Symbiosis255 Doty Final

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SYMBIOSIS(2009) 47, 2333 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 Singleton

1, Zareen Khan

1,

Azra Vajzovic1, and James T. Staley

2

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. [email protected];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, andAcinetobacter. 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

syntheticnitrogenfertilizersthatcanhavenegativeeffects

*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 (Dbereiner, 1977;

Dbereiner, 1992; Dbereiner 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,


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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, includingAzoarcus andHerbaspirillum are at least

suspected of providing fixed nitrogen to their non-

leguminous plant hosts (Reinhold-Hurek and Hurek, 1998).

Further investigation of diazotrophic endophytic bacteriawill 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 occurin 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 inour 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


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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 Enterobactersp. YRL01 ++ ND

WP9 Burkholderia sp. H801 ++ + +

WP19 Acinetobacter calcoaceticus ++ +

WW1 Acinetobactersp. 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 -80C in glycerol.

Growth on nitrogen-limited medium

Isolates from frozen stocks were streaked onto YMA

plates and incubated at 28C. Isolated colonies were then

streaked onto Ashbys 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 119.75 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 growthwas assessed after four days. Ashby's NFM contains the

following (g/l): K2HPO4, 0.20; MgSO47H2O, 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 Ashbys 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 MgSO47H2O, 20 mg

CaCl2, and 2 ml trace mineral solution (Xin et al., in

review) (515.3 mg/l FeSO47H2O, 158.1 mg/l ZnSO47H2O,150.0 mg/l MnSO4, 27.6 mg/l CuSO45H2O, 28.1 mg/l

CoCl26H2O, 16.1 mg/l Na2MoO42H2O, 24.7 mg/l H3BO3,

24.9 mg/l KI, 11 mg/l NiCl26H2O, 67.5 mg/l

Al2(SO4)318H2O, 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 formulatedfor 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.,


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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 nifHprimers were

used in the first round of PCR. One microliter of the 25 l

sample was then used in the nested PCR reaction using the

internal nifHprimers 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 ARBsoftware 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 0.15% (v/v)

and incubated for 37 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 tosurvive 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 Ashbys 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 highpercentage 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

fromPopulus 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,


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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

Azotobactercontrol 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 ATCCAzotobactermedium.

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,

andRhizobium.

0.01

0.1

1

020

40

60

80

100

120

OD600

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

OD600

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 -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 ofPantoea sp. P101, an isolatefound 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 ofPseudomonas 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.

BothRahnella strains showed the strongest growth on NFM

agar. Rahnella aquatilis, a plant-associated bacterium withbiocontrol 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 nifHgene have not been previously reported for this

strain.

A

B

C


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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 ofHerbaspirillum (98%; 1477/1492 bases).

Like Burkholderia, Herbaspirillum is classified in the -

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


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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 genusPseudomonas. The 16S rRNA gene sequences of

WW6 and WW8 had 9799% identity with Pseudomonas

sp. H9zhy, and share 96% identity with each other

(1470/1516 bases). The 16S rRNA sequence of WW13matched 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 ofnifH 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 nifPCR product. The nifHgene 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 nifHsequence 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, andFrankia , an actinobacterium. Perhaps

most strikingly, WP-B and WP9 were both most closelyrelated 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

toPseudomonas 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

Theacetylenereductionassayisanindirecttestof


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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 othernifH-containing isolates were also tested

in this assay, there was no clear ethylene production. Theethylene production by the 5 positive strains varied, with

theBurkholderia 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 nitrogenfixation 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 ofBuchnera 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 42endophytes 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 inthese 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


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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.

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