Journal of Experimental Marine Biology and Ecology 291 (2003) 199 218 www.elsevier.

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Effects of light reduction on growth of the submerged macrophyte Vallisneria americana and the community of root-associated $ heterotrophic bacteria
Janis C. Kurtz, Diane F. Yates, John M. Macauley, Robert L. Quarles, Fred J. Genthner, Cynthia A. Chancy, Richard Devereux *
National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, U.S. Environmental Protection Agency, 1 Sabine Island Drive, Gulf Breeze, FL 32561, USA Received 22 August 2002; received in revised form 5 December 2002; accepted 25 February 2003

Abstract A shading experiment was conducted over a growing season to measure the effects of light reduction on Vallisneria americana in Perdido Bay on the Florida Alabama border and to determine the response of heterotrophic bacteria in the rhizosphere. Plants subjected to 92% light reduction showed the most pronounced effects in chlorophyll a concentration, above- and below-ground biomass, and leaf dimensions. These results further suggested that the V. americana life cycle, as exhibited in temperate waters, was impaired. Heterotrophic bacteria were enumerated and identified (i) from the roots and sediments of fully illuminated plants and from unvegetated sediments at three intervals and (ii) from the roots of plants that have been subjected to 92% light reduction for 3 months. Up to two orders of magnitude greater numbers of bacteria were enumerated from root samples than sediment samples on a dry weight basis. Bacteria enumerated from the roots of plants subjected to light reduction (1.3 F 1.1 108 CFU g 1) were significantly higher than numbers of bacteria enumerated from the roots of fully illuminated plants (4.8 F 1.8 107 g 1 in the summer) or sediment samples (1.4 F 0.03 106 g 1). This suggests the roots of seagrasses stressed by light reduction provided more nutrients for bacterial growth. Higher percentages of Gram-negative bacteria were isolated from roots (up to 85% in the fall) than sediments (0 15%). Examination of isolates for traits characteristic of rhizosphere bacteria (siderophore production, formation of the phytohormone indole-3-acetic acid, and antifungal activity) did not show a clear distinction between

Contribution number 1169 from the NHEERL Gulf Ecology Division. * Corresponding author. Tel.: +1-850-934-9346; fax: +1-850-934-2401. E-mail address: devereux.richard@epa.gov (R. Devereux).

0022-0981/03/$ - see front matter. Published by Elsevier Science B.V. doi:10.1016/S0022-0981(03)00120-5

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root-associated and sediment isolates. Taxonomic identifications of root-associated bacteria based on MIDI analysis of fatty acid methyl esters were consistent with bacteria known to be associated with other plants or found at oxic anoxic interfaces. In addition, the bacterial identifications showed most species were associated with only roots or only sediments. These results support studies suggesting seagrass rhizospheres harbor distinct bacterial communities. Published by Elsevier Science B.V.
Keywords: Seagrass; Light reduction; Rhizosphere; Bacteria

1. Introduction Seagrasses provide essential habitats for many estuarine species, stabilize sediments, and support active microbial communities vital to ecosystem nutrient cycles (Herbert, 1999; de Wit et al., 2001). Widespread loss of seagrasses has been documented throughout the Gulf of Mexico and other ecosystems around the world (Dennison et al., 1993; Livingston et al., 1998; Donnelly and Herbert, 1999; Seddon et al., 2000). Light reduction linked to degraded water quality is considered a primary factor leading to the decline of seagrasses (Dennison et al., 1993), yet other factors such as salinity, temperature, sediment characteristics, water-borne toxicants, and wave energy also influence seagrass coverage (Livingston et al., 1998; Azzoni et al., 2001; Koch, 2001). Hemminga (1998) characterized light reduction on seagrasses as resulting in a cascade of effects to above-ground and below-ground biomass. Reduced light decreases photosynthesis which shifts the balance in CO2 metabolism from fixation and oxygen synthesis in the leaves toward respiration and oxygen consumption in the roots. Less oxygen and sucrose subsequently are available to the roots and rhizomes. The diminished translocation of oxygen below ground allows accumulation of sulfides which may reach toxic levels (Terrados et al., 1999; Erskine and Koch, 2000; Koch and Erskine, 2001; Holmer and Laursen, 2002). Although seagrass roots can enter nightly periods of anaerobiosis (Smith et al., 1984a, 1988; Kraemer and Alberte, 1995), sustained conditions of light reduction can prolong anaerobic root metabolism which leads to the depletion of root sucrose reserves. Carbon is lost from the roots as fermentation products. Eventually, the deleterious effects of light reduction on root-rhizome activities contribute to plant death. Seagrass roots are important for the acquisition of plant nutrients and the microbial communities associated with the roots are important for making nutrients available (Barko et al., 1991; Waisel and Agami, 1996; Hemminga, 1998; Donnelly and Herbert, 1999). Seagrass roots release organic carbon which in some instances has been directly linked to bacterial production (Moriarty et al., 1986; Pollard and Moriarty, 1991; Holmer et al., 2001). Most studies on bacterial-seagrass root interactions have focused on nitrogen cycling. A significant portion of seagrass nitrogen requirements may be fulfilled through nitrogen fixation in the rhizosphere, especially by sulfate-reducing bacteria (Patriquin and Knowles, 1972; Capone, 1982; Capone and Budin, 1982; Capone and Taylor, 1980; Smith and Hayasaka, 1982; Welsh et al., 1996; Welsh, 2000; Nielsen et al., 2001). Deamination of amino acids by rhizosphere bacteria may also provide seagrasses with nitrogen (Smith

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et al., 1984a,b). In addition, bacteria and mycorrhizal fungi can be important for providing seagrasses with phosphorous and other nutrients (Craven and Hayasaka, 1982; Barko et al., 1991; Wigand and Stevenson, 1997). Plant root exudates can selectively stimulate and inhibit microbial growth and thus foster the establishment of specific microbial communities (Klein et al., 1990). Bacterial rhizosphere communities can vary in composition according to plant species, growth stage, root exudates, and available carbon source (deBrito Alvarez et al., 1995; Rooney Varga et al., 1997; Hines et al., 1999; Marschner et al., 2001). Rhizoplane bacteria obtained from the roots of Zostera marina demonstrated chemotaxis toward root exudates, in particular toward amino acids (Wood and Hayasaka, 1981). Rhizosphere bacteria in more thoroughly studied terrestrial and agricultural systems have been shown to supply nutrients, protect against colonization by pathogens, promote plant growth through the production of phytohormones, and influence root physiology (Klein et al., 1990; Costacurta and Vanderleyden, 1995; Glick, 1995; Andrews and Harris, 2000; Whipps, 2001). Considering that light limitation affects seagrass root metabolism and could alter the composition of root exudates and translocation of oxygen to the sediments, it is possible that light limitation could also impact the rhizosphere bacterial community. Alteration of seagrass rhizosphere bacterial communities could be an addition to the cascading effects of light reduction, limiting the availability of nutrients and possibly rendering plants susceptible to opportunistic pathogens. However, beyond nitrogen fixation, only a few studies have addressed whether there are specific bacterial-seagrass root associations and the possibility that such interactions are important for seagrass growth and maintenance (Blotnick et al., 1980; Schmidt and Hayasaka, 1985; Glazebrook et al., 1996). The objectives of our study were to measure the effects of light reduction on the wild celery Vallisneria americana Michaux and to compare identifications and phenotypic traits among heterotrophic bacteria isolated from roots of fully illuminated and light-limited V. americana and from vegetated and unvegetated sediments. Our results indicated the rhizosphere bacteria responded to plant light limitation and provide further evidence that seagrass rhizospheres harbor distinct bacterial communities.

2. Materials and methods 2.1. Experimental site and design Upper Perdido Bay, on the Florida Alabama border, contains lush V. americana beds that are under tidal influence from the Gulf of Mexico. At the end of March, 1998, a V. americana bed on the southeastern shore approximately 15 3 m with a depth at mean high tide of 1.5 m was selected for the study. The site receives full sunlight throughout the day. The latitude and longitude coordinates, derived with a GPS unit without differential corrections, were 30.25.181N and 87.24.360W in decimal minutes. Light exclusion shades were constructed using 1.0 m2 PVC frames affixed with commercially available shade cloth at densities to reduce the amount of available total ambient light by 79% and 92%. PVC posts were driven into the sediment to mark 12

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experimental plots and provide support for the shade frames. Treatments were randomly assigned to each of the 12 plots: four replicate plots were used for each light exclusion treatment and four were left uncovered as controls. Shade frames were attached to the posts of the assigned plots at a height 1.0 m above the sediment. A side curtain (1.0 m 20 cm) of the same density cloth was attached to the southern facing side of the frame to control the angular entry of light as the sun moved across the southern horizon. A spade was driven into the sediment around the perimeter of each plot to sever rhizome connections of shoots in the plot with those outside the area. Shade screens were inspected and cleaned weekly in place with a clean laboratory brush; heavy fouling did not occur during the experiment. A LI-193 SA spherical light sensor (Licor, Lincoln, NE) was placed at the height of the canopy in each of two replicates of the treatment and control plots. Another sensor was placed on a post out of the water to monitor ambient surface light. Light energy was measured every three seconds during daylight and a mean value was recorded every half hour. The sensors were adjusted on a weekly basis to remain at the height of the canopy as the length of the plant leaves increased. Ambient water temperature and salinity measurements were also recorded each week. The experiment was planned to continue into the fall. Unfortunately, the shade frames were destroyed by Hurricane Georges at the end of September. 2.2. Plant measurements Each experimental plot was divided into 50 10 cm2 grids centered beneath the shade frame, and each grid was randomly selected to be sampled only once during the study. Above- and below-ground plant biomass, leaf length and width, leaf chlorophyll a (chl a) concentration, shoot and leaf density, and the carbon and nitrogen content of aboveand below-ground portions were measured in March and July. A 7.6-cm diameter coring device was used to collect the samples to a depth of 10 cm. Plant material rinsed free of sediment was sealed in plastic bags and stored on ice for transportation to the laboratory where the contents of each bag were separated into above- and below-ground portions, and the total number of leaves and shoots were determined. Thirty leaves were randomly selected and their lengths and widths were measured to the nearest millimeter. The leaves were then gently scraped with a razor blade to remove epiphytes. Chlorophyll was extracted from 10 of the 30 leaves and chl a concentrations were determined spectrophotometrically as described by Macauley et al. (1988). The remaining leaves and below-ground biomass were dried at 110 jC for 48 h and weighed. The dried material was then ground to a fine powder in a Tekmar analytical mill and portions weighed for carbon and nitrogen determination using a Carlo Erba NA1500 C:H:N analyzer. Results for plant measurements are reported on a dry weight basis. Leaf length, width, and chlorophyll content were measured monthly during most of the study. For these measurements, a pair of scissors was used to sample the above-ground portion within a grid and the below-ground portion was left undisturbed. Dunnetts t-test was used to compare treatments to the controls at each month of the study. Significant differences were determined at the a = 0.05 level.

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2.3. Enumeration and isolation of bacteria Samples were collected from areas receiving full illumination and from a nearby unvegetated area in April, July, and November 1998. Sediment cores were obtained in 60 cc syringe barrels and placed into sterilized plastic centrifuge bottles. Roots were obtained from 7.6-cm diameter cores and rinsed free of sediments upon collection using water from the site and placed into plastic bags. The samples were brought to the laboratory on ice and processed within 2 h. Samples were also collected in July from a plot subjected to 92% light reduction. Roots and stolons were separated from shoots and surrounding sediment and rinsed free of loose sediment and organic material with sterile phosphate buffered saline (PBS; 100 mM sodium phosphate pH 7.0) amended with NaCl to adjust the buffers salt concentration to the salinity of the site at time of sampling. Root samples were obtained by trimming the stolons to within 3.5 mm of the root attachments. The percent dry weights for root, sediment, and leaf samples were determined by drying a known amount to a constant weight at 60 jC in a vacuum oven. Rinsed roots (1 g wet weight) and sediment samples (1 g wet weight) were homogenized in a sterilized blender jar at low speed in salinity adjusted PBS, serially diluted in the same buffer, and spread onto 1/10 strength Trypticase Soy Agar (TSA) (Difco Laboratories; Detroit, MI) containing 0.5% NaCl. The plates were incubated at 30 jC for 7 days. Total colony-forming units (CFU) and colony types, as observed with a dissecting microscope, were enumerated on plates having between 30 and 300 colonies. Representative colonies from these plates, and colonies of differing morphology or pigmentation appearing on plates inoculated with lower dilutions, were selected and streaked onto fresh plates which were then incubated at 30 jC. This process was repeated until pure bacterial cultures were obtained. Isolates were stored frozen in 15% glycerol at 70 jC. 2.4. Bacterial identifications and distribution The Gram reaction (Difco) was determined with bacterial colonies obtained from 16- to 24-h-old plates and cell shape was recorded during microscopic observation. Oxidase (Oxidase Test Kit; bioMerieux Vitek; Hazelwood, MO), catalase (Loewen, 1984), and glucose fermentation tests (O/F-test; MacFadden, 1980) were conducted using standard procedures. Isolates were identified by analysis of their fatty acid profiles after growth on TSA for 24 h at 28 jC. Approximately 40 mg of bacteria were harvested from the plates and whole cell fatty acids were saponified, methylated, and extracted (1996 MIDI Manual, MIDI; Newark, DE). The extracts were analyzed on a Hewlett-Packard 5890 Series II gas chromatograph equipped with a flame ionization detector. Fatty acid methyl esters were identified and profiles generated using the Microbial Identification System Software (MIDI). Isolate profiles were compared to those of bacteria in the MIDI TSBA4.0 library (January, 1999 update) and a similarity index of greater than 0.3, with a separation between species of 0.1, was considered to provide a reliable identification.

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Isolates that could not be identified with the MIDI library were analyzed by 16S rDNA sequence comparison. DNA was extracted from overnight cultures grown on TSA by using the Wizard Genomic DNA Purification KIT (Promega; Madison, WI). Nearly fulllength 16S rDNAs were obtained by PCR using primers fD1 and rP2 (Weisberg et al., 1991). PCR products were purified with Microcon PCR Centrifugal Filter cartridges (Fisher Scientific; Norcross, GA). Sequences of purified PCR products were determined by the ICBR DNA Core Sequencing Laboratory at the University of Florida using previously described primers (Dorsch and Stackebrandt, 1992). 16S rRNA sequences of the most closely related known bacteria were retrieved from GenBank by performing Basic Local Alignment Search Tool (BLAST) search using the National Center for Biotechnology Information (NCBI) database. Sequences were aligned and similarities between sequences were obtained using the MegAlign program available with the DNASTAR package (DNASTAR; Madison, WI). 2.5. Determination of bacterial phenotypic traits Isolates were screened for traits common to rhizosphere bacteria. Siderophores are ironchelating compounds produced by bacteria that enable them to compete against other bacteria and fungi for available iron (Neilands, 1995). Isolates were tested for siderophore production by examining growth on indicator plates containing a complex of Chrome Azurol-S (Sigma, St. Louis, MO), iron (III), and hexadecyltrimethylammonium bromide (Sigma) (Schwyn and Neilands, 1987). A strain was considered positive for siderophores if an orange halo appeared around a colony indicating removal of iron from the complex and release of free dye. Indole-3-acetic acid (IAA) is a plant growth-promoting compound that can be produced by bacteria (Costacurta and Vanderleyden, 1995). To test for IAA production, isolates were grown at 28 jC for 3 days with shaking in LB broth amended with 5.0-mM tryptophan. 1.2 ml of each culture was centrifuged to remove the cells and 1.0 ml of the supernatant solution was mixed with 1.0 ml of Salkowski reagent (4.5 g FeC13 l 1, 10.8 M H2SO4) and incubated in the dark for 30 min (Glickman and Dessaux, 1995). Absorption spectra were recorded on a Hewlett Packard diode array spectrophotometer and absorption at 530 nm was compared to a standard curve prepared with IAA (Sigma) ranging from 0.4 to 200 Ag ml 1. Bacterial supernatant solutions causing a red to brown color and an increase in absorption at 530 nm were considered positive for IAA. Isolates were tested for the capacity to inhibit growth of Lindra marinera, (ATCC 18637), an estuarine ascomycete known to be associated with leaves Thalassia sp. (Meyers, 1969), or the plant-pathogenic fungus Pythium aphanidermatum (isolate 9021-6 kindly provided by J. Brantner, Univ. of Minnesota). Fungi were grown on BY+ (filtered, 33 ppt seawater with 0.1% yeast extract, 0.1% peptone, and 0.5% glucose) agar plates. Agar blocks (1 1 cm) were excised from established plates of fungi and placed onto the center of fresh BY+ plates. After mycelial growth reached a diameter of 2.5 3 cm (3 5 days), bacteria from eight liquid cultures were inoculated onto a plate by streaking from the perimeter of the plate into the zone of fungal growth. Plates were incubated at 25 jC for 3 7 days. A strain was considered positive when a clear zone of fungal inhibition was observed around the bacterial growth.

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3. Results 3.1. Plant measurements In situ light measurements showed that the shade coverings caused consistent 79% and 92% reductions in the amount of light available to the experimental plots throughout the study period. Ambient water temperatures ranged from 17.6 jC in April to 35.6 jC in July and to 23.3 jC in November. The pH varied from 6.17 to 8.96 during the study with a mean value of 7.24. The salinity at the site gradually increased from 0 ppt at initiation of the experiment in the spring to 4.3 ppt in July. The salinity spiked to 17.0 ppt in August and returned to around 5.0 ppt in November following the hurricane. Effects of light reduction on chl a leaf concentrations are shown in Fig. 1. Mean leaf chl a concentrations increased from April to May in both the control (from 3.8 to 7.0 mg chl a g 1) and 79% light reduction treatment (from 3.8 to 5.9 mg chl a g 1). The chl a concentration in the leaves from the 92% light reduction treatment did not show a significant increase for the same period. From May to July, chl a in the control leaves declined to spring values (4.0 mg chl a g 1). However, leaves from both light reduction treatments contained 6.1 mg chl a g 1 in July, a significantly (a = 0.05) higher concentration than in the July control leaves and in leaves at the beginning of the experiment. In July, both the above- and below-ground biomass values for the 79% and 92% light reduction treatments were significantly lower than that of the controls, but not from each other (Table 1). Carbon and nitrogen concentrations in below-ground biomass did not differ significantly from the controls. The mean length to width ratios of leaves from the 92% light reduction treatments were significantly lower than the control.

Fig. 1. Chlorophyll a concentrations in V. americana leaves measured over the course of the experiment.

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Table 1 Vallisneria americana measurements taken in July 1999 Treatment Dry weight biomass Above ground (g) Control 79% light reduction 92% light reduction 3.35 (1.36) 1.02* (0.49) 0.48* (0.21) Below ground (g) 1.47 (0.74) 0.75* (0.38) 0.67* (0.17) Above/ below 2.28 1.36* 0.72* Carbon (mg C mg 1) Above ground 0.33 (0.02) 0.32 (0.03) 0.30 (0.02) Below ground 0.20 (0.03) 0.24 (0.03) 0.26 (0.03) Nitrogen (mg N mg 1) Above ground 0.025 (0.001) 0.027 (0.002) 0.026 (0.002) Below ground 0.017 (0.004) 0.024 (0.004) 0.026 (0.002) Leaves Number 71 (29) 48 (24) 40 (23) Length (mm) 246.6 (57.3) 160.1 (32.1) 119.5 (26.8) Width (mm) 6.3 (0.9) 4.9 (0.5) 4.2 (1.1) Length/ width 39.6 (2.8) 33.1 (5.4) 27.8* (3.6)

Standard deviations are in parentheses and values significantly different from the control (a = 0.05) using Dunnetts test are indicated with an asterisk.

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3.2. Bacterial enumerations Numbers of bacteria, determined on the basis of CFU g 1 dry weight (roots or sediment), are presented in Table 2. CFU of bacteria obtained from roots were one to two orders of magnitude higher than those obtained from the sediment samples. CFU from roots of fully illuminated plants were from 1.9 F 0.4 107 to 4.8 F 1.8 107 g 1 whereas CFU from sediments ranged from 1.1 F 1.9 105 g 1 for the unvegetated sediment in July to 1.4 F 0.03 106 g 1 for the vegetated sediment in November. The highest numbers of bacteria were obtained in July from the roots of plants subjected to 92% light reduction (1.6 F 1.1 108 g 1) and are significantly higher than the numbers of bacteria obtained from the roots of fully illuminated plants (4.8 F 1.8 107 g 1) at the same time. Clear seasonal differences in bacterial numbers within sample type was limited to a significantly higher number of CFU obtained from the vegetated sediment in November (1.4 F 0.03 106 g 1) relative to that obtained during the previous April (1.4 F 0.1 105 g 1). Bacterial numbers obtained from the roots of fully illuminated plants showed a small, but significant decrease in November. There was also an apparent increase in bacteria in the vegetated sediment from April to July, but insufficient countable replicate plates due to overgrowth by spreading colonies were obtained in July to determine whether this increase was significant. 3.3. Isolate identifications One hundred thirty-seven bacterial isolates selected from plates used to determine CFU were identified. The majority of isolates obtained from sediment samples were Grampositive bacteria (Tables 3 and 4) and were identified primarily as Bacillus, Brevibacillus and Paenibacillus species. In contrast, an average of 57% (27/47) of all isolates obtained from root samples were Gram-negative bacteria. These were identified as species of Achromobacter, Aeromonas, Methlylobacterium, Phenylobacterium, Pseudomonas, and Vibrio. In addition to these isolates, bacteria obtained on plates inoculated with root samples at lower dilutions included Pantoea ananas, Aeromonas sobria, and Azospirillum spp. With the exception of Pseudomonas chlororaphis, which was isolated from both roots

Table 2 Numbersa of bacteria obtained from Vallisneria americana roots and sediment samples during 1998 Sample type Plant (roots) 95% Light excluded plant (roots) Vegetated sediment (control) Non-vegetated sediment
a b

April 4.4 ( F 0.5) 107 NDb 1.4 ( F 0.4) 105 3.5 ( F 0.1) 105

July 4.8 ( F 1.8) 107 1.6 ( F 1.1) 108 7.2 105c 1.1 ( F 1.9) 105d

November 1.9 ( F 0.4) 107 NDb 1.4 ( F 0.03) 106 7.2 ( F 3.6) 105d

CFU g 1 dry weight. Not determined. c Insufficient useable replicates for determining variance due to overgrowth. d Range.

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Table 3 Identification of rhizosphere bacteria isolated from Vallisneria americana roots and sediment during 1998 Sample Roots April, bacterial identification (N)a Aeromonas sobria (1) Bacillus cereus (2) Bacillus sp Bch1.b (1) Brevundimonas sp.b (1) Microbacterium sp.b (1) Pseudomonas chlororaphis (1) Pseudomonas sp.b (1) Sanguibacter suareziib (1) Roots from NDc 92% light Excluded plants July, bacterial identification (N)a Achromobacter sp.b (1) Bacillus pumilus (1) Enterococcus sp. (1) Methylobacterium organophilum (2) Pseudomonas alcaligenes (2) Pseudomonas balearica (4) Staphylococcus epidermidis/warneri (1) unidentified (1) November, bacterial identification (N)a Achromobacter sp.b (1) Bacillus cereus (1) Bacillus megaterium (1) Methylobacterium/ Paracoccus (NG) (1) Pseudomonas alcaligenes (1) Pseudomonas alcaligenes/ mendocina (6) Pseudomonas pseudoalcaligenesb (1) Pseudomonas sp. (1) Vibrio furnissii (1) NDc

Vegetated sediment

Non-vegetated sediment

Bacillus cereus (2) Bacillus lentimorbus (1) Bacillus marinus (1) Bacillus megaterium (3) Bacillus psychrophilus/ megaterium (1) Bacillus sp. (1) Bacillus GC group 22 (1) Brevibacillus brevis (1) Brevibacillus laterosporus (1) Marine Bacillusb (1) Paenibacillus glucanlyticus (1) Pseudomonas chlororaphis/ putida (1) Actinomadura yumaensis (1) Bacillus cereus (5) Bacillus licheniformis (1) Bacillus marinus (1) Bacillus megaterium (2) Bacillus pumilus (8) Bacillus sphaericus (1) Bacillus/brevibacillus (1)

Achromobacter sp.b (4) Bacillus cereus (1) Bacillus mycoides (1) Phenylobacterium immobile (1) Pseudomonas balearica (4) Sinorhizobium sp. (1) Bacillus flexus (1) Bacillus megaterium (1) Bacillus pumilus (2) Bacillus sphaericus (2) Curtobaterium/ Microbacterium (1) Marine Bacillusb (1) Paenibacillus polymyxa (2)

Bacillus Bacillus Bacillus Bacillus Bacillus

cereus (4) licheniformis (1) marinus (1) mycoides (1) sp. (2)

Bacillus sp.b (1) Brevibacillus brevis (1) Paenibacillus macerans (2) Stenotrophomonas maltophiliab (1)

Bacillus cereus (4) Bacillus lentimorbus (1) Bacillus megaterium (1) Bacillus pumilus (2) Bacillus sphaericus (3) Bacillus sp. (1) Brevibacillus sp. (1) Brevibacillus centrosporus (1)

Bacillus cereus (3) Bacillus marinus (1) Paenibacillus azotofixans (1)

J.C. Kurtz et al. / J. Exp. Mar. Biol. Ecol. 291 (2003) 199218 Table 3 (continued) Sample Non-vegetated sediment April, bacterial identification (N)a Bacillus sp. (1) Brevundimonas vesicularis (1) Frigoribacterium sp.b (1) Shewanella putrifaciens (1) Sphingomonas sp. (1) Stenotrophomonas maltophila (1) unidentified (1) July, bacterial identification (N)a Kocuria varians (1) Paenibacillus sp. (1) Pseudoaltermonas haloplanktis (2) November, bacterial identification (N)a

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Identifications are based on fatty acid analysis unless otherwise indicated. Only identifications of isolates from plates containing 30 300 colonies are reported. a (N) total number of isolates with same identification. b Identification by 16S rDNA sequence comparison. c (ND) Not determined.

and vegetated sediments in April, specific species of Gram-negative bacteria were found only associated with root samples or sediment samples. Gram-positive bacteria isolated from roots included some of the same Bacillus species obtained from sediment samples and, in addition, bacteria belonging to the genera Microbacterium, Sanguibacter, Enterococcus, and Staphylococcus that were obtained only from root samples.
Table 4 Percentages of isolated bacteria demonstrating a tested phenotypic trait Sample % Gram-negative (N Gram-negative/ N tested) % Siderophore positive (N positive/ N tested) 100 (9/9) 73 (11/15) 100 (22/22) 100 (7/7) 100 (7/7) 77 (7/9) 94 (15/16) 91 (10/11) 78 (7/9) % IAA (N positive/ N tested) % Antifungal vs. Lindra marinera (N positive/ N tested) 43 (3/7) 77 (10/13) 62 (13/21) 75 (6/8) 84 (5/6) 40 (4/10) 56 (9/16) 75 (9/12) 71 (5/7) % Antifungal vs. Pythium aphanidermatum (N positive/ N tested) 33 (2/6) 13 (2/15) 17 (4/23) 30 (3/10) 0 (0/7) 10 (1/10) 0 (0/16) 0 (0/12) 0 (0/9)

Roots, April Vegetated sediment, April Unvegetated sediment, April Roots, July Roots, 92% light reduction, July Vegetated sediment, July Unvegetated sediment, July Roots, November Vegetated sediment, November Unvegetated sediment November

45 (4/9) 7 (1/15) 15 (4/26) 31 (4/13) 67 (8/12) 0 (0/10) 11 (2/18) 85 (11/13) 0 (0/13)

88 (7/8) 64 (9/14) 76 (16/21) 71 (5/7) 100 (6/6) 100 (7/7) 94 (15/16) 84 (10/12) 100 (9/9)

0 (0/5)

75 (3/4)

100 (2/2)

67 (2/3)

0 (0/4)

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3.4. Phenotypic traits Isolates were tested for their ability to produce siderophores, form IAA and inhibit the growth of fungi (Table 4). Most of the bacteria tested were positive for siderophores. Ninety seven percent of all isolates (33/34) examined from roots and 95% of isolates (40/ 42) tested from unvegetated sediments produced siderophores in comparison to siderophore production by 76% of isolates (25/33) from vegetated sediment. Eighty-four percent of the bacteria tested (86/102) produced IAA, however there were no clear differences in the percentages between season or source of isolates. Sixty-four percent of the isolates tested (66/103) inhibited growth of L. marinera. There were no distinct differences in the percentages between season or source of isolates. In contrast, fewer of the bacterial isolates (11%; 12/111) inhibited growth of the plant-pathogenic fungus P. aphanidermatum and none of the isolates obtained in November inhibited growth of this fungus.

4. Discussion 4.1. Response of V. americana to light reduction The minimum light requirements of seagrasses range from about 4% to 30% of incident light (Duarte, 1991; Dennison et al., 1993). V. americana has been reported to maintain photosynthesis at depths having 0.5% of surface irradiation and grow well at depths receiving 10% of surface light (Korschgen and Green, 1988). In this study, V. americana grew in light reduced to 21% and 8% of the incident level although effects of light reduction on biomass were apparent at both levels. The salinity in upper Perdido Bay was 0 ppt in March and steadily increased to 4.26 ppt during July, but reached 17.0 ppt in August because of drought conditions. Twilley and Barko (1990) reported V. americana is capable of tolerating up to 12 ppt, while Kraemer et al. (1999) observed V. americana distribution to be limited by an upper tolerance of 15 ppt. Even though salinity in Perdido Bay exceeded levels considered tolerated by V. americana, the observed effects can be attributed to light reduction rather than changes in salinity, pH, conductivity, or other environmental factors since all fully illuminated and light-excluded plots at the experimental site were exposed to the same ambient conditions and plant measurements were taken when the salinity was normal. However, it is possible that the observed effects from light reduction were exacerbated by multiple stressors. Responses of V. americana to light reduction in Perdido Bay included decreased production of above- and below-ground biomass with a shortening and narrowing of leaves (Table 1). Similar results were described by Blanch et al. (1998) who studied the effects of light reduction due to turbidity on V. americana from the River Murray in South Australia over 120 days, a period comparable to that used in our experiment. The decreased growth response was attributed to depressed carbon assimilation (net assimilation rate) with lowered irradiance in accordance to a standard P I curve (Blanch et al., 1998). Changes in leaf morphology were shown to optimize exposure to light at the canopy surface as an adaptation to turbid conditions. In addition, Blanch et al. (1998)

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noted that V. americana in the River Murray conserved below-ground biomass relative to above-ground biomass, potentially to maximize leaf production when turbidity is alleviated. A similar response was seen in V. americana in Perdido Bay where leaf biomass in the 92% light reduction treatment was reduced sevenfold relative to the control while root biomass was reduced only by a half. Thus, our results confirm the findings of Blanch et al. (1998) and extend observations of effects from light reduction on V. americana to geographically separated populations. Light reduction may also effect the growth cycle of V. americana. First, control plants and plants exposed to 72% light reduction were observed in July to have female flowers, with the attached seed pods, that were absent in the 92% light reduction treatment. Development of reproductive tissue in greenhouse-grown V. americana was dependent on attaining a threshold dry weight (0.75 g) although a threshold rule was not strongly held in the field (Titus and Hoover, 1991). V. americana exposed to 92% light reduction apparently had not attained sufficient energy reserves to initiate development of reproductive tissues. Second, V. americana in more northern habitats, such as the Upper Mississippi River, form winter buds from stolons grown near the close of the growing season followed by disintegration of the remaining above- and below-ground tissues (Titus and Hoover, 1991). Dawes and Lawrence (1989) determined that the stolon of V. americana in central and southern Florida is not used for carbohydrate storage as the plants are not exposed to low temperatures. V. americana in Perdido Bay does not produce winter buds but does enter a state of reduced above-ground biomass (dormancy) with limited chlorophyll during the fall and winter. The decrease in chl a concentrations observed with control plants in July could be an indication the plants had initiated dormancy. Increasing chl a concentrations in seagrasses subjected to light reduction is recognized as a response to increase the photosynthetic efficiency of the leaves (Longstaff et al., 1999). However, retention of high chl a levels in July for V. americana subjected to light reduction suggests dormancy was delayed because sufficient carbon reserve or biomass had not been attained. 4.2. Enumeration of heterotrophic bacteria Plate counts of heterotrophic bacteria were used as an approximation of the relative numbers of bacteria between the different sample types and different periods over the growing season (Table 2). CFU of heterotrophic bacteria were higher with vegetated sediment samples than with unvegetated sediment samples. This was expected since bacterial activities in seagrass beds are high due to the increased availability of organic matter and nutrients (Pollard and Moriarty, 1991; Blackburn et al., 1994; Lopez et al., 1995, 1998; Hansen et al., 2000). Previous studies have also found higher numbers of bacteria and greater microbial biomass in seagrass bed sediments in comparison with unvegetated areas (Patriquin and Knowles, 1972; Donnelly and Herbert, 1999; Ku et sel al., 1999; Boschker et al., 2000; Holmer et al., 2001). On a dry weight basis, the highest numbers of bacteria were obtained from V. americana roots rather than sediments. These results indicate the roots of V. americana offer a habitat that supports greater bacterial growth than the surrounding sediment. Bacteria extensively colonize seagrass root-rhizome tissues and attain densities higher than

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in sediments (Smith et al., 1984a,b; Schmidt and Hayasaka, 1985; Blaabjerg and Finster, 1998; Glazebrook et al., 1996; Donnelly and Herbert, 1999; Kusel et al., 1999). However, total numbers of bacteria are probably higher in the sediments since the roots represent a smaller portion of below-ground mass on a unit volume basis (Blaabjerg and Finster, 1998). Since organic carbon released from seagrass roots can be utilized by bacteria (Moriarty et al., 1986; Holmer et al., 2001), rhizoplane and intercellular bacteria have immediate access to root exudates for growth (Smith et al., 1984a,b). As proposed, these bacteria could respond to changes in root physiology (Blotnick et al., 1980) as evidenced by the rapid increased activity of sediment bacteria when seagrasses or benthic algae commence photosynthesis (Moriarty and Pollard, 1982; Boschker et al., 2000; Welsh, 2000). Bacterial CFU obtained from roots of plants subjected to 92% light reduction were half an order of magnitude higher than those obtained from fully illuminated plants at any of the sampling times. This suggests more nutrients were available to bacteria on the roots of plants subjected to light reduction as would be expected in keeping with loss of carbon through the roots as an effect of light reduction on seagrasses, as outlined by Hemminga (1998). In the fall, bacterial CFU significantly increased in vegetated sediments whereas bacterial CFU in unvegetated sediments remained unchanged, and CFU of bacteria obtained from roots showed a decrease. This suggests input of organic matter to the vegetated sediments, but not the unvegetated sediments, possibly from decaying phytoplankton, epiphytes, or leaf litter trapped by the seagrasses (Koch, 2001). Stabile carbon isotope analysis has indicated bacteria in seagrass bed sediments located in a temperate climate utilized organic carbon derived from benthic algae rather than the seagrasses (Boschker et al., 2000). However, in a tropical setting, stable carbon isotope analysis indicated bacteria in seagrass bed sediments utilized organic carbon derived from the seagrasses (Holmer et al., 2001). The fact that bacteria enumerated from roots in the fall did not increase suggests there was no increased exudation of carbon from the roots, and that nutrients entering the sediments from detritus did not become available to the bacteria on the roots. This is supported by observations of Pereg et al. (1994) that different compartments of a Halophila stipulacea seagrass bed (roots-rhizomes, leaves, and sediments) contain distinct types of nitrogen-fixing bacteria. 4.3. Bacterial identifications Identification of bacterial isolates was undertaken to gain insight into whether the composition of bacterial communities on the roots were similar to or different from those in the sediments. Most studies comparing seagrass rhizosphere bacterial communities to unvegetated sediment bacterial communities have focused on enumeration of bacteria having a certain physiology such as nitrogen fixation (Blotnick et al., 1980; Donnelly and Herbert, 1999; Hansen et al., 2000; Welsh, 2000). An aim of our study was to determine if seagrass root-associated bacteria had physiological traits that could be beneficial to the plants. Bacterial identifications indicated that the root-associated bacteria were markedly different from those in sediments, including the vegetated sediments. The most striking

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difference was the dominance of Gram-negative bacteria isolated from roots compared to the dominance of Gram-positive isolates from sediments. In addition, among the bacterial species isolated, most were obtained either from roots or sediments but not both. This further points to compartmentalization between seagrass roots and the surrounding sediments as suggested by Pereg et al. (1994). The seagrass root isolates included many known to be associated with plants, and others that have been found associated with Zostera noltii (Cifuentes et al., 2000). Achromobacter spp., Micrococcus spp., Azospirillum spp., and Pantoea spp. have all been previously found associated plants or seeds and are either beneficial or harmless (Mergaert et al., 1993; Bacon et al., 2002). P. chlororaphis, isolated in April, is capable of denitrification and has been found in the rhizosphere of the emergent plant Glyceria maxima where it proliferates upon addition of nitrate (Nijburg et al., 1997). Other denitrifying bacteria previously obtained from seagrass rhizospheres include bacteria identified as Alcaligenes spp., Cytophaga spp., Moraxella spp. and, as also found during our study, Pseudomonas spp. and a Vibrio or Photobacterium sp. (Sheih and Yang, 1997). An isolate identified as A. sobria was obtained in April. Aeromonas species are capable of nitrate reduction with ammonium as the end product (Macfarlane and Herbert, 1984). Nitrate-respiring bacteria had also been found associated with Z. noltii and are important in seagrass bed sediments (Cifuentes et al., 2000; Herbert, 1999; Welsh, 2000; de Wit et al., 2001). The capability to fix nitrogen is widespread among many different bacteria. Nitrogen fixers previously isolated from the seagrass rhizosphere include sulfate reducers and clostridia (Patriquin and Knowles, 1972; Nielsen et al., 1999), Klebsiella sp. (Schmidt and Hayasaka, 1985), Vibrio spp., and Photobacterium spp. (Sheih et al., 1989). Sinorhizobium spp., isolated from the roots of shaded plants in July, are common nitrogen-fixing soil bacteria capable of forming root nodules in certain plant species (Widmer et al., 1999). Other isolates were identified as bacteria having physiology that would be advantageous for growth on seagrass roots where one would expect an oxic/anoxic interface. Methylobacterium species are capable of oxidizing methane (Patt et al., 1974). Kusel et al. (1999) showed archaea, quite possibly methanogens colonize the epidermis of Halodule wrightii roots which could provide methane for methanotrophs in the rhizosphere. In addition, Methylobacterium species are capable of fixing nitrogen (Sy et al., 2001) and producing cytokinins (Lidstrom and Chistoserdova, 2002). Pseudomonas balearica is capable of oxidizing thiosulfate under denitrifying conditions and could be important in mediating sulfide oxidation (Sorokin et al., 1999). The most curious identification was that of an April isolate as a Sanguibacter sp. Sanguibacter spp. are coryneform bacteria that have so far only been isolated from the blood of apparently healthy cattle (Pascual et al., 1996). However, it is becoming clear that some closely related bacteria are able to thrive in both plant and animal hosts (Cao et al., 2001). These identifications are consistent with views that bacterial-seagrass root interactions are important to plant productivity (Blotnick et al., 1980; Barko et al., 1991; Welsh, 2000) and further suggest that interactions between bacteria and seagrass roots might be similar to those described in better studied terrestrial plants (Klein et al., 1990; Andrews and Harris, 2000; Whipps, 2001).

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4.4. Bacterial phenotypic traits High percentages of the isolates examined from both roots and sediments possessed physiological traits considered important for rhizosphere (Table 4). Although the traits were common among all isolates, it does not necessarily mean that they are unimportant to interactions between bacteria and seagrass roots. A wide variety of bacteria can produce plant growth-promoting, phytohormonelike compounds such as IAA (Costacurta and Vanderleyden, 1995). Bacteria from terrestrial rhizospheres appear to have a greater potential to synthesize and release auxins, e.g. IAA, as secondary metabolites than bacteria in root-free soil because a rich supply of substrates is available in the rhizosphere environment (Rossi et al., 1984; Frankenberger and Arshad, 1995; Lebuhn et al., 1997). The potential for bacterial IAA production may be different in aquatic sediments where nutrients are available from detrital organic matter. Maruyama et al. (1989) measured IAA in marine sediment porewater at concentrations on the order of 10 10 10 6 mol l 1. Established seagrass tissue cultures have been shown to benefit from auxins and other plant growth-promoting compounds (Koch and Durako, 1991; Durako and Kuss, 1994) and it is possible that some of these might be furnished in nature by bacteria. Differences were noted in the proportion of bacterial isolates displaying inhibitory activity toward the two fungi; higher percentages of isolates inhibited the growth of L. marinera than P. aphanidermatum. This result is not surprising as these two organisms are not related. L. marinera is a true fungus, a saprophyte found on marine plants (Meyers, 1969). P. aphanidermatum is an Oomycete, a mycelial protoctist which favors warm temperatures. Unlike L. marinera, P. aphanidermatum is an aggressive, cosmopolitan pathogen possessing a wide host-range causing blights and rots (Agrios, 1997). Although not recognized as a V. americana pathogen, we hypothesized that bacteria inhibiting P. aphanidermatum may have the potential of providing a disease protective function, whereas those inhibiting the growth of L. marinera may do so to reduce competition for available nutrients. Acknowledgements We thank David A. Flemer for his enthusiastic interest and guidance, Lyndsey Brown for help with collecting field samples, Joe James for assistance with the anti-fungal assays and Tim Franklin for assistance with IAA analysis. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the US EPA. [SS] References
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