SYMBIOSIS (2009) 47, 23–33 ©2009 Balaban, Philadelphia/Rehovot ISSN 0334-5114

Diazotrophic endophytes of native black cottonwood and willow Sharon L. Doty1*, Brian Oakley2,4, Gang Xin3,5, Jun Won Kang1, Glenda Singleton1, Zareen Khan1, Azra Vajzovic1, and James T. Staley2

1College of Forest Resources, UW Box 352100, University of Washington, Seattle, WA 98195, USA, Tel. +1-206-616-6255, Fax. +1-206-543-3254, Email. sldoty@u.washington.edu; 2Department of Microbiology and 3Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195, USA; 4Current address: Department of Biological Sciences, Microbiology Research Group, University of Warwick, Coventry, CV4 7AL, UK; 5Current address: Hydranautics, Oceanside, CA 98058, USA (Received December 20, 2007; Accepted May 25, 2008)

Abstract Poplar and willow are economically-important, fast-growing tree species with the ability to colonize nutrient-poor environments. To initiate a study on the possible contribution of endophytes to this ability, we isolated bacteria from within surface-sterilized stems of native poplar (Populus trichocarpa) and willow (Salix sitchensis) in a riparian system in western Washington state. Several of the isolates grew well in nitrogen-limited medium. The presence of nifH, a gene encoding one of the subunits of nitrogenase, was confirmed in several of the isolates including species of Burkholderia, Rahnella, Sphingomonas, and Acinetobacter. Nitrogenase activity (as measured by the acetylene reduction assay) was also confirmed in some of the isolates. The presence of these diazotrophic microorganisms may help explain the ability of these pioneering tree species to grow under nitrogen limitation. Keywords: Endophyte, nitrogen fixation, poplar, willow, Salicaceae

1. Introduction Most plants in their native environments depend on

symbioses with microorganisms for their existence (Hirsch, 2004). The interior of plants provides a habitat for a wide range of bacteria and fungi, both termed endophytes, that benefit the plant host by increasing nutrient acquisition, stress tolerance, pathogen resistance, seed germination, seedling length, and aiding in phytoremediation of environmental pollutants (Reis et al., 2000; Cook et al., 1995; Siciliano et al., 2001; Nejad and Johnson, 2000; Hirsch, 2004; Mastretta et al., 2006; Ryan et al., 2008; Doty 2008). The focus of most endophyte research has been on crop plants, emphasizing nitrogen-fixing (diazotrophic) endophytes, with the goal of decreasing dependency on synthetic nitrogen fertilizers that can have negative effects

*The author to whom correspondence should be sent.

on the environment (Cocking, 2005; Sturz et al., 2000). Nitrogen fixed biologically by plant-symbiotic bacteria is ecologically friendly and has been effectively exploited for important leguminous crop species. Although associations of diazotrophic bacteria with non-leguminous plants such as grasses have been known for decades (Döbereiner, 1977; Döbereiner, 1992; Döbereiner and Pedrosa, 1987), they have been less studied in other crop plants except for a few cases; for example, associative bacteria of some tropical species of rice and maize (Reis et al., 2000; Cocking, 2005). A more complete understanding of the diversity and function of diazotrophic microorganisms, especially those that have symbiotic relationships with commercially important non-leguminous plant species, is of great value for research and application.

For years, it was thought that nodule formation was a requirement for effective transfer of fixed atmospheric nitrogen to plants for growth. Because inducing non-legume crop plants to produce effective nodules is difficult,

24 S.L. DOTY ET AL.

research into biological nitrogen fixation without nodule formation became a new focus (Cocking, 2005). For example, a well-studied diazotrophic endophyte is Gluconacetobacter diazotrophicus of sugarcane. Inoculation with nif (nitrogen fixing-deficient) mutants of this organism resulted in reduced sugarcane growth, strongly suggesting that fixed nitrogen is transferred to the plant under normal symbiotic conditions (Sevilla et al., 2001). This bacterium is capable of secreting nearly half of its fixed nitrogen in a form that the plant can utilize. The ability of G. diazotrophicus to fix nitrogen in the aerobic environment of the stem is attributed to “respiratory protection”, whereby the extremely rapid respiration of high levels of sucrose from metabolism within the sugarcane stem leads to a microaerobic environment that is needed for the oxygen-sensitive nitrogenase enzyme (Flores-Encarnacion et al., 1999). Other examples of endophytic bacteria, including Azoarcus and Herbaspirillum are at least suspected of providing fixed nitrogen to their non-leguminous plant hosts (Reinhold-Hurek and Hurek, 1998). Further investigation of diazotrophic endophytic bacteria will lead to a more complete understanding of the contributions of these bacteria to plants.

Cottonwood (Populus sp.) and willow (Salix sp.) are important early-successional trees with rapid growth, deep roots, and the ability to grow in nutrient-poor environments (Stettler et al., 1996). Cottonwoods and other poplar species are of economic value for several reasons. They are grown in short-rotation plantations for the production of pulp and paper, and for lumber and fuel throughout the world. Together with willows, they also have multiple environmental uses including phytoremediation of pollutants, carbon sequestration, soil stabilization along river banks, and renewable energy production. Recently, black cottonwood (Populus trichocarpa) was chosen as a model tree species for genomics research due to its small genome size, fast growth, high transformation frequency, simple vegetative propagation, and ease in tissue culture (Boerjan, 2005). Willow trees are used extensively as a source of biofuel in several European countries. A better understanding of the endophytes of cottonwood and willow will help increase our knowledge of the roles of the microbial community in tree plantations and in their native environment. It could lead to a significant reduction in the need for chemical fertilizers and to an improvement in overall plant growth, disease resistance, and phyto-remediation potential.

Only recently has research begun on the endophytic bacteria of cottonwood. In 2001, the first discovery of a nitrogen-fixing endophyte, Rhizobium tropici bv populus, within hybrid poplar was reported (Doty et al., 2005). A novel methane-utilizing species named Methylobacterium populi sp. nov. was also isolated from a hybrid poplar, P. deltoides x nigra DN34 (van Aken et al., 2004a). This isolate is able to degrade nitro-substituted explosives, an

ability that may promote the use of poplar in the remediation of contaminated sites at military training ranges (van Aken et al., 2004b). Many other endophyte sequences were also identified during the sequencing of the poplar genome (Tuskan, 2006). Recently, a paper was published on the diversity of endophytes of hybrid poplar grown under field conditions (Ulrich et al., 2008). In 2004, Germaine and colleagues reported that endophytes of poplar could be labeled with green fluorescent protein (by expressing the gfp gene) and re-introduced, demonstrating that specific bacteria can be introduced into plants (Germaine et al., 2004). In a ground-breaking study, the concept of engineering endophytes for phytoremediation was proven to be successful (Barac et al., 2004; Taghavi et al., 2005). These two studies not only demonstrated the concept of endophyte-assisted phytoremediation, but also showed that horizontal gene transfer to native poplar endophytes can occur in planta (Taghavi et al., 2005).

Studies of the endophytic populations of poplar and willow will not only be of potential use in enhancing plantation growth or phytoremediation, but also of value in our understanding of how these pioneer species are able to colonize rocky substrates in riparian environments containing little organic material. Based on our earlier work on identifying a Rhizobium species in greenhouse-grown hybrid cottonwood (Doty et al., 2005), we hypothesized that nitrogen-fixing bacteria may be endophytes of poplar and willow in their native habitat. In this paper, we report the identification of a diazotrophic community within these tree species that may lead to an explanation of how these trees survive in nutrient-poor areas.

2. Materials and Methods Collection of endophytes

Cuttings of young poplar and willow were collected

from Three Forks Park alongside the Snoqualmie River in Western Washington. The Three Forks Park area at the Snoqualmie River near the towns of North Bend and Snoqualmie is used by University of Washington (UW) researchers as an example of a near-natural riverine system (Fig. 1). The site is regularly disturbed by flooding which exposes bare mineral soils and gravel bars on which riparian cottonwoods and willows commonly establish (Braatne et al., 1996). Cuttings (approx. 8 cm) were placed in flasks of Nitrogen-Free Medium (NFM, Qubit Systems) and allowed to sprout. Cuttings of the new growth were collected, surface-sterilized with 10% bleach for 10 minutes and 1% Iodophor for 5 minutes, rinsed three times, and sections were then placed on MS (Murashige and Skoog, 1962) plates. Non-sectioned cuttings of the new growth did not result in bacterial growth; therefore, the growth is most likely due to endophytes being exposed to the medium from

DIAZOTROPHIC ENDOPHYTES 25

Figure 1. Cottonwood and willow plants growing in the rocky substrate at Three Forks Fork in Snoqualmie, Washington.

Table 1. Bacterial endophyte isolates of black cottonwood and willow.

Isolate Closest 16S rDNA Growth nifH Acet. name match on NFM red.

WP-B Burkholderia vietnamiensis ++ + + WP-C Pantoea sp. – ND ND WP2 Pseudomonas graminis – ND – WP5 Rahnella sp. CDC 2987-79 +++ + + WP7 Enterobacter sp. YRL01 ++ ND – WP9 Burkholderia sp. H801 ++ + + WP19 Acinetobacter calcoaceticus ++ + – WW1 Acinetobacter sp. PHD-4 ++ ND – WW2 Herbaspirillum +++ ND + WW4 Stenotrophomonas sp. LQX-11 + ND – WW5 Sphingomonas yanoikuyae +++ + – WW6 Pseudomonas sp. H9zhy + + + WW7 Sphingomonas sp. ZnH-1 +++ + – WW8 Pseudomonas sp. H9zhy – ND – WW11 Sphingomonas yanoikuyae +++ ND – WW12 Sphingomonas sp. ZnH-1 +++ ND – WW13 Pseudomonas sp. WAI-21 ++ ND –

WP, wild poplar isolates from P. trichocarpa; WW, wild willow isolates from Salix sitchensis; NFM, nitrogen-free medium; ND, not determined; Acet. red., acetylene reduction assay.

the cut sites. Morphologically-distinct colonies were streak-purified on yeast mannitol agar (YMA) plates. Cultures were then frozen at -80ºC in glycerol.

Growth on nitrogen-limited medium

Isolates from frozen stocks were streaked onto YMA

plates and incubated at 28ºC. Isolated colonies were then streaked onto Ashby’s Nitrogen Free Medium (NFM)

Table 2. Nitrogen-free medium (NFM) from Qubit. Working solution was prepared using a 1:2000 dilution of the following stock solutions. Final pH was adjusted to 6.8.

M g/l

1 0.514 KH2PO4 69.9 2 0.114 K2HPO4 19.84 3 1.004 K2SO4 174.7 4 0.486 MgSO4 · 7H2O 119.7 5 0.492 MgCl2 · 6H2O 100 6 1.496 CaCl2 · H2O 219.8 7 0.02 MnSO4 · H2O 3.38 8 0.002 CuSO4 · 5 H2O 0.5 9 0.002 ZnSO4 · 7H2O 0.55 10 0.062 H3BO3 3.83 11 0.001 NaMoO4 · 2H2O 0.24 12 0.0004 CoSO4 · 6.5H2O 0.11 13 0.076 Fe from Fe Sequestrine

containing 20 g/l sucrose as the carbon source, and growth was assessed after four days. Ashby's NFM contains the following (g/l): K2HPO4, 0.20; MgSO4·7H2O, 0.20; NaCl, 0.20; K2SO4, 0.10; Ca2CO3, 5.00; agar, 15.00. After autoclaving, 20 ml/l of Hutner's salts (Hutner, 1972) and 10 ml/l of vitamin solution (Staley, 1968) were added. For growth curve assays, a different medium was chosen due to the high level of precipitants in Ashby’s medium. The American Type Culture Collection (ATCC) Medium #240 is a nitrogen-free medium for growth of Azotobacter (www.atcc.org). One liter of the broth included 50 mg K2HPO4, 150 mg KH2PO4, 200 mg MgSO4·7H2O, 20 mg CaCl2, and 2 ml trace mineral solution (Xin et al., in review) (515.3 mg/l FeSO4·7H2O, 158.1 mg/l ZnSO4·7H2O, 150.0 mg/l MnSO4, 27.6 mg/l CuSO4·5H2O, 28.1 mg/l CoCl2·6H2O, 16.1 mg/l Na2MoO4·2H2O, 24.7 mg/l H3BO3, 24.9 mg/l KI, 11 mg/l NiCl2·6H2O, 67.5 mg/l Al2(SO4)3·18H2O, and 3432.8 mg/l Na2EDTA). The pH of the broth was adjusted to 7.0 with 1 M NaOH. A homogeneous inoculum from the isolated colonies in the modified ATCC NFM was used to inoculate 25 ml of ATCC NFM containing 1% sucrose. Growth was monitored by measuring optical density at 600 nm. All flasks were washed thoroughly and treated with acid (10% hydrochloric acid) followed by three rinses in E-Pure water prior to autoclaving. Some of the endophytes grew better in Nitrogen-Free Murashige and Skoog Medium formulated for plants (Caisson MSP007; www.caissonlabs.com) whereas others grew better in Qubit Systems nitrogen-free medium (Table 2).

Identification of endophytes

Genomic DNA was prepared from individual isolates,

and PCR was performed using the universal 16S rDNA primers, 8F and 1492, as described previously (Doty et al.,

26 S.L. DOTY ET AL.

2005). The 1.5 Kb PCR products were purified using a gel extraction kit (Qiagen) and subcloned into pGEM T Easy (Promega). The 16S rRNA gene was sequenced using the T7 and SP6 primer sites on the vector by the University of Washington Biochemistry Department Sequencing Facility using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI3730 XL sequencer (Applied Biosystems). DNA sequences were assembled using the Seqman software (DNA STAR Inc.) and analyzed using BLAST (Altschul et al., 1997). Taxonomic determination was based upon the maximum score. All had an E-value of 0.0. The maximum value, maximum identity, and query coverage, respectively, were as follows: WPB (2599, 99%, 99%), WPC (2659, 99%, 99%), WP2 (2565, 98%, 100%), WP5 (2610, 99%, 100%), WP7 (2589, 98%, 100%), WP9 (2630, 99%, 99%), WP19 (2607, 99%, 100%), WW1 (2693, 99%, 99%), WW2 (2619, 99%, 99%), WW4 (2682, 99%, 99%)), WW5 (2536, 99%, 99%), WW6 (2560, 97%, 99%), WW7 (2554, 99%, 99%), WW8 (2672, 99%, 99%), WW11 (2594, 99%, 99%), WW12 (2574, 99%, 99%), and WW13 (2645, 99%, 99%).

Cloning of nitrogenase gene fragments

Genomic DNA from some of the isolates was subjected

to nested nifH PCR using the technique of Burgmann (Burgmann et al., 2004). The universal nifH primers were used in the first round of PCR. One microliter of the 25 µl sample was then used in the nested PCR reaction using the internal nifH primers as described (Burgmann et al., 2004). The 371 bp products were gel-purified, cloned into pGEM T Easy, and sequenced. Raw sequence data were edited using Sequencher (Gene Codes, Ann Arbor, MI), and the sequences were subsequently incorporated into the ARB software package (Ludwig et al., 2004) for phylogenetic analysis. Translated DNA sequences were aligned manually and used for phylogenetic tree reconstruction using maximum-likelihood methods.

Acetylene reduction assay

The acetylene reduction assay was used for examining

the nitrogen-fixing activity of the bacterial isolates, and was performed according to the method previously described earlier (Kessler and Leigh, 1999). Sixteen ml of NFM agar containing 3% sucrose and 1.5% noble agar (BD, Franklin Lakes, New Jersey) was added into each 27-ml Balch test tube. Bacterial cultures were grown in YPD (Yeast extract/Peptone/Dextrose) broth for 24 hours; the cells were then pelleted and grown in NFM with 3% sucrose for another 24 hours. Twenty ml of each bacterial isolate culture (adjusted to an OD600 = 0.7) were stabbed into the NFM agar (about 1 cm deep) in the Balch test tubes before sealing. Acetylene gas was injected into the head space (11 ml) of the test tubes at a final concentration of 0.1–5% (v/v)

and incubated for 3–7 days at 30oC. The ethylene peak was identified on a gas chromatograph using a column at 85oC containing a mixture of Poropak N and Poropak Q attached to a flame ionization detector. Positive nitrogen fixation activity of bacterial cultures was demonstrated by increased ethylene concentration over time in the acetylene reduction assay.

3. Results and Discussion Poplar and willow endophyte isolations

In earlier studies, we found that surface-sterilized hybrid

cottonwood stems from plants grown in fertilized soil commonly contained the endophytic bacterium, Rhizobium tropici bv. populus (Doty et al., 2005). Because this species is known to fix atmospheric nitrogen and because the native environment of cottonwood is nutrient-poor, we speculated that cottonwood may have the ability to establish symbiotic relationships with nitrogen-fixing microbes in order to survive in nitrogen-limited settings. To test this hypothesis, we began studying the endophytes of black cottonwood (Populus trichocarpa) and of sitka willow (Salix sitchensis) in their joint native habitat along the Snoqualmie River in Western Washington.

Growth in nitrogen-limited medium

As a first screen for the ability to fix nitrogen, we

streaked the isolates onto Ashby’s nitrogen-free medium with either glucose or sucrose as the carbon source. Because sucrose is transportable in plants, we speculated that the endophytes might prefer this carbon source. A high percentage of the endophytes were able to grow on this nitrogen-free medium. The isolates from black cottonwood at the Snoqualmie River site are designated by a “WP” (Wild Poplar) to differentiate them from our earlier isolates from Populus trichocarpa x deltoides hybrid poplar (PTD). A designation of “WW” refers to isolates from sitka willow (Wild Willow). A majority of the endophyte isolates from native willow at the Snoqualmie River site grew on medium lacking ammonium and nitrate. The best-growing isolates on plates were the black cottonwood endophytes WPB, WP4, WP5, WP7, and WP9 and the willow endophytes WW5, WW9, and WW11. Growth was confirmed by incubating the strains in nitrogen-free broth and monitoring optical density over time. Representative growth curves are shown in Fig. 2. The endophytes grew at different rates in different formulations of nitrogen-free medium, and no one medium was best for all the strains. Azotobacter vinelandii, a known aerobic nitrogen-fixing bacteria, and Agrobacterium tumefaciens strain C58, a plant-associated bacterium known to not contain the nitrogenase gene, were included for comparison. The poplar endophyte, WPB,

DIAZOTROPHIC ENDOPHYTES 27

grew rapidly in multiple experiments, but growth rates leveled off after approximately one day of growth (Fig. 2A). This appears to be due to acid production from this strain (Gang Xin, unpublished). In multiple experiments, the willow endophytes grew well, and much faster than the Azotobacter control strain (Figs. 2A and 2B). The willow endophytes had sustained growth even in week-long experiments, and reached higher optical densities in NFMS (Caisson Labs) than in the ATCC Azotobacter medium.

Identification of endophytes

Isolates were identified by sequencing of the 16S rRNA

gene. As shown in Table 1 and Fig. 3, BLAST searches revealed close matches (up to 99%) to known plant-associated microbes including Burkholderia, Rahnella, Pseudomonas, Acinetobacter, Pantoea, Herbaspirillum, and Rhizobium.

0.01

0.1

1

0

20

40

60

80

100

120

OD

600

Time (hrs)

Azotobacter

C58

WPB

WW9

B

B

B B

BB BJ

J

J J J J J

HH

HH H

H H

F

F

F

F

F

F F

3

3

33

3

33

1

1

1 1

1

1 1

>

>

>

>

>

>

>

0.01

0.1

1

10

0 10 20 30 40 50 60 70 80

OD

600

Time (hrs)

B C58

J Azotobacter

H WPB

F WP19

3 WW2

1 WW5

> WW6

0.01

0.1

1

0 12 24 36 48

OD

60

0

Time (hr)

Azotobacter

WPB

WP5

WP7

WP9

WP19

Figure 2. Growth of bacteria in nitrogen-free medium with sucrose as the carbon source. Azotobacter vinelandii was included as a positive control, and Agrobacterium tumefaciens strain C58 served as the negative control. A) Growth in ATCC NFM. B) Growth in NFMS (Caisson). C) Growth in NFM (Quibit).

The 16S rRNA gene sequence of isolate WPB (accession number EU563934) was most closely related to Burkholderia vietnamiensis (99% identity; 1485/1491), and WP9 is closely related to Burkholderia sp. H801 (99% identity; 1463/1465). For years, it was believed that nitrogen fixation was limited in the genus Burkholderia to only the species, B. vietnamiensis, originally isolated from rice in Vietnam, but now it is recognized that nitrogen fixing ability is common in this genus (Caballero-Mellado et al., 2004). Burkholderia has been isolated from tissues of a variety of non-legumes including maize, sugarcane, sorghum and coffee plants (Caballero-Mellado et al., 2004). Burkholderia are in the β-class of Proteobacteria, and their discovery within nodules in 2001 ended the dogma that only bacteria of the alpha subdivision were able to nodulate legumes (Chen et al., 2003; Moulin et al., 2001). Some Burkholderia sp. are also excellent PCB-degraders, making this endophyte a candidate for endophyte-assisted phytoremediation (Fain and Haddock, 2001).

The 16S rRNA gene sequence of WPC was closely related (1488/1499) to that of Pantoea sp. P101, an isolate found in a study of diazotrophic endophytes from grasses (Riggs et al., 2002). It was also closely related to Enterobacter sp. YRL01 and sp. J11 (both 1488/1498). WP2 was closely related to that of Pseudomonas graminas, a yellow-pigmented, plant-associated bacterium identified from grasses (Behrendt et al., 1999). Unlike the other endophytes isolated from cottonwood, WPC and WP2 did not grow on nitrogen-free medium.

The 16S rRNA gene sequences of WP5 (99%; 1469/1483), as well as an epiphyte isolate WP4 from poplar leaves, are closely related to those of Rahnella aquatilis. Both Rahnella strains showed the strongest growth on NFM agar. Rahnella aquatilis, a plant-associated bacterium with biocontrol properties on fruit (Calvo et al., 2007), has been shown to fix nitrogen in the rhizosphere of wheat and maize (Berge et al., 1991). It was recently isolated from seeds of Norway spruce where it was shown to have growth-promoting effects (Cankar et al., 2005).

The 16S rRNA sequence of WP7 is closely related to Pantoea agglomerans (98% identity; 1464/1481). Pantoa agglomerans is a known diazotrophic endophyte of rice, and has been shown not only to fix nitrogen but also produce phytohormones and promote plant growth (Feng et al., 2006). This species is also an endophyte of sweet potato stem where it was shown to be diazotrophic by the acetylene reduction assay (Asis and Adachi, 2004).

The sequence of the 16S rRNA fragment of WP19 had 99% identity with Acinetobacter calcoaceticus type strain NCCB22016. Acinetobacter calcoaceticus is a common soil bacterium, and was found in an analysis of endophytic bacteria from soybean (Kuklinsky-Sobral et al., 2005). However, growth in nitrogen-free medium and the presence of the nifH gene have not been previously reported for this strain.

A

B

C

28 S.L. DOTY ET AL.

The 16S rRNA sequence of the willow endophyte,

WW1, had 99% identity (1496/1498) with Acinetobacter sp. PHD-4, a phenol-degrading bacterial species (Wang et al., 2007). The 16S rDNA of isolate WW2 was a close match to that of Herbaspirillum (98%; 1477/1492 bases). Like Burkholderia, Herbaspirillum is classified in the β-Proteobacteria Class. This genus includes known nitrogen-fixing endophytes, and has been found in a variety of non-legumes including maize, wheat, oat, and sugarcane. For example, it was demonstrated to fix nitrogen in planta in wild rice (Elbeltagy et al., 2001).

The 16S rRNA sequence of the willow endophyte, WW4, had 99% identity (1496/1505) with Stenotrophomonas sp. LQX-11. Stenotrophomonas was the

Figure 3. Phylogenetic relationships of endo-phytic bacteria (bold font) based on 16S rRNA gene sequences. Additional taxa shown represent a non-redundant list of isolates most closely related to each endophyte. Tree was reconstructed using the maximum-likelihood method of PhyML implemented in ARB (Ludwig et al., 2004) based on nucleotide alignment by the NAST algorithm (DeSantis et al., 2006) with a maximum frequency filter excluding positions with <50% homology.

dominant genus in a study of rice endophytes (Sun et al. 2008). This genus may have potential use for bioremediation as some species are able to degrade phenolic compounds such as p-nitrophenol and 4-chlorophenol (Liu et al., 2007).

There were several isolates from willow (WW5, WW7, WW9, WW11, WW12) that matched Sphingomonas species. The genus Sphingomonas contains strictly aerobic, chemo-heterotrophic, usually yellow pigmented bacteria that contain glycosphingolipids as cell envelope components and belong to the α-4-subgroup of the Proteobacteria (Takeuchi et al., 2001). In addition to being isolated from a wide variety of environments, sphingomonads have also been detected in rather high cell

DIAZOTROPHIC ENDOPHYTES 29

densities on the surfaces of various plants (Kim et al., 1998). The genus Sphingomonas is becoming increasingly of interest in environmental microbiology because various xenobiotic-degrading organisms belong to this group. Sphingomonas strains have been described that degrade compounds such as PCPs (Ederer et al., 1997), PAHs (Khan et al., 1996; Rentz et al., 2005), chlorinated phenols (Yrjala et al., 1998), herbicides (Zipper et al., 1996), and a variety of benzofurans (Harms et al., 1995) and aromatic hydrocarbons (Zylstra and Kim, 1997). Sphingomonas yanoikuyae was identified in rhizoplane bacteria from paddy rice, and the authors suggested that some of the bacteria might have a role in nitrogen fixation (Hashidoko et al., 2006). Adhikari and colleagues (2001) reported nitrogen fixation by Sphingomonas spp. among the rhizosphere of rice plants.

Three willow isolates were identified as species within the genus Pseudomonas. The 16S rRNA gene sequences of WW6 and WW8 had 97–99% identity with Pseudomonas sp. H9zhy, and share 96% identity with each other (1470/1516 bases). The 16S rRNA sequence of WW13 matched Pseudomonas sp. WAI-21 (99%; 1492/1505). Beneficial Pseudomonas strains are frequently found associated with plants where they act as Plant Growth Promoting Bacteria (PGPB) by suppressing growth of

Figure 4. Phylogenetic relationships of nifH sequences retrieved from endophytic bacteria (bold font). Tree was reconstructed using the maximum-likelihood method of ProML implemented in ARB (Ludwig et al., 2004) based on manual alignment of amino-acid sequences.

pathogens or by producing plant growth hormones. Nitrogen-fixing Pseudomonas isolates were identified from rice plants (Muthukumarasamy et al., 2007). These P. putida isolates from rice contained nif genes and were positive in the acetylene reduction assay. In addition, there are several reports of plant-growth-promoting Pseudomonas species that enhance phytoremediation of trichloroethylene and polychlorinated biphenyls (recently reviewed in Zhuang et al., 2007). WAI-21, to which WW13 was most related, was identified as a strain that degrades the organophosphate pesticide Ethion (Foster et al., 2004). Analysis of nifH sequences

The nested PCR approach to clone nifH from

environmental samples was used successfully on 8 of the isolates from cottonwood and willow (Table 1 and Fig. 3). Since this method requires that the appropriate nitrogenase-specific primers for the species are known, we designated a negative result as “not determined” rather than “minus” where the strains did not yield a nif PCR product. The nifH gene sequences did not always align with the 16S rRNA matches (Minerdi et al., 2001). The five isolates sequenced here belonged to either alpha, beta, or gamma-proteobacteria on the basis of 16S sequencing, but the nifH phylogeny suggests a more complicated evolutionary history. For example, WP19 is most closely related to a Gamma-proteobacterium, but its nifH sequence belongs to a clade containing sequences from alpha and beta-proteobacteria. Similarly, WW5 is most closely related to Sphingomonas, an alpha-proteobacterium, but its nifH sequence is most similar to those from Anabaena, a cyanobacterium, and Frankia, an actinobacterium. Perhaps most strikingly, WP-B and WP9 were both most closely related to Burkholderia, a Beta-proteobacterium, but the nifH sequences from these two strains belonged to completely separate clades. This incongruence has been noted in other nitrogen-fixing bacteria and can best be explained by horizontal gene transfer of the nif genes (Minerdi et al., 2001). Certainly in the case of WP-B and WP19, there appear to have been multiple horizontal transfer events.

WW6 and WW8, both identified as most closely related to Pseudomonas sp. H9zhy, but only 96% identical to each other, had different phenotypes. WW6 grew on nitrogen-free medium, had the nifH gene, and was positive in the acetylene reduction assay. However, WW8 did not grow on the nitrogen-free medium, and was negative for both the nifH PCR and the acetylene reduction assay. Further research is needed to determine if WW6 perhaps harbors a plasmid which WW8 lacks that enables it to fix nitrogen.

Acetylene reduction assay

The acetylene reduction assay is an indirect test of

30 S.L. DOTY ET AL.

Figure 5. Acetylene reduction assay. Ethylene produced by bacterial endophytes after 72 hours of exposure to acetylene. nitrogenase assay that takes advantage of the non-specific activity of the nitrogenase enzyme to reduce acetylene to ethylene gas that can be quantified by gas chromatography. Isolates that were positive in the acetylene reduction assay were WPB, WP5, WP9, WW2, and WW6 (Fig. 5). Although the other nifH-containing isolates were also tested in this assay, there was no clear ethylene production. The ethylene production by the 5 positive strains varied, with the Burkholderia isolate, WPB, having the highest ethylene production.

4. Conclusions

This initial study of the endophytes of cottonwood and

willow in their native habitat revealed the presence of several microbes that grow on nitrogen-free medium. These growth experiments were all performed under aerobic conditions. It is quite possible that some endophytes require a microaerobic environment for efficient nitrogen fixation since nitrogenase is oxygen-sensitive. Furthermore, some may require an association with the plant before nitrogen fixation occurs. Nevertheless, these data show that both poplar and willow harbor microorganisms that grow well under nitrogen-limited conditions.

Surprisingly, none of the endophytes we isolated from cottonwood were identical to any of the endophytes of willow, even though both tree species were growing at the same site within a meter of each other. This differential “recruitment” of endophytes has been noted in other studies of endophyte populations from plants growing in the same location, especially on contaminated sites (Siciliano et al., 2001). This finding is consistent with a co-evolutionary process whereby the endophytic bacteria may have evolved in a coordinated fashion with the host plants in a manner similar to that of Buchnera and aphids (Moran et al., 1993).

Although many of the isolates grew well on nitrogen-free medium, not all were confirmed to contain the nitrogenase gene or to have acetylene reduction activity. The nested PCR technique for nifH gene amplification

requires a strong degree of sequence identity that may be lacking in some of the isolates. A negative result from nifH PCR therefore does not necessarily mean that the gene is absent, but simply that adequate primers have not yet been utilized. There have also been reports of microbes that can grow in nitrogen-free medium yet were negative in the acetylene reduction assay. One possible explanation is that the test conditions may not be optimized for these isolates even though other isolates showed acetylene-reducing activity under the same conditions. Alternatively, the capacity to reduce acetylene might not be essential for a functional nitrogenase. For example, Gadkari et al. showed that the nitrogenase of Streptomyces thermoautotrophicus did not reduce acetylene and was not inhibited by acetylene (Gadkari et al., 1992). The nitrogenase enzyme was purified from this organism and verified to be unable to reduce ethine or ethene (Ribbe et al., 1997). Furthermore, Brighnigna and colleagues demonstrated that some epiphytic isolates could grow in nitrogen-free medium yet they were acetylene reduction negative (Brighnigna et al., 1992). Ozawa et al. described the isolation of 42 endophytes from which the nifH gene fragment could be isolated and could grow on nitrogen-free medium yet were negative for the acetylene reduction assay (Ozawa et al., 2003). Therefore, this indirect assay for nitrogen fixation may not be an absolute determinant for nitrogen fixation.

This preliminary study is an initial survey of the endophytes of black cottonwood and sitka willow in their native habitat. Nonetheless, our small collection of isolates has already yielded important information on some of the diversity of poplar endophytes. Several of the isolates are related to strains with important pollutant degradation ability; therefore they may be useful in phytoremediation studies. The high frequency of diazotrophic bacteria in these non-leguminous trees points to an as yet unexplored symbiosis with trees. The harboring of nitrogen-fixing microorganisms within tree stems may be an adaptation to the harsh environment in which these colonizing trees germinate: nutrient-poor gravel with frequent flooding. These tree seedlings must rapidly take root and draw from a source of nitrogen. We interpret the evidence for multiple horizontal transfers of nifH genes as support for the hypothesis that acquisition of these genes and the ability to grow diazotrophically is an important ecological event that has conferred a selective advantage on the bacterial strains found in our study. The presence of endophytes may play a vital role in the biology of poplar. It is necessary to next determine if nitrogen fixation by these microbes is occurring within poplar and willow, and if the fixed nitrogen is utilized by these plants.

A paper was recently published on the diversity of endophytes within four clones of poplar grown at two different sites (Ulrich et al., 2008). The authors reported the identification of a diverse group of endophytes from 53 taxa. They noted that the four poplar clones harbored four

DIAZOTROPHIC ENDOPHYTES 31

distinct endophytic populations, further supporting the hypothesis that plant genotype plays a role in determining which bacteria can colonize the host. The authors detected a high abundance, up to 21% of the 16S rRNA gene clones, of bacteria belonging to the Sphingomonas genus, indicating that this genus may play an important role in poplar. In our study of willow endophytes, Sphingomonas isolates (WW5, 7, 9, 11, and 12) were the most abundant, and all of these grew vigorously in nitrogen-free medium. Experiments to determine if these isolates help willow plants to grow in nitrogen-free medium are underway.

Acknowledgements

We thank Megan Dosher and Jessica LaTourelle for

their help with the original screening of the endophytes. We gratefully acknowledge the guidance and encouragement of Reinhard F. Stettler. This research was not funded by grants, but supported by the UW College of Forest Resources. REFERENCES Adhikari, T.B., Joseph, C.M., Yang, G., Phillips, D.A., and

Nelson, L.M. 2001. Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice. Canadian Journal of Microbiology 47: 916–924.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402.

Asis, C.A., Jr. and Adachi, K. 2004. Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Entero-bacter asburiae from sweetpotato stem in Japan. Letters in Applied Microbiology 38: 19–23.

Barac, T., Taghavi, S., Borremans, B., Provoost, A., Oeyen, L., Colpaert, J.V., Vangronsveld, J., and van der Lelie, D. 2004. Engineered endophytic bacteria improve phyto-remediation of water-soluble, volatile, organic pollutants. Nature Bio-technology 22: 583–588.

Behrendt, U., Ulrich, A., Schumann, P., Erler, W., Burghardt, J., and Weyfarth, W. 1999. A taxonomic study of bacteria isolated from grasses: a proposed new species, Pseudomonas graminis sp nov. International Journal of Systematic Bacteriology 49: 297–308.

Berge, O., Heulin, T., Achouak, W., Richard, C., Bally, R., and Balandreau, J. 1991. Rahnella aquatilis, a nitrogen-fixing enteric bacterium associated with the rhizosphere of wheat and maize. Canadian Journal of Microbiology 37: 195–203.

Boerjan, W. 2005. Biotechnology and the domestication of forest trees. Current Opinion in Biotechnology 16: 1–8.

Braatne, J.H., Rood, S.B., and Heilman, P.E. 1996. Life history, ecology, and conservation of riparian cottonwoods in North America. In: Biology of Populus and its Implications for Management and Conservation. Stettler, R.F., Bradshaw, H.D., Heilman, P.E., and Hinckley, T.M., eds. NRC Research Press, Ottawa, pp. 57–85.

Brighnigna, L., Montaini, P., Favilla, F., and Trejo, A.C. 1992. The role of the nitrogen-fixing bacterial microflora in the epiphytism of Tillandsia (Bromeliaceae). American Journal of Botany 79: 723–727.

Burgmann, H., Widmer, F., Sigler, W.V., and Zeyer, J. 2004. New molecular screening tools for the analysis of free-living diazotrophs in soil. Applied and Environmental Microbiology 70: 240–247.

Caballero-Mellado, J., Martinez-Aguilar, L., Paredes-Valdez, G., and Estrada-de Los Santos, P. 2004. Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species. International Journal of Systematic and Evolutionary Microbiology 54: 1165–1172.

Calvo, J., Calvente, V., de Orellano, M.E., Benuzzi, D., and Sanz de Tosetti, M.I. 2007. Biological control of postharvest spoilage caused by Penicillium expansum and Botrytis cinerea in apple by using the bacterium Rahnella aquatilis. International Journal of Food Microbiology 113: 251–257.

Cankar, K., Kraigher, H., Ravnikar, M., and Rupnik, M. 2005. Bacterial endophytes from seeds of Norway spruce (Picea abies L. Karst). FEMS Microbiology Letters 244: 341–345.

Chen, W.M., Moulin, L., Bontemps, C., Vandamme, P., Bena, G., and Boivin-Masson, C. 2003. Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature. Journal of Bacteriology 185: 7266–7272.

Cocking, E.C. 2005. Intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers. In vitro Cellular and Developmental Biology-Plant 41: 369–373.

Cook, R.J., Thomashow, L.S., Weller, D.M., Fujimoto, D., Mazzola, M., Bangera, G., and Kim, D.-S. 1995. Molecular mechanisms of defense by rhizobacteria against root disease. Proceedings of the National Academy of Sciences USA 92: 4197–4201.

DeSantis, T.Z., Hugenholtz, P., Keller, K., Brodie, E.L., Larsen, N., Piceno, Y.M., Phan, R., and Andersen, G.L. 2006. NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucleic Acids Research 34: 394–399.

Döbereiner, J. 1992. History and new perspectives of diazotrophs in association with non-leguminous plants. Symbiosis 13: 1–13.

Döbereiner, J. and Pedrosa, F.O. 1987. Nitrogen Fixing Bacteria In Non-Leguminous Crop Plants. Science Tech Publishers, Madison, WI, USA.

Döbereiner, J. 1977. N2 fixation associated with non-leguminous plants. Basic Life Science 9: 451–461.

Doty, S.L. 2008. Tansley Review: Enhancing phytoremediation through the use of transgenics and endophytes. New Phytologist doi: 10.1111/j.1469–8137.2008.02446.x.

Doty, S.L., Dosher, M.R., Singleton, G.L., Moore, A.L., van Aken, B., Stettler, R.F., Strand, S.E., and Gordon, M.P. 2005. Identification of an endophytic Rhizobium in stems of Populus. Symbiosis 39: 27–36.

Ederer, M.M., Crawford, R.L., Herwig, R.P., and Orser, C.S. 1997. PCP degradation is mediated by closely related strains of the genus Sphingomonas. Molecular Ecology 6: 39–49.

Elbeltagy, A., Nishioka, K., Sato, T., Suzuki, H., Ye, B., Hamada, T., Isawa, T., Mitsui, H., and Minamisawa, K. 2001. Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Applied and Environmental Microbiology 67: 5285–5293.

Fain, M.G. and Haddock, J.D. 2001. Phenotypic and phylogenetic characterization of Burkholderia (Pseudomonas) sp. strain LB400. Current Microbiology 42: 269–275.

32 S.L. DOTY ET AL.

Feng,Y., Shen, D., and Song, W. 2006. Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates. Journal of Applied Microbiology 100: 938–945.

Flores-Encarnacion, M., Contreras-Zentella, M., Soto-Urzua, L., Aguilar, G.R., Baca, B.E., and Escamilla, J.E. 1999. The respiratory system and diazotropic activity of Acetobacter diazotrophicus PAL5. Journal of Bacteriology 181: 6987–6995.

Foster, L.J., Kwan, B.H., and Vancov, T. 2004. Microbial degradation of the organophosphate pesticide, Ethion. FEMS Microbiology Letters 240: 49–53.

Gadkari, D., Morsdorf, G., and Meyer, O. 1992. Chemolitho-autotrophic assimilation of dinitrogen by Streptomyces thermoautotrophicus UBT1: identification of an unusual N2-fixing system. Journal of Bacteriology 174: 6840–6843.

Germaine, K., Keogh, E., Garcia-Cabellos, G., Borremans, B., van der Lelie, D., Barac, T., Oeyen, L., Vangronsveld, J., Moore, F.P., Moore, E.R.B., Campbell, C.D., Ryan, D., and Dowling, D.N. 2004. Colinisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiology Ecology 48: 109–118.

Harms, H., Wilkes, H., Wittich, R., and Fortnagel, P. 1995. Metabolism of hydroxydibenzofurans, methoxydibenzofurans, acetoxydibenzofurans, and nitrodibenzofurans by Sphingomonas sp. strain HH69. Applied and Environmental Microbiology 61: 2499–2505.

Hashidoko, Y., Hayashi, H., Hasegawa, T., Purnomo, E., Osaki, M., and Tahara, S. 2006. Frequent isolation of sphingomonads from local rice varieties and other weeds grown on acid sulphate soil in South Kalimantan, Indonesia. Tropics 154: 395.

Hirsch, A.M. 2004. Plant-microbe symbioses: A continuum from commensalism to parasitism. Symbiosis 37: 345–363.

Hutner, S.H. 1972. Inorganic nutrition. Annual Review of Microbiology 26: 313–346.

Kessler, P.S. and Leigh, J.A. 1999. Genetics of nitrogen regulation in Methanococcus maripaludis. Genetics 152: 1343–1351.

Khan, A.A., Wang, R.F., Cao, W.W., Franklin, W., and Cerniglia, C.E. 1996. Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing. International Journal of Systematic Bacteriology 46: 466–469.

Kim, H., Nishiyama, M., Kunito, T., Senoo, K., Kawahara, K., Murakami, K., and Oyaizu, H. 1998. High population of Sphingomonas species on plant surface. Journal of Applied Microbiology 85: 731–736.

Kuklinsky-Sobral, J., Welington, L.A., Mendes, R., Pizzirani-Kleiner, A.A., and Azevedo, J.L. 2005. Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide. Plant and Soil 273: 91–99.

Liu, Z., Yang, C., and Qiao, C.L. 2007. Biodegradation of p-nitrophenol and 4-chlorophenol by Stenotrophomonas sp. FEMS Microbiology Letters 277: 150–156.

Ludwig, W., Strunk, O., Westram, R., and et al. 2004. ARB: A software environment for sequence data. Nucleic Acids Research 14: 358–366.

Mastretta, C., Barac, T., Vangronsveld, J., Newman, L., Taghavi, S., and van der Lelie, D. 2006. Endophytic bacteria and their potential application to improve the phytoremediation of contaminated environments. Biotechnology and Genetic Engineering 23: 175–207.

Minerdi, D., Fani, R., Gallo, R., Boarino, A., and Bonfante, P. 2001. Nitrogen fixation genes in an endosymbiotic Burkholderia strain. Applied and Environmental Microbiology 67: 725–732.

Moran, N.A., Munson, M.A., Baumann, P., and Ishikawa, H. 1993. A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts. Proceedings of the Royal Society of London B253: 167–171.

Moulin, L., Munive, A., Dreyfus, B., and Boivin-Masson, C. 2001. Nodulation of legumes by members of the b-subclass of Proteobacteria. Nature 411: 948–950.

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiology of the Plant 15: 473–497.

Muthukumarasamy, R., Kang, U.G., Park, K.D., Jeon, W.T., Park, C.Y., Cho, Y.S., Kwon, S.W., Song, J., Roh, D.H., and Revathi, G. 2007. Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants. Journal of Applied Microbiology 102: 981–991.

Nejad, P. and Johnson, P.A. 2000. Endophytic bacteria induce growth promotion and wilt disease suppression in oilseed rape and tomato. Biological Control 18: 208–215.

Ozawa, T., Ohwaki, A., and Okumura, K. 2003. Isolation and characterization of diazotrophic bacteria from the surface-sterilized roots of some legumes. Scientific Report of the Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University 55: 29–36.

Reinhold-Hurek, B. and Hurek, T. 1998. Life in grasses: diazotrophic endophytes. Trends in Microbiology 6: 139–144.

Reis, V.M., Baldani, J.I., Baldani, V.L.D., and Döbereiner, J. 2000. Biological dinitrogen fixation in gramineae and palm trees. Critical Reviews in Plant Sciences 10: 227–247.

Rentz, J.A., Alvarez, P.J.J., and Schnoor, J.L. 2005. Benzo[a]pyrene co-metabolism in the presence of plant root extracts and exudates: Implications for phytoremediation. Environmental Pollution 136: 477–484.

Ribbe, M., Gadkari, D., and Meyer, O. 1997. N2 fixation by Streptomyces thermoautotrophicus involves a molybdenum-dinitrogenase and a manganese-superoxide oxidoreductase that couple N2 reduction to the oxidation of superoxide produced from O2 by a molybdenum-CO dehydrogenase. Journal of Biological Chemistry 272: 26627–26633.

Riggs, P.J., Moritz, R.L., Chelius, M.K., Dong, Y., Iniguez, A.L., Kaeppler, S.M., Casler, M.D., and Triplett, E.W. 2002. Isolation and characterization of diazotrophic endophytes from grasses and their effects on plant growth. In: 13th International Congress on Nitrogen Fixation. Hamilton, Ontario, Canada. Nitrogen Fixation: A Global Perspective. pp. 263–267.

Ryan, R.P., Germaine, K., Franks, A., Ryan, D.J., and Dowling, D.N. 2008. Bacterial endophytes: recent developments and applications. FEMS Microbiology Letters 278: 1–9.

Sevilla, M., Burris, R.H., Gunapala, N., and Kennedy, C. 2001. Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif- mutant strains. Molecular Plant Microbe Interactions 14: 358–366.

Siciliano, S.D., Fortin, N., Mihoc, A., Wisse, G., Labelle, S., Beaumier, D., Ouellette, D., Roy, R., Whyte, L.G., Banks, M.K., Schwab, P., Lee, K., and Greer, C.W. 2001. Selection of specific endophytic bacterial genotypes by plants in response to soil contamination. Applied and Environmental Microbiology 6: 2469–2475.

DIAZOTROPHIC ENDOPHYTES 33

Staley, J.T. 1968. Prosthecomicrobium and Ancalomicrobium: new prosthecate freshwater bacteria. Journal of Bacteriology 95: 1921–1942.

Stettler, R.F., Bradshaw, H.D., Heilman, P.E., and Hinckley, T.M. 1996. Biology of Populus and its Implications for Management and Conservation. NRC Research Press, Ottawa.

Sturz, A.V., Christie, B.R., and Nowak, S. 2000. Bacterial endophytes: Potential role in developing sustainable systems of crop production. Critical Reviews of Plant Science 19: 1–30.

Sun, L., Qiu, F., Zhang, X., Dai, X., Dong, X., and Song, W. 2008. Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microbial Ecology 55: 415–424.

Taghavi, S., Barac, T., Greenberg, B., Borremans, B., Vangronsveld, J., and van der Lelie, D. 2005. Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene. Applied and Environmental Microbiology 71: 8500–8505.

Takeuchi, M., Hamana, K., and Hiraishi, A. 2001. Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. International Journal of Systematic Evolutionary Microbiology 51: 1405–1417.

Ulrich, K., Ulrich, A., and Ewald, D. 2008. Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiology and Ecology 63: 169–180.

van Aken, B., Peres, C.M., Doty, S.L., Yoon, J.M., and Schnoor, J.L. 2004a. Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides x nigra

DN34). International Journal of Systematic and Evolutionary Microbiology 54: 1191–1196.

van Aken, B., Yoon, J.M., and Schnoor, J.L. 2004b. Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phyto-symbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34). Applied and Environmental Microbiology 70: 508–517.

Wang, Y.D., Dong, X.J., Wang, X., Hong, Q., Jiang, X., and Li, S.P. 2007. Isolation of phenol-degrading bacteria from natural soil and their phylogenetic analysis. Huan Jing Ke Xue 28: 623–626.

Yrjala, K., Suomalainen, S., Suhonen, E.L., Kilpi, S., Paulin, L., and Romantschuk, M. 1998. Characterization and reclassification of an aromatic- and chloroaromatic-degrading Pseudomonas sp., strain HV3, as Sphingomonas sp. HV3. International Journal of Systematic Bacteriology 48: 1057–1062.

Zhuang, X., Chen, J., Shim, H., and Bai, Z. 2007. New advances in plant growth-promoting rhizobacteria for bioremediation. Environmental International 33: 406–413.

Zipper, C., Nickel, K., Angst, W., and Kohler, H.P. 1996. Complete microbial degradation of both enantiomers of the chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy) propionic acid] in an enantioselective manner by Sphingomonas herbicidovorans sp. nov. Applied and Environmental Microbiology 62: 4318–4322.

Zylstra, G.J. and Kim, E. 1997. Aromatic hydrocarbon degradation by Sphingomonas yanoikuyae B1. Journal of Industrial Microbiology and Biotechnology 19: 408–414.

34 S.L. DOTY ET AL.