have also influenced the rate of nutrient re-lease within the river reaches. Also, the 3 plants are known toproduce several defensive chemicals. CP and MA produce fur-anocoumarins and podophyllotoxin, respectively (Berenbaumand Feeny 1981; Moraes et al. 2000; Bedir et al. 2006),and GM produces various allelochemicals, including flavo-noids, glycosides, and glucosinolate, which have been effec-tively employed to suppress microbial growth in theenvironment (Cipollini 2002).FISH results revealed that bacterial members belonging to

the Cytophaga–Flavobacterium and Gammaproteobacteriaconstituted a large proportion of total bacterial numbers, ac-counting for more than 50% of the leaf-associated popula-tions on both MA and CP at the PCC site. Conversely, atthe KR site, members of Betaproteobacteria and Gammapro-teobacteria, and to a lesser extent the Cytophaga–Flavobacterium, accounted for more than half the total bacte-rial populations on the 3 plants. The relatively high occur-rences of these 3 taxa within the assemblages are notsurprising, given their high propensity for similar organicmatter sources in aquatic systems (Eiler et al. 2003). Simi-larly, clone libraries constructed based on 16S rRNA sequen-ces were found to be also dominated numerically bymembers of these bacterial taxa at the PCC site (Olapade etal. 2011), again further validating previous suggestions re-garding their global distribution and ubiquity in freshwaterbiofilm assemblages (e.g., Brümmer et al. 2000; Olapadeand Leff 2004).Specifically, the population size of E. coli cells accounted

for between 60% and 100% (at the PCC site) and only be-tween 0.46% and 0.80% (at the KR site) of Gammaproteo-bacteria numbers on the three plants. In contrast,occurrences of E. faecalis cells were relatively low and onlymade up smaller proportions of the Gammaproteobacterianumbers within the plant-associated assemblages at the 2sites. This particular result is probably an accurate reflectionof indigenous bacterial population dynamics at the 2 sitesand further validates past studies that have also reported nu-tritional support and sheltering of fecal indicator bacterialpopulations by various aquatic plants (Whitman et al. 2003,2005; McNamara and Leff 2004; Olapade et al. 2006;Olapade and Weage 2010). In general, this study suggeststhat the 3 plants examined mostly enhanced rather than sup-pressed bacterial colonization and development on their sur-

Fig. 3. Abundance of Enterococcus faecalis (A) and Escherichiacoli (B) within the plant-associated assemblages in Pierce CedarCreek (PCC) and Kalamazoo River (KR) during the study period.

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faces while resident in the 2 freshwater systems used for thestudy. More importantly, the overall results indicated thatcombinations of several variables and complex factors, in-cluding plant characteristics, bacterial nutritional properties,and site-specific hydrodynamic conditions, probably collec-tively influenced the development of the plant-associated as-semblages found within the 2 freshwater environments. Theresults from this study further validate the increasingly prev-alent paradigm that has suggested that epiphytic bacterial as-semblages are probably under the influences of and probablysubsequently respond to multiple variables and conditions inaquatic milieus.

AcknowledgementsThis study was supported partly by grants from the foun-

dation for undergraduate research and creative activities(FURSCA, Albion College) and Tri beta (BBB) society toKG and by the Albion College Hewlett-Mellon faculty devel-opment (FDC) fund to OAO. We thank Erin Goldman, SheilaLyons-Sobaski, Lori Duff, Dave Carey, Freyja Davis, andKurt Hellman for their various contributions and support dur-ing the study.

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