Degradation, Phytoprotection and Phytoremediation of Phenanthrene

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Degradation, Phytoprotection and Phytoremediation of Phenanthrene by Endophyte Pseudomonas putida, PD1 Zareen Khan, David Roman, Trent Kintz, May delas Alas, Raymond Yap, and Sharon Doty* School of Environmental and Forest Sciences, College of the Environment, University of Washington, Seattle 98195-2100, United States S Supporting Information *

ABSTRACT: Endophytes have been isolated from a large diversity of plants and have been shown to enhance the remediation efficiency of plants, but little information is available on the influence of endophytic bacteria on phytoremediation of widespread environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs). In this study we selected a naturally occurring endophyte for its combined ability to colonize plant roots and degrade phenanthrene in vitro. Inoculation of two different willow clones and a grass with Pseudomonas putida PD1 was found to promote root and shoot growth and protect the plants against the phytotoxic effects of phenanthrene. There was an additional 25−40% removal of phenanthrene from soil by the willow and grasses, respectively inoculated with PD1 when compared to the uninoculated controls. Fluorescent microscopy using fluorescent protein tagging of PD1 confirmed the presence of bacteria inside the root tissue. Inoculation of willows with PD1 consistently improved the growth and health when grown in hydroponic systems with high concentrations of phenanthrene. To our knowledge this is the first time that the inoculation of willow plants has been shown to improve the degradation of PAHs and improve the health of the host plants, demonstrating the potential wide benefit to the field of natural endophyte-assisted phytoremediation.

1. INTRODUCTION The increasing industrialization of the global economy over the last century has greatly increased the presence of anthropogenic substances such as polycyclic aromatic hydrocarbons(PAHs) in the environment posing a threat to human and ecosystem health.1−3 The U.S. Environmental Protection Agency has listed 16 PAHs as priority pollutants because of their carcinogenicity and persistence based on toxicity and potential for human exposure.4 A broad range of physical, chemical and biological methods has been applied for remediation of water and soil contaminated with these hydrophobic chemicals.5−8 Phytoremediation is an emerging green technology where plants are grown in the presence of contaminated soil, sediment, surface, and groundwater to enhance the decomposition or removal of inorganic and organic contaminants.9 In the case of phytoremediation of organic pollutants in soil, plants are used to take up or enhance the decomposition into non- or less-toxic forms. Additionally, plants can improve the soil structure by increasing aeration, humidity and also promote microbial growth. Release of exudates by plant roots can contribute to increased contaminant bioavailability and oxidation/degradation rates of recalcitrant molecules such as PAHs.10−13 However, successful field phytoremediation has been limited due to the high phytotoxicity of PAHs causing growth inhibition, reduced transpiration, chlorosis and wilting.14,15 Microbes that reside in the inner tissues of living plants without causing apparent negative symptoms of infection have been termed endophytes16 and have been demonstrated to play © XXXX American Chemical Society

a key role in host plant adaptation in polluted environments. In the plant−endophyte symbiosis, endophytes receive the carbohydrates from plants and, in return, they can improve the plants’ resistance to external stresses such as contaminants, temperature extremes, water and nutrient limitations, salt, and pathogens.17 Pollutant degrading endophytes have been isolated from plants growing in contaminated areas. For instance, petroleum degrading bacteria were isolated from plants growing in petroleum-contaminated soil, with a preference for the bacteria in the root interior.18 Another study isolated endophytic bacteria from English oak and common ash growing on a trichloroethylene (TCE) contaminated site and found that the majority of the bacterial community showed increased tolerance to TCE and enhanced TCE degradation capacity in some of the strains.19 A study done by van Aken et al.20 indicated that Methylobacterium populum BJ001, isolated from poplar trees is able to degrade energetic compounds such as 2,4,6-trinitrotoluene, hexahydro1,3,5-trinitro- 1,3,5-triazine (RDX), and hexahydro-1,3,5trinitro-1,3,5-triazine. Mineralization of about 60% of RDX to carbon dioxide was observed within 2 months time. Recently, Oliveira et al.21 have isolated three strains from Cerrado plants exhibiting the capacity for degradation of different fractions of petroleum, diesel oil, and gasoline. Kang et al.22 reported a Received: August 11, 2014 Revised: September 29, 2014 Accepted: October 2, 2014

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novel endophyte Enterobacter sp. strain PDN3 that was isolated from a hybrid poplar and showed high tolerance to TCE. Without the addition of inducers, PDN3 rapidly metabolized TCE to chloride ion, and nearly 80% TCE (55.3 uM) was dechlorinated to chloride ions within 5 days. In recent years, much more attention has been focused on the application of endophytic bacteria for enhancing phytoremediation. Barac et al.23 demonstrated that a genetically modified endophyte strain of Burkholderia cepacia was able to increase the tolerance of yellow lupine to toluene. The recombinant strain decreased phytovolatilization of toluene from the plant into the atmosphere by 70% in laboratory scale experiments. Germaine et al.14 described inoculation of pea plants (Pisum sativum) with Pseudomonas. putida VM1450, a genetically modified bacterial endophyte that naturally possessed the ability to degrade 2,4-dichlorophenoxyacetic acid (organochlorine herbicide). The inoculated plants had a higher degradation capacity of up to 40% for 2,4-dichlorophenoxyacetic acid from the soil. In a different study the same authors showed that inoculation of pea plants with an endophytic napthalene degrading bacterium Pseudomonas putida VM1441 protected the host plant from phytotoxic effects of naphthalene, increased seed germination, increased transpiration rates and removed 40% more naphthalene from artificially contaminated soils than did uninoculated plants.15 The first in situ inoculation of poplar trees growing on a TCE contaminated site with TCE-degrading strain Pseudomonas putida W619-TCE was done by Weyens et al.24 The inoculation resulted in a 90% reduction of TCE evapotranspiration under the field conditions. Another study demonstrated the use of engineered endophytes for improving phytoremediation of environments contaminated by organic pollutants and toxic metals.25 The yellow lupine was inoculated with B. cepacia VM1468 possessing (a) the pTOM-Bu61 plasmid coding for constitutive TCE degradation and (b) the ncc-nre Ni resistance/sequestration. Inoculation with B. cepacia VM1468 into plants resulted in a decrease in Ni and TCE phytotoxicity, which was reflected by a 30% increase in root biomass and up to a 50% decrease in the activities of enzymes involved in antioxidative defense in the roots. Ho et al.26 isolated several endophytic bacteria from reed and water spinach, that utilized aromatic compounds as the sole carbon source. They selected Achromobacter xylosidans strain F3B and inoculated A.thaliana plants and showed that the strain improved phytoremediation of phenolic pollutants. Unfortunately, only a very small number of PAH-degrading endophytes have been isolated thus far and despite evidence from studies demonstrating how plant-endophyte symbiosis can be exploited to improve plants’ resistance to pollutant stress, no research has been done on utilizing endophytic bacteria to reduce PAH contamination with fast growing trees. Willows are ideal for phytoremediation applications because of their extensive root system, adaptability to extreme environments, rapid growth, high water uptake, and high biomass.27−33 Thus, willows have the potential to be immensely advantageous if used with endophytes on PAH contaminated sites. Overall, growing willows with PAH-degrading endophytes could have multiple applications, including phytoremediation, carbon sequestration, and biomass production.27,29−33 The main objective of the study was to determine if there is a potential to enhance phytoremediation of PAHs using endophytes. Studies were conducted to screen poplar and willow endophytes for their ability to grow on PAHs and

degrade phenanthrene in vitro, to examine effects of endophyte inoculation on willow and grasses grown in the presence of phenanthrene, to verify colonization, and finally to investigate uptake of phenanthrene from soil. Willow clone S-365 was chosen because it has proven effectiveness in phytoremediation applications,27 needs minimum maintenance and has extensive root surface area, and grasses were selected because they grow relatively fast and have a greater potential for application on public recreational areas contaminated with pollutants.

2. MATERIALS AND METHODS 2.1. Chemicals and Solvents. Analytical grade naphthalene, phenanthrene, pyrene were purchased from SigmaAldrich. All solvents (acetonitrile, dichloromethane) were of HPLC grade. 2.2. Screening of Poplar and Willow Endophytes for Growth in the Presence of Naphthalene, Phenanthrene, Pyrene. Endophytes from poplar and willow were isolated using standard methods.34 To determine if the endophytes were able to utilize the PAHs, isolates were grown in test tubes under batch cultivation conditions. Three PAHs were selected based on the degree of toxicity. Naphthalene, phenanthrene, and pyrene were dissolved in 1% (w/v) isopropanol in sterile test tubes to give a concentration of 0.1 g L−1. After the solvent was evaporated, 5 mL of mineral medium35 per test tube was added. Dilutions were made in mineral medium to a final concentration of 5 ppm naphthalene, 1 ppm phenanthrene, and 0.1 ppm pyrene. The tubes were inoculated with the endophytes to an initial O.D600 of 0.1. The tubes were sealed and shaken at room temperature for 10 days, and the growth was measured using a spectrophotometer at wavelength of 600 nm (O.D600). 2.3. Phenanthrene Degradation in Liquid Cultures by Strain PD1. The best growing strain PD1, selected from the screening experiment was tested for degradation of phenanthrene. Briefly, the isolate was grown overnight in MG/L broth,36 harvested and washed thrice with minimal medium (M9) containing: Na2HPO4 6.0 g l−1; NaCl 0.5 g l−1; KH2OP4 3.0 g L−1; NH4Cl 1.0 g L−1; MgSO4·7H2O 0.5 g L−1; peptone 2.0 g L−1, pH 7.2 and then adjusted to an OD600 of 0.5 using a spectrophotometer. Phenanthrene was added at a concentration of 5 μg mL−1 to 125 mL Erlenmeyer flasks with screw caps containing 25 mL of M9 medium. The flasks were covered with foil and shaken at 30 °C for 3 days. The experiment was done in triplicate and the controls were grown similarly but without the endophyte. At time intervals of 24 h, 100 μL of sample was aliquoted out and extracted as follows. To the sample, 10 μL of d10 phenanthrene (internal standard) was added, vortexed, followed by 500 μL of methylene chloride. The solution was vortexed and the methylene chloride layer was pulled off and injected into a Shimadzu QP 2010 gas chromatograph quadrupole mass spectrometer equipped with a methyl silicone column (30 × 0.32 × 5). Helium was the carrier gas with a total flow of 48.2 mL/min. The column temperature was held at 150 °C, programmed to 300 °C at a rate of 50 °C/ min, and held at 300 °C for 6 min. Injector and analyzer temperatures were 260 °C and 300 °C respectively. The mass spectrophotometer was operated in the SIM mode. 2.4. Identification of PD1. For the 16S rDNA analysis, genomic DNA was extracted and 16S rDNA was PCRamplified using genomic DNA as template and bacterial universal primers.37 The PCR was performed in a thermocycler at 95 °C for 10 min followed by 30 cycles of 95 °C for 1 min, B

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53 °C for 1 min, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The amplification products were purified using a DNA purification kit (Qiagen) and the sequencing was done by GENEWIZ Inc. (Seattle, WA). The DNA sequences obtained were analyzed with the basic sequence alignment BLAST program38 run against the nucleotide collection (nr/nt) database from the National Center for Biotechnology Information (www.ncbi.nlm.nih. gov/BLAST). The 16S rDNA sequences of the strain has been deposited in GenBank under accession no. KF443801. 2.5. PD1 Salt and Temperature Stress Experiments. To test the capacity of PD1 to grow under salt stress and temperature stress, pure cultures were exposed to varied salt concentrations and two temperature extremes. PD1 was streaked on MG/L plates and incubated at 4 °C and 37 °C. To assess the effect of salt stress, PD1 was grown on LuriaBertani (LB) plates containing 1%, 1.5%, 2%, 4% sodium chloride. 2.6. Phytoprotection and Phytoremediation Experiments. 2.6.1. Preparation of Inoculant Solution. Pseudomonas putida PD1 was grown in MG/L broth at 30 °C in a rotary shaker (200 rpm) for 24 h, cells were then collected by centrifugation (8000 rpm for 10 min), diluted in 0.5X Hoaglands solution39 and adjusted to a final OD600 of 0.1. 2.6.2. Willow Hydroponic Experiments. Two similar hydroponic experiments were conducted using two different Salix species- Salix purpurea clone 94006 and Salix discolor clone S-365 kindly provided by J. Isebrands. These two species were chosen due to wide differences in the robustness of each plant in polluted environments. S365 is already being used in remediation practices.27 12−15 cm size cuttings obtained from greenhouse grown plants were placed in 1000 mL beakers filled to capacity with 0.5× Hoaglands, wrapped in foil and allowed to grow under plant growth lights programmed at an 18 h photoperiod and a 20 °C/14 °C day/night temperature cycle, until they had developed a viable and healthy root system. Plants within each experiment were separated, with one-half being inoculated with PD1 (as described previously) and the other half left as noninoculated controls. After 24 h of cocultivation with the bacteria, the solution was replaced with 0.5× Hoaglands solution and the endophytes were allowed to colonize for 2 weeks. The plants were next placed into individual flasks containing a phenanthrene- saturated solution of 0.5× Hoaglands. 2.6.3. Willow Soil Experiment. Preparation of Contaminated Soil. A stock solution of phenanthrene was prepared in pure ethanol and used for spiking the soil according to the procedure described by Binch et al.40 Phenanthrene was added with only 25% of the total quantity of dry soil and kept closed for a few minutes to disperse. Then the solvent was allowed to volatilize in a fumehood and the dosed soil was mixed with the rest of the nonpolluted soil using a trowel to obtain a final phenanthrene concentration of 100 mg kg−1 of potting mixture. The same protocol was used with solvent without phenanthrene as a control. Experimental Design. Rooted S-365 cuttings (inoculated and uninoculated) and contaminated soil with no plants were placed in a fumehood equipped with plant growth lights programmed at an 18 h photoperiod, and a 20 °C/14 °C day/ night temperature cycle for a period of 30 days. Each of these treatments was carried out in triplicate. Plants were watered and were also rearranged randomly every time they were watered.

Analysis. Three soil samples from each pot were taken, thoroughly mixed and air-dried overnight in the fumehood. Phenanthrene was extracted from the soils using the procedure from Germaine et al.15 Five milliliters of HPLC-grade acetonitrile were added to 2 g soil samples and vortexed vigorously for 5 min. The samples were then centrifuged for 5 min at 13 K rpm, the supernatant was carefully removed and centrifuged once more. The supernatant was then filtered through a 0.22 μm filter before HPLC analysis. Extractions were done in triplicate for each soil treatment. The extraction procedure was tested for efficiency of recovery. The recovery averaged at 95% (n = 7) for phenanthrene. Concentrations of phenanthrene were quantitatively analyzed by a Shimadzu HPLC equipped with a Restek Pinnacle II PAH (150 × 3.0 mm) column, following the EPA method 610 with a modification that included an isocratic solvent instead of a gradient. The mobile phase was acetonitrile−water-phophate buffer (20 mM, pH 7.1) (80:20:0.1), the flow rate was 1 mL/ min and detection was by UV absorbance at 254 nm. A retention time of 4 min was observed under these conditions. 2.6.4. Grass Experiments. Pennington Pacific Northwest Mix perennial rye grass was chosen in the study mainly because of the potential of using this technology at local PAHcontaminated sites and also due to its fast growth. Two by six cell packs were used for this experiment and three different levels of phenanthrene were added. Group A had 50 mg kg−1, group B had 130 mg kg−1 and group C had 200 mg kg−1 of phenanthrene in soil. Each cell pack received 1g of seeds distributed evenly, three of which were inoculated with 1 mL of PD1 culture per cell, and the other three were uninoculated with media without the inoculum. The experiment was conducted in a fumehood equipped with plant growth lights programmed at an 18 h photoperiod, and a 20 °C/14 °C day/ night temperature cycle. Plants were watered and were also rearranged randomly every time they were watered. Tillers were counted at day 13. Post treatment phenanthrene concentrations were extracted from soil and analyzed using the protocol described above. 2.7. Construction of Red Fluorescent Protein Labeled PD1 and Verification of Colonization. The rf p plasmid (kindly provided by Sam Miller (UW Microbiology) was introduced into Pseudomonas putida, PD1 via triparental mating usin E. coli DH5 α containing red fluorescent protein (rfp) as the donor, and E. coli HB101 (pRK2073) as the helper strain following standard protocols.41 After the triparental mating, the bacterial cells were plated onto MGL agar plates containing carbenicillin (50 μg mL−1) and gentamycin (100 μg mL−1). After streak purification of the rf p-labeled PD1, fluorescent microscopy was performed using a Zeiss Imager M2 equipped with an AxioCam MRM and recorded with Zeiss AxioVision software (Carl Zeiss, LLC,USA) and checked for rf p expression by viewing under the red channel and acquired with Zeiss filter set 00 (excitation, 530 to 585 nm; beam splitter, 600 nm; emission, >615 nm).To evaluate endophytic colonization with the rf p-labeled PD1, rooted willow S-365 cuttings were inoculated using procedures as described above. The plants were surface sterilized with 0.1% sodium hypochlorite for 5 min, rinsed several times with sterile water and the roots were observed under the microscope.

3. RESULTS AND DISCUSSION 3.1. Degradation of Phenanthrene by PD1. Three willow endophytes- WW1, WW3, WW11, and three poplar C

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Figure 3. Amount of phenanthrene accumulated in the grass tillers at different phenanthrene concentrations. A, 50 mg kg−1; B, 130 mg kg−1; C, 200 mg kg−1. Error bars represent ± SD(n = 6).

3 days and further degradation did not occur after 4 days of incubation. The bacteria were identified by 16S rDNA sequencing to be Pseudomonas putida (99% similarity). The genus Pseudomonas exhibits diverse metabolic capacities, which allow it to survive in different ecological niches,43 including sites contaminated with aromatic hydrocarbons. Some strains have been used in bioremediation of phenanthrene contaminated sites,44 however remediation is not at satisfactory rates most likely due to the difficulties in generating high biomass for meaningful remediation due to competition by other microbes, apart from other challenges.45 Using endophyte-assisted phytoremediation is a sustainable approach which could assist in improving the degradation efficiency of recalcitrant PAHs. 3.2. Salt and Temperature Stress. Growth of PD1 on lactose broth plates was evaluated at four different concentrations of sodium chloride. It was observed that PD1 grew normally within 2 days on plates containing 1%, 1.5%, and 2% sodium chloride and 2 days longer on plates containing 4% sodium chloride. High temperature did not affect growth of PD1 on plates as it took 2 days to grow at 37 °C; however, the growth was slow at 4 °C (3 weeks) when compared to growth at 30 °C. In the field, the growth of plants may be inhibited by a variety of biotic and abiotic stresses, including extreme temperature and high salt salt concentrations.46 The ability of this isolate to grow under a range of environmental conditions could make the entire phytoremediation process more efficacious. 3.3. Inoculation of Willows and Grasses with Endophyte PD1 Provides Phytoprotection. To determine whether the phenanthrene degrading bacterium, PD1 provided phytoprotection against phenanthrene, two week old inoculated plants were exposed to lethal concentrations of phenanthrene in hydroponic solution. There were striking differences between the control plants and those inoculated with the endophyte PD1. At the end of 19 days, all the uninoculated S-94006 control plants were stressed and lost leaves, while the PD1 inoculated plants were still healthy, with many having new growth (Figure 1 of the Supporting Information (SI)) and there was a statistically significant change in the weight between inoculated and control plants. The inoculated plants were healthy, 5 cm taller and had a weight gain of 1.5 g (P = 0.013), whereas the uninoculated plants expressed no growth, lost weight (60% of phenanthrene removed from the media after D

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Figure 4. Effect of phenanthrene exposure on growth of willow clone S-365 after one month. Photographs of inoculated plants on the left and uninoculated plants on the right.

and denser root systems compared to their uninoculated counterparts that showed signs of wilting, chlorosis, leaf senescence, and browning of roots. Images taken at the end of the experiment show a distinct trend in the health of the plants (Figure 2 of the SI) with the inoculated plants being healthier than the uninoculated controls. In the second trial of the experiment, there was statistical significance (p = 0.01) in the differences in the shoot growth, with the PD1 inoculated plants having an average growth of 22 cm, while the uninoculated control plants only having an average of 6 cm shoot growth. Another important observation was that there was a negative effect of addition of phenanthrene on the transpiration of most of the uninoculated plants whereas some of the inoculated controls transpired normally indicating that endophyte PD1 protected the plants against the toxic effects of phenanthrene (data not shown). One possible mechanism to explain the protection against phenanthrene in the inoculated plants could be its detoxification in planta. This has been shown by other studies where phytoprotection was conferred because of the ability of the added endophyte degrading the pollutant inside plant tissues.14,15,24 The stimulated plant growth in the inoculated plants in the presence of high levels of phenanthrene could be also due to endophytic traits that can alter contaminants toxicity. For example, through producing phytohormones such as auxins and cytokinins, iron chelators, siderophores, organic acids and various degrading enzymes50,51 or redox changes. A recent study was done by Sheng et al.52 where a pyrene degrading endophyte was characterized for its capacity to produce indole acetic acid (IAA), solubilize phosphate and make siderophores. These authors speculated that the inoculation of plants with the pyrene degrader protected the plants against the inhibitory effects of high concentrations of pyrene and enhanced its removal from soil by production of IAA, siderophore, or inorganic phosphate solubilization or by pyrene degradation in soil.

Figure 5. Amount of phenanthrene removed in soils spiked with 100 mg kg−1 of phenanthrene in different treatment groups- Uninoculated Willow S-365, inoculated with Pseudomonas putida PD1 and unplanted controls. Error bars represent ± SD(n = 6).

and healthy and grew 2.8 cm longer, while the uninoculated control plants had solution that was brown and root systems that were brown and with no growth. The experiments using the S-365 clone were conducted using the same protocols except that these experiments lasted about two months, but with similar results noticed for plants inoculated with PD1. The reason for the longer duration of the experiments using the S-365 is that it is naturally a much more robust plant, as was shown in other phytoremediation studies.47−49 This experiment was repeated twice with similar results. During the first trial, the PD1 inoculated plants had an average shoot growth of 6 cm compared to the noninoculated control plants which averaged only 0.8 cm shoot growth. The differences in total weight change over the length of the experiment was also striking, with the PD1 inoculated plants averaging a gain of 3.2 g vs the 0.5 g gained by uninoculated control plants. The willow S365 plants that were inoculated with PD1 tolerated the contaminant stress, had higher survival, E

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control plants (P < 0.05). At the lower level of dosing, a similar trend was observed but it was not statistically significant. For the plant tissue samples there was 40−50% more phenanthrene accumulated with highly significant amounts by the group that received the highest phenanthrene dose (Figure 3). Even though the endophyte seemed to protect the grass plants against the phytotoxic effects of phenanthrene and enhanced its removal from soil, it did not seem to degrade it in planta during the time frame of this experiment. Since the endophyte was able to degrade phenanthrene in vitro and considering how healthy the inoculated plants were at the time of harvest perhaps degradation may have occurred if the experiment had been conducted longer. Also it is possible that the cell density of the inoculant may not have been sufficient for effective degradation, as was shown in other studies where the degradation rates were proportional to the cell counts.15 Other studies have also reported that the successful use of endophytic bacteria in combating organic contamination of plants requires the presence of high counts of contaminantdegrading bacteria inside of the plant tissues.53 In experiments conducted with the willow clone S-365, wiithin 2 weeks of planting in the soil spiked with phenanthrene at 100 mg kg−1 level, the uninoculated plants started showing signs of stress with leaves wilting and turning chlorotic and death occurring after 4 weeks, whereas all the inoculated plants continued to stay healthy (Figure 4). This clearly demonstrates that the endophyte PD1 protected the plants against the phytotoxic effect of phenanthrene. To test if it was enhancing phytoremediation, triplicate soil samples were taken from each of the pots and analyzed individually. In the control experiment with no plants the phenanthrene levels were significantly (P ≤ 0.05) reduced to 15−25 mg kg−1 soil. This constituted a 25% reduction in the level of phenanthrene present in the soil, and this loss is thought to be due to photooxidation/volatilization of phenanthrene from the soil.54,55 In soil that had been planted with uninoculated plants, there had been a further reduction of phenanthrene levels by up to an additional 25%. This reduction may have been brought about by sorption of phenanthrene to the roots, plant metabolism56,57 or enhanced degradation through the rhizospheric bacteria and/or promotion of indigenous phenanthrene degraders.58,59 Soil containing plants that were inoculated with PD1 underwent the most dramatic decrease in the phenanthrene levels (Figure 5). There was a statistically significant difference between this treatment and each of the other two treatments (P ≤ 0.05). Compared with the control soil, 40% more phenanthrene was removed from the soil and compared with uninoculated plants 25% more phenanthrene was removed (P ≤ 0.05). Attempts to detect phenanthrene concentrations in plant organs did not give any significant results because of lack of sufficient tissue material for analysis. Red fluorescent PD1 could be visualized on plates and inside the roots. Sites of lateral root emergence were the most heavily colonized regions and are generally believed to be the principal sites of plant infection by bacteria (Figure 6a,b). No red fluorescence was detected in roots from uninoculated control plants (data not shown). By using a natural microbial symbiont of poplar trees, we have demonstrated a dramatic reduction in the phytotoxicity of phenanthrene and improved removal of pollutant from soils. Although the endophyte was isolated from poplar, it functioned well in both Salix, a family member with Populus, and in grasses,

Figure 6. Visualization of inoculated endophyte rf p-Pseudomonas putida PD1 within root tissues of Willow S-365. After inoculation with rf p-PD1, colonization was observed in plant roots. Bacterial cells with red fluorescence were displayed as bright red rods inside roots. (A) Colonization at sites of lateral root emergence (B) Interior views of a section of willow root. Arrows indicate representative rf p tagged cells.

3.4. Willow and Grasses Inoculated with PD1 Demonstrate an Increased Ability to Remove Phenanthrene from Soil. In the grass experiments, there were significant differences in plant germination in the presence of phenanthrene with uninoculated seeds taking 5 days longer to germinate than inoculated seeds. It was also apparent that phenanthrene was inhibiting plant growth as significant differences in the heights and number of tillers were observed in the uninoculated control and the inoculated groups during the early stages of plant development, more noticeable in the plants that were exposed to the highest level of phenanthrene (Figure 3 of the SI). After 13 days of growth, grass plants inoculated with strain PD1 grew approximately 2.5 cm taller, producing 100 more tillers than the control group. Grass plants that were inoculated with Pseudomonas putida PD1 also removed 40% more phenanthrene than the uninoculated F

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making endophyte-assisted phytoremediation widely applicable for different environments.

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ASSOCIATED CONTENT

S Supporting Information *

The additional figures addressing supporting data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 206-616-6255; fax:206-685-0790; e-mail: sldoty@uw. edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Tom DeLuca and Jud Isebrands for critical reading of this manuscript. We also thank undergraduate researchers Habiba Mohammad, Zahra Mohammad, Sherry Xheng, and Rehmatullah Arif for their help in the experiments. This work was financially supported by NIH/NIEHS-SBIR Grant No. 2R44ES020099-02.

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